MEDICAL UNIVERSITY 0 OF VIENNA Evaluation of Cartilage Repair Tissue in the Knee and Ankle Joint using Sodium Magnetic Resonance Imaging at 7 Tesla

Doctoral thesis at the Medical University ofVienna

for obtaining the academic degree

Doctor of Philosophy

Submitted by

Mag. Stefan Zbyn

Supervisor: Univ.-Prof. Dr.med.univ. Siegfried Trattnig High Field MR Center Department of Biomedical Imaging and Image-guided Therapy Medical University ofVienna, Waehringer Guertel 18-20 A-1090 Vienna, Austria

Vienna, October2015

DECLARATION

DECLARATION

I, Stefan Zbyn, declare that this PhD thesis entitled “Evaluation of Cartilage Repair Tissue in the Knee and Ankle Joint using Sodium Magnetic Resonance Imaging at 7 Tesla” was accomplished at High Field MR Center, Department of Biomedical Imaging and Image- Guided Therapy, Medical University of Vienna. I declare that all the presented data are results of original work carried out by me under the supervision of Professor Siegfried Trattnig. I have clearly specified my own contribution and acknowledged the work of all my colleagues from the High Field MR Center. I declare that work of others is quoted and the literature used in this doctoral thesis is accurately cited in the text and in the References section. This thesis, which I submit to the Medical University of Vienna for obtaining the Doctor of Philosophy academic degree, has never been submitted or published at this or at any other University.

Signed:

Date: IV TABLE OF CONTENTS

TABLE OF CONTENTS

DECLARATION...... iii

TABLE OF CONTENTS...... v

ABSTRACT...... ix

ZUSAMMENFASSUNG...... xi

PUBLICATIONS ARISING FROM THIS THESIS...... xiii

ABBREVIATIONS...... xiv

LIST OF FIGURES AND TABLES...... xvii

ACKNOWLEDGMENTS...... xix

1. INTRODUCTION...... 1

1.1 Theory of Nuclear Magnetic Resonance...... 1

1.1.1 Properties of Nucleus...... 1

1.1.2 Interaction between Spins and Static Magnetic Field ...2

1.1.2.1 Quantum-mechanical Model...... 2

1.1.2.2 Macroscopic Magnetization in a Magnetic Field ...3

1.1.2.3 Semi-classical Model - Equation of Motion...... 5

1.1.3 Interaction between Spins and Radiofrequency Field ...5

1.1.4 Relaxation Mechanisms and the Bloch Equations...... 8

1.1.4.1 Origins of Relaxations...... 8

1.1.4.2 Bloch Equations...... 9

1.1.4.3 Magnetic Field Gradients...... 9

1.1.4.4 T2*Relaxation...... 10

1.1.5 Sodium Nuclear Magnetic Resonance...... 10

1.1.5.1 NMR Sensitivity...... 10

V TABLE OF CONTENTS

1.1.5.2 Nuclear Quadrupolar Interaction...... 11

1.1.5.3 Nuclear Quadrupolar Interaction as Perturbationof the Zeeman Coupling...... 13

1.1.5.4 Relaxation Processes of Quadrupolar Nucleii...... 15

1.1.5.5 Relaxation of Sodium Nuclei in Tissues...... 18

1.1.5.6 Comparison between Proton and Sodium Relaxation Times...... 19

1.1.5.7 Proton and Sodium T2* RelaxationTimes ...... 20

1.2 Magnetic Resonance Imaging Techniques...... 21

1.2.1 Frequency Encoding...... 22

1.2.2 Phase Encoding...... 22

1.2.3 Fourier I Cartesian Methods...... 22

1.2.4 Resolution of Image...... 24

1.2.5 Three Dimensional Acquisition...... 26

1.2.6 Sodium MR Imaging...... 26

1.2.7 Gradient Echo...... 27

1.2.8 Gradient Echo with Variable Echo Time Scheme...... 28

1.2.9 Optimization of Cartesian Gradient Echo Techniques...... 30

1.3 Structure and Function of Articular Cartilage...... 31

1.3.1 Synovial Joints...... 31

1.3.2 Macroscopic View of Cartilage...... 33

1.3.3 Zonal Architecture of Cartilage...... 33

1.3.4 Molecular Composition of Cartilage...... 35

1.3.4.1 Chondrocytes...... 36

1.3.4.2 Water...... 36

1.3.4.3 Collagen...... 37

1.3.4.4 Proteoglycans...... 37

1.3.5 Biomechanical Implication of Cartilage Composition...... 38

1.4 Cartilage Injury and Repair...... 40

1.4.1 Mechanical Injury of Cartilage...... 40

1.4.2 Intrinsic Response of Cartilage to Injury...... 41

vi TABLE OF CONTENTS

1.4.3 Measures of Knee and Ankle Function...... 42

1.4.4 Surgical Methods for Treatment of Cartilage Defects...... 44

1.4.4.1 Debridement...... 44

1.4.4.2 Pridie Drilling and Microfracture...... 45

1.4.4.3 Mosaicplasty...... 46

1.4.4.4 Periosteal Grafts...... 47

1.4.4.5 Autologous Chondrocyte Implantation...... 48

1.4.4.6 Stem Cells in Cartilage Tissue Engineering...... 51

1.4.5 Differences between Knee and Ankle Cartilage...... 51

1.5 MR Imaging of Cartilage and Repair Tissue...... 53

1.5.1 Morphological Assessment of Cartilage Defects and Repair Tissue...... 53

1.5.1.1 Morphological MR Imaging Sequences...... 53

1.5.1.2 MR Imaging Based Morphological Scoring of Cartilage Defects...... 57

1.5.1.3 MR Imaging Based Morphological Evaluation of Cartilage Repair...... 57

1.5.2 Biochemical MR Imaging of Cartilage Defects and Repair Tissue...... 58

1.5.2.1 Ti Delayed Gadolinium-enhanced MR Imaging...... 59

1.5.2.2 Tip Mapping...... 61

1.5.2.3 Chemical Exchange Saturation Transfer Imaging...... 62

1.5.2.4 Paper 5: Evaluation of Cartilage Repair and Osteoarthritis with Sodium MRI.... 63

1.6 Aims of the Thesis...... 75

2. RESULTS...... 76

2.1 Prologue...... 76

2.2 Paper 1:23Na MRI at 7 T after Knee MACT...... 76

2.3 Interlude 1 ...... 87

2.4 Paper 2: Evaluation of native hyaline cartilage and repair tissue with 23Na MRI at 7 T..... 87

2.5 Interlude 2...... 97

2.6 Paper 3:23Na MRI of Ankle Joint in Specimens, Volunteers, and Patients at 7 T...... 97

3. DISCUSSION...... 1 07

3.1 General Discussion...... 1 07

vii TABLE OF CONTENTS

3.2 Paper 1:23Na MRI at 7 T after Knee MACT...... 108

3.3 Paper 2: Evaluation of native hyaline cartilage and repair tissue with 23Na MRI at 7 T.....110

3.4 Paper 3:23Na MRI of Ankle Joint in Specimens, Volunteers, and Patients at 7 T...... 113

3.5 Conclusion and Future Prospects...... 116

4. MATERIALS AND METHODS...... 118

4.1 Subjects and Specimens...... 118

4.1.1 Subjects...... 118

4.1.2 Human Specimens...... 120

4.2 Clinical Outcome...... 120

4.3 MR Imaging...... 120

4.3.1 Morphological MR Imaging...... 121

4.3.2 Ti Delayed Gadolinium-enhanced MR Imaging...... 122

4.3.3 Sodium Imaging...... 122

4.4 Postprocessing and Evaluations of MR Images...... 124

4.4.1 Morphological Evaluation of Repair Tissue...... 124

4.4.2 Evaluation of Postcontrast Ti Maps...... 126

4.4.3 Postprocessing of Sodium Images...... 126

4.4.4 Evaluation ofSodium Images...... 127

4.5 Histochemical Analyses of Cartilage Samples...... 129

4.6 Statistical Analyses...... 130

5. REFERENCES...... 132

6. APENDIX...... 152

6.1 Paper 4: Sodium MR Imaging of Articular Cartilage Pathologies...... 152

6.2 Curriculum Vitae...... 163

6.3 Scientific Publications...... 165

viii ABSTRACT

ABSTRACT

Articular cartilage of adults shows no or very limited intrinsic capacity for self-repair. Since untreated chondral defects often progress to osteoarthritis, symptomatic defects should be treated. Different cartilage repair procedures have been developed with the goal to restore joint function and prevent further cartilage degeneration by providing repair tissue of the same structure, composition, and biomechanical properties as native cartilage. Various cartilage repair procedures have been developed; including bone marrow stimulation (BMS) techniques such as microfracture (MFX), cell-based techniques such as matrix-associated autologous chondrocyte transplantation (MACT), and others. Since biopsies of cartilage repairtissue are invasive and cannot be repeated, a noninvasive method is needed that could follow-up the quality of cartilage and repair tissue. Negatively charged glycosaminoglycans (GAG) are very important for cartilage function as they attract positive ions such as sodium. The high concentration of ions in cartilage is responsible for osmotic pressure providing cartilage its resilience to compression. Since GAGs are counterbalanced by sodium ions, sodium magnetic resonance imaging (MRI) was validated as a sensitive method for the in vivo evaluation of GAG concentration in native cartilage but not for repair tissue. Thus, the main goal of this thesis was to optimize and validate sodium 7 Tesla MRI for the evaluation of cartilage repair tissue quality in patients after different cartilage repair surgeries in the knee and anklejoint. In our studies, sodium MRI was used for the first time for the clinical evaluation of cartilage repair tissue. A strong correlation found between sodium imaging and dGEMRIC (another GAG-sensitive technique) in patients after MACT on femoral cartilage proved sensitivity of sodium MRI to GAG changes in native cartilage and repair tissue in vivo. Comparison between BMS and MACT patients showed significantly lower sodium values in repair tissue than in native femoral cartilage. Although morphological appearance of repair tissue was similar in BMS and MACT patients, our sodium MRI results suggest higher GAG content, and thus repair tissue of higher quality, in patients after MACT than in patients after

ix ABSTRACT

BMS treatment. Sufficiently high sensitivity of sodium imaging to changes in the GAG content in thin ankle cartilage was demonstrated by a strong correlation observed between the sodium results and the histochemically evaluated GAG content in cadaver ankle samples. In contrast to our findings from femoral cartilage of knee joint, MFX and MACT in talar cartilage of ankle joint seems to produce repair tissue with a similar GAG content and similar morphological appearance, however, of lower quality than native cartilage. Sodium MRI provides information on the GAG concentration that is crucial for proper function of repair tissue and might be therefore beneficial in non-invasive evaluation of efficacy of cartilage repair surgery and in assessment of new cartilage repair procedures.

x ZUSAMMENFASSUNG

ZUSAMMENFASSUNG

Die Gelenksknorpel von Erwachsenen weisen kein oder ein nur sehr limitiertes intrinsisches Vermögen zur Selbstreparatur auf. Da unbehandelte Knorpeldefekte sich oft zur Gelenksarthrose entwickeln, sollten symptomatische Defekte behandelt werden. Unterschiedliche Knorpelreparaturverfahren wurden mit dem Ziel entwickelt, die Gelenksfunktion wiederherzustellen und eine weitere Degeneration des Knorpels zu verhindern indem Reparaturgewebe mit der gleichen Struktur, Zusammensetzung, und biomechanischen Eigenschaften wie der native Knorpel verwendet wird. Verschiedene Knorpelreparaturverfahren wie etwa Knochenmarksstimulationstechniken (BMS) einschliesslich Mikrofrakturierung (MFX) oder zell-basierte Techniken wie beispielsweise die Matrix-assozierte Autologe Chondrozyten Transplantation (MACT) wurden entwickelt. Da Biopsien des Knorpelersatzgewebes invasiv sind und nicht beliebig wiederholt werden können ist eine nicht-invasive Methode notwendig, die es erlaubt die Qualität des Knorpels und des Reparaturgewebes im zeitlichen Verlauf zu verfolgen. Negativ geladene Glykosaminoglykane (GAG) sind sehr wichtig für die Knorpelfunktion, da sie positive Ionen wie etwa Natrium anziehen. Die hohe Konzentration von Ionen im Knorpel ist für den hohen osmotischen Druck verantwortlich, der dem Knorpel seine hohe Belastbarkeit gegen Kompression verschafft. Da GAG durch Natrium-Ionen kompensiert werden, wurde die Natrium-Magnetresonanzbildgebung (MRI) als eine sensitive Methode für die in vivo Evaluierung der GAG Konzentration in nativem Knorpel, jedoch nicht in Reparaturgewebe, validiert. Das Hauptziel dieser Doktorarbeit ist deshalb die Natrium 7 Tesla MRI für die Evaluierung von Knorpelreparaturgewebequalität in Patienten nach unterschiedlichen Knorpelreparationstechniken im Knie und im Sprunggelenk zu optimieren und zu validieren. In unseren Studien wurde Natrium MRI weltweit erstmals für die klinische Evaluierung von Knorpelreparaturgewebe verwendet. Die starke Korrelation zwischen Natriumbildgebung und dGEMRIC (eine andere GAG-sensitive Technik) in Patienten nach MACT des femoralen Knorpels beweisen die Sensitivität der Natriumbildgebung für Änderungen des GAG Gehaltes

xi ZUSAMMENFASSUNG in nativem Knorpel und Reparaturgewebe in vivo. Ein Vergleich zwischen BMS und MACT Patienten zeigte signifikant niedrigere Natriumwerte in Reparaturgewebe als in nativem femoralem Knorpel. Obwohl das morphologische Erscheinungsbild von Reparaturgewebe in BMS und MACT Patienten ähnlich war, deuten unsere Natrium MRI Resultate auf einen höheren GAG Gehalt hin, und daher auf Reparaturgewebe von höherer Qualität in Patienten nach MACT im Gegensatz zu Patienten nach BMS Behandlung. Eine ausreichend hohe Sensitivität der Natrium MRI für Änderungen im GAG Gehalt in dünnen Gelenksknorpeln wie dem Sprunggelenk wurde durch eine starke Korrelation gezeigt, die zwischen den Natriumresultaten und dem histochemisch evaluierten GAG Gehalt in Kadavergelenkspräparaten beobachtet wurde. Im Gegensatz zu unserer Ergebnissen beim femoralen Knorpel des Kniegelenkes, scheint MFX und MACT beim Knorpel Reparaturgewebe des Talus am Sprunggelenk einen ähnlichen GAG Gehalt und ähnlichen morphologischen Erscheinungsbild zu produzieren, allerdings von niedrigerer Qualität als bei nativem Knorpel. Natrium MRI liefert Informationen über die GAG Konzentration, welche entscheidend für die ordentliche Funktion des Reparaturgewebes ist und welches deshalb vorteilhaft für die nicht-invasive Evaluierung der Effizienz von Knorpelreparaturoperationen und der Beurteilung von neuen Knorpelreparaturverfahren ist.

xii PUBLICATIONS ARISING FROM THIS THESIS

PUBLICATIONS ARISING FROM THIS THESIS

1) Trattnig S., Welsch G.H., Juras V., Szomolanyi P., Mayerhoefer M.E., Stelzeneder D., Mamisch T.C., Bieri O., Scheffler K., Zbvn S. 23Na MR Imaging at 7 T after Knee Matrix-associated Autologous Chondrocyte Transplantation: Preliminary Results Radiology. 2010; 257(1): 175-184. Original Research

2) Zbvn S., Stelzeneder D., Welsch G.H., Negrin L.L., Juras V., Mayerhoefer M.E., Szomolanyi P., Bogner W., DomayerS.E., Weber M., Trattnig S. Evaluation of native hyaline cartilage and repair tissue after two cartilage repair surgery techniques with (23)Na MR imaging at 7 T: initial experience Osteoarthritis Cartilage. 2012; 20(8): 837-845. Original Research

3) Zbvn S., Brix M.O., Juras V., Domayer S.E., Walzer S.M., Mlynarik V., Apprich S., Windhager R., Trattnig S. Sodium MR Imaging of Ankle Joint in Cadaver Specimens, Volunteers and Patients after Different Cartilage Repair Techniques at 7T - Initial Results InvestRadiol. 2015; 50(4): 246-254. Original Research

4) Zbvn S., Mlynarik V., Juras V., Szomolanyi P., Trattnig S. Sodium MR Imaging of Articular Cartilage Pathologies Curr Radiol Rep. 2014; 2(41): 1-10. Review Paper

5) Zbvn S., Mlynarik V., Juras V., Szomolanyi P., Trattnig S. Evaluation of cartilage repair and osteoarthritis with sodium MRI NMRBiomed. 2015; DOI: 10.1002/nbm.3280. Review Paper

xiii ABBREVIATIONS

ABBREVIATIONS

1H nucleus of hydrogen 23Na nucleus of sodium 2D/3D two-dimensional / three-dimensional

ACI autologous chondrocyte implantation AOFAS american orthopaedic foot and ankle society score

BLOKS Boston-Leeds osteoarthritis knee score BMS bone marrow stimulation BW bandwidth

CNR contrast-to-noise ratio CROAKS cartilage repair osteoarthritis knee score

DESS double-echo steady-state dGEMRIC Ti delayed gadolinium-enhanced magnetic resonance imaging of cartilage

EFG electric field gradient

FCD fixed charge density FOV field-of-view FSE fast spin-echo

GAG glycosaminoglycan gag-CEST glycosaminoglycan chemical exchange saturation transfer GRE gradient echo

xiv ABBREVIATIONS

IKDC international knee documentation committee ICRS international cartilage repair society IR inversion recovery

KOOS knee injury and osteoarthritis outcome score KOSS knee osteoarthritis scoring system

MACI matrix-associated autologous chondrocyte implantation MACT matrix-associated autologous chondrocyte transplantation MFX microfracture MOAKS magnetic resonance imaging osteoarthritis knee score MOCART magnetic resonance observation of cartilage repair tissue MR magnetic resonance MRI magnetic resonance imaging

NMR nuclear magnetic resonance

OA osteoarthritis

PD proton-density PG proteoglycans

RF radiofrequency ROI region-of-interest

SNR signal-to-noise ratio SPACE sampling perfection with application optimized contrasts using different flip angle evolutions SSFP steady state free precession

Ti longitudinal relaxation time constant t2 transverse relaxation time constant TAS Tegner activity score TE echo time

XV ABBREVIATIONS

TPI twisted projection imaging TR repetition time true-FISP fast imaging with steady-state precession

UTE ultra-short echo time vTE variable echo time vTE-GRE gradient echo sequence employing variable echo time scheme

WOMAC Western Ontario and McMaster universities osteoarthritis WORMS whole-organ magnetic resonance imaging scoring

xvi LIST OF FIGURES AND TABLES

LIST OF FIGURES AND TABLES

Figures

Fig. 1.1. The nuclear spin energy levels for a spin-1/2 nucleus...... 4 Fig. 1.2. The spin magnetic moments...... 5 Fig. 1.3 : Excitation ofthe magnetization...... 6 Fig. 1.4 : Precession ofthe magnetization and of a magnetic moment...... 7 Fig. 1.5 : Zeeman energy levels for1H and 23Na at7 Tesla...... 12 Fig. 1.6: Energy level diagram of a 23Na in different motional regimes...... 16 Fig. 1.7: Comparison of magnetization relaxation in cartilage between 1H and 23Na...... 19 Fig. 1.8: Dependency of 23Na T2* constants on Bo field inhomogeneity and T2...... 20 Fig. 1.9: Diagram of Cartesian 3D gradient echo pulse sequence...... 23 Fig. 1.10: Comparison of symetric and asymmetric 3D gradient echo sequence...... 28 Fig. 1.11: Diagram ofCartesian vTE gradient echo pulse sequence...... 29 Fig. 1.12: Illustration ofthe Median-Sagittal cut through the right knee joint...... 32 Fig. 1.13: Illustration ofthe Sagittal cut through the ankle joint...... 32 Fig. 1.14: Schematic diagram of the structure of healthy articular cartilage...... 34 Fig. 1.15: Organization of macromolecules in extracellular matrix of cartilage...... 36 Fig. 1.16: Schematic sketch of a proteoglycan aggregate...... 38 Fig. 1.17: Scheme of processes in matrix of cartilage during rest and load...... 39 Fig. 1.18: Schematic illustration of debridement procedure...... 45 Fig. 1.19: Schematic representation of microfracture technique...... 46 Fig. 1.20: Schematic illustration of mosaicplasty procedure...... 47 Fig. 1.21: Schematic representation of method using periosteal graft...... 48 Fig. 1.22: Scheme oftwo steps of ACI technique employing the periosteal patch...... 49 Fig. 1.23: The third generation of ACI techniques - the NeoCart® implant...... 50 Fig. 1.24: High-resolution 3 T MR images of a man in the follow-up after MACT repair...... 54 Fig. 1.25: High-resolution 3D DESS images of a man after MFX ofthe patella...... 55

xvii LIST OF FIGURES AND TABLES

Fig. 1.26: High-resolution 3D true-FISP images ofthe MACT repairtissue...... 56 Fig. 1.27: A 3D dual flip angle dGEMRIC images of a MFX repairtissue...... 60 Fig. 1.28: Morphologic and gagCEST images of a patient after ACT...... 63 Fig. 4.1: Magnetom 7 T MR whole body system and 23Na-only knee coils...... 123 Fig. 4.2 : Illustration of segmentation of talar cartilage in the ankle sample...... 130

Tables

Table 1.1: Ti and T2 relaxation times for1H in various musculosceletal tissues at 3 T...... 8 Table 1.2: Comparison ofthe NMR properties influencing the 23Na and the 1H MR signal.. 11 Table 4.1: Parameters of morphological sequences...... 121 Table 4.2: Measurement parameters of the 23Na 3D GRE sequences...... 124 Table 4.3: MOCART scoring system...... 125

xviii ACKNOWLEDGMENTS

ACKNOWLEDGMENTS

I am greatly indebted to my thesis supervisor, Prof. Siegfried Trattnig for his excellent guidance and support during my study at Medical University of Vienna. I would like to thank Prof. Reinhard Windhager and Prof. Heinz Redl, members of my doctoral thesis committee, for their time and kind help.

I would also like to thank all my colleagues from the High Field MR Center and from the Department of Biomedical Imaging and Image-Guided Therapy, especially to Dr. Vladimir Mlynarik, Dr. Pavol Szomolanyi and Dr. Vladimir Juras for their help, fruitful scientific discussions and collaborations. Thank you all for making my stay at MUW inspiring, illuminating and exciting.

Many thanks goes also to my family, especially to my wife, Martina, my children, Simona, Filip and Adam, for all of their constant love, support, encouragement and patientce. And last, but certainly not least, I would like to thank my mother, Eliska and my father Stefan for their never ending support, without which my education would not have been possible.

xix XX INTRODUCTION

1. INTRODUCTION

1.1 Theory of Nuclear Magnetic Resonance

In this chapter, the basic principles of the Nuclear Magnetic Resonance (NMR) are presented, with emphasis on sodium NMR. For more detailed description, the reader is referred to the cited texts and books [1-3],

1.1.1 Properties of Nucleus

The nuclear spin quantum number I defines fundamental property of the nucleus. I can be zero, half integer or integer value. If I is zero, nucleus will not interact with a magnetic field. If I is not equal to zero, the interaction between a magnetic field and a spin is described as a quantum mechanical phenomenon. The nuclear spin is associated with an angular momentum P by:

p=hl [1.1] where h = h Un and h is Planck's constant. The rotating electric charge of the nucleus creates a magnetic field around it represented by magnetic dipole moment p , defined as:

p = yp = [1.2] where / is the gyromagnetic ratio. This is a constant and a physical characteristic of specific nucleus. Nuclei of atoms with an odd number of protons or neutrons or both possess non-zero magnetic dipole moment. The nucleus of hydrogen (1H) has a single proton and its gyromagnetic ratioyH is 2.6752x108. 1H has the greatest NMR sensitivity and it is the most commonly imaged nucleus in NMR. Unlike 1H, the nucleus of sodium (23Na) has twelve

1 INTRODUCTION neutrons and eleven protons and its gyromagnetic ratio YNa is 6.7266x107. After 1H, 23Na has the second highest NMR sensitivity in living tissues.

1.1.2 Interaction between Spins and Static Magnetic Field 1.1.2.1 Quantum-mechanical Model

When a nucleus which posses magnetic dipole moment is placed in an external magnetic field B , the magnetic moments of the sample align with B The interaction of the magnetic field with the magnetic moment describes the Hamilton operator:

=-juB = -yhIB [1.3]

If an external magnetic field is time-independent, homogeneous, and oriented in the z direction

Bo = (0,0,Bo), the Hamilton operator can be simplified to:

Hz =-yhIzBQ [1.4]

Based on the quantum mechanical description, only a limited number of magnetic moment orientations is possible with respect to the direction of the magnetic field Bo. The splitting of the energy levels in the presence of a magnetic field is called the Zeeman effect.

The nucleus with spin quantum number / can be found in 2/ + 1 possible energy levels , with the following energy difference between two neighboring levels:

E = Em -Em-i = ytä, = M [1-5]

Protons with I = 1/2, are limited to two energy levels, whereas 23Na with I = 3/2 can have four different energy levels with a energy difference of AE. The constant energy difference \F, is directly proportional to the gyromagnetic ratio and to the external magnetic field strength. Due to smaller gyromagnetic ratio of 23Na, \F, for 23Na will be four times smaller than that of protons at the same field strength.

Besides the Zeeman effect, the external magnetic field Bo results in a precession of the spinning nucleus about the axis of Bo. This precession of the spinning nucleus in the external field is analogous to the wobbling of a toy top that spins about its axis, but also

2 INTRODUCTION wobbles about the vertical due to the earth’s gravitational field [4], The evolution of the system in time describes the Schrödinger equation:

ZÄ = #j ¥(,)) [1.6] where | Y(Y)) is an arbitrary state and 77 is the Hamilton operator. If the Hamiltonian operator is constant in time, the Schrödinger equation results in:

|W)) = WtlW)) [1.7] where the evolution operator £7 (f) is defined as:

jj _ e-i&J! * _ evBoa2 _ e«Z2 [1.8]

Expected rotation angle about the axis of Bo is defined as a = -yBot. In the external magnetic field, nuclei in all states precess about the axis of Bo at the Larmor frequency o = yB0. In an

MR system with the field strength of 7 T, the Larmor frequency of 1H is 297.2 MHz, and the Larmor frequency of 23Na is 78.6 MHz.

1.1.2.2 Macroscopic Magnetization in a Magnetic Field

Let's consider a population of N spins in a homogeneous magnetic field B, in thermal equilibrium at temperature T. In this case, a population difference between the energy states describes the Boltzmann probability factor:

p = ±e kT = -e kT [1.9] m Z Z where k is the Boltzmann constant (k = 1.38 x 10 z; JIK). To normalise the Boltzmann probability factor to the values between 0 and 1, the sum of probabilities is introduced:

I Z = £ e kT [1.10] m=-I The individual energy levels have different populations. In thermal equilibrium, the absolute value of the macroscopic magnetisation is described as:

/ e m=-I

3 INTRODUCTION

At room temperature Em « kT, the exponent can be approximated as e* «1 + x , and the equation 2.16 is simplified into the Curie Law:

N \T V1 n_ h2?21 (i+1) b0 m m=-I [1-12] M 0 V 7 kT 21 +1 V 3kT

The population difference between the energy states and - can be calculated as follows:

2

According to the quantum-mechanical description, a nucleus of I = 1/2 can be found in two energy levels which corresponds to two spin states m = +1/2 (ji parallel with B) and m = -1/2 (//antiparallel to B) (Fig. 1.1). The angle 9 between //and Bo is defined as: cos9 = m I(i +1) .Therefore, we can introduce a model where the nuclei in two states are distributed on the surface of two cones, and rotate about B with the Larmor frequency (Fig.

1.2). In the case of a 23Na, the magnetic moments will be distributed on four cones at four discrete angles 0 as defined before.

Fig. 1.1. a) The nuclear spin energy diagram for a spin-1/2 nucleus as a function of the external magnetic field strength B. The difference in energy levels between the two spin states corresponds to electromagnetic radiation in the radiofrequency range, b) The lower energy level (a spin state) corresponds to magnetic moments parallel with B, while spins in the higher energy level (ß spin state) have an antiparallel alignment with

4 INTRODUCTION

Fig. 1.2. The spin magnetic moments li precesses about Bo with the angle 0. The spins are distributed in two possible orientations [2],

1.1.2.3 Semi-classical Model - Equation of Motion

For the macroscopic magnetization M at room temperature Em << kT, the predictions of the classical model agree with the predictions of the quantum theory. The equation of motion of the macroscopic magnetization in the magnetic field reads:

= M(t) xyB(t) [1.14]

The magnetization M precesses about the direction ofthe external magnetic field B with an angular frequency a = yB0.

1.1.3 Interaction between Spins and Radiofrequency Field

In order to tilt the magnetization M from the equilibrium state, application of a radiofrequency (RF) pulse is necessary. Although the basic quantum mechanical model shown in Figure 1.2 is convenient to demonstrate the distribution ofthe spin angular moments in states with discrete energies, it is not very suitable to describe the interaction of the spins with external magnetic fields. Therefore the semi-classical model of the net macroscopic magnetization vector M will be used in further discussions.

Tilted magnetization M can be split into two components. The longitudinal component of magnetization parallel with Bo field is static, whereas the transversal component of

5 INTRODUCTION magnetization precesses in the plane perpendicular to Bo and produces its own rotating field, which induces an oscillating voltage in the RF coil. The RF pulse refers to a short application

(duration from several ps to several ms) of an oscillating magnetic field B- with a frequency similar to the Larmour frequency, which is applied in a plane perpendicular to Bo. The applied

Bx field can be described as:

B1 = B1(x cosat+y sin^f) [1-15]

The Hamilton operator then expands to:

H = -yhI(B, +BJ [1.16]

Similarly, the equation of motion ofthe macroscopic magnetization [1.14] expands to:

= M(t)x/[.xB1cosföf + v/), sinpy + zB0] [1-17]

The torque of B field applied in the presence of magnetization M results in a rotation of M towards the transverse plane (Fig. 1.3).

Fig. 1.3 : Excitation of the magnetization M by 90° from the z axis. Application of B- field results in a rotation towards the transverse plane and a simultaneous precession at the Larmor frequency about z axis due to

Applied 5, field can tilt the magnetization M to the transverse plane (90° excitation RF pulse) or even invert it to the -z axis (180° inversion RF pulse). The transverse magnetization induces an electromotive force in the receiver RF coil and this force gives rise to the NMR signal.

6 INTRODUCTION

By introducing a rotating frame of reference which rotates with a frequency a about the Bo field, the equation [1.17] becomes time-independent and can be written in the form:

d\'l - - = M ^yBef [1.18] dt

The magnetic field Bef in the rotating frame of reference does not have high-frequency components, B = (B..Q.Bo. Correspondingly, in the rotating frame, magnetization 7 rotates about Bef with the frequency aef =/| Bef\. This frequency is much slower than the resonance frequency (of the order of 0 to100 kHz) (Fig. 1.4).

Fig. 1.4: a) Precession of the magnetization M and b) precession of a magnetic moment in the rotating frame of reference.

At resonance condition (« = yB0), the z component of Bef is cancelled and the magnetization precesses about the direction of Si (conventially aligned with x axis) (Fig. 1.4 a). When the constant B field is applied for a time tp , the magnetization will be flipped around the x axis by an angle a:

a = yB,tp [1.19]

7 INTRODUCTION

1.1.4 Relaxation Mechanisms and the Bloch Equations 1.1.4.1 Origins of Relaxations

When assuming only the interaction of the magnetic moment with Bo and B. fields,

M will precess about Bo at a constant angle a after RF excitation (equation [1.19]). Due to the interactions of spins with their environment and amongst themselves, the precessing magnetic moment progressively realigns with A> and returns to its equilibrium state A/ . Two main types of spin relaxations occur: Spin-lattice or longitudinal relaxation: the exchange of energy between the spins and their surroundings, the longitudinal component (parallel to £0) of M, Mz, recovers towards its equilibrium value Mo with a time constant Ti.

Spin-spin or transverse relaxation: the nucleus experience besidesBo and Bx fields also magnetic field from any other neighboring nuclei. This dipole-dipole interaction causes slight variation of the precession rate of individual nuclei which results in the loss of phase coherence. This leads to the reduction ofthe transverse component of M (Mwhich decays to zero with a time constant T2.Value of T2 is always smaller than or equal to Ti. Since 1H has the greatest NMR sensitivity, very high concentration in tissues and a natural abundance of 99.98%, 1H is the most commonly observed nucleus. Depending on the chemical binding, 1H nuclei have different resonance frequencies and relaxation times. The large range of different relaxation times in different biological tissues provides the contrast between measured tissues in the NMR image. Moreover, changes in the relaxation characteristics of the tissue can refer to pathological processes.

Table 1.1 shows typical 1H relaxation times of tissues in the human join at3T.

Tissue Ti (ms) T2(ms) Muscle 1420 31,7 Cartilage 1240 36,9 Synovial fluid 3620 767 Subcutaneous fat 371 133 Marrow fat 365 133

Table 1.1: Ti and T2 relaxation times for1H in various musculosceletal tissues at 3 T [5],

8 INTRODUCTION

1.1.4.2 Bloch Equations

To reflect the relaxation processes, the Bloch equations can be derived by incorporating of the Ti and T2 constants into the equation of motion of the macroscopic magnetisation (equation [1.14]):

■ x Bj [1-20] dt

dMy = y[M xB) y [1-21] dt 'y B

dMz M 0 - Mz ■ = r (M x B); [1-22] dt B These equations describe the interactions for biological tissues and liquids. However, tensors are used for description of relaxation processes in solids. For a homogeneous static magnetic field B = (0,0, A ), the solutions for the longitudinal magnetization Mz (t) and the transversal magnetization M = Mx + iMv are following:

Mz(t) = M0-(M0 -Mz(Zo))e T1 [1-23]

ikat----- (*) = Mxy (t0)e T 2 [1-24] whereM(t = t0) = (MX(t0),My(t0),Mz(r0)), A« « -®rot, and ®rot describing the frequency of the rotating frame of reference.

1.1.4.3 Magnetic Field Gradients

For recording ofthe NMR images, it is necessary to use magnetic field gradients:

ö dn , g;, G! [1-25] Sr ’ Sy 5z j

When applying a field gradient in a specific direction, the magnetic field strength varies according to B(r, t) = Bo + rG(t). As a result, the Larmor frequency varies in G direction and becomes time dependent:

®(r, t) = x( Bo + rG(t)) [1.26]

9 INTRODUCTION

When assuming the effects of the relaxation processes, the gradients and the RF field B. applied along the x direction, the Bloch equations become: d\I Mx' ■ = yG (Z *- [1-27] dt B dM. M. —r- = ~rG(t)rMx, + yBxMz [1-28] dt /'

dMz. Mo -Mz ------= -yB.M ,------[1-29] dt 1 y B,

1.1.4.4 T2* Relaxation

Although the shim coils in the NMR systems allow to make the Bo field in the examined region more homogeneous, some variation in Bo field are present in the measured volume.

This imperfection of B field result in an additional line broadening in the NMR spectrum

(usually few Hz), which implies a more rapid decay of the Mxy than that caused by T2 relaxation mechanisms alone. The resultant T2* constant can be written as:

1 2_ + -^AB * [1-30] 22 2T2 2^ where AB is the inhomogeneity of magnetic field in Bo. direction. The T2* time constant is always smaller than the T2.

1.1.5 Sodium Nuclear Magnetic Resonance 1.1.5.1 NMR Sensitivity

At room temperature, the population difference between the energy states and 2

at 1.5T for 1H is 10.5-10'6, according to equation [1.13], For 23Na it is 2.8-10"6, that is about four times smaller compared to 1H. Comparison of relative concentration, natural abundance and MR sensitivity of the 1H signal and the 23Na signal is summarized in Table

10 INTRODUCTION

1.2. As shown here, the 23Na MR sensitivity (~ yl (I +1)) is just 9.3% ofthe 1H MR sensitivity.

Additionally, the sodium in vivo concentration is about 320 times lower than the 1H in vivo concentration in femoral cartilage. As a result of these factors, the 23Na signal is ~3400 times lower than the 1H signal in cartilage. As the 23Na and the 1H concentrations are tissue dependent, the difference in MR signals vary in different tissues types.

Natural Gyromagnetic MR In vivo In vivo Nucleus abundance ratio sensitivity concentration signal (%) (Hz/T) relat. to 1H (M) relat. to 1H

1H 99.99 42500 1.000 ~83 1

23Na 100.00 11200 0.093 ~0.26£ 2.91 ■ 10-4

Table 1.2: Comparison of some of the NMR properties influencing the 23Na and the 1H MR signal in the articular cartilage. The sodium signal in articular cartilage is about 3400 times lower compared to that of 1H (£ [6]).

1.1.5.2 Nuclear Quadrupolar Interaction

The 1H nucleus has a spin quantum number I = 1/2, which gives rise oftwo energy levels m = -1/2,+1/2 in the presence ofa magnetic field (see chapter 1.1.2.1). On the other hand, 23Na has a spin I = 3/2, which result in four energy levels described by the magnetic quantum numbers m = -3/2,-1/2,+1/2, +3/2 (Fig. 1.5). Since the energy difference is proportional to Bo and to the gyromagnetic ratio y, at the same Bo, \E for 23Na will be about four times smaller compared to 1H.

11 INTRODUCTION

Fig. 1.5 : Zeeman energy levels fora) 1H and b) 23Na at field strength of 7 Tesla.

Since 23Na have a spin quantum number I = 3/2, in addition to the magnetic dipole moment q, it also exhibits an electric quadrupole moment Q. This feature applies for all nuclei with spin number greater than 1/2. Because of the non-spherically symmetric distribution ofthe electric charge in the nucleus, an electric quadrupole moment Q that does not depend on the exact charge is created. When a nucleus with quadrupolar moment is in an interaction with an electric field gradient (EFG) (for example the electrons around the nucleus), quadrupolar interaction affect the energy spectrum. The Hamilton operator (a quantum mechanical expression) of the quadrupolar interaction reads [7]:

where e is an elementary charge, q represents EFGs, q is asymmetry parameter and I represent the total angular momentum operators of the nucleus, with the raising and lowering operators I+ = Ix + ily and I_ = Ix -ily. The strength of quadrupolar coupling depends on the magnitude of the EFG eq generated by the environment of the nucleus, and on the parameter

e2 qO eQ . The coupling strength is usually given as the quadrupolar coupling constant Co =—— h . This constant depends on the environment of the nucleus, and for 23Na is ~ 2 MHz [7],

12 INTRODUCTION

1.1.5.3 Nuclear Quadrupolar Interaction as Perturbation of the Zeeman Coupling

When a spin with angular momentum I is in a static magnetic field Bo, the Zeeman interaction results in creating of 2/ +1 energy levels (see chapter 1.1.2.1) defined as:

[1-32]

The difference between two adjacent energy levels(m -1, m) can be expressed in frequency units as:

o' . x = (m-1 //, m-1)-(m //, m) = oo [1-33]

Equation [1.33] indicates equally spaced energy levels of a free spin in a static magnetic field Bo; with the separation between two consecutive levels equal to o. In the MR spectrum, the central line (-1/2,1/2) is located at o = yB0. The quadrupolar interaction can be described by the Hamiltonian , and the resulting Hamilton operator for 23Na become:

H = Ho + + T/ß [1.34] with eigenvalues:

Em = Eo+ Eq [1.35]

When assuming the quadrupolar interaction in a reference frame that rotates with Larmor frequency « relative to the main frame of reference, becomes time-dependent. By averaging the HQ (t) over a Larmorperiod 2^/®0, the interaction becomes time-independent and the first-order perturbation terms can be written as:

[1-3SI with the resulting energy eigenvalues:

> -,(I'IH [1-37] where

13 INTRODUCTION

(3cos2 ß-l) + -^sin2 ßcos2a [1-38] where a and ß are the Euler angles that describe the direction of Bo in the principal axis system of the EFG tensor. The resulting energies are proportional to the square of the magnetic quantum number(in).

The energy levels are shifted by an EQ (equation [1.37]), and the resonance frequency shift in the spectrum associated with the transition (m-1,m) reads [8]:

?Q 76 (i - 2« )r0 [1-39] " 4 j (21 -1) 3

The sodium MR spectrum consists of 27 peaks. The central peak corresponding to the -) | transition is located at o. Two satellite peaks are placed symmetrically with respect to the inner peak, and their distance from central peak depends on the strength of the quadrupolar interaction. Equations [1.37] and [1.38] suggest that the energy differences, and thus also the position ofthe satellite peaks, depend on the direction ofthe symmetry axis with respect to the Bo field. When neglecting the asymmetry parameter 7, term (3 cos2 ß-1) modulates the frequency shift. This term vanishes at the Magic Angle (ß = 54.7°) and all the peaks collapse into one peak [9], The orientation angle is homogeneously distributed in isotropic liquids, and in most in-vivo tissues. As a result, the quadrupolar interaction is averaged to zero and only one peak, the superposition of three Lorenzian peaks of possibly different line width, can be observed. The quantum-mechanical calculations demonstrate that the inner transition

1 , corresponds to the central peak, and illustrates 40% ofthe total 23Na signal 2

3 intensity. Each ofthe outer transitions corresponds to 2 the satellite peaks, and illustrates 30% ofthe total signal intensity.

14 INTRODUCTION

1.1.5.4 Relaxation Processes of QuadrupolarNucleii

In nucleus with I = 3/2 ,the relative energies of the four spin states are influenced by EFG effects from the molecular environment of the nucleus; where the fluctuations of EFG in time are due to thermal motions. When assuming an axial EFG tensor (7 = 0) in equations

[1.38] and [1.39], the time-averaged quadrupolar interaction frequency ä>Q is proportional to:

®q « e2Q(q (3cos2 ß-l)^ [1.40]

The fluctuations in the strength and/or the orientation of the EFG tensor can be characterized, according to Debye model, by a correlation time tc . Correlation time is the minimum time the EFG tensor spends in a particular state. For 23Na at A = IT, the Larmor period (2^/®0) is about 3 ns. The energy level pattern of a 23Na depends on its motional regime. The following four motional regimes are possible for sodium (in biological tissues we can find only first three regimes) [3], Fast Isotropic Motion. In this case, the rapid tumbling of molecules containing sodium is resulting in a very rapid fluctuation ofthe EFG tensor. Thus the correlation time tc is much smaller than o, which is usually in the order of nanoseconds and « r. «1. As a result, quadrupolar interaction frequency ä>Q is ‘motion-narrowed’ to zero. In this extreme narrowing condition, the Zeeman interaction alone will produce four distinct energy levels, all separated with frequency . The three possible single-quantum coherences (transitions) give rise to three isochronous spectral peaks of equal line widths which lead to single resonance peak at

0 (type A in Fig. 1.6). Both T1 and T2 relaxation times decay mono-exponentially with R1 =

R2 = R3, where R1 and R3 is for relaxation rate of satellite transitions and R2 stands for central transitions. This type of spectrum can be observed in saline (NaCI) solutions such as synovial or cerebro-spinal fluid.

15 INTRODUCTION

Fig. 1.6: Energy level diagram of a 23Na in different motional regimes.

Slow Isotropic Motion. In this regime, sodium ions are in the interaction with macromolecules, which result in slower motion of ions and produce a slower modulation of coQ than in previous case. In this situation, correlation time is longer co0tc «1, so the quadrupolar interaction dominates the relaxation and the satellite and central transitions have different relaxation rates. Thus, Ti and T2 relaxation constants are bi-exponential, with faster relaxation rate of satellite transitions; Ri and R3> R2. The outer transitions encompass 60% of the MR signal and create the fast relaxation component. The inner transition encompass 40% of the MR signal and produces the slow relaxation component. However, coqtc «1, so that co 0 and therefore the shift of fast and slow decaying components from co is very small; smaller than the line widths and so very difficult to detect. Hence, the observed single-quantum spectrum contains three isochronous homogeneous peaks. The narrow inner peak

l\ l\ +2 ■ l'' +- w +) I and 2/ 2/

1\ 3\ ------/ 2/ 2/ exponential relaxation can create multiple quantum coherences that can be detected with quantum filtered sequences. In this regime, triple quantum filtered 23Na spectroscopy is very useful tool for estimating bi-exponential transverse relaxation times, and to acquire MR signal only from sodium ions in the interaction with macromolecules (mainly intracellular sodium).

16 INTRODUCTION

Slow Anisotropic Motion. In this case, sodium the ions are bound to proteins such as the structural proteins like collagen fibers in connective tissues (cartilage, tendon), or the proteins in cell membranes. This regime is present in ordered biological tissues, where the motion of molecules is restricted and thus slow r. > 1). As ä>Q 0, a very small line splitting, which is obscured by the larger line-widths of single quantum resonances, is present in the spectrum (type B in Fig. 1.6). However, Ti and T2 decay is bi-exponential. Moreover, the prevailing presence of sodium in isotropic motion in surrounding fluids obscures the detection of sodium bounded to proteins. Since the measurement of motion-restricted sodium in biological tissues is difficult using conventional single quantum MR, alternative concepts are needed. Although the residual coQ is comparable to line widths, and thus it does not result to line splitting, it creates double quantum coherence. As a result, double quantum filtered signal can be used to assess the degree of ordering in the tissue. Fast Anisotropic Motion. Sodium ions in this regime can be found in the sample that is anisotropic on the macroscopic scale (such as single crystals or oriented liquid crystals). Since co0Tc »1, the time-averaged frequency of quadrupolar interaction ä>Q does not become zero and the EFG tensor has preferred orientation. Moreover, g>qtc »1, so all energy levels are shifted by coQ , which results in four energy levels with three different frequency separations between them (type C in Fig. 1.6). The transitions between these energy levels result in three resonance lines (a central and two satellite lines) separated by coQ . Similar to previous cases,

Ti and T2 decay is bi-exponential. When the components of bi-exponential relaxation cannot be resolved, weighted average of the relaxation times can be observed as:

1 0.2 0.8 — —------1------[1.41] 2T1 2T1 fast 1T1 slow and

_£ _ 0,6 0,4 [1-42] yT2 2T fast 2T slow The above described cases are applicable for single pool of 23Na under the quadrupolar relaxation. Sodium ions in biological tissues can be found in various pools simultaneously. The ions in these pools can relax with different time constants and can exchange between different pools. Thus, a superposition of relaxation time constants from different environments is observed and may result into multi-exponential decay.

17 INTRODUCTION

1.1.5.5 Relaxation of Sodium Nuclei in Tissues

The relaxation times in biological tissues are influenced by motion of the molecules, molecular structure and interactions between molecules and sodium ions. Many macromolecules contain charged groups, such as negatively charged carboxyl or sulfate groups. These groups attract and bind counter charged ions or groups such as Na+, Cl-, or Li+. As described in previous section, bound sodium ions are rotating slower compared to free ions, which result in increase of their correlation time tc and in shortening of the relaxations constants. Moreover, sodium ions bonded to macromolecules can experience cross-relaxation with the nuclei of macromolecules, which result in further shortening of the relaxation times. Macromolecules with charged sites result in electrostatic field gradients. Because the tumbling of macromolecules is relatively slow, EFG are not averaged, and this cause s decrease in relaxation time constants. An anisotropic motion due to the increased size and mass of macromolecules (e.g. collagen) is also influencing the relaxation times in tissues. In ordered biological structures, this property introduces an additional parameter - orientation ofthe macromolecule axis with respect to Bo. Sodium ions bounded or interacting with such macromolecules are in restricted anisotropic motion resulting in shortening of relaxation times. Exchange: Sodium ions in tissues can exist in multiple compartments, such as free, bound, intracellular or extracellular sodium. Assuming different relaxation time constants in different compartments, the exchange of ions between these environments can have significant influence on the observed relaxation times. The rate of exchange between two pools can be described as slow, intermediate or fast, when compared to the difference between their respective relaxation rates. In slow exchange, two pools are considered to be independent, thus bi-exponential relaxation is observed. However, the bi-exponential decay can be distinguished only if the population in each pool is significant and the two relaxation times are sufficiently different from each other. When fast exchange takes place, observed relaxation time is the weighted average of the relaxation times from the two compartments. When the exchange rate is intermediate, multi-exponential curves can be observed.

18 INTRODUCTION

1.1.5.6 Comparison between Proton and Sodium Relaxation Times

1H and 23Na longitudinal (Ti) and transverse (T2) relaxation time constants differ between each other. Previous relaxation times measurements in femoral cartilage of healthy volunteers at 7T revealed 1H Ti of 1259 ms, T2 of 56.3 ms (this is average T2 value; T2 change between cartilage surface and cartilage-bone interface), T2* of 19.7 ms [10] and 23Na of 21.0 ms, T*fast of 0.8 ms and T*sIow of 14.8 ms [11], Fig. 1.7 compares the relaxation processes for

1H and 23Na in cartilage based on these results. As demonstrated, both Ti and T2* relaxation times are much faster for 23Na than for 1H.

T, of 23Na and 1H

T2*of23Na and 1H

Fig. 1.7: Comparison ofT-i recovery of longitudinal magnetisation (upper image) and T2 decay of transverse magnetization (lower image) in cartilage between 1H (green curves) and 23Na (red curves) at 7 Tesla. Relaxation times were taken from references [10, 11],

19 INTRODUCTION

1.1.5.7 Proton and Sodium T2* Relaxation Times

Because 23Na MR signal decay with very short T2 constants, gradient echo (GRE) based techniques are usually used for sodium measurements. Since these techniques do not compensate for the spatial inhomogeneities of the main magnetic field Bo, a transversal magnetisation decay with the time constant T2*. Nevertheless, the 23Na T2 values are important parameters that need to be measured. It can be demonstrated that, for short T2 values and small field inhomogeneities of few pT, T2* differ only very slightly from T2. Since AB is the same for 1H and 23Na, the strength of the field inhomogeneity can be estimated from the 1H T2* values. The influence of a typical range of AB values (0.2 - 10.0 pT) on the drift of 23Na T2* is demonstrated on Fig. 1.8.

Fig. 1.8: Dependency of 23Na T2* constants on Bo field inhomogeneity and T2.

From equation [1.30] we can derive the relationship between T2, T2* and Bo field inhomogeneity:

20 INTRODUCTION

z, T =■ 1 2 [1-43] i+z, / Aß 2^ By using proton T2 and T2* values, it is possible to assess Aß, which can be used for the estimation of 23Na T2 constants from the experimentally calculated T2* values. For example, Welsh et al. reported the mean proton T2 of 56.3 ms and the mean T2* of 19.7 ms for in vivo measurements of femoral cartilage at 7T [10], which results in an average field inhomogeneity of Aß - 0.12^Z. Assuming the same Aß for sodium measurements, the 23Na Z2,z of 0.8 ms would result in T2fast of 0.81 ms and the 23Na T2slow of 14.8 ms would result in T2slow of 16.99 ms. In this case T2* amounts to 25.3ms and 0.99ms respectively. This example demonstrates that the 23Na T2* measurements based on GRE sequences yields values that are not very different from T2 values.

1.2 Magnetic Resonance Imaging Techniques

Basic principles of Magnetic Resonance Imaging (MRI) and specific features of sodium MRI are discussed in this part.

An additional magnetic field (field gradient) in the same direction as ß0 =(0,0, ß0) and with the amplitude that varies linearly with position along a chosen axis is needed for MRI.

When a field gradient G (r) is applied in a given direction r , the strength of magnetic field varies according to:

Bz (r ) = Bo + fG, [1.44]

An MR imaging system can generate gradients along any of the three orthogonal axes x, y or z, using three gradient coils. The application of field gradients enables to use techniques for spatial localization of MR signal such as frequency encoding and phase encoding.

21 INTRODUCTION

1.2.1 Frequency Encoding

Frequency encoding technique allows encoding of the spatial information in one direction. When a gradient is switched on along x axis (without loss of generality) during the acquisition of MR signal, the spins precess at a frequency:

x) = 7/t (*) = /( + xGx) [1-45]

Thus the frequency encoding gradient generates a time-dependent and a position-dependent phase shift ofthe magnetisation vector in the rotating frame of reference. The MR signal then reads: 5 (k ) = fMxy (x, y, t0) e-+°»dxdy [1.46]

MR signal is proportional to the density of given nuclei in the object and can be obtained by the Fourier Transform of the acquired signal. The one-dimensional projection of the measured sample is the Fourier transform of the signal as it evolves with time in the presence of a frequency encoding gradient.

1.2.2 Phase Encoding

To encode the signal in another spatial direction (y), a constant gradient is applied perpendicular to readout (frequency encoding) direction before data acquisition. This results in an additional phase Oy = Gy ■ y in the MR signal that is constant over the readout period:

S (t) = JMxy (x, y, t0)evdxdy [1.47]

In order to obtain a projection of the magnetisation on the axis y, the experiment has to be repeated N times with incrementing the strength of Gy gradient.

1.2.3 Fourier / Cartesian Methods

Measured raw data represent the so-called k-space, which is the inverse Fourier transformation of the image space. Due to Fourier transformation, each point in k-space is

22 INTRODUCTION contributing to each point in the reconstructed image space (and vice versa). A path in k- space can be traced (Fig. 1.9).

Fig. 1.9 : Cartesian three-dimensional Gradient Echo pulse sequence. Readout gradient GP encodes MR signal in x direction, one phase encoding gradient GP encodes signal in y direction, and second phase encoding gradient Gs encodes signal in z (slice) direction. Amplitudes ofthe phase encoding gradients Gs and GP changing in subsequent scans.

After a non-selective RF pulse is applied, the readout dephaser shifts k from (0, 0, 0) to (-kmax, 0, 0). Simultaneously, gradients with variable amplitude Gy and Gz perform the spatial encoding in y and z (slice) direction, which takes k to a new starting point k = (-kmsa, ky, kz), with ky,kz e[-^max, +£max] ■ During the MR signal acquisition, the frequency encoding gradient

Gx achieves spatial encoding in the x direction. The k vector travels horizontally at a constant velocity from k = (-kmax, k.k.,') to k = (+kmax, ky,kz). After the repetition time (TR), the time between two following excitation pulses, a new line is acquired using the different Gy magnitude (for the same slice) or using the different Gz magnitude (for different slice). The signal is sampled in the k-space along lines parallel to the k.axis. Each k-space line represents one digitally sampled gradient echo in one TR. The calculation of the image slices is accomplished through a three-dimensional (3D) Fourier transform along all three encoding directions. This results in a matrix of R xP x S complex data points.

23 INTRODUCTION

In fact, each k-space point contributes to each image point. The central data points in the k-space are responsible for the contrast in the image and also comprise the information about the coarse structure of measured object. On the other hand, peripheral k-space points yield higher spatial frequencies and offer information about the fine details (resolution). In order to optimize usage of the dynamic range of the gradient coils, they are switched evenly between -Gmax and +Gmax. Thus the central k-space points are recorded when the amplitude ofthe phase encoding gradients is low, so phase coherence ofthe spins is highest (resulting in highest intensity). The time between the centre of the excitation pulse and the central k- space data acquisition is defined as the echo time (TE). The setting of TE and TR is influencing the image contrast. The acquisition speed of MR imaging sequence is determined by how fast the k-space data can be acquired. The time demanding aspect of Cartesian data sampling is the large number of phase encoding steps needed to create an image. For a standard GRE technique, a single line of k-space data is acquired after each excitation. Thus the scan time (Tacq) is defined as:

1Tacg = N‘i'K -TR-11V Ne.,acq [1-48]

Where NPE is the number of phase encoding steps, TR stands for the repetition time and Nacq is the number of averages.

1.2.4 Resolution of Image

The signal in MR imaging is digitized and represented by a finite number of data points. For example, if R time domain data points is sampled with an interval At, the total acquisition time is Tacq = RAt. Only limited number of frequencies can be represented by the digitized oscillatory data. The waveforms oscillating with periods shorter than 2At will appear to oscillate with a period longer than 2At (aliasing). The Nyquist (sampling) theorem says that the highest frequency contained in the MR signal that can be measured is vmax = 1/2AZ [2],

The relationship between the image resolution Ax and the frequency resolution Av during data sampling is given by the Tacq.

Gx Ax [1.49]

24 INTRODUCTION

2x 1 i Ax = [1-50] ^^xTacq 2kmax

v where Ax = ^~GxAr is the width of a k-space step in readout direction. The field-of-view 2n

During phase encoding, direction the gradient strength vary between -Gy and +Gy.

Therefore it is necessary to perform P phase encoding steps with AG = to encode P12 FOV = PRy in the phase direction. Similarly, S phase encoding steps are needed to encode set of images in third direction. When assuming an isotropic image resolution (FOV is a square), the momentum of the maximum phase encoding gradient needs to be only half the momentum of the readout gradient Gytp = GxTacq, Thus when tp is the phase encoding gradient duration, Gy 2tp = GxTacq, and the maximum strength of phase encoding gradient is:

G = 2xR = PAG y ~ yFOVt 2 [1-52]

The width of k-space step is AA =— AG / and the resolution Ay in phase encoding y 2n y p direction reads: 1 2x Ay = [1-53] M Rky yGf. The resolution in third-dimension (“slice encoding”) is calculated in the same manner as the phase encoding resolution Az = Ay. The equations [1.51] and [1.52] suggest, that the spatial resolution (at given TaCq, tp) depends on the maximum of the gradient strength (limited by the gradient coil performance) and the gyromagnetic ratio y. For fixed gradient strength, greater y allows higher resolution. Therefore in order to achieve in 23Na images the same resolution as for 1H images, four times stronger gradients are needed. Since sodium concentration in the body is much lower compared to 1H concentration, the limiting factor for image resolution in sodium in vivo measurements is to acquire sufficient signal-to-noise ratio (SNR) in feasible measurement times, which result in images with low resolution.

25 INTRODUCTION

1.2.5 Three Dimensional Acquisition

The 3D Fourier transform is used to reconstruct a 3D data set. Since each excitation of the 3D volume contributes to the final SNR, the SNR of this technique is higher than the SNR ofthe multi-slice method. The SNR is then proportional to:

= AxAyAz^, • Ny • Nz • Tacq [1.54] where AxAyAz is the volume of one voxel, Nacq is the number of averages, N, and Nz is the number of phase encoding steps and Tacq is the readout time. Additionally, non-selective RF pulses with duration of few microseconds can be used in the 3D acquisition compared to much longer slice selective RF pulses used for two-dimensional (2D) multi-slice techniques; thus 3D methods acquire signal at shorter TEs. Moreover, 3D data sets recorded with isotropic resolution can be reconstructed in any direction in the post-processing. Total measurement time then takes:

Tmeasurement = Ny acq-TR • N'y • Ny z Lm 1 ■*'*<1551

In the case of very long Ti times, and thus also very long TR, the 2D multi-slice techniques might be more effective, as the time between two RF excitation in the same slice can be used to excite other slices.

1.2.6Sodium MR Imaging

Since the 23Na MR signal in cartilage is about 3400 times smaller than that of 1H, and 23Na relaxation times are much shorter than 1H relaxation constants, the optimization of sodium MRI sequences aims to maximize the SNR in sodium images. As discussed in section 1.2.4, the minimum spatial resolution Axmm depends on the maximum strength of gradients Gmax and the gyromagnetic ratio /. Therefore to facilitate comparable resolution between sodium and 1H images, the gradient strengths of all gradients in sodium sequence must be scaled by 4 « I /23Na.

Minimizing echo time. The 23Na MR signal in the cartilage decays bi-exponentially; with the short component T*fast of 0.8 ms and long component T*slow of 14.8 ms, as measured in

26 INTRODUCTION vivo at 7T [11], To collect 23Na signal from both T* components, it is necessary to use sequence with TE shorter than T*fast. In order to minimize TE of GRE techniques, some sequence parameters can be optimized: - 3D excitation, which allows shorter RF pulses - shortening of RF pulse duration (restricted by specific absorption rate limits) - increasing readout bandwidth (BW) (limited by the gradient hardware and the SNR loss) - employing asymmetric readout [12] More detailed description the optimization is presented in section 1.2.9. Point Spread Function and Readout Bandwidth. The NMR signal decays with T* during the acquisition of data. This leads to an inhomogeneous weighting of the k-space and to a decrease in image resolution due to the attenuation of the outer k-space points (higher frequencies). To reduce blurring, the readout time Tread should be smaller than T*, and thus the readout BW must be larger than the line width of the free induction decay 1/r2* [13]:

BW > JT [1-56]

Assuming a T* of 0.8 ms, the required readout BW should be larger than 1250Hz.

1.2.7 Gradient Echo

GRE is a class of pulse sequences used in applications that require high acquisition speed. Since T2 decay of 23Na signal is very fast, GRE based sequences are the most appropriate for sodium MRI. In GRE sequences, echo is formed using gradient reversal on the frequency-encoded axis. To get the echo at the centre of the ADC acquisition, the frequency encoding gradient is preceded by the readout dephaser whose momentum equals half the readout gradient momentum (Fig. 1.9). In case TR of sequence is not long enough to allow for complete decay of the transverse magnetization (TR < 4 x TE) by the end of TR, remnant transverse magnetisation can cause imaging artefacts. To prevent this problem, the remnant magnetisation can be spoiled by destroying phase coherence between two following TR periods. Spoiling can be achieved by two different methods. Gradient spoiling rely on randomly varying gradient pulses

27 INTRODUCTION after data acquisition (Fig. 1.9). RF spoiling is performed by randomly jittering the phase of the RF pulses. In latter case, the phase of the receiver must be changed to follow the phase shift of the transmitter. The steady state signal (magnetization) ofthe GRE sequence is given by equation:

TR \ TE 1 - e sin a [1-57] 5 = 5,0 _TR 1 - cos a- e Tl where 50 is the signal in equilibrium. The maximum signal S for a given TR, TE and ?j is obtained at the Ernst Angle aE [14]:

TR \ aE = arccos e [1-58]

The GRE sequence available on most MR systems offers two type of data acquisition. The echo can be acquired either in the center of readout gradient (full or symmetric echo) or before the center of the readout gradient (partial or asymmetric echo). When using asymmetric echo option, the TE can be reduced when compared to symmetric echo case (Fig. 1.10). In asymmetric acquisition, missing data are estimated during image reconstruction.

Fig. 1.10: Comparison of 3D GRE sequence with echo acquired in the center of readout gradient A) and asymmetric echo B). Please notice the TE reduction in the asymmetric case.

1.2.8 Gradient Echo with Variable Echo Time Scheme

Several approaches can be helpful in shortening the TE of Cartesian sampling techniques, such as asymmetric RF pulses, asymmetric readout, optimal gradient switching

28 INTRODUCTION patterns by using maximum slew rates and gradient amplitudes, sampling at gradient ramp, or employing variable echo time (vTE) scheme [15], The vTE concept is varying the duration of the phase encoding gradient, rather than its amplitude. As a result, the TE in the k-space center (requiring small gradient moments for slice and phase encoding) is significantly shortened, when compared to the outer k-space parts (Fig. 1.11). Moreover, the signal contrast is defined by the lower spatial frequencies of k-space [16], In work by Deligianni et al., authors present the Fourier-encoded GRE sequence employing vTE scheme (vTE-GRE) in combination with high asymmetric readout (with maximum asymmetry of 8%, where 100% is fully sampled echo) and optimized gradient slew rate and switching patterns [17], The vTE- GRE sequence allowed for considerably shorter TE compared to conventional GRE. A projection onto convex sets algorithm was combined with vTE-GRE for image reconstruction from partially sampled echoes [18],

Fig.1.11 : For vTE-GRE sequences, the TE is a function of the prephasing gradient moments along the phase (ky) and slice (kz) encoding direction. The minimum TE (7iEmin) is achieved at the k-space center. As long as only the amplitude of ky, kz prephasing gradient moments is changing, TEm\n can be maintained (indicated by kmin in the plot). With an increase in duration time of ky, kz prephasing gradient moments, the TE starts to increase as well (for ky, kz>km\n). Reproduced from [17], 29 INTRODUCTION

1.2.9 Optimization of Cartesian Gradient Echo Techniques

Since the 23Na T2 time is very short, the GRE sequence is the most suitable technique for sodium MRI. Due to the short Ti of 23Na, gain of signal can be achieved by fast averaging of GRE sequence with short TR. Moreover, 3D methods with shorter non-selective RF pulses allow for shorter TEs compared to 2D techniques with longer selective RF pulses. Additionally, 3D methods offer intrinsically better SNR. The 3D Cartesian sampling encodes the k-space in the kx and ky directions (Fig. 1.9). The amplitude ofthe phase encoding gradients GP,GS varies at each subsequent TR, so that the starting point in ky direction varies from -k,max to +kymax. The TE is defined as the interval between the middle of RF pulse and the acquisition of point kx= 0 in the k-space. The minimum possible TE is thus limited by the RF pulse duration, the length of phase encoding gradients and the length of readout dephaser (Fig. 1.9). To shorten the TEof GRE based sequence, following parameters can be optimized: RF pulse duration. Shortening the RF pulse for a 2D method is limited by the deterioration ofthe slice profile. For a 3D method, the shortening of non-selective RF pulse is limited by the SAR limits, which are related to the TR of the sequence. In other words, the longer is the TR, the shorter RF pulse can be applied. Duration of gradients. This can be shortened by employing the maximum available slew-rates for readout dephaser and phase encoding gradients. To prevent peripheral nerve stimulation, the slew-rates are limited by the scanner (slew rate at Siemens 7T is 200 mT/m/ms). When measuring the image with lower resolution, lower gradient strengths can be used for space encoding and also SNR is improved. Asymmetric readout. As defined before, the TE is the time between the centre of the RF pulse and the acquisition of kx= 0. To reduce TE, it is possible to implement an asymmetric readout. When asymmetric readout is used, the integral of the readout dephasing gradient is smaller than the half of the readout gradient, and thus the echo is refocused before the middle of the acquisition window. This result in omission of some ofthe k-space data before the echo, which can result in artifacts such as Gibbs ringing. The missing data are calculated during image reconstruction. Time of acquisition (readout time). In the GRE based sequences, the shorter is the acquisition time (Tacq = 1/BW), the shorter is the TE. However, shortening of acquisition time also reduces the SNR in the image (equation [1.54]). The SNR loss can be partially

30 INTRODUCTION compensated by decreasing the TR while increasing the number of averages. Since the T2 decay of 23Na signal during acquisition results in blurring in the image, the decrease of acquisition time also minimize blurring. Minimal duration of acquisition time is limited by hardware - readout gradient strength.

1.3 Structure and Function of Articular Cartilage

In this chapter, basic information about the macroscopic and microscopic structure as well as the function of articular cartilage will be discussed. More details on this topic can be found in the following review papers [19-21],

1.3.1 Synovial Joints

The joint is complex structure that achieves biomechanical tasks associated with skeletal motion. Articular cartilage covers the end of long bones in the joints and provides a smooth surface and the distribution of the load. The joint components are in close interaction and the resultant distribution of load in thejoint is influenced by the anatomy and mechanical properties of joint components. In synovial joints, such as knee or upper ankle joint (Fig. 1.12, Fig. 1.13), synovial fluid nourishes and lubricates articular cartilage. Ligaments constrain the motion of skeletal elements. Bones provide the transmission of skeletal load and allow the transportation of macromolecules to cartilage. In the knee, menisci help in distributing the load. Abnormalities of ligaments, menisci (e.g. tears) or bones can lead to changes in distribution of cartilage load which may result in degeneration of articular cartilage [22, 23],

31 INTRODUCTION

Femur Patellar art -"-, cartilage Patella —

Femoral art. cartilage

Lig. patellae Lig. cruciatum anterius

Corpus adiposum " infrapatellare Tibial articular cartilage

Fig. 1.12: Illustration ofthe Median-Sagittal cut through the right knee joint. Reproduced from the “Thieme” online picture database (www.thiemebilddatenbankanatomie.de).

Lig. talo- calcaneum Tibia interosseum Articulatio talocruralis Articulatio talocaicaneonaviculare Tibial cartilage (art. talocruralis)

Talar cartilage (art. talocruralis)

Talus

Articulatio talocalcanea Talar cartilage (art. talocalcanea) Calcaneal cartilage (art. talocalcanea) Calcaneus

Fig. 1.13: Illustration of the Sagittal cut through the ankle joint. Reproduced from the “Thieme” online picture database (www.thiemebilddatenbankanatomie.de).

In order to understand cartilage disease, monitoring of various joint structure is required. MR imaging is capable of imaging various joint elements (ligaments, menisci, subchondral bone) in one imaging session [22, 24], In addition, several kinematic studies

32 INTRODUCTION proved the ability of monitoring the movement of joint structures [25], Finally, novel MR imaging methods do not reflect only the morphological aspects of cartilage, but also offer a measure of cartilage biochemical composition and its functional integrity.

1.3.2 Macroscopic View of Cartilage

Articular cartilage is specialized connective tissue located in diarthrodial joints. Native articular cartilage is hyaline cartilage having thickness of 2 to 4 millimeters. Its main task is to promote the load transmission with a low frictional coefficient and to maintain a smooth interface for articulation. Cartilage does not have blood vessels, lymphatics, and nerves, therefore its nutrition is provided by diffusion from the synovial fluid and chondrocytes depend mainly on anaerobic metabolism. Since the intrinsic healing and repair capacity of cartilage is limited, healthy cartilage is important for correct functioning of the whole joint. Articular cartilage as an organ can be evaluated by assessing its morphological appearance, its ability to bear up normal mechanical loads, and its biochemical composition. During the process of cartilage degeneration, changes in mechanical and biochemical properties advance alterations in gross morphology of cartilage [20],

1.3.3 Zonal Architecture of Cartilage

The structure of mature articular cartilage is highly ordered. The cartilage is composed of several layers which differ in architectural structure and key components. From joint cavity into the direction of cartilage-bone interface, cartilage can be divided into superficial zone, transition zone, radial or deep zone and calcified zone (Fig. 1.14). This specific organization results from complex process during embryogenesis, which is called endochondral ossification. Superficial (tangential) zone. This zone represents 10% - 20% of cartilage thickness and forms gliding surface for articulation. The superficial layer provides the tensile properties to the cartilage, which help to resist the tensile, compressive, and sheer stress imposed on cartilage by articulation. In this zone, the tightly packed collagen fibers (mainly type II and IX) are aligned parallel to the joint surface. The superficial zone contains a high number of

33 INTRODUCTION elongated inactive chondrocytes (Fig. 1.14). The integrity of this layer is crucial for the protection of deeper layers. Transition (middle) zone. This zone makes up 40% - 60% of the cartilage volume and can be seen as a functional and anatomical bridge between the deep and superficial layers. The transition layer helps cartilage resist compressive forces. This zone contains obliquely organized thicker collagen fibrils and lower density of chondrocytes with intermediate shape (Fig. 1.14). Deep (radial) zone. This zone represents about 30% of articular cartilage thickness and provides the greatest resistance to compressive stress. In this zone, collagen fibrils have the largest diameter and are aligned perpendicular to the cartilage-bone interface. The spheroidal-like chondrocytes are arranged in columns, parallel to the collagen fibers (Fig. 1.14). This layer has the lowest water content and the highest proteoglycan (PG) concentration. Calcified cartilage. The tidemark creates border between the deep zone and the calcified cartilage. The calcified layer is securing the collagen fibers of the deep zone to subchondral bone and thus providing fixation of cartilage to bone as well as a mechanical transition between the two tissues. The chondrocytes in this layer are rare and hypertrophic.

Artimilor C'lirfar'O

Tidemark Tidemark A B Fig. 1.14: Schematic diagram of the structure of healthy articular cartilage. Organization of the cells in the cartilage zones A. Architecture of the collagen fibers in the cartilage zones B. STZ stands for superficial (tangential) zone. Reproduced from [26],

34 INTRODUCTION

In addition to zonal variations in cartilage structure, there is variability in composition and organization of the extracellular matrix based on the distance from chondrocytes. Thus the extracellular matrix can be classified as pericellular, territorial, and interterritorial matrix. The pericellular matrix is a thin layer situated immediately around the cells, and it contains mainly PGs and only very little collagen. Outside this region, the territorial matrix composed of basketlike web of thin collagen fibrils is protecting chondrocytes against mechanical stress. Further away from chondrocytes is the interterritorial region, which is the largest of all regions. It contains large collagen fibers and much more PGs when compared to previous regions. The orientation of collagen fibers in this region depends on their proximity from cartilage-bone interface. Of all the regions, interterritorial matrix is contributing most to the cartilage biomechanical properties [27],

1.3.4 Molecular Composition of Cartilage

Different types of cartilage tissue are present in human body and based on their molecular composition they can be histologically classified as hyaline, elastic cartilage and fibrocartilage [28], Native articular cartilage is the hyaline type of cartilage, which is the predominant type of cartilage in the body. Cartilage contains chondrocytes nested in an extracellular matrix. The matrix is composed of macromolecules such as collagen, PG, noncollagenous proteins, lipids and others (Fig. 1.15) [29], The cartilage zones are characterized by a different organization of the collagen fiber network, and by differences in the type and concentration of PGs and chondrocytes (see chapter 1.3.3).

35 INTRODUCTION

Fig. 1.15: Schematic diagram ofthe organization of macromolecules in extracellular matrix of articular cartilage. Reproduced from [30],

1.3.4.1 Chondrocytes

Chondrocytes comprise about 2% of the total cartilage volume and vary in size, number, and, shape across the cartilage zones. In the superficial zone, the chondrocytes are smaller, flatter and more densely distributed with a greater density when compared to deeper zones (Fig. 1.14). The extracellular matrix is strongly influenced by the function of chondrocytes. These cells synthesize, maintain and repair the extracellular matrix while the matrix state is directly influencing their function. Changes in chemical and mechanical environment of chondrocytes can alter cellular synthesis or even directly damaged the chondrocytes. The amount of chondrocytes decrease, they rarely divide and their cellular processes slow down when skeletal maturity is achieved [29], These factors contribute to the limited intrinsic capacity of mature cartilage the heal defects caused mainly by injury.

1.3.4.2 Water

In hyaline cartilage, water makes up 60% to 80% of its wet weight. The relative content of water is increasing from the deep zone (~65%) to the superficial zone (80%). Most of water is situated in interfibrillar space, and it appears in form of gel with dissolved ions (e.g. sodium, calcium, chloride, potassium). After applying pressure to cartilage, most of water may move

36 INTRODUCTION through the matrix and across the cartilage surface [31], Water flow contributes to cartilage material properties, provides joint lubrication, and helps to transport nutrients to chondrocytes through diffusion. Articular cartilage has a high frictional resistance against the movement of water across the matrix. This mechanism allows cartilage to cope with high loads.

1.3.4.3 Collagen

The most abundant macromolecule in the extracellular matrix of cartilage is collagen. It makes up about 15% of cartilage wet weight. More than 90% of collagen in the cartilage is of type II. This component creates long fibers that are interwined with PG macromolecules. Other types of collagen macromolecules (such as III, V, VI, VII, IX, X, XI, XII, XIV) occur in small quantities and help stabilize the collagen network [32], All collagen types contain a region containing three polypeptide chains arranged into triple helix that stabilizes the matrix and provides cartilage with important tensile and shear properties.

1.3.4.4 Proteoglycans

PGs can exist either as protein monomers or as aggregates of monomers linked to fibers of hyaluronic acid using specialized proteins (Fig. 1.16). Each PG monomer is linked to a protein core via one or several sulfated glycosaminoglycans (GAG) side chains [29], The GAGs side chains consist of more than 100 monosaccharides and contain high concentration of anionic charge (mainly due to carboxyl and sulfate groups), which give PGs to two important characteristics. The PGs bind cations (such sodium) and they are hydrophilic. Additionally, the GAGs remain separated because of negative charge repulsion. A variety of PGs is present in the articular cartilage. The aggregating PGs, including aggrecan, and versican, makes 5% to 7% of cartilage wet weight. The main and the largest PG is aggrecan, which contains over 100 keratin sulfate and chondroitin sulfate side chains [29], Aggrecan is situated mainly between the fibrils of the extracellular matrix and is responsible for osmotic properties of cartilage. The nonaggregating PGs, such as decorin, biglycan, and fibromodulin, form less than 5% of cartilage wet weight. These PGs are much smaller than aggrecan. Decorin and fibromodulin are responsible for interfibril interactions and they interact with the collagen II. Biglycan may interact with type VI collagen and is usually located near chondrocytes.

37 INTRODUCTION

Protein Core

Chondroitin Sulfate Chains

Keratan Sulfate/^ Chains Protei

Hyaluronic Acid

Fig. 1.16: Schematic sketch of a PG aggregate composed of a central hyaluronic acid chain with the PG monomers attached via link proteins. Each monomer consists of multiple GAGs (such as keratan sulfate, chondroitin sulfate) bound to a protein core. Reproduced from [33],

1.3.5 Biomechanical Implication of Cartilage Composition

PGs occupy much greater volume in solution than they do in native cartilage. PGs in cartilage seems to be “compressed” by the collagen network, and thus only partially hydrated. In theory, damaged collagen network could result in the expansion of the PGs and in the absorption of more water by cartilage, causing the swelling of the extracellular matrix. Similar behavior can be seen in chondromalacia patella. The model of compressed and partially hydrated matrix also helps understand the nutrition and mechanical properties of cartilage. To explain some of the properties, we can view the cartilage as medium composed of two phases: a solid phase consisting of extracellular matrix (collagen, PGs, and other glycoproteins) and a fluid phase consisting of water and electrolytes [34], The relationship between interstitial fluid and PG aggregates is responsible for compressive resilience of cartilage. The close spacing between negatively charged GAGs result in negative electrostatic repulsion forces and contributes to substantial osmotic pressure in the cartilage [34, 35], During joint loading, interstitial fluid flows slowly out ofthe extracellular matrix, and when the compressive load is removed, fluid return back into the cartilage tissue. Thus the fluid phase of cartilage “protects” the solid phase from the shock of rapid joint loading (Fig. 1.17) [34],

38 INTRODUCTION

Fig. 1.17: Schematic representation of processes in matrix of cartilage during rest and load. Negative electrostatic repulsion forces and high osmotic pressure (both provided by GAGs) together with high water content help cartilage absorb stress. Reproduced from [36],

Articular cartilage has viscoelastic properties and during constant load exhibits time- dependent behavior. In the first seconds of loading, up to 75% of the compressive stress is supported by the fluid phase. When loading last longer (100 to 1000 of seconds) equilibrium is reached, and the exit of tissue fluid stops. In this case, all ofthe compressive stress is borne by the solid phase. Moreover, fluid flow during cartilage loading and the interaction between the fluid and solid phases are important for supplying the nutrients into the cartilage, and transporting the products of metabolism away [34], Joint motion and load are therefore important for maintaining normal cartilage structure and function.

39 INTRODUCTION

1.4 Cartilage Injury and Repair

This chapter summarizes basic information about cartilage injury, its respond to damage and about treatment options for repair of chondral defects. Additional information on this topic can be found in the following papers [19, 37-39],

1.4.1 Mechanical Injury of Cartilage

Epidemiology of cartilage injuries. Two retrospective studies reviewed a large number of arthroscopies reported 66% (with 11 % of full thickness lesions) [40] and 63% [41] incidence of articular cartilage defects. These studies highlighted an underestimated incidence of chondral lesions. Based on arthroscopy, focal chondral lesions were classified by Outerbridge into different stages [42], Untreated large lesions ultimately lead to premature end-stage arthritis with the most often treatment being prosthetic replacement of the articular surface (knee arthroplasty). This treatment is appropriate mainly for elderly people (older than 50 years), with a sedentary life style. Pathomechanisms of cartilage degradation by mechanical overload. Mechanical loading in a physiological range influences cartilage synthesis [43] and is very important for the cartilage health. The cartilage tissue can atrophy without mechanical stimulation. On the other hand, a major cause of cartilage destruction is a mechanical injury that often develops into the secondary osteoarthritis (OA). Moreover, obesity-related abnormality in joint loading [44], joint laxity [45], or altered joint geometry [46] have been also identified as risk factors for the cartilage degeneration and OA. Mechanical injury can result in direct damage to the extracellular matrix, or in a reduction of biosynthetic activity of chondrocytes and in expression of enzymes responsible for matrix degradation. Animal studies demonstrated that high impact loads to the joint can result in loss of tissue integrity, mechanical properties, or to cell death [47], Moreover, various in vitro models have described that a mechanical injury often lead to cartilage tissue swelling, decrease in shear and compressive stiffness (due to collagen network disruption), decrease of chondrocyte biosynthetic activity, chondrocyte death by necrosis and apoptosis, progressive PG decrease and consequent loss of cartilage properties, and increased expression of several matrix-degrading enzymes such ADAM-TS5 and matrix- metalloproteinases (MMP-1, MMP-2, MMP-3, MMP-9, MMP-13) [48], Although all triggers,

40 INTRODUCTION mediators and pathways of cartilage degradation remain unknown, activation of antioxidative defense mechanisms, together with the inhibition of MMPs and of angiogenetic factors might have potential therapeutic effect against cartilage destruction.

1.4.2 Intrinsic Response of Cartilage to Injury

A typical tissue response to injury relies on a cascade of inflammation, necrosis, repair, scar tissue remodeling and it requires vascular supply. Hyaline cartilage is an avascular tissue, and thus its response to injury differ from the classic response. Intrinsic repair capacity of cartilage is therefore very low [29, 49, 50], Additionally, the chondrocytes are immobilized in a collagen network, and cannot migrate from healthy tissue to the site of injury. Cartilage defects can be divided into two basic categories. 1. ) Damage of the extracellular matrix, without destructing the cells. In this case, if the capacity of chondrocytes to synthesize new PGs is greater than the loss of matrix, cartilage tissue will be restored. 2. ) Destruction of the matrix and cells, due to intensive or penetrating injury. In this case, results of the repair response depend on several factors. Defect depth. According to the depth, cartilage lesion is classified as osteochondral defect, full thickness chondral defect or partial thickness (flap) defect. The chondral defects further increase in depth and size and do not repair on their own [49, 51], On the other hand, when osteochondral defect extends into the subchondral vascular bone marrow that provides mesenchymal progenitor cells into the defect, fibrocartilage-like repair tissue is created. However, fibrocartilage-like repair tissue is structurally and biomechanically inferior to the hyaline cartilage, and thus not ideal for a load-bearing function. Defect size. Small defects, with area less than 1 cm2, do not affect stress distribution so much, and are less likely to progress. Patient’s age. Population of chondrocytes, its synthetic and mitotic activities, as well as cartilage hydration decline with age [51], Younger rabbits (5 weeks) have demonstrated a better repair response for chondral lesions than older animals (4 months). Depth of injury is also strongly influenced by age. Children and teenagers usually get osteochondral lesions, whereas adults usually develop chondral lesions, probably due to the well-developed calcified zone. Thus while osteochondral lesions usually heal without any problem in the children, they heal very rarely in adults.

41 INTRODUCTION

Defect location. Repair response depends on whether the defect is located in loaded or unloaded part of the cartilage. Reduced loading (or immobilization) results in reduction of smaller PGs, in reduction of synthesis and aggregation of GAGs (reversible to a certain limit), and irreversible disruption of collagen network.

1.4.3 Measures of Knee and Ankle Function

In most cases, damage to the cartilage occurs after a trauma to the knee (e.g. twisting injury) and it is often associated with damage to other knee structures (e.g. ligament, meniscus). Usual symptoms of cartilage injury are similar to meniscus tear, i.e., pain around the joint and during movement, swelling, locking (piece of cartilage obstruct from the smooth movement), pseudo-locking and catching. Measures of knee function reported by patients with knee problems are important from research as well as from clinical context. Aspects important to patients include pain, quality of life, function, and activity level. Since the gold standard of patient outcome measure does not exist, many scoring systems of patient-reported outcome are currently in use. International Knee Documentation Committee (IKDC) Subjective Knee Evaluation Form [52], The IKDC form detects deterioration or improvement mainly in function, but also in symptoms, and in sports activities due to knee problems. Achieved score is between 0 and 100, where 100 represents no limitation in daily activities and no symptoms. The IKDC form has been proved to be sensitive to changes resulting from surgical interventions, presenting its usefulness in certain patient populations. International Cartilage Repair Society (ICRS) Form [53], The ICRS form is based on the IKDC form and has supplementary questions regarding the functional status, the activity level, as well as forms for classification and mapping of the injury. This form is often used to record demographic data, the history of symptoms, pain, functional score, characteristics of the chondral defect, and the findings of the clinical examination. Knee Injury and Osteoarthritis Outcome Score (KOOS) [54], The KOOS form evaluates patients’ knee problems over short- and long-term period - from weeks to decades. The maximal score of 100 points indicates no problems, while the minimal score of 0 represents extreme problems. The KOOS form is well established and reliable patient- reported scoring of knee function for patients with various types of knee injuries and for people with OA. Instead of total, aggregate score, individual scores for each subscale can be used.

42 INTRODUCTION

Following five subscales can be used: Symptoms - 7 questions; Pain - 9 questions; Function in daily living - 17 questions; Function in sports and recreational activities - 5 questions; Quality of life - 4 questions. This option enhances clinical interpretation, the impact of different types of surgeries on different subscales, and guarantees the validity of this form for different groups. Lvsholm Knee Scoring Scale [55], This scale determines outcome after ligament surgery on the knee, especially the symptoms of instability. It includes five subjective and three functional subcriteria. The score ranges from 0 to 100, and can be categorized as excellent (95-100), good (84-94), fair (65-83), or poor (less than 65) [56], The Lysholm scale was validated in many studies and is considered sensitive to changes following surgical and nonsurgical intervention. Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) [57, 58], This measure evaluates the treatment response or the course of disease in patients with OA. The higher score, the worse is the pain, physical function, or stiffness of the knee/hip joint. The WOMAC is simple, quick, and one ofthe most often used patient-reported scoring system. It offers the option to use the scores from each subscale, which enhances the interpretation of the measure. Teqner Activity Score (TAS) [56], This score is a standardized system of grading sporting and working activities. Several observations suggest that limitations in function scores evaluated by Lysholm scale could be masked by an overall decrease in activity level. To overcome this limitation, TAS was developed [56], Assigned score is based on the level of patients’ activity, and ranges from 0 to 10. The more demanding activities, the higher is the TAS score. The TAS was designed to consider the effect of activity level on symptoms, such as pain relief by avoiding irritating activities. Modified Cincinnati Knee Rating System [59], This system rates the knee function and the limitations in managing daily activities. Maximum score of 10 points refers to unlimited full functionality. This scoring system can be used also for ankle joint. The American Orthopaedic Foot and Ankle Society score (AOFAS) [60], The AOFAS score evaluates a perception of function and pain in patients. The maximum score is 100 points; 50 points for function, 40 for pain and 10for alignment.

43 INTRODUCTION

1.4.4 Surgical Methods for Treatment of Cartilage Defects

The goal of cartilage repair surgery techniques is to allow a pain-free motion of the joint by restoring the surface and function of articular cartilage. Additionally, surgical procedures aim to prevent from further cartilage degeneration by providing repair tissue that has the same structure, composition, and mechanical properties as native cartilage [61], Chondral defects can be treated with different surgical interventions. Currently used cartilage repair protocols can be divided in two groups: i) bone marrow stimulating (BMS) techniques, ii) reconstructive techniques [62], i) Microfracture (MFX), debridement (abrasion), and Pridie drilling can be classified as BMS techniques. These procedures expose chondral lesion to material coming from the bone marrow through the holes which penetrate the subchondral plate. ii) Reconstructive techniques such as mosaicplasty or autologous chondrocyte implantation use donor-derived osteochondral fragments or chondrocytes (mostly autografts) to reconstruct the area of cartilage defect. These methods require a suitable donor tissue. Although the results in the literature vary between the patient groups, the cartilage repair tissue usually does not have identical structure, composition and thus mechanical properties as the native hyaline cartilage. However, most of cartilage repair techniques have improved the patient symptoms and joint movements [49], Untreated osteochondral defects often lead to early-onset of OA in adults. Symptomatic defects should be therefore treated. While isolated chondral defects are not very common (4% of arthroscopies), 40-70% of defects is accompanied by ligament and/or meniscus injuries [50],

1.4.4.1 Debridement

Mechanical overloading of cartilage in a symptomatic chondral defect has a detrimental effect on adjacent and opposing cartilage [63], Removal of damaged cartilage has been shown to decrease the symptoms for five or more years [64], The aim of debridement is to remove all damaged cartilage and to abrade the calcified layer in order to form the base for creation of new tissue (Fig. 1.18). Although the patient outcome was improved after this procedure, the results of treatment gradually deteriorated over the next five years [55],

44 INTRODUCTION

Fig. 1.18: Schematic illustration of debridement procedure. Reproduced from www.medimagery.net.

1.4.4.2 Pridie Drilling and Microfracture

MFX was introduced by Steadman [65] and this procedure is a modification of the previously developed Pridie drilling technique [66], Both procedures rely on accurate debridement of all damaged cartilage down to the subchondral bone plate. Afterwards, multiple holes are created in the subchondral bone in the area of defect, by a surgical drill (Pridie drilling), or by an arthroscopic awl (MFX) (Fig. 1.19). Finally, the repair site is filled with the super clot, which is the optimal medium for pluripotent bone marrow cells which migrate into defect area. The cells should in ideal case differentiate into chondrogenic cells, which are able to produce cartilage [67], However, the result of these techniques is usually the formation of fibrous repairtissue. Advantages of MFX over Pridie drilling comprise reduced thermal harm of subchondral bone and the formation of a rougher surface with easier adherence of repair tissue. Additionally, curved awl penetrates subchondral bone plate easier than a drill during an arthroscopic procedure. There are currently no studies which compare the outcome of Pridie drilling with MFX. Hunziker et al. published acceptable clinical results of BMS techniques during the five years after surgery, which afterwards decline with time [68], Steadman et al reported that 80% of patients which received MFX seven years ago rated themselves as improved outcomes [69],

45 INTRODUCTION

They observed greater improvement in patients younger than 35 years compared to patients with age between 35 and 45 years [69], Knutsen et al showed that most of the repair tissue biopsies from MFX patients two years after surgery were composed mainly from fibrocartilage that degenerates with time [70], The rehabilitation protocol with early joint motion with reduced weight-bearing is important part of the treatment.

© Mod:magery nol Fig. 1.19: Schematic representation of MFX technique. Reproduced from www.medimagery.net.

1.4.4.3 Mosaicplasty

Osteochondral grafting or mosaicplasty, was introduced in 1993 [71], In this repair technique, cylindrical osteochondral plugs are taken from affected joint with a cylindrical cutting device and placed into the osteochondral lesion to fill the defect with an autograft “mosaic”. Plugs are usually harvested from the nonload-bearing areas of affected joint (e.g. peripheries of femoral condyle). To maximise defect filling, plugs of different sizes can be taken (Fig. 1.20). This treatment can be performed as an open procedure, or arthroscopically.

46 INTRODUCTION

Fig. 1.20: Schematic illustration of mosaicplasty procedure. A. procurement of osteochondral graft; B. placement and transplantation of graft at repair site; C. final appearance of an autograft “mosaic” at repair site and the holes after harvested osteochondral cylinders at donor site. Reproduced from [72],

This method allows treatment of chondral and osteochondral defects in the same way. Another benefit of this method is that the defect is filled with hyaline cartilage. The treatment area is recommended to be between 1 and 4 cm2 [73], There are some limitations of this technique. For example, production of a smooth convex cartilage surface is not easy. The thickness ofthe cartilage in the plugs is usually different from that in the defect area, and thus the subchondral layer reconstitution may not be optimal. Additionally, good lateral integration is rare [74] and the synovial fluid may pass through the subchondral layer and result in formation of a cyst. Since access to the cartilage surface needs to be perpendicular, the treatment of lesions in the tibial plateau and patella is difficult [75], Hangody and Fules reported excellent or good results in 92%, 79% and 87% of patients who received mosaicplasty of the femoral condyle, patellofemoral joint and tibial plateau, respectively [73], Unfortunately, authors did not report survival of the grafts in the patients with the longest follow-up period.

1.4.4.4 Periosteal Grafts

Periosteal grafts are taken from the perichondrial tissue and grafted to the area of chondral defect (Fig. 1.21). An advantage of this approach is that both chondrogenesis and osteogenesis can occur in periosteum [76],

47 INTRODUCTION

Fig. 1.21: Schematic representation of method using periosteal graft. Reproduced from www.medimagery.net.

Lorentzon et al combined periosteal grafting with drilling of the subchondral bone in defect and reported promising results in patellar lesions [77], In this study with a mean follow­ up period of 42 months, 25 patients had excellent or good outcome and only 1 patient had a poor result. Another study showed good clinical results 4 years after periosteal grafting [78], However, poor clinical results were observed in all patients 12 years after repair surgery. A serious long term problem is the calcification of the periosteal grafts. Another paper reported results from a heterogeneous group of 37 patients, who received combination of periosteal grafting with a silicon membrane for treatment of lesions of the hip or knee [79], With a mean follow-up of 10.5 years, the clinical results were reported as excellent in 11 cases, good in 18 and poor in 8 patients.

1.4.4.5 Autologous Chondrocyte Implantation

Autologous chondrocyte implantation (ACI) was the first approach of cell engineering in the treatment of cartilage lesions [80, 81], ACI is a two-step procedure. In the first arthroscopy, pieces of cartilage are taken from a nonload-bearing donor site of the cartilage (Fig. 1.22 A). Chondrocytes are afterwards cultured in vitro. In the second step, the

48 INTRODUCTION autologous chondrocyte cells in a suspension are injected into a chondral defect (Fig. 1.22 B) [82], In the first generation of ACI, a patch of autologous periosteum served as coverage for the implanted chondrocytes. In the second generation of ACI, cultured chondrocytes were implanted beneath a membrane of collagen type I and III collagen instead of periosteum [83], The periosteal and the collagen membrane are sutured to adjacent cartilage and sealed with fibrin glue. In the third generation of ACI. manufactured collagen membrane based cell carriers (e.g. matrix-associated ACI - MACI) stabilize and provide the chondrocytes in the repair area [84], More advanced third generation of ACIs use chondrocytes cultured in 3D scaffolds before implanting into the defect area (e.g. matrix-associated autologous chondrocyte transplantation - MACT) (Fig. 1.23) [85], The advantage of third generation procedures is a possibility of implantation using arthroscopy or only a small incision [82],

Femur r cm j' Nai bone] nah bone)

Cartilage Periosteum biopsy sutured (sewn) for lab over defect are i Area Injured MM of knee Cartilage cells of knee articular injected into cartilage cartilage cartilage biopsy defect area biopsy

Periosteum from tibia (shin bone) Fibula Tibia Fibula Medimegery.net shin bone) Med megery net Fig. 1.22: Schematic representation of two steps of ACI technique employing the periosteal patch. A. is showing cartilage biopsy, B. describes injection of cultured chondrocytes into the defect area. Reproduced from www.medimagery.net.

49 INTRODUCTION

Fig. 1.23: The third generation of ACI techniques - the NeoCart® implant. Reproduced from [86],

Good or excellent clinical outcome was reported in 70% of ACI patients with a mean treatment follow-up of 39 months [80], Eleven out of fifteen biopsies from repair site on femoral condylar had a hyaline-like repair tissue. Durable long-term results of up to 11 years after the ACI treatment of osteochondral lesions were published [87], Although a hyaline-like repair tissue was found in some of the ACI specimens, this tissue is not morphologically or histochemically identical to native hyaline cartilage [88], Moreover, fibrocartilage can be also found in some of the samples. It is well known that parameters such as lesion size, age, and activity level influence the outcome of cartilage repair surgery [89], Lesions that are larger than 2.5 cm2 should be treated with osteochondral autologous transplantation or ACI. On the other hand, MFX is a good treatment option for lesions smaller than 2.5 cm2. Knutsen et al. reported that both MFX and ACI provide good results in 77% of the patients at 5 years follow-up, with no significant difference in the clinical outcome [90], Although authors did not observe significant differences in histological properties of repair tissue between ACI and MFX patients two years after surgery, ACI patients showed more often hyaline-like repair tissue than MFX patients [70], Zeifang and coworkers compared twenty one patients with full-thickenss cartilage defects which were randomly selected for either the original periosteal flap ACI procedure, or for the MACI technique [91], They did not find any significant difference in the efficacy of the procedure or the patient outcome when compared these techniques 12 and 24 months after repair surgery.

50 INTRODUCTION

1.4.4.6 Stem Cells in Cartilage Tissue Engineering

Since chondrocytes harvested from elderly patients have much lower potential for cartilage repair than cells from young ones, the aging of cells is a problem for all autologous cell-based repair techniques applied to aged patients [92], This issue was the motivation for exploration of more active cell types, such as stem cells, for cartilage repair. The application of stem cells derived from bone marrow for cartilage tissue engineering was reported in many works [93, 94], The main disadvantage of cartilage tissue engineered from the bone marrow- derived stem cells are the inferior mechanical properties of its matrix in comparison with chondrocyte-seeded constructs [95, 96], However, a study comparing 72 patients after chondrocyte-based or after bone marrow-derived stem cells based ACI reported similar outcome in both groups of patients [97], In addition, it has been shown that although patients younger than 45 years had significantly better outcome than older patients receiving chondrocyte-based ACI, the age did not influence the patient outcome when using the bone marrow-derived stem cells. Various studies reported that adipose-derived stem cells (e.g. infrapatellar fat pad) are able to differentiate into chondrocytes and produce cartilage matrix with improved mechanical properties [98, 99], Unfortunately, the chondrogenic potential of adipose-derived stem cells is lower than that of bone marrow-derived cells. Moreover, the chondrogenic potential of cell from other tissues (synovium, muscle, periosteum) is even lower than potential of stem cells from bone marrow or adipose tissue [100], In any case, this field of research is still relatively new and the implantation of stem cells with osteogenic and chondrogenic potential might become very useful for the treatment of osteochondral defects in the near future.

1.4.5 Differences between Knee and Ankle Cartilage

OA, even in individuals with generalized OA, does not affect all joints equally. The most affected joints are knee, hip, spine and metatarsophalangeal, as well as interphalangeal joints of the hand. Degeneration in these joints very often progresses to the disease. In asymptomatic joints (e.g. shoulder, elbow, wrist), degeneration of the cartilage also occur, however, it seems to be nonprogressive. Several studies showed that asymptomatic OA with

51 INTRODUCTION radiographical signs occurs 8 to 9 times more often in the knee joint than in the ankle joint [101, 102], While about 10% of population will develop symptomatic OA in the knee, this disease is very rare in the ankle joint (< 0.1% of population) [103], Moreover, studies on cadaver samples demonstrated that full-thickness cartilage defects develop about 20 times more frequently in knee joints than in ankle (talocrural) joints [104-106], There are various biomechanical [107, 108] and anatomical differences between the ankle and knee joints that could help to explain less common degenerative changes in the ankle than in the knee. While the ankle (talocrural) joint is very stable with movement limited to extension/flexion, the knee is less stable joint where a mixture of flexion/extension and rotation movement is possible. Since surface of cartilages in ankle is exposed to higher loads per cm2 than the knee cartilage in normal walking [109], it is not surprise that the ankle cartilage have properties that protect it from the higher loads. Differences in susceptibility to OA between joints might be also explained by the imbalance between cartilage degeneration and repair [110], In fact, human knee and ankle cartilages response differently to various catabolic substances [111, 112], Eger et al. reported that the ankle is less responsive to catabolic stimulation and more responsive to anabolic stimulation following treatment with lnterleaukin-1ß (proinflammatory cytokine), or with osteogenic protein 1 that helps to restore matrix [113], Cartilage in the ankle is far more cellular and has lower water content than knee cartilage [113-115], Additionally, chondrocytes from the ankle show higher rates of PG and collagen synthesis than chondrocytes from the knee cartilage [114, 115], These properties are responsible for reduced permeability and increased stiffness of ankle cartilage compared to knee cartilage. Moreover, the underlying bones in these joints also respond differently to degenerative changes. These reports suggest that ankle cartilage may have greater capacity for repair and seems to be less responsive to catabolic stimulation. Moreover, these biochemical differences might also help to understand the biomechanical differences, such as lower dynamic stiffness, lower equilibrium modulus, and higher hydraulic permeability in the knee compared to the ankle [116], The differences in biochemical properties of chondrocytes between knee and ankle joints thus might play a critical role in different prevalence of OA in ankle and knee.

52 INTRODUCTION

1.5 MR Imaging of Cartilage and Repair Tissue

Recent developments are providing a variety of approaches for noninvasive monitoring of native cartilage, cartilage repair tissue and the process of chondral degradation in vitro and in vivo [117, 118], This chapter briefly review the state-of-the-art MR imaging methods for evaluating and following of the cartilage defects treatment. Although this chapter focus on the repair of chondral defects, the described methods are helpful for understanding and evaluation of all kinds of chondral degeneration in different joints (e.g. knee, hip, ankle). Discussed MR imaging techniques are capable to assess the morphological appearance as well as molecular composition of cartilage tissue.

1.5.1 Morphological Assessment of Cartilage Defects and Repair Tissue

Both sufficient SNR and spatial resolution are essential for the evaluation of cartilage defects, for the follow-up after cartilage repair surgery, and for the evaluation of other joint structures, such as subchondral bone, ligaments, or menisci. Optimal MRI hardware, such as MR systems with high (3 T) or ultra-high field (7 T) strength and dedicated array (multi­ element) RF coils are able to improve intrinsic SNR per unit of time. Moreover, array RF coils enable parallel imaging, which can decrease the measurement time by a factor oftwo or more by using parallel acquisition techniques. Adequate characterization of these structures using high resolution images is very important for the planning of individual therapy. The same MR imaging sequences can be used for standard morphological evaluation of cartilage repair tissue as well as for the evaluation of native cartilage or for OA [119],

1.5.1.1 Morphological MRImaging Sequences

The most widely used MR imaging sequences are 2D proton-density (PD)-weighted fast spin-echo (FSE), and 3D fat-suppressed gradient-echo (GRE) techniques [119-121], These sequences have proved high accuracy, specificity, and sensitivity for detecting chondral lesions in the knee. New, usually GRE-based, 3D sequences provide high resolution isotropic MR images, which can be reformatted in any arbitrary plane. To this group of

53 INTRODUCTION sequences we can include double-echo steady-state (DESS), fast imaging with steady-state precession (true-FISP), and 3D isotropic fast spin-echo based techniques such as 3D FSE or PD Sampling Perfection with Application of optimized Contrasts using different flip angle Evolutions (SPACE) have been introduced. 2D FSE. This sequence can provide Ti-, T2-, intermediate-, and PD-weighted contrast in images. PD-weighted 2D FSE with or without fat saturation and using intermediate TE of 40-60 ms, that minimizes the magic angle effect, is the workhorse in cartilage and joint imaging in general. The FSE sequence uses differences in T2 relaxation times of cartilage and synovial fluid, and provides excellent contrast between them (cartilage has lower signal intensity than fluid). This sequence visualizes thickness, surface changes (fibrillations), and abnormal internal structure of the cartilage [122], This technique is also useful for evaluation of the changes in bone marrow, menisci and ligaments. Due to refocusing 180-degree RF pulses, FSE sequence is not so prone to artifacts caused by local changes in magnetic susceptibility, which is very useful in postoperative assessment of cartilage repair tissue (Fig. 1.24 A). A dual 2D FSE version provides images with shorter and longer TEs (Fig. 1.24 C, D).The 3D version of FSE technique has not yet replaced 2D FSE in clinical practice.

Fig. 1.24: High-resolution 3 T MR images of a 37-year-old man in the follow-up after MACT repair (arrows) showing good morphological outcome. A. Sagittal proton-density weighted 2D FSE (TR/TE = 2400/28 ms) with ultrahigh in-plane resolution (0.23x0.23 mm2) achieved in 6:01 minutes. B. Coronal Ti-weighted turbo inversion recovery magnitude sequence (TR/TE

54 INTRODUCTION

= 7690/41 ms) with high resolution (0.59x0.59x3.0 mm3) acquired in 2:19 minutes. C, D. Sagittal T2-weighted dual 2D FSE sequence (TR/TE1/TE2 = 5120/9.5/124 ms) with high resolution (0.4x0.4x3.0 mm3) measured in 6:35 minutes. Reproduced from [123],

3D GRE (3D FLASH). This technique visualizes chondral defects using the differences in Ti relaxation time between the cartilage and the fluid (cartilage has higher signal intensity than fluid) (Fig. 1.24 B). It can be measured with isotropic resolution and reformatted in any plane. Measured with high resolution, this sequence is not prone to partial-volume artifacts. The 3D GRE technique with fat suppression exhibits high contrast-to-noise ratios (CNR), an accurate assessment of cartilage surface, volume, and its regional distribution in the knee joint, which results in high reproducibility of cartilage segmentation [124, 125], The quantitative MR parameters based on cartilage segmentation (e.g. volume or thickness), seem to be sensitive for detecting and evaluating cartilage degeneration [126], However, this sequence is sensitive to susceptibility artifacts, it provides only low contrast between cartilage and synovial fluid, and it is not optimal for assessment of bone marrow. 3D DESS. This sequence acquires two or more gradient echoes which are combined in one image. It offers high SNR, shorter measurement times than 3D GRE, and high contrast between fluid and cartilage. High resolution images minimize partial-volume artifacts and isotropic voxels can be used for multiplanar reformatting (Fig. 1.25). This sequence allowed to detect the cartilage volume changes with high precision in the in follow-up study of patient with OA [127], The drawbacks of DESS are sensitive to susceptibility artifacts and poor assessment of bone marrow.

Fig. 1.25: High-resolution isotropic (0.6x0.6x0.6 mm3) 3D DESS images (TR/TE = 15.1/5.1 ms) of a 43-year-old man after MFX of the patella cartilage (arrows) obtained with water excitation in 5:38 minutes at 3 T. From left to right, sagittal, coronal, and axial multiplanar reconstruction images. Reproduced from [123],

55 INTRODUCTION

3D true-FISP. This sequence provides higher SNR and CNR than the 3D FLASH technique [128-130], Contrast in images depends on T2/T1 ratio of tissue. This allows higher spatial resolution (Fig. 1.26), and thus improvement of the segmentation (volumetric assessment) accuracy [128], It might also be used for isotropic MRI measurements and reconstruct in multiple planes. 3D true-FISP with water-excitation showed similar sensitivity, specificity, and accuracy for the preoperative detection of cartilage defects as a 3D FLASH and a 3D DESS sequences [130], In comparison with standard 2D sequences, an isotropic (0.6 mm3) 3D true-FISP demonstrated improved performance in diagnosing cartilage defects, meniscal tears, and anterior cruciate ligament abnormalities [131], Banding artifacts may occur at very long TR.

Fig. 1.26: High-resolution isotropic (0.4x0.4x0.4 mm3) 3D true-FISP images (TR/TE = 8.9/3.8 ms) with water excitation obtained in 6:46 minutes at 3 T, to visualize the MACT repair tissue (arrows). 3D set reconstructed in the A. sagittal, B. coronal and C, D. axial plane. D. represents additional possibility for diagnosis of menisci. Reproduced from [123],

56 INTRODUCTION

1.5.1.2 MR Imaging Based Morphological Scoring of Cartilage Defects

MRI can provide an insight into the morphological changes of cartilage (focal or diffuse cartilage loss, cartilage fissuring, defect depth), and of other tissues in the joint (ligaments, menisci). Various MR-based semiquantitative scoring systems were proposed for evaluation of cartilage lesions. A comprehensive whole-organ MRI (WORMS) scoring system contains parameters for the evaluation of cartilage morphology, subchondral bone marrow changes, ligament and meniscal abnormalities, synovitis, etc [132], The WORMS showed high interobserver agreement among two trained readers (>0.98 for cartilage abnormalities and >0.80 for most features) at 1.5T. Another compartment-based scoring methods like a knee osteoarthritis scoring system (KOSS) [133] and recently Boston-Leeds osteoarthritis knee score (BLOKS) [134] were introduced for evaluating changes in the knee cartilage associated with OA. The BLOKS system includes features of cartilage, ligaments, osteophytes, synovitis, bone-marrow lesions, and effusions, and showed a good interreader reliability for cartilage morphology. The maximum bone-marrow lesion size in the BLOKS had a positive linear correlation with pain. Additionally, the baseline bone-marrow lesion size was associated with cartilage loss in both the WORMS and BLOKS systems. These scoring systems have also limitations, especially in subscales, which may need to be redeveloped [135],

1.5.1.3 MR Imaging Based Morphological Evaluation of Cartilage Repair

Sequences used for assessment of cartilage defects (Fig. 1.24, Fig. 1.25, Fig. 1.26) can also be useful for morphological evaluation of the success of cartilage repair techniques [136-139], MOCART - Magnetic resonance Observation of CArtilage Repair Tissue - scoring system offers a very comprehensive semiquantitative MR imaging assessment of cartilage repairtissue [136-138, 140], The reliability and validity of this scoring system has been tested on the assessment of MACT repair tissue in the knee [137], Nine variables of MOCART include assessment of: 1) defect filling, 2) border zone to the adjacent cartilage, 3) surface of the repairtissue, 4) structure ofthe repairtissue, 5) MRI signal intensities in the repairtissue (compared to the native cartilage), 6) subchondral lamina, 7) subchondral bone, 8) adhesions, and 9) joint effusion. An excellent interobserver agreement was found for eight of the nine score variables (>0.81). MOCART has been already used for the longitudinal evaluation of all cartilage repair techniques [141-144], Moreover, different isotropic 3D MR sequences have

57 INTRODUCTION been recently used for the evaluation of repair tissue with 3D MOCART [145], Authors demonstrated benefits of isotropic 3D MR sequences, such as multiplanar reconstruction, and a reduced measurement time, where the 3D PD-SPACE sequence gave the best results. CROAKS - Cartilage Repair OsteoArthritis Knee Score - is a scoring system that combines MOCART scoring tool with MRI osteoarthritis knee score (MOAKS) system [146], In CROAKS evaluation, the joint is divided into 14 subregions according to MOAKS system [147]: 2 regions in patella, 6 in femur, and 6 in tibia. All subregions influenced by repair surgery are evaluated using the cartilage repair part of CROAKS, based on the MOCART. This part uses the variables from MOCART, except of joint effusion that is scored using MOAKS system. The rest of subregions (unaffected by cartilage repair) is evaluated using the whole joint part of CROAKS, based on the MOAKS tool developed for the whole joint evaluation of common OA features. Following features are scored: 1) cartilage area, 2) percentage of cartilage affected by full thickness cartilage loss, 3) size, type and number of bone marrow lesions, 4) meniscus morphology, 5) meniscus extrusion, 6) osteophytes, 7) anterior and posterior cruciate ligament, 8) effusion, 9) infrapatellar synovitis, and 10) intercondylar synovitis. CROAKS system can be applied for the evaluation of knee cartilage before and after surgery with good to excellent reliability in a cross-sectional manner. However, the applicability of CROAKS in longitudinal studies needs to be yet proven.

1.5.2Biochemical MR Imaging of Cartilage Defects and Repair Tissue

In addition to morphological MRI techniques that provide basis for diagnosis and follow-up evaluation of cartilage defects and repair tissue, biochemical MR imaging methods enable to study the cartilage ultrastructure, and to quantify functionally important molecules in cartilage. These methods are very interesting for the evaluation of cartilage repair, as they offer possibility to image functionally relevant cartilage molecules and thus evaluate cartilage repair maturation after surgery in vivo [148-151], Different biochemical MRI techniques have been exploited for their use in the evaluation of cartilage degeneration and cartilage repair tissue. The most frequently used biochemical MRI technique is the T2 mapping of cartilage that is sensitive to changes in PG- collagen matrix and water content and to tissue anisotropy which allows for evaluation of

58 INTRODUCTION cartilage zonal variations [152-155], T2* mapping can be acquired in shorter measurement time and provides information that is comparable to T2 [123, 153, 154, 156], Diffusion- weighted imaging probes molecular motion in the tissue that is also affected by density and orientation of the PG-collagen matrix and by water content [157-159], Another group of biochemical MRI sequences focuses on visualization of GAGs that provide a fixed charge density (FCD), which is important to the cartilage function, especially tensile strength. Since the GAG content has been reported to decrease in early stages of cartilage degeneration [160] or in cartilage repair tissue [161], GAG sensitive techniques may provide valuable information about the tissue composition. Several MRI techniques such as Ti delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC), Tip, glycosaminoglycan chemical exchange saturation transfer (gag-CEST), or sodium imaging have been proposed to be sensitive to changes in the GAG content. The following chapters will focus only on the GAG sensitive techniques.

1.5.2.1 Ti Delayed Gadolinium-enhanced MR Imaging

This GAG sensitive method relies on the intravenous administration of a negatively charged contrast agent (gadolinium diethylenetriaminepentaacetate anion) which diffuses into articular cartilage and equilibrates in inverse relation to the FCD. More contrast agent accumulates in the areas of cartilage with lower GAG, than in areas with higher GAG concentration, which leads to a decrease in Ti relaxation times. Since Ti values in cartilage are determined by the contrast agent concentration, dGEMRIC can be used for monitoring GAG content of cartilage in vitro and in vivo [162-166]. Diffusion of the contrast agent into cartilage is relatively slow, therefore post-contrast Ti measurements are performed about 90 minutes after intravenous administration of the contrast agent. Since Ti values differ between repair tissue and native cartilage already before contrast administration, the precontrast Ti values are measured as well [161], Delta relaxation rate is calculated from precontrast and postcontrast Ti values (AR-i = 1/ Tipostcontrast - 1/ Tiprecontrast). The relative delta relaxation rate index (AR-i of repair tissue I ARi of native cartilage) was reported to correlate with the GAG content in the biopsy samples from repair tissue [161], GAG concentration was measured by digesting biopsy samples with chondroitinase ABC and quantifying chondroitine sulfates by liquid chromatography combined with fluorometry. However, later study showed a high correlation between ARi and postcontrast Ti which implies that precontrast Ti mapping is not necessary for the repair tissue assessment [167], This reduce costs, patient discomfort,

59 INTRODUCTION simplifies the measurement, and makes dGEMRIC more attractive for cartilage repair follow­ up. Since standard inversion recovery (IR) for Ti mapping of dGEMRIC is time consuming, development of fast 3D Ti mapping techniques with shorter acquisition increased clinical applicability ofdGEMRIC [149, 168, 169], dGEMRIC has been used to assess the relative GAG content of repair tissue in patients who received different cartilage repair procedures. Two studies reported that the relative GAG content in repair tissue 10 (or more) months after ACI is comparable with adjacent reference cartilage [170, 171], These studies did not measure Ti maps before contrast medium application. In contrast to these first results, a significantly lower relative GAG concentration has been reported in repair tissue more than 1 year after treatment [149, 172], This result could be partially explained by histologic findings suggesting that patients after MACT may have different types of repairtissue (hyaline-like to fibrous tissue) overtime [70, 173, 174], Previous histologic studies also showed that most of the changes in repair tissue can be found within the first year after surgery. To evaluate the maturation of repair tissue, Trattnig et al. compared dGEMRIC results between early (3 to 13 months) and late (19 to 42 months) postoperative MACT patients [149], Significantly lower Ti values were found in repair tissue than in reference cartilage in both groups. However, the differences were not significant between the early and late group. Another dGEMRIC study with 20 age- and postoperative interval matched patients after MFX or ACI reported a significantly lower relative GAG content in MFX (AR-i = 3.39) than in MACT (AR-i = 2.18) repairtissue [175] (Fig. 1.27). This is in accordance with histological evaluations of biopsies, which shown that MACT patients develop more often hyaline-like repair tissue, while fibrous-like repair tissue predominates in MFX patients [70, 173, 174],

Fig. 1.27: Images of a repair tissue (situated between white arrows) in femoral cartilage of a 36-year-old male patient 31 months after MFX acquired with a 3D, dual flip angle dGEMRIC

60 INTRODUCTION technique, a: Color-coded Ti values from repair tissue and native cartilage before contrast administration. The Ti values in repair tissue are similar to native cartilage, b: Post-contrast color-coded Ti values from repair tissue and normal cartilage. The post-contrast Ti values are smaller in the repair tissue than in healthy cartilage. Reproduced from [175],

The main limitation of dGEMRIC is the requirement for an exogenous contrast agent. Another limitation is the need of exercise and a very long time (about 60 minutes) delay between the Ti mapping and contrast agent administration, which is necessary for reaching a steady-state distribution of the negatively charged contrast agent in cartilage. Moreover, the distribution of contrast agent depends on time, cartilage thickens, level of activity and other variables that make comparison of dGEMRIC results between patients very difficult. These limitations diminish the clinical attractiveness of this method.

1.5.2.2 Tip Mapping

Since Tip relaxation time is sensitive to protons attached to large macromolecules, direct relationship between Tip values and GAG concentration was proposed. First studies of bovine cartilage after enzymatic degradation showed increase of Tip values with decreasing GAG concentration [176-179], Moreover, Tip could differentiate between healthy volunteers and patients with OA in vivo [180, 181], Although Tip was able to distinguish between early, moderate and severe OA in cartilage samples from total knee replacements [182], Tip was not compared to histochemically evaluated GAG content in this study. Another studies using cartilage specimens from patients after total knee replacement found no correlation between Tip and GAG content calculated from biochemical analyses [183, 184], These studies were limited by the fact that cartilage samples at late stages of the OA process may not show the inverse relation between Tip and GAG content. However, recent results indicate thatT-ip does not only reflect the GAG concentration, but is also strongly affected by changes in dipolar interaction related to the collagen fiber architecture that alter T2 relaxation times. This fact was demonstrated by a relatively strong correlation between Tip and T2 values in several cartilage studies [184-187], A broader use of Tipin the clinical routine has been limited by its low specificity so far [181], Additionally, to our best knowledge, there is no study employing Tip for the evaluation of cartilage repair tissue so far. Most studies focused on the evaluation of Tip as a biomarker for OA.

61 INTRODUCTION

1.5.2.3 Chemical Exchange Saturation Transfer Imaging

Chemical exchange saturation transfer (CEST) is a method that generates contrast in MR images based on chemical exchange between water protons and exchangeable protons bound to macromolecules. The CEST technique is based on an observation of decrease in the bulk water MR signal after exchangeable protons are selectively pre-saturated by RF irradiation and afterwards go through the chemical exchange with bulk water protons [188], The most common CEST measurement is the acquisition of multiple sets of images with pre­ saturation at different offset frequencies around the water line, and one set of reference data without saturation or with saturation at frequency with a very large offset [189], The amide and hydroxyl protons of GAGs have exchange properties that can be used for CEST experiments [190], Experiments in bovine cartilage samples demonstrated that CEST contrast mediated by GAGs (gagCEST) can be a useful biomarker for the evaluation of cartilage GAG concentration [191, 192], The gagCEST imaging benefits from increasing frequency differences at ultra-highfield strengths (7 Tesla and above), where the RF spillover is reduced. On the other hand, this method requires good shimming, correction for Bo homogeneity and compensation for the patient movement during the scans. Initial 7 T experiments demonstrated the sensitivity of gagCEST contrast to the GAG concentration in articular cartilage and in tissue after cartilage repair surgery. The usefulness of gagCEST imaging for the evaluation of GAG content was demonstrated in several papers. Schmitt et al. compared gagCEST and sodium values in patients after MFX and after MACT [150], The observed correlation between the two compared techniques validated sensitivity of gagCEST to changes in GAG content of cartilage. Moreover, gagCEST can differentiate between native cartilage and cartilage repair tissue of patients (Fig. 1.28). In another study, significantly lower gagCEST values were also found in repair tissue of patients after autologous osteochondral transplantation [193], The clinical outcome of these patients did not correlate with morphological evaluations or with the results of sodium and gagCEST imaging. This finding demonstrate that biochemical MR imaging provides unique insight into the composition of native cartilage and repair tissue. The gagCEST technique seems to be especially useful for detecting early pathological changes that coincide with a primary loss of GAG content from cartilage.

62 INTRODUCTION

Fig. 1.28: Images of a patient 9.4 years after ACT at the medial femoral condyle. A: Morphologic proton-density weighted fast spin-echo image. B: Color-coded gagCEST image overlaid on morphological image. Colorbar stands for gagCEST asymmetries in [%]. Lower values reflect lower GAG content. Reproduced from [193],

gagCEST seems to reflect the GAG concentration in cartilage more specifically compared to T2 and Tip mapping. This advantage can be used for detection of cartilage degeneration in early stages, when morphological changes are not visible. It also does not require administration of a contrast agent or a complicated imaging protocol like dGEMRIC. However, gagCEST also has limitations. For example, the acquisition and evaluation of gagCEST data is a quite complex process. A successful gagCEST measurement depends on a optimization of the saturation scheme to provide maximal efficiency of the CEST effect. Moreover, this method is influenced by various effects, such as homogeneity of Bo and Si magnetic fields, RF spillover and others that need to be corrected to avoid errors in the gagCEST maps. These limitations complicate the clinical application of this method.

1.5.2.4 Paper 5: Evaluation of Cartilage Repair and Osteoarthritis with Sodium MRI

Authors: Stefan Zbvn, Vladimir Mlynarik, Vladimir Juras, Pavol Szomolanyi, Siegfried Trattnig

This review paper was published in NMR in Biomedicine in 2015 (DOI: 10.1002/nbm.3280). It summarizes the literature available in November 2014 concerning

63 INTRODUCTION clinical application of sodium MRI for the evaluation of cartilage affected by OA and cartilage repair tissue. Furthermore, review provides basic information about cartilage composition, pathologies, treatment techniques, sodium MR properties, sodium MRI pulse sequences, evaluation of GAG concentration, and overview of clinical sodium MRI studies. A reproduction of this review in PhD-thesis was permitted by the publisher - John Wiley & Sons, Ltd.

64 NMR Special issue review article IN BIOMEDICINE

Received: 2 December 2014, Revised: 20 January 2015, Accepted: 29 January 2015, Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI: 10.1002/nbm.3280 Evaluation of cartilage repair and osteoarthritis with sodium MRI a,c Stefan Zbyna,b*, Vladimfr Miynärika,b, Vladimir Juras z Pavol Szomolanyia,c and Siegfried Trattniga,b

The growing need for early diagnosis and higher specificity than that which can be achieved with morphological MRI is a driving force in the application of methods capable of probing the biochemical composition of cartilage tissue, such as sodium imaging. Unlike morphological imaging, sodium MRI is sensitive to even small changes in cartilage glycosaminoglycan content, which plays a key role in cartilage homeostasis. Recent advances in high- and ultrahigh- field MR systems, gradient technology, phase-array radiofrequency coils, parallel imaging approaches, MRI acquisi­ tion strategies and post-processing developments have resulted in many clinical in vivo sodium MRI studies of car­ tilage, even at 3 T. Sodium MRI has great promise as a non-invasive tool for cartilage evaluation. However, further hardware and software improvements are necessary to complete the translation of sodium MRI into a clinically fea­ sible method for 3-T systems. This review is divided into three parts: (i) cartilage composition, pathology and treat­ ment; (ii) sodium MRI; and (iii) clinical sodium MRI studies of cartilage with a focus on the evaluation of cartilage repair tissue and osteoarthritis. Copyright © 2015 John Wiley & Sons, Ltd.

Keywords: sodium MRI; musculoskeletal; cartilage repair; osteoarthritis; sodium concentration; proteoglycans

INTRODUCTION quality and for the assessment of OA. In the following sections, we provide a short description of cartilage composition, pathol­ Articular cartilage has very limited intrinsic repair capacity in ogy and treatment, an overview of sodium MRI, and of clinical adults. Mechanical injury of articular cartilage thus often leads di­ sodium MRI studies for the evaluation of cartilage repair tissue rectly to cartilage defects or indirectly to cartilage degeneration. and OA. Patients with persistent pain and a full thickness cartilage defect usually receive surgical treatment. Different procedures have been proposed for the repair of cartilage defects. However, the common goal of all of these techniques is to replace the defect with a new tissue with a structure, composition and properties identical to those of native articular cartilage (1). Non-invasive * Correspondence to: S. Zbyn, Department of Biomedical Imaging and Image- Guided Therapy, Medical University of Vienna, Waehringer Guertel 18-20, assessment of repair tissue quality therefore provides valuable A-1090 Vienna, Austria. information about the efficacy of different repair surgery tech­ E-mail: [email protected] niques during follow-up. Osteoarthritis (OA) is a leading cause of chronic disability in a S. Zbyn, V. Mlynarik, V. Juras, P. Szomolanyi, S. Trattnig the elderly population and is the most common form of arthritis High-Field MR Center, Department of Biomedical Imaging and Image-Guided Therapy, Medical University of Vienna/Vienna General Hospital, Vienna, in synovial joints (2). Although radiography and morphological Austria MRI are able to detect structural changes in cartilage caused by OA, these techniques are insensitive to biochemical changes as­ b S. Zbyn, V. Mlynarik, S. Trattnig sociated with early OA, before morphological changes appear CD Laboratory for Clinical Molecular MR Imaging, Vienna, Austria (3). Therefore, there is a great demand for non-invasive methods c V. Juras, P. Szomolanyi capable of detecting early OA. Department of Imaging Methods, Institute of Measurement Science, Slovak Various MRI methods can provide biochemical information Academy of Sciences, Bratislava, Slovakia from osteoarthritic cartilage or from repair tissue. The most Abbreviations used: ACI, autologous chondrocyte implantation; BMS, bone frequently used techniques are T2 (4), T2* (5) and T1p (6) map­ marrow stimulation; DESS, double-echo steady-state; dGEMRIC, delayed ping, delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) gadolinium-enhanced MRI of cartilage; FCD, fixed charge density; GAG, glycos- (7), glycosaminoglycan chemical exchange saturation transfer aminoglycan; gagCEST, glycosaminoglycan chemical exchange saturation (gagCEST) (8) and sodium imaging (9). Additional musculoskele­ transfer; GRE, gradient echo; MACT, matrix-associated autologous chondro­ tal applications of sodium imaging have been focused on skele­ cyte transplantation; MFX, microfracturing; MOCART, MR observation of carti­ lage repair tissue; OA, osteoarthritis; PG, proteoglycan; RF, radiofrequency; ROI, tal muscles (10) and the Achilles tendon (11). region of interest; SNR, signal-to-noise ratio; TPI, twisted projection imaging; This review discusses advances in sodium MRI of cartilage as a UTE, ultrashort TE; vTE, variable TE; WURST, wideband uniform rate smooth potential biomarker for the evaluation of cartilage repair tissue truncation.

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CARTILAGE COMPOSITION, PATHOLOGY AND marrow-stimulating techniques; (ii) auto-/allo-grafting tech­ TREATMENT niques; and (iii) advanced cell-based repair techniques.

Main cartilage components Bone marrow-stimulating techniques

Cartilage consists of an extracellular matrix and of a small Pridie drilling (19) and microfracturing (MFX) (20) can be viewed amount of chondrocytes [~2% of cartilage w/w (mass fraction as marrow-stimulating procedures. These repair techniques relative to its wet weight)]. The main matrix components are create multiple holes in the subchondral bone in the defect area water, collagen and proteoglycans (PGs) (12). About one-third to fill it with material coming from the bone marrow. Ideally, the of the water is trapped in the collagen network. The rest of the cells from the bone marrow will differentiate into chondrogenic water contains dissolved ions (such as sodium) and may flow cells that produce hyaline-like repair tissue (21). However, through the extracellular matrix after application of pressure on Knutsen et al. (22) showed that most of the biopsies from repair the cartilage (13). The high frictional resistance of cartilage tissue of patients 2 years after MFX were composed mainly of against water flow helps the joint to withstand heavy loads. fibrocartilage tissue. Bone marrow-stimulating techniques usu­ Type II collagen forms fibers intertwined with PGs and is the ally result in the formation of a low-quality fibrous repair tissue main macromolecule in the hyaline cartilage (10-20% of carti­ that degenerates with time. lage w/w) (14). Collagen fibers provide cartilage with shear and tensile strength. Auto-/allo-grafting techniques PGs in cartilage are present as monomers (<5% of cartilage w/w) or as aggregates attached to hyaluronic acid fibers (5-7% Osteochondral grafting (mosaicplasty) (23) and periosteal of cartilage w/w). Each PG monomer contains at least one gly- grafting (24) use an autograft to fill the lesion. In mosaicplasty, cosaminoglycan (GAG) side chain attached to a central protein cylindrical osteochondral plugs are harvested from non-load­ core (12). The GAGs contain many negatively charged sulfate bearing areas of an affected joint, and placed into the and carboxyl groups, and thus provide cartilage with a negative osteochondral defect. Mosaicplasty is advantageous because fixed charge density (FCD). The negative FCD attracts positively the lesion is filled with hyaline cartilage and the method allows chargedions(mainlysodium),which arethusin equilibriumwith the treatment of both osteochondral and chondral defects. The the PGs in cartilage. Osmotic pressure, created by the high ionic limitationsofmosaicplastyincludethesuboptimalreconstitution concentration, pumps water into cartilage and gives cartilage ofthesubchondrallayer,theproblematicproductionofasmooth unique properties, such as compressive stiffness (15). cartilage surface and the high chance of insufficient integration of the osteochondral plugs into the adjacent cartilage (25). Cartilage injury and repair Advanced cell-based repair techniques Chondrocytes are immobilized in an extracellular matrix and cannot migrate to the injury site. Thus, hyaline cartilage, as an Advanced cell-based repair techniques, such as autologous avascular tissue, has very low intrinsic reparative capacity chondrocyte implantation (ACI), rely on donor-derived (12,16,17). Moreover, cartilage injury can result in direct damage chondrocytes(usuallyautografts)forcartilage defectreconstruc- to the extracellular matrix, or in damage mediated by tion. ACI was the first technique to use cell engineering for the chondrocytes via the expression of matrix-degrading enzymes treatmentofcartilagelesions(26,27).In thethirdACIgeneration, and/or a reduction in their biosynthetic activity. Animal studies also known as matrix-associated autologous chondrocyte trans­ havedemonstratedthathigh-impactloadstothejointcan cause plantation (MACT), cartilage pieces are taken from a non-load­ the deterioration of mechanical properties, a loss of tissue integ­ bearing donor site of an affected joint, and extracted rity or the death of chondrocytes (18). chondrocytes are cultured in three-dimensional scaffolds The result of the injury to cartilage depends on many factors, in vitro for several weeks (28). In the next step, the cultured cells such as the patient's age, depth, location and size of the defect, seededonascaffold,suchasacollagenmembrane,collagengel, etc. Although isolated chondral defects are found in only 4% of fibrin gel or others, are implanted into the defect. Although arthroscopies, 40-70% of arthroscopies have found cartilage de- these cell-based cartilage repair surgery techniques can produce fectsincombinationwith ligamentand/ormeniscusinjuries(17). a hyaline-like repair tissue, this tissue is not morphologically or All parts of the knee joint are closely connected; thus, cartilage histochemically identical to native hyaline cartilage, and health is very important for the health of the whole knee. fibrocartilage can also be found in the repair tissue (29). Al­ Untreated osteochondral defects often lead to an early onset though no significant differences in the histological quality of of OA, and therefore symptomatic defects in adults should be repair tissue were found between patients treated with ACI treated with cartilage repair techniques. and MFX 2 years after surgery, patients treated with ACI more often showed hyaline-like repair tissue than did those treated Cartilage repair techniques with MFX (22). Cartilage repair surgery techniques aim to restore the cartilage Osteoarthritis surface and function to enable pain-free motion of the joint and to prevent further cartilage degeneration. To achieve these Knee OA is a degenerative disease associated with changes in aims,cartilagerepairtissueshouldideallyhavethesamecompo- the whole joint, such as the degradation of cartilage, ligaments sition,structure andmechanical propertiesasnative hyalinecar- and menisci, changes in subchondral bone and synovial inflam­ tilage (1). Different surgical interventions have been developed mation (30). There are many obstacles in the study of OA, such for the treatment of articular cartilage defects. Cartilage repair as heterogeneous etiology, variability in disease progression techniques can thus be divided into three main groups: (i) bone and the often long time required to see morphological changes

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66 NMR EVALUATION OF CARTILAGE REPAIR AND OSTEOARTHRITIS WITH SODIUM MRI IN BIOMEDICINE in a joint. The diagnosis of OA is based on clinical symptoms (loss isotropic motion; (ii) slow isotropic motion; (iii) slow anisotropic of function, pain), the patient's medical history and radiographic motion; and (iv) fast anisotropic motion (15). The conventional evaluation of the joint space width (30). The treatment aims to single-quantum sodium MRI can distinguish between fast and relieve symptoms and improve knee function. End-stage knee slow motional regimes only. OA commonly requires joint replacement. Sodium in a liquid environment (such as synovial fluid) is rep­ Although radiography is the current standard for the evalua­ resentative of fast isotropic motion; thus, the static quadrupolar tion of structural changes in the joint, it has limited sensitivity interaction is averaged to zero. Longitudinal (T1) and transverse and precision (31). Morphological MRI is a non-invasive alterna­ (T2) relaxation of sodium in liquids is mono-exponential, with a tive that provides a comprehensive evaluation of the whole joint T1 value of about 62ms and a T2* value of about 28 ms (44,45). and detects the presence of OA with higher specificity than does Sodium in tissues is in either slow anisotropic (such as in a tis­ radiography or arthroscopy (32). The semi-quantitative and sue containing collagen fibers) or slow isotropic motion. The quantitative morphological MRI of knee OA includes the assess­ quadrupolar interaction dominates the relaxation of sodium in ment of cartilage thickness and volume (33), synovitis, synovial tissues, and longitudinal (T1) and transverse (T2) relaxations are fluid effusions, lesions in bone marrow and meniscal damage. bi-exponential. The two T1 components have similar relaxation Eckstein et al. (34) have demonstrated that the loss of thickness times of about 20 ms. The short T2 component (T2*SHORT) has a in the medial femorotibial cartilage compartment is a sensitive value in the range of 0.5-1.4ms, and the long T2 component quantitative parameter that correlates with radiographic joint (T2*LONG) hasa value in the rangeof11.4-14.8ms(46).Asshown space width, and seems to be a strong predictor of the need by Staroswiecki et al. (47), sodium T2* values in cartilage are not for knee replacement. Quantitative morphological MRI may be significantly different between 3 and 7 T. a viable alternative to radiography for the evaluation of changes As the gyromagnetic ratio of sodium is 70.808 x 106 rad/T/s, in knee OA. However, radiography and morphological MRI are sodium MR sensitivity is only 9.3% that of proton sensitivity. In insensitive to biochemical changes in the cartilage that occur addition, the sodium in vivo concentration in healthy cartilage in early OA stages and precede the morphological changes (3). is about 320 times lower than that of the proton concentration. The detection of early OA stages, when the disease is poten­ As a result of all of the above-mentioned factors, the sodium tially reversible, is of major interest in cartilage imaging and in signal-to-noise ratio (SNR) in cartilage is about 3400 times the clinical development of potential disease-modifying OA smallerthanthatofprotonMRI.Thus,sodiumimagesare,ingen- drugs. GAG molecules play a central role in cartilage homeosta­ eral,acquiredwith lowerSNR(between 15and40),lowerresolu- sis, which is altered in a pathological condition (35,36). The early tion (2-5mm) and longer measurement times (10-30min) than stages of OA can be related to changes in the organization and are proton images. Generally, sodium imaging is challenging composition of the extracellular matrix, such as a decrease in and requires an MR system with multinuclear capabilities, dedi­ GAG content. However, all the mechanisms associated with early cated radiofrequency (RF) coils and, preferably, ultrahigh-field OA are not entirely understood. Although most studies have (7-T) strength for higher SNR. shown a decreased GAG content in early OA (37,38), there are data that have reported an elevated (39-41) or unchanged Sodium MRI pulse sequences (42,43) GAG concentration. As the bi-exponential T2 values of sodium in tissues are very short, sequences suitable for the acquisition of sodium images SODIUM MRI at very short TEs are recommended. The first in vivo sodium im­ ages of cartilage were acquired by Reddy et al. (9), with Cartesian MR properties of sodium (Fig. 1a) three-dimensional gradient echo (GRE) sequences at 4T. The sodium nucleus has a spin number of 3/2 and exhibits a In order to increase SNR in Cartesian GRE imaging, TE can be re­ quadrupole moment, which results in specific MR properties of duced by using a non-selective RF pulse, an asymmetric readout this nucleus. The quadrupole moment arises from the non­ (partial echo) and optimal gradient switching patterns, together spherically symmetric distribution of the electric charge around with a variable TE (vTE) train (TE reduction towards the k-space the sodium nucleus. When the sodium nucleus interacts with center) (48). These approaches were combined in the vTE-GRE an electric field gradient (as a result of varying electron density sequence(49),andproducedhigh-qualitysodiumimages(Fig.2). surrounding the nucleus), the quadrupolar interaction affects The main advantages of Cartesian GRE sequences are their its spin energy levels. Based on the molecular environment, so- robustness and fewer image artifacts compared with non­ diumcanbefoundinthefollowingfourmotionalregimes:(i)fast Cartesian (e.g. radial) sequences.

Figure 1. Schematic representation of basic k-space sampling strategies: (a) Cartesian; (b) radial projection; (c) spiral projection.

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Another form of fluid suppression providing additional infor- mationonthedegreeofanisotropyincartilageistriple-quantum filtering (64). As demonstrated by Kotecha et al. (65), sodium triple-quantum coherence spectroscopy can be used to investi­ gate anisotropy and growth dynamics in cartilage tissue- engineered constructs. However, multiple quantum-filtered sequences suffer from even lower SNR than that of conventional single-quantum sodium MRI, and have not yet been used for the in vivo evaluation of cartilage pathologies (64,66,67). Although fluid suppression helps to minimize contamination Figure 2. Examples of knee images from a healthy volunteer acquired from fluid, the decrease in cartilage signal caused by the partial at 7 T. (a) Morphological proton density-weighted two-dimensional turbo volume effect from bone or menisci is still present. Moon et al. spin echo image with fat suppression (resolution, 0.4 x 0.4 x 2.5 mm3; ac­ (68) therefore proposed a compensation for the signal reduction quisition time, 4:17 min). (b) Corresponding sodium variable TE (vTE) gra- caused by partial volume effects based on cartilage thickness dientecho(GRE)image(resolution,1.4x1.5x3.0mm3;acquisitiontime, measurements from morphological proton images. As co­ 28:14 min.). registered morphological and sodium images are necessary for the proposed signal compensation, and thus more accurate A shorter TE (below 1 ms) can be obtained with non-Cartesian quantification of sodium concentration in cartilage, dual-tuned ultrashort TE (UTE) imaging techniques by sampling the data proton/sodium coils are very useful. from the center towards the periphery of k space, using either a radial (Fig. 1b) or spiral (Fig. 1c) trajectory. The most common Validation of sodium MRI for the evaluation of cartilage GAG type of UTE sequence for sodium MRI is the three-dimensional content radial projection sequence (50). The SNR and time efficiency of this sequence can be further improved by sampling k space The negative FCD ofcartilage is proportional to the GAG concen­ tration, which is in equilibrium with positively charged sodium more homogenously along the projections, and thus also im­ ionsincartilage(69).SodiumMRIisthereforeasensitivemethod proving the point spread function of acquisition. This is used in for the evaluation of GAG content in cartilage. Sodium MRS was the density-adapted radial projection technique, where the den­ the first method used to explore the relationship between so­ sity of acquisition points along the projections is greater in outer dium signal intensity and PG concentration in enzymatically k-space parts (51). Another approach with efficient k-space sam­ pling is twisted projection imaging (TPI) (52,53). Many other treated (trypsin, papain) cartilage (70-72). It was shown that so­ dium in cartilage is 100% MR visible, and that the FCD of carti­ methods have been inspired by TPI: three-dimensional cones lage can be calculated from the sodium concentration when (54), an acquisition-weighted stack of spirals (55) and FLORET using the ideal Donnan theory (73). Moreover, sodium concen­ (56).Thenon-CartesiandataofUTEsequencesareusuallyrecon- tration measured by sodium imaging has been demonstrated structed using regridding reconstruction (57-59) or non-uniform fast Fourier transform algorithms (60,61). Although the UTE se­ to be proportional to the cartilage GAG content (74-76). These quences sample the sodium signal at shorter TE (greater SNR, papers validated sodium MRI as a feasible method for the direct and non-invasive evaluation of GAG content in cartilage. lesser T2-weighted images) than Cartesian sequences, they are also more prone to artifacts arising from the static field inhomo­ geneity, gradient imperfections and off-resonance effects (62). Quantification of sodium concentration, FCD and GAG content

Partial volume artifacts and fluid suppression For the evaluation of FCD or GAG concentration in cartilage, sodium signal intensities need to be converted into sodium con­ Asaresultofthegenerallylowresolutionofsodiumimages,par- centrations (44). This is generally performed by measuring the tial volume effects from surrounding tissues, such as bone or subjecttogetherwithaseriesofagarose/salinecalibrationphan- synovialfluid(sodiumconcentration of140-150mmol/L),can in­ toms with known sodium concentrations (usually between 100 fluence the accuracy of quantitative sodium MRI results. To min­ and 350 mM), as well as relaxation times, similar to those in imize the contamination of the cartilage signal by synovial fluid cartilage (usually 6-10% agar gels). A linear regression of the or joint effusion, various techniques can be employed. sodium signal intensities of phantoms corrected for their relaxa­ SodiumMRIwithinversionrecoverypreparationhasbeenpro- tion times against their sodium concentrations provides a cali­ posed to suppress signal from synovial fluid in the knee. Sodium bration curve (Fig. 3a). Sodium signal intensities from cartilage inversion recovery benefits from the significantly longer T1 relax­ are then fitted to the calibration curve to create a sodium ation time of sodium in synovial fluid compared with that in car­ concentration map (Fig. 3b). As cartilage comprises about 75% tilage tissue (45). This technique, in general, produces images water, each value in the concentration map is divided by 0.75 with lower SNR in cartilage and requires a longer measurement (75,76). The sodium concentration in healthy cartilage is usually time than that of conventional sodium MRI. between 240 and 280 mM (75,76). When assuming that the ideal Weighted subtraction of sodium images acquired at shorter Donnan equilibrium conditions hold for cartilage, FCD can be and longer TE, using double-echo sodium MRI, can also be used calculated from the sodium concentration according to the fol­ for fluid suppression (63). This method is based on the fact that lowing equation: sodium ions have a much longer T2* value in fluid than in carti­ lage. Although double-echo sodium imaging can provide images sF] with higher SNR in comparison with those obtained by inversion FCD(mM) = [Na [NaJar] recovery, it is also more sensitive to susceptibility artifacts. [NaJar]

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Figure 3. Quantification of sodium concentration. (a) Mean calibration curve obtained from 10% agarose/saline calibration phantoms of different con­ centration (100-250 mM) and mean background noise evaluated on four consecutive images. Circles represent measured signal intensities and error bars their standard deviations. (b) Color-coded sodium concentration map of a healthy volunteer calculated from a variable TE (vTE) gradient echo (GRE) image acquired at 7 T. Before calculation of the sodium concentrations, the signal intensities in the sodium image were corrected for the inho­ mogeneous B1 of the radiofrequency (RF) coil, T1 and T2* relaxation times in cartilage and partial volume effect, as proposed by Moon et al. (68). Color scale represents the sodium concentration in cartilage. where [Na^] is the sodium concentration in the synovial fluid OA cartilage. These results are encouraging for further sodium MRI studies of cartilage, even at 3 T. (typically 140-150 mM) and [NaJar] is the sodium concentration in cartilage (73). In healthy human cartilage, FCD ranges from about -158 to -182 mM. Knowing the FCD, the GAG concentra­ tion can be calculated. When assuming 2mol ofnegative charge CLINICAL SODIUM MRI STUDIES per mole of chondroitin sulfate (one from carboxyl and one from Cartilage repair thesulfate group),anda molecularweightofchondroitin sulfate of 502.5 g/mol, the GAG concentration can be calculated as (73): Several clinical sodium MRI studies for the evaluation of tissue after cartilage repair surgery have shown promising results. The first sodium images of patients after cartilage repair were [GAG](mg/L) = FCD(mM)*502-5(mg/mM) published in 2010 by Trattnig et al. (79). The authors measured 12 patients with a mean time of 56 months after MACT in the femoral cartilage, and compared the results of sodium imaging at7Twith dGEMRIC(80) -acontrastagent-based,GAG-sensitive technique at 3 T. A three-dimensional GRE sequence was used Repeatability of sodium MRI in cartilage at 3 and 7 T for sodium imaging with a sodium-only birdcage knee coil. The The expected linear increase in sodium SNR with the magnetic sodium-normalized values were significantly lower in the repair field strength was demonstrated by Staroswiecki et al. (47). The tissue (mean ±standard deviation, 174±53) than in the authorsshowedabouta2.3timeshighersodiumSNRincartilage reference cartilage (267± 42). dGEMRIC measurements also at 7 T than at 3 T. However, Madelin et al. (77) reported only revealed significantly lower post-contrast T1 values in repair about a 1.6 times higher SNR at 7T relative to 3 T. The authors tissue (510±195ms) compared with those in reference cartilage of the latter paper also demonstrated high repeatability and re­ (756± 188ms). Moreover, a strong correlation was found producibility of concentration quantification using a radial se­ between sodium imaging and dGEMRIC. The authors concluded quence with and without fluid suppression at 3 and 7 T. All the that sodium MRI allows the differentiation between MACT repair root-mean-square coefficients of variation in the quantification tissue and native cartilage in patients without contrast agent of sodium concentration were similar for 3 T (without fluid sup­ application. pression, 9.1%; with fluid suppression, 11.1%) and 7 T (without For the validation of gagCEST imaging as a new method sen­ fluid suppression, 10.7%; with fluid suppression, 12.3%). Jordan sitive to changes in GAG content, Schmitt et al. (8) compared et al. (78) compared the variability of quantitative proton MRI gagCEST results with sodium MRI in patients with femoral carti­ techniques by measuring T1p (with a CubeQuant sequence) and lage repair at 7 T. Results from five patients treated with MFX T2 (with a double-echo, steady-state sequence, DESS) relaxation and seven with MACT showed a strong correlation between times with normalized sodium signals, measured by three­ sodium and gagCEST values, and demonstrated the sensitivity dimensional cones, in the knee cartilage of healthy volunteers of this method to the cartilage GAG content. in vivo at 3 T. The measurements were obtained at baseline, after In a paper by zbyn et al. (81), sodium imaging at 7 T was used 1 day, 5 months and 1 year. The mean intra-subject root-mean- to compare the quality of repair tissue in femoral cartilage be­ square coefficients of variation were 4.6-6.1% for T1p mapping, tween twodifferentrepairprocedures:bonemarrowstimulation 6.4-10.7% for T2 mapping and 11.3-12.9% for sodium imaging. (BMS) techniques (Pridie drilling and MFX) and MACT. For more The values reported above demonstrate that the repeatability accurate comparison, each patient treated with BMS was ofsodiumMRIiscomparablewith thatofT2mappingwith DESS. matched with one patient treated with MACT according to age Moreover, the calculated coefficients of variation are substan­ (mean, 37 years), postoperative interval (mean, 33 months) and tially lower than the changes expected in patients with ad­ defect location. Sodium MRI was performed with a three­ vanced OA, suggesting that quantitative proton and sodium dimensional GRE sequence using a sodium-only birdcage knee methods are sufficiently sensitive for the in vivo evaluation of coil. Sodium-normalized signal intensities were significantly

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69 NMR IN BIOMEDICINE s. ZBVN etal. lower in the repair tissue than in the corresponding reference Cartilage defects can also occur in the ankle joint, mostly after cartilage for both groups. However, the main finding was that injury, or in patients with osteochondritis dissecans. Cartilage re­ sodium-normalized signal intensities were significantly higher pair procedures used in the knee joint can also be performed in in repair tissue after MACT (210±36) than after BMS (164±31). the ankle joint. However, there are metabolic, biochemical and Nevertheless, the morphological appearance of the repair tissue, biomechanical differences between knee and ankle cartilage. An- evaluated by the MR observation of cartilage repair tissue otherstudybyZbynetal.(86)testedthefeasibilityofsodiumMRI (MOCART) scoring system (82), was not significantly different be- fortheevaluation ofrepairtissuein verythin anklecartilage.The tweenpatientstreatedwith BMSandMACT.Theseresultssuggest authorsmeasuredcadaveranklesamples,volunteers,sixpatients ahigherGAGcontentinrepairtissueofpatientstreatedwithMACT treated with MFX and six patients treated with MACTusing a so­ than with BMS,and therefore a repairtissue ofhigherqualityafter dium phase-arrayknee coil at 7T. Patients treated with MFX and MACT. Sodium MRI is able to distinguish between repair tissues MACT had a similar age, body mass index and defect size. Both with different GAG content (Fig. 4), and thus can non-invasively repair techniques resulted in significantly lower mean sodium- evaluatetheperformanceofnewcartilagerepairtechniques. corrected signal intensities in repair tissue (MFX, 243±58; MACT, Chang et al. (83) compared sodium MRI, with and without fluid 193±33) than in reference cartilage (MFX, 317±44; MACT, suppression, as methods for the non-invasive evaluation of carti­ 295 ± 32) (Fig. 5). Sodium-corrected signal intensities and lage repair tissue at 7T. The knees of11 patients after different MOCART scores in repair tissue did not differ between patients cartilage repair procedures (MFX, MACT, osteochondral grafting, treated with MFX and MACT. No significant difference in juvenile cartilage implantation) in patellar or femoral cartilage, sodium-corrected signal intensities was found between the refer- and with a median follow-up of 26 weeks, were measured with encecartilageofthepatientsandthecartilageofthevolunteers. a radial UTE sequence. A sodium-only birdcage knee coil was This study demonstrates the feasibility of quantitative sodium used for signal acquisition, and fluid suppression was achieved MRI in vivo for the evaluation of the thin cartilage of the ankle using an inversion recovery preparation with an adiabatic and subtalar joints at 7 T. Both MFX and MACT produced repair inversion pulse (84). Sodium concentrations were not signifi­ tissue with lower sodium values, and thus of lower quality com­ cantly different between the repair tissue and native cartilage pared with native cartilage. Moreover, MFX and MACTproduced when using a sequence without fluid suppression. This may be repair tissue with similar GAG content and similar morphological attributabletotheheterogeneityofthemeansodiumconcentra- appearance in patients with a similar surgical outcome. tionsin therepairtissuebecauseofthevarietyofcartilagerepair techniques used in this study. However, when using fluid sup­ Osteoarthritis pression, the mean sodium concentration in the repair tissue (108.9±29.8mmol/L) was significantly lower than that in adja­ After validation by in vitro studies and studies on animal models cent native cartilage (204.6±34.7mmol/L) and in cartilage from (87), sodium MRI was used forthe evaluation ofpatients with OA. a completely different knee compartment (249.9±44.6mmol/L). Wheaton et al. (76) measured the knees of nine healthy asymp­ Thus, fluid-suppressed sodium MRI seems to be more accurate tomatic volunteers and three patients with symptoms of early in evaluating sodium concentration in the repair tissue. In addi­ OA. Sodium images were acquired with the UTE radial sequence tion, the sodium concentration in adjacent native cartilage was using a surface coil at 4T. The mean sodium concentration in the significantly lower than that in cartilage in different knee com- patellaeofvolunteerswas 254mmol/L,which corresponded toa partments.This is in agreementwith the findings ofin vitro stud- mean FCD of -182 mmol/L. Subjects with OA symptoms revealed ies,whichdemonstratedthattheamountofviablechondrocytes cartilage regions with a significantly lower FCD (-108 to - increases with the distance from the site of injury (85). 144 mmol/L) when compared with the FCD of volunteers. These

Figure 4. Comparison of morphological and sodium 7-T images between patient after microfracturing (MFX) (top) and patient after ma­ trix-associated autologous chondrocyte transplantation (MACT) (bottom). Figure 5. Proton and corresponding sodium 7-T images from a refer­ (a, b) Proton density-weighted turbo spin echo images with fat suppres­ ence site (top) and a repair site (bottom) of the ankle joint in a patient sion. (c, d) Color-coded sodium three-dimensional gradient echo (GRE) after microfracturing (MFX) cartilage repair. (a, b) Proton density- images with color scale representing the sodium signal intensity values. weighted images with fat suppression. (c, d) Color-coded sodium maps The lower sodium values in repair tissue than in reference cartilage of corrected signal intensities in cartilage and repair tissue. The lower so­ should be noted. The area of cartilage repair tissue is located between dium values in repair tissue than in reference cartilage should be noted. the arrows. Cartilage repair tissue is situated between the arrows.

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authors very probably included signal from both cartilage and synovial fluid in the femorotibial ROIs, perhaps because of low resolution and blurring in sodium images. The effect of the contamination from synovial fluid on sodium concentrations in cartilage was studied by Madelin et al. (89). These authors calculated cartilage sodium concentrations in 28 patients with OA and 19 healthy volunteers using 7-T sodium MRI with and without fluid suppression. The first 12 subjects were measured with a sodium-only birdcage knee coil, and the rest with a home-built, eight-channel, double-tuned proton/sodium knee coil. Inversion recovery with an adiabatic inversion WURST (wideband uniform rate smooth truncation) pulse was used for fluid suppression in the UTE radial sequence (84). The sodium concentration was evaluated in the patellar, femorotibial lateral and femorotibial medial cartilage compart­ ments on four consecutive slices. The mean sodium concentra­ tion over all cartilage compartments measured without Figure 6. Sodium concentration maps from healthy control (top) and fluid suppression was similar in patients with OA (174mmol/L) from patient with osteoarthritis (OA) (bottom) acquired at 7 T. Compari­ and healthy subjects (192 mmol/L). When fluid suppression was son of standard sodium images without fluid suppression (R3D, left) with used, the difference between the mean sodium concentration images obtained with fluid suppression using an inversion recovery tech­ nique (IRW, right). The difference in cartilage sodium concentration in patients with OA (~194mmol/L) and healthy subjects between the control and patient with OA is higher in fluid-suppressed (~243 mmol/L) increased (Fig. 6). The mean sodium concentration maps than in maps without fluid suppression. Originally published by in cartilage calculated from fluid-suppressed images was a sig­ Madelin et al. (89) and reprinted with permission. nificant predictor of OA and early OA (only patients with a Kellgren-Lawrence score of 1 or 2). The cartilage sodium con­ centration obtained with fluid-suppressed MRI at 7 T might be findings suggest that sodium MRI can be useful for the monitor­ a potential biomarker for OA. ing of changes in the GAG content of OA cartilage. The very promising results of sodium MRI for OA evaluation at Theinitial7-TsodiumimagesfrompatientswithOAwerepub- 7 T encouraged the use of this technique at 3 T. Newbould et al. lishedbyWang etal.(88).Sodiumimagesfromfivesubjectswith (90) were the first to test the stability and reproducibility of the clinically diagnosed OA and five asymptomatic volunteers were sodium signal in the knee cartilage in healthy volunteers and measured with the UTE radial sequence, using a sodium bird­ patients with OA at 3 T. The authors measured 15 patients with cage knee coil. The sodium concentration was calculated in three OA and five volunteers, twice in the same day, using a cartilage regions of interest (ROIs): patellar, medial femorotibial T1-weighted three-dimensional cone sequence in combination and lateral femorotibial. The mean sodium concentration in the with a dual-tuned quadrature knee coil. The within-subject cartilage of volunteers was between 240 and 280mmol/L. The variability of the median sodium concentration was similar in sodium concentration was significantly lower (30-60% lower) healthy age-matched volunteers (2.0%) and in subjects with OA in patients with OA compared with volunteers. The authors (3.6%). Sodium MRI may thus be a more specific marker of carti­ concluded that sodium imaging may be a useful tool for physio­ lage degeneration, and may be useful for the measurement of logical OA imaging and clinical diagnosis. Unfortunately, the the treatment response in clinical trials of OA.

Figure 7. Example three-dimensional dataset of sodium concentration maps overlaid on the morphological three-dimensional, double-echo, steady- state (DESS) images from 3 T. High sodium concentrations can be found in articular cartilage. Color scale represents the sodium concentration. Reproduced from Newbould et al. (91).

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71 NMR IN BIOMEDICINE s. zbVn etal.

In another study by Newbould et al. (91), T1-weighted sodium in OA cartilage and cartilage repair tissue may be different from MRI using three-dimensional cones was used to map longitudi­ those in hyaline cartilage. The quantification ofsodium concentra­ nal and cross-sectional differences in OA knee cartilage at 3 T tion in OA cartilage and in cartilage repair tissue, using T1 and T2* (Fig. 7). The authors compared sodium concentrations in knee values from native cartilage,might diverge from the ‘true' sodium cartilage between 28 subjects with OA and 19 age- and concentrations.Unfortunately,themeasurementofsodiumrelaxa- gender-matched healthy controls at baseline and at 3 and tion times is much more time-demanding than ‘morphological' so- 6 months. The knee injury and OA outcome score, the knee pain diumimaging,andrelaxationtimescouldchangewiththeprogress by visual analog score and the Kellgren-Lawrence score all sig­ ofOAoronmaturationofrepairtissue.Correctionforwatercontent nificantly separated patients with OA from healthy controls. In mayalsoinfluence the accuracyofsodium concentration quantifi­ contrast with previous sodium MRI studies of patients with OA, cation. In native hyaline cartilage, water represents between 65% the authors found a significantly (8%) higher sodium concentra­ and80%oftotalweight(12,13,18).However,thewatercontentin tion in the knees of subjects with OA than in healthy controls at OA cartilage or in repair tissue in the first year after surgery may allthreetimepoints.Thisincreasewasmoststronglyobservedin be slightly higher compared with that in native cartilage (92,93). the lateral tibial (18% higher), lateral femoral (12% higher) and In any case, a knowledge of cartilage relaxation times and water medial patellar (11% higher) regions. Newbould et al. suggested content might improve the sensitivity and specificity of sodium that the decrease in sodium concentration found in previous MRI in evaluating the degenerative changes in cartilage. studies (87,88) could have been attributed to the much lower Recentnewtechnicaldevelopmentshaveenabledthetransla- age of volunteers compared with patients with OA, and to the er­ tionofsodiumMRIfrominvitrostudiestothefirstclinicalinvivo roneous and biased placement of ROIs that included synovial applications. Advances in high- and ultrahigh-field MR systems fluid. This study did not find any significant changes in concen­ (47,94), dedicated RF phase-array coils (68,95) and optimized tration over time for the subjects with OA or healthy controls, sodium MRI sequences (55,96) have resulted in sodium images which validated the stability of the sodium content measure- with higher SNR, higher spatial resolution and shorter measure­ ments.TheresultsofthisstudyindicatethatT1-weightedsodium ment times. However, further hardware and software improve­ MRI is able to distinguish between subjects with OA and healthy ments are needed to complete the translation of sodium MRI controls in a reproducible manner. into a clinically feasible method for non-invasive cartilage evalu­ Sodium imaging seems to be sensitive for the detection of ation on 3-T systems. changesbetweenhealthyandOAcartilage,andthusmightbeause- In summary, recent clinical applications ofsodium imaging for ful method for the non-invasive evaluation of cartilage response to the evaluation of cartilage repair and knee OA have demon­ OAdrugs.However,furthersodiumMRIstudiesareneededtoclarify strated the great promise of this non-invasive GAG-sensitive the changes in GAG/sodium concentration associated with OA. method, and will motivate further interesting studies and techni­ cal developments in this field.

CONCLUSIONS Acknowledgements

Recent clinical studies in patients with OA and patients after car­ The authors thank Lasse Räsänen for help with the images. tilage repair have demonstrated that sodium MRI is a non­ Funding support was provided by the Austrian Science Fund invasive and quantitative method forthe assessment ofcartilage (FWF) projects P 1652 B19, P 23481 B19 and P 25246 B24. GAGconcentration,whichplaysakeyroleincartilagehomeosta- The authors have no conflicts of interest to declare. sis. As native reference cartilage (adjacent or more distant from the repair site) is available in sodium images of repair tissue, the absolute sodium concentration is not a critical parameter, and ra­ REFERENCES tios between native and repair sodium signal intensities can also 1. BuckwalterJ,MowV.CartilageRepairinOsteoarthritis.W.B.Saunders be compared among patients from different studies. From this Co.: Philadelphia, PA; 1992. point of view, sodium MRI of OA cartilage is more demanding 2. Lawrence RC, Felson DT, Helmick CG, Arnold LM, Choi H, Deyo RA, and requires measurements with agarose/saline calibration Gabriel S, Hirsch R, Hochberg MC, Hunder GG, Jordan JM, Katz JN, phantoms to quantify sodium concentration. In addition, carti­ Kremers HM, Wolfe F, National Arthritis Data W. Estimates of the prevalenceofarthritisandotherrheumaticconditionsintheUnited lage is usually thinner in patients with OA than in healthy volun­ States. Part II. Arthritis Rheum. 2008; 58(1): 26-35. teers, and the partial volume effect is therefore more pronounced 3. Burstein D, Bashir A, Gray ML. MRI techniques in early stages of car­ in OA applications. This issue can be minimized by combining tilage disease. Invest. Radiol. 2000; 35(10): 622-638. fluid-suppressed sequences, such as inversion recovery (84), or 4. Baum T, Joseph GB, Karampinos DC, Jungmann PM, Link TM, Bauer JS. Cartilage and meniscal T2 relaxation time as non-invasive bio­ heavily T1-weighted sodium MRI (91), with a compensation for marker for knee osteoarthritis and cartilage repair procedures. the signal reduction based on measurements of cartilage thick­ Osteoarthr. Cartilage, 2013; 21(10): 1474-1484. ness from morphological proton images (68). Moreover, using 5. Andreisek G, Weiger M. T2* mapping of articular cartilage: current dual-tunedproton/sodiumRFcoils,sodiumMRIcanbecombined status of research and first clinical applications. Invest. Radiol. 2014; 49(1): 57 62. with proton techniques (e.g. morphological imaging,T2 mapping, - 6. Duvvuri U, Reddy R, Patel SD, Kaufman JH, Kneeland JB, Leigh JS. diffusion-weighted imaging) to assess other important cartilage T1rho-relaxation in articular cartilage: effects of enzymatic degrada­ components (e.g. watercontent, collagen matrix). A combination tion. Magn. Reson. Med. 1997; 38(6): 863-867. of different biomarkers might provide more accurate insight into 7. Bashir A, Gray ML, Burstein D. Gd-DTPA(2-) as a measure ofcartilage cartilage degeneration or maturation of cartilage repair tissue. degradation. Magn. Reson. Med. 1996; 36(5): 665-673. 8. Schmitt B, zbyn S, Stelzeneder D, Jellus V, Paul D, Lauer L, Bachert P, Previous in vitro studies of enzymatically induced (trypsin, Trattnig S. Cartilage quality assessment by using glycosaminoglycan papain) PG depletion in articular cartilage have shown changes in chemicalexchangesaturationtransferand(23)NaMRimagingat7T. T1andT2*relaxationtimes(70-72).Thus,T1andT2*relaxationtimes Radiology, 2011; 260(1): 257-264.

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Cartilage, 2000; 8(4): 288-293. surgery: outcome evaluation by using noninvasive cartilage bio­ 75. Shapiro EM, Borthakur A, Gougoutas A, Reddy R. 23Na MRI accurately markers based on quantitative MRI techniques? BioMed Res. Int. measures fixed charge density in articular cartilage. Magn. Reson. 2014; DOI: 10.1155/2014/840170. Med. 2002; 47(2): 284-291. 94. Regatte RR, Schweitzer ME. Ultra-high-field MRI of the musculoskele­ 76. Wheaton AJ, Borthakur A, Shapiro EM, Regatte RR, Akella SV, tal system at 7.0 T. J. Magn. Reson. Imaging, 2007; 25(2): 262-269. Kneeland JB, Reddy R. Proteoglycan loss in human knee cartilage: 95. Brown R, Madelin G, Lattanzi R, Chang G, Regatte RR, Sodickson DK, quantitation with sodium MR imaging—feasibility study. Radiology, Wiggins GC. Design of a nested eight-channel sodium and four- 2004; 231(3): 900-905. channel proton coil for 7 T knee imaging. Magn. Reson. Med. 2013; 77. Madelin G, Babb JS, Xia D, Chang G, Jerschow A, Regatte RR. Repro­ 70(1): 259-268. ducibility and repeatability of quantitative sodium magnetic reso­ 96. Madelin G, Chang G, Otazo R, Jerschow A, Regatte RR. Compressed nance imaging in vivo in articular cartilage at 3 T and 7 T. Magn. sensing sodium MRI of cartilage at 7 T: preliminary study. J. Magn. Reson. Med. 2012; 68(3): 841-849. Reson. 2012; 214: 360-365.

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74 INTRODUCTION

1.6 Aims of the Thesis

The main goal of this thesis is to contribute to the current knowledge about the cartilage repair tissue produced by different repair techniques in femoral cartilage ofthe knee and talar cartilage ofthe ankle joint. In particular, sodium MRI was used for the noninvasive evaluation of GAG content in native cartilage and repair tissue. This is important information since sufficiently high GAG concentration is important for maintaining compressive stiffness and thus also cartilage function and health. Sodium MRI is a challenging method that suffers from low SNR that results in images acquired with low resolution (e.g. 2x2x3 cm3). Thus, the first aim was to optimize GRE sequence for sodium MRI of cartilage in the knee and to validate the sensitivity of our MRI protocol to changes in the GAG content. The most important part of MRI protocol optimization was to maximize sodium SNR per unit of time. This was possible by using short RF pulses, short TE and TR values, and a flip angle that is set to the Ernst angle optimized for cartilage. Forverifying sensitivity ofthe sodium MRI protocol to GAG changes in cartilage repair tissue, sodium MRI results were compared with dGEMRIC - other GAG sensitive technique. One of most often used cartilage repair procedures are MACT and BMS techniques. Although more advanced MACT techniques produce hyaline-like repair tissue more often than relatively simple BMS techniques, their role in cartilage repair is not clearly defined yet. Thus, the second part of the thesis aimed to use GAG-sensitive sodium MRI protocol for the evaluation of biochemical and biomechanical properties of repair tissue produced by MACT and BMS repair surgery procedures in femoral cartilage. There are various differences in biochemical composition and in biomechanical properties between the knee and ankle joints. It is therefore not surprising that the findings on cartilage repair tissue produced in femoral cartilage might not be valid for the cartilage of ankle joint. Therefore, the last aim was to evaluate repair tissue properties after different cartilage repair procedures in the ankle. Since cartilage in the ankle is thinner that the cartilage in the knee, which result in more pronounced partial volume artifacts, validation of sodium MRI sensitivity was a crucial task. To address this concern, histochemically evaluated GAG content of cartilage from cadaver ankle samples was compared with sodium MRI results. As a next step, sodium MRI was measured in MFX and MACT patients in order to compare the GAG content in repair tissue produced by these repairtechniques.

75 RESULTS

2. RESULTS

2.1 Prologue

Several scientific papers on follow-up of patients after cartilage repair surgery were previously published by our group. Different morphological and biochemical methods (Diffusion-weighted imaging, dGEMRIC, T2 and T2* mapping) have been used for the evaluation of cartilage repair. Although sodium MRI was already shown to be sensitive to changes in the cartilage GAG content, it was not used before for the evaluation of cartilage repair. The launch of the 7 T MRI systems brought exciting possibilities and new challenges to in vivo MRI of cartilage repair tissue. While conventional proton MRI at 7 T is accompanied by many challenges (e.g. increased RF power deposition, homogeneity of RF coils, susceptibility artifacts), sodium MRI primarily profits from the ultra-high-field strength - mainly from higher intrinsic SNR compared to 3 T. The SNR gain enables to acquire sodium images with higher resolution and/or in shorter measurement times. The aim of paper 1 was therefore to validate sensitivity of our sodium MRI protocol by comparing sodium data with dGEMRIC results (another GAG-specific method) and to evaluate repair tissue properties in patients after MACT on the femoral condyle [194], My contribution to paper 1 was measurement of sodium relaxation times in cartilage of volunteers, optimization of the sodium sequences, measurements of patients, postprocessing and ROI evaluations, statistical analyses, writing and editing of manuscript.

2.2 Paper 1:23Na MRI at7T after Knee MACT

A permission for reproduction of this paper in PhD-thesis was granted by the publisher - Radiologic Society of North America [194],

76 Note: This copy is for your personal, non-commercial use only. To order presentation-ready copies for distribution to your colleagues or clients, contact us at www.rsna.org/rsnarights. ORIGINAL

23Na MR Imaging at 7 T after RESEARCH

Knee Matrix-associated n

Autologous Chondrocyte MUSCULOSKELETAL Transplantation: Preliminary Results1

IMAGING Siegfried Trattnig, MD Purpose: Goetz H.Welsch, MD To evaluate the feasibility of sodium 7-T magnetic reso­ nance (MR) imaging in repaired tissue and native carti­ Vladimir Juras, PhD lage of patients after matrix-associated autologous chon­ Pavol Szomolanyi, PhD drocyte transplantation (MACT) and compare results with Marius E. Mayerhoefer, MD delayed gadolinium-enhanced MR imaging of cartilage David Stelzeneder, MD (dGEMRIC) at 3 T. Tallal C. Mamisch, MD Oliver Bieri, PhD Materials and Ethical approval was provided by the local ethics com- Klaus Scheffler, PhD Methods: mittee; written informed consent was obtained from all Stefan Zbyri, MSc patients. Six women and six men (mean age, 32.8 year 6 8.2 [standard deviation] and 32.3 years 6 12.7, respec­ tively) were included. Mean time between MACT and MR was 56 months 6 28. A variable three-dimensional (3D) gradient-echo (GRE) dual-flip-angle technique was used for T1 mapping before and after contrast agent ad­ ministration at 3 T. All patients were also examined at 7 T (mean delay, 70.5 days 6 80.1). A sodium 23-only transmit-receive knee coil was used with the 3D GRE sequence. A statistical analysis of variance and Pearson correlation were applied.

Results: Mean signal-to-noise ratio (SNR) was 24 in native carti­ lage and was 16 in transplants ( P , .001). Mean sodium signal intensities normalized with the reference sample were 174 6 53 and 267 6 42 for repaired tissue in the cartilage transplant and healthy cartilage, respectively ( P , .001). Mean postcontrast T1 values were 510 msec 6 195 and 756 msec 6 188 for repaired tissue and healthy cartilage, respectively ( P = .005). Mean score of MR ob­ servation of cartilage repair tissue was 75 6 14. Asso­ ciation between postcontrast T1 and normalized sodium signal values showed a high Pearson correlation coefficient ( R) of 0.706 ( P = .001). A high correlation of R = 0.836 ( P = .001) was found between ratios of normalized sodium values and ratios of T1 postcontrast values.

1 From the Department of Radiology, MR Centre-High Conclusion: With the modified 3D GRE sequence at 7 T, a sufficiently Field MR, Medical University of Vienna, Lazarettgasse 14, A-1090 Vienna, Austria. Received February 4, 2010; high SNR in sodium images was achieved, allowing for dif­ revision requested March 10; final revision received April 27; ferentiation of repaired tissue from native cartilage after accepted April 29; final version accepted May 18. Sup­ MACT. A strong correlation was found between sodium ported by the Austrian Science Funds: FWF-TRP-Projekt imaging and dGEMRIC in patients after MACT. L243-B15, FWF-Projekt P18110-B15, VIACLIC (WWTF) FA102A0017, and the Slovak Scientific Grant Agency VEGA q RSNA, 2010 2/0142/08. Address correspondence to S.T. (e-mail: [email protected]).

q RSNA, 2010

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77 MUSCULOSKELETAL IMAGING: Sodium MR Imaging after Knee Chondrocyte Transplantation Trattnig et al

rticular cartilage consists primarily MR imaging that is sufficient for the Cartilage-repair surgery is a dynamic of an extracellular matrix made detection of even small (approximately research field, with the need for reliable of mostly type II collagen, pro­ 5%) changes in the PG content. In these and objective monitoring, to evaluate Ateoglycan (PG), chondrocytes, and waexperiments,­ sodium spectroscopic evalu­ and compare various surgical treatment ter (1). PG is made of a linear protein ation of the articular cartilage was per­ options. The aim of our study was to core to which polysaccharides, known formed to evaluate the single-quantum evaluate the feasibility of sodium imag­ as glycosaminoglycan (GAG), are at­ sodium signal, effective T1, and the two ing at 7 T indicative of GAG content in tached. PG provides both compressive components of the biexponential T2, repaired tissue and in native cartilage and tensile strength to the matrix and fast T2 (T2f) and slow T2 (T2s), through of patients after MACT and to compare links restrict fluid flow through the a triple-quantum method on cartilage the results with those of dGEMRIC matrix, providing increased resistance specimens that have undergone serial at 3 T. to structural deformation (2,3). proteolytic enzymatic degradation. The sulfate and carboxyl groups of Generally, sodium imaging is chal­ Materials and Methods the GAG impart a negative fixed-charge lenging and requires the MR system to density (FCD) to the matrix. These neg­ have multinuclear capabilities and dedi­ One of the authors (T.C.M.) is a research ative ions attract positive counterions cated sodium coils, preferably a very consultant for Siemens Healthcare. (eg, sodium) and water molecules. The high field strength to allow for sufficient loss of PG from the extracellular matrix signal-to-noise ratio (SNR), and a high Subjects has been hypothesized to be the event enough spatial resolution to depict de­ Ethical approval for this study was pro­ that initiates the onset of osteoarthritis fined areas of articular cartilage. vided by the local ethics commission, (OA) (4,5) and results in a reduction Chondral injury has very limited ca­ and written informed consent was ob­ of FCD in cartilage. Maroudas et al (6) pability for self-repair, and the subse­ tained from all patients. showed that the FCD of cartilage is cor­ quent degeneration often leads to OA, related to the GAG content of cartilage. which may result in the need for total Because the FCD is counterbalanced by knee arthroplasty (3). Surgical options the sodium ions, a loss of PG (and, con­ for cartilage repair include bone marrow Published online before print sequently, GAG and FCD) owing to car­ stimulation techniques, such as micro­ 10.1148/radiol.10100279 tilage degeneration results in the loss of fracture (13-15), osteochondral graft Radiology 2010; 257:175-184 sodium ions from the tissue (1). transplantation (mosaicplasty, osteo­ Sodium magnetic resonance (MR) chondral autologous transplantation) Abbreviations: ACI = autologous chondrocyte implantation imaging has been validated as a quan­ (16-18), and autologous chondrocyte dGEMRIC = delayed gadolinium-enhanced MR imaging of titative method of computing FCD and, implantation (ACI) (19-22). Newer ACI cartilage hence, GAG content in healthy humans techniques are often referred to as FCD = fixed-charge density (7-9). The major advantage of sodium scaffold-guided or matrix-associated au­ GAG = glycosaminoglycan MR imaging of cartilage is its high spec­ tologous chondrocyte transplantation GRE = gradient echo ificity to PG content without the need (MACT), because biomaterials that are MACT = matrix-associated autologous chondrocyte transplantation for any exogenous contrast agent, such based on collagen, hyaluronan, polylac- MOCART = Magnetic Resonance Observation of Cartilage as that in delayed gadolinium-enhanced tides, or other materials, are used as Repair Tissue MR imaging of cartilage (dGEMRIC) scaffolds for cell growth (23-27). OA = osteoarthritis (10). Moreover, the low sodium content One goal of repair techniques is the PG = proteoglycan (,50 mmol/L) of surrounding structures formation of hyalinelike repaired tissue, ROI = region of interest in the joint enables visualization of the which is histologically characterized by SE = spin echo SNR = signal-to-noise ratio cartilage with very high tissue contrast. the organized type II collagen fiber net­ 3D = three-dimensional The results of experiments with con­ work and a GAG content that should trolled cartilage degradation (11,12) comprise 5%-10% of the cartilage Author contributions: have confirmed a sensitivity of sodium matrix (28). Guarantors of integrity of entire study, S.T., G.H.W., T.C.M., K.S., S.Z.; study concepts/study design or data acquisition or data analysis/interpretation, all Advance in Knowledge: authors; manuscript drafting or manuscript revision Implication for Patient Care: for important intellectual content, all authors; approval n We found a strong correlation of final version of submitted manuscript, all authors; between sodium imaging and n Sodium MR imaging at 7 T indic­ literature research, S.T., G.H.W., V.J., P.S., S.Z.; clinical delayed gadolinium-enhanced MR ative of glycosaminoglycan con­ studies, S.T., G.H.W., D.S., T.C.M., O.B., K.S., S.Z.; imaging of cartilage in patients tent allows a nonenhanced statistical analysis, S.T., G.H.W., V.J., T.C.M., S.Z.; after matrix-associated autolo­ assessment of the potential per­ and manuscript editing, S.T., G.H.W., V.J., P.S., M.E.M., O.B., K.S., S.Z. gous chondrocyte formance of cell-based cartilage- transplantation. repair surgical techniques. See Materials and Methods for pertinent disclosures.

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78 MUSCULOSKELETAL IMAGING: Sodium MR Imaging after Knee Chondrocyte Transplantation Trattnig et al

From February 2009 to October in three cases, and the trochlear region in (Table 1). The same patients were also 2009, six women (mean age, 32.8 years one case. The mean time between MACT examined with a 3-T MR whole-body 6 8.2 [standard deviation]; range, 22­ and MR imaging was 56 months 6 28 system (TimTrio; Siemens Healthcare) 44 years) and six men (mean age, (range, 11-102 months). equipped with a dedicated eight-channel 32.3 years 6 12.7; range, 23-55 years) knee coil (Invivo, Gainesville, Fla). who had undergone MACT were included MR Imaging A dual-flip-angle 3D GRE technique in the study. All patients were chosen Examinations were performed with a (Table 1) was used for T1 mapping be­ from a larger cohort of 45 patients 7-T MR whole-body system (Magne- fore and after contrast agent administra­ (mean age, 34.7 years 6 13.2; range, tom; Siemens Healthcare, Erlangen, tion (dGEMRIC) (30). The postcontrast 20-55 years) who had undergone 3-T Germany). Sodium measurements were MR imaging protocol comprised a bolus MR imaging as part of the standard performed by using a sodium 23 (23Na)- injection of 0.2 mmol per kilogram body MR follow-up after MACT. Immedi­ only 78.61-MHz circularly polarized weight of gadopentetate dimeglumine ately after the follow-up examination or transmit-receive knee coil (Stark Con­ (Magnevist; Bayer Schering Pharma, through telephone recruitment within trast, Erlangen, Germany) with an in­ Berlin, Germany). After injection, the 4 weeks, all patients were then asked ner diameter of 19 cm. To enable com­ patient exercised the knee by walking up to participate in the study and undergo parison between subjects, a reference and down stairs for 20 minutes. Postcon­ an additional sodium protocol at 7-T sample containing 308 mmol/L NaCl trast MR imaging was performed 90 min­ MR imaging. All patients with implanted solution was fixed to the inner surface utes after administration of the contrast metal devices were excluded from the of the sodium coil to serve for normal­ agent (10). The exercise and delay after study. Further exclusion criteria were ization of the signal owing to different the injection were necessary to allow the contraindications to contrast agent- coil filling caused by various knee sizes contrast agent to penetrate the articular enhanced MR imaging, advanced OA, between subjects. cartilage and cartilage-repair tissue. The meniscal tear, knee joint instability as­ All sodium measurements were per­ corresponding T1 maps were calculated sessed by using standard clinical testing formed with a modified three-dimensional with the built-in software (MapIt; Sie­ (29), ligament injuries excluded on the (3D) gradient-echo (GRE) sequence mens Healthcare). The mean time be­ basis of morphologic MR evaluations, that allows operating on both proton tween the 3- and 7-T measurements was and surgical procedures in the knee joint and sodium resonance frequencies and 70.5 days 6 80.1 (range, 0-210 days). prior to the study. Two imaged patients allows adjustment of the pulse length were excluded: a 60-year-old woman of the excitation pulse. In fact, we were Calculation and Comparison of Sodium with very thin cartilage owing to severe able to overcome exceeding specific Content with Postcontrast T1 Values OA and a 53-year-old man in whom im­ absorption rate limit by adjusting the All region-of-interest (ROI) evaluations aging was not successfully accomplished pulse length of the rectangular excita­ were performed by one author (S.T., with owing to technical problems. tion pulse to 970 microseconds. After 15 years of experience in musculoskele­ The location of the cartilage trans­ the flip angle calibration and localizer tal radiology) with an in-house, custom- plant was the medial femoral condyle in image acquisition, a 3D GRE sequence written evaluation tool for data analy­ eight cases, the lateral femoral condyle optimized for sodium imaging was used sis programmed in IDL (Interactive

Table 1

MR Imaging Parameters

Flip Angle Bandwidth Field of Section Acquisition Sequence TR/TE (msec) (degrees) (Hz/pixel) View (mm2) Matrix Size Thickness (mm) No. of Sections Time (min:sec)

3 T Proton-density-weighted turbo SE 2400/38 160 244 120 3 120 512 3 512 2.0 32 6:11 Dual fast SE 5120/9.5, 67 140 203 180 3 180 448 3 448 3.0 30 6:46 Turbo inversion-recovery magnitude* 7690/41 150 250 150 3 150 256 3 256 3.0 36 2:35 Dual- flip-angle 3D GRE 15/3.94 5, 26 490 160 3 160 384 3 384 3.0 22 3:46 7 T T2-weighted double-echo steady 16.02/5 22 283 160 3 160 192 3 192 0.83 120 7:16 state Proton-density-weighted turbo SE 2500/11 160 240 160 3 160 320 3 320 3.0 12 5:52 23Na 3D GRE 10/3.77 56 170 199 3 199 128 3 64 3.0 48 30:45

Note.—SE = spin echo, TE = echo time, TR = repetition time. * Inversion time was 220 msec.

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Table 2 sample from all patients and the mean Cartilage-Repair Tissue Assessment with MOCART: Grading and Score Scale sodium signal intensity from the refer­ ence sample of the evaluated patient. All Variable and Class Score SNR values were calculated as mean Degree of defect repair and defect filling signal intensity in the ROI divided by the Complete (on a level with adjacent cartilage) 20 standard deviation of an ROI selected Hypertrophy (over the level of the adjacent cartilage) 15 in the signal-free area. Incomplete (under the level of the adjacent cartilage; underfilling) The cartilage transplant location . 50% of the adjacent cartilage 10 was verified by using proton-density , 50% of the adjacent cartilage 5 weighted turbo SE and T2-weighted 3D Subchondral bone exposed (complete delamination or dislocation and/or loose body) 0 double-echo steady-state sequences at Integration to border zone 7 T (Table 1). In cases of unclear delin­ Complete (complete integration with adjacent cartilage) 15 eation, surgical reports were also used. Incomplete (incomplete integration with adjacent cartilage), demarcating border visible (splitlike) 10 To assure comparability between 3-T Defect visible dGEMRIC and 7-T sodium MR imaging, , 50% of the length of the repair tissue 5 ROIs of similar area were drawn in the . 50% of the length of the repair tissue 0 cartilage-repair tissue and the native Surface of the repair tissue cartilage on comparable sections in a Surface intact (lamina splendens intact) 10 side-by-side evaluation. To calculate the Surface damaged (fibrillations, fissures, and ulcerations) area of the ROI, image resolution was , 50% of repair tissue depth 5 .50% of repair tissue depth or total degeneration 0 multiplied by the number of pixels in the Structure of the repair tissue ROI. Comparable to the sodium evalu­ Homogeneous 5 ation at 7 T, an ROI analysis was also Inhomogeneous or cleft formation 0 performed in the repaired tissue and Signal intensity of the repair tissue the control cartilage on the dGEMRIC Dual T2-weighted fast SE maps. From the number of pixels in se­ Isointense 15 lected ROIs, the area covered by the ROI Moderately hyperintense 5 was calculated according to the image Markedly hyperintense 0 resolution. The mean difference between 3D GRE with fat suppression the area of the ROI from the sodium im­ Isointense 15 age and the area of the ROI from the T1 Moderately hypointense 5 map was only 5% (range, 1%-12%). Markedly hypointense 0 To eliminate systematic errors and Subchondral lamina for better (dimensionless) comparabil­ Intact 5 ity of both methods, we calculated the Not intact 0 ratios of normalized sodium values and Subchondral bone ratios of postcontrast T1 values. The val­ Intact 5 ues of the control cartilage were divided Edema, granulation tissue, cysts, sclerosis 0 by the values of the repaired tissue, and Adhesions a normalized sodium ratio, as well as a No 5 dGEMRIC T1 ratio was thus defined. In Yes 0 Effusion addition, the mean sodium SNR and the No 5 mean contrast-to-noise ratio between Yes 0 native cartilage and transplanted tissue Maximum score 100 in sodium images were calculated.

Comparison of MOCART Scoring with Sodium Content and Postcontrast Data Language; Research Systems, normalization of the mean cartilage so­ T1 Values Boulder, Colo). Cartilage sodium signal dium signal from the ROIs in cartilage- The morphologic condition of the re­ was measured from a region covering repair tissue and native cartilage. Signal paired tissue was assessed with the Mag­ the cartilage-repair tissue and an equally intensity was normalized by multiplying netic Resonance Observation of Carti­ sized region of normal native cartilage. mean sodium signal intensity values from lage Repair Tissue (MOCART) scoring Owing to the various sizes of the pa­ ROIs placed in cartilage with the ratio system, as introduced by Marlovits tients' knees, correction for different between the highest sodium signal inten­ et al (31). To each evaluated variable, a filling of the coil was performed by the sity of the ROI placed in the reference point score was subscribed according to

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80 MUSCULOSKELETAL IMAGING: Sodium MR Imaging after Knee Chondrocyte Transplantation Trattnig et al

Figure 1 (for women, 32.8 years; for men, 32.3 17 years; P = .958) and postoperative in­ terval (for women, 51 months; for men, 61 months; P = .572). The mean SNR in native cartilage was 24 (range, 17-30), and in transplants, it was 16 (range, 11-29) ( P , .001). The contrast-to- noise ratio between native cartilage and transplanted tissue in sodium images was in the range of 1-16 (mean, 7). The values of SNR for native cartilage and the repaired tissue of transplants in each patient are shown in Figure 1. The mean normalized sodium signal intensity values were 174 6 53 (range, 117-309) for the repaired tissue within the cartilage transplant and 267 6 42 (range, 219-350) for control cartilage 5 -I------1------(Table 3). We found significantly lower Transplanted Native normalized sodium values of the re­ Cartilage paired tissue, in comparison with val­ Figure 1: Plot represents comparison of mean sodium SNR values from ROIs ues of the control cartilage ( P , .001). in transplanted areas (left) and corresponding ROIs in native cartilage (right) in Comparable to the sodium results, the 12 patients. Each line represents one patient. All mean SNR values from native dGEMRIC evaluation also revealed sig­ cartilage are higher than values from corresponding transplanted areas, except nificantly higher mean postcontrast T1 for one case where only a minor change was observed in both mean sodium SNR and T1 values between transplanted and native cartilage. values ( P = .005) for the control cartilage of 756 milliseconds 6 188, compared with that of the cartilage-repair tissue at Table 2. The morphologic MR protocol Statistical Analysis 510 milliseconds 6 195 (Table 3). The was prepared at 3 and 7 T and was identi­ For statistical analysis, quantitative evalu­ mean MOCART score of the transplants cal for all patients. It consisted of a sagit­ ation was performed with analysis of vari­ in the presented patient population was tal high-spatial-resolution proton-density ance by using a three-way univariate anal­ 75 points 6 14 (range, 45-95 points) weighted turbo SE sequence, a sagittal ysis of variance with two inner-subject (Table 3). The normalized sodium ratio T2-weighted dual fast SE sequence, and factors and one random factor. We per­ (control cartilage-repair tissue ratio) a coronal T1-weighted turbo inversion- formed a power analysis. As we assumed was 1.62 6 0.43 (range, 1.02-2.42). recovery magnitude sequence measured a large effect (e = 1), a power analysis re­ With comparable results, the normal­ at 3 T (Table 1). Moreover, a sagittal vealed 12 patients to be enough to obtain ized postcontrast T1 dGEMRIC ratio T2-weighted double-echo steady-state a power of 85%. A Pearson correlation was 1.56 6 0.40 (range, 1.03-2.45). sequence and a sagittal proton-density- coefficient was calculated to evaluate the The normalized sodium values weighted turbo SE sequence were re­ associations between the dGEMRIC and and the postcontrast T1 values of the corded at 7 T (Table 1). sodium imaging technique and between dGEMRIC evaluation showed a signifi­ MOCART scoring and dGEMRIC and cant correlation of R = 0.706 ( P = .001, Interreader Agreement sodium imaging techniques. The strength R2 = 0.50) (Fig 2a). Furthermore, a To evaluate the interreader agreement, of linear association between the vari­ highly significant correlation of R = 0.836 all ROIs in all patients that were used ables can be classified according to the ( P = .001, R2 = 0.70) was found between for evaluations were selected inde­ correlation coefficients as low ( R , 0.5), the ratios of normalized sodium and the pendently by one author (M.E.M., with moderate ( R = 0.5-0.7), and strong ratios of postcontrast T1 values (Fig 2b). 6 years of experience in general radiol­ ( R . 0.7). Statistical software (SPSS 17.0 Comparison of MOCART scores with ogy) with the use of the same home- for Windows; SPSS Institute, Chicago, Ill) postcontrast T1 values from transplants built tool. To evaluate the differences was used, and P , .05 was considered and MOCART scores with normalized between the readers' ROIs, a paired to indicate a significant difference. sodium signal intensities revealed cor­ t test was used and the Pearson correla­ relation of R = 0.541 ( P = .070, R2 = tion coefficient was calculated. The data 0.29) and R = 0.678 ( P = .015, R2 = Results points in each correlation were linearly 0.46), respectively. However, when we fitted, and the coefficient of determina­ When comparing sexes, no significant compared MOCART scores with the tion ( R2) was calculated. difference could be found between age ratios between ROIs from native and

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Table 3

Comparison of MOCART Scores with Postcontrast T1 Values and Sodium Values in the Study Cohort

T1 Mapping Sodium Imaging

Subject No. and Tissue Type ROI Location MOCART Score Postcontrast T1 (msec) Pixel Count ROI Area (mm2) Signal Intensity* Pixel Count ROI Area (mm2)

1

Note.—LFC = lateral femoral condyle, MFC = medial femoral condyle, N = native tissue, T = transplanted tissue. * Signal intensity = normalized sodium signal intensity.

transplant regions for postcontrast T1 nificant difference between the readers ( R = 0.974, P = .001, R2 = 0.95) and and normalized sodium values, moder­ neither in the postcontrast T1 values when we compared sodium signal inten­ ate correlation of R = 0.629 ( P = .028, from native ( P = .158) and transplanted sities in ROIs from native ( R = 0.685, R2 = 0.40) and a strong correlation of cartilage ( P = .420), nor in the sodium P = .014, R2 = 0.47) and transplanted car­ R = 0.813 ( P = .001, R2 = 0.66) were signal intensities from ROIs selected in tilage ( R = 0.847, P = .001, R2 = 0.72). found (Fig 3). native cartilage ( P = .087) and trans­ An example of comparison among plants ( P = .724). Moreover, strong cor­ Discussion morphologic image, T1 map, and sodium relations were found between the read­ image can be seen on Figure 4. ers when we compared postcontrast T1 With the application of a modified 3D The paired t test used for inter­ values from native cartilage ( R = 0.878, GRE sequence for sodium imaging with reader comparison did not show any sig­ P = .001, R2 = 0.77) and transplants a 7-T whole-body system in patients who

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Figure 2 correction of images (33). However, lim­ ited excitation of a surface coil restricts the field of view in the superficial region of the knee joint, thereby limiting the practical use of this coil to the patella and the wrist (34). For the evaluation of the femoral condyle cartilage layers, a volume coil is necessary. We achieved a mean SNR of 24 in native cartilage and of 16 in transplants within approxi­ mately 30 minutes by using a volume coil. These SNRs of native cartilage are well within the values reported in a Post-contrast T1 [ms] Post-contrast T1 Ratio [-] study (33) in which the investigators used a. b. a surface coil. Figure 2: (a) Plot describes correlation of normalized sodium signal intensities with postcontrast T1 The development of new scaffolds values. Each of 24 points represents values derived from ROIs selected from repaired tissue and native car­ and cell-based therapies for cartilage tilage. Line on a and b represents linear regression of data points. (b) Plot shows relationship between ratios repair are designed to encourage the of normalized sodium signal intensities and ratios of postcontrast T1. Each dot represents the ratio between formation of hyalinelike tissue. Hya­ native cartilage and transplanted areas from the same section in one patient, calculated from normalized line tissue requires a significantly high sodium images and corresponding T1 maps. A high Pearson correlation coefficient of 0.836 was obtained. amount of GAG, which should be pro­ duced by the cells transferred either by classic ACI or seeded on different scaf­ folds in the newer generation of MACT (35-38). In our patient group, sodium imaging allowed differentiation between various normalized sodium signal inten­ sities in repaired tissue and in healthy cartilage, which is indicative of the GAG content. Several histologic follow-up studies performed after cartilage repair surgery have shown that the histologic outcome of repaired tissue can vary from hya­ linelike tissue to mixed fibrous-hyaline to fibrous tissue, with the latter tissue lacking a significant amount of GAG (20,25,35,36). The results of the pres­ ent study, in patients who underwent (R = 0.629, P = .028, R 2 = 0.40). Each dot represents the ratio between native cartilage and transplanted MACT, are comparable and suggest an areas from the same section in one patient, calculated from T1 maps. Line on a and b represents linear overall lower sodium content in repaired regression of data points. (b) Plot of correlation between MOCART scores and normalized sodium ratio tissue compared with that in control (R = 0.813, P = .001, R2 = 0.66). Each dot represents ratio of normalized sodium signal intensities acquired cartilage. This may imply a lower GAG from native cartilage and repaired tissue in one patient. content; however, so far, the correla­ tion between sodium content and GAG concentration has been shown only for have undergone MACT, we achieved sitivity of surface coils, as compared with healthy and trypsin-degraded cartilage an SNR that was sufficiently high to a 17-cm-diameter circular-volume coil, but not for repaired tissue after different visualize repaired tissue in patients af­ a similar SNR (approximately 12) can cartilage-repair surgeries. The different ter MACT of the femoral condyle and be achieved in a shorter image time relaxation times in repaired tissue may trochlear cartilage layer. Reports on (22 versus 30 minutes) with the smaller lead to an overestimation or underesti­ sodium imaging of the knee joint have voxel size (2.75 times smaller) (12,33). mation of the sodium content. so far focused mostly on the patella, It has been previously reported that the Although the reference standard owing to its greater cartilage thick­ advantages of increased sensitivity and in the monitoring of patients after ness and accessibility for surface coils the closer proximity of the surface coil cartilage-repair surgeries is biopsy with (8,11,32). Because of the increased sen­ outweigh the cost of the postprocessing histologic analysis (20,21,27,28,35,36),

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a. b. Figure 4: (a) Conventional proton-density-weighted fast SE image in a 22-year-old man obtained 11 months after MACT with (b) corresponding T1 postcontrast map, (c) T1 postcontrast map with 3331 color-coded femoral cartilage, (d) corresponding . 1 sodium image, and (e) color-coded sodium image. Arrows mark the borders of the transplanted area. Note that, on conventional MR images, an excellent 1 167 filling of the defect and integration of the transplant is visible although the sodium content is low.

0-

• d. e. this standard has obvious practical to be specific for the GAG content in faster 3D sequences were developed limitations. For ethical reasons, only articular cartilage (39-42). Of these and clinically used, the robustness of a very limited amount of cartilage tis­ techniques, only dGEMRIC has been each of these techniques for T1 map­ sue can be harvested during biopsy and clinically applied to patients after car­ ping is still controversial (45,47,48). The because the composition in repaired tilage-repair surgery and has shown dual-flip-angle technique used in our tissue can vary, particularly in larger different results after different cartilage- study has been validated in phantom transplants, the accuracy of this tech­ repair surgeries such as ACI (43), MACT studies and in vivo in comparison with nique is limited. Morphologic MR imag­ (44,45), and MACT as compared with inversion-recovery sequences in other ing has shown no correlation with the microfracture (46). studies (45,47,48). biochemical composition of repaired Problems with dGEMRIC are the We found a strong correlation be­ tissue (38). However, in our study, we need for intravenous contrast agent ad­ tween sodium imaging and dGEMRIC found a strong correlation between the ministration, usually in a double dose, in repaired tissue and native cartilage MOCART scores and the sodium ratio and the time delay between contrast in patients after MACT. ROI evalua­ values. This correlation is based on only agent administration and imaging, since tions were reproducible, with no signifi­ 12 data points and has to be verified sufficient time is necessary for the con­ cant differences and strong correlation in further studies with a larger cohort trast agent to diffuse through the car­ ( R . 0.685) between two independent of patients. In recent years, several MR tilage layer completely (10). Moreover, readers. Sodium imaging has clinically techniques, such as dGEMRIC, sodium T1 mapping that is based on standard demonstrated a correlation with the imaging, T1r imaging, and most recently inversion-recovery techniques is time GAG concentration in native articular chemical exchange saturation transfer, consuming and limited to a few sections cartilage and allows direct quantifica­ or CEST, were developed and proved in a two-dimensional technique. Although tion of GAG concentration (8,9). This

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84 MUSCULOSKELETAL IMAGING: Sodium MR Imaging after Knee Chondrocyte Transplantation Trattnig et al

is explained by the negatively charged tween the dGEMRIC and sodium im­ lar cartilage. Biochim Biophys Acta 1969; side chains of PG, which are counter­ aging examinations. 177(3):492-500. balanced by sodium ions. However, so­ Furthermore, we did not calculate 7. Lesperance LM, Gray ML, Burstein D. dium imaging is technically challenging absolute sodium concentrations. Al­ Determination of fixed charge density in because it requires special hardware though there are publications about ab­ cartilage using nuclear magnetic resonance. with multinuclear capability, special solute quantification of sodium concen­ J Orthop Res 1992;10(1):1-13. coils tuned for sodium resonance, and trations, they assume constant sodium 8. Reddy R, Insko EK, Noyszewski EA, an very high field strength (.3 T) to relaxation times in evaluated cartilage Dandora R, Kneeland JB, Leigh JS. Sodium provide sufficient SNR in a clinically ac­ for the correction of sodium signal in­ MRI of human articular cartilage in vivo. Magn Reson Med 1998;39(5):697-701. ceptable time. The problem with sodium tensity (33,34). is its low sensitivity. The MR sensitivity In summary, with the application of 9. Borthakur A, Mellon E, Niyogi S, Witschey for 23Na is only 9.2% that of hydrogen 7 T and a modified 3D GRE sequence, W, Kneeland JB, Reddy R. Sodium and 1 (1H), and the in vivo 23Na concentra­ sufficiently high SNR for sodium imag­ T1rho MRI for molecular and diagnostic imaging of articular cartilage. NMR Biomed tion is approximately 360 times lower ing of both cartilage transplants and na­ 2006;19(7):781-821. than the in vivo 1H proton concentra­ tive cartilage was achieved. For the first tion, which results in a 23Na signal that time to our knowledge, a strong cor­ 10. Burstein D, Velyvis J, Scott KT, et al. Pro­ tocol issues for delayed Gd(DTPA)(2-)- is approximately 4000 times lower than relation was shown between the GAG­ enhanced MRI (dGEMRIC) for clinical eval­ the 1H signal (49). content sensitive techniques in cartilage uation of articular cartilage. Magn Reson The requirements of a very high-field- transplants and native cartilage with Med 2001;45(1):36-41. strength MR system operating at 7 T two different methods, sodium imaging 11. Insko EK, Kaufman JH, Leigh JS, Reddy R. make it unlikely that sodium imaging at 7 T and dGEMRIC at 3 T, in patients Sodium NMR evaluation of articular carti­ will gain widespread use. Sodium imag­ after MACT. lage degradation. Magn Reson Med 1999; ing at 7 T allows evaluation of the per­ 41(1):30-34. Acknowledgments: The authors thank Vladimir formance of new cell-based cartilage- Mlynarik, PhD, Laboratory of Functional and 12. Borthakur A, Shapiro EM, Beers J, repair surgical techniques without the Metabolic Imaging, Ecole Polytechnique Feder- Kudchodkar S, Kneeland JB, Reddy R. Sensi­ need for contrast agents and helps to ale de Lausanne, Lausanne, Switzerland, for his tivity of MRI to proteoglycan depletion in car­ comments and advice on the sodium imaging. We tilage: comparison of sodium and proton MRI. improve and optimize faster dGEMRIC also thank Ronald Dorotka, MD, Department Osteoarthritis Cartilage 2000;8(4):288-293. methods that can be applied at lower of Orthopedics, Medical University of Vienna, field strengths without special hard­ Vienna, Austria, for his help in recruiting patients. 13. Steadman JR, Rodkey WG, Rodrigo JJ. Mi­ ware. In addition, 7-T MR imaging helps crofracture: surgical technique and rehabili­ to examine the specificity of new tech­ tation to treat chondral defects. Clin Orthop niques, such as diffusion-weighted im­ References Relat Res 2001;(391 suppl):S362-S369. aging, T1r imaging, and, most recently, 1. Mankin HJ. Biochemical and metabolic as­ 14. Steadman JR, Briggs KK, Rodrigo JJ, Kocher gagCEST. pects of osteoarthritis. Orthop Clin North MS, Gill TJ, Rodkey WG. Outcomes of mi­ The limitations of our study were Am 1971;2(1):19-31. crofracture for traumatic chondral defects the small number of patients and the of the knee: average 11-year follow-up. Ar­ 2. Roughley PJ, Lee ER. Cartilage proteogly­ throscopy 2003;19(5):477-484. fact that we did not perform a correla­ cans: structure and potential functions. tion with immunohistochemical quanti­ Microsc Res Tech 1994;28(5):385-397. 15. Steadman JR, Rodkey WG, Briggs KK. fication of the GAG concentration. Microfracture to treat full-thickness chondral 3. Buckwalter JA, Mankin HJ. Articular carti­ defects: surgical technique, rehabilitation, and Another limitation was the variabil­ lage: degeneration and osteoarthritis, repair, outcomes. J Knee Surg 2002;15(3):170-176. ity between dGEMRIC at 3 T and so­ regeneration, and transplantation. Instr 16. Jakob RP, Franz T, Gautier E, Mainil-Varlet dium imaging examinations at 7 T. The Course Lect 1998;47:487-504. P. Autologous osteochondral grafting in the dGEMRIC method used in our study, knee: indication, results, and reflections. and that was based on a dual-flip-angle 4. Lohmander LS. Articular cartilage and os­ teoarthrosis: the role of molecular markers Clin Orthop Relat Res 2002;(401):170-184. technique, is sensitive to B1 inhomoge­ to monitor breakdown, repair and disease. neities, which are even worse at higher 17. Hangody L, Feczko P, Bartha L, Bodo G, Kish J Anat 1994;184(Pt 3):477-492. G. Mosaicplasty for the treatment of articular field strengths. Therefore, we performed defects of the knee and ankle. Clin Orthop dGEMRIC only at 3 T. Another limi­ 5. Grushko G, Schneiderman R, Maroudas A. Some biochemical and biophysical param­ Relat Res 2001;(391 suppl)S328-S336. tation is the relatively long interval eters for the study of the pathogenesis of 18. Brittberg M, Lindahl A, Nilsson A, Ohlsson C, between dGEMRIC and sodium imag­ osteoarthritis: a comparison between the Isaksson O, Peterson L. Treatment of deep ing examinations. From members of processes of ageing and degeneration in hu­ cartilage defects in the knee with autologous our patient cohort, who underwent man hip cartilage. Connect Tissue Res 1989; chondrocyte transplantation. N Engl J Med dGEMRIC examinations, we recruited 19(2-4):149-176. 1994;331(14):889-895. patients who were willing to participate 6. Maroudas A, Muir H, Wingham J. The 19. Petersen L, Brittberg M, Lindahl A. Autolo­ in an additional sodium measurement. correlation of fixed negative charge with gous chondrocyte transplantation of the ankle. This explains the different intervals be­ glycosaminoglycan content of human articu­ Foot Ankle Clin 2003;8(2):291-303.

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20. Peterson L, Brittberg M, Kiviranta I, ation rate in dGEMRIC evaluation of carti­ Detection of changes in articular cartilage Akerlund EL, Lindahl A. Autologous chon­ lage repair tissue. Invest Radiol 2009;44(9): proteoglycan by T(1rho) magnetic resonance drocyte transplantation: biomechanics and 598-602. imaging. J Orthop Res 2005;23(1):102-108. long-term durability. Am J Sports Med 2002; 31. Marlovits S, Singer P, Zeller P, Mandl I, 41. Bashir A, Gray ML, Hartke J, Burstein D. 30(1):2-12. Haller J, Trattnig S. Magnetic resonance ob­ Nondestructive imaging of human cartilage 21. Brittberg M. Autologous chondrocyte trans­ servation of cartilage repair tissue (MOCART) glycosaminoglycan concentration by MRI. plantation. Clin Orthop Relat Res 1999; for the evaluation of autologous chondro­ Magn Reson Med 1999;41(5):857-865. (367 suppl)S147-S155. cyte transplantation: determination of inter­ 42. Ling W, Regatte RR, Navon G, Jerschow A. observer variability and correlation to clini­ Assessment of glycosaminoglycan concentra­ 22. Nehrer S, Breinan HA, Ramappa A, et al. cal outcome after 2 years. Eur J Radiol 2006; tion in vivo by chemical exchange-dependent Chondrocyte-seeded collagen matrices im­ 57(1):16-23. planted in a chondral defect in a canine model. saturation transfer (gagCEST). Proc Natl Biomaterials 1998;19(24):2313-2328. 32. Shapiro EM, Borthakur A, Gougoutas A, Acad Sci U S A 2008;105(7):2266-2270. Reddy R. 23Na MRI accurately measures 43. Gillis A, Bashir A, McKeon B, Scheller A, 23. Dorotka R, Bindreiter U, Macfelda K, fixed charge density in articular cartilage. Gray ML, Burstein D. Magnetic resonance Windberger U, Nehrer S. Marrow stimula­ Magn Reson Med 2002;47(2):284-291. tion and chondrocyte transplantation using imaging of relative glycosaminoglycan dis­ a collagen matrix for cartilage repair. Os­ 33. Wheaton AJ, Borthakur A, Shapiro EM, tribution in patients with autologous chon­ teoarthritis Cartilage 2005;13(8):655-664. et al. Proteoglycan loss in human knee drocyte transplants. Invest Radiol 2001; cartilage: quantitation with sodium MR 36(12):743-748. 24. Campoccia D, Doherty P, Radice M, Brun imaging—feasibility study. Radiology 2004; P, Abatangelo G, Williams DF. Semisyn­ 231(3):900-905. 44. Pinker K, Szomolanyi P, Welsch GC, et al. thetic resorbable materials from hyaluro- Longitudinal evaluation of cartilage com­ 34. Borthakur A, Shapiro EM, Akella SV, nan esterification. Biomaterials 1998;19(23): position of matrix-associated autologous 2101-2127. Gougoutas A, Kneeland JB, Reddy R. Quan­ chondrocyte transplants with 3-T delayed tifying sodium in the human wrist in vivo by gadolinium-enhanced MRI of cartilage. AJR 25. Nehrer S, Domayer S, Dorotka R, Schatz using MR imaging. Radiology 2002;224(2): Am J Roentgenol 2008;191(5):1391-1396. K, Bindreiter U, Kotz R. Three-year clinical 598-602. outcome after chondrocyte transplantation 45. Trattnig S, Marlovits S, Gebetsroither S, 35. Steinwachs M, Kreuz PC. Autologous chon­ using a hyaluronan matrix for cartilage re­ et al. Three-dimensional delayed gadolinium- drocyte implantation in chondral defects pair. Eur J Radiol 2006;57(1):3-8. enhanced MRI of cartilage (dGEMRIC) for in of the knee with a type I/III collagen mem­ vivo evaluation of reparative cartilage after 26. Chu CR, Coutts RD, Yoshioka M, Harwood brane: a prospective study with a 3-year fol­ matrix-associated autologous chondrocyte FL, Monosov AZ, Amiel D. Articular carti­ low-up. Arthroscopy 2007;23(4):381-387. transplantation at 3.0T: preliminary results. lage repair using allogeneic perichondrocyte- 36. Marcacci M, Berruto M, Brocchetta D, J Magn Reson Imaging 2007;26(4):974-982. seeded biodegradable porous polylactic acid et al. Articular cartilage engineering with (PLA): a tissue-engineering study. J Biomed 46. Trattnig S, Mamisch TC, Pinker K, et al. Hyalograft C: 3-year clinical results. Clin Mater Res 1995;29(9):1147-1154. Differentiating normal hyaline cartilage from Orthop Relat Res 2005;(435):96-105. post-surgical repair tissue using fast gradient 27. Grigolo B, Lisignoli G, Piacentini A, et al. 37. Knutsen G, Engebretsen L, Ludvigsen TC, echo imaging in delayed gadolinium-enhanced Evidence for redifferentiation of human et al. Autologous chondrocyte implantation MRI (dGEMRIC) at 3 Tesla. Eur Radiol 2008; chondrocytes grown on a hyaluronan-based compared with microfracture in the knee. 18(6):1251-1259. biomaterial (HYAff 11): molecular, immuno­ A randomized trial. J Bone Joint Surg Am 47. McKenzie CA, Williams A, Prasad PV, Burstein histochemical and ultrastructural analysis. 2004;86-A(3):455-464. Biomaterials 2002;23(4):1187-1195. D. Three-dimensional delayed gadolinium- 38. Tins BJ, McCall IW, Takahashi T, et al. enhanced MRI of cartilage (dGEMRIC) at 28. Behrens P, Bitter T, Kurz B, Russlies M. Autologous chondrocyte implantation in 1.5T and 3.0T. J Magn Reson Imaging 2006; Matrix-associated autologous chondrocyte knee joint: MR imaging and histologic fea­ 24(4):928-933. transplantation/implantation (MACT/MACI): tures at 1-year follow-up. Radiology 2005; 48. Kimelman T, Vu A, Storey P, McKenzie C, 5-year follow-up. Knee 2006;13(3):194-202. 234(2):501-508. Burstein D, Prasad P. Three-dimensional 29. Kakarlapudi TK, Bickerstaff DR. Knee in­ 39. Wheaton AJ, Casey FL, Gougoutas AJ, T1 mapping for dGEMRIC at 3.0 T using stability: isolated and complex. Br J Sports et al. Correlation of T1rho with fixed charge the Look Locker method. Invest Radiol Med 2000;34(5):395-400. density in cartilage. J Magn Reson Imaging 2006;41(2):198-203. 2004;20(3):519-525. 30. Trattnig S, Burstein D, Szomolanyi P, Pinker 49. Regatte RR, Schweitzer ME. Novel contrast K, Welsch GH, Mamisch TC. T1(Gd) gives 40. Wheaton AJ, Dodge GR, Borthakur A, mechanisms at 3 Tesla and 7 Tesla. Semin comparable information as Delta T1 relax- Kneeland JB, Schumacher HR, Reddy R. Musculoskelet Radiol 2008;12(3):266-280.

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86 RESULTS

2.3 Interlude 1

Results from the paper 1 showed that sodium MRI can detect the differences in GAG content between native cartilage and repair tissue of patients after MACT [194], Furthermore, sensitivity of sodium protocol to changes in the cartilage GAG concentration was verified by observing the correlation between dGEMRIC and sodium results. Although these findings are important, the question if sodium imaging is able to distinguish between repair tissues of different biochemical composition is clinically more relevant. Several different cartilage repair techniques has been developed for treatment of chondral defects, such as BMS techniques or more advanced MACT. The goal of all these procedures is to create a repair tissue that has composition, structure, and properties similar to native hyaline cartilage and thus restore joint function and prevent from OA. Since GAGs play a key role in cartilage homeostasis, function and biomechanical properties, noninvasive evaluation of the GAG content can offer unique information on the repair tissue. The goal of all cartilage repair surgery methods is the development of a high GAG content during the repair tissue maturation, which should achieve a similar level compared to healthy hyaline cartilage. Since sodium imaging correlates directly with the GAG concentration, noninvasive longitudinal assessment of GAG content in repair tissue is possible. The aim of paper 2 was therefore to compare sodium results from native cartilage and repair tissue on femoral condyle between patients treated by BMS techniques and patients after MACT [151], For better comparability between cartilage repair techniques, each BMS patient was matched to a MACT patient according to age, defect location and follow-up interval criteria. My contribution to paper 2 was the measurement of BMS and MACT patients, postprocessing and ROI analyses, statistical evaluations, writing and editing the manuscript.

2.4 Paper 2: Evaluation of native hyaline cartilage and repair tissue with 23Na MRI at 7T

Reproduction of article in this thesis was granted by the publisher - Elsevier [151],

87 Osteoarthritis and Cartilage 20 (2012) 837e845

Evaluation of native hyaline cartilage and repair tissue after two cartilage repair surgery techniques with 23Na MR imaging at 7 T: initial experience

S. Zbyn ft*, D. Stelzeneder f, G.H. Welsch f§, L.L. Negrin ||, V. Juras f{, M.E. Mayerhoefer f, P. Szomolanyiy{, W. Bognery, S.E. Domayerz, M. Webery, S. Trattnigy y MR Centre-High field MR, Department of Radiology, Medical University of Vienna/Vienna General Hospital, Vienna, Austria z Department of Orthopedic Surgery, Medical University of Vienna/Vienna General Hospital, Vienna, Austria x Department of Trauma Surgery, University Hospital of Erlangen, Erlangen, Germany | Department of Trauma Surgery, Medical University of Vienna/Vienna General Hospital, Vienna, Austria { Department of Imaging Methods, Institute of Measurement Science, Slovak Academy of Sciences, Bratislava, Slovakia article info summary

Article history: Objective: To compare the sodium normalized mean signal intensity (NMSI) values between patients after Received 19 July 2011 bone marrow stimulation (BMS) and matrix-associated autologous chondrocyte transplantation (MACT) Accepted 24 April 2012 cartilage repair procedures. Methods: Nine BMS and nine MACT patients were included. Each BMS patient was matched with one Keywords: MACT patient according to age [BMS 36.7 ± 10.7 (mean ± standard deviation) years; MACT 36.9 ± 10.0 Sodium MRI years], postoperative interval (BMS 33.5 ± 25.3 months; MACT 33.2 ± 25.7 months), and defect location. 7T All magnetic resonance imaging (MRI) measurements were performed on a 7 T system. Proton images Spoiled gradient echo Knee cartilage served for morphological evaluation of repair tissue using the magnetic resonance observation of Cartilage repair tissue cartilage repair tissue (MOCART) scoring system. Sodium NMSI values in the repair area and morpho­ Repair surgery efficacy logically normal cartilage were calculated. Clinical outcome was assessed right after MRI. Analysis of covariance, t-tests, and Pearson correlation coefficients were evaluated. Results: Sodium NMSI was significantly lower in BMS (P = 0.004) and MACT (P = 0.006) repair tissue, compared to reference cartilage. Sodium NMSI was not different between the reference cartilage in MACT and BMS patients (P = 0.664), however it was significantly higher in MACT than in BMS repair tissue (P=0.028).BetterclinicaloutcomewasobservedinBMSthaninMACTpatients.Therewasnodifference between MOCART scores for MACT and BMS patients (P = 0.915). We did not observe any significant correlation between MOCART score and sodium repair tissue NMSI (r = -0.001; P = 0.996). Conclusions: Our results suggest higher glycosaminoglycan (GAG) content, and therefore, repair tissue of better quality in MACT than in BMS patients. Sodium imaging might be beneficial in non-invasive evaluation of cartilage repair surgery efficacy. © 2012 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Introduction chondrocyte implantation (ACI)6 and matrix-associated autologous chondrocyte transplantation (MACT)7, have been developed. BMS Injuries of articular cartilage are one of the most common types techniques produce bleeding from the subchondral bone to of injuries in orthopedic practice1. The articular cartilage of adults promote clot formation and a subsequent healing cascade forms shows no or minimal potential for self-healing of chondral defects scar tissue that fills the chondral defect. MACT techniques are based that exceed a critical size2 and such defects often progress to on the cultivation of chondrocytes on a polymer scaffold for several osteoarthritis (OA)3. Therefore, various treatment procedures, such weeks, which are then implanted into a chondral defect. The goal of as bone marrow stimulation (BMS) techniques including micro­ cartilage repair procedures is to restore joint function and prevent fracture (MFX)4 and subchondral drilling5, or advanced cell-based OA by providing repair tissue that has structure, composition, and cartilage repair surgery techniques, including autologous biomechanical properties similar to native articular cartilage8. Articular cartilage consists of small amount of chondrocytes * Address correspondence and reprint requests to: S. Zbyn, MR Centre-Highfield MR, embedded in a large extracellular matrix which is created by water Department of Radiology, Medical University of Vienna/Vienna General Hospital, molecules, collagen fibers and proteoglycan macromolecules made Lazarettgasse14,A-1090Vienna,Austria.Tel: 431-40400-6468;Fax: 431-40400-6475. of a protein core with attached glycosaminoglycan (GAG) side E-mail address: [email protected] (S. Zbyn).

1063-4584/$ — see front matter © 2012 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.joca.2012.04.020

88 838 S. Zbyn et al. / Osteoarthritis and Cartilage 20 (2012) 837—845 chains9,10. The negatively charged residues of the sulfate and International Cartilage Repair Society classification. All evaluated carboxyl groups of GAG attract positive counter-ions (such as criteria were assessed preoperatively with radiographs and sodium) and water molecules, and provide strong electrostatic and conventionalMRIofthekneejoint,andverifiedanddocumentedat osmotic forces responsible for the functional and structural prop­ the time of cartilage repair surgery. The mean defect size was erties of cartilage11. Therefore, a lot of effort has been made to 2.5 ± 0.8 cm2 (range, 1.5—3.6 cm2) in BMS and 5.0 ± 1.3 cm2 (range, develop methods for the non-invasive monitoring of the GAG 3.2—7.3 cm2) in MACT patients. content in native cartilage and cartilage repair tissue, such as Both employed BMS techniques, subchondral drilling5, and delayed gadolinium-enhanced magnetic resonance imaging (MRI) MFX4, are marrow stimulating techniques which rely on the same of cartilage (dGEMRIC)12,13 or sodium imaging14,15. biological principles and therefore are producing comparable type Based on the fact that GAG molecules are counterbalanced by of repair tissue. To our best knowledge, no systematic comparison sodium ions, sodium imaging was successfully used for the evalu­ of the cartilage repair outcome between these techniques was done ation of GAG (and hence, proteoglycan) content in the cartilage of yet. The following three-dimensional scaffolds, seeded with healthy humans16—18 and, recently, in patients after MACT19. autologous chondrocytes, were used in MACT treatment: Hyalog- Therefore, the aim of this study was to compare sodium raft C (Fidia Advanced Biopolymers, Abano Terme, Italy); CaReS normalized mean signal intensities (NMSIs) at 7 T, suggestive of (Arthro Kinetics, Esslingen, Germany); or BioCart II (ProChon GAG content, between native cartilage and repair tissue of patients Biotech, Woburn, MA). All MACT and BMS patients followed the after BMS and MACT repair procedures. same established protocols for the rehabilitation after repair surgery on the femoral condyle or trochlea21,22. Materials and methods Clinical outcome Patients The International Knee Documentation Committee (IKDC) This cross-sectional study was approved by the Institutional Subjective Knee Form and the Modified Cincinnati Knee Rating Review Board and written informed consent was obtained from all were used to evaluate clinical outcome after cartilage repair patients prior to enrollment in the study. From our database of surgery. The IKDC Subjective Knee Form measures symptoms, about 135 follow-up patients, 15 BMS patients (six women, nine function, and sports activity in patients with pathologies in the men; mean age, 38.1 ± 14.2 years; age range, 21.4—67.3 years; three knee joint23. Higher scores (maximum 100 points) are denoting subchondral drillings, and 12 MFX patients), and29 MACT patients greater levels of function and lower knee symptoms. (10 women, 19 men; mean age, 35.8 ± 11.8 years; age range, The Modified Cincinnati Knee Rating evaluates limitations in 22.4—60.6 years) agreed to undergo additional sodium MRI at 7 T daily life, ranging from severe limitations to unlimited full func­ between July 2009 and September 2010. From this patient cohort, tionality (maximum 10 points) and gives an overview ofmanaging the best matching MACT partner was assigned to each BMS patient. daily activities24. Only pairs of patients with age difference of less than 3.5 years, postoperative interval difference of less than 3 months and similar MRI defect location were included in this study. Matching criteria passed nine BMS patients [two Pridie drilling, seven MFX patients; All MRI measurements were carried out on a 7 T whole body four women, five men; mean age, 36.7 ± 10.7 (mean ± standard system (Magnetom, Siemens Healthcare, Erlangen, Germany) using deviation) years; age range, 21.4—57.7 years; mean postoperative a 28-channel knee coil (Quality Electrodynamics LLC, Cleveland, interval, 33.5 ± 25.3 months], and nine MACT patients (three OH) and a 23Na-only circularly polarized knee coil (Stark Contrast, women, six men; mean age, 36.9 ± 10.0 years; age range, 24.6—56.0 Erlangen, Germany). Normalization sample containing 308 mmol/L years; mean postoperative interval, 33.2 ± 25.7 months). The NaCl solution was fixed to the sodium coil to serve for normaliza­ difference between matched patients ranged between 0.1 and 3.1 tion of the sodium signal. years for the age and 0—2.8 months for the postoperative interval. For the morphological evaluation of cartilage repair tissue The repair tissue was situated at the medial femoral condyle (five served a proton density-weighted two-dimensional turbo spin BMS and five MACT patients), lateral femoral condyle (four BMS echo (2D-TSE) sequence with fat suppression in the sagittal and and two MACT patients), or the trochlea region (two MACT coronal plane, and a T1-weighted three-dimensional gradient echo patients). The mean body mass index (BMI) was 25.1 ± 2.4 in BMS (3D-GRE) sequence (Table I). The total measurement time for and 23.8 ± 2.4 in MACT patients. morphological imaging was about 13 min. Only patients with single symptomatic full-thickness cartilage All sodium measurements were acquired with an optimized defect caused either by trauma or pre-existing osteochondritis 3D-GRE sequence. To comply with the specific absorption rate dissecans were included in the study. Exclusion criteria were limits and minimize echo time, the excitation pulse length was advanced OA, meniscal tear, knee ligament injuries, metallic 970 ms(Table I). The readout length was 5.88 ms. The total implants, and knee joint instability that were evaluated with measurement time for sodium imaging with the nominal resolu­ standard clinical testing20. Patients with advanced OA were defined tion of 3.11 x 1.55 x 3.0 mm3, including flip angle calibration, was as patients with cartilage defect of grade 3 or 4 according to the less than 34 min.

Table I Parameters of proton and sodium MR sequences used in presented study

Sequence TR/echo time Flip angle Bandwidth Field of view Matrix Resolution Section thickness No. of No. of Acquisition time (TE) (msec) (degrees) (Hz/pixel) (mm2) size (mm2) (ST) (mm) slices averages (TA) (min:sec) Sagittal 2D-TSE 3,400/26 125 243 160 x 129 448 x 360 0.36 x 0.36 3.0 20 1 4:20 Coronal 2D-TSE 2,400/24 122 243 160 x 159 448 x 377 0.36 x 0.42 3.0 15 1 1:21 1H-3D-GRE 8.3/3.6 8 450 160 x 135 384 x 324 0.42 x 0.42 0.45 224 1 6:09 23Na-3D-GRE 10/3.77 56 170 199 x 199 128 x 64 1.55 x 3.11 3.0 48 60 30:45

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Fig. 1. Sagittal proton density-weighted 2D-TSE MR image with fat suppression (left); sagittal sodium 3D-GRE image (middle); and color-coded sagittal sodium 3D-GRE image (right) in a 43-year-old woman obtained 42 months after an MFX procedure. Cartilage repair tissue is situated between the two arrows. Red contours in the middle image represent the ROI analysis of repair tissue (right contour) and reference cartilage (left contour). Please note that repair tissue voxels situated closest to the repair tissue—native cartilage interface are not included into the ROI evaluations. Color scale represents the sodium signal intensity values.

Evaluation of proton MRI ROIs. The reference ROI was defined in morphologically normal­ appearing cartilage that was exposed to similar weight-bearing The proton images served for the morphological evaluation of conditions as the corresponding repair tissue. A minimum cartilage and bone after repair surgery using the magnetic reso­ distance of 8 mm was maintained between the repair and reference nance observation of cartilage repair tissue (MOCART) scoring ROIs. Both, repair and reference ROIs were defined in accordance system25. A senior musculoskeletal radiologist (ST, with 15 years of with the corresponding proton morphological images and the experience) assigned a MOCART score to each repair site of each intraoperative documentation. The sizes of both ROIs were kept as patient. MOCART is a reproducible grading system that has been similar as possible, with a mean area of 24.6 ± 7.6 mm2 (range, applied to different cartilage repair techniques19,25,26. A point score 12.1—38.7 mm2) in the reference ROIs, and a mean area of was subscribed to each variable, with a maximum score of 100 26.1 ± 7.4 mm2 (range, 15.5—38.7 mm2) in the repair ROIs (Figs. 1 points representing excellent morphological outcome of repair and 2). tissue, and 0 representing poor outcome. The signal-to-noise ratio (SNR) was calculated by dividing the mean signal intensity from the reference or repair ROI by the Evaluation of sodium MRI standard deviation of the signal intensity in a ROI defined in the signal-free area of the same image. The NMSI values were calcu­ According to the morphological images and the intraoperative lated by multiplying the mean sodium signal from the reference or documentation, one sodium image containing the largest amount repair ROIs with the normalization factor. This factor is the ratio of of cartilage repair tissue was selected and used for evaluation. All the highest sodium signal in the normalization sample, over all regions-of-interest (ROI) were defined by a senior musculoskeletal patients, to the sodium signal in the normalization sample of the radiologist (ST, with 15 years of experience) in consensus with an patient under consideration. For better comparability of cartilage orthopedic surgeon with a special interest in musculoskeletal MRI repair techniques, we calculated the sodium repair-to-reference (GHW, with 10 years of experience). For ROI analyses, sodium signal intensity ratio, characterized as the ratio between the image was rescaled to resolution of 2D-TSE images and overlaid signal intensity from the repair ROI and the corresponding refer­ with corresponding 2D-TSE image in picture editing tool (Adobe ence ROI. Photoshop CS2, version 9.0). The ROIs defined on 2D-TSE image To evaluate reproducibility of sodium measurements, 20- were transferred to sodium image and evaluated using the JiveX year-old woman after MACT on medial femoral condyle was DICOM Viewer (JiveX 4.3, VISUS Technology Transfer GmbH, measured twice. When comparing the NMSI values between Bochum, Germany). To avoid partial volume artifacts between the measurements, the mean coefficient of variation was 2.25% repair tissue and surrounding native cartilage, the border region (range 0.32—3.81%) and the mean difference 3.12% (range between repair and native tissue was excluded from the repair 0.46—5.25%).

Fig. 2. Sagittal proton density-weighted 2D-TSE MR image with fat suppression (left); sagittal, sodium 3D-GRE image (middle); and color-coded sagittal sodium 3D-GRE image (right) in a 35-year-old woman obtained 50.6 months after MACT surgery. Cartilage repair tissue is situated between the two arrows. Red contours in the middle image represent the ROI analysis of repair tissue (left contour) and reference cartilage (right contour). Please note that repair tissue voxels situated closest to the repair tissue-native cartilage interface are not included into the ROI evaluations. Color scale represents the sodium signal intensity values.

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Statistical analyses NMSI of 270 ± 36 (range, 227—322) in reference cartilage and 210 ± 36 (range, 165—277) in repair tissue (Fig. 3). The mean To compare the sodium NMSI between the repair and the sodium NMSI values from repair tissue were lower compared to reference regions, by taking different type of surgery, as well as age corresponding values from reference cartilage for each patient and and follow-up interval into account, an analysis of covariance was for both types of treatment techniques (Fig. 4). In each matched performed by using a two-way repeated measures analysis of pair of patients, the sodium repair-to-reference signal intensity covariance (ANCOVA) with four covariates (surgery, age, follow-up ratio was higher in patients after MACT, compared to BMS patients interval and BMI), and one within-subject effect (tissue type). All (Fig. 5). post hoc NMSI comparisons were achieved with a two-tailed paired A two-way repeated measures ANCOVA did not reveal signifi­ t-test and were corrected for multiple test errors according to cant influence of surgery (P = 0.124), age (P = 0.095), follow-up Bonferroni—Holm. A Pearson correlation coefficient (r) was deter­ interval (P = 0.059), BMI (P = 0.248) or tissue type (P = 0.117) on mined to evaluate the associations between results of sodium sodium NMSI values. However, the ANCOVA showed statistically imaging and MOCART scoring, and between sodium imaging and significant influence of surgery type (P = 0.026), but no significant patient's age and postoperative interval. The strength of association influence of age (P = 0.201), follow-up interval (P = 0.294) or BMI was classified as none correlation (r < 0.3), low (r = 0.3—0.5), (P = 0.624) on the difference in NMSI values between reference moderate (r = 0.5—0.7), or strong (r > 0.7). All statistical evalua­ cartilage and repair tissue. A post hoc paired samples t-test revealed tions were performed using the SPSS software (SPSS 15.0 for significantly lower sodium NMSI in repair tissue compared to cor­ Windows; SPSS Inc., Chicago, IL) and a P-value equal to or less than responding reference cartilage in the BMS patients (P = 0.004), as 0.05 was considered statistically significant. well as in the MACT subjects (P = 0.006) (Fig. 3). There was no significant difference in the sodium NMSI of reference cartilage when comparing patients after MACT and BMS treatment with Results paired t-test (P = 0.664) (Fig. 3). Significantly higher sodium NMSI was observed in repair tissue after the MACT procedure, compared Sodium MRI results to the BMS techniques (P = 0.028) using a paired t-test (Fig. 3). Similarly, a paired t-test showed significantly higher sodium repair- The mean sodium SNR value in the BMS patients was 20.6 ± 4.8 to-reference signal intensity ratio in MACT patients when (range, 13.2—26.1) in reference cartilage and 12.0 ± 2.5 (range, compared to BMS patients (P = 0.003). 7.3—15.1) in repair tissue. The patients after the MACT procedure showed mean sodium SNR of 21.8 ± 3.0 (range, 19.4—28.2) in Clinical outcome and morphological evaluations reference cartilage and 17.0 ± 3.2 (range, 11.3—23.7) in repair tissue. The mean sodium NMSI value was 279 ± 47 (range, 205—336) in The mean IKDC Subjective Knee Form score was 78.8 [standard reference cartilage and 164 ± 31 (range, 127—216) in repair tissue of errorofthemean(SE)=11.2;95%confidenceinterval(CI)=47.7to BMS patients (Fig. 3). The MACT patients revealed mean sodium 109.9] for five BMS and 64.1 (SE = 9.6; CI = 37.3 to 90.9) for five MACT patients. The mean Cincinnati Knee Rating was 8.0 (SE = 0.7; CI =6.0to10.0) forfiveBMSand 6.0(SE=1.2;CI=2.7 to9.3)for five MACT patients. No statistically significant difference was observed between BMS and MACT patients when comparing the results of IKDC Subjective Knee Form (P = 0.408) or the Modified Cincinnati Knee Rating (P = 0.275) using paired t-test. However, BMS patients had in average 14.7 points more (CI = -29.5 to 58.9) in IKDC Subjective Knee Form scoring and two points more (CI = -2.4 to 6.4) in Cincinnati Knee Rating (Fig. 6). These differ­ ences were considered as clinically relevant. No significant differ­ ence in BMI values was observed between BMS and MACT patients using paired t-test (P = 0.076). The mean MOCART score was 75.0 ± 16.6 points (range, 50—100 points) in BMS and 73.9 ± 16.7 points (range, 55—95 points) in MACT patients. A paired t-test did not show a significant difference betweentheMOCARTscoresofMACTandBMSpatients(P=0.915).

Linear associations

There was no significant correlation between clinical outcome scores and sodium NMSI in repair tissue (IKDC scoring: r = -0.382; P = 0.276; CI = -0.815 to 0.326; Cincinnati rating: r = -0.521; P = 0.123; CI = -0.866 to 0.162). Medium association was found between clinical outcome scores and sodium repair-to-reference Fig. 3. Graph of mean sodium NMSI values from reference cartilage (middle) and from signal intensity ratio (IKDC scoring: r = -0.502; P = 0.139; repair tissue produced by BMS (left) and MACT (right) techniques. Note the significant decrease in mean sodium NMSI of repair tissue after BMS, as well as after MACT CI = -0.860 to 0.187; Cincinnati rating: r = -0.549; P = 0.100; compared to corresponding values from reference cartilage. Although there was no CI = -0.876 to 0.123) [Fig. 7(a and b)]. No linear association significant difference in the sodium NMSI of reference cartilage between patients after between the MOCART score and sodium NMSI from repair tissue MACT and BMS treatment using independent samples t-test (P = 0.662), significantly (r = -0.001; P = 0.996; CI = -0.468 to 0.466), or between the higher sodium NMSI was observed in repair tissue after a MACT procedure compared MOCART score and the sodium repair-to-reference signal intensity to the BMS techniques with independent samples t-test. Error bars stand for 95% CIs, * represent the signi ficant difference with P = 0.010, ** stands for P = 0.002, *** are ratio (r = 0.232; P = 0.354; CI = -0.263 to 0.631) was observed assigned to P < 0.001, and n represents the number of independent observations. [Fig. 7(c)]. Low correlation was observed between sodium NMSI in

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Fig. 4. Graphs comparing the mean sodium NMSI values between the reference cartilage and the repair tissue of patients after BMS (a) and MACT (b) repair techniques. Each pair of bars represents one patient. The patients are ordered according to the follow-up interval between surgery and sodium MR imaging plotted on the x-axis. Note the matching follow­ up interval between corresponding BMS and MACT patients.

repair tissue and postoperative follow-up interval (r = -0.359; Relatively simple and low-cost BMS techniques are limited to P = 0.143; CI = -0.707 to 0.130), and no association was observed the treatment of smaller, isolated chondral defects (1 —3cm2)2 . between sodium repair-to-reference signal intensity ratio and More sophisticated cartilage repair surgery techniques, such as postoperative follow-up interval (r = -0.096; P = 0.704; MACT, allow treatment of larger (up to 8 cm2) defects28.MACT CI = -0.539 to 0.388) [Fig. 7(d)]. relies on three-dimensional biodegradable scaffolds that produce hyaline-like repair tissue7,29,30. Conversely, BMS techniques fill the Discussion defect mostly with fibrocartilaginous repair tissue, which lacks the structural, biomechanical, and biochemical properties of native hyaline cartilage4,22,31,32. The GAG content comprises 3—10% ofthe The aim of this study was to compare the sodium NMSI values from native cartilage and repair tissue between the patients after extracellular matrix in native cartilage9 and provides cartilage with MACT and BMS cartilage repair procedures. Significantly lower functional and structural properties11. Under ideal conditions, sodium NMSI values in repair tissue than in native cartilage found repair tissue produced by the cartilage repair techniques should, in MACT and BMS patients indicate lower GAG content in repair over time, develop and maintain GAG content similar to hyaline tissue than in native cartilage. Higher sodium NMSI values cartilage. Despite the mentioned advantages of MACT, its role as an observed in MACT patients suggest higher GAG content, and alternative to BMS techniques is not thoroughly defined yet33. therefore, repair tissue of better quality than the repair tissue after Therefore, the ability to track changes in native cartilage and repair BMS techniques. However, higher sodium NMSI values in repair tissue non-invasively is crucial for understanding of the impact of tissuedidnotresultinbetterclinicaloutcomeoftheMACTpatients therapeutic procedures. in this study. Using sodium MRI at 7 T in patients after different cartilage repair surgeries, we found significantly lower sodium NMSI in repair tissue after BMS and MACT treatment compared to corre­ sponding reference native cartilage. Moreover, MACT repair tissue demonstrated significantly higher sodium NMSI than repair tissue after BMS. Sodium MRI has been validated as a quantitative method for the calculation of GAG (and hence, proteoglycan) concentration for healthy17,34 and trypsin-degraded cartilage15,35, but not for repair tissue after different cartilage repair surgeries. On the other hand, dGEMRIC has been validated as a method for measuring the GAG concentration in native cartilage and repair tissue36,37. Recently, Trattnig et al. demonstrated a strong correlation of sodium imaging with dGEMRIC in native and repair tissue19. Thus, it seems reasonable to assume that sodium imaging is indicative of GAG content, both, in native cartilage and in repair tissue. Our findings may indicate higher GAG content in MACT repair tissue compared to repair tissue after BMS techniques. Furthermore, the presented data suggest a lower GAG content in repair tissue after both techniques when compared to the native reference cartilage. Similarly, significantly lower sodium NMSI in MACT repair tissue compared to reference cartilage was reported previously19. Fig. 5. Bar plot comparing the sodium repair-to-reference signal intensity ratios To our knowledge, there has been no prior sodium imaging between the matched pair of patients after BMS and MACT repair surgeries. Each pair study comparing the patients after different cartilage repair of bars in the plot represents the comparison between two matched patients with procedures. However, prior dGEMRIC and histological studies of different types of repair surgery. Sodium repair-to-reference signal intensity ratios are fi ordered according to the mean follow-up interval between surgery and sodium MR cartilage repair can be compared with our ndings. Varying results imaging of matched patients. Note that all repair-to-reference signal intensity ratios of from previous histological studies might be explained by the MACT patients are higher compared to BMS patients in each matched pair of patients. limited size of tissue harvested from the whole repair tissue area,

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Type of Repair Surgery Type of Repair Surgery

Fig. 6. Plots represent comparison of the IKDC Subjective Knee form scores (left) and the Modified Cincinnati Knee Rating (right) between BMS and MACT patients. Each line represents one pair of matched patients. The patients after BMS showed better clinical results in four cases, whereas only one patient after MACT had better clinical outcome than corresponding BMS patient. which might not be of homogeneous histological quality. Although MACT repair tissue, over the 1-year period after repair surgery51,52. Knutsen et al. did not observe a significant difference between A larger longitudinal study would be necessary to adequately histological evaluation of repair tissue produced by MFX and ACI, evaluate GAG changes after repair surgery. hyaline-like repair tissue was observed more frequently in ACI Clinical applicability of sodium imaging is limited because it patients but without significant differences in the clinical outcome requires special hardware with multinuclear capability, sodium when compared to MFX patients33. Conversely, the repair tissue coils and preferably very high field strength (>3 T) to provide after ACI, using characterized cell therapy, demonstrated statisti­ sufficient SNR in a clinically acceptable time. The last issue may be cally significant improvement of structural quality36, and resulted overcome by employing projection imaging or 3D cones techniques in a statistically significant improvement of clinical outcome when which may allow to transfer sodium MRI from 7 T to clinical 3T compared to MFX37. Similarly, Gudas et al. reported only fibro- systems53. Other available GAG-sensitive techniques, such as cartilage in biopsies from repair tissue after MFX38, and other dGEMRIC, T1 r and GAG chemical exchange saturation transfer studies showed 27—75% of biopsies with hyaline-like cartilage in (gagCEST), have also certain limitations. Major limitation of tissue after MACT39—41. Since fibrocartilage demonstrates a lower dGEMRIC is the need of intravenous contrast agent and the time GAGcontentcomparedtonativehyalinecartilage42,43,theresultsof delay between its administration and the MRI12. Although T1 r is our study correspond well with this data. In contrast to studies affected by GAG concentration, it is also influenced by other reported above, we observed better clinical outcome in BMS than in relaxation mechanisms, especially by dipolar one54,55. The gagCEST MACT patients. Thus, higher sodium NMSI values in repair tissue, is sensitive to patient motion and static magnetic field inhomoge­ suggestive of higher GAG content, did not result in better clinical neities which require postprocessing corrections56. Sodium outcome of the MACT patients in this study. Although GAGs imaging can be seen as reference standard for GAG content strongly influence the functional properties of cartilage, it seems assessment and can help evaluate specificity of new GAG-sensitive there is no direct relationship between the GAG content and the techniques56. patient's clinical outcome44. Although measured NMSI values are proportional to GAG The dGEMRIC technique12 has been shown to be sensitive to the content,onlyabsolutequantificationofsodiumcontentcanprovide GAG content of cartilage45—47. Previous dGEMRIC studies reported information about the GAG concentration in native cartilage and a significant difference between reference cartilage and repair repair tissue. For this calculation, it is necessary to correct sodium tissue in MACT patients at different follow-up periods48,49. Similar signal for longitudinal relaxation (T1) and for short (T2f) and long to the presented results, dGEMRIC was able to differentiate (T2s) components of the biexponential transverse relaxation decay between different types of cartilage repair tissues, and found lower and to use a calibration curve for assigning the signal intensity to GAG content in repair tissue after MFX compared to MACT13. a sodium concentration34. We found no linear association between the morphological Mainly due to short biexponential transversal relaxation times MOCART score and biochemical sodium imaging. Similarly, Tins and much lower concentration of sodium than proton nuclei in the et al. found no correlation between MRI parameters and graft articular cartilage, sodium images are in general acquired with low histological appearance50. resolution in order to achieve sufficiently high SNR. Consequently, In presented study, we found no or only low linear association ROI evaluations of sodium images are prone to partial volume between the follow-up interval and sodium values from BMS and errors, which could result in overestimation of sodium signal in MACT repairtissue. Similarly, no significant difference was found in repair tissue. This possible source of error was overcome by a dGEMRIC study that compared two groups of MACT patients; excluding repair voxels situated on the interface between repaired 3—13 months and 19—42 months after repair surgery49. However, tissue and native cartilage from the repair ROIs. Also voxels con­ histological and clinical data suggest an increase in GAG content of taining both, cartilage and synovial fluid could result in

93 S. Zbyn et al. / Osteoarthritis and Cartilage 20 (2012) 837—845 843

Cincinnati Knee Rating Scores [a.u.]

c

Fig. 7. (a) As the plot demonstrates, medium association was observed between the IKDC Subjective Knee Form score and the sodium repair-to-reference signal intensity ratio of BMS and MACT patients (r = -0.502; P = 0.139; CI = -0.860 to 0.187). (b) Similarly, medium association was observed between the Cincinnati Knee Rating and the sodium repair- to-reference signal intensity ratio (r = -0.549; P = 0.100; CI = -0.876 to 0.123). (c): Plot represents the association between the MOCART score and sodium repair-to-reference ratio of BMS and MACT patients. Low association was observed (r = 0.232; P = 0.354; CI = -0.263 to 0.631). (d): Plot depicts relationship between sodium NMSI values from repair tissue and the follow-up interval between repair surgery and MR imaging. Low correlation was observed (r = -0.359; P = 0.143; CI = -0.707 to 0.130). Lines on plots (a—d) represent the linear regression of data points obtained from patients after BMS (blue dots) and MACT (red dots) procedures.

overestimated sodium signal. The contribution of synovial fluid to different follow-up intervals and the lack of direct histological sodium signal was minimized using repetition time (TR) of 10 ms, evaluation of GAG content in repair tissue. Due to ethical guide­ which resulted in heavily T1-weighted images. Another limitation lines, histological samples can be taken only if the patient has pain of presented study is unknown sodium relaxation times in the in the operated knee or if there is a new trauma. Since this is very repair tissue. Time demanding relaxation measurements were not rare, it is very difficult to obtain histological evaluation of GAG added to protocol and sodium relaxations times in repair tissue content in repair tissue. have not yet been published. An estimate of changes in relaxation With the assumption that histological evaluations of biopsies times between repair tissue and native cartilage may provide from repair tissue reveal more fibrocartilage after BMS tech- in vitro relaxation study57. In this study, GAG depletion resulted in niques33,38, and more hyaline-like tissue after MACT39—41, our prolongation of T1 and T2s times and shortening of T2f time. This results may indicate that sodium MRI is able to distinguish not only change could decrease presented NMSI values in addition to their between native hyaline cartilage and repair tissue, but also decrease due to GAG loss. When correcting repair NMSI for the between the different qualities ofrepair tissue after MACTand BMS decreasecausedbytherelaxationtimeschange,weobservedagain techniques. As our results suggest, the MACT treatment provides significantly higher sodium in the MACT repair tissue than in the higher GAG content, and therefore, repair tissue of better quality BMS repair tissue (P = 0.043) using a paired t-test. On the other compared to the BMStechniques, and that sodium MRI at 7 Tmight hand, changes in relaxation times make presented T1-weighted be beneficial in the non-invasive evaluation of cartilage repair sodium imaging more sensitive to small changes in GAG surgery efficacy. content35 and therefore more attractive for clinical practice. Another limitation of our preliminary study is the low number of Author contributions patients. Only nine pairs were satisfactorily matched for age, follow-up interval, and defect location and clinical evaluations The authors declare the following contributions to the prepa­ were available only from five pairs. Further limitations are the ration of the manuscript: Study conception and design (Welsch,

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Mayerhoefer, Szomolanyi, Bogner, Trattnig); collection and 13. Trattnig S, Mamisch TC, Pinker K, Domayer S, Szomolanyi P, assembly of data (Zbyn, Negrin, Domayer); analysis and interpre­ Marlovits S, et al. Differentiating normal hyaline cartilage from tation of data (Zbyn, Stelzeneder, Juras, Weber, Trattnig); drafting of post-surgical repair tissue using fast gradient echo imaging in the manuscript (Zbyn, Stelzeneder, Trattnig); critical revision of the delayed gadolinium-enhanced MRI (dGEMRIC) at 3 Tesla. Eur manuscript for important intellectual content (Welsch, Negrin, Radiol 2008;18:1251—9. Juras, Mayerhoefer, Szomolanyi, Bogner, Domayer, Weber); final 14. Lesperance LM, Gray ML, Burstein D. Determination of fixed approval of the article (Zbyn, Stelzeneder, Welsch, Negrin, Juras, charge density in cartilage using nuclear magnetic resonance. Mayerhoefer, Szomolanyi, Bogner, Domayer, Weber, Trattnig); J Orthop Res 1992;10:1—13. obtaining of funding (Bogner, Domayer, Trattnig). All authors take 15. Borthakur A, Shapiro EM, Beers J, Kudchodkar S, Kneeland JB, responsibility for the integrity of the work. Reddy R. Sensitivity of MRI to proteoglycan depletion in cartilage: comparison of sodium and proton MRI. Osteoar­ Conflict of interest thritis Cartilage 2000;8:288—93. None of the authors have any conflict of interest relating to the 16. Reddy R, Insko EK, Noyszewski EA, Dandora R, Kneeland JB, submitted manuscript. Leigh JS. Sodium MRI of human articular cartilage in vivo. Magn Reson Med 1998;39:697—701. 17. Borthakur A, Shapiro EM, Akella SV, Gougoutas A, Kneeland JB, Acknowledgments Reddy R. Quantifying sodium in the human wrist in vivo by using MR imaging. Radiology 2002;224:598—602. Funding for this study was provided by the Vienna Spots of 18. Borthakur A, Mellon E, Niyogi S, Witschey W, Kneeland JB, Excellence des Wiener Wissenschafts-und Technologie-Fonds Reddy R. Sodium and T1rho MRI for molecular and diagnostic (WWTF) — Vienna Advanced Imaging Center — VIACLIC, OeNB imaging of articular cartilage. NMR Biomed 2006;19:781 —821. Jubilaeumsfond #13834, OeNB Jubilaeumsfond #13209 and Slovak 19. Trattnig S, Welsch GH, Juras V, Szomolanyi P, Mayerhoefer ME, Scientific Grant Agency VEGA 2/0090/11. The study sponsors did Stelzeneder D, et al. 23Na MR imaging at 7 T after knee matrix- not have a role in study design, the collection and analysis of data, associated autologous chondrocyte transplantation prelimi­ interpretation of results and decision to publish. nary results. Radiology 2010;257:175—84. 20. Kakarlapudi TK, Bickerstaff DR. Knee instability: isolated and References complex. Br J Sports Med 2000;34:395—400. 21. Hambly K, Bobic V, Wondrasch B, Van Assche D, Marlovits S. 1. Curl WW, Krome J, Gordon ES, Rushing J, Smith BP, Autologous chondrocyte implantation postoperative care and Poehling GG. Cartilage injuries: a review of 31,516 knee rehabilitation: science and practice. Am J Sports Med arthroscopies. Arthroscopy 1997;13:456—60. 2006;34:1020—38. 2. Mankin HJ. The response of articular cartilage to mechanical 22. Steadman JR, Rodkey WG, Briggs KK. Microfracture to treat injury. J Bone Joint Surg Am 1982;64:460—6. full-thickness chondral defects: surgical technique, rehabili­ 3. Buckwalter JA, Mankin HJ. Articular cartilage: degeneration tation, and outcomes. J Knee Surg 2002;15:170—6. and osteoarthritis, repair, regeneration, and transplantation. 23. Irrgang JJ, Anderson AF, Boland AL, Harner CD, Kurosaka M, Instr Course Lect 1998;47:487—504. Neyret P, et al. Development and validation of the interna­ 4. Steadman JR, Rodkey WG, Rodrigo JJ. Microfracture: surgical tional knee documentation committee subjective knee form. technique and rehabilitation to treat chondral defects. Clin Am J Sports Med 2001;29:600—13. Orthop Relat Res 2001;(391 Suppl):S362—9. 24. Noyes FR, Barber SD, Mooar LA. A rationale for assessing sports 5. Pridie KH. A method of resurfacing osteoarthritic knee joints. activity levels and limitations in knee disorders. Clin Orthop J Bone Joint Surg Br 1959;41B:618—9. Relat Res 1989:238—49. 6. Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, 25. Marlovits S, Singer P, Zeller P, Mandl I, Haller J, Trattnig S. Peterson L. Treatment of deep cartilage defects in the knee Magnetic resonance observation of cartilage repair tissue with autologous chondrocyte transplantation. N Engl J Med (MOCART) for the evaluation of autologous chondrocyte 1994;331:889—95. transplantation: determination ofinterobserver variabilityand 7. Grigolo B, Lisignoli G, Piacentini A, Fiorini M, Gobbi P, correlation to clinical outcome after 2 years. Eur J Radiol Mazzotti G, et al. Evidence for redifferentiation of human 2006;57:16—23. chondrocytes grown on a hyaluronan-based biomaterial 26. Trattnig S, Millington SA, Szomolanyi P, Marlovits S. MR (HYAff 11): molecular, immunohistochemical and ultrastruc­ imaging of osteochondral grafts and autologous chondrocyte tural analysis. Biomaterials 2002;23:1187—95. implantation. Eur Radiol 2007;17:103—18. 8. Buckwalter J, Mow V. Cartilage Repair in Osteoarthritis. Phil­ 27. Steinwachs MR, Guggi T, Kreuz PC. Marrow stimulation tech­ adelphia, PA: WB Saunders Co; 1992. niques. Injury 2008;39(Suppl 1):S26—31. 9. Mankin HJ, Mow VC, Buckwalter JA. Form and function of 28. Nehrer S, Minas T. Treatment of articular cartilage defects. articular cartilage. In: Simon SR, Ed. Orthopedic Basic Sciences. Invest Radiol 2000;35:639—46. Philadelphia, PA: American Academy of Orthopedic Surgeons; 29. Marlovits S, Zeller P, Singer P, Resinger C, Vecsei V. Cartilage 1994:3—44. repair: generations of autologous chondrocyte transplantation. 10. Roughley PJ, Lee ER. Cartilage proteoglycans: structure and Eur J Radiol 2006;57:24—31. potential functions. Microsc Res Tech 1994;28:385—97. 30. Marcacci M, Berruto M, Brocchetta D, Delcogliano A, 11. Maroudas AI. Balance between swelling pressure and collagen Ghinelli D, Gobbi A, et al. Articular cartilage engineering with tension in normal and degenerate cartilage. Nature Hyalograft (R) C — 3-year clinical results. Clin Orthop Relat Res 1976;260:808—9. 2005:96—105. 12. Burstein D, Velyvis J, Scott KT, Stock KW, Kim YJ, Jaramillo D, 31. Steadman JR, Briggs KK, Rodrigo JJ, Kocher MS, Gill TJ, et al. Protocol issues for delayed Gd(DTPA)(2-)-enhanced MRI: Rodkey WG. Outcomes of microfracture for traumatic chondral (dGEMRIC) for clinical evaluation of articular cartilage. Magn defects of the knee: average 11-year follow-up. Arthroscopy Reson Med 2001;45:36—41. 2003;19:477—84.

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32. Steadman JR, Miller BS, Karas SG, Schlegel TF, Briggs KK, proteoglycans with Gd-DTPA-enhanced T1 imaging. Magn Hawkins RJ. The microfracture technique in the treatment of Reson Med 2002;48:640—8. full-thickness chondral lesions of the knee in National Football 46. Burstein D, Gray ML. Is MRI fulfilling its promise for molecular League players. J Knee Surg 2003;16:83—6. imaging of cartilage in arthritis? Osteoarthritis Cartilage 33. Knutsen G, Engebretsen L, Ludvigsen TC, Drogset JO, 2006;14:1087—90. Grontvedt T, Solheim E, et al. Autologous chondrocyte 47. Watanabe A, Wada Y, Obata T, Ueda T, Tamura M, Ikehira H, implantation compared with microfracture in the knee — et al. Delayed gadolinium-enhanced MR to determine glycos- a randomized trial. J Bone Joint Surg Am 2004;86A:455—64. aminoglycan concentration in reparative cartilage after 34. Shapiro EM, Borthakur A, Gougoutas A, Reddy R. 23Na MRI autologous chondrocyte implantation: preliminary results. accurately measures fixed charge density in articular cartilage. Radiology 2006;239:201—8. Magn Reson Med 2002;47:284—91. 48. Pinker K, Szomolanyi P, Welsch GC, Mamisch TC, Marlovits S, 35. Insko EK, Kaufman JH, Leigh JS, Reddy R. Sodium NMR evalu­ Stadlbauer A, et al. Longitudinal evaluation of cartilage ation of articular cartilage degradation. Magn Reson Med composition of matrix-associated autologous chondrocyte 1999;41:30—4. transplants with 3-T delayed gadolinium-enhanced MRI of 36. Saris DB, Vanlauwe J, Victor J, Haspl M, Bohnsack M, Fortems Y, cartilage. AJR Am J Roentgenol 2008;191:1391 —6. et al. Characterized chondrocyte implantation results in better 49. Trattnig S, Marlovits S, Gebetsroither S, Szomolanyi P, structural repair when treating symptomatic cartilage defects Welsch GH, Salomonowitz E, et al. Three-dimensional delayed of the knee in a randomized controlled trial versus micro­ gadolinium-enhanced MRI of cartilage (dGEMRIC) for in vivo fracture. Am J Sports Med 2008;36:235—46. evaluation of reparative cartilage after matrix-associated 37. Saris DB, Vanlauwe J, Victor J, Almqvist KF, Verdonk R, autologous chondrocyte transplantation at 3.0 T: preliminary Bellemans J, et al. Treatment of symptomatic cartilage results. J Magn Reson Imaging 2007;26:974—82. defects of the knee: characterized chondrocyte implantation 50. Tins BJ, McCall IW, Takahashi T, Cassar-Pullicino V, Roberts S, results in better clinical outcome at 36 months in Ashton B, et al. Autologous chondrocyte implantation in knee a randomized trial compared to microfracture. Am J Sports joint: MR imaging and histologic features at 1-year follow-up. Med 2009;37(Suppl 1):10S—9S. Radiology 2005;234:501—8. 38. Gudas R, Stankevicius E, Monastyreckiene E, Pranys D, 51. Peterson L, Brittberg M, Kiviranta I, Akerlund EL, Lindahl A. Kalesinskas RJ. Osteochondral autologous transplantation Autologous chondrocyte transplantation — biomechanics and versus microfracture for the treatment of articular cartilage long-term durability. Am J Sports Med 2002;30:2—12. defects in the knee joint in athletes. Knee Surg Sports Trau- 52. Henderson IJP, Tuy B, Connell D, Oakes B, Hettwer WH. matol Arthrosc 2006;14:834—42. Prospective clinical study of autologous chondrocyte implan­ 39. Bartlett W, Skinner JA, Gooding CR, Carrington RW, tation and correlation with MRI at three and 12 months. J Bone Flanagan AM, Briggs TW, et al. Autologous chondrocyte Joint Surg Br 2003;85B:1060—6. implantation versus matrix-induced autologous chondrocyte 53. Staroswiecki E, Bangerter NK, Gurney PT, Grafendorfer T, implantation for osteochondral defects of the knee: a prospec­ Gold GE, Hargreaves BA. In vivo sodium imaging of human tive, randomised study. J Bone Joint Surg Br 2005;87:640—5. patellar cartilage with a 3D cones sequence at 3 T and 7 T. 40. D'Anchise R, Manta N, Prospero E, Bevilacqua C, Gigante M. J Magn Reson Imaging 2010;32:446—51. Autologous implantation of chondrocytes on a solid collagen 54. Mlynarik V, Szomolanyi P, Toffanin R, Vittur F, Trattnig S. scaffold: clinical and histological outcomes after 2 years of Transverse relaxation mechanisms in articular cartilage. follow-up. J Orthop Traumatol 2005;6:36—43. J Magn Reson 2004;169:300—7. 41. Zheng MH, Willers C, Kirilak L, Yates P, Xu J, Wood D, et al. 55. Keenan KE, Besier TF, Pauly JM, Han E, Rosenberg J, Smith RL, Matrix-induced autologous chondrocyte implantation (MACI): et al. Prediction of glycosaminoglycan content in human biological and histological assessment. Tissue Eng cartilage by age, T1rho and T2 MRI. Osteoarthritis Cartilage 2007;13:737—46. 2011;19:171—9. 42. Minas T, Nehrer S. Current concepts in the treatment of 56. Schmitt B, Zbyn S, Stelzeneder D, Jellus V, Paul D, Lauer L, et al. articular cartilage defects. Orthopedics 1997;20:525—38. Cartilage quality assessment by using glycosaminoglycan 43. Ghivizzani SC, Oligino TJ, Robbins PD, Evans CH. Cartilage injury chemical exchange saturation transfer and (23)Na MR imaging and repair. Phys Med Rehabil Clin N Am 2000;11:289—307. at 7 T. Radiology 2011;260:257—64. 44. Gray ML. Toward imaging biomarkers for glycosaminoglycans. 57. Borthakur A, Shapiro EM, Beers J, Kudchodkar S, Kneeland JB, J Bone Joint Surg Am 2009;91(Suppl 1):44—9. Reddy R. Effect of IL-1beta-induced macromolecular depletion 45. Nieminen MT, Rieppo J, Silvennoinen J, Toyras J, Hakumaki JM, on residual quadrupolar interaction in articular cartilage. Hyttinen MM, et al. Spatial assessment of articular cartilage J Magn Reson Imaging 2002;15:315—23.

96 RESULTS

2.5 Interlude 2

The findings revealed in paper 2 showed that the sensitivity of sodium MRI is sufficient for differentiating between repair tissue produced by MACT and BMS techniques [151], Our results indicate that the MACT procedure provides a repair tissue with higher GAG content, thus with better biomechanical properties, than the BMS treatment. This finding is underlined by the fact that in each matched pair of BMS and MACT patients, the subject after MACT had higher sodium values than the BMS patient. Therefore, sodium MRI at 7 T seems to be a sensitive method for the noninvasive evaluation of the efficacy of different cartilage repair techniques and for the follow-up of repair tissue. The same cartilage repair procedures used in knee joints can also be applied for treating defects of the ankle joint. Although our sodium results from paper 2 indicate that MACT provide repair tissue with better biomechanical properties than MFX in knee joint, it might not be true also for cartilage in ankle. There are various biochemical, biomechanical, and metabolic differences between cartilage in knee and in anklejoint, for example, a greater repair capacity of ankle cartilage than knee cartilage [115], Moreover, sodium MRI of ankle joint have not been published before. Therefore, the aims of paper 3 were: a) to validate sodium imaging methodology by comparing histochemical analysis of GAG content with MRI results in cadaver ankle samples; b) to assess sodium values in ankle cartilage of volunteers; c) to compare sodium values in repairtissue ofthe MFX and MACT patients [195], My contribution to paper 3 includes of MRI measurements of ankle samples, volunteers and patients, image postprocessing and ROI analyses, statistical evaluation of data, writing and editing of manuscript.

2.6 Paper 3: 23Na MRI of Ankle Joint in Specimens, Volunteers, and Patients at 7T

A permission to reuse this article in PhD-thesis was provided by the publisher - Wolters Kluwer Health Lippincott Williams & Wilkins [195],

97 ORIGINAL ARTICLE

Sodium Magnetic Resonance Imaging of Ankle Joint in Cadaver Specimens, Volunteers, and Patients After Different Cartilage Repair Techniques at 7 T Initial Results

Stefan Zbyn, MSc, * Martin O. Brix, MD, f Vladimir Juras, PhD, *j Stephan E. Domayer, MD, PhD, f Sonja M. Walzer, MD, f Vladimir Mlynarik, PhD,* Sebastian Apprich, MD, *f Kai Buckenmaier, PhD,§ Reinhard Windhager, MD,f and Siegfried Trattnig, MD*

the talar cartilage than in the calcaneal cartilage of subtalar joint (P <0.001).Data Objectives: The goal of cartilage repair techniques such as microfracture (MFX) from the cadaver ankle samples showed a strong linear relationship between the or matrix-associated autologous chondrocyte transplantation (MACT) is to sodium values and the histochemically determined GAG content (r = 0.800; produce repair tissue (RT) with sufficient glycosaminoglycan (GAG) content. P < 0.001; R2 =0.639). Sodium magnetic resonance imaging (MRI) offers a direct and noninvasive Conclusions: This study demonstrates the feasibility of in vivo quantification evaluation of the GAG content in native cartilage and RT. In the femoral cartilage, of sodium cSI, which can be used for GAG content evaluation in thin cartilages this method was able to distinguish between RTs produced by MFX and MACT of ankle and subtalar joints at 7 T. A strong correlation observed between the having different GAG contents. However, it needs to be clarified whether sodium histochemically evaluated GAG content and the sodium values proved the MRI can be useful for evaluating RT in thin ankle cartilage. Thus, the aims of this sufficient sensitivity of sodium MRI to changes in the GAG content of carti­ 7-T study were (1) to validate our sodium MRI protocol in cadaver ankle samples, lages in the ankle. Both MFX and MACT produced RT with lower sodium cSI (2) to evaluate the sodium corrected signal intensities (cSI) in cartilage of and, thus, of lower quality compared with reference cartilage in the patients volunteers, (3) and to compare sodium values in RT between patients after or in the volunteers. Our results suggest that MFX and MACT produce RT with MFX and MACT treatment. similar GAG content and similar morphological appearance in patients with Materials and Methods: Five human cadaver ankle samples as well as ankles similar surgery outcome. Sodium MRI at 7 T allows a quantitative evaluation of 9 asymptomatic volunteers, 6 MFX patients and 6 MACT patients were mea­ of RT quality in the ankle and may thus be useful in the noninvasive as­ sured in this 7-T study. Sodium values from the ankle samples were compared with sessment of new cartilage repair procedures. histochemically evaluated GAG content. In the volunteers, sodium cSI values were calculated in the cartilages of ankle and subtalar joint. In the patients, sodium Key Words: sodium MRI, ankle joint, 7 T, cartilage repair, glycosaminoglycan cSI in RT and reference cartilage were measured, morphological appearance of content RT was evaluated using the magnetic resonance observation of cartilage repair (Invest Radiol 2015;50: 246-254) tissue (MOCART) scoring system, and clinical outcome before and after surgery was assessed using the American Orthopaedic Foot and Ankle Society score and Modified Cincinnati Knee Scale. All regions of interest were defined on morpho­ efects of talar cartilage are common, and most of them are associated with ankle sprains.1 Patients with persistent pain in logical images and subsequently transferred to the corresponding sodium images. D Analysis of variance, t tests, and Pearson correlation coefficients were evaluated. the ankle joint usually receive surgical treatment. Currently, micro­ Results: In the patients, significantly lower sodium cSI values were found in fracture (MFX) is the treatment of choice for smaller primary RT than in reference cartilage for the MFX (P = 0.007) and MACT patients osteochondral talar lesions.2 Especially in larger cartilage defects, (P = 0.008). Sodium cSI and MOCART scores in RT did not differ between cell-based techniques such as autologous chondrocyte implantation the MFX and MACT patients (P = 0.185). No significant difference in sodium (ACI) and matrix-associated autologous chondrocyte transplantation (MACT) are applied to the ankle.3,4 Several knee studies suggest that cSI was found between reference cartilage of the volunteers and the patients (P = 0.355). The patients showed significantly higher American Orthopaedic cell-based repair techniques, rather than MFX, lead more often in Foot and Ankle Society and Modif ied Cincinnati scores after treatment than hyaline-like repair tissue (RT) and could provide a better clinical they did before treatment. In the volunteers, sodium cSI was signif icantly higher outcome.5,6 However, it should be noted that there are metabolic, in the tibial cartilage than in the talar cartilage of ankle joint (P = 0.002) and in biochemical, and biomechanical differences between knee and ankle cartilage and that ankle cartilage has a greater capacity for repair when compared with knee cartilage.7 Although MFX procedures are more cost- Received for publication June 20, 2014; and accepted for publication, after revision, October 8, 2014. effective, are less technically demanding, and have lower complica­ From the *High-Field MR Center of Excellence, Department of Biomedical Imaging tion rates than cell-based techniques do, their role in the ankle repair and Image-guided Therapy, and fDepartment of Orthopaedic Surgery, Medical is not strictly defined yet. University ofVienna, Vienna, Austria; ^Department of Imaging Methods, Institute The negatively charged glycosaminoglycans (GAGs) provide ofMeasurement Science, Slovak Academy of Sciences, Bratislava, Slovakia; strong electrostatic and osmotic forces responsible for the functional and §High-Field MR Center, Max Planck Institute for Biological Cybernetics, Tübingen, Germany. and structural properties of cartilage. Because the noninvasive moni­ Conflicts of interest and sources of funding: Supported by the Vienna Science and toring of the GAG content in cartilage is of great interest, magnetic Technology Fund (Project WWTF-LS11-018), by the Austrian Science Fund resonance imaging (MRI) techniques such as delayed gadolinium- (FWF) P 25246 B24, by the Vienna Spots of Excellence of the Vienna Science enhanced MRI of cartilage (dGEMRIC),8,9 GAG chemical exchange and Technology Fund (WWTF), Vienna Advanced Imaging Center (VIACLIC FA102A0017), and by DACH (SNF 320030E-134474). saturation transfer,10 Tip mapping,11 or sodium imaging12 have been The authors report no conflicts ofinterest. developed for the noninvasive evaluation of the native cartilage and Reprints: Stelan Zbyn, MSc, High-Field MR Center ofExcellence, Department ofBiomedical cartilage RT. Imaging and Image-guided Therapy, Medical University of Vienna, Waehringer Owing to low sodium concentration, very short transverse Guertel 18-20, A-1090 Vienna, Austria. E-mail: [email protected]. Copyright © 2014 Wolters Kluwer Health, Inc. All rights reserved. relaxation times, and the need for special multinuclear hardware, ISSN: 0020-9996/15/5004-0246 sodium MRI is a challenging method. However, recent hardware (7-T

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systems, phase-array coils) and software developments (measurement The Modified Cincinnati Knee Rating evaluates limitations in daily and image reconstruction techniques) resulted in many exciting in vivo life and sports, ranging from severe limitations (zero points) to full cartilage studies.13 Sodium MRI is very sensitive to even small changes functionality (10 points). in cartilage GAG content and can be thus used as a reference technique for the evaluation of other GAG-sensitive techniques.10 Ankle Specimens The negatively charged GAGs in cartilage are counterbalanced by Eight human cadaver fresh-frozen ankle joints (midtibia to foot the positively charged sodium ions. Therefore, a change in GAG content is base) were obtained from a local anatomy department with the ap­ reflected by a change of signal in sodium image.14 The evaluation of GAG proval of the university institutional review board. On the basis of content in the knee cartilage using sodium imaging was successfully morphological MRI, talar and tibial cartilage in the ankle samples achieved in healthy humans15 and patients.16,17 Sodium MRI of the knee was evaluated according to the guidelines of International Cartilage was able to distinguish not only between native hyaline cartilage and RT Repair Society. Specimens with very thin cartilage due to advanced but also between RT after MACT and RT after bone marrow stimulation osteoarthritis or with full-thickness defects (International Cartilage repair techniques.18 Although a sodium MRI study of the ankle cartilage RepairSocietygradeof3or4)wereexcludedfromfurtherevaluations. has not been published yet, an article on sodium imaging of the wrist cartilage Thus, only 5 ankle samples, from 3 women and 2 men with median suggests that this method could be applied in joints smaller than the knee.19 ageof46years(range,41-60years),wereusedforfurtherevaluations. The aims of this 7-T sodium MRI study were (1) to validate a sodium MRI protocol by comparing MRI results with histochemical Magnetic Resonance Imaging analysis of GAG content in ankle samples, (2) to evaluate the sodium All MRI measurements were performed on a 7-T whole-body cSI indicating GAG content in tibial and talar cartilage of healthy system (MAGNETOM; Siemens Healthcare, Erlangen, Germany). volunteers, (3) and to compare sodium cSI values in RT between the Proton images were recorded using a 28-channel knee array coil patients after MFX and MACT treatment. (Quality Electrodynamics LLC, Cleveland, OH). Sodium images were acquired with a 15-channel sodium-only knee array coil (Tx birdcage/ MATERIALS AND METHODS Rx array) (QED, Quality Electrodynamics LLC, Cleveland, OH) and reconstructed online with adaptive combine algorithm using signal Subjects intensities from each coil element. Special care was taken to position the ankles of the subjects and the ankle samples in the same way for This cross-sectional feasibility study was approved by the both proton and sodium measurements, which was made easier by institutional review board, and written informed consent was obtained identical housing ofboth coils. from all patients and volunteers. Proton density-weighted 2-dimensional (2D) turbo spin echo Nine asymptomatic volunteers without any pain, ankle mal­ (TSE) images with fat suppression in the sagittal and coronal planes alignment (evaluated through the clinical examination), or history of were used for the morphological evaluation. In addition to product trauma or surgery were recruited to obtain baseline sodium values in sequences, a work-in-progress TSE sequence optimized for ultrahigh- ankle cartilages. This group consisted of 6 women and 3 men with field systems (WIP 729C; Siemens Healthcare, Erlangen, Germany) mean (SD) age of 28.7 (5.5) years (range, 23 39 years) and mean body - providing higher resolution was used for subject measurements. mass index (BMI) (SD) of 23.9 (2.7) kg/m2 (range, 19.8-27.7 kg/m2). Sodium images were acquired with a standard 3-dimensional From the cohort of more than 70 patients treated with MFX, gradient echo (3D-GRE) sequence optimized for sodium imaging Pridie drilling, ACI or MACT technique, 6 MFX patients and of cartilage. The small acquisition bandwidth of 80 hertz per pixel 6 MACT patients with similar age, BMI, and defect size agreed to (readout length of 12.5 milliseconds), together with asymmetric undergo additional MRI examination at 7 T. Inclusion criteria were readout, was used to maximize signal-to-noise ratio in the sodium as follows: a singular osteochondral defect (Hepple 3 and 4) or images. Although sodium signal in cartilage exhibits a biexponential a symptomatic deep chondral singular defect (Outerbridge grade 3 T2* decay with short and long components, a relatively long echo time or 4) with stable adjacent cartilage. Exclusion criteria were used for sodium MRI minimized signal contribution from the short progressed osteoarthritis or rheumatoid arthritis, kissing lesions, component. As a result, the broadening of the point-spread-function and instability or malalignment of the joint. Briefly, a marrow duetoaveryfastrelaxationoftheshortT2*componentwasminimized. stimulation-based MFX technique was carried out as described by Sodium MRI protocols used for the subjects and the ankle samples Steadman et al.20 A 3-dimensional scaffold seeded with autologous were identical (Table 1). chondrocytes was used for MACT treatment: Hyalograft C (Fidia Advanced Biopolymer, Abano, Italy) in 4 patients, CaReS (Arthro Kinetics, Esslingen, Germany) in 1 patient, and Carticel (Genzyme Biosurgery, Evaluation of Proton MRI Cambridge, MA) in 1 patient.21 Proton images served for the morphological evaluation of carti­ In the 6 MFX patients (2 women, 4 men), the mean (SD) age lage repair site after surgery using the magnetic resonance observation at MRI was 35.8 (10.0) years, the mean (SD) postoperative interval of cartilage repair tissue (MOCART) scoring system.24 The MOCART was 99.5 (33.1) months, the mean (SD) BMI was 27.0 (5.5 kg/m2), is a reproducible grading system that has been applied to different and the mean (SD) defect size was 1.26 (0.41) cm2.Inthe6MACT cartilage repair techniques.16,18 Scores ofzero and 100 points represent patients (4 women, 2 men), the mean (SD) age at MRI was 35.0 (6.0) poor and excellent outcomes, respectively. Moreover, the thickness years, the mean (SD) postoperative interval was 85.7 (23.9) months, of reference cartilage and RT was measured in the proton images, in the mean(SD) BMIwas 26.1 (3.1) kg/m2, andthe mean(SD) defect the region where sodium cSI values were evaluated in the MFX and size was 1.53 (0.45) cm2. MACT patients. Five measurements of tissue thickness were made in equidistant positions over each region. The mean thickness of refer­ Clinical Outcome ence cartilage and RT was calculated for each patient. The American Orthopaedic Foot and Ankle Society (AOFAS) score22 and the Modified Cincinnati Knee Scale23 were evaluated Postprocessing of Sodium Images before and after the surgery to monitor the clinical outcome. The Cylindrical phantom filled with a homogenous saline solution AOFAS score reflects a patient's perception of pain and function. was measured to correct sodium images for spatially inhomogeneous The maximum is 100 points (50, function; 40, pain; 10 alignment). sodium knee array coil. First, phantom images were acquired using

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the same geometry as that for the images of the subjects and samples. Second, signal intensities of the phantom images were inverted (1/signal cn cm on intensity) and rescaled to the values of 1 to 4095 to produce a correction matrix. Finally, the corrected images were obtained by multiplying the original image data with the correction matrix. All images were corrected using in-house written IDL (Interactive Data Language; Research Systems, Inc, Boulder, CO) and Matlab (MATLAB 7.7; The Mathworks, Natick, MA) scripts. Sodium cSI maps of cartilage were calculated from the sodium images corrected for inhomogeneous B1 field. For the correction of sodium signal intensities in cartilage, the subjects were measured together with 10% wt/wt agarose gel phantoms having different concentrations of sodium (100, 150, 200, 250, and 300 mmol/L), as previously described by Shapiro et al.25 After correcting the signal intensity of the phantoms for their T1 and T2* relaxation times (T1, 30.1 milliseconds; T2*, 6.2 milliseconds) and for a 50-degree flip angle, a calibration curve was obtained. Sodium cSI maps were calculated using linear regression, p p p p p cn cn cn cn cn and they were corrected for the mean sodium T1 and T2* of native cartilage taken from previously published measurements (T1, 20 milliseconds; T2*, 4.5 milliseconds)12,19,26 and for the 50-degree e flip angle. Because the average water content from our histochemical iß© nt o o o evaluations was 70%, similar to 75% reported by Treppo et al,27 the values * s ® S in the final sodium cSI maps were divided by a factor of 0.7 (Figs. 1, 2). m m tp cn cn

LLl Histochemical Analyses —I > CO Tibial and talar cartilage from the human ankle samples was < ■> divided into lateral, central, and medial compartments (Fig. 3). From

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FIGURE 1. Proton and corresponding sodium images of the ankle joint in a 23-year-old healthy woman. A, Sagittal proton density-weighted 2D-TSE image with fat suppression with lower resolution (0.31 x 0.31 x 3.0 mm3). B, Sagittal proton density-weighted 2D-TSE image with fat suppression (WIP 729C; Siemens, Erlangen, Germany) with higher resolution (0.27 x 0.27 x 1.5 mm3). C, Sagittal sodium 3D-GRE image with redcontoursrepresentingtheROIevaluationsofcartilagesintheankleandsubtalarjoints.D,Color-codedcSImapofsodiumincartilages overlaid on the corresponding morphological image. each cartilage compartment, 4 to 6 cartilage pieces ofapproximately a mixed-model ANOVA with the treatment type and the tissue type 1-mm3 volume were harvested in the anterior-posterior direction for (repair, reference) as a factor. Post hoc comparison was achieved with the biochemical evaluations. Each piece ofcartilage was weighed, dried 2-tailed t tests corrected according to Bonferroni-Holm. The dif­ at 60°C for 24 hours, weighed again to obtain wet and dry weights, and ference in sodium cSI between the lateral and medial sides of all 4 followed by enzymatic digestion. Water content was calculated from cartilage types and between global cSI of all 4 cartilages in the the dry and wet weights. For quantitative biochemical analysis of GAG volunteers was evaluated by 2-way repeated measures ANOVAwith content in the cartilage samples, Blyscan Sulfated Glycosaminoglycan side and cartilage type as a factor. Post hoc Bonferroni-corrected Assay kit (Biocolor Ltd, Belfast, Ireland) was used28 and optical absor­ comparisons were then calculated to show differences in sodium bance of single-tissue digestion at 656 nm was performed in duplicates cSI between cartilages of ankle and subtalar joint. Differences in on a microplate spectrophotometer (Fluostar-Optima; BMG-Labtech, the MOCART and clinical outcome scores between the MFX and Offenburg, Germany). The sulfated GAG content was then estimated MACT patients were evaluated with independent samples t tests. using a standard curve determined from calibration measurements of A Pearson correlation coefficient (r) was determined to eval­ chondroitin-4 sulfate. Finally, the total GAG content in each cartilage uate linear relation between 2 variables. The strength of association compartment was calculated as the ratio between the sum of GAG was classified as noncorrelation (r <0.3),low(r =0.3-0.5), weights from all cartilage pieces and the sum of wet weights from all moderate(r=0.5-0.7), orstrong(r >0.7). Allstatisticalevaluations cartilage pieces and expressed as a percentage. were performed using the SPSS software (IBM SPSS Statistics 21.0), and a P value 0.05 was considered statistically significant. Statistical Analyses To compare sodium cSI in tibial and talar reference cartilage RESULTS between the volunteers as well as the MFX and MACT patients, 1-way analysis of variance (ANOVA) with the treatment type as a factor was Patients calculated. For comparing sodium cSI from repair and reference Independent-samples t test did not reveal any significant dif­ cartilage between and within the MFX and MACT group, we used ferences in age, postoperative interval, BMI, or the defect size between

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FIGURE 2. Proton and corresponding sodium images from the medial side (upper row) and from the lateral side (lower row) of the ankle joint in a 35-year-old man obtained 54 months after the MFX treatment. Cartilage RT is situated between arrows. A and B, Sagittal proton density-weighted 2D-TSE image with fat suppression with lower resolution (0.31 x 0.31 x 3.0 mm3). C and D, Sagittal proton density-weighted 2D-TSE image with fat suppression (WIP 729C; Siemens) with higher resolution (0.27 x 0.27 x 1.5 mm3). Eand F, Sagittal sodium 3D-GRE image with red contours representing the ROI evaluations of cartilage RT, tibial, and talar cartilage in ankle joint. G and H, Color-coded sodium cSI maps in cartilage and RT overlaid on the corresponding morphological images. Please note the lower sodium cSI values in RT than that in reference cartilage on the contralateral side of the ankle joint. the MFX and MACT groups. One-way ANOVA did not show any sig­ between clinical outcome scores after the surgery and sodium cSI nificant differences in sodium cSI between the volunteers as well as the in RT (AOFAS: r = -0.207, P = 0.519, R2 = 0.043; Cincinnati rating: MFX and MACT patients for both tibial (P = 0.300) and talar reference r = 0.136, P = 0.672, R2 = 0.019). cartilage (P = 0.391). The mixed-model ANOVA found a significant The mean (SD) thickness measured in the proton images was difference in cSI between talar reference cartilage and talar RT in the smaller in reference cartilage (MFX, 1.27 [0.22] mm; MACT, 1.22 ankle joint (P < 0.001). Post hoc paired t tests revealed significantly [0.11] mm) than that in RT (MFX, 1.82 [0.42] mm; MACT, 1.69 lower sodium cSI in talar RT than that in talar reference cartilage in [0.53] mm). No linear correlation was found between sodium cSI and the MFX (P = 0.007) and MACT patients (P = 0.008) (Fig. 4). The tissue thickness in reference cartilage (r = 0.232, P = 0.468, R2 = difference between the MFX and MACT patients was not statistically 0.054) and in RT (r = 0.011; P =0.973;R2 <0.001)(Fig.6B). significant in talar reference cartilage (P = 0.355) as well as in talar RT (P = 0.185) with independent-samples t tests (Fig. 5A). The mean Volunteers sodium cSI values from RT and reference cartilage of the patients and Sodium cSI values were calculated for the medial and lateral the volunteers are in Table 2. sides of all 4 cartilages of ankle and subtalar joint in the volunteers The mean MOCART evaluations showed that majority of the (Table 3). Although 2-way repeated-measures ANOVA did not show patients had complete filling of the defect (MFX, 5; MACT, 5) with any significant differences in sodium cSI between the lateral and medial visible demarcating border of RT (MFX, 4; MACT, 5), with surface sides of tibial and talar cartilage of ankle joint as well as talar and damaged to less than 50% of depth (MFX, 3; MACT, 5), and with isointense signal intensity of RT (MFX: 5, MACT: 3). Independent- samples t test did not show significant difference in the MOCART scores between the MFX and MACT patients (P = 0.341). All patients showed significantly higher AOFAS scores after the treatment than before the treatment (MFX, P <0.001;MACT,P = 0.009). Similarly, the Modified Cincinnati Knee Rating scores were significantly higher after the treatment than before the treatment (MFX, P =0.018;MACT, P= 0.006).Nostatisticallysignificantdifferencewasobservedbetween the MFX and MACT patients when comparing the results of AOFAS (pretreatment, P = 0.950; posttreatment, P = 0.745) or the Modified Cincinnati Knee Rating (pretreatment, P = 0.666; posttreatment, P = 0.829) using independent samples t tests. Table 2 shows the mean FIGURE 3. Each talar cartilage of the human ankle samples was divided scores of MOCART and clinical outcome evaluations. into lateral, central, and medial segments. Dots represent cartilage pieces(4-6)ofapproximately1-mm3volume,whichweresampledinthe There was a medium negative correlation between postoperative anterior-posterior direction from each cartilage compartment for the interval and sodium cSI in RT (r = -0.590; P = 0.043; R2 = 0.349) biochemical evaluations. Tibial cartilage was divided and sampled (Fig. 6A). No linear association was found between MOCART scores similarly to talar cartilage. Figure 3 can be viewed online in color at and sodium cSI in RT (r = -0.255; P = 0.424; R2 = 0.065) as well as www.investigativeradiology.com.

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FIGURE4. A,GraphsindicatingthesodiumcSIvaluesfornativetalarcartilageandtalarRTforeachMFX(A)andMACT(B)patient.Eachlinerepresents 1 patient.AllmeansodiumcSIvaluesfromtheROIsplacedinnativecartilagearehigherthanthevaluesfromthecorrespondingRTareas.Both cartilagerepairsurgeriesresultedinsignificantlylowersodiumcSIvaluescomparedwithnativecartilage(MFX,P=0.007;MACT,P=0.008).Figure4can be viewed online in color at www.investigativeradiology.com. calcaneal cartilage of the subtalar joint (P = 0.695), there was a sig­ of the status of native articular cartilage in volunteers and that of car­ nificant difference between all 4 types of cartilages in ankle and sub­ tilage RT in patients after MFX and MACT treatment. talar joint (P = 0.007). Post hoc comparisons revealed significant Because of ethical reasons, there is a lack of histological data differenceinmeansodiumcSIbetweentibialandtalarcartilageofankle concerning RT composition after MFX and MACT.4 It is therefore of joint (P = 0.002), between tibial cartilage of ankle joint and calcaneal substantial interest to develop and optimize biochemical MRI tech­ cartilage of subtalar joint (P = 0.050), and between calcaneal cartilage niques for the noninvasive evaluation of native cartilage and quality andtalarcartilageofsubtalarjoint(P<0.001)(Fig. 5B). Allothercom- of RT in the ankle. Morphological29 and biochemical MRI techni­ parisons were not statistically significant. ques such as T2 and T2* mapping,30-32 diffusion-weighted imaging,33 or dGEMRIC34 have provided interesting data about cartilage and Ankle Samples RT in the ankle.15Although sodium MRI was successfully used for Fromthe5anklesamples,15ROIsfromtalarcartilageand15ROIs theevaluationofRTqualityinthekneejoint,16-18toourknowledge, from tibial cartilage were obtained. The mean (SD) sodium NSI value in it has not yet been applied for the evaluation of ankle. thetibialcartilagewas63.8(12.2)(range,50.2-90.3)andintalarcartilage Although sodium MRI is challenging and requires multinuclear was 60.1 (13.8) (range, 43.5-87.6). The mean (SD) GAG content was hardware, dedicated array coils, and preferably very high field strength 6.7% (1.0%) of wet weight (range, 5.2%-8.0%) in tibial and 6.8% (>3 T), recent in vivo studies demonstrated its clinical potential for the (1.4%) of wet weight (range, 3.3%-8.8%). A strong linear relationship evaluation ofcartilage repair and osteoarthritis.13 In addition, sodium im­ wasobservedbetweenthesodiumNSIandthehistochemicallyassessed aging seems to be more sensitive to subtle changes in GAG content than GAGcontent(r=0.800;P<0.001;R2=0.639)(Fig.6C). do other GAG-sensitive techniques that have also specific limitations. The dGEMRIC is limited by the need for intravenous contrast agent and the time delay between its administration and the MRI.8,9,34 The DISCUSSION T1 p maps are affected not only by GAG concentration but also by dipolar This study demonstrates, for the first time, the feasibility of in relaxation mechanisms.11 The GAG chemical exchange saturation trans­ vivo sodium MRI of the native cartilage and cartilage RT in the ankle fer is sensitive to patient motion and static magnetic field inhomogeneities, joint at 7 T. Sodium MRI offers quantitative biochemical evaluation which require sophisticated postprocessing corrections.10

A MFX Volunteers MACT B Ankle Joint Subtalar Joint FIGURE 5. A, Mean sodium cSI values from talar reference cartilage and talar RT of the MFX and MACT patients as well as those from talar referencecartilageofthevolunteers.NotethesignificantdecreaseinmeansodiumcSIofrepairtissueafterMFXandMACTcomparedwiththe corresponding values from reference cartilage. There was no significant difference in the sodium cSI of reference cartilage between the MFX and MACT patients and the volunteers. No significant difference in the sodium cSI was observed between MFX and MACT RT. B, Mean sodium cSI from tibial and talar cartilage of the ankle joint and that from calcaneal and talar cartilage of the subtalar joint from all 9 volunteers. Error bars stand for standard deviations, asterisk (*) represents the significant difference, and “n” stands for the number of independent observations. Figure 5 can be viewed online in color at www.investigativeradiology.com.

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TABLE 2. Sodium Concentration, Morphological Score, and Clinical Outcome in the Patients and the Volunteers

Sodium cSI (a.u.) MOCART AOFAS Score Cincinnati Score Tibial RC Talar RC Talar RT Score Preoperative Postoperative Preoperative Postoperative MFX Mean (SD) 348 (54) 317 (44) 243 (58) 75.8 (16.6) 43.8 (10.3) 88.0 (10.4) 2.8 (1.5) 7.2 (0.8) Range 299-438 266-377 166-311 45-95 35-55 75-100 1-4 6-8 MACT Mean (SD) 317 (35) 295 (32) 193 (33) 65.0 (20.7) 45.4 (21.6) 90.2 (10.3) 3.2 (1.5) 7.4 (1.8) Range 280-365 264-330 158-248 40-85 15-75 76-100 1-5 5-10 Volun. Mean (SD) 352 (43) 320 (42) Range 285-407 253-376

a.u. indicates arbitrary units; AOFAS, American Orthopaedic Foot and Ankle Society; cSI, corrected signal intensity; MACT, matrix-associated autologous chondrocyte transplantation; MFX, microfracture; MOCART, magnetic resonance observation of cartilage repair tissue; RC, reference cartilage; RT, repair tissue; volun., volunteers.

To validate the sensitivity of sodium MRI to the GAG content The presented 7-T sodium MRI data indicate a significantly in thin ankle cartilages, measurements on the human cadaver ankle lower GAG content in RTafter MFX and MACT compared with native samples were first performed. A strong correlation observed between cartilage, which can reflect less-favorable mechanical properties of RT. the histochemically evaluated GAG content and sodium NSI proved This is in agreement with previous sodium studies in the knee, which the sufficient sensitivity of this method to changes in the GAG content reported lower GAG concentration in RT of patients after MFX and of tibial and talar cartilage and encouraged us to use sodium MRI for MACT than in reference cartilage.16 18 In the ankle, dGEMRIC, which the in vivo evaluation of ankle cartilage. We found significantly lower is sensitive to cartilage and RT GAG content, also showed a lower sodium cSI in talar cartilage RT after both surgical procedures when GAG content in RT after matrix-induced chondrogenesis than that compared with reference cartilage in the patients or in the volunteers. in reference cartilage.34 The sodium cSI values in tibial and talar reference cartilage of In the previous knee studies, differences in the biochemical pro­ the patients were not significantly different from the values mea­ perties of RT from the 2 repair procedures were manifested by differ­ sured in the volunteers. Sodium cSI in RT did not differ significantly ences in sodium values18 and in T2 relaxation times.39,40 In contrast between the MFX and MACT patients, and the same results were to these reports, we did not observe any significant difference in observed for AOFAS and Modified Cincinnati Knee scores before sodium cSI between the MFX and MACT RT in ankle joint. This dis­ and after the surgery. Both scores were higher after cartilage repair crepancy between knee and ankle studies could be explained by in each MFX and MACT patient. In addition, sodium cSI values in differences in the biochemical composition of the knee and ankle the cartilages of asymptomatic volunteers were determined in the cartilage andbyagreatercapacityofankle cartilage forrepaircom- ankle and subtalar joints. pared with the knee joint.7 Furthermore, recent T2 mapping study At present, it is not clear whether clinical outcome after cartilage at 7 T also did not find significant differences between MFX and repair of the talar dome is different for MFX and MACT. Excellent MACT cartilage RT in talus.31 outcomes at short term and midterm have been reported after MFX The fact that the sodium cSI didnot correlate withthe MOCART in the ankle.22,35,36 The first short-term and midterm results also report score or with the clinical outcome scores suggests that sodium MRI a good or excellent outcome after MACT.3,4,37 A few studies that provides complementary information to morphological and clinical directly compared the outcomes after MFX and MACT/ACI achieved results. Although the cartilage GAG content and clinical outcome (pain, a comparable outcome at midterm.31,33,38 This is in accordance with function) might not be directly related, only RT with sufficient con­ our observations of similar AOFAS and Modified Cincinnati Knee centration of GAGs can provide long-lasting functional and structural scores for the MFX and MACT patients. substitution for native cartilage. Because both types of cartilage repair

FIGURE6. A,PlotrepresentsthemediumlinearassociationbetweenthesodiumcSI valuesandpostoperativeintervaloftheMFX(circles)andMACT (triangles)patients(r=-0.590;P=0.043;R2=0.349).Dataarefittedbylinearregressionline,y=-1.2534x+160.51.B,Nolinearassociation wasfoundbetweenthesodiumcSIvaluesandtissuethicknessforreferencecartilage(circles)(r=0.232;P=0.468;R2=0.054)andforRT(triangles) (r=0.011;P=0.973;R2<0.001).C,PlotdemonstratesastronglinearcorrelationbetweenthesodiumNSIandthebiochemicallyassessed GAG contentfromtalarcartilage(circles)and fromtibial(triangles)cartilageofthehumananklesamples(r=0.800;P<0.001;R2=0.639).Dataarefitted bylinearregressionline,y=0.0727x+2.1965.Figure6canbeviewedonlineincoloratwww.investigativeradiology.com.

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ACKNOWLEDGMENTS TABLE 3. Sodium Concentrations in the Cartilages of the Volunteers The authors thank Michael Weber for his helpful critical revision of the statistical analysis and Claudia Kronnerwetter for her help with Sodium cSI (arbitrary units) MRI measurements. Ankle Joint Subtalar Joint Tibial Talar Talar Calcaneal REFERENCES Medial Mean (SD) 353 (42) 320 (43) 362 (21) 311 (13) 1. Ronga M, Grassi FA, Montoli C, et al. Treatment of deep cartilage defects Range 278-408 251-374 336-409 294-335 of the ankle with matrix-induced autologous chondrocyte implantation Lateral Mean (SD) 352 (45) 320 (43) 361 (22) 310 (13) (MACI). Foot Ankle Surg. 2005;11:29-33. Range 289-407 254-378 337-413 294-336 2. Zengerink M, Struijs PA, Tol JL, et al. Treatment of osteochondral lesions of Global Mean (SD) 352 (43) 320 (42) 362 (21) 311 (13) the talus: a systematic review. Knee Surg Sports Traumatol Arthrosc.2010;18: 238-246. Range 285-407 253-376 336-411 294-335 3. Giannini S, Battaglia M, Buda R, et al. Surgical treatment of osteochondral lesions of the talus by open-field autologous chondrocyte implantation: a 10-year follow-up clinical and magnetic resonance imaging T2-mapping techniques showed a decrease of sodium cSI in RT with time after evaluation. Am J Sports Med. 2009;37:112S-118S. surgery, sodium MRI might be useful for the evaluation ofrelationship 4. Giannini S, Buda R, Vannini F, et al. Arthroscopic autologous chondrocyte between the GAG content and RT resilience over time. Although mid­ implantation in osteochondral lesions of the talus: surgical technique and results. term and long-term MFX RT seems to be more prone to degeneration, Am J Sports Med. 2008;36:873-880. further long-term follow-up studies with sodium MRI will be needed 5. Knutsen G, Drogset JO, Engebretsen L, et al. A randomized trial comparing for assessing optimal cartilage repair algorithms for ankle joint in autologous chondrocyte implantation with microfracture. J Bone Joint Surg Am. 2007;89A:2105-2112. clinical routine. 6. Saris DB, Vanlauwe J, Victor J, et al. Characterized chondrocyte implantation To achieve a sufficiently high signal-to-noise ratio, sodium images results in better structural repair when treating symptomatic cartilage defects of are, in general, acquired with low resolution. Because the cartilage the knee in a randomized controlled trial versus microfracture. Am J Sports Med. layers inthe ankle are thin(mean[SD] thicknesses oftalarcartilage 2008;36:235-246. are approximately1.35 [0.22]mminmales and1.11 [0.28]mmin 7. Kuettner KE, Cole AA. Cartilage degeneration in different human joints. females41) and in-plane resolution in sodium images is 1.79 x Osteoarthritis Cartilage. 2005;13:93-103. 1.79mm2,partialvolumeeffectsfromthesurroundingtissuescould 8. Trattnig S, Mamisch TC, Pinker K, et al. Differentiating normal hyaline car­ tilage from post-surgical repair tissue using fast gradient echo imaging in cause underestimation (in the presence of bone in a voxel) or over­ delayed gadolinium-enhanced MRI (dGEMRIC) at 3 tesla. Eur Radiol. 2008; estimation (in the presence of synovial fluid) of sodium signal in­ 18:1251-1259. tensities and thus affect the accuracy of the presented results. To 9. Trattnig S, Burstein D, Szomolanyi P, et al. T1(Gd) gives comparable information minimize this limitation, the phase encoding was set to the anterior­ as Delta T1 relaxation rate in dGEMRIC evaluation of cartilage repair tissue. posterior direction and ROIs were selected carefully in the morpho­ Invest Radiol. 2009;44:598-602. logical images and transferred to the corresponding sodium images. 10. Schmitt B, Zbyn S, Stelzeneder D, et al. Cartilage quality assessment by using glycosaminoglycan chemical exchange saturation transfer and (23)Na MR imaging Moreover, because ROIs containing many pixels covering the whole at 7 T. Radiology. 2011;260:257-264. native cartilage or repair site were selected (in the volunteers, ROIs 11. Keenan KE, Besier TF, Pauly JM, et al. Prediction of glycosaminoglycan from 2 slices were averaged together), the overall partial volume effect content in human cartilage by age, T1rho and T2 MRI. Osteoarthritis Cartilage. on mean sodium cSI was averaged and similar in different types of 2011;19:171-179. cartilage and in different patients. As demonstrated in Figure 6B, the 12. Wheaton AJ, Borthakur A, Shapiro EM, et al. Proteoglycan loss in human knee difference in sodium cSI values observed between reference cartilage cartilage: quantitation with sodium MR imaging—feasibility study. Radiology. 2004;231:900-905. and RT is not substantially affected by the tissue thickness and the 13. Zbyn S, Mlynarik V, Juras V, et al. Sodium MR imaging of articular cartilage corresponding partial volume effects. In addition, sodium cSI values pathologies. Curr Radiol Rep.2014;2:41. are relative numbers; thus, the correction for possible systematic bias 14. Mankin HJ. Biochemical and metabolic aspects of osteoarthritis. Orthop Clin due to partial volume effect is not necessary. North Am. 1971;2:19-31. Another limitation of the present data is that we used the same 15. Borthakur A, Mellon E, Niyogi S, et al. Sodium and T1rho MRI for molecular mean T1 and T2* relaxation times measured in the cartilage ofhealthy and diagnostic imaging of articular cartilage. NMR Biomed. 2006;19:781-821. volunteers12,19,26 to correct the sodium maps in native cartilage and 16. Trattnig S, Welsch GH, Juras V, et al. 23Na MR imaging at 7 T after knee matrix- RTofpatients.AlthoughRTmighthavedifferentrelaxationtimescom- associated autologous chondrocyte transplantation preliminary results. Radiology. 2010;257:175-184. pared with native cartilage, these data are not available yet. Furthermore, 17. Chang G, Madelin G, Sherman OH, et al. Improved assessment of cartilage relaxation times in RT could change even among patients with the same repair tissue using fluid-suppressed Na inversion recovery MRI at 7 Tesla: type of cartilage repair. An additional limitation of our preliminary preliminary results. Eur Radiol. 2012;22:1341-1349. study is the low number of patients. However, the number of subjects 18. Zbyn S, Stelzeneder D, Welsch GH, et al. Evaluation of native hyaline cartilage was sufficient to demonstrate significant difference in sodium cSI and repair tissue after two cartilage repair surgery techniques with 23Na MR values between RT and native cartilage. Another source of bias in our imaging at 7 T: initial experience. Osteoarthritis Cartilage. 2012;20:837-845. 19. Borthakur A, Shapiro EM, Akella SV, et al. Quantifying sodium in the human measurements was a different follow-up interval of patients. wrist in vivo by using MR imaging. Radiology. 2002;224:598-602. Becauseofthepreviouslymentionedlimitations(mainlypartial 20. Steadman JR, Rodkey WG, Rodrigo JJ. Microfracture: surgical technique volume artifacts), we did not evaluate the sodium concentrations in the and rehabilitation to treat chondral defects. Clin Orthop Relat Res.2001; native cartilage and RT. However, calculated sodium cSI values should 391:S362-S369. be close to the expected sodium concentrations in the ankle cartilage. 21. Grigolo B, Lisignoli G, Piacentini A, et al. Evidence for redifferentiation of Inconclusion,sodiumMRIat7Tallowsquantitativeassessment human chondrocytes grown on a hyaluronan-based biomaterial (HYAff 11): of native cartilage and cartilage RT. In contrast to the knee, both MFX molecular, immunohistochemical and ultrastructural analysis. Biomaterials. 2002; 23:1187-1195. andMACTleadtoRTwithsimilarsodiumcSIvaluesbutdifferentfrom 22. Saxena A, Eakin C. Articular talar injuries in athletes: results of microfracture and that in the native cartilage. Because sodium concentration in cartilage autogenous bone graft. Am J Sports Med. 2007;35:1680-1687. is proportional to the cartilage GAG content, sodium MRI might be 23. Noyes FR, Barber SD, Mooar LA. A rationale for assessing sports activity levels beneficial in the noninvasive evaluation of cartilage repair surgery. and limitations in knee disorders. Clin Orthop Relat Res. 1989;246:238-249.

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24. Marlovits S, Singer P, Zeller P, et al. Magnetic resonance observation of cartilage 34. Wiewiorski M, Miska M, Kretzschmar M, et al. Delayed gadolinium-enhanced repair tissue (MOCART) for the evaluation of autologous chondrocyte trans­ MRI of cartilage of the ankle joint: results after autologous matrix-induced plantation: determination of interobserver variability and correlation to clinical chondrogenesis (AMIC)-aided reconstruction of osteochondral lesions of the outcome after 2 years. Eur J Radiol. 2006;57:16-23. talus. Clin Radiol. 2013;68:1031-1038. 25. Shapiro EM, Borthakur A, Dandora R, et al. Sodium visibility and quantitation in 35. Becher C, Driessen A, Hess T, et al. Microfracture for chondral defects of the intact bovine articular cartilage using high field (23)Na MRI and MRS. JMagn talus: maintenance of early results at midterm follow-up. Knee Surg Sports Reson. 2000;142:24-31. Traumatol Arthrosc. 2010;18:656-663. 26. Madelin G, Jerschow A, Regatte RR. Sodium relaxation times in the knee joint in 36. Kuni B, SchmittH, Chloridis D, etal. Clinical and MRI results aftermicrofracture vivo at7 T. NMR Biomed. 2012;25:530-537. of osteochondral lesions of the talus. Arch Orthop Trauma Surg. 2012;132: 27. Treppo S, Koepp H, Quan EC, et al. Comparison of biomechanical and bio­ 1765-1771. chemical properties of cartilage from human knee and ankle pairs. JOrthop 37. Aurich M, Bedi HS, Smith PJ, et al. Arthroscopic treatment of osteochondral Res. 2000;18:739-748. lesions of the ankle with matrix-associated chondrocyte implantation: early 28. Pietila K, Kantomaa T, Pirttiniemi P, et al. Comparison of amounts and properties clinical and magnetic resonance imaging results. Am J Sports Med. 2011;39: of collagen and proteoglycans in condylar, costal and nasal cartilages. Cells 311-319. Tissues Organs. 1999;164:30-36. 38. Brix M, Chiari C, Nehrer S, et al. Cartilage repair of the ankle with micro­ 29. Notohamiprodjo M, Kuschel B, Horng A, et al. 3D-MRI of the ankle with fracturing or autologous chondrocyte implantation/matrix associated autologous optimized 3D-SPACE. Invest Radiol. 2012;47:231-239. chondrocyte implantation: follow up from 1 to 14 years. Osteoarthritis Cartilage. 30. Battaglia M, Vannini F, Buda R, et al. Arthroscopic autologous chondrocyte 2012;20:S130-S131. implantation in osteochondral lesions of the talus: mid-term T2-mapping MRI 39. Welsch GH, Trattnig S, Domayer S, et al. Multimodal approach in the use of evaluation. Knee Surg Sports Traumatol Arthrosc. 2011;19:1376-1384. clinical scoring, morphological MRI and biochemical T2-mapping and diffusion- 31. Domayer SE, Apprich S, Stelzeneder D, et al. Cartilage repair of the ankle: first weighted imaging in their ability to assess differences between cartilage repair results of T2 mapping at 7.0 T after microfracture and matrix associated tissue after microfracture therapy and matrix-associated autologous chondrocyte autologous cartilage transplantation. Osteoarthritis Cartilage. 2012;20:829-836. transplantation: apilotstudy. Osteoarthritis Cartilage. 2009;17:1219-1227. 32. Andreisek G, Weiger M. T2* mapping of articular cartilage: current status of 40. Welsch GH, Mamisch TC, Domayer SE, et al. Cartilage T2 assessment at 3-T MR research and first clinical applications. Invest Radiol. 2014;49:57-62. imaging: in vivo differentiation of normal hyaline cartilage from reparative 33. Apprich S, Trattnig S, Welsch GH, et al. Assessment of articular cartilage re­ tissue after two cartilage repair procedures - Initial experience. Radiology. 2008; pair tissue after matrix-associated autologous chondrocyte transplantation or the 247:154-161. microfracture technique in the ankle joint using diffusion-weighted imaging at 41. Sugimoto K, Takakura Y, Tohno Y, et al. Cartilage thickness of the talar dome. 3tesla.Osteoarthritis Cartilage. 2012;20:703-711. Arthroscopy. 2005;21:401-404.

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Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. 106 DISCUSSION

3. DISCUSSION

3.1 General Discussion

The main goal of all cartilage repair techniques is to restore joint function and provide repair tissue that has in the ideal case the same structure, composition, and biomechanical properties as native cartilage. The reference standard for the biochemical evaluation of cartilage repair tissue is histologic analysis of biopsy samples [85, 196-198], However, this approach has many limitations. For example, due to ethical reasons, only small piece of repair tissue can be harvested. Since biochemical composition of tissue can vary within repair area, the accuracy of biopsy is limited, especially in larger repair sites. In addition, since the composition of repair tissue changes over time, repeated biopsies are not ideal for treatment follow-up. Due to these limitations, there is a lack of data providing information about histological composition of the tissue after different cartilage repair techniques. Development of various MRI methods thus brought new possibilities for a non-invasive evaluation of different biochemical parameters of repairtissue. Morphological and biochemical (e.g. T2 and T2* mapping, dGEMRIC, diffusion-weighted imaging and most recently gag­ CEST) MRI techniques have provided interesting data about native cartilage and repairtissue in the knee and ankle joint. GAGs are responsible for unique biomechanical properties of cartilage. Therefore only repair tissue with sufficiently high GAG concentration can provide long-lasting structural and functional replacement of native cartilage. Methods sensitive to changes in tissue GAG content, such as sodium MRI, therefore provide important information about the composition and biomechanical properties of repair tissue. The main advantage of noninvasive sodium MRI is its direct association with the GAG concentration that provides this method with high sensitivity (probably highest among all MR methods) to even subtle changes in cartilage GAG/PG content. This was confirmed by results from controlled cartilage degradation, which suggested that sodium MRI is able to detect about 5% change in PG concentration [199, 200], Sodium MRI can therefore serve as a reference standard for evaluation of the GAG concentration and for assessing specificity of

107 DISCUSSION newly developed GAG-sensitive MRI methods [150], Recent patient studies demonstrated a clinical potential of sodium imaging for the noninvasive evaluation OA and for the assessment of biomechanical properties of cartilage repair tissue [201], The general limitation of sodium MRI is its relatively low resolution and special hardware requirements. The sodium MR signal in cartilage is about 3400 times lower compared to that of signal from 1H. In order to maintain sufficient SNR, sodium images are usually reconstructed with relatively low resolution and acquired in longer measurement times than proton images. Moreover, sodium MRI requires hardware with multinuclear capability, dedicated sodium array coils, and preferably 7 T (or at least 3 T) MR system to acquire images with sufficient SNR in a clinically tolerable measurement time. Current clinical applicability of sodium imaging is limited due to hardware and measurement time requirements. However, new array coils and advanced MR imaging techniques may help to transfer sodium imaging to clinical 3-T MR systems [202], Other GAG-sensitive MRI techniques, such as Tip, dGEMRIC or gagCEST, also suffer from some limitations (for details see chapter 1.5.2). This doctoral thesis focuses on the validation and application of sodium MRI for the evaluation of cartilage repair tissue properties. Paper 1 and 2 discuss evaluation of cartilage repair tissue in the knee joint, paper 3 describes assessment of repair tissue in the ankle joint. In addition, papers 2 and 3 compare the biochemical properties of repair tissue between two different types of cartilage repair procedures, which is particularly interesting for the evaluation of newly introduced cartilage repair procedures.

3.2 Paper 1:23Na MRI at 7T after Knee MACT

The aim of paper 1 was to assess the feasibility of 7-T sodium MRI for the evaluation of native cartilage and repair tissue of MACT patients and to compare sodium results with those of dGEMRIC [194], By using a 3D GRE sequence in combination with volume knee coil at 7 T, we were able to acquire sufficient SNR to differentiate between native cartilage (SNR ~24) and repair tissue (SNR ~16) of patients after MACT on the femoral cartilage. SNR values obtained in 30 minutes in our study are comparable to values from patella reported by Wheaton et al. who used a surface coil at 4T [203], Previous papers on sodium MRI of knee focused mostly on the imaging of patellar cartilage [204, 205], This was caused mainly by the thicker cartilage layers and easier accessibility for surface RF coils compared to femoral or tibial cartilage.

108 DISCUSSION

Higher sensitivity of a surface coil enables to obtain sodium images with a similar SNR, but in a shorter time and with higher resolution, than images acquired with a volume knee coil [203], However, complicated postprocessing corrections of sodium images and field of view restricted only to superficial regions of the joints limit the use of surface coils to the patella. For imaging of femoral and tibial cartilage, a volume RF coil is required. Various histologic studies demonstrated that repaired tissue can vary from fibrous to mixed fibrous-hyaline to hyaline-like type of tissue [70, 85, 87, 90] . In addition, the fibrous type contains significantly lower amount of GAGs compared to the hyaline-like type of tissue. The results of paper 1 are in agreement with previous histological findings and suggest lower sodium concentration in repair tissue after MACT compared to that in native cartilage. These findings suggest a lower GAG concentration. However, relaxation times in repair tissue may differ from those in native cartilage, which could eventually lead to an overestimation or underestimation of the sodium concentration based on sodium images measured with a relatively long TE (~3.8 ms) and short TR (~10 ms) in this study. Unfortunately, it was not possible to further shorten the TE of 3D GRE sequence. At the same time, prolonging of TR to approximately 100 ms would lead to a decrease of SNR in sodium images. In general, most studies did not find correlation between morphologic MRI evaluations and biochemical composition of cartilage repairtissue (e.g. [174]). In contrast to these studies, our results showed a strong correlation between the sodium ratios and the MOCART scores. However, only 12 data points were used to evaluate this relationship, thus further studies with a larger cohort of patients should be conducted to verify our observation. In recent years, various MRI methods, like dGEMRIC [162], sodium imaging [204], Tip imaging [206], or gag-CEST [150], were proposed for the evaluation of GAG content in cartilage. Among these methods, dGEMRIC has been successfully applied to patients after different cartilage repair surgery techniques such as ACI [171], MACT [149, 172], or MACT and MFX [175] and has shown ability to distinguish between repair tissues produced after different surgeries. Unfortunately, dGEMRIC requires intravenous administration of a contrast agent (usually a double dose), and a long waiting time period necessary for passive diffusion of the contrast agent into cartilage before MRI scans [163], Instead of time consuming Ti mapping based on standard inversion-recovery method, we used a faster dual-flip-angle method that has been validated in phantom and in vivo studies [149, 168, 169], As demonstrated in previous studies, sodium MRI allows direct quantification of GAG concentration in native cartilage [3, 204], However, it has not yet been applied for the evaluation of cartilage repair tissue. In our study, we observed high reproducibility of regions-

109 DISCUSSION of-interest (ROI) evaluations in sodium images, with strong correlation and no significant differences between two readers. Additionally, we observed a strong correlation between dGEMRIC and sodium values in native cartilage and in MACT repair tissue, which is important for validation of our sodium MRI protocol forevaluation of repair tissue. 7-T sodium MRI could therefore be helpful for evaluation of the specificity and for optimization of new GAG-sensitive methods, such as gagCEST that can be used without specialized MR hardware. This study has some limitations. Only 12 patients were included in this study and it was not possible to compare our data with immunohistochemical quantification of the GAG content in cartilage and repair tissue. Another limitation was the fact that dGEMRIC was measured at 3 T and sodium MRI at 7 T. This was caused by high sensitivity of dual-flip-angle technique on Si inhomogeneities, which are more pronounced at 7 Tesla. dGEMRIC was therefore measured at 3 T. Another limitation was a relatively long and varying time interval between dGEMRIC and sodium MRI. Unfortunately, not all patients were willing to participate in 7-T sodium MRI immediately after the dGEMRIC measurement. Furthermore, we did not calculate absolute sodium concentrations. Since our sodium images were Ti- and T2- weighted, and the relaxation times in repair tissue were unknown, we could not calculated absolute sodium concentrations in cartilage and repair tissue. To our best knowledge, this is the first paper that applies sodium MRI for the evaluation of cartilage repair tissue. In conclusion, 3D GRE sequence optimized for cartilage imaging at 7 T provided sufficiently high sodium SNR for evaluation of native cartilage and repair tissue. A strong correlation between dGEMRIC at 3 T and sodium imaging at 7 T was observed in patients after MACT. In addition, sodium values in repair tissue were significantly lower than values in native cartilage. Sodium MRI at 7 T therefore might be useful for the noninvasive evaluation of the performance of surgery techniques for cartilage repair.

3.3 Paper 2: Evaluation of native hyaline cartilage and repair tissue with 23Na MRI at 7 T

The aim of paper 2 was to investigate whether sodium MRI is able to differentiate between the repair tissues produced by MACT and by BMS techniques in femoral cartilage [151],

110 DISCUSSION

Relatively simple and low-cost BMS procedures fill the defect mostly with fibrocartilage that have inferior structural, biochemical, and biomechanical properties compared to hyaline cartilage [69, 207, 208], More sophisticated and more expensive MACT repairtechniques use 3D-biodegradable scaffolds that provide hyaline-like repair tissue [85, 209], However, the role of MACT and BMS techniques in cartilage repair is not completely defined yet [70], The ability to observe changes in GAG content of repair tissue and native cartilage in a non-invasive way is therefore crucial for understanding of the impact of cartilage repair procedures. We found significantly lower sodium values in repair tissue than in reference cartilage of patients after BMS and MACT treatment. In addition, significantly higher sodium values were found in MACT repair tissue than in repair tissue after BMS techniques. In paper 1 (mentioned above), a strong correlation was observed between sodium and dGEMRIC results from MACT patients. Therefore, it is reasonable to presume that sodium MRI is able to detect changes in GAG concentration of native cartilage and repair tissue. Recent results therefore suggest that the MACT repair tissue has in general higher GAG content, and thus better biomechanical properties, than the repair tissue after BMS techniques. Additionally, both surgical techniques seems to produce repair that still has lower GAG content than native cartilage, which is in agreement with findings in paper 1. A limited amount of tissue harvested from repair area, which might not contain only one type of tissue and thus may not be representative of the whole transplant, could partly explain varying results of previous histological studies. Although Knutsen et al. observed hyaline-like repairtissue more often in patients after ACI, histological evaluations did not result in significant differences between repair tissue after MFX and ACI [70], On the other hand, characterized ACI resulted in repair tissue of superior histological quality (chondrocyte phenotype and tissue structure) when compared to MFX [210], Different studies showed hyaline-like tissue in 27 to 75% of biopsies from MACT patients [84, 211, 212], On the other hand, Gudas etal. found only fibrocartilage in biopsies from MFX repairtissue [213], Assuming that fibrocartilage (usually produced by MFX) has lower GAG concentration than hyaline-like repair tissue (often result of MACT) [214, 215], sodium results of paper 2 correspond well to previous histological findings. Since dGEMRIC proved to be sensitive to the GAG concentration in cartilage and in repair tissue, our sodium findings can be compared to previous dGEMRIC papers. Previous dGEMRIC papers also demonstrated a higher GAG content in reference cartilage than in repair tissue of MACT patients at different follow-up time-points [149, 172], In addition, the

111 DISCUSSION dGEMRIC studies also indicated a higher GAG content in repair tissue of MACT patients compared to patients after MFX [175], Saris et al. reported a significantly improved clinical outcome of patients after characterized ACI when compared to MFX patients [216], In our study, however, patients after the BMS treatment showed similar (statistically non-significantly different) clinical outcome when compared to the MACT patients. Although sufficient GAG concentration is important for correct cartilage function, patient’s clinical outcome (such as joint function, or pain) does not seem to be directly affected by the GAG concentration in repair tissue [217], This study has several limitations. Although presented sodium values are proportional to the GAG concentration, quantification of sodium concentration is necessary for reliable calculation of the GAG concentration in cartilage and repair tissue. For calculating sodium concentration, correction of sodium signal intensities for Ti, T2fasf and T2s,Ow relaxation times are necessary [205], Since sodium relaxations times have not yet been measured in repair tissue, this was not possible. Unfortunately, relaxation measurements are very time demanding and relaxation times may be different in various types of repair tissue. Anyhow, previous in vitro relaxation study [218] can provide approximate assessment of how much can different sodium relaxation times in repair tissue influence our sodium results. In this study, depletion of GAGs from cartilage lead to decrease in T2fasf time and increase in Ti and T2s/ow times. Even when the measured sodium values were corrected for the signal decrease due to the different relaxation times, we found significantly higher sodium values in the repair tissue after MACT than after BMS treatment. Moreover, change in relaxation times associated with cartilage degradation enhances contrast between native and degraded cartilage in Ti- weighted sodium images [199] which makes sequence even more attractive for clinical applications. Since resolution in sodium images is in general low, ROI evaluations are susceptible to partial volume effects, which could lead to over- or under-estimation of sodium signal. In order to minimize this error, we excluded voxels situated close to the interface between reference cartilage and repaired tissue from the ROIs. Similarly, voxels close to bone or synovial fluid were excluded from ROIs. Since we measured strongly Ti-weighted sodium images (TR of 10 ms), the contribution of signal from synovial fluid was minimized. A relatively low number of patients could be considered as another limitation of this study. However, nine pairs of patients matched for follow-up interval, age, and defect location was enough to observe significant differences between MFX and MACT patients. Another limitation is that sodium results were not compared to gold-standard histological evaluations of GAG

112 DISCUSSION concentration in repair tissue. Unfortunately, we were not able to obtain histological samples from repair tissue for GAG analysis due to strict ethical guidelines. To our knowledge, this is the first study that uses sodium MRI for comparing the repair tissue properties between patients after MACT and BMS treatment. Considering previous histological evaluations of repair tissue (BMS often result in fibrocartilage while MACT in hyaline-like repair tissue), our results suggest that sodium MRI can distinguish between the qualitatively different types of repair tissue produced by MACT and BMS repair. Our findings indicate that the MACT repair provides repair tissue with better biomechanical properties (with higher GAG concentration) than the BMS techniques. Sodium imaging at7 T might be useful for non-invasive assessment of biochemical properties in cartilage repair tissue and for the comparison of efficacy between different repair surgery techniques.

3.4 Paper 3: 23Na MRI of Ankle Joint in Specimens, Volunteers, and Patients at 7T

The aim of paper 3 was to compare the biochemical properties of repair tissues in patients after MACT and patients after MFX treatment of ankle cartilage using sodium MRI at 7 T [195]. The optimal treatment of cartilage defects in the talar dome is still a topic of discussion; and difference in clinical outcome between MFX and MACT is unclear. In various studies, good to excellent outcomes of patients after MFX [60, 219, 220] and after MACT [198, 221, 222] have been reported at short-term and mid-term. Direct comparison of the outcomes between MFX and MACT or ACI patients also showed similar results at midterm [223-225], These results are in agreement with our clinical findings. The MFX and MACT patients included in our study demonstrated comparable AOFAS and Modified Cincinnati Knee scores. Although the evaluation of repair tissue using sodium MRI was successfully demonstrated in the knee joint (e.g. paper 1 and paper 2 of this thesis), this method has not yet been applied in ankle. In first step, human cadaver ankle samples were measured in order to evaluate the sensitivity of sodium imaging to GAG content changes in thin cartilage layers. We found a strong correlation between the sodium values and histochemically assessed GAG concentration that suggests sufficient sensitivity of our sodium protocol for observation of

113 DISCUSSION differences in the GAG content of talar and tibial cartilage in the ankle joint. This finding encouraged further in vivo sodium measurements ofankle joint. Similarly to our previous results in the knee (paper 1 and paper 2 of this thesis), we observed significantly higher sodium values in talar reference cartilage than in repairtissue in patients after MFX and MACT. This indicates a significant decrease of GAG concentration and therefore less-favorable biomechanical properties of repair tissue than those of reference cartilage. In another ankle study, dGEMRIC (another method sensitive to changes in GAG content) also showed a lower GAG concentration in repair tissue produced by matrix-induced chondrogenesis in cartilage repair surgery than that in native cartilage [226], Since sodium values in talar and tibial reference cartilage were not different between groups of the patients and the asymptomatic volunteers, we can consider the selected reference cartilage in patients as representative of healthy normal cartilage. Unlike our previous knee study (paper 2 of this thesis), we did not observe significantly different sodium values between the MACT and MFX patients. Moreover, AOFAS and Modified Cincinnati Knee scores were not significantly different between MFX and MACT patients before surgery as well as after the cartilage repair. Successful cartilage repair in each MACT and MFX patient was demonstrated by an increase in both type of clinical scores when comparing status before and after cartilage repair. Several knee studies reported different biochemical properties between the repair tissues produced by MACT and MFX that were manifested by differences in T2 relaxation times [227, 228] as well as in sodium values (paper 2 of this thesis). However, sodium MRI did not show any significant difference between the repairtissue produced by MACT and MFX in ankle. Different findings from knee and ankle may be related to differences in the biochemical composition of cartilage from these two joints. Ankle cartilage has for instance greater capacity for repair than articular cartilage in the knee joint [115], Additionally, a recent 7 T study employing T2 mapping of talar cartilage also did not report any significant differences between repair tissue after MACT and MFX [223], Sodium values reported in our study did not correlate either with the AOFAS, the Modified Cincinnati Knee Rating, or the MOCART score. These findings suggest that sodium imaging offers information that is different from that provided by clinical outcome scores or by morphological MRI. Both surgery techniques (MACT and MFX) demonstrated lower sodium values in repair tissue of patients with longer follow-up period than in patients with shorter follow-up period. Sodium imaging could be used to explore the relationship between the GAG concentration and the resilience of repair tissue over time. Since repair tissue after MFX

114 DISCUSSION seems to be more prone to degeneration at mid-term and long-term, long-term follow-up studies employing sodium imaging might help to assess optimal clinical algorithms for cartilage repair surgery in the ankle joint. One ofthe limitations in this study was the low spatial resolution of evaluated sodium images (1.79 x 1.79 x 3.0 mm3). Cartilage layers in the ankle joint are thin (mean thickness of talar cartilage is about 1.35 mm in males and 1.11 mm in females [229]), which leads to partial volume artifacts from the surrounding structures and could result in overestimation (due to synovial fluid in a voxel) or underestimation (due to bone in a voxel) of sodium MR signal and thus affect accuracy of results. In order to minimize this type of artifact, ROI selection was made on morphological images and then transferred to the corresponding sodium images. Additionally, because selected ROIs covered the whole repair site or native cartilage on a selected slice, the impact of overall partial volume artifacts on mean sodium values was averaged and thus similar in different ROIs in different patients. This is illustrated by Figure 6B, where the difference in sodium values found between repair tissue and reference cartilage is not substantially influenced by the cartilage thickness in ROI. Moreover, since reported sodium results are relative numbers, it is not necessary to correct the results for systematic bias caused by partial volume artifacts. Another limitation in our results is the fact that although Ti and T2* relaxation times in repair tissue are probably slightly different from those in native cartilage, they are still unknown. This is mainly due to very time demanding relaxation time measurements but also because relaxation times in repair tissue could depend on a type of repair surgery technique as well as the follow-up interval. Thus, we used the Ti and T2* relaxation times of cartilage of heathy volunteers [11, 203, 230] for correcting values in repair tissue as well as native cartilage in the sodium maps. Limitations also include a relatively low number of patients and different follow-up interval in the evaluated patients. Since previously mentioned limitations (e.g. partial volume effects) could affect sodium MR signal, we did not evaluate the sodium concentrations in the repair tissue or reference cartilage. Nevertheless, the presented sodium values should be similar to the sodium concentrations expected in the repair tissue and native cartilage. In conclusion, this is the first study demonstrating feasibility of quantitative biochemical evaluation of native cartilage and repair tissue in the ankle joint by using in vivo sodium MRI at 7 T. In contrast to our previous knee study (paper 2 of this thesis), both MACT and MFX repair techniques provided repair tissue with similar sodium values. However, both types of repair tissue were of inferior biochemical (and thus also biomechanical) properties compared

115 DISCUSSION with that in reference cartilage. Since sodium concentration in native cartilage and repair is proportional to the GAG concentration, sodium imaging can offer the unique information on the biomechanical properties of cartilage repair tissue in ankle joint and allows the follow-up of patient in a noninvasive way.

3.5 Conclusion and Future Prospects

In conclusion, the papers presented in this thesis demonstrate that sodium MRI at 7T is a useful technique that can provide the unique biochemical and biomechanical information on the cartilage repair tissue produced by different repair surgery procedures in knee and ankle joints. We validated our MR protocols by comparing sodium results with other GAG­ sensitive methods or with histochemically assessed GAG concentration and proved their sensitivity to even small changes in cartilage GAG content. Although histologic analysis of biopsy samples is regarded as a reference standard for the evaluation of cartilage repair tissue, the main advantage of sodium imaging is its noninvasive character. Sodium MRI can be therefore used in larger cohorts as well as for regular follow-up of patients after cartilage repair, which is not possible by biopsy due to ethical reasons. As shown in this thesis, sodium MRI enables to distinguish not only between repair tissue and native cartilage, but also between qualitatively different types (i.e. with different GAG concentration) of repair tissues that are created by different surgery techniques. While our results from knee suggest that MFX results in repair tissue of lower biomechemical quality (lower GAG concentration) than the MACT technique, these procedures provide repair tissue with similar GAG content in the anklejoint. Unfortunately, cartilage repair surgery technique that will provide repairtissue with the GAG concentration similar to the concentration found in native cartilage has not been developed so far, leaving room for new repair procedures. Thus, noninvasive sodium imaging is perfectly suited for the long-term in vivo evaluation of the performance of new and promising surgery procedures developed for cartilage repair. Sodium MRI has a potential to detect early biochemical changes associated with cartilage degeneration. Since biochemical changes precede morphological alterations, sodium imaging may be able to detect cartilage degeneration before the damage becomes irreversible. However, the sensitivity of sodium measurements needed to be further improved before being used for detection of small changes in cartilage sodium or GAG concentration. This goal can be achieved by using more advanced acquisition and postprocessing

116 DISCUSSION techniques that will increase SNR and resolution in sodium images and therefore decrease the influence of partial volume artifacts on results. The easiest way to increase SNR is to decrease TE by using radial (center-out) acquisition schemes. Although radial sequences (such as radial projection imaging, twisted projection imaging, or 3D spirals) provide images with higher intrinsic SNR, they are very sensitive to off-resonance effects, imperfections in gradient waveforms and Bo inhomogeneities that often results in blurring, especially in imaging of knee at 7 T. On the other hand, more robust Cartesian sampling suffers from excessive signal loss caused by much longer TE used in GRE sequences. Since the newly developed vTE-GRE sequence combines robustness of Cartesian sequence with relatively short TE (~ 1 ms), it seems to be currently the optimal method for sodium MRI of cartilage. We plan to use this sequence for the evaluation of cartilage degeneration process in patients after injury over a period of six months. vTE-GRE will be also used to evaluate whether a systemic disease such as type 1 diabetes mellitus could influence the cartilage biochemical and biomechanical properties. Another project we are currently working on is the implementation of coherent steady state free precession (SSFP) sequences (such as true-FISP) for sodium imaging of cartilage. Although these techniques can provide significantly higher SNR compared to spoiled sequences, they have not yet been investigated for sodium MRI. The main disadvantages of SSFP sequences seems to be banding artifacts and the dependence of the sodium signal on T1/T2 factor of measured tissue.

117 MATERIALS AND METHODS

4. MATERIALS AND METHODS

4.1 Subjects and Specimens

Papers 1 to 3 were based on cross-sectional studies that were approved by the institutional review board. Written informed consent was obtained from all patients and volunteers before their participation in the study.

4.1.1 Subjects

Exclusion criteria for patients and volunteers were metallic implants, pain in the evaluated joint, advanced OA or rheumatoid arthritis (grade 3 or 4 chondral defect according to ICRS classification), kissing lesions, ligament injuries of the joint or meniscal tear (based on morphological MRI), and joint malalignment or joint instability (evaluated using clinical examination [231]). Furthermore, subjects with contraindications to MR examination (e.g. metallic implants, pregnancy, claustrophobia) and to application of the contrast agent (only for paper 1) were excluded from the study. Only volunteers without pain, trauma or surgery in the knee or ankle joint participated in the study. Additionally, only patients with single symptomatic chondral (Outerbridge grade 3 or 4) or osteochondral defect (Hepple stage 3 or 4) with stable cartilage adjacent to the defect site that was usually caused by trauma were enrolled in the studies. Paper 1. All MACT patients were recruited from a cohort of 45 follow-up patients (mean age ± standard deviation, 34.7 ± 13.2 year; range, 20 - 55 years) who had undergone 3 T MRI. 14 MACT patients agreed to participate in our study and underwent an additional sodium and morphological MR measurements at 7T between February and October 2009. Two patients were excluded: a 53-year-old man due to technical problems and a 60-year-old woman due to severe OA. Finally, six men (mean age, 32.3 ± 12.7 years; age range, 23 - 55

118 MATERIALS AND METHODS years) and six women (mean age, 32.8 ± 8.2 years; age range, 22 - 44 years) were included into further evaluations. The MACT repair tissue was located at the medial femoral condyle (eight patients), at the lateral femoral condyle (three patients) or at the trochlea (one patient). The mean postoperative interval was 56 ± 28 months (range, 11-102 months). Paper 2. From a large cohort of 135 follow-up patients, 29 patients after MACT and 15 patients after BMS cartilage repair were willing to participate in our study. From this group of patients, best matching BMS - MACT pairs were selected according to following criteria: difference between postoperative intervals was less than three months, age difference was less than 40 months and similar location of cartilage repair tissue. Finally, nine patients after MACT (six men, three women; mean age, 36.9 ± 10.0 years; age range, 24.6 - 56.0 years; mean postoperative interval, 33.2 ± 25.7 months; mean body mass index, 23.8 ± 2.4 kg/m2; defect size range, 3.2 - 7.3 cm2) and nine patients after BMS cartilage repair (seven MFX patients, two Pridie drilling patients; five men, four women; mean age, 36.7 ± 10.7 years; age range, 21.4 - 57.7 years; mean postoperative interval, 33.5 ± 25.3 months; mean body mass index, 25.1 ± 2.4 kg/m2; defect size range, 1.5 - 3.6 cm2) were selected and underwent MRI measurements at 7 T between July 2009 and September2010. The repairtissue was located at the medial femoral condyle (five MACT and five BMS patients), at the lateral femoral condyle (two MACT and four BMS patients) or at the trochlea (two MACT patients). For MACT treatment, the following 3D scaffolds were used: Hyalograft C (Fidia Advanced Biopolymers, Abano Terme, Italy), CaReS (Arthro Kinetics, Esslingen, Germany), or BioCart II (ProChon Biotech, Woburn, MA). All patients followed the same rehabilitation protocols after cartilage repair surgery [207, 232], Both, Pridie drilling and MFX, rely on bone marrow stimulation which results in a similar type of repair tissue. Patients after Pridie drilling and after MFX were therefore included in the same BMS group. Paper 3. In order to acquire baseline sodium data from ankle cartilages, nine asymptomatic volunteers without trauma or surgery of ankle joint (three men, six women; mean age, 28.7 ± 5.5 years; age range, 23 - 39 years; mean body mass index, 23.9 ± 2.7 kg/m2) were included into this study. Additionally, patients of similar age, size of repair tissue and body mass index were recruited form a cohort of about 70 patients after cartilage repair of talar dome. Six MFX patients (four man, two women; mean age, 35.8 ± 10.0 years; mean postoperative interval, 99.5 ± 33.1 months; mean body mass index, 27.0 ± 5.5 kg/m2; mean defect size, 1.26 ± 0.41 cm2) and six MACT patients (two man, four women; mean age, 35.0 ± 6.0 years; mean postoperative interval, 85.7 ± 23.9 months; mean body mass index, 26.1 ± 3.1 kg/m2; mean defect size, 1.53 ± 0.45 cm2) received additional 7 T MRI of anklejoint. For

119 MATERIALS AND METHODS

MACT treatment, Hyalograft C scaffold (Fidia Advanced Biopolymer, Abano, Italy) was used in four patients, CaReS scaffold (Arthro Kinetics, Esslingen, Germany) in one patient, and Carticel scaffold (Genzyme Biosurgery, Cambridge, MA) also in one patient [233],

4.1.2Human Specimens

Human specimens were used only in paper 3. Eight fresh-frozen samples of ankle joints (mid-tibia to foot base) from human cadavers were provided by the Department of anatomy at Medical University of Vienna. Based on findings in morphological images, specimens with advanced osteoarthritis or with chondral defects (ICRS grade 3 or 4) situated on talar or tibial cartilage were excluded from further evaluations. Therefore only five ankle samples (from two men and three women; median age, 46 years; age range, 41 - 60 years) were included into evaluations.

4.2 Clinical Outcome

In papers 2 and 3, the IKDC Subjective Knee Form, the Modified Cincinnati Knee Rating or the AOFAS score were used for the evaluation of clinical outcome in patients after cartilage repair. The IKDC Subjective Knee Form evaluates the level of knee function, symptoms, and managing of daily and sport activities of patients with problems in the knee [52], Scores are between 0 and 100 points, where later denotes no symptoms and no limitations in patients’ activities. The Modified Cincinnati Knee Rating reflects the knee function and the limitations in managing daily activities [59], Scores are between 0 and 10 points, where 10 refers to unlimited functionality. The AOFAS score measures function of ankle joint and perception of pain in patients [60], The maximum score is 100 points (50 points for function, 40 for pain and 10 for alignment of the ankle joint). Higher scores suggest better function and lower pain.

4.3 MR Imaging

120 MATERIALS AND METHODS

4.3.1 Morphological MR Imaging

All morphological images were acquired using either a 3 T (paper 1; TimTrio) or a 7 T (paper 1, 2 and 3; Magnetom) MR whole-body system (both from Siemens Healthcare, Erlangen, Germany). A 1H-only receive-only eight-channel knee phased array coil (paper 1; Invivo, Gainesville, FL, USA) ora 1H-only transmit/receive 28-channel knee phased array coil (paper 2 and 3; Quality Electrodynamics LLC, Cleveland, OH, USA) were used. For better comparison, subjects were positioned in the same way for morphological and sodium MRI measurements. The identical housing of the 23Na-only 15-channel knee coil and the 1H-only 28-channel knee coil was therefore very helpful.

Paper - Flip Band­ Nominal Acquisitio TR/TE MR Sequence angle width resolution n time (ms) system (deg.) (Hz/pix.) (mm3) (min:sec)

1 - s. PD-w. TSE 2400 / 38 160 244 0.2x0.2x2. 6:11

3T s. Dual TSE 5120/10, 67 140 203 0.4x0.4x3. 6:46

c. TRIM 7690 / 41 150 250 0.6x0.6x3. 2:35

1 - s. T2-w. DESS 16/5 22 283 0.8x0.8x0. 7:16

7T s. PD-w. TSE 2500/ 11 160 240 0.5x0.5x3. 5:52

2- s. PD-w. TSE 3400 / 26 125 243 0.4x0.4x3. 4:20

7T c. PD-w. TSE 2400 / 24 122 243 0.4x0.4x3. 1:21

s. T-i-w. GRE 8.3/3.6 8 450 0.4x0.4x0. 6:09

3- s. PD-w. TSE 4000 / 26 145, 243 0.3x0.3x3. 3:18 170 7T c. PD-w. TSE 3000 / 25 179 243 0.3x0.3x3. 2:29

hr PD-w. TSE 4610/24 140 254 0.3x0.3x1. 5:10

Table 4.1: Parameters of morphological sequences. Note: s. is for sagittal plane, c. is for coronal plane, hr is for high resolution and TRIM is for turbo inversion recovery magnitude.

121 MATERIALS AND METHODS

Morphological appearance of cartilage and repair tissue was evaluated by using different 1H sequences. The backbone of morphological assessment was a sagittal PD- weighted 2D turbo spin echo sequence with fat suppression. Other sequences, such as DESS and GRE sequences, were also used. More details on morphological sequences can be found in Table 4.1. The acquisition of different morphological sequences and localizers took from 10 to 15 minutes per patient.

4.3.2 Ti Delayed Gadolinium-enhanced MR Imaging

In paper 1, sodium results were compared with the findings from dGEMRIC measurements performed at 3 T (TimTrio; Siemens Healthcare, Erlangen, Germany) using a 1H-only eight-channel knee phased array coil (Invivo, Gainesville, FL, USA). ForTi mapping of cartilage before and after contrast agent administration, we used a dual-flip-angle 3D GRE sequence with the following parameters: (TR= 15 ms, TE= 3.94 ms, flip angles= 5 and 26 degrees, BW= 490 Hz/pixel, nominal resolution= 0.4x0.4x3.0 mm3, acquisition time= 3:46 min) [167], The postcontrast Ti maps were obtained 90 minutes after the intravenous injection [163] of a bolus of 0.2 mmol per kilogram body weight of gadopentetate dimeglumine contrast agent (Magnevist; Bayer Schering Pharma, Berlin, Germany). After injection, patients were asked to walk up and down stairs for 20 minutes. The exercise and time delay facilitated the transport of contrast agent molecules into cartilage and repair tissue. Unfortunately, not all patients were willing to undergo 7 T exams right after the 3 T scans. Thus, the time between 3 T and 7 T examinations ranged between 0 and 210 days (mean time, 70.5 days; standard deviation, 80.1 days).

4.3.3Sodium Imaging

All sodium MRI measurements were acquired on a 7 T MR whole body system (Magnetom; Siemens Healthcare, Erlangen, Germany) with a bore size of 60 cm and an SC 72 gradient system (maximal gradient strength of 70 mT/m with a gradient slew rate of 200 T/m/ms) (Fig. 4.1 A). In paper 1 and 2, sodium images were recorded using a 23Na-only birdcage transmit-receive knee coil (Stark Contrast, Erlangen, Germany) (Fig. 4.1 B). Sodium

122 MATERIALS AND METHODS images in paper 3 were acquired with a 23Na-only 15-channel phased array knee coil (with birdcage transmit coil) (Quality Electrodynamics LLC, Cleveland, OH) (Fig. 4.1 C). For more accurate comparison between subjects, a reference sample containing 308 mmol/L NaCI solution was attached to both sodium coils during the measurements and served as a sodium MR signal intensity reference. All sodium images were reconstructed directly on the MR console. MR signals from the array coil elements were reconstructed using adaptive combine algorithm.

Fig. 4.1 : Hardware for sodium MR imaging. A: Magnetom 7 T MR whole body system (Siemens Healthcare, Erlangen, Germany). B: 23Na-only birdcage transmit-receive knee coil (Stark Contrast, Erlangen, Germany). C: 23Na-only 15-channel phased array knee coil (Quality Electrodynamics LLC, Cleveland, OH).

All sodium images were recorded with a standard 3D GRE sequence using measurement parameters optimized for cartilage (Table 4.2). The length of an excitation pulse was set to comply with specific absorption rate limits and to minimize TE (970 ps in paper 1, 2 and 950 ps in paper 3). We used asymmetric readout in combination with a relatively small acquisition BW (readout length range, 5.9 - 12.5 ms) to improve the SNR per unit of time in

123 MATERIALS AND METHODS sodium sequences. Due to the small BW, sodium sequences were measured with a relatively long TE (between 3.8 ms and 8.3 ms), which resulted in the minimal contribution of a fast decaying signal (2^) to the total sodium signal. Therefore, the broadening of the point- spread-function caused by 2^ component was also minimal. The total sodium MRI protocol including localizers and the calibration ofthe reference pulse took about 35 minutes.

TR/TE Flip angle BW Nominal Acquisition Paper (ms) (degrees) (Hz/pixel) resolution (mm3) time (min:sec)

1 10/3.77 56 170 1.6x3.1x3.0 30:45

2 10/3.77 56 170 1.6x3.1x3.0 30:45

3 17/8.34 50 80 1.8x1.8x3.0 31:53

Table 4.2: Measurement parameters ofthe 23Na 3D GRE sequences that were used in papers 1,2 and 3.

4.4 Postprocessing and Evaluations of MR Images

4.4.1 Morphological Evaluation of Repair Tissue

In all papers, MOCART scoring system was applied for the morphological evaluation of the cartilage repair site (including surrounding bone) of all patients [137], MOCART is a semiquantitative reproducible grading system based on morphological MR images that has been used for the evaluation of repair tissue after different cartilage repair surgery techniques in the knee [138, 234] and anklejoint [224, 235], A senior musculoskeletal radiologist assigned a point score to each variable in the MOCART scoring system [137], The MOCART score offers a point scale with different weighting to each variable. The score of 100 is a maximum and it refers to excellent morphological outcome.

124 MATERIALS AND METHODS

MOCART Scoring System Variable Classes Score Degree of defect repair Complete (on a level with adjacent cartilage) 20 and defect fining Hypertrophy (over the level of the adjacent cartilage) 15 Incomplete (under the level of the adjacent cartilage) >50% of the adjacent cartilage 10 <50% of the adjacent cartilage 5 Subchondral bone exposed (complete delamination or 0 Integration to border Complete (complete integration with adjacent cartilage) 15 zone Incomplete (incomplete integration with adjacent cartilage) 10 Defect visible <50% of the length of the repair tissue 5 >50% of the length of the repair tissue 0 Surface of the repair Surface intact (lamina splendens intact) 10 tissue Surface damaged (fibrillations, fissures and ulcerations) <50% of repair tissue depth 5 >50% of repair tissue depth or total 0 Structure of the repair Homogeneous 5 tissue Inhomogeneous or cleft formation 0 Signal intensity of the Dual 12 w. FSE repair tissue Isointense 15 Moderately hyperintense 5 Markedly hyperintense 0 3D-GKE Isointense 15 Moderately hypointense 5 Markedly hypointense 0 Subchondral lamina Intact 5 Not intact 0 Subchondral bone Intact 5 Oedema, granulation tissue, cysts, sclerosis 0 Adhesions No 5 Yes 0 Effusion No effusion 5 Effusion 0 Mai score 100

Table 4.3: MOCART scoring system. Note: FSE stands for fast spin echo and GRE is for gradient echo.

125 MATERIALS AND METHODS

In paper 3, morphological images of patients were additionally used to measure the thickness of repair tissue and reference cartilage. The mean thickness was calculated from five measurements in the same regions were sodium values were assessed.

4.4.2 Evaluation of Postcontrast Ti Maps

From dGEMRIC measurements in paper 1, the postcontrast Ti maps were calculated directly on the MR console using the Maplt software (Siemens Healthcare, Erlangen, Germany). The location of repair tissue on Ti maps was verified by using morphological images and surgical reports. ROIs were defined by a senior musculoskeletal radiologist (Prof. Siegfried Trattnig) in the repair tissue and reference cartilage using an in-house written IDL script for data analysis (Interactive Data Language; Research Systems, Boulder, CO, USA). To improve comparison between postcontrast Ti values from 3 T and sodium data from 7 T, the corresponding sections were selected and evaluated side-by-side. In postcontrast Ti maps and in sodium images, ROIs with similar area were selected in reference cartilage and in repair tissue. The mean difference in ROIs area between the postcontrast Ti maps and the sodium images was 5% (range, 1% - 12%). Moreover, to minimize systematic errors, a dGEMRIC Ti ratio was calculated by dividing values from reference cartilage by values from repair tissue.

4.4.3 Postprocessing of Sodium Images

Due to spatially inhomogeneous Si field of a 15-channel phased array knee coil used in paper 3, sodium images had to be corrected before further evaluation. However, this was not the case for paper 1 and 2, where the birdcage type of the coil with a homogenous Si field was used. For correcting sodium images, we used images of a homogenous saline phantom of cylindrical shape. First, sodium images of phantom were measured with the same geometry and orientation as the images of volunteers, patients and samples. Correction matrices were then calculated by inverting sodium signal intensities from phantom images and rescaling them to the values from 1 to 4095. At the end, the original sodium images were multiplied

126 MATERIALS AND METHODS with the corresponding correction matrices to provide corrected sodium images. All corrections were performed automatically on a pixel-by-pixel basis using scripts written in IDL (Interactive Data Language; Research Systems, Inc, Boulder, CO, USA) and Matlab (The Mathworks, Natick, MA, USA). Since the evaluation of sodium concentration from Ti- and T2-weighted images is not as accurate as that from density-weighted images (TE < 1 ms, TR > 100 ms), we calculated so-called corrected signal intensities that should be close to actual sodium concentrations in the cartilages in paper 3. To obtain corrected signal intensities, five 10% wt/wt agarose gel phantoms with different sodium concentrations (100, 150, 200, 250, and 300 mmol/L) were measured together with each subject. From the images corrected for the inhomogeneous Si field, equilibrium magnetization Mo in phantoms and in subjects was calculated by correcting sodium signal intensities for a 50-degree flip angle and for their relaxation times (phantoms: Ti= 30.1 ms, T2*= 6.2 ms, all subjects: Ti= 20 ms, T2*= 4.5 ms). Since relaxation times ofthe cartilage repair tissue were not measured until now, we used the previously published relaxation times of healthy native cartilage [11, 203, 230] for the evaluation of the reference cartilage and the repair tissue. A linear calibration curve was then calculated by plotting corrected sodium signal intensities from phantoms against their sodium concentrations. Corrected signal intensities of cartilage and repair tissue were evaluated using linear regression analysis. Finally, these signal intensities were divided by an average water content in the cartilage (70%) that was obtained from histochemical analyses of ankle specimens. Treppo et al. reported a similar water content of 75% [116],

4.4.4 Evaluation ofSodium Images

All ROIs in reference cartilage and in repair tissue of patients were defined by a senior musculoskeletal radiologist (Prof. Siegfried Trattnig). In all papers, SNR is defined as mean signal intensity from a ROI divided by the standard deviation of the signal intensity in the empty (MR signal-free) area. Normalized sodium signal intensities were calculated by multiplying the mean signal intensity from a ROI with the normalization factor. This factor was defined as the ratio between the highest mean signal of reference sample from all patient measurements and the mean signal of reference sample in the evaluated patient.

127 MATERIALS AND METHODS

Paper 1. All ROIs were selected according to morphological images and drawn in a script written in IDL (Interactive Data Language; Research Systems, Boulder, CO, USA). In each patient, a repair ROI was defined to cover the whole cartilage repair tissue and another ROI was selected in equally sized region of normal native cartilage. SNR values were then calculated for repair tissue and for reference cartilage of each patient. CNR was calculated as difference of signal intensities between native cartilage and repair tissue divided by the standard deviation of the signal in the empty (MR signal-free) area. For better comparison among patients, normalized sodium signal intensities were evaluated in reference cartilage and repairtissue. A normalized sodium ratio, defined as the ratio between normalized sodium values from reference cartilage and from repair tissue, was also calculated. For evaluation of the inter-reader agreement, another radiologist (Dr. Marius Mayerhöfer) reevaluated independently all patients using the same IDL script. Paper 2. One sodium image containing the largest repair tissue area and the corresponding morphological image were selected in each patient. ROI covering the whole area of repair tissue and ROI in morphologically normal appearing cartilage with weight­ bearing similar to those in repair tissue were defined on the selected morphological images. Special care was taken to define the reference ROIs (mean area, 24.6 ± 7.6 mm2; range, 12.1 - 38.7 mm2) of similar size as the repair ROIs (mean area, 26.1 ± 7.4 mm2; range, 15.5 - 38.7 mm2). Furthermore, resolution of sodium images was rescaled to the resolution of morphological images using picture editing software (Adobe Photoshop CS2; Adobe Systems Incorporated, USA). The rescaled sodium images were then overlaid with corresponding morphological images and the ROIs were transferred to sodium images and evaluated in the JiveX viewer (JiveX 4.3, VISUS Technology Transfer GmbH, Bochum, Germany). Sodium SNR values and normalized mean signal intensities were calculated for reference cartilage and repairtissue in each patient. The sodium repair-to-reference signal intensity ratio, defined as a ratio of signal intensity from the repair ROI and the signal intensity from the reference ROI, was also evaluated. For estimating the reproducibility of sodium measurements, one patient was measured twice. Comparison of normalized mean signal intensities showed good reproducibility of measurements, with the mean difference of 3.12% (range, 0.46 - 5.25%) and the mean coefficient of variation of 2.25% (range, 0.32 - 3.81%). Paper 3. All ROIs were selected on morphological images. Maps of sodium corrected signal intensities (described in the previous chapter) were rescaled to match spatial resolution of morphological images and overlaid with morphological images. Finally, the ROIs were transferred to maps of sodium corrected signal intensities and mean values were calculated

128 MATERIALS AND METHODS in the JiveX Viewer (JiveX 4.3; VISUS Technology Transfer GmbH, Bochum, Germany). For each patient, one sodium image with the largest repair tissue area and the corresponding morphological image were selected and a repair ROI covering the whole area of repair tissue was defined. Additionally, two reference ROIs were defined on the side (lateral or medial) opposite to the side where cartilage repair was performed. Reference ROIs were selected to cover the whole morphologically normal tibial and talar cartilage on the side (medial or lateral) opposite to the cartilage repair site. Special care was taken to prevent from including synovial fluid into the ROIs. Three sodium corrected signal intensities were calculated for each patient. For each volunteer, two contiguous morphological images from the medial side and two from the lateral side were used for evaluations. In each image, four ROIs were selected to encompass the whole cartilage: talar and tibial cartilage of the ankle joint, calcaneal and talar cartilage of the subtalar joint. The sodium corrected signal intensities from the same type of cartilage at the same side were averaged. Thus, eight values per volunteer was obtained. When comparing with patients, the sodium values form medial and lateral side of volunteers were averaged together. For each ankle specimen, two morphological images from the medial, two from the central and two from the lateral side of the joint were chosen. In each image, two ROIs covering the whole cartilage were selected: one in talar and one in tibial cartilage of ankle joint. Normalized sodium signal intensities were calculated and the values from the same type of cartilage at the same side (medial, central, lateral) were averaged. Thus, six values were calculated in each ankle specimen.

4.5 Histochemical Analyses of Cartilage Samples

For the biochemical evaluations, four to six pieces of cartilage (volume ~1 mm3) were harvested from each (medial, central, lateral) segment of talar and tibial cartilage of all ankle samples (Fig. 4.2). Each cartilage piece was weighted to obtain wet weight. Next, the cartilage pieces were dried at 60°C for 24 hours and weighted again to calculate dry weight. Finally, wet and dry weights served for the evaluation of water content in talar and tibial cartilage. For the evaluation of GAG content, all cartilage pieces were enzymatically digested. Optical absorbance of the digested tissue at 656 nm was evaluated on a microplate spectrophotometer (Fluostar-Optima; BMG-Labtech, Offenburg, Germany), using the Blyscan Sulfated Glycosaminoglycan Assay kit (Biocolor Ltd, Belfast, Ireland). Finally, a calibration

129 MATERIALS AND METHODS measurements of chondroitin-4 sulfate provided a standard curve that was used for calculation of the sulfated GAG concentration. Finally, the GAG concentration in one cartilage segment was calculated as the ratio of the sum of GAG concentrations to the sum of wet weights of all pieces ofthe cartilage in one compartment and expressed in percent.

Fig. 4.2 : Illustration of a medial, central and lateral segment of talar cartilage in the ankle sample. For the biochemical evaluations, four to six cartilage pieces (blue dots) were harvested from each cartilage segment. In a similar way, tibial cartilage was divided and harvested [195],

4.6 Statistical Analyses

The SPSS software (v. 15 and 17, SPSS Institute, Chicago, IL, USA; v. 21.0, IBM SPSS Statistics) was used for statistical analyses and a p-value < 0.05 was regarded as statistically significant. For the evaluation of linear association between two variables (e.g. sodium values and MOCART scores), a Pearson correlation coefficient (r) and a coefficient of determination (R2) were calculated. Based on the value of correlation coefficient, the strength of linear association between variables was classified as strong (r > 0.7), moderate (r = 0.5 - 0.7), low (r = 0.3 - 0.5), or as none correlation (r < 0.3). Paper 1. Assuming a large effect (e= 1), a power analysis showed that 12 patients will be enough to achieve a statistical power of 85%. A three-way univariate analysis of variance with one random factor and two inner-subject factors was used for the evaluation of differences between repair tissue and reference cartilage. A paired t-test value and the

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Pearson correlation coefficient were determined for the evaluation ofthe agreement between two readers. Paper 2. A two-way repeated measures analysis of covariance with one within-subject effect (tissue type) and four covariates (treatment type, age, follow-up interval, and body mass index) was employed for the evaluation of differences in sodium values between reference cartilage and repair tissue of patients after MACT and BMS surgery techniques. A two-tailed paired t-test was used for all post hoc evaluations of sodium values. A Bonferroni-Holm correction was applied to compensate for errors resulting from performing multiple tests. Paper 3. A one-way analysis of variance with one factor (treatment type) was evaluated for comparing sodium values of talar and tibial reference cartilage between volunteers, MACT and MFX patients. A mixed-model analysis of variance with two factors (tissue type, and treatment type) was done in order to compare the sodium values between reference cartilage and repair tissue of MACT and MFX groups. A set of two-tailed t-tests with a Bonferroni-Holm correction was used for post hoc comparison. A two-way repeated measures analysis of variance with two factors (cartilage type and side) was performed to evaluate differences in sodium values between the medial and lateral sides and among four different types of cartilage (talar and tibial cartilage of the ankle joint, calcaneal and talar cartilage of the subtalar joint) in volunteers. A Bonferroni-Holm corrected t-tests were calculated in post hoc evaluations. Independent samples t-tests were calculated to compare clinical scores and MOCART scores between the MACT and MFX groups.

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151 APENDIX

6. APENDIX

6.1 Paper 4: Sodium MR Imaging of Articular Cartilage Pathologies

This review article was published as a part of Cartilage Zrnag/ng Topical Collection in Current Radiology Reports, 2014. Since the review was published as open access, it was not necessary to obtain a permission for reproduction in this PhD-thesis [201],

152 Curr Radiol Rep (2014) 2:41 DOI 10.1007/s40134-014-0041-4

CARTILAGE IMAGING (S TRATTNIG, SECTION EDITOR)

Sodium MR Imaging of Articular Cartilage Pathologies

Stefan Zbyn • Vladimir Mlynarik • Vladimir Juras • Pavol Szomolanyi • Siegfried Trattnig

Published online: 20 February 2014 © The Author(s) 2014. This article is published with open access at Springerlink.com

Abstract Many studies have proved that noninvasive Mechanical injury is a major cause of articular cartilage sodium MR imaging can directly determine the cartilage destruction in young patients. Different surgical cartilage GAG content, which plays a central role in cartilage repair techniques have been developed for treatment of homeostasis. New technical developments in the recent cartilage defects. One of the goals of these procedures is to decade have helped to transfer this method from in vitro to replace the defect with a newly produced tissue that has an pre-clinical in vivo studies. Sodium imaging has already identical structure, composition and biomechanical prop­ been applied for the evaluation of cartilage and repair tis­ erties as native articular cartilage [1]. Thus, noninvasive sue in patients after various cartilage repair surgery tech­ biochemical MR imaging methods might be useful for the niques and in patients with osteoarthritis. These studies evaluation of repair tissue and efficacy of different repair showed that this technique could be helpful not only for surgery techniques. assessment of the cartilage status, but also predictive for Osteoarthritis (OA) is a leading cause of chronic dis­ osteoarthritis. However, due to the low detectable sodium ability in the elderly population and the most common form MR signal in cartilage, sodium imaging is still challenging, of arthritis in synovial joints [2]. Although radiography or and further hardware and software improvements are nec­ morphological MR imaging may be useful for assessing essary for translating sodium MR imaging into clinical structural changes in knee OA and can indicate the need for practice, preferably to 3T MR systems. knee joint replacement, both techniques are insensitive to biochemical changes in the cartilage, which occur in early

Keywords Sodium MR imaging • Cartilage • Repair stages of OA before morphological changes appear [3]. tissue • Osteoarthritis Various MR techniques have been developed for non­ invasive biochemical evaluation of articular cartilage and to repair tissue. The most prominent methods are T2 Introduction mapping, delayed gadolinium-enhanced magnetic reso­ nance imaging of cartilage (dGEMRIC) [4], T1q mapping Since the intrinsic repair capacity of articular cartilage is [5], glycosaminoglycan chemical exchange saturation very limited, cartilage health is very important for the transfer (gagCEST) [6] imaging and sodium MR imaging health of the whole joint. [7]. Besides articular cartilage, sodium imaging was also used for evaluation of other musculoskeletal tissues such as the Achilles tendon [8] or skeletal muscles [9]. The goal of this review is to describe the advances in This article is part of the Topical Collection on Cartilage Imaging. sodium MR imaging of cartilage as a potential biomarker

S. Zbyn (&) • V. Mlynarik • V. Juras • P. Szomolanyi • for evaluation of OA and for examining the efficacy of S. Trattnig cartilage repair surgery. In the following chapters, we High Field MR Center, Department of Biomedical Imaging and provide a short description of the cartilage composition, an Image-Guided Therapy, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria overview of sodium MR properties and sodium pulse e-mail: [email protected] sequences, evaluation of the glycosaminoglycan (GAG)

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153 41 Page 2 of 10 Curr Radiol Rep (2014) 2:41 content in cartilage, basic information and recent sodium femoral cartilage is about 320 times lower than the proton imaging studies on repair tissue and OA cartilage. one. Moreover, sodium in biological tissues exhibits very short biexponential transversal relaxation times (T2). As a result of these factors, the sodium signal-to-noise ratio Composition of Normal Cartilage (SNR) in cartilage is about 3,400 times smaller compared to the proton SNR. Thus, sodium MR imaging is chal­ Articular cartilage contains a small amount of chondro­ lenging, and sodium images are acquired with lower SNR cytes (*2 % of total cartilage volume) nested in an (10-40), lower resolution (2-5 mm) and longer measure­ extracellular matrix. The main components of the matrix ment times (10-30 min) than proton images. are water, collagen and proteoglycans [10]. Sodium has a spin number of 3/2, which results in The relative concentration of water increases from about specific MR properties. In addition to the magnetic dipole 65 % in the deep zone up to 80 % in the superficial zone of moment, sodium also exhibits a quadrupole moment, cartilage. About 30 % of water is trapped in the collagen which arises from the non-spherically symmetric distribu­ fiber network; the rest appears as a gel with dissolved tion of the electric charge in the nucleus. When a nucleus inorganic ions (such as sodium), and most of it can flow with the quadrupole moment interacts with an electric field through the extracellular matrix by applying pressure to the gradient (e.g., the gradient formed by electrons surrounding cartilage [11]. High frictional resistance against water flow the nuclei in a molecule), a quadrupolar interaction takes is one of the mechanisms that helps cartilage to withstand place and affects the NMR properties of sodium. Based on heavy loads. the molecular environment of sodium, the following four Collagen contributes 10-20 % of cartilage wet weight. motional regimes are possible: (1) fast isotropic motion, (2) Type II collagen is the principal molecular component slow isotropic motion, (3) slow anisotropic motion and (4) (90-95 % of total collagen) in hyaline cartilage and forms fast anisotropic motion [13]. Multiple quantum filtered fibers intertwined with proteoglycans. Other collagen types sequences have been proposed to distinguish between dif­ are present in much smaller amounts in the matrix and help ferent sodium environments. However, these sequences to stabilize the type II collagen network [12]. Collagen suffer from even lower SNR compared to conventional fibers are responsible for tensile and shear strength in single-quantum sodium MR imaging [14-16]. In biological cartilage. tissues, sodium nuclei can be found only in the first three Proteoglycans (PG) can exist either as protein mono­ regimes. Using conventional sodium MR imaging, we can mers (\5 % of cartilage wet weight) or aggregates of only distinguish between two different sodium motional monomers attached to hyaluronic acid fibers via special­ regimes—fast and slow. ized link proteins (5-7 % of cartilage wet weight). Each Sodium in the fluid is in fast isotropic motion. The rapid PG monomer contains one or multiple sulfated GAG side tumbling of sodium ions results in a very rapid fluctuation chains covalently attached to a central protein core [10]. of the electric field gradient orientation, and thus quadru- The most common PG in cartilage is aggrecan with polar interaction is ‘averaged' to zero. In this environment, 100-150 GAG side chains. The GAGs contain a high both T1 (*63 ms) and T2* (*34 ms) relaxation times concentration of negatively charged sulfate and carboxyl decay mono-exponentially. groups, and thus provide negative fixed charge density Sodium in tissues is either in slow isotropic or slow (FCD) to the cartilage. This results in two important anisotropic (ordered structures, e.g., collagen fibers) physical properties of PGs. The negative FCD attracts motion. In these cases, quadrupolar interaction dominates positively charged ions (mainly sodium), and thus sodium the relaxation, and T1 and T2 decay is bi-exponential, with ions are in balance with the PG content in the cartilage. a short T2 component (T2*SHORT)of*0.9 ms, long T2 Since PGs are hydrophilic, the water molecules are pumped component (T2*LONG)of*13 ms and two T1 components by osmotic pressure into the cartilage. Additionally, the PG with similar relaxation times of *20 ms. In this regime, macromolecules remain separated because of the strong multiple quantum filtered sequences can be used to deter­ electrostatic repulsive force between GAGs. Through this mine the degree of order in the tissue. mechanism, PGs are responsible for the compressive stiffness of cartilage [13]. Pulse Sequences for Sodium MR Imaging

Sodium MR Properties Due to the very short bi-exponential T2 values of sodium in tissues, sequences suitable for acquiring images at short The sodium MR sensitivity is 9.3 % of the proton MR echo times (TE) are useful for sodium MR imaging. The sensitivity, and the sodium in vivo concentration in healthy first sodium images of articular cartilage in vivo were

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Fig. 1 Schematic representation of a Cartesian, b radial projection and c twisted projection k-space trajectories acquired by Reddy et al. [7] using Cartesian 3D gradient negative FCD of cartilage is in equilibrium with positively echo sequences (GRE) with TE of 2 ms at 4T. The charged sodium ions, sodium MR imaging was proposed to Cartesian trajectory is illustrated in Fig. 1a. In order to be a sensitive method for the evaluation of the GAG con­ increase SNR in the GRE images, TE can be minimized by tent in the cartilage. In early works, sodium MR spec­ using a nonselective radiofrequency pulse and an asym­ troscopy was employed to explore the relationship between metric readout (partial echo). To further reduce TE in the the sodium signal and PG content of enzymatically treated GRE sequence, optimal gradient switching patterns toge­ (trypsin, papain) cartilage [34-36]. Lesperance et al. [37] ther with the variable echo time (vTE) train (dynamically found that sodium in cartilage is 100 % MR visible, and by reduced TE toward the k-space center) [17] were recently using the ideal Donnan theory, they estimated the FCD of combined in the vTE-GRE sequence [18] and used for cartilage from the sodium content measured by MR spec­ sodium MR imaging [19]. The main advantage of Cartesian troscopy. Later studies have demonstrated that the sodium GRE sequences is their robustness. content in cartilage measured by sodium imaging is pro­ More advanced non-Cartesian ultra-short echo time (UTE) portional to the cartilage GAG content [38-40]. Thus, imaging techniques can acquire sodium images with TE sodium MR imaging can be useful for direct and nonin­ below 1 ms. Short TE in UTE sequences is achieved by vasive evaluation of the GAG content in native, OA car­ sampling the data from the center toward the periphery of the tilage and cartilage repair tissue. k-space using either radial (Fig. 1b) or spiral trajectories For the evaluation of the GAG concentration and FCD (Fig. 1c). The most widely used type of UTE sequence for in the cartilage, signal intensities from sodium images need sodium MR imaging is a 3D radial projection technique [20]. to be converted into sodium concentration values [41]. The The SNR of this method was further improved by the density- quantification of the sodium concentration is performed by adapted radial projection technique, where the density of measuring the subject together with agarose/saline phan­ acquisition points along the projections is modified, and thus toms of known sodium concentration (usually the k-space is sampled in a more efficient way [21]. Twisted 100-350 mM) (Fig. 2a) and of relaxation times similar to projection imaging (TPI) is another approach for sodium MR cartilage relaxation times (usually 6-10 % agar of the imaging with efficient k-space sampling and improved SNR phantom's wet weight). Sodium signal intensity from [22, 23]. Many other techniques, such as 3D cones [24], phantoms corrected for their relaxation times is then plot­ acquisition-weighted stack of spirals [25] and FLORET [26], ted against their sodium concentration, and a calibration were inspired by TPI. The data acquired with non-Cartesian curve is obtained by linear fitting of plotted data (Fig. 2b). UTE sequences are reconstructed by using regridding Sodium intensities from cartilage are then pixel-by-pixel reconstruction [27-29] or nonuniform fast Fourier transform fitted to the calibration curve to produce a tissue sodium algorithms[30,31].AlthoughtheUTEsequencescanprovide concentration map. Since the water fraction in cartilage is moreSNRthanCartesiansequences,theyarealsomoreprone about 75 %, the values in the concentration maps are to artifacts rising from gradient imperfections and off-reso­ divided by a factor of 0.75 [39, 40]. The sodium concen­ nance effects [32]. tration in the healthy cartilage was found to be 240-280 mM [39, 40]. For correct evaluation of sodium concentration, sodium Evaluation of GAG Content with Sodium MR Imaging relaxation times in the cartilage need to be calculated. Madelin et al. [42] measured sodium relaxation times using It was shown that the negative FCD of cartilage correlates the UTE radial sequence in knees of eight healthy volun­ with the GAG concentration of cartilage [33]. Since the teers at 7T. Relaxation times were evaluated in four knee

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Fig. 2 Quantification of sodium concentration in bovine cartilage. 3D data set. Circles represent signal intensities measured from four a Sagittal sodium 3D-GRE image of bovine patella and four saline/ saline/agarose phantoms. Error bars indicate the 95 % confidence agarose phantoms of different concentrations (150-300 mmol/l). intervals. From [41], with permission b Mean calibration curve obtained from four different slices of a cartilage regions (patellar, trochlear, femorotibial medial The result of the cartilage response to injury depends on and femorotibial lateral cartilage), and the authors reported several factors such as defect depth and location, the similar T1 (*20 ms), but different T2*SHORT (0.5-1.4 ms) patient's age, defect size, etc. Usual symptoms of cartilage and T2*LONG (11.4-14.8 ms) in different regions. As injury are local pain, swelling, locking, pseudo-locking and shown by Staroswiecki et al. [43], T2* values were not catching. Isolated chondral defects were found in 4 % of significantly different between 3T and 7T. This study also arthroscopies, while a much higher percentage (40-70 %) verified a linear increase of SNR with the magnetic field of defects has been found in combination with ligament strength and showed about 2.3-times higher sodium SNR in and/or meniscus injuries [46]. Thus, the cartilage health is cartilage at 7T compared to 3T. On the other hand, Madelin very important for the health of the whole joint. Since et al. [44] showed that the SNR efficiency is only *1.4 untreated osteochondral defects in adults often lead to early times higher at 7T compared to 3T. This study also dem­ onset of OA, symptomatic defects should be treated. onstrated high reproducibility and repeatability of sodium The goal of cartilage repair surgery techniques is to quantification using radial sequences with and without fluid restore the cartilage surface and function, to allow pain- suppression at 3T and 7T, which is comparable to other free motion of the joint and to prevent further cartilage proton-based MR imaging techniques for assessing artic­ degeneration by providing cartilage repair tissue that has ular cartilage. This is a very promising result for further 3T the same composition, structure and mechanical properties studies on sodium imaging of cartilage. as native articular cartilage [1]. Articular cartilage defects are currently treated with a number of different surgical interventions, which can be divided into three groups: (1) bone marrow-stimulating techniques, (2) auto-/allo-graft- Cartilage Injury and Repair ing techniques and (3) advanced cell-based repair techniques. Background (a) Pridie drilling [48] and microfracture (MFX) [49] can Hyaline cartilage is avascular tissue. Thus, its response to be considered marrow-stimulating techniques. These injury differs from that of other tissues, and its intrinsic procedures create multiple holes in the subchondral reparative capacity is very low [10, 45, 46]. In addition, the bone in the defect area to fill it with the material immobilized chondrocytes cannot migrate from healthy car­ coming from the bone marrow. Ideally, the cells tilage to the injury site. Mechanical injury can cause direct should differentiate into chondrogenic cells that damage to the extracellular matrix, or the damage can be produce cartilage [50]; however, these techniques mediated by chondrocytes via reduction of biosynthetic usually result in the formation of fibrous repair tissue. activityandexpressionofmatrix-degradingenzymes.Studies Knutsen et al. [51] noted that most of the biopsies on animal models have shown that high impact loads to the from repair tissue of patients 2 years after MFX were joint can lead to a loss of tissue integrity, degradation of composed mainly of fibrocartilage tissue, which mechanical properties or cell death [47]. degenerates with time.

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(b) Osteochondral grafting (mosaicplasty) [52] and peri­ appearing reference cartilage (267 ± 42). Similarly, osteal grafting [53] rely on the filling of lesions with dGEMRIC measurements showed significantly lower an autograft. In mosaicplasty, cylindrical osteochon­ postcontrast T1 values in repair tissue (510 ± 195 ms) than dral plugs are taken from non-load-bearing areas of in reference cartilage (756 ± 188 ms). Moreover, a strong an affected joint and placed into the osteochondral correlation was found between sodium imaging and defect. Benefits of mosaicplasty are that the lesion is dGEMRIC in MACT patients. The authors concluded that filled with mature hyaline cartilage and that it allows sodium imaging allows for differentiation of repaired tissue treatment of both osteochondral and chondral defects. from native cartilage in patients after MACT repair without Limitations of this technique are problematic produc­ the application of contrast agent. tion of a smooth convex cartilage surface, suboptimal To validate and evaluate the potential of the gagCEST reconstitution of the subchondral layer and usually technique as a biomarker for GAG content in cartilage, insufficient lateral integration of the repair site [54]. Schmitt et al. [6] compared sodium imaging with gagCEST (c) Advanced cell-based repair techniques such as autol­ in five MFX and seven MACT patients. A strong correla­ ogous chondrocyte implantation (ACI) use donor- tion between sodium and gagCEST values proved the derived chondrocytes (mostly autografts) to recon­ sensitivity of this method to GAG content in native carti­ struct the cartilage defect. ACI was the first cell lage and repair tissue. engineering approach to the treatment of cartilage In the study by Zbyn et al. [61 •], sodium MR imaging at lesions [55, 56]. In the third generation of ACI, such 7T was used to evaluate repair tissue after two different as in matrix-associated autologous chondrocyte trans­ types of cartilage repair techniques: bone marrow stimu­ plantation (MACT), the pieces of cartilage are taken lation (BMS) techniques (Pridie drilling and MFX) and from a non-load-bearing donor site, and extracted MACT. For more accurate comparison between repair chondrocytes are cultured in 3D scaffolds (matrices) techniques, each MACT patient was matched with one in vitro for several weeks [57]. In the second step, the BMS patient according to age (mean, *37 years), post­ cultured cartilage is implanted into the chondral operative interval (mean, *33 months) and defect loca­ defect. Although the ACI can produce a hyaline-like tion. Sodium images were measured with 3D-GRE repair tissue in some specimens, this tissue is not sequences using a sodium-only birdcage knee coil, and histochemically or morphologically identical to hya­ ROIs were drawn in the reference cartilage and cartilage line cartilage, and fibrocartilage can be found in some repair tissue. For both the BMS and MACT groups of of the samples [58]. Although there were no signif­ patients, sodium normalized values were significantly icant differences in the histological quality of repair lower in repair tissue than in reference cartilage. However, tissue between the patients after ACI and MFX the main finding of this study was that sodium normalized 2 years after surgery, ACI patients more often showed values were significantly higher in MACT (210 ± 36) than hyaline-like repair tissue than MFX patients [51]. in BMS (164 ± 31) repair tissue. On the other hand, the morphological appearance of the repair tissue, evaluated by the MOCART scoring system, was not different between Sodium MR Imaging of Cartilage Repair BMS and MACT patients. The results suggest a higher GAG content and therefore repair tissue of higher quality Several studies used sodium MR imaging for the evalua­ in MACT than in BMS patients. Sodium imaging can tion of tissue after cartilage repair surgery. The first sodium distinguish between repair tissues with different GAG MR images of patients after MACT cartilage repair were contents (Fig. 3) and thus can be useful for non-invasive published by Trattnig et al. [59*] in 2010. The authors evaluation of the performance of new cartilage repair measured 12 patients with a mean time of 56 months after techniques. MACT surgery in femoral cartilage and compared the Due to the low resolution of sodium images, partial results of sodium imaging at 7T with dGEMRIC (another volume effects from surrounding tissues such as bone or GAG-sensitive MR technique) at 3T. The mean value of synovial fluid (sodium concentration of 140-150 mmol/l) the magnetic resonance observation of cartilage repair influence the accuracy of the sodium content measurements tissue (MOCART) score [60], a scoring system for the in cartilage. To minimize contamination from synovial evaluation of the morphological appearance of repair tis­ fluid, triple quantum filtering techniques [16], inversion sue, was 75 (range, 45-95). A 3D-GRE sequence opti­ recovery (IR) methods [62] or relaxation-weighted sodium mized for sodium imaging was used together with a imaging [63] can be employed. The goal of the study by sodium-only birdcage knee coil. The sodium normalized Chang et al. [64*] was to evaluate cartilage repair and values were significantly lower in repair tissue native tissue using 7T sodium MR imaging with and (mean ± standard deviation: 174 ± 53) than in normal­ without fluid suppression. After different cartilage repair

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Osteoarthritis

Background

Knee OA is associated with structural changes in the whole joint, including degradation of cartilage, ligaments, menisci, subchondral bone changes and synovial inflam­ mation [67]. The diagnosis of OA is based on patient's medical history, clinical symptoms (loss of function, pain) and radiographic evidence (joint space width) [67]. The goal of a treatment is to improve the knee function and relief from symptoms. The most frequent treatment of end­ stage arthritis is knee joint replacement. Although radiography is the current standard for evaluating structural modifications in joints in trials of potential disease-modifying OA drugs, it has many limi­ tations (e.g., the sensitivity and precision) [68]. Morpho­ Fig. 3 Proton density-weighted MR images with fat suppression (left logical MR imaging is a non-invasive alternative that column) and color-coded sodium 3D-GRE images (right column) detects the presence of OA with high specificity compared from a patient after MFX treatment (upper row) and a subject after to radiography or arthroscopy, and provides comprehen­ MACT surgery (lower row). The area of cartilage repair tissue is located between the arrows. Color scale represents the sodium signal sive evaluation of the whole joint [69]. MR imaging of intensity values knee OA includes assessment of the cartilage thickness and volume [70], synovitis, synovial fluid effusions, lesions in the bone marrow and meniscal damage. A loss procedures (e.g., MFX, osteochondral grafting, MACT, of thickness in medial compartment cartilage is a sensi­ juvenile cartilage implantation) and with a median follow­ tive quantitative parameter that correlates with radio­ up of 26 weeks (range 12-151 weeks), 11 patients were graphic joint space width and seems to be a strong measured with a radial UTE sequence using a sodium-only predictor of the need for knee replacement [71]. Although birdcage knee coil. Fluid suppression was achieved using MR imaging may be a viable alternative to radiography an IR preparation with an adiabatic inversion pulse [65]. for the assessment of structural changes in knee OA and The sodium concentration was calculated in repair tissue, prediction of the knee replacement, both techniques are adjacent native cartilage and native cartilage in the knee insensitive to biochemical changes in the cartilage, which compartment not involved in surgery. Sodium concentra­ occurs in early stages of OA before the morphological tions were not significantly different between repair tissue changes [3]. and both types of native cartilage when using sequences Detection of early events in OA, when the disease without fluid suppression. This could be caused by heter­ process is potentially reversible, is of major interest in ogeneity of mean sodium concentrations in repair tissue cartilage imaging. GAG molecules have been considered due to the variety of repair procedures. However, when to play a central role in cartilage homeostasis [72]. The using fluid suppressed sequences, the mean sodium con­ equilibrium between synthesis and degradation of carti­ centration in repair tissue (108.9 ± 29.8 mmol/l) was sig­ lage matrix molecules is altered in pathologic conditions nificantly lower compared to adjacent native cartilage [73]. For instance, increased levels of aggrecan (204.6 ± 34.7 mmol/l) or native cartilage in a different 393ARGS fragments in the synovial fluid of patients knee compartment (249.9 ± 44.6 mmol/l). Thus, fluid with OA and knee injury have been proposed to reflect suppressed sodium imaging seems to be more accurate in early pathology [74, 75]. Early stages of OA are sug­ the assessment of sodium concentration in repair tissue. gested to be characterized by changes in the organization Additionally, a significantly lower sodium concentration and composition of the extracellular matrix, such as a was found in adjacent native cartilage when compared to decrease in cartilage GAG content. However, the native cartilage in different knee compartments. This is in mechanisms associated with early OA are not entirely accord with in vitro studies that demonstrated that the understood, and there are conflicting data on whether the number of viable chondrocytes increases with the distance GAG content is elevated [76-78], unchanged [79, 80]or from the site of injury [66]. decreased [81, 82] in early OA.

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Sodium MR Imaging of OA Cartilage

After validation in in vitro studies and in studies on animal models [83], sodium MR imaging was performed on vol­ unteers and OA patients. Wheaton et al. [40] measured sodium images with UTE radial sequences in nine healthy asymptomatic volunteers and three patients with symptoms of early OA using a surface coil at 4T. The mean sodium content measured in the patellae of the nine healthy volun­ teers was 254 mmol/l, which corresponded to a mean FCD measurement of -182 mmol/l. Sodium maps for the sub­ jects with symptoms of OA revealed cartilage regions with significantly lower FCD (-108 to -144 mmol/l) when compared to the FCD of healthy volunteers. These results suggest that sodium MR imaging might be a useful method for monitoring the changes in GAG content of OA cartilage. The first 7T sodium MR images from OA patients were published by Wang et al. [84*]. Sodium images were acquired from five asymptomatic volunteers and five clin­ Fig. 4 Sodium concentration maps from a control subject (upper row) and from a patient with OA (lower row). Sodium images were ically diagnosed OA subjects using UTE radial sequences. acquired with fluid suppression using IR (IRW) and without fluid The sodium concentration was calculated in three ROIs: suppression (R3D). Note that the difference in cartilage sodium patellar, medial femorotibial and lateral femorotibial car­ concentration between the control and OA patient is higher with the tilage. The mean sodium concentration in cartilage of fluid-suppressed radial sequence than with the radial sequence without fluid suppression. From [85"], with permission volunteers ranged from 240 to 280 mmol/l. The sodium concentration in OA patients was significantly smaller, 30-60 % lower compared to volunteers. The authors con­ cluded that sodium imaging may be a useful for physio­ increased. The mean sodium concentration in cartilage logic OA imaging and clinical diagnosis. Unfortunately, from IR with WURST was found to be a significant pre­ due to very thin cartilage (especially in OA patients), low dictor of OA and early OA (only patients with a Kellgren- resolution and blurring in sodium images, the authors were Lawrence score of 1 or 2). An example of sodium MR not able to distinguish among patellar, femoral and tibial imaging of OA cartilage with and without fluid suppression cartilage, and the evaluated ROIs probably included signal is shown in Fig. 4. Evaluation of the cartilage sodium from both cartilage and synovial fluid. concentration with fluid-suppressed MR imaging at 7T is a To evaluate contamination from synovial fluid, Madelin potential biomarker for OA. et al. [85"] calculated the cartilage sodium concentration in 19 healthy volunteers and 28 OA patients using 7T sodium imaging with and without fluid suppression. The Conclusions first eight volunteers and six patients were measured with a sodium-only birdcage knee coil; the rest of the subjects Recent studies proved that sodium MR imaging can directly, were measured with a homemade eight-channel double­ in a noninvasive and quantitative manner, assess the cartilage tuned proton/sodium knee coil. Measurements with fluid GAG content, which plays a central role in cartilage homeo­ suppression were achieved by using an IR preparation with stasis in native and OA cartilage as well as in repair tissue. an adiabatic inversion WURST pulse in the UTE radial Sincenativereferencecartilage(adjacentormoredistantfrom sequence [65]. The cartilage sodium concentration was the repair site) is available in sodium imaging of repairtissue, evaluated in the patellar, femorotibial lateral and femoro- the sodium concentration is not the critical parameter, and tibial medial ROIs on four consecutive sodium maps. The ratios between native and repair sodium signal intensities can mean sodium concentration over all cartilage ROIs mea­ also be compared between patients. From this point of view, sured with radial sequence without fluid suppression was sodium imaging of OA cartilage is more demanding and similar between healthy subjects (192 mmol/l) and OA requires quantification of the sodium concentration. Addi­ patients (174 mmol/l). When fluid suppression was tionally, OA cartilage is thinner than native cartilage, and applied, the difference between the mean sodium concen­ partial volume artifacts are therefore more pronounced in OA tration over all cartilage ROIs in healthy subjects applications. This issue of sodium imaging can be overcome (*243 mmol/l) and in OA patients (*194 mmol/l) by introducing fluid-suppressed sequences, which allow

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159 41 Page 8 of 10 Curr Radiol Rep (2014) 2:41 evaluation of sodium signal from cartilage without contami­ Human and Animal Rights and Informed Consent This article nation coming from synovial fluid [65]. Moreover, by using does not contain any studies with human or animal subjects performed by the author. dual-tuned proton/sodium radiofrequency coils, sodium imaging can be combined with other MR techniques (e.g., morphological imaging, T2 mapping, diffusion-weighted imaging) to assess other cartilage components (e.g., water References content, collagen matrix). Such a combination of biomarkers might provide more accurate insight into cartilage degenera- Papers of particular interest, published recently, have been tionormaturationofrepairtissue.However,acquisitiontimes highlighted as: of sodium imaging must be reduced to acquire a complete * Of importance imaging protocol in less than 1 h. ** Of major importance Previous in vitro studies showed changes in T1 and T2* relaxation times of cartilage after enzymatically induced 1. Buckwalter J, Mow V. Cartilage repair in osteoarthritis. Phila­ (trypsin, papain) PG depletion [34-36]. Thus, different T1 delphia: WB Saunders Co; 1992. and T2* relaxation times can also be expected in OA carti­ 2. Lawrence RC, Felson DT, Helmick CG, et al. Estimates of the prevalence of arthritis and other rheumatic conditions in the lage and repair tissue. The general limitation of the sodium United States Part II. Arthritis Rheum. 2008;58(1):26-35. concentrationquantificationistheuseofsodiumT1 andT2* 3. Burstein D, Bashir A, Gray ML. MRI techniques in early stages values from native cartilage also for quantification of the of cartilage disease. Invest Radiol. 2000;35(10):622-38. concentration in OA cartilage and in repair tissue, which 4. Bashir A, Gray ML, Burstein D. Gd-DTPA(2-) as a measure of cartilage degradation. Magn Reson Med. 1996;36(5):665-73. might result in bias from ‘true' sodium concentrations. To 5. Duvvuri U, Reddy R, Patel SD, et al. T1rho-relaxation in articular overcome this limitation might be difficult as the measure­ cartilage: effects of enzymatic degradation. Magn Reson Med. ment of sodium relaxation times is more time consuming 1997;38(6):863-7. than morphological sodium imaging. However, the infor­ 6. SchmittB,ZbynS,StelzenederD,etal.Cartilagequalityassessment by using glycosaminoglycan chemical exchange saturation transfer mation on cartilage relaxation times might improve the and (23)Na MR imaging at 7 T. Radiology. 2011;260(1):257-64. sensitivity and specificity of sodium imaging in evaluating 7. Reddy R, Insko EK, Noyszewski EA, et al. Sodium MRI ofhuman degenerative changes in cartilage. articular cartilage in vivo. Magn Reson Med. 1998;39(5):697-701. Sodium MR imaging is a challenging method. However, 8. Juras V, Zbyn S, Pressl C, et al. Sodium MR imaging of Achilles tendinopathy at 7 T: preliminary results. Radiology. new technical developments in the recent decade have 2012;262(1):199-205. enabled transferring this technique from in vitro to pre­ 9. Weber MA, Nagel AM, Wolf MB, et al. Permanent muscular clinical in vivo studies. The advent of whole-body ultra- sodium overload and persistent muscle edema in Duchenne high-field MR systems [43, 86], dedicated radiofrequency muscular dystrophy: a possible contributor of progressive muscle degeneration. J Neurol. 2012;259(11):2385-92. phase-arraycoils [87] and optimized MR sequences [65] has 10. Buckwalter JA, Mankin HJ. Articular cartilage.1. Tissue design provided higher SNR, higher spatial resolution in the images and chondrocyte-matrix interactions. J Bone Joint Surg Am. and/or shorter measurement times. Moreover, compressed 1997;79A(4):600-11. sensing applications for the acceleration of sodium imaging 11. Mow VC, Ratcliffe A, Poole AR. Cartilage and diarthrodial joints as paradigms for hierarchical materials and structures. Biomate­ acquisition with undersampling are very promising [88, 89]. rials. 1992;13(2):67-97. However, more research is necessary on both software and 12. Poole AR, Kojima T, Yasuda T, et al. Composition and structure hardware to translate sodium MR imaging of cartilage into of articular cartilage—a template for tissue repair. Clin Orthop clinical practice, preferably to 3T MR systems. Relat Res. 2001;391:S26-33. 13. Borthakur A, Mellon E, Niyogi S, et al. Sodium and T1rho MRI for molecular and diagnostic imaging of articular cartilage. NMR Acknowledgments Funding support provided by the Austrian Biomed. 2006;19(7):781-821. Science Fund (FWF) P 25246 B24 and by the Vienna Spots of 14. Wimperis S, Wood B. Triple-quantum sodium imaging. J Magn Excellence of the Vienna Science and Technology Fund (WWTF): Reson. 1991;95(2):428-36. Vienna Advanced Imaging Center-VIACLIC FA102A0017. 15. Hancu I, Boada FE, Shen GX. Three-dimensional triple-quantum- filtered (23)Na imaging of in vivo human brain. Magn Reson Open Access This article is distributed under the terms of the Med. 1999;42(6):1146-54. Creative Commons Attribution License which permits any use, 16. Borthakur A, Hancu I, Boada FE, et al. In vivo triple quantum distribution, and reproduction in any medium, provided the original filtered twisted projection sodium MRI of human articular carti­ author(s) and the source are credited. lage. J Magn Reson. 1999;141(2):286-90. 17. Ying K, Schmalbrock P, Clymer B. Echo-time reduction for Compliance with Ethics Guidelines submillimeter resolution imaging with a 3D phase encode time reduced acquisition method. Magn Reson Med. 1995;33(1):82-7. Conflict of Interest Stefan Zbyn, Vladimir Mlynarik, Vladimir 18. Deligianni X, Bar P, Scheffler K, et al. High-resolution Fourier- Juras, Pavol Szomolanyi and Siegfried Trattnig declare that they have encoded sub-millisecond echo time musculoskeletal imaging at 3 no conflicts of interest. Tesla and 7 Tesla. Magn Reson Med. 2013;70(5):1434-9.

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evaluation of autologous chondrocyte transplantation: determi­ disease than glycosaminoglycan or aggrecan levels: a cross-sec­ nation of interobserver variability and correlation to clinical tional study. Arthritis Res Ther. 2009;11(3):R92. outcome after 2 years. Eur J Radiol. 2006;57(1):16-23. 76. Adams ME, Brandt KD. Hypertrophic repair of canine articular 61. * Zbyn S, Stelzeneder D, Welsch GH, et al. Evaluation of native cartilage in osteoarthritis after anterior cruciate ligament tran­ hyaline cartilage and repair tissue after two cartilage repair sur­ section. J Rheumatol. 1991;18(3):428-35. gery techniques with 23Na MR imaging at 7 T: initial experience. 77. Matyas JR, Ehlers PF, Huang D, et al. The early molecular natural Osteoarthritis Cartilage. 2012; 20(8): 837-45. Relevant pre­ history of experimental osteoarthritis. I. Progressive discoordinate clinical study using sodium MR imaging for the evaluation of expression of aggrecan and type II procollagen messenger RNA in cartilage repair tissue and OA cartilage. the articular cartilage of adult animals. Arthritis Rheum. 62. Rong P, Regatte RR, Jerschow A. Clean demarcation of cartilage 1999;42(5):993-1002. tissue 23Na by inversion recovery. J Magn Reson. 2008;193(2): 78. Lorenzo P, Bayliss MT, Heinegard D. Altered patterns and syn­ 207-9. thesis of extracellular matrix macromolecules in early osteoar­ 63. Nagel AM, Bock M, Hartmann C, et al. The potential of relax­ thritis. Matrix Biol. 2004;23(6):381-91. ation-weighted sodium magnetic resonance imaging as demon­ 79. Maroudas A. Glycosaminoglycan turn-over in articular cartilage. strated on brain tumors. Invest Radiol. 2011;46(9):539-47. Philos Trans R Soc Lond B Biol Sci. 1975;271(912):293-313. 64. * Chang G, Madelin G, Sherman OH, et al. Improved assessment 80. Lorenz H, Wenz W, Ivancic M, et al. Early and stable upregu­ of cartilage repair tissue using fluid-suppressed Na inversion lation of collagen type II, collagen type I and YKL40 expression recovery MRI at 7 Tesla: preliminary results. Eur Radiol. 2012; levels in cartilage during early experimental osteoarthritis occurs 22(6): 1341-9. Relevant pre-clinical study using sodium MR independent of joint location and histological grading. Arthritis imaging for the evaluation of cartilage repair tissue and OA Res Ther. 2005;7(1):R156-65. cartilage. 81. Grushko G, Schneiderman R, Maroudas A. Some biochemical 65. MadelinG,LeeJS,InatiS,etal.SodiuminversionrecoveryMRIof and biophysical parameters for the study of the pathogenesis of the knee joint in vivo at 7T. J Magn Reson. 2010;207(1):42-52. osteoarthritis: a comparison between the processes of ageing and 66. Tochigi Y, Buckwalter JA, Martin JA, et al. Distribution and degeneration in human hip cartilage. Connect Tissue Res. progression of chondrocyte damage in a whole-organ model of 1989;19(2-4):149-76. human ankle intra-articular fracture. J Bone Joint Surg Am. 82. Yagi R, McBurney D, Laverty D, et al. Intrajoint comparisons of 2011;93(6):533-9. gene expression patterns in human osteoarthritis suggest a change 67. Bijlsma JW, Berenbaum F, Lafeber FP. Osteoarthritis: an update in chondrocyte phenotype. J Orthop Res. 2005;23(5):1128-38. with relevance for clinical practice. Lancet. 2011;377(9783): 83. Wheaton AJ, Borthakur A, Dodge GR, et al. Sodium magnetic 2115-26. resonance imaging of proteoglycan depletion in an in vivo model 68. Hunter DJ, Guermazi A. Imaging techniques in osteoarthritis. PM of osteoarthritis. Acad Radiol. 2004;11(1):21-8. R. 2012;4(5 Suppl):S68-74. 84. * Wang L, Wu Y, Chang G, et al. Rapid isotropic 3D-sodium 69. Menashe L, Hirko K, Losina E, et al. The diagnostic performance MRI of the knee joint in vivo at 7T. J Magn Reson Imaging. of MRI in osteoarthritis: a systematic review and meta-analysis. 2009; 30(3): 606-14. Relevant pre-clinical study using sodium Osteoarthr Cartil. 2012;20(1):13-21. MR imaging for the evaluation of cartilage repair tissue and OA 70. Eckstein F, Wirth W. Quantitative cartilage imaging in knee cartilage. osteoarthritis. Arthritis. 2011;2011:475684. 85. ** Madelin G, Babb J, Xia D, et al. Articular cartilage: evaluation 71. Eckstein F, Kwoh CK, Boudreau RM, et al. Quantitative MRI with fluid-suppressed 7.0-T sodium MR imaging in subjects with measures of cartilage predict knee replacement: a case-control and subjects without osteoarthritis. Radiology. 2013; 268(2): study from the osteoarthritis initiative. Ann Rheum Dis. 481-91. Relevant pre-clinical study using sodium MR imaging for 2013;72(5):707-14. the evaluation of cartilage repair tissue and OA cartilage. 72. Heinegard D. Proteoglycans and more—from molecules to biol­ 86. Regatte RR, Schweitzer ME. Ultra-high-field MRI of the mus­ ogy. Int J Exp Pathol. 2009;90(6):575-86. culoskeletal system at 7.0T. J Magn Reson Imaging. 2007; 73. Heathfield TF, Onnerfjord P, Dahlberg L, et al. Cleavage of fi- 25(2):262-9. bromodulin in cartilage explants involves removal of the N-ter- 87. Qian Y, Zhao T, Wiggins GC, et al. Sodium imaging of human minal tyrosine sulfate-rich region by proteolysis at a site that is brain at 7 T with 15-channel array coil. Magn Reson Med. sensitive to matrix metalloproteinase-13. J Biol Chem. 2012;68(6):1807-14. 2004;279(8):6286-95. 88. Lustig M, Donoho D, Pauly JM, Sparse MRI. The application of 74. Struglics A, Larsson S, Pratta MA, et al. Human osteoarthritis compressed sensing for rapid MR imaging. Magn Reson Med. synovial fluid and joint cartilage contain both aggrecanase and 2007;58(6):1182-95. matrix metalloproteinase-generated aggrecan fragments. Osteo- 89. Madelin G, Chang G, Otazo R, et al. Compressed sensing sodium arthr Cartil. 2006;14(2):101-13. 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162 APENDIX

6.2 Curriculum Vitae

Name: Stefan Zbyn

Telephone: +43 1 40400 64670

E-mail: [email protected]

Marital status: Married, three children

Nationality: Slovak

Birth date: 24th June, 1981

University Education:

09/2005-present: PhD studies in Medical Physics at the Medical University of Vienna, Vienna, Austria. Doctoral thesis: “Clinical Application ofSodium Magnetic Resonance Imaging for Evaluation of Articular Cartilage at 7 Tesla” at the High Field MR Centre. Supervisor: Prof. Siegfried Trattnig.

1999-2005: MSc studies in Biomedical Physics at the Faculty of Mathematics, Physics and Informatics, Comenius University, Bratislava, Slovakia. Diploma thesis: “Measurement of Rate constants of Creatine Kinase Reaction using Magnetic Resonance as a Factor of Brain Damage in Different Pathologies at Rats and Humans”. Supervisor: Vladimir Mlynarik, PhD.

Appointments and Professional Activities:

07/2005-present: Research fellow at the High Field MR Centre, Department of Biomedical Imaging and Image-guided Therapy, Medical University of Vienna, Austria. Focused on clinical applications of sodium MRI for evaluation of cartilage, intervertebral discs, tendon, kidneys, and breast cancer.

24-26/9/2009: ESMRMB Lectures on MR: Rapid Imaging: Echo Generation and Manipulation, Magdeburg, Germany.

163 APENDIX

19-23/2/2007: Training in IDEA Sequence Programming, Vienna, Austria.

13-15/10/2005: ESMRMB Lectures on MR: 1H MR Spectroscopy, Prague, Czech Rep.

10/2004-12/2004: International exchange program of SAIA. Measurements of patients for diploma thesis at High Field MR Centre, Vienna, Austria.

2003-2004: Animal measurements for diploma thesis at Derer Faculty Hospital, Bratislava, Slovakia.

Invited Lectures and Awards:

4-6/10/2015: Invited plenary lecture entitled “Multiparametric MRI of Breast Cancer: from 3T to 7T” at ESMRMB annual meeting, Edinburgh, United Kingdom.

1-5/6/2015: First price for the best presentation at the High Field Systems and Applications Study Group, and the Magna cum Laude ISMRM Merit Award for presentation entitled “Relaxation-Weighted Sodium MRI of Breast Lesions at 7T”, ISMRM annual meeting, Toronto, ON, Canada.

10-16/6/2014 The Summa cum Laude ISMRM Merit Award for presentation entitled “Sodium MRI of Articular Cartilage with Improved SNR Using Coherent SSFP Imaging at7T”, ISMRM-ESMRMBjoint meeting, Milan, Italy.

The Magna cum Laude ISMRM Merit Award for presentation entitled “Sodium MRI of Cartilage RepairTissue in the Ankle Joint at 7T”, ISMRM- ESMRMBjoint meeting, Milan, Italy.

4-6/10/2012: Invited educational lecture entitled “Instrumental and methodological issues of sodium imaging and its applications in MSK” at ESMRMB annual meeting, Lisbon, Portugal.

Reviewerfor Scientific Journals:

Investigative Radiology; Osteoarthritis and Cartilage; Journal of Magnetic Resonance Imaging; Magnetic Resonance Materials in Physics, Biology and Medicine; Journal of Tissue Science and Engineering

164 APENDIX

6.3 Scientific Publications

First-author Publications

1) Zbvn S., Mlynarik V., Juras V., Szomolanyi P., Trattnig S. Evaluation of cartilage repair and osteoarthritis with sodium MRI. NMR Biomed. 2015; DOI: 10.1002/nbm.3280. Review.

2) Zbvn S., Brix M.O., Juras V., Domayer S.E., Walzer S.M., Mlynarik V., Apprich S., Buckenmaier K., Windhager R., Trattnig S. Sodium Magnetic Resonance Imaging of Ankle Joint in Cadaver Specimens, Volunteers and Patients after Different Cartilage Repair Techniques at 7T - Initial Results. Invest Radiol. 2015; 50(4): 246-254.

3) Zbvn S., Krssak M., Memarsadeghi M., Gholami B., Haitel A., Weber M., Helbich T., Trattnig S., Moser E., Gruber S. Technical Note: Evaluation of the Uncertainties in (Choline+Creatine)/Citrate Ratios Measured by Proton MR Spectroscopic Imaging in Patients Suspicious for Prostate Cancer. Fortschr Röntgenstr. 2014; 186(7): 698-702.

4) Zbvn S., Mlynarik V., Juras V., Szomolanyi P., Trattnig S. Sodium MR Imaging of Articular Cartilage Pathologies. Curr Radiol Rep. 2014; 2(41): 1-10. Review.

5) Zbvn S., Stelzeneder D., Welsch G.H., Negrin L.L., Juras V., Mayerhoefer M.E., Szomolanyi P., Bogner W., Domayer S.E., Weber M., Trattnig S. Evaluation of native hyaline cartilage and repair tissue after two cartilage repair surgery techniques with (23)Na MR imaging at 7T: initial experience. Osteoarthritis Cartilage. 2012; 20(8): 837­ 845.

Last-author Publications

1) Haneder S., Juras V., Michaely H.J., Deligianni X., Bieri O., Schoenberg S.O., Trattnig S., Zbvn S. In vivo sodium (23Na) imaging of the human kidneys at 7 T: Preliminary results. Eur Radiol. 2014; 24(2): 494-501.

165 APENDIX

2) Trattnig S., Welsch G.H., Juras V., Szomolanyi P., Mayerhoefer M.E., Stelzeneder D., Mamisch T.C., Bieri O., Scheffler K., Zbvn S. 23Na MR Imaging at 7 T after Knee Matrix- associated Autologous Chondrocyte Transplantation: Preliminary Results. Radiol. 2010; 257(1): 175-184.

Co-author Publications

1) Juras V., Bohndorf K., Heule R., Kronnerwetter C., Szomolanyi P., Hager B., Bieri O., Zbvn S., Trattnig S. A comparison of multi-echo spin-echo and triple-echo steady-state T2 mapping for in vivo evaluation of articular cartilage. Eur Radiol, 2015 Sep; DOI: 10.1007/ S00330-015-3979-6.

2) Trattnig S., Ohel K., Mlynarik V., Juras V., Zbvn S., Korner A. Morphological and compositional monitoring of a new cell-free cartilage repair hydrogel technology - GelrinC by MR using semi-quantitative MOCART scoring and quantitative T2 index. Osteoarthritis Cartilage. 2015; DOI: 10.1016/j.joca.2015.07.007.

3) Schoenbauer E., Szomolanyi P., Shiomi T., Juras V., Zbvn S., Zak L., Weber M., Trattnig S. Cartilage evaluation with biochemical MR imaging using in vivo Knee compression at 3T-comparison of patients after cartilage repair with healthy volunteers. J Biomech. 2015; DOI: 10.1016/j.jbiomech.2015.06.016.

4) Trattnig S., Bogner W., Gruber S., Szomolanyi P., Juras V., Robinson S., Zbvn S., Haneder S. Clinical applications at ultrahigh field (7 T). Where does it make the difference? NMR Biomed. 2015; DOI: 10.1002/nbm.3272. Review.

5) Widhalm H.K., Apprich S., Welsch G.H., Zbvn S., Sadoghi P., VekszlerG., Hamböck M., Weber M., Hajdu S., Trattnig S. Optimized cartilage visualization using 7-T sodium (23Na) imaging after patella dislocation. Knee Surg Sports Traumatol Arthrose. 2014; DOI: 10.1007/S00167-014-3455-x.

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6) Juras V., Apprich S., Zbvn S., Zak L., Deligianni X., Szomolanyi P., Bieri O., Trattnig S. Quantitative MRI analysis of menisci using biexponential T2 * fitting with a variable echo time sequence. Magn Reson Med. 2014; 71(3): 1015-1023.

7) Riegler G., Deligianni X., Juras V., Zbyn S., Apprich S., Szomolanyi P., Weber M., Bieri O., Trattnig S. Quantitative T2*-Mapping of the Knee Using a Spoiled Gradient Echo Sequence at 3 Tesla: Preliminary Results. Journal of Pharmacy and Pharmacology. 2014; 2(1): 59-69.

8) Pachowsky M.L., Trattnig S., Apprich S., Mauerer A., Zbyn S., Welsch G.H. Impact of different coils on biochemical T2 and T2* relaxation time mapping of articular patella cartilage. Skeletal Radiol. 2013; 42(11): 1565-1572.

9) Raghuraman S., Mueller M.F., Zbvn S., Baer P., Breuer F.A., Friedrich K.M., Trattnig S., Lanz T., Jakob P.M. 12-channel receive array with a volume transmit coil for hand/wrist imaging at 7 T. JMagn Reson Imaging. 2013; 38(1): 238-244.

10) Juras V., Apprich S., Pressl Ch., Zbvn S., Szomolanyi P., Domayer S., Hofstaetter J., Trattnig S. Histological correlation of 7 T multi-parametric MRI performed in ex-vivo Achilles tendon. EurJ Radiol. 2013; 82(5): 740-744.

11) Juras V., Zbvn S., Pressl Ch., Valkovic L., Szomolanyi P., Frollo I., Trattnig S. Regional variations of T2* in healthy and pathologic achilles tendon in vivo at 7 Tesla: Preliminary results. Mag Reson Med. 2012; 68(5): 1607-1613.

12) Trattnig S. , Zbvn S., Schmitt B., Friedrich K., Juras V., Szomolanyi P., Bogner W. Advanced MR methods at ultra-high field (7 Tesla) for clinical musculoskeletal applications. EurRadiol, 2012; 22(11): 2338-2346.

13) Noebauer-Huhmann I.M., Juras V., Pfirrmann C.W., Szomolanyi P., Zbyn S., Messner A., Wimmer J., Weber M., Friedrich K.M., Stelzeneder D., Trattnig S. Sodium MR Imaging of the Lumbar Intervertebral Disk at 7 T: Correlation with T2 Mapping and Modified Pfirrmann Score at 3 T- Preliminary Results. Radiology. 2012; 265(2): 555-564.

167 APENDIX

14) Juras V., Zbvn S., Pressl Ch., Domayer S.E.R., Hofstaetter J.G., Mayerhoefer M.E., Windhager R., Trattnig S. Sodium MR Imaging of Achilles Tendinopathy at 7 T: Preliminary Results. Radiology, 2012; 262(1): 199-205.

15) FuegerB.J., Helbich T.H., Schernthaner M.,, Zbvn S., Linhart H.-G., Stiglbauer A., Doan A., Pinker K., Heinz G., Padhani A.R., Brader P. Stellenwert der multiparametrischen Magnetresonanztomographie beim Prostatakarzinom Importance of multiparametric magnetic resonance tomography for prostate cancer. Radiologe. 2011; 51(11): 947-954.

16) Schmitt B., Zbvn S., Stelzeneder D., Jellus V., Paul D., Lauer L., Bachert P., Trattnig S. Cartilage Quality Assessment by Using Glycosaminoglycan Chemical Exchange Saturation Transfer and Na MR Imaging at 7 T. Radiology. 2011; 260(1): 257-264.

17) Welsch G.H., Apprich S., Zbvn S., Mamisch T.C., Mlynarik V., Scheffler K., Bieri O., Trattnig S. Biochemical (T2, T2* and magnetisation transfer ratio) MRI of knee cartilage: feasibility at ultra-high field (7T) compared with high field (3T) strength. Eur Radiol. 2011; 21(6): 1136-1143.

18) Juras V., Zbvn S., Szomolanyi P., Trattnig S. Regression error estimation significantly improves the region-of-interest statistics of noisy MR images. Medical Physics. 2010; 37(6): 2813-2821.

19) Stadler M., Anderwald Ch., Pacini G., Zbvn S., Promintzer-Schifferl M., Mandl M., Bischof M., Gruber S., Nowotny P., Luger A., Prager R., Krebs M. Chronic peripheral hyperinsulinemia in type 1 diabetic patients after successful combined pancreas kidney transplantation does not affect ectopic lipid accumulation in skeletal muscle and liver. Diabetes, 2010; 59(1): 215-218.

20) Kasparova S., Zbvn S., Zilka N., Horecky J., Liptaj T., Mlynarik V., Juranek 1.31P magnetic resonance spectroscopy study of brain creatine kinase in rats with moderate and severe hypertension and chronic cerebral hypoperfusion. Biologia - Section Cellular and MolecularBiology. 2005; 60(17): 81-84.

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