Marseille, France Berlin, Germany Marseille, France
Master Thesis (Travail de Fin d’Études)
From April, 9th 2012 to September, 28th 2012
Mechanical and structural characterization of the spinal entheses: a multimodal approach
Quentin Léguillette
Final-year student, Promotion 2012
Specialization:
Mechanical Modelling of Materials and Structures (M3S)
Academic tutor : Laboratory tutors :
Thierry Désoyer Kay Raum Jean-Philippe Berteau
Cécile Baron Quentin Léguillette Promotion 2012
Table of contents
Acknowledgements ...... 3 Glossary ...... 4 Abstract ...... 5 Résumé ...... 6 General introduction...... 7 1- Presentation of the laboratories and of the work environment ...... 8 1-1- Involved institutes in this partnership ...... 8 1-1-1- The Julius Wolff Institute ...... 8 1-1-2- The Institute of Movement Sciences ...... 9 1-2- Work environment ...... 10 2- Assignment realization ...... 11 2-1- Clinical introduction to the problem ...... 11 2-1-1- General anatomical recalls ...... 12 2-1-2- State of art about entheses ...... 14 2-2- Aim of the assignment ...... 19 2-3- Experimental characterization of spinal entheses ...... 21 2-3-1- Materials ...... 21 2-3-2- Methods ...... 23 2-3-3- Results ...... 33 2-3-4- Discussion ...... 50 2-3-5- Synthesis ...... 51 2-4- Numerical modeling of spinal entheses ...... 52 2-4-1- Presentation of the generic model ...... 52 2-4-2- Developed models: description and results ...... 53 2-4-3- Discussion ...... 58 2-4-4- Prospects: description of the forthcoming models ...... 59 Technical and scientific conclusions ...... 60 Personal perspectives ...... 61 Bibliography ...... 62 Appendices ...... 64
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Acknowledgements
Before starting this report, I would like to thank warmly the following people who played an important role during my master internship:
First of all, I express my entire gratitude to my supervisor in Berlin, Herr Professor Doktor Kay Raum, for having let me the opportunity to realize my internship within the Julius Wolff Institute. Besides that, Kay, thank you so much for your precious advices and your will to integrate me as much as possible in the ISS team.
Of course, I would also like to thank my two other laboratory tutors, Cécile Baron from the GIBOC team in Marseille and Jean-Philippe Berteau from the Institute of Biomechanics in the Technische Universität Hamburg Harburg, without whom this partnership and thus this internship would never have existed. Thank you for having given me the possibility to work on such an interesting topic!
A special thank to Peter Varga for the many rewarding discussions about the development of the finite-element models of the spinal entheses, it was a real pleasure to work with you.
In my daily work I also had the chance to work with Daniel Rohrbach, who I would like to kindly thank for his precious help on Matlab and his availability. My gratitude also goes to Martin Schöne, Nils Männicke and Susanne Schrof for their constant very good mood, their practical tricks and advices and their excellent team spirit. I also would like to thank Anne Slawig and the Johannes team. I could not forget to thank Anke Kadow- Romacker as well for her availability and her concern about my samples.
Finally, I would like to thank the following people for their precious help in their respective fields and their availability: Marzena Princ, Gabriela Korus and Zienab Kronbach, Mario Thiele and Paul Zaslansky.
Last but not least, I also express my gratitude to the European Commission for the Erasmus scholarship for internship I was granted.
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Glossary
Arthritis : inflammation of a joint, usually accompanied by pain, swelling, and stiffness, and resulting from infection, trauma, degenerative changes, metabolic disturbances, or other causes.
Fibrosis : formation of fibrous tissue
Joint capsule : sac-like fibrous tissue that envelopes a synovial joint.
Sacroiliac joint : joint formed by the sacrum and ilium where they meet on either side of the lower back
Syndesmophyte : bony growth attached to a ligament, found between adjacent vertebrae in ankylosing spondylitis
Proprioception : unconscious perception of movement and spatial orientation arising from stimuli within the body itself
Thoracolumbar fascia : sheet of connective tissue covering the deep muscles of the back
Enthesitis : Traumatic disease occurring at the point of attachment of skeletal muscles to bone, where recurring stress causes inflammation and often fibrosis and calcification
Process : projection or outgrowth of tissue from a larger body
Collagen : fibrous protein constituent of bone, cartilage, tendon, and other connective tissue
Cytoplasm : material between the nuclear and cell membranes; includes fluid organelles, and various membranes
Fibroblast : large, flat, oval cell found in connective tissue and responsible for the formation of fibers
Aggrecan : glycoprotein with heteropolysaccharide side chains, major component of cartilage
Acetabulum : cup-shaped cavity at the base of the hipbone into which the ball-shaped head of the femur fit
Periodontal ligament : connective-tissue layer covering the cementum of a tooth and holding it in place in the jawbone
Cementum : thin layer of calcified (tough calcium deposits) tissue covering the dentine of the root
Endochondral ossification : formation of bone in which a cartilage template is gradually replaced by a bone matrix, as in the formation of long bones
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Abstract
Low back pain treatments represent today a serious public healthcare issue and are associated with many joint diseases of the vertebral column, or spondyarthropathies. Among them, ankylosing spondylitis (AS) causes destruction and fusion of the spine and sacroiliac joints. In this specific disease, transitional tissues performing the attachment of spinal ligaments to the bone, also called spinal entheses, are believed to play a primary role in the ligament calcification process, which results in pain and can lead to a complete loss of the spine´s mobility.
Accordingly there is a need for further knowledge of these transitional tissues in order to provide a better understanding of the operating mechanisms of AS and find treatment suggestions. The objective of this research project was the gain of both structural and mechanical properties of spinal entheses in order to model the ligament calcification process occurring during AS.
To achieve this goal, firstly we experimentally tested spinal entheses samples -extracted from growing pigs- with three techniques, Scanning Acoustic Microscopy, histology and Micro Computerized Tomography; secondly we developed two two- dimensional finite-element models of spinal entheses by using the properties deduced from the experimental step. Each model included a healthy and a pathological version. The first model used literature data, and the second the mechanical properties deduced from the experimental phase. For each pathological version, the models predicted a higher stretching of the interspinous ligament, which may lead to the onset of low back pain since it provokes the disrupting of the pain receptors in this ligament.
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Résumé
Le traitement des douleurs dans le bas du dos ou lombalgies représente à l´heure actuelle un enjeu de santé publique considérable et est associé à de nombreuses maladies articulaires de la colonne vertébrale, ou spondylarthropathies. Parmi ces dernières, la spondylarthrite ankylosante provoque la destruction et la fusion de la colonne vertébrale ainsi que des joints sacro-iliaques. On pense que pour cette maladie spécifique, les tissus de transition permettant d´attacher les ligaments spinaux à l´os, également appelés enthèses spinales, jouent un rôle crucial dans le processus de calcification des ligaments, qui mène à l’apparition de lombalgies et dans le pire des cas à une perte complète de la mobilité du rachis.
Une connaissance approfondie de ces tissus de transition est donc une étape indispensable pour une meilleure connaissance des mécanismes de fonctionnement de la spondylarthrite ankylosante, elle-même préalable à la découverte de traitements efficaces et innovants. L’objectif de ce projet de recherche était l´obtention de propriétés structurelles et mécaniques d´enthèses spinales afin de pouvoir modéliser le phénomène de calcification des ligaments intervenant dans la spondylarthrite ankylosante.
Pour atteindre ce but, nous avons dans un premier temps testé expérimentalement des échantillons d´enthèses spinales – extraites de porcs en croissance - à l´aide de 3 techniques : microscopie acoustique, histologie et microtomographie assistée par ordinateur. Dans un second temps, nous avons développé deux modèles éléments finis des enthèses spinales en utilisant les propriétés déduites de l´étape expérimentale. Chaque modèle comportait deux versions, une saine et une pathologique. Le premier modèle utilisait des valeurs de la littérature et le second les propriétés mécaniques déduites de la phase expérimentale. Pour chaque version pathologique, les modèles ont prédit un étirement du ligament interépineux plus important, ce qui suggère l’apparition de lombalgies car un étirement trop important peut perturber les récepteurs de la douleur dans ce ligament.
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General introduction
As part of my education at the generalist engineer school École Centrale Marseille, I had the opportunity to perform my master internship as an engineer for almost 6 months. Being particularly interested in the public healthcare issues, I chose to do mine in the field of biomechanics, and more specifically in the research and development area.
This master internship is the result of a partnership between two institutes: ‹ The Julius Wolff Institute in Berlin ‹ The Institute of Movement Sciences in Marseille, and more specifically the GIBOC team
I spent my entire internship time in the premises of the Julius Wolff Institute in Berlin.
My assignment during this internship was the determination of the structural and mechanical properties of the entheses, which could be briefly described as transitional tissues performing the attachment of a tendon or a ligament to bone. These transitional tissues present various structural properties and above all display mechanical graded properties. I focused more particularly on spinal entheses, located in the lower region of the back, which are believed to be involved in many diseases associated with low back pain, among them ankylosing spondylitis.
My work consisted of 2 main parts; first an experimental phase during which I used several methods to characterize both structural and mechanical properties of these entheses, and second a numerical phase where I used the results of the previous step to develop two two-dimensional finite-element models of both healthy and pathological spinal entheses.
In this report, I will first briefly present the framework of my assignment; then I will focus on the assignment itself, beginning with a clinical introduction followed by a detailed description of both experimental and numerical phases of my work. This description will of course include the results of these experiments as well as the subsequent discussion. Finally, I will set forth my conclusions regarding the obtained results.
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1- Presentation of the laboratories and of the work environment
1-1- Involved laboratories in this partnership
1-1-1- The Julius Wolff Institute
The Julius Wolff Institute was established in 2008, founded by a merger of the research laboratory of the Center of Musculoskeletal Surgery of the Charité (CMSC) and the Clinical Biomechanics Laboratory of the Charité.
The institute stands in the tradition of Julius Wolff, the founder of the orthopedic clinic in the Charité. The theory of Julius Wolff – the so-called "Wolff's law" – shows the correlation between mechanics and medicine, explaining that the form and structure of bone constantly adapts to mechanical loading.
This theory is still highly relevant for musculoskeletal research today. The institute takes Wolff’s findings as a starting point and investigates the influence of mechanical stimulation on biological reactions. For example, stresses and strains are studied on the musculoskeletal system of patients and athletes. Other important fields of interest of the Institute are the regeneration of bone, cartilage, muscles and ligaments and the analyses of the direct loadings in joints and bones.
Patient care benefits from this research, which also stimulates innovations for implants in the area of joint replacement as well as fracture healing. Through a better understanding of biological processes during healing, new therapies and products for accelerating healing processes can be developed.
Concerning its inner structure, the Julius Wolff Institute is organized into seven research areas: Loading & Movement, Spine, Basic Bone Research, Bone Healing, Cell Behaviour in Regeneration, Stimulation of Healing and finally Imaging, Simulation & Stimulation. Each research team consists of principal investigators as well as research assistants, PhD students and postgraduates from various disciplines including engineering, chemistry and biology. A tight collaboration between the teams, as well as with the CMSC, ensures an active and interdisciplinary exchange of research ideas and findings within the institute. Further information about the Institute can be found here: http://jwi.charite.de/en/institute/
I did my master thesis in the “Imaging, Simulation & Stimulation” group, which mainly concerns itself with ultrasound spectroscopy and acoustic microscopy. A fundamental aspect of its research is the development and application of novel diagnostic tools dedicated for assessment of functional properties/biomechanical competence of musculoskeletal tissues (bone, muscle, cartilage, cells) under normal, pathological, and healing conditions.
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However, since no single technology provides a comprehensive view of the interactions between composition, structure and the resulting functional behavior of the organ (e.g. the resistance to fracture of bone), the group usually combines acoustic methods with other innovative technologies (e.g. synchrotron radiation µCT, Raman spectroscopy, nanoindentation, in-vivo ultrasound, Finite Element Analysis). Another important aspect of its work is the utilization of the elastic interaction of waves with matter to stimulate healing.
The increasing complexity of this research field requires an effective interaction between various traditional research disciplines. Therefore, the Q-BAM lab consists of physicists, engineers, physicians, but also biologists and computer engineers. Moreover, the lab has strong and tight collaborations with national and international experts in the field, e.g. within the French-German research network "Ultrasound assessment of bone strength from the tissue level to the organ level". Further information about the group can be found here: http://jwi.charite.de/en/research/imaging_simulation_and_stimulation/
1-1-2- The Institute of Movement Sciences
The Institute of Movement Sciences is a Joint Research Unit (UMR) of the Aix- Marseille University in association with the scientific Institutes - Institute of Biological Sciences (INSB), - Institute for Humanities and Social Sciences (INSHS), - Institute for Information Sciences and Technologies (INS2I) - Institute for Engineering and Systems Sciences (INSIS) of the CNRS.
About 110 people (scientists, academics, ITA / IATOS and PhD) are working at the Institute. The research themes involve mechanical, physiological, neuronal, psychological and sociological determinants of movement production in living beings, particularly humans. The Institute includes 6 research teams: - Plasticity of neural and muscular system - Comportements perceptivo-moteurs - Contextes, Motivation et Comportements - Movement performance and modeling - Biorobotics - Modeling of the osteo-articular and cardiovascular system and two transverse research axes: Motor deficiency & Risk and security.
One of the partners of this master thesis internship is the research team “Modeling of the osteoarticular and cardiovascular system” (the so-called GIBOC team).
This team project combines complementary skills brought by clinicians, biomechanicians and scientists specialized in materials and structures on a single theme related to the study and characterization of the healthy, degenerative, pathological and repaired osteoarticular system. This group mainly concerns itself with major public health issues, such as the long-term behavior of orthopedic implants or osteoporosis, a disease which affects every one in three women after menopause. Further information about the group can be found here: http://www.laps.univ-mrs.fr/spip.php?rubrique37
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1-2- Work environment
At the Julius Wolff Institute like in many other labs, a special attention is paid to the rules for safeguarding good scientific practice. Every new member of the Institute is made aware of the crucial importance of these rules, the golden rule in this field being intellectual honesty both towards oneself and towards others. Moreover, every research newcomer receives a laboratory notebook, in which he or she is required to record the daily advancement of his/her work.
The work atmosphere is also one of the major preoccupations at the Julius Wolff Institute: for example, recognition of the achievements and successes of each co-worker is important for providing both a mentally stimulating as well as pleasant atmosphere. Enjoying work is indeed essential, so that every member of the laboratory can identify himself/herself with the Julius Wolff Institute and its exciting challenges. In addition, several events aimed at strengthen the existing ties between coworkers are organized; for example, during my internship, I could attend to an excursion to Lübenau, where I had the opportunity to get in touch with all members of the institute in a relaxed atmosphere, while we kayaked an entire day.
It is in these excellent work conditions that my master internship took place.
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2- Assignment realization
2-1- Clinical introduction to the problem
The human lumbar spine is one of the major locations of low back pain which can be caused by spinal injury, aging, genetic disorder or long-term mechanical actions. In the United States, the total cost of low back pain treatments reached $200 billion in 2005 [1] . There is also a high cost in terms of lost working hours associated with such pain.
Ankylosing spondylitis (AS) is a chronic, systemic inflammatory arthritis and enthesopathy ( i.e. pathology of the site of ligament insertion into bone) that causes destruction and ankylosis (fusion) of the spine and sacroiliac joints [2] .
This disease, which approximately strikes a person in a thousand, typically appears in the third and fourth decades of life, and males are affected twice as frequently as females [3] . The most common symptom is by far low back pain, first complaint in 75% of people with AS, which may be difficult to distinguish from other causes of spinal pain, particularly in primary care practice. This may explain why diagnostic delay averages 8 to 9 years from onset of symptoms. One of the imaging signs is the inflammatory joint disease with proliferative bony changes and chronic inflammation at the entheses. This latter induces fibrosis and then ossification of the ligaments and joint capsule, ultimately causing ankylosis of the affected joints.
AS can decrease the spine's flexibility, and eventually lead to a total loss of mobility. This process begins with the inflammation of the bone around the edges of the sacroiliac joints. Once enough bone damage has occurred, the body deposits calcium around the damaged area. These calcium deposits spread to ligaments and discs between the vertebrae, thus leading to fusion of the spine. The term “bamboo spine” [2] describes the typical appearance of the spine under X-rays when AS has caused complete fusion of the vertebral column, with syndesmophytes and ossification extending to the posterior ligaments.
The role of the posterior ligamentous system (posterior longitudinal, interspinous and supraspinous ligaments) in proprioception, essential both in static and dynamic function of the spine [4] , is to act as force transducers, translating the tension of the thoracolumbar fascia, developed in the extremities and torso, into the lumbar vertebral column. Moreover, these spinal ligaments are also involved in the pain onset, as they include pain receptors, which get disrupted and result in pain when the ligaments are stretched [5] . Enthesitis occurring in those areas of the spine diminishes the ability of the spine to move, of the thoracolumbar fascia to influence the alignment of the lumbar vertebrae, and thereby increases their risk of destructive injury [6] .
The calcification of the entheses is the final step before the apparition of the “bamboo spine”. This phenomenon decreases the length of the visco-elastic part of the ligaments and no mechanical properties are available in the literature to describe the resulting phenomenon of diminishing the ability of the spine to move.
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2-1-1- General anatomical recalls
The most important model coordinate system in the medical field is the anatomical space. To describe the standard anatomical position of a human, three planes are usually used (i.e. transverse, coronal and sagittal), which are detailed as follows (see fig 1A):
- The transverse or axial plane is parallel to the ground and separates the head (superior) from the feet (inferior) - The coronal plane is perpendicular to the ground and separates the front (anterior) from the back (posterior) - The sagittal plane separates the left from the right
A B
Fig.1: A- Anatomical space and B- overview of the human spine and its different regions
The human spinal column is situated in the dorsal aspect of the torso and usually consists of 33 vertebrae (see fig 1B). In the upper part of the spinal column, it is possible to distinguish three types of regions: the cervical, thoracic and lumbar regions, which consist respectively of 7, 12 and 5 vertebrae (for further information, see appendix 1 page 64).In its lower part, 5 fused vertebrae form the sacrum and the 4 coccygeal bones form the tailbone.
Although their shape can be very different depending on their position in the spinal column, vertebrae exhibit a common architecture (see fig.2). Indeed a vertebra is the main bone component of the spine and consists of a vertebral body and a vertebral arch. The vertebral arch, which consists itself of a pair of pedicles and a pair of laminae, encloses the vertebral foramen (opening) and supports seven processes: the inferior articular, superior articular, transverse and spinous processes.
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Fig.2: Top and side view of a human vertebra
In order to better understand the articulation mechanisms between vertebrae, let us introduce the concept of the motion segment (see fig.3) [7] . As it can be seen on theses schematic views, a motion segment consists of two vertebrae and the connecting soft tissues. This diagram shows a vertebral body (VB), pedicle (P), superior art icular process (SAP), inferior articular process (AP) and spinous process (SP) . The vertebral bodies are connected by the anterior longitudinal ligament (ALL), the posterior longitudinal ligament (PLL) and the intervertebral disc which consists of a hollow cylinder, the nucleus pulposus (NP), surrounded by a series of cylindrical layers (or lamellae), the annulus fibrosus (AF).
Fig.3: Schematic diagram s of a human motion segment [7]
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The disc is separated from the vertebral body by a thin cartilage end-plate (EP) on the caudal (=inferior) and cranial (=superior) surfaces. Each pedicle is attached to a lamina to form the neural arch. On each side of the arch, the laminae of the upper and lower vertebrae are joined by a ligamentum flavum (LF). There is a spinous process (SP) at the junction of the two laminae. The spinous processes of the upper and lower vertebrae are joined by the interspinous ligament (ISL) and the supraspinous ligament (SSL) . The zygapophyseal joints, also called facet joints form between the superior articular process (SAP) of the lower vertebra and the inferior articular process (AP) of the upper vertebrae.
In the frame on our study we will mainly concern ourselves with the biomechanical properties of the entheses relative to the interspinous ligament, which is colored in red on figure 3.
An enthesis can be defined as a transitional tissue which basically performs a function of anchorage between a tendon or a ligament and a bone. Such tissues displaying mechanical graded properties are believed to play an important role in painful back diseases such as AS.
2-1-2- State of art about entheses
Entheses are sites of stress concentration at the region where tendons and ligaments attach to bone. A special attention should be paid to the structure-function correlations of entheses on both the hard and the soft tissue sides of the junction.
∑ Classification of entheses and ´enthesis organ´ concept
It is possible to distinguish 2 types of attachment sites according to their structure: the fibrous and the fibrocartilaginous entheses . While the former can be associated with indirect attachments, the latter equate to direct attachments because they present an absence of periosteum, which can be defined as the dense fibrous membrane covering the surface of bones except at the joints and serving as an attachment for muscles and tendons. Here we are going to focus on fibrocartilaginous entheses, in which AS is localized.
Fibrocartilaginous entheses present 4 zones of tissue : pure dense fibrous connective tissue, uncalcified fibrocartilage, calcified fibrocartilage and bone. The difficulty of defining with any degree of precision where such an enthesis begins and ends can already be seen.
Figure 4 below shows an histological section of a typical fibrocartilaginous enthesis, the Achilles tendon, stained with Masson's trichrome, a 3-colors staining protocol used in histology which aims at distinguishing cells from surrounding connective tissue. It stains erythrocytes (=red blood cells) and muscle fibers red, collagen blue or green, cytoplasm light red or pink, and cell nuclei dark brown to black.
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Fig.4: Histological section of a typical fibrocartilaginous enthesis (Achilles tendon) showing the 4 zones of tissue at the bone -tendon interface
(D): dense fibrous connective tissue (UF): uncalcified fibrocartilage (CF): calcified fibrocartilage (B): bone
(T): tidemark (basophilic line) separating the two fibrocartilage zones
Scale bar: 300µm
Fig.5: Schematic representation of a fibrocartilaginous enthesis
The enthesis organ concept enables a bett er understanding of ankylosing spondylitis , which provides the most striking examples of entheseal involvement in inflammator y joint disease [8] . It was proposed by Benjamin and McGonagle [9], [10] , who defined an ´enthesis organ´ as a collection of related tissues at and near the enthesis, which serve a common function of stress dissipation. The typical application example of this concept is the attachment of the Achilles tendon to the calcaneus.
∑ Structure , function and m echanical properties of fibrocartilaginous entheses [10]
The organization of such entheses depends on the stresses acting on it. Modifications of this organization in AS can disturb the motion of the associated bone.
Structure
In a fibrocartilaginous enthesis, the fibrocartilage cells in the zone of uncalcified fibrocartilage are arranged in longitudinal rows between parallel bundles of collagen fib ers (see fig.6) . It has been suggested that if rows of fibrocartilage cells are present at an enthesis, they reflect the prior alignment of the fibroblasts from which they differenciate.
Calcified fibrocartilage is typically less cellular than its uncalcified equivalent, probably because the deposition of calcium salts in the extracellular matrix leads to cell death.
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Fig.6: Photomicrograph of fibrocartilage. Note the rows of chondrocytes separat ed by collagen fibers
Fibrocartilage differentiation from tendon/ligament cells at the enthesis involves changes in cell shape and cell -cell interactions. The cells lose contact with their neighbours, round-up and enlarge and express cartilage markers, es pecially type II collagen and aggrecan.
A common feature of normal entheses is adipose tissue, which should not be regarded as a sign of degeneration. It contributes indeed to the increase in surface area of attachment sites, promotes movement between ten don/ligament and bone, and forms part of an enthesis organ that dissipates stress. The presence of numerous nerve endings in fat at attachment sites suggests that it has a mechanosensory role and this could account for the rich innervation of many entheses .
The molecular composition of the extracellular matrix (ECM) of the enthesis is very likely to be directly linked to the mechanical loadings at the soft/hard tissue interface. Indeed entheses undergo local compressive stresses, especially at sites with an oblique insertional angle of the tendon or ligament fibres. These compressive stresses induce a functional adaptation of the molecular composition of the ECM, which leads to the apparition of cartilage-related molecules. Those specific molecules are produced in addition to the classical components of dense connective tissue, and depending on the level of the compressing stresses involved, they may even replace them completely.
Type-I collagen is the prevailing collagen of tendons, ligaments and bone, w hereas type II-collagen is a special feature of fibrocartilaginous tissue, both calcified and uncalcified. In entheses of particularly cartilaginous, it can happen that type -I collagen disappears and is replaced by type II-collagen. As a consequence, a ga p between two tissues containing type I - collagen appears; it is the so-called “gap -phenomenon” (see fig.7).
Ligament or Fibrocartilaginous Bone tendon tissue
Ty pe 1 Collagen Type 2 Collagen Type 1 Collagen
Fig.7: Illustration of the gap phenomenon
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The function of entheses: anchorage and stress dissipation
The main goal of an enthesis is to enable the attachment of the ligament or tendon to the bone. Such a function is central to any force transmission. A great number of comparisons between entheses and other biological interfaces can be drawn. For example, entheses can be compared to the root systems of a tree. Like tendons and ligaments, plants are subject to mechanical forces created by their static loading, the influence of the wind, and the slope of the ground. Only a very small proportion of the plant actually participates in securing firm skeletal anchorage, and it is the same with tendons and ligaments. In both cases, the necessary anchorage is performed with a minimum investment in structural material.
It is also worth emphasizing the fact that fibrocartilage in entheses manages to do exactly what scientists are currently trying to do: creating materials with graded mechanical properties in order to provide a more efficient resistance to damage than their homogeneous counterparts. Thus entheses are able to balance widely different elastic moduli: although tendon and bone have similar tensile strength, the elastic modulus of bone (approximately 21 GPa) is about 10 times larger than that of tendon.
It has been suggested that pathologies occur in the regions where strain levels are lowest. The regions most vulnerable to damage at entheses are likely to be initially stress- shielded. Fibrocartilage is an adaptation to compression and/or shear and is generally more conspicuous in the deepest parts of entheses. Considering the facts that clinically recognizable enthesopathy occurs more frequently in the deep than in the superficial part of an enthesis and that the deep part of an attachment site is compressed by the superficial part, it seems pertinent to take into account these compressive forces to understand enthesopathies, and not only tensile failure.
For example, on the very short human acetabular ligament, which has conspicuous fibrocartilaginous entheses, the rapid increase of tensile stress during load bearing is likely to produce biologically relevant shear stress that acts as the mechanical stimulus for fibrocartilage formation.
Mechanical properties of fibrocartilaginous entheses
Two main difficulties explaining the lack of attempts to study biomechanical aspects of entheses in relation to junctional properties should be taken into account; first, practical difficulties of recording strain levels within such a small volume and tissue and second, the transitional nature of the region without clear well-established boundaries.
Nevertheless, some studies reported mechanical values for entheses: for example, Ho et al [11] characterized the tooth enthesis by means of AFM and AFM-based indentation and reported for this tooth enthesis an elastic modulus between 1 and 3 GPa. Hurng et al [12] also used AFM-based nanoindentation to characterize tooth entheses and found a range in elastic modulus values for periodontal ligament (PDL)-bone and PDL-cementum entheses in 150-380 μm wide PDL-complex of 0.1-1.0 and 0.1-0.6 GPa.
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In the same vein, Sano et al [13] used SAM to realize a speed of sound mapping of the enthesis relative to the rabbit supraspinous ligament. An average value was calculated for each of the 4 zones (see table 1). However, since the velocity measurements were not combined with mass density measurements, mechanical properties can not be derived from this study.
Speed of sound [m/s]
Tendon proper 1661 - 1695 Non-mineralized fibrocartilage 1575 - 1609 Mineralized fibrocartilage 1731 - 1765 Bone 1731 - 1765 Table 1: Speed of sound values for the different zones of the rabbit supraspinous ligament enthesis
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2-2- Aim of the assignment
As described below, the main purpose of this internship was the gain of both structural and mechanical properties of the entheses , in order to model the ligament calcification process occurring during AS . To achieve this goal, firstly we have experimentally tested pig spinal entheses samples with 3 techniques, Scanning Acoustic Microscopy (SAM)- based measurements, histology t echniques and μCT measurements; secondly, we have developed two two-dimensional -finite-element models of spinal entheses by using the mechanical properties deduced from the experimental step.
During the whole project, we made the assumption that our investigated entheses presented the following structure, that is to say a structure of fibrocartilaginous entheses (see fig.8):
Fig. 8: Assumed structure of the investigated entheses
We used 2 different sets of samples of spinal entheses extracted from pigs . The first set was aimed at characterizing the mechanical propert ies of the hard tissue zone, (B) and (CF) zones, and providing structural properties of the whole entheses, while the second was aimed at characterizing the mechanical propert ies of the mechanical prop erties of the soft tissue zones, (UF) and (L) zones.
Experimental tests:
Following experiments were carried out on the samples of the set 1:
‹ First, SAM measurements with a 50 MHz transducer to deduce me chanical properties of the hard tissue zones ‹ Then several types of histological staining and immunohistochemical labelling to localize the different zones constitutive of the enthesis. ‹ Finally, μCT measurements on 1 sample to gain structural properties of the entheses
Following experiments were carried out on the samples of the set 2:
‹ First, SAM measurements with a 5 MHz confocal device to obtain speed of sound maps in the entheses ‹ Then, density measurements with a pycnometer
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The following diagram summarizes all the measurements conducted during the experimental phase of the internship:
Fig.9: O verview of the conducted experiments during the experimental phase
Numerical model:
Once all these experiments were carried out , two two-dimensional finite-element models taking into account their results w ere developed with Abaqus CAE . Those models were used to simulate the ligament calcification process occurring in AS. Therefore, each model included two versions, a healthy one and a pathological one. In the first model we used values from the literature and in the second values from the experimental step.
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2-3- Experimental characterization of spinal entheses
2-3-1- Materials
∑ Presentation of the samples
Two different sets of pig spine samples sent from the Institute of Biomechanics of the Technische Universität Hamburg -Harburg were used to carry out our experiments.
The first set consisted of 4 samples extracted from a pig thoracic spine and more precisely of 2 subsets of 2 samples each, the former subset including entheses samples relative to the supraspinous ligament (1A and 1B) and the latter entheses samples relative to the interspinous ligament (2A and 2B).
A B
Fig. 10: Set 1 samples, subset 1 on the left (A) and subset 2 on the right (B)
The second set consisted of 4 samples extracted from 2 consecutive motion segments of a pig lumbar spine . These samples included entheses relative to the interspinous ligament.
Fig.11: Set 2 samples with a green line indicating the used cutting plane to get thinner sections
∑ Preparation of the samples
Set 1: The samples of the set 1 were prepared using a 5 -step protocol, summarized on the following diagram (for further info rmation, see appendix 2 on page 65):
Dehydration Grinding PMMA Extraction Fixation and and embedding defatting polishing
Fig.12: Schematic diagram of the p reparation protocol of the set 1 samples
First, t he 4 samples were roughly extracted from the thoracic spine of a growing pig aged of 6 months. Then they were placed in tightly closed glass jars, where they soaked in a fixation solution. At the end of this step, the samples wer e maintained under a constant water flow during a half hour.
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They were then dehydrated and defatted (see table 2); the procedure was accomplished by hand on a shaker. Afterwards they were prepared for the PMMA embedding (see table 3).
Liquid Duration Temperature 70% alcohol 3 days Room Temperature 80% alcohol 3 days Room Temperature 96% alcohol 3 days Room Temperature 96% alcohol 3 days Room Temperature absolute alcohol 3 days Room Temperature absolute alcohol 3 days Room Temperature absolute alcohol 3 days Room Temperature xylol 2 hours Room Temperature Table 2: Description of the dehydration and defatting steps
Preinfiltration 1 – 2 days Room Temperature Infiltration 4 – 5 days ca. 4°C Polymerization Overnight ca. 4°C Table 3: Description of the PMMA-embedding step
After being placed in the appropriate orientation, the samples were soused with the polymerization mixture in an embedding plastic vessel until ca. 2-3 mm under the edge of the samples. The vessel was then sealed and the polymerization took place at 4°C overnight. It was important to ensure that no air bubbles appeared. The sample was agitated with a pair of tweezers until they were gone.
After the embedding of the samples, it was still necessary to use a band saw in order to obtain more precise slices of our regions of interest (ROI). Once this latter step done, the samples had to be polished to be able to undergo correct SAM-measurements. This operation was performed using a sample preparation system, the Phoenix 4000 of Buehler®.
The samples were first grinded for a few minutes with a 4000 µm SiC paper under a constant flow of water. The same operation was repeated with a thinner 500 µm SiC paper. Then the embedded samples were polished for several minutes with a 6 µm hard synthetic cloth and a water-based monocrystalline diamond suspension including 6 µm diamond particles as abrasive. The very same operations were then repeated on a 3 µm (resp. 1 µm) hard synthetic cloth and a water-based monocrystalline diamond suspension including 3 µm Fig.13: Sample preparation machine (resp. 1 µm) diamond particles as abrasive. (Phoenix 4000, Buehler) The samples displayed then a sufficient smoothness and a satisfying homogeneous shine; thus they were ready to be examined by SAM.
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Set 2:
These 4 samples were extracted from a pig lumbar spine and were conserved in formalin. They were then sawed in order to minimize their thickness, which should be between 1 and 3 mm. Finally they were rehydrated in PBS during one day before the speed of sound measurements by means of the SAM 5 MHz confocal device.
2-3-2- Methods
To characterize our entheses samples, several experimental methods, both destructive and non destructive and originating from various specialized fields of biology and physics, were applied. They provided complementary information on our samples, mainly on a mechanical and on a structural level.
∑ 50 MHz Scanning Acoustic Microscopy (SAM) measurements
Functioning principles of SAM
Acoustic microscopy is a relatively recent field, in which many applications were developed in the last decades. This technique is usually used to measure material properties which could not be obtained with other processes, such as traditional microscopy for example. In addition to afford an enlarged visualization of opaque materials, acoustic microscopy also provides information on their elastic properties.
One of its major advantages is the fact that this process is both non invasive and non destructive, making it sometimes more suitable than destructive micromechanical examinations, among them nanoindentation. To top it all, acoustic microscopy is able to provide both 2D and 3D structural and mechanical information on the investigated materials. Last but not least, a very interesting aspect of this method is the possibility of a so-called multi-scale data processing, based on the use of a range of usable ultrasound frequencies.
Scanning acoustic microscopy (SAM) uses the piezoelectric effect as well as high focused transducers to generate and measure high resolution pulses. Frequencies between a range of 50 MHz and 2 GHz are particularly adapted to the investigation of local nano- and microscale elasticity of the bone tissue matrix [14] . However in this frequency range, only single element transducers are currently available; therefore acoustic microscopes usually require mechanical scanning for the translation of the transducer.
SAM presents different modes, depending on the employed scanning type. The most important scanning types are A-, B- and C-scans. While an A-scan records at a point a time-resolved signal, a B-scan corresponds to the acquisition of this time-resolved signal at fixed intervals along a line, e.g. the -axis. By combining these two modes a C-scan can be obtained, during which the transducer is being linearly moved in the -direction, thus recording several B-scans one after the other.
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By calculating with this method a reference value for every signal acqui red, it becomes then possible to produce 2D gray -scaled images, which will be used for the further data processing. Figure 14 below illustrates step by step the acquisition of a C -scan for a time-resolved detection. z y x
Fig.14: Recording process of a C-scan. a) Linear scanning of the transducer S, b) recording in fixed intervals of a
time-resolved signal and calculation of S a, c) all S a put together produce a gray -scale image.
In addition to the ability of moving the transducer in the -direction, it is also possible to realize measurements depending on an additional axis, for example a rotation axis or a z-axis. The corresponding modes are written or . , ,
Description of t he Scanning Acoustic Microscope
Th e SAM used to conduct our measurements consists of several hardware components, which communicate through various interfaces and a standard PC -system. The figure 15 below provides an overview of the main components of this device.
Fig.15: schematic di agramm of a Scanning Acoustic Microscope [14]
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The major components of this SAM are a motion controller, a three-axis scanning stage, a Pulser/Receiver, an A/D-card and a transducer, which is fixed to the three-axis scanning stage and linked to the Pulser/Receiver. The positioning of the transducer is performed with a scanner, which three axes can scan a region of 60x60x26 mm 3 with a precision of 0.1 μm. The x-axis is thereby equipped with an optical incremental encoder, which produces every 4 μm a TTL-signal. This enables a precise position assignment and measurement during the continuous scanning process.
The control of these axes is realized by means of a three-axis motion controller MC2000 (ITK Dr. Kassen GmbH, Lahnau, Germany), this latter being linked with the PC via a RS.232 interface and communicating via the programming language VENUS1 (ITK Dr. Kassen GmbH, Lahnau, Germany). The generation of pulses as well as the acquisition is implemented in the triple coupled system consisting of the ultrasound transducer, the Pulser/Receiver and the A/D-card. For the experiments described in this internship, a transducer V605 50 MHz of the firm Valpey Fisher was used. This transducer basically consists of a piezoelectric ceramic PZT5A and a silicon delay-block, which is polished at its end in a spherical concave shape. This provokes the focusing after traversing the near ultrasound field and provides a lateral resolution of approximately 23 μm.
The employed Pulser/Receiver is a Panametrics PR5900 with a 200 MHz receiving bandwidth. It produces short negative spike pulses with a rise time between 1.1 and 2 ns and a voltage amplitude between 77 and 175 V.
For the data collection and the A/D-transformation, a Gage CompuScope 12400 card with an analogue bandwith of 200 MHz, a sampling rate of 400 MS/s and a memory depth of 12 bits is used. It provides a quick data transfer, which is essential for the continuous scanning routine.
In addition to these components, which are linked to a PC, there are still other external devices. The most important here is the water tank, which temperature is controlled in a range of 20-60°C with a precision of 0.1 °C by a temperature controlled stage and thermal resistors. This is of significant importance for the gain of the reflection coefficient, because the time of flight as well as the ultrasound attenuation strongly depends on temperature.
Preparation of the acquisition
To carry out the SAM measurements, it was necessary to use an ultrasound coupling medium between the 50 MHz transducer and the sample, which contributes to the transmission of ultrasonic waves. In our case, we chose water, mainly because of its low attenuation. To avoid the formation of air bubbles during the measurements, which are likely to stick to the transducer and lead to wrong results, distilled water was degassed in an ultrasonic bath with a vacuum pump. Once poured into the ultrasonic bath, the distilled and degassed water was held to a constant temperature of 26.6°C. In order to eliminate the air bubbles in the pores of the samples embedded in PMMA, these latter received the same treatment than the coupling fluid and were degassed in the ultrasonic bath before starting the measurements.
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Once the scanning region set thanks to the SAM 200 software, the samples could undergo a C-scan procedure. The chosen spatial increment size in the x- and y-directions was 16 μm, thus resulting in measurements of approximately one hour. Moreover, complementary B(z) scans with an increment of 4 μm in the z-axis direction were performed both in a bone area and in an area including embedding material.
Immersion of the embedded samples in water during the acoustic inspection causes diffusion of water into resin and tissue. However, in our case, the resulting swelling could be neglected because the sample was embedded properly and because the duration of the exposure to water was short, that is to say inferior to 2 hours for 50MHz measurements. Nevertheless, it should be noted that repetitive wet-dry cycles result in a remarkable increase of the surface roughness [14] .
Acoustic impedance mapping
The acoustic impedance of a medium is defined by the relation: (1) where ρ is the mass density of the medium expressed in kg/m3, c the speed of sound in it, expressed in m/s and Z the acoustic impedance of the medium expressed in rayl
Under the condition of normal impedance, (when the sample surface is perpendicular to the sound beam axis), acoustic impedances are defined by the following relation:
2
where R is the confocal reflection coefficient and Z1 (resp Z 2) the acoustic impedance of the coupling fluid (resp. the investigated material) expressed in rayl
The estimation of the acoustic impedance from the confocal reflection amplitude requires very stable measurement conditions as well as a sophisticated calibration. All influences that potentially have an effect on the measured voltage in addition to the variations caused by the reflection coefficient have to be excluded or compensated. A summary of the most prominent involved effects can be found on the array of the appendix 3 page 66.
To access to the acoustic impedance distribution on a sample surface, the first thing to do is to establish the link between the confocal reflection amplitude V and the confocal reflection coefficient R. It can be obtained at a given temperature by performing a calibration using several homogeneous isotropic and non-dispersive materials.
(3)
To achieve this goal, speed of sound and mass density of these materials and the coupling fluid should be determined by a low frequency substitution method and by Archimedes’ principle, respectively. From equation (2), the corresponding reflection coefficient R can be calculated. Finally, the relation between the reflection coefficient R and the measured voltage V is obtained by linear regression (see fig.16).
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Fig.16: impedance calibration
Thanks to this calibration, acoustic impedance images can be obtained from the acquired C-scans and B(z)-scans, these latter acting as a correction.
Raum [14] found a relation which enables the estimation of the elastic coefficient c ii from a given impedance Z (see fig.17):
Fig.17: Relationship between acoustic impedance and elastic stiffness for different materials
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From these elastic coefficients c ii it is then possible to deduce engineering constants, assuming that all independant coefficients are known. For example, the conversion between c33 and E 3 is :
1 1 2 4 1 where is the Po isson’s ration in the cross -sectional plane ( – plane) and and are the Poisson ratios in the longitudinal section (i.e., and planes). In order to simplify that kind of equations, a few hyp othesis are usually made, according to the assumed structure of the investigated material (e.g. isotropic transverse).
The figure 18 below summarizes the obtaining process of th ese engineering constants from the SAM measurements:
Fig.18: Derivation of Young’s moduli from the measurement of the confocal reflection amplitude
∑ Histology staining
Once the SAM measurements with the 50 MHz were conducted, the 4 sa mples of the set 1 needed to be prepared to undergo several types of histology staining, in order to localize precisely the different types of tissue within the entheses samples. Indeed, histology being a destructive method, it was suitable to conduct the SAM measurements first.
First of all, the size of the samples was increased by adding two types of PMMA (see fig.19), so that samples displayed a size adapted to the cutting of histological slides.
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Fig.19: “Enlargement of the samples”, a) Re -embedding in a larger amount of PMMA b) &c) addition of a yellow fast polymerization PMMA resin to increase the height of the samples.
Vertical sections, perpendicular to the surface of the sample, were then cut by means of a steel knife mounted in a microtome (see fig.20). The thickness of these histological slides was 6 μm, and twelve of them were realized for each of the four samples.
Fig.20 : Overview of the microtome with the steel knife mounted in it
The tissue slices were laid on a glass slide immerged in a 70% ethanol solution, to prevent the drying out of the tissue slice and the eventual apparitio n of cracks. Finally, a thin film washed with a stretching liquid was laid on the top of the tissue slice. The aim of this stretching liquid wa s to avoid that the sample wound ed on itself because of its low thickness. The samples were then covered with thi n foil.
Three types of histology staining were perform ed: Movat’s pentachrome, Safranin - O/von Kossa and Safranin-O/Light green staining. The detailed protocols of these staining can be found in the appendix 4 on page 67.
‹ The Movat’s pentachrome staining , which enables a differentiation of hard and soft tissue components , was aimed at distinguishing mineralized and unmineralized cartilage and bone tissue.
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Feature Color Mineralized bone tissue, collagen bright yellow Cartilage tissue yellow Elastic fibers red Osteoid dark-red Mineralized cartilage blue-green Table 4: Correspondence feature – color for Movat’s pentachrome staining
‹ The Safranin-O/von Kossa staining should highlight the difference between mineralized and unmineralized tissue and provide a good visualization of the so-called tidemark or mineralization front.
Feature Color Cartilage intensive red Bone green Connective tissue green Table 5: Correspondence feature – color for Safranin-O/Light green staining
‹ The Safranin-O/Light green staining was used to differentiate the cartilage from the bone tissue.
Feature Color Mineralized bone and cartilage black Unmineralized bone pale red Hyaline cartilage intensive red Table 6: Correspondence feature – color for Safranin-O/von Kossa staining
The pictures were acquired by means of a light microscope Leica DMRB equipped with a camera AxioCam MRc (Zeiss) and the software AxioVision. In our case, using the objective 2.5x and 5.0x turned out to be sufficient.
∑ Immunohistochemical labelling
Type 1 and type 2 collagen staining were performed on the sample 1A of the set 1. The main objective of this experiment was the visualization of the gap phenomenon described page 16. It was also a mean of confirming the results obtained with the histological staining, and more particularly the Safranin-O/Light green staining, given that the only zones in which type 2 collagen is present in the entheses are mineralized and unmineralized cartilage. The detailed protocol for these two staining can be found in the appendix 5 on page 69.
The pictures were acquired by means of a light microscope Leica DMRB equipped with a camera AxioCam MRc (Zeiss) and the software AxioVision. In our case, using the objective 2.5x and 5.0x turned out to be sufficient.
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∑ Micro C omputerized Tomography (μCT) measurements
In order to gain additional information on the structural properties of the pig spinal entheses, the half of the sample 1A displaying the most important area of transitional tissue was scanned with a micro-CT scan ner of Skyscan with a resolution of about 2 μm (see fig.21). The sample was stuck to the holder by using a temporary resin acrylic (Bosworth Trim) .
Fig.21: Inside of the microCT scanner device. The scanned part of the sample is surrounded in red
∑ Speed of sound measurements with the 5 MHz SAM confocal device
The aim of these measurements was the gain of speed of sound in the soft tissue zones of the entheses. To achieve this goal, the samples of the set 2 were investigated by means of a 5 MHz SAM confo cal device. The design of the device was pretty much the same than for the 50 MHz measurements, except for three main differences (see fig.2 2): - Two transducers with a frequency of 5 MHz instead 50 MHz (RA5.06.09 and RA5.04.09) , which provided a resolution of 0.5 mm , were confocally aligned in through-transmission mode. - These transducers were not tight to the three -axis scanning stage, but directly laid in the coupled fluid tank, whereas the examined sample was bound to the three-axis scanning stage . - The cou pling fluid tank was filled with a phosphate buffered saline solution (PBS), which was beforehand degassed in the ultrasonic bath with the vacuum pump.
Fig.22: Experimental setup for the SAM 5 MHz confocal measurements
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Two types of measurements were performed: through transmission (TT) and pulse- echo (PE) measurements. While the former required both transducers, the latter only necessitated only one transducer. The RA5.06.09 being slightly more sensitive than the other, it was used as the receiv er for the measurements in TT mode. Only B(z) -scans with a spatial increment of 0.1 mm in the z -axis direction and a spatial increment of 0.1 mm in the x-axis direction were recorded.
The experimental set up can be schematized by the following figure:
Fig.2 3: Top view of the 5MHz SAM confocal device
To determine the speed of sound (SOS) in our sample, we have the following system of equations:
(5) (6)
(7) ∆ 10 (8) (9) ∆
with: the thickness of the sample the distance between both transducers the distance between the sample and the transducer 1 the distance between the sample and the transducer 2 the speed of sound in PBS the time of flight in PBS to travel the distance (without sample) the time of flight in PBS to travel twice the distance the time of flight in PBS to travel twice the distance the time of flight in PBS to travel the distance with the sample in the middle
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∑ Mass density measurements
The aim of these measurements was the gain of mass density values for the components of the soft tissue part of the entheses, in other words the unmineralized cartilage and ligament zones. The used device was a pycnometer and the measuring principle is based on Archimede’s principle.
Fig.24: a pycnometer with: the weight of the empty pycnometer the weight of the pycnometer filled with distilled water the weight of the pycnometer with the solid body the weight of the pycnometer filled with distilled water with the solid body the mass density of distilled water,
Then it is possible to access to the mass density of the investigated solid body:
11
2-3-3- Results
∑ 50 MHz Scanning Acoustic Microscopy (SAM) measurements
After processing data, we obtained for each of the 4 samples of the set 1 an acoustic impedance map and a time of flight (TOF) map. This latter was above all aimed at controlling that the acquisition of the sample occurred in correct conditions, that is to say that the surface of the investigated sample was really perpendicular to the sound beam axis and in the focal plane of sound field.
In order to check that quickly, I developed a customized colormap which took into account the focus position of the transducer according to the temperature. This theoretical value was placed in the middle of the color bar and associated with the green color. TOF higher than this value were plotted in red, indicating a bigger distance between the transducer and the sample, and TOF smaller than this value were plotted in pink, revealing a shorter distance between the transducer and the sample.
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Results for the SAM measurements for the 4 samples of the set 1 (see description page 21):
Fig.25: Acoustic impedance map of the sample 1A [MRayl]
0 17.7 1 17.6
2 17.5 3 17.4
4 17.3
0 5 10 15 20 25 30 35 Fig.26: TOF map of the sample 1A [μs]
Fig.27: Acoustic impedance map of the sample 1B [MRayl]
0 17.7
17.6 2 17.5
4 17.4
17.3 6
0 5 10 15 20 25 30 35 40 Fig.28: TOF map of the sample 1B [μs]
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Fig.29: Acoustic impedance map of the sample 2A [MRayl]
0 17.7
1 17.6 2
17.5 3
4 17.4
5 17.3 6
0 5 10 15 20 25 Fig.30: TOF map of the sample 2A [μs]
Fig.31: Acoustic impedance map of the sample 2B 0 17.7 1
2 17.6
3 17.5 4
5 17.4 6 17.3 7
8 0 5 10 15 20 25 30 Fig.32: TOF map of the sample 2B [μs]
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- The samples 1A and 1B displayed a very similar structure (see fig.25 and 2 7): on one of the longest edge of the sample, an external layer of cortical bone could be seen. This layer was external to an oval region made of trabecular bone. An interesting feature was the surrounding of this external layer by a thin layer. Because ligament surrounded this thin layer, we first thought it co nsisted of mineralized cartilage but the histology revealed it was actuall y a trabecular bone layer. Both tissues seemed to smoothly overlap, resulting in a decreasing stiffness and an increasing porosity when moving worm outside the sample. The following figure illustrates this transition:
Embedding material (PMMA) Ligament External trabecular layer External cortical layer
Internal trabecular layer
Fig.33: Zoom on the longest edge of the acoustic impedance map of the sample 1B
- The sample 2A presented the particularly to display a second cortical layer on its upper long edge, but only one of them, i.e. the lowest on the acoustic impedance map picture (see fig.29), showed the external trabecular layer also seen on the samples 1A and 1B.
- Unlike the other samples, the sample 2B did not present a very thick cortical layer and did not seem to o wn an external trabecular layer (see fig. 31)
- All samples were surrounded by a ligament layer , and three of them (1A, 1B and 2B) conspicuously displayed at least one other transitional zone on a corner. From the center of the sample to the involve d corner, the structure was the following: first a bony structure with a decreasing stiffness and a decreasing porosity, then a cartilage region (a little triangle - shaped), which was finally surrou nded by a ligament zone. By looking closer on some samples, it was even possible to see two zones into the cartilage region.
Bony structure with decreasing Cartilage Cartilage region Ligament stiffness and poro sity region (zone 1) (zone 2) layer Fig.3 4: Zoom on the right half of the acoustic impedance map of the sample 2B
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Gain of local properties of the set 1 samples
To achieve this goal, a custom region of interest (ROI) -tracking software was developed. It was based on a threshold segmentation technique and enabled the gain of the local microelastic coefficient c 33 and the local porosity of the set 1 samples.
For the samples 1A, 1B and 2A, a ROI-system consisting of three different zones corresponding to the external trabecular, cortical and internal trabecular layers was applied.
Fig.35: Successive three-storey ROI-system applied on the sample 1A
As it can be seen on this picture, for some ROIs the content of the storey was not representative of the corresponding material, therefore only the most suited ROIs were used for the data processing. These “good” ROIs were mostly to be found in the middle of the sample, as the following picture shows it:
Fig.36: Boundaries of the selected ROIs on the sample 1A for the further data processing
For the 3 samples 1A, 1B and 2A, the data concerning the external trabecular layer were ignored in a first time in order to simplify the problem and mostly because of numerical difficulties: indeed the height of this layer was really small and consisted of very few pixels, which led to aberrant values of local porosity and stiffness reported by the software.
For the sample 2B, a ROI-system consisting of two different zones corresponding to the external cortical and internal trabecular layers was applied (see fig.37).
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Fig.37: Successive two-storey ROI-system applied on the sample 1A
Finally, the following results were obtained for local microelastic coefficient c 33 and the local porosity of the set 1 samples:
Fig.38: Comparison of the local elastic coefficient c 33 in the cortical and internal trabecular bone layers of the set 1 samples
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Fig.39: Comparison of the local porosity in the cortical and internal trabecular bone layers of the set 1 samples
The table 7 below summarizes the obtained results:
Median elastic Median porosity coefficient c 33 (GPa) cortical trabecular cortical trabecular Sample Processed ROIs bone bone bone bone 1A 17 20.6 20.9 0.280 0.835 1B 25 21.6 23.5 0.255 0.776 2A 28 20.3 23.3 0.230 0.798 2B 22 15.2 21.7 0.597 0.820 Table 7: Obtained results with the 50 MHz SAM measurements
It should be noted that in spite of some really different values for local porosity and elastic coefficient for the cortical layer of the sample 2B (highlighted in yellow), the 4 samples seem to present similar values for these two parameters, both for cortical and trabecular regions.
Here it would seem appropriate to exclude the values surrounded in yellow, mostly because as we observed it earlier, the cortical layer of the sample was not as developed as the one in the other samples.
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Gain of homogenized properties of the set 1 samples
We used an Asymptotic Homogenization (AH) algorithm to gain a map of the homogenized elastic coefficient C33 , from which an average value was extracted by applying the same ROI-system than before. This value was then ready to be used as an input parameter for the material properties of the developed finite-element models. The homogenization process took into account the local porosity of the samples in the calculations.
Fig.40: Map of the homogenized elastic coefficient C33 for the sample 1A
Fig.41: Map of the homogenized elastic coefficient C33 for the sample 1B
Fig.42 : Map of the homogenized elastic coefficient C33 for the sample 2A
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Fig.43 : Map of the homogenized elastic coefficient C33 for the sample 2B
Fig.44: Successive three-storey ROI system applied on the sample 1A
Fig.45: Comparison of the homogenized elastic coefficient C33 in the cortical and internal trabecular bone layers of the set 1 samples
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Once again, it would be reasonable to exclude the value of the homogenized C 33 obtained in the cortical layer of the sample 2B. The table 8 below summarizes the obtained results: Median elastic
coefficient C 33 (GPa) cortical trabecular Sample Processed ROIs bone bone 1A 17 10.2 6.2 1B 25 (23 cortical) 12.2 7.6 2A 28 (23 cortical) 12.3 7.1 2B 22 7.4 6.0
Table 8: Summary of the obtained results for the homogenized C 33
The homogenization process, which takes into account the local porosity of the material, yields the compound properties of cortical and trabecular tissues. Because of its high porosity at the macroscopic scale, trabecular bone is softer than cortical bone, which is consistent with the existing literature.
Finally, we gained the following mean values for the cortical and internal trabecular bone areas:
Cortical bone C33 = 11.6 GPa Trabecular bone C33 = 6.7 GPa
∑ Histology staining
The sample 2B provided the most representative interface structure. Therefore, following analysis is described for this sample.
Fig.46: Overview of the Movat’s pentachrome staining for a slide extracted from the sample 2B
With the Movat’s pentachrome staining (see fig.46) it is already possible to see that the ligament layer surrounds the entire sample and that the cartilage is located in both corners of the histological slide. Furthermore, by looking closer to the right corner, it is possible to observe that this cartilage area seems to consist of approximately three zones: first a mineralized cartilage zone in blue-green (zone A on fig.47), then a thin unmineralized cartilage zone in yellow (zone B on fig.47) and finally an extended zone of unmineralized
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fibrocartilage in yellow and pale green (zone C on fig.47). Moreover, this figure shows well the smooth transition between all these different kinds of tissues.
Zone A Zone C
Zone B
Fig.47: Zoom on the right (and bigger) corner of the Movat’s pentachrome staining of the sample 2B
In addition to provide the location of the cartilage and bone areas in the sample, thus confirming the results obtained by the previous staining, the Safranin-O/Light green staining (see fig.48) exhibits an interesting feature: by looking closer to the same corner than before (see fig.49), a phenomenon of endochondral ossification can be observed. This process, which corresponds to the formation of bone tissue, involves the presence of cartilage cells (chondrocytes) in the primary center of ossification. The centripetal aspect of this process can be well visualized on the left side of the figure 49.
Fig.48: Overview of the Safranin-O/Light green staining for a slide extracted from the sample 2B
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Fig.49: Zoom on the right (and bigger) corner of the Safranin-O/Light green staining of the sample 2B
Finally, the Safranin-O/von Kossa staining (see fig.50) allows for a clear visualization of the areas occupied by mineralized and unmineralized tissues without distinction between bone and cartilage tissue.
Fig.50: Overview of the Safranin-O/von Kossa staining for a slide extracted from the sample 2B
A zoom on the right side of the sample (see fig.51) enables to see clearly the limit between the mineralized and unmineralized tissue areas, the so-called mineralization front or tidemark.
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Fig.51: Zoom on the right (and bigger) corner of the Safranin-O/von Kossa staining of the sample 2B
By comparing the pictures obtained from the Movat’s pentachrom and Safranin- O/Light green staining, one could remark that the mineralized area of the Safranin-O/von Kossa staining does not correspond to the mineralized cartilage area of the Movat’s pentachrom staining. This would be later discussed.
To assess the importance of the surface area of the mineralized cartilage tissue, both histological slides of the Safranin-O/von Kossa and Safranin-O/Light green should be superimposed and the thickness of the mineralized cartilage layer could be gained.
∑ Immunohistochemical labelling
Fig.52: Overview of the type 1 collagen labelling for a slide extracted from the sample 1A
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Fig.53: Overview of the type 2 collagen labelling for a slide extracted from the sample 1A
For the type 1 collagen (see fig.52), the areas including this specific component are labelled in dark purple, whereas for the type 2 collagen (see fig.53), the areas including it are labelled in pink. It is worth noting that the phenomenon of endochondral ossification can be well seen on the figure 53.
∑ μCT measurements
These measurements enabled a three-dimensional visualization of the mineralized tissues of the sample (see fig.21). In figure 54-A), it is possible to see the global structure already seen with the 50 MHz SAM measurements and histology staining. In figure 54-B), we see how the transition between the internal layer of cortical bone and the mineralized cartilage is smoothly made.
A B
Fig.54: Micro-CT pictures of the upper part of the sample 1A, A) overview of the entire scanned domain, B) zoom on the transition zone between the internal trabecular bone layer and mineralized cartilage
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Fig.55: Micro-CT picture of a cross-section of the sample 1A including the cortical bone layer on the left
∑ Speed of sound measurements with the 5 MHz SAM confocal device
As previously described, these measurements allow for a speed of sound mapping of the set 2 samples. Unfortunately, only one sample led to a satisfying result: this could be explained by the fact that the other samples displayed a too irregular thickness variation or the interfaces were not perpendicular to the section planes. These factors are indeed really crucial for through transmission measurements.
The processing of the raw data was a complex issue. In fact two kinds of ultrasonic waves propagated: a fast wave which was led through trabeculae and a slow wave which was led through the coupling fluid, here the PBS solution. The measured voltage of the fast wave was significantly smaller than the voltage of the fast wave, thus making the detection of this wave difficult. It explains for example why on figure 56, the speed of sound in the trabecular bone area was similar to that measured in water. At the temperature of measurement (35.2°C), the speed of sound in PBS was approximately 1540 m/s.
Fig.56: Speed of Sound map in a set 2 sample
On figure 55 the cortical layer region could be clearly recognized and corresponded to the red area. This material belonging to the stiffest of the enthesis, it displayed speed of sound values of about 3000 m/s. The figure 56 also showed a transitional green zone between bone and cartilage, in which the speed of sound was about 2250 m/s. Then there were a small layer of cartilage and finally an important ligament area. Our goal was the obtainment of speed of sound values for the cartilage and ligament zones of the enthesis. To achieve it, two ROIs corresponding to these two zones were drawn (see fig.57), and several properties of them were calculated (see table 9).
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Fig.57: ROI masks of the cartilage and ligament areas used for the calculations of SOS in these regions
Mean value Median value Standard Minimum Maximum Tissue [m/s] [m/s] deviation [m/s] value [m/s] value [m/s] Cartilage 1792 1807 96 1527 2349 Ligament 1592 1588 30 1540 1746 Table 9: Speed of sound values obtained in a set 2 sample
The mean speed of sound in the cartilage region was a little higher than in the ligament region, but the value for this latter region seemed to be “more trustable” first because of the bigger size of the selected area and second because of the significantly smaller standard deviation.
In addition, we selected several paths from the beginning of the transitional zone tissue between bone and cartilage until the ligament zone (see fig.58). We plotted then an average speed of sound profile curve along the selected paths (see fig.59).
Fig.58: Paths selection between the bone-cartilage transitional area and the ligament zone
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Fig.59: Average speed of sound profile along the selected paths
On this speed of sound profile, three different areas can be distinguished: the first 2 mm correspond to the transitional green zone between bone and cartilage, the 3 following mm to the cartilage layer, and the rest to the ligamentous zone. A smooth transition is observed between these two last zones.
∑ Mass density measurements
The speed of sound map defined the boundaries of the cartilage and ligament areas. Using it, a small section of each zone was extracted from the sample and dried. However, even if the calibration measurement with a piece of PMMA worked out very well, these measurements did not provide correct mass densities for cartilage and ligament. The obtained values were 1.01 g/cm 3 for cartilage and 0.58 g/cm 3 for ligament. The ligament mass density value was indeed particularly disturbing because the piece of ligament (as well as the piece of cartilage) did sink in water and should therefore have a density higher than this of water! This could probably be due to a swelling of the tissue which may have caused this artifact in the measurements.
It is known that ligament almost exclusively consist of parallel collagen bundles. The mass density of collagen being estimated between 1200 and 1600 kg/m 3, it seems 3 reasonable to take ρ ligament = 1200 kg/m for the continuation of our study. Concerning the cartilage, Leicht & Raum [15] found for cartilage an impedance between 2.1 and 2.5 Mrayl depending on the mineralization of the tissue. The most part of cartilage being unmineralized, it seems reasonable to estimate it thanks to equation (1) to 2.1 10 1170 / 1792
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2-3-4- Discussion
∑ SAM 50 MHz measurements
It should be noted that for the 4 samples of the set 1, the highest values of acoustic impedance (and thus elastic stiffness) were found in the internal trabecular bone area, which is unusual. We were expecting these highest values to be found in the cortical bone area. This would maybe have a link with the fact that the examined samples were extracted from growing pigs aged of 6 months.
After the application of the asymptotic homogenization algorithm, which took into account the local porosity of the materials, higher values of homogenized elastic coefficient C33 were found in the cortical bone area than in the inner trabecular bone area. These values reflected the elastic stiffness of the tissue and its inner inclusions.
The architectural differences between the 4 samples of the set 1 could be explained by the cutting step of the samples. In the same vein, the external layer of trabecular bone, which was found on only one large edge of three samples, may be due to the fact that the cutting step may have included a small part of another vertebra feature.
∑ Histology staining
By comparing the pictures obtained from the Movat’s pentachrom and Safranin- O/Light green staining, one could remark that the mineralized area of the Safranin-O/von Kossa staining (see fig.51) does not correspond to the mineralized cartilage area of the Movat’s pentachrom staining (see fig.47).
This could be explained by the fact that the chemical dyes involved in both staining processes are not the same and thus present different sensibility to their respective targets: for example the dye(s) in Movat’s pentachrome linked to the staining of mineralized cartilage tissue seem to be much more sensitive than the dye relative to the staining of mineralized tissue in Safranin-O/von Kossa staining.
∑ Immunohistochemical labeling
The type 1 and type 2 collagen labelling are usually used with paraffin-embedded samples, or more preferably with frozen slices of samples. In our case, it was not sure at all that this experiment would work because the sample was embedded in plastic and more precisely PMMA, but against all odds we managed to obtain some “correct” labelling which were similar to the results of the previous histology staining.
In particular, both histology staining and immunohistochemical labelling revealed the existence of an endochondral ossification phenomenon inside the sample. This is due to the fact that the pig samples we investigated on were extracted from growing pigs. This observation suggested that entheses can act as growth plates for apophyses at tendon and ligament attachment sites, which was in agreement with the previous work of Knese & Biermann [16].
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In a similar perspective, Gao et al. [17] conducted a developmental study of the medial collateral ligament of the rat knee joint and showed by exploiting age-related changes of labelling in types I and II collagen that the cartilage at the enthesis is initially derived from that of the embryonic bone rudiment. Equally, however, they showed that this hyaline cartilage is eroded during endochondral ossification and replaced by enthesis fibrocartilage that develops within the adjacent ligament by fibroblast metaplasia.
∑ Speed of sound measurements with the 5 MHz SAM confocal device
The mean speed of sound in the cartilage region (1792 m/s) was a little higher than in the ligament region (1592 m/s), but the value for this latter region seemed to be “more trustable” first because of the bigger size of the selected area and second because of the significantly smaller standard deviation. Nevertheless, the speed of sound value in ligament was coherent with the precedent findings: Topp & O’Brien [18] found indeed speed of sound values in rat skeletal muscle in a range of 1560 to 1590 m/s.
The observed smooth transition between the cartilage and the ligament areas on the speed of sound profile (see fig.59) suggested an improved balance of the different elastic moduli of both zones in comparison to a direct transition which could be modeled with a step function. This should logically lead to a better resistance to damage.
2-3-5- Synthesis
All the experiments which were carried out in this part enabled the gain of structural as well as mechanical properties of the spinal entheses of growing pigs.
The SAM 50MHz measurements, the histology slides, the immunohistochemical labellings and the micro-CT measurements contributed to the obtainment of structural properties of these spinal entheses of growing pigs. Moreover they brought out the occurring phenomenon of endochondral ossification. Another interesting finding was the existence of an external layer of trabecular bone between the cortical bone and the ligament layers.
The SAM 50MHz & 5MHz (with the confocal device) provided us mechanical properties of these growing pigs’ spinal entheses:
• The SAM 50 MHz measurements permitted us to gain local values of porosity and elastic coefficient c33 both in cortical and trabecular bone areas, and also values of the homogenized elastic coefficient C33 in these two regions.
• The SAM 5 MHz measurements allowed for the obtainment of speed of sound values in both cartilage and ligament regions. By combining these values with their respective mass density, the impedance and thus the elastic coefficient C 33 of these two areas could be deduced.
These results formed a solid basis for the development of finite-element models of spinal entheses in growing pigs.
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2-4- Numerical modeling of spinal entheses
Thanks to the previous experiments, it was possible to build two-dimensional finite- element models of spinal entheses in growing pigs. The models were created with Abaqus CAE. Each model consisted of two versions: a healthy one and a pathological one reproducing the ligament calcification phenomenon observed in AS.
2-4-1- Presentation of the generic model
The first step in the realization of the successive two-dimensional finite-element models with an increasing level of complexity was the creation of a so-called “generic” model, from which only the material properties should be changed when testing a new and more thorough model. Moreover, this “generic” model should facilitate the comparison between all these successive two-dimensional finite-element models.
To achieve this goal, we made several hypotheses:
• Structural consideration : first of all, for the sake of simplicity, we willingly excluded the external layer of trabecular bone tissue from our modeling. It was mostly due to our lack in properties of this specific area. Therefore we consider that our entheses consisted of five distinct materials: - cortical bone - trabecular bone - mineralized cartilage - unmineralized cartilage - ligament
• Geometrical consideration : because of the observed structure of the samples on the impedance pictures as well as on the histological slides, it was decided to use symmetry conditions to model only one quarter of the full enthesis (see fig.60). The length of this quarter was 12 mm and its height 2 mm.
Cortical bone layer Ligament layer Internal trabecular bone layer Mineralized cartilage Unmineralized cartilage
Fig.60: Chosen geometry of the right upper quarter of the spinal enthesis of a growing pig
• Theoretical framework consideration: we decided to work in the framework of linear elasticity, again for simplicity purposes. Moreover, we assumed that all materials were isotropic. The only input parameters for the material properties of the different zones were the Young’s modulus and the Poisson’s ratio.
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• Boundary conditions: the symmetry conditions determined the boundary conditions on the left (X-symmetry) and lower side (Y-symmetry) of the model The hypothesis of plane stress was also made. A tensile load of 0.25 N was applied on the right (therefore external) side of the enthesis quarter. This value was chosen because other numerical finite-element models used a load of 15 N for a bending of 20° of the entire interspinous ligament. Given that here we had only one slice including the ligament, this value had to be normalized by the length of the interspinous ligament, estimated to be about 30 mm (see fig.61). Moreover the symmetry conditions imposed us to divide the normalized value by 2.
« 1 »
Spinous process Vertebral body
F Interverte bral disc
L
Fig.61: Determination of the tensile load applied on the models
• Mesh: quadratic elements with exact integration were used here. The shape of the element was quadrilateral with a typical size of about 50 μm.
It is important to note here that the measured c 33 , which led to the gain of the homogenized C 33 , and which corresponds to c zz in our case, does not correspond to the c ii of the traction direction, c 11 or c xx . This is one of the reasons why the isotropy hypothesis was made.
2-4-2- Developed models: description and results
The main parameter we focused on was the strain component in the traction direction. It was a relevant clinical indicator since the interspinous ligament includes pain receptors, which get disrupted and result in pain when the ligaments are stretched [5] .
In particular we plotted this strain component along a specific path (see fig.62). The expected result was a smooth transition between the different zones.
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Fig.62: Selected path to plot the strain component in the traction direction
∑ Model 1
This model exclusively used values taken from the existing literature (see table 10), [19–22] . No distinction was made between the mineralized and unmineralized cartilage zones. The calcification phenomenon (typical of AS) was taken into account into increasing (3 times higher) the value of the Young’s modulus of the cartilage in the pathological version.
Cortical Trabecular Mineralized Unmineralized Interspinous Tissue Parameter bone bone cartilage cartilage ligament Healthy Young's modulus [MPa] 18600 500 7 5 model Poisson's ratio 0.3 0.3 0.45 0.45 Pathological Young's modulus [MPa] 18600 500 21 5 model Poisson's ratio 0.3 0.3 0.45 0.45 Table 10: Material properties of the different tissues of the enthesis used in model 1
We obtained the following distribution of the strain component in the traction direction for the healthy and pathological versions:
Fig.63: Repartition of the strain component in the traction direction for the healthy model
Fig.64: Repartition of the strain component in the traction direction for the pathological model
When plotting the strain component in the traction direction along the selected path (see fig.62), we observed the following curves:
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healthy
pathological
[mm] Fig.65: Strain component of the traction direction along the selected path (see fig.62)
It is worth nothing that both pathological and healthy versions displayed similar strain profiles (see fig.65) in the traction direction: this profile reminds a step function, with a clear distinction of three different levels. The first level corresponded to the trabecular bone, in which the strain almost remained constant (almost null). The second level was relative to the cartilage region and also remained pretty constant in the entire zone. The third level corresponded to the ligament zone and the strain progressively decreased when moving outside of the sample, with a slight ascent at the end.
Because the cartilage zone was assigned a higher Young’s modulus value in the pathological version than in the healthy version, the strain of this material naturally became lower in the pathological version and approximately diminished from a maximum of 2.25% to a maximum of 1%.
However, the ligament had the same mechanical properties for both versions; therefore the strain in this zone was more important in the pathological version. Indeed the ligament took the most important part of the load, due to the increased stiffness of the cartilage region. The gap in terms of Young’s modulus between the two materials passed from 5 to 7 MPa to 5 to 21 MPa, thus explaining the bigger difference between the strains in cartilage and ligament areas. The maximal strain recorded in the ligament zone was 3.0% in the healthy version and 4.7% in the pathological version, thus being multiplied by more than one and a half. Such stretching increase suggests a possible activation of the pain receptors in the interspinous ligament, which may lead to low back pain onset.
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∑ Model 2
This model used the homogenized values of Young’s modulus for the cortical and trabecular bone areas, and the Young’s modulus value of the ligament zone was derived from the measured speed of sound in this zone. We could have used the Young’s modulus value we found for the cartilage area, but in order to make a distinction between the mineralized and the unmineralized cartilage regions, we used the values found by Leicht & Raum [15] . The second model included consequently original data for three of the five materials.
To calculate the values of the Young’s moduli from the elastic coefficient C 33 , we use the Hooke’s law for isotropic materials:
1 2 1 12 1 The modeling of the ligament calcification process was performed by increasing the Young’s modulus of the unmineralized cartilage zone in the pathological version (see table 11 below). This reflected the expansion of the mineralization front in the enthesis.
Cortical Trabecular Mineralized Unmineralized Interspinous Tissue Parameter bone bone cartilage cartilage ligament Healthy Young's modulus [MPa] 8600 5200 1300 980 920 model Poisson's ratio 0.3 0.3 0.45 0.45 Pathological Young's modulus [MPa] 8600 5200 1300 1100 920 model Poisson's ratio 0.3 0.3 0.45 0.45 Table 11: Material properties of the different tissues of the enthesis used in model 2
We obtained the following distribution of the strain component in the traction direction for the healthy and pathological versions:
Fig.66: Repartition of the strain component in the traction direction for the healthy model
Fig.67: Repartition of the strain component in the traction direction for the pathological model
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When plotting the strain component in the traction direction along the selected path (see fig.62), we observed the following curves:
healthy
pathological
[mm] Fig.68: Strain component of the traction direction along the selected path (see fig.61)
Once again, both pathological and healthy versions displayed similar strain profiles (see fig.68) in the traction direction. These profiles also reminded a step function, with three different levels. The first level corresponded to the trabecular bone, the second to the unmineralized cartilage region and the third to the ligament zone. One major difference with the previous model was the transition between the trabecular bone and the unmineralized cartilage zones, which was a little less “direct” than before, thanks to the addition of the thin mineralized cartilage zone.
Another interesting feature was the evolution of the strain in the trabecular bone area: unlike the previous model, on the considered scale, it did not remain constant in the entire zone but instead slowly increased until the beginning of the mineralized cartilage area.
Exactly as for the previous model, because the unmineralized cartilage zone was assigned a higher Young’s modulus value in the pathological case, the strain of this material naturally became lower in the pathological version and approximately diminished from a maximum of almost 0.015% to a maximum of 0.013%.
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In the portion of the curve corresponding to the ligament, we found again a decreasing trend of the strain in the traction direction when moving outside the sample with also a slight ascent at the end of the sample. This time however, the difference in this zone in terms of maximal strain between the pathological and healthy versions was much less important than for the previous model: we observed indeed a maximal strain of 0.014% in the healthy version and of 0.015% in the pathological version. Even if this difference was really tiny, it still reflected a higher stretching of the ligament, which is a condition of the activation of the pain receptors and may lead to low back pain onset.
2-4-3- Discussion
The important differences in Young’s moduli between the first and the second model (see tables 10 and 11) could be explained by the fact that the values of the first model were taken from literature publications where the materials were measured mechanically, while the second data exclusively included data deduced from ultrasonic measurements.
While the first model predicted “reasonable” values for the strain component in the traction direction, it also presented the drawback of not distinguishing the mineralized cartilage from the unmineralized cartilage. The second model predicted really low strain values but at least included this distinction between both tissues.
However, the maximal strain values found in the different zones of our models should also be used carefully, as most of them are obtained at interfaces between different materials.
Concerning the load conditions, it should not be forgotten that the entheses samples were extracted from pigs, which are quadruped animals, while humans are bipeds. This factor has naturally a huge influence on the load conditions, and this should be taken into account before drawing any comparison. Moreover those pigs were growing pigs of 6 months, which also has an additional influence on the load conditions.
Last but not least, the developed models included many hypotheses, among them the isotropy of the investigated materials, which is questionable given the structure of our samples. Moreover, it should be highlighted that the axis where the traction was applied (x- axis) was not the same as the beam sound axis for the SAM 50 MHz measurements (z-axis). In order to get a three-dimensional model of pig spinal entheses, measurements in at least the three directions x, y and z should be conducted, which could be problematic considering the size of our samples.
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2-4-4- Prospects: description of the forthcoming models
To improve the currently existing models, several tracks could be explored:
• The natural extension of the model 2 would be to give as an input parameter for the material properties of the mineralized and unmineralized cartilage regions a stiffness curve representing the evolution of the Young’s modulus across the distance. For example, a curve derived from the impedance curve in the paper of Leicht & Raum [15] would seem reasonable. The possibility to use the histological slides, and more particularly the results of the Movat’s pentachrome staining (where 3 zones are distinguishable) in order to create a stiffness scale associating a given color with a specific stiffness could be successful.
• In the same vein, the “stiffness transition” between the cortical and trabecular bone areas could also be improved by implementing as an input parameter for the material properties a similar stiffness curve representing the evolution of the Young’s modulus across the distance, thus realizing a smooth transition between both materials, unlike the current abrupt transition in the models 1 and 2.
• Moreover, an improved modeling of the ligament calcification process in several steps could be realized. For example, we could progressively increase the Young’s moduli in the unmineralized cartilage zone, thus mimicking the phenomenon of expansion of the mineralization front. However, the exact development of this process is not known and a progressive stiffening of the mineralized cartilage zone could also be considered. The question is then to know if these two stiffening occur simultaneously or one after the other.
• Finally, another option would consist in integrating the observed external layer in the model. However, a special attention should be paid to the transitions between on one hand this zone and the cortical layer and on the other hand this zone and the ligament layer.
In all cases, all those improvements of the model should be introduced carefully and above all successively, in order to be able to determine the respective influence of each change on the model.
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Technical and scientific conclusions
The purpose of this master internship was the characterization of the spinal entheses of growing pigs by means of a wide range of experimental methods (among them the Scanning Acoustic Microscopy, the histology techniques and the micro-CT scanning), in order to model the ligament calcification process occurring during AS.
By combining all these experimental methods from various scientific fields, we were able to gain the mechanical properties of the spinal entheses of growing pigs, as well as their structural properties.
These data were then used as a basis for the creation of two two-dimensional finite- element models of these entheses. The purpose of these two models was a better understanding of the mechanism of AS, and more specifically the modeling of the ligament calcification process. The first model included no original data, while the second included original data for 3 of the 5 considered materials. Every model consisted of a healthy and a pathological version which were compared. For both models, a higher stretching of the interspinous ligament was predicted in the pathological version when comparing it with the healthy version. This higher stretching tends to disrupt pain receptors within this ligament and thus may lead to low back pain.
In addition, further improvement methods were suggested.
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Personal perspectives
This master internship really represented the perfect junction between my education at the École Centrale Marseille, where I chose for the final year the specialization “Mechanical Modeling of Materials and Structures” and my complementary research master “Heterogeneous Materials”. Indeed, this internship combined an experimental aspect with several non destructive testing methods, which I had had for some of them the occasion to discover them in the framework of the NDT course of my research master, and a numerical aspect which was mainly addressed in my specialization courses at the École Centrale Marseille.
I truly enjoyed the multidisciplinary aspect of this internship: the wide range of involved fields in this project was in my opinion an excellent example of a concrete mission scenario for a generalist engineer. Indeed the assignment not only aggregated several fields of physics and biology, but also appealed to the informatics field. Once again, it was an occasion to note the increasing importance of this latter discipline in the daily work environment, especially for data processing. Moreover, it was a unique opportunity to discover fields I was not particularly (sometimes not at all) familiar with.
Due to a misunderstanding between both partners, the investigated samples arrived with an important delay relative to the beginning of the internship, which first somewhat limited my progress. However the positive side of this schedule error was that I had the opportunity to experience the whole sample preparation process, since the reception of fresh samples until the polishing of embedded samples.
In the same vein, it permitted me to become more aware of the difficulty regarding the transfer of material, data or information from a country (or a place) to another. Thus I realized the fundamental importance of a global vision of the assignment, which requires thinking beyond the step by step process, especially when you have to work with people from several different teams or services. In this way I could begin to develop various anticipation skills.
This internship was a good occasion to deepen my knowledge of the world of research; it was above all an amazing opportunity to widen my horizons by discovering the German research conception. It was also a chance to evolve in an international environment where several languages were daily spoken, which was particularly profitable for the improvement of my English and German skills.
Despite a slight feeling of frustration at not having been able to completely end the mission incumbent upon me, and this exclusively because of logistic factors, I am still satisfied with the work I produced for the bibliographical, experimental and numerical parts.
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Appendices
Appendix 1: Description of the different types of vertebrae in the human spine
In the upper part of the spinal column, it is possible to distinguish three types of regions: the cervical, thoracic and lumbar regions, which consist respectively of 7, 12 and 5 vertebrae.
A B C Schematic view of a cervical (A), thoracic (B) and lumbar (C) vertebra
‹ Cervical vertebrae
These 7 vertebrae, which are abbreviated C1 through C7 (top to bottom), protect the brain stem and the spinal cord, support the skull, and allow for a wide range of head movement, such as coupling between inclination and axial rotation.Together, the atlas (C1) and axis (C2) enable the head to rotate and turn. The other cervical vertebrae (C3-C7) are shaped like boxes with small spinous processes (finger-like projections) that extend from the back of the vertebrae.
‹ Thoracic vertebrae
Beneath the last cervical vertebra are 12 thoracic vertebrae abbreviated T1-T12 (top to bottom). T1 is the smallest and T12 is the largest thoracic. The thoracic vertebrae are larger than the cervical bones and have longer spinous processes. Rib attachments add to strength and stability the thoracic spine. The rib cage and ligaments limit range of motion and protect many vital organs.
‹ Lumbar vertebrae
The 5 lumbar vertebrae, abbreviated L1-L5 (top to bottom), are the largest and carry most of the body’s weight. They present a more massive vertebral body, which is wider from side to side than from front to back and a little thicker in front than in back. The lumbar spine allows more range of motion than the thoracic spine, but less than the cervical spine. These motions are facilitated by an important height of the intervertebral discs. Lumbar facet joints enable significant flexion and extension movement, but limit rotation.
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Appendix 2: Additional information about the set 1 samples preparation procedure
Composition of the fixation solution:
Formaldehyde 36 % 324 ml Ethyl alcohol (or Methyl alcohol 100%) 540 ml Barbital Natrium Puffer pH 7,4/0,1 molar 130 ml Glucose 6 g
Description of the used solutions for the PMMA-embedding step:
Before the embedding, the base solution needed to be destabilized. To reach this goal, a chromatographic column was filled with 50g of active, neutral aluminium oxide 90 and then traversed by the base solution Technovit 9100 (material n°1). One column filling was sufficient for the destabilization of 3-4L of the base solution. The storage of the base solution should be performed at 4°C or at -15 -20°C in an amber bottle.
The preinfiltration was realized by mixing 200 mL of the base solution with 1 g Hardener 1 (material n°3). At room temperature, the duration of the preinfiltration was 1-2 days. The solution could be frozen at -20°C and was then up to 3 months.
The infiltration was made by mixing 250 mL of the base solution with 20 g of PMMA- Pulver and 1 g of Hardener 1. The infiltration lasted 4-5 days. The solution could be frozen at -20°C and was then up to 3 months.
Before performing the polymerization, the first thing to do was to produce the stock solutions A and B.
Stock solution A: In a 500 ml flask, 80 g of powder (material n°2) were added gradually with destabilized base solution. The mixture was stirred at room temperature with a magnetic stirrer until the powder was dissolved. Then 3 g of Hardener 1 (material n° 3) were added, stirred again and finally filled with the base solution to 500 ml. The resulting solution should have a gelatinous consistency and can be stored at 4 °- - 20 ° C in an amber bottle.
Stock solution B: In a 50 mL volumetric flask, 4 mL of Hardener 2 (material n° 4) were introduced; then 30 mL of unstabilized base solution were added, while the solution was being mixed on a magnetic stirrer. Then, 2 x 1 mL Regler ( material n° 5) were added and the flask was filled with the base solution to 50 ml. Then again the solution was well mixed . The resulting solution should be fluid and can be stored at 4 ° - - 20 ° C in a n amber bottle.
Before the polymerization, both stock solutions were mixed as follows: 9 volumes of stock solution A 1 volume of stock solution B
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Appendix 3: Prominent involved effects during SAM measurements [14]
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Appendix 4: Histology staining protocoles
Three different stainings were performed here: first a Movat´s pentachrome staining, then a Safranin-O / Lightgreen staining and finally a Van Kossa / Safranin O staining.
‹ Movat´s pentachrome staining
1- A) Sections were deparaffinized in xylol for 2 x 10 min B) Rehydration in descending concentrations of ethanol, 2 min each C) Section brought into aqua bidest for 2 min 2- Staining in 1% alcian blue solution for 10 min 3- Wash in running tap water for 5 min 4- Incubation in alkaline alcohol solution (10 mL 25% ammoniac on 180 mL 96% alcohol) for 60 min 5- Wash in running tap water for 10 min 6- Rinse quickly in aqua bidest 7- Staining in Weigert´s iron hematoxylin (iron hematoxylin A and B in proportion 1:1) for 10 min 8- Rinse quickly in aqua bidest 9- Wash in running tap water for 15 min 10- Staining in Brillant Crocein-Fuchsin for 10 min 11- Rinse quickly in 0.5% acetic acid 12- Staining in 5% phosphomolybdic acid for 15 min 13- 3 x 100% EtOH for 5 min 14- Staining in Saffron du Gatinais in 100% EtOH for 60 min 15- 3 x 100% EtOH for 3 min 16- Deffating in xylol, 2 x 5 min 17- Mount in xylene-soluble mounting medium
Results:
Feature Color Mineralized bone tissue, collagen bright yellow Cartilage tissue (depending on the fixation) reddish to yellow Cytoplasm reddish Elastic fibers red Osteoid dark-red Mineralized cartilage blue-green Acidic glycosaminoglycans bright light blue Cell nuclei blue-black
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‹ Safranin-O / Lightgreen staining (cartilage staining)
1- Sections were deplastified (resp. deparaffinized for 3 x 30 min (resp. 3 x 5min)
2- Rehydration in descending concentrations of ethanol and brought into aqua bidest, 2 min each 3- Staining in Safranin-O for 8-10 min 4- Rinse in aqua bidest, 5 times 5- Incubation in picric acid for 10 min 6- Rinse quickly in aqua bidest 7- Rinse in 1% acetic acid 8- Staining in Lightgreen for 8-10 min 9- Rinse in 1 % acetic acid 10- 100% EtOH, 2 x 3 min 11- Xylol, 2 x 5 min 12- Mount in xylene-soluble mounting medium
Results: Feature Color Cartilage intensive red Cell nuclei red Bone green Connective tissue green
‹ Van Kossa / Safranin O staining
1- Sections were immersed in MEA (1-acetoxy-2-methoxy-ethane) for 3 x 30 min 2- Rehydration in descending concentrations of ethanol and brought into aqua bidest, 2 min each 3- Incubation in 5% aqueous silver nitrate solution for 10 min 4- Rinse in aqua bidest, 3 times 5- Incubation in sodium carbonate / formaldehyde solution for 2 min 6- Wash in running tap water for 10 min 7- Incubate in 5% sodium thiosulfate for 5 min 8- Wash in running tap water for 10 min 9- Rinse in aqua bidest 10- Staining in Safranin O for 8 min 11- 96% EtOH, 2 x 2min 12- 100% EtOH, 2 x 2min 13- Xylene, 2 x 5 min 14- Mount in xylene-soluble mounting medium
Results: Feature Color Mineralized bone and cartilage black Unmineralized bone pale red Hyaline cartilage intensive red Nuclei red
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Appendix 5: Immunohistochemical labelling protocole
Collagen II-Färbung (Versuch) an PMMASchnitten mit Coll II Firma Quartett # 203 150 2101, Mouse
1.) Entplasten in MEA 3x 15 Min 2.) Absteigende Alkoholreihe bis A.dest je 2 Min 3.) Spülen in TBS, pH: 7,4 plus 0,1% Triton 2x 5 Min 4.) Demaskierung: a) 0,02% Hyaluronidase 2 Std/37°C
b) 0,1% Pepsin in 0,01N HCl 30 Min/37°C
5.) Spülen in TBS/Triton 2x 5 Min
6.) Blocking mit NS Horse 5%/TBS 30Min/RT
7.) Prim. AK Coll II ( 1:50) in Antibody-Diluent über Nacht/4°C
8.) Spülen in TBS/Triton 2x 5 Min
9.) Sek.Ak Anti-Mouse, Rat adsorbed, Host Horse, Biotinlyated 30 Min/RT
(2% Normalserum Horse/2%AK in TBS)
10.) Spülen in TBS/Triton 2x 5 Min
11.) AB-Komplex (Kit AK 5000: 5ml TBS plus 1Trpf Lsg A und B) 50 Min/RT
12.) Spülen in TBS/Triton 2x 5 Min
13.) Spülen mit Chromogenpuffer 2x 5 Min
14.) Visualisierung mit Alkaline Phosphatase Kit SK 5100 mikroskop. Kontrolle!
15.) Spülen in TBS/Triton 2x 5 Min
16.) Spülen in A. dest kurz
17.) Kernfärbung mit Hämatoxylin nach Mayer 5 Min
(Alternative: Hämatoxylin nach Gill)
18.) Bläuen in Leitungswasser 5 Min
19.) Eindeckeln in Aquatex
Normalserum Horse: Firma Biozol # S-2000
Anti-Mouse, Rat adsorbed, made in Horse: Firma Biozol # BA-2001
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