Integrative Studies on Cartilage Tissue Engineering and Joint Homeostasis Marijn Rutgers

Integrative studies on cartilageIntegrative tissue engineering and joint homeostasis

Integrative studies on cartilage tissue engineering and joint homeostasis Marijn Rutgers

Marijn Rutgers

Integrative studies on cartilage tissue engineering and joint homeostasis

Marijn Rutgers The investigations in this thesis were performed at the Orthopaedics department of the University Medical Center Utrecht, the Netherlands.

Research support was provided by the Reumafonds and the Anna Foundation.

Publication and distribution of this thesis was kindly supported by the Anna Fonds Leiden, Arthrex, BISLIFE Foundation, ChipSoft, DePuy Synthes, Eemland Orthopedie Techniek, George In der Maur orthopedische schoentechniek, Leuk Orthopedie, Livit Orthopedie, Mathys Orthopaedics, MRI Centrum, Nederlandse Orthopedische Vereni- ging, Reumafonds, het Rugcentrum, TiGenix NV.

© M. Rutgers, 2014. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by means, without prior written permission of the author.

ISBN/EAN: 978-94-6169-504-8

Cover design: Optima Grafische Communicatie and Marijn Rutgers

Printed by: Optima Grafische Communicatie, Rotterdam, The Netherlands Integrative studies on cartilage tissue engineering and joint homeostasis

Integratief onderzoek naar kraakbeen tissue engineering en gewrichtshomeostase (met een samenvatting in het Nederlands)

Proefschrift

ter verkrijging van de graad van doctor aan de Universiteit Utrecht, op gezag van de rector magnificus, prof. dr. G.J. van der Zwaan, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op donderdag 17 april 2014 des middags te 12.45 uur

door

Marinus Rutgers geboren op 5 maart 1980 te Den Haag Promotoren: Prof. dr. W.J.A. Dhert Prof. dr. D.B.F. Saris

Co-promotor: Dr. L.B. Creemers Contents

Chapter 1 General introduction 7 Based on: Joint injury and osteoarthritis: soluble mediators in the course and treatment of cartilage pathology. Rutgers M, Saris DB, Auw Yang KG, Dhert WJ, Creemers LB. Immunotherapy. 2009 May;1(3):435-45

Chapter 2 Outline and aims of this thesis 21

Chapter 3 Effect of collagen type I or type II on chondrogenesis by 27 cultured human articular chondrocytes Rutgers M, Saris DB, Vonk LA, van Rijen MH, Akrum V, Langeveld D, van Boxtel A, Dhert WJ, Creemers LB. Tissue Eng Part A. 2013 Jan;19(1-2):59-65

Chapter 4 PTH decreases in vitro cartilage regeneration without affecting 41 hypertrophic differentiation Rutgers M, Vonk LA, van Rijen MH, Akrum V, Tryfonidou MA, van Boxtel A, Dhert WJ, Saris DB, Creemers LB. Manuscript in preparation

Chapter 5 Similar regenerative capacity of juvenile versus adult talar 53 chondrocytes despite high expression of hypertrophic markers Rutgers M, Saris DB, Vonk LA, van Rijen MH, Akrum V, van Boxtel A, Sakkers R, Dhert WJ, Creemers LB. Submitted

Chapter 6 Cytokine profile of autologous conditioned serum for 67 treatment of osteoarthritis, in vitro effects on cartilage metabolism and intra-articular levels after injection Rutgers M, Saris DB, Dhert WJ, Creemers LB. Arthritis Res Ther. 2010;12(3):R114 Letter to Editor: Moser C; 81 Response: Rutgers M, Saris DB, Creemers LB 82

Contents 5 Chapter 7 Osteoarthritis treatment using a cytokine-based intra- 85 articular therapy – comparison of clinical results to earlier placebo treatment Rutgers M, Creemers LB, Auw Yang KG, Raijmakers NJ, Dhert WJ, Saris DB. Submitted

Chapter 8 Evaluation of histological scoring systems for tissue- 97 engineered, repaired and osteoarthritic cartilage Rutgers M, van Pelt MJ, Dhert WJ, Creemers LB, Saris DB Osteoarthritis and Cartilage, 18(1):12-23

Chapter 9 dGEMRIC as a tool for measuring changes in cartilage quality 115 following high tibial osteotomy: a feasibility study Rutgers M, Bartels LW, Tsuchida AI, Castelein RM, Dhert WJ, Vincken KL, van Heerwaarden RJ, Saris DB. Osteoarthritis and Cartilage, 20(10):1134-41

Chapter 10 Summary 129

Chapter 11 Discussion 135

Addendum References 145 Dutch summary 175 List of abbreviations 183 Publications and presentations 187 Acknowledgements 195

6 Contents General introduction Based on: Joint injury and osteoarthritis: soluble mediators in the course and treatment of cartilage pathology

Marijn Rutgers, Daniël B Saris, K Gie Auw Yang, Wouter J Dhert and Laura B Creemers

Immunotherapy. 2009 May;1(3):435-45

Cartilage damage and its consequences The consequences of injury to the cartilaginous surface of the knee joint are often unknown to those active and often otherwise healthy individuals at the time they undergo their trauma. As cartilage lesions do not have an intrinsic repair capacity178, over the following months to years the impact of pain and disability may influence work, sports and other activities. Through an alteration in joint homeostasis355 and biomechanical loading157, cartilage lesions may accelerate the onset of osteoarthritis 134, 429. A patient with a painful cartilage lesion may subsequently become a patient with invalidating osteoarthritis in the future. Over the past decades, the emphasis in treatment of cartilage lesions has shifted. Initially, the chondrocyte itself was the subject of investigations. More and more, the entire joint is considered influential in the process of cartilage repair16, 357. Before mov- ing on, background information on the components that influence joint homeostasis is provided below.

Chondrocytes and extracellular matrix metabolism Chondrocytes reside within the a-neural, non-vascularized cartilage matrix in cavities called ‘lacunae’. Mature human chondrocytes average 13 μm in size and represent 10- 15% of the cartilage volume52, 232. The other 85% of the cartilage volume consists of water, collagens and proteoglycans (the extracellular matrix or ECM)73. Proteoglycans are com- posed of a protein core with covalently bound glycosaminoglycan side chains, of which the most important are chondroitin sulfate and keratan sulfate197. Hyaluronic acid (HA) is an unsulfated glycosaminoglycan. Although it is not covalently bound to the protein core and thus not part of a proteoglycan, it is involved in formation of proteoglycan aggregates through aggrecan– HA-link protein complexes52. Exchange of low-molecular-weight components (<20 kDa) between the cartilage matrix and synovial fluid (SF) is dependent on biomechanical loading197. However, proteolytic activity (observed in normal and in degenerative cartilage) enables other, larger matrix components, such as proteoglycans and HA, to be cleaved into smaller fragments and released into the SF52. Fibronectin fragments (Fn-fs) released following proteolytic activity play a direct role in cartilage turnover by a) either enhancing cartilage anabolic activity (at low concentrations) or stimulation of catabolic cytokine release240, b) upregulation of matrix metalloproteinase (MMP) expression and c) enhancing degradation of proteoglycans169. Increased levels of Fn-fs in SF may be considered as a ‘signal’ of increased catabolism, and have been observed in patients with rheumatoid arthritis (RA) as well as OA59. In addition to cytokine-mediated cartilage degeneration, it was demonstrated that activation of the discoidin domain receptor (DDR)-2 by native collagen plays a role through increased expression of the cartilage- degrading MMP-13 in cultured chondrocytes447. In addition, DDR-2 protein was increased at the immunohistochemical level in the femoral heads of OA patients446.

General introduction 9 Another ‘signal’ of impending catabolism is the expression of markers of hypertrophic differentiation by chondrocytes (Runt-related transcription factor 2 (Runx2)/Core-binding factor alfa-1(Cbfa1)) or later on, by collagen type X expression in the matrix. When uninterrupted, hypertrophic differentiation eventually leads to osteoblast invasion and cartilage degradation. An important factor in maintenance of the chondrocyte in its proliferating, non-hypertrophic state is parathyroid hormone related-peptide (PTHrP). This peptide stimulates Sox9 expression through binding to the PTH/PTHrP receptor and subsequent activation of protein kinase A91.

Synovium Synovial tissue consists of a synovial membrane (facing the joint capsule) and a fibrous layer containing loose connective tissue52, and it is the main contributor to changes in SF composition187. The synovial membrane consists of one or more layers of epitheloid cells, which cover a layer of fibrous tissue containing type A and type B cells196. The A cells resemble macrophages and can be released from the synovial membrane into the SF. However, the majority of the cells in the synovial membrane are ‘B’ cells, which resemble fibroblasts and secrete proteins, glycoproteins and hyaluronic acid into the SF159. The fibrous tissue layer contains adipocytes, mast cells, nerve fibers, vascular endo- thelial cells and, occasionally, lymphocytes. Capillaries in the fibrous tissue layer enable exchange of nutrients.

Synovial fluid The composition and volume of SF varies between individuals and between different joints within an individual. The average healthy human knee contains between 1.1 and 6.7 ml158, 340 of SF, which is distributed over the femoral and tibial articulating surfaces throughout the joint cavity in a layer just 10–20 μm thick52. Traumatic changes to the joint such as rupture of an anterior cruciate ligament, meniscal tears or dislocation of a cartilage or osteochondritis dissecans fragments, may change the SF composition. This SF change may occur either gradually following increased SF production by type B synovial cells, or rapidly through intra-articular hemorrhaging. Although leukocytes are usually only present at low concentrations in SF, they have been mentioned to contribute to intra-articular cytokine and growth factor levels416. In contrast to the relatively a-cellular healthy SF, blood contains high amounts of leukocytes, as well as erythrocytes and thrombocytes. Breaching of the blood–SF barrier may affect SF composition, not only through entrance of erythrocytes, leukocytes and monocytes into the joint and subsequent cartilaginous damage339, but also through the presence of pro-inflammatory cytokines in blood.

10 Chapter 1 D

Cytokines Cytokines are a category of soluble or cell surface molecules that play a role in cellular communication through paracrine, autocrine and endocrine signalling, and they provide a network controlling both innate and specific immune responses337. In addition, cytokines may be involved in tissue degradation, for example, during remodeling of the periodontal ligament133, 147, wound healing136, corneal destruction following keratitis260 or bone loss during osteolytic pathologies39. In the intra-articular environment, cytokines and growth factors may diffuse from the SF towards chondrocytes, where they may bind to the cell surface and stimulate or inhibit the release of other proteins. Vice versa, chondrocytes release cytokines and growth factors into the surrounding matrix, which are transferred into the SF in part through fluid flow generated by hydrostatic pressure from joint loading52. In cartilage pathology, the relatively low turnover and remodeling of cartilage matrix components141 is disturbed, resulting in a net loss of cartilage matrix components and deterioration of structural and functional properties of the cartilage143. Cytokines contribute to this process by stimulating and producing proteolytic enzymes and other catabolic cytokines52, or by inhibiting proteoglycan453 or collagen synthesis142. Cytokines are considered to play an important part in the pathogenesis and progression of osteoarthritis (OA)42, 130, 245.

Synovial fibroblasts

IL-1RA, IL-4, IL-10, IL-13, OPG, bFGF Activated IL-1β, TNF-α, IL-6, IL-7, IL-8, LIF T cells

IL-17, OSM, OPG, IFN-γ Synovium

Synovial fluid Cartilage degradation

Extracellular cartilage matrix NO Fibronectin fragments MMP

IL-1RA, IL-4, IL-10, IL-13 TGF-β, IGF-β, FGF TIMP IL-1β, TNF-α, IL-6, IL-7, IL-8, LIF Chondrocyte

Figure 1: Cytokines in the intra-articular environment345. LIF: Leukocyte-inhibitory factor; MMP: Matrix Metalloproteinase; NO: Nitric oxide; OPG: Osteoprotegerin; OSM: Oncostatin M; TIMP: Tissue inhibitor of matrix metalloproteinase

General introduction 11 Chondroprotective, chondromodulatory and chondrodestructive cytokines Although the majority of cytokines exert multiple effects on cartilage and are involved in a balance of stimulatory as well as inhibitory effects, they can be differentiated between a) cartilage-degrading, proinflammatory cytokines (catabolic, or ‘chondrodestructive’), b) cytokines with both destructive as well as protective functions (‘chondromodulatory’)141 and c) cartilage-protecting, anti-inflammatory cytokines (anabolic, anticatabolic or ‘chondroprotective’). Of the chondrodestructive cytokines, IL-1β318, 381, TNF-α437 and their synergistic interactions have been studied most thoroughly160, 162. These cytokines stimulate upregulation of various cartilage-degrading MMPs387, a process that leads to cleavage and subsequent loss of proteoglycans from the extracellular matrix (ECM). On the other hand, proteoglycan synthesis is inhibited52, 107. IL-1α and IL-1β exhibit an amino acid homology of 22% and they both bind to the biologically active IL-1 receptor (IL-1R). This binding leads to transcription of many proinflammatory genes, including IL-6 and IL-810. Although not as frequently studied as IL-1β, IL-1α is also present in OA SF, albeit at low concentra- tions93. Both cytokines are thought to exert more or less similar effects in cartilage degeneration11 and they are used interchangeably in research. Three other cytokines with chondrodestructive effects in the intra-articular environment, IFN-γ163, IL-17194 and oncostatin-M98, were shown to act synergistically with IL-1 in stimulation of production of MMPs and aggrecanases220. The amount of IL-7 production by osteoarthritic chondrocytes is higher in comparison with healthy chondrocytes240; and IL-7 may be involved in Fn-f-mediated osteoarthritic degeneration. IL-8 has been shown to promote release of IL-6162 and to promote hypertrophic differentiation270. Leukocyte-inhibitory factor (LIF), produced by activated T cells in the synovium, has been shown to enhance IL-1β and IL-8 production in chondrocytes244. IL-6 has been described to have chondrodestruc- tive108 as well as chondroprotective162, 400 actions. Its ‘chondromodulatory’ effects are mediated through increased production and attraction of inflammatory cells293 on the one hand, and through stimulated production of TGF-β417 and metalloproteinase tissue inhibition (TIMP)-1243 on the other hand. IL-6 production is stimulated by IL-1β162, 293. Ba- sic FGF (bFGF) may inhibit IGF-1-mediated proteoglycan production 239 and induce MMP expression418, although it may also stimulate chondrocyte proliferation151 and TIMP-1 production418. Anti-inflammatory cytokines, such as IL-4, -10 and -136, were shown to have diverse effects, including inhibition of IL-1, TNF-α secretion and stimulation of IL-1 receptor antagonist (IL-1RA/IRAP) expression. IL-1RA competes with IL-1 in binding to the IL-1 receptor. Prevention of IL-1 binding inhibits target cell activation 10, thus inhibiting cartilage degradation 13, 118, 146, 380. Growth factors may also mediate proliferation or differentiation of chondrocytes. TGF-β, a so-called cysteine-knot cytokine369, stimulates IL-1RA expression and proliferation and differentiation of chondrocytes228, 409; however, only few studies report on the presence

12 Chapter 1 Table 1. Chondrodestructive, chondromodulatory and chondroprotective cytokines in the intra-articular environment345 Chondrodestructive Chondromodulatory Chondroprotective IL-1α, IL-1β IL-6 IL-1RA TNF-α Basic FGF IL-4 IL-7 IL-10 IL-8 IL-13 IL-17 TGF-β Oncostatin M IGF-1 IFN-γ Osteoprotegerin Leucocyte-inhibitory factor of TGF-β in the intra-articular environment327, 361. At high concentrations, TGF-β may lead to osteophyte formation358. IGF is considered as an important mediator in the repair of cartilage defects111, 145 as it is a potent mitogen and differentiation factor steering progenitor cells toward a cartilaginous pathway 128, 401. Osteoprotegerin216, 375 was found to stimulate IL-1RA expression, and increased levels of osteoprotegerin have been reported in OA392.

Synovial fluid content in acute trauma Acute trauma is associated with higher SF cytokine levels than the amounts commonly found in healthy or OA patients. In vivo, changes in the intra-articular cytokine level following trauma have been studied following naturally acquired articular injuries127, 181, 231, immobilization of a joint 122, biochemically induced injury442, or mechanically induced intra-articular trauma111, 433. In patients, cytokine levels have often been studied following rupture of the anterior cruciate ligament55, 165, 185 or following meniscus or cartilage trauma 325, 336. In both in vivo and clinical studies, high levels of a variety of cytokines were seen shortly after intra-articular trauma, often followed by a gradual decrease. In animals, IL-6 levels increased the most frequently 127, 231, as well as IL-1β, TNF-α, MMP-3127 and TGF-β433. In humans, IL-6 55, 165, 185, IL-1RA 55, 185, 255, IL-1β185, 255, IL-855, 185, IL-10 and TNF-α185 have been reported to increase following injury. It is unclear whether these acute post-traumatic changes affect cartilage turnover in a positive 336 or negative way 452. It is also unclear whether these changes implicate an initial repair response followed by a ‘turning point’, after which the environment of the chronically injured joint becomes unfavorable for cartilage integrity336.

General introduction 13 Table 2. Average cytokine levels of chondroprotective, chondromodulatory and chondrodestructive cytokines in serum of healthy patients, and in synovial fluid (SF) of healthy and OA patients345. + indicates that levels were obtained of osteoarthritic synovial fluid. LIF: Leukocyte-inhibitory factor; OPG: Osteoprotegerin; OSM: Oncostatin M Cytokine Healthy serum (pg/ml) Reference SF (pg/ml) Reference IL-1 < 5.0 17, 432 < 3.0 37, 170, 226 IL-4 < 20 261, 268 0.1 226 IL-6 < 12.5 261, 331, 432 0.5 (0.7) 226 IL-7 < 10.0 88 1.1 (2)+ 411 IL-8 < 15 349 89.9 (45.88)+ 37 IL-10 < 10 432 21 (2.0)+ 177 IL-13 < 20 230 0.4 (1.4)+ Rutgers M, unpublished data IL-17 39.9 (2.3) 156 0 (0-84)+ 2 IFN-γ < 15 230 16.0 + 361 TNF-α < 3.0 261 < 5.0 170, 226 OPG 5.4-6.5 258 8935 (9303)+ Rutgers M, unpublished data OSM < 1.0 331 4.5 (2-20)+ 253, 301 LIF 3.8 (3.4) 331 < 15.0+ 301 IL-1RA 73-175 268, 432 614 (292-1951)+ 328 TGF-β 20580 85 581 (257)+ Rutgers M, unpublished data IGF-1 186000 75 82000 (10000)+ 394

Synovial fluid content in OA The osteoarthritic joint demonstrates changes in cartilage, synovium, subchondral bone and SF52, 177. Expression of IL-1 in chondrocytes is increased at the mRNA386 level and at the immunohistochemical level279, but expression in the synovial membrane does not seem higher than in healthy individuals177, 379. Although IL-1β is usually considered an important mediator for OA development, low (<10 pg/ml) or undetectable IL-1β levels were found in several studies focusing on cytokine levels in SF of OA patients37, 289, 361, 416. In general, the role of IL-1 in OA may have been overstated. Although it is commonly suggested that IL-1 exerts its effects at very low concentrations, possibly in synergy with other pro-inflammatory cytokines, there is not much support for this assumption. While there are studies that mention IL-1β concentrations as low as 10 pg/ml are capable of inhibiting proteoglycan synthesis in vitro33, 176, commonly, IL-1β concentrations of at least 1000 pg/ml were necessary to induce cartilage damage. Studies demonstrating synergistic effects of IL-1β with IFN-γ163, TNF-α160, IL-1764 or oncostatin-M98 also involve much higher IL-1β concentrations (100 pg/ml and higher) than in OA SF, which do not support a synergistic effect at low IL-1β levels. Animal models using IL-1β to induce OA also report concentrations 1000-fold higher than those found in OA SF 235, 372, further supporting the need for high levels to induce intra-articular effects. In addition, study

14 Chapter 1 of OA in animals usually involves models in which OA onset is ‘accelerated’ based on surgical183 or biochemical405 destruction of part of the cartilage, complete or incomplete meniscectomy283 or transsection of a cruciate ligament 67, 321. Depending on the time after ‘induction’ of OA, this ‘OA model’ may, in fact, resemble a post-trauma situation rather than an OA setting. Thus, the in vitro and in vivo models used for OA may not adequately simulate human OA and overstate the importance of IL-1β. Similar remarks can be made regarding TNF-α, which is also low in OA SF416. In addition to IL-1α, increased production of IL-8 and IL-10 was observed in synovial membranes as well as in the cartilage and subchondral bone of patients with OA, while subchondral bone also expressed TNF-α, MMP-3, MMP-9 and TIMP-1177. However, major increases in SF cytokine levels, as observed in patients with RA, are not observed in OA93, 170, 361, 416. It may, therefore, be that other SF components are responsible for cartilage degeneration in OA, either alone or in combination with the cytokines mentioned earlier. Although the OA SF concentrations of IL-1β and TNF-α alone do not seem high enough to induce cartilage degeneration, SF of patients with chronically injured knees inhibited chondrogenesis in a chick limb bud bioassay, while SF of patients with acutely injured knees stimulated chondrogenesis336.

Treatment of cartilage lesions Treatment of a knee cartilage lesion ideally restores full joint motion, reduces pain and improves functional performance. In theory, by restoring cartilage surface integrity, this procedure may postpone or even prevent osteoarthritis onset157, 429. The most widely used cartilage repair procedure is microfracture385. In microfracture, multiple perfora- tions or ‘microfractures’ are made into the subchondral bone plate. The mesenchymal stem cells thus released from the underlying bone marrow are expected to differentiate into matrix producing cells with chondrocyte-like characteristics. These cells ideally fill the defect with a collaginous tissue, thus improving knee function, reducing pain, and preventing development of osteoarthritis. However, over the past decades the regenerated tissue has been shown not to consist of hyaline cartilage, but of fibrocartilaginous tissue, at its best containing some collagen type II121 and macroscopically resembling the surrounding cartilage222, 356. The Autologous Chondrocyte Implantation technique (ACI), described first by Brittberg in 199450, is another commonly used procedure aiming at restoring cartilage surface integrity. In two sequential surgical interventions, chondrocytes are harvested, and after an in vitro expansion phase re-implanted into the cartilage defect. Prior to re- implantation, cells may be screened for their chondrogenic potential and selectively processed356. Various techniques exist to re-implant the cells and keep them in place, including a periosteal flap (1st generation ACI), a collagen membrane (2nd generation ACI), or the use of a collagen type I/III matrix (3rd generation ACI)256. Although the clinical results of this procedure are ‘good’ to ‘excellent’ in up to 90% of patients275, 316, biopsies

General introduction 15 taken from the repaired cartilage frequently display remaining fibrocartilaginous char- acteristics32, 356.

Treatments for osteoarthritis When a patient with early osteoarthritis (OA) symptoms seeks treatment, symptomatic treatment will be attempted first. Of the oral symptomatic therapies, nonsteroidal anti- inflammatory drugs (NSAIDs)360 and cyclooxygenase (COX)-2-specific inhibitors have proven to be effective in reduction of OA symptoms, while COX-2 inhibitors also demonstrate chondroprotective actions90, 259. Although COX-2-specific inhibitors are associated with a lower incidence of gastrointestinal complaints than NSAIDs83, they are associated with a higher cardiovascular morbidity382 and are thus currently prescribed less frequently. When unsuccessful, disease-modifying OA drugs may be applied in specific patient populations. In OA patients, the combination of oral glucosamine and chondroitin sulfate was more effective in reduction of OA symptoms than placebo, in patients with moderate to severe knee pain72. Although the evidence for clinical effectiveness of hyaluronic acid (HA) treatment in OA is conflicting14, 277, it currently seems that intra-articular HA supplementation is comparable in efficacy to systemic forms of active intervention (NSAIDs and physical therapy). In addition, the effects of HA treatment are more prolonged than intra-articular corticosteroids for patients with OA of the knee29. Although each intra-articular injection of course poses a risk of infection, this risk is considered extremely low (4.6 per 100,000 injections)308. In the acute post-trauma setting, improved rehabilitation after ACL reconstruction was demonstrated after intra-articular administration of HA174. When treatments are unsuccessful in reducing OA symptoms (including symptomatic treatments, or treatments aiming to restore SF viscosity or supplement ECM components), the final ‘gold standard’ is prosthetic joint replacement. Although effective to reduce pain in the majority of patients, placement of a knee prosthesis is considered a fairly invasive procedure with a significant rehabilitation period. Short and long term surgical complications (infection, arthrofibrosis, aseptic loosening)371 are low in prevalence, but they may be severe in nature with possible life-long consequences.

Cytokine-modulating therapies In rheumatoid arthritis (RA), targeting of IL-1 and TNF-α has proved successful for reduction of disease-related symptoms74, 280. In OA, inhibition of cartilage degradation has been most frequently attempted by targeting IL-120, 68, 449. In vivo, this targeting has seemed to be successful. Reduction of OA symptoms, histological parameters and preservation of articular cartilage quality were demonstrated after intra-articular injection of synoviocytes transduced with the IL-1RA gene in equine and canine OA models118, 314, and after injection of IL-1RA plasmids into a rabbit knee joint with OA induced by meniscectomy. However, in the only clinical OA trial carried out until now, intra-articular injec-

16 Chapter 1 tion of recombinant IL-1RA did not lead to improvement of OA symptoms68. Thus far, no compelling evidence exists that IL-1RA therapy is effective for treatment of OA in human patients. A more complex treatment based on intra-articular IL-1RA administration is Orthokine® (Orthogen, Düsseldorf, Germany). This treatment involves first preparing autologous conditioned serum (ACS) by in vitro incubation of the patient’s whole blood in the presence of glass beads. The resulting product, containing increased levels of IL-1RA as well as IL-4 and IL-10268, is injected into the joint. Although infrequently investigated, both in vivo studies118, 120 examining the effect of Orthokine® on OA, and clinical stud- ies20, 449 comparing Orthokine® with HA or placebo demonstrate either little or moderate improvement of OA symptoms. In the study demonstrating the greatest improvement of OA symptoms, the Orthokine®-treated cohort was injected with ACS six times while the placebo and HA cohort were injected only three times20. In this particular study, more frequent injections may have led to a greater improvement in OA symptoms for two reasons. First, a regression analysis of 174 randomized, controlled clinical OA trials demonstrated that placebo effects increase significantly with increased invasiveness of a treatment procedure (i.e., ‘injection’ in contrast to, for example, an oral therapy)465. Thus, the additional injections during ACS treatment may have caused an improvement in OA symptoms merely due to the ‘invasiveness’ of the treatment. Second, since joint lavage proved successful to reduce OA symptoms in rabbits with surgically induced OA124, the three additional injections may have been responsible for a ‘lavaging’ effect, thus also resulting in improvement of OA symptoms. The exact composition of the intra-articular injected Orthokine® is unknown, and thus the basis for the in vivo mechanisms responsible for clinically observed effects is also unknown120, 449. Although several IL-1RA-based therapies focus on the role of IL-1, their therapeutic relevance is open to discussion. IL-1RA levels in OA SF (614 pg/ml328) may already be high enough to block endogenous IL-1β13, and it may not therefore be necessary to elevate this baseline level exogenously, as for example by using ACS (2015 pg/ml 432 and 10254 pg/ml 268) IL-1RA. Moreover, exogenously added IL-1RA may be mostly unavailable to the cartilage due to rapid clearance from the joint. Although the theory behind ACS preparation and its effects seem interesting, the lack of knowledge on the exact composition of the product and the in vivo clinical effects demands further investigation before broad clinical application. Although TNF-α inhibition has gained a definite place in RA treatment43, only a few reports exist on successful improvement of OA symptoms in patients treated with TNF-α inhibi- tors150, 388. Osteoarthritic SF and osteoarthritic synovium supernatants upregulated p55 TNF-α receptors in human chondrocytes431. TNF-α antagonists decreased production of TNF-α by rabbit synovial cells in vitro, and suppressed TNF-α-mediated nitric oxide (NO) production while stimulating IL-1RA synthesis422. As TNF-α levels are strongly elevated in SF after trauma, TNF-α therapy in orthopedics may be promising for intra-articular trauma. However, TNF-α therapy may increase the risk of infections and malignancies44.

General introduction 17 Imaging of cartilage disorders New, non-invasive imaging techniques are improving the ability to evaluate the success of cartilage influencing therapies. An important development in this field has been the introduction of the delayed gadolinium-enhanced MRI of cartilage (dGEMRIC), which depicts intra-articular proteoglycan distribution without having to perform an invasive procedure such as a biopsy or arthroscopy54, 456. The technique is based on the negative ionic charge of proteoglycans as well as of the intravenously administered contrast agent (gadolinium (Gd(DPTA)2−). Using voxel by voxel curve fitting, an average T1 is calculated of different regions of interest. A high T1 relaxation time after Gd administration thus is indicative of high glycosaminoglycan content267. dGEMRIC has been used successfully in the in vitro and in vivo environment, and is finding its way into daily clinical use in orthopaedic and rheumatologic clinical patient evaluation291, 377, 414.

Healthy

T1Gd (msec) 800

700

600

500

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300

200

T1Gd (msec) Damaged Figure 2: delayed Gadolinium-Enhanced MRI of Cartilage (dGEMRIC), displaying proteoglycan distribution throughout the knee of a patient prior to a high tibial osteotomy.

Improving outcome of cartilage tissue engineering while addressing joint homeostasis The exchange of cytokines and growth factors between cartilage, synovial fluid and synovium is important for the maintenance of a healthy joint environment. Once altered due to trauma or OA, chondroprotective and chondrodestructive mediators are found

18 Chapter 1 at altered concentrations, further influencing cartilage integrity. Early treatment of cartilage lesions through ACI or microfracture may influence joint homeostasis and prevent osteoarthritis onset. However, after ACI the repaired tissue still displays fibrocartilaginous characteristics, po- tentially preventing a successful long-term outcome. The outcome of (M)ACI treatment, as well as of ‘disease-modifying OA drug’ administration, is often only slightly superior to existing alternative treatments. Follow-up of patients treated for cartilage defects may be hampered by refusal to undergo a follow-up biopsy to assess histological quality. Optimizing the outcome of cartilage repair, as well as improving the joint environment after trauma and in OA, thus should be further explored.

General introduction 19

Outline and aims of this thesis

General aim

Good to excellent clinical results are achieved in up to 90%275, 316 of patients treated by (matrix-induced) autologous chondrocyte implantation ((M)ACI), with hyaline cartilage formation in up to 75% of patients161, 468. Although currently considered the best treatment option for femoral cartilage defects larger than 4 cm2 406, increasing the percentage of patients in which a defect is filled with hyaline cartilage may even further improve clinical outcomes for large as well as smaller (>2 cm2 27) cartilage defects. Influencing cell phenotype may improve the chondrocyte’s regenerative capacity and result in higher quality regenerated cartilage. Maintenance of the hyaline covering for a prolonged period of time is likely to further improve clinical superiority and benefit cost-effectiveness of the procedure71. However, stimulating the chondrocyte alone is not sufficient, because cytokines and growth factors from the surrounding extracellular matrix, synovium and synovial fluid all influence the chondrocyte’s matrix-forming capacity. A broader approach is required, taking into account the interaction between joint homeostasis and therapies aiming to maintain or restore damaged cartilage. The overall aim of this thesis therefore is:

‘to enhance chondrocyte regenerative capacity during ACI by exogenous modulation of cell phenotype and by addressing joint homeostasis’

In this thesis the following are investigated: external and internal factors influencing cartilage matrix metabolism, the fundamental and clinical basis of a joint-influencing therapy, and histological and radiological evaluation tools. Seven specific aims that address the individual thesis research questions are listed within the text below.

External factors influencing cartilage matrix metabolism Cellular metabolism is modulated by extrinsic as well as intrinsic (innate or cell-specific) factors. During preparation of chondrocytes for (M)ACI, the surface on which the cells grow may influence neocartilage quality through cell-matrix interactions. By coating the membrane on which chondrocytes are replanted with collagens, the quality of the regenerated matrix may be increased.

Aim 1: Is the quality of in vitro generated neocartilage enhanced by the collagen coating of the culture undersurface (Chapter 3)?

An undesirable effect of cartilage repair procedures may be the onset of hypertrophic differentiation, in which chondrocyte proliferation and matrix production are inhibited, and osteoblast invasion is stimulated91. Parathyroid hormone (PTH) has been demonstrated to inhibit this process in mesenchymal stem cells (MSCs)284. PTH addition to

Outline and aims of this thesis 23 articular chondrocytes may also prevent hypertrophic differentiation during cartilage repair, while stimulating proteoglycan production.

Aim 2: Is the chondrocyte matrix metabolism enhanced and hypertrophic differentiation also inhibited by addition of PTH to in vitro cultured chondrocytes (Chapter 4)?

Internal factors influencing cartilage matrix metabolism Cell-specific innate factors may also influence extracellular matrix formation. For - ex ample, young lambs were shown to be capable of spontaneously repairing artificially created cartilage defects, while older animals were incapable of this repair286. Current data on the possible advantageous effects of juvenile cells are conflicting.

Aim 3: Do chondrocytes of young human donors produce higher quality cartilage than those of older donors (Chapter 5)?

Principles of a joint homeostasis-influencing therapy Cytokines and growth factors play important roles in joint homeostasis. Interleukin-1 receptor antagonist (IL-1RA) has been shown to counteract the chondrodegradative effects of intra-articular IL-1 in rheumatologic patients74. The ‘Autologous Conditioned Serum’ (ACS) therapy is based on intra-articular administration of autologous IL-1RA to osteoarthritic patients. ACS is incubated with glass beads and then injected into the knee joint. During the incubation period, IL-1RA levels have been shown to rise268. ACS therapy resulted in varying degrees of OA symptom reduction20, 449. Surprisingly, fundamental information is unknown about the possible content of other cytokines and growth factors in the product, and also about its in vitro effects on cartilage metabolism.

Aim 4: What is the exact composition of ACS, what are the effects of ACS on cartilage proteoglycan (PG) metabolism in vitro, and are cytokine levels maintained in the joint after injection (Chapter 6)?

Using patient-reported outcome measures (PROMs) to evaluate placebo effects of ACS treatment The effectiveness of ACS seems limited 20, 449, and in both randomized controlled trials performed thus far also the ‘placebo’ treated patients show significant clinical improvement. Administering ACS to previously placebo-treated patients may distinguish ‘real’ treatment effects from placebo effects.

Aim 5: In patients treated with placebo in an earlier osteoarthritis trial, does the current treatment with ACS result in increased clinical scores (Chapter 7)?

24 Chapter 2 Past and present tools to evaluate the success of cartilage-improving interventions Histological quality is considered an important outcome parameter of in vitro as well as in vivo cartilage-influencing therapies. Several scoring systems exist, hindering communication between clinicians and researchers. Consensus is required.

Aim 6: Which histological scoring system should be used when investigating osteoarthritic, in vivo repaired and tissue-engineered cartilage (Chapter 8)?

In patients with medial compartment osteoarthritis of the knee, the high tibial osteotomy (HTO) has been shown to be effective in reducing knee pain51. Realignment of the limb results in transfer of the weight bearing load from the degenerative medial compartment to the relatively uninvolved lateral compartment. The effects of HTO on cartilage quality are largely unknown. Delayed gadolinium enhanced magnetic resonance imaging of cartilage (dGEMRIC) enables non-invasive assessment of cartilage glycosaminoglycan content, eliminating the need for a second-look arthroscopy.

Aim 7: Does dGEMRIC detect relevant changes in cartilage glycosaminoglycan content following HTO, in patients with medial compartment OA of the knee (Chapter 9)?

Outline and aims of this thesis 25

Effect of collagen type I or type II on chondrogenesis by cultured human articular chondrocytes

Marijn Rutgers, Daniël B. Saris, Lucienne A. Vonk, Mattie H. van Rijen, Vanessa Akrum, Danielle Langeveld, Antonette van Boxtel, Wouter J. Dhert and Laura B. Creemers

Tissue Eng Part A. 2013 Jan;19(1-2):59-65 Abstract

Introduction: Current cartilage repair procedures using autologous chondrocytes rely on a variety of carriers for implantation. Collagen types I and II are frequently used and valuable properties of both were shown earlier in vitro, although a preference for either was not demonstrated. Recently however, fibrillar collagens were shown to promote cartilage degradation. The goal of this study was to evaluate the effects of collagen type I and type II coating on chondrogenic properties of in vitro cultured human chondrocytes, and to investigate if collagen-mediated cartilage degradation occurs.

Methods: Human chondrocytes of 8 healthy cartilage donors were isolated, expanded, and cultured on culture well inserts coated with either collagen type I, type II, or no coating (control). After 28 days of redifferentiation culture, safranin O and immunohistochemical staining for collagen types I, II, X and Runx2/Cbfa1 were performed and glycosaminoglycan (GAG) and DNA content and release were examined. Further, expression of collagen type I, type II, type X, MMP13, Runx2/Cbfa1, DDR2, α2 and β1 integrin were examined by RT-PCR.

Results: The matrix, created by chondrocytes grown on collagen type I and II coated membranes, resembled cartilage more than when grown on non-coated membranes as reflected by histological scoring. Immunohistochemical staining did not differ between the conditions. GAG content as well as GAG/DNA were higher for collagen type II-coated cartilage constructs than control. GAG release was also higher on collagen type I and II coated constructs. Expression of collagen type X was higher of chondrocytes grown on collagen type II compared to controls, but no collagen X protein could be demonstrated by immunohistochemistry. Effects of collagen coating on neither DDR2 nor MMP-13 gene expression were found. No differences were observed between collagen types I and II.

Conclusion: Chondrocyte culture on collagen type I or II promotes more active matrix production and turnover. No significant differences between collagen types I and II were observed, nor were hypertrophic changes more evident in either condition. The use of collagen type I or II coating for in vitro models thus seems a sound basis for in vivo repair procedures.

28 Chapter 3 Introduction

Cartilage defects predispose to early osteoarthritis96, 134. Autologous chondrocyte transplantation (ACT) has shown to be a promising treatment option to restore hyaline-like cartilage and is assumed to postpone the onset of osteoarthritis symptoms356. During this procedure, chondrocytes are isolated from non-load-bearing cartilage in the affected joint and expanded in vitro before reimplantation. Novel improvements of this technique in addition involve the use of carriers on which the cells are seeded, and are referred to as matrix assisted autologous chondrocyte implantation (MACI). Several materials have been proposed as carriers for the expanded cells, but the current MACI procedures all involve the use of collagen-based carriers. However, it seems that little evidence has supported the choice of a specific collagen type, i.e. either type I, II or type III collagen, in any of these scaffolds. Some studies have suggested that collagen type II may be more suitable. This collagen was shown to increase matrix production and induce a more chondrocyte-like morphology in comparison with collagen type I287, 288, using canine chondrocytes seeded in 3-D collagen sponges. Furthermore, the expression of chondrocyte-specific genes was higher when bovine MSCs were cultured in a collagen type II hydrogel compared to MSCs cultured in collagen type I46. Upregulation of these chondrogenic markers may be mediated by binding of α2β-1 integrins, which seems essential for maintaining the chondrocyte phenotype110, 238. However, recently a host of literature has suggested that the presence of native fibrillar collagens in the vicinity of chondrocytes may induce a catabolic response leading to cartilage degradation. Collagen II coating of cell culture surfaces induced upregulation of the discoidin domain receptor 2 (DDR 2) and matrix metalloproteinase 13 (MMP 13) expression in human chondrocytes210, which are thought to induce a downward spiral of matrix degradation and hypertrophy446, 467. Denatured collagen was not capable of inducing a suchlike response447. To further elucidate the role of collagen as chondrocyte carrier, the aim of the current study was to evaluate the effects of collagen type I and II on overall chondrogenesis, DDR2 activation and hypertrophic differentiation.

Methods

Chondrocyte isolation and expansion Healthy human femoral cartilage was obtained post-mortem of 4 male donors and 4 female donors. 5 cartilage samples were Collins grade 0 and 3 samples were grade 1. The mean age of the patients was 67 years (range: 47 years – 83 years). Tissue samples were used with patients’ informed consent and through institutional approval (University Medical Center

Effect of collagen type I or type II on chondrogenesis 29 Utrecht, the Netherlands). The cartilage was digested overnight in Dulbecco’s modified Eagle Medium (DMEM) (42430, Invitrogen, Carlsbad, USA) containing 0.1% collagenase (CLS2, Worthington, Lakewood, USA) and 1% penicillin/streptomycin (15140, Invitrogen). Isolated chondrocytes were washed 3 times in phosphate-buffered saline (PBS) and expanded at 5000 cells/cm2 in monolayer in two passages in DMEM containing 1% penicillin/streptomycin, 10% fetal bovine serum (FBS, DE14-801F, Cambrex Bio Science Verviers, Belgium) and 10 ng/ml bFGF (223-FB, R&D Systems, Minneapolis, USA). At 80% confluency, cells were trypsinized and passaged, and used at passage 2 for redifferentiation.

Redifferentiation culture As an in vitro model for regeneration, a 24 well transwell system was used as described previously200. After two passages, chondrocytes were washed 3 times in phosphate- buffered saline (PBS) and seeded on Millipore culture inserts (Millipore BV, Amsterdam, the Netherlands) with hydrophilic poly-tetrafluoroethylene (PTFE) membrane at 720.000 cells/filter (corresponding with a total DNA amount of 8.4 x 106 pg DNA/filter189) and cultured in DMEM supplemented with 2% ITSx (51500, Invitrogen), 2% ascorbic acid (A8960, Sigma-Aldrich, Zwijndrecht, the Netherlands), 2% human serum albumin (HS-440, Sera- care Life Sciences, Milford, USA), 1% penicillin / streptomycin and 10 ng/ml TGF-beta2 (302-B2, R&D Systems). Cells were cultured on the inserts for 28 days in duplo as described earlier 47, 200. The insert membranes were coated with 0.125 mg/ml collagen type I in 0.1 M acetic acid (C9791, Sigma-Aldrich) or 0.125 mg/ml collagen type II in 0.1 M acetic acid (C9301, Sigma-Aldrich). Control inserts were treated with 0.1 M acetic acid (control). Ef- ficient collagen coating was verified by immunohistochemistry for collagen types I and II (Supplementary Figs S1 and S2). Before seeding with chondrocytes, all filters were washed for 30 minutes with sterile PBS. At 28 days of culture, regenerated tissues were washed 3 times in phosphate-buffered saline (PBS) and fixed in 10% neutral buffered formalin or snap-frozen in liquid nitrogen and stored at -20 for GAG analysis or at -80 for RNA analysis.

Histological analysis Histological evaluation was performed on 5 µm deparaffinised sections using safranin O/fast green staining as described by Rosenberg341. Histological quality was assessed using the Bern Score149, a score designed specifically for tissue engineered cartilage, assessing darkness and uniformity of Safranin O staining, the amount and organization of the extracellular matrix and cell morphology. The maximum score is 9, representing healthy hyaline cartilage.

Immunohistochemistry Immunohistochemical staining was performed for collagen types I, II and X, and core- binding factor α1 (Cbfa1/Runx-2) as markers of hypertrophic- and re-and dedifferentia-

30 Chapter 3 tion. To this end, sections were incubated with PBS/BSA 5% (collagen type I, type II and X) and incubated in 10% egg white, rinsed and then blocked with 5% skim milk in TBS-T (Cbfa1) to correct for aspecific binding, then incubated overnight at 4°C with monoclonal antibodies against collagen type I (20 μg/ml, CP-17, Calbiochem, USA), collagen type II (1:100, ascites II-II6B3, Developmental Studies Hybridoma Bank (DSHB), USA), collagen type X (1:50 X53, cell culture supernatant, Quartett, Germany) and Cbfa 1 (1.4 μg/ml, D130-3, MBL, Woburn, USA in 5%PBS/BSA. Subsequently the samples were incubated with a biotinylated sheep anti-Mouse (1:200, RPN1001V, GE Healthcare, Belgium) followed by peroxidase labeled streptavidin (1:500, IM0309 Beckman Coulter) (Collagen I and X) or with goat anti Mouse HRP (1:100, P0447 DAKO, the Netherlands)(Collagen II and Runx2/Cbfa1) all for 1 hr at room temperature in 5% PBS/BSA. Diaminobenzidine was used to obtain a signal. Juvenile growth plate cartilage was used as a positive control for type X and cbfa1 production. Sections were incubated with isotype antibodies as negative controls. Finally, the sections were counterstained with hematoxylin.

Alcian Blue precipitation and DNA assay In vitro constructs were digested in 2% papain (P3125, Sigma-Aldrich) in 50 mM phosphate buffer, 2 mM N-acetylcysteine, and 2 mM Na2-EDTA (pH 6.5) at 65°C for 2 h. Part of the digest was used to measure DNA content and part was used for the quantification of glycosaminoglycan content as a measure of proteoglycan content, using an Alcian Blue precipitation assay (described below). Glycosaminoglycans (GAGs) were precipitated from the chondrocyte digests as well as from the culture medium (at each medium change) and stained with an Alcian blue dye solution (Alcian blue 8GX, 05500, Sigma-Aldrich), saturated in 0.1 M sodium acetate 440 buffer, containing 0.3 M MgCl2 (pH 6.2) for 30 min at 37°C . The blue staining of the medium was quantified photospectrometrically from the change in absorbance at 620 nm, using chondroitin sulphate (Sigma) as a reference. DNA was stained with the fluorescent dye Hoechst 33258 (Sigma) and fluorescence was measured on the Cytofluor (Bio-Rad) 208, using calf thymus DNA (Sigma) as a reference.

PCR For RNA isolation, all steps were performed on dry ice using instruments pretreated with RNAzap and distilled water. After separation of the cartilage from the transwell membranes using a scalpel (Swann-Morton, Sheffield England), the cartilage samples were crushed in a 2-ml Eppendorf tube and pestled using liquid nitrogen with RLT-mercapto- ethanol buffer and a 21G syringe until a homogenous mass was obtained. RNA isolation was done using the RNEasy Micro Kit (74004; Qiagen, the Netherlands) according to the manufacturer’s protocol using DNAse treatment. RNA concentration was measured spectrophotometrically using the NanoDrop® ND-1000 spectrophotometer (NanoDrop

Effect of collagen type I or type II on chondrogenesis 31 Technologies, Wilmington, USA), sufficient RNA purity was assumed if the 260/280nm ratio was higher than 1.9. Subsequently, 500 ng of RNA was reverse transcribed using the iScript cDNA synthesis Kit (Bio-Rad, Hercules, CA). RT-PCR was performed according to the manufacturer’s protocol using the following TaqMan® Gene Expression Assays (Ap- plied Biosystems): Collagen type I (Hs00164004_m1), Collagen type II (Hs00264051_m1), Collagen type X (Hs00166657_m1), Runx2/Cbfa1 (Hs00231692_m1, recognizes all three isoforms of Runx2), MMP 13 (Hs00942589_m1), DDR2 (Hs00178815_m1), α2 integrin (ITGA2, Hs00158127_m1) and β1 integrin (ITGB1, Hs00559595_m1), corrected for beta- actin (Hs99999903_m1). All analysis was performed in technical duplicates except for RNA quantification, which was performed in triplicate.

Statistics Statistical analyses were performed using the statistical software SPSS 15.0 for Windows (SPSS Inc., Chicago, Il, USA). The comparison between the three types of coatings was performed using repeated measurement analysis. Gene expression comparison for collagen type X was compared using Friedmans test. After performing a Bonferroni correction, p≤0.05 (*) was considered statistically significant. All figures show mean values ± standard error of the mean. Histological slides shown are representative of all donors.

Results

Bern score Macroscopically, chondrocytes grown on non-coated membranes formed a more ir- regularly shaped cartilage matrix than cartilage generated on collagen type I or type II coated inserts. Histologically, 6 out of 8 tissue cultures grown on non-coated inserts demonstrated a more aggregated appearance.

7 * 6 * 5 4 3

Bernscore 2 1 0 control coll I coll II Figure 1: Histological scoring of the in vitro generated cartilage using the Bern score149. 1-A: Collagen type I (p=0.01) as well as collagen type II (p=0.05) coated membranes promoted chondrocytes to generate a matrix with more cartilage-like histological properties than when cultured on non- coated (control) membranes. No differences were observed between collagen types I and type II.

32 Chapter 3 1-B: Safranin-O stained histological slides of the in vitro generated cartilage after 28 days of culture on the different membranes, at 40x, 100x and 200x magnification. The more intense safranin-O staining on collagen-coated membranes is clearly demonstrated.

control collagen I collagen II control collagen I collagen II

1 1 Collagen Collagen

type I type X 2 2 IHC IHC

3 3

1 1 Collagen Runx2/ type II Cbfa1 IHC 2 2 IHC

3 3 Figure 2. Immunohistochemical staining for collagen types I, II, collagen type X and Runx2 at the end of the 28-day in vitro culture for 3 human chondrocyte donors at 400x magnification. Collagen type I and II staining were more donor-dependent than dependent of the coating conditions and no differences between either condition were detected. Immunohistochemical staining for collagen type X and Runx2/Cbfa1 was low or absent for all conditions in all donors.

Effect of collagen type I or type II on chondrogenesis 33 Histological quality of in vitro cultured cartilage (Bern score) was higher for collagen type I and col II vs. control (p<0.01 and p=0.05, respectively) [Figure 1-A and 1-B].

Immunohistochemistry Immunohistochemistry for collagen type I and II demonstrated a variable strong staining in all conditions, however this seemed donor-specific rather than dependent on coating and no significant differences were observed. No collagen type X production was observed in any of the conditions. Low Cbfa1 staining was observed under all conditions [Figure 2].

GAG and DNA content No visible loss of cells was noted after seeding of the inserts immediately after plating, nor after the first medium change. Upon comparison of the separate coating procedures after 28 days, DNA content did not differ significantly between the conditions (10.5 x 106 pg DNA/filter for collagen type I coated filters (corresponding with 910.000 cells), 10.9 x 106 pg DNA/filter (corresponding with 940.000 cells) for collagen type II coated filters and 8.2 x 106 pg DNA/filter (corresponding with 710.000 cells) for control filters; p=0.16). Total GAG content was higher in tissues grown on collagen type II (80 μg ± 11 μg) compared to cells cultured on uncoated scaffolds (50 μg ± 11 μg; p<0.01). The amount of GAG normalized per DNA was higher for collagen type II-coated scaffolds compared to controls (p=0.02) [Figure 3-A]. Total GAG release was higher on collagen type I (139 μg ± 33 μg; p<0.01) and II coated filters (116 μg ± 26 μg; p=0.05) vs. control. Total proteoglycan production (final GAG content + total amount of GAG released during culture) was higher for collagen type I vs. control (p=0.01) and collagen II vs. control (p=0.02). When release was expressed as percentage of total production, no differences were observed between collagen type I or II and controls (p=0.60) [Figure 3-B].

PCR ECM and hypertrophic markers Collagen type X expression was higher for collagen type II vs. control (p=0.03). Collagen type II expression was nonsignificantly higher in type II coated constructs (p=0.09). DDR2 expression appeared higher for collagen types I and II, although this did not reach statistically significant levels. No differences were observed in expression of collagen type I, collagen type II, MMP-13, Runx2/Cbfa1, α2 integrin and β1 integrin expression [Figure 4].

34 Chapter 3 10.0 control * collagen type I 300 collagen type II * 8.0 250 *

6.0 * 200 * * 150

4.0 GAG ug * 100 p =0.16 2.0 50

content relative to control 0.0 0 GAG content Total GAG GAG production GAG DNA GAG/DNA release (release + cont) Content at 28 days, relative to control Absolute GAG quantity Figure 3‑A. GAG, DNA and GAG/DNA content, rela- Figure 3‑B. Absolute GAG content, GAG release and tive to the control inserts. GAG content was higher total GAG production (in μg/scaffold). Total GAG re- in cartilage on collagen type II-coated constructs lease was higher during culture of chondrocytes on than on non-coated constructs (p<0.01). Collagen collagen type I (p<0.01) and type II (p=0.05) coated type I did not differ significantly from control constructs than when cultured on non-coated (p=0.06). No significant differences were observed (control) constructs. Total glycosaminoglycan pro- between collagen types I and type II. DNA content duction during the 28-day culture, consisting of the did not differ significantly between the conditions final GAG content added to the total GAG released (p=0.16). GAG/DNA was higher for collagen type II during culture, was higher when cultured on col- coated constructs compared to controls (p=0.02). lagen type I (p=0.01) as well as when cultured on collagen type II coated constructs (p=0.02) in comparison to control. No differences were observed between collagen types I and II (p=0.60).

Figure 4. RNA expression of collagen type I, type II and type X, MMP13, DDR2, Runx2/Cbfa1, α2 integrin (ITGA2) and β1 integrin (ITGB1) at the end of the 28-day redifferentiation culture. Collagen type X expression was higher in chondrocytes cultured on type II coated constructs (p=0.03) than in chondrocytes cultured on non-coated constructs. For none of the other genes significant differences in expression were found between control, collagen type I or collagen type II coating.

Effect of collagen type I or type II on chondrogenesis 35 Discussion

The current study aimed at determining the effect of collagen I and II on chondrocyte regeneration, with respect to extracellular matrix production and degradation, but also to examine possible activation of DDR2-mediated degradation. Cartilage regenerated on collagen type II was characterized by a higher final GAG content, higher GAG/DNA ratio and morphologically resembled hyaline-like cartilage more than cartilage generated on non-coated constructs, although no clear difference was observed between collagen types I and II. Further, total GAG production was higher in the collagen coated scaffolds compared to controls. Although the release of GAGs into the culture medium was also higher, the release expressed as percentage of total production did not differ significantly, thus suggesting that the increased release into the medium was partly due to the release of newly produced GAGs that could not be retained in the matrix, rather than degraded GAGs. At the protein level, collagen production appeared to vary between the donors, which was reflected by the mRNA levels. Hypertrophic differentiation was not evident at the protein level either, although at the mRNA level collagen type X gene expression was increased on coated scaffolds. However, MMP-13 expression was not increased, which was in line with the lack of a clear upregulation of DDR2 in the current study. MRNA for α2 and β1 integrins was clearly detected but was not affected by culture on the different coatings. To what extent the collagen-mediated stimulatory effect on GAG production was collagen-specific cannot be stated with full certainty. The effect may be related to mere cell attachment to collagen as proteinaceous substrate. Integrin-mediated attachment and signaling could also be responsible238 as α2 and β1 integrin were clearly shown to be expressed in the current study. However, even if these integrins would have been present at the protein level, inhibition of their expression would have provided firm evidence for this explanation. The differential morphology of cartilage grown on non-coated inserts suggested that a lack of cell-matrix or cell-protein adherence may have led to some aggregation of cells, a principle used in pellet culture models of chondrogenic regeneration. In a previous study, however, no differences in cartilage matrix production were observed450 between these two culture models, suggesting the collagen coatings as such had played a role in improving cartilage regeneration rather than the inhibition of aggregation. The collagen types I and II used in the current study were from bovine and chicken origin, respectively. Although ideally collagens from the same animal source would have been used, the currently used collagens both have a long history in in vitro cell culture use 47, 272, 352, 365, 391, 450. However, differential effects using collagens from other species cannot be excluded. Despite the recently postulated effects of fibrillar collagens on cartilage degradation, no significant upregulation of DDR2 or its downstream target gene MMP-13 were observed,

36 Chapter 3 nor did GAG degradation during culturing seem to be affected by the use of fibrillar collagens for coating. Although collagen release was not determined in the current study, the lack of any difference in MMP-13 levels and in GAG content suggests the absence of an effect on collagen degradation. Although no DDR2 activation was noted in the current study, this may have occurred during the early culture periods, its expression wearing off by the end of the period. Even so, apparently, these effects are not strong enough to affect the net production of cartilaginous tissue, which was actually highest in collagen type I and II-coated constructs. While the increase in collagen type X expression on collagen type II coated scaffolds suggested some hypertrophic differentiation, at the histological level this was difficult to confirm. However, a discrepancy between mRNA levels and protein production frequently occurs99, 359, with sometimes large effects on mRNA levels being translated into smaller or even opposite effects on protein production 123, 359, 427. Overall, the high inter-donor variability in immunohistochemical staining for collagen I and II is in accordance rather than in contrast with the mRNA levels found.

Why, in contrast with previous results287, no clear difference in matrix production was observed between the two collagen types is not clear, but was reflected in the α2β1 integrin receptor expression, which was similar in all conditions at 28 days. However, expression may have been different at earlier stages of culture, or when the chondrocytes first came into contact with the collagens. The common drawback of in vitro studies is that their outcome is usually based on the results of one or a limited number of timepoints, which for cartilage regeneration is usually limited to a period of 21-28 days. It is not known at which time point in vitro regeneration reflects the final effects found in vivo, typically analyzed several months or years after implantation. It would be worthwhile to study the relation between regeneration in vitro and in vivo and further define which stage of in vitro regeneration best predicts in vivo cartilage formation. In addition, the lack of more time points in these and other studies also precludes a clear picture of the course of the redifferentiation process, which may be suboptimal and re- flect fibrocartilage formation22. However, given the identical starting point of this study, i.e. dedifferentiated chondrocytes, it can be concluded that collagen coating positively affected their phenotype at least in the course of the 4 weeks studied. Another explanation for the difference observed between collagen I and II in the study by Nehrer et al287, may be the more extensive cell-matrix contact area between the 3D collagen gel and the canine chondrocytes in this particular study. This may have been responsible for more cell surface receptor-matrix interaction, thereby maintaining specific cell characteristics. Although the currently used filter culture model is a 2D rather than a 3D model, also MACI procedures are in essence 2D culture conditions, as commonly cells seeded on carriers attach to the surface of carrier pores rather than being surrounded by the ma-

Effect of collagen type I or type II on chondrogenesis 37 terials as is the case with for example hydrogel encapsulation. As on MACI membranes, a ‘multilayered cell sheet’ is formed in the current culture model 49, 131, 138, 139, 342. However, most MACI procedures employ a collagen type I / III membrane and therefore the current culture set up is not completely comparable.

In conclusion, fibrillar collagens type I and II do not seem to have any lasting negative influences on in vitro cultured human chondrocytes with regard to synthesis of tissue engineered cartilage. Collagen type II coating seems slightly preferable to collagen type I, although both collagen types are preferable to no collagen coating. Future studies may include analysis of the effects of collagen type III, which is currently also used in MACI constructs.

38 Chapter 3 Supplementary figures

Coll coated staining - Coll coated staining +

Uncoated staining - Uncoated staining +

Supplementary Figure S1. Immunohistochemistry for collagen type I, on a collagen type I-coated filter. Upper left collagen type I-coated filter, isotype control; upper right collagen type I-coated filter, positive collagen type I staining. Lower left: uncoated filter, isotype control. Lower right: uncoated filter, negative staining for collagen type I.

Effect of collagen type I or type II on chondrogenesis 39 Coll coated staining - Coll coated staining +

Uncoated staining - Uncoated staining +

Supplementary Figure S2. Immunohistochemistry for collagen type II, on a collagen type II-coated filter. Upper left collagen type II-coated filter, isotype control; upper right collagen type II-coated filter, positive collagen type II staining. Lower left: uncoated filter, isotype control. Lower right: uncoated filter, negative staining for collagen type II.

40 Chapter 3 PTH decreases in vitro cartilage regeneration without affecting hypertrophic differentiation

Marijn Rutgers, Lucienne A. Vonk, Mattie H. van Rijen, Vanessa Akrum, Marianne A. Tryfonidou, Antonette van Boxtel, Wouter J. Dhert, Daniël B. Saris and Laura B. Creemers

Manuscript in preparation Abstract

Introduction Regenerated cartilage after Autologous Chondrocyte Implantation (ACI) may be of suboptimal quality, in part due to postulated hypertrophic changes. Parathyroid Hor- mone-related peptide (PTHrP), has been shown to inhibit hypertrophic differentiation in mesenchymal stem cells (MSCs) and enhance cartilage growth during development. It is unclear if human articular chondrocytes respond in the same fashion.

Methods Healthy human articular chondrocytes were isolated from 6 human donors, followed by in vitro culture on Millipore transwells with addition of PTH at 0.1 and 1.0 μM from days 0, 9 or 21 onwards up to the end of the culture at 28 days. At the end of culture, histological analysis was performed to evaluate cartilage quality (Bern score), as well as glycosaminoglycan (GAG) and DNA production and release. Immunohisto-chemical and gene expression analysis were performed for collagen types I, II X and Runx2 and gene expression analysis were also performed for DDR2 and MMP13.

Results PTH inhibited cell proliferation (DNA content, p=0.027) regardless of concentration and timing of addition, resulting in lower amounts of GAG (p=0.001) and a lower Bern score (p=0.005) in comparison to controls. Immunohistochemical staining for collagen type I was higher and collagen type II was lower when PTH was added, which was reflected by a decrease in mRNA for collagen type II expression (p<0.001). No effects were noted on Runx2 and collagen X expression.

Discussion PTH addition to healthy articular chondrocytes does not affect hypertrophic differentiation, while negatively influencing GAG and DNA content. Inhibition of hypertrophy may be more relevant after microfracture procedures involving endogenous MSCs than in ACI departing from healthy chondrocytes.

42 Chapter 4 Introduction

Autologous chondrocyte implantation (ACI) is a commonly applied treatment for cartilage trauma in patients with medium-sized defects. Ideally the chondrocytes, transplanted after in vitro culture, become matrix-producing cells which fill the void with hyaline neocartilage. More recent versions of ACI are based on the use of collagen membranes and gels as carrier of the expanded cells. However, variable results have been obtained with regard to cartilage quality190, with frequently observed filling of the defect with fibrous tissue50, 212 and even hypertrophic differentiation464, a process in which cartilage degrades and is replaced by bone. One of the factors crucial in maintenance of the chondrocytic phenotype in native cartilage is parathyroid hormone related-peptide (PTHrP).This peptide stimulates Sox9 expression through binding to the PTH/PTHrP receptor and subsequent activation of protein kinase A91. PTHrP is necessary to maintain chondrocytes in their proliferating state, necessary for cartilage formation and prevention of hypertrophic differentiation and subsequent bone formation91. Moreover, PTHrP plays an important role in early development and limb growth9. PTH is assumed to have similar effects as PTHrP435, 461, as it shares the same N-terminus and binds to the same PTH/PTHrP receptor320, 402. In adipose tissue derived mesenchymal stem cells207 and bone marrow stromal cells (BMSC)198, 207, 284, PTHrP/PTH administration effectively stimulated chondrogenic differentiation and prevented hypertrophic differentiation. In rabbit growth plate chondrocytes, PTH suppressed collagen type X expression and increased proteoglycan production186. In rabbit knees with osteochondral defects, intra-articular PTH administration during the first weeks after introduction of the defect indeed stimulated regeneration, although intermittent administration was more effective 223, 224. PTHrP/PTH signaling may stimulate cartilage formation by increasing proliferation through cyclin-D induction462. However, the direction of the effects on proliferation appears to be dependent on dose and cell maturation241, 415. Since osteochondral defect healing is most likely MSC-mediated, the in vivo stimulation of chondrogenesis appeared to be in line with the in vitro effects found. However, (M)ACI is based on the use of expanded chondrocytes. Although these have shown to have stem cell-like properties as well80, their expression profiles are still more chondrocyte-like than those of undifferentiated MSCs34. Application of PTH in vivo for enhanced regeneration and prevention of hypertrophic differentiation in combination with (M)ACI hence requires insight in the effects of PTH signaling on chondrocyte- mediated regeneration. Therefore we currently examined the effects of PTH in a culture model based on expanded chondrocytes seeded on collagen carriers. To mimic different phases of maturation, PTH was added at different time points and concentrations.

Effect of PTH on chondrogenesis 43 Methods

Human chondrocytes Healthy human femoral cartilage was obtained postmortem of 3 male and 3 female donors (mean age 68, range 47 – 83 years). Only cartilage which was macroscopically intact (Collins grade 0 or 177) was used, and all samples were embedded and Safranin O- stained to exclude low-quality cartilage samples. 4 cartilage samples were Collins grade 0 and 2 samples were grade 1. Tissue samples were used through institutional approval (University Medical Center Utrecht). Investigations were performed in accordance with the guidelines set out in the Helsinki declaration.

Chondrocyte isolation and expansion The cartilage was digested overnight in Dulbecco’s modified Eagle Medium (DMEM) (42430, Invitrogen, Carlsbad, USA) containing 0.1% collagenase (CLS2, Worthington, Lakewood, USA) and 1% penicillin/streptomycin (15140, Invitrogen). Isolated chondrocytes were washed 3 times in phosphate-buffered saline (PBS) and expanded at 5000 cells/cm2 in monolayer in two passages in DMEM containing 1% penicillin/streptomycin, 10% fetal bovine serum (FBS, DE14-801F, Cambrex Bio Science Verviers, Belgium) and 10 ng/ml bFGF (223-FB, R&D Systems, Minneapolis, USA). At 80% confluency, cells were trypsinized and passaged. Pellets of 500.000 chondrocytes were snap-frozen in liquid nitrogen for RNA analysis. Cells at passage 2 were used for redifferentiation culture.

Redifferentiation culture As an in vitro model for regeneration, a 24 well transwell system was used as described previously200. After two passages, chondrocytes were trypsinized, washed 3 times in phosphate-buffered saline (PBS) and seeded on Millipore culture inserts (Millipore BV, Amsterdam, the Netherlands) with a hydrophilic poly-tetrafluoroethylene (PTFE) membrane at 1.6 x106 cells/cm2 and cultured in DMEM supplemented with 2% ITSx (51500, Invitrogen), 2% ascorbic acid (A8960, Sigma-Aldrich, Zwijndrecht, the Nether- lands), 2% human serum albumin (HS-440, Seracare Life Sciences, Milford, USA), 100 units/ml penicillin with 100 μg/ml streptomycin and 10 ng/ml TGF-beta2 (302-B2, R&D Systems). The insert membranes had been coated with 0.125 mg/ml collagen type II in 0.1 M acetic acid (C9301, Sigma-Aldrich200). Efficient collagen coating was verified by immunohistochemistry344. All filters were washed for 30 minutes with sterile PBS before seeding. PTH was added at 0.1 or 1.0 μM to the culture medium, from days 0 (d0, n=4 donors), 9 (d9, n=6) or 21 (d21, n=6) onwards, or no PTH was added (control, n=6). The choice of PTH concentrations was based on an earlier study198. After 28 days, the regenerated tissues were washed 3 times in phosphate-buffered saline (PBS) and fixed in 10% neutral

44 Chapter 4 buffered formalin or snap-frozen in liquid nitrogen and stored at -20 ºC for GAG and DNA analysis or at -80 ºC for RNA analysis.

Histological analysis Histological evaluation of 28-day cultured tissue was performed on 5 µm deparaffinised sections using safranin O/fast green staining as described by Rosenberg341. Histological quality was assessed using the Bern Score149, a score designed specifically for tissue engineered cartilage, assessing intensity and uniformity of Safranin O staining, the amount and organization of the extracellular matrix and cell morphology. The maximum score is 9, representing healthy hyaline cartilage.

GAG and DNA analysis Day 28 in vitro constructs were digested in 2% papain (P3125, Sigma-Aldrich) in 50 mM phosphate buffer, 2 mM N-acetylcysteine, and 2 mM Na2-EDTA (pH 6.5) at 65°C for 2 h. Part of the digest was used to measure DNA content and part was used for the quantification of glycosaminoglycan content as a measure of proteoglycan content, using an Alcian Blue precipitation assay. Glycosaminoglycans (GAGs) were precipitated from the tissue digests as well as from the culture medium (at each medium change) with an Alcian blue dye solution (Alcian blue 8GX, 05500, Sigma-Aldrich), saturated in 0.1 M sodium acetate buffer, containing 0.3 440 M MgCl2 (pH 6.2) for 30 min at 37°C . The blue staining of the medium was quantified photospectrometrically from the change in absorbance at 620 nm, using chondroitin sulphate (Sigma) as a reference. DNA was quantified by staining with the fluorescent dye Hoechst 33258 (Sigma), followed by fluorescence measurement on the Cytofluor (Bio-Rad) 208, using calf thymus DNA (Sigma) as a reference.

PCR For RNA isolation, all steps were performed on dry ice using instruments pretreated with RNAzap and distilled water. After separation of the neocartilage from the transwell membranes using a scalpel (Swann-Morton, Sheffield England), the cartilage samples were crushed in a 2-ml Eppendorf tube and pestled using liquid nitrogen with RLT-mercapto- ethanol buffer (Sigma) and a 21G syringe until a homogeneous mass was obtained. RNA isolation was done using the RNEasy Micro Kit (74004; Qiagen, the Netherlands) according to the manufacturer’s protocol using DNAse treatment. RNA concentration was measured spectrophotometrically using the NanoDrop® ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, USA), sufficient RNA purity was assumed if the 260/280nm ratio was higher than 1.9. Subsequently, 500 ng of RNA was reverse transcribed using the iScript cDNA synthesis Kit (Bio-Rad, Hercules, CA). RT-PCR was performed according to the manufacturer’s protocol using the following TaqMan®

Effect of PTH on chondrogenesis 45 Gene Expression Assays (Applied Biosystems): Collagen type I (Hs00164004_m1), Collagen type II (Hs00264051_m1), Collagen type X (Hs00166657_m1), Runx2/Cbfa1 (Hs00231692_m1, recognizes all three isoforms of Runx2), MMP 13 (Hs00942589_m1) and DDR2 (Hs00178815_m1), corrected for beta-actin (Hs99999903_m1). All analysis was performed in technical duplicates except for RNA quantification, which was performed in triplicate.

Immunohistochemistry Immunohistochemical staining was performed for collagen types I, II and X, and core- binding factor α1 (Cbfa1/Runx-2) as differentiation and/or hypertrophic markers. To this

end, sections were incubated with 0.3% H2O2 in PBS and PBS/BSA 5% (collagen type I, type II, type X) or TBS/BSA 5% (Cbfa1) to correct for aspecific binding, then incubated overnight at 4°C with monoclonal antibodies against collagen type I (20 μg/ml, CP-17, Calbiochem, USA), collagen type II (1:100, ascites II-II6B3, Developmental Studies Hybridoma Bank (DSHB), USA), collagen type X (1:50 X53, cell culture supernatant, Quartett, Germany) and Cbfa 1 (5 μg/ml, SC-101145, Santa Cruz, USA) in 5%PBS/BSA. Subsequently the samples were incubated with a biotinylated sheep anti-mouse (1:200, RPN1001V, GE Healthcare, Belgium) followed by peroxidase labeled streptavidin (1:500, IM0309 Beckman Coulter) (Collagen I and X) or with goat anti mouse-HRP (1:100, P0447 DAKO, the Netherlands)(Col- lagen II and Runx2/Cbfa1) all for 1 hr at room temperature in 5% PBS/BSA. Diaminoben- zidine was used to obtain a signal. Juvenile growth plate cartilage was used as a positive control for type X and Cbfa1 production. Sections were incubated with isotype antibodies as negative controls. Finally, the sections were counterstained with hematoxylin.

Statistics Statistical analyses were performed using the statistical software IBM SPSS 20 for Windows (IBM SPSS Inc., Chicago, Il, USA). Data were tested for normality using the Kolmogorov–Smirnov test and for homogeneity of variances using Levene’s test of equality of variances. Subsequently, a mixed model analysis was performed to detect the influence of PTH concentration as well as timing of PTH addition. After performing a Bonferroni correction, p≤0.05 (*) was considered statistically significant. All figures show mean values ± 95% confidence interval.

Results

GAG and DNA content, GAG production and release Addition of PTH at either 0.1 or 1.0 μM decreased GAG content (p=0.001) irrespective of the time point at which it was added, while DNA content also decreased (p=0.030),

46 Chapter 4 140 day 0 day 0 20 * 120 * day 9 day 9 day 21 100 day 21 15 80 60 10 40 5 20

GAGcontent(ug/filter) 0 0 DNA content(ug/filter) DNA 0.1 uM 1.0 uM control 0.1 uM 1.0 uM control PTH concentration PTH concentration

Figure 1A-C: GAG, DNA and GAG/DNA content af- 25 day 0 ter 28 days of culture. Graphs show means ± 95% day 9 20 C-I. 1-A: GAG content at day 28. Addition of PTH re- day 21 sulted in a lower GAG content than when no PTH 15 was added (p=0.001). No differences were observed 10 between the PTH concentrations nor were time-de- control pendent effects observed. 1-B: Total DNA content 5 after 28 days of culture was decreased by addition GAG/DNArelative to 0 of PTH at 0.1 and 1.0 μM (p=0.030). A time-depen- 0.1 uM 1.0 uM control dent increase in DNA content was observed when PTH concentration PTH was added later during the culture (p=0.027), with no difference between 0.1 and 1.0 μM. 1-C: GAG/DNA at day 28 did not differ significantly between the conditions and was independent of PTH concentration or timing of its addition. which was most pronounced when PTH was added later during the culture (p=0.027). No differences were observed between the PTH concentrations. GAG per DNA after 28 days was not influenced by addition of PTH or timing (p=0.482) [Figure 1a-c]. GAG release nor total production (calculated by adding up the total amount of GAGs released into the medium and the GAG content of the regenerated tissue) were altered by addition of PTH. Relative GAG release (release as percentage of total production) did not differ between the different PTH concentrations or control (p=0.656) [Figure 2].

Bern score PTH addition resulted in lower Bern scores for both concentrations of PTH and all time points compared to controls (p=0.005) [Figure 3].

Immunohistochemistry Collagen type I expression appeared slightly lower and collagen type II expression slightly higher, in PTH treated cultures. Only very weak staining was observed for collagen type X under all conditions. Cbfa1 expression was present in 3 out of 6 donors, however this was independent of PTH addition [Figure 4].

Effect of PTH on chondrogenesis 47 500 day 0 400 day 9 day 0 400 day 21 300 day 9 300 day 21 200 200

100 100 Total GAG production (ug) Total GAG production

0 (ug) release GAG Total 0 0.1 µM 1.0 µM control 0.1 µM 1.0 µM control PTH concentration PTH concentration

day 0 100 day 9 80 day 21 60 Figure 2: Total GAG production (2-A), Total GAG release (2-B) and GAG release as % of total pro- 40 duction (2-C). GAG production was dependent production) 20

on timing of addition of PTH (p=0.042) but not Releasetotal (% of 0 dependent on PTH concentration. Graphs show 0.1 µM 1.0 µM control means ± 95% C-I. PTH concentration

day 0 8 * day 9 7 day 21 6 5 4 3

Bernscore 2 1 0 0.1 uM 1.0 uM control PTH concentration

Figure 3: Bern score of 28 day in vitro culture chondrocytes. PTH addition resulted in lower Bern scores, compared to controls (p=0.005). No differences were observed between the different PTH concentrations. Graphs show means ± 95% C-I.

PCR Collagen type II expression was decreased by addition of either 0.1 or 1.0 μM PTH (p<0.001). No effects of PTH administration were observed on collagen type I, collagen type X, DDR2, MMP13 or Runx2 expression [Figure 5].

48 Chapter 4 Figure 4: Histological sections of chondrocytes cultured for 28 days in the presence of 0.1 μM PTH from day 0, 9 or 21 onwards, in the presence of 1.0 μM PTH from day 0, 9 or 21 onwards or without PTH (control) at 400x magnification. The Bern score was highest in the control group (figure 2-a). Collagen type I staining appeared slightly lower and collagen type II expression slightly higher, in the absence of PTH (control). Collagen type X was virtually absent under all conditions. Cbfa1 expression was present in 3 out of 6 donors, however this was independent of PTH addition. No effects of timing were observed.

7.0 0.1 day 0 0.1 day 9 0.1 d 21 6.0 1.0 day 0 1.0 day 9 5.0 1.0 day 21 control

4.0

3.0

2.0 *

Gene expression relative to control 1.0

0.0 Col I Col II DDR2 MMP13 ColX Runx2

Figure 5: Gene expression of chondrocytes after 28 days redifferentiation culture. Collagen type II expression was reduced by PTH addition to the redifferentiation culture (p<0.001). Graphs show means ± 95% C-I.

Effect of PTH on chondrogenesis 49 Discussion

In the current study, the influence of PTH addition on regeneration and hypertrophic differentiation was analyzed in in vitro cultured human articular chondrocytes. PTH addition did not affect hypertrophic differentiation at the protein nor mRNA level. Ad- dition of PTH negatively influenced cell proliferation and collagen type II expression independent of PTH concentration, leading to a lower total matrix content and reduced histological quality after 28 days of culture. The absence of an effect on hypertrophic differentiation in the expanded chondrocytes was surprising giving the previously found effects on MSC-mediated chondrogenic differentiation. Although a diminished responsivity to PTH in chondrocytes has been reported, 186, 198, this is not supported by the effect on proliferation, matrix content and collagen type II expression found. Most likely, the degree of hypertrophic differentiation level was too low, which was supported by the lack of immunohistochemical detection of hypertrophic differentiation markers. In MSC-based chondrogenic differentiation, hypertrophic differentiation has been demonstrated at the mRNA as well as the immunohistochemical level34, 284. Collagen type II expression was inhibited by PTH. Decreased collagen type II expression following systemic PTH administration was also observed in avian154 and rat402 chondrocytes at 0.1 μM. Using lower concentrations of PTH186 than currently used may prevent collagen type II inhibition. In an earlier MSC study, PTH levels higher than 10−3 μM inhibited chondrogenic differentiation 434, while at lower concentrations collagen type II was present throughout the matrix. The current inhibitory effect of PTH on cellular proliferation was unexpected. In the growth plate, PTHrP increases proliferation of prehypertrophic chondrocytes, thereby inhibiting further maturation. PTHrP-mediated activation of cyclin D1 gene expression and promotion of chondrocyte proliferation is believed to occur through transcription factors ATF2 and CREB25. Previous data showed that PTH increased the mitogenic 35 2− response as well as SO4 incorporation of chick growth plate chondrocytes, but did not affect (healthy) non-expanded articular chondrocytes81. Increased proliferation may thus only occur in ‘maturing’ growth plate or osteoarthritic chondrocytes, but not in ‘immature’ resting or proliferating articular chondrocytes. The dedifferentiated chondrocytes currently used may be considered ‘immature’, which would at least explain an absence of increased proliferation. During the 28-day culture period in chondrogenic medium containing TGF-beta, the chondrocytes ‘matured’ and formed cartilaginous tissue. Addition of PTH to the ‘more mature’ chondrocytes, from day 21 onwards, still inhibited proliferation, albeit at a lower extent. The inhibited proliferation, as observed when PTH was added from day 0 onwards, may be related to binding of PTH to a different receptor. For example, binding of tuberoinfundibular peptide of 39 residues (TIP39, a member of the PTH family) to the type 2 PTH receptor inhibited articular chondro-

50 Chapter 4 cyte proliferation 309. In addition, although PTH and PTHrP share the same amino acid sequence, possibly intracellular receptor-independent signaling may occur402. However, we have compared these two peptides in pilot experiments and found the same effects (data not shown) on chondrogenesis. When considering PTH for repair of cartilage defects clinically, intermittent PTH administration seems favorable to continuous administration. In humans, intermittent administration of PTH (1-84) was shown to prevent osteoporosis458, while in vivo it improved the quality of repaired cartilage in a rabbit cartilage defect model224. In contrast, continuous systemic exposure may result in osteomalacia, focal bone resorption, and peritrabecular bone marrow fibrosis242. However, repeated intra-articular injections pose a risk for intra- articular infection. Recently, attempts were made to overcome these issues by using an intra-articular controlled release model 105. Controlled release of PTH from PLGA microspheres was compared to intermittent (1x/ 3 days) injection in a papain-induced rat OA model. Similar protective effects against GAG loss and collagen type X expression were observed. However, the inhibition of regeneration found in the current study would argue against the use of PTH in defect treatment, even in cartilage repair procedures based on combinations of chondrocytes and MSCs 26.

In conclusion, PTH seems to have an inhibitory effect on the regenerative potential of healthy human articular chondrocytes while hypertrophic differentiation was not influenced. PTH may be more suitable for inhibition of hypertrophic differentiation in cartilage repair based on growth plate chondrocytes or MSCs.

Effect of PTH on chondrogenesis 51

Similar regenerative capacity of juvenile versus adult talar chondrocytes despite high expression of hypertrophic markers

Marijn Rutgers, Daniël B. Saris, Lucienne A. Vonk, Mattie H. van Rijen, Vanessa Akrum, Antonette van Boxtel, Ralph J. Sakkers, Wouter J. Dhert and Laura B. Creemers

Submitted Abstract

Objective: Juvenile cartilage is presumed to have better repair characteristics than adult cartilage. It is unclear if this is related to the juvenile chondrocyte phenotype or to the juvenile joint environment. This study aimed at comparing regenerative capacity of juvenile and adult talus chondrocytes.

Methods: Human talar chondrocytes of juvenile (n=5) and adult (n=10) donors were cultured on collagen-coated inserts for 28 days. GAG and DNA analysis was performed at 35 day 9 and 28, GAG synthesis was evaluated by SO4 incorporation, PCR (collagen types I, II, X, Cbfa1/Runx2, MMP13, DDR2), immunohistochemical and histological staining (Bern score) were performed. Data were compared to adult femoral chondrocyte donors (n=8).

Results: GAG and DNA production in tissue generated by juvenile cells were higher on day 9 (p<0.01) but not on day 28. Expression of DDR2, MMP 13, collagen X and Cbfa1/ Runx2 was higher in juvenile tissue than in adult talar neotissue (p<0.02). The Bern score was similar, however was lower compared to tissue generated by adult femoral cells (p<0.001).

Conclusion: Juvenile talar chondrocytes initially regenerated faster than adult chondrocytes, however this did not result in higher matrix production. The higher expression of hypertrophic differentiation markers in juvenile tissue suggested degenerative changes, which were not reflected by protein expression or histology. The lack of a clear difference in regenerative capacity between adult and juvenile cells suggests the difference in outcome of ACI in young patients is related to the joint environment rather than to chondrocyte phenotype.

54 Chapter 5 Introduction

For restoration of cartilage defects using autologous chondrocyte implantation (ACI), in vitro engineered cartilage quality frequently lacks hyaline characteristics212, 329. Dedif- ferentiation towards a more fibroblast-like phenotype137 and subsequent hypertrophic differentiation396, initiated during the proliferation phase prior to implantation, may be responsible for less favorable clinical outcomes. In humans, clinical results of cartilage repair procedures tend to be best in young, active patients215, 276. Although this may suggest that the characteristics of younger chondrocytes are responsible for this difference, it cannot be excluded that the intra-articular environment of the older joint is less amenable to regeneration. If indeed regeneration would depend on the characteristics of the cells used, elucidation of the mechanism behind an increased regenerative repair capacity in juvenile cells may lead to the discovery of new regenerative targets. More- over, this would also open up new avenues towards ACI by using cartilage of juvenile donors for repair of adult cartilage defects, as is already being done 63. Several studies on animal tissue-derived chondrocytes have suggested that the chondrogenic potential of juvenile cells is higher compared to adult cells, independent of the articular environment. Isolated chondrocytes of fetal and young animals displayed higher proliferation rates, glycosaminoglycan (GAG) and collagen production399, higher expression of TGF-β receptors40 and increased collagen type II content410 in comparison to those of older animals. However, for human chondrocytes, many contradictory results were found. Usually higher cell proliferation rates were observed for juvenile cells1, 21, but differences in matrix production were variable. In agarose or polyethylene-glycol (PEG)- derived hydrogel cultures based on juvenile chondrocytes, both similar and higher basal proteoglycan production seemed to be found compared to older donors1, 378. Also in pellet culture of chondrocytes from donors below 40 years of age the same production rate per cell was found as in pellets from older donors21. However, here a stronger response of juvenile cells was noted to expansion in the presence of growth factors (TGFβ1, FGF-2, PDGF) in terms of matrix production during subsequent differentiation than adult cells21. None of the previous studies have cultured cells in the presence of collagen, while the ACI or MACI repair procedures involve the use of collagen membranes. Although exposure to collagens, postulated to induce hypertrophic differentiation and subsequent cartilage degeneration210, did not affect tissue regeneration by adult femoral chondrocytes cultured on collagen 344, juvenile cells may be affected. We therefore aimed at evaluating if human juvenile chondrocytes produce more hyaline-like cartilage than adult human chondrocytes, using talar chondrocytes, in a culture model based on the use of collagen type II. In addition, regeneration of adult talar chondrocytes was compared to adult femoral chondrocytes to verify to what extent chondrocyte behavior from these cartilage surfaces differ.

Chondrogenesis by juvenile and adult talar chondrocytes 55 Methods

Human chondrocytes Talar cartilage of five juvenile patients (mean age 9.9, range 1.5-16 years) was acquired as redundant material during talar resection because of an uncorrectable foot and ankle deformity. Adult talar cartilage was obtained from adult tissue donors (mean age 43, range 21-53 years), registered as bone tissue donor at the Dutch Transplantation Foundation (Nederlandse Transplantatie Stichting, NTS) [Table 1]. Adult femoral cartilage was obtained post-mortem from 4 male donors and 4 female donors (mean age 67, range 47– 83 years). For both juvenile and adult donors, only cartilage which was macroscopically intact (Collins grade 0 or 177) was used, and from all donors samples were paraffin-embedded for safranin O staining to exclude low-quality cartilage samples [Appendix 1]. Anonymous use of redundant tissue for research purposes is part of the standard treatment agreement with patients in the University Medical Center Utrecht, and according to the national guidelines ‘code of conduct for the proper secondary use of human tissue’ 408. Approval for the use of donor material was obtained through the

Table 1: Talus and femur cartilage donor characteristics Juvenile talus Adult talus Adult femur n= 5 10 8 Donor characteristics Age (yrs) Underlying disease Age (yrs) Cause of death Age (yrs) Cause of death 1.5 FA 21 Cardiac (sudden) 59 UN 5 AHD 38 Cardiac (VF) 73 UN 13 AC 48 Cardiac (MI) 69 UN 14 HMSN 53 Resp. insuff. 47 UN 16 HMSN 36 Cardiac (MI) 72 UN 39 Cardiac (MI) 83 UN 41 Cardiac (MI) 73 UN 49 Cardiac (MI) 58 UN 53 Cardiac (VF) 52 Cardiac (MI) Age (mean) 9.9years 43 years 67 years (distribution) (range: 1.5 – 16 years) (range: 21-53 years) (range: 47– 83 years) Male : female 3 : 2 8 : 2 4 : 4 FA= fibula aplasia requiring a Syme amputation AHD= anterior horn cell disease with clubfoot deformity AC= Arnold-Chiari malformation with lumbar myelomeningocele and clubfoot deformity HMSN= hereditary motor and sensory neuropathy with clubfoot deformity VF= ventricular fibrillation MI= myocardial infarction UN= unknown due to medical ethical restrictions during acquisition of donor tissue

56 Chapter 5 Netherlands Bone Foundation (NBF). Investigations were performed in accordance with the guidelines set out in the Helsinki declaration.

Chondrocyte isolation and expansion The cartilage was digested overnight in Dulbecco’s modified Eagle Medium (DMEM) (42430, Invitrogen, Carlsbad, USA) containing 0.1% collagenase (CLS2, Worthington, Lakewood, USA) and 1% penicillin/streptomycin (15140, Invitrogen). Isolated chondrocytes were washed 3 times in phosphate-buffered saline (PBS) and expanded at 5000 cells/cm2 in monolayer in two passages in DMEM containing 1% penicillin/streptomycin, 10% foetal bovine serum (FBS, DE14-801F, Cambrex Bio Science Verviers, Belgium) and 10 ng/ml bFGF (223-FB, R&D Systems, Minneapolis, USA). At 80% confluency, cells were trypsinized and passaged. Pellets of 500.000 chondrocytes were snap-frozen in liquid nitrogen for RNA analysis. Cells at passage 2 were used for redifferentiation.

Redifferentiation culture As an in vitro model for regeneration, a 24 well transwell system was used as described previously200. After two passages, chondrocytes were washed 3 times in phosphate- buffered saline (PBS) and seeded on Millipore culture inserts (Millipore BV, Amsterdam, the Netherlands) with hydrophilic poly-tetrafluoroethylene (PTFE) membrane at 1.6 x106 cells/cm2 and cultured in DMEM supplemented with 2% ITSx (51500, Invitrogen), 2% ascorbic acid (A8960, Sigma-Aldrich, Zwijndrecht, the Netherlands), 2% human serum albumin (HS-440, Seracare Life Sciences, Milford, USA), 1% penicillin / streptomycin and 10 ng/ml TGF-β2 (302-B2, R&D Systems). The insert membranes had been coated with 0.125 mg/ml collagen type II in 0.1 M acetic acid (C9301, Sigma-Aldrich). Efficient collagen coating was verified by immunohistochemistry344. All filters were washed for 30 minutes with sterile PBS before seeding. Cells were cultured on the inserts for 28 days in duplicate as described earlier450. At 9 days of culture, 3 filters were used to measure 35 2− 35 SO4 incorporation (Na2 SO4, carrier-free; Perkin Elmer, Boston, MA, USA) in order to quantify proteoglycan incorporation as well as GAG and DNA content (method described below). The remaining filters were cultured for 28 days, after which the regenerated tissues were washed 3 times in phosphate-buffered saline (PBS) and fixed in 10% neutral buffered formalin or snap-frozen in liquid nitrogen and stored at -20 for GAG and DNA analysis or at -80 for RNA analysis.

Chondrogenesis by juvenile and adult talar chondrocytes 57 and organization of the extracellular matrix and cell morphology. The maximum score is 9, representing healthy hyaline cartilage.

GAG and DNA analysis Day 9 and day 28 in vitro neocartilage was digested in 2% papain (P3125, Sigma-Aldrich)

in 50 mM phosphate buffer, 2 mM N-acetylcysteine, and 2 mM Na2-EDTA (pH 6.5) at 65°C for 2 h. Part of the digest was used to measure DNA content and part was used for the quantification of glycosaminoglycan content as a measure of proteoglycan content, using an Alcian Blue precipitation assay. Glycosaminoglycans (GAGs) were precipitated from the tissue digests as well as from the culture medium (at each medium change) and stained with an Alcian blue dye solution (Alcian blue 8GX, 05500, Sigma-Aldrich), saturated in 0.1 M sodium acetate buffer, 440 containing 0.3 M MgCl2 (pH 6.2) for 30 min at 37°C . The blue staining of the medium was quantified by photospectrometry from the change in absorbance at 620 nm, using chondroitin sulphate (Sigma) as a reference. DNA was stained with the fluorescent dye Hoechst 33258 (Sigma) and fluorescence was measured on the Cytofluor (Bio-Rad) 208, using calf thymus DNA (Sigma) as a reference.

35 SO4 incorporation 35 2− 35 2− At day 9 of the culture, SO4 incorporation (Na2 SO4 , carrier-free; Perkin Elmer, Bos- ton, MA, USA) was measured in order to quantify proteoglycan incorporation by means 35 2− of a 4 h incubation in culture medium containing 20 µCi of SO4 . 3 separate filters per donor were used. Filters were then rinsed in plain culture medium during three changes and stored until analysis. Digestion of the sample for GAG and DNA analysis 35 2− was performed as mentioned earlier, followed by quantification of SO4 incorporation using a scintillation counter (Tri-carb 1900CA, Packard, Ramsey, MN). Results were 35 normalized to DNA content. Day 9 SO4 incorporation was measured in juvenile and adult talar cartilage only.

PCR For RNA isolation, all steps were performed on dry ice using instruments pre-treated with RNAzap and distilled water. After separation of the neocartilage from the transwell membranes using a scalpel (Swann-Morton, Sheffield England), the cartilage samples were crushed in a 2-ml Eppendorf tube and pestled using liquid nitrogen with RLT-mercapto- ethanol buffer and a 21G syringe until a homogenous mass was obtained. RNA isolation was done using the RNEasy Micro Kit (74004; Qiagen, the Netherlands) according to the manufacturer’s protocol using DNAse treatment. RNA concentration was measured spectrophotometrically using the NanoDrop® ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, USA), sufficient RNA purity was assumed if the 260/280nm

58 Chapter 5 ratio was higher than 1.9. Subsequently, 500 ng of RNA was reverse transcribed using the iScript cDNA synthesis Kit (Bio-Rad, Hercules, CA). RT-PCR was performed according to the manufacturer’s protocol using the following TaqMan® Gene Expression Assays (Ap- plied Biosystems): Collagen type I (Hs00164004_m1), Collagen type II (Hs00264051_m1), Collagen type X (Hs00166657_m1), Runx2/Cbfa1 (Hs00231692_m1, recognizes all three isoforms of Runx2), MMP 13 (Hs00942589_m1) and DDR2 (Hs00178815_m1), corrected for beta-actin (Hs99999903_m1).

Immunohistochemistry Immunohistochemical staining was performed for collagen types I, II and X, and core- binding factor α1 (Runx2/Cbfa1) as markers of hypertrophic- and re-and dedifferen- tiation. To this end, sections were incubated with PBS/BSA 5% (collagen type I, type II and X) and TBS/BSA 5% (Runx2/Cbfa1) to correct for aspecific binding, then incubated overnight at 4°C with monoclonal antibodies against collagen type I (20 μg/ml, CP-17, Calbiochem, USA), collagen type II (1:100, ascites II-II6B3, Developmental Studies Hy- bridoma Bank (DSHB), USA), collagen type X (1:50 X53, cell culture supernatant, Quar- tett, Germany) and Runx2/Cbfa1 (5 μg/ml, SC-101145, Santa Cruz, USA) in 5%PBS/BSA. Subsequently the samples were incubated with a biotinylated sheep anti-Mouse (1:200, RPN1001V, GE Healthcare, Belgium) followed by peroxidase labelled streptavidin (1:500, IM0309 Beckman Coulter) (Collagen I, X and Runx2/Cbfa1) or with goat anti Mouse HRP (1:100, P0447 DAKO, the Netherlands)(Collagen II) all for 1 hr at room temperature in 5% PBS/BSA. Diaminobenzidine was used to obtain a signal. Juvenile growth plate cartilage was used as a positive control for type X and Runx2/Cbfa1 production. Sections were incubated with isotype antibodies as negative controls. Finally, the sections were counterstained with haematoxylin.

Statistics All analysis was performed in technical duplicates except for RNA quantification, which was performed in triplicate. Statistical analyses were performed using the statistical software SPSS 20 for Windows (SPSS Inc., Chicago, Il, USA). Data were tested for normality using the Kolmogorov–Smirnov test and for homogeneity of variances using Levene’s test of equality of variances followed by ANOVA. Nonparametric comparisons were performed using the Wilcoxon signed ranks test. Spearman’s rank test was used for correlation analysis of cytokine with GAG and DNA levels at day 9 and day 28. After performing a Bonferroni correction, p≤0.05 (*) was considered statistically significant. All figures show mean values ± 95% confidence interval.

Chondrogenesis by juvenile and adult talar chondrocytes 59 Results

GAG content, production and release, and DNA content On day 9, GAG (p=0.006) and DNA (p=0.011) content, but not GAG/DNA, were higher for juvenile chondrocytes. On day 28, GAG, DNA and GAG/DNA did not differ between juvenile and adult chondrocytes. No differences were observed between adult femoral and adult talar chondrocytes [Figure 1A-C]. Cumulative GAG release (p=0.31), total produced GAGs (consisting of the sum of GAGs in the tissue at the end of culture with the amount of GAGs released into the medium, p=0.73), and release as percentage of total GAG production (p=0.73), did not differ between juvenile and adult chondrocytes. Cumulative GAG release (p=0.03) and total GAG production (p=0.03) were higher in adult femoral chondrocytes than adult talar chondrocytes [Figure 1-D].

120 GAG 18 DNA 16 100 14 80 12 10 60 p=0.011 p=0.006 8 40 6 DNA (ug/filter) DNA GAG(ug/filter) 4 20 2 0 0 day 9 day 28 day 9 day 28

400 GAG release juvenile talus 25 GAG/DNA 350 adult talus adult femur 20 300 250 p=0.03 15 200 p=0.03

10 GAG (ug) 150 100 5 GAG/DNA (ug/ug) 50 0 0 day 9 day 28 Total GAG release Total GAG production

Figure 1 A-D: GAG and DNA content, and GAG release of juvenile and adult talar chondrocytes, and of adult femur chondrocytes. GAG (fig. 1-A) and DNA (fig. 1-B) content at day 9 and 28 of juvenile and adult talar chondrocytes. At day 9, GAG (p=0.006) as well as DNA (p=0.011) content was higher in juvenile talar chondrocytes than in adult chondrocytes. At day 28, no differences were observed. No differences were observed in GAG, DNA or GAG/DNA (fig 1-C) between adult talus and adult femoral chondrocytes. Figure 1-D: GAG release during culture. Adult femoral chondrocytes had a higher release (p=0.03) and total production (p=0.03) of GAGs than adult talar chondrocytes. No differences existed between GAG release of juvenile and adult talar chondrocytes. Bars show means +/− 95% confidence intervals.

60 Chapter 5 35 SO4 incorporation To verify whether the increased GAG content in juvenile cultures was reflected in active 35 35 synthesis, on day 9, total SO4 incorporation and SO4 incorporation/DNA were determined. Both were higher in adult chondrocytes compared to juvenile chondrocytes (p<0.01) [Figure 2].

250 35-S incorporation 200 p<0.001 juvenile adult 150

100 (nmol/hr/DNA) 50 35-Sincorporation/DNA

0 day 9

35 2− Figure 2. SO4 (35-S) incorporation on day 9 was higher for adult talar chondrocytes than juvenile chondrocytes (p<0.001). Bars show means +/− 95% confidence intervals.

Immunohistochemistry A strong staining for collagen types I and II was observed in juvenile, adult talar as well as adult femoral in vitro engineered cartilage. Positive staining for Runx2/Cbfa1 was observed in all conditions. Staining for collagen type X varied between donors, however, overall weak to no staining was observed, and was independent of the cell type used for regeneration [Figure 3-A].

Bern score The Bern score of juvenile talar regenerated tissue was equal to adult talar regenerated tissue (p=0.92), and for both age groups this was lower than of femoral cartilage (p<0.001) [Figure 3-B].

PCR The expression of collagen type X, MMP13, Runx2 and DDR2 was higher in juvenile chondrocytes (Col X p=0.008, MMP 13 p=0.008, Runx2 p=0.002, DDR2 p=0.019) than in any other type of adult chondrocyte. The expression of collagen type I (p=0.06) and collagen type II (p=0.94) did not differ significantly between any of the cell sources. Adult talar chondrocytes displayed lower levels of collagen type X (p<0.001) and Runx2 (p<0.001) than adult femoral chondrocytes [Figure 4].

Chondrogenesis by juvenile and adult talar chondrocytes 61 Figure 3 A–B: Histological and immunohistochemical staining of regenerated neocartilage.

Figure 3-A: Safranin O and immunohistochemical staining for collagen types I, II, X and Cbfa1/Runx2 of in vitro engineered cartilage by juvenile talus, adult talus and adult knee chondrocytes after 28 days of culture. Femoral chondrocytes produced the highest quality cartilage according to the Bern score (below). No differences were observed between staining for collagen type I, type II, type X and Cbfa1/Runx2. Histological slides shown are representative of all donors. 9 Bern score 8 p<0.001 7 6 p=0.92 5 4 3 Bernscore 2 1 0 juvenile talus adult talus adult femur Figure 3-B: Bern score of juvenile and adult talus and adult femoral regenerated cartilage. Tissue engineered cartilage of femoral chondrocytes was of higher quality than of juvenile or adult talar chondrocytes (p<0.001). Bars show means +/− 95% confidence intervals.

62 Chapter 5 p=0.019 juvenile talus 10000.0 p=0.008 adult talus adult femur 1000.0 p=0.002 p<0.001

100.0 p<0.001

p=0.008 10.0

1.0

0.1 0 Col1 Col2 ColX MMP13 DDR2 Runx2 Expression, relative to femoral chondrocytes

Figure 4: RNA expression after 28 days of culture of juvenile talar and adult talar chondrocytes, relative to adult femur chondrocytes. Expression of Collagen type X (p=0.008), MMP13 (p=0.008), DDR2 (p=0.019) and Runx2 (p=0.002) were higher in juvenile chondrocytes than in adult chondrocytes. Adult talar chondrocytes displayed lower levels of collagen type X (p<0.001) and Runx2 (p<0.001) than adult femoral chondrocytes. Results are displayed relative to adult femoral RNA expression, on a logarithmic scale. Bars show means +/− 95% confidence intervals.

Discussion

In this study, we demonstrated that juvenile chondrocytes do not have better regenerative capacities than adult cells. Although in the first stage of regeneration increased matrix production was found through higher cell proliferation, at the end of the culture period this did not result in higher total matrix production. Surprisingly, at this early time point, the synthetic activity by adult chondrocytes appeared higher. This discrepancy could not be explained by a higher release at this time point. Several markers of hypertrophic differentiation (collagen type X, Runx2) were expressed at higher levels by juvenile chondrocytes, concomitantly with an increase in expression of MMP13 and DDR2. However, these altered expression levels were not reflected at the protein level as determined by histology or by immunohistochemical staining. Further, a clear histological difference was found between adult talar and adult femoral chondrocytes, with the latter producing a matrix more similar to hyaline cartilage, however, this was not reflected by proteoglycan content nor mRNA levels for hypertrophic differentiation markers. The higher initial proliferation rates of juvenile chondrocytes, accounting for the higher matrix content at the early stage in regeneration, are in line with previous data on 2D proliferation rates364. This proliferation-dependent increase in matrix production was also in accordance with previous findings for chondrocytes cultured in PEG gels378, although here the difference was noted at 28 days of culture, while in our study, the difference had disappeared by that time. Possibly the current 2-D dimensions of the

Chondrogenesis by juvenile and adult talar chondrocytes 63 regenerating tissue limited further cell growth, for example by competition for oxygen and nutrients38. The higher sulfate incorporation rate in adult cells at the initial stage of regeneration contrasted with the lack of difference in the amount of PGs deposited per cell at that time point, and could not be explained by an increase in GAG release in juvenile cultures. This may be explained by an increase in GAG release at this time point. However, there was no difference in release profiles at this time point, with overall release even appearing lower in adult cell culture, although this did not reach statistical significance. On the other hand, it should be borne in mind that labeled incorporation assays only provide very short term information, i.e. incorporation could have shown opposing effects before or after the moment of analysis. Increased gene expression for DDR2 and several hypertrophic markers, including Runx2, collagen type X and MMP13 was apparent in young talar chondrocytes while hardly detectable in adult cells. Overexpression of hypertrophic differentiation genes is related to cartilage degradation and osteoarthritis404, 467. As gene expression of Runx2 and collagen type X currently did not result in altered immunohistochemical staining in the young cells, it may be that other stimuli are required for an actual altered protein production to occur as is observed in osteoarthritis467. An alternative explanation for the increased hypertrophic differentiation gene expression may be that juvenile cells are more susceptible to the effects of collagen type II binding, which has been shown to promote pro-MMP-13 expression210. It must be noted that Runx2 has shown to be differentially spliced, with one transcript induced before the onset of chondrogenic differentiation, and the other one associated with hypertrophic differentiation and osteogenesis463. As the primers used in the current study could not distinguish between these isoforms, it cannot be excluded that the increase concerned the isoform related to the early stages of chondrogenesis. Another explanation for the differences found between studies comparing juvenile to adult chondrocytes, may be the cut-off age for defining ‘young’. Our juvenile group con- sisted of donors from 1.5 to 16 years of age, with an average of 9.9 yrs. An even younger cut-off age does not seem likely to make a difference, as no differences in cell density or GAG content were observed between fetal and young bovine chondrocytes399. For human chondrocytes, a greater increase in GAG production by juvenile chondrocytes after expansion in the presence of PDGF, TGF and bFGF compared to adult cells, was observed up to an age of 40 years21. Although expansion in the presence of bFGF and redifferentiation with TGF-β is a standard part of the currently used culture procedure, no differences were found. Differences between talar and femoral chondrocytes seemed greater than between juvenile and adult talar chondrocytes. Talar chondrocytes produced tissue of lower histological quality than femoral chondrocytes, although proteoglycan content did not

64 Chapter 5 differ. In contrast to our findings, increased GAG synthesis of talar chondrocytes has also been observed175. Also talar chondrocytes were described to be less responsive to catabolic stimuli than knee chondrocytes101, 225. However, this concerned freshly isolated rather than passaged cells and in addition the current study did not involve catabolic stimulation56. Given the decreased expression of both collagen type X and Runx2 in adult talar compared to adult femoral chondrocytes, it is tempting to speculate that this may explain the higher susceptibility of the femoral joint to osteoarthritis development compared to the talar joint76, 214. However, also here no effects were observed at the protein level. Possibly a second stimulus after elevation of Runx2 and collagen type X expression is required for the cells to actually enter the hypertrophic pathway. Other factors than those analyzed here, as for example cytokines and growth factors from the synovial fluid, may contribute to this process. Several considerations should be taken into account. First, it is not clear to what extent talus chondrocytes from clubfeet can be considered as normal, even though no histological abnormalities were found prior to culture (Appendix 1). The age of the current adult talar chondrocyte donors (mean age 43 years) was lower than of the femoral chondrocyte donors (mean age 67 years) and adult cartilage was of post-mortem donors. Despite this observation, apparently femoral chondrocytes still are more regenerative cells. As cartilage is not dependent on blood supply, and the cartilage of post-mortem donors was isolated within 8 hours of decease, it is not clear if adult chondrocytes would experience negative effects on GAG synthesis. At least activation of matrix degrading enzymes does not seem to be affected by a post-mortem delay of up to 24 hours347. Moreover, this again would have resulted in less rather than more cartilage formation by juvenile cells compared to adult cells.

In conclusion, juvenile chondrocytes may not have a larger regenerative capacity than adult chondrocytes, other than achieving regeneration at a faster pace. The initially higher GAG content of neocartilage from juvenile donors most likely is related to their higher proliferation rates, which is in line with previous observations. However, the per- sistent elevation of markers of hypertrophic differentiation in young talus cells, as well as elevated markers of chondrocyte degradation and absence of GAG and DNA differences after 28 days may indicate a different phenotype, which in the end may have implications for regeneration in vivo in combination with other cues, such as biomechanical loading and the presence of synovial fluid factors. Further investigation of the juvenile and adult joint environment may explain why a higher expression of hypertrophic differentiation markers does not lead to decreased matrix production.

Chondrogenesis by juvenile and adult talar chondrocytes 65 Appendix 1

Safranin O stained histological sections of 4 juvenile and 4 adult talar cartilage donors before chondrocyte isolation at 100x and 400x magnification.

66 Chapter 5 Cytokine profile of autologous conditioned serum for treatment of osteoarthritis, in vitro effects on cartilage metabolism and intra- articular levels after injection

Marijn Rutgers, Daniël B. Saris, Wouter J. Dhert and Laura B. Creemers

Arthritis Res Ther. 2010;12(3):R114 Abstract

Introduction: Intra-articular administration of autologous conditioned serum (ACS) recently demonstrated some clinical effectiveness in treatment of osteoarthritis (OA). The current study aims to evaluate the in vitro effects of ACS on cartilage proteoglycan (PG) metabolism, its composition and the effects on synovial fluid (SF) cytokine levels following intra-articular ACS administration.

Methods: The effect of conditioned serum on PG metabolism of cultured OA cartilage explants was compared to unconditioned serum. The effect of serum conditioning on levels of interleukin-1beta (IL-1β), IL-4, IL-6, IL-10, IL-13, interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), osteoprotegerin (OPG), oncostatin M (OSM), interleukin-1 receptor (IL-1ra) and transforming growth factor beta (TGF-β) were measured by multiplex ELISA. As TNF-α levels were found to be increased in conditioned serum, the effect of TNF-α inhibition by etanercept on PG metabolism was studied in cartilage explants cultured in the presence of conditioned serum. Furthermore, cytokine levels in SF were measured three days after intraarticular ACS injection in OA patients to verify their retention time in the joint space.

Results: PG metabolism was not different in the presence of conditioned serum compared to unconditioned serum. Levels of the anti-inflammatory cytokines IL-1ra, TGF-β, IL-10 as well as of pro-inflammatory cytokines IL-1β, IL-6, TNF-α and OSM were increased. IL-4, IL-13 and IFN-γ levels remained similar, while OPG levels decreased. TNF-α inhibition did not influence PG metabolism in cartilage explant culture in the presence of conditioned serum. Although OPG levels were higher and TGF-β levels were clearly lower in ACS than in SF, intraarticular ACS injection in OA patients did not result in significant changes in these cytokine levels.

Conclusions: ACS for treatment of osteoarthritis contains increased levels of anti-inflammatory as well as pro-inflammatory cytokines, in particular TNF-α, but conditioned serum does not seem to have a net direct effect on cartilage metabolism, even upon inhibition of TNF-α. The fast intraarticular clearance of cytokines in the injected ACS may explain the limited effects found previously in vivo.

68 Chapter 6 Introduction

Osteoarthritis (OA)-associated cartilage degradation is mediated in part by cytokines and growth factors, excreted into the intraarticular environment by synoviocytes, activated immune cells, or by the articular cartilage itself 143, 379. Therapies interfering with these cytokines may influence disease progression and improve the patient’s quality of life. A pivotal role in the progression of OA has been assigned to the pro-inflammatory cytokine interleukin-1β (IL-1β), which induces a cascade of inflammatory and catabolic events including the expression of cartilage degrading matrix metalloproteinases (MMP) 387, 141 nitric oxygen (NO) production and prostaglandin E2 (PGE2) release , while inhibiting proteoglycan and collagen synthesis 107, 381. The number of type-1 IL-1 receptors is significantly increased in OA chondrocytes 257 and synovial fibroblasts 351, increasing the susceptibility for IL-1α and IL-1β mediated effects. In addition, it was suggested that in OA synovium, a relative deficit in IL-1ra-production exists 379. As intraarticular administration of recombinant human interleukin-1 receptor antagonist has been shown to alleviate symptoms in several animal models of OA and rheumatoid arthritis 60, 118, 314, intraarticular treatment with IL-1ra was also suggested as a feasible treatment for patients with OA. One example of a disease-modifying osteoarthritis-drug (DMOAD) based on blocking the intraarticular effects of IL-1 associated with OA, is autologous conditioned serum (ACS or Orthokine®; Orthogen, Düsseldorf, Germany). Autologous conditioned serum (ACS) treatment consists of six repetitive injections of ACS over a period of 21 days. ACS is prepared from whole blood that is incubated in the presence of glass beads to initiate monocyte activation 12, 268. The resulting conditioned serum (ACS), has been shown to contain increased levels of IL-1ra as well as IL-4 and IL-10 268. In horses with arthroscopi- cally induced osteochondral defects, ACS treatment demonstrated a reduction in lame- ness and a decrease in synovial membrane hyperplasia 120. ACS treatment of human OA patients, however, demonstrated limited to moderate clinical effects 20, 449. Despite the fact that this approach has already been introduced in the clinic, the mechanisms by which administration of this product may result in reduction of OA symptoms is not yet fully understood 20, 120, 432. Although the primary goal of ACS treatment is alleviation of OA symptoms, one of the mechanisms may be enhancement of cartilage integrity through the inhibition of inflammatory activity, in particular with respect to Il-1 signaling. In fact, the direct effect of the entire blend of known and unknown factors present in ACS on cartilage metabolism in human OA cartilage has not been described. Moreover, only limited data are available on the actual composition of the conditioned serum. Besides IL-1ra, growth factors, such as transforming growth factor beta 1 (TGF-β1), which stimulates chondrocyte proliferation 45, 269, are upregulated during incubation 432. Of several pro-inflammatory cytokines like IL-1β, tumor necrosis factor-alpha (TNF-α) 252, 437 and IL-6 108, of the last of which also anti-inflammatory effects have been described 162, it is not

Autologous conditioned serum – in vitro characteristics 69 entirely clear if their levels remain equal or are upregulated during incubation 268, 432. As a consequence of monocyte activation during incubation of blood, anti-inflammatory cytokines such as IL-13, which was shown to inhibit the production of IL-1β and enhance production of IL-1ra 95, and osteoprotegerin (OPG) 216, which protected cartilage in a murine model of surgically induced osteoarthritis from further degeneration 375, may be upregulated. Also pro-inflammatory cytokines oncostatin-M (OSM) 220 and interferon- gamma (IFN-γ) 163 which act synergistically with IL-1β to stimulate production of MMPs and aggrecanases, may be upregulated together with the anti-inflammatory cytokines. Even if the composition of ACS would be favorable to cartilage regeneration, it is still unknown if the intraarticular injected cytokines are present long enough in the knee joint to exert their actions. The intraarticular availability of adequate levels of IL-1ra is important, as IL-1β is considered to be active at low concentrations and relatively high levels of IL-1ra are required to inhibit the effects of IL-1β 13. In vivo, increased IL-1ra levels were found in equine synovial fluid 35 days after the last (of four) injections with ACS 120. The current study aims to evaluate the direct in vitro effect of conditioned serum on cartilage proteoglycan metabolism, to further evaluate the composition of ACS and to examine to what extent intraarticular injection of ACS is reflected in cytokine level changes in human osteoarthritic synovial fluid.

Materials and methods

Preparation of conditioned serum To prepare conditioned serum, 35 ml of whole blood was acquired through venapunc- tion and aspirated in six polypropylene syringes (5 ml) containing glass beads (Orthogen, Düsseldorf, Germany). The syringes were incubated at 37°C for six hours. After incubation, the blood was centrifuged at 1,000 x g for 10 minutes, and serum was aspirated and stored at -80°C until further use. Control syringes containing whole blood (5 ml without glass beads) were centrifuged and serum was stored at -80°C.

Effect of conditioned serum on proteoglycan metabolism To measure the effects of conditioned serum on proteoglycan metabolism, 48 full thickness osteoarthritic cartilage explants were taken of the femoral condyles of OA patients undergoing a total knee arthroplasty (Kellgren-Lawrence grade III) and satisfying the OA criteria of the American College of Rheumatology 8. The explants were cultured in the presence of conditioned serum (n = 24) or non-conditioned control serum (n = 24) of healthy serum donors. The cartilage was washed, cut into cubes of approximately 3 x 3 x 3 mm, weighed and cultured for 16 days in Dulbecco’s Modified Eagles Medium containing 1% penicillin/streptomycin, 1% ascorbic acid (ASAP) and either 25% conditioned

70 Chapter 6 Table 1. Patient characteristics of cartilage explant experiments (CS, conditioned serum; OA, osteoarthritis). Experiment series Cartilage donor OA grade Cartilage explants/ Serum donor (Kellgren-Lawrence) condition control vs CS 56 year old male III 24 (48) 26 year old male 65 year old female IV 24 (48) 33 year old male 76 year old male III 24 (48) 30 year old male control vs CS vs 54 year old male III 8 (24) 27 year old male etanercept 55 year old male III 8 (24) 27 year old male 44 year old male IV 8 (24) 26 year old female

serum or 25% control (non-stimulated) serum. The experiment was repeated with two other OA cartilage and serum donor combinations (Table 1).

Effect of TNF-α inhibition by etanercept on proteoglycan metabolism in the presence of conditioned serum In another series of three experiments comparing the effect of conditioned serum and unconditioned serum on in vitro cartilage metabolism, Etanercept (Enbrel®, Wyeth Phar- maceuticals Inc., Collegeville, PA, USA) was added to full-thickness cartilage explants of femoral condyles of OA patients undergoing a knee replacement surgery and cultured in vitro. The explants were cultured with 25% control serum (n = 8), 25% conditioned serum (n = 8) and 25% conditioned serum with etanercept (1 μg/ml etanercept, n = 8). This concentration was based on a previous publication showing that this concentration was capable of inhibiting the activity of 40 ng/ml of TNFα 422. 35 35 2− S incorporation was measured by means of a four-hour incubation with SO4 , on Day 4 for all conditions (see below). The medium released on Days 4, 8, 12 and on Day 16 was analyzed for proteoglycan release, including release of newly synthesized PGs. The experiment was repeated with two other OA cartilage and serum donor combinations (Table 1).

35S incorporation

35 2− 35 At Days 4, 8 and 12 of the culture, SO4 incorporation (Na2 SO4, carrier-free; Perkin Elmer, Boston, MA, USA) was measured in order to quantify proteoglycan incorporation 35 2− by means of a four-hour incubation in culture medium containing 20 µCi of SO4 . For the control as well as for the conditioned serum cultured cartilage explants, eight separate cartilage explants were used for each incorporation time point. Explants were then rinsed in plain culture medium during three 45-minute changes, and the culture of these explants was continued in isotope-free medium until the end of culture on Day 16. At Days 4, 8, 12 and 16, conditioned media were collected to quantify the release of 35 2− newly synthesized PG. SO4 incorporation was quantified using a scintillation counter

Autologous conditioned serum – in vitro characteristics 71 (Tri-carb 1900CA, Packard, Ramsey, MN, USA), and results were normalized to DNA content and weight of the sample.

Alcian blue immunoprecipitation and DNA assay On Day 16, all cartilage explants were washed three times in phosphate-buffered saline (PBS) at 4°C. Explants were then digested in 2% papain (Sigma, St. Louis, MO, USA) in

50 mM phosphate buffer, 2 mM N-acetylcysteine, and 2 mM Na2-EDTA (pH 6.5) at 65°C for two hours. Part of the digest was used to measure DNA content and part was used for the quantification of the glycosaminoglycan content as a measure of proteoglycan content using an Alcian Blue precipitation assay (described below). Another part was 35 2− used to measure SO4 activity. Glycosaminoglycans (GAGs) were precipitated from the explant digests as well as from the culture medium and stained with an Alcian blue dye solution (Alcian blue 8GX, Sigma-Aldrich, Zwijndrecht, The Netherlands), saturated in 0.1M sodium acetate buffer, 440 containing 0.3M MgCl2 (pH 6.2) for 30 minutes at 37°C . The blue staining of the medium was quantified photospectrometrically from the change in absorbance at 620 nm, using chondroitin sulphate (Sigma) as a reference. DNA was stained with the fluorescent dye Hoechst 33258 (Sigma) and fluorescence was measured on the Cytofluor (MTX Lab Systems, Vienna, VA, USA)208, using calf thymus DNA (Sigma) as a reference.

Composition of autologous conditioned serum (ACS) Whole blood was obtained from 22 OA patients meeting the American College of Rheumatology criteria for OA (mean age 52 years, range 35 to 72). ACS for intraarticular treatment was prepared by whole blood incubation in the presence of ACS-specific glass beads. Unconditioned serum was taken as control.

Multiplex ELISA Multiplex ELISA was used for measurement of cytokine levels in conditioned and unconditioned serum and in SF. Earlier validation studies showed high correlation of multiplex ELISA readings with conventional ELISA 94 and demonstrated that multiplex ELISA is suitable for SF analysis 93. The cytokines measured were IL-1β, IL-4, IL-6, IL-10, IL-13, IFN-γ, OSM and OPG. Measurements and data analysis were performed using the Bio-Plex system in combination with the Bio-Plex Manager software version 3.0 using five parametric curve fitting (Bio-Rad Laboratories, Hercules, CA, USA). Coating antibodies for IL-1β, IL-6, IL-10 and TNF-α were provided by Strathman Biotec (Hannover, Germany); coating antibodies for IL-4, OPG and OSM by R&D Systems (Abingdon, UK), coating antibody for IL-13 by National Institute for Biological Standards and control (Potters Bar, UK) and coating antibody for IFN-γ by BD Biosciences (San Diego, CA, USA). The recombinant proteins for IL-1β, IL-6, IL-10 and IL-13 were provided by Sanquin (Amsterdam, The Netherlands), for

72 Chapter 6 IL-4 and IFN-γ by eBioscience (San Diego, CA, USA), for TNF-α by BD Biosciences, for OPG by R&D Systems (Abingdon, UK) and for OSM by Biocarta (Hamburg, Germany).

Preparation of recombinant cytokine mixes, covalent coupling of the captured antibodies to the microspheres and preparation of detection antibodies were performed as described previously 93, 94. For determination of cytokine profiles in SF, aliquots of 200 µl were first pre-treated with 20 µl of hyaluronidase (0.5 mg/ml, type IV-S, Sigma-Aldrich, Zwijndrecht, The Netherlands) for 30 minutes at 37°C, spun over 0.22 µm nylon membrane (Spin-X column; Corning, The Netherlands) and diluted with High-Performance ELISA-buffer (Sanquin Blood Supply Foundation, Utrecht, Netherlands) at a 1:2 dilution. Recombinant protein standards and calibration curves were prepared in serum diluents (R&D Systems). A mix containing 1,000 coupled microspheres per cytokine (total volume of 10 µl/well) was added to the standard, sample or blank, and incubated for 60 minutes. Next, a 10 µl mix of biotinylated antibodies (final concentration 16.6 µg/ml for each antibody) was added to each well and incubated for an additional 60 minutes. Beads were washed in PBS containing 1% BSA and 0.5% Tween 20 (pH 7.4) in order to remove residual sample and unbound antibodies. After incubation for 10 minutes with 0.5 µg/ml streptavidin R-phyco- erythrin (BD Biosciences) and washing twice with 1% BSA and 0.5% Tween 20 (pH 7.4), the fluorescence intensity of the beads was measured in 100 µl High Performance ELISA buffer (Sanquin). Measurements and data analysis were performed using the Bio-Plex system in combination with the Bio-Plex Manager software version 3.0 using five parametric curve fitting (Bio-Rad Laboratories). To eliminate the possibility of inter-assay variability, control and conditioned serum samples were measured in duplo in the same assay.

ELISA IL-1ra and TGF-β1 levels were measured using commercially available ELISA kits (Quanti- kine®, DRA00 and DB100B, R&D Systems), following the manufacturer’s protocol.

Analysis of cytokines in synovial fluid after ACS injection Twenty-two OA patients were treated with six consecutive injections of ACS at Days 0, 3, 7, 10, 14 and 21, according to the ACS treatment schedule. To this end, a 21 gauge needle was inserted into the knee joint through a lateral supra-patellar approach. After aspiration of the SF, 2 ml of ACS was injected into the joint through a 0.22 mm sterile nitrocellulose filter (Millex®, Millipore Express, Carrigtwohill, Co. Cork, Ireland). The knee was flexed and extended manually to ensure thorough distribution of the serum throughout the joint. Within 30 minutes after aspiration, the aspirated SF was centrifuged for 10 minutes at 1,000 x g and the aspirate and the residual serum samples were stored at -80°C until further analysis. Treatment of patients with ACS was performed in compliance with the Helsinki Dec- laration. Written informed consent was given by all participants, and approval by the

Autologous conditioned serum – in vitro characteristics 73 Medical Ethics Committee (University Medical Center Utrecht, The Netherlands; trial registration ID 03-232/G-O) was obtained before initiation of the trial.

Statistical analysis SPSS version 15.0 for Windows (SPSS Inc., Chicago, IL, USA) was used for data analysis. Paired t-tests were used to compare cytokine levels in ACS and control serum of OA patients (n = 22 patients), and independent t-tests were used to compare PG content, DNA content and PG/DNA for each of the in vitro experiments. Analysis of variance (ANOVA) was performed on the pooled data of the experiments, with randomized block design to correct for inter-donor variability. Repeated measurement analysis was used to identify changes in SF cytokine levels during treatment. Comparisons between different treatments (control, ACS, Etanercept) were followed by a Bonferroni correction. P-values less than 0.05 were considered statistically significant. Graphs show mean values with standard deviation (SD).

Results

Proteoglycan metabolism of cartilage explant culture An average of 40% of explant proteoglycan (PG) was released into the culture medium in 25% conditioned serum or in 25% control serum (Figure 1). PG release, PG content at the end of the culture, nor 35S incorporation on Days 4, 8 or 12 differed between OA cartilage explants cultured in either condition, measured with independent t-tests and ANOVA (Figures 1 and 2).

9 Control 7 Control 8 CS 6 CS 7 5 6 5 4 4 3 3 (nmol /hr*mg) (nmol 2

2 35-Sincorporation 1 1 0 0 ug GAG release ug cartilage mg / day 4 day 8 day 12 day 16 day 4 day 8 day 12 Figure 1‑A. Proteoglycan release during culture of Figure 1‑B. PG incorporation rate on Days 4, 8 and 35 2− cartilage explants. A similar amount of proteogly- 12 of the culture, measured using SO4 incorpora- cans was released into the culture medium by ex- tion (n = 8 per timepoint). Results are representa- plants cultured with unstimulated (n = 24) or condi- tive of three separate experiments with different tioned serum (CS; n = 24). OA cartilage donor – serum donor combinations (mean +/− SD).

74 Chapter 6 25 0.20

20 0.15 15 0.10 10 0.05 5 ug GAG / mg cartilage GAG mg / ug ug DNA cartilage mg ug / 0 0.00 Control CS Control CS

200

150 Figure 2. Proteoglycan and DNA content during culture of cartilage explants (mean +/− SD). 100 Proteoglycan and DNA content of cartilage explants after culturing with unconditioned serum 50 (n = 24) or conditioned serum (CS, n = 24 cartilage DNA GAG ug / ug explants). (a) Proteoglycan content. (b) DNA con- 0 tent, (c) PG / DNA ratio. Control CS

Proteoglycan metabolism upon TNF-α inhibition by etanercept Addition of etanercept to conditioned serum or control serum did not alter PG release, PG incorporation and final PG or DNA content after culture measured with independent t-tests and ANOVA (Figures 3 and 4).

4.0 Control 4.5 CS 4.0 Etanercept 3.5 3.0 3.0 2.5 2.0 2.0 1.5 1.0 1.0

(nmol/hr*mg cartilage) (nmol/hr*mg 0.5 35-S incorporation day 4 35-Sincorporation 0.0 0.0

GAG release (ug) / mg cartilage GAG mg release/ (ug) day 4 day 8 day 12 day 16 Control CS Etanercept Figure 3‑A. Proteoglycan release and incorporation Figure 3‑B. Proteoglycan incorporation on Day 4, 35 2− (mean +/− SD) in the presence of unconditioned se- measured by SO4 incorporation (n = 8). Results rum (control, n = 8), conditioned serum (CS, n = 8) or are representative for three separate experiments conditioned serum with Etanercept (n = 8). with different OA cartilage donor – serum donor combinations.

Autologous conditioned serum – in vitro characteristics 75 25 0.30

20 0.25 0.20 15 0.15 10 0.10 5 0.05 ug DNA cartilage mg ug / ug GAG / mg cartilage GAG mg / ug 0 0.00 Control CS Etanercept Control CS Etanercept

250

Figure 4. Proteoglycan and DNA content during 200 culture in unconditioned, conditioned serum or conditioned serum with Etanercept (mean +/− 150 SD) Proteoglycan (PG) metabolism in the pres- 100 ence of unconditioned serum (control, n = 8), conditioned serum (CS, n = 8) or conditioned serum DNA GAG ug / ug 50 with etanercept (n = 8). (a) PG content, (b) DNA 0 content, (c) PG / DNA ratio. Control CS Etanercept

Cytokines in unconditioned serum and ACS Serum levels of IL-10 and IL-1ra increased after conditioning (3.0-fold and 7.9-fold, respectively) (P <0.01). Of the other anti-inflammatory cytokines, TGF-β1 was upregulated (14.9-fold) and OPG was downregulated (2.8-fold) (both P <0.001). Pro-inflammatory cytokines IL-1β, OSM and TNF-α were upregulated (20.9-fold, 2.9 fold and 10.2-fold, respectively) (all P <0.01) while IFN-γ levels did not change. IL-6 levels were upregulated

100000 control ** ACS

10000 **

1000 ** **

* 100 ** ** log (pg/ml) log

cytokinelevel 10 **

0 1 4 3 β 6 γ α ra 1 β - 1 1 - -1 - PG IL - - IL SM IL F- O IL IL O IL IFN- TNF- TG Figure 5. Cytokine levels in serum of 22 OA patients, before incubation (control) and after six hours of incubation in the presence of glass beads (ACS). Note the increase in anti-inflammatory cytokines (IL-1ra, TGF-β1, IL-10) and pro-inflammatory cytokines (IL-1β, IL-6, IFN-γ, OSM, TNF-α) after incubation. OPG levels were decreased. All values are displayed as mean ± SD in pg/ml. * P <0.01; ** P <0.001.

76 Chapter 6 19.3 fold (P <0.001). IL-4 levels and IL-13 levels were below detection limits in non- stimulated serum as well as in ACS (Figure 5).

Cytokines in synovial fluid before and after treatment Sufficient amounts of SF for all treatment time points were available for analysis in 14 patients. To verify whether this implied a bias in the ensuing experiments, clinical grade of OA and baseline serum cytokine levels were compared between this group of patients and the eight patients from whom no SF could be aspirated. No statistically significant differences between these patients and the other group of eight patients were noted (Table 2).

Table 2. Clinical scores and serum cytokine levels in patients with sufficient and with insufficient SF for analysis. Pre-treatment clinical OA and baseline serum characteristics (mean +/− SD) of patients whose synovial fluid were analyzed, were similar to those of patients whose synovial fluid were not analyzed due to insufficient amounts of SF in the knee at time of aspiration. KOOS, Knee and Osteoarthritis Outcome Score; KSCRS, Knee Society Clinical Rating Scale; SF, synovial fluid; OA, osteoarthritis; IL-4, interleukin-4; IL-6, interleukin-6; IL-10, interleukin-10; IL-13, interleukin-13; IFN-γ, interferon gamma; OSM, oncostatin M; TNF-α, tumor necrosis factor alpha; OPG, osteoprotegerin; IL-1ra, interleukin-1 receptor antagonist. KOOS KSCRS Baseline serum cytokine levels score score IL-1 IL-4 IL-6 IL-10 IL-13 TNFα IFNγ OSM OPG IL-1ra SF available and analysed 46 79 0.2 0.0 8.5 1.5 0.0 0.0 2.2 13 376 180 (14 patients) (10) (19) (0.7) (0.0) (28) (2.7) (0.1) (0.2) (2.2) (27) (128) (137) No SF available 51 75 0.2 0.0 0.1 0.1 0.1 14 16 14 371 276 (8 patients) (15) (15) (0.6) (0.0) (0.2) (0.2) (0.2) (29) (44) (29) (229) (288)

Levels of IL-1β, IL-4, IL-13, TNF-α, and IFN-γ were low or undetectable in SF before and during treatment with ACS. IL-6, OSM, OPG, IL-10, TGF-β and IL-1ra were detectable in synovial fluid, but only OPG and TGF-β levels differed significantly from ACS levels. The levels of OPG in SF at baseline were higher than in ACS (14,476 pg/ml vs. 134 pg/ml, P <0.001), but had not changed significantly three days after injection of the serum. Baseline synovial fluid TGF-β levels were lower than in ACS (580.7 vs. 21,670.9 pg/ml, P <0.001), but did not change significantly after ACS injection either (Figure 6).

Discussion

Disease-modifying drugs for conservative treatment of osteoarthritis have proven effective in a variety of randomized controlled clinical trials 72, 195. Although autologous conditioned serum (ACS, Orthokine®) proved slightly to moderately effective for alleviation of OA symptoms up to two years after treatment in human OA patients 20, 449, many aspects of this therapy have remained unclear so far.

Autologous conditioned serum – in vitro characteristics 77 100000 IL-1raIL-1raIL-1ra IL-1raTGF-TGF-βTGF-β βTGF-βIL-10IL-10IL-10IL-10 IL-6IL-6IL-6IL-6 OSMOSMOSMOSM OPGOPGOPGOPG

ACS ACS ACS SF SFSF → SF → ** ** 10000

1000

** 100 ** ACS and SF and ACS log log (pg/ml) Control serum, Control cytokine levels (pg/ml) levels cytokine 10

synovial fluid cytokinesynovialfluid level * 1 * * * * 0 3 7 10 14 21 Control0ACS00 3 3 3Synovial 7 7 7Fluid (day 10 10of 10aspiration) 14 14 14 21 21 21 DayDay Dayof of aspiration ofaspiration aspiration

Figure 6. Control serum (control), autologous conditioned serum (ACS) and synovial fluid cytokine levels (mean +/− SD) of IL-1RA, TGF-β1, IL-10, IL-6, OSM and OPG during treatment with ACS in 14 OA patients. The large symbols next to the y-axis correspond to the levels of these cytokines in control serum and the injected ACS. TGF-β1 levels in SF were lower than in the injected ACS, and OPG levels in SF were higher than in ACS (P <0.01). During the course of treatment, no significant changes in cytokine levels occurred despite repeated ACS injection (SF was aspirated before each of the six injections with ACS, at t = 0, Day 3, Day 7, Day 10, Day 14 and Day 21).

In vitro, conditioned serum does not seem to have a direct effect on cartilage metabolism compared to unstimulated serum. In line with earlier studies, IL-1ra levels of ACS in the current study were upregulated, although the reported relative increases in conditioned serum differed an order of a magnitude with those from the current study 268, 432. Also IL- 10 levels were upregulated two-fold as found earlier 141, but IL-4 was hardly detectable. It is not known to what extent this is related to the change in the manufacturer’s protocol, in which the conditioning period is reduced to six hours 432, as opposed to the 24 hours initially included in the preparation protocol 120, 449. Allegedly, most of the cytokine production occurs after six hours. Moreover, although this has not been argued as such by the manufacturer, long incubation periods at body temperature are known to reduce the bioactivity of most cytokines, while their immunoreactivity as determined by ELISA is still retained 92. In particular Il-10, one of the anti-inflammatory cytokines upregulated in ACS, has been shown to have a half-life of several hours under these conditions 389. This may also represent another explanation for the limited effects found in vivo thus far. More important, however, pro-inflammatory cytokines, in particular IL-1β and TNF-α, were found to be significantly upregulated in the current ACS study, in contrast to previous results 432. As, unlike for IL-1, the increased TNF-α levels were not counterbalanced by an increase in levels of natural inhibitors, and TNF-α has been postulated to have degenerative effects on cartilage 160, 437, this may have explained the limited effects found in OA patients treated with conditioned serum. However, blocking the action of TNF-α 422

78 Chapter 6 did not result in a net positive effect of conditioned serum on matrix metabolismin vitro, suggesting that, if any, in vivo effects of the TNFα in the injected ACS would have been indirect. It is not clear to what extent the increased levels of IL-6, OSM and lower levels of OPG in conditioned serum may have had a pro-inflammatory effect, but as conditioned serum addition did not result in decreased sulphate incorporation after four days of cartilage explant culture, or a lower PG content after 16 days of culture, conditioning of serum is not likely to have any effect on OA cartilage. These findings were strength- ened by the large number of explants used per experiment, and by repeating both experiments in a total of six different OA donors. Nevertheless, it cannot be excluded that factors present in conditioned serum, either known or as yet undiscovered, play a role in vivo by inducing other mediators, not determined in the current study, in the joint space. With respect to the role of Il-1 signaling in OA, in the one human clinical study using recombinant IL-1ra as a treatment for OA, a single injection into the knee joint did not result in an improvement of OA symptoms 193. This may have been due to fast clearance from the joint space. Injection of ACS led to an increase of IL-1ra SF levels in osteoarthritic equine knee joints during ACS treatment in vivo 120. However, in the current study, IL-1ra levels did not increase during the course of the treatment, even though the interval between injection and measurement was shorter than in the former study (3 days vs. 7 and 35 days 120). The fast clearance of injected cytokines from the joint found in the current study suggests that any in vitro net effect would still have been difficult to reproduce in vivo. Continuous intraarticular availability of IL-1ra may be more effective. In vivo injection of synoviocytes transduced with the IL-1ra gene into a canine knee joint after sectioning of the anterior cruciate ligament 314 and intraarticular injection of IL-1ra plasmid into a rabbit knee joint after meniscectomy resulted in reduction of OA clinical symptoms (histological parameters, preservation of articular cartilage quality) 106. Even- tually these long term approaches may be more effective than the limited number of injections of this treatment, but currently they are not practically feasible in a clinical setting. Nevertheless, even if IL-1ra levels are increased in the synovial fluid in vivo, it is uncertain if these IL-1ra levels correlate with OA symptoms or disease progression, as the ratio of IL-1ra to IL-1β in the SF of human OA subjects were shown not to correlate with pain or with the Lequesne OA index 328. With respect to the upregulation of IL-1β, its role in progression of OA may actually be disputed 68. In our study, IL-1β levels in OA SF were extremely low, which is in line with previous reports 94, 361, 416. Although there are studies in which IL-1β inhibited proteoglycan synthesis in vitro at concentrations as low as 10 pg/ml 176, commonly IL-1β concentrations of at least 1,000 pg/ml are used to induce detectable cartilage damage 235, 372. Studies demonstrating synergistic effects of IL-1β with IFN-γ, TNF-α, IL-17 or Oncostatin- M 98 also departed from IL-1β concentrations much higher than detected in OA synovial fluid and hence synergistic effects of low IL-1β levels with other cytokines in the current

Autologous conditioned serum – in vitro characteristics 79 study do not seem likely. Moreover, the baseline IL-1ra levels in the SF of the currently studied OA patients were already in the effective range to block IL-1β 13. Although it may be argued that the patients with sufficient amounts of SF may have differed from the group as a whole, this is contradicted by the observation that serum cytokine levels as well as the clinical response of the patients with sufficient amounts of SF were similar to the patients with insufficient amounts of SF. A high standard deviation in synovial fluid cytokine levels was encountered, as is common in OA. Increasing the number of subjects may decrease this standard deviation and possibly enable subgroup analysis (for example, by progression of OA according to radiological parameters (dGEMRIC) or by clinical parameters (KOOS scores). However, as the group of patients was already small, this would have even further reduced the likelihood of finding statistically significant changes. Future evaluation of intraarticular cytokine changes following ACS injection might include SF analysis earlier after injection, which would give further insight on intraarticular half-life. Also, effects of ACS on synovium, either alone or in coculture with cartilage explants may be studied. Even though multiplex ELISA showed good to excellent correlation with ELISA 94, separate ELISAs may give slightly more accurate information about absolute cytokine levels. Although the present in vitro data show no effect of ACS and a short intraarticular half life, a recent clinical study demonstrated a two-year lasting improvement of ACS treatment compared to hyaluronic acid and placebo treatment20. However, it must be noted that the treatment regimens differed, with six injections of ACS being compared to three injections of hyaluronic acid or placebo. Moreover, as none of the clinical trials carried out so far included unconditioned serum as a placebo, it can actually not be excluded that injection of serum without prior conditioning per se has a beneficial effect on proteoglycan metabolism in vivo. In conclusion, ACS is a mix of counteracting growth factors and cytokines that does not have a direct effect on cartilage metabolism and probably has a minimal influence in the joint space, given the fast disappearance of cytokines from the synovial fluid after injection. Development of new intraarticular therapies may focus on their prolonged presence in the joint space.

80 Chapter 6 Response to: Cytokine profile of autologous conditioned serum for treatment of osteoarthritis, in vitro effects on cartilage metabolism and intra-articular levels after injection

Carsten Moser

Arthritis Res Ther 2010, 12:410 We welcome the interest of Rutgers and colleagues343 in providing additional information on the mode of action of autologous conditioned serum (ACS). Studies such as the one they published in a recent issue of Arthritis Research & Therapy are important because the Orthokine ACS treatment (Orthogen, Düsseldorf, Germany) has been shown to be safe and effective in a number of clinical studies and is used widely in Europe. Randomized controlled trials have confirmed the efficacy of this treatment in osteoarthritis of the knee and lumbar radiculopathy. Additional human studies show promise in treating muscle injury and tunnel widening after anterior cruciate ligament (ACL) reconstruction. All clinical and preclinical studies confirm an excellent risk-to-benefit ratio. We would like to offer some comments concerning the paper of Rutgers and colleagues. The method of producing ACS has been carefully optimized to enrich for anti-inflammatory cytokines, such as interleukin (IL)-1Ra, IL-10, and IL-13, while keeping low the concentration of inflammatory cytokines, such as IL-1β and tumor necrosis factor-alpha (TNF-α). The ACS preparation of Rutgers and colleagues had increased concentrations of IL-1β and TNF-α, and this raises questions about the composition of the product that was tested. Nevertheless, despite the elevated concentrations of these two cytokines, there was no adverse effect of ACS on proteoglycan turnover in the cartilage explant cultures. This suggests that anti-inflammatory and possibly chondroprotective ingredients within ACS pre dominate. Further studies using additional controls and autologous instead of heterologous serum would be interesting. The third component of the study by Rutgers and colleagues measured cytokine levels in synovial fluids before and after treatment with ACS. No changes were found, but it should be noted that Orthokine should be used only when knee effusion has been removed effectively. The described possible aspiration of synovial fluid after treatment with ACS raises questions about the selection of these patients or the composition of the injected ACS. In this regard, it is worth noting that another study86, 87 involving the injection of Orthokine ACS into knees after ACL plasty showed evidence of reduced IL-1β content in synovial fluid. This reduction corresponded with improved pain and function and with reduced tunnel widening. Clearly, the mechanisms through which ACS brings about clinical improvement are incompletely understood and should be the subject of additional research.

Autologous conditioned serum – in vitro characteristics 81 Response to: Cytokine profile of autologous conditioned serum for treatment of osteoarthritis, in vitro effects on cartilage metabolism and intra-articular levels after injection – authors’ reply

Marijn Rutgers, Daniël B Saris, Wouter J Dhert and Laura B Creemers

Arthr Res Ther 2010, 12:411 We thank Moser for his comments282 on the paper343 we published in a previous issue of Arthritis Research & Therapy. To dispel any concerns about the proper use of the product, the autologous conditioned serum (ACS) in this study was prepared in a GMP (good manufacturing practice) facility in strict accordance with the guidelines supplied by the manufacturer (Orthogen, Düsseldorf, Germany) and was injected six times at 3-day intervals in accordance with the instructions given. As recommended, immediately before injection of ACS, synovial fluid was carefully aspirated to minimize ACS dilution. This synovial fluid was used to determine the cytokine levels before and during ACS treatment. We showed that, 3 days after injection, cytokine levels were at baseline and had no secondary effects on other cytokines. This finding is not in conflict with that of Darabos and colleagues87, whose publication is cited by Moser; upon careful reading of that publication, none of the effects of ACS injection, including the alleged decrease in synovial fluid levels of interleukin-1 (IL-1), turned out to be statistically significant. We certainly agree with Moser that in vitro and in vivo studies should be well controlled. However, in the case of ACS, the use of an autologous product is not required; this autologous product is devoid of cells, which are the only factors capable of evoking an immune response upon transfer of human materials to human recipients. We have used unconditioned serum as controls in our in vitro studies because serum in general seems to be more amenable to cartilage health than either saline or the synovial fluid the tissue is exposed to in vivo452. In this setup, we could not demonstrate any effect of conditioning the serum. The observation that IL-1 and tumor necrosis factor-alpha levels were upregulated in ACS, in contrast to the levels found previously upon characterization of ACS, may be due to the use of healthy subjects in the latter case, whereas we characterized the ACS prepared from osteoarthritis (OA) patients, the intended target population. Blood from OA patients was recently shown to contain cytokine profiles different from those of healthy subjects, suggesting a differential immune status70. Unconditioned serum, despite being the best control for ACS, has never been used in any in vivo study or clinical trial. Until now, the trials carried out with ACS have either used saline449 or compared ACS with other treatments. However, the number of injections per treatment type was always dissimilar in these latter trials. For example, in

82 Chapter 6 the OA trial carried out by Baltzer and colleagues20, six injections of ACS yielded more clinical improvement than three injections of hyaluronic acid, but the effect of multiple lavage sessions removing proinflammatory cytokines present in the synovial fluid cannot be ruled out here. In addition, concerns about the blinding of the patients to their treatment may be raised. In particular, in OA, more invasive treatment shows a stronger placebo effect than non-invasive therapies do465. It is questionable whether ACS has any positive effects with regard to in vitro and in vivo chondroprotection and clinical outcome. This notion is further corroborated by the fact that ACS therapy (Orthokine; Orthogen), to our knowledge, is not registered in any European country and that its ‘wide use’ is limited to clinical trials and some private clinics. We would encourage investigators to set up ACS-based randomized controlled trials with full patient and observer blinding and with unconditioned serum as control in order to provide a final answer to whether this treatment merits registration.

Autologous conditioned serum – in vitro characteristics 83

Osteoarthritis treatment using Autologous Conditioned Serum – comparison of clinical results to earlier placebo treatment

Marijn Rutgers, Laura B. Creemers, K. Gie Auw Yang, Natasja J. Raijmakers, Wouter J. Dhert, Daniël B. Saris

Submitted Abstract

Background and purpose: Autologous Conditioned Serum (ACS) is a disease-modifying drug for treatment of knee osteoarthritis. It is unclear if earlier demonstrated clinical improvement after ACS-treatment outweighs the frequent intra-articular injections, as placebo treatment proved nearly as effective. The authors hypothesized that treatment with ACS after placebo would result in an even greater improvement of osteoarthritis symptoms than placebo-treatment alone.

Methods: 20 out of 74 patients treated with placebo in the course of a previous trial, were now treated with ACS and followed for 12 months. Clinical improvement of the 20 ACS-treated patients was measured using knee-specific clinical scores, as was ‘response shift’. Patients that did not opt for further treatment were interviewed about their mo- tives.

Results: Pain from injections, time involved in treatment and limited proven effectiveness withheld patients from undergoing additional ACS-treatment after placebo. Although the Visual Analogue Score for pain (VAS) did improve early after treatment (p=0.02), clinical results one year after ACS-treatment were similar to those after placebo- treatment. Response shift measurement demonstrated adaptation of the osteoarthritis patients to their disease (Knee Injury and Osteoarthritis Outcome Score, p=0.02).

Interpretation: Intra-articular osteoarthritis treatment with ACS áfter placebo did not result in greater clinical improvement than placebo-treatment only. For a large number of placebo-treated patients, the combination of limited effectiveness and the laborious nature of ACS therapy were important motivators to refrain from ACS-treatment. Considering its limited advantage over placebo, critical consideration of ACS application seems required.

86 Chapter 7 Introduction

Effective conservative treatment of osteoarthritis (OA) of the knee should improve symptoms of osteoarthritis and enhances quality of life. Both oral-, topical and intra- articular therapies for treatment of OA have been studied in detail in a variety of prospective randomized clinical trials69, 72, 195, 263, 317. In particular, disease-modifying OA drugs (DMOAD) have gained attention. Autologous conditioned serum (ACS, Orthokine®) is one of these DMOADs. ACS treatment demonstrated to lead to a limited improvement of OA symptoms in two randomized controlled trials19, 449. ACS therapy consists of 6 consecutive intra-articular injections with autologous serum, after incubation of whole blood in the presence of glass beads for 6 hours. The proposed working mechanism of the resulting product is by intra-articular inhibition of the pro- inflammatory cytokine interleukin-1 (IL-1) through injection of autologous incubated serum containing increased levels of IL-1 receptor antagonist (IL-1ra)268. Until now, ACS has been studied in two human19, 449 and one equine study120. The results of these trials are in favor of ACS, however the differences between placebo and ACS treatment are either marginal, or the study design does not enable adequate comparison of treatment results. As the therapy is time-consuming for patients as well as physicians, and as clinical relevance of the effect and understanding of the exact biological mechanisms of this therapy remain disputed120, 343, 449, the applicability of this product should be studied more extensively. The placebo-treated patients of an earlier clinical trial449 formed an ideal cohort to address this question, and in the current trial these patients were given the option to receive the ‘proven effective’ ACS. To correct for an inevitably occurring placebo bias265, in addition to the Knee Injury and Osteoarthritis Outcome Score (KOOS338), Knee Society Clinical Rating Scale (KSCRS182), Western Ontario and McMaster Universities Osteoarthri- tis Index for OA (WOMAC28) and Visual Analogue Score for pain (VAS322), the ‘response shift’ tool was used, which corrects for the patient’s adaptation to a decline in health status due to ‘disease-related’ disability218. While aiming at further investigating the effects of ACS and distinguishing treatment- related effects from placebo effects, we hypothesized that a similar or even greater improvement would be achieved when these patients were treated with ACS. We further evaluated how patients had experienced their earlier intra-articular placebo treatment in case they did not opt for ACS treatment. In addition to clinical scores, response shift was measured to examine the degree of adaptation to OA.

Autologous conditioned serum – comparison to earlier placebo 87 Methods

Study design All placebo-treated patients who completed follow-up in a previous ACS trial449 (n=74) were informed about their placebo treatment as well as about the results of the trial by mail, explaining that a ‘moderate’ effect of ACS treatment was seen as compared to placebo. These patients were given the option to participate in a new trial, in which they would now be treated with ACS, and patients who decided not to participate were asked to motivate their decision. A power analysis was performed on the participating patients. The study was performed at the University Medical Center Utrecht according to the guidelines set out in the Helsinki declaration, and was approved by the local medical ethics committee (Extension to public trial register ISRCTN44912979). Written informed consent was obtained from all participants before inclusion in the trial.

Patients All patients that participated were older than 18 years and had evidence of OA, as defined by clinical (pain, stiffness, disability) and radiological criteria (Kellgren– Lawrence index grade I-III). Exclusion criteria were poor general health as judged by the orthopaedic surgeon, physical conditions that would interfere with evaluation of the affected knee, OA grade IV, or participation in other trials within 3 months of inclusion. Out of the 74 placebo-treated patients that had completed the former study449, 60 patients responded to the follow-up request (81 %). Of these patients, 20 patients opted for treatment with ACS and 40 patients decided not to be treated with ACS (Figure 1). Baseline clinical scores of the 20 participants were similar to the scores of the 40 patients who decided not to receive treatment (Appendix 1).

Figure 1: Overview of patient enrolment. Placebo-treated patients were given the option to receive ACS treatment; 20 opted for treatment. These patients were now treated with ACS and followed for 12 months. Reasons for not participating in ACS treatment are also given in the figure.

88 Chapter 7 Intervention For preparation of the ACS-serum, 60 ml of venous blood was obtained from each patient and aspirated in 6 separate ACS-syringes (Orthogen, Düsseldorf, Germany) containing glass beads. The syringe was gently inverted to ensure complete mixing and maximal contact of beads and blood. These syringes were subsequently incubated at 37°C in the local Good Medical Practice (GMP) laboratory. After a 6-hour incubation period, syringes were spun at 2655 x g for 10 minutes and 2 ml of serum was aspirated and transferred aseptically into a 5-ml syringe. For each patient, 6 separate syringes containing 2 ml of ACS were stored at -20°C until treatment. A sample of the remaining serum was used for microbacterial and viral screening. The 6 intra-articular injections with ACS were performed within a 21-day time frame. On day 0, 3, 7, 10, 14 and 21, the patients were placed in a supine position, the knee was disinfected with alcohol and draped in a sterile fashion. A sterile 21-gauge needle was placed into the supra-patellar pouch of the affected knee joint, through a supra-lateral approach. Remaining synovial fluid was aspirated from the knee joint to minimize dilution. Through the same needle, 2 ml of ACS was injected into the knee joint, using a sterile 0.22 µm pore size anti-bacterial filter.

Study outcomes and follow-up At baseline (before ACS treatment), and at 3, 6, 9 and 12 months after treatment, patients completed the VAS for pain (0 indicating no pain, 100 indicating extreme pain), the KOOS (sub-domains for Pain, Symptoms, Function, Sports and Quality of Life (QOL) and the KSCRS (0 indicating severe disability and 100 indicating no disability). The 24- item WOMAC score was deduced from the separate KOOS items. The scores at 3 and at 12 months were used to distinguish ‘short term’ and ‘extended’ treatment effects, respectively. Questionnaires were sent to the patients by mail or filled out during clinical follow-up, without assistance from the independent study physician to ensure that responses were based entirely on self assessment.

Response shift During treatment with a ‘proven effective’ substance, patients are inevitably susceptible to placebo bias. From the ‘response shift theory’367, a simple test has been developed which may distinguish placebo effects from ‘real’ treatment effects. The test is called the ‘then-test’ and has been used in orthopaedic326, 466 as well as non-orthopaedic research settings218, 366, 424, 439. Treatment effects, which are otherwise obscured by the patient’s adaptation towards their ‘disease-related disability’, may now be revealed218, 326. At 12 months after ACS treatment, patients were asked to retrospectively assess their health situation at the start of the treatment (‘then-test’). A distinction can then be made between ‘unadjusted treatment effect’ (post-test – pre-test) and ‘adjusted treatment effect’

Autologous conditioned serum – comparison to earlier placebo 89 3 3 - 1 Unadjusted Treatment Effect 1 3 - 2 Adjusted Treatment Effect

Clinical score Clinical 1 - 2 Response shift

Before treatment After treatment Figure 2: Response shift measurement using the ‘then-test’. When measured at the end of the treatment (‘3’), patients may rate their initial performance differently (‘2’), as compared to their perception of performance at the actual start of the treatment (‘1’).

(post-test – then-test). It was hypothesized that the ‘adjusted treatment effect’ would be larger than the ‘unadjusted treatment effect’ (Figure 2). Apart from the ‘response shift’ analysis, follow-up procedures were similar to follow-up during earlier placebo treatment. Patients undergoing additional interventions due to knee OA during follow-up were considered treatment failures. Data sets of these patients, as well as data sets of patients undergoing (non-) surgical procedures of the knee not related to knee OA during follow-up, were completed using the ‘last observation carried forward’ method. For example, if a patient experienced a trauma to the knee requiring arthroscopic intervention at 10 months follow-up, the scores observed at 9 months were transferred to 12 months. Finally, to enable comparison of the results of the current cohort to the general population, KOOS subscale values are plotted against an age-matched control group derived from a previous publication310.

Statistical Methods SPSS version 15.0 for Windows (SPSS Inc., Chicago, IL, USA) was used for data analysis. The power of the study was 73% (20 patients, Delta 20%, standard deviation 33%). Data were tested for normality using the Kolmogorov-Smirnov test and for homogeneity of variances using Levene’s test of equality of variances. Assuming normality and equal variances, continuous variables were analyzed using paired samples T-tests at 3 months (‘short term effects’) and at 12 months (‘extended effects’), followed by a Bonferroni correction. When both the placebo treatment in the previous trial and the ACS treatment of the current study yielded a significant improvement, the size of the effect was compared

90 Chapter 7 using paired samples T-tests. 12 Month clinical scores after ACS treatment were compared to baseline clinical scores before placebo treatment. P-values less than 0.05 were considered statistically significant. Graphs show mean values ± 95% confidence-interval (95% C-I).

Results

Patients A total of 20 patients enlisted for the ACS treatment, and were followed until the pre- defined 12 months’ endpoint. Of the patients that did not participate, reasons not to enroll in the second trial were amongst others an acceptable level of pain after placebo treatment, pain from earlier injections, the laborious nature (frequency/ logistics) of the treatment, lack of conviction by earlier study results or an other operative or non- operative procedure in the meantime (Figure 1). The mean age of the 20 patients was 50 years (range 34 – 70 years), 14 of the patients were male and 6 were female. The left knee was treated in 5 patients, the right knee in 12 patients and both knees in 3 patients. No treatment failures occurred. Two out of 20 patients experienced a knee trauma during the 12-months follow-up; one patient had an ACL reconstruction and one patient underwent an arthroscopy in which microfracturing was combined with nettoyage of the lateral meniscus. Their scores were completed using the last-follow up carried forward method. Treatment with ACS started within an average of 14 months after completion of the ACS trial (6-25 months).

Placebo treatment: improvement of all clinical scores at 3- and 12 months During treatment in the previous trial449, the 20 placebo-treated patients reported improvement on all clinical outcome scores at 3 months (VAS p=0.036, KSCRS p=0.036, KOOS total p=0.002 and WOMAC p=0.012) as well as 12 months (VAS p=0.030, KSCRS p=0.014, KOOS total p=0.004 and WOMAC p=0.008). At the start of ACS treatment, baseline VAS pain (p=0.175) and KSCRS (p=0.162) scores did not differ from before earlier placebo treatment, however KOOS total (p=0.002) as well as WOMAC (p=0.001) scores had improved (Figure 3).

ACS treatment: improvement of VAS pain at 3 months During ACS treatment in the current trial, VAS pain improved after 3 months (p=0.022) but not after 12 months (p=0.550). The improvement did not differ from previous placebo treatment (p=0.942). In contrast to the response to earlier placebo treatment, improvement was seen after ACS treatment at 3 nor at 12 months for KSCRS, KOOS overall, KOOS individual or WOMAC scores after ACS treatment (Figure 3).

Autologous conditioned serum – comparison to earlier placebo 91 Before p=0.01 Before 100 treatment 100 treatment p=0.94 p=0.04 3 months p=0.04 p=0.02 3 months 80 p=0.03 80 12 months 12 months 60 60

40 KSCRS 40 VAS for pain

20 20

0 0 Placebo ACS Placebo ACS

100 Before 100 Before treatment treatment p<0.01 3 months 3 months 80 80 p=0.01 p<0.01 12 months 12 months p<0.01 60 60

40 WOMAC 40 KOOS overall

20 20

0 0 Placebo ACS Placebo ACS Figure 3: Clinical scores before, and 3 and 12 months after placebo and ACS treatment (n=20). (A) VAS for pain, (B) KSCRS, (C) KOOS overall score, (D) WOMAC. Patients reported improvement on all scores during placebo treatment, on short term (3 months) as well as during extended follow-up (12 months). For ACS treatment, improvement is only reported on VAS (pain) after 3 months of treatment. The KSCRS (B) was the only score that demonstrated improvement after the combination of placebo and ACS treatment. Bars represent mean ± 95% C-I.

Before Placebo ACS 12 months Placebo 12 months Age-matched control 100

40 KOOS subscores 20

0 Symptoms Pain ADL Sports QOL Figure 4: KOOS subscores in relation to scores of healthy historical controls310. 12 months after ACS treatment, KOOS subscores do not differ from those, 12 months after placebo, but they are still remarkably decreased in comparison to the healthy control scores. Bars represent mean KOOS subscore values (n=20).

92 Chapter 7 Overall improvement Clinical scores after 12 months of ACS treatment were similar to those after 12 months of placebo treatment. Taking the initially (non-significantly) lower scores before ACS treatment into account, this indicates that some sort of relapse took place after placebo treatment, followed by a ‘renewed’ improvement (significant for VAS pain p=0.022 at 3 months). However, clinical scores were still remarkably lower than those of age matched healthy, historical controls310 (figure 4).

Response shift: improvement in KOOS, 12 months after ACS treatment Patients report an improvement in KOOS overall score 12 months after ACS treatment, when asked to rate their health in retrospect using the then-test (p=0.015) (Figure 5). In- terestingly, in retrospect, patients seemed to rate their initial health (all outcomes) lower than before the actual ACS-treatment, but this difference was not statistically significant.

100

80 p=0.02 60

40 KOOS overall 20

0 Before treatment 12 months after ('then test') treatment Figure 5: When evaluated in retrospect, patients report an improvement in KOOS overall score after ACS treatment (n=20). Bars represent mean ± 95% C-I.

Discussion

There is a great need for effective conservative treatment strategies for osteoarthritis of the knee, as well as a need for better understanding of the biological aspects of disease and therapy. Disease-modifying osteoarthritis drugs (DMOADs) aim at diminishing OA progression in vivo while improving clinical signs and symptoms. Autologous conditioned serum (ACS) treatment improved these symptoms to a moderate extent in an earlier trial,449 while placebo treatment (physiological saline) also resulted in significant improvement. During this study we aimed to record the placebo patients’ experiences with the earlier treatment and to evaluate if further improvement in clinical signs and symptoms of osteoarthritis occurred after ACS treatment.

Autologous conditioned serum – comparison to earlier placebo 93 The greatest improvement in KOOS, KSCRS and WOMAC occurred during placebo treatment, and additional ACS treatment did not further improve clinical scores. The VAS score for pain was the only score to show a temporary improvement after ACS. Baltzer et al19 demonstrated an improvement in clinical symptoms lasting up to two years, when comparing 6 intra-articular ACS injections to 3 injections with hyaluronan or saline. The authors conclude that although clinical effects have been demonstrated, it remains to be determined how these ‘disease-modifying’ effects result from in vivo chondroprotective or chondroregenerative mechanisms. Parallel to the conduction of the current trial, effects of ACS on synovial fluid composition and on cartilage metabolism were studied343. The presence of chondrodegradative cytokines in the product and the absence of in vitro effectiveness, as well as unknown effects of IL-1ra on pain and inflammation68, demand for further in vitro and in vivo investigation of the product. Overall, patients scored their health status higher before treatment, than when retrospectively scoring their before-treatment status áfter treatment. The KOOS overall score was the only score that improved significantly after ACS treatment, measured using the ‘response shift’. The currently observed adaptation of OA patients to their disease seems similar to that observed earlier, when response shift was used as evaluation tool18, 326, 466. Regarding the ‘then-test’ methodology, the possible influence of ‘recall bias’ (i.e. recall of a previous state is influenced by the patient’s current state) and its influence on ‘then- test’ outcome should be considered when drawing conclusions330, 334, 419. However, the authors believe that response shift may be valuable as outcome measure in addition to validated outcome measures as the KOOS, VAS and WOMAC, to increase knowledge on long term implications of OA as chronic disease. Indeed, KOOS and WOMAC clinical scores were higher at the start of ACS treatment than before earlier placebo treatment, challenging ACS to achieve a similar improvement. Apparently, placebo treatment had some sort of longer lasting effect. This is in line with earlier observations460, which describe that placebo effects in intra-articular OA treatment may be responsible for up to 79% of OA symptoms reduction237. The experienced ‘invasiveness’ of the application subsequent injections may be responsible465. A pure effect of the repetitive ‘flushing’ of the joint with physiological saline during the injections seems disputable, as in a previous comparison of arthroscopic lavage with debridement or placebo surgery281, no greater reduction of OA symptoms was observed after lavage. On the other hand, the limited response to ACS may result from further progression of the osteoarthritic disease, as well as from low patient expectations after conclusion of the previous treatment episode, which may have masked improvement. Limitations of this study include the relatively small patient number opting for ACS treatment after completion of the earlier trial, resulting in a slightly lower power (73%) than commonly used (80%) and possibly leading to a selection bias. Amongst the reasons given, a substantial number of patients reported that they had already reached an

94 Chapter 7 acceptable level of pain after placebo treatment, that the injections were too painful or the patients believed that the frequency and logistics of the injections would not outweigh the benefit of treatment. However, the baseline scores of the currently studied patients did not differ from the group as a whole (Appendix 1), thus suggesting that the 20 patients in the current study may be considered representative of the entire cohort. In conclusion, patients that experienced improvement in clinical scores after intra- articular injections with physiological saline did not experience a similar improvement after subsequent treatment with ACS. Although there may be several explanations for the absence of further improvement, the invasiveness of the injection therapy and the resulting placebo effect may be of greater importance in reduction of OA symptoms than the effects of ACS-incubated serum itself.

Autologous conditioned serum – comparison to earlier placebo 95 Appendix 1

70 Participants (n=20) Entire cohort (n=74)

30 KOOS subscore 20

0 KOOS pain KOOS sympt. KOOS ADL KOOS sports KOOS QOL Clinical scores of the 20 patients that opted for ACS treatment after their earlier placebo treatment, did not differ from the clinical scores of patients that decided not to participate. Bars represent mean ± 95% C-I.

96 Chapter 7 Evaluation of histological scoring systems for tissue-engineered, repaired and osteoarthritic cartilage

Marijn Rutgers, Maarten J. van Pelt, Wouter J. Dhert, Laura B. Creemers and Daniël B. Saris

Osteoarthritis and Cartilage. 2010;18(1):12-23 Abstract

Objective: Regeneration of hyaline cartilage has been the focus of an increasing number of research groups around the world. One of the most important outcome measures in evaluation of its success is the histological quality of cartilaginous tissue. Currently, a variety of histological scoring systems is used to describe the quality of osteoarthritic, in vivo repaired or in vitro engineered tissue. This review aims to provide an overview of past and currently used histological scoring systems, in an effort to aid cartilage researchers in choosing adequate and validated cartilage histological scoring systems.

Methods: Histological scoring systems for analysis of osteoarthritic, tissue engineered and in vivo repaired cartilage were reviewed. The chronological development as well as the validity and practical applicability of the scoring systems are evaluated.

Results: The Histological Histochemical Grading System (HHGS) or a HHGS-related score is most often used for evaluation of osteoarthritic cartilage, however the OARSI Osteoarthritis Cartilage Histopathology Assessment System seems a valid alternative. The O’Driscoll score and the ICRS II score may be used for in vivo repaired cartilage. The ‘Bern score’ seems most adequate for evaluation of in vitro engineered cartilage.

Conclusion: A great variety of histological scoring systems exists for analysis of osteoarthritic or normal, in vivo repaired or tissue-engineered cartilage, but only few have been validated. Use of these validated scores may considerably improve exchange of information necessary for advances in the field of cartilage regeneration.

98 Chapter 8 Introduction

The histological quality of cartilage is considered to be one of the most important outcome tools to objectify severity of cartilage pathology and success of its treat- ment50, 212, 356. Over the past decades, as knowledge on osteoarthritic (OA), in vivo repaired and tissue-engineered cartilage increased, the number of systems evaluating histological characteristics of these cartilaginous tissue types increased simultaneously. One of the earliest systems for the grading of ‘OA cartilage’ was developed in 1949 by Collins78, who proposed a macroscopic system for classification of osteoarthritic changes of the human patella. This macroscopic system was succeeded by a microscopic system for analysis of OA cartilage by Mankin in 1971, who developed the now well-known His- tological-Histochemical Grading System (HHGS254). From then, methods enabling study of cartilage characteristics in vitro as well as in vivo expanded, and in 1986 O’Driscoll295 presented a scoring system for analysis of a ‘new’ type of cartilaginous tissue, commonly referred to as ‘in vivo repaired’ cartilage. The first report on successful cartilage regeneration in human clinical practice was presented in 1994 by Brittberg50, and the hyaline-like characteristics of the repaired tissue inspired researchers world-wide to further improve results from this procedure. Consequently, yet another category of cartilage histology developed, being that of the ‘tissue engineered’ cartilage. While introduction of new scoring systems in all categories continued149, 248, 297, and in part due to disadvantages of earlier systems303, 304, the Osteoarthritis Research Society International (OARSI) as well as the International Cartilage Research Society (ICRS) established committees to develop standardized histological grading systems for qualitative description of various cartilage characteristics248, 323. The variety of available scoring systems in the field of cartilage research may obscure the choice of the appropriate scoring system for a specific research setting. Further, it is often not clear which system should be applied to answer a specific research question, nor is it clear if a scoring system has been validated for this application. This may be important when comparing one’s results to that of other cartilage researchers, or when aiming at validating methods to analyze proteoglycan content of cartilage non- invasively148. This review provides an overview of existing histological scores for evaluation of osteoarthritic, in vivo repaired, and in vitro regenerated cartilage, and discusses validity and applicability of each of these systems. (Figure 1)

Histological scoring systems 99           

1949 1959 1969 1979 1989 1999 2009        

  Figure 1: Chronological development of cartilage histological scores. OA scores are presented in blue, in vivo cartilage repair scores in red, and in vitro tissue engineering scores in green.

Methodology in histological scoring of cartilage

A system for evaluation of cartilage histological properties should be comprehensive but also applicable to researchers with only basic knowledge of (cartilage) histology, regardless of the tissue type analyzed. Intra- as well as inter-observer variation should be low and the score system is preferably validated. The importance of various characteristics for the ideal cartilage scoring system when developing a scoring system for in vivo repaired cartilage was recognized by Pritzker and was summarized as ‘simplicity, utility, scalability, extendibility and comparability’323. Further, each scoring system should be used for its designated tissue type: scoring systems for OA cartilage should focus on degenerative features of healthy or diseased (OA) tissue, scoring systems for in vivo cartilage repair should focus on the degree in which a cartilage defect is successfully repaired, and scoring systems for tissue engineered cartilage should focus on the quality of newly generated cartilage after in vitro cartilage tissue engineering. Before choosing which scoring system is to be used, some basic knowledge of tissue pre- treatment and subsequent staining characteristics is required. To enable visualization of cartilaginous tissue, an ethanol-formaldehyde fixation most adequately preserves morphology305. Glycosaminoglycans are best visualized using a ‘Safranin-O’ staining341. When used in combination with a ‘Fast-Green FCF/Hematoxylin’ counterstaining, collagens and cell nuclei may be visualized as well. The ‘Alcian Blue’ staining also stains glycosaminoglycans, but has been reported to yield less reproducible results. In combination with a ‘Picosirius Red’ staining, the Alcian Blue staining may visualize collagenous structures179. For both the Safranin O as well as the Alcian Blue staining, incorporation of control cartilage samples during the histochemical staining process prevents over- or underestimation of the glycosaminoglycan content due to variation in fixation and staining circumstances. A variety of tissue characteristics may be assessed by a scoring system after histological staining of the tissue. Each characteristic may be scored separately in a quantitative

100 Chapter 8 fashion, or in combination with other characteristics in a categorical fashion, which may influence the statistical design and evaluation of the experiment. In a quantitative score, features considered important by the designer of the score like ‘structure of the tissue’ or ‘proteoglycan content’, may be emphasized through a higher contribution to the eventual score254. The resulting score may thus constitute the sum of the individual parameters, for example: a tissue is scored as 5 out of a maximum of 8 points, if 2 points are obtained for ‘intensity of Safranin O-staining’, 2 points for ‘cellularity’, and 1 point for ‘structural integrity’. The score may also be the result of multiplication of the abovementioned sum of parameters with ‘stage’ (extent of the involved cartilage surface)323, thus obtaining a score describing the entire tissue area. Quantitative scores with a broad numerical range like the O’Driscoll score for in vivo repaired cartilage296, may increase the likelihood of finding statistically significant differences between different groups, but may also be susceptible to a larger inter-observer variation. Only few of the currently existing scoring systems have been ‘validated’. Using a validated scoring system improves reliability of observations and improves comparability of these observations with results from other research groups. It is therefore important to know not only if a score has been validated, but also how the ‘validation’ was performed. Validation of a score may occur through comparison to already validated macroscopic scoring systems303, to other already validated histological systems84, to automated (and validated) histomorphometric systems297 or to biochemical parameters149. Of these validation methods, correlation to proteoglycan content (‘biochemical parameters’) is often considered important, as proteoglycan content is considered one of the major features of cartilage integrity in healthy, repaired or tissue engineered cartilage. Despite the number of scoring systems, only the HHGS for OA cartilage and the Bern score for tissue engineered cartilage have been validated by biochemical analysis149, 254. Vice versa, existing histological scores have been used as a tool to promote or validate emerging techniques analyzing cartilage quality, as for example radiographic measure- ments350, MRI266, 333 and new T2 and contrast-enhanced cartilage imaging techniques (dGEMRIC)430.

All ‘semi-quantitative’ histological scoring systems are observer-dependent and thus subjective. Automated computerized histomorphometry has been reported to enable objective, accurate and reproducible analysis of cartilage characteristics. However, use of computerized histomorphometry may be limited by the high costs or by lack of technical expertise297, 298. This review was based on a search for literature in which histological scoring systems were introduced, applied or discussed. PubMed, Embase and Cochrane Library were used for the search (syntax: histological AND (scoring* OR grading* OR assessment* OR scale*) AND cartilage. Last search: March 31st, 2009).

Histological scoring systems 101 Osteoarthritis (OA)

Histological-Histochemical Grading System (HHGS) / Mankin score The Histological-Histochemical Grading System (HHGS) or Mankin score for evaluation of osteoarthritic cartilage was originally proposed by Mankin in 1971254, and although developed for the assessment of human articular cartilage it has also been used for grading of animal cartilage. There is much confusion about the use of the ‘Mankin score’, ‘HHGS’ or ‘modified Mankin score’ (the last of which there are many). As Mankin in 1971 referred to his score as the ‘histological-histochemical grading system’ or ‘HHGS’, we have decided to do the same. All other systems should be referred to as a ‘modified Mankin system’ or a ‘modified HHGS’ with specific remarks on the altered parameters. The HHGS identifies ‘cartilage structure’, ‘cell distribution’, ‘Safranin-O staining’ and ‘tidemark integrity’ as separate sub-items. The sum of the separate scores ranges from 0 (normal) to 14 (severe osteoarthritis). The HHGS was correlated to a macroscopical 303 35 2− 254 score , to biochemical parameters and to SO4 incorporation . Although frequently used, the HHGS score has been criticized for its questionable reproducibility and inadequate assessment of ‘mild’ and ‘moderate’ osteoarthritis303, 304. Further, a lower score may result from scoring features like ‘pannus’ and ‘surface irregularities’, characteristics which may also be found in healthy, non-osteoarthritic tissue304. Moreover, the system does not evaluate the extent to which the cartilage surface is affected by the degenerative process84, 323. As cartilage often contains different regions with varying quality, omit- ting the aspect of ‘extent’ may over- or under-estimate the overall quality of the tissue. Moreover, the HHGS was demonstrated to have a significant intra- and inter-observer variability84, 303, 304, 407 (Table 1).

Table 1: Intra- and inter-observer variation of the HHGS Year Author Assessment Result Comment ’92 Van der Sluijs407 Intra- and inter-observer- Adequate More strict criteria do not lead to variation higher reproducibility ’97 Ostergaard304 Intra- and inter-observer- Inadequate HHGS inadequately discriminates variation; validated against between macroscopically normal other score and OA cartilage ’99 Ostergaard303 Intra- and inter-observer- Moderate HHGS is valid for normal and severe variation; validation OA cartilage, not for macroscopically according to macroscopical mild and moderate changes. appearance Experience not important. ’07 Custers84 Intra- and inter-observer- Excellent Reproducibility is experience- variation; comparison to dependent OARSI system.

102 Chapter 8 OARSI Osteoarthritis Cartilage Histopathology Assessment System With the objective to design a more useful method than the HHGS to assess osteoarthritis histopathology for wide application in clinical and experimental osteoarthritis assessment, the OARSI Working Group developed the OARSI Osteoarthritis Cartilage Histopathology Assessment System323. The OARSI system assesses the severity and the extent of cartilage surface involvement in the local osteoarthritic process. In contrast to the HHGS and most other OA scores, the OARSI system emphasizes the extent of cartilage damage over the articular surface through a ‘stage’ component’, in addition to damage analyzed at several levels of the cartilage layer (i.e. ‘depth’ and ‘local cartilage damage’). ‘Grade’ (0 points for ‘normal’ up to 6 points for ‘severe’) and ‘stage’ (0 points for ‘no osteoarthritis activity seen’ up to 4 points for ‘>50% of articular surface affected’) can be used separately or can be combined in an overall score by multiplication. The OARSI system was anticipated to be more adequate for the assessment of mild osteoarthritis and it was expected that the OARSI system could be applied more consistently by less experienced observers than the HHGS323. In a study comparing the OARSI system with the HHGS, a similar reproducibility was found, however the reliability of the OARSI system was higher and indeed observer experience seemed to be less important when using the OARSI system84. Nevertheless, the authors reported that the staging component of the OARSI system was difficult to determine with certainty due to the precision required for estimation of the surface extent of osteoarthritic lesions323. This is demonstrated in figure 2B, where interpretation of ‘surface extent’ depends on which ‘grade’ is considered most important (i.e. the irregular area in the superficial zone of figure 2B is of a higher ‘grade’ (grade 2.0) but present in only 10-25% of the surface (stage 2) while areas with cell death (lower ‘grade’, i.e. grade 1.5) are present in more than 25% of the cartilage surface (higher ‘stage’, i.e. Stage 3). Although we used the OARSI system to illustrate differences in outcome between HHGS and OARSI, the OARSI system has not yet been validated for application on human articular cartilage, nor has it been validated through correlation to macroscopic or biochemical parameters (Figure 2a-c).

Other osteoarthritis scores Besides the HHGS and the OARSI system, several other simple systems have been used to grade osteoarthritis31, 79, 109, 112, 173, 307, 350, 448. These scores are often referred to as ‘modified Mankin’ systems. In essence, all of these systems assess similar parameters as the HHGS, but the parameters are either scored in a different fashion, or an overall score is applied in stead of separate subscores. Most of these scores were only used in the study in which they were introduced, and none were validated (Table 2).

Histological scoring systems 103 Figure 2: Safranin O/Fast Green stained histological sections of human knee articular cartilage (femoral condyle, 5 μm sections). Sections were scored with the HHGS254 and with the OARSI scale323 in small (left) and larger magnification (right images, # and *).

A A

FigureFigure 2A: 2A: good good quality quality cartilage cartilage of a 45-year (HHGS old male 2 points, tissue donorOARSI without grade history 0 stage of traumatic 0) or inflammatory joint diseases. FigureB 2A: good quality cartilage (HHGS 2 points, OARSI grade 0 stage 0) B Figure 2A: good quality cartilage (HHGS 2 points, OARSI grade 0 stage 0)

Figure 2B: moderate quality cartilage (HHGS 6 points, OARSI grade 2.0 stage 2)

FigureFigure 2B :2B: moderate moderate quality quality cartilage cartilage of a 57 year(HHGS old female, 6 point obtaineds, OARSI during grade a unicompartmental 2.0 stage 2) joint replacement.C Note the altered structure of the cartilage in comparison to figure 2A, the slight surface irregularities and the onset of cell clone formation. C Figure 2B: moderate quality cartilage (HHGS 6 points, OARSI grade 2.0 stage 2)

C 4 mm 50 µm 50 µm

Figure 2C: low quality OA cartilage4 mm (HHGS 9 points, OARSI50 µ gradem 4.0 stage 4)50 µm

Figure 2C: low quality OA cartilage (HHGS 9 points, OARSI grade 4.0 stage 4) 4 mm 50 µm 50 µm

FigureFigure 2C: 2C: low lowquality quality cartilage OA with cartilage fibrous (HHGS tissue characteristics, 9 points, OARSI demonstrating grade 4.0matrix stage loss, 4)surface abra- sions and extensive cloning.

104 Chapter 8 Table 2: Modifications of the HHGS and other OA scores. Year Authors Comment ’69 Otte307 Also known as ‘Fassbender’; modified by Saal350. Only grades cartilage surface structure. High correlation with ‘surface structure’ category of HHGS ’83 Colombo79 The Colombo score is similar to the HHGS and was modified by Hotta171 ’87-’07 Several129, 132, 152, Varying modifications, however ‘tidemark integrity’ is a frequently discussed or 153, 192, 204, 206, 217, 227, excluded category 229, 233, 236, 353, 354, 397

’91-’97 Bendele31, These scores directly apply an overall score of severity to the cartilage section. Fremont112, Bendele includes a staging component. Huang classifies into 4 grades: Grade II, for Huang173 example, comprises flaking, superficial fibrillation, chondrocyte enlargement and hyalinization. ’94 Foland109 Measures fibrillation, chondrocyte necrosis, chondrone formation and focal loss of cells. Modified by Frisbie119. ’05 Yagi448 Scores matrix depletion and cellularity quantitatively.

Cartilage repair

O’Driscoll score The first ‘cartilage repair’ score was used to assess the quality of cartilage repair in rabbits after periosteal grafting of cartilage trauma295, 296. This score specific for cartilage repair, which is now often referred to as the O’Driscoll score, encompassed four major categories (‘nature of the predominant tissue’, ‘structural characteristics’, ‘freedom from cellular changes of degeneration’ and ‘freedom from degenerative changes in adjacent cartilage’) and was frequently used for cartilage analysis in animal studies168, 355, 420. A high intra- and inter-observer reproducibility was demonstrated, when used to analyze goat articular cartilage278. However, the many different subitems make it a somewhat lengthy score. The O’Driscoll score is one of the few scores in which ‘integration’ of the repair tissue with its surroundings is assessed (Figure 3a and 3b), however the additionally added (or subtracted points) do not greatly affect the eventual score. The O’Driscoll score has frequently been used in modified versions (Table 3).

Pineda scale In an effort to create a more compact score to evaluate cartilage repair, Pineda319 developed a new system which was applied to assess the natural healing capacity of defects drilled into rabbit knee articular cartilage. The Pineda scale contains four categories: ‘percent filling of the defect’, ‘reconstitution of osteochondral junction’, ‘matrix staining’ and ‘cell morphology’. Although it was hypothesized that a simple score as the Pineda scale would have a higher reproducibility than the O’Driscoll score, the more comprehensive O’Driscoll score demonstrated a similar intra- and inter-observer variation. The correlation with the Pineda scale was high (correlation coefficient of 0.71)278.

Histological scoring systems 105 Figure 3: Safranin O/Fast Green stained histological sections of adult female Dutch Milk goat knee articular cartilage, after a microfracture procedure (5 μm sections). Sections were scored with the O’Driscoll score6 and the ICRS II score48 in small (left) and larger magnification (right images, # and *). ICRS II score categories are abbreviated as follows: overall; staining; morphology; clusters; tidemark; subchondral bone; surface architecture; basal integration; cells; abnormal calcification; vascularisation; mid deep zone; surface assessment. The microfractured area is between the dotted lines.

FigureFigure 3A: highhigh quality quality repair repaired tissue cartilage; (O’Driscoll assessment score: 22 points;of ‘bonding ICRS ofII score: the repaired o 87, s 76,cartilage m 73, cwith 11, itst 60, sur - roundings’b 77, a 79, isi 92, possible e 80, f with75, v the77, dO’Driscoll 81, u 70) score, while the ICRS II score gives more detailed information aboutFigureB specific 3A: high repair quality features repair tissue as for (O’Driscollexample basalscore: integration. 22 points; ICRS II score: o 87, s 76, m 73, c 11, t 60, b 77, a 79, i 92, e 80, f 75, v 77, d 81, u 70) B Figure 3A: high quality repair tissue (O’Driscoll score: 22 points; ICRS II score: o 87, s 76, m 73, c 11, t 60, b 77, a 79, i 92, e 80, f 75, v 77, d 81, u 70) B

Figure 3B: moderate quality repair tissue (O’Driscoll score: 15 points; ICRS II score: o 65, s 82, m 66, c 88, t 55, b 26, a 64, i 83, e 70, f 51, v 71, d 38, u 63)

FigureFigure 3B:3B: moderate quality quality repair repaired tissue cartilage; (O’Drisco thell score: ICRS 15II score points; acknowledges ICRS II score: the o 65, more s 82, intense m 66, coverall 88, t 55,C b 26, a 64, i 83, e 70, f 51, v 71, d 38, u 63) Safranin O staining in this section than in the previous ‘high quality’ section (figure 3A), while the O’Driscoll score only provides a total score. Note the hypercellularity in the microfractured part of the tissue (#) in C comparisonFigure 3B: moderate to the cellular quality cloning repair tissue in the (O’Drisco non-microfracturedll score: 15 points; part (*). ICRS II score: o 65, s 82, m 66, c 88, t 55, b 26, a 64, i 83, e 70, f 51, v 71, d 38, u 63)

C 1 mm 50 µm 50 µm

Figure 3C: low quality repair tissue (O’Driscoll1 mm score: 13 points; ICRS II score:50 µm o 48, s 7, m 53, c 12, t50 12, µm b 59, a 90, i 8, e 80, f 49, v 77, d 31, u 51)

Figure 3C: low quality repair tissue (O’Driscoll score: 13 points; ICRS II score: o 48, s 7, m 53, c 12, t 12, b 59, a 90, i 8, e 80, f 49, v 77, d 31, u 51) 1 mm 50 µm 50 µm

FigureFigure 3C: low qualityquality repair repaired tissue cartilage; (O’Driscoll weak score: Safranin 13 points; O staining ICRS II and score: an oabsent 48, s 7, tidemark m 53, c 1are2, treflected 12, b in59, the a 90,ICRS i 8,II score,e 80, f while 49, v 77,these d 31, features u 51) are not discussed in the O’Driscoll score. While the O’Driscoll score hardly differs from the previous section (figure 2B), significant differences exist between the ICRS II subcat- egories of these sections.

A commonly used modification of the Pineda scale was introduced in 1994 by Wakitani426 to study the characteristics of repaired full-thickness cartilage defects in rabbits. This ‘Wakitani score’ assesses ‘surface regularity’, ‘thickness of reparative cartilage compared with surrounding cartilage’ and ‘integration of donor cartilage

106 Chapter 8 Table 3: Most cartilage repair scores (other than the Pineda, Oswestry, Knutsen and ICRS scores) are based on modifications of the O’Driscoll score or HHGS. Year Authors Comment ’95-’08 Several30, 53, 61, O’Driscoll category modifications; most important new or altered categories: 113-115, 117, 251, 290, 292, ‘reconstitution of subchondral bone’, ‘tidemark formation’, ‘basal integration of the 324, 363, 383, 412, 457 graft’ and ‘signs of inflammation’ ’97-’07 Several57, 89, 145, 403 Includes a score which evaluates focal chondral defect treatment using a score based on HHGS403. As the O’Driscoll score, this score evaluates cartilage repair integration. Further, macroscopical evaluation is used in combination with histological evaluation. ’01 Mainil-Varlet251 O’Driscoll category modifications; ‘chondrocyte clustering’ is considered as a feature of repair tissue and not as a degenerative feature ’08 Nettles290 Combination of O’Driscoll score and ICRS-I (Visual Histological Assessment Scale). Although various ‘new’ scales combine features of earlier scales, this is one of the only systems that explicitly combines two scales. with adjacent cartilage’, instead of ‘percent filling of the defect’ and ‘reconstitution of osteochondral junction’ in the O’Driscoll score. Both the Pineda and Wakitani score have been modified frequently125, 201, 285, 286, 306, 370, 454, however solely the Pineda scale was validated278.

The Oswestry Score In contrast to animal studies, study of articular cartilage repair in humans is limited by availability and size of the biopsy of the repaired tissue. For example, the category ‘bonding to the adjacent cartilage’ (O’Driscoll score) may only be scored when a biopsy is taken from a transitional zone between ‘new’ and ‘old’ cartilage, requiring either a large biopsy or a full joint explant. This issue was recognized by Roberts333, and as harvesting of large size biopsies including this transitional zone is usually not desirable as this may affect repair of the damaged area, the authors proposed a grading system for small biopsies of repaired human cartilage. The subsequently developed ‘OsScore’ comprises 7 parameters (‘tissue morphology’, ‘matrix staining’, ‘surface architecture’, ‘chondrocyte clusters’, ‘mineral’, ‘blood vessels’ and ‘basal integration’) and has a maximum score of 10 points. The OsScore demonstrated a high inter-observer variability, and an excellent correlation with the O’Driscoll score (r=0.9)333.

Knutsen score In addition to these scoring systems, less extensive scoring systems were used for histological analysis of cartilage repair. Knutsen212 categorized cartilage biopsy samples using a hematoxylin-eosin staining and describes categories as ‘hyaline’ (≥60% hyaline cartilage), ‘fibrocartilage-hyaline mixture’ (40-60% hyaline cartilage), ‘fibrocartilage’ (≥60% fibrocartilaginous tissue) or as a sample ‘without repair tissue’. In 2000 Peterson315, using multiple staining methods, used a similar system to evaluate the results of the

Histological scoring systems 107 autologous chondrocyte transplantation, describing the tissue as ‘hyaline-like’, ‘fibrous’ or ‘mixed’. Neither of these scores was validated.

ICRS Interestingly, the ICRS Histological Endpoint Committee introduced the ICRS Visual His- tology Score in 2003, arguing that for more easy and reliable comparison of histological assessment a system based on visual patterns is preferable over a verbal description- based system248. A web-based catalogue of cartilage repair images was set up as a reference for scoring, and to enable discrimination of the individually scored features, instead of summarizing all the individual subscores (I–VI) to create a total score, the score values (0-3) for the different categories (‘surface’, ‘matrix’, ‘cell distribution’, ‘cell population’, ‘subchondral bone’, ‘cartilage mineralization’) are described in the final score (i.e. I:3 ; II:3 ; III:1 ; IV:2 ; V:1 ; VI:3). To our knowledge no studies exist that have evaluated the validity and reliability of the ICRS Visual Histology Score. However, the OsScore and the ICRS score were found to correlate fairly well332. The ICRS VHS was modified by Chang66, by addition of categories for the staining of type I collagen and type II collagen. A new histological scoring system has been developed and validated by the Histology Work- ing Group of the ICRS249, 250. This “ICRS II’ score addresses various aspects of cartilage repair and was first applied clinically in a large prospective randomized trial in which the clinical and histological results of microfracture and ACI were compared356. The ICRS II score contains several categories which are subdivided in 13 categories each scored on a 100-mm visual analogue scale. A 100-mm VAS scale enables evaluation of subtle differences and facilitates statistical comparison of the individual cartilage characteristics322. The categories of the ICRS II score are, besides ‘overall assessment’ (bad – good) ‘matrix -staining metachromasia’ (no metachromasia – full metachromasia), ‘tissue morphology’ (fibril presence viewed under polarized light; full thickness collagen fibers – normal cartilage birefringence), ‘chondrocytes clusters’ (4 or more grouped cells; absent – present), ‘calcification front/tidemark’ (no calcification front – tidemark), ‘subchondral bone abnormalities/cellular infiltration’ (abnormal – normal/no infiltrates), ‘architecture of the surface’ (delamination/loosening/disruptions – smooth surface), ‘basal integration’ (no basal integration – basal integration), ‘cell morphology’ (no round/oval cells – mostly round/oval cells), ‘abnormal calcification/debris’ (present – absent), ‘vascularisation within the repair tissue’ (present – absent), ‘mid-deep zone assessment’ (bad – good) and ‘surface/superficial assessment’ (bad – good) (Figure 3)(Table 4). A 14th category (‘inflammation’) may be included when a scaffold has been used during the cartilage repair procedure, as for example during matrix-assisted chondrocyte implantation. Similar to most other cartilage repair scores, this score does not evaluate integration of the repair tissue with its surroundings, which is due to the often limited size of a biopsy in the clinical setting.

108 Chapter 8 Table 4: Parameters assessed by some major cartilage repair scores. Mankin (Uhl) Mankin Modified OsScore ICRS I (VHS) Knutsen ICRS II (VAS) O’Driscoll Pineda Wakitani 319 333 212 426 295 248 Parameters of the different 250 403 grading systems cell morphology x x x x x x x x matrix staining x x x x x surface regularity x x x x x x structural integrity x x thickness / defect filling x x x osteo-chondral junction x x x adjacent bonding x x x basal integration x x cellularity x clustering/distribution x x x x adjacent cartilage degeneration x mineral x x x blood vessels x x x subchondral bone x x viability cell population x x inflammation x cartilage plug quality x

When comparing the O’Driscoll score and the ICRS II score in figure 3, differences in ‘intensity of matrix staining’ are not reflected in O’Driscoll subscores, while the ICRS II score does distinguish the more intense Safranin O staining in the ‘moderate’ quality tissue, even though the ‘good quality’ cartilage has a higher O’Driscoll score (figure 3A and 3B). Further, little difference exists between total O’Driscoll scores for ‘moderate’ and ‘low’ quality repaired tissue (figures 3B and 3C), while the ICRS II subscores are obviously different (i.e. staining and basal integration).

Tissue engineered cartilage

As in OA and in vivo repaired cartilage, the quality of tissue engineered cartilage is described using macroscopic morphological outcome parameters, immunohistochemical staining, quantitative histomorphometry, analysis of overall cellularity, DNA content, glycosaminoglycan content and collagen content116, 246, 247, 294, 298, 312, 376. Few histological scoring systems are available for the evaluation of tissue engineered cartilage.

Histological scoring systems 109 Figure 4: Safranin O/Fast Green-stained histological sections of tissue engineered (TE) cartilage (healthy human knee articular chondrocytes, passage 2, cultured for 28 days in medium containing transforming growth factor β2). Sections are scored using the Bern score149 and the O’Driscoll score297.

A A A

FigureFigure 4A: 4A: TE cartilage good qualityof high quality; TE cartilage note the intense (O’Driscoll Safranin sc O orestaining 3, andBern high score amount 9) of matrix, relative to the rounded, chondrocyte-like cells. FigureB 4A: good quality TE cartilage (O’Driscoll score 3, Bern score 9) FigureB 4A: good quality TE cartilage (O’Driscoll score 3, Bern score 9) B

Figure 4B: moderate quality TE cartilage (O’Driscoll score 2, Bern score 6)

FigureFigure 4B :4B: TE cartilage moderate of moderate quality quality; TE notecartilage the less (O’Driscolintense Safraninl score O staining 2, Bern and the score combination 6) of fibroblast-likeC cells and chondrocytes, resulting in a lower Bern score. Figure 4B: moderate quality TE cartilage (O’Driscoll score 2, Bern score 6) C C

1 mm 50 µm

1 mm 50 µm Figure 4C: low quality TE cartilage (O’Driscoll score 1, Bern score 5) 1 mm 50 µm Figure 4C: low quality TE cartilage (O’Driscoll score 1, Bern score 5) FigureFigure 4C: 4C: TE cartilage low quality of low quality.TE cartilage a thin matrix (O’Driscoll and hardly sco any reSafranin 1, Bern O staining score leads 5) to an even lower Bern score in figure 4C. The Bern score obviously facilitates more subtle distinction of slight changes in tissue quality through its broader range (Bern score: 3-9 vs. O’Driscoll: 0-3).

110 Chapter 8 In 2001, O’Driscoll correlated a simple four-category subjective scoring system to an automated histomorphometry program determining the intensity of Safranin O staining297. The score, in essence a modification of the Mankin score, showed a good correlation with the automated system and the authors suggest that this system is a good alternative to a computerized system. However, this automated system only focused on proteoglycan ‘density’, and did not evaluate characteristics as, for example, cell morphology. Another grading system developed exclusively for the evaluation of in vitro engineered cartilage was presented in 2006149. This ‘Bern score’ showed a good correlation of cartilage quality with glycosaminoglycan content as measured biochemically and by automated histomorphometry. The Bern score was validated for the assessment of pellet-cultured cartilage, and has been used several times thus far140, 205, 450‑452. Im et al180 modified the Bern score by addition of ‘immunohistochemical staining for type II collagen’ to the first scoring parameter. The influence of this modification on the validity of the original score was not evaluated. An advantage of the Bern score, is that it covers a broader score range and thus gives more detailed information on tissue characteristics, than for example the O’Driscoll score. (Figure 4)

Discussion

Evaluation of cartilage quality through histological analysis significantly contributes to the assessment of the extent of cartilage damage or the success of cartilage regenera- tion50, 212, 356. The development of in vitro tissue engineering techniques further extends knowledge of cartilage structure and composition. When evaluating cartilage characteristics, choosing the appropriate histological scoring system is important for analysis of cartilage pathology, evaluation of the result of its in vivo treatment or for further in vitro tissue engineering research. For each of these categories, different histological scoring systems exist. Healthy cartilage is most often distinguished from osteoarthritic cartilage using the HHGS254 or a HHGS-related score, even though reproducibility and validity of this score have been disputed303, 304. Nevertheless, in our experience it is an adequate system for analysis of degenerative cartilage characteristics due to its overall comprehensive character. The widespread use of this system further facilitates comparison of one’s histological data with that of other cartilage researchers. The many modifications of the HHGS necessitate the use of adequate terminology; ‘HHGS’ or ‘Mankin score’ for the original 1971 score, and ‘modified Mankin’ or ‘modified HHGS’ for scores in which a modification has been made, even if only one category is omitted. An alternative for the HHGS is the OARSI system323, which seems easier in use for inexperienced scorers84, 323 but has been used infrequently thus far. When the entire articular

Histological scoring systems 111 surface is included in a tissue section, as for example in joints of small animals, the influence of surface extent or ‘stage’ in the eventual score provides additional information. As the OARSI system seems more robust and the reliability is higher than the HHGS84, this system will be preferable in most cases. Of the various scores available for analysis of in vivo repaired cartilage, the ICRS II score249 seems most suitable, as it is validated, comprehensive and describes each cartilage characteristic individually. Further, this score proved valuable during analysis of biopsies in the course of a recent randomized controlled clinical trial356. As biopsies of human cartilage are often small and do not include a border region between repaired cartilage and its surroundings, one may choose to comment on this separate from the score. Alternatively, the O’Driscoll score295 may be used. For evaluation of in vitro engineered cartilage, the ‘Bern score’149 is preferred as it is validated and adequately distinguishes between characteristics specific for tissue- engineered cartilage140, 205, 450‑452. Sporadically, authors use two different histological scoring systems in a parallel fashion, or combine the scoring systems into one, in essence creating a new scoring system290. The difficulty of combining scoring systems is that the mutual parameters of each scoring system (for example: proteoglycan staining) are over-emphasized in the eventual score, thus decreasing the ‘impact’ of separate categories of the individual scores. Using scoring systems in a parallel fashion results in more information on the histological characteristics of the assessed tissue, however using one comprehensive validated system is preferable over using several non-validated systems. Although validation is one of the major motivations for the application of a particular scoring method, the ideal validation method remains disputable. Ideally, a histological score is validated by comparison to several parameters important to cartilage, i.e. by comparison to (validated) biochemical, biomechanical, macroscopical and (functional) imaging techniques. Of these parameters, regardless of the tissue type (OA, repaired or tissue engineered cartilage), correlation to biochemical parameters seems important. Nevertheless, biomechanical properties are important for repaired cartilage as well and biomechanical testing techniques as for example ‘indentation stiffness’ may add to the qualitative description of the tissue413. However, these techniques may have their limitations (i.e. analysis of cartilage repair after microfracture using indentation stiffness is limited due to outgrowth of subchondral bone)209. The ideal combination of analysis techniques therefore remains to be developed. In the future, not only uniformity in the application of histological scores, but also in the use of validation techniques will aid development of improved cartilage analysis techniques. Even when adequately validated, additive information on macroscopical, biochemical and biomechanical aspects of the tissue will yield a more complete picture of tissue quality403, 413.

112 Chapter 8 Histological scoring of cartilage

Osteoarthritic in vivo repaired Tissue engineered cartilage cartilage cartilage

Grading x Grading only Simple Comprehensive staging

OARSI O'Driscoll HHGS Bern Simple OA Bendele (2001) scores

Animal Human

Simple Comprehensive Simple Comprehensive

O'Driscoll OsScore Semi- Visual Pineda (1986) Knutsen quantitative assessment Peterson score scale

ICRS I ICRS II (VHS) (VAS) Figure 5: Choice of an appropriate scoring system may be aided by a flow diagram.

In conclusion, a variety of histological scoring systems exists for analysis of osteoarthritic or normal, in vivo repaired or in vitro tissue-engineered cartilage, but only few have been validated. For each category, specific validated histological scores emerge as most suitable for application. Use of these validated scores may considerably improve exchange of information necessary for advances in the field of cartilage regeneration and thus stimulate uniformity enabling comparison of results of different cartilage research groups. (Figure 5)

Histological scoring systems 113

dGEMRIC as a tool for measuring changes in cartilage quality following high tibial osteotomy: a feasibility study

Marijn Rutgers, L. Wilbert Bartels, Anika I. Tsuchida, René M. Castelein, Wouter J. Dhert, Koen L. Vincken, Ronald J. van Heerwaarden and Daniël B. Saris

Osteoarthritis and Cartilage. 2012;20(10):1134-41 Abstract

Objective: The high tibial osteotomy (HTO) is an effective strategy for treatment of painful medial compartment knee osteoarthritis. Effects on cartilage quality are largely unknown. Delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC) enables non-invasive assessment of cartilage glycosaminoglycan content. This study aimed to evaluate if dGEMRIC could detect relevant changes in cartilage glycosaminoglycan content following HTO.

Design: Ten patients with medial compartment osteoarthritis underwent a dGEMRIC scan prior to HTO, and after bone healing and subsequent hardware removal. A dGEMRIC index (T1Gd) was used for changes in cartilage glycosaminoglycan content, a high T1Gd indicating a high glycosaminoglycan content and vice versa. Radiographic analysis included mechanical axis and tibial slope measurement. Clinical scores [Knee Osteoarthritis Outcome Scale (KOOS), visual analogue score (VAS) for pain, Knee Society Clinical Rating System (KSCRS)] before, 3 and 6 months after HTO and after hardware removal were correlated to T1Gd changes.

Results: Overall a trend towards a decreased T1Gd, despite HTO, was observed. Before and after HTO, lateral femoral condyle T1Gd was higher than medial femoral condyle (MFC) T1Gd and tibial cartilage T1Gd was higher than that of femoral cartilage (P < 0.001). The MFC had the lowest T1Gd before and after HTO. Clinical scores all improved significantly (P < 0.01), KOOS Symptoms and QOL were moderately related to changes in MFC T1Gd.

Conclusions: dGEMRIC effectively detected differences in cartilage quality within knee compartments before and after HTO, but no changes due to HTO were detected. Hard- ware removal post-HTO seems essential for adequate T1Gd interpretation. T1Gd was correlated to improved clinical scores on a subscore level only. Longer follow-up after HTO may reveal lasting changes. ClinicalTrials.gov registration ID: NCT01269944.

116 Chapter 9 Introduction

In patients with medial compartment osteoarthritis of the knee, there is ‘silver level evidence’51 that the high tibial osteotomy (HTO) leads to reduction in pain and improvement in knee function. The goals of HTO, as first described by Jackson and Waugh in 1961188, are realignment of the limb, resulting in a transfer of weight bearing load from the degenerative medial compartment to the relatively uninvolved lateral compartment. Ideally, the need for a total knee arthroplasty is thus delayed for several years or even abolished. Over the past decade, HTO has regained in appreciation due to improved indications and guidelines, a better understanding of knee biomechanics, improved surgical techniques and implant characteristics and higher numbers of young active patients with symptomatic osteoarthritis of the knee48, 444. In contrast to the uniform application of HTO in clinical practice and the substantial biomechanical evidence to support this, the effects of HTO on cartilage quality are still largely unknown. A few in vivo studies have attempted to describe the effect of mechanical axis realignment on cartilage quality of the knee joint. During a ‘second look arthroscopy’ in 58 knees, partial or complete coverage with fibrocartilage was observed in 55% of the femorotibial joint surfaces 18 months after HTO199. A repair with ‘white scattering fibrocartilage’ was achieved in 34%, and 3 knees showed no regenerative change. In an earlier arthroscopic evaluation of 54 knee joints after HTO, it was observed that if adequate mechanical axis correction was obtained, repair of the ulcerated region was initiated by the surviving cartilage in the affected area and the cartilage bordering the affected area126. At one and one-half to two years after osteotomy, the ulcerated region was completely covered with fibrous and membranous tissue. Histological biopsies taken after HTO in two other studies demonstrated mild safranin O and collagen type II staining219, 425. However, performing a routine arthroscopy or taking intra-articular biopsies as a follow- up method is not a desirable method to determine cartilage quality after HTO. A non- invasive technique to quantitatively depict cartilage glycosaminoglycan content, and thus cartilage quality, is the dGEMRIC (delayed-gadolinium enhanced MRI of cartilage) technique. Since its first description in 200154, the dGEMRIC technique has been used successfully in orthopaedic and rheumatologic research as well as in clinical prac- tice291, 377, 414. The dGEMRIC technique is based on the electrical polarity of gadolinium (Gd(DPTA)2− ions) and glycosaminoglycans in the joint. After intravenous injection of the gadolinium, the Gd(DPTA)2− ions diffuse into the joint, and subsequently distribute between the negatively charged glycosaminoglycan side chains of the cartilage in an inverse relationship54. Due to electrical polarity, a high concentration of glycosaminoglycans results in a lower concentration of also negatively charged Gd(DPTA)2− ions and vice versa. As the local concentration of Gd(DPTA)2− ions within the cartilage is related to the longitudinal relaxation time (T1, ms), the value of T1 after administration of gadolinium

dGEMRIC in HTO patients 117 (T1Gd) is representative of local cartilage glycosaminoglycan content. A high T1 relaxation time after Gd administration thus is indicative of high glycosaminoglycan content. The aim of this study was to examine if changes in cartilage quality caused by a HTO, in patients with medial compartment osteoarthritis of the knee, were detectable with dGEMRIC and were correlated to changes in clinical parameters of osteoarthritis.

Methods

Patients Ten patients with medial compartment osteoarthritis of the knee were included in the study. Medial compartment osteoarthritis of the knee was defined as clinical osteoarthritis symptoms (pain, morning stiffness, decreased range of motion) in combination with radiographic features of medial compartment osteoarthritis (joint space narrowing of the medial articular facet, medial sclerosis on AP and/or lateral x-rays). Patients with previous surgery of the knee, total meniscectomy or anterior cruciate ligament (ACL) lesions were excluded. The indication for HTO surgery was further based on weightbearing radiographs of the complete lower extremity, in which a varus leg axis was seen in all patients. All included patients underwent a medial opening wedge HTO. In short, an osteotomy of the proximal tibia was performed, starting from the medial side. Next, the medial collateral ligament fibers were decompressed followed by realignment of the leg towards a slight valgus axis. The patient’s body weight was thus shifted from the medial articular facet to the lateral facet, where the cartilage is presumed to be relatively unaffected4. The newly achieved mechanical axis was maintained using a locking compression plate and screws. No bone graft was added. The HTO was followed by a standardized regime of limited load bearing with range-of-motion (ROM) exercises and subsequently controlled weight bearing. The study was performed at the University Medical Center Utrecht and the Sint Maartenskliniek Woerden, the Netherlands, according to the guidelines set out in the Helsinki declaration, approved by the local medical ethics committee (local reference: 07.117) and registered accordingly (NCT01269944). Written informed consent was obtained from all participants before inclusion in the trial.

Primary endpoint: change in cartilage quality after HTO All patients underwent two dGEMRIC examinations. The first dGEMRIC was performed before HTO. The second dGEMRIC was performed after bone healing and subsequent removal of HTO hardware. Hardware removal was required to obtain adequate image quality, to avoid overwhelming metal-induced artifacts [Figure 1]. A minimum period of 9 months was chosen

118 Chapter 9 Figure 1. Influence of high tibial osteotomy (HTO) hardware artifacts on dGEMRIC scan result. Left: dGEM-

RIC scan of a HTO patient with hardware in situ. Gross artifacts are seen influencing T1Gd measurements. Right: dGEMRIC scan in a HTO patient, in which hardware was removed prior to MRI. The right image is representative of all HTO patients, in whom hardware was removed. between HTO and removal of hardware, to allow for adequate bone healing. The ‘post’ MRI was made at a minimum of 1 month after removal of the hardware, to ensure an adequate period of full weight bearing between hardware removal and MRI. dGEMRIC Prior to the MRI, patients were injected intravenously with Magnevist® (Bayer HealthCare Pharmaceuticals Inc, Germany) at a dose of 0.2 mmol/kg body weight and were asked to walk a standardized route under supervision of a study investigator for at least 10 minutes. The walking route included several flights of steps, to facilitate uptake of the Magnevist® by the articular cartilage. After 90 minutes, MRI scanning of the affected knee joint was performed on a clinical 1.5-T MRI scanner (Achieva, Philips Healthcare, Best, The Netherlands) using a dedicated 8-element sense knee coil (Philips Healthcare, Best, the Netherlands) as a receiver. The pulse sequence used was a 3D sagittal tran- sient field echo (TFE) with 5 different inversion times (50, 150, 350, 650 and 1650 ms), resembling a previously described protocol267. The acquired voxel size was 0.625 x 0.625 x 3 mm3, 36 partitions were acquired with an in plane acquisition matrix of 256 x 232, resulting in a field of view (FOV) of 160mm (cranio-caudal) x 145mm (anterior-posterior) x 118mm (right-left). As a 3D acquisition was used, the acquired voxels were contiguous in the partition direction, so there was no gap between the slices as is often used with multiple 2D acquisition schemes. The repetition time was 10 ms with an echo time of 4.3 ms and a flip angle of 20˚. Including survey scans and a reference scan to measure the receive coil sensitivity profile, the total examination time was about 25 minutes. The average T1 after administration of contrast agent (T1Gd) per ROI was calculated using voxel by voxel curve fitting with the Levenberg-Marquardt optimization method, using in-house developed software (imageXplorer).

dGEMRIC in HTO patients 119 T1Gd measurement in femoral condyle and tibial plateau cartilage The original 3D scan was used to draw four Regions Of Interest’s (ROIs) of cartilage only. The sagittal slices of the 350ms sequence were used as they provided optimal visual distinction between the cartilage and surrounding tissues (bone, meniscus, synovium). The following ROI’s were distinguished: medial femoral condyle (MFC), lateral femoral condyle (LFC), medial tibial plateau (MTP) and lateral tibial plateau (LTP). Depending on the width of the knee, 4 or 5 adjacent sagittal slices, depicting a cartilage width of 12 or 15 mm within the selected femoral or tibial compartment, were selected to create the

ROI [Figure 2-A]. Using these ROI’s, a ‘T1Gd map’ of the corresponding knee compartment was created [Figure 2-B]. The average T1 of each ROI, and of all ROI’s combined, was used to compare the glycosaminoglycan content in the different knee compartments

Fig 2-A Fig 2-B 1000 ms

A P M 500 ms

Fig 2-C 0 ms

Figure 2: 3D regions of interest (ROI’s) were generated of various sections of the knee and used for T1Gd comparison before and after HTO. Figure 2-A: Designation of the cartilaginous area on a sagittal slice of the lateral femoral condyle and tibial plateau. Depending on the width of the knee, 4 or 5 adjacent sagittal slices were used to create the separate 3D ROI’s: the medial femoral condyle (MFC), the lateral femoral condyle (LFC), the medial tibial plateau (MTP) and the lateral tibial plateau (LTP). Figure 2-B: T1-mapping

was performed in each ROI. This section demonstrates T1Gd distribution throughout a sagittal slice, used

for creation of the LFC and LTP ROI. Using these sagittal slices, a mean T1Gd could be calculated for each ROI. Figure 2-C: A sagittal slice, used for creation of the LFC ROI is demonstrated. The MFC and LFC ROI’s were divided into three different sub-ROI’s, using the anterior and posterior meniscal border as anatomical landmark: Anterior (A), Middle load bearing (M) and Posterior (P) femoral cartilage.

120 Chapter 9 before and after HTO. To assure that the ROI’s were in the same area before- and after HTO, in addition to positioning the knee in the same knee coil as the scan before HTO, the center of the knee, defined as the slice depicting the sagittal crossing of the anterior cruciate ligament (ACL) and the posterior cruciate ligament (PCL), was used as anatomical reference. ROI measurements were performed by two different observers to enable reproducibility testing.

T1Gd distribution throughout the femoral condyles To distinguish smaller cartilage regions within the abovementioned ROI’s, the MFC and LFC ROI were divided in an anterior, a middle (load bearing) and a posterior sub-ROI. The anterior meniscus served as the border between the anterior and middle (load bearing) sub-ROI, the posterior meniscus served as a border between the middle (load bearing) and posterior cartilage sub-ROI264 [Figure 2-C].

Secondary endpoint: change in clinical scores To evaluate clinical changes, all patients filled out the VAS (visual analogue score for pain)322, Knee Osteoarthritis Outcome Scale (KOOS)338 and the Knee Society Clinical Rating System (KSCRS)182 before HTO, 3 and 6 months after HTO, and after subsequent hardware removal. Correlation analysis between changes in clinical scores (KOOS, KSCRS and VAS) and changes in T1Gd (MFC, LFC, MTP and LTP) after HTO were performed using the statistical analysis described below.

Radiographic analysis Anteroposterior (AP) and lateral views of the knee were acquired to depict the grade of knee OA. Weightbearing radiographs of the complete lower extremity were acquired to measure the pre- and postoperative mechanical leg axis defined as the angle between the femoral and tibial mechanical axes. Furthermore, the posterior tibial slope (the angle between the slope of the tibial plateau and the line drawn perpendicular to the tibial anatomic axis, in lateral view) was measured semi-automatically as described earlier335 and compared before and after HTO. Measurements of femorotibial angle and tibial slope were performed using Philips Image Viewer Image Viewer R 11.4. Correlation analysis between the change in mechanical axis and tibial slope before and after HTO, and changes in T1Gd within the MFC, LFC, MTP and LTP after HTO were performed using statistical analysis described below.

Statistics Statistical analyses were performed using the statistical software SPSS 15.0 for Windows (SPSS Inc., Chicago, Il, USA). Data was tested for normality using the Kolmogorov–Smirnov test and for homogeneity of variances using Levene’s test of equality of variances. As-

dGEMRIC in HTO patients 121 suming normality and equal variances, T1Gd values before and after HTO were compared using paired T-testing. Clinical scores were compared using repeated measurement testing. Pearson’s correlation coefficient was used for correlation analysis of changes in MFC,

LFC, MTP and LTP T1Gd (% of T1Gd before HTO) with changes in clinical scores (% of score before HTO), mechanical axis and tibial slope. Inter-observer and intra-observer agree-

ment of T1Gd within the ROI’s were evaluated by two observers in three knee joints using intra-class correlation (ICC) testing. Data were expressed as means ± 95 % confidence intervals (C.I.). P≤0.05 (*) was considered statistically significant.

Results

Patient characteristics 8 male patients and 2 female patients with medial compartment osteoarthritis of the knee were included. Mean age was 53 years (range, 43-64 years), all patients were Cauca- sian. Kellgren-Lawrence score was grade II (1 patient), grade III (7 patients) and grade IV (2 patients). Mean change in knee alignment after HTO was 10.0˚ (range, 7.8˚ -15.3˚). The posterior tibial slope increased by 1.3˚ (from 8.7˚ to 10.0˚). In one patient, hardware was removed 22 months after HTO due to delayed bone healing as seen on x-rays, followed by the dGEMRIC [Table 1].

Table 1: Patient characteristics Patients 8 male, 2 female Age 53 years (range; 43 – 64 years) Weight 85.8 kg (range; 72.0 – 110 kg) Length 1.78 m (range; 1.66 – 1.95 m) Body Mass Index 28.2 (range; 25.5 – 32.1) Osteoarthritis grade (Kellgren-Lawrence) 1 grade II, 7 grade III, 2 grade IV Time HTO – hardware removal 12 months (range; 10 – 22 months)

° varus before HTO 7.1° (range; 2.0° / 13.8°) ° varus after HTO -3.6° (range; -5.0° / 1.0°) ° change in mechanical axis after HTO 10.0° (range; 7.0° /15.3°)

° posterior tibial slope before HTO 8.6° (range; 5.1° / 12.0°) ° posterior tibial slope after HTO 9.9° (range; 6.5° / 15.3°) ° change in posterior tibial slope 1.3° (range; -0.3° / 3.4°)

122 Chapter 9 Differences in 1T Gd before and after HTO

Intra-class correlation (ICC) reliability analysis for inter-observer agreement in T1Gd was 0.934 (95% CI 0.816-0.977), for the intra-observer agreement it was 0.960 (95% CI 0.885-0.986), demonstrating an excellent reproducibility. Following HTO and hardware removal, the mean T1Gd of all ROI’s combined was lower than before HTO (before: 533, 95% CI 474-632; after: 466, 95% CI 399-533; p=0.11) [Figure 3-a]. Similar mean lower values were observed for the individual compartments (MFC p=0.148, LFC p=0.532, MTP p=0.118, LTP p=0.197). Interestingly, the postoperative femoral cartilage T1Gd values seemed to resemble their pre-operative values to a greater extent (95% medial, 97% lateral) than the tibial cartilage values (85% medial, 84% lateral) [Figure 3-b]. Within the medial femoral condyle, posterior cartilage T1Gd decreased following HTO (p=0.047).

No correlation was detected between changes in T1Gd within MFC, LFC, MTP or LTP and changes in mechanical axis or tibial slope (MFC p=0.73 r=-0.13, LFC p=0.30 r=-0.39, MTP p=0.38 r=-0.32, LTP p=0.48 r=-0.27).

* 700 600 * 500 400 * * 300 T1(ms) 200 100 0 pre-HTO post-HTO

Figure 3-A. Mean T1Gd values of all compartments combined, before and after high tibial osteotomy (HTO) in 10 patients (before vs. after, p=0.11). Bars represent means ± 95% CI.

Figure 3-B. Mean T1Gd values of the ROI’s before and after high tibial osteotomy (HTO) in 10 patients. The decrease in T1Gd was higher in tibial (15% decrease in medial tibial plateau T1Gd, 16% decrease in lateral tibial plateau T1Gd) than in femoral cartilage (5% decrease in medial femoral condyle T1Gd, 3% decrease in lateral femoral condyle T1Gd), although the differences were not significant within the separate ROI’s.

Before and after HTO, medial femoral cartilage had a lower T1Gd than lateral femoral cartilage (before HTO p=0.007; after HTO p=0.015). Tibial cartilage had a consistent higher T1Gd than femoral cartilage in the medial as well as lateral compartment (p<0.001). Bars represent means ± 95% CI. MFC=medial femoral condyle, LFC=lateral femoral condyle, MTP=medial tibial plateau, LTP=lateral tibial plateau.

Regional differences in 1T Gd distribution

Before HTO, T1Gd of medial femoral cartilage (mean 379, 95% CI 332-426) was lower than that of lateral femoral cartilage (mean 423, 95% CI 387–459; p=0.007). Femoral cartilage had a lower T1Gd than the opposing tibial cartilage (MTP mean 748, 95% CI 547-909, LTP mean 662, 95% CI 571-753; p<0.001). Within the medial femoral condyle surface,

dGEMRIC in HTO patients 123 cartilage of the anterior aspect of the femoral condyle (mean 371, 95% CI 330-412) had

a lower T1Gd than the posterior femoral cartilage (mean 407, 95% CI 461-453) (p=0.036).

Middle (load bearing) cartilage had the lowest T1Gd in the medial (mean 364, 95% CI 304-424) as well as lateral femoral condyle surface (mean 412, 95% CI 380-444).

After HTO, medial femoral cartilage T1Gd (mean 356, 95% CI 325-387) was lower than that of the lateral femoral cartilage (mean 409, 95% CI 369-449; p=0.015) and femoral

cartilage had a lower T1Gd than tibial cartilage (MTP mean 567, 95% CI 440-694; LTP mean 531, 95% CI 383-679; p=0.008), similar to before HTO [Figure 3-b]. In the MFC, anterior

cartilage T1Gd (mean 365, 95% CI 334-396; p=0.001) and posterior cartilage T1Gd (mean 376, 95% CI 333-419; p=0.001) were greater than middle (load bearing) femoral cartilage (mean 306, 95% CI 276-336). In the lateral femoral condyle, no significant differences between the sub-ROI’s were observed post-HTO.

Improvement of clinical scores Clinical scores improved significantly following HTO; the VAS for pain, the KSCRS and KOOS scores for pain, symptoms, ADL and QOL all improved (p<0.01) except for KOOS sports (p=0.15). A moderate correlation existed between improvement in KOOS subscore for Symptoms (r=0.63, p=0.05) and KOOS Quality of Life (r=0.63, p=0.05) with %

change in MFC T1Gd, and a weak correlation existed for improvement in KOOS overall

score (r=0.35, p=0.03) with % change in MFC T1Gd [Table 2]. No significant correlations

were present for clinical scores and changes in LFC, MTP or LTP T1Gd.

Table 2: Clinical scores (KOOS, KSCRS, VAS) of HTO patients (n=10), and correlation of change in clinical

scores to change in medial femoral condyle (MFC) T1Gd. The clinical scores are displayed as mean ± 95% C.I. Before 3 months 6 months After p-value Pearson’s r High Tibial after HTO after HTO hardware (Δ clin. score vs.

Osteotomy removal Δ MFC T1Gd) (HTO) (>9 months) KOOS Pain 48 (37-58) 59 (52-66) 76 (68-85) 82 (72-91) p<0.001 n.s. KOOS Symptoms 56 (45-66) 62 (52-72) 74 (63-84) 79 (68-89) p=0.002 r=0.63, p=0.05 KOOS ADL 58 (47-70) 67 (57-76) 75 (66-85) 83 (73-92) p=0.008 n.s. KOOS Sports 34 (11-58) 36 (19-52) 42 (27-57) 55 (37-73) p=0.157 n.s. KOOS QOL 31 (24-39) 32 (22-41) 47 (32-62) 58 (44-73) p=0.004 r=0.69, p=0.03 KOOS Overall 46 (35-57) 51 (45-57) 63 (53-72) 71 (60-83) P=0.001 r=0.35, p=0.04

KSCRS 76 (68-83) 75 (66-85) 90 (84-96) 90 (83-99) p=0.008 n.s. VAS 63 (52-74) 37 (27-47) 20 (11-29) 11 (6-17) p<0.001 n.s.

124 Chapter 9 Discussion

Although the HTO seems to have regained popularity48 as treatment for medial compartment osteoarthritis of the knee due to effectiveness in pain reduction, its effect on cartilage quality is still largely unknown. In the current study, dGEMRIC was used to detect changes in cartilage quality before and after HTO, however no changes due to HTO were detected. Pre-existing differences between medial and lateral compartment cartilage remained and in all investigated knee compartments cartilage degradation continued despite HTO. In the study of Parker et al313, dGEMRIC was also used to evaluate changes following HTO. Interestingly, several findings are similar to ours. Lateral femoral cartilage had a higher T1 than medial femoral cartilage pre and postoperatively, and changes following HTO varied per patient but overall a decrease in T1 was observed. Similar to the observation of Parker et al, the medial tibial plateau had a slightly higher T1Gd than the lateral tibial plateau. In the study of Parker, the greatest decrease in T1 was observed at 6 months, when patients had just resumed weight bearing. Loss of GAG due to the immobilization was mentioned as a plausible cause. In the current study, we removed the hardware after bone healing prior to the second MRI, which in our opinion decreases the risk of inadequate T1 measurements due to metal-induced artifacts [Figure 1]. The postoperative scan was made at a minimum of 9 months after HTO, when the patients had resumed full weight bearing of their knee joints. T1Gd was calculated of medial and lateral femoral and tibial cartilage, as well as of multiple sub-ROI’s, which provides additional information about the sub-regional T1Gd distribution on sagittal slices throughout the femoral surface. In addition, correlations were measured between T1Gd and clinical scores, mechanical axis and tibial slope. In contrast to expectations, clinical scores improved without similar improvement in

T1Gd, except for a moderate correlation of MFC T1Gd with the KOOS subscores for ‘Symp- toms’ and ‘Quality of Life’. In an earlier HTO study, clinical improvement was also shown not to correlate with repair tissue quality299. It is unclear why a low correlation often exists between clinical symptoms of OA and histological15, 211, radiographic390 or MRI100, 234 parameters of cartilage quality. The clinical importance of cartilage quality is well docu- mented in longitudinal follow-up series, in which the prevalence of knee pain is shown to correlate with cartilage volume decrease443 and radiographic severity of OA166. In this study, clinical symptoms may have been influenced by changes in serum markers of cartilage metabolism35. Mere placebo effects of the HTO seem unlikely, as improvement in pain and knee function following HTO have been described in systematic review of several clinical trials51. It can be stated with reasonable certainty that following HTO the medial compartment is covered with (a thin layer) of fibrocartilage199, 219, 300, 425, which has inferior biomechanical

dGEMRIC in HTO patients 125 5, 36 properties in comparison to hyaline cartilage . The low T1Gd values currently observed can well be seen in the light of these findings. However, it remains difficult to explain

why T1Gd actually decreases. The intra-articular environment may be of influence, and at the time of osteotomy contains a variety of chondrodegradative cytokines345. Follow- ing osteotomy, increases in proteoglycan epitopes299, FGF-2302 as well as procollagen peptides213 have been observed, which would be in favor of cartilage regeneration143, 345. Nevertheless, chondrodegradative cytokines present in the subchondral bone of the osteoarthritic knee177 are still present and may continue to inhibit cartilage regeneration and overrule regeneration. Further, the femoral chondrocytes may have become hypertrophic during the development of osteoarthritis421 and thus produce lower amounts of glycosaminoglycans, or no glycosaminoglycans at all. It may thus take longer for the non-hypertrophic chondrocytes to compensate for the loss in GAG production and resume matrix formation. Minimum duration before hardware removal was 10 months, with an average of 12 months in our study. One patient had hardware removed at 22 months owing to doubtful bone healing on radiography. Time to bony healing is reported to vary from 12 weeks103 to 1 year, assessed by radiographs, CT, and MRI imaging48. Although in theory complete remodeling of the medial cortex may not yet have occurred, full weight bearing was initiated early in all patients and no adverse events after hardware removal were observed.

The mean mechanical leg axis in this patient group was in accordance with the recommended valgus leg alignment post-HTO274, 373. The mean change in femorotibial angle was 10 degrees which seems to be large enough to expect significant changes in cartilage loading patterns represented in the dGEMRIC measurements. Nevertheless, a

correlation between change in mechanical axis and change in T1Gd was not detected. Possibly, the variation in degrees of axial realignment outweighs the subtle changes in

T1Gd. In any case, it can be assumed that the medial knee compartment was adequately decompressed by releasing the distal fibers of the medial collateral ligament4. The increase in tibial slope after HTO is in agreement with previous observations428. One would expect that an increase in tibial slope decreases forces directed to the posterior 3 femoral cartilage , leading to at least steady or increased T1Gd, instead of a decrease as observed in the current study. Possibly, as the entire medial condyle was unloaded, the regional changes were greater than changes due to unloading of the posterior cartilage, caused by decreased contact pressure as a result of the increase in tibial slope.

Differences between medial and lateral compartment T1Gd are not unique to osteoarthritic knees or those with a varus or valgus axis. Healthy subjects with varying activity levels398, medial meniscectomy patients 1-6 years after meniscectomy104 and ACL-defi- 291 cient patients have been reported to have a lower medial compartment T1Gd. While

126 Chapter 9 441 studying osteoarthritis patients, Williams et al found a lower T1Gd in the compartment with the narrowest joint space width. The lowest T1Gd was observed medially in knees with a varus mechanical leg axis, while a lower lateral T1Gd was observed in knees with a valgus mechanical leg axis. As the lowest T1Gd values were currently observed medially before and after HTO, considering the influence of altered cartilage stress due to an axial realignment262, changes in T1Gd may possibly occur during longer follow-up. There are several limitations to this study. First, this study focuses on relatively short term effects of mechanical axis change after HTO. Ideally, a control cohort with longer follow-up of non-operatively treated osteoarthritis of the medial knee compartment would have been included to evaluate the natural progress of cartilage degeneration. Although the patient number of this feasibility study is relatively small, this is the first study in which the hardware was removed before performing a post-HTO dGEMRIC. From our experience [Figure 1] it seems advisable to take hardware artifacts into account when using dGEMRIC for follow-up. The general consensus is that osteotomy provides best results in mild to moderate unicompartmental osteoarthritis48, 373. Although out of 10 patients two had radiological grade IV osteoarthritis, the remaining population was representative for the general HTO population. In future studies, an interesting control group may be formed by patients undergoing joint distraction in late stage osteoarthritis. Recently, Intema et al184 demonstrated that joint distraction during 2 months resulted in an increase in joint space width and cartilage thickness assessed by MRI, while clinical scores increased, similar to the HTO. Due to the characteristics of the MRI method used, cartilage quality was not evaluated. dGEMRIC as a tool to evaluate cartilage changes has gained attention during the past decade and is increasingly being used to gain fundamental information about cartilage-related disorders, formerly requiring invasive procedures as arthroscopy291 and histological biopsies. From this feasibility study it can be concluded that dGEMRIC can effectively be applied to assess cartilage quality of HTO patients, provided the hardware is removed after consolidation of the osteotomy. Although unaffected by HTO, this study confirms that the cartilage glycosaminoglycan content of the medial femoral condyle is lower than that of the lateral condyle, and that femoral cartilage glycosaminoglycan content is lower than that of the tibial plateau. Further, differences in T1Gd between anterior, middle (load bearing) and posterior femoral cartilage are demonstrated. Although dGEMRIC should be further investigated and its value for assessment of cartilage quality compared to other techniques such as T1 Rho395, dGEMRIC seems a valuable method to longitudinally evaluate changes in cartilage quality following cartilage related surgery97, 414. This feasibility study is valuable for future studies, in which dGEMRIC will be used to compare effects of knee distraction versus HTO versus total knee replacement.

dGEMRIC in HTO patients 127

Summary

In this thesis, the influence of various internal and external factors on cell phenotype was assessed, and the therapeutic effects of joint homeostasis-modulating therapies were evaluated. Fundamental and clinical approaches were used to improve knowledge on how to obtain an optimal environment for re-implantation of chondrocytes during autologous chondrocyte implantation (ACI).

External factors influencing cartilage matrix metabolism Collagen is the membrane material predominantly used to cover a chondrocyte defect before injection of chondrocytes in ACI. In the matrix-assisted ACI (MACI), collagen is used as bearer of chondrocytes during implantation. Concerns have risen that hypertrophic differentiation may occur due to contact between collagen and the chondrocytes, but it is unknown if hypertrophic differentiation occurs during (M)ACI. Hypertrophic differentiation is an undesirable process in which remodeling and mineralization of the extracellular matrix (ECM) occurs, eventually leading to invasion of blood vessels and apoptotic chondrocyte death. In Chapter 3 an in vitro model resembling the MACI was used to evaluate the influence of a culture insert coated with collagen type I, type II, or a control (with no coating) on femoral chondrocyte regenerative capacity. Chondrocytes of 8 healthy human donors were isolated, expanded and cultured for 28 days on collagen- coated or uncoated inserts. The regenerated neocartilage was analysed for biochemical and histological composition, immunohistochemical staining and chondrogenic gene expression. Neocartilage quality was highest when a collagen type II coating was used. This result was deduced from the higher histological scores, glycosaminoglycan (GAG) and DNA content in comparison with the results when no coating was used. A higher turnover also seemed to take place, as GAG release into the culture inserts was higher for both collagens. However, net degradation was not observed, suggesting that this release reflected an inability to retain newly synthesized proteoglycans. This result was supported by a lack of differences in expression of MMP13 and DDR2. The similar α2β1-integrin expression between collagen types I and II, and the absence of significant differences in GAG and DNA between the two collagen types, support the use of both collagen types as a two- or three-dimensional scaffold for cartilage re-implantation. In addition to influencing cell-matrix interactions, another approach to prevent the onset of hypertrophic differentiation in ACI may be through addition of external factors during the in vitro culture phase or after implantation of the chondrocytes in vivo. In mesenchymal stromal cells (MSCs), parathyroid hormone (PTH) was shown to effectively inhibit hypertrophic differentiation while stimulating chondrogenic differentiation. The influence of PTH on healthy articular chondrocytes in a MACI-like environment is unknown. In Chapter 4, using healthy articular femoral chondrocytes of 6 human do-

Summary 131 nors, the influence of parathyroid hormone (PTH) addition on enhancement of cartilage matrix metabolism and inhibition of hypertrophic differentiation was assessed using the culture model described above. In order to evaluate PTH effects, PTH was added at 0.1 and 1.0 µM from day 0, day 9 or day 21 up to the end of the culture time on day 28. At 0.1 and 1.0 µM, PTH resulted in a decreased DNA content in comparison with the control, while GAG/DNA concentrations were maintained. Expression of collagen type II was lower after addition of PTH. Markers of hypertrophic differentiation core-binding factor alpha 1 (Cbfa-1/Runx-2) and collagen type X were not influenced. Thus it is concluded that PTH-mediated inhibition of hypertrophic differentiation and subsequent matrix quality enhancement is more applicable to MSCs than to the healthy articular chondrocytes used here.

Internal factors influencing cartilage matrix metabolism In addition to external factors, internal factors may also influence chondrocyte regenerative capacity. Earlier, cartilage from young animals was shown to possess an intrinsic repair capacity, while that of older animals did not. Published data on the in vitro regenerative capacity of young chondrocytes thus far are ambiguous. Neverthe- less, recent reports show that juvenile cartilage is already being used as an allograft for talus cartilage defect repair. In Chapter 5, we therefore aimed to evaluate the in vitro regenerative capacity of young talar chondrocytes, in comparison to that of adult talar and femoral chondrocytes. Although regeneration was faster, as shown by a higher GAG and DNA content at day 9 for the chondrocytes from young donors, there was no difference between final GAG and DNA content at 28 days in cartilage generated by juvenile and adult talar chondrocytes. Expression of hypertrophic differentiation markers (Cbfa1/ Runx2, collagen type X) was highest in young talar chondrocytes and adult femoral chondrocytes, while histological quality was the highest in cartilage generated by femoral chondrocytes. Apparently, expression of markers for hypertrophic differentiation may not necessarily result in lower quality cartilage. The discrepancy between these observations and the earlier observed spontaneous regeneration of cartilage defects in young animals indicates that external factors (for example, joint homeostasis) may play a larger role than internal cellular factors.

Principles of a joint homeostasis-influencing therapy In order to improve the signs and symptoms of cartilage degeneration, various intra- articular therapies have been developed. The goal of these therapies is to modify joint homeostasis through external factors. In autologous conditioned serum (ACS), autologous blood with increased concentrations of Interleukin-1 Receptor Antagonist (IL-1RA) is injected into the OA joint. This therapy aims at lowering proposedly elevated levels of IL-1 in the synovial fluid of OA patients. However, the exact composition of ACS and its

132 Chapter 10 effects on cartilage metabolism are unknown. This information does seem relevant, as ACS may contain chondrodegradative factors in addition to chondroprotective factors. Nevertheless, ACS has already been the subject of two clinical trials, which demonstrated varying effectiveness in reduction of the signs and symptoms of OA patients. In order to elucidate the ACS mechanism, the following were evaluated in 22 osteoarthritis patients in Chapter 6: the cytokine composition of ACS, its effect on the proteoglycan metabolism of cartilage explants, and the synovial fluid (SF) cytokine levels after intra-articular administration. The cytokine content appeared to consist of not only the chondroprotective cytokines IL-1RA, IL-10 and TGF-β, but also of chondrodegradative cytokines IL-1 β, Oncostatin-M and TNF-α. Proteoglycan metabolism of cartilage explant cultures was not different in the presence of ACS when compared to unconditioned serum. However, inhibition of TNF-α did not affect the proteoglycan metabolism of cartilage explants cultured with ACS. In comparison with the baseline, ACS injection did not alter SF cytokine levels. The equivocal clinical results of ACS therapy are thus reflected in this in vitro study, demanding further development of ACS before clinical application.

Using patient-reported outcome measures (PROMs) to evaluate placebo effects of ACS treatment An injectable therapy for osteoarthritis may exert desired or undesired non-pharmacological effects on a patient’s signs and symptoms. Consecutively administering placebo and ACS to the same patient may aid in distinguishing pharmacological from placebo effects. In Chapter 7, ACS was administered to 20 OA patients who had earlier received intra-articular injections with physiological saline (placebo). ACS did not lead to a similar improvement in OA signs and symptoms, as reported during earlier placebo treatment. Although the VAS for pain improvement was similar to that after placebo treatment, the following results did not improve: knee injury and osteoarthritis outcome score (KOOS), knee society clinical rating scale (KSCRS) and Western Ontario and McMaster Universi- ties osteoarthritis index (WOMAC). The study further demonstrates that ‘response shift’ aids in understanding the development of a patient’s longitudinal perception of disease implications. It illustrates the ability of OA patients to adapt to the consequences of his or her illness, since a more positive clinical score is given than when analysed in retrospect after treatment.

Past and present tools to evaluate the success of cartilage improving interventions Histological quality is considered an important outcome parameter for the evaluation of cartilage tissue, either when directly isolated from an OA knee (after experimental treatment of an artificially (in vivo) created cartilage defect), or after in vitro creation of neocartilage. Thus far, over 20 histological scores have been developed. Most histo-

Summary 133 logical scores are non-validated, or they are sometimes inadequately applied to types of tissue that they were not intended to evaluate. In Chapter 8, we presented an overview of histological scores for osteoarthritic, in vivo repaired and tissue-engineered cartilage. The goal of this overview was to improve knowledge on the existing validated scores and to provide a basis for consensus between research societies. The Histological-His- tochemical Grading System (HHGS) or the Osteoarthritis Research Society International (OARSI) system (which seems easier in use for inexperienced scorers) are recommended for evaluation of normal to osteoarthritic cartilage tissue. The ICRS-II score is the most suitable for in vivo repaired cartilage. For evaluation of in vitro engineered cartilage, the ‘Bern score’ is preferred. Obtaining a biopsy to assess histological cartilage quality is generally undesirable in clinical studies, especially when the cartilage quality is to be evaluated at multiple timepoints after an intervention. MRI techniques such as the delayed Gadolinium-Enhanced MRI of Cartilage enable non-invasive evaluation of cartilage proteoglycan content as an indicator of cartilage quality. In high tibial osteotomy (HTO) for treatment of medial compartment osteoarthritis of the knee, the mechanical leg axis is altered, thus unloading the damaged medial femoral condyle and loading the relatively unaffected lateral femoral condyle. In Chapter 9, dGEMRIC is used to measure the effect of the HTO on cartilage proteoglycan content in a pilot study of 10 patients with medial compartment osteoarthritis of the knee. Clinically, all 10 patients improved following surgery, even though neither femoral nor tibial cartilage quality changed significantly. Differences in proteoglycan distribution between medial and lateral femoral condyles were demonstrated prior to HTO as well as afterwards. Provided that the hardware is removed, dGEMRIC thus seems applicable for longitudinal follow-up of cartilage quality in HTO patients.

134 Chapter 10 Discussion

Discussion

The following promising targets are recommended for further exploration: a) optimizing the outcome of cartilage repair by modulating cell phenotype, b) influencing joint homeostasis after trauma and during OA, and c) selecting existing and new evaluation tools for evaluation of cartilage repair. The basis for these recommendations is discussed below, together with background information on several relevant issues.

The influence of scaffold coating and composition During in vivo repair of cartilage defects, several materials can be used to keep re-implanted chondrocytes in a cartilage defect and support the regenerating tissue. Ideally, the material used ensures maintenance of chondrogenic gene expression, or it may even reverse the de-differentiation which often occurs during the earlier in vitro culturing phase. In the autologous chondrocyte implantation (ACI), a collagen membrane (second generation ACI) is sutured into the surrounding cartilage to cover the re-implanted chondrocytes. In MACI (third generation ACI), a collagen membrane is used as to bear the chondrocytes. After inversion of the membrane/chondrocyte combination, it is glued to the subchondral bone using fibrin glue. The current investigations have concluded that collagen types I and II may both be used with comparable chondrogenic action. Practi- cal considerations may play a role in choosing collagen type I and not collagen type II, as the former may be obtained at a lower price from most manufacturers. Although one might argue that we focused on cell-matrix interactions in an in vitro setting, these same interactions are likely to occur when chondrocytes are exposed to the collagen membrane sutured into the defect. Earlier concerns regarding accelerated degradation due to onset of hypertrophic differentiation and increased DDR2 expression have not been confirmed in the current investigations. It is somewhat unclear why collagen type III is used in the collagen type I/III MACI procedure. No reports seem to exist in which collagen type III alone stimulates chondrogenic differentiation. Although collagen type III was shown to be expressed during early transformation of an osteoid/fibrin clot to undifferentiated mesenchyme during spontaneous healing of cartilage defects271, the tissue eventually became fibrocartilaginous. During comparisons of the collagen type II and type I/III, ‘more roundish cells and higher production of proteoglycans’ were observed on collagen type II membranes, in comparison to collagen type I/III membranes131. However, it is hypothesized that ‘less differentiated chondrocytes, like those we found on the type I/III scaffolds’ are more ‘mo- tile and capable of dividing’ and thus might lead to ‘better fixation of the biocomposite to the cartilage defect’. The biocomposite is mentioned to be ‘so far developed that it can be used for the repair of human knee cartilage defects, and this is currently being established in a clinical trial’102, 131.

Discussion 137 Our investigation did not focus on the structural properties of the membrane, and the collagen-coated polytetrafluoroethylene (PTFE) membrane that we used may have evoked different cell-matrix interactions than a pure collagen membrane. Also, our model was in fact two-dimensional (2-D). Even though a multi-layered cell sheath was formed (as in MACI49), growth of the chondrocytes into the membrane did not occur. 3-D structures may be preferable to 2-D structures since the contact surface area between chondrocytes and surrounding matrix is greater. In the 3-D hydrogel “CaReS”, (Cartilage Regeneration System, Arthro Kinetics Biotechnology, Austria) 362, chondrocytes are grown in a collagen type I matrix and cultured in autologous serum prior to re-implantation. Gene expression reveals higher levels of collagen type II and aggrecan, with a more spherical morphology (instead of elongated) than that for cells grown on MACI membranes in autologous serum7. Although improvement in clinical362 and radiologi- cal436 parameters was observed, no long term follow-up or histological biopsy results are available thus far. Nevertheless, 3-D hydrogels seem promising.

The influence of cell phenotype on hypertrophic differentiation Hypertrophic differentiation is the process in which chondrocytes deviate from their matrix producing and proliferating role towards a phase in which they express signals that stimulate osteoblast and osteoclast invasion. This process is influenced by growth factors (fibroblast growth factor, FGF), hormones (parathyroid hormone related peptide, PTHrP) and secreted polypeptides (Indian hedgehog, IHH), expressed through an alteration in transcription factors (Cbfa1/Runx2). It results in altered matrix composition (collagen type X). During the development of osteoarthritis, hypertrophic differentiation occurs and it is partially responsible for cartilage degradation and osteophyte formation. However, hypertrophic differentiation has also been mentioned to endanger cartilage repair pro- cedures464. During our observations, this did not seem to play a major role. Collagen type X expression was negligible at the immunohistochemical level, even though gene expression of Cbfa1/Runx2 and collagen type X occurred. The absence of overwhelming hypertrophic differentiation may be explained by the source of chondrocytes, because we used healthy and not OA chondrocytes in our investigations. Low or absent Cbfa1/ Runx2 and collagen type X expression was observed. The cause may be adequate blocking of the hypertrophic differentiation pathway in healthy chondrocytes by intrinsic factors404, even after multiple passages 393, 450. However, instances may occur when chondrocytes are harvested from a joint that displays some degenerative features, or when only osteoarthritic chondrocytes are available for harvesting in ACI. PTH(rP) pre- treatment may be applicable in these instances, as osteoarthritic chondrocytes have been shown to express collagen type X, expanded or non-expanded451.

138 Chapter 11 Inhibiting hypertrophic differentiation may also be interesting during microfracture. The MSCs which migrate into the defect form fibrocartilage with hypertrophic characteristics 41, 202, 198, 207. Thus inhibiting this process may improve cartilage quality. Recently, hypertrophic differentiation during microfracture was effectively inhibited by the use of thrombospondin-1 in a miniature pig cartilage defect model135; however, chondrogenesis was limited. PTH treatment during microfracture may possibly inhibit hypertrophic differentiation while stimulating chondrogenesis. The cell source is apparently important in order to inhibit hypertrophic differentiation. In earlier reports, juvenile cells have been postulated to generate higher quality neocartilage than adult cells, although overall data are conflicting. Allologous transplantation of these cells, which are thought to have ‘absent immunoreactivity’, has been mentioned to have therapeutic potential for treatment of talar cartilage defects1. In our investigations, juvenile chondrocytes expressed high levels of hypertrophic differentiation markers. The quality of the repaired cartilage was not inferior, and faster regeneration was observed at day 9 in comparison to adult chondrocytes. The relevance of the higher expression of hypertrophic differentiation markers remains unclear. Three possible explanations are as follows: a) hypertrophic differentiation is essential during juvenile chondrocyte proliferation, b) juvenile chondrocytes react differently in an in vitro environment, or c) in a juvenile joint environment more factors circulate which inhibit this process. Our observations and others published earlier show conflicting data. It is noteworthy that an allologous product based on ‘juvenile minced cartilage’ (DeNovo ET, ISTO Technologies, St. Louis, Missouri) is being applied clinically for talus cartilage defect repair1, 155, apparently disregarding these concerns.

Modulating joint homeostasis after trauma The intra-articular environment is probably at least as important for maintenance of cartilage integrity as is the cell phenotype for cartilage repair. After trauma an altered joint environment influences chondrocyte regenerative capacity452. A ‘normal’ protective intra-articular environment may turn into a chondrodestructive environment with high levels of chondroprotective (IL-10, IL-1RA) cytokines as well as chondrodegradative (IL-1β, TNF-α) cytokines62, 185. It may possibly even predispose to early onset OA. Altering cytokine levels may reverse this process and prevent cartilage degradation, and it is thus commonly applied in rheumatoid arthritis74, 280. However, developing the ideal mix of cartilage-stimulating or degradation-blocking cytokines and growth factors is not sufficient. Adequate therapeutic levels may not be reached due to a short half-life and fast clearance from the joint. This is currently demonstrated for IL-1RA. Injection of ACS containing IL-1RA at >1000pg/ml did not elevate the synovial fluid IL-1RA levels, even after repeated injections343. In another study of 11 patients with an anterior cruciate ligament rupture, a significant reduction of IL-1 was not reached even though IL-1RA proved

Discussion 139 effective in reducing pain and improving knee function in comparison to placebo221. Furthermore, cytokines and growth factors may exert adverse effects if they enter the systemic circulation, which is a concern for example in the case of IGF (increased risk of prostate cancer 65). A carrier is thus needed to ensure adequate delivery and release of the cytokines into the joint. For example, retention of IGF-1 in the extracellular matrix was enhanced after intra-articular injection by adding a heparin-binding domain273. Gelatin hydrogel microspheres are studied for bone morphogenetic protein (BMP) delivery in bone tissue engineering203 and for MSC and TGF-β delivery in cartilage tissue engineering311. These microspheres may also be considered for intra-articular cytokine delivery after trauma. The role of some cytokines in cartilage regeneration is still under investigation. IL-6 has been postulated to have chondrodegradative as well as chondroprotective effects. Re- cently, IL-6 was shown to have a modest anabolic effect on GAG production by healthy chondrocytes, while inhibition decreased GAG content of OA cartilage explants400. IL-8 had earlier been shown to promote release of IL-6162 and induce hypertrophic differentiation270. It has now been demonstrated that IL-8 has a minor degenerative effect on cartilage regeneration, while not influencing hypertrophy [Tsuchida et al, submitted]. Oncostatin-M is also present in OA synovial fluid and its degradative role through interaction with IL-1 has been described98. Oncostatin-M was shown to have its receptor expressed throughout the depth of OA cartilage; and it is not capable of causing cartilage degradation independent of factors in synovial fluid [Beekhuizen et al, submitted]. However, its inhibition resulted in a higher matrix production, suggesting a role in inhibiting matrix synthesis. Altogether, increasing intra-articular chondroprotective cytokine levels or inhibiting chondrodestructive cytokines after trauma may protect cartilage from a ‘second hit’ due to an altered joint environment.

Modulating joint homeostasis in OA Disease-modifying OA drugs (DMOADs) may be administered systemically or through intra-articular administration. They ideally inhibit cartilage degradation and delay or even cancel the need for surgical intervention. At a minimum, the aim is a reduction in OA signs and symptoms. This goal may be reached through interaction with cytokines and growth factors, hormones or through influencing nociception. Corticosteroid injection has been shown to result in short term pain relief, but it is not indicated for long-term treatment of OA164. Hyaluronic acid (HA) injection has and is being extensively investigated and opinions on its effectiveness continue to vary. It was formerly considered more effective for pain reduction than physical therapy or NSAIDs for patients with mild to moderate OA29, 423, reportedly through an improvement in joint viscoelastic properties 144. Novell studies report that the effect may be small and irrel- evant348. They have led to an adjustment of the AAOS and Dutch Orthopaedic Society

140 Chapter 11 (NOV) recommendations, against its use. Dutch insurance companies now do not regularly re-imburse its prescription. Fundamental investigations on HA nociceptive activity continue, however459. In CHO cells (Chinese Hamster Ovary, a commonly used cell line) expressing the ‘kappa-opoid receptor’ (located in the synovial lining of the joint374), HA with a medium molecular weight (i.e., 200kDa) selectively activated this receptor, acting as an agonist and diminishing pain. Cytokine-modulating therapies thus are not as successful in OA as they are in rheumatoid arthritis (RA)74, 280. Most cytokines including IL-1β and TNF-α are present at low levels in OA synovial fluid, whereas both cytokines are present at higher levels in RA37, 289, 361, 438. Moreover, IL-1RA levels are already high in OA synovial fluid328, 343, 345. Thus targeting IL-1β through IL-1RA may not be effective 68. One of the postulated advantages of autologous conditioned serum (ACS) is that it contains other chondroprotective cytokines, IL-4 and IL-10, in addition to IL-1RA alone. Clinically, two randomized controlled trials demonstrated limited effectiveness20, 449 of ACS. In both our study343 and another recent study58, ACS had the same effect as unconditioned serum on proteoglycan degradation even though adequate IL-1RA levels were obtained in the serum. Other findings included the presence of chondrodegradative as well as chondroprotective factors in ACS, and fast clearance of IL-1RA from the joint. Currently, newer versions of ACS have been developed using borosilicate beads (IRAP-II, Arthrex Inc., Florida, USA) and tested using human and equine serum23, 172. Nevertheless, an upregulation of IL-1β is also found in this newer version of ACS. A recently described small chemical compound with therapeutical potential for OA is kartogenin (KGN). In MSCs191 it stimulated expression of chondrogenic markers collagen type II, aggrecan and lubricin, while it did not affect matrix metalloproteinases MMP3 and MM13, hypertrophic differentiation marker collagen type X or aggrecanases. In the same study, using chondrocytes and cartilage explants, it inhibited TNF-α and Oncostatin-M induced NO and glycosaminoglycan release. In mouse models of OA, intra-articular injection of KGN resulted in a decrease in superficial and midzone cartilage fibrillation and diminished pain. Its action is proposed to be through binding to filamin A, resulting in a CBF-β related RUNX transcription regulation455. Although OA synovial fluid contains few cells (unpublished observations), progenitor cells from surrounding tissues may indeed be susceptible to the advantageous effects of kartogenin.

Fundamental and clinical evaluation tools to evaluate successful cartilage regeneration Fundamental and/or clinical evaluation tools may be used to evaluate the success of a cartilage-regenerating therapy. For assessment of cartilage quality, ideally a biopsy of the repaired cartilage is taken, processed and assessed histologically. Scoring systems facilitate systematically assessing tissue characteristics. A review is currently presented of

Discussion 141 scoring systems for degrading cartilage, in vivo repaired cartilage and tissue engineered cartilage. We hereby also focused on validation of the scores and formulated recommendations for which scoring systems should be used for different types of tissue346. Meanwhile, the International Cartilage Repair Society (ICRS) has published a guideline on obtaining, processing and analyzing cartilage samples in cartilage repair research167. Using the guideline together with the scoring system recommendations, comparison of cartilage repair procedure outcomes is improved for in vitro as well as in vivo research. Another way to evaluate cartilage quality is through non-invasive imaging techniques. An advantage of these techniques is that the cartilage remains intact, in contrast to when a biopsy is taken for histological assessment. Furthermore, the regenerating cartilage may be ‘followed’ in vivo at multiple timepoints. dGEMRIC is a technique which has been validated by comparison to biochemical data24, 445, and more than a decade of clinical experience with dGEMRIC has been obtained. In the current study, it proved applicable to patients who had undergone an HTO and had their hardware removed. Overall however, a longer follow-up period would be desirable to monitor patients for long-term changes. Other cartilage imaging techniques as T1ρ, sodium imaging or diffusion-weighted im- aging82, are increasingly being investigated as additive or alternative to dGEMRIC. It is important to realize that these techniques are not necessarily ‘better’ than the others and they may focus on different structures of cartilage (i.e. collagens vs. proteoglycans). In addition, each imaging technique has its drawback. For example, dGEMRIC involves i.v. infusion with gadolinium contrast as well as a 1.5 hr. delay between injection and imaging. Thus far however, dGEMRIC remains one of the most widely used techniques to visualize longitudinal changes in proteoglycan content. Patient-reported outcome measures (PROMs) may be used for clinical evaluation of the success of a cartilage repair procedure. A new perspective in orthopaedic patient- reported outcome measures is the use of ‘response shift’ measurement. In short, ‘response shift’ refers to the change in response that a patient gives, when asked how he would evaluate his health before a given treatment in comparison to his current health evaluation. More specifically, response shift is defined as a change in the meaning of one’s self-evaluation of a target construct as a result of 1) change in the respondent’s internal standards of measurement (scale recalibration); 2) a change in the respondent’s values (scale reprioritization); or 3) redefinition of the target construct (reconceptualiza- tion)384. Improvement in application of response shift measurement would include using external clinical measures and using a control group in which response shift would not be expected to occur368. In osteoarthritis research response shift primarily has been used to detect changes in outcome after knee prosthesis placement326, 466. In the future its application may also aid in improving patient expectations, timing and cost-effectiveness of cartilage regenerating therapies.

142 Chapter 11 Future Perspectives In conclusion, future therapies may focus on interfering with joint homeostasis after trauma and in osteoarthritis, and in optimizing cell-based cartilage regeneration. Con- trolling the maintenance of chondromodulatory factors in the joint may be as important as the discovery of their individual characteristics. Giving re-implanted cells a starting advantage by optimizing their (3-D) surroundings may be essential to create high quality neocartilage. While hypertrophic differentiation may form a threat during microfracture or during a 1-stage procedure, it does not seem to influence ACI. As long as an optimal intra-articular environment is created, the outcome of cartilage repair techniques is likely to be further enhanced.

Discussion 143

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References 173

Dutch Summary – Nederlandse Samenvatting

Introductie en doelstelling Pijn, zwelling en stijfheid zijn vaak de belangrijkste symptomen die een patiënt ervaart na het oplopen van een kraakbeenletsel van de knie. Na een trauma kunnen factoren in de gewrichtsvloeistof, zoals cytokines, het resterende gezonde kraakbeen negatief be- ïnvloeden en het begin inluiden van een uitgebreide verstoring van kraakbeenaanmaak en afbraak. Op den duur kan een beperkt kraakbeendefect, met initieel geringe klachten, uitgroeien tot een uitgebreider kraakbeendefect of zelfs tot algehele gewrichtsslijtage (artrose), met invaliderende pijn- en stijfheidsklachten tot gevolg. Er zijn enkele therapieën die tot doel hebben het kraakbeenoppervlak te herstellen. De meest frequent toegepaste therapie is ‘microfracture’, waarbij na het arthroscopisch creëren van kleine gaatjes in het subchondrale bot, het kraakbeendefect wordt opgevuld door vrijgekomen stamcellen die een nieuwe ‘matrix’ aanmaken. Een nadeel van deze therapie is dat het nieuw gegenereerde weefsel meestal niet dezelfde samenstelling heeft als gezond hyalien kraakbeen, maar eerder lijkt op een bindweefsel-kraakbeen mengsel. Daarnaast is deze therapie voornamelijk geschikt voor kleinere kraakbeendefecten. Een andere therapie is de in 1994 voor het eerst beschreven ‘autologe chondrocyttrans- plantatie’ (ACI). Bij deze therapie worden kraakbeencellen uit een onbelast deel van de knie geïsoleerd, in vitro vermenigvuldigd en teruggeplaatst in het kraakbeendefect. De cellen worden op hun plaats gehouden door een collageen membraan als bedekking. Deze therapie leidt tot een verbetering van klinische (vermindering van pijn en verbetering van functie) en histologische (kwaliteit van het nieuwgevormde weefsel) scores. Toch is de kraakbeenkwaliteit ook bij ACI vaak nog suboptimaal.

Onderzoek naar kraakbeenregeneratie richt zich daarom op het optimaliseren van de kwaliteit van geregenereerd kraakbeen. Waar initieel de nadruk lag bij het in vitro of in vivo creëren van optimaal kraakbeen, komt er nu steeds meer aandacht voor het optimaliseren van de omgeving waarin het kraakbeen wordt teruggeplaatst. De samen- werking tussen chondrocyten, extracellulaire matrix en de gewrichtsomgeving wordt ook wel ‘gewrichtshomeostase’ genoemd. Dit proefschrift beschrijft studies gericht op: ‘het optimaliseren van de regeneratieve capaciteit van de chondrocyt tijdens ACI, door beïnvloeding van het cellulaire phenotype en door het moduleren van de gewrichtshomeostase’.

De invloed van externe factoren op kraakbeen metabolisme De invloed van collageen op de regeneratieve capaciteit van humane femur-chondrocyten werd onderzocht in Hoofdstuk 3. Uit eerdere studies bleek, dat in vitro contact tussen chondrocyten en collageen tot hypertrophe differentiatie en kraakbeenafbraak zou leiden. Bij acht donoren werden chondrocyten uit gezond kraakbeen geïsoleerd en gekweekt op collageen type I, type II of ongecoate PTFE membranen. Dit kweekmodel

Dutch Summary 177 werd gebruikt om de (M)ACI te simuleren. Collageen type II coating van de membraan leidde tot nieuwgevormd kraakbeen met de hoogste histologische en biochemische kwaliteit. Zowel collageen type I en II coating zorgden voor hogere histologische scores, glycosaminoglycanen (GAG) en DNA gehalte in vergelijking met ongecoate membranen. De omzet van GAGs was bij beide collagenen ook hoger, zoals bleek uit de hogere ‘release’ van GAGs in het kweekmedium. Ondanks deze verschillen was er geen verschil in MMP13 en DDR2 expressie (beide markers voor degradatie) tussen de collageen ge- coate en de ongecoate membranen. Bovendien was de expressie van α2β1-integrine bij collageen type I en II vergelijkbaar. Zowel collageen type I als type II kunnen dus worden gebruikt als onderlaag voor chondrocyt re-implantatie.

Eén van de processen die de kraakbeenkwaliteit bij ACI in gevaar zou kunnen brengen is hypertrophe differentiatie. Daarom werd in Hoofdstuk 4 de invloed van parathyroid hormone (PTH) op stimulering van kraakbeenaanmaak en op de remming van hypertrophe differentiatie geanalyseerd. Toevoeging van 0.1 en 1.0 µM PTH aan femorale chondrocyten van 6 humane donoren met gezond kraakbeen leidde tot een lager DNA gehalte dan bij de controle conditie, hoewel de GAG/DNA ratio vergelijkbaar was. Col- lageen type II expressie was lager na toevoeging van PTH. De markers van hypertrophe differentiatie, core-binding factor alpha 1 (Cbfa-1/Runx-2) en collageen type X, werden niet beïnvloed. Blijkbaar zijn gezonde humane chondrocyten minder gevoelig dan andere celtypes voor de effecten van PTH op hypertrophe differentiatie.

De invloed van interne factoren op kraakbeenmetabolisme Jong kraakbeen herstelt vermoedelijk beter dan volwassen kraakbeen. In Hoofdstuk 5 werd de regeneratieve capaciteit van juveniele talus chondrocyten vergeleken met die van volwassen talus en femurchondrocyten. Hoewel juveniele chondrocyten van vijf humane talus donoren op dag 9 van de in vitro kweek kraakbeen genereerden met een hoger GAG en DNA gehalte dan volwassen talus chondrocyten, verschilde het uitein- delijke GAG en DNA gehalte op dag 28 van de kweek niet. De expressie van markers voor hypertrophe differentiatie (Cbfa1/Runx2 en collageen type X) was het hoogste in juveniele talus chondrocyten en in volwassen femurchondrocyten, terwijl de histologische kwaliteit van door volwassen femurchondrocyten geregenereerd kraakbeen het hoogst was. Blijkbaar resulteert een hogere expressie van markers voor hypertrophe differentiatie niet noodzakelijkerwijs in een lagere kraakbeenkwaliteit. De discrepantie tussen het eerder geobserveerde spontane kraakbeenherstel bij jonge dieren en onze bevindingen, suggereert dat het intra-articulaire milieu mogelijk een grotere rol speelt tijdens de regeneratie van kraakbeen dan het cellulaire phenotype alleen.

178 Dutch Summary De fundamentele basis van een gewrichtshomeostase beïnvloedende therapie In Hoofdstuk 6 werd de fundamentele basis van autoloog geconditioneerd serum (ACS, een intra-articulaire therapie voor artrose) onderzocht. Eerdere klinische toepassing van deze therapie leidde tot wisselend succes. Bij 22 patiënten met artrose werden de cytokine-samenstelling van ACS, het effect van ACS op het proteoglycanen metabolisme van kraakbeen explant kweken, en intra-articulaire cytokineniveaus na toediening van ACS onderzocht. ACS bleek niet alleen kraakbeenbeschermende cytokines zoals IL-1RA, IL-10 en TGF-β te bevatten, maar ook kraakbeenbeschadigende cytokines zoals IL-1 β, Oncostatin-M en TNF-α. Proteoglycanen (PG) metabolisme van kraakbeen explants verschilde niet na toevoeging van ACS of ongeconditioneerd serum. Remming van TNF-α, in aanwezigheid van ACS, had geen invloed op het PG metabolisme. In vergelijking met het cytokineniveau voorafgaand aan ACS, leidde ACS niet tot een verandering in intra- articulaire cytokineniveaus tijdens de behandeling. Een solide fundamentele basis voor toepassing van ACS lijkt niet aanwezig.

Gebruik van ‘patient-reported outcome measures’ (PROMs) om placebo effecten van ACS behandeling te detecteren In Hoofdstuk 7 werden placebo effecten van ACS behandeling onderzocht. ACS werd toegediend aan 20 patiënten met artrose, die in het kader van een eerdere studie waren behandeld met fysiologisch zout. De ‘visual analogue scale’ (VAS) voor pijn verbeterde na toediening van ACS net als na eerdere toediening van het placebo, maar de ‘knee injury and osteoarthritis outcome score (KOOS)’, de ‘knee society clinical rating scale (KSCRS)’ en de ‘Western Ontario and McMaster Universities osteoarthritis index (WOMAC)’ verbeterden niet, in tegenstelling tot na eerdere placebo toediening. In deze studie werd aangetoond dat patiënten zich in de loop der tijd aanpassen aan de beperkingen die artrose met zich mee brengt, aangezien patiënten in retrospect na behandeling hun klachten vooráfgaand aan behandeling beter scoren, dan op het moment zelf. Dit wordt ‘repsonse shift’ genoemd, en illustreert de aanpassing in perceptie die artrose patiënten doormaken.

Oude en nieuwe methodes om het succes van kraakbeen-beïnvloedende therapieën te evalueren In Hoofdstuk 8 is een overzicht gegeven van histologische scores voor artrotisch, in vivo geregenereerd en tissue-engineered kraakbeen. Het doel van deze review was om onderscheid te maken tussen gevalideerde en niet-gevalideerde scores. De ‘Histological-Histochemical Grading System (HHGS)’ en de ‘Osteoarthritis Research Society International (OARSI) system’ worden aanbevolen voor evaluatie van gezond tot artrotisch kraakbeen. De ICRS-II score lijkt het meest geschikt voor in vivo geregenereerd

Dutch Summary 179 kraakbeen. Voor evaluatie van in vitro geregenereerd kraakbeen lijkt de ‘Bern score’ de voorkeur te hebben.

In Hoofdstuk 9 werd in een ‘pilot study’ het effect van de valgiserende tibiakoposteo- tomie (HTO) op kraakbeen proteoglycanen content gemeten met behulp van een non- invasieve MRI methode, de ‘delayed Gadolinium Enhanced MRI of Cartilage’ (dGEMRIC). De klinische scores van alle tien patiënten verbeterden postoperatief, ondanks verschillen in femorale of tibiale kraakbeenkwaliteit. Tussen mediale en laterale femurcondyl werden wel verschillen in proteoglycan verspreiding gedemonstreerd, zowel vóór als na HTO. dGEMRIC lijkt toepasbaar voor het vervolgen van kraakbeenkwaliteit bij patiënten met mediale compartimentsgonartrose, mits het osteosynthesemateriaal wordt verwij- derd in het geval er een HTO is verricht. In deze studie wordt bevestigd dat dGEMRIC een goed alternatief is voor het vervolgen van veranderingen in kraakbeenkwaliteit, ten opzichte van invasieve methodes zoals arthroscopie of biopsie.

Implicaties van de data en toekomstperspectieven Onderzoek naar de beïnvloeding van gewrichtshomeostase, zowel na trauma als in OA, lijkt belangrijke aanknopingspunten te bieden voor toekomstige therapieën. Hierbij is het niet alleen van belang om relevante cytokines en groeifactoren te identificeren, maar ook om er voor te zorgen dat deze na intra-articulaire toediening in voldoende hoge concentraties in het gewricht aanwezig blijven om hun invloed te kunnen uitoe- fenen, zonder tot systemische toxiciteit te leiden. Een gedegen fundamentele basis is van belang bij klinische toepassing van gewrichtsmodulerende therapieën, getuige de controversiële resultaten van ACS. Bij het herstel van kraakbeendefecten, bijvoorbeeld in het kader van ACI, is niet alleen de celkwaliteit van belang, maar ook de drager waarin de cel wordt teruggeplaatst, en de omgeving waarin cel en matrix terechtkomen. 3-dimensionale structuren bieden in dit perspectief mogelijk voordelen ten opzichte van de huidige, veelal 2-dimensionale dragers. Het remmen van hypertrophe differentiatie door middel van PTH lijkt met name zinvol indien artrotische chondrocyten of mesenchymale stamcellen (MSCs) worden gebruikt, en minder relevant bij ACI. Ondanks dat juveniel kraakbeen reeds als basis voor kraakbeenherstel van de talus wordt gebruikt, lijkt hier onvoldoende fundamentele onderbouwing voor. Het volgen van het proces van kraakbeenherstel in de patiënt wordt vergemakkelijkt door gevalideerde beeldvormende technieken zoals de dGEMRIC. Nieuwere methodes zoals T1ρ, ‘sodium imaging’ en ‘diffusion weighted imaging’ bieden aanvullende infor- matie maar zijn op zich nog niet superieur ten opzichte van dGEMRIC. Bovenstaande ontwikkelingen op het gebied van kraakbeenherstel en voorkomen van artrose zijn veelbelovend en lijken in toenemende mate hun weg te vinden naar in vivo

180 Dutch Summary en humane studies. Inspirerende ontwikkelingen zijn die naar interventies in het intra- articulaire milieu, zowel vroeg posttraumatisch als bij reeds ontwikkelde artrose, en die naar het optimaliseren van de omgeving van de teruggeplaatste cellen bij behandeling van een lokaal kraakbeendefect.

Dutch Summary 181

List of abbreviations

ACI /ACT Autologous chondrocyte implantation / transplantation ACS Autologous conditioned serum ADAMTs A disintegrin and metalloproteinases with thrombospondin motifs bFGF Basic fibroblast growth factor BMP Bone morphogenetic protein BMSC Bone marrow stromal cell COL Collagen COX-2 Cyclooxygenase-2 CS Chondroitin sulphate DDR2 Discoidin domain receptor 2 dGEMRIC delayed Gadolinium-Enhanced MRI of Cartilage DMEM Dulbecco’s modified Eagle’s medium DMMB Dimethylmethylene blue DMOAD Disease-modifying OA drug DNA Deoxyribonucleic acid ECM Extracellular matrix EDTA Ethylenediaminetetraacetic acid ELISA Enzyme-linked immunosorbent assay FBS Fetal bovine serum GAG Glycosaminoglycan HTO High tibial osteotomy IFNγ Interferon γ IGF Insuline-like growth factor IL Interleukin IL-1RA Interleukin-1 receptor antagonist ITS-X Insuline-transferrine-selenium-X KOOS Knee injury and Osteoarthritis Outcome Score KSCRS Knee Society Clinical Rating Scale KS Keratan sulphate MACI Matrix-assisted autologous chondrocyte implantation MMP Matrix metalloproteinases MRI Magnetic resonance imaging MSC Mesenchymal stromal cell n.s. non-significant NSAID Nonsteroidal anti-inflammatory drug OA Osteoarthritis OPG Osteoprotegerin OSM Oncostatin M PBS Phosphate buffered saline

List of abbreviations 185 PCR Polymerase chain reaction PG Proteoglycan PTFE Poly-tetra-fluoro-ethylene QOL Quality of Life RA Rheumatoid arthritis RNA Ribonucleic acid SD Standard deviation SF Synovial fluid SOX9 (Sex determing region Y)-box9 TIMP Tissue inhibitor of metalloproteinases TGFβ Transforming growth factor β TNFα Tumor necrosis factor α VAS Visual Analogue Score WOMAC Western Ontario and McMasters Universities Index

186 List of abbreviations Publications and presentations

Scientific Publications

Rutgers M, Saris DBF, Vonk LA, van Rijen MH, Akrum V, van Boxtel A, Dhert WJ, Creemers LB. PTH decreases in vitro cartilage regeneration without affecting hypertrophic differentiation. Manuscript in preparation

Rutgers M, Saris DBF, Vonk LA, van Rijen MH, Akrum V, van Boxtel A, Sakkers R, Dhert WJ, Creemers LB. Similar regenerative capacity of juvenile versus adult talar chondrocytes despite high expression of hypertrophic markers. Submitted

Rutgers M, Creemers LB, Auw Yang KG, Raijmakers NJ, Dhert WJA, Saris DBF. Osteoar- thritis treatment using a cytokine-based intra-articular therapy – comparison of clinical results to earlier placebo treatment. Submitted

Rutgers M, Bartels LW, Tsuchida AI, Castelein RM, Dhert WJ, Vincken KL, van Heerwaarden RJ, Saris DB. dGEMRIC as a tool for measuring changes in cartilage quality following high tibial osteotomy: a feasibility study. Osteoarthritis and Cartilage 2012: 20(10)1134-41

Tsuchida AI, Beekhuizen M, Rutgers M, van Osch GJM, Bekkers JEJ, Bot AGJ, Geurts B, Dhert WJA, Saris DBF, Creemers LB. Interleukin 6 is elevated in synovial fluid of patients with focal cartilage defects and stimulates cartilage matrix production in an in vitro regeneration model. Arthr Res Ther 2012;14:R262

Rutgers M, Saris DB, Vonk LA, van Rijen MH, Akrum V, Langeveld D, van Boxtel A, Dhert WJ, Creemers LB. Effect of Collagen Type I or Type II on Chondrogenesis by Cultured Human Articular Chondrocytes. Tissue Eng Part A. 2012 Sep 13

Rutgers M, Saris DBF, Dhert WJA, Creemers LB. Cytokine profile of autologous conditioned serum for treatment of osteoarthritis, in vitro effects on cartilage metabolism and intra-articular levels after injections. Arthritis Res Therapy 2010;12(3):R114

Vasquez O, Rutgers M, Ring DC, Walsh M, Egol KA. Fate of the ulnar nerve following operative fixation of distal humerus fractures. J Orthop Trauma 2010;24(7):395-9

Rutgers M, van Pelt MJ, Dhert WJA, Creemers LB, Saris DBF. Evaluation of Histological Scoring Systems for Tissue-Engineered, Repaired and Osteoarthritic Cartilage. Osteoar- thritis and Cartilage 2010: 18;12-23

Publications and presentations 189 Rutgers M, Jupiter J and Ring D. Isolated Posttraumatic Ulnar Translocation of the Radio- carpal Joint. Journal of Hand and Microsurgery 2009; 1(2):108-112

Rutgers M, Saris DBF, Auw Yang KG, Dhert WJA, Creemers LB. From joint Injury to osteoarthritis: Soluble mediators in the course and treatment of cartilage pathology. Immunotherapy 2009:1(3)435-445

Rutgers M, Mudgal CS, Shin R. Combined fractures of distal radius and scaphoid. J Hand Surg Eur Vol. 2008 Aug;33(4):478-83

Souer S, Rutgers M, Andermahr J, Jupiter JB, Ring D. Perilunate Fracture–Dislocations of the Wrist: Comparison of temporary screw versus K-wire fixation. J Hand Surg Am 2007; 32A(3):318-325

Rutgers M and Ring D. Functional fracture bracing of diaphyseal humeral fractures. J Orthop Trauma 2006; 20(9):597-601

Book Chapters Rutgers M en van Dijk CN. Nonunion of lateral talar process. In: Concepts and Cases in Nonunion Treatment. Marti RK en Kloen P. Dubendorf: AO Publishing 2011: 2.16

Rutgers M en van Dijk CN. Nonunion of the anterior calcaneal process. In: Concepts and Cases in Nonunion Treatment. Marti RK en Kloen P. Dubendorf: AO Publishing 2011: 2.16

Podium presentations Nederlandse Orthopaedische Vereniging, jaarvergadering (Amsterdam) 2013. Rutgers M, Saris DBF, Vonk LA, van Rijen MH, Akrum V, van Boxtel A, Sakkers R, Dhert WJ, Creemers LB. Limited differences in cartilage regeneration between juvenile and adult human talar chondrocytes.

Nederlandse Orthopaedische Vereniging, jaarvergadering (Den Haag) 2012. AI Tsu- chida, M Beekhuizen, M Rutgers, AGJ Bot, B Geurts, JEJ Bekkers, WJA Dhert, LB Creemers, DBF Saris. The role of IL6 in osteoarthritis and cartilage regeneration

Nederlandse Vereniging voor Matrix Biologie (Lunteren) 2011. AI Tsuchida, M Beekhuizen, M Rutgers, AGJ Bot, B Geurts, JEJ Bekkers, WJA Dhert, LB Creemers, DBF Saris. The role of IL6 in osteoarthritis and cartilage regeneration

190 Publications and presentations Nederlandse Orthopaedische Vereniging, jaarvergadering (Groningen) 2011. Rut- gers M, Tsuchida AI, Heerwaarden RJ van, Castelein RM, Dhert WJA, Bartels LW, Vincken KL, Saris DBF. delayed Gadolinium Enhanced MRI of Cartilage (dGEMRIC) to measure changes in cartilage following high tibial osteotomy

Nederlandse Orthopaedische Vereniging, voorjaarsvergadering (Utrecht) 2009. Rutgers M, Saris DBF, Heerwaarden RJ van, Castelein RM, Dhert WJA, Bartels LW, Vincken KL. Non-invasief meten van kraakbeenkwaliteit in de knie met behulp van delayed Gadolinium-Enhanced MRI of Cartilage (dGEMRIC)

International Cartilage Repair Society Annual Meeting (Miami) 2009. Rutgers M, Saris DBF, Langeveld DA, Dhert WJA, Creemers LB. Collagen type II scaffold coating positively influences composition of in vitro regenerated cartilage.

Nederlandse Vereniging voor Matrix Biologie (Lunteren) 2009. Rutgers M, Saris DBF, Langeveld DA, Dhert WJA, Creemers LB. Collagen type II scaffold coating positively influences composition of in vitro regenerated cartilage.

Nederlandse Orthopaedische Vereniging, jaarvergadering (Den Bosch) 2009. Rutgers M, Creemers LB, Castelein RM, Dhert WJA, Saris DBF. Autoloog geconditioneerd serum voor de behandeling van artrose; klinische en fundamentele aspecten van de behandeling

Symposium Experimenteel Onderzoek Heelkundige Specialismen (Utrecht) 2008. Rutgers M, Creemers LB, Castelein RM, Dhert WJA, Saris DBF. Autoloog geconditioneerd serum voor de behandeling van artrose

International Cartilage Repair Society Basic skills lab course (Utrecht) 2008. Rutgers M, Saris DBF, Dhert WJA, Creemers LB. Histological scoring systems for Tissue Engineered, Repaired and Osteoarthritic Cartilage.

Nederlandse Vereniging voor Matrix Biologie, jaarvergadering (Lunteren) 2008. Rutgers M, Saris D, Dhert W, Creemers L. Cytokines in serum and synovial fluid of patients, treated with autologous conditioned serum.

American Academy of Orthopaedic Surgeons, annual meeting (San Diego, CA) 2007. Rutgers M, Ruijs A, Torrano JP, Jupiter JB, Ring D. Transposition vs release and protection of the ulnar nerve in treatment of distal humerus fractures.

Publications and presentations 191 American Society for Surgery of the Hand, annual meeting (Washington DC) 2006. Rutgers M, Shin R, Jupiter JB, Ring D, Mudgal CS. Combined fractures of distal radius and scaphoid.

Nederlandse Orthopaedische Vereniging, jaarvergadering (Amsterdam) 2006. Rutgers M, Ruijs A, Torrano JP, Jupiter JB, Ring D. Transposition vs release and protection of the ulnar nerve in treatment of distal humerus fractures.

New England Orthopaedic Society annual meeting (Boston, MA) 2005. Rutgers M, Shin R, Jupiter JB, Ring D, Mudgal CS et al. Combined fractures of distal radius and scaphoid.

New England Hand Society, annual meeting (Sturbridge, MA) 2005. Rutgers M, Shin R, Jupiter JB, Ring D, Mudgal CS et al. Combined fractures of distal radius and scaphoid.

Poster presentations Traumadagen (Amsterdam) 2011. Kolk A, Rutgers M, Vreeburg ME, Kropman RHJ, Hammacher ER. Behandeling van geimpacteerde greenstick fracturen bij kinderen met drukverband.

International Cartilage Repair Society Annual Meeting (Barcelona) 2010. Rutgers M, Tsuchida AI, Bartels LW, Heerwaarden RJ van, Creemers LB, Castelein RM, Dhert WJA, Vincken KL, Saris DBF. Change in cartilage quality following opening wedge valgus tibial osteotomy, measured using dGEMRIC

International Cartilage Repair Society Annual Meeting (Miami) 2009. Rutgers M, Saris DBF, Langeveld DA, Dhert WJA, Creemers LB. Influence of parathyroid hormone on proteoglycan metabolism of in vitro cultured chondrocytes

Orthopaedic Research Society annual meeting (San Francisco, CA) 2008. Rutgers M, Saris D, Dhert W, Creemers L. Cytokines in serum and synovial fluid of patients, treated with autologous conditioned serum.

International Cartilage Repair Society annual meeting (Warsaw) 2007. Rutgers M, Saris DBF, Castelein R, Creemers L, Dhert WJA. Synovial fluid composition in relation to intra-articular knee joint disorders.

192 Publications and presentations International Cartilage Repair Society annual meeting (Warsaw) 2007. Rutgers M, Saris DBF, Castelein R, Creemers L, Dhert WJA. CBFA-1 expression in different in vitro chondrocyte culture models.

Orthopaedic Research Society annual meeting (San Diego, CA) 2007. Rutgers M, Saris D, Schoots S, Auw Yang KG, Sasaguri K, Castelein R, Creemers LB, Dhert W. Cbfa-1 expression in different in vitro chondrocyte culture models.

American Academy for Orthopaedic Surgeons Annual Meeting (San Diego, CA) 2007. Souer S, Rutgers M, Andermahr J, Jupiter JB, Ring D. Perilunate fracture–dislocations of the wrist: comparison of temporary screw versus K-wire fixation.

American Academy of Orthopaedic Surgeons annual meeting (San Diego, CA) 2007. Rutgers M, Ring D. Functional fracture bracing of diaphyseal humerus fractures.

American Academy of Orthopaedic Surgeons annual meeting (San Diego, CA) 2007. Rutgers M, Shin R, Jupiter JB, Ring D, Mudgal CS. Combined fractures of distal radius and scaphoid.

Dutch Society for Biomaterials and Tissue Engineering (Lunteren) 2006. Rutgers M, Saris D, Schoots SGM, Auw Yang KG, Castelein R, Sasaguri K, Creemers L, Dhert W. Cbfa1 expression in different in vitro chondrocyte culture models.

Symposium Experimenteel Onderzoek Heelkundige Specialismen (Groningen) 2006. Rutgers M, Saris D, Schoots SGM, Auw Yang KG, Castelein R, Sasaguri K, Creemers L, Dhert W. Cbfa1 expression in different in vitro chondrocyte culture models.

Federation for European Societies for Surgery of the Hand (Glasgow) 2006. Rutgers M, Shin R, Jupiter JB, Ring D, Mudgal CS. Combined fractures of distal radius and scaphoid.

Publications and presentations 193

Acknowledgements – Dankwoord

Geachte prof. dr. W.J.A. Dhert, beste Wouter, Het verzorgen van de fundamentele vorming van een jonge orthopaed in opleiding lijkt mij geen makkelijke opdracht. Onder jouw begeleiding lukt het keer op keer en is bovendien de onderzoeksgroep enorm in grootte toegenomen. De visie die hiervoor nodig is en het vertrouwen dat je in de onderzoekers stelt waardeer ik zeer. Net als je deelname aan alle sociale onderzoeks- en niet-onderzoeksgerelateerde evenementen. Dank.

Geachte prof. dr. D.B.F. Saris, beste Daan, Sinds kort weet ik dat de snelheid en scherpte die je toepast bij de begeleiding van onderzoekers ook naar voren komt in je begeleiding van de AIOS. Ik heb je leren ken- nen als een vernieuwingsgezinde gedreven opleider en onderzoeksbegeleider, die een keten creëert van alleen maar sterke schakels. Ik zie het als een voorrecht om bij de kraakbeen-groep in Utrecht te promoveren. Dank.

Geachte dr. L.B. Creemers, beste Laura, Aan jou heb ik mijn fundamentele vorming te danken. Je scherpe blik is zichtbaar en onzichtbaar merkbaar in de fundamenten van het Utrechtse kraakbeenonderzoek. Ook jij hebt enorm veel tijd in deze studies gestoken. Nachtelijke revisies ten spijt, het is zo ver! Dank voor je expertise en kennis, geduld en vertrouwen.

Beste Mattie, Levensgevaarlijk, al die artsen op het lab. Dank voor je hulp, geduld en vriendschap.

Prof. dr. J.M. van Laar, Prof. dr. M. Kon, Prof. dr. S.K. Bulstra, Prof. dr. H.B.J. Karpe- rien, Dr. P.M. van der Kraan, Hartelijk dank voor de tijd die u heeft genomen voor de beoordeling van het manuscript en uw deelname aan de leescommissie.

Bijzondere dank aan Peter Kloen, David Ring, Remco Karthaus, René Castelein

Immunologie Wilco de Jager, Berend Prakken, Ineke Slaper

Internationaal Deborah Burstein, Bruce Caterson, Sally Roberts, Chaitanya Mudgal, Robbert Shin, Jesse Jupiter, Laura O’Brien

ISI Wilbert Bartels, Koen Vincken, Anneke Hamersma

Onderzoekscoördinatoren Natasja Raijmakers, Jessika Ouwerkerk en Nienke Buisman

Acknowledgements 197 Orthopaeden NL Jan-Willem Louwerens, Ronald van Heerwaarden, Paul Bertelink

Reumatologie Nathalie Jansen, Floris Lafeber, Kim Jacobs, Marieke van Vianen, Simon Mastbergen, Femke Intema

Staf en assistenten van de afdeling chirurgie van het St. Antonius Ziekenhuis, afdeling orthopaedie van het Onze Lieve Vrouwe Gasthuis, van het Diakonessenhuis en van het UMCU. In het bijzonder dank aan Peter Go, Michiel Segers, Jolanda Vonk, Rudolf Poolman, Trisha Mol, Geert vd Werf, Arthur de Gast, Ralph Sakkers en Charles Vogely.

Studenten en actieve participatoren Danielle Langeveld, Thalitha de Hoop, Maarten van Pelt, Nick Vinken, Vanessa van Akrum, Antonette van Boxtel, Lucienne Vonk

Poli Joyce, Alberdine, Alfred, Rachida, Jacqueline, Attie

Vrienden, collega’s, medewerkers, in het bijzonder Huub Olthof, Elmer Tegel, Maarten van Wijk. Frans, Brigitte, Dorothée, Stéphanie en Charlotte Vreede, Marnix en Allard. Yde Engelsma. Job Doornberg, Anneluuk Lindenhovius, Marieke Broekman, Sebastiaan Souer, Daan van Poll. Pieter Prins, Maud Roodbaard en Ingrid Boele. Gie Auw Yang, Roel Custers, Diyar Delawi, Dirk-Jan Moojen, Moyo Kruyt, Jan-Willem Kouwenhoven, Diederik Kempen, Joost Rutges, Ruth Geuze, Anneloes Westerveld, Natalja Fedorovich, Michiel Janssen, Joris Bekkers, Michiel Beekhuizen, Fiona Wegman, Anika Tsuchida, Floris Groen (koning!), Just vd Linde, Karlijn van Overvest, Rik de Groot, Eline van Amerongen, Sylvia Visser, Antoinette Zielman, Angelina Bentvelzen-Peek, Simone Sienema, Jacqueline Al- blas, Ruud Hiensch, Yvonne vd Helm, Danny van Caspel, Paul Westers, José, Niels Blanken, Arien Dijkstra, Marc Falke, Robbert van Dijk, Arnard van der Zwan, Agnita Stadhouder, Jorrit-Jan Verlaan, Derek van Deurzen.

Alle patiënten die hebben deelgenomen aan de klinische studies.

Beste Derk en Frank, You guys know it all, dank voor jullie steun; geen ´major revision´ kan tegen onze goede vriendschap op! Op alle mooie avonturen die nog gaan komen!

Lieve Marien en Agnes, Bedankt voor de afgelopen 34 jaar liefde, steun, vertrouwen en alle andere investerin- gen die een zoon met zich mee brengt. Dank voor alle zorg voor Reinoud. Zonder jullie was dit boekje er nooit geweest. Lieve Michiel, je bent de allerbeste broer en vriend. Mireille en Hidde, wat een grote familie zijn we aan het worden! Dank.

198 Acknowledgements Lieve kleine Reinoud, Dit boek is ook een beetje voor jou (en dan bedoel ik niet om op te eten of te verscheu- ren!) Voorlezen zal ik in de toekomst vaker, maar wel uit een ander boek! Er staan je nog zó veel mooie dingen te wachten!

Lieve Christine, Zoals bij een wetenschappelijk artikel de laatste auteur dankzij zijn of haar toewijding, begeleiding of ondersteuning de laatste plaats in de auteurslijst verdient, zo bedank ik ook jou hier als laatste. Je positieve blik, tijd, liefde en ondersteuning hebben in essenti- ële mate bijgedragen aan de afronding van dit boekje. We gaan onverstoorbaar verder, maar weet dat er geen groter geluk bestaat dan deze reis met jou te mogen maken. Ik ben reuze trots op jou. Lieverd, dank je wel.

Acknowledgements 199

Curriculum Vitae

Marijn Rutgers was born in The Hague on March 5th, 1980. His childhood was spent in Cairo (Egypt) and Bonn (Germany) as the son of a Dutch diplomat. In 1988 he returned to the Netherlands and graduated in 1998 from the Rijnlands Lyceum in Oegstgeest. Being used to travelling, he chose to study Medicine at the RijksUniversiteit Groningen. In 2003 he went abroad for an elective on general surgery in the Nyanga Hospital in Zimbabwe, and studied Spanish in Guatemala. He fulfilled his internships at the Medisch Spectrum Twente in Enschede, where he developed an affection for orthopaedic surgery, and at the Sint Elizabeth Hospitaal in Willemstad, Curaçao. Returning to the Netherlands, he decided to follow a career in orthopaedic surgery. After an elective at the AMC in Amsterdam under supervision of prof. dr. C.N. van Dijk and at Ziekenhuis Hilversum (dr. G.H.R. Albers), he travelled to Boston in 2005 and undertook several research projects at the Hand & Upper Extremity Service of the Massachusetts General Hospital under supervision of dr. D. Ring.

In 2006 he returned to live in Amsterdam in the proximity of his friends and applied for a PhD program at the Orthopaedics department of the University Medical Center in Utrecht. The foundation for the current thesis was made during fundamental and clinical investigations performed between 2006 and 2009, under supervision of prof. dr. W.J.A. Dhert, prof. dr. D.B.F. Saris and dr. L.B. Creemers. In 2009 he started his training to become an orthopaedic surgeon in the St. Antonius Ziekenhuis (dr. P.M.N.Y.H. Go), continued in 2012 in orthopedics at the Onze Lieve Vrouwe Gasthuis (dr. R. Poolman), in 2013 at the Diakonessenhuis Utrecht (dr. A. de Gast) and has currently returned to the UMCU for the 5th year of his residency. Despite all the commuting, he was happy to encounter Christine Vreede, who he married in 2012. Marijn enjoys long distance running and kitesurfing and currently lives with Christine and their 1-year old son Reinoud in The Hague. Integrative studies on cartilage tissue engineering and joint homeostasis Marijn Rutgers