Oxygen tension and biomechanical signals are factors that regulate inflammatory mechanisms in chondrocytes. We examined whether low oxygen tension influenced the cells response to TNFα and dynamic compression. Chondrocyte/agarose constructs were treated with varying concentrations of TNFα (0.1 to 100 ng/ml) and cultured at 5% and 21% oxygen tension for 48 hours. In separate experiments, constructs were subjected to dynamic compression (15%) and treated with TNFα (10 ng/ml) and/or L-NIO (1 mM) at 5% and 21% oxygen tension using an ex-vivo bioreactor for 48 hours. Markers for catabolic activity (NO, PGE2) and tissue remodelling (GAG, MMPs) were quantified by biochemical assay. ADAMTS-5 and MMP-13 expression were examined by real-time qPCR. 2-way ANOVA and a post hoc Bonferroni-corrected t-test were used to analyse data. TNFα dose-dependently increased NO, PGE2 and MMP activity (all p < 0.001) and induced MMP-13 (p < 0.05) and ADAMTS-5 gene expression (p < 0.01) with values greater at 5% oxygen tension than 21%. The induction of catabolic mediators by TNFα was reduced by dynamic compression and/or L-NIO (all p < 0.001), with a greater inhibition observed at 5% than 21%. The stimulation of GAG synthesis by dynamic compression was greater at 21% than 5% oxygen tension and this response was reduced with TNFα or reversed with L-NIO. The present findings revealed that TNFα has dose-dependent catabolic activities and increased production of inflammatory mediators at low oxygen tension. Dynamic compression or the NOS inhibitor downregulated the inflammatory effects induced by TNFα, linking both types of stimuli to reparative activities. Future therapeutics should develop oxygen-sensitive antagonists which are directed to interfering with the TNFα induced pathways.

Les rétinoïdes (dérivés de la vitamine A) ont montré des propriétés anti-arthritiques intéressantes dans des modèles expérimentaux d’arthropathie. Nous avons étudié leur effet sur l’inhibition de cytokines pro-inflammatoires et de métalloprotéases matricielles (MMP) induites par l’IL-1b dans les fibroblastes synoviaux et les chondrocytes humains et de rat. En conditions inflammatoires, les fibroblastes synoviaux produisent de fortes quantités de cytokines inflammatoires dans l’articulation (TNF-a, IL-1b, IL-6). L’acide all-trans rétinoïque (ATRA, agoniste RAR), et l’acide 9-cis rétinoïque (co-agoniste RAR/RXR) inhibent l’expression de ces cytokines dans les fibroblastes synoviaux de rat stimulés par l’IL-1. L’ATRA inhibe également la phosphorylation d’ERK1/2 induite par l’IL-1, sans affecter celles de p38 ou de JNK, et diminue l’activation des facteurs de transcription AP-1 et NF-IL-6 induits par l’IL-1. L’effet de l’ATRA sur les cytokines inflammatoires n’est pas potentialisé par un agoniste RXR, ni reproduit par des agonistes sélectifs des isotypes RAR, et n’est pas antagonisé par la répression des isotypes RAR, démontrant ainsi des effets indépendants de RAR ou RXR. En revanche, l’effet inhibiteur de l’ATRA sur AP-1, sur NF-IL-6 et sur la production d’IL-6 est reproduit par un antagoniste d’ERK1/2. En conditions inflammatoires, les chondrocytes produisent de grandes quantités de MMP et de nitrites. L’ATRA inhibe l’expression d’iNOS et la production de nitrites induites par l’IL-1 dans les chondrocytes de rat et humains. Cependant, l’activité MMP totale est réduite uniquement dans les chondrocytes humains, et cette différence n’est pas due à une modulation variable de l’activation d’AP-1. Ceci démontre que les rétinoïdes ont un impact important sur les paramètres inflammatoires des cellules articulaires, mais qu’il varie suivant l’espèce, suggérant que l’extrapolation des résultats obtenus à partir des cellules de rat est délicate. / Retinoids (molecules derivated from vitamin A) used in experimental models of arthropathies, have shown interesting anti-arthritic properties. We have studied retinoid effects on the inhibition of pro-inflammatory cytokines and matrix metalloproteases (MMP) induced by IL-1b in rat and human synovial fibroblasts and chondrocytes. In pro-inflammatory conditions, synovial fibroblasts produce large quantities of inflammatory cytokines in the joint (TNF-a, IL-1b, IL-6). Both all-trans retinoic acid (ATRA, agonist of RAR) and 9-cis retinoic acid (9-cis RA, co-agonist of RAR and RXR) inhibit the expression of these cytokines in rat synovial fibroblasts stimulated by IL-1. ATRA also inhibit the phosphorylation of ERK1/2 induced by IL-1, without affecting that of p38 or JNK, and decrease activation of transcription factors AP-1, NF-kB and NF-IL-6 induced by IL-1. The effect of ATRA on inflammatory cytokines expression is not potentiated by RXR agonist, nor reproduces by selective RAR agonists, and is not antagonized by RAR isotypes silencing, showing that the effects of ATRA are independent of RAR or RXR. However, the inhibitory effect of ATRA on AP-1 and on NF-IL-6 is reproduced by a ERK1/2 antagonist. In pro-inflammatory conditions, chondrocytes produce large amounts of MMP and nitrites. ATRA inhibits iNOS expression and nitrites production induced by IL-1 in rat and human chondrocytes. However, total MMP activity is only inhibited in human chondrocytes, and this difference is not the consequence of a variable modulation of AP-1 activation. These results showed that retinoids have an important impact on inflammatory parameters on articular cells, but they varied regarding to the species, suggesting that extrapolation of results obtained from rat cells to human is difficult.

The ability of articular cartilage to self-repair after injury is limited due to the nature of the tissue. Biological repair is a promising treatment for cartilage injuries but success is limited by the ability to integrate with native cartilage. An in vitro model can be developed to investigate factors that regulate cartilage repair. A tissue engineered cartilage construct was placed into a host bovine osteochondral explant and cultured for 4 and 8 weeks. This same construct was cultured under stimulated and unstimulated conditions for 2 and 4 weeks. Autologous osteochondral implants served as controls. Integration was evaluated histologically, biochemically, biomechanically and for changes in gene expression. The tissue-engineered implants integrated over time whereas the autologous implants did not. Mechanical stimulation and prolonged incubation improved integration between implant and host tissue. An in vitro model of repair-native cartilage integration has been developed which is suitable for further study of tissue integration.

The ability of articular cartilage to self-repair after injury is limited due to the nature of the tissue. Biological repair is a promising treatment for cartilage injuries but success is limited by the ability to integrate with native cartilage. An in vitro model can be developed to investigate factors that regulate cartilage repair. A tissue engineered cartilage construct was placed into a host bovine osteochondral explant and cultured for 4 and 8 weeks. This same construct was cultured under stimulated and unstimulated conditions for 2 and 4 weeks. Autologous osteochondral implants served as controls. Integration was evaluated histologically, biochemically, biomechanically and for changes in gene expression. The tissue-engineered implants integrated over time whereas the autologous implants did not. Mechanical stimulation and prolonged incubation improved integration between implant and host tissue. An in vitro model of repair-native cartilage integration has been developed which is suitable for further study of tissue integration.

One major barrier that prevents cartilage integration following mosaic arthroplasty is the presence of a zone of chondrocyte death (ZCD) that is generated upon osteochondral graft harvest, which can extend up to 400 μm into the cartilaginous portion of the graft. In order for cartilage integration to occur, chondrocytes must be present at the graft periphery; however chondrocyte migration through the ZCD to the graft periphery is inhibited by the dense extracellular matrix (ECM) of cartilage. The purpose of this study was to develop a method for increasing the number of chondrocytes within the ZCD and at the periphery of a cartilage graft. This method used a combination of collagenase treatment (as a means of degrading the ECM within the ZCD) and chondrocyte chemotaxis (as a means of improving chondrocyte migration into the ZCD and to the cartilage periphery). Results indicate that treating bovine articular cartilage with 0.6 % collagenase for 10 min decreased with extent of the ZCD by approximately 35% (collagenase: 109 ± 13 μm; control: 175 ± 13 μm). Each of the chemotactic agents tested (PDGF-bb, bFGF, and IGF-I) were found to induce bovine chondrocyte chemotaxis at concentrations of 25 ng/mL in modified Boyden chamber experiments. However, in bovine articular cartilage samples that were pre-treated with collagenase (0.6% for 10 min), supplementation with 25 ng/mL of either PDGF-bb or bFGF had no apparent effect on the ZCD relative to samples treated only with collagenase (PDGF-bb: 85 ± 10 μm; bFGF: 88 ± 10 μm). Alternatively, bovine articular cartilage samples pre-treated with collagenase (0.6% for 10 min) and supplementation with 25 ng/mL IGF-I resulted in an approximately 65% reduction in the ZCD relative to samples treated only with collagenase (IGF-1: 38 ± 5 μm). Thus, treating osteochondral grafts with collagenase and IGF-1 induces chondrocyte repopulation of the zone of chondrocyte death generated by osteochondral graft harvesting, and could enhance cartilage integration after implantation. / Thesis (Master, Chemical Engineering) -- Queen's University, 2009-12-21 20:16:05.815

In vitro grown cartilage is functionally inferior to native tissue, and improvements in its quality should be attempted so it can be used therapeutically. In these studies we investigated the effects of cell shape on tissue quality through alteration of substrate geometry and application of mechanical stimuli. Articular chondrocytes were isolated and cultured on the surface Ti-6Al-4V substrates with various geometries. When cultured on fully porous titanium alloy substrates, chondrocyte spreading was enhanced over those grown on substrates with solid bases. Chondrocytes which remained round did not synthesize significant amounts of matrix and were thus unable to form cartilaginous tissue. In contrast, chondrocytes which were directed to spread to a limited amount, resulting in a polygonal morphology, accumulated significantly more matrix molecules and in time formed cartilage-like tissue. Computational fluid dynamics analyses demonstrated that cells on fully porous substrates experience time-dependent shear stresses that differ from those experienced by cells on substrates with solid bases where media flow-through is restricted. Integrin-blocking experiments revealed that integrins are important regulators of cell shape, and appeared to influence the accumulation of collagen and proteoglycans by chondrocytes. Furthermore, compressive mechanical stimulation induced a rapid, transient increase in chondrocyte spreading by 10 minutes, followed by a retraction to pre-stimulated size within 6 hours. This has been shown to be associated with increased accumulation of newly synthesized proteoglycans. Blocking the α5β1 integrin, or its β1 subunit, inhibited cell spreading and resulted in a partial inhibition of compression-induced increases in matrix accumulation, thereby substantiating the role of β1 integrins in this process. These results suggest that both fluid induced shear forces and compressive forces regulate chondrocyte matrix accumulation by altering cell morphology, which is mediated by integrins. Identifying the molecular mechanisms that influence chondrocyte shape and thus tissue formation may ultimately lead to the development of a tissue that more closely resembles native articular cartilage.

Articular cartilage provides a near frictionless surface for the articulating ends of bones. Cartilage functions to lubricate and transmit compressive forces resulting from joint loading and impact. If damaged, whether by traumatic injury or disease, cartilage lacks the ability for self-repair. This study explores the production of scaffoldree cartilage and investigates the effect of Tissue Growth Technologies’ CartiGen Bioreactor on the cartilage. Chondrocyte and bone marrow-derived stem cell (BMSC) attachment to chitosan is also investigated in hopes of producing a bilayered construct for osteochondral repair. Results demonstrate that culturing of scaffoldree cartilage in the CartiGen bioreactor resulted in an enhancement of the scaffoldree cartilage’s biomechanical and biochemical properties and that the chitosan microspheres were able to successfully support porcine chondrocyte and BMSC attachment. Results from both studies are encouraging for future work involving tissue engineered cartilage.

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Matrix Extracellular Phosphoglycoprotein (MEPE) is a member of a family of proteins called small integrin-binding ligand, N-linked glycoproteins (SIBLINGs) which play key roles in biomineralisation. Altered MEPE expression is associated with several phosphate and bone-mineral metabolic disorders such as oncogenic osteomalacia and hypophosphatemic rickets. Despite this, it remains undetermined what impact MEPE has on the growth plate; the cartilage anlagen from which endochondral ossification, the process responsible for linear bone growth, occurs. The work of this thesis has characterised the ATDC5 cell line and the metatarsal organ culture as useful in vitro models of endochondral ossification. These will prove vital in the pursuit of underpinning the molecular mechanisms involved in endochondral bone growth. These models form the basis of the further studies in this thesis examining the role of MEPE within this highly orchestrated process. Before such role can be defined, this thesis details the spatial and temporal localisation patterns of MEPE in 10-day- and 4-week-old murine growth plates. More specifically, MEPE protein and mRNA were preferentially expressed by the hypertrophic chondrocytes as shown by immunohistochemistry and in situ hybridisation respectively. Microdissection of the murine growth plate confirmed this. Localisation of the cleavage product of MEPE, a 2.2kDa acidic serine- and aspirate-rich motif (ASARM) peptide, followed a similar pattern of expression. The localisation of MEPE to sites of mineralisation serves to strengthen its potential role in chondrocyte matrix mineralisation. This thesis identified this role in both mineralising ATDC5 cells and the metatarsal organ culture. The ASARM peptide was found to be the functional component of MEPE and this function was dependent upon its post-translational phosphorylation. Phosphorylated (p)ASARM peptides significantly inhibited chondrocyte matrix mineralisation without altering the proliferation or differentiation of the chondrocyte cells, or their ability to produce an extracellular matrix. mRNA analysis by qPCR indicted a feedback system by which the pASARM peptide functions to allow the release of further ASARM peptides. Moreover, the pASARM peptide inhibited mRNA expression of markers of vascular angiogenesis highlighting a novel mechanism by which they may inhibit chondrocyte matrix mineralisation. This thesis also determines the regulatory cross-talk between the chondrocytes of the murine growth plate, with the most abundant bone cell type, the osteocyte. This cross-talk inhibits chondrocyte matrix mineralisation and is attributed to sclerostin, an osteocyte-specific secretory protein. Furthermore, it is shown that sclerostin acts through the MEPE-ASARM axis to regulate chondrocyte matrix mineralisation and thus endochondral ossification. The work described herein has characterised and validated in vitro models of growth plate chondrocyte matrix mineralisation and has used these to identify the role of MEPE within chondrocyte matrix mineralisation.