Extracellular Vesicles in the Synovial Joint: Is there a Role in the Pathophysiology of Osteoarthritis?

The role of extracellular vesicles (EV) in osteoarthritis has become the focus of much research. These vesicles were isolated from several cell types found in synovial joint including chondrocytes and synovium. As articular cartilage is an avascular tissue surrounded by synovial fluid, it is believed that EV might play a crucial role in the homeostasis of cartilage and also could hold key information in the pathogenesis of osteoarthritis. This is thought to be due to activation of pro-inflammatory factors leading to a catabolic state and degradation of cartilage. In addition, due to the nature of articular cartilage lacking neuronal innervation, knowledge of EV can contribute to identification of novel biomarkers in this debilitating condition. This can be either directly isolated from aspirate of synovial fluid or from peripheral blood. Finally, EVs are known to shuttle important signalling molecules which can be utilised as unique modality in transferring therapeutic compounds in a cell free manner.

Chondrogenesis of mesenchymal stem cells for cartilage tissue engineering.

Despite its remarkable ability to resist mechanical loading, articular cartilage is not capable of mounting a useful reparative reaction in response to damage caused by trauma or disease. As a result numerous surgical and medical approaches have been developed to aid the healing of articular cartilage. Despite the success of surgical techniques such as microfracture, recently attentions have been turned to cell based therapies such as autologous chondrocyte implantation (ACI). ACI has produced encouraging results, however better results may be achievable through an evolution of this surgical approach. Since the first generation of ACI techniques changes have been made in the technique e.g. the introduction of collagen membranes instead of periosteal flaps, and more recently the use of collagen scaffolds for cellular delivery. The procedure has also moved on from being performed as an open operation and can now be performed arthroscopically. Despite these advances the procedure still uses chondrocytes harvested from the joint being repaired. These cells are vulnerable to dedifferentiation during the required in vitro expansion, and as a result may not be capable of producing repair tissue once implanted back into the joint. Mesenchymal stem cells (MSCs) may provide a dedifferentiation resistant alternative to chondrocytes. MSCs would also allow for the use of one arthroscopic operation on the affected joint, as opposed to the two operations that are currently required for ACI.

Interleukin-1ß enhances cartilage-to-cartilage integration.

The failure of cartilages to fuse, particularly in the case of articular cartilage under conditions of repair is due to morphological and structural constraints of the tissue. Factors that impede integration include, non-vascularisation, low cellularity, and proteoglycan in the surrounding extracellular matrix acting as a natural barrier to cellular migration. We hypothesised that brief activation of a catabolic cascade by cytokines followed by culture under anabolic conditions would promote tissue fusion in a ring-disk model of cartilage integration. Our results show that transient exposure to 10 ng mL(-1) interleukin-1ß, followed by two weeks post-culture under anabolic conditions, enhanced cartilage-cartilage integration compared to untreated explants. Quantitative PCR analysis of catabolism-related genes ADAMTS4 and MMP13 showed both were transiently upregulated and these findings correlated with evidence of extracellular matrix remodelling. At the level of histology, we observed chondrocytes readily populated the interfacial matrix between fused explants in interleukin-1ß treated explants, whereas in control explants this region was relatively acellular in comparison. Catabolic cytokine treated explants exhibited 29-fold greater adhesive strength (0.859 MPa versus 0.028 MPa, P 〈 0.05) than untreated counterparts. Collectively, our results demonstrate that a single short catabolic pulse followed by an anabolic response is sufficient to generate mechanically robust, integrative cartilage repair.

Pellet culture model for human primary osteoblasts.

In vitro monolayer culture of human primary osteoblasts (hOBs) often shows unsatisfactory results for extracellular matrix deposition, maturation and calcification. Nevertheless, monolayer culture is still the method of choice for in vitro differentiation of primary osteoblasts. We believe that the delay in mature ECM production by the monolayer cultured osteoblasts is determined by their state of cell maturation. A functional relationship between the inhibition of osteoblast proliferation and the induction of genes associated with matrix maturation was suggested within a monolayer culture model for rat calvarial osteoblasts. We hypothesize, that a pellet culture model could be utilized to decrease initial proliferation and increase the transformation of osteoblasts into a more mature phenotype. We performed pellet cultures using hOBs and compared their differentiation potential to 2D monolayer cultures. Using the pellet culture model, we were able to generate a population of cuboidal shaped central osteoblastic cells. Increased proliferation, as seen during low-density monolayer culture, was absent in pellet cultures and monolayers seeded at 40,000 cells/cm2. Moreover, the expression pattern of phenotypic markers Runx2, osterix, osteocalcin, col I and E11 mRNA was significantly different depending on whether the cells were cultured in low density monolayer, high density monolayer or pellet culture. We conclude that the transformation of the osteoblast phenotype in vitro to a more mature stage can be achieved more rapidly in 3D culture. Moreover, that dense monolayer leads to the formation of more mature osteoblasts than low-density seeded monolayer, while hOB cells in pellets seem to have transformed even further along the osteoblast phenotype.

The role of surface microtopography in the modulation of osteoblast differentiation.

The osteoinductive and conductive capabilities of commercially pure titanium and its alloys is well documented, as is their ability to provide long-term stability for permanent implantable devices. Fracture fixation in paediatric and trauma patients generally requires transient fixation after which the implant becomes redundant and requires removal. Removal can be complicated due to excessive bony over-growth which is encouraged by the standard micro-rough implant surface. We have shown in vivo that removal related morbidity can be significantly reduced with surface polishing, a technique which reduces the micro-roughness of clinically available materials. However, tissue integration at the bone-implant interface requires activation of key regulatory pathways which influences osteoblastic differentiation and maturation therefore we do not believe this effect to be purely mechanical. To elucidate potential mechanisms by which surface polishing exerts its effect on bone regeneration this study assessed in vitro the effect of surface polishing commercially pure titanium on cell growth, morphology and on the regulation of core binding factor 1, osterix, collagen I, alkaline phosphatase, bone sialoprotein and osteocalcin for primary rat calvarial osteoblasts. Results indicate that polishing differentially influences osteoblast differentiation in a surface dependent manner and that these changes are potentially linked to surface dependent morphology, but not to differences in cell proliferation.

Use of stem cells in the biological repair of articular cartilage.

IMPORTANCE OF THE FIELD: Articular cartilage is avascular, aneural, and renowned for its poor capacity to repair after damage. For decades scientists and clinicians have deliberated over the potential to repair or regenerate articular cartilage and to date many techniques have been used in an attempt to create the best possible repair tissue. AREAS COVERED IN THIS REVIEW: This review article summarises surgical interventions that have been developed since the late 1940's; covering conservative strategies, invasive techniques and touching upon latest advancements involving stem cells and tissue engineering. WHAT WILL THE READER GAIN: The reader will gain a sound understanding into the history and background of strategies that have developed in attempts to reverse clinical symptoms of damaged or diseased articular cartilage. The article provides an insight into the plethora of potential repair mechanisms, and reviews future developments involving stem cells and biomaterials. TAKE HOME MESSAGE: Although work is still in its infancy, the use of stem cells in the biological repair of articular cartilage provides a promising outlook onto future developments; advancing from strategies and techniques that are already in use.

Surface polishing positively influences ease of plate and screw removal.

Difficulties removing temporary fracture fixation devices due to excessive bony on-growth results in extended surgical time leading to excessive blood loss, debris contamination and potentially refracture. Commercially available locking plates and screws are manufactured for clinics with a micro-rough surface, which contributes to the excessive bony on-growth reported. We have applied polishing technology to commercially pure titanium locking compression plates (LCP) and titanium-6%aluminium-7%niobium (TAN) plates and screws to assess if it can alleviate problems with strong bony overgrowth. Samples were implanted for 6, 12 and 18 months in a bilateral sheep tibia non fracture model and assessed for screw removal torque, percentage of bone contact and tissue-material response. Both electropolishing (p=0.001) and paste polishing (p=0.010) of TAN screws significantly reduced the mean torque required for removal compared to their micro-rough counterparts. This was accompanied by a trend for a lower percentage of bone contact for polished screws. This difference in bone contact was significant for paste polished TAN screws (p<0.001 parallel but not electropolished TAN screws (p=0.066). Ex vivo, soft tissue removal was much easier (approximately five minutes) for polished constructs, which was difficult and at least four times longer for standard micro-rough constructs. We suggest that polishing of locked plate/screw systems will improve ease of removal and reduce implant related removal complications encountered due to excessive strong bony on-growth while maintaining biocompatibility and implant stability. Future studies aim to assess the potential of this technology in the next level of complication, a fracture model.

Fibroblast growth factor-2 induced chondrocyte cluster formation in experimentally wounded articular cartilage is blocked by soluble Jagged-1.

INTRODUCTION: Basic fibroblast growth factor (FGF2) is a mitogen for articular chondrocytes. Cell death frequently occurs upon cartilage wounding and is evident during the progression of osteoarthritis. We hypothesised that incubation of wounded articular cartilage with exogenously added FGF2 would enhance cartilage repair, replacing dead cells through increased cell proliferation. METHODS: Articular cartilage from the metacarapalphalangeal joint of immature bovine steers was wounded in situ, then incubated in vitro in the continual presence or absence of FGF2. Cellular proliferation was expressed as a ratio of cell density of a fixed area between wounded and adjacent cartilage. Immunolabelling revealed the incorporation of bromodeoxyuridine and localisation of collagen type VI and Notch1 epitopes. gamma-secretase inhibitor N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester and soluble Jagged1 ligand (sJ1) were used to analyse the function of Notch signalling in this wound model. RESULTS: FGF2 induced cellular proliferation at the margins of wounded articular cartilage, where proliferative chondrocytes adopted a cluster configuration. Collagen type VI protein was expressed by chondrocytes in clusters, as was Notch1. Cellular proliferation was not affected by inhibition of gamma-secretase dependent Notch1 signalling. Binding of sJ1 to Notch1 receptors in FGF2 treated cartilage inhibited proliferation. CONCLUSION: Addition of FGF2 induces rapid chondrocyte proliferation in wounded cartilage, chondrocytes adopt a cluster morphology and also express Notch1. Binding of sJ1 to Notch1 causes apoptosis overriding a proliferative response. This study may shed some light on the significance of increased Notch1 expression and its localisation in chondrocyte clusters in osteoarthritic cartilage.

Clonal chondroprogenitors maintain telomerase activity and Sox9 expression during extended monolayer culture and retain chondrogenic potential.

OBJECTIVE: Articular cartilage contains mesenchymally derived chondroprogenitor cells that have the potential to be used for stem cell therapy. The aim of this study was to characterise the growth kinetics and properties of in vitro expanded cloned chondroprogenitors and determine if critical determinants of the progenitor phenotype were maintained or lost in culture. METHODS: Chondroprogenitors were isolated from immature bovine metacarpalphalangeal joints by differential adhesion to fibronectin. Cloned colonies were expanded in vitro up to 50 population doublings (PD). Growth characteristics were assessed by cell counts, analysis of telomere length, telomerase activity, expression of senescence-associated beta-galactosidase activity and real-time quantitative polymerase chain reaction to analyse the gene expression patterns of sox9 and Notch-1 in chondroprogenitors. RESULTS: Cloned chondroprogenitors exhibited exponential growth for the first 20 PD, then slower linear growth with evidence of replicative senescence at later passages. Mean telomere lengths of exponentially growing chondroprogenitors were significantly longer than dedifferentiated chondrocytes that had undergone a similar number of PD (P<0.05). Chondroprogenitors also had 2.6-fold greater telomerase activity. Chondroprogenitors maintained similar sox9 and lower Notch-1 mRNA levels compared to non-clonal dedifferentiated chondrocytes. Chondroprogenitors were induced to differentiate into cartilage in 3D pellet cultures, immunological investigation of sox9, Notch-1, aggrecan and proliferating cell nuclear antigen (PCNA) expression showed evidence of co-ordinated growth and differentiation within the cartilage pellet. CONCLUSION: Clonal chondroprogenitors from immature articular cartilage provide a useful tool to understand progenitor cell biology from the perspective of cartilage repair. Comparisons with more mature progenitor populations may lead to greater understanding in optimising repair strategies.

Cartilage integration: evaluation of the reasons for failure of integration during cartilage repair. A review.

Articular cartilage is a challenging tissue to reconstruct or replace principally because of its avascular nature; large chondral lesions in the tissue do not spontaneously heal. Where lesions do penetrate the bony subchondral plate, formation of hematomas and the migration of mesenchymal stem cells provide an inferior and transient fibrocartilagenous replacement for hyaline cartilage. To circumvent the poor intrinsic reparative response of articular cartilage several surgical techniques based on tissue transplantation have emerged. One characteristic shared by intrinsic reparative processes and the new surgical therapies is an apparent lack of lateral integration of repair or graft tissue with the host cartilage that can lead to poor prognosis. Many factors have been cited as impeding cartilage:cartilage integration including; chondrocyte cell death, chondrocyte dedifferentiation, the nature of the collagenous and proteoglycan networks that constitute the extracellular matrix, the type of biomaterial scaffold employed in repair and the origin of the cells used to repopulate the defect or lesion. This review addresses the principal intrinsic and extrinsic factors that impede integration and describe how manipulation of these factors using a host of strategies can positively influence cartilage integration.

Oxidative stress induces expression of osteoarthritis markers procollagen IIA and 3B3(-) in adult bovine articular cartilage.

OBJECTIVE: Oxidative stress occurs when the metabolic balance of a cell is disrupted through exposure to excess pro-oxidant. Whilst it is known that unregulated production or exposure to exogenous sources of pro-oxidants induces chondrocyte cell death and degrades matrix components in vitro, relatively little is known of the effects of pro-oxidants on articular cartilage in situ. The objective of this study was to determine if a single exposure to the pro-oxidant hydrogen peroxide (H(2)O(2)) induces a degenerative phenotype. METHODS: Articular cartilage explants were obtained from skeletally mature bovine steers and exposed to a single dose of hydrogen peroxide (0.1-1.0 mM) and cultured for up to 21 days. Cell death, and sulfated glycosaminoglycan loss into the medium and gene expression were quantitatively determined. Adoption of an abnormal chondrocyte phenotype was analyzed through the expression of 3B3(-), nitrotyrosine and procollagen type IIA epitopes in cartilage explants. RESULTS: Cell death occurred primarily at the surface zone of cartilage in a dose-dependent manner in H(2)O(2) treated explants, and supplementation of standard serum-free medium with insulin-selenium-transferrin significantly reduced cell death (>fourfold). Nitric oxide synthase-2 gene expression and proteoglycan loss increased in oxidant treated explants in a concentration-dependent manner. Antibody labeling to 3B3(-), procollagen type IIA and nitrotyrosine was present in all treated explants but absent in untreated explants. CONCLUSIONS: This study demonstrates that a single exposure to high levels of pro-oxidant causes the expression of genes and antibody epitopes that are associated with early degenerative changes observed in experimental osteoarthritis.

The development of synovial joints.

During vertebrate evolution, successful adaptation of animal limbs to a variety of ecological niches depended largely on the formation and positioning of synovial joints. The function of a joint is to allow smooth articulation between opposing skeletal elements and to transmit biomechanical loads through the structure, and this is achieved through covering the ends of bones with articular cartilage, lubricating the joint with synovial fluid, using ligaments to bind the skeletal elements together, and encapsulating the joint in a protective fibrous layer of tissue. The diversity of limb generation has been proposed to occur through sequential branching and segmentation of precartilaginous skeletal elements along the proximodistal axis of the limb. The position of future joints is first delimited by areas of higher cell density called interzones initially through an as yet unidentified inductive signal, subsequently specification of these regions is controlled hierarchically by wnt14 and gdf5, respectively. Joint-forming cell fate although specified is not fixed, and joints will fuse if growth factor signaling is perturbed. Cavitation, the separation of the two opposing skeletal elements, and joint morphogenesis, the process whereby the joint cells organize and mature to establish a functional interlocking and reciprocally shaped joint, are slowly being unraveled through studying the plethora of molecules that make up the unique extracellular matrix of the forming structure. The joint lining tissue, articular cartilage, is avascular, and this limits its reparative capacity such that arthritis and associated joint pathologies are the single largest cause of disability in the adult population. Recent discoveries of adult stem cells and more specifically the isolation of chondroprogenitor cells from articular cartilage are extending available therapeutic options, though only with a more complete understanding of synovial joint development can such options have greater chances of success.

Mechanically loaded ex vivo bone culture system 'Zetos': systems and culture preparation.

This paper introduces the culture preparation of ovine, bovine and human cancellous bone cores to be used in an explants model Zetos. The three dimensional (3D) bone cores were prepared and evaluated for all three animals. Bone cells in vivo constantly interact with each other, migratory cells, surrounding extracellular matrix (ECM) and interstitial fluid in a microenvironment, which continuously responds to various endogenous and exogenous stimuli. The Zetos system was designed to culture and mechanically load viable cancellous bone explants in their near natural microenvironment. This 3D ex vivo system bridges the current gap between in vitro and in vivo methods. One aim of this work was to compare the macro and micro-architecture of ovine, bovine and human cancellous bone tissue in preparation for culture within the Zetos system in order to determine the optimal source of experimental material. A second aim was to optimise the preparations of the bone cores as well as develop techniques involved during tissue maintenance. Bone core response was visualised using histological and immunohistochemical methods. The results demonstrate that cancellous bone explants vary greatly in trabecular density and bone volume depending on species, age and location. Sheep and human samples displayed the greatest variation between bones cores when compared to bovine. Even cores taken from the same animal possessed very different characteristics. The histology demonstrated normal bone and cell structure after the core preparation. Immunohistochemistry results demonstrated antigen retention after preparation methods.

Differential regulation of GDF-5 and FGF-2/4 by immobilisation in ovo exposes distinct roles in joint formation.

Members of the fibroblast growth factor (FGF) family and growth and differentiation factor 5 (GDF-5) have been implicated in joint specification, but their roles in subsequent cavity formation are not defined. Cavity formation (cavitation) depends upon limb movement in embryonic chicks and factors involved in joint formation are often identified by their expression at the joint-line. We have sought support for the roles of FGF-2, FGF-4, and GDF-5 in cavitation by defining expression patterns, immunohistochemically, during joint formation and establishing whether these are modified by in ovo immobilisation. We found that FGF-2 exhibited low level nuclear expression in chondrocytes and fibrocartilage cells close to presumptive joints, but showed significantly higher expression levels in cells at, and directly bordering, the forming joint cavity. This high-level joint line FGF-2 expression was selectively diminished in immobilised limbs. In contrast, we show that FGF-4 does not exhibit differential joint-line expression and was unaffected by immobilisation. GDF-5 protein also failed to show joint-line selective labelling, and although immobilisation induced a cartilaginous fusion across presumptive joints, it did not affect cellular GDF-5 expression patterns. Examining changes in GDF-5 expression in response to a direct mechanical strain stimulus in primary embryonic chick articular surface (AS) cells in vitro discloses only small mechanically-induced reductions in GDF-5 expression, suggesting that GDF-5 does not exert a direct positive contribution to the mechano-dependent joint cavitation process. This notion was supported by retroviral overexpression of UDPGD, a characteristic factor involved in hyaluronan (HA) accumulation at presumptive joint lines, which was also found to produce small decreases in AS cell GDF-5 expression. These findings support a direct mechano-dependent role for FGF-2, but not FGF-4, in the cavitation process and indicate that GDF-5 is likely to influence chondrogenesis positively without contributing directly to joint cavity formation.

The ultrastructure of mouse articular cartilage: collagen orientation and implications for tissue functionality. A polarised light and scanning electron microscope study and review.

Adult mouse articular cartilage (AC) has not been thoroughly described using high resolution imaging techniques, despite the fact that the availability of knockout mice with specific extracellular matrix (ECM) mutations have renewed interest in using the mouse as a model for a variety of different human conditions. With osteoarthritis affecting millions of people worldwide, investigations into the structure and, therefore, the ability of AC to act as a load-bearing tissue, are crucial for developing treatments and prevention techniques to limit the degree of severity in this condition. Cryofixation and formaldehyde fixation as well as chemical digestion of the uncalcified regions of AC were used in combination with bright field light, polarised light and scanning electron microscopy to image the structure of adult mouse AC. Chemical digestion of the tissue revealed unique insights into the structure of mouse AC and the high cellular density of the tissue. Tightly packed sheets of collagen fibrils formed the territorial matrix (TM) of the deep zone. These were observed closely surrounding the chondrons, after applying both chemical and cryofixation techniques. The interterritorial matrix (IM), in contrast, was more isotropically arranged. The results of the study have implications for the interpretation of biomechanical functionality of mouse AC with probable applications to other species.

Current strategies for articular cartilage repair.

Defects of articular cartilage that do not penetrate to the subchondral bone fail to heal spontaneously. Defects that penetrate to the subchondral bone elicit an intrinsic repair response that yields a fibrocartilaginous repair tissue which is a poor substitute for hyaline articular cartilage. Many arthroscopic repair strategies employed utilise this intrinsic repair response to induce the formation of a repair tissue within the defect. The goal, however, is to produce a repair tissue that has the same functional and mechanical properties of hyaline articular cartilage. To this end, autologous osteochondral transfer can provide symptomatic relief. This technique involves the excision of healthy cartilage plugs from 'non-load bearing' regions of the joint for implantation into the defect. Cell based transplantation methods currently involve the transplantation of expanded autologous chondrocytes to the defects to form a repair tissue. This technique again involves the excision of healthy cartilage from the joint for expansion. Current research is exploring the potential use of mesenchymal stem cells as a source for tissue engineering, as well as the combination of cells with biodegradable scaffolds. Although current repair strategies improve joint function, further research is required to prevent future degeneration of repair tissue.

Cell-cell contact and membrane spreading in an ultrasound trap.

An ultrasonic standing wave trap [Langmuir 19 (2003) 3635] in which the morphologies of 2-D latex-microparticle aggregates, forming a pressure node plane, were characterised has been applied here to different cell suspensions with increasing order of specificity of cross-linking molecule, i.e. polylysine with chondrocytes; wheat germ agglutinin (WGA) with erythrocytes and surface receptors on neural cells. The outcome of initial cell-cell contact, i.e. whether the cells stuck at the point of contact (collision efficiency = 1) or rolled around each other (collision efficiency = 0), was monitored in situ by video-microscopy. The perimeter fractal dimensions (FD) of 2-D hexagonally symmetric, closely packed aggregates of control erythrocytes and chondrocytes were 1.16 and 1.18, respectively while those for the dendrititc aggregates formed initially by erythrocytes in 0.5microg/ml WGA and chondrocytes in 20 microg/ml polylysine were 1.49 and 1.66. The FDs for control and molecularly cross-linked cells were typical of reaction-limited aggregation (RLA) and transport diffusion-limited aggregation (DLA), respectively. The FDs of the aggregates of cross-linked cells decreased with time to give more closely packed aggregates without clear hexagonal symmetry. Suspensions of neural cells formed dendritic aggregates. Spreading of inter-cellular membrane contact area occurred over 15 min for both erythrocyte and neural cell dendritic aggregates. The potential of the technique to characterise and control the progression of cell adhesion in suspension away from solid substrata is discussed.

The cellular responses of articular cartilage to sharp and blunt trauma.

OBJECTIVE: To determine the response of immature articular cartilage to both sharp and blunt trauma in terms of cell death, cell proliferation and matrix synthesis. DESIGN: Blunt wounds were made with a trephine in full depth immature bovine articular cartilage explants which were cut in half through the center of the trephine wound with a sharp scalpel to produce blunt and sharp trauma on the same explant. Explants were maintained in culture for up to 10 days. Prior to fixation at days 2, 5 and 10, medium was supplemented with 10 microCi ml-1 35S-sulphate, [3H]-proline or [3H]-thymidine for 24h to assess matrix synthesis and cell proliferation. Cell death was assessed using a Live/Dead label. RESULTS: In the case of blunt wounds, a band of cell death was observed adjacent to the lesion edge. Microautoradiography demonstrated little radiolabel incorporation and, therefore, no new matrix synthesis or cell proliferation within this region. In contrast, wounds made with a sharp scalpel showed restricted cell death, with radiolabel incorporation adjacent to the lesion edge at all time points. This demonstrated not only chondrocyte proliferation and new matrix synthesis at the wound margin, but also an up-regulation of matrix synthesis adjacent to the lesion edge. CONCLUSIONS: In terms of clinical relevance, the use of sharp precise instruments during the surgical management of cartilage defects may be necessary to reduce cell death and promote matrix elaboration at the lesion edge in order to facilitate successful integration.

A mechanism underlying the movement requirement for synovial joint cavitation.

Many studies have highlighted the importance of movement-induced mechanical stimuli in the development of functional synovial joints. However, such phenomenological results have failed to provide a full explanation of the mechanism essential for the morphogenesis of fluid-filled joint cavities. We have previously demonstrated that the large glycosaminoglycan hyaluronan (HA), in association with its principal cell surface receptor CD44, plays a major role during the morphogenesis of chick joints. We have taken cells from the surface of recently cavitated joints and subjected them to a brief period of dynamic mechanical strain (3800 microE for 10 min) and measured changes in HA synthesis/release, CD44 expression and HA synthase gene expression. In addition, we subjected cells to matrix depletion prior to the application of mechanical strain in order to examine any potential modulatory function of the ECM during the cell response to strain. Removal of the cell-associated HA-containing matrix with hyaluronidase significantly increased the release of HA into tissue culture media over 24 h and is associated with increased CD44 expression, alterations in HA synthase gene expression and enhanced binding of HA to the cell surface. Such changes in HA release were shown to be blocked by addition of exogenous HA and synergistically enhanced by the application of dynamic mechanical strain. These results show that cell-matrix interactions modify the response of embryonic cells to mechanical strain and provide further insight into the mechano-dependent mechanism of joint cavity morphogenesis.

The distribution of Notch receptors and their ligands during articular cartilage development.

We examined the distribution of Notch family members and their ligands during the development of articular cartilage and the growth plate. Notch 1 was expressed by the chondrocytes of the developing articular surface but became increasingly restricted to the deeper layers after birth whilst expression of this family member was restricted to hypertrophic chondrocytes in the growth plate. Notch 2 and 4, Delta and Jagged 2 showed a broadly similar distribution, being present throughout the articular cartilage during development and becoming increasingly restricted to deeper layers with age. Hypertrophic chondrocytes within the growth plate also expressed Notch 2 and 4, Delta and Jagged 2 (which was also expressed in prehypertrophs). Notch 3 and Jagged 1 were absent from developing articular cartilage but were present in deeper layers at later time points (> 1 month) and both receptor and ligand were expressed in hypertrophic chondrocytes at all ages examined. These results highlight the complex Notch signalling interactions that result in the formation of the heterogeneous articular cartilage and allow for the co-ordinated ossification and elongation of the growth plate. Mechanisms by which these processes are controlled are discussed in light of recent advances in the understanding of Notch signalling pathways.