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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.

Articular cartilage undergoes cycles of compressive loading during joint movement, leading to its cyclical deformation and recovery. This loading is essential for chondrocytes to perform their normal function of maintenance of the extracellular matrix. Various lines of evidence suggest the involvement of the cytoskeleton in load sensing and response. The purpose of the present study is to describe the 3-dimensional (3D) architecture of the cytoskeleton of chondrocytes within their extracellular matrix, and to examine cytoskeletal responses to experimentally varied mechanical conditions. Uniformly sized explants of articular cartilage were dissected from adult rat femoral heads. Some were immediately frozen, cryosectioned and labelled for filamentous actin using phalloidin, and for the focal contact component vinculin or for vimentin by indirect immunofluorescence. Sections were examined by confocal microscopy and 3D modelling. Actin occurred in all chondrocytes, appearing as bright foci at the cell surface linked to an irregular network beneath the surface. Cell surface foci colocalised with vinculin, suggesting the presence of focal contacts between the chondrocyte and its pericellular matrix. Vimentin label occurred mainly in cells of the deep zone. It had a complex intracellular distribution, with linked networks of fibres surrounding the nucleus and beneath the plasma membrane. When cartilage explants were placed into organ culture, where in the absence of further treatments cartilage imbibes fluid from the culture medium and swells, cytoskeletal changes were observed. After 1 h in culture the vimentin cytoskeleton was disassembled, leading to diffuse labelling of cells. After a further hour in culture filamentous vimentin label reappeared in deep zone chondrocytes, and then over the next 48 h became more widespread in cells of the explants. Actin distribution was unaffected by culture. Further experiments were performed to test the effects of load on the cytoskeleton. Explants were placed in culture and immediately subjected to static uniaxial radially unconfined compressive loads of 0.5, 1, 2 or 4 MPa for 1 h using a pneumatic loading device. Loads greater than 0.5 MPa maintained the vimentin organisation over the culture period. At 0.5 MPa, the chondrocytes within the explant behaved as in free-swelling culture. The rapid change in vimentin organisation probably relates to rapid swelling of the explants—under free-swelling conditions, these reached their maximum swollen size in just 15 min of culture. The chondrocytes' response to change in tissue dimensions, and thus to their relationship to their immediate environment, was to disassemble their vimentin networks. Loading probably counteracts the swelling pressure of the tissue. Overall, this work suggests that chondrocytes maintain their actin cytoskeleton and modify their vimentin cytoskeleton in response to changing mechanical conditions.

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.

The myogenic potential of chick limb mesenchyme from stages 18-25 was assessed by micromass culture under conditions conductive to myogenesis, and was measured as the proportion of differentiated (muscle myosin-positive) mononucleated cells detected. It was found that similar myogenic potentials existed in mesenchyme from whole limbs between stages 18 and 19, but this potential was halved by stage 20. At stage 21, proximal mesenchyme showed significantly more myogenesis than distal mesenchyme, but this difference was abolished by stage 22. Thereafter, myogenesis was increasingly restricted from the distal mesenchyme, whilst the potential in more proximal regions did not significantly increase after stage 23. When the ratio between total limb myoblasts which differentiated on days 1 and 4 of culture was analysed, it was found that two distinct peaks existed at stages 20 and 23. The significance of these ratio peaks is unclear, but may be related to different proliferative potentials of the pre-myoblasts at these stages.

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.

To determine the response of immature articular cartilage to both sharp and blunt trauma in terms of cell death, cell proliferation and matrix synthesis.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.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.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.

To investigate the differences between chondrocytes of the superficial and underlying zones of articular cartilage at the level of gene expression.Messenger RNA (mRNA) was isolated from chondrocytes harvested from the superficial and deep zones of immature bovine articular cartilage. This mRNA was reverse transcribed, radiolabeled, and then each complementary DNA (cDNA) sample was used to screen duplicate filters of a bovine chondrocyte cDNA library. By comparing autoradiographic signals on matching filter sets, clones exclusively expressed in the superficial zone of articular cartilage were isolated and characterized further.Of the superficial-specific gene clones isolated, 25% were found to be a single gene product, clusterin. Northern hybridization was used to show that clusterin is expressed specifically in the superficial zone of articular cartilage and that its expression is up-regulated in mature cartilage. In situ hybridization was used to precisely localize clusterin transcripts in articular cartilage, where it was found that clusterin expression was confined to the articular surface in both immature and mature samples.The discovery of clusterin expression at the articular cartilage surface extends previous observations that superficial articular chondrocytes are highly specialized cells. Clusterin is a multifunctional, secreted glycoprotein that has been shown to be expressed in diverse locations that have in common a tissue-fluid boundary. Additionally, clusterin has been implicated in regulating complement activation and cell death in injured and degenerating tissues.

Growth/differentiation factor-5 (GDF-5) has been shown to be essential for normal appendicular skeletal and joint development in humans and mice. In brachypod, a Gdf-5 gene mouse mutant, the defect is first apparent during early chondrogenesis, with the cartilage blastema already reduced in size by E12.5. This defect is associated with changes in the expression of cell surface molecules.To understand further how GDF-5 controls cartilage formation, we first mapped the expression of the Gdf-5 gene during skeletal development (please note that the abbreviation for the gene is given in italics and the abbreviation for the protein expressed by the gene is given in capital letters). Subsequently, we over-expressed GDF-5 in the developing chick embryo using a replication competent retrovirus, RCAS(BP). We determined its effects on skeletal development by histological examination and its effects on early growth by autoradiography of proliferating cells. In addition, we examined the effect of GDF-5 on chondrogenic differentiation using micromass and single cell suspension cultures of limb mesenchymal cells,These studies show that the Gdf-5 gene is expressed in the early cartilage condensation, the perichondrium, and the joint interzone. Over-expresSion of GDF-5 in chick limb buds, during the condensation stage or later when the skeletal elements have formed, increased the size of the affected elements. In both cases, the increase in size was associated with an increase in cell number and, at later stages, this was correlated with an increase in S-phase cells. In vitro studies showed that GDF-5 could increase cell adhesiveness, and this may be a mechanism through which GDF-5 initiates condensation formation.These studies show that GDF-5 acts at two stages of skeletal development and by two distinct mechanisms. First, GDF-5 promotes the initial stages of chondrogenesis by promoting cell adhesion, which is consistent with the expression of Gdf-5 in the cartilage condensation. Second, GDF-5 can increase the size of the skeletal elements by increasing proliferation within the epiphyseal cartilage adjacent to its expression within the joint interzone.

To determine the cellular and matrix responses to experimental wounding of articular cartilage.Immature and mature bovine articular cartilage was used as an in vitro model system to study the cellular responses to cartilage wounding. Explant cultures were wounded centrally with a trephine and maintained for up to 10 days. TUNEL labeling together with ultrastructural analyses were used to assess the nature of the observed cell death. In vitro labeling with 3H-thymidine was used to detect cell proliferation, and 2 antibodies (COL2-3/4M and BC-13) were used to detect changes in matrix turnover.Cell death was observed as a response to wounding and was considered to be a combination of necrosis and apoptosis. In immature tissue, cell death was more pronounced, particularly in the articular surface region. Within the area of cell death, many cells that did not die subsequently underwent proliferation. The collagenous network showed evidence of denaturation in the area of the wound, but "aggrecanase" activity was not detected.There are 2 contrasting, but related, responses to cartilage wounding--apoptosis and proliferation. In order to improve cartilage repair, future studies need to elucidate the regulatory mechanisms that determine these responses.

The magnetic resonance imaging appearance of the proximal interphalangeal joints of cadavers was correlated with histology of the same specimen allowing many small-scale features to be identified that might otherwise have been misinterpreted. It enabled the magnetic resonance signal to be understood at a tissue and cellular level, allowing identification of synovial folds extending from the extensor tendon and volar plate, the entheses of the proper collateral ligament, the epitenon of the flexor tendons and the presence of osteophytes and sites of cartilage erosion. The main difficulties in matching two-dimensional magnetic resonance images with histology were the differing section thicknesses of the two methods and shrinkage of histological specimens. There are many advantages in producing high resolution three-dimensional datasets—the magnetic resonance section thickness is reduced and the individual components of the joint can be viewed simultaneously in two or more planes. A unique magnetic resonance atlas of three dimensional joint structure is presented.

There have been many reports on the histological development of mammalian diarthrodial or synovial joints. While these are useful for comparative purposes, they tell us little of the cellular basis of joint morphogenesis which must underlie a number of morphogenetic defects. The process of joint morphogenesis is complex and can be subdivided into a number of facets and this report will focus on 2 of them. First, the process of joint cavitation in the chick metatarsophalangeal joint, where we propose that the selective secretion of hyaluronan into the presumptive cavity plays a central role. Secondly, the development of articular cartilage where we have used the South American opossum Monodelphis domestica as a model for mammalian development. Like most marsupials, the young are born at a much earlier developmental stage than eutherian mammals. Using antibodies which detect proliferating chondrocytes and those synthesizing insulin-like growth factors 1 and 2 and insulin-like growth factor 1 binding protein, we report that the majority of growth (as assessed by these indicators) appears appositional.

The mechanisms essential for generating diarthrodial joint cavities between skeletal elements in developing limbs remain enigmatic. Histochemical localization of hyaluronan (HA) at joint interzones concomitant with cavitation led to the postulation that HA may be pivotal in this process. HA synthesis involves the transfer of UDP-glucuronate and UDP-N-acetyl glucosamine to nascent HA by HA synthase. Uridine diphosphoglucose dehydrogenase (UDPGD) activity is responsible for the prior conversion of UDP-glucose to UDP-glucuronate. We have assessed the relationship between the appearance of HA and enzyme activities (quantitatively where possible) involved in HA synthesis during metatarsophalangeal joint development in embryonic chicks. Microspectrophotometric assessment of UDPGD activity using an in situ biochemical assay indicated that cells immediately adjacent to forming cavities contained increased UDPGD activity, which was subsequently maintained after cavitation. Immunocytochemistry showed that high levels of expression of HA synthase were localized to these same cells. In addition, radiolabeled sulfate autoradiography showed that cells bordering developing cavities incorporated relatively little sulfate, suggesting that UDP-glucuronate is utilized in the synthesis of undersulfated or non-sulfated glycosaminoglycans. These results indicate that the differentiation of cells bordering presumptive spaces may involve alterations associated specifically with differential synthesis of HA, which appears to be a primary event in joint cavity formation.

There have been many reports on the histological development of mammalian diarthrodial or synovial joints. While these are useful for comparative purposes, they tell us little of the cellular basis of joint morphogenesis which must underlie a number of morphogenetic defects. The process of joint morphogenesis is complex and can be subdivided into a number of facets and this report will focus on 2 of them. First, the process of joint cavitation in the chick metatarsophalangeal joint, where we propose that the selective secretion of hyaluronan into the presumptive cavity plays a central role. Secondly, the development of articular cartilage where we have used the South American opossum Monodelphis domestica as a model for mammalian development. Like most marsupials, the young are born at a much earlier developmental stage than eutherian mammals. Using antibodies which detect proliferating chondrocytes and those synthesising insulin-like growth factors 1 and 2 and insulin-like growth factor 1 binding protein, we report that the majority of growth (as assessed by these indicators) appears appositional.

The causative mechanism of tendon calcification ('calcifying tendinitis') is unknown. In this report, pathological human tendon samples were examined to give morphological and ultrastructural detail of the calcified regions and these findings were compared with those from normal tendon. Selected specimens were cryosectioned to enable histochemical and immunohistochemical comparison of the occurrence and distribution of specific matrix molecules in diseased and normal tendon tissues. The lack of collagen type II and alkaline phosphatase in the pathological regions suggests that the calcification process is not mediated through an endochondral transition. In contrast, the pathological areas were characterised by widespread labelling for chondroitin-4-sulphate/dermatan sulphate and intense pericellular localisation of chondroitin-6-sulphate.

Hyaluronan, a common connective tissue component, influences cell adhesion, migration and cytodifferentiation in vitro, and because of these properties has been postulated to have a role in morphogenesis. Its role in the development of cartilage-associated structures, such as joints, has yet to be defined. Using a biotinylated hyaluronan-binding region-link protein complex, free hyaluronan binding sites have been localised in the joint region concomitant with the first signs of cavitation (Stage 37), whereafter it is localised in the joint space and is maintained here as this enlarges. The application of our results is discussed in the context of a primary role for hyaluronan in joint cavity formation.

The morphogenetic capacity of prechondrogenic mesenchyme from two developmentally distinct sources was investigated in high density micromass cultures. We confirmed an earlier report (WeissMoscona, 1958) that scleral mesenchyme formed cartilage sheets whilst limb bud mesenchyme formed distinct cartilage nodules. It was thus suggested by these authors that this morphogenesis was tissue type specific. However, by varying cell density at inoculation (which controls cell configuration) and by varying the relative amount of prechondrogenic mesenchyme present in cultures we found that dramatic changes in morphogenesis could be brought about. Viewed in these terms we suggest that cartilage morphogenesis in vitro is dependent on cell configuration and the presence of non-chondrogenic cell types and hence is not necessarily a function of an intrinsic morphogenetic potential of the constituent cells.