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Objective: To investigate the immunolocalisation of beta-dystroglycan (beta-DG) and specific matrix metalloproteinases (MMPs)-3, -9, -13 and a disintegrin like and metalloproteinase thrombospondin type 1 motif 4 (ADAMTS-4) within the joint tissues of patients with osteoarthritis (OA) and unaffected controls. Design: Cartilage, synovium and synovial fluid were obtained from the hip joints of five osteoarthritic (patients undergoing total hip replacement) and five control hip joints (patients undergoing hemiarthroplasty for femoral neck fracture). The samples were analysed for beta-DG protein using Western blot technique and by immunohistochemistry for tissue distribution of beta-DG, MMP-3, -9, -13, and ADAMTS-4. Results: beta-DG was detected in the smooth muscle of both normal and osteoarthritic synovial blood vessels. Importantly, beta-DG was detected in endothelium of blood vessels of OA synovium, but not in the control endothelium. In the endothelium of osteoarthritic synovial blood vessels, beta-DG co-localised with MMP-3 and -9. MMP-13 and ADAMTS-4 showed no endothelial staining, and only weak staining of the vascular smooth muscle was found. In contrast, we did not detect beta-DG protein in cartilage or synovial fluid. Conclusions: beta-DG has been shown to have a role in angiogenesis, and our results demonstrate for the first time that there are clear differences in beta-DG staining between OA and control synovial blood vessels. The specific immunolocalisation of beta-DG within endothelium of inflamed OA blood vessels and its co-localisation with MMP-3 and -9, reported to have pro-angiogenic roles and believed to be involved in beta-DG cleavage, may also suggest that beta-DG plays a role in angiogenesis accompanying OA. [ABSTRACT FROM AUTHOR]

Epidemiological studies suggest that poor growth during fetal life and infancy is associated with decreased bone mass in adulthood. However, theses observations have not, to date, been corroborated in animal models. To address this issue we evaluated the influence of maternal protein restriction on bone mass and growth plate morphology among the adult offspring, using a rat model. Maternal protein restriction resulted in a reduction in bone area and BMC, but not BMD, among the offspring in late adulthood. The widened epiphyseal growth plate in the protein-restricted offspring is compatible with the programming of cartilage and bone growth by maternal nutrition in early life. [ABSTRACT FROM AUTHOR]

The effectiveness of Na-beta-, and Ca-glycerophosphates (GPs) in inducing mineralization was tested during long-term organ culture of femurs from 14-day-old chick embryos. When bones were incubated with Na-GP, a 66% rise in inorganic phosphate level was measured in the medium, supporting the notion that provision of a substrate for alkaline phosphatase (ALP) increased available phosphate. On the other hand, if the concentrations of Ca2+ were raised, available inorganic phosphate was decreased. Similarly, increases in inorganic phosphate decreased available calcium. Both GPs induced mineralization in bone and cartilage, but more matrix was mineralized with Ca-GP. However, the induction of mineralization by GPs was accompanied by dystrophic calcification, reduction of matrix formation and ALP activity, and increased release of lactate dehydrogenase into the culture medium. The new osteoid, which formed during culture, mineralized in the absence of GPs without the above adverse effects provided the culture period was longer than 15 days. The described organ culture system therefore facilitates studies of the mechanism of bone mineralization without the disadvantages of GP addition. [ABSTRACT FROM AUTHOR]

This study has examined the osteogenic and chondrogenic differentiation of human foetal femur-derived cells in 3-dimensional pellet cultures. After culture for 21-28 days in osteogenic media, the pellets acquired a unique configuration that consisted of an outer fibrous layer, an osteoid-like shell surrounding a cellular and cartilaginous region. This configuration is typical to the cross section of the foetal femurs at the same age and was not observed in pellets derived from adult human bone marrow stromal cells. Time course study showed that after 7-14 days, the cells of the inner cellular region were viable, proliferated rapidly, and were immuno-positive for c-myc, as well as for bone sialoprotein and type I collagen. After 21-28 days, the cells accumulated at the inner edge of the osteoid shell. The direction of osteoid formation thus differed from that of periosteal bone formation. Following micro-dissection of the human foetal femurs into epiphyses, bone cylinder and hypertrophic cartilage, epiphyseal chondrocytes and osteoblasts both gave rise to osteoid-shell forming cells. These studies demonstrate the developmental plasticity of human foetal skeletal and epiphyseal chondrocytes and suggest that the microenvironment modulates lineage commitment and matrix formation. Furthermore, this ex vivo model offers a new approach to delineate human bone development as well as a model with potential application for evaluation of therapeutic compounds for bone formation.

Objective: To review the current knowledge of the mechanism of DNA methylation, its association with transcriptional silencing, possible mechanisms of hyper- and hypomethylation and how epigenetic changes may relate to the pathogenesis of osteoarthritis (OA)., Methods: Journal literature was searched using Pubmed. Since there are very few publications directly on epigenetic phenomena in OA, the search was extended to give an overview of epigenetic mechanisms as they relate to the molecular mechanisms of the disease., Results: While the epigenetics of cancer cells have been intensively investigated, little attention has so far been paid as to whether epigenetic changes contribute to the pathology of non-neoplastic diseases such as OA. This review explains the mechanisms of DNA methylation, its role in transcriptional regulation, and possible demethylation mechanisms that may be applicable to OA. Preliminary evidence suggests that changes in DNA methylation, together with cytokines, growth factors and changes in matrix composition, are likely to be important in determining the complex gene expression patterns that are observed in osteoarthritic chondrocytes., Conclusion: Early evidence points to a role of epigenetics in the pathogenesis of OA. Since epigenetic changes, although heritable at the cellular level, are potentially reversible, epigenetics could be a new molecular target for therapeutic intervention, especially early in the disease.

Objective: Collagen fibril degeneration involves initially the cleavage within the triple helix by the collagenases 1 (MMP-1) and 3 (MMP-13), but then mainly involves also the gelatinases A (MMP-2) and B (MMP-9). The objective of this study was to determine the quantitative expression levels as well as the distribution in normal and osteoarthritic cartilage of gelatinase B and in cultured articular chondrocytes with and without stimulation by Il-1Beta., Methods: Conventional and real-time quantitative PCR technology and immunohistochemistry were used to determine gelatinase B expression on the mRNA and protein level., Results: Conventional PCR analysis could demonstrate the presence of gelatinase B mRNA only in osteoarthritic chondrocytes. Real-time quantitative PCR confirmed the increased expression of gelatinase B mRNA expression in osteoarthritic chondrocytes. No significant up-regulation of gelatinase B was observed by Il-1Beta. Immunostaining for gelatinase B showed the presence of gelatinase B in a subset of normal and in a large portion of osteoarthritic chondrocytes with a more extended distribution in the latter., Conclusion: In osteoarthritic cartilage destruction, gelatinase B is involved in collagen destruction though still at a very much lower level than gelatinase A. Only a very small subset of normal adult articular chondrocytes express gelatinase B in vivo suggesting that gelatinase B unlike gelatinase A is hardly or only very focally involved in physiological collagen turnover.

Six calcaneal fragments from patients aged 2, 3, 4, and 5 years with relapsed talipes, and two normal feet from a 40-week-old stillborn fetus were studied. All tissue was sectioned in the sagittal or coronal plane and stained using alcian blue and sirius red to distinguish cartilage and bone. Immunocytochemistry was performed to illustrate collagen types I and II. Within the clubfoot calcaneum, there were fewer chondrocytes and a diminished number of cartilage canals. Although a growth plate was present, the zones of differentiated chondrocytes were not apparent and the chondrocytes were smaller and flatter. The alcian blue staining within the spherical physis was paler than normal, suggesting that the amount of extracellular proteoglycans was reduced. Overall, the growth plate region of the talipes calcaneum resembled that of a permanent cartilage, like articular cartilage. Abnormalities were also seen in the ossification center. Cartilage spicules were rare, and developing bone frequently abutted directly onto the growth plate cartilage. The relative absence of a primary spongiosa suggested that the physis was virtually inactive and endochondral bone formation was retarded. These findings are consistent with the hypothesis that an intrinsic primary growth disorder causes the formation of a small hypoplastic bone and, subsequently, a smaller foot.

The efficient elimination of apoptotic cells depends on heterophagocytosis by other cells, which is difficult or impossible when the dying cells are embedded in an extracellular matrix. This situation is exemplified by the epiphyseal chondrocytes during the development of the chondroepiphyses of long bones. A detailed ultrastructural study identified an unusual type of epiphyseal chondrocyte which contained a very dark nucleus with irregular patches of condensed chromatin and a crenated nuclear membrane. The cytosol consisted of excessively expanded endoplasmic reticulum lumen, containing "islands" of cytoplasm and organelles. Since these cells appeared to be "in limbo," neither viable nor dead, they are referred to as "paralyzed" cells. By studying cells of intermediate morphologies, we were able to demonstrate the sequence of events leading to cell paralysis. It is proposed that the paralysis represents an intermediate state in the physiological cell death of epiphyseal chondrocytes in which destruction is orderly and avoids a inflammatory, potentially locally destructive, reaction. The cell is rendered paralyzed in terms of function but impotent in respect of damaging consequences. Paralysis is compared and contrasted with apoptosis, autophagocytosis, and necrosis and may represent another mode of programmed cell death in situations where cells are immature and/or where phagocytosis by neighboring cells is difficult.

The epiphyses of femurs from 7.5-15 day chicken embryos were studied by electron microscopy. Several forms of aberrant cell cycles were present: (1) in the perichondrium, polyploid metaphases, segmentating large (giant) cells, and mitotic catastrophe (midway between mitosis and apoptosis) were observed; (2) in the resting zone, premature chromosome condensation was found; (3) in the proliferative zone, approximately 5% of divisions were aberrant, representing most often mitosis restitution from metaphase and more seldom from the anaphase; (4) in all layers, 'dark chondrocytes' representing a premortal form of hypersecretory cells undergoing often a-mitotic nuclear segmentation were present. Many of the aberrations of cell cycle were combined with cell death. These deviations omitting or adapting the cell cycle check-points represent evidently the normal epigenetic mechanisms of development and repair. At the same time, by origin and appearances they seem very close to the loss of the growth control displayed by malignant tumours. This connection is briefly analysed in view of some current concepts of carcinogenesis.

The activities of acid and alkaline phosphatases were localized by enzyme histochemistry in the chondroepiphyses of 5 week old rabbits. Using paraformaldehyde-lysine-periodate as fixative, the activity of acid phosphatase was particularly well preserved and could be demonstrated not only in osteoclasts, but also in chondrocytes as well as in the cartilage and early endochondral matrices. The acid phosphatase in the chondrocytes and the matrix was tartrate-resistant, but inhibited by 2 mM sodium fluoride, whereas for osteoclasts 50-100 mM sodium fluoride were required for inhibition. Simultaneous localisation of both acid and alkaline phosphatase activities was possible in tissue that had been fixed in 85% ethanol and processed immediately. In the growth plates of the secondary ossification centre and the physis, there was a sequential localisation of the two phosphatases associated with chondrocyte maturation. The matrix surrounding immature epiphyseal chondrocytes or resting/proliferating growth plate chondrocytes contained weak acid phosphatase activity. Maturing chondrocytes were positive for alkaline phosphatase which spread to the matrix in the pre-mineralizing zone, in a pattern that was consistent with the known location of matrix vesicles. The region of strong alkaline phosphatase activity was the precise region where acid phosphatase activity was reduced. With the onset of cartilage calcification, alkaline phosphatase activity disappeared, but strong acid phosphatase activity was found in close association with the early mineral deposition. Acid phosphatase activity was also present in the matrix of the endochondral bone, but was only found in early spicules which had recently mineralised. The results suggest that alkaline phosphatase activity is required in preparation of mineralization, whereas acid phosphatase activity might have a contributory role during the early progression of mineral formation.

Many events occur concurrently during the initiation of the secondary ossification center in the cartilaginous epiphyses of long bones. We have investigated the chronology of interactions between the vascular system and epiphyseal chondrocytes by culturing explanted heads of femurs and humeri from pre- and neonatal rabbits on the chorioallantoic membrane (CAM) of growing chick embryos. We confirmed that, on the whole, the epiphyseal cartilage was resistant to vascular invasion, whereas the physeal growth plate was resorbed. However, new CAM-derived cartilage canals occasionally penetrated through the articular surface. This caused death of those chondrocytes in the immediate vicinity of the canal but no further reaction. If explants already contained a bony epiphysis and were halved prior to culture, CAM-derived vessels were attracted to the spongiosa. From there they pushed into the uncalcified cartilage, indicating that calcification was not a prerequisite for vascular invasion. Where at least two vessels were in apposition, a new pseudo-ossification center was initiated: chondrocytes became hypertrophic and the matrix calcified. This suggests that cumulative release of diffusible factors from more than one vessel was the trigger for chondrocyte hypertrophy, which, in turn, led to the initiation of the bony epiphysis. CAM cultures thus provide an experimental model for both the quiescent angiogenesis of cartilage canal formation and the reactionary angiogenesis associated with chondrocyte hypertrophy. By exploiting the different anatomy of CAM-derived vascularity, events that occur concurrently in vivo can be specially separated in CAM culture.

Growth plate chondrocytes of embryonic chick femurs were examined by electron microscopy, cytophotometry and autoradiography. Apart from the well-described 'light' chondrocyte, a different 'dark' type of chondrocyte was present, comprising 10 - 35% of the cell population. They were found at all stages of chondrocyte differentiation and in all ages of the femurs studied. Well developed rough endoplasmatic reticulum and Golgi complex, many secretory vesicles, energetically active mitochondria and a lot of glycogen, indicating high activity of the cytoplasm, were combined with low RNA synthesis, gentle margination and scattered compaction of the chromatin. DNA cytometry revealed that most of dark cells were diploid, but 15 - 30% were tetraploid, with the absence of an S-phase. Substantial loss of DNA was found in about 10% of dark chondrocytes. The TUNEL reaction demonstrated a limited number of DNA strand breaks. Advanced dark cells possessed the nuclear features of both apoptosis and necrosis. Besides chromomeric-chromonemic compaction, a chromatin arrangement similar to that of prometaphase and metaphase, as well as amitotic nuclear segregation, all of them degenerative, were found. Our interpretation is that the dark chondrocytes undergo an aberrant type of cell death which may be combined with aberrant cell cycle. Cell death of dark chondrocytes is preceded by a pre-mortal burst of secretion.

A detailed histological study of the growth plates from 9- to 20-day-old embryonic chick long bones was carried out with the aim of clarifying the long-debated question of the fate of the hypertrophic chondrocytes. Since resorption in chick bones does not occur synchronously across the plate as it does in mammals, specialized regions develop and the fate of the chondrocyte depends on its location within the growth plate. Where resorption took place, as at the sites of primary vascular invasion or at the main cartilage/marrow interface, chondrocytes underwent apoptosis before the lacunae were opened. In addition, spontaneous apoptosis of chondrocytes occurred at apparently random sites throughout all stages of chondrocyte differentiation. In older chick bones, a thick layer of endochondral bone matrix covered the cartilage edge. This consisted of type I collagen and the typical noncollagenous bone proteins but, in addition, contained tartrate-resistant acid phosphatase in the mineralized matrix. Where such matrix temporarily protected the subjacent cartilage from resorption, chondrocytes differentiated to bone-forming cells and deposited bone matrix inside their lacunae. At sites of first endochondral bone formation, some chondrocytes underwent an asymmetric cell division resulting in one daughter cell which underwent apoptosis, while the other cell remained viable and re-entered the cell cycle. This provided further support for the notion that chondrocytes as well as marrow stromal cells give rise to endochondral osteoblasts.

Transdifferentiation of hypertrophic chondrocytes into osteogenic cells was induced in 14 day chick embryo femurs by cutting through the region of hypertrophic cartilage. The process was studied in organ culture, using electron microscopy, staining for alkaline phosphatase, immunocytochemistry of collagen type I and proliferative cell nuclear antigen, and in situ localization of DNA strand-breaks. In addition, DNA and RNA synthesis were studied by 3[H]-T and 3[H]-U radioautography. Loss of ECM components from the cut edge occurred in culture. During the 12 day period necessary for transdifferentiation we observed phenotypic instability and bi-potentiality, the death of some cells and the gradual promotion of the osteoblastic phenotype in the survivors. Transition from chondrocytic to osteoblastic phenotype progressed stepwise, through variable mosaic intermediates, and involved a few cell cycles including asymmetric (differential) divisions. Proliferating and apoptotic cells were found in close proximity. As judged by the relative proportion of apoptotic cells and composition of the surrounding intralacunar matrix, negative selection of intermediate cell types displaying chondrocytic and altered mosaic phenotypes occurred. When the osteoblastic lineage was finally established, apoptotic cells were no longer present. Our hypothesis is that after disruption of cell-cell or cell-matrix interactions and lack of growth factors certain cells are selected and channelled through proliferation into the new stable phenotype. This process is targeted by the environment through a set of pre-determined steps.

We studied the endochondral ossification that occurs during the transition of soft to hard callus during fracture healing in the rabbit. During this process, parts of the cartilaginous soft callus are invaded by capillaries, and new bone is laid down onto the central unresorbed cartilage struts. We found that the chondrocytes within these cartilage struts changed phenotype and became bone-forming cells which directly replaced the central cartilage core with bone matrix. We have termed this bone "lacunar" bone to distinguish it from the "vascular" bone laid down by osteoblasts. With time the lacunar bone spread beyond the confines of the lacunae and gradually replaced all the cartilage matrix that was originally present in the early endochondral spicules. The lacunar bone could still be distinguished from the vascular bone as follows: (1) it was woven bone, whereas vascular bone was lamellar bone; (2) it contained acid phosphatase activity, whereas vascular bone did not; and (3) it had strong antigenicity for bone sialoprotein, whereas this noncollagenous protein was undetectable in vascular bone. Eventually the hard callus was resorbed and remodeled, but at an interim period of endochondral ossification the direct replacement of cartilaginous callus by the formation of lacunar bone is a rapid mechanism by which the mechanical strength of fracture callus is increased.

Trans-differentiation of hypertrophic chondrocytes into bone-forming cells was observed when femurs from 14-day-old chick embryos were cut through the region of hypertrophic cartilage and the separated pieces were cultured for 2-18 days. Inside many chondrocytic lacunae a new matrix was present which had the staining characteristics of bone matrix including birefringence and the capacity to mineralize. The cells within the lacunae had the characteristics of osteoblasts, such as alkaline phosphatase activity and positive immunocytochemical staining for osteocalcin, osteonectin, osteopontin and type I collagen. Chondrocyte necrosis and empty lacunae were only observed immediately at the cut edge, and in that region no bone-forming cells were present inside the lacunae. Where bone-matrix was present, the lacunae had remained intact, the cells were viable and no evidence of cell migration was observed. This suggested that the bone-forming cells had originated from the hypertrophic chondrocytes. The temporal sequence of events was followed closely. Two days following the cut only a few chondrocytes showed a positive reaction for osteocalcin, osteonectin, osteopontin and the type I collagen. At that time no such reaction product was observed in the chondrocytes of uncut femurs. Many hypertrophic chondrocytes divided, as shown by tritiated thymidine incorporation. The rate of cell division increased between 2-6 days, when several smaller basophilic cells were present inside the lacuna instead of the single hypertrophic chondrocyte. These cells expressed alkaline phosphatase activity, were positive for fibronectin, the above non-collagenous bone proteins and type I collagen. The bone matrix that was observed after 6-18 days was initially confined to the inside of the chondrocytic lacunae, but later spread beyond the lacunar confines. The bone proteins were still associated with the bone-forming cells, but fibronectin was absent when matrix formation was evident. Mineralization of the intra-lacunar osteoid took place after 12-18 days. It is speculated that the trans-differentiation was initiated by disruptions of the normal cell-cell associations.

We describe the histology of a specimen taken from an amputated leg seven months after a 15 cm bone gap in the tibia had been closed by bone transport. Lengthening appeared to have occurred by repeated minor trauma to the bone, with the fractured trabeculae in sufficiently close contact for the repair process to proceed. Osteogenesis did not occur through a cartilage phase, but the fracture gaps were bridged by collagen fibres, around which new bone formed. Microfractures had repaired by primary healing with woven bone and with no microcallus. Small regions of bone were necrotic. Resorption of the necrotic bone and remodelling of the immature bundle and woven bone were still at an early stage, suggesting that complete remodelling in man may take years rather than months.

Bone organ culture is an experimental system in which skeletal cells remain within their extracellular matrix but are removed from systemic influences. Femurs from 14-day-old chick embryos, which contain bone and cartilage matrix in approximately equal proportions, were cultured for up to 9 days in a serum-free medium. Cell proliferation, differentiation into chondrocytes and osteoblasts, formation of bone and cartilage matrix, and in vitro mineralization as well as bone and cartilage resorption were assessed using histologic and analytic methods. Particular attention was paid to the differences between cartilage and bone growth and to interpreting analytic data in the light of histologic observations. The first 2 days of culture represented an "adaptation" period, characterized by the release of intracellular enzymes into the culture medium, probably as a consequence of cell breakdown. Days 3-9 in culture represented a period of "steady growth" during which skeletal cells continued to multiply in the absence of fetal serum and to secrete large amounts of bone and cartilage matrix. De novo mineralization could be induced by Ca-beta-glycerophosphate, but calcium deposits in tissues other than bone and cartilage were also induced. Resorption of bone or cartilage matrix was virtually absent. Bone organ culture facilitates the study of bone and cartilage formation at an intermediate level of organization and thereby provides the necessary link between in vivo studies and investigations at the cellular level.