Identification and location of bone-forming cells within cartilage canals on their course into the secondary ossification centre

Osteoblasts and osteocytes derive from the same precursors, and osteocytes are terminally differentiated osteoblasts. These two cell types are distinguishable by their morphology, localization and levels of expression of various bone cell-specific markers. In the present study on the chicken femur we investigated the properties of the mesenchymal cells within cartilage canals on their course into the secondary ossification centre (SOC). We examined several developmental stages after hatching by means of light microscopy, electron microscopy, immunohistochemistry and in situ hybridization. Cartilage canals appeared as extensions of the perichondrium into the developing distal epiphysis and they were arranged in a complex network. Within the epiphysis an SOC was formed and cartilage canals penetrated into it. In addition, they were successively incorporated into the SOC during its growth in the radial direction. Thus, the canals provided this centre with mesenchymal cells and vessels. It should be emphasized that regression of cartilage canals could never be observed in the growing bone. Outside the SOC the mesenchymal cells of the canals expressed type I collagen and periostin and thus these cells had the characteristics of preosteoblasts. Periostin was also expressed by numerous chondrocytes. Within the SOC the synthesis of periostin was down-regulated and the majority of osteoblasts were periostin negative. Furthermore, osteocytes did not secret this protein. Tissue-non-specific alkaline phosphatase (TNAP) staining was only detectable where matrix vesicles were present. These vesicles were found around the blind end of cartilage canals within the SOC where newly formed osteoid started to mineralize. The vesicles originated from osteoblasts as well as from late osteoblasts/preosteocytes and thus TNAP was only expressed by these cells. Our results provide evidence that the mesenchymal cells of cartilage canals express various bone cell-specific markers depending on their position. We suggest that these cells differentiate from preosteoblasts into osteocytes on their course into the SOC and consider that cartilage canals are essential for normal bone development within the epiphysis. Furthermore, we propose that the expression of periostin by preosteoblasts and several chondrocytes is required for adhesion of these cells to the extracellular matrix.     

Design, morphometry and development of the secondary osteonal system in the femoral shaft of the rabbit

The architecture of the diaphyseal bone is closely correlated with the cortical vessel network, whose pattern develops in the course of growth. Various methods have been applied to clarify the three-dimensional anatomy of the cortical canal system, but there is still disagreement about the geometry, blood supply, flux dynamics and factors controlling canal geometry during bone growth and remodeling. A modification of the currently employed dye-injection method was applied to study the vessel network of the whole hemi-shaft of the rabbit femur in mature bones (8-month-old rabbits) and growing bones (1.5-month-old rabbits). The cortical vascular tree of the hemi-shaft of the femur was injected with black China ink and observed in full-thickness specimens of the cortex. The same specimens were then processed for histology. A comparative study of the middle diaphysis (mid-shaft) with the distal extremity (distal shaft) was performed in both young and old rabbit femurs. The longitudinally oriented pattern of the vessel network was seen to develop in the diaphysis of mature femurs, while at the extremity of the shaft of the same specimen the network showed a reticular organization without a dominant polarization. The vessels were significantly higher in the mid-shaft than in the distal shaft of the old femurs (P < 0.0001), as was their diameter (P < 0.05). In the group of young rabbits at mid-shaft level the longitudinally oriented pattern of the vessel network was not yet completely developed, without their being significant differences in length and diameter between the mid-shaft and distal shaft. The differentiation of the mid-shaft from the distal shaft was confirmed histologically by the presence, in the latter, of longitudinal calcified cartilage septa between osteons. This pattern of structural organization and development of the intracortical vascular network has not been previously reported. The cells primarily involved in polarization of the remodeling process were the osteoclasts at the top of the cutting cones advancing from the proximal and distal metaphyses toward the mid-shaft. This suggests, first, a relationship with the longitudinally oriented structures already present in the cortex near the metaphysis (the calcified cartilage septa) and then with the columns of interosteonic breccia, which were formed as a secondary effect of the longitudinal polarization of the remodeling process. Our observations did not enable us to substantiate the model of two different systems, one of longitudinal vessels (Havers) and the other of connecting transversal vessels (Volkmann), but suggested instead that there is a network whose loops lengthen in the direction of the major bone axis in the course of growth and secondary modeling. The associated morphology supported the view that the type of structural organization of the tubular bone cortex is primarily determined by an inherited constitutional factor rather than by mechanical strains.     

Basement membrane composition of cartilage canals during development and ossification of the epiphysis

Background: Cartilage canals are perichondral invaginations of blood vessels and connective tissue that are found within the epiphyses of most mammalian long bones. Functionally, they provide a means of transport of nutrients to the hyaline cartilage, a mechanism for removal of metabolic wastes, and a conduit for stem cells that are capable of initiating and sustaining ossification of the chondroepiphysis. Morphological and biomolecular changes of the chondroepiphyses appear to potentiate vascular invasion and enable regional formation of secondary centers of ossification within the chondroepiphyses of developing bones. Methods: As both cell migration and vascular invasion are anchorage dependent processes, antibodies to laminin and Type IV collagen were used to assess compositional changes in the basement membrane of cartilage canals accompanying epiphyseal ossification. Results: Differences in chronological appearance, as well as, in distribution between the two components were noted in the chondroepiphysis. Laminin was distributed throughout the connective tissue of cartilage canal at all stages of developement, and not limited to an association with the vascular lumen. Type IV collagen was not Present during the initial perichondral invagination. Although staining for Type IV collagen was later acquired, its distribution was restricted to a discontinuous rimming of the periphery of the canal, and a diffuse presence within the intra-canalicular mesenchyme. Conclusions: Concurrent with chondrocyte hypertrophy and mineralization of the hyaline matrix, rapid changes in both the morphology of the vessel and distribution of the antibodies were detected. In addition to the presence of laminin at the interface of the endothelium and the hyaline matrix, a wide distribution within the connective tissue components of the newly ossifying matrix of epiphyseal bone could be detected. Type IV collagen remained closely associated with the lumens of the intra-canalicular vessels throughout the transition. Following ossification of the secondary center, staining for Type IV collagen could then be detected in the boneforming regions of transforming matrix as well, clearly delineating the individual vessels within the newly formed marrow spaces. This suggests that bone formation is intimately related to vessel staining for collagen type IV, and that acquired vessel competence is a facet of endochondral bone formation that results from provisional matrix changes. Furthermore, the data suggests that during bone formation under tension, basement membrane deposition can be demonstrated without an intermediary hyaline matrix hypertrophic chondrocyte phase. This data was interpreted to suggest that chondrocyte hypertrophy at the growth plate may be a reaction to vascular invasion, that in turn, stimulates adjacent chondrocyte proliferation. © 1995 Wiley-Liss, Inc.     

Ossification in the human calcaneus: a model for spatial bone development and ossification

Perichondral bone, the circumferential grooves of Ranvier and cartilage canals are features of endochondral bone development. Cartilage canals containing connective tissue and blood vessels are found in the epiphysis of long bones and in cartilaginous anlagen of small and irregular bones. The pattern of cartilage canals seems to be integral to bone development and ossification. The canals may be concerned with the nourishment of large masses of cartilage, but neither their role in the formation of ossification centres nor their interaction with the circumferential grooves of Ranvier has been established. The relationships between cartilage canals, perichondral bone and the ossification centre were studied in the calcaneus of 9 to 38-wk-old human fetuses, by use of epoxy resin embedding, three-dimensional computer reconstructions and immunhistochemistry on paraffin sections. We found that cartilage canals are regularly arranged in shells surrounding the ossification centre. Whereas most of the shell canals might be involved in the nourishment of the cartilage, the inner shell is directly connected with the perichondral ossification groove of Ranvier and with large vessels from outside. In this way the inner shell canal imports extracellular matrix, cells and vessels into the cartilage. With the so-called communicating canals it is also connected to the endochondral ossification centre to which it delivers extracellular matrix, cells and vessels. The communicating canals can be considered as inverted 'internal' ossification grooves. They seem to be responsible for both build up intramembranous osteoid and for the direction of growth and thereby for orientation of the ossication centre.     

Inhibiting MMP activity prevents the development of endometriosis in the chicken chorioallantoic membrane model

BACKGROUND: Matrix metalloproteinases (MMPs) are essential for extracellular matrix remodelling and may contribute to the development of endometriosis. Transplantation of endometrium onto the chicken chorioallantoic membrane (CAM) results in endometriosis-like lesion formation, a process that requires extensive tissue remodelling. We investigated the expression of a wide range of MMPs in menstrual endometrium, endometriosis-like lesions in CAMs, in peritoneal endometriosis and in endometriosis in the rectovaginal space, as well as the function of MMPs in early lesion formation in the CAM model. METHODS: Expression of MMPs was evaluated by immunohistochemistry and MMP function was studied in the CAM by inhibiting MMP activity during lesion formation. RESULTS: Nearly all MMPs were present in all tissues studied. No significant differences in the expression of a majority of MMPs were found in endometriosis-like lesions in CAMs when compared with human endometriosis. Inhibition of MMP-1, -2, -3, -7 and -13 activities significantly impaired endometriosis-like lesion formation in CAMs. CONCLUSIONS: The MMP expression profiles of experimentally induced endometriosis in CAMs and human endometriosis are similar. The prevention of endometriosis-like lesion formation in the CAM by inhibiting MMP activity strongly suggests that MMPs have a function in the early development of endometriotic lesions.     

Microvascular features and ossification process in the femoral head of growing rats

In the epiphysis of long bones, different patterns of development of ossification processes have been described in different species. The development of the vascularisation of the femoral head has not yet been fully clarified, although its role in the ossification process is obvious. Our aim was to investigate ossification and vascular proliferation and their relationship, in growing rat femoral heads. Male Wistar rats aged ～ 1, 5 and 8 wk and 4, 8 and 12 mo were used. Light microscopy frontal sections and vascular corrosion casts observed by scanning electron microscopy were employed. In the rat proximal femoral epiphysis, ossification develops from the medullary circulation of the diaphysis, quickly extending to the neck and the base of the head. Hypertrophic chondrocytes occupy the epiphyseal cartilage, and a physeal plate with regular cell columns is present. Starting from about the end of the third month one or more points of fibrovascular outgrowth, above the physeal line, can be observed in each sample. They are often placed centrally or, sometimes, peripherally. The fibrovascular outgrowths penetrate deeply into the cartilage and extend laterally. At age 8 mo, large fibro-osseous peduncles connect the epiphysis to the diaphyseal tissue. At 12 mo, the entire epiphysis appears calcified with an almost total absence of residual cartilage islands. This situation differs in man and in other mammals due both to differing thickness of the cartilage and to the presence of more extensive sources of blood vessels other than the diaphyseal microcirculation, as supplied by the teres ligament and Hunter's circle. In young rats, subchondral vessels and the synovial fluid could play a role in feeding the ossifying cartilage. Later, a loss of resistance of the physis due to marked degeneration of the cell columns, and extensive chondrocyte hypertrophy permit fibrovascular penetration starting from diaphyseal vessels rather than neighbouring vascular territories, such as those of the periosteum and capsule.     

尺骨远端次级骨化中心X线与MRI下出现时间的对比研究

目的：探讨儿童尺骨远端次级骨化中心在MRI与X线下出现的时间以及形态的差异。方法：57名健康男孩志愿者，按年龄分成5个组，从5岁组到9岁组。每组分别用1．5T超导型磁共振机采用FLASH2d梯度回波加脂肪抑制序列行右腕关节扫描及CR射片。观察尺骨远端在MRJ及X线片上的不同表现。结果：尺骨远端骺软骨在MRI下呈高信号表现，外形近似于成熟的尺骨远端，骺软骨在X线下不显影；尺骨远端次级骨化中心最早在MRI下表现为一条低信号带，位于高信号表现的骺软骨中，平行于干骺端，此信号带在X线下不能显影；尺骨远端次级骨化中心出现后在MRJ下呈粟粒状的低信号表现，位置靠近干骺端，并非骺软骨中央，骨化中心与干骺端呈两种信号强度，X线下骨化中心表现一粟粒状影。结论：尺骨远端次级骨化中心在MRJ下的出现时间较X线下早，最早在MRJ下表现为一条低信号的临时钙化带。     

Epiphyseal and physeal cartilage vascularization: A light microscopic and tritiated thymidine autoradiographic study of cartilage canals in newborn and young postnatal rabbit bone

The vascular pattern of newborn and early postnatal epiphyseal and physeal cartilage is integral to long bone development and differs from later postnatal patterns. In the present study, we supplement light microscopic histology with tritiated thymidine autoradiography to help assess the position of cartilage canals and the dynamics of cartilage vascularity in relation to growth. Tritiated thymidine labeling studies to assess cell proliferation activity were done by using 2 μc/g body weight intraperitoneal injections into newborn and 3-, 4-, and 7-day-old New Zealand white rabbits that were killed 1 hr after the injection. Proximal humeral, distal femoral, and third metatarsal epiphyses were assessed by routine histology and serial section autoradiography. Cartilage canals were seen in each epiphysis. Transphyseal vessels were seen in each epiphysis continuous from the epiphysis to the metaphysis or were present within the physis traversing the proliferating and hypertrophic cell zones. Histologic sections showed vessels from the perichondrium continuous with those of the epiphyseal cartilage canals at proximal humeral, distal femoral, and metatarsal epiphyses. Serial sections showed vascular buds and connective tissue cells lying in indentations at the periphery of and present within the epiphyseal cartilage. Autoradiographic studies showed extensive labeling of vessel wall cells and surrounding connective tissue cells of the cartilage canals (a) within the epiphyseal cartilage, (b) traversing the physis, and (c) within the epiphyseal cartilage but continuous with the perichondrial vessels. The labeling was always far more extensive than in the surrounding chondrocytes and was always present throughout the entire extent of the canals. In conclusion, the cell labeling activity strongly supports an active dynamic phenomenon underlying the vascularization of epiphyseal and physeal cartilage. Anat. Rec. 252:140–148, 1998. © 1998 Wiley-Liss, Inc.     

Bone development in the femoral epiphysis of mice: the role of cartilage canals and the fate of resting chondrocytes.

In mammals, the exact role of cartilage canals is still under discussion. Therefore, we studied their development in the distal femoral epiphysis of mice to define the importance of these canals. Various approaches were performed to examine the histological, cellular, and molecular events leading to bone formation. Cartilage canals started off as invaginations of the perichondrium at day (D) 5 after birth. At D 10, several small ossification nuclei originated around the canal branched endings. Finally, these nuclei coalesced and at D 18 a large secondary ossification centre (SOC) occupied the whole epiphysis. Cartilage canal cells expressed type I collagen, a major bone-relevant protein. During canal formation, several resting chondrocytes immediately around the canals were active caspase 3 positive but others were freed into the canal cavity and appeared to remain viable. We suggest that cartilage canal cells belong to the bone lineage and, hence, they contribute to the formation of the bony epiphysis. Several resting chondrocytes are assigned to die but others, after freeing into the canal cavity, may differentiate into osteoblasts.     

Transient local presence of nerve fibers at onset of secondary ossification in the rat knee joint

In view of recent evidence that nerves may be involved in bone formation, the present study examines the local occurrence of axons at the onset of secondary ossification center formation in the knee region of developing rats. Radiographic and histological examination showed that secondary ossification center formation commenced at day 10. At day 15 the epiphyseal ossification had reached a relatively mature state. As seen by light microscopy, cartilage canals first appeared at day 5, reaching the epiphyseal center by day 9. Axons exhibiting a neurofilament-like immunoreactivity emerged from the perichondrial plexa into the cartilage canals. Many calcitonin gene-related peptide (CGRP)-immunoreactive and substance P (SP)-immunoreactive axons were found in the canals, as well as in the perichondrium. Axons with tyrosine hydroxylase-like immunoreactivity were not found in the canals, but such fibers occurred in relation to blood vessels at other sites. The canal-related axons disappeared between days 13 and 15, and the canals themselves did not persist beyond bone formation. As seen in the electron microscope, an individual canal contained 3–10 unmyelinated Schwann cell-enclosed axons with diameters of 0.1–2.0 m. These observations show that putative sensory unmyelinated axons with CGRP-and SP-like immunoreactivity are transiently present during initiation of bone formation in developing epiphyses. Whether there is a causal relation between transient innervation and osteogenesis remains to be determined.     

Inorganic phosphate transport in matrix vesicles from bovine articular cartilage

Aims: In mineralizing tissues such as growth plate cartilage extracellular organelles derived from the chondrocyte membrane are present. These matrix vesicles (MV), possess membrane transporters that accumulate Ca²⁺ and inorganic phosphate (P_i), and initiate the formation of hydroxyapatite crystals. MV are also present in articular cartilage, and hydroxyapatite crystals are believed to promote cartilage degradation in osteoarthritic joints. This study characterizes P_i transport in MV derived from articular cartilage. Methods: Matrix vesicles were harvested from collagenase digests of bovine articular cartilage by serial centrifugation. P_i uptake by MV was measured using radioactive phosphate (³³[P]HPO). The Na⁺ dependence, pH sensitivity and effects of P_i analogues that inhibit P_i transport were determined. Results: P_i uptake was temperature‐sensitive and comprised Na⁺‐dependent and Na⁺‐independent components. The Na⁺‐dependent component saturated at high extracellular P_i concentrations, with a K_m of 0.16 mm . In Na⁺‐free solutions, uptake did not fully saturate implying that carrier‐mediated uptake is supplemented by a diffusive pathway. Uptake was inhibited by phosphonoacetate and arsenate, although a fraction of Na⁺‐independent P_i uptake persisted. Total P_i uptake was maximal at pH 6.5, and reduced at more acidic or alkaline values, representing inhibition of both components. Conclusion: These properties are highly similar to those of P_i uptake by chondrocytes, suggesting that MV inherit P_i transporters of the chondrocyte membrane from which they are derived. Na⁺‐independent P_i uptake has not previously been described in MV from growth plate cartilage and is relatively uncharacterized, but warrants further attention in articular cartilage, given its likely role in initiating inappropriate mineral formation.     

An Immunohistochemical Study of Matrix Components in Early‐Stage Vascular Canals Within Mandibular Condylar Cartilage in Midterm Human Fetuses

Matrix components of vascular canals (VCs) in human fetal mandibular condylar cartilage (15–16 weeks of gestation) were analyzed by immunohistochemistry. Prevascular canals (PVCs), consisting of spindle‐shaped cells without capillary invasion, were observed within the cartilage. Intense immunoreactivity for collagen type I, weak immunoreactivity for aggrecan and tenascin‐C, weak hyaluronan (HA) staining, and abundant argyrophilic fibers in PVCs indicated that they contain noncartilaginous fibrous connective tissues that was different from those in the perichondrium/periosteum. These structural and immunohistochemical features of PVCs are different from those of previously reported cartilage canals of the long bone. Capillaries entered the VCs from the periosteum and ascended through VCs. Following capillary invasion, loose connective tissue had formed in the lower part of VCs, and immunoreactivity for collagen types I and III, tenascin‐C, and HA staining was evident in the matrix of loose connective tissue. No chondroclasts or osteogenic cells were seen at the front of capillary invasion, although small, mononuclear tartrate‐resistant acid phosphatase (TRAP)‐positive cells were present. Meanwhile, TRAP‐positive, multinucleated chondroclasts and flattened, osteoblast‐like cells were observed in the loose connective tissue at the lower part of VCs. These results may indicate slow progress of endochondral ossification in human fetal mandibular condyle. Further, unique matrix components in PVCs/VCs, which were different from those in cartilage canals in long bone, may reflect the difference of speed of endochondral ossification in cartilage canals and human fetal mandibular condyles. Anat Rec, 298:1560–1571, 2015. © 2015 Wiley Periodicals, Inc.     

VEGF and its role in the early development of the long bone epiphysis

In long bones of murine species, undisturbed development of the epiphysis depends on the generation of vascularized cartilage canals shortly after birth. Despite its importance, it is still under discussion how this event is exactly regulated. It was suggested previously that, following increased hypoxia in the epiphyseal core, angiogenic factors are expressed and hence stimulate the ingrowth of the vascularized canals. In the present study, we tested this model and examined the spatio‐temporal distribution of two angiogenic molecules during early development in mice. In addition, we investigated the onset of cartilage hypertrophy and mineralization. Our results provide evidence that the vascular endothelial growth factor is expressed in the epiphyseal resting cartilage prior to the moment of canal formation and is continuously expressed until the establishment of a large secondary ossification centre. Interestingly, we found no expression of secretoneurin before the establishment of the canals although this factor attracts blood vessels under hypoxic conditions. Epiphyseal development further involves maturation of the resting chondrocytes into hypertrophic ones, associated with the mineralization of the cartilage matrix and eventual death of the latter cells. Our results suggest that vascular endothelial growth factor is the critical molecule for the generation of the epiphyseal vascular network in mice long bones. Secretoneurin, however, does not appear to be a player in this event. Hypertrophic chondrocytes undergo cell death by a mechanism interpreted as chondroptosis.     

The hip joint: the fibrillar collagens associated with development and ageing in the rabbit

The fibrillar collagens associated with the articular cartilages, joint capsule and ligamentum teres of the rabbit hip joint were characterised from the 17 d fetus to the 2-y-old adult by immunohistochemical methods. Initially the putative articular cartilage contains types I, III and V collagens, but when cavitation is complete in the 25 d fetus, type II collagen appears. In the 17 d fetus, the cells of the chondrogenous layers express type I collagen mRNA, but not that of type II collagen. Types III and V collagens are present throughout life, particularly pericellularly. Type I collagen is lost. In all respects, the articular cartilage of the hip joint is similar to that of the knee. The joint capsule contains types I, III and V collagens. In the fetus the ligamentum teres contains types I and V collagens and the cells express type I collagen mRNA; type III collagen is confined mainly to its surface and insertions. After birth, the same distribution remains, but there is more type III collagen in the ligament, proper. The attachment to the cartilage of the head of the femur is marked only by fibres of type I collagen traversing the cartilage; the attachment cannot be distinguished in preparations localising types III and V collagens. The attachment to the bone at the lip of the acetabulum is via fibres of types I and V collagens and little type III is present. The ligament is covered by a sheath of types III and V collagens. Type II collagen was not located in any part of the ligamentum teres. The distribution of collagens in the ligamentum teres is similar to that in the collateral ligaments of the knee. Its insertions are unusual because no fibrocartilage was detected.     

人星形细胞瘤中单核细胞在血管生成过程中的作用

目的探讨单核细胞在人星形细胞瘤血管生成(Angiogenesis)过程中的作用.方法应用硷性纤维母细胞生长因于(bFGF)的抗体,通过免疫组织化学手段,在组织学切片上探讨单核细胞和bFGF在人星形细胞瘤血管生成过程中的作用.结果1.bFGF在血管内皮细胞中的表达强度随血管密度增加而增加,并与血管密度之间存在回归直线,P＜0.01.2.28.3%的切片中单核细胞表达bFGF,表达bFGF的单核细胞可见于血管内,或沿血管壁呈规则排列,也可成堆靠近血管壁,并使血管呈出芽生长.结论肿瘤内新生的血管内皮细胞可产生bFGF;单核细胞不但产生bFGF,而且参与肿瘤内的血管生成,它可能是血管内皮细胞的前期细胞.     

Localization of tartrate-resistant acid phosphatase (TRAP), membrane type-1 matrix metalloproteinases (MT1-MMP) and macrophages during early endochondral bone formation

Endochondral bone formation, the process by which most parts of our skeleton evolve, leads to the establishment of the diaphyseal primary (POC) and epiphyseal secondary ossification centre (SOC) in long bones. An essential event for the development of the SOC is the early generation of vascularized cartilage canals that requires the proteolytic cleavage of the cartilaginous matrix. This in turn will allow the canals to grow into the epiphysis. In the present study we therefore initially investigated which enzymes and types of cells are involved in this process. We have chosen the mouse as an animal model and focused our studies on the distal part of the femur during early stages after birth. The formation of the cartilage canals was promoted by tartrate-resistant acid phosphatase (TRAP) and membrane type-1 matrix metalloproteinases (MT1-MMP). In addition, macrophages and cells containing numerous lysosomes contributed to the establishment of the canals and enabled their further advancement into the epiphysis. As development continued, the SOC was formed, and in mice aged 10 days a distinct layer of type I collagen (= osteoid) was laid down onto the cartilage scaffold. The events leading to the establishment of the SOC were compared with those of the POC. Basically these processes were quite similar, and in both ossification centers, TRAP-positive chondroclasts resorbed the cartilage matrix. However, occasionally co-expression of TRAP and MT1-MMP was noted in a small subpopulation of this cell type. Furthermore, numerous osteoblasts expressed MT1-MMP from the start of endochondral ossification, whereas others did not. In osteocytogenesis, MT1-MMP has been shown to be critical for the establishment of the cytoplasmic processes mediating the communication between osteocytes and bone-lining cells. Considering the well-known fact that not all osteoblasts transform into osteocytes, and in accordance with the present data, we suggest that MT1-MMP is needed at the very beginning of osteocytogenesis and may additionally determine whether an osteoblast further differentiates into an osteocyte.     

Postnatal development of the collagen matrix in rabbit tibial plateau articular cartilage

Changes in the 3-dimensional arrangement of the articular cartilage matrix during growth of the rabbit tibial plateau were studied. Knees from newborn, and 1, 2 and 6 wk-old rabbits were compared with those of adults by light and electron microscopy. The specimens were fixed, embedded en bloc in epoxy resin and sectioned vertically/coronally through the point where the articular cartilage was thickest in the adult medial tibial plateau. At birth, the proximal tibial epiphysis was cartilaginous, but nascent articular cartilage was recognisable as a densely cellular layer covering the tibial condyle. Within 30 μm of the articular surface, the chondrocytes were flattened and collagen fibres ran among these cells in a direction parallel to the surface. Deeper in the articular cartilage, rounded cells were evenly distributed within a random collagen fibril network. At the centre of the plateau, the tangential layer changed little during growth, whereas the subjacent cellular layer grew in thickness and steadily achieved a more vertical character in the organisation of its constituent collagen and cellular elements. At 1 wk, cells were separated into clusters by acellular regions filled with collagen fibrils. At 2 wk, cells within the forming radial zone were aligned in columns bracketed by vertical collagen fibres. Continuity of these vertical fibres with those in the tangential surface layer was evident at this age. The chondrocytes were surrounded by fibrous capsules typical of chondrons. By 6 wk, the bases of the radial collagen fibres in the very centre of the condyle had calcified, as had the adjacent hypertrophic hyaline cartilage. A solid subchondral plate and tidemark did not appear until skeletal maturity. From birth to age 6 wk, maximum thickness of the layer identified as primordial articular cartilage increased from 0.13 mm to 0.70 mm, and was 1.5 mm in the adult. Throughout growth, however, the thickness of the tangential layer in the centre of the plateau never exceeded 0.05 μm. In the patella, femoral head and peripheral tibial plateau, cartilage development followed the same general sequence. In contrast to the central tibial plateau, the tangential layer also grew in thickness, but at a slower rate than that of the radial zone. At all ages, the developing articular cartilage was structurally distinct from the deeper hyaline cartilage which contributed to growth of the ossification centre through enchondral ossification. The collagen matrix of articular cartilage acquires a characteristic, orderly 3-dimensional structure soon after birth. Growth in cartilage thickness occurs primarily through enlargement of the radial zone.     

Fetal and postnatal development of the patella, patellar tendon and suprapatella in the rabbit; changes in the distribution of the fibrillar collagens

The development of the patella, its associated tendons, and suprapatella of the rabbit knee joint is described from the 17 d fetus to the mature adult. The patellar tendon (ligament) with the patella on its posterior surface is seen in the 17 d fetus and is fully developed by 1 postnatal wk. It is composed of bundles of types I and V collagens separated by endotenons of types III and V collagens. Anteriorly there is an epitenon of types III and V collagens while synovium and a fat pad cover its posterior surface. In the 25 d fetus, the patella is cartilaginous and is separated from the femoral condyles. The cartilage contains type II collagen, but types I, III and V collagens are found along the articular surface. Ossification starts 1 postnatal wk and at 6 wk only the articular cartilage remains. In addition to type II, types III and V collagens are located around the chondrocyte lacunae. The long anterior junction between the patella and its tendon is fibrocartilaginous at 1 wk, but as ossification proceeds this is replaced by bone. Types I and V collagens are found in this region. The suprapatella on the posterior surface of the quadriceps tendon is first seen 1 wk postnatally as an area of irregularly organised fibres and chondrocyte-like cells. Types I, II, III and V collagens are present from 3 wk onwards. It is compared with the fibrocartilage of other tendons that are under compression. The arrangement of the collagens in the patellar tendon is discussed in relation to its use as a replacement for injured anterior cruciate ligaments. It is suggested that the structural differences between the patellar tendon and anterior cruciate ligament preclude the translocated tendon acquiring mechanical strength similar to that of a normal cruciate ligament. The designation 'patellar ligament' as opposed to 'patellar tendon' is questioned. It is argued that the term patellar tendon reflects its structure more accurately than patellar ligament.     

Variation in mammalian proximal femoral development: comparative analysis of two distinct ossification patterns

The developmental anatomy of the proximal femur is complex. In some mammals, including humans, the femoral head and greater trochanter emerge as separate ossification centres within a common chondroepiphysis and remain separate throughout ontogeny. In other species, these secondary centres coalesce within the chondroepiphysis to form a single osseous epiphysis much like the proximal humerus. These differences in femoral ontogeny have not been previously addressed, yet are critical to an understanding of femoral mineralization and architecture across a wide range of mammals and may have key implications for understanding and treating hip abnormalities in humans. We evaluated femora from 70 mammalian species and categorized each according to the presence of a 'separate' or 'coalesced' proximal epiphysis based on visual assessment. We found that ossification type varies widely among mammals: taxa in the 'coalesced' group include marsupials, artiodactyls, perissodactyls, bats, carnivores and several primates, while the 'separate' group includes hominoids, many rodents, tree shrews and several marine species. There was no clear relationship to body size, phylogeny or locomotion, but qualitative and quantitative differences between the groups suggest that ossification type may be primarily an artefact of femoral shape and neck length. As some osseous abnormalities of the human hip appear to mimic the normal morphology of species with coalesced epiphyses, these results may provide insight into the aetiology and treatment of human hip disorders such as femoroacetabular impingement and early-onset osteoarthritis.     

Germ layer differentiation during early hindgut and cloaca formation in rabbit and pig embryos

Relative to recent advances in understanding molecular requirements for endoderm differentiation, the dynamics of germ layer morphology and the topographical distribution of molecular factors involved in endoderm formation at the caudal pole of the embryonic disc are still poorly defined. To discover common principles of mammalian germ layer development, pig and rabbit embryos at late gastrulation and early neurulation stages were analysed as species with a human‐like embryonic disc morphology, using correlative light and electron microscopy. Close intercellular contact but no direct structural evidence of endoderm formation such as mesenchymal–epithelial transition between posterior primitive streak mesoderm and the emerging posterior endoderm were found. However, a two‐step process closely related to posterior germ layer differentiation emerged for the formation of the cloacal membrane: (i) a continuous mesoderm layer and numerous patches of electron‐dense flocculent extracellular matrix mark the prospective region of cloacal membrane formation; and (ii) mesoderm cells and all extracellular matrix including the basement membrane are lost locally and close intercellular contact between the endoderm and ectoderm is established. The latter process involves single cells at first and then gradually spreads to form a longitudinally oriented seam‐like cloacal membrane. These gradual changes were found from gastrulation to early somite stages in the pig, whereas they were found from early somite to mid‐somite stages in the rabbit; in both species cloacal membrane formation is complete prior to secondary neurulation. The results highlight the structural requirements for endoderm formation during development of the hindgut and suggest new mechanisms for the pathogenesis of common urogenital and anorectal malformations.