The role of hypertrophic cartilage in endochondral ossification.

Normal stages of histogenesis of long bones show that the hypertrophy of cartilage cells is the pre-requisite for the perichondrium to take up osteoblastic activity, (Fell 1925, Lutfi 1971). Cooper (1965) found the cartilage cells from epihysis of the long bones of chick failed to induce chondrogenesis in somites in mice and chick whereas flat cells and early Peripheral cells could do same. Fell and Landauer (1935) noticed that in avian phocomelia the hypertrophied cartilage cells fail to hypertrophy leading subsequently to deformities of long bones. Presently an attempt is made to analyse this process further by culture experiments. It is found that complete tibial rudiment or part of it grows normally in vitro with good differentiation of various zones and the development of osteoid tissue. However it is noticed that when cartilage and the associated perichondrial tissues are grown separately, there is no patterned growth of cartilage and the absence of development of osteoid tissue in either types of cultures. The role of perichondrium and cartilage is discussed in the light of experimental findings.     

Matrix remodeling during endochondral ossification

Endochondral ossification, the process by which most of the skeleton is formed, is a powerful system for studying various aspects of the biological response to degraded extracellular matrix (ECM). In addition, the dependence of endochondral ossification upon neovascularization and continuous ECM remodeling provides a good model for studying the role of the matrix metalloproteases (MMPs) not only as simple effectors of ECM degradation but also as regulators of active signal-inducers for the initiation of endochondral ossification. The daunting task of elucidating their specific role during endochondral ossification has been facilitated by the development of mice deficient for various members of this family. Here, we discuss the ECM and its remodeling as one level of molecular regulation for the process of endochondral ossification, with special attention to the MMPs.     

Differential actions of VEGF-A isoforms on perichondrial angiogenesis during endochondral bone formation

During endochondral bone formation, vascular invasion initiates the replacement of avascular cartilage by bone. We demonstrate herein that the cartilage-specific overexpression of VEGF-A₁₆₄ in mice results in the hypervascularization of soft connective tissues away from cartilage. Unexpectedly, perichondrial tissue remained avascular in addition to cartilage. Hypervascularization of tissues similarly occurred when various VEGF-A isoforms were overexpressed in the chick forelimb, but also in this case perichondrial tissue and cartilage were completely devoid of vasculature. However, following bony collar formation, anti-angiogenic properties in perichondrial tissue were lost and perichondrial angiogenesis was accelerated by VEGF-A₁₄₆, VEGF-A₁₆₆, or VEGF-A₁₉₀. Once the perichondrium was vascularized, osteoclast precursors were recruited from the circulation and the induction of MMP9 and MMP13 can be observed in parallel with the activation of TGF-β signaling. Neither perichondrial angiogenesis nor the subsequent cartilage vascularization was found to be accelerated by the non-heparin-binding VEGF-A₁₂₂ or by the VEGF-A₁₆₆ΔE₁₆₂-R₁₆₆ mutant lacking a neuropilin-binding motif. Hence, perichondrial angiogenesis is a prerequisite for subsequent cartilage vascularization and is differentially regulated by VEGF-A isoforms.     

Transforming growth factor-beta 1, -beta 2 and -beta 3 in cartilage and bone cells during endochondral ossification in the chick.

The localization of TGF-beta 1, -beta 2 and -beta 3 was studied in the growth plate, epiphysis and metaphysis of the tibiotarsus of three-week-old chicks. The different TGF-beta isoforms were localized to hypertrophic chondrocytes, chondroclasts, osteoblasts and osteoclasts using immunohistochemical staining analysis with specific TGF-beta antibodies. TGF-betas in osteoclasts and chondroclasts were restricted to those cells located on the respective matrices. TGF-beta 3 localization was mainly cytoplasmic in the transitional (early hypertrophic) chondrocytes, but nuclear staining was also detected in some proliferating chondrocytes. The cell-specific localization of these TGF-beta isoforms supports the hypothesis that TGF-beta has a role in the coupling of new bone formation to bone and cartilage matrix resorption during osteochondral development and suggests that TGF-beta may be a marker of chondrocyte differentiation. TGF-beta localization preceded a marked increase in type II collagen mRNA expression in transitional chondrocytes, suggesting a role for TGF-beta in the induction of synthesis of extracellular matrix.     

Merging the old skeletal biology with the new. I. Intramembranous ossification, endochondral ossification, ectopic bone, secondary cartilage, and pathologic considerations

Skeletogenesis and chondrogenesis result from a sequence of events involving epithelial-mesenchymal interaction, condensation, and differentiation. Types of bone and cartilage formation include: (1) intramembranous ossification, (2) endochondral ossification, (3) combined endochondral and intramembranous ossification, (4) heterotopic bone and cartilage formation, and (5) secondary cartilage formation. Pathologic conditions with bone and cartilage include: (1) benign and malignant tumors and (2) reactive osseous and cartilaginous metaplasia.     

The localization of thiamine pyrophosphatase activity in Meckel's cartilage cells during endochondral ossification

Summary The cytochemical distribution of thiamine pyrophosphatase (TPPase) activity in Meckel's cartilage cells of the mouse embryo has been studied during the endochondral ossification. All the cartilage cells contain reaction product within the Golgi apparatus. In immature chondrocytes, at the reserve cell zone, TPPase activity is restricted to several inner cisternae of independent Golgi apparatus. In mature cells at the proliferative cell zone, several Golgi complexes form a Golgi network connecting with each other by the TPPase positive tubular stalks. Golgi cisternae, condensing vacuoles and vesicles also contain reaction product. In the hypertrophic chondrocytes located in the calcifying zone, their disorganized Golgi apparatus still retain reaction product. Some chondrocytes, even those located within calcified or opened lacunae, exhibit intact structures and normal cytochemical enzyme distribution. These data indicate the possibility that some chondrocytes may survive and contribute the formation of mandible.     

The localization of thiamine pyrophosphatase activity in Meckel's cartilage cells during endochondral ossification

The cytochemical distribution of thiamine pyrophosphatase (TPPase) activity in Meckel's cartilage cells of the mouse embryo has been studied during the endochondral ossification. All the cartilage cells contain reaction product within the Golgi apparatus. In immature chondrocytes, at the reserve cell zone, TPPase activity is restricted to several inner cisternae of independent Golgi apparatus. In mature cells at the proliferative cell zone, several Golgi complexes form a Golgi network connecting with each other by the TPPase positive tubular stalks. Golgi cisternae, condensing vacuoles and vesicles also contain reaction product. In the hypertrophic chondrocytes located in the calcifying zone, their disorganized Golgi apparatus still retain reaction product. Some chondrocytes, even those located within calcified or opened lacunae, exhibit intact structures and normal cytochemical enzyme distribution. These data indicate the possibility that some chondrocytes may survive and contribute the formation of mandible.     

Perlecan modulates VEGF signaling and is essential for vascularization in endochondral bone formation

Perlecan (Hspg2) is a heparan sulfate proteoglycan expressed in basement membranes and cartilage. Perlecan deficiency (Hspg2(-/-)) in mice and humans causes lethal chondrodysplasia, which indicates that perlecan is essential for cartilage development. However, the function of perlecan in endochondral ossification is not clear. Here, we report the critical role of perlecan in VEGF signaling and angiogenesis in growth plate formation. The Hspg2(-/-) growth plate was significantly wider but shorter due to severely impaired endochondral bone formation. Hypertrophic chondrocytes were differentiated in Hspg2(-/-) growth plates; however, removal of the hypertrophic matrix and calcified cartilage was inhibited. Although the expression of MMP-13, CTGF, and VEGFA was significantly upregulated in Hspg2(-/-) growth plates, vascular invasion into the hypertrophic zone was impaired, which resulted in an almost complete lack of bone marrow and trabecular bone. We demonstrated that cartilage perlecan promoted activation of VEGF/VEGFR by binding to the VEGFR of endothelial cells. Expression of the perlecan transgene specific to the cartilage of Hspg2(-/-) mice rescued their perinatal lethality and growth plate abnormalities, and vascularization into the growth plate was restored, indicating that perlecan in the growth plate, not in endothelial cells, is critical in this process. These results suggest that perlecan in cartilage is required for activating VEGFR signaling of endothelial cells for vascular invasion and for osteoblast migration into the growth plate. Thus, perlecan in cartilage plays a critical role in endochondral bone formation by promoting angiogenesis essential for cartilage matrix remodeling and subsequent endochondral bone formation.     

Parathyroid hormone-related peptide-depleted mice show abnormal epiphyseal cartilage development and altered endochondral bone formation

To elucidate the role of PTHrP in skeletal development, we examined the proximal tibial epiphysis and metaphysis of wild-type (PTHrP-normal) 18- 19-d-old fetal mice and of chondrodystrophic litter mates homozygous for a disrupted PTHrP allele generated via homologous recombination in embryonic stem cells (PTHrP-depleted). In the PTHrP-normal epiphysis, immunocytochemistry showed PTHrP to be localized in chondrocytes within the resting zone and at the junction between proliferative and hypertrophic zones. In PTHrP-depleted epiphyses, a diminished [3H]thymidine-labeling index was observed in the resting and proliferative zones accounting for reduced numbers of epiphyseal chondrocytes and for a thinner epiphyseal plate. In the mutant hypertrophic zone, enlarged chondrocytes were interspersed with clusters of cells that did not hypertrophy, but resembled resting or proliferative chondrocytes. Although the overall content of type II collagen in the epiphyseal plate was diminished, the lacunae of these non-hypertrophic chondrocytes did react for type II collagen. Moreover, cell membrane-associated chondroitin sulfate immunoreactivity was evident on these cells. Despite the presence of alkaline phosphatase activity on these nonhypertrophic chondrocytes, the adjacent cartilage matrix did not calcify and their persistence accounted for distorted chondrocyte columns and sporadic distribution of calcified cartilage. Consequently, in the metaphysis, bone deposited on the irregular and sparse scaffold of calcified cartilage and resulted in mixed spicules that did not parallel the longitudinal axis of the tibia and were, therefore, inappropriate for bone elongation. Thus, PTHrP appears to modulate both the proliferation and differentiation of chondrocytes and its absence alters the temporal and spatial sequence of epiphyseal cartilage development and of subsequent endochondral bone formation necessary for normal elongation of long bones.     

Tenascin-W inhibits proliferation and differentiation of preosteoblasts during endochondral bone formation

We identified a cDNA encoding mouse Tenascin-W (TN-W) upregulated by bone morphogenetic protein (Bmp)2 in ATDC5 osteo-chondroprogenitors. In adult mice, TN-W was markedly expressed in bone. In mouse embryos, during endochondral bone formation TN-W was localized in perichondrium/periosteum, but not in trabecular and cortical bones. During bone fracture repair, cells in the newly formed perichondrium/periosteum surrounding the cartilaginous callus expressed TN-W. Furthermore, TN-W was detectable in perichondrium/periosteum of Runx2-null and Osterix-null embryos, indicating that TN-W is expressed in preosteoblasts. In CFU-F and -O cells, TN-W had no effect on initiation of osteogenesis of bone marrow cells, and in MC3T3-E1 osteoblastic cells TN-W inhibited cell proliferation and Col1a1 expression. In addition, TN-W suppressed canonical Wnt signaling which stimulates osteoblastic differentiation. Our results indicate that TN-W is a novel marker of preosteoblasts in early stage of osteogenesis, and that TN-W inhibits cell proliferation and differentiation of preosteoblasts mediated by canonical Wnt signaling.     

p107 and p130 Coordinately regulate proliferation,Cbfa1 expression,and hypertrophic differentiation during endochondral bone development

During endochondral bone development, both the chondrogenic differentiation of mesenchyme and the hypertrophic differentiation of chondrocytes coincide with the proliferative arrest of the differentiating cells. However, the mechanisms by which differentiation is coordinated with cell cycle withdrawal, and the importance of this coordination for skeletal development, have not been defined. Through analysis of mice lacking the pRB-related p107 and p130 proteins, we found that p107 was required in prechondrogenic condensations for cell cycle withdrawal and for quantitatively normal alpha1(II) collagen expression. Remarkably, the p107-dependent proliferative arrest of mesenchymal cells was not needed for qualitative changes that are associated with chondrogenic differentiation, including production of Alcian blue-staining matrix and expression of the collagen IIB isoform. In chondrocytes, both p107 and p130 contributed to cell cycle exit, and p107 and p130 loss was accompanied by deregulated proliferation, reduced expression of Cbfa1, and reduced expression of Cbfa1-dependent genes that are associated with hypertrophic differentiation. Moreover, Cbfa1 was detected, and hypertrophic differentiation occurred, only in chondrocytes that had undergone or were undergoing a proliferative arrest. The results suggest that Cbfa1 links a p107- and p130-mediated cell cycle arrest to chondrocyte terminal differentiation.