Relationship between the chondrocyte maturation cycle and the endochondral ossification in the diaphyseal and epiphyseal ossification centers

The chondrocyte maturation cycle and endochondral ossification were studied in human, fetal cartilage Anlagen and in postnatal meta‐epiphyses. The relationship between the lacunar area, the inter‐territorial fibril network variations, and calcium phosphorus nucleation in primary and secondary ossification centers were assessed using light microscopy and scanning electron microscopy (SEM) morphometry. The Anlage topographic, zonal classification was derived from the anatomical nomenclature of the completely developed long bone (diaphysis, metaphyses and epiphyses). A significant increase in the chondrocyte lacunar area was documented in the Anlage of epiphyseal zones 4 and 3 to zone 2 (metaphysis) and zone 1 (diaphysis), with the highest variation from zone 2 to zone 1. An inverse reduction in the intercellular matrix area and matrix interfibrillar empty space was also documented. These findings are consistent with the osmotic passage of free cartilage water from the interfibrillar space into the swelling chondrocytes, which increased the ion concentrations to a critical threshold for mineral precipitation in the matrix. The mineralized cartilage served as a scaffold for osteoblast apposition both in primary and secondary ossification centers and in the metaphyseal growth plate cartilage, though at different periods of bone Anlage development and with distinct patterns for each zone. All developmental processes shared a common initial pathway but progressed at different rates, modes and organization in diaphysis, metaphysis and epiphysis. In the ossification phase the developing vascular supply appeared to play a key role in determining the cortical or trabecular structure of the long bones. J. Morphol. 277:1187–1198, 2016. © 2016 Wiley Periodicals, Inc.     

Role of tartrate-resistant acid phosphatase (TRAP) in long bone development

Tartrate resistant acid phosphatase (TRAP) was shown to be critical for skeleton development, and TRAP deficiency leads to a reduced resorptive activity during endochondral ossification resulting in an osteopetrotic phenotype and shortened long bones in adult mice. A proper longitudinal growth depends on a timely, well-coordinated vascularization and formation of the secondary ossification center (SOC) of the long bones epiphysis. Our results demonstrate that TRAP is not essential for the formation of the epiphyseal vascular network. Therefore, in wild type (Wt) controls as well as TRAP deficient (TRAP(-/-)) mutants vascularised cartilage canals are present from postnatal day (P) five. However, in the epiphysis of the TRAP(-/-) mice cartilage mineralization, formation of the marrow cavity and the SOC occur prematurely compared with the controls. In the mutant mice the entire growth plate is widened due to an expansion of the hypertrophic zone. This is not seen in younger animals but first detected at week (W) three and during further development. Moreover, an enhanced number of thickened trabeculae, indicative of the osteopetrotic phenotype, are observed in the metaphysis beginning with W three. Epiphyseal excavation was proposed as an important function of TRAP, and we examined whether TRAP deficiency affects this process. We therefore evaluated the marrow cavity volume (MCV) and the epiphyseal volume (EV) and computed the MCV to EV ratio (MCV/EV). We investigated developmental stages until W 12. Our results indicate that both epiphyseal excavation and establishment of the SOC are hardly impaired in the knockouts. Furthermore, no differences in the morphology of the epiphyseal bone trabeculae and remodeling of the articular cartilage layers are noted between Wt and TRAP(-/-) mice. We conclude that in long bones, TRAP is critical for the development of the growth plate and the metaphysis but apparently not for the epiphyseal vascularization, excavation, and establishment of the SOC.     

Development of the cartilage canals and the secondary center of ossification in the distal chondroepiphysis of the prenatal human femur.

The cartilaginous epiphysis of the distal femur is vascularized by a network of cartilage canals during prenatal development. The vascular invasion of the epiphysis begins at approximately eight to ten weeks of gestation with the initiation of cartilage canal formation. A complex vascular system develops within the canals and is well defined by fourteen weeks of gestation. The vascular system is fully developed several months prior to the development of the secondary center of ossification. The formation of the secondary center of ossification within the distal femoral epiphysis is preceded by changes that occur simultaneously within both the chondrocytes in the central portion of the epiphysis and the vascular and perivascular elements contained within the cartilage canals in the central portion of the epiphysis. These concurrent changes in the cellular morphology of the central chondrocytes and in the cellular structure of the central cartilage canals appear to be linked with the initiation of the process of osteogenesis.     

Gene expression and distribution of connective tissue growth factor (CCN2/CTGF) during secondary ossification center formation.

CCN2/connective tissue growth factor (CCN2/CTGF) is a critical signaling modulator of mesenchymal tissue development. This study investigated the localization and expression of CCN2/CTGF as a factor supporting angiogenesis and chondrogenesis during development of secondary ossification centers in the mouse tibial epiphysis. Formation of the secondary ossification center was initiated by cartilage canal formation and blood vessel invasion at 7 days of age, and onset of ossification was observed at 14 days. In situ hybridization showed that CCN2/CTGF mRNA was distinctively expressed in the region of the cartilage canal and capsule-attached marginal tissues at 7 days of age, and distinct expression was also observed in proliferating chondrocytes around the marrow space at 14 days of age. Immunostaining showed that CCN2/CTGF was distributed broadly around the expressed cells located in the central region of the epiphysis, where the chondrocytes become hypertrophic and the cartilage canal enters into the hypertrophic mass. Furthermore, an overlapping distribution of metalloproteinase (MMP)9 and CCN2/CTGF was found in the secondary ossification center. These findings suggest that the CCN2/CTGF is involved in establishing epiphyseal vascularization and remodeling, which eventually determines the secondary ossification center in the developing epiphysial cartilage.     

Development and growth of long bones in European water frogs (Amphibia: Anura: Ranidae), with remarks on age determination

Differentiation and development of long bones were studied in European water frogs: Rana lessonae, R. ridibunda, and R. esculenta. The study included premetamorphic larvae (Gosner Stage 40) to frogs that were 5 years old. Femora, metatarsal bones, and proximal phalanges of the hindlimb exhibit the same pattern of periosteal bone differentiation and the same pattern of growth. Longitudinal and radial growth of these bones was studied by examination of the diaphyses and epiphyses, particularly where the edge of periosteal bone is inserted into the epiphysis. The periosteum seems to be responsible for both longitudinal and radial growth. Investigation of the formation, length, and arrangement of lines of arrested growth reveals that the first line is present only in the middle 25-35% of the length of the diaphysis of an adult bone; therefore, only the central portion of the diaphysis should be used for age estimation in skeletochronological studies. Comparison of the shapes and histological structures of epiphyses in the femur, metatarsal bones, and phalanges revealed that epiphyseal cartilages are composed of an inner and outer part. The inner metaphyseal cartilage has distinct zones and plugs the end of the periosteal bone cylinder; its role in longitudinal growth is questioned. The outer epiphyseal cartilage is composed of articular cartilages proper, in addition to lateral articular cartilages. Differences in the symmetry of the lateral articular cartilages of distal epiphyses of the femur and toes may reflect adaptations to different kinds of movements at the knee and in the foot.     

A mathematical model of epiphyseal development: hypothesis on the cartilage canals growth

The role of cartilage canals is to transport nutrients and biological factors that cause the appearance of the secondary ossification centre (SOC). The SOC appears in the centre of the epiphysis of long bones. The canal development is a complex interaction between mechanical and biological factors that guide its expansion into the centre of the epiphysis. This article introduces the ‘Hypothesis on the growth of cartilage canals’. Here, we have considered that the development of these canals is an essential event for the appearance of SOC. Moreover, it is also considered to be important for the transport of molecular factors (RUNX2 and MMP9) at the ends of such canals. Once the canals are merged in the centre of the epiphysis, these factors are released causing hypertrophy of adjacent cells. This RUNX2 and MMP9 release occurs due to the action of mechanical loads that supports the epiphysis. In order to test this hypothesis, we use a hybrid approach using the finite element method to simulate the mechanical stresses present in the epiphysis and the cellular automata to simulate the expansion of the canals and the hypertrophy factors pathway. By using this hybrid approach, we have obtained as a result the spatial–temporal patterns for the growth of cartilage canals and hypertrophy factors within the epiphysis. The model is in qualitative agreement with experimental results previously reported by other authors. Thus, we conclude that this model may be used as a methodological basis to present a complete mathematical model of the processes involved in epiphyseal development.     

Senile features in the skeleton of an aged Japanese monkey

The senile features in the skeleton of a male Japanese monkey, who is presumed to be about 40 years old, were examined in comparison with younger individuals. As for the skull, every part is constructed solidly, and the sutures around the temporal and parietal bones are for the most part closed. In the dentition many of the front teeth are destroyed or lost, and the cheek teeth are severely worn. In the vertebrae, the annular epiphyseal discs unite completely with the body at its anterior and posterior surfaces, and the porosity and deformation of the bodies are remarkable. The hip bones, in the pelvis, unite with each other by solid ossification of the pubic symphysis. The long bones of the anterior and posterior limbs are marked by their general thickness, the rugged increase of bone around the joints, especially in the arms, and the complete union of each epiphysis with the shaft through the ossification of the epiphyseal cartilage. These senile features furnish a clue as to the establishment of a criterion for age estimation in Japanese monkeys.This observation was briefly reported inMonkey Vol. 13, No. 1, 1969, and Fig. 9 was used there.     

The epiphyseal cartilage and growth of long bones in Rana catesbeiana.

The structure of the epiphyseal cartilage of the bullfrog Rana catesbeiana and its role in the growth of long bones were examined. The epiphyseal cartilage was inserted into the end of a tubular bone shaft, defining three regions: articular cartilage, lateral articular cartilage and growth cartilage. Joining the lateral cartilage to the bone was a fibrous layer of periosteum, rich in blood vessels. Osteoblasts with alkaline phosphatase activity were found on the surface of the periosteal bone, which presented a fibrous non-mineralised tip. The growth cartilage was inside the bone. The proliferative chondrocytes presented perpendicular separation of daughter cells and there was no columnar arrangement of the cells. Furthermore, chondrocyte hypertrophy was not associated with either calcification or endochondral ossification, in apparent contrast to the avian and mammalian models. Finally, there was no reinforcement system capable of directing cell volume increase into longitudinal growth. Since bone extension depends on the intramembranous ossification of the periosteum, the growth cartilage is inside and not at the end of the bone and the cells in the growth cartilage show no columnar arrangement and separate in a direction perpendicular to the long bone axis, we conclude that the growth cartilage mainly contributes to the radial expansion of the bone.     

Growth and differentiation of fetal rat limb buds transplanted in athymic (nude) mice

Limb buds of day 14 rat fetuses were cut into pieces and transplanted into the subcutaneous tissue of athymic (nude) mice. In day 14 fetal limbs, mesenchymal cells have begun to condense to form cartilaginous anlage, but no cartilage has been formed. Within 7 days after grafting, masses of hyaline cartilage developed. Numerous osteoblasts appeared, and new bone formation began by 14 days. By 20 days, osteoclasts appeared, and the formation of bone trabeculae and marrow cavities progressed. The cytological characteristics of chondrocytes, osteoblasts and osteoclasts were essentially the same as those seen in vivo. Many grafts developed into long bones, having the diaphysis and epiphysis. The mode of chondrogenesis and osteogenesis in the grafts was histologically similar to the corresponding process in vivo, although the differentiation was slower in the grafted limbs. Since the grafted limb buds showed remarkable growth and tissue differentiation for at least several weeks, this heterotransplantation system would be of potential use for the study of bone formation and resorption as well as for developmental toxicological studies.     

Mechanism of the reduction of Meckel's cartilage in man

The mechanism of reduction of the anterior end of Meckel's cartilage was studied in human embryos, with the following findings: 1. Meckel's cartilage is surrounded, from the outside and from below, by newly formed mandibular bone over the extent of the insertion of the musculus mylohyoideus. 2. Blood vessels from the newly formed bone penetrate Meckel's cartilage and break it down in the same way as in enchondral ossification of cartilaginous models of other bones. 3. The anlagen of the musculus mylohyoideus and musculus genioglossus are at first inserted on Meckel's cartilage; further muscle fibres, formed on the under surface of the two muscles, are inserted on the newly formed bone of the rudimentary mandible. Parallel to this process, the fibres on the upper surface of the muscles, which were originally inserted on Meckel's cartilage, disappear. The two processes combined lead to transposition of the insertions of the two muscles from Meckel's cartilage to the mandible. 4. In the area of the resorbed Meckel's cartilage, a minimum number of bone trabeculae are formed at the time of its resorption. The space left by Meckel's cartilage is taken over chiefly by the primitive medullary cavity of the rudimentary mandible, medially to the canal for the nerve and blood vessels.     

Bone growth aud development of secondary ossification centers of extremities in the cynomolgus monkey (Macaca fascicularis).

The development of so-called long bones in the extremity has been studied roentgenographically in forty-seven males and fifty-one females cynomolgus monkeys bred and reared at the National Institute of Health. The age of the females ranged from five months to eight years and nine months, and that of the males was from four months to seven years. In addition, the fetuses of six to twenty weeks of gestation age were examined for the time of appearance of ossification centers. As the biological parameters concerning body growth, the body weight and the bone length were measured and the secondary ossification centers were scrutinized and assessed the maturity process on the basis of the criteria that divided the state into eleven stages. Also the allometric analyses of body weight against bone length was conducted. Most of the secondary ossification centers except the proximal fibulal epiphysis appeared during the period from the prenatal stage (15-20 weeks of gestationage) to the postnatal one (several months of age). From four to five months of age, many ossification centers had developed to some extent. But, the appearance of proximal fibulal epiphysis was delayed and often lacking until 10 months of age in female and one year and three months of age in male. The earliest epiphyseal fusion was observed at the distal humeral epiphysis in both sexes. The latest epiphyseal fusion was observed at the distal ulnal epiphysis in both sexes and at the distal ulnal and radial epiphyses in female. From this study, the time of fusion was at five and three guarters years of age in females and at six and a half years of age in males. As a result, it is suggested that the estimation of animal's age might be put to practical use by introducing the assessing method that the score was given from the observation of the secondary ossification center.     

Retinol-binding protein 4 is expressed in chondrocytes of developing mouse long bones: implications for a local role in formation of the secondary ossification center

Retinol-binding protein 4 (Rbp4) is the major carrier of retinol in the bloodstream, a retinoid whose metabolites influence osteogenesis, chondrogenesis and adipogenesis. Rbp4 is mainly produced in the liver where it mobilizes hepatic retinol stores to supply other tissues. However, Rbp4 is also expressed in several extrahepatic tissues, including limbs, where its role is largely unknown. This study aimed to identify the cellular localization of Rbp4 to gain insight into its involvement in limb development and bone growth. Using immunohistochemistry, we discovered that Rbp4 was present in a variety of locations in developing embryonic and postnatal mouse hindlimbs. Rbp4 was present in a restricted population of epiphyseal chondrocytes and perichondral cells correlating to the future region of secondary ossification. With the onset of secondary ossification, Rbp4 was detected in chondrocytes of the resting zone and in chondrocytes that bordered invading cartilage canals and the expanding front of ossification. Rbp4 was less abundant in proliferating chondrocytes involved in primary ossification. Our data implicate the involvement of chondrocytic Rbp4 in bone growth, particularly in the formation of the secondary ossification center of the limb.     

A mathematical model of epiphyseal development: hypothesis of growth pattern of the secondary ossification centre

This paper introduces a 'hypothesis about the growth pattern of the secondary ossification centre (SOC)', whereby two phases are assumed. First, the formation of cartilage canals as an event essential for the development of the SOC. Second, once the canals are merged in the central zone of the epiphysis, molecular factors are released (primarily Runx2 and MMP9) spreading and causing hypertrophy of adjacent cells. In addition, there are two important molecular factors in the epiphysis: PTHrP and Ihh. The first one inhibits chondrocyte hypertrophy and the second helps the cell proliferation. Between these factors, there is negative feedback, which generates a highly localised and stable pattern over time. From a mathematical point of view, this pattern is similar to the patterns of Turing. The spread of Runx2 hypertrophies the cells from the centre to the periphery of the epiphysis until found with high levels of PTHrP to inhibit hypertrophy. This mechanism produces the epiphyseal bone-plate. Moreover, the hypertrophy is inhibited when the cells sense low shear stress and high pressure levels that maintain the articular cartilage structure. To test this hypothesis, we solve a system of coupled partial differential equations using the finite element method and we have obtained spatio-temporal patterns of the growth process of the SOC. The model is in qualitative agreement with experimental results previously reported by other authors. Thus, we conclude that this model can be used as a methodological basis to present a complete mathematical model of the whole epiphyseal development.     

A mathematical model of epiphyseal development: hypothesis of growth pattern of the secondary ossification centre

This paper introduces a ‘hypothesis about the growth pattern of the secondary ossification centre (SOC)’, whereby two phases are assumed. First, the formation of cartilage canals as an event essential for the development of the SOC. Second, once the canals are merged in the central zone of the epiphysis, molecular factors are released (primarily Runx2 and MMP9) spreading and causing hypertrophy of adjacent cells. In addition, there are two important molecular factors in the epiphysis: PTHrP and Ihh. The first one inhibits chondrocyte hypertrophy and the second helps the cell proliferation. Between these factors, there is negative feedback, which generates a highly localised and stable pattern over time. From a mathematical point of view, this pattern is similar to the patterns of Turing. The spread of Runx2 hypertrophies the cells from the centre to the periphery of the epiphysis until found with high levels of PTHrP to inhibit hypertrophy. This mechanism produces the epiphyseal bone-plate. Moreover, the hypertrophy is inhibited when the cells sense low shear stress and high pressure levels that maintain the articular cartilage structure. To test this hypothesis, we solve a system of coupled partial differential equations using the finite element method and we have obtained spatio-temporal patterns of the growth process of the SOC. The model is in qualitative agreement with experimental results previously reported by other authors. Thus, we conclude that this model can be used as a methodological basis to present a complete mathematical model of the whole epiphyseal development.     

Expression profile of Xenopus banded hedgehog, a homolog of mouse Indian hedgehog, is related to the late development of endochondral ossification in Xenopus laevis

Late development of endochondral ossification occurs at the boundary between the growth cartilage and bone marrow during the formation of long bones in Xenopus laevis. Since the Indian hedgehog (Ihh) is involved in endochondral ossification in mouse, we investigated the expression of Xenopus banded hedgehog (X-bhh), which is a homolog of mouse Ihh. RT-PCR analysis demonstrated that the X-bhh mRNA was detected from an early stage of limb formation to formation of femurs in mature frogs, and it was associated with the expression of Xenopus-ptc1 (X-ptc1), Xenopus-gli1 (X-gli1), Xenopus-type II collagen (X-col II), Xenopus-runx2 (X-runx2), and Xenopus-osteocalcin (X-ocn) mRNAs. In situ hybridization revealed that chondrogenic cells observed at early limb development expressed X-bhh and X-gli1. At later stages of limb development, chondrocytes, located slightly away from the boundary between the cartilage and bone marrow, expressed the X-bhh, X-ptc1, and X-gli1 mRNAs; however, the mesenchymal cells at the boundary failed to express these mRNAs. The X-bhh, X-ptc1, and X-gli1 mRNAs as well as those of X-runx2 and X-ocn were expressed by the mesenchymal cells in the periosteal region at the tip of the cortical bone, indicating an intimate relationship between X-bhh expression and bone formation in this region. Considered collectively, the present study suggests that X-bhh evolutionally acquired the function to induce osteogenesis; however, the expression profile of X-bhh in epiphysis is closely related to the late development of endochondral ossification in X. laevis.     

Phagocytosis of dying chondrocytes by osteoclasts in the mouse growth plate as demonstrated by annexin-V labelling

Endochondral ossification in the epiphyseal growth plate of long bones is associated with programmed cell death (PCD) of a major portion of the chondrocytes. Here we tested the hypothesis that at the ossification front of the epiphyseal growth plate osteoclasts preferentially phagocytose chondrocytes that are undergoing PCD. We injected biotin-labelled annexin-V (anx-V-biotin, an early marker of PCD) intravenously in young adult mice. After 30 min of labelling, long bones were recovered and the tissue distribution examined of anx-V-biotin-labelled cells in the growth plate using ABC-peroxidase histochemistry. Positive staining for anx-V-biotin was detected in hypertrophic chondrocytes still present in closed lacunae at some distance from the ossification front. At the ossification front, chondrocyte lacunae were opened and close contacts were seen between tartrate-resistant acid phosphatase-positive osteoclasts and hypertrophic cartilage cells. Osteoclasts were significantly more frequently in contact with anx-V-biotin-labelled chondrocytes than with unlabelled chondrocytes. Osteoclasts also contained labelled and unlabelled phagocytic fragments within their cytoplasm. We conclude that in the growth plate osteoclasts preferentially phagocytose hypertrophic chondrocytes that are dying, suggesting these dying cells may signal osteoclasts for their removal.     

The pathology of acute chondro-osseous injury in the child.

Skeletal tissues from children sustaining acute skeletal trauma were analyzed with detailed radiologic and histologic techniques to assess the failure patterns of the developing skeleton. In the physis- and epiphysis-specific fracture propagation varied, usually going through the portion of the hypertrophic zone adjacent to the metaphysis. However, the physeal fracture in types 1 and 2 sometimes involved the germinal zone. There may also be microscopic propagation at oblique angles from the primary fracture plane, splitting cell columns apart longitudinally. The cartilage canals supplying the germinal zone appear to be "weak" areas into which the fracture may propagate, especially in infancy. Incomplete type 1 physeal fractures, which cannot be detected by routine radiography, may occur. Types 1, 2, and 4 physeal injuries may be comminuted. In type 3 injuries, discrete segments of physis that include the germinal zone may "adhere" to the metaphysis, separating the cells from their normal vascularity. In types 2 and 3, comminution may occur at the site of fracture redirection from the physis. Direct type 5 crushing of the physeal germinal zone does not occur, even in the presence of significant pressure-related changes within other areas of the epiphysis. Type 7 separation between cartilage and bone at any chondro-osseous epiphyseal interface may occur, but is similarly impossible to diagnose radiographically. In the metaphysis torus, fractures result from plastic deformation of the cortex, coupled with a partial microfracturing that may be difficult to visualize with clinical radiography. Some of the energy absorption may also be transmitted to the physis, causing metaphyseal hemorrhage adjacent to the growth plate and variable microscopic damage within the physis. In the diaphysis, the greenstick fracture is associated with longitudinal tensile failure through the developing osteons of the "intact" cortex. The inability of these failure patterns to "narrow" after the fracture force dissipates is the probable cause of retained bowing (plastic deformation). In both torus and greenstick fractures, the fractured bone ends show micro-splitting through the osteoid seams. In the diaphysis, metaphysis, and epiphyseal ossification center there may be areas of focal hemorrhage and microfracture that correlate with the reported MRI phenomenon of "bone bruising." Again, such injury cannot be diagnosed during routine radiography.     

Comparative morphometric analysis of the reaction of the rat bone to dehydration and burn trauma

The epiphyseal cartilage and diaphysis of the tibia have been studied at dehydration and burn trauma. Dehydration produces certain disturbances in growth and formation of the bone, and burn trauma--development of dystrophic changes.     

The physical and mechanical effects of suspension-induced osteopenia on mouse long bones.

The present investigation addresses the extent of tail-suspension effects on the long bones of mice. The effects are explored in both sexes, in both forelimb and hindlimb bones, and in both diaphyseal and metaphyseal/epiphyseal bones. Two weeks of suspension provided unloading of the femora and tibiae and an altered loading of the humeri. Whole-bone effects included lower mass (approximately 10%) and length (approximately 4%) in the bones of suspended mice compared to controls. The geometric and material properties of the femora were considered along the entire length of the diaphysis and in the metaphysis/epiphysis portions as a unit. Geometric effects included lower cross-sectional cortical area (16%), cortical thickness (25%) and moment of inertia (21%) in the femora of suspended mice; these differences were observed in both distal and proximal portions of the femur diaphysis. The relative amount of bone comprising the middle 8 mm of the diaphysis was greater (3%) in the control mice than in the suspended mice. Significant mass differences between the group in the metaphysis/epiphysis were not observed. Material effects included lower %ash (approximately 2%) in the femora and tibiae as well as in the humeri of suspended mice compared to controls. With respect to the measured physical and material properties, suspension produced similar bone responses in male and female mice. The effects of suspension are manifested largely through geometric rather than through material changes.     

Study of differential collagen synthesis during development of the chick embryo by immunofluorescence: II. Localization of type I and type II collagen during long bone development

The transition of type I and type II collagens during cartilage and bone development in the chick embryo was studied by immunofluorescence using antibodies against type I or type II collagens. Type II collagen was found in all cartilaginous structures which showed metachromatic staining. Type I collagen appeared in the perichondrium of the tibia at stage 28 and was also found in osteoid, periosteal and enchondral bone after decalcification, periosteum, and tendons, ligaments, and capsules.Using the immunohistological method it was possible to identify specific collagen types in areas undergoing rapid proliferation and collagen transition, such as diaphyseal and epiphyseal perichondrium, or in enchondral osteogenesis. During enchondral ossification type I collagen is deposited onto the eroded surface of cartilage. It partially diffuses into the cartilage matrix forming a “hybrid” collagen matrix with type II collagen, which is a site for subsequent ossification. During appositional growth of diaphyseal cartilage and differentiation of epiphyseal perichondrium into articular cartilage, perichondral cells switch from type I to type II collagen synthesis when differentiating into chondroblasts. In the transition zones, chondroblasts are imbedded in a “hybrid” matrix consisting of a mixture of type I and type II collagens.