Integrins and extracellular matrix proteins in the human childhood and adolescent growth plate

Interaction of chondrocytes with the surrounding matrix significantly influences differentiation and growth. These processes involve cell surface proteins, particularly integrins. The aim of this study was to compare the expression of integrins (alpha1, alpha2, alpha3, alpha5, alpha6, alphav, beta1, beta3, and beta5 subunits) together with matching binding proteins in human childhood and adolescent growth plate cartilage using immunohistochemistry. Integrin beta1 was detected in all chondrocytes of the growth plate cartilage, beta3 only in osteoclasts of the opening zone, and beta5 in hypertrophic chondrocytes and osteoblasts. Integrin alpha1, alpha2, and alpha5 subunits were expressed by chondrocytes in the proliferative and hypertrophic zone as well as in osteoblasts and osteoclasts. Integrin av and alpha6 subunits were present in chondrocytes of all zones, alpha3 only in osteoclasts. Collagen type II and fibronectin were seen throughout the growth plate, collagen type X in the hypertrophic zone, collagen type I in the ossifying trabecules. Laminin was expressed by chondrocytes in the resting zone and more weakly in the proliferative zone, collagen VI was present in the pericellular and interterritorial matrix in all zones of the growth plate. These results differ from previous reports on the distribution of integrins in the fetal growth plate. However, there was no difference in integrin expression in children before and during puberty. Our results indicate that integrin expression is not influenced by endocrine factors during sexual maturation and suggest that the process of skeletal maturation is not regulated via altered integrin expression.     

The extracellular matrix of cartilage in the growth plate before and during calcification: Changes in composition and degradation of type II collagen

Summary Calcification occurs in the extracellular matrix of the hypertrophic zone of the growth plate when the extra-cellular matrix volume is reduced to a minimum and alkaline phosphatase content is maximal. The present study shows that significant quantitative and qualitative changes occur in the composition and structure of macromolecules in the extracellular matrix before and during calcification in the proximal tibial growth plate of the bovine fetus. These were detected in part by using microchemical and microimmuno-chemical analyses of sequential transverse frozen sections at chemical analyses of sequential transverse frozen sections at defined sites throughout the growth plate. Concentrations of matrix molecules in the extracellular matrix have not previously been determined biochemically. They were measured per unit matrix volume by using combined immunochemical/chemical-histomorphometric analyses. The concentrations within the extracellular matrix of the C-propeptide of type II collagen, aggregating proteoglycan (aggrecan), and hyaluronic acid all progressively increased in the maturing and hypertrophic zones, being maximal (or near maximal) at the time of initiation of mineralization. These results for proteoglycan are contrary to some earlier reports of a loss of proteoglycan prior to mineralization which measured the tissue content of proteoglycan rather than that present in the extracellular matrix, the volume of which is progressively reduced as the growth plate matures. The C-propeptide data provides a quantitative confirmation of previous immunohistochemical studies. Total collagen concentration (measured as hydroxyproline) in the extracellular matrix initially increased through the proliferating and maturing zones but then rapidly decreased in the hypertrophic zone. Immunohistochemical studies revealed that this is associated with the unwinding of the triple helix of type II collagen (previously shown to result from cleavage) which starts in pericellular sites in the zone of maturation (when type X collagen is first synthesized) and then extends throughout the hypertrophic zone. The significance of these matrix changes in the development and mineralization of the growth plate is discussed.     

P-glycoprotein is expressed in the mineralizing regions of the skeleton

Using oligonucleotide primers specific for the human MDR 1 gene, we were able to identify a specific amplicon using RT-PCR from total bovine growth plate chondrocyte RNA. The identification of MDR mRNA in growth plate chondrocytes led us to examine the precise distribution of MDR P-glycoprotein in bone and cartilage. We applied two monoclonal antibodies (C219 and C494) to human fetal, neonatal, and childhood growth plates and bone. In growth plates, P-glycoprotein was detected at high levels in a perilacunar distribution in the calcifying zone and at lower levels in hypertrophic, but not proliferative or reserve zone, chondrocytes. P-glycoprotein was also observed in perichondrial chondrocytes, in perivascular chondrocytes and matrix in the fetal cartilage anlage, and in osteoblasts and the surface osteoid matrix of newly formed bone trabeculae in the primary spongiosa. The recently described chloride channel of P-glycoprotein suggests a potential role of P-glycoprotein in growth plate chondrocyte hypertrophy. D. C. Mangham is supported by the Wechsler fellowship     

Characterization of growth plate mitochondria

The growth plate chondrocyte plays a central role in growth plate function. The purpose of this study was to characterize the respiratory and calcium transport properties of isolated mammalian growth plate chondrocytes and mitochondria obtained from these cells and to quantitate the mitochondrial weight and volume fraction in each zone of the growth plate. A new method was developed for isolation of mitochondria from chondrocyte suspensions. Isolated chondrocyte mitochondria demonstrated an eightfold increase in oxygen consumption in response to calcium and a two- to threefold increase in oxygen consumption in response to adenosine diphosphate. Similar responses were observed in chondrocytes treated with digitonin. The mitochondrial protein content of the growth plate and hyaline cartilage chondrocytes is significantly less than hepatocytes. Conversely, the chondrocyte mitochondrial cytochrome aa3 content is similar to mitochondria from a wide variety of sources. A zonal analysis of the growth plate demonstrates an increase in the mitochondrial weight (protein) fraction from the reserve to the hypertrophic zone whereas the mitochondrial volume fraction decreases from the reserve to the hypertrophic zone. The findings of this study emphasize the dependence of chondrocytes on glycolysis as a prime energy source and support the concept that chondrocyte mitochondria have become specialized in the process of matrix calcification.     

Differences in matrix vesicle concentration among growth plate zones

We studied the proximal tibial growth plates of 15-day-old mice to determine if matrix vesicle concentration varies among growth plate zones or between the pericellular and territorial matrix compartment and the interterritorial matrix compartment. Growth plates were examined by electron microscopy and divided into five zones: reserve zone (RZ), upper proliferative zone (UPZ), lower proliferative zone (LPZ), upper hypertrophic zone (UHZ), and lower hypertrophic zone (LHZ) which included the calcifying zone. We measured the diameter and volume fraction of matrix vesicles and calculated their numerical density and volume per cell and number per cell in the pericellular and territorial matrix and in the interterritorial matrix of each zone. In the pericellular and territorial matrix compartment, the matrix vesicle concentration progressively decreased from the RZ to the LHZ. Changes in matrix vesicle concentration in the interterritorial matrix followed a different pattern. Between the RZ and the UPZ, matrix vesicle numerical density declined slightly and then increased to peak values in the LPZ and UHZ, followed by a decline between the UHZ and the LHZ. These changes in matrix vesicle concentration paralleled previously reported changes in intramitochondrial calcium content, suggesting that matrix vesicle production in growth plate may be related to intracellular calcium concentration. The existence of the maximum concentration of matrix vesicles in the LPZ and UHZ longitudinal septa which do not mineralize followed by a decline in matrix vesicle concentration in the LHZ longitudinal septa which mineralize suggests that a high concentration of matrix vesicles may be needed to prepare the matrix for mineralization or to initiate mineralization and that matrix vesicles are depleted during mineralization.     

Short-term glucocorticoid treatment of piglets causes changes in growth plate morphology and angiogenesis

OBJECTIVE: Glucocorticoid treatment of children often leads to growth retardation, and the precise target(s) in the growth plate responsible for this effect are unknown. Angiogenesis is an important part of the endochondral ossification process, and VEGF expressed in the growth plate is essential for proper angiogenesis to occur. Since glucocorticoid treatment down-regulates VEGF expression in cultured chondrocytes, we hypothesized that in vivo glucocorticoid treatment could result in VEGF down-regulation in the growth plate and disturbed angiogenesis, thus contributing to the growth retardation. DESIGN: We treated 6-week-old prepubertal piglets (10 kg) for 5 days with prednisolone (50 mg/day). Tibial growth plate sections were studied for apoptosis and the expression of VEGF protein and mRNA and MMP-9 protein. Capillaries in the metaphysis were visualized by CD31 immunostaining. Growth plate morphology (width of various zones) was determined by interactive measurements on hematoxylin/eosin stained sections and apoptotic cells were detected by TUNEL assay. RESULTS: In the prednisolone-treated animals, the total width of the growth plate decreased to 81% of controls (P<0.02), which was explained by a decrease of the width of the proliferative zone to 73% (P<0.05). The treatment had no effect on the orderly organization of the chondrocyte columns. In the growth plates of control animals, apoptosis was shown in 5.8% of the hypertrophic chondrocytes and was limited to the terminal hypertrophic chondrocytes. In prednisolone-treated animals, 40.5% of the hypertrophic chondrocytes was apoptotic (P<0.02), with apoptotic chondrocytes also appearing higher in the hypertrophic zone. We observed fewer capillaries and loss of their parallel organization in the metaphysis in the prednisolone-treated animals. The capillaries were shorter and chaotic in appearance. In contrast to controls, in prednisolone-treated animals VEGF mRNA and protein could not be detected in the hypertrophic zone of the growth plate. Trabecular bone length in the primary spongiosa was also diminished by the treatment. No changes were observed in the expression pattern of MMP-9, a matrix metalloproteinase, which is also important for angiogenesis and bone formation. CONCLUSIONS: These results indicate that short-term glucocorticoid treatment of growing piglets severely disturbs the width of the growth plate, apoptosis of chondrocytes, VEGF expression by hypertrophic chondrocytes, the normal invasion of blood vessels from the metaphysis to the growth plate and bone formation at the chondro-osseous junction. These effects could alter the dynamics of endochondral ossification and thus contribute to glucocorticoid-induced growth retardation.     

Localization of Matrix Metalloproteinases, (MMPs) Their Tissue Inhibitors, and Vascular Endothelial Growth Factor (VEGF) in Growth Plates of Children and Adolescents Indicates a Role for MMPs in Human Postnatal Growth and Skeletal Maturation

Numerous studies have focused on the expression, regulation, and biological significance of matrix metalloproteinases (MMPs) in the growth plate. Findings in mouse knockout models and in vitro data from various species indicate that MMPs not only degrade extracellular matrix components but may regulate the activity of local growth factors. In this study we investigated the presence, distribution, and activity of various MMPs and inhibitors, tissue transglutaminase (tTG or TG2) and vascular endothelial growth factor (VEGF) in the human child and adolescent growth plates by means of immunohistochemistry and gelatin zymography. Tissue was derived during orthopedic surgery (epiphysiodesis) in two prepubertal and four pubertal patients.MMP-2 and MMP-14 were present in reserve cell chondrocytes. MMP-14 was the most prominent MMP within all zones of the growth plate including proliferating chondrocytes. MMP-1 and MMP-13 (collagenases 1 and 3), MMP-9 (gelatinases B), MMP-10, and MMP-11 (stromelysins) and VEGF were positive in hypertrophic chondrocytes and osteoblasts. MMP-2 showed the same expression pattern but was negative in osteoblasts. Osteoclasts stained positive for MMP-9, MMP-2, and TG2. Tissue inhibitor of MMP (TIMP)-1 was present in all zones of the growth plate, osteoblasts, and osteoclasts; TIMP-2 was found in hypertrophic chondrocytes and osteoblasts. In summary, the presence of MMPs, TIMPs, TG2, and VEGF in our study indicated that the MMPs are relevant in growth plate physiology during the postnatal period in humans. The specific location of MMP expression within the growth plate may be the basis for further studies on the role of MMPs in the local regulation of chondrocyte differentiation, proliferation, and ossification at the chondroosseus junction.     

p21 and Parathyroid Hormone-Related Peptide in the Growth Plate

It is essential for terminal chondrocytes to die before the conversion of calcified cartilage to bone. We have previously demonstrated that apoptosis occurred in the terminal hypertrophic chondrocyte of the growth plate. However, the essential mechanism by which the differentiation of chondrocytes is regulated has not yet been characterized. The purpose of this study was to investigate the mechanism for regulating chondrocyte differentiation. We focused on PTHrP and p21 which regulated the differentiation of chondrocytes and investigated how these factors interacted with each other in chondrocyte differentiation in the growth plate. PTHrP was strongly positive on immunostaining at the interface between the proliferating and the upper zone of the hypertrophic chondrocytes, whereas p21 was negative. On the other hand, p21 was positive in the lower zone of hypertrophic chondrocytes. Furthermore, PTHrP up-regulated the cell proliferation and down-regulated the expression of the p21 messengers in SW-1353 chondrosarcoma cells. These findings indicated that PTHrP might be a negative regulator for p21 in the differentiation of chondrocytes. Received: 1 March 2000 / Accepted: 25 May 2000 / Online publication: 2 November 2000     

The role of hepatocyte growth factor in growth plate cartilage

To investigate the physiological role of hepatocyte growth factor (HGF) in endochondral bone formation, we examined the expression of HGF and its receptor c-met and the effects of HGF on growth plate chondrocytes. HGF was highly expressed in the prehypertrophic zone and hypertrophic zone in rat costal growth plate cartilage. The expression of HGF increased in rabbit chondrocytes as they matured in culture. Conversely, c-met expression was down regulated along maturation of growth plate chondrocytes. HGF had weak stimulatory effects on DNA and proteoglycan synthesis of growth plate chondrocytes. However, HGF strongly inhibited expression of terminal differentiation-related phenotypes, such as type X collagen and alkaline phosphatase (APase) synthesis and cartilage matrix mineralization. When HGF was removed from the cultures, cells quickly expressed type X collagen and APase. Once chondrocytes differentiated to mature chondrocytes, HGF did not inhibit further differentiation of these cells. These results suggested that HGF is a negative regulator of terminal differentiation of growth plate chondrocytes.. Received: Feb. 12, 1998 / Accepted: March 12, 1998     

Morphometric Changes in the Epiphyseal Plate of the Growing and Young Adult Male Rat After Long-Term Salmon Calcitonin Administration

The function of the epiphyseal plate is related to the differentiation and maturation of the chondrocytes, especially of the hypertrophic zone. Salmon calcitonin exerts a positive effect on chondrocytes of different types of cartilage, e.g., articular cartilage, osteochondral callus formation, and the epiphyseal plate. In the present study, the effect of long-term daily salmon calcitonin treatment upon epiphyseal plate function was examined in 80 male Wistar rats aged 12 weeks at the beginning of the experiment. A daily dose of 6 IU of salmon calcitonin enhanced the number of the chondrocytes of the hypertrophic zone of the upper tibial epiphyseal plate, increased the mean thickness of the epiphyseal plate, and accelerated the longitudinal growth of long bones. It was found that the peripheral growth of the epiphyseal plate was delayed after calcitonin treatment in comparison with the placebo-treated animals. The most effective period for calcitonin treatment on epiphyseal plate function seems to be the late accelerated period of growth, i.e., puberty. In conclusion, long-term salmon calcitonin treatment has a beneficial effect on longitudinal skeletal growth and this effect remains throughout the adult life of the animal. Salmon calcitonin does not enlarge the surface of the epiphyseal plate.     

Cartilage abnormalities are associated with abnormal Phex expression and with altered matrix protein and MMP-9 localization in Hyp mice

X-linked hypophosphatemic rickets (HYP) in humans is caused by mutations in the PHEX gene. This gene mutation is also found in Hyp mice, the murine homologue of the human disease. At present, it is unknown why loss of Phex function leads to cartilage abnormalities in Hyp mice. In the present study, we compared in wild-type and Hyp mice Phex protein localization in cartilage of developing long bone as well as localization of skeletal matrix proteins and matrix metalloproteinase-9 (MMP-9). Also compared were chondrocyte apoptosis in the growth plate, mineralization and cartilage remnant retention in the metaphysis, and chondroclast/osteoclast characteristics in the primary spongiosa. Phex protein was detected in proliferating and hypertrophic chondrocytes in growth plate cartilage of wild-type mice, but not in Hyp mice. Hyp mice exhibited a widened and irregular hypertrophic zone in growth plate cartilage showing hypomineralization, increased cartilage remnants from the growth plate in both metaphyseal trabecular and cortical bone, and fewer and smaller chondroclasts/osteoclasts in the primary spongiosa. Increased link protein and C-propeptide of type II procollagen of Hyp mice reflected the increase in chondrocytes and matrix in the cartilaginous growth plate and in bone. In addition, growth plate osteocalcin and bone sialoprotein levels were decreased, while osteonectin was increased, in hypertrophic chondrocytes and cartilage matrix in Hyp mice. MMP-9 in hypertrophic chondrocytes was also reduced in Hyp mice and fewer apoptotic hypertrophic chondrocytes were detected. These findings suggest that Phex may control mineralization and removal of hypertrophic chondrocytes and cartilage matrix in growth plate by regulating the synthesis and deposition of certain bone matrix proteins and proteases such as MMP-9.     

A poptosis of hypertrophic chondrocytes in rat cartilaginous growth plate

Calcifying cartilages undergo endochondral ossification, a process in which cartilage is replaced by bone. These tissues contain chondrocytes that proliferate, leading, to differentiation and hypertrophy. Recent histological and biochemical studies suggest that hypertrophic chondrocytes undergo apoptosis. We investigated the process of this cell death to determine when fragmentation of DNA, a hallmark of apoptosis, occurs during cellular commitment to hypertrophy, and to test the hypothesis that the chondrocytes are intrinsically programmed to undergo apoptosis. End-labeling of fragmented DNA of rat proximal tibiae revealed that a majority of hypertrophic cells bore fragmented DNA, indicating that apoptosis was in progress in this zone. In pelleted chondrocyte cultures isolated from, rat rib growth plates and employed in an in vitro model of a growth plate, hypertrophic cells were also positive for end-labeling. Gel electrophoresis of DNA isolated from the chondrocyte cultures at 1–3 weeks yielded the ladder formation characteristic of apoptosis. We conclude that the chondrocytes in the growth plate are programmed to self-annihilate by apoptosis and that the apoptotic process is closely associated with the commitment to hypertrophy.     

Distribution of type X collagen in tibiotarsi of broiler chickens with vitamin D deficiency

Summary Type X collagen is a significant component of the extracellular matrix of the hypertrophic zone of physeal cartilage, but its precise role in endochondral ossification has not been determined. The concentration of type X collagen increases in physeal cartilage in chicks with vitamin D deficiency. The purpose of our study was to determine whether defective endochondral ossification due to vitamin D deficiency was associated with abnormalities in the distribution of type X collagen in the proximal tibiotarsus of chicks. To accomplish this, we induced vitamin D deficiency in broiler chicks and sequentially evaluated the pattern of type X collagen immunoreactivity in the proximal tibiotarsus using a monoclonal antibody specific for chicken type X collagen. Type X collagen immunoreactivity was present in the matrix of the prehypertrophic zone, hypertrophic zone, cartilage cores of the primary spongiosa, and within the chondrocytes of the prehypertrophic and early hypertrophic zones in vitamin D-deficient and D-replete chicks. However, rachitic chicks exhibited two consistent differences in type X collagen immunoreactivity: hypertrophic chondrocytes in the late hypertrophic zone and primary spongiosa contained intracellular type X collagen; and type X collagen was concentrated into laminated aggregates in the pericellular and territorial matrices in the late hypertrophic zone and primary spongiosa. We conclude from these findings that (1) normal serum concentrations of 1,25(OHD)₂D₃ and 25OHD₃ are not required for type X collagen production; (2) type X collagen production does not decrease in the most mature zones of the physes in chicks with vitamin D deficiency; and (3) newly secreted type X collagen accumulates in the pericellular and territorial matrices of the late hypertrophic zone and primary spongiosa of rachitic chicks, perhaps because it is not readily incorporated into the interterritorial matrix.     

Lectin-binding histochemistry of intracellular and extracellular glycoconjugates of the reserve cell zone of growth plate cartilage

The distribution of intracellular and extracellular lectin-binding glycoconjugates of the reserve cell zone of growth plate cartilage was studied in the distal radial growth plate of 4-week-old Yucatan swine using a postembedment method on Epon-embedded sections. Direct comparisons were made to articular, tracheal, and auricular cartilages not involved in endochondral ossification. All patterns of lectin binding that in the growth plate were restricted to the reserve cell zone were also patterns characteristic of tracheal, articular, and auricular cartilages. These included: (a) pericellular binding with peanut agglutinin (PNA) without prior digestion with neuraminidase; (b) pericellular binding with wheat germ agglutinin (WGA) at 24 h; (c) intracellular cytoplasmic binding to concanavalin A (CON-A), Lens culinaris agglutinin (LCA), and Lotus tetragonobolus agglutinin (LTA) after periodic acid oxidation; and (d) a lack of pericellular binding with CON-A and ricin agglutinin 1 (RCA-1) after periodic acid oxidation. We conclude that reserve zone chondrocytes lack specific phenotypic markers as defined by lectin-binding affinity that are found in the cellular zones of the growth plate that undergo calcification and vascularization. The reserve zone has identical lectin-binding affinities to the three structural cartilages used as controls. One interpretation of these results is that the reserve zone may not be involved directly in endochondral ossification, but may have a structural function in growth plate cartilage.     

Transforming growth factor-β1 and fibroblast growth factors in rat growth plate

Chondrocytes in the growth plate progress in an orderly fashion from resting through proliferating to hypertrophic cells. In the region of hypertrophic chondrocytes, the cartilage is invaded by capillary loops and endochondral ossification is initiated. It is currently believed that growth factors may regulate the proliferation and maturation of chondrocytes and the synthesis of extracellular matrix in the growth plate. The ordered sequence of proliferation and differentiation observed in the growth plate provides a unique opportunity to study the role of acidic fibroblast growth factor, basic fibroblast growth factor, and transforming growth factor-β1 in the regulation of these processes. In this study, expression of the mRNA of these growth factors was examined using total RNA extracted from the physis and epiphysis of rat tibias. Transforming growth factor-β1 mRNA was detected by Northern hybridization. Expression of the genes encoding acidic and basic fibroblast growth factors was demonstrated by polymerase chain reaction amplification. In addition, using polyclonal antibodies against these growth factors, we localized them by immunohistochemical analysis. Strong intracellular staining with a predominantly nuclear pattern was observed in chondrocytes from the proliferating and upper hypertrophic zones. In contrast, chondrocytes in the resting zone stained only faintly for the presence of these growth factors. Some chondrocytes in the resting zone adjacent to the proliferating zone stained with these antibodies, and the antibodies also stained cells in the zone of Ranvier, which regulates latitudinal bone growth. Lastly, the location of transforming growth factor-β1 was examined further with use of a polyclonal antipeptide antibody specific for its extracellular epitope. Interestingly, extracellular staining for transforming growth factor-β1 was observed only around chondrocytes in the hypertrophic zone. These results suggest a role for these growth factors in the regulation of proliferation and maturation of chondrocytes and in endochondral ossification.     

Visualization of living terminal hypertrophic chondrocytes of growth plate cartilage in situ by differential interference contrast microscopy and time-lapse cinematography

The functional unit within the growth plate consists of a column of chondrocytes that passes through a sequence of phases including proliferation, hypertrophy, and death. It is important to our understanding of the biology of the growth plate to determine if distal hypertrophic cells are viable, highly differentiated cells with the potential of actively controlling terminal events of endochondral ossification prior to their death at the chondro-osseous junction. This study for the first time reports on the visualization of living hypertrophic chondrocytes in situ, including the terminal hypertrophic chondrocyte. Chondrocytes in growth plate explants are visualized using rectified differential interference contrast microscopy. We record and measure, using time-lapse cinematography, the rate of movement of subcellular organelles at the limit of resolution of this light microscopy system. Control experiments to assess viability of hypertrophic chondrocytes include coincubating organ cultures with the intravital dye fluorescein diacetate to assess the integrity of the plasma membrane and cytoplasmic esterases. In this system, all hypertrophic chondrocytes, including the very terminal chondrocyte, exist as rounded, fully hydrated cells. By the criteria of intravital dye staining and organelle movement, distal hypertrophic chondrocytes are identical to chondrocytes in the proliferative and early hypertrophic cell zones.     

Induction of bone by xenografts of rabbit growth plate chondrocytes in the nude mouse

Summary Subcutaneous transplantation of growth plate chondrocytes isolated enzymatically from the proximal tibia of 6-week-old rabbits into athymic (nu/nu) mice resulted in the formation of cartilaginous nodules. Calcification of the matrix was first seen after 48 hrs, and endochrondral ossification at 12 days. The mineral first occurred about hypertrophic cells. Histochemical alkaline phosphatase activity was concentrated in pericellular collars at the same location. Immunofluorescence examination with rabbit anti-mouse lymphocyte serum disclosed that the bulk of the osteoblasts was derived from the mouse. A small quantity of mouse antigen was present in the cartilage matrix at its junction with bone. It presumably diffused into the cartilaginous interface from the host, but the possibility that some chondrocytes were of murine origin has not been excluded. Five of six grafts of cells grown to confluence in monolayer culture for 10 to 14 days became ossified. The ability to induce mineralization declined in subculture. Chondrocytes killed by heating to 56° did not induce calcified cartilage or bone. Supported by grant AM17258-09 from the National Institutes of Health.     

Linear relationship between the volume of hypertrophic chondrocytes and the rate of longitudinal bone growth in growth plates

In this study, we tested the hypothesis that hypertrophic cell volume varies directly with the rate of longitudinal bone growth. The volume of hypertrophic chondrocytes (using stereological techniques) and longitudinal bone growth per 24 h (using oxytetracycline labeling techniques) were measured in the proximal and distal radial growth plates and the proximal and distal tibial growth plates of 21- and 35-day-old hooded rats and 21- and 35-day-old Yucatan pigs. We demonstrated a high coefficient of correlation (rats 0.98, pigs 0.83) between the final volume of hypertrophic chondrocytes and the rate of longitudinal bone growth over a wide range of growth rates and volumes of hypertrophic chondrocytes. In addition, we demonstrated a positive linear relationship between the rate of longitudinal bone growth and the final volume of hypertrophic chondrocytes. The slope of the regression line was different for rats than for pigs. The relationship was independent of the location of the growth plate in the animal and the age of the animal. The data suggest that mechanisms regulating volume changes in hypertrophic chondrocytes may exist and that chondrocytic volume increase is a major determinant of the rate of longitudinal bone growth. However, the relative contribution of cellular hypertrophy to longitudinal bone growth may be different in rats than in pigs.     

Ultrastructural Immunolocalization of Cartilage Oligomeric Matrix Protein (COMP) in Porcine Growth Cartilage

Cartilage oligomeric matrix protein (COMP) is a macromolecule of yet unknown function with restricted distribution among tissues. In the present study, the ultrastructural localization of COMP in porcine immature joint cartilage and growth plate cartilage was semiquantitatively delineated. Tissues were fixed in a mixture of low concentration glutar- and paraformaldehyde, embedded at low temperature, and subjected to immunocytochemistry using polyclonal antibodies raised against bovine COMP. Protein A-coated colloidal gold was used for detection. The most intense immunolabeling for COMP was noted in the proliferative zones of the growth cartilages. Here the concentration of immunomarker was higher in the territorial compartment than in the pericellular and interterritorial areas. A low concentration of COMP was observed in the resting and hypertrophic zones. The immunolabeling for COMP did not differ between the three matrix compartments of these zones. Supported by previous data obtained by in situ hybridization, the concentration of immunolabeling in the proliferative zone indicates a high rate of COMP synthesis in proliferative chondrocytes. Hence, COMP may be considered as a marker for normal differentiation into proliferative chondrocytes. Received: 28 November 1995 / Accepted: 15 October 1996