Mice Deficient in NF-κB p50 and p52 or RANK Have Defective Growth Plate Formation and Post-natal Dwarfism

NF-κBp50/p52 double knockout (dKO) and RANK KO mice have no osteoclasts and develop severe osteopetrosis associated with dwarfism. In contrast, Op/Op mice, which form few osteoclasts, and Src KO mice, which have osteoclasts with defective resorptive function, are osteopetrotic, but they are not dwarfed. Here, we compared the morphologic features of long bones from p50/p52 dKO, RANK KO, Op/Op and Src KO mice to attempt to explain the differences in their long bone lengths. We found that growth plates in p50/p52 dKO and RANK KO mice are significantly thicker than those in WT mice due to a 2-3-fold increase in the hypertrophic chondrocyte zone associated with normal a proliferative chondrocyte zone. This growth plate abnormality disappears when animals become older, but their dwarfism persists. Op/Op or Src KO mice have relatively normal growth plate morphology. In-situ hybridization study of long bones from p50/p52 dKO mice showed marked thickening of the growth plate region containing type 10 collagen-expressing chondrocytes. Treatment of micro-mass chondrocyte cultures with RANKL did not affect expression levels of type 2 collagen and Sox9, markers for proliferative chondrocytes, but RANKL reduced the number of type 10 collagen-expressing hypertrophic chondrocytes. Thus, RANK/NF-κB signaling plays a regulatory role in post-natal endochondral ossification that maintains hypertrophic conversion and prevents dwarfism in normal mice.     

3β-Hydroxysterol-Delta24 reductase plays an important role in long bone growth by protecting chondrocytes from reactive oxygen species

Desmosterolosis is an autosomal recessive disease caused by mutations in the 3β-hydroxysterol-Delta24 reductase (DHCR24) gene, with severe developmental anomalies including short limbs. We utilized DHCR24 knockout (KO) mice to study the underlying bone pathology. Because the KO mice died within a few hours after birth, we cultured metatarsal bones from newborn mice. The growth of bones from KO mice was significantly retarded after 1 week of culture. Absence of proliferating chondrocytes in the growth plate and abnormal hypertrophy of prehypertrophic chondrocytes were observed in the bones from KO mice. Hypertrophic differentiation was evidenced by higher expression of Indian hedgehog, alkaline phosphatase, and matrix metalloproteinase 13. Since elevated levels of reactive oxygen species (ROS) during chondrogenesis are known to inhibit proliferation and to initiate chondrocyte hypertrophy in the growth plate, and since DHCR24 acts as a potent ROS scavenger, we hypothesized that the abnormal chondrocyte proliferation and differentiation in KO mice were due to decreased ROS scavenging activity. Treatment with an antioxidant, N-acetyl cysteine, could correct the abnormalities observed in the bones from KO mice. Treatment of bones from wild-type mice with U18666A, a chemical inhibitor of DHCR24, resulted in short broad bones with a disrupted proliferating zone. Treatment of ATDC cells with hydrogen peroxide (H₂O₂) induced hypertrophic changes as evidenced by the expression of the marker genes specific for hypertrophic chondrocyte differentiation. H₂O₂-induced hypertrophic change was prevented by adenoviral delivery of DHCR24. Induction of chondrocyte differentiation in ATDC cells by insulin was associated with increased ROS production that was markedly enhanced by treatment of ATDC5 cells with DHCR24 siRNA. This is the first demonstration that DHCR24 plays an important role in long bone growth by protecting chondrocytes from ROS     

Thyroid hormones regulate hypertrophic chondrocyte differentiation and expression of parathyroid hormone-related peptide and its receptor during endochondral bone formation.

Hypothyroidism in children causes developmental abnormalities in bone and growth arrest, while thyrotoxicosis accelerates growth rate and advances bone age. To determine the effects of thyroid hormones on endochondral bone formation, we examined epiphyseal growth plates in control, hypothyroid, thyrotoxic, and hypothyroid-thyroxine (hypo-T4)-treated rats. Hypothyroid growth plates were grossly disorganized, contained an abnormal matrix rich in heparan sulfate, and hypertrophic chondrocyte differentiation failed to progress. These effects correlated with the absence of collagen X expression and increased parathyroid hormone-related protein (PTHrP) messenger RNA (mRNA) expression. In thyrotoxic growth plates, histology essentially was normal but PTHrP receptor (PTHrP-R) mRNA was undetectable. PTHrP is a potent inhibitor of hypertrophic chondrocyte differentiation that acts in a negative feedback loop with the secreted factor Indian hedgehog (Ihh) to regulate endochondral bone formation. Thyroid hormone receptor alpha1(TRalpha1), TRalpha2, and TRbeta1 proteins were localized to reserve zone progenitor cells and proliferating chondrocytes in euthyroid rat cartilage; regions in which PTHrP and PTHrP-R expression were affected by thyroid status. Thus, dysregulated Ihh/PTHrP feedback loop activity may be a key mechanism that underlies growth disorders in childhood thyroid disease.     

Leptin regulates chondrocyte differentiation and matrix maturation during endochondral ossification

Leptin has been suggested to mediate a variety of actions, including bone development, via its ubiquitously expressed receptor (Ob-Rb). In this study, we investigated the role of leptin in endochondral ossification at the growth plate. The growth plates of wild-type and ob/ob mice were analyzed. Effects of leptin on chondrocyte gene expression, cell cycle, apoptosis and matrix mineralization were assessed using primary chondrocyte culture and the ATDC5 cell differentiation culture system. Immunohistochemistry and in situ hybridization showed that leptin was localized in prehypertrophic chondrocytes in normal mice and that Ob-Rb was localized in hypertrophic chondrocytes in normal and ob/ob mice. Growth plates of ob/ob mice were more fragile than those of wild-type mice in a mechanical test and were broken easily at the chondro-osseous junction. The growth plates of ob/ob mice showed disturbed columnar structure, decreased type X collagen expression, less organized collagen fibril arrangement, increased apoptosis and premature mineralization. Leptin administration in ob/ob mice led to an increase in femoral and humeral lengths and decrease in the proportional length of the calcified hypertrophic zone to the whole hypertrophic zone. In primary chondrocyte culture, the matrix mineralization in ob/ob chondrocytes was stronger than that of wild-type mice; this mineralization in both types of mice was abolished by the addition of exogenous leptin (10 ng/ml). During ATDC5 cell differentiation culture, exogenous leptin at a concentration of 1-10 ng/ml (equivalent to the normal serum concentration of leptin) altered type X collagen mRNA expression and suppressed apoptosis, cell growth and matrix calcification. In conclusion, we demonstrated that leptin modulates several events associated with terminal differentiation of chondrocytes. Our finding that the growth plates of ob/ob mice were fragile implies a disturbance in the differentiation/maturation process of growth plates due to depletion of leptin signaling in ob/ob mice. These findings suggest that peripheral leptin signaling plays an essential role in endochondral ossification at the growth plate.     

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     

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     

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.     

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.     

S-100 protein in human cartilage lesions

S-100 protein is an acidic calcium-binding protein that was originally isolated from the mammalian central nervous system in 1965. Initially, S-100 protein was thought to be specific to neuroectodermal tissues, but its presence in chondrocytes was recently reported. This study is an analysis of the distribution of S-100 protein in lesions of human cartilage and its possible significance. Several cartilaginous tumors, both benign and malignant, as well as normal epiphyseal growth plates, were examined for S-100 protein by the immunoperoxidase technique. Each cartilaginous lesion that was examined showed immunoreactivity for S-100 protein. The staining product was noted only intracellularly. The highest intensity of staining was seen in the hypertrophic chondrocytes of the zone of provisional calcification in the growth plate and in the large chondrocytes located adjacent to areas of matrix mineralization in cartilaginous tumors. In normal epiphyseal growth plates, the intensity of staining increased in chondrocyte cytoplasm as one moved from the proliferating columnar chondrocytes through the zone of hypertrophic chondrocytes to the hypertrophic, degenerating chondrocytes in the zone of provisional calcification. In cartilaginous tumors, the cells of enchondroma and of the cartilaginous cap of osteochondroma were more immunoreactive than those of chondromyxoid fibroma. In benign chondroblastoma, the chondroblasts were less reactive than the chondrocytes in areas of chondroid matrix production. The latter areas of chondroblastomas showed stronger immunoreactivity in the matrix-enclosed cells adjacent to areas of mineral deposition. Among conventional chondrosarcomas, grade-I tumors showed greater immunoreactivity of the chondrocyte cytoplasm than did those of a higher grade, in which chondroid matrix production was less abundant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth plate cartilage formation and resorption are differentially depressed in growth retarded uremic rats

To characterize the modifications of growth plate in individuals with growth impairment secondary to chronic renal failure, young rats were made uremic by subtotal nephrectomy (NX) and, after 14 d, their tibial growth plates were studied and compared with those of sham-operated rats fed ad libitum (SAL) or pair-fed with NX (SPF). NX rats were growth retarded and severely uremic. Growth plate height (mean +/- SD) was much greater (P<0.05) in NX (868.4+/-85.4 microm) than SAL (570.1+/-93.5 microm) and SPF (551.9+/-99.7 microm) rats as a result of a higher (P<0.05) hypertrophic zone (661.0+/-89.7 versus 362.8+/-71.6 and 353.0+/-93.9 microm, respectively). The increased size of the growth plate was associated with a greater number of chondrocytes and modifications in their structure, particularly in the hypertrophic zone adjacent to bone. In this zone, chondrocytes of NX animals were significantly (P<0.05) smaller (12080.4+/-1158.3 microm3) and shorter (34.1+/-2.5 microm) than those of SAL (16302.8+/-1483.4 microm3 and 37.8+/-2.0 microm) and SPF (14465.8+/-1521.0 microm3 and 36.3+/-1.8 microm). The interface between the growth plate cartilage and the metaphyseal bone appeared markedly irregular in NX rats. Kinetics of chondrocytes was also modified (P<0.05) in the NX rats, which had lower cell turnover per column per day (5.4+/-0.9), longer duration of hypertrophic phase (89.0+/-15.2 h), and reduced cellular advance velocity (7.4+/-2.2 microm/h) compared with SAL (8.0+/-1.6, 32.1+/-6.7 h, and 11.3+/-2.7 microm/h) and SPF (7.2+/-1.1, 34.8+/-5.1 h, and 10.1+/-2.5 microm/h). Cell proliferation was no different among the three groups. Because the growth plates of SPF and SAL rats were substantially not different, modifications observed in the NX rats cannot be attributed to the nutritional deficit associated with renal failure. These findings indicate that chronic renal failure depresses both the activity of the growth plate cartilage by altering chondrocyte hypertrophy and the replacement of cartilage by bone at the metaphyseal end. The two processes are differentially depressed since cartilage resorption is more severely lowered than cartilage enlargement and this leads to an accumulation of cartilage at the hypertrophic zone.     

In vivo dynamic bone growth modulation is less detrimental but as effective as static growth modulation

Longitudinal bone growth, which occurs in growth plates, has important implications in pediatric orthopedics. Mechanical loads are essential to normal bone growth, but excessive loads can lead to progressive deformities. In order to compare the effects of in vivo static and dynamic loading on bone growth rate and growth plate histomorphometry, a finely controlled, normalized and equivalent compression was applied for a period of two weeks on the seventh caudal vertebra (Cd7) of rats during their pubertal growth spurt. The load was sustained (0.2MPa, 0.0Hz) in the static group and sinusoidally oscillating (0.2MPa±30%, 0.1Hz) in the dynamic group. Control and sham (operated but no load applied) groups were also studied. Cd7 growth rate was statistically reduced by 19% (p<0.001) for both static and dynamic groups when compared to the sham group. Loading effects on growth plate histomorphometry were greater in the static than dynamic groups with significant reductions (p<0.001) observed for growth plate thickness, proliferative chondrocyte number per column and hypertrophic chondrocyte height in the static group when compared to the sham group. Significant differences (p<0.01) were also found between static and dynamic groups for growth plate thickness and proliferative chondrocyte number per column while the difference nearly reached significance (p=0.014) for hypertrophic chondrocyte height. This in vivo study shows that static and dynamic loading are equally effective in modulating bone growth of rat caudal vertebrae. However, dynamic loading causes less detrimental effects on growth plate histomorphometry compared to static loading. This knowledge is greatly relevant for the improvement and/or development of new minimally invasive approaches, which are based on the local modulation of bone growth, to correct several progressive musculoskeletal deformities.     

Quantitation of chondrocyte performance in growth-plate cartilage during longitudinal bone growth

The longitudinal growth of bone depends on the activities of individual chondrocytes of the growth plate. Each chondrocyte remains in a fixed location throughout its life, and there accomplishes all of its functions. Although a cell may perform several or all of its activities simultaneously, one of these will usually predominate during a particular phase of its life. The two most prominent stages are those of cellular proliferation and hypertrophy (including the mineralization of matrix) before the resorption of tissue during vascular invasion. By applying recently developed stereological procedures and improved methods for the fixation of cartilage, we compared cellular shape modulation, various ultrastructural parameters (surface areas or volumes of endoplasmic reticulum, Golgi membranes, and mitochondria), the production of matrix, and cellular turnover for proliferating and hypertrophic chondrocytes within the proximal tibial growth plate of the rat. By the late hypertrophic stage, fourfold and tenfold increases in the mean cellular height and volume, respectively, and a threefold increase in the mean volume of the matrix per cell were achieved. The high metabolic activity of hypertrophic cells was reflected by a twofold to fivefold increase in the mean cellular surface area of rough endoplasmic reticulum, the Golgi membranes, and the mean cellular mitochondrial volume. Rates of longitudinal growth were determined by fluorochrome labeling and incident-light fluorescence microscopy. Using these values and the stereological estimators describing cellular height, the rates of cellular turnover were calculated. The rapid progression of the vascular invasion front was found to eliminate, for each column of cells, one chondrocyte every three hours; that is, eight cells a day. The maintenance of a steady-state structure for growth-plate cartilage in rats in a steady state of growth thus necessitates efficient compensation for these losses, which is achieved by a high rate of cellular proliferation and rapid hypertrophy.     

PTH Receptor Signaling in Osteoblasts Regulates Endochondral Vascularization in Maintenance of Postnatal Growth Plate

Longitudinal growth of postnatal bone requires precise control of growth plate cartilage chondrocytes and subsequent osteogenesis and bone formation. Little is known about the role of angiogenesis and bone remodeling in maintenance of cartilaginous growth plate. Parathyroid hormone (PTH) stimulates bone remodeling by activating PTH receptor (PTH1R). Mice with conditional deletion of PTH1R in osteoblasts showed disrupted trabecular bone formation. The mice also exhibited postnatal growth retardation with profound defects in growth plate cartilage, ascribable predominantly to a decrease in number of hypertrophic chondrocytes, resulting in premature fusion of the growth plate and shortened long bones. Further characterization of hypertrophic zone and primary spongiosa revealed that endochondral angiogenesis and vascular invasion of the cartilage were impaired, which was associated with aberrant chondrocyte maturation and cartilage development. These studies reveal that PTH1R signaling in osteoblasts regulates cartilaginous growth plate for postnatal growth of bone. © 2014 American Society for Bone and Mineral Research.     

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.     

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.     

Estrogen deficiency leads to decrease in chondrocyte numbers in the rabbit growth plate

Background In the pubertal growth plate, sex hormones play important roles in regulating the proliferation, differentiation, maturation, and programmed death of chondrocytes. Although many studies have been reported on the regulation of estrogen in long-bone growth, some of the mechanisms have remained unclear, including its role in cell kinetics in growth plate chondrocytes. The aim of this study was to clarify the effect of a deficiency of estrogen on growth plate chondrocytes. Methods We obtained growth plates of the femoral head from normal and ovariectomized Japanese white rabbits at 10, 15, 20, and 25 weeks of age. The effects of estrogen deficiency on the cell kinetics of growth plate chondrocytes were investigated immunohistochemically using antibodies for an apoptotic marker, caspase-3, and for proliferating cell nuclear antigen (PCNA). Results Both the length of the femur and the height of the growth plate in the ovariectomized rabbits tended to be larger than those in the normal rabbits. There were fewer chondrocytes in the ovariectomized rabbits than in the normal ones. Caspase-3-positive cells were detected mainly in the hypertrophic zone, whereas PCNA-positive cells were found in the proliferating to upper hypertrophic zones. The ovariectomized rabbits showed a higher caspase-3-positive rate at 20 weeks of age and a lower PCNA-positive ratio in all age groups than the normal rabbits. Conclusions This study indicated that ovariectomy led to a decreased number of growth plate chondrocytes, which resulted from decreased cell-proliferating ability and probably acceleration of the number of chondrocytes undergoing apoptosis.     

Development of Avian Tibial Dyschondroplasia: Gene Expression and Protein Synthesis

Age-dependent gene expression and protein synthesis associated with chondrocyte differentiation were evaluated in the epiphyseal growth plates of normal and tibial dyschondroplasia (TD)-afflicted chickens. In the normal growth plate, collagen type II gene is expressed mainly by chondrocytes at the upper zone of the growth plate and by the chondrocytes in the articular cartilage. Collagen type X and osteopontin (OPN) genes are expressed in the lower zone of the growth plate and in the zone of cartilage-to-bone transition. No age-dependent changes in the pattern of OPN and collagen type II or X gene expression were observed up to 20 days of age. In the TD-afflicted growth plates, the lesion is enlarged with age, and chondrocytes expressing the collagen type II gene were observed in the hypertrophic zone as early as 8 days posthatching. Abnormal expression of OPN and collagen type X genes was also observed starting at 13 days of age. At day 20, the entire TD lesion—which was significantly enlarged—was surrounded by collagen type II, collagen type X, and OPN expressing cells. The level of OPN in TD was reduced with increasing age, and at 20 days almost no OPN could be detected in either the upper or the lower hypertrophic zones. The level of bone sialoprotein (BSP) also diminished with increasing age in the TD growth plates. In contrast to OPN, the age-dependent reduction in BSP levels was mainly in the lower hypertrophic zone (LHZ), and at 20 days of age, BSP was barely detected in the LHZ, whereas in the upper hypertrophic zone, the levels of BSP were similar to those in normal growth plate. In summary, our results suggest that the primary event of the TD lesion occurs in cells of proliferative phenotype within the hypertrophic zone. These cells divide and form the TD lesion, which consists of cells that do not express the genes associated with hypertrophy. Received: 11 June 1997 / Accepted: 11 May 1998