Expression of parathyroid hormone-related peptide and insulin-like growth factor I during rat fracture healing.

Parathyroid hormone-related peptide (PTHrP) and insulin-like growth factor I (IGF-I) are both involved in the regulation of bone and cartilage metabolisms and their interaction has been reported in osteoblasts. To investigate the interaction of PTHrP and IGF-I during fracture healing, the expression of mRNA for PTHrP and IGF-I, and receptors for PTH/PTHrP and IGF were examined during rat femoral fracture healing using an in situ hybridization method and an immunohistochemistry method, respectively. During intramembranous ossification, PTHrP mRNA, IGF-I mRNA and IGF receptors were detected in preosteoblasts, differentiated osteoblasts and osteocytes in the newly formed trabecular bone. PTH/PTHrP receptors were markedly detected in osteoblasts and osteocytes, but only barely so in preosteoblasts. During cartilaginous callus formation, PTHrP mRNA was expressed by mesenchymal cells and proliferating chondrocytes. PTH/PTHrP receptors were detected in proliferating chondrocytes and early hypertrophic chondrocytes. IGF-I mRNA and IGF receptor were co-expressed by mesenchymal cells, proliferating chondrocytes, and early hypertrophic chondrocytes. At the endochondral ossification front, osteoblasts were positive for PTHrP and IGF-I mRNA as well as their receptors. These results suggest that IGF-I is involved in cell proliferation or differentiation in mesenchymal cells, periosteal cells, osteoblasts and chondrocytes in an autocrine and/or paracrine fashion. Furthermore, PTHrP may be involved in primary callus formation presumably co-operating with IGF-I in osteoblasts and osteocytes, and by regulating chondrocyte differentiation in endochondral ossification.     

Tie2 ligands angiopoietin-1 and angiopoietin-2 are coexpressed with vascular endothelial cell growth factor in growing human bone

Angiogenesis is essential for bone growth and repair. Recent studies have shown that the endothelial-specific mitogen vascular endothelial growth factor (VEGF) is a key regulator of vascular invasion into the growth plate in infant and adolescent animals. In order to identify mechanisms regulating VEGF-induced angiogenesis in growing bone, we have investigated the expression of the angiopoietins (Ang-1 and Ang-2) in human neonatal ribs. Ang-1 and Ang-2 exhibited similar patterns of staining in the growing rib. In the cartilage, expression of Ang-1 and Ang-2 increased with chondrocyte maturation. Ang-1, Ang-2, and VEGF were not detected in the resting zone except adjacent to vascular canals, and maximum expression was detected at the cartilage bone interface. In the cartilage, Ang-2 was more highly expressed than Ang-1 or VEGF, with staining observed in the proliferating, hypertrophic, and mineralized zones. In the bone, Ang-1, Ang-2, and VEGF were detected in modeling and remodeling sites. Ang-1 was detected in the majority of osteoblasts, osteoclasts, and in some marrow space cells. Ang-2 was expressed at variable levels by osteoblasts and osteoclasts in modeling and remodeling bone. VEGF was detected in cells at bone surfaces and in the marrow spaces. Strong staining for VEGF was observed in osteoblasts and osteoclasts in modeling and remodeling bone. In the perichondrium, Ang-1, Ang-2, and VEGF were most highly expressed adjacent to the hypertrophic zone and at sites of bone collar formation. In the periosteum, Ang-1, Ang-2, and VEGF expression colocalized with alkaline phosphatase expression. These observations provide the first evidence for the expression of the angiopoietins in growing human bone in vivo. The distribution of Ang-1, Ang-2, and VEGF indicate these factors may play key roles in the regulation of angiogenesis at sites of endochondral ossification, intramembranous ossification, and bone turnover in the growing human skeleton.     

The critical role of the epidermal growth factor receptor in endochondral ossification

Loss of epidermal growth factor receptor (EGFR) activity in mice alters growth plate development, impairs endochondral ossification, and retards growth. However, the detailed mechanism by which EGFR regulates endochondral bone formation is unknown. Here, we show that administration of an EGFR-specific small-molecule inhibitor, gefitinib, into 1-month-old rats for 7 days produced profound defects in long bone growth plate cartilage characterized by epiphyseal growth plate thickening and massive accumulation of hypertrophic chondrocytes. Immunostaining demonstrated that growth plate chondrocytes express EGFR, but endothelial cells and osteoclasts show little to no expression. Gefitinib did not alter chondrocyte proliferation or differentiation and vascular invasion into the hypertrophic cartilage. However, osteoclast recruitment and differentiation at the chondro-osseous junction were attenuated owing to decreased RANKL expression in the growth plate. Moreover, gefitinib treatment inhibited the expression of matrix metalloproteinases (MMP-9, -13, and -14), increased the amount of collagen fibrils, and decreased degraded extracellular matrix products in the growth plate. In vitro, the EGFR ligand transforming growth factor α (TGF-α) strongly stimulated RANKL and MMPs expression and suppressed osteoprotegerin (OPG) expression in primary chondrocytes. In addition, a mouse model of cartilage-specific EGFR inactivation exhibited a similar phenotype of hypertrophic cartilage enlargement. Together our data demonstrate that EGFR signaling supports osteoclastogenesis at the chondro-osseous junction and promotes chondrogenic expression of MMPs in the growth plate. Therefore, we conclude that EGFR signaling plays an essential role in the remodeling of growth plate cartilage extracellular matrix into bone during endochondral ossification.     

Immunohistochemical localization of parathyroid hormone/parathyroid hormone-related peptide receptor in rat tibiae

After developing antisera against the extracellular part of the parathyroid hormone (PTH)/parathyroid hormone-related peptide (PTHrP) receptor (PTH/PTHrP receptor), we examined the receptor's localization in rat tibiae. Immunoreactivity for the PTH/PTHrP receptor was intense on chondrocytes in the late proliferating zone and the early hypertrophic zone in the growth plate. Active bone surface osteoblasts also showed intense immunoreactivity for the PTH/PTHrP receptor along their plasma membranes. In other osteoblastic cells, the PTH/PTHrP receptor was detected on spindle-shaped mononuclear cells located between the blood vessels and osteoblasts, osteocytes, and flat osteoblasts. Some stromal cells in bone marrow and bone marrow cells with a round profile also showed immunoreactivity. However, we could not detect immunoreactivity on osteoclasts. It is still unclear whether the PTH/PTHrP receptor exists on osteoclast precursor cells. However, these data confirmed the stage-dependent changes in PTH/PTHrP receptor levels on chondrocytes in the growth plate and on osteoblastic cell lineage, suggesting that the effect of PTH and/or PTHrP on mineral homeostasis as well as on skeletal growth and development is well mediated by chondrocytes and osteoblasts.This study was partly supported by a grant from the Japanese Ministry of Education, Science and Culture to I.K. (No. 06771577) and O.H. (No. 06404063)     

Effects of acute 5-fluorouracil chemotherapy and insulin-like growth factor-I pretreatment on growth plate cartilage and metaphyseal bone in rats

With the intensified use of chemotherapy and improved survival rates for childhood malignancies, it has become increasingly apparent that some children or adult survivors show poor bone growth and develop osteoporosis. As a step to investigate underlying mechanisms, this project examined short-term effects in rats of chemotherapy agent 5-fluorouracil (5-FU) on cell proliferation, apoptosis, and bone formation at tibial growth plate cartilage and its adjacent bone-forming region metaphysis. In addition, since insulin-like growth factor (IGF-I) is important for bone growth, we examined whether IGF-I pretreatment would potentially protect growth plate cartilage and bone cells from chemotherapy damage. Two days after a single high dose of 5-FU injection, proliferation of growth plate chondrocytes and metaphyseal osteoblasts/preosteoblasts was dramatically suppressed, and apoptosis was induced among osteoblasts and preosteoblasts. As a result, there was a reduction in the chondrocyte number and zonal height at the proliferative zone and a decline in the number of osteoblasts and preosteoblasts on the metaphyseal trabecular bone surface. At day 2, no obvious deleterious effects were observed on the height of the growth plate hypertrophic zone and the bone volume fraction of the metaphyseal primary spongiosa trabeculae. At day 10, while cell proliferation and growth plate structure returned to normal, there were slight decreases in trabecular bone volume, body length increase, and tibial length. While pretreatment with 1-week IGF-I systemic infusion did not attenuate the suppressive effect of 5-FU on proliferation in both growth plate and metaphysis, it significantly diminished apoptotic induction in metaphysis. These results indicate that growth plate cartilage chondrocytes and metaphyseal bone cells are sensitive to chemotherapy drug 5-FU and that IGF-I pretreatment has some anti-apoptotic protective effects on metaphyseal bone osteoblasts and preosteoblasts.     

甲状旁腺激素和甲状旁腺激素相关蛋白在正常及骨性关节炎软骨中作用机制的研究进展

目的综述在正常和骨性关节炎(osteoarthritis,OA)的关节软骨及软骨下骨中,甲状旁腺激素(parathyroid hormone,PTH)和甲状旁腺激素相关蛋白(parathyroid hormone-related protein,PTHrP)的作用机制研究进展。方法广泛查阅近年来有关PTH和PTHrP对正常和OA关节软骨作用机制的文献,并进行总结与分析。结果 PTH和PTHrP可抑制OA软骨细胞的肥大分化及凋亡,促进其增殖,从而对OA软骨细胞起到保护作用;OA软骨下骨成骨细胞对PTH的反应下降。结论 PTH、PTHrP可能通过多种信号通路参与软骨降解和软骨下骨重塑,并对OA进展起到延缓和保护作用。     

Deficiency of insulin receptor substrate-1 impairs skeletal growth through early closure of epiphyseal cartilage.

Morphological analyses in and around the epiphyseal cartilage of mice deficient in insulin receptor substrate-1 (IRS-1) showed IRS-1 signaling to be important for skeletal growth by preventing early closure of the epiphyseal cartilage and maintaining the subsequent bone turnover at the primary spongiosa. Introduction: IRS-1 is an essential molecule for intracellular signaling by IGF-I and insulin, both of which are potent anabolic regulators of cartilage and bone metabolism. To clarify the role of IRS-1 signaling in the skeletal growth, morphological analyses were performed in and around the epiphyseal cartilage of mice deficient in IRS-1 (IRS-1(-/-)), whose limbs and trunk were 20-30% shorter than wildtype (WT) mice. MATERIALS AND METHODS: The epiphyseal cartilage and the primary spongiosa at proximal tibias of homozygous IRS-1(-/-) and WT male littermates were compared using histological, immunohistochemical, enzyme cytohistochemical, ultrastructural, and bone histomorphometrical analyses. RESULTS: In and around the WT epiphyseal cartilage, IRS-1 and insulin-like growth factor (IGF)-1 receptors were widely expressed, whereas IRS-2 was weakly localized in bone cells. Chronological observation revealed that height of the proliferative zone and the size of hypertrophic chondrocytes were decreased in WT mice as a function of age, and these decreases were accelerated in the IRS-1 (-/-) cartilage, whose findings at 12 weeks were similar to those of WT at 24 weeks. In the IRS-1(-/-) cartilage, proliferating chondrocytes with positive proliferating cell nuclear antigen (PCNA) or parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor immunostaining had almost disappeared by 12 weeks. Contrarily, TUNEL+ apoptotic cells were increased in the hypertrophic zone, at the bottom of which most of the chondrocytes were surrounded by the calcified matrix, suggesting the closure of the cartilage. In the primary spongiosa, bone volume, alkaline phosphatase (ALP)+ osteoblasts, TRACP+ osteoclasts, and the osteopontin-positive cement line were markedly decreased. Bone histomorphometrical parameters for both bone formation and resorption were significantly lower in IRS-1(-/-) mice, indicating the suppression of bone turnover. CONCLUSION: The IRS-1(-/-) epiphyseal cartilage exhibited insufficient proliferation of chondrocytes, calcification of hypertrophic chondrocytes, acceleration of apoptosis, and early closure of the growth plate. Thus, the data strongly suggest that IRS-1 signaling is important for the skeletal growth by preventing early closure of the epiphyseal cartilage and by maintaining the subsequent bone turnover at the primary spongiosa.     

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     

Inhibition of growth plate angiogenesis and endochondral ossification with diminished expression of MMP-13 in hypertrophic chondrocytes in FGF-2-treated rats

Fibroblast growth factors (FGFs)/fibroblast growth factor receptor-3 signaling interferes with endochondral bone growth. However, the exact mechanisms by which FGFs inhibit endochondral ossification remain to be elucidated. In the present study, we utilized immunohistochemical techniques to clarify the effects of FGF-2 on the proximal tibial growth plate cartilage, when injected systemically into growing rats. In the FGF-2-treated rats, the growth plate was obviously thickened and, in the lowermost part, the hypertrophic chondrocytes were flattened, with an irregular arrangement. The connection of the cartilage columns and trabecular bone was disrupted. FGF-2 treatment stimulated the proliferation of chondrocytes and permitted their differentiation, but inhibited vascular invasion and resorption of the cartilage matrix. Expression of matrix metalloproteinase-13 (MMP-13) was detected in the chondrocytes in the last row of the hypertrophic zone of the growth plate in control animals. The immunoreactivity of MMP-13 was diminished in the regions where endochondral ossification was disturbed in the FGF-2-treated rats. Because MMP-13 has potent proteolytic activity on cartilage components, the FGF-2 signal may inhibit angiogenesis and endochondral ossification of the growth plate by the suppression of MMP-13 expression in hypertrophic chondrocytes. Received: March 17, 2001 / Accepted: November 16, 2001     

Localization of Pigment Epithelium-Derived Factor in Growing Mouse Bone

Pigment epithelium-derived factor (PEDF) is a potent anti-angiogenic factor found in a wide range of fetal and adult tissues, where it is thought to play a role in the regulation of angiogenesis during development. The temporal expression of PEDF during endochondral bone formation has not previously been reported. In this study, we analysed the expression pattern of PEDF in growing mouse hindlimbs from newborn day one through to maturation at week 9, using immunohistochemistry and in situ hybridization. PEDF expression was demonstrated in chondrocytes within the resting, proliferative and upper hypertrophic zones of the epiphyseal growth plate. The pattern of expression was consistent throughout the developmental stages of the mouse. In addition, PEDF was expressed by osteoblasts lining the bone spicules in the ossification zone of metaphyseal bone, as well as by osteoblasts lining cortical periosteum. These novel results demonstrate that PEDF is developmentally expressed in both cartilage and bone cells during endochondral bone formation, and strongly suggest that it may play a regulatory role in the processes of chondrocyte and osteoblast differentiation, endochondral ossification, and bone remodelling during growth and development of long bones.     

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.     

Wnt-mediated reciprocal regulation between cartilage and bone development during endochondral ossification

The role of Wnt signaling is extensively studied in skeletal development and postnatal bone remodeling, mostly based on the genetic approaches of β-catenin manipulation. However, given their independent function, a requirement for β-catenin is not the same as that for Wnt. Here, we investigated the effect of Wnt proteins in both tissues through generating cartilage- or bone-specific Wls null mice, respectively. Depletion of Wls by Col2-Cre, which would block Wnt secretion in the chondrocytes and perichondrium, delayed chondrocyte hypertrophy in the growth plate and impaired perichondrial osteogenesis. Loss of Wls in chondrocytes also disturbed the proliferating chondrocyte morphology and division orientation, which was similar to the defect observed in Wnt5a null mice. On the other hand, inactivation of Wls in osteoblasts by Col1-Cre resulted in a shorter hypertrophic zone and an increase of TRAP positive cell number in the chondro-osseous junction of growth plate, coupled with a decrease in bone mass. Taken together, our studies reveal that Wnt proteins not only modulate differentiation and cellular communication within populations of chondrocytes, but also mediate the cross regulation between the chondrocytes and osteoblasts in growth plate.     

Parathyroid hormone [PTH(1-34)] and parathyroid hormone-related protein [PTHrP(1-34)] promote reversion of hypertrophic chondrocytes to a prehypertrophic proliferating phenotype and prevent terminal differentiation of osteoblast-like cells.

The effects of parathyroid hormone/parathyroid hormone-related protein (PTH/PTHrP) on late events in chondrocyte differentiation were investigated by a dual in vitro model where conditions of suspension versus adhesion culturing are permissive either for apoptosis or for the further differentiation of hypertrophic chondrocytes to osteoblast- like cells. Chick embryo hypertrophic chondrocytes maintained in suspension synthesized type II and type X collagen and organized their extracellular matrix, forming a tissue highly reminiscent of true cartilage, which eventually mineralized. The formation of mineralized cartilage was associated with the expression of alkaline phosphatase (ALP), arrest of cell growth, and apoptosis, as observed in growth plates in vivo. In this system, PTH/PTHrP was found to repress type X collagen synthesis, ALP expression, and cartilage matrix mineralization. Cell proliferation was resumed, whereas apoptosis was blocked. Hypertrophic chondrocytes cultured in adherent conditions in the presence of retinoic acid underwent further differentiation to osteoblast-like cells (i.e., they resumed cell proliferation, switched to type I collagen synthesis, and produced a mineralizing bone-like matrix). In this system, PTH addition to culture completely inhibited the expression of ALP and matrix mineralization, whereas cell proliferation and expression of type I collagen were not affected. These data indicate that PTH/PTHrP inhibit both the mineralization of a cartilage-like matrix and apoptosis (mimicked in the suspension culture) and the production of a mineralizing bone-like matrix, characterizing further differentiation of hypertrophic chondrocytes to osteoblasts like cells (mimicked in adhesion culture). Treatment of chondrocyte cultures with PTH/PTHrP reverts cultured cells in states of differentiation earlier than hypertrophic chondrocytes (suspension), or earlier than mineralizing osteoblast-like cells (adhesion). However, withdrawal of hormonal stimulation redirects cells toward their distinct, microenvironment-dependent, terminal differentiation and fate.     

Immunohistochemical localization of interleukin 1 in human growth cartilage

Although it has been reported that interleukin 1 (IL-1) stimulate chondrocytes to produce collagenase and proteoglycanase in vitro, IL-1 producing cells and the function of IL-1 have not been demonstrated in osteocartilaginous tissue in vivo. Immunohistochemical studies of human cartilaginous epiphysis and growth cartilage demonstrated that IL-1 was detected in: (1) chondrocytes surrounding cartilage canal, (2) hypertrophic chondrocytes in cartilaginous epiphysis, (3) chondrocytes at the hypertrophic and calcified zones in the growth cartilage of actively growing bone. In contrast, few hypertrophic chondrocytes showed positive reactions to IL-1 in growth plates nearing physiologic closure. Furthermore, IL-1 was detected in chondrocytes cultured from human growth cartilage. These results show that IL-1 is produced by matured chondrocytes of human growth cartilage in vivo. Chondrocyte-derived IL-1 might play a key role in the hypertrophy of chondrocytes, in the vascularization of cartilage and in the formation of bone.     

Expression of mouse HtrA1 serine protease in normal bone and cartilage and its upregulation in joint cartilage damaged by experimental arthritis

Levels of HtrA1 protein in cartilage have been reported to elevate in joints of human osteoarthritis patients. To understand roles of HtrA1 in normal osteogenesis as well as in pathogenesis of arthritis, we examine HtrA1 expression pattern during bone and cartilage development and in articular cartilage affected by experimental arthritis. HtrA1 is not expressed in mesenchymal or cartilage condensations before initiation of ossification. When ossification begins in the condensations, the expression of HtrA1 starts in chondrocytes undergoing hypertrophic differentiation near the ossification center. Hypertrophic chondrocytes found in adult articular cartilage and epiphyseal growth plates also express HtrA1. When arthritis is induced by injection of anti-collagen antibodies and lipopolysaccharide, resting chondrocytes proceed to terminal hypertrophic differentiation and start expressing HtrA1. These data suggest that hypertrophic change induces HtrA1 expression in chondrocytes both in normal and pathological conditions. HtrA1 has been reported to inhibit TGF-beta signaling. We show that HtrA1 digests major components of cartilage, such as aggrecan, decorin, fibromodulin, and soluble type II collagen. HtrA1 may, therefore, promote degeneration of cartilage by inducing terminal hypertrophic chondrocyte differentiation and by digesting cartilage matrix though its TGF-beta inhibitory activity and protease activity, respectively. In bone, active cuboidal osteoblasts barely express HtrA1, but osteoblasts which flatten and adhere to the bone matrix and osteocytes embedded in bone are strongly positive for HtrA1 production. The bone matrix shows a high level of HtrA1 protein deposition akin to that of TGF-beta, suggesting a close functional interaction between TGF-beta and HtrA1.     

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.     

Wnt gene expression in the post-natal growth plate: regulation with chondrocyte differentiation

Longitudinal growth of long bones occurs at the growth plate by endochondral ossification. In the embryonic mouse, this process is regulated by Wnt signaling. Little is known about which members of the Wnt family of secreted signaling proteins might be involved in the regulation of the postnatal growth plate. We used microdissection and real-time PCR to study mRNA expression of Wnt genes in the mouse growth plate. Of the 19 known members of the Wnt family, only six were expressed in postnatal growth plate. Of these, Wnts -2b, -4, and -10b signal through the canonical beta-catenin pathway and Wnts -5a, -5b, and -11 signal through the noncanonical calcium pathway. The spatial expression for these six Wnts was remarkably similar, showing low mRNA expression in the resting zone, increasing expression as the chondrocytes differentiated into the proliferative and prehypertrophic state and then (except Wnt-2b) decreasing expression as the chondrocytes underwent hypertrophic differentiation. This overall pattern is broadly consistent with previous studies of embryonic mouse growth cartilage suggesting that Wnt signaling modulates chondrocyte proliferation and hypertrophic differentiation. We also found that mRNA expression of these Wnt genes persisted at similar levels at 4 weeks, when longitudinal bone growth is waning. In conclusion, we have identified for the first time the specific Wnt genes that are expressed in the postnatal mammalian growth plate. The six identified Wnt genes showed a similar pattern of expression during chondrocyte differentiation, suggesting overlapping or interacting roles in postnatal endochondral bone formation.     

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.     

Differential effects of parathyroid hormone on chick growth plate and articular chondrocytes

Summary Parathyroid hormone (PTH) binds specifically to the hypertrophic region of growth plate cartilage [16]. This specific binding suggests a role for this hormone in chondrocyte maturation. Enzymatically isolated chick articular and growth plate chondrocytes grown in monolayer culture were used to assay the direct effects of PTH on chondrocytes. The articular chondrocytes were unresponsive to PTH. The growth plate chondrocytes, however, demonstrated a marked mitogenic response to PTH, with a 39-fold increase of [³H]-thymidine incorporation into DNA. PTH also affected matrix production by the growth plate chondrocytes causing a twofold stimulation of proteoglycan synthesis as determined by the rate of ³⁵SO₄ incorporated into matrix macromolecules. Furthermore, PTH depressed collagen synthesis as measured by [³H]-proline incorporation. PTH caused a 12-fold increase in intracellular cAMP in growth plate chondrocytes but no increase in the articular cells. This specificity of PTH for growth plate chondrocytes suggests a possible regulatory role in enchondral ossification.