Endochondral Bone Growth Research Articles

Genetic basis for skeletal disease. CNP therapy for achondroplasia

By using transgenic and knockout mice, we have elucidated that C-type natriuretic peptide (CNP) is a potent stimulator of endochondral bone growth. Furthermore, loss-of-function mutations in the gene coding for guanylyl cyclase-B (GC-B) , the specific receptor for CNP, have been proved to cause impaired skeletal growth in humans. Following these results, we have started to translate the stimulatory effect of CNP on endochondral bone growth into the therapy for patients with achondroplasia, and have shown that targeted overexpression of CNP in cartilage or systemic administration of CNP reverses the impaired skeletal growth of mice model of achondroplasia.

Circulating C-Type Natriuretic Peptide (CNP) Rescues Chondrodysplastic CNP Knockout Mice from Their Impaired Skeletal Growth and Early Death

C-type natriuretic peptide (CNP) is a potent stimulator of endochondral bone growth through a subtype of membranous guanylyl cyclase receptor, GC-B. Although its two cognate natriuretic peptides, ANP and BNP, are cardiac hormones produced from heart, CNP is thought to act as an autocrine/paracrine regulator. To elucidate whether systemic administration of CNP would be a novel medical treatment for chondrodysplasias, for which no drug therapy has yet been developed, we investigated the effect of circulating CNP by using the CNP transgenic mice with an increased circulating CNP under the control of human serum amyloid P component promoter (SAP-Nppc-Tg mice). SAP-Nppc-Tg mice developed prominent overgrowth of bones formed through endochondral ossification. In organ culture experiments, the growth of tibial explants of SAP-Nppc-Tg mice was not changed from that of their wild-type littermates, exhibiting that the stimulatory effect on endochondral bone growth observed in SAP-Nppc-Tg mice is humoral. Then we crossed chondrodysplastic CNP-depleted mice with SAP-Nppc-Tg mice. Impaired endochondral bone growth in CNP knockout mice were considerably and significantly recovered by increased circulating CNP, followed by the improvement in not only their longitudinal growth but also their body weight. In addition, the mortality of CNP knockout mice was greatly decreased by circulating CNP. Systemic administration of CNP might have therapeutic potential against not only impaired skeletal growth but also other aspects of impaired growth including impaired body weight gain in patients suffering from chondrodysplasias and might resultantly protect them from their early death.

Endothelial nitric oxide synthase deficiency in mice results in reduced chondrocyte proliferation and endochondral bone growth

Nitric oxide (NO) and aberrant chondrocyte differentiation have both been implicated in the pathogenesis of osteoarthritis, but whether these processes are connected is unknown, and the role of specific NO synthase (NOS) enzymes in chondrocyte physiology is unclear. This study was undertaken to examine the effects of inactivation of endothelial cell NOS (eNOS) on cartilage development in mice. Skeletal growth and development of mice carrying a null mutation in the eNOS gene was compared with that of their control littermates. In situ analyses were complemented by experiments with primary chondrocytes and tibial explants from these mice. Mice that were deficient in eNOS showed increased fatality and reduced bone growth, with hypocellular growth plates and a marked reduction in the number of proliferating chondrocytes. In vitro studies demonstrated lower chondrocyte numbers and reduced endochondral bone growth in mutant mice, suggesting that the role of eNOS signaling in chondrocyte proliferation is cell autonomous. Reduced chondrocyte numbers appear to be caused by decreased cyclin D1 and increased p57 expression in mutant mice, resulting in slower cell cycle progression and earlier cell cycle exit. In addition, expression of early chondrocyte markers such as SOX9 was reduced, and prehypertrophic markers were expressed prematurely in mutant mice. Our findings identify a novel and important role of eNOS in chondrocyte proliferation and endochondral bone growth and demonstrate that loss of eNOS results in premature cell cycle exit and prehypertrophic chondrocyte differentiation during cartilage development.

Bone vascularization and trabecular bone formation are mediated by PKBalpha/Akt1 in a gene‐dosage‐dependent manner: In vivo and ex vivo MRI

PKBalpha/Akt1, a protein kinase, is a major mediator of angiogenic signaling. The purpose of this study was to determine the role of PKB alpha/Akt1 in bone vascularization and development. For that aim, macromolecular dynamic contrast enhanced MRI was applied to examine in vivo vascular changes in long bones of 40-day-old growing PKB alpha/Akt1-deficient, heterozygous, and wild-type mice. Ex vivo microMRI and microCT were applied to monitor the impact of PKB alpha/Akt1 gene dosage on trabecular bone formation during endochondral bone growth. PKB alpha/Akt1-deficient mice and, remarkably, also heterozygous mice showed significantly reduced blood volume fraction in the humerus compared to wild-type mice. Moreover, PKB alpha/Akt1-deficient mice showed a more severe vascular deficiency with reduced permeability. microCT and microMRI of trabeculae revealed impaired bone formation in both PKB alpha/Akt1-deficient and heterozygous mice, whereas cortical bone parameters were only reduced in PKB alpha/Akt1-deficient mice. Reduction of metaphyseal blood vessel invasion, concomitant with aberrant trabeculae and shorter long bones, demonstrates a gene-dose-dependent role for PKB alpha/Akt1 in regulation of overall size and endochondral bone growth. MRI proved to provide high sensitivity for in vivo detection of subtle gene dose effects leading to impaired bone vascularity and for uncovering changes in trabecular bone.

Interaction between cartilage oligomeric matrix protein and extracellular matrix protein 1 mediates endochondral bone growth

In an effort to define the biological functions of COMP, a functional genetic screen was performed. This led to the identification of extracellular matrix protein 1 (ECM1) as a novel COMP-associated partner. COMP directly binds to ECM1 both in vitro and in vivo. The EGF domain of COMP and the C-terminus of ECM1 mediate the interaction between them. COMP and ECM1 colocalize in the growth plates invivo. ECM1 inhibits chondrocyte hypertrophy, matrix mineralization, and endochondral bone formation, and COMP overcomes the inhibition by ECM1. In addition, COMP-mediated neutralization of ECM1 inhibition depends on their interaction, since COMP largely fails to overcome the ECM1 inhibition in the presence of the EGF domain of COMP, which disturbs the association of COMP and ECM1. These findings provide the first evidence linking the association of COMP and ECM1 and the biological significance underlying the interaction between them in regulating endochondral bone growth.

Control of chondrocyte gene expression by actin dynamics: a novel role of cholesterol/Ror-α signalling in endochondral bone growth

Control of chondrocyte gene expression by actin dynamics: a novel role of cholesterol/Ror-α signalling in endochondral bone growth

Regulation of Gene Expression by PI3K in Mouse Growth Plate Chondrocytes

BackgroundEndochondral ossification, the process through which long bones are formed, involves chondrocyte proliferation and hypertrophic differentiation in the cartilage growth plate. In a previous publication we showed that pharmacological inhibition of the PI3K signaling pathway results in reduced endochondral bone growth, and in particular, shortening of the hypertrophic zone in a tibia organ culture system. In this current study we aimed to investigate targets of the PI3K signaling pathway in hypertrophic chondrocytes.Methodology/Principal FindingsThrough the intersection of two different microarray analyses methods (classical single gene analysis and GSEA) and two different chondrocyte differentiation systems (primary chondrocytes treated with a pharmacological inhibitor of PI3K and microdissected growth plates), we were able to identify a high number of genes grouped in GSEA functional categories regulated by the PI3K signaling pathway. Genes such as Phlda2 and F13a1 were down-regulated upon PI3K inhibition and showed increased expression in the hypertrophic zone compared to the proliferative/resting zone of the growth plate. In contrast, other genes including Nr4a1 and Adamts5 were up-regulated upon PI3K inhibition and showed reduced expression in the hypertrophic zone. Regulation of these genes by PI3K signaling was confirmed by quantitative RT-PCR. We focused on F13a1 as an interesting target because of its known role in chondrocyte hypertrophy and osteoarthritis. Mouse E15.5 tibiae cultured with LY294002 (PI3K inhibitor) for 6 days showed decreased expression of factor XIIIa in the hypertrophic zone compared to control cultures.Conclusions/SignificanceDiscovering targets of signaling pathways in hypertrophic chondrocytes could lead to targeted therapy in osteoarthritis and a better understanding of the cartilage environment for tissue engineering.

Translational research of C-type natriuretic peptide (CNP) into skeletal dysplasias

By using transgenic and knockout mice, we have elucidated that C-type natriuretic peptide (CNP) is a potent stimulator of endochondral bone growth. In humans, loss-of-function mutations in the gene coding for guanylyl cyclase-B (GC-B), the specific receptor for CNP, have been proved to be the cause of acromesomelic dysplasia, type Maroteaux, one form of human skeletal dysplasias. Following these results, we have started to translate the stimulatory effect of CNP on endochondral bone growth into the therapy for patients with skeletal dysplasias. We have shown that targeted overexpression of CNP in cartilage or systemic administration of CNP reverses the impaired skeletal growth of mice model of achondroplasia, the most common form of human skeletal dysplasias.

Septoclast Deficiency Accompanies Postnatal Growth Plate Chondrodysplasia in the Toothless ( tl) Osteopetrotic, Colony-Stimulating Factor-1 (CSF-1)-Deficient Rat and Is Partially Responsive to CSF-1 Injections

The septoclast is a specialized, cathepsin B-rich, perivascular cell type that accompanies invading capillaries on the metaphyseal side of the growth plate during endochondral bone growth. The putative role of septoclasts is to break down the terminal transverse septum of growth plate cartilage and permit capillaries to bud into the lower hypertrophic zone. This process fails in osteoclast-deficient, osteopetrotic animal models, resulting in a progressive growth plate dysplasia. The toothless rat is severely osteopetrotic because of a frameshift mutation in the colony-stimulating factor-1 (CSF-1) gene (Csf1(tl)). Whereas CSF-1 injections quickly restore endosteal osteoclast populations, they do not improve the chondrodysplasia. We therefore investigated septoclast populations in Csf1(tl)/Csf1(tl) rats and wild-type littermates, with and without CSF-1 treatment, at 2 weeks, before the dysplasia is pronounced, and at 4 weeks, by which time it is severe. Tibial sections were immunolabeled for cathepsin B and septoclasts were counted. Csf1(tl)/Csf1(tl) mutants had significant reductions in septoclasts at both times, although they were more pronounced at 4 weeks. CSF-1 injections increased counts in wild-type and mutant animals at both times, restoring mutants to normal levels at 2 weeks. In all of the mutants, septoclasts seemed misoriented and had abnormal ultrastructure. We conclude that CSF-1 promotes angiogenesis at the chondroosseous junction, but that, in Csf1(tl)/Csf1(tl) rats, septoclasts are unable to direct their degradative activity appropriately, implying a capillary guidance role for locally supplied CSF-1.

Femoral Head Deformation and Repair Following Induction of Ischemic Necrosis

Ischemic necrosis of the femoral head can be induced surgically in the piglet. We used this model to assess femoral head deformation and repair in vivo by sequential magnetic resonance imaging and by correlating end-stage findings with histologic assessments. Ischemic necrosis of the femoral head was induced in ten three-week-old piglets by tying a silk ligature around the base of the femoral neck (intracapsular) and cutting the ligamentum teres. We used magnetic resonance imaging with the piglets under general anesthesia to study the hips at forty-eight hours and at one, two, four, and eight weeks. Measurements on magnetic resonance images in the midcoronal plane of the involved and control sides at each time documented the femoral head height, femoral head width, superior surface cartilage height, and femoral neck-shaft angle. Histologic assessments were done at the time of killing. Complete ischemia of the femoral head was identified in all involved femora by magnetic resonance imaging at forty-eight hours. Revascularization began at the periphery of the femoral head as early as one week and was underway in all by two weeks. At eight weeks, magnetic resonance imaging and histologic analysis showed deformation of the femoral head and variable tissue deposition. Tissue responses included (1) vascularized fibroblastic ingrowth with tissue resorption and cartilage, intramembranous bone, and mixed fibro-osseous or fibro-cartilaginous tissue synthesis and (2) resumption of endochondral bone growth. At eight weeks, the mean femoral head measurements (and standard error of the mean) for the control compared with the ligated femora were 10.4 +/- 0.4 and 4.8 +/- 0.4 mm, respectively, for height; 26.7 +/- 0.8 and 31.2 +/- 0.8 mm for diameter; 1.1 +/- 0.1 and 2.3 +/- 0.1 mm for cartilage thickness; and 151 degrees +/- 2 degrees and 135 degrees +/- 2 degrees for the femoral neck-shaft angle. Repeated-measures mixed-model analysis of variance revealed highly significant effects of ligation in each parameter (p < 0.0001). Magnetic resonance imaging allows for the assessment of individual hips at sequential time periods to follow deformation and repair. There was a variable tissue response, and histologic assessment at the time of killing was shown to correlate with the evolving and varying magnetic resonance imaging signal intensities. Femoral head height on the ischemic side from one week onward was always less than the initial control value and continually decreased with time, indicating collapse as well as slowed growth. Increased femoral head width occurred relatively late (four to eight weeks), indicating cartilage model overgrowth concentrated at the periphery.

Control of chondrocyte gene expression by actin dynamics: a novel role of cholesterol/Ror-α signalling in endochondral bone growth

Elucidating the signalling pathways that regulate chondrocyte differentiation, such as the actin cytoskeleton and Rho GTPases, during development is essential for understanding of pathological conditions of cartilage, such as chondrodysplasias and osteoarthritis. Manipulation of actin dynamics in tibia organ cultures isolated from E15.5 mice results in pronounced enhancement of endochondral bone growth and specific changes in growth plate architecture. Global changes in gene expression were examined of primary chondrocytes isolated from embryonic tibia, treated with the compounds cytochalasin D, jasplakinolide (actin modifiers) and the ROCK inhibitor Y27632. Cytochalasin D elicited the most pronounced response and induced many features of hypertrophic chondrocyte differentiation. Bioinformatics analyses of microarray data and expression validation by real-time PCR and immunohistochemistry resulted in the identification of the nuclear receptor retinoid related orphan receptor-α (Ror-α) as a novel putative regulator of chondrocyte hypertrophy. Expression of Ror-α target genes, (Lpl, fatty acid binding protein 4 [Fabp4], Cd36 and kruppel-like factor 5 [Klf15]) were induced during chondrocyte hypertrophy and by cytochalasin D and are cholesterol dependent. Stimulation of Ror-α by cholesterol results in increased bone growth and enlarged, rounded cells, a phenotype similar to chondrocyte hypertrophy and to the changes induced by cytochalasin D, while inhibition of cholesterol synthesis by lovastatin inhibits cytochalasin D induced bone growth. Additionally, we show that in a mouse model of cartilage specific (Col2-Cre) Rac1, inactivation results in increased Hif-1α (a regulator of Rora gene expression) and Ror-α+ cells within hypertrophic growth plates. We provide evidence that cholesterol signalling through increased Ror-α expression stimulates chondrocyte hypertrophy and partially mediates responses of cartilage to actin dynamics.

Therapeutic potential of CNP for skeletal dysplasias

Background We had revealed that C-type natriuretic peptide (CNP) is a potent stimulator of endochondral bone growth by using transgenic and knockout mice: transgenic mice with targeted overexpression of CNP in cartilage develop prominent skeletal overgrowth [1], whereas mice depleted with CNP exhibit impaired endochondral bone growth [2]. We planned to translate this effect of CNP into the therapy for skeletal dysplasias, of which the effective drug therapy is not established yet. Here we investigated the effect of CNP on the impaired bone growth of mice model of achondroplasia, the most common form of skeletal dysplasias.

ADAMTS-7, a Direct Target of PTHrP, Adversely Regulates Endochondral Bone Growth by Associating with and Inactivating GEP Growth Factor

ADAMTS-7, a metalloproteinase that belongs to ADAMTS family, is important for the degradation of cartilage extracellular matrix proteins in arthritis. Herein we report that ADAMTS-7 is upregulated during chondrocyte differentiation and demonstrates the temporal and spatial expression pattern during skeletal development. ADAMTS-7 potently inhibits chondrocyte differentiation and endochondral bone formation, and this inhibition depends on its proteolytic activity. The cysteine-rich domain of ADAMTS-7 is required for its interaction with the extracellular matrix, and the C-terminal four-thrombospondin motifs are necessary for its full proteolytic activity and inhibition of chondrocyte differentiation. ADAMTS-7 is an important target of canonical PTHrP signaling, since (i) PTHrP induces ADAMTS-7, (ii) ADAMTS-7 is downregulated in PTHrP null mutant (PTHrP-/-) growth plate chondrocytes, and (iii) blockage of ADAMTS-7 almost abolishes PTHrP-mediated inhibition of chondrocyte hypertrophy and endochondral bone growth. ADAMTS-7 associates with granulin-epithelin precursor (GEP), an autocrine growth factor that has been implicated in tissue regeneration, tumorigenesis, and inflammation. In addition, ADAMTS-7 acts as a new GEP convertase and neutralizes GEP-stimulated endochondral bone formation. Collectively, these findings demonstrate that ADAMTS-7, a direct target of PTHrP signaling, negatively regulates endochondral bone formation by associating with and inactivating GEP chondrogenic growth factor.

The effects of socioeconomic status on endochondral and appositional bone growth, and acquisition of cortical bone in children from 19th century Birmingham, England

Endochondral growth, appositional growth, and acquisition of cortical bone thickness in the femur are investigated in subadult skeletons (N = 43, dental age range birth to 12 years) from the 19(th)-century AD burial site of St. Martin's churchyard, Birmingham, England. Endochondral growth is monitored using diaphyseal femoral length. Appositional growth is monitored using radiographic midshaft mediolateral width and acquisition of cortical bone using combined mediolateral cortical thickness measured at the midshaft from radiographs. The methodology involves plotting these variables against dental age. Growth is compared in children of differing socioeconomic status. Higher and lower status individuals are identified in the assemblage by their burial in brick vaults in the case of the former and in earth-cut graves in the case of the latter. The relationships between bone dimensions and dental age are described using a polynomial regression procedure, and analysis of regression residuals is used to evaluate differences in bone dimension-for-dental age between the two status groups. Results show that lower socioeconomic status individuals had lower cortical thickness-for-dental age than those of higher status. This was interpreted as likely reflecting poorer nutrition in the children of lower socioeconomic backgrounds. There was no patterning with respect to socioeconomic status in femur diaphyseal length or midshaft width. The results support the idea that, for skeletal populations, growth in cortical thickness may be a more sensitive indicator of adverse conditions in childhood than growth in bone length or width.

Bone morphogenetic protein–2 activity is regulated by secreted phosphoprotein–24 kd, an extracellular pseudoreceptor, the gene for which maps to a region of the human genome important for bone quality

The material properties of bone are the sum of the complex and interrelated anabolic and catabolic processes that modulate formation and turnover. The 2q33-37 region of the human genome contains quantitative trait loci important in determining the broadband ultrasound attenuation (an index of trabecular microarchitecture, bone elasticity, and susceptibility to fracture) of the calcaneus, but no genes of significance to bone metabolism have been identified in this domain. Secreted phosphoprotein–24 kd ( SPP24 or SPP2) is a novel and relatively poorly characterized growth hormone–regulated gene that maps to 2q37. The purpose of this review is to summarize the status of research related to spp24 and how it regulates bone morphogenetic protein (BMP) bioactivity in bone. SPP24 codes for an extracellular matrix protein that contains a high-affinity BMP-2–binding transforming growth factor– β receptor II homology 1 loop similar to those identified in fetuin and the receptor itself. SPP24 is transcribed primarily in the liver and bone. High levels of spp24 (a hydroxyapatite-binding protein) are found in bone, and small amounts are found in fetuin-mineral complexes. Full-length secretory spp24 inhibits ectopic bone formation, and overexpression of spp24 reduces murine bone mass and density. Spp24 is extremely labile to proteolysis, a process that regulates its bioactivity in vivo. For example, an 18.5-kd degradation product of spp24, designated spp18.5, is pro-osteogenic. A synthetic cyclized Cys 1-to-Cys 19 disulfide-bonded peptide (BMP binding peptide) corresponding to the transforming growth factor– β receptor II homology 1 domain of spp24 and spp18.5 binds BMP-2 and increases the rate and magnitude of BMP-2–mediated ectopic bone formation. Thus, the mechanism of action of spp18.5 and spp24 may be to regulate the local bioavailability of BMP cytokines. SPP24 is regulated by growth hormone and 3 major families of transcription factors (nuclear factor of activated T cells, CCAAT/enhancer-binding protein, Cut/Cux/CCAAT displacement protein) that regulate mesenchymal cell proliferation, embryonic patterning, and terminal differentiation. The gene contains at least 2 single nucleotide polymorphisms. Given its mechanism of action and sequence variability, SPP24 may be an interesting candidate for future studies of the genetic regulation of bone mass, particularly during periods of BMP-mediated endochondral bone growth, development, and fracture healing.

The role of matrix metalloproteinases (MMP's) and osteoclasts during endochondral bone growth

The role of matrix metalloproteinases (MMP's) and osteoclasts during endochondral bone growth

α-MSH Counteracts the Suppressive Effect of Ultraviolet B on Nrf1/2 and Nrf-Dependent Gene Expression in Human Skin

α-MSH Counteracts the Suppressive Effect of Ultraviolet B on Nrf1/2 and Nrf-Dependent Gene Expression in Human Skin

Systemic Administration of C-Type Natriuretic Peptide as a Novel Therapeutic Strategy for Skeletal Dysplasias

Skeletal dysplasias are a group of genetic disorders characterized by severe impairment of bone growth. Various forms of them add to produce a significant morbidity and mortality, yet no efficient drug therapy has been developed to date. We previously demonstrated that C-type natriuretic peptide (CNP), a member of the natriuretic peptide family, is a potent stimulator of endochondral bone growth. Furthermore, we exhibited that targeted overexpression of a CNP transgene in the growth plate rescued the impaired bone growth observed in a mouse model of achondroplasia (Ach), the most frequent form of human skeletal dysplasias, leading us to propose that CNP may prove to be an effective treatment for this disorder. In the present study, to elucidate whether or not the systemic administration of CNP is a novel drug therapy for skeletal dysplasias, we have investigated the effects of plasma CNP on impaired bone growth in Ach mice that specifically overexpress CNP in the liver under the control of human serum amyloid P component promoter or in those treated with a continuous CNP infusion system. Our results demonstrated that increased plasma CNP from the liver or by iv administration of synthetic CNP-22 rescued the impaired bone growth phenotype of Ach mice without significant adverse effects. These results indicate that treatment with systemic CNP is a potential therapeutic strategy for skeletal dysplasias, including Ach, in humans.

Systemic Administration of C-Type Natriuretic Peptide as a Novel Therapeutic Strategy for Skeletal Dysplasias

Skeletal dysplasias are a group of genetic disorders characterized by severe impairment of bone growth. Various forms of them add to produce a significant morbidity and mortality, yet no efficient drug therapy has been developed to date. We previously demonstrated that C-type natriuretic peptide (CNP), a member of the natriuretic peptide family, is a potent stimulator of endochondral bone growth. Further we exhibited that targeted overexpression of a CNP transgene in the growth plate rescued the impaired bone growth observed in mice model of achondroplasia (Ach), the most frequent form of human skeletal dysplasias, leading us to propose that CNP may prove to be an effective treatment for this disorder. In the present study, in order to elucidate whether or not the systemic administration of CNP is the novel drug therapy for skeletal dysplasias, we have investigated the effects of plasma CNP on impaired bone growth in Ach mice that specifically overexpress CNP in the liver under the control of human serum amyloid P component promoter, or in those treated with a continuous CNP infusion system. Our results demonstrated that increased plasma CNP from the liver or by intravenous administration of synthetic CNP-22 rescued the impaired bone growth phenotype of Ach mice without significant adverse effects. These results indicate that treatment with systemic CNP is a potential therapeutic strategy for skeletal dysplasias, including Ach, in humans.

Bone growth during rapamycin therapy in young rats

BackgroundRapamycin is an effective immunosuppressant widely used to maintain the renal allograft in pediatric patients. Linear growth may be adversely affected in young children since rapamycin has potent anti-proliferative and anti-angiogenic properties.MethodsWeanling three week old rats were given rapamycin at 2.5 mg/kg daily by gavage for 2 or 4 weeks and compared to a Control group given equivalent amount of saline. Morphometric measurements and biochemical determinations for serum calcium, phosphate, iPTH, urea nitrogen, creatinine and insulin-growth factor I (IGF-I) were obtained. Histomorphometric analysis of the growth plate cartilage, in-situ hybridization experiments and immunohistochemical studies for various proteins were performed to evaluate for chondrocyte proliferation, chondrocyte differentiation and chondro/osteoclastic resorption.ResultsAt the end of the 2 weeks, body and tibia length measurements were shorter after rapamycin therapy associated with an enlargement of the hypertrophic zone in the growth plate cartilage. There was a decrease in chondrocyte proliferation assessed by histone-4 and mammalian target of rapamycin (mTOR) expression. A reduction in parathyroid hormone/parathyroid hormone related peptide (PTH/PTHrP) and an increase in Indian hedgehog (Ihh) expression may explain in part, the increase number of hypertrophic chondrocytes. The number of TRAP positive multinucleated chondro/osteoclasts declined in the chondro-osseous junction with a decrease in the receptor activator of nuclear factor kappa β ligand (RANKL) and vascular endothelial growth factor (VEGF) expression. Although body and tibial length remained short after 4 weeks of rapamycin, changes in the expression of chondrocyte proliferation, chondrocyte differentiation and chondro/osteoclastic resorption which were significant after 2 weeks of rapamycin improved at the end of 4 weeks.ConclusionWhen given to young rats, 2 weeks of rapamycin significantly decreased endochondral bone growth. No catch-up growth was demonstrated at the end of 4 weeks, although markers of chondrocyte proliferation and differentiation improved. Clinical studies need to be done to evaluate these changes in growing children.