Stanniocalcin 1 acts as a paracrine regulator of growth plate chondrogenesis

During embryogenesis, the expression of mammalian stanniocalcin (STC1) in the appendicular skeleton suggests its involvement in the regulation of longitudinal bone growth. Such a role is further supported by the presence of dwarfism in mice overexpressing STC1. Yet, the STC 1 inhibitory effect on growth may be related to both postnatal metabolic abnormalities and prenatal defective bone formation. In our study, we used an organ culture system to evaluate the effects of STC on growth plate chondrogenesis, which is the primary determinant of longitudinal bone growth. Fetal rat metatarsal bones were cultured in the presence of recombinant human STC (rhSTC). After 3 days, rhSTC suppressed metatarsal growth, growth plate chondrocyte proliferation and hypertrophy/differentiation, and extracellular matrix synthesis. In addition, rhSTC increased the number of apoptotic chondrocytes in the growth plate. In cultured chondrocytes, rhSTC increased phosphate uptake, reduced chondrocyte proliferation and matrix synthesis, and induced apoptosis. All these effects were reversed by culturing chondrocytes with rhSTC and phosphonoformic acid, an inhibitor of phosphate transport. The rhSTC-mediated inhibition of metatarsal growth and growth plate chondrocyte proliferation and hypertrophy/differentiation was abolished by culturing metatarsals with rhSTC and phosphonoformic acid. Taken together, our findings indicate that STC1 inhibits longitudinal bone growth directly at the growth plate. Such growth inhibition, likely mediated by an increased chondrocyte phosphate uptake, results from suppressed chondrocyte proliferation, hypertrophy/differentiation, and matrix synthesis and by increased apoptosis. Last, the expression of both STC1 and its binding site in the growth plate would support an autocrine/paracrine role for this growth factor in the regulation of growth plate chondrogenesis.     

Bortezomib Is Cytotoxic to the Human Growth Plate and Permanently Impairs Bone Growth in Young Mice

Bortezomib, a novel proteasome inhibitor approved for the treatment of cancer in adults, has recently been introduced in pediatric clinical trials. Any tissue-specific side effects on bone development have to our knowledge not yet been explored. To address this, we experimentally studied the effects of bortezomib in vivo in young mice and in vitro in organ cultures of rat metatarsal bones and human growth plate cartilage, as well as in a rat chondrocytic cell line. We found that bortezomib while efficiently blocking the ubiquitin/proteasome system (UPS) caused significant growth impairment in mice, by increasing resting/stem-like chondrocyte apoptosis. Our data support a local action of bortezomib, directly targeting growth plate chondrocytes leading to decreased bone growth since no suppression of serum levels of insulin-like growth factor-I (IGF-I) was observed. A local effect of bortezomib was confirmed in cultured rat metatarsal bones where bortezomib efficiently caused growth retardation in a dose dependent and irreversible manner, an effect linked to increased chondrocyte apoptosis, mainly of resting/stem-like chondrocytes. The cytotoxicity of bortezomib was also evaluated in a unique model of cultured human growth plate cartilage, which was found to be highly sensitive to bortezomib. Mechanistic studies of apoptotic pathways indicated that bortezomib induced activation of p53 and Bax, as well as cleavage of caspases and poly-ADP-ribose polymerase (PARP) in exposed chondrocytes. Our observations, confirmed in vivo and in vitro, suggest that bone growth could potentially be suppressed in children treated with bortezomib. We therefore propose that longitudinal bone growth should be closely monitored in ongoing clinical pediatric trials of this promising anti-cancer drug.     

Site-1 protease is essential to growth plate maintenance and is a critical regulator of chondrocyte hypertrophic differentiation in postnatal mice

Site-1 protease (S1P) is a proprotein convertase with essential functions in lipid homeostasis and unfolded protein response pathways. We previously studied a mouse model of cartilage-specific knock-out of S1P in chondroprogenitor cells. These mice exhibited a defective cartilage matrix devoid of type II collagen protein (Col II) and displayed chondrodysplasia with no endochondral bone formation even though the molecular program for endochondral bone development appeared intact. To gain insights into S1P function, we generated and studied a mouse model in which S1P is ablated in postnatal chondrocytes. Postnatal ablation of S1P results in chondrodysplasia. However, unlike early embryonic ablations, the growth plates of these mice exhibit a lack of Ihh, PTHrP-R, and Col10 expression indicating a loss of chondrocyte hypertrophic differentiation and thus disruption of the molecular program required for endochondral bone development. S1P ablation results in rapid growth plate disruption due to intracellular Col II entrapment concomitant with loss of chondrocyte hypertrophy suggesting that these two processes are related. Entrapment of Col II in the chondrocytes of the prospective secondary ossification center precludes its development. Trabecular bone formation is dramatically diminished in the primary spongiosa and is eventually lost. The primary growth plate is eradicated by apoptosis but is gradually replaced by a fully functional new growth plate from progenitor stem cells capable of supporting new bone growth. Our study thus demonstrates that S1P has fundamental roles in the preservation of postnatal growth plate through chondrocyte differentiation and Col II deposition and functions to couple growth plate maturation to trabecular bone development in growing mice.     

Impairment of bone healing by insulin receptor substrate-1 deficiency

Insulin receptor substrate-1 (IRS-1) is an essential molecule for intracellular signaling of insulin-like growth factor (IGF)-I and insulin, both of which are potent anabolic regulators of bone and cartilage metabolism. To investigate the role of IRS-1 in bone regeneration, fracture was introduced in the tibia, and its healing was compared between wild-type (WT) mice and mice lacking the IRS-1 gene (IRS-1(-/-) mice). Among 15 IRS-1(-/-) mice, 12 remained in a non-union state even at 10 weeks after the operation, whereas all 15 WT mice showed a rigid bone union at 3 weeks. This impairment was because of the suppression of callus formation with a decrease in chondrocyte proliferation and increases in hypertrophic differentiation and apoptosis. Reintroduction of IRS-1 to the IRS-1(-/-) fractured site using an adenovirus vector significantly restored the callus formation. In the culture of chondrocytes isolated from the mouse growth plate, IRS-1(-/-) chondrocytes showed less mitogenic ability and Akt phosphorylation than WT chondrocytes. An Akt inhibitor decreased the IGF-I-stimulated DNA synthesis of chondrocytes more potently in the WT culture than in the IRS-1(-/-) culture. We therefore conclude that IRS-1 deficiency impairs bone healing at least partly by inhibiting chondrocyte proliferation through the phosphatidylinositol 3-kinase/Akt pathway, and we propose that IRS-1 can be a target molecule for bone regenerative medicine.     

Smad4 is required for the normal organization of the cartilage growth plate

Smad4 is the central intracellular mediator of transforming growth factor-beta (TGF-beta) signals. To study the role of Smad4 in skeletal development, we introduced a conditional mutation of the gene in chondrocytes using Cre--loxP system. We showed that Smad4 was expressed strongly in prehypertrophic and hypertrophic chondrocytes. The abrogation of Smad4 in chondrocytes resulted in dwarfism with a severely disorganized growth plate characterized by expanded resting zone of chondrocytes, reduced chondrocyte proliferation, accelerated hypertrophic differentiation, increased apoptosis and ectopic bone collars in perichondrium. Meanwhile, Smad4 mutant mice exhibited decreased expression of molecules in Indian hedgehog/parathyroid hormone-related protein (Ihh/PTHrP) signaling. The cultured mutant metatarsal bones failed to response to TGF-beta1, while the hypertrophic differentiation was largely inhibited by Sonic hedgehog (Shh). This indicated that Ihh/PTHrP inhibited the hypertrophic differentiation of chondrocytes independent of the Smad4-mediated TGF-beta signals. All these data provided the first genetic evidence demonstrating that Smad4-mediated TGF-beta signals inhibit the chondrocyte hypertrophic differentiation, and are required for maintaining the normal organization of chondrocytes in the growth plate.     

Phosphate regulates embryonic endochondral bone development

Phosphate is required for terminal differentiation of hypertrophic chondrocytes during postnatal growth plate maturation. In vitro models of chondrocyte differentiation demonstrate that 7 mM phosphate, a concentration analogous to that of the late gestational fetus, activates the mitochondrial apoptotic pathway in hypertrophic chondrocytes. This raises the question as to whether extracellular phosphate modulates chondrocyte differentiation and apoptosis during embryonic endochondral bone formation. To address this question, we performed investigations in the mouse metatarsal culture model that recapitulates in vivo bone development. Metatarsals were cultured for 4, 8, and 12 days with 1.25 and 7 mM phosphate. Metatarsals cultured with 7 mM phosphate showed a decrease in proliferation compared to those cultured in 1.25 mM phosphate. This decrease in proliferation was accompanied by an early enhancement in hypertrophic chondrocyte differentiation, associated with an increase in FGF18 expression. By 8 days in culture, an increase caspase‐9 activation and apoptosis of hypertrophic chondrocytes was observed in the metatarsals cultured in 7 mM phosphate. Immunohistochemical analyses of embryonic bones demonstrated activation of caspase‐9 in hypertrophic chondrocytes, associated with vascular invasion. Thus, these investigations demonstrate that phosphate promotes chondrocyte differentiation during embryonic development and implicate a physiological role for phosphate activation of the mitochondrial apoptotic pathway during embryonic endochondral bone formation. J. Cell. Biochem. 108: 668–674, 2009. © 2009 Wiley‐Liss, Inc.     

Resveratrol Treatment Delays Growth Plate Fusion and Improves Bone Growth in Female Rabbits

Trans-resveratrol (RES), naturally produced by many plants, has a structure similar to synthetic estrogen diethylstilbestrol, but any effect on bone growth has not yet been clarified. Pre-pubertal ovary-intact New Zealand white rabbits received daily oral administration of either vehicle (control) or RES (200 mg/kg) until growth plate fusion occurred. Bone growth and growth plate size were longitudinally monitored by X-ray imaging, while at the endpoint, bone length was assessed by a digital caliper. In addition, pubertal ovariectomized (OVX) rabbits were treated with vehicle, RES or estradiol cypionate (positive control) for 7 or 10 weeks and fetal rat metatarsal bones were cultured in vitro with RES (0.03 µM–50 µM) and followed for up to 19 days. In ovary-intact rabbits, sixteen-week treatment with RES increased tibiae and vertebrae bone growth and subsequently improved final length. In OVX rabbits, RES delayed fusion of the distal tibia, distal femur and proximal tibia epiphyses and femur length and vertebral bone growth increased when compared with controls. Histomorphometrical analysis showed that RES-treated OVX rabbits had a wider distal femur growth plate, enlarged resting zone, increased number/size of hypertrophic chondrocytes, increased height of the hypertrophic zone, and suppressed chondrocyte expression of VEGF and laminin. In cultured fetal rat metatarsal bones, RES stimulated growth at 0.3 µM while at higher concentrations (10 μM and 50 μM) growth was inhibited. We conclude that RES has the potential to improve longitudinal bone growth. The effect was associated with a delay of growth plate fusion resulting in increased final length. These effects were accompanied by a profound suppression of VEGF and laminin expression suggesting that impairment of growth plate vascularization might be an underlying mechanism.     

Annexin V/beta5 integrin interactions regulate apoptosis of growth plate chondrocytes

Apoptosis of terminally differentiated chondrocytes allows the replacement of growth plate cartilage by bone. Despite its importance, little is known about the regulation of chondrocyte apoptosis. We show that overexpression of annexin V, which binds to the cytoplasmic domain of beta5 integrin and protein kinase C alpha (PKCalpha), stimulates apoptotic events in hypertrophic growth plate chondrocytes. To determine whether the balance between the interactions of annexin V/beta5 integrin and annexin V/active PKCalpha play a role in the regulation of terminally differentiated growth plate chondrocyte apoptosis, a peptide mimic of annexin V (Penetratin (Pen)-VVISYSMPD) that binds to beta5 integrin but not to PKCalpha was used. This peptide stimulated apoptotic events in growth plate chondrocytes. Suppression of annexin V expression using small interfering ribonucleic acid decreased caspase-3 activity and increased cell viability in Pen-VVISYSMPD-treated growth plate chondrocytes. An activator of PKC resulted in a further decrease of cell viability and further increase of caspase-3 activity in Pen-VVISYSMPD-treated growth plate chondrocytes, whereas inhibitors of PKCalpha led to an increase of cell viability and decrease of caspase-3 activity of Pen-VVISYSMPD-treated cells. These findings suggest that binding of annexin V to active PKCalpha stimulates apoptotic events in growth plate chondrocytes and that binding of annexin Vto beta5 integrin controls these interactions and ultimately apoptosis.     

Uncoupling of chondrocyte death and vascular invasion in mouse galectin 3 null mutant bones

Galectin 3 is a beta-galactoside binding protein which localizes to the cytoplasm of proliferative, mature, and hypertrophic chondrocytes in the growth plate cartilage of developing long bones. To elucidate the function of galectin 3 during bone development, we examined the epiphyseal femurs and tibias of fetal mice carrying a null mutation for the galectin 3 gene. Detailed histological and ultrastructural studies identified abnormalities in the cells of the proliferative, mature, and hypertrophic zones and in the extracellular matrix of the hypertrophic zone, as well as a reduction in the total number of hypertrophic chondrocytes. The expression patterns of several chondrocyte and bone cell markers were analyzed and revealed a subtle modification of Ihh expression in the galectin 3 mutant growth plate. A striking difference was observed at the chondrovascular junction where many empty lacunae are present. In addition, large numbers of condensed chondrocytes exhibiting characteristic signs of cell death were found in the late hypertrophic zone, indicating that the rate of chondrocyte death is increased in the mutants. These results suggest a role for galectin 3 as a regulator of chondrocyte survival. In addition, this unique phenotype shows that the elimination of chondrocytes and vascular invasion can be uncoupled and indicates that galectin 3 may play a role in the coordination between chondrocyte death and metaphyseal vascularization.     

Synthesis and secretion of Ch 21 protein in embryonic chick skeletal tissues

We reported the identification, purification and characterization of a low molecular weight protein (Ch 21) expressed in vitro by differentiating chondrocytes at a late stage of development and observed in vivo in the growth plate region of the long bones at the border between hypertrophic cartilage and newly formed bone (Descalzi Cancedda, F., P. Manduca, C. Tacchetti, P. Fossa, R. Quarto, R. Cancedda, J. Cell Biol. 107, 2455-2463 (1988]. In this article, the synthesis and location of Ch 21 protein in the chick embryo tibia at late stage of development were further investigated. Ch 21 was observed in the cartilage matrix surrounding marrow cavities and in the prearticular outer layer by immunolocalization. In addition, the timing of Ch 21 appearance during the tibia development and its distribution in the growth plate region was better defined. We first observed presence of Ch 21 in the perichondral mid-diaphyseal sleeve of 7-day-old tibia. Ch 21 antibodies stained also the newly formed bone. Synthesis and secretion in the culture medium of Ch 21 protein was observed when bone fragments or cultured osteoblasts isolated from 19-day-old embryo tibiae were labeled in vitro. A search for the presence of Ch 21 in the chick embryo sternum was performed. The synthesis of Ch 21, both in the presumptive calcification cranial portion and in the permanent cartilaginous caudal portion of the sternum, was shown by metabolic labeling of tissue slices.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proepithelin stimulates growth plate chondrogenesis via nuclear factor-kappaB-p65-dependent mechanisms

Proepithelin, a previously unrecognized growth factor in cartilage, has recently emerged as an important regulator for cartilage formation and function. In the present study, we provide several lines of evidences in proepithelin-mediated induction of cell proliferation, differentiation, and apoptosis in the metatarsal growth plate. Proepithelin-mediated stimulation of metatarsal growth and growth plate chondrogenesis was neutralized by pyrrolidine dithiocarbamate, a known NF-κB inhibitor. In rat growth plate chondrocytes, proepithelin induced NF-κB-p65 nuclear translocation, and nuclear NF-κB-p65 initiated its target gene cyclin D1 to regulate chondrocyte functions. The inhibition of NF-κB-p65 expression and activity (by p65 short interfering RNA (siRNA) and pyrrolidine dithiocarbamate, respectively) in chondrocytes reversed the proepithelin-mediated induction of cell proliferation and differentiation and the proepithelin-mediated prevention of cell apoptosis. Moreover, the inhibition of the phosphatidylinositol 3-kinase and Akt abolished the effects of proepithelin on NF-κB activation. Finally, using siRNA and antisense strategies, we demonstrated that endogenously produced proepithelin by chondrocytes is important for chondrocyte growth in serum-deprived conditions. These results support the hypothesis that the induction of NF-κB activity of in growth plate chondrocytes is critical in proepithelin-mediated growth plate chondrogenesis and longitudinal bone growth.     

Stimulation of chondrocyte hypertrophy by chemokine stromal cell-derived factor 1 in the chondro-osseous junction during endochondral bone formation

During endochondral bone formation, chondrocytes undergo differentiation toward hypertrophy before they are replaced by bone and bone marrow. In this study, we found that a G-protein coupled receptor CXCR4 is predominantly expressed in hypertrophic chondrocytes, while its ligand, chemokine stromal cell-derived factor 1 (SDF-1) is expressed in the bone marrow adjacent to hypertrophic chondrocytes. Thus, they are expressed in a complementary pattern in the chondro-osseous junction of the growth plate. Transfection of a CXCR4 cDNA into pre-hypertrophic chondrocytes results in a dose-dependent increase of hypertrophic markers including Runx2, Col X, and MMP-13 in response to SDF-1 treatment. In organ culture SDF-1 infiltrates cartilage and accelerates growth plate hypertrophy. Furthermore, a continuous infusion of SDF-1 into the rabbit proximal tibial physis results in early physeal closure, which is accompanied by a transient elevation of type X collagen expression. Blocking SDF-1/CXCR4 interaction suppresses the expression of Runx2. Thus, interaction of SDF-1 and CXCR4 is required for Runx2 expression. Interestingly, knocking down Runx2 gene expression results in a decrease of CXCR4 mRNA levels in hypertrophic chondrocytes. This suggests a positive feedback loop of stimulation of chondrocyte hypertrophy by SDF-1/CXCR4, which is mediated by Runx2.     

Chondrocyte hypertrophy in skeletal development,growth, and disease

Most of our bones form through the process of endochondral ossification, which is tightly regulated by the activity of the cartilage growth plate. Chondrocyte maturation through the various stages of growth plate physiology ultimately results in hypertrophy. Chondrocyte hypertrophy is an essential contributor to longitudinal bone growth, but recent data suggest that these cells also play fundamental roles in signaling to other skeletal cells, thus coordinating endochondral ossification. On the other hand, ectopic hypertrophy of articular chondrocytes has been implicated in the pathogenesis of osteoarthritis. Thus, a better understanding of the processes that control chondrocyte hypertrophy in the growth plate as well as in articular cartilage is required for improved management of both skeletal growth disorders and osteoarthritis. This review summarizes recent findings on the regulation of hypertrophic chondrocyte differentiation, the cellular mechanisms involved in hypertrophy, and the role of chondrocyte hypertrophy in skeletal physiology and pathophysiology. Birth Defects Research (Part C) 102:74–82, 2014. © 2014 Wiley Periodicals, Inc.     

Fit optimisation of a distal medial tibia plate

An iterative method for the fit optimisation of a pre-contoured fracture fixation plate for a given bone data set is presented. Both plate shape optimisation and plate fit quantification are conducted in a virtual environment utilising computer graphical methods and 3D bone and plate models. Two optimised shapes of the undersurface of an existing distal medial tibia plate were generated based on a dataset of 45 3D bone models reconstructed from computed tomography image data of Japanese tibiae. The existing plate shape achieved an anatomical fit on 13% of tibiae from the dataset. Modified plate 1 achieved an anatomical fit for 42% and modified plate 2 a fit for 67% of the bones. If either modified plate 1 or plate 2 is used, then the anatomical fit can be increased to 82% for the same dataset. Issues pertaining to any further improvement in plate fit/shape are discussed.