Growth Plate Defects Research Articles

Mef2c regulates bone mass through Sost-dependent and -independent mechanisms

Mef2c is a transcription factor that mediates key cellular behaviors that promote endochondral ossification and bone formation. Previously, Mef2c has been shown to regulate Sost transcription via its osteocyte-specific enhancer, ECR5, and conditional deletions of Mef2cfl/fl with either Col1-Cre or Dmp1-Cre produced generalized high bone mass (HBM) consistent with Van Buchem Disease phenotypes. However, Sost−/−; Mef2cfl/fl; Dmp1-Cre mice produced a significantly higher bone mass phenotype that Sost−/− alone suggesting that Mef2c modulates bone mass through additional mechanisms, independent of Sost. To identify new Mef2c transcriptional targets important in bone metabolism, we profiled gene expression by single-cell RNA sequencing in subpopulations of cells isolated from Mef2cfl/fl; Dmp1-Cre and Mef2cfl/fl; Bglap-Cre femurs, both strains exhibiting similar high bone mass phenotypes. However, we found Mef2cfl/fl; Bglap-Cre to also display a growth plate defect characterized by an expansion of several osteoprogenitor subpopulations. Differential gene expression analysis identified a total of 96 up- and 2434 down- regulated genes in Mef2cfl/fl; Bglap-Cre and 176 up- and 1041 down- regulated genes in Mef2cfl/fl; Dmp1-Cre bone cell subpopulations compared to wildtype mice. Mef2c deletion affected the transcriptomes across several cell types including mesenchymal progenitors (MP), osteoprogenitors (OSP), osteoblast (OB), and osteocyte (OCY) subpopulations. Several energy metabolism genes such as Uqcrb, Ndufv2, Ndufs3, Ndufa13, Ndufb9, Ndufb5, Cox6a1, Cox5a, Atp5o, Atp5g2, Atp5b, Atp5 were significantly down regulated in Mef2c-deficient OBs and OCYs, in both strains. Binding motif analysis of promoter regions of differentially expressed genes identified Mef2c binding in Bone Sialoprotein (BSP/Ibsp), a gene known to cause increased trabecular BV/TV in the femurs of Ibsp−/− mice. Immunohistochemical analysis confirmed the absence of Ibsp protein in OBs and OCYs. These findings suggests that the HBM in Sost−/−; Mef2cfl/fl; Dmp1-Cre is caused by a multitude of transcriptional changes in genes that regulate bone formation, two of which are Sost and Ibsp.

Suppressor of fused-restrained Hedgehog signaling in chondrocytes is critical for epiphyseal growth plate maintenance and limb elongation in juvenile mice.

Hedgehog (Hh) signaling plays multiple critical roles in regulating chondrocyte proliferation and differentiation during epiphyseal cartilage development. However, it is still unclear whether Hh signaling in chondrocytes is required for growth plate maintenance during juvenile growth, and whether sustained activation of Hh signaling in chondrocytes promotes limb elongation. In this study, we first utilized Hh reporter mice to reveal that Hh signaling was activated in resting and columnar chondrocytes in growth plates of juvenile and adult mice. Next, we genetically modulated Hh signaling by conditionally deleting Smo or Sufu in all or a subpopulation of growth plate chondrocytes, and found that ablation of either Smo or Sufu in chondrocytes of juvenile mice caused premature closure of growth plates and shorter limbs, whereas Osx-Cre-mediated deletion of either of these two genes in prehypertrophic chondrocytes did not lead to obvious growth plate defects, indicating that Hh signaling mainly functions in resting and/or columnar chondrocytes to maintain growth plates at the juvenile stage. At the cellular level, we found that chondrocyte-specific ablation of Smo or Sufu accelerated or suppressed chondrocyte hypertrophy, respectively, whereas both decreased chondrocyte proliferation and survival. Thus, our study provided the first genetic evidence to establish the essential cell-autonomous roles for tightly-regulated Hh signaling in epiphyseal growth plate maintenance and limb elongation during juvenile growth.

Mesenchymal Stem Cell Exosomes Promote Growth Plate Repair and Reduce Limb-Length Discrepancy in Young Rats.

The objective of this study was to examine the therapeutic effects of human mesenchymal stromal/stem cell (MSC) exosomes in a rat model of growth plate injury. A growth plate defect was surgically created on the distal part of the right femur of 40 female Sprague-Dawley rats. A single intra-articular injection of 100 µg of MSC exosomes in 100 µL of phosphate-buffered saline solution (PBS), or an equivalent volume of PBS alone, was administered to the right knee immediately after surgery. At 4 and 8 weeks post-treatment, limb length was measured with micro-CT, and tissue repair was assessed with histological, immunohistochemical, and histomorphometric analyses. A single injection of MSC exosomes significantly increased limb length from 3.29 ± 0.07 cm at 4 weeks to 3.37 ± 0.11 cm at 8 weeks (p = 0.047). However, no improvement in limb length was observed in the PBS control group. The limb-length discrepancy between the involved limb and the contralateral limb in the exosome-treated group was significantly less than the discrepancy in the PBS-treated group at both 4 weeks (2.52% ± 1.30% versus 4.11% ± 0.93%; p = 0.006) and 8 weeks (5.27% ± 2.11% versus 8.06% ± 2.56%; p = 0.016). Consistent with the reduced limb-length discrepancy, the exosome-treated defects displayed significantly more chondrocytes (p < 0.05) and a higher area percentage with deposition of sulphated glycosaminoglycan (p < 0.05) and collagen II (p < 0.05) than PBS-treated defects at 8 weeks. However, bone bridge formation was not inhibited in either group. A single intra-articular injection of MSC exosomes significantly enhanced physeal repair and reduced limb-length discrepancy but did not inhibit bone-bridge formation. This proof-of-concept study demonstrates for the first time the potential use of MSC exosomes as a minimally invasive cell-free therapeutic to promote physeal repair and reduce limb-length discrepancy following growth plate injuries.

Age-Progressive and Gender-Dependent Bone Phenotype in Mice Lacking Both Ebf1 and Ebf2 in Prrx1-Expressing Mesenchymal Cells

Ebfs are a family of transcription factors regulating the differentiation of multiple cell types of mesenchymal origin, including osteoblasts. Global deletion of Ebf1 results in increased bone formation and bone mass, while global loss of Ebf2 leads to enhanced bone resorption and decreased bone mass. Targeted deletion of Ebf1 in early committed osteoblasts leads to increased bone formation, whereas deletion in mature osteoblasts has no effect. To study the effects of Ebf2 specifically on long bone development, we created a limb bud mesenchyme targeted Ebf2 knockout mouse model by using paired related homeobox gene 1 (Prrx1) Cre. To investigate the possible interplay between Ebf1 and Ebf2, we deleted both Ebf1 and Ebf2 in the cells expressing Prrx1. Mice with Prrx1-targeted deletion of Ebf2 had a very mild bone phenotype. However, deletion of both Ebf1 and Ebf2 in mesenchymal lineage cells lead to significant, age progressive increase in bone volume. The phenotype was to some extent gender dependent, leading to an increase in both trabecular and cortical bone in females, while in males a mild cortical bone phenotype and a growth plate defect was observed. The phenotype was observed at both 6 and 12 weeks of age, but it was more pronounced in older female mice. Our data suggest that Ebfs modulate bone homeostasis and they are likely able to compensate for the lack of each other. The roles of Ebfs in bone formation appear to be complex and affected by multiple factors, such as age and gender.

Roles of apoptotic chondrocyte-derived CXCL12 in the enhanced chondroclast recruitment following methotrexate and/or dexamethasone treatment.

Intensive use of methotrexate (MTX) and/or dexamethasone (DEX) for treating childhood malignancies is known to cause chondrocyte apoptosis and growth plate dysfunction leading to bone growth impairments. However, mechanisms remain vague and it is unclear whether MTX and DEX combination treatment could have additive effects in the growth plate defects. In this study, significant cell apoptosis was induced in mature ATDC5 chondrocytes after treatment for 48 h with 10-5 M MTX and/or 10-6 M DEX treatment. PCR array assays with treated cells plus messenger RNA and protein expression confirmation analyses identified chemokine CXCL12 having the most prominent induction in each treatment group. Conditioned medium from treated chondrocytes stimulated migration of RAW264.7 osteoclast precursor cells and formation of osteoclasts, and these stimulating effects were inhibited by the neutralizing antibody for CXCL12. Additionally, while MTX and DEX combination treatment showed some additive effects on apoptosis induction, it did not have additive or counteractive effects on CXCL12 expression and its functions in enhancing osteoclastic recruitment and formation. In young rats treated acutely with MTX, there was increased expression of CXCL12 in the tibial growth plate, and more resorbing chondroclasts were found present at the border between the hypertrophic growth plate and metaphysis bone. Thus, the present study showed an association between induced chondrocyte apoptosis and stimulated osteoclastic migration and formation following MTX and/or DEX treatment, which could be potentially or at least partially linked molecularly by CXCL12 induction. This finding may contribute to an enhanced mechanistic understanding of bone growth impairments following MTX and/or DEX therapy.

Targeted truncation of the ADAM17 cytoplasmic domain in mice results in protein destabilization and a hypomorphic phenotype

A disintegrin and metalloprotease 17 (ADAM17) is a cell-surface metalloprotease that serves as the principle sheddase for tumor necrosis factor α (TNFα), interleukin-6 receptor (IL-6R), and several ligands of the epidermal growth factor receptor (EGFR), regulating these crucial signaling pathways. ADAM17 activation requires its transmembrane domain, but not its cytoplasmic domain, and little is known about the role of this domain in vivo. To investigate, we used CRISPR-Cas9 to mutate the endogenous Adam17 locus in mice to produce a mutant ADAM17 lacking its cytoplasmic domain (Adam17Δcyto). Homozygous Adam17Δcyto animals were born at a Mendelian ratio and survived into adulthood with slightly wavy hair and curled whiskers, consistent with defects in ADAM17/EGFR signaling. At birth, Adam17Δcyto mice resembled Adam17−/− mice in that they had open eyes and enlarged semilunar heart valves, but they did not have bone growth plate defects. The deletion of the cytoplasmic domain resulted in strongly decreased ADAM17 protein levels in all tissues and cells examined, providing a likely cause for the hypomorphic phenotype. In functional assays, Adam17Δcyto mouse embryonic fibroblasts and bone-marrow-derived macrophages had strongly reduced ADAM17 activity, consistent with the reduced protein levels. Nevertheless, ADAM17Δcyto could be stimulated by PMA, a well-characterized posttranslational activator of ADAM17, corroborating that the cytoplasmic domain of endogenous ADAM17 is not required for its rapid response to PMA. Taken together, these results provide the first evidence that the cytoplasmic domain of ADAM17 plays a pivotal role in vivo in regulating ADAM17 levels and function.

A SMOC2 variant inhibits BMP signaling by competitively binding to BMPR1B and causes growth plate defects.

Endochondral ossification is the major process of long bone formation, and chondrogenesis is the final step of this process. Several studies have indicated that bone morphogenetic proteins (BMPs) are required for chondrogenesis and regulate multiple growth plate features. Abnormal BMP pathways lead to growth plate defects, resulting in osteochondrodysplasia. The SPARC-related modular calcium binding 2 (SMOC2) gene encodes an extracellular protein that is considered to be an antagonist of BMP signaling. In this study, we generated a mouse model by knocking-in the SMOC2 mutation (c.1076T>G), which showed short-limbed dwarfism, reduced, disorganized, and hypocellular proliferative zones and expanded hypertrophic zones in tibial growth plates. To determine the underlying pathophysiological mechanism of SMOC2 mutation, we used knock-in mice to investigate the interaction between SMOC2 and the BMP-SMAD1/5/9 signaling pathway in vivo and in vitro. Eventually, we found that mutant SMOC2 could not bind to COL9A1 and HSPG. Furthermore, mutant SMOC2 inhibited BMP signaling by competitively binding to BMPR1B, which lead to defects in growth plates and short-limbed dwarfism in knock-in mice.

High-Phosphate Diet Improved the Skeletal Development of Fam20c-Deficient Mice

FAM20C (family with sequence similarity 20 – member C) is a protein kinase that phosphorylates secretory proteins, including the proteins that are essential to the formation and mineralization of calcified tissues. Previously, we reported that inactivation of Fam20c in mice led to hypophosphatemic rickets/osteomalacia along with increased circulating fibroblast growth factor 23 (FGF23) levels and dental defects. In this study, we examined whether a high-phosphate (hPi) diet could rescue the skeletal defects in Fam20c-deficient mice. Fam20c conditional knockout (cKO) mice were generated by crossing female Fam20c-floxed mice (Fam20cfl/fl) with male Sox2-Cre;Fam20cfl/+ mice. The pregnant female Fam20cfi/fl mice were fed either a normal or hPi diet until the litters were weaned. The cKO and control offspring were continuously given a normal or hPi diet for 4 weeks after weaning. Plain X-ray radiography, micro-CT, histology, immunohistochemistry (FGF23, DMP1, OPN, and SOX9), and in situ hybridization (type II and type X collagen) analyses were performed to evaluate the effects of an hPi diet on the mouse skeleton. Plain X-ray radiography and micro-CT radiography analyses showed that the hPi diet improved the shape and mineral density of the Fam20c-deficient femurs/tibiae, and rescued the growth plate defects in the long bone. Histology analyses further demonstrated that an hPi diet nearly completely rescued the growth plate-widening defects in the long bone and restored the expanded hypertrophic zone to nearly normal width. These results suggested that the hPi diet significantly improved the skeletal development of the Fam20c-deficient mice, implying that hypophosphatemia partially contributed to the skeletal defects in Fam20c-deficient subjects.

Constitutively-active FGFR3 disrupts primary cilium length and IFT20 trafficking in various chondrocyte models of achondroplasia.

Fibroblast growth factor receptor 3 (FGFR3) gain-of-function mutations cause dwarfisms, including achondroplasia (ACH) and thanatophoric dysplasia (TD). The constitutive activation of FGFR3 disrupts the normal process of skeletal growth. Bone-growth anomalies have been identified in skeletal ciliopathies, in which primary cilia (PC) function is disrupted. In human ACH and TD, the impact of FGFR3 mutations on PC in growth plate cartilage remains unknown. Here we showed that in chondrocytes from human (ACH, TD) and mouse Fgfr3Y367C/+ cartilage, the constitutively active FGFR3 perturbed PC length and the sorting and trafficking of intraflagellar transport (IFT) 20 to the PC. We demonstrated that inhibiting FGFR3 with FGFR inhibitor, PD173074, rescued both PC length and IFT20 trafficking. We also studied the impact of rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) pathway. Interestingly, mTOR inhibition also rescued PC length and IFT20 trafficking. Together, we provide evidence that the growth plate defects ascribed to FGFR3-related dwarfisms are potentially due to loss of PC function, and these dwarfisms may represent a novel type of skeletal disorders with defective ciliogenesis.

Fibroblasts derived from patients with opsismodysplasia display SHIP2-specific cell migration and adhesion defects.

The SH2 domain containing inositol phosphatase 2 (SHIP2) dephosphorylates PI(3,4,5)P3 to generate PI(3,4)P2, a lipid involved in the control of cell migration and adhesion. The INPPL1 gene that encodes SHIP2 has been found to be mutated in several cases of opsismodysplasia (OPS), a rare autosomal recessive chondrodysplasia characterized by growth plate defects and delayed bone maturation. Reported mutations often result in premature stop codons or missense mutations in SHIP2 catalytic domain. SHIP2 biochemical properties are known from studies in cancer cells; its role in endochondral ossification is unknown. Here, we report two novel mutations in the INPPL1 gene and show that cell migration is very much decreased in fibroblasts derived from three OPS patients as compared with control individuals. In contrast, cell adhesion on fibronectin is increased in OPS fibroblasts. An inhibitory effect on migration was also observed when normal fibroblasts were incubated in the presence of a SHIP2 competitive inhibitor. We conclude that both migration and adhesion are very much disrupted in OPS-derived fibroblasts. It is suggested that signaling events linked to migration and particularly to adhesion, which are lost in OPS patients, would prevent normal endochondral ossification.

P38α MAPK Regulates Lineage Commitment and OPG Synthesis of Bone Marrow Stromal Cells to Prevent Bone Loss under Physiological and Pathological Conditions.

SummaryBone marrow-derived mesenchymal stromal cells (BM-MSCs) are capable of differentiating into osteoblasts, chondrocytes, and adipocytes. Skewed differentiation of BM-MSCs contributes to the pathogenesis of osteoporosis. Yet how BM-MSC lineage commitment is regulated remains unclear. We show that ablation of p38α in Prx1+ BM-MSCs produced osteoporotic phenotypes, growth plate defects, and increased bone marrow fat, secondary to biased BM-MSC differentiation from osteoblast/chondrocyte to adipocyte and increased osteoclastogenesis and bone resorption. p38α regulates BM-MSC osteogenic commitment through TAK1-NF-κB signaling and osteoclastogenesis through osteoprotegerin (OPG) production by BM-MSCs. Estrogen activates p38α to maintain OPG expression in BM-MSCs to preserve the bone. Ablation of p38α in BM-MSCs positive for Dermo1, a later BM-MSC marker, only affected osteogenic differentiation. Thus, p38α mitogen-activated protein kinase (MAPK) in Prx1+ BM-MSCs acts to preserve the bone by promoting osteogenic lineage commitment and sustaining OPG production. This study thus unravels previously unidentified roles for p38α MAPK in skeletal development and bone remodeling.

FGF signaling in the osteoprogenitor lineage non-autonomously regulates postnatal chondrocyte proliferation and skeletal growth.

Fibroblast growth factor (FGF) signaling is important for skeletal development; however, cell-specific functions, redundancy and feedback mechanisms regulating bone growth are poorly understood. FGF receptors 1 and 2 (Fgfr1 and Fgfr2) are both expressed in the osteoprogenitor lineage. Double conditional knockout mice, in which both receptors were inactivated using an osteoprogenitor-specific Cre driver, appeared normal at birth; however, these mice showed severe postnatal growth defects that include an ∼50% reduction in body weight and bone mass, and impaired longitudinal bone growth. Histological analysis showed reduced cortical and trabecular bone, suggesting cell-autonomous functions of FGF signaling during postnatal bone formation. Surprisingly, the double conditional knockout mice also showed growth plate defects and an arrest in chondrocyte proliferation. We provide genetic evidence of a non-cell-autonomous feedback pathway regulating Fgf9, Fgf18 and Pthlh expression, which led to increased expression and signaling of Fgfr3 in growth plate chondrocytes and suppression of chondrocyte proliferation. These observations show that FGF signaling in the osteoprogenitor lineage is obligately coupled to chondrocyte proliferation and the regulation of longitudinal bone growth.

Transplantation of a Scaffold-Free Cartilage Tissue Analogue for the Treatment of Physeal Cartilage Injury of the Proximal Tibia in Rabbits.

PurposeThe purpose of this study was to investigate the effects of transplantation of an in vitro-generated, scaffold-free, tissue-engineered cartilage tissue analogue (CTA) using a suspension chondrocyte culture in a rabbit growth-arrest model.Materials and MethodsWe harvested cartilage cells from the articular cartilage of the joints of white rabbits and made a CTA using a suspension culture of 2×107 cells/mL. An animal growth plate defect model was made on the medial side of the proximal tibial growth plate of both tibias of 6-week-old New Zealand white rabbits (n=10). The allogenic CTA was then transplanted onto the right proximal tibial defect. As a control, no implantation was performed on the left-side defect. Plain radiographs and the medial proximal tibial angle were obtained at 1-week intervals for evaluation of bone bridge formation and the degree of angular deformity until postoperative week 6. We performed a histological evaluation using hematoxylin-eosin and Alcian blue staining at postoperative weeks 4 and 6.ResultsRadiologic study revealed a median medial proximal tibial angle of 59.0° in the control group and 80.0° in the CTA group at 6 weeks. In the control group, statistically significant angular deformities were seen 3 weeks after transplantation (p<0.05). On histological examination, the transplanted CTA was maintained in the CTA group at 4 and 6 weeks postoperative. Bone bridge formation was observed in the control group.ConclusionIn this study, CTA transplantation minimized deformity in the rabbit growth plate injury model, probably via the attenuation of bone bridge formation.

Ingénierie tissulaire utilisant des cellules souches mésenchymateuses sur charpentes de Chitosan dans les lésions traumatiques de la plaque de croissance. Étude préliminaire chez le lapin

BackgroundGrowth plate injury in children could produce limb length discrepancy and angular deformity. Removal of damaged physis or bony bar and insertion of spacers produced variable results and for large defects in young children, the treatment is challenging. In this study, we used tissue-engineered mesenchymal stem cells (MSC-based Chitosan scaffold) for restoration of the damaged physis. The usage of Chitosan as a spacer was also investigated. Materials and methodsAn experimental model of growth arrest was created by removing lateral 50 % of distal femoral physis of fourteen 4-week-old albino rabbits. The left side growth plate defects were filled with MSC-based Chitosan scaffold in 10 and scaffold alone in 4 rabbits. For all the rabbits, right side defects were left alone as the control limb. After 3months, femoral bones were harvested and gross inspection and radiology for measurement of angulations were done; histological study for evaluation of regeneration of physis was also done. ResultsThe hemi-physeal resection procedures were successful and all of the operated limbs showed angular deformities. There was a trend toward less angular deformity in cases in which more concentration of MSCs with Chitosan scaffold was used. In cases of transfer of MSCs with concentration of less than 1.5 millions, mixed results were observed and angular deformities were not reduced. Transfer of Chitosan alone yielded poor results. ConclusionIn this study, we have developed an in vitro construction of a transplantable tissue-engineered disk, using natural Chitosan scaffold and MSCs. We investigated the efficacy of these disks for repairing the defect of growth plate cartilage at distal femoral physis. Our results showed that the beneficial effect of these cells on scaffold appeared in more concentration of cells. Level of evidenceLevel III. Low power comparative study.

Tissue engineering strategy using mesenchymal stem cell-based chitosan scafolds in growth plate surgery: A preliminary study in rabbits

BackgroundGrowth plate injury in children could produce limb length discrepancy and angular deformity. Removal of damaged physis or bony bar and insertion of spacers produced variable results and for large defects in young children, the treatment is challenging. In this study, we used tissue-engineered mesenchymal stem cells (MSC-based chitosan scaffold) for restoration of the damaged physis. The usage of chitosan as a spacer was also investigated. Materials and methodsAn experimental model of growth arrest was created by removing lateral 50% of distal femoral physis of fourteen 4-week-olds albino rabbits. The left side growth plate defects were filled with MSC-based chitosan scaffold in 10 and scaffold alone in 4 rabbits. For all the rabbits, right-side defects were left alone as the control limb. After 3 months, femoral bones were harvested and gross inspection and radiology for measurement of angulations were done; histological study for evaluation of regeneration of physis was also done. ResultsThe hemiphyseal resection procedures were successful and all of the operated limbs showed angular deformities. There was a trend toward less angular deformity in cases in which more concentration of MSCs with chitosan scaffold was used. In cases of transfer of MSCs with concentration of less than 1.5 millions, mixed results were observed and angular deformities were not reduced. Transfer of chitosan alone yielded poor results. ConclusionIn this study, we have developed an in vitro construction of a transplantable tissue-engineered disk, using natural chitosan scaffold and MSCs. We investigated the efficacy of these disks for repairing the defect of growth plate cartilage at distal femoral physis. Our results showed that the beneficial effect of these cells on scaffold appeared in more concentration of cells. Level of evidenceLevel III. Low power comparative study.

RECQL4 Regulates p53 Function In Vivo During Skeletogenesis

RECQ DNA helicases play critical roles in maintaining genomic stability, but their role in development has been less well studied. Rothmund-Thomson syndrome, RAPADILINO, and Baller-Gerold syndrome are rare genetic disorders caused by mutations in the RECQL4 gene. These patients have significant skeletal developmental abnormalities including radial ray, limb and craniofacial defects. To investigate the role of Recql4 in the developing skeletal system, we generated Recql4 conditional knockout mice targeting the skeletal lineage. Inactivation of Recql4 using the Prx1-Cre transgene led to limb abnormalities and craniosynostosis mimicking the major bone findings in human RECQL4 patients. These Prx1-Cre(+) ;Recql4(fl/fl) mice as well as Col2a1-Cre(+) ;Recql4(fl/fl) mice exhibited growth plate defects and an increased p53 response in affected tissues. Inactivation of Trp53 in these Recql4 mutants resulted in genetic rescue of the skeletal phenotypes, indicating an in vivo interaction between Recql4 and Trp53, and p53 activation as an underlying mechanism for the developmental bone abnormalities in RECQL4 disorders. Our findings show that RECQL4 is critical for skeletal development by modulating p53 activity in vivo.

Col10a1 gene expression and chondrocyte hypertrophy during skeletal development and disease

The type X collagen gene, COL10A1, is specifically expressed by hypertrophic chondrocytes during endochondral ossification. Endochondral ossification is a well-coordinated process that involves a cartilage intermediate and leads to formation of most of the skeleton in vertebrates during skeletogenesis. Chondrocyte hypertrophy is a critical stage of endochondral ossification linking both bone and cartilage development. Given its specific association with chondrocyte hypertrophy, type X collagen plays essential roles in endochondral ossification. It was previously shown that transgenic mice with mutant type X collagen develop variable skeleton-hematopoietic abnormalities indicating defective endochondral ossification, while mutations and abnormal expression of human COL10A1 cause abnormal chondrocyte hypertrophy that has been seen in many skeletal disorders, including skeletal chondrodysplasia and osteoarthritis. In this review, we summarized the skeletal chondrodysplasia with COL10A1 gene mutation that shows growth plate defect. We also reviewed recent studies that correlate the type X collagen gene expression and chondrocyte hypertrophy with osteoarthritis. Due to its significant clinical relevance, the type X collagen gene regulation has been extensively studied over the past two decades. Here, we focus on recent progress characterizing the cis-enhancer elements and their binding factors that together confer hypertrophic chondrocyte-specific murine type X collagen gene (Col10a1) expression. Based on literature review and our own studies, we surmise that there are multiple factors that contribute to hypertrophic chondrocyte-specific Col10a1 expression. These factors include both transactivators (such as Runx2, MEF2C etc.) and repressors (such as AP1, NFATc1, Sox9 etc.), while other co-factors or epigenetic control of Col10a1 expression may not be excluded.

Effect of thiram on avian growth plate chondrocytes in culture

Thiram is a dithiocarbamate pesticide that causes tibial dyschondroplasia (TD), a growth plate defect, in poultry. Deaths of transitional zone chondrocytes appear to interrupt endochondral bone development leading to the broadening of growth plate. The mechanism of action of thiram on chondrocytes is not well understood. Since proteins play major roles in different aspects of cell's metabolism, growth, and survival, the objective of this study was to find whether thiram produces proteomic changes that could impair the development of chondrocytes. The chondrocytes, isolated from proximal tibial growth plates, were cultured with or without a sub-lethal concentration of thiram for 48 hr, and the cell proteins were extracted, and subjected to 2-D gel electrophoresis. The gel images were compared and statistically evaluated using Melanie software to identify differentially expressed protein spots. Of a total of 72 identifiable spots 3 were down-regulated and 2 up-regulated in thiram treated chondrocytes. In-gel trypsin digestion of the protein spots followed by their characterization by matrix-assisted laser desorption ionization-time-of- flight (MALDI-TOF) mass spectrometry identified 25 spots comprising of 23 proteins. Two of 3 down-regulated proteins were identified as a heat shock protein 70 (HSP 70) and a GALE (UDP-galactose-4 epimerase) protein isoform I. The up-regulated proteins were Serpin H1, a protein involved in collagen metabolism and a redox sensor NmrA-like (NMRAL) family domain protein-1. Both GALE and NMRAL proteins are implicated in energy metabolism and redox regulation whereas the HSP 70 protects cells against stress, and implicated in chondrocyte hypertrophy, an important event in endochondral bone formation. The failure of chondrocyte protective mechanisms such as associated with protection against cellular stress and energy metabolism appear to be the likely cause for chondrocyte death induced by thiram.

Treatment of growth plate injury using IGF-I-loaded PLGA scaffolds

Growth plate fracture can lead to retarded growth and unequal limb length, which may have a lifelong effect on a person's physical stature. The goal of this research was to develop an in vivo tissue-engineering approach for the treatment of growth plate injury via localized delivery of insulin-like growth factor I (IGF-I) from cell-free poly(lactic-co-glycolic acid) (PLGA) scaffolds. Mass loss and drug release studies were conducted to study the scaffold degradation and IGF-I release patterns. In vitro cell studies showed that rat bone marrow stromal cells seeded on the porous scaffolds colonized the pores and deposited matrix within the scaffolds. These in vitro evaluations were followed by a proof-of-concept animal study involving implantation of scaffolds in proximal tibial growth plate defects in New Zealand white rabbits. Histological analysis of tissue sections from the in vivo studies showed regeneration of cartilage, albeit with disorganized structure, at the site of implantation of IGF-I-releasing scaffolds; in contrast, only bone was formed in empty defects and those treated with IGF-free scaffolds. The present findings show the potential for treating growth plate injury using in vivo tissue engineering techniques.

The Therapeutic Effect of Bone Marrow-Derived Stem Cell Implantation After Epiphyseal Plate Injury Is Abrogated by Chondrogenic Predifferentiation

The goal of this study was to determine the effects of chondrogenic predifferentiation on the ability of bone marrow-derived stromal cells (BMSCs) delivered to growth plate defects to restore growth function. Chondrogenesis was induced with transforming growth factor (TGF)-β1 treatment in high-density monolayer cultures of BMSCs in vitro. The predifferentiated or undifferentiated BMSCs were either seeded into agarose gels for continued in vitro culture, or injected into growth plate defects via an in situ gelling agarose. Predifferentiated BMSCs had higher Sox-9, type II collagen, and aggrecan mRNA levels compared to undifferentiated cells after high-density monolayer culture. After transfer to agarose gels, predifferentiated cells did not produce a cartilaginous matrix, even with continued TGF-β1 stimulation, whereas undifferentiated cells produced a cartilaginous matrix in this system. Three-dimensional images of the growth plate created from microcomputed tomography scans showed that delivery of either predifferentiated or undifferentiated cells to defects resulted in a decrease in mineralized tether formation (fusion) in the growth plate tissue surrounding the defect to normal levels. Limb length discrepancy between injured and control limbs was corrected after treatment with undifferentiated, but not predifferentiated, cells. These results indicate that cell therapy may be an effective treatment to reduce growth dysfunction after growth plate injury, perhaps by maintaining the health of the uninjured growth plate tissue, and that the cell differentiation state plays a role in restoring the growth potential of the injured limb.