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Legg-Calvé-Perthes disease (LCPD) involves femoral head osteonecrosis caused by disrupted blood supply, leading to joint deformity and early osteoarthritis. This study investigates the role of miRNA-223-5p in regulating hypoxia-induced apoptosis and enhancing osteogenesis in bone marrow mesenchymal stem cells (BMSCs). Utilizing a juvenile New Zealand white rabbit model of LCPD established through femoral neck ligation, we transfected BMSCs with miR-223-5p mimics, inhibitors, and controls, followed by hypoxic exposure. The impact of miR-223-5p on BMSC apoptosis was assessed using qPCR, Western blotting, and dual-luciferase reporter assays, focusing on the Wnt/β-catenin signaling pathway. In vivo, we evaluated the effects of transplanting miR-223-5p-overexpressing BMSCs into the LCPD model. Our results indicate that miR-223-5p is downregulated under hypoxic conditions. Overexpression of miR-223-5p in BMSCs inhibited hypoxia-induced apoptosis and activated the Wnt/β-catenin pathway by directly targeting CHAC2. In vivo, miR-223-5p-overexpressing BMSCs enhanced femoral head osteogenesis and reduced necrosis in the LCPD model. These findings suggest that miR-223-5p inhibits hypoxia-induced apoptosis in BMSCs by targeting CHAC2 and activating the Wnt/β-catenin pathway, proposing miR-223-5p as a promising target for improving bone repair in ischemic conditions., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Yang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Lateral Meningocele Syndrome (LMS), a disorder associated with NOTCH3 pathogenic variants, presents with neurological, craniofacial and skeletal abnormalities. Mouse models of the disease exhibit osteopenia that is ameliorated by the administration of Notch3 antisense oligonucleotides (ASO) targeting either Notch3 or the Notch3 mutation. To determine the consequences of LMS pathogenic variants in human cells and whether they can be targeted by ASOs, induced pluripotent NCRM1 and NCRM5 stem (iPS) cells harboring a NOTCH36692-93insC insertion were created. Parental iPSCs, NOTCH36692-93insC and isogenic controls, free of chromosomal aberrations as determined by human CytoSNP850 array, were cultured under conditions of neural crest, mesenchymal and osteogenic cell differentiation. The expected cell phenotype was confirmed by surface markers and a decline in OCT3/4 and NANOG mRNA. NOTCH36692-93insC cells displayed enhanced expression of Notch target genes HES1, HEY1, 2 and L demonstrating a NOTCH3 gain-of-function. There was enhanced osteogenesis in NOTCH36692-93insC cells as evidenced by increased mineralized nodule formation and ALPL, BGLAP and BSP expression. ASOs targeting NOTCH3 decreased both NOTCH3 wild type and NOTCH36692-93insC mutant mRNA by 40% in mesenchymal and 90% in osteogenic cells. ASOs targeting the NOTCH3 insertion decreased NOTCH36692-93insC by 70-80% in mesenchymal cells and by 45-55% in osteogenic cells and NOTCH3 mRNA by 15-30% and 20-40%, respectively. In conclusion, a NOTCH3 pathogenic variant causes a modest increase in osteoblastogenesis in human iPS cells in vitro and NOTCH3 and NOTCH3 mutant specific ASOs downregulate NOTCH3 transcripts associated with LMS., Competing Interests: PJ and MC are paid employees of Ionis Pharmaceuticals. Please note that the synthesis and applications of antisense oligonucleotides may be covered by patent(s) filed by Ionis Pharmaceuticals. Individuals wanting to obtain antisense oligonucleotides from Ionis Pharmaceuticals are required to contact Ionis Pharmaceuticals directly. This does not alter our adherence to PLOS ONE policies on sharing data and materials.” as detailed in http://journals.plos.org/plosone/s/competing-interests, (Copyright: © 2025 Canalis et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objective: In this experiment, we screened key miRNAs involved in the dexamethasone-induced decrease in osteogenic capacity of mouse precursor osteoblasts MC3T3-E1 over and investigated their specific regulatory mechanisms., Methods: In this experiment, cell counting kit assay was utilized to act on MC3T3-E1 cells at 0, 5μM, 10μM, 15μM concentrations of dexamethasone for 24h, 48h and 72h to observe the changes in cell viability in order to select the appropriate dexamethasone concentration. Apoptosis and reactive oxygen species were detected by flow cytometry. The transcription of osteogenesis-related genes (Runx2, ALP, OCN, OPN, OPG, COL1A1) and protein expression levels (Runx2, ALP, OCN, OPN) were detected by Western Blot and qRT-PCR to validate the changes in cellular osteogenesis. The differentially expressed miRNAs related to MC3T3-E1 osteogenic differentiation after dexamethasone action were screened out. The expression levels of selected target miRNAs were verified in the experimental group and the control group by qRT-PCR. The miRNA inhibitor was transfected to knock down miRNA in dexamethasone-induced MC3T3-E1 injury. Alkaline phosphatase staining and flow cytometry were performed to detect apoptosis and reactive oxygen species changes. transcript and protein expression levels of osteogenesis-related genes in mouse MC3T3-E1 were detected by qRT-PCR and Western blot experiments. By miRNA target gene prediction, luciferase reporter gene experiments, qRT-PCR and Western blot experiments were used to verify whether the selected target miRNAs targeted the target gene., Results: First, it was determined that 10μM dexamethasone solution was effective in inducing a decrease in osteogenic function in mouse MC3T3-E1 by CCK8 experiments, which showed a significant decrease in alkaline phosphatase activity, a decrease in calcium nodules as shown by alizarin red staining, an increase in apoptosis and reactive oxygen species as detected by flow cytometry, as well as a decrease in the expression of osteogenesis-related genes and proteins. Five target miRNAs were identified: miR-706, miR-296-3p, miR-7011-5p, miR-145a-3p, and miR-149-3p. miR-145a-3p, which had the most pronounced and stable expression trend and was the most highly expressed miRNA, was chosen as the target of this experiment by qRT-PCR analysis. -145a-3p, as the subject of this experiment. Knockdown of miR-145a-3p in MC3T3-E1 cells after dexamethasone action significantly improved the expression of their impaired osteogenic indicators. It was shown that after knocking down the target miRNA, alkaline phosphatase staining was significantly increased compared with the dexamethasone-stimulated group and approached the level of the blank control group. Meanwhile, the expression of osteogenic function-related proteins and genes also increased in the dexamethasone-stimulated group after knocking down miR-145a-3p, and approached the level of the blank control group. A direct targeting relationship between miR-145a-3p and Runx2 was indeed confirmed by luciferase reporter gene assays, qRT-PCR and Western blot experiments., Conclusions: The results indicated that dexamethasone impaired the osteogenic differentiation ability of MC3T3-E1 cells by inducing the up-regulation of miR-145a-3p expression. MiR-145a-3p inhibited the osteogenic differentiation ability of MC3T3-E1 cells by targeting and suppressing the expression level of Runx2 protein. Inhibition of miR-145a-3p levels significantly improved the osteogenic differentiation ability of MC3T3-E1 cells., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Wu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Osteoblasts, the key cells responsible for bone formation and the maintenance of skeletal integrity, originate from a diverse array of progenitor cells. However, the mechanisms underlying osteoblast differentiation from these multiple osteoprogenitors remain poorly understood. To address this knowledge gap, we developed a comprehensive framework to investigate osteoblast differentiation at multiple scales, encompassing cells, genes, and gene modules. We constructed a reference atlas focused on differentiation, which incorporates various osteoprogenitors and provides a seven-level cellular taxonomy. To reconstruct the differentiation process, we developed a model that identifies the transcription factors and pathways involved in differentiation from different osteoprogenitors. Acknowledging that covariates such as age and tissue type can influence differentiation, we created an algorithm to detect differentially expressed genes throughout the differentiation process. Additionally, we implemented methods to identify conserved pseudotemporal gene modules across multiple samples. Overall, our framework systematically addresses the heterogeneity observed during osteoblast differentiation from diverse sources, offering novel insights into the complexities of bone formation and serving as a valuable resource for understanding osteogenesis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Han et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

MIR125B, particularly its 5p strand, is apparently involved in multiple cellular processes, including osteoblastogenesis and osteoclastogenesis. Given that MIR125B is transcribed from the loci Mir125b1 and Mir125b2, three mature transcripts (MIR125B-5p, MIR125B1-3p, and MIR125B2-3p) are generated (MIR125B-5p is common to both); however, their expression profiles and roles in the bones remain poorly understood. Both primary and mature MIR125B transcripts were differentially expressed in various organs, tissues, and cells, and their expression patterns did not necessarily correlate in wild-type (WT) mice. We generated Mir125b2 knockout (KO) mice to examine the contribution of Mir125b2 to MIR125B expression profiles and bone phenotypes. Mir125b2 KO mice were born and grew normally without any changes in bone parameters. Interestingly, in WT and Mir125b2 KO, MIR125B-5p was abundant in the calvaria and bone marrow stromal cells. These results indicate that the genetic ablation of Mir125b2 does not impinge on the bones of mice, attracting greater attention to MIR125B-5p derived from Mir125b1. Future studies should investigate the conditional deletion of Mir125b1 and both Mir125b1 and Mir125b2 in mice., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ogasawara et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Fracture non-unions affect many patients worldwide, however, known risk factors alone do not predict individual risk. The identification of novel biomarkers is crucial for early diagnosis and timely patient treatment. This study focused on the identification of microRNA (miRNA) related to the process of fracture healing. Serum of fracture patients and healthy volunteers was screened by RNA sequencing to identify differentially expressed miRNA at various times after injury. The results were correlated to miRNA in the conditioned medium of human bone marrow mesenchymal stromal cells (BMSCs) during in vitro osteogenic differentiation. hsa-miR-1246, hsa-miR-335-5p, and miR-193a-5p were identified both in vitro and in fracture patients and their functional role in direct BMSC osteogenic differentiation was assessed. The results showed no influence of the downregulation of the three miRNAs during in vitro osteogenesis. However, miR-1246 may be involved in cell proliferation and recruitment of progenitor cells. Further studies should be performed to assess the role of these miRNA in other processes relevant to fracture healing., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Bella et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Entheses are classified into three types: fibrocartilaginous, fibrous, and periosteal insertions. However, the mechanism behind the development of fibrous entheses and periosteal insertions remains unclear. Since both entheses are part of the temporomandibular joint (TMJ), this study analyzes the TMJ entheses. Here, we show that SOX9 expression is negatively regulated during TMJ enthesis development, unlike fibrocartilage entheses which are modularly formed by SCX and SOX9 positive progenitors. The TMJ entheses was adjacent to the intramembranous bone rather than cartilage. SOX9 expression was diminished during TMJ enthesis development. To clarify the functional role of Sox9 in the development of TMJ entheses, we examined these structures in TMJ using Wnt1Cre;Sox9flox/+ reporter mice. Wnt1Cre;Sox9flox/+ mice showed enthesial deformation at the TMJ. Next, we also observed a diminished SOX9 expression area at the enthesis in contact with the clavicle's membranous bone portion, similar to the TMJ entheses. Together, these findings reveal that the timing of SOX9 expression varies with the ossification development mode., Competing Interests: The authors declare no competing or financial interests., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Osteoclasts are crucial in the events leading to bone metastasis of lung cancer. Interleukin-17A (IL-17A) affects osteogenesis by regulating the survival of osteoclast precursors (OCPs) and is enriched in lung cancer cells. However, how factors derived from tumor cells that metastasize to bone affect osteoclastogenesis remains poorly understood. We examined whether IL-17A derived from lung cancer cells affects osteoclast differentiation by regulating OCP apoptosis. IL-17A expression was inhibited in A549 non-small cell lung cancer cells using RNA interference. Compared with conditioned medium (CM) from A549 cells (A549-CM), CM from IL-17A-deficient A549 cells (A549-si-CM) suppressed osteoclastogenesis. The mRNA expression of osteoclast-specific genes was downregulated following A549-si-CM treatment. Furthermore, A549-si-CM promoted osteoclast precursor apoptosis at an early stage of osteoclastogenesis, which was related to the promotion of caspase-3 expression by A549-si-CM during osteoclast differentiation. In vivo experiments also showed that inhibition of IL-17A expression in A549 cells reduced osteoclast activation and bone tissue destruction. Collectively, our results indicate that IL-17A deficiency inhibits lung cancer-induced osteoclast differentiation by promoting apoptosis of osteoclast precursors in the early stage of osteoclast formation and that IL-17A is a potential therapeutic target for cancer-associated bone resorption in patients with lung cancer., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Thoracic ossification of the ligamentum flavum (TOLF) is a heterotopic ossification of spinal ligaments, leading to serious myelopathy. TOLF underlying mechanisms are not well understood. Our iTRAQ analysis have identified ten inflammatory factors related to TOLF, including l. We found that PTGR1 expressions increased in TOLF by RT-PCR and western blot in this study. Both cell proliferation and differentiation are important for the process of bone formation. In our previous study, we demonstrated that TOLF primary cells grew faster than control cells. It was reported that knockdown of PTGR1 inhibited cell proliferation. We hypothesize that PTGR1 may participate in cell proliferation in TOLF. To test this hypothesis, TOLF primary cells were treated for 24h with PTGR1. We observed that PTGR1 increased cell proliferation. The effect of PTGR1 on cell proliferation related genes was examined in TOLF primary cells. Our results showed that PTGR1 was able to activate expressions of c-Myc and CyclinD1. Moreover, blocking JNK pathway by selective JNK inhibitor SP600125 eliminated the positive effect of PTGR1 on c-Myc expression, indicating that PTGR1 activated the expression of c-Myc via JNK pathway. Our new findings suggest that PTGR1 is involved in cell proliferation of TOLF., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Chondromodulin (Cnmd) is a glycoprotein known to stimulate chondrocyte growth. We examined in this study the expression and functional role of Cnmd during distraction osteogenesis that is modulated by mechanical forces. The right tibiae of the mice were separated by osteotomy and subjected to slow progressive distraction using an external fixator. In situ hybridization and immunohistochemical analyses of the lengthened segment revealed that Cnmd mRNA and its protein in wild-type mice were localized in the cartilage callus, which was initially generated in the lag phase and was lengthened gradually during the distraction phase. In Cnmd null (Cnmd-/-) mice, less cartilage callus was observed, and the distraction gap was filled by fibrous tissues. Additionally, radiological and histological investigations demonstrated delayed bone consolidation and remodeling of the lengthened segment in Cnmd-/- mice. Eventually, Cnmd deficiency caused a one-week delay in the peak expression of VEGF, MMP2, and MMP9 genes and the subsequent angiogenesis and osteoclastogenesis. We conclude that Cnmd is necessary for cartilage callus distraction., Competing Interests: The authors have that no competing interests exist., (Copyright: © 2023 Yukata et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Bone formation and loss are the characteristic clinical manifestations of leprosy, but the mechanisms underlying the bone remodeling with Mycobacterium leprae (M. leprae) infection are unclear., Methodology/principal Findings: Osteocytes may have a role through regulating the differentiation of osteogenic lineages. To investigate osteocyte-related mechanisms in leprosy, we treated osteocyte-like cell with N-glycosylated muramyl dipeptide (N.g MDP). RNA-seq analysis showed 724 differentially expressed messenger RNAs (mRNAs) and 724 differentially expressed circular RNA (circRNAs). Of these, we filtered through eight osteogenic-related differentially expressed genes, according to the characteristic of competing endogenous RNA, PubMed databases, and bioinformatic analysis, including TargetScan, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes. Based on these results, we built a circRNA-microRNA (miRNA)-mRNA triple network. Quantitative reverse-transcription polymerase chain reaction and western blots analyses confirmed decreased Clock expression in osteocyte-like cell, while increased in bone mesenchymal stem cells (BMSCs), implicating a crucial factor in osteogenic differentiation. Immunohistochemistry showed obviously increased expression of CLOCK protein in BMSCs and osteoblasts in N.g MDP-treated mice, but decreased expression in osteocytes., Conclusions/significance: This analytical method provided a basis for the relationship between N.g MDP and remodeling in osteocytes, and the circRNA-miRNA-mRNA triple network may offer a new target for leprosy therapeutics., Competing Interests: The authors have declared that no competing interests exist.

The evolution of complex skeletal traits in primates was likely influenced by both genetic and environmental factors. Because skeletal tissues are notoriously challenging to study using functional genomic approaches, they remain poorly characterized even in humans, let alone across multiple species. The challenges involved in obtaining functional genomic data from the skeleton, combined with the difficulty of obtaining such tissues from nonhuman apes, motivated us to consider an alternative in vitro system with which to comparatively study gene regulation in skeletal cell types. Specifically, we differentiated six human (Homo sapiens) and six chimpanzee (Pan troglodytes) induced pluripotent stem cell lines (iPSCs) into mesenchymal stem cells (MSCs) and subsequently into osteogenic cells (bone cells). We validated differentiation using standard methods and collected single-cell RNA sequencing data from over 100,000 cells across multiple samples and replicates at each stage of differentiation. While most genes that we examined display conserved patterns of expression across species, hundreds of genes are differentially expressed (DE) between humans and chimpanzees within and across stages of osteogenic differentiation. Some of these interspecific DE genes show functional enrichments relevant in skeletal tissue trait development. Moreover, topic modeling indicates that interspecific gene programs become more pronounced as cells mature. Overall, we propose that this in vitro model can be used to identify interspecific regulatory differences that may have contributed to skeletal trait differences between species., Competing Interests: The authors have declared that no competing interests exist.

Epigenetic mechanisms regulate osteogenic lineage differentiation of mesenchymal stromal cells. Histone methylation is controlled by multiple lysine demethylases and is an important step in controlling local chromatin structure and gene expression. Here, we show that the lysine-specific histone demethylase Kdm1A/Lsd1 is abundantly expressed in osteoblasts and that its suppression impairs osteoblast differentiation and bone nodule formation in vitro. Although Lsd1 knockdown did not affect global H3K4 methylation levels, genome-wide ChIP-Seq analysis revealed high levels of Lsd1 at gene promoters and its binding was associated with di- and tri-methylation of histone 3 at lysine 4 (H3K4me2 and H3K4me3). Lsd1 binding sites in osteoblastic cells were enriched for the Runx2 consensus motif suggesting a functional link between the two proteins. Importantly, inhibition of Lsd1 activity decreased osteoblast activity in vivo. In support, mesenchymal-targeted knockdown of Lsd1 led to decreased osteoblast activity and disrupted primary spongiosa ossification and reorganization in vivo. Together, our studies demonstrate that Lsd1 occupies Runx2-binding cites at H3K4me2 and H3K4me3 and its activity is required for proper bone formation., Competing Interests: The authors have declared that no competing interests exist.

Rothmund-Thomson syndrome (RTS) is an autosomal recessive genetic disorder characterized by poikiloderma, small stature, skeletal anomalies, sparse brows/lashes, cataracts, and predisposition to cancer. Type 2 RTS patients with biallelic RECQL4 pathogenic variants have multiple skeletal anomalies and a significantly increased incidence of osteosarcoma. Here, we generated RTS patient-derived induced pluripotent stem cells (iPSCs) to dissect the pathological signaling leading to RTS patient-associated osteosarcoma. RTS iPSC-derived osteoblasts showed defective osteogenic differentiation and gain of in vitro tumorigenic ability. Transcriptome analysis of RTS osteoblasts validated decreased bone morphogenesis while revealing aberrantly upregulated mitochondrial respiratory complex I gene expression. RTS osteoblast metabolic assays demonstrated elevated mitochondrial respiratory complex I function, increased oxidative phosphorylation (OXPHOS), and increased ATP production. Inhibition of mitochondrial respiratory complex I activity by IACS-010759 selectively suppressed cellular respiration and cell proliferation of RTS osteoblasts. Furthermore, systems analysis of IACS-010759-induced changes in RTS osteoblasts revealed that chemical inhibition of mitochondrial respiratory complex I impaired cell proliferation, induced senescence, and decreased MAPK signaling and cell cycle associated genes, but increased H19 and ribosomal protein genes. In summary, our study suggests that mitochondrial respiratory complex I is a potential therapeutic target for RTS-associated osteosarcoma and provides future insights for clinical treatment strategies., Competing Interests: The authors have declared that no competing interests exist.

Mammalian Hedgehog (HH) signalling pathway plays an essential role in tissue homeostasis and its deregulation is linked to rheumatological disorders. UBR5 is the mammalian homologue of the E3 ubiquitin-protein ligase Hyd, a negative regulator of the Hh-pathway in Drosophila. To investigate a possible role of UBR5 in regulation of the musculoskeletal system through modulation of mammalian HH signaling, we created a mouse model for specific loss of Ubr5 function in limb bud mesenchyme. Our findings revealed a role for UBR5 in maintaining cartilage homeostasis and suppressing metaplasia. Ubr5 loss of function resulted in progressive and dramatic articular cartilage degradation, enlarged, abnormally shaped sesamoid bones and extensive heterotopic tissue metaplasia linked to calcification of tendons and ossification of synovium. Genetic suppression of smoothened (Smo), a key mediator of HH signalling, dramatically enhanced the Ubr5 mutant phenotype. Analysis of HH signalling in both mouse and cell model systems revealed that loss of Ubr5 stimulated canonical HH-signalling while also increasing PKA activity. In addition, human osteoarthritic samples revealed similar correlations between UBR5 expression, canonical HH signalling and PKA activity markers. Our studies identified a crucial function for the Ubr5 gene in the maintenance of skeletal tissue homeostasis and an unexpected mode of regulation of the HH signalling pathway., Competing Interests: The authors have declared that no competing interests exist.

Runx1 is highly expressed in osteoblasts, however, its function in osteogenesis is unclear. We generated mesenchymal progenitor-specific (Runx1f/fTwist2-Cre) and osteoblast-specific (Runx1f/fCol1α1-Cre) conditional knockout (Runx1 CKO) mice. The mutant CKO mice with normal skeletal development displayed a severe osteoporosis phenotype at postnatal and adult stages. Runx1 CKO resulted in decreased osteogenesis and increased adipogenesis. RNA-sequencing analysis, Western blot, and qPCR validation of Runx1 CKO samples showed that Runx1 regulates BMP signaling pathway and Wnt/β-catenin signaling pathway. ChIP assay revealed direct binding of Runx1 to the promoter regions of Bmp7, Alk3, and Atf4, and promoter mapping demonstrated that Runx1 upregulates their promoter activity through the binding regions. Bmp7 overexpression rescued Alk3, Runx2, and Atf4 expression in Runx1-deficient BMSCs. Runx2 expression was decreased while Runx1 was not changed in Alk3 deficient osteoblasts. Atf4 overexpression in Runx1-deficient BMSCs did not rescue expression of Runx1, Bmp7, and Alk3. Smad1/5/8 activity was vitally reduced in Runx1 CKO cells, indicating Runx1 positively regulates the Bmp7/Alk3/Smad1/5/8/Runx2/ATF4 signaling pathway. Notably, Runx1 overexpression in Runx2-/- osteoblasts rescued expression of Atf4, OCN, and ALP to compensate Runx2 function. Runx1 CKO mice at various osteoblast differentiation stages reduced Wnt signaling and caused high expression of C/ebpα and Pparγ and largely increased adipogenesis. Co-culture of Runx1-deficient and wild-type cells demonstrated that Runx1 regulates osteoblast-adipocyte lineage commitment both cell-autonomously and non-autonomously. Notably, Runx1 overexpression rescued bone loss in OVX-induced osteoporosis. This study focused on the role of Runx1 in different cell populations with regards to BMP and Wnt signaling pathways and in the interacting network underlying bone homeostasis as well as adipogenesis, and has provided new insight and advancement of knowledge in skeletal development. Collectively, Runx1 maintains adult bone homeostasis from bone loss though up-regulating Bmp7/Alk3/Smad1/5/8/Runx2/ATF4 and WNT/β-Catenin signaling pathways, and targeting Runx1 potentially leads to novel therapeutics for osteoporosis., Competing Interests: The authors have declared that no competing interests exist.

Chondrocytes proliferate and mature into hypertrophic chondrocytes. Vascular invasion into the cartilage occurs in the terminal hypertrophic chondrocyte layer, and terminal hypertrophic chondrocytes die by apoptosis or transdifferentiate into osteoblasts. Runx2 is essential for osteoblast differentiation and chondrocyte maturation. Runx2-deficient mice are composed of cartilaginous skeletons and lack the vascular invasion into the cartilage. However, the requirement of Runx2 in the vascular invasion into the cartilage, mechanism of chondrocyte transdifferentiation to osteoblasts, and its significance in bone development remain to be elucidated. To investigate these points, we generated Runx2fl/flCre mice, in which Runx2 was deleted in hypertrophic chondrocytes using Col10a1 Cre. Vascular invasion into the cartilage was similarly observed in Runx2fl/fl and Runx2fl/flCre mice. Vegfa expression was reduced in the terminal hypertrophic chondrocytes in Runx2fl/flCre mice, but Vegfa was strongly expressed in osteoblasts in the bone collar, suggesting that Vegfa expression in bone collar osteoblasts is sufficient for vascular invasion into the cartilage. The apoptosis of terminal hypertrophic chondrocytes was increased and their transdifferentiation was interrupted in Runx2fl/flCre mice, leading to lack of primary spongiosa and osteoblasts in the region at E16.5. The osteoblasts appeared in this region at E17.5 in the absence of transdifferentiation, and the number of osteoblasts and the formation of primary spongiosa, but not secondary spongiosa, reached to levels similar those in Runx2fl/fl mice at birth. The bone structure and volume and all bone histomophometric parameters were similar between Runx2fl/fl and Runx2fl/flCre mice after 6 weeks of age. These findings indicate that Runx2 expression in terminal hypertrophic chondrocytes is not required for vascular invasion into the cartilage, but is for their survival and transdifferentiation into osteoblasts, and that the transdifferentiation is necessary for trabecular bone formation in embryonic and neonatal stages, but not for acquiring normal bone structure and volume in young and adult mice., Competing Interests: The authors have declared that no competing interests exist.

Chondrocytes, comparable to many cells from the connective tissue, dedifferentiate and end up in a similar fibroblastoid cell type, marked by the loss of the specific expression pattern. Here, chondrocytes isolated from osteoarthritic (OA) patients were investigated. The OA chondrocytes used in this work were not affected by the loss of specific gene expression upon cell culture. The mRNA levels of known cartilage markers, such as SOX5, SOX6, SOX9, aggrecan and proteoglycan 4, remained unchanged. Since chondrocytes from OA and healthy tissue show different DNA methylation patterns, the underlying mechanisms of cartilage marker maintenance were investigated with a focus on the epigenetic modification by DNA methylation. The treatment of dedifferentiated chondrocytes with the DNA methyltransferase inhibitor 5-aza-2´-deoxycytidine (5-aza-dC) displayed no considerable impact on the maintenance of marker gene expression observed in the dedifferentiated state, while the chondrogenic differentiation capacity was compromised. On the other hand, the pre-cultivation with 5-aza-dC improved the osteogenesis and adipogenesis of OA chondrocytes. Contradictory to these effects, the DNA methylation levels were not reduced after treatment for four weeks with 1 μM 5-aza-dC. In conclusion, 5-aza-dC affects the differentiation capacity of OA chondrocytes, while the global DNA methylation level remains stable. Furthermore, dedifferentiated chondrocytes isolated from late-stage OA patients represent a reliable cell source for in vitro studies and disease models without the need for additional alterations., Competing Interests: The authors have declared that no competing interests exist.

Osteoporosis is a genetic disease characterized by progressive reductions in bone mineral density (BMD) leading to an increased risk of fracture. Over the last decade, genome-wide association studies (GWASs) have identified over 1000 associations for BMD. However, as a phenotype BMD is challenging as bone is a multicellular tissue affected by both local and systemic physiology. Here, we focused on a single component of BMD, osteoblast-mediated bone formation in mice, and identified associations influencing osteoblast activity on mouse Chromosomes (Chrs) 1, 4, and 17. The locus on Chr. 4 was in an intergenic region between Wnt4 and Zbtb40, homologous to a locus for BMD in humans. We tested both Wnt4 and Zbtb40 for a role in osteoblast activity and BMD. Knockdown of Zbtb40, but not Wnt4, in osteoblasts drastically reduced mineralization. Additionally, loss-of-function mouse models for both genes exhibited reduced BMD. Our results highlight that investigating the genetic basis of in vitro osteoblast mineralization can be used to identify genes impacting bone formation and BMD., Competing Interests: The authors have declared that no competing interests exist.

The strength of bone depends on bone quantity and quality. Osteocalcin (Ocn) is the most abundant noncollagenous protein in bone and is produced by osteoblasts. It has been previously claimed that Ocn inhibits bone formation and also functions as a hormone to regulate insulin secretion in the pancreas, testosterone synthesis in the testes, and muscle mass. We generated Ocn-deficient (Ocn-/-) mice by deleting Bglap and Bglap2. Analysis of Ocn-/-mice revealed that Ocn is not involved in the regulation of bone quantity, glucose metabolism, testosterone synthesis, or muscle mass. The orientation degree of collagen fibrils and size of biological apatite (BAp) crystallites in the c-axis were normal in the Ocn-/-bone. However, the crystallographic orientation of the BAp c-axis, which is normally parallel to collagen fibrils, was severely disrupted, resulting in reduced bone strength. These results demonstrate that Ocn is required for bone quality and strength by adjusting the alignment of BAp crystallites parallel to collagen fibrils; but it does not function as a hormone., Competing Interests: The authors have declared that no competing interests exist.

Fibrous dysplasia (FD) of bone is a complex disease of the skeleton caused by dominant activating mutations of the GNAS locus encoding for the α subunit of the G protein-coupled receptor complex (Gsα). The mutation involves a substitution of arginine at position 201 by histidine or cysteine (GsαR201H or R201C), which leads to overproduction of cAMP. Several signaling pathways are implicated downstream of excess cAMP in the manifestation of disease. However, the pathogenesis of FD remains largely unknown. The overall FD phenotype can be attributed to alterations of skeletal stem/progenitor cells which normally develop into osteogenic or adipogenic cells (in cis), and are also known to provide support to angiogenesis, hematopoiesis, and osteoclastogenesis (in trans). In order to dissect the molecular pathways rooted in skeletal stem/progenitor cells by FD mutations, we engineered human skeletal stem/progenitor cells with the GsαR201C mutation and performed transcriptomic analysis. Our data suggest that this FD mutation profoundly alters the properties of skeletal stem/progenitor cells by pushing them towards formation of disorganized bone with a concomitant alteration of adipogenic differentiation. In addition, the mutation creates an altered in trans environment that induces neovascularization, cytokine/chemokine changes and osteoclastogenesis. In silico comparison of our data with the signature of FD craniofacial samples highlighted common traits, such as the upregulation of ADAM (A Disintegrin and Metalloprotease) proteins and other matrix-related factors, and of PDE7B (Phosphodiesterase 7B), which can be considered as a buffering process, activated to compensate for excess cAMP. We also observed high levels of CEBPs (CCAAT-Enhancer Binding Proteins) in both data sets, factors related to browning of white fat. This is the first analysis of the reaction of human skeletal stem/progenitor cells to the introduction of the FD mutation and we believe it provides a useful background for further studies on the molecular basis of the disease and for the identification of novel potential therapeutic targets., Competing Interests: The authors have declared that no competing interests exist.

Craniosynostosis is the premature fusion of the sutures of the calvaria and is principally designated as being either syndromic (demonstrating characteristic extracranial malformations) or non-syndromic. While many forms of syndromic craniosynostosis are known to be caused by specific mutations, the genetic etiology of non-syndromic, single-suture craniosynostosis (SSC) is poorly understood. Based on the low recurrence rate (4-7%) and the fact that recurrent mutations have not been identified for most cases of SSC, we propose that some cases of isolated, single suture craniosynostosis may be polygenic. Previous work in our lab identified a disproportionately high number of rare and novel gain-of-function IGF1R variants in patients with SSC as compared to controls. Building upon this result, we used expression array data from calvarial osteoblasts isolated from infants with and without SSC to ascertain correlations between high IGF1 expression and expression of other osteogenic genes of interest. We identified a positive correlation between increased expression of IGF1 and RUNX2, a gene known to cause SSC with increased gene dosage. Subsequent phosphorylation assays revealed that osteoblast cell lines from cases with high IGF1 expression demonstrated inhibition of GSK3β, a serine/threonine kinase known to inhibit RUNX2, thus activating osteogenesis through the IRS1-mediated Akt pathway. With these findings, we have utilized established mouse strains to examine a novel model of polygenic inheritance (a phenotype influenced by more than one gene) of SSC. Compound heterozygous mice with selective disinhibition of RUNX2 and either overexpression of IGF1 or loss of function of GSK3β demonstrated an increase in the frequency and severity of synostosis as compared to mice with the RUNX2 disinhibition alone. These polygenic mouse models reinforce, in-vivo, that the combination of activation of the IGF1 pathway and disinhibition of the RUNX2 pathway leads to an increased risk of developing craniosynostosis and serves as a model of human SSC., Competing Interests: The authors have declared that no competing interests exist.

Periodontal ligament stem cells (PDLSC) play an important role in periodontal tissue homeostasis/turnover and could be applied in cell-based periodontal regenerative therapy. Bacterial supernatants secreted from diverse periodontal bacteria induce the production of cytokines that contribute to local periodontal tissue destruction. However, little is known about the impact of whole bacterial toxins on the biological behavior of PDLSC. Therefore this study investigated whether proliferation, migration, inflammatory cytokines expression and transcriptional profile would be affected by exposure to endotoxins from bacterial species found in the subgingival plaque. PDLSC were cultured with the following bacterial supernatants: S. mutans, S. anginosus, P. intermedia, F. nucleatum, P. gingivalis and T. denticola. These supernatants were prepared in dilutions of 1:1000, 1:500, 1:300 and 1:50. Using quantitative RT-PCR, gene expression of selected inflammatory cytokines (IL-6, IL-8 and IL-1β) and cell-surface receptors (TLR2, TLR4) showed upregulation of ≈2.0- to 3.0-fold, when exposed to P. intermedia, F. nucleatum, P. gingivalis and T. denticola. However, supernatants did not affect proliferation (MTT) and migration (wound scratch assays) of PDLSC. Next generation RNA sequencing confirmed modified lineage commitment of PDLSC by stimulating chondrogenesis, adipogenesis and inhibition of osteogenesis under P. gingivalis supernatant treatment compared to control. Taken together, this study shows stem cell immunomodulatory response to different periodontal bacteria supernatant and suggests that stem cell transcriptional capacity, migration/proliferation and osteogenesis may differ in the presence of those pathogens. These results bring into question stem cell contribution to periodontal tissue regeneration and onset of inflammation., Competing Interests: The authors have declared that no competing interests exist.

ASAP1 is a multi-domain adaptor protein that regulates cytoskeletal dynamics, receptor recycling and intracellular vesicle trafficking. Its expression is associated with poor prognosis for a variety of cancers, and promotes cell migration, invasion and metastasis. Little is known about its physiological role. In this study, we used mice with a gene-trap inactivated ASAP1 locus to study the functional role of ASAP1 in vivo, and found defects in tissues derived from mesenchymal progenitor cells. Loss of ASAP1 led to growth retardation and delayed ossification typified by enlarged hypertrophic zones in growth plates and disorganized chondro-osseous junctions. Furthermore, loss of ASAP1 led to delayed adipocyte development and reduced fat depot formation. Consistently, deletion of ASAP1 resulted in accelerated chondrogenic differentiation of mesenchymal cells in vitro, but suppressed osteo- and adipogenic differentiation. Mechanistically, we found that FAK/Src and PI3K/AKT signaling is compromised in Asap1GT/GT MEFs, leading to impaired adipogenic differentiation. Dysregulated FAK/Src and PI3K/AKT signaling is also associated with attenuated osteogenic differentiation. Together these observations suggest that ASAP1 plays a decisive role during the differentiation of mesenchymal progenitor cells., Competing Interests: The authors have declared that no competing interests exist.

Nonautologous bone grafts have limited osteoinductive potential and thus there is substantial interest in reconstituting these graft materials with osteogenic factors such as bone morphogenic protein 2 (BMP2). However, one limitation of this approach is that BMP2 is typically weakly bound to the graft, which can lead to side effects associated with BMP2 dissemination. In the current study we added a hydroxyapatite (HA)-binding domain onto BMP2 to increase coupling to the graft surface. A sequence consisting of eight glutamate residues (E8) was inserted into the C-terminus of BMP2, and the recombinant protein (rBMP2-E8) was expressed in E. coli. Compared with rBMP2, rBMP2-E8 displayed markedly enhanced binding to HA disks and was better retained on the disks following exposure to vigorous wash steps. Furthermore, rBMP2-E8 was purified using a heparin column, and evaluated for its capacity to stimulate osteoblastic cell signaling. Treatment of SAOS2 cells with rBMP2-E8 induced SMAD 1/5 activation, confirming that the protein retains activity. Collectively these results suggest that the E8 domain serves as an effective tool for improving rBMP2 coupling to graft materials. The increased retention of rBMP2-E8 on the graft surface is expected to prolong BMP2's osteoinductive activity within the graft site, while simultaneously reducing off-target effects., Competing Interests: The authors have declared that no competing interests exist.

Bone mineral density (BMD) is a strong predictor of osteoporotic fracture. It is also one of the most heritable disease-associated quantitative traits. As a result, there has been considerable effort focused on dissecting its genetic basis. Here, we performed a genome-wide association study (GWAS) in a panel of inbred strains to identify associations influencing BMD. This analysis identified a significant (P = 3.1 x 10-12) BMD locus on Chromosome 3@52.5 Mbp that replicated in two separate inbred strain panels and overlapped a BMD quantitative trait locus (QTL) previously identified in a F2 intercross. The association mapped to a 300 Kbp region containing four genes; Gm2447, Gm20750, Cog6, and Lhfp. Further analysis found that Lipoma HMGIC Fusion Partner (Lhfp) was highly expressed in bone and osteoblasts. Furthermore, its expression was regulated by a local expression QTL (eQTL), which overlapped the BMD association. A co-expression network analysis revealed that Lhfp was strongly connected to genes involved in osteoblast differentiation. To directly evaluate its role in bone, Lhfp deficient mice (Lhfp-/-) were created using CRISPR/Cas9. Consistent with genetic and network predictions, bone marrow stromal cells (BMSCs) from Lhfp-/- mice displayed increased osteogenic differentiation. Lhfp-/- mice also had elevated BMD due to increased cortical bone mass. Lastly, we identified SNPs in human LHFP that were associated (P = 1.2 x 10-5) with heel BMD. In conclusion, we used GWAS and systems genetics to identify Lhfp as a regulator of osteoblast activity and bone mass., Competing Interests: The authors have declared that no competing interests exist.

Osteocytes, cells forming an elaborate network within the bones of most vertebrate taxa, are thought to be the master regulators of bone modeling, a process of coordinated, local bone-tissue deposition and removal that keeps bone strains at safe levels throughout life. Neoteleost fish, however, lack osteocytes and yet are known to be capable of bone modeling, although no osteocyte-independent modeling regulatory mechanism has so far been described. Here, we characterize a novel, to our knowledge, bone-modeling regulatory mechanism in a fish species (medaka), showing that although lacking osteocytes (i.e., internal mechanosensors), when loaded, medaka bones model in mechanically directed ways, successfully reducing high tissue strains. We establish that as in mammals, modeling in medaka is regulated by the SOST gene, demonstrating a mechanistic link between skeletal loading, SOST down-regulation, and intense bone deposition. However, whereas mammalian SOST is expressed almost exclusively by osteocytes, in both medaka and zebrafish (a species with osteocytic bones), SOST is expressed by a variety of nonosteocytic cells, none of which reside within the bone bulk. These findings argue that in fishes (and perhaps other vertebrates), nonosteocytic skeletal cells are both sensors and responders, shouldering duties believed exclusive to osteocytes. This previously unrecognized, SOST-dependent, osteocyte-independent mechanism challenges current paradigms of osteocyte exclusivity in bone-modeling regulation, suggesting the existence of multivariate feedback networks in bone modeling-perhaps also in mammalian bones-and thus arguing for the possibility of untapped potential for cell targets in bone therapeutics., Competing Interests: The authors have declared that no competing interests exist.

Background: After bone resorption, ions and degraded organic components are co-released into the extracellular space. Ions and growth factors, although different in their biological nature, induce a common and coordinated chemotactic effect. Conditioned media has been used successfully in bone regeneration by promoting endogenous cell recruitment. Likewise, calcium alone act as a paracrine chemotactic signal, inducing the host's undifferentiated progenitor cell infiltration into the implanted biomaterials. The aim of the present study was to compare the chemotactic effect of calcium and conditioned media in primary calvarial cells., Methods: The chemotactic cell response was evaluated in vitro using an agarose spot and a wound healing assay. In addition, we used a calvarial bone explant model ex-vivo. The healing potential was also tested through an in vivo model, a critical-size calvarial bone defect in mice. For the in vivo experiment, cell-free calcium-containing or conditioned media-containing scaffolds were implanted, and MSC's seeded scaffolds were used as positive control. After seven weeks post-implantation, samples were retrieved, and bone regeneration was evaluated by μCT and histological analysis. Osteogenic gene expression was evaluated by qPCR., Results: We found that chemotactic cell migration in response to either calcium or conditioned media was equivalent in vitro and ex vivo. Accordingly, μCT analysis showed that bone regeneration induced by the MSC's seeded scaffolds was similar to that obtained with cell-free calcium or conditioned media-containing scaffolds. Pre-treatment with SB202190, a highly selective p38 inhibitor, abrogated the chemotactic effect induced by conditioned media. In contrast, p38 activity was not essential for the calcium-induced chemotaxis. Moreover, BAPTA-AM treatment, a cytosolic calcium chelator, decreased the chemotactic effect and the expression of key osteogenic genes induced by calcium or conditioned media., Conclusion: We show that calcium ions alone not only mimic the conditioned media chemotactic effect, but also induce an osteogenic effect similar to that produced by transplanted MSC's in vivo. Furthermore, the chemotactic effect induced by conditioned media is calcium and p38 dependent. The rise in cytosolic calcium might integrate the different signaling pathways triggered by conditioned media and extracellular Ca2+. This calcium-driven in situ bone regeneration is a promising and convenient alternative to promote endogenous cell recruitment into the injured bone site. This pre-clinical cell-free and growth factor-free approach might avoid the disadvantages of the ex vivo cell manipulation., Competing Interests: The authors have declared that no competing interests exist.

Bone remodeling involves the coordinated actions of osteoclasts, which resorb the calcified bony matrix, and osteoblasts, which refill erosion pits created by osteoclasts to restore skeletal integrity and adapt to changes in mechanical load. Osteoblasts are derived from pluripotent mesenchymal stem cell precursors, which undergo differentiation under the influence of a host of local and environmental cues. To characterize the autocrine/paracrine signaling networks associated with osteoblast maturation and function, we performed gene network analysis using complementary "agnostic" DNA microarray and "targeted" NanoString nCounter datasets derived from murine MC3T3-E1 cells induced to undergo synchronized osteoblastic differentiation in vitro. Pairwise datasets representing changes in gene expression associated with growth arrest (day 2 to 5 in culture), differentiation (day 5 to 10 in culture), and osteoblast maturation (day 10 to 28 in culture) were analyzed using Ingenuity Systems Pathways Analysis to generate predictions about signaling pathway activity based on the temporal sequence of changes in target gene expression. Our data indicate that some pathways involved in osteoblast differentiation, e.g. Wnt/β-catenin signaling, are most active early in the process, while others, e.g. TGFβ/BMP, cytokine/JAK-STAT and TNFα/RANKL signaling, increase in activity as differentiation progresses. Collectively, these pathways contribute to the sequential expression of genes involved in the synthesis and mineralization of extracellular matrix. These results provide insight into the temporal coordination and complex interplay between signaling networks controlling gene expression during osteoblast differentiation. A more complete understanding of these processes may aid the discovery of novel methods to promote osteoblast development for the treatment of conditions characterized by low bone mineral density., Competing Interests: The authors have declared that no competing interests exist.

Parathyroid hormone (PTH) exerts dual effects, anabolic or catabolic, on bone when administrated intermittently or continuously, via mechanisms that remain largely unknown. PTH binding to cells induces PTH-responsive genes including primary response genes (PRGs). PRGs are rapidly induced without the need for de novo protein synthesis, thereby playing pivotal roles in directing subsequent molecular responses. In this study, to understand the role of PRGs in mediating osteoblastic cellular responses to PTH, we investigated whether various durations of PTH differentially induce PRGs in primary osteoblasts and MC3T3-E1. Nurr1 and RANKL, PRGs known for their anabolic and catabolic roles in bone metabolism respectively, presented distinctive transient vs. sustained induction kinetics. Corroborating their roles, maximum induction of Nurr1 was sufficiently achieved by brief PTH in as little as 30 minutes and continued beyond that, while maximum induction of RANKL was achieved only by prolonged PTH over 4 hours. Our data suggested distinctive regulatory mechanisms for Nurr1 and RANKL: PKA-mediated chromatin rearrangement for transcriptional regulation of both PRGs and ERK-mediated transcriptional regulation for RANKL but not Nurr1. Lastly, we classified PRGs into two groups based on the induction kinetics: The group that required brief PTH for maximum induction included Nur77, cox-2, and Nurr1, all of which are reported to play roles in bone formation. The other group that required prolonged PTH for maximum induction included IL-6 and RANKL, which play roles in bone resorption. Together, our data suggested the crucial role of PRG groups in mediating differential osteoblastic cellular responses to intermittent vs. continuous PTH. Continued research into the regulatory mechanisms of PKA and ERK for PRGs will help us better understand the molecular mechanisms underlying the dual effects of PTH, thereby optimizing the current therapeutic use of PTH for osteoporosis., Competing Interests: The authors have declared that no competing interests exist.

Osteoporosis is associated with chronic iron overload secondary to hereditary hemochromatosis (HH), but the causative mechanisms are incompletely understood. The main objective of this study was to investigate the role of dietary iron on osteoporosis, using as biological model the Hfe-KO mice, which have a systemic iron overload. We showed that these mice show an increased susceptibility for developing a bone loss phenotype compared to WT mice, which can be exacerbated by an iron rich diet. The dietary iron overload caused an increase in inflammation and iron incorporation within the trabecular bone in both WT and Hfe-KO mice. However, the osteoporotic phenotype was only evident in Hfe-KO mice fed the iron-enriched diet. This appeared to result from an imbalance between bone formation and bone resorption driven by iron toxicity associated to Hfe-KO and confirmed by a decrease in bone microarchitecture parameters (identified by micro-CT) and osteoblast number. These findings were supported by the observed downregulation of bone metabolism markers and upregulation of ferritin heavy polypeptide 1 (Fth1) and transferrin receptor-1 (Tfrc), which are associated with iron toxicity and bone loss phenotype. In WT mice the iron rich diet was not enough to promote a bone loss phenotype, essentially due to the concomitant depression of bone resorption observed in those animals. In conclusion the dietary challenge influences the development of osteoporosis in the HH mice model thus suggesting that the iron content in the diet may influence the osteoporotic phenotype in systemic iron overload conditions., Competing Interests: The authors have declared that no competing interests exist.

Polymeric biomaterials composed of extracellular matrix components possess osteoconductive capacity that is essential for bone healing. The presence of collagen and the ability to undergo physicochemical modifications render these materials a suitable alternative in bone regenerative therapies. The objective of this study was to evaluate the osteogenic capacity of collagen-based matrices (native and anionic after alkaline hydrolysis) made from bovine intestinal serosa (MBIS). Twenty-five animals underwent surgery to create a cranial defect to be filled with native and anionic collagen matrixes, mmineralized and non mineralized. The animals were killed painlessly 6 weeks after surgery and samples of the wound area were submitted to routine histology and morphometric analysis. In the surgical area there was new bone formation projecting from the margins to the center of the defect. More marked bone neoformation occurred in the anionic matrices groups in such a way that permitted union of the opposite margins of the bone defect. The newly formed bone matrix exhibited good optical density of type I collagen fibers. Immunoexpression of osteocalcin by osteocytes was observed in the newly formed bone. Morphometric analysis showed a greater bone volume in the groups receiving the anionic matrices compared to the native membranes. Mineralization of the biomaterial did not increase its osteoregenerative capacity. In conclusion, the anionic matrix exhibits osteoregenerative capacity and is suitable for bone reconstruction therapies., Competing Interests: The authors have declared that no competing interests exist.

Periodontal tissue damage, accompanied by the degradation and destruction of periodontal tissue collagen, is one of the most clinically common complications and difficulty self-repair in patients with diabetes. Human periodontal ligament stem cells (PDLSC) are the undifferentiated mesenchymal cells that persist in the periodontal ligament after development of periodontal tissue and the ability of PDLSC osteogenic differentiation is responsible for repairing periodontal tissue defects. However, the reasons of high glucose environment in diabetic patients inhibiting PDLSC to repair periodontal tissues are unclear. To address these issues, we propose exposing PDLSC to high-sugar mimics the diabetic environment and investigating the activity of osteogenic differentiation and adipogenic differentiation of PDLSC. At the cellular level, high glucose can promote the adipogenic differentiation and inhibit osteogenic differentiation to decrease the self-repair ability of PDLSC in periodontal tissues. Mechanistically at the molecular level, these effects are elicited via regulating the mRNA and protein expression of C/EBPβ, PPAR-γ., Competing Interests: The authors have no conflict of interest.

Tissue calcification is an important physiological process required for the normal structure and function of bone. However, ectopic or excessive calcification contributes to diseases such as chondrocalcinosis, to calcium deposits in the skin or to vascular calcification. SMOC2 is a member of the BM-40/osteonectin family of calcium-binding secreted matricellular proteins. Using osteoprogenitor MC3T3-E1 cells stably overexpressing SMOC2, we show that SMOC2 inhibits osteogenic differentiation and extracellular matrix mineralization. Stable Smoc2 knockdown in these cells had no effect on mineralization suggesting that endogenous SMOC2 is not essential for the mineralization process. Mineralization in MC3T3-E1 cells overexpressing mutant SMOC2 lacking the extracellular calcium-binding domain was significantly increased compared to cells overexpressing full length SMOC2. When SMOC2 overexpressing cells were cultured in the presence of extracellular calcium supplementation, SMOC2's inhibitory effect on calcification was rescued. Our observations were translationally validated in primary human periosteal-derived cells. Furthermore, SMOC2 was able to impair mineralization in transdifferentiated human umbilical vein endothelial cells. Taken together, our data indicate that SMOC2 can act as an inhibitor of mineralization. We propose a possible role for SMOC2 to prevent calcification disorders., Competing Interests: The authors have declared that no competing interests exist.

Osteoporosis has been associated with pulmonary silicosis in California horses exposed to soils rich in cytotoxic silica dioxide crystals, a syndrome termed silicate associated osteoporosis (SAO). The causal mechanism for the development of osteoporosis is unknown. Osteoporotic lesions are primarily located in bone marrow-rich sites such as ribs, scapula and pelvis. Gene transcription patterns within bone marrow and pulmonary lymph nodes of affected horses may offer clues to disease pathobiology. Bone marrow core and tracheobronchial lymph node tissue samples harvested postmortem from affected and unaffected horses were examined histologically and subjected to RNA sequencing (RNA-seq). Sequenced data were analyzed for differential gene expression and gene ontology. Metatranscriptomic and metagenomic assays evaluated samples for infectious agents. Thirteen of 17 differentially expressed transcripts in bone marrow were linked to bone and cartilage formation such as integrin binding bone sialoprotein (log2FC = 3.39, PFDR = 0.013) and chondroadherin (log2FC = 4.48, PFDR = 0.031). Equus caballus solute carrier family 9, subfamily A2 (log2FC = 3.77, PFDR = 0.0034) was one of the four differentially expressed transcripts linked to osteoclast activity. Osteoblasts were hyperplastic and hypertrophic in bone marrow from affected horses. Biological pathways associated with skeletal morphogenesis were significantly enriched in affected horses. The 30 differentially expressed genes in affected lymph nodes were associated with inflammatory responses. Evidence of infectious agents was not found. The SAO affected bone marrow molecular signature demonstrated increased transcription and heightened activation of osteoblasts. Increased osteoblastic activity could be part of the pathological mechanism for osteoporosis or a compensatory response to the accelerated osteolysis. Transcriptome data offer gene targets for inquiries into the role of osteocytes and osteoblasts in SAO pathogenesis. Viral or bacterial infectious etiology in SAO is less likely based on metatranscriptomic and metagenomic data but cannot be completely ruled out., Competing Interests: The authors have declared that no competing interests exist.

Craniofacial abnormalities, including facial skeletal defects, comprise approximately one-third of all birth defects in humans. Since most bones in the face derive from cranial neural crest cells (CNCCs), which are multipotent stem cells, craniofacial bone disorders are largely attributed to defects in CNCCs. However, it remains unclear how the niche of CNCCs is coordinated by multiple gene regulatory networks essential for craniofacial bone development. Here we report that tumor suppressors breast cancer 1 (BRCA1) and breast cancer 2 (BRCA2) are required for craniofacial bone development in mice. Disruption of Brca1 in CNCC-derived mesenchymal cells, but not in epithelial-derived cells, resulted in craniofacial skeletal defects. Whereas osteogenic differentiation was normal, both osteogenic proliferation and survival were severely attenuated in Brca1 mutants. Brca1-deficient craniofacial skeletogenic precursors displayed increased DNA damage and enhanced cell apoptosis. Importantly, the craniofacial skeletal defects were sufficiently rescued by superimposing p53 null alleles in a neural crest-specific manner in vivo, indicating that BRCA1 deficiency induced DNA damage, cell apoptosis, and that the pathogenesis of craniofacial bone defects can be compensated by inactivation of p53. Mice lacking Brca2 in CNCCs, but not in epithelial-derived cells, also displayed abnormalities resembling the craniofacial skeletal malformations observed in Brca1 mutants. Our data shed light on the importance of BRCA1/BRCA2 function in CNCCs during craniofacial skeletal formation.

We have previously investigated the physiological role of C-type natriuretic peptide (CNP) on endochondral bone growth, mainly with mutant mouse models deficient in CNP, and reported that CNP is indispensable for physiological endochondral bone growth in mice. However, the survival rate of CNP knockout (KO) mice fell to as low as about 70% until 10 weeks after birth, and we could not sufficiently analyze the phenotype at the adult stage. Herein, we generated CNP KO rats by using zinc-finger nuclease-mediated genome editing technology. We established two lines of mutant rats completely deficient in CNP (CNP KO rats) that exhibited a phenotype identical to that observed in mice deficient in CNP, namely, a short stature with severely impaired endochondral bone growth. Histological analysis revealed that the width of the growth plate, especially that of the hypertrophic chondrocyte layer, was markedly lower and the proliferation of growth plate chondrocytes tended to be reduced in CNP KO rats. Notably, CNP KO rats did not have malocclusions and survived for over one year after birth. At 33 weeks of age, CNP KO rats persisted significantly shorter than wild-type rats, with closed growth plates of the femur in all samples, which were not observed in wild-type rats. Histologically, CNP deficiency affected only bones among all body tissues studied. Thus, CNP KO rats survive over one year, and exhibit a deficit in endochondral bone growth and growth retardation throughout life.

Human mesenchymal stem cell (hMSC)-derived exosomes have shown regenerative effects, but their role in osteogenesis and the underlying mechanism are yet to be determined. In this study, we examined the time-course secretion of exosomes by hMSCs during the entire process of osteogenic differentiation. Exosomes derived from hMSCs in various stages of osteogenic differentiation committed homotypic cells to differentiate towards osteogenic lineage, but only exosomes from late stages of osteogenic differentiation induced extracellular matrix mineralisation. Exosomes from expansion and early and late stages of osteogenic differentiation were internalised by a subpopulation of hMSCs. MicroRNA profiling revealed a set of differentially expressed exosomal microRNAs from the late stage of osteogenic differentiation, which were osteogenesis related. Target prediction demonstrated that these microRNAs enriched pathways involved in regulation of osteogenic differentiation and general mechanisms how exosomes exert their functions, such as "Wnt signalling pathway" and "endocytosis". Taken together, the results show that MSCs secrete exosomes with different biological properties depending on differentiation stage of their parent cells. The exosomal cargo transferred from MSCs in the late stage of differentiation induces osteogenic differentiation and mineralisation. Moreover, it is suggested that the regulatory effect on osteogenesis by exosomes is at least partly exerted by exosomal microRNA.

Wnt3a is a major regulator of bone metabolism however, very few of its target genes are known in bone. Wnt3a preferentially signals through transmembrane receptors Frizzled and co-receptors Lrp5/6 to activate the canonical signaling pathway. Previous studies have shown that the canonical Wnt co-receptors Lrp5 and Lrp6 also play an essential role in normal postnatal bone homeostasis, yet, very little is known about specific contributions by these co-receptors in Wnt3a-dependent signaling. We used high-throughput sequencing technology to identify target genes regulated by Wnt3a in osteoblasts and to elucidate the role of Lrp5 and Lrp6 in mediating Wnt3a signaling. Our study identified 782 genes regulated by Wnt3a in primary calvarial osteoblasts. Wnt3a up-regulated the expression of several key regulators of osteoblast proliferation/ early stages of differentiation while inhibiting genes expressed in later stages of osteoblastogenesis. We also found that Lrp6 is the key mediator of Wnt3a signaling in osteoblasts and Lrp5 played a less significant role in mediating Wnt3a signaling.

Gorlin syndrome is an autosomal dominant inherited syndrome that predisposes a patient to the formation of basal cell carcinomas, odontogenic keratocysts, and skeletal anomalies. Causative mutations in several genes associated with the sonic hedgehog (SHH) signaling pathway, including PTCH1, have been identified in Gorlin syndrome patients. However, no definitive genotype-phenotype correlations are evident in these patients, and their clinical presentation varies greatly, often leading to delayed diagnosis and treatment. We generated iPSCs from four unrelated Gorlin syndrome patients with loss-of-function mutations in PTCH1 using the Sendai virus vector (SeVdp(KOSM)302). The patient-derived iPSCs exhibited basic iPSC features, including stem cell marker expression, totipotency, and the ability to form teratomas. GLI1 expression levels were greater in fibroblasts and patient-derived iPSCs than in the corresponding control cells. Patient-derived iPSCs expressed lower basal levels than control iPSCs of the genes encoding the Hh ligands Indian Hedgehog (IHH) and SHH, the Hh acetyltransferase HHAT, Wnt proteins, BMP4, and BMP6. Most of these genes were upregulated in patient-derived iPSCs grown in osteoblast differentiation medium (OBM) and downregulated in control iPSCs cultured in OBM. The expression of GLI1 and GLI2 substantially decreased in both control and patient-derived iPSCs cultured in OBM, whereas GLI3, SHH, and IHH were upregulated in patient-derived iPSCs and downregulated in control iPSCs grown in OBM. Activation of Smoothened by SAG in cells grown in OBM significantly enhanced alkaline phosphatase activity in patient-derived iPSCs compared with control iPSC lines. In summary, patient-derived iPSCs expressed lower basal levels than the control iPSCs of the genes encoding Hh, Wnt, and bone morphogenetic proteins, but their expression of these genes strongly increased under osteogenic conditions. These findings indicate that patient-derived iPSCs are hypersensitive to osteogenic induction. We propose that Hh signaling is constituently active in iPSCs from Gorlin syndrome patients, enhancing their response to osteogenic induction and contributing to disease-associated abnormalities.

Skeletal development and mineralization are essential processes driven by the coordinated action of neural signals, circulating molecules and local factors. Our previous studies revealed that the novel neuropeptide Pth4, synthesized by hypothalamic cells, was involved in bone metabolism via phosphate regulation in adult zebrafish. Here, we investigate the role of pth4 during skeletal development using single-cell resolution, two-photon laser ablation of Pth4:eGFP-expressing cells and confocal imaging in vivo. Using a stable transgenic Pth4:eGFP zebrafish line, we identify Pth4:eGFP-expressing cells as post-mitotic neurons. After targeted ablation of eGFP-expressing cells in the hypothalamus, the experimental larvae exhibited impaired mineralization of the craniofacial bones whereas cartilage development was normal. In addition to a decrease in pth4 transcript levels, we noted altered expression of phex and entpd5, genes associated with phosphate homeostasis and mineralization, as well as a delay in the expression of osteoblast differentiation markers such as sp7 and sparc. Taken together, these results suggest that Pth4-expressing hypothalamic neurons participate in the regulation of bone metabolism, possibly through regulating phosphate balance during zebrafish development.

Tissue engineering using suitable mesenchymal stem cells (MSCs) shows great potential to regenerate bone defects. Our previous studies have indicated that human amnion-derived mesenchymal stem cells (HAMSCs) could promote the osteogenic differentiation of human bone marrow mesenchymal stem cells (HBMSCs). Human adipose-derived stem cells (HASCs), obtained from adipose tissue in abundance, are capable of multi-lineage differentiation. In this study, the effects of HAMSCs on osteogenic and angiogenic differentiation of HASCs were systematically investigated. Proliferation levels were measured by flow cytometry. Osteoblastic differentiation and mineralization were investigated using chromogenic alkaline phosphatase activity (ALP) activity substrate assays, Alizarin red S staining, real-time polymerase chain reaction (real-time PCR) analysis of osteogenic marker expression, and Western blotting. We found that HAMSCs increased the proliferation and osteoblastic differentiation of HASCs. Moreover, enzyme-linked immunosorbent assay (ELISA) and human umbilical vein endothelial cells (HUVECs) tube formation suggested HAMSCs enhanced angiogenic potential of HASCs via secretion of increased vascular endothelial growth factor (VEGF). Thus, we conclude that HAMSC might be a valuable therapeutic approach to promote HASCs-involved bone regeneration.

Epithelial-mesenchymal interactions drive embryonic fusion events during development, and perturbations of these interactions can result in birth defects. Cleft palate and neural tube defects can result from genetic defects or environmental exposures during development, yet very little is known about the effect of chemical exposures on fusion events during human development because of a lack of relevant and robust human in vitro assays of developmental fusion behavior. Given the etiology and prevalence of cleft palate and the relatively simple architecture and composition of the embryonic palate, we sought to develop a three-dimensional culture system that mimics the embryonic palate and could be used to study fusion behavior in vitro using human cells. We engineered size-controlled human Wharton's Jelly stromal cell (HWJSC) spheroids and established that 7 days of culture in osteogenesis differentiation medium was sufficient to promote an osteogenic phenotype consistent with embryonic palatal mesenchyme. HWJSC spheroids supported the attachment of human epidermal keratinocyte progenitor cells (HPEKp) on the outer spheroid surface likely through deposition of collagens I and IV, fibronectin, and laminin by mesenchymal spheroids. HWJSC spheroids coated in HPEKp cells exhibited fusion behavior in culture, as indicated by the removal of epithelial cells from the seams between spheroids, that was dependent on epidermal growth factor signaling and fibroblast growth factor signaling in agreement with palate fusion literature. The method described here may broadly apply to the generation of three-dimensional epithelial-mesenchymal co-cultures to study developmental fusion events in a format that is amenable to predictive toxicology applications.

BRIL (bone-restricted IFITM-like), is a short transmembrane protein expressed almost exclusively in osteoblasts. Although much is known about its bone-restricted gene expression pattern and protein biochemical and topological features, little information is available for BRIL physiological function. Two autosomal dominant forms of osteogenesis imperfecta (OI) are caused by distinct, but recurrent mutations in the BRIL gene. Yet, the underlying mechanisms by which those mutations lead to OI are still poorly understood. A previous report indicated that BRIL knockout (KO) mice had bone deformities, shortened long bones, and reproductive problems. Here we generated and systematically analyzed the skeletal phenotype of a new global Bril KO/LacZ knockin mouse model. KO mice reproduced and thrived normally up to 12 month of age. The skeletal phenotype of KO and WT littermates was assessed at embryonic (E13.5 to E18.5) and postnatal (2 days, 3 weeks, 3 months and 8 months) time-points. Embryos from E13.5 through to E18.5 showed significant X-Gal staining in all skeletal elements without any apparent patterning anomalies. Although bone deformities were never observed at any postnatal ages, minor and transient differences were noted in terms of bone length and static uCT parameters, but not systematically across all ages and genders. These changes, however, were not accompanied by significant alteration in bone material properties as assessed by a 3-point bending test. In addition, no changes were detected in circulating serum markers of bone turnover (P1NP, CTX-I, and osteocalcin). Gene expression monitoring also revealed no major impact of the loss of BRIL. Further, when mice were challenged with a surgically-induced fracture in tibia, bones repaired equally well in the KO mice as compared to WT. Finally, we showed that BRIL C-terminus is not a bona fide binding site for calcium. In conclusion, our in depth analysis suggest that skeletal patterning, bone mass accrual and remodeling in mice proceeded independent of BRIL.

Mesenchymal stem cell (MSC)-mediated tissue engineering represents a promising strategy to address adipose tissue defects. MSCs derived from Wharton's jelly of the umbilical cord (WJCMSCs) may serve as an ideal source for adipose tissue engineering due to their abundance, safety profile, and accessibility. How to activate the directed differentiation potentials of WJCMSCs is the core point for their clinical applications. A thorough investigation of mechanisms involved in WJCMSC adipogenic differentiation is necessary to support their application in adipose tissue engineering and address shortcomings. Previous study showed, compared with periodontal ligament stem cells (PDLSCs), WJCMSCs had a weakened adipogenic differentiation potentials and lower expression of insulin-like growth factor binding protein 2 (IGFBP2). IGFBP2 may be involved in the adipogenesis of MSCs. Generally, IGFBP2 is involved in regulating biological activity of insulin-like growth factors, however, its functions in human MSCs are unclear. Here, we found IGFBP2 expression was upregulated upon adipogenic induction, and that IGFBP2 enhanced adipogenic differentiation of WJCMSCs and BMSCs. Moreover, IGFBP2 increased phosphorylation of c-Jun N-terminal kinase (p-JNK) and p-Akt, and activated JNK or Akt signaling significantly promoted adipogenic differentiation of MSCs. Furthermore, inhibitor-mediated blockage of either JNK or Akt signaling dramatically reduced IGFBP2-mediated adipogenic differentiation. And the JNK inhibitor, SP600125 markedly blocked IGFBP2-mediated Akt activation. Moreover, IGFBP2 was negatively regulated by BCOR, which inhibited adipogenic differentiation of WJCMSCs. Overall, our results reveal a new function of IGFBP2, providing a novel insight into the mechanism of adipogenic differentiation and identifying a potential target mediator for improving adipose tissue engineering based on WJCMSCs.

The anaphylatoxin receptor C5aR1 plays an important role not only in innate immune responses, but also in bone metabolism and fracture healing, being highly expressed on immune and bone cells, including osteoblasts and osteoclasts. C5aR1 induces osteoblast migration, cytokine generation and osteoclastogenesis, however, the exact role of C5aR1-mediated signaling in osteoblasts is not entirely known. Therefore, we hypothesized that osteoblasts are essential target cells for C5a and that fracture healing should be disturbed in mice with an osteoblast-specific C5aR1 overexpression (Col1a1-C5aR1). Osteoblast activity in vitro, bone phenotype and fracture healing after isolated osteotomy and after combined osteotomy with additional thoracic trauma were analyzed. The systemic and local inflammatory reactions were analyzed by determining C5a and IL-6 concentrations in blood, bronchoalveolar lavage fluid and fracture callus and the recruitment of immune cells. In vitro, osteoblast proliferation and differentiation were similar to wildtype cells, and phosphorylation of p38 and expression of IL-6 and RANKL were increased in osteoblasts derived from Col1a1-C5aR1 mice. Bone phenotype and the inflammatory reaction were unaffected in Col1a1-C5aR1 mice. Fracture healing was significantly impaired as demonstrated by significantly reduced bone content, bone mineral density and flexural rigidity, possibly due to significantly increased osteoclast numbers. C5aR1 signaling in osteoblasts might possibly affect RANKL/OPG balance, leading to increased bone resorption. Additional trauma significantly impaired fracture healing, particularly in Col1a1-C5aR1 mice. In conclusion, the data indicate that C5aR1 signaling in osteoblasts plays a detrimental role in bone regeneration after fracture.

To improve the safe use of allograft bone, decellularization techniques may be utilized to produce acellular scaffolds. Such scaffolds should retain their innate biological and biomechanical capacity and support mesenchymal stem cell (MSC) osteogenic differentiation. However, as allograft bone is derived from a wide age-range, this study aimed to determine whether donor age impacts on the ability an osteoinductive, acellular scaffold produced from human bone to promote the osteogenic differentiation of bone marrow MSCs (BM-MSC). BM-MSCs from young and old donors were seeded on acellular bone cubes from young and old donors undergoing osteoarthritis related hip surgery. All combinations resulted in increased osteogenic gene expression, and alkaline phosphatase (ALP) enzyme activity, however BM-MSCs cultured on old donor bone displayed the largest increases. BM-MSCs cultured in old donor bone conditioned media also displayed higher osteogenic gene expression and ALP activity than those exposed to young donor bone conditioned media. ELISA and Luminex analysis of conditioned media demonstrated similar levels of bioactive factors between age groups; however, IGF binding protein 1 (IGFBP1) concentration was significantly higher in young donor samples. Additionally, structural analysis of old donor bone indicated an increased porosity compared to young donor bone. These results demonstrate the ability of a decellularized scaffold produced from young and old donors to support osteogenic differentiation of cells from young and old donors. Significantly, the older donor bone produced greater osteogenic differentiation which may be related to reduced IGFBP1 bioavailability and increased porosity, potentially explaining the excellent clinical results seen with the use of allograft from aged donors.

Gamma-glutamyl carboxylase (GGCX) gene mutation causes GGCX syndrome (OMIM: 137167), which is characterized by pseudoxanthoma elasticum (PXE)-like symptoms and coagulation impairment. Here, we present a 55-year-old male with a novel homozygous deletion mutation, c.2,221delT, p.S741LfsX100, in the GGCX gene. Histopathological examination revealed calcium deposits in elastic fibers and vessel walls, and collagen accumulation in the mid-dermis. Studies of dermal fibroblasts from the patient (GGCX dermal fibroblasts) demonstrated that the mutated GGCX protein was larger, but its expression level and intracellular distribution were indistinguishable from those of the wild-type GGCX protein. Immunostaining and an enzyme-linked immunosorbent assay showed an increase in undercarboxylated matrix gamma-carboxyglutamic acid protein (ucMGP), a representative substrate of GGCX and a potent calcification inhibitor, indicating that mutated GGCX was enzymatically inactive. Under osteogenic conditions, calcium deposition was exclusively observed in GGCX dermal fibroblasts. Furthermore, GGCX dermal fibroblast cultures contained 23- and 7.7-fold more alkaline phosphatase (ALP)-positive cells than normal dermal fibroblast cultures (n = 3), without and with osteogenic induction, respectively. Expression and activity of ALP were higher in GGCX dermal fibroblasts than in normal dermal fibroblasts upon osteogenic induction. mRNA levels of other osteogenic markers were also higher in GGCX dermal fibroblasts than in normal dermal fibroblasts, which including bone morphogenetic protein 6, runt-related transcription factor 2, and periostin (POSTN) without osteogenic induction; and osterix, collagen type I alpha 2, and POSTN with osteogenic induction. Together, these data indicate that GGCX dermal fibroblasts trans-differentiate into the osteogenic lineage. This study proposes another mechanism underlying aberrant calcification in patients with GGCX syndrome.

Background and Aim: The biological functions of non-coding RNAs (ncRNAs) have been widely identified in many human diseases. In the present study, the relationship between long non-coding RNA HOTAIR and microRNA-17-5p (miR-17-5p) and their roles in osteogenic differentiation and proliferation in non-traumatic osteonecrosis of femoral head (ONFH) were investigated., Methods: The expression levels of HOTAIR and miR-17-5p in the mesenchymal stem cells (MSCs) derived from patients with non-traumatic ONFH and osteoarthritis (OA) were examined by real-time PCR. BMP-2 induced human MSCs from bone marrow (hMSC-BM) were used for osteogenic differentiation., Results: It was observed that the expression level of miR-17-5p was lower and the level of HOTAIR was higher in samples of non-traumatic ONFH compared with OA. HOTAIR downregulation induced by si-HOTAIR led to the increase of miR-17-5p expression and the decrease of miR-17-5p target gene SMAD7 expression. The values of osteogenic differentiation markers, including RUNX2 and COL1A1 mRNA expression and ALP activity, were also elevated by si-HOTAIR. However, the increase of these values was canceled by miR-17-5p inhibitor or SMAD7 upregulation., Conclusion: HOTAIR played a role in regulating osteogenic differentiation and proliferation through modulating miR-17-5p and its target gene SMAD7 in non-traumatic ONFH.

To date, mutations within the coding region and translocations around the SOX9 gene both constitute the majority of genetic lesions underpinning human campomelic dysplasia (CD). While pathological coding-region mutations typically result in a non-functional SOX9 protein, little is known about what mechanism(s) controls normal SOX9 expression, and subsequently, which signaling pathways may be interrupted by alterations occurring around the SOX9 gene. Here, we report the identification of Stat3 as a key modulator of Sox9 expression in nascent cartilage and developing chondrocytes. Stat3 expression is predominant in tissues of mesodermal origin, and its conditional ablation using mesoderm-specific TCre, in vivo, causes dwarfism and skeletal defects characteristic of CD. Specifically, Stat3 loss results in the expansion of growth plate hypertrophic chondrocytes and deregulation of normal endochondral ossification in all bones examined. Conditional deletion of Stat3 with a Sox9Cre driver produces palate and tracheal irregularities similar to those described in Sox9+/- mice. Furthermore, mesodermal deletion of Stat3 causes global embryonic down regulation of Sox9 expression and function in vivo. Mechanistic experiments ex vivo suggest Stat3 can directly activate the expression of Sox9 by binding to its proximal promoter following activation. These findings illuminate a novel role for Stat3 in chondrocytes during skeletal development through modulation of a critical factor, Sox9. Importantly, they further provide the first evidence for the modulation of a gene product other than Sox9 itself which is capable of modeling pathological aspects of CD and underscore a potentially valuable therapeutic target for patients with the disorder.