Transient downregulation of NR4A1 leads to impaired osteoblast differentiation through the TGF-ß pathway, and Elesclomol (STA-4783) rescues this phenotype.

Bone formation is regulated by numerous factors, such as transcription factors, cytokines, and extracellular matrix molecules. Human hormone nuclear receptors (hHNR) are a family of ligand-regulated transcription factors that are activated by steroid hormones, such as estrogen and progesterone, and various lipid-soluble signals, including retinoic acid, oxysterols, and thyroid hormone. We found that an hHNR called NR4A1 was the most highly expressed after human MSC differentiation into osteoblasts by whole-genome microarray. NR4A1 knockout decreased the osteoblastic differentiation of hMSCs in terms of ALPL expression and key marker gene expression. Whole-genome microarray analysis further confirmed the decrease in key pathways when we knocked down NR4A1. Further studies with small molecule activators identified a novel molecule called Elesclomol (STA-4783), which could activate and enhance osteoblast differentiation. Elesclomol activation of hMSCs also induced the gene expression of NR4A1 and rescued the phenotype of NR4A1 KD. In addition, Elesclomol activated the TGF-ß pathway by regulating key marker genes. In conclusion, we first identified the role of NR4A1 in osteoblast differentiation and that Elesclomol is a positive regulator of NR4A1 through activation of the TGF-ß signalling pathway.

Inhibition of TGF-ß type I receptor by SB505124 down-regulates osteoblast differentiation and mineralization of human mesenchymal stem cells.

Numerous signaling pathways are well-known in osteoblastic differentiation of human bone marrow mesenchymal stem cells (hBMSCs), including transforming growth factor-beta (TGF-ß) signaling pathway, which sends signals through specific type I and II serine/threonine kinase receptors. However, the key role of TGF-ß signaling during bone formation and remodeling is yet to be studied. A TGF-ß type I receptor inhibitor, SB505124, discovered through a screening of a small molecule library for their effect of osteoblast differentiation of hBMSCs. Alkaline phosphatase quantification and staining were tested as indicators of osteoblastic differentiation and Alizarin red staining was tested as an indicator of in vitro mineralization. Changes in gene expressions were assessed using qRT-PCR. SB505124 showed significant inhibition of the osteoblast differentiation of hBMSCs, as confirmed by reduced alkaline phosphatase, in vitro mineralization, and downregulation of osteoblast-associated gene expression. To further understand the molecular mechanisms involved in the inhibition of the TGF-ß type I receptor, we assessed the effects on signature genes of several signaling pathways identified in the osteoblast differentiation of hBMSCs. SB505124 downregulated gene expression of many genes linked to osteoblast-related signaling pathways including TGF-ß, insulin, focal adhesion, Notch, Vitamin D, interleukin (IL)-6, osteoblast signaling, and cytokines and inflammatory. We report TGF-ß type I receptor inhibitor (SB505124) is a potent inhibitor of osteoblastic differentiation of hBMSCs that could be a valuable innovative therapeutic tool to cure bone disorders with increased bone formation, besides its potential use to treat patients with cancer and fibrosis.

Inhibition of GSK-3ß Enhances Osteoblast Differentiation of Human Mesenchymal Stem Cells through Wnt Signalling Overexpressing Runx2.

Small-molecule-inhibitor-based bone differentiation has been recently exploited as a novel approach to regulating osteogenesis-related signaling pathways. In this study, we identified 1-Azakenpaullone, a highly selective inhibitor of glycogen synthase kinase-3ß (GSK-3ß), as a powerful inducer of osteoblastic differentiation and mineralization of human mesenchymal stem cells (MSCs). GSK-3ß is a serine-threonine protein kinase that plays a major role in different disease development. GSK-3ß is a key regulator of Runx2 activity in osteoblastic formation. We evaluated alkaline phosphatase activity and staining assays to assess osteoblast differentiation and Alizarin Red staining to assess the mineralization of cultured human MSCs. Gene expression profiling was assessed using an Agilent microarray platform, and bioinformatics were performed using Ingenuity Pathway Analysis software. Human MSCs treated with 1-Azakenpaullone showed higher ALP activity, increased in vitro mineralized matrix formation, and the upregulation of osteoblast-specific marker gene expression. Global gene expression profiling of 1-Azakenpaullone-treated human MSCs identified 1750 upregulated and 2171 downregulated mRNA transcripts compared to control cells. It also suggested possible changes in various signaling pathways, including Wnt, TGFß, and Hedgehog. Further bioinformatics analysis employing Ingenuity Pathway Analysis recognized significant enrichment in the 1-Azakenpaullone-treated cells of genetic networks involved in CAMP, PI3K (Complex), P38 MAPK, and HIF1A signaling and functional categories associated with connective tissue development. Our results suggest that 1-Azakenpaullone significantly induced the osteoblastic differentiation and mineralization of human MSCs mediated by the activation of Wnt signaling and the nuclear accumulation of ß-catenin, leading to the upregulation of Runx2, a key transcription factor that ultimately promotes the expression of osteoblast-specific genes. Thus, 1-Azakenpaullone could be used as an osteo-promotor factor in bone tissue engineering.

Evaluation of a hyaluronic acid hydrogel (Restylane Lyft) as a scaffold for dental pulp regeneration in a regenerative endodontic organotype model.

Scaffolds are crucial elements for dental pulp regeneration. Most of the currently used scaffolds in regenerative endodontic procedures (REPs) are unsuitable for chairside clinical use. This study aimed to evaluate the effect of an injectable synthetic scaffold (Restylane Lyft) on human bone marrow mesenchymal stem cell (hBMSC) viability, proliferation, and osteo/dentinogenic differentiation in a regenerative endodontic organotype model (REM). hBMSC were loaded in an REM either alone (hBMSC group) or mixed with the Restylane Lyft scaffold (Restylane/hBMSC group) and cultured in basal culture medium (n = 9/group). hMSC on culture plates served as controls. Cell viability and proliferation were measured using AlamarBlue assay. The loaded REM was cultured in an osteogenic differentiation medium to measure alkaline phosphatase activity (ALP) and examine the expression of the osteo/dentinogenic markers using real-time reverse transcriptase polymerase chain reaction. Cell viability in all groups increased significantly over 5 days. The Restylane/hBMSC group showed significantly higher ALP activity and dentin sialophosphoprotein, osteocalcin, and bone sialoprotein genes expression than the hBMSC and the control groups. Restylane Lyft, a hyaluronic acid (HA) injectable, FDA-approved hydrogel, maintained cell viability and proliferation and promoted osteo/dentinogenic differentiation of hBMSC when cultured in an REM. Henceforth, it could be a promising chairside scaffold material for REPs.

Silver Nanoparticles Alone or in Combination with Calcium Hydroxide Modulate the Viability, Attachment, Migration, and Osteogenic Differentiation of Human Mesenchymal Stem Cells.

This study aimed to evaluate the effect of silver nanoparticles (AgNPs) alone or in combination with calcium hydroxide (Ca(OH)2) on the proliferation, viability, attachment, migration, and osteogenic differentiation of human mesenchymal stem cells (hMSCs). Different concentrations of AgNPs alone or mixed with Ca(OH)2 were prepared. Cell proliferation was measured using AlamarBlue, and hMSCs attachment to dentin disks was evaluated using scanning electron microscopy. Live-dead imaging was performed to assess apoptosis. Wound healing ability was determined using the scratch-migration assay. To evaluate osteogenic differentiation, the expression of Runt-related transcription factor (RUNX2), Transforming growth factor beta-1 (TGF-ß1), Alkaline Phosphatase (ALP), and Osteocalcin (OCN) were measured using real-time reverse transcriptase polymerase chain reaction. ALP staining and activity were also performed as indicators of osteogenic differentiation. AgNPs alone seemed to favor cell attachment. Lower concentrations of AgNPs enhanced cell proliferation. AgNP groups showed markedly less apoptosis. None of the medicaments had adverse effects on wound closure. The expression of TGF-ß1 was significantly upregulated in all groups, and OCN was highly expressed in the AgNP groups. AgNPs 0.06% showed the most enhanced ALP gene expression levels, activity, and marked cytochemical staining. In conclusion, AgNPs positively affect hMSCs, making them a potential biomaterial for various clinical applications.

Assessment of the effect of silica calcium phosphate nanocomposite on mesenchymal stromal cell differentiation and bone regeneration in critical size defect.

OBJECTIVE: The research was designed to assess silica calcium phosphate nanocomposite (SCPC) biocompatibility and bioactivity as an osteoinductive scaffold and cell carrier. Consequently, the ability of cell seeded SCPC implant to regenerate a critical size defect in rat calvarium. MATERIALS AND METHODS: The study was conducted in two parts. A series of in vitro experiments on bone marrow stromal cells (MSCs) seeded in the SCPC scaffold evaluated cell attachment, proliferation and osteogenic differentiation. In the second part, a cell seeded SCPC construct was implanted in rat calvarium and bone regeneration was assessed by histological examination to evaluate the newly formed bone quality and the residual graft volume. RESULTS: In vitro experimentation revealed that MSCs cultured on SCPC maintained viability and proliferation when seeded into the SCPC. Scanning electron microscopy demonstrated cell adhesion and calcium appetite formation, MSCs differentiated towards the osteogenic lineage as indicated by the upregulation of RUNX2, ALP, Col1a1 markers. Histological examination showed regeneration from the periphery and core of the defect with new bone formation at different stages of maturation. CONCLUSION: Regenerative medicine delivers promising solutions and technologies for application in craniofacial reconstruction. SCPC scaffold has the potential to be used as a cell carrier to achieve stem cell-based bone regeneration, which provides a viable alternative for treatment of challenging critical size defect.

Evaluation of the regenerative potential of decellularized skeletal muscle seeded with mesenchymal stromal cells in critical-sized bone defect of rat models.

BACKGROUND: The morbidities and complications reported in the reconstruction of large bony defects have inspired progression in the field of bioengineering, with a recent breakthrough for the use of decellularized skeletal muscle grafts (DSMG). AIM: To assess the osteogenic potentials of seeded DSMG in vitro and to investigate bone regeneration in critical size defect in vivo. MATERIALS AND METHODS: Assessment of cell viability and characterization was carried out on seeded DSMG for different intervals in vitro. For in vivo experiments, histological analysis was performed for rat cranial defects for the following groups: (A) non-treated DSMG and (B) seeded DSMG after a period of 8 weeks. RESULTS: The in vitro experiment demonstrated the lack of cytotoxicity and inert properties of seeded DSMG; these facilitated the osteogenic differentiation and significant gene expressions, particularly of COL1A1, RUNX2, and OPN (1.9174 ± 0.11673, 1.1806 ± 0.02383, and 1.1802 ± 0.00775, respectively). In the in vivo experiment, superior results were detected in the seeded DSMG group which showed highly vascularized and cellular dense connective tissue with deposited bone matrix and multiple scattered islets of newly formed bone. CONCLUSION: Our results demonstrated the promising aspects of DSMG; however, there is a lack of studies to support further implications.

JAK2 Inhibition by Fedratinib Reduces Osteoblast Differentiation and Mineralisation of Human Mesenchymal Stem Cells.

Several signalling pathways, including the JAK/STAT signalling pathway, have been identified to regulate the differentiation of human bone marrow skeletal (mesenchymal) stem cells (hBMSCs) into bone-forming osteoblasts. Members of the JAK family mediate the intracellular signalling of various of cytokines and growth factors, leading to the regulation of cell proliferation and differentiation into bone-forming osteoblastic cells. Inhibition of JAK2 leads to decoupling of its downstream mediator, STAT3, and the subsequent inhibition of JAK/STAT signalling. However, the crucial role of JAK2 in hBMSCs biology has not been studied in detail. A JAK2 inhibitor, Fedratinib, was identified during a chemical biology screen of a small molecule library for effects on the osteoblastic differentiation of hMSC-TERT cells. Alkaline phosphatase activity and staining assays were conducted as indicators of osteoblastic differentiation, while Alizarin red staining was used as an indicator of in vitro mineralised matrix formation. Changes in gene expression were assessed using quantitative real-time polymerase chain reaction. Fedratinib exerted significant inhibitory effects on the osteoblastic differentiation of hMSC-TERT cells, as demonstrated by reduced ALP activity, in vitro mineralised matrix formation and downregulation of osteoblast-related gene expression, including ALP, ON, OC, RUNX2, OPN, and COL1A1. To identify the underlying molecular mechanisms, we examined the effects of Fedratinib on a molecular signature of several target genes known to affect hMSC-TERT differentiation into osteoblasts. Fedratinib inhibited the expression of LIF, SOCS3, RRAD, NOTCH3, TNF, COMP, THBS2, and IL6, which are associated with various signalling pathways, including TGFß signalling, insulin signalling, focal adhesion, Notch Signalling, IL-6 signalling, endochondral ossification, TNF-α, and cytokines and inflammatory response. We identified a JAK2 inhibitor (Fedratinib) as a powerful inhibitor of the osteoblastic differentiation of hMSC-TERT cells, which may be useful as a therapeutic option for treating conditions associated with ectopic bone formation or osteosclerotic metastases.

Tankyrase inhibitor XAV-939 enhances osteoblastogenesis and mineralization of human skeletal (mesenchymal) stem cells.

Tankyrase is part of poly (ADP-ribose) polymerase superfamily required for numerous cellular and molecular processes. Tankyrase inhibition negatively regulates Wnt pathway. Thus, Tankyrase inhibitors have been extensively investigated for the treatment of clinical conditions associated with activated Wnt signaling such as cancer and fibrotic diseases. Moreover, Tankyrase inhibition has been recently reported to upregulate osteogenesis through the accumulation of SH3 domain-binding protein 2, an adaptor protein required for bone metabolism. In this study, we investigated the effect of Tankyrase inhibition in osteoblast differentiation of human skeletal (mesenchymal) stem cells (hMSCs). A Tankyrase inhibitor, XAV-939, identified during a functional library screening of small molecules. Alkaline phosphatase activity and Alizarin red staining were employed as markers for osteoblastic differentiation and in vitro mineralized matrix formation, respectively. Global gene expression profiling was performed using the Agilent microarray platform. XAV-939, a Tankyrase inhibitor, enhanced osteoblast differentiation of hBMSCs as evidenced by increased ALP activity, in vitro mineralized matrix formation, and upregulation of osteoblast-related gene expression. Global gene expression profiling of XAV-939-treated cells identified 847 upregulated and 614 downregulated mRNA transcripts, compared to vehicle-treated control cells. It also points towards possible changes in multiple signaling pathways, including TGFß, insulin signaling, focal adhesion, estrogen metabolism, oxidative stress, RANK-RANKL (receptor activator of nuclear factor κB ligand) signaling, Vitamin D synthesis, IL6, and cytokines and inflammatory responses. Further bioinformatic analysis, employing Ingenuity Pathway Analysis identified significant enrichment in XAV-939-treated cells of functional categories and networks involved in TNF, NFκB, and STAT signaling. We identified a Tankyrase inhibitor (XAV-939) as a powerful enhancer of osteoblastic differentiation of hBMSC that may be useful as a therapeutic option for treating conditions associated with low bone formation.

Hedgehog Signaling Inhibition by Smoothened Antagonist BMS-833923 Reduces Osteoblast Differentiation and Ectopic Bone Formation of Human Skeletal (Mesenchymal) Stem Cells.

BACKGROUND: Hedgehog (Hh) signaling is essential for osteoblast differentiation of mesenchymal progenitors during endochondral bone formation. However, the critical role of Hh signaling during adult bone remodeling remains to be elucidated. METHODS: A Smoothened (SMO) antagonist/Hedgehog inhibitor, BMS-833923, identified during a functional screening of a stem cell signaling small molecule library, was investigated for its effects on the osteoblast differentiation of human skeletal (mesenchymal) stem cells (hMSC). Alkaline phosphatase (ALP) activity and Alizarin red staining were employed as markers for osteoblast differentiation and in vitro mineralization capacity, respectively. Global gene expression profiling was performed using the Agilent® microarray platform. Effects on in vivo ectopic bone formation were assessed by implanting hMSC mixed with hydroxyapatite-tricalcium phosphate granules subcutaneously in 8-week-old female nude mice, and the amount of bone formed was assessed using quantitative histology. RESULTS: BMS-833923, a SMO antagonist/Hedgehog inhibitor, exhibited significant inhibitory effects on osteoblast differentiation of hMSCs reflected by decreased ALP activity, in vitro mineralization, and downregulation of osteoblast-related gene expression. Similarly, we observed decreased in vivo ectopic bone formation. Global gene expression profiling of BMS-833923-treated compared to vehicle-treated control cells, identified 348 upregulated and 540 downregulated genes with significant effects on multiple signaling pathways, including GPCR, endochondral ossification, RANK-RANKL, insulin, TNF alpha, IL6, and inflammatory response. Further bioinformatic analysis employing Ingenuity Pathway Analysis revealed significant enrichment in BMS-833923-treated cells for a number of functional categories and networks involved in connective and skeletal tissue development and disorders, e.g., NFκB and STAT signaling. CONCLUSIONS: We identified SMO/Hedgehog antagonist (BMS-833923) as a powerful inhibitor of osteoblastic differentiation of hMSC that may be useful as a therapeutic option for treating conditions associated with high heterotopic bone formation and mineralization.

Notch Signaling Inhibition by LY411575 Attenuates Osteoblast Differentiation and Decreased Ectopic Bone Formation Capacity of Human Skeletal (Mesenchymal) Stem Cells.

BACKGROUND: Chemical biology approaches using small molecule inhibitors targeting specific signaling pathways are useful tools to dissect the molecular mechanisms governing stem cell differentiation and for their possible use in therapeutic interventions. METHODS: Stem cell signaling small molecule library functional screen was performed employing human bone marrow skeletal (mesenchymal) stem cells (hBMSCs). Alkaline phosphatase (ALP) activity and formation of mineralized matrix visualized by Alizarin red staining were employed as markers for osteoblastic differentiation. Global gene expression profiling was conducted using the Agilent microarray platform, and data normalization and bioinformatics were performed using GeneSpring software. Pathway analyses were conducted using the Ingenuity Pathway Analysis (IPA) tool. In vivo ectopic bone formation was performed using hBMSC mixed with hydroxyapatite-tricalcium phosphate granules that were implanted subcutaneously in 8-week-old female nude mice. Hematoxylin and eosin staining and Sirius red staining were performed to identify bone formation in vivo. RESULTS: Among the tested molecules, LY411575, a potent γ-secretase and Notch signaling inhibitor, exhibited significant inhibitory effects on osteoblastic differentiation of hBMSCs manifested by reduced ALP activity, mineralized matrix formation, and decreased osteoblast-specific gene expression as well as in vivo ectopic bone formation. Global gene expression profiling of LY411575-treated cells revealed changes in multiple signaling pathways, including focal adhesion, insulin, TGFß, IL6, and Notch signaling, and decreased the expression of genes associated with functional categories of tissue development. Among the affected signaling networks were TGFß1, SPP1, and ERK regulatory networks. CONCLUSIONS: We identified γ-secretase inhibitor (LY411575) as a potent regulator of osteoblastic differentiation of hBMSC that may be useful as a therapeutic option for treating conditions associated with ectopic bone formation.

Stem cell library screen identified ruxolitinib as regulator of osteoblastic differentiation of human skeletal stem cells.

BACKGROUND: Better understanding of the signaling pathways that regulate human bone marrow stromal stem cell (hBMSC) differentiation into bone-forming osteoblasts is crucial for their clinical use in regenerative medicine. Chemical biology approaches using small molecules targeting specific signaling pathways are increasingly employed to manipulate stem cell differentiation fate. METHODS: We employed alkaline phosphatase activity and staining assays to assess osteoblast differentiation and Alizarin R staining to assess mineralized matrix formation of cultured hBMSCs. Changes in gene expression were assessed using an Agilent microarray platform, and data normalization and bioinformatics were performed using GeneSpring software. For in vivo ectopic bone formation experiments, hMSCs were mixed with hydroxyapatite-tricalcium phosphate granules and implanted subcutaneously into the dorsal surface of 8-week-old female nude mice. Hematoxylin and eosin staining and Sirius Red staining were used to detect bone formation in vivo. RESULTS: We identified several compounds which inhibited osteoblastic differentiation of hMSCs. In particular, we identified ruxolitinib (INCB018424) (3 µM), an inhibitor of JAK-STAT signaling that inhibited osteoblastic differentiation and matrix mineralization of hMSCs in vitro and reduced ectopic bone formation in vivo. Global gene expression profiling of ruxolitinib-treated cells identified 847 upregulated and 822 downregulated mRNA transcripts, compared to vehicle-treated control cells. Bioinformatic analysis revealed differential regulation of multiple genetic pathways, including TGFß and insulin signaling, endochondral ossification, and focal adhesion. CONCLUSIONS: We identified ruxolitinib as an important regulator of osteoblast differentiation of hMSCs. It is plausible that inhibition of osteoblast differentiation by ruxolitinib may represent a novel therapeutic strategy for the treatment of pathological conditions caused by accelerated osteoblast differentiation and mineralization.