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.

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.

Concurrent targeting of BMI1 and CDK4/6 abrogates tumor growth in vitro and in vivo.

Despite recent advances in cancer management and therapy, resistance to cytotoxic medications remains a major clinical challenge; hence, combination-based anti-cancer treatment regimens are currently gaining momentum. PTC-209 reduced BMI1 protein expression, while palbociclib inhibited CDK4, Rb, and pRbSer795 protein expression in MDA-MB-231 cells. PTC-209 and palbociclib exhibited dose-dependent cytotoxic effects against MDA-MB-231 (breast), HCT116 (colon), and PC-3 (prostate) models, which was more profound in the combination group. Transcriptome and pathway analyses revealed inhibition of insulin signaling, focal adhesion, DNA damage response, and Wnt/pluripotency signaling pathways as well as cell proliferation, and cellular movement functional categories by PTC-209. Transcriptome and pathway analyses revealed palbociclib to mainly affect cell cycle progression and survival. Upstream analysis identified several networks affected by PTC-209 (EZH2, IFNB1, TRIB3, EGFR, SREBF1, IL1A, ERG, TGFB1, MAX, MNT) and palbociclib (RABL6, MITF, RARA, TAL1, AREG, E2F3, FOXM1, ESR1, ERBB2, and E2F). PTC-209 and palbociclib reduced colony and sphere formation, cell migration, and cell viability, which was further enhanced in the combination group. Concordantly, combination of PTC-209 and palbociclib exhibited more profound effects on MDA-MB-231 tumor formation in vivo. Our data suggest concurrent targeting of BMI1 and CDK4/CDK6 might provide novel therapeutic opportunity for breast, colon, and prostate cancer.