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 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.

Profiling of G-Protein Coupled Receptors in Adipose Tissue and Differentiating Adipocytes Offers a Translational Resource for Obesity/Metabolic Research.

G protein-coupled receptors (GPCRs) are expressed essentially on all cells, facilitating cellular responses to external stimuli, and are involved in nearly every biological process. Several members of this family play significant roles in the regulation of adipogenesis and adipose metabolism. However, the expression and functional significance of a vast number of GPCRs in adipose tissue are unknown. We used a high-throughput RT-PCR panel to determine the expression of the entire repertoire of non-sensory GPCRs in mouse white, and brown adipose tissue and assess changes in their expression during adipogenic differentiation of murine adipocyte cell line, 3T3-L1. In addition, the expression of GPCRs in subcutaneous adipose tissues from lean, obese, and diabetic human subjects and in adipocytes isolated from regular chow and high-fat fed mice were evaluated by re-analyzing RNA-sequencing data. We detected a total of 292 and 271 GPCRs in mouse white and brown adipose tissue, respectively. There is a significant overlap in the expression of GPCRs between the two adipose tissue depots, but several GPCRs are specifically expressed in one of the two tissue types. Adipogenic differentiation of 3T3-L1 cells had a profound impact on the expression of several GPCRs. RNA sequencing of subcutaneous adipose from healthy human subjects detected 255 GPCRs and obesity significantly changed the expression of several GPCRs in adipose tissue. High-fat diet had a significant impact on adipocyte GPCR expression that was similar to human obesity. Finally, we report several highly expressed GPCRs with no known role in adipose biology whose expression was significantly altered during adipogenic differentiation, and/or in the diseased human subjects. These GPCRs could play an important role in adipose metabolism and serve as a valuable translational resource for obesity and metabolic research.

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.

A New Procedure in Bone Engineering Using Induced Adipose Tissue.

Background: Osteoporosis is associated with a metabolic imbalance between adipogenesis and osteogenesis. We hypothesized that implanting a carrier for differentiated stem cells and signaling molecules inside adipose tissues could be used to enable transdifferentiation between cells, upregulate osteogenesis, and support bone formation, which may regain the balance between osteogenesis and adipogenesis. Methodology: A CL1 human mesenchymal stem cell line was grown in an osteogenic medium to differentiate into osteoblasts, and the differentiated cells were then exposed to an adipogenic medium to stimulate differentiation into adipocytes. Osteogenic and adipogenic differentiation were confirmed by the following assays: alkaline phosphatase staining, Nile red Staining, and quantitative real-time polymerase chain reaction (qPCR). The ratio of adipocytes to osteocytes for both cases was calculated. To evaluate bone induction in vivo, a calcium sulfate/hydroxyapatite cement was prepared in a syringe and then seeded with 106 cells/mL of rat bone marrow stromal cells (rMSCs) and covered with 1 mL of tissue culture media containing 0.1 mg of bone morphogenetic protein 7 (BMP-7). The construct was injected into the abdominal fat tissue of 10 male Sprague-Dawley rats. Results: The conversion of osteocytes to adipocytes was 20-fold greater than the reverse conversion, and the area of bone regeneration was 15.7 ± 3.7%, the area of adipose tissue was 65.8 ± 13.1%, and the area of fibrous tissue was 18.3 ± 7.8%. Conclusion: Adipogenic interconversion and associated bone formation demonstrate the potential of a new therapy for balancing osteogenesis and adipogenesis.

The use of muscle extracellular matrix (MEM) and SCPC bioceramic for bone augmentation.

BACKGROUND: Bone augmentation is a challenging problem in the field of maxillofacial surgery. OBJECTIVE: In this study, we prepared and evaluated muscle extracellular matrix (MEM) after adding silica calcium phosphate composite (SCPC) seeded with human bone marrow mesenchymal cells (hBMSCs). We then investigated bone augmentation in vivo using the prepared MEM-SCPC. MATERIALS AND METHODS: hBMSCs were seeded on MEM-SCPC, and MEM was characterized. Calvarial bone grafts were prepared using nude mice (n = 12) and grafted separately in two experimental groups: grafts with MEM (control, n = 4) and grafts with MEM-SCPC-hBMSCs (experimental group, n = 8) for 8 weeks. Micro-computed tomography (micro-CT) and histological analysis were then performed. RESULTS: Micro-CT analysis demonstrated a thinner trabeculae in grafted defects than normal native bone, with a high degree of anisotropy. Quantitative histomorphometric assessment showed a higher median bone percentage surface area of 80.2% ± 6.0% in the experimental group. CONCLUSION: The enhanced bone formation and maturation of bone grafted with MEM-SCPC-hBMSCs suggested the potential use of this material for bone augmentation.

Cytotoxicity of Different Concentrations of Three Root Canal Sealers on Human Mesenchymal Stem Cells.

This study assessed the dose-dependent effect on the cytotoxicity of BioRoot RCS (BR) and Endosequence BC (BC) sealers in human bone marrow mesenchymal stem cells (hMSCs) compared to those of the AH Plus sealer. Cells were exposed to different dilutions of extracts from freshly prepared sealers (1:2, 1:8, 1:32). Unexposed cells acted as the negative control. Cytotoxicity was evaluated by an alamar blue assay. Cell morphology was analyzed by using scanning electron microscopy after exposure to the different sealers' extracts. Statistical analysis was performed using a one-way analysis of variance and the Bonferroni post hoc test (p < 0.05). The cytotoxicities of BC and BR were less than that of AH Plus. In the presence of 1:2 BR, the cell proliferation was significantly lower than the control. At 1:8 and 1:32 concentrations, both the tricalcium silicate sealers led to similar cellular proliferation. Cells in BC and BR sealers' extracts spread better than those in AH Plus extract.