TGF-β1, a multifunctional regulator of cell growth and differentiation, is the most abundant bone matrix growth factor. During differentiation of human bone stromal cells (hBMSCs), which constitute bone marrow osteoblast (OS) and adipocyte (AD) progenitor cells, continuous TGF-β1 (10 ng/ml) treatment enhanced OS differentiation as evidenced by increased mineralised matrix production. Conversely, pulsed TGF-β1 administration during the commitment phase increased mature lipid-filled adipocyte numbers. Global gene expression analysis using DNA microarrays in hBMSCs treated with TGF-β1 identified 1587 up- and 1716 down-regulated genes in OS-induced, TGF-β1-treated compared to OS-induced hBMSCs (2.0 fold change (FC), p < 0.05). Gene ontology (GO) analysis revealed enrichment in ‘osteoblast differentiation’ and ‘skeletal system development-associated’ genes and up-regulation of several genes involved in ‘osteoblastic-differentiation related signalling pathways’. In AD-induced, TGF-β1-treated compared to AD-induced hBMSCs, we identified 323 up- and 369 down-regulated genes (2.0 FC, p < 0.05) associated with ‘fat cell differentiation’, ‘fatty acid derivative biosynthesis process’, ‘fatty acid derivative metabolic process’, and ‘inositol lipid-mediated’. Serpin peptidase inhibitor, clade B (ovalbumin), member 2 (SERPINB2) was down-regulated 3-fold in TGF-β1-treated hBMSCs. siRNA-mediated SERPINB2 inhibition enhanced OS and AD differentiation. Thus, TGF-β signalling is important for hBMSC OS and AD differentiation and SERPINB2 is a TGF-β-responsive gene that plays a negative regulatory role in hBMSC differentiation.

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-3β (GSK-3β), as a powerful inducer of osteoblastic differentiation and mineralization of human mesenchymal stem cells (MSCs). GSK-3β is a serine-threonine protein kinase that plays a major role in different disease development. GSK-3β 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, TGFβ, 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 β-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.

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-β1), 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-β1 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.

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.

Dental caries is a highly prevalent chronic disease that leads to dental pulp inflammation. It is treated by removing the damaged tooth structure and applying a material that promotes resolution of pulpal inflammation. Tumor necrosis factor superfamily 14 (TNFSF14) is an immunomodulatory cytokine and a member of the TNF superfamily. This study aimed to evaluate the effect of TNFSF14 on the levels of inflammatory cytokines involved in pulpal inflammation using lipoteichoic acid (LTA)-induced human dental pulp stem cells (hDPSCs). hDPSCs were cultured and induced with LTA, followed by treatment with TNFSF14 at 25 and 50 ng/mL. Cellular viability was evaluated using the Alamar Blue assay. The levels of inflammatory cytokines IL-6, IL-8, IL-10, and TNF-α were quantified using reverse transcription–quantitative polymerase chain reaction (RT–qPCR) and enzyme-linked immunosorbent assay (ELISA). TNFSF14 at 25 and 50 ng/mL significantly reduced the mRNA and protein levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-8, and increased the anti-inflammatory cytokine IL-10. In addition, TNFSF14-treated groups enhanced cell viability. Adding TNFSF14 to LTA-induced hDPSCs regulated the production of inflammatory cytokines by lowering the levels of IL-6, IL-8, and TNF-α and elevating IL-10 levels.