Fibroblast growth factors (FGFs) are growth factors that were initially identified as proteins that stimulate fibroblast proliferation. The aim of the present study was to examine the effects of FGF-4 on the morphology, cellular viability and osteogenic differentiation of stem cell spheroids. Stem cell spheroids were generated using concave microwells in the presence of FGF-4 at concentrations of 0, 50, 100 and 200 ng/ml. Cellular viability was qualitatively assessed by a fluorometric live/dead assay using a microscope and quantitatively determined by using Cell Counting Kit-8. Furthermore, alkaline phosphatase activity and calcium deposition were determined to assess osteogenic differentiation. Reverse transcription-quantitative PCR (RT-qPCR) was performed to evaluate the mRNA expression levels of Runt-related transcription factor 2 (RUNX2) and bone γ-carboxyglutamate protein (BGLAP). Spheroidal shapes were achieved in the microwells on day 1 and a significant increase in the spheroid diameter was observed in the 200 ng/ml FGF-4 group compared with the control group on day 1 (P<0.05). The results regarding viability using Cell Counting Kit-8 in the presence of FGF-4 at 50, 100 and 200 ng/ml at day 1 were 98.0±2.5, 106.2±17.6 and 99.5±6.0%, respectively, when normalized to the control group (P>0.05). Furthermore, the alkaline phosphatase activity was significantly elevated in the 200 ng/ml group, when compared with the control group. The RT-qPCR results demonstrated that the mRNA expression levels of RUNX2 and BGLAP were significantly increased at 200 ng/ml. Therefore, the present results suggested that the application of FGF-4 maintained cellular viability while enhancing the osteogenic differentiation of stem cell spheroids, at least partially by regulating RUNX2 and BGLAP expression levels.

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The present study was performed to evaluate the effects of short-term application of bone morphogenetic protein-7 (BMP-7) on human gingiva-derived mesenchymal stem cells with next-generation sequencing. Human gingiva-derived stem cells were treated with a final concentration of 100 ng/ml BMP-7 and the same concentration of a vehicle control. mRNA sequencing and data analysis were performed along using gene ontology and pathway analysis. RT-qPCR of mRNA of collagen I, Sp7, IBSP and western blot analysis of collagen I, osterix and bone sialoprotein was also performed. A total of 25,737 mRNAs were identified to be differentially expressed. Regarding osteoblast differentiation, 14 mRNAs were upregulated and 10 were downregulated when the results of the BMP-7 at 3 h were compared with the control at 3 h. The expression of collagen I was increased following the application of BMP-7 at 3 h, and this increase was also observed following western blot analysis. The effects of BMP-7 on stem cells were evaluated with mRNA sequencing, and the expression was validated with RT-qPCR and western blot analysis. The short-term application of BMP-7 produced an increased expression of collagen I, which was associated with target genes selected for osteoblast differentiation. This study may provide novel insights into the role of BMP-7 using mRNA sequencing.

Stem cells have the characteristics of long-term self-renewal and plasticity and the ability to differentiate into specialized cells. Stem cells are widely recognized as potential tools for use in the development of novel therapeutic strategies. The aim of the current study was to investigate the effect of demographic factors on adipogenic and chondrogenic differentiation in bone marrow-derived stem cell (BMSC) spheroids. Age- and gender-associated alterations in the adipogenic and chondrogenic differentiation potential of BMSCs were examined. Human BMSCs were isolated from male and female participants in their 20s, 30s and 50s. Cell morphology and relative values of adipogenesis and chondrogenesis were examined by measuring the relative intensity of oil red O and Alcian blue staining, respectively. Cell morphology alterations in BMSCs isolated from male and female participants in their 20s, 30s and 50s and grown in adipogenic media were very similar. In addition, there were no significant differences in the relative values of adipogenesis in BMSCs for the 20s, 30s and 50s age groups on day 8 and 16. Similarly, no significant differences were observed in the relative values of adipogenesis in BMSCs for the male and female groups on day 8 and 16. Cell morphology changes in BMSCs isolated from male and female participants in their 20s, 30s and 50s and grown in chondrogenic media were very similar. In addition, there were no significant differences in the relative values of chondrogenesis in BMSCs for the 20s, 30s and 50s age groups on day 8, however there was a significant difference observed in the relative values of chondrogenesis in BMSCs on day 16 for the 30s and 50s age groups, compared with the 20s age group. Furthermore, no significant differences were observed in the relative values of adipogenesis in BMSCs for the male and female groups on day 8 and 16. The current study demonstrated that there were no significant differences in the adipogenic and chondrogenic differentiation potential of BMSCs isolated from healthy male donors vs. healthy female donors. Similarly, no significant differences were observed in the adipogenic differentiation potential of BMSCs isolated from different age groups on day 8. However, there was a significant increase in the chondrogenic differentiation potential of BMSCs isolated from participants in their 30s and 50s, compared with BMSCs isolated from participants in their 20s on day 16.

Bone morphogenetic protein 2 (BMP-2) is a growth factor that is used to induce osteogenic differentiation in stem cells. The present study assessed the effects of BMP-2 on stem cell spheroid morphology, viability and osteogenic differentiation. Stem cell spheres were constructed and treated with BMP-2 at predetermined concentrations (0-100 ng/ml) using concave microwells. Cell viability was qualitatively and quantitatively analyzed via microscopy and a water-soluble tetrazolium salt assay kit, respectively. Alkaline phosphatase activity was assessed and an anthraquinone dye for calcium deposit evaluation was performed to determine osteogenic differentiation. The expressions of (runt-related transcription factor 2) and collagen 1 were also determined via quantitative PCR. Spherical shapes were formed using concave microwells on day 1, which were maintained up to day 21. On day 1, the relative cell viability of 0, 10 and 100 ng/ml BMP-2 treated cells was 100.0±1.9, 97.3±4.4 and 101.3±2.6%, respectively. Significantly higher values for alkaline phosphatase activity were determined in the 100 ng/ml treated group when compared with the control group. Additionally, Runx2 mRNA levels were significantly higher in the 100 ng/ml BMP-2 group compared with the control group, as determined via quantitative PCR. The results of the present study indicated that BMP-2 enhanced the differentiation of stem cell spheres, which was demonstrated by increased alkaline phosphatase activity and Runx2 expression.