Fibroblast growth factor-4 maintains cellular viability while enhancing osteogenic differentiation of stem cell spheroids by partially regulating RUNX2 and BGLAP expression

Background

Fibroblast growth factor (FGF) is a growth factor that was initially identified as a protein stimulating fibroblast proliferation. A number of studies showed that it also participates in multiple biological processes including cellular proliferation differentiation. Some studies have demonstrated stimulating effect of FGF-4 on proliferation of mesenchymal stem cells. Dental stem cells, including gingiva-derived stem cells, are promising candidates for restoring lost periodontal tissues.

Aim/Hypothesis

The aim of this study was to examine the effects of FGF-4 on the morphology, cellular viability, and osteogenic differentiation of gingiva-derived stem cells and whether these effects would be regulated through RUNX2 and BGLAP expression.

Material and Methods

Three-dimensional cell spheroids were made using concave microwells in the presence of FGF-4 at concentration of 0 ng/ml, 50 ng/ml, 100 ng/ml, and 200 ng/ml. Cellular viability was determined qualitatively and quantitatively using a microscope and a Cell-Counting Kit-8 (CCK-8) assay. Alkaline phosphatase activity and Alizarin Red S staining were used to assess osteogenic differentiation. Quantification by real-time polymerase chain reaction was conducted for the evaluation of expression of RUNX2 and BGLAP.

Results

Spheroidal shape was achieved in the microwells on day 1. The average spheroid diameters at day 1 for FGF-4 at 0 ng/ml, 50 ng/ml, 100 ng/ml, and 200 ng/ml were 189.1 ± 1.8 μm, 188.9 ± 2.4 μm, 186.4 ± 2.6 μm, and 225.6 ± 8.7 μm, respectively. The relative Cell Counting Kit-8 (CCK-8) assay values at day 1 were 0.311 ± 0.011, 0.305 ± 0.008, 0.331 ± 0.055, and 0.310 ± 0.019 for 0 ng/ml, 50 ng/ml, 100 ng/ml, and 200 ng/ml FGF-4 concentration, respectively. There were significantly higher values of alkaline phosphatase activity at 200 ng/ml group. Quantitative real-time polymerase chain reaction revealed that mRNA levels of RUNX2 and BGLAP were significantly higher at 200 ng/ml group.

Conclusion and Clinical Implications

In this study, addition of FGF-4 was seen to enhance osteogenic potential of gingiva-derived stem cells, which could be partially explained by increased expression of RUNX2 and BGLAP. Though additional researches regarding effect of FGF-4 on stem cells are needed, present study showed possibility of FGF-4 application for increased bone regeneration. Use of FGF-4 with gingiva-derived stem cells could be a promising biomaterial for tissue regeneration.