Modulation of proliferation and differentiation of gingiva‑derived mesenchymal stem cells by concentrated growth factors: Potential implications in tissue engineering for dental regeneration and repair

Abstract

The aim of the present study was to evaluate the proliferation and osteogenic differentiation ability of gingiva‑derived mesenchymal stem cells (GMSCs) cultured with different concentrations of concentrated growth factors (CGF). GMSCs were isolated from gingival connective tissues and characterized by flow cytometry, immunofluorescence staining and immunohistochemical staining. Cell proliferation activity was determined by the MTT assay, and the effect of CGF on MCSCs was detected with the Cell Counting Kit (CCK)‑8 assay. Mineralization induction was evaluated by alkaline phosphatase (ALP)‑positive cell staining and mineralized nodule formation assay. Dentin matrix acidic phosphoprotein (DMP)1, dentin sialophosphoprotein (DSPP), bone morphogenetic protein (BMP)2 and runt‑related transcription factor (RUNX)2 mRNA and protein expression were evaluated by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) analysis and western blotting. The flow cytometry, immunofluorescence staining and immunohistochemical staining results indicated that the cultured cells were GMSCs. The MTT assay results revealed that the third‑generation gingival stem cells exhibited the highest proliferative capacity, and the CCK‑8 results indicated that 10% CGF achieved the most prominent promotion of GMSC proliferation. ALP activity analysis and mineralized nodule assay demonstrated that CGF may successfully induce osteogenic differentiation of GMSCs, whereas RT‑qPCR and western blot analyses demonstrated that CGF is involved in the differentiation of GMSCs by regulating the expression of DMP1, DSPP, BMP2 and RUNX2 (P<0.05). In conclusion, CGF were demonstrated to promote the proliferation and osteogenic differentiation of GMSCs. Therefore, CGF may be applied in tissue engineering for tooth regeneration and repair.

Figure 1

Culture of gingiva-derived mesenchymal stem cells in vitro (magnification, ×100). (A) Primary culture: The cells exhibited a long fusiform or polygonal shape. (B) Subculture: The cells exhibited consistent morphology, including fusiform shape with a plump cell body, clear nucleus and fibroblast-like phenotype.

Figure 2

Characterization of GMSCs. (A) Flow cytometry analysis indicated that human GMSCs expressed CD73, CD105 and CD90, but not CD45. (B) Expression of stem cell markers in GMSCs. Cells cultured in a 24-well plate were fixed and stained with specific antibodies against human vimentin, CD90, CD73 and cytokeratin (CK). The cells were incubated with rhodamine- or fluorescein isothiocyanate-conjugated secondary antibodies and then observed under a fluorescence microscope (magnification, ×200). (C) Immunofluorescence staining revealed that the cytoplasm of CD90- and vimentin-stained cells exhibited red fluorescence, and the cytoplasm of S100A4-stained cells exhibited green fluorescence. The nuclei of all cells exhibited blue fluorescence, whereas the cytokeratin-stained cells exhibited no fluorescence (magnification, ×200). GMSCs, gingiva-derived mesenchymal stem cells.

Figure 3

Analysis of proliferative activity of GMSCs. The MTT assay results demonstrated that GMSCs exhibited logarithmic growth on days 3-4 following inoculation, and the cell number tended to stabilize on days 7-8. The third-generation cells exhibited strong proliferative capacity. GMSCs, gingiva-derived mesenchymal stem cells; OD, optical density.

Figure 4

CGF. (A) The following blood fractions were obtained by centrifugation: i) A superior phase represented by the serum (platelet-poor plasma; PPP); ii) an interim phase represented by a large and dense polymerized fibrin clot containing CGF, white blood cells and stem cells; and iii) the lower red blood cell (RBC) layer. (B) The CGF gel exhibited a light yellow gelatinous, translucent, soft, elastic, smooth surface. (C) The Cell Counting Kit-8 assay results demonstrated that the optical density (OD) value increased gradually over time. Compared with the control group, 10% CGF significantly promoted the proliferation of GMSCs. ^*P<0.05. CGF, concentrated growth factors; GMSCs, gingiva-derived mesenchymal stem cells.

Figure 5

Effect of CGF on GMSC osteogenic differentiation at different time points. (A) Effect of CGF on alkaline phosphatase activity in GMSCs at different time points. Data are presented as the mean ± standard deviation. ^*P<0.05 indicated pure mineralization group vs. control group; ^**P<0.01 indicated experimental group vs. control group; ^#P<0.05 indicated experimental group at 14 days vs. experimental group at 7 days; ^##P<0.01 indicated experimental group at 21 days vs. experimental group at 7 days. (B) Mineralized nodule formation analysis. The Alizarin Red S staining results indicated that mineralized nodules appeared in the experimental group earlier, and at a higher number and density. Osteogenesis was most marked on the 21st day after mineralization induction (magnification, ×200). CGF, concentrated growth factors; GMSCs, gingiva-derived mesenchymal stem cells.

Figure 6

mRNA expression of DSPP, DMP1, BMP2 and RUNX2 on the 21st day after mineralization induction. The RT-qPCR results indicated that the (A) DSPP, (B) DMP1, (C) BMP2 and (D) RUNX2 mRNA expression were significantly increased. ^*P<0.05 indicated pure mineralization group vs. control group; ^**P<0.01 indicated experimental group vs. control group. DSPP, dentin sialophosphoprotein; DMP1, dentin matrix acidic phosphoprotein 1; BMP2, bone morphogenetic protein 2; RUNX2, runt-related transcription factor 2; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Figure 7

Protein expression of DSPP, DMP1, RUNX2 and BMP2 on the 21st day after mineralization induction. The western blotting results indicated that the DSPP, DMP1, BMP2 and RUNX2 protein expression was significantly increased. ^*P<0.05 indicated pure mineralization group vs. control group; ^**P<0.01 indicated experimental group vs. control group. DSPP, dentin sialophosphoprotein; DMP1, dentin matrix acidic phosphoprotein 1; BMP2, bone morphogenetic protein 2; RUNX2, runt-related transcription factor 2.