Fig. 1

GMSC isolation and characterization. a Gingival tissues were digested and cultured for 14–21 days to obtain a selected population of GMSCs. b Colony-forming unit fibroblast (CFU-F) assay. Cells were plated at densities of 500 or 1000 cells/well. After 14 days, the cells were fixed, and stained with crystal violet. Groups of 50 or more cells were scored as colonies. The data are expressed as number of colonies, and are presented as the mean values ± SEM of three independent experiments, each performed in duplicate. c GMSC mRNA was extracted and PCR analysis of CD34, CD45, CD90, and CD105 was performed. Representative image of 2% agarose gel electrophoresis is shown. d GMSCs were fixed to determine the CD90 expression by immunofluorescence staining with anti-CD90 antibody. DAPI was used to label cell nuclei. Representative images are shown. e, f Expression of CD90 in GMSC and HuDe cells were evaluated by Western blot and a representative image (e) and quantitative analysis performed using ImageJ (f) are reported. The data were expressed as the percentage of optical density (OD) versus control set to 100% and represent the mean values ± SEM of three different experiments. The significance of the differences was determined by student t test: ***P ≤ 0.001 versus GMSCs. g, h The osteogenic differentiation potential was evaluated using Alizarin Red staining. GMSCs were cultured in osteogenic differentiation media for 21 days; at the end, the cells were stained and a representative photograph (10× magnified images) of mineral nodules was captured. Top represents the mineralization when GMSC were mantained in growth medium, bottom after 21 days of differentiation. (g). The amount of Alizarin Red S was quantified. The data are expressed as the percentage of osteogenic differentiation versus cells cultured in proliferation medium, and are presented as the mean values ± SEM of three independent experiments, each performed in duplicate. The significance of the differences was determined by student t test: ***P ≤ 0.001 vs. the control (CTRL)