The Effect Of Basic Fibroblast Growth Factor On Cell Proliferation, Apoptosis And Osteogenic Differentiation Of Human Gingival Fibroblasts In Vitro

ObjectiveTo observe the effects of basic fibroblast growth factor(b FGF) on osteogenic differentiation ability, cell proliferation and apoptosis of human gingival fibroblasts(h GFs), and explore the function of b FGF on the process of osteogenic induction in vitro.MethodsWith informed consent from donors, fresh gum gingival tissues were isolated from donors(14-20 years old) for impacted wisdom teeth extraction(without caries or periodontal diseases). Tissues were thoroughly rinsed with phosphate buffer solution(PBS) containing 100U/L penicillin and 100 μg/L streptomycin.1) The human gingival-derived fibroblasts were cultured in vitro and the 3rd passages of cells were used in this study. Groups were as follows: normal medium as group 1, normal medium with 10 μg/L b FGF as group 2,osteogenic medium as group 3 and osteogenic medium with 10 μg/L b FGF as group 4.2) MTT method was used to evaluate the proliferation of h GFs under different culture conditions.3) Acridine orange/ethidium bromide(AO/EB) double fluorescence staining was used to detect the apoptosis of h GFs under different culture conditions.4) ALP activity test and Alizarin red staining were applied to investigate osteogenic ability of h GFs under different culture conditions.5) Reverse transcription-polymerase chain reaction(RT-PCR) was used for analysis of osteogenic related genes runt-related transcription factor 2(Runx2)、alkaline phosphatase(ALP) and collagen type I(Col I) of h GFs under different culture conditions.6) Data analysis was performed using statistical software(version V8, SAS).Data were showed as mean±SD. Independent t-text was used for MTT and apoptosis results, analysis of variance(ANONA) was used for other results.P<0.05 was considered statistically significant.Results:1) The human gingival fibroblasts were cultured with tissue-explant method. The primary cultured cells grew relatively slowly, after about 5-8 days there were cells migrated from the tissue blot. After passaging, cells began to converge in the long fusiform.2) 10 μg/L b FGF could increase cell proliferation of h GFs both in normal and osteogenic medium(P<0.01).3) The apoptotic cells in every group increased as time passed by. 10 μg/L bFGF could inhibit cell apoptosis of h GFs in both normal and osteogenic medium from 3rd day to 7th days(P<0.05), while in the normal culture of 9th to 11 th days and osteogenic culture of 11 th day, 10 μg/L b FGF showed promoting effect on apoptosis(P<0.05).4) HGFs could be induced into osteogenic differentiation and form mineralized nodule in osteogenic medium. However, 10 μg/L b FGF had no effects on ALP activity and mineralized nodule formation of h GFs during osteogenic differentiation.5) The results of RT-PCR showed that: the expression level of Runx 2 in osteogenic medium was higher than that in normal medium(P<0.05), while10 μg/L b FGF had no effects on its expression. The expression levels of ALP and Col I in osteogenic medium were higher than those in normal medium(P<0.05), and 10 μg/L b FGF could only promote the expression levels of ALP and Col I in normal medium(P<0.05).Conclusion:1) 10 μg/L b FGF promotes the proliferation of h GFs. In the early days of culture, 10 μg/L b FGF inhibit cell apoptosis of h GFs, while it shows promoting effect on apoptosis at a later date.2) 10 μg/L b FGF has no effects on osteogenic differentiation of h GFs.