The Effect Of EGF And Bfgf Of Proliferation And Differentiation On Human Dental Pulp Stem Cells

Objective:Just like most of the stem cells, human dental pulp stem cells (hDPSCs) have capacity of multi-potential differentiation. They can form new functional cells to keep the dynamic balance between the growth and recession of dental tissues. Some of the studies show that either in the development of dentin and dental tissues or during the restoration of dental pulp lesion, pulp cells accompany proliferation and differentiation. In addition, a variety of growth factors are up-regulated. But there are seldom researches on effect of growth factors EGF (Epidermal growth factor) and bFGF (basic fibroblast growth factor) in the proliferation and differentiation of hDPSCs. In this study the effect of EGF, bFGF and EGF&bFGF were observed in vitro on the proliferation and differentiation of hDPSCs. This may help us further understand of the possible mechanism of EGF and bFGF in promoting the proliferation and differentiation of somatic DPSCs and also could provide some experimental foundation and theoretical basis for the transplantation of human dental pulp stem cells in regeneration of teeth. Methods:1. Cryopreserved hDPSCs were thawed routinely and cultivated for24hours then divided into experimental groups and control group. In the experimental groups, the growth factors EGF and bFGF were added in the hole of culture plate according to different concentrations (5,10,20,50,100ng/ml). No growth factor was put in the control group. Each group has five parallel holes. Metromethyl Thiazolyl blue (MTT) was used to measure the optical density (OD) of each group at wavelength of490nm four days later. Compared the effect of different concentrations EGF and bFGF on the growth of hDPSCs and selected the optimal concentration.2. Cultivated the subculture DPSCs at the concentration of1×104/ml for24hours then changed culture medium and took the optimal concentration growth factor to each experimental group (EGF group, bFGF group and EGF&bFGF group). None growth factor was put in the control group. Each group has five parallel holes. Determined OD values of each group at0,1,3,5and7day and analyzed the proliferation of human dental pulp stem cells in each group;3. Cryopreserved hDPSCs were thawed routinely and added, the cells was grouped as above.The optimal concentration of EGF, bFGF and EGF&bFGF for cultivation. Detected the ALP activity of hDPSCs at1,3,5,7,14days later and analyzed the role of EGF, bFGF, EGF&bFGF on differentiation of the dental pulp stem cells.4. Statistical analysis:The result of experiment was statistically analyzed with software SPSS17.0All data were expressed by M±SD, P<0.05, indicated statistical significance. Results:1The optimal concentration of EGF and bFGF are50ng/ml and20ng/ml respectively;2(1) The OD values of the experimental groups and the control group increased with time from0to7days;(2) There was no statistical difference of OD values between the EGF experimental group and the bFGF group after added growth factor at0day and1day compared with the control group (P>0.05), However, the OD values were significant higher than that of the control group at3,5,7day (P<0.05).(3) Among the OD values of EGF&bFGF group, EGF group, bFGF group and the control group, there was no obvious difference after added growth factor at0,1day.(P>0.05), but the OD values of EGF&bFGF group was significantly higher than other groups at3,5,7day (P<0.05);3.(1) The ALP activity of experimental groups and the control group increased with time from1to14days;(2) There were no significant difference of ALP activity among the EGF group, the EGF&bFGF group and the control group at1and3day after added the growth factors, but the ALP activity were significantly higher than that of the control group at5,7,14day;(3)There were no significant difference of ALP activity between the bFGF group and the control group from1day to14day (P>0.05);(4) The ALP activity of the EGF&bFGF group was higher than that of the EGF group and bFGF group. But there was no statistical difference to EGF group(P>0.05).Conclusion:1. The optimal concentration of the growth factor EGF and bFGF are50ng/ml and20ng/ml respectively;2. The growth factor EGF can promote the proliferation and differentiation of dental pulp stem cells effectively. The growth factor bFGF could promote the proliferation of dental pulp stem cells, but the differentiation is not obvious yet;3. EGF and bFGF seem to have a synergistic or superimposed effect on the proliferation of dental pup stem cells, but no synergistic effect in differentiation.