Abstract: Objective： To explore suitable concentration of recombinant human transforming growth factor β1 (rhTGF-β1) usage and study the effect of rhTGF-β1 on differentiation of dental pulp stem cells (DPSCs). Methods: DPSCs were isolated from the undiseased third molars of people aged 18-25 years and cultured according to instructions in vitro. Different concentrations (1 , 6 , 10 μg/L) of rhTGF-β1 were added to the culture medium to examine DPSCs proliferation by CCK-8 (cell counting kit-8) assay.The suitable concentration was then selected. For differentiation, the DPSCs were incubated for 7 or 14 days with rhTGF-β1 supplemented with osteo/odontoblastic induction medium containing 10 nmol/L dexamethasone, 10 mmol/L b-glycerophosphate, 50 g/L ascorbate phosphate, 10 nmol/L 1,25-dihydroxyvitamin D3 and 10% fetal bovine serum. The cells were then washed 3 times with phosphate-buffered saline and sonicated with 1%Triton X-100 for 30 minutes on ice. Cellular alkaline phosphatase (ALP) activity was assayed with p-nitrophenyl phosphate as the substrate. The enzyme activity was expressed as p-nitrophenyl produced per milligram of protein ［bicinchoninic acid (BCA) protein assay kit］. To examine mineral nodule formation, the cultured cells were fixed in 4% paraformaldehyde and washed in water, and the mineralization of the extracellular matrix was assayed by 1% alizarin red S staining and elution of staining was examined as optical density (D) under microplate reader. The mean difference was considered significant at 0.05 and 95% confidence interval. Results: The DPSCs had ty-pical fibroblast morphology and could form mineral nodules after being cultured with osteo/odontoblstic induction medium for 14 days. 6 μg/L rhTGF-β1 significantly promoted the DPSCs proliferation on the 3rd and 5th days. After the incubation of osteo/odontoblastic induction medium, the DPSCs with the 6 μg/L rhTGF-β1 increased ALP activities compared with the control; D values in the 6 μg/L rhTGF-β1 group was 0.31±0.03, while the control group was 0.02±0.01(P<0.05). The total protein content in the 6 μg/L  rhTGF-β1 group was (2 775.46±83.54) mg/L, and the control group was (1 432.20±110.83) mg/L (P<0.05). To eliminate the cells proliferation influence, relative ALP activities, which was  defined as the total ALP divided by the total protein content, the 6μg/L rhTGF-β1 group was 6 times higher than the control group. Alizarin red S staining showed increased mineral nodule formation in the rhTGF-β1 group. The elution of staining under microplate reader also showed more optical density in the 6 μg/L rhTGF-β1-treated cells （0.83±0.02） than that in the control groups (0.55±0.05, P<0.05). Conclusion: 6 μg/L rhTGF-β1 could significantly promote DPSCs proliferation and odontoblastic differentiation in vitro.

Key words: Dental pulp stem cells, Recombinant human transforming growth factor β1, Odontoblast, Differentiation