The Effects Of Nerve Growth Factor And Basic Fibroblast Growth Factor On Proliferation And Differentiation Of Human Dental Pulp Cells In Vitro

Background: Nerve growth factor(NGF) has the capability to promote the proliferation and differentiation of human dental papilla mesenchyme cells in virto. Basic fibroblast growth factor(bFGF) has the capability to promote the proliferation of human dental pulp cells(HDPC) in vitro, too. However, it has not been reported whether NGF and bFGF have the effects on proliferation and differentiation of HDPC in vitro.Objective: To evaluate the effects of NGF,bFGF, and combination of NGF and bFGF on proliferation and differentiation of HDPC in vitro.Methods: 1. Cell culture: Human dental pulp cells were cultured by using an explant technique. Normal human premolars and impacted third molars were collected from patients (10–25 years of age). The pulp tissue was removed aseptically and minced into small fragments(0.5 mm×0.5mm) which were incubated in DMEM supplemented with 20% fetal bovine serum and antibiotics. When HDPC covered about 80% floor of culture bottle, the confluent cells were detached with 0.25% trypsin and aliquots of separated cells were passaged . Their morphology and growth condition were observed under inverted light microcopy. The cytokeratin and vimentin were immunohistochemically detected to identify the cell phenotype. Von Kossa staining was to determine the ability of mineralization after the fifth passage of HDPC was cultured up to 30d consecutively.2. Thiazoly blue(MTT) colorimetric assay analysis :Cell cultures between the fifth and eighth passages were used in this study. 100mL HDPC(2×104/ml) per well were seeded to 96-well plates and left 48 hours to grow. The medium was exchanged for 1%FBS DMEM another 24 hours. Then various elutes(experiment groups) and 1%FBS DMEM(control group) in 100μl volumes were added respectively and the cells were treated for 48 hours . After treatment, 20μl MTT solution (5mg/mL)was added to each well and incubated for another 4 hours. All of the media were then discarded and 150μl dimethyl sulfoxide was added to each well. The optical density was read with an ELISA reader at a wavelength of 490 nm3. Alkaline phosphatase(ALP) activity analysis: The same method of Cell culture was adopted just like 2. 100μl 0.2%Triton X-100 was added and incubated overnight at 4℃. On the second day 100μl reagent of ALP kit was added and treated at 37℃ for 30 minutes. Then, 50μl 0.1%M/L NaOH was added to stop the treatment. The optical density was read with an ELISA reader at a wavelength of 410 nm. Results1. 30 times of cell primary culture was performed totally: 7 polluted, 5 succeeded and the others failed. The success rate was 21.74%. The growth curve was drawn and the double time of HDPC is about 67 hours. The immunohistochemical study revealed that vimentin staining was positive and cytokeratin staining was negative. Some of the long-term cultured HDPC formed mineralized nodules and Von Kossa staining was positive.2. When compared with control group, (1)10U/ml NGF significantly promoted the proliferation of HDPC(P<0.05). 1U/ml NGF and 100U/ml NGF also did the same effects but there was no statistically significant difference(P＞0.05). (2)100U/mLNGF significantly increased the ALP activity of HDPC but 1U/mLNGF and 10U/mLNGF hardly any effects on it. (3)10μg/L bFGF significantly promoted the proliferation of HDPC(P<0.05) but it simultaneously inhibited the ALP activity of it(P＞0.05).(4)Combination of NGF and bFGF(10U/mL NGF＋10μg/L bFGF,5U/mL NGF＋5μg/L bFGF) not only significantly promoted the proliferation of HDPC(P<0.05) but also increased the ALP activity(P<0.05). Conclusion 1. 10U/mLNGF promoted the proliferation and 100U/mLNGF increased the ALP activity of HDPC significantly. 2. Although it significantly promoted the proliferation of HDPC, 10μg/L bFGF might inhibit the differentiation of it. 3. Combination of NGF and bFGF(10U/mL NGF＋10μg/L bFGF,5U/mL NGF＋5μg/L bFGF) significantly promoted the proliferation and differentiation of HDPC simultaneously.