Effects Of Tea Polyphenols On Human Alveolar Osteoblasts Cell Proliferation And Expression Of Osteoblast-related Genes

Objective: By detecting the different concentrations of tea polyphenols(TP) on healthy human alveolar osteoblasts(HAOB) cell proliferation and expression of osteoblast-related genes, runt related transcription factor 2(RUNX2), osterix(OSX), tissue-nonspecific alkaline phosphatase(TNAP), collagen type Ⅰ alpha 1(COL1A1), osteocalcin(OCN) and bone morphogenetic protein 2(BMP2), the effect of TP on healthy HAOB cell proliferation and its mechanism was studied in order to seek a simple, safe and reliable method to maintain periodontal health and repair periodontal destruction, and further study the TP on oral disease and bone metabolism-related disease providing experimental foundation and theoretical basis.Methods:1. Through the modified digestion of tissue cultivation, the healthy HAOB cells were cultured in vitro, observed its morphology, and identified using alkaline phosphatase(ALP)staining and alizarin red staining method.2. The effect of TP on healthy HAOB cell proliferation was detected by the cell counting kit-8(CCK-8) assay.3. The expression of osteoblast-related genes, RUNX2, OSX, TNAP, COL1A1, OCN and BMP2, was detected by the real-time fluorescence quantitative polymerase chain reaction(RTFQ PCR).Results:1. Using the modified digestion of tissue cultivation, the healthy HAOB was cultured in vitro, and observed that HAOB swam out from bone’s grain about 5 days, which were conformed the morphological features, a lot of triangular and fusiform cell, larger volume,an oval nucleus, 1 ~ 3 nucleolus and without contact inhibition. The HAOB was identifiedthe positive expression of 90% cells using ALP staining method. And the HAOB was identified the positive expression of cells using alizarin red staining method.2. TP promoted significantly the proliferation of HAOB in 48 h by CCK-8 assay, and it was stronger than that in 24 h and 72 h(P < 0.05). And the 100 μg/ml of TP promoted significantly the proliferation of HAOB in 24 h, 48 h and 72 h(P < 0.05). However, the lower concentration of TP had slight promoted proliferation effect on the HAOB in 24 h and 72 h, even 102 μg/ml of TP had inhibited(P < 0.05). And the 10-4 μg/ml TP ~ 100 μg/ml TP promoted proliferation effect with the concentration-dependent.3. The relative expression quantity of RUNX2 was approximate according with the second part of the proliferation. The relative expression quantity of OSX, COL1A1, OCN and BMP2 with 1.0 μg/ml TP in 72 h was promoted, it was higher than that in 24 h and 48 h.The trend of relative expression quantity of COL1A1 and OCN was similar.Conclusion: Using the modified digestion of tissue cultivation, the healthy HAOB was cultured in vitro. The different concentrations of TP on the proliferation of HAOB had different influence. The 10-4 μg/ml ~ 100 μg/ml(0.0001 μg/ml ~ 1 μg/ml) of TP promoted proliferation effect with the concentration-dependent. And the 100 μg/ml(1 μg/ml) of TP promoted significantly the proliferation of HAOB in 24 h, 48 h and 72 h. However, the higher concentration of TP inhibited proliferation effect on the HAOB. And TP promoted significantly the proliferation of HAOB in 48 h, and it was stronger than that in 24 h and72 h. TP on the proliferation of HAOB might be positive correlation of RUNX2. And with1.0 μg/ml(1 μg/ml) TP in 72 h might be promoted the expression quantity of OSX,COL1A1, OCN and BMP2 after the third day.