The Combined Effects Of Total Flavones Of Rhizoma Drynariae And Icariin On Proliferation And Alkaline Phosphatase Activity Of Human Periodontal Ligament Cells

Background:Periodontitis is a common disease,which is the main reason leading to the loss of adults teeth in our country.As the periodontitis developing,periodontium is destructed,periodontal attachment is lost,alveolar bone is resorped,teeth become mobile and even lost.Alveolar bone resorption and attachment loss are considered to be main pathological change of periodontitis and main reason of the teeth loss.How to promote the cells differentiation which possess regenerative potential to generate the functional periodontal tissues and promote alveolar bone restoration become the key subject for therapy of periodontal disease.The traditional Chinese medicine therapy for periodontal disease has a long history in China. It is our advantage to treat periodontitis by developing and using herbs. Drynaria fortunei and Epimedium brevicornum Maxim which can invigorate the kidney are widely used in the osteonosus and traumatism.They can invigorate the kidney and strengthen the bones and muscles.They can also promote osteogenesis function of human periodontal ligament cells to generate the functional tissues. According to the principle of mutual promotion and mutual enhancement in the compatibility theory of traditional Chinese medicine and the theory of renal deficiency resulting in loose tooth, we will study the effects of the combination of Drynaria fortunei and Epimedium brevicornum Maxim on human periodontal ligament cells and discuss the possibility of the combination of traditional Chinese medicine in invigorating the kidney in order to lay a foundation of developing traditional Chinese medicine with independent intellectual property for curing periodontitis.Objective:To investigate the combined effects of total flavones of rhizoma drynariae(TFRD) and Icariin(ICA) on the proliferation and alkaline phosphatase(ALP) activity of human periodontal ligament cells(HPDLCs).Methods:1 Adopt tissue explants culture technique for the culture of primary HPDLCs,then the primary cells were passaged after morphological and immunohistochemical identification.2 Take 4th to 6th generations of HPDLCs for experiments.The cells were divided into the TFRD experimental groups with the dose of 0.1mg/L, 1mg/L, 10mg/L and the ICA experimental groups with the dose of 0.1mg/L, 0.01mg/L, 0.001mg/L.Besides,set a blank control group without drugs.The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT) colorimetric method was used to observe the effects of different concentration groups of TFRD and ICA on the proliferation of HPDLCs after 6 days in order to obtain the optimal concentration experimental group. The enzyme kinetics method was used to observe the effects of different concentration groups of TFRD and ICA on the ALP activity of HPDLCs after 6 days in order to obtain the optimal concentration experimental group.3 Take 4th to 6th generations of HPDLCs for experiments. The cells were divided into the optimal TFRD concentration group, the optimal ICA concentration group and the optimal TFRD+ICA concentration group. Besides,set a blank control group without drugs and a positive control group with recombinant human bone morphogenetic protein-2(rhBMP-2) with the dose of 0.2mg/L. The MTT colorimetric method was used to observe the effects of each experimental groups with drugs on the proliferation of HPDLCs after 6 days. The enzyme kinetics method was used to observe the effects of each experimental groups with drugs on the ALP activity of HPDLCs after 6 days.Results:1 The primary human periodontal ligament cells are cultured and passaged successfully.2 The TFRD groups with the dose of 0.1mg/L,1mg/L could promote the proliferation of HPDLCs and the most efficient concentration group was the dose of 1mg/L. All the concentration groups of TFRD could promote the ALP activity of HPDLCs and the most efficient concentration group was the dose of 1mg/L.The ICA groups with the dose of 0.1mg/L,0.01mg/L could promote the proliferation and the ALP activity of HPDLCs and the most efficient concentration group was the dose of 0.01mg/L. The TFRD group with the dose of 10mg/L could not promote the proliferation of HPDLCs and The ICA group with the dose of 0.001mg/L could not promote the proliferation and the ALP activity of HPDLCs.3 The optimal concentration groups of TFRD,ICA and TFRD+ICA could promote the proliferation and the ALP activity of HPDLCs.Among them,significantly greater effects of the proliferation and the ALP activity could be observed in TFRD+ICA groups compared with TFRD groups or ICA groups. All the optimal concentration groups of TFRD,ICA and TFRD+ICA could not compare with rhBMP-2 groups as the positive control in promoting the proliferation and the ALP activity.Conclusions:TFRD and ICA for use together could be observed the synergism.