The Effect Of MS-275 On The Proliferation And Differentiation Of Human Dental Pulp Stem Cells

Dental caries have captured people’s attention as one of the common diseases in oral medicine. The dentin is demineralized by cariogenic bacteria and their toxic products, resulting in defect of the hard tissue. Further, the pulp encaged in dentin is injured by the strong and continuous stimulation through dentinal tubule. When the pulp injury is severe, the mesenchymal stem cells in pulp which have been identified as dental pulp stem cells(DPSCs) would migrate and adhere to the injury, differentiate into odontalblast and secret dentin matrix to repair the tissue, which makes a massy foundation of cytobiology for the reparation and generation of teeth.Histone deacetylase(HDAC)is a kind of enzymes that catalyze histone deacetylation. HDAC not only plays an important role in the regulation of histone acetylation level which is related with the generation and development of tumor, but also in regulation of stem cells proliferation and differentiation. Our previous study found that during the differentiation of h DPSCs, the expression of HDAC1 declined obviously, which indicated that HDAC1 may participated in the differentiation of h DPSCs.MS-275(Entinostat, SNDX-275), which belongs to toluamide, is a specific inhibitor of HDAC1. It could selectively inhibit class I HDAC, including the HDAC1. MS-275 can also regulate acetylation level of histone, consequently influence the expression of tumor related genes. Compared with other HDACs, the hypotoxic and antineoplastic property of MS-275 make it an important antineoplastic drug in clinical applications. MS-275 inhibits the proliferation of the tumor cells by regulating cyclin. Besides, MS-275 also plays an important role in neural differentiation of embryonic stem cells.Tissue engineering(TE) technology has been widely used in generating tissue and organ for regenerative medicine. The appropriate application of different materials is the key point of successful TE. Recently nano-materials are widely used in tissue regeneration.The nano-fibrous gelatin(NF-gelatin) is a new scaffold material which is similar to natural dentin in terms of three demention(3D) physical structure and chemical composition. It has been reported that NF-gelatin could promote adhesion, proliferation, migration and differentiation of stem cells, which makes a grander prospect for TE. The aim of our study is to investigate the effect of MS-275 on proliferation and differentiation of h DPSCs, therefore further explore the function of HDAC1. The results will provide a laboratory basis for the repararion and regeneration of the injured teeth. The principle results are as follows:1. Isolation, cultivation and identification of h DPSCsDental pulp tissues of healthy third molars or extracted tooth for the orthodontic reason were collected from patients aged from 18 to 22 with informed consent. Tissue block and enzymatic digestion methods were used to isolate and culture primary h DPSCs. After limiting dilution selecting monoclonal cells and expending culture, h DPSCs were obtained. Flow Cytometry was used to identify the cell surface makers. Alizarin red and oil red O tests were applied to identify the osteogenic and adipogenic ability of h DPSCs. All these results confirmed that we successfully established h DPSCs lines.2. The effect of MS-275 on the proliferation and differentiation of h DPSCs in vitroOur previous study found that the expression of HDAC1 declined obviously during the osteogenic /odontogenic differentiation of h DPSCs. In order to further explore the function of HDAC1, we chose its specific inhibitor, MS-275, as the object of the study. MS-275 with different concentrations(20 μM, 2 μM and 0.2 μM) were applied to stimulate h DPSCs on two dimensional environment. The MTT results showed that different concentrations of MS-275 all inhibited the proliferation of h DPSCs and e20μM MS-275 was found toxic. After induction of osteogenic differentiation, we found that both 2 μM and 0.2 μM MS-275 could promote the expression of osteogenic/odontogenic related genes such as ALP, DSPP, OCN and BSP, while 2 μM MS-275 showed a stronger effect.3. The effect of MS-275 on proliferation and differentiation of h DPSCs cultured on NF-gelatin scaffoldWe seeded h DPSCs on NF-gelatin scaffold and treated with 20 μM MS-275, 2 μM MS-275 and 0.2 μM MS-275, respectively. MTT results showed that in 3D environment, MS-275 with all different concentrations inhibited the proliferation of h DPSCs and only 20 μM MS-275 was found toxic. After osteogenic induction, 2 μM and 0.2 μM MS-275 treatment promoted the formation of calcium nodule and the expression of osteogenic related genes and proteins such as ALP, DSPP, OCN and BSP, while the effect of 2 μM MS-275 was more obvious. The cell-scaffold complexes were transplanted subcutaneously in nude mice showed that comparing to the control group, 2 μM MS-275 could promote the formation of collagenous fibers as showed by HE and Masson’s staining.Von Kossa staining showed that 2 μM MS-275 could promote the formation of hard mineralized tissue. X-ray measurement and Micro CT analysis showed that 2 μM MS-275 could promote the formation of hard mineralized tissue.In conclusion, our study confirmed that a certain concentration of HDAC1 specific inhibitor MS-275 could inhibit the proliferation of h DPSCs and promote the osteogenic/odontogenic differentiation, which indicated that HDAC1 plays an important role in the proliferation and differentiation of h DPSCs. It laid a laboratory basis for the pulp injury reparation and teeth regeneration in the future.