| Ultimate goal of periodontal treatment is the regeneration of periodontal tissues and the formation of new periodontal attachment apparatus. But periodontal disease results in the loss of the periodontium, which includes cementum, periodontal ligament, and bone, so it is difficult for periodontium to repair or reconstruct. And then tissue engineering is supposed to be able to solve the problem. Marrow stromal cells (MSCs) can be used as seeded cells in periodontal tissue engineering, due to its' advantages of easy to obtain, growing stably, powerful capability of proliferation and orientationaly differentiation and easily surviving after transplantation.Many investigations have indicated that it is undouted that enamel matrix proteins (EMPs) successfully promoted the regeneration of periodontal tissues. EMPs not only induced formation of acellular cementum, but also could promote regeneration of periodontal ligament and alveolar bone. And the regeneration process was mimic to natural histogenesis.However, relatively little information has been reported with regard to biological effects of EMPs on MSCs in vitro. So we had done some work about this, and we hope to provide a basic data for applying EMPs and MSCs in periodontal tissue regeneration in the future.In our study, we cultured SD rat marrow stromal cells (rMSCs) in vitro, extracted EMPs whose main component is amelogenin from porcine tooth germ, and identified it's biological activity. Then we observed some effects of EMPs on rMSCs, including synthesis of extracellular matrix, cellular differentiation and production of growth factor. There are four parts involved in our experiment, as followed:1. Culture of rMSCs in vitro and extraction and identification of EMPsThe bone marrow of tibia and fibula of rat was drawn to gain MSCs, and then the cell cion was cultured under the standard culture condition. Teeth germ were dissected from fresh jaws of porcine with age 4-6 months, and EMPs was extracted by acetic acid method. Cell proliferation ability was assessed with varied concentration and time of EMPs or without EMPs by MTT method, and the proliferation of rMSCs was regarded as an index of identificating biology activity of EMPs. The result showed that the proteins we got possesed typical molecular weight distribution of amelogenin by using SDS-PAGE, that is to say, the molecular weight distribution was collected in 20 kDa 13 kDa and 5 kDa. According to our experiments, EMPs could promote rMSCs proliferation significantly at the concentration of 50-200mg/L, and mininus effective concentretion was 50 mg/L, and the strongest effective concentration was 100mg/L. Under the effects of EMPs at 100 mg/L, rMSCs proliferation was enhanced at 1st day, and achieve to peak at 5th day. These results suggested that EMPs we extracted had good biology activity.2. Effects of EMPs on synthesis of type I collagen and type III collagen by rMSCs in vitroTo investigate rMSCs' synthesis ability of type I collagen and type HI collagen following treatment with enamel matrix proteins immunocytochemistry and image analysis were carried. The results showed that EMPs promoted the ability of rMSCs' synthesis of collagen, and under the effects of EMPs at 100mg / L, stain of type I collagen was strongest at 7th day , but stain of type III collagen was strongestat 5th day.3. Effects of enamel matrix proteins on synthesis of non-collagenous protein by rMSCs in vitroImmunocytochemistry and image analysis were carried to investigate rMSCs' synthesis ability of non-collegenous proteins following treatment with enamel matrix proteins. The results showed that under the effects of EMPs, rMSCs' synthesis of OPN had been enhanced at 3th day, rMSCs' synthesis of BSP had been enhanced at 5th day, and rMSCs' synthesis of ON had been enhanced significantly with time prolonging. OPN, BSP and ON are important marks of differentiation to osteoblastic, and they play important role in biomineralization. Our results indicated that EMPs could promote rMSCs' differentiation to osteoblas... |
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