| OBJECTIVE:Study confirmed that enamel matrix proteins (Enamel matrix proteins, EMPs) can promote the regeneration of periodontal tissues including alveolar bone, and commercialization of products have been used in clinical. The purposes of this study are 1. To ensure that amelogenin is the main component of EMPs which were extracted by acetic acid and analyzed by SDS-PAGE; 2. To clarify EMPs affects on bone regeneration in skull defect of nomal rats; 3. To clarify EMPs affects on bone regeneration in skull defect of diabetic rats.METHOD:1. EMPs were extracted from porcine tooth germs by acetic acid and analysed by SDS-PAGE.2.20 Wister rats, male, were randomly divided into two groups,10 each. After anesthetized by Chloral Hydrate, rats were cut the hair and disinfected on the roof of skull. A about 5-6cm incision of "U" -shape was done on the forehead of rat, then the surgical area of the crinial bone was fully exposed. Full thickness cranial plate defects in a diameter of 5mm were created on both sides of the skull of rats. The implants were inserted in the defects:the control side inserted by 100mg/ml PGA(vector of EMPs, propylene glycol alginate), experimental side by 10mg/ml EMPs+100mg/ml PGA, then sew up the incision. Benzylpenicillin sodium was injected in the following three days to prevent from inflammation. Experimental animals were observed and recorded after surgery. After 4 weeks,8 weeks,10 rats were sacrificed by anesthesia overdose respectively and the specimen were examined by X-ray and histological examination.3.20 Wister rats, male, were raised for 1 week. After overnight fasting, the diabetic rats were induced by Streptozotocin(STZ) (60mg/kg). If the blood glucose examinated is over 16.7mmol/L 1 week later, the model is established successfully. During the model building, one rat died and then other 24 diabetic rats were randomly divided in two groups,12 each. Following the surgery and other processes were the same as the second step, while the blood glucose need be examined before pre-sacrificed to make sure the model was still the diabetic rats.RESULT:1. Most of the bands are blow 25kDa protein molecular weight and the dominant bands are 20kDa,13 kDa, and smaller molecular weight range, which are the highest band of 20 kDa.2. Wound healing is great in normal rats, no inflammation, no infection, no rejection reaction. X ray showed:at 4week, the scope of bone defects of the experimental side is no more narrower than the control side, but boundary of defects in the experimental side has becomed vague; at 8week, the scopes of bone defects of both sides are reduced, while the experimental side is more significant and most has been repaired. The Mean and Standard deviation of relative bone density:at 4week, the experimental side 31.8±2.9, the control side 25.8±2.85, statistical difference is between the both(P<0.05); at 8week, the experimental side 39.4±3.3, the control side 28.6±3.2, statistical difference is between the both(P<0.05). The observation of histology indicated:at 4week, there is a lot of fibrous connective tissue and extensive infiltration of inflammatory cells on the control side, while a small amount of osteoblasts exist; on the experimental side, there is a lot of dense fibrous connective tissue, in which you can observe a large number of cord-like and mesh-like bone matrix, widespread osteoblasts and some Chondro-like structures. At 8week, there is a lot of loose fibrous connective tissue, extensived inflammatory cells, a amound of osteoblasts on the control side and some bone-like and chondro-like structures on the bouncary of bone defect(?)on the experimental side, a large number of non-mature and mature bone can be found, which form woven bones and some lamellar bones and the bone marrow-like structures exist in some areas. 2. There is occasional lack of energy and the symptoms of more food, polydipsia, polyuria in the diabetic rats and some died from infecton. X ray indicated:at 4week and 8week, the scopes of bone defects on both experimental side and control side have no significant narrowing and the center and the boncrary of defects do not exist high-density images. The Mean and Standard deviation of relative bone density:at 4week, the experimental side 31.8±6.8, the control side 29.8±6.0, no statistical difference is between the both sides(P>0.05); at 8week, the experimental side 34.6±4.6, the control side 31.7±2.6, no statistical difference is between the both sides(P>0.05). The observation of histology indicated: at 4week and 8week, the defects of the control side and the experimental side are filled with the thin fibrous connective tissue and there are few osteoblasts and no new bone formation in the defects.CONCLUSION:1. Amelogenin is the main component of EMPs which were extracted from porcine tooth germs by acetic acid.2. lOmg/ml of EMPs in combination with PGA carrier can promote healing of skull defects in normal rats and is favorable for the bone regeneration.3. The effect of EMPs (10mg/ml) in combination with PGA on bone regeneration in skull defect of diabetic rats is little, and the concentration of EMPs and/or whether or not EMPs works need us to further study. |
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