| Objective: The purpose of this study was to compare the difference among silk fibroin /graphene oxide/Simvastatin(SF/GO/Sim)composite membrane,silk fibroin/graphene oxide(SF/GO)composite membrane,silk fibroin/Simvastatin(SF/Sim)composite membrane and silk fibroin(SF)membrane in promoting calvarial bone repair in rats and investigate the effect of silk fibroin/graphite Simvastatin composite membrane on bone defect repair in rats.In addition,the another purpose of this study was to observe the general reaction of the GO by local application,and to evaluate the biological safety of important organs(liver,spleen and kidney)for biological safety evaluation.Methods: 1.A total of 48 SD rats aged 8 weeks were randomly divided into 4 groups(n = 12)to establish bilateral 5mm diameter calvarial defect model.The experimental group(group A)was implanted with SF/GO/Sim composite membrane and the control groups were respectively implanted with SF/GO composite membrane(group B),SF/Sim composite membrane(group C)and SF membrane(group D).Half of the animals in each group were sacrificed at 8 weeks,the skull specimens after decalcification were taken for pathological analysis of bone formation by Van-Gieson staining(VG staining);the other half of the animals with sequential fluorescent labeling were killed in 12 weeks after operation.The skull specimens were observed,the hard tissue sections were prepared and the fluorescence labeling was observed by laser confocal microscope,the amount of new bone formation in the direction of defect center and lateral direction of skull were measured by using the Image Pro PLUS software and Van-Gieson staining(VG staining).The liver,spleen and kidney specimens of the experimental animals of group A and group B when killed in 12 weeks after operation were observed.HE staining was performed to evaluate the biological safety of the local GO.2.The experimental data was processed by IBM SPSS Statistics 20 statistical analysis software,and the data were expressed by (?)±s.Two-way classification ANOVA test was used for statistical analysis of the differences among the experimental groups,and the results were statistically different when P < 0.05.Results: Specimen observation: The membrane materials of each group were in good condition without shedding.The edge of bone tissue specimen at 8 weeks was blurring,the defect texture is soft and it is in certain toughness.We can use angle forceps press it and the specimen will rebound when the external force was removed.The rebound time of experimental group was shorter than that of the control groups,and there was no significant difference between group B and group C.The texture of group D is most soft so it’s slow to rebound.In the group at 12 weeks,the bone tissue was closely connected with the surrounding bone tissue,the edge of bone defect and membrane material boundary is not clear;the edge of bone defect of the experimental group was more irregular than that of the control groups,and the defect range of the experimental group decreased significantly;the defect texture is hard,but its hardness is less than the hardness of the autogenous bone;the defect texture is also tough,we can use angle forceps press it hard and the specimen will rebound rapidly when the external force was removed.The hardness of the defect area was stronger than that of the control groups.The rebound time of experimental group was shorter than that of the control groups,but the difference was not significant;there was no significant difference in rebound time between group B and group C,and the rebound time of group B and group C are all faster than group D.The color and texture of liver,spleen and kidney specimen had no significantly abnormal,the specimens’ capsule was without tension,and the edge of the specimens was not edema or round-blunt.VG staining results of animal specimens: At the end of the 8 week,each group had different levels of material residue,the residual material in the experimental group A and the control group B were higher than those in control group C and group D.The number of cells was rich in the observation range in each groups,the fibers in the experimental group A were the thickest and the most uniform contact with the membrane material comparison with other groups.The fibers in control group B and group C were thicker,and the fibers in group D were the thinest.At 12 weeks,there was no residual material in the four groups and the GO particles distributed uniformly throughout the defect area in group A and group B.The number of cells in each group was significantly lower than that in 8 weeks groups.The red dyed collagen fibers were spread over the whole defect area and interwoven into dense mesh.The fibers in control group B and group C showed loose reticular structure and collagen fibers in group D were interwoven into bundles.Results of fluorescence measurements for the specimens at 12 weeks: The new bone formation is mainly from the defect edge to the defect center.The amount of bone growth in the direction of the defect center and the lateral direction in the experimental group A was(290.546±23.256)μm,(102.014±11.196)μm respectively.In the control group B,the amount of bone growth in the direction of the defect center and the lateral direction was(146.470±29.482)μm,(96.898±13.351 m)μm;in the control group C,the amount of bone growth in the direction of the defect center and the lateral direction was(155.501±13.594)μm,(94.008±25.003)μm,and the amount of bone growth in the direction of the defect center and the lateral direction in the experimental group D was(109.526±24.025)μm,(61.128±15.512)μm respectively.In the center of the defect direction,the main effects of Simvastatin and graphene oxide were 95.025 μm and 85.994 μm respectively,the difference was statistically significant(P < 0.05),and the two factors have interaction effect(P < 0.05);the main effect of Simvastatin and graphene oxide to the lateral direction were 18.998 μm and 21.888 μm respectively,the difference was statistically significant(P < 0.05),but there was no interaction effect between them(P > 0.05).HE histopathological staining results of liver,spleen and kidney: No foreign bodies were deposited in each tissue under the microscope;the morphology of each tissue was normal;and there was no hyperemia and inflammatory cell infiltration in all tissues.Conclusion:Silk fibroin/graphene oxide/Simvastatin composite membrane can promote cranial defect repair;the short-term observation of local application of 0.5wt %GO has no damage to important organs(liver,spleen and kidney)of rats. |
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