| Objective:The aim of this study is to explore the repair effect of BMP-2 loaded chitosan temperature-sensitive hydrogel on bone defects around implants through in vivo experiments,and to provide clinical theoretical guidance for repairing the bone defects.Methods:1.Preparation of hydrogel and determination of the ratio of constituents: four temperature-sensitive hydrogel systems of chitosan with different concentrations were prepared by physical crosslinking.Then the coagulation time of each ratio at 37℃ was determined with inversion method,meanwhile,the ratio of constituents of hydrogel system which appropriate for clinical surgery was determined.2.In vivo study on the repair effect of chitosan temperature-sensitive hydrogel loaded with BMP-2 on bone defects around the implants: two standard implantation cavities were prepared at the proximal end of the femur on one side of the rabbits,and then the edge of the top of implantation cavities were created a bone defect area about 3 mm.After the 4.1*10mm Straumann implant was implanted,drugs were injected into the bone defect area.According to the different drug,implant + CS/temperaturesensitive hydrogel alone was used as the control group,and implant + CS/thermosensitive hydrogel loaded with BMP-2 was used as the experimental group.Another side of the rabbit was treated at the same way.Two rabbits were sacrificed at 3 weeks,6weeks and 9 weeks respectively after operation,and then the gross specimen was observed.Meanwhile,the distribution of osteoblasts,the maturation of collagen fibers and bone tissue,and the formation of new bone were observed by HE staining,Masson staining,toluidine blue staining,and methylene blue-acid magenta staining.By observing the osteogenesis in the defect area under HE microscopy,after that Image Pro Plus 6.0,SPSS 11.5 and Prism statistical software were used to perform the comparison between groups and within groups.Result:1.The prepared hydrogel system is sol-like,translucent,and has a certain fluidity.At 37 °C,the coagulation time of the hydrogel gradually decreased with the increase of β-GP.When the mixed concentration ratio of CS / β-GP is 2.5% / 60%,the coagulation time is 180 ± 15 s,which is appropriate ratio for animal experiments.2.General observation of animals: Within 48 hours after operation,rabbits have a slightly worse spirit,poor appetite,and reduced activity.After 3 days,they consumed food and water normally,and their activities gradually increased.After 7 days,the sutures gradually fell off,no swelling and exudation appeared in the wound.3.HE staining results: There were no obvious inflammatory cell infiltration in the bone defect area at each time point in the two groups.At 3 weeks after operation,a small amount of new bone was seen in both control group and experimental group.Meanwhile,some osteoblasts with blue cytoplasm were linearly arranged along the sparse trabeculae.At 6 weeks after operation,there were more new trabeculae in the control and experimental groups,and there were osteoblasts arranged around the trabeculae.The experimental group had more calcium deposits in the trabecula than the control group.At 9 weeks after operation,a large number of thick and mature bone trabeculae were seen in the experimental group,and the area of pale blue calcium salt deposition area was larger than that in the control group.A large number of osteoblasts were arranged around the new bone trabeculae,but the control group was relatively loose.4.Masson staining results: At 3 weeks after operation,the maturity level of new bone tissue was low in both groups.At 6 weeks after operation,the color of the new bone tissue in both group changed from blue to red,and the new bone in the experimental group gradually matured when compared with control group.At 9 weeks after operation,the trabeculae of the new bone in the experimental group were large and distributed in a patch-like shape,and the new bone was largely red-stained and the maturity reached a high level.However,the new bone and their maturity level in the control group increased slightly compared to 6 weeks,and the arrangement was relatively sparse.5.Toluidine blue staining results: At 3 weeks after operation,a small amount of new bone tissue was seen in both groups,the new bone trabecula was loose,and there were some osteoblasts arranged around the new bone.At 6 weeks after operation,new bone tissue increased in both groups.Also,in the experimental group,the new trabeculae were thick and distributed in a sheet shape,and the osteogenesis activity was active.At 9 weeks after operation,the amount of new bone in the experimental group and the control group reached a high level,and the trabecular in the experimental group showed a reticular distribution.6.Methylene blue-acid fuchsine staining results: At 3 weeks after operation,the staining showed that there were some new trabecular in both group,and the trabecular bones were sparse.At 6 weeks after operation,more trabecular was seen in both groups,and some osteoblasts arranged around the trabecular bone.Also,the gap between the trabecula of the new bone in the experimental group was reduced.At 9 weeks after operation,the experimental group showed more mature and thick trabeculae,which were distributed in a sheet shape.The control group showed that the new trabeculae increased compared with that at 6 weeks,and the neonatal trabeculae were loosely arranged.7.Statistical analysis results: By observing the osteogenesis in the defect area under HE microscopy,the percentage of new bone area in the experimental group and the control group were 12.06 ± 1.75% and 10.23 ± 1.00% after 3 weeks;26.92 ± 2.75% and 21.55 ± 1.85% after 6 weeks;46.53 ± 3.56% and 34.54 ± 2.41% after 9 weeks.At each time point,the percentage of new bone area in the experimental group was larger than that in the control group,and the difference was statistically significant(P <0.01).When comparing within groups,the percentage of new bone area in both increased with time,and the difference was statistically significant(P <0.01).Conclusion:1.Chitosan temperature-sensitive hydrogel system is prepared by chitosan and β-glyceryl phosphate as raw materials.It is sol-like at room temperature,translucent,and has a certain fluidity.At 37℃,when the mixed concentration ratio of CS / β-GP is 2.5% / 60%,the coagulation time is suitable,and it is appropriate ratio for animal experiments.2.BMP-2 loaded chitosan temperature-sensitive hydrogel showed good biocompatibility after being injected into the bone defect around the implant,meanwhile,it can induce the formation of new bone and promote the maturation of new bone.It has certain value for clinical application. |
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