壳聚糖/β-磷酸三钙/rhBMP-2可注射性复合体修复兔下颌骨缺损的实验研究
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摘要
目的:研究壳聚糖(chitosan,CS)/β-磷酸三钙(beta-Tricalcium phosphate,β-TCP)作为人重组骨诱导蛋白(recombinant human bone morphogenetic protein-2,rhBMP-2)注射性载体修复兔下颌骨缺损的效果及机制,探讨该复合体作为注射性骨替代物的可行性。为临床应用提供理论依据。
方法:将24只新西兰大白兔共48处缺损完全随机分为4组:CS/β-TCP/rhBMP-2组、CS/β-TCP组、自体骨移植组、空白对照组。每组12侧骨缺损,分2、4、8周三个时间点,每个时间点4侧骨缺损,2、4、8周各取8只兔,进行大体观察、X线检查、苏木精-伊红(hematoxylin eosin,HE)染色、Ⅰ型胶原(CollagenⅠ,COLⅠ)免疫组织化学染色、骨密度测量、扫描电镜(scanning electronmicroscope,SEM)观察及图像统计学分析。
结果:(1)大体观察显示,各实验组骨缺损区骨痂量和骨痂厚度随时间延长而逐渐增多增厚,且各时间点,CS/β-TCP/rhBMP-2组的骨痂量与自体骨组相当,而多于其它组。(2)X线显示,各组缺损区透光区面积随时间延长而逐渐减小,术后2周,CS/β-TCP/rhBMP-2组和自体骨组修复区边缘和中心有散在的小片状阻射影像,术后4周,空白对照组、CS/β-TCP组修复区中心才出现散在的小片状阻射影像,术后8周,CS/β-TCP/rhBMP-2组和自体骨组缺损区无透光区,其他组缺损区透光区也较4周有所减小。(3)组织学观察显示,各时间点CS/β-TCP/rhBMP-2组、自体骨组两组骨基质均分别较CS/β-TCP组和空白对照组多,且CS/β-TCP/rhBMP-2组和自体骨组骨基质无明显差别。空白对照组最差。(4)术后骨密度测量分析,各组材料骨修复区随时间的增长密度逐渐增大,8周时CS/β-TCP/rhBMP-2组与CS/β-TCP组以及BC组之间骨密度有显著性差异。(5)SEM结果显示,术后4周,材料与周围骨床呈纤维骨性结合,材料已开始降解;术后8周,CS/β-TCP/rhBMP-2组材料与周围骨床形成骨性结合,间隙基本消失:其他实验组间隙也有所减小,材料降解明显,界面处不能看到完整材料结构。(6)图像统计学分析显示,术后不同组间成骨面积比比较有显著差异(F=53.292,P=0.000),CS/β-TCP/rhBMP-2组(均数为22.96%)与自体骨组(均数为22.63%)之间无显著性差异,两组均优于空白对照组(均数为10.35)、CS/β-TCP(均数为10.73)组。不同观察时间之间有显著性差异(F=128.984,P=0.000),随着观察时间的延长,成骨面积比例升高。
结论:CS/β-TCP/rhBMP-2可注射组织工程骨成骨效果与自体骨相当,明显优于CS/β-TCP组、空白对照组,可以加快骨缺损的修复过程,促进早期愈合,具有良好的临床应用前景。
Objective:To evaluate the repaired efficacy and mechanism of scaffolds for chitosan / beta-Tricalcium phosphate(CS/β-TCP) as an injectable bone tissue engineering scaffold material of recombinant human bone morphogenetic protein(rhBMP-2) on mandible of rabbits.To approach the possibilities of applications as an injectable bone surrogate and provide theoretical evidence for the clinical practices.
Methods:Twenty-four healthy New Zealand White Rabbits were selected,and the 15 mm×6 mm bilateral mandibular defects were made by dental drill.Then the animals were randomly divided into 4 groups composed of CS/β-TCP /rhBMP-2 group,CS/β-TCP group,autograft group and blank contrast group,the defects were made synchronously,implanted with differert materials according to different groups. 2、4 and 8 weeks after operation the specimen were harvested and examined by gross observation,X-ray observation,histological observation,including hematoxylin eosin (HE) staining,collagenⅠ(COLⅠ)immunochemical staining and scanning electron microscope(SEM) and Statistical analysis of images.
Result:(1)Gross observation demonstrated that the size and thickness of osteotylus in every defect increased gradually.And,in every experimental time,the size of osteotyluses in CS/β-TCP/rhBMP-2 group is equivalent with autograft group,there were more osteotyluses than others.(2)X-ray observation demonstrated that the area of photic zone in every group diminished gradually.2 weeks after operation,there were some opacity areas in the edge and the center of the recovery area in CS/β-TCP/rhBMP-2 group and autograft group,4 weeks after operation,there were some opacity areas in the center of the recotery area in CS/β-TCP group and BC group,8 weeks after operation,there was no photic zone in CS/β-TCP/rhBMP-2 group and autograft group,while,the photic zone in other groups also decreased.(3)Histological observation demonstrated that,in every experimental time, there were more bone matrixs in CS/β-TCP/rhBMP-2 group and autograft group than which in CS/β-TCP group and BC group.The bone matrixs in CS/β-TCP/rhBMP-2 group have not significant difference with autograft group.There are least bone matrixs in BC group.(4) The bone density of each group gradually increased with time.The bone density of CS/β-TCP/rhBMP-2 group was significant difference from CS/β-TCP group and BC group.(5) ESEM demonstrated that,4 weeks after operation,the bone bed and the materials were connected with fibrous bone,the materials degraded.8 weeks after operation,the bone bed and the materials in CS/β-TCP/rhBMP-2 group were connected with bone,the gap between them disminished,the degradation of the materials were so obvious that the complete structure of materials couldn't be found.(6) Statistical analysis of images demonstrated that,There was significant difference among the areas of bone regeneration of all groups(F=53.292 P=0.000).There was no significant differentce between CS/β-TCP/rhBMP-2 group(Mean=22.96%) and autograft group (Mean=22.63%).The two groups was better than blank contrast group (Mean=10.35%) and CS/β-TCP group(Mean=10.73%).There was significant differentce among all weeks,the proportion of area of bone regeneration increased with time.
Conclusion:Osteogenic effect of injectable CS/β-TCP/rhBMP-2 composite in repairing the rabbit mandible defects is considerable with autograft group.CS/β-TCP/rhBMP-2 group,which compared with CS/β-TCP group and blank contrast group,could advance the reparative process of bone defect,promote the earlier period healing,which had good foreground of clinical application.
方法:将24只新西兰大白兔共48处缺损完全随机分为4组:CS/β-TCP/rhBMP-2组、CS/β-TCP组、自体骨移植组、空白对照组。每组12侧骨缺损,分2、4、8周三个时间点,每个时间点4侧骨缺损,2、4、8周各取8只兔,进行大体观察、X线检查、苏木精-伊红(hematoxylin eosin,HE)染色、Ⅰ型胶原(CollagenⅠ,COLⅠ)免疫组织化学染色、骨密度测量、扫描电镜(scanning electronmicroscope,SEM)观察及图像统计学分析。
结果:(1)大体观察显示,各实验组骨缺损区骨痂量和骨痂厚度随时间延长而逐渐增多增厚,且各时间点,CS/β-TCP/rhBMP-2组的骨痂量与自体骨组相当,而多于其它组。(2)X线显示,各组缺损区透光区面积随时间延长而逐渐减小,术后2周,CS/β-TCP/rhBMP-2组和自体骨组修复区边缘和中心有散在的小片状阻射影像,术后4周,空白对照组、CS/β-TCP组修复区中心才出现散在的小片状阻射影像,术后8周,CS/β-TCP/rhBMP-2组和自体骨组缺损区无透光区,其他组缺损区透光区也较4周有所减小。(3)组织学观察显示,各时间点CS/β-TCP/rhBMP-2组、自体骨组两组骨基质均分别较CS/β-TCP组和空白对照组多,且CS/β-TCP/rhBMP-2组和自体骨组骨基质无明显差别。空白对照组最差。(4)术后骨密度测量分析,各组材料骨修复区随时间的增长密度逐渐增大,8周时CS/β-TCP/rhBMP-2组与CS/β-TCP组以及BC组之间骨密度有显著性差异。(5)SEM结果显示,术后4周,材料与周围骨床呈纤维骨性结合,材料已开始降解;术后8周,CS/β-TCP/rhBMP-2组材料与周围骨床形成骨性结合,间隙基本消失:其他实验组间隙也有所减小,材料降解明显,界面处不能看到完整材料结构。(6)图像统计学分析显示,术后不同组间成骨面积比比较有显著差异(F=53.292,P=0.000),CS/β-TCP/rhBMP-2组(均数为22.96%)与自体骨组(均数为22.63%)之间无显著性差异,两组均优于空白对照组(均数为10.35)、CS/β-TCP(均数为10.73)组。不同观察时间之间有显著性差异(F=128.984,P=0.000),随着观察时间的延长,成骨面积比例升高。
结论:CS/β-TCP/rhBMP-2可注射组织工程骨成骨效果与自体骨相当,明显优于CS/β-TCP组、空白对照组,可以加快骨缺损的修复过程,促进早期愈合,具有良好的临床应用前景。
Objective:To evaluate the repaired efficacy and mechanism of scaffolds for chitosan / beta-Tricalcium phosphate(CS/β-TCP) as an injectable bone tissue engineering scaffold material of recombinant human bone morphogenetic protein(rhBMP-2) on mandible of rabbits.To approach the possibilities of applications as an injectable bone surrogate and provide theoretical evidence for the clinical practices.
Methods:Twenty-four healthy New Zealand White Rabbits were selected,and the 15 mm×6 mm bilateral mandibular defects were made by dental drill.Then the animals were randomly divided into 4 groups composed of CS/β-TCP /rhBMP-2 group,CS/β-TCP group,autograft group and blank contrast group,the defects were made synchronously,implanted with differert materials according to different groups. 2、4 and 8 weeks after operation the specimen were harvested and examined by gross observation,X-ray observation,histological observation,including hematoxylin eosin (HE) staining,collagenⅠ(COLⅠ)immunochemical staining and scanning electron microscope(SEM) and Statistical analysis of images.
Result:(1)Gross observation demonstrated that the size and thickness of osteotylus in every defect increased gradually.And,in every experimental time,the size of osteotyluses in CS/β-TCP/rhBMP-2 group is equivalent with autograft group,there were more osteotyluses than others.(2)X-ray observation demonstrated that the area of photic zone in every group diminished gradually.2 weeks after operation,there were some opacity areas in the edge and the center of the recovery area in CS/β-TCP/rhBMP-2 group and autograft group,4 weeks after operation,there were some opacity areas in the center of the recotery area in CS/β-TCP group and BC group,8 weeks after operation,there was no photic zone in CS/β-TCP/rhBMP-2 group and autograft group,while,the photic zone in other groups also decreased.(3)Histological observation demonstrated that,in every experimental time, there were more bone matrixs in CS/β-TCP/rhBMP-2 group and autograft group than which in CS/β-TCP group and BC group.The bone matrixs in CS/β-TCP/rhBMP-2 group have not significant difference with autograft group.There are least bone matrixs in BC group.(4) The bone density of each group gradually increased with time.The bone density of CS/β-TCP/rhBMP-2 group was significant difference from CS/β-TCP group and BC group.(5) ESEM demonstrated that,4 weeks after operation,the bone bed and the materials were connected with fibrous bone,the materials degraded.8 weeks after operation,the bone bed and the materials in CS/β-TCP/rhBMP-2 group were connected with bone,the gap between them disminished,the degradation of the materials were so obvious that the complete structure of materials couldn't be found.(6) Statistical analysis of images demonstrated that,There was significant difference among the areas of bone regeneration of all groups(F=53.292 P=0.000).There was no significant differentce between CS/β-TCP/rhBMP-2 group(Mean=22.96%) and autograft group (Mean=22.63%).The two groups was better than blank contrast group (Mean=10.35%) and CS/β-TCP group(Mean=10.73%).There was significant differentce among all weeks,the proportion of area of bone regeneration increased with time.
Conclusion:Osteogenic effect of injectable CS/β-TCP/rhBMP-2 composite in repairing the rabbit mandible defects is considerable with autograft group.CS/β-TCP/rhBMP-2 group,which compared with CS/β-TCP group and blank contrast group,could advance the reparative process of bone defect,promote the earlier period healing,which had good foreground of clinical application.
引文
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