组织工程骨及引导性骨再生修复兔桡骨节段性骨缺损的实验研究
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摘要
目的
主要研究具有引导性骨再生作用的n-HA/CS/CMC膜与n-HA/CS/CMC组织工程骨单独及联合应用修复节段性骨缺损的可行性。为新型n-HA/CS/CMC膜与n-HA/CS/CMC组织工程骨多孔材料能否应用于临床提供理论基础，并探讨引导性骨组织再生膜与组织工程骨联合应用对骨缺损修复的影响。
实验方法
1，体外实验：取新西兰兔骨髓经密度梯度离心法分离兔骨髓间充质干细胞及成骨诱导分化培养成骨细胞，比较BMSCs及成骨细胞生长情况,碱性磷酸酶染色及钙质染色检测成骨诱导细胞。将BMSCs及成骨细胞以1.0×105／ml分别接种到n-HA/CS/CMC三元多孔复合材料及n-HA/PA66上，进行体外共培养，观测细胞生长及粘附情况，MTT法检测细胞生长及增殖。
2，体内实验：制作新西兰兔双侧桡骨10mm节段性骨缺损模型，实验根据植入不同材料分为A、B、C组。A组分为AL组和AR组，AL组左侧桡骨缺损处植入n-HA/CS/CMC+BMSCs，外包n-HA/CS/CMC膜，AR组右侧桡骨缺损植入n-HA/CS/CMC+成骨细胞，外包n-HA/CS/CMC膜； B组： BL组左侧桡骨缺损植入n-HA/CS/CMC+BMSCs+BMSCs，外不包膜，BR组右侧桡骨缺损植入n-HA/PA66+BMSCs，外包n-HA/CS/CMC膜；C组：CL组左侧桡骨缺损周围包绕n-HA/CS/CMC膜，CR组右侧桡骨缺损区不填充任何植入物，为空白对照组。术后进行大体观察和X线检查，组织学观察，植入区BMP及VEGF浓度检测。
结果
1，体外实验：MSCs及成骨细胞在n-HA/CS/CMC三元多孔复合材料及n-HA/PA66上贴附、生长良好。
2，体内实验：经大体观察、X线、组织学及BMP、VEGF检测发现，术后4周后A、B各组及CL组骨缺损区均有新骨生成，成骨量随着时间的推移而增加，且AL组、AR组及BR组较BL和CL组成骨更早，局部成骨诱导因子浓度更高。
结论
1，体外实验：n-HA/CS/CMC（纳米羟基磷灰石/壳聚糖/羧甲基纤维素）三元多孔复合材料可作为理想的构建组织工程骨载体进行骨缺损修复研究。
2，体内实验：BMSCs或成骨细胞复合n-HA/CS/CMC三元多孔复合材料构建的组织工程骨可作为节段性骨缺损的修复材料，n-HA/CS/CMC三元复合膜有引导性骨再生作用，n-HA/CS/CMC三元多孔复合组织工程骨与n-HA/CS/CMC复合膜联合应用更有利于骨缺损修复。
Objective
To study the feasibility for repairing segmental defects with single orcombination of n-HA/CS/CMC composite membrane and n-HA/CS/CMCtri-component composite. To provide the rational for the clinical applicationof the new n-HA/CS/CMC composite membrane and n-HA/CS/CMCtri-component composite, and implore the effects on the repairing bonedefects with combined application of guided bone regeneration membraneand tissue engineering bone.
Methods
Experiment in vitro: BMSCs were isolated by density gradientcentrifugation from the bone marrow of New Zealand rabbits, and theosteoblasts were differentiated from BMSCs by bone induction. The growthstates and growth curve of BMSCs and osteoblast cells were detected andcompared. The osteoblasts were identified using alkaline phosphatase andcalcium steining. Then, the BMSCs and osteoblasts were inoculated with n-HA/CS/CMC tri-component porous composite and n-HA/PA66by aconcentration of1.0×105/ml, and cultured in vitro. The growth state andadhesion of cells were observed, and the growth and proliferation of cellswere examined by MTT, and the adhesion between cells and material wereobserved by HE staining.
Experiment in vivo: The experimental model of10mm bone segmentaldefect were produced in New Zealand white rabbits with both radius, andwere divided into group A, group B and group C according to transplantmaterials. Group A was divided into two groups, the group AL and GroupBL, and the segmental defect on the left side of the former group wasimbedded with n-HA/CS/CMC+BMSCs which was coated with amembrane of n-HA/CS/CMC, while the segmental defect on the right side ofthe latter group was imbedded with n-HA/CS/CMC+osteoblasts, which wascoated with a membrane of n-HA/CS/CMC. Group B: the segmental defecton the left side of the group BL was imbedded withn-HA/CS/CMC+BMSCs+BMSCs without membrane coated, while thesegmental defect on the right side of the group BR was imbedded withn-HA/PA66+BMSCs with a membrane of n-HA/CS/CMC coated. Group C:the segmental defect on the left side of the group CL was coateded with amembrane of n-HA/CS/CMC, while the segmental defect on the right side ofthe group CR taken as the control group was imbedded with nothing.Postoperative gross observation, X-ray evaluation, histological examination and concentration tests of BMP and VEGF in the implanting region wascarried out.
Results
Experiment in vitro: MSCs and osteoblasts presented a good adhesionand growth in the n-HA/CS/CMC tri-component porous composite andn-HA/PA66.
Experiment in vivo: according to gross observation, X-ray evaluation,histological examination and concentration tests of BMP and VEGF, newbone formed and increased as time went on in the radial segmental defect inall the rabbits from group A, group B and group CL4weeks postoperatively.In addition, new bone formation developed earlier and the regionalconcentration of the osteoinductive factor was much higher in the group AL,group AR and group BR than group BL and group CL.
Conclusions
Experiment in vitro: n-HA/CS/CMC tri-component porous compositecould be considered as ideal carriers for constructing tissue engineering bonein bone defects repairing study.
Experiment in vivo: The tissue engineering bone constructed byBMSCs or osteoblasts and n-HA/CS/CMC tri-component porous compositecan be taken as repairing material for the segmental bone defects. Then-HA/CS/CMC composite membrane can guide bone regeneration. And thecombined use of n-HA/CS/CMC n-HA/CS/CMC tri-component porous composite tissue engineering bone and n-HA/CS/CMC membrane canenhance bone defects repairing.
主要研究具有引导性骨再生作用的n-HA/CS/CMC膜与n-HA/CS/CMC组织工程骨单独及联合应用修复节段性骨缺损的可行性。为新型n-HA/CS/CMC膜与n-HA/CS/CMC组织工程骨多孔材料能否应用于临床提供理论基础，并探讨引导性骨组织再生膜与组织工程骨联合应用对骨缺损修复的影响。
实验方法
1，体外实验：取新西兰兔骨髓经密度梯度离心法分离兔骨髓间充质干细胞及成骨诱导分化培养成骨细胞，比较BMSCs及成骨细胞生长情况,碱性磷酸酶染色及钙质染色检测成骨诱导细胞。将BMSCs及成骨细胞以1.0×105／ml分别接种到n-HA/CS/CMC三元多孔复合材料及n-HA/PA66上，进行体外共培养，观测细胞生长及粘附情况，MTT法检测细胞生长及增殖。
2，体内实验：制作新西兰兔双侧桡骨10mm节段性骨缺损模型，实验根据植入不同材料分为A、B、C组。A组分为AL组和AR组，AL组左侧桡骨缺损处植入n-HA/CS/CMC+BMSCs，外包n-HA/CS/CMC膜，AR组右侧桡骨缺损植入n-HA/CS/CMC+成骨细胞，外包n-HA/CS/CMC膜； B组： BL组左侧桡骨缺损植入n-HA/CS/CMC+BMSCs+BMSCs，外不包膜，BR组右侧桡骨缺损植入n-HA/PA66+BMSCs，外包n-HA/CS/CMC膜；C组：CL组左侧桡骨缺损周围包绕n-HA/CS/CMC膜，CR组右侧桡骨缺损区不填充任何植入物，为空白对照组。术后进行大体观察和X线检查，组织学观察，植入区BMP及VEGF浓度检测。
结果
1，体外实验：MSCs及成骨细胞在n-HA/CS/CMC三元多孔复合材料及n-HA/PA66上贴附、生长良好。
2，体内实验：经大体观察、X线、组织学及BMP、VEGF检测发现，术后4周后A、B各组及CL组骨缺损区均有新骨生成，成骨量随着时间的推移而增加，且AL组、AR组及BR组较BL和CL组成骨更早，局部成骨诱导因子浓度更高。
结论
1，体外实验：n-HA/CS/CMC（纳米羟基磷灰石/壳聚糖/羧甲基纤维素）三元多孔复合材料可作为理想的构建组织工程骨载体进行骨缺损修复研究。
2，体内实验：BMSCs或成骨细胞复合n-HA/CS/CMC三元多孔复合材料构建的组织工程骨可作为节段性骨缺损的修复材料，n-HA/CS/CMC三元复合膜有引导性骨再生作用，n-HA/CS/CMC三元多孔复合组织工程骨与n-HA/CS/CMC复合膜联合应用更有利于骨缺损修复。
Objective
To study the feasibility for repairing segmental defects with single orcombination of n-HA/CS/CMC composite membrane and n-HA/CS/CMCtri-component composite. To provide the rational for the clinical applicationof the new n-HA/CS/CMC composite membrane and n-HA/CS/CMCtri-component composite, and implore the effects on the repairing bonedefects with combined application of guided bone regeneration membraneand tissue engineering bone.
Methods
Experiment in vitro: BMSCs were isolated by density gradientcentrifugation from the bone marrow of New Zealand rabbits, and theosteoblasts were differentiated from BMSCs by bone induction. The growthstates and growth curve of BMSCs and osteoblast cells were detected andcompared. The osteoblasts were identified using alkaline phosphatase andcalcium steining. Then, the BMSCs and osteoblasts were inoculated with n-HA/CS/CMC tri-component porous composite and n-HA/PA66by aconcentration of1.0×105/ml, and cultured in vitro. The growth state andadhesion of cells were observed, and the growth and proliferation of cellswere examined by MTT, and the adhesion between cells and material wereobserved by HE staining.
Experiment in vivo: The experimental model of10mm bone segmentaldefect were produced in New Zealand white rabbits with both radius, andwere divided into group A, group B and group C according to transplantmaterials. Group A was divided into two groups, the group AL and GroupBL, and the segmental defect on the left side of the former group wasimbedded with n-HA/CS/CMC+BMSCs which was coated with amembrane of n-HA/CS/CMC, while the segmental defect on the right side ofthe latter group was imbedded with n-HA/CS/CMC+osteoblasts, which wascoated with a membrane of n-HA/CS/CMC. Group B: the segmental defecton the left side of the group BL was imbedded withn-HA/CS/CMC+BMSCs+BMSCs without membrane coated, while thesegmental defect on the right side of the group BR was imbedded withn-HA/PA66+BMSCs with a membrane of n-HA/CS/CMC coated. Group C:the segmental defect on the left side of the group CL was coateded with amembrane of n-HA/CS/CMC, while the segmental defect on the right side ofthe group CR taken as the control group was imbedded with nothing.Postoperative gross observation, X-ray evaluation, histological examination and concentration tests of BMP and VEGF in the implanting region wascarried out.
Results
Experiment in vitro: MSCs and osteoblasts presented a good adhesionand growth in the n-HA/CS/CMC tri-component porous composite andn-HA/PA66.
Experiment in vivo: according to gross observation, X-ray evaluation,histological examination and concentration tests of BMP and VEGF, newbone formed and increased as time went on in the radial segmental defect inall the rabbits from group A, group B and group CL4weeks postoperatively.In addition, new bone formation developed earlier and the regionalconcentration of the osteoinductive factor was much higher in the group AL,group AR and group BR than group BL and group CL.
Conclusions
Experiment in vitro: n-HA/CS/CMC tri-component porous compositecould be considered as ideal carriers for constructing tissue engineering bonein bone defects repairing study.
Experiment in vivo: The tissue engineering bone constructed byBMSCs or osteoblasts and n-HA/CS/CMC tri-component porous compositecan be taken as repairing material for the segmental bone defects. Then-HA/CS/CMC composite membrane can guide bone regeneration. And thecombined use of n-HA/CS/CMC n-HA/CS/CMC tri-component porous composite tissue engineering bone and n-HA/CS/CMC membrane canenhance bone defects repairing.
引文
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