联合基因转染兔骨髓间充质干细胞促进组织工程骨形成的实验研究
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摘要
颅颌面部骨缺损是平、战时意外事故的常见损伤,临床需做骨缺损修复的病例很多。骨形态发生蛋白(Bone Morphogenetic Proteins,BMPs)对骨原细胞的分化起决定性作用,是促进成骨的重要因子之一;血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)是在创伤愈合过程中促进血管再生发挥着重要作用的生长因子,是最强的促血管生成因子。VEGF和BMP参与颅面部骨缺损修复过程中的不同阶段,二者具有协同作用。
本研究在创建兔颅骨缺损动物模型的基础上,对兔骨髓间充质干细胞(bonemarrow mesenchymal stem cells,BMSCs)进行体外的分离和培养,在传代后采用阳离子脂质体介导的转染方式,将外源性VEGF165及BMP2通过前期已成功构建的hVEGF165与hBMP2真核细胞共表达载体pIRES-hBMP2-hVEGF165导入到BMSCs中,使其正常表达,以提高BMSCs的成骨能力,再将转染后的骨髓间充质干细胞置入聚乳酸可吸收材料内,随材料一起植入骨缺损部位,观察组织工程骨形成,骨缺损修复成骨情况。本实验通过大体形态学观察、X线检查、组织学检查和免疫组化等方法检测联合基因转染后BMSCs促进颅骨缺损修复重建的效果。
结果:
1.大体形态学改变:4周时转染组颅骨缺损区开始被新生骨组织代替,表面有皮质骨生成;对照组颅骨缺损区无明显骨痂形成,表面被肉芽组织包裹。8周时转染组骨缺损区有大量骨痂生成,质地变硬;对照组仅在骨缺损区边缘可见部分骨痂形成。12周时转染组骨缺损区基本被新生骨覆盖,质地较硬。对照组骨缺损区边缘有较多骨痂生成,中心区域无明显骨质覆盖。
2.x线检查:术后2周时转染组开始出现模糊骨痂,中心区域有散在点状密度增强。对照组缺损边界明显,未见明显骨痂生成。术后4周时转染组缺损区出现明显骨痂,中心区域出现片状密度增强。二组缺损边缘出现骨痂,中心区域未见骨痂。8周时转染组缺损区基本被新生骨痂包裹。对照组边缘区域骨痂增多。12周转染组缺损区基本消失,密度较周围正常略低。对照组缺损面积变小,但中心无新骨形成。
3.组织学变化:转染组术后2周时有大量纤维性骨痂生成,骨缺损附近骨外膜内层的间充质细胞分裂增殖,向成骨细胞和软骨细胞转化,肉芽组织中软骨细胞和成骨细胞数目很多,软骨岛形成。4周时纤维性骨痂、软骨骨痂与骨性骨痂混杂在一起,以纤维性骨痂和软骨骨痂为主,有较多的新生血管,并出现软骨内骨化,可见部分软骨骨化,形成桥接骨痂。8周时成骨细胞、纤维母细胞减少、纤维骨痂消失,代之以软骨骨痂和骨性骨痂。12周时骨组织结构趋于完善,骨髓腔基本形成。对照组仅在骨缺损附近有成骨反应和少量新骨生成。
4.VEGF165表达情况
转染组VEGF在术后2周时,软骨细胞和成骨细胞呈阳性表达,基质内阳性表达,4周时软骨细胞分裂增生,呈团状排列,表达达到高峰,呈强阳性。对照组2周时在新生的软骨细胞和成骨细胞内呈阳性表达,4周时仅部分细胞呈阳性表达。
5.BMP2蛋白表达情况
转染组BMP2在术后2周时,在原始骨痂、成骨细胞、骨端骨细胞、新形成的软骨基质内呈阳性。4周时,新形成的软骨细胞出现阳性染色,软骨基质也呈阳性。幼稚软骨细胞、骨痂表面成骨细胞呈阳性。对照组2周时新生的软骨细胞和成骨细胞呈阳性表达,4周时仅部分细胞呈阳性表达。
结论:
1.通过pIRES-hBMP2-hVEGFl65重组质粒体外经脂质体转染兔骨髓间充质干细胞促进组织工程骨形成的研究,我们初步实现从体外研究向体内研究的深入,并进一步阐明在新骨生成过程中,BMP2与VEGF165具有协同作用,转基因BMSCs在转染后4周甚至更长时间内可促进表达BMP2和VEGF165。
2.pIRES-hBMP2-hVEGFl65真核细胞共表达载体转染使BMSCs中外源性BMP2、VEGF165基因稳定表达,向成骨细胞定向分化的能力增强,可促进颅骨缺损的修复。
3.本实验为运用骨组织工程技术修复巨大骨缺损提供了初步的实验依据。
Cranio-maxillo-facial bone defect is common both in daily life and during the war. So many cinical bone defect cases also needed to be reconstructed . Bone Morphogenetic Proteins (BMPs) play a critical role in the differentiation of osteoprogenitor cells and the bone formation process. Vascular Endothelial Growth Factor (VEGF) is the recently discovered cellular growth factor related to the angiogenesis which is important in the wound healing process. VEGF and BMP-2 have synergistic interaction in ossification of the cranium bone defect in different healing stages.
In this study, an rabbit canium defect model was established , by the prophase research of vector containing combinations of condensed plasmid DNA encoding for hBMP2 and hVEGF165. The cultured rabbit bone marrow mesenchymal stem cells (BMSCs) were transfected with cation liposome-mediated transfection method to express the exogenous VEGF and BMP-2. The transfected BMSCs could improve the bone regeneration. Then we put the bone marrow mesenchymal stem cells (BMSCs) into the absorbtable polylatic acid, all of them were implantded into the bone defect area together,so we can observe the regeneration of the defect bone. The reconstruction of cranium bone defect was detected in the transfected rBMSCs by gross observation , X-ray examination , histological test and immunohistochemistry examination.
Results
1. The gross morphological alternation
The cranium bone defect in the transfected group was replaced with neogenetic bone tissue with the generation of os integumentable 4 weeks later. No obvious osteotylus was found in the control group, however the defect area was encapsuled with granulation tissue. A large amount of lipid bony callus formed in the cranium bone defect in the transfected group 8 weeks later. Partial bone callus could be found merely encircled the cranium bone defect in the control group. The cranium bone defect in the transfected group was completely replaced with neogenetic rigid bone 12 weeks later. The callus encircled the cranium bone defect proliferated in bigger number without the cental converase of the bone in the control group.
2. X-ray examination
The undefined bone callus appeared in the transfected group 2 weeks later, with scattered spot-like density enhancement in the center. The defected margin exposed evidently without the formation of osteotylus in the control group. The callus emerged in cranium bone defect 4 weeks later in the transfected group , lamellar density enhancement could be found in the center. While in the control group the marginal callus increased . The cranium bone defect area in the transfected group vanished basically still with higher bone density than perimeter 12 weeks later. The cranium bone defect area declined without central bone formation in the control group.
3. histological alteration
Considerable fibrous osteotylus could be found at cranium bone defect area 2 weeks after the occurence of the bone defect .Mesenchymal stem cells in the medial of the periosteum around the cranium bone defect area proliferated, converting into massive osteoblasts and chondrocytes. Come out with the cartilaginous insula. The chondral tissue regenerated rapidly to fill the interspace of the cranium bone defect area. Fibrous osteotylus , chondral osteotylus and osseous osteotylus mixed together at the bone defect area 4 weeks later, by the majority of fibrous osteotylus and chondral osteotylus together with more neogenetic vascellum. With the appearance of the of intral-chondral ossification, partial chondral ossification formed bridge-grafting could be seen. The osteoblasts , fibroblasts declined . The fibrous osteotylus disappreared replaced by the chondral osteotylus and osseous osteotylus 8 weeks later. The bone construction proned to be perfect at the 12th week and the medullary canal was formed basically.
4. The expression of VEGF165
In the transfected group, we found that VEGF was positively expressed in the chondrocytes , osteoblasts and matrix at the 2nd week in the bone defect area. The chondrocytes proliferated and arranged in the clusters. The expression of VEGF reached peak value and was hadro-positive 4 weeks later. While the expression of VEGF was postive in the neogenetic chondrocytes and osteoblasts 2 weeks later , only partial reserved positive 4 weeks later after the occurence of the defect in the control group.
5.The expression of BMP2
In the transfected group, BMP2 was expressed in the osteoblasts, osteocytes and regenerated cartilage matrix, granular matrix in the primary callus at the bone defect area 2 weeks later after the bone defect. Strong expression of BMP-2 was observed in the regenerated chondrocytes and cartilage matrix, junior chondral cells and osteoblasts at the surface of the callus 4 weeks later. While the expression of BMP2 was postive in the neogenetic chondrocytes and osteoblasts 2 weeks later , only partial reserved positive 4 weeks later after the occurence of the defectin the control group.
Conclusions
1.The constructed pIRES-hBMP2-rVEGF165 can be successfully transfected into BMSCs of rabbits by bangosome. So this way can faciliate the bone ossification in tissue engineering.we can also advance our reserch from vitro study to vivo study primarily with this cation liposome-mediated transfection method. VEGF and BMP-2 have synergistic interaction in ossification. BMP2-VEGF165 can be steadily expressed in the transfected BMSCs both immediately and 4 weeks later.
2. The differentiation abilities of BMSCs is enhanced, so to promote the reconstruction of canium bone defect.
3. This study can provide an experimental base for rectification enormous bone defect in tissue engineering way.
本研究在创建兔颅骨缺损动物模型的基础上,对兔骨髓间充质干细胞(bonemarrow mesenchymal stem cells,BMSCs)进行体外的分离和培养,在传代后采用阳离子脂质体介导的转染方式,将外源性VEGF165及BMP2通过前期已成功构建的hVEGF165与hBMP2真核细胞共表达载体pIRES-hBMP2-hVEGF165导入到BMSCs中,使其正常表达,以提高BMSCs的成骨能力,再将转染后的骨髓间充质干细胞置入聚乳酸可吸收材料内,随材料一起植入骨缺损部位,观察组织工程骨形成,骨缺损修复成骨情况。本实验通过大体形态学观察、X线检查、组织学检查和免疫组化等方法检测联合基因转染后BMSCs促进颅骨缺损修复重建的效果。
结果:
1.大体形态学改变:4周时转染组颅骨缺损区开始被新生骨组织代替,表面有皮质骨生成;对照组颅骨缺损区无明显骨痂形成,表面被肉芽组织包裹。8周时转染组骨缺损区有大量骨痂生成,质地变硬;对照组仅在骨缺损区边缘可见部分骨痂形成。12周时转染组骨缺损区基本被新生骨覆盖,质地较硬。对照组骨缺损区边缘有较多骨痂生成,中心区域无明显骨质覆盖。
2.x线检查:术后2周时转染组开始出现模糊骨痂,中心区域有散在点状密度增强。对照组缺损边界明显,未见明显骨痂生成。术后4周时转染组缺损区出现明显骨痂,中心区域出现片状密度增强。二组缺损边缘出现骨痂,中心区域未见骨痂。8周时转染组缺损区基本被新生骨痂包裹。对照组边缘区域骨痂增多。12周转染组缺损区基本消失,密度较周围正常略低。对照组缺损面积变小,但中心无新骨形成。
3.组织学变化:转染组术后2周时有大量纤维性骨痂生成,骨缺损附近骨外膜内层的间充质细胞分裂增殖,向成骨细胞和软骨细胞转化,肉芽组织中软骨细胞和成骨细胞数目很多,软骨岛形成。4周时纤维性骨痂、软骨骨痂与骨性骨痂混杂在一起,以纤维性骨痂和软骨骨痂为主,有较多的新生血管,并出现软骨内骨化,可见部分软骨骨化,形成桥接骨痂。8周时成骨细胞、纤维母细胞减少、纤维骨痂消失,代之以软骨骨痂和骨性骨痂。12周时骨组织结构趋于完善,骨髓腔基本形成。对照组仅在骨缺损附近有成骨反应和少量新骨生成。
4.VEGF165表达情况
转染组VEGF在术后2周时,软骨细胞和成骨细胞呈阳性表达,基质内阳性表达,4周时软骨细胞分裂增生,呈团状排列,表达达到高峰,呈强阳性。对照组2周时在新生的软骨细胞和成骨细胞内呈阳性表达,4周时仅部分细胞呈阳性表达。
5.BMP2蛋白表达情况
转染组BMP2在术后2周时,在原始骨痂、成骨细胞、骨端骨细胞、新形成的软骨基质内呈阳性。4周时,新形成的软骨细胞出现阳性染色,软骨基质也呈阳性。幼稚软骨细胞、骨痂表面成骨细胞呈阳性。对照组2周时新生的软骨细胞和成骨细胞呈阳性表达,4周时仅部分细胞呈阳性表达。
结论:
1.通过pIRES-hBMP2-hVEGFl65重组质粒体外经脂质体转染兔骨髓间充质干细胞促进组织工程骨形成的研究,我们初步实现从体外研究向体内研究的深入,并进一步阐明在新骨生成过程中,BMP2与VEGF165具有协同作用,转基因BMSCs在转染后4周甚至更长时间内可促进表达BMP2和VEGF165。
2.pIRES-hBMP2-hVEGFl65真核细胞共表达载体转染使BMSCs中外源性BMP2、VEGF165基因稳定表达,向成骨细胞定向分化的能力增强,可促进颅骨缺损的修复。
3.本实验为运用骨组织工程技术修复巨大骨缺损提供了初步的实验依据。
Cranio-maxillo-facial bone defect is common both in daily life and during the war. So many cinical bone defect cases also needed to be reconstructed . Bone Morphogenetic Proteins (BMPs) play a critical role in the differentiation of osteoprogenitor cells and the bone formation process. Vascular Endothelial Growth Factor (VEGF) is the recently discovered cellular growth factor related to the angiogenesis which is important in the wound healing process. VEGF and BMP-2 have synergistic interaction in ossification of the cranium bone defect in different healing stages.
In this study, an rabbit canium defect model was established , by the prophase research of vector containing combinations of condensed plasmid DNA encoding for hBMP2 and hVEGF165. The cultured rabbit bone marrow mesenchymal stem cells (BMSCs) were transfected with cation liposome-mediated transfection method to express the exogenous VEGF and BMP-2. The transfected BMSCs could improve the bone regeneration. Then we put the bone marrow mesenchymal stem cells (BMSCs) into the absorbtable polylatic acid, all of them were implantded into the bone defect area together,so we can observe the regeneration of the defect bone. The reconstruction of cranium bone defect was detected in the transfected rBMSCs by gross observation , X-ray examination , histological test and immunohistochemistry examination.
Results
1. The gross morphological alternation
The cranium bone defect in the transfected group was replaced with neogenetic bone tissue with the generation of os integumentable 4 weeks later. No obvious osteotylus was found in the control group, however the defect area was encapsuled with granulation tissue. A large amount of lipid bony callus formed in the cranium bone defect in the transfected group 8 weeks later. Partial bone callus could be found merely encircled the cranium bone defect in the control group. The cranium bone defect in the transfected group was completely replaced with neogenetic rigid bone 12 weeks later. The callus encircled the cranium bone defect proliferated in bigger number without the cental converase of the bone in the control group.
2. X-ray examination
The undefined bone callus appeared in the transfected group 2 weeks later, with scattered spot-like density enhancement in the center. The defected margin exposed evidently without the formation of osteotylus in the control group. The callus emerged in cranium bone defect 4 weeks later in the transfected group , lamellar density enhancement could be found in the center. While in the control group the marginal callus increased . The cranium bone defect area in the transfected group vanished basically still with higher bone density than perimeter 12 weeks later. The cranium bone defect area declined without central bone formation in the control group.
3. histological alteration
Considerable fibrous osteotylus could be found at cranium bone defect area 2 weeks after the occurence of the bone defect .Mesenchymal stem cells in the medial of the periosteum around the cranium bone defect area proliferated, converting into massive osteoblasts and chondrocytes. Come out with the cartilaginous insula. The chondral tissue regenerated rapidly to fill the interspace of the cranium bone defect area. Fibrous osteotylus , chondral osteotylus and osseous osteotylus mixed together at the bone defect area 4 weeks later, by the majority of fibrous osteotylus and chondral osteotylus together with more neogenetic vascellum. With the appearance of the of intral-chondral ossification, partial chondral ossification formed bridge-grafting could be seen. The osteoblasts , fibroblasts declined . The fibrous osteotylus disappreared replaced by the chondral osteotylus and osseous osteotylus 8 weeks later. The bone construction proned to be perfect at the 12th week and the medullary canal was formed basically.
4. The expression of VEGF165
In the transfected group, we found that VEGF was positively expressed in the chondrocytes , osteoblasts and matrix at the 2nd week in the bone defect area. The chondrocytes proliferated and arranged in the clusters. The expression of VEGF reached peak value and was hadro-positive 4 weeks later. While the expression of VEGF was postive in the neogenetic chondrocytes and osteoblasts 2 weeks later , only partial reserved positive 4 weeks later after the occurence of the defect in the control group.
5.The expression of BMP2
In the transfected group, BMP2 was expressed in the osteoblasts, osteocytes and regenerated cartilage matrix, granular matrix in the primary callus at the bone defect area 2 weeks later after the bone defect. Strong expression of BMP-2 was observed in the regenerated chondrocytes and cartilage matrix, junior chondral cells and osteoblasts at the surface of the callus 4 weeks later. While the expression of BMP2 was postive in the neogenetic chondrocytes and osteoblasts 2 weeks later , only partial reserved positive 4 weeks later after the occurence of the defectin the control group.
Conclusions
1.The constructed pIRES-hBMP2-rVEGF165 can be successfully transfected into BMSCs of rabbits by bangosome. So this way can faciliate the bone ossification in tissue engineering.we can also advance our reserch from vitro study to vivo study primarily with this cation liposome-mediated transfection method. VEGF and BMP-2 have synergistic interaction in ossification. BMP2-VEGF165 can be steadily expressed in the transfected BMSCs both immediately and 4 weeks later.
2. The differentiation abilities of BMSCs is enhanced, so to promote the reconstruction of canium bone defect.
3. This study can provide an experimental base for rectification enormous bone defect in tissue engineering way.
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