共查询到14条相似文献,搜索用时 140 毫秒
1.
基因修饰的组织工程骨修复节段性骨缺损及相关免疫学研究 总被引:1,自引:0,他引:1
目的:观察骨形态发生蛋白-2(BMP-2)基因修饰的组织工程骨修复节段性骨缺损效果及异种骨支架体内应用的安全性。方法:①制备去抗原牛松质骨块(BCB),植入小鼠股四头肌袋内,术后行淋巴细胞转化试验和组织学观察。②在腺病毒载体介导下将BMP-2基因导入兔骨髓间质干细胞后,种植到BCB支架中,构建基因修饰的组织工程骨。于兔双侧桡骨中段造成15mm骨缺损,采用5种方法进行处理:BMP-2基因转染细胞+B(B(A组);未转染细胞+重组BMP-2+BCB(B组);对照基因转染细胞+BCB(C组);未转染细胞+B(B(D组);单纯BCB(E组)。术后4、8、12周行X线、组织学和生物力学检测。结果:①BCB具有较低的抗原性和良好的组织相容性;②A组术后4周诱导生成软骨组织并向编织骨转化,12周骨缺损修复,髓腔再通,新骨强度明显优于其他各组(P〈0.01)。结论:BMP-2基因修饰的组织工程骨是修复节段性骨缺损的好方法。 相似文献
2.
基因修饰的生物可降解人工骨修复骨缺损的血管化研究 总被引:5,自引:0,他引:5
目的评价转染骨形成蛋白2(bone morphogenetic protein 2,BMP-2)基因的骨髓间充质干细胞(marrow mesenchymal stem cells,MSCs)复合生物可降解人工骨修复兔桡骨缺损的血管化过程,观察BMP-2基因对促进移植骨血管化的影响。方法利用携带BMP-2基因的腺病毒载体(adenovirus carrying BMP-2 gene,Ad—BMP-2)转染MSCs及复合人工骨制备。取新西兰大耳白兔60只制成双侧桡骨中段1.5cm骨缺损模型,随机分为4组,每组15只(30侧)。各组采用不同材料植入缺损,A组:Ad—BMP-2转染MSCs+聚乳酸/聚己内酯(polylactic acid/polycaprolactone,PLA/PCL);B组:β-半乳糖酐酶基因的重组腺病毒转染MSCs+PLA/PCL;C组:未转染MSCs+PI,A/PCL;D组:单纯PLA/PCL。分别于术后4、8和12周各组处死5只动物,行X线片、微血管分布、组织学、透射电镜观察,并行血管内皮生长因子(vascular endothelial growth factor,VEGF)表达检测及微血管计数。结果A组4周时见移植骨内片状成骨影,有较多新生血管长入,支架孔隙内充满软骨痂,功能活跃的成骨细胞围绕微血管生长,VEGF表达及微血管数均明显高于其它各组,且差异有统计学意义(P〈0.01);8周时移植骨内成骨逐渐增多,微血管迂曲扩张并相互连接,软骨痂转变为小梁骨;12周时皮质骨连续,髓腔再通,微血管呈规则地纵向排列。B、C组成骨能力较弱,血管再生缓慢,12周时骨缺损得到初步修复,微血管沿新生骨小梁孔隙分布。D组各时间点新生血管少见,12周时骨端硬化,缺损区被纤维组织填充。结论BMP-2基因转染可通过上调VEGF表达,间接诱导移植骨血管化,促进种子细胞成活,加速新骨形成。 相似文献
3.
骨形成蛋白2基因修复兔桡骨缺损的实验研究 总被引:2,自引:0,他引:2
目的观察携带人骨形成蛋白2(bone morphogenetic protein 2,BMP-2)基因的腺病毒载体(adenovirus carrying BMP-2 gene,Ad—BMP2),通过纤维蛋白凝胶与牛松质骨支架(bovine cancellous bone,BCB)复合,修复骨缺损的效果。方法将60只新西兰大耳白兔随机分为4组,每组15只。制成双侧桡骨中段1.5cm骨缺损模型,采用4种材料植入修复。A组:Ad-BMP-2+BCB;B组:重组BMP-2+BCB;C组:携带D-半乳糖酐酶基因的腺病毒对照载体(adenovirus carrying β—galgene,Ad—Lacz)+BCB;D组:单纯BCB支架。修复术后各组于4、8和12周各处死动物5只取材,行X线片、组织学、生物力学和免疫组织化学染色检查。结果A、B两组骨缺损均得到了修复,但术后各时间点,A组在成骨活跃程度、新生骨量、力学强度及BMP-2表达等方面均明显优于B组;C、D两组均无新骨形成。结论BMP-2直接基冈治疗,操作简便、骨诱导能力强,是修复节段性骨缺损的有效方法。 相似文献
4.
目的评价骨形态发生蛋白2(BMP-2)基因强化的组织工程骨联合显微外科方法修复长段骨缺损的效果。方法分离培养兔骨髓基质干细胞,经BMP-2基因转染后复合异种骨支架体外构建基因强化的组织工程骨(GEB)。建立兔双侧桡骨缺损(2.5cm长)模型,采用5种方法修复。A:GEB加带血管蒂骨膜移植;B:GEB加血管束植入;C:GEB加游离骨膜移植;D:GEB;E:单纯支架。术后4、8、12周行X线、组织学、生物力学测定和微血管墨汁灌注等观察血管形成及成骨情况。结果A组血运建立最快,B组血管束早期即发出分支向移植骨内长入,C组4周时游离骨膜成活并发出微小血管,D组在BMP-2基因诱导下成骨速度和质量优于E组,12周时骨缺损部分修复,但中央区成骨不良,而E组12周时形成骨不连,缺损区内被纤维组织填充。在细胞成活率、生物力学性能、VEGF表达水平等方面,均表现为A〉B〉C〉D〉E,差异有统计学意义(P〈0.05)。结论BMP-2基因强化的组织工程骨联合显微外科方法修复长段骨缺损,既提供了血运,又提供了有效的成骨诱导因子,是治疗长段骨缺损较为理想的方法。其中,带血管蒂骨膜联合移植修复效果最佳;血管束植入法血供重建较快,方便临床应用。 相似文献
5.
腺病毒介导的骨形态发生蛋白2基因转染对成骨及血管化的影响 总被引:9,自引:2,他引:9
目的 观察骨形态发生蛋白2(BMP-2)基因转染人骨髓基质干细胞(hBMSC)对诱导成骨及血管化的影响,方法 基因转染后,检则骨钙素、Ⅰ型胶原和血管内皮生长因子(VEGF)的表达。并利用转染后的培养液上清诱导小鼠成纤维细胞(L929)。然后将转染后细胞接种到PLA/PCL(聚乳酸/聚己内酯)支架上,然后扫描电镜观察。结果 BMP-2基因转染后,可诱导hBMSC有L929骨钙索、Ⅰ型胶原呈阳性表达,VEGF的表达明显增高。转染细胞在支架中上生长良好,能量谱仪测得钙质分泌。蛋白印迹法检测到培养液中有BMP-2蛋白产生。结论 BMP-2基因转染可诱导hBMSC成骨转化,且通过上调VEGF表达促进血管化,复合转染后细胞构建的组织工程骨对治疗骨缺损具有重要意义。 相似文献
6.
基因修饰的组织工程骨联合带血管蒂骨膜移植修复长段骨缺损的研究 总被引:4,自引:0,他引:4
目的评价骨形态发生蛋白2(bone morphogenetic protein2,BMP-2)基因修饰的组织工程骨联合带血管蒂骨膜移植修复长段骨缺损的效果。方法分离培养兔骨髓基质干细胞,经BMP-2基因转染后复合异种骨支架体外构建基因修饰的组织工程骨(gene modified tissue engineering bone,GMB)。建立兔双侧桡骨缺损(长2.5cm)模型,采用5种方法修复。A组:GMB+带血管蒂骨膜移植;B组:GMB+血管束植入;C组:GMB+游离骨膜移植;D组:GMB;E组:单纯支架。于术后第4、8、12周行X线、组织学、生物力学测定和微血管墨汁灌注等观察血管形成及成骨情况。结果①A组血运建立快,第8周时即可修复骨缺损,其修复机制包括膜内成骨和软骨成骨两种机制;②B组血管束发出分支向移植骨内长入,但中心区成骨缓慢,第12周时骨缺损得到完全修复;③C组第4周时游离骨膜成活并发出微小血管,第8周时形成薄层外骨痂,第12周时骨缺损基本修复;④D组在BMP-2基因诱导下成骨速度和质量优于E组,可在第12周时使骨缺损部分修复,但中心区呈"空心"现象;而E组第12周时形成骨不连,缺损区内被纤维组织填充。结论带血管蒂骨膜与BMP-2基因修饰的组织工程骨联合移植,既提供了血运又提供了骨膜成骨细胞,同时具有良好的骨生成、骨诱导和骨引导作用,是治疗节段性骨缺损较为理想的方法。 相似文献
7.
重组PcDNA3.1-hBMP-2转染骨髓基质干细胞及复合异种骨支架体外构建组织工程骨 总被引:3,自引:2,他引:1
目的:构建人骨形态发生蛋白-2(human bone morphogenetic protein,,hBMP-2)真核表达载体PcDNA3.1-hBMP-2,转染兔骨髓基质干细胞(bane marrow stromal cells,BMSCs),种植去抗原牛松质骨(bovine cancellous bone,BCB)支架体外构建组织工程骨。方法:蛋白印迹法检测转染后细胞BMP-2的表达,碱性磷酸酶(ALPase)活性检测分析基因转染对细胞分化的影响。然后将转染后细胞接种到BCB支架上,扫描电镜观察细胞贴附、生长状况。结果:转染后,BMSCs表达BMP-2,ALP活性明显增高。扫描电镜见转染细胞分布均匀,伸展良好。结论:在脂质体介导下,BMP-2基因可导入细胞且稳定表达基因产物促进自身增殖分化,转染后细胞在支架材料上贴附生长良好,为进一步应用携带BMP-2基因的人工骨修复骨缺损奠定了实验基础。 相似文献
8.
转染人血管内皮生长因子基因的组织工程骨修复眼眶壁骨缺损的实验研究 总被引:1,自引:0,他引:1
目的探讨以转染人血管内皮生长因子(Vascular endothelial growth factor,VEGF)基因的骨髓基质干细胞(Bone marrow stromal cells,BMSCs)构建的组织工程骨在犬眼眶壁骨缺损中的修复效果。方法体外分离扩增犬自体BMSCs至第2代,用腺病毒转染VEGF基因。采用Real-time PCR和Western Blot检测目的基因和蛋白表达情况。细胞接种在珊瑚支架材料上构建组织工程骨,Elisa检测转基因细胞在支架材料上的蛋白持续表达情况。成年比格犬24只,双侧眼眶内侧壁制造直径12mm圆形骨缺损模型,随机分为4组:A组植入转染VEGF的组织工程骨,B组植入未转染基因的组织工程骨,C组植入单纯珊瑚材料,D组为旷置组。分别在手术后4周、12周、24周取材,行大体观察、Micro-CT分析、免疫组化方法检测血管形成情况,以及组织学观察和组织形态学检测比较骨缺损修复效果。结果VEGF基因修饰的BMSCs能够高表达目的基因和蛋白,构建组织工程骨后能够在体外持续分泌VEGF蛋白22d。C组和D组均未修复眶壁骨缺损。A组在骨缺损修复过程中,4周时的新生血管形成量和新生骨体... 相似文献
9.
重组pcDNA3.1-hBMP-2转染人骨髓基质干细胞和对其增殖及血管内皮生长因子表达的影响 总被引:3,自引:2,他引:3
目的构建人骨形成蛋白-2(hBMP-2)真核表达载体pcDNA3.1-hBMP-2,转染人骨髓基质干细胞(MSCs),探讨基因转染对其增殖和血管内皮生长因子(VEGF)表达的影响. 方法利用重组DNA和基因克隆技术构建重组载体pcDNA3.1-hBMP-2;细胞培养和基因转染技术体外转染人MSCs;免疫细胞化学、原位杂交和蛋白印迹法检测细胞BMP-2的表达;通过流式细胞仪和VEGF探针原位杂交分析其对细胞增殖和VEGF表达的影响. 结果转染后细胞在mRNA水平和蛋白质水平均表达BMP-2;转染后S期细胞比例增多,提示细胞DNA的合成增加;BMP-2基因转染上调细胞VEGF的表达. 结论在脂质体介导下,pcDNA3.1-hBMP-2转染MSCs获得成功.基因转染后能促进细胞增殖并将通过使VEGF的表达增加促进血管再生,为进一步骨缺损的基因治疗及构建组织工程骨奠定了实验基础. 相似文献
10.
骨形态发生蛋白2基因治疗与缓释载体修复骨缺损的比较研究 总被引:5,自引:3,他引:2
[目的]比较骨形态发生蛋白2(BMP-2)基因治疗与生长因子缓释方法修复节段性骨缺损效果。[方法]于兔双侧桡骨中段造成1.5cm骨缺损,采用4种方法修复:A组植入转基因骨髓间质干细胞(MSCs)与PLA/PCL(聚乳酸/聚己内酯)支架的复合物;B组植入单纯MSCs与含重组BMP-2的PLA/PCL缓释载体的复合物;C组植入单纯MSCs与PLA/PCL复合物;D组植入单纯PLA/PCL。术后4、8、12周行X线、组织学、生物力学和骨密度等检测,[结果]A组体内植入4周后,成骨细胞和间质细胞呈BMP-2强阳性表达;其成骨速度及成骨质量均明显优于B组,12周时骨缺损完全修复、C组成骨能力较弱,而D组则无新骨形成,残留骨缺损。[结论]BMP-2基因治疗是修复节段性骨缺损的好方法。 相似文献
11.
The objective of this study was to compare the efficacy of adenoviral and lentiviral regional gene therapy in a rat critical sized femoral defect model. The healing rates and quality of bone repair of femoral defects treated with syngeneic rat bone marrow cells (RBMCs) transduced with either lentiviral vector (Group I) or adenoviral vector (Group II) expressing bone morphogenetic protein-2 (BMP-2) gene were assessed. RBMCs transduced with the adenoviral vectors produced more than 3 times greater (p < 0.001) BMP-2 when compared to RBMCs transduced with lentiviral vectors in an in vitro evaluation. Serial bioluminescent imaging demonstrated short duration luciferase expression (less than 3 weeks) in defects treated with RBMCs co-transduced with two adenoviral vectors (Group IV; adenovirus expressing BMP-2 and luciferase [Ad-BMP-2 + Ad-Luc]). In contrast, the luciferase signal was present for 8 weeks in defects treated with RBMCs co-transduced with two lentiviral vectors (Group III; lentivirus expressing BMP-2 and luciferase gene [LV-BMP-2 + LV-Luc]). There were no significant differences with respect to the radiological healing rates (p = 0.12) in defects treated with lentiviral versus adenoviral mediated BMP-2 gene transfer. Biomechanical testing of healed Group I femoral specimens demonstrated significantly higher energy to failure (p < 0.05) when compared to Group II defects. Micro CT analysis revealed higher bone volume/tissue volume fraction (p = 0.04) in Group I defects when compared to Group II defects. In conclusion, prolonged BMP-2 expression associated with lentiviral mediated gene transfer demonstrated a trend towards superior quality of bone repair when compared to adenoviral mediated transfer of BMP-2. These results suggest that the bone repair associated with regional gene therapy is influenced not just by the amount of protein expression but also by duration of protein production. This observation needs validation in a more biologically challenging environment where differences in healing rates and quality of bone repair are more likely to be significantly different. 相似文献
12.
目的 用人骨形态发生蛋白 2腺病毒表达载体 (Ad -BMP - 2 )转染的人骨髓基质干细胞 (hBMSC) ,复合PLA/PCL(聚乳酸 /聚己内酯 )生物降解支架体外构建组织工程骨。方法 用Ad -BMP - 2转染体外培养的成人BMSC ,免疫组化、原位杂交染色和蛋白印迹方法检测细胞BMP - 2的表达 ,并通过流式细胞仪和ALP活性检测分析其对细胞增殖、分化的影响。然后将转染后细胞接种到PLA/PCL支架上 ,扫描电镜观察细胞贴附、生长状况。结果 转染后 ,hBMP - 2基因在mRNA水平和蛋白水平均有表达 ;S期细胞比例和ALP活性明显增高。扫描电镜见转染细胞分布均匀 ,伸展良好。结论 Ad-BMP - 2可高效转染hBMSC ,且促进细胞增殖及成骨转化。转染后细胞在PLA/PCL上生长良好 ,BMP - 2基因治疗的组织工程骨构建成功 相似文献
13.
This study investigated the effects of dual delivery of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2 (BMP-2) for bone regeneration in a rat cranial critical size defect. Four groups of scaffolds were generated with VEGF (12 microg), BMP-2 (2 mug), both VEGF (12 microg) and BMP-2 (2 microg), or no growth factor released from gelatin microparticles incorporated within the scaffold pores. These scaffolds were implanted within an 8 mm rat cranial critical size defect (n=8-9 for each group). At 4 and 12 weeks, implants were retrieved and evaluated by microcomputed tomography (microCT) and histological scoring analysis. Additionally, 4 week animals were perfused with a radiopaque material to visualize and quantify blood vessel formation. Histological analysis revealed that for all groups at 4 weeks, a majority of the porous scaffold volume was filled with vascularized fibrous tissue; however, bone formation appeared most abundant in the dual release group at this time. At 12 weeks, both dual release and BMP-2 groups showed large amounts of bone formation within the scaffold pores and along the outer surfaces of the scaffold; osteoid secretion and mineralization were apparent, and new bone was often in close or direct contact with the scaffold interface. MicroCT results showed no significant difference among groups for blood vessel formation at 4 weeks (<4% blood vessel volume); however, the dual release group showed significantly higher bone formation (16.1+/-9.2% bone volume) than other groups at this time. At 12 weeks, dual release and BMP-2 groups exhibited significantly higher bone formation (39.7+/-14.1% and 37.4+/-18.8% bone volume, respectively) than either the VEGF group or blank scaffolds (6.3+/-4.8% and 7.8+/-7.1% bone volume, respectively). This work indicates a synergistic effect of the dual delivery of VEGF and BMP-2 on bone formation at 4 weeks and suggests an interplay between these growth factors for early bone regeneration. For the doses investigated, the results show that the addition of VEGF does not affect the amount of bone formation achieved by BMP-2 at 12 weeks; however, they also indicate that delivery of both growth factors may enhance bone bridging and union of the critical size defect compared to delivery of BMP-2 alone. 相似文献
14.
Combined angiogenic and osteogenic factor delivery enhances bone marrow stromal cell-driven bone regeneration. 总被引:14,自引:0,他引:14
Yen-Chen Huang Darnell Kaigler Kevin G Rice Paul H Krebsbach David J Mooney 《Journal of bone and mineral research》2005,20(5):848-857
Bone formation is a coordinated process involving various biological factors. We have developed a scaffold system capable of sustained and localized presentation of osteogenic (BMP-4) and angiogenic (VEGF) growth factors and human bone marrow stromal cells to promote bone formation at an ectopic site. Combined delivery of these factors significantly enhanced bone formation compared with other conditions. INTRODUCTION: Tissue regeneration entails complex interactions between multiple signals and materials platforms. Orchestrating the presentation of these signals may greatly enhance the regeneration of lost tissue mass. Bone formation, for example, is dependent on the signaling of BMPs, molecules initiating vascularization (e.g., vascular endothelial growth factor [VEGF]), and osteogenic precursor cells capable of responding to these cues and forming bone tissue. It was hypothesized that combined and concerted delivery of these factors from biodegradable scaffolds would lead to enhanced bone formation. MATERIALS AND METHODS: Poly(lactic-co-glycolic acid) scaffolds containing combinations of condensed plasmid DNA encoding for BMP-4, VEGF, and human bone marrow stromal cells (hBMSCs) were implanted into the subcutaneous tissue of SCID mice. Implants (n = 6) were retrieved at 3, 8, and 15 weeks after implantation. Bone and blood vessel formation was determined qualitatively and quantitatively by methods including histology, immmunostaining, and muCT. RESULTS: Scaffolds delivering VEGF resulted in a prominent increase in blood vessel formation relative to the conditions without VEGF. BMP-4 expression in scaffolds encapsulating condensed DNA was also confirmed at the 15-week time-point, showing the characteristic of long-term delivery in this system. Combined delivery of all three types of factors resulted in a significant increase in the quantity of regenerated bone compared with any factor alone or any two factors combined, as measured with DXA, X-ray, and histomorphometric analysis. Furthermore, bone formed with all three factors had elastic moduli significantly higher than any other condition. CONCLUSIONS: Concerted delivery of BMP-4, VEGF, and hBMSCs promoted greater bone formation relative to any single factor or combination of two factors. Materials systems that allows multifactorial presentation more closely mimic natural developmental processes, and these results may have important implications for bone regeneration therapeutics. 相似文献