Prefabrication of a Vascularized Bone Graft With Beta Tricalcium Phosphate Using an In Vivo Bioreactor

We aimed to introduce an in vivo bioreactor‐vascular pedicle threaded through the central portion of a scaffold in which a vascularized bone graft was prefabricated using adenoviral human BMP‐2 gene (AdBMP2)‐modified bone marrow mesenchymal stem cells (BMSCs), beta tricalcium phosphate (β‐TCP), a vessel bundle, and muscularis membrane(group A). As controls, Adβgal‐BMSCs/β‐TCP granules, vessel bundle, and the muscularis membrane (group B); BMSCs/β‐TCP granules, vessel bundle, and muscularis membrane (group C); and β‐TCP granules, vessel bundle, and muscularis membrane (group D) were prepared. Formation of bone tissue and a vascular network was assessed by microangiography and histological methods 4 weeks after prefabrication. New cartilage and bone tissue in the space between β‐TCP granules (mainly endochondral bone) were confirmed by histology, and a de novo vascular network circulating from the vessel bundle through newly formed bone tissue was observed in group A. Formation of bone or cartilage was not observed in the control groups. We concluded that the in vivo bioreactor is a promising method for prefabrication of vascularized functional bone.     

Evolution of an in vivo bioreactor.

The ideal bone graft substitute requires osteoconductive, osteoinductive, and osteogenic components. This study introduces an "in vivo bioreactor," a model in which pluripotent cells are recruited from circulating blood to a vascularized coralline scaffold supplemented with bone morphogenetic protein-2 (BMP-2). The bioreactor generates new, ectopic host bone with the capability of vascularized tissue transfer. More importantly, bone is reproducibly formed in a closed and malleable environment. In a rat model, the superficial inferior epigastric vessels were isolated, ligated, and then threaded through a prefabricated coral cylinder (hydroxyapatite, ProOsteon 500). Experimental groups were characterized by the following variables: (1) with/without incorporation of vascular pedicle; (2) with/without addition of BMP-2 (0.02 mg/cm3). Scaffolds were harvested 6 weeks after implantation, embedded and sectioned. Tissue samples were decalcified, fixed, and stained with H&E, trichrome green, and CD31/PECAM-1 (a marker of endothelial cells). Vascularized coral scaffolds supplemented with BMP-2 presumably recruited circulating mesenchymal stem cells to generate bone. Bone formation was quantified through histological analysis, and reported as a percentage, area bone/area cross section scaffold x 100. Mean bone formation was 11.30%+/-1.19. All scaffolds supplied by the vascular pedicle, regardless of BMP-2 supplementation, demonstrated neo-vascular ingrowth. Scaffolds lacking a pedicle showed no evidence of vascular ingrowth or bone formation. This paper introduces a model of a novel "in vivo bioreactor" that has future clinical and research applications. The tissue engineering applications of the "bioreactor" include treatment of skeletal defects (nonunion, tumor post-resection reconstruction). The bioreactor also may serve as a unique model in which to study primary and metastatic cancers of bone.     

Engineering Vascularized Bone Graft With Osteogenic and Angiogenic Lineage Differentiated Bone Marrow Mesenchymal Stem Cells

Tissue‐engineered bone provides a promising method for the rehabilitation of acquired bone defects and congenital deformities. However, generating a vascular supply to the engineered graft remains a major challenge. We report a novel strategy to engineer vascularized bone grafts with osteogenic and angiogenic lineage differentiated marrow mesenchymal stem cells (MSCs). MSCs were expanded to form an osteogenic cell sheet using a continuous culture method and a scraping technique under osteogenic culture conditions. Another portion of MSCs was directed to differentiate into highly proliferative endothelial progenitor cells (EPCs), which were then seeded onto the cell sheets. Cell sheet–EPC complexes were implanted subcutaneously in nude mice. Cell sheets without EPCs were also implanted as a control. The mice were sacrificed, and the samples were harvested for evaluation consisting of micro‐CT scanning, histological analysis and scanning electronic microscopy 4 and 8 weeks after implantation. The results showed that cell sheets were composed of viable cells and extracellular matrix and showed apparent mineralization. The obtained EPCs could express the specific antigen marker of CD31 and form capillary‐like structures in vitro. The osteogenic cell sheet–EPC complexes yielded well‐vascularized bone grafts 4 and 8 weeks after implantation. Both bone density and vascular density were significantly higher in the cell sheet–EPC complex group than in the control group. The results demonstrated that the introduction of EPCs could not only generate a vascular network but also increase bone formation for cell sheet‐based bone engineering. These findings suggest that the strategy of engineering bone grafts with osteogenic and angiogenic lineage differentiated MSCs has great potential for clinical applications to repair large bone defects.     

组织工程骨膜异体体内成骨修复兔骨缺损的初步观察

目的探讨以兔BMSCs和猪SIS复合构建的组织工程骨膜，在异体兔体内成骨的可行性。方法取新西兰大白兔BMSCs与SIS复合构建组织工程骨膜。选2月龄新西兰大白兔12只，制备双侧桡骨1．5～2．0cm缺损模型。随机选一侧植入组织工程骨膜，作为实验组；另一侧仅植入单纯SIS，作为对照组。术后观察动物一般情况，4周后摄X线片观察，取骨缺损中段标本行HE及Masson染色观察。结果两组动物术后饮食及日常活动基本正常：伤口无红肿、溢脓等：伤肢基本能负重行走。x线片观察：实验组骨缺损处有长条状新生骨形成，密度与正常骨相同，新生骨桥接骨缺损；对照组骨缺损无骨形成征象，骨缺损处密度与周围软组织影相近。组织学观察：实验组骨缺损处有新骨形成，骨组织中可见血管腔及不规则髓腔样结构；未见明显异物巨噬细胞及淋巴细胞浸润征象：对照组骨缺损处仅为胶原瘢痕组织，无骨组织形成。结论以SIS和BMSCs构建的组织工程骨膜在同种异体体内可以成骨，有修复骨缺损的可行性。     

血管束植入组织工程骨修复兔股骨缺损对血管内皮生长因子表达的影响

目的 探讨血管束植入组织工程骨修复兔股骨缺损对局部血管内皮生长因了(VEGF)表达的影响.方法 将兔自体骨髓基质下细胞经诱导后与β-磷酸三钙材料复合,植入制备的兔股骨缺损处,其中实验组在材料侧槽中植入股骨的动静脉血管束,对照组则单纯植入组织工稗骨,分别于术后2、4、8、12周通过形态学检测新生骨量,免疫组织化学方法检测新生骨中VEGF的表达量.结果 随着时间进展,各组成骨量逐渐增加,差异有统计学意义(P<0.05),并且从第4周开始实验组成骨量高于对照组,差异有统计学意义(P<0.05);符时问点实验组新生骨中VEGF的表达最均高于对照组,差异有统汁学意义(P<0.05),且4周时达到最人值.结论 血管束植入组织工程骨可明显增加新生骨中VEGF的表达并能促进骨缺损的修复.     

Repair of bone defect with vascularized tissue engineered bone graft seeded with mesenchymal stem cells in rabbits

This study evaluated the results of repair of the radius defect with a vascularized tissue engineered bone graft composed by implanting mesenchymal stem cells (MSCs) and a vascular bundle into the xenogeneic deproteinized cancellous bone (XDCB) scaffold in a rabbit model. Sixty-four rabbits were used in the study. Among them, four rabbits were used as the MSCs donor. Other 57 rabbits were divided into five groups. In group one (n = 9), a 1.5 cm bone defect was created with no repair. In group two (n = 12), the bone defect was repaired by a XDCB graft alone. In group three (n = 12), the defect was repaired by a XDCB graft that included a vascular bundle. In group four (n = 12), the defect was repaired by a XDCB graft seeded with MSCs. In group five (n = 12), the defect was repaired by a XDCB graft including a vascular bundle and MSCs implantation. The rest three rabbits were used as the normal control for the biomechanical test. The results of X-ray and histology at postoperative intervals (4, 8, and 12 weeks) and biomechanical examinations at 12 weeks showed that combining MSCs and a vascular bundle implantation resulted in promoting vascularization and osteogenesis in the XDCB graft, and improving new bone formation and mechanical property in repair of radius defect with this tissue engineered bone graft. These findings suggested that the vascularized tissue engineered bone graft may be a valuable alternative for repair of large bone defect and deserves further investigations.     

血管束植入组织工程骨修复兔股骨缺损对血管内皮生长因子表达的影响

目的 探讨血管束植入组织工程骨修复兔股骨缺损对局部血管内皮生长因了(VEGF)表达的影响.方法 将兔自体骨髓基质下细胞经诱导后与β-磷酸三钙材料复合,植入制备的兔股骨缺损处,其中实验组在材料侧槽中植入股骨的动静脉血管束,对照组则单纯植入组织工稗骨,分别于术后2、4、8、12周通过形态学检测新生骨量,免疫组织化学方法检测新生骨中VEGF的表达量.结果 随着时间进展,各组成骨量逐渐增加,差异有统计学意义(P<0.05),并且从第4周开始实验组成骨量高于对照组,差异有统计学意义(P<0.05);符时问点实验组新生骨中VEGF的表达最均高于对照组,差异有统汁学意义(P<0.05),且4周时达到最人值.结论 血管束植入组织工程骨可明显增加新生骨中VEGF的表达并能促进骨缺损的修复.     

血管束植入组织工程骨修复兔股骨缺损对血管内皮生长因子表达的影响

目的 探讨血管束植入组织工程骨修复兔股骨缺损对局部血管内皮生长因了(VEGF)表达的影响.方法 将兔自体骨髓基质下细胞经诱导后与β-磷酸三钙材料复合,植入制备的兔股骨缺损处,其中实验组在材料侧槽中植入股骨的动静脉血管束,对照组则单纯植入组织工稗骨,分别于术后2、4、8、12周通过形态学检测新生骨量,免疫组织化学方法检测新生骨中VEGF的表达量.结果 随着时间进展,各组成骨量逐渐增加,差异有统计学意义(P<0.05),并且从第4周开始实验组成骨量高于对照组,差异有统计学意义(P<0.05);符时问点实验组新生骨中VEGF的表达最均高于对照组,差异有统汁学意义(P<0.05),且4周时达到最人值.结论 血管束植入组织工程骨可明显增加新生骨中VEGF的表达并能促进骨缺损的修复.     

血管束植入组织工程骨修复兔股骨缺损对血管内皮生长因子表达的影响

目的 探讨血管束植入组织工程骨修复兔股骨缺损对局部血管内皮生长因了(VEGF)表达的影响.方法 将兔自体骨髓基质下细胞经诱导后与β-磷酸三钙材料复合,植入制备的兔股骨缺损处,其中实验组在材料侧槽中植入股骨的动静脉血管束,对照组则单纯植入组织工稗骨,分别于术后2、4、8、12周通过形态学检测新生骨量,免疫组织化学方法检测新生骨中VEGF的表达量.结果 随着时间进展,各组成骨量逐渐增加,差异有统计学意义(P<0.05),并且从第4周开始实验组成骨量高于对照组,差异有统计学意义(P<0.05);符时问点实验组新生骨中VEGF的表达最均高于对照组,差异有统汁学意义(P<0.05),且4周时达到最人值.结论 血管束植入组织工程骨可明显增加新生骨中VEGF的表达并能促进骨缺损的修复.     

血管束植入组织工程骨修复兔股骨缺损对血管内皮生长因子表达的影响

目的 探讨血管束植入组织工程骨修复兔股骨缺损对局部血管内皮生长因了(VEGF)表达的影响.方法 将兔自体骨髓基质下细胞经诱导后与β-磷酸三钙材料复合,植入制备的兔股骨缺损处,其中实验组在材料侧槽中植入股骨的动静脉血管束,对照组则单纯植入组织工稗骨,分别于术后2、4、8、12周通过形态学检测新生骨量,免疫组织化学方法检测新生骨中VEGF的表达量.结果 随着时间进展,各组成骨量逐渐增加,差异有统计学意义(P<0.05),并且从第4周开始实验组成骨量高于对照组,差异有统计学意义(P<0.05);符时问点实验组新生骨中VEGF的表达最均高于对照组,差异有统汁学意义(P<0.05),且4周时达到最人值.结论 血管束植入组织工程骨可明显增加新生骨中VEGF的表达并能促进骨缺损的修复.     

血管束植入组织工程骨修复兔股骨缺损对血管内皮生长因子表达的影响

目的 探讨血管束植入组织工程骨修复兔股骨缺损对局部血管内皮生长因了(VEGF)表达的影响.方法 将兔自体骨髓基质下细胞经诱导后与β-磷酸三钙材料复合,植入制备的兔股骨缺损处,其中实验组在材料侧槽中植入股骨的动静脉血管束,对照组则单纯植入组织工稗骨,分别于术后2、4、8、12周通过形态学检测新生骨量,免疫组织化学方法检测新生骨中VEGF的表达量.结果 随着时间进展,各组成骨量逐渐增加,差异有统计学意义(P<0.05),并且从第4周开始实验组成骨量高于对照组,差异有统计学意义(P<0.05);符时问点实验组新生骨中VEGF的表达最均高于对照组,差异有统汁学意义(P<0.05),且4周时达到最人值.结论 血管束植入组织工程骨可明显增加新生骨中VEGF的表达并能促进骨缺损的修复.     

血管束植入组织工程骨修复兔股骨缺损对血管内皮生长因子表达的影响

目的 探讨血管束植入组织工程骨修复兔股骨缺损对局部血管内皮生长因了(VEGF)表达的影响.方法 将兔自体骨髓基质下细胞经诱导后与β-磷酸三钙材料复合,植入制备的兔股骨缺损处,其中实验组在材料侧槽中植入股骨的动静脉血管束,对照组则单纯植入组织工稗骨,分别于术后2、4、8、12周通过形态学检测新生骨量,免疫组织化学方法检测新生骨中VEGF的表达量.结果 随着时间进展,各组成骨量逐渐增加,差异有统计学意义(P<0.05),并且从第4周开始实验组成骨量高于对照组,差异有统计学意义(P<0.05);符时问点实验组新生骨中VEGF的表达最均高于对照组,差异有统汁学意义(P<0.05),且4周时达到最人值.结论 血管束植入组织工程骨可明显增加新生骨中VEGF的表达并能促进骨缺损的修复.     

血管束植入组织工程骨修复兔股骨缺损对血管内皮生长因子表达的影响

目的 探讨血管束植入组织工程骨修复兔股骨缺损对局部血管内皮生长因了(VEGF)表达的影响.方法 将兔自体骨髓基质下细胞经诱导后与β-磷酸三钙材料复合,植入制备的兔股骨缺损处,其中实验组在材料侧槽中植入股骨的动静脉血管束,对照组则单纯植入组织工稗骨,分别于术后2、4、8、12周通过形态学检测新生骨量,免疫组织化学方法检测新生骨中VEGF的表达量.结果 随着时间进展,各组成骨量逐渐增加,差异有统计学意义(P<0.05),并且从第4周开始实验组成骨量高于对照组,差异有统计学意义(P<0.05);符时问点实验组新生骨中VEGF的表达最均高于对照组,差异有统汁学意义(P<0.05),且4周时达到最人值.结论 血管束植入组织工程骨可明显增加新生骨中VEGF的表达并能促进骨缺损的修复.     

血管束植入组织工程骨修复兔股骨缺损对血管内皮生长因子表达的影响

目的 探讨血管束植入组织工程骨修复兔股骨缺损对局部血管内皮生长因了(VEGF)表达的影响.方法 将兔自体骨髓基质下细胞经诱导后与β-磷酸三钙材料复合,植入制备的兔股骨缺损处,其中实验组在材料侧槽中植入股骨的动静脉血管束,对照组则单纯植入组织工稗骨,分别于术后2、4、8、12周通过形态学检测新生骨量,免疫组织化学方法检测新生骨中VEGF的表达量.结果 随着时间进展,各组成骨量逐渐增加,差异有统计学意义(P<0.05),并且从第4周开始实验组成骨量高于对照组,差异有统计学意义(P<0.05);符时问点实验组新生骨中VEGF的表达最均高于对照组,差异有统汁学意义(P<0.05),且4周时达到最人值.结论 血管束植入组织工程骨可明显增加新生骨中VEGF的表达并能促进骨缺损的修复.     

血管束植入组织工程骨修复兔股骨缺损对血管内皮生长因子表达的影响

目的 探讨血管束植入组织工程骨修复兔股骨缺损对局部血管内皮生长因了(VEGF)表达的影响.方法 将兔自体骨髓基质下细胞经诱导后与β-磷酸三钙材料复合,植入制备的兔股骨缺损处,其中实验组在材料侧槽中植入股骨的动静脉血管束,对照组则单纯植入组织工稗骨,分别于术后2、4、8、12周通过形态学检测新生骨量,免疫组织化学方法检测新生骨中VEGF的表达量.结果 随着时间进展,各组成骨量逐渐增加,差异有统计学意义(P<0.05),并且从第4周开始实验组成骨量高于对照组,差异有统计学意义(P<0.05);符时问点实验组新生骨中VEGF的表达最均高于对照组,差异有统汁学意义(P<0.05),且4周时达到最人值.结论 血管束植入组织工程骨可明显增加新生骨中VEGF的表达并能促进骨缺损的修复.     

Differential analysis of peripheral blood‐ and bone marrow‐derived endothelial progenitor cells for enhanced vascularization in bone tissue engineering

For tissue engineering applications, effective bone regeneration requires rapid neo‐vascularization of implanted grafts to ensure the survival of cells in the early post‐implantation phase. Incorporation of autologous endothelial progenitor cells (EPCs) for the promotion of primitive vascular network formation ex vivo has offered great promise for improved graft survival, enhanced rate of vascularization and bone regeneration in vivo. For clinical usage, identification of an optimal EPC isolation source from the patient is critical. We have, for the first time, characterized and directly compared EPCs from rabbit peripheral blood and bone marrow (PB‐EPCs and BM‐EPCs, respectively). PB‐EPCs outperformed BM‐EPCs on all measures. PB‐EPCs displayed typical endothelial cell markers, such as CD31, as well as high angiogenic potential in three‐dimensional extracellular matrix in vitro. Furthermore, PB‐EPCs cultured simultaneously with mesenchymal stem cells, displayed significantly enhanced expression levels of key osteogenic and vascular markers, including alkaline phosphatase, bone morphogenetic protein 2, and vascular endothelial growth factor. On the contrary, putative BM‐EPCs did not express CD31, and instead, expressed key smooth muscle markers. BM‐EPCs further failed to display vasculogenic activity. Hence, the highly angiogenic PB‐derived EPCs may serve as an ideal cell population for enhanced vascularization and success of engineered bone tissue. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1507–1515, 2012     

骨髓基质干细胞复合小肠黏膜下层体内异位成骨的研究

目的 观察骨髓间充质干细胞复合小肠黏膜下层构建组织工程骨膜的异位成骨能力.方法 将常规方法培养的BMSCs与SIS复合(M1组)及进行成骨诱导培养BMSCs与SIS复合(M2组)植入兔(n=8)背部肌袋内作为实验组,以单纯SIS作为阴性对照.术后4、8、12周观察其成骨情况.结果 BMSCs在SIS支架上黏附、生长良好.植入体内4、8、12周,M2、M1、SIS组成骨量分别为(25.08±0.79)%、(18.81±0.42)%和(13.98±1.86)%,各指标M2组高于M1或SIS组(P<0.05),M1组高于SIS组(P<0.05).随着时间的延长,两实验组成骨量逐渐增加(P<0.05),SIS组的成骨量变化不明显(P>0.05). 结论 BMSCs与SIS复合构建组织工程骨膜在体内有异位成骨作用,而且经过成骨诱导的组织工程骨膜成骨作用更加明显.     

带血管蒂骨膜、骨瓣移植和血管束植入修复骨缺损的实验研究

目的 将松质骨移植到骨缺损处,再将带血管蒂骨膜,骨瓣移植和血管束植入,以探讨不同方法对骨缺损修复过程的影响。方法 制作兔桡骨骨缺损模型,移植松质骨后,分别用带血管蒂的骨膜,骨瓣移植和血管束植入,单纯松质骨移植作对照,术后４,８,１６周进行大体标本,Ｘ线,光镜及电镜观察。结果 骨膜移植组血运建立快,成骨面积大,骨缺损修复快,质量最好。骨瓣移植组是通过带血管蒂的骨瓣移植增加了血液供应,成骨较快且好;但     

骨髓间充质干细胞膜片复合磷酸三钙陶瓷构建工程化骨组织的体内成骨研究

目的：探讨骨髓间充质干细胞膜片复合磷酸三钙支架材料构建组织工程骨的可行性。方法：将兔骨髓间充质干细胞高密度接种于普通培养皿,在成骨诱导条件下连续培养2周,获得细胞膜片,修剪成长方形,并由一端卷起包裹圆柱状的磷酸三钙材料。静置孵育24h后将构建物移植到裸鼠背部皮下。术后6周取材,进行大体观察、组织学检查、组织定量学分析。结果：所获骨髓间充质干细胞膜片有多层细胞组成,保留了细胞外基质。实验组在材料表面及其孔隙内有较多的骨质形成;单一材料组空隙内为纤维组织,未见骨或软骨样组织;单一膜片组见片状编织骨形成。结论：骨髓间充质干细胞膜片在体内具有良好的成骨能力,可作为细胞释放载体与磷酸三钙支架复合构建骨组织。本研究为骨组织工程构建提供了新的方法。     

The evaluation of bone formation of the whole-tissue periosteum transplantation in combination with beta-tricalcium phosphate (TCP)

We investigated the osteogenic potential of a combination graft of beta-tricalcium phosphate (TCP) and periosteum in the rat calvarial defect model. The combination beta-TCP and periosteum graft was grafted into rat calvarial defects; the newly formed bone in the defect was studied histologically and radiographically and compared with periosteum grafts and TCP grafts. Ten days after combination grafting, the grafted periosteum showed cell proliferation and Runx2 immunoreaction; 20 days after grafting, new bone formation was seen around the beta-TCP; and 30 days after grafting, new bone developed and actively replaced beta-TCP, while radiography showed calcified areas. Total bone formation of the combination periosteum and beta-TCP graft was significantly increased compared with single grafts of beta-TCP or periosteum (P < 0.01). The combination graft of periosteum and beta-TCP showed marked bone formation in rat calvarial defects. This result suggests that combination grafts may be effective for repairing bone defects.