Feasibility of Autologous Bone Marrow Mesenchymal Stem Cell–Derived Extracellular Matrix Scaffold for Cartilage Tissue Engineering

Extracellular matrix (ECM) materials are widely used in cartilage tissue engineering. However, the current ECM materials are unsatisfactory for clinical practice as most of them are derived from allogenous or xenogenous tissue. This study was designed to develop a novel autologous ECM scaffold for cartilage tissue engineering. The autologous bone marrow mesenchymal stem cell–derived ECM (aBMSC‐dECM) membrane was collected and fabricated into a three‐dimensional porous scaffold via cross‐linking and freeze‐drying techniques. Articular chondrocytes were seeded into the aBMSC‐dECM scaffold and atelocollagen scaffold, respectively. An in vitro culture and an in vivo implantation in nude mice model were performed to evaluate the influence on engineered cartilage. The current results showed that the aBMSC‐dECM scaffold had a good microstructure and biocompatibility. After 4 weeks in vitro culture, the engineered cartilage in the aBMSC‐dECM scaffold group formed thicker cartilage tissue with more homogeneous structure and higher expressions of cartilaginous gene and protein compared with the atelocollagen scaffold group. Furthermore, the engineered cartilage based on the aBMSC‐dECM scaffold showed better cartilage formation in terms of volume and homogeneity, cartilage matrix content, and compressive modulus after 3 weeks in vivo implantation. These results indicated that the aBMSC‐dECM scaffold could be a successful novel candidate scaffold for cartilage tissue engineering.     

三维支架与肋软骨细胞体外及体内培养构建组织工程化气管

目的 探索建立适合替代气管的组织工程化气管模型.方法 分离2月龄新西兰兔肋软骨细胞并传代二次后,以5×107/ml浓度分别种于PLGA和涤纶支架,体外环境中培养2周.将支架-软骨细胞模型植入裸鼠脊柱两侧皮下,培养4、6、8周后取出,HE染色、PAS染色、扫描电镜等观察软骨组织发育、新生毛细血管、整体组织结构层次.结果 种植软骨细胞的支架经体外和体内培养,软骨细胞及基质生长分泌旺盛,支架与上皮间存在紧密的纤维连接,含有丰富的新生毛细血管.涤纶-软骨细胞模型和PLGA-软骨细胞模型可以形成完整的组织结构.结论 肋软骨细胞种植于PLGA和涤纶支架材料上,经体外及体内培养构建的组织工程化气管模型符合气管替代要求.

Abstract：

Objective To investigate the effect of a tissue engineered trachea for replacement fabricated using three dimensional scaffold and chondrocytes by in vitro and in vivo culturing. Methods Rib chondrocytes were isolated and expanded to two passages, then seeded in PLGA or Dacron scaffold at density of 5 × 107/ml. Cultured in vitro for two weeks, the chondrocytes-scaffold model was planted under dorsal skin between nude mice's spine. Histology of cartilage, neovascularization and organizational structure were observed with HE staining, PAS staining and electron microscopic scan were performed after 4,6,8 weeks in vivo. Results Organized structure were observed in both PLGA-chondrocyte model and dacron-chondrocyte model with cartilage formation, neovascularization and tight fibrous connective tissue between scaffold and skin after in vitro and in vivo culture. Conclusion Tissue engineered trachea fabricated using rib chondrocytes and PLGA or dacron scaffold with in vitro and in vivo culture meets the requirement of trachea replacement.     

Tissue engineering of trachea-like cartilage grafts by using chondrocyte macroaggregate: experimental study in rabbits

Abstract:  Treatment and management of tracheal defects remain challenges in head and neck surgery. The purposes of this study were to explore a novel strategy to fabricate tissue-engineered trachea by using chondrocyte macroaggregate, and evaluate the feasibility of creating tracheal cartilage equivalents grown in the shape of cylindrical structure without scaffold. Chondrocytes from rabbit cartilage were expanded and seeded into a culture dish at high density to form mechanically stable chondrocyte macroaggregate. Once the chondrocyte macroaggregate was harvested by scrapping technique, it was wrapped around a silicon tube and implanted subcutaneously into the cell donor rabbit. Eight weeks later, specimens were harvested and analyzed for gross appearance, and histological, biochemical, and biomechanical properties. These values were compared with native rabbit cartilage. It was found that expanded chondrocytes could be harvested as a coherent cellular macroaggregate and could be fabricated into a tubelike graft. After in vivo implantation, cartilage-like tissue with cylindrical structure was regenerated successfully. Histological analysis showed engineered trachea cartilage consisted of evenly spaced lacunae embedded in a matrix rich in proteoglycans; type II collagen was also highly expressed in this engineered trachea cartilage. In a conclusion, based on the chondrocyte macroaggregate strategy, tracheal cartilage equivalents with cylindrical shape could be successfully reconstructed. This construct has advantages of high cell-seeding efficiency, good nutritional perfusion, and minimal inflammatory reaction, which provided a highly effective cartilage graft substitute and could be useful in many situations of trachea–cartilage loss encountered in clinical practice.     

骨髓基质细胞膜片复合聚乳乙醇酸支撑体体外构建管状软骨

目的 研究利用骨髓基质细胞膜片复合聚乳乙醇酸(ploy of lactic-co-glycolic acid,PLGA)支撑体,在生物反应器条件下体外构建管状软骨的可行性.方法 分离兔骨髓基质细胞,高密度连续培养,转化生长因子-1诱导构建成干细胞膜片,制作圆柱状PLGA支撑体,将细胞膜片均匀缠绕在表面.静置孵育14 d,使细胞膜片与PLGA相互贴附后,进入生物反应器动态培养8周后,取出标本.从大体形态、组织学结构、蛋白多糖含量以及生物力学性能等方面评价形成软骨的理化特性.结果 通过此策略构建的软骨外观与天然软骨组织非常相似,保持着良好的管状外形,颜色呈乳白色,有光泽,质地均匀,弹性好,具有中等偏软的硬度.组织学结果显示总体结构呈现软骨样结构,HE染色可见软骨样细胞分布于细胞陷窝之中,周围是均匀的细胞外基质,番红-0染色可见细胞外基质着色为鲜红色,提示蛋白多糖含量丰富,有大量软骨基质物产生.结论 细胞膜片复合支撑体策略能形成管状形态的软骨组织,为气管软骨的再造提供了新的方法,有可能解决气管缺损的临床难题.     

自体骨髓间质干细胞外基质支架在体外软骨组织工程中的应用

 目的 探讨利用自体骨髓间质干细胞外基质(autologous bone marrow mesenchymal stem cell-derived extracellular matrix,aBMSC-dECM)支架体外制备组织工程软骨的可行性。方法 取2周龄新西兰大白兔5只,分离、培养骨髓间质干细胞,原代培养4周,收集其分泌的细胞外基质,制备aBMSC-dECM支架。对支架行扫描电镜和HE染色观察。分离培养自体软骨细胞,植入支架内,48 h后对细胞-支架复合物行Live-Dead染色。分别于种植后1、2、4和6周对细胞-支架复合物(组织工程软骨)进行大体观察、体积测量、HE染色、Safranin-O染色、Ⅱ型胶原免疫组织化学染色、Real-Time PCR检测和抗压强度测试。对照为atelocollagen支架。结果 aBMSC-dECM支架呈三维多孔状海绵样结构,孔隙分布均匀,连通性较好,孔径(304.4±108.2) ?滋m,孔隙率93.3%±4.5%。与atelocollagen支架组比较,aBMSC-dECM支架组组织工程软骨呈乳白色,表面光滑有弹性,随观察时间延长体积逐渐增大,软骨细胞数量、蛋白聚糖和Ⅱ型胶原含量逐渐增多,Ⅱ型胶原及Aggrecan的mRNA持续高表达,抗压强度持续增高。结论 aBMSC-dECM支架有利于维持软骨细胞活性和生物学功能,促进组织工程软骨形成。     

PLGA–PTMC–Cultured Bone Mesenchymal Stem Cell Scaffold Enhances Cartilage Regeneration in Tissue‐Engineered Tracheal Transplantation

The treatment of long‐segment tracheal defect requires the transplantation of effective tracheal substitute, and the tissue‐engineered trachea (TET) has been proposed as an ideal tracheal substitute. The major cause of the failure of segmental tracheal defect reconstruction by TET is airway collapse caused by the chondromalacia of TET cartilage. The key to maintain the TET structure is the regeneration of chondrocytes in cartilage, which can secrete plenty of cartilage matrices. To address the problem of the chondromalacia of TET cartilage, this study proposed an improved strategy. We designed a new cell sheet scaffold using the poly(lactic‐co‐glycolic acid) (PLGA) and poly(trimethylene carbonate) (PTMC) to make a porous membrane for seeding cells, and used the PLGA–PTMC cell‐scaffold to pack the decellularized allogeneic trachea to construct a new type of TET. The TET was then implanted in the subcutaneous tissue for vascularization for 2 weeks. Orthotopic transplantation was then performed after implantation. The efficiency of the TET we designed was analyzed by histological examination and biomechanical analyses 4 weeks after surgery. Four weeks after surgery, both the number of chondrocytes and the amount of cartilage matrix were significantly higher than those contained in the traditional stem‐cell–based TET. Besides, the coefficient of stiffness of TET was significantly larger than the traditional TET. This study provided a promising approach for the long‐term functional reconstruction of long‐segment tracheal defect, and the TET we designed had potential application prospects in the field of TET reconstruction.     

An animal model study for tissue-engineered trachea fabricated from a biodegradable scaffold using chondrocytes to augment repair of tracheal stenosis

Introduction

We have designed an engineered graft fabricated from a biodegradable scaffold using chondrocytes and applied this construct to augment repair of tracheal stenosis. This study investigated the feasibility of using such tissue-engineered airways with autologous chondrocytes in a rabbit model.

Material and Methods

Chondrocytes were isolated and expanded from the auricular cartilage of New Zealand white rabbits, then seeded onto composite 3-layer scaffolds consisting of a collagen sheet, a polyglycolic acid mesh, and a copolymer (l-lactide/?-caprolactone) coarse mesh. The engineered grafts were implanted into a 0.5 × 0.8-cm defect created in the midventral portion of the cervical trachea. Gelatin sponges that slowly released basic fibroblast growth factor (b-FGF) were then placed on the constructs, which were retrieved 1 or 3 months after implantation.

Results

The biodegradable scaffold seeded with chondrocytes could maintain airway structure up to 3 months after implantation. Tracheal epithelial regeneration occurred in the internal lumen of this composite scaffold. Three months after implantation, staining of the sections showed cartilage accumulation in the engineered tracheal wall.

Conclusion

This composite biodegradable scaffold may be useful for developing engineered trachea. A gelatin sponge slowly releasing b-FGF might enhance chondrogenesis.     

组织工程气管体内构建的研究进展

气管替代物利用体内天然环境对细胞贴覆生长及支架性能改善,促进移植物血管化,诱导免疫耐受和提高术后生存率有指导意义。内外层壁覆有干细胞和气道上皮细胞的脱细胞气管,利用天然生物反应器于体内预培养促组织工程气管成熟的方法,以实现黏膜的再上皮化、软骨细胞形成和血管化,在此基础上进行原位移植,对于长段气管病损有良好的临床应用前景。现就组织工程气管体内构建的意义及研究现状予以综述。     

兔骨髓间质干细胞用于构建组织工程软骨组织的初步报告

目的 采用组织工程方法,以培养后的兔骨髓间质干细胞（MSC）制成人工软骨培养物,经体内外培养后发育出成活的软骨组织。方法 抽取兔人经密度梯度离心得到单个核细胞,再经体外分离、培养获得兔骨髓MSC。向MSC培养液内加入地塞米松、转化生长因子－β1（TGF－β1）和维生素C进行软骨起源诱导培养3周,部分细胞开始转变为圆形并分泌基质。将诱导后的细胞与牛Ⅰ型胶原及人纤维蛋白按一定的比例混合,制成软骨样的培养物并分别做体内外培养。结果 体外培养2周后,培养物内大部分细胞已萎缩消失。但剩余的少量细胞成活,形成类似的软骨陷窝并分泌甲苯胺蓝异染的软骨基质。体内移植培养3周后,培养物已发育成颗粒状成熟的软骨组织。结论 骨髓间质干细胞可用于组织工程软骨组织的构建,是一种非常有前途的人工软骨组织构建中的功能细胞。     

组织工程骨软骨复合物的构建与形态学观察

目的探讨采用组织工程技术构建骨软骨复合物的可行性。方法将骨髓基质细胞(BMSCs)成诱导软骨后接种于快速成形的三维支架材料聚乳酸／聚羟乙酸共聚物(PLGA)构建组织工程软骨，经成骨诱导的BMSCs接种于聚乳酸／聚羟乙酸共聚物／磷酸三钙(PLGA／TCP)构建组织工程骨，在体外分别培养2周后，将两种工程化组织及两者以无损伤线缝合形成的组织工程骨软复合体分别植入自体股部肌袋，术后8周取材，行组织学观察。结果术后组织学观察表明。组织工程软骨在体内可形成软骨组织组织工程骨在体内可形成骨组织，两者的复合体在体内可形成骨软骨复合物。结论以骨髓基质细胞为种子细胞、以快速成形的生物降解材料为支架体外构建的组织工程骨软骨复合物，可在体内形成骨软骨组织，有望用于骨软骨缺损的修复。     

Fetal tissue engineering: in utero tracheal augmentation in an ovine model

Background/Purpose: This study was aimed at comparing fetal tissue engineering with autologous free grafting in an ovine model of in utero tracheal repair. Methods: Chondrocytes were isolated from both elastic and hyaline cartilage specimens harvested from fetal lambs and expanded in vitro. Cells were seeded dynamically onto biodegradable scaffolds, which then were maintained in a rotating bioreactor for 6 to 8 weeks. Constructs subsequently were implanted into fetal tracheas (n = 15), in a heterologous fashion (group I). In group II, fetuses (n = 5) received autologous free grafts of elastic cartilage harvested from the ear as tracheal implants. In vivo specimens were harvested for histologic analysis at different time-points postimplantation. Results: In the 12 of 15 surviving fetuses of group I, all constructs were found to resemble normal hyaline cartilage, engraft well despite their heterologous origin, and display time-dependent epithelialization derived from the native trachea. All autologous free grafts were engrafted and epithelialized at birth, retaining histologic characteristics of elastic cartilage, but were more deformed than engineered constructs. Of the lambs allowed to reach term, 5 of 5 in the engineered group and 4 of 5 in the free graft group could breathe spontaneously. Conclusions: (1) Tissue-engineered cartilage, as well as autologous free grafts, can be implanted successfully into the fetal trachea, resulting in engraftment and function. (2) Engineered cartilage provides enhanced structural support after implantation into the fetal trachea when compared with free grafts. Prenatal tracheoplasty may prove useful for the treatment of severe congenital tracheal malformations. J Pediatr Surg 37:1000-1006.     

壳聚糖与Ⅱ型胶原复合制作组织工程软骨支架及其性能研究

目的探讨采用壳聚糖与Ⅱ型胶原复合制作新型组织工程软骨三维多孔支架的方法,并对其理化性能进行检测. 方法将精制88%脱乙酰度壳聚糖溶于0.2 mol/L醋酸溶液制成2%溶液,高纯度猪Ⅱ型胶原溶于0.5 mol/L醋酸溶液制成1%溶液,两者按4∶1(重量比)充分搅拌混合,采用冷冻干燥法制成壳聚糖与Ⅱ型胶原复合支架.采用碳化二亚胺/ N-羟基琥珀酰亚胺对支架进行交联,力学测定比较支架交联前后的强度变化,扫描电镜观察其超微结构,于2、4、6和8周经溶菌酶体外降解实验测定其体外降解性. 结果制备的复合支架成形良好,交联后其力学强度明显增加.扫描电镜显示壳聚糖与Ⅱ型胶原成分在支架内分布均匀,支架内孔洞相互连通似海绵状,孔径100～250 μm.各时间段复合支架体外降解较单纯壳聚糖支架快. 结论壳聚糖与Ⅱ型胶原复合成功地制作成了三维多孔复合支架.其理化性能及体外降解测定,可以作为支架载体,应用于组织工程软骨的构建.     

Repair of large segmental bone defects using bone marrow stromal cells with demineralized bone matrix

Objective: The aim of the present study was to evaluate the effect of tissue‐engineered constructs on repair of large segmental bone defects in goats. Methods: Allogenic demineralized bone matrix (aDBM) was seeded with autologous marrow stromal cells (aMSC) for seven days to construct DBM–MSC grafts prior to implantation. 24 goats were randomly divided into three groups (eight in each). In each group, 3 cm diaphyseal femoral defects were created unilaterally, and subsequently filled with the DBM‐MSC grafts, DBM alone and an untreated control, respectively. Radiological analysis and biomechanical evaluation were performed at 12 and 24 weeks after operation. Results: Obvious increases in radiological scoring and biomechanical strength were found in the DBM‐MSC group when compared to the DBM group. X‐ray examination showed excellent bone healing in the DBM‐MSC group, whereas only partial bone repair was seen in the DBM group, and no healing in untreated controls. Histologically, a tendency to bone regeneration and remodeling was far more obvious for the DBM‐MSC group than the DBM only and untreated controls. Conclusion: Our results strongly suggest that transplantation of bone MSC within a DBM could have advantages for the bone repair of large segmental defects.     

Orderly osteochondral regeneration in a sheep model using a novel nano‐composite multilayered biomaterial

The objective of this article was to investigate the safety and regenerative potential of a newly developed biomimetic scaffold when applied to osteochondral defects in an animal model. A new multilayer gradient nano‐composite scaffold was obtained by nucleating collagen fibrils with hydroxyapatite nanoparticles. In the femoral condyles of 12 sheep, 24 osteochondral lesions were created. Animals were randomized into three treatment groups: scaffold alone, scaffold colonized in vitro with autologous chondrocytes and empty defects. Six months after surgery, the animals were sacrificed and the lesions were histologically evaluated. Histologic and gross evaluation of specimens showed good integration of the chondral surface in all groups except for the control group. Significantly better bone regeneration was observed both in the group receiving the scaffold alone and in the group with scaffold loaded with autologous chondrocytes. No difference in cartilage surface reconstruction and osteochondral defect filling was noted between cell‐seeded and cell‐free groups. In the control group, no bone or cartilage defect healing occurred, and the defects were filled with fibrous tissue. Quantitative macroscopic and histological score evaluations confirmed the qualitative trends observed. The results of the present study showed that this novel osteochondral scaffold is safe and easy to use, and may represent a suitable matrix to direct and coordinate the process of bone and hyaline‐like cartilage regeneration. The comparable regeneration process observed with or without autologous chondrocytes suggests that the main mode of action of the scaffold is based on the recruitment of local cells. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:116–124, 2010     

Tissue engineered tracheal prosthesis with acceleratedly cultured homologous chondrocytes as an alternative of tracheal reconstruction

BACKGROUND: Autologous tissue is an ideal substitue for an extensive tracheal reconstruction, but it is rarely feasible in clinical situations. Many tracheal prosthesis had been used for such an instances, but unfortunately it is still problematic. Dislocation, local infection, hemorrage, and luminal stenosis can cause prosthetic failure. To achieve clinically available autologous tracheal prosthesis, it is necessary that we have to get phenotypically functioning chondrocytes, rapid differentiation of harvested autologous chondrocytes, and the survival of free grafted cultured chondrocytes. METHODS: In this study, we investigated isolation and culture method of the chondrocytes using the rabbit costal cartilage, and the cells were characterized microscopically and biochemically first. Then we have used cultured rabbit chondrocytes to investigate the role of growth factors upon the proliferation and regulation of the cultured chondrocytes. We have examined the effect of peptide growth factors on DNA and proteoglycan synthesis to the rabbit chondrocyte. The effects of IGF-I and basic FGF were investigated individually. Secondly, acceleratedly cultured chondrocytes were embeded to polymer (PLGA) scaffold in bioreactor, and implanted to defected rabbit trachea. Six weeks later, the rabbits were sacrificed and examined their histologic characteristics. RESULTS: The harvested chondrocytes from costal arch grew well and were amplified successfully maitaining their own phenotypes. Its embedding to PLGA scaffold was accomplished successfully. The implanted tracheal prosthesis maintains its physical integrity well, but the histologic examination revealed non-viable chondrocytes. The epithelial linings were good. CONCLUSIONS: The tissue engineered tracheal prosthesis can be a promising alternative of good functional air way tube in short term experiment, but biologically not vital yet. Further investigations are necessary to see the survival of free grafted chondrocytes and the long term results.     

Tissue‐Engineered Tracheal Reconstruction Using Three‐Dimensionally Printed Artificial Tracheal Graft: Preliminary Report

Three‐dimensional printing has come into the spotlight in the realm of tissue engineering. We intended to evaluate the plausibility of 3D‐printed (3DP) scaffold coated with mesenchymal stem cells (MSCs) seeded in fibrin for the repair of partial tracheal defects. MSCs from rabbit bone marrow were expanded and cultured. A half‐pipe‐shaped 3DP polycaprolactone scaffold was coated with the MSCs seeded in fibrin. The half‐pipe tracheal graft was implanted on a 10 × 10‐mm artificial tracheal defect in four rabbits. Four and eight weeks after the operation, the reconstructed sites were evaluated bronchoscopically, radiologically, histologically, and functionally. None of the four rabbits showed any sign of respiratory distress. Endoscopic examination and computed tomography showed successful reconstruction of trachea without any collapse or blockage. The replaced tracheas were completely covered with regenerated respiratory mucosa. Histologic analysis showed that the implanted 3DP tracheal grafts were successfully integrated with the adjacent trachea without disruption or granulation tissue formation. Neo‐cartilage formation inside the implanted graft was sufficient to maintain the patency of the reconstructed trachea. Scanning electron microscope examination confirmed the regeneration of the cilia, and beating frequency of regenerated cilia was not different from those of the normal adjacent mucosa. The shape and function of reconstructed trachea using 3DP scaffold coated with MSCs seeded in fibrin were restored successfully without any graft rejection.     

Mechanical properties and structure‐function relationships of human chondrocyte‐seeded cartilage constructs after in vitro culture

Autologous Chondrocyte Implantation (ACI) is a widely recognized method for the repair of focal cartilage defects. Despite the accepted use, problems with this technique still exist, including graft hypertrophy, damage to surrounding tissue by sutures, uneven cell distribution, and delamination. Modified ACI techniques overcome these challenges by seeding autologous chondrocytes onto a 3D scaffold and securing the graft into the defect. Many studies on these tissue engineered grafts have identified the compressive properties, but few have examined frictional and shear properties as suggested by FDA guidance. This study is the first to perform three mechanical tests (compressive, frictional, and shear) on human tissue engineered cartilage. The objective was to understand the complex mechanical behavior, function, and changes that occur with time in these constructs grown in vitro using compression, friction, and shear tests. Safranin‐O histology and a DMMB assay both revealed increased sulfated glycosaminoglycan (sGAG) content in the scaffolds with increased maturity. Similarly, immunohistochemistry revealed increased lubricin localization on the construct surface. Confined compression and friction tests both revealed improved properties with increased construct maturity. Compressive properties correlated with the sGAG content, while improved friction coefficients were attributed to increased lubricin localization on the construct surfaces. In contrast, shear properties did not improve with increased culture time. This study suggests the various mechanical and biological properties of tissue engineered cartilage improve at different rates, indicating thorough mechanical evaluation of tissue engineered cartilage is critical to understanding the performance of repaired cartilage. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2298–2306, 2017.

     

Arterial Tissue Regeneration for Pediatric Applications: Inspiration From Up‐to‐Date Tissue‐Engineered Vascular Bypass Grafts

The need for a valid replacement for autologous tissues in vascular surgery has led to the development of tissue‐engineered vascular grafts (TEVGs). Currently, only three kinds of TEVG have been used in clinical trials: synthetic scaffold‐based TEVGs, self‐assembled grafts, and decellularized exogenous tissues. This review presents the current options in the construction of TEVG and the changes that have occurred in the design following the clinical experience while focusing on the potential for pediatric applications. The emerging trend in the field, which is also pertinent for pediatric applications, is a shift from the development of vascular analogues to implants composed of scaffolds with autologous cellular components. Designs of such implants are currently being fine‐tuned so that a natural, functional tissue can gradually take over the role of scaffolds to stimulate the host's regenerative capacity and maintain the physiological homeostasis.     

组织工程化骨修复胸壁缺损的实验研究

目的　探索利用组织工程化骨修复胸壁缺损的可行性。方法　实验借助组织工程的基本原理和方法 ,利用猪骨脱细胞基质作为可降解支架材料 ,同时体外扩增自体骨髓间质干细胞作为种子细胞 ,体外构建组织工程化骨组织修复实验用北格犬双侧胸壁各 5cm× 5cm缺损 ,同时在构建的组织工程化骨组织中设计“辅助灌流系统” ,定时注入DMEM培养液、BMP等细胞因子和自体骨髓间质干细胞 ,即保证组织工程化骨中细胞早期营养 ,又维持了局部成骨微环境 ,并同时保证了种子细胞来源。结果　 5只实验动物胸廓未见反常呼吸 ,病理切片证明局部骨组织形成 ;单纯材料对照组可降解支架材料基本吸收 ,伴纤维组织形成。结论　组织工程化骨具有良好的生物相容性 ,并具有一定的强度 ,可防止反常呼吸 ,是一种理想的胸壁缺损修复材料     

Promotion of osteogenesis in tissue‐engineered bone by pre‐seeding endothelial progenitor cells‐derived endothelial cells

In addition to a biocompatible scaffold and an osteogenic cell population, tissue‐engineered bone requires an appropriate vascular bed to overcome the obstacle of nutrient and oxygen transport in the 3D structure. We hypothesized that the addition of endothelial cells (ECs) may improve osteogenesis and prevent necrosis of engineered bone via effective neovascularization. Osteoblasts and ECs were differentiated from bone marrow of BALB/c mice, and their phenotypes were confirmed prior to implantation. Cylindrical porous polycaprolactone (PCL)‐hydroxyapatite (HA) scaffolds were synthesized. ECs were seeded on scaffolds followed by seeding of osteoblasts in the EC‐OB group. In the OB group, scaffolds were only seeded with osteoblasts. The cell‐free scaffolds were denoted as control group. A 0.4‐cm‐long segmental femur defect was established and replaced with the grafts. The grafts were evaluated histologically at 6 weeks postimplantation. In comparison with the OB group, the EC‐OB group resulted in a widely distributed capillary network, osteoid generated by osteoblasts and absent ischemic necroses. Pre‐seeding scaffold with ECs effectively promoted neovascularization in grafts, prevented the ischemic necrosis, and improved osteogenesis. The integration of bone marrow‐derived ECs and osteoblasts in porous scaffold is a useful strategy to achieve engineered bone. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1147–1152, 2008