组织工程化静脉瓣构建的关键技术
|
摘要
原发性瓣膜功能不全是临床上常见病，多发病，静脉瓣移植被认为是最后的选择。但自体静脉瓣移植来源有限，限制了静脉瓣移植的应用，应用细胞生物学与工程学原理开发出具有生物活性、无免疫原性的组织工程化静脉瓣是修复与功能重建深静脉功能不全的理想方案。
目前构建组织工程化静脉瓣研究处尚处于起步阶段。Teebken等（2003）应用组织工程学原理，用受体静脉壁来源的肌纤维母细胞和内皮细胞构建组织工程化静脉瓣，但是肌纤维母细胞没能长入支架内部；该课题组在2009年又以人大隐静脉内皮细胞为种子细胞，大隐静脉脱细胞支架为支架材料，构建组织工程化静脉瓣，三维立体培养8天，发现细胞能在瓣膜两侧和血管壁表面生长并内皮化，但该研究血管壁内没种植细胞，没有进行体内移植。
我们实验室在国家、军队和上海市基金的资助下，近年来一直进行组织工程化静脉瓣的研究，我们已[1]利用骨髓来源的多能成体祖细胞（Multipotent adult progenitor cells，MAPC）和内皮祖细胞（Endothelial prigenitor cell，EPC）作为种子细胞，以绵羊脱细胞静脉瓣支架为支架材料，成功构建了组织工程化静脉，并进行了犬、绵羊在体效用性研究，及安全性评价。但是，静脉瓣在体内长期功能欠佳，我们认为，其原因可能与瓣膜的内皮化程度、内皮细胞体内黏附强度、脱细胞支架材料在制备过程中损伤、变性等因素有关。拟提高组织工程化静脉瓣的质量，缩短与生理性静脉瓣的差距，优化构建程序，有必要对组织工程化静脉瓣的构建中所用材料和构建技术进行再探讨。在种子细胞研究方面，我们实验室利用骨髓源性MAPC和EPC为种子细胞，成
功构建了组织工程化静脉瓣，但是，细胞分离培养过程复杂，需采用全骨髓血培养加免疫磁珠分选，免疫磁珠价格昂贵、不经济，细胞分选过程复杂，易污染。为优化组织工程化静脉瓣构建程序、降低成本，有必要对种子细胞的种类及分离培养方法进行再研究。在支架材料制备方法上，先前的研究采用了两种不同的脱细胞支架制备方法，但其中哪一种方法较好还未曾研究。近期有研究报道的冻融+生物酶的方法，在其他组
织脱细胞支架制备中对支架材料结构损伤小，但目前该方法还未见在静脉瓣膜脱细胞支架制备中应用。在支架材料制备方法上，我们先前的研究采用TritonX-100+NH4OH+DNase+RNase的脱细胞方法，支架无细胞残留，纤维连续，支架内布满大小不等的孔隙，无免疫原性。但利用该支架材料构建的组织工程化静脉瓣体内长期功能欠佳，这是否与该脱细胞方法有关尚不得而知。近期研究报道，冻融+生物酶的方法在其他组织脱细胞中发现对支架材料结构损伤小，利用该方法制备的瓣膜脱细胞支架是否能提高组织工程化静脉瓣体内长期功能，也值得研究。
在种子细胞种植方法上，我们先前的研究采用多点注射和加压灌注的方法，程序复杂、需要较高的专门技术、且发现利用该方法构建的组织工程化静脉瓣体内超过6个月，有内皮细胞脱落、血栓形成的现象，严重影响了组织工程化静脉瓣的体内长期功能，迫切需要寻找新的方法。
本研究针对我们组织工程化静脉瓣研究中遇到到的问题，拟通过简化种子细胞诱导培养方法，获取活性好，纯度高的种子细胞；获得一种脱细胞完全、对支架材料结构损害较小的组织工程化静脉瓣脱细胞支架的方法；以及研发平滑肌细胞种植器，提高平滑肌细胞粘附率等三个方面的研究；以提高组织工程化静脉瓣的构建效果，改善组织工程化静脉瓣远期性能。
第一部分同时培养兔骨髓源性EPCs和SPCs
研究目的：同时分离和培养兔骨髓来源的EPCs和平滑肌祖细胞（Smoothprogenitor cells,SPCs），研究其生物学特性，评估其作为组织工程化静脉瓣种子细胞的可能性。
材料和方法：密度梯度离心法获取兔骨髓血单个核细胞沉淀，分别用含5%FBS的EGM-2完全培养基向EPC方向诱导培养；用含5%FBS，20ng/mlPDGF-BB，不含VEGF的EBM-2培养基向SPC方向诱导培养。细胞培养48h后首次换液，相差显微镜下观察细胞形态特征，透射电镜观察两类细胞超微结构的特点。诱导第7天，14天细胞免疫荧光、流式细胞仪检测EPCs/SPCs表面标志阳性率表达情况；检测细胞摄取DiI-ac-LDL和结合FITC-UEA-1功能，以及在MatriGel上成血管功能情况，将第3代细胞进行冻存和复苏，测定细胞冻存前后的细胞活性变化。
结果：EPCs生物学特性：EPCs培养10天左右，细胞单层融合呈“铺路石”状；EPC表达CD34，VEGFR-2，弱表达CD133；透射电镜可见EPCs胞浆内特征性W-P小体；细胞生物学功能检测可见EPCs在Matrigel上呈现血管状；EPCs具有摄取DiI-ac-LDL和结合FITC-UEA-1的功能。冻存细胞复苏前后细胞生长特性方面无明显变化；SPCs生物学特性：SPCs培养14天左右出现血管平滑肌特异的生长特征“峰—谷”样生长特性；表达CD34，SMA，不表达Ⅷ和VEGF-2；在透射电镜下可见细胞内含有与细胞纵轴平行排列的肌丝；无摄取DiI-ac-LDL和结合FITC-UEA-1功能；在Matrigel上
无血管状结构形成。
结论：骨髓血梯度密度离心得到的单个核细胞，在不同诱导培养基的诱导下，可同时获到高纯度的EPCs和SPCs，SPCs可自然分化为平滑肌样细胞，不需要经向平滑肌细胞诱导分化，省时、经济、不易污染。
第二部分不同方法制备组织工程化静脉瓣脱细胞支架
研究目的：比较三种不同方法制备的带瓣静脉脱细胞支架的组织学，生物学特性，以期获得一种较好的带瓣静脉脱细胞支架材料。
材料和方法:采用以下三种不同方法制备带瓣静脉脱细胞支架。
1.脱氧胆酸钠组：Beagle犬带瓣静脉，浸入4%去氧胆酸钠溶液，4℃振荡1h进行脱细胞处理，然后于37℃以50mL生理盐水反复冲洗，得到脱细胞带瓣静脉支架，于4℃PBS液中保存备用；
2.Triton组：Beagle犬带瓣静脉，浸入0.5%Triton-100+0.05%NH4OH溶液，4℃振摇3d；超纯水4℃振摇3d；DNase+RNase处理（37℃）12h；超纯水漂洗，将脱细胞支架60CO辐照消毒，-80℃保存备用；
3.冻融+生物酶组：Beagle犬带瓣静脉，浸入4℃低渗液浸泡11小时，-80℃3小时，37℃水浴30min，PBS振荡冲洗。0.05％胰酶+0．02％EDTA处理8h，DNase0.2mg/mL、Nase0.02mg/mL消化8h，PBS漂洗，上述步骤重复3次，支架材料冷冻干燥，辐照消毒，-80℃保存备用；随机选取各组支架材料检测，病理切片分别行HE染色观察组织结构；扫描电镜观察支架材料表面及内部超微结构；透射电镜DAPI染色观察DNA残留；体外EPCs细胞种植检测细胞相容性。
结果：三种脱细胞方法均能彻底去除细胞，DAPI荧光检测显示各组支架材料细
胞核，均无DNA成分残留；HE染色及扫描电镜显示，冻融+生物酶组胶原纤维排列整齐，未见明显的胶原纤维结构改变，其他两组可见胶原纤维断裂，及结构紊乱现象；与其他两组脱细胞支架材料相比冻融+生物酶组支架皮下埋置炎细胞浸润较少，EPCs与冻融生物酶组支架材料粘附性好。
结论：结合渗透压改变的反复冻融加上低浓度胰酶，核酸酶脱细胞法，既可以较彻底除去带瓣膜静脉细胞成分，又保留了较完整的细胞外基质结构，具有良好的组织和细胞相容性，是较理想的带瓣膜静脉脱细胞支架制备方法。
第三部分平滑肌种植器构建组织工程化静脉瓣
研究目的：研制平滑肌细胞种植器，并利用该装置种植SPCs，加压灌注旋转种植EPCs，以提高细胞的种植率，构建性能良好的组织工程化静脉瓣。
材料和方法：
平滑肌细胞种植器的研制：改革传统的从官腔内种植平滑肌细胞的方法，利用真空吸引的作用将平滑肌细胞从官腔外壁较均匀的种植平滑肌细于在内膜下。
组织工程化静脉瓣构建：选第3代SPCs/EPCs为种子细胞，实验组用自制的平滑肌种植器种植SPCs；对照组用加压灌注多点注射的方法种植SPCs。培养三天后，以加压灌注种植EPCs，继续培养四天。SPCs种植4h，冰冻切片，DAPI染色观察SPCs种植密度；24h扫描电镜、甲苯胺蓝染色观察SPCs在支架材料上的粘附情况；MTT检测、细胞粘附实验检测种子细胞在支架材料内的增殖性能。一周后HE染色，免疫组织化学染色观察构建的组织工程化静脉瓣的组织学结构。
结果：平滑肌种植器种植的细胞密度较均匀、支架材料内膜层结构破坏较小、种植平滑肌细胞的数量可进行量化控制；在无菌的细胞悬液筒中进行，减少了构建过程中的污染几率。种植4h后DAPI染色显示，实验组SPCs种植密度均匀，对照组细胞种植密度不均匀，仅局部有细胞聚集。扫描电镜显示，实验组血管外表面细胞粘附较多，已有少量的细胞外基质分泌，支架材料表面平滑；对照组有少量细胞粘附，支架材料表面有蜂窝状。甲苯胺蓝染色显示，实验组SPCs在支架材料上粘附较多，细胞密度较大。MTT、细胞粘附实验显示，实验组细胞在支架材料上有较好的增殖活性，与对照组相比差别有统计学意义。组织学检查可见实验组内皮细胞已经完全覆盖支架材料的内表面，较对照组内皮化完整。结论：平滑肌细胞种植装置是有效的平滑肌种植工具，可高效、均匀种植SPCs，
种植的SPCs产生的细胞外基质，可修复脱细胞支架材料表面结构，细胞外基质所含的生长因子，促进EPCs的粘附增殖，促进了支架材料的内皮化。
Primary valve dysfunction is commonly seen in clinical practice. Venous valve transplantation is often considered as the last choice. However, autologous venous valve transplantation is unsatisfactory due to limited source. To develop a graft bearing immunologically tolerated tissue-engineered venous valve that will be incorporated into a native vessel and restore normal valve function for the treatment of deep venous insufficiency, a manufactured, tissue-engineered, nonimunogenic venous valve that remains patent and competent over time is an attractive alternative to direct venous valves transplantation for the treatment of deep venous insufficiency.
Currently, studies in tissue engineered venous valve are still at the primary stage. In2003, Teebken et al. applied the theories of tissue engineering, used the allochthonous decellularized venous valves as scaffolds, and seeded myofibroblasts and EC to construct tissue engineered venous valves. But myofibroblasts could not grow into the walls of scaffolds. After being replanted, the grafts could not undertake the long term function. Teebken et al.(2009) took the great saphenous vein endothelial cells as seed cells, the decellularized great saphenous vein as scaffolds to construct tissue engineered venous valves. After three-dimensional culture for8days, they found that cells could grow on the valve and on both sides of the vessel wall. However, this study did not plant cells in the vessel wall in transplantation in vivo.
With the support of national, army and Shanghai municipal government foundations, our laboratory has been focusing on the study of tissue-engineered venous valves in recent years. We have used canine bone marrow-derived multipotent adult progenitor cells (Multipotent Adult Progenitor Cells, MAPC)/endothelial progenitor cells (Endothelial Prigenitor cells, EPC) as seed cells, and sheep acellular venous valve stents as scaffolds to successfully construct the tissue engineered vein, and test its functions in dogs and sheep in vivo. However, the long term function of venous valve in vivo is poor. We believe that this may be related to the seed cells, scaffolds, implantation methods of seed cells in the scaffolds, etc. It needs further study on the technologies of tissue engineered venous valve construction to improve the quality of tissue engineered venous valve and shorten the gap with the physiologic vein valve.
In seed cell research, Teebken et al. selected peripheral mature cells, which is not suitable due to the cell activity. We selected bone marrow-derived stem cells as seed cells with desirable cell activity. However, the seed cell isolation and culture are complicated and require bone marrow blood culture and immunomagnetic bead selection, which is time-consuming, expensive and easily contaminated during the cell separation.
In scaffold material preparation, the previous studies applied two different methods of acellular scaffold preparation. Which method is better has not been studied. The recently reported freezing and thawing+enzyme method was found to have little damage on acellular scaffolds in other tissues decellularization. But it is rarely applied in valve decellularization.
In seed cells implantation, the cell adhesion on blood vessels and valve surface is not enough, the seeded cells are easily to fall off, which remain the difficulty in the construction of tissue engineered vessels and valves and constrain the development of cardiovascular tissue engineering.
In this study we aim to resolve the problem in tissue-engineered veinous valve, to simplify the seed cells induced cultivation method, to obtain good activity, high purity seed cells; to obtain a good performance cell scaffold materials that structure damage little and decellariat completely, And the development of smooth muscle cell planting implement, improving the smooth muscle cell adhesion rate in the three aspects of the research; In order to improve the construction of tissue-engineered venous valve, and to improve tissue engineering venous valve long-term performance.
Part Ⅰ. Rabbit bone marrow-derived EPCs and SPCs culture
Objective:To isolate and culture rabbit bone marrow-derived EPCs and smooth muscle progenitor cells (SPCs) to study their biological properties and assess the possibility as the seed cells for tissue-engineered venous valves.
Materials and Methods:Density gradient centrifugation was used to obtain bone marrow blood mononuclear cells, which were sedimentated and cultured with EGM-2complete medium containing5%FBS to be induced to EPC and were cultured with EBM-2medium without VEGF containing5%FBS,20ng/ml PDGF-BB for SPC induction. First medium fluid update was carried out48h later and cell of the3rd generation underwent cryopreservation and recovery and the cell activity was measured before and after the cryopreservation. The cell morphology was observed under phase contrast microscopy and ultrastructures were observed under transmission electron microscopy. EPCs/SPCs surface marker-positive rates were detected7days and14days after the induction with immunofluorescence and flow cytometry. The uptake of DiI-ac-LDL and binding to FITC-UEA-1as well as vessel genesis on MatriGel were measured.
Results:Biological features of EPS:EPCs were cultured for10days and the cells fused as monolayer, showing a "stepping stone" appearance. EPC expressed CD34, VEGFR-2and weakly expressed CD133. Under the transmission electron microscope, WP bodies could be seen within the EPCs cytoplasm. Biological functions detect showed visible EPCs grew on the Matrigel in a blood vessel-like form and could uptake DiI-ac-LDL and bind to FITC-UEA-1. No significant changes in cell growth before and after recovery. Biological features of SPCs:SPCs was cultured for14days and showed specific features of the vascular smooth muscle growth, namely,"peak-valley" growth way. SPCs expressed CD34and SMA without Ⅷ and VEGF-2expression. Myofilaments, parallel with the cell longitudinal axis, could be seen under the transmission electron microscope. SPCs could not uptake DiI-ac-LDL and bind to FITC-UEA-1and did not form vessel-like structures on the Matrigel.
Conclusion:Mononuclear cells could be obtained through density gradient centrifugation of the bone marrow blood, which could be cultured and induced to EPCs and SPCs of high purity. SPCs could naturally differentiate into smooth muscle-like cells, without the need to induce MAPCs to differentiate into smooth muscle cells. Compared to separate isolation, culture and differentiation induction of EPCs and MAPCs, this was time-saving, economic and not easily contaminated.
Part Ⅱ. Preparation of tissue-engineered venous valve acellular scaffolds with different methods
Objective:To compare the biological features of tissue-engineered venous valve acellular scaffolds constructed with3methods
Materials and Methods:To prepare tissue-engineered venous valve acellular scaffolds with3methods:
Sodium deoxycholate group:The veins with valves from Beagle dogs were immersed in the4%sodium deoxycholate, oscillated at4℃for1h for decellularization and then repeatedly washed with37℃50mL normal saline. The acellular veins with valves were obtained and stored in4℃PBS.
Triton group:The veins with valves from Beagle dogs were immersed in0.5%Triton-100+0.05%of NH40H solution and oscillated at4℃for3d, shaken in ultrapure water at4℃for3d, treated with DNase+RNase treatment (37℃) for12h, washed with ultrapure water and disinfected with60CO irradiation and preserved at-80℃.
Freezing and thawing+enzyme group:Scaffold materials in each group were randomly selected. HE staining was performed to observe the microstructure, a scanning electron microscope was used to observe the surface and internal ultrastructure, TEM plus DAPI staining were used to detect DNA residues, in vitro EPCs cell cultivation was used to examine cell compatibility and acellular scaffolds were subcutaneously embedded to test the histocompatibility.
Results:These three methods all could completely remove the cells. DAPI fluorescent examination showed no residual DNA components in the scaffolds. HE staining and scanning electron microscopy showed that collagen fibers were arranged orderly and there were no significant collagen fiber structure changes in the freezing and thawing+enzyme group. Collagen fiber breakage and structural disorder were detected in other two groups. Compared with other two groups, in the freezing and thawing plus enzyme group, subcutaneously embedded scaffolds showed less infiltration of inflammatory cells less and better EPCs adhesion to the scaffolds.
Conclusion:Repeated freezing and thawing combined with osmotic pressure changes and low concentrations of trypsin, ribonuclease could thoroughly remove cells in the rabbit vein with valves, preserve relatively complete extracellular matrix and maintain good tissue and cell compatibility, indicating that is was an ideal method for decellularization.
Part Ⅲ. Smooth muscle planting device for construction of tissue engineered venous valve
Objective:To develop a smooth muscle planting device to seed SPCs and EPCs, elevate cell planting rate and establish tissue-engineered venous valves with good functions.
Materials and Methods:To develop a smooth muscle planting device SPCs/EPCs of the3rd generation were selected as the seed cells. SPCs were seeded with the device in the experimental group and were implanted with the pressurized perfusion multi-point injection method in the control group. After3days, EPCs were planted with the pressurized perfusion multi-point injection method and were cultured for4days. After4hours, the SPCs were made into frozen sections, and DAPI was used to observe SPCs planting density.24h scanning electron microscopy and toluidine blue staining were used to monitor the implanting condition of SPCs on the scaffolds. MTT assay, cell adhesion assay were used to detect the cell proliferation performance. A week after HE staining, immunohistochemical staining was used to observe the histological structure of the constructed tissue-engineered venous valves.
Results:Four hours after the implantation, DAPI staining showed uniform density of SPCs in the experimental group and uneven density in the control group with local cell aggregation. Scanning electron microscopy showed that more cells adhering to the extravascular surface in the experimental group with a small amount of extracellular matrix secretion and a smooth surface of the scaffolds. In the control group, a small number of cells adhered to the scaffolds, which was honeycombed. Toluidine blue staining showed that more SPCs adhered to the scaffolds in the experimental group with higher cell density in the experimental group. MTT and cell adhesion experiments showed that cells in the experimental group had higher proliferative activity and the difference was statistically significant compared with the control group.
Conclusion:The smooth muscle planting device worked in effectively and evenly planting SPCs, which then secreted extracellular matrix and restore the surface structure of the scaffolds, thus promoting the adhesion and proliferation of EPCs
目前构建组织工程化静脉瓣研究处尚处于起步阶段。Teebken等（2003）应用组织工程学原理，用受体静脉壁来源的肌纤维母细胞和内皮细胞构建组织工程化静脉瓣，但是肌纤维母细胞没能长入支架内部；该课题组在2009年又以人大隐静脉内皮细胞为种子细胞，大隐静脉脱细胞支架为支架材料，构建组织工程化静脉瓣，三维立体培养8天，发现细胞能在瓣膜两侧和血管壁表面生长并内皮化，但该研究血管壁内没种植细胞，没有进行体内移植。
我们实验室在国家、军队和上海市基金的资助下，近年来一直进行组织工程化静脉瓣的研究，我们已[1]利用骨髓来源的多能成体祖细胞（Multipotent adult progenitor cells，MAPC）和内皮祖细胞（Endothelial prigenitor cell，EPC）作为种子细胞，以绵羊脱细胞静脉瓣支架为支架材料，成功构建了组织工程化静脉，并进行了犬、绵羊在体效用性研究，及安全性评价。但是，静脉瓣在体内长期功能欠佳，我们认为，其原因可能与瓣膜的内皮化程度、内皮细胞体内黏附强度、脱细胞支架材料在制备过程中损伤、变性等因素有关。拟提高组织工程化静脉瓣的质量，缩短与生理性静脉瓣的差距，优化构建程序，有必要对组织工程化静脉瓣的构建中所用材料和构建技术进行再探讨。在种子细胞研究方面，我们实验室利用骨髓源性MAPC和EPC为种子细胞，成
功构建了组织工程化静脉瓣，但是，细胞分离培养过程复杂，需采用全骨髓血培养加免疫磁珠分选，免疫磁珠价格昂贵、不经济，细胞分选过程复杂，易污染。为优化组织工程化静脉瓣构建程序、降低成本，有必要对种子细胞的种类及分离培养方法进行再研究。在支架材料制备方法上，先前的研究采用了两种不同的脱细胞支架制备方法，但其中哪一种方法较好还未曾研究。近期有研究报道的冻融+生物酶的方法，在其他组
织脱细胞支架制备中对支架材料结构损伤小，但目前该方法还未见在静脉瓣膜脱细胞支架制备中应用。在支架材料制备方法上，我们先前的研究采用TritonX-100+NH4OH+DNase+RNase的脱细胞方法，支架无细胞残留，纤维连续，支架内布满大小不等的孔隙，无免疫原性。但利用该支架材料构建的组织工程化静脉瓣体内长期功能欠佳，这是否与该脱细胞方法有关尚不得而知。近期研究报道，冻融+生物酶的方法在其他组织脱细胞中发现对支架材料结构损伤小，利用该方法制备的瓣膜脱细胞支架是否能提高组织工程化静脉瓣体内长期功能，也值得研究。
在种子细胞种植方法上，我们先前的研究采用多点注射和加压灌注的方法，程序复杂、需要较高的专门技术、且发现利用该方法构建的组织工程化静脉瓣体内超过6个月，有内皮细胞脱落、血栓形成的现象，严重影响了组织工程化静脉瓣的体内长期功能，迫切需要寻找新的方法。
本研究针对我们组织工程化静脉瓣研究中遇到到的问题，拟通过简化种子细胞诱导培养方法，获取活性好，纯度高的种子细胞；获得一种脱细胞完全、对支架材料结构损害较小的组织工程化静脉瓣脱细胞支架的方法；以及研发平滑肌细胞种植器，提高平滑肌细胞粘附率等三个方面的研究；以提高组织工程化静脉瓣的构建效果，改善组织工程化静脉瓣远期性能。
第一部分同时培养兔骨髓源性EPCs和SPCs
研究目的：同时分离和培养兔骨髓来源的EPCs和平滑肌祖细胞（Smoothprogenitor cells,SPCs），研究其生物学特性，评估其作为组织工程化静脉瓣种子细胞的可能性。
材料和方法：密度梯度离心法获取兔骨髓血单个核细胞沉淀，分别用含5%FBS的EGM-2完全培养基向EPC方向诱导培养；用含5%FBS，20ng/mlPDGF-BB，不含VEGF的EBM-2培养基向SPC方向诱导培养。细胞培养48h后首次换液，相差显微镜下观察细胞形态特征，透射电镜观察两类细胞超微结构的特点。诱导第7天，14天细胞免疫荧光、流式细胞仪检测EPCs/SPCs表面标志阳性率表达情况；检测细胞摄取DiI-ac-LDL和结合FITC-UEA-1功能，以及在MatriGel上成血管功能情况，将第3代细胞进行冻存和复苏，测定细胞冻存前后的细胞活性变化。
结果：EPCs生物学特性：EPCs培养10天左右，细胞单层融合呈“铺路石”状；EPC表达CD34，VEGFR-2，弱表达CD133；透射电镜可见EPCs胞浆内特征性W-P小体；细胞生物学功能检测可见EPCs在Matrigel上呈现血管状；EPCs具有摄取DiI-ac-LDL和结合FITC-UEA-1的功能。冻存细胞复苏前后细胞生长特性方面无明显变化；SPCs生物学特性：SPCs培养14天左右出现血管平滑肌特异的生长特征“峰—谷”样生长特性；表达CD34，SMA，不表达Ⅷ和VEGF-2；在透射电镜下可见细胞内含有与细胞纵轴平行排列的肌丝；无摄取DiI-ac-LDL和结合FITC-UEA-1功能；在Matrigel上
无血管状结构形成。
结论：骨髓血梯度密度离心得到的单个核细胞，在不同诱导培养基的诱导下，可同时获到高纯度的EPCs和SPCs，SPCs可自然分化为平滑肌样细胞，不需要经向平滑肌细胞诱导分化，省时、经济、不易污染。
第二部分不同方法制备组织工程化静脉瓣脱细胞支架
研究目的：比较三种不同方法制备的带瓣静脉脱细胞支架的组织学，生物学特性，以期获得一种较好的带瓣静脉脱细胞支架材料。
材料和方法:采用以下三种不同方法制备带瓣静脉脱细胞支架。
1.脱氧胆酸钠组：Beagle犬带瓣静脉，浸入4%去氧胆酸钠溶液，4℃振荡1h进行脱细胞处理，然后于37℃以50mL生理盐水反复冲洗，得到脱细胞带瓣静脉支架，于4℃PBS液中保存备用；
2.Triton组：Beagle犬带瓣静脉，浸入0.5%Triton-100+0.05%NH4OH溶液，4℃振摇3d；超纯水4℃振摇3d；DNase+RNase处理（37℃）12h；超纯水漂洗，将脱细胞支架60CO辐照消毒，-80℃保存备用；
3.冻融+生物酶组：Beagle犬带瓣静脉，浸入4℃低渗液浸泡11小时，-80℃3小时，37℃水浴30min，PBS振荡冲洗。0.05％胰酶+0．02％EDTA处理8h，DNase0.2mg/mL、Nase0.02mg/mL消化8h，PBS漂洗，上述步骤重复3次，支架材料冷冻干燥，辐照消毒，-80℃保存备用；随机选取各组支架材料检测，病理切片分别行HE染色观察组织结构；扫描电镜观察支架材料表面及内部超微结构；透射电镜DAPI染色观察DNA残留；体外EPCs细胞种植检测细胞相容性。
结果：三种脱细胞方法均能彻底去除细胞，DAPI荧光检测显示各组支架材料细
胞核，均无DNA成分残留；HE染色及扫描电镜显示，冻融+生物酶组胶原纤维排列整齐，未见明显的胶原纤维结构改变，其他两组可见胶原纤维断裂，及结构紊乱现象；与其他两组脱细胞支架材料相比冻融+生物酶组支架皮下埋置炎细胞浸润较少，EPCs与冻融生物酶组支架材料粘附性好。
结论：结合渗透压改变的反复冻融加上低浓度胰酶，核酸酶脱细胞法，既可以较彻底除去带瓣膜静脉细胞成分，又保留了较完整的细胞外基质结构，具有良好的组织和细胞相容性，是较理想的带瓣膜静脉脱细胞支架制备方法。
第三部分平滑肌种植器构建组织工程化静脉瓣
研究目的：研制平滑肌细胞种植器，并利用该装置种植SPCs，加压灌注旋转种植EPCs，以提高细胞的种植率，构建性能良好的组织工程化静脉瓣。
材料和方法：
平滑肌细胞种植器的研制：改革传统的从官腔内种植平滑肌细胞的方法，利用真空吸引的作用将平滑肌细胞从官腔外壁较均匀的种植平滑肌细于在内膜下。
组织工程化静脉瓣构建：选第3代SPCs/EPCs为种子细胞，实验组用自制的平滑肌种植器种植SPCs；对照组用加压灌注多点注射的方法种植SPCs。培养三天后，以加压灌注种植EPCs，继续培养四天。SPCs种植4h，冰冻切片，DAPI染色观察SPCs种植密度；24h扫描电镜、甲苯胺蓝染色观察SPCs在支架材料上的粘附情况；MTT检测、细胞粘附实验检测种子细胞在支架材料内的增殖性能。一周后HE染色，免疫组织化学染色观察构建的组织工程化静脉瓣的组织学结构。
结果：平滑肌种植器种植的细胞密度较均匀、支架材料内膜层结构破坏较小、种植平滑肌细胞的数量可进行量化控制；在无菌的细胞悬液筒中进行，减少了构建过程中的污染几率。种植4h后DAPI染色显示，实验组SPCs种植密度均匀，对照组细胞种植密度不均匀，仅局部有细胞聚集。扫描电镜显示，实验组血管外表面细胞粘附较多，已有少量的细胞外基质分泌，支架材料表面平滑；对照组有少量细胞粘附，支架材料表面有蜂窝状。甲苯胺蓝染色显示，实验组SPCs在支架材料上粘附较多，细胞密度较大。MTT、细胞粘附实验显示，实验组细胞在支架材料上有较好的增殖活性，与对照组相比差别有统计学意义。组织学检查可见实验组内皮细胞已经完全覆盖支架材料的内表面，较对照组内皮化完整。结论：平滑肌细胞种植装置是有效的平滑肌种植工具，可高效、均匀种植SPCs，
种植的SPCs产生的细胞外基质，可修复脱细胞支架材料表面结构，细胞外基质所含的生长因子，促进EPCs的粘附增殖，促进了支架材料的内皮化。
Primary valve dysfunction is commonly seen in clinical practice. Venous valve transplantation is often considered as the last choice. However, autologous venous valve transplantation is unsatisfactory due to limited source. To develop a graft bearing immunologically tolerated tissue-engineered venous valve that will be incorporated into a native vessel and restore normal valve function for the treatment of deep venous insufficiency, a manufactured, tissue-engineered, nonimunogenic venous valve that remains patent and competent over time is an attractive alternative to direct venous valves transplantation for the treatment of deep venous insufficiency.
Currently, studies in tissue engineered venous valve are still at the primary stage. In2003, Teebken et al. applied the theories of tissue engineering, used the allochthonous decellularized venous valves as scaffolds, and seeded myofibroblasts and EC to construct tissue engineered venous valves. But myofibroblasts could not grow into the walls of scaffolds. After being replanted, the grafts could not undertake the long term function. Teebken et al.(2009) took the great saphenous vein endothelial cells as seed cells, the decellularized great saphenous vein as scaffolds to construct tissue engineered venous valves. After three-dimensional culture for8days, they found that cells could grow on the valve and on both sides of the vessel wall. However, this study did not plant cells in the vessel wall in transplantation in vivo.
With the support of national, army and Shanghai municipal government foundations, our laboratory has been focusing on the study of tissue-engineered venous valves in recent years. We have used canine bone marrow-derived multipotent adult progenitor cells (Multipotent Adult Progenitor Cells, MAPC)/endothelial progenitor cells (Endothelial Prigenitor cells, EPC) as seed cells, and sheep acellular venous valve stents as scaffolds to successfully construct the tissue engineered vein, and test its functions in dogs and sheep in vivo. However, the long term function of venous valve in vivo is poor. We believe that this may be related to the seed cells, scaffolds, implantation methods of seed cells in the scaffolds, etc. It needs further study on the technologies of tissue engineered venous valve construction to improve the quality of tissue engineered venous valve and shorten the gap with the physiologic vein valve.
In seed cell research, Teebken et al. selected peripheral mature cells, which is not suitable due to the cell activity. We selected bone marrow-derived stem cells as seed cells with desirable cell activity. However, the seed cell isolation and culture are complicated and require bone marrow blood culture and immunomagnetic bead selection, which is time-consuming, expensive and easily contaminated during the cell separation.
In scaffold material preparation, the previous studies applied two different methods of acellular scaffold preparation. Which method is better has not been studied. The recently reported freezing and thawing+enzyme method was found to have little damage on acellular scaffolds in other tissues decellularization. But it is rarely applied in valve decellularization.
In seed cells implantation, the cell adhesion on blood vessels and valve surface is not enough, the seeded cells are easily to fall off, which remain the difficulty in the construction of tissue engineered vessels and valves and constrain the development of cardiovascular tissue engineering.
In this study we aim to resolve the problem in tissue-engineered veinous valve, to simplify the seed cells induced cultivation method, to obtain good activity, high purity seed cells; to obtain a good performance cell scaffold materials that structure damage little and decellariat completely, And the development of smooth muscle cell planting implement, improving the smooth muscle cell adhesion rate in the three aspects of the research; In order to improve the construction of tissue-engineered venous valve, and to improve tissue engineering venous valve long-term performance.
Part Ⅰ. Rabbit bone marrow-derived EPCs and SPCs culture
Objective:To isolate and culture rabbit bone marrow-derived EPCs and smooth muscle progenitor cells (SPCs) to study their biological properties and assess the possibility as the seed cells for tissue-engineered venous valves.
Materials and Methods:Density gradient centrifugation was used to obtain bone marrow blood mononuclear cells, which were sedimentated and cultured with EGM-2complete medium containing5%FBS to be induced to EPC and were cultured with EBM-2medium without VEGF containing5%FBS,20ng/ml PDGF-BB for SPC induction. First medium fluid update was carried out48h later and cell of the3rd generation underwent cryopreservation and recovery and the cell activity was measured before and after the cryopreservation. The cell morphology was observed under phase contrast microscopy and ultrastructures were observed under transmission electron microscopy. EPCs/SPCs surface marker-positive rates were detected7days and14days after the induction with immunofluorescence and flow cytometry. The uptake of DiI-ac-LDL and binding to FITC-UEA-1as well as vessel genesis on MatriGel were measured.
Results:Biological features of EPS:EPCs were cultured for10days and the cells fused as monolayer, showing a "stepping stone" appearance. EPC expressed CD34, VEGFR-2and weakly expressed CD133. Under the transmission electron microscope, WP bodies could be seen within the EPCs cytoplasm. Biological functions detect showed visible EPCs grew on the Matrigel in a blood vessel-like form and could uptake DiI-ac-LDL and bind to FITC-UEA-1. No significant changes in cell growth before and after recovery. Biological features of SPCs:SPCs was cultured for14days and showed specific features of the vascular smooth muscle growth, namely,"peak-valley" growth way. SPCs expressed CD34and SMA without Ⅷ and VEGF-2expression. Myofilaments, parallel with the cell longitudinal axis, could be seen under the transmission electron microscope. SPCs could not uptake DiI-ac-LDL and bind to FITC-UEA-1and did not form vessel-like structures on the Matrigel.
Conclusion:Mononuclear cells could be obtained through density gradient centrifugation of the bone marrow blood, which could be cultured and induced to EPCs and SPCs of high purity. SPCs could naturally differentiate into smooth muscle-like cells, without the need to induce MAPCs to differentiate into smooth muscle cells. Compared to separate isolation, culture and differentiation induction of EPCs and MAPCs, this was time-saving, economic and not easily contaminated.
Part Ⅱ. Preparation of tissue-engineered venous valve acellular scaffolds with different methods
Objective:To compare the biological features of tissue-engineered venous valve acellular scaffolds constructed with3methods
Materials and Methods:To prepare tissue-engineered venous valve acellular scaffolds with3methods:
Sodium deoxycholate group:The veins with valves from Beagle dogs were immersed in the4%sodium deoxycholate, oscillated at4℃for1h for decellularization and then repeatedly washed with37℃50mL normal saline. The acellular veins with valves were obtained and stored in4℃PBS.
Triton group:The veins with valves from Beagle dogs were immersed in0.5%Triton-100+0.05%of NH40H solution and oscillated at4℃for3d, shaken in ultrapure water at4℃for3d, treated with DNase+RNase treatment (37℃) for12h, washed with ultrapure water and disinfected with60CO irradiation and preserved at-80℃.
Freezing and thawing+enzyme group:Scaffold materials in each group were randomly selected. HE staining was performed to observe the microstructure, a scanning electron microscope was used to observe the surface and internal ultrastructure, TEM plus DAPI staining were used to detect DNA residues, in vitro EPCs cell cultivation was used to examine cell compatibility and acellular scaffolds were subcutaneously embedded to test the histocompatibility.
Results:These three methods all could completely remove the cells. DAPI fluorescent examination showed no residual DNA components in the scaffolds. HE staining and scanning electron microscopy showed that collagen fibers were arranged orderly and there were no significant collagen fiber structure changes in the freezing and thawing+enzyme group. Collagen fiber breakage and structural disorder were detected in other two groups. Compared with other two groups, in the freezing and thawing plus enzyme group, subcutaneously embedded scaffolds showed less infiltration of inflammatory cells less and better EPCs adhesion to the scaffolds.
Conclusion:Repeated freezing and thawing combined with osmotic pressure changes and low concentrations of trypsin, ribonuclease could thoroughly remove cells in the rabbit vein with valves, preserve relatively complete extracellular matrix and maintain good tissue and cell compatibility, indicating that is was an ideal method for decellularization.
Part Ⅲ. Smooth muscle planting device for construction of tissue engineered venous valve
Objective:To develop a smooth muscle planting device to seed SPCs and EPCs, elevate cell planting rate and establish tissue-engineered venous valves with good functions.
Materials and Methods:To develop a smooth muscle planting device SPCs/EPCs of the3rd generation were selected as the seed cells. SPCs were seeded with the device in the experimental group and were implanted with the pressurized perfusion multi-point injection method in the control group. After3days, EPCs were planted with the pressurized perfusion multi-point injection method and were cultured for4days. After4hours, the SPCs were made into frozen sections, and DAPI was used to observe SPCs planting density.24h scanning electron microscopy and toluidine blue staining were used to monitor the implanting condition of SPCs on the scaffolds. MTT assay, cell adhesion assay were used to detect the cell proliferation performance. A week after HE staining, immunohistochemical staining was used to observe the histological structure of the constructed tissue-engineered venous valves.
Results:Four hours after the implantation, DAPI staining showed uniform density of SPCs in the experimental group and uneven density in the control group with local cell aggregation. Scanning electron microscopy showed that more cells adhering to the extravascular surface in the experimental group with a small amount of extracellular matrix secretion and a smooth surface of the scaffolds. In the control group, a small number of cells adhered to the scaffolds, which was honeycombed. Toluidine blue staining showed that more SPCs adhered to the scaffolds in the experimental group with higher cell density in the experimental group. MTT and cell adhesion experiments showed that cells in the experimental group had higher proliferative activity and the difference was statistically significant compared with the control group.
Conclusion:The smooth muscle planting device worked in effectively and evenly planting SPCs, which then secreted extracellular matrix and restore the surface structure of the scaffolds, thus promoting the adhesion and proliferation of EPCs
引文
[1]Pavcnik D,Barry T,Hans A,et al. Percutaneous bioprosthetic venous valve:Along—term study in sheep[J]. J Vase Surg,2002,35(3):598-603.[2]Pavcnik D, Kaufman J, Uchida B, et al. Second generation pereutaneous bioprosthetic valve:A short—term study in sheep[J].2004,40(6):1223-1227.[3]Tissue-engineered bioprosthetic venous valve:a long-term study in sheep. Eur J Vase Endovasc Surg,2003,25(4):305-312.[4]袁建明,管松晖,党瑞山,杨向群,沈曼茹,张传森.绵羊骨髓来源内皮祖细胞的培养与鉴定.2010,14(36)：62-66[5]沈曼茹,袁建明,张传森等.细胞外基质材料的安全性能.中国组织工程研究与临床康复,2010,14(38)：7145-7148.[6]Roh, J.D., Sawh-Martinez. Tissue-engineered vascular grafts transform into mature blood vessels via an inflammation-mediated process of vascular remodeling. Proceedings of the National Academy of Sciences,2010,107(10):4669-4674.[1]Wen yu, Yuan jian-ming, Dang rui-shan, Shen man-ru, Zhang chuan-sen. Experimental study of Tissue-Engineered vein valve in Canine. Tissue engineering,2011.10(5)21-24.[2]Teebken OE, Puschmann C, Aper T, Haverich A, Mertsching H. Tissue-engineered bioprosthetic venous valve:a long-term study in sheep. Eur J Vase Endovasc Surg2003;25:305-12.[3]Arany, P.R, D.J. Mooney, At the edge of translation-materials to program cells for directed differentiation. Oral Diseases,2011.17(3):241-251.[4]Takayuki Asahara, Toyoaki Murohara, Alison Sullivan, etal.Isolation of putative progenitor endothelial cells for angiogenesis. Science,1997.275(5302):964-967.[5]Eggermann K, liche S, Jarmy G,etal. Endothelial progenitor cell culture and differentiation in vitro:a methodological comparison using human umbilical cord blood. Cardiovascular research,2003.58(2):478.-486[6]Shi.Q, Rafii.S,Hong-De Wu,.M, et al.Evidence for circulating bone marrow-derived endothelial cells. Blood,1998.92(2):362.-367[7]Satoshi Shintani, Toyoaki Murohara, Hisao Ikeda, et al., Augmentation of postnatal neovascularization with autologous bone marrow transplantation. Circulation,2001.103(6):897-903.[8]Urbich, C. and S. Dimmeler, Endothelial progenitor cells. Circulation research,2004.95(4):343-353.[9]Simper, D.talboerger, P.G.Panetta, C.J.et al. Smooth muscle progenitor cells in human blood. Circulation,2002.106(10):1199-1204.[10]Ferreira. L,S.Gerecht.S,Shieh.H.F,et al.. Vascular progenitor cells isolated from human embryonic stem cells give rise to endothelial and smooth muscle like cells and form vascular networks in vivo. Circulation research,2007.101(3):286.-294[11]Sone, M, Itoh, H, Yamahara. K,et al. Pathway for differentiation of human embryonic stem cells to vascular cell components and their potential for vascular regeneration. Arteriosclerosis, Thrombosis, and Vascular Biology,2007.27(10):2127-2134.[12]Huang.H,Zhao, X,Chen, L,et al.Differentiation of human embryonic stem cells into smooth muscle cells in adherent monolayer culture. Biochemical and biophysical research communications,2006.351(2):321-327[13]Cesselli, D.Beltrami, A.P.Rigo, S et al. Multipotent progenitor cells are present in??human peripheral blood. Circulation research,2009.104(10):1225-1234.[14]Daniel, J.M.Bielenberg, W.Stieger, P et al., Time-course analysis on the differentiation of bone marrow-derived progenitor cells into smooth muscle cells during neointima formation. Arteriosclerosis, thrombosis, and vascular biology,2010.30(10):1890-1896.[15]Kucia, M., J. Ratajczak, M. Ratajczak. Bone marrow as a source of circulating CXCR4+tissue-committed stem cells. Biology of the Cell,2005.97:133-146.[16]Kucia, M.Reca, R.Jala, VR, et al. Bone marrow as a home of heterogenous populations of nonhematopoietic stem cells. Leukemia,2005.19(7):1118-1127.[17]Reca, R.Wysoczynski, M.Yan, J, et al., The role of third complement component (C3) in homing of hematopoietic stem/progenitor cells into bone marrow. Current Topics in Complement,2006:35-51.[18]张林,张岩,杨向群等.骨髓间质细胞中组织定向干细胞的鉴定.解剖学杂志,2008.31(001)：8-10.[19]Kurpinski, K.Park, J.Thakar, R.G, et al.Regulation of vascular smooth muscle cells and mesenchymal stem cells by mechanical strain. MOLECULAR AND CELLULAR BIOMECHANICS,2006.3(1):21.[20]周预,刘峒,黄靖香等.兔半月板脱细胞基质的制备.中国医学科学院学报，2011(01).62-65[21]Simper, D.talboerger, P.G.Panetta, C.J.et al. Smooth muscle progenitor cells in human blood. Circulation,2002.106(10):1199-1204.[22]Frid, M.G., V.A. Kale, K.R. Stenmark. Mature vascular endothelium can give rise to smooth muscle cells via endothelial-mesenchymal transdifferentiation. Circulation research,2002.90(11):1189-1196.[23]Hong, J.H.Song, C.Shin, Y. et al., Estrogen induction of smooth muscle differentiation of human prostatic stromal cells is mediated by transforming growth factor-B. The Journal of urology,2004.171(5):1965-1969.[24]韩雅玲,肖艳平,齐岩梅等.胚胎血管发育早期PDGF-BB对血管平滑肌细胞趋化的影响.中国病理生理杂志,2008.24(2)：251-256.[25]王卫东,陈正堂.血管内皮祖细胞研究进展.中国微循环,2002.6(1)：6-8.[26]Gong, Z, L.E. Niklason.Small-diameter human vessel wall engineered from bone marrow-derived mesenchymal stem cells (hMSCs). The FASEB Journal,2008.22(6):1635-1648.[27]Gehling, U.M.Ergiin, S.Schumacher, U. et al., In vitro differentiation of endothelial??cells from AC133-positive progenitor cells. Blood,2000.95(10):3106-3112.[28]Peichev, M.Naiyer, A.J.Pereira,D.et al.Expression of VEGFR-2and AC133by circulating human CD34+cells identifies a population of functional endothelial precursors. Blood,2000.95(3):952-958.[29]Tanzi, M.C. S. Fare, Adipose tissue engineering:state of the art, recent advances and innovative approaches. Expert Review of Medical Devices,2009.6(5):533-551.[30]Levenberg, S.Zoldan, J.Basevitch, Y., et al. Endothelial potential of human embryonic stem cells. Blood,2007.110(3):806.-814[31]Pujol, B.F.Lucibello, F.C.Gehling, U.M. et al..Endothelial-like cells derived from human CD14positive monocytes. Differentiation,2000.65(5):287-300.[1]Terada, S., Sato, M., Sevy, A., and Vacanti, J.P. Tissue engineering in the twenty-first century. Yonsei Medical Journal,2000,41(6):685-691.[2]Borschel, G.H., Huang, Y.C., Calve, S. Tissue engineering of recellularized small-diameter vascular grafts. Tissue Engineering,2005,11(5-6):778-786.[3]Cho, S.W., Park, H.J., Ryu, J.H., Kim.Vascular patches tissue-engineered with autologous bone marrow-derived cells and decellularized tissue matrices. Biomaterials,2005,26(14):1915-1924.[4]Teebken, O., Puschmann, C., Aper, T., et al. Tissue-engineered bioprosthetic venous valve:a long-term study in sheep. European journal of vascular and endovascular surgery,2003,25(4):305-312.[5]温昱,MAPC/EPC性组织工程化静脉瓣,2008,第二军医大学.[6]Schmidt, C.E. and Baier, J.M. Acellular vascular tissues:natural biomaterials for tissue repair and tissue engineering. Biomaterials,2000,21(22):2215-2231.[7]韩雪峰,杨大平.曲拉通.X—100对制备脱细胞血管基质影响的实验研究.中华外科杂志，2002,40(001)：27-29.[8]Brown, B., Lindberg, K., Reing, J. The basement membrane component of biologic scaffolds derived from extracellular matrix. Tissue engineering,2006,12(3):519-526.[9]Barkan, D., Green, J.E.,Chambers, A.F. Extracellular matrix:a gatekeeper in the transition from dormancy to metastatic growth. European Journal of Cancer,2010,46(7):1181-1188.[10]Nelson, C.M, Bissell, M.J. Extracellular matrix, scaffolds, and signaling:tissue architecture regulates development, homeostasis, and cancer. Annual review of cell and developmental biology,2006,22:287.[11]熊猛,鲁开化,商庆新等.血管组织工程基质材料及管形支架的制备.西北国防医学杂志,2004,25(001)：3-6.[12]Allen, R.A., Seltz, L.M., Jiang, H. Adrenal extracellular matrix scaffolds support adrenocortical cell proliferation and function in vitro. Tissue Engineering Part A,2010,16(11):3363-3374.[13]Blank, R, Boskey, A. Genetic Collagen Diseases:Influence of Collagen Mutations on Structure and Mechanical Behavior. Collagen:structure and mechanics,2008:447.[14]Xu, C.C., Chan, R.W., Weinberger, D.G., Efune, G.Pawlowski, K.S. A bovine acellular scaffold??for vocal fold reconstruction in a rat model. Journal of Biomedical Materials Research Part A,2010,92(1):18-32.[15]Parekh, A., Mantle, B., Banks, J., Swarts, J.D., Badylak, S.F., Dohar, J.E., and Hebda, P.A. Repair of the tympanic membrane with urinary bladder matrix. The Laryngoscope,2009,119(6):1206-1213.[16]Valentin, J.E., Turner, N.J., Gilbert, T.W., and Badylak, S.F. Functional skeletal muscle formation with a biologic scaffold. Biomaterials,2010,31(29):7475-7484.[17]Nagase, H., Visse, R., and Murphy, G. Structure and function of matrix metalloproteinases and TIMPs. Cardiovascular research,2006,69(3):562-573.[18]Vorotnikova, E., McIntosh, D., Dewilde, A., Zhang, J., Reing, J.E., Zhang, L., Cordero, K., Bedelbaeva, K., Gourevitch, D., and Heber-Katz, E. Extracellular matrix-derived products modulate endothelial and progenitor cell migration and proliferation in vitro and stimulate regenerative healing in vivo. Matrix Biology,2010,29(8):690-700.[19]Zhang, Q., Raoof, M., Chen, Y., Sumi, Y., Sursal, T., Junger, W., Brohi, K., Itagaki, K., and Hauser, C.J. Circulating mitochondrial DAMPs cause inflammatory responses to injury. Nature,2010,464(7285):104-107.[20]Baluk, P., Hashizume, H., and McDonald, D.M. Cellular abnormalities of blood vessels as targets in cancer. Current opinion in genetics&development,2005,15(1):102-111.[21]Nagata, S., Hanayama, R., and Kawane, K. Autoimmunity and the clearance of dead cells. Cell,2010,140(5):619-630.[1]温昱,MAPC/EPC性组织工程化静脉瓣,2008,第二军医大学.[2]Hsu.S., Tsai.I. The effect of dynamic culture conditions on endothelial cell seeding and retention on small diameter polyurethane vascular grafts. Medical engineering physics,2005,27(3):267-272.[3]Roh. J.D, Nelson.G.N., Brennan.M.P., et al. Small-diameter biodegradable scaffolds for functional vascular tissue engineering in the mouse model. Biomaterials,2008,29(10):1454-1463.[4]Shimizu, K,Ito. A., Arinobe, M., et al.Effective cell-seeding technique using magnetite nanoparticles and magnetic force onto decellularized blood vessels for vascular tissue engineering. Journal of bioscience and bioengineering,2007,103(5):472-478.[5]Badylak SF, Valentin JE, Ravindra AK, McCabe GP, Stewart-Akers AM. Macrophage phenotype as a determinant of biologic scaffold remodeling. Tissue Engineering Part A2008;14(11):1835-42.[6]Gilbert TW, Sellaro TL, Badylak SF. Decellularization of tissues and organs.Biomaterials2006;27(19):3675-83.[7]HoboK, ShimizuT, SekineH,etal. Therapeutic angiogen-esis using tissue engineered human smooth muscle cellsheets[J]. ArteriosclerThromb Vase Bio,12008,28(4):637-643.[1]Cho, S.W., Park, H.J., Ryu, J.H., et al. Vascular patches tissue-engineered with autologous bone marrow-derived cells and decellularized tissue matrices. Biomaterials,2005,26(14):1915-1924.[2]Brown, B., Lindberg, K., Reing, J., et al. The basement membrane component of biologic scaffolds derived from extracellular matrix. Tissue engineering,2006,12(3):519-526.[3]Gilbert, T.W., Sellaro, T.L., Badylak, S.F. Decellularization of tissues and organs. Biomaterials,2006,27(19):3675-3683.[4]Badylak, S.F. The extracellular matrix as a biologic scaffold material. Biomaterials,2007,28(25):3587-3593.[5]Woods, T. Gratzer, P.F. Effectiveness of three extraction techniques in the development of a decellularized bone-anterior cruciate ligament-bone graft. Biomaterials,2005,26(35):7339-7349.[6]Cho, S.W., Lim, S.H., Kim, I.K., et al. Small-Diameter Blood Vessels Engineered With Bone Marrow-Derived Cells. Annals of surgery,2005,241(3):506.[7]Crapo, P.M., Gilbert, T.W., Badylak, S.F. An overview of tissue and whole organ decellularization processes. Biomaterials,2011.[8]Uygun, B.E., Soto-Gutierrez, A., Yagi, H., et al. Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix. Nature medicine,2010,16(7):814-820.[9]Cox, B. and Emili, A. Tissue subcellular fractionation and protein extraction for use in mass-spectrometry-based proteomics. Nature protocols,2006,1(4):1872-1878.[10]Elder, B.D., Kim, D.H., Athanasiou, K.A. Developing an articular cartilage decellularization process toward facet joint cartilage replacement. Neurosurgery,2010,66(4):722.[11]Alhamdani, M.S.S., Schro" der, C., Werner, J., et al. Single-step procedure for the isolation of proteins at near-native conditions from mammalian tissue for proteomic analysis on antibody microarrays. Journal of Proteome Research,2010,9(2):963-971.[12]Cebotari, S., Tudorache, I., Jaekel, T., et al.. Detergent decellularization of heart valves for tissue engineering:toxicological effects of residual detergents on human endothelial cells. Artificial Organs,2010,34(3):206-210.[13]Nakayama, K.H., Batchelder, C.A., Lee, C.I., et al. Decellularized rhesus monkey kidney as a three-dimensional scaffold for renal tissue engineering. Tissue Engineering Part A,2010,16(7):2207-2216.[14]FRAKENBURG, E.P., GOLDSTEIN, S.A., BAUER, T.W., et al. Biomechanical and Histological Evaluation of a Calcium Phosphate Cement*. The Journal of Bone and Joint Surgery (American),1998,80(8):1112-24.[15]Lin, P., Chan, W.C.W., Badylak, et al. Assessing porcine liver-derived biomatrix for hepatic tissue engineering. Tissue engineering,2004,10(7-8):1046-1053.[16]Yang, M., Chen, C.Z., Wang, X.N., et al. Favorable effects of the detergent and enzyme extraction method for preparing decellularized bovine pericardium scaffold for tissue engineered heart valves.??Journal of Biomedical Materials Research Part B:Applied Biomaterials,2009,91(1):354-361.[17]Petersen, T.H., Calle, E.A., Zhao, L., et al. Tissue-engineered lungs for in vivo implantation. Science,2010,329(5991):538-541.[18]Reing, J.E., Brown, B.N., Daly, K.A., et al. The effects of processing methods upon mechanical and biologic properties of porcine dermal extracellular matrix scaffolds. Biomaterials,2010,31(33):8626-8633.[19]Vorotnikova, E., McIntosh, D., Dewilde, A., et al. Extracellular matrix-derived products modulate endothelial and progenitor cell migration and proliferation in vitro and stimulate regenerative healing in vivo. Matrix Biology,2010,29(8):690-700.[20]Allen, R.A., Seltz, L.M., Jiang, H., et al. extracellular matrix scaffolds support adrenocortical cell proliferation and function in vitro. Tissue Engineering Part A,2010,16(11):3363-3374.[21]Nagata, S., Hanayama, R., Kawane, K. Autoimmunity and the clearance of dead cells. Cell,2010,140(5):619-630.