CRTER杂志“组织工程”栏目本月组稿重点

组织工程肝脏研究的组稿重点 组织工程肝脏支架生物评价研究组织工程肝脏纳米纤维支架研究透明质酸支架构建工程化肝组织脱细胞化肝脏生物衍生支架研究可注射型组织工程化肝脏的构建微载体及其在肝细胞培养的研究肝细胞构建组织工程化肝脏组织肝脏细胞和组织重建的影响因素端粒和端粒酶与肝细胞的永生化人工肝技术基础研究和临床研究组织工程肝脏与生物反应器研究微重力影响下的肝细胞形态研究     

以胶原凝胶为支架原位构建可注射型工程化肝

背景：为了解决肝移植供体的不足，最具潜力的肝组织工程技术悄然兴起，为肝组织的修复与功能重建开辟了新的前景。目的：探索一种以新生大鼠肝细胞为种子细胞，以胶原凝胶为支架材料，可原位注射的工程化肝组织构建方法。设计、时间及地点：以动物为观察对象的实验方法探索，于2007—03／2008—02在解放军总医院普通外科研究所完成。材料：成年雌性SD大鼠及出生24h以内的雄性新生SD大鼠。方法：以胰酶消化法获取新生大鼠肝细胞，以PKH26标记后与液态胶原复合，采用注射方法植入大鼠肝脏。主要观察指标：于肝细胞／胶原凝胶复合物植入当天及植入后第3，7天序贯取样、切片，分别行苏木精-伊红染色和自蛋白免疫组织化学染色后对“类肝组织”进行形态学观察，并观察植入细胞的示踪荧光。结果：①以胶原为基质构建的工程化肝组织能方便地植入到肝脏。②荧光显微镜观察到移植区域主要由PKH26阳性细胞组成。③苏木精伊红染色显示，移植区域肝细胞在胶原凝胶中可存活、生长。④免疫组织化学染色证实植入肝细胞能够表达自蛋白。结论：以新生大鼠肝细胞，胶原凝胶为基础构建可原位注射的工程化肝组织是可行的，这种方法对于发展以组织工程为基础的原位肝脏重建具有良好的应用前景。     

细胞外基质凝胶支架混合人脐静脉来源内皮细胞构建组织工程化的内皮细胞片层

背景:由于血管内皮细胞是形成血管网络的主要功能细胞,因此通过在支架复合体中复合血管内皮细胞的方式促进再造心肌的血管化是目前研究的热点。目的:以细胞外基质凝胶为支架混合人脐静脉内皮细胞,观察内皮细胞在细胞外基质凝胶中的生长过程及发育情况。设计、时间及地点:观察实验,于2006-11/2008-01在解放军军事医学科学院基础医学研究所组织工程研究室实验室完成。材料:取健康剖腹产孕妇的脐带分离培养人脐静脉内皮细胞。取新生SD大鼠鼠尾制备Ⅰ型液态胶原溶液。方法:将2.4g/L液态Ⅰ型胶原与2×M199培养基等比例混合,用0.1mol/LNaOH调为中性,加入分离培养3d的人脐静脉内皮细胞,使其终浓度为4×109L-1,再加入200g/L的Matrigel基质凝胶。最后加入有静态拉伸力的组织工程化片层所需的模具中,构建组织工程化内皮细胞片层。主要观察指标:体外培养20d后光学显微镜、常规苏木精-伊红染色、免疫组织化学染色和透射电镜观察。结果:显微镜下观察可见内皮细胞在细胞外基质凝胶中形成明显的管腔样、分支样、类似血管样结构。通过组织学和免疫组织化学的检测,证实其确为来源于人脐静脉中分离培养的内皮细胞。电镜观察可见随着时间的延长,在组织工程化内皮细胞片层中,内皮细胞可形成管腔,细胞间有紧密连接。结论:以细胞外基质凝胶为支架混合人脐静脉内皮细胞构建的组织工程化内皮细胞片层中具有类似血管样结构。     

碱性成纤维细胞生长因子影响脂肪干细胞的血管内皮迁移

背景：良好血运机制的建立是工程化组织成功植入的关键。 目的：观察外源性碱性成纤维细胞生长因子对植入小鼠皮下的人脂肪来源干细胞-透明质酸钠复合物中人脂肪来源干细胞向血管内皮迁移情况的影响。 方法：由吸脂术所得的脂肪组织中分离提取人脂肪来源干细胞进行培养传代,取第3代人脂肪来源干细胞进行cm-dil荧光标记后制成 5×10^9 L-1的细胞悬液,碱性成纤维细胞生长因子配成2 mg/L的工作液。将由0.25 mL透明质酸钠凝胶、0.2 mL细胞悬液和0.05 mL工作液/DMEM混合制成的碱性成纤维细胞生长因子组/对照组移植物分别植入小鼠背部左右两侧皮下作自身对照,6周后取材,行苏木精-伊红染色和免疫荧光标记血管内皮细胞进行观察分析。 结果与结论：植入处未出现结节、坏死及液化,取材时无凝胶残留。苏木精-伊红染色见标本性质多为脂肪组织及疏松结缔组织。荧光显微镜下仍可见标记人脂肪来源干细胞的cm-dil荧光,其与标记小鼠血管内皮的FITC荧光重合数碱性成纤维细胞生长因子组多于对照组（P〈0.05）。提示碱性成纤维细胞生长因子促进透明质酸钠支架中的人脂肪来源干细胞向血管内皮迁移、分化。     

小肠黏膜下层细胞及内皮细胞 平滑肌细胞构建组织工程血管的初步研究

目的初步探究小肠黏膜下层(SIS)细胞及内皮细胞、平滑肌细胞构建组织工程血管的实验方法、条件及可行性。方法取20只家兔(6~8周大),于体外分离培养内皮细胞、平滑肌细胞并标记,最后进行组织工程化血管的自体移植。结果实验结果表明于培养板中合成的支架材料呈现为白色、均匀质地;内皮细胞及平滑肌细胞可较好附着并植入SIS材料。结论小肠SIS及内皮细胞、平滑肌细胞构建组织工程血管具有可行性,并具有十分重要的医学应用价值。     

壳聚糖-生物蛋白胶用于工程化血管构建的初步研究

目的探讨壳聚糖管和生物蛋白胶作为工程化血管支架的可行性。方法血管内皮细胞和平滑肌细胞取自大鼠主动脉,体外扩增后,血管内皮细胞被接种在壳聚糖管的内表面;平滑肌细胞与生物蛋白胶混合后接种在壳聚糖管的外表面构建工程化血管。采用倒置显微镜观察、免疫组化染色和扫描电镜观察方法对工程化血管进行评价。结果血管内皮细胞在壳聚糖管的内壁形成连续的单层细胞层,覆盖在壳聚糖支架管腔内壁。平滑肌细胞在生物蛋白胶中成活生长,并形成三维的立体结构。结论壳聚糖生物蛋白胶可潜在地作为工程化血管支架。     

转染血管内皮细胞生长因子骨髓间充质干细胞与牛松质骨支架复合组织工程骨的血管形成

背景:小块组织工程骨植入动物体内后,早期依靠组织液的渗透可获得营养,但大块组织工程骨的营养仅靠组织液的渗透是远远不够的,必须通过血管再生来获得。目的:观察转染血管内皮细胞生长因子表达载体骨髓间充质干细胞复合组织工程骨植入动物体内后的血管形成能力。方法:制作日本大耳白兔双侧尺骨中段骨缺损模型,左侧尺骨缺损植入转染血管内皮细胞生长因子表达载体的自体骨髓间充质干细胞复合脱钙脱脂去蛋白的牛松质骨支架组织工程骨为实验组,右侧尺骨缺损植入自体骨髓间充质干细胞复合脱钙脱脂去蛋白的牛松质骨支架组织工程骨为对照组。术后12周行X射线摄片观察、大体标本观察、苏木精-伊红染色和Masson染色组织切片观察、MiFas图像分析系统定量分析。结果与结论:实验组与对照组尺骨缺损处均有连续骨痂形成;组织切片经苏木精-伊红染色、Masson三色法染色及MiFas图像分析系统定量分析可见实验组和对照组均有大量的新生骨,但实验组新生血管明显多于对照组(P<0.01),且血管较粗大,而且与新生骨接近。说明将转染血管内皮细胞生长因子表达载体的骨髓间充质干细胞复合在组织工程骨上植入动物体内可明显促进新生血管的形成。     

转染血管内皮细胞生长因子骨髓间充质干细胞与牛松质骨支架复合组织工程骨的血管形成

背景：小块组织工程骨植入动物体内后,早期依靠组织液的渗透可获得营养,但大块组织工程骨的营养仅靠组织液的渗透是远远不够的,必须通过血管再生来获得。目的：观察转染血管内皮细胞生长因子表达载体骨髓间充质干细胞复合组织工程骨植入动物体内后的血管形成能力。方法：制作日本大耳白兔双侧尺骨中段骨缺损模型,左侧尺骨缺损植入转染血管内皮细胞生长因子表达载体的自体骨髓间充质干细胞复合脱钙脱脂去蛋白的牛松质骨支架组织工程骨为实验组,右侧尺骨缺损植入自体骨髓间充质干细胞复合脱钙脱脂去蛋白的牛松质骨支架组织工程骨为对照组。术后12周行X射线摄片观察、大体标本观察、苏木精-伊红染色和Masson染色组织切片观察、MiFas图像分析系统定量分析。结果与结论：实验组与对照组尺骨缺损处均有连续骨痂形成;组织切片经苏木精-伊红染色、Masson三色法染色及MiFas图像分析系统定量分析可见实验组和对照组均有大量的新生骨,但实验组新生血管明显多于对照组（P〈0.01）,且血管较粗大,而且与新生骨接近。说明将转染血管内皮细胞生长因子表达载体的骨髓间充质干细胞复合在组织工程骨上植入动物体内可明显促进新生血管的形成。     

人胎盘间充质干细胞促进血管生成

背景：构建组织工程化骨组织的同时,促进种子细胞与材料复合物内血液供应的重建成为研究的关键。胎盘间充质干细胞可以作为骨组织工程研究的种子细胞,研究其分化为血管内皮细胞以及促进血管生成有着重要意义。目的：观察胎盘间充质干细胞在体外分化为血管内皮细胞以及体内促血管生成作用。方法：分离培养人胎盘间充质干细胞,鉴定其表面抗原,经血管内皮生长因子和人碱性成纤维细胞生长因子联合体外诱导胎盘来源间充质干细胞向血管内皮细胞定向分化,诱导后细胞通过内皮细胞标志物KDR、v-WF染色鉴定。8只新西兰大白兔制成桡骨中段1．5cm长的骨缺损模型,分别植入人胎盘间充质干细胞,丝素蛋白／羟基磷灰石和丝素蛋白,羟基磷灰石进行对照。植入后4,12周分别行大体观察、组织学观察和X射线观察,比较骨缺损修复以及血管生成情况。结果与结论：胎盘间充质干细胞的诱导分化形态明显改变,胞体逐步回缩,立体感增强,内皮细胞标志物KDR、v-WF染色结果阳性。胎盘来源间充质干细胞与丝素蛋白,羟基磷灰石材料复合培养植入后,4周时新骨已开始形成,12周时有部分新生骨组织形成板层骨,骨小梁形成,内可见新生血管形成,而对照组支架材料降解较慢,未见新生血管。说明胎盘来源间充质干细胞体外可以分化为血管内皮细胞,与丝素蛋白,羟基磷灰石材料联合移植能够促进移植物内血管生成,较好的修复骨缺损。     

用新生大鼠肝细胞体外构建工程化肝组织

目的:在供体肝短缺的情况下,构建可植入的工程化肝组织具有重要的临床意义.实验尝试以新生大鼠肝细胞为种子细胞体外构建工程化肝组织,以期进一步的体内植入.方法:实验于2007-04/08在解放军总医院普通外科研究所完成.①实验材料:SPF级、出生24 h以内的雄性SD新生大鼠.②实验过程:采用胰酶消化法获取新生大鼠肝细胞;以2×L-DMEM液(添加地塞米松10 μg/L、表皮生长因子20 μg/L、肝细胞生长因子40 μg/L及胰岛素0.04 U/mL)与液态鼠尾胶原等比例混合;再将肝细胞与胶原凝胶复合构建细胞/凝胶复合物,接种于培养板进行培养.③实验评估:培养后第1,3,5,7,9天,采用相差显微镜观察、四甲基偶氮唑盐比色法、苏木精-伊红染色和免疫组织化学染色分别对工程化组织的生长情况及组织形态特征进行观察,并对工程化组织的白蛋白合成功能及尿素的代谢水平进行评价.结果:①肝细胞与胶原凝胶复合后,细胞均匀的分布在复合物中.在整个培养过程中,肝细胞保持着稳定的细胞形态.②四甲基偶氮唑盐检测显示肝细胞活性在生长初期平缓下降,直至第5天时仍然保持75%的细胞活性,之后快速下降.③体外培养5 d后,相差显微镜下观察显示肝细胞在胶原凝胶中呈三维立体生长,并保持肝细胞胞体圆形,核大而圆的特异形态;免疫组织化学证实这些肝细胞抗白蛋白抗体染色呈强阳性.④对培养上清中白蛋白和尿素的含量测定表明胶原凝胶中的肝细胞在培养初期保持着稳定的代谢合成功能.结论:用新生大鼠肝细胞及胶原构建出一种有功能的工程化肝组织模型,这种模型可以应用于今后的工程化肝组织研究.     

预分化脂肪干细胞与纤维蛋白胶和磷酸钙骨水泥自体异位构建血管化移植物

背景：预构骨皮瓣研究启发人们构建预构血管化骨进行游离移植来替代带血管蒂游离自体骨移植修复大段骨缺损的想法。目的：设计一种基于预分化脂肪干细胞、纤维蛋白胶和多孔磷酸钙骨水泥支架复合体的血管化移植物。方法：将体外分离培养的大鼠脂肪干细胞在条件培养基中进行血管内皮细胞定向分化,经鉴定活性后,复合至纤维蛋白胶和多孔磷酸钙骨水泥构建血管化组织工程支架。将血管化组织工程支架、纤维蛋白胶/多孔磷酸钙骨水泥支架及多孔磷酸钙骨水泥支架分别植入SD大鼠股四头肌肌袋内,植入后2,4周进行组织学检测、血管定量分析和Western blot检测。结果与结论：向血管内皮细胞分化的脂肪干细胞与纤维蛋白胶和多孔磷酸钙骨水泥共培养7 d,可见细胞密度适中,与支架组织结合较好。植入实验中,各组支架孔隙中充填有纤维血管组织和脂肪组织,血管化组织工程组支架孔隙中均长入大量血管,并有小动脉长入,不同时间点的血管直径和数量及血管内皮生长因子C的表达量均优于纤维蛋白胶/多孔磷酸钙骨水泥组和多孔磷酸钙骨水泥组（P〈0.01）。表明构建的血管化组织工程支架能够实现可靠迅速血管化。     

Efficient in vivo vascularization of tissue‐engineering scaffolds

The success of tissue engineering depends on the rapid and efficient formation of a functional blood vasculature. Adult blood vessels comprise endothelial cells and perivascular mural cells that assemble into patent tubules ensheathed by a basement membrane during angiogenesis. Using individual vessel components, we characterized intra‐scaffold microvessel self‐assembly efficiency in a physiological in vivo tissue engineering implant context. Primary human microvascular endothelial and vascular smooth muscle cells were seeded at different ratios in poly‐L ‐lactic acid (PLLA) scaffolds enriched with basement membrane proteins (Matrigel) and implanted subcutaneously into immunocompromised mice. Temporal intra‐scaffold microvessel formation, anastomosis and perfusion were monitored by immunohistochemical, flow cytometric and in vivo multiphoton fluorescence microscopy analysis. Vascularization in the tissue‐engineering context was strongly enhanced in implants seeded with a complete complement of blood vessel components: human microvascular endothelial and vascular smooth muscle cells in vivo assembled a patent microvasculature within Matrigel‐enriched PLLA scaffolds that anastomosed with the host circulation during the first week of implantation. Multiphoton fluorescence angiographic analysis of the intra‐scaffold microcirculation showed a uniform, branched microvascular network. 3D image reconstruction analysis of human pulmonary artery smooth muscle cell (hPASMC) distribution within vascularized implants was non‐random and displayed a preferential perivascular localization. Hence, efficient microvessel self‐assembly, anastomosis and establishment of a functional microvasculture in the native hypoxic in vivo tissue engineering context is promoted by providing a complete set of vascular components. Copyright © 2010 John Wiley & Sons, Ltd.     

Liver sinusoidal endothelial cells are not permissive for adenovirus type 5

Adenoviral vectors are known to transduce hepatocytes in normal liver tissue with high efficiency. The aim of this study was to investigate whether sinusoidal endothelial cells, which separate hepatocytes from the bloodstream in the sinusoidal lumen, are permissive for infection by adenoviruses. We show here that microvascular liver sinusoidal endothelial cells are not infected by adenovirus type 5 in vivo or in vitro unless high MOIs are used. In contrast, macrovascular endothelial cells from aorta are efficiently infected by adenovirus type 5. In addition, Kupffer cells, similar to sinusoidal endothelial cells, are not infected by adenovirus type 5. Liver sinusoidal endothelial cells do not express the integrin receptor alpha(v)beta3, which is required for efficient infection by adenoviruses. Our results demonstrate that hepatocytes are the main cell population of the liver that is infected by adenovirus type 5.     

A scanning electron microscopic study on the liver of mice

The three-dimensional fine structures of several tissue components of the liver in normal mice were studied by scanning electron microscopy. The tissue components observed were as follows: hepatocytes, sinusoidal endothelial cells, the Kupffer cells, fat-storing cells, reticulin fibers and epithelial cells of the bile duct. Two types of fenestrations were found in the sinusoidal endothelial cells. One was smaller and clustered, and the other larger and scattered. Both of them were distributed equally throughout the hepatic lobule. Intercellular gaps were found at the endothelial junction. The Kupffer cell which was localized in a large gap between the endothelial cells was characterized by numerous villous projections, and by the absence of fenestrations which were observed in the endothelial cells. Fat-storing cells were located between hepatocytes. They elongated their processes into the space of Disse, but never protruded into the sinusoidal lumen. They were clearly distinguished from the endothelial cells and the Kupffer cells by their morphological feature and location. No transitional form was seen among the endothelial cells, the Kupffer cells, and the fat-storing cells.     

血管组织工程实验研究

目的获得血管组织工程的实验依据。方法获取大鼠血管内皮（VEC）和平滑肌细胞（VSMCs）,进行培养扩增,然后接种到血管组织工程支架上;将VSMCs-血管支架移植到大鼠体内,观察移植后大鼠活体和组织病理反应。结果培养的细胞符合血管种子细胞的形态特征;VSMCs接种到血管组织工程支架上获得成功;VSMCs-血管支架移植到大鼠体内10d后,支架被纤维组织包裹,支架内见VSMCs生长,支架中央管腔存在,但与正常血管结构有很大差别,而且不具备血管功能。结论利用血管组织工程支架构建的大鼠VSMCs-血管支架移植到大鼠皮下是可行的;人工构建血管具有类似血管雏形,但尚不具备血管的正常结构。     

Hydrogel surfaces to promote attachment and spreading of endothelial progenitor cells

Endothelialization of artificial vascular grafts is a challenging process in cardiovascular tissue engineering. Functionalized biomaterials could be promising candidates to promote endothelialization in repair of cardiovascular injuries. The purpose of this study was to synthesize hyaluronic acid (HA) and heparin‐based hydrogels that could promote adhesion and spreading of endothelial progenitor cells (EPCs). We report that the addition of heparin into HA‐based hydrogels provides an attractive surface for EPCs promoting spreading and the formation of an endothelial monolayer on the hydrogel surface. To increase EPC adhesion and spreading, we covalently immobilized CD34 antibody (Ab) on HA–heparin hydrogels, using standard EDC/NHS amine‐coupling strategies. We found that EPC adhesion and spreading on CD34 Ab‐immobilized HA–heparin hydrogels was significantly higher than their non‐modified analogues. Once adhered, EPCs spread and formed an endothelial layer on both non‐modified and CD34 Ab‐modified HA–heparin hydrogels after 3 days of culture. We did not observe significant adhesion and spreading when heparin was not included in the control hydrogels. In addition to EPCs, we also used human umbilical cord vein endothelial cells (HUVECs), which adhered and spread on HA–heparin hydrogels. Macrophages exhibited significantly less adhesion compared to EPCs on the same hydrogels. This composite material could possibly be used to develop surface coatings for artificial cardiovascular implants, due to its specificity for EPC and endothelial cells on an otherwise non‐thrombogenic surface. Copyright © 2012 John Wiley & Sons, Ltd.     

Comparison of sulphated glycosaminoglycan and hyaluronate synthesis and secretion in cultured hepatocytes, fat storing cells, and Kupffer cells

The extracellular matrix of normal liver contains several types of proteoglycans including heparan sulphate, chondroitin sulphate isomers, dermatan sulphate, and the glycosaminoglycan, hyaluronic acid. In the present study both the synthesis and secretion as well as the pattern of radioactively labeled proteoglycans and hyaluronic acid of hepatocytes, fat-storing cells (Ito cells), and Kupffer cells maintained in monolayer cultures under mostly identical conditions were compared to assess their relative contribution to hepatic proteoglycan synthesis. Fat-storing cells were identified as the main type of cell producing and secreting proteoglycan and hyaluronic acid. More than 70% of labeled proteoglycan and hyaluronic acid were secreted into the medium. Heparan sulphate is the main type of proteoglycan in hepatocytes, whereas in the medium of fat-storing cells, chondroitin sulphate and dermatan sulphate comprise the major fractions. Hyaluronic acid was not detectable in hepatocyte cultures and found only in low amounts in the medium of Kupffer cells. The results point to a stringent quantitative and qualitative cellular compartmentation of proteoglycan synthesis in liver with fat-storing cells as the most important cell type for matrix proteoglycan and hyaluronic acid production.     

大鼠烫伤后早期肝脏的变化及其机制

目的：观察大鼠烫伤后早期肝功能变化及肝窦内皮细胞（ＳＥＣ）在其中的作用。方法：制备大鼠烫伤模型，检测伤后２、１２、２４小时血清丙氨酸转氨酶、胆红素、红细胞压积、丙二醛（ＭＤＡ）、内皮素和透明质酸，肝组织ＭＤＡ、ＡＴＰ和肝脏有效血流量等指标，并与对照组比较。结果：伤后２小时血中丙氨酸转氨酶、透明质酸含量明显增高，肝组织ＭＤＡ含量增多，而肝组织ＡＴＰ水平显著下降；伤后肝脏有效血流量低于伤前；血清内皮素含量明显升高，与肝血流量下降呈明显负相关。结论：肝脏损伤和某些毒素的作用与烫伤后肝血流量减少有关；后者则可能与血容量减少、血液再分配有关，其中肝窦内皮细胞的改变起重要作用。