应用婴幼儿骨髓间充质干细胞体外构建组织工程化心肌

目的探讨以先天性心脏病(congenital heart disease,CHD)婴幼儿骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)作为种子细胞于体外构建组织工程化心肌组织的可行性。方法分离培养CHD婴幼儿胸骨BMSCs,经5-氮杂胞苷诱导分化为心肌细胞,种植到可降解的载体——聚乳酸/羟基乙酸的共聚物(polyactic acid/glycolic acid homoconjugate,PLGA)上,加入含有10%胎牛血清的LG-DMEM培养液,摇床培养1周后种植到裸鼠背部皮下,分别在种植后1、2、3周处死裸鼠。实验终止后通过光学显微镜、常规HE染色、蛋白质印记(Western Blotting)和透射电镜观察等对构建的组织工程化心肌组织进行评价。结果骨髓间充质干细胞经定向诱导分化后与PLGA构建的细胞-支架复合体培养1周后可见大部分细胞存活,有大量的细胞外基质分泌,植入裸鼠体内后3周取出标本大体观察可见组织样结构形成,Western blot检测结果表明构建的心肌组织有心房利钠肽(ANP)和心肌特异性肌球蛋白重链(MHC)的表达,透射电镜下可见体外构建的工程化心肌具有肌丝,缝隙连接和闰盘样结构形成。结论以CHD要幼儿BMSCs为种子细胞,PLGA为支架构建组织工程化心肌可行,构建的组织工程化心肌具有自体心肌的特性。     

应用胶原凝胶构建组织工程化皮肤的研究

目的探讨以胶原凝胶为支架材料构建组织工程化皮肤的可行性。方法体外分离、培养人皮肤表皮细胞和成纤维细胞;利用自制的胶原蛋白制成胶原凝胶作为组织工程支架材料;在成功构建人工真皮的基础上种植表皮细胞,构建复合人工皮肤;采用HE染色与免疫组织化学的方法对复合人工皮肤进行组织学检测。结果HE染色可见,构建的复合人工皮肤具有表皮和真皮双层结构;免疫组织化学染色显示,Ⅳ型胶原、纤维连接蛋白和层粘连蛋白阳性,在形态结构上与正常皮肤相似。结论培养的人表皮细胞和成纤维细胞种植于胶原凝胶支架上,气-液界面培养可构建出具有类似正常皮肤结构的组织工程化皮肤。     

应用胶原凝胶构建组织工程化皮肤的研究

目的探讨以胶原凝胶为支架材料构建组织工程化皮肤的可行性。方法体外分离、培养人皮肤表皮细胞和成纤维细胞；利用自制的胶原蛋白制成胶原凝胶作为组织工程支架材料；在成功构建人工真皮的基础上种植表皮细胞，构建复合人工皮肤；采用HE染色与免疫组织化学的方法对复合人工皮肤进行组织学检测。结果HE染色可见，构建的复合人工皮肤具有表皮和真皮双层结构；免疫组织化学染色显示，Ⅳ型胶原、纤维连接蛋白和层粘连蛋白阳性，在形态结构上与正常皮肤相似。结论培养的人表皮细胞和成纤维细胞种植于胶原凝胶支架上，气一液界面培养可构建出具有类似正常皮肤结构的组织工程化皮肤。     

聚磷酸钙纤维／左旋聚乳酸软骨支架复合自体骨髓间充质干细胞构建组织工程化人工关节软骨

目的：应用组织工程学技术,体外初步构建组织工程化人工关节软骨。方法：制备三维多孔软骨支架材料CPP／PLLA,体外诱导兔MSCs向软骨细胞表型分化,免疫组织化学染色检测软骨特异性Ⅱ型胶原表达,将诱导细胞与软骨支架材料CPP／PLLA复合,体外培养构建人工关节软骨,1周后终止培养,扫描电镜观察组织工程化人工软骨的微观结构;同时将构建人工软骨移植于兔大腿皮下,3周后处死动物,甲苯胺蓝染色观察。结果：扫描电镜观察可见该复合材料CPP／PLLA为高孔隙率的网状、连通、微孔结构,微孔分布均匀,孔径大小为300～400μm之间;兔MSCs经体外软骨表型定向诱导后,Ⅱ型胶原免疫组化染色阳性。诱导后的MSCs可在支架材料内良好贴附生长,细胞被分泌的胶原基质包裹;从体内获取的培养物组织切片观察可见大量的软骨细胞生成,甲苯胺蓝染色阳性。结论：经软骨起源诱导后的MSCs与CPP／PLLA复合培养可以构建自体软骨移植的替代物,为应用软骨组织工程方法修复关节软骨缺损和功能重建提供一种新材料,具有较大的潜在应用价值。     

聚磷酸钙纤维和左旋聚乳酸软骨支架复合自体骨髓间充质干细胞构建组织工程化人工关节软骨

目的应用组织工程学技术，体外初步构建组织工程化人工关节软骨。方法制备三维多孔软骨支架材料CPP／PLLA，体外诱导兔MSCs向软骨细胞表型分化，免疫组织化学染色检测软骨特异性Ⅱ型胶原表达，将诱导细胞与软骨支架材料CPP／PLLA复合，体外培养构建人工关节软骨，1周后终止培养，扫描电镜观察组织工程化人工软骨的微观结构；同时将构建人工软骨移植于兔大腿皮下，3周后处死动物，甲苯胺蓝染色观察。结果扫描电镜观察可见该复合材料CPP／PLLA为高孔隙率的网状、连通、微孔结构，微孔分布均匀，孔径大小为300～400Ⅳn之间；兔MSCs经体外软骨表型定向诱导后，Ⅱ型胶原免疫组化染色阳性。诱导后的MSCs可在支架材料内良好贴附生长，细胞被分泌的胶原基质包裹；从体内获取的培养物组织切片观察可见大量的软骨细胞生成，甲苯胺蓝染色阳性。结论经软骨起源诱导后的MSCs与CPP／PLLA复合培养可以构建自体软骨移植的替代物，为应用软骨组织工程方法修复关节软骨缺损和功能重建提供一种新材料，具有较大的潜在应用价值。     

体外构建皮肤基底膜的组织学特征

目的观察制备的组织工程皮肤基底膜的组织学特征。方法取门诊正常儿童包皮环切术之包皮，采用胰蛋白酶胶原酶顺序消化得到角质形成细胞（KC）和成纤维细胞（Fb）悬液。制备复方壳多糖组织工程皮肤，浸没培养3d后，继续行气液界面培养。将培养7、10、15d的复方壳多糖组织工程皮肤用中性甲醛溶液固定后石蜡包埋、切片，行HE及高碘酸-雪夫（PAS）染色，并用免疫组织化学染色法观察基底膜的重要成分：Ⅳ型胶原、Ⅶ型胶原及层黏连蛋白（LN）的存在情况。结果HE染色可见培养的组织工程皮肤表皮结构分化良好，大致可分为基底层、棘层和角质层，各层均有数量不等的扁平梭形细胞。PAS染色显示真皮表皮间有一均匀红染的条带。免疫组织化学染色结果显示，Ⅳ型胶原、Ⅶ型胶原及LN呈阳性表达。结论复方壳多糖组织工程皮肤基底膜构建良好。     

人体尿道粘膜上皮细胞的连续培养

目的：探讨人尿道粘膜上皮细胞体外培养技术，为构建组织工程化尿道提供种子细胞。方法：取尿道下裂患者尿道粘膜，经中性蛋白酶和胰蛋白酶消化，获取粘膜上皮细胞接种于含8％胎牛血清培养基中连续培养，观察细胞形态变化及生、增殖过程。结果：体外培养细胞长满后呈上皮细胞特有的铺路石样外观，体外可连续传9-10代，生长期50-60d。细胞角质蛋白免疫组织化学染色阳性，透射电镜下可见上皮细胞间特有的桥粒结构。结论：尿道粘膜上皮细胞可在体外连续培养，4代以内的培养细胞可用于组织工程化尿道的构建。     

离心力下构建组织工程纤维环的实验研究

目的 探讨离心力对体外构建组织工程纤维环的影响. 方法 取兔胸腰段椎间盘纤维环组织,经胰蛋白酶和Ⅱ型胶原酶消化后细胞原代培养,体外扩增至第3代,5×10<'7>/mL的细胞悬液种植于聚乳酸-羟基乙酸(PLGA)无纺网支架上.实验组在离心力下培养(相对离心力:85.86×g,每天2次,每次离心15min),对照组静态培养.每组5个标本,培养4周后取出纤维环细胞与支架复合物,行大体标本观察,组织学和免疫组织化学染色,检测Ⅰ型胶原分泌情况. 结果 体外培养4周后,实验组与对照组均为类纤维环样组织,实验组纤维环厚度和组织弹性均优于对照组,Ⅰ型胶原免疫组织化学染色,实验组阳性染色面积为(45.39±6.78)%,对照组为(33.53±4.58)%,两者差异有统计学意义(P<0.01).实验组细胞排列较对照组更具方向性. 结论 离心力刺激纤维环细胞增加Ⅰ型胶原的分泌,有利于在体外构建组织工程纤维环.     

力学刺激促进骨髓基质干细胞体外软骨分化

目的 探讨离心力刺激对猪骨髓基质干细胞(BMSCs)体外成软骨分化的作用,以及对三维支架上构建组织工程化软骨的影响,明确力学刺激与细胞分化及组织形成的关系,为体外软骨构建提供适当参数.方法 抽取8周龄猪髂嵴骨髓,应用贴壁法分选单个核细胞,体外培养扩增后获得第2 代BMSCs,以5.0×107/cm3 的细胞密度接种到聚羟基乙酸(PGA)制成的圆柱形三维支架上,7 d后分成4组,在不同的力学条件及诱导条件下培养.8周后取材,行相关检测.结果 力学诱导组形成的组织呈白色,细腻光泽,形状规则,体积无明显改变,有良好的弹性和硬度,并具有典型的软骨陷窝结构和大量软骨特异性细胞外基质 ,Ⅱ型胶原及丰富的聚合蛋白多糖(GAG)成分.GAG含量为(6.0±1.2) mg/g,抗压强度为 (2.2±0.8) kPa,弹性模量为(7.4±1.6) kPa.各项指标均明显优于其他各组.结论 力学刺激有利于促进BMSCs成软骨分化,并在三维支架材料上构建组织工程化软骨.     

骨质疏松大鼠骨髓基质细胞膜片的体外构建研究

目的探讨骨质疏松大鼠骨髓基质细胞膜片的构建及基本生物学特性。方法建立大鼠绝经后骨质疏松症(PMOP)模型,分离培养骨质疏松大鼠骨髓基质细胞(O-r BMSCs),并以O-rBMSCs为种子细胞构建细胞膜片。通过倒置显微镜、HE染色、扫描电镜及透射电镜对细胞膜片进行形态学检测;以免疫组织化学染色检测细胞外基质相关蛋白的表达情况。结果成功建立了大鼠PMOP模型,体外分离培养的O-rBMSCs具有多向分化潜能。显微镜下可见细胞复层生长,采用富含维生素C的培养基连续培养10 d左右即可获得白色膜样结构,该细胞膜片具有较好的弹性,HE染色及扫描电镜观察发现O-rBMSCs膜片由多层细胞及丰富的细胞外基质组成。透射电镜下可观察到细胞间连接。免疫组织化学染色示O-rBMSCs膜片高表达Ⅰ型胶原、纤维连接蛋白。结论 O-rBMSCs体外采用富含维生素C的培养基连续培养可构建细胞膜片,该细胞膜片富含细胞外基质,有望应用于骨质疏松机体的组织再生。     

Construction of a unidirectionally beating 3-dimensional cardiac muscle construct.

BACKGROUND: Cardiac tissue engineering aims to construct cardiac tissue with characteristics similar to those of the native tissue. Engineered cardiac tissues (ECTs) can be constructed using synthetic scaffold or liquid collagen. We report an initial study using our own newly designed cardiac muscle device to construct heart tissue. We investigated the effects of cell seeding density and collagen quantity on the formation of liquid collagen-based cardiac muscle. METHODS: We obtained cardiac myocytes from neonatal rats mixed with collagen type I and matrix factors cast in circular molds to form circular strands. Cell densities (0.1 x 10(7) to 6 x 10(7)) and collagen quantity (0.3 to 1.0 ml/ECT) were tested. Cell gross morphology, cell orientation, spatial distribution and ultrastructure were evaluated using histologic analyses, confocal laser scanning microscopy and transmission electron microscopy. RESULTS: Histologic analyses of ECTs revealed that cardiac cells reconstituted longitudinally oriented, cardiac bundles with morphologic features characteristic of the native tissue. Confocal and electron microscopy demonstrated that, using optimized cell density and collagen quantity, we made ECTs with characteristic features similar to those of native differentiated myocardium. CONCLUSIONS: ECTs comparable to native cardiac tissue can be engineered under optimized conditions. This construct is a first step in the development of cardiac tissue engineered in vitro, and may be used as a basis for studies of cardiac development, drug testing and tissue replacement therapy.     

In vitro engineering of heart muscle: artificial myocardial tissue

INTRODUCTION: Myocardial infarction followed by heart failure represents one of the major causes of morbidity and mortality, particularly in industrialized countries. Engineering and subsequent transplantation of contractile artificial myocardial tissue and, consequently, the replacement of ischemic and infarcted areas of the heart provides a potential therapeutic alternative to whole organ transplantation. METHODS: Artificial myocardial tissue samples were engineered by seeding neonatal rat cardiomyocytes with a commercially available 3-dimensional collagen matrix. The cellular engraftment within the artificial myocardial tissues was examined microscopically. Force development was analyzed in spontaneously beating artificial myocardial tissues, after stretching, and after pharmacologic stimulation. Moreover, electrocardiograms were recorded. RESULTS: Artificial myocardial tissues showed continuous, rhythmic, and synchronized contractions for up to 13 weeks. Embedded cardiomyocytes were distributed equally within the 3-dimensional matrix. Application of Ca(2+) and epinephrine, as well as electrical stimulation or stretching, resulted in enhanced force development. Electrocardiographic recording was possible on spontaneously beating artificial myocardial tissue samples and revealed physiologic patterns. CONCLUSIONS: Using a clinically well-established collagen matrix, contractile myocardial tissue can be engineered in vitro successfully. Mechanical and biologic properties of artificial myocardial tissue resemble native cardiac tissue. Use of artificial myocardial tissues might be a promising approach to reconstitute degenerated or failing cardiac tissue in many disease states and therefore provide a reasonable alternative to whole organ transplantation.     

毛囊单位促进组织工程皮肤形成的体外实验研究

目的 通过插入分离的毛囊单位,构建带有皮肤附属器的组织工程皮肤,探讨毛囊对组织工程皮肤形成的作用.方法 将头皮分离成毛囊单位,插入由Ⅰ型鼠尾胶原、成纤维细胞和角质形成细胞构建的组织工程皮肤模型中,浸没培养1周,气-液界面培养3周.通过镜下观察、HE染色和免疫组化检测组织工程皮肤和毛囊的生长状态.结果 相对于悬浮培养的毛囊,实验组毛囊体外生长期延长,结构更完整.HE染色显示,实验组组织工程皮肤可见毛囊和皮脂腺存在.免疫组化染色显示,带有毛囊的组织工程皮肤中可见反映基底膜完整性的Laminin和Ⅳ型胶原呈线状连续分布于表皮、真皮连接处;而反应表皮成熟度的CK4和CK10/13则呈阳性分布于基底上层非角化细胞.结论 复合毛囊单位可以促进组织工程皮肤表皮组织的分化和成熟.本方法为组织工程皮肤的构建和毛囊的体外培养提供了新的思路和方法.     

漏斗胸肋软骨基质的形态学与组织化学研究

目的：了解漏斗胸肋软骨基质形态学与组织化学特征。方法：对19例漏 斗胸和14例同年龄儿童肋软骨基质的胶原进行电镜观察并对Ⅱ型胶原进行免疫组化染色,对基质中蛋白多糖进行PAS和Safranin-O染色。将所有染色结果进行图像分析。结果：与对照组相比,电镜下病变组肋软骨基质中胶原分布不均,排列紊乱;Ⅱ型胶原免疫组化染色发现肋软骨深层Ⅱ型胶原分布不均,但浅,深层Ⅱ型胶原的染色强度没有明显改变;PAS和Safranin-O染色发现基质中蛋白多糖的含量与分布均无明显改变。结论：漏 斗胸肋软骨基质中蛋白多糖含量与分布及Ⅱ型胶原的含量无明显改变,而基质中的胶原分布与排列异常,后者可能与漏斗胸肋软骨生物力学性能降低有关。     

Characterization of the urethral plate and the underlying tissue defined by expression of collagen subtypes and microarchitecture in hypospadias

Objectives: In hypospadia patients, the urethral plate and the underlying tissue were previously thought to be the main cause of penile curvature and, because of this, they used to be excised to correct the curvature. Currently, they are preserved as they are not thought to cause penile curvature anymore. The aim of the present histology study was to elucidate the characteristic structure of the tissue beneath the urethral plate. Methods: The experimental group consisted of 27 hypospadiac patients with moderately severe penile curvature, who underwent one‐stage urethroplasty after dividing the urethral plate. Excised tissues were observed under light microscopy and transmission electron microscopy (TEM). Furthermore, the presence of collagen subtypes I, III and IV was examined with immunohistochemical staining and western blotting. Results: Light microscopy showed the existence of many massed and intertwined collagen fibers and vessels that resembled those of the cavernous sinus. TEM showed the existence of many collagen fibers, capillary vessels and other structures. Immunohistochemical staining showed collagen subtype I in the interfascicular space and collagen fibers were densely stained. Collagen subtype IV was found in the basement membrane of vessels, but collagen subtype III was not detected. The same results were obtained by western blotting. Conclusions: The tissue beneath the urethral plate was considered to originate from the corpus spongiosum penis. The distribution of collagen subtypes suggests that the presence of the tissue might affect ventral penile curvature. Long‐term follow up is required after one‐stage hypospadias repair with preservation of the urethral plate and the underlying tissue.     

凝胶包埋脂肪基质细胞接受振荡载荷模式构筑工程化软骨的研究

目的 观察构建工程化软骨生物学行为,评估扩增软骨种子细胞的方法.方法 软骨诱导化脂肪基质细胞(ADSCs),接种至nβ-TCP/Cs/PCL支架构建工程化软骨:A组(凝胶+振荡培养)、B组(凝胶+静态培养)、C组(振荡培养)、D组(静态培养);每组48块构建物,各阶段每组随机取6块,第1、4、8、12、16、20、24、28天,电镜、共聚焦观察,检测存活率、细胞增殖、Ⅱ型胶原(Col-Ⅱ)、DNA及氨基葡聚糖(GAG)含量.结果 A组细胞生长旺盛、细胞外基质丰富,存活率高于其他组(86.39±5.05,P<0.05),增殖活力、Col-Ⅱ、DNA及GAG含量均高于其他组(0.57±0.12,71.30±2.51,73.21±1.38,81.25±1.29,P<0.05).结论 凝胶包埋及振荡方式能扩增种子细胞及优化软骨构建质量.     

Cardiac tissue engineering

Recent progress in implantations of differentiated cardiac and non-cardiac cells as well as adult stem cells into the heart suggests that the irreversible loss of viable cardiac myocytes that occurs during myocardial infarction can be at least partly substituted. We evaluated an alternative approach by reconstituting cardiac tissue grafts in vitro and implanting them as spontaneously and coherently contracting tissues. For this purpose we have optimized a method to generate ring-shaped three-dimensional engineered heart tissue (EHT) in vitro from neonatal rat cardiac myocytes. When subjected to isometric force measurements in organ baths, electrically stimulated EHTs exhibit a Frank-Starling behavior, a positive inotropic response to increases in extracellular calcium, a positive inotropic and lusitropic response to isoprenaline, and a negative inotropic response to the muscarinic agonist carbachol ('accentuated antagonism'). Twitch tension under maximal calcium amounts to 1-2 mN/ mm2. Importantly, passive (resting) tension is low, yielding a ratio of active/passive tension of approximately 1.5 under basal and 14 under maximal calcium. Morphologically, EHTs represent a highly interconnected three-dimensional network of cardiac myocytes resembling loose cardiac tissue with a high fraction of binucleated cardiac myocytes, strong eosin staining and elongated centrally located nuclei. Electron microscopy demonstrated well developed sarcomeric structures, T-tubules, SR vesicles, T-tubule-SR-junctions, all types of intercellular connective structures, and a basement membrane. Thus, EHTs comprise functional and morphological properties of intact, ventricular myocardium. First implantation experiments of EHTs in the peritoneum of Fischer 344 rats showed that EHTs survived for at least 14 days, maintained a network of differentiated cardiac myocytes, and were strongly vascularized. Thus, EHTs may serve as material for a novel tissue replacement approach.     

Immunohistochemical identification of vascular endothelial growth factor in pig latissimus dorsi musculocutaneous flaps following ischemia-reperfusion injury

Vascular Endothelial Growth Factor (VEGF), a potent angiogenic, mitogenic and vascular permeability enhancing protein, appears to improve survival of ischemic flaps independent of its route of administration. The purpose of this study was to examine VEGF protein expression in biopsies of surgical flaps with immunohistochemical techniques. In 6 male Yorkshire-type pigs, 10 cm x 15 cm Latissimus dorsi musculocutaneous flaps were elevated bilaterally. Flap zones I, II, and III were established according to their distance from the vascular pedicle. After isolation of the thoracodorsal artery and vein, one flap was randomly assigned to ischemia by temporary occlusion of the vascular pedicle. Ischemia (4 hours) was followed by 2 hours of reperfusion (ischemia group, n = 6). The contralateral (nonischemic) flap served as a control (control group, n =6). Skin and muscle biopsies of flaps were taken at the end of the protocol for immunohistochemical staining using a VEGF antihuman monoclonal antibody.Epidermis of flap skin did not demonstrate VEGF-positive staining, but the dermis and subcutaneous tissue did. Muscle components of biopsies demonstrated staining of interfascicular septa and staining of myocytes. A semi-quantitative scoring system with a scale of 0 to 3 was used for grading of immunohistochemical staining.In skin, areas adjacent to the flap showed an overall mean VEGF staining score of 0.7. All zones of ischemic flaps showed increased mean immunohistochemical staining for VEGF (scores = 1.2, 1.6, and 1.4 in zones I, II, and III, respectively). In muscle, however, only zone I showed increased VEGF immunohistochemical staining from 0.7 in adjacent areas to 1.7 in ischemic flaps. The results indicate only moderate endogenous up-regulation of VEGF in flaps, supporting the utilization of exogenous VEGF as an adjunct in microsurgical therapy.     

心脏跳动中二尖瓣置换术心肌组织黏附分子的变化

目的观察心脏跳动中二尖瓣置换术围术期心肌细胞间黏附分子-1（ICAM-1）和血管细胞间黏附分子-1（VCAM-1）的表达。方法30例风湿性二尖瓣病变病人分为两组，试验组（浅低温下阻断主动脉）16例与对照组（中度低温阻断主动脉）14例。分别于CPB前、CPB后30min留取右心房标本，采用免疫组织化学技术检测心肌细胞上ICAM-1和VCAM-1的表达。结果与CPB前比较较，两组CPB后心肌细胞上ICAM-1和VCAM．1的表达明显升高（P〈0．01）。与对照组相比，试验组CPB后ICAM-1和VCAM-1的表达明显降低（P〈0．01）。结论心脏跳动中二尖瓣置换术对心肌细胞上黏附分子的影响较停跳手术少，减轻了术后的炎症反应。     

冷冻保存人脐带血管细胞培育组织工程心血管补片

目的 探讨应用液氮冷冻保存的人脐带动脉壁肌成纤维细胞(HUAMs)制备组织工程心血管补片的可行性.方法 将液氮冷冻保存的人脐带动脉壁肌成纤维细胞种植在聚-4-羟基丁酸酯(P4HB)聚合材料构建的补片支架材料上,制备组织工程心血管补片.体外静态培育21 d,对补片组织进行组织学、扫描电镜检查.免疫组织化学分析组织样本中细胞外基质(胶原蛋白)的合成.结果 苏木素-伊红(HE)染色显示,肌成纤维细胞较好地黏附于聚合材料上,大部分细胞保持生长活力,并浸润生长入支架材料内部,形成组织.免疫组织化学显示补片材料中有胶原蛋白合成.结论 液氮冷冻保存的人类脐带肌成纤维细胞可作为种子细胞,用于制备人组织工程心血管补片.