Fibrin gel as a three dimensional matrix in cardiovascular tissue engineering.

OBJECTIVE: In tissue engineering, three-dimensional biodegradable scaffolds are generally used as a basic structure for cell anchorage, cell proliferation and cell differentiation. The currently used biodegradable scaffolds in cardiovascular tissue engineering are potentially immunogenic, they show toxic degradation and inflammatory reactions. The aim of this study is to establish a new three-dimensional cell culture system within cells achieve uniform distribution and quick tissue development and with no toxic degradation or inflammatory reactions. METHODS: Human aortic tissue is harvested from the ascending aorta in the operation room and worked up to pure human myofibroblasts cultures. These human myofibroblasts cultures are suspended in fibrinogen solution and seeded into 6-well culture plates for cell development for 4 weeks and supplemented with different concentrations of aprotinin. Hydroxyproline assay and histological studies were performed to evaluate the tissue development in these fibrin gel structures. RESULTS: The light microscopy and the transmission electron microscopy studies for tissue development based on the three-dimensional fibrin gel structures showed homogenous cell growth and confluent collagen production. No toxic degradation or inflammatory reactions could be detected. Furthermore, fibrin gel myofibroblasts structures dissolved within 2 days in medium without aprotinin, but medium supplemented with higher concentration of aprotinin retained the three-dimensional structure and had a higher collagen content (P<0.005) and a better tissue development. CONCLUSIONS: A three-dimensional fibrin gel structure can serve as a useful scaffold for tissue engineering with controlled degradation, excellent seeding effects and good tissue development.     

Tissue engineering in cardiovascular surgery: new approach to develop completely human autologous tissue.

OBJECTIVE: In cardiovascular tissue engineering, three-dimensional scaffolds serve as physical supports and templates for cell attachment and tissue development. Currently used scaffolds are still far from ideal, they are potentially immunogenic and they show toxic degradation and inflammatory reactions. The aim of this study is to develop a new method for a three-dimensional completely autologous human tissue without using any scaffold materials. METHODS: Human aortic tissue is harvested from the ascending aorta in the operation room and worked up to pure human myofibroblasts cultures. These human aortic myofibroblasts cultures (1.5x10(6) cells, passage 3) were seeded into 15-cm culture dishes. Cells were cultured with Dulbecco' s modified Eagle's medium supplemented with 1 mM L-ascorbic acid 2-phosphate for 4 weeks to form myofibroblast sheets. The harvested cell sheets were folded to form four-layer sheets. The folded sheets were then framed up and cultured for another 4 weeks. Tissue development was evaluated by biochemical assay and light and electron microscopy. RESULTS: After 4 weeks of culture in ascorbic acid supplemented medium, myofibroblasts formed thin cell sheets in culture dishes. The cell sheets presented in a multi-layered pattern surrounded by extracellular matrices. Cultured for additional 4 weeks on the frames, the folded sheets further developed into more solid and flexible tissues. Light microscopy documented a structure resembling to a native tissue with confluent extracellular matrix. Under transmission electron microscope, viable cells and confluent bundles of striated mature collagen fibers were observed. Hydroxyproline assays showed significant increase of collagen content after culturing on the frames and were 80.5% of that of natural human pericardium. CONCLUSIONS: Improved cell culture technique may render human aortic myofibroblasts to a native tissue-like structure. A three-dimensional completely autologous human tissue may be further developed on the base of this structure with no show toxic degradation or inflammatory reactions.     

复合可降解基质膜片制备及血管内皮细胞在其吸附生长的实验研究

目的　制备几种复合可降解基质膜片 ,观察血管内皮细胞在其上的生长情况。方法　用胶原酶消化法分离牛主动脉血管内皮细胞 (VEC) ,另采用两步盐析法提取牛骨 型胶原。将交联胶原包埋处理聚羟基乙酸 (PGA)、聚乳酸 (PL A)及聚β羟基丁酸 (PHB)形成可降解基质材料膜片 ,并接种牛 VEC,采用 MTT法比较 VEC在几种可降解基质材料膜片的生长情况。结果　胶原、PGA/胶原、PL A/胶原膜质地均匀柔韧 ,固定成形 ,具有一定弹性和吸水性 ;MTT比色实验表明 VEC在胶原、PGA/胶原、PL A/胶原上均生长良好 ,优于 PHB/胶原组。尤以 PGA/胶原所形成的基质材料固定成形 ,弹性和韧性好 ,VEC在其上贴附生长良好。结论　通过生物材料与人工可降解材料有机结合 ,PGA/胶原物理性能互补 ,是组织工程再造血管较理想的基质材料之一。     

血管平滑肌细胞在几种基质膜片上生长情况及三种接种培养方法的比较

目的 筛选适合于组织工程血管构建的可降解基质材料及最佳的接种培养方式。方法 将血管平滑肌细胞接种于几种现有的可降解基质材料上，采用MTT法观察细胞在几种基质材料上的生长情况；比较静态、水平摇床、旋转接种三种接种培养方式。结果 细胞相容性比较好的材料有：PGA、胶原加黏多糖、脱钙骨等。三种接种方法的接种率为：静态1．5％。水平摇床7．2％，旋转接种培养53．5％。结论 PGA、胶原加黏多糖、脱钙骨三种材料适合于组织工程血管再造的应用。而三种接种培养方式以旋转接种培养的效率最高。     

Living patches engineered from human umbilical cord derived fibroblasts and endothelial progenitor cells.

OBJECTIVE: A major shortcoming in contemporary congenital heart surgery is the lack of viable replacement materials with the capacity of growth and regeneration. Here we focused on living autologous patches engineered from human umbilical cord derived fibroblasts and endothelial progenitor cells (EPCs) as a ready-to-use cell source for paediatric cardiovascular tissue engineering. METHODS: EPCs were isolated from 20 ml fresh umbilical cord blood by density gradient centrifugation and myofibroblasts were harvested from umbilical cord tissue. Cells were differentiated and expanded in vitro using nutrient media containing growth factors. Before seeding, cell-phenotypes were assessed by immuno-histochemistry. Biodegradable patches fabricated from synthetic polymers (PGA/P4HB) were seeded with myofibroblasts followed by endothelialization with EPCs. All patches were cultured in a perfusion bioreactor. A subgroup of patches was additionally stimulated by cyclic strain. Analysis of the neo-tissues comprised histology, immuno-histochemistry, extracellular matrix (ECM) analysis and biomechanical testing. RESULTS: Endothelial phenotypes of EPCs before seeding were confirmed by Ac-Dil-LDL, CD 31, von-Willebrand-Factor and eNOS staining. Histology of the seeded patches demonstrated layered viable tissue formation in all samples. The cells in the newly formed tissues expressed myofibroblast markers, such as desmin and alpha-SMA. The EPCs derived neo-endothelia showed constant endothelial phenotypes (CD 31, vWF). major constituents of ECM such as collagen and proteoglycans were biochemically detected. Stress-strain properties of the patches showed features of native-analogous tissues. CONCLUSIONS: Living tissue engineered patches can be successfully generated from human umbilical cord derived myofibroblasts and EPCs. This new cell source may enable the tissue engineering of versatile, living, autologous replacement materials for congenital cardiac interventions.     

脱细胞天然支架生物力学性能变化及预处理改善组织相容性的实验研究

目的观察脱细胞猪主动脉瓣的生物力学性能变化,探讨不同预处理改善天然支架组织相容性的效果。方法新鲜猪主动脉瓣经酶加去污剂法去除细胞,力学测试仪检测其最大负荷、最大应力、最大应变和弹性模量的变化,苏木精-伊红（HE）染色、Ⅰ型胶原免疫组化染色和扫描电镜观察其病理形态学变化;将脱细胞瓣膜分别予磷酸缓冲液、多聚赖氨酸和未灭活胎牛血清包被处理,然后种植大鼠主动脉肌成纤维细胞,甲基噻唑基四唑试验检测细胞黏附率,HE染色和扫描电镜观察形态学变化。结果酶加去污剂法能完全脱去瓣膜细胞,基本维持胶原纤维的空间结构,但其最大负荷、最大应力及弹性模量下降,最大应变上升（P〈0．05）;胎牛血清预处理去细胞瓣能显著提高肌成纤维细胞的黏附率,促进细胞生长、分化和增殖,并在瓣膜表面形成连续的细胞层（F值=129．26,P=0．000）。结论酶加去污剂法可较完全去除猪主动脉瓣膜细胞并保持细胞外基质的三维结构,但其生物力学性能有所下降;胎牛血清预处理能改善脱细胞瓣天然支架的细胞黏附、生长和繁殖。     

不同支架及接种培养方法对前脂肪细胞生长情况的影响

目的 通过对体外培养前脂肪细胞并分别与聚乳酸-羟基乙酸共聚物（PLGA）支架、胶原支架以及透明质酸支架复合,研究3种不同支架材料的细胞相容性.方法 切取成年女性腹部皮下脂肪组织,采用胶原酶消化的方法分离培养人前脂肪细胞,采用3-（4,5-二甲基噻唑-2）-2,5-二苯基四氮唑臭盐（MTT）法观察3种不同支架材料的细胞相容性,并以透明质酸作为支架,考察静态和水平摇床两种接种培养方式对前脂肪细胞在支架上接种率的影响.结果 前脂肪细胞可以在体外成功地分离、培养,能定向分化为脂肪细胞,并可以进行传代扩增.前脂肪细胞在PLGA、胶原以及透明质酸3种材料上生长良好,其中透明质酸支架以及水平摇床接种培养效果更佳.结论 透明质酸更加适宜作为组织工程化脂肪的支架材料,水平摇床的接种培养可提高细胞与支架材料之间的接种率,优于静态培养.     

人脐带间充质干细胞与蚕丝素多孔支架的体外复合培养

目的观察蚕丝素多孔支架对人脐带间充质干细胞（human umbilical cord mesenchymal stem cells,hUCMSCs）吸附作用及支架对hUCMSCs形态、功能及活性的影响,为脂肪组织工程支架选择提供实验依据。方法将hUCMSCs制成细胞悬液接种在蚕丝素多孔支架,荧光倒置相差显微镜、扫描电镜和四甲基偶氮唑蓝（MTT）法观察hUCMSCs的吸附和生长情况。结果培养1～2d后可见hUCMSCs与蚕丝素多孔支架充分附着。培养5～7d细胞生长增殖十分活跃,10d左右时,蚕丝素多孔支架孔中hUCMSCs成片状融合。荧光倒置相差显微镜和扫描电镜见细胞与支架黏附良好并有大量基质分泌,且活性指标与正常培养的hUCMSCs比较差异无统计学意义（P〉0．05）。结论蚕丝素多孔支架对hUCMSCs具有良好的吸附作用,并能维持其正常形态、功能及活性,蚕丝素多孔支架是hUCMSCs三维立体培养时的良好天然支架。     

旋转系统下三维培养成纤维细胞-PGA复合物的实验研究

目的　观察微重力旋转培养系统对细胞种植于支架和细胞 支架复合物体外培养的影响。　方法　分离培养兔皮肤成纤维细胞 ,实验用第 2代细胞。 ( 1)分别用静止接种和动态接种方式 ,以 2× 10 6/cm3 的细胞密度接种于PGA网架 ,静止接种即滴加细胞悬液于PGA网架上 ,动态接种是把细胞悬液与PGA网架置于旋转细胞培养系统旋转接种 ,分别于 2、4、8、12、2 4h消化下网架上细胞后计数 ,通过计算细胞吸附率了解细胞吸附情况 ;( 2 )将相同细胞密度静止接种的细胞—PGA复合物分别置于旋转和静止条件下培养 ,于 1、3、5、7、14、2 1dMTT法检测细胞增殖 ;用倒置相差显微镜和扫描电镜观察细胞生长、基质形成及其细胞与PGA纤维结合状况的变化。　结果　细胞吸附量随时间延长而逐渐升高 ,动态组在接种后 8、12、2 4h细胞吸附率显著高于静止接种组 ,分别为 ( 46 70 %±2 16 %)比 ( 31 5 0 %± 3 5 4%) ;( 5 6 36 %± 3 18%)比 ( 34 2 8%± 3 16 %) ;和 ( 6 6 32 %± 4 6 0 %)比( 37 38%± 4 6 6 %)。在 3周培养中旋转培养组细胞增殖快于静止组 ,而且细胞在网架中分布比静止组均匀 ,并伴有活跃的细胞基质分泌。　结论　旋转培养系统具有促进细胞吸附增殖分化和在支架内均匀分布的特点 ,是培养细胞支架复合物进行组织工     

种植人体活性细胞的生物心脏瓣膜

目前,种植人体活性细胞的生物心脏瓣膜主要有组织工程心脏瓣膜和种植人体活性细胞的猪主动脉瓣两种。组织工程心脏瓣膜是在人体可吸收的聚二醇酸纤维支架上种植人体同种活性细胞,先种植成纤维细胞,再种植单层内皮细胞包裹瓣叶。种植人体活性细胞的猪主动脉瓣是在清除原有细胞的组织内重建人体同种活性细胞。清除新鲜猪主动脉瓣呐原有细胞的方法是将瓣膜先经高、低渗溶液处理,然后用酶溶液处理。细胞经培养分离后,将成纤维细胞植入经处理的瓣膜组织,再植入内皮细胞。种植人体活性细胞的生物心脏瓣膜不会促使受者产生有害的免疫反应,并具有再生能力。     

经胶原包埋、修饰的聚羟基乙酸与软骨细胞体外培养的实验研究

目的：探讨经胶原包埋、修饰后的聚羟基乙酸（poloyglycolic acid简称PGA）作为组织技术中细胞培养支架的可行性；方法：将用胶原包埋的PGA和没有包埋的PGA分别与软骨细胞共同置于二氧化碳培养箱中培养，观察二者的亲水性，对细胞的吸附能力和细胞分泌基质的能力进行比较；结果：以胶原包埋的PGA和没有包埋的PGA亲水性没有明显的差异，二者亲水性均不强，而前者对细胞的吸附能力和细胞在其上面生长、分泌基质的能力明显增强；结论：以胶原包埋、修饰的PGA作为组织工程技术中细胞生长的支架，具有很大的应用前景。     

Endothelial Cell Lining of Bioprosthetic Heart Valve Material

In this in vitro study, the growth properties of cultured endothelial cells on conventionally treated pericardial valve material were measured. These data were compared to endothelial cell proliferation on an alternatively treated valve material. This alternative preservation procedure was developed in order to bind free, residual glutaraldehyde in the valve tissue by reaction with L-glutamic acid. In order to optimize endothelial cell attachment and proliferation, fibronectin and fibrillar collagen type I were tested as surface precoating substances. Cell viability of the seeded cells was evaluated by means of proliferation kinetics, antithrombotic activity, and morphological appearance. Endothelial cell death occurred within the first 2 days after seeding on conventionally treated valve tissue, independent of the type of precoating. On alternatively treated tissue, regular endothelial cell proliferation was observed. Precoating with fibrillar collagen markedly increased endothelial cell attachment and proliferation as compared to fibronectin. Maintenance of antithrombotic activity of the seeded cells was proven by regular release of prostacyclin.     

聚羟基烷酸酯与绵羊骨髓基质干细胞相容性的研究

目的评价聚羟基烷酸酯(PHBV)作为组织工程支架与绵羊骨髓基质干细胞(BMSCs)的生物相容性。方法原代培养绵羊BMSCs,传至2～3代后,接种至PHBV膜和泡沫样三维支架上,光镜和扫描电镜观察细胞形态,计数1、2、6 h时的细胞黏附率;并以接种至培养板上细胞为对照组,每日细胞计数,绘制生长曲线;按培养液量与支架体积10 mL／cm3为标准浓度制备浸提液,并制备标准浓度1／16～16倍的浸提液,以MTT法检测细胞毒性;流式细胞仪分析接种到材料上的细胞周期,计算增殖指数;BMSCs接种于PHBV三维支架上4、8、12 d,以Hoechst33258荧光法定量测定细胞内DNA含量, BCA法测定蛋白质含量。结果第3代BMSCs接种至PHBV膜上2 h后即大部黏附,黏附率75．6％,与对照组相比差异无统计学意义,绘制生长曲线见细胞生长与对照组无差异;MTT法检测见9个浓度梯度的浸提液毒性均为0级;光镜和扫描电镜观察见细胞接种于PHBV膜上2 h后大部分黏附,3 d后伸展良好,呈纺锤形或梭形,在三维支架的孔隙内立体生长,1周开始细胞间连接,3周广泛连接,分泌大量基质;流式细胞分析见接种于材料上的细胞周期无变化;接种至PHBV三维支架上的细胞内DNA、蛋白质浓度与对照组比较无差异。结论PHBV作为BMSCs的组织工程支架材料,具有良好的生物相容性。     

Cell characterization of porcine aortic valve and decellularized leaflets repopulated with aortic valve interstitial cells: the VESALIO Project (Vitalitate Exornatum Succedaneum Aorticum Labore Ingenioso Obtenibitur)

BACKGROUND: Heart valve bioprostheses for cardiac valve replacement are fabricated by xeno- or allograft tissues. Decellularization techniques and tissue engineering technologies applied to these tissues might contribute to the reduction in risk of calcification and immune response. Surprisingly, there are few data on the cell phenotypes obtained after cellularizing these naturally-derived biomaterials in comparison to those expressed in the intact valve. METHODS: Aortic valve interstitial cells (VIC) were used to repopulate the corresponding valve leaflets after a novel decellularization procedure based on the use of ionic and nonionic detergents. VIC from leaflet microexplants at the third passage were utilized to repopulate the decellularized leaflets. Intact, decellularized and repopulated valve leaflets and cultured VIC were examined by immunocytochemical procedures with a panel of antibodies to smooth muscle and nonmuscle differentiation antigens. Intact and cellularized leaflets were also investigated with Western blotting and transmission electron microscopy, respectively. RESULTS: Myofibroblasts and smooth muscle cells (SMC) were mostly localized to the ventricularis of the leaflet whereas fibroblasts were dispersed unevenly. Cultured VIC were comprised of myofibroblasts and fibroblasts with no evidence of endothelial cells and SMC. Two weeks after VIC seeding into decellularized leaflets, grafted cells were found penetrating the bioscaffold. The immunophenotypic and ultrastructural properties of the grafted cells indicated that a VIC heterogeneous mesenchymal cell population was present: fibroblasts, myofibroblasts, SMC, and endothelial cells. CONCLUSIONS: VIC seeding on detergent-treated valve bioscaffolds has the cellular potential to reconstruct a viable aortic valve.     

MTT法检测国产PGA支架对人成纤维细胞增殖的影响

目的 检测国产PGA支架对瘢痕、皮肤和肌腱成纤维细胞增殖的影响.方法 体外培养人瘢痕、皮肤和肌腱成纤维细胞,取第二代细胞接种到96 孔板,以不同浓度的国产PGA支架浸提液培养,用MTT法检测成纤维细胞在国产PGA上的增殖情况.结果 不同浸提液培养的瘢痕、皮肤和肌腱成纤维细胞的生长曲线均无明显差异.结论 国产PGA支架对瘢痕、皮肤和肌腱成纤维细胞的增殖无明显影响,该材料可以用于以成纤维细胞为种子的组织构建.     

组织工程血管模型体外构建的实验研究

目的：在体外环境下，探索构建分别含内皮细胞，平滑肌细胞及成纤维细胞的三层结构的组织工程化血管，三种细胞相互作用，相互支持，形成一个在形态和功能与正常血管近似的组织工程化血管。方法：通过用胶原分别包埋处理的三个管径大小不同，但能相互嵌套的聚羟基乙酸（PGA）管形支架，并种植人脐静脉内皮细胞，人血管平滑肌细胞，人成纤维细胞进行三维立体生长培养，观察细胞生长分化情况。采用酶消化法和组织块法分离培养人脐静脉内皮细胞，人血管平滑肌细胞，人成纤维细胞并传代，纯化，将聚羟基乙酸（PGA）无纺网用胶原溶液包埋，真空冷冻干燥，形成多孔状PGA＋胶原管型支架；接种3代－7代人脐静脉内皮细胞，人血管平滑肌细胞及人成纤维细胞与其内腔面，采用动脉旋转培养技术体外培养3周，行扫描电镜观察。结果：构建后的血管模型，层与层结构紧密，内皮细胞，平滑肌细胞及成纤维细胞分泌细胞外基质，相互进行物质交换，类似于生理条件。结论：该支架具有一定弹性和韧性，在培养液中，能较长时间保持形状。此方法的进一步应用组织工程方法构筑具有分层结构的人造血管打下基础。     

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.     

Preseeding with autologous fibroblasts improves endothelialization of glutaraldehyde-fixed porcine aortic valves

OBJECTIVE: This study represents the development of a treatment and seeding procedure to improve endothelial cellular adhesion on glutaraldehyde-fixed valves. METHODS: Porcine aortic valves were fixed with 0.2% glutaraldehyde. Wall pieces of these valves had either no additional treatment (n = 4), incubation in M199 Earle (1x), with sodium carbonate at 2.2 g/L without l-glutamine for 24 hours (n = 4), or additional pretreatment with 5%, 10%, or 15% citric acid (three groups, n = 4 each). Thereafter the pieces were washed and buffered to a physiologic pH. This was followed by seeding of human endothelial cells (5 x 10(6) cells). On the basis of the results of these pilot tests, complete glutaraldehyde-fixed aortic roots treated with 10% citric acid were subjected to cell seeding. The valves were seeded with endothelial cells (4.3 x 10(6) cells) either alone (n = 4) or in combination with preseeding of autologous fibroblasts (2.4 x 10(7) cells, n = 4). After each seeding procedure specimens of the free wall of the grafts were taken. In addition, one leaflet was taken for histologic examination after endothelial cell seeding, after 7 days, and after 21 days. Finally, two commercially available stentless aortic valve prostheses (Freestyle; Medtronic, Inc, Minneapolis, Minn) were treated with 10% citric acid and seeded with human fibroblasts and endothelial cells. Specimen were taken according to the glutaraldehyde-fixed aortic roots. Specimen of all experiments were examined with scanning electron microscopy. Frozen sections were stained immunohistochemically for collagen IV, factor VIII, and CD31. RESULTS: On untreated glutaraldehyde-fixed aortic wall pieces, only poor adhesion (24%) was seen. No viable cells were found after 1 week. Cellular adhesion was best on aortic wall pieces pretreated with 10% citric acid. After 7 days, the cells formed a confluent layer. Endothelial cell seeding on citric acid-treated complete aortic valves showed 45% adhesion, but no confluent layer was found after 1 week. Preseeding of these valves with autologous fibroblasts resulted in an endothelial cellular adhesion of 76% and a confluent endothelial cell layer after 7 days. The layer remained stable for at least 21 days. Results of staining for collagen IV, factor VIII, and CD31 were positive on the luminal side of these valves, indicating the synthesis of matrix proteins and viability of the cells. Pretreatment of commercially available porcine valves with 10% citric acid and preseeding with autologous fibroblasts followed by endothelial cell seeding resulted in an adhesion of 78%. The cells formed a confluent cell layer after 7 days. CONCLUSIONS: Pretreatment of glutaraldehyde-fixed porcine aortic valves with citric acid established a surface more suitable for cellular attachment. Preseeding these valves with autologous fibroblasts resulted in a confluent endothelial cell layer on the luminal surface. Flow tests and animal experiments are necessary for further assessment of durability and shear stress resistance.     

Fetal tissue engineering: in vitro analysis of muscle constructs

Background/purpose: This study was aimed at examining the impact of different tissue engineering techniques on fetal muscle construct architecture.Methods: Myoblasts from ovine specimens of fetal skeletal muscle were expanded in culture and their growth rates determined. Cells were seeded at different densities onto 3 scaffold types, namely polyglycolic acid (PGA) treated with poly-l-lactic acid (PLLA), a composite of PGA with poly-4-hydroxybutyrate (P4HB), and a collagen hydrogel. Constructs were maintained in a bioreactor and submitted to histologic, scanning electron microscopy, and DNA analyses at different time-points. Statistical analysis was by the likelihood ratio and paired Student’s t tests (P < .05).Results: Fetal myoblasts proliferated at faster rates than expected from neonatal cells. Cell attachment was enhanced in the PGA/PLLA matrix and collagen hydrogel when compared with the PGA/P4HB composite. Necrosis was observed at the center of all constructs, directly proportional to cell seeding density and time in the bioreactor.Conclusions: Fetal myoblasts can be expanded rapidly in culture and attach well to PGA/PLLA, as well as collagen hydrogel but less optimally to PGA/P4HB. Excessive cell seeding density and bioreactor time may worsen final construct architecture. These findings should be considered during in vivo trials of muscle replacement by engineered fetal constructs.     

Human umbilical cord cells for cardiovascular tissue engineering: a comparative study.

OBJECTIVE: Tissue engineering of viable, autologous cardiovascular replacements with the potential to grow, repair and remodel represents an attractive approach to overcome the shortcomings of available replacements for the repair of congenital cardiac defects. Currently, vascular myofibroblast cells represent an established cell source for cardiovascular tissue engineering. Cell isolation requires the invasive harvesting of venous or arterial vessel segments prior to scaffold seeding, a technique which may not be preferable, especially in pediatric patients. This study evaluates cells isolated from human umbilical cord artery, umbilical cord vein and whole cord as alternative autologous cell sources for cardiovascular tissue engineering. METHODS: Cells were isolated from human umbilical cord artery (UCA), umbilical cord vein (UCV), whole umbilical cord (UCC) and saphenous vein segments (VC), and were expanded in culture. All three expanded cell groups were seeded on bioabsorbable copolymer strips and grown in vitro for 28 days. Isolated cells were characterized by flow cytometry, histology, immunohistochemistry, proliferation assays and compared to VC. Morphological analysis of the seeded polymer strips included histology, immunohistochemistry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transmission electron microscopy (TEM), scanning electron microscopy (SEM) and uniaxial stress testing. RESULTS: UCA, UCV and UCC demonstrated excellent cell growth properties comparable to VC. Following isolation, all three cell groups showed myofibroblast-like morphology and characteristics by staining positive for alpha-smooth muscle actin (ASMA) and vimentin. Histology and immunohistochemistry of seeded polymers showed good tissue and extracellular matrix formation containing collagen I, III and elastin. TEM showed viable myofibroblasts and the deposition of collagen fibrils and progressive growing tissue formation, with a confluent surface, was observed in SEM. No difference was found among the mechanical properties of UCA, UCV, UCC and VC tissue engineered constructs. CONCLUSIONS: Tissue engineering of cardiovascular constructs by using UCA, UCV and UCC is feasible in an in vitro environment. Cell growth, morphology, characteristics and tissue formation were comparable between UCA, UCV, UCC and VC. UCC represent an attractive, readily available autologous cell source for cardiovascular tissue engineering offering the additional benefits of utilizing juvenile cells and avoiding the invasive harvesting of intact vascular structures.