Shinoka T, Ma P X, Shum-Tim D, Breuer C K, Cusick R A, Zund G, Langer R, Vacanti J P, Mayer J E
Department of Cardiovascular Surgery, Children's Hospital, Boston, Mass, USA.
Circulation. 1996 Nov 1;94(9 Suppl):II164-8.
We have previously reported the successful creation of tissue-engineered valve leaflets and the implantation of these autologous tissue leaflets in the pulmonary valve position. This study was designed to trace cultured cells that were seeded onto a biodegradable polymer with the use of a 1,1'-dioctadecyl-3,3,3' 3'-tetramethylindo-carbocyanine perchlorate (Di-1) cell-labeling method. We also examined the time-related biochemical, biomechanical, and histological characteristics and evolution of these tissue constructs.
Mixed cell populations of endothelial cells and fibroblasts were isolated from explanted ovine arteries. Endothelial cells were selectively labeled with an acetylated low density lipoprotein marker and separated from fibroblasts with the use of a fluorescence-activated cell sorter. A synthetic biodegradable scaffold consisting of polyglycolic acid fibers was seeded first with fibroblasts, then coated with endothelial cells. Using these methods, we implanted autologous cell/polymer constructs in six animals. In two additional control animals, a leaflet of polymer was implanted without prior cell seeding. In each animal, cardiopulmonary bypass was used to completely resect the right posterior leaflet of the pulmonary valve and replace it with an engineered valve leaflet with (n = 6) or without (n = 2) prior cultured cell seeding. The animals were killed either after 6 hours or after 1, 6, 7, 9, or 11 weeks, and the implanted valve leaflets were examined histologically, biochemically, and biomechanically. 4-Hydroxyproline assays were performed to determine collagen content. Leaflet strength was evaluated in vitro with a mechanical tester Factor VIII and elastin stains were done to verify histologically that endothelial cells and elastin, respectively, were present. Animals receiving leaflets made from polymers without cell seeding were killed and examined in a similar fashion after 8 weeks. In the control animals, the acellular polymer leaflets were completely degraded, with no residual leaflet tissue at 8 weeks. The tissue-engineered valve leaflet persisted in each animal in the experimental group. 4-Hydroxyproline analysis of the constructs showed a progressive increase in collagen content. Immunohistochemical staining demonstrated elastin fibers in the matrix and factor VIII on the surface of the leaflet. The cell-labeling experiments demonstrated that the cells on the leaflets had persisted from the in vitro seeding of the leaflets.
In the tissue-engineered heart valve leaflet, transplanted autologous cells generated a proper matrix on the polymer scaffold in a physiological environment at a period of 8 weeks after implantation.
我们之前曾报道成功构建了组织工程瓣膜小叶，并将这些自体组织小叶植入肺动脉瓣位置。本研究旨在利用1,1'-二辛基-3,3,3',3'-四甲基吲哚羰花青高氯酸盐（Di-1）细胞标记方法追踪接种到可生物降解聚合物上的培养细胞。我们还研究了这些组织构建物随时间变化的生化、生物力学和组织学特征及演变情况。
从取出的羊动脉中分离出内皮细胞和成纤维细胞的混合细胞群。用乙酰化低密度脂蛋白标记物选择性标记内皮细胞，并使用荧光激活细胞分选仪将其与成纤维细胞分离。首先将由聚乙醇酸纤维组成的合成可生物降解支架接种成纤维细胞，然后再覆盖内皮细胞。采用这些方法，我们将自体细胞/聚合物构建物植入6只动物体内。在另外2只对照动物中，植入未经预先接种细胞的聚合物小叶。在每只动物中，使用体外循环完全切除肺动脉瓣的右后小叶，并用预先接种（n = 6）或未接种（n = 2）培养细胞的工程瓣膜小叶进行替换。在6小时后或1、6、7、9或11周后处死动物，对植入的瓣膜小叶进行组织学、生化和生物力学检查。进行4-羟脯氨酸测定以确定胶原蛋白含量。用机械测试仪在体外评估小叶强度，进行因子VIII和弹性蛋白染色以分别从组织学上验证内皮细胞和弹性蛋白的存在。接受未接种细胞的聚合物制成的小叶的动物在8周后处死并以类似方式进行检查。在对照动物中，无细胞聚合物小叶在8周时完全降解，没有残留的小叶组织。在实验组的每只动物中，组织工程瓣膜小叶持续存在。构建物的4-羟脯氨酸分析显示胶原蛋白含量逐渐增加。免疫组织化学染色显示基质中有弹性纤维，小叶表面有因子VIII。细胞标记实验表明，小叶上的细胞从体外接种小叶后一直持续存在。
在组织工程心脏瓣膜小叶中，移植的自体细胞在植入后8周的生理环境中在聚合物支架上生成了合适的基质。
