Falke German, Yoo James J, Kwon Tae Gyun, Moreland Robert, Atala Anthony
Laboratory for Tissue Engineering and Cellular Therapeutics, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Tissue Eng. 2003 Oct;9(5):871-9. doi: 10.1089/107632703322495529.
We explored the feasibility of developing corporal tissue, consisting of human cavernosal smooth muscle and endothelial cells in vivo, using three-dimensional acellular collagen matrices, which are similar in architecture to native corpora. Acellular collagen matrices were derived from processed donor rabbit corpora, using cell lysis techniques. Human corpus cavernosal muscle and endothelial cells were seeded on the acellular matrices. A total of 80 matrices, 20 without cells and 60 with cells, were implanted subcutaneously in athymic mice. An additional 36 matrices seeded with cells were maintained in culture for up to 4 weeks. Hydroxyproline quantification, Western blot analysis, RT-PCR, and scanning electron microscopy of the matrices, with and without cells, were performed at various time points. Animals were killed 3 days and 1, 2, 3, 4, 6, and 8 weeks after implantation. Immunocytochemical and histological analyses were performed to confirm the muscle and endothelial phenotype. Organ bath studies were performed in order to determine the degree of tissue contraction. Western blot analysis detected alpha-actin, myosin, and tropomyosin proteins from human corporal smooth muscle cells. Expression of muscarinic acetylcholine receptor (mAChR) subtype m4 mRNA was demonstrated by RT-PCR from corporal muscle cells before and 8 weeks after seeding. The implanted matrices showed neovascularity into the sinusoidal spaces by 1 week after implantation. Increasing organization of smooth muscle and endothelial cells lining the sinusoidal walls was observed at 2 weeks and continued with time. The matrices were covered with the appropriate cell architecture 4 weeks after implantation. The matrices showed a stable collagen concentration over 8 weeks, as determined by hydroxyproline quantification. Immunocytochemical studies using alpha-actin and factor VIII antibodies confirmed the presence of corporal smooth muscle and endothelial cells, both in vitro and in vivo, at all time points. There was no evidence of cellular organization in the control matrices. Organ bath studies showed that the cell-seeded corporal tissue matrices responded to electrical field stimulation, whereas the unseeded implants failed to respond. This study demonstrates that human cavernosal smooth muscle and endothelial cells seeded on three-dimensional acellular collagen matrices derived from donor corpora are able to form well-vascularized corporal tissues in vivo.
我们利用三维脱细胞胶原基质探索了在体内培育由人海绵体平滑肌和内皮细胞组成的阴茎组织的可行性，这种基质的结构与天然海绵体相似。脱细胞胶原基质由经过处理的供体兔海绵体通过细胞裂解技术获得。将人海绵体肌和内皮细胞接种到脱细胞基质上。总共80个基质，20个无细胞的和60个有细胞的，皮下植入无胸腺小鼠体内。另外36个接种细胞的基质在培养中维持长达4周。在不同时间点对有细胞和无细胞的基质进行羟脯氨酸定量、蛋白质印迹分析、逆转录聚合酶链反应（RT-PCR）和扫描电子显微镜检查。植入后3天以及1、2、3、4、6和8周处死动物。进行免疫细胞化学和组织学分析以确认肌肉和内皮表型。进行器官浴研究以确定组织收缩程度。蛋白质印迹分析检测到人阴茎平滑肌细胞的α-肌动蛋白、肌球蛋白和原肌球蛋白蛋白。通过RT-PCR在接种前和接种8周后的阴茎肌细胞中证实了毒蕈碱型乙酰胆碱受体（mAChR）亚型m4 mRNA的表达。植入的基质在植入后1周显示有新血管长入窦状间隙。在2周时观察到窦状壁内衬的平滑肌和内皮细胞组织化增加，并随时间持续。植入后4周基质被合适的细胞结构覆盖。通过羟脯氨酸定量测定，基质在8周内显示出稳定的胶原浓度。使用α-肌动蛋白和因子VIII抗体的免疫细胞化学研究在所有时间点均证实了体外和体内阴茎平滑肌和内皮细胞的存在。对照基质中没有细胞组织化的证据。器官浴研究表明，接种细胞的阴茎组织基质对电场刺激有反应，而未接种的植入物无反应。这项研究表明，接种到人供体海绵体来源的三维脱细胞胶原基质上的人海绵体平滑肌和内皮细胞能够在体内形成血管化良好的阴茎组织。
