Huang Hui-min, Wu Shao-feng, Ren Hong
Department of Cardiothoracic Surgery, Shanghai Children's Medical Center, Xinhua Hospital, Shanghai Jiao Tong University, Shanghai 200127, China.
J Zhejiang Univ Sci B. 2006 May;7(5):351-6. doi: 10.1631/jzus.2006.B0351.
Endothelial and smooth muscle cells were used as seeding cells and heterogeneous acellularized matrix was used as scaffold to construct the tissue-engineered graft.
A 2 weeks piglet was selected as a donor of seeding cells. Two-centimetre length of common carotid artery was dissected. Endothelial cells and smooth muscle cells were harvested by trypsin and collagenase digestion respectively. The isolated cells were cultured and expanded using routine cell culture technique. An adult sheep was used as a donor of acellularized matrix. The thoracic aorta was harvested and processed by a multi-step decellularizing technique to remove the original cells and preserve the elastic and collagen fibers. The cultured smooth muscle cells and endothelial cells were then seeded to the acellularized matrix and incubated in vitro for another 2 weeks. The cell seeded graft was then transplanted to the cell-donated piglet to substitute part of the native pulmonary artery.
The cultured cells from piglet were characterized as endothelial cells by the presence of specific antigens vWF and CD31, and smooth muscle cells by the presence of specific antigen alpha-actin on the cell surface respectively with immunohistochemical technique. After decellularizing processing for the thoracic aorta from sheep, all the cellular components were extracted and elastic and collagen fibers kept their original morphology and structure. The maximal load of acellular matrix was decreased and 20% lower than that of untreated thoracic aorta, but the maximal tensions between them were not different statistically and they had similar load-tension curves. Three months after transplantation, the animal was sacrificed and the graft was removed for observation. The results showed that the inner surfaces of the graft were smooth, without thrombosis and calcification. Under microscopy, a great number of growing cells could be seen and elastic and collagen fibers were abundant.
Cultured self-derived endothelial and smooth muscle cells could be used as seeding cells and heterogeneous acellularized matrix could be used as scaffold in constructing tissue-engineered graft.
以内皮细胞和平滑肌细胞作为种子细胞，以异种脱细胞基质作为支架构建组织工程移植物。
选取2周龄仔猪作为种子细胞供体。解剖出2厘米长的颈总动脉。分别用胰蛋白酶和胶原酶消化法获取内皮细胞和平滑肌细胞。采用常规细胞培养技术对分离出的细胞进行培养和扩增。选取成年绵羊作为脱细胞基质供体。获取胸主动脉，采用多步脱细胞技术进行处理，去除原有细胞，保留弹性纤维和胶原纤维。然后将培养的平滑肌细胞和内皮细胞接种到脱细胞基质上，再在体外培养2周。将接种细胞的移植物移植到提供细胞的仔猪体内，替代部分天然肺动脉。
采用免疫组织化学技术，仔猪培养细胞分别通过细胞表面存在特异性抗原血管性血友病因子（vWF）和CD31鉴定为内皮细胞，通过存在特异性抗原α-肌动蛋白鉴定为平滑肌细胞。对绵羊胸主动脉进行脱细胞处理后，所有细胞成分均被去除，弹性纤维和胶原纤维保持其原始形态和结构。脱细胞基质的最大负荷降低，比未处理的胸主动脉低20%，但两者之间的最大张力在统计学上无差异，且具有相似的负荷-张力曲线。移植3个月后，处死动物并取出移植物进行观察。结果显示，移植物内表面光滑，无血栓形成和钙化。显微镜下可见大量生长的细胞，弹性纤维和胶原纤维丰富。
培养的自体来源内皮细胞和平滑肌细胞可作为种子细胞，异种脱细胞基质可作为构建组织工程移植物的支架。
