He Hongbing, Shirota Toshihiko, Yasui Hisataka, Matsuda Takehisa
Department of Biomedical Engineering, Graduate School of Medicine, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
J Thorac Cardiovasc Surg. 2003 Aug;126(2):455-64. doi: 10.1016/s0022-5223(02)73264-9.
We sought to fabricate a compliant engineered vascular graft (inner diameter of approximately 4.5 mm and length of 6 cm) lined with endothelial progenitor cells derived from circulating peripheral canine blood and to verify its nonthrombogenicity potential in vivo.
Autologous circulating endothelial progenitor cells derived from the peripheral veins of 6 adult mongrel dogs were isolated by using a density gradient method. The cells were proliferated in vitro in EGM-2 culture medium, prelined on the luminal surface of in situ-formed collagen type I meshes as an extracellular matrix, and wrapped with a segmented polyurethane thin film with multiple micropores as a compliant scaffold. After canine carotid arteries were bilaterally implanted with these grafts for 1 and 3 months, microscopic observation, histologic staining, and immunochemical staining were performed to evaluate morphogenesis.
After 33.3 +/- 10.5 days of culture in vitro, 4.2 +/- 1.2 x 10(6) endothelial progenitor cells were obtained. Eleven of the 12 engineered vascular grafts were patent. The grafts possessed smooth, glistening, and ivory-colored luminal surfaces at the predetermined observation period up to 3 months. The intimal layer was covered with confluent, cobblestone-like monolayered cells that were positively stained with factor VIIIB-related antigen. The thickness of the neoarterial walls was approximately 300 microm at 3 months after implantation. A few smooth muscle cells were observed in the medial tissue, and fibroblasts dominated the adventitial tissue.
Circulating endothelial progenitor cells could be a substitute source of endothelial cells for endothelialization on small-diameter-vessel prostheses to ensure nonthrombogenicity.
我们试图构建一种顺应性工程血管移植物（内径约4.5毫米，长度6厘米），其内衬源自犬循环外周血的内皮祖细胞，并在体内验证其抗血栓形成潜力。
采用密度梯度法从6只成年杂种犬的外周静脉中分离出自体循环内皮祖细胞。细胞在EGM-2培养基中体外增殖，预先内衬于原位形成的I型胶原网的管腔表面作为细胞外基质，并用具有多个微孔的分段聚氨酯薄膜包裹作为顺应性支架。将这些移植物双侧植入犬颈动脉1个月和3个月后，进行显微镜观察、组织学染色和免疫化学染色以评估形态发生。
体外培养33.3±10.5天后，获得4.2±1.2×10⁶个内皮祖细胞。12个工程血管移植物中有11个通畅。在长达3个月的预定观察期内，移植物的管腔表面光滑、有光泽且呈象牙色。内膜层覆盖着汇合的、鹅卵石样的单层细胞，这些细胞被因子VIIIB相关抗原阳性染色。植入后3个月，新生动脉壁厚度约为300微米。在中层组织中观察到少量平滑肌细胞，成纤维细胞在外膜组织中占主导。
循环内皮祖细胞可作为小直径血管假体内皮化的内皮细胞替代来源，以确保抗血栓形成。
