Department of Developmental and Stem Cell Biology, The Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada.
J Biomed Mater Res A. 2010 Sep 15;94(4):1205-15. doi: 10.1002/jbm.a.32777.
Successful tissue engineering requires appropriate recellularization and vascularization. Herein, we assessed the regenerative and angiogenic effects of porcine bladder acellular matrix (ACM) incorporated with hyaluronic acid (HA) and vascular endothelial growth factor (VEGF) in mouse and porcine models. Prepared HA-ACMs were rehydrated in different concentrations of VEGF (1, 2, 3, 10, and 50 ng/g ACM). Grafts were implanted in mice peritoneum in situ for 1 week. Angiogenesis was quantified with CD31 and Factor VIII immunostaining using Simple PCI. Selected optimal VEGF concentration that induced maximum vascularization was then used in porcine bladder augmentation model. Implants were left in for 4 and 10 weeks. Three groups of six pigs each were implanted with ACM alone, HA-ACM, and HA-VEGF-ACM. Histological, immunohistochemical (Uroplakin III, alpha-SMA, Factor VIII), and immunofluorescence (CD31) analysis were performed to assess graft regenerative capacity and angiogenesis. In mouse model, statistically significant increase in microvascular density was demonstrated in the 2 ng/g ACM group. When this concentration was used in porcine model, recellularization increased significantly from weeks 4 to 10 in HA-VEGF-ACM, with progressive decrease in fibrosis. Significantly increased vascularization, coupled with increased urothelium and smooth muscle cell (SMC) regeneration, was observed in HA-VEGF grafts at week 10 in the center and periphery, compared with week 4. HA-VEGF grafts displayed highest in vivo epithelialization, neovascularization, and SMCs regeneration. A total of 2 ng/g tissue VEGF when incorporated with HA proved effective in stimulating robust graft recellularization and vascularization, coordinated with increased urothelial bladder development and SMC augmentation into bundles by week 10.
成功的组织工程需要适当的再细胞化和血管化。在此，我们评估了猪膀胱去细胞基质（ACM）与透明质酸（HA）和血管内皮生长因子（VEGF）结合在小鼠和猪模型中的再生和血管生成作用。制备的 HA-ACM 在不同浓度的 VEGF（1、2、3、10 和 50 ng/g ACM）中进行水合。将移植物原位植入小鼠腹膜中 1 周。使用 Simple PCI 通过 CD31 和因子 VIII 免疫染色来量化血管生成。然后选择诱导最大血管化的最佳 VEGF 浓度用于猪膀胱增强模型。植入物放置 4 周和 10 周。每组 6 只猪分别植入 ACM 单独、HA-ACM 和 HA-VEGF-ACM。进行组织学、免疫组织化学（尿路上皮蛋白 III、α-SMA、因子 VIII）和免疫荧光（CD31）分析，以评估移植物的再生能力和血管生成。在小鼠模型中，2 ng/g ACM 组的微血管密度显著增加。当该浓度用于猪模型时，在 HA-VEGF-ACM 中，从第 4 周到第 10 周，细胞再殖显著增加，纤维化逐渐减少。与第 4 周相比，在第 10 周，HA-VEGF 移植物的中心和外围观察到明显增加的血管化，同时伴随尿路上皮和平滑肌细胞（SMC）再生增加。在体内，HA-VEGF 移植物的上皮化、新生血管形成和 SMC 再生最高。当与 HA 结合时，总共 2ng/g 组织的 VEGF 被证明可以有效地刺激强大的移植物再细胞化和血管化，到第 10 周时，与增加的尿路上皮膀胱发育和 SMC 增加到束中协调一致。
