Department of Urology, Nanjing First Hospital, Nanjing Medical University, Nanjing, China.
Department of Urology, Nanjing First Hospital, Nanjing Medical University, Nanjing, China.
Cytotherapy. 2023 Dec;25(12):1317-1330. doi: 10.1016/j.jcyt.2023.08.005. Epub 2023 Oct 6.
Cell failure and angiogenesis are the key to bladder wall regeneration. Three-dimensional (3D) culture using porous gelatin microspheres (GMs) as a vehicle promotes stem cell proliferation and improves the paracrine capacity of cells. This study aimed to evaluate the therapeutic potential of GMs constructed from adipose-derived mesenchymal stromal cells (ADSCs) (ADSC-GMs) combined with bladder acellular matrix (BAM) in tissue-engineered bladders.
Isolation of ADSCs, flow cytometry, scanning electron microscopy and cell counting kit-8, β-galactosidase and enzyme-linked immunosorbent assays were performed in vitro to compare two-dimensional (2D) and 3D cultures. In the in vivo study, male Sprague-Dawley rats were randomly divided into three groups: the BAM replacement alone (BAM) group, ADSCs grown on BAM in replacement (ADSC) group and ADSC-GMs combined with BAM followed by replacement (ADSC-GM) group. Bladder function assessed by urodynamics after 12 weeks of bladder replacement, and the rats were sacrificed at 4 and 12 weeks for further experiments.
The in vitro results showed that GM culture promoted ADSC proliferation, inhibited apoptosis and delayed senescence compared with those in the 2D culture. In addition, ADSC-GMs increased the secretion of the angiogenic factors vascular endothelial growth factor, platelet-derived growth factor-BB, and basal fibroblast growth factor. In vivo experiments revealed that ADSC-GMs adhered to the BAM for longer than ADSCs. Moreover, ADSC-GMs significantly promoted the regeneration of bladder vessels and smooth muscle, thereby facilitating the recovery of bladder function. The expression of phosphorylated protein kinase B (AKT) and phosphorylated endothelial nitric oxide synthase (eNOS) was significantly greater in the ADSC-GMs group compared with the BAM and ADSCs groups.
ADSC-GMs increased retention of ADSCs on the BAM, thereby promoting the regeneration and functional recovery of the bladder tissue. ADSC-GMs promoted angiogenesis by activating the AKT/eNOS pathway.
细胞衰竭和血管生成是膀胱壁再生的关键。使用多孔明胶微球（GMs）作为载体的三维（3D）培养可促进干细胞增殖并提高细胞的旁分泌能力。本研究旨在评估由脂肪间充质基质细胞（ADSCs）构建的 GMs（ADSC-GMs）与膀胱去细胞基质（BAM）结合在组织工程膀胱中的治疗潜力。
体外分离 ADSC，流式细胞术、扫描电子显微镜和细胞计数试剂盒-8、β-半乳糖苷酶和酶联免疫吸附试验比较二维（2D）和 3D 培养。在体内研究中，雄性 Sprague-Dawley 大鼠随机分为三组：BAM 单独替代组（BAM）、BAM 上生长的 ADSCs 替代组（ADSC）和 ADSC-GMs 结合 BAM 替代组（ADSC-GM）。12 周膀胱替代后通过尿动力学评估膀胱功能，4 周和 12 周时处死大鼠进行进一步实验。
体外结果表明，GM 培养促进 ADSC 增殖，抑制凋亡，延缓衰老，与 2D 培养相比。此外，ADSC-GMs 增加了血管内皮生长因子、血小板衍生生长因子-BB 和碱性成纤维细胞生长因子等血管生成因子的分泌。体内实验表明，ADSC-GMs 比 ADSCs 更能粘附在 BAM 上。此外，ADSC-GMs 显著促进了膀胱血管和平滑肌的再生，从而促进了膀胱功能的恢复。与 BAM 和 ADSCs 组相比，ADSC-GMs 组磷酸化蛋白激酶 B（AKT）和磷酸化内皮型一氧化氮合酶（eNOS）的表达明显更高。
ADSC-GMs 增加了 ADSCs 在 BAM 上的保留，从而促进了膀胱组织的再生和功能恢复。ADSC-GMs 通过激活 AKT/eNOS 通路促进血管生成。
