Drewa Tomasz, Joachimiak Romana, Kaznica Anna, Wisńiewska-Skopińska Joanna, Sionkowska Alina, Sir Jan, Sarafian Victoria, Łysik-Miśkurska Joanna
Zakład Inzynierii Tkankowej, Katedra Biologii Medycznej, Uniwersytet im. M. Kopernika, Bydgoszcz, Polska.
Polim Med. 2008;38(2):33-42.
To develop a tissue-engineered bladder wall replacement with elements obtained from non-urinary tract components is an atractive idea. The aim of this study was to compare growth of hair follicles epithelial stem cells and mesenchymal stem cells to urothelial cells and fibroblasts cells on scaffold prepared from rat collagen type I.
Wistar rats were used in experiment. Rat urothelial cells, hair follicles epithelial stem cells, mesenchymal stem cells and 3T3 cells were cultivated in DMEM (Sigma) supplemented with 10% (or 20% for hair follicles cells) of Fetal Bovine Serum (FBS). Epithelial cell cultures were suplemented with EGF (10 ng/ml; Sigma). Cells were stained using anti-cytokeratine (Clone MMF) and anti-cytokeratine 7. Anti-CD34 and anti-p63 staining were done. Collagen scaffold was prepared from tendoms of Wistar rat's tails. 6-well plates were covered with collagen scaffold. 25 x 10(3) of cells were seeded on each well and cultured for a week. Cells in the controls were seeded on polystyrene surface. After a week cell viability was assessed using MTT test (Sigma). Each experiment was triplicated. Photo documentation was prepared. The differences between means were compared using t-Student test.
There were 106.5 +/- 23.4 x 10(3) and 310.7 +/- 60.7 x 10(3) of 3T3 fibroblasts growing on polystyrene and collagen, respectively (p < 0.05). The initial cell number was 25.0 x 10(3). Urothelial cells expressed epithelial markers. There were 40.0 +/- 4.2 x 10(3) and 4.5 +/- 1.8 x 10(3) urothelial cells growing on polystyrene and collagen, respectively after 7 days of culture (p < 0.01). There were 118.5 +/- 19.7 x 10(3) and 114.1 +/- 33.2 x 0(3) of mesenchymal stem cells growing on polystyrene and collagen, respectively (NS). Hair follicles epithelial cells expressed epithelial markers and were slightly positive for CD34 and p63. There were 292.5 +/- 33.3 x 10(3) and 167.4 +/- 24.9 x 10(3) of hair follicles epithelial cells growing on polystyrene and collagen, respectively (p < 0.05). Collagen scaffold decreased proliferation of follicle epithelial and urothelial cells.
Hair follicles epithelial stem cells and mesenchymal stem cells can be potentially used in tissue-engineering, with the guarantee of the sufficient cell number for transplantation. It seems that construction in vitro of urinary bladder walls from elements obtained from non-urinary tract tissues is feasible.
利用非泌尿道成分获得的元件开发组织工程膀胱壁替代物是一个有吸引力的想法。本研究的目的是比较毛囊上皮干细胞和间充质干细胞与尿路上皮细胞和成纤维细胞在由大鼠I型胶原制备的支架上的生长情况。
实验使用Wistar大鼠。大鼠尿路上皮细胞、毛囊上皮干细胞、间充质干细胞和3T3细胞在补充有10%（毛囊细胞为20%）胎牛血清（FBS）的DMEM（Sigma）中培养。上皮细胞培养物补充有表皮生长因子（10 ng/ml；Sigma）。使用抗细胞角蛋白（克隆MMF）和抗细胞角蛋白7对细胞进行染色。进行抗CD34和抗p63染色。胶原支架由Wistar大鼠尾巴的肌腱制备。6孔板用胶原支架覆盖。每孔接种25×10³个细胞并培养一周。对照组的细胞接种在聚苯乙烯表面。一周后使用MTT试验（Sigma）评估细胞活力。每个实验重复三次。制备照片记录。使用t检验比较均值之间的差异。
在聚苯乙烯和胶原上生长的3T3成纤维细胞分别为106.5±23.4×10³和310.7±60.7×10³（p<0.05）。初始细胞数为25.0×10³。尿路上皮细胞表达上皮标志物。培养7天后，在聚苯乙烯和胶原上生长的尿路上皮细胞分别为40.0±4.2×10³和4.5±1.8×10³（p<0.01）。在聚苯乙烯和胶原上生长的间充质干细胞分别为118.5±19.7×10³和114.1±33.2×10³（无显著性差异）。毛囊上皮细胞表达上皮标志物，并且对CD34和p63呈弱阳性。在聚苯乙烯和胶原上生长的毛囊上皮细胞分别为292.5±33.3×10³和167.4±24.9×10³（p<0.05）。胶原支架降低了毛囊上皮细胞和尿路上皮细胞的增殖。
毛囊上皮干细胞和间充质干细胞在组织工程中具有潜在应用价值，能保证有足够数量的细胞用于移植。似乎利用非泌尿道组织获得的元件在体外构建膀胱壁是可行的。
