Department of Urology, Urological Diseases Research Center, Boston Children's Hospital, Boston, Massachusetts, United States of America.
PLoS One. 2013;8(2):e56237. doi: 10.1371/journal.pone.0056237. Epub 2013 Feb 7.
Silk-based biomaterials in combination with extracellular matrix (ECM) coatings were assessed as templates for cell-seeded bladder tissue engineering approaches. Two structurally diverse groups of silk scaffolds were produced by a gel spinning process and consisted of either smooth, compact multi-laminates (Group 1) or rough, porous lamellar-like sheets (Group 2). Scaffolds alone or coated with collagen types I or IV or fibronectin were assessed independently for their ability to support attachment, proliferation, and differentiation of primary cell lines including human bladder smooth muscle cells (SMC) and urothelial cells as well as pluripotent cell populations, such as murine embryonic stem cells (ESC) and induced pluripotent stem (iPS) cells. AlamarBlue evaluations revealed that fibronectin-coated Group 2 scaffolds promoted the highest degree of primary SMC and urothelial cell attachment in comparison to uncoated Group 2 controls and all Group 1 scaffold variants. Real time RT-PCR and immunohistochemical (IHC) analyses demonstrated that both fibronectin-coated silk groups were permissive for SMC contractile differentiation as determined by significant upregulation of α-actin and SM22α mRNA and protein expression levels following TGFβ1 stimulation. Prominent expression of epithelial differentiation markers, cytokeratins, was observed in urothelial cells cultured on both control and fibronectin-coated groups following IHC analysis. Evaluation of silk matrices for ESC and iPS cell attachment by alamarBlue showed that fibronectin-coated Group 2 scaffolds promoted the highest levels in comparison to all other scaffold formulations. In addition, real time RT-PCR and IHC analyses showed that fibronectin-coated Group 2 scaffolds facilitated ESC and iPS cell differentiation toward both urothelial and smooth muscle lineages in response to all trans retinoic acid as assessed by induction of uroplakin and contractile gene and protein expression. These results demonstrate that silk scaffolds support primary and pluripotent cell responses pertinent to bladder tissue engineering and that scaffold morphology and fibronectin coatings influence these processes.
丝基生物材料与细胞外基质（ECM）涂层结合，被评估为用于细胞接种的膀胱组织工程方法的模板。通过凝胶纺丝工艺制备了两种结构不同的丝支架，分别由光滑、致密的多层（第 1 组）或粗糙、多孔的层状片（第 2 组）组成。单独的支架或涂覆有 I 型或 IV 型胶原蛋白或纤连蛋白的支架被独立评估，以评估其支持原代细胞系（包括人膀胱平滑肌细胞（SMC）和尿路上皮细胞）以及多能细胞系（如小鼠胚胎干细胞（ESC）和诱导多能干细胞（iPS）细胞）附着、增殖和分化的能力。AlamarBlue 评估显示，与未涂覆的第 2 组对照和所有第 1 组支架变体相比，纤连蛋白涂覆的第 2 组支架促进了原代 SMC 和尿路上皮细胞附着的最高程度。实时 RT-PCR 和免疫组织化学（IHC）分析表明，纤连蛋白涂覆的两组丝均允许 SMC 收缩分化，这是通过 TGFβ1 刺激后α-肌动蛋白和 SM22α mRNA 和蛋白表达水平的显著上调来确定的。IHC 分析显示，在对照和纤连蛋白涂覆组上培养的尿路上皮细胞中观察到上皮分化标志物细胞角蛋白的明显表达。通过 alamarBlue 评估 ESC 和 iPS 细胞在丝基质上的附着，结果显示纤连蛋白涂覆的第 2 组支架与所有其他支架配方相比，促进了最高水平的附着。此外，实时 RT-PCR 和 IHC 分析表明，纤连蛋白涂覆的第 2 组支架促进了 ESC 和 iPS 细胞向尿路上皮和平滑肌谱系分化，这是通过诱导尿路上皮蛋白和收缩基因和蛋白表达来评估的。这些结果表明，丝支架支持与膀胱组织工程相关的原代和多能细胞反应，并且支架形态和纤连蛋白涂层影响这些过程。
