Department of Chemical and Biochemical Engineering, Faculty of Engineering, The University of Western Ontario, London, Ontario, Canada.
Tissue Eng Part A. 2011 Jun;17(11-12):1561-71. doi: 10.1089/ten.TEA.2010.0593. Epub 2011 Mar 30.
Due to the important structural and signaling roles of elastin in vascular stability, engineered human vascular tissues must incorporate elastin. However, despite considerable progress toward engineering of elastin-containing vascular tissues from animal cells, currently engineered vascular tissues using human cells largely lack elastin. In this study, we evaluated the effect of scaffold topography (two dimensional [2D] vs. three dimensional [3D]) on elastogenesis in adult human coronary artery smooth muscle cells (HCASMCs). We report that elastin gene expression by HCASMCs was increased by twofold after 4 days of culture in porous 3D polyurethane scaffolds. Transforming growth factor β1 (TGF-β1) further increased elastin gene expression in 3D cultures but not in 2D cultures. To evaluate if gene expression is translated into elastin synthesis, both 2D and 3D cultures were analyzed using Western blots. We show that only HCASMCs in 3D scaffolds produced elastin, suggesting that scaffold geometry itself is an important cue for elastogenesis. Moreover, TGF-β1 enhanced elastin synthesis in 3D, but had no effect on cells grown on 2D surfaces. TGF-β1, known to induce vascular smooth muscle cells (VSMC) differentiation, upregulated contractile VSMC marker proteins smooth muscle-α-actin and calponin in cells on 2D surfaces. Interestingly, in 3D scaffolds, TGF-β1 failed to upregulate these differentiation marker proteins for at least 7 days, but did so in cells cultured for 14 days, whereas elastin synthesis was not affected. To our knowledge this study is the first to successfully demonstrate that adult human VSMC can produce elastin when seeded on 3D scaffolds and to directly compare the effect of scaffold topography on elastin synthesis. Knowledge about the conditions required to regulate the phenotype of human VSMCs is paramount to engineer elastin-containing autologous human vascular substitutes.
由于弹性蛋白在血管稳定性方面具有重要的结构和信号作用，因此工程化的人血管组织必须包含弹性蛋白。然而，尽管在从动物细胞工程化包含弹性蛋白的血管组织方面取得了相当大的进展，但目前使用人细胞工程化的血管组织在很大程度上缺乏弹性蛋白。在这项研究中，我们评估了支架形貌（二维[2D]与三维[3D]）对成人冠状动脉平滑肌细胞（HCASMC）弹性蛋白生成的影响。我们报告说，HCASMC 在多孔 3D 聚氨酯支架中培养 4 天后，弹性蛋白基因表达增加了两倍。转化生长因子β1（TGF-β1）进一步增加了 3D 培养物中的弹性蛋白基因表达，但在 2D 培养物中没有增加。为了评估基因表达是否转化为弹性蛋白合成，我们使用 Western blot 分析了 2D 和 3D 培养物。我们表明，只有在 3D 支架中的 HCASMC 才会产生弹性蛋白，这表明支架几何形状本身是弹性蛋白生成的重要线索。此外，TGF-β1 增强了 3D 中的弹性蛋白合成，但对在 2D 表面生长的细胞没有影响。TGF-β1 已知可诱导血管平滑肌细胞（VSMC）分化，上调细胞在 2D 表面的收缩性 VSMC 标志物蛋白平滑肌-α-肌动蛋白和钙调蛋白。有趣的是，在 3D 支架中，TGF-β1 至少在 7 天内未能上调这些分化标志物蛋白，但在培养 14 天的细胞中上调了这些蛋白，而弹性蛋白合成不受影响。据我们所知，这项研究首次成功地证明了成年人类 VSMC 可以在 3D 支架上产生弹性蛋白，并直接比较支架形貌对弹性蛋白合成的影响。了解调节人 VSMC 表型所需的条件对于工程化包含弹性蛋白的自体人血管替代品至关重要。
