Department of Mechanical and Materials Engineering, University of Nebraska-Lincoln, Lincoln, NE 68588, USA.
Acta Biomater. 2013 Aug;9(8):7737-45. doi: 10.1016/j.actbio.2013.04.013. Epub 2013 Apr 12.
While the potential of nanofibers as tissue engineering scaffolds has been demonstrated, very little has been revealed as regards the molecular mechanism by which cells sense and respond to nanofibers. It was hypothesized that RhoA kinase (ROCK), one of the vital cell tension signaling cascades, plays a role in regulating cell alignment on nanofibers. To test this, unidirectionally aligned and randomly distributed nanofibers, both with an average diameter of ∼130nm, were fabricated with poly(l-lactic acid) (PLLA). A flat PLLA film was used as the control. Mesenchymal stem cells (MSCs, C3H10T1/2) displayed high fidelity in cell orientation along aligned nanofibers, and showed an increased cell spreading area on random nanofibers. Interestingly, cells cultured on aligned nanofibers displayed significantly greater ROCK expression relative to cells on a flat surface, as assessed by immunoblotting. To further test the role of ROCK, MSCs with ROCK small hairpin RNA (shRNA) were established. It is notable that, even when ROCK was stably knocked down via shRNA, cells could still display preferred orientation along aligned nanofibers. However, MSCs with shRNA-ROCK displayed a significantly decreased cell major axis length following aligned nanofibers compared with shRNA vector control, suggesting that ROCK may be involved in cell elongation on aligned nanofibers. Along with the reduction in cell length, cell area was decreased with ROCK silencing. These cell morphological changes induced by shRNA-ROCK were generally maintained on a flat surface and random nanofibers. A pharmacological ROCK inhibitor, Y-27632, produced results similar to those of shRNA-ROCK. The data on the role of ROCK in regulating cell alignment on nanofibers may provide a new mechanistic insight into nanofiber control of cells.
虽然纳米纤维作为组织工程支架的潜力已经得到了证明，但关于细胞如何感知和响应纳米纤维的分子机制却知之甚少。研究假设 RhoA 激酶（ROCK）是细胞张力信号级联反应的关键之一，在调节细胞在纳米纤维上的排列中发挥作用。为了验证这一点，用聚（L-乳酸）（PLLA）制备了定向排列和随机分布的纳米纤维，平均直径约为 130nm。使用 PLLA 平膜作为对照。间充质干细胞（MSCs，C3H10T1/2）在沿着定向纳米纤维的方向上表现出高度的细胞取向保真度，并且在随机纳米纤维上显示出增加的细胞扩展面积。有趣的是，通过免疫印迹评估，与在平面上的细胞相比，培养在定向纳米纤维上的细胞显示出显著更高的 ROCK 表达。为了进一步测试 ROCK 的作用，建立了 ROCK 短发夹 RNA（shRNA）的 MSCs。值得注意的是，即使通过 shRNA 稳定敲低 ROCK，细胞仍然可以沿着定向纳米纤维显示出优先取向。然而，与 shRNA 载体对照相比，具有 shRNA-ROCK 的 MSCs 在定向纳米纤维后表现出细胞长轴长度显著降低，表明 ROCK 可能参与细胞在定向纳米纤维上的伸长。随着细胞长度的降低，细胞面积也随着 ROCK 沉默而减小。shRNA-ROCK 诱导的这些细胞形态变化在平面和随机纳米纤维上基本保持不变。ROCK 药理学抑制剂 Y-27632 的结果与 shRNA-ROCK 的结果相似。关于 ROCK 在调节细胞在纳米纤维上排列中的作用的数据，可能为纳米纤维控制细胞的机制提供新的见解。
