Gao Jing, Raghunathan Vijay Krishna, Reid Brian, Wei Dongguang, Diaz Rodney C, Russell Paul, Murphy Christopher J, Zhao Min
Department of Dermatology, School of Medicine, University of California Davis, Sacramento, CA 95817, USA; School of Life Science, Yunnan Normal University, Kunming, Yunnan 650500, People's Republic of China.
Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California Davis, Davis, CA 95616, USA.
Acta Biomater. 2015 Jan;12:102-112. doi: 10.1016/j.actbio.2014.10.007. Epub 2014 Oct 13.
Directed migration of corneal epithelial cells (CECs) is critical for maintenance of corneal homeostasis as well as wound healing. Soluble cytoactive factors and the intrinsic chemical attributes of the underlying extracellular matrix (ECM) participate in stimulating and directing migration. The central importance of the intrinsic biophysical attributes of the microenvironment of the cell in modulating an array of fundamental epithelial behaviors including migration has been widely documented. Among the best measures of these attributes are the intrinsic topography and stiffness of the ECM and electric fields (EFs). How cells integrate these multiple simultaneous inputs is not well understood. Here, we present a method that combines the use of (i) topographically patterned substrates (mean pore diameter 800nm) possessing features that approximate those found in the native corneal basement membrane; and (ii) EFs (0-150mVmm(-1)) mimicking those at corneal epithelial wounds that the cells experience in vivo. We found that topographic cues and EFs synergistically regulated directional migration of human CECs and that this was associated with upregulation of matrix metalloproteinase-3 (MMP3). MMP3 expression and activity were significantly elevated with 150mVmm(-1) applied-EF while MMP2/9 remained unaltered. MMP3 expression was elevated in cells cultured on patterned surfaces against planar surfaces. The highest single-cell migration rate was observed with 150mVmm(-1) applied EF on patterned and planar surfaces. When cultured as a confluent sheet, EFs induced collective cell migration on stochastically patterned surfaces compared with dissociated single-cell migration on planar surfaces. These results suggest significant interaction of biophysical cues in regulating cell behaviors and will help define design parameters for corneal prosthetics and help to better understand corneal wound healing.
角膜上皮细胞（CECs）的定向迁移对于维持角膜稳态以及伤口愈合至关重要。可溶性细胞活性因子和下层细胞外基质（ECM）的内在化学特性参与刺激和引导迁移。细胞微环境的内在生物物理特性在调节包括迁移在内的一系列基本上皮细胞行为中的核心重要性已得到广泛记录。这些特性的最佳衡量指标包括ECM的内在拓扑结构和硬度以及电场（EFs）。细胞如何整合这些多个同时输入的信息尚不清楚。在此，我们提出一种方法，该方法结合使用（i）具有与天然角膜基底膜中发现的特征近似的特征的地形图案化基质（平均孔径800nm）；以及（ii）模拟细胞在体内角膜上皮伤口处经历的EFs（0 - 150mVmm(-1)）。我们发现地形线索和EFs协同调节人CECs的定向迁移，并且这与基质金属蛋白酶-3（MMP3）的上调有关。施加150mVmm(-1)的EF时，MMP3的表达和活性显著升高，而MMP2/9保持不变。在图案化表面上培养的细胞与平面表面上培养的细胞相比，MMP3表达升高。在图案化和平面表面上施加150mVmm(-1)的EF时，观察到最高的单细胞迁移率。当作为汇合片层培养时，与平面表面上的解离单细胞迁移相比，EFs在随机图案化表面上诱导集体细胞迁移。这些结果表明生物物理线索在调节细胞行为方面存在显著相互作用，并将有助于确定角膜假体的设计参数，并有助于更好地理解角膜伤口愈合。
