Pins George D, Bush Katie A, Cunningham Lawrence P, Campagnola Paul J
Department of Biomedical Engineering, Worcester Polytechnic Institute, Worcester, Massachusetts 01609, USA.
J Biomed Mater Res A. 2006 Jul;78(1):194-204. doi: 10.1002/jbm.a.30680.
We use multiphoton excited (MPE) photochemistry to fabricate patterned extracellular matrices (ECM) and to investigate the morphology of human dermal fibroblasts adhered to the resulting photocrosslinked linear structures of fibronectin (FN), fibrinogen (FG), and bovine serum albumin (BSA). These proteins were chosen to systematically investigate the roles of topography and ECM biochemistry on cell spreading, as fibroblasts bind directly to both FN and FG at RGD sites through known integrins, whereas BSA provides no comparable ECM cues for cell binding. MPE crosslinked patterns are created from parallel linear structures 600 nm in width, 200 microm in length, and spaced by either 10 or 40 microm. Immunofluorescence staining of FN and FG was used to assay the functionality of crosslinked proteins. The metrics of orientation, elongation, and cell perimeter were used to quantitate the resulting cellular behavior on the crosslinked protein patterns. These parameters all reflect statistical differences for cells on BSA, relative to the similar statistical behavior on fibronectin and fibrinogen. Cells on the BSA patterns are constrained by physical guidance and orientation between linear structures. In contrast, cells adhered on both FN and FG had a greater propensity to spread across adjacent structures, indicating the importance of cell matrix interactions. Focal adhesion staining of cells adhered to the protein structures revealed similar trends. These findings are consistent with our hypothesis that these crosslinked matrix protein structures are expected to direct cell adhesion and spreading and that the topography and ECM cues lead to different forms of guidance.
我们利用多光子激发（MPE）光化学技术制备图案化的细胞外基质（ECM），并研究人类真皮成纤维细胞附着于纤连蛋白（FN）、纤维蛋白原（FG）和牛血清白蛋白（BSA）经光交联形成的线性结构后的形态。选择这些蛋白质是为了系统地研究拓扑结构和ECM生物化学对细胞铺展的作用，因为成纤维细胞通过已知的整合素在RGD位点直接与FN和FG结合，而BSA则不能为细胞结合提供类似的ECM信号。MPE交联图案由宽度为600 nm、长度为200 µm且间距为10或40 µm的平行线性结构构成。通过对FN和FG进行免疫荧光染色来检测交联蛋白的功能。使用取向、伸长率和细胞周长等指标来定量细胞在交联蛋白图案上产生的行为。相对于在纤连蛋白和纤维蛋白原上的类似统计行为，这些参数均反映了细胞在BSA上的统计差异。线性结构之间的物理引导和取向限制了细胞在BSA图案上的行为。相比之下，附着在FN和FG上的细胞更倾向于跨相邻结构铺展，这表明细胞-基质相互作用的重要性。对附着于蛋白质结构的细胞进行的粘着斑染色显示出类似的趋势。这些发现与我们的假设一致，即这些交联的基质蛋白结构有望指导细胞粘附和铺展，并且拓扑结构和ECM信号会导致不同形式的引导。
