The Ohio State University, Department of Biomedical Engineering, 270 Bevis Hall 1080 Carmack Rd., Columbus, OH 43210, USA.
Acta Biomater. 2013 Aug;9(8):7651-61. doi: 10.1016/j.actbio.2013.04.002. Epub 2013 Apr 17.
A three-dimensional (3-D) cell culture system that allows control of both substrate stiffness and integrin binding density was created and characterized. This system consisted of two self-assembling peptide (SAP) sequences that were mixed in different ratios to achieve the desired gel stiffness and adhesiveness. The specific peptides used were KFE ((acetyl)-FKFEFKFE-CONH2), which has previously been reported not to support cell adhesion or MVN formation, and KFE-RGD ((acetyl)-GRGDSP-GG-FKFEFKFE-CONH2), which is a similar sequence that incorporates the RGD integrin binding site. Storage modulus for these gels ranged from ∼60 to 6000Pa, depending on their composition and concentration. Atomic force microscopy revealed ECM-like fiber microarchitecture of gels consisting of both pure KFE and pure KFE-RGD as well as mixtures of the two peptides. This system was used to study the contributions of both matrix stiffness and adhesiveness on microvascular network (MVN) formation of endothelial cells and the morphology of human mesenchymal stem cells (hMSC). When endothelial cells were encapsulated within 3-D gel matrices without binding sites, little cell elongation and no network formation occurred, regardless of the stiffness. In contrast, matrices containing the RGD binding site facilitated robust MVN formation, and the extent of this MVN formation was inversely proportional to matrix stiffness. Compared with a matrix of the same stiffness with no binding sites, a matrix containing RGD-functionalized peptides resulted in a ∼2.5-fold increase in the average length of network structure, which was used as a quantitative measure of MVN formation. Matrices with hMSC facilitated an increased number and length of cellular projections at higher stiffness when RGD was present, but induced a round morphology at every stiffness when RGD was absent. Taken together, these results demonstrate the ability to control both substrate stiffness and binding site density within 3-D cell-populated gels and reveal an important role for both stiffness and adhesion on cellular behavior that is cell-type specific.
创建并描述了一种允许控制基底刚度和整合素结合密度的三维（3-D）细胞培养系统。该系统由两种自组装肽（SAP）序列以不同比例混合组成，以达到所需的凝胶刚度和粘附性。使用的特定肽是 KFE（（乙酰基）-FKFEFKFE-CONH2），先前报道它不支持细胞粘附或 MVN 形成，而 KFE-RGD（（乙酰基）-GRGDSP-GG-FKFEFKFE-CONH2）是一种类似的序列，它包含 RGD 整合素结合位点。这些凝胶的储能模量范围为 60 至 6000Pa，取决于其组成和浓度。原子力显微镜显示，由纯 KFE 和纯 KFE-RGD 以及两种肽的混合物组成的凝胶具有类似细胞外基质的纤维微观结构。该系统用于研究基质刚度和粘附性对内皮细胞微血管网络（MVN）形成和人骨髓间充质干细胞（hMSC）形态的影响。当内皮细胞被包裹在没有结合位点的 3-D 凝胶基质中时，无论基质的刚度如何，细胞的伸长都很少，并且没有形成网络。相比之下，含有 RGD 结合位点的基质促进了强大的 MVN 形成，并且这种 MVN 形成的程度与基质刚度成反比。与没有结合位点的相同刚度的基质相比，含有 RGD 功能化肽的基质导致网络结构的平均长度增加了约 2.5 倍，这被用作 MVN 形成的定量测量。当存在 RGD 时，基质中存在 hMSC 会增加细胞突起的数量和长度，而在不存在 RGD 时，会导致细胞呈现圆形形态。总之，这些结果表明能够在 3-D 细胞填充凝胶中控制基质刚度和结合位点密度，并揭示了刚度和粘附性对细胞行为的重要作用，这种作用具有细胞类型特异性。
