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Modulating stem cell-substrate Interactions and differentiation by controlling substrate topography via microphase separation

机译：通过微相分离控制基质形貌，从而调节干细胞-基质的相互作用和分化

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Introduction: Biomaterials used in regenerative medicine are required to direct the differentiation of human mesenchymal stem cells (hMSCs). Equally important is the maintenance of multipotency of hMSCs for clinical and Industrial use. In this work, we generated a range of surface textures by phase-separation (polymer demixing) for modulating stem cell-substrate interactions and hMSC differentiation. In combination with 3D printing, this process can be a powerful tool for fabricating scaffolds for tissue engineering. Materials and Methods: Surface patterns were fabricated by varying the ratio of two immiscible polymers poly(DTE carbonate) (PDTEC) and polystyrene (PS). Polymers were spin-coated onto coverslips to obtain phase-separated films. PS was selectively removed to obtain patterned PDTEC surfaces. Substrates were cultured with hMSCs in a 1:1 ratio of osteogenic and adipogenic induction media so as not to restrict lineage commitment. Cytoskeletal F-actin was fluorescently stained with Alexa conjugated Phalloidin, and phosphorylated focal adhesions (pFAK) were immunolabeled. High-resolution confocal images were used to analyze the morphological features and cytoskeletal anisotropy. Results and Discussion: Reproducible surface patterns (Fig. 1 A) were obtained at each of the PDTEC:PS compositions. There were three categories of surface patterns: flat (DO, D100), continuous (D40, D60), and discontinuous (D20, D80). The × in Dx refers to the wt% PDTEC. DO is the uncoated substrate, D100 is the uniform PDTEC film, D20 had islands and D80 had pits.The height of the features were ～7μm, their widths were ～16,48,68 and 151 μm in D20, D40, D60, and D80, respectively. Figure 1. (A) Bright field optical micrographs of the surface patterns with uniform chemistry, but varying topography. (B) Differentiation commitment of hMSCs on varying surface topographies exposed to mixed differentiation media for 14 days. (C) Quantification of cytoskeletal anisotropy of hMSCs at 72hrs in differentiation media. (D) Quantification of focal adhesion major-axis length. Results are given as mean ± s.e.; statistics by Tukey's ANOVA; * is p ＜ 0.05. hMSCs cultured in differentiation induction media were assayed at 14 days to assess lineage commitment. Continuous topographies favored hMSCs differentiation, whereas discontinuous topographies supported hMSC multipotency (Fig. 1B). There were no significant differences between DO and D100, indicating that observed changes in lineage commitment are due to topographic features, and not to surface chemistry. Examination of the hMSCs 72 hours post-seeding showed no significant changes in area or aspect ratio, indicating that cell morphology was not constrained by surface topography. Continuous features promoted cytoskeletal anisotropy (Fig. 1C). Cytoskeletal major-axis length measurements showed that continuous features promote the development of mature pFAK, and discontinuous features do not (Fig. 1D). These data indicate that the observed long-term differentiation profiles can be predicted from early changes in cytoskeletal and pFAK organization. Conclusions: Tunable substrate textures can be easily and reproducibly produced on surfaces of devices with complex shapes such as bone screws, plates and scaffolds via microphase separation. Discontinuous patterns promote multipotency in hMSC. Actin organization and pFAK maturity can be used as reliable predictors of sternness.
机译：简介：再生医学中使用的生物材料是指导人类间充质干细胞（hMSCs）分化的必需物质。同样重要的是保持临床和工业用途的hMSC的多能性。在这项工作中，我们通过相分离（聚合物分解）产生了一系列表面纹理，以调节干细胞-底物的相互作用和hMSC的分化。结合3D打印，此过程可以成为制造用于组织工程的支架的强大工具。材料和方法：通过改变两种不混溶的聚合物聚（碳酸DTE）和聚苯乙烯（PS）的比例制成表面图案。将聚合物旋涂在盖玻片上以获得相分离的膜。选择性地除去PS以获得图案化的PDTEC表面。用成骨和成脂诱导培养基的比例为1：1的hMSCs培养底物，以便不限制谱系承诺。用Alexa偶联的鬼笔环肽对细胞骨架的F-肌动蛋白进行荧光染色，并对磷酸化的粘着斑（pFAK）进行免疫标记。高分辨率共聚焦图像用于分析形态特征和细胞骨架各向异性。结果与讨论：在每种PDTEC：PS组合物上均获得了可重现的表面图案（图1A）。共有三类表面图案：平坦的（DO，D100），连续的（D40，D60）和不连续的（D20，D80）。 Dx中的×表示PDTEC的重量％。 DO是未涂层的基材，D100是均匀的PDTEC膜，D20有岛状物，D80有凹坑.D20，D40，D60和D20中特征的高度分别为〜7μm，宽度分别为〜16、48、68和151μm。 D80分别。图1.（A）具有均匀化学组成但形貌变化的表面图案的明场光学显微照片。 （B）hMSCs在暴露于混合分化培养基14天的不同表面形貌上的分化承诺。 （C）在分化培养基中在72小时时定量hMSC的细胞骨架各向异性。 （D）定量粘着长轴长度。结果以平均值±s.e.给出; Tukey ANOVA的统计数据; *是p ＜0.05。在第14天测定在分化诱导培养基中培养的hMSC，以评估谱系承诺。连续的地形有利于hMSCs的分化，而不连续的地形则支持hMSC的多能性（图1B）。 DO和D100之间没有显着差异，表明观察到的谱系承诺变化是由于地形特征引起的，而不是由于表面化学性质引起的。播种后72小时检查hMSCs，发现面积或长宽比无明显变化，表明细胞形态不受表面形貌的限制。连续特征促进了细胞骨架的各向异性（图1C）。细胞骨架长轴长度测量显示，连续的特征促进成熟的pFAK的发展，而不连续的特征则不能（图1D）。这些数据表明，可以从细胞骨架和pFAK组织的早期变化中预测观察到的长期分化情况。结论：可通过微相分离在具有复杂形状的设备（如骨螺钉，板和支架）的表面上轻松且可复制地产生可调整的基材纹理。不连续模式促进hMSC中的多能性。肌动蛋白的组织和pFAK的成熟度可以用作可靠的严厉预测指标。

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