Introduction: Biomaterials used in regenerative medicine are required to direct the differentiation of human mesenchymal stem cells (hMSCs)[1]. 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[2]. 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. 1A) were obtained at each of the PDTEC:PS compositions. There were three categories of surface patterns: flat (D0, D100), continuous (D40, D60), and discontinuous (D20, D80). The x in Dx refers to the wt% PDTEC. D0 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 D0 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[3] 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 stemness.
The National Resource for Polymeric Biomaterials funded by the National Institutes of Health (NIH grant EB001046)
References:
[1] Pittenger, M.F., et al., “Multilineage potential of adult human mesenchymal stem cells”, Science (1999) 284: 143-7.
[2] Dalby, M.J., Pasqui, D. and Affrossman, S., “Cell response to nano-islands produced by polymer demixing: a brief review”, IEEE Proc.-Nanobiotechnol. (2004) 151:53-61.
[3] Kim, Dong-Hwee, and Denis Wirtz. "Focal adhesion size uniquely predicts cell migration." The FASEB Journal (2013) 27: 1351-1361.