Introduction: We seek to develop a biomaterial surface with improved fibroblast cell attachment and alignment using polymeric wrinkles. Previously, shape memory polymers (SMP) have been used to form wrinkled coatings that align adipose stem cells in vitro[1] while polyelectrolyte multi-layers (PEMs) have been shown to improve fibroblast cell attachment on patterned surfaces[2]. Here, we combine these approaches by using an SMP to buckle PEM coatings to a desired wrinkled state. This new system allows study of cell alignment and motility dynamically with only a small change in temperature. We report here the effects of wrinkle wavelength and amplitude on surface properties and fibroblast cell-surface interactions.
Materials and Methods: An acrylic shape memory polymer was synthesized by UV-initiated polymerization and rectangular specimens were strained and fixed uniaxially. PEMs were then applied to the strained SMPs using sequential spin-coating of poly(sodium-4-styrenesulfonate) (PSS) and poly(allylamine hydrochloride) (PAH) from aqueous solutions. Next, the coated SMPs were thermally triggered to contract, wrinkling the PEM coatings. Wrinkle amplitude, wavelength, surface roughness and water contact angle were measured by conventional means. C3H10T1/2 mouse fibroblast cells were seeded at varying densities onto the wrinkled substrates along with flat controls (with and without PEM coatings). After 24 h of culture, cell nuclei and actin filaments were stained and imaged by fluorescence microscopy. Further, the density, orientation and orientation degree of nuclei relative to the wrinkle direction were determined using custom MATLAB software[3].
Results: Upon SMP recovery, PEMs buckled to form wrinkles with nano-sized amplitude and micro-sized wavelengths, which modified the hydrophobicity of the surface to a level compatible with cell attachment. Increasing PEM thickness led to strong increases in both wrinkle amplitude and wavelength (Figure 1).
Fibroblast cells attached more favorably on SMP surfaces with PEM, indicating positive correlation with surface hydrophobicity. After 24 h of culture, both cell nuclear density and degree of alignment in the direction of the wrinkle angle increased for all seeding densities (Figure 2; p < 0.05).
Discussion: These results show that PEM wrinkles can be prepared by SMP contraction and that fibroblasts attach and align well on SMP-PEM wrinkled topographies. As fibroblast cells are essential in wound-healing and move faster on wrinkled topographies[3], the present patterned surfaces may be useful to enhance directionality of fibroblast migration to close a wound.
Conclusion: We have introduced a new dynamic cell culture system that combines SMPs with PEMs. We observed that shape recovery of the SMP induces wrinkles and that the change in surface chemistry and topography synergistically improves cell attachment and induces cell alignment.
NSF IGERT; NSF GRFP; Syracuse Biomaterials Institute
References:
[1] P. Yang, R. Baker, J. H. Henderson, and P.T. Mather, In vitro wrinkle formation via shape memory dynamically aligns adherent cells, Soft Matter, 2013, 9, 4705-4714
[2] S. Kidambi, L. Sheng, M. L.Yarmush, M. Toner, I. Lee, and C. Chan, “Patterned co-culture of primary hepatocytes and fibroblasts using polyelectrolyte multilayer templates,” Macromolecular Bioscience, 2007, 13:8, 344-353
[3] R. Baker, M. E. Brasch, L.M. Manning, and J. H. Henderson, “Automated, contour-based tracking and analysis of cell behaviour over long time scales in environments of varying complexity and cell density,” The Royal Society Interface, 2014, 11, 1-13