Introduction: Cellulose nanocrystals (CNCs) have been targeted as environmentally friendly dopants for thin films and bulk materials due to their biocompatibility, biodegradability, and high tensile strength[1]. Biocomposite films incorporating CNCs have been extensively studied; however, challenges remain in determining the mechanical properties of thin films in a humidity-independent manner. One route to access this information is to compressively buckle biocomposite films on a PDMS substrate, and then measure the periodicity of the resulting structures to extract a Young’s modulus. If a sufficiently large compressive stress is applied to a thin film, it undergoes wrinkling that results in micro/nanostructured surfaces, which are of interest due to their enhanced surface area and potential anti-microbial properties[2]. Here, we show that CNC-loaded biocomposite films formed through layer-by-layer (LbL) deposition can be irreversibly structured via compressive stress, which is generated by thermal shrinking of a shape-memory polymer (SMP). By characterizing the morphology of the structured surfaces, we determine the Young’s modulus of the CNC biocomposite films in a humidity-independent manner. We also show the application of the structured surfaces as platforms for the growth of murine RAW 264.7 cells.
Materials and Methods: Biocomposite films were fabricated on polystyrene (PS) substrates through LbL deposition of alternating layers of CNCs and xyloglucan (XG) or polyethyleneimine (PEI). Films were assessed using white light interferometry (WLIM) and variable angle spectroscopic ellipsometry (VASE), for surface roughness and thickness. LbL films were structured by heat shrinking of PS at 135 °C.[2] Structured films were assessed using WLIM, SEM, and 2D fast Fourier transform (2D FFT) analysis, for roughness, morphology, and periodicity. 2D FFT analyzed the edges of wrinkled films to determine their periodicity. To calculate the Young’s modulus, the film thickness was plotted against the structure wavelength. Unstructured and structured films were then used as substrates to grow murine RAW 264.7 cells. Following fixation, cells were imaged with epifluorescence microscopy by using the dyes 4′,6-diamidino-2-phenylindole (DAPI) and phalloidin-AlexaFluor 488 to stain the nucleus and actin filaments.
Results and Discussion: WLIM characterization of unstructured LbL films showed that despite changes in thickness, surface roughness was constant at 5.09 + 0.07 nm, while VASE analysis showed that film thickness increased linearly with the number of layers deposited onto the substrates. At the same number of layers, CNC-PEI films were thicker than CNC-XG films. After structuring, WLIM, SEM, and 2D FFT analysis showed that thicker films had higher roughness, larger structures, and higher wavelength values – this was also seen for CNC-PEI films compared to CNC-XG films, at the same number of layers. Young’s moduli indicated that CNC-XG films were stiffer than CNC-PEI films, with values of 39 + 1 GPa and 32 + 1 GPa. These biocomposite films were then used as a support for cell growth. Epifluorescence microscopy indicated that RAW 264.7 cells grew similarly on both unstructured and structured films, where they exhibited both round and “fried egg” morphologies.
Conclusion: We continue to explore this structuring approach as a reliable and humidity-independent way of determining the Young’s modulus of CNC biocomposite thin films. Thus far, we demonstrate that biocomposite structures incorporating CNCs can be fabricated on PS substrates in a reproducible and stable manner. The structured surfaces remain intact under varying conditions including elevated temperatures, changes in relative humidity, following lifting-off of the structured films from the PS substrate, and culturing of cells onto the structured films. Using these structures as a platform for cell growth is highly desirable due to the biocompatibility and anionic charge of CNCs, and thus, these structures can be further explored for potential applications in areas such as in vitro studies.
References:
[1] Kan, K. H., and Cranston, E. D. (2013) Mechanical testing of thin film nanocellulose composites using buckling mechanics. TAPPI J. 12, 9-17.
[2] Gabardo, C. M., Zhu, Y., Soleymani, L., and Moran‐Mirabal, J. M. (2013) Bench‐top fabrication of hierarchically structured high‐surface‐area electrodes. Adv. Funct. Mater. 23, 3030-3039.