Cellulose nanowhiskers for skeletal muscle engineering

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Abstract

Prior work has shown that spin-coating tunicin cellulose nanowhiskers onto a glass surface creates a highly oriented surface that supports the adhesion, spreading and proliferation of myotubes.Building on this work, this project aimed to develop culture surfaces with biologically active topography and tuneable stiffness with the aim of better mimicking native muscle tissue. The ultimate aim is to develop biomaterials that can direct the differentiation of mesenchymal stem cells. Cellulose nanocrystals (CNWs) from Ascidiella spp were isolated and characterised. Polyelectrolyte multilayers (PEMs) are nanocomposite films formed from the sequential deposition of oppositely charged polymers and offer a flexible method of building films with a variety of chemical compositions and physical properties. CNWs were used in combination with chitosan to create PEMs using a combination of two well-established, low-cost and facile production methods, dip-coating and spin-coating. The resulting PEM was shown to be a nanoporous substrate that was stable under cell culture conditions. It robustly allowed the attachment, alignment and myogenic differentiation of the immortalised C2C12 myoblast cell line. Proteomic analysis of the ECM produced by C2C12 cells in response to the substrate showed that cells cultured on CNW-chitosan PEMs secreted increased fibronectin, tenascin-c, elastins and collagen I, an expression pattern that is consistent with a more developmental, rather than mature, muscle ECM. The thickness and mechanical stiffness of the PEM films could be tuned by replacing replacing increasing volume fractions of CNWs with poly(4-sodium styrene sulfonate) (PSS). The thickness of the dry films increased with increasing CNW content, increasing from 20 nm for films containing 12 bilayers of PSS and chitosan to 100 nm for films containing 12 bilayers of CNW and chitosan. The compressive stiffness of hydrated films decreased with increasing CNW content, from 1.67 ± 0.73 MPa, to 1.06 ± 0.24 MPa. Unfortunately, PSS-modified PEMs proved to be cytotoxic to cells.The response of bone marrow stem cells to the substrates showed that mesenchymal stem cells were contact guided by the CNWs, but did so by avoiding the material, thus being better guided by substrates where CNWs were present at a low surface density than substrates where it was present at a high density. When cultured directly on PEMs, MSCs expressed myogenin, a key marker of terminal muscle differentiation, which was suggestive, but not definitive, of a potential of the biomaterial to direct the myogenic differentiation of MSCs.

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