Skeletal muscle has a high capacity for self-regeneration, however, this is limited when there is a significant loss of tissue. The development of cell based constructs using biomaterials that can stimulate cell proliferation and differentiation, could support the bodyâs natural systems during regeneration. Cellulose nanowhiskers (CNWs) are rod like nanomaterials, typically 5 to 20 nm in diameter, and can range from 100âs of nanometres to several microns in length. The high aspect ratio of the CNWs could provide contact guidance suitable for directing cells of highly structural tissues, like skeletal muscle. This work explores the potential of multilayer substrates composed of alternating polyelectrolyte CNW and chitosan, with an oriented CNW top layer to promote the alignment and differentiation of myoblasts in to myotubes. Multilayer substrates were assembled through the dip coating of CNW and chitosan solutions on to glass coverslips. CNWs are produced through the partial acid hydrolysis of cellulose. The use of sulphuric acid results in a residual negative charge caused by the addition of sulphate ester groups on the whiskers. As a polyanion, CNWs can be layered with an oppositely charged polycation, chitosan, to form stable multilayer films. CNWs can then be spin coated on to the multilayer substrates forming a radial oriented nanotopography. Once formed, the capabilities of the substrates to promote cell growth and differentiation were investigated using C2C12s, an immortalised mouse myoblast cell line. The cells showed distinct signs of differentiation through the immunofluorescence staining of key myogenic components. Broad alignment of myotubes was observed on the oriented CNW multilayers whereas only local alignment formed on the substrates without the CNWs. Other cell types also showed a morphological cell response to these substrates, as human skeletal muscle cells (hSkMCs) and bone marrow derived mesenchymal stem cells (BM-MSCs) both showed alignment and elongation on the oriented CNWs. To further encourage the proliferation and differentiation of cells, key extracellular matrix (ECM) proteins, such as fibronectin and laminin, were adsorbed onto the substrates. Both proteins showed increased cell spreading and proliferation compared to untreated controls for C2C12 and BM-MSCs. These results are extremely promising for development of nanotopographical substrates, and pave the way for more complex 3D tissue scaffolds to be developed.