Chitosan Films Incorporating Cellulose Nanofibers or Carbon Nanotubes Differentially Modulate Early Responses of Stem Cells from Human Exfoliated Deciduous Teeth

Early stem cell-material interactions are critically governed by interfacial physicochemical cues. In this study, chitosan films incorporating cellulose nanofibers (CNFs) or carbon nanotubes (CNTs) were investigated as distinct nanomaterial strategies to modulate the morphology and viability of stem cells from human exfoliated deciduous teeth (SHED). The films were prepared by solvent casting followed by ionic crosslinking and characterized in terms of surface morphology, chemical structure, swelling behavior, and wettability. CNF-containing films exhibited a granular distribution of nanoscale surface features and reduced apparent wettability, whereas CNT-containing films displayed an irregular distribution of nanoscale surface features with greater topographical complexity. SHED responses were evaluated through qualitative morphological assessment, quantitative cell-spreading analysis, metabolic activity assays (Alamar Blue and MTT), and membrane integrity testing (Trypan Blue). CNF/Ch films restricted cell spreading and were associated with reduced metabolic activity and membrane integrity. In contrast, CNT/Ch films supported enhanced cell spreading, maintained high Alamar Blue-derived metabolic activity, and exhibited recovery of membrane integrity after 72 h. Collectively, these findings demonstrate that CNFs and CNTs impart distinct interfacial cues to chitosan films, leading to differential early SHED responses and identifying CNT/Ch as the most supportive formulation among those tested under the present conditions. This work underscores the importance of nanomaterial-driven surface features in guiding early stem cell-material interactions and provides insights for the design of chitosan-based substrates for regenerative medicine applications.