Nanostructured PCL/MWCNT Composite Scaffolds Promote Cell Adhesion and Mineralized Matrix Formation for Bone Regeneration: SHED and MC3T3 Evaluation

Abstract The development of biomimetic scaffolds capable of simultaneously supporting stem cell viability and enhancing osteogenic differentiation remains a major challenge in bone tissue engineering. This study evaluated the effects of porous polycaprolactone (PCL)/multiwalled carbon nanotube (MWCNT) composite scaffolds containing different MWCNT concentrations on cytocompatibility, cellular adhesion, morphology, and osteogenic mineralization using stem cells from human exfoliated deciduous teeth (SHED) and MC3T3-E1 preosteoblasts. Porous PCL scaffolds containing 0.5, 1.5, and 2.5 wt% MWCNTs were fabricated using a solvent casting–salt leaching technique. Scaffold cytocompatibility was assessed by Alamar Blue assays after 1, 3, and 7 days of culture. Cell attachment and morphology were examined by scanning electron microscopy (SEM). Osteogenic differentiation was evaluated by Alizarin Red S staining at 7, 14, and 21 days to quantify extracellular matrix mineralization under osteogenic medium. Statistical analyses of cytocompatibility were performed in triplicate using one-way analysis of variance followed by Tukey's post hoc test (p All PCL/MWCNT scaffolds exhibited excellent cytocompatibility above the ISO 10993-5 threshold. SEM demonstrated progressive cell spreading, extensive filopodia formation, and intimate cell–scaffold interactions, with more pronounced adhesion on MWCNT-containing scaffolds than on pristine PCL. Mineralization increased in a time-dependent manner in both SHED and MC3T3-E1 cultures. Among all formulations, the 1.5 wt% MWCNT scaffold consistently produced the greatest calcium-rich mineralized matrix, whereas higher MWCNT loading (2.5 wt%) did not provide additional osteogenic benefits, suggesting concentration-dependent optimization of scaffold bioactivity. Incorporation of MWCNTs significantly enhanced the biological performance of porous PCL scaffolds by promoting cell attachment and extracellular matrix mineralization while preserving cytocompatibility. The 1.5 wt% MWCNT formulation demonstrated the optimal balance between biocompatibility and osteogenic activity.