Quaternary ammonium-functionalized carbon nanotubes/alginate nanocomposite hydrogels support myoblast growth and differentiation

Carbon nanotube (CNT) composite hydrogels are promising materials for tissue engineering due to the biocompatibility of the matrix and the electrical conductivity of the filler. However, specific chemical functionalization of CNT is required to disperse them homogeneously within an aqueous matrix. Therefore, we synthesized and characterized (through XPS, TGA, Raman, DLS and Zeta Potential) a water-soluble CNT derivative (fCNT) bearing quaternary ammonium groups, and we used it to prepare alginate-based nanocomposite hydrogels. We employed two different gelation techniques (external and internal) to obtain two plain hydrogels (HG1 and HG2 respectively) and two fCNT-filled composites (HG1-fCNT and HG2-fCNT respectively) and we compared the physical properties of the four different materials. Hydrogels were characterized through SEM microscopy and elastic modulus measurements. Moreover, an exhaustive investigation of the electrical properties of the hydrogel samples was carried on by cyclic voltammetry, chronoamperometry and electrochemical impedance spectroscopy in the range of 1-104 Hz. Notably, the addition of fCNT enhanced the conductivity of the hydrogel from internal gelation (HG2-fCNT) by more than one order of magnitude, thereby making it a promising candidate as a scaffold for electroactive tissues such as muscle. The observed features are even more appealing when considering active electrostimulation of cells. The stability of the scaffolds in the culture medium was improved by an enhanced calcium concentration. Finally, the behavior of the four hydrogels as scaffolds for muscle tissue engineering was investigated through comprehensive studies of myoblast viability, proliferation, and differentiation. While similar and satisfying behaviours were observed for all samples in terms of cytocompatibility and proliferation, a relevant improvement in differentiation was obtained by the addition of fCNT in the case of HG2-fCNT. These outcomes hint at the feasibility of using the fCNT combined with the alginate hydrogel in order to support the myoblasts growth and proliferation.