3D-Printed, Biomimetic, Conductive MXene-Microfiber Hyaluronic Acid Composite Scaffolds Enhance the Axonal Growth-Promoting Characteristics of Electrical Stimulation in a Microarchitecture-Dependent Manner

Neurotrauma is associated with losses in motor and sensory function and no effective reparative treatment exists. The complex pathophysiology of neurotrauma (including inflammation, scarring and poor neuronal regrowth) suggests that a multi-faceted approach may be required to direct effective repair. Electrical stimulation-based therapies exhibit multifunctional benefits (e.g. axonal growth, neuronal differentiation) and conductive biomaterials can enhance the delivery of electrical stimulation. However, existing conductive biomaterials suffer from significant trade-offs between neurocompatibility, poor processability, and limited conductivity. Ti₃C₂T_x titanium carbide nanosheets, known as MXenes, are a class of highly conductive (&gt;10⁷ S/m) biocompatible 2D nanomaterials. It was hypothesized that functionalizing a 3D-printed polycaprolactone (PCL) microfiber scaffold with MXene nanosheets would produce highly conductive MXene-PCL microfiber architectures whose tunable electroconductive properties could be used to enhance tissue engineering scaffolds for neurotrauma repair applications<br/><br/>Melt-electrowriting was used to 3D print orthogonal PCL microfiber architectures of varying fiber densities (Low, Medium and High) which were coated with a Ti₃C₂T_x MXene ink (1 mg/ml), resulting in composite MXene-PCL microfiber architectures. 2-probe conductivity measurements demonstrated that the bulk electrical conductivity of these microfibrous architectures could be varied in a controlled manner from approximately 0.081 ±0.053 S/m to 18.87 ±2.94 S/m - depending on the microfiber density and layer of MXene coating. The MXene microfiber architectures were filled with a previously developed biomimetic macroporous neurotrophic hyaluronic acid-collagen type-IV/fibronectin biomaterial, designed to mimic the stiffness and composition of neural tissues (Woods et al. 2022). Scanning electron microscopic analysis of the resulting MXene-HA composite scaffolds indicated a macroporous microstructure and uniaxial mechanical testing indicated the scaffolds exhibited biomimetic stiffnesses between approximately 587 Pa and 3.25 kPa, depending on MXene-PCL fiber density. Confocal microscopic analysis of immunolabelled neurons following 7 days of continuous electrical stimulation (200 mV/mm, 12 Hz) indicated that MXene-HA scaffolds enhanced the average maximum axonal length projected from neurons in a manner dependent on both the conductivity of the microfiber scaffolds (p&lt;0.05) and microfiber density (p&lt;0.05).<br/><br/>To assess the capacity of the MXene-HA scaffolds to deliver electrical stimulation within more complex multicellular systems, primary murine neural stem cells were cultured to form neurospheres containing mixed populations of neuronal and glial precursor cells. Confocal microscopic analysis of stimulated neurospheres indicated upregulation of βIII-tubulin, a key marker of neuronal differentiation, on the MXene-HA scaffolds compared to inert controls and “High” density MXene-HA architectures promoted enhanced axonal growth (approx. 2-fold increase) from the neurospheres compared to “Low” density MXene-HA scaffolds.<br/><br/>These results describe the development of highly tunable electroconductive scaffolds which combine the excellent electroconductivity of 2D MXene nanosheets with cutting-edge biomimetic tissue engineering scaffolds through microscale 3D printing. These composite MXene-HA scaffolds demonstrate that MXene-PCL microfiber architectures can enhance the delivery of electrical stimulation to neurons. Furthermore, the controlled spatial distribution of these conductive microfibers was shown to play a key role in directing the electrical stimulation. These biomimetic electroconductive MXene-microfiber hyaluronic acid composite scaffolds overcome existing challenges associated with the use of conductive biomaterials and hold significant clinical potential for neurotrauma repair.<br/>Reference:<br/>I. Woods et al., Adv. Healthcare Mater. 2022, 11, 2101663.