D16 - A Multifunctional Piezoelectric–Hydrogel Biomaterial for Peripheral Nerve Regeneration

Introduction: : The peripheral nervous system (PNS) is more susceptible to injury than the central nervous system (CNS) due to its limited protective barriers. Peripheral nerve injuries often result in partial or complete loss of sensory and motor functions in large gap injuries, and the intrinsic regenerative capacity of the PNS is typically insufficient for full recovery without surgical intervention. Although autografts remain the clinical gold standard, their use is constrained by donor site morbidity, limited availability, and the requirement for additional surgery. In the search for alternatives, nerve guidance conduits (NGCs) have emerged as promising tools to support and enhance nerve regeneration. Nevertheless, NGCs are still in their early stages of development, with one of the most critical challenges being their insufficient provision of the appropriate mechanical, biochemical, and electrical cues necessary for cellular proliferation, differentiation, and migration—fundamental processes in the construction of new tissue. In an effort to address this signaling insufficiency, we have developed a novel, multifunctional scaffold that integrates a porous hyaluronic acid (HA)-based hydrogel with a sheet of biofunctionalized piezoelectric polyvinylidene fluoride-trifluoroethylene (PVDF-TrFE) fibers, creating a dynamic microenvironment conducive to peripheral nerve repair.

Materials and

Methods: : The scaffold is composed of two layers. One layer is made of glycidyl methacrylate hyaluronic acid (GMHA) hydrogel with a methacrylation degree of approximately 16 percent. This hydrogel was fabricated by adding glycidyl methacrylate to the HA backbone in the presence of triethylamine, followed by UV treatment at a wavelength of ~365 nm. The second layer is made of PVDF-TrFE, which is a piezoelectric material, capable of producing electrical signals (useful for neural cell growth) in response to external mechanical forces. This PVDF-TrFE layer was biofunctionalized by incorporating different concentrations of decellularized extracellular matrix (dECM) derived from fibroblasts. For this, fibroblast-derived ECM was decellularized, lyophilized, digested, and mixed into the precursor solution of PVDF-TrFE to form the blend solution. This blend of PVDF-TrFE and dECM is electrospun and collected on a rotating drum to produce aligned fibers. The biofunctionalized (bioactive) piezoelectric layer was characterized using scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS). Primary Schwann Cells were cultured on the bioactive scaffold, and their morphology was analyzed through fluorescence imaging. DAPI and phalloidin were used to stain cell nuclei and F-actin filaments, respectively. Fourier transform analysis was used to evaluate the alignment of the cells on the scaffolds. Additionally, scaffolds were surgically implanted into rat models to analyze foreign body response at 7- and 28-day post-implant (DPI) using H&E staining.

Results, Conclusions, and Discussions:: Results and discussions
Our findings demonstrate enhanced cell alignment on the bioactive biomaterial. After a 24-hour growth period, cells on all scaffolds with various concentrations exhibited elongated morphologies and aligned along the direction of the fibers (Figure 1, A). At 24 hours, cells plated on the 0% and 0.4% scaffolds maintained their highly aligned structures. Fourier transform analysis showed higher alignment for the cells grown on scaffolds with 0.4 % dECM (Figure 1, B). Stiffness and porosity were within the optimal range for peripheral nerve tissue in the hydrogel portion of the scaffold. The addition of the hydrogel layer onto the bioactive scaffold showed cell infiltration throughout the scaffolds. H&E staining following surgical implantation revealed mild foreign body reaction at 7 DPI (Figure 2, A-C). The ratio of counted foreign body giant cells to the total number of nuclei cells near the scaffold showed a reduction from 0% to 0.4% dECM (Figure 2, D). By 28 DPI, vasculature and nerve growth were seen near bioactive scaffolds. These findings suggest that incorporating dECM proteins into the PVDF-TrFE scaffold may help reduce undesirable capsule development. The bi-layer scaffold introduced in this study demonstrates the potential of optimizing mechanical properties, electrical stimulation, and biochemical cues to promote peripheral nerve regeneration. It shows great promise in supporting cell migration, maintaining scaffold integrity under physiological forces, and presenting a superior alternative to current clinical approaches.
Conclusion
Within the proposed biomaterial, each layer contributes a distinct function to the scaffold. The hydrogel layer serves as a biodegradable base that supports cell growth and differentiation, while also functioning as a diffusive medium that facilitates the migration and distribution of cells and biochemical cues necessary for tissue formation. The second layer, composed of piezoelectric fibers, provides mechanical and electrical stimuli through its elongated, aligned morphology and electrical signal production, working in conjunction with biochemical signals derived from the dECM component. Taken together, the scaffold supported cell growth, and the bioactive piezoelectric scaffold showed limited inflammation in vivo and supported nerve and vascular growth near the scaffold.

Acknowledgements and/or References (Optional)::