(628d) The Design and Fabrication of a Piezoelectric Biomaterial for Nerve Repair

Introduction. Severe peripheral nervous system (PNS) damage often leads to a prolonged cessation of full functionality. While traumatic injuries can be repaired with clinically accepted solutions such as nerve grafts, nerve guidance conduits, and neurorrhaphies, there is a distinct risk for complications such as inflammatory response, improper reinnervation, and donor site morbidity. Researchers are thus intrigued by the potential of alternative biomaterial-based therapeutics to induce regeneration of PNS tissue. Electrospun fibrous scaffold are commonly used to influence the repair of soft tissue and provide a practical supplement to existing technology. In particular, polyvinylidene fluoride - triflouroethylene (PVDF-TrFE) can be electrospun into a piezoelectric fibrous mat that creates a favorable regenerative microenvironment for PNS applications. Here we demonstrate that PVDF-TrFE scaffolds can be functionalized with extracellular matrix (ECM) and fabricated with precise mechanical, chemical, and electrical properties to influence the behavior of Schwann cells and fibroblasts, two cells that are essential to the native PNS repair cascade. Further, we demonstrate that the unique piezoelectric capacity of PVDF-TrFE can be modulated without the need for direct stimulation, thereby introducing a non-invasive mechanism of control that can readily translate to a clinical model.

Materials and Methods. 20% (w/v) PVDF-TrFE was dissolved in a (6:4) Dimethyl Formamide (DMF) – Acetone solution and subjected to electrospinning for 1 hour. Scaffolds were produced in an aligned direction with tunable porosity (Fig 1A). Decellularized ECM (dECM) was derived from a culture of NIH 3T3 fibroblast cells and lyophilized before enzymatic digestion in a 1 mg mL^-1 solution of pepsin in 0.1 M HCL. Scaffolds were then functionalized by immersing the digested dECM solution into the PVDF-TrFE precursor and electrospinning as normal. Schwann cells and fibroblasts were both seeded at densities of 75 cells mm^-2 and cultured over multiple time points. Immunoflouresence staining, western blot, scanning electron microscopy, and confocal microscopy were used to analyze changes in cell phenotype and relevant protein expression.

Results and Discussion. Scaffolds were fabricated with uniform alignment and an average fiber diameter of 1.0046 ± 0.038 µm and 76.33% porosity, allowing ample space for the attachment of cells. Piezoelectricity of the scaffolds was examined indirectly by measuring the crystallinity of PVDF-TrFE and observed an 88.49% β-phase configuration as derived from Fourier Transform Infrared Spectroscopy and X-Ray diffraction spectra. The piezoelectric capacity was further ensured by direct deformation and ultrasound (US) as monitored by an Axopatch 200B system. All scaffolds, both functionalized and non-functionalized scaffolds allowed for the adhesion, alignment, elongation, and proliferation of both cells (Fig 1B). Incorporation of dECM into the functionalized scaffolds augmented the adhesive and proliferative phenotypes of Schwann cells (Fig 1C,D) while additionally facilitating the expression of favorable regenerative markers such as c-jun, p75 NTR, NGF, and GDNF.

Conclusions. PVDF-TrFE scaffolds offer immense potential for peripheral nerve regeneration. Here we have designed a novel method for optimization of electrospun scaffolds with supplemented dECM modifications that augment the regenerative microenvironment. The unique piezoelectric stimulation and corresponding analysis likewise confer a distinct advantage in understanding the implementation of electroactive biomaterials for tissue engineering. The culmination of such findings provide an ample segue to an emphatically appealing clinical implementation model.

Figure 1. PVDF-TrFE can be electrospun into aligned scaffolds for nerve repair. (A) SEM image of aligned PVDF-TrFE scaffold without modifications. (B) Immunoflouresence image of Schwann cells with induced alignment from scaffold fibers. (C) Schwann cell densities quantified over multiple time points while cultured on scaffolds with functionalized Schwann cell and fibroblast co-culture derived dECM (d1:1, d1:3) and non-functionalized scaffolds (Control). (D) Immunoflouresence image of Schwann cells representing the same induced effect as quantified in (C).