Graduate Student University of Cincinnati Lima, Ohio, United States
Introduction: Traumatic injury to the peripheral nervous system (PNS) currently lacks adequate solutions for full recovery. Nerve guidance conduits (NGCs) offer a promising solution; however, these are still severely limited in effectiveness for certain injuries. These limits stem from the lack of physical, biochemical, and electrical cues present in the conduit material. The extracellular matrix (ECM) acts as a guide for PNS cells and axons to direct cell function in regeneration. Our work seeks to enhance PNS regeneration by combining cell-derived ECM (dECM) with piezoelectric polyvinylidene fluoride-trifluorethylene (PVDF-TrFE) through a blend electrospinning process to create a multi-cue, biofunctionalized scaffold. Scaffolds were then evaluated in their ability to drive favorable cell and tissue response in a wound environment.
Materials and
Methods: PVDF-TrFE scaffolds were constructed from a polymer solution consisting of 20% PVDF-TrFE powder dissolved in (6:4) DMF-acetone. Scaffolds were biofunctionalized using bulk electrospinning where fibroblast-derived ECM was decellularized, lyophilized, digested, and thoroughly mixed into the precursor to form a homogenous solution. Scaffolds of varying dECM concentrations were blend-electrospun and characterized with scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS). Cell metabolic activity was measured upon early adhesion through an MTT colorimetric assay. Schwann cell morphology was analyzed through immunofluorescence imaging after being cultured on bioactive scaffolds for 3 and 24 h. Additionally, scaffolds were surgically implanted into rat models to analyze foreign body response at 7 and 28 days post implant (DPI) using H&E staining.
Results, Conclusions, and Discussions: Biofunctionalized scaffold groups possessed the following weight:volume concentrations of dECM: 0.2 %, 0.3 %, and 0.4 % dECM. 0 % dECM PVDF-TrFE scaffolds were used as controls. Metabolic activity tests at 3 h showed cells on all scaffold types present with similar metabolic levels; however, scaffolds with 0.3 % and 0.4 % dECM possessed slightly higher rates than their counterparts. Along with increased metabolic activity, SCs undergo a transdifferentiation process following injury where myelinating gene expression patterns in SCs downregulate as repair-related phenotypes are upregulated. Further, SC morphology changes to elongate, creating regeneration tracks necessary to guide bridging axons. These differences studied on dECM scaffolds showed primary SCs on 0 % and 0.4 % scaffolds presented with statistically significant elongated shapes along the direction of nanofibers (Fig. A, B). Alignment and cell shape was not as pronounced on 0.2 % and 0.3 % scaffolds. After a 24 h growth period, cells on all scaffold types displayed lengthened morphologies (Fig. C, D) and aligned in the direction of nanofibers with all aligned scaffolds statistically different than unaligned controls. At 24 h, 0.2 % and 0.3 % scaffolds possessed a greater alignment index at 24 h opposed to 3 h. We hypothesize the chemical signaling present due to the presence of morphogenic ECM in a biofunctionalized scaffold along with the stiffness changes caused by the biofunctionalization process is a fundamental component to promoting the differentiation of Schwann cell morphology towards a regenerative state. In subcutaneous testing in rat, H&E staining showed limited macrophage infiltration and foreign body reaction at 7 DPI. Whereas minor capsular formation around stitch sites were visible after 7 DPI, the notable absence of a capsule around our material is sign initial inflammatory reactions are diminished. At 28 DPI, slight inflammation presents around the material in addition to a thin, but less organized capsule formation, suggesting the dECM proteins incorporated into a PVDF-TrFE scaffold may mitigate unwanted capsular formation. Overall, the culminating in vitro and in vivo work represented here demonstrates this biofunctionalized scaffold can drive favorable tissue response in a wound environment.
