Schwann cell-laden PVDF-TrFE conduits for spinal cord repair

摘要

Introduction: The goal in this study was to develop a piezoelectric polyvinylidenefluoride-trifluoroethylene (PVDF-TrFE) conduit containing Schwann cells (SCs) for spinal cord repair. Piezoelectric materials can generate electrical activity with minute deformation. PVDF-TrFE aligned fibers facilitate axon extension by contact guidance in vitro'. This study first evaluated the conduit design where PVDF-TrFE aligned fibers were fabricated into hollow tubes or tubes filled with aligned fibers. Then, fiber filled conduits were either coated with or without Matrigel and loaded with SCs and implanted into a transected spinal cord. We hypothesized that the fibers within the filled tubes would facilitate SC survival without Matrigel and direct axon growth. Eliminating Matrigel, derived from mouse sarcoma cells, will make this approach more clinically relevant. Materials and Methods: Fabrication; PVDF-TrFE aligned fibers were fabricated via electrospinning. Conduits were formed as hollow tubes or tubes filled with aligned fibers (filled tubes) (Exp1). Filled tubes were pre-coated with or without Matrigel overnight and seeded with 3 × 106 GFP-SCs and incubated for 2h prior to transplantation (Exp2). See Table 1 for list of experimental groups. Transplantation: Laminectomy was performed on adult Fischer rats followed by complete transection at T8. The hollow (Exp1) or filled (Exp1&2) tubes were inserted between the cord stumps and GFP-SCs in DMEM with or without Matrigel were injected into the tubes. Tissue analysis: The rats were perfused at 3 (Exp1) or 6 (Exp2) wks; cryostat 20pm sagittal sections were stained with antibodies against GFP, GFAP (astrocyte), RT97 (general axon), and CGRP (Calcitonin gene-related peptide, sensory neuron). Table 1. Experimental groups of Exp 1 and 2. Results and Discussion: Exp1 (Fig 1): More SCs appeared to be in hollow than in filled tubes possibly due to (1) SC survival supported by the Matrigel and (2) less space within the lumen of filled tubes. RT97+ axons were associated with SCs in both tubes and along the rostral/caudal axis on the inner fibers, suggesting directed axon growth. CGRP+ sensory axons were observed only at the caudal host-SC interface in hollow tubes but were closely associated with SCs along the caudal/rostral axis in filled tubes. Fig 1. (Exp 1) Confocal fluorescent images of: SC bridges 3 wks post-transplantation in hollow (A) and filled (B) conduits (scale bar - 200μm); RT97+ axons (general axon marker) closely associated with transplanted GFP-SCs in the middle of the transplant in hollow (C) and filled (D) conduits (scale bar =100 μm). CGRP+ axons at the caudal host/SC interface in hollow (E) conduits and In between the layers at the caudal end of filled (F) conduits. Exp2 (Fig 2): The group without Matrigel (DD) appeared to have the fewest SCs and axons. SC survival appeared to be similar amongst the rest of the groups. More axons appeared to be regenerated into the SC bridges when Matrigel was injected with SCs during transplantation than with pre-coating (DM vs MD). Similar to Exp1, contact guidance of RT97+ and CGRP+ axons was observed among all the groups. Fig 2. (Exp 2) Confocal fluorescent images of SC bridges 6 weeks post-transplantation in groups MM (A), MD (B), DM (C), and DD (D) (scale bar = 200μm). Conclusion: This is the first study to investigate filled PVDF-TrFE tubes with SCs for spinal cord repair. The filled fibers enabled contact guidance for directed axon regeneration along the rostral/caudal axis regardless of the use of Matrigel but Matrigel injection enhanced the number of axons entering the fiber filled conduits and SC survival.