Schwann cells seeded in acellular nerve grafts improve functional recovery

Abstract

Introduction: This study evaluated whether Schwann cells (SCs) from different nerve sources transplanted into cold-preserved acellular nerve grafts (CP-ANGs) would improve functional regeneration compared with nerve isografts.

Methods: SCs isolated and expanded from motor and sensory branches of rat femoral and sciatic nerves were seeded into 14mm CP-ANGs. Growth factor expression, axonal regeneration, and functional recovery were evaluated in a 14-mm rat sciatic injury model and compared with isografts.

Results: At 14 days, motor or sensory-derived SCs increased expression of growth factors in CP-ANGs versus isografts. After 42 days, histomorphometric analysis found CP-ANGs with SCs and isografts had similar numbers of regenerating nerve fibers. At 84 days, muscle force generation was similar for CP-ANGs with SCs and isografts. SC source did not affect nerve fiber counts or muscle force generation.

Conclusions: SCs transplanted into CP-ANGs increase functional regeneration to isograft levels; however SC nerve source did not have an effect.

Keywords: cell transplantation; growth factor; nerve regeneration; peripheral nerve injury; reinnervation.

Figure 1. Sensory and motor-derived Schwann cells increase growth factor expression at 2 weeks

qRT-PCR was used to determine the gene expression level of each marker with the values normalized to β-actin. The groups injected with sensory and motor-derived SCs showed greater expression of all growth factors examined compared to the isograft and acellular CP-ANG groups. The mRNA fold difference was calculated versus the acellular CP-ANG. ** the dotted line at 2 is the threshold value for upregulation versus the CP group. Error bars represent the standard deviation (n = 3). * denotes P< 0.05 when compared to isograft, ^ denotes P< 0.05 when compared to sciatic, # denotes P< 0.05 when compared to motor, $ denotes P< 0.05 when compared to acellular CP-ANG.

Figure 2. SC gene expression patterns remain dysregulated 2 weeks after transplantation in CP-ANGs

qRT-PCR was used to determine the gene expression of each marker with the values normalized to β-actin. MBP, a sensory marker, showed increased expression in the motor group when compared to all other groups. PRKCi, a motor marker, showed increased expression in the sensory group. The mRNA fold difference was calculated versus the acellular CP-ANG. ** the dotted line at 2 is the threshold value for upregulation versus the CP group. Error bars represent the standard deviation (n = 3). * denotes P< 0.05 when compared to isograft, & denotes P< 0 05 when compared to sensory, ^ denotes P< 0.05 when compared to sciatic, # denotes P< 0.05 when compared to motor, $ denotes P< 0.05 when compared to acellular CP-ANG.

Figure 3. Histomorphometry on the midgraft of the regenerating nerve grafts 6 weeks post-transplantation

(A) The isograft group had a higher number of total myelinated nerve fibers compared to all other groups. (B) All groups had regenerated fibers of similar width. (C) A higher percentage of neural tissue was observed in the isograft group compared to all other groups. Error bars represent the standard deviation (n = 8). * denotes P<0.05 versus sensory and motor. ^ denotes P<0.05 versus sciatic, # denotes P < 0.05 versus CP.

Figure 4. Histomorphometry in the distal segment of nerve grafts 6 weeks post-transplantation

(A) The groups injected with sensory, motor, and sciatic nerve-derived SCs, showed nerve regeneration similar to the positive control (isograft group). These 4 groups had more total myelinated nerve fibers than the acellular CP-ANG (CP). (B) The isograft group regenerated thicker fibers (an indicator of fiber maturity) than the motor, sensory, and CP groups. (C) A higher percentage of neural tissue was observed in the isograft group compared to all other groups, but the groups with injected SCs (sensory, motor, and sciatic nerve-derived) all had more neural tissue than the CP group. Error bars represent the standard deviation (n = 8). * denotes P<0.05 versus sensory and motor. ^ denotes P<0.05 versus sciatic, # denotes P < 0.05 versus CP.

Figure 5. Light micrographs of nerve distal to the grafts 6 weeks post-transplantation

The isograft group shows more myelinated nerve fibers (white arrows) than the acellular CP-ANG (CP) group. By visual inspection, the groups injected with sciatic, motor, and sensory-derived SCs closely approximate the isograft, in contrast to the CP group. Scale bar = 30 μm.

Figure 6. Evoked muscle force measurements in EDL reveal similar functional recovery in distal musculature 12 weeks after implantation of CP-ANGs seeded with SCs

Specific force measurements demonstrate the positive effect of SC supplementation on the neuroregenerative capacity of CP-ANGs seeded with SCs. Specific tetanic force measurements normalized to EDL muscle mass demonstrate that isografts and CP-ANGs supplemented with either sensory or motor-derived SCs support increased reinnervation of distal musculature compared to acellular CP-ANGs (CP). Observation of normal tetanic responses in all EDL muscles innervated by repaired sciatic nerve confirms normal function of regenerated motor axons and corresponding motor units. Error bars represent the standard deviation (n = 6). * - denotes P < 0.05 versus isograft, # - denotes P < 0.05 versus CP.