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Axonal Regeneration Across Long Peripheral Nerve Defects Using Acellular Nerve Allografts
Ying Yan, MD/PhD; Dan Hunter, RA; Susan E. Mackinnon, MD; Philip J. Johnson, PhD
Washington University in St. Louis, St. Louis, MO, USA
It has been demonstrated in both preclinical and clinical studies that the regenerative potential of conduits is limited to 30mm in humans. This distance has been defined as the conduit’s critical gap length and influences their clinical application. In contrast, the critical gap length for acellular nerve allografts (ANA) has not been defined. The current study was designed to explore the regenerative limits of ANAs, the mechanisms that limit regeneration in long graft ANAs, and to define the critical gap length beyond which they fail to support axonal regeneration. In a pilot study, we evaluated 40 mm isografts and ANAs 22 weeks after rat sciatic nerve transection and reconstruction. Histomorphometric analysis of 40mm ANA demonstrated significantly fewer nerve fibers (~5,000) compared to the isografts (~12,000). While the regeneration in ANAs was limited, axonal fibers presented across this gap length indicating that 40mm was not the regenerative limit and prompting us to extend our experimental gap length to 60mm. We have demonstrated the ability to increase the gap length of nerve defects up to 60 mm in the rat sciatic nerve by interposition of multiple long nerve grafts. Three variable isograft and ANA lengths of 20, 40, and 60 mm at two lengths of time (10 and 20 weeks) after graft placement were used to evaluate histomorphometric and muscle force recovery. To investigate the mechanism for limited the nerve regeneration in ANAs, expression of β-galactosidase, a marker of cellular senescence, was assayed in isografts and ANAs after transplantation. After 10 weeks, the histomorphometric and muscle force analysis of the isograft demonstrated a consistent decrease in the number of regenerating fibers and muscle force output with increased graft distance. The 10 week isograft data illustrates the significant increase in regenerative challenge posed by the 60mm defect. Expression of β-galactosidase in tissue of 40mm ANAs was found to be increased in the distal portion of graft when compared with 40mm isografts which exhibited limited expression. These results suggest that SCs in the distal portions of the 40 mm graft may have reached their proliferative limit and thus implicate limited SC proliferation as a likely mechanism underlying limited nerve regeneration in the long ANAs.
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