A Detergent-Free Strategy for Preparing Acellular Nerve Allografts
Carolyn Clark, BA1, Megumi Ishii, MD1, Ting-Hsien Chuang, PhD2, Elisabeth Orozco, M.S.2, Ahmed S Suliman, MD1 and Sameer B. Shah, PhD2, 1University of California, San Diego, San Diego, CA, 2University of California, San Diego, La Jolla, CA
Introduction: Peripheral nerve injury remains an unsolved clinical challenge, with sensorimotor recovery especially poor for larger nerve gaps. To this date, autologous grafts (autografts) remain the gold standard for repairing transected nerves. Nerve allografts avoid several complications of autografts and have now been successfully applied in several clinical scenarios with promising efficacy. Despite this progress, there remains the potential for improvement. A potential shortcoming of several allograft protocols is the use of detergents, which ensure comprehensive breakdown and removal of cellular material, but also require long treatment times to remove residue and may destabilize graft durability. Detergent-free methods, on the other hand, may result in insufficient removal of cellular material, raising the likelihood of rejection by the host as well as physical and biological barriers to the pathfinding of regenerating axons.
Materials and Methods: We developed a new detergent-free approach for creating nerve allografts that avoids the above limitations. Rat sciatic nerves were subject to sequential cycles of sodium hydroxide treatment to disrupt cells and membranes; nucleases to destabilize nucleic acids; and agitation to clear debris. Preparation of acellular grafts was complete in less than 24 hours. Grafts were characterized using immunohistochemical approaches to evaluate clearance of cellular material and structural integrity; spectrophotometric methods to evaluate clearance of nucleic acids; and Schwann cell implantation into the graft to evaluate biocompatibility.
Results: Trichrome labeling and anti-laminin immunolabeling demonstrated preserved extracellular matrix and basal lamina structure. Spectrophotometry demonstrated nucleic acid reduction of over 90% compared to untreated controls. Schwann cells transfected with green fluorescent protein (GFP) were injected into the acellular grafts ex vivo, and repopulated the scaffold, demonstrating cyto-compatibility of the graft. Outcomes were superior or comparable to other published allograft fabrication approaches.
Conclusions: Our findings demonstrate the feasibility of generating biocompatible, detergent-free nerve allografts, with comparable cellular and nucleic acid clearance to other preparation methods as well as retained structural stability. Future directions will evaluate the use of these grafts in pre-clinical regenerative applications.
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