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Acellular Grafts for Peripheral Nerve Repair

Acellular Grafts for Peripheral Nerve Repair. Research Update. Peripheral Nerve Injury. Injury can occur due to mechanical, chemical, thermal or pathological damage Accounts for 2.8% of total trauma injuries More than 200,000 surgical procedures annually in the United States

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Acellular Grafts for Peripheral Nerve Repair

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  1. Acellular Grafts for Peripheral Nerve Repair Research Update

  2. Peripheral Nerve Injury • Injury can occur due to mechanical, chemical, thermal or pathological damage • Accounts for 2.8% of total trauma injuries • More than 200,000 surgical procedures annually in the United States • Failure of treatment leads to loss of muscle function, impaired sensation, and painful neuropathy

  3. Essential Components for PNS Regeneration • Structural retention of nerve cable • Presence of ECM proteins • Collagen, Laminin, Fibronectin, Fibrin • Neurotrophic factors • NGF, BDNF, CNTF, GDNF • Regenerative cells like Schwann cells, fibroblasts and macrophages

  4. Treatment Options • Surgical reconnection of severed ends in case of small injuries (5- 10 mm transection) • Autologous grafts in case of larger injuries (>10mm) • Use of healthy nerve from other area • Limited graft availability • Functional loss at the donor site • Allografts and Xenografts • Immunosuppression required • Nerve Guide Conduits • Generally made of biocompatible and degradable polymers • Often functionalized with ECM proteins, growth factors, regenerative cells

  5. Acellular Grafts • Serve as a natural scaffold for regeneration • Consist of the structural proteins of native tissue • Reduced immunogenic response due to removal of cellular antigens • Amenable to functionalization with growth factors and regeneration promoting cells • Explored for repair blood vessels, skin and heart valves

  6. Decellularization Methods for Nerves • Thermal decellularization • Lead to cell removal and non-immunogenecity • Remnants of destroyed cells present • Fracture of basal lamina tubes • Radiation • Structural retention is observed • Unable to extract the cellular debris • Chemical Treatments • No structural preservation of ECM structure

  7. Chemical Decellularization: Sondell Method • Tissue treated with distilled water for 7 hours • Washed with Triton X -100 for 16hrs • Treated with sodium deoxycholate for 24 hrs • Detergent washes are repeated • Final wash in distilled water • Stored in phosphate buffer at 4oC

  8. Optimized Decellularization Method • Tissue washed in distilled water for 7 hours • Washed in 125 mM SB-10 in 10 mM phosphate buffer for 15 hours • Washed in buffer for 15 minutes • Treated with 0.6 mM SB-16 and 0.14% Triton X-200 for 24 hours • Washed in buffer three times for 15 minutes • Treated with SB-10 solution for 7 hours and washed with buffer • Subsequent wash with SB-16 and Triton X-200 for 15 hours • Final wash in 10 mM phosphate buffer three times for 15 minutes • Stored at 4oC before subsequent use

  9. Characterization of Decellularized Nerve Fresh Nerve Acellular Nerve Blue: DAPI Red: Schwann cell Green: NF

  10. Scanning Electron Microscopy Acellular Nerve Fresh Nerve Sondell Nerve

  11. Collagen Fibers in Acellular Nerve

  12. Functionalization with NGF • Important for maintenance, survival, and differentiation of sensory neurons • Present in peripheral nerve and levels increase after the injury • Several studies on the functionalization of NGCs with NGF and other neurotrophic factors • Functionalization of OA graft would combine topographical and biological cues

  13. Functionalization with NGF Acellular Nerve Fresh Nerve NGF is washed off in the decellularization process

  14. Functionalization with NGF Decellular nerve soaked in NGF solution of 50µg/ml at 4oC for 5 days Blue: DAPI Red: NGF First end of nerve

  15. Functionalization with NGF Middle of nerve Blue: DAPI Red: NGF Second end of nerve

  16. Future Directions • Structural Characterization • TEM • Mechanical Characterization • Tensile and compressive strength of decellular nerve and its comparison with fresh nerve • Biological Functionalization • Functionalization of decellular nerve with GDNF, CNTF and their combinations • Functionalization of decellular nerve with Schwann cells

  17. Future Directions • NGF release from the graft • Qualitative and quantitative characterization of release • Culture of functionalized nerves with DRGs to determine the regenerative potential • Crystal templated gels made from collagen, laminin and fibronectin and their characterization

  18. Acknowledgements • Prof Christine Schmidt • Dr Zin Khaing • Schmidt lab members

  19. Thank you !

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