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Functional and Structural Repair of Peripheral Nerve Injury by Adipose-Derived Stem Cells: An Experimental Study

Functional and Structural Repair of Peripheral Nerve Injury by Adipose-Derived Stem Cells: An Experimental Study. Hiroshi Mizuno, MD, A. Cagri Uysal, MD, Hakan Orbay, MD, Kyoko Kobe, MD, Hiko Hyakusoku, MD Department of Plastic and Reconstructive Surgery, Nippon Medical School

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Functional and Structural Repair of Peripheral Nerve Injury by Adipose-Derived Stem Cells: An Experimental Study

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  1. Functional and Structural Repair of Peripheral Nerve Injury by Adipose-Derived Stem Cells: An Experimental Study Hiroshi Mizuno, MD, A. Cagri Uysal, MD, Hakan Orbay, MD, Kyoko Kobe, MD, Hiko Hyakusoku, MD Department of Plastic and Reconstructive Surgery, Nippon Medical School Tokyo, Japan

  2. In human adults, peripheral nervous tissue is capable of healing and regeneration. INTRODUCTION Neural tissue must heal with true regeneration, because healing by scar will not re-establish electrical connectivity.

  3. INTRODUCTION Adipose derived stem cells(ASCs) were proven to have a positive effect on skin wound healing processes. Control ASCs 5mm Healing-impaired db/db mouse Nambu M et al, Ann Plast Surg 62; 317, 2009 100µm

  4. INTRODUCTION Clinical Treatment of Radiotherapy Tissue Damage by Adipose-Derived Adult Stem Cells Rigotti G et al, Plast Reconstr Surg, 119: 1409, 2007

  5. INTRODUCTION • Adipose derived stem cells might; • Control and increase the growth hormones and cytokines • Differentiate into specialised cells • Increase vascularity

  6. OBJECTIVE We have performed an experimental study on rats to find out the effect of adipose derived stem cells (ASCs) on primary peripheral nerve repair.

  7. MATERIALS AND METHODS DMEM+10% FBS 1st culture Adipose 2nd culture The ASCs were gathered from Fisher rat. 3rd culture After three passage in control medium (DMEM, 10% FBS), the cells were ready to be injected. ASCs Every injection included 1x107 cells.

  8. Labelling: Fate of the Stem Cells The cells were labelled with DiI and Hoechst staining before the injection for tracing. Every injection included 1x107 stem cells Hoechst Wavelength 310µm DiI Wavelength 560µm

  9. Six fisher rats Primary incision and nerve coaptation to sciatic nerve Over the coapted nerve Experimental: fibrin glue with ASCs Control: fibrin glue only MATERIALS AND METHODS

  10. MATERIALS AND METHODS

  11. MATERIALS AND METHODSEvaluation • Walking gate analysis (3rd and 6th months) • Sciatic function index (SFI) • Open electroneurography (ENG) (6th month) • Histology • Immunohistochemistry • DiI and Hoescht • Anti – S100 protein antibody • Anti – VEGF antibody • Electron microscopy

  12. MATERIALS AND METHODSWalking Gate Analysis

  13. MATERIALS AND METHODSSciatic Function Index Intermediary toe spread (ITS) Toe spread (TS) Print length (PL) SFI=118.9 x TSF - 51.2 x PLF - 7.5 TSF (toe spread factor) = (TS experimental – TS normal) / TS normal PLF (print lenghth factor ) = (PL experimental – PL normal) /PL normal Inserra et al, Microsurgery 1988

  14. RESULTS Sciatic function index (SFI) at 3rd month * p<0.05 *

  15. RESULTS Sciatic function index (SFI) at 6th month * p<0.05 *

  16. RESULTSElectroneurography (ENG) at 6th month

  17. RESULTSElectroneurography (ENG) at 6th month * * p<0.05 ** ** p<0.05

  18. RESULTSHistology DiI & hematoxylen HE Hoechst & hematoxylen Anti – S100& hematoxylen

  19. RESULTS Histology Double positive endothelial(e) cells in the epineurium(EP) EP EP e e e e DiI & hematoxylen x100 Hoescht & hematoxylen x100

  20. RESULTSImmunohistochemistry Anti - VEGF control ASCs * * p<0.05

  21. RESULTSElectron Microscopy Control ASCs x4000

  22. RESULTSElectron Microscopy Control ASCs x16000 * A myelinated fiber indicates the presence of a schwann cell. The myelinated vs total fiber percentage.(* p<0.05)

  23. CONCLUSIONS • ASCs improve primary nerve healing by; • Direct effects: • Differentiation into Schwann cell • Differentiation into endothelial cell • Indirect effects: • Growth factors • Fibroblast, macrophage • ASCs are much abundant and easy to harvest and use when compared to Schwann cell seeding. • ASCs might be helpful in clinical cases of peripheral nerve repair.

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