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

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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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slide1

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

slide2
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.

introduction
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

introduction4
INTRODUCTION

Clinical Treatment of Radiotherapy Tissue Damage by Adipose-Derived Adult Stem Cells

Rigotti G et al, Plast Reconstr Surg, 119: 1409, 2007

introduction5
INTRODUCTION
  • Adipose derived stem cells might;
    • Control and increase the growth hormones and cytokines
    • Differentiate into specialised cells
    • Increase vascularity
objective
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.

slide7

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.

slide8

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

slide9
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

slide11

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
slide13

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

slide14

RESULTS

Sciatic function index (SFI) at 3rd month

* p<0.05

*

slide15

RESULTS

Sciatic function index (SFI) at 6th month

* p<0.05

*

slide18

RESULTSHistology

DiI &

hematoxylen

HE

Hoechst &

hematoxylen

Anti – S100& hematoxylen

slide19

RESULTS Histology Double positive endothelial(e) cells in the epineurium(EP)

EP

EP

e

e

e

e

DiI & hematoxylen x100

Hoescht & hematoxylen x100

slide22

RESULTSElectron Microscopy

Control

ASCs

x16000

*

A myelinated fiber indicates the presence of a schwann cell.

The myelinated vs total fiber percentage.(* p<0.05)

slide23

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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