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


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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.


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INTRODUCTION healing and regeneration.

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


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INTRODUCTION healing and regeneration.

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

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


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INTRODUCTION healing and regeneration.

  • Adipose derived stem cells might;

    • Control and increase the growth hormones and cytokines

    • Differentiate into specialised cells

    • Increase vascularity


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OBJECTIVE healing and regeneration.

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


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MATERIALS AND METHODS healing and regeneration.

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.


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Labelling: Fate of the Stem Cells healing and regeneration.

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


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Six fisher rats healing and regeneration.

Primary incision and nerve coaptation to sciatic nerve

Over the coapted nerve

Experimental: fibrin glue with ASCs

Control: fibrin glue only

MATERIALS AND METHODS


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MATERIALS AND METHODS healing and regeneration.


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MATERIALS AND METHODS healing and regeneration. Evaluation

  • 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


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MATERIALS AND METHODS healing and regeneration. Walking Gate Analysis


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MATERIALS AND METHODS healing and regeneration. Sciatic 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


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RESULTS healing and regeneration.

Sciatic function index (SFI) at 3rd month

* p<0.05

*


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RESULTS healing and regeneration.

Sciatic function index (SFI) at 6th month

* p<0.05

*


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RESULTS healing and regeneration. Electroneurography (ENG) at 6th month


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RESULTS healing and regeneration. Electroneurography (ENG) at 6th month

*

* p<0.05

**

** p<0.05


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RESULTS healing and regeneration. Histology

DiI &

hematoxylen

HE

Hoechst &

hematoxylen

Anti – S100& hematoxylen


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RESULTS healing and regeneration. Histology Double positive endothelial(e) cells in the epineurium(EP)

EP

EP

e

e

e

e

DiI & hematoxylen x100

Hoescht & hematoxylen x100


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RESULTS healing and regeneration. Immunohistochemistry Anti - VEGF

control

ASCs

*

* p<0.05


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RESULTS healing and regeneration. Electron Microscopy

Control

ASCs

x4000


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RESULTS healing and regeneration. Electron Microscopy

Control

ASCs

x16000

*

A myelinated fiber indicates the presence of a schwann cell.

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


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CONCLUSIONS healing and regeneration.

  • 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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