Basic Study
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Stem Cells. Jul 26, 2015; 7(6): 956-975
Published online Jul 26, 2015. doi: 10.4252/wjsc.v7.i6.956
Evaluation of biodegradable electric conductive tube-guides and mesenchymal stem cells
Jorge Ribeiro, Tiago Pereira, Ana Rita Caseiro, Paulo Armada-da-Silva, Isabel Pires, Justina Prada, Irina Amorim, Sandra Amado, Miguel França, Carolina Gonçalves, Maria Ascensão Lopes, José Domingos Santos, Dina Morais Silva, Stefano Geuna, Ana Lúcia Luís, Ana Colette Maurício
Jorge Ribeiro, Tiago Pereira, Ana Rita Caseiro, Miguel França, Ana Lúcia Luís, Ana Colette Maurício, Departmento de Clínicas Veterinárias and +UPVet, Instituto de Ciências Biomédicas de Abel Salazar (ICBAS) and Centro de Estudos de Ciência Animal (CECA), Instituto de Ciências, Tecnologias e Agroambiente da Universidade do Porto (ICETA), Rua de Jorge Viterbo Ferreira, 4050-313 Porto, Portugal
Paulo Armada-da-Silva, Sandra Amado, Centro Interdisciplinar de Estudo de Performance Humana (CIPER), Faculdade de Motricidade Humana (FMH), Universidade de Lisboa (UL), Estrada da Costa, 1499-002 Cruz Quebrada, Dafundo, Portugal
Isabel Pires, Justina Prada, CECAV and Departamento de Ciências Veterinárias, Universidade de Trás-os-Montes e Alto Douro (UTAD), 5001-801 Vila Real, Portugal
Irina Amorim, Departmento de Patologia e de Imunologia Molecular, Instituto de Ciências Biomédicas de Abel Salazar, Rua de Jorge Viterbo Ferreira, 4050-313 Porto, Portugal
Irina Amorim, Instituto Português de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugal
Sandra Amado, UIS-IPL: Unidade de Investigação em Saúde da Escola Superior de Saúde de Leiria, Instituto Politécnico de Leiria, 2411-901 Leiria, Portugal
Carolina Gonçalves, Maria Ascensão Lopes, José Domingos Santos, CEMUC, Departamento de Engenharia Metalúrgica e Materiais, Faculdade de Engenharia da Universidade do Porto (FEUP), Rua Dr. Roberto Frias, 4200-465 Porto, Portugal
Dina Morais Silva, Biosckin, Molecular and Cell Therapies S.A. TecMaia, Rua Engenheiro Frederico Ulrich 2650, 4470-605 Maia, Portugal
Stefano Geuna, Neuroscience Institute of the Cavalieri Ottolenghi Foundation and Department of Clinical and Biological Sciences, University of Turin, 10124 Turin, Italy
Author contributions: Ribeiro J and Pereira T equally contributed; all authors contributed to this manuscript.
Supported by System of Incentives for Research and Technological development of QREN in the scope of project n° 38853/2013 - DEXGELERATION: “Soluções avançadas de regeneração óssea com base em hidrogéis de dextrin”; by the European Community FEDER fund through ON2 - O Novo Norte - North Portugal Regional Operational Program 2007-2013; by Project n° 34128 - BEPIM II: “Microdispositivos biomédicos com capacidade osteointegrativa por μPIM”; funded by AdI, and by the program COMPETE - Programa Operacional Factores de Competitividade, projects Pest-OE/AGR/UI0211/2011; PTDC/CVT/103081/2008; and CDRsp’s Strategic Project - UI-4044-2011-2012 (Pest-OE/EME/UI4044/2011) funding from FCT.
Institutional review board statement: The article describes a basic research study involving animal subjects and was reviewed and was approved by the Instituto de Ciências Biomédicas Abel Salazar (ICBAS) - University of Porto (UP) and CECA-ICETA scientific boards. All procedures involving the experimental animals were performed with the approval of the Veterinary Authorities of Portugal in accordance with the European Communities Council Directive of November 1986 (86/609/EEC), and the NIH guidelines for the care and use of laboratory animals have been observed.
Institutional animal care and use committee statement: The article describes a basic research study involving animal subjects and was approved by the Veterinary Authorities of Portugal in accordance with the European Communities Council Directive of November 1986 (86/609/EEC), and the NIH guidelines for the care and use of laboratory animals have been observed. Also all the authors involved in the in vivo tasks have a degree in Veterinary Medicine and are accredited by the Veterinary Authorities of Portugal (Direcção Geral de Alimentação e Veterinária - DGAV) and by Felasa - Category C for working with laboratory animals. Humane end points were always followed in accordance to the OECD Guidance Document on the Recognition, Assessment and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation (2000). Adequate measures were taken to minimize pain and discomfort taking into account human endpoints for animal suffering and distress. Animals were housed for two weeks before entering the experiment.
Conflict-of-interest statement: The authors state that they do not have any conflict of interests, including commercial, personal, political, intellectual, or religious interests that are related to the work submitted for consideration of publication.
Data sharing statement: This work is original in that it has not been published before or submitted for publication elsewhere, and will not be submitted elsewhere before a decision has been taken as to its acceptability in this Journal where you are Editor. Each author meets the criteria for authorship and assumes the corresponding responsibility. Technical appendix, statistical code, and dataset available from the corresponding author at Dryad repository, who will provide a permanent, citable and open-access home for the dataset is provided.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Ana Colette Maurício, Professor, Departmento de Clínicas Veterinárias and +UPVet, Instituto de Ciências Biomédicas de Abel Salazar (ICBAS) and Centro de Estudos de Ciência Animal (CECA), Instituto de Ciências, Tecnologias e Agroambiente da Universidade do Porto (ICETA), Rua de Jorge Viterbo Ferreira, n° 228, 4050-313 Porto, Portugal. ana.colette@hotmail.com
Telephone: +351-220-428000
Received: February 2, 2015
Peer-review started: February 2, 2015
First decision: March 6, 2015
Revised: April 17, 2015
Accepted: May 5, 2015
Article in press: May 6, 2015
Published online: July 26, 2015
Processing time: 180 Days and 12.9 Hours
Abstract

AIM: To study the therapeutic effect of three tube-guides with electrical conductivity associated to mesenchymal stem cells (MSCs) on neuro-muscular regeneration after neurotmesis.

METHODS: Rats with 10-mm gap nerve injury were tested using polyvinyl alcohol (PVA), PVA-carbon nanotubes (CNTs) and MSCs, and PVA-polypyrrole (PPy). The regenerated nerves and tibialis anterior muscles were processed for stereological studies after 20 wk. The functional recovery was assessed serially for gait biomechanical analysis, by extensor postural thrust, sciatic functional index and static sciatic functional index (SSI), and by withdrawal reflex latency (WRL). In vitro studies included cytocompatibility, flow cytometry, reverse transcriptase polymerase chain reaction and karyotype analysis of the MSCs. Histopathology of lung, liver, kidneys, and regional lymph nodes ensured the biomaterials biocompatibility.

RESULTS: SSI remained negative throughout and independently from treatment. Differences between treted groups in the severity of changes in WRL existed, showing a faster regeneration for PVA-CNTs-MSCs (P < 0.05). At toe-off, less acute ankle joint angles were seen for PVA-CNTs-MSCs group (P = 0.051) suggesting improved ankle muscles function during the push off phase of the gait cycle. In PVA-PPy and PVA-CNTs groups, there was a 25% and 42% increase of average fiber area and a 13% and 21% increase of the “minimal Feret’s diameter” respectively. Stereological analysis disclosed a significantly (P < 0.05) increased myelin thickness (M), ratio myelin thickness/axon diameter (M/d) and ratio axon diameter/fiber diameter (d/D; g-ratio) in PVA-CNT-MSCs group (P < 0.05).

CONCLUSION: Results revealed that treatment with MSCs and PVA-CNTs tube-guides induced better nerve fiber regeneration. Functional and kinematics analysis revealed positive synergistic effects brought by MSCs and PVA-CNTs. The PVA-CNTs and PVA-PPy are promising scaffolds with electric conductive properties, bio- and cytocompatible that might prevent the secondary neurogenic muscular atrophy by improving the reestablishment of the neuro-muscular junction.

Keywords: Nerve regeneration; Neurotmesis; Tube-guides; Mesenchymal stem cells; Carbon nanotubes; Functional assessment; Gait biomechanical analysis; Histomorfometry; Polyvinyl alcohol

Core tip: The rat sciatic injury neurotmesis injury model is an appropriate model to evaluate the nerve regeneration when electric conductive tube-guides of polyvinyl alcohol (PVA)-carbon nanotubes (CNTs) and PVA-polypyrrole (PPy) associated to mesenchymal stem cells (MSCs) are used as scaffolds. The results obtained revealed that treatment with MSCs associated to PVA-CNTs tube-guides induced an increased number of regenerated fibers and thickening of the myelin sheet. Functional and kinematics analysis revealed positive synergistic effects brought by MSCs and PVA-CNTs. The PVA-CNTs and PVA-PPy are promising scaffolds with electric conductive properties, bio- and cytocompatible that might prevent the secondary neurogenic muscular atrophy by improving the reestablishment of the neuro-muscular junction.