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Open Access Research

Decellularized silk fibroin scaffold primed with adipose mesenchymal stromal cells improves wound healing in diabetic mice

Stefania Elena Navone18*, Luisa Pascucci2, Marta Dossena1, Anna Ferri1, Gloria Invernici1, Francesco Acerbi3, Silvia Cristini1, Gloria Bedini1, Valentina Tosetti1, Valentina Ceserani1, Arianna Bonomi4, Augusto Pessina4, Giuliano Freddi5, Antonio Alessandrino5, Piero Ceccarelli2, Rolando Campanella6, Giovanni Marfia7, Giulio Alessandri1 and Eugenio Agostino Parati1

Author Affiliations

1 The Cellular Neurobiology Laboratory, Cerebrovascular Diseases Unit, IRCCS Foundation Neurological Institute “C. Besta”, Milan, Italy

2 Department of Veterinary Medicine, University of Perugia, Perugia, Italy

3 Neurosurgery Department, IRCCS Foundation Neurological Institute “C. Besta”, Milan, Italy

4 Department of Public Health, Microbiology, Virology, University of Milan, Milan, Italy

5 Innovhub-SSI, Div. Stazione Sperimentale per la Seta, Milan, Italy

6 Neurosurgery Department, S. Carlo Borromeo Hospital, Milan, Italy

7 Laboratory of Experimental Neurosurgery and Cell Therapy, Neurosurgery Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico Milano, Milan, Italy

8 Current address: Laboratory of Experimental Neurosurgery and Cell Therapy, Neurosurgery Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico Milano, via Francesco Sforza, 28 20122 Milan, Italy

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Stem Cell Research & Therapy 2014, 5:7  doi:10.1186/scrt396

Published: 14 January 2014

Abstract

Introduction

Silk fibroin (SF) scaffolds have been shown to be a suitable substrate for tissue engineering and to improve tissue regeneration when cellularized with mesenchymal stromal cells (MSCs). We here demonstrate, for the first time, that electrospun nanofibrous SF patches cellularized with human adipose-derived MSCs (Ad-MSCs-SF), or decellularized (D-Ad-MSCs-SF), are effective in the treatment of skin wounds, improving skin regeneration in db/db diabetic mice.

Methods

The conformational and structural analyses of SF and D-Ad-MSCs-SF patches were performed by scanning electron microscopy, confocal microscopy, Fourier transform infrared spectroscopy and differential scanning calorimetry. Wounds were performed by a 5 mm punch biopsy tool on the mouse’s back. Ad-MSCs-SF and D-Ad-MSCs-SF patches were transplanted and the efficacy of treatments was assessed by measuring the wound closure area, by histological examination and by gene expression profile. We further investigated the in vitro angiogenic properties of Ad-MSCs-SF and D-Ad-MSCs-SF patches by affecting migration of human umbilical vein endothelial cells (HUVECs), keratinocytes (KCs) and dermal fibroblasts (DFs), through the aortic ring assay and, finally, by evaluating the release of angiogenic factors.

Results

We found that Ad-MSCs adhere and grow on SF, maintaining their phenotypic mesenchymal profile and differentiation capacity. Conformational and structural analyses on SF and D-Ad-MSCs-SF samples, showed that sterilization, decellularization, freezing and storing did not affect the SF structure. When grafted in wounds of diabetic mice, both Ad-MSCs-SF and D-Ad-MSCs-SF significantly improved tissue regeneration, reducing the wound area respectively by 40% and 35%, within three days, completing the process in around 10 days compared to 15–17 days of controls. RT2 gene profile analysis of the wounds treated with Ad-MSCs-SF and D-Ad-MSCs-SF showed an increment of genes involved in angiogenesis and matrix remodeling. Finally, Ad-MSCs-SF and D-Ad-MSCs-SF co-cultured with HUVECs, DFs and KCs, preferentially enhanced the HUVECs’ migration and the release of angiogenic factors stimulating microvessel outgrowth in the aortic ring assay.

Conclusions

Our results highlight for the first time that D-Ad-MSCs-SF patches are almost as effective as Ad-MSCs-SF patches in the treatment of diabetic wounds, acting through a complex mechanism that involves stimulation of angiogenesis. Our data suggest a potential use of D-Ad-MSCs-SF patches in chronic diabetic ulcers in humans.