Abstract
IntroductionSilk 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.MethodsThe 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.ResultsWe 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.ConclusionsOur 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.
Highlights
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)
In order to investigate whether SF patches could support attachment, proliferation and maintenance of the phenotypic profile of adipose tissue derived MSCs (Ad-MSCs), we seeded and cultured them on SF patches for seven days
Confocal light microscopy further demonstrated that the Ad-MSCs cultured on SF patches maintained their phenotypic profile and were positive for the mesenchymal markers CD146 (Figure 1E) and CD44 (Figure 1F) and for the integrin CD49d (Figure 1G)
Summary
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). Cell-based therapies are slowly gaining ground in routine medical care and, especially, in wound management of skin. They offer promise for the repair and/or replacement of damaged tissue and the restoration of lost functionality because they possess many of the criteria necessary for wound healing [2,3]. Reports increasingly suggest that multipotent mesenchymal stromal cells (MSCs), isolated from different tissue sources, confer benefits in vivo as tissue restorative agents. They act by either releasing trophic factors or through their multilineage differentiation properties [3,4]. Adipose tissue derived MSCs (Ad-MSCs) are currently under investigation to treat skin wounds [5] because of their abundance, easy isolation and in vitro expansion
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