Mesh-like electrospun membrane loaded with atorvastatin facilitates cutaneous wound healing by promoting the paracrine function of mesenchymal stem cells

Abstract

Background: Functional electrospun membranes are promising dressings for promoting wound healing. However, their microstructure and drug loading capacity need further improvements. It is the first time to design a novel mesh-like electrospun fiber loaded with atorvastatin (ATV) and investigated its effects on paracrine secretion by bone marrow-derived mesenchymal stem cells (BMSCs) and wound healing in vivo.

Methods: We fabricated a mesh-like electrospun membrane using a copper mesh receiver. The physical properties of the membranes were evaluated by SEM, FTIR spectroscopy, tensile strength analysis, and contrast angle test. Drug release was measured by plotting concentration as a function of time. We tested the effects of conditioned media (CM) derived from BMSCs on endothelial cell migration and angiogenesis. We used these BMSCs and performed RT-PCR and ELISA to evaluate the expressions of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF) genes and proteins, respectively. The involvement of FAK and AKT mechanotransduction pathways in the regulation of BMSC secretion by material surface topography was also investigated. Furthermore, we established a rat model of wound healing, applied ATV-loaded mesh-like membranes (PCL/MAT) seeded with BMSCs on wounds, and assessed their efficacy for promoting wound healing.

Results: FTIR spectroscopy revealed successful ATV loading in PCL/MAT. Compared with random electrospun fibers (PCL/R) and mesh-like electrospun fibers without drug load (PCL/M), PCL/MAT induced maximum promotion of human umbilical vein endothelial cell (HUVEC) migration. In the PCL/MAT group, the cell sheet scratches were nearly closed after 24 h. However, the cell sheet scratches remained open in other treatments at the same time point. The PCL/MAT promoted angiogenesis and led to the generation of longer tubes than the other treatments. Finally, the PCL/MAT induced maximum gene expression and protein secretion of VEGF and b-FGF. As for material surface topography effect on BMSCs, FAK and AKT signaling pathways were shown to participate in the modulation of MSC morphology and its paracrine function. In vivo, PCL/MAT seeded with BMSCs significantly accelerated healing and improved neovascularization and collagen reconstruction in the wound area compared to the other treatments.

Conclusions: The mesh-like topography of fibrous scaffolds combined with ATV release creates a unique microenvironment that promotes paracrine secretion of BMSCs, thereby accelerating wound healing. Hence, drug-loaded mesh-like electrospun membranes may be highly efficacious for wound healing and as artificial skin. It is a promising approach to solve the traumatic skin defect and accelerate recovery, which is essential to developing functional materials for future regenerative medicine.

Keywords: Atorvastatin; Bone marrow stem cells; Electrospun fibrous; Mesh-like topography; Paracrine secretion; Tissue engineering; Wound healing.

Fig. 1

The experimental designed to investigate the influence of the fiber morphology and atorvastatin (ATV) loaded in the fiber on the paracrine secretion function of BMSCs. The scaffolds for cell culture included electrospun fibers (EFs) in random, a mesh-like organization, and the mesh-like organization with atorvastatin loaded in, which were designated as PCL/R, PCL/M and PCL/MAT, and the cultures on EFs were compared to the BMSCs cultured on polystyrene microplate (MP). Then the PCL/MAT membranes seeded with BMSCs were used as the artificial skin in vivo wound healing

Fig. 2

a–c SEM images of PCL/R, PCL/M, and PCL/MAT, respectively. SEM micrographs of electrospun fibers at a magnification of 100X (scale bar = 500 µm) and a magnification of 5000X (scale bar = 10 µm). d FTIR spectra of ATV powder, PCL/M and PCL/MAT; e Water droplet in contact with PCL/R, PCL/M, and PCL/MAT. f The stress–strain curves, g Young's modulus and h tensile strength. **Indicates significant difference of p < 0.01. ***Indicates significant difference of p < 0.001

Fig. 3

Release of atorvastatin from PCL/MAT electrospun fibers after incubation in PBS at 37 °C

Fig. 4

a Cell proliferation examined by a CCK-8 assay after BMSCs were cultured on different membranes for 1, 3, and 5 days. b–d Fluorescence microscopy images of BMSCs cultured on the surfaces of different samples PCL/R, PCL/M, and PCL/MAT for 3 days, respectively. (Red: cytoskeleton stained with rhodamine phalloidin; blue: nuclei stained with DAPI). **p < 0.01. ***p < 0.001

Fig. 5

a Schematic diagram illustrated the effect of PCL/MAT scaffolds on the paracrine action of BMSCs and the experiment above. b The effects of the conditioned mediums (CMs) derived from BMSCs cultures on the HUVECs migration for up to 24 h and (d) HUVECs tube formation, c quantified results of b, e quantified results of the total tube length of d. f, g gene expression levels and secretion of b-fgf, vegf for different groups. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 6

a Representative Western blot images and semiquantitative analysis of FAK and AKT signaling pathway protein expression b in MSCs cultured on the two kinds of scaffolds and c in MSCs cultured on the mesh-like scaffold with FAK inhibitor. The activation of FAK and the downstream AKT pathways was observed in MSCs cultured on the mesh-like scaffold. d, e Inhibitory effects of FAK and AKT on paracrine factor expression in MSCs cultured on the mesh-like scaffold detected via RT-PCR analysis and ELISA test. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 7

a Images on the day of surgery show that the initial wound covered with different dressings and the posterior images of the wounds at different time points (7 and 14 days) after surgery were sequentially arranged. b, c Statistics of the wound area at different time periods (7 and 14 days), which was used to characterize the wound healing of Blank, Gauze dressing, PCL/MAT, Chitosan dressing, PCL/R + BMSCs, PCL/M + BMSCs, and PCL/MAT + BMSCs dressing. *p < 0.05, **p < 0.01

Fig. 8

a–g H&E staining images of different groups of wound areas and detailed images of H&E staining corresponding to each group. h–n Masson's stained images of wound areas in each group and detailed images of Masson's staining corresponding to each group. The red arrow indicates the blood vessel. Blank, Gauze dressing, PCL/MAT, Chitosan dressing, PCL/R + BMSCs, PCL/M + BMSCs, and PCL/MAT + BMSCs dressing, respectively

Fig. 9

a–g Immunofluorescence staining and h quantification of CD31 detected in wounds to evaluate angiogenesis. *p < 0.05; **p < 0.01; ***p < 0.001. Blank, Gauze dressing, PCL/MAT, Chitosan dressing, PCL/R + BMSCs, PCL/M + BMSCs, and PCL/MAT + BMSCs dressing, respectively

Fig. 10

Schematic diagram illustrating the effect of mesh-like structure signals combined with atorvastatin chemical signals on the paracrine action of BMSCs and the PCL/MAT-BMSCs membrane works as barrier protection on wound area