Novel nanofibrous membrane-supporting stem cell sheets for plasmid delivery and cell activation to accelerate wound healing

Abstract

The integration of biomaterials with cells for high overall performances is vitally important in tissue engineering, as scaffold-free cell sheet lacks enough mechanical performance and cell viability while cell-free scaffold possesses limited biological functions. In this study, we propose a new strategy to strengthen cell sheets and enhance cell activity for accelerating wound healing based on a novel sandwich structure of cell sheet-plasmid@membrane-cell sheet (CpMC). Specifically, the CpMC contains two adipose-derived stem cell (ADSC) sheets on outer surfaces and an electrospun gelatin/chitosan nanofibrous membrane (NFM) encapsulating vascular endothelial growth factor (VEGF) plasmids in between. The physicochemical properties of NFM including swelling, stiffness, strength, elasticity, and biodegradation can be tailored by simply adjusting the ratio between gelatin and chitosan to be 7:3 which is optimal for most effectively supporting ADSCs adhesion and proliferation. The swelling/biodegradation of NFM mediates the sustained release of encapsulated VEGF plasmids into adjacent ADSCs, and NFM assists VEGF plasmids to promote the differentiation of ADSCs into endothelial, epidermal, and fibroblast cells, in support of the neoangiogenesis and regeneration of cutaneous tissues within 2 weeks. The proposed membrane-supporting cell sheet strategy provides a new route to tissue engineering, and the developed CpMC demonstrates a high potential for clinical translation.

Keywords: cell sheet; chitosan; gelatin; stem cell; sustained release; tissue engineering; wound healing.

SCHEME 1

Schematic illustration of the fabrication of the sandwich structure of cell sheet‐plasmid@membrane‐cell sheet (CpMC) with electrospun gelatin/chitosan nanofibrous membrane (NFM) and two‐layer adipose‐derived stem cell (ADSC) sheets for wound healing (b), and the mechanisms for skin regeneration (a)

FIGURE 1

Characterization of electrospun nanofibrous membrane (NFM) with different gelatin/chitosan proportions. (a) Microstructure of different gelatin/chitosan NFM observed by scanning electron microscope (SEM). Scale bars: 20 μm. (b) Young's modulus of different gelatin/chitosan NFM before and after cross‐linking. (c) Water absorption ability of all the gelatin/chitosan NFM. (d) Degradation properties of all the gelatin/chitosan NFM

FIGURE 2

Adhesion and proliferation of adipose‐derived stem cells (ADSCs) on nanofibrous membrane (NFM) with different gelatin/chitosan proportions. (a) Spreading of ADSCs on different NFM after 3 days cultivation was observed by scanning electron microscope (SEM). Scale bars: 20 μm. (b) Morphology of ADSCs on different NFM after 14 days cultivation observed by confocal laser scanning microscope. Scale bars: 200 μm. (c) Cell proliferation on different NFM was tested by CCK‐8

FIGURE 3

Releasing and expression properties of plasmid from plasmid@nanofibrous membrane (NFM). (a) Ultrastructure of plasmid loaded NFM observed by scanning electron microscope (SEM). Scale bars: 5 μm. (b) The compositional distribution of plasmid@NFM was further investigated by energy dispersive X‐ray spectroscopy (EDX) mapping. (c) Release profile of plasmid from plasmid@NFM. (d) Spreading of adipose‐derived stem cells (ADSCs) on plasmid@NFM after one‐week cultivation observed by SEM. Scale bars: 20 μm. (e) Proliferation of ADSCs on plasmid@NFM after 10 days cultivation observed by Confocal Laser Scanning Microscope. Scale bars: 100 μm. (f) Construction and observation of cell sheet‐plasmid@membrane‐cell sheet (CpMC) by macroscopy, microscopy, and multiphoton microscopy. Scale bars: 100 μm

FIGURE 4

Differentiation of adipose‐derived stem cells (ADSCs) into endothelial‐like cells on plasmid@nanofibrous membrane (NFM) after 3 weeks cultivation. *p < 0.05, *p < 0.001 versus ADSC group. (a) Expression of endothelial specific marker CD31 in differentiated ADSCs tested by immunofluorescence staining; scale bar: 100 μm. (b) Expression of endothelial specific markers (vWF, CD31, and VE‐cadherin) examined by western blotting. (c) Endothelial specific genes detected by real‐time quantitative polymerase chain reaction (qRT‐PCR). (d) The concentration of NO released from differentiated ADSCs detected with DAF‐FM Diacetate. ADSC: ADSCs only (control group). NFM: ADSCs cultured on NFM. plasmid@NFM: ADSCs cultured on plasmid@NFM. HMEC, human microvascular endothelial cell as positive control

FIGURE 5

In vivo wound healing using nanofibrous membrane (NFM), plasmid@NFM, cell sheet‐membrane‐cell sheet (CMC) and cell sheet‐plasmid@membrane‐cell sheet (CpMC). (a) Images of wound beds healed using different membrane and cell sheets. (b) Histological appearance of wounds harvested on day of healing. Scale bars: 100 μm. (c) Masson's trichrome staining of wounds harvested on day of healing. Scale bars: 100 μm. (d) Changes in wound size among different group during healing. **p < 0.01. (e) Thickness of neo‐skin (epidermal and dermal layers) was measured according to histological appearance. **p < 0.01. (f) The mechanical property of neo‐skin compared with normal skin. **p < 0.01. Control: wound without transplantation; NFM: transplantation of NFM alone; plasmid@NFM: transplantation of plasmid@NFM; CMC: transplantation of CMC; CpMC: transplantation of CpMC

FIGURE 6

Angiogenesis examination of neo‐skins at the day of healing. (a) Immunofluorescent staining of CD‐31 (green) and α‐SMA (red) at the wound area after healing. Scale bar: 200 μm. Blue color indicates the nuclei. (b) Quantified data of average diameter of capillary at the wound area with different treatments. **p < 0.01. (c) Quantified data of capillary number at the wound area with different treatments. *p < 0.05, **p < 0.01. (d) Protein expressions (VEGF, CD31, α‐SMA) in the wound bed at the day of healing by western blot analysis. *p < 0.05, **p < 0.01