In this study, we demonstrated a simple and effective technique to construct multi-layer cell sheets using a natural electrospun gelatin/chitosan nanofibrous and ADSCs for accelerating wound healing. The architecture of electrospun nanofibrous made from natural biodegradable and biocompatible material (gelatin and chitosan) was similar to real tissue, which can satisfy physiological requirements. However, the strength properties of electro spun chitosan fibers are weak [19], and gelatin is sensitive to degradation [20, 21]. In this work, the blending of chitosan with gelatin can improve the mechanical properties of chitosan fibers, what’s more, chitosan is a hard electrospinning biomaterial, and gelatin can increase the spinnability of chitosan. The blend electro spun method also creates a new component without the need for synthesizing a new co-polymer. Therefore, we combined gelatin with different ratios of chitosan to fabricate electrospun gelatin/chitosan nanofibrous.
The advantages of the electrospinning technique include the production of very thin nanofibrous membranes with large specific growth surface, which allows for cell proliferation and functionalization. It is reported that both fiber diameter and pore size of electrospun nanofibrous membrane affect cell proliferation and differentiation [22]. The morphology of gelatin/chitosan nanofibrous clearly demonstrates that gelatin concentration was very important in obtaining fine and large gelatin/chitosan fibers, more gelatin in membrane exhibited much higher mechanical strength than the composite containing more chitosan. The mechanical property of skin is important to its structure, appearance and functionality [23], so the mechanical strength of electrospun nanofibrous is also important for the cell sheets construction and wound healing. However, electrospun nanofibrous of natural biomaterials usually possess inferior mechanical properties and fast degradation rates, crosslinking is a common approach for improving the mechanical and degradation properties of natural biomaterials for tissue engineering applications [24]. A crosslinking treatment of gelatin/chitosan nanofibrous with glutaraldehyde effectively optimizes mechanical properties and modifies biodegradation rate. Our crosslinked gelatin/chitosan (7:3) electrospun nanofibrous showed the highest mechanical strength because this fiber was uniform and had medium-size in diameter, both of which are important factors dictating the mechanical properties of electro spinning [25].
The property of nanofibrous biodegradation is beneficial for skin regeneration as it can support and regulate skin regeneration [20]. Gelatin absorbed an amount of water and made the nanofibrous more hydrophilic and sensitive to degradation, while more chitosan exhibited slower degradation, which may inhibit wound healing [26]. So the crosslinked electrospun gelatin/chitosan (7:3) nanofibrous with appropriate biodegradation may be more suitable for skin regeneration. Additionally, the degradation products of gelatin and chitosan are relatively non-toxic small molecules, which can easily be excreted directly or after entry and exit from various metabolic pathways [20, 27].
Natural biomaterials usually possess better biocompatibility and biofunctionality, we indicated that electrospun gelatin/chitosan nanofibrous had good biocompatibility with adipose-derived stem cells (ADSCs). ADSC is a promising adult stem cell for clinical therapy, because it can be easily isolated from adipose tissue, and has strong proliferation and differentiation ability [28]. The electrospun gelatin/chitosan nanofibrous (7:3) with appropriate mechanical strength and biodegradation can accelerate more ADSC adhesion and proliferation. Studies indicated that culture surface with higher stiffness favored cell adhesion and spreading [29, 30], and hydrophilic surfaces favored BMSC spreading [31], which are consistent with our observation. The electrospun gelatin/chitosan nanofibrous was shown to not be toxic at all compositions.
The electrospun gelatin/chitosan nanofibrous are positively charged, which can be a promising nonviral vector for gene transfection. Negatively charged molecules can be ionically bound to positively charged free amino groups of chitosan, which can protect molecules from the destruction caused by enzymes or other harmful factors [18]. Some researchers combined cationized gelatin with plasmid DNA and impregnated the complex into a collagen/polyglycolic acid scaffold to enhance the formation of engineered bone tissue [32]. Endogenous angiogenic factors, vascular endothelial growth factor (VEGF), are naturally produced in response to tissue hypoxia and during wound healing [33]. Because of inadequate secretion of VEGF, this restorative process is insufficient to prevent tissue ischemia and necrosis, exogenous delivery of VEGF is necessary for wound healing. After impregnation of plasmid DNA VEGF into electrospun gelatin/chitosan nanofibrous (7:3), the positively charged gelatin and chitosan firmly immobilize negatively charged plasmid DNA, the release of plasmid DNA was unlikely to be simple diffusion through the surface of nanofibrous, it will be completely released only when the nanofibrous is degraded to generate water-soluble fragments. The electrostatic interaction between the negatively charged cell membrane and the positively charged polymer attaches the gelatin/chitosan-plasmid complex to ADSCs, which promote the long-term expression of VEGF.
The electrospun gelatin/chitosan nanofibrous (7:3) with plasmid VEGF can promote ADSC proliferation and VEGF expression, so it could be a good supporting nanofibrous membrane for sandwich-like cell sheets construction. Cell sheet was established with a confluence layer of cell populations on a temperature responsive surface [10, 34]. Our previous study suggested that at least 5 days of culture was necessary for sufficient matrix deposition, and then the thick cell sheet was easily to be peeled off from culture surface with PVDF membrane [35]. Even so, it was hard to transplant intact cell sheets without PVDF membrane because they were too thin and fragile. Using biodegradable electrospun gelatin/chitosan nanofibrous as supporting membrane to replace of PVDF membrane, it was much easier to operate and form ADSC- nanofibrous -ADSC sandwich-like cell sheets, and transplant for wound healing.
A mouse dorsal wound healing model was used to investigate the skin regeneration potential of sandwich-like cell sheets. The whole wound healing process included cell adhesion, proliferation as well as migration, and eventually leads to skin regeneration [36]. Our in vivo skin defect models demonstrated that complete wound closure and re-epithelialization was only observed after implanting sandwich-like cell sheets, and the time of wound healing was faster than the case of no transplantation. In contrast, the treatment with electrospun gelatin/chitosan nanofibrous only had no obvious beneficial effect compared to the no-transplantation group with respect to wound closure, and both of them lack of a complete epithelial layer. It may be reasoned that the ADSC adding to electrospun gelatin/chitosan nanofibrous promoted its biomechanical features, and provided a better wound healing environment. So both ADSCs and nanofibrous were the key elements for wound healing and skin regeneration. Although wound closed almost at the same time after transplantation of cell sheets and cell sheets with plasmid VEGF, the wound skin transplanting of cell sheets with plasmid had thicker and complete epithelial layer than that of cell sheets only, which indicate that VEGF in the nanofibrous can promote wound healing and skin regeneration. Of course, it will have better repairing effects if we differentiated ADSCs into fibroblasts and epidermal cells before seeding on the nanofibrous, it will be our next work.