Study On Electrospun PVA/Chitosan Hybrid Nanofibrous Scaffolds For Tissue-engineered Skin

Skin grafting is an effective way for the coverage of the wounds, which are caused by burn and trauma, et al. It can not only reduce the fluid loss and risk of infection, but also improve wound repair and healing. Many natural skin substitutes such as xenografts. allografts and autografts have been used for wound healing. However, these naturally derived skin substitutes cannot accomplish skin regeneration due to limited donor sites, risk of infection and immunologic rejection reaction. Dermal substitutes act as a promising application for clinical arena. The disadvantages of dermal substitutes are lack of vascular structures and susceptible to infection, moreover, the process of vascularization is too slow to assure nutrition of the overlying epidermis resulting in epidermal necrosis or graft loss. Therefore, a novel dermal substitute which could simulate the physical structure of the skin extracellular matrix possesses the abilities of faster vascularization and infectious resistance, which are vital for the treatment of large and deep burn wounds and skin defects.Many studies have demonstrated that electrospun polymer nanofibrous scaffolds structure can be designed to create ECM analogs composed of nanoscale fibers with mimicking structure and function to native ECM. Much attention has been drawn to the use of electrospun natural and synthetic nanofibrous scaffolds for biomedical fields. Thus, the electrospinning technique has become the dominant technology recently. In order to fabricate dermal substitutes with both antibacterial activity and fast revascularization abilities, an appropriate material should be concerned at first. Chitosan is a natural polymer which has many advantages including biocompatibility, biodegradability and antibacterial properties. Meanwhile, it can enhance the wound healing rate. However, there is still a great challenge to be electrospun alone in tissue engineering field. Polyvinyl alcohol (PVA) is introduced as a biocompatible and biodegradable polymer that has good fiber-forming properties. And, hence, nanofibrous chitosan and PVA hybrid scaffolds have been electrospun to meet the needs mentioned above.In this study, the PVA/chitosan hybrid nanofibrous scaffolds was fabricated in the following parameters of electrospinning process:A fixed electrical potential of 15 kilovolt high-voltage, the distance between the needle tip and the aluminum foil collector was 20cm, chitosan:PVA mass ratio was 1:500, the solution feeding rate was 0.1ml/h. Scanning electron microcopy (SEM) showed a smooth surface of the fiber and uniform of fiber diameter, which ranged between 200nm-300nm, and high porosity and high surface-to-volume ratio. However, whether the nanofibrous scaffolds could meet the requirements of tissue-engineered skin was not known yet, so we compared PVA/chitosan hybrid nanofibrous scaffolds with PVA nanofibrous scaffolds in three aspects of degradation behavior, antibacterial activity and the effect on regeneration in vivo and in vitro studies.(1) The degradation properties and cell compatibility of PVA and PVA/Chitosan were investigated through in vivo and in vitro studies. The samples were placed in phosphate-buffer saline (PBS). The bottles were incubated at 37.0℃for different amounts of times. The third day, the seventh day, and the fourteenth day after the samples were placed in the bottles, they were taken out from the solution, and rinsed by distilled water. The drying method of placing in room environment overnight was taken. Dry samples were sputtered with gold for observation of scaffold morphology by SEM. Meanwhile, each sample was grafted into rat subcutaneous tissue, to investigate the degradation behaviors and inflammation response in the surrounding tissues around the site of grafting at specified time intervals. On the 3rd day, 7th day,14th day and 28thafter the operation,6 rats were taken randomly from each group at each time point for sampling biopsy and performing H & E staining.(2) Antibacterial activity of PVA and PVA/chitosan hybrid nanofibrous scaffolds was investigated. Antibacterial test (Kirby-Bauer method) and bacteria penetration test were used to evaluate the antibacterial activity of the scaffolds. Samples were analyzed after the tests by SEM to observe the morphological changes of scaffolds.(3) The PVA/chitosan hybrid nanofibrous scaffolds and PVA nanofibrous scaffolds were wrapped outside the outer acellular dermal matrix to form a "sandwich" composite dermal substitutes. The pure swine acellular dermal matrix and two "sandwich" complex set of dermal substitutes were implanted subcutaneously into Wistar rats. On the 3rd day,7th day and 14th day after the operation,6 rats were taken randomly from each group at each time point for sampling biopsy and endothelial cell CD31 immunohistochemical staining in acellular dermal matrix. Masson's trichrome staining was performed on the PVA/chitosan hybrid nanofibrous scaffolds group to observe ingrowth of new collagen fibrils and matrix which may imply the process of new tissue replacement. The staining of vascular endothelial cells CD31 immunohistochemical staining was performed PVA nanofibrous scaffolds group and PVA/chitosan hybrid nanofiber scaffolds group to observe the formation of neovascularizations.Test Results and Conclusions:(1) Degradation in vitro the fibrous scaffolds changes in morphology of both groups were very significant. The appearance of the fibers changed from swelling to "dissolved" adhesion, and ultimately to "rupture" and disappearance. H & E staining of nanofibrous scaffolds after degradation showed that the structural integrity was not observed and it was difficult to identify the margins of the original nanofibrous scaffolds. Ingrowths of new collagen fibrils and matrix were observed between both groups nanofibers after 7 days of degradation. After 14 days of degradation, less discernable nanofibrous scaffolds and residual fragments of undegraded remained in surrounding tissue. And H & E staining showed no significant inflammatory cell infiltration in graft area and no formation of foreign-body giant cells.(2) Antibacterial activity:our result showed that PVA/chitosan hybrid nanofibrous scaffolds group has antibacterial activity for Staphylococcus aureus, Pseudomonas aeruginosa and E. coli. SEM showed that the uniform fiber profile was unclear, pores were uneven, the criss-cross three-dimensional structure of fibers could be observed, and microorganisms' debris was on the surface of scaffolds.(3) Regeneration:The vascular endothelial CD31 immunohistochemical staining result of the three groups showed that the number of vascular endothelial cells within swine acellular dermal matrix in PVA/chitosan hybrid nanofibrous scaffolds group were significantly more than the other two groups (P＜0.05). The neovascularizations of vascular endothelial CD31 immunohistochemical staining result showed that the PVA/chitosan hybrid nanofibrous scaffolds group has significant difference with the PVA nanofibrous scaffolds group (P<0.05). As Masson's trichrome staining result of PVA/chitosan nanofibrous scaffolds group showed, the ingrowths of cells was noted firstly, followed by the formation of new collagen fibrils and gradual degradation of the scaffolds, then the residual scaffolds'fragments were surrounded by a loose connective tissue, and eventually replaced by the neonatal tissue.Conclusion:PVA/chitosan hybrid nanofibrous scaffolds have good biocompatibility, appropriate biodegradation rate, excellent antibacterial activity and apparent ability to improve wound repair. This study provided useful help and great contribution to fabricate the real nano-dimensional tissue-engineered scaffolds which have infectious resistance and ability of neovascularization finally.