The Study On Effects And Mechanisms Of SF/CS Scaffold As Tissue Engineered Skin Substitutes On Wound Healing

?Objective?The aim of this study is to clarify whether the SF/CS scaffold meets the needs of tissue engineered artificial skin substitutes and clinical medical standards and whether it has a significant role in promoting wound healing,and to explore its underlying mechanisms.?Methods?Part I:In this section,different ratios of SF/CS composite scaffolds were constructed by freeze-drying method.Then,they were characterized by scanning electron microscopy(SEM),Fourier transform infrared spectroscopy(FITR),powder diffraction(XRD),swelling ratio,water stability,degradability,and mechanical test to screen out the proportion of scaffolds with the best physicochemical properties,the best stability,and the most suitable for cell growth,so as to meet the needs of skin tissue engineering.Part II:In this section,we selected several common biological methods such as cytotoxicity,systemic acute toxicity,skin sensitization,intradermal stimulation and hemolysis test according to “biological evaluation of medical devices(GB/T16886-ISO10993)”.And the biosafety of the composite scaffolds were evaluated to see if they met the medical standards of biomaterials,so as to satisfy the clinical application requirements of skin tissue engineering.Part III: In this part of the study,in order to verify whether SF/CS scaffolds can accelerate wound healing,we constructed a full-thickness skin wound model on mouse and performed statistical analysis on the wound area and skin tissue pathological sections at different time points to evaluate its repair effect.Then,we explore the mechanism by western blot and immunohistochemistry to detect the differential expression of related proteins.?Results?Part ?:1.There are no obvious differences in the appearance of the composite scaffold in each group.All samples are white,stable,solid,and have no special smell.2.5SF / 5CS and 4SF / 6CS scaffolds have obvious advantages over other groups in pore cross-linking degree and uniformity of pore size.Thus,they are more suitable for cell adhesion,migration and proliferation.3.5SF / 5CS and 4SF / 6CS scaffolds have the highest stability,suitable degradation and good water permeability.4.The compression modulus of 5SF / 5CS and 4SF / 6CS scaffolds are at an appropriate level,in line with the flexibility and tensile properties of artificial skin.Above all,we conclude that 5SF/5CS and 4SF/6CS scaffolds are more stable in three-dimensional structure and have better physicochemical properties,which is more suitable for the study of subsequent skin tissue engineering.Part ?:1.The 5SF / 5CS scaffold has better cytocompatibility than the 4SF / 6CS scaffold,it can promote more cell adhesion and growth.So we used 5SF/5CS scaffold in the follow-up studies.2.The extract of SF/CS scaffold had no toxic effect on cells and animals,so the SF/CS scaffold was non-toxic.3.SF / CS scaffold's extract had no skin sensitization,intradermal stimulation and hemolysis.In summary,we concluded that the SF/CS scaffold material has good cytocompatibility,qualified biosafety,meet the clinical application standards of biomaterials,and possess the possibility of clinical application.Part ?: 1.SF/CS scaffold can significantly accelerate wound healing and shorten the time of wound healing.2.Compared with the control group,the wounded skin of the SF/CS scaffold treatment group had thicker epidermis,more collagen deposition,and better wound repair.And there is less capillary congestion and inflammatory response.3.The expression of CD31,VEGF,COX-2,and TNF-? in the wounded skin of the SF/CS scaffold treatment group had no significant difference with the control group.However,the expression of p-Akt and Bcl-2 related to cell proliferation and apoptosis was significantly higher.Therefore,we inferred that SF/CS scaffold significantly accelerated wound healing may not by promoting angiogenesis,but may be related to promoting cell proliferation and inhibiting the expression of downstream pro-apoptotic signaling factors.?Conclusions?1.SF and CS can be mixed in different ratios to obtain a three-dimensional porous scaffold.When SF and CS are mixed in a ratio of 1:1,the structure of it is the most stable and the physicochemical properties are the most superior,which meets the needs of skin tissue engineering.2.The SF/CS scaffold has no toxicity,hemolysis and skin irritation response,while has good cytocompatibility,qualified biosafety,which meets the clinical application standards of biomaterials,and possess the possibility of clinical application.3.The SF/CS scaffold can effectively promote the repair of skin wounds in mouse,shorten the time of wound healing,and improve wound healing.The mechanism is mainly related to promoting the proliferation of skin-related cells,and inhibiting apoptosis.