| Currently,traditional autologous split skin grafts(SSGs)are considered as a significant clinical treatment for serious full-thickness skin defects.However,several problems are still great challenges,such as limitation of available donor skin,secondary surgery damage,etc.In addition,burn wounds and severe traumatic wounds are prone to cause tissue infection and necrosis during their healing process,which are liable to induce scar contracture and in turn cause local dysfunction.Generally,engineering skin substitutes represent a prospective source of advanced therapy in repairing severe traumatic wounds.At present,many researches are focused on the scaffolds as skin substitutes because they possess microporous structure and template support effect.However,there are some disadvantages in the scaffold,for example,the poor mechanics performance leading to pore structure is easy to collapse in the process of curing the defects.In this study,a steady biomimetic scaffold was prepared using biocompatible silk fibroin(SF)and permeable sodium alginate(SA)by freeze-drying recombination technology,in which asymmetric bilayer structure and porous morphology were formed.This bilayer scaffold mimicking normal skin tissue structure is expected to overcome the shortcomings of the single scaffold.Large numbers of domestic and foreign studies have shown that both of SF and SA have potential for use in wound dressing or artificial skin,and the composites consisted of proteins and polysaccharides are similar to the extracellular matrix,which improve the biocompability of the materials.Thus,it is quite important to study the efficiency and influence mechanism of the bilayer SF/SA porous biocomposites for repairing the full-thickness skin defects.Firstly,SF/SA biocomposites were prepared as “dermis” layer of the bilayer material through a facile freeze-dried method.The properties such as stability,mechanical property,swelling behavior and conformational structure were studied systematically.The results indicated that the tensile strength value of SF/SA boicomposites improved slightly as the blends ratio of SF/SA reached to 75/25,in which the swelling degree also improved constantly with the increase of SA content.The swelling equilibrium time for each group of composites was about 6 hours.SEM and Micro-CT 3D image demonstrated that various pore-dimeters of SF/SA composites with a continuous phase and interconnected network porous structure can be obtained.When the precooled temperature was –40 °C,the vertical-section of the composites showed a long and narrow morphology with large diameter,parallel to the axial approximation;while the cross-section of the composites showed an irregular ellipse morphology with pore diameter of 100-200 ?m,in which micropores can be found on the hole-wall with porosity more than 85%.This kind of pore structure is beneficial to cell ingrowth and material transportation.The conformational structures analysis results manifested that EDC crosslinking didn't change the conformational structure of SF in the biocomposites,appeared mainly as random coil and silk I structure.The DSC thermal analysis result indicated that the compatibility of the two materials improved by EDC crosslinking.On the basis of the study above,we selected the better performance of two kinds of proportion of composite porous materials(SF/SA 75/25,SF/SA 50/50)as the "dermal layer",and the SA films prepared by casting as the "epidermal layer" to prepare the bilayer SF/SA biocomposites scaffolds.Through the SEM and the Micro-CT 3D image,the vertical-section of the composites showed directed larger pores while the cross-section of the composites showed equally distributed and interconnected pores with diameter of 100-120 ?m.The film and porous biomaterials were closely integrated,which enhanced the mechanical properties of scaffolds.The tensile strength of the bilayer SF/SA 50/50 @ SA film biocomposites was improved from 48±7 kPa corresponding to monolayer material to 598±69 kPa.Secondly,to provide experimental basis of biocompatibility in vitro for the biocomposites as a full-thichness skin defects substitute,properties including biodegradable,cytotoxic and cytocompatibility of the SF/SA biocomposites were further studied.In vitro enzymatic degradation results showed that introduction of SA can inhibit the degradation of these SF/SA biocomposites by collagen enzymes.The degradation rate of SF/SA 50/50 porous materials was the slowest one,and the pore structure of scaffolds can still be maintained after 18 days of degradation with thinner pore wall,increased number of micropores and microfibers.The degradation results in vivo indicated the rate of SF/SA biocomposites in vivo was faster than that in vitro,and the degradation rate of SF scaffolds were the slowest one in vivo.There was no cytotoxicity to L929 cell for the biocomposites extracted liquid when the SA content is lower than 50% in the SF/SA biocomposites.However,the extracted liquid of biocomposites in which the content of SA is 75% and the pure SA scaffold caused a small amount of cell apoptosis during the early growth stage,while there was no significant effect on the proliferation of cells in the later logarithmic growth stage.The introduction of appropriate SA content could enhance the proliferation of L929 cells and increase the secretion of collagen of cells in SF/SA composite porous materials.The results showed that the SF/SA 75/25 and SF/SA 50/50 scaffolds showed the best cytocompatibility.At last,animal model of full-thickness skin defect was built by removing the full-thickness skin on the back part of the SD rat using surgical scissors;and then investigated the healing effect for deeply burned traumatic wounds by transplanting various scaffolds(SA film,SF/SA 75/25 scaffold,SF/SA 50/50 scaffold,SF/SA 75/25 scaffold@SA film and SF/SA 50/50 scaffold@SA film).Besides,the influencing mechanism on wound healing caused by different materials during the healing process was also preliminarily discussed.It was concluded that the bilayer scaffold effectively facilitated healing of the dorsal skin defects.Pathological section results proved that neotissue repaired by SF/SA 50/50 scaffold @SA film was similar to normal skin tissues after 3 weeks transplant operation,including the formation of collagen bundles,distribution of cell and cell extracellular matrix and reconstruction of vessels.As dermal layers of the skin,SF/SA biocomposites could induce various kinds of cells ingrowth and normal differentiation,which accelerated newborn of blood capillary and formation of good granulation tissue at the traumatic area,while the biomaterial was prone to degrade under the complex healing micro environment.Besides,SA film supplied a good protection for neotissue,which isolated the outside infection and reduced the risks of inflammation.The above result showed that the bilayer SF/SA biocomposites scaffold possesses several advantages,including excellent mechanical property,suitable microporous for cell ingrowth,superior swelling property,controllable degradability and excellent cytocompatibility.In addition,the influencing mechanism of the bilayer scaffold facilitating the dorsal full-thickness skin defects of the SD rat was also preliminarily revealed in this study.This biomimetic bilayer scaffold with favorable healing effect for deep skin wounds is a promising candidate for repairing skin tissue engineering applications,furthermore,it is beneficial for enriching the biomaterial system for soft tissue engineering. |
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