Fabrication And Application Study Of Hybrid Scaffold For Dermal Tissuereconstructionbased Onbioactive Glass/Chitosan/Silkfibroin Composite

The development of novel skin tissue engineering scaffold for the rapid healing and functional restoration of skin defects,especially those impaired wounds,has been a critical issue in the medical and biomaterials research area.Bioactive glass(BG),chitosan(CHI)and silk fibroin(SF)have been used in skin wound healing because of their distinct advantages.However,there are many deficiencies of using single material for biomedical application,such as the unstable thermostability,fast degradation rate,poor 3D structure and low bioactivity.As we know,few study focused on the composites of the above three materials.In this study,we fabricated a bioactive glass/chitosan/silk fibroin scaffold through the freeze drying technology,which we hope to solve the problems caused by single use of materials and can be a suitable candidate for skin wound healing.The construction and their wound healing mechanism of bioactive glass/ chitosan/silk fibroin composite scaffolds have been fully investigated by various methods.In order to obtain the scaffolds with good physical and chemical property,proper degradation rate and fast vascularization rate,firstly,the fabrication technology of the scaffolds was optimized through the analysis of FTIR,SEM,thermostability,pore size distribution and porosity.In vitro,the biocompatibility of the composite scaffolds was also studied by co-culturing with 3T3 cells.The cell proliferation and migration were monitored through the test of MTT and immunofluorescence.In vivo,a full-thickness skin wound rat model was established.The proliferation of fibroblasts,epithelial cells,endothelial cells,the wound healing times and rates,and the angiogenesis and vascularization markers of CD31 and ?-SMA of wounds treated by the composite scaffolds was investigated by gross observation,HE staining,Masson staining and immunostaining.Taken together,this research systematically studied the healing ability of the bioactive glass/chitosan/silk fibroin scaffolds(2: 8),which have great potential in wound healing application with faster vascularization,shortened healing time and faster healing rate.The specific work is stated as following:Firstly,the composite scaffolds was fabricated through the freeze drying methods by combining the sol-gel technology prepared 58 S bioactive glass and chitosan/silk fibroin(CHI/SF and BG/CHI/SF)with different ratios(1:9 to 5:5).Through the and porosity test,we found that the pore size of pure SF scaffold,CHI/SF(3:7)and CHI/SF(2:8)ranged from 100 to 120?m with high porosity(>90%),and the thermostability test showed that CHI/SF(2:8)scaffold was obviously better than the CHI/SF(3:7).Meanwhile,the water absorption index of CHI/SF(2:8)was statically higher than pure SF,i.e.4.89±0.338 to 1.12±0.104.With the incorporation of bioactive component BG,no obvious change of the pore size or the porosity was found on SF based scaffold,and BG can uniformly distribute within the pore wall of the scaffold.Moreover,the thermostability was enhanced with the addition of BG,as well as the roughness and the specific surface area.Therefore,the BG/CHI/SF(2:8)obtained in our study should be more suitable for skin wound healing.Secondly,the cytocompatibity of the composite scaffolds was evaluated by co-culturing the scaffolds with the 3T3 fibroblasts cell line.Cell proliferation and migration on the scaffolds was measured by MTT test and confocal microscope.Results revealed that the four scaffolds of BG/CHI,BG/SF,CHI/SF and BG/CHI/SF showed none cytotoxicity,and at the later stage of culturing(3 days and later),cells incubated with BG/CHI/SF exhibited faster proliferation rate than other scaffolds,and can migrate into the internal of the scaffold.For further investigation the wound healing effect and the related mechanism of the composite scaffold,the in vivo full-thickness skin wound model was established.Four kinds of scaffolds of BG/CHI,BG/SF,CHI/SF and BG/CHI/SF were used to treat the wounds.Results showed that all of the four scaffolds can degrade with the increase of healing time,and can maintain the wounds in moisture,which resulted in a good microenvironment for the wounds.Besides,CHI/SF and BG/CHI/SF can reduce the inflammatory response of the wounds at the early time of healing.Compared to other three scaffolds,the wound healing time of BG/CHI/SF scaffold has shortened 40%.Histology results showed that BG/CHI/SF scaffold can induce the proliferation of fibroblasts,the formation of granulation tissue and more number of newly-formed blood vessels in the wound site.In addition,we evaluated the stimulating ability of BG/CHI,BG/SF,CHI/SF and BG/CHI/SF on the newly-formed and mature blood vessels in the regenerated tissue.Immunostaining results showed that all the four scaffolds enhanced the expression of CD31 and ?-SMA,indicating that all the scaffolds can stimulate the angiogenesis and vascularization process of wound healing.Based on the statistical analysis of vessels numbers,during the whole healing period,the expressions of CD31 and ?-SMA in BG/CHI/SF group was significantly higher than other three groups.More newly-formed and mature vessels can be found in granulation tissue,suggesting that BG/CHI/SF can accelerate the angiogenesis and vascularization process of the tissue,which should be beneficial for the wound healing process.In conclusion,BG/CHI/SF composite scaffolds possess excellent wound healing ability,which has great application potential in skin tissue engineering.