Preparation Of Composite Nanofibers Containing Silicon/Magnesium And Their Application In Skin/Bone Tissue Regeneration

Electrospun organic nanofiber materials are widely used in tissue engineering because of their topological structure simulating natural extracellular matrix（ECM）.However,organic nanofiber materials have disadvantages such as fast degradation and weak mechanical strength.The organic/inorganic composite nanofibers prepared by the combination of inorganic materials are expected to solve their shortcomings and be better applied in tissue engineering.Organic/inorganic composite nanofiber materials combine the outstanding advantages of inorganic nanoparticles or nanofibers,and show better properties in mechanical properties,drug encapsulation and cell responsiveness,which is beneficial to its application in tissue engineering.Preparation of organic/inorganic composite nanofiber scaffolds:On the one hand,compared with organic nanofibers,composite nanofibers effectively enhance the mechanical properties of materials;On the other hand,inorganic components can significantly promote cell adhesion,migration,proliferation and differentiation.Therefore,it is of great significance to develop multi-functional organic/inorganic composite nanofiber scaffolds.The inorganic components in the traditional organic/inorganic composite nanofiber materials are usually distributed in the form of particles in the nanofiber,while the inorganic nanofiber has the fiber network structure and microenvironment similar to the natural extracellular matrix,which is more conducive to cell growth than the inorganic nanoparticles.In this study,flexible inorganic nanofiber membranes were prepared by electrospinning and template agent.The organic/inorganic composite nanofiber materials can be better used in soft and hard tissue regeneration.Therefore,based on the excellent physical and chemical properties and biological activities of organic/inorganic nanofiber composite materials,functional scaffolds with different inorganic components were prepared in this paper and applied to wound healing and bone defect regeneration.The specific research content and results can be divided into the following five parts:（1）Bacterial infections can cause serious secondary damage to wounds and prevent healing.We prepared polycaprolactone/gelatin（PCL/gelatin）nanofiber membranes doped with magnesium oxide（MgO）nanoparticles by electrospinning and investigated their potential in inhibiting bacterial infection in wound dressings.PCL/gelatin/MgO nanofiber membrane has good elasticity and flexibility similar to natural skin tissue,and has good hydrophilicity,which is conducive to its close adhesion to the wound surface in the wet state.The antibacterial ability of nanofiber membrane was proportional to the amount of MgO nanoparticles,among which the nanofiber membrane containing 0.5 percent of MgO nanoparticles could inhibit the growth of 98%of Escherichia coli,90%of Staphylococcus aureus and 94%of Staphylococcus epidermidis.Animal experiments show that PCL/gelatin/MgO nanofiber membranes can regulate macrophage phenotype,down-regulate inflammatory response,contribute to reducing inflammation and create a favorable microenvironment for wound healing.Specifically,the release of magnesium ions(Mg²⁺)from PCL/gelatin/MgO nanofiber membranes stimulates macrophages to transform into the pro-healing M2 phenotype.In addition,PCL/gelatin/MgO nanofiber membranes can promote angiogenesis by up-regulating the expression of vascular endothelial growth factor（VEGF）in endothelial cells.PCL/gelatin/MgO nanofiber membranes coordinate keratinocyte phenotypes and reduce the expression of inflammatory cytokine interleukin 1-α（IL-1α）,promoting skin hair follicle regeneration.These results indicate that PCL/gelatin/MgO nanofiber membrane can inhibit bacterial infection,reduce inflammation,promote extracellular matrix production and epithelialization,and elucidate the mechanism of its enhancement of hair follicle regeneration,which proves that PCL/gelatin/MgO nanofiber membrane has the potential to be used as an infectious wound dressing.（2）A large number of studies have shown that insufficient angiogenesis is the main problem affecting the wound healing process of diabetes mellitus.Therefore,the preparation of nano-fiber membranes with the ability to promote wound angiogenesis is of great significance for the wound healing of diabetic patients.In the previous study,the prepared PCL/gelatin/MgO nanofiber membrane was proved to promote angiogenesis by releasing Mg²⁺,so we applied PCL/gelatin/MgO nanofiber membrane to the healing of diabetic wounds.In vitro cell experiments have shown that PCL/gelatin/MgO nanofiber membranes can promote the proliferation of human umbilical vein endothelial cells and up-regulate the production of VEGF.Subcutaneous implantation studies in rat models showed that PCL/gelatin/MgO nanofiber membranes showed a faster degradation curve than PCL/gelatin nanofiber membranes.PCL/gelatin/MgO nanofiber membrane can form more blood vessels one week after implantation,and with the passage of time,a large number of new capillaries are enriched in the degraded nanofiber membrane.PCL/gelatin/MgO nanofiber membranes significantly accelerate diabetic wound healing by inhibiting inflammatory response,promoting angiogenesis and promoting granulation formation.In conclusion,the results show that PCL/gelatin/MgO nanofiber membranes with angiogenic activity are important for diabetic wound healing.（3）In the previous chapter,it was found that the hydrolysis process of MgO nanoparticles supported by PCL/gelatin/MgO nanofiber membranes would lead to the increase of ambient p H and ion release.In order to solve the above problems,in this study,a magnesium ion chelated PCL/gelatin/MgAC nanofiber membrane material was prepared by adding excess acetic acid to convert MgO nanoparticles into magnesium acetate（MgAC）,and its application effect in diabetic wound healing was studied.The coordination of Mg²⁺and gelatin not only effectively solved the problem of increasing the ambient p H caused by the hydrolysis of MgO nanoparticles,but also enhanced the mechanical properties of nanofiber membranes.In addition,slowly released Mg²⁺promoted the proliferation of human umbilical vein endothelial cells and upregulated the growth factors that promote angiogenesis.In the skin defect model of diabetic rats,PCL/gelatin/MgAC nanofiber membrane can reduce the inflammatory response,up-regulate the M1-to-M2-type conversion of macrophages,promote the formation of granulation tissue and mature epidermal tissue,and thus promote the healing of diabetic wounds.（4）In the previous study,it was found that PCL/gelatin/MgAC nanofiber membrane still had too much acetic acid residue,resulting in acidic p H in the early stage.On the other hand,compared with inorganic nanoparticles,the construction of inorganic nanofiber membrane can better simulate the natural extracellular matrix structure to promote cell growth.Therefore,in this study,we prepared magnesium-doped silica bioactive glass（SiO₂/MgO）nanofibers with excellent flexibility by controlling the composition ratio and chain structure,and used them as infectious wound dressings.The results show that SiO₂/MgO nanofiber membrane has good flexibility and hydrophilicity,which makes it in close contact with the wound surface.In vitro evaluation showed that Si⁴⁺and Mg²⁺bioactive substances released by nanofiber membranes promoted cell proliferation and angiogenesis.It has also been shown to regulate levels of inflammatory responses in mice’s macrophages.In addition,in vitro studies demonstrated that the flexible SiO₂/MgO nanofiber membranes had good antibacterial activity against both Gram-positive and Gram-negative strains.The application of flexible SiO₂/MgO nanofiber membrane in full-layer skin defect wound of mice inoculated with Staphylococcus aureus can also effectively inhibit bacterial infection.Both gene expression and histological/immunohistochemical analysis confirmed that flexible SiO₂/MgO nanofiber membranes could induce pro-inflammatory factor down-regulation,anti-inflammatory factor up-regulation and angiogenesis enhancement.In summary,the flexible SiO₂/MgO nanofiber membranes were shown to contribute to the rapid healing of infected wounds.（5）Due to the limitations of two-dimensional inorganic nanofiber membrane in application,three-dimensional nanofiber scaffolds are needed for bone defect regeneration.Therefore,the preparation of a scaffold that can completely fit the defect site and induce osteogenesis and angiogenesis at the same time is of great significance for the healing of bone defects.The aim of this study was to prepare superelastic organic/inorganic composite aerogel scaffolds by mixing silica（SiO₂）nanofibers and polylactic acid/gelatin（PLA/gel）nanofibers.The contents of SiO₂ nanofibers in the scaffolds were 0 wt%（PLA/gel）,20 wt%（PLA/gel/SiO₂-L）,40wt%（PLA/gel/SiO₂-M）and 60 wt%（PLA/gel/SiO₂-H）,respectively.The PLA/gel/SiO₂-M support has excellent elasticity and good mechanical properties.In vitro experiments showed that silica ions released by PLA/gel/SiO₂-M scaffolds could promote the differentiation of bone marrow mesenchymal stem cells into osteoblasts,thus enhancing the activity of alkaline phosphatase and the expression of bone-related genes.Meanwhile,the released silicon ions also promote the proliferation of human umbilical vein endothelial cells and the expression of vascular endothelial growth factor,thus promoting angiogenesis.Evaluation of these scaffolds in a rat skull defect model showed that the PLA/gel/SiO₂-M scaffolds had significant potential to promote osteogenesis and angiogenesis,and had greater bone regeneration capacity than the blank control defect group（no scaffolds）or the PLA/gel scaffolds.Overall,superelastic scaffolds containing flexible SiO₂nanofibers can simultaneously induce osteogenesis and angiogenesis,which will have a wide range of applications in bone tissue engineering.In summary,different materials based on organic/inorganic hybrid nanofibers were prepared by electrospinning technology in this paper.And according to the characteristics of the material gradually optimized to obtain the best performance.Specifically,PCL/gelatin/MgO nanofiber membranes were prepared first,and the shortcomings of ion release were found in the study.By optimizing the conversion of MgO nanoparticles to magnesium acetate,the magnesium element exists in the nanofibers in the form of metal ion chelation,thus avoiding the problem of ion release.Considering that inorganic nanofibers can better simulate the structure of natural extracellular matrix and solve the problem of p H change,flexible SiO₂/MgO nanofiber membranes were prepared.Subsequently,in order to be applied in bone tissue regeneration,the two-dimensional nanofiber membrane was transformed into three-dimensional organic/inorganic composite nanofiber scaffolds.