Study On The Heparin Modified Silk Fibroin Scaffold For Dermal Tissue Engineering And Its Promotion On Angiogenesis

Promoting the rapid formation of blood vessels network in dermal tissue engineering scaffold plays an important role in improving the survival rate of scaffold in vivo and the in situ induced regeneration of dermal tissue.Silk fibroin(SF)materials have a wide prospects of development and application when they are used as scaffolds for dermal regeneration because of their excellent biocompatibility,biodegradation and no obvious immunogenicity.In order to enhance the angiogenesis of the three-dimensional SF materials as dermal regenerative scaffolds and thereby accelerate the regeneration of dermal tissue.In this study,heparin was grafted on the surface of SF materials via covalent bonding.On the one hand,the effect of the heparin modified SF materials on the anticoagulant activity was investigated,and on the other hand,the effect of SF materials modified by heparin on the adsorption and accumulatation of VEGF and the adhesion and proliferation of vascular endothelial cells were also studied in vitro.The heparin modified SF scaffolds were implanted into dorsal full-thickness wounds in Sprague-Dawley rats to investigate their effect on inbiting the formation of blood clots,enhancing the adsorption of VEGF and thereby promoting the migration,adhesion and proliferation of vascular endothelial cells.The dual effects of anticoagulant activity and the VEGF adsorption ability of the heparin modified SF materials on subsequent angiogenesis and the regeneration of dermal tissue were investigated.Firstly,the appearance of the regenerated SF membrane is transparent and the surface is dense,making it easy to observe and analyze its surface composition and structural changes.Heparin was firmly immobilized on the SF membrane with a high density by covalent bonding by the double induction method of combining low-temperature N₂plasma treatment and carbodiimide activation.The effect of the heparin modified SF membranes on the anticoagulant activity,the adsorption of VEGF and the adhesion and proliferation of vascular endothelial cells was investigated.The results showed that the densities of amino groups and heparin introduced onto the SF membranes surface reached approximately 162.4 nmol/mg and 7.8?g/cm²,respectively,as the working power was 90W and treatment time was 9 min.The in vitro experimental results suggested that the heparin modified SF membranes could not only significantly prolong the coagulation time and reduce the adhesion of platelets,but also promote the adsorption of VEGF and the adhesion and proliferation of vascular endothelial cells compared to pristine or plasma-treated SF membranes.Secondly,three-dimensional SF scaffolds were prepared by freeze drying method,then heparin was grafted on the surface of SF scaffolds using carbodiimide as a cross-linking agent.To investigate whether the heparin modified SF scaffolds have similar effects as heparinized SF membranes in vitro and whether they could inhibit the formation of blood clots and promote the migration and adhesion of tissue repair cells in vivo.The data gathered from FTIR and XPS suggested that the heparin could be immobilized on the SF scaffolds surface via amido bond with the aid of carbodiimide.The amount of grafted heparin was approximately 12?18?g/mg.Heparin immobilized on the SF scaffolds surface released slowly and sustainably in saline,and the relative release of heparin was25?30%after 14 days.The in vitro experimental results showed that SF scaffolds modified by heparin could significantly reduce the adsorption of plasma protein,prolong coagulation time and promote the adsorption of VEGF,thereby enhancing the adhesion and proliferation of endothelial cells.The in vivo experimental results of implanting SF scaffolds into dorsal full-thickness wounds in Sprague-Dawley rats suggested that heparin modified SF scaffolds could significantly reduce the the adhesion of platelets and the deposition of fibrin and inhibit the formation of blood clots in the scaffolds,thereby promoting the immigration of tissue repair cells towards the inside of the scaffolds.Finally,the SF scaffolds modified by heparin were implanted into dorsal full-thickness wounds in Sprague-Dawley rats as dermal regeneration scaffolds to investigate their effect on angiogenesis and the regeneration of dermal tissue.The qualitative and quantitative analysis results by method of combining intravital fluorescent staining and tissue optical clearing technique indicated that the heparin modified SF scaffolds could significantly accelerate the regeneration of blood vessels network after 2 weeks.The immunohistochemical analysis results of CD31 and VEGF suggested that the positive expression rates of CD31 and VEGF and the blood vessel density of the heparin modified SF scaffolds were significantly higher than those of the unmodified SF scaffolds after 2weeks.HE and Masson staining indicated that the number of blood vessels and the deposition of collagen in the heparin modified SF scaffolds significantly increased,and the rate of dermal regeneration and wound healing was significantly accelerated after 2?4weeks.The heparin modified SF scaffolds could significantly inhibit the formation of blood clots in the scaffolds,providing physical channels for the immigration of tissue repair cells towards the inside of the scaffolds,and at the same time,the scaffolds could enhance the adsorption of VEGF,creating a good micro-environment for the adhesion,migration and proliferation of tissue repair cells,thereby promoting the formation of blood vessels network and the regeneration of dermal tissue.