Silk Fibroin Scaffolds With A Micro/nano-fibrous Architecture For Promoting Vascularization And Dermal Regeneration

Rapid vascularization to construct an in vivo microcirculation system is a key challenge in dermal tissue engineering field.In vitro prevascularization is a potential and effective method for promoting dermal regeneration.Therefore,the microstructure of 3D scaffolds should be further optimized to promote the adhesion,proliferation,growth and migration of endothelial cells for achieving rapid vascularization after dermal implantation.On the other hand,suitable seeding cells with high cell viability and without ethical restrictions are required for achieving vascularization for dermal tissue engineering.In this study,SF scaffolds with 3D nanofibrous structure are considered to promote dermal regeneration based on the hypothesis that the micro-/nano-fiber structure can be used as instructive cues to direct cellular responses.SF porous scaffold was used as a template,and the carbodiimide-activated SF solution poured into the pore space of pre-fabricated template scaffolds and then assembled into a micro-/nano-fibrous network within the pore space of the template scaffolds by chemical crosslinking,rapid freezing and lyophilization.The pre-fabricated porous SF scaffolds provided a suitable macroporous structure and mechanical support for tissue regeneration,and the subsequently introduced micro-/nano-fibrous networks provided a phycial microenvironment mimicking the fibrillar collagen framework in the natural dermis for cell growth.The dermal scaffold with a nanofibrous microenvironment should be able to enhance the viability of seeding cells and tissue repair cells,and thus promote the regeneration of dermal tissue.On the other hand,human umbilical cord mesenchymal cells(h UC-MSCs)derived from Wharton's jelly can be expanded in culture and induced to form several different types of cells.Moreover,the applications of h UC-MSCs will help to avoid several ethical restrictions and the pain of stem cell harvest.Therefore,h UC-MSCs may therefore prove to be a new source of seeding cells for tissue engineering.The endothelial differentiation can provide a new strategy for in vitro prevascularization.The in vitro culture and prevascularization of the differentiated endothelial cells in SF scaffolds with fibrous microstructure can promote vascularization and dermal regeneration after implalntation.First,a novel SF scaffold containing micro/nano fibers was prepared by a facile two-step freeze drying technology,which provided a microenvironment mimicking the fibrillar collagen framework in the natural dermis for cell growth.Carbodiimide-activated SF solution was diluted and poured into the pore space of pre-fabricated porous SF scaffolds and then assembled into a micro-/nano-fibrous network within the pore space of the porous scaffolds after liquid nitrogen freezing and lyophilization.Chemical crosslinking yielded a macromolecular network through the intermolecular covalent bonds and induced the rearrangement and stretch of SF molecule chains in solution,resulting in the conformational changes of SF from the random coil to ?-sheet structure.The ice crystal shear and internal stress of the SF macromolecules caused the SF macromolecular chains to stretch,and then induced SF macromolecules to realign and assemble into micro/nano fibers.The diameter and density of micro-/nano-fibers could be regulated by the concentration of the poured SF solution.The average diameter of the fibers increased from 350 nm to 406 nm and then to 511 nm with increasing SF solution concentration from 0.05% to 0.1% and then to 0.2%.However,the amount of fibers was significantly reduced when the concentration was over 0.4 wt%,and many sheet-like structures formed.In this study,the fibrous network prepared by 0.2 wt% SF solution was distributed evenly within the macropores of the scaffold.Based on the aim of this study,0.2 wt% SF solution was chosen for subsequent experiments.Secondly,dermal fibroblast cells and umbilical vein endothelial cells were cultured to investigate the effect of fibrous microenvironment on cell behaviors.We used laser scanning confocal microscope,scanning electron microscope,and cell viabitliy tests to analyse the effect of the micro/nano fibrous structure within scaffolds on cell attachment,proliferation,shape and growth.The results demonstrated that the micro/nano fibrous structure in scaffolds provided more anchor points for cell adhesion,and was able to guide cell migration within the space of pores.These micro/nano fibers provided a suitable 3D microenviroment for cell growth,thus promoted the adhesion and proliferation of tissue repair cells involving dermal regeneration,and guided cell growth and migration within the space of pores.Further,MSCs were successfully isolated from Wharton's jelly of human umbilical cord.We detected the phenotype and characteristics of isolated cells by morphological observation,surface antigen markers and cell differentiation,and the results demonstrated that the isolated h UC-MSCs have phenotype and characteristics of mesenchymal stem cells.Then,the h UC-MSCs were successfully induced into endothelial-like cells by in vitro expansion and differentiation.The immunofluorescence CD31 staining,Real time-PCR and Di I-Ac-LDL uptake of the induced cells showed that the differentiated cells possessed phenotype and characteristics of endothelial cells.Furthermore,in vitro culture and angiogenic activity of differentiated cells was determined by Matrigel tube formation assay,indicating that the h UC-MSCs-differentiated endothelial cells showed excellent angiogenic activity.The results of in vitro culture in the SF scaffolds showed that the differentiated endothelial cells were able maintain the phenotype and angiogenic activity of endothelial cells after long-term culture.Moreover,the introduction of the micro-/nano-fibers within SF porous scaffolds significantly enhanced cell attachment,proliferation and migration by providing 3D topographic cues,and thus promoted the angiogenic ability.Finally,the SF scaffolds were implanted into dorsal full-thickness wounds of Sprague-Dawley rats as dermal equivalents to evaluate the effect of the fibrous microstructure and seeding cells on dermal tissue reconstruction.The porous SF scaffold,SF scaffold with fibrous microstructure,and these scaffolds with pre-clutured seeding cells were implanted into dermal defects,respectively.The in vivo results demonstrated that the micro/nano fibers within SF scaffolds promoted the viabitliy of fibroblasts and endothelial cells,accelerated collagen matrix deposition,and enhanced the expression of VEGF and b FGF,thus promoted the tissue neogenesis and angiogenic activity.The differentiated endothelial cells pre-cultured in the SF scaffolds were able to survive in vivo,and achieve rapid vascularization.The rapid vascularization of SF scaffolds provided enough nutrients and oxygen for tissue repair cells,thus provided a suitable microenviroment for tissue neogenesis.Moreover,a suitable microenviroment could recruit tissue repair cells including fibroblasts and endothelial cells,to migrate into scaffolds and secrete ECM.This study developed a facile two-step freeze-drying method to prapare SF scaffold containing micro-/nano-fibers.The results demonstrated that the micro-/nano-fibers within SF scaffolds significantly enhanced the attachment and proliferation of dermal tissue repair cells by providing adhesion sties and physical guidance cues.Moreover,the presence of micro-/nano-fibers was able to direct cell migration within the pore space of pores.Furthermore,in vitro prevascularization was achieved by inducing endothelial differentiation of h UC-MSCs.The synergistic effect of fibrous microstructure within SF scaffolds and in vitro prevascularization of h UC-MSCs-differentiated endothelial cells successfully promoted in vivo vascularization and tissue neogenesis,further promoted dermal reconstruction.This new fibrous SF scaffold provides potential for dermal tissue regeneration.