Preparation And Biological Evaluation Of PLLA/POSS/pDNAs Porous Composite Nanofibers For Promoting Vascularization And Regeneration Of Tissue

Vascularization of deep skin defects is a key challenge for the healing of tissue with blood vessel structures.Therefore,developing a tissue repair materials with vascular-like structure,suitable mechanical strength,good biocompatibility and biodegradable for vascularization promotion has important scientific significance and application prospect.1.In this study,through controlling the molar ratios of functionalized silsesquioxane（POSS）to PLLA,different kinds of arm length ofocta-armed POSS were obtained,and combined with PLLA to prepare nanofibers.The mechanical properties and biocompatibility properties were analyzed and studied,and the results show that POSS nanoparticles could significantly improve the interaction with PLLA.The mechanical and tough properties of matrix fiber materials are significantly improved by POOS-PLLA₈ nanoparticles.In addition,the proliferation,spreading and adhesion of human umbilical vein endothelial cells can be promoted by good biocompatibility of composite porous nanofibers.2.POSS nanoparticles,known for the organic-inorganic structure and biocompatibility,were incorporated into PLLA matrix and processed by electrospinning into nanofibers as a delivery vehicle of plasmid DNA encoding angiopoietin（pANG）.pANG released from the nanofibrous scaffold showed sustained delivery over 35 days and maintained high transfection efficiency.A full-thickness skin wound model was used to evaluate the pANG delivery efficacy and Aniogenesis capability of the plasmid DNA-based polymeric scaffold in vivo.The results indicated that the pANG loaded PLLA/POSS scaffold effectively promoted Angiogenesis and dermal wound healing.In addition,such fibers can be networked to form scaffold materials for tissue engineering after integration with endothelial cells,thereby providing the potential for the in vitro vascularization of functional constructs in tissue engineering.3.In this research,to accelerate wound regeneration,complexes of trimethyl chitosan chloride（TMC）,multiple plasmid DNAs（pDNAs）encoding ANG and basic fibroblast growth factor（bFGF）were integrated into porous nanofibers of poly（L-lactic acid）（PLLA）/polyhedral oligomeric silsesquioxane（POSS）;these nanofibers were used to treat deep skin defects.The PLLA/POSS/pDNA nanofibers produced a notable positive influence on the transfection of HUVECs in vitro,and the PLLA/POSS/pANG/pbFGF（Fab）composite nanofibers showed significantly higher expression of ANG and vinculin than the nanofibers without pDNA or the nanofibers loaded with pANG or pbFGF only.The four different composite nanofibers were further transplanted onto deep burn wounds on the backs of rats in vivo.The results indicated that the Fab nanofibers had observably higher neovasculature regeneration.This finding revealed that Fab not only prevented the inflammatory response but also accelerated wound regeneration.All of the results showed that pANG and pbFGF have positive effects on promoting tissue repair.Therefore,an even higher level of vascularization can be realized when ANG and bFGF are both present to achieve desirable interactions for wound repair.4.The pANG and p BMP-2 plasmid-loaded PLLA/POSS nanofibers（p NF）were combined with PEGDA/CS gel to obtain a three-dimensional scaffold material with dual gene activity and used for skull.The results show that the compression and mechanical properties of the PEGDA/CS gel are not affected by the addition of the composite fibers.In addition,the PEGDA/CS/p NF fiber scaffold was implanted in the rat skull defect for 12 weeks,and a good healing effect was obtained.The PEGDA/CS/p NF double gene scaffold can not only induce new bone growth and angiogenesis,but also be used for clinical treatment of bone defects.5.In this study,two targeting polypeptides,Arg-Glu-Asp-Val（REDV）and cell-penetrating peptides-localization signals of nuclear（CPP-LSN）,were linked to two ends of an amphiphilic block copolymer（PEI-PLMD-PEI）,and self-assembled to form positively charged REDV/CL-PEI-PLMD-PEI（R/CL-PPP）nanoparticles,which could bind to the negatively charged pANG by electrostatic interaction;the polypeptides were finally loaded onto PLLA/POSS porous fibers to prepare targeted porous nanofibers（TF）and targeted nanofiber bundles（TFB）.The effects of the porous fibers on the transfection,spreading,proliferation,cell morphology and expression of related factors in human umbilical vein endothelial cells（HUVECs）were investigated under the action of a flow shearing force.The results showed that the PLLA/POSS nanofibers could maintain the stable release of R/CL-PPP nanoparticles for nearly 45 days.Under the action of the flow shear force and dual-targeted gene nanoparticles,the HUVECs in the TFB group had a trend of growth along the direction of the fiber bundle,and the cells could cover the surface of the fiber bundle,producing a vascular structure.These results provided a potential strategy to design HUVEC-specific gene carriers and use flow shear to enhance endothelialization.In summary,PLLA/POSS composite nanoparticle can continuously and stably deliver the released gene,which can be used as a potential application gene-active stent for clinical treatment of defect tissue repair and Angiogenesis.