Engineered Exosomes Mediated Gene-activated Materials Induce Vascularized Bone Regeneration

Natural bone tissue is a highly vascularized tissue that can self-repair in a certain extent,but it cannot heal by itself when the size of bone defect exceeds its critical defect size.Therefore,the critical bone defect is one of severe clinical problems to be resolved at present.Due to the large volumes of critical bone repair materials,the center zone of newly formed bone tissue has a limited vasculature that may cause the regional necrosis and result in the failure of repair eventually.Thus,it is of importance for basic research and clinical application to design and construct the bone regeneration repair materials for enhancing vascularization.Gene-activated matrix(GAM)that integrates tissue engineering with gene therapy to create new materials has received more and more attentions.The gene form of growth factor has been used to replace the traditional protein form of growth factor to effectively avoid many problems involving burst release of protein to cause short duration of effective function,the inactivation of protein growth factors to affect the poor efficiency,systemic toxicity caused by initial rapid release,and high cost to be produced.Based on above comparison,the GAM exhibits great advantages and has been extentively applied in tissue engineering.In this thesis,the gene form of Vascular Endothelial Growth Factor(VEGF)was in place of traditional VEGF protein by gene-activated materials.The exosome was selected as a gene carrier due to its excellent biocompatibility,lower toxicity and higher efficiency of cell uptake and then combined with porous bone scaffolds.Thus,herein a series of vascularized bone materials with dual functions on angiogenesis and osteogenesis had been designed and constructed to solve severe clinical problems of insufficient vascularization in the central zone of repair materials for critical bone defect.It is also laying the theoretical foundation for the development of next generation of bone regeneration materials.The main contents of this thesis include the followed three aspects:(1)To develop a vascularized bone regeneration repair material based on the exosomes as gene delivery vehicles and applied to construct the cranial defect.Firstly,the exosomes derived from chondrogenic progenitor cell line were extracted as a delivery vehicle of VEGF plasmid to construct engineered exosomes.These engineered exosomes not only can utilize as gene delivery vehicle,but also can develop their differentiation function from their source cells.Then,the electrospun film of SF/PCL was prepared by electrospinning technique.The resultant electrospun film of SF/PCL was able to mimic the extracellular matrix.Furthermore,it was modified with p DA to improve the adhesion ability of engineered exosomes.Finally,the electrospun film of SF/PCL with engineered exosomes was applied as a novel vascularized bone material for the repair of cranial defects in rats.Both in vitro and in vivo investigations confirmed that the engineered exosomes exhibited dual functions on angiogenesis and osteogenesis to well promote the critical bone defect repair.(2)The vascularized bone materials based on exosomes were further optimized and expanded the application on segmental bone defect repair.In order to solve inappropriate connection between exosomes and materials which leads to the burst release of exosomes in the early stage,the connection mode was optimized by introducing the specific exosomal anchor peptide.The PCL porous scaffolds were prepared by 3D printing technology,and the exosomal anchor peptide was further modified to specifically bind the engineered exosomes onto PCL porous scaffolds.This specific flexible connection enabled exosomes to be well adsorbed on the material interface,playing the role of in-situ cell uptake and insitu controlled release of VEGF.This vascularized bone material was used to repair the rat radial defect and the results showed the effective bone regeneration.It is further verified that the vascularized bone materials based on engineered exosomes may provide a new strategy for the repair of critical bone defects.(3)To design a vascularized bone material based on the exosome-mimetics as a gene delivery vehicle and applied on the cranial defect.In order to overcome the drawbacks of complicated extraction procedures,long extraction cycle and low yield of exosomes,a novel exosome-mimetic was extracted by building molecular sieves and using the approach of gradient mechanical extrusion.The exosome-mimetic employs similar morphology,size and surface protein markers with exosomes,but the yield of exosome-mimetics was about100 times than the exosomes extracted with the routine strategy.The coaxial electrospun film of CS/PLA with the core-shell structure was fabricated by coaxial electrospinning technique.In addition,to strengthen the connection of exosome-mimetics with coaxial electrospun film,we further optimized the connection mode using biotin-streptavidin system,the binding strength of which was between physical adsorption and covalent bonding.Exosome-mimetics and coaxial electrospun film were both modified with biotin,the streptavidin as a bridge was integrated with each together.Animal evaluation also indicated that the material exhibited a good repaired effect.The use of exosome-mimetics further enriched the application of gene-activated vascularized bone regeneration materials in bone tissue engineering.In summary,a series of engineered exosomes mediated vascularized bone materials were designed and constructed in this thesis.The vascularized bone materials can effectively promote the repair of critical bone defects and overcome the clinical problems on the necrosis of segmental bone defect due to the lack of internal vascularization.From two aspects of angiogenesis and osteogenesis,here we further provide an alternative material system for the development on a new generation of vascularized bone materials.