Mechanism And Application Study On A Novel Hydrogel Promoting Endogenous Regeneration In Mandibular Defect Through Capturing Neutrophil-derived Exosomes

BackgroundJawbone defects are prone to occur both in peacetime and wartime.They not only cause physiological dysfunctions such as breathing,speech,chewing,and swallowing,but also lead to facial deformities,and even severe psychological disorders in patients.The repair and reconstruction therapy for jawbone defects is a highly complex systematic treatment with the goal of restoring the morphology and function of the maxillofacial region.Therefore,the treatment of jaw bone defects has always been an important issue in the fields of oral and maxillofacial surgery and military medicine.Endogenous bone regeneration technology utilizes endogenous stem cells and acellular biomaterial scaffolds to promote bone tissue regeneration.It is considered a safe,effective,convenient,and economical method for treating bone defects,and has become a trend in the treatment of jawbone defects.In the early stage of self-healing,the inflammatory response and vascular generation of bone are crucial for bone defect regeneration.Neutrophils(PMNs)are one of the most abundant white blood cells in the body,playing a crucial role in the early stages of tissue regeneration,particularly in initiating bone defect regeneration.Clearance of neutrophils can directly lead to the failure of bone defect regeneration repair.Exosomes,as an important intercellular communication medium,can regulate various biological processes such as inflammation,tissue regeneration,and aging.They are widely used as natural endogenous nanoscale drugs or drug-targeted delivery vehicles to promote the regeneration of various tissues such as bones,cartilage,liver,and heart.Therefore,we believe that endogenous exosomes,especially neutrophil-derived exosomes(PMN-Exos),may also play a very important role in regulating vascular generation and osteogenic cell differentiation in the early stage of self-healing of bone defects.This provides a new perspective and direction for the treatment of jaw bone defects based on endogenous bone regeneration technology.ObjectiveThis study attempts to demonstrate the important role of endogenous exosomes,especially neutrophil-derived exosomes,in promoting endogenous bone regeneration by designing a simple exosome-capturing hydrogel scaffold(ECS).It also elucidates the molecular mechanisms by which PMN-Exos promote endogenous bone regeneration through participating in early vascularization and new bone formation,aiming to provide a simple,effective,and feasible method and strategy for promoting endogenous bone regeneration therapy for jawbone defects.MethodsThe study consists of four main experimental sections,utilizing techniques such as hydrogel synthesis,exosomes isolation,animal surgery,single-cell RNA sequencing,proteomic sequencing,mi RNA microarray sequencing,and cell biology and molecular biology experimental methods.The study systematically explores the role and mechanism of PMN-Exos in promoting endogenous bone defects from four aspects: material construction,animal model validation,cell function detection,and molecular mechanism exploration.(1)In the first part,a chitosan-based exosome-capturing hydrogel scaffold(ECS)was constructed,and the role of exosomes in promoting bone regeneration was verified using ECS.Firstly,ECS was synthesized through the Schiff base reaction between phosphatidylserine-modified chitosan and terminally benzaldehyde-modified F127.Then,the capturing properties of ECS for exosomes both in vitro and in vivo were demonstrated using scanning electron microscopy(SEM)and nanoparticle tracking analysis(NTA)techniques.Finally,after the biosafety assessment,ECS was implanted into a critical mandibular defect model in rats,and experiments such as imaging and tissue section staining were conducted at 4 and 8 weeks to evaluate the role of ECS in endogenous regeneration.(2)In the second part,the types of endogenous exosomes locally enriched after ECS implantation were detected and analyzed,demonstrating the role of PMN-Exos in local vascular formation and bone regeneration promotion.Firstly,a small animal mandibular injury healing model was established,and through single-cell sequencing technology and flow cytometry,the types of endogenous exosomes captured by ECS were analyzed.Subsequently,PMN-Exos from rat bone marrow-derived neutrophils were isolated from the culture supernatant using a 30% sucrose cushion ultra-high-speed centrifugation method,and the vesicles were characterized using NTA,transmission electron microscopy(TEM),and western blotting.Finally,through a rat critical mandibular defect model,imaging,tissue staining,and angiography experiments were conducted to demonstrate that PMN-Exos could accelerate bone defect regeneration by promoting vascular formation in the mandibular defect area.(3)In the third part,the effects of PMN-Exos on the vascular-related functions of EPCs in vitro and their molecular mechanisms were explored.Firstly,subpopulation analysis of single-cell sequencing data from mandibular injury healing samples was performed,and EPCs derived from rat bone marrow were isolated and cultured.The impact of PMN-Exos on the proliferation,migration,and in vitro capillary formation of EPCs was then investigated.Finally,using proteomic and mi RNA chip sequencing technologies,along with relevant in vitro cell angiogenesis function experiments,the molecular mechanisms by which PMN-Exos promote vascular formation in bone defect areas were elucidated.(4)In the fourth part,the effects of PMN-Exos on the osteogenic differentiation function of BMSCs and their molecular mechanisms were explored.Firstly,mesenchymal stem cells(BMSCs)derived from rat bone marrow were isolated and cultured,and their characterization was performed through flow cytometry and multi-directional differentiation potential testing.Subsequently,the impact of PMN-Exos on the proliferation,migration,and osteogenic differentiation of BMSCs in vitro was assessed.Finally,using proteomic sequencing technology and in vivo osteogenesis experiments with PMN-Exos integrated cell membrane sheets,the mechanisms by which PMN-Exos affect the osteogenic differentiation function of BMSCs were revealed.Results(1)Successfully constructed ECS and demonstrated its role in promoting endogenous regeneration of mandibular bone defects.Compared with control groups,the phosphatidylserine-modified chitosan hydrogel scaffold(ECS)exhibited better exosomecapturing performance and biocompatibility.After implanting ECS into a rat model of mandibular bone defect,the local endogenous exosome quantity significantly increased within 6 hours and persisted for at least 48 hours.Following implantation into rat mandibular bone defects for 4 weeks,ECS significantly promoted the generation of new blood vessels and new bone in the critical defect area.(2)Identified the main types of exosomes captured by ECS and validated their role in promoting endogenous mandibular regeneration.Single-cell RNA sequencing analysis and flow cytometry results revealed that over 75% of the endogenous exosomes captured by ECS originated from PMNs.Rat bone marrow-derived PMN-Exos were successfully isolated and loaded onto ECS,leading to abundant perfusable blood vessel formation in the defect healing area at 4 weeks post-implantation.At 8 weeks,PMN-Exos increased the amount of new bone formation from around 37% to approximately 42%.(3)Successfully isolated rat bone marrow-derived EPCs and elucidated the molecular mechanism by which PMN-Exos promote vascular generation through influencing EPC proliferation.In vitro,PMN-Exos had a significant promoting effect on the proliferation of EPCs by targeting and inhibiting the expression levels of Smad4 m RNA and protein in EPCs through the highly expressed mi RNA455-3p,without significant effects on EPC migration,differentiation,or tube formation.(4)BMSCs derived from rat bone marrow were successfully isolated,and the molecular mechanism by which PMN-Exos promote osteogenic differentiation of BMSCs was preliminarily revealed.BMSCs can efficiently uptake PMN-Exos and,by increasing the expression of key proteins Sod2 and Gja1 associated with osteogenic differentiation,significantly enhance the proliferation and osteogenic differentiation potential of BMSCs.ConclusionThe use of ECS to locally enrich endogenous exosomes can achieve endogenous bone regeneration in jawbone defects.The mechanism is that PMN-Exos can dual-regulate vascular and bone formation by influencing the proliferation of endothelial progenitor cells(EPCs)and the osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs).This is a simple,secure,and effective method to improve endogenous bone regeneration therapy for jawbone defects.Innovations(1)A novel material for exosome transformation application.In this study,we synthesized a new material with broad-spectrum exosome-capturing capabilities and demonstrated its ability to promote endogenous bone regeneration by enriching neutrophilderived exosomes in vivo,providing a new material for the transformation application of exosomes.(2)A new mechanism of neutrophil-derived exosomes participating in bone defect regeneration.This study first confirmed the significant role of PMN-Exos in promoting endogenous bone regeneration in bone defects and elucidated the molecular mechanisms by which PMN-Exos regulate vasculogenesis and osteogenesis by targeting both EPCs and BMSCs,revealing a new role and mechanism of neutrophil-derived exosomes in bone defect regeneration.(3)A novel method to promote endogenous regeneration of mandibular defects.This study first demonstrated the use of a naturally phosphatidylserine-modified chitosan hydrogel scaffold to achieve endogenous regeneration of critical mandibular defects through local enrichment of exosomes,providing a new method to guide clinical treatment of bone defect regeneration.