The Research On Effect And Mechanism Of A Microenvironment-responsive DNA Hydrogel Loaded With Dental-derived Exosomes In Bone Regeneration Of Diabetic Rats

Objective:The regeneration of bone defects in patients with diabetes mellitus(DM)is remarkably impaired by metabolic disorder,hyperglycemia,over-expressed proinflammatory cytokines and proteinases(such as matrix metalloproteinases,MMPs)via disturbing the bone homeostasis.With the increasing incidence of diabetes and suboptimal individual glycemic control,a large number of diabetic patients suffer from poor bone healing.However,the traditional treatments for bone defects are limited by insufficient vascularization,adverse effects of pathological microenvironment,etc.To address this clinical issue,this research aimed to investigate the effect and related mechanism of stem cells from apical papilla-derived exosomes(SCAP-Exo)on angiogenesis and osteogenesis,and evaluate the ability of the microenvironment-responsive PEG/DNA hybrid hydrogel loaded with SCAP-Exo to promote vascularized bone regeneration in diabetic rats.Methods:1.Stem cells from apical papilla(SCAPs)and dental pulp stem cells(DPSCs)were isolated and cultured by enzyme digestion;multilineage differentiation ability was verified after osteogenic,chondrogenic and adipogenic induction;stem cell surface markers were detected by flow cytometry.The gradient centrifugation was used to extract SCAP-Exo and DPSC-Exo,and the two exosomes were identified by TEM,NTA,and Western Blot.The effects of SCAP-Exo and DPSC-Exo on the proliferation,migration and differentiation of HUVECs and MC3T3-E1 cells were compared by CCK-8,Transwell,and q RT-PCR methods.2.The effects of SCAP-Exo on the migration and differentiation of HUVECs and MC3T3-E1 cells under physiological and DM conditions were verified by CLSM,Transwell,q RT-PCR,tube formation assay,ALP staining,and ARS staining.3.The mi RNAs involved in angiogenesis and osteogenesis in SCAP-Exo were explored by mi RNA sequencing technology,target gene prediction and functional enrichment analysis;the results were verified in HUVECs and MC3T3-E1 cells by q RT-PCR and mi RNA mimics transfection methods.4.The viscoelastic,self-healing,and mechanical properties of the polyethylene glycol(PEG)/DNA hybrid hydrogel were detected by rheological tests;the pore structure and SCAP-Exo distribution were detected by SEM and CLSM;the responsiveness to MMP-9 was detected by the exosome release rate assay in different conditions.5.The STZ was used to induce diabetic rats.Micro-CT,HE staining and immunofluorescence staining were used to verify the effect of PEG/DNA hybrid hydrogel loaded with SCAP-Exo on the vascularized bone regeneration in diabetic rats.Results:1.SCAPs and DPSCs were successfully isolated and cultured,and the cells showed the typical stem cell morphology of spindle shape;SCAPs and DPSCs had the ability of multilineage differentiation;SCAPs and DPSCs positively expressed stem cell surface markers,and negatively expressed hematopoietic stem cell and leukocyte surface markers.SCAP-Exo and DPSC-Exo were successfully extracted and displayed typical disk shape with bilayer membrane structure;the particle size range of SCAP-Exo and DPSC-Exo was 100-150 nm;SCAP-Exo and DPSC-Exo positively expressed exosomes marker proteins,and negatively expressed endoplasmic reticulum protein.Compared with DPSC-Exo,SCAP-Exo had better ability to promote the migration,differentiation of HUVECs and MC3T3-E1 simultaneously.2.SCAP-Exo could be successfully internalized by HUVECs and MC3T3-E1 cells.SCAP-Exo could promote the migration,differentiation of HUVECs and MC3T3-E1 under physiological condition,and 10 μg/ml of SCAP-Exo was considered as the optimal concentration;SCAP-Exo could promote the migration,differentiation of HUVECs and MC3T3-E1 under DM condition.3.Highly expressed mi R-126-5p and mi R-150-5p in SCAP-Exo might play a role in angiogenesis and osteogenesis respectively;the expression of mi R-126-5p and mi R-150-5p was upregulated after the internalization of SCAP-Exo by HUVECs and MC3T3-E1;the expression of angiogenesis-related genes in HUVECs and osteogenesis-related genes in MC3T3-E1 were upregulated respectively after transfected with mi R-126-5p mimics or mi R-150-5p mimics.4.The microenvironment-responsive PEG/DNA hybrid hydrogel was successfully prepared;the hydrogel had great viscoelastic,self-healing,and mechanical properties;the pore structure was uniform with a diameter of about 500 nm,SCAP-Exo was uniformly loaded in the hydrogel;the hydrogel was responsive to MMP-9 and could release SCAP-Exo in a controlled manner.5.It was confirmed that the bone healing ability of diabetic rats was impaired;the PEG/DNA hybrid hydrogel loaded with SCAP-Exo promoted vascularized bone regeneration in diabetic rats.Conclusion:In this research,SCAP-Exo was verified to promote angiogenesis and osteogenesis in vitro.The microenvironment-responsive PEG/DNA hybrid hydrogel system loaded with SCAP-Exo was developed.This smart system could achieve controlled release of dual-functional SCAP-Exo by responding to the elevated MMP-9 in diabetic microenvironment.The effect of this system on promoting regeneration of diabetic bone defects was further verified in vivo.It provides a promising alternative therapy for diabetic patients with poor bone healing.