Versatile Hypoxic Extracellular Vesicles Laden in an Injectable and Bioactive Hydrogel for Accelerated Bone Regeneration

Abstract

Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) have emerged as an appealing alternative to cell therapy in regenerative medicine. Unlike bone marrow MSCs (BMSCs) cultured in vitro with normoxia, bone marrow in vivo is exposed to a hypoxic environment. To date, it remains unclear whether hypoxia preconditioning can improve the function of BMSC‐derived EVs and be more conducive to bone repair. Herein, it is found that hypoxia preconditioned BMSCs secrete more biglycan (Bgn)‐rich EVs via proteomics analysis, and these hypoxic EVs (Hypo‐EVs) significantly promote osteoblast proliferation, migration, differentiation, and mineralization by activating the phosphatidylinositide 3‐kinase/protein kinase B pathway. Subsequently, an injectable bioactive hydrogel composed of poly(ethylene glycol)/polypeptide copolymers is developed to improve the stability and retention of Hypo‐EVs in vivo. The Hypo‐EVs‐laden hydrogel shows continuous liberation of Hypo‐EVs for 3 weeks and substantially accelerates bone regeneration in 5‐mm rat cranial defects. Finally, it is confirmed that Bgn in EVs is a pivotal protein regulating osteoblast differentiation and mineralization and exerts its effects through paracrine mechanisms. Therefore, this study shows that hypoxia stimulation is an effective approach to optimize the therapeutic effects of BMSC‐derived EVs and that injectable hydrogel‐based EVs delivery is a promising strategy for tissue regeneration.

Energy metabolism as therapeutic target for aged wound repair by engineered extracellular vesicle

“…EV is composed of proteins, RNAs, lipid, etc., and these different composites in EV can play distinct roles in regulating biological behavior. Some molecules in EV can exert positive effects on cell, while some can cause adverse effects, thus there exists an optimum concentration range, the result of which was consistent with previous reports ( 61 , 62 ).…”

Mesenchymal Stem Cell-Derived Extracellular Vesicles in Bone-Related Diseases: Intercellular Communication Messengers and Therapeutic Engineering Protagonists

“… [ 55 ] ADSCs Coupled dextran sulfate via click chemistry mediated by metabolic glycoengineering Collagen-induced arthritis mouse model Intravenous injection Targeted accumulation in the inflamed joints; Reprogrammed macrophage phenotype with a 10-fold less dose [ 104 ] Neoplastic diseases BMSC-EVs Mixing with Doxorubicin hydrochloride Tumor xenograft mouse model Intravenous injection Enhanced therapeutic efficiency against osteosarcoma [ 105 ] BMSCs Transfected miR-206 mimic by Lipofectamine 3000 Tumor xenograft mouse model Intravenous injection Suppressed osteosarcoma progression [ 106 ] hMSCs Transfected has-miR-150 by Lipofectamine 2000 Tumor xenograft rat model Injection into the intradermal left axilla. Inhibited migration and invasion of osteosarcoma cells and the growth of transplanted tumors [ 107 ] Traumatic diseases SHEDs Hypoxia preconditioning Rat calvarial defect model Injection into defects with microspheres Continuous release of EVs for 21 days and promoted vascularized bone regeneration [ 108 ] SHED-EVs Solution adsorption to lyophilized PMS-PDA microspheres BMSCs Hypoxia preconditioning Rat calvarial defect model Injection into defects with hydrogel Enhanced osteogenic differentiation and expedited bone regeneration [ 109 ...…”

“… [ 114 ] BMSC-EVs Solution adsorption to β-TCP scaffold and lyophilization Rat calvarial defect model Implantation of the scaffold Promoted bone regeneration and neovascularization. [ 115 ] BMSC-EVs Solution adsorption to hierarchical MBG scaffold and lyophilization Rat calvarial defect model Implantation of the scaffold Continuous release EV release and promoted bone regeneration [ 116 ] hGMSC-EVs PEI-mediated immobilization to PLA scaffold Rat calvarial defect model Implantation of the scaffold Promoted bone regeneration [ 117 ] ADSC-EVs PDA-assisted solution adsorption to PLGA/PDA scaffold Mouse calvarial defect model Implantation of the scaffold Controlled release of EVs for 8 days and promoted bone regeneration [ 118 ] BMSC-EVs Encapsulation by injectable thermosensitive mPEG-PA Hydrogel Rat calvarial defect model Injection into defects with hydrogel Continuous release of EVs for 3 weeks and accelerated bone regeneration [ 109 ] DPSC-EVs Combined with PLGA-PEG-PLGA triblock copolymer microspheres by microfluidic fabrication and attached to the PLLA scaffold by solvent-wetting Mouse calvarial defect model Implantation of the scaffold Controlled release of EVs and accelerated bone regeneration [ 119 ] BMSC-EVs Encapsulation by SIS-CA/fusion peptides hydrogel …”

“…They highlighted the importance of biglycan, a crucial protein in the ECM, in promoting osteoblast differentiation and mineralization. 109 However, Liu et al demonstrated that exosomes derived from hypoxia-elicited human umbilical cord MSCs (hUMSCs) facilitated bone fracture healing by improved production of exosomal miR-126. 72 In a study by Zhang et al, they identified four microRNAs (hsa-miR-181c-5p, hsa-miR-18a-3p, hsa-miR-376a-5p, and hsa-miR-337-5p) of hypo-EVs that mediated cartilage repair in rat osteoarthritis model.…”

Thermo-induced physically crosslinked polypeptide-based block copolymer hydrogels for biomedical applications

“…In a recent study by Yu, Yu, Liu and co-workers, hypoxic extracellular vesicles (EVs) derived from BMSCs were encapsulated into an mPEG- b -PLAla hydrogel [ 108 ]. The hypoxic EVs-loaded polypeptide hydrogel showed sustained release of the EVs for 3 weeks in vitro .…”