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

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.

[1]  M. Heke,et al.  Stem cell-based therapy for human diseases , 2022, Signal Transduction and Targeted Therapy.

[2]  Hancheng Wang,et al.  Sustained Release of Nitric Oxide and Cascade Generation of Reactive Nitrogen/Oxygen Species via an Injectable Hydrogel for Tumor Synergistic Therapy , 2022, Advanced Functional Materials.

[3]  G. Wang,et al.  Research and clinical translation of trilayer stent-graft of expanded polytetrafluoroethylene for interventional treatment of aortic dissection , 2022, Regenerative biomaterials.

[4]  Zhi-Qin Zhao,et al.  The effect of AKT in extracellular matrix stiffness induced osteogenic differentiation of hBMSCs. , 2022, Cellular signalling.

[5]  Bin Liu,et al.  A 3D bioprinted nano-laponite hydrogel construct promotes osteogenesis by activating PI3K/AKT signaling pathway , 2022, Materials today. Bio.

[6]  Yusheng Shi,et al.  Enhanced tissue regeneration through immunomodulation of angiogenesis and osteogenesis with a multifaceted nanohybrid modified bioactive scaffold , 2022, Bioactive materials.

[7]  A. Sabino,et al.  Preconditioning Methods to Improve Mesenchymal Stromal Cell-Derived Extracellular Vesicles in Bone Regeneration—A Systematic Review , 2022, Biology.

[8]  H. Cao,et al.  Effects of BMSC-Derived EVs on Bone Metabolism , 2022, Pharmaceutics.

[9]  Chuanglong He,et al.  3D bioprinted gelatin/gellan gum-based scaffold with double-crosslinking network for vascularized bone regeneration. , 2022, Carbohydrate polymers.

[10]  N. Chiba,et al.  HIF‐1α plays an essential role in BMP9‐mediated osteoblast differentiation through the induction of a glycolytic enzyme, PDK1 , 2022, Journal of cellular physiology.

[11]  Si Wang,et al.  Dynamically Bioresponsive DNA Hydrogel Incorporated with Dual-Functional Stem Cells from Apical Papilla-Derived Exosomes Promotes Diabetic Bone Regeneration. , 2022, ACS applied materials & interfaces.

[12]  Q. Cai,et al.  Hierarchical Therapeutic Ion‐Based Microspheres with Precise Ratio‐Controlled Delivery as Microscaffolds for In Situ Vascularized Bone Regeneration , 2022, Advanced Functional Materials.

[13]  Shaobo Ruan,et al.  Light‐induced high‐efficient cellular production of immune functional extracellular vesicles , 2022, Journal of extracellular vesicles.

[14]  Changsheng Liu,et al.  Fabrication and evaluation of a BMP-2/dexamethasone co-loaded gelatin sponge scaffold for rapid bone regeneration , 2022, Regenerative biomaterials.

[15]  Yuming Zhao,et al.  Bioinspired porous microspheres for sustained hypoxic exosomes release and vascularized bone regeneration , 2022, Bioactive materials.

[16]  Xiao Zhang,et al.  Exosomes derived from hypoxia preconditioned mesenchymal stem cells laden in a silk hydrogel promote cartilage regeneration via the miR-205-5p/PTEN/AKT pathway. , 2022, Acta biomaterialia.

[17]  Zhi-hao Li,et al.  A comparative analysis of the osteogenic capacity of osteoblasts from newborn and two-week-old rats. , 2022, Acta histochemica.

[18]  Changqing Zhang,et al.  Bone Mesenchymal Stem Cell-Derived sEV-Encapsulated Thermosensitive Hydrogels Accelerate Osteogenesis and Angiogenesis by Release of Exosomal miR-21 , 2022, Frontiers in Bioengineering and Biotechnology.

[19]  S. Cha,et al.  Folic acid pretreatment and its sustained delivery for chondrogenic differentiation of MSCs. , 2022, Journal of controlled release : official journal of the Controlled Release Society.

[20]  Xiufeng Xiao,et al.  Osteoblast/Osteoclast and Immune Cocktail Therapy of an Exosome/Drug Delivery Multifunctional Hydrogel Accelerates Fracture Repair. , 2022, ACS nano.

[21]  Yingjia Sun,et al.  Extracellular vesicles derived from neural EGFL-Like 1-modified mesenchymal stem cells improve acellular bone regeneration via the miR-25-5p-SMAD2 signaling axis , 2022, Bioactive materials.

[22]  Jinlin Song,et al.  A Logic‐Based Diagnostic and Therapeutic Hydrogel with Multistimuli Responsiveness to Orchestrate Diabetic Bone Regeneration , 2021, Advanced materials.

[23]  Y. Lai,et al.  3D-printed NIR-responsive shape memory polyurethane/magnesium scaffolds with tight-contact for robust bone regeneration , 2021, Bioactive materials.

[24]  Rui Zhu,et al.  Mechanically reinforced injectable bioactive nanocomposite hydrogels for in-situ bone regeneration , 2021, Chemical Engineering Journal.

[25]  Kun Wang,et al.  Engineering Extracellular Vesicles Restore the Impaired Cellular Uptake and Attenuate Intervertebral Disc Degeneration. , 2021, ACS nano.

[26]  Xiaowei Yang,et al.  Decisive Influence of Hydrophobic Side Chains of Polyesters on Thermoinduced Gelation of Triblock Copolymer Aqueous Solutions , 2021, Macromolecules.

[27]  Shuyun Liu,et al.  Magnetic resonance imaging for non-invasive clinical evaluation of normal and regenerated cartilage , 2021, Regenerative biomaterials.

[28]  Sushant P Sahu,et al.  Multimodal Label‐Free Monitoring of Adipogenic Stem Cell Differentiation Using Endogenous Optical Biomarkers , 2021, Advanced functional materials.

[29]  Xuesi Chen,et al.  Biofunctionalized composite scaffold to potentiate osteoconduction, angiogenesis, and favorable metabolic microenvironment for osteonecrosis therapy , 2021, Bioactive Materials.

[30]  Xin Zhao,et al.  Bone-a-Petite: Engineering Exosomes towards Bone, Osteochondral, and Cartilage Repair. , 2021, Small.

[31]  M. J. Wood,et al.  Extracellular vesicles as a next-generation drug delivery platform , 2021, Nature Nanotechnology.

[32]  Zhiyun Lu,et al.  Tetraphenylethylene-conjugated polycation covered iron oxide nanoparticles for magnetic resonance/optical dual-mode imaging , 2021, Regenerative biomaterials.

[33]  L. Deng,et al.  Gradient bimetallic ion–based hydrogels for tissue microstructure reconstruction of tendon-to-bone insertion , 2021, Science Advances.

[34]  Yi Zhang,et al.  Surface engineering of titania nanotubes incorporated with double-layered extracellular vesicles to modulate inflammation and osteogenesis , 2021, Regenerative biomaterials.

[35]  Eric A. Appel,et al.  Translational Applications of Hydrogels , 2021, Chemical reviews.

[36]  Ibrahim T. Ozbolat,et al.  Intra‐Operative Bioprinting of Hard, Soft, and Hard/Soft Composite Tissues for Craniomaxillofacial Reconstruction , 2021, Advanced functional materials.

[37]  Dan Lin,et al.  Optimized BMSC-derived osteoinductive exosomes immobilized in hierarchical scaffold via lyophilization for bone repair through Bmpr2/Acvr2b competitive receptor-activated Smad pathway. , 2021, Biomaterials.

[38]  Yu Su,et al.  Current Advances and Challenges of Mesenchymal Stem Cells-Based Drug Delivery System and Their Improvements. , 2021, International journal of pharmaceutics.

[39]  Zhirong Liu,et al.  Manipulation of Stem Cells Fates: The Master and Multifaceted Roles of Biophysical Cues of Biomaterials , 2021, Advanced Functional Materials.

[40]  Yanfei Liu,et al.  Biomimetic cell-adhesive ligand-functionalized peptide composite hydrogels maintain stemness of human amniotic mesenchymal stem cells , 2021, Regenerative biomaterials.

[41]  Andrew L. Ferguson,et al.  Accelerated polymerization of N-carboxyanhydrides catalyzed by crown ether , 2021, Nature Communications.

[42]  S. Hofmann,et al.  Cell Sources for Human In vitro Bone Models , 2021, Current Osteoporosis Reports.

[43]  Y. You,et al.  Extracellular vesicles derived from hypoxic glioma stem-like cells confer temozolomide resistance on glioblastoma by delivering miR-30b-3p , 2021, Theranostics.

[44]  Jiaming Sun,et al.  Simple application of adipose-derived stem cell-derived extracellular vesicles coating enhances cytocompatibility and osteoinductivity of titanium implant , 2020, Regenerative biomaterials.

[45]  L. Deng,et al.  Bioinspired Functional Black Phosphorus Electrospun Fibers Achieving Recruitment and Biomineralization for Staged Bone Regeneration. , 2020, Small.

[46]  P. Ma,et al.  Scaffolds with Controlled Release of Pro-Mineralization Exosomes to Promote Craniofacial Bone Healing without Cell Transplantation. , 2020, Acta biomaterialia.

[47]  Xiaowei Yang,et al.  Sustained release of lipophilic gemcitabine from an injectable polymeric hydrogel for synergistically enhancing tumor chemoradiotherapy , 2020 .

[48]  Jiulong Zhang,et al.  Visualizing the In Vivo Evolution of an Injectable and Thermosensitive Hydrogel Using Tri‐Modal Bioimaging , 2020, Small Methods.

[49]  S. Varderidou-Minasian,et al.  Mesenchymal stromal/stem cell-derived extracellular vesicles in tissue repair: challenges and opportunities , 2020, Theranostics.

[50]  R. Lanza,et al.  Next-generation stem cells — ushering in a new era of cell-based therapies , 2020, Nature Reviews Drug Discovery.

[51]  D. Mooney,et al.  Biomaterials Functionalized with MSC Secreted Extracellular Vesicles and Soluble Factors for Tissue Regeneration , 2020, Advanced functional materials.

[52]  X. Niu,et al.  Human ESC-sEVs alleviate age-related bone loss by rejuvenating senescent bone marrow-derived mesenchymal stem cells , 2020, Journal of extracellular vesicles.

[53]  V. Moreno-Manzano,et al.  Human adipose-derived mesenchymal stem cells accelerate decellularized neobladder regeneration , 2019, Regenerative biomaterials.

[54]  Jin Fan,et al.  Hypoxic mesenchymal stem cell-derived exosomes promote bone fracture healing by the transfer of miR-126. , 2019, Acta biomaterialia.

[55]  M. Nagano,et al.  Rab5-mediated endosome formation is regulated at the trans-Golgi network , 2019, Communications Biology.

[56]  Xuesi Chen,et al.  Electroactive composite scaffold with locally expressed osteoinductive factor for synergistic bone repair upon electrical stimulation. , 2019, Biomaterials.

[57]  Wei Zhang,et al.  Extracellular IL-37 promotes osteogenic differentiation of human bone marrow mesenchymal stem cells via activation of the PI3K/AKT signaling pathway , 2019, Cell Death & Disease.

[58]  shanliang song,et al.  Biomimetic Composite Scaffolds to Manipulate Stem Cells for Aiding Rheumatoid Arthritis Management , 2019, Advanced Functional Materials.

[59]  Oded Shoseyov,et al.  A BMP/activin A chimera is superior to native BMPs and induces bone repair in nonhuman primates when delivered in a composite matrix , 2019, Science Translational Medicine.

[60]  Xuesi Chen,et al.  Chiral Polypeptide Thermogels Induce Controlled Inflammatory Response as Potential Immunoadjuvants. , 2019, ACS applied materials & interfaces.

[61]  Bin Wang,et al.  HIF-1α inducing exosomal microRNA-23a expression mediates the cross-talk between tubular epithelial cells and macrophages in tubulointerstitial inflammation. , 2019, Kidney international.

[62]  C. Théry,et al.  Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication , 2019, Nature Cell Biology.

[63]  Aijun Wang,et al.  Engineering mesenchymal stem cells to improve their exosome efficacy and yield for cell-free therapy , 2018, Journal of extracellular vesicles.

[64]  T. Komori.  Runx2, an inducer of osteoblast and chondrocyte differentiation , 2018, Histochemistry and Cell Biology.

[65]  Shi-Cong Tao,et al.  Modularized Extracellular Vesicles: The Dawn of Prospective Personalized and Precision Medicine , 2018, Advanced science.

[66]  R. Schiffelers,et al.  Cellular uptake of extracellular vesicles is mediated by clathrin‐independent endocytosis and macropinocytosis , 2017, Journal of controlled release : official journal of the Controlled Release Society.

[67]  Wei Zhang,et al.  HIF-1-mediated production of exosomes during hypoxia is protective in renal tubular cells. , 2017, American journal of physiology. Renal physiology.

[68]  Wenjun Wang,et al.  The PI3K/Akt pathway: a critical player in intervertebral disc degeneration , 2017, Oncotarget.

[69]  Hongwei Liang,et al.  Pyruvate kinase type M2 promotes tumour cell exosome release via phosphorylating synaptosome-associated protein 23 , 2017, Nature Communications.

[70]  Lin Yu,et al.  Controlled release of liraglutide using thermogelling polymers in treatment of diabetes , 2016, Scientific Reports.

[71]  A. Theocharis,et al.  Extracellular matrix structure. , 2016, Advanced drug delivery reviews.

[72]  Zhenhua Li,et al.  Effects of cell-cell contact and oxygen tension on chondrogenic differentiation of stem cells. , 2015, Biomaterials.

[73]  Driton Vllasaliu,et al.  Mechanisms of nanoparticle internalization and transport across an intestinal epithelial cell model: effect of size and surface charge. , 2014, Molecular pharmaceutics.

[74]  D. Zhu,et al.  Lentiviral delivery of biglycan promotes proliferation and increases osteogenic potential of bone marrow-derived mesenchymal stem cells in vitro , 2013, Journal of Molecular Histology.

[75]  R. Jahn,et al.  Membrane Fusion Intermediates via Directional and Full Assembly of the SNARE Complex , 2012, Science.

[76]  Fanxin Long,et al.  Building strong bones: molecular regulation of the osteoblast lineage , 2011, Nature Reviews Molecular Cell Biology.

[77]  M. Yamauchi,et al.  Role of glycosaminoglycans of biglycan in BMP-2 signaling. , 2011, Biochemical and biophysical research communications.

[78]  Qiang Zhou,et al.  Cellular Internalization of Exosomes Occurs Through Phagocytosis , 2010, Traffic.

[79]  G. Burnstock,et al.  Hypoxia stimulates vesicular ATP release from rat osteoblasts , 2009, Journal of cellular physiology.

[80]  H. Lee,et al.  Enzymatically degradable temperature-sensitive polypeptide as a new in-situ gelling biomaterial. , 2009, Journal of controlled release : official journal of the Controlled Release Society.

[81]  D. Prockop,et al.  Concise Review: Mesenchymal Stem/Multipotent Stromal Cells: The State of Transdifferentiation and Modes of Tissue Repair—Current Views , 2007, Stem cells.

[82]  Woo-Kul Lee,et al.  Gelation Behavior of Poly(ethylene glycol) and Polycaprolactone Triblock and Multiblock Copolymer Aqueous Solutions , 2006 .

[83]  R. Bruick,et al.  Oxygen sensing in the hypoxic response pathway: regulation of the hypoxia-inducible transcription factor. , 2003, Genes & development.