A Targeting Trained Immunity Nanofiber Scaffold for Large Bone Defect Repair

Abstract

Modulating trained immunity while simultaneously initiating regenerative cues presents a significant challenge in large bone defect therapy. This study introduces a cell-free approach utilizing a 3D microenvironment-responsive scaffold to orchestrate immune reprogramming. To mitigate maladaptive trained immunity and activate regenerative signaling, a composite fibrous scaffold is functionalized with immune-engineered exosomes derived from inflammation-primed mesenchymal stem cells (PSS-iEXO) in a reactive oxygen species (ROS)-responsive manner. The PSS-iEXO scaffolds incorporate boronic ester linkages as ROS-sensitive moieties, enabling rapid, dynamic, and “on-demand” exosome release in response to elevated ROS levels characteristic of the early inflammatory phase post-injury, thereby initiating regeneration. In vitro and in vivo analyses reveal that these scaffolds precisely target and modulate maladaptive trained immunity, reprogramming immune responses by shifting macrophage polarization from a hyperactivated type I phenotype to a balanced state while promoting CD4⁺ regulatory T cell activation—both critical for coupling angiogenesis and osteogenesis. Mechanistic insights highlight the role of engineered exosomes in enhancing mitochondrial function and oxidative phosphorylation in macrophages, establishing a cell-free immune-regenerative niche for large bone defect therapy.

Graphical Abstract

Schematic diagram of the fabrication, function, and mechanism of ROS-responsive 3D electrospun nanofiber scaffolds loaded with immunoengineered exosomes (PSS-iEXO) for promoting large bone repair.

Keywords

3D electrospun nanofibrous scaffold / Exosomes / Microenvironment responsive / Trained immunity / Bone regeneration

References

[1] Xiong Y, Mi BB, Lin Z, Hu YQ, Yu L, Zha KK, Panayi AC, Yu T, Chen L, Liu ZP. et al.. The role of the immune microenvironment in bone, cartilage, and soft tissue regeneration: from mechanism to therapeutic opportunity. Mil Med Res, 2022, 9: 65

[2] Loebel C, Burdick JA. Engineering stem and stromal cell therapies for musculoskeletal tissue repair. Cell Stem Cell, 2018, 22: 325-339.

[3] Koushik TM, Miller CM, Antunes E. Bone tissue engineering scaffolds: function of multi-material hierarchically structured scaffolds. Adv Healthc Mater, 2023, 12. e2202766

[4] Vermeulen S, Tahmasebi Birgani Z, Habibovic P. Biomaterial-induced pathway modulation for bone regeneration. Biomaterials, 2022, 283. 121431

[5] Dec P, Modrzejewski A, Pawlik A. Existing and novel biomaterials for bone tissue engineering. Int J Mol Sci. 2022;24.

[6] Maruyama M, Rhee C, Utsunomiya T, Zhang N, Ueno M, Yao Z, Goodman SB. Modulation of the inflammatory response and bone healing. Front Endocrinol (Lausanne), 2020, 11: 386.

[7] Miron RJ, Bohner M, Zhang Y, Bosshardt DD. Osteoinduction and osteoimmunology: emerging concepts. Periodontol, 2000, 2024(94): 9-26

[8] Ochando J, Mulder WJM, Madsen JC, Netea MG, Duivenvoorden R. Trained immunity—basic concepts and contributions to immunopathology. Nat Rev Nephrol, 2023, 19: 23-37.

[9] Locati M, Curtale G, Mantovani A. Diversity, mechanisms, and significance of macrophage plasticity. Annu Rev Pathol, 2020, 15: 123-147.

[10] Dominguez-Andres J, Dos Santos JC, Bekkering S, Mulder WJM, van der Meer JWM, Riksen NP, Joosten LAB, Netea MG. Trained immunity: adaptation within innate immune mechanisms. Physiol Rev, 2023, 103: 313-346.

[11] Netea MG, Quintin J, van der Meer JW. Trained immunity: a memory for innate host defense. Cell Host Microbe, 2011, 9: 355-361.

[12] Rahmani NR, Belluomo R, Kruyt MC, Gawlitta D, Joosten LAB, Weinans H, Croes M. Trained innate immunity modulates osteoblast and osteoclast differentiation. Stem Cell Rev Rep, 2024, 20: 1121-1134.

[13] Zindel J, Kubes P. DAMPs, PAMPs, and LAMPs in immunity and sterile inflammation. Annu Rev Pathol, 2020, 15: 493-518.

[14] Kelly B, O'Neill LA. Metabolic reprogramming in macrophages and dendritic cells in innate immunity. Cell Res, 2015, 25: 771-784.

[15] Schlundt C, Fischer H, Bucher CH, Rendenbach C, Duda GN, Schmidt-Bleek K. The multifaceted roles of macrophages in bone regeneration: a story of polarization, activation and time. Acta Biomater, 2021, 133: 46-57.

[16] Hao Z, Chen T, Wang Y, Feng Q, Chen J, Li H, Wang J, Wang Z, Zhang Z, Chen R. et al.. Self-assembling peptide nanofibers anchored parathyroid hormone derivative for bone tissue engineering. Adv Fiber Mater, 2024, 6: 583-606.

[17] Cui J, Yu B, Li D, Fu Z, Yang X, Jiang L, Wang X, Lin K. Remodeling electrophysiological microenvironment for promoting bone defect repair via electret hybrid electrospun fibrous mat. Adv Fiber Mater, 2024, 6: 1855-1873.

[18] Chen R, Wang Y, Yu C, Zhang X, Wang Y, Yu T, Wu T. Bioactive glass-reinforced hybrid microfibrous spheres promote bone defect repair via stem cell delivery. Adv Fiber Mater, 2025, 7: 240-253.

[19] Liu S, Cheng S, Chen B, Xiao P, Zhan J, Liu J, Chen Z, Liu J, Zhang T, Lei Y, Huang W. Microvesicles-hydrogel breaks the cycle of cellular senescence by improving mitochondrial function to treat osteoarthritis. J Nanobiotechnol, 2023, 21: 429.

[20] Dong L, Li L, Song Y, Fang Y, Liu J, Chen P, Wang S, Wang C, Xia T, Liu W, Yang L. MSC-derived immunomodulatory extracellular matrix functionalized electrospun fibers for mitigating foreign-body reaction and tendon adhesion. Acta Biomater, 2021, 133: 280-296.

[21] Hu S, Li Z, Shen D, Zhu D, Huang K, Su T, Dinh PU, Cores J, Cheng K. Exosome-eluting stents for vascular healing after ischaemic injury. Nat Biomed Eng, 2021, 5: 1174-1188.

[22] Sun H, Xu J, Wang Y, Shen S, Xu X, Zhang L, Jiang Q. Bone microenvironment regulative hydrogels with ROS scavenging and prolonged oxygen-generating for enhancing bone repair. Bioact Mater, 2023, 24: 477-496

[23] Huang Q, Qu Y, Tang M, Lan K, Zhang Y, Chen S, Li W, Gu L. ROS-responsive hydrogel for bone regeneration: controlled dimethyl fumarate release to reduce inflammation and enhance osteogenesis. Acta Biomater, 2025, 195: 183-200.

[24] Lai JJ, Chau ZL, Chen SY, Hill JJ, Korpany KV, Liang NW, Lin LH, Lin YH, Liu JK, Liu YC. et al.. Exosome processing and characterization approaches for research and technology development. Adv Sci (Weinh), 2022, 9. e2103222

[25] Zhang J, Chen L, Wang J, Lei Y, Huang Y, Xu J, Hu N, Huang W, Cui W, Luo X. Multisite captured copper ions via phosphorus dendrons functionalized electrospun short nanofibrous sponges for bone regeneration. Adv Funct Mater. 2023;33:2211237.

[26] Gu X, Ding F, Williams DF. Neural tissue engineering options for peripheral nerve regeneration. Biomaterials, 2014, 35: 6143-6156.

[27] Ferreira AV, Alarcon-Barrera JC, Dominguez-Andres J, Bulut O, Kilic G, Debisarun PA, Roring RJ, Ozhan HN, Terschlusen E, Ziogas A. et al.. Fatty acid desaturation and lipoxygenase pathways support trained immunity. Nat Commun, 2023, 14: 7385.

[28] Chen C, Chang ZH, Yao B, Liu XY, Zhang XW, Liang J, Wang JJ, Bao SQ, Chen MM, Zhu P, Li XH. 3D printing of interferon gamma-preconditioned NSC-derived exosomes/collagen/chitosan biological scaffolds for neurological recovery after TBI. Bioact Mater, 2024, 39: 375-391

[29] Kim J, Lee SK, Jung M, Jeong SY, You H, Won JY, Han SD, Cho HJ, Park S, Park J. et al.. Extracellular vesicles from IFN-gamma-primed mesenchymal stem cells repress atopic dermatitis in mice. J Nanobiotechnol, 2022, 20: 526.

[30] Aazmi A, Zhang D, Mazzaglia C, Yu M, Wang Z, Yang H, Huang YYS, Ma L. Biofabrication methods for reconstructing extracellular matrix mimetics. Bioact Mater, 2024, 31: 475-496

[31] Huang J, Tan Q-C, Bai H, Wang J, Makvandi P, Khan MA, Hu X, Wu Z. Harnessing immunomodulation for efficient bone Regeneration: Bioengineered black phosphorus-incorporated Self-Healing hydrogel. Chem Eng J. 2023;470:144117.

[32] Mader M, Jerome V, Freitag R, Agarwal S, Greiner A. Ultraporous, compressible, wettable polylactide/polycaprolactone sponges for tissue engineering. Biomacromol, 2018, 19: 1663-1673.

[33] Zhang Y, Sun N, Zhu M, Qiu Q, Zhao P, Zheng C, Bai Q, Zeng Q, Lu T. The contribution of pore size and porosity of 3D printed porous titanium scaffolds to osteogenesis. Biomater Adv, 2022, 133. 112651

[34] Jung SH, Hwang BH, Shin S, Park EH, Park SH, Kim CW, Kim E, Choo E, Choi IJ, Swirski FK. et al.. Spatiotemporal dynamics of macrophage heterogeneity and a potential function of Trem2(hi) macrophages in infarcted hearts. Nat Commun, 2022, 13: 4580.

[35] Chen M, Zhang Y, Zhou P, Liu X, Zhao H, Zhou X, Gu Q, Li B, Zhu X, Shi Q. Substrate stiffness modulates bone marrow-derived macrophage polarization through NF-kappaB signaling pathway. Bioact Mater, 2020, 5: 880-890

[36] Niu Y, Wang Z, Shi Y, Dong L, Wang C. Modulating macrophage activities to promote endogenous bone regeneration: biological mechanisms and engineering approaches. Bioact Mater, 2021, 6: 244-261

[37] Zhao J, Andreev I, Silva HM. Resident tissue macrophages: key coordinators of tissue homeostasis beyond immunity. Sci Immunol., 2024, 9: eadd1967.

[38] Van den Bossche J, Baardman J, Otto NA, van der Velden S, Neele AE, van den Berg SM, Luque-Martin R, Chen HJ, Boshuizen MC, Ahmed M. et al.. Mitochondrial dysfunction prevents repolarization of inflammatory macrophages. Cell Rep, 2016, 17: 684-696.

[39] Li K, Yan G, Huang H, Zheng M, Ma K, Cui X, Lu D, Zheng L, Zhu B, Cheng J, Zhao J. Anti-inflammatory and immunomodulatory effects of the extracellular vesicles derived from human umbilical cord mesenchymal stem cells on osteoarthritis via M2 macrophages. J Nanobiotechnol, 2022, 20: 38.

[40] Pang L, Jin H, Lu Z, Xie F, Shen H, Li X, Zhang X, Jiang X, Wu L, Zhang M. et al.. Treatment with mesenchymal stem cell-derived nanovesicle-containing gelatin methacryloyl hydrogels alleviates osteoarthritis by modulating chondrogenesis and macrophage polarization. Adv Healthc Mater, 2023, 12. e2300315

[41] Nakao Y, Fukuda T, Zhang Q, Sanui T, Shinjo T, Kou X, Chen C, Liu D, Watanabe Y, Hayashi C. et al.. Exosomes from TNF-alpha-treated human gingiva-derived MSCs enhance M2 macrophage polarization and inhibit periodontal bone loss. Acta Biomater, 2021, 122: 306-324.

[42] Ferreira JR, Teixeira GQ, Santos SG, Barbosa MA, Almeida-Porada G, Goncalves RM. Mesenchymal stromal cell secretome: influencing therapeutic potential by cellular pre-conditioning. Front Immunol, 2018, 9: 2837.

[43] Gao Q, Jia F, Li X, Kong Y, Tian Z, Bi L, Li L. Biophysical cues to improve the immunomodulatory capacity of mesenchymal stem cells: the progress and mechanisms. Biomed Pharmacother, 2023, 162. 114655

[44] Yang Y, Wu Y, Yang D, Neo SH, Kadir ND, Goh D, Tan JX, Denslin V, Lee EH, Yang Z. Secretive derived from hypoxia preconditioned mesenchymal stem cells promote cartilage regeneration and mitigate joint inflammation via extracellular vesicles. Bioact Mater, 2023, 27: 98-112

[45] Jalilian E, Massoumi H, Bigit B, Amin S, Katz EA, Guaiquil VH, Anwar KN, Hematti P, Rosenblatt MI, Djalilian AR. Bone marrow mesenchymal stromal cells in a 3D system produce higher concentration of extracellular vesicles (EVs) with increased complexity and enhanced neuronal growth properties. Stem Cell Res Ther, 2022, 13: 425.

[46] Liu K, Li L, Chen J, Li Y, Wen W, Lu L, Li L, Li H, Liu M, Zhou C, Luo B. Bone ECM-like 3D printing scaffold with liquid crystalline and viscoelastic microenvironment for bone regeneration. ACS Nano, 2022, 16: 21020-21035.

[47] Heng BC, Bai Y, Li X, Lim LW, Li W, Ge Z, Zhang X, Deng X. Electroactive biomaterials for facilitating bone defect repair under pathological conditions. Adv Sci (Weinh), 2023, 10. e2204502

[48] Wu M, Chen G, Li YP. TGF-beta and BMP signaling in osteoblast, skeletal development, and bone formation, homeostasis and disease. Bone Res, 2016, 4: 16009.

[49] Diomede F, Marconi GD, Fonticoli L, Pizzicanella J, Merciaro I, Bramanti P, Mazzon E, Trubiani O. Functional relationship between osteogenesis and angiogenesis in tissue regeneration. Int J Mol Sci. 2020;21:3242.

[50] Yang N, Liu Y. The role of the immune microenvironment in bone regeneration. Int J Med Sci, 2021, 18: 3697-3707.

[51] Mulder WJM, Ochando J, Joosten LAB, Fayad ZA, Netea MG. Therapeutic targeting of trained immunity. Nat Rev Drug Discov, 2019, 18: 553-566.

[52] Diefenhardt P, Nosko A, Kluger MA, Richter JV, Wegscheid C, Kobayashi Y, Tiegs G, Huber S, Flavell RA, Stahl RAK, Steinmetz OM. IL-10 receptor signaling empowers regulatory T cells to control Th17 responses and protect from GN. J Am Soc Nephrol, 2018, 29: 1825-1837.

[53] Patel S, Schmidt KF, Farhoud M, Zi T, Jang SC, Dooley K, Kentala D, Dobson H, Economides K, Williams DE. In vivo tracking of [(89)Zr]Zr-labeled engineered extracellular vesicles by PET reveals organ-specific biodistribution based upon the route of administration. Nucl Med Biol, 2022, 112–113: 20-30.

[54] Jiang H, Lou C, Jiang L, Lin C, Wang W, Yan Z, Yu J, Cai T, Lin S, Wang J. et al.. Simvastatin-enhanced bioinspired exosome mimetics regulate osteogenesis and angiogenesis for the treatment of glucocorticoid-induced osteonecrosis of the femoral head. Chem Eng J, 2023, 472. 144729

[55] Qin L, Yang J, Su X, Xilan L, Lei Y, Dong L, Chen H, Chen C, Zhao C, Zhang H. et al.. The miR-21-5p enriched in the apoptotic bodies of M2 macrophage-derived extracellular vesicles alleviates osteoarthritis by changing macrophage phenotype. Genes Dis, 2023, 10: 1114-1129.

[56] Xiao H, Zou Y, Wang J, Wan S. A review for artificial intelligence based protein subcellular localization. Biomolecules. 2024;14:409.

[57] Van den Bossche J, O'Neill LA, Menon D. Macrophage immunometabolism: where are we (going)?. Trends Immunol, 2017, 38: 395-406.

[58] Benmoussa K, Garaude J, Acin-Perez R. How mitochondrial metabolism contributes to macrophage phenotype and functions. J Mol Biol, 2018, 430: 3906-3921.

[59] Lloberas J, Munoz JP, Hernandez-Alvarez MI, Cardona PJ, Zorzano A, Celada A. Macrophage mitochondrial MFN2 (mitofusin 2) links immune stress and immune response through reactive oxygen species (ROS) production. Autophagy, 2020, 16: 2307-2309.

[60] Tur J, Pereira-Lopes S, Vico T, Marin EA, Munoz JP, Hernandez-Alvarez M, Cardona PJ, Zorzano A, Lloberas J, Celada A. Mitofusin 2 in macrophages links mitochondrial ROS production, cytokine release, phagocytosis, autophagy, and bactericidal activity. Cell Rep, 2020, 32. 108079

[61] Wang Y, Li N, Zhang X, Horng T. Mitochondrial metabolism regulates macrophage biology. J Biol Chem, 2021, 297. 100904

[62] Willenborg S, Sanin DE, Jais A, Ding X, Ulas T, Nuchel J, Popovic M, MacVicar T, Langer T, Schultze JL. et al.. Mitochondrial metabolism coordinates stage-specific repair processes in macrophages during wound healing. Cell Metab, 2021, 33(2398–2414e2399

[63] El Kasmi KC, Stenmark KR. Contribution of metabolic reprogramming to macrophage plasticity and function. Semin Immunol, 2015, 27: 267-275.

[64] Ryan DG, Murphy MP, Frezza C, Prag HA, Chouchani ET, O'Neill LA, Mills EL. Coupling Krebs cycle metabolites to signalling in immunity and cancer. Nat Metab, 2019, 1: 16-33.

[65] Chen W, Zhao H, Li Y. Mitochondrial dynamics in health and disease: mechanisms and potential targets. Signal Transduct Target Ther, 2023, 8: 333.

[66] Zhou J, Duan M, Wang X, Zhang F, Zhou H, Ma T, Yin Q, Zhang J, Tian F, Wang G, Yang C. A feedback loop engaging propionate catabolism intermediates controls mitochondrial morphology. Nat Cell Biol, 2022, 24: 526-537.

[67] Cogliati S, Frezza C, Soriano ME, Varanita T, Quintana-Cabrera R, Corrado M, Cipolat S, Costa V, Casarin A, Gomes LC. et al.. Mitochondrial cristae shape determines respiratory chain supercomplexes assembly and respiratory efficiency. Cell, 2013, 155: 160-171.

[68] Nolfi-Donegan D, Braganza A, Shiva S. Mitochondrial electron transport chain: oxidative phosphorylation, oxidant production, and methods of measurement. Redox Biol, 2020, 37. 101674

[69] Zickermann V, Wirth C, Nasiri H, Siegmund K, Schwalbe H, Hunte C, Brandt U. Structural biology. Mechanistic insight from the crystal structure of mitochondrial complex I. Science, 2015, 347: 44-49.

[70] Rodriguez-Nuevo A, Torres-Sanchez A, Duran JM, De Guirior C, Martinez-Zamora MA, Boke E. Oocytes maintain ROS-free mitochondrial metabolism by suppressing complex I. Nature, 2022, 607: 756-761.

[71] Guo H, Rubinstein JL. Structure of ATP synthase under strain during catalysis. Nat Commun, 2022, 13: 2232.

[72] Xu J, Ji L, Ruan Y, Wan Z, Lin Z, Xia S, Tao L, Zheng J, Cai L, Wang Y. et al.. UBQLN1 mediates sorafenib resistance through regulating mitochondrial biogenesis and ROS homeostasis by targeting PGC1beta in hepatocellular carcinoma. Signal Transduct Target Ther, 2021, 6: 190.

[73] Chang X, Li Y, Cai C, Wu F, He J, Zhang Y, Zhong J, Tan Y, Liu R, Zhu H, Zhou H. Mitochondrial quality control mechanisms as molecular targets in diabetic heart. Metabolism, 2022, 137. 155313

[74] Rosario FJ, Gupta MB, Myatt L, Powell TL, Glenn JP, Cox L, Jansson T. Mechanistic target of rapamycin complex 1 promotes the expression of genes encoding electron transport chain proteins and stimulates oxidative phosphorylation in primary human trophoblast cells by regulating mitochondrial biogenesis. Sci Rep, 2019, 9: 246.

[75] Halling JF, Pilegaard H. PGC-1alpha-mediated regulation of mitochondrial function and physiological implications. Appl Physiol Nutr Metab, 2020, 45: 927-936.

[76] Fan H, Ding R, Liu W, Zhang X, Li R, Wei B, Su S, Jin F, Wei C, He X. et al.. Heat shock protein 22 modulates NRF1/TFAM-dependent mitochondrial biogenesis and DRP1-sparked mitochondrial apoptosis through AMPK-PGC1alpha signaling pathway to alleviate the early brain injury of subarachnoid hemorrhage in rats. Redox Biol, 2021, 40. 101856

[77] Zheng Q, Liu H, Zhang H, Han Y, Yuan J, Wang T, Gao Y, Li Z. Ameliorating mitochondrial dysfunction of neurons by biomimetic targeting nanoparticles mediated mitochondrial biogenesis to boost the therapy of Parkinson's disease. Adv Sci (Weinh), 2023, 10. e2300758

[78] Ikeda G, Santoso MR, Tada Y, Li AM, Vaskova E, Jung JH, O'Brien C, Egan E, Ye J, Yang PC. Mitochondria-rich extracellular vesicles from autologous stem cell-derived cardiomyocytes restore energetics of ischemic myocardium. J Am Coll Cardiol, 2021, 77: 1073-1088.

Funding

Natural Science Foundation of Chongqing(CSTB2022NSCQ-BHX0683)

National Natural Science Foundation of China-Joint Fund Project(U22A20284)

National Natural Science Foundation of China(12472320)

Postdoctoral Special Funding Project of Chongqing Human Resources and Social Security Bureau(2112012726787861)

Innovative Project for Doctoral Students of the First Affiliated Hospital of Chongqing Medical University(CYYY-BSYJSCXXM-202308)

RIGHTS & PERMISSIONS

Donghua University, Shanghai, China