Coral-inspired immunoreprogramming scaffold reverses the “immune-freeze” microenvironment to promote bone regeneration in steroid-induced osteonecrosis of the femoral head

Abstract

The core pathological mechanism of steroid-induced osteonecrosis of the femoral head (SONFH) is an “immune freeze” microenvironment—where corticosteroids continuously drive macrophages toward a pro-inflammatory M1 phenotype, inhibiting the conversion to reparative M2 macrophages, thereby impairing bone regeneration. To address this bottleneck, this study was inspired by the hierarchical pore structure of coral. Using low-temperature deposition 3D printing technology, we constructed an immunoreprogramming biomimetic scaffold that integrates multi-walled carbon nanotubes (MWCNT) and nano-hydroxyapatite (nHA) (MWCNT bionic scaffold). The scaffold leverages the active immune regulatory function of MWCNT to activate the PI3K-AKT signaling pathway, driving macrophages to transition from the M1 to M2 phenotype, effectively breaking the “immunological freeze” state and reshaping the pro-regenerative bone immune microenvironment. Meanwhile, the nHA component provides a biomimetic mineralization matrix and sustained release of calcium and phosphate ions, synergizing with the nanofiber structure of MWCNT to promote the migration and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and angiogenesis. In vivo experiments confirm that scaffold implantation reverses the local “immune freeze” state, drives macrophage polarization toward the M2 phenotype, reduces inflammatory responses, and enhances the maturation and functional vascular network formation of new bone matrix in bone defect areas, ultimately achieving bone structural reconstruction. The coral-inspired immunoreprogramming strategy proposed in this study provides a new strategy for targeting the regulation of the pathological microenvironment in SONFH.

References

[1] Weinstein RS. Glucocorticoid-induced osteonecrosis. Endocrine, 2012, 41: 183-190

[2] Duan P, et al. . Correction: Exosomal miR-1a-3p derived from glucocorticoid-stimulated M1 macrophages promotes the adipogenic differentiation of BMSCs in glucocorticoid-associated osteonecrosis of the femoral head by targeting Cebpz. J. Nanobiotechnol., 2024, 22 694

[3] Ma M, et al. . Osteoimmunology and osteonecrosis of the femoral head. Bone Jt. Res, 2022, 11: 26-28

[4] Zhang Q, Sun W, Li T, Liu F. Polarization behavior of bone macrophage as well as associated osteoimmunity in glucocorticoid-induced osteonecrosis of the femoral head. J. Inflamm. Res., 2023, 16: 879-894

[5] Kong N, et al. . An injectable self-adaptive polymer as a drug carrier for the treatment of nontraumatic early-stage osteonecrosis of the femoral head. Bone Res, 2022, 10: 28

[6] Tsubosaka M, et al. . Preclinical models for studying corticosteroid-induced osteonecrosis of the femoral head. J. Biomed. Mater. Res. B Appl. Biomater., 2024, 112 e35360

[7] Quan H, et al. . Application of biomaterials in treating early osteonecrosis of the femoral head: Research progress and future perspectives. Acta Biomater., 2023, 164: 15-73

[8] Lai Y, et al. . Porous composite scaffold incorporating osteogenic phytomolecule icariin for promoting skeletal regeneration in challenging osteonecrotic bone in rabbits. Biomaterials, 2018, 153: 1-13

[9] Sun Y, et al. . Polydopamine grafting polyether ether ketone to stabilize growth factor for efficient osteonecrosis repair. Sci. Rep., 2025, 15 3697

[10] Long J, et al. . Regulation of osteoimmune microenvironment and osteogenesis by 3D-printed PLAG/black phosphorus scaffolds for bone regeneration. Adv. Sci., 2023, 10 e2302539

[11] Mondal S, et al. . Hydroxyapatite: a journey from biomaterials to advanced functional materials. Adv. Colloid Interface Sci., 2023, 321 103013

[12] Biedrzycka A, Skwarek E. Composites of hydroxyapatite and their application in adsorption, medicine and as catalysts. Adv. Colloid Interface Sci., 2024, 334 103308

[13] Zhang, J., Cao, J., Liu, Y. & Zhao, H. Advances in the pathogenesis of steroid-associated osteonecrosis of the femoral head. Biomolecules14, 667 (2024).

[14] Li H, et al. . Nitrogen-doped multiwalled carbon nanotubes enhance bone remodeling through immunomodulatory functions. ACS Appl. Mater. Interfaces, 2021, 13: 25290-25305

[15] Ding K, et al. . Multiwalled carbon nanotubes-reprogrammed macrophages facilitate breast cancer metastasis via NBR2/TBX1 Axis. ACS Nano, 2024, 18: 11103-11119

[16] Lin R, et al. . Multi-walled carbon nanotubes reversing the bone formation of bone marrow stromal cells by activating M2 macrophage polarization. Regen. Biomater., 2023, 10 rbad042

[17] E Silva, E. P. et al. In vivo study of conductive 3D printed PCL/MWCNTs scaffolds with electrical stimulation for bone tissue engineering. Bio-Design Manufact.4, 190–202 (2021).

[18] Luo S, et al. . Advances in electroactive biomaterials: Through the lens of electrical stimulation promoting bone regeneration strategy. J. Orthop. Transl., 2024, 47: 191-206

[19] Liu J, et al. . Functional cobalt-doped hydrogel scaffold enhances concurrent vascularization and neurogenesis. J. Nanobiotechnol., 2025, 23 179

[20] Liu L, et al. . Biomimetic bone tissue engineering hydrogel scaffolds constructed using ordered CNTs and HA induce the proliferation and differentiation of BMSCs. J. Mater. Chem. B, 2020, 8: 558-567

[21] Shimizu M, et al. . Carbon nanotubes induce bone calcification by bidirectional interaction with osteoblasts. Adv. Mater., 2012, 24: 2176-2185

[22] Gupta B, Sharma PK, Malviya R. Carbon nanotubes for targeted therapy: safety, efficacy, feasibility and regulatory aspects. Curr. Pharm. Des., 2024, 30: 81-99

[23] Green DW, Ben-Nissan B, Yoon KS, Milthorpe B, Jung HS. Natural and synthetic coral biomineralization for human bone revitalization. Trends Biotechnol., 2017, 35: 43-54

[24] Wan MC, et al. . Biomaterials from the sea: future building blocks for biomedical applications. Bioact. Mater., 2021, 6: 4255-4285

[25] Luo, Z. et al. Coral-inspired bioactive porous adhesive for fractured bone repair. Adv. Funct. Mater.35, 2507592 (2025).

[26] Ying, G. et al. Bioprinted injectable hierarchically porous gelatin methacryloyl hydrogel constructs with shape-memory properties. Adv. Funct. Mater.30 (2020).

[27] Liu W, et al. . Low-temperature deposition manufacturing: a novel and promising rapid prototyping technology for the fabrication of tissue-engineered scaffold. Mater. Sci. Eng. C. Mater. Biol. Appl, 2017, 70: 976-982

[28] Nie, P. et al. Preparation and tribological properties of polyimide/carboxyl-functionalized multi-walled carbon nanotube nanocomposite films under seawater lubrication. Tribol. Lett.58, 7 (2015).

[29] Luo Y, Li D, Zhao J, Yang Z, Kang P. In vivo evaluation of porous lithium-doped hydroxyapatite scaffolds for the treatment of bone defect. Biomed. Mater. Eng., 2018, 29: 699-721

[30] Liang HF, et al. . Biomimetic structural protein based magnetic responsive scaffold for enhancing bone regeneration by physical stimulation on intracellular calcium homeostasis. Adv. Health Mater., 2023, 12 e2301724

[31] Li XR, et al. . 3D cryo-printed hierarchical porous scaffolds provide immobilization of surface-functionalized sleep-inspired small extracellular vesicles: synergistic therapeutic strategies for vascularized bone regeneration based on macrophage phenotype modulation and angiogenesis-osteogenesis coupling. J. Nanobiotechnol., 2024, 22 764

[32] Tabia Z, Bricha M, El Mabrouk K, Vaudreuil S. Manufacturing of a metallic 3D framework coated with a bioglass matrix for implant applications. J. Mater. Sci., 2020, 56: 1658-1672

[33] Khoramgah MS, et al. . Freeze-dried multiscale porous nanofibrous three-dimensional scaffolds for bone regeneration. Bioimpacts, 2020, 10: 73-85

[34] Bai L, et al. . Multifunctional scaffold comprising metal-organic framework, hydrogel, and demineralized bone matrix for the treatment of steroid-induced femoral head necrosis. Small, 2025, 21 e2407758

[35] Ding T, Kang W, Li J, Yu L, Ge S. An in situ tissue engineering scaffold with growth factors combining angiogenesis and osteoimmunomodulatory functions for advanced periodontal bone regeneration. J. Nanobiotechnol., 2021, 19 247

[36] Wang J, et al. . BMP-2 functional polypeptides relieve osteolysis via bi-regulating bone formation and resorption coupled with macrophage polarization. NPJ Regen. Med., 2023, 8: 6

[37] Wu M, et al. . Smart-responsive multifunctional therapeutic system for improved regenerative microenvironment and accelerated bone regeneration via mild photothermal therapy. Adv. Sci., 2024, 11 e2304641

[38] Li Z, et al. . Advances in experimental models of osteonecrosis of the femoral head. J. Orthop. Transl., 2023, 39: 88-99

[39] Dempster DW, et al. . Standardized nomenclature, symbols, and units for bone histomorphometry: a 2012 update of the report of the ASBMR Histomorphometry Nomenclature Committee. J. Bone Miner. Res., 2013, 28: 2-17

[40] van Gaalen SM, et al. . Use of fluorochrome labels in in vivo bone tissue engineering research. Tissue Eng. B Rev., 2010, 16: 209-217

[41] Meng X, et al. . An impaired healing model of osteochondral defect in papain-induced arthritis. J. Orthop. Transl., 2021, 26: 101-110

[42] Mehl J, et al. . External mechanical stability regulates hematoma vascularization in bone healing rather than endothelial YAP/TAZ mechanotransduction. Adv. Sci. (Weinh.), 2024, 11e2307050

[43] Yu H, et al. . Improved tissue-engineered bone regeneration by endothelial cell-mediated vascularization. Biomaterials, 2009, 30: 508-517

[44] Lin CS, Xin ZC, Dai J, Lue TF. Commonly used mesenchymal stem cell markers and tracking labels: Limitations and challenges. Histol. Histopathol., 2013, 28: 1109-1116

[45] Liang S, et al. . The coupling of reduced type H vessels with unloading-induced bone loss and the protection role of Panax quinquefolium saponin in the male mice. Bone, 2021, 143 115712

[46] Vergadi E, Ieronymaki E, Lyroni K, Vaporidi K, Tsatsanis C. Akt Signaling Pathway in Macrophage Activation and M1/M2 Polarization. J. Immunol., 2017, 198: 1006-1014

[47] Covarrubias AJ, Aksoylar HI, Horng T. Control of macrophage metabolism and activation by mTOR and Akt signaling. Semin Immunol., 2015, 27: 286-296

[48] Luyendyk JP, et al. . Genetic analysis of the role of the PI3K-Akt pathway in lipopolysaccharide-induced cytokine and tissue factor gene expression in monocytes/macrophages. J. Immunol., 2008, 180: 4218-4226

[49] Ruckerl D, et al. . Induction of IL-4Ralpha-dependent microRNAs identifies PI3K/Akt signaling as essential for IL-4-driven murine macrophage proliferation in vivo. Blood, 2012, 120: 2307-2316

[50] Na N, et al. . Carbamylated erythropoietin regulates immune responses and promotes long-term kidney allograft survival through activation of PI3K/AKT signaling. Signal Transduct. Target Ther., 2020, 5: 194

[51] Teo BK, et al. . Nanotopography modulates mechanotransduction of stem cells and induces differentiation through focal adhesion kinase. ACS Nano, 2013, 7: 4785-4798

[52] Liu YC, Zou XB, Chai YF, Yao YM. Macrophage polarization in inflammatory diseases. Int J. Biol. Sci., 2014, 10: 520-529

[53] Hu K, Olsen BR. The roles of vascular endothelial growth factor in bone repair and regeneration. Bone, 2016, 91: 30-38

[54] Parithimarkalaignan S, Padmanabhan TV. Osseointegration: an update. J. Indian Prosthodont Soc., 2013, 13: 2-6

[55] Bouxsein ML, et al. . Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. J. Bone Miner. Res, 2010, 25: 1468-1486

[56] Qin L, et al. . Phytomolecule icaritin incorporated PLGA/TCP scaffold for steroid-associated osteonecrosis: Proof-of-concept for prevention of hip joint collapse in bipedal emus and mechanistic study in quadrupedal rabbits. Biomaterials, 2015, 59: 125-143

[57] Chen Y, Chen Z, Lei K, Ding J, Yu L. Surface modification of polyetheretherketone for boosted osseointegration: a review. Biomater. Transl., 2025, 6: 181-201

[58] Kinaret PAS, Scala G, Federico A, Sund J, Greco D. Carbon nanomaterials promote M1/M2 macrophage activation. Small, 2020, 16 e1907609

[59] Pei, B., Wang, W., Dunne, N. & Li, X. Applications of carbon nanotubes in bone tissue regeneration and engineering: superiority, concerns, current advancements, and prospects. Nanomaterials9, 1501 (2019).

[60] Nazarnezhad, S., Baino, F., Kim, H. W., Webster, T. J. & Kargozar, S. Electrospun nanofibers for improved angiogenesis: promises for tissue engineering applications. Nanomaterials10, 1609 (2020).

[61] Rambhia KJ, Ma PX. Controlled drug release for tissue engineering. J. Control Release, 2015, 219: 119-128

[62] Pierce TP, et al. . A current review of core decompression in the treatment of osteonecrosis of the femoral head. Curr. Rev. Musculoskelet. Med, 2015, 8: 228-232

[63] Karageorgiou V, Kaplan D. Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials, 2005, 26: 5474-5491

[64] Singh R, et al. . Engineered nanomaterials for immunomodulation: a review. ACS Appl. Bio. Mater., 2024, 7: 727-751

[65] Hosseini FS, et al. . Graphene oxide in bone regenerative engineering: current challenges and future perspectives. ACS Bio. Med. Chem. Au, 2025, 5: 350-364

[66] Petek D, Hannouche D, Suva D. Osteonecrosis of the femoral head: pathophysiology and current concepts of treatment. EFORT Open Rev., 2019, 4: 85-97

[67] Li D, et al. . Delivery of MiR335-5p-pendant Tetrahedron DNA nanostructures using an injectable heparin lithium hydrogel for challenging bone defects in steroid-associated osteonecrosis. Adv. Health Mater., 2022, 11 e2101412

[68] Qin L, et al. . Multiple bioimaging modalities in evaluation of an experimental osteonecrosis induced by a combination of lipopolysaccharide and methylprednisolone. Bone, 2006, 39: 863-871

[69] Roy A, et al. . De novo design of highly selective miniprotein inhibitors of integrins alphavbeta6 and alphavbeta8. Nat. Commun., 2023, 14 5660

Funding

Health Department of Sichuan Province (Sichuan Province Department of Health)(2025ZNSFSC1789)

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The Author(s)