Immunoprotective Multifunctional Nanofiber–Hydrogel Janus Membranes for Advanced Ectopic Cartilage Regeneration

Abstract

Cartilage tissue engineering presents a promising strategy for generating transplantable neocartilage capable of structurally and functionally restoring damaged tissues. However, the neglect of inflammatory responses and the absence of a pro-chondrogenic microenvironment lead to suboptimal regeneration outcomes during in vivo maturation. Guided cartilage regeneration (GCR) is a promising strategy for addressing this challenge through the integration of a physical barrier, an immunomodulatory capacity, and a pro-regenerative microenvironment. In this study, we developed a novel Janus GCR membrane featuring an FGF18-loaded dopamine-modified methacrylated hyaluronic acid hydrogel as the inner layer to promote cartilage-specific extracellular matrix synthesis, combined with a curcumin-grafted oxidized bacterial cellulose nanofiber outer layer that provides both an effective physical barrier and immunomodulatory function. Subsequent proof-of-concept experiments in subcutaneous ectopic models using nude mice and rabbits demonstrated that, following in vitro pre-culture and in vivo maturation, low-seeding-density constructs encapsulated with the GCR membrane achieved robust in vivo cartilage regeneration with properties comparable to those derived from high-cell seeding densities. The GCR membrane effectively modulated the local immune microenvironment and suppressed the invasion of host fibrous connective tissue, even in an aseptic inflammatory microenvironment. More importantly, the modularly assembled toroidal cartilage encapsulated with the GCR membrane successfully matured within the highly vascularized muscle, ultimately enabling the construction of a functional biomimetic trachea for segmental tracheal defect repair. Overall, the nanofiber–hydrogel Janus membrane attenuated local inflammation and fostered an immune and regenerative milieu conducive to ectopic cartilage formation, offering an advanced biomaterial design for next-generation GCR technologies.

Graphical Abstract

Keywords

Cartilage tissue engineering / Immunomodulation / Nanofiber–hydrogel membrane / Electrospinning / Janus / Artificial trachea

References

[1] Shim DW, Lee KM, Lee D, Kim JS, Jung YS, Oh SS, Lee SW, Lee JW, Kim BS. Osteochondral repair with autologous cartilage transplantation with or without bone grafting: a short pilot study in mini-pigs. Cartilage, 2025, 16: 61

[2] Tao B, Xu TT, Yu L, Zhang L, Cao GQ, Gong NJ, Zhou GD, Xiao KY, Huo YQ, Xia HT. A simple and low-cost method to develop porous egg white scaffolds with controllable shape for cartilage regeneration. Compos Part B Eng, 2025, 295: 112192

[3] Wang G, Lin W, Yan W, Zhang C, Dang W, Meng X, Li Z, Han M, Ao R, Hu X, An Y. Self-assembled hADSCs/hNSCs spheroids combined with 3D printed M-shaped GelMA/Pu scaffolds: creating histologically biomimetic engineered cartilage that meets the characteristics of alar cartilage. Biomaterials, 2025, 326: 123691

[4] Wang Q, Ran X, Wang J, Wang S, Zhang P, Gao E, Bai B, Zhang J, Zhou G, Lei D. Elastic fiber-reinforced silk fibroin scaffold with a double-crosslinking network for human ear-shaped cartilage regeneration. Adv Fiber Mater, 2023, 5: 1008

[5] Cheng P, Yang J, Wu S, Xie L, Xu Y, Xu N, Xu Y. Temporal modulation of inflammation and chondrogenesis through dendritic nanoparticle-mediated therapy with diclofenac surface modification and strontium ion encapsulation. J Biomater Sci Polym Ed, 2024, 35: 2049

[6] Wang Z, Wang Y, Chen Z, Gao E, Guo L, Li J, Zheng S, Yi J, Chen Z-S, Tao B. Dual-pedicle tissue-engineered trachea promotes biomimetic cartilaginous framework, vascularization, and epithelial lining for long-segment tracheal reconstruction. Adv Sci, 2025, 12: e14724

[7] Gao EJ, Wang ZM, Zhao SH, Zhu XS, Xu SS, Xu Y, Wang YG, Xu N, Tao B. Core-shell structured nanomembrane with sequential immune and vascular isolation effects followed by chondrogenic induction to promote stable stem cell-based subcutaneous cartilage regeneration in large animals. Chem Eng J, 2024, 498: 154878

[8] Zhang X, Ying HF, Wang TT, Zhou GD, Xu Y, Cao YL. Modulatory effect of three cartilaginous niches on cartilage regeneration from different chondrocyte sources in a goat model. Cell Tissue Bank, 2025, 26: 35

[9] Chen ZY, Zheng AX, Wang JP, Li Y, Zhao SH, Xu Y, Kong WQ, Lu J. A time-programmed therapy for enhanced cartilage regeneration in immunocompetent animals. Mater Des, 2025, 260: 115062

[10] Wei CZ, Lin MY, Bo QT, Dai WF, Ding JH, Chen R. Enhancing the maturity of in vitro engineered cartilage from Wharton’s jelly-derived photo-crosslinked hydrogel using dynamic bioreactors and its in vivo outcomes in animal models. Regen Biomater, 2025, 12: rbaf037

[11] Li S, Wang R, Huang L, Jiang Y, Xing F, Duan W, Cen Y, Zhang Z, Xie H. Promotion of diced cartilage survival and regeneration with grafting of small intestinal submucosa loaded with urine-derived stem cells. Cell Prolif, 2024, 57: e13542

[12] Gordon CR, Alghoul M, Goldberg JS, Habal MB, Papay F. Diced cartilage grafts wrapped in AlloDerm for dorsal nasal augmentation. J Craniofac Surg, 2011, 22: 1196

[13] Guo Z, Zhu X, Xu L, Zhu J, Zhang X, Yang Y, Song N. Immuno-isolation strategy with Tacrolimus-loaded nanofilm promotes stable stem cell-based cartilage regeneration. Adv Funct Mater, 2025, 35: 2414567

[14] Yuan S, Feng YHZ, Wang HC, Chen SM, Li JH, Zhu YB, Yu SH, Wang ZL. Sword and board in one: a bioinspired nanocomposite membrane for guided bone regeneration. Adv Mater, 2025, 37: 2504577

[15] Huang L, Wu T, Sun J, Lin X, Peng Y, Zhang R, Gao Y, Xu S, Sun Y, Zhou Y, Duan B. Biocompatible chitin-based Janus hydrogel membranes for periodontal repair. Acta Biomater, 2024, 190: 219

[16] Yang D, Xu ZL, Huang D, Luo Q, Zhang CL, Guo JM, Tan L, Ge LM, Mu CD, Li DF. Immunomodulatory multifunctional Janus collagen-based membrane for advanced bone regeneration. Nat Commun, 2025, 16: 4264

[17] Lei Q, He D, Ding L, Kong F, He P, Huang J, Guo J, Brinker CJ, Luo G, Zhu W, Yu Y. Microneedle patches integrated with biomineralized Melanin nanoparticles for simultaneous skin tumor photothermal therapy and wound healing. Adv Funct Mater, 2022, 32: 2113269

[18] Chen QC, Bai L, Wan GY, Hao YF, Yang X, Zhang HT. Multifunctional MeHA hydrogel for living materials delivery with enhanced cartilage regeneration. Front Bioeng Biotechnol, 2025, 13: 1545773

[19] Chu B, Chu YF, He JM, Lin ZW, Chen CS, Wang S, Liu WQ, Li XL. A nature-inspired multifunctional adhesive for cartilage tissue-biomaterial integration. Soft Matter, 2024, 20: 2017

[20] Antunes BP, Vainieri ML, Alini M, Monsonego-Ornan E, Grad S, Yayon A. Enhanced chondrogenic phenotype of primary bovine articular chondrocytes in Fibrin–Hyaluronan hydrogel by multi-axial mechanical loading and FGF18. Acta Biomater, 2020, 105: 170

[21] Firoozi N, Kang YQ. Immobilization of FGF on Poly(xylitol dodecanedioic Acid) polymer for tissue regeneration. Sci Rep, 2020, 10: 10419

[22] Huang TY, Sun HY, Chen JG, Liu X, Pan B, He LR, Jiang HY. A single dose of local injection of adipose stem cells promotes ectopic cartilage regeneration by modulating inflammatory response and enhancing cartilage extracellular matrix synthesis in a porcine model. Curr Stem Cell Res Ther, 2023, 18: 237

[23] Fu LW, Feng Q, Chen YJ, Fu JZ, Zhou XJ, He CL. Nanofibers for the immunoregulation in biomedical applications. Adv Fiber Mater, 2022, 4: 1334

[24] Davlet M, Smyrnova K, Pogrebnjak A. Advanced biomaterials in tissue engineering: a critical review of nanocomposites based on bacterial cellulose, MXenes, hydroxyapatite, and metal particles for regenerative medicine. Adv Colloid Interface Sci, 2025, 345: 103634

[25] Zhao FL, Liu GD, Guan YJ, Li JF, Wang TY, Zhao JM, He W, Zhang LY, Meng HY, Xu WJ, Wang Y, Zheng YD. An electromechanical converted bacterial cellulose based composite film for repairing peripheral nerve injury through mimicking physiological electrical signal. Adv Fiber Mater, 2025, 7: 1929

[26] Rybak D, Du JT, Nakielski P, Rinoldi C, Kosik-Koziol A, Zakrzewska A, Wu HY, Li J, Li XR, Yu YL, Ding B, Pierini F. NIR-light activable 3D printed platform nanoarchitectured with electrospun plasmonic filaments for on demand treatment of infected wounds. Adv Healthc Mater, 2025, 14: 2404274

[27] Talouki PY, Tamimi R, Rudi SG. A comprehensive review of curcumin-based scaffolds in cartilage tissue engineering. Stem Cell Res Ther, 2025, 16: 528

[28] Zhang YL, Xiang Y, Zhang J, Huang HF, Tan H, Chen S, Ma JH, You ZW, Zhu SH. Rhizome-inspired liquid-management membrane to promote wound healing. Adv Fiber Mater, 2026, 8: 73

[29] Zhou HL, Zhao YF, Zha XJ, Zhang ZM, Zhang LL, Wu YK, Ren RY, Zhao ZH, Yang W, Zhao LX. A Janus, robust, biodegradable bacterial cellulose/Ti3C2Tx MXene bilayer membranes for guided bone regeneration. Biomater Adv, 2024, 161: 213892

[30] Ma X, Chen Y, Huang J, Lv P, Wei Q. In situ formed active and intelligent bacterial cellulose/cotton fiber composite containing curcumin. Cellulose, 2020, 27: 9371

[31] Zhang KY, Wei Z, Xu XY, Feng Q, Xu JB, Bian LM. Efficient catechol functionalization of biopolymeric hydrogels for effective multiscale bioadhesion. Mat Sci Eng C Mater Biol Appl, 2019, 103: 109835

[32] Kherani S, Noel CW, Nazir T, Bitar MA. Irradiated homologous costal cartilage grafts for single-stage open airway reconstruction in severe subglottic stenosis for children under the age of one. Int J Pediatr Otorhinolaryngol, 2020, 136: 110167

[33] Wu H, Wu Q, Liang C, Hua J, Meng L, Nakielski P, Lu C, Pierini F, Xu L, Yu Y, Luo Q. Immunoregulatory electrospinning fiber mediates macrophage energy metabolism reprogramming to promote burn wound healing. Mater Today Bio, 2025, 35: 102430

[34] Vichare R, Kulahci Y, Mccallin R, Zor F, Selek FN, Liu L, Crelli C, Troidle A, Herneisey M, Nichols JM, Shepherd AJ, Gorantla VS, Janjic JM. Theranostic nanoemulsions suppress macrophage-mediated acute inflammation in rats. J Nanobiotechnol, 2025, 23: 80

[35] Kuang Y, Hua B, Ye X, Zhao Y, Yu M, Liu X. Dual-functional ROS-responsive hydrogel alleviates temporomandibular joint osteoarthritis by enhancing cartilage repair and mitigating inflammation. Mater Today Bio, 2025, 33: 102103

[36] Sun W, Yang Y, Wang L, Tang H, Zhang L, She Y, Xiao X, Hu X, Feng Q, Chen C. Utilization of an acellular cartilage matrix-based photocrosslinking hydrogel for tracheal cartilage regeneration and circumferential tracheal repair. Adv Funct Mater, 2022, 32: 2201257

[37] Zhang Y, Cai R, Li J, Wu X. The immunosuppressive niche established with a curcumin-loaded electrospun nanofibrous membrane promotes cartilage regeneration in immunocompetent animals. Membranes, 2023, 13: 335

[38] Xu Y, Li D, Yin ZQ, He AJ, Lin MM, Jiang GN, Song X, Hu XF, Liu Y, Wang JP, Wang XY, Duan L, Zhou GD. Tissue-engineered trachea regeneration using decellularized trachea matrix treated with laser micropore technique. Acta Biomater, 2017, 58: 113

[39] Xu Y, Dai J, Zhu XS, Cao RF, Song N, Liu M, Liu XG, Zhu JJ, Pan F, Qin LL, Jiang GN, Wang HF, Yang Y. Biomimetic trachea engineering via a modular ring strategy based on bone-marrow stem cells and atelocollagen for use in extensive tracheal reconstruction. Adv Mater, 2022, 34: 2106755

[40] Gao Y, Wang J, Dai W, Li S, Liu Q, Zhao X, Fu W, Xiao Y, Guo L, Fan Y, Zhang X. Collagen-based hydrogels induce hyaline cartilage regeneration by immunomodulation and homeostasis maintenance. Acta Biomater, 2024, 186: 108

[41] Shen Y, Tu T, Yi B, Wang X, Tang H, Liu W, Zhang Y. Electrospun acid-neutralizing fibers for the amelioration of inflammatory response. Acta Biomater, 2019, 97: 200

[42] Bonato A, Fisch P, Ponta S, Fercher D, Manninen M, Weber D, Eklund KK, Barreto G, Zenobi-Wong M. Engineering inflammation-resistant cartilage: bridging gene therapy and tissue engineering. Adv Healthc Mater, 2023, 12: 2202271

[43] Wei F, Liu SY, Chen MX, Tian GZ, Zha KK, Yang Z, Jiang SP, Li MZ, Sui X, Chen ZW, Guo QY. Host Response to biomaterials for cartilage tissue engineering: key to remodeling. Front Bioeng Biotechnol, 2021, 09: 664592

[44] Deng H, Zhang A, Pang DRR, Xi Y, Yang Z, Matheson R, Li G, Luo H, Lee KM, Fu Q, Zou Z, Chen T, Wang Z, Rosales IA, Peters CW, Yang J, Coronel MM, Yolcu ES, Shirwan H, Garcia AJ, Markmann JF, Lei J. Bioengineered omental transplant site promotes pancreatic islet allografts survival in non-human primates. Cell Rep Med, 2023, 4: 100959

[45] Xu N, Yuan YC, Ding LP, Li JF, Jia JZ, Li Z, He DF, Yu YL. Multifunctional chitosan/gelatin@tannic acid cryogels decorated with in situ reduced silver nanoparticles for wound healing. Burns Trauma, 2022, 10: tkac019

[46] Jia LT, Zhang PL, Ci Z, Zhang W, Liu Y, Jiang HY, Zhou GD. Immune-inflammatory responses of an acellular cartilage matrix biomimetic scaffold in a xenotransplantation goat model for cartilage tissue engineering. Front Bioeng Biotechnol, 2021, 9: 667161

[47] Xu N, Gao Y, Li Z, Chen Y, Liu M, Jia J, Zeng R, Luo G, Li J, Yu Y. Immunoregulatory hydrogel decorated with Tannic acid/Ferric ion accelerates diabetic wound healing via regulating Macrophage polarization. Chem Eng J, 2023, 466: 143173

[48] Kanazawa S, Fujihara Y, Sakamoto T, Asawa Y, Komura M, Nagata S, Takato T, Hoshi K. Tissue responses against tissue-engineered cartilage consisting of chondrocytes encapsulated within non-absorbable hydrogel. J Tissue Eng Regen Med, 2013, 7: 1

[49] Liu Y, Li D, Yin ZQ, Luo XS, Liu W, Zhang WJ, Zhang ZY, Cao YL, Liu Y, Zhou GD. Prolonged precultivation alleviates post-implantation inflammation and promotes stable subcutaneous cartilage formation in a goat model. Biomed Mater, 2017, 12: 015006

[50] Wu J, Deng J, Theocharidis G, Sarrafian TL, Griffiths LG, Bronson RT, Veves A, Chen J, Yuk H, Zhao X. Adhesive anti-fibrotic interfaces on diverse organs. Nature, 2024, 630: 360

[51] Wang H, Wu X, Chen L, Tong H, Hu X, He A, Li C, Guo X, Fu Y, Zhang T. Dynamic Col-HZ Hydrogel with efficient delivery of bioactivator promotes ECM deposition and cartilage formation. Mater Today Bio, 2025, 31: 101623

[52] Sueyoshi S, Chitose S, Sato K, Fukahori M, Kurita T, Umeno H. Stable tracheal regeneration using organotypically cultured tissue composed of autologous chondrocytes and epithelial cells in beagles. Ann Oto Rhinol Laryn, 2019, 128: 585

[53] Li YQ, Liu YQ, Xun XW, Zhang W, Xu Y, Gu DY. Three-dimensional porous scaffolds with biomimetic microarchitecture and bioactivity for cartilage tissue engineering. ACS Appl Mater Interfaces, 2019, 11: 36359

[54] Zhan JD, Chen ZL, Liu JY, Pang QM, Lei MJ, Liu JC, Song Y, Huang W, Dong LL. A targeting trained immunity nanofiber scaffold for large bone defect repair. Adv Fiber Mater, 2025, 7: 1423

[55] Li Y, Xun X, Duan L, Gao E, Li J, Lin L, Li X, He A, Ao H, Xu Y, Xia H. Cartilage structure-inspired nanofiber-hydrogel composite with robust proliferation and stable chondral lineage-specific differentiation function to orchestrate cartilage regeneration for artificial tracheal construction. Bioact Mater, 2025, 47: 136

[56] Xie BW, Ma HB, Yang FY, Chen HG, Guo YN, Zhang HX, Li TF, Huang XG, Zhao YT, Li XJ, Du JJ. Development and evaluation of 3D composite scaffolds with piezoelectricity and biofactor synergy for enhanced articular cartilage regeneration. J Mater Chem B, 2024, 12: 10416

[57] Zheng S, Xu L, Bo Q, Gao E, Zheng E, Xie L, Zhao B, Yi J, Li Y, Xu Y, Wang Y, Tao B. Xanthohumol bulk-modified polyurethane for tracheal repair: a ‘killing two birds with one stone’ strategy for tailorable mechanics and durable anti-inflammatory efficacy. Mater Today Bio, 2025, 32: 101831

Funding

National Natural Science Foundation of China(82302395)

RIGHTS & PERMISSIONS

Donghua University, Shanghai, China