Synergistic Integration of Immune Regulation and Bioactive Guidance Cues in Multi-Channel Nanofibrous Nerve Guidance Conduits for Accelerated Peripheral Nerve Regeneration

Abstract

Peripheral nerve injury presents a significant clinical challenge due to the limited regenerative capacity of the injured nerves, often resulting in permanent functional deficits. A key obstacle to effective nerve regeneration is the inability to modulate the inflammatory response, guide axonal elongation, and promote myelination. To address these challenges, we developed a multi-channel nerve guidance conduit (NGC) that integrated immune-modulating drug with gradient cues to enhance peripheral nerve regeneration. The inner tubes of the conduit were composed of degradable electrospun gelatin methacryloyl/collagen (GelMA/COL) fibers loaded with 1400W, an inducible nitric oxide synthase (iNOS) inhibitor. The outer tube consisted of electrospun polycaprolactone (PCL) fibers decorated with a density gradient of collagen particles encapsulating acidic fibroblast growth factor (aFGF). The release of 1400W enhanced macrophage activity and promoted their polarization from the pro-inflammatory M1 phenotype to the reparative M2 phenotype, thereby creating a pro-regenerative microenvironment conducive to nerve repair. The incorporation of gradient cues guided and promoted Schwann cell migration and neurite extension in vitro. In a rat sciatic nerve injury model, the conduit significantly improved nerve regeneration by sequentially modulating the inflammatory response and guiding axonal elongation, providing both spatial support and biological activity. Furthermore, the conduit promoted organized nerve fiber alignment, enhanced myelination, and achieved functional recovery outcomes that closely resembled those of the autograft. These findings suggest that the integration of immune-regulatory drug release, gradient cues, and a multi-channel structure presents a promising strategy for enhancing peripheral nerve repair.

Keywords

Multi-channel nerve guidance conduit / Nitric oxide synthase inhibitor / Immune regulation / Gradient cues / Peripheral nerve regeneration

References

[1] VijayavenkataramanS, NaghiehS, McInnesAD, SchreyerDJ, ChenX. Nerve guide conduits for peripheral nerve injury repair: a review on design, materials and fabrication methods. Acta Biomater, 2020, 10654

[2] SarkerMD, NaghiehS, McInnesAD, SchreyerDJ, ChenXB. Regeneration of peripheral nerves by nerve guidance conduits: Influence of design, biopolymers, cells, growth factors, and physical stimuli. Prog Neurobiol, 2018, 171125

[3] XueJJ, WuT, QiuJC, RutledgeS, TanesML, XiaYN. Promoting cell migration and neurite extension along uniaxially aligned nanofibers with biomacromolecular particles in a density gradient. Adv Funct Mater, 2020, 302002031

[4] XieJW, LiXR, XiaYN. Putting electrospun nanofibers to work for biomedical research. Macromol Rapid Commun, 2008, 291775

[5] ChenD, ZhangY, NiCJ, MaC, YinJ, BaiH, LuoYW, HuangFH, XieT, ZhaoQ. Drilling by light: ice-templated photo-patterning enabled by a dynamically crosslinked hydrogel. Mater Horiz, 2019, 61013

[6] Li GC, Zheng TT, Wu LL, Han Q, Lei YF, Xue LJ, Zhang LZ, Gu XS, Yang YM. Bionic microenvironment-inspired synergistic effect of anisotropic micro-nanocomposite topology and biology cues on peripheral nerve regeneration. Sci. Adv. 2021; 7: eabi5812.

[7] FangZW, GeXM, ChenX, XuY, YuanWE, OuyangYM. Enhancement of sciatic nerve regeneration with dual delivery of vascular endothelial growth factor and nerve growth factor genes. J Nanobiotechnol, 2020, 1846

[8] WangSR, ZhuCL, ZhangB, HuJX, XuJH, XueCB, BaoSX, GuXK, DingF, YangYM, GuXS, GuY. BMSC-derived extracellular matrix better optimizes the microenvironment to support nerve regeneration. Biomaterials, 2022, 280121251

[9] GaoYS, DaiCL, ZhangM, ZhangJY, YinL, LiWH, ZhangKY, YangYM, ZhaoYH. Biomimetic silk fibroin hydrogel for enhanced peripheral nerve regeneration: synergistic effects of graphene oxide and fibroblast exosome. Adv Funct Mater, 2024, 342314610

[10] LiCY, MengFQ, YangZJ, PengJ, GuoYW, NaJ, ShiQS, LiuY, WangY, ZhengLS, FanYB. Micropattern-based stem cell gym: mechanical memory enhanced stemness maintenance of human dental pulp stem cells and nerve regeneration. Adv Funct Mater, 2023, 332302829

[11] LiY, LvS, YuanHP, YeG, MuWB, FuY, ZhangX, FengZY, HeY, ChenW. Peripheral nerve regeneration with 3D printed bionic scaffolds loading neural crest stem cell derived Schwann cell progenitors. Adv Funct Mater, 2021, 312010215

[12] MaoRY, YuB, CuiJJ, WangZY, HuangXT, YuHB, LinKL, ShenSGF. Piezoelectric stimulation from electrospun composite nanofibers for rapid peripheral nerve regeneration. Nano Energy, 2022, 98107322

[13] HanQ, GuanWC, SunSL, ZhengTT, WuLL, GaoHX, LiuYQ, YangYM, LiGC. Anisotropic topological scaffolds synergizing non-invasive wireless magnetic stimulation for accelerating long-distance peripheral nerve regeneration. Chem Eng J, 2024, 496153809

[14] XuDY, FuSQ, ZhangH, LuWC, XieJD, LiJL, WangH, ZhaoYJ, ChaiRJ. Ultrasound-responsive aligned piezoelectric nanofibers derived hydrogel conduits for peripheral nerve regeneration. Adv Mater, 2024, 362307896

[15] PiW, RaoF, CaoJW, ZhangMR, ChangT, HanYZ, ZhengYF, LiuSY, LiQY, SunXY, ShaoY. Sono-electro-mechanical therapy for peripheral nerve regeneration through piezoelectric nanotracts. Nano Today, 2023, 50101860

[16] BaiH, ZhangSQ, YangHR, WangJ, ChenHL, LiJ, LiL, YangQ, PengB, ZhuZY, NiSY, LiuKY, LeiW, TaoTH, FengYF. Advanced nerve regeneration enabled by neural conformal electronic stimulators enhancing mitochondrial transport. Bioact Mater, 2024, 39287

[17] ZhaoRL, JiangLH, DuJ, XuBT, LiA, WangW, ZhaoSC, LiXL. Fluffy sponge-reinforced electrospun conduits with biomimetic structures for peripheral nerve repair. Composites Part B-Eng, 2022, 230109482

[18] ZhangCY, GongJX, ZhangJY, ZhuZY, QianY, LuKJ, ZhouSY, GuTY, WangHM, HeY, YuMF. Three potential elements of developing nerve guidance conduit for peripheral nerve regeneration. Adv Funct Mater, 2023, 332302251

[19] WuS, ShenW, GeXM, AoF, ZhengY, WangYG, JiaXN, MaoYY, LuoYL. Advances in large gap peripheral nerve injury repair and regeneration with bridging nerve guidance conduits. Macromol Biosci, 2023, 232300078

[20] ZhangFC, MiaoY, LiuQY, LiSY, HeJH. Changes of pro-inflammatory and anti-inflammatory macrophages after peripheral nerve injury. RSC Adv, 2020, 1038767

[21] YanZW, YeTB, YangL, JiangHQ, ChenC, ChenSL, QianY, FanCY. Nanobiology dependent therapeutic convergence between biocompatibility and bioeffectiveness of graphene oxide quantum dot scaffold for immuno-inductive angiogenesis and nerve regeneration. Adv Funct Mater, 2023, 332211709

[22] ScheibJ, HökeA. Advances in peripheral nerve regeneration. Nat Rev Neurol, 2013, 9668

[23] OishiY, ManabeI. Macrophages in inflammation, repair and regeneration. Int Immunol, 2018, 30511

[24] LiJJ, YaoYX, WangY, XuJS, ZhaoD, LiuMT, ShiSR, LinYF. Modulation of the crosstalk between Schwann cells and macrophages for nerve regeneration: a therapeutic strategy based on a multifunctional tetrahedral framework nucleic acids system. Adv Mater, 2022, 342202513

[25] WculekSK, DunphyG, Heras-MurilloI, MastrangeloA, SanchoD. Metabolism of tissue macrophages in homeostasis and pathology. Cell Mol Immunol, 2022, 19384

[26] ChenX, GeXM, QianY, TangHZ, SongJL, QuXH, YueB, YuanWE. Electrospinning multilayered scaffolds loaded with melatonin and Fe₃O₄ magnetic nanoparticles for peripheral nerve regeneration. Adv Funct Mater, 2020, 302004537

[27] YangHC, LiQ, LiLM, ChenSC, ZhaoY, HuYR, WangL, LanXQ, ZhongLM, LuD. Gastrodin modified polyurethane conduit promotes nerve repair via optimizing Schwann cells function. Bioact Mater, 2022, 8355

[28] ZigrnondRE, EchevarriaFD. Macrophage biology in the peripheral nervous system after injury. Prog Neurobiol, 2019, 173102

[29] HuangTC, WuHL, ChenSH, WangYT, WuCC. Thrombomodulin facilitates peripheral nerve regeneration through regulating M1/M2 switching. J Neuroinflammation, 2020, 17240

[30] YangPX, PengY, DaiX, JieJ, KongDL, GuXS, YangYM. Bionic peptide scaffold in situ polarization and recruitment of M2 macrophages to promote peripheral nerve regeneration. Bioact Mater, 2023, 3085

[31] Van den BosscheJ, BaardmanJ, OttoNA, van der VeldenS, NeeleAE, van den BergSM, Luque-MartinR, ChenHJ, BoshuizenMCB, AhmedM, HoeksemaMA, de VosAF, de WintherMPJ. Mitochondrial dysfunction prevents repolarization of inflammatory macrophages. Cell Rep, 2016, 17684

[32] BaoTY, LiN, ChenH, ZhaoZN, FanJ, TaoYF, ChenCL, WanMM, YinGY, MaoC. Drug-loaded zwitterion-based nanomotors for the treatment of spinal cord injury. ACS Appl Mater Interfaces, 2023, 1532762

[33] AndrabiSM, SharmaNS, KaranA, ShahriarSMS, CordonB, MaB, XieJW. Nitric oxide: physiological functions, delivery, and biomedical applications. Adv Sci, 2023, 102303259

[34] IkegamiY, ShafiqM, AishimaS, IjimaH. Heparin/growth factors-immobilized aligned electrospun nanofibers promote nerve regeneration in polycaprolactone/gelatin-based nerve guidance conduits. Adv Fiber Mater, 2023, 5554

[35] ZhuL, JiaSJ, LiuTJ, YanL, HuangDG, WangZY, ChenS, ZhangZP, ZengW, ZhangY, YangH, HaoDJ. Aligned PCL fiber conduits immobilized with nerve growth factor gradients enhance and direct sciatic nerve regeneration. Adv Funct Mater, 2020, 302002610

[36] JinBH, YuYL, LouCH, ZhangXD, GongBW, ChenJH, ChenXX, ZhouZH, ZhangLQ, XiaoJ, XueJJ. Combining a density gradient of biomacromolecular nanoparticles with biological effectors in an electrospun fiber-based nerve guidance conduit to promote peripheral nerve repair. Adv Sci, 2023, 102203296

[37] CharrasG, SahaiE. Physical influences of the extracellular environment on cell migration. Nat Rev Mol Cell Biol, 2014, 15813

[38] SenGuptaS, ParentCA, BearJE. The principles of directed cell migration. Nat Rev Mol Cell Biol, 2021, 22529

[39] WangJ, XiongH, ZhuTH, LiuY, PanHB, FanCY, ZhaoXL, LuWWJ. Bioinspired multichannel nerve guidance conduit based on shape memory nanofibers for potential application in peripheral nerve repair. ACS Nano, 2020, 1412579

[40] MaY, WangH, WangQQ, CaoXD, GaoHC. Piezoelectric conduit combined with multi-channel conductive scaffold for peripheral nerve regeneration. Chem Eng J, 2023, 452139424

[41] YuTH, AoQ, AoTR, AhmadMA, WangAJ, XuYX, ZhangZT, ZhouQ. Preparation and assessment of an optimized multichannel acellular nerve allograft for peripheral nerve regeneration. Bioeng Transl Med, 2022, 8e10435

[42] XueJJ, XieJW, LiuWY, XiaYN. Electrospun nanofibers: new concepts, materials, and applications. Acc Chem Res, 2017, 501976

[43] XueJJ, WuT, DaiYQ, XiaYN. Electrospinning and electrospun nanofibers: methods, materials, and applications. Chem Rev, 2019, 1195298

[44] PawelecKM, YoonC, GigerRJ, SakamotoJ. Engineering a platform for nerve regeneration with direct application to nerve repair technology. Biomaterials, 2019, 216119263

[45] YeWS, LiHB, YuK, XieCQ, WangP, ZhengYT, ZhangP, XiuJF, YangY, ZhangF, HeY, GaoQ. 3D printing of gelatin methacrylate-based nerve guidance conduits with multiple channels. Mater Des, 2020, 192108757

[46] PuttacharySK, SharmaS, VermaS, YangY, PutraM, ThippeswamyA, LuoD, ThippeswamyT. 1400W, a highly selective inducible nitric oxide synthase inhibitor is a potential disease modifier in the rat kainate model of temporal lobe epilepsy. Neurobiol Dis, 2016, 93184

[47] LiuP, PengJ, HanGH, DingX, WeiS, GaoG, HuangK, ChangF, WangY. Role of macrophages in peripheral nerve injury and repair. Neural Regener Res, 2019, 141335

[48] HuangYJ, WuLW, ZhaoYS, GuoJ, LiRY, MaSC, YingZX. Schwann cell promotes macrophage recruitment through IL-17B/IL-17RB pathway in injured peripheral nerves. Cell Rep, 2024, 43113753

[49] Atochina-VassermanEN, BeersMF, KadireH, TomerY, InchA, ScottP, GuoCJ, GowAJ. Selective inhibition of inducible NO synthase activity in vivo reverses inflammatory abnormalities in surfactant protein D-deficient mice. J Immunol, 2007, 1798090

[50] GuoCJ, Atochina-VassermanE, AbramovaH, GeorgeB, ManojVP, ScottP, GowA. Role of NOS2 in pulmonary injury and repair in response to bleomycin. Free Radical Biol Med, 2016, 91293

[51] LiuQY, XieLH, ChenW. Recombinant bovine FGF1 promotes muscle satellite cells mitochondrial fission and proliferation in serum-free conditions. Food Res Int, 2024, 175113794

[52] CattinAL, BurdenJJ, Van EmmenisL, MackenzieFE, HovingJJ, GarciaCV, GuoYP, McLaughlinM, RosenbergLH, QueredaV, JamecnaD, NapoliI, ParrinelloS, EnverT, RuhrbergC, LloydAC. Macrophage-induced blood vessels guide schwann cell-mediated regeneration of peripheral nerves. Cell, 2015, 1621127

[53] KabatAM, PearceEJ. Inflammation by way of macrophage metabolism. Science, 2017, 356488

[54] WangJ, ChengY, WangHY, WangYH, ZhangKH, FanCY, WangHJ, MoXM. Biomimetic and hierarchical nerve conduits from multifunctional nanofibers for guided peripheral nerve regeneration. Acta Biomater, 2020, 117180

[55] WangXQ, ChenS, ChenXL, WuJ, HuangZH, WangJ, ChenFP, LiuCS. Biomimetic multi-channel nerve conduits with micro/nanostructures for rapid nerve repair. Bioact Mater, 2024, 41577

[56] Nazeri N, Derakhshan MA, Mansoori K, Ghanbari H. Improvement of sciatic nerve regeneration by multichannel nanofibrous membrane-embedded electro-conductive conduits functionalized with laminin. J. Mater. Sci.: Mater. Med. 2022; 33: 50.

Funding

National Key R&D Program of China(2022YFC2408200)

National Natural Science Foundation of China(52073014)

Key Program of Beijing Natural Science Foundation(Z200025)

Fundamental Research Funds for the Central Universities(buctrc202020)

Natural Science Foundation of Zhejiang Province(LQN25H090021)

BJAST Budding Talent Program(23CE-BGS-02)

RIGHTS & PERMISSIONS

Donghua University, Shanghai, China