Jammed Microfiber Inks Enable Aligned 3D Printing for Epigenetic Muscle Regeneration

Abstract

Fiber alignment in the extracellular matrix provides critical topographical cues for tissue regeneration, yet the underlying epigenetic mechanisms remain poorly understood. Here, we developed 3D-printable hydrogel microfiber inks to systematically investigate how aligned topography drives muscle regeneration through epigenetic priming. Gelatin-norbornene (GelNB) microfibers (approximately 300 μm in diameter) were fabricated via ultraviolet crosslinking, fragmented, and jammed to create shear-thinning inks for extrusion-based 3D printing. Aligned microfiber scaffolds induced alignment of C2C12 murine skeletal myoblasts with increased nuclear aspect ratios and chromatin decondensation. An assay for transposase-accessible chromatin using sequencing revealed rapid chromatin remodeling within 24 h, with enrichment of master myogenic regulators and mechanosensitive transcription factors at newly accessible sites. RNA-seq confirmed widespread differential gene expression, demonstrating that physical alignment drives chromatin accessibility changes that enable transcriptional activation of muscle differentiation programs. In vivo validation using a volumetric muscle loss model revealed that compared with the perpendicular alignment of control groups, the groups with scaffolds with horizontally aligned fibers achieved superior muscle regeneration, with enhanced tissue recovery and reduced fibrosis. Epigenetic changes observed in vitro were confirmed in vivo, validating the mechanistic link between topographical cues and chromatin remodeling during tissue regeneration. These findings establish that 3D-printed aligned microfiber topography systematically controls epigenetic mechanisms to guide muscle regeneration, providing a powerful platform for the development of regenerative fibrous biomaterials.

Graphical Abstract

Keywords

Gelatin-norbornene / Microfibers / Alignment / 3D printing / Topography / Epigenetic regulation

References

[1] Moroni L, Burdick JA, Highley C, Lee SJ, Morimoto Y, Takeuchi Set al. . Biofabrication strategies for 3D in vitro models and regenerative medicine. Nat Rev Mater. 2018, 3. 21

[2] Wang C, Wang Y, Gu Y, Zhu Y, Yin R, Li Yet al. . SPI1 facilitates microfracture-mediated cartilage regeneration in the elderly by enhancing bone marrow stromal cells ctemness. J Tissue Eng. 2025.

[3] Wang Y, Liu A, Zhang X, Lyu Y, Rong X, Niu Cet al. . Microfluidic organ-on-a-chip for modeling coronary artery disease: recent applications, limitations and potential. J Tissue Eng. 2025.

[4] Sanz-Horta R, Matesanz A, Gallardo A, Reinecke H, Jorcano JL, Acedo Pet al. . Technological advances in fibrin for tissue engineering. J Tissue Eng. 2023.

[5] Kang Y, Kalaskar DM, Player DJ. In vitro models of muscle spindles: From traditional methods to 3D bioprinting strategies. J Tissue Eng.. 2023.

[6] Fuchs B, Mert S, Hofmann D, Kuhlmann C, Birt A, Wiggenhauser PSet al. . Bioactivated scaffolds promote angiogenesis and lymphangiogenesis for dermal regeneration in vivo. J Tissue Eng. 2025.

[7] Hinton TJ, Jallerat Q, Palchesko RN, Park JH, Grodzicki MS, Shue HJet al. . Three-dimensional printing of complex biological structures by freeform reversible embedding of suspended hydrogels. Sci Adv. 2015, 1. e1500758

[8] Wan W, Wang X, Zhang R, Li Y, Wu H, Liu Yet al. . Construction of artificial lung tissue structure with 3D-inkjet bioprinting core for pulmonary disease evaluation. J Tissue Eng. 2025.

[9] Grigoryan B, Paulsen SJ, Corbett DCet al. . Multivascular networks and functional intravascular topologies within biocompatible hydrogels. Science. 2019, 364. 458

[10] Bhattacharjee T, Zehnder SM, Rowe KG, Jain S, Nixon RM, Sawyer WGet al. . Writing in the granular gel medium. Sci Adv. 2015, 1. e1500655

[11] Highley CB, Rodell CB, Burdick JA. Direct 3D printing of shear-thinning hydrogels into self-healing hydrogels. Adv Mater. 2015, 275075.

[12] Wu W, Deconinck A, Lewis JA. Omnidirectional printing of 3D microvascular networks. Adv Mater. 2011, 23H178

[13] Mirdamadi E, Tashman JW, Shiwarski DJ, Palchesko RN, Feinberg AW. FRESH 3D bioprinting a full-size model of the human heart. ACS Biomater Sci Eng. 2020, 66453.

[14] Nguyen DG, Funk J, Robbins JB, Crogan-Grundy C, Presnell SC, Singer Tet al. . Bioprinted 3D primary liver tissues allow assessment of organ-level response to clinical drug induced toxicity in vitro. PLoS ONE. 2016, 11. e0158674

[15] Kang D, Hong G, An S, Jang I, Yun WS, Shim JHet al. . Bioprinting of multiscaled hepatic lobules within a highly vascularized construct. Small. 2020, 16905505

[16] Faksawat K, Limsuwan P, Naemchanthara KElsevier Ltd. 3D printing technique of specific bone shape based on raw clay using hydroxyapatite as an additive material. Appl Clay Sci. 2021, 214. 106269

[17] Wu Y, Wang L, Guo B, Ma PX. Interwoven aligned conductive nanofiber yarn/hydrogel composite scaffolds for engineered 3D cardiac anisotropy. ACS Nano. 2017, 115646.

[18] Basurto IM, Muhammad SA, Gardner GM, Christ GJ, Caliari SR. Controlling scaffold conductivity and pore size to direct myogenic cell alignment and differentiation. J Biomed Mater Res A. 2022, 1101681.

[19] Cheng Y, Zhu S, Pang SW. Directing Cell Migration with Patterned Nanostructures. In: Proceedings of the IEEE International Conference on Micro Electro Mechanical Systems (MEMS). Institute of Electrical and Electronics Engineers Inc.; 2021. pp. 579–582.

[20] Kwon KW, Park H, Song KH, Choi J-C, Ahn H, Park MJet al. . Nanotopography-guided migration of T cells. J Immunol. 2012, 1892266.

[21] Song KH, Park SJ, Kim DS, Doh J. Sinusoidal wavy surfaces for curvature-guided migration of Tlymphocytes. Biomaterials. 2015, 51151.

[22] Tang QY, Qian WX, Xu YH, Gopalakrishnan S, Wang JQ, Lam YWet al. . Control of cell migration direction by inducing cell shape asymmetry with patterned topography. J Biomed Mater Res A. 2015, 1032383.

[23] Werner M, Blanquer SBG, Haimi SP, Korus G, Dunlop JWC, Duda GNet al. . Surface curvature differentially regulates stem cell migration and differentiation via altered attachment morphology and nuclear deformation. Adv Sci. 2017, 4. 1600347

[24] Lou HY, Zhao W, Li X, Duan L, Powers A, Akamatsu Met al. . Membrane curvature underlies actin reorganization in response to nanoscale surface topography. Proc Natl Acad Sci USA. 2019, 11623143.

[25] Bhattacharjee P, Cavanagh BL, Ahearne M. Influence of micropatterned substrates on keratocyte phenotype. Sci Rep. 2020, 10. 6679

[26] He FL, Li DW, He J, Liu YY, Ahmad F, Liu YLet al. . A novel layer-structured scaffold with large pore sizes suitable for 3D cell culture prepared by near-field electrospinning. Mater Sci Eng C Mater Biol. 2018, 8618.

[27] Morelli S, Piscioneri A, Salerno S, Chen CC, Chew CH, Giorno Let al. . Microtube array membrane bioreactor promotes neuronal differentiation and orientation. Biofabrication. 2017, 92. 025018

[28] Wang X, Li S, Yan C, Liu P, Ding J. Fabrication of RGD micro/nanopattern and corresponding study of stem cell differentiation. Nano Lett. 2015, 151457.

[29] Xie C, Gao Q, Wang P, Shao L, Yuan H, Fu Jet al. . Structure-induced cell growth by 3D printing of heterogeneous scaffolds with ultrafine fibers. Mater Des. 2019, 181. 108092

[30] Chu WS, Park H, Moon S. Novel fabrication of 3-D cell laden micro-patterned porous structure on cell growth and proliferation by layered manufacturing. Bioengineering. 2023, 10. 1092

[31] Wang B, Shi J, Wei J, Tu X, Chen Y. Fabrication of elastomer pillar arrays with elasticity gradient for cell migration, elongation and patterning. Biofabrication. 2019, 11. 045003

[32] Lei X, Miao S, Wang X, Gao Y, Wu H, Cheng Pet al. . Microgroove cues guiding fibrogenesis of stem cells via intracellular force. ACS Appl Mater Interfaces. 2023, 1516380.

[33] Downing TL, Soto J, Morez C, Houssin T, Fritz A, Yuan Fet al. . Biophysical regulation of epigenetic state and cell reprogramming. Nat Mater. 2013, 121154.

[34] Jeong B, Yoon J, Ahn J, Lee B, Park S, Kim Jet al. . Eco-fabricated nanowave-textured implants drive microtubule-assisted nuclear mechanotransduction and chromatin modification: biophysical priming for osteogenesis and bone regeneration. Adv Funct Mater. 2025, 35. e03422

[35] Uhler C, Shivashankar GV. Regulation of genome organization and gene expression by nuclear mechanotransduction. Nat Rev Mol Cell Biol. 2017, 18717.

[36] Kim HS, Taghizadeh A, Taghizadeh M, Kim HW. Advanced materials technologies to unravel mechanobiological phenomena. Trends Biotechnol. 2024, 42179.

[37] Zhou W, Lin J, Wang Q, Wang X, Yao X, Yan Yet al. . Chromatin-site-specific accessibility: a microtopography-regulated door into the stem cell fate. Cell Rep. 2025, 44. 115106

[38] Zhang C, Wang X, Zhang E, Yang L, Yuan H, Tu Wet al. . An epigenetic bioactive composite scaffold with well-aligned nanofibers for functional tendon tissue engineering. Acta Biomater. 2018, 66141.

[39] Ahn H, Kim KJ, Park SY, Huh JE, Kim HJ, Yu WR. 3D braid scaffolds for regeneration of articular cartilage. J Mech Behav Biomed Mater. 2014, 3437.

[40] Cooper JA, Lu HH, Ko FK, Freeman JW, Laurencin CT. Fiber-based tissue-engineered scaffold for ligament replacement: design considerations and in vitro evaluation. Biomaterials. 2005, 261523.

[41] Su Y, Li Z, Zhu H, He J, Wei B, Li D. Electrohydrodynamic fabrication of triple-layered polycaprolactone dura mater substitute with antibacterial and enhanced osteogenic capability. Chin J Mech Eng Addit Manuf Front. 2022, 1100026

[42] Pramanick A, Hayes T, Sergis V, McEvoy E, Pandit A, Daly AC. 4D bioprinting shape-morphing tissues in granular support hydrogels: sculpting structure and guiding maturation. Adv Funct Mater.. 2024, 35. 2414559

[43] Yin Z, Chen X, Song H, Hu J, Tang Q, Zhu Tet al. . Electrospun scaffolds for multiple tissues regeneration invivo through topography dependent induction of lineage specific differentiation. Biomaterials. 2015, 44173.

[44] Sahoo S, Ang LT, Cho-Hong Goh J, Toh SL. Bioactive nanofibers for fibroblastic differentiation of mesenchymal precursor cells for ligament/tendon tissue engineering applications. Differentiation. 2010, 79102.

[45] Wang Z, Lee WJ, Koh BTHet al. . Functional regeneration of tendons using scaffolds with physical anisotropy engineered via microarchitectural manipulation. Sci Adv.. 2018, 4. eaat4537

[46] Chai M, Zhong W, Yan S, Ye T, Zheng R, Yang Zet al. . Diffusion-induced phase separation 3D printed scaffolds for dynamic tissue repair. BMEMat.. 2024, 2. e12108

[47] Shafiee A, Seyedjafari E, Sadat Taherzadeh E, Dinarvand P, Soleimani M, Ai J. Enhanced chondrogenesis of human nasal septum derived progenitors on nanofibrous scaffolds. Mater Sci Eng C Mater Biol Appl. 2014, 40445.

[48] Fisher MB, Henning EA, Söegaard N, Esterhai JL, Mauck RL. Organized nanofibrous scaffolds that mimic the macroscopic and microscopic architecture of the knee meniscus. Acta Biomater. 2013, 94496.

[49] Orr SB, Chainani A, Hippensteel KJ, Kishan A, Gilchrist C, Garrigues NWet al. . Aligned multilayered electrospun scaffolds for rotator cuff tendon tissue engineering. Acta Biomater. 2015, 24117.

[50] Lee JW, Song KH. Fibrous hydrogels by electrospinning: novel platforms for biomedical applications. J Tissue Eng. 2023.

[51] Wang S, Wang X, Jia M, Liu W, Gu Q. Thickening tissue by thinning electrospun scaffolds for skeletal muscle tissue engineering. BMEMat. 2024, 2. e12084

[52] Gwiazda M, Kumar S, Świeszkowski W, Ivanovski S, Vaquette C. The effect of melt electrospun writing fiber orientation onto cellular organization and mechanical properties for application in anterior cruciate ligament tissue engineering. J Mech Behav Biomed Mater. 2020, 104. 103631

[53] Lee H, Kim WJ, Lee JU, Yoo JJ, Kim GH, Lee SJ. Effect of hierarchical scaffold consisting of aligned dECM nanofibers and poly(lactide- co-glycolide) struts on the orientation and maturation of human muscle progenitor cells. ACS Appl Mater Interfaces. 2019, 1139449.

[54] Kim WJ, Kim M, Kim GH. 3D-printed biomimetic scaffold simulating microfibril muscle structure. Adv Funct Mater. 2018, 28. 1800405

[55] Kessel B, Lee M, Bonato A, Tinguely Y, Tosoratti E, Zenobi-Wong M. 3D bioprinting of macroporous materials based on entangled hydrogel microstrands. Adv Sci. 2020, 7. 2001419

[56] Highley CB, Song KH, Daly AC, Burdick JA. Jammed microgel inks for 3D printing applications. Adv Sci. 2019, 6. 1801076

[57] Moon D, Song KH, Doh J. Jammed microgels fabricated via various methods for biological studies. Korean J Chem Eng. 2023, 40267.

[58] Kim B, Lee B, Mandakhbayar N, Kim Y, Song Y, Doh Jet al. . Effect of lyophilized gelatin-norbornene cryogel size on calvarial bone regeneration. Mater Today Bio. 2023, 23. 100868

[59] Bae HJ, Shin SJ, Jo SB, Li CJ, Lee DJ, Lee JHet al. . Cyclic stretch induced epigenetic activation of periodontal ligament cells. Mater Today Bio. 2024, 26. 101050

[60] Klarmann GJ, Piroli ME, Loverde JR, Nelson AF, Li Z, Gilchrist KHet al. . 3D printing a universal knee meniscus using a custom collagen ink. Bioprinting. 2023, 31. e00272

[61] Petersen W, Tillmann B. Collagenous fibril texture of the human knee joint menisci. Anat Embryol. 1998, 197317.

[62] Liu Z, Wang H, Yuan Z, Wei Q, Han F, Chen Set al. . High-resolution 3D printing of angle-ply annulus fibrosus scaffolds for intervertebral disc regeneration. Biofabrication. 2023, 15. 015015

[63] Martin JT, Milby AH, Chiaro JA, Kim DH, Hebela NM, Smith LJet al. . Translation of an engineered nanofibrous disc-like angle-ply structure for intervertebral disc replacement in a small animal model. Acta Biomater. 2014, 102473.

[64] Li YM, Ji Y, Meng YX, Kim YJ, Lee H, Kurian AGet al. . Neural tissue-like, not supraphysiological, electrical conductivity stimulates neuronal lineage specification through calcium signaling and epigenetic modification. Adv Sci. 2024, 11. 2400586

[65] Singh RK, Kurian AG, Sagar V, Park I, Park JH, Lee Het al. . Coordinated biophysical stimulation of MSCs via electromagnetized Au-nanofiber matrix regulates cytoskeletal-to-nuclear mechanoresponses and lineage specification. Adv Funct Mater. 2023, 33. 2304821

[66] Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012, 9357.

[67] Quinlan AR, Hall IM. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010, 26841.

[68] Gentleman RC, Carey VJ, Bates DMet al. . Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 2004, 5. R80

Funding

National Research Foundation of Korea(RS-2021-NR061595)

RIGHTS & PERMISSIONS

Donghua University, Shanghai, China