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RESEARCH ARTICLE

Fibrous bioinks for bioprinting anisotropic micro-and nanoscale scaffolds: A novel strategy for in vitro skeletal muscle engineering

Gerardina Ruocco1,2,3† ,  Elena Marcello1,2,3†* ,  Camilla Paoletti1,2,3 ,  Massimo Salvi2,4 ,  Alice Zoso1,2,3 ,  Mattia Spedicati1,2,3 ,  Irene Carmagnola1,2,3‡ ,  Valeria Chiono1,2,3‡
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1 Department of Mechanical and Aerospace Engineering, Politecnico di Torino, Turin, Italy
2 PolitoBIOMed Lab, Politecnico di Torino, Turin, Italy
3 Interuniversity Center for the Promotion of 3Rs Principles in Teaching and Research, Pisa, Italy
4 Department of Electronics and Telecommunications, Politecnico di Torino, Turin, Italy
†These authors contributed equally to this work.
IJB, 025440455 https://doi.org/10.36922/IJB025440455
Received: 2 July 2025 | Accepted: 10 November 2025 | Published online: 10 November 2025
© 2025 by the Author(s). This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ )
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Abstract

Replicating skeletal muscle architecture remains challenging in 3D bioprinting, as conventional bioinks lack multiscale directional cues. Herein, we propose a next-generation fibrous bioink composed of fragmented electrospun gelatin fibers (f-GFs), uniformly embedded in an alginate/gelatin hydrogel matrix (f-ALG/Gel). Upon microextrusion bioprinting, shear-induced f-GF alignment enabled the fabrication of microfilament-based scaffolds with intrinsic anisotropy. The resulting constructs exhibited high shape fidelity, favorable viscoelastic properties, and physiologically relevant stiffness (Young’s modulus: 16.1 ± 1.7 kPa). In vitro studies using C2C12 murine myoblasts demonstrated that the embedded f-GFs provided strong topographical guidance, enhancing cell alignment and myogenesis. After 14 days of culture, the f-ALG/Gel scaffolds supported a 2.5-fold increase in myotube fusion index and length, alongside reduced angular dispersion. These effects were achieved without the need for biochemical induction with a differentiation medium, underscoring the key role of structural cues at the micro- and nanoscale in C2C12 differentiation and maturation. In conclusion, this work proposes a scalable, cell-compatible strategy to recapitulate the hierarchical organization of skeletal muscle tissue within 3D-printed constructs. The platform holds broad potential for applications in regenerative medicine, skeletal muscle tissue modeling, and the engineering of cultured meat.

Graphical abstract
Keywords
Anisotropy
3D bioprinting
Fibrous bioinks
Skeletal muscle
Topographical cue
Funding
This work was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation Programme (Grant Agreement No. 772168), through the BIORECAR ERC Consolidator project (www.biorecar.polito.it); and under the European Union’s Research and Innovation Programme (Grant Agreement No. 101158332) for the ERC-2023-POC EMPATIC project.
Conflict of interest
Elena Marcello serves as the Editorial Board Member of the journal, but did not in any way involve in the editorial and peer-review process conducted for this paper, directly or indirectly. Other authors declare they have no competing interests.
References
  1. Hulett NA, Scalzo RL, Reusch JEB. Glucose uptake by skeletal muscle within the contexts of type 2 diabetes and exercise: An integrated approach. Nutrients. 2022;14(3):647. doi: 10.3390/nu14030647
  2. Kim J, Kim IU, Lee ZF, Sim G, Jeon JS. Strategic approaches in generation of robust microphysiological 3D musculoskeletal tissue system. Adv Funct Mater. 2024;34(52). doi: 10.1002/adfm.202410872
  3. García-Lizarribar A, Villasante A, Lopez-Martin JA, et al. 3D bioprinted functional skeletal muscle models have potential applications for studies of muscle wasting in cancer cachexia. Biomater Adv. 2023;150:213426. doi: 10.1016/j.bioadv.2023.213426
  4. Zhuang P, An J, Chua CK, Tan LP. Bioprinting of 3D in vitro skeletal muscle models: A review. Mater Des. 2020;193:108794. doi: 10.1016/j.matdes.2020.108794
  5. Volpi M, Paradiso A, Costantini M, Świȩszkowski W. Hydrogel-based fiber biofabrication techniques for skeletal muscle tissue engineering. ACS Biomater Sci Eng. 2022;8(2):379-405. doi: 10.1021/acsbiomaterials.1c01145
  6. Lou H, Lu H, Zhang S, et al. Highly aligned myotubes formation of piscine satellite cells in 3D fibrin hydrogels of cultured meat. Int J Biol Macromol. 2024;282:136879. doi: 10.1016/j.ijbiomac.2024.136879
  7. Li X, Sim D, Wang Y, et al. Fiber-based biomaterial scaffolds for cell support towards the production of cultivated meat. Acta Biomater. 2025;191:292-307. doi: 10.1016/j.actbio.2024.11.006
  8. Sabetkish S, Currie P, Meagher L. Recent trends in 3D bioprinting technology for skeletal muscle regeneration. Acta Biomater. 2024;181:46-66. doi: 10.1016/j.actbio.2024.04.038
  9. Schwab A, Hélary C, Richards RG, Alini M, Eglin D, D’Este M. Tissue mimetic hyaluronan bioink containing collagen fibers with controlled orientation modulating cell migration and alignment. Mater Today Bio. 2020;7:100058. doi: 10.1016/j.mtbio.2020.100058
  10. Mehmood H, Kasher PR, Barrett-Jolley R, Walmsley GL. Aligning with the 3Rs: Alternative models for research into muscle development and inherited myopathies. BMC Vet Res. 2024;20(1):477. doi: 10.1186/s12917-024-04309-z
  11. Kang MS, Lee SH, Park WJ, Lee JE, Kim B, Han DW. Advanced techniques for skeletal muscle tissue engineering and regeneration. Bioengineering. 2020;7(3):99. doi: 10.3390/bioengineering7030099
  12. Trovato F, Imbesi R, Conway N, Castrogiovanni P. Morphological and functional aspects of human skeletal muscle. J Funct Morphol Kinesiol. 2016;1(3):289-302. doi: 10.3390/jfmk1030289
  13. Nikkhah M, Edalat F, Manoucheri S, Khademhosseini A. Engineering microscale topographies to control the cell– substrate interface. Biomaterials. 2012;33(21):5230-5246. doi: 10.1016/j.biomaterials.2012.03.079
  14. Wang L, Wu Y, Guo B, Ma PX. Nanofiber yarn/hydrogel core–shell scaffolds mimicking native skeletal muscle tissue for guiding 3D myoblast alignment, elongation, and differentiation. ACS Nano. 2015;9(9):9167-9179. doi: 10.1021/acsnano.5b03644
  15. Mueller C, Trujillo‐Miranda M, Maier M, Heath DE, O’Connor AJ, Salehi S. Effects of external stimulators on engineered skeletal muscle tissue maturation. Adv Mater Interfaces. 2021;8(1). doi: 10.1002/admi.202001167
  16. Ostrovidov S, Hosseini V, Ahadian S, et al. Skeletal muscle tissue engineering: Methods to form skeletal myotubes and their applications. Tissue Eng Part B Rev. 2014;20(5):403-436. doi: 10.1089/ten.teb.2013.0534
  17. Kim H, Jang J, Park J, et al. Shear-induced alignment of collagen fibrils using 3D cell printing for corneal stroma tissue engineering. Biofabrication. 2019;11(3):035017. doi: 10.1088/1758-5090/ab1a8b
  18. Moncal KK, Ozbolat V, Datta P, Heo DN, Ozbolat IT. Thermally-controlled extrusion-based bioprinting of collagen. J Mater Sci Mater Med. 2019;30(5):55. doi: 10.1007/s10856-019-6258-2
  19. Prendergast ME, Davidson MD, Burdick JA. A biofabrication method to align cells within bioprinted photocrosslinkable and cell-degradable hydrogel constructs via embedded fibers. Biofabrication. 2021;13(4). doi: 10.1088/1758-5090/AC25CC
  20. Chaudhuri O, Cooper-White J, Janmey PA, Mooney DJ, Shenoy VB. Effects of extracellular matrix viscoelasticity on cellular behaviour. Nature. 2020;584(7822):535-546. doi: 10.1038/s41586-020-2612-2
  21. Lloyd EM, Hepburn MS, Li J, et al. Multimodal three-dimensional characterization of murine skeletal muscle micro-scale elasticity, structure, and composition: Impact of dysferlinopathy, duchenne muscular dystrophy, and age on three hind-limb muscles. J Mech Behav Biomed Mater. 2024;160:106751. doi: 10.1016/j.jmbbm.2024.106751
  22. Choi S, Lee KY, Kim SL, et al. Fibre-infused gel scaffolds guide cardiomyocyte alignment in 3d-printed ventricles. Nat Mater. 2023;22(8):1039-1046. doi: 10.1038/s41563-023-01611-3
  23. Kamaraj M, Rezayof O, Barer A, et al. Development of silk microfiber-reinforced bioink for muscle tissue engineering and in situ printing by a handheld 3d printer. Biomater Adv. 2025;166:214057. doi: 10.1016/j.bioadv.2024.214057
  24. Li T, Hou J, Wang L, et al. Bioprinted anisotropic scaffolds with fast stress relaxation bioink for engineering 3D skeletal muscle and repairing volumetric muscle loss. Acta Biomater. 2023;156:21-36. doi: 10.1016/j.actbio.2022.08.037
  25. Stola GP, Paoletti C, Nicoletti L, et al. Internally-crosslinked alginate dialdehyde/alginate/gelatin-based hydrogels as bioinks for prospective cardiac tissue engineering applications. Int J Bioprint. 2024;10(6):544-566. doi: 10.36922/ijb.4014
  26. Tonda-Turo C, Gentile P, Saracino S, et al. Comparative analysis of gelatin scaffolds crosslinked by genipin and silane coupling agent. Int J Biol Macromol. 2011;49(4):700-706. doi: 10.1016/j.ijbiomac.2011.07.002
  27. Tonda-Turo C, Cipriani E, Gnavi S, et al. Crosslinked gelatin nanofibres: Preparation, characterisation and in vitro studies using glial-like cells. Mater Sci Eng: C. 2013;33(5):2723-2735. doi: 10.1016/j.msec.2013.02.039
  28. Giuntoli G, Muzio G, Actis C, et al. In-vitro characterization of a hernia mesh featuring a nanostructured coating. Front Bioeng Biotechnol. 2021;8. doi: 10.3389/fbioe.2020.589223
  29. John JV., McCarthy A, Wang H, et al. Engineering biomimetic nanofiber microspheres with tailored size, predesigned structure, and desired composition via gas bubble–mediated coaxial electrospray. Small. 2020;16(19). doi: 10.1002/smll.201907393
  30. Carmagnola I, Chiono V, Ruocco G, et al. PLGA membranes functionalized with gelatin through biomimetic mussel-inspired strategy. Nanomaterials. 2020;10(11):2184. doi: 10.3390/nano10112184
  31. Salgado-Delgado AM, González-Mondragón EG, Hernández-Pérez R, Salgado-Delgado R, Santana-Camilo JA, Olarte-Paredes A. Obtention and characterization of GO/Epoxy and GO-GPTMS/epoxy nanocompounds with different oxidation degrees and ultrasound methods. C (Basel). 2023;9(1):28. doi: 10.3390/c9010028
  32. Sitthiracha M, Kilmartin PA, Edmonds NR. Novel organic– inorganic hybrid materials based on epoxy-functionalized silanes. J Solgel Sci Technol. 2015;76(3):542-551. doi: 10.1007/s10971-015-3804-3
  33. Wang X, Wang M, Xu Y, Yin J, Hu J. A 3D-printable gelatin/alginate/ε-poly-l-lysine hydrogel scaffold to enable porcine muscle stem cells expansion and differentiation for cultured meat development. Int J Biol Macromol. 2024; 271:131980. doi: 10.1016/j.ijbiomac.2024.131980
  34. Yeong WY, Yu H, Lim KP, et al. Multiscale topological guidance for cell alignment via direct laser writing on biodegradable polymer. Tissue Eng Part C Methods. 2010;16(5):1011-1021. doi: 10.1089/ten.tec.2009.0604
  35. Guven S, Chen P, Inci F, Tasoglu S, Erkmen B, Demirci U. Multiscale assembly for tissue engineering and regenerative medicine. Trends Biotechnol. 2015;33(5):269-279. doi: 10.1016/j.tibtech.2015.02.003
  36. Kerativitayanan P, Carrow JK, Gaharwar AK. Nanomaterials for engineering stem cell responses. Adv Healthc Mater. 2015;4(11):1600-1627. doi: 10.1002/adhm.201500272
  37. Rizzi R, Bearzi C, Mauretti A, Bernardini S, Cannata S, Gargioli C. Tissue engineering for skeletal muscle regeneration. Muscles Ligaments Tendons J. 2012;2(3):230-234.
  38. Griffith LG, Swartz MA. Capturing complex 3D tissue physiology in vitro. Nat Rev Mol Cell Biol. 2006;7(3):211-224. doi: 10.1038/NRM1858
  39. Dutta SD, Ganguly K, Jeong MS, et al. Bioengineered lab-grown meat-like constructs through 3D bioprinting of antioxidative protein hydrolysates. ACS Appl Mater Interfaces. 2022;14(30):34513-34526. doi: 10.1021/acsami.2c10620
  40. Yi H, Forsythe S, He Y, et al. Tissue-specific extracellular matrix promotes myogenic differentiation of human muscle progenitor cells on gelatin and heparin conjugated alginate hydrogels. Acta Biomater. 2017;62:222-233. doi: 10.1016/j.actbio.2017.08.022
  41. Rao KM, Kim HJ, Won S, Choi SM, Han SS. Effect of grape seed extract on gelatin-based edible 3D-hydrogels for cultured meat application. Gels. 2023;9(1):65. doi: 10.3390/gels9010065
  42. Valliant EM, Romer F, Wang D, et al. Bioactivity in silica/ poly(γ-glutamic acid) sol–gel hybrids through calcium chelation. Acta Biomater. 2013;9(8):7662-7671. doi: 10.1016/j.actbio.2013.04.037
  43. Li X, Wang X, Yao D, et al. Effects of aligned and random fibers with different diameter on cell behaviors. Colloids Surf B Biointerfaces. 2018;171:461-467. doi: 10.1016/j.colsurfb.2018.07.045
  44. Tian F, Hosseinkhani H, Hosseinkhani M, et al. Quantitative analysis of cell adhesion on aligned micro- and nanofibers. J Biomed Mater Res A. 2008;84(2):291-299. doi: 10.1002/jbm.a.31304
  45. Martino F, Perestrelo AR, Vinarský V, Pagliari S, Forte G. Cellular mechanotransduction: From tension to function. Front Physiol. 2018;9. doi: 10.3389/fphys.2018.00824
  46. Davidenko N, Schuster CF, Bax D V., et al. Evaluation of cell binding to collagen and gelatin: A study of the effect of 2D and 3D architecture and surface chemistry. J Mater Sci Mater Med. 2016;27(10):148. doi: 10.1007/s10856-016-5763-9
  47. Abdin M, Salama MA, Riaz A, Akhtar HMS, Elsanat SY. Enhanced the entrapment and controlled release of syzygium cumini seeds polyphenols by modifying the surface and internal organization of alginate‐based microcapsules. J Food Process Preserv. 2021;45(1). doi: 10.1111/jfpp.15100
  48. Levato R, Jungst T, Scheuring RG, Blunk T, Groll J, Malda J. From shape to function: The next step in bioprinting. Adv Mater. 2020;32(12). doi: 10.1002/adma.201906423
  49. Tirella A, Orsini A, Vozzi G, Ahluwalia A. A phase diagram for microfabrication of geometrically controlled hydrogel scaffolds. Biofabrication. 2009;1(4):045002. doi: 10.1088/1758-5082/1/4/045002
  50. Nam SY, Park SH. ECM based bioink for tissue mimetic 3d bioprinting. Adv Exp Med Biol. 2018; 1064:335-353. doi: 10.1007/978-981-13-0445-3_20
  51. Malda J, Visser J, Melchels FP, et al. 25th anniversary article: Engineering hydrogels for biofabrication. Adv Mater. 2013;25(36):5011-5028. doi: 10.1002/adma.201302042
  52. Sakai S, Yoshii A, Sakurai S, Horii K, Nagasuna O. Silk fibroin nanofibers: A promising ink additive for extrusion three-dimensional bioprinting. Mater Today Bio. 2020;8:100078. doi: 10.1016/j.mtbio.2020.100078
  53. Jessop ZM, Al-Sabah A, Gao N, et al. Printability of pulp derived crystal, fibril and blend nanocellulose-alginate bioinks for extrusion 3D bioprinting. Biofabrication. 2019;11(4):045006. doi: 10.1088/1758-5090/ab0631
  54. Markstedt K, Mantas A, Tournier I, Martínez Ávila H, Hägg D, Gatenholm P. 3D bioprinting human chondrocytes with nanocellulose–alginate bioink for cartilage tissue engineering applications. Biomacromolecules. 2015;16(5):1489-1496. doi: 10.1021/acs.biomac.5b00188
  55. Seiti M, Mazzoldi EL, Pandini S, et al. FRESH 3D bioprinting of alginate – cellulose – gelatin constructs for soft tissue biofabrication. Procedia CIRP. 2024;125:42-47. doi: 10.1016/j.procir.2024.08.008
  56. Distler T, McDonald K, Heid S, Karakaya E, Detsch R, Boccaccini AR. Ionically and enzymatically dual cross-linked oxidized alginate gelatin hydrogels with tunable stiffness and degradation behavior for tissue engineering. ACS Biomater Sci Eng. 2020;6(7):3899-3914. doi: 10.1021/acsbiomaterials.0c00677
  57. Sonaye SY, Ertugral EG, Kothapalli CR, Sikder P. Extrusion 3D (bio)printing of alginate-gelatin-based composite scaffolds for skeletal muscle tissue engineering. Materials. 2022;15(22):7945. doi: 10.3390/ma15227945
  58. Sonnleitner D, Schrüfer S, Berglund L, Schubert DW, Lang G. Correlating rheology and printing performance of fiber-reinforced bioinks to assess predictive modelling for biofabrication. J Mater Res. 2021;36(19):3821-3832. doi: 10.1557/s43578-021-00276-5
  59. Hausmann MK, Rühs PA, Siqueira G, et al. Dynamics of cellulose nanocrystal alignment during 3D printing. ACS Nano. 2018;12(7):6926-6937. doi: 10.1021/acsnano.8b02366
  60. Chaturvedi V, Dye DE, Kinnear BF, van Kuppevelt TH, Grounds MD, Coombe DR. Interactions between skeletal muscle myoblasts and their extracellular matrix revealed by a serum free culture system. PLoS One. 2015;10(6):e0127675. doi: 10.1371/journal.pone.0127675
  61. Denes LT, Riley LA, Mijares JR, et al. Culturing C2C12 myotubes on micromolded gelatin hydrogels accelerates myotube maturation. Skelet Muscle. 2019;9(1):17. doi: 10.1186/s13395-019-0203-4
  62. Jensen JH, Cakal SD, Li J, et al. Large-scale spontaneous self-organization and maturation of skeletal muscle tissues on ultra-compliant gelatin hydrogel substrates. Sci Rep. 2020;10(1):13305. doi: 10.1038/s41598-020-69936-6
  63. Spedicati M, Zoso A, Mortati L, Chiono V, Marcello E, Carmagnola I. Three-dimensional microfibrous scaffold with aligned topography produced via a combination of melt-extrusion additive manufacturing and porogen leaching for in vitro skeletal muscle modeling. Bioengineering. 2024;11(4):332. doi: 10.3390/bioengineering11040332
  64. Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell. 2006;126(4):677-689.doi: 10.1016/j.cell.2006.06.044
  65. Cui W, Huang Y, Chen L, et al. Tiny yet tough: Maximizing the toughness of fiber-reinforced soft composites in the absence of a fiber-fracture mechanism. Matter. 2021;4(11):3646-3661. doi: 10.1016/j.matt.2021.08.013
  66. Heher P, Maleiner B, Prüller J, et al. A novel bioreactor for the generation of highly aligned 3D skeletal muscle-like constructs through orientation of fibrin via application of static strain. Acta Biomater. 2015;24:251-265. doi: 10.1016/j.actbio.2015.06.033
  67. Schwab A, Levato R, D’Este M, Piluso S, Eglin D, Malda J. Printability and shape fidelity of bioinks in 3D bioprinting. Chem Rev Am Chem Soc. 2020;120(19):11028-11055. doi: 10.1021/acs.chemrev.0c00084
  68. Heck T, Faccio G, Richter M, Thöny-Meyer L. Enzyme-catalyzed protein crosslinking. Appl Microbiol Biotechnol. 2013;97(2):461-475. doi: 10.1007/s00253-012-4569-z
  69. Kashiwagi T, Yokoyama K ichi, Ishikawa K, et al. Crystal Structure Of Microbial Transglutaminase From Streptoverticillium mobaraense. J Biol Chem. 2002;277(46):44252-44260. doi: 10.1074/jbc.M203933200
  70. Kitzmann M, Carnac G, Vandromme M, Primig M, Lamb NJC, Fernandez A. The muscle regulatory factors MyoD and Myf-5 undergo distinct cell cycle–specific expression in muscle cells. J Cell Biol. 1998;142(6):1447-1459. doi: 10.1083/jcb.142.6.1447
  71. Yang HS, Lee B, Tsui JH, et al. Electroconductive nanopatterned substrates for enhanced myogenic differentiation and maturation. Adv Healthc Mater. 2016;5(1):137-145. doi: 10.1002/adhm.201500003
  72. Niknezhad SV, Mehrali M, Khorasgani FR, et al. Enhancing volumetric muscle loss (VML) recovery in a rat model using super durable hydrogels derived from bacteria. Bioact Mater. 2024;38:540-558. doi: 10.1016/j.bioactmat.2024.04.006
  73. Kim K, Jin S, Shin M. Effect of 3D‐printable anisotropic fibrous hydrogels on fabricating artificial skeletal muscle constructs. Adv Ther (Weinh). 2024;7(1). doi: 10.1002/adtp.202300170
  74. Lee S, Kim W, Kim G. Efficient myogenic activities achieved through blade-casting-assisted bioprinting of aligned myoblasts laden in collagen bioink. Biomacromolecules. 2023;24(11):5219-5229. doi: 10.1021/acs.biomac.3c00749
  75. Li Q, Yu S, Wang Y, et al. Programmable embedded bioprinting for one-step manufacturing of arterial models with customized contractile and metabolic functions. Trends Biotechnol. 2025;43(4):918-945. doi: 10.1016/j.tibtech.2024.11.019

 

 

 

 

 

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International Journal of Bioprinting, Electronic ISSN: 2424-8002 Print ISSN: 2424-7723, Published by AccScience Publishing
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