Optimization of 3D bioprinting of mouse preosteoblasts using nanofibrillated cellulose hydrogels

Abstract

Development and optimization of advanced bioink formulations for living tissue-engineered scaffolds remain a challenging task. Herein, a nanofibrillated cellulose (NFC)-composited gelatin methacryloyl (G)/alginate (A) formulation (G/A/NFC100) was prepared for 3D bioprinting of mouse preosteoblasts MC3T3-E1, whereby the G/A formulations with a mixed NFC/microfibrillated cellulose and without NFC were included for comparison. The rheological properties of G/A formulation were enhanced by the addition of NFC, as evidenced by a decreased viscosity index characterizing shear thinning behavior from 0.52 (G/A) to 0.19 (G/A/NFC100). To construct 3D scaffolds with excellent shape fidelity while minimizing shear damage to cells during extrusion, the bioprinting conditions of the formulations were optimized based on the parameter optimization index. The G/A/NFC100 scaffold printed at a printing speed of 2 mm/s and a dispensing pressure of 30 kPa from a 27-gauge nozzle displayed a high shape fidelity (printability index of 0.883). The mechanical stability of the crosslinked 20-layered G/A/NFC100 structures were demonstrated by three consecutive press-relax cycles. The successful bioprinting of mouse preosteoblasts using the G/A/NFC100 formulation translated into an increased cell viability (above 97.64%) up to 21 days post-bioprinting. These results emphasize the exceptional potential of NFC-composited G/A formulation for bioprinting of bone tissue analogues for biomedical applications. In addition, the long-term controlled release of ampicillin (67.42% after 72 h) by G/A/NFC100 scaffolds demonstrates the feasibility of utilizing porous cellulose fibers as drug-delivery carriers to enable multifunctionality in bone tissue repair.

Keywords

3D bioprinting / Bioink formulation / Cell viability / Hydrogel / Nanofibrillated cellulose / Printability

Funding

We thank the financial support provided by the UKRI (Horizon Europe Guarantee scheme, 10066793), and FiberLean Technologies for kindly providing microfibrillated cellulose samples. Z.J.Z. thanks the financial support given by the Engineering and Physical Science Research Council (EP/V029762/1). N.L. acknowledges the Chinese Scholarship Council for the awarded scholarship (CSC201906950042).

Conflict of Interest

The authors declare they have no competing interests.

References

[1] Fang Y, Guo Y, Liu T, et al. Advances in 3D bioprinting. Chin J Mech Eng Addit Manuf Front. 2022;1(1):100011. doi: 10.1016/j.cjmeam.2022.100011

[2] Zhang B, Gao L, Ma L, Luo Y, Yang H, Cui Z. 3D bioprinting: a novel avenue for manufacturing tissues and organs. Engineering. 2019;5(4):777-794. doi: 10.1016/j.eng.2019.03.009

[3] Sharma C, Raza MA, Purohit SD, et al. Cellulose-based 3D printing bio-inks for biomedical applications: a review. Int J Biol Macromol. 2025;305:141174. doi: 10.1016/j.ijbiomac.2025.141174

[4] Mo Q, Huang L, Sheng Y, et al. Crosslinking strategy and promotion role of cellulose as a composite hydrogel component for three-dimensional printing – a review. Food Hydrocoll. 2024;154:110079. doi: 10.1016/j.foodhyd.2024.110079

[5] You P, Sun H, Chen H, et al. Composite bioink incorporating cell-laden liver decellularized extracellular matrix for bioprinting of scaffolds for bone tissue engineering. Biomater Adv. 2024;165:214017. doi: 10.1016/j.bioadv.2024.214017

[6] Ouiyangkul P, Hirun N, Suknuntha K, Tantishaiyakul V. Development and characterization of 3D bioprintable and mechanically reinforced hydrogel based on gellan gum/ methylcellulose/cellulose nanocrystals. Polym Adv Technol. 2024;35(1):e6206. doi: 10.1002/pat.6206

[7] Suamte L, Tirkey A, Barman J, Jayasekhar Babu P. Various manufacturing methods and ideal properties of scaffolds for tissue engineering applications. Smart Mater Manuf. 2023;1:100011. doi: 10.1016/j.smmf.2022.100011

[8] Mohammed A, Jiménez A, Bidare P, et al. Review on engineering of bone scaffolds using conventional and additive manufacturing technologies. 3D Print Addit Manuf. 2023;11(4):1418-1440. doi: 10.1089/3dp.2022.0360

[9] Li N, Guo R, Zhang ZJ. Bioink formulations for bone tissue regeneration. Front Bioeng Biotechnol. 2021;9. doi: 10.3389/fbioe.2021.630488

[10] Yang J, Wang J, Yang Y, et al. 3D-printed bioactive scaffolds: an emerging strategy for the regeneration of infectious bone defects. Int J Bioprint. 2024;11(2):79-138. doi: 10.36922/ijb.4986

[11] Zhao T, Liu Y, Wu Y, Zhao M, Zhao Y. Controllable and biocompatible 3D bioprinting technology for microorganisms: fundamental, environmental applications and challenges. Biotechnol Adv. 2023;69:108243. doi: 10.1016/j.biotechadv.2023.108243

[12] Chen A, Wang W, Mao Z, et al. Multimaterial 3D and 4D bioprinting of heterogenous constructs for tissue engineering. Adv Mater. 2024;36(34):2307686. doi: 10.1002/adma.202307686

[13] Liu S, Yu J-M, Gan Y-C, et al. Biomimetic natural biomaterials for tissue engineering and regenerative medicine: new biosynthesis methods, recent advances, and emerging applications. Mil Med Res. 2023;10(1):16. doi: 10.1186/s40779-023-00448-w

[14] Tarsitano M, Ming CLC, Idais D, et al. Sericin improves alginate-gelatin hydrogels’ mechanical properties, porosity, durability, and viability of fibroblasts in cardiac spheroids. Int J Bioprint. 2024;11(1):327-346. doi: 10.36922/ijb.5678

[15] Jahani A, Nourbakhsh MS, Ebrahimzadeh MH, Mohammadi M, Yari D, Moradi A. Biomolecules- loading of 3D-printed alginate-based scaffolds for cartilage tissue engineering applications: a review on current status and future prospective. Arch Bone Jt Surg. 2024;12(2):92-101. doi: 10.22038/abjs.2023.73275.3396

[16] Liu F, Jiang J, Zhe M, Yu P, Xing F, Xiang Z. Alginate-based 3D bioprinting strategies for structure–function integrated tissue regeneration. J Mater Chem B. 2025;13(40):12765-12811. doi: 10.1039/D5TB01489A

[17] de Souza JR, Rahimnejad M, Mendes Soares IP, et al. 3D printing β-TCP-laden GelMA/alginate interpenetrating-polymer-network biomaterial inks for bone tissue engineering. Bioprinting. 2025;49:e00413. doi: 10.1016/j.bprint.2025.e00413

[18] Aldana AA, Valente F, Dilley R, Doyle B. Development of 3D bioprinted GelMA-alginate hydrogels with tunable mechanical properties. Bioprinting. 2021;21:e00105. doi: 10.1016/j.bprint.2020.e00105

[19] Seddiqi H, Oliaei E, Honarkar H, et al. Cellulose and its derivatives: towards biomedical applications. Cellulose. 2021;28(4):1893-1931. doi: 10.1007/s10570-020-03674-w

[20] Tabatabaei Hosseini BS, Meadows K, Gabriel V, Hu J, Kim K. Biofabrication of cellulose-based hydrogels for advanced wound healing: a special emphasis on 3D bioprinting. Macromol Biosci. 2024;24(5):2300376. doi: 10.1002/mabi.202300376

[21] Singh P, Baniasadi H, Gupta S, et al. 3D-printed cellulose nanocrystals and gelatin scaffolds with bioactive cues for regenerative medicine: advancing biomedical applications. Int J Biol Macromol. 2024;278:134402. doi: 10.1016/j.ijbiomac.2024.134402

[22] Jiao H, Shi Y, Sun J, et al. Sawdust-derived cellulose nanofibrils with high biosafety for potential bioprinting. Ind Crops Prod. 2024;209:118025. doi: 10.1016/j.indcrop.2024.118025

[23] Carvalho JPF, Lameirinhas NS, Teixeira MC, et al. All-cellulose hydrogel-based bioinks for the versatile 3D bioprinting of different cell lines. Biomacromolecules. 2025;26(3):1761-1770. doi: 10.1021/acs.biomac.4c01546

[24] Lameirinhas NS, Carvalho JPF, Teixeira MC, et al. Nanocomposite hydrogel-based bioinks composed of a fucose-rich polysaccharide and nanocellulose fibers for 3D-bioprinting applications. Bioprinting. 2025;45:e00382. doi: 10.1016/j.bprint.2024.e00382

[25] Li N, Qi S, Buccoli L, et al. Multiscale mechanical properties and enhancement mechanism of cellulose-composited hydrogels. Carbohydr Polym. 2025;357:123421. doi: 10.1016/j.carbpol.2025.123421

[26] Li N, Bassett DC, Zhang ZJ. Microfibrillated cellulose (MFC)-composite formulations for 3D bioprinting with excellent printability, mechanical strength, and biological functionality. Chem Eng J. 2025:169037. doi: 10.1016/j.cej.2025.169037

[27] Gatenholm P, Martinez H, Karabulut E, et al. Development of nanocellulose-based bioinks for 3D bioprinting of soft tissue. In: Ovsianikov A, Yoo J, Mironov V, eds. 3D Printing and Biofabrication. New York, NY: Springer International Publishing; 2016:1-23. doi: 10.1007/978-3-319-45444-3_14

[28] Webb B, Doyle BJ. Parameter optimization for 3D bioprinting of hydrogels. Bioprinting. 2017;8:8-12. doi: 10.1016/j.bprint.2017.09.001

[29] Lewicki J, Bergman J, Kerins C, Hermanson O. Optimization of 3D bioprinting of human neuroblastoma cells using sodium alginate hydrogel. Bioprinting. 2019;16:e00053. doi: 10.1016/j.bprint.2019.e00053

[30] Ouyang L, Yao R, Zhao Y, Sun W. Effect of bioink properties on printability and cell viability for 3D bioplotting of embryonic stem cells. Biofabrication. 2016;8(3):035020. doi: 10.1088/1758-5090/8/3/035020