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. 2025 Mar 21;6(1):103583.
doi: 10.1016/j.xpro.2024.103583. Epub 2025 Jan 24.

Protocol for the fabrication of self-standing (nano)cellulose-based 3D scaffolds for tissue engineering

Tamilselvan Mohan  1 , Matej Bračič  2 , Doris Bračič  3 , Florian Lackner  4 , Chandran Nagaraj  5 , Andreja Dobaj Štiglic  3 , Rupert Kargl  6 , Karin Stana Kleinschek  7
Affiliations

Protocol for the fabrication of self-standing (nano)cellulose-based 3D scaffolds for tissue engineering

Tamilselvan Mohan et al. STAR Protoc. .

Abstract

Three-dimensional (3D) and porous scaffolds made from nanocellulosic materials hold significant potential in tissue engineering (TE). Here, we present a protocol for fabricating self-standing (nano)cellulose-based 3D scaffolds designed for in vitro testing of cells from skin and cartilage tissues. We describe steps for preparation of nanocellulose ink, scaffold formation using 3D printing, and freeze-drying. We then detail post-processing procedures to enhance mechanical properties, stability, and biocompatibility. This protocol offers researchers a framework for developing versatile and sustainable biomaterials for regenerative medicine. For complete details on the use and execution of this protocol, please refer to Mohan et al.1 and Štiglic et al.2.

Keywords: Biotechnology and bioengineering; Chemistry; Material sciences; Tissue Engineering.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Illustration of the direct-ink-writing (DIW) printing of scaffolds, freeze-drying, dehydrothermal (DHT) post-treatment procedures, and the cross-linking mechanism among the scaffold components NFC, nanofibrillated cellulose; CMC, carboxymethyl cellulose; CA, citric acid.
Figure 2
Figure 2
Preparation of three-component ink system from the combination of nanofibrillated cellulose (NFC), carboxymethyl cellulose (CMC), and citric acid (CA)
Figure 3
Figure 3
Illustration of direct-ink-writing 3D printing methodology: printing syringe preparation, loading into printing head, scaffold design using bioscaffolder 3.2 software, and printing of scaffolds DIW 3D printing.
Figure 4
Figure 4
Illustrates the meticulous process of transforming 3D-printed scaffolds, starting with their placement in a freeze-drying unit and followed by their transfer to a vacuum oven for crosslinking through dehydrothermal treatment
Figure 5
Figure 5
Illustrates the neutralization process of the 3D-printed scaffold using sodium hydroxide, followed by thorough rinsing with water to ensure complete removal of residual alkali
Figure 6
Figure 6
SEM micrographs of NFC/CMC scaffolds crosslinked with different amounts of citric acid
See this image and copyright information in PMC

References

    1. Mohan T., Dobaj Štiglic A., Beaumont M., Konnerth J., Gürer F., Makuc D., Maver U., Gradišnik L., Plavec J., Kargl R., Stana Kleinschek K. Generic Method for Designing Self-Standing and Dual Porous 3D Bioscaffolds from Cellulosic Nanomaterials for Tissue Engineering Applications. ACS Appl. Bio Mater. 2020;3:1197–1209. doi: 10.1021/acsabm.9b01099. - DOI - PubMed
    1. Štiglic A.D., Gürer F., Lackner F., Bračič D., Winter A., Gradišnik L., Makuc D., Kargl R., Duarte I., Plavec J., et al. Organic acid cross-linked 3D printed cellulose nanocomposite bioscaffolds with controlled porosity, mechanical strength, and biocompatibility. iScience. 2022;25 doi: 10.1016/j.isci.2022.104263. - DOI - PMC - PubMed
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    1. Mohan T., Maver T., Štiglic A.D., Stana-Kleinschek K., Kargl R. In: Fundamental Biomaterials: Polymers. Thomas S., Balakrishnan P., Sreekala M.S., editors. Woodhead Publishing; 2018. 6 - 3D bioprinting of polysaccharides and their derivatives: From characterization to application; pp. 105–141. - DOI
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