Researchers Develop Cellulose-Based 3D Scaffolds That Mimic Natural Tissue Architecture

By Pooja Toshniwal PahariaReviewed by Lily Ramsey, LLMApr 9 2025

In a recent study published in STAR Protocols, researchers developed a protocol for creating self-standing three-dimensional (3D) scaffolds from cellulose-based substances. These scaffolds can mimic the architecture and functions of natural tissues such as skin and cartilage.

The protocol leverages the unique properties of nano-fibrillated cellulose (NFC) to produce scaffolds with adjustable porosity, structural integrity, and mechanical stability, all of which are necessary for enabling cell development and tissue regeneration.

​​​​​​​Image Credit: Gorodenkoff/Shutterstock.com

Introduction

Advanced biological engineering scaffolds are becoming increasingly essential to reproducing the structure and features of biological tissues. Self-standing three-dimensional scaffolds can replicate the extracellular matrix (ECM), a network of proteins and carbohydrates that support and shape cells and tissues in the human body.

The ECM is vital for cell attachment, proliferation, and communication. The scaffolds hold promising potential in tissue engineering applications since they provide a strong structural framework and environment favorable for cell growth.

Cellulose-based materials are superior to other biomaterials for constructing scaffolds with high renewability, biocompatibility, and adaptability. NFC-based hydrogels improve cell viability and can be 3D printed into complex shapes.

About the Study

In the present study, researchers present a protocol for creating self-standing (nano)cellulose-based 3D scaffolds for in vitro testing of cells from skin and cartilage tissues.

The technique includes nanocellulose ink production, 3D printing, freeze-drying, and cross-linking. Post-processing procedures improve mechanical characteristics, stability, and biocompatibility.

The researchers created three-dimensional scaffolds by combining nano-fibrillated cellulose, citric acid (CA), and carboxymethyl cellulose (CMC). They mixed 1.5g of NFC with 6g of CMC and 5.0 to 10 CA by weight to create an ink with superior shear thinning capabilities. The prepared ink could be continuously released to generate three-dimensional structures. The ink was prepared in an hour and stored at 6-8°C for further use.

To print 3D scaffolds, the researchers inserted the ink into a 10 mL plastic syringe and mounted the syringe on a mounting ring, which they capped with a metal top and screwed firmly. They also inserted the gas tubing onto the metal cover.

The team used a circular printing model with a 5.0 cm radius and a maximum height of 10 mm. They printed the scaffold on a polystyrene petri dish with a diameter of 5.0 cm. After printing, they closed the petri dish and sealed it with stretch foil.

The direct-ink-writing (DIW) process of 3D printing involved software-enabled layer-by-layer prints over four hours to produce structures with remarkable form integrity.

Printing parameters were 100 edge corners, 220-260 kPa dispensing pressure, 500-900 mm strand distances, 0.2 mm strand height, and 15 mm/s printing speed. The procedure required a temperature of 22-24 °C.

The 3D-printed structures were freeze-dried and cross-linked by dehydrothermal (DHT) treatment for over 48 hours. Freeze-drying eliminates excess water from scaffolds while leaving their porous structure intact.

Cross-linking of carboxylic and hydroxyl groups of CMC and NFC with CA improves resistance against degradation while preserving the structure’s porosity and microstructure. DHT treatment enables cross-linking without chemical additives.

After dehydrothermal treatment, the scaffolds were exposed to 0.1M sodium hydroxide for 60 minutes to neutralize excess citric acid and prevent aberrant cell behavior during cell testing in vitro.

Procedural Challenges and Potential Solutions

During DIW printing of three-dimensional structures, the nozzle may clog due to particle size and the dispersibility of NFC fibers. To prevent this, the 3D printing ink should have a homogeneous consistency without visible nano-fibrillated cellulose fibers.

Performing an ink test and ultrasonicating NFC before printing could prevent nozzle obstruction. The ink may not extrude from the syringe due to gas leakage.

In such a scenario, tightly secure the mounting ring, cartridge, gas tube, and metal lid attachments. Also, ensure that the cartridge pressure is correct.

If the freeze-dried scaffold form appears flat, repeat the operation while maintaining pressure below 10 bar and a temperature below 24°C. If the finished scaffolds turn yellow due to dehydrothermal treatment, reduce the room temperature to 90°C from 120°C.

Neutralized scaffolds with cross-linked structures could have a lower pH between 4.0 and 5.0. To address pH concerns, immerse the scaffolds in a 0.10 M sodium hydroxide solution for an extended duration. To ensure complete neutralization, one can double the neutralization time.

Conclusions

The study highlights the development of self-standing nanocellulose-based scaffolds that can replicate biological tissues in three dimensions. The procedure involves preparing a three-component ink, creating 3D-printed scaffolds using the ink, and cross-linking individual components by dehydrothermal treatment.

NFC offers high strength and surface area for functionalization, whereas CMC improves hydrogel characteristics, improving print quality and stability. Citric acid increases the mechanical stability and biocompatibility of the scaffolds.

The technique can generate biocompatible structures with high mechanical strength and customized porosity for cell adhesion and proliferation. Future research should focus on improving cross-linking conditions and increasing scalability for clinical use.