A Brief Overview on Polysaccharide-Based Hydrogels in 3D Bioprinting for Biomedical Applications: Cases of Cellulose, Chitosan, and Lignin ^†

Abstract

Three-dimensional (3D) bioprinting has become one of the most advanced and useful innovations that allows the creation of personalized macroscopic and microscopic constructs at different scales that match a patient’s anatomy. Intensive research efforts are currently underway to develop highly printable and biocompatible materials. Among the variety of bioprinting materials (i.e., biomaterial inks), naturally derived hydrogels have attracted great interest due to their beneficial properties in terms of biocompatibility, cost-effectiveness, and biodegradability. In this proceeding paper, we provide an overview of the formulation and use of three functional polysaccharides as ink-based hydrogels. First, 3D bioprinting is summarized as revolutionary technology that is able to create cell-laden structures layer by layer in a specific pattern that mimics native tissue and organs. Cellulose, chitosan, and lignin are presented below, followed by an overview of their applicability in 3D bioprinting, focusing on printability and the resulting printed 3D structures as illustrated in various published figures. In the same way, a comparative overview of 3D bioprinting applications is summarized. Finally, a section dedicated to comparisons, limitations, and crosslinking strategies is provided. It is worth noting that this proceedings paper provides a brief overview rather than a comprehensive review, as it is limited by page constraints and is based on the content of our poster presented at the 1st International Online Conference on Bioengineering.

1. Introduction

Three-dimensional (3D) bioprinting has revolutionized tissue engineering and regenerative medicine fields due to the increasing demand for organ and tissue replacement [1,2]. It involves the creation of patient-specific structures using cell-laden inks or bioinks based on natural or synthetic polymers. Three-dimensional bioprinting techniques are generally grouped into four categories, namely extrusion-based, droplet-based, laser-assisted, and vat-based polymerization bioprinting [3,4]. Finding appropriate bioprinting material is the crucial step in the bioprinting process, as it should provide a microenvironment that can support cell growth, proliferation, and differentiation [5]. Hydrogels show great promise for bioink preparation from both their rheological and biological properties [6]. Moreover, their porous structure and capacity to absorb water and biological fluids without being dissolved, enabling oxygen and nutrient exchange, make them the most used biomaterials in this field [7]. Furthermore, it is possible to tune hydrogels properties, such as viscosity, to reach the different 3D bioprinting methods requirements. Recently, researchers tended to use natural-based hydrogels, either polysaccharide- or protein-based, thanks to their interesting properties such as biodegradability, biocompatibility, and low cytotoxicity. This is the case with cellulose, chitosan, and lignin biopolymers [4].

Cellulose is the most abundant biopolymer in nature; it has various advantages over others, such as good mechanical and barrier properties [8]. In addition, chitosan biopolymer constitutes a promising candidate for the preparation of hydrogels for application in this field thanks to its beneficial properties, such as antimicrobial activity and its structural resemblance to natural glycosaminoglycans (GAG) [9]. Furthermore, due to the non-cytotoxicity, biocompatibility, biodegradability, mechanical strength, and reactivity of the lignin biopolymer, it has been considered an excellent candidate to manufacture hydrogels for 3D bioprinting applications [10]. The capacity of 3D bioprinting of these biopolymer-based hydrogels has been demonstrated in the regeneration of different damaged tissues, including cartilage, bone, muscle, skin, blood vessels, and other biological tissues.

This study—as a complementary proceeding paper of the poster presented at the 1st International Online Conference on Bioengineering [11]—provides an overview on the formulation and use of cellulose-, chitosan-, and lignin-based hydrogels for 3D bioprinting applications. It does not follow the structure of a full systematic review. The selection of references is illustrative rather than exhaustive, and the paper is intended as a brief overview.

2. Three-Dimensional Bioprinting

Bioprinting, a computer-assisted technology, is able to create cell-laden structures layer by layer in a specific pattern that mimics native tissue and organs [4,12,13]. As human anatomy is so complicated, various bioprinting methods are developed to figure out the challenges of different applications. Furthermore, the utilization of each method also depends on bioink properties [14]. With regard to that, 3D bioprinting methods can be divided into four groups, including extrusion bioprinting, which allows the formation of unbroken filaments; droplet-based bioprinting, which generates droplets of bioinks and gradually stacks them in 3D structures; laser-assisted bioprinting, which uses laser energy in the form of impulses to transfer bioinks to a substrate; and vat-polymerization-based bioprinting, which uses a photocurable bioink and ultraviolet or infrared radiation for its crosslinking [2,14]. Generally, as presented in Figure 1, the process of 3D bioprinting consists of several steps, namely pre-bioprinting (Steps 1 and 2), cells and biomaterial inks preparation (Step 3), the bioprinting process (Step 4), and post-bioprinting/applications (Steps 5 and 6) [3,15].

Selection and preparation of the convenient bioinks is highly challenging; biocompatibility and biofunctionality are important requirements of a bioink, among others. It should provide an adequate microenvironment that promotes cell attachment, proliferation, and differentiation [5]. For the development of such bioink, several research studies have explored the potential of hydrogels [16], which can be defined as a 3D, crosslinked, hydrophilic, polymeric network created from natural or synthetic polymers [17]. However, natural-based hydrogels have attracted more attention due to not only biocompatibility, biodegradability, and non-toxicity but also other specific properties such as antibacterial and antioxidant properties as a function of the employed polymers [4].

3. Polysaccharide-Based Hydrogels in 3D Bioprinting

3.1. Cellulose

Cellulose, a semi-crystalline material, is the most abundant biopolymer in nature. It is found in a wide variety of living species, including plants, animals (e.g., urochordates) [18,19], and some bacteria [20]. This biopolymer exhibits highly desired properties such as mechanical robustness, hydrophilicity, and biocompatibility [21]. Cellulose consists of repeating cellobiose units, made of β-D-gluco-hexopyranosyl-(1→4)-β-D-gluco-hexopyranose. Due to the large number of polar hydroxyl groups on the glycopyranosyl ring, numerous intermolecular and intramolecular hydrogen bonds are formed. This arrangement leads to an amorphous region and a robust crystalline region, which gives the plant walls rigidity and insolubility in water and most organic solvents, which limits its use [22,23]. To expand its application, different chemical modifications are introduced, such as esterification and etherification, which are based on introducing functionalities into the cellulose backbone, resulting in materials with highly desirable properties [24,25]. Cellulose ethers, especially carboxymethyl cellulose (CMC), ethyl cellulose (EC), methyl cellulose (MC), and hydroxypropylmethyl cellulose (HPMC), are the most widely used for the preparation of hydrogel-based bioinks for biomedical applications [22].

In 2021, Gospodinova et al. have developed a bioink using hydroxyethyl cellulose (HEC) blended with sodium alginate for a cervical tumor model [26]. The high viscosity (over 2000 Pa·s) and thixotropic behavior of HEC contribute to its good printability, with the resulting bioink exhibiting a cell viability of up to 81.5% after one day of incubation. Similarly, a cellulose-based hydrogel composed of nanofibrillated cellulose combined with gellan gum and crosslinked with calcium chloride was developed by Lameirinhas et al. in 2023 [27]. This system exhibited improved properties suitable for use as a bioink in 3D bioprinting [27]. In another study, CMC-based bioinks alone were also investigated, revealing acceptable viscosities (20–80 Pa·s), good printability, and a cell viability of 86% after 23 days of incubation (Figure 2) [28]. Its characteristics suggest suitability for applications in 3D bioprinting technologies.

3.2. Chitosan

Chitosan is a chitin-derived biopolymer, which is found in the exoskeletons of crustaceans like shrimp, crab, and lobster. Chitosan is a partially or fully deacetylated chitin; its degree of deacetylation can range from 40% to 98%. It is a cationic heteropolymer composed of linear chains of β (1→4)-linked D-glucosamine and N-acetyl-D-glucosamine units [29,30]. It possesses highly desired characteristics such as biocompatibility, biodegradability, muco-adhesion, and anti-microbial properties, as well as structural resemblance with GAG, one of the components of the extracellular matrix (ECM) that interacts with collagen fibers and favors cell adhesion [31]. Therefore, chitosan has attracted significant interest in 3D bioprinting and tissue engineering. It has been successfully employed in bone and cartilage tissue engineering, where it is often blended with other polymers such as HEC, cellulose nanocrystals, or polycaprolactone to enhance its mechanical properties and printability.

For instance, according to Kamal et al. in 2018 [32] chitosan scaffolds have improved osseointegration and bone healing compared to the xenograft. Chitosan was blended with HEC and cellulose nanocrystal to prepare a printable and biocompatible bioink for bone tissue engineering and regeneration applications. As a result, the obtained biomaterial presented a viscosity range from 106 to 258 Pa·s and promoted osteogenic differentiation, which was demonstrated by the accelerated activity of alkaline phosphatase, calcium mineralization, and collagen formation in ECM. Moreover, to predict the shape fidelity of the scaffolds, the yield stress of the bio-inks was characterized, showing values in the range of 400 to 585 Pa. The developed formulations also exhibited a high percentage of storage modulus recovery, exceeding 75% [33]. Similarly, a thermosensitive chitosan-based hydrogel was used for generating 3D constructs for bone tissue engineering [34]. Zinc oxide nanoparticles were added to this formulation to favor their antimicrobial property. Chitosan-based bioinks were also used for cartilage regeneration, as demonstrated by Kim et al. in 2021 [35]. In this study, chitosan was blended with polycaprolactone to improve its mechanical properties. The obtained material showed enhanced in vivo chondrogenic performance for tracheal tissue engineering (Figure 3).

3.3. Lignin

Lignin is one of the three major components of the cell wall of lignocellulosic biomass, attached to cellulose and hemicellulose polymers, and accounting for 10–25% of its composition [36]. It is an amorphous, highly crosslinked aromatic biopolymer consisting of three repeating units, namely, p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S), which make it the principal recalcitrant component of lignocellulosic biomass [37]. Thanks to their key properties, such as biodegradability, biocompatibility, low cytotoxicity, antioxidant, and antimicrobial activities, lignin has attracted significant interest for the development of hydrogel-based bioinks for biomedical applications [10,38].

In 2020, Jiang et al. developed a printable bioink using alkali lignin with Pluronic F127 [39]. The utilization of lignin biopolymer in this formulation offers advanced properties such as high stiffness, water stability, thermostability, and UV-blocking performance. Lignin was also combined with gellan gum to form a bio-printable hydrogel for cartilage tissue repair (Figure 4). The results of the study conducted by Bonifacio et al. in 2022 [40] demonstrated that it improves the chondrogenic potential—with >70% viable cells—compared to hydrogels made up of gellan gum only [40]. On the other hand, Dominguez et al. proposed, in 2019, a combination of lignin biopolymer with poly(lactic acid) via hot melt extrusion to print personalized wound dressings [41].

3.4. Comparative Overview of Cellulose, Chitosan, and Lignin for 3D Bioprinting Applications

To better understand their suitability as bioink components, a comparative analysis of cellulose, chitosan, and lignin is presented in

Table 1. Comparison of structural, functional, and bioprinting-related attributes of cellulose, chitosan, and lignin.

Property/Application	Cellulose	Chitosan	Lignin
Source	Most abundant biopolymer; found in plants, animals, and some bacteria	Derived from chitin in crustacean exoskeletons (e.g., shrimp, crab, lobster)	Found in the cell walls of lignocellulosic biomass (plants); alongside cellulose and hemi-cellulose
Chemical Structure	Linear chains of β(1→4)-linked β-D-anhydroglucpyranose (cellobiose units)	Linear β(1→4)-linked D-glucosamine and N-acetyl-D-glucosamine units	Aromatic polymer composed of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units
Crystallinity	Semi-crystalline (amorphous and crystalline regions)	Mostly amorphous	Highly amorphous
Charge	Neutral	Cationic (positively charged)	Generally neutral to slightly anionic
Solubility	Insoluble in water and most solvents without modification	Soluble in acidic aqueous solutions	Limited solubility; requires chemical modification or suitable blending
Key Properties	Mechanical robustness, hydrophilicity, biocompatibility	Biocompatibility, biodegradability, muco-adhesion, antimicrobial, ECM mimicry	Biodegradability, biocompatibility, antioxidant, antimicrobial, UV-blocking
Modifiability	Chemically modifiable (e.g., esterification, etherification to CMC, HPMC, etc.)	Degree of deacetylation can be tuned; blends well with other materials	Requires chemical modification (e.g., alkali lignin) for better processability
Typical Bioink Components	HEC, CMC, EC, MC, HPMC; often blended with alginate, gelatin, chitosan	Often blended with HEC, cellulose nanocrystals, polycaprolactone	Often combined with Pluronic F127, gellan gum, PLA
Biomedical Applications	Hydrogels for tissue scaffolding, bone regeneration, cervical tumor models	Bone and cartilage tissue engineering, tracheal implants, wound healing	Cartilage regeneration, wound dressings, antioxidant delivery
Printability	Good, especially with derivatives (e.g., HEC shows thixotropic behavior)	Printable when blended; thermosensitive hydrogels and composite scaffolds show good extrusion	Printable when combined with other polymers (e.g., Pluronic F127 or gellan gum)
Biological Performance	High cell viability (e.g., 86% with CMC after 23 days); supports proliferation	Promotes osteogenesis, ECM formation, and chondrogenesis; good antimicrobial profile	Enhances chondrogenesis, UV protection, water/thermal stability

. The comparison is based on key criteria relevant to 3D bioprinting, including chemical structure, crystallinity, surface charge, solubility, modifiability, and compatibility with crosslinking strategies. Additionally, printability characteristics, biological performance, and typical biomedical applications are included to provide a comprehensive perspective.

4. Comparison, Limitations, and Crosslinking Strategies

4.1. Comparison

Cellulose derivatives such as carboxymethyl cellulose (CMC) and hydroxyethyl cellulose (HEC) are widely used in bioink formulations due to their excellent viscosity control, thixotropic behavior, and printability. These materials support high cell viability and proliferation, making them suitable for applications like bone tissue regeneration and tumor modeling. However, native cellulose requires chemical modification to become water-processable.

Chitosan, a cationic polymer, exhibits good biocompatibility, bioactivity, and antimicrobial properties, with inherent muco-adhesion and ECM-mimicking behavior. Its printability improves significantly when blended with other polymers or additives. Chitosan-based bioinks are especially useful in bone and cartilage tissue engineering, supporting osteogenesis and chondrogenesis, though they may require reinforcement to improve mechanical strength.

Lignin, while not a conventional polysaccharide, offers antioxidant, UV-blocking, and antimicrobial functions in bioink systems. It is not inherently printable but performs well when combined with other polymers like Pluronic F127 or gellan gum. Lignin-enhanced bioinks are used in cartilage regeneration and wound dressing, contributing to improved mechanical stability and biological performance.

4.2. Limitations

Despite its favorable mechanical properties and biocompatibility, native cellulose has poor solubility in water and most organic solvents, making it unsuitable for direct use in bioinks. Chemical modification is often necessary, which introduces complex processing steps and potential variability in material properties. Even modified celluloses, such as CMC and HEC, can suffer from batch-to-batch inconsistency in viscosity and gelation behavior, affecting print fidelity and resolution. Moreover, cellulose-based bioinks can exhibit limited shear-thinning behavior unless properly formulated, which may compromise extrusion stability and fine structure formation during printing.

Chitosan’s pH-dependent solubility poses a significant challenge, as it dissolves only in acidic conditions, which may be incompatible with certain cell types or bioactive agents. Additionally, its mechanical strength and shape retention are generally poor when used alone, often requiring blending with synthetic or reinforcing agents to achieve structural stability. Chitosan bioinks may also exhibit low print resolution and slow gelation, limiting the fabrication of complex geometries. Furthermore, the degree of deacetylation and molecular weight of chitosan can vary significantly between sources, leading to reproducibility issues in printing performance and biological outcomes.

Lignin’s highly branched and aromatic structure results in limited solubility and processability, making it difficult to formulate as a standalone bioink. It is typically used as an additive rather than a primary matrix component. The heterogeneous composition of lignin, which depends on the source and extraction method, leads to significant batch-to-batch variability in molecular weight, functional groups, and reactivity. This inconsistency can negatively affect rheological properties, print resolution, and crosslinking behavior. Additionally, lignin lacks intrinsic bioactivity for supporting cell adhesion and proliferation, often requiring functionalization or blending with biologically active materials to enhance cellular responses.

4.3. Crosslinking Strategies

Crosslinking is a critical strategy in enhancing the mechanical strength, stability, and biological functionality of polysaccharide-based bioinks used in 3D bioprinting.

For cellulose derivatives, ionic crosslinking (e.g., with multivalent cations like Ca²⁺ when blended with alginate) and chemical crosslinking using agents such as silanolate, epichlorohydrin, or glutaraldehyde are common, providing tunable stiffness and print fidelity. Photocrosslinking is also employed when cellulose is modified with photoreactive groups (e.g., methacrylated cellulose), enabling spatial and temporal control of gelation. In the case of chitosan, crosslinking can be achieved through ionic interactions (e.g., with sodium tripolyphosphate (TPP)) to form polyelectrolyte complexes, or via chemical crosslinkers like genipin or glutaraldehyde to enhance stability and reduce degradation rates.

Thermosensitive gelation, especially in chitosan/β-glycerophosphate systems, is another method that facilitates mild in situ crosslinking suitable for encapsulating cells.

Lignin, due to its aromatic and polyphenolic structure, typically requires chemical crosslinking—for example, via oxidative coupling or blending with UV-curable polymers like Pluronic F127 diacrylate for photopolymerization.

These crosslinking approaches improve print resolution, shape retention, and long-term mechanical integrity of the printed constructs, making them essential for the effective use of these natural polymers in tissue engineering applications.

5. Conclusions

Naturally derived biopolymers such as cellulose, chitosan, and lignin show great promise for 3D printing for biomedical applications due to their biocompatibility, biodegradability, and low cytotoxicity. Furthermore, these materials mimic the extracellular matrix, providing a conducive environment for cell proliferation and differentiation. However, they present some limitations, such as limited tunability of mechanical and rheological properties, which can affect their stability and shape fidelity. To overcome these limitations and develop bioinks that satisfactorily meet the requirements of bioprinting, researchers have optimized the crosslinking strategies and the bioink formulation by chemical modification or integrating synthetic polymers.