Chitosan-based inks for 3D printing and bioprinting

Abstract

The advent of 3D-printing/additive manufacturing in biomedical engineering field has introduced great potential for the preparation of 3D structures that can mimic native tissues. This technology has accelerated the progress in numerous areas of regenerative medicine, especially led to a big wave of biomimetic functional scaffold developments for tissue engineering demands. In recent years, the introduction of smart bio-inks has created growing efforts to facilitate the preparation of complex and homogeneous living-cell-containing 3D constructs. In the past decade, a considerable body of literature has been created on identifying an ideal bioinspired-ink with excellent printability, cell viability, bioactivity, and mechanical properties. This state-of-the-art review article briefly outlines 3D-printing/bioprinting techniques applied for chitosan-based bio-inks, their resources, crosslinking methods, characteristics, reasons for their superiority over other bio-inks, and challenges of commercialization; this is followed by a comprehensive description of the full potential and the key indicators of success in terms of 3D bio-printing of such bio-inks as platforms for tissue regeneration, advanced biosensors, drug delivery, and wastewater treatment. Next, the restrictions and challenges of chitosan bio-inks are highlighted. In this work, we also discussed about developing a coherent research strategy based on combination of microfluidics-based lab-on-a-chip (organ-on-a-chip) platforms with 3D-bioprinting which enables designing of self-healing scaffolds. And finally, the potential of smart inks based on chitosan for 4D bioprinting of more detailed and practical engineered tissues and artificial organs is reviewed.

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1. Introduction

Bioprinters, unlike 3D-printers, are suited to printing gel-based materials and biological materials (in particular bio-inks) and, additionally, they can perform noncontact droplet printing.^1–3 In other words, 3D-printing of either biological inks (bio-inks) or cell-laden inks is considered as a new process called 3D-bioprinting. During the past few years, bioprinting has become a new approach to manufacture various kinds of 3D framework with different bioprintable inks for tissue engineering and drug delivery applications.⁴ The proposed scaffolds’ features are similar to living body parts.⁵ There is a multitude of reasons why bioprinting has been attracting attention of researchers for biomedical applications (see Fig. 1). The advantages of using 3D-bioprinting could be listed as: (a) controlling the system and the process, which allows for biomolecules and cells to be directly encapsulated in the bioprinted network, (b) post-modification of 3D-bioprinted constructs, which is frequently found to be problematic for scaffolds using conventional techniques, and (c) bioprinting as one of the appealing processes to open a new window into regenerative medicine through enabling a high resolution of organ printing.^6,7

Fig. 1 Yearly evolution of papers on 3D-printing and 3D-bioprinting based on the number of publications. These data were extracted on July, 12 2021 from the Scopus database.^5,8

According to Fig. 1, along with development of 3D-printing, the application of 3D bioprinting in biomedical engineering has sharply developed. Surpassing the number of research studies based on 3D-bioprinting from five hundred by 2020 (according to the Scopus database) gives us a great hope for possible successful achievements in the near future. Materials used in 3D-bioprinting should have printing potential without deterioration in their mechanical stability and shape integrity, as well as supporting cell viability and differentiation.⁹ To achieve a construct with improved physical stability, chemical, physical, or enzymatic crosslinking of the bio-ink is required. Besides, bio-ink formulations should support cell adhesion and proliferation combined with growth factors and cell media to enable cells to flourish in the bioprinted constructs while mimicking the target tissue architecture. From a bio-ink perspective, finding natural or synthetic bioprintable materials with essential features such as suitable mechanical stability to withstand physical stress and improved integrity of their structure and network within in vivo microenvironments is the major challenge. Recent advances in 3D-bioprinting for biomedical engineering can respond to the emerging needs through emergence of a wide range of hydrogels based on natural or synthetic precursors and more accurate 3D-bioprinting methodologies (Fig. 2).¹⁰

Fig. 2 Timeline of the most significant steps forward in the development of 3D-bioprinting.^11–14

Due to the countless disadvantages of using synthetic polymers, such as poor biocompatibility and cellular adhesion as well as mechanical instability along with producing noxious by-products during the degradation process, indisputably natural polymers are considered as the better choice for 3D-printing demands.^15–17 Given the information proposed in Table 1, there are multiple concerns regarding the use of biopolymers as inks. The most significant groups of natural polymers are marine polysaccharides (e.g., alginate, chitosan, agarose, etc.). Disadvantages like the immunogenicity of fibrin, shape inconsistency of collagen, high viscosity of agarose, rheology optimization requirements of silk fibroin, and low cell attachment and low protein absorption of alginate push us to make use of alternative biopolymers. Chitosan, a polycationic biocompatible natural polymer, possesses some unique merits for use in bioprinting. Chitosan-containing solutions remain stable under physiological conditions and show appropriate values of viscosity, applicable for bioprinting applications.^18–23 Moreover, chitosan supports proper cell proliferation and differentiation. Cells cultured on chitosan scaffolds show high viability.^24,25 Thus, chitosan resins and hydrogels meet these requirements; furthermore, chitosan hydrogels can be tuned to be good mimics of the native extracellular matrix (ECM) of tissues.²⁵ However, this natural polymer has shown some drawbacks in tissue engineering, including a slow gelation rate and weak mechanical strength.^26,27 Here, it is vital to note that the physical stability and mechanical strength of a bio-ink require it to be a liquid or semi-solid material that can be chemically or physically crosslinked. Thus, chitosan-based biomaterials can be modified by a variety of approaches like coupling with methacrylic anhydride (methacrylation of backbone), to improve the possibility of a stronger crosslinking. Indeed, the crosslinking of chitosan hydrogels facilitates the process of overcoming its inherent minor disadvantages and makes it a biomaterial of choice for bioprinting due to its attractive portfolio of properties mentioned above. As a consequence, it is rational to use chitosan and chitosan derivatives for applications in tissue engineering to replace or fix bone, cartilage, and skin.²⁸

Table 1 Currently used 3D-bioprinting bio-inks for engineered body tissues^8,19

Bio-ink	Chemical structure	Printing process	Application	Bio-ink properties		Ref.
Chitosan (natural polymer)		Extrusion-based bioprinting (layer-by-layer deposition), laser-based bioprinting (e.g. stereolithography)	Bone tissue, vascular tissue, cartilage tissue (e.g., human ear), skin tissue	Stability under physiological conditions, appropriate range of solution viscosity values, cell viability, cell proliferation and differentiation, applicability, printability		29–34
GelMA (functionalized natural polymer)		Extrusion-based bioprinting (layer-by-layer (LBL)) deposition, laser-based bioprinting droplet-based bioprinting	Articular cartilage, bone regeneration, cardiac myocytes and fibroblasts	Good cell adhesion, low mechanical properties, low viscosity at low shear rates		35–37
Alginate (natural polymer)		Extrusion-based bioprinting (layer-by-layer (LBL)) deposition, laser-based bioprinting droplet-based bioprinting	Bone, vascular tissue, cartilage	Fast crosslinking ability, non-toxic, non-immunogenic, quick rate of degradation, various cellular responses due to different resources, elevated hydrophilic nature which causes low cell attachment and absorbability of protein		10 and 38–41
Gelatin (natural polymer)		Extrusion-based bioprinting	Human nasal inferior turbinate tissue, cartilage, vascular tissue	Possessing inherent signaling molecules for cell adhesion, thermoresponsive, good cellular proliferation, low immunogenicity, mechanically weak at physiological temperatures		42–44
Silk fibroin (natural polymer)		Extrusion-based bioprinting (e.g., inkjet)	Musculoskeletal tissue, human bone marrow mesenchymal stem cells, human nasal inferior turbinate tissue	Ease of structural modification, controlled degradation, crosslinking, and co-polymerization required to optimize the rheology of the bio-ink for more optimal printability		45 and 46
Pluronic F-127 (synthetic polymer)		Extrusion-based bioprinting	Fibroblasts, vascularized tissue (vessel formation), neural and glial tissues	Excellent degradation, good printability, and bioprintability, nonimmunogenic, heat needed for printability, low mechanical properties, fast gelation at 37 °C to avoid cell sedimentation, small shear forces during the printing process to preserve cell viability, sufficient concentration of polymer to allow quick supply of nutrients and oxygen to encapsulated cells and removal of waste		47–49
Agarose (natural polymer)		Extrusion-based bioprinting	Vascular tissue	Low cell adhesion and spreading, cell viability, non-toxic, non-degradable, gel at low temperatures, high viscosity which makes it not suitable for inkjet printing		50 and 51
Fibrin (natural polymer)		Droplet-based bioprinting	Neural stem cells, adipose tissue, bone, cardiac tissue, ocular tissue, cartilage, skin, liver, tendons, nervous tissue, and ligaments	Possesses inherent signaling molecules for cell adhesion, low mechanical properties, infectious transmission, non-shear-thinning behavior		10, 52 and 53
Collagen type I (natural polymer)		Extrusion-based bioprinting, droplet-based bioprinting	Vascular tissue, skin tissue	Appropriate cell adhesion, minimal immunological reactions, slow gelation rate for bioprinting, immense structural changes by volume shrinkage, poor mechanical properties		10
Hyaluronan gel (natural polymer)		Extrusion-based bioprinting, laser-based bioprinting	Bone tissue, cartilage, vascular tissue	Biocompatibility, ability to form flexible hydrogels, poor mechanical properties, gel often contains impurities, mechanically weak without modification		54 and 55
PEG (synthetic polymer)		Extrusion-based bioprinting, droplet-based bioprinting	Articular cartilage	Nonimmunogenic, high cell viability, considerable cell proliferation rate, biocompatibility, poor mechanical stability without modification, low cell adhesion		56 and 57

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In this review, we highlighted the key reasons for selecting chitosan-based inks for 3D-printing and bioprinting; moreover, a wide array of applications of these bioprinted-based scaffolds in various branches of biomedical engineering are described to understand chitosan's full potential. Besides, the remained challanges in the way of using chitosan-based bio-inks were fully disscused. Indeed, quantifying the characteristics of chitosan-based bio-inks and comparing them with the ideal bio-ink is such an important issue and results in an understanding of the best way to exploit their full strengths and to overcome the existing constraints through modification processes. In this study, we have tried to cover the most recent papers, which have expanded the description and important analysis of documented methods of functionalization, concentrating on their results and contributions towards 3D-printable bio-inks based on chitosan. At the end, we also explained the future directions of 3D-bioprinting based on chitosan.

2. 3D-Bioprinting and the role of chitosan

3D-bioprinting represents a fast-growing field of science with a strong commitment to mimicking applicable artificial tissues and organs. This technology provides the means of designing and generating structures to replace damaged tissues and impaired organs with patients’ own cells. The main ingredients of bioprinting processes are bioprinters, 3D-bioprinting modalities, and bio-inks. Bio-inks usually are 3D-printable bio-based material solutions, applied in living cell bioprinting, which are classified into two big groups of (a) scaffold-based bio-inks (including microcarriers, hydrogels, cell aggregates, and decellularized matrix) and (b) scaffold-free bio-inks (tissue strands, tissue spheroids, cell pellets).⁵⁸ To facilitate the synthesis of efficient and applicable bio-inks, interdisciplinary collaborations of scientists working in different disciplines such as chemistry, materials science, mechanical engineering, biology, and biomedical engineering are advantageous. Indeed, an applicable bioprinting modality needs to be adapted to the specific requirements of the intended printing process and to be suited to appropriate printing of a wide range of biomaterials. In general, it is considered to be more compatible with novel emerging bio-inks, in particular those with characteristics that build current constructs (see Fig. 3).⁵⁹

Fig. 3 An overview of different aspects of 3D-bioprinting.⁶⁸ Bioprinting as a multi-disciplinary science required a collaborative work between chemists, engineers, and biologists to study tissue maturation and design new, suitable scaffold-free/scaffold-based bio-inks to apply them through three primary 3D-bioprinting methods (extrusion-, droplet-, and laser-based methods) in order to prepare applicable, exquisite cellular/acellular 3D-bioprinted constructs for biomedical uses.

According to the literature, bio-inks could be printed through three main bioprinting methods:

1. Extrusion-based (powered by pneumatic, mechanical, or solenoid drivers),

2. Laser-based (stereolithography, mask-image projection, laser-induced forward transfer, beam scanning, laser-guided direct writing, selective laser sintering),

3. Droplet-based (inkjet-based, multi-jet modeling, electrohydrodynamic jetting, laser assisted-droplets, and pneumatic pressure assisted) bioprinting.^60–62

Extrusion-based bioprinting methods (EBB) are most commonly used in the topic of chitosan-based bio-inks for 3D-scaffold construction (Table 2). Laser-based bioprinting techniques (LBB) have been rarely used in this area, and on the other hand, the droplet-based bioprinting method (DBB) needs to be investigated.

Table 2 Comparison of various kinds of 3D-printing/bioprinting approaches for preparation of engineered tissue scaffolds^3,12,60,61

Additive manufacturing categories (3D-printing)				3D bioprinting				Features	Ref.
Printing modality	Material	Description	Chitosan 3D-printability	Bioprinting modality		Material	Chitosan 3D bioprintability
Binder jetting	Metals, ceramics, polymers, & powders (plastics & sands)	Powered layers are joined together by adhesive liquid binder	Applicable	Laser-based bioprinting	Laser-induced forward transfer	Cells in media	Applicable	Low mechanical properties, high preparation time, high precision, weak structural integrity, very good resolution, time-consuming process, single-cell level printability, low scalability, high printing cost, high cell densities printable, process often requires high-intensity UV light and long post-processing (potentially problematic for cells)	10, 69 and 70
					Stereolithography-based bioprinting	Hydrogels	Applicable	Smooth surface finish, fast printing, high spatial resolution, low cost, good quality of vertical printing, fair cell density (<10^8 cells per mL), high cell viability, layer-by-layer printing process	10, 69 and 70
Powder bed fusion (DMLS, EBM, SLS, SLM)	Powders (plastics & metals)	Thermal energy selectively fuses regions of a powder bed	N/A	Droplet-based bioprinting	Granule-based medium-assisted	Cells in media		To increase printability of bio-inks with weak printability and a poor crosslinking density	71
Material jetting	Polymers & gels	Droplets of build material are selectively deposited	N/A		Inkjet-based	Liquids and hydrogels	Applicable	Low preparation time, low mechanical properties with poor structure integrity, high throughput (scalable), high cell viability, moderate accuracy, affordable moderate precision, low printing cost, low cell densities can be printed, inferior droplet size control, an important condition for low viscous bio-inks	69 and 72
Sheet lamination (LOM, UC)	Paper, plastic, wood, metals like aluminum, & composites	Sheets of material are bonded to form an object	N/A
Material extrusion (FDM, bioplotting)	Polymers & gels	Inks extrude through nuzzle tips	Applicable		Multi-jet modeling	Hard and soft plastics, elastomers	Applicable	High resolution, high accuracy, suitable for small cell-culturing approach (microfluidic channels and chip), fast printing, ease of multi-material preparation at the same time, high cost, current resolution of microchannels printing	73
Directed energy deposition (LENS)	Metals	Focused thermal energy is utilized to produce a melt pool powders on a base platform	N/A	Extrusion-based bioprinting		Hydrogels & cell aggregates	Applicable	High mechanical properties, fair preparation time, very good structural integrity poor fidelity, multi-nozzle multi-material printability, cells undergo shear stress at the nozzle tip, fair printing cost, viscous bio-inks, and high cell densities could be applied, cell structure distortion and often inferior resolution, good quality of vertical	10, 69, 70 and 72

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2.1. Extrusion-based

Using extrusion-based deposition system, working with air pressure (pneumatic) or mechanical forces (pistons or screws), bio-inks can be extruded precisely in three dimensions to form the desired 3D patterns in bioprinting. In general, these bioprinters are capable of quickly producing scaffolds with a resolution of around 100 to 200 μm.⁶³ Extrusion-based bioprinted architectures with deposited cells and matrix proteins within hydrogel networks are focused on distinctive design, porosity, biomechanics, and composition, in which every factor is separately monitored to create structures with high accuracy from the submicron to the few-millimeter level, depending on the application. Bioprinting based on extrusion is the most commonly used method for 3D-bioprinting of chitosan-based bio-inks (Table 2).

2.2. Droplet-based

Droplet-based bioprinting differs from extrusion-based methods by a discontinuous application of microdroplets in a high-throughput manner, driven by various energy sources. Inkjet-based bioprinting techniques are the most prominent subclass of droplet-based bioprinting and are very cost-efficient. Cell-laden bio-inks can be printed with a speed of 10⁴ to 10⁵ drops per second at a high resolution of approximately down to 50 μm. Although some papers suggested possible cell damage related to both temperature and mechanical stresses during the printing process, surprisingly, suitable cooling systems prevent cell apoptosis.^64–66 Inkjet-based bioprinting with single- and multi-jet techniques is suitable for the preparation of both cellular and acellular 3D-bioprinted chitosan-based scaffolds, especially for printing prevascular structures.

2.3. Laser-based

Laser-based modalities, as non-contact methods, are usually referred to as techniques making use of laser pulses to produce a droplet that will be deposited on the print surface that can eventually achieve a single-cell resolution. Cells printed via this method, in comparison with the extrusion-based modality, experience less mechanical stress, leading to superior viability. However, their operating speed is relatively lower than that of extrusion-based modalities.^63,67 On the other hand, laser-based bioprinting allows for printing hydrogels with a high viscosity – above the viscosity limit of droplet-based bioprinting processes. Although a lack of accurate information on the impact of laser pulses on living cells, along with expensive machinery, has limited the use of laser-based methods, applicability of this approach in the past, especially stereolithography (SLA) and laser-induced forward transfer (LIFT), has been reported for scaffold biofabrication based on high viscous chitosan-based bio-inks.³¹

3. Reasons for choosing chitosan

There are a range of natural and synthetic polymers suitable for 3D-printing and bioprinting. Some examples of printable biopolymers are gelatin,⁷⁴ silk,⁷⁵ collagen,⁷⁶ alginate⁷⁷ and their functionalized types (e.g., gelatin methacryloyl (GelMA)⁷⁸ and nano-cellulose⁷⁹). There are also some well-known synthetic polymers,⁸⁰ such as polyester-based, Pluronic®-based, polyhydroxy acid-based, polyurethane-based and PEG-based, thermoplastics, and silicones, used as 3D-bioprinting inks.^81–83 For applications in medicine such as drug/gene/DNA delivery,^84,85 biosensors,⁸⁶ tissue engineering,⁸⁷ cancer therapy,^88,89 and diagnosis,^90,91 the distinctive structural features require a high amount of hydrophilicity and sufficient porosity (interconnected) of the biomaterials for better growth and migration of the cells.⁹² The chitosan-based printed bio-inks possess unique characteristics such as excellent cell/matrix interactions, mimicking the native tissue structure, providing a microenvironment for oxygen and nutrition exchanges, and favorable immune responses following implantation.^93–95 The presence of chitosan in bio-ink compositions could impart high antimicrobial activities. The ability for electron penetration into the negative shell membrane of bacteria from the electrostatic interaction of protonated NH₃⁺ in chitosan leads to bacterial death or restricts their growth.^96,97 Moreover, chitosan-based printed platforms exhibited biomolecule adaption, stable biocompatibility, and biological activity even after various post-printing modifications.^98,99

3.1. Cell viability, migration, and proliferation

Crucial factors of basic 3D-bioprinted structures are proliferation, matrix remodeling, and cell motility. As the cells are surrounded by a hydrogel, after the 3D-bioprinting process they have to be capable of proliferating into complicated anatomically related 3D cellular architectures. Wang et al.¹⁰⁰ suggested new hydroxyethyl-chitosan/cellulose scaffolds with bubble-like porous media. The chemical and physical structure of bio-inks controls the deposition of the released proteins, including protein signals. Cells can release morphogens to migrate to the surrounding. Then, a 3D-bioprinted hydrogel matrix supplies mechanical and architectural signals that direct cells for alignment. The cell motility may be influenced by various micro-environmental situations, including the geometry of the construct and the hydrophilicity of the material. The bio-ink, therefore, has a significant effect on setting the destiny of the surrounding cells.¹⁰¹ With the aid of integrin molecules, the cells stick to extracellular matrix proteins like fibronectin. The organization of protein collections and special adhesions is created while integrins are connected to the ligand.¹⁰²

Since gelatin includes the Arg–Gly–Asp-like sequence to enhance its biological activity, it was combined with chitosan by Huang et al.¹⁰³ Chitosan in chitosan/gelatin constructs is capable of creating insoluble ionic collections that contain gelatin, which is negatively charged.^103,104 This can have an impact on the integrin ligands. The organization of protein collections and cell/matrix adhesions happens while the integrins are connected to the ligands.¹⁰⁵ Therefore, it has a notable effect on the tissue remodeling procedure.

Bio-inks should demonstrate a low viscous behavior in order to adequately maintain the shear stress levels under moderate extrusion pressures to balance good cell viability and thin printing (high resolution).¹⁰⁶ For 3D-bioprinting, various variables such as the diameter of the nozzle, the temperature, and the viscosity of the bio-inks have a direct effect on the shear stresses experienced by the bio-ink and, thus, by the embedded cells. Hence, controlling and balancing between the parameters involved in bioprinting is essential, where high-viscosity bio-inks and narrow nozzles are used to enhance the ultimate printer resolution, which, however, is associated with high shear stresses that might lead to cell-membrane damage and, thus, cell apoptosis.¹⁰⁷

Bioprinting based on 3D extrusion most crucially focuses on tailoring the bio-ink to preserve cell viability by preventing damage to the cell membrane from process-induced mechanical impairments such as excessive shear stresses. This is achieved by an extremely hydrated environment to prevent drying and a physiological environment after printing. In this context, multi-nozzle bioprinting has a poor performance due to the low cell viability caused by external forces during extrusion. Furthermore, while a high viscosity (caused by a higher molar mass or crosslinking) leads to a superior post-printing physical stability, it lowers the cell viability because of the higher shear stresses. Moreover, for bioprinting, methods based on photocuring, the exposure of cells to excessive amounts of light even in the visible light range, might negatively impact cell functions as well as cell viability. Thus, cell viability is a key indicator for judging the processing properties of the extruded bio-inks. Accordingly, it is necessary to optimize crosslinking and viscosity to produce a bio-ink that is compatible with the cells and supplies high-quality scaffold constructions.

Whether cells are used for test procedures such as examining novel scaffolds or to be placed in patients, viability experiments are essential to analyze. Typically, viability experiments are conducted via Trypan blue exclusion approaches, live/dead assays, and MTT assays. It is crucial to preserve a high degree of cell viability throughout and after the bioprinting process. Up to now, according to the literature, low cell viability in printed scaffolds is one of the essential challenges.¹⁰⁸ To address this deficiency, scientists add biopolymers (e.g., chitosan) to the inks to enhance the viscosity and, thus, the printing resolution. Although the presence of such biopolymers could improve the printability of bio-inks, cell viability might be reduced, in particular for high polymer contents. For instance, Chavanne et al.¹⁰⁹ applied an adapted 3D-bioprinting process to prepare cylinder-shaped hydroxyapatite–chitosan with 40 wt% lactic acid (LA) for bone regeneration applications, where the authors used assessed chitosan concentration and LA addition to improve the hydrophilicity, rheological properties, and gelation time. A scaffold with 20 wt% chitosan and 40 wt% LA had a superior printability, and furthermore a post-treatment of the scaffolds with 10 wt% lactic acid leads to collapsed pores and a smooth chitosan cover layer on the constructs.

Some methods are available to improve cell viability: for example, in the case of a loss of integrity after printing, a modification of the ECM after decellularization could lead to increased structural viability.^28,110 After seeding, cells cause ECM degradation because of the generation of matrix metalloproteinases. The most crucial disadvantage of this approach is the required step of ECM extraction, which is a complicated and risky procedure, which leads to very high costs for this tissue engineering technique.²⁸

Some works focus on introducing nanostructured cavities into bio-inks. Results indicate that permeability could be enhanced by the nanoporosity of the printed structure,^111,112 which, however, includes poorly understood connections between nanostructure characteristics and their influence on cell functions. Kyle et al.¹¹³ attempted to understand the printability procedure by analyzing the effects of bio-ink features and printing parameters on cell viability. They understood that printability affects cell viability in three ways: (1) the printing temperature, (2) the concentration of bio-ink, and (3) the holding time. Therefore, bioprinting parameter optimization of polymeric-based hydrogels has a severe impact on the printability of cell-embedded bio-inks.^113,114 Poorly chosen operational parameters during and after printing such as crosslinking, temperature, and pH alteration can cause severe damage to the cell membranes and biological molecules (e.g., proteins and growth factors). Furthermore, crucial factors of a basic 3D-bioprinted structure are proliferation, matrix remodeling, and cell motility. Considering that an ink surrounds the cells after bioprinting, they have to be capable of proliferating into complicated anatomically related 3D cellular architectures. Accordingly, the ink has an essential effect in determining the destiny of the surrounded cells. The bio-ink's chemical structure adjusts the deposition of released proteins, including signaling proteins. Once cells released morphogens, they can migrate to the surrounding. Then, the 3D-bioprinted hydrogel matrix supplies mechanical and architectural signals that direct the cells for alignment. The cells’ mobility may be influenced by various micro-environmental situations, including the geometry of the construct and the hydrophilicity of the material. These factors need to be optimized to create scaffolds with geometries and stiffnesses similar to human tissues or organs. Some essential parameters of bio-inks are applied in 3D-bioprinting are as follows:¹¹⁵

(1) The rheology of bio-ink, such as viscosity, plays a pivotal role in the printing process. For example, when the viscosity of bio-ink is increased more than what it should be, it acts as a gel and leads to cell death, nozzle clogging, and the ejection of self-supporting filamentous frameworks. The initial viscosity of bio-ink should help the uniform distribution of cells and facilitate their mixing process without a sharp reduction in cell viability.

(2) For extrusion-based printing, bio-inks should possess low viscosity to enable the facile flow of bio-ink through the bioprinter with a narrow-sized nozzle. Meanwhile, after printing, the bio-ink must have the ability to maintain the 3D structure without shrinkage or collapse.

(3) The gelation time of bio-inks should be optimized not only to help the self-supportive networks but also to prevent a nonuniform distribution of cells and nozzle plugging.

(4) The surface tension of bio-inks should be optimized to allow better detachment from the tip of the nozzle and gentle filament extrusion.

(5) The ability of hydrogels to contain water is crucial for cell viability and readily transportation of nutrients, oxygen, and waste exchange.

(6) The extrudate/filament diameter is related to the rheology and flow rate of the bio-ink, the nozzle diameter, and the extrusion pressure.

3.2. Biomimicry

Cell modeling is possible by the printing of cell-laden bio-inks; nevertheless, following extracellular generation, digestion, and the deterioration of 3D-printed scaffolds and growth of encapsulated cells are all problems that need thoughtful consideration. Commonly applied cell-laden bio-inks have various innate drawbacks, including limited interactions of cells, migration, partial growth, and settlement of immobilized cells within the bio-ink network for better recapitulation of native tissue.¹¹⁶ The possibility of such problems emerging increases upon increase of bio-ink concentration and viscosity. The both homo- and hetero-cells-laden chitosan-based bio-inks exhibit excellent biomimetic properties due to an inability to immobilize an exogenous network. Chitosan-based 3D-bioprinted scaffolds recapitulate the real tissue with high exactness and maintain cell morphology and stability over long periods.¹⁰⁸ Microcarrier-laden bio-inks also enable excellent interactions between cells because of an intrinsic extreme cell density. Recent investigations have proved that chitosan-based bio-inks could be excellent applicants for 3D-bioprinting demands.¹¹⁷

3.3. Biodegradability and mechanical stability

Degradation of bio-inks directly depends on their composition, element concentration, added particles, temperature, and other external conditions. External stimuli including enzymes, environment pH value, electrical and magnetic fields, and temperature can influence the external-responsive polymers and readily dissolve them, which changes the 3D-printed structure. The biodegradation degree of the scaffolds plays a pivotal role in cell-laden constructs and restricts the selection of bio-ink composition, because the cells should implant within the 3D scaffolds with improved biodegradability which could reshape their environment.^118,119

According to the literature, the biodegradation of chitosan-based scaffolds is mainly influenced by the concentration, molecular weight, degree of deacetylation, and swelling behavior of chitosan.¹²⁰ In PBS/lysozyme solutions, due to the low tendency of amine groups to lysozyme, highly deacetylated chitosan degraded at a low rate, which was determined by gravimetric degradation techniques.¹²¹

Chitosan-based bio-inks possess adequate mechanical properties to keep encapsulated cells alive and provide a suitable microenvironment similar to the native tissue which is gradually replaced by the extracellular matrix generated by the cells.¹²² Indeed, in the first stage of neo-tissue creation, cell accumulates do not possess sufficient mechanical stability; subsequently, they will be able to aid each other using cell–cell adhesion mediated by cadherin accompanied by extracellular matrix deposition. Nanoparticles and microcarriers could play a role as an environment for cells and ensure the structural stability of the bioprinted constructs. The mechanical characteristics of the bioprinted scaffolds affect their permeability and diffusion properties for the required oxygen, nutrient, and waste exchange.¹⁰

3.4. Immunogenicity

In the accurate preparation and effective implantation of bioprinted chitosan scaffolds for various medical demands, immunogenicity regularly poses a significant barrier by causing an immune response. The implantation of inappropriate bio-inks can cause a series of infections. Recent in vivo and in vitro studies show that 3D-printed scaffolds composed of biopolymers such as chitosan complexes cause a low-level immune response in conjunction with a high-level cell attachment and excellent cell viability.¹¹ 3D-printed structures created with bio-ink composed of hydroxybutyl chitosan/oxidized chondroitin sulfate activated only a minimum amount of pro-inflammatory gene expression of macrophages and restricted intense immune responses within a week for RAW 264.7 cell line samples.¹²³

3.5. Feasibility and affordability

Indeed, recent reports related to bioprinting of hydrogels indicate the practicability and possibility of various chitosan composites for utilization in a wide range of biomedical engineering sub-branches. Crosslinking mechanisms of chitosan intensively influenced the bioprinting of hydrogels and selecting of the suitable crosslinking methods capable of facilitating bioprinting processes.¹⁰ The excellent ability of printed chitosan-containing constructs to adapt to human tissue/organs, and the improved efficacy of cells and hydrophobic/hydrophilic drug delivery, have made them excellent candidates for clinical trials. A large number of in vivo studies on 3D-printable chitosan-based bio-inks proved that these materials could be the key to answering difficulties in tissue engineering.^10,13,124

While a few kinds of bio-based polymer (e.g., collagen, fibrin, Matrigel) are expensive, most bio-inks, especially those that are chitosan-based and those made of a combination of natural and synthetic polymers (e.g., PEG, Pluronic F-127, microcarriers) are affordable for 3D-bioprinting demands. Chitosans with various molar masses, deacetylation degrees, and reasonable prices are available on the market. Moreover, cell-laden bio-inks, especially ECM-based bio-inks, are more costly to prepare and need very advanced techniques for their preparation and storage.⁹⁵ Indeed, bio-inks consist of plentiful cells and the number of cells is mostly related to their size and the rate of extracellular matrix deposition as well. The growth of cells in the required large quantities is not only not cost-efficient but also time-consuming and labor-intensive. Decellularized matrix-based extracellular bio-inks are not cheap because a significant amount of real tissue is required to form a low amount of bio-ink (Fig. 4).¹⁰

Fig. 4 Characteristics of excellent bio-inks and 3D-printed structures. (A) Shows the cell encapsulation process in the bio-ink, preparation for 3D-bioprinting, and finally, an SEM image of a Y79 cell-laden 3D-printed alginate/pluronic-based scaffold.¹²⁵ (B) Microphotographs of HaCaT cells seeded on 3D-printed chitosan structures with a chitosan film at the base and with Neutral Red staining after 20 days. (B. 4) Microphotographs of Nhdf and HaCaT cells after 5 weeks seeding them together on a 3D chitosan construct with a film of chitosan at the base upon Neutral Red staining.¹²⁶ (C. 1) The combination of the biodegradation and tissue regeneration mechanism of cell-laden 3D structures. (C. 2) The biodegradation and extracellular matrix replacement of HeLa cells encapsulated in a hyaluronic-acid-based hollow fiber after 15 days. (C. 3) Presents fiber-shaped tissue regeneration of red-fluorescing HeLa cells coated with green-fluorescing 10T1/2 cells after biodegradation of scaffolds.¹²⁷

3.6. Advantage of chitosan bio-inks

An “ideal bio-ink” for 3D-bioprinting is a bio-based material with excellent (a) printability, possessing characteristics which make a material printable (e.g., appropriate viscosity, crosslinking mechanisms), must be able to withstand forces applied during the printing process and have a suitable structural post-printing stability, (b) bioactivity (biocompatibility, high post-printing cell viability, encouragement of cellular adhesion, growth and proliferation, and good porosity to foster nutrient transportation), and (c) mechanical properties (strong enough to withstand forces experienced by native tissue, resilient and biodegradable).¹²⁸

Selection of the right bio-ink for the 3D-bioprinting process requires biomaterials with optimized specific characteristics (e.g., physical parameters). Thus, successful implementation of 3D-bioprinting processes requires a properly optimized rheological profile of the utilized bio-ink. A solution with too high viscosity cannot be printed properly without applying excessive pressure, and subsequently will block the extrusion discharge tips, leading to a breakdown of the printing process. However, printing with very low viscosity bio-ink will also fail, as the desired shapes cannot be obtained with sufficient accuracy and longevity. Therefore, to control surface-tension-driven flows to avoid droplet formation driven by surface-tension and drawing straight filaments from the bio-ink solutions, a viscous material with appropriate viscosity is required. According to the literature, bio-inks with viscosity between 30 mPa.s and 6 × 10⁷ mPa.s are needed to apply micro-extrusion bioprinting techniques.^30,70 At the same time, the concentration of additives in the bio-ink matrix determines the crosslink density of the resulting network. This could result in controlled mechanical properties, resulting in controlled in vivo degradation.

Besides other noticeable characteristics of excellent bio-inks, especially those used in 3D extruder-based bioprinting systems, they have a highly hydrated, physiological structure to ensure cell viability, to avoid drying, and to preserve the cell membrane integrity against various types of mechanical damage during the different steps of the printing process (e.g., shear stress and abnormal pressures).

Among all 3D-bioprintable bio-inks, only a number have obtained approval from the Food and Drug Administration (FDA), a federal agency of the United States, which is necessary for them to be applied for medical use. Fortunately, the application of chitosan-based biomaterials is commonly accepted for use in drug delivery and tissue engineering. Nowadays, understanding the requirements for FDA approval, however, requires more appropriate precautions in additional clinical trials involving chitosan-based drug carriers. Thus, a limited number of companies such as West Pharmaceutical Services, Inc. are currently working on trials for new drug delivery systems based on chitosan's derivatives to develop FDA-approved drug carriers.¹²⁹ A broad viscosity range, various routes to crosslinking, and controllable mechanical properties of chitosan-based bio-inks by introducing numerous types of reinforcing additives allow for tailoring chitosan-based bio-inks to be easily printable. Moreover, chitosan-based hydrogels have shown excellent cell viability and high bioactivity. The combination of these versatile combinable and adjustable parameters allows chitosan-based bio-inks to reach the top of the list of ink candidates for 3D-bioprinting applications.

In the following discussion, the authors have tried to give readers a more comprehensive insight into the full potential of chitosan-based bioprinted platforms by focusing on the individual characteristics and applications in the context of 3D scaffolds. Collating such information will ensure a deep understanding of interactions between the cells and the chitosan matrix, and it will help to build new regenerative approaches based on chitosan-based 3D-bioprinted artificial tissues. In Table 3, a comprehensive list of 3D-printed scaffolds with different printing approaches and applications in the biomedical field are tabulated to present an overview of a vast number of publications regarding the use of chitosan-based bio-inks in recent years.

Table 3 Recent paper related to printed chitosan-based inks/bio-inks, 3D-bioprinting characteristics, and their applications

Bio-ink	3D-bioprinting method/process conditions	Goal/application	Ref.
Chitosan	Extrusion-based bioprinting	To prepare scaffold with high flexibility and organized microfiber networks to promote cell growth	34
PCL-DA/PEG-DA/chitosan	Visible light 3D-printing	Promoting cell adhesion and cell differentiation onto a new platform	130
Chitosan/gelatin 2.5–7.5%	Extrusion of different percentage composites	Fibroblasts grew well but fidelity was low	131
Collagen/chitosan	Extrusion and printing	For axon regeneration and neurological recovery	132
Chitosan and PEG-diacrylate	SLA with Irgacure 819	Poor resolution of features, weak mesenchymal stromal cell (MSC) engraftment/survival	31
Hepatocytes combined with gelatin/alginate/chitosan hybrid system	Double-nozzle low-temperature deposition	Liver physiological simulation systems	133
Chitosan and raffinose in acetic acid	Printed at −14 °C, gelation in KOH solution, macrofibre size, pore size of 200 μm	Keratinocytes and fibroblasts	126 and 134
Polyelectrolyte/chitosan/gelatin	Using a 6-dispensible regenHU 3D-bioprinter, printed onto a 27 °C bed	Skin tissue regeneration	135
Chitosan/PVA/HA	The crosslinked fluid was sprayed on the scaffold, printing speed 10 mm/s	Hydroxyapatite-based scaffolds for hard-tissue engineering	136
Collagen/chitosan into gelatin	Binder jetting technique, collagen/chitosan combination bioprinted into dry gelatin	Poor resolution, good growth of neural stem cells, acceptable degradation rate (90% of the network in 12 weeks)	137
Chitosan/gelatin/HA	Extrusion of different percentage composites	P3 bone mesenchymal stem cell (BMSC)-loaded scaffold for osteochondral tissue regeneration	138
Bioceramic (brushite)/chitosan	Multi-jet 3D-printing	A bioceramic scaffold utilized as a drug delivery agent for bone tissue regeneration	139
Chitosan-coated alginate	Printing of CaCl2 through a coaxial extrusion method and crosslinking by EDC and genipin	Hepatocyte (HepaRG) culture, anisotropic mechanical properties of a scaffold	140
Alginate, xanthan, chitosan, gelatin, κ-carrageenan, and GelMA	Extrusion of layer-by-layer of Kca2 and GelMA10 with separated syringes	Study of characteristics and 3D-printability	141
Gelatin/chitosan	Extruding the polymer at the ambient temperature associated with vacuum freeze-drying	Increase growing of stem cells	142
PLA/chitosan, PLA grafted maleic anhydride/chitosan	—	Nontoxic antibacterial scaffold for tissue engineering applications	143
Hydroxybutyl chitosan	Extrusion of hydroxybutyl chitosan	Thermoresponsive gel, chondrocytes, grew, cartilage tissue, and cardiac fibroblasts	144 and 145
Chitosan-g-oligo	SLA 3D-printing	Treating spinal cord injuries and other neuronal degenerative diseases	146
BMSC-laden gelatin/sodium alginate/carboxymethyl chitosan	Homogeneous plotting temperature controlled at 15 °C, printing speed 1 to 10 mm s^−1	Antibacterial scaffold for bone mesenchymal stem cell (BMSC) delivery	98
Chitosan-g-oligolactides	Two photon-induced microstereolithography (laser stereolithography)	Study of the characterization and mechanical properties of scaffolds	147
Chitosan, PCL diacrylate, PEG-diacrylate	SLA 3D-printing	Faster growth of fibroblasts (L929)	130
Chitosan/HA/glioma stem cell	3D-printing via extrusion-based bioprinting	Cancer treatment	148 and 149
CPC containing chitosan/dextran/BSA	Extrusion of cement by 3D plotting system BioScaffolder	Loading of the CPC paste, loaded protein, better efficiency of growth factors	150
Silver-loaded/lactose-modified chitosan coated on bisphenol-A-di methacrylate and triethyleneglycol dimethacrylate/E-glass	Inkjet-based printing	It could be used as an antibacterial implant for bone tissue reformation	151
N-Carboxyethyl chitosan–oxidized dextran	Direct-write or extrusion of hollow hydrogel fibers	Printing different-scale cost-efficient vasculatures	152
Chitosan coated on PLA 3D-printed scaffold	Extrusion-based printing at 200 °C, cylindrical shape design with pore sizes of 0.3 × 0.3 mm^2	Tissue engineering: to promote growth of human fibroblast cells	106
Chitosan-coated hydroxyapatite composite	Extrusion-based bioprinting with printing air pressure of 0.35 MPa and printing speed 8 mm s^−1	Using as drug (rhodamine B and bovine serum albumin) carrier agent	153
Chitosan and its allyl substituted derivatives	SLA bioprinting with a printing speed of 1.5 m s^−1	No application mentioned	33
N,O-Carboxymethyl chitosan-Ca^2+-polyphosphate complex	Printing was performed at 25 °C using a pressure of 1.4 bar and a printing speed of 18 mm s^−1	Alternative tissue-engineering solutions	154
Carboxymethyl (CMC)-chitosan	Direct-write printing of CMC following ionic crosslinking	Human neural development	155
Polycaprolactone/chitosan	Electrohydrodynamic (E-jet) 3D-printing	To promote regeneration of cartilage tissue, blood vessels, and skin using human embryonic stem cell-derived fibroblasts cells	156
Chitosan-coated 5-FU loaded-alginate tablet	Printing of CaCl2 with 5-FU loaded-alginate through a hot extrusion method	Controlled drug delivery	157
Chitosan complexation with serum proteins	Extruded by a printing rate of 150 mm min^−1	Facile cell encapsulation can be performed, study of the mechanical stability of the prepared scaffold	122
Tricalcium phosphate-chitosan/collagen	SFF printing, plasma-sterilization, low-temperature gas plasma	Enhancement of bone tissue formation and bone parts replacement	158
Chitosan/eggshell membrane-derived calcium phosphate	Extrusion-based printing via layer-by-layer deposition	Bone graft application	159
Chitosan, chitosan/pectin, chitosan/genipin	Extrusion of chitosan-based bio-inks into sodium hydroxide solution	Promoting osteoblast proliferation and mineralization
Hyaluronate/chitosan/adipic acid dihydrazide/ATDC5 chondrocyte	3D-printing via extrusion-based bioprinting	Potential application in tissue engineering	160
Chitosan–gelatin	3D-printing using microreplication and freeze-drying techniques	Highly porous scaffold with average 100 μm pore size suitable for hepatocyte culture	161
Quaternized chitosan-grafted polylactide-co-glycolide/hydroxyapatite scaffold (PLGA/HA/HACC)	—	To boost the regeneration of impaired bone tissue	162

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4. Chitin/chitosan

In 1811, Henri Braconnot introduced the term fongine for the very first time, which was later referred to chitin in 1823, when Odier extracted it from the elytrum of the cockchafer beetle. This word is taken from the Greek word χιτών (chitón), meaning coat of armor. Indeed, Braconnot and Odier are credited as the finders of chitin from natural resources, which is well known these days as the first superabundant marine polysaccharide, and also it has taken second place among all existing polysaccharides in nature.¹⁶³ Chitin is a structural polysaccharide that is almost always associated with proteins.¹⁶⁴ Charles Rouget successfully designed and performed a process in 1859 in order to hydrolyse the acetamide group of chitin by boiling it in concentrated KOH to achieve acetate ions and an –NH₂ group in order to produce a polymer with improved characteristics.¹⁶⁵ Later, this product was called chitosan by Felix Hoppe-Seyler in 1894. Generally, the deacetylation degree is determined by the ratio of glucosamine to the N-acetylated glucosamine units existing in the structure of chitosan. So far, three major deacetylation processes of chitin have been reported: (a) chemical deacetylation, (b) enzymatic deacetylation, and (c) microwave-assisted deacetylation (see Fig. 5). Deacetylation of chitin, the linear amino polysaccharide, at various levels leads to chitosan, a semi-synthetic commercial amino polysaccharide, with different properties; however, owing to the possibility of depolymerization and side reactions, the product is rarely completely deacetylated (100%). The standard deacetylation degree of chitosan for biomedical demands should be around 75 to 98%, as provided by the pharmaceutical industries. Various sources of chitin and, subsequently, chitosan exist in nature from animal, fungi, and plant sources. Importantly, on an industrial scale, the two primary resources of chitosan are referred to as crustaceans and fungal mycelia.¹⁶⁶

Fig. 5 Discovered resources of chitin and chitosan.^169,170 In this graphic, six primary natural resources of chitin from both animal and vegetable sources are illustrated. Chitin is mainly extracted from these resources through a chemical or biological extraction method, including series processes of demineralization, deproteination (chemical or enzymatic), decolorization, and fermentation; however, using different resources requires different orders of these steps. As the last step, chitosan is produced from chitin by deacetylation via chemical, enzymatic, or microwave-assisted methods.

4.1. Animal resources

The predominant source of chitosan, crustaceans, stems from parts of marine species like shrimp shells, crab shells and the shells of freshwater lobster (crayfish), etc. It is commercially feasible to extract chitosan from seafood waste, in particular if it involves carotenoid recovery (e.g., crustacean shells). Insect cuticles as in honeybees and crayfishes, terrestrial crustaceans including Annelida, and squid pens, etc. are other animal resources for the extraction of chitin/chitosan (see Fig. 5).^167,168

4.2. Vegetable and fungi resources

Besides the animal sources, chitin can be found in some vegetable resources such as mushroom envelopes, green algae cell walls, or yeasts (see Fig. 5). In general, applications of vegetable-extracted chitosan for biomedical demands have been prioritized over crustacean because their healthcare privilege and less allergenic side effects as well. Furthermore, mushroom and other fungi-based sources of chitin provide several advantages, guaranteeing that the resulting chitosan has more constant properties. Indeed, chitosan obtained from mushrooms usually has a narrower molecular mass distribution than sea-animal-based resources, and it can also vary in molecular weight as well as in terms of the deacetylation degree.¹⁶⁶

5. Chitosan-based inks for 3D-bioprinting

5.1. Challenges and limitations of 3D-bioprinting of chitosan-based inks

Although great strides in mimicking native tissues by chitosan have been taken so far and many successes have been achieved, there is still a great deal of effort necessary to create artificial organs which work as human spare parts. To deal with this challenge, inspite of bioprinting techniques, the characteristics of bio-ink are required to be optimized. For instance, the concentration of hydrogels in bio-ink solutions, the molar mass of the applied hydrogels for the preparation of ink, along with the final chemical composition of the printing solution, which is mainly affected by additional agents (e.g., reinforced additives, polymers, nanoparticles, etc.), are indispensable factors controlling the printability of bio-inks. Nowadays, chitosan is available on the market with various molar masses (low, medium, and high molar mass) and a wide range of deacetylation degrees. Using chitosan with different molar masses as a precursor for bioprinting will lead to different printing results; to be more precise, printing 3D patterns using low molar mass chitosan will result in superior dimensional resolution, printing fidelity, and more mechanically rigid constructs.³¹

The deacetylation degree is defined as the proportion of existing glucosamine to N-acetylated glucosamine in the chitosan structure. Furthermore, chitosan is known as a pH-sensitive biopolymer, soluble in weakly acidic solutions, especially in acetic acid. The solvent pH value (acid/water volume ratio of the solvent) controls solubility of chitosan and viscosity of the chitosan hydrogel solution (e.g., a more viscous hydrogel is achieved in solutions with higher acid to water ratios). Needless to say, both the deacetylation degree of chitosan and the pH value of the solvent medium directly affect the solubility of chitosan, and the solubility directly influences mechanical/rheological properties of chitosan hydrogel solution such as its viscosity; flexibility; reactivity; and heat conductivity, and indirectly affects the characeristics of the final printed platform in terms of its swelling, and tensile strength; the pore volume, porosity, and its specific surface area. Moreover, it controls the biological characteristics by modifying adsorption, antioxidant, bioavailability, and bioactivity properties as well.^31,171

3D-bioprinting of dilute solutions not only decreases the printability of the utilized inks, but also the prepared 3D structures have poor mechanical stability and low printing fidelity. On the other hand, 3D constructs printed with viscous bio-inks and higher crosslinker densities have a better chance of higher printing fidelity using 3D-bioprinted devices. This entails making use of bio-inks with high viscosity, which are not recommended for biomedical applications (especially as implants, oral delivery systems, etc.) because of their low bioactivity (e.g., serious biocompatibility problems).^60,109,172 The density of living cells in the bio-ink also affects the viscosity of the hydrogel. Suspended cells within the matrix of the bio-ink, along with the other existing ingredients, tend to aggregate and precipitate. Aggregates also may cause blocking of the nozzle, which can additionally lead to excessive mechanical stress on the cells, causing the above-discussed decreases in cell viability, and non-uniform droplets.

The gelation rate of bio-inks is another crucial parameter, involved in the shape stability/instability of 3D-printed platforms. Pure chitosan solutions take too long to form gel (ca. 10 min). Therefore, initially, insufficient mechanical behavior and inadequate bioprintability made chitosan an unsuitable bio-ink for the production of complex 3D frameworks when applied in its pure form.¹⁰ Fortunately, nowadays, excellent progress has been made in new, practical methods of in situ crosslinking of bio-inks, such as aerogel crosslinking, and printing in a crosslinker pool. Emerging effective natural crosslinkers (e.g., genipin for crosslinking of chitosan-based bio-inks), molecular weight, controlling printing temperature, and various additives (e.g., various types of nanoparticles and salts), together could help to improve the gelation speed of chitosan-based bio-inks, up to a point.¹⁷³

The development of bioprinting techniques also plays a significant role in paving the way to an improved preparation of 3D networks with higher resolution and higher printing speed. Hydrogels capable of 3D-bioprinting through different bioprinting methods are needed to fulfill specific requirements; for example, generally, hydrogels with a viscosity below 10 mPa.s are welcome for the inkjet 3D-bioprinting process.³⁰ In addition, inkjet-based bioprinting requires a homogeneous solution with appropriate viscosity, as a higher viscosity means that an inkjet printer requires more energy to eject a microdrop, which may damage the laden cells. Furthermore, bio-inks with too high a viscosity may not only impede the sufficient transport of oxygen and nutrition to the cells but also reduce cell motility. On the other hand, only high-viscosity structures can maintain their structure and shapes for hours.¹⁷⁴ One of drawbacks of chitosan for inkjet-based bioprinting uses is its too high a viscosity. The addition of additives can tackle this problem, but it is not an ideal solution, as useful additives such as surface-active substances can adversely affect the survival of cells.

According to thermogravimetric results (TGA), polysaccharide chains like chitosan start to decompose at temperatures >230 °C;^175,176 therefore, adding reinforcing additives such as magnetic particles, carbon-based materials, and other nanoparticles should be performed at lower temperatures. In SLA-based bioprinters, which uses UV light as the energy source for proceeding with the printing process, if they exceed the predetermined threshold of the required photo-initiator concentration, the radiation may cause serious damage to the viability of encapsulated living cells and to cell adhesion as well.

After successful bioprinting of chitosan bio-ink, still there are a number of challenges and factors which should be considered. The 3D construct networks, due to adsorption of moisture (hydration) and other possibilities, have an excellent potential for swelling.²⁵ Subsequently, porosity and pore volumes may dramatically decline mainly because of the struts’ post-processing shrinkage (channels may be plugged, or pore diameters could be smaller than required). Anticipating these phenomena in designing 3D patterns before the printing process, and implementing viscous bio-inks with a higher density of crosslinker, could reduce the damage.³²

5.2. Evolution of chitosan derivatives for 3D-bioprinting

There is no question that the main parameters that are directly involved in the development of 3D-bioprinting are (a) printability (e.g., printing fidelity, resolution, construct, and shape stability) and (b) cell encapsulation (e.g., cell viability, proliferation, differentiation, and tissue formation).¹⁷⁷ Like 3D-printing, 3D-bioprinting is affected by many other parameters such as mechanical strength and elasticity, shear-thinning properties, and also biocompatibility. Increasing demands for new and efficient bio-inks induced extensive research on material properties to identify their unsuitable characteristics and find an ideal bio-ink for 3D-bioprinting applications.^25,93

Chitosan, as a prominent biopolymer, with some modification, meets almost all these requirements, and in order to achieve more adequate bio-inks, a wide range of chitosan-based materials has emerged in recent years. Firstly, neat chitosan bio-ink was used to produce 3D scaffolds, and with the passage of time, researchers have developed a variety of chitosan derivatives to be 3D-printed for in vivo body use. So far, many published reports have validated that both physical and chemical approaches are applicable to crosslink the polysaccharide chains of chitosan.³⁰

In the last few years, extensive application of extrusion-based methods (“direct-write” techniques) to prepare chitosan-based 3D scaffolds is a sign of its significant benefits for potential tissue engineering applications, mostly owing to its clear-cut processing approach leading to the appearance of simplicity and greater diversity of this technique.^152,155 Direct-write compatible bio-inks have been developed for diverse uses from micro stents to cellular scaffolding. Generally, incorporation of cellular inclusion may also be taken into account, and owing to the harmfulness of curing agents, biological components are usually introduced in a separated step. For instance, by adding silk particles to a chitosan scaffold, not only a 5-fold increase in compressive modulus will occur, but also it leads to enhancing the accuracy of the bioprinting process and the stability of the prepared scaffolds as well.¹⁷⁸ Extrusion of chitosan–HA and silk into sodium hydroxide/ethanol and a methanol bath, respectively, is a case in point. In recent reports, in order to facilitate crosslinking processes shortly after the time of writing, jammed soft granular gels were utilized for better printing in a direct-write fashion, also letting colloidal systems and cells remain supportless inside the gel solution. Currently, chitosan-based biomaterials are widely crosslinked by NaOH in extrusion-based bioprinting (EBB) methods for bioprinting perfusable vessel-like microfluidic channels.¹⁷⁹ At the same time, a hollow tubular framework was achieved by coaxial nozzle 3D-printers, where the polymer from the outside core and the crosslinker solution from the inner core were co-printed.¹⁵² Laser-based printing (especially SLA) mostly requires bio-inks with photosensitive properties and limited cytotoxicity of additives, such as photo-crosslinkers, which might lead to possible cell or DNA damage along with the lasers’ UV/near to UV light.¹⁸⁰ So, the application of this method for the preparation of chitosan-based 3D patterns has had only limited success.^31,140

So far, many types of research associated with the bioprinting of marine polysaccharide biopolymers such as chitosan, alginate, agarose, and their blends have been reported. In this regard, a multi-nozzle 3D-bioprinting process working in low pressure/ambient temperature operational conditions was designed to be applied for the deposition of bioprintable polymers such as alginate and living cells simultaneously.

To minimize the cell viability reduction caused by mechanical forces and deformations during bioprinting, the rheology of bio-ink was precisely examined.¹⁸¹ The results of mechanical tests have showed an influence of the following parameters on cell viability: the geometry of the nozzle, pressure dispensing, material concentration, and material flow rate.^182,183 Campos et al.^44,184 have performed a full study on the impact of bioprinted agarose-based materials, from pure agarose to different blend forms of it with collagen and chitosan polymers, on human MSCs and the differentiation of cells into the osteogenic or adipogenic lineage. In this report, a live/dead cell viability above 95% survival was unconditionally observed even after three weeks. The findings validated that the cells tend to differentiate in entirely different ways in stiff collagen and soft agarose matrix. In a rigid collagen matrix, for instance, the division of the cells happens in the osteogenic lineage, while in a soft agarose matrix, the cells primarily vary in adipogenic tissues. In another study, Demirtaş et al., for the very first time, could successfully prepare a 3D-printed platform based on a blend of cells with the composition of chitosan and hydroxyapatite (HA). In this way, the properties of 3D-printed MC3T3-E1 cell-laden chitosan and alginate hydrogel plus their composite form with nanostructured bone-like (HA) particles, which were bioprinted through an extruder-based approach, were analyzed and compared in full. The outcome of their research indicated that cell proliferation, cell survival, and osteogenic differentiation were appreciably promoted with the existence of hydroxyapatite in the alginate and chitosan hydrogel structure.³⁰

Han and Yan¹⁸⁵ tried to control the thermo-reversible sol–gel transition property of a self-healing supramolecular chitosan-based hydrogel using various amounts of 2D sheets of graphene oxide (GO) through electrostatic interactions between them. By varying the fraction of GO, which acts as the crosslinker, in the chitosan matrix and also the operational temperature, the gelation could be tuned over a wide range. The outcomes of their work show that, at room temperature, a mixture containing 0.2 wt% GO and 8 wt% chitosan still remained a solution; however, it will become a gel by increasing the GO amount to 0.3 wt%. In fact, the storage modulus G′ increased more with higher concentrations of chitosan and GO. Also, they successfully synthesized an optimized room temperature chitosan/GO composite, which could be utilized in various fields such as biomedical and environmental engineering. Hu et al.¹⁸⁶ introduced a new type of electroactive biopolymer by crosslinking of chitosan with aniline pentamer (AP) through a mixture of acetic acid, dimethyl sulfoxide, and dimethylformamide solution. In this report, considerable PC-12 differentiation was reported on platforms containing AP; however, using pure chitosan samples leads to only low cell differentiation after five days. Complex networks were developed by the cells that were observed on both prepared platforms with 4.9% and 9.5% AP. In this study, the sample with around 5 wt% AP exhibited the best performance, in terms of cell differentiation; further increases of the AP amount lead to significantly worse results in PC-12 cell differentiation. Lee and coworkers¹²² showed the capability of chitosan–catechol to be used as a bio-ink for 3D-printing. Chitosan amino groups are changed by a reaction with the catechol's carboxylic acid. The utilization of chitosan–catechol bio-ink led to 3D constructs in regular culture media with quick complexation with serum proteins. Furthermore, the combination of metal/catechol, including small amounts of vanadyl ions in a ratio to catechol of 0.0005, significantly improved the mechanical stability and bioprintability of embedded-cell bio-inks and also illustrated almost 90% of cell viability. In the presence of vanadium, the resultant conjugated biopolymer undergoes ionotropic gelation, creating a bio-ink that is extrudable.

5.3. Crosslinking of chitosan

To improve the properties of polymers, in particular their mechanical stability, they usually are crosslinked through one of the (a) chemical (e.g., Schiff base formation, covalent crosslinking), (b) enzymatic and (c) physical (e.g., ionic, photo and thermal crosslinking) crosslinking approaches.^10,187 Undoubtedly, many features of hydrogel-based scaffolds, including degradability, rate of drug release from the matrix of the bio-ink, viability, proliferation, migration of embedded cells, and release speed of loaded agents such as growth factors and biological agents, will be affected after a crosslinking process. Crosslinking of chitosan will result in higher structural stability and will provide the living cells and other biological agents with more effective environmental conditions. Covalent and ionic crosslinking (e.g., polyelectrolyte solution and multivalent ions) as chemical methods are the most crucial techniques for interconnecting chitosan polysaccharide chains.¹⁸⁸

From a bioprinting perspective (Table 4), due to the soft and moldable nature of bio-inks, most of these solutions need to be in situ crosslinked during the printing process to be capable of maintaining their structural integrity and designed 3D shape. Usually, the selection of a suitable crosslinking strategy depends on the rheological properties of the utilized bio-ink.⁶⁰ In this regard, direct interactions between chitosan chains through their complexes with other polymers such as PVA, alginate, and, more importantly, gelatin, are another common way to achieve physical chitosan-based hydrogels with elevated rheological properties.^136,138,140 Indeed, considerable enhancement in the bioactivity of chitosan, as well as an improvement in cell migration and cell adhesion, is the result of an appropriate combination of chitosan and gelatin; moreover, the hydrophilicity of gelatin improves water retention and leads to promotion of the transportation of oxygen and nutrients to the embedded cells. Accordingly, by raising the concentration of gelatin in chitosan/gelatin/genipin blends, the created environment helps to improve cell proliferation.¹⁸⁸ Using polyaldehydes (e.g., o-phthaldialdehyde) and, more importantly, glutaraldehyde to react with the amino groups of chitosan represents another popular approach to chitosan hydrogels, albeit with the possibility of existing cell cytotoxicity, so this method has been rarely utilized for the cross-linking of chitosan-based bio-inks for biomedical demands.^189–191 Likewise, using enzymes for the gelation of chitosan-based hydrogels through enzymatic cross-linking has been reported,^192,193 but it is not a popular approach for 3D-bioprinting and bioprinting applications.

Table 4 An overlook of printing principles and strategies for crosslinking of utilized bio-inks for extrusion-based 3D-bioprinting of cell-laden filaments as the building blocks^68,194

Crosslinking methods for 3D-bioprinting	Chitosan 3D-bioprintability	Method	Ref.
	✓	To improve the viscosity of bio-ink, and thus, to enhance the accuracy of the bioprinting	123
	No data	With the aid of complete temperature control of the bioprinting process, is mostly credited for pre-bioprinting of some thermoresponsive bio-inks and is applicable for or post-bioprinting	195
	✓	To immediately introduce crosslinker along with bio-inks by spraying crosslinker solution on printed filaments	141
	✓	Using a dual-needle or core–shell nozzle (coaxial needle) to extrude crosslinker and bio-ink solution simultaneously and to stabilize the printed filaments	140
	No data	Immersing the printing area into a pool of crosslinker agent; thus printed bio-ink, which possess a higher density in comparison to crosslinker, extrusion is performed underwater and the written stent is held in the bath until the printing is over; the temperature and viscosity of the crosslinking batch has a significant impact on bioprinting process	195
	No data	Make use of crosslinker to create aerosol conditions to enhance quick bio-ink gelation on the printing surface	196

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5.3.1. Crosslinking with naturally derived materials. Day by day, the application of natural-based materials for crosslinking of a wide array of hydrogels is, because of a smaller number of noxious by-products and the provision of more cell viability, enjoying full attention among researchers. Nowadays, genipin has attracted a great deal of interest in the preparation of chitosan-based 3D-bioprinted constructs. Indeed, there are many reports related to the use of genipin as a natural crosslinker for crosslinking of chitosan-based gels and chitosan/gelatin hydrogels as well. The in vivo cell cytotoxicity assay of genipin, which was carried out on mice, has indicated excellent results (1000 times less toxic) in comparison with glutaraldehyde, as one of the most popular classical chitosan crosslinkers, owing to genipin's bioderived nature.¹⁹⁷ High mechanical stability of the prepared gelatin/chitosan/genipin 3D structure is found after 24 hours of the crosslinking process at ambient temperature, proving that this material could be a great candidate for segment replacement in load-bearing and calcified human tissues, such as menisci and cartilage.

5.3.2. Photo-crosslinking using UV light. Photo-crosslinking has developed as a great strategy to tackle numerous bioprinting problems as the illumination is minimally invasive. In addition, spatiotemporal control of the light application makes it feasible to crosslink living cell-containing hydrogels, while cell viability still remains excellent. The crucial factor in utilizing photo-crosslinking in bioprinting-based preparation is the wavelength of the exposure light. The wavelength regime, which is usually UV light, is essential to ensure integrity of the structure in the 3D-printed structure. Therefore, an imminent challenge in improving new crosslinking is minimizing the light exposure during printing scaffolds. Some studies have shown that chitin and chitosan speed up wound healing and the repair process. To date, several grades of chitin and chitosan are commercialized for wound dressing, but they are mostly combined with polyester or cotton in the form of granules, filaments, or sponges composites. Moreover, chitosan hydrogel, as an occlusive dressing, is capable of contributing to healing of the impaired tissue, in suitable humid healing surroundings.^7,198

Continuous UV light irradiation has been commonly used to start the photo-crosslinking of printed bio-inks, in particular in inkjet-based printers. The BioPen has been introduced in recent years to decrease the overall exposure to UV light.¹⁹⁹ This is accomplished by projecting the illumination to the particular point at which the bio-ink has already been extruded. The BioPen is capable of 3D-bioprinting line-shaped cell-laden UV-crosslinked bio-ink for the surgical reconstruction of tissues.²⁰⁰ Among all extrusion-based strategies, continuous crosslinking can be considered as the simplest one; however, severe over-exposure, which can impair the cell, has to be avoided when UV light is utilized. The application of this method for chitin and chitosan hydrogels has been limited to producing 2D scaffolds. Although some reports have justified the use of a photo-crosslinking approach to generate 3D constructs,^16,201,202 according to the best knowledge of the authors, there are no data on the preparation of chitosan-based 3D-bioprinted scaffolds through a photo-crosslinking methodology.⁷

5.4. Optimization of chitosan bio-ink rheology

As already mentioned in this study, excellent bioprinting directly depends on the rheological characteristics of the bio-ink. Therefore, the bio-ink should possess printable viscosity, which enables extrusion through the very tiny nozzles at low pressures (<4 bars). Designing bio-inks from an appropriate selection of biopolymers can fulfill the rheological requirements for 3D-bioprinting that include non-Newtonian viscoelastic behavior, typically accompanied by high elasticity, signified by a high storage modulus G′ (as a function of the angular frequency), at high shear rates [small gamma, Greek, dot above], and high viscosity at low shear rates γ.¹¹⁵ Mostly for successful extrusion-based bioprinting, control of bio-ink viscosity as one of the significant parameters, which affects the printability, depends on controlling the temperature or shear-thinning for various printing techniques. It is evident that bio-inks must be in the liquid phase and possess uniform composition in conjunction with the desired viscosity and shear-thinning properties for preventing nozzle clogging. Bio-ink viscosity affects the 3D-bioprinting procedure mainly at the dispenser tip, the most critical spot at the time of extrusion.⁹³

High mechanical stability, superior capability to protect the cells, and elevated ability to precisely design pore shape and size in 3D scaffolds and printed implants need optimized viscoelastic behavior. Therefore, various copolymers and methods are applied to change and optimize the rheological properties of chitosan-based bio-inks and prepare them for 3D-bioprinting. The relatively high viscosity of chitosan-based bio-inks not only prolongs their printing time to several hours (depending on the demands) but might also lead to nozzle clogging of small-diameter nozzle-based printing techniques. In addition, low bio-ink viscosity causes a failure to obtain a 3D-printed construct with precisely defined dimensions. Demirtaş et al.³⁰ studied and optimized the rheological and thermal gelation of chitosan and chitosan/hydroxyapatite hydrogels while using them as cell-laden bio-inks for bone tissue regeneration. The mentioned hydrogels exhibit a temperature-induced gelation behavior since fast heating leads to a quick increase in storage modulus G′. In detail, following the first increase in G′, at the optimum temperature, the moduli became constant. For temperature sweep tests, the hydrogels were heated up from a specific temperature to a fixed predetermined temperature, in order to identify the G′ and loss modulus G′′. Dynamic-mechanical measurements with a rotational rheometer as a function of frequency were applied to assess the bio-inks’ viscosity and modulus functions. During thermal gelation at body temperature, the G′ of chitosan increased 13-fold, while for chitosan/hydroxyapatite a 29-fold increase was observed. By increasing the oscillation frequency, the storage moduli increased steadily for all bio-inks, which is typical of rather elastic shear-thinning materials. The loss moduli for all bio-inks were lower than the storage moduli, which means that the elastic nature of the materials dominates, which is typical of viscoelastic gels. In addition, adding glycerol phosphate disodium salt and NaOH to the chitosan bio-inks raised the viscosity of the hydrogels as a result of internal gelation. Increasing shear rates γ led to a reduction of dynamic viscosity η for all bio-inks. Fig. 6 illustrates an overview of the distance between the existing bio-inks and an ideal one.

Fig. 6 The biofabrication window. From the more conventional bio-inks, there has been a major shift in confrontation considering the correlation between the type and biocompatibility of biomaterials (top left and bottom right).^194,203,204

5.5. Mussel-inspired chitosan derivatives

In oceans, special proteins are released by mussels and utilized as a bioglue for sticking to a surface. These proteins contain moieties, which are known as DOPA. Most prominently, the adhesion of these materials to rocks and other substrates under harsh oceanic conditions (e.g., destructive waves, tide-dependent water levels, leading to submersion and exposure to atmospheric conditions following a regular pattern) has been assigned to the existence of the catechol groups available in DOPA.²⁰⁵ Regardless of the exact chemistry of these materials, their versatility is caused by ‘catechol’ moieties in the structure of the proteins.^122,205

The modification of chitosan chains with catechol units can open new doors in the medical field and be considered as an effective bioadhesive polymer for biomedical demands (see Fig. 7). Drug and gene delivery, as well as cancer thermotherapy, are listed as the most attractive fields of interest of mussel-inspired chitosan-based biomaterials in biomedical engineering. Moreover, many reports have been conducted related to the application of mussel-inspired chitosan-based structures for hemostatic materials, tissue engineering, and biosensors as well. Using mussel-inspired chemistry to modify chitosan-based bio-inks leads to the high promotion of chitosan's water solubility even for neutral aqueous solutions, and will result in improved mechanical stability, enhanced tissue adhesion, and excellent biocompatibility.²⁰⁶ However, recent research on functionalized chitosan–catechol materials has demonstrated that the use of glycol/chitosan–catechol instead of chitosan–catechol as a hemostatic agent had a better result. Seminal research, published by Park et al.,²⁰⁷ deals with making use of mussel-inspired chemistry for the preparation of a 3D-printed-chitosan construct applicable for a wide array of uses in the biomedical field. Using ethylene glycol groups gives chitosan–catechol improved anti-biofouling properties, making it a promising alternative product from cellular toxicity and in vivo skin irritation aspects.

Fig. 7 Schematic illustration of the molecular structure of (A) chitosan, (B) chitosan–catechol, (C) chitosan–catechol complex with vanadyl(IV) ions and (D) glycol chitosan–catechol.^8,122,204

Inspired by mussel-chemistry, Lee et al.¹²² tried to make use of an incorporated vanadyl ion/chitosan–catechol combination, as a bio-ink, for successful 3D-bioprinting through a direct writability technique. They found that the presence of a small number of vanadyl ions in the mixture of catechol-functionalized chitosan could greatly increase its printability, mechanical properties, and also it indicated acceptable cell viability. This methodology for the preparation of a cell-encapsulated chitosan-based bio-ink in fetal bovine serum (FBS) in culture medium justified that it could be an excellent approach for the manufacturing of efficient 3D bio-ink crosslinked without any external stimuli. They also showed that the bioprinting process in a medium containing 20% FBS could achieve more stable 3D networks (see Fig. 7). In another study, Wang et al.¹⁰⁰ suggested a straightforward mussel-inspired strategy to load Ag nanoparticles and construct chitosan–polyurethane coatings to endow polyethersulfone with dual antibacterial features. This conveys the possibility of preparing various hybrid biomaterials with particular functionalities. Existing Ag nanoparticles as an antibacterial agent could appreciably elevate the antimicrobial properties of prepared chitosan–catechol 3D structures.

6. Biomedical applications

3D networks using chitosan-based bio-inks have a significant number of applications in tissue engineering (from hard to soft tissues), advanced biosensors, and drug delivery, which are considered to be the most conspicuous branches of biomedical engineering. In the following discussions, the authors of this work have given a comprehensive insight into the printing of the various chitosan-based 3D scaffolds applicable in each mentioned section.

6.1. Tissue engineering

6.1.1. Bone tissue. Wang et al.²⁰⁸ used a mixture of ethanol and sodium hydroxide (NaOH) to facilitate gelation of chitosan and chitosan–hydroxyapatite composites prepared by a robotic dispensing rapid prototyping mechanism. In this technique, inks are extruded into a dispensing solution via a Teflon-lined nozzle and made a 3D construct with interconnected pores and high cell attachment. The role of NaOH is to maintain the shape of cuboid construct under hydrostatic pressure. At a high concentration of NaOH, rapid precipitation and low mobility of chitosan assist attachment of a small layer, while in low concentrations the shape fidelity and perception kinetics are changed. They found the factors involved in the preparation of the scaffolds, such as the initial height, flow rate, and pressure, which are firmly dependent on ink viscosity. Therefore, by monitoring and controlling the involved factors in various compositions they achieved an optimum composition. The degree of the constructs’ biocompatibility was analyzed by in vivo bone-cell culturing. The SEM images taken three weeks after cell seeding exhibited excellent adhesion, healthy morphology, and homogeneous cell distribution.

Demirtaş et al.³⁰ illustrated that cells could be blended and printed with chitosan solution and a composite of the solution with nanostructured bone-like hydroxyapatite (HA). Analysis of cell proliferation and viability revealed that, using bio-solutions for printing, cells can be viable after printing. In addition, it is proved that chitosan, as a bio-printing solution, is printable. Moreover, it is shown that the existence of HA in chitosan hydrogel can enhance cell survival and osteogenic differentiation.

Wang et al.¹⁰⁰ suggested new hydroxyethyl chitosan-based scaffolds and cellulose. HECS/CEL has a porous framework which facilitates chemical crosslinking, freeze-drying, and silica-leaching approaches, such that by generating macro- and micropores using freeze-drying, it makes possible particulate porogen procedures. Bioanalysis illustrated that, since the HECS/CEL scaffolds are contemplated to be an excellent medium for applying in bone tissue engineering, they can aid the spreading, attachment, proliferation, and viability of osteoblastic MC3T3-E1 cells. Wang et al.²⁰⁸ investigated beta-glycerophosphate as an osteogenic medium supplement, which is utilized as a weak foundation for the simultaneous gelation of pure chitosan at physiological temperatures and pH values. However, DNA content decreased by 50% within 12 days in chitosan. When collagen was used, DNA content increased two times. The presence of chitosan boosted osterix in both media with or without osteogenic supplements. Chitosan–collagen composites are potentially useful for cell delivery and encapsulation, or can be considered as in situ gelling materials for tissue amendment.^209–211 Because cell viability must be maintained when the gel is prepared, the injectable biomaterials’ cytocompatibility is a crucial factor in cell encapsulation.²¹² Collagen and chitosan, which are nature-derived materials, have been investigated widely because of their capability of integration with adjacent ECM and supporting cell growth.^213–215 It was illustrated that the high viability of cells in chitosan gels without additional components connected to the β-GP was inhabited by fibro-chondrocytes. Wang et al.²⁰⁸ reported, however, that calcium was deposited after a while. However, calcium secretion took place because of cell necrosis, which was reflected in decrease in DNA content over time. They reported higher expression, higher calcium content, and incremented ALP activity upon increasing the chitosan content in the composite of chitosan–collagen.

6.1.2. Cartilage tissue. Li et al.¹²³ 3D-printed a homogeneous pore-sized, cell-laden implant by hydroxybutyl chitosan/oxidized chondroitin sulfate-based ink in various architectures and applied it for regeneration of cartilage tissue. In vivo and in vitro studies disclosed that the proposed implant had anti-inflammatory activity, good ability for cell delivery, non-cytotoxicity, high mechanical properties similar to cartilage tissue, and biocompatibility. The controlled shape and capability of multifunctionality besides its other characteristics made it a suitable applicant for cartilage tissue regeneration.

In a similar work on cartilage tissue regeneration, Morris et al.³¹ prepared an ear-shaped, interconnected, uniform, controlled internal pore-size (50 μm) scaffold through 3D-bioprinting of chitosan and polyethylene glycol diacrylate ink. They optimized its mechanical properties, printability, and cell attachment ability through monitoring and manipulating the involved operational parameters such as feed-ratios, photo-initiator concentrations, and chitosan molecular weight. They found that the best ratios of chitosan to PEGDA were between 5 : 1 and 10 : 1. Needless to say, the presence of chitosan imparts hydrophilicity and good swelling properties to the scaffold (see Fig. 8). Ye et al.²¹⁶ studied infrapatellar fat pad adipose stem cells embedded in a chitosan-based 3D-printed construct and studied its capacity in cartilage tissue engineering. The in vitro survey revealed that embedded cells in the construct network in the chondrogenic environment hindered the emergence of cartilaginous-shape tissue. However, in the non-chondrogenic environment, fibroblastic morphology emerged. They found that scaffold pore size impacts on cell adhesion and proliferation. Their obtained outcomes proved that the presence of TGFβ3 and BMP6 in the scaffold architecture remarkably improved the repair process of damaged cartilage.

Fig. 8 Chitosan-based 3D structures used in bone and cartilage regeneration. (A.a1) 3D-printed quaternized chitosan (HACC)-grafted polylactide-co-glycolide (P)/hydroxyapatite (HA) construct (P/HA/HACC) applied for bone regeneration of a femoral shaft bone defect made in a rat (A.a2) and femoral condyle local bone defect made in a white rabbit (A.a3). (A.b) X-ray image of the right femoral condyle of a rabbit at week 4 and week 8 after implantation of (P/HA/HACC) scaffold with remarkable regeneration after week 8. (A.c) Bone volume regenerated per total bone volume for all the scaffolds after 8 weeks of implantation. (A.d) Lateral X-ray image of the left femoral shaft defects of a rat; bone regeneration after 8 weeks shown by the blue arrow. (A.e) Bone volume regenerated per total bone volume at week 2, week 4, and week 8 after the implantation of various 3D-printed scaffolds.¹⁶² (B.a) 3D-printed, ear-shaped low molar mass chitosan/polyethylene glycol diacrylate with a ratio of 1 : 7.5. (B.b) SEM image of the mentioned scaffolds clearly presenting the micropores of the construct. (B.c and B.d) Images with low and high magnification of the scaffold (H&E stained), showing an excellent cell distribution.³⁰

Reed et al.²¹⁷ applied an amalgamation of directional freezing and 3D-printing to the controlled design of micro/macro-interconnected pores and channels of an acellular chitosan–alginate construct. This 3D scaffold not only possesses high compression resistance and acceptable swelling ability but also inhibits the denaturation process of loaded drug and protects encapsulated growth factors due to its preparation procedure in a neutral pH environment. Such constructs with lamellar pores parallel and perpendicular struts were used to recapitulate the real cartilage tissue better, showing improved swelling absorption, and accelerated cell migration at their full height and diameter, as well as promoting the regeneration cycle.

Madihally et al.¹¹⁷ mixed gelatin with chitosan in a mixture of deionized water and hydrochloric acid for facile and inexpensive 3D-bioprinting. Gelation ensures the enhancement of cell/chitosan interactions for application as a highly efficient thermogelling bio-ink. Subsequently, to improve its characteristics and to form hydrogels at 37 °C, they added β-glycerophosphate and cells to the ink. They concluded that a cooling mixture, vacuum degassing, and centrifugation could prevent printer clogging due to undesired gelation, the presence of substantial contamination, agglomeration, and aggregation. The authors found that a reduction in fiber size, enhancement of the rheology of the solution, and acceleration of the gelation process occurred with increments in the chitosan concentration.

6.1.3. Neural system and vascular network. Vascular network and neural system repairs are the most challenging issues and inseparable parts of tissue regeneration. Bio-nature scaffolds such as chitosan-based 3D-printed networks have exhibited a high potential for regeneration of the neural and vascular systems. Gu et al.¹⁵⁵ designed a new, printable bio-ink based on an agarose/alginate/carboxymethyl-chitosan complex for application in neural tissue regeneration. The prepared bio-ink possesses outstanding features such as nontoxicity, low-time gelation, elevated adhesive properties, an excellent environment for cell migration and proliferation, high stability, and a suitable porous medium for encapsulating cells. Adding carboxymethyl-chitosan to the alginate based-ink enhanced the permeability, porosity, hydrophilicity, and cell growth/viability of a 3D-printed scaffold. Besides, they applied silver particles to optimize the rheology of the bio-ink. Such 3D scaffolds not only facilitate the repair and operation of human neural systems but they can also be tailored to be applied for other tissue regeneration demands.

Zhang et al.¹⁷⁷ used a 3D-bioprinter to create microfluidic cellular vessel-like channels with chitosan/alginate ink through direct printing. The delicate channels with a wall thickness of <200 μm were crosslinked by NaOH solution. An increase in the chitosan concentration (>4%) decreased cell viability, owing to the need for more printing pressure due to increasing solution viscosity. However, a low chitosan concentration (2%) led to weak mechanical properties. Accordingly, solutions with a chitosan concentration in the range of 2.5 to 3% were selected as optimum for bioprinting. The polymer dispensing rate had a direct relationship with wall thickness. Overall, the channels made by chitosan and alginate had excellent cell viability and could be applied for vessel network regeneration.

6.1.4. Skin tissue. Distinctive characteristics of chitosan, such as hydrophilicity, biodegradability, and hemostasis features, have made this biopolymer a suitable candidate for skin regeneration demands.²¹⁸ Moreover, chitosan enhanced wound healing by imparting fibroblast growth, making it an appropriate environment for introducing polymorphonuclear leukocytes into damaged tissue.^219,220 Moreover, good swelling properties of chitosan-based 3D scaffolds ensure cell viability and a reduction in scar formation. Ng et al.¹³¹ optimized the healing and antimicrobial characteristics of chitosan-gelatin complexes for 3D-bioprinting of robust skin wound dressings. They selected chitosan instead of collagen due to its excellent printability and ready functionalization ability. Chitosan facilitated inflammatory cell migration, nutrient and oxygen penetration and resulted in cytokine formation, which accelerates wound healing.²²¹ The carboxylate group of gelatin reacted with an ammonium group in the chitosan and formed a polyelectrolyte ink, which had a gelation temperature below room temperature. The rheological properties, storage capacity, biocompatibility, and physical and chemical stability of this polyelectrolyte ink were comprehensively surveyed. A high ink viscosity led a sharp reduction in cell proliferation and viability and, ultimately, to cell death.

Long et al.²²⁰ embedded lidocaine hydrochloride as a medicine for accelerating wound healing in extrusion-based 3D-printed chitosan/pectin structures. The printed structures exhibit improved printability and strong wet-adhesion to the skin. Due to the hydrophilicity and high porosity of the printed ink, the construct provided an excellent microenvironment similar to the real tissue for exchanging nutrients, oxygen, and wastes. Hafezi et al.¹²⁴ designed a wound dressing based on genipin-crosslinked chitosan, plasticized by PEG and glycerol. By 3D-bioprinting, they controlled the geometry and the interconnectivity of the printed porous medium. The nontoxicity of the printed scaffold was confirmed by 90% viability of the skin fibroblast cell lines after two days. The in vitro study of the designed dressing exhibits acceptable swelling and ability for drug release, enhanced biocompatibility, cell attachment, and proliferation.

Thein-Han et al.¹⁰⁵ described a favorable cellular response for buffalo embryonic stem cells encapsulated in 3D composite scaffolds consisting of biodegradable chitosan–gelatin for tissue engineering based on stem cells. The biological reaction of cell growth on the constructs suggested that mixing gelatin in the chitosan enhanced cellular efficacy. 3D-printed chitosan–gelatin scaffolds can be considered to be excellent contenders for tissue engineering. The outcomes depicted that the constructs of the skin from this proposed method had high biocompatibility. In strongly visualized GFP-expressing cell–chitosan–gelatin scaffolding systems, both cell proliferation and cell spreading are shown to be superior compared with mere chitosan. A correlation, including cell viability and replication, shows that a combination of gelatin and chitosan improves the potency of cells. Studies including analyses by fluorescence and flow cytometry and histological observations of the structures implicate a pluripotency of the polygonally connected cells after 28 days culture time. The research emphasizes that chitosan–gelatin scaffolds are excellent 3D constructs for cell beds and application in tissue regeneration.

A composite of hyaluronic acid, collagen, and chitosan with a new crosslinking procedure (a mixture of different crosslinking approaches) has been employed by Zhang et al.²²¹ to create a bio-ink to make 3D cell-laden builds using 3D bioprinting for skin tissue engineering. Fibroblasts were distributed into the bio-ink to generate 3D cell-laden living constructs. A bio-ink composition of chitosan of 8 wt%, hyaluronic acid of 0.5 wt%, and 10 wt% of collagen was selected to make skin-imitating constructs. The morphology of the constructs has a layered structure like human skin. The tensile strength for chitosan/HA/collagen (CS/HA/Col) was 29.9 kPa, and this value for CS/HA/Col–tyrosinase was 39.1 kPa. The Young's modulus for CS/HA/Col was 0.170 MPa, and for CS/HA/Col–tyrosinase was 0.313 MPa. These values show that CS/HA/Col–tyrosinase proposed an enhanced mechanical strength that is similar to human skin (1.46 ± 0.26 MPa (ref. 222)) because of chemically-induced crosslinking using tyrosinase. The rate of degradation of CS/HA/Col was 81.99%. This value for CS/HA/Col–tyrosinase in thirty-five days was 55.34%.

In comparison with a non-crosslinked CS/HA/Col scaffold, a tyrosinase pre-chemical crosslinked hydrogel (CS/HA/Col–tyrosinase) shows a significantly slower degradation of these biocompatible constructs of the skin, featuring a high cell survival rate. The outcomes depicted that the constructs of the skin from this proposed method have good mechanical features and biocompatibility. Moreover, with increase in the tyrosinase concentration the mechanical strenght increases and degradation rate decreases. Hence, the new crosslinking method (by tyrosinase) is more desirable. Fig. 9 shows examples of how chitosan-based bio-inks could be effective for the regeneration of soft tissues such as neural and skin tissue.

Fig. 9 Chitosan-based 3D-printed structures for soft tissue regeneration. (A.a) 3D structure made by collagen/chitosan bio-ink. (A.b and A.c) SEM image of the 3D construct, exhibiting interconnected pores with a specific shape and diameter. This bio-ink was applied by two different methods: freeze-drying (C/C) and 3D-bioprinting (3D–C/C). HE analysis shows that 3D-printed scaffolds could repair the injured tissue and made smaller cavities and a more linearly ordered structure in comparison with C/C (A.d, e, f).¹³² (B.a, b, c) SEM image of seeded 3D chitosan scaffolds supporting normal human dermal fibroblasts (NHDF) and the human keratinocyte cell line HaCaT. (B.d) Wound healing images in the modified and non-modified groups.¹²⁶ (C.a) Microfluidics-based synthesis of a 3D, cell-laden chitosan structure in a glass capillary. (C.b) Fluorescent microscopic image of viable human umbilical vein endothelial cells inside the tubular microgels at day 0. On the third day (C.c), the HUVECs exhibited spiny structures and started to disperse through the pipes, and after a week, the cells survived on the inner face of the tubular microgels (C, d).¹⁵²

6.2. Drug delivery

3D-printing methodologies and applying various compositions of 3D-printable ink allow for overcoming the existing shortcomings such as burst and off-target release of drugs. An effective technique is magnetizing printed constructs by adding magnetic particles. For instance, magnetic particles like Fe₃O₄, Fe₂O₃, CoFe₂O₄, MnFe₂O₄, and ZnFe₂O₄ are widely used in biomedical technology because of their remote operating potential, mobility, active movement, accurate target localization abilities, and external control on the cargo carriers.

In an intriguing work, a high-resolution 3D-printed magnetic micro-swimmer-shaped methacrylamide chitosan crosslinked by PEG was applied to carry doxorubicin as a chemotherapeutic drug using a photocleavable crosslinker and controlled release of the drug by an external light stimulus. The movement, direction, and path of the micro-swimmers can be monitored and controlled. The presence of chitosan in the bio-ink ensures the biocompatibility and biodegradability of the carrier over a span of 204 h.²²³

Yan et al.²²⁴ 3D-printed carboxymethyl chitosan with snake gourd root/astragalus polysaccharide patches in various structures (circular, square, and rectangular) by a melt-extruding 3D-printer and surveyed their controlled-release ability of bovine serum albumin for possible diabetes therapy. Their observation justified that the shapes of the utilized patches influenced their efficiency; to be more precise, the circular patches exhibited a higher release rate than square and rectangular patches. Besides, the net drug release in the acidic region was more pronounced than the alkaline region. The development of Schiff bases between the (–NH₂) on carboxymethyl chitosan and the aldehyde groups on oxidized polysaccharides throughout the gelation procedure induces a sharp decrease in the toxicity of the printed constructs and increases the viability and cell proliferation.

Since direct ink writing offers easy composition and encapsulation of hydrophobic/hydrophilic drugs or proteins into 3D-printed constructs, Sommer et al.²²⁵ applied this technique to 3D-bioprinting of chitosan-modified silica-nanoparticle-stabilized emulsion, which contains hydrophobic compounds in the specific location of its structure, helping the controlled release of the drugs (see Fig. 10). In another report, 3D-printed bioceramic scaffolds coated with chitosan hydrochloride enable the transport and delivery of bioactive molecules such as rhBMP-2, heparin, and vancomycin with minimum biological activity reduction in printed shapes in comparison with the initial state before the printing process. The presence of the chitosan hydrochloride not only increased the biocompatibility of the carrier but also caused the change in the profile release of vancomycin from 1^st order burst release to 0^th order continual release in the range of 0.68 to 0.96% h⁻¹.¹³⁹

Fig. 10 Drug-delivery and sensor applications of chitosan-based 3D-printed constructs. (A.a) Preparation pathway of silica–chitosan complex soft materials by 3D-bioprinting of stable emulsions. The oil droplets were applied to encapsulate hydrophobic drugs or to model pores after the structural consolidation. (A.b) 3D-printed structure with non-volatile oil–water emulsion stabilized by chitosan–silica for carrying both hydrophobic and hydrophilic compounds.²²⁸ (B.a–c) Healing process of a self-healing, chitosan-based material, which was used for 3D-printing on an APS-contained gelatin membrane to create a sensor (B.d). Resistance variation of the attached sensor on the index finger was assessed as the finger was subjected to repeated bending and relaxing from 20°, 45°, and 90° (B.e).

6.3. Biosensors

Recently, preparation of conductive constructs as sensors for application in health monitoring systems and biomimetic prostheses has gained significant attention. 3D-printing of sensors imparts efficient sensing ability and tailors nanocomposite mechanical and electrical characteristics.^226,227 In a seminal work, Darabi et al.²²⁸ 3D-printed a chitosan-based, conductive, self-healing hydrogel with improved stretchability (1500%) and repairability in 2 minutes under ambient conditions by appropriate, two-step physical/chemical crosslinking for human motion detection. The fast self-healing property of the printed structure stems from the strong ionic interactions between ferric ions, NH groups of polypyrrole, –OH and NH₂ groups of chitosan, and carboxylic groups of poly(acrylic acid). This amazing sensor can record and monitor the pulse rate, breathing rate, and biceps and finger flexion by conductivity changing under compression. In another report, Wu et al. 3D-printed a mixture of chitosan and CNT with high resolution (30 μm) to achieve a structure that was self-healing at room temperature when exposed to water vapor. The synthesized magnetic sensor could retain its electrical and mechanical characteristics after the repair process. 3D constructs with various pore shapes (diamond and square) after neutralization in an alkaline environment (sodium hydroxide) exhibit remarkable stretchability and flexibility. A high conductivity (∼1450 S m⁻¹), stretchability (strain at break ∼180%), and flexibility of the printed material make it an excellent applicant for identifying defects in human elbow movements.

7. Other applications of chitosan

7.1. Water treatment

To fight the upcoming water crisis, removing toxic organic compounds and heavy metals from wastewater has the utmost priority among environmentalists.²²⁹ Applying biocompatible materials with maximum efficiency, as well as maintaining their nontoxic and eco-friendly features, lies at the center of attention.²³⁰ The presence of (–OH) and (–NH₂) in the main chain of chitosan and its capability to mix with synthetic polymers to form fibers through electrospinning increased its utilization in wastewater treatment demands.^231,232 In recent years, the number of publications on applying chitosan with different compositions and shapes for the removal of nitrate, phosphate, heavy metals, and dye molecules from aqueous medium has skyrocketed.^233–235 Nevertheless, there has been only one report of 3D-printed chitosan-based adsorbents for application in the removal of the pollutants from water, owing to the relatively lower demands of dimensional precision and the high costs associated with 3D-bioprinting. In this work, Zhou et al.²³⁵ synthesized a PLA@GO/chitosan bionic filter with a 3D-bioprinting and double-freezing methodology. They designed an efficient, biocompatible filter with the help of 3D-bioprinting for the remediation of crystal violet in aqueous solution (see Fig. 12a–e).

Fig. 11 Variations in human tissue stiffness. The suitable stiffness for cartilage tissue regeneration is in the range of 1 to 10 MPa.²³⁹

Fig. 12 Other applications of chitosan in 3D-printed structures. (a–c) Mechanism of adsorption of organic and heavy metal ions with chitosan from aqueous media. (d and e) Schematic bionic construct design for effective crystal violet uptake assisted by a 3D-printed polylactic acid-graphene oxide/chitosan filter.²³⁵ (f and g) Application of chitosan in the coating of 3D structures like PLGA/hydroxyapatite to enhance cell viability and attachment for bone regeneration demands. (h and i) Image of 3D scaffold alginate and chitosan-coated alginate scaffold, respectively. (j and k) SEM images of alginate- and chitosan-coated alginate scaffolds, respectively. (l) Higher cell viability of the chitosan-coated in comparison with bare alginate scaffold using DNA as a cell sample.²³⁷

7.2. Chitosan coatings on 3D-printed structures

Chitosan has been broadly applied as a coating on various 3D-printed constructs to enhance their functionality and cell attachment as well as to provide them with ability to immobilization of biological molecules such as growth factors and different drugs. For instance, Lee et al.²³⁶ enhanced mouse bone marrow MSC growth by indirect 3D-printed chitosan. First, a 3D gelatin model was printed and infiltrated by PCL; subsequently, gelatin was dissolved in an aqueous medium, and the surviving materials covered with chitosan. In the end, the PCL was dissolved in the chloroform, and a 3D chitosan structure was produced. Lin et al.²³⁷ applied chitosan to coat a 3D-printed alginate fiber, which was loaded with diclofenac to increase biocompatibility, bone-cell proliferation ability, and to enhance the drug release profile. The adsorption/desorption behavior of diclofenac enhanced with chitosan, and therefore remarkably restricted interleukin-6 and tumor necrosis factor-α discharge from macrophages. Ainola et al.²³⁸ utilized glutaraldehyde crosslinked electrospun chitosan/PEO to support a 3D-printed PCL grid for chondrogenesis (i.e., the development process of cartilage tissue). This supporting coat improved cell/matrix interactions and chondrogenesis. Wu et al.¹⁵⁶ designed PLA/CS, and PLA-g-MA/CS complexes with a height of 123 μm and width of 21 μm through a 3D-bioprinting technique, and subsequently characterized and surveyed their biological activities such as antimicrobial properties. The authors also observed that the printed structures exhibit improved stiffness (elastic modulus), and tensile strength of 13.93 MPa and 1.47 MPa, respectively. Stiffness (elastic modulus) represents the amount of force required to deform a material to a certain extent elastically. Fig. 11 depicts the stiffness of various human tissues/organs with their determined elastic modulus.

Since the presence of a vast amount of living cells is needed for successful 3D-bioprinting, induced pluripotent stem cells are considered as ideal candidates for this matter, so that many scientists have applied this technique in recent years.²³⁹ Wong et al.²⁴⁰ could preserve and grow a large amount of stem cell markers without a significant reduction in their proliferation speed in chitosan sheets. Moreover, they embedded cells in a biodegradable, 3D-printed polyurethane construct and studied its impact on cell viability, but they did not survey the effect of the chitosan/polyurethane printed structure.

Also, PLGA/hydroxyapatite was printed by Yang et al.²⁴¹ and covalently covered with quaternized chitosan. Due to the presence of quaternized chitosan in the printed scaffold it showed antimicrobial and osteoconductive properties. In vivo studies on several rabbit femoral condyle defects and many rat femoral defects showed a significant increase in the volume of regenerated bones. Furthermore, the scaffold deterioration rate occurred similar to the progression of infection, impacting the bone generation in the infected bone models. Tsai et al.²⁴² 3D-printed titanium scaffolds by a selective laser melting (SLM) process and then applied chitosan/magnesium–calcium silicate as a coating agent to add the bioactivity and other needed characteristics to the printed scaffold, such as hydrophilicity, mechanical stability, cell attachment, proliferation and more similarity with real bone tissue. The in vivo study showed that the coated 3D construct had a high potential for bone regeneration.

8. Future directions of 3D bioprinting based on chitosan

The accuracy and similarity of 3D bio-printed tissues/organs depend on the constructional signals of the environmental medium. In this regard, 3D bio-printing outdoes old-fashioned tissue engineering in many terms owing to its extraordinary ability to integrate various intricate architectures with improved mechanical properties and high alignment resolution in conjunction with great cell/cell sustainability to make an artificial tissue very close to real ones. However, current reports show that 3D bioprinting could not mimic sophisticated organs with complex vascular or neural networks (e.g., heart and brain) similar to real organs.^243,244 Furthermore, since present studies on finding high-efficient medicines and lasting cures for sophisticated cancerous tumors or chronic wounds are mainly based on animal model investigations or monolayer cell cultures, which possess different gene expression, cell function, vascular network, epigenetics, cell/cell and cell/matrix interaction compared with human 3D tissues, more accurate answers will have to be found solely by analyzing the efficiency of the designed materials and drugs on 3D models sufficiently similar to real organs.^245–247 Therefore, the advent of microfluidics in biomedical engineering triggered developing organ-on-a-chip systems to remedy the disadvantages of traditional techniques (e.g., gel confinement and lithography) for creating complicated organ/tissue models with a high extent of similarity to the architectural, microenvironmental and functional complexity of real organs on the way to achieve more efficient diagnostics, and therapies.^69,248 As mentioned before, while 3D-bioprinters can produce complex organs with precise control over spatial factors, they still suffer from restrictions such as printing resolution, ideal bio-ink composition and properties, and limited co-printing ability of different cell types. On the other hand, organ-on-a-chip platforms not only ensure that designed structures have physiological, mechanical, and chemical cues in vitro but also have the ability to implement multiple experiments on a single chip (unlike many single animal experiments).²⁴⁹ Organ-on-a-chip devices mostly consist of microchannels and sophisticated architecture to simulate the structure of native organs/tissues. This technology is not only applied for mechanistic surveys but also used as proof of concept for drug-analyzing investigations. However, they suffer from fully controlling soft tissue-like construct characteristics. Scientists firmly believe that a combination of lab-on-a-chip and 3D-bioprinting could tackle the existing disadvantages that both techniques are facing and providing a unique methodology for recapitulating more sophisticated organs with high exactness.^4,244–251Fig. 13 shows some advantages of combining 3D-bioprinting and organ-on-a-chip approaches.^{70,183,250–258}

Fig. 13 Benefits of combining 3D-bioprinting with organ-on-a-chip technology.

Recently, applications of chitosan in chips, microfluidic devices, biosensors, and sophisticated tissues like neural networks are established by a few scientists.^{161,259–262} Distinctive characteristics of chitosan such as the ability to prepare films and nanofiber shapes, biocompatibility and biodegradability, elevated biological activity with nontoxicity could make it an excellent choice for application as a matrix for immobilizing biomolecules, enzymes, various cells, nanoparticles, etc. on microfluidics-based chips.^263,264 Wang et al.²⁶⁵ electrodeposited chitosan films with various thicknesses in a freestanding microfluidic channel made by dry SU-8 sheet as a polymer with high chemical stability, to enhance its biological activity through capillary effects for the immobilization of enzymes. Oxygen plasma treatment was used to change the hydrophobic characteristics of the SU-8 surface to hydrophilic and facilitate the adhesion and penetration of chitosan solution to the channel, which is applied to create a compact portable biosensor. In terms of diagnosis and in-depth cellular assessment, carboxyl betaine acrylamide functionalized electrospun chitosan nanofiber was coated with HA for utilization in a microfluidic chip for the uptake and discharge of circulating tumor cells by Wang et al.²⁶⁵ Their prepared platform exhibited high capability (91%) in uptake of A549, a human lung cancer cell line. Since circulating tumor cells were found in patients with lung and breast cancer, their investigation could open the door to a better diagnosis of cancer. While 3D-bioprinting for organ-on-a-chip technology has been performed for various bio-ink compositions in different areas (see Table 5), according to the best knowledge of the authors, chitosan-based structures have not yet been implemented in this technique. Hence, it is a new and challenging topic for bioengineering scientists to use and survey the possible future applications of 3D-bioprinted chitosan-based bio-inks for organ-on-a-chip approaches.

Table 5 Various bio-inks applied in 3D-bioprinting for lab-on-a-chip

Microfluidic chip substrate	Bio-ink composition	Bioprinting method	Application and description	Ref.
Polydimethylsiloxane (PDMS)	Cell suspended in biopolymer media	Extrusion-based bioprinting with multi-nozzle tips	Cancer therapy; investigating drug metabolism on MDA-MB-231 cells (human breast adenocarcinoma)	266
Poly-methyl methacrylate (PMMA)	Mixture of gelatin and fibrinogen	Extrusion-based	Albumin adsorption analyzing, and cyclosporine A testing on a prepared kidney tissue model	267
PDMS	Cells suspended in medium	Extrusion-based bioprinting with four-nozzle tips	Study of cancer cells in a co-cultured microfluidic media	249
Glass and PDMS	Alginate	Extrusion-based	Study of in vivo human response to drug administrations	181
PDMS	Cell suspended in biopolymer medium	Droplet-based	Investigation of anti-cancer drug responses of cancer cells	268
PMMA and PDMS	Mixture of gelatin and GelMA	Extrusion-based	In vitro drug analysis using a liver-on-a-chip platform	269
Polycaprolactone (PCL)	Tracheal mucosa extracellular matrix	Direct 3D-bioprinting	Preparation of in vitro airway coupled with a naturally-derived blood vessel	270
—	Collagen (type I)	Extrusion-based (with two coaxial nozzles)	Preparation of a 3D vascular network	271
Stereolithography resin	GelMA	Inkjet-based	Study of cancer cells in a co-cultured microfluidic platform	272
Glass	Gelatin fibrinogen	Extrusion-based (with four nozzles)	Preparation of thick vascularized tissues through the printing of bone marrow and neonatal dermal fibroblasts stem cells	273

---

4D-bioprinting, as an updated version of 3D-bioprinting, imparts dynamic behavior to the bioprinted scaffolds under external stimuli like water, light, temperature, pH, biological activity, or magnetic and electric fields, by adding time as the fourth dimension.²⁷⁴ 4D bioprinting has already been applied in many biomedicine fields like biosensors, bioactuators and biorobotics, and tissue engineering, and so far, many attempts have been made at 4D bioprinting of bio-inks based on various polymeric complexes.^275,276 Of course, considering the possible effects of a dynamic reshaping of 4D scaffolds on cell viability and proliferation, the combination of smart cell-laden bio-inks composed of natural polymers like chitosan with nanoparticles could result in the preparation of complex dynamic tissue constructs as a future perspective in medical applications of 4D bioprinting.²⁷⁷ In this regard, drug delivery, tissue engineering, cancer therapy, and wastewater purification using 4D printed grafts could be considered as a new application perspective of chitosan-based biomaterials. Moreover, there is enthusiasm and a need for the synthesis of rapidly self-healing scaffolds for a wide range of requests in biomedicine due to their extreme mechanical/physical stabilities and their long lifetime after induction of structural wounds.⁸⁸ The characteristic of self-healing constructs is that they sense external alterations and assimilate to them by changing their features and how they work. It is a proven fact that this wonderful characteristic of a scaffold enormously depends on the mechanical robustness of the precursors.²⁷⁸ According to previous reports, prepared chitosan-based self-healing constructs could not completely fulfill the mechanical strength requirements for the biomedical engineering industries. However, many different mechanisms and procedures, such as adding magnetic and conductive nanoparticles to their composition, have been applied to increase the self-healing and mechanical properties.^279–282 Lately, rapid, room temperature conductive chitosan-based frameworks have been prepared for biomedical engineering fields.²²⁸

9. Commercialization

Fortunately, the commercialization of chitosan polymers with a broad range of deacetylation degrees and molecular weights paves the way for a wide range of requirements, including regenerative medicine and biomedical engineering. Although the application of chitosan for wound healing and dietary demands has been approved by the FDA and some European regulatory agencies, it has not succeeded yet in gaining FDA approval for cancer therapy, gene delivery, and drug delivery uses.^283,284 Unique features of chitosan, including nontoxicity, affordability (abundancy and cost-efficient factors), excellent bioactivity and mechanical properties drives researchers to keep using of chitosan in cancer research and delivery systems. Accordingly, many published papers and patented chitosan-based materials have been reported.¹⁹⁰ The introduction of an injectable pre-gelled paste, called BST-Gel®, based on a chitosan/mineral composite, for cartilage tissue repair is a case in point.²⁸⁵ Reaxon®, SlimMED™, and ChitoDot® are other examples of commercial chitosan-based patented products that are applicable for different functionalities from peripheral nerve repair to treatment of overweight and antibacterial hemostatic dressings to stop bleeding, respectively.^286–288 Lately, a new methacrylate chitosan-based ink was invented to prepare a biodegradable tissue 3D construct utilized as a quick healing agent for small to large bone fractures.^289,292 So far, many patented chitosan-based inks/bio-inks for different functionalities have been manufactured all around the world, giving new hope for the fast-burgeoning commercialized chitosan-based 3D scaffolds for biomedical applications.^290,291

10. Conclusions

Conventional 3D scaffolds such as gels and fibrous matrices applied in tissue regeneration have failed to recapitulate native tissues with high exactness mostly due to suffering from a non-uniform distribution of structural pores, which restricts the ease of oxygen penetration, exchange of food and wastes. On the other hand, 3D bioprinting as a low-cost technique can offer facile preparation of sophisticated human organs and tissues holding vascular systems, neural networks, or osteochondral tissue with high resolution, anatomical similarities, and functional dynamics identical to the real tissues. Among all suitable materials for bio-inks (e.g., combinations of various synthetic and biopolymers along with a wide array of microcarriers and nanoparticles), chitosan-based bio-inks as biodegradable and affordable inks with an elevated degree of printability, cytocompatibility, biocompatibility, cell attachment/proliferation/migration, antimicrobial activity, and mechanical stability have been captured a lot of attentions. Chitosan-based bio-inks have been applied to prepare high-resolution 3D patterns with tailorable pore volume size, interconnected lamellae, and the capability to support the lineage commitment of encapsulated cells for utilization in bone, cartilage, vascular systems, neural networks, and skin tissue regeneration. Despite the fact that many challenges still remain in the use of chitosan for creating more complex 3D constructs, mainly due to chitosan's nature and the restrictions in currently existing bioprinting techniques. A plethora of attempts have been made to improve the printability of chitosan-based bio-inks, and, finally, to gain FDA approval for the utilization of these 3D-printed scaffolds in tissue engineering and drug delivery demands. Furthermore, chitosan-based 3D-printed structures exhibited extreme potential to take up high amounts of hazardous contamination such as organic pollutants and heavy metal ions from wastewater pathways and for the adsorption/desorption of hydrophilic/hydrophobic drugs by different mechanisms for environmental purification and drug delivery demands as well. Finally, in this study, the possible open door for 3D-bioprinting of chitosan for organ-on-a-chip systems in order to recapitulate complex organs containing sophisticated vascular or neural networks with high fidelity is pointed out.

Statement of contributions

A.T., M.T., and M. K. Y. equally contributed to the writing of the original manuscript. M.R.S., F.J.S., P.Z., S.H.R., J.D.R. and G.N. contributed to the search and also drafting the manuscript. M.R.S., M.M. and U.S.S. supervised the project, edited the manuscript, and approved the final version.

Conflicts of interest

The authors declare that they have no competing interests.

References

1. I. Matai, G. Kaur, A. Seyedsalehi, A. McClinton and C. T. Laurencin, Progress in 3D bioprinting technology for tissue/organ regenerative engineering, Biomaterials, 2020, 226, 119536.
2. B.-J. de Gans, P. C. Duineveld and U. S. Schubert, Inkjet Printing of Polymers: State of the Art and Future Developments, Adv. Mater., 2004, 16(3), 203–213,  DOI:10.1002/adma.200300385.
3. E. Tekin, P. J. Smith and U. S. Schubert, Inkjet printing as a deposition and patterning tool for polymers and inorganic particles, Soft Matter, 2008, 4(4), 703–713,  10.1039/B711984D.
4. S. Das and B. Basu, An Overview of Hydrogel-Based Bioinks for 3D Bioprinting of Soft Tissues, J. Indian Inst. Sci., 2019, 1–24.
5. N. Ahmad, P. Gopinath and R. Dutta, 3D printing technology in nanomedicine, Elsevier, 2019.
6. Y. S. Zhang, et al., Bioprinting 3D microfibrous scaffolds for engineering endothelialized myocardium and heart-on-a-chip, Biomaterials, 2016, 110, 45–59.
7. S. Knowlton, B. Yenilmez, S. Anand and S. Tasoglu, Photocrosslinking-based bioprinting: examining crosslinking schemes, Bioprinting, 2017, 5, 10–18.
8. L. Valot, J. Martinez, A. Mehdi and G. Subra, Chemical insights into bioinks for 3D printing, Chem. Soc. Rev., 2019, 48(15), 4049–4086.
9. R. Seyedmahmoud, et al., Three-Dimensional Bioprinting of Functional Skeletal Muscle Tissue Using Gelatin Methacryloyl-Alginate Bioinks, Micromachines, 2019, 10(10), 679.
10. M. Hospodiuk, M. Dey, D. Sosnoski and I. T. Ozbolat, The bioink: a comprehensive review on bioprintable materials, Biotechnol. Adv., 2017, 35(2), 217–239.
11. S. V. Murphy, A. Skardal and A. Atala, Evaluation of hydrogels for bio–printing applications, J. Biomed. Mater. Res., Part A, 2013, 101(1), 272–284.
12. K. Karzyński, et al., Use of 3D bioprinting in biomedical engineering for clinical application, Med. Stud./Stud. Med., 2018, 34(1), 93–97.
13. T. J. Kean and M. Thanou, Utility of chitosan for 3D Printing and Bioprinting, in Sustainable Agriculture Reviews 35, Springer, 2019, pp. 271–292.
14. B. Duan, State-of-the-art review of 3D bioprinting for cardiovascular tissue engineering, Ann. Biomed. Eng., 2017, 45(1), 195–209.
15. O. R. Young, F. Berkhout, G. C. Gallopin, M. A. Janssen, E. Ostrom and S. Van der Leeuw, The globalization of socio-ecological systems: an agenda for scientific research, Global Environ. Change, 2006, 16(3), 304–316.
16. R. F. Pereira and P. J. Bártolo, 3D bioprinting of photocrosslinkable hydrogel constructs, J. Appl. Polym. Sci., 2015, 132(48), 42458.
17. X. Wang, et al., 3D bioprinting technologies for hard tissue and organ engineering, Materials, 2016, 9(10), 802.
18. M. Shabnam, et al., Chitosan in Biomedical Engineering: A Critical Review, Curr. Stem Cell Res. Ther., 2019, 14(2), 93–116,  DOI:10.2174/1574888X13666180912142028.
19. S. Manouchehri, et al., Electroactive bio-epoxy incorporated chitosan-oligoaniline as an advanced hydrogel coating for neural interfaces, Prog. Org. Coat., 2019, 131, 389–396.
20. B. Bagheri, et al., Self-gelling electroactive hydrogels based on chitosan–aniline oligomers/agarose for neural tissue engineering with on-demand drug release, Colloids Surf., B, 2019, 184, 110549.
21. M. A. Salati, et al., Agarose-Based Biomaterials: Opportunities and Challenges in Cartilage Tissue Engineering, Polymers, 2020, 12(5), 1150.
22. M. K. Yazdi, et al., Agarose-based biomaterials for advanced drug delivery, J. Controlled Release, 2020, 523–543.
23. P. Zarrintaj, et al., Agarose-based biomaterials for tissue engineering, Carbohydr. Polym., 2018, 187, 66–84.
24. Y. Sun, Y. You, W. Jiang, Q. Wu, B. Wang and K. Dai, Generating ready-to-implant anisotropic menisci by 3D-bioprinting protein-releasing cell-laden hydrogel-polymer composite scaffold, Appl. Mater. Today, 2020, 18, 100469.
25. M. Sahranavard, A. Zamanian, F. Ghorbani and M. H. Shahrezaee, A critical review on three dimensional-printed chitosan hydrogels for development of tissue engineering, Bioprinting, 2020, 17, e00063.
26. B. Bagheri, et al., Tissue engineering with electrospun electro-responsive chitosan-aniline oligomer/polyvinyl alcohol, Int. J. Biol. Macromol., 2020, 147, 160–169.
27. G. Mahmodi, et al., From microporous to mesoporous mineral frameworks: An alliance between zeolite and chitosan, Carbohydr. Res., 2020, 489, 107930.
28. S. Tarassoli, Z. Jessop, S. Kyle and I. Whitaker, Candidate bioinks for 3D bioprinting soft tissue, in 3D Bioprinting for Reconstructive Surgery, Elsevier, 2018, pp. 145–172.
29. C. Liu, et al., Bioprinted chitosan and hydroxyapatite micro-channels structures scaffold for vascularization of bone regeneration, J. Biomater. Tissue Eng., 2017, 7(1), 28–34.
30. T. T. Demirtaş, G. Irmak and M. Gümüşderelioğlu, A bioprintable form of chitosan hydrogel for bone tissue engineering, Biofabrication, 2017, 9(3), 035003.
31. V. B. Morris, S. Nimbalkar, M. Younesi, P. McClellan and O. Akkus, Mechanical properties, cytocompatibility and manufacturability of chitosan: PEGDA hybrid-gel scaffolds by stereolithography, Ann. Biomed. Eng., 2017, 45(1), 286–296.
32. C. R. Almeida, T. Serra, M. I. Oliveira, J. A. Planell, M. A. Barbosa and M. Navarro, Impact of 3-D printed PLA-and chitosan-based scaffolds on human monocyte/macrophage responses: unraveling the effect of 3-D structures on inflammation, Acta Biomater., 2014, 10(2), 613–622.
33. T. Akopova, et al., Solid state synthesis of chitosan and its unsaturated derivatives for laser microfabrication of 3D scaffolds, in IOP Conf. Ser. Mater. Sci. Eng., 2015, vol. 87, p. 012079.
34. Q. Wu, M. Maire, S. Lerouge, D. Therriault and M. C. Heuzey, 3D printing of microstructured and stretchable chitosan hydrogel for guided cell growth, Adv. Biosyst., 2017, 1(6), 1700058.
35. R. Gauvin, et al., Microfabrication of complex porous tissue engineering scaffolds using 3D projection stereolithography, Biomaterials, 2012, 33(15), 3824–3834.
36. D. Chimene, K. K. Lennox, R. R. Kaunas and A. K. Gaharwar, Advanced bioinks for 3D printing: a materials science perspective, Ann. Biomed. Eng., 2016, 44(6), 2090–2102.
37. P. Koti, N. Muselimyan, E. Mirdamadi, H. Asfour and N. A. Sarvazyan, Use of GelMA for 3D printing of cardiac myocytes and fibroblasts, J. 3D Print. Med., 2019, 3(1), 11–22.
38. M. Guvendiren, J. Molde, R. M. Soares and J. Kohn, Designing biomaterials for 3D printing, ACS Biomater. Sci. Eng., 2016, 2(10), 1679–1693.
39. E. Axpe and M. L. Oyen, Applications of alginate-based bioinks in 3D bioprinting, Int. J. Mol. Sci., 2016, 17(12), 1976.
40. A. Tabriz, M. Hermida, N. Leslie and W. Shu, Three-dimensional bioprinting of complex cell laden alginate hydrogel structures, Biofabrication, 2015, 7, 45012.
41. S. Khalil and W. Sun, Bioprinting endothelial cells with alginate for 3D tissue constructs, J. Biomech. Eng., 2009, 131(11), 111002.
42. S. Das, et al., Bioprintable, cell-laden silk fibroin–gelatin hydrogel supporting multilineage differentiation of stem cells for fabrication of three-dimensional tissue constructs, Acta Biomater., 2015, 11, 233–246.
43. S. Chawla, A. Kumar, P. Admane, A. Bandyopadhyay and S. Ghosh, Elucidating role of silk-gelatin bioink to recapitulate articular cartilage differentiation in 3D bioprinted constructs, Bioprinting, 2017, 7, 1–13.
44. D. F. Duarte Campos, et al., The stiffness and structure of three-dimensional printed hydrogels direct the differentiation of mesenchymal stromal cells toward adipogenic and osteogenic lineages, Tissue Eng., Part A, 2015, 21(3–4), 740–756.
45. Z. Zheng, et al., 3D bioprinting of self–standing silk–based bioink, Adv. Healthcare Mater., 2018, 7(6), 1701026.
46. J. B. Costa, J. Silva-Correia, J. M. Oliveira and R. L. Reis, Fast setting silk fibroin bioink for bioprinting of patient–specific memory–shape implants, Adv. Healthcare Mater., 2017, 6(22), 1701021.
47. C. M. Smith, et al., Three-dimensional bioassembly tool for generating viable tissue-engineered constructs, Tissue Eng., 2004, 10(9–10), 1566–1576.
48. M. Müller, J. Becher, M. Schnabelrauch and M. Zenobi-Wong, Nanostructured Pluronic hydrogels as bioinks for 3D bioprinting, Biofabrication, 2015, 7(3), 035006.
49. A. P. Haring, et al., Process-and bio-inspired hydrogels for 3D bioprinting of soft free-standing neural and glial tissues, Biofabrication, 2019, 11(2), 025009.
50. Y. Zhao, et al., Three-dimensional printing of Hela cells for cervical tumor model in vitro, Biofabrication, 2014, 6(3), 035001.
51. C. Norotte, F. S. Marga, L. E. Niklason and G. Forgacs, Scaffold-free vascular tissue engineering using bioprinting, Biomaterials, 2009, 30(30), 5910–5917.
52. T. Xu, C. A. Gregory, P. Molnar, X. Cui, S. Jalota, S. B. Bhaduri and T. Boland, Viability and electrophysiology of neural cell structures generated by the inkjet printing method, Biomaterials, 2006a, 27, 3580–3588.
53. T. A. Ahmed, E. V. Dare and M. Hincke, Fibrin: a versatile scaffold for tissue engineering applications, Tissue Eng., Part B, 2008, 14(2), 199–215.
54. A. Skardal, J. Zhang and G. D. Prestwich, Bioprinting vessel-like constructs using hyaluronan hydrogels crosslinked with tetrahedral polyethylene glycol tetracrylates, Biomaterials, 2010, 31(24), 6173–6181.
55. L. Pescosolido, et al., Hyaluronic acid and dextran-based semi-IPN hydrogels as biomaterials for bioprinting, Biomacromolecules, 2011, 12(5), 1831–1838.
56. L. Hockaday, et al., Rapid 3D printing of anatomically accurate and mechanically heterogeneous aortic valve hydrogel scaffolds, Biofabrication, 2012, 4(3), 035005.
57. X. Cui, K. Breitenkamp, M. Finn, M. Lotz and D. D. D'Lima, Direct human cartilage repair using three-dimensional bioprinting technology, Tissue Eng., Part A, 2012, 18(11–12), 1304–1312.
58. I. T. Ozbolat, Scaffold-based or scaffold-free bioprinting: competing or complementing approaches?, J. Nanotechnol. Eng. Med., 2015, 6(2), 024701.
59. H. Su, C.-A. H. Price, L. Jing, Q. Tian, J. Liu and K. Qian, Janus particles: design, preparation, and biomedical applications, Mater. Today Bio, 2019, 4, 100033.
60. Q. Wu, D. Therriault and M.-C. Heuzey, Processing and properties of chitosan inks for 3D printing of hydrogel microstructures, ACS Biomater. Sci. Eng., 2018, 4(7), 2643–2652.
61. C. B. Highley, 3D Bioprinting Technologies, in 3D Bioprinting in Medicine, Springer, 2019, pp. 1–66.
62. D. Tan, A. Nokhodchi and M. Maniruzzaman, 3d and 4d printing technologies: innovative process engineering and smart additive manufacturing, in 3D and 4D Printing in Biomedical Applications: Process Engineering and Additive Manufacturing, 2019, pp. 25–52.
63. J. Jang, J. Y. Park, G. Gao and D.-W. Cho, Biomaterials-based 3D cell printing for next-generation therapeutics and diagnostics, Biomaterials, 2018, 156, 88–106.
64. N. Ashammakhi, et al., Advancing frontiers in bone bioprinting, Adv. Healthcare Mater., 2019, 8(7), 1801048.
65. G. Gao and X. Cui, Three-dimensional bioprinting in tissue engineering and regenerative medicine, Biotechnol. Lett., 2016, 38(2), 203–211.
66. X. Cui, D. Dean, Z. M. Ruggeri and T. Boland, Cell damage evaluation of thermal inkjet printed Chinese hamster ovary cells, Biotechnol. Bioeng., 2010, 106(6), 963–969.
67. Z. Galliger, C. D. Vogt and A. Panoskaltsis-Mortari, 3D bioprinting for lungs and hollow organs, Transl. Res., 2019, 211, 19–34.
68. L. Ouyang, Study on Microextrusion-based 3D Bioprinting and Bioink Crosslinking Mechanisms, Springer, 2019.
69. H.-G. Yi, H. Lee and D.-W. Cho, 3D printing of organs-on-chips, Bioengineering, 2017, 4(1), 10.
70. S. V. Murphy and A. Atala, 3D bioprinting of tissues and organs, Nat. Biotechnol., 2014, 32(8), 773–785.
71. Y.-J. Choi, H.-G. Yi, S.-W. Kim and D.-W. Cho, 3D cell printed tissue analogues: a new platform for theranostics, Theranostics, 2017, 7(12), 3118.
72. H. Cui, M. Nowicki, J. P. Fisher and L. G. Zhang, 3D bioprinting for organ regeneration, Adv. Healthcare Mater., 2017, 6(1), 1601118.
73. N. Bhattacharjee, A. Urrios, S. Kang and A. Folch, The upcoming 3D-printing revolution in microfluidics, Lab Chip, 2016, 16(10), 1720–1742.
74. G. Cidonio, et al., Osteogenic and angiogenic tissue formation in high fidelity nanocomposite Laponite-gelatin bioinks, Biofabrication, 2019, 11(3), 035027.
75. Y. P. Singh, A. Bandyopadhyay and B. B. Mandal, 3D Bioprinting Using Cross-Linker-Free Silk–Gelatin Bioink for Cartilage Tissue Engineering, ACS Appl. Mater. Interfaces, 2019, 11(37), 33684–33696.
76. A. Mazzocchi, M. Devarasetty, R. Huntwork, S. Soker and A. Skardal, Optimization of collagen type I-hyaluronan hybrid bioink for 3D bioprinted liver microenvironments, Biofabrication, 2018, 11(1), 015003.
77. O. Jeon, Y. B. Lee, T. J. Hinton, A. W. Feinberg and E. Alsberg, Cryopreserved cell-laden alginate microgel bioink for 3D bioprinting of living tissues, Mater. Today Chem., 2019, 12, 61–70.
78. J. Yin, M. Yan, Y. Wang, J. Fu and H. Suo, 3D bioprinting of low-concentration cell-laden gelatin methacrylate (GelMA) bioinks with a two-step cross-linking strategy, ACS Appl. Mater. Interfaces, 2018, 10(8), 6849–6857.
79. P. Apelgren, et al., In vivo human cartilage formation in three-dimensional bioprinted constructs with a novel bacterial nanocellulose bioink, ACS Biomater. Sci. Eng., 2019, 5(5), 2482–2490.
80. E. Abelardo, Synthetic material bioinks, in 3D Bioprinting for Reconstructive Surgery, Elsevier, 2018, pp. 137–144.
81. Z. Wu, X. Su, Y. Xu, B. Kong, W. Sun and S. Mi, Bioprinting three-dimensional cell-laden tissue constructs with controllable degradation, Sci. Rep., 2016, 6(1), 1–10.
82. F. P. Melchels, et al., Hydrogel-based reinforcement of 3D bioprinted constructs, Biofabrication, 2016, 8(3), 035004.
83. W. Jia, et al., Direct 3D bioprinting of perfusable vascular constructs using a blend bioink, Biomaterials, 2016, 106, 58–68.
84. Z. Shariatinia and A. Mazloom-Jalali, Chitosan nanocomposite drug delivery systems designed for the ifosfamide anticancer drug using molecular dynamics simulations, J. Mol. Liq., 2019, 273, 346–367.
85. B. dos Santos Rodrigues, S. Lakkadwala, D. Sharma and J. Singh, Chitosan for gene, DNA vaccines, and drug delivery, in Materials for Biomedical Engineering, Elsevier, 2019, pp. 515–550.
86. H. Guan, D. Gong, Y. Song, B. Han and N. Zhang, Biosensor composed of integrated glucose oxidase with liposome microreactors/chitosan nanocomposite for amperometric glucose sensing, Colloids Surf., A, 2019, 574, 260–267.
87. A. Sadeghi, F. Moztarzadeh and J. A. Mohandesi, Investigating the effect of chitosan on hydrophilicity and bioactivity of conductive electrospun composite scaffold for neural tissue engineering, Int. J. Biol. Macromol., 2019, 121, 625–632.
88. X. Zhang, et al., Dual-responsive nanoparticles based on chitosan for enhanced breast cancer therapy, Carbohydr. Polym., 2019, 221, 84–93.
89. S. Niu, et al., A novel chitosan-based nanomedicine for multi-drug resistant breast cancer therapy, Chem. Eng. J., 2019, 369, 134–149.
90. M. Ul-Islam, et al., Development of three-dimensional bacterial cellulose/chitosan scaffolds: Analysis of cell-scaffold interaction for potential application in the diagnosis of ovarian cancer, Int. J. Biol. Macromol., 2019, 137, 1050–1059.
91. V. Medawar-Aguilar, et al., Serological diagnosis of Toxoplasmosis disease using a fluorescent immunosensor with chitosan-ZnO-nanoparticles, Anal. Biochem., 2019, 564, 116–122.
92. J. Groll, et al., Biofabrication: reappraising the definition of an evolving field, Biofabrication, 2016, 8(1), 013001.
93. S. Derakhshanfar, R. Mbeleck, K. Xu, X. Zhang, W. Zhong and M. Xing, 3D bioprinting for biomedical devices and tissue engineering: A review of recent trends and advances, Bioact. Mater., 2018, 3(2), 144–156.
94. A. Mazzocchi, S. Soker and A. Skardal, 3D bioprinting for high-throughput screening: Drug screening, disease modeling, and precision medicine applications, Appl. Phys. Rev., 2019, 6(1), 011302.
95. J. Gopinathan and I. Noh, Recent trends in bioinks for 3D printing, Biomater. Res., 2018, 22(1), 11.
96. R. Goy, D. de Britto and O. Assis, A review of the antimicrobial activity of chitosan, PolÌmer CiÍnc, ed: Tecnol, 2009.
97. G. Cruz, et al., Antimicrobial activity of chitosan composites against bacterial strains isolated from goat meat and cheese, J. Pharm. Health Care Sci., 2019, 1173(1), 012005.
98. J. Huang, et al., BMSCs-laden gelatin/sodium alginate/carboxymethyl chitosan hydrogel for 3D bioprinting, RSC Adv., 2016, 6(110), 108423–108430.
99. D. Chimene, R. Kaunas and A. K. Gaharwar, Hydrogel bioink reinforcement for additive manufacturing: a focused review of emerging strategies, Adv. Mater., 2020, 32(1), 1902026.
100. Y. Wang, J. Qian, N. Zhao, T. Liu, W. Xu and A. Suo, Novel hydroxyethyl chitosan/cellulose scaffolds with bubble-like porous structure for bone tissue engineering, Carbohydr. Polym., 2017, 167, 44–51.
101. S. Bose, S. Vahabzadeh and A. Bandyopadhyay, Bone tissue engineering using 3D printing, Mater. Today, 2013, 16(12), 496–504.
102. M. A. Aronow, L. C. Gerstenfeld, T. A. Owen, M. S. Tassinari, G. S. Stein and J. B. Lian, Factors that promote progressive development of the osteoblast phenotype in cultured fetal rat calvaria cells, J. Cell. Physiol., 1990, 143(2), 213–221.
103. Y. Huang, S. Onyeri, M. Siewe and A. Moshfeghian, In vitro characterization of chitosan–gelatin scaffolds for tissue engineering, Biomaterials, 2005, 7616–7627.
104. S. Muench, et al., Printable ionic liquid-based gel polymer electrolytes for solid state all-organic batteries, Energy Storage Mater., 2020, 25, 750–755.
105. W. Thein-Han, J. Saikhun, C. Pholpramoo, R. Misra and Y. Kitiyanant, Chitosan–gelatin scaffolds for tissue engineering: Physico-chemical properties and biological response of buffalo embryonic stem cells and transfectant of GFP–buffalo embryonic stem cells, Acta Biomater., 2009, 5(9), 3453–3466.
106. J. Wang, et al., Surface entrapment of chitosan on 3D printed polylactic acid scaffold and its biomimetic growth of hydroxyapatite, Compos. Interfaces, 2019, 26(5), 465–478.
107. A. Blaeser, D. F. Duarte Campos, U. Puster, W. Richtering, M. M. Stevens and H. Fischer, Controlling shear stress in 3D bioprinting is a key factor to balance printing resolution and stem cell integrity, Adv. Healthcare Mater., 2016, 5(3), 326–333.
108. T. Billiet, B. V. Gasse, E. Gevaert, M. Cornelissen, J. C. Martins and P. Dubruel, Quantitative Contrasts in the Photopolymerization of Acrylamide and Methacrylamide–Functionalized Gelatin Hydrogel Building Blocks, Macromol. Biosci., 2013, 13(11), 1531–1545.
109. P. Chavanne, et al., 3D printed chitosan/hydroxyapatite scaffolds for potential use in regenerative medicine, Biomed. Eng./Biomed. Tech., 2013, 58(SI-1-Track-C), 4069.
110. L. T. Saldin, M. C. Cramer, S. S. Velankar, L. J. White and S. F. Badylak, Extracellular matrix hydrogels from decellularized tissues: structure and function, Acta Biomater., 2017, 49, 1–15.
111. S. Yuan, D. Strobbe, J.-P. Kruth, P. Van Puyvelde and B. Van der Bruggen, Super-hydrophobic 3D printed polysulfone membranes with a switchable wettability by self-assembled candle soot for efficient gravity-driven oil/water separation, J. Mater. Chem. A, 2017, 5(48), 25401–25409.
112. H. Montazerian, et al., Permeability and mechanical properties of gradient porous PDMS scaffolds fabricated by 3D-printed sacrificial templates designed with minimal surfaces, Acta Biomater., 2019, 96, 149–160.
113. S. Kyle, Z. M. Jessop, A. Al-Sabah and I. S. Whitaker, Biofabrication: ‘Printability’ of Candidate Biomaterials for Extrusion Based 3D Printing: State-of-the-Art (Adv. Healthcare Mater. 16/2017), Adv. Healthcare Mater., 2017, 6(16), 1700264.
114. E. DeSimone, K. Schacht, T. Jungst, J. Groll and T. Scheibel, Biofabrication of 3D constructs: Fabrication technologies and spider silk proteins as bioinks, Pure Appl. Chem., 2015, 87(8), 737–749.
115. S. Chawla, S. Midha, A. Sharma and S. Ghosh, Silk–based bioinks for 3D bioprinting, Adv. Healthcare Mater., 2018, 7(8), 1701204.
116. S. Van Belleghem, et al., Hybrid 3D Printing of Synthetic and Cell–Laden Bioinks for Shape Retaining Soft Tissue Grafts, Adv. Funct. Mater., 2020, 30(3), 1907145.
117. K. D. Roehm and S. V. Madihally, Bioprinted chitosan-gelatin thermosensitive hydrogels using an inexpensive 3D printer, Biofabrication, 2017, 10(1), 015002.
118. J. E. Trachtenberg, P. M. Mountziaris, J. S. Miller, M. Wettergreen, F. K. Kasper and A. G. Mikos, Open–source three–dimensional printing of biodegradable polymer scaffolds for tissue engineering, J. Biomed. Mater. Res., Part A, 2014, 102(12), 4326–4335.
119. G. D. Nicodemus and S. J. Bryant, Cell encapsulation in biodegradable hydrogels for tissue engineering applications, Tissue Eng., Part B, 2008, 14(2), 149–165.
120. M. Rinaudo, Chitin and chitosan: properties and applications, Prog. Polym. Sci., 2006, 31(7), 603–632.
121. A. Lončarević, M. Ivanković and A. Rogina, Lysozyme-induced degradation of chitosan: the characterisation of degraded chitosan scaffolds, J. Tissue Repair Regener., 2017, 1(1), 12.
122. D. Lee, et al., Chitosan-catechol: a writable bioink under serum culture media, Biomater. Sci., 2018, 6(5), 1040–1047.
123. C. Li, et al., Controllable fabrication of hydroxybutyl chitosan/oxidized chondroitin sulfate hydrogels by 3D bioprinting technique for cartilage tissue engineering, Biomed. Mater., 2019, 14(2), 025006.
124. F. Hafezi, N. Scoutaris, D. Douroumis and J. Boateng, 3D printed chitosan dressing crosslinked with genipin for potential healing of chronic wounds, Int. J. Pharm., 2019, 560, 406–415.
125. P. Shi, W. Y. Yeong, A. Laude and E. Y. S. Tan, Hybrid three-dimensional (3D) bioprinting of retina equivalent for ocular research, Int. J. Bioprint., 2017, 138–146.
126. C. Intini, et al., 3D-printed chitosan-based scaffolds: An in vitro study of human skin cell growth and an in vivo wound healing evaluation in experimental diabetes in rats, Carbohydr. Polym., 2018, 199, 593–602.
127. M. Khanmohammadi, S. Sakai and M. Taya, Fabrication of single and bundled filament-like tissues using biodegradable hyaluronic acid-based hollow hydrogel fibers, Int. J. Biol. Macromol., 2017, 104, 204–212.
128. A. Parak, P. Pradeep, L. C. du Toit, P. Kumar, Y. E. Choonara and V. Pillay, Functionalizing bioinks for 3D bioprinting applications, Drug Discovery Today, 2019, 24(1), 198–205.
129. M. Yadav, P. Goswami, K. Paritosh, M. Kumar, N. Pareek and V. Vivekanand, Seafood waste: a source for preparation of commercially employable chitin/chitosan materials, Bioresour. Bioprocess., 2019, 6(1), 8.
130. Y.-L. Cheng and F. Chen, Preparation and characterization of photocured poly (ε-caprolactone) diacrylate/poly (ethylene glycol) diacrylate/chitosan for photopolymerization-type 3D printing tissue engineering scaffold application, Mater. Sci. Eng., C, 2017, 81, 66–73.
131. W. L. Ng, W. Y. Yeong and M. W. Naing, Polyelectrolyte gelatin-chitosan hydrogel optimized for 3D bioprinting in skin tissue engineering, Int. J. Bioprint, 2016, 2(1), 53–62.
132. Y. Sun, et al., 3D printing collagen/chitosan scaffold ameliorated axon regeneration and neurological recovery after spinal cord injury, J. Biomed. Mater. Res., Part A, 2019, 107(9), 1898–1908.
133. S. Li, Z. Xiong, X. Wang, Y. Yan, H. Liu and R. Zhang, Direct fabrication of a hybrid cell/hydrogel construct by a double-nozzle assembling technology, J. Bioact. Compat. Polym., 2009, 24(3), 249–265.
134. L. Elviri, et al., Highly defined 3D printed chitosan scaffolds featuring improved cell growth, Biomed. Mater., 2017, 12(4), 045009.
135. W. L. Ng, W. Y. Yeong and M. W. Naing, Development of polyelectrolyte chitosan-gelatin hydrogels for skin bioprinting, Procedia CIRP, 2016, 49, 105–112.
136. N. M. Ergul, et al., 3D printing of chitosan/poly (vinyl alcohol) hydrogel containing synthesized hydroxyapatite scaffolds for hard-tissue engineering, Polym. Test., 2019, 79, 106006.
137. F. Fu, et al., Magnetic resonance imaging-three-dimensional printing technology fabricates customized scaffolds for brain tissue engineering, Neural Regener. Res., 2017, 12(4), 614.
138. X. Hu, et al., 3D Bio-printing of CS/Gel/HA/Gr hybrid osteochondral scaffolds, Polymers, 2019, 11(10), 1601.
139. E. Vorndran, U. Klammert, A. Ewald, J. E. Barralet and U. Gbureck, Simultaneous immobilization of bioactives during 3D powder printing of bioceramic drug–release matrices, Adv. Funct. Mater., 2010, 20(10), 1585–1591.
140. C. Colosi, et al., Rapid prototyping of chitosan-coated alginate scaffolds through the use of a 3D fiber deposition technique, J. Mater. Chem. B, 2014, 2(39), 6779–6791.
141. H. Li, Y. J. Tan, S. Liu and L. Li, Three-dimensional bioprinting of oppositely charged hydrogels with super strong interface bonding, ACS Appl. Mater. Interfaces, 2018, 10(13), 11164–11174.
142. H. Chen, Y. Liu, Z. Jiang, W. Chen, Y. Yu and Q. Hu, Cell–scaffold interaction within engineered tissue, Exp. Cell Res., 2014, 323(2), 346–351.
143. C.-S. Wu, Modulation, functionality, and cytocompatibility of three-dimensional printing materials made from chitosan-based polysaccharide composites, Mater. Sci. Eng., C, 2016, 69, 27–36.
144. X. Wang, C. Wei, B. Cao, L. Jiang, Y. Hou and J. Chang, Fabrication of multiple-layered hydrogel scaffolds with elaborate structure and good mechanical properties via 3D printing and ionic reinforcement, ACS Appl. Mater. Interfaces, 2018, 10(21), 18338–18350.
145. Y. Tsukamoto, T. Akagi, F. Shima and M. Akashi, Fabrication of orientation-controlled 3D tissues using a layer-by-layer technique and 3D printed a thermoresponsive gel frame, Tissue Eng., Part C, 2017, 23(6), 357–366.
146. K. Bardakova, et al., 3D printing biodegradable scaffolds with chitosan materials for tissue engineering, in IOP Conf Ser: Mater Sci Eng., 2018, vol. 347, p. 012009.
147. D. Fan, U. Staufer and A. Accardo, Engineered 3D Polymer and Hydrogel Microenvironments for Cell Culture Applications, Bioengineering, 2019, 6(4), 113.
148. K. Wang, F. M. Kievit, A. E. Erickson, J. R. Silber, R. G. Ellenbogen and M. Zhang, Culture on 3D chitosan–hyaluronic acid scaffolds enhances stem cell marker expression and drug resistance in human glioblastoma cancer stem cells, Adv. Healthcare Mater., 2016, 5(24), 3173–3181.
149. B. A. Lindborg, et al., A chitosan-hyaluronan-based hydrogel-hydrocolloid supports in vitro culture and differentiation of human mesenchymal stem/stromal cells, Tissue Eng., Part A, 2015, 21(11–12), 1952–1962.
150. A. R. Akkineni, Y. Luo, M. Schumacher, B. Nies, A. Lode and M. Gelinsky, 3D plotting of growth factor loaded calcium phosphate cement scaffolds, Acta Biomater., 2015, 27, 264–274.
151. S. Nganga, et al., Inkjet printing of Chitlac-nanosilver—a method to create functional coatings for non-metallic bone implants, Biofabrication, 2014, 6(4), 041001.
152. J. Jung, K. Kim, S. C. Choi and J. Oh, Microfluidics-assisted rapid generation of tubular cell-laden microgel inside glass capillaries, Biotechnol. Lett., 2014, 36(7), 1549–1554.
153. S. Chen, Y. Shi, Y. Luo and J. Ma, Layer-by-layer coated porous 3D printed hydroxyapatite composite scaffolds for controlled drug delivery, Colloids Surf., B, 2019, 179, 121–127.
154. W. E. Müller, et al., A new printable and durable N, O-carboxymethyl chitosan–Ca²⁺–polyphosphate complex with morphogenetic activity, J. Mater. Chem. B, 2015, 3(8), 1722–1730.
155. Q. Gu, et al., Functional 3D neural mini–tissues from printed gel–based bioink and human neural stem cells, Adv. Healthcare Mater., 2016, 5(12), 1429–1438.
156. Y. Wu, et al., Fabrication and evaluation of electrohydrodynamic jet 3D printed polycaprolactone/chitosan cell carriers using human embryonic stem cell-derived fibroblasts, J. Biomater. Appl., 2016, 31(2), 181–192.
157. M. Osorio, E. Martinez, T. Naranjo and C. Castro, Recent Advances in Polymer Nanomaterials for Drug Delivery of Adjuvants in Colorectal Cancer Treatment: A Scientific-Technological Analysis and Review, Molecules, 2020, 25(10), 2270.
158. K. Haberstroh, et al., Bone repair by cell–seeded 3D–bioplotted composite scaffolds made of collagen treated tricalciumphosphate or tricalciumphosphate–chitosan–collagen hydrogel or PLGA in ovine critical–sized calvarial defects, J. Biomed. Mater. Res., Part B, 2010, 93(2), 520–530.
159. P. Dadhich, et al., A simple approach for an eggshell-based 3D-printed osteoinductive multiphasic calcium phosphate scaffold, ACS Appl. Mater. Interfaces, 2016, 8(19), 11910–11924.
160. S. W. Kim, D. Y. Kim, H. H. Roh, H. S. Kim, J. W. Lee and K. Y. Lee, Three-dimensional bioprinting of cell-laden constructs using polysaccharide-based self-healing hydrogels, Biomacromolecules, 2019, 20(5), 1860–1866.
161. N. J. Vickers, Animal communication: when I'm calling you, will you answer too?, Curr. Biol., 2017, 27(14), R713–R715.
162. Y. Yang, et al., Dual-functional 3D-printed composite scaffold for inhibiting bacterial infection and promoting bone regeneration in infected bone defect models, Acta Biomater., 2018, 79, 265–275.
163. G. Crini, Historical review on chitin and chitosan biopolymers, Environ. Chem. Lett., 2019, 1–21.
164. M. Thanou, et al., Renewable resources for functional polymers and biomaterials: polysaccharides, proteins and polyesters, Royal Society of Chemistry, 2011.
165. C. Rouget, Des substances amylacées dans les tissus des animaux, spécialement des Articulés (chitine), Comp. Rend., 1859, 48, 792–795.
166. F. Croisier and C. Jérôme, Chitosan-based biomaterials for tissue engineering, Eur. Polym. J., 2013, 49(4), 780–792.
167. N. M. Mahmoodi, M. Oveisi, A. Taghizadeh and M. Taghizadeh, Synthesis of pearl necklace-like ZIF-8@chitosan/PVA nanofiber with synergistic effect for recycling aqueous dye removal, Carbohydr. Polym., 2020, 227 DOI:10.1016/j.carbpol.2019.115364.
168. G. Mahmodi, P. Zarrintaj, A. Taghizadeh, M. Taghizadeh, S. Manouchehri, S. Dangwal, A. Ronte, M. R. Ganjali, J. D. Ramsey and S.-J. Kim, From microporous to mesoporous mineral frameworks: An alliance between zeolite and chitosan, Carbohydr. Res., 2020, 489 DOI:10.1016/j.carres.2020.107930.
169. N. Morin-Crini, E. Lichtfouse, G. Torri and G. Crini, Fundamentals and applications of chitosan, in Sustainable Agriculture Reviews 35, Springer, 2019, pp. 49–123.
170. S. Ahmed and S. Ikram, Chitosan: derivatives, composites and applications, John Wiley & Sons, 2017.
171. J. C. Roy, F. Salaün, S. Giraud, A. Ferri, G. Chen and J. Guan, Solubility of chitin: solvents, solution behaviors and their related mechanisms, Solubility Polysaccharides, 2017, 10, 110–130.
172. A. Tirella, A. Orsini, G. Vozzi and A. Ahluwalia, A phase diagram for microfabrication of geometrically controlled hydrogel scaffolds, Biofabrication, 2009, 1(4), 045002.
173. F. Ganji and M. Abdekhodaie, A.-Ramazani SA J Sol—Gel, Sci. Technol., 2007, 42, 47–53.
174. R. R. Jose, M. J. Rodriguez, T. A. Dixon, F. Omenetto and D. L. Kaplan, Evolution of bioinks and additive manufacturing technologies for 3D bioprinting, ACS Biomater. Sci. Eng., 2016, 2(10), 1662–1678.
175. B. Lim, C. Poh, C. Voon and H. Salmah, Rheological and thermal study of chitosan filled thermoplastic elastomer composites, in Applied Mechanics and Materials, Trans Tech Publ, 2015, vol. 754, pp. 34–38.
176. A. Kohut, A. Voronov and W. Peukert, An effective way to stabilize colloidal particles dispersed in polar and nonpolar media, Langmuir, 2007, 23(2), 504–508.
177. Y. Zhang, Y. Yu and I. T. Ozbolat, Direct bioprinting of vessel-like tubular microfluidic channels, J. Nanotechnol. Eng. Med., 2013, 4(2), 0210011–0210017.
178. J. Zhang, et al., 3D printing of silk particle-reinforced chitosan hydrogel structures and their properties, ACS Biomater. Sci. Eng., 2018, 4(8), 3036–3046.
179. C. Marques, G. Diogo, S. Pina, J. M. Oliveira, T. Silva and R. Reis, Collagen-based bioinks for hard tissue engineering applications: a comprehensive review, J. Mater. Sci.: Mater. Med., 2019, 30(3), 1–12.
180. R. Chang, J. Nam and W. Sun, Direct cell writing of 3D microorgan for in vitro pharmacokinetic model, Tissue Eng., Part C, 2008, 14(2), 157–166.
181. S. Khalil and W. Sun, Biopolymer deposition for freeform fabrication of hydrogel tissue constructs, Mater. Sci. Eng., C, 2007, 27(3), 469–478.
182. P. S. Gungor-Ozkerim, I. Inci, Y. S. Zhang, A. Khademhosseini and M. R. Dokmeci, Bioinks for 3D bioprinting: an overview, Biomater. Sci., 2018, 6(5), 915–946.
183. C. Christou, K. Philippou, T. Krasia-Christoforou and I. Pashalidis, Uranium adsorption by polyvinylpyrrolidone/chitosan blended nanofibers, Carbohydr. Polym., 2019, 219, 298–305.
184. J. Hu, et al., Electroactive aniline pentamer cross-linking chitosan for stimulation growth of electrically sensitive cells, Biomacromolecules, 2008, 9(10), 2637–2644.
185. D. Han and L. Yan, Supramolecular Hydrogel of Chitosan in the Presence of Graphene Oxide Nanosheets as 2D Cross-Linkers, ACS Sustainable Chem. Eng., 2014, 2(2), 296–300.
186. C. A. Murphy, J. B. Costa, J. Silva-Correia, J. M. Oliveira, R. L. Reis and M. N. Collins, Biopolymers and polymers in the search of alternative treatments for meniscal regeneration: state of the art and future trends, Appl. Mater. Today, 2018, 12, 51–71.
187. M. Nourbakhsh, et al., Fabricating an electroactive injectable hydrogel based on pluronic-chitosan/aniline-pentamer containing angiogenic factor for functional repair of the hippocampus ischemia rat model, Mater. Sci. Eng., C, 2020, 117, 111328.
188. N. Islam, H. Wang, F. Maqbool and V. Ferro, In vitro enzymatic digestibility of glutaraldehyde-crosslinked chitosan nanoparticles in lysozyme solution and their applicability in pulmonary drug delivery, Molecules, 2019, 24(7), 1271.
189. G. Kumar, J. Bristow, P. Smith and G. Payne, Enzymatic gelation of the natural polymer chitosan, Polymer, 2000, 41(6), 2157–2168.
190. E. Hafemann, R. Battisti, C. Marangoni and R. A. Machado, Valorization of royal palm tree agroindustrial waste by isolating cellulose nanocrystals, Carbohydr. Polym., 2019, 218, 188–198.
191. J.-P. Méricq, D. Bouyer, D. Wlodarczyk, L. Soussan and C. Faur, Modeling of enzymatic chitosan gelation: Tools for monitoring chitosan gelation and urea hydrolysis kinetics, React. Funct. Polym., 2019, 144, 104337.
192. A. M. Heimbuck, et al., Development of Responsive Chitosan–Genipin Hydrogels for the Treatment of Wounds, ACS Appl. Bio Mater., 2019, 2(7), 2879–2888.
193. X. Du, 3D bio-printing review, in IOP Conf. Ser.: Mater. Sci. Eng., 2018, vol. 301, p. 012023.
194. S. Kyle, Z. M. Jessop, A. Al-Sabah and I. S. Whitaker, Printability’ of candidate biomaterials for extrusion based 3D printing: state–of–the–art, Adv. Healthcare Mater., 2017, 6(16), 1700264.
195. I. T. Ozbolat, 3D bioprinting: fundamentals, principles and applications, Academic Press, 2016.
196. K. Obara, et al., Photocrosslinkable chitosan hydrogel containing fibroblast growth factor-2 stimulates wound healing in healing-impaired db/db mice, Biomaterials, 2003, 24(20), 3437–3444.
197. C. D. O'Connell, et al., Development of the Biopen: a handheld device for surgical printing of adipose stem cells at a chondral wound site, Biofabrication, 2016, 8(1), 015019.
198. S. Duchi, et al., Handheld co-axial bioprinting: application to in situ surgical cartilage repair, Sci. Rep., 2017, 7(1), 1–12.
199. J. Lammel-Lindemann, I. A. Dourado, J. Shanklin, C. A. Rodriguez, L. H. Catalani and D. Dean, Photocrosslinking-based 3D printing of unsaturated polyesters from isosorbide: A new material for resorbable medical devices, Bioprinting, 2020, 18, e00062.
200. G. Choi and H. J. Cha, Recent advances in the development of nature-derived photocrosslinkable biomaterials for 3D printing in tissue engineering, Biomater. Res., 2019, 23(1), 18.
201. D. Thomas, Z. M. Jessop and I. S. Whitaker, 3D bioprinting for reconstructive surgery: techniques and applications, Woodhead Publishing, 2017.
202. S. Kyle, Z. Jessop, S. Tarassoli, A. Al-Sabah and I. Whitaker, Assessing printability of bioinks, in 3D Bioprinting for Reconstructive Surgery, Elsevier, 2018, pp. 173–189.
203. A. I. Neto, et al., Nanostructured polymeric coatings based on chitosan and dopamine–modified hyaluronic acid for biomedical applications, Small, 2014, 10(12), 2459–2469.
204. H. Ji, S. Hong and H. Lee, Bio-inspired adhesive catechol-conjugated chitosan for biomedical applications: A mini review [J], Acta Biomater., 2015, 27, 101–115.
205. E. Park, J. Lee, K. M. Huh, S. H. Lee and H. Lee, Toxicity–Attenuated Glycol Chitosan Adhesive Inspired by Mussel Adhesion Mechanisms, Adv. Healthcare Mater., 2019, 8(14), 1900275.
206. R. Wang, et al., Mussel-inspired chitosan-polyurethane coatings for improving the antifouling and antibacterial properties of polyethersulfone membranes, Carbohydr. Polym., 2017, 168, 310–319.
207. E. Park, et al., Toxicity-attenuated glycol chitosan adhesive inspired by mussel adhesion mechanisms, Adv. Healt. Mat., 2019, 8(14), 1900275 https://doi.org/10.1002/adhm.201900275 .
208. L. Wang and J. P. Stegemann, Thermogelling chitosan and collagen composite hydrogels initiated with β-glycerophosphate for bone tissue engineering, Biomaterials, 2010, 31(14), 3976–3985.
209. J. Si, Y. Yang, X. Xing, F. Yang and P. Shan, Controlled degradable chitosan/collagen composite scaffolds for application in nerve tissue regeneration, Polym. Degrad. Stab., 2019, 166, 73–85.
210. S. Ji, E. Almeida and M. Guvendiren, 3D bioprinting of complex channels within cell-laden hydrogels, Acta Biomater., 2019, 95, 214–224.
211. A. G. Sontyana, A. P. Mathew, K.-H. Cho, S. Uthaman and I.-K. Park, Biopolymeric in situ hydrogels for tissue engineering and bioimaging applications, Tissue Eng. Regener. Med., 2018, 15(5), 575–590.
212. A. Gilarska, J. Lewandowska-Łańcucka, W. Horak and M. Nowakowska, Collagen/chitosan/hyaluronic acid–based injectable hydrogels for tissue engineering applications–design, physicochemical and biological characterization, Colloids Surf., B, 2018, 170, 152–162.
213. C. Arakawa, R. Ng, S. Tan, S. Kim, B. Wu and M. Lee, Photopolymerizable chitosan–collagen hydrogels for bone tissue engineering, J. Tissue Eng. Regener. Med., 2017, 11(1), 164–174.
214. R. Patel, et al., Effect of dietary advanced glycation end products on mouse liver, PLoS One, 2012, 7(4), e35143.
215. S. Ahmed, A. Ali and J. Sheikh, A review on chitosan centred scaffolds and their applications in tissue engineering, Int. J. Biol. Macromol., 2018, 116, 849–862.
216. K. Ye, R. Felimban, k. Traianedes, S. E. Moulton, G. G. Wallace, J. Chung, A. Quigley, P. F. Choong and D. E. Myers, Chondrogenesis of infrapatellar fat pad derived adipose stem cells in 3D printed chitosan scaffold, PloS one, 2014, 9(6) DOI:10.1371/journal.pone.0099410.
217. S. Reed, G. Lau, B. Delattre, D. D. Lopez, A. P. Tomsia and B. M. Wu, Macro-and micro-designed chitosan-alginate scaffold architecture by three-dimensional printing and directional freezing, Biofabrication, 2016, 8(1), 015003.
218. H. Ueno, M. Murakami, M. Okumura, T. Kadosawa, T. Uede and T. Fujinaga, Chitosan accelerates the production of osteopontin from polymorphonuclear leukocytes, Biomaterials, 2001, 22(12), 1667–1673.
219. A. Rasool, S. Ata and A. Islam, Stimuli responsive biopolymer (chitosan) based blend hydrogels for wound healing application, Carbohydr. Polym., 2019, 203, 423–429.
220. J. Long, A. E. Etxeberria, A. V. Nand, C. R. Bunt, S. Ray and A. Seyfoddin, A 3D printed chitosan-pectin hydrogel wound dressing for lidocaine hydrochloride delivery, Mater. Sci. Eng., C, 2019, 104, 109873.
221. H. Zhang, K. Yang, G. Liu, S. Zhu, R. Yin and W. Zhang, 3D bioprinting of multi-biomaterial/crosslinked bioink for skin tissue engineering, in Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress, 2016.
222. J. van Kuilenburg, M. A. Masen and E. van der Heide, Contact modelling of human skin: What value to use for the modulus of elasticity?, Proc. Inst. Mech. Eng., Part J., 2013, 227(4), 349–361.
223. U. Bozuyuk, O. Yasa, I. C. Yasa, H. Ceylan, S. Kizilel and M. Sitti, Light-triggered drug release from 3D-printed magnetic chitosan microswimmers, ACS Nano, 2018, 12(9), 9617–9625.
224. J. Yan, Y. Wang, X. Zhang, X. Zhao, X. Ma, X. Pu, Y. Wang, F. Ran, Y. Wang and F. Leng, Snakegourd root/Astragalus polysaccharide hydrogel preparation and application in 3D printing, Int. J. Biol. Macromol., 2019, 121, 309–316.
225. M. R. Sommer, L. Alison, C. Minas, E. Tervoort, P. A. Rühs and A. R. Studart, 3D printing of concentrated emulsions into multiphase biocompatible soft materials, Soft Matter, 2017, 13(9), 1794–1803.
226. T. H. Van Osch, J. Perelaer, A. W. de Laat and U. S. Schubert, Inkjet printing of narrow conductive tracks on untreated polymeric substrates, Adv. Mater., 2008, 20(2), 343–345.
227. S. Wünscher, R. Abbel, J. Perelaer and U. S. Schubert, Progress of alternative sintering approaches of inkjet-printed metal inks and their application for manufacturing of flexible electronic devices, J. Mater. Chem. C, 2014, 2(48), 10232–10261,  10.1039/C4TC01820F.
228. M. A. Darabi, et al., Skin–inspired multifunctional autonomic–intrinsic conductive self–healing hydrogels with pressure sensitivity, stretchability, and 3D printability, Adv. Mater., 2017, 29(31), 1700533.
229. J. Xiong, Ø. Borg, E. A. Blekkan and A. Holmen, Hydrogen chemisorption on rhenium-promoted γ-alumina supported cobalt catalysts, Catal. Commun., 2008, 9(14), 2327–2330.
230. M. Rajamani and K. Rajendrakumar, Chitosan-boehmite desiccant composite as a promising adsorbent towards heavy metal removal, J. Environ. Manage., 2019, 244, 257–264.
231. G. Sargazi, et al., Chitosan/polyvinyl alcohol nanofibrous membranes: towards green super-adsorbents for toxic gases, Heliyon, 2019, 5(4), e01527.
232. M. B. Kasiri, Application of chitosan derivatives as promising adsorbents for treatment of textile wastewater, in The Impact and Prospects of Green Chemistry for Textile Technology, Elsevier, 2019, pp. 417–469.
233. S. Sessarego, S. C. Rodrigues, Y. Xiao, Q. Lu and J. M. Hill, Phosphonium-enhanced chitosan for Cr(VI) adsorption in wastewater treatment, Carbohydr. Polym., 2019, 211, 249–256.
234. J. Desbrières and E. Guibal, Chitosan for wastewater treatment, Polym. Int., 2018, 67(1), 7–14.
235. G. Zhou, et al., Three-dimensional polylactic acid@ graphene oxide/chitosan sponge bionic filter: highly efficient adsorption of crystal violet dye, Int. J. Biol. Macromol., 2018, 113, 792–803.
236. J. Lee, B. Choi, B. Wu and M. Lee, Customized biomimetic scaffolds created by indirect three-dimensional printing for tissue engineering. Biofabrication, Biofabrication, 2013, 5(4), 045003.
237. H. Y. Lin, T. W. Chang and T. K. Peng, Three–dimensional plotted alginate fibers embedded with diclofenac and bone cells coated with chitosan for bone regeneration during inflammation, J. Biomed. Mater. Res., Part A, 2018, 106(6), 1511–1521.
238. M. Ainola, et al., A bioactive hybrid three-dimensional tissue-engineering construct for cartilage repair, J. Biomater. Appl., 2016, 30(6), 873–885.
239. A. M. Handorf, Y. Zhou, M. A. Halanski and W.-J. Li, Tissue stiffness dictates development, homeostasis, and disease progression, Organogenesis, 2015, 11(1), 1–15.
240. C.-W. Wong, Y.-T. Chen, C.-L. Chien, T.-Y. Yu, S.-P. Rwei and S.-h. Hsu, A simple and efficient feeder-free culture system to up-scale iPSCs on polymeric material surface for use in 3D bioprinting, Mater. Sci. Eng., C, 2018, 82, 69–79.
241. Y. Yang, et al., Anti-infective efficacy, cytocompatibility and biocompatibility of a 3D-printed osteoconductive composite scaffold functionalized with quaternized chitosan, Acta Biomater., 2016, 46, 112–128.
242. C.-H. Tsai, C.-H. Hung, C.-N. Kuo, C.-Y. Chen, Y.-N. Peng and M.-Y. Shie, Improved bioactivity of 3D printed porous titanium alloy scaffold with chitosan/magnesium-calcium silicate composite for orthopaedic applications, Materials, 2019, 12(2), 203.
243. N. Noor, A. Shapira, R. Edri, I. Gal, L. Wertheim and T. Dvir, Tissue Engineering: 3D Printing of Personalized Thick and Perfusable Cardiac Patches and Hearts (Adv. Sci. 11/2019), Adv. Sci., 2019, 6(11), 1970066.
244. G. D. Brusko, E. M. Cox and M. Y. Wang, 3-dimensional printing a novel framework for spinal cord injury repair, Neurosurgery, 2019, 85(2), E192–E193.
245. S.-L. Ryan, et al., Drug discovery approaches utilizing three-dimensional cell culture, Assay Drug Dev. Technol., 2016, 14(1), 19–28.
246. Q. Pi, et al., Digitally tunable microfluidic bioprinting of multilayered cannular tissues, Adv. Mater., 2018, 30(43), 1706913.
247. D. E. Ingber, Reverse engineering human pathophysiology with organs-on-chips, Cell, 2016, 164(6), 1105–1109.
248. Q. Yang, Q. Lian and F. Xu, Perspective: fabrication of integrated organ-on-a-chip via bioprinting, Biomicrofluidics, 2017, 11(3), 031301.
249. K. Fetah, et al., The emergence of 3D bioprinting in organ-on-chip systems, Prog. Biomed. Eng., 2019, 1(1), 012001.
250. J. Ma, Y. Wang and J. Liu, Bioprinting of 3D tissues/organs combined with microfluidics, RSC Adv., 2018, 8(39), 21712–21727.
251. D.-H. T. Nguyen, et al., Biomimetic model to reconstitute angiogenic sprouting morphogenesis in vitro, Proc. Natl. Acad. Sci. U. S. A., 2013, 110(17), 6712–6717.
252. D. Huh, G. Hamilton and D. Ingber, Program B, From three-dimensional cell culture to organs-on-chips, Dongeun Trends Cell. Biol., 2011, 21, 745–754.
253. D. Choudhury, S. Anand and M. W. Naing, The arrival of commercial bioprinters–Towards 3D bioprinting revolution, Int. J. Bioprint., 2018, 4(2), 139.
254. I. T. Ozbolat and Y. Yu, Bioprinting toward organ fabrication: challenges and future trends, IEEE Trans. Biomed. Eng., 2013, 60(3), 691–699.
255. C. Colosi, et al., Microfluidic bioprinting of heterogeneous 3D tissue constructs using low–viscosity bioink, Adv. Mater., 2016, 28(4), 677–684.
256. F. Yu, et al., A perfusion incubator liver chip for 3D cell culture with application on chronic hepatotoxicity testing, Sci. Rep., 2017, 7(1), 1–16.
257. F. Yu, et al., On chip two-photon metabolic imaging for drug toxicity testing, Biomicrofluidics, 2017, 11(3), 034108.
258. J. Rouwkema and A. Khademhosseini, Vascularization and angiogenesis in tissue engineering: beyond creating static networks, Trends Biotechnol., 2016, 34(9), 733–745.
259. P. H. Dykstra, V. Roy, C. Byrd, W. E. Bentley and R. Ghodssi, Microfluidic electrochemical sensor array for characterizing protein interactions with various functionalized surfaces, Anal. Chem., 2011, 83(15), 5920–5927.
260. X. Luo, et al., Programmable assembly of a metabolic pathway enzyme in a pre-packaged reusable bioMEMS device, Lab Chip, 2008, 8(3), 420–430.
261. X. Luo, et al., Design optimization for bioMEMS studies of enzyme-controlled metabolic pathways, Biomed. Microdevices, 2008, 10(6), 899–908.
262. Y. Cheng, et al., Electroaddressing functionalized polysaccharides as model biofilms for interrogating cell signaling, Adv. Funct. Mater., 2012, 22(3), 519–528.
263. Z. Wang, L. He, J. Lv and M. Kimura, Electrodeposition of thin chitosan membrane in freestanding SU-8 microfluidic channel for molecular addressing by capillary effect, Mater. Res. Express, 2019, 6(4), 045403.
264. K. Balagangadharan, S. Dhivya and N. Selvamurugan, Chitosan based nanofibers in bone tissue engineering, Int. J. Biol. Macromol., 2017, 104, 1372–1382.
265. M. Wang, Y. Xiao, L. Lin, X. Zhu, L. Du and X. Shi, A microfluidic chip integrated with hyaluronic acid-functionalized electrospun chitosan nanofibers for specific capture and nondestructive release of CD44-overexpressing circulating tumor cells, Bioconjugate Chem., 2018, 29(4), 1081–1090.
266. Q. Hamid, C. Wang, Y. Zhao, J. Snyder and W. Sun, A three-dimensional cell-laden microfluidic chip for in vitro drug metabolism detection, Biofabrication, 2014, 6(2), 025008.
267. K. A. Homan, et al., Bioprinting of 3D convoluted renal proximal tubules on perfusable chips, Sci. Rep., 2016, 6, 34845.
268. S. Mi, et al., A novel controllable cell array printing technique on microfluidic chips, IEEE Trans. Biomed. Eng., 2019, 66(9), 2512–2520.
269. N. S. Bhise, et al., A liver-on-a-chip platform with bioprinted hepatic spheroids, Biofabrication, 2016, 8(1), 014101.
270. J. Y. Park, et al., Development of a functional airway-on-a-chip by 3D cell printing, Biofabrication, 2018, 11(1), 015002.
271. Q. Gao, et al., 3D bioprinting of vessel-like structures with multilevel fluidic channels, ACS Biomater. Sci. Eng., 2017, 3(3), 399–408.
272. Q. Hamid, C. Wang, J. Snyder, S. Williams, Y. Liu and W. Sun, Maskless fabrication of cell-laden microfluidic chips with localized surface functionalization for the co-culture of cancer cells, Biofabrication, 2015, 7(1), 015012.
273. L. E. Bertassoni, et al., Hydrogel bioprinted microchannel networks for vascularization of tissue engineering constructs, Lab Chip, 2014, 14(13), 2202–2211.
274. S. Bakarich, R. Gorkin III, M. in het Panhuis and G. M. Spinks, ACS Appl. Mater. Interfaces, 2014, 6, 15998–16006.
275. Y. Luo, X. Lin, B. Chen and X. Wei, Cell-laden four-dimensional bioprinting using near-infrared-triggered shape-morphing alginate/polydopamine bioinks, Biofabrication, 2019, 11(4), 045019.
276. A. Lapomarda and G. Vozzi, 4D Bioprinting as New Tissue Engineering Perspective, Biosci., Biotechnol. Res. Asia, 2019, 16(1), 15.
277. I. Lukin, et al., Can 4D bioprinting revolutionize drug development?, Taylor & Francis, 2019.
278. F. Ding, H. Li, Y. Du and X. Shi, Recent advances in chitosan-based self-healing materials, Res. Chem. Intermed., 2018, 44(8), 4827–4840.
279. F. Gang, et al., Robust magnetic double-network hydrogels with self-healing, MR imaging, cytocompatibility and 3D printability, Chem. Commun., 2019, 55(66), 9801–9804.
280. X. Chen, et al., Magnetic and self-healing chitosan-alginate hydrogel encapsulated gelatin microspheres via covalent cross-linking for drug delivery, Mater. Sci. Eng., C, 2019, 101, 619–629.
281. B. Guo, J. Qu, X. Zhao and M. Zhang, Degradable conductive self-healing hydrogels based on dextran-graft-tetraaniline and N-carboxyethyl chitosan as injectable carriers for myoblast cell therapy and muscle regeneration, Acta Biomater., 2019, 84, 180–193.
282. B. Fu, B. Cheng, X. Bao, Z. Wang, Y. Shangguan and Q. Hu, Self–healing and conductivity of chitosan–based hydrogels formed by the migration of ferric ions, J. Appl. Polym. Sci., 2019, 136(34), 47885.
283. T. Kean and M. Thanou, Biodegradation, biodistribution and toxicity of chitosan, Adv. Drug Delivery Rev., 2010, 62(1), 3–11.
284. A. Babu and R. Ramesh, Multifaceted applications of chitosan in cancer drug delivery and therapy, Mar. Drugs, 2017, 15(4), 96.
285. C. Chaput and A. Chenite, Injectable in situ self-forming mineral-polymer hybrid composition and uses thereof, ed: Google Patents, 2014.
286. J.-B. D'Huart and C. Dallas, Cactaceae-based formulation having the property of fixing fats and method for obtaining same, ed: Google Patents, 2004.
287. V. G. Varanasi, A. Ilyas, P. R. Kramer, T. Azimaie, P. B. Aswath and T. Cebe, In vivo live 3D printing of regenerative bone healing scaffolds for rapid fracture healing, ed: Google Patents, 2019.
288. S. McCarthy, B. McGrath and E. Winata, Absorbable tissue dressing assemblies, systems, and methods formed from hydrophilic polymer sponge structures such as chitosan, ed: Google Patents, 2012.
289. J. K. Smith, A. C. Parker, J. A. Jennings, B. T. Reves and W. O. Haggard, Methods for producing a biodegradable chitosan composition and uses thereof, ed: Google Patents, 2017.
290. X. Liu, et al., Responsive Materials: 3D Printing of Living Responsive Materials and Devices (Adv. Mater. 4/2018), Adv. Mater., 2018, 30(4), 1870021.
291. C. A. Custódio, A. del Campo, R. L. Reis and J. F. Mano, Smart instructive polymer substrates for tissue engineering, in Smart Polymers and their Applications, Elsevier, 2019, pp. 411–438.
292. A. Lee, et al., 3D bioprinting of collagen to rebuild components of the human heart, Science, 2019, 365(6452), 482–487.

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Footnote

† Mosen Taghizadeh, Ali Taghizadeh and Mohsen Khodadadi Yazdi equally contributed to this work. Masoud Mozafari currently at Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, Canada.

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