*3.1. Resource of Chitosan*

Chitin is one of the most abundant natural polymers in organism [27]. It is the second most abundant natural polymer next to cellulose, consisting of 2-acetamido-2-deoxy-d-glucose through a (1-4) linkage, extracted from the shells of marine crustaceans, insects, or fungi. Thus, the structural formula of chitin is poly-(1-4)-*<sup>N</sup>*-acetyl-glucosamine. Because of its facility to generate long-chain polymer structure, chitin is vitally important for some biological structure formation, such as cell walls in fungi and yeast, and exoskeleton of many invertebrates in shrimps and crabs. The extractive product is white, slightly pearly luster, and translucent sheet solid. Because of its insolubility in water and most of the organic solvents, chitin has limited applications in biomedical fields.

Chitosan, a derivate of chitin, is a linear polysaccharide, or carbohydrate polymer, derived from partial deacetylation of natural chitin. The deacetylation of chitin is conducted by chemical hydrolysis in alkaline conditions, using concentrated alkali water, or enzymatic hydrolysis with chitin deacetylase [28–30]. As its origin chitin, chitosan is highly available in nature.

### *3.2. Physico-Chemical Properties of Chitosan*

Chitosan is a linear carbohydrate polymer derived from chitin with a structural similarity to glycosaminoglycan, a component of ECMs (Figure 3) [31–38]. Its chemical name is (1,4) -2-amino-2-deoxy-beta-*<sup>D</sup>*-glucan, a copolymer of randomly located (1-4)-2-amino-2-deoxy-d-glucan (d-glucosamine) and (1-4)-2-acetamido-2-deoxy-d-glucan (*N*-acetyl d-glucosamine) units. The number of amino groups as a ratio between d-glucosamine to the sum of d-glucosamine and *N*-acetyl d-glucosamine is indicated as a deacetylation degree (DD) and ordinarily should be larger than 60%. Some chitosans (e.g., 50% deacetylation degree) are water-soluble, while most of the chitosans are acid-soluble. Thus, chitosan can be recognized as a semi-natural positively charged polysaccharide at acidic conditions. Chitosan molecules can be modified through changing the functional groups, such as OH, and NH2 by -COCH3, -CH3, -CH2COOH, -SO3H, -PO(OH)2, etc.

**Figure 3.** Chitosan generated by deacylation of chitin can be chemically modified through changing the functional groups, R1, R2, R3 in the derivatives represents -COCH3, -CH3, -CH2COOH, -SO3H, -PO(OH)2, etc. Image reproduced with permission from [31].

### *3.3. Prominent Characteristics of Chitosan for 3D Bioprinting*

The biophysical characters of chitosan relay on a few factors, such as the molecular weight, DD, and purity of the molecules. Compared with animal derived natural polymers, such as collagen, gelatin, and fibrin, the degradation rate of chitosan is relatively slow. The biophysical properties of chitosan can be easily adjusted through changing the deacetylation rate of the original chitin and the molecular weight of the product.

Chitosan molecules possess several important properties, such as biocompatibility, biodegradability, antibacterial activity, non-antigenicity, and bioadsorbility. The solubility of chitosan in diluted acids is pH dependent through protonation of amino groups of the d-glucosamine residues. The availability of protonated amino groups enables chitosan to form complexes with metal ions, natural or synthetic anionic poly(acrylic acid) polymers, lipids, proteins, and deoxyribonucleic acid (DNA) [39–41]. The cationic feature of chitosan molecules favors the formation of gel particles through electrostatic interactions, with, e.g., sodium sulfate employed as a precipitant [39–41].

Stress should be given to the cationic feature of chitosan molecules. Few polymers in nature possess such special characters. The positive charged chitosan molecules can interact with hydrophobic components, giving rise to amphiphilic particles with grea<sup>t</sup> self-assembly and encapsulation capabilities. The polycationic nature of chitosan at a mild acidic condition allows

the immobilization of negatively charged enzymes, proteins, and DNA for gene delivery and other composite biomaterial formation [42,43]. It has proven that appropriate interactions between chitosan molecules and drugs can produce expected pharmacological e ffect at the target site. The hydrophilic structure of chitosan promotes almost all cell types to adhere and proliferate on its sca ffolds and makes chitosan a good candidate for tissue repair and organ 3D bioprinting.

### **4. Chitosan-Base Polymers in Tissue Repair and 3D Bioprinting**

### *4.1. Antimicrobial Activities for Skin Regeneration*

Chitosan and its derivations have shown a lot of distinctive characters in skin regeneration with antimicrobial activities. The mechanisms of antibiosis for Gram positive and Gram-negative bacteria are di fferent. The di fferences are closely related to the composition of the bacterial walls [44,45]. It is supposed that chitosan presents antimicrobial activities through e ffectively interacting with the outer membrane or cytoderm of the bacteria, probably, electrostatic attraction and osmotic pressure taken upon the dominant roles in the interactions.

In Gram-positive bacteria, such as staphylococcus and streptococcus, the cytoderm consists of peptidoglycan with a thickness of 20–80 nm. *N*-acetylmuramic acids in the cytoderm can absorb negatively charged teichoic acids through covalent linkages. At the same time, lipopolyteichoic acids form covalent bonds with the cytoplasmic membrane. When teichoic acids form a layer of high-density charges in the cytoderm, the cytoderm is strengthened and the ion transportation through the outer surface layers can be restrained. Compared with Gram-positive bacteria, the peptidoglycan stratum in the cytoderm of Gram-negative bacteria is relatively thin, which lies over the cytoplasmic membrane. This complexity is further covered by an additional outer envelope membrane, with the fundamental elements of lipoprotein and lipopolysaccharide.

In acidic condition (especially, pH <6), the quaternary ammonium groups (R-NH3+) in chitosan molecules can competitively integrate with the divalent metal ions, such as Ca2+ and Mg<sup>2</sup>+. To maintain the balance of voltage, polyanions often passively bind with the cytoderm leave. This binding can lead to the cytoderm to be hydrolyzed and cause the leakage of intracellular components [44,45]. The hydrolysis of peptidoglycans also results in an increased electrical interaction, leading to the enhancement of solute conductivity and cytoplasmic β-galactosidase release in the cell suspensions [46–49].

Thus, the reason of chitosan a ffecting the permeability of the outer cytoderm is through forming an ionic type of bonding and preventing the intracellular transport of nutrients into the cells. This type of tunnel also increases the internal osmotic pressure, leading to the apoptosis of cells because of the lack of nutrients [50]. Some other studies have shown that chitosan can penetrate the multilayered (murein cross-linked) bacterial walls as well as the cytoplasmic membrane. Chitosan destroys the bacterial cells by binding to the DNA which prevents DNA transcription and interrupts protein and m-ribonucleic acid (mRNA) synthesis [50]. The destructiveness is highly dependent on the capability of chitosan to penetrate the multilayered cell wall and the cytoplasmic membrane. Nevertheless, chitosan has generally been regarded as a membrane disruptor rather than a penetrator during the antimicrobial activities.

Skin regeneration is a complex procedure that contains four dynamic phases—hemostasis, inflammation, proliferation, and tissue remodeling [51]. This dynamic process involves vascular stimulators, ECM components, soluble factors, and various cells. The treatment of skin injuries needs to ensure a very high degree of protection, strong anti-inflammatory e ffect, and minimum scar formation.

Chitosan solutions can be made into porous sca ffolds with intrinsic antimicrobial properties. The antimicrobial e ffect of the porous sca ffolds can be further promoted through adding other antimicrobial agents for wound healing. In one study, a chitosan-cordycepin hydrogel was prepared via adsorbing negatively charged cordycepin onto the positively charged chitosan molecules without adding any cross-linking agen<sup>t</sup> [52]. In another study, polyethylene terephthalate was 3D printed with a chitosan solution [53]. Each layer of the textile polyethylene terephthalate-chitosan was loaded

with chlorhexidine. The stability of the 3D printed porous sca ffold was enhanced by a heating system, which also extended the delivery time of chlorhexidine up to seven weeks. Chitosan with other natural polymers can be coprinted into asymmetric membranes, and normally, the lower layer can directly contact the damaged skin [54]. The 3D printed membranes presented e fficient antimicrobial capability against methicillin resistant staphylococcus aureus (MRSA) strains during the skin repair processes using a mouse model. The skin repair e ffect is similar to the commercially available products [55].

In addition to antimicrobial function, chitosan participates in all phases of skin regeneration in many ways. Chitosan molecules can e ffectively promote the migration of neutrophils, increasing the secretion of IL-8, a potent neutrophil chemokine [56]. This reaction is in correlation with the level of *N*-acetylation [57]. Moreover, chitosan can a ffect the expression of growth factors by increasing transforming growth factor-1 (TGF-1) expression in early post-injury phase and decreasing it in later stages through binding themselves to anionic growth factors [58,59]. Especially, chitosan molecules with high DD stimulate the proliferation of dermal fibroblasts, allowing fibrous tissue formation and re-epithelialization [60,61]. These unique properties of chitosan molecules make them favorable candidates in skin regeneration.

### *4.2. Hemostatic Activity for Wound Healing*

Some chitosan molecules with specific molecular weight and DD demonstrate powerful hemostatic capability, which is independent of the coagulation pathway of the host [62–64]. The amine groups in the chitosan molecules can interact directly with coagulation factors, promoting the initiation of coagulation. When the DD of chitosan is 68.36%, chitosan molecules in a solution tend to form mesh-like structures and act with vascular components directly. Whereas higher DD results in stronger hydrogen bonds and crystalline structures within chitosan chains that have limited interaction with red blood cells [65]. The interactions of chitosan molecules with polyelectrolytes can be enhanced when the molecular weight of chitosan is increased, so as to the procoagulation processes [66,67]. There are several chitosan-containing hemostatic products, such as Celox ®, HemCon ®, Axiostat ®, Chitoflex ®, and Chitoseal ®, available in the market, which have been approved by the Food and Drug Administration of the United States (FDA) [68].

It is found that 3D bioprinted chitosan/collagen films are useful in wound healing. The host tissues had anaphylaxis reactions to allogenic source collagens. It is necessary to prepare more biocompatible chitosan/collagen substitutes for wound healing in the future. Human keratin-chitosan membrane produced through UV-crosslinking has shown the potential as wound dressing with improved mechanical properties [69]. Chitosan-chondroitin sulfate-based polyelectrolyte complex has shown strong hemostatic capability beside antimicrobial e ffect for wound healing applications [70]. 3D printed chitosan with positive charged bioactive agents, such as growth factors and cytokines, can promote the wound healing capability. In an attempt, nanoparticles of chitosan generated by ionotropic gelation with tripolyphosphate were loaded with granulocyte-macrophage colony-stimulating factor (GM-CSF). The complexity was freeze-dried afterward, leading to the production of nanocrystalline cellulose–hyaluronic acid combination [71,72]. Polycaprolactone nanofibers loaded with chitosan NPs containing GM-CSF accelerated wound closure phenomenon [73]. 3D printing of chitosan combined with peptides presents the ability of wound closure as well. Bioprinting of cell-laden chitosan hydrogels, containing Ser-Ile-Lys-Val-Ala-Val-chitosan macromers can e ffectively induce various types of collagen expression, prompting angiogenesis with markers of TGF-1 [74]. Meanwhile, the inflammatory factors, which are not conducive for wound healing, such as TNFα, IL-1β, and IL-6 mRNA in a mouse skin wound model were significantly inhibited [75]. In some other studies, chitosans were used to enhance the a ffinity of growth factors. A 3D printed chitosan sca ffold containing heparin-like polysaccharide (2- *N*, 6- *O*-sulfated) demonstrated an enhanced capability to attract vascular endothelial cells and induce the secretion of growth factors because of the high sulfonation degree [76].

### *4.3. Three-Dimensional Constructs for Bone Rehabilitation*

Traditionally, chitosan membrane is one of the commonly used biomaterials in biomedical and clinical applications. It can be prepared through various technologies, such as electrospinning, thermal induced phase separation and self-assembly. During electrospinning, chitosan fibers are deposited irregularly to form non-woven fibrous membranes. The physical structures of the non-woven fibrous membranes are similar to those of natural ECMs. The shortages of the non-woven fibrous membranes to be used as tissue engineering sca ffolds are the small pore sizes and weak mechanical strengths. On the one side, the pore sizes of the fibrous membranes are too small to let cells grow in [77]. For example, Sajesh et al. prepared a chitosan fibrous membrane through electrospinning [78]. The tensile strength of the fibrous membrane was 10 MPa, the average pore size was 5 um, and the porosity was over 80%. Shalumon found that in a high chitosan-contenting membrane, the tensile strength was only 1.5 MPa, which was lower than that of a commonly clinically used bone regeneration membrane [79]. The tensile strength of chitosan fibrous membrane needs to be improved. The pore size of chitosan membranes prepared through chitosan molecule self-assembly is also small, and the diameter of the pores is easily a ffected by the concentration of the chitosan molecules, solution pH, temperature, and other factors. Some scholars used sodium chloride as pore-forming agen<sup>t</sup> to obtain large pores in the chitosan membranes via thermal induced phase separation.

Current research shows that calcium phosphate, carbon nanotubes, and hydroxyapatite can increase the mechanical properties of the chitosan sca ffolds to some degree [80]. For example, Matinfar et al. mixed chitosan and carboxymethyl cellulose (CMC), and reinforced with whisker-like biphasic and triphasic calcium phosphate fibers as bone repair sca ffolds [81]. The composite chitosan/CMC were obtained by freeze drying. The composite sca ffolds exhibited desirable microstructures with high porosity (61–75%) and interconnected pores in range of 35–200 μm. Addition of CMC to chitosan solution led to a significant improvement in the mechanical properties (up to 150%) but did not a ffect the water uptake ability and biocompatibility. The composite chitosan/CMC sca ffolds reinforced with 50 wt% triphasic fibers were superior in terms of mechanical and biological properties and showed compressive strength and modulus of 150 kPa and 3.08 MPa, respectively, which is up to 300% greater than pure chitosan sca ffolds. Bi et al. prepared a chitosan-containing composite sca ffold and seeded with osteoblasts. It was found that a large number of osteoblasts adhered on the sca ffold and proliferated inside the go-through pores [82]. When the chitosan-containing composite sca ffold was implanted into rats with skull-parietal bone loss, new bone formed at the edge of the bone loss site and the center of the sca ffold in 2 weeks. After five weeks' implantation, new bone mass was significantly higher than that of the blank control.

With the introduction of 3D printing technologies in tissue engineering, the physical, biochemical, and physiological properties of the 3D printed chitosan sca ffolds can be greatly improved. In a 3D printed chitosan sca ffolds, osteoblasts grew along the computer controlled go-through channels and formed trabecula structures. Meanwhile blood vessels are easy to form along the go-through channels with the addition of endothelial cells [83]. Emphasis should be given to those growth factors, i.e., polypeptides, that can bind to specific cell membrane receptors to control cell destiny and regulate cell functions. Osteoinductive growth factors include vascular endothelial factor (VEGF), bone morphogenetic protein (BMP), platelet-derived growth factor (PDGF), and TGF, etc. Kjalarsdttir et al. cultured mouse fibroblasts with BMP-2 encapsulated in chitosan microsphere through a ion cross-linking method. The results showed that the encapsulation rate of chitosan microspheres was over 80% with a slow growth factor releasing rate. The sustainable releasing time attained 30 days. When the rhBMP-2 adsorbed chitosan microspheres were compounded on collagen sponge sca ffolds and implanted into rabbits with radial segmental defects, the rhBMP-2-adsorbed chitosan microsphere sca ffolds had more new bone mass than that of the control rhBMP-2/collagen sca ffolds 12 weeks after the implantation, indicating that chitosan microspheres as carriers could e ffectively maintain the biological activity of rhBMP-2 [84]. The chitosan molecules can inhibit the secretion of osteoclasts and promote the proliferation of osteoblasts, thus promoting the bone tissue repair e ffect. When the

VEGF-containing chitosan microspheres were implanted into rat peritoneal adipose tissues, two weeks later, the number of endothelial cells and erythrocytes in the rats was significantly higher than that of the controls. These results sugges<sup>t</sup> that the chitosan-containing 3D sca ffolds together with growth factors can e ffectively promote large bone repair rate [85].

In some other researches, the positive charge amino groups in the chitosan molecules can combine with negative charged DNA molecules to form nanoparticles through polyelectrolyte actions. Foreign genes can be transfected into the cells of the body and play some roles in the cell behaviors. For example, Zeng et al. prepared nanoparticles via the reaction of mercaptan-organized chitosan and recombinant plasmid polyelectrolyte. When the recombinant plasmids containing *BMP-4* and *VEGFR1* genes were implanted into rabbits with radius defect, the experimental group had faster bone defect repair speed and more new bone mass compared with the controls [86]. Similarly, chitosan/polyacrylic acid nanofibers had been used as e ffective carriers of DNA plasmids. These researches have elaborated the active roles of chitosan molecules in bone tissue rehabilitation processes at molecular and cellular levels.

For large bone repair, a series of pioneering work have been done by the corresponding author of this article herself before 2000 through chemical modification of chitosans (Figures 4–6) [31–36]. For example, several large bone repair materials have been created by adding phosphorylated chitin (P-chitin), phosphorylated chitosan (P-chitosan), and disodium (1 <sup>→</sup>4)-2-deoxy-2-sulfoamino-β-*D*-glucopyranuronan (S-chitosan) as the additives of biodegradable calcium phosphate cement (CPC) systems. The large bone repair materials are biocompatible, bioabsorbable, osteoconductive and/or osteoinductive. In vitro and in vivo experiments have shown that the bone repair rates and e ffects are directly related to the functional groups on the chitosan-based molecules and polymer concentrations in the CPCs. There are many di fferent bone repair manners with these materials: some new trabeculae form directly after body fluid infiltration of the implants (Figure 5) [33]; some new trabeculae form following chondrocytes disappearing around the implants (Figure 6) [32]; some new trabeculae form after fibroblast-like cells being swallowed up [36]. The biodegradation rates of the materials have negative relationships with the P-chitin, P-chiosan, and S-chitosan contents. Most of the low concentration samples degrade in 16 weeks. While the high concentration samples disappear around 22 weeks. The fastest bone repair rates comes from those samples containing low concentrations of P-chitin and P-chitosan. Especially, the P-chitosan contained CPCs possess excellent biocompatibilities which can be transferred to trabeculae straightly after body fluid infiltration without any vise reactions or adverse e ffects, such as hematoma, inflammation, fibrous encapsulation, tissue necrosis, and excessive growth. The degradation rates of P-chitin and P-chitosan contained samples can be adjusted to match the ingrowth speeds of new trabeculae. A mild foreign-body reaction appears in the high P-chitin content samples during the early implantation stages which do not impair the final bone repair e ffects.

Later in 2003, the chitosan-based polymers have been 3D printed into hybrid large bone repair sca ffolds with synthetic polymers, such as poly(lactic acid-co-glycolic acid) (PLGA), and mineral salts, such as calcium phosphate (TCP). Some of them have multiple functions, such as promoting osteoblast growth and inhibiting osteoclast activity (Figure 7) [87]. Di fferent CAD models have been utilized to manufacture the hybrid sca ffolds. Optimal fabrication parameters have been systematically studied through manipulating the processing materials. Furthermore, the microscopic structures, water absorbability, and mechanical properties of the hybrid sca ffolds can be easily adjusted through adding di fferent amount of P-chitin, P-chitosan, and S-chitosan. These hybrid sca ffolds have been proven to be promising candidates for large hard tissue and organ manufacturing and restoration with later animal tests.

**Figure 4.** X-ray radiographs of large bone repair in rabbits with phosphorylated chitosan (P-chitosan) containing biodegradable calcium phosphate cements (CPCs): (**a**) 1 week (0.12 g/mL P-chitosan); (**b**) 4 weeks (0.14 g/mL P-chitosan); (**c**) 12 weeks (0.12 g/mL P-chitosan); (**d**) 12 weeks (0.07 g/mL P-chitosan); (**e**) 12 weeks (0.02 g/mL P-chitosan); (**f**) 22 weeks (0.12 g/mL P-chitosan). Image reproduced with permission from [33].

**Figure 5.** Tissue responses to different samples containing different concentrations of phosphorylated chitosan (P-chitosan) at different time points after implantation: (**a**) 1 week (0.12 g/mL P-chitosan), Masson Trichroism (M-T) staining; (**b**) 4 weeks (0.12 g/mL P-chitosan), M-T staining; (**<sup>c</sup>**–**<sup>e</sup>**) 12 weeks (0.02 g/mL P-chitosan); (**f**) 12 weeks (0.12 g/mL and 0.05 g/mL P-chitosan respectively), M-T and haematoxylin-eosin staining; (**g**) 22 weeks (0.12 g/mL P-chitosan) M-T staining; (**h**) 12 weeks (0.02 g/mL P-chitosan), a back scattered scanning electron microscopy (BSE) image; (**i**) 12 weeks (0.02 g/mL P-chitosan), a BSE image. Image reproduced with permission from [33].

**Figure 6.** Tissue responses to different samples containing different concentrations of phosphorylated chitin (P-chitin) at different time points after implantation: (**<sup>a</sup>**,**b**) 1 week (0.14 g/mL P-chitin), Masson Trichroism (M-T) and Giemsa staining respectively; (**<sup>c</sup>**,**d**) 4 weeks (0.14 g/mL P-chitin), M-T and haematoxylin-eosin staining respectively; (**<sup>e</sup>**,**f**) 4 weeks (0.08 g/mL P-chitin); (**g**) 12 weeks (0.08 g/mL P-chitin) M-T staining; (**h**) 22 weeks (0.14 g/mL P-chitin); (**i**) 12 weeks (0.02 g/mL P-chitin), a back scattered scanning electron microscopy image. Image reproduced with permission from [32].

**Figure 7.** Graphical description of large bone repair scaffolds made in Tsinghua University, the corresponding author's laboratory in 2007: (**a**) a double-nozzle low-temperature 3D bioprinter; (**b**) working state of the double nozzles; (**c**) a grid computer-aided design (CAD) model containing two material systems; (**d**) a sample made from chitosan/gelatin and polyurethane; (**e**) a sample containing both P-chitosan and S-chitosan made via the double-nozzle low-temperature 3D bioprinter; (**f**) a computerized tomography of the 3D printed sample of (**e**). Image reproduced with permission from [87].
