**1. Introduction**

The incidence of bone disorders has increased, as a result of the aging population coupled with increased obesity and poor physical activity, drawing extensive attention to bone repair medicine research [1,2]. When the bone disorder exceeds the critical size defect (>2 cm), the bone tissue cannot heal by itself and clinical treatment is required [2]. Bone grafting is one of the most common methods for bone regeneration, with over two million bone graft procedures conducted worldwide annually. Numerous types of bone grafts have been used in bone tissue engineering in the last few decades; however, increasing attention is directed towards the biomimetic approach in scaffold design, where molecular, structural and biological compatibility with complex native bone tissue is achieved [3]. When fundamental limitations of biomaterials of first and second generations were recognized, studies shifted to the biomimetic approach and biomaterials that stimulate specific cellular responses at the level of molecular biology [4]. For the successful development of biomimetic scaffolds for bone regeneration, and the role of inorganic and organic phases in the bone tissue, a detailed understanding of the bone composition is essential.

Bone is a heterogeneous composite material consisting of a mineral phase, calciumdeficient carbonated hydroxyapatite (CDHAp, Ca10−x(PO4)6−x(HPO4)x(OH)2−x), and organic phase, consisting of ~90% collagen type I, ~5% non-collagenous proteins, ~2% lipids and water [5–7]. The various ionic substitutions (e.g., Mg2+, Sr2+, Na<sup>+</sup> , K<sup>+</sup> , CO<sup>3</sup> <sup>2</sup>−) in the biological CDHAp structure result in a remarkably complex crystal structure with unique biological properties [7]. Proteins in the bone extracellular matrix can be divided into (i) structural proteins (collagen and fibronectin) and (ii) proteins with specialized functions (e.g., regulation of collagen fibril diameter, signaling molecules, growth factors, enzymes) [8]. Although these proteins are present in the bone structure in a relatively small amount of the total protein mass, they modulate a wide variety of bone key role functions such as regulation of mineralization, cell adhesion and bone resorption/remodeling [6]. Cortical bone is a dense outer surface of bone that forms a protective layer around the inner part, spongy or trabecular bone, in which the main metabolism functions occur [5]. The building blocks of bone tissue are mineralized collagen fiber, composite biomaterial of collagen type I and nano-sized CDHAp. The CDHAp crystals are deposited in parallel with the collagen fibers, and they are later formed by self-assembly of the collagen triple helix [7,9]. Type I collagen is a right-handed helix composed of three left-handed helix polypeptide chains with nonhelical ends, with molecular dimensions of ~300 nm in length and ~1.5 nm in diameter. The collagen triple helix is stabilized via direct inter-chain hydrogen bonds and inter- and intra-chain water-mediated hydrogen bonds [10]. Along with type I collagen, osteocalcin is the next most abundant protein within the bone organic matrix and plays a major role as a structure-directing molecule. It is assumed that osteocalcin mediates the nucleation and growth of platelet-shaped (~50 × 25 × 2 nm) CDHAp crystals [11]. Figure 1 shows a hierarchical structure of typical bone at various length scales. On the macro-length scale, the structure of cortical or compact bone consists of circles in cross-section (Haversian systems) with osteonic canals, while the trabecular part of the bone has a highly porous structure. On the nano-length scale, the structure framework is collagen fibers composed of bundles (triple helix) of mineralized collagen fibers [9]. hough these proteins are present in the bone structure in a relatively small amount of the total protein mass, they modulate a wide variety of bone key role functions such as regulation of mineralization, cell adhesion and bone resorption/remodeling [6]. Cortical bone is a dense outer surface of bone that forms a protective layer around the inner part, spongy or trabecular bone, in which the main metabolism functions occur [5]. The building blocks of bone tissue are mineralized collagen fiber, composite biomaterial of collagen type I and nano-sized CDHAp. The CDHAp crystals are deposited in parallel with the collagen fibers, and they are later formed by self-assembly of the collagen triple helix [7,9]. Type I collagen is a right-handed helix composed of three left-handed helix polypeptide chains with nonhelical ends, with molecular dimensions of ~300 nm in length and ~1.5 nm in diameter. The collagen triple helix is stabilized via direct inter-chain hydrogen bonds and inter- and intra-chain water-mediated hydrogen bonds [10]. Along with type I collagen, osteocalcin is the next most abundant protein within the bone organic matrix and plays a major role as a structure-directing molecule. It is assumed that osteocalcin mediates the nucleation and growth of platelet-shaped (~50 × 25 × 2 nm) CDHAp crystals [11]. Figure 1 shows a hierarchical structure of typical bone at various length scales. On the macrolength scale, the structure of cortical or compact bone consists of circles in cross-section (Haversian systems) with osteonic canals, while the trabecular part of the bone has a highly porous structure. On the nano-length scale, the structure framework is collagen fibers composed of bundles (triple helix) of mineralized collagen fibers [9].

proteins (collagen and fibronectin) and (ii) proteins with specialized functions (e.g., regulation of collagen fibril diameter, signaling molecules, growth factors, enzymes) [8]. Alt-

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**Figure 1.** The hierarchical structure of bone at various length scales. Adapted from [12] with permission from Elsevier. **Figure 1.** The hierarchical structure of bone at various length scales. Adapted from [12] with permission from Elsevier.

As biological apatites are characterized by various ionic substitutions that are crucial for bone metabolism, numerous studies have focused on the synthesis and characterization of biomimetic ionic-substituted hydroxyapatite, which is used as a bioactive phase in biomaterials for bone regeneration [12]. As a lot of efforts are put into mimicking the inorganic phase of the bone tissue, the same efforts are directed towards mimicking the organic phase. Combination of these mimicking biomaterials leads to composite material with a complex structure similar to natural bone tissue. Naturally derived polymers (e.g., collagen, gelatin, chitosan, glycosaminoglycans, silk fibrin) have been widely used in a variety of tissue engineering applications, as they can mimic a natural extracellular matrix. As natural polymers are building components of biological tissues, they demonstrate excellent biocompatibility in vivo and present a range of ligands and peptides that facilitate cell adhesion and osteogenic differentiation [3]. One of the most widely studied biopolymers is chitosan, a natural aminopolysaccharide with a unique structure and multidimensional properties suitable for a wide range of applications in biomedicine [13]. Along with bone tissue engineering applications, chitosan has been widely applied in drug delivery and gene therapy because of its excellent biocompatibility and biodegradability under As biological apatites are characterized by various ionic substitutions that are crucial for bone metabolism, numerous studies have focused on the synthesis and characterization of biomimetic ionic-substituted hydroxyapatite, which is used as a bioactive phase in biomaterials for bone regeneration [12]. As a lot of efforts are put into mimicking the inorganic phase of the bone tissue, the same efforts are directed towards mimicking the organic phase. Combination of these mimicking biomaterials leads to composite material with a complex structure similar to natural bone tissue. Naturally derived polymers (e.g., collagen, gelatin, chitosan, glycosaminoglycans, silk fibrin) have been widely used in a variety of tissue engineering applications, as they can mimic a natural extracellular matrix. As natural polymers are building components of biological tissues, they demonstrate excellent biocompatibility in vivo and present a range of ligands and peptides that facilitate cell adhesion and osteogenic differentiation [3]. One of the most widely studied biopolymers is chitosan, a natural aminopolysaccharide with a unique structure and multidimensional properties suitable for a wide range of applications in biomedicine [13]. Along with bone tissue engineering applications, chitosan has been widely applied in drug delivery and gene therapy because of its excellent biocompatibility and biodegradability under physiological conditions [14]. In addition, the chitosan structure allows chemical and mechanical modifications in order to obtain novel properties, functions and applications [13]. Prior to the development of scaffolds with appropriate regenerative properties, the in vivo regenerative process steps need to be taken into account. After scaffold implantation (1), proteins are absorbed in the scaffold interface (2), followed by infiltration of immune cells (3), the release of chemical signals by immune cells to recruit stem cells (4), microenvironment remodeling (5) and vascularization (6), as schematically shown in Figure 2 [14,15]. regenerative process steps need to be taken into account. After scaffold implantation (1), proteins are absorbed in the scaffold interface (2), followed by infiltration of immune cells (3), the release of chemical signals by immune cells to recruit stem cells (4), microenvironment remodeling (5) and vascularization (6), as schematically shown in Figure 2 [14,15].

physiological conditions [14]. In addition, the chitosan structure allows chemical and mechanical modifications in order to obtain novel properties, functions and applications [13]. Prior to the development of scaffolds with appropriate regenerative properties, the in vivo

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**Figure 2.** In vivo bone regeneration process after scaffold implantation. Created with BioRender.com (accessed on 30.06.2022.). **Figure 2.** In vivo bone regeneration process after scaffold implantation. Created with BioRender.com (accessed on 30 June 2022).

Constant progress in bone tissue engineering is ensuring the development of novel functional biomaterials that can solve current challenges in the field. However, more efforts are required to ensure the reproducibility of developed biomaterials and standardization of characterization methods, which could increase the ability to compare biomaterials properties conducted in different studies. Joined efforts and frequent analysis of available literature and design requirements could increase the development of scaffolds with appropriate characteristics. In this short review paper, which is organized into several sections, the author first outlines general knowledge about natural bone tissue and natural polymers, followed by chitosan structure and characteristics. The third section provides a summary of requirements for bone scaffold development (biocompatibility, porosity and pore size distribution, mechanical strength, biodegradation) with a focus on chitosan-based materials. The fourth section provides a summary of different methods for the preparation of chitosan-based biomaterials (molded macroporous, fiber-based, hydrogel, microspheres and 3D-printed scaffolds). The last section provides relevant and recent viewpoints from the literature on the composite scaffolds based on chitosan and calcium phosphates, calcium silicate and bioactive glass. Current trends in the design of chitosan-Constant progress in bone tissue engineering is ensuring the development of novel functional biomaterials that can solve current challenges in the field. However, more efforts are required to ensure the reproducibility of developed biomaterials and standardization of characterization methods, which could increase the ability to compare biomaterials properties conducted in different studies. Joined efforts and frequent analysis of available literature and design requirements could increase the development of scaffolds with appropriate characteristics. In this short review paper, which is organized into several sections, the author first outlines general knowledge about natural bone tissue and natural polymers, followed by chitosan structure and characteristics. The third section provides a summary of requirements for bone scaffold development (biocompatibility, porosity and pore size distribution, mechanical strength, biodegradation) with a focus on chitosan-based materials. The fourth section provides a summary of different methods for the preparation of chitosan-based biomaterials (molded macroporous, fiber-based, hydrogel, microspheres and 3D-printed scaffolds). The last section provides relevant and recent viewpoints from the literature on the composite scaffolds based on chitosan and calcium phosphates, calcium silicate and bioactive glass. Current trends in the design of chitosan-based scaffolds are highlighted and future perspectives are discussed.

### **2. Chitosan Structure and Characteristics**

**2. Chitosan Structure and Characteristics**  Chitosan is a partially deacetylated derivate of chitin, one of the most abundant polymers in nature found in the shells of crustaceans and walls of fungi. It is composed of randomly distributed β-(1-4)-linked D-glucosamine (glucosamine) and *N*-acetyl-D-glucosamine (*N*-acetylglucosamine) structure units, structurally similar to glycosaminoglycan, a key component of the bone matrix and cell surface which modulates the bioavailability and activity of various osteoclastic and osteogenic factors [5,16–18]. Deacetylation of chitin is almost never complete and the chitosan chain still contains amide groups to Chitosan is a partially deacetylated derivate of chitin, one of the most abundant polymers in nature found in the shells of crustaceans and walls of fungi. It is composed of randomly distributed β-(1-4)-linked D-glucosamine (glucosamine) and *N*-acetyl-Dglucosamine (*N*-acetylglucosamine) structure units, structurally similar to glycosaminoglycan, a key component of the bone matrix and cell surface which modulates the bioavailability and activity of various osteoclastic and osteogenic factors [5,16–18]. Deacetylation of chitin is almost never complete and the chitosan chain still contains amide groups to some extent [16]. The degree of deacetylation (DD, %) is defined as the molar fraction of glucosamine in the chitosan composed of *N*-acetylglucosamine and glucosamine structure units [19]. The DD of chitosan is defined as low (55–70%), middle (70–85%), high (85–95%) or ultrahigh (95–100%), where ultrahigh is difficult to achieve [20]. In Figure 3, the

based scaffolds are highlighted and future perspectives are discussed.

structures of chitin, chitosan and protonated chitosan as a water-soluble poly-electrolyte are shown.

shown.

ware.

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some extent [16]. The degree of deacetylation (DD, %) is defined as the molar fraction of glucosamine in the chitosan composed of *N*-acetylglucosamine and glucosamine structure units [19]. The DD of chitosan is defined as low (55–70%), middle (70–85%), high (85–95%) or ultrahigh (95–100%), where ultrahigh is difficult to achieve [20]. In Figure 3, the structures of chitin, chitosan and protonated chitosan as a water-soluble poly-electrolyte are

**Figure 3.** Structure of chitin, chitosan and protonated chitosan (water-soluble poly-electrolyte). The structures were obtained in the ChemDraw 7.0 (PerkinElmer, Massachusetts, United States) soft-**Figure 3.** Structure of chitin, chitosan and protonated chitosan (water-soluble poly-electrolyte). The structures were obtained in the ChemDraw 7.0 (PerkinElmer, Waltham, MA, USA) software.

Chitosan has poor solubility in physiological solvents (e.g., water) due to its strong intermolecular hydrogen bonding and it is considered a strong base due to primary amino groups with a pKa value of 6.3 [13,16]. Chitosan solution can be obtained in acidic aqueous (pH < 6) media, which protonate chitosan amino groups, rendering the polymer positively charged and thereby overcoming associative forces between chains [13,16,21]. If the pH of chitosan solution increases above 6, chitosan amino groups become deprotonated and the polymer chain loses its charge, which leads to insolubility. The solubility is highly dependent on the degree of the deacetylation, the used deacetylation method and molecular weight. The solubility of chitosan can be increased by chemical modifications possible at two hydroxyl functional groups in the polymer chain [13]. The detailed review paper by Upadhyaya et al. [22] provides an overview of the water-soluble carboxymethyl chitosan as a modification of the non-soluble chitosan. The highly desired properties of biomaterials designed for applications in the human organism are antibacterial properties without harmful effects on healthy cells. The polycationic nature of the chitosan chain is essential for antibacterial activity. The most probable pathway of chitosan antibacterial activity is by binding to the negatively charged bacterial cell wall (disruption of the cell membrane), followed by attachment to DNA (inhibition of DNA replication) and subsequently cell death. Electrostatic interaction between the polycationic structure and the predominantly anionic components of the microorganisms' surface, such as Gram-negative lipopolysac-Chitosan has poor solubility in physiological solvents (e.g., water) due to its strong intermolecular hydrogen bonding and it is considered a strong base due to primary amino groups with a pKa value of 6.3 [13,16]. Chitosan solution can be obtained in acidic aqueous (pH < 6) media, which protonate chitosan amino groups, rendering the polymer positively charged and thereby overcoming associative forces between chains [13,16,21]. If the pH of chitosan solution increases above 6, chitosan amino groups become deprotonated and the polymer chain loses its charge, which leads to insolubility. The solubility is highly dependent on the degree of the deacetylation, the used deacetylation method and molecular weight. The solubility of chitosan can be increased by chemical modifications possible at two hydroxyl functional groups in the polymer chain [13]. The detailed review paper by Upadhyaya et al. [22] provides an overview of the water-soluble carboxymethyl chitosan as a modification of the non-soluble chitosan. The highly desired properties of biomaterials designed for applications in the human organism are antibacterial properties without harmful effects on healthy cells. The polycationic nature of the chitosan chain is essential for antibacterial activity. The most probable pathway of chitosan antibacterial activity is by binding to the negatively charged bacterial cell wall (disruption of the cell membrane), followed by attachment to DNA (inhibition of DNA replication) and subsequently cell death. Electrostatic interaction between the polycationic structure and the predominantly anionic components of the microorganisms' surface, such as Gram-negative lipopolysaccharide and cell surface proteins, plays a key role in antibacterial activity [23].

charide and cell surface proteins, plays a key role in antibacterial activity [23]. As previously mentioned, protein adsorption is the first step to take place upon implantation. Protein adsorption occurs within a few minutes or even seconds after scaffold As previously mentioned, protein adsorption is the first step to take place upon implantation. Protein adsorption occurs within a few minutes or even seconds after scaffold implantation and the cells that reach the biomaterial surface no longer attach directly to the biomaterial but to the adsorbed protein layer. Through cell membrane-bound receptors or ligands, cells identify bioactive binding sites on the protein layer and behave according to the stimuli received [15]. As a natural positive-charged polysaccharide, protonable amino groups on the chitosan backbone electrostatically interact with the various negatively charged proteins [24]. Electrostatic interactions between biomaterial and proteins depend on the biomaterials' surface and protein charges, which are a function of pH and the solution ionic content. Usually, at low pH, proteins are positively charged, whereas at

high pH they are negatively charged [15]. Bovine serum albumin protein is often used as a model protein for biomaterial characterization regarding protein adsorption capacity, because of its high stability, availability at high purity and water solubility [25]. Interactions between BSA protein and chitosan chain depend on the pH and the interaction mechanism is highly complex. BSA protein is negatively charged at neutral pH and the electrostatic interaction of BSA with chitosan is governed by the following two factors: (i) the interaction between protonated chitosan amino groups and the dissociated carboxyl groups of BSA and (ii) the repulsion of the protonated amino groups of chitosan and BSA, as explained by Kim et al. [26]. The protein adsorption capacity of scaffolds needs to be determined, as protein adsorption is the first and crucial step after biomaterial implantation. However, to develop and design a suitable scaffold for bone tissue regeneration, numerous requirements need to be addressed.

### **3. Requirements for Bone Scaffold Development**

Scaffolds for bone tissue regeneration must be biocompatible, non-toxic and biodegradable, with an ability to mold into various geometries and forms suitable for cell seeding, migration, growth and differentiation. The structure should mimic the porous and phase structure of the natural bone while maintaining suitable mechanical properties [27,28].

### *3.1. Biocompatibility*

Biocompatibility is one of the essential requirements for materials used in tissue engineering applications. Biocompatible materials do not produce a toxic or immunological response in the human body [5]. In almost all published papers, chitosan is described as a non-toxic and biocompatible biopolymer safe for use in the human organism as a scaffold or drug carrier. However, the biocompatibility must be confirmed by biological evaluation for each chitosan-based biomaterial, as they might have different physicochemical characteristics due to different biogenic sources, chitosan type, molecular weight, DD of chitosan and different phases incorporated into the chitosan to obtain composite biomaterials with multifunctional characteristics. In addition, non-cytotoxicity is commonly assessed for 3 or 7 days of cell culture; however, the extended time period of evaluation should be considered. Along with the required extended cell culture time, the appropriate cell lines for bone applications should be used.

### *3.2. Porosity and Pore Size Distribution*

Porosity, pore size distribution and pore diameter are some of the most important factors for efficient cell attachment, migration, vascularization and tissue regeneration [29]. Cortical bone has a low porosity of 5–10%, whereas trabecular bone has a porosity of 50–90% [5]. During bone regeneration, interconnected pores in the scaffold are essential for the efficient diffusion of nutrient, oxygen and metabolic waste [30]. In order to design a functional scaffold, along with the porosity in the range of 50–90%, micro- (<20 µm) and macroporosity (>100–400 µm) need to be considered [5]. Microporosity is crucial for cell seeding and retention, capillaries growth, vascularization and cell-matrix interactions. Macroporosity promotes osteogenesis by enhancing cell migration, cell–cell network formation, vascularization, nutrient supply and metabolic waste diffusion [3,30]. Oh et al. [31] systematic study on pore size gradient scaffolds has shown that 380–405 µm pore size has better cell growth for chondrocytes and osteoblasts, whereas the scaffolds with 186–200 µm pore size were better for fibroblasts' growth. In addition, scaffolds with 290–310 µm pore size showed faster new bone formation than those of other pore sizes. Zhou et al. [32] obtained chitosan-based scaffolds with different bioactive phases, hydroxyapatite and whitlockite, and pore size of ~105 µm. In vivo studies have shown new bone formation within the scaffolds, meaning that pores of ~105 µm meet the requirements for efficient cell seeding and bone ingrowth. An innovative approach to obtain a multi-layered chitosan scaffold with a gradient of pore size (160–275 µm) for osteochondral defect repair was developed by Pitrolino et al. [33]. Osteogenic and chondrogenic differentiation of human mesenchymal

stem cells (MSCs) preferentially occurred in selected layers of the scaffold in vitro, driven by the distinct pore gradient and material composition. In the study by Ressler et al. [34], a multi-substituted (Sr2+, Mg2+, Zn2+ and SeO<sup>3</sup> <sup>2</sup>−) calcium phosphate/chitosan composite scaffold with a pore size in the range of 20–350 µm and a porosity of ~75% was prepared by the freeze-gelation method. The requirements for micro- and macroporosity were successfully achieved by adjusting the polymer concentration in the starting solution. Different pore size distributions in these studies indicate that, by using different preparation methods and chitosan concentration of the starting solution, the pore size distribution and porosity can be adjusted and controlled. The pore size distribution and porosity should be some of the main scaffold characteristics considered prior to scaffold development. If the pores are mainly micropores, seeded cells can clog the pores on the scaffold surface and disable diffusion, tissue ingrowth and regeneration. If the pores are mainly macropores, seeding of the cell would not be efficient and that might lead to parts of the scaffold where tissue regeneration is not possible.

### *3.3. Mechanical Strength*

The mechanical strength is a critical feature in bone regeneration and it is primarily controlled by pore volume and characteristics of used materials [22]. Optimum balance between porosity, pore size distribution and mechanical properties requirements is still a major challenge in the development of the scaffold. The compressive strength of a trabecular bone is 2–12 MPa, whereas for the cortical bone it is 100–230 MPa [35–37]. The mechanical properties of scaffolds for load-bearing applications should be such to successfully replace hard bone tissue [30]. The mechanical characteristics of chitosan scaffolds are significantly lower than the compressive strength and modulus of natural bone tissue. Reported compressive modulus and strength differ depending on the scaffold characteristics, but fall in the ranges of 0.0038–2.56 MPa [38]. Due to poor mechanical properties, chitosan-based scaffolds can be used for non-load-bearing applications, mainly as support for osteoblast cells to adhere, proliferate and differentiate into mature bone cells, producing mineralized extracellular matrix or as drug carriers [39]. Poor mechanical properties limit chitosanbased scaffolds to small bone loss in non-load-bearing implantation areas and improvement of such biomaterials is needed if they would be used for load-bearing applications [39,40]. An innovative approach to improve the mechanical properties of hydroxyapatite/chitosan scaffolds was reported by Rogina et al. [40]. A 3D-printed poly (lactic acid) (PLA) construct was used as a mechanical support, where large pores of 960 ± 50 µm allowed enough space to form a porous composite hydrogel by freeze-gelation technique. PLA and PLA/chitosan scaffolds show similar linear region behavior under loading with a modulus of 32.3 ± 5.4 and 27.3 ± 3.2 MPa, respectively, whereas composite scaffolds based on PLA and hydroxyapatite/chitosan hydrogel possessed lower stiffness with the modulus of 16.4 ± 2.5 MPa [40]. Depending on the application of chitosan-based scaffolds, mechanical properties are one of the main characteristics that should be considered during scaffold design. The design of a scaffold with appropriate porosity, pore diameter and mechanical properties is still a challenge, as these parameters are correlated and their compensation is required.

### *3.4. Biodegradation*

The ideal scaffold for bone regeneration should degrade at the same rate as the new tissue formation. If the rate of degradation is higher than the regeneration rate, the scaffold cannot provide support for the host tissue and the regeneration would not be efficient. At physiological conditions, chitosan undergoes physical (e.g., swelling, cracking, dissolution) and chemical (e.g., depolymerization, oxidation, non-enzymatic and enzymatic hydrolysis) degradation. Hydrolytic degradation of the glycosidic bonds between polysaccharide units occurs at a higher rate, making non-enzymatic hydrolytic mechanisms a minor part of chitosan degradation [41]. Chitosan can be enzymatically degraded in vivo by lysozyme, a polycationic protein present in the extracellular matrix in human bone tissue [30]. Lysozyme breaks the chitosan chain by cleaving the glycosidic bonds between polysaccharide units in

the polymer. As a result, the molecular weight of the polymer is reduced until eventual solubility and removal of degradation products occur. The degradation products are nontoxic, mainly composed of glucosamine and saccharide, which can then be easily extracted from the body without interference with organs. The degradation rate by each mechanism is related to the degree of crystallinity, which is controlled by the DD, where higher DD results in a lower degradation rate due to closer chain packing and hydrogen bonding [5]. The lysozyme concentration in the extracellular matrix of human tissues can increase up to 1000-fold the amount usually found in serum (0.95–2.45 µm) [42–44]. Therefore, it is important to determine the degradation rate of chitosan scaffolds at lower and higher concentrations of lysozyme to examine scaffold stability under physiological conditions. tion products are non-toxic, mainly composed of glucosamine and saccharide, which can then be easily extracted from the body without interference with organs. The degradation rate by each mechanism is related to the degree of crystallinity, which is controlled by the DD, where higher DD results in a lower degradation rate due to closer chain packing and hydrogen bonding [5]. The lysozyme concentration in the extracellular matrix of human tissues can increase up to 1000-fold the amount usually found in serum (0.95–2.45 μm) [42–44]. Therefore, it is important to determine the degradation rate of chitosan scaffolds at lower and higher concentrations of lysozyme to examine scaffold stability under physiological conditions. **4. Chitosan Three-Dimensional Scaffolds** 

The ideal scaffold for bone regeneration should degrade at the same rate as the new tissue formation. If the rate of degradation is higher than the regeneration rate, the scaffold cannot provide support for the host tissue and the regeneration would not be efficient. At physiological conditions, chitosan undergoes physical (e.g., swelling, cracking, dissolution) and chemical (e.g., depolymerization, oxidation, non-enzymatic and enzymatic hydrolysis) degradation. Hydrolytic degradation of the glycosidic bonds between polysaccharide units occurs at a higher rate, making non-enzymatic hydrolytic mechanisms a minor part of chitosan degradation [41]. Chitosan can be enzymatically degraded in vivo by lysozyme, a polycationic protein present in the extracellular matrix in human bone tissue [30]. Lysozyme breaks the chitosan chain by cleaving the glycosidic bonds between polysaccharide units in the polymer. As a result, the molecular weight of the polymer is reduced until eventual solubility and removal of degradation products occur. The degrada-

### **4. Chitosan Three-Dimensional Scaffolds** Due to chitosan's physical and chemical properties, various types of scaffolds

Due to chitosan's physical and chemical properties, various types of scaffolds (molded macroporous, fiber-based, hydrogel, microspheres and 3D-printed) can be obtained (Figure 4) for specific treatments that require unique properties. Increasing attention has been gained by 3D-printed chitosan-based scaffolds in recent years, as this technique enables the biofabrication of patient-personalized scaffolds with highly complex geometries. In recent years, a few high-quality review papers on the 3D printing of chitosan, including bioprinting, were published by Rajabi et al. [45], Taghizadeh et al. [46] and Yadav et al. [47]. (molded macroporous, fiber-based, hydrogel, microspheres and 3D-printed) can be obtained (Figure 4) for specific treatments that require unique properties. Increasing attention has been gained by 3D-printed chitosan-based scaffolds in recent years, as this technique enables the biofabrication of patient-personalized scaffolds with highly complex geometries. In recent years, a few high-quality review papers on the 3D printing of chitosan, including bioprinting, were published by Rajabi et al. [45], Taghizadeh et al. [46] and Yadav et al. [47].

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*3.4. Biodegradation* 

**Figure 4.** Different designs of chitosan-based three-dimensional scaffolds. Created with BioRender.com (accessed on 30.06.2022.). **Figure 4.** Different designs of chitosan-based three-dimensional scaffolds. Created with BioRender. com (accessed on 30 June 2022).

### *4.1. Molded Macroporous Scaffolds 4.1. Molded Macroporous Scaffolds*

Compared to the fibers, hydrogel, microspheres and 3D-printed scaffolds, molded chitosan scaffolds are the most studied. The most commonly used method is phase separation and lyophilization, where molded chitosan solution is frozen to allow phase separation [48]. As acetic acid is most commonly used for dissolving chitosan, after the lyophilization the neutralization of chitosan acetate salt is required to prevent scaffold dissolution in the aqueous media. The freeze-gelation method is similar to a previously explained method where after phase separation due to freezing, scaffolds are placed in the gelation solution of sodium hydroxide and ethanol below the chitosan freezing temperature. Fol-Compared to the fibers, hydrogel, microspheres and 3D-printed scaffolds, molded chitosan scaffolds are the most studied. The most commonly used method is phase separation and lyophilization, where molded chitosan solution is frozen to allow phase separation [48]. As acetic acid is most commonly used for dissolving chitosan, after the lyophilization the neutralization of chitosan acetate salt is required to prevent scaffold dissolution in the aqueous media. The freeze-gelation method is similar to a previously explained method where after phase separation due to freezing, scaffolds are placed in the gelation solution of sodium hydroxide and ethanol below the chitosan freezing temperature. Following the gelation, scaffolds are washed with ethanol and lyophilized (dried). A combination of the described methods can be used [34,40]. To obtain the desired pore dimension and shape, the polymer concentration, freezing speed and freezing temperature need to be adjusted. In addition, prior to the step phase separation/lyophilization method, porogens can be added to the chitosan solution. The porogens are later leached from the scaffold, leading to additional porosity. When porogens are used without combination with the separation/lyophilization method, the drawback is that this method leads to a lack of control over the interconnectivity of pores inside the scaffold structure. Further, the gas foaming technique can be used alone or in combination with porogens to obtain an open porosity of scaffolds. The high-pressure carbon dioxide (CO2) is allowed to saturate the polymeric solution, which causes clusters in the solution and induces porosity [30].
