*4.3. Hydrogel*

Conventional methods for applications in tissue engineering (pre-formed hydrogels and scaffolds) face the problem of surgical implantation, increasing the risk of infections and improper scaffold shape and size [54]. In the last decade, smart injectable hydrogels have gained increasing attention because they can be used in minimally invasive treatments [55]. Detailed review papers, regarding mechanisms of injectable hydrogel formation and application in adipose, bone, cartilage, intervertebral discs and muscle tissue engineering were published by Sivashanmugam et al. [54] and Gasperini et al. [56]. Smart injectable gelling systems are liquid at room temperature, and then form gels when injected into the fractured location, filling the complex shape of the defect [55]. Such hydrogels should shorten the surgical operation time, minimize the damage effects of large muscle retraction, reduce the size of scars and lessen post-operative pain, allowing patients to achieve rapid recovery in a cost-effective manner [57]. Hydrogels can be used in non-load-bearing applications to carry and protect cells, proteins, growth factors or drugs, and ensure adequate permeability for the transport of cells' nutrients and metabolites [58]. A highly important characteristic of injectable hydrogels is gelation time, as slow gelation can cause delocalized gel formation due to the gel precursor diffusion [59]. Hydrogels derived from naturally occurring polysaccharides mimic many features of the extracellular matrix. Therefore, they can direct the migration, growth and organization of encapsulated cells during tissue regeneration [16]. As previously mentioned, chitosan is a pH-responsive polymer, as in mild acids it is soluble and upon neutralization it forms a hydrogel. This occurs due to the removal of repulsive electrostatic interactions during the neutralization process, thereby allowing the amino groups to interact via intermolecular hydrogen bonding [46]. The anionic nature of most human tissues, due to the presence of glycosaminoglycans in the extracellular matrix and the cationic character of chitosan, allows adherence of these hydrogels to tissue sites [19]. In addition, as the cells are negatively charged, positively charged scaffolds are expected to provide a more suitable environment for attachment due to ionic or electrostatic interactions [54]. Due to the polycationic nature of chitosan, pH and thermally induced physical cross-linked hydrogels are highly interesting, as they can be obtained without using cross-linking agents that might be toxic to the human organism. Glycerophosphate salts are widely used for obtaining pH and thermo-sensitive injectable chitosan hydrogels; however, sodium bicarbonate (NaHCO3) can also be used as a gelling agent [60,61]. In situ synthesized hydroxyapatite in a chitosan solution (10 ◦C) was used to obtain pH-responsive hydrogel at 37 ◦C. A slightly acidic environment of prepared composite solution favors NaHCO<sup>3</sup> dissociation that releases HCO<sup>3</sup> − ions responsible for carbon dioxide production and pH increases (Figure 5). Although the sol-gel transitions in chitosan solutions with NaHCO<sup>3</sup> as a gelling agent appeared to be thermally sensitive upon the temperature increase, these systems performed the pH-induced gelation process. The decrease in the apparent charge density of chitosan molecules allows the formation of the three-dimensional chitosan network due to physical junctions of hydrogen bonds [61].

glycosaminoglycans in the extracellular matrix and the cationic character of chitosan, allows adherence of these hydrogels to tissue sites [19]. In addition, as the cells are negatively charged, positively charged scaffolds are expected to provide a more suitable environment for attachment due to ionic or electrostatic interactions [54]. Due to the polycationic nature of chitosan, pH and thermally induced physical cross-linked hydrogels are highly interesting, as they can be obtained without using cross-linking agents that might be toxic to the human organism. Glycerophosphate salts are widely used for obtaining pH and thermo-sensitive injectable chitosan hydrogels; however, sodium bicarbonate (Na-HCO3) can also be used as a gelling agent [60,61]. In situ synthesized hydroxyapatite in a chitosan solution (10 °C) was used to obtain pH-responsive hydrogel at 37 °C. A slightly acidic environment of prepared composite solution favors NaHCO3 dissociation that releases HCO3− ions responsible for carbon dioxide production and pH increases (Figure 5). Although the sol-gel transitions in chitosan solutions with NaHCO3 as a gelling agent appeared to be thermally sensitive upon the temperature increase, these systems performed the pH-induced gelation process. The decrease in the apparent charge density of chitosan molecules allows the formation of the three-dimensional chitosan network due to physical

**Figure 5.** Preparation of physically cross-linked chitosan-hydroxyapatite (Cht-HA) hydrogel, with permission from Elsevier [61]. **Figure 5.** Preparation of physically cross-linked chitosan-hydroxyapatite (Cht-HA) hydrogel, with permission from Elsevier [61].

### *4.4. Microspheres 4.4. Microspheres*

junctions of hydrogen bonds [61].

Due to biocompatibility and biodegradability, chitosan microsphere systems have been proposed for use as injectable bone-filling (non-load-bearing) biomaterial and/or drug delivery matrices [62]. Chitosan microspheres for drug delivery and preparation methods were summarized in the review paper by Mitra and Dey [63]. Cell microcarriers in the form of injectable scaffolds offer advantages similar to ones characteristic of injectable hydrogels that repair complex-shaped tissue defects with minimal surgical intervention [64]. Wang et al. [65] fabricated collagen/chitosan-based microspheres (diameter of 200 μm) via the emulsification method by using glutaraldehyde as a cross-linking agent. Due to biocompatibility and biodegradability, chitosan microsphere systems have been proposed for use as injectable bone-filling (non-load-bearing) biomaterial and/or drug delivery matrices [62]. Chitosan microspheres for drug delivery and preparation methods were summarized in the review paper by Mitra and Dey [63]. Cell microcarriers in the form of injectable scaffolds offer advantages similar to ones characteristic of injectable hydrogels that repair complex-shaped tissue defects with minimal surgical intervention [64]. Wang et al. [65] fabricated collagen/chitosan-based microspheres (diameter of 200 µm) via the emulsification method by using glutaraldehyde as a cross-linking agent. Obtained microspheres have shown stability for at least 90 days and good biological properties by supporting attachment and proliferation of the cells. As previously described, chitosan degradation products are not toxic for cells and the human organism; however, to obtain stable chitosan microspheres, chemical cross-linking is required to cross-link amino groups in the chitosan chain. As suggested by Fang et al. [64], residual cross-linking agents in microspheres might have a toxic effect towards cells, surrounding tissue and the human organism. Complete removal of unreacted cross-linking agents from obtained scaffolds remains a challenge. To overcome these drawbacks of chemically cross-linked chitosan, the introduction of a bioactive polyanionic biopolymer to interact electrostatically with the amino groups of chitosan to form polyelectrolyte complexes (PEC) has been proposed. Fang et al. [64] obtained poly(L-glutamic acid)/chitosan PEC porous microspheres by electrostatic interactions. It was determined that the pore size distribution, porosity, structure and stability of microspheres are dependent on freezing temperature and polymer concentration (Figure 6). An additional study with an approach free of toxic cross-linking agents was conducted by Huang et al. [66]. Highly porous chitosan microspheres were prepared through an emulsion-based thermally induced phase method with an average diameter of microspheres of ~150 µm and with interconnected pores in the range of 20–50 µm. Obtained microspheres showed excellent biocompatibility with multidirectional cell–cell interactions. Another approach to avoid cross-linking agents to produce stable chitosan-based microspheres is through physical cross-linking via chelation interactions of copper and zinc with chitosan, as recently reported by Lonˇcarevi´c et al. [67] and Rogina et al. [68]. The studies highlight the alternative approach to produce stable chitosan-based microspheres by using simple complexation reactions through transition metal ions. interactions of copper and zinc with chitosan, as recently reported by Lončarević et al. [67] and Rogina et al. [68]. The studies highlight the alternative approach to produce stable chitosan-based microspheres by using simple complexation reactions through transition metal ions.

Obtained microspheres have shown stability for at least 90 days and good biological properties by supporting attachment and proliferation of the cells. As previously described, chitosan degradation products are not toxic for cells and the human organism; however, to obtain stable chitosan microspheres, chemical cross-linking is required to cross-link amino groups in the chitosan chain. As suggested by Fang et al. [64], residual cross-linking agents in microspheres might have a toxic effect towards cells, surrounding tissue and the human organism. Complete removal of unreacted cross-linking agents from obtained scaffolds remains a challenge. To overcome these drawbacks of chemically cross-linked chitosan, the introduction of a bioactive polyanionic biopolymer to interact electrostatically with the amino groups of chitosan to form polyelectrolyte complexes (PEC) has been proposed. Fang et al. [64] obtained poly(L-glutamic acid)/chitosan PEC porous microspheres by electrostatic interactions. It was determined that the pore size distribution, porosity, structure and stability of microspheres are dependent on freezing temperature and polymer concentration (Figure 6). An additional study with an approach free of toxic cross-linking agents was conducted by Huang et al. [66]. Highly porous chitosan microspheres were prepared through an emulsion-based thermally induced phase method with an average diameter of microspheres of ~150 μm and with interconnected pores in the range of 20–50 μm. Obtained microspheres showed excellent biocompatibility with multidirectional cell–cell interactions. Another approach to avoid cross-linking agents to produce stable chitosan-based microspheres is through physical cross-linking via chelation

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**Figure 6.** Scanning electron micrographs of porous chitosan microspheres fabricated from chitosan at a concentration of 2% at different temperatures. Reprinted from [64] with permission from Elsevier. **Figure 6.** Scanning electron micrographs of porous chitosan microspheres fabricated from chitosan at a concentration of 2% at different temperatures. Reprinted from [64] with permission from Elsevier.

For usage as a bone-filling biomaterial, the drawbacks of pure chitosan microspheres are lack of bone-binding ability and burst release problems. To overcome limitations, Ding et al. [62] proposed that obtaining composite microspheres based on chitosan and hydroxyapatite can lead to an increase in bone-binding ability. In vivo studies on apatite-coated chitosan microsphere conducted by Xu et al. [69] showed bone formation after 7 days. Further, hydroxyapatite/sodium alginate/chitosan composite microspheres, reported by Bi et al. [70], were prepared by an emulsion cross-link technique where calcium ions were used as a cross-linking agent. However, the microspheres as microcarriers often only enabled cell attachment and growth on the surface due to low or closed porosity [62,66]. Although multiple chitosan-based microspheres have been developed, minority studies report highly porous microspheres with open porosity that enable cell migration and cell– For usage as a bone-filling biomaterial, the drawbacks of pure chitosan microspheres are lack of bone-binding ability and burst release problems. To overcome limitations, Ding et al. [62] proposed that obtaining composite microspheres based on chitosan and hydroxyapatite can lead to an increase in bone-binding ability. In vivo studies on apatitecoated chitosan microsphere conducted by Xu et al. [69] showed bone formation after 7 days. Further, hydroxyapatite/sodium alginate/chitosan composite microspheres, reported by Bi et al. [70], were prepared by an emulsion cross-link technique where calcium ions were used as a cross-linking agent. However, the microspheres as microcarriers often only enabled cell attachment and growth on the surface due to low or closed porosity [62,66]. Although multiple chitosan-based microspheres have been developed, minority studies report highly porous microspheres with open porosity that enable cell migration and cell– cell interactions in the 3D environment. However, even with low or closed porosity, the advantage of microspheres for use in biomedical applications over a granular approach for bone repair is a larger specific surface area, which can improve cell adhesion and proliferation [70]. In addition, chitosan microspheres can be used as a filler component in molded scaffolds based on other polymers [71], or microspheres can be molded to obtain highly porous scaffolds [72].

### **5. Chitosan Composite Scaffolds**

The extracellular matrix in natural bone tissue supports cell attachment, proliferation and differentiation. Scaffolds for bone regeneration should mimic the natural ECM as much as possible. In particular, integration of multiple stimuli in scaffolds including physical (e.g., porosity, pore size distribution, topography, stiffness) and biochemical (e.g., growth factors, key role elements, genes, proteins) factors similar to natural bone tissue will improve scaffold efficacy [3,12]. MSCs are used in regenerative medicine because of their potential for self-renewal and multipotency. Multipotent stem cells can differentiate into multiple lineages (e.g., myocyte, adipocyte, osteoblast, chondrocyte, neuron), as schematically shown in Figure 7 [73,74]. Biomaterials can direct MSC attachment, proliferation and differentiation into different cell types and this can be controlled by the optimization of material characteristics such as composition, geometry, pore size, porosity, topography, stiffness, etc. [75]. Chitosan and its polymer-based composites are often used for the development of materials due to the similarity of the polysaccharide structure to the glycosaminoglycans

of cartilage and as it can direct MSCs differentiation towards chondrocyte cell type (chondrogenesis) [76–78]. However, in order for chitosan-based scaffolds to mimic natural bone tissue and stimulate differentiation into osteoblast cell types (osteogenesis), chitosan is often combined with inorganic phases. Calcium phosphates, calcium silicates and bioactive glass are among the most studied bioactive components within the chitosan matrix to mimic naturally occurring mineral phases and stimulate the osteogenic differentiation of cells. An innovative approach was reported by Pitrolino et al. [33] and Erickson et al. [79], who obtained a multi-layered chitosan scaffold for osteochondral defect repair with the incorporation of the highly porous layer based on chitosan and hydroxyapatite for bone regeneration. saccharide structure to the glycosaminoglycans of cartilage and as it can direct MSCs differentiation towards chondrocyte cell type (chondrogenesis) [76–78]. However, in order for chitosan-based scaffolds to mimic natural bone tissue and stimulate differentiation into osteoblast cell types (osteogenesis), chitosan is often combined with inorganic phases. Calcium phosphates, calcium silicates and bioactive glass are among the most studied bioactive components within the chitosan matrix to mimic naturally occurring mineral phases and stimulate the osteogenic differentiation of cells. An innovative approach was reported by Pitrolino et al. [33] and Erickson et al. [79], who obtained a multi-layered chitosan scaffold for osteochondral defect repair with the incorporation of the highly porous layer based on chitosan and hydroxyapatite for bone regeneration.

cell interactions in the 3D environment. However, even with low or closed porosity, the advantage of microspheres for use in biomedical applications over a granular approach for bone repair is a larger specific surface area, which can improve cell adhesion and proliferation [70]. In addition, chitosan microspheres can be used as a filler component in molded scaffolds based on other polymers [71], or microspheres can be molded to obtain

The extracellular matrix in natural bone tissue supports cell attachment, proliferation and differentiation. Scaffolds for bone regeneration should mimic the natural ECM as much as possible. In particular, integration of multiple stimuli in scaffolds including physical (e.g., porosity, pore size distribution, topography, stiffness) and biochemical (e.g., growth factors, key role elements, genes, proteins) factors similar to natural bone tissue will improve scaffold efficacy [3,12]. Mesenchymal stem cells (MSCs) are used in regenerative medicine because of their potential for self-renewal and multipotency. Multipotent stem cells can differentiate into multiple lineages (e.g., myocyte, adipocyte, osteoblast, chondrocyte, neuron), as schematically shown in Figure 7 [73,74]. Biomaterials can direct MSC attachment, proliferation and differentiation into different cell types and this can be controlled by the optimization of material characteristics such as composition, geometry, pore size, porosity, topography, stiffness, etc. [75]. Chitosan and its polymer-based composites are often used for the development of materials due to the similarity of the poly-

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highly porous scaffolds [72].

**5. Chitosan Composite Scaffolds** 

**Figure 7.** Mesenchymal stem cells differentiation. Created with BioRender.com (accessed on 2 July 2022).

### *5.1. Calcium Phosphates*

Calcium phosphates (e.g., hydroxyapatite, α-tricalcium phosphate, β-tricalcium phosphate) are among the most studied bioactive phases combined with the chitosan matrix. Inspired by natural bone tissue, numerous studies are focused on the scaffolds, where chitosan provides an organic matrix mimicking naturally occurring collagen, while calcium phosphate crystals mimic naturally occurring minerals (apatite). Numerous studies have confirmed the osteogenic properties of chitosan/calcium phosphate-based scaffolds. Rogina et al. [80,81] confirmed that compressive strength and swelling capacity measured in physiological conditions have shown that the critical hydroxyapatite portion, which improves chitosan properties, does not exceed 30 wt%, while further studies in perfusion conditions confirmed the best influence of hydroxyapatite on hMSC proliferation and osteoinduction on composite scaffolds with 30% of hydroxyapatite. A higher apatite fraction indicated poor mineralization of hMSCs extracellular matrix. Siddiqui et al. [82] prepared composite scaffolds based on chitosan and β-tricalcium phosphate cross-linked with genipin, confirming osteogenic differentiation during 21 days of cell culture. In recent years, to mimic the chemical composition of natural apatite, calcium phosphates were substituted with various key role ions and combined with chitosan. Ran et al. [83] obtained Mg-, Zn-, Sr- and Si-doped hydroxyapatite/chitosan hydrogels and confirmed that the Sr-chitosan/hydroxyapatite hydrogel exhibited the highest proliferation potential among

the cultured cells compared to the samples with other ions. To further enhance osteogenic properties and improve mechanical properties, Zhang et al. [84] obtained composite scaffolds based on silk fibroin, carboxymethyl chitosan, strontium substituted hydroxyapatite and cellulose. Mansour et al. [85] prepared a chitosan-based scaffold loaded with Ag/Mgco-substituted hydroxyapatite. Ghorbani et al. [86] prepared an electrospun scaffold (fiber diameter 210 nm) based on PCL, chitosan and zinc-doped hydroxyapatite, showing a positive effect on cell attachment and proliferation. Further, the synergic effect of Sr2+, Mg2+ , Zn2+ and SeO<sup>3</sup> <sup>2</sup><sup>−</sup> ions was confirmed in perfusion conditions. It has been determined that ions have a significant influence on the expression of characteristic bone genes (alkaline phosphatase, bone sialoprotein, collagen type I and dentin matrix protein I), phosphate deposits and newly formed bone tissue. Recent studies have shown better osteogenic properties of substituted calcium phosphate/chitosan scaffolds compared to the non-substituted scaffolds [34]. Therefore, further studies can be focused on using substituted calcium phosphates with chitosan and other polymers, such as collagen, which can further increase osteogenic properties.
