*5.2. Calcium Silicate*

Along with calcium phosphates and silicates, calcium silicates are promising biocompatible ceramic materials that can provide a microenvironment suitable for bone tissue regeneration. Along with Ca2+ ions, silicate ions have a key role in the bone regeneration process, as they can regulate MEK and PKC pathways [87]. In a recent study by Zhou et al. [88], it has been demonstrated that calcium silicate had significantly greater osteoinductive capacity both in vitro and in vivo compared with the traditional clinically used β-tricalcium phosphate bioceramics. Further, ionic substitutions are not only studied for calcium phosphates. In recent years, increasing attention has been directed towards substituted calcium silicates, especially for substitutions with Sr2+ ions [89–91]. Due to the positive effect of calcium silicates on bone regeneration, significant efforts have been directed toward obtaining scaffolds based on chitosan and calcium silicates. Peng et al. developed a lanthanum- [87] and gadolinium [92] -doped mesoporous calcium silicate/chitosan scaffold, which supports the adhesion, proliferation and differentiation of MSCs. Lin et al. [93] enhanced calcium silicate properties by obtaining composite scaffolds based on chitosan to ensure the antibacterial properties of the scaffolds. Mukherjee et al. [94] reported improved osteoblast function (viability, adhesion and proliferation) on titanium implant surfaces coated with a nanocomposite based on apatite, wollastonite (CaSiO3) and chitosan. A significant increase in the expression of osteocalcin and mineralization, compared to a non-treated substrate, confirmed the biocompatibility of the composite coating and its ability to initiate early osseointegration. Further, Genasan et al. [95] confirmed that the addition of calcium silicate (40% *w*/*w*) into gellan-chitosan scaffolds induces osteogenic differentiation of mesenchymal stromal cells where significant depositions of minerals, along with the expression of osteogenic genes, including BMP2, Run2, osteocalcin and osteonectin, were detected. Even though the calcium silicate-based scaffolds have shown desired properties for bone regeneration, more studies are focused on scaffold development and characterization based on calcium phosphates. Future studies should be focused on the development of scaffolds based on calcium silicates and substituted calcium silicates within the chitosan matrix and should be compared to similar scaffolds based on calcium phosphates and chitosan.

### *5.3. Bioactive Glass*

Along with calcium phosphates, bioactive glasses are used as bioactive fillers in chitosan-based scaffolds to increase cell response and osteogenic properties. Bioactive glasses are widely used for bone tissue regeneration due to their chemical interactions in vivo, where osteointegration is promoted by the formation of a calcium phosphate layer [96]. When included in the chitosan matrix, bioactive glasses enhance the metabolic activity of cells and mineralization [97]. Saatchi et al. [98] reported chitosan/polyethylene oxide nanofibrous scaffolds containing different amounts of cerium-doped bioactive glass. It has been determined that increasing the content of cerium-doped bioactive glass, cell adhesion and spreading have been enhanced. Further, fibroblast cells spread across the composite scaffold and took a 3D shape; however, there was no sign of cell expansion on the polymer scaffold without cerium-doped bioactive glass. In addition, composites based on chitosan and bioactive glasses are used as coating materials on AZ91 Mg alloy [99], 316 L stainless steel [100], WE43 Mg alloy [101,102] and Ti-6Al-4V [103] to improve the biocompatibility and bioactivity of metallic substrates for biomedical applications. To prevent the formation of biofilm on orthopedic implants, coatings based on chitosan and bioactive glasses can be combined with different drugs (e.g., vancomycin) to prevent the adhesion and proliferation of bacteria, as reported by Zarghami et al. [104]. Further, Sergi et al. [105] prepared a composite based on commercial passive gauzes, chitosan and bioactive glass doped with Sr2+, Mg2+ and Zn2+ ions for wound healing. It was determined that wound dressings with obtained composite material showed higher bioactivity compared to wound dressings with pure chitosan. The release of Sr2+, Mg2+ and Zn2+ ions enhanced cell proliferation and wound healing rate. A composite system based on chitosan and doped bioactive glass could be further examined for bone tissue engineering applications. In addition to a research paper, a detailed review paper by Sergi et al. [106] provided an overview of studies on bioactive glasses and natural polymer composites for medical devices for both soft and hard tissues. Due to different ions present in the bioactive glasses, further studies should provide additional comparative studies and a better understanding of the significance of each ionic component in bioactive glasses and its influence on the osteogenic properties of chitosan scaffolds.

### **6. Conclusions and Future Perspective**

Various chitosan-based materials have been designed and reported in the literature. However, more efforts are required to address current challenges to bring developed biomaterials to clinical use and application. Prior to the scaffold design, the researcher should consider all requirements for in vivo studies, clinical trials and mass productions. *ISO 10993 Biological evaluation of medical devices—Part 1: Evaluation and testing within a risk management process* should be considered prior to biological evaluation. Further, the characterization of chitosan-based materials for bone regeneration is not standardized. Even if there is a large number of papers on chitosan-based scaffolds as potential materials for bone regeneration, different methods for characterization are used. Therefore, the results of different studies cannot be properly compared and a final conclusion on material potential cannot be conducted. In recent years, the standardization of protocols and regulation of biomaterials has become highly required in order to improve technology transfer and increase the amount of commercially available products [107–109]. An additional challenge characteristic for naturally derived polymers is that the different properties depend on the source and preparation method. This further disables comparison between obtained scaffolds from different studies. With joint efforts from researchers by following requirements for biomaterials design and characterization, more developed biomaterials could be translated to clinical trials and be approved for commercial use.

**Funding:** This research received no external funding.

**Acknowledgments:** Smart Servier Medical Art (http://smart.servier.com/, accessed on 30 June 2022) and BioRender.com are gratefully acknowledged.

**Conflicts of Interest:** The author declares no conflict of interest.

### **References**

