Among the various natural polymers, for biomedical applications, water-soluble polymers with outstanding biological properties are preferred over polymers soluble only in organic and inorganic solvents [1]. Chitosan is a polysaccharide and a chemical derivative of chitin [2]. Chitins are polymers based on the disaccharide 2-acetamido-2- deoxy-β-D-glucose linked by β (1→ 4) linkage. Chitosan is a heteropolymer (Figure 1) that exhibits both glucosamine and acetylglucosamine groups because deacetylation is incomplete [3].

Figure 1. Schematic representation of the structure of chitosan polymer

Chitosan properties include physicochemical and biological properties. Chitosan is biodegradable and hemostatic. In addition to its numerous biological properties such as antimicrobial activity, hemostasis, macrophage immune responses, ability to stimulate pro-inflammatory and anti-inflammatory cytokines, lipids, growth factors and various chemokines, polyelectrolyte complex formation, mucoadhesive strength and higher fragility of tablets, it is preferred for various biomedical products [4]. Chitosan has found its way into various medical applications including tissue regeneration, where the properties of nanoparticles have made them interesting for the fabrication of nanocomposites, especially for bone tissue regeneration [5]. Cells cultured on chitosan- based nanocomposites proliferate rapidly and such scaffolds promote cell differentiation without any growth factors, which is very beneficial for tissue regeneration [6]. Therefore, for tissue engineering, the development of a substrate or biomaterial that mimics the cellular environment is a key factor [7]. When fabricating a substrate scaffold for tissue regeneration, many characteristics such as cytotoxicity, mechanical properties, as well as healing efficiency should be considered. The basis for selecting a suitable biomaterial for bone tissue engineering is a thorough understanding of bone anatomy and the healing process. Bone healing is a complex process, and the rate of healing and success varies from one individual to another. In the context of selecting suitable biomaterials, chitosan nanocomposites with nanofillers such as hydroxyapatite are widely used for bone tissue engineering applications. Hydroxyapatite has a hexagonal crystal structure, specifically with the space group P63/m. It can be described as a structure in which calcium (Ca) ions form chains along the c-axis, and phosphate (PO4) and hydroxyl (OH) groups fill the spaces between them (Figure 2).

Hydroxyapatite (HAp) is a biocompatible and bioactive ceramic nanocomposite that is widely used in tissue engineering due to its similarity to the mineral component of bone [8].

Figure 2. Structure of hydroxyapatite

Furthermore, synthetic HAp has been the most stable calcium phosphate in in vitro biological studies because it is nontoxic, induces minimal inflammatory response, and is osteogenic [9-12]. The biocompatibility of synthetic HAp has been previously investigated in various studies [13-16]. Lin et al. (2009) reported that HAp is biocompatible and enhances the induction of osteogenesis and osteogenic differentiation of mouse stem cells [17].

Jing et al. (2021) evaluated the cytotoxicity and biocompatibility of a biofunctional HAp gradient coating on MC3T3-E1 osteoblasts using an in vitro cell proliferation assay. Overall, the experiments showed that the HAp-based coating was not cytotoxic and was favorable for osteoblast proliferation and facilitated uniform cell distribution on the biomaterial [13]. Considering these factors, HAp has emerged as a suitable alternative in the field of bone regeneration, so nanohydroxyapatite is a very promising option for bone regeneration due to its chemical composition and crystal structure, which closely resembles bone minerals [19, 18].

The extracellular matrix of bone has both inorganic and organic components. The inorganic part, consisting of calcium, calcium carbonate, and phosphate, known as hydroxyapatite, is the source of bone strength. While the organic part, which is mostly composed of type I collagen, provides flexibility and adhesion to tissue and cells, respectively. Decellularized bone is often used as a scaffold in bone tissue engineering due to its ability to remove cellular components and antigens, osteogenic and biomechanical properties, as well as its physiological similarity to bone matrix [20]. The main function of bone is to provide load-bearing capacity for the skeleton and protect internal organs. Bone undergoes a continuous cycle of resorption and remodeling [22, 21].

Although our body can continuously repair bone tissue defects, larger defects require medical treatment. Therefore, considering the above, this study aimed to investigate the effect of scaffolds containing chitosan and hydroxyapatite in bone tissue engineering by freeze-drying method as a systematic review. Evaluation of this issue is needed, as it can determine the direction of future research.

The research design was a systematic review using the Open Science Framework (OSF) for systematic reviews. This study retrieved and screened articles related to chitosan and hydroxyapatite-containing scaffolds, bone tissue engineering, and freeze-drying methods from international databases using relevant keywords. The study was conducted in June 2025 and was approved by the review board.

Selection criteria for this study

Studies that met the following criteria were included in this study: including studies of chitosan and nanohydroxyapatite-containing scaffolds in their matrix in the field of tissue engineering and bone tissue, reviewed, and published in English. The reviewers collectively determined the exclusion criteria, which included studies published in languages other than English; clinical reports, opinions, editorials, review articles; guidelines, consensus documents, or expert position papers; opinions, letters, short reports, proceedings, or protocol studies; Publications with incomplete data; and meta- analysis articles and studies lacking full text were excluded. Studies that met at least one of the exclusion criteria were excluded from this analysis. Also, during the database search, it was found that although the authors used nanohydroxyapatite in their studies, they tended to refer to it as hydroxyapatite in general, which was not a problem in selection. Therefore, the articles were manually screened to confirm the HAp structure [23].

References

To find relevant articles, several international databases were searched, including PubMed, Web of Science (Clarivate Analytics), Scopus, and the Cochrane Library and Embase. All studies were conducted up to 15 June 2025. To find more relevant articles, Google Scholar was also manually searched, as well as the reference lists of the articles included in the systematic review.

Literature search strategy

Search strategy the databases listed in the (References) section were searched using a variety of relevant keywords to find articles. Keywords included "hydroxyapatite"[Mesh], "hydroxyapatite and tissue engineering"[tw], "hydroxyapatite and bone tissue engineering by freeze-drying"[tw], "hydroxyapatite and chitosan by freeze-drying"[tw], “chitosan"[Mesh], “chitosan"[tw], “chitosan and tissue engineering"[tw], "chitosan and bone tissue engineering by freeze-drying"[Mesh], "scaffold"[Mesh], "hydroxyapatite and chitosan scaffolds"[tw]. Various tags such as text word (tw) and medical subject heading (MeSH) were used in the search.

Methodological quality assessment

The methodological quality of published articles was assessed using the Risk of Bias tool. Each area was rated as having low, moderate, serious, or critical risk of bias. No data category was used unless there were insufficient data reported to make a judgment [24]. The overall risk of bias judgment is determined based on the interpretation of each domain level, with low risk indicating that the study is comparable to a well-conducted randomized trial for all domains assessed. Moderate risk of bias indicates that the study evidence is suitable for a non-randomized study but is not comparable to a randomized trial (low or moderate risk of bias for all domains). Serious risk of bias indicates that there are important problems (serious risk of bias in at least one domain, but not at critical risk of bias in any domain). Critical risk of bias indicates that the study has difficulty providing any useful evidence [24]. Any disagreement about the inclusion or exclusion of a study was resolved through discussion between the authors. Risk of bias was assessed based on quality assessment.

Risk of bias assessment

A systematic assessment of the procedural quality of each study in the article was performed. The assessment criteria were chosen to focus on critical information about the use of chitosan and nanohydroxyapatite-containing scaffolds. In the evaluation of study design, implementation and analysis, criteria were applied, and studies were assigned scores on a scale of 0 to 5, with higher scores indicating higher study quality and lower risk of bias. The risk of bias assessment was performed according to the following scoring system. Scores of 0-1 indicate high risk, 2-3 indicate moderate risk and 4-5 indicate low risk.

Selection process

Retrieved articles were first entered into Endnote software. Then, duplicate articles were removed and in the next step, titles and abstracts of remaining screening articles and irrelevant articles were removed. Then, the full text of remaining screening articles and relevant articles were included in the systematic review. Two authors independently performed all these steps.

Data collection process

Extracted variables included the name of the first author, year of publication of the article, scaffold composition, morphological characteristics of scaffolds, methods examined and main results. All these steps were performed independently by two authors, and disagreements between authors were resolved by discussion with the third author.

Study Selection

The initial search of the included databases yielded 278 articles that were potentially relevant to the proposed review. The total number of eligible articles retrieved in this study was 81. 72 titles, abstracts, and full texts remained for further screening after removing duplicate records. Of these, 63 did not meet the inclusion criteria and were therefore excluded (Figure 3). Finally, 9 articles were selected for qualitative synthesis (Table 1).

Figure 1. Flowchart of literature search for studies included in the systematic review

General characteristics of the reviewed studies

The studies included in the systematic review demonstrate the effectiveness of scaffolds containing chitosan and hydroxyapatite in bone tissue engineering. The general characteristics of the studies are presented in Table 1. A total of 9 studies were used. The technique used to fabricate the scaffolds was freeze-drying. All studies concluded that the evaluated chitosan and hydroxyapatite-containing biomaterials can be successfully used in bone tissue engineering.

Table 1. Studies reviewed in the research

Main results of the study A detailed description of the selected studies are provided in Table 1. The aim of this review was to evaluate studies on scaffolds made from chitosan and hydroxyapatite by freeze-drying. In all studies [25-33], the main focus of the investigation of scaffolds containing chitosan and hydroxyapatite was their use in tissue engineering and bone tissue by the new freeze-drying method and only scaffolds made by the freeze-drying method were considered. Therefore, the researchers approached this issue from a broader perspective and investigated the use of scaffolds containing chitosan and hydroxyapatite in bone tissue engineering by the freeze-drying method, where the lower freeze-drying temperature results in denser and more compact scaffold structures due to the slower formation of ice crystals. These scaffolds exhibit an interconnected porous architecture that is critical for nutrient diffusion and cell penetration. Key improvements in compressive strength and porosity were observed, making these scaffolds suitable for bone tissue engineering applications.

Shahbazarab et al. (2018) showed that these scaffolds containing chitosan and hydroxyapatite were non-toxic to cells and cells attached to the pore walls within the scaffolds. The results regarding physicochemical properties and superior cell compatibility suggest that scaffolds containing chitosan and hydroxyapatite could be potential candidate materials for tissue engineering [25]. Also, Zhang et al. (2015) showed that not only the freeze-drying method has a significant effect on the properties of the complex scaffolds, but also the concentration of chitosan has a significant effect on the properties of the complex scaffolds [28].

Teimouri et al. (2015) synthesized chitin, gelatin and nanohydroxyapatite nanocomposite scaffolds using freeze-drying method. The results proved that these scaffolds could be suitable candidates for tissue engineering applications [31]. Abd- Khorsand et al. (2017), the pore size and porosity of the scaffolds could be effectively tuned by selecting appropriate amounts of hydroxyapatite. The results of mechanical property measurements showed that the scaffolds could basically maintain their strength in the dry state and had sufficient mechanical properties close to that of cancellous bone. This study showed that the fabricated nanocomposite scaffolds were suitable for bone tissue engineering [27]. Therefore, hydroxyapatite can be used as one of the closest synthetic composites to natural bone in hard tissue engineering. Also, chitosan also has unique properties such as biocompatibility, biodegradability, antimicrobial activity, wound healing and acceleration of healing, and non-toxicity due to the presence of amino groups and its naturalness [34]. Also, most bone injuries that occur in the body heal spontaneously and with minimal treatment. With this multiple healing, the reasons for some cases that do not heal on their own become more necessary. Today, tissue engineering techniques and the use of various types of scaffolds are used to achieve faster healing of large bone fractures. Also, the cultivation of osteogenic cells on scaffolds, which has been investigated in several studies, seems to be effective for reconstruction [35]. Therefore, hydroxyapatite and chitosan can be used to fabricate bone composites by freeze-drying and free radical polymerization, and the fabricated bone composites may have potential applications in the field of bone tissue engineering scaffolds.

This review discusses the research on the use of scaffolds containing chitosan, hydroxyapatite, and other compounds that enhance their properties for bone tissue engineering by freeze-drying. Current engineering technologies allow the fabrication of complex synthetic biomaterials that exhibit properties almost as good as natural bone and are highly biocompatible. Chitosan, on the other hand, is a naturally occurring polysaccharide that is mainly obtained from the shells of crustaceans. This polymer has a high level of biocompatibility and biodegradability, and has antimicrobial properties and supports wound healing.

Chitosan can be used to produce scaffolds in bone tissue engineering due to its ability to form porous structures. Numerous studies have been conducted on the use of chitosan in combination with other materials added to it with the aim of improving its biological and mechanical properties. The combination of chitosan with hydroxyapatite has been widely tested. Freeze-drying is a simpler method than other methods but has better advantages than other methods in the field of bone tissue engineering.

Hydroxyapatite is the main mineral component of bone and has been used for bone tissue improvement and in bone graft substitutes. We believe that the osteogenic capacity of scaffolds can be further enhanced by improving their degradation characteristics. When fabricating a substrate scaffold for tissue regeneration, many characteristics such as cytotoxicity, mechanical properties, as well as healing efficiency should be considered. In recent years, there has been a rapid and increasing development in the use of bioactive materials in tissue engineering applications. Hence, there has been an increasing demand for materials with suitable physical, biological, and mechanical properties as well as predictable degradation behavior.

Chitosan-based materials are a unique biopolymer, exhibiting outstanding properties in research, and are ideal bioactive materials due to their outstanding properties. The basis for selecting a suitable biomaterial for bone tissue engineering is a thorough understanding of bone anatomy and the healing process. Bone healing is a complex process, and the rate of healing and success varies from person to person. Recent advances in bone tissue engineering utilize three-dimensional (3D) processing technologies that provide mechanical, cellular, and molecular environments for the repair, maintenance, or regeneration of damaged bone tissue. In terms of selecting suitable biomaterials, chitosan nanocomposites with nanofillers such as hydroxyapatite, bioactive glass, zeolite, copper nanoparticles, and carbon fillers are widely used for bone tissue engineering applications and are applied in the form of thin films, fibrous meshes, scaffolds, and hydrogels. Therefore, the use of bioactive and biocompatible materials, such as chitosan-doped hydroxyapatite nanofibers synthesized by freeze- drying method, shows promise as a strategy in tissue engineering.

The number of studies selected for this review on the effect of freeze-dried chitosan- hydroxyapatite scaffolds in bone engineering is limited, indicating the need for further extensive research. Chitosan-based scaffolds and their modifications allow for the treatment of larger and more complex bone defects without complications and treatment failures. Scaffolds that exhibit beneficial mechanical signals through physical cues, such as stiffness and other mechanical properties, can generate internal mechanical forces to facilitate cell differentiation. Cells are more likely to differentiate into bone cells when grown on stiffer substrates. Therefore, advanced fabrication methods (freeze-drying, electrospinning, 3D bioprinting) allow for the design of customized scaffolds.

This study aimed to overcome the limitations of mechanical performance, strength, and clinical application, with the aim of making them effective in bone tissue engineering.

This systematic review describes promising developments in freeze-dried chitosan- hydroxyapatite scaffolds for bone tissue engineering applications. Freeze-dried chitosan-hydroxyapatite scaffolds have emerged as effective candidates for bone tissue engineering, offering significant benefits based on the review of studies. This review highlights the diverse approaches and materials used in the development of chitosan- and apatite-based scaffolds for bone regeneration. The reviewed studies demonstrate a wide range of cost-effectiveness. Freeze-dried chitosan- and hydroxyapatite-based scaffolds have been shown to enhance scaffold properties, such as mechanical strength, bioactivity, and cellular permeability. It is noteworthy that the incorporation of hydroxyapatite into chitosan improves scaffold performance. However, the differences in the rate of degradation indicate that further research is needed. The mechanical properties of scaffolds have been carefully evaluated, and several studies support the claim that the addition of hydroxyapatite with chitosan significantly increases the compressive strength. These results emphasize the need for a comprehensive evaluation, including the integration of morphological and mechanical evaluations, in order to optimize the design of scaffolds for effective bone tissue engineering. Therefore, further research is needed to explore the potential of scaffolds containing chitosan and hydroxyapatite. It is of great importance to disseminate knowledge about these biomaterials in order to increase their use in the implant field.

Acknowledgement: The authors would like to present their gratitude to the Azerbaijan Medical University (Azərbaycan Tibb Universiteti), Baku, Azerbaijan for supporting this study.

Author Contributions: All authors contributed to the study conception and design. Material preparation, data collection and analysis. All authors read and approved the final manuscript.

Conflict of Interest: The authors declare that they have no competing interests.

Funding: This research has received no external funding

Data Availability: The data that support the findings of this study are available on request from the corresponding author: Sahar.Hefzollesan@amu.edu.az

Ethical approval: None.

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