Bone Regeneration Capabilities of Scaffolds Containing Chitosan and Nanometric Hydroxyapatite—Systematic Review Based on In Vivo Examinations

In this systematic review, the authors aimed to investigate the state of knowledge on in vivo evaluations of chitosan and nanometric hydroxyapatite (nanohydroxyapatite, nHAp) scaffolds for bone-tissue regeneration. In March 2024, an electronic search was systematically conducted across the PubMed, Cochrane, and Web of Science databases using the keywords (hydroxyapatite) AND (chitosan) AND (scaffold) AND (biomimetic). Methodologically, the systematic review followed the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) protocol to the letter. Initially, a total of 375 studies were screened, and 164 duplicates were removed. A further 188 articles were excluded because they did not correspond to the predefined topics, and an additional 3 articles were eliminated due to the inability to obtain the full text. The final compilation included 20 studies. All publications indicated a potential beneficial effect of the scaffolds in in vivo bone defect repair. A beneficial effect of hydroxyapatite as a scaffold component was observed in 16 studies, including greater mechanical resistance, cellular differentiation, and enhanced bone damage regeneration. The addition of chitosan and apatite ceramics, which combined the strengths of both materials, had the potential to become a useful bone-tissue engineering material.


Introduction
Bone is a metabolically active tissue that undergoes constant rebuilding and remodeling throughout the lifespan of an individual [1].This implies that it has the capacity for regeneration, which can occur following injury [2,3].However, as the organism ages or is depleted of vital substrates, this ability is diminished, or the process becomes imbalanced [4].Furthermore, natural processes are inadequate in cases of larger bone defects.The critical size threshold for a bone defect is approximately 2 cm or greater than 50% loss of the circumference of the bone.A tissue deficit may result in nonunion, malunion, or pathological fracture [5].In such situations, grafting materials become immensely useful.Nevertheless, allogeneic, xenogeneic, and autologous materials exhibit relatively high degradation rates, with a concomitant reduction in bioactivity over time.One of the objectives of bone-tissue engineering is to develop functional alternatives that induce osteoconduction, provide mechanical stability, and integrate into the bone structure [6].
Apatite is a ubiquitous mineral found in the rocks of the Earth, the soil of the Moon, meteorites from Mars, and the hard tissues of vertebrates [7].Apatite structures have the general formula of Ca 10 (PO 4 ) 6 X 2 , where X can most commonly be substituted by a hydroxyl group, fluoride, or chlorine (Figure 1) [8].Apatite structures are very versatile and can be chemically modified.In the context of apatite ceramics, this affects their solubility, hardness, brittleness, strain, thermal stability, and optical properties such as birefringence [9], making apatite materials a useful substance in bone-tissue engineering.Among various apatite compounds present within the human body, hydroxyapatite (HAp), which contains hydroxyl groups, is the most prevalent.It is capable of forming a direct chemical bond with surrounding tissues, has osteoconductive properties, and is nontoxic, non-inflammatory, and non-immunogenic [10].In addition to its application in the medical field, where it is employed in the repair and regeneration of bone and teeth, hydroxyapatite has a number of applications in the industrial sector.These include the production of fertilizers and pharmaceutical products, protein chromatography, and water treatment processes [11].Incorporating apatite ceramics into bone-tissue engineering materials is a popular practice today.Its porosity is highly desirable, and it creates a space for cells to migrate [7].Its similarity to natural bone makes the material highly compatible [12].Nevertheless, the utilization of pure HAp is currently constrained by its unfavorable brittleness [11].Consequently, novel composite materials have been developed with the objective of accentuating the benefits of HAp while simultaneously mitigating its inherent limitations.
induce osteoconduction, provide mechanical stability, and integrate into the bone structure [6].
Apatite is a ubiquitous mineral found in the rocks of the Earth, the soil of the Moon, meteorites from Mars, and the hard tissues of vertebrates [7].Apatite structures have the general formula of Ca10(PO4)6X2, where X can most commonly be substituted by a hydroxyl group, fluoride, or chlorine (Figure 1) [8].Apatite structures are very versatile and can be chemically modified.In the context of apatite ceramics, this affects their solubility, hardness, brittleness, strain, thermal stability, and optical properties such as birefringence [9], making apatite materials a useful substance in bone-tissue engineering.Among various apatite compounds present within the human body, hydroxyapatite (HAp), which contains hydroxyl groups, is the most prevalent.It is capable of forming a direct chemical bond with surrounding tissues, has osteoconductive properties, and is non-toxic, non-inflammatory, and non-immunogenic [10].In addition to its application in the medical field, where it is employed in the repair and regeneration of bone and teeth, hydroxyapatite has a number of applications in the industrial sector.These include the production of fertilizers and pharmaceutical products, protein chromatography, and water treatment processes [11].Incorporating apatite ceramics into bone-tissue engineering materials is a popular practice today.Its porosity is highly desirable, and it creates a space for cells to migrate [7].Its similarity to natural bone makes the material highly compatible [12].Nevertheless, the utilization of pure HAp is currently constrained by its unfavorable brittleness [11].Consequently, novel composite materials have been developed with the objective of accentuating the benefits of HAp while simultaneously mitigating its inherent limitations.Bone tissue can be defined as a nanocomposite, predominantly comprising nanohydroxyapatite and type-I collagen [13].This is the rationale behind the extensive utilization of nanohydroxyapatite in grafting materials.Nanoparticles constitute a class of materials that display unique properties compared to their bulk and molecular counterparts.They have the capacity to elicit a biological response and augment mechanical properties [14].The nano form of the materials has been demonstrated to promote the differentiation of bone-marrow-derived mesenchymal stem cells into osteoblasts, with this process being selectively triggered by integrin receptors [15].A number of techniques may be employed in the synthesis of nanohydroxyapatite.These include the preparation of nanohydroxyapatite powder in a solid-state reaction, sol-gel methods, hydrothermal route and co-precipitation methods, mechano-chemical method, microwave irradiation, and ultrasonic-assisted process [16,17].
Biopolymers, otherwise known as natural polymers, constitute a group of macromolecules that are formed under natural conditions by living organisms.These polymers are distinguished by their high degree of stability.The most widely known examples of biopolymers are cellulose and starch.Nevertheless, there is a growing interest in more intricate hydrocarbon polymers produced by bacteria and fungi, particularly polysaccharides such as xanthan, curdlan, pullulan, chitin, chitosan, and hyaluronic acid [18].Chitosan represents a notable exception within this group, offering a range of advantageous properties that align with those required in medical applications.These Bone tissue can be defined as a nanocomposite, predominantly comprising nanohydroxyapatite and type-I collagen [13].This is the rationale behind the extensive utilization of nanohydroxyapatite in grafting materials.Nanoparticles constitute a class of materials that display unique properties compared to their bulk and molecular counterparts.They have the capacity to elicit a biological response and augment mechanical properties [14].The nano form of the materials has been demonstrated to promote the differentiation of bonemarrow-derived mesenchymal stem cells into osteoblasts, with this process being selectively triggered by integrin receptors [15].A number of techniques may be employed in the synthesis of nanohydroxyapatite.These include the preparation of nanohydroxyapatite powder in a solid-state reaction, sol-gel methods, hydrothermal route and co-precipitation methods, mechano-chemical method, microwave irradiation, and ultrasonic-assisted process [16,17].
Biopolymers, otherwise known as natural polymers, constitute a group of macromolecules that are formed under natural conditions by living organisms.These polymers are distinguished by their high degree of stability.The most widely known examples of biopolymers are cellulose and starch.Nevertheless, there is a growing interest in more intricate hydrocarbon polymers produced by bacteria and fungi, particularly polysaccharides such as xanthan, curdlan, pullulan, chitin, chitosan, and hyaluronic acid [18].Chitosan represents a notable exception within this group, offering a range of advantageous properties that align with those required in medical applications.These include biocompatibility, biodegradability, mucoadhesion, anticholesterolemic, hemostatic, antimicrobial, and even antitumoral effects [19], which are not exhibited by other materials from this group at all, or not to the same extent.Chitosan is a natural polymer derived from chitin-the second most abundant biopolymer in nature after cellulose.Chitin is found in crustaceans, insects, and fungi [20].The monomer of chitosan is β-1,4-D-glucosamine, which differs only slightly from the monomer of chitin, N-acetyl-D-glucosamine, by the absence of the acetyl group [20] (Figure 2).While the use of chitin is quite limited due to its insolubility and intractable nature, chitosan has much better properties and can, therefore, be used for a variety of purposes.It has a better solubility profile, less crystallinity, and is amenable to chemical modification due to the presence of functional groups such as hydroxyl, acetamido, and amine [21].Chitosan is widely used in the field of medicine due to its many desirable properties.It has been reported that chitosan is used as a wound dressing, a material for repairing broken nerve endings, a carrier for various volume-expanding or slow-release drugs, an anti-tumor remedy, and a material for bone or cartilage engineering [22].The combination of chitosan and apatite ceramics, intertwining both their strengths, has a great potential for becoming a useful bone-and cartilage-tissue engineering material [23].The composite can act as a material with the potential to facilitate multi-lineage stem-cell differentiation and even localized gene delivery [24,25].Inquiry into how to further enhance, by modifications or additions, such an organic-nonorganic union is even more essential for developing a new reliable bone deficit filler [26].A summary and an evaluation of the topic are needed, as it could set the path for future research.
polymer derived from chitin-the second most abundant biopolymer in nature after cellulose.Chitin is found in crustaceans, insects, and fungi [20].The monomer of chitosan is β-1,4-D-glucosamine, which differs only slightly from the monomer of chitin, N-acetyl-D-glucosamine, by the absence of the acetyl group [20] (Figure 2).While the use of chitin is quite limited due to its insolubility and intractable nature, chitosan has much better properties and can, therefore, be used for a variety of purposes.It has a better solubility profile, less crystallinity, and is amenable to chemical modification due to the presence of functional groups such as hydroxyl, acetamido, and amine [21].Chitosan is widely used in the field of medicine due to its many desirable properties.It has been reported that chitosan is used as a wound dressing, a material for repairing broken nerve endings, a carrier for various volume-expanding or slow-release drugs, an anti-tumor remedy, and a material for bone or cartilage engineering [22].The combination of chitosan and apatite ceramics, intertwining both their strengths, has a great potential for becoming a useful bone-and cartilage-tissue engineering material [23].The composite can act as a material with the potential to facilitate multi-lineage stem-cell differentiation and even localized gene delivery [24,25].Inquiry into how to further enhance, by modifications or additions, such an organic-nonorganic union is even more essential for developing a new reliable bone deficit filler [26].A summary and an evaluation of the topic are needed, as it could set the path for future research.

Focused Question
The systematic review followed the PICO (Population/Patient/Problem; Intervention; Comparison; Outcome) framework [27], as follows (Figure 3): cellulose.Chitin is found in crustaceans, insects, and fungi [20].The monomer of chitosan is β-1,4-D-glucosamine, which differs only slightly from the monomer of chitin, N-acetyl-D-glucosamine, by the absence of the acetyl group [20] (Figure 2).While the use of chitin is quite limited due to its insolubility and intractable nature, chitosan has much better properties and can, therefore, be used for a variety of purposes.It has a better solubility profile, less crystallinity, and is amenable to chemical modification due to the presence of functional groups such as hydroxyl, acetamido, and amine [21].Chitosan is widely used in the field of medicine due to its many desirable properties.It has been reported that chitosan is used as a wound dressing, a material for repairing broken nerve endings, a carrier for various volume-expanding or slow-release drugs, an anti-tumor remedy, and a material for bone or cartilage engineering [22].The combination of chitosan and apatite ceramics, intertwining both their strengths, has a great potential for becoming a useful bone-and cartilage-tissue engineering material [23].The composite can act as a material with the potential to facilitate multi-lineage stem-cell differentiation and even localized gene delivery [24,25].Inquiry into how to further enhance, by modifications or additions, such an organic-nonorganic union is even more essential for developing a new reliable bone deficit filler [26].A summary and an evaluation of the topic are needed, as it could set the path for future research.

Focused Question
The systematic review followed the PICO (Population/Patient/Problem; Intervention; Comparison; Outcome) framework [27], as follows (Figure 3):  The PICO question is: among scaffolds containing chitosan and nanohydroxyapatite (population), do in vivo tests (investigated condition) showcase enhanced bone-tissue regeneration (outcome) compared to different compositions and forms of biomaterial (comparison condition)?

Protocol
The article selection process for the systematic review was outlined carefully, following the PRISMA flow diagram [28] and presented in Figure 4.The systematic review was registered on the Open Science Framework (OSF) under the following address: https: //doi.org/10.17605/OSF.IO/69W8Y (access date: 6 June 2024).

Protocol
The article selection process for the systematic review was outlined carefully, following the PRISMA flow diagram [28] and presented in Figure 4.The systematic review was registered on the Open Science Framework (OSF) under the following address https://doi.org/10.17605/OSF.IO/69W8Y (access date: 6 June 2024).

Eligibility Criteria
All studies included in the systematic review were required to meet specific criteria including the investigation of scaffolds containing chitosan and nanohydroxyapatite in their matrix in the scope of bone-tissue regeneration, in vivo examination, and publication in the English language.The reviewers collectively established exclusion criteria, which included studies published in languages other than English, clinical reports, opinions, editorial papers, review articles, and studies lacking a full-text version.During the database search, it was noted that, although authors utilize nanohydroxyapatite in their studies they tend to refer to it generally as hydroxyapatite.Therefore, articles were manually screened to verify the structure of HAp and include only those studies in which it was present in the nanometric form (at least one of the dimensions is nanometrically sized) [29].Hence, the search string presented in Section 2.4.included (hydroxyapatite) instead of (nanohydroxyapatite).

Eligibility Criteria
All studies included in the systematic review were required to meet specific criteria, including the investigation of scaffolds containing chitosan and nanohydroxyapatite in their matrix in the scope of bone-tissue regeneration, in vivo examination, and publication in the English language.The reviewers collectively established exclusion criteria, which included studies published in languages other than English, clinical reports, opinions, editorial papers, review articles, and studies lacking a full-text version.During the database search, it was noted that, although authors utilize nanohydroxyapatite in their studies, they tend to refer to it generally as hydroxyapatite.Therefore, articles were manually screened to verify the structure of HAp and include only those studies in which it was present in the nanometric form (at least one of the dimensions is nanometrically sized) [29].Hence, the search string presented in Section 2.4.included (hydroxyapatite) instead of (nanohydroxyapatite).

Information on Sources, Study Selection, and Search Strategy
A search was conducted in March 2024 using the PubMed, Scopus, and Web of Science (WoS) databases.In the Scopus database, the search results were refined to include only titles, abstracts, and keywords.In PubMed, they were limited to titles and abstracts.In WoS, the results were restricted solely to abstracts.The search criteria were based on the following keywords: (hydroxyapatite) AND (chitosan) AND (scaffold) AND (biomimetic).All searches were conducted in accordance with the established eligibility criteria, and only articles with available full-text versions were considered.

Data Collection and Data Items
The selected articles were evaluated to ascertain whether they met the predefined criteria.Subsequently, the data were collated and organized in a standardized format.

Assessing the Risk of Bias of Individual Studies
At the stage of study selection, the titles and abstracts of each study were independently checked by the authors to minimize the potential for reviewer bias.The level of agreement among the reviewers was determined using the Cohen κ test.Any discrepancies in opinion regarding the inclusion or exclusion of a study were resolved through discussion between the authors.The risk of bias was evaluated based on the quality assessment.

Quality Assessment (QA)
Two independent evaluators (P.J.P. and T.H.) conducted a systematic appraisal of the procedural quality of each study within the article.The assessment criteria were selected to focus on critical information regarding the use of scaffolds containing chitosan and nanohydroxyapatite.In evaluating the study design, implementation, and analysis, criteria were applied, including a minimum in vivo test-group size of 10 animals, the presence of a control group in in vivo examination, a detailed description of the biomaterial composition used in the study, a description of the effect of the scaffold on the process of bone regeneration, and a description of the potential clinical applicability of the biomaterial.The studies were assigned points on a scale of 0 to 5, with a higher score indicating superior study quality and a lower risk of bias.The evaluation of the risk of bias was conducted according to the following scoring system.A score of 0-1 indicates a high risk, 2-3 denotes a moderate risk, and 4-5 signifies a low risk.Any discrepancies in the scoring were resolved through a comprehensive discussion until a consensus was reached [27,[30][31][32][33][34][35].

Study Selection
The initial database search across PubMed, Scopus, and WoS yielded 375 articles that were potentially relevant for the presented review.Following the removal of duplicates, 211 articles underwent screening.The initial screening of titles and abstracts resulted in the exclusion of 188 articles that did not involve a comparison of analysis between different specialists.Subsequently, 23 articles underwent further full-text analysis, during which 3 articles were excluded for not meeting the inclusion criteria.In conclusion, a total of 20 articles were included in this review.The considerable heterogeneity among the included studies precludes the possibility of conducting a meta-analysis.

General Characteristics of the Included Studies
The studies included in the systematic review present the influence of scaffolds containing at least chitosan and nanohydroxyapatite on in vivo bone-tissue regeneration.The general characteristics of the studies are presented in Table 1.A total of eleven studies employed rats as subjects for in vivo examinations, while four studies used rabbits.Five of the studies were conducted on mice.The techniques employed in the manufacture of scaffolds included co-precipitation, freeze gelation, and biomimetic processes.The authors frequently utilized the freeze-drying method.All of the studies concluded that the evaluated biomaterials may be successfully used in bone-tissue engineering.Sun et al. [47] Three g of chitosan powder was added to 97 g of acetic acid.Then, the Zn-UHANW suspension was added.
After mixing, it was frozen at −20

Chitosan, silk fibroin, nanohydroxyapatite
The nanofibers obtained a "beads on a string" surface morphology with an average fiber diameter of 266 ± 47 nm.
Before implantation, CS/SF/30%nHAP NMS were cultured with hMSCs for 14 days in cell-culture medium.The cell-free and hMSC-free scaffold was implanted into the subcutaneous pocket on both sides of the back of the mouse.The animals were sacrificed after 4 and 8 weeks.
This scaffold is an excellent tool for bone-tissue engineering based on µ-CT and histological analysis.

Jiang et al. [52]
The membrane was mechanically perforated with a pore size of 300 µm and an inter-pore spacing of 1.0 mm.It was then tightly rolled in a concentric manner.The jacket was eroded with acetic acid and attached to the scaffold.A cylindrical scaffold was prepared.
Chitosan (CS), sodium carboxymethyl cellulose (CMC), and nanohydroxyapatite (n-HA) The scaffold showed a rough surface The spiral-cylindrical scaffolds were cut into pieces 10 mm long and 3 mm in diameter.A defect was created in the central part of the rabbits' left radius, filled with a spiral-cylindrical scaffold and secured with sutures.The animals were sacrificed at 4, 8, and 12 weeks after surgery The manufactured scaffold has the potential to treat large bone defects, and its ability to heal segmental bone defects of critical size is currently being tested.

Main Study Outcomes
A detailed characterization of the selected studies is presented in Table 2.The objective of this review was to compare and assess studies concerning scaffolds made primarily of chitosan and apatite.In the majority of studies [36][37][38][39]41,42,[44][45][46][47][50][51][52][53], the primary focus of the examination of said scaffolds was their use in regeneration and new bone formation.Three groups of researchers approached the subject from a broader perspective, examining the use of CS/HA scaffolds in bone-tissue engineering in general [44,54,55].Korpayev's approach [40] involved the development of multi-layered osteochondral mimetic constructs without the need for growth factors, with the conclusion of the research suggesting a potentially higher elastic modulus.An innovative approach can be found in the studies of Huang et al. [43].Researchers composed and examined a minimally invasive injectable biomimetic gel scaffold that provides a biocompatible environment for bone-marrow stem cell survival and can serve as a cell carrier.A similar approach regarding hydrogels in tissue engineering was employed by Ju et al. [56].Good vascularization is essential for uninterrupted bone regeneration, and neovascularization is an important parameter in bone regeneration.Yu et al. [49] achieved notable effects by incorporating copper ions into the scaffold.The composition of the materials examined differed throughout the articles analyzed.Two studies by Korpayev et al. [41] and Kong et al. [54] focused on scaffolds made solely of pure chitosan and hydroxyapatite.Furthermore, Korpayev et al. [40] did not assess a reference sample; instead, they focused on the different properties of the various layers of the material.In the other article, the main substrates have been either enhanced or an additional component has been added to the mixture.In two articles [36,38], the chitosan phase was subjected to carboxymethylation.In turn, hydroxyapatite has been modified in three studies by the addition of lanthanum [37], zinc [47], and copper [48].In addition to the aforementioned substrates, other materials were employed in the studies, including B-cyclodextrin [36], icariin [39,42], genipin [41], and collagen [38,40,43,44,50].Furthermore, others employed poly (lactic-co-glycolic acid) [45], Fe 3 O 4 [46], BMP2-derived peptide [44], copper [49], and silk fibroin [51].Other materials that have been investigated include sodium carboxymethyl cellulose [52], bone-marrow mesenchymal stem cells [53], and hyaluronic acid oligosaccharides [55].
The biomimetic nature of the scaffolds provides appropriate conditions for bone reconstruction, as indicated in studies [36,37,41,46,47,51].The porous structure and physical and chemical properties comparable to human tissue provide an environment for the formation of new bone.A study by Chen et al. found that the evaluated materials effectively initiated bone repair processes in vivo [44].These processes showcase the biomimetic nature of the CS/HAp scaffolds and their ability to promote in vivo bone-tissue regeneration.
Six studies [36,37,43,44,50,54] did not investigate hydroxyapatite particles and instead focused on the overall influence and performance of the implanted scaffold.Those researchers who focused on HA unanimously attest that hydroxyapatite and its modifications enhance the properties of the scaffolds.It is said to enhance alkaline phosphatase activity [46], mechanical properties [39,47], bioactivity [39], cellular infiltration and activity [44,53,55], and osseointegrative capability [41].Furthermore, it can facilitate the resolution of inflammation [46].As stated by Yu [49], the morphology of HA particles renders them suitable for use as drug-delivery vessels.Furthermore, as demonstrated by Lai [51], hydroxyapatite has the potential to facilitate the differentiation of bone-marrow stem cells.Lai [51] additionally posited that the impact of HA is contingent upon its concentration within the scaffold.It was also demonstrated that modified hydroxyapatite can be an effective addition to the scaffold.Frohbergh [41] reports delayed degradation profiles when adding HA, whereas Chen et al. [44] state that HA facilitates said degradation.This discrepancy represents a notable inconsistency identified by the authors of this review, and it is a topic that merits further investigation.
Twelve of the selected studies included an evaluation of the mechanical properties of the scaffolds [36,37,39,40,42,[44][45][46][47]49,51,55].Jolly et al. reported that a BCHD3 scaffold containing carboxymethyl chitosan, nanohydroxyapatite, β-cyclodextrin, and date-seed extract exhibited a compressive modulus of 1533 MPa, which corresponds to the mechanical properties of human cortical bone [36].The scaffold reported by Yin et al. (La-doped CS/nHAp) matched the mechanical strength of trabecular bone [37].Hu et al. [39] put an emphasis on the increase of compressive strength of the scaffold with a greater addition of HAp (up to 3 wt.%).Korpayevs' study reported a gradient increase in compressive strength for a multilayer scaffold, indicating the regeneration of osteochondral defects [40].Wu et al. [42] stated that the addition of icariin to the CS/HAp scaffold decreased the elastic modulus and fracture strength, but icariin itself boosted the scaffolds' osteoconductive and osteoinductive potentials.On the other hand, the addition of Fe 3 O 4 to the CS/HAp matrix enhanced compressive resistance [46], while the doping of HAp microspheres with Cu did not affect the compressive mechanical properties significantly [49].Subsequent studies indicated that the addition of HAp to the polymer-based matrix increased compressive strength [44,45,47,55].This conclusion is persistent throughout the studies evaluated in the presented systematic review.Finally, Lai et al. [51] implied that the homogenous incorporation of apatite ceramics into the matrix enhances mechanical stability.Therefore, side-by-side morphological and mechanical assessment is beneficial for concluding the relation between the structure and its properties.

Discussion
This review discussed research relevant to the use of scaffolds containing chitosan, hydroxyapatite, and other compounds that enhance their properties for bone-tissue regeneration.The current engineering technologies enable the construction of synthetic, complex biomaterials that exhibit properties nearly as good as those of natural bone and are highly biocompatible.In cases of tooth loss and subsequent alveolar atrophy, bone-substitute materials play a significant role in the treatment process.Composite grafts offer the advantages of autografts and allografts in a single surgical procedure, combining the best of both techniques.Such a graft may be constructed by combining a synthetic scaffold with biological elements, thereby stimulating cell infiltration and new bone formation [58].The rising incidence of craniofacial cancer further contributes to the formation of critical bone defects.Although allogeneic, xenogeneic, and autogenous materials have been used for years to treat patients, there is a need to develop new materials to address the increasing challenges faced by clinicians in treating bone defects.The number of articles selected for this review concerning materials based on chitosan-hydroxyapatite scaffolds for in vivo bone regeneration is limited, indicating a need for further extensive research.Chitosanbased scaffolds and their modifications allow for the treatment of increasingly larger and more complex bone defects without causing complications and therapeutic failures.
Biomimetic bone-substitute materials are characterized by a morphology and physical and chemical properties similar to human bone.After application, the material takes over the function of the autologous bone, enabling the regeneration of the defect.These materials are intended to resemble natural bone as much as possible.As mentioned previously, HAp used in scaffolds is also a component of natural bone.Research conducted by Jolly et al. [36] and Huang et al. [43] show that scaffolds containing HAp have a biomimetic character and proadhesive properties.They caused an osteogenic effect and revascularization of the newly formed bone, which suggests a high biomimetic potential.A histological analysis of the collected samples showed a high degree of resorption and replacement of the bone-substitute material with newly formed bone.The lack of immunological reaction and inflammation, caused by the significant similarity of the material to natural bone, additionally emphasizes the biomimetic potential of the scaffolds.
The structure and morphology of scaffolds are important, as noted by Kong et al. [54] in their research.The multilayer nanohydroxyapatite-chitosan scaffold, compared to the uniform nanohydroxyapatite/chitosan scaffold, has much larger pores and leads to greater biocompatibility, prevents the formation of fibrous tissue in the defect, and ensures better delivery of nutrients to the newly formed bone.In the analyzed studies, the average scaffold structure had a porous structure, with an average pore size ranging from 30-90 µm [48] to even 200-400 µm in research by Sun et al. [47].
For several years, the use of autogenous materials necessitated the collection of material from another site, posing a potential risk of complications at the donor site.Currently, widely available bone-substitute materials serve as good alternatives for treating patients needing bone defect reconstruction.Their use not only reduces the risk of complications but also significantly accelerates and improves the procedure.Detailed characterization of these materials allows for better prediction of possible complications and the implementation of appropriate treatment regimens at the onset of symptoms.
Surface-functionalized, controlled/sustained release, preprogrammed release, stimuliresponsive, and gene delivery are the five main categories of biomolecule delivery platforms [59].The role of growth factors (GFs) in the bone repair process has been extensively documented [60].There have been numerous studies on the effects of growth factors in enhancing the in vitro chondrogenic potential and in vivo cartilage regeneration [61][62][63][64].The utilization of growth factors is also employed in the regeneration of bone tissue [63,65,66].Scaffolds based on chitosan and hydroxyapatite allow for the delivery and controlled release of drugs, as shown by Zeng et al. [67] in their research.This enables the adoption of new strategies for the treatment of critical bone defects and chronic bone disease processes.In vivo, animal studies demonstrated the biocompatibility of the obtained scaffolds and the osteoconductive effect.This produced significantly better results in the treatment of bone defects compared to the control group.The physical properties of the scaffolds allowed for obtaining a stable structure for osteoblasts and mineral components of the newly formed bone.The objective of utilizing substitute materials is to facilitate biological integration while stimulating the body's intrinsic capacity for tissue regeneration.
Most of the analyzed articles were published in journals related to chemistry and biomaterials.It is appropriate to widely disseminate knowledge about biomaterials used to regenerate bone defects.This approach may increase interest in this topic among doctors and researchers who deal with the treatment of bone defects that exceed the regenerative capabilities of the human body on a daily basis.The increasing role of regenerative methods is worth mentioning in the context of dentistry, especially dental surgery.This field encompasses the repair, restoration, and replacement of defective or non-functional tissues that have been lost due to various diseases, regressive changes, congenital defects, or damage.

Figure 1 .
Figure 1.Possibilities of structural modifications of HAp.

Figure 1 .
Figure 1.Possibilities of structural modifications of HAp.

Figure 2 .
Figure 2. Outlook on structure of chitosan polymeric chain.

Figure 3 .
Figure 3. PICO framework of the presented study.

Figure 2 .
Figure 2. Outlook on structure of chitosan polymeric chain.

Figure 2 .
Figure 2. Outlook on structure of chitosan polymeric chain.

Figure 3 .
Figure 3. PICO framework of the presented study.Figure 3. PICO framework of the presented study.

Figure 3 .
Figure 3. PICO framework of the presented study.Figure 3. PICO framework of the presented study.

Figure 4 .
Figure 4. PRISMA 2020 flow diagram of the presented study.

Figure 4 .
Figure 4. PRISMA 2020 flow diagram of the presented study.

Table 1 .
General characteristics of the selected studies.

Table 2 .
Detailed characteristics of the included studies.