Biomaterials Based on Chitosan and Its Derivatives and Their Potential in Tissue Engineering and Other Biomedical Applications—A Review

In the times of dynamically developing regenerative medicine, more and more attention is focused on the use of natural polymers. This is due to their high biocompatibility and biodegradability without the production of toxic compounds, which means that they do not hurt humans and the natural environment. Chitosan and its derivatives are polymers made most often from the shells of crustaceans and are biodegradable and biocompatible. Some of them have antibacterial or metal-chelating properties. This review article presents the development of biomaterials based on chitosan and its derivatives used in regenerative medicine, such as a dressing or graft of soft tissues or bones. Various examples of preparations based on chitosan and its derivatives in the form of gels, films, and 3D structures and crosslinking products with another polymer are discussed herein. This article summarizes the latest advances in medicine with the use of biomaterials based on chitosan and its derivatives and provides perspectives on future research activities.


Introduction
Tissue engineering is focused on the creation of tissues to repair or replace diseased or damaged organs. Recently, natural polymers have become of increasing interest due to rapidly developing medical applications. This is due to their biodegradability and non-toxicity. They also mimic tissue structure better than synthetic polymers due to their physicochemical similarity. The development of new products based on tissue-mimicking biopolymers that are more robust, non-toxic, and biodegradable is a key issue that will guarantee rapid growth in the development of tissue engineering. Biomimetic natural polymers and hybrid polymer materials have the advantage of combining desired functions with tailored morphology and superior chemical and physical stability. These polymeric materials aim to cover all aspects of the subject, including, for instance, the design of hybrid materials, films, gel, sponge, nanocomposites, and hydrogels, without forgetting studies of structure-property relationships, production of materials with precise structural control and advanced properties, and applications of bioinspired polymers for various fields including tissue engineering, drug delivery systems, or wound dressings [1][2][3][4].
The main problems of the resulting materials made from single polymers are insufficient mechanical properties and too rapid biodegradability. Therefore, mixtures of polymers and the use of a cell-free tissue matrix started to be used. Silk fibroin [5][6][7], collagen [8,9], hyaluronate [10,11], or gelatin [12] were used for this purpose. The materials obtained should be biodegradable and the biodegradation products must be non-toxic and removed from the body without any effect on other tissues. Furthermore, the materials should support cell adhesion, migration, and proliferation through appropriate porosity, pore size, and their appropriate combination. The physicochemical and mechanical properties should be as similar as possible to those of the tissue to be replaced and should be strong enough the materials should support cell adhesion, migration, and proliferation through appropriate porosity, pore size, and their appropriate combination. The physicochemical and mechanical properties should be as similar as possible to those of the tissue to be replaced and should be strong enough to allow its implantation during surgery [13,14]. These materials can take the form of thin films, hydrogels, membranes, 3D structures, fibers, and nanofibers.
Herein, we reviewed various examples of chitosan-based biomaterials, mixed with other polymers and cross-linked with chemical agents, in biomedical applications based on previous research.

Chitosan and Its Derivatives
Chitosan (CS) (poly(β-(1,4)-2-amino-2-deoxy-D-glucopyranose) is a natural polymer obtained by partial deacetylation of chitin in an alkaline medium ( Figure 1). Chitin was produced from the exoskeletons of crustaceans. Chitosan also occurs naturally in the cell walls of some fungi. Chitosan is a polymer with a degree of deacetylation of at least 60%. The polymer's molecular weight and the degree of deacetylation determine its properties such as biodegradability, biocompatibility, viscosity, hydrophilicity, and antibacterial or antifungal properties. The major disadvantage of chitosan is its lack of solubility in water [15][16][17][18][19][20][21]. The most important properties are shown in Figure 2. Chitosan is the only naturally occurring polysaccharide classified as a cationic polyelectrolyte, which allows it to interact with different types of molecules. The polymer's positive charge is responsible for its antibacterial properties, attaching to the negatively charged cell membrane of various microorganisms [14,21]. The most important properties are shown in Figure 2. Chitosan is the only naturally occurring polysaccharide classified as a cationic polyelectrolyte, which allows it to interact with different types of molecules. The polymer's positive charge is responsible for its antibacterial properties, attaching to the negatively charged cell membrane of various microorganisms [14,21].
Carboxymethyl chitosan (CMC) is a chitosan derivative in which the carboxymethyl group is attached to either an amino group or a hydroxyl group (Figure 1). This chitosan derivative is water soluble and this is one of the main reasons for the increased interest in this polymer by researchers. It can be obtained in many types: Ncarboxymethyl chitosan N,N-carboxymethyl chitosan, N,O-carboxymethyl chitosan, and O-carboxymethyl chitosan. During the substitution reaction, the listed types of derivatives or their mixtures can be obtained [22,23]. CMC is characterized by high viscosity, biocompatibility, and biodegradability, and is non-toxic. It also has antimicrobial activity, with O-carboxymethyl chitosan showing greater activity due to the more abundant presence of amino groups. Carboxymethyl chitosan shows improved physicochemical and biological properties relative to chitosan. The properties of CMC are influenced by the average molecular weight, degree of deacetylation, and degree of substitution. In addition, CMC has antioxidant activity, antibacterial or antifungal properties, and the ability to chelate metals [22][23][24][25][26]   Carboxymethyl chitosan (CMC) is a chitosan derivative in which the carboxymethyl group is attached to either an amino group or a hydroxyl group (Figure 1). This chitosan derivative is water soluble and this is one of the main reasons for the increased interest in this polymer by researchers. It can be obtained in many types: Ncarboxymethyl chitosan N,N-carboxymethyl chitosan, N,O-carboxymethyl chitosan, and O-carboxymethyl chitosan. During the substitution reaction, the listed types of derivatives or their mixtures can be obtained [22,23]. CMC is characterized by high viscosity, biocompatibility, and biodegradability, and is non-toxic. It also has antimicrobial activity, with O-carboxymethyl chitosan showing greater activity due to the more abundant presence of amino groups. Carboxymethyl chitosan shows improved physicochemical and biological properties relative to chitosan. The properties of CMC are influenced by the average molecular weight, degree of deacetylation, and degree of substitution. In addition, CMC has antioxidant activity, antibacterial or antifungal properties, and the ability to chelate metals [22][23][24][25][26] Chitosan acetate is obtained by reaction with acetic acid in an aqueous-ethanol environment. It is water soluble and its solution is more stable than chitosan dissolved in acetic acid, while retaining the physicochemical and biological properties of chitosan. It exhibits stronger antimicrobial activity against Gram-positive bacteria than against Gramnegative bacteria. It is used as a dressing material (Chitopack C ® ) and a hemostat (Hemcon Bandage ® ) approved by the FDA [27].

Chitosan and Its Derivatives in Medicine
Due to its properties, chitosan and its derivatives can be used in the production of dressing materials, in the manufacture of drugs as a controlled-release active substance Chitosan acetate is obtained by reaction with acetic acid in an aqueous-ethanol environment. It is water soluble and its solution is more stable than chitosan dissolved in acetic acid, while retaining the physicochemical and biological properties of chitosan. It exhibits stronger antimicrobial activity against Gram-positive bacteria than against Gramnegative bacteria. It is used as a dressing material (Chitopack C ® ) and a hemostat (Hemcon Bandage ® ) approved by the FDA [27].

Chitosan and Its Derivatives in Medicine
Due to its properties, chitosan and its derivatives can be used in the production of dressing materials, in the manufacture of drugs as a controlled-release active substance carrier, or in tissue engineering involving soft tissues, nerves, cartilage, bones, or arteries. Studies on the use of chitosan are summarized in Table 1 and studies on its derivatives are in Table 2.    Good drug delivery, non-cytotoxic to the cornea, good degradability [103] The team of Fangsong Zhang et al. [30] used two chemical agents, glutaraldehyde, genipin, and a physical agent, ultraviolet light, to crosslink nerve extracellular matrix/chitosan scaffolds. Scaffolds cross-linked with genipin were characterized by higher porosity and regular structure in contrast to scaffolds cross-linked with glutaraldehyde and UV. The degree of crosslinking for genipin-crosslinked and glutaraldehyde-crosslinked scaffolds were similar to each other. Genipin-crosslinked scaffolds had the lowest degree of cytotoxicity and the highest histocompatibility, with good mechanical properties.
Another team, Jie Xu et al. [31], prepared a scaffold based on decellularized extracellular matrix, gelatin, and chitosan cross-linked EDC/NHS. The resulting scaffolds were characterized by a high modulus of elasticity and biodegradability. The obtained scaffolds are not cytotoxic and provided a good substrate for cell proliferation. The scaffolds were also characterized by antibacterial properties (E. Coli, S. Aureus). The scaffolds obtained could be used in skin tissue engineering.
A scaffold for use in muscle tissue engineering is a project by the team of Weiguang Zhao et al. [32]. They used genipin as a crosslinking agent and electrospun cellulose acetate nanofibers that were incorporated into a chitosan/fibroin silk cryogel scaffold. The resulting scaffolds were characterized by larger pores and roughness than the cryogel scaffold itself. They are also a good substrate for smooth muscle cell proliferation, which showed a higher potential for the expression of genes related to muscle contraction. They also exhibit good mechanical properties.
Scaffold for use in cartilage tissue engineering is a study by Christian E. G. Garcia et al. [33]. The properties of chitosan in two forms were compared: thin film and electrospinning scaffold chitosan/poly (ethylene oxide) (PEO). PEO of two different molecular weights was used and different weight ratios of Cs/PEO were applied. Some of the materials obtained were neutralized in order to compare the effect of neutralization on the properties of the scaffolds. The scaffolds after neutralization were characterized by better adhesion of chondrocyte cells and better proliferation; the worst properties were characterized by the chitosan film.
Nihui Zhang and her team [65] used genipin solutions with different concentrations (2.5%, 5%, 10%, and 15%) to produce carboxymethyl chitosan hydrogels. Analysis of HSFs cell proliferation and adhesion showed that the best cell adhesion and proliferation were obtained for the hydrogel with the highest amount of genipin, and the worst for the hydrogel with the lowest proportion of genipin. The best properties for promoting wound healing and reducing the appearance of scars in vivo tests were obtained for the hydrogel carboxymethyl chitosan/genipin 5% (v/v). This was confirmed by an in vivo test using female rats. With the additional contribution of aloe vera gel, wound healing results improved even further. In conclusion, genipin-crosslinked chitosan hydrogels are promising candidates for use as a dressing to accelerate wound healing.
Yalei Liu et al. [66] used polyvinyl alcohol, carboxymethyl chitosan, silver nanoparticles, and borax as a crosslinking agent to produce a hydrogel. The resulting hydrogel, due to its dual crosslinking (hydrogen bonds and borate ester bonds), has self-healing properties and is characterized by good mechanical properties. It also exhibits antibacterial properties, as confirmed by a test with E.coli and S. aureus bacteria. A cytotoxicity test was also performed using L929 cells, which showed that the resulting scaffolds were non-toxic.
Guozhu Chang et al. [67] produced a carboxymethyl chitosan/carboxymethyl cellulose hydrogel using heparin and glutaraldehyde as a crosslinking agent. This allowed the fabrication of a self-healing hydrogel. It is biocompatible with cells and its ability to release drugs has also been studied. An in vivo study was also performed on rats with diabetes, where its effect on accelerating open wound healing was confirmed. It can be concluded that the resulting hydrogel has the potential to be used as a material to accelerate diabetic wound healing.
To form antimicrobial scaffolds, A. Mishra's team [68] used carboxymethyl chitosan, zinc, and genipin. Carboxymethyl chitosan/genipin/Zn scaffolds were obtained. Wet compression analysis showed that the carboxymethyl chitosan/genipin/Zn scaffold was more robust than the non-cross-linked scaffold. Degradation testing was carried out under enzymatic and non-enzymatic conditions. The resulting scaffold also showed good stability. An adhesion and proliferation test was performed using dental pulp stem cells; in addition, a biocompatibility test against red blood cells was performed, which confirmed its good biological properties. An antibacterial test was also performed (Pseudomonas aeruginosa ATCC 25619, S. aureus ATCC 9144, S. aureus ATCC 25923, and Staphylococcus epidermidis ATCC 155). No biofilm formed on the surface of the scaffold carboxymethyl chitosan/genipin/Zn. In conclusion, scaffold carboxymethyl chitosan/genipin/Zn can find application in dental tissue engineering due to its antibacterial properties.
Summarizing the data overview on the use of chitosan and its derivatives in tissue engineering (Figure 3a,b), it can be written that the most research on chitosan-based materials concerned bone tissue engineering and the least concerned dental tissue engineering. For materials based on chitosan derivatives, the greatest interest in use was in skin tissue engineering and the least in tissue engineering applications.
Summarizing the data overview on the use of chitosan and its derivatives in tissue engineering (Figure 3a,b), it can be written that the most research on chitosan-based materials concerned bone tissue engineering and the least concerned dental tissue engineering. For materials based on chitosan derivatives, the greatest interest in use was in skin tissue engineering and the least in tissue engineering applications.

Conclusions
The use of chitosan and its derivatives in medicine offers a huge opportunity in the further development of regenerative medicine. The use of different forms of polymers such as films, hydrogel scaffolds, or the use of strongly developing ways of producing materials such as electrospinning and 3D printing open another door to the medicine of the future. Owing to continuous development, we are able to produce biomaterials that mimic the structure, morphology, and function of various organs such as blood vessels nerves, soft tissues, or bones. Further research using other solvents, new mixtures, or using a different cross-linking agent may bring us even closer to a perfectly mimicking tissue biomaterial. A constant challenge is to produce in the spirit of green production and ecology in a closed loop using natural polymers where their extraction will not adversely affect the environment.

Conclusions
The use of chitosan and its derivatives in medicine offers a huge opportunity in the further development of regenerative medicine. The use of different forms of polymers such as films, hydrogel scaffolds, or the use of strongly developing ways of producing materials such as electrospinning and 3D printing open another door to the medicine of the future. Owing to continuous development, we are able to produce biomaterials that mimic the structure, morphology, and function of various organs such as blood vessels, nerves, soft tissues, or bones. Further research using other solvents, new mixtures, or using a different cross-linking agent may bring us even closer to a perfectly mimicking tissue biomaterial. A constant challenge is to produce in the spirit of green production and ecology in a closed loop using natural polymers where their extraction will not adversely affect the environment.