**4.1 Physio-mechanical parameters of chitosan**

Chitosan is a polysaccharide composed of randomly distributed acetylated and deacetylated units of D-glucosamine. Chitosan exists in five different crystal forms, four of which are hydrated and one is anhydrous. Microcrystalline chitosan is characterized by better biodegradability and bioactivity.

Most of the properties of chitosan depend on two parameters: degree of deacetylation and molecular weight distribution. Depending on the source and method of preparation, the deacetylation degree varies from 30 to 95%, and the molecular weight from 300 to over 1000 kDa [43]. The solubility of chitosan strongly depends on the deacetylation degree, which translates into the number of free amino groups. Chitosan is soluble in acidic solutions due to its susceptibility to protonation and formation of ammonium salts. It is soluble in acetic, formic, citric, lactic and hydrochloric acid and insoluble in most organic solvents. Chitosan, as a biodegradable polymer, is easily broken down by microorganisms into simple chemical compounds such as carbon dioxide (CO2) and ammonia (NH3). Like other biopolymers, it is susceptible to many chemical and physical factors leading to its degradation. The degradation process also depends on the degree of deacetylation and the molecular weight of the polymer [3, 5].

Chitosan has many valuable properties, such as: biocompatibility, biodegradability, non-toxicity, the ability to create polycations in an acidic environment, the possibility of modification, high affinity for metals, dyes and proteins, hydrophilicity, ability to create membranes and others [3, 5, 44]. These features make it applicable in medicine and pharmacy, in various industries, in environmental protection, including water treatment and separation processes. [5, 45, 46]. Chitosan also has a number of properties that limit its use in certain areas. It swells strongly in water (especially in an acidic environment), and in the swollen state it is characterized by low mechanical strength. The use of chitosan is also limited due to its high viscosity. The reduction of the viscosity of chitosan solutions can be achieved by increasing the deacetylation degree while reducing the molecular weight and increasing the temperature or ionic strength [5, 47]. The key problem with the use of chitosan is its susceptibility to external factors (humidity and temperature) and processing conditions (heating or sterilization), which can affect its structure and cause its degradation. Parameters such as molecular weight or the presence of impurities have a significant impact on the processing of chitosan [48]. This causes difficulties in maintaining the stability of chitosan (no changes in molecular weight) for a long time at room temperature [49]. The influence of many factors, such as increased temperature, the presence of strong acids, mechanical shear or radiation, on the molecular weight of chitosan was demonstrated. It is also believed that high molecular weight chitosan is more stable. The lack of reproducibility in the processing of chitosan is also due to significant differences in molecular weight, deacetylation degree and purity level depending on the source of the raw material. The level of chitosan purity may affect both biological properties, such as biodegradability or immunogenicity, as well as its solubility and stability [48, 50].

#### **4.2 Biological parameters of chitosan**

Chitosan is a non-toxic polysaccharide containing randomly distributed acetylated and deacetylated units of D-glucosamine. The results of many studies confirm the antibacterial effect of chitosan. The mechanism explaining this feature is unknown [51]. The antimicrobial activity of chitosan is strongly dependent on many factors, such as molecular weight [52], degree of deacetylation (DD), pH of the dissolving medium and its ionic strength. Stronger antibacterial activity was observed with a high degree of deacetylation [53] and a low molecular weight of chitosan [54]. The antibacterial activity of chitosan is also associated with the form of the polymer (hydrogels, membranes, dissolved form) and the presence of other compounds [55]. One of the factors responsible for the antibacterial activity of chitosan is its cationic nature. The positively charged ammonium groups of chitosan may interact with negatively charged components of the bacterial cell wall, causing damage to the cell membrane and destruction of bacterial cells (a mechanism proposed for high molecular weight chitosan) [56]. Ultimately, this causes the formation of an impermeable layer around the bacterial cell, affecting permeability and transport to the cell [57, 58]. It has been suggested that low molecular weight chitosan can penetrate bacterial cell walls and eventually enter the cytoplasm and bind to DNA affecting DNA transcription, mRNA synthesis and finally protein biosynthesis [59].

The difference in the hydrophilicity and the negative charge of the cell surface of the bacteria makes gram-negative bacteria interact more strongly with chitosan, resulting in higher antibacterial activity against them.

The antibacterial activity of chitosan or its derivatives on gram-negative bacteria has been demonstrated for various strains: *Escherichia coli*, *E. coli* K88, *E. coli* ATCC 25922, *E. coli* O157, *Pseudomonas aeruginosa*, *Proteus mirabilis*, *Salmonella enteritidis*, *S. choleraesuis* ATCC 50020, *S. typhimurium*, *S. typhimurium* ATCC 50013, *Enterobacter aerogenes*, *Listeria monocytogenes* [60–64]. The antibacterial activity of chitosan or its derivatives on gram-positive bacteria has been demonstrated for: *Staphylococcus aureus*, *S. aureus* ATCC 25923, *Corynebacterium*, *Staphylococcus epidermidis*, *Enterococcus faecalis*, *Bacillus cereus*, *Bacillus megaterium*. This indicates a broad spectrum of chitosan activity. It was also found that the effectiveness of chitosan binding to the bacterial cell wall depends on the pH value. At low pH, chitosan shows better adsorption to the bacterial cell wall due to the increased positive charge of protonated amino groups [65–67].

The antifungal activity of chitosan also depends on its molecular weight and degree of acetylation. It was found that chitosan shows antifungal activity against selected phytopathogenic fungi *Penicillium* sp. in citrus [68], *Botrytis cinerea* in cucumber [69], *Phytophthora infestans* [70], *Alternaria solani* and *Fusarium oxysporum* [71]. Chitosan is also active against fungal species pathogenic to humans, while being non-toxic to human cells. The antifungal activity of chitosan or its derivatives has been demonstrated against: *Candida albicans*, *Candida parapsilosis*, *Candida krusei*, *Candida glabrata*, *Penicillium digitatum*, *Penicillium italicum*, *Fusarium proliferatum*, *Hamigera avellanea*, *Aspergillus fumigatus*, *Rhizopus stolonifer*, *Cryptus stolofattans*. The suggested mechanism of action is to create a permeable layer by chitosan which disrupts fungal growth [72–75]. It is believed to be related to the activation of defense processes, including chitinse accumulation, synthesis of proteinase inhibitors, callus synthesis and the lignification process [76].

The antiviral activity of chitosan derivatives is also suggested. The research focuses mainly on HIV. Peptide-chitosan conjugates (GlnMetTrp-chitosan and TrpMetGlnchitosan) show a protective effect on C8166 cells by counteracting the cytolytic effects of the HIV-1RF strain. These derivatives have the ability to suppress HIV-induced syncytium formation and reduce HIV load without inhibiting HIV-1 reverse transcriptase and protease *in vitro* [77]. Sulfated low molecular weight chitosan derivatives inhibit HIV-1 replication, HIV-1 induced syncytium formation, lytic activity and p24 antigen production. These derivatives are believed to influence the binding of HIV-1 gp120 to the CD4 cell surface receptor [78]. In comparative studies, chitosan conjugates with

**145**

*Modulating the Physicochemical Properties of Chitin and Chitosan as a Method of Obtaining…*

thioglycolic acid, lactic acid, PEG and antiviral drugs, significantly higher efficacy

Recent studies have shown that chitosan and its derivatives exhibit anti-tumor activity in both *in vitro* and *in vivo* models. It was found that chitosan derivatives increased the secretion of interleukin-1 and 2, influencing the maturation and infiltration of cytotoxic T lymphocytes [86]. The results have been confirmed in an *in vivo* study [87]. Moreover, a direct cytotoxic effect on neoplastic cells was found by inducing apoptosis. For A375, SKMEL28 and RPMI7951 cancer cell lines it was found to reduce cell adhesion, inhibit proliferation, inhibit specific caspases, regulate the activity of Bax as well as Bcl-2 and Bcl-XL, induce CD95 receptor expression through greater susceptibility to chitosan-induced apoptosis by FasL [88]. In the case of the PC3 A549, HepG2 and LCC cell lines, in the presence of chitosan, inhibition of the growth of neoplastic cells and inhibition of MMP-9 expression, hampering of cells in the S phase and reduction of the rate of DNA synthesis, p21 and PCNA regulation were found [89]. HepG2 and LCC xenografts in a mouse model showed inhibition of tumor growth and a reduction in the number of metastatic colonies at the dose of 500 mg/kg [90]. Carboxymethylated chitosan [91] inhibited hydrogen peroxideinduced apoptosis in Schwann cells by reducing caspase-3, −9 and Bax activity and

Various biological properties of chitosan also include good adhesion to cells, macrophage activation, stimulation of fibroblast proliferation, stimulation of cytokinin production, stimulation of type IV collagen synthesis, promoting angiogenesis processes, haemostatic properties [92–95]. Moreover, it has a positive effect on granulation and epithelialization, and reduces scar formation. It is believed that many of the listed biological activities of chitosan are related to its unique feature, namely its cationic nature. Chitosan molecules with a positive charge interact with negatively charged erythrocytes and thrombocytes activating the extrinsic coagula-

Chemical modification of chitosan allows to modulate the biological activity of chitosan, for example, heparin inactivation or antiviral activity. Chitosan can be in the form of: gel, sponge, fiber or porous composition with ceramics, collagen or gelatin. Chitosan is used as a component of wound healing dressings, while in the case of scaffolds, it is usually used with other natural polymers (hyaluronic acid, alginic acid, poly-L-lactidic acid, elastin, collagen, gelatin) or additives (hydroxy-

Chitosan increases the inflow of phagocytic cells (segmented granulocytes and macrophages) to the site of infection, stimulates the migration and proliferation of endothelial cells and fibroblasts. The effect of chitosan on the proliferation of fibroblasts depends on the degree of deacetylation and molecular weight. Forms with a higher degree of deacetylation and lower molecular weight stimulate fibroblast proliferation to a greater extent [98–123]. Chitosan is widely researched for its use in bone and cartilage reconstruction. It has the ability to create porous structures, which makes it used in tissue engineering, orthopedics and bone regeneration. It has also been used in drug delivery systems or therapeutic substances (DNA plasmids, siRNA, nanosilver), for the production of surgical sutures, wound healing

An interesting chitosan feature is also its ability to bind with mucus and cross epithelial barriers, so that its use as an adjuvant or auxiliary adjuvant in vaccines is considered. It is also included among the auxiliary substances that enable the

It is an excellent metal ion complexing agent. This parameter is useful due to the immobilization of metal ions with antibacterial activity and enabling their

*DOI: http://dx.doi.org/10.5772/intechopen.95815*

increasing Bcl-2 activity.

tion, effectively stopping bleeding.

apatite, calcium phosphate, ceramic components) [95–97].

dressings and artificial internal organs [124–150].

controlled release, depending on the needs [97].

preparation of various forms of drugs with specific properties.

was observed compared to the use of the drug alone [79–85].

#### *Modulating the Physicochemical Properties of Chitin and Chitosan as a Method of Obtaining… DOI: http://dx.doi.org/10.5772/intechopen.95815*

thioglycolic acid, lactic acid, PEG and antiviral drugs, significantly higher efficacy was observed compared to the use of the drug alone [79–85].

Recent studies have shown that chitosan and its derivatives exhibit anti-tumor activity in both *in vitro* and *in vivo* models. It was found that chitosan derivatives increased the secretion of interleukin-1 and 2, influencing the maturation and infiltration of cytotoxic T lymphocytes [86]. The results have been confirmed in an *in vivo* study [87]. Moreover, a direct cytotoxic effect on neoplastic cells was found by inducing apoptosis. For A375, SKMEL28 and RPMI7951 cancer cell lines it was found to reduce cell adhesion, inhibit proliferation, inhibit specific caspases, regulate the activity of Bax as well as Bcl-2 and Bcl-XL, induce CD95 receptor expression through greater susceptibility to chitosan-induced apoptosis by FasL [88]. In the case of the PC3 A549, HepG2 and LCC cell lines, in the presence of chitosan, inhibition of the growth of neoplastic cells and inhibition of MMP-9 expression, hampering of cells in the S phase and reduction of the rate of DNA synthesis, p21 and PCNA regulation were found [89]. HepG2 and LCC xenografts in a mouse model showed inhibition of tumor growth and a reduction in the number of metastatic colonies at the dose of 500 mg/kg [90]. Carboxymethylated chitosan [91] inhibited hydrogen peroxideinduced apoptosis in Schwann cells by reducing caspase-3, −9 and Bax activity and increasing Bcl-2 activity.

Various biological properties of chitosan also include good adhesion to cells, macrophage activation, stimulation of fibroblast proliferation, stimulation of cytokinin production, stimulation of type IV collagen synthesis, promoting angiogenesis processes, haemostatic properties [92–95]. Moreover, it has a positive effect on granulation and epithelialization, and reduces scar formation. It is believed that many of the listed biological activities of chitosan are related to its unique feature, namely its cationic nature. Chitosan molecules with a positive charge interact with negatively charged erythrocytes and thrombocytes activating the extrinsic coagulation, effectively stopping bleeding.

Chemical modification of chitosan allows to modulate the biological activity of chitosan, for example, heparin inactivation or antiviral activity. Chitosan can be in the form of: gel, sponge, fiber or porous composition with ceramics, collagen or gelatin. Chitosan is used as a component of wound healing dressings, while in the case of scaffolds, it is usually used with other natural polymers (hyaluronic acid, alginic acid, poly-L-lactidic acid, elastin, collagen, gelatin) or additives (hydroxyapatite, calcium phosphate, ceramic components) [95–97].

Chitosan increases the inflow of phagocytic cells (segmented granulocytes and macrophages) to the site of infection, stimulates the migration and proliferation of endothelial cells and fibroblasts. The effect of chitosan on the proliferation of fibroblasts depends on the degree of deacetylation and molecular weight. Forms with a higher degree of deacetylation and lower molecular weight stimulate fibroblast proliferation to a greater extent [98–123]. Chitosan is widely researched for its use in bone and cartilage reconstruction. It has the ability to create porous structures, which makes it used in tissue engineering, orthopedics and bone regeneration. It has also been used in drug delivery systems or therapeutic substances (DNA plasmids, siRNA, nanosilver), for the production of surgical sutures, wound healing dressings and artificial internal organs [124–150].

An interesting chitosan feature is also its ability to bind with mucus and cross epithelial barriers, so that its use as an adjuvant or auxiliary adjuvant in vaccines is considered. It is also included among the auxiliary substances that enable the preparation of various forms of drugs with specific properties.

It is an excellent metal ion complexing agent. This parameter is useful due to the immobilization of metal ions with antibacterial activity and enabling their controlled release, depending on the needs [97].

*Chitin and Chitosan - Physicochemical Properties and Industrial Applications*

the antibacterial effect of chitosan. The mechanism explaining this feature is

unknown [51]. The antimicrobial activity of chitosan is strongly dependent on many factors, such as molecular weight [52], degree of deacetylation (DD), pH of the dissolving medium and its ionic strength. Stronger antibacterial activity was observed with a high degree of deacetylation [53] and a low molecular weight of chitosan [54]. The antibacterial activity of chitosan is also associated with the form of the polymer (hydrogels, membranes, dissolved form) and the presence of other compounds [55]. One of the factors responsible for the antibacterial activity of chitosan is its cationic nature. The positively charged ammonium groups of chitosan may interact with negatively charged components of the bacterial cell wall, causing damage to the cell membrane and destruction of bacterial cells (a mechanism proposed for high molecular weight chitosan) [56]. Ultimately, this causes the formation of an impermeable layer around the bacterial cell, affecting permeability and transport to the cell [57, 58]. It has been suggested that low molecular weight chitosan can penetrate bacterial cell walls and eventually enter the cytoplasm and bind to DNA affecting DNA transcription, mRNA synthesis and finally protein biosynthesis [59].

The difference in the hydrophilicity and the negative charge of the cell surface of the bacteria makes gram-negative bacteria interact more strongly with chitosan,

The antibacterial activity of chitosan or its derivatives on gram-negative bacteria has been demonstrated for various strains: *Escherichia coli*, *E. coli* K88, *E. coli* ATCC 25922, *E. coli* O157, *Pseudomonas aeruginosa*, *Proteus mirabilis*, *Salmonella enteritidis*, *S. choleraesuis* ATCC 50020, *S. typhimurium*, *S. typhimurium* ATCC 50013, *Enterobacter aerogenes*, *Listeria monocytogenes* [60–64]. The antibacterial activity of chitosan or its derivatives on gram-positive bacteria has been demonstrated for: *Staphylococcus aureus*, *S. aureus* ATCC 25923, *Corynebacterium*, *Staphylococcus epidermidis*, *Enterococcus faecalis*, *Bacillus cereus*, *Bacillus megaterium*. This indicates a broad spectrum of chitosan activity. It was also found that the effectiveness of chitosan binding to the bacterial cell wall depends on the pH value. At low pH, chitosan shows better adsorption to the bacterial cell wall due to the increased positive

The antifungal activity of chitosan also depends on its molecular weight and degree of acetylation. It was found that chitosan shows antifungal activity against selected phytopathogenic fungi *Penicillium* sp. in citrus [68], *Botrytis cinerea* in cucumber [69], *Phytophthora infestans* [70], *Alternaria solani* and *Fusarium oxysporum* [71]. Chitosan is also active against fungal species pathogenic to humans, while being non-toxic to human cells. The antifungal activity of chitosan or its derivatives has been demonstrated against: *Candida albicans*, *Candida parapsilosis*, *Candida krusei*, *Candida glabrata*, *Penicillium digitatum*, *Penicillium italicum*, *Fusarium proliferatum*, *Hamigera avellanea*, *Aspergillus fumigatus*, *Rhizopus stolonifer*, *Cryptus stolofattans*. The suggested mechanism of action is to create a permeable layer by chitosan which disrupts fungal growth [72–75]. It is believed to be related to the activation of defense processes, including chitinse accumulation, synthesis of proteinase inhibitors, callus synthesis and the lignification process [76].

The antiviral activity of chitosan derivatives is also suggested. The research focuses mainly on HIV. Peptide-chitosan conjugates (GlnMetTrp-chitosan and TrpMetGlnchitosan) show a protective effect on C8166 cells by counteracting the cytolytic effects of the HIV-1RF strain. These derivatives have the ability to suppress HIV-induced syncytium formation and reduce HIV load without inhibiting HIV-1 reverse transcriptase and protease *in vitro* [77]. Sulfated low molecular weight chitosan derivatives inhibit HIV-1 replication, HIV-1 induced syncytium formation, lytic activity and p24 antigen production. These derivatives are believed to influence the binding of HIV-1 gp120 to the CD4 cell surface receptor [78]. In comparative studies, chitosan conjugates with

resulting in higher antibacterial activity against them.

charge of protonated amino groups [65–67].

**144**

Chitosan can also be an environmentally friendly agent used to obtain textiles with antibacterial properties. Attempts were made to introduce chitosan powder into cotton and polyester-cotton fabric. Chitosan was introduced after the fabric surface was activated by 20% NaOH. The performed studies confirmed that chitosan is well implemented in fabrics made of a cotton and polyester/cotton blend [151].

## **4.3 Chitosan in dressing materials**

Due to its physicochemical and biological properties, chitosan and its derivatives are considered to be versatile biomaterials with various biological activities [152–159].

Chitosan and its derivatives as materials with antimicrobial activity and low immunogenicity are widely used in wound healing. They provide a three-dimensional matrix for tissue growth, activate macrophages and stimulate cell proliferation [160]. Chitosan improves the activity of polymorphonuclear leukocytes, macrophages and fibroblasts, which increase granulation and organization of repaired tissues [161]. Its degradation to N-acetyl-β-D-glucosamine stimulates the proliferation of fibroblasts, supports regular collagen deposition, and also stimulates the synthesis of hyaluronic acid at the wound site. These properties accelerate healing and prevent scarring [162]. The development of chitosan formation in the form of nanofibers with the assumed adhesive properties allowed to obtain a material useful for the creation of dressing materials [163]. Chitosan nanofibers obtained by electrospinning method are porous, have high tensile strength, large surface area combined with an ideal rate of water vapor and oxygen transfer. They are also compatible with stem cells derived from adipose tissue, which is beneficial for wound healing [164, 165].

A characteristic feature of chitosan dressings is their ability to effectively control bleeding [166]. The most important element of hemostasis is blood clotting, which leads to the formation of a clot consisting mainly of the fibrin network and platelets embedded in it. This process prevents further loss of fluid and electrolytes from the wound and reduces contamination of the wound. There is erythema around the wound, swelling, pain and locally increased temperature. Inflammation widens local blood vessels, which facilitates the penetration of macrophage cells and fibroblasts into the wound, which cleanse the wound of tissue residues, vascular clots and pathogenic bacteria. In the next phase of healing, fibroblasts synthesize collagen and other proteins needed to build and regenerate connective tissue and rebuild damaged blood vessels. In the course of scar formation, type III collagen fibers transform into type I collagen until they reach the balance characteristic of healthy skin and are necessary to restore skin continuity. The final remodeling process leads to a significant increase in the mechanical strength of the wound. The haemostatic effect of chitosan has been clearly documented. Chitosan in the form of a non-woven fabric has a positive effect on each stage of wound healing. The unique features of chitosan include: macrophage activation, stimulation of fibroblast proliferation, absorption of growth factors, stimulation of cytokinin production, stimulation of type IV collagen synthesis, support for angiogenesis processes, antibacterial and hemostatic properties. The positive effect of chitosan on granulation tissue, epidermis and reduction of scar formation has been proven. Like chitin, chitosan is susceptible to enzymatic biodegradation which produces biologically active oligosaccharides. The positively charged chitosan molecules react with negatively charged erythrocytes and thrombocytes to activate the external clotting pathway and effectively block bleeding. At the same time, chitosan can serve as a carrier of specific medicinal substances (DNA plasmids, siRNA, nanosilver particles), which enhance its positive effect on the healing process. Chitosan has also been found to significantly increase the adhesion and aggregation of platelets in the process of hemostasis [167, 168].

**147**

**Figure 10.**

*Tromboguard® dressing structure.*

*Modulating the Physicochemical Properties of Chitin and Chitosan as a Method of Obtaining…*

Currently, there are many chitosan materials available on the market that are used to heal wounds in patients undergoing plastic surgery [169], skin grafting [170, 171] and endoscopic sinus surgery [172]. Chitosan-containing materials in the form of nonwovens, nanofibers, composites, films and sponges are: HemCon®, GuardaCare®, ChitoFlex®, ChitoGauze®, Celox™ Granules, Celox™ Gauze, Chito-Seal™, Clo-SurPLUS PAD Tegasorb™, Tegaderm™ ChiGel, ChitopackC®, and

Haemostatic dressings also include Tromboguard® - a multi-layer dressing made of three layers: semi-permeable polyurethane foil, hydrophilic polyurethane foam, and a layer containing chitosan. The film layer protects the dressing against seepage, allowing the wound environment to remain moist, ensuring optimal air permeability to its interior and creating a barrier against external factors. Polyurethane foam is a load-bearing layer and has strong absorbent properties thanks to the "pore-inpore" structure. The polyurethane layer is responsible for storing exudate and keeping it outside the wound surface, ensuring adequate wound moisture. Additionally,

The active layer, which is created by a unique composition of chitosan and alginates, activates the blood coagulation process, significantly reducing bleeding time. By reacting on the wound surface with erythro- and thrombocytes, chitosan significantly shortens the bleeding time. Calcium alginate accelerates the natural clotting process, and sodium alginate - by absorbing wound discharge - creates a layer of gel on the surface of the dressing that prevents it from sticking to the wound. Alginates are resorbable, non-toxic, non-carcinogenic, non-allergic and haemostatic [177]. When used as dressing materials, it is important that during contact with the wound, part of the alginate dressing passes in the form of a gel, which prevents the wound surface from drying out, and thus creates the possibility of creating a favorable, moist environment within the skin lesion [178]. At the same time, hemostatic properties result in a faster wound healing process and allow for more effective scarring. Patients also benefit from using these dressings to reduce pain when changing them. A significant advantage of using alginate-containing dressings is the elimination of the dressing sticking to the wound and high absorbency.

The Tromboguard® dressing (**Figure 10**) is used to stop bleeding in the case of: traumatic wounds, postoperative wounds, skin graft collection sites in surgery and reconstructive surgery - including combustiology, wounds requiring emergency care, gunshot and puncture wounds, wounds resulting from traffic accidents. It is characterized by a quick hemostatic effect (stops bleeding in 3 minutes), an antibacterial effect inside the product (protecting the dressing against the growth of microorganisms), and effective blood absorption even under pressure. It is not

it is a layer that protects the wound against mechanical damage.

*DOI: http://dx.doi.org/10.5772/intechopen.95815*

TraumaStat™ [173–176].

irritating, sensitizing and cytotoxic.

### *Modulating the Physicochemical Properties of Chitin and Chitosan as a Method of Obtaining… DOI: http://dx.doi.org/10.5772/intechopen.95815*

Currently, there are many chitosan materials available on the market that are used to heal wounds in patients undergoing plastic surgery [169], skin grafting [170, 171] and endoscopic sinus surgery [172]. Chitosan-containing materials in the form of nonwovens, nanofibers, composites, films and sponges are: HemCon®, GuardaCare®, ChitoFlex®, ChitoGauze®, Celox™ Granules, Celox™ Gauze, Chito-Seal™, Clo-SurPLUS PAD Tegasorb™, Tegaderm™ ChiGel, ChitopackC®, and TraumaStat™ [173–176].

Haemostatic dressings also include Tromboguard® - a multi-layer dressing made of three layers: semi-permeable polyurethane foil, hydrophilic polyurethane foam, and a layer containing chitosan. The film layer protects the dressing against seepage, allowing the wound environment to remain moist, ensuring optimal air permeability to its interior and creating a barrier against external factors. Polyurethane foam is a load-bearing layer and has strong absorbent properties thanks to the "pore-inpore" structure. The polyurethane layer is responsible for storing exudate and keeping it outside the wound surface, ensuring adequate wound moisture. Additionally, it is a layer that protects the wound against mechanical damage.

The active layer, which is created by a unique composition of chitosan and alginates, activates the blood coagulation process, significantly reducing bleeding time. By reacting on the wound surface with erythro- and thrombocytes, chitosan significantly shortens the bleeding time. Calcium alginate accelerates the natural clotting process, and sodium alginate - by absorbing wound discharge - creates a layer of gel on the surface of the dressing that prevents it from sticking to the wound. Alginates are resorbable, non-toxic, non-carcinogenic, non-allergic and haemostatic [177]. When used as dressing materials, it is important that during contact with the wound, part of the alginate dressing passes in the form of a gel, which prevents the wound surface from drying out, and thus creates the possibility of creating a favorable, moist environment within the skin lesion [178]. At the same time, hemostatic properties result in a faster wound healing process and allow for more effective scarring. Patients also benefit from using these dressings to reduce pain when changing them. A significant advantage of using alginate-containing dressings is the elimination of the dressing sticking to the wound and high absorbency.

The Tromboguard® dressing (**Figure 10**) is used to stop bleeding in the case of: traumatic wounds, postoperative wounds, skin graft collection sites in surgery and reconstructive surgery - including combustiology, wounds requiring emergency care, gunshot and puncture wounds, wounds resulting from traffic accidents. It is characterized by a quick hemostatic effect (stops bleeding in 3 minutes), an antibacterial effect inside the product (protecting the dressing against the growth of microorganisms), and effective blood absorption even under pressure. It is not irritating, sensitizing and cytotoxic.

**Figure 10.** *Tromboguard® dressing structure.*

*Chitin and Chitosan - Physicochemical Properties and Industrial Applications*

**4.3 Chitosan in dressing materials**

Chitosan can also be an environmentally friendly agent used to obtain textiles with antibacterial properties. Attempts were made to introduce chitosan powder into cotton and polyester-cotton fabric. Chitosan was introduced after the fabric surface was activated by 20% NaOH. The performed studies confirmed that chitosan is well

Due to its physicochemical and biological properties, chitosan and its derivatives are considered to be versatile biomaterials with various biological activities [152–159]. Chitosan and its derivatives as materials with antimicrobial activity and low immunogenicity are widely used in wound healing. They provide a three-dimensional matrix for tissue growth, activate macrophages and stimulate cell proliferation [160]. Chitosan improves the activity of polymorphonuclear leukocytes, macrophages and fibroblasts, which increase granulation and organization of repaired tissues [161]. Its degradation to N-acetyl-β-D-glucosamine stimulates the proliferation of fibroblasts, supports regular collagen deposition, and also stimulates the synthesis of hyaluronic acid at the wound site. These properties accelerate healing and prevent scarring [162]. The development of chitosan formation in the form of nanofibers with the assumed adhesive properties allowed to obtain a material useful for the creation of dressing materials [163]. Chitosan nanofibers obtained by electrospinning method are porous, have high tensile strength, large surface area combined with an ideal rate of water vapor and oxygen transfer. They are also compatible with stem cells derived from adipose tissue, which is beneficial for wound healing [164, 165]. A characteristic feature of chitosan dressings is their ability to effectively control bleeding [166]. The most important element of hemostasis is blood clotting, which leads to the formation of a clot consisting mainly of the fibrin network and platelets embedded in it. This process prevents further loss of fluid and electrolytes from the wound and reduces contamination of the wound. There is erythema around the wound, swelling, pain and locally increased temperature. Inflammation widens local blood vessels, which facilitates the penetration of macrophage cells and fibroblasts into the wound, which cleanse the wound of tissue residues, vascular clots and pathogenic bacteria. In the next phase of healing, fibroblasts synthesize collagen and other proteins needed to build and regenerate connective tissue and rebuild damaged blood vessels. In the course of scar formation, type III collagen fibers transform into type I collagen until they reach the balance characteristic of healthy skin and are necessary to restore skin continuity. The final remodeling process leads to a significant increase in the mechanical strength of the wound. The haemostatic effect of chitosan has been clearly documented. Chitosan in the form of a non-woven fabric has a positive effect on each stage of wound healing. The unique features of chitosan include: macrophage activation, stimulation of fibroblast proliferation, absorption of growth factors, stimulation of cytokinin production, stimulation of type IV collagen synthesis, support for angiogenesis processes, antibacterial and hemostatic properties. The positive effect of chitosan on granulation tissue, epidermis and reduction of scar formation has been proven. Like chitin, chitosan is susceptible to enzymatic biodegradation which produces biologically active oligosaccharides. The positively charged chitosan molecules react with negatively charged erythrocytes and thrombocytes to activate the external clotting pathway and effectively block bleeding. At the same time, chitosan can serve as a carrier of specific medicinal substances (DNA plasmids, siRNA, nanosilver particles), which enhance its positive effect on the healing process. Chitosan has also been found to significantly increase the adhesion and aggregation of platelets in

implemented in fabrics made of a cotton and polyester/cotton blend [151].

**146**

the process of hemostasis [167, 168].

Tests of operational parameters: tensile strength, the ability to adapt to the injury site or the transmission of moisture vapors have shown that this dressing has a tensile strength (for porous materials) of min. 75 kPa (according to PN-EN ISO 1798), which corresponds to the value recommended for dressing materials, and vapor permeability (transmission of moisture vapor) of min. 400 g/m3 /24h.

The results of clinical trials have demonstrated the high haemostatic efficacy of Tromboguard®. The high effectiveness and durability of the antihaemorrhagic effect was confirmed 24 hours after application, which allowed the introduction of an absorbent foam dressing [179] and a three-layer hemostatic dressing to the market [96, 97].
