INTRODUCTION:

Polymers now play a major role in all biological applications. Utilizing the advantages provided by novel polymeric materials such as blends and composites has led to numerous noteworthy technological advancements¹. Biopolymers comprise polysaccharides such as cellulose and starch and animal protein-based polymers such as chitin, chitosan, wool, silk, gelatin, and collagen. Carbohydrate-based polymers, in particular, have been shown to have a highly promising industrial application in a variety of forms². Although chitosan is a basic polysaccharide, most commercial polysaccharides, including cellulose, dextran, pectin and alginic acids, carrageenan, agarose, starch, heparin,and agar–agar, are neutral or acidic. Chitosan becomes soluble in water at acidic pH as amino groups are protonated; at neutral or basic pH, it has free amino groups and is also insoluble in water. The distribution of free amino and N-acetyl groups determines the solubility³. With an acidic pH, chitosan is a linear polyelectrolyte. Having one charge for every glucose, it has a high charge density.

The inherent viscosity of polyelectrolytes is significantly influenced by pH and ionic strength⁴. Polyelectrolyte complexes were created by surface-reacting chitosan in an aqueous acid solution with polyanion aqueous solutions (heparin, sodium alginate, carboxymethyl chitin, and polyacrylic acid). Chitin is a key structural element found in the cell walls of fungi and yeast as well as in the exoskeletons of crab and shrimp shells. Chitin occurs in nature as organized macrofibrils. Because chitin is not easily dissolved in typical solvents, it is frequently transformed into chitosan, a more deacetylated derivative⁵. Both chitosan and chitin are non-toxic, biocompatible, and biodegradable. Additionally, chitosan and chitin function as moisturizing and antibacterial substances⁶. Wound healing is a specific biological process related to the general phenomenon of growth and tissue regeneration. A number of cellular and matrix components work together during the interdependent and overlapping phases of the wound healing process to restore the integrity of damaged tissue and replace lost tissue³. It has been suggested that the wound healing process consists of five overlapping stages that entail intricate cellular and molecular mechanisms. Homeostasis, inflammation⁷, migration, proliferation, and maturation are the terms used to characterize these stages⁸. Many reports have described the advantageous effects of chitin and chitosan for wound healing using in vitro, animal, clinical, pre-clinical, theranostic, and clinical studies. Applications for nano-chitin and nano-chitosan have expanded as a result of this technology's advancement. The biological characteristics of chitin and chitosan for wound healing are outlined in this review. Furthermore, new studies and advancements using nanochitin and nanochitosan for wound healing are discussed.

Figure 1: Illustration of different types of wounds and complications/problems associated with associated with acute and chronic wound

MECHANISM INVOLVED IN WOUND HEALING:

Hemostasis is the first stage of wound healing and serves as the foundation for subsequent phases⁹. The hemostasis process consists of four phases from a physiological perspective. First phase, the reflex vasoconstriction is triggered by the wound during the vasoconstriction stage. Second phase, platelet plugs are created by platelet aggregation. Third phase, the coagulation system is activated to generate fibrin clots. The coagulation system has three pathways: extrinsic, intrinsic, and common coagulation. The pathway of extrinsic coagulation initiates when blood vessels are injured and factor III is released. Factor VII, upon being activated by factor III, combines with factor-III and Ca2C to produce activated factor X (AFX). After blood vessel injury, factor XII is activated, exposing collagen fibres and initiating the intrinsic coagulation cascade. Following the activation of factor XII, factor XI activates factor XI, which then activates factor IX. Factor X is activated by a combination of Ca₂C, AF₃, AFV, and PF₃. AFX produced by the intrinsic and extrinsic coagulation pathways is used by the common coagulation pathway. Fourth phase, AFX combines with Ca₂C, PF₃, and AFV to produce a prothrombin activator, which activates thrombin[4]. Fibrinogen can be converted by thrombin into fibrin, which then forms a network to clump platelets, leukocytes, and erythrocytes to create fibrin clots that assist in the coagulation process. Once the wound stops bleeding, plasminogen activators transform inactive plasminogen into active plasmin, which causes the fibrin clot to dissolve. To avoid clogging of vessels, blood clots are dissolved in this stage¹⁰. Chitosan helps to stop bleeding and promote haemostasis, both of which contribute to the wound healing process during the homeostasis phase. It promotes erythrocyte and platelet aggregation necessary to initiate the clotting process. Furthermore, the haemostatic properties of chitosan help in the development of clots to halt bleeding, helping the earliest stage of wound healing¹⁰.At the "Inflammation" stage chitosan reduces inflammation during wound healing by helping to remove microorganisms and decreasing the inflammatory response. Hydrogels and other chitosan-based materials can help during the inflammatory phase by allowing macrophages and other inflammatory cells to remove necrotic tissue and germs from the wound. In addition, the anti-inflammatory properties of chitosan could help in modulating the inflammatory phase of wound healing^10,11. At the "Proliferation" stage, epithelial cells divide and spread out, forming the epithelial tissue that covers the wound. Granulation tissue grows more quickly in chitosan hydrogel, ultimately filling a tissue gap. Chitosan can enhance the proliferation stage of wound healing by improving cell proliferation as well as tissue regeneration¹². Chitosan has shown the tensile strength of wound, which supports its function during the proliferation phase of wound healing¹³. The final stage is remodeling, which completes the entire skin restoration operation. Chitosan has improved wound healing by enhancing collagenase activity, particularly in fibroblasts. Collagen, an essential part of an extracellular matrix (EM) in tissues, is broken down by the enzyme collagenase. Chitosan can promote remodeling of the EM, which might result in the development of new tissue and the restoration of tissue function, by increasing the activity of the enzyme collagenase¹².

Figure 2: (A)Schematic diagram to illustrate the mechanisms of chitosan to promote wound healing. (B) illustration of action of chitosan in wound healing and showing wound state before and after treatment with chitosan.

Stimulates Cell Proliferation and Angiogenesis:

Chitosan may speed up the overall process of wound healing by boosting angiogenesis, stimulating cell proliferation, lowering local growth factors and angiogenesis-inducing substances in contact with the wound¹⁴. Chitosan has the ability to absorb osmotic fluid and a wide range of inflammatory components. It can also prevent wound injury from becoming severe and buffer the inflammatory response of the wound, all of which assist in creating a reasonably mild growing environment that is better for the development of vascular endothelial cells (VEC)¹⁵. Chang et al. found that injectable chitosan-hyaluronic acid (CS-HA) hydrogel containing adipose-derived stem cells (ADSCs) dramatically accelerated wound closure. The composite hydrogel boosted cell proliferation and promoted keratinocyte migration by upregulating VEGF, chemotactic factors, and ECM-remodeling matrix metaloproteinases¹⁶. Chitosan-gelatin hydrogels suppress bacteria while simultaneously stimulating cell proliferation, neovascularization, granulation tissue development, wound healing, and active substance delivery ^17,18,¹⁴.

Promotes Haemostasis:

It is important to consider both the hemostatic and analgesic effects of chitosan while considering its ability to heal wounds. The distinct hemostatic characteristics of chitosan do not depend on regular clotting cascades¹⁹. Furthermore, chitosan may interact with neutrophils and macrophages, regulating fibroplasia and re-epithelialization process ²⁰. Chitosan can quickly coagulate blood because of its ability to connect with red blood cells; therefore, it has recently received regulatory approval for use in dressings and other hemostatic treatments^21,22. Moreover, chitosan regulates the function of inflammatory cells, thus promoting granulation and organization²³. Chitosan-based nanoparticles aid in the overall healing process of wounds by promoting hemostasis, demonstrating antibacterial activity, and scavenging free radicals. Ong et al. enhanced chitosan dressing by combining polyphosphate and silver to improve hemostatic and antibacterial properties²⁴. Both in vitro and animal experiments were conducted. The ideal chitosan-polyphosphate formulation (CSPP) was found to speed up blood clotting, increase platelet adhesion, produce thrombin faster, and absorb more blood than compared to chitosan alone. When compared with CSPP in vitro, silver-loaded CSPP had noticeably higher bactericidal activity, often killing P. aeruginosa completely and killing S. aureus with a mortality rate > 99.99%. An in vivo animal experiment revealed that silver dressing also significantly decreased mortality in a P. aeruginosa-infected wound model in mice, from 90% to 14.3%. Wound healing was not impeded, even though the dressing exhibited severe cytotoxicity toward cultured fibroblasts²⁴.

Promotes Skin regeneration:

Chitosan is a desirable substance for skin wound healing because studies have demonstrated that it promotes skin regeneration. As soon as the skin is injured, tissue renewal and skin restoration begin. The development of granulation tissues, which are composed of new capillaries, fibroblasts, and inflammatory cells, is an essential phase of skin restoration. Granulation tissue can promote epidermal regeneration and replenish the injured area. Research has revealed that chitosan can accelerate skin wound regeneration by stimulating the proliferation of inflammatory cells (macrophages), capillaries, and fibroblasts. Chitosan has the ability to stimulate macrophages to secrete cytokines, including PDGF, IL-1, and transforming growth factor-b (TGF-b). TGF-b stimulates collagen secretion and fibroblast proliferation by encouraging macrophage migration to injured regions²⁵. PDGF promotes angiogenesis, fibroblast migration, fibroblast proliferation, and the production of collagen, proteoglycans, and glycosaminoglycans during skin regeneration, resulting in the development of granulation tissue. By promoting angiogenesis, fibroblast proliferation, and collagen production, IL-1 also assists wound healing. Chitosan can also cause fibroblasts to secrete more IL-8, which can quicken the inflammatory process and promote angiogenesis. The influence of chitosan on fibroblast proliferation depends on the degree of deacetylation and molecular weight. A higher degree of deacetylation and a lower molecular weight of chitosan have a more significant influence on fibroblast proliferation²⁶.

GROWTH FACTORS INVOLVED IN WOUND HEALING:

Growth factors (GFs) are regulatory peptides secreted by platelets, endothelial cells, inflammatory cells, and fibroblasts. GFs can encourage the formation of the extracellular matrix (ECM) and promote cell migration, differentiation, and proliferation ^27,28,29. Chronic wounds produce fewer GFs at different phases of healing than normal wound healing³⁰. Chitosan is important for transporting GFs to the region of a wound, which promotes wound healing. Studies have found that GFs, like epidermal growth factor (EGF), can be efficiently transported by chitosan nanoparticles. Research has demonstrated that the presence of EGF in chitosan nanoparticles can prevent the degradation of GF, maintain its release, and improve its penetration into the skin, ultimately resulting in faster wound healing³¹. Chitosan has also been used to treat wounds because of its hemostatic and mucoadhesive³² qualities, as well as its capacity to encourage tissue growth and differentiation³³. Furthermore, by promoting the formation of fibroblasts and capillaries, chitosan has been found to speed up skin wound healing, emphasizing its role in delivering GFs to the site of injury²⁵. Consequently, chitosan acts as an effective means of administering GFs to wounds, thereby promoting improved wound healing. Chitosan-albumin hydrogel microspheres containing EGF showed sustained release in rats for almost three weeks after subcutaneous implantation³⁴. Pulat et al. produced a chitosan–polyacrylamide hydrogel containing EGF. The composite facilitated the proliferation of fibroblast cells for a longer duration than that of free EGF³⁵. Moreover, rhEGF was prepared and tested in diabetic rats using DDS-like, sodium carboxymethyl chitosan hydrogel³⁶, chitosan-alginate beads³⁷, and pluronic chitosan hydrogels³⁸. According to these findings, the chitosan-based formulations significantly increased the rate of healing and improved the quality of healing by releasing rhEGF at the wound sites in a controlled manner. Fibroblast GF (FGF) may promote angiogenesis by triggering the activation of fibroblasts and capillary endothelial cells³⁹. To provide stable release of FGF at the wound site, chitosan-based hydrogels were developed with its incorporation. According to numerous studies, adding aFGF⁴⁰ and bFGF^39,41 to hydrogels based on chitosan was an effective means to speed up the healing of chronic wounds. Rapid angiogenesis is essential for skin regeneration because it can reduce the risk of infection, eliminate metabolic waste, transfer nutrients and oxygen, and promote regeneration⁴². Improved wound healing and angiogenesis could be achieved by adding Vascular endothelial GF (VEGF) inside the dressing and gradually releasing it, all without causing reactive or granulomatous inflammatory reactions⁴³. GFs are classified into various families according to their respective characteristics. The growth factor families that are most important for wound healing are VEGF, platelet-derived GF( PDGF), TGF-b, EGF, and FGF^44,45,⁴⁶. Table 1 provides an overview of the origins and some important functions of these GFs involved in wound healing.

PHARMACOLOGICAL ACTIVITIES OF CHITOSAN:

It has been observed that the biodegradable and biocompatible polymer chitosan supports wound healing through various pharmacological effects. Some of these attributes are as follows:

Accelerates wound healing:

Several studies have been conducted on the potential benefits of chitosan as a wound-healing accelerator, and there is strong evidence that chitosan can positively influence each step of wound healing. Chitosan and its derivatives can accelerate wound healing by improving the function of inflammatory cells like polymorphonuclear leukocytes (PMN), osteoblasts/fibroblasts, and macrophages⁴⁷. Santos et al. investigated the impact of chitosan-derived membranes on PMN activation in humans. PMNs were obtained from the peripheral blood of healthy participants and cultured to develop into chitosan membranes. The findings indicated that PMNs, when exposed to chitosan-based membranes, release comparable quantities of lysozyme compared with the control group (PMNs devoid of materials). Furthermore, the results revealed that the components do not promote the production of free radicals(O2− or HO−)⁴⁸. Another in vitro study of chitosan on human keratinocytes and dermal fibroblasts revealed that chitosan stimulated fibroblast proliferation, which may interact with GFs found in the serum and enhance their proliferation effect. The degree of deacetylation (DOD) had significant effects on the proliferation activity of chitosan and exhibited greater efficacy²⁶. Chitosan also improves the tensile strength of wounds. The positive effects of chitosan on wound healing may be altered by characteristics such as molecular weight ⁴⁹^,⁵⁰,⁵¹, degree of deacetylation⁵², and chitosan state⁵³.

Antimicrobial activity:

Chitosan is one of the powerful antimicrobial (AM) agents because of its cationic nature. AM agents kill or inhibit the growth of bacteria. Because of its unique characteristics, chitosan is ideal for use in the formulation of AM edible films. Chitosan binds or chelates with gram-negative bacterial endotoxins, reducing their acute toxicity. Chitosan’s strong chelating capacity may eliminate the need for external chelating chemicals such as EDTA when combined with AM compounds such as nisin to inhibit gram-negative bacteria. Chitosan has been used as an AM agent in several studies, including in vivo and in vitro interactions in different forms (composites, films, and solutions) because of its ability to inhibit various bacteria, fungi, yeasts, and viruses. Chitosan-dependent AM activity was observed against different microorganisms including bacteria, fungi, and algae.⁵⁴. Intrinsic factors such as chitosan type, polymerization degree, host, natural nutrient content, substrate composition, and environmental conditions (such as substrate activity in water or moisture) influence antimicrobial effects. Photo-cross-linked electrospun mats with quartinized chitosan (QCS) successfully inhibited the growth of gram-positive and gram-negative bacteria. The resulting chitosan-based mats showed potential for wound dressing⁵⁵.The cationic group(amino) present in chitosan binds to the anionic groups, thereby inhibiting the growth of microorganisms. Bacterial infections can impede the healing process of a burn site during its infectious phase and may result in severe complications such as sepsis. The combination of chitosan and minocycline hydrochloride improves wound healing and reduces the number of bacteria⁵⁶. Tara et al. designed chitosan-coated noisomes loaded with tetracycline hydrochloride (CS-TCH) for the treatment of bacterial infections associated with skin wounds, urinary catheters, and dental implants⁵⁷. It showed high encapsulation efficiency (EE), enhanced stability, controlled release, negligible in vitro toxicity toward fibroblast human cells, and improved AM activity compared with uncoated TCH niosomes. CS-TCH provided sustained release for up to 72 h, exhibited 2-fold more anti-biofilm and anti-bacterial activity against S. aureus, P. aeruginosa, and E. coli than the uncoated TCH niosomes and 4-fold more than TCH solution. CS-TH and TH niosomes downregulated the expression of some biofilm genes such as icaA, ndvB, and csgA in tested bacteria, thus confirming its enhanced antibacterial activity than normal TH.

According to the literature, there are some main generally recognized mechanisms of action of chitosan against microorganisms, such as

Cell membrane/cell wall disruption:

Electrostatic interactions between positively charged chitosan molecules and negatively charged cell membranes are likely to be responsible for this effect.⁵⁸^,59,⁶⁰^,⁶¹.

Interaction with microbial DNA-products resulting from the hydrolysis of chitosan interact with microbial DNA, thereby impacting the process of protein synthesis by inhibiting mRNA⁶².

Chelation of nutrients through chitosan: Significant chelating activity of chitosan toward different metallic ions such as Cu²⁺,Ni²⁺, Fe²⁺, Co²⁺, Zn²⁺whenever the pH is greater than its pKa may explain why it inhibits microbial development⁶³.

Development of a dense polymeric film over the cell exterior surface: Chitosan can settle and produce a dense polymer layer on the cell surface, blocking nutrition and oxygen intake and thereby inhibiting the development of aerobic bacteria⁶⁴Various materials are produced on the basis of the type of wound and the mechanism of healing. They comprise synthetic polymers like silicon and polyurethane rubber and natural polymers such as collagen, alginate, gelatin, and chitosan. Bioactive dressings are of better quality than synthetic dressings.⁶⁵.

Anti-inflammatory activity:

Anti-inflammatory refers to a drug or treatment that lowers inflammation. Nearly half of analgesics are anti-inflammatory drugs that work by reducing inflammation and relieving pain, in contrast to opioids that act on central nervous system(CNS). Chitosan is a promising material for biomedical applications, including drug delivery, wound healing, antimicrobial, hemostatic agent, fat binder, and hypocholesterolemic effects, as indicated by many research studies published in recent years⁵⁴. Chitosan therapy, administered intraperitoneally with acetic acid, decreases pain associated with inflammation in a dose-dependent manner. Bradykinin is a key chemical associated with pain. A study found that administering a chitosan-acid (CSA) acetic solution reduced bradykinin levels in peritoneal lavage fluid compared with 0.5% acetic acid solution, indicating that chitosan may have analgesic benefits. Chitosan developed for wound management can provide analgesia by cooling and relaxing an open wound. Chitosan provided effective pain relief when applied topically to open wounds, including burns, ulcers, skin grafts, and abrasions⁶⁶. The anti-inflammatory activity of chitosan makes it effective for treating chronic inflammation at wound sites. Watersoluble chitosan inhibits the release and expression of proinflammatory cytokines (example, TNF-α and IL-6) and inducible nitric oxide (NO) synthase present in astrocytes, the primary neuroglial cells in the CNS. It is also crucial for cytokine-mediated inflammation⁶⁷. N-acetylglucosamine, an anti-inflammatory medicine, is produced in the body from glucose. It is found in both glycosaminoglycans and glycoproteins. Chito-oligosaccharides with a molecular weight of 5 kDa were shown to be more effective anti-inflammatory agents than the nonsteroidal anti-inflammatory drug (NSAID) indomethacin⁶⁸. Chitosan lowers inflammation by reducing the expression of prostaglandin E2, cyclooxygenase-2 proteins, and proinflammatory cytokines such as TNF-α and IL-1β. However, chitosan therapy enhances the expression of IL-10, an anti-inflammatory cytokine⁶⁹. Nurshen et al. designed chitosan-Zn complex (CS-Zn) films to improve chitosan’s biological performance by incorporating Zn as an antibacterial agent and a biologically active ion. No toxic response was observed when the stromal cell line ST-2 came into indirect contact with these CS-Zn films. The dissolving solution of CSZn increased wound closure by 88% compared with the positive control group (70%). CS-Zn showed effective antibacterial properties against S. aureus, with a minor increase (~30%) in VEGF secretion and slight reduction in NO evolution⁷⁰. In an in vitro study by Li et al., Curcumin (Cur) and CSNPs were found to reduce NF-κB expression as well as TNF-α and IL-6 levels. This showed that Cur-CSNPs reduced macrophage-induced inflammation. Cur-CSNPs were applied to the wounds of diabetic rats to test these findings. The experimental group showed lower inflammatory cell infiltration, higher new blood vessel development, and better collagen distribution⁷¹.

PRECLINICAL APPLICATIONS OF CHITOSAN IN WOUND HEALING:

Wounds can be caused by various injuries and proper cleaning and dressing of wounds are critical for preventing infections and additional harm⁷². Healing of the skin is the most prevalent form of wound recovery. It starts shortly after damage to the outermost skin layer and can persist for an extended period, often spanning years⁷³. This is a dynamic procedure that involves well-coordinated cellular, humoral, and molecular mechanisms. Inflammation, proliferation, and remodeling are the three interconnected phases. Any disruption in this sequence can result in irregular wound healing⁷⁴. Chronic wound development is a difficult issue for both healthcare providers and patients. Chitosan and its derivatives have been extensively employed in various biomedical applications, including wound healing. They stimulate rapid skin regeneration and wound healing. For the management of wounds, various dressing materials made from chitosan and chitin have been designed. Chitin and chitosan are interesting biopolymers for wound care because of their positive intrinsic characteristics and great potential for wound healing⁷⁵. The hemostatic effect of chitin is thought to be related to vasoconstriction, including the mobilization of erythrocytes, platelets, and clotting factors to the region of the damage⁷⁶. Chitosan is a polycation that possesses agglutinating and binding properties, as well as the ability to assist wound healing²³.

The solvent casting technique was used to create nanocomposite films of bentonite-chitosan for wound healing applications. The hydrophilic characteristics of bentonite improved the water absorption and mechanical strength of the film. The prepared films showed enhanced activity against gram-negative and gram-positive bacteria, indicating their potential usage as wound dressing⁷⁷. Cell behavior is significantly influenced by chitosan membranes containing immobilized RGD sequences. When L929 murine fibroblasts were exposed to these membranes in vitro, cell adhesion increased over time. RGD sequences are essential for cell adhesion, spreading, and proliferation. By binding to integrin and activating the Rho GTPase pathway, RGD promotes cell adhesion. Cell adhesion and biocompatibility were improved when RGD was immobilized on chitosan 3-D scaffolds. Functionalized chitosan functions as an extracellular matrix substitute, boosting cell adherence, spreading, and proliferation⁷⁸. The hydrogel film is made from chitosan and carboxyl-modified hypromellose. The cross linked films exhibited a higher thickness and better water absorption than neat chitosan fill. Hydrogels are appropriate for biomedical applications because of their outstanding biocompatibility and physical features that are similar to those of living tissues⁷⁹. Electrospinning was used to create biocompatible chitosan/sericin composite nanofibres. Furthermore, the hybrid nanofibres demonstrated good bactericidal action against both gram-positive and gram-negative bacteria and the ability to induce cell proliferation. These nanofibers can be used in wound dressings. Cell viability was greater than 90% for 24 h for all test concentrations of up to 250 g/mL, demonstrating the nontoxicity and good biocompatibility of composite nanofibers toward cells. Interestingly, after 72 h of incubation, all cell viability was greater than 100% when compared with the control group⁸⁰. When combined with chitosan, the asymmetric membrane serves an important function in wound healing. The outer layer of the asymmetric membrane serves as a protective barrier, preventing the wound from being damaged by external factors such as germs and chemical agents. The inner porous portion of the membrane acts as a template for cell adhesion, migration, and proliferation, thereby enhancing wound healing. It improves hemostasis, modulates inflammatory cell activity, protects against bacterial contamination, promotes tissue growth, and increases fibroblast proliferation, deposition of collagen, and angiogenesis⁸². By heating a combination of chitosan and graphene oxide, researchers created an injectable and self-healing hydrogel that produced no contaminants or waste. The mechanical characteristics of the hydrogel can be altered by varying the dosage of graphene oxide. It also demonstrated a high degree of adhesion and compatibility with blood. In vivo experiments on rats revealed excellent hemostatic and wound regeneration properties in liver hemorrhage and in a full-thickness skin wound model⁸³. By combining hydroxy butyl chitosan with chitosan, a porous material was formed using vacuum freeze-drying. In comparison to sponges made of chitosan and hydroxy butyl chitosan, this hydrophilic and macroporous sponge exhibited increased porosity (approximately 85%), higher water-absorbing capacity (about 25 times), improved flexibility, and a reduced blood-clotting index.

The composite sponge absorbs blood moisture, increasing its concentration, viscosity and clogging capillaries. It was nontoxic to cells, encouraged fibroblast development, and demonstrated substantial antibacterial effectiveness with a bacterial reduction of over 99.99%. The composite sponge aided wound healing in rats by enhancing epithelial cell adhesion, penetration, skin gland development, and re-epithelialization⁸⁴. The wound healing benefits of a chitosan-derived gel incorporating vitexin were studied in vitro and in vivo. In vitro studies with NIH 3T3 and HaCaT cells revealed that the vitexin/chitosan formulation significantly increased cell proliferation. In an in vivo model of excisional wounds, a chitosan-based gel containing vitexin accelerated wound healing and stimulated skin regeneration. A histological investigation revealed increased skin regeneration and cell proliferation⁸⁴. Hydrogel, membrane, and sponge dressings were prepared using carboxymethyl-chitosan (CMCS) cross-linked with genipin. The CMCS sponge dressing outperformed the other sponge dressings in terms of water absorption, gaseous permeability, hemostatic efficacy, and skin fibroblast proliferation. The sponge dressing elevated the levels of α-smooth muscle actin (α-SMA) and transforming growth factor-1 (TGF-1), whereas the CMCS hydrogel enhanced the expression of matrix metalloproteinase-1 (MMP-1).In vivo experiments revealed that the CMCS sponge dressing resulted in the quickest wound closure as well as epithelization, resulting in a well-structured epidermis and organized collagen in the dermis⁸⁵. For wound healing applications, a CS composite sponge dressing loaded using iturin-AgNPs was created and tested. Iturin-AgNPs have better antibacterial properties at lower concentrations than typical AgNPs. The CS dressing with iturin-AgNPs demonstrated greater antibacterial efficacy and improved wound healing in vivo compared with commercial wound dressings incorporating AgNPs. Wound healing promotion mechanisms included improved re-epithelialization, collagen synthesis, and antimicrobial activity. Importantly, iturin-AgNPs loaded with CS dressing were not harmful to mouse organs⁸⁶. Mardi et al. reported that starting on day 7, the topical use of TP chitosan NPs demonstrated a gradual reduction within wounds. On day 21, TP-NP significantly sped up wound healing compared to both the untreated and groups treated with vehicle, resulting in almost complete healing. remarkably, the designed TP-NPs formula demonstrated much better healing activities than the plain TP group, as measured by percentage wound contraction ⁸⁷.

THERANOSTIC APPLICATIONS OF CHITOSAN IN WOUND HEALING:

A smart dressing for diabetic wound healing was created using graphene quantum dot-decorated luminous porous silicon (GQDs@PSi), therapeutic peptides, and CS films. GQD ornamentation increased peptide loading, and PSi nanochannels protected peptides from destruction by matrix metalloproteinases (MMP). Owing to PSi oxidation, the GQDs@PSi exhibited H₂O₂-responsive ratiometric fluorescence alteration and drug release. In diabetic wounds, smart dressings release drugs under acidic and oxidative conditions. Cell proliferation and migration, particularly diabetic wound healing, were improved in vitro and in vivo, as well as in real-time monitoring associated with the wound healing process by measuring the rate of fluorescence discoloration^88,89. A polyacrylamide-quaternary ammonium chitosan-based carbon quantum dots (CQDs) red hydrogel was used to create a unique wound dressing method. These hydrogels can detect wound pH levels while decreasing bacterial infection and aiding in wound healing. Smartphone photos can be transformed into real-time wound pH level signals, allowing the remote assessment of wound dynamics. Hydrogels are hemostatic, sticky, moisture-retaining, antimicrobial, and skin repair. These multifunctional hydrogels have great potential as smart wound-dressing tools for skin regeneration⁹⁰. In a study by Kaikai et al., polyacrylamide (PAM)-quaternary ammonium chitosan QCS-phenol red(P) hydrogel-treated mouse wounds exhibited a smaller area than the control group. After a 14-day treatment, the PAM-QCS-carbon quantum dot (C)-P hydrogels led to the complete regeneration of the entire wound layer, suggesting their effectiveness in wound healing. The ability of hydrogels to securely bind to the wound surface, promptly stop bleeding, absorb exudates, and reduce bacterial growth, combined with their pH-dependent real-time monitoring capacity, highlights their potential as effective theranostic dressings for wound healing⁹⁰. The effects of integrating prostaglandin E2 (PGE₂) into a chitosan hydrogel (CS+PGE₂ hydrogel) on tissue restoration were studied using a mouse wound healing model. The CS hydrogel increased PGE₂ release, which improved the tissue regeneration. Molecular imaging, notably bioluminescence imaging, demonstrated that sustained PGE₂ production enhanced M2 macrophage polarization, resulting in decreased inflammation. The CS+PGE₂ hydrogel also increased angiogenesis during the early stages of tissue repair. During wound healing, the hydrogel efficiently controlled the inflammatory process, regeneration (angiogenesis), and remodelling (fibrosis) stages. By monitoring cellular and molecular activities in real-time, molecular imaging can help guide tissue restoration and aid in the development of successful therapies⁹⁰.

CLINICAL APPLICATIONS OF CHITOSAN IN WOUND HEALING:

The efficiency of chitosan dressing compared with conventional gauze dressing in managing postoperative bleeding wounds in both patients and rats was investigated. Chitosan dressings have demonstrated beneficial properties such as biocompatibility and fast blood coagulation. It showed strong antibacterial activity and prevented bacterial growth for up to eight days after surgery. Furthermore, chitosan dressings stimulate the formation of beneficial bacteria in patients, thereby boosting skin immunity and improving wound healing. These findings imply that chitosan dressings serve as beneficial antibacterial and procoagulant alternatives that promote wound healing by creating a favorable habitat for beneficial microorganisms in both clinical and experimental contexts⁹¹. Damour et al. described the first clinical application of a collagen-chitosan- glycosaminoglycan-based artificial dermis. Preclinical testing in rats and mice was followed by clinical testing in the human volunteers. Preliminary investigations in rodents have indicated inadequate blood supply and fibrous tissue. To address these limitations, researchers assessed the application of an over-grafted cultured epidermis consisting of collagen–chitosan– glycosaminoglycans in patients with deep burn wounds. The wounds were excised early and treated using autologous cultured autografts. Histological study results 10 days after grafting demonstrated homogeneous partial colonization of fibroblasts to complete colonization after 21 days ^92,93. Furthermore, one study found that the recommended dermal material made of collagen, chitosan, and glycosaminoglycans assists in promoting vascularization and colonization^93,94. The researchers evaluated the efficacy of chitosan-based membranes compared to traditional dressings (Bactigras®). Wound healing, adhesion, re-epithelialization, and hemostasis were aided by the chitosan membranes. Because of their porosity, meshed chitosan membranes have been shown to promote faster healing and are more suitable than non-meshed chitosan membranes⁵³. NG et al. created carboxymethyl chitosan composite hydrogels and conducted in vitro experiments to assess their influence of these composite hydrogels on the growth of human skin and hypertrophic scar fibroblasts. The results demonstrated the impressive mechanical characteristics and favorable biological attributes of chitosan-based hydrogels, indicating their potential utility in the advancement of wound dressings. The survival percentage of NS-FB cells grown with the hydrogel extract was 93%, demonstrating good cytocompatibility and the capacity to stimulate cell proliferation. HS-FB cells survived at a rate above 88 % with no negative side effects⁹⁵.

CONCLUSION:

Wounds are very prevalent and can be caused by many different types of trauma, including secondary injuries, ulcers, microbial infections, and tissue dehydration. Therefore, from a clinical perspective, it is crucial to develop functional wound dressings that may help in the healing process. Chitosan is a type of natural polysaccharide that is frequently used as a wound care material because of its hemostatic, antimicrobial, and granulation tissue growth-promoting properties. This study well explained the pharmacological effect of chitosan and their various applications such as pre-clinical, theranostic, and clinical particularly for wound dressings. Owing to its antibacterial and anti-inflammatory properties, chitosan has been demonstrated to be a promising biomaterial for wound healing. The use of chitosan biomaterials would be cost-effective and improve patient compliance. Although several results have been reported, these materials still require thorough and uniform characterization. To accomplish these objectives, it is necessary to modify the physical characteristics of the identified systems. Despite the abundance of research on chitosan or modified chitosan (blends, compositions, and derivatives) for wound healing, a number of issues still need to be investigated. It is anticipated that this paper will offer insights into the application of these significant chitosan-based materials to researchers conducting research in pharmaceutical and biological sciences.

REFERENCES:

1. Bura AR. Effect of Wound Healing Potential of Plumeria obtusa (Champa) Spray. Asian J Pharm Res. 2018; 8(4): 231. doi:10.5958/2231-5691.2018.00039.4

2. Jacob J, Haponiuk JT, Thomas S, Gopi S. Biopolymer based nanomaterials in drug delivery systems: A review. Mater Today Chem. 2018; 9: 43-55. doi:https://doi.org/10.1016/j.mtchem.2018.05.002

3. Thiruchelvi R, Priyadharshini S, Mugunthan P, Rajakumari K. Collagen–Zinc Oxide Nanoparticles (ZnO NPs) Composites for Wound Healing–A Review. Res J Pharm Technol. 2022; 15(6): 2838-2844. http://dx.doi.org/10.52711/0974-360X.2022.00474

4. Gobinath T, Kaviya K, Ravichandran S. Chitosan/Ulva lactuca polysaccharide Hydrogel containing copper (II) beneficial for Biofilm-associated wound infection: Formulation Characterizations and in vitro study. Res J Pharm Technol. 2020; 13(9): 4224-4230. http://dx.doi.org/10.5958/0974-360X.2020.00746.5

5. Hamzah ML. Formulation and evaluation of Flurbiprofen nanogel. Res J Pharm Technol. 2020; 13(11): 5183-5188. http://dx.doi.org/10.5958/0974-360X.2020.00906.3

6. Ratnaparkhi MP, Andhale RS, Karnawat GR. Nanofibers-a newer technology. Res J Pharm Technol. 2021; 14(4): 2321-2327. http://dx.doi.org/10.52711/0974-360X.2021.00410.

7. Paswan SK, Srivastava S, Rao CV. Incision wound healing, anti-inflammatory and analgesic activity of amaranthus spinonous in wistar rats. Res J Pharm Technol. 2020; 13(5): 2439-2444. doi:10.5958/0974-360X.2020.00437.0

8. Beanes SR, Dang C, Soo C, Ting K. Skin repair and scar formation: the central role of TGF-β. Expert Rev Mol Med. 2003; 5(8): 1-22. https://doi.org/10.1017/S1462399403005817.

9. Budhy TI, Rahayu RP, Prathama FA. Potential of Anadara granosa Nanoparticles to improve the Expression of the Fibroblast Growth Factor-2 (FGF-2) in Chronic wound of Hyperglycemia conditions. Res J Pharm Technol. 2022; 15(12): 5757-5760.http://dx.doi.org/10.52711/0974-360X.2022.00971.

10. Feng P, Luo Y, Ke C, et al. Chitosan-Based Functional Materials for Skin Wound Repair: Mechanisms and Applications. Front Bioeng Biotechnol. 2021; 9(February). doi:10.3389/fbioe.2021.650598

11. Kalaivani C, Kuppusamy G, Karri VVSR. Simvastatin loaded polycaprolactone-collagen scaffolds for the treatment of diabetic foot ulcer. Res J Pharm Technol. 2019; 12(6): 2637-2644. http://dx.doi.org/10.5958/0974-360X.2019.00441.4.

12. Salva E, Alan S, Karakoyun B, Çakalağaoğlu F, Özbaş S, Akbuğa J. Investigation of therapeutic effects in the wound healing of chitosan/pGM-CSF complexes. Brazilian J Pharm Sci. 2022; 58: 1-15. doi:10.1590/s2175-97902022e19668

13. Jangde RK, Khute S. Design and Development of Ciprofloxacin Lipid Polymer Hybrid Nanoparticle by Response Surface Methodology. Res J Pharm Technol. 2020; 13(7): 3249-3256. http://dx.doi.org/10.5958/0974-360X.2020.00576.4.

14. Liu H, Wang C, Li C, et al. A functional chitosan-based hydrogel as a wound dressing and drug delivery system in the treatment of wound healing. RSC Adv. 2018; 8(14): 7533-7549. doi:10.1039/c7ra13510f

15. Hu J, Lin Y, Cui C, et al. Clinical efficacy of wet dressing combined with chitosan wound dressing in the treatment of deep second-degree burn wounds: A prospective, randomised, single-blind, positive control clinical trial. Int Wound J. 2023; 20(3): 699-705. doi:10.1111/iwj.13911

16. Liu H, Ding J, Wang C, et al. Intra-articular transplantation of allogeneic BMMSCs rehabilitates cartilage injury of antigen-induced arthritis. Tissue Eng Part A. 2015; 21(21-22): 2733-2743. https://doi.org/10.1089/ten.tea.2014.0666.

17. Huang X, Zhang Y, Zhang X, Xu L, Chen X, Wei S. Influence of radiation crosslinked carboxymethyl-chitosan/gelatin hydrogel on cutaneous wound healing. Mater Sci Eng C. 2013; 33(8): 4816-4824. https://doi.org/10.1016/j.msec.2013.07.044.

18. Lin Y, Lin J, Li T, et al. Dressing with epigallocatechin gallate nanoparticles for wound regeneration. Wound Repair Regen. 2016; 24(2): 287-301.https://doi.org/10.1111/wrr.12372.

19. Sharma D, Rajput J, Kaith BS, Kaur M, Sharma S. Synthesis of ZnO nanoparticles and study of their antibacterial and antifungal properties. Thin Solid Films. 2010; 519(3): 1224-1229.https://doi.org/10.1016/j.tsf.2010.08.073.

20. Diegelmann RF, Dunn JD, Lindblad WJ, Cohen IK. Analysis of the effects of chitosan on inflammation, angiogenesis, fibroplasia, and collagen deposition in polyvinyl alcohol sponge implants in rat wounds. Wound Repair Regen. 1996; 4(1): 48-52. https://doi.org/10.1046/j.1524-475X.1996.40109.x

21. Kozen BG, Kircher SJ, Henao J, Godinez FS, Johnson AS. An alternative hemostatic dressing: comparison of CELOX, HemCon, and QuikClot. Acad Emerg Med. 2008; 15(1): 74-81. https://doi.org/10.1111/j.1553-2712.2007.00009.x.

22. Millner RWJ, Lockhart AS, Bird H, Alexiou C. A New Hemostatic Agent: Initial Life-Saving Experience With Celox (Chitosan) in Cardiothoracic Surgery. Ann Thorac Surg. 2009; 87(2): e13-e14. doi:10.1016/j.athoracsur.2008.09.046

23. Ueno H, Mori T, Fujinaga T. Topical formulations and wound healing applications of chitosan. Adv Drug Deliv Rev. 2001; 52(2): 105-115.https://doi.org/10.1016/S0169-409X(01)00189-2

24. Ong SY, Wu J, Moochhala SM, Tan MH, Lu J. Development of a chitosan-based wound dressing with improved hemostatic and antimicrobial properties. Biomaterials. 2008; 29(32): 4323-4332. doi:10.1016/j.biomaterials.2008.07.034

25. Feng P, Luo Y, Ke C, et al. Chitosan-based functional materials for skin wound repair: Mechanisms and applications. Front Bioeng Biotechnol. 2021; 9: 650598. https://doi.org/10.3389/fbioe.2021.650598.

26. Howling GI, Dettmar PW, Goddard PA, Hampson FC, Dornish M, Wood EJ. The effect of chitin and chitosan on the proliferation of human skin fibroblasts and keratinocytes in vitro. Biomaterials. 2001; 22(22): 2959-2966. doi:https://doi.org/10.1016/S0142-9612(01)00042-4

27. Dvorak HF. Vascular permeability to plasma, plasma proteins, and cells: an update. Curr Opin Hematol. 2010; 17(3): 225. https://doi.org/10.1097%2FMOH.0b013e3283386638.

28. Nedelec B, De Oliveira A, Saint-Cyr M, Garrel DR. Differential effect of burn injury on fibroblasts from wounds and normal skin. Plast Reconstr Surg. 2007; 119(7): 2101-2109. https://doi.org/ 10.1097/01.prs.0000260592.31969.06

29. Guo S al, DiPietro LA. Factors affecting wound healing. J Dent Res. 2010; 89(3): 219-229. https://doi.org/10.1177/0022034509359125

30. Moura LIF, Dias AMA, Carvalho E, de Sousa HC. Recent advances on the development of wound dressings for diabetic foot ulcer treatment—A review. Acta Biomater. 2013; 9(7): 7093-7114. https://doi.org/10.1016/j.actbio.2013.03.033

31. Montazeri S, Rastegari A, Mohammadi Z, et al. Chitosan nanoparticle loaded by epidermal growth factor as a potential protein carrier for wound healing: In vitro and in vivo studies. IET nanobiotechnology. 2023; 17(3): 204-211. doi:10.1049/nbt2.12116

32. Mishra A, Sahu G, Kumar A, et al. Underlining the pharmaceutical aspects associated with the development of pH responsive hydrogel. Res J Pharm Technol. 2017; 10(4): 1261-1268. http://dx.doi.org/10.5958/0974-360X.2017.00224.4.

33. Vijayan A, Sabareeswaran A, Kumar GSV. PEG grafted chitosan scaffold for dual growth factor delivery for enhanced wound healing. Sci Rep. 2019; 9(1): 1-12. doi:10.1038/s41598-019-55214-7

34. Amsden B. Novel biodegradable polymers for local growth factor delivery. Eur J Pharm Biopharm. 2015; 97: 318-328. https://doi.org/10.1016/j.ejpb.2015.06.008.

35. Pulat M, Kahraman AS, Tan N, Gümüşderelioğlu M. Sequential antibiotic and growth factor releasing chitosan-PAAm semi-IPN hydrogel as a novel wound dressing. J Biomater Sci Polym Ed. 2013; 24(7): 807-819. https://doi.org/10.1080/09205063.2012.718613.

36. Hajimiri M, Shahverdi S, Esfandiari MA, et al. Preparation of hydrogel embedded polymer-growth factor conjugated nanoparticles as a diabetic wound dressing. Drug Dev Ind Pharm. 2016; 42(5): 707-719. https://doi.org/10.3109/03639045.2015.1075030.

37. Sukumar N, Ramachandran T, Kalaiarasi H, Sengottuvelu S. Characterization and in vivo evaluation of silk hydrogel with enhancement of dextrin, rhEGF, and alginate beads for diabetic Wistar Albino wounded rats. J Text Inst. 2015; 106(2): 133-140. https://doi.org/10.1080/00405000.2014.906100.

38. Choi JS, Yoo HS. Pluronic/chitosan hydrogels containing epidermal growth factor with wound‐adhesive and photo‐crosslinkable properties. J Biomed Mater Res Part A. 2010; 95(2): 564-573. https://doi.org/10.1002/jbm.a.32848.

39. Park CJ, Clark SG, Lichtensteiger CA, Jamison RD, Johnson AJW. Accelerated wound closure of pressure ulcers in aged mice by chitosan scaffolds with and without bFGF. Acta Biomater. 2009; 5(6): 1926-1936. https://doi.org/10.1016/j.actbio.2009.03.002.

40. Wang W, Lin S, Xiao Y, et al. Acceleration of diabetic wound healing with chitosan-crosslinked collagen sponge containing recombinant human acidic fibroblast growth factor in healing-impaired STZ diabetic rats. Life Sci. 2008; 82(3-4): 190-204. https://doi.org/10.1016/j.lfs.2007.11.009.

41. Obara K, Ishihara M, Fujita M, et al. Acceleration of wound healing in healing‐impaired db/db mice with a photocrosslinkable chitosan hydrogel containing fibroblast growth factor‐2. Wound repair Regen. 2005; 13(4): 390-397. https://doi.org/10.1111/j.1067-1927.2005.130406.x.

42. Guo L, Wang W, Chen Z, Zhou R, Liu Y, Yuan Z. Promotion of microvasculature formation in alginate composite hydrogels by an immobilized peptide GYIGSRG. Sci China Chem. 2012; 55: 1781-1787. https://doi.org/10.1007/s11426-012-4513-1.

43. Ribeiro MP, Morgado PI, Miguel SP, Coutinho P, Correia IJ. Dextran-based hydrogel containing chitosan microparticles loaded with growth factors to be used in wound healing. Mater Sci Eng C. 2013; 33(5): 2958-2966. https://doi.org/10.1016/j.msec.2013.03.025.

44. Kiwanuka E, Junker J, Eriksson E. Harnessing growth factors to influence wound healing. Clin Plast Surg. 2012; 39(3): 239-248. https://doi.org/10.1016/j.cps.2012.04.003

45. Behm B, Babilas P, Landthaler M, Schreml S. Cytokines, chemokines and growth factors in wound healing. J Eur Acad Dermatology Venereol. 2012; 26(7): 812-820.https://doi.org/10.1111/j.1468-3083.2011.04415.x.

46. Gainza G, Villullas S, Pedraz JL, Hernandez RM, Igartua M. Advances in drug delivery systems (DDSs) to release growth factors for wound healing and skin regeneration. Nanomedicine Nanotechnology, Biol Med. 2015; 11(6): 1551-1573. https://doi.org/10.1016/j.nano.2015.03.002.

47. Dai T, Tanaka M, Huang YY, Hamblin MR. Chitosan preparations for wounds and burns: antimicrobial and wound-healing effects. Expert Rev Anti Infect Ther. 2011; 9(7): 857-879. doi:10.1586/eri.11.59

48. Santos TC, Marques AP, Silva SS, et al. In vitro evaluation of the behaviour of human polymorphonuclear neutrophils in direct contact with chitosan-based membranes. J Biotechnol. 2007; 132(2): 218-226. doi:https://doi.org/10.1016/j.jbiotec.2007.07.497

49. Kojima K, Okamoto Y, Kojima K, et al. Effects of chitin and chitosan on collagen synthesis in wound healing. J Vet Med Sci. 2004; 66(12): 1595-1598.https://doi.org/10.1292/jvms.66.1595.

50. Minagawa T, Okamura Y, Shigemasa Y, Minami S, Okamoto Y. Effects of molecular weight and deacetylation degree of chitin/chitosan on wound healing. Carbohydr Polym. 2007; 67(4): 640-644.https://doi.org/10.1016/j.carbpol.2006.07.007.

51. Alsarra IA. Chitosan topical gel formulation in the management of burn wounds. Int J Biol Macromol. 2009; 45(1): 16-21. doi:10.1016/j.ijbiomac.2009.03.010

52. Alsarra IA. Chitosan topical gel formulation in the management of burn wounds. Int J Biol Macromol. 2009; 45(1): 16-21. https://doi.org/10.1016/j.ijbiomac.2009.03.010.

53. Azad AK, Sermsintham N, Chandrkrachang S, Stevens WF. Chitosan membrane as a wound‐healing dressing: characterization and clinical application. J Biomed Mater Res Part B Appl Biomater An Off J Soc Biomater Japanese Soc Biomater Aust Soc Biomater Korean Soc Biomater. 2004; 69(2): 216-222. https://doi.org/10.1002/jbm.b.30000.

54. Ahmed S, Ikram S. Chitosan & its derivatives: a review in recent innovations. Int J Pharm Sci Res. 2015; 6(1): 14-30. http://dx.doi.org/10.13040/IJPSR.0975-8232.6(1).14-30.

55. Jayakumar R, Prabaharan M, Nair S V, Tamura H. Novel chitin and chitosan nanofibers in biomedical applications. Biotechnol Adv. 2010; 28(1): 142-150. https://doi.org/10.1016/j.biotechadv.2009.11.001.

56. Boateng JS, Matthews KH, Stevens HNE, Eccleston GM. Wound healing dressings and drug delivery systems: a review. J Pharm Sci. 2008; 97(8): 2892-2923. https://doi.org/10.1002/jps.21210.

57. Pourseif T, Ghafelehbashi R, Abdihaji M, et al. Chitosan -based nanoniosome for potential wound healing applications: Synergy of controlled drug release and antibacterial activity. Int J Biol Macromol. 2023; 230: 123185. doi:https://doi.org/10.1016/j.ijbiomac.2023.123185

58. Pasquina LW, Santa Maria JP, Walker S. Teichoic acid biosynthesis as an antibiotic target. Curr Opin Microbiol. 2013; 16(5): 531-537. https://doi.org/10.1016/j.mib.2013.06.014.

59. Severino R, Ferrari G, Vu KD, Donsì F, Salmieri S, Lacroix M. Antimicrobial effects of modified chitosan based coating containing nanoemulsion of essential oils, modified atmosphere packaging and gamma irradiation against Escherichia coli O157: H7 and Salmonella Typhimurium on green beans. Food Control. 2015; 50: 215-222. https://doi.org/10.1016/j.foodcont.2014.08.029

60. Tamara FR, Lin C, Mi FL, Ho YC. Antibacterial effects of chitosan/cationic peptide nanoparticles. Nanomaterials. 2018; 8(2): 88. https://doi.org/10.3390/nano8020088

61. Beck BH, Yildirim‐Aksoy M, Shoemaker CA, Fuller SA, Peatman E. Antimicrobial activity of the biopolymer chitosan against Streptococcus iniae. J Fish Dis. 2019; 42(3): 371-377. https://doi.org/10.1111/jfd.12938.

62. Archana D, Singh BK, Dutta J, Dutta PK. Chitosan-PVP-nano silver oxide wound dressing: in vitro and in vivo evaluation. Int J Biol Macromol. 2015; 73: 49-57.

63. Chien RC, Yen MT, Mau JL. Antimicrobial and antitumor activities of chitosan from shiitake stipes, compared to commercial chitosan from crab shells. Carbohydr Polym. 2016; 138: 259-264. https://doi.org/10.1016/j.carbpol.2015.11.061.

64. Tayel AA, Moussa S, Opwis K, Knittel D, Schollmeyer E, Nickisch-Hartfiel A. Inhibition of microbial pathogens by fungal chitosan. Int J Biol Macromol. 2010; 47(1): 10-14.https://doi.org/10.1016/j.ijbiomac.2010.04.005

65. Devi N, Dutta J. Preparation and characterization of chitosan-bentonite nanocomposite films for wound healing application. Int J Biol Macromol. 2017;104:1897-1904.https://doi.org/10.1016/j.ijbiomac.2017.02.080

66. Okamoto Y, Kawakami K, Miyatake K, Morimoto M, Shigemasa Y, Minami S. Analgesic effects of chitin and chitosan. Carbohydr Polym. 2002; 49(3): 249-252.https://doi.org/10.1016/S0144-8617(01)00316-2.

67. Aoyagi S, Onishi H, Machida Y. Novel chitosan wound dressing loaded with minocycline for the treatment of severe burn wounds. Int J Pharm. 2007; 330(1-2): 138-145. https://doi.org/10.1016/j.ijpharm.2006.09.016.

68. Kim MS, Sung MJ, Seo SB, Yoo SJ, Lim WK, Kim HM. Water-soluble chitosan inhibits the production of pro-inflammatory cytokine in human astrocytoma cells activated by amyloid β peptide and interleukin-1β. Neurosci Lett. 2002; 321(1-2): 105-109. https://doi.org/10.1016/S0304-3940(02)00066-6

69. Spindola H, Fernandes J, De Sousa V, et al. Anti-inflammatory effect of chitosan oligomers. N Biotechnol. 2009; 25: S9. https://doi.org/10.1016/j.nbt.2009.06.025

70. Mutlu N, Liverani L, Kurtuldu F, Galusek D, Boccaccini AR. Zinc improves antibacterial, anti-inflammatory and cell motility activity of chitosan for wound healing applications. Int J Biol Macromol. 2022; 213: 845-857. doi:https://doi.org/10.1016/j.ijbiomac.2022.05.199

71. Li F, Shi Y, Liang J, Zhao L. Curcumin-loaded chitosan nanoparticles promote diabetic wound healing via attenuating inflammation in a diabetic rat model. J Biomater Appl. 2019;34(4):476-486. https://doi.org/10.1177/0885328219860929

72. Nahar K, Hossain K, Khan TA. Alginates: The Wonder Molecule and its Gelling Techniques. Res J Pharm Technol. 2017; 10(9): 3195-3204.http://dx.doi.org/10.5958/0974-360X.2017.00568.6

73. Oliviya CR. Production of ecofriendly silver nanoparticle and evaluation of its potential antimicrobial activity. Res J Pharm Technol. 2015; 8(10): 1374-1378.http://dx.doi.org/10.5958/0974-360X.2015.00246.2.

74. Reinke JM, Sorg H. Wound repair and regeneration. Eur Surg Res Eur Chir Forschung Rech Chir Eur. 2012; 49(1): 35-43. doi:10.1159/000339613

75. Singh R, Shitiz K, Singh A. Chitin and chitosan: biopolymers for wound management. Int Wound J. 2017; 14(6): 1276-1289. doi:10.1111/iwj.12797

76. Kulling D, Vournakis JN, Woo S, et al. Endoscopic injection of bleeding esophageal varices with a poly-N-acetyl glucosamine gel formulation in the canine portal hypertension model. Gastrointest Endosc. 1999; 49(6): 764-771. https://doi.org/10.1016/S0016-5107(99)70298-1

77. Patrulea V, Ostafe V, Borchard G, Jordan O. Chitosan as a starting material for wound healing applications. Eur J Pharm Biopharm. 2015; 97: 417-426. doi:10.1016/j.ejpb.2015.08.004

78. Jiang Q, zhou W, Wang J, Tang R, Zhang D, Wang X. Hypromellose succinate-crosslinked chitosan hydrogel films for potential wound dressing. Int J Biol Macromol. 2016; 91: 85-91. doi:10.1016/j.ijbiomac.2016.05.077

79. Zhao R, Li X, Sun B, et al. Electrospun chitosan/sericin composite nanofibers with antibacterial property as potential wound dressings. Int J Biol Macromol. 2014; 68: 92-97. https://doi.org/10.1016/j.ijbiomac.2014.04.029

80. Ahmed S, Ikram S. Chitosan Based Scaffolds and Their Applications in Wound Healing. Achiev Life Sci. 2016; 10(1): 27-37. doi:https://doi.org/10.1016/j.als.2016.04.001

81. Miguel SP, Moreira AF, Correia IJ. Chitosan based-asymmetric membranes for wound healing: A review. Int J Biol Macromol. 2019; 127: 460-475. doi:10.1016/j.ijbiomac.2019.01.072

82. Feng W, Wang Z. Shear-thinning and self-healing chitosan-graphene oxide hydrogel for hemostasis and wound healing. Carbohydr Polym. 2022; 294: 119824. doi:https://doi.org/10.1016/j.carbpol.2022.119824

83. Hu S, Bi S, Yan D, et al. Preparation of composite hydroxybutyl chitosan sponge and its role in promoting wound healing. Carbohydr Polym. 2018; 184: 154-163. doi:https://doi.org/10.1016/j.carbpol.2017.12.033

84. Bektas N, Şenel B, Yenilmez E, Özatik O, Arslan R. Evaluation of wound healing effect of chitosan-based gel formulation containing vitexin. Saudi Pharm J. 2020; 28(1): 87-94. doi:https://doi.org/10.1016/j.jsps.2019.11.008

85. Wang D, Zhang N, Meng G, He J, Wu F. The effect of form of carboxymethyl-chitosan dressings on biological properties in wound healing. Colloids Surfaces B Biointerfaces. 2020; 194: 111191. doi:https://doi.org/10.1016/j.colsurfb.2020.111191

86. Zhou L, Zhao X, Li M, et al. Antibacterial and wound healing–promoting effect of sponge-like chitosan-loaded silver nanoparticles biosynthesized by iturin. Int J Biol Macromol. 2021; 181: 1183-1195. doi:https://doi.org/10.1016/j.ijbiomac.2021.04.119

87. Algandaby MM, Esmat A, Nasrullah MZ, et al. LC-MS based metabolic profiling and wound healing activity of a chitosan nanoparticle-loaded formula of Teucrium polium in diabetic rats. Biomed Pharmacother. 2023; 168: 115626. https://doi.org/10.1016/j.biopha.2023.115626

88. Cui Y, Duan W, Jin Y, Wo F, Xi F, Wu J. Graphene quantum dot-decorated luminescent porous silicon dressing for theranostics of diabetic wounds. Acta Biomater. 2021; 131: 544-554. doi:https://doi.org/10.1016/j.actbio.2021.07.018

89. Zhang S, Liu Y, Zhang X, et al. Prostaglandin E(2) hydrogel improves cutaneous wound healing via M2 macrophages polarization. Theranostics. 2018; 8(19): 5348-5361. doi:10.7150/thno.27385

90. Zheng K, Tong Y, Zhang S, et al. Flexible bicolorimetric polyacrylamide/chitosan hydrogels for smart real‐time monitoring and promotion of wound healing. Adv Funct Mater. 2021; 31(34): 2102599. https://doi.org/10.1002/adfm.202102599

91. Wang CH, Cherng JH, Liu CC, et al. Procoagulant and Antimicrobial Effects of Chitosan in Wound Healing. Int J Mol Sci. 2021; 22(13). doi:10.3390/ijms22137067

92. Vescovali C, Damour O, Shahabedin L, et al. Epidermalization of an artificial dermis made of collagen. Ann Mediterrian Burn Club. 1989; 2: 137139.

93. Shivakumar P, Gupta MS, Jayakumar R, Gowda DV. Prospection of chitosan and its derivatives in wound healing: Proof of patent analysis (2010–2020). Int J Biol Macromol. 2021; 184(May): 701-712. doi:10.1016/j.ijbiomac.2021.06.086

94. Damour O, Gueugniaud PY, Berthin-Maghit M, et al. A dermal substrate made of collagen-GA-chitosan for deep burn coverage: first clinical uses. Clin Mater. 1994; 15(4): 273-276. https://doi.org/10.1016/0267-6605(94)90057-4

95. Zhang C, Yang X, Hu W, Han X, Fan L, Tao S. Preparation and characterization of carboxymethyl chitosan/collagen peptide/oxidized konjac composite hydrogel. Int J Biol Macromol. 2020; 149: 31-40. https://doi.org/10.1016/j.ijbiomac.2020.01.127

96. Mo X, Cen J, Gibson E, Wang R, Percival SL. An open multicenter comparative randomized clinical study on chitosan. Wound repair Regen Off Publ Wound Heal Soc [and] Eur Tissue Repair Soc. 2015; 23(4): 518-524. doi:10.1111/wrr.12298

97. Shaik J, Garlapati R, Nagesh B, Sujana V, Jayaprakash T, Naidu S. Comparative evaluation of antimicrobial efficacy of triple antibiotic paste and calcium hydroxide using chitosan as carrier against Candida albicans and Enterococcus faecalis: An in vitro study. J Conserv Dent. 2014; 17(4): 335-339. doi:10.4103/0972-0707.136444

98. Liu J, Shen H. Clinical efficacy of chitosan-based hydrocolloid dressing in the treatment of chronic refractory wounds. Int Wound J. 2022; 19(8): 2012-2018. doi:10.1111/iwj.13801

99. Shao Y, Zhou H. Clinical evaluation of an oral mucoadhesive film containing chitosan for the treatment of recurrent aphthous stomatitis: a randomized, double-blind study. J Dermatolog Treat. 2020; 31(7): 739-743. doi:10.1080/09546634.2019.1610548

100. Valentine R, Athanasiadis T, Moratti S, Hanton L, Robinson S, Wormald PJ. The efficacy of a novel chitosan gel on hemostasis and wound healing after endoscopic sinus surgery. Am J Rhinol Allergy. 2010; 24(1): 70-75. doi:10.2500/ajra.2010.24.3422

Received on 18.01.2024 Modified on 11.03.2024

Research J. Pharm. and Tech 2024; 17(10):5102-5112.

DOI: 10.52711/0974-360X.2024.00784

S. No.	Growth Factors	Source	Function
1	EGF	Fibroblasts, macrophages, and platelets	In acute wounds, proliferation and cell motility increase, whereas in chronic wounds, they decrease.
2	VEGF	Endothelial cells, fibroblasts, neutrophils, macrophages, and platelets	Angiogenesis and granulation tissue development increase in acute wounds but decrease in chronic wounds.
3	Hepatocyte growth factor (HGF)	Fibroblasts	Re-epithelialization, granulation tissue development, angiogenesis, and suppression of inflammation.
4	FGF	Endothelial cells, macrophages, and fibroblasts	Keratinocyte mitogen, angiogenesis and fibroblast mitogen, and mitogen.
5	IGF	Hepatocytes, neutrophils, fibroblasts, and skeletal muscle	Promotes the growth of fibroblasts and the re-epithelialization of wounds.
6	PDGF	Endothelial cells, fibroblasts, keratinocytes, macrophages, and platelets	Chemotaxis, inflammation, granulation tissue development, matrix remodeling, elevated levels in acute wounds, and lowered levels in chronic wounds.
7	TGF-b1, TGF-b2	Keratinocytes, macrophages, lymphocytes, platelets, and fibroblasts	Re-epithelialization and inflammation, fibrosis, granulation tissue development, and tensile strength, with increasing levels in acute wounds and decreasing levels in chronic wounds.

S. No.	Disease/condition	Study design	Application	Effect	Ref.
1	Surgical wound	Interventional	Chitosan Dressing	In patients with surgical wounds, the study demonstrated better procoagulant and antibacterial qualities of chitosan dressings compared to normal gauze-type surgical dressings.	91
2	Burn and scald wound	Prospective, randomised, single-blind, positive controlled trial	Chitosan Dressing	The results indicated that the chitosan dressing effectively treated both the burn and scald wounds. It has the potential to considerably accelerate the healing process, relieve pain and infection from wounds, and successfully treat scar hyperplasia.	15
3	Chronic wounds (pressure ulcers, diabetic foot ulcers, vascular ulcers, and infectious wounds)	Open multicenter comparative prospective randomized controlled trial	KA01 chitosan dressing(next-generation)	In the treatment of chronic wounds, the novel chitosan wound dressing proved to be more effective than the control dressing. The reduction in wound area, depth, pain level upon dressing removal, and control of wound exudate are indicators of the effectiveness of chitosan dressing over vaseline gauze dressing.	96
4	Periapical lesions (in single rooted teeth)	Interventional	Chitosan + calcium hydroxide (Ca(OH)₂), Chitosan + triple antibiotic (TAP) paste	The combined therapy of TAP + CS and Ca(OH)₂ + CS showed better results than the combination of medicaments with saline. The results of this study showed that TAP and Ca(OH)₂combined with chitosan as a carrier exhibited potent antibacterial activity against E. faecalis and C. albicans.	97
5	Chronic refractory wounds	Retrospective study	Chitosan based hydrocolloid dressing	In treating chronic refractory patients, the use of hydrocolloid dressings based on chitosan is effective in decreasing pain, promoting wound healing, relieving itching, and lowering the overall costs and dressing change frequency.	98
6	Canker sores or recurrent aphthous stomatitis (RAS)	Randomized, parallel-controlled, double-blind controlled trial	Chitosan mucoadhesive oral film	Chitosan mucoadhesive film accelerates ulcer healing and can be utilized as a drug carrier for treating mouth ulcers.	99
7	Surgical wound (endoscopic sinus surgery)	Randomized controlled trial	Chitosan gel	Chitosan gel is instantly hemostatic during ESS and reduces adhesion development, thereby treating some of the most prevalent problems of sinus surgery.	100