Volume 6 Issue 1
Feb.  2021
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Recent Advancements in Applications of Chitosan-based Biomaterials for Skin Tissue Engineering

  • The use of polymer based composites in the treatment of skin tissue damages, has got huge attention in clinical demand, which enforced the scientists to improve the methods of biopolymer designing in order to obtain highly efficient system for complete restoration of damaged tissue. In last few decades, chitosan-based biomaterials have major applications in skin tissue engineering due to its biocompatible, hemostatic, antimicrobial and biodegradable capabilities. This article overviewed the promising biological properties of chitosan and further discussed the various preparation methods involved in chitosan-based biomaterials. In addition, this review also gave a comprehensive discussion of different forms of chitosan-based biomaterials including membrane, sponge, nanofiber and hydrogel that were extensively employed in skin tissue engineering. This review will help to form a base for the advanced applications of chitosan-based biomaterials in treatment of skin tissue damages.
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Recent Advancements in Applications of Chitosan-based Biomaterials for Skin Tissue Engineering

    Corresponding author: Fazli Wahid, fazli.wahid@fbse.paf-iast.edu.pk
  • a. Department of Biotechnology, COMSATS University Islamabad, Abbottabad Campus 22060, Pakistan
  • b. Department of Pharmacy, Women Institute of Learning, Abbottabad, Pakistan
  • c. Department of Biomedical Sciences, Pak-Austria Fachhochschule: Institute of Applied Sciences and Technology, Mang, Khanpur Road, Haripur, Pakistan

Abstract: The use of polymer based composites in the treatment of skin tissue damages, has got huge attention in clinical demand, which enforced the scientists to improve the methods of biopolymer designing in order to obtain highly efficient system for complete restoration of damaged tissue. In last few decades, chitosan-based biomaterials have major applications in skin tissue engineering due to its biocompatible, hemostatic, antimicrobial and biodegradable capabilities. This article overviewed the promising biological properties of chitosan and further discussed the various preparation methods involved in chitosan-based biomaterials. In addition, this review also gave a comprehensive discussion of different forms of chitosan-based biomaterials including membrane, sponge, nanofiber and hydrogel that were extensively employed in skin tissue engineering. This review will help to form a base for the advanced applications of chitosan-based biomaterials in treatment of skin tissue damages.

1.   Introduction
  • Tissue engineering is an advanced area of reparative medicine that emerged from the field of biomaterials development. Basically, this technique repairs, improves and maintains the function of injured tissue or organ by combining cells, biologically active molecules and scaffolds. The goal of tissue engineering is to bring together functional constructs that provide biological support to damaged tissue/organ for its proper restoration and regeneration (Rahmani Del Bakhshayesh et al., 2018). For example, in the treatment of burn wounds, multiple therapeutic strategies have been explored where split and full skin graft are commonly used. Although, being the available treatment option, grafting poses a risk of tissue/organ rejection and infection transmission, as well as, requires additional surgical process and provides difficulty in finding healthy donor tissues (Boyce and Lalley, 2018). In the current scenario, tissue engineering provides a promising hope to patients having chronic and hard to heal injuries (Zulkifli et al., 2017). A tissue can be engineered by using cells (reparative cells mainly embryonic stem cell that helps in formation of functional tissue), scaffold (biomaterial that helps in providing support for cells growth and proliferation) or mediators (include bioactive molecules such as growth factors or cytokines that direct the cells to assemble in proper shape and functioning tissue) (De Isla et al., 2010). Of these, in clinical practices, the use of human cells or tissues are highly controlled by various regulatory laws, while, scaffolds (biomaterials) are free of such regulations and conditions. American National Institute of Health defines biomaterials as any active substance or combination of active substances, extracted naturally or modified synthetically, that could be used partial or whole for the tissue or organ replacement (Halim et al., 2016). Several polymers such as chitosan, cellulose, collagen, fibronectin, laminin, polycaprolactone, polyethylene glycol and polyurethane have been extensively reported as biomaterials for scaffold preparation (Ghosal et al., 2017; Khalid et al., 2020). In tissue engineering, biomaterial serves as an extracellular matrix for cellular organization, helping in the attachment, proliferation and differentiation of seeded living cells (Sah and Rath, 2016). Therefore, an important avenue of biomaterial selection for tissue engineering is that it must promote regeneration by actively transporting cell population and therapeutic agents, along with that, it must provide structural scaffolding having sufficient mechanical strength. Moreover, ideally the material should degrade at the site of implantation at a comparable rate of growth (Wahid et al., 2018). Along with tissue engineering applications, lignocellulosic biomass deserves special attention in different fields due their properties and availability. It was found that cellulose derived from natural or bacterial source can be designed to develop electronic and energy devices (Dutta et al., 2017). Similarly, lignocellulosic biomass has the potential to be chemically transformed into highmethylfurfural in a single step (Chen et al., 2021). Thereby, providing renewable raw material to the chemical industries for the synthesis of fuels (Binder and Raines, 2009; Liao et al., 2018). Moreover, hemicellulose based products are applicable in drug delivery systems. Thus, lignocellulosic materials can be considered as the materials of future.

    Chitosan is a biological derived material and the 2nd most abundant polymer after cellulose. It is a cationic polymer derived from chitin by partial deacetylation process and proved as a suitable candidate for tissue engineering applications based on its peculiarities like biocompatibility, non-toxicity, and biodegradability along with biological properties such as antimicrobial, antioxidant, anticancer, anti-inflammatory, hemo-compatible and hemostatic activities (Naveed et al., 2019). Several studies reported that chitosan, due to its functional groups, can be easily modified with other bioactive molecules, in order to impart additional properties to the tissue construct. Moreover, the molecules of chitosan facilitates three-dimensional cells growth and proliferation as well as organizes deposition of collagen, thereby, ensuring rapid healing (Sharma and Batra, 2019). Commonly, membranes/film, hydrogel, sponges and fiber forms of chitosan biomaterial have been investigated for wound healing or tissue engineering applications (Jayakumar et al., 2011). Sources, biological properties, synthesis methods and applications of chitosan are depicted in Fig. 1.

    Figure 1.  A detailed scheme of sources, preparation methods, biological activities, different types of scaffolds and applications of chitosan in tissue engineering. (a) Biomaterials, like, chitosan, cellulose, collagen, fibronectin, laminin and others are the suitable candidate and can be engineered in a scaffold form to support the cells and provide natural environment for growth; (b) Chitosan, is obtained from fungal, aquatic and terrestrial source by chemical or enzymatic treatment methods; (c) It has different biological activities like anti-oxidant, anti-inflammatory, hemocompatible, anti-fungal and anti-microbial activities; (d) The chitosan scaffolds can be made through different methods such as solvent casting, compression molding, freeze drying and others; (e) The scaffolds can be made in different forms such as membrane, hydrogel, sponges etc. that have (f) diverse tissue engineering applications such as skin tissue engineering.

    This review highlights the characteristic biological properties of chitosan that make it an excellent biomaterial to be employed in skin tissue engineering. Various applied methods in synthesis of chitosan biomaterials for skin tissue engineering have been extensively elaborated in this review. Comprehensive details of chitosan-based biomaterials mainly used in skin tissue engineering have been presented in detail. It is anticipated that this study will act as base information for the development of future chitosan-based products in tissue engineering and regenerative medicine.

2.   Chitosan
  • Chitosan is a semi-crystalline polysaccharide, containing N-acetyl-D-glucosamine and D-glucosamine residues arranged in linear dimension. It is cationic in nature, due to the presence of amino group (-NH2) in its structure. This positive charge helps the formation of extracellular matrix by attracting negatively charged molecules, like proteoglycans (Sivashankari and Prabaharan, 2016). In addition, hydroxyl group (-OH) is also present in the structure and attracts positively charged molecules to enhance the bonding (Rahmani Del Bakhshayesh et al., 2018). Besides electrostatic attractions, these functional groups help in modification of chitosan thus improving its mechanical and biological properties to bring novel functional properties and promising practical application (Ruiz and Corrales, 2017). The chemical structure of chitosan is presented in Fig. 2.

    Figure 2.  Molecular representation of chitosan polymer. Chitosan possesses N-acetyl-D-glucosamine and D-glucosamine residues linked in linear dimension.

  • Chitosan was first discovered in 1811, in mushroom by a French researcher Henri Braconnot. Afterward, researchers found out its alternative source, from fungi, aquatic species and other terrestrial living organisms (Halim et al., 2016) as detailed in Table 1. Among these, crustaceans and fungal mycelia are widely used at industrial scale, whereas, animals as a source organism find limitations, such as seasonal providence, expensive extraction procedure and less production. On the other hand, mushroom-based chitosan extraction has advantages of maximum and environmentally controlled production (Dhillon et al., 2013).

    Biological sources of chitosan References
    Fungi sources
      Absidia glauca (Hu et al., 2004)
      Aspergillus niger (Pochanavanich and Suntornsuk, 2002; Di Mario et al., 2008)
      Auricularia auricula-judae (Pochanavanich and Suntornsuk, 2002; Berger et al., 2016)
      Candida albicans (yeast) (Kaya et al., 2015a)
      Cunninghamella elegans (Yen et al., 2007)
      Fomitopsis pinicola (de Queiroz Antonino et al., 2017)
      Lentinula edodes (shiitake stipes) (Di Mario et al., 2008)
      Litopenaeus vannamei Boone (shrimp shells) (Kleekayai and Suntornsuk, 2011)
      Pleurotus ostreatus
      Rhizopus oryzae
    Aquatic sources
      Ceriodaphnia Quadrangula (Kaya et al., 2014b)
      Daphnia longispina (Kaya et al., 2014a)
      Paeneaus indicus (prawan) (Pochanavanich and Suntornsuk, 2002; Berger et al., 2016)
      Parapenaeus longirostris (Limam et al., 2011)
      Potamon potamios (Bolat et al., 2010)
      Pranulirus orntus (lobster) (Oduor-Odeto et al., 2007)
      Sepia sp. (cuttlefish) (Sagheer et al., 2009)
      Shylla cerrata (crab) (Oduor-Odeto et al., 2007)
      Squilla mantis (Limam et al., 2011)
      Thenus orientalis (lobster) (Sagheer et al., 2009)
    Terrestrial sources
      Calliphora erthrocephala (blue bottlefly) (Oduor-Odeto et al., 2007)
      Calliptamus barbarous (grass hopper) (Kaya et al., 2015b)
      Geolycosa vultuosa (spider specie) (Kaya et al., 2014c)
      Hogna radiate (spider specie) (Kaya et al., 2014c)
      Leptinotarsa decemlineata (Kaya et al., 2014d)
      Mesobuthus gibbosus (scorpion) (Kaya et al., 2016)
      Oedaleus decorus (grass hopper) (Kaya et al., 2015b)

    Table 1.  Various reported sources including fungi, aquatic and terrestrial for chitosan.

    There are two extraction methods of chitosan, including, chemical treatment and enzymatic treatment. The chemical method is widely applied due to its cost-effectiveness and large mass production. The process of chemical deacetylation is used to obtain chitosan from crustaceans (Cheung et al., 2015) as illustrated in Fig. 3. This method involves demineralization, deproteinization, decolorization and deacetylation. In this process, crustaceans shells are demineralized by employing hydrogen chloride and deproteinized using sodium hydroxide to remove minerals and protein residues, respectively. Following this, treatments with potassium manganese oxide remove pigmented molecules, resulting in clear chitin. Finally, the chitin is subjected to alkaline treatment to form chitosan.

    Figure 3.  Schematic presentation of chitosan synthesis from the source via chemical and enzymatic method. The chemical method specifically employs the alkali treatment while enzymatic method uses protease and chitin deacetylase in the reaction.

    In the second method, chitosan is enzymatically produced by using chitin deacetylase (Tsigos et al., 2000). Chitin deacetylase can be obtained from various fungal, bacterial, and some insect species (Zhao et al., 2010). In mechanism, the enzyme hydrolyzes the N-acetamide bond presented in the chitin molecule (Zhao et al., 2010) as depicted in Fig. 4. Prior to hydrolysis of chitin, it is physically and chemically modified, as crude chitin is poor substrate for enzyme. Therefore, chitin is first processed as reprecipitation, glycolation and depolymerization (Yamada et al., 2008).

    Figure 4.  Presentation of enzymatic reaction involved in chitosan synthesis. Chitin is converted into chitosan in the presence of chitin deacetylase.

  • Chitosan polysaccharide shows diverse biological properties such as anti-microbial, anti-oxidant, anti-inflammatory and anti-cancer activity, due to the presence of active functional groups like amino, hydroxyl and carboxyl groups in its structure (Kim, 2018). Followings are the important biological properties of chitosan that make it a suitable and effective polysaccharide for skin tissue engineering applications.

  • Chitosan is well known for its action against wide-range of microbes due to its poly-cationic structure that makes it a promising antimicrobial candidate (Goy et al., 2016). In its structure, the positively charged amino group interacts electrostatically with the negatively charged microbial membranes, resulting in the leakage of cellular contents and finally cell death. In the membrane, lipo-polysaccharides and cell surface proteins are the main targets of interactions (Kong et al., 2010). Several factors such as molecular weight, degree of deacetylation, concentration of chitosan and pH of solution affect the antimicrobial activity of chitosan. For instance, chitosan with high molecular weight and degree of acetylation has better antimicrobial activity. Along with that, bactericidal activity of chitosan also depends on hydrophilicity and content of negatively charged moieties present on the cell membrane of microbes, as gram-negative bacteria is more affected by chitosan as compared with gram-positive bacteria (Vilar et al., 2016). Furthermore, various studies demonstrated the chitosan antifungal activity against Candida albicans and Fusarium solani. Chitosan fungicidal mechanism involves penetration of chitosan molecule inside hyphae part of fungus body, followed by disruption of the enzyme structure, that are essential for fungus growth (Basseri et al., 2019). Antibacterial and antifungal activity of chitosan against various microbes are listed in Table 2.

    Antimicrobial activity of chitosan> References
      Bacillus subtilis (Du et al., 2009)
      Escherichia coli (Abdel-Rahman et al., 2015)
      Klebsiella pneumoniae (Ma et al., 2016)
      Lactobacillus brevis (No et al., 2002)
      Listeria monocytogenes (No et al., 2006)
      Methicillin-resistant Staphylococcus aureus (Ma et al., 2016)
      Pseudomonas aeruginosa (Eldin et al., 2008)
      Salmonella paratyphi (Islam et al., 2011)
      Salmonella typhimurium (No et al., 2002)
      Staphylococcus aureus (Chang et al., 2015)
      Streptococcus mutans (de Paz et al., 2011)
      Vancomycin-resistant Enterococcus sp. (Ma et al., 2016)
      Vibrio cholera (Ma et al., 2016)
      Aiternaria solani (Zhong et al., 2007)
      Alternaria alternate (Ziani et al., 2009)
      Aspergillus niger (Ziani et al., 2009)
      Candida albicans (Yien et al., 2012)
      Fusarium oxysporum (Saharan et al., 2015)
      Fusarium solani (Ing et al., 2012)
      Phomopsis asparagi (Zhong et al., 2007)

    Table 2.  Bioactive potential of chitosan against bacteria and fungi.

  • Free radicals such as oxygen superoxide, hydroxyl radical and proton, are continuously produced as by-products during metabolic reactions in humans. These radical ions are known to cause damage to several biomolecules including lipids of cell membrane, cytoplasmic proteins and DNA. To counter the damaging effect of radicals, antioxidants function by scavenging the ions, prevent their production or decompose the structure. Chitosan also acts as an antioxidant agent by forming stable radicals with its functional groups (Younes and Rinaudo, 2015; Chang et al., 2018). The antioxidant activity of chitosan is also affected by its molecular weight. As reported in a study, the formation of carbonyl groups was significantly inhibited in the presence of low molecular weight chitosan as compared with high molecular weight chitosan (Tomida et al., 2009). It was found that low molecular weight chitosan has less compact structure and possesses weak intermolecular hydrogen bonds giving more free space to hydroxyl and amino groups in comparison to high molecular weight chitosan, thereby possesses more potential to form stable radicals (Chien et al., 2007). Moreover, Hajji and his colleague extracted chitosan from shrimp waste, crab shells and cuttlefish bones, each having different degree of deacetylation, i.e., 88%, 83% and 95%, respectively. The experimental results suggested that the chitosan with high deacetylation degree displayed highest scavenging activity (Hajji et al., 2015). Similarly, in another study, chitosan derivative had been tested in-vitro and in-vivo using high-fat diet animal model to analyze the radical scavenging activities. The study revealed a positive impact on mice health with the addition of chitosan in the diet. It was found that chitosan addition enhanced the activity of certain enzymes including catalase, glutathione peroxidase and superoxide in serum, stomach and liver of animal model. As described, due to the scavenging ability of chitosan, stable radicals were formed and enzymatic activity was restored (Qu and Han, 2016). Therefore, chitosan can be a used as dietary supplement to improve the human health.

  • Inflammation is a protective response of our body to control wound related infections, however in case of aberrant healing, long term inflammation may cause oxidative stress, leading to damage of the own cell/tissue of the body (Rajitha et al., 2016). Various studies confirmed the anti-inflammatory properties of chitosan. It has been suggested that the anti-inflammatory activity of chitosan is due to the presence of charged moieties in its structure, which negatively regulates the pro-inflammatory reactions.

    In a study, anti-inflammatory activity of chitosan was examined by analyzing expression levels of pro-inflammatory cytokines in-vitro on rat basophilic leukemia cells (RBL-2H3 cells) and in-vivo on asthma mice model. The in-vitro results indicated that chitosan treatment reduced the expression of interleukins (IL-4, IL-13) and tumor necrosis factor alpha (TNF-α) to 3.33, 1.67 and 1.67 folds, respectively. Similarly, study of mice model also displayed low expression of IL-4, IL-13 and TNF-α in the lung tissue (Chung et al., 2012). Likewise, another study found out that chitosan can inhibit macrophages to express prostaglandin E2 gene (PGE2) that is involved in stimulation of cytokine release from inflammatory cells. Furthermore, anti-inflammatory effect was exerted in a dose-dependent manner and more the chitosan amount, less IL-8 was produced. Chitosan exhibited effect on suppressing the production of IL-8 RAW 264.7 macrophage cells (Yang et al., 2010). Similarly, another study revealed a new anti-inflammatory mechanism of chitosan, where chitosan was found to inhibit cyclooxygenase pathway by lowering cyclooxygenase-2 (COX-2) expression, both on transcriptional and translational level (Fernandes et al., 2010). Moreover, the treatment of macrophage cells with chitosan leads to a significant reduction in expression of pro-inflammatory cytokines, including cluster of differentiation 86 molecule (CD-86) and major histocompatibility complex class II molecule (MHC-II), whereas, anti-inflammatory markers such as IL-10 and transforming growth factor beta 1 (TGF- β1) level have been increased in the cell. However, in the dendritic cell, chitosan acted as a positive regulator of pro-inflammatory cytokines and increased the production of CD-86, TNF-α and IL-1β. The study concluded that chitosan possess different pattern of action depending upon the type of cells (Oliveira et al., 2012). Likewise, chitosan nanoparticle was tested on lipopolysaccharide (LPS)-induced Caco-2 cells to evaluate its anti-inflammatory activity. The result revealed that chitosan inhibited the expression of TNF-α, macrophage migration inhibitory factor (MIF), IL-8 and monocyte chemoattractant protein-1 (MCP-1) that were known to be stimulated by the presence of lipopolysaccharide (Tu et al., 2016). Therefore, chitosan exerts its anti-inflammatory effect by targeting the pro-inflammatory cytokines and repressing their expression in order to limit inflammatory process.

  • Hemocompatibility is defined as the interaction of foreign material with the blood, showing least adverse effects. While, synthesizing a biomaterial for human body, it is essential to select a biocompatible material having reliable interaction with the body cells (Dash et al., 2011). Any biomaterial is referred as hemocompatible when it shows less than 5% hemolysis (Kronenthal, 2013). Chitosan is reported of having good hemocompatible activities (Balan and Verestiuc, 2014). In a study, chitosan nanoparticles were tested against human erythrocytes to evaluate its hemocompatable properties. It was found that the neutralized pH solution of chitosan particles showed less hemolytic activity of 2.56% and better agglutination (de Lima et al., 2015). Likewise, an injectable chitosan-based hydrogel, when administrated to rats showed no significant change in hematology pattern. Furthermore, the serum alanine aminotransferase and blood ureic nitrogen levels of treated group were not altered as compared with control group. The result proved that chitosan-based material have good hemo-compatibility characteristics and no hepatotoxicity (Zhou et al., 2011). In a study, hemocompatibility and blood clotting of chitosan-TiO2-pectin composites were evaluated. In hemocompatibility testing, chitosan material displayed 1.14% of hemolysis, making good hemocompatible material. Further, the chitosan composite induced blood clotting and showed significant hemostatic properties (Archana et al., 2013). Similarly, the chitosan composite with nickel and titanium alloy has been investigated for hemolytic activities. The chitosan, in integrated form possess better hemocompatibility as compared with NiTi alone (Dong et al., 2009). Therefore, chitosan may serve as a suitable material to be used in designing advanced constructs for tissue engineering.

3.   Methods Used in Preparation of Chitosan Biomaterials
  • Biomaterials serve as a framework having architectural similarity with the native extracellular matrix, to ensure cell growth and regeneration. Over the years, different biomaterials were explored having suitable characteristic properties for tissue engineering. Moreover, numerous processing techniques and scaffold designs have been developed in order to obtain improved form of biomaterial constructs. Widely used techniques for the synthesis of chitosan biomaterials are solvent casting, compression molding method, freeze-drying, electrospinning, microwave assisted, and dense gas foaming method.

  • The solvent casting technique, also known as solvent evaporation and tape casting method, is used to develop chitosan membranes having ultrafine pores. In this method, polymer is dissolved in acidic solution, at room temperature. To obtain homogenous solution of the polymer, it is stirred overnight in shaking incubator. The homogenized solution is pass through nylon cloth to remove unmixed particle or impurities. On clean polystyrene petri plates, the viscous solution is poured and oven dried. The dried membranes are peeled off from the petri plate and stored for experimentation (Azad et al., 2004). The structural quality of membranes can be enhanced by using different solvent system or by adding useful compounds. For instance, chitosan dissolved in lactic acid gives more flexible and transparent membranes, rather that glacial acetic acid dissolved membranes. Moreover, addition of plasticizers like glycerol, polyethylene glycol and sorbitol further improve the tensile strength of membranes making it suitable for skin tissue engineering applications (Campos et al., 2015; Madni et al., 2019). Furthermore, chitosan membrane can be loaded with various bioactive molecules to obtain multifunctional properties. Despite being an easy and cost effective method, this system allows only 2 dimensional cell growth (Pandey et al., 2017).

  • Similar to solvent casting, compression molding method also formulates chitosan in a membrane form. However, in this method, first the conditioning is done by drying chitosan and choline chloride in drying oven. The dried powder is finely grinded and mixed by mortar and pestle for at least 15 min. The grinded material is again oven dried at 70 ℃ and later treated with aqueous acetic solution in slow fashion. Finally, the hydraulic tool presses the soften material into membrane form (Galvis-Sánchez et al., 2016). Due to the simplicity and cost effectiveness, this method has been employed at the lab scale. While, the process is not applicable at large scale as some content of the material is lost during hydraulic hot press, as well as, the morphology of the material is also disrupted. Therefore, this process do not find wide range of applications for biomaterial preparation (Park and Lee, 2012).

  • Freeze-drying is the low temperature dehydration process in which the sample is freeze, pressure is lowered and ice is removed by sublimation. Freeze drying yields high quality product as low temperature do not alter the original structure of the sample. The freeze-drying method can be applied for the preparation of porous scaffolds including hydrogel and sponge material (Garg et al., 2012b). In this technique, the polymer is dissolved in water or organic solvent that follows the emulsification process of water phase. Then, the solution is casted into mould and cooled below its triple point. The freezing step is critical as it ensures product stability and appropriate crystallization. Afterward, the freeze dried mixture is lyophilized, by applying enough pressure and heat to sublime the ice. Slow drying results in the formation of porous structure (Garg et al., 2012a). In case of hydrogel system, the polymeric chains are arranged in an irregular pattern having less interconnected pore structure, whereas, sponge system possess regular pattern of highly interconnecting pore of approximately same size (Zhang et al., 2013).

    Freeze drying method finds promising application in preparation of biomaterials, as the process do not require high temperature, laborious steps and expensive machinery. Importantly, the process allows development of scaffolds having characteristic to facilitate growth of cells in 3 dimension. However, the process finds limitation due to the formation of irregular pores. To overcome this problem, the freezing rate and pH of the solution can be adjusted to produce uniform pore sized material (Garg et al., 2012b). Moreover, the addition of different cross-linkers can enhance the mechanical characteristics of the material and maintain the physical structure during conduction of different tests (Ehterami et al., 2019). For instance, glutaraldehyde, genipin, and tripolyphosphate cross-linkers were majorly reported in chitosan-based scaffold (Ahmadi et al., 2015; Sacco et al., 2018).

  • Electrospinning technique produce ultrafine, nanometer sized fibers of the biomaterial and organize in a structure mimicking the native extracellular matrix. Therefore, the developed matrix allows cell adhesion and growth similar to natural system (Blakeney et al., 2011; Oryan and Sehvieh, 2017). For the preparation of chitosan scaffolds via electrospinning technique, it is first solubilized in aqueous acetic or formic acid solution. Then, the chitosan is homogenously blend with other polymer(s) and filled in the syringe having needle of 21 gauge blunt end. A high voltage of 16 kV is applied to the polymer solution and collector, which allows solution to extrude from nozzle forming a jet. The fibrous non-woven web is detached from cylindrical mandrel and processed with deionized water to eliminate the solvent. Afterward, the resulted fibers are air dried at 37 ℃ for overnight. Prior to use in cell culture, these fiber mats are sterilized with UV radiation (Prasad et al., 2015). Usually, chitosan is blended with other polymer(s), as it is difficult to electro-spin single chitosan (Hohman et al., 2001; Duan et al., 2004). Moreover, blending of chitosan with hydrophilic polymer may disrupt its morphology when soaked in phosphate buffer saline. Therefore, to solve the problem, different cross-linkers such as glutathione, genipin and glutaraldehyde have been widely employed to electrospun fibers of chitosan (Duan et al., 2006).

  • Microwave assisted technique utilized the energy of microwave radiation in order to generate cross linked polymeric chains. This method is suitable for the preparation of scaffolds having characteristics of a hydrogel. This can be utilized to prepare chitosan-based hydrogel systems for tissue engineering. In this method, chitosan is first mixed with polyvinyl alcohol to obtain homogenous solution. The mixture is irradiated in microwave oven at 800 W for about 90 s. The viscous solution is lyophilized in freeze drier to remove water content under pressure and heat (Hiep et al., 2016). The prepared hydrogels possess chains interlinked with each other. As this method avoids the use of chemical cross-linker therefore, the prepared hydrogel may cause less toxicity to the living system (Cook et al., 2012; Visentin et al., 2014).

  • Dense gas foaming method synthesize highly porous hydrogel systems by using supercritical gas. For this purpose, polymeric solution is injected into Teflon mould of high pressure vessel. The vessel is stabilized to attain thermal equilibrium, and then optimized carbon dioxide (CO2) gas is pressurized for specific period of time. The pressure is released from the vessel and collected sample is washed with water. Then, the sample is treated with 100 mmol/L of Tris-phosphate buffer saline (PBS) for 1 h. Prepared hydrogel is washed again and stirred in PBS for later use (Annabi et al., 2009). Supercritical CO2 gas allows the creation of uniform sized pores in the polymer matrix system (Pini et al., 2007). Whereas, this method is not suitable for preparation of chitosan hydrogel as CO2 is less soluble in acidic solution, thereby, no sufficient pores are formed. To overcome this problem, co-solvent system comprising of dilute acid and ethanol are used that promote the solubility of CO2 into aqueous polymer solution to form porous hydrogels (Ji et al., 2011).

    Stability of polymer is the foremost parameter to be considered while constructing biomaterials for practical applications. Materials composed of pure chitosan including membranes, hydrogels and nanofibers structure show less mechanical strength and get damage during handling. To overcome such problems, several physical and chemical cross-linkers can be used. Cross-linker improves the tensile strength of materials and enhances the stability through inter-connecting networks (Sami El-banna et al., 2019). In physical cross-linker, the anionic molecules forms hydrogen bond, hydrophobic interaction, or ionic complexation in chitosan polymer. Calcium ions, copper ions, sodium sulfate, sodium triphosphate (Tripolyphospahte-TTP), laponite, and tannic acid are examples of physical cross-linkers (Ahmed and Aljaeid, 2016; Domalik-Pyzik et al., 2019). This method is highly efficient and results in the safe production of materials for biomedical application. The chemical cross-linker leads to the permanent bond formation in the synthesized biomaterials. Genipin, glutaraldehyde, glyoxal, and N, N-methylene-bisacrylamide are commonly used chemical cross-linker in chitosan-based materials (Domalik-Pyzik et al., 2019). In some cases, second polymer are also used instead of cross-linker to enhance the materials stability and make it a multifunctional dressing system for tissue engineering applications (Madni et al., 2019)

4.   Applications of Chitosan-based Biomaterials in Skin Tissue Engineering
  • Skin is the largest organ of the body, which functions as the first line of defense against external factors including pathogens or mutagenic substances (Kabashima et al., 2019). The damage to the skin due to any thermal, chemical or electrical stimuli causes cutaneous complications and in adverse case may lead to chronic and hard to heal injuries. Available treatment options and skin grafting techniques show delayed and improper healing, as well as, stimulates negative immunogenic responses. In this arena, tissue engineering finds promising solution as it mimics natural system in morphology, hence promotes effective healing process. This technique also allows three dimensional cell growth and proliferation, moreover, fabrication of growth factors in biomaterials supports the proper cell differentiation and migration of skin cells (Pereira and Bártolo, 2016; Liu et al., 2018; Shpichka et al., 2019). Chitosan-based functional constructs are suitable for tissue engineering as chitosan is non-toxic, biodegradable and biocompatible, and it can be modified to develop multifunctional constructs having morphology similar to natural matrix.

  • The potential of chitosan-based membranous constructs are extensively explored to find its tissue engineering capabilities. For instance, a study designed the chitosan film fabricated with titanium dioxide nanoparticle having potential structural and functional regenerative properties. The membrane construct possessed good mechanical, crystallinity and flexible properties. Along with that, membranes showed antibacterial activities against Staphylococcus aureus. Application of chitosan membrane on mouse fibroblast L929 cells, revealed rapid growth, reduced oxidative stress and apoptosis as compared with control group, having plastic surface. Furthermore, protein expression analysis confirmed the presence of fibroblast associated markers having role in survival and growth of L929 cells on membrane surface (Behera et al., 2017). Similarly, in another study, chitosan membranes were prepared and loaded with glycerol and antibacterial agents. The addition of glycerol provided long-term stability to membranes while antibacterial agents acted against Escherichia coli and S. aureus growth. The in-vitro dermal fibroblast culture assay showed enhanced proliferation of fibroblast cells on the membranes. The results suggested that prepared membrane has potential to be applied as antimicrobial dressing system for skin burn treatment (Ma et al., 2017). Furthermore, in another study, chitosan-aloe vera-curcumin membrane was investigated for promotion of skin tissue regeneration in full thickness wound. In-vitro assays showed appropriate physicochemical characteristics and antimicrobial activities against tested microbes. Moreover, mouse fibroblast NIH-3T3 cells cultured on membrane, efficiently modulate the proliferation of cells. Thus, curcumin loaded membrane composite has capability to promote effective wound healing, along with pathogen control (Liu et al., 2019). Another study examined the wound healing potential of chitosan-polyvinyl alcohol membrane incorporated with ibuprofen. The in-vitro drug release assay revealed sustained drug release property of membrane, where 80% of drug was released after 12 h and complete release was obtained in 3 days. Further, biocompatibility test confirmed that membrane showed no adverse effect to normal human dermal fibroblast cells (NHDF), as well as, promoted the adherence and growth of the cultured cells. Histological microphotographs showed complete epithelialization and less inflammation in ibuprofen loaded membrane treated samples (Morgado et al., 2017). Similarly, biocompatible chitosan-silk microfibers membrane was prepared that were having suitable mechanical properties. The membrane showed no toxicity toward L929 cells and promoted cell proliferation. Moreover, in-vivo experiment conducted on burn mice model demonstrated that application of membrane caused reduction in inflammatory response, promoted uniform distribution of fibroblast and displayed sufficient collagen deposition (Xu et al., 2015). In another study, xanthan-chitosan membrane was examined as bioactive dressing system for dermo-epidermal wounds. Moreover, mesenchymal stem cells (MSC) were seeded on to the membrane surface which presented good adherence properties. The in-vivo healing assay confirmed that the MSC embedded chitosan membranes displayed higher healing percentage (96.49% ± 1.62%) as compared with control (Bellini et al., 2015). Likewise, another study examined the anti-inflammatory performances of chitosan-hyaluronan-edaravone membrane during wound healing experiment. The in-vitro antioxidant test revealed that edaravone incorporation enhanced free-radical scavenging activity of membrane. In addition, in-vivo analysis on rat skin showed that membranes induced less inflammatory response and supported the migration of fibroblast, keratinocytes and endothelial cells, therefore, promoting effective wound healing (Tamer et al., 2018).

    In one study, Chen et al. (2019) prepared chitosan-hydroxylated lecithin complexed iodine-sodium alginate composite system and tested on burn wound rat model. The fabricated membrane was synthesized by microwave drying technique. The incorporation of iodine helped in improving the stability of hydroxylated lecithin in membrane and also enhanced the complex formation. Additionally, the composite membrane possessed good mechanical and swelling characteristics with superior antimicrobial property against Gram-positive and Gram-negative bacteria. Further, the fabricated chitosan-based composite membrane displayed a high repairing property in deep partial-thickness rat burn model. In Masson's staining test, a high ratio of vascular endothelial growth factor (VEGF) was observed that corresponded to platelets growth factors and involved in stimulation of capillary vessels development (Chen et al., 2019). Similarly, in another study, chitosan film was fabricated with agarose polymer. The resulting membrane was slightly acidic, having pH of 5.98, matching the skin pH. Further, the composite membrane possessed high exudate absorption capacity and elastic deformation. In addition, the membrane showed sensitivity in stimulated enzymatic environment which depicted the biodegradation of film at wound site and helped in release of active compounds at the wound site for healing process. The MTT assay showed that fabricated membrane was non-toxic to foreskin cells (BJ fibroblast cells) and their numbers were almost similar throughout the experiment length. Moreover, fluorescent staining revealed that chitosan-based membrane supported the proliferation and growth of seeded fibroblast cells. The fibroblast cells possessed typical morphology and extensive cytoskeleton network structure which confirmed its adhesion to the membrane (Vivcharenko et al., 2020). The experimental evidences showed that chitosan-based membrane material acts as an efficient modulators of the healing cells and due to the intrinsic antimicrobial potential of chitosan, thus it can be a promising candidate for developing membranes for skin tissue engineering.

  • Polysaccharide-based hydrogel system has captured huge attention in tissue engineering application as the morphology of hydrogel is similar in characteristic with natural tissues. Such hydrogel scaffolds possess high porosity, high water uptake and swelling capability, satisfactory mechanical property, biocompatibility and biodegradability (Ma, 2008). Therefore, hydrogel based implants find promising application in the field of skin tissue engineering. Chitosan-based hydrogels have been widely reported in skin tissue engineering applications. However, chitosan blends with other natural or synthetic polymer are more effective than simple chitosan hydrogel, as the later possess low mechanical strength. Moreover, cross-linkers are also found to improve the mechanical strength of chitosan-based hydrogels (Fu et al., 2018). In a study, chitosan hydrogel crosslinked with glutaraldehyde and genipin was prepared. The hydrogel possessed high porosity level with average size of 60-80 µm, where the presence of crosslinking agent maintain porosity of chitosan hydrogel. The hydrogels were analyzed for their cell growth capacities using human skin fibroblast cells GM3348. The in-vitro cell proliferation assay revealed that enhancing cellular growth was observed on the surface of chitosan hydrogel as compared with control. Moreover, histological analysis demonstrated that cells also penetrated inside the scaffold, showing increased number of fibroblast cells at day 7 (Ji et al., 2011).

    In a study, a biocompatible hydrogel constituting chitosan and alkali lignin was synthesized. In the prepared hydrogel system, lignin acted as a crossing agent by making an electrostatic interaction with chitosan through its phenoxide groups, therefore, avoiding the use of cross-linker. The interconnected pores inside chitosan-lignin hydrogel have (750.4 ± 277.9) μm average length and (200.2 ± 59.3) μm average breadth size. The biocompatibility assay demonstrated that mesenchymal stem cells showed enhanced adhesion on the scaffold surface. In scratch wound healing assay, chitosan-lignin hydrogel system promoted migration of mouse fibroblast 3T3 cell line across the scratch area. However, prolong incubation hours showed a fully-covered scratch area populated by fibroblast cell. The obtained hydrogel provided an inexpensive and sustainable hydrogel construct for skin regeneration (Ravishankar et al., 2019). Likewise, a gelatin-chitosan loaded with skin cell (human dermal fibroblast) was investigated for wound healing potential in rat. The scaffold was highly porous in structure, having highly interconnected pore percentage. The scanning electron microscopy (SEM) and histological assays confirmed that human dermal fibroblast cells showed attachment and migration on the scaffold. Moreover, the images of Masson's trichrome (MT) staining showed that human dermal fibroblast produced collagen fibers that were found inside the scaffold structure. Also, in-vivo experiments on rat displayed enhanced re-epithelization of skin wound treated with human dermal fibroblast loaded hydrogel (Pezeshki-Modaress et al., 2014). Similarly, another study reported a novel chitosan hydrogel integrated with phenytoin capsule loaded with polycaprolactone and sorbatin monostearate. Application of this system on cutaneous injuries enhanced the deposition of collagen fibers and increased the number of fibroblast cells at wound site. It was found that nanocarrier system imparted adhesion characteristics to chitosan hydrogel, whereas, the hydrogel reduced the systemic absorption risk of phenytoin into the skin (Cardoso et al., 2019).

    A chitosan hydrogel fabricated with oxygenated fluorinated methacrylamide was tested for its regenerative potentials. Histological study of hydrogel treated diabetic rat skin showed increased collagen fiber contents, improved re-epithelization and neovascularization. The hydrogel was reported to be novel as it provided oxygen at the wound site (Patil et al., 2019). Similarly, in another study, alginate-chitosan hydrogel fabricated with hesperidin was examined on wounded rat model. Hydrogel contained 91.2% ± 5.33% porosity and weight loss assay indicated its biodegradability as 80% of hydrogel contents release after 14 days. Moreover, the time-killing method showed the bactericidal property of hydrogel, and the cytotoxicity assay revealed the non-toxic effect on murine fibroblast 3T3. The in-vivo wound healing experiment demonstrated 98% ± 1.87% wound closure in hydrogel treated group while, only 59.1% ± 2.6% wound closure was observed in control group. The hydrogel was found to control inflammation and promote fibroblast proliferation causing rapid epidermal layer formation, tissue granulation and remodeling. The study concluded that the prepared hydrogel can be an effective treatment option for human skin injuries (Bagher et al., 2020). In a similar study, chitosan-polyethylene glycol hydrogel impregnated with silver nanoparticle was tested to treat chronic diabetic wound. In morphology the hydrogel showed high porosity and swelling characteristics. The addition of silver nanoparticles increased the antimicrobial activity of hydrogel against E. coli, Pseudomonas aeruginosa, Bacillus subtilis and S. aureus. In addition, the hydrogel also exhibited strong antioxidant activity due to the presence of amino group of chitosan and silver nanoparticle. In wound healing study, the hydrogel showed significant keratinocytes migration, better re-epithelialization and higher wound contraction as compared with the control group (Masood et al., 2019). Therefore, chitosan being a base polymer can be impregnated with various useful agents to develop multifunctional hydrogel system to encounter various challenges faced by the patients having acute and chronic cutaneous skin injuries.

  • Sponges are the scaffolds having soft and flexible nature and possess three-dimensional, interconnected porous structure. The highly porous arrangement of sponges closely mimic the extracellular matrix and facilitate adhesion, interaction, growth and proliferation of cells. Sponges have the capability to retain their porous structure, making it suitable in skin tissue engineering applications (Han et al., 2014). For instance, chitosan sponge conjugated with carboxy-methyl konjac glucomannan was synthesized to examine its wound healing potential. The sponge promoted granulation and dermis layer formation in Institute of Cancer Research mice (ICR mice-Albino strain). Moreover, the healed skin of chitosan sponge treated group showed new blood vessel, sebaceous glands and mature hair follicles (Xie et al., 2018). Similarly, another study reported novel chitosan-gelatin sponge skin tissue engineering applications. The sponge showed a uniform sized of pores of 120-140 μm. In addition, the internal morphology of sponge promoted the proliferation of fibroblast cells in a fusiform manner. The sponge showed biocompatibility with human keratinocyte (HaCaT). Further, in-vivo experiment concluded that composite sponge system helped in maturation of blast cells into fibroblast and formation of granulation tissue (Han et al., 2014). In another study, a biocompatible chitosan-gelatin sponge loaded with curcumin was investigated for its wound healing capabilities. The sponge was designed in a manner to maintain the gelatin content lower than chitosan because the higher gelatin concentration can reduce the pore size and uptake of water content. The sponge was found to be non-toxic to mouse fibroblast L929 cell-line. The in-vivo healing study revealed more wound closure (99.49%) in chitosan-gelatin composite sponge as compared with control group (94.30%). Moreover, the histopathological analysis showed more granulation and alignment of collagen as compared to untreated group (Nguyen et al., 2013). In another study, a three-dimensional sponge was prepared by using combination of silk fibroin and chitosan. The mechanical strength of sponge was improved by increasing the concentration of chitosan. Biocompatibility testing on mouse fibroblast 3T3 cell showed biocompatible nature of chitosan composite sponge. However, the sponge was found to promote healing pattern by increasing the fibroblast proliferation and migration (Sionkowska and Płanecka, 2013). Moreover, in another study, chitosan sponge loaded with dermal fibroblast was constructed and tested full thickness rabbit wound model. The in-vivo application of sponge showed that it promoted re-epithelization by increasing migration of propagating cells and enhanced collagen deposition. The chitosan grafts also enhanced the angiogenesis and formation of hyperkeratotic dermis (Deepa et al., 2013). In a similar study, chitosan sponge was incorporated with basic fibroblast growth factor to obtain novel tissue engineering construct. The prepared sponge contained large number of pores, also higher concentration of chitosan improved tensile strength and promoted sustained release of basic fibroblast growth factor (bFGF). A novel chitosan-based sponge was prepared that having excellent biomechanical properties and was able to provide growth factor at the wound site (Ikeda et al., 2014). In another study, the prepared collagen-chitosan sponge biomaterial was investigated in skin wound healing of diabetic rats. The fabricated sponge was loaded with mesenchymal stem cells. The in-vivo experimental testing in diabetic mice model suggested that application of sponge on wounded skin aided in reducing inflammation and enhanced angiogenesis, thus accelerated the wound closure. This study showed that sponge composite comprised of collagen-chitosan- mesenchymal stem cells may act as a promising skin substitute for compromised skin (Tong et al., 2016). Another study reported the synthesis of highly porous biocompatible chitosan sponge integrated with lithium chloride. The prepared sponge enhanced the healing process. The healed skin showed normal skin morphology such as thicker epidermis and hair follicle regeneration. Moreover, mRNA expression study revealed that the sponge treated tissues showed sustained expression of β-catenin, a vital player in wound healing and dermis formation (Yuan et al., 2020). In a study, Ran et al. (2019) synthesized porous chitosan sponge and impregnated it with Sanghuangporus sanghuang polysaccharides and silver nanoparticles. The sponge possessed an internal pore size of 50-100 μm. In addition, it showed good water retention and swelling characteristics. The chitosan sponge material also displayed antibacterial activity against tested microbes (S. aureus and E. coli). Furthermore, cytotoxicity assay revealed that the biomaterial displayed a negligible toxicity against mouse fibroblast L929 cell line. In wound healing test, the treated group with sponge biomaterial showed better wound contraction and tissue growth as compared to control group (Aquacel®Ag) (Ran et al., 2019). Thus, chitosan-based sponges are able to promote healing process by providing favorable environment for growth of new cells, while impregnation of additional compounds augment the healing process by activating or repressing healing related genes. Therefore, chitosan sponges may be promising candidate in developing tissue constructs for damaged skin tissues.

  • Fiber based biomaterials was improved type as it supported three dimensional growth and proliferation of cells, and bio-mimicking the natural healing system. Therefore, various blends of chitosan were formulated to harness useful properties of the composite for the healing process. For instance, fibrous mat of chitosan blend with polycaprolactone (PCL) were prepared for potential applications in wound healing. The experimental results indicated that fibers supported adhesion and spreading of cells on its surface, along with that, cells were impregnated in the fibers, as well. This chitosan fiber may serve as a biological substitute in skin tissue engineering (Prasad et al., 2015). Similarly, another composition of chitosan-graft-polycaprolactone nanofibers were prepared to obtain effective tissue regenerative construct. The composite showed 100% hydrophilicity and 85.5% porosity, allowing more cell migration and exchange of gases. The cell culture test showed faster growth rate of L929 cells grown on composites compared to PCL fibers. It was suggested that amino group of chitosan immobilized various biomolecules such as growth factors, peptides and polysaccharides, thereby providing nutrients to the growing cells (Chen et al., 2011). Another study reported the preparation of polyvinyl alcohol-chitosan-starch nanofibers scaffold. The composite nanofibers showed uniform bead-free nanofibrous structure and water absorption characteristics. The scaffold was found to be effective against both gram positive and gram negative bacteria. The biocompatibility test showed that an excellent growth and proliferation of mouse fibroblast L929 cell-line was observed on the composites. The presence of starch and chitosan promoted migration of L929 cells covering scratched area (wound gap). Thus, the nanofibrous composite limited the pathogens and promoted healing process (Adeli et al., 2019). Likewise, chitosan composite fabricated with collagen microfibers was prepared and tested for healing activities on cell lines using 3T3 fibroblasts and HaCaT keratinocytes. The chitosan-collagen fibers possessed 0.3-15 μm diameter pores. It was found that the fibers promoted healing process by supporting cell attachment and spreading in a sheath-like structure. Presence of collagen up-regulated integrin and intracellular signaling cascade, facilitating good attachment of cells on chitosan-collagen fibers (Sarkar et al., 2013). In another study, the silk fibroin-hydroxybutyl chitosan nanofiber was crosslinked by two different cross linkers i.e., genicin and glutaraldehyde. Both types displayed similar physicochemical properties of fibers, while, genipin added fibers displayed low cytotoxicity. Similarly, histological analysis showed potentials of wound healing due to higher rate of fibroblast cell proliferation (Zhang et al., 2010). The chitosan and gelatin were fabricated in a nanofiber having 100-220 nm diameter fibers. The composite nanofibers showed enhanced tensile strength as compared with separate chitosan and gelatin nanofibers. The nanofibers possessed suitable tensile strength which may serve as a promising materials for skin tissue engineering (Dhandayuthapani et al., 2010). In addition, nanofiber of chitosan coating polylactic acid-collagen-aloe vera has been investigated for potential applications in skin tissue engineering. The synthesized chitosan-polylactic acid-collagen nanofibers possessed 67.5% porosity and higher water uptake rate as compared with control polylactic acid-collagen. The cell culture assay indicated that nanofibrous scaffold could provide optimal environment for proliferation of cells (Salehi et al., 2016). Similarly, the nanofiber based scaffold of chitosan-polyvinyl alcohol was prepared and investigated for skin fibroblast cell attachment. This nanofiber was also reinforced with halloysite nanotubes, in order to enhance the hydrophilicity property of nanofibers. The composite was found to be biocompatible and enhanced the fibroblast proliferation. This made the prepared composite system an effective candidate for skin tissue engineering (Koosha et al., 2019). In a study, a nanofiber of chitosan and polyvinyl alcohol incorporated with carboxymethyl chitosan nanoparticle was developed and investigated as wound dressing system. The nanofibers effectively improved the bioavailability, biological activity of bioactive compound and further controlled the degradation. In addition, the composite nanofibers showed promising antimicrobial activity against S. aureus and E. coli. The histopathological assay revealed that chitosan-based nanofiber had improved collagen deposition and re-epithelialization pattern in wound model. The study revealed that the fabricated nanofiber showed dual functions of antibacterial and wound healing (Zou et al., 2020). In another study, Abid et al. (2019) investigated the development of chitosan and polyethylene oxide nanofiber. The fabricated fibers were impregnated with zinc oxide nanoparticle and ciprofloxacin. The loading of zinc oxide nanoparticles enhanced the thermal stability of nanofibers. The composite nanofiber system displayed 68% and 65% of S. aureus and E. coli colonies reduction. Furthermore, the cytotoxicity test revealed that co-polymer nanofiber system showed a higher rate of biocompatibility (> 82.5%) against keratinocytes and human dermal fibroblast cell lines. The study suggested that the synthesized nanofiber could act as promising chitosan-based biomaterial in controlling infection and additionally would help in improvement of healing process (Abid et al., 2019).

5.   Conclusions
  • Based upon the chitosan inherited peculiarities including hemocompatibility, hemostatic and biodegradability properties and diverse source for extraction, have widen the applications of chitosan in biomedical domain. The presence of amino, hydroxyl and carboxyl functional groups in chitosan assist in easy formation of composite system with other natural and synthetic materials. This enhances the biological and mechanical properties of chitosan that may be exploited for the treatment of acute and chronic wounds and other skin tissue engineering applications. Furthermore, preparation techniques such as freeze-drying, electrospinning, solvent casting, microwave assisted technique and dense gas foaming method can be used for the development of suitable chitosan-based biomaterials based upon the nature of treatment for damaged skin tissue. Among other biomaterials, nanofibers and hydrogels based chitosan proved to have an exceptional characteristic that is important for skin tissue engineering such as high water uptake, high porosity, improved mechanical properties. The porous chitosan hydrogel and nanofiber system support the attachment and proliferation of skin cells. However, there is still need of development of an ideal material that would fully bio-mimic the natural tissue functions and help in reducing the time for full tissue recovery.

6.   Future Perspectives
  • In the past decade, tissue engineering has made remarkable progress and is still evolving. It is now possible to synthesize living tissues and understand the complexities of organ systems for the clinical use. This field is advancing with every new technique and trend including, new stem-cell resources, vascular engineering, microfluidics-based physiological platforms, smart biomaterials and many more. However, this field is facing some challenges like, the immunogenicity of tissue engineered scaffolds, lack of ideal bioinks, engineering vasculature and innervation in bioengineered tissues and regulatory issues. The coming decade is anticipated to develop more breakthroughs and material products accepted at clinical level.

Conflict of Interest
  • There are no conflicts to declare.

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