Effectiveness of chitosan scaffold in skin, bone and cartilage healing

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Abstract

Chitosan (CS) is a carbohydrate biopolymer, which has been known as a biological material in promoting the healing process of soft and hard connective tissues. It is biocompatible, biodegradable, bioactive, non-toxic, non-expensive and non-immunogenic, with antibacterial capability. Additionally, the capacity of forming complexes with other anionic biomaterials and molecules offers CS the characteristics to be used in biomedical applications. Therefore, this natural polysaccharide has widely been used as a wound dressing and in bone and cartilage regeneration because of these considerable properties. However, some studies have showed limited value in application of CS in tissue regeneration. It has been used alone or in combination with other biopolymers, bioceramics, or promotive growth factors to enhance regeneration of the soft and hard connective tissues. This study has reviewed various forms of CS including hydrogels, sponges, films, and nanofiber membranes. The role of CS alone or in combined form with other materials has also been reviewed in healing and regeneration of the cutaneous, cartilage and bone wounds. In addition, the present study has attempted to clarify the controversies and conflicts regarding effectiveness of CS in the healing process.

Introduction

One of the main concerns and challenges for physicians and surgeons is finding a method to improve the healing and regeneration of tissue defects [1]. Many factors such as traumas, tumors, burns, accidents, arthritis, and large fractures cause wound, cartilage and bone defects [2], [3], [4]. The tissue ability to repair is limited in the cases of massive defects and thus it requires surgical intervention [2], [3]. To overcome this problem, the cosmetic and orthopedic surgeons and researchers in the field of regenerative medicine have tested various materials and methods including grafts, natural or synthetic biomaterials, various types of stem cells as well as the reconstructive materials such as growth factors, statins, and glycosaminoglycans (GAGs) [2], [3], [4], [5]. Unfortunately, these compounds have some disadvantages that have limited their use [3], [6]. Researches to find an appropriate method to substitute the grafts continue and one of the methodologies which has received much attention by researchers and surgeons is tissue engineering and regenerative medicine [3], [8]. It attempts to design and fabricate a three-dimensional (3D) scaffold augmented by stem cells and promotive growth factors as the promising substitute to bone grafts with no or minimal disadvantages, in comparison to the bone grafts [9], [10], [11], [12]. Selection of an appropriate material for scaffold fabrication is a critical stage in tissue engineering [3], [6]. In general, the materials used for the scaffold fabrication may be natural (biological) or synthetic [3]. Some samples of the natural materials for scaffolding are collagen, gelatin (Gel), alginate, chitin and its deacetylated derivate i.e. chitosan (CS), hyaluronic acid, and GAGs [3], [4], [5], [6], [10]. Among these, CS is a linear natural carbohydrate biopolymer that exhibits structural similarity to GAGs of the extracellular matrix (ECM) which have important role in cell–cell adhesion [13], [14], [15].

Chitosan has a hydrophilic surface that promotes cell adhesion, proliferation and differentiation and it has high hydrophilic capacity, therefore it may maintain and attract fluids and cells to the defect site [13]. It has extensively been used in the field of tissue engineering due to its beneficial properties such as bioactivity, biocompatibility, biodegradability, and high availability in various forms such as hydrogels, sponges, fibers, sheets, films and other structures [5], [15]. Therefore, CS seems to satisfy most of the cosmetic and orthopedic surgeons and its properties support its candidature to be applied in the field of tissue engineering and regenerative medicine [7], [12], [15]. Due to the positively-charged amino groups in the CS structure, CS is a mucoadhesive, hemostatic biomaterial with the ability to bind cell membranes [12]. In addition, CS is capable of making scaffold with desired shapes with appropriately interconnected porosity [5], [12]. Given the ability of CS-based scaffold materials in the controlled delivery of the loaded molecules or growth factors in the injured area, they are regarded as suitable candidates for tissue engineering and regenerative medicine, in combined form [9], [11], [12]. Since some studies have reported the inefficacy of CS scaffold alone in the healing process [6], [16], [17], to enhance and augment the properties of CS for tissue engineering applications, other polymers, and inorganic materials are added to CS [6], [12], [14], [15], [17], [18]. For instance, Nandi et al. [8] investigated the effectiveness of porous CS alone and in combination with insulin-like growth factor-1 and bone morphogenetic protein (BMP)-2 in the repair of tibial bone defect in rabbits. They showed that porous CS in combination with growth factors can be successfully used in healing of bone defects. On the other hand, CS has poor mechanical properties, which can be eliminated by adding other biomaterials. Moreover, when using CS to make composites, it should be noticed that high degree of deacetylation (DD) and molecular weight retard its biodegradation rate [4], [5], [15]. The present study has reviewed the structure, properties and different types of CS scaffold, and also its effects on wound, cartilage and bone healing. Positive points and conflicts regarding the use of CS in the field of regenerative process has been addressed in this review.

Chitosan, poly(β-(1–4)-linked-2-amino-2-deoxy-O-glucose), is a linear polysaccharide with a β-1, 4-linked polymer of glucosamine (2-amino-2-deoxy-β-d-glucose) and N-acetylglucosamine and also it has one to four glycosidic bonds [2], [4], [7], [18]. It is a chitin derivative, produced from partial deacetylation of chitin through enzymatic hydrolysis by chitin deacetylase, or chemical hydrolysis under alkaline condition that was firstly discovered by Henri Braconnot in 1811 [2], [19], [20].Chitin can be isolated from various natural sources including mushrooms, cell wall of green algae and fungi, and the exoskeleton or shells of crustaceans, cephalopods and mollusks [2], [4]. It has two forms including α-chitin and β-chitin [21].

To extract the chitin and then CS from the dried shell wastes, they should be washed by water, deproteinized by NaOH, demineralized by HCL, washed again and finally dried. The CS is then deacetylated, using NaOH at about 90°C for 2 h and is purified by application of the following steps: 1) removal of insoluble compounds by filtration, 2) re-precipitation of CS with NaOH solution, and 3) de-metallization of CS [21].

The physical properties of CS depend on the molecular weight (10,000 to 1 million Dalton), DD (50–95%), sequence of the acetamido and amino groups and purity of the product [8]. The linear polyamine, and reactive amino and hydroxyl groups are the major chemical components of CS [20]. Chitosan is soluble in aqueous acidic medium and DD lower than 0.5 [21]. It is naturally viscous in nature and its solubility can be improved by decreasing the molecular weight, or chemical introduction of a hydrophilic functional group [22]. The degree of viscosity determines the functional properties of CS and the extent of water solubility, owing to the pK value of the amino group, results in protonation from acidic to neutral solutions [4], [15], [20]. Solubility in different media, optical characteristics, metal chelation, solution formation, polyoxy salt formation, and film formation abilities are some other properties of chitin and CS [21].

Chitosan is cheap, biocompatible, biodegradable, bioactive, non-toxic, biologically renewable, non-antigenic, non-immunogenic, and antibacterial and it can stimulate the activity of growth factors [2], [4], [11], [12], [15]. It exists in various forms or shapes including filaments, granules, films, capsules, tablets, gels, powder, microspheres, paste, membrane, sponge, bioactive fibers, nanofibers, filaments, granules, coating mesh, solutions, and porous scaffold as well as composite with other materials [4], [5], [12], [13], [18], [20], [23], [24], [25]. For example, capsules are used as delivery vehicle, tablets are used in compressed diluents as disintegrating agent and excipient, and sponges are used as wound dressing as artificial skin and also for drug delivery [21], [26]. Flakes, fibers, powder, films, microparticles and spongers are the solid forms of CS which are used in tissue engineering [27], [28].

This carbohydrate biopolymer can interact with the surrounding biological environment without adverse impacts on the host body and thus, CS can be a biomaterial for specific biomedical applications after some chemical modifications [21]. Overall, CS is applied as a biomaterial in biomedical-pharmaceutical tissue engineering of bone, cartilage, liver, nerve and cutaneous wound healing, drug delivery, gene therapy, bio-imaging and green chemistry [29], [30]. Chitosan has various applications and it is used in biomedical engineering, artificial organs, biotechnology, production of cosmetics and scaffolds for repairing the injured tissues. It is also applied as space-filling implant, orthopedic material, bone substitute, as a drug delivery vehicle in biomedical and pharmaceutical applications, and also as a carrier for immobilized enzymes, proteins, genes and cells [18], [21], [22], [31], [32], [33], [34], [35]. The glucan content of CS has antioxidant and anti-microbial activities [4], [20]. In fact, because of the amino groups of CS which are cationized in acidic-neutral media, the 2-amino-2-deoxyglucose units can bring the polymer into solution by salt formation. The low molecular weight CS has anti-tumor, anti-fungal, anti-microbial and immunostimulating properties [33].

It can bind to red blood cells to facilitate clot formation [2], and also can stimulate the release of transforming growth factor (TGF)-α1 and platelet-derived growth factor (PDGF)-AB [12], [36]. It is normally used as a hemostatic bandage in USA and European countries to stop bleeding quickly [3]. In fact, the presence of the positive charges results in hemostatic activity of CS [37]. Chitosan may also bind to fat molecules and decrease the cholesterol level; however, its mechanism of action is still unknown [29]. Chitosan can block nerve endings and result in pain reduction [37]. This analgesic effect of CS in the inflamed region is because of the protonation of the amino groups of the D-glucosamine residues and gradual release of N-acetyl-b-d-glucosamine, a byproduct of CS depolymerization which causes fibroblast proliferation and increases the level of natural hyaluronic acid (HA) synthesis in the wound region [37], [38], [39].

Chitosan resembles the proxy structure of GAGs which are the major ingredients of ECM [18]. Chitosan can increase infiltration of inflammatory cells and it can increase the angiogenic and hemostatic activities of platelets [38]. It can also promote the organization of granulation tissue by modulating the function of inflammatory cells, decreasing treatment frequency, accelerating collagen type III production by enhancing fibroplasia, giving up a painless wound, protecting wound surface and ability to activate host defenses to prevent infections [2], [26], [38], [39].

Chitosan can prevent wound dehydration and contamination, when it is used as a semipermeable biological dressing [2]. It also has immunological activity and can act as an accelerator of wound healing [13]. It increases the production of osteopontin which promotes infiltration and attachment of a variety of cell types and has a role in granulomatous inflammation and also can inhibit infection by stimulating the macrophages to release cytokines [2], [13]. Chitosan can agitate the migration of polymorphonuclear cells and stimulate neutrophils and macrophages to phagocytize and produce leukoterian B4 and interleukin (IL)-1, TGF-B1 and PDGF to precipitate the re-epithelization [23], [26]. Chitosan can bind with the DNA of microorganism and inhibit the mRNA and protein synthesis and multiplication of the organisms [23]. Because of the cationic nature of CS, it is used as an antimicrobial agent in the prevention and treatment of infections [2]. In fact, antibacterial activity of CS is related to the interaction between its positive-charged amino groups and the anionic groups of lipoteichoic acids in Gram-positive and lipopolysaccharide in Gram-negative bacteria that results in disruption of the microbial membrane [2], [40]. The molecular weight, ionic strength, DD and pH of the dissolving medium are some factors that could influence the antimicrobial effects [2]; for instance, the interaction between CS and microbial proteins, in acidic pH, is minimum [41].

The glycosaminoglycan, proteoglycans, and other negatively charged molecules available in the ECM can react with the N-acetyl glucosamine and glucosamine of CS [42]. The muco-adhesion is because the negatively charged mucin binds to the protonable amino groups of CS [37]. Acylation, alkylation, reductive alkylation, Schiff base formation, tosylation, N-carboxyalkylation, O-carboxymethylation, and graft copolymerization are some process that could be chemically adjusted with CS because of the hydroxyl and amino groups of CS derivatives [43]. Grafting, chemical cross-linking, casting, melt-blending and chemical functionalization are some methods used in fabrication of CS-based hybrids or composite scaffolds [4], [20], [13]. Moreover, electrospinning, wet spinning, particle aggregation, freeze-drying by lyophilization of a frozen CS solution, rapid prototyping and melt-based approaches are other methods in production of CS scaffolds [13], [21].

Application of scaffolds made by carboxymethyl-CS, CS and magnesium gluconate have previously been investigated [18]. These scaffolds have uniform porosity with highly interconnected 50–250 μm pores. The authors showed that CS and carboxymethyl-CS have a similar structure, but with opposite electric charge. Therefore, they interact strongly with each other and form polyelectrolyte network structures which improve fibroblast and osteoblast adhesion by releasing magnesium ions in aqueous medium [18].

Section snippets

Types of chitosan scaffolds

Chitosan has been used in the 2D-scaffolds for dressing of wounds and in 3D-scaffolds tested in tissue engineering [4], [18], [20]. The CS hydrogels, foams and sponges are used in the synthesis and fabrication of the 3D-scaffolds [20]. The 2D-scaffolds are in forms of films and nanofiber porous membranes manufactured to cover and dress the external wounds [4], [39]. The porous scaffolds manufactured as 3D templates result in enhanced regeneration by promoting matrix deposition, carrying

Application of chitosan scaffolds in cutaneous wound healing

Wound healing is a dynamic process consisting of five phases, including hemostasis, inflammation, migration, proliferation and maturation which are sequential and overlapping cascade [2], [38]. The stages of bedsore and burn wounds are different; they have infection, necrosis, agglutination, proliferation, and epidermis formation periods. The non-severe skin wounds may be treated by ointment and wound dressing [66]. The small defects can heal by surgical reconstruction, while larger or

Application of chitosan scaffolds in bone healing

Some situations such as traumas, bone tumor resections, compound or pathologic fractures, osteotomies, osteomyelitis or chronic bone infections causing massive bone defects which need intervention of the orthopedic surgeons to use good methodologies and treat and reconstruct the defects [3], [75], [76]. Some complications such as massive bone loss, non-unions, and delayed unions may occur in the process of bone repair [3]. After a bone fracture, cytokines and growth factors are released by

Role of chitosan scaffolds in cartilage healing

Due to lack of vascularization in articular cartilage and the limitation on proliferation capacity of chondrocytes, the catabolic response of chondrocytes to pathological mediators, anisotropic, non-linear, non-homogeneous and viscoelastic proteins, the regeneration capacity of articular cartilage is so limited [82], [103], [104]. The normal human meniscal tissue contains chondroitin-6-sulfate (60%), dermatan sulfate (20–30%), chondroitin-4-sulfate (10–20%), and keratan sulfate (15%) which do

Discussion and conclusion

Although a huge background exists in the literature that have suggested CS as an acceptable biomaterial for tissue engineering applications [17], [39], [83], [85], [103], [112], [113], [114], there are several research works that showed ineffectiveness of CS in tissue regeneration [6], [16], [79], [115], [118]. The oppose studies have showed that the healing potential of CS alone is doubtable because of its low biodegradability and also limited osteoconductivity (in the field of bone healing)

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