Versatility of Chitosan-Based Biomaterials and Their Use as Scaffolds for Tissue Regeneration

Chitosan is a naturally occurring polysaccharide obtained from chitin, present in abundance in the exoskeletons of crustaceans and insects. It has aroused great interest as a biomaterial for tissue engineering on account of its biocompatibility and biodegradation and its affinity for biomolecules. A significant number of research groups have investigated the application of chitosan as scaffolds for tissue regeneration. However, there is a wide variability in terms of physicochemical characteristics of chitosan used in some studies and its combinations with other biomaterials, making it difficult to compare results and standardize its properties. The current systematic review of literature on the use of chitosan for tissue regeneration consisted of a study of 478 articles in the PubMed database, which resulted, after applying inclusion criteria, in the selection of 61 catalogued, critically analysed works. The results demonstrated the effectiveness of chitosan-based biomaterials in 93.4% of the studies reviewed, whether or not combined with cells and growth factors, in the regeneration of various types of tissues in animals. However, the absence of clinical studies in humans, the inadequate experimental designs, and the lack of information concerning chitosan's characteristics limit the reproducibility and relevance of studies and the clinical applicability of chitosan.


Introduction
Tissue engineering represents a recent area of multidisciplinary research that applies the knowledge of materials engineering and biology, with the aim of reconstructing or regenerating damaged biological tissue in clinical/pathological situations such as lesions, infections, traumas, and sequelae resulting from tumors or systemic disorders. To this end, it was sought to develop biological substitutes capable of restoring, maintaining, or improving organ and tissue function. Tissue engineering approaches generally involve using a combination of cells, bioactive molecules, and biomaterials. The cells are responsible for the synthesis of the matrix of new tissue. Bioactive molecules, such as cytokines and growth factors, promote cell proliferation, differentiation, and migration. Biomaterials act as a scaffold for the new tissue, providing an environment that favors cell growth and differentiation [1,2].
All tissue is basically made up of an extracellular matrix (ECM) and cell components. The ECM is the structure upon which the cells establish and organize themselves, providing the environment and architecture specific for cell growth and proliferation, functioning as a reservoir of water, nutrients, and growth factors essential to cells [3,4]. In this regard, when the regeneration of tissue is desired, the presence is required of a structure that acts as a temporary matrix, mimicking the ECM, allowing cell proliferation and tissue synthesis, until regeneration is complete [5,6]. This structure, the scaffold,  [12] and Lanza et al. 2014 [2]. may be defined as a porous, three-dimensional, solid biomaterial that acts as a temporary ECM during tissue regeneration [7]. The biomaterials used as scaffolds in tissue regeneration are of fundamental importance and may determine the success or failure of any tissue engineering approach [8,9]. Like the ECM, scaffolds must promote vascularization and provide a three-dimensional infrastructure that permits new tissue to form. Also, they must be able to act as a carrier of biologically relevant molecules and, ideally, modulate their biological responses and the regeneration process ( Figure 1). Thus, the use of a biomaterial as a scaffold for tissue engineering assumes that it has certain characteristics that make it similar to the extracellular matrix [9][10][11][12].
In tissue engineering, the scaffold must have a temporary structure that undergoes gradual biodegradation over a period of time consistent with the speed of new tissue formation, allowing the regenerated tissue to replace it. To this end, it is necessary to possess certain essential characteristics, such as biocompatibility, biodegradability, and mechanical performance and surface properties such as porosity, which favor cell adhesion [7,10]. The materials most commonly used for tissue regeneration include calcium phosphate ceramics like hydroxyapatite (HA) and beta-tricalcium phosphate ( -TCP), synthetic polymers such as polyglycolic acid (PGA) and poly(lactic-co-glycolic) acid (PLGA), and naturally occurring biodegradable polymers such as collagen, hyaluronic acid, silk fibroin, gelatin, and chitosan [5,[13][14][15].
Chitosan is a polysaccharide obtained from abundant sources in nature and has been regarded as having great potential for application as a biomaterial for tissue regeneration. It is obtained through the deacetylation of chitin, a polymer found in the exoskeleton of crustaceans, mollusks, and insects and on the cell walls of fungi [16]. Chitosan (CS) has a chemical structure with a linear chain composed of units of D-glucosamine and N-acetyl-glucosamine joined together via (1-4) glycosidic bonds ( Figure 2). In this structure, there are three reactive functional groups: one amine (NH 2 ) and two hydroxylic groups (OH). These groupings, particularly the amine, give CS a cationic nature due to the protonation of amino groups (NH 3 +) in acidic medium. This peculiar cationic nature gives CS a great affinity for anionic biomolecules, such as sialic acid, sulfonic acid, and the glycosaminoglycans (GAGs) abundantly present in mucous secretions and extracellular matrix [17,18]. In fact, CS is considered to be the only natural polysaccharide with this characteristic [19]. By way of ionic interactions, CS has the capacity to bond with mucous tissue, called mucoadhesiveness [10,[20][21][22]. This property is of great interest to tissue engineering as the possibility of using a scaffold that interacts with the GAGs and tissue's proteoglycans may facilitate the incorporation of  Figure 2: Chemical structure of chitin/chitosan. The index represents the number of repeat units of glucosamine in the chain and the number of repeat units of acetyl-glucosamine in the chain ( + indicating the degree of polymerization and / + being the degree of acetylation). When is more than 50%, the polymer is called chitosan. Content of NH 2 increases its reactivity [16,25].
cytokines and tissue growth factors, as a large number of these factors have an affinity with GAGs [23,24]. The singular physicochemical and biological characteristics of CS have prompted numerous studies investigating its application as a scaffold for tissue regeneration [26][27][28][29][30][31]. Biocompatibility, biodegradability, mucoadhesiveness, absence of toxicity, capacity to form three-dimensional porous structures, ease of handling, and low cost are just some of the benefits mentioned by the authors, which make CS a suitable biomaterial with great potential for application in tissue engineering. Moreover, the reactivity of CS also makes it possible to modify its structure through the substitution of its functional groups, which allows it to be combined with other synthetic or natural polymers, forming the so-called polymer blends, or composites with calcium and phosphate ceramics. These modifications enable the characteristics of CS to be modulated, such as solubility, biodegradability, and mechanical performance, depending upon the type of tissue to be regenerated, which makes it an extremely versatile biomaterial for application in tissue engineering [17,[32][33][34][35][36].
There is, however, a great diversity in the characteristics of CS used in the studies for tissue regeneration, either for the structure of the scaffolds, such as membranes, sponges, films, and nanofibrils, or for the physicochemical parameters of chitosan. The degree of acetylation (DA) and molecular weight (MW), for instance, are the two major chemical properties of CS and influence other physicochemical and biological characteristics, like solubility, crystallinity, mechanical performance, biocompatibility, and biodegradation [19,[37][38][39][40]. The lack of standardization in the studies, particularly with regard to the physicochemical characteristics of CS, makes it difficult to establish a consensus as to its applicability as a scaffold for tissue engineering. The present study proposes a systematic review of the literature on the use of chitosan in tissue engineering, with a critical approach to studies on animals and humans, with the aim of establishing a relationship between the methodologies used and results obtained in tissue regeneration, so as to make a contribution to the understanding of the application of this material and to demonstrate its potential use.

Methodology
For this systematic review, a search was conducted of the PubMed electronic database, through the US National Library of Medicine portal (https://www.ncbi.nlm.nih.gov/ pubmed/) by two independent researchers, in accordance with the selection criteria. For the search, the following keywords were employed: chitosan, scaffold, tissue engineering, and regeneration. The initial study produced 478 articles. The search was restricted to the last five years and only preclinical studies (with animals) or clinical studies (humans) were used, resulting in 246 articles. After a critical reading of the titles, abstracts, and, where necessary, the articles' methodology, only articles published in English were included. The following exclusion criteria were then applied: (i) Literature reviews (ii) Case studies (iii) Studies in which CS was not used to evaluate tissue regeneration (iv) Abstracts or studies where the full content was not accessible The final selection resulted in 61 articles that were studied, catalogued, and grouped in tables, according to the type of tissues that was involved. A flowchart of the bibliographic research is displayed in Figure 3.

Results
The selected articles were analysed and the results are shown in Tables 1-5, which list, according to the type of tissues  involved, most relevant information such as the scaffold characteristics, main physicochemical properties of CS (when  available), combinations with other polymers, incorporation of growth factors and/or cells, the in vitro and in vivo methodology evaluated, the main results in terms of tissue regeneration, and the authors' major conclusions.
As for the characteristics of the scaffolds, the majority of studies involved CS combined with other biomaterials (85.25%), including HA (16%), collagen (14%), gelatin (10%), silk fibroin (10%), PLGA (8%), multiple combinations between them (20%), or forming other polymer blends (22%). Scaffolds of natural CS were used in 14.75% of the studies [26,28,31,33,59,64,74,75,79]. The physicochemical characteristics of CS varied greatly. The molecular weight ranged from 22 [74] to 1,800 [73] kilodaltons (kDa), while the degree of acetylation ranged from 2% [54] to 40% [81]. In almost one-half of the studies reviewed (47.5%), the molecular weight and degree of acetylation of the chitosans used were not specified. When these parameters were mentioned, 46.9% of the chitosans exhibited low molecular weight (up to 150 kDa), 37.5% were of average molecular weight (150 to 700 kDa), and 12.6% had a high molecular weight (in excess of 700 kDa). As for the macrostructure of the scaffolds, these were primarily used in the form of sponges (24.6%), membranes (19.6%), conduits or tubules (13.1%), or even as nanofibrils, hydrogels, microspheres, powder, and paste (11.4%) or were unspecified (31.3%). In 63.9% of the studies, there was an incorporation into the scaffolds of bioactive molecules such as growth factors, genetic factors, peptides, extracellular matrix, or cell components such as stem cells and other cells related to tissue involved in regeneration, or even drugs.
As far as the study outline is concerned, 75.4% used in vitro and in vivo assay methodologies in their investigation of scaffold activities with CS, while in 24.6% of studies only in vivo assays were performed. The in vitro analyses consisted of tests on the cytocompatibility of the scaffolds in cell cultures, evaluating cell adhesion, proliferation, viability, and differentiation. The in vivo assays investigated the effects of scaffolds on tissue regeneration and/or their biocompatibility through macroscopic, radiographic, tomographic, and histological analyses and biomolecular tests such as PCR and immunoassays to detect specific tissue markers involved in regeneration. With regard to the use of comparison groups, 59% of the studies used negative control groups, and 32.8% did not use negative control groups, while in 8.2% of studies the control was not specified in the methodology. In 11.5% of studies, both positive and negative controls were employed, while in 8.2% only positive controls were employed. As for sample size, 21.3% of studies used, in their in vivo assays, samples where was less than 4 sample units; 36.1% had a value of between 4 and 6; in 13.1% was between 8 and 10 and 13.1% had greater than 10. The sample size was not specified in the methodology in 16.4% of the studies reviewed.
The interrelationship between the scaffolds and their effects on tissue regeneration reveals that, in 93.4% of the studies, scaffolds using CS promoted in vivo or ex vivo tissue regeneration. Only 6.6% of the studies [27,41,71,85] showed no evidence of the scaffolds having a positive influence on tissue regeneration.

Discussion
Regenerative medicine has been highlighted as one of the most promising fields of scientific research towards which the most recent advances in biological sciences are converging, such as tissue engineering, stem-cell technology, and genetic therapy. The progress achieved in the area of tissue engineering is due, for the most part, to the huge volume of studies using biomaterials that offer potential for regeneration of almost all tissues and organs in the human body. In this context, chitosan has received a great attention from the scientific research literature. Its favorable characteristics as a biomaterial, such as biocompatibility, biodegradability, and, above all, chemical affinity with biological molecules, make it an attractive material for tissue regeneration applications. Also, its chemical reactivity allows its properties to be modulated, making it an extremely versatile material [7,19].
In the present review, the versatility of CS applications can be observed. It was used in scaffolds for the regeneration of bone, cartilage, skin, nerve, periodontal, vascular, colorectal, mammary, and tympanic membrane tissues. However, though CS is considered to be a biomaterial of great potential for over a decade [16], it is somewhat surprising that none of the studies reviewed in this work has applied CS in clinical trials on human beings. In fact, in the only study where CS was evaluated on human tissue, this was implanted in cutaneous tissue ex vivo or, in other words, removed from individuals who had been subjected to breast or abdomen reduction surgery [68]. This paucity of clinical studies may be explained by researchers' difficulties in obtaining ethical research approval for the use of this material, as there are no regulations for the use of CS as a biomaterial for human tissue regeneration. In the USA, the regulatory agency FDA only approves the use of CS in human beings for the bandaging of skin wounds or hemostatic dressings, while, in other countries such as Japan, Italy, Finland and Brazil, its use is approved as a food supplement and in the cosmetics industry [17,25,35].
Most of CS's versatility may be explained by the numerous possibilities of modification in its chemical structure through The Scientific World Journal 5 The CS-based scaffolds were shown to be biocompatible and promoted bone regeneration in vivo, particularly in the presence of SC 6 The Scientific World Journal

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The Scientific World Journal  In vivo: the CS+SF+SC scaffold promoted almost complete repair of the defects and positive HT and IHC results; CS+SF scaffold demonstrated better results than the control, but not as good as in the group with SC CS+SF scaffold showed itself to be effective as SC carrier and capable of being used in the regeneration of cartilage tissue ( < 0.05)   The Scientific World Journal the substitution of its functional groups. The combination of CS with other natural polymers, such as collagen, gelatin, and silk fibroin, synthetic polymers such as PLGA, and compounds of calcium and phosphate, such as HA, permits an improvement in specific properties like solubility, biodegradation, and mechanical performance of CS as a function of the tissue to be regenerated [10]. In fact, some studies have clearly shown that the association of CS with collagen [42,44,68], silk fibroin [73], and HA [45,57,58] was capable of promoting greater tissue regeneration than pure CS, as in these studies pure or combined CS scaffolds were implanted, making it possible to establish a comparison between them. However, in the vast majority of studies evaluating CS combined with other biomaterials, the favorable results obtained cannot be attributed to the associations, as pure CS was neither evaluated nor used as a control [8,30,32,41,46,48,49,53,54,62,63,67,71,76,77,80,81,83,86,88]. Another aspect which merits some attention is the great variability in the chemical characteristics of CS used in the studies, such as molecular weight and degree of deacetylation. As it is a polymer which can be obtained from a variety of sources, CS may actually present a large variation in MW (ranging from 30 to 2000 kDa) and in DA (5 to 46%) resulting from the different processing conditions [7,21]. However, it should be stressed that these properties drive important physical characteristics of chitosan, such as solubility, crystallinity, absorption of water, and mechanical performance, and also biological characteristics, such as biodegradation, antimicrobial activity, mucoadhesiveness, and biocompatibility [7,10,21,36,61]. Wu et al. [38] and Zheng et al. [39], for instance, demonstrated that the in vitro bioactivity of CS on macrophages was dependant on its MW. Accordingly, it is probable that these parameters may influence the behavior of CS as a biomaterial for tissue regeneration. Thus, the use of such diverse chitosans, such as MW 22 kDa [74] and 1800 kDa [73], though both studies investigated regeneration in nerve tissue, renders any comparison of results unviable. In addition, the fact that MW and DA have not been specified in 47.5% of the studies raises speculation that the authors have overlooked the significance of these parameters. In fact, the lack of information on the physicochemical characteristics and their effects on biological properties of chitosan-based scaffolds is a matter of concern in the literature, since most of the useful applications of CS are determined by DA, MW, and the nature and fraction of substituents as pendant groups [19]. In the present review, only two studies accounted for the variation of MW in the methodology and interpretation of results. In the study by Cao et al. [49], chitosans of different molecular weights were evaluated in vitro and, based on the results, the 98 kDa CS was selected for the in vivo assay. Denost et al. [26] used layers of CS of different MW to fabricate the scaffolds, due to the different characteristics they presented, corroborating the importance of this parameter on the biological activity of CS.
The use of comparison standards (control groups) is essential when investigating a particular experimental material. It is important to compare its effects with other already familiar or used materials (positive control) or with no treatment at all (negative control). In the present review, only 11.5% of the studies used positive and negative controls to compare with the experimental scaffolds [30,33,48,73,76,77,80]. As many as 20 studies (32.8%) did not use negative controls for comparison. Bearing in mind that in some studies the results showed that tissue regeneration also occurred in negative control groups [31,41,48,65], the absence of a negative control could cast doubt on the relevance of the results obtained with regard to the actual effectiveness of the material being investigated.
Sample size is an important consideration in the design of many studies because of its effect on statistical power. Statistical power is the probability that a statistical test will indicate a significant difference when there truly is one. In this review, some studies used small samples for their in vivo assays, with = 3 [32, 46-48, 61, 65, 68, 81], = 2 [27,56,69], and even = 1 [72]. It is important to point out that the value of considered in this study is always the number of animals used for each treatment condition. For instance, the study by Sundaramurthi et al. [65] mentions a sample of 30 animals. However, the animals were subdivided into 5 groups and 2 evaluation periods, totaling 10 groups, resulting in three animals for each condition evaluated ( = 3). Simões et al. [72] used 4 rats that were evaluated at 4 different points in time, resulting in just 1 animal per period ( = 1). Small samples, such as those mentioned in these studies, may compromise the results obtained, thus diminishing the reliability of the statistical differences found or omitting actual differences that could be observed with an increased sample size [32,48,69]. Given the importance of the sample size in the study outline and its influence on the results, it is significant to note that 16.4% of the studies reviewed do not clearly specify this information in their methodologies. The omission of this piece of information makes it difficult to interpret the results and establish a comparison between them.
The overall results concerning tissue regeneration with the use of CS-based scaffolds seem to indicate that they favored the regenerative in vivo process in 93.4% of the works reviewed. Although this information may suggest the effectiveness of CS, it is important to consider that quite a lot of the analysed studies used inadequate experimental designs (such as the absence of proper control groups, small sample size, and lack of comparisons between CS alone or combined with other biomaterials). For this reason, it is not possible to unequivocally confirm that the regeneration was due to the presence of chitosan. However, these results strongly suggest that CS fulfils the function of a scaffolding biomaterial as a reservoir of bioactive factors, such as stem cells and growth factors, showing biocompatibility, biodegradation, adsorption capacity, and gradual release of incorporated factors in most of the studies. Biological activity, with regard to the induction of tissue regeneration, seems to be attributed to the cell components and/or growth factors incorporated to the CS-based scaffolds and not to CS itself. In 83.33% of the studies in which scaffolds were compared, either with or without these components, the results showed a better regenerative performance with the presence of cells and/or growth factors. In other studies, there was no difference in results, neither with nor without bioactive factors [29,56,80], or there was no comparison between them [46,62,81]. Therefore, it cannot 22 The Scientific World Journal be asserted, from the results presented, that chitosan possesses per se activity that induces tissue regeneration, as some authors suggest [7,10,16,22,25]. Nevertheless, some studies showed that pure CS, without the addition of bioactive factors, may be effective in promoting regeneration on cutaneous [64], nerve [79], colorectal [26], bone [55], periodontal ligament [33], and tympanic membrane [31] tissues.

Conclusion
The critical analysis of the studies reviewed confirms the potential for the application of chitosan as a biomaterial in tissue engineering, considering the great versatility of this polymer in the regeneration of various types of tissue in preclinical studies. Scaffolds composed of CS combined with other biomaterials, cells, and growth factors were found to be effective in promoting in vivo tissue regeneration. Nevertheless, the large chemical variability of the CS employed, the omission of precise information about its characteristics, and the methodological limitations of the studies make it difficult to reproduce and to establish a standardization for clinical application. Further studies are required with the aim of defining chitosan's ideal physicochemical characteristics for application with each type of tissue and, thus, propose a protocol for its use in clinical studies in order to confirm its effectiveness in tissue regeneration.

Conflicts of Interest
The authors declare that there are no conflicts of interest regarding the publication of this paper.