Keywords

1 Introduction

A look at the world population reveals that the most common chronic problem associated with man is loss of tissue and organ damage. This can be cured by organ transplantation using tissue engineering techniques. The main problem associated with organ transplantation is shortage of suitable donors. This circumstance demands the need of a suitable scaffold wherein autologous cells can be grown under optimum conditions in vitro and subsequently transplanted back into the human body. This will obviate the need to wait for a donor and, on the other hand, will also increase the patients’ comfort and compliance. The very fundamental of tissue engineering is the requirement for a scaffold material with specific characteristics that provides a temporary artificial matrix for cell seeding [1]. One of the most important characteristics of a scaffold material is that it should provide an ideal site for cell attachment and proliferation, leading to further tissue engineering. The extracellular matrix (ECM) not only provides the physical support for cells but also regulates their proliferation and differentiation. Therefore, scaffolds need to be developed for sustaining in vitro tissue reconstruction as well as for in vivo cell-mediated tissue regeneration. Repair of tissue defects can only be possible if the cells are supplied with such an ECM substitute [2]. A scaffold is a support, either natural or artificial, that maintains tissue contour. Substances that are frequently used for scaffold preparation are natural polymers, synthetic polymers, or ceramics with adsorbed proteins or immobilized functional groups [3]. Natural polymers have drawn the attention of various researchers because of their outstanding biocompatibility properties. Biodegradable materials have gained more attention because they have the advantage of allowing new tissue to take over their load-bearing or other functions without creating any potential chronic problems associated with the presence of biostable implants [4]. The paradigm of tissue engineering consists of seeding cells on a scaffold made of either a synthetic or natural polymer blend, maturing the tissue in vitro, and finally implanting the construct at the desired site in the patient as an artificial prosthesis [5, 6]. Overall, the strategy of tissue engineering [7] generally involves the following steps:

  1. 1.

    Identify, isolate, and produce an appropriate cell source in sufficient amount

  2. 2.

    Synthesize a scaffold with the desired shape and dimension, which will subsequently be used as a cell carrier

  3. 3.

    Seed the cells uniformly onto or into the carrier and incubate for a predetermined time in a bioreactor

  4. 4.

    Implant the cell-seeded carrier in a proper animal model. Depending on the site and the structure, vascularization may be necessary

2 Scaffolds

Tissue scaffolds are synthetic bioresorbable polymers that act as functional substitutes for missing or malfunctioning human tissues and organ. The primary role of a scaffold is to provide a temporary substrate to which the transplanted cells can adhere [8]. The most important factors to be considered with respect to nutrient supply to transplanted and regenerated cells are porosity, pore size and pore structure for porous scaffolds with a large surface-area-to volume ratio, and void volume. Optimization of these parameters is desirable for attachment, growth, maximal cell seeding, ECM production, and vascularization. Pores of the same diameter are preferable in scaffolds in order to yield high surface area per volume, provided the pore size is greater than the diameter of a cell in suspension [1, 2].

Thus, scaffolds provide physical support to cells, and pores provide space for remodeling of tissue structures. The major challenge associated with the development of scaffolds is the organization of cells and tissue in a three-dimensional (3D) configuration so that molecular signals are presented in an appropriate spatial and temporal fashion in a common manner that promotes the individual cells to grow and form the desired tissue structures [9, 10].

2.1 Factors Governing the Design of Scaffolds

During design of a scaffold for real-life applications, we must pay attention in choosing the scaffold material, body acceptability, mechanical properties, surface chemistry, and porosity. The porosity, morphology, and mechanical strength of scaffolds are governed by various factors. Some of the factors governing the designing of scaffolds are discussed below.

2.1.1 Materials

During design of scaffolds for tissue engineering applications, one must emphasize the selection of suitable materials. The materials should be biocompatible and biodegradable (i.e., they can be degraded into harmless products, leaving the desired living tissue). Some of the materials used for fabricating scaffolds include natural polymers, synthetic polymers, ceramics, metals, and hybrids of these materials [11]. Metals and ceramics are not a good choice for tissue engineering applications because they are not biodegradable (except for bioceramics such as α-tricalcium phosphate and β-tricalcium phosphate) and because their processability is very limited. For these reasons, natural polymers have gained increased attention because they are biodegradable and biocompatible. One of the major drawbacks exhibited by scaffolds made up of natural polymers is their poor mechanical properties. These problems associated with natural polymers can be circumvented by using synthetic resorbable polymers such as poly(α-hydroxy esters), polyanhydrides, polyorthoesters, and polyphosphazens. Polyglycolic acid (PGA), polylactic acid (PLA), polydioxanone, and copolymers thereof are the only FDA-approved synthetic and degradable polymers.

2.1.2 Porosity and Surface Area

Scaffolds should be highly porous and the pores should be interconnected to favor tissue integration and vascularization. Scaffolds should have appropriate surface chemistry to provide the necessary initial support for the attachment and proliferation of cells, and for the retention of their differentiated functions [12]. The porosity and pore size of the scaffold play crucial roles in the regeneration of a specific tissue. For instance, scaffolds with pore size less than 150 μm have been successfully used for regeneration of skin in burn patients [13]. Angiogenesis is a requirement for some scaffold application scenarios and can be unpleasantly affected by material porosity. Pore morphology can also affect scaffold degradation kinetics and the mechanical properties of the developing tissue [14]. The degree of interconnectivity has a greater influence on osteoconduction than does the actual pore size [15]. Highly porous materials facilitate the easy diffusion of nutrients to, and waste products from, the implant. Similarly, the larger the surface area of the scaffold, the more it favors cell attachment and growth [16].

2.1.3 Mechanical Properties and Processability

The scaffold should possess good mechanical strength so that it can be used for the reconstruction of hard, load-bearing tissues such as bone and cartilage. The biomaterial should be easily processed so that it can be easily fabricated into different shapes and sizes to meet the needs of the desired tissue reconstruction. The scaffold’s architecture plays a vital role in maintaining its dimensional stability [15]. The scaffolds should have sufficient structural integrity that matches the mechanical properties of native tissue [17]. The external shape of the scaffold is also extremely important from the clinical point of view because the final anatomical shape of a regenerated tissue is basically dependent on the shape of the associated scaffold [18]. The mechanical properties of scaffold in tissue-engineering applications are of great importance due to the necessity of the structural stability to withstand stress incurred during culturing in vitro and implanting in vivo. In addition, the mechanical properties can significantly affect the specific biological functions of cells within the engineered tissue [19].

2.2 Scaffold Fabrication Techniques

Scaffolds can be fabricated by using different types of methodologies such as fiber bonding, salt leaching, gas-induced foaming, phase separation, electrospinning, solid freeform fabrication, and molecular self assembly [15, 17]. Some of the fabrication techniques are discussed below.

2.2.1 Salt Leaching

Salt leaching is one of the simplest fabrication methods for producing scaffolds with controllable porosity and pore size using various biodegradable polymers. The process for the manufacture of solid polymer–porogen constructs consists of combination of a suitable porogen with a solution of polymer in an appropriate mold. The porogen is then leached out to form porous sponges [20]. The traditional methods generally employ a solid porogen within a 3D polymer matrix to create well-defined pore size, pore structure, and total scaffold porosity. Murphy et al. [21] has introduced a modified method for producing porous, biodegradable tissue engineering scaffolds with improved pore interconnectivity. They fabricated a 3D porous scaffold by using a copolymer of 85:15 poly(lactide-co-glycolide) (PLG) via a solvent casting and particulate leaching process. They partially fused the NaCl crystals via treatment in 95% humidity to create the interconnecting pores, prior to the formation of a 3D polymer scaffold. This technique allows scaffolds for tissue engineering to be formed with minimal laboratory equipment and polymer amounts. Several recent modifications to this method demonstrate the tremendous pace of improvement in the manufacture of scaffolds with precise chemical, physical, and biological properties.

2.2.2 Phase Separation

Phase separation is one of the most popular techniques for fabricating porous scaffolds for tissue engineering applications. In this process, phase separation is induced by decreasing the temperature of a polymer solution, which results into two different phases, one having a high polymer concentration (polymer-rich phase) and one having a low polymer concentration (polymer-lean phase). The solvent from the polymer-lean phase is later removed by extraction, evaporation, or sublimation to leave behind open pores. The polymer in the polymer-rich phase solidifies into the skeleton of the polymer foam. This separation can be categorized into two types on the basis of the crystallization temperature of the solvent in the polymer solution. One type is solid–liquid phase separation and the other is liquid–liquid phase separation. When the solvent crystallization temperature is higher than the liquid–liquid phase separation temperature, then it can be separated by lowering the temperature and the process is known as solid–liquid phase separation. This process consists of crystallization of solvent, and the polymer is expelled from the solvent crystallization front. However, when the solvent crystallization temperature is much lower than the phase separation temperature, a liquid–liquid phase separation takes place on decreasing the temperature of the polymer solution. Phase separation is relatively a simple technique for the fabrication of scaffolds having highly organized structures [22].

2.2.3 Solid Freeform Fabrication

Control over internal architecture and interconnectivity is a tough task for researchers. These days, the solid freeform technique (SFF) has attracted the attention of researchers. SFF is a collective term for a group of techniques that can rapidly produce highly complex 3D physical objects using data generated by computer-aided design (CAD) systems, computer-based medical imaging modalities, digitizers, and other data makers. The technique involves in the manufacture of objects in a layer-by-layer fashion from the 3D computer design of the object [23]. Some of the advantages [24] of using SFF technique are listed below:

  • In SFF scaffolds, the 3D interconnection of the scaffold can be maintained at a wide porosity level

  • Using computerized tomography (CT) or magnetic resonance image (MRI) as the data source, scaffolds can be made with an external geometry conforming to the patients’ anatomic structure, and thus the external geometry of the scaffolds can also be designed and customized to fulfill the need of the tissue engineer to construct scaffolds for specific tissues

  • Scaffolds with a distinct material and design domain can be fabricated by using SFF techniques

Finite element analysis (FEA) and CAD can be combined with manufacturing technologies such as SFF to allow virtual design, characterization, and production of scaffold that is optimized for tissue replacement. This makes it possible to design and manufacture very complex tissue scaffold structures with functional components that are difficult to fabricate.

2.2.4 Supercritical Fluid Drying

Recently, various types of supercritical fluid processing methods have been developed for the production of microparticles, foams, fibers, and aerogels [25–27]. Fabrication of scaffolds using supercritical fluid has been reported recently by several researchers [28–30]. The rapid expansion of supercritical solutions (RESS) and the gas anti-solvent technique (GAS) are widely used for the formation of microparticles and fibers. In RESS, a supercritical solution is rapidly expanded, which leads to a rapid decrease of the polymer solubility in the supercritical fluid (SCF) and, finally, to the formation of microparticles or nanoparticles with narrow size distribution. In GAS, a polymer solution is expanded into a SCF, which acts as an anti-solvent since it is not miscible with polymer but is miscible with the organic solvent. Because the solvent is miscible with the SCF, it expands, resulting in the reduction of solvent capacity to support polymer dissolution [25]. The author’s laboratory [29, 30] have prepared chitosan scaffolds using supercritical carbon dioxide (scCO2). In the first step, the hydrogels were prepared and treated with organic solvent(s) and then placed in the chamber of a supercritical fluid reactor to undergo solvent exchange. Thereafter, the temperature and pressure were raised. Thus, the continuous flow of scCO2 through the sample replaced all the organic solvent with CO2 to obtain a porous chitosan scaffold.

2.2.5 Hydrothermal Preparation

The use of a hydrothermal bomb for preparation of a metal organic framework is a well-known technique in inorganic chemistry [31]. However, the use of a hydrothermal bomb for the preparation of scaffold is very rare. The final mixture with the appropriate composition for scaffold preparation is sealed in a PTFE-lined acid digestion bomb and heated at 40°C for 8 h under autogeneous pressure. After that, the bomb is kept at room temperature to cool the product, which is then frozen at −20°C. Finally, the product is vacuum dried to obtain the desired scaffolds [32–34] (Dutta PK et al., unpublished results).

2.2.6 Electrospinning

Another important scaffold fabrication technique is that of electrospun nanofibers. Electrospun nanofibers could be used to mimic the nanofibrous structure of the ECM in native tissue [35–37]. Electrospinning involves the ejection of a charged polymer fluid onto an oppositely charged surface. This technique is used to create polymeric fibers with diameters in the nanometer range. In electrospinning, a charge is applied to a polymer solution or melt, which is ejected toward an oppositely charged target. The body of the polymer solution or melt becomes charged, and electrostatic repulsion counteracts the surface tension so the droplets become stretched. At a critical point, a stream of liquid erupts from the surface. This point of eruption is known as the “Taylor cone.” When the applied voltage is increased beyond a threshold value, the electric forces in the droplet overcome the opposing surface tension forces and a narrow charged jet is ejected from the tip of the Taylor cone [38]. The commonly used polymers for the electrospinning method of fabrication are the aliphatic polyesters [39]. Preparation of chitosan scaffolds by electrospinning has been mentioned by various researchers [37, 40–42]. Duan et al. [43] developed a nanofibrous composite membrane of poly(lactide-co-glycolic acid) (PLGA)–chitosan/poly(vinyl alcohol) (PVA) by simultaneous electrospinning of PLGA and chitosan/PVA from two different syringes and mixing on a rotating drum to prepare a nanofibrous composite membrane, which was then crosslinked with glutaraldehyde (GA). The obtained composite membrane was cytocompatible for fibroblastic cells.

3 Chitosan as a Scaffolding Material

Among the naturally derived polymers such as gelatin, collagens, glycosaminoglycan (GAG), starch, and alginate, chitosan, a partially deacetylated derivative of chitin, is chemically similar to GAG and has many desirable properties that make it a suitable candidate for use as a tissue engineering scaffold. Fabricating the hybrid scaffolds by combining natural polymers with synthetic polymers and ceramics is the best method, because the hybrid scaffolds possess both the mechanical strength of synthetic polymers and the biodegradability of natural polymers [15].

The principal derivative of chitin, chitosan, has gained more attention as a scaffold in tissue regeneration due to: (1) the possibility of large scale production and low cost; (2) its positively charged and reactive functional groups that enable it to form complexes with anionic polymers, including proteins that help to regulate cellular activity, [44]; and (3) its antibacterial properties [45]. Apart from this, chitosan is hemocompatible and non-immunogenic, and is degradable into non-toxic oligosaccharides inside the body due to the action of lysozymes. But, chitosan lacks the tensile strength required to match that of several natural tissues [46, 47]. It has been reported that chitosan-based biomaterials do not lead to any inflammatory or allergic reaction following implantation, injection, topical application, or ingestion in the human body [48]. Chitosan possesses wound-healing properties and favors both soft and hard tissue regeneration [49, 50]. By contrast, many synthetic polymers exhibit physicochemical and mechanical properties comparable to those of the biological tissues that they are required to substitute, but are not sufficiently bioactive [51]. Polyesters such as PLA, PLGA, and polycaprolactone (PCL) can be reproduced with specific molecular weights, block structures, degradable linkages, and crosslinking modes, and have excellent mechanical strength [52, 53]. Thus, the lack of mechanical strength of chitosan scaffolds can be resolved by incorporation of inorganic materials so that the hybrid material possesses improved mechanical and biological properties. Many inorganic materials such as calcium carbonate, calcium phosphate, and silica have been studied for the preparation of chitosan–inorganic composites [54].

3.1 Structural Analysis and Characterization of Chitosan

The structure of chitosan plays an important role if it is to serve as a scaffold material for application in tissue engineering. The biocompatibility of chitosan is attributed to its chemical properties. The polysaccharide unit of chitosan resembles the structure of GAGs, which are a major component of ECM of bone and cartilage and, hence, chitosan could be an attractive candidate for an ECM substitute [55]. The cationic nature of chitosan facilitates pH-dependent electrostatic interaction with anionic GAGs, proteoglycans, and other negatively charged molecules. This property is of particular interest in tissue engineering because it makes chitosan suitable in various shapes and sizes, i.e., porous scaffolds [14], planar membranes [56], and hydrogels [57], for specific interactions with growth factors, receptors, and adhesion proteins [58]. The cell adhesion, proliferation, and differentiation properties of chitosan are attributed to its hydrophilic nature, and its compact aggregated polymeric chains are helpful in providing stability to the scaffolds in terms of size and morphology during cell culture [59].

The physical and mechanical properties of chitosan can be ameliorated by using graft copolymerization and crosslinking. Chitosan forms aldimines and ketimines with aldehydes and ketones, respectively. Upon hydrogenation with simple aldehydes, chitosan produces N-alkyl chitosan [60]. The physicochemical and biological properties [61] as well as conformational structures [62] of chitosan are very effective for biomedical applications.

3.2 Role of Molecular Weight and Degree of Deacetylation

The molecular weight (Mw) and degree of deacetylation (DD) of chitosan play pivotal roles in dictating the biological properties of chitosan scaffolds. Notably, the DD itself influences many of the properties of chitosan, namely mechanical properties, biodegradability, immunological activity, wound-healing properties, and osteogenesis enhancement [63–71]. Chitosan scaffolds with higher DD showed higher cell proliferation, lower biodegradation rate, and higher mechanical strength. One of the studies in this direction was done by Hsu et al. [71]. They investigated the role of DD and Mw of chitosan in terms of hydrophilicity, degradation, mechanical properties, and biocompatibility by seeding fibroblastic cells and immortalized rat chondrocytes (IRC) on chitosan films of differing DD and Mw. They observed that in the chitosan films having similar Mw, the higher the DD of chitosan, the smaller was the elongation of chitosan films; with similar DD, a higher Mw led to higher tensile strength. The results of degradation studies showed that for chitosan with the higher average Mw, higher DD led to a higher degradation rate. However, the result for chitosan films with the lower average Mw was found to be opposite, i.e., higher DD led to slower degradation. The average Mw has also some significant effect on degradation rate. For chitosan films having similar DDs, higher average Mw led to the higher degradation rate. The acetyl group, –NHCOCH3 of chitosan plays an important role in deciding the degradation rate. Chitosan with lower DD have more –NHCOCH3 groups and might be more amorphous and degrade faster. The results showed that with the lower average Mw, lower DD led to higher degradation rate of chitosan films. They got the inference from their study that hydrophilicity and biocompatibility of chitosan films were affected by DD. However, the rate of degradation and the mechanical properties were found to be affected by Mw.

Another study in this direction was performed by Chatelet et al. [72]. They investigated the effect of DD on the biological properties of chitosan films by culturing keratinocytes and fibroblasts on chitosan films having different DDs. They found that DD has no significant effect on the in vitro cytocompatibility of chitosan films towards keratinocytes and fibroblasts. They demonstrated that the lower the DD of chitosan, the lower was the cell adhesion on the films, and found that keratinocyte proliferation increases when the DD of chitosan films increases. They concluded from their study that the DD plays a key role in cell adhesion and proliferation, but does not change the cytocompatibility of chitosan.

4 Application of Chitosan for Regeneration of Various Types of Tissue

Chitosan scaffolds may find application in regeneration of skin tissue, liver tissue, bone and cartilage tissue, cardiac tissue, corneal tissue, and vascular tissue to mention a few [73]. A brief account of its application in various branches of tissue engineering is described in this section.

4.1 Skin Tissue

Dermal wounds are very widespread in man. The skin damage can be caused by heat, chemicals, electricity, ultraviolet, nuclear energy, or disease. In the case of wounds that extend entirely through the dermis such as full-thickness burns or deep ulcers, as a result of many skin substitutes such as xenografts, allografts, and autografts have been employed for wound healing. However, the disadvantages of these approaches include the limited availability of skin grafts in severely burned patients and the problems of disease transmission and immune response [52, 53]. One of the good alternatives to skin grafts for curing skin damage is to develop a tissue-engineered skin equivalent. Polymeric tissue scaffolds made of PLGA, collagen, and chitosan are currently being employed for tissue reconstruction [74, 75]. An ideal scaffold for skin tissue engineering should possess the characteristics of excellent biocompatibility, suitable microstructure such as 100–200 μm mean pore size and porosity above 90%, controllable biodegradability, and suitable mechanical properties [76–78]. A brief account of work done by some researchers in the field of skin tissue engineering is given in Table 1.

Table 1 Work done in the field of skin tissue engineering
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There are complications in skin tissue engineering for cases of severe burn (third degree burns). Composite skin substitutes are applied to patients suffering from extensive burns, but slow cell ingrowths and insufficient vascularization has made it unreliable for curing the people suffering from third degree burns [81]. Consequently, some researchers are leaning towards the approach of tissue engineering, which utilizes both engineering and life science disciplines, to promote skin regeneration and to sustain and recover skin function [82].

In this direction, some good results-oriented data was reported by Liu et al. [81]. The work demonstrated the fabrication and effect of controlled-release of fibroblast growth factor (bFGF) from chitosan–gelatin microspheres (CGMSs) loaded with bFGF, where human fibroblasts were cultured on the chitosan–gelatin scaffold itself. The comparative study looked at cell morphology, cell proliferation, GAG synthesis, and gene expression with respect to loading of bFGF on the chitosan–gelatin scaffold. The DNA assay result indicated that the DNA content of human fibroblasts seeded on the scaffolds with and without bFGF-CGMS increased with culture time. The cell proliferation was 1.47 times higher over a period of 2 weeks on the scaffolds with bFGF-CGMS than on scaffolds without. GAG production was also higher on scaffolds with bFGF-CGMS than on the chitosan–gelatin scaffolds. Scanning electron microscopy (SEM) observations were also in accordance with the suitability of scaffolds containing bFGF for skin tissue engineering. They indicated that human fibroblasts attached and spread well on the scaffolds with bFGF-CGMS. Overall, the results indicated that chitosan–gelatin scaffolds with bFGF have a good potential as tissue engineering scaffolds to improve skin regeneration efficacy and to promote vascularization.

Very recently, Dhandayuthapani et al. [83] reported the development of novel chitosan–gelatin blend nanofiber systems for skin tissue engineering applications. In this study, they were able to electrospin defect-free chitosan, gelatin, and chitosan–gelatin blend nanofibers with smooth morphology and diameters of 120–200 nm, 100–150 nm, and 120–220 nm, respectively, by optimizing the process and solution parameters. Chitosan and gelatin formed completely miscible blends, as evidenced from differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy measurements. The tensile strength of the chitosan–gelatin blend nanofibers (37.91 ± 4.42 MPa) was significantly higher than that of the gelatin nanofibers (7.23 ± 1.15 MPa) (p < 0.05) and comparable with that of normal human skin.

4.2 Bone and Cartilage Tissue

Scaffold serves as a temporary skeleton inserted into the sites of defective or lost bone to support and stimulate bone tissue regeneration while it gradually degrades and is replaced by new bone tissue [84–87]. Both bioactive ceramics and polymers are used as scaffolding materials. Bioceramics have chemical compositions resembling bone and they also allow osteogenesis. The major drawback of using bioceramics as scaffolding material are its brittle nature and low degradation rates. However, the biodegradation rates and mechanical properties of biopolymers can be tailored to a certain extent for specific applications. Biopolymers are particularly amenable for implantation and can be easily fabricated into desired shapes [15, 88]. Chitosan is widely used as scaffolding for the regeneration of bone tissue because of its osteocompatible and osteoconductive properties, and can enhance bone formation both in vitro and in vivo [51]. The scaffolds should possess excellent mechanical properties. The work done in the field of bone tissue engineering is outlined in Table 2.

Table 2 Work done in the field of bone tissue engineering
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As far as chitosan is concerned for cartilage tissue engineering applications, the rate of biodegradation of the scaffold (used to organize cells in vitro) plays a crucial role. The presence of non-biodegradable articles in soft tissue often causes acute foreign body reactions elicited by the body’s immune system that can result in severe inflammation and soreness around the implant site. Many studies have reported that chitin and chitosan are biodegradable polymers and that they degrade in vivo mainly through their susceptibility to enzymatic hydrolysis mediated by lysozyme, which is ubiquitous in the human body. However, this action is dependent on factors such as pH, type of chitin or chitosan, and chitosan preparation method. The use of chitosan as scaffolding material for cartilage tissue has been reported by many researchers [2, 101, 102]. Composite chondroitin-6-sulfate/dermatan sulfate/chitosan scaffolds were reported to be used for articular cartilage regeneration [103]. A brief account of work done in cartilage tissue engineering is described in Table 3.

Table 3 Work done in the field of cartilage tissue engineering
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4.3 Liver Tissue

Liver is one of the most important and complex organs, serving several essential functions in the body. A biohybrid artificial liver using isolated hepatocytes and polymer scaffolds is expected to be an alternative method of treatment for liver failure because the shortage of suitable donors and costly surgical procedure has limited the use of liver transplantation. For this approach, various scaffolds have been used and it has been shown that the scaffolding material is crucial for control of cell adhesion, growth, and tissue reconstruction [107]. For the culture of anchorage-dependent cells such as hepatocytes, scaffolds require specific interaction with ECM components, growth factor, and the cell surface receptor to ensure cell survival, differentiation, and function [108]. This must be taken into account during the design and selection of polymeric materials for liver tissue-engineering. Calcium alginate sponge has been used for hepatocyte culture [109]. However, the alginate sponge is mechanically unstable due to ion exchange of Ca2+ with monovalent cations, and it lacks the cell-adhesive signals that are necessary to preserve long-term hepatocyte function and to suppress apoptosis. Application of chitosan scaffolds in liver tissue engineering is described in Table 4.

Table 4 Application of chitosan scaffolds to liver tissue engineering
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4.4 Cardiac Tissue

Myocardial infarction is one of the major public health concerns and the leading cause of death all over the world [118]. Human myocardium lacks the possibility of regeneration after myocardial infarction [119]. This results in a progressive loss of functional myocardium and a successive enlargement of the left ventricular cavity, thus impairing cardiac function [120]. The loss of viable myocardium is irreversible and, if extensive, could result in heart failure. The only available treatment of end-stage heart failure is heart transplantation. Shortage of donor hearts and immunological rejection of the transplanted tissue has limited transplantation to certain patients only. One good alternative for the treatment of heart failure is the replacement of damaged tissue with a tissue-engineered graft generated using cells and biodegradable scaffolds [121]. An ideal scaffold for cardiac tissue engineering should be (1) highly porous with large interconnected pores, (2) hydrophilic, (3) structurally stable, (4) degradable, and (5) elastic (to enable transmission of contractile forces) [122, 123]. The main focus of cardiac tissue engineering is on the development of 3D heart muscle that can be utilized to augment the function of failing myocardium. Table 5 gives glimpses of work done on cardiac tissue engineering applications.

Table 5 Work done on cardiac tissue engineering applications
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4.5 Vascular Tissue

Vascular diseases, such as blood vessel damage, atherosclerosis, and aneurysms, remain an obstacle for clinicians because of limited donor sites and the immune response to allograft and xenograft. Tissue-engineered blood vessel is an optimal alternative for blood vessel substitution. Vascular transplantation has been commonly used for the treatment of vascular diseases. An ideal scaffold for vascular tissue engineering should use a biocompatible polymer with suitable degradation rate and biological qualities that interact favorably with blood and cells. A variety of biodegradable polymers, like poly(glycerol-sebacate), PLA or PLGA, as well as collagen and chitosan have been evaluated as scaffolds to support the regeneration of tissue-engineered vascular graft. More detail work in this direction is presented in Table 6.

Table 6 Work done in the field of vascular tissue engineering
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4.6 Corneal Tissue

In the human body, the eye is the most delicate and remarkable organ. The cornea is the transparent part of the eye that covers the iris, pupil, and anterior chamber. It has five distinct anatomical layers. From anterior to posterior, the five layers are corneal epithelium, Bowman’s layer, corneal stroma, Descemet’s membrane, and corneal endothelium. Due to some hereditary diseases, infection, or injury, the cornea becomes opacified and results in loss of vision. According to the World Health Organization, over 10 million individuals are blinded from corneal scarring. Corneal transplantation is the best way to overcome this kind of defect. In the USA, more than 40,000 corneas are transplanted successfully each year. Most patients receiving a corneal transplant suffer from corneal scarring or decompensation due to keratoconus, bullous keratopathy, corneal scars from trauma, Fuchs endothelial dystrophy, and stromal corneal dystrophies such as lattice, granular, or macular dystrophy. In addition to these, in much of the developing world, religious and cultural factors, lack of general education, and the absence of eye banking facilities prevent widespread cadaveric donation for corneal transplantation, leading to the need for an alternative to cadaveric corneal transplantation. Thus, shortage of donor corneal tissue has drawn the attention of researchers towards keratoprosthesis for the treatment of corneal blindness. The ideal keratoprosthesis would be inert and not rejected by the patient’s immune system, inexpensive, and maintain long-term clarity. In addition, it would be quick to implant, easy to examine, and allow an excellent view of the retina [131]. Due to its good optical transmittance, chitosan is widely used in corneal tissue engineering scaffolds and corneal regeneration; its transparency is above 85% at 400 nm [132]. Some more details of work in this field are given in Table 7.

Table 7 Work done in the field of corneal tissue engineering
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4.7 Nerve Tissue

The nervous system plays a vital role in maintaining body functions. Nervous tissue is composed of two main cell types: neurons, which transmit impulses, and the neuralgia, which assist propagation of nerve impulses as well as provide nutrients to the neurons. The nervous system is a complex, sophisticated system that regulates and coordinates the basic functions and activities of our body. Overall, it plays the role of headmaster in giving instructions to all parts of the body. Yet the nervous system is complex and is vulnerable to various disorders. Nervous system disorders cause many diseases such as Alzheimer’s disease, brain cancer and brain tumors, Meningitis, Parkinson’s disease etc. In recent years, researchers are devoting efforts to cure these diseases by regenerating nervous tissue. Some studies showed that chitosan promotes survival and neurite outgrowth of neural cells in vitro [137–141]. In most of the studies, a nerve guidance conduit is employed for peripheral nerve regeneration. In general, a suitable material for peripheral nerve regeneration should possess the following properties [142, 143]:

  1. 1.

    It must be biocompatible

  2. 2.

    It must allow diffusion transport of nutrients while preventing external cells from entering the conduit

  3. 3.

    The material must be degraded slowly enough to maintain a stable support structure for the entire regeneration process, but it should not remain in the body much longer than needed to prevent later compression of the nerve

  4. 4.

    The material must be able to support cell adhesion and cell spreading on its surface

Chitosans have these biological properties and can serve as the main materials for artificial nerve conduits. Neural stem cells (NSCs) have drawn the interest of researchers because of their potential for neural regeneration [144]. Recently, Scanga et al. [145] reported that chitosan had the greatest surface amine content and the lowest equilibrium water content, which probably contributed to the greater viability of neural precursor cells (NPCs or stem and progenitor cells) as observed over 3 weeks in culture. Plating intact NPC colonies revealed greater cell migration on chitosan relative to the other hydrogels. Importantly, long-term cultures on chitosan showed no significant difference in total cell counts over time, suggesting no net cell growth. Together, these findings reveal chitosan as a promising material for the delivery of adult NPC cell-based therapies. Table 8 focuses on the development of chitosan-based biomaterials for neural tissue regeneration and neural stem cell implantation.

Table 8 Chitosan for neural tissue regeneration and neural stem cell implantation
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4.8 Some Other Applications

Apart from the above use of chitosan as scaffolding material in tissue engineering, it also finds application in periodontal tissue engineering [154, 155] and disc tissue engineering [156]. The other uses of chitosan as scaffolding material may find applications in esophageal tissue, dental tissue and breast tissue for organ-specific tissue regeneration.

5 Conclusions

Among other natural polysaccharides, chitosan is a very promising and versatile biomaterial due to the ease with which this material can be manipulated to fit certain circumstances. This discovery has opened several avenues of thought concerning chitosan as a biopolymer for tissue engineering applications. The fundamental principles of tissue engineering are based on living cells, signal molecules, and scaffold. Tissue engineering involves repair of injured body parts and restores their functions by using laboratory-grown tissues, materials, and artificial implants. In this chapter, we have mainly concentrated on the selection of chitosan as scaffolding material and looked at different types of scaffold fabrication techniques as well as the factors governing the design of scaffolds for various tissue engineering applications. It is hoped that this article will act as a research guide for beginners on the use of chitosan as a scaffold for the regeneration of various types of tissues and organs. The description of methods for producing tissue engineering scaffolds may also be useful for practitioners to understand the physicochemical and biological properties of chitosan scaffolds in their real-life application.