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Tissue engineering has leaped forward in a short period thereafter and this clearly demonstrates the importance of this field. The research areas covered by this engineering science is wide and includes skin, nerves, bone, bone marrow, cartilage, blood vessels, corneal epithelia, arteries, heart valves and so on. Studies on skin and cartilage have been done the most and desirable results were first performed on these. (5,6)Other tissues such as blood vessels and myocardium are difficult to engineer upon and is hampered by various factors such as complex 3D structure, multiple cell types and high cell density. Currently, tissue engineering is slowed down by the difficulties in growing thick tissues without intrinsic capillary network (7-8). Due to this, tissues have poor availability to nutrients, oxygen and waste removal and the thickness of engineered tissues is limited to 100-200µm(9,10). There are also external issues this field faces, such as cost, governmental regulations, ethics and acceptance by public(11). The ultimate goal of tissue engineering is to create and replace portions/complete organs and tissues like bone, cartilage, heart, kidney, pancreas and liver. This implementation would in turn reduce the need for donors in organ replacement and ease development of new drugs by providing models based on human cells for development (11). The overall development of tissue engineering would not only help meet its goals but also eventually eliminate organ transplants helping the human health a long way (11).
In a methodical tissue engineering approach, living cells and biomolecules are incorporated into a pre-selected scaffold and then kept in the bioreactor. The living cells proliferate within the bioreactor, in vitro and allows engineered tissues to reach some degree of function before they can be transplanted. Ideally, cells grow and start to make tissues on the scaffold. After transplantation into the body, scaffold supports further cell function, proliferation or differentiation and allows blood vessel infiltration, while the scaffold itself biodegrades. The chosen scaffold is envisioned to degrade completely after the cells differentiate into the desired tissue and the tissue starts functioning properly. There are three main approaches to tissue engineering(12). Implantation of freshly isolated or cultured cells for treatment of diseased or damaged tissue comes under first approach. While this approach helps desired cell manipulations, complications and morbidity due to surgery, this approach is often limited by the cells being washed out from the site of injection and inability of implanted cells to maintain proper function. The second approach is stimulation of tissue regeneration, in situ by implanting scaffolds or injecting tissue-inducing substances at the site of injured tissue. This requires purification of tissue-inducing molecules and choosing appropriate delivery methods. The next approach requires implantation of functional tissues engineered from cells and scaffolds in vitro. This method requires optimization of cell ratio and density and mechanical and biochemical properties of scaffolds. Depending on cell source and method of implantation, immunoreaction(14) can occur which in itself is a vast topic.
CELLS SCAFFOLD MATERIAL BIOREACTOR Implantation into patients for repair
Templates for tissue engineering (Scaffolds)
In tissue engineering, biomaterials are natural or synthetic materials that serves as scaffold that come in direct contact with cells or tissue. Scaffolds are required for adherence and support for cell and tissue growth, in order to repair or replace organ structure. Scaffolds should be biodegradable, biocompatible, nonimmunogenic, non-toxic and also mechanically compatible with the native tissue. Further, they should also be generated quickly and can be sterilized and implanted with ease. Biomaterial selection is an very important criteria and the most important factors while choosing them are material (mechanical, electrical and optical) and biological properties. The desired material properties of chosen scaffold can be attained by altering physical and chemical properties of scaffold. This desired property is dependant on the tissue being repaired or replaced. A single biomaterial mostly cannot satisfy the conditions for engineering multiple types of tissues, as different type of tissues have different properties .
Mechanical strength or mechanical integrity is an important criteria while choosing scaffold. They should withstand handling during transplantation and also provide mechanical support during tissue regeneration.
Scaffold degradation rate is another property to be verified before being chosen. Scaffold should provide support for cells to grow and deposit extra-cellular matrix (ECM) and degrade while the living tissue replaces it. Hence, this property depends on the regeneration rate of tissue being repaired or replaced. Another point to be taken care of is degradation byproducts as they can interfere with tissue growth or cause inflammation. Hence, they should also be non toxic and removable.
Porosity of scaffold is another factor affecting the cell proliferation and differentiation. It also plays a role in nutrient and metabolite transport. Pores need to be fully interconnected for mass transfer. Higher the porosity, better the cell infiltration. Scaffold porosity allows vascular ingrowth by overcoming diffusional limitations for delivering nutrients and oxygen and removal of wastes(15). Less porous scaffolds help in mechanical load bearing and also preferred in cell differentiation.
The properties of biomaterials should also let scaffold attain various desired shapes required for appropriate tissue regeneration(16). The scaffold properties should also accomadate fill irregular defects. Rapid prototyping through computer aided design (CAD) can be used to design and manufacture customized chin implants (17,18) or three dimensional vascular microcapillary structures.
Apart from the required material properties, scaffold should support cells perform proper biological functions. Scaffolds have to be biomimetic and similar to ECM of tissue to be replaced. Scaffolds should cause minimal immune and foreign body reactions, if at all. Scaffolds should direct the cells to grow into 3D tissues and also allow cell adhesion, survival, proliferation, differentiation, migration and organization, both in vitro and in vivo. The cells grown on scaffolds shouldn't have any morphological differences and also ECM should be produced by cells.
Scaffolds for tissue engineering are classified as (1) fibrous (2) porous and (3) hydrogel. Each of these scaffolds are obtained by different approaches and they are used as per as the demand of the cells. Scaffolds are further categorised based on their chemical composition as (1) natural and (2)synthetic.
Fibrous biomaterials are being used for numerous tissue engineering applications. Fiber structure and diameter can influence the cells growing on these scaffolds. Though difficult to control and characterize the regulation of organization and cell activity of cells on these scaffolds, it is observed that pore size is a considerable factor (19). Nanofibrous scaffolds are obtained via electrospinning, self-assembly and phase separation.
Porous scaffolds have large surface area for cell adhesion. Porous scaffolds have been successfully used to grow cells into functional tissues (20). Porosity of scaffolds and inter-connectivity of pores within these scaffolds are important for host-cell infiltration, cell proliferation and differentiation (21). These type of scaffolds are prepared by using freeze-drying, particulate leaching, phase separation and solid freefoam separation (10,15,21).
Hydrogels are gels made of polymer chains that swell in aqueous solutions. They are fabricated by forming a covalently bound polymer network and the crosslinking can be induced thermally, chemically or photochemically. Hydrogels are biocompatible as they have large water content due to swelling.
Cells are the prime ingredients of an engineered tissue. Cells have many different functions, depending on the specific system. Cells are chosen for tissue engineering depending on their (a) expandability, to be available in sufficient amounts (b) ability to survive and maintain function for a required period of time (3) and (c) compatibility, to avoid immune reactions(2).
The source for cells required for tissue engineering, falls under 3 types (a) autologous (b) allogenic or (c) xenogenic. Autologous cells are obtained from the patient receiving implantation and the advantage is decreased chances for rejection. However, disadvantages are that these cells are not available or in less amounts for maintaining cell lines and the patient has to undergo additional pain and perhaps face donor site infection.
Allogenic cells are isolated from a donor of the same species. Compared to autologous, allogenic cells have higher chances for immunological rejections in patients. However, allogenic cells can be alternative cell source when dealing with ill, elderly or patients who have genetic diseases.
Xenogenic cells are taken from a donor of different species. Animal sources should be reliable and abundant source of cells should be available for tissue engineering. However, these cell sources are controversial because animal pathogens could get transmitted to humans.
Chitosan is a natural polymer obtained from renewable sources such as shell of shellfish and wastes of seafood industry. The history of chitosan was first recorded in 1859 when Rouget explained about deacetylated form of chitosan(23). Chitin is the source material for chitin and is the next most abundant organic material after cellulose, produced via biosynthesis. Chitin is seen in exoskeleton of animals especially in crustacean, molluscs and insects. It is also the principle fibrillar polymer in cell walls of certain fungi(24).
Chitosan is a linear polysaccharide, made of glucosamine and N-acetyl glucosamine units linked by β(1-4) glycosidic bonds. The content of glucosamine is termed as degree of deacetylation(DD). The molecular weight ranges from 300 to 1000kd, with a DD from 30% to 95%, depending on the source and preparation methods(25). In crystalline form, chitosan is normally insoluable in aqueous solutions above pH 7. Chitosan is soluable in dilute acids of pH<6.0 because the protonated free amino groups on glucosamine facilitates solubility of the molecule(26). Generally, chitosan has three types of reactive functional groups, an amino group as well as both primary and secondary hydroxyl groups at the C(2), C(3) and C(6) positions respectively. These groups allows modification of chitosan for producing various scaffolds for tissue engineering applications. The chemical nature of chitosan provides many possibilities for covalent and ionic modifications which allows extensive adjustments of mechanical and biological properties, as required for the scaffold. The presence of NH2 groups is the reason for greater potential of chitosan over chitin in different applications. Chitosan is the only pseudonatural cationic polymer and therefore henca a novel functional material(27,28).
Diagram of chitosan
Chitosan is commonly prepared by deacetylation of chitin using 40-50% aqueous alkali solution at 100-1600C, resulting in chitosan having DD of 95%. For complete deacetylation, alkaline treatment can be repeated. β- chitin gets deacetylated at a lower temperature than α-chitin, around 800C and also provides nearly colourless products via suppression of colouration processes. Infrared spectroscopy is relatively a quick method for qualitative evaluation of DD via estimation of absorption ratios. The determaination of DD can be evaluated via various methods and has been well explained in reference-(29). As mentioned earlier, chitosan is obtained mainly via deacetylation of chitin. Isolation of chitin is affected by its source. Usually, the obtained raw material is crushed, washed with detergent or water and cut into small pieces. It has been demonstrated that β-chitin is more susceptible to deacetylation than α-chitin(30). Chitin is also thermally stable than chitosan.
Chitosan has a wide range of applications. Some of the potential applications of this biopolymer are in the areas of medicine, drug delivery, water treatment, membranes, hydrogels, adhesives, antioxidants, biosensors, and food packaging.
Chitosan has antioxidant properties. Two tpes of chitosan, B or C, have been prepared by alkaline N-deacetylation of crude chitin B or C for different durations of 60, 90 and 120 min(31). An another research(32) reported antioxidant property of chitosan with different molecular weights(30,90 and 120 kDa) in salmon.
Biological adhesives- introduction of azide and lactose moieties into chitosan provides better water solubility at neutral pH. This has been used as biological adhesives for soft tissues. It is photo-cross-linkable by UV irradiation, thereby producing an insoluable hydrogel within 60s. The obtained material has great potential as a biological adhesive in medical use(33). Another research performed application of dilute chitosan solutions gelled by melB tyrosinasecatalyzed reaction with 3,4-dihydroxyphenethylamine (dopamine). The obtained adhesive property is related to the increased viscosity of modified chitosan. Adhesive strength was found to increase on increasing molecular weight of chitosan samples used and their amino group concentration. Hence, these reactions of dopamine can be utilized to provide water-resistant adhesive properties to dilute chitosan solution(34).
Biofilms- Biodegradable flexible composite films have been synthesized from corn starch and chitosan(35). These films are biofilms with homogenous matrix, stable structure, good water barrier and mechanical properties. Chitosan and poly(Lactic acid) (PLA) has been used for novel biodegradable films by solution mixing and film casting(36). These films exhibit s interesting qualities in bioactive packaging due to antimicrobial activity of chitosan and excellent mechanical properties of PLA. Though these films offer advantage in preventing surface growth of mycotoxinogen strains, their physiochemical properties limit their further usage as packaging material.
Coating, biosensors and surface conditioners- Electrophoretic deposition(EPD) has been used for fabrication of nanocomposite silica-chitosan coatings(37). Good binding and film poring property of chitosan helps in formation of relatively thick coating of uptp 100µm. This process occurs at room temperature and therefore, problems due to sintering at high temperature can be avoided.
Chitosan is used as coating material for fruits. Studies on effects of edible chitosan coating on quality and shelf life of mango provided positive results (38). Chitosan is used in electrochemistry and biosensors. Natural oligosaccharide-derived ionic fluids have been made from 1-ethy-3-methylimidazolium hydroxide and carboxymethylated chitosan by acid-base neutralization reaction(39) and results showed that ionic fluids with low molecular weight have good ionic conductivity and thermal stability.
Antibacterial properties and food packaging- Chitosan is antimicrobial against a wide range of target organisms. Activity varies considerably depending on the type of chitosan being used. Though literature records vary, generally, yeasts and moulds are the most sensitive group, followed by gram-positive bacteria and finally gram negative bacteria(60). New chitosan derivatives with much higher antimicrobial activity have been synthesised(40). The antimicrobial activities of acetyl, chloroacetyl and benzoyl thiourea derivatives of chitosan against four bacterial species were also studied. The results show that antimicrobial property of these derivatives are much better than that of parent chitosan. Further, the antifungal activity of chloroacetyl thiourea derivatives of chitosan are significantly higher than those of acetyl and benzoyl thiourea derivatives. In another study, the antimicrobial effect of a commercial chitosan with high deacetylation degree (94%) and low molecular weight on different psychotrophic spoilage organisms and food pathogens has been quantified. In this study, influence of food components such as starch, whey, protein and oil on antimicrobial effect of chitosan was studied. The chitosan coating for controlling fruit decay have been studied. The results show that Gram-negative bacteria are very sensitive to the applied chitosan while Gram-positive bacteria is highly variable(41).
Water treatment- Use of chitosan as an adsorbent has attained focus recently in water treatment industries due to its high content of amino and hydroxyl functional groups(42). Chitosan shows high potential for adsorption of dyes, metal ions and proteins along with the properties mentioned earlier. Therefore, it could be a good candidate for removing pollutants from water and wastewater. Since chitosan forms gel below pH 5.5, use of chitosan as adsorbent for dye removal will be limited. Researches are beginning to show cross-linked chitosan being prepared and stabilised in acid medium.
As tissue supporting material- Chitosan-based scaffolds possess certain special properties for use in tissue engineering. The application of chitosan and its derivatives for artificial organs have been well recorded.
Healing of a skin wound is complicated and includes a wide range of cellular, molecular, physiological and biological processes. Wound dressing is the important step in wound management. However, wound repairs in case of acute, chronic, more extensive wounds or skin loss would be impossible unless some skin substitutes are used. The aim for skin tissue engineering is to rapidly produce a construct that offers complete regeneration of functional skin. This should allow many normal functions like barrier formation, defence against UV irradiation, thermoregulation and aesthetic functions(43). In past decades, many skin substitutes have been used such as xenograft, allografts and autografts for wound healing. However, there were limitations observed due to antigenicity or limitations of donor sites, and therefore the skin substitutes cannot accomplish purpose of skin recovery and not used widely(44). The main role of skin substitutes is to promote wound healing by stimulating the host to produce various cytokines which play very big role in preventing inflammation, dehydration and promotes tissue granulation in wound healing.
Chitosan as tissue substitute for skin has various advantages for wound healing like hemostasis, accelerating tissue regeneration and stimulates fibroblast synthesis of collagen(45). This has resulted in many studies using chitosan as skin substitute. Chitosan in the form of chitosan-cotton accelerate woubd healingby promoting infiltration of polymorphonuclear cells (PMN) cells at the wound sites. This is a very important requirement in rapid wound healing(46). It has been demonstrated in another study that incorporation of chitosan to basic fibroblast growth factor accelerated rate of healing(47). The deacetylated chitosan were found to be biologically more active than chitin and less deacetylated chitosan in another study(48). All these results are closely related to the electrostatic interaction of chitosan with anionic GAG, depending on the pH of the environment and DD of chitosan. The GAG is widely distributed within the body and is well known to bind and modulate cytokines and other growth factors. Further experiments have been carried out to achieve rapid wound healing by combining chitosan with other material. Polyelectrolyte complex (PEC) membranes were made by combining chitosan with alginate which are biodegradable. These biodegradable membranes express greater stability to pH changes and therefore very effective as controlled release membranes than individual chitosan or alginate(49). PEC membranes were used to demonstrate accelerated wound healing of incisional wounds in rat model. Another study used fabricated porous chitosan/collagen scaffold by crosslinking them with glutaraldehyde and freeze drying to increase biostability and biocompatibility(50). This study also reported the reduced cytotoxicity of glutaraldehyde and suggested it to be due to presence of chitosan.
Chitosan is the most potent choice as scaffold for skin replacement due to its physio-chemical and biological properties. Due to limitation of donor sites thestudy for biomaterials as skin replacements have high potential and the role of chitosan as a skin substitute would further attain focus.
Bone tissue engineering utilises the biodegradable substitute as a temporary skelton inserted into the defective sites of skeleton or lost bone sites for support and simulation of bone tissue regeneration. Meanwhile these substitues gradually degrade and becomes replaced by new bone tissues. While treating for vertebral fracture or related conditions, the selected materials as bone cements must posses- proper injectibility, a rapid setting time, bioactivity, a rapid setting time, appropriate stiffnessand radio-pacity(51). Polymers and bioactive ceramics have been developed and analysed for use as tissue engineering scaffolds. These bioactive ceramics are chemically similar to natural bone and allows osteogenesis to occur and is able to provide a bony contact or bonds with host bones(52). However disadvantages of these bioceramics are- low biodegradability and brittleness and limits the use of bioceramics as bone substitutes.
Various polymers via natural and syntetic sources have been researched upon as a bone substitute. Chitosan has been used extensively as a substitute in bone tissue engineering due to it's ability to promote growth and mineral rich matrix deposition via osteoblasts in culture. Chitosan reduces local inflammation as its biocompatible and also has advantages like biodegradability and ablity to be molded into porous structure helping osteoconduction(53). Chitosan- calcium phosphate composites (CP) have been studied extensively for this purpose in bone engineering. Beta-tricalcium phosphate (β-TCP) and hydroxyapatite (HA) of CP bioceramics have been well demonstrated for bone repair and regeneration due to their similarity in chemical composition with inorganic components of the bone. A study using CP-bioceramics embedded with chitosan sponges have demonstrated to enhance mechanical property of ceramic phase via matrix reinforcement while preserving osteoblast phenotype(54). Another study using gentamycin conjugated with macroporous chitosan scaffolds reinforced with β-TCP showed MG63 osteoblast cells to be attached on the surface of scaffolds , supporting their proliferation and migration into the pore walls. Good mechanical and superior biocompatible properties of sintered HA which is similar to Calcium-HA, makes it a good substitute for bone and teeth implant. The invivo effect of HA-chitosan materials through its applications on tibia surface after peristoneum removal showed new bone formation after one week and continued during a 20-week follow up, supporting this material for further clinical studies as bone filling material (55). Phase-separation technique to fabricate biomimetic HA/chitosan-gelatin network composites in form of 3D-porous scaffolds have shown improved adhesion, proliferation and adhesion of rat calvaria osteoblasts on these scaffolds which are highly porous in nature(54).
Chitosan has also been experimented as an adjuvant to improve injectability of the cement while keeping physiochemical properties suitable for surgical application, setting time convenient for surgery, mechanical properties suited for operation and low disintegration of the cement in biological fluids. Octo-CP obtained from Calcium phosphate cements (CPC) along with chitosan demonstrated the improvement in injectability and strength of cement(56).
Lack of or few donor organs for orthotopic liver transplantation worldwide have increased the focus on requirement for new therapies for acute and chronic liver diseases(57). Bioartificial liver (BAL) is a emerging field for the application of tissue engineering to treat fulminant hepatic failure (FHF). The prime goal is to develop a BAL device in which plasma of patient is circulated extracorporeally through a bioreactor that houses metabolically active liver cells. There are certain parameters to be verified for BAL devices- proper choice of cell sources such as primary hepatocytes, hepatic cell lines and liver stem cells, The primary hepatocyte of these cells represent the most direct approach to BAL devices. The current researches primarily focuses on developing BAL devices in which hepatocytes are optimally maintained so that they carry out many activities as possible(58). The hepatocytes are anchorage dependant and are highly sensitive to ECM milieu for their viability and differentiated functions. Therefore, BAL devices requires suitable ECM and porous scaffolds with large surface to volume ratio are relevant as they are involved in cell attachment.
Chitosan as a biomaterial for liver tissue engineering is very promising due to its various properties. Chitosan as scaffold for hepatocyte culture is possible because its structure is very similar to GAGs, which are components of liver ECM(59). It has been demonstrated by Chupa et.al that chitosan and chitosan complexes with GAGs had potential for the design of new biologically active biomaterials which can modulate activities of vascular endothelial and smooth muscle cells in vitro and in vivo. The micro-structure of porous scaffold provided large surface area for cells to adhere and facilitate nutrient and oxygen transportation(59). Chitosan/collagen matrix (CCM) was prepared by crosslinking agent EDC in NHS buffer system(61). The EDC croos-linked CCM showed moderate mechanical strength , good hepatocyte compatibility and high blood compatibility.
Stem cells (http://stemcells.nih.gov/info/basics/basics1.asp)
As refered above, promise of cellular therapy lies in repair of damaged organs and tissues in vivo and also in generating tissue constructs in vitro for the required transplantation. However, the medical advances in this field is restricted by the lack of available donar cell sources. Stem cells have thus gained high importance in regards with this restriction. Stem cells are unique as they have the special ability to develop into different cell types in the body during early life and growth. Further, stem cells are also seen in many tissues as a part of internal repair system, dividing essentially without limit to replenish other cells till the person dies. When a stem cell divides, each new cell has the potential to either remain as a stem cell or differentiate to become a cell type with specific functions like brain cell or muscle cell. The success of cellular treatments is dependant on creating reserves of undifferentiated stem cells and later leading them to differentiate to a selection of specialised cells in an efficient and measured level. In recent years, different type of biomaterials have been used in stem cell cultures, mainly to provide conductive microenvironment for their growth and differentiation and eventually provide the stem cell niche. Tissue engineering techniques are utilised to incorporate growth factors and morphogenetic factors, which would help induce lineage commitment of stem cells into cultures with scaffolding materials (both synthetic and natural).
Natural biomaterials such as collagen, hydroxyapatite etc. are used as scaffolds for stem cells as they provide suitable microenvironment, are biodegradable , biocompatible and have similar properties to that of native tissue. However, unlike synthetic polymers, these natural biomaterials have limited control over physiochemical properties, difficulty in modifying degradation rates and problems with purification when isolating these materials from various sources(62). Chitosan, obtained from natural sources, commercially is however very well suited for stem cell growth and proliferation as it overcomes the disadvantages and are well-characterized and have reproducible, well-controlled properties(63).
Owing to the features of chitosan, many works have been carried out on stem cells using chitosan as the scaffold. A study combined chitosan with coralline, exoskeleton of marine species or coral, as a composite scaffold to study MSC-osteogenesis, since coral is composed of calcium carbonate, which is a component found in bone. In another study, chitosan was modified to be thermo-responsive, by incorporating hydroxybutyl groups onto polymer backbone which undergoes sol-gel transistion when exposed to 370C(65). This modified chitosan was used in encapsulation and in vitro culture of hMSGCs to finally develop an injectable cell biomaterial composite for degenerative disk diseases. MSCs were efficiently encapsulated within these chitosan gels with minimal cell toxicity and gene expression of bone specific markers were successfully shown(65). Another study (66) utilized Carboxy methyl chitosan (CMCS), which is chitosan derivatized with carboxyl groups for promoting osteoblast and stem cell differentiation. CMCS is more bioactive than chitosan and is found to chelate calcium from a mineralizing solution containing calcium and phosphate. CMCS was used as non-coated and coated with hydroxyapaptite(HAP)-coated scaffolds and supported attachment, proliferation and differentiation of the osteoblasts and directed stem cell differentiation to osteoblasts. The HAP coated CMCS enhanced these effects on the osteoblasts and stem cells(66). Neural stem cells (NSCs), canditates for regeneration of spinal cords and peripheral nerves, was derived from fetal rat cortices and cultured on chitosan to evaluate the cell affinity on chitosan(67). The NSCs grew and proliferated well on chitosan films and most of them differentiated to neuron-like cells after 4 days of culture.