Polysaccharides As Cell Carriers For Tissue Engineering .

S32Vol. 63Bačáková et al.CelluloseCellulose is a structural polysaccharideconsisting of a linear chain of several hundred to over tenthousand β(1 4) linked D-glucose units. It wasdiscovered and isolated from green plants by Payen(1838) (for a review, see O'Sullivan 1997). It is the mostabundant biopolymer on Earth, synthesized by herbswoody plants, many forms of algae, fungi and somespecies of bacteria, namely Acetobacter xylinum.Bacterial cellulose is identical to plant cellulose inchemical structure, but it can be produced luloses, and does not require intensivepurification processes. In addition, it is remarkable for itsmechanical strength and biocompatibility, and it hastherefore often been applied in tissue engineering (for areview, see Helenius et al. 2006, Petersen and Gatenholm2011). In addition to vascular tissue engineering, which isdiscussed below, cellulose has also been used forengineering a variety of other tissues, such as bone (Shi etal. 2012a), cartilage (Andersson et al. 2010), skin(Kingkaew et al. 2010), skeletal muscle (Dugan et al.2013), cardiac muscle (Entcheva et al. 2004) and heartvalves (Mohammadi 2011). Cellulose has also been usedfor constructing nanofibrous three-dimensional carriersfor liver cells (Bhattacharya et al. 2012), forencapsulating and immunoisolating Langerhans islets(Risbud et al. 2003), for creating tubes for regeneratingdamaged peripheral nerves (Kowalska-Ludwicka et al.2013), and also for creating carriers for delivery anddifferentiation of mesenchymal stem cells (Gu et al.2010) and neural stem cells (Mothe et al. 2013) for neuraltissue regeneration. Even lignin, a macromoleculecrosslinking different plant polysaccharides includingcellulose, induced differentiation of embryonic stem cellsinto neuroectodermal cells, namely ocular cells andneural cells (Inoue et al. 2013). Cellulose acetate in theform of porous membranes has been applied forconstructing a bioartificial renal tubule system (Sato et al.2005), and in the form of electrospun porousmicrofibrous three-dimensional scaffolds, for potentialurinary bladder reconstruction (Han and Gouma 2006).Microporous scaffolds made of bacterial cellulose andseeded with human urine-derived stem cells supported theformation of a multilayered urothelium, and thus theseconstructs hold promise for creating tissue-engineeredurinary conduits for urinary reconstruction and diversion(Bodin et al. 2010).Chitin and amine. It is the main component of the cellwalls of fungi, the exoskeletons of arthropods such asinsects and crustaceans (e.g. crabs, lobsters and shrimps),the radulas of mollusks, and the beaks and internal shellsof cephalopods, including squid and octopus. Chitin itselfhas been used only relatively rarely for tissueengineering. For example, composite scaffolds containingchitin, pectin and CaCO3 nanoparticles were tested withfibroblasts for potential use in tissue engineeringand controlled drug delivery (Kumar et al. esponsivehydrogelscaffoldspromotedodontogenic differentiation of human dental pulp cells,and thus they proved to be promising materials for dentinregeneration (Park et al. 2013).However, an important derivative of chitin, usedfor engineering a wide range of tissues and organs, ischitosan. To date, about 1400 papers concerning chitosanand tissue engineering can be found in the PubMeddatabase. Chitosan is a linear polysaccharide composedof randomly distributed β-(1-4)-linked D-glucosamine(deacetylated unit) and N-acetyl-D-glucosamine.Chitosan, particularly in the form of nanofibrousscaffolds, in combination with other polymers, ceramic orcarbon nanoparticles, growth factors and other bioactivemolecules, has been applied for reconstructing almost alltissues, such as bone (Frohbergh et al. 2012), bloodvessels (Du et al. 2012), heart valves (Hong et al. 2009),myocardium (Hussain et al. 2013), liver (Wang et al.2005, Mareková et al. 2013), pancreatic islets (Deng etal. 2011), kidney (Gao et al. 2012), urinary bladder(Drewa et al. 2008), skin (Lin et al. 2013) or the centraland peripheral nervous system (Shokrgozar et al. 2011,Hu et al. 2013).PectinsPectins are a family of complex polysaccharidesthat contain 1,4-linked α-D-galactosyluronic acidresidues. They are present in most primary cell walls andin the non-woody parts of terrestrial plants. Due to theirsimple and cytocompatible gelling mechanism, pectinshave recently been exploited for various biomedicalapplications, including drug and gene delivery, woundhealing and tissue engineering (for a review, see Munarinet al. 2012). Nanostructured pectin films deposited ontitanium, glass and polystyrene substrates promoted theadhesion, growth and osteogenic differentiation of murine

2014preosteoblastic MC3T3-E1 cells and primary ratosteoblasts (Kokkonen et al. 2007, 2008, 2012). Pectingels, particularly those functionalized with RGDcontaining oligopeptides, which serve as ligands forintegrin adhesion receptors, have been proposed asinjectable carriers for osteoblasts for bone tissueregeneration, due to their ability to keep immobilizedcells viable and differentiating (Munarin et al. 2011). Forpotential bone tissue engineering, pectins have also beenused in combination with other natural and syntheticpolymers, such as chitosan (Coimbra et al. 2011) orpolylactide (Liu et al. 2004).AlginateAlginate, also called alginic acid, is an anionicpolysaccharide distributed widely in the cell walls ofbrown algae, where through binding with water it forms aviscous gum. Alginate is a linear copolymer withhomopolymeric blocks of (1-4)-linked β-D-mannuronateand its C-5 epimer α-L-guluronate residues.Alginate has been used for engineering andregeneration of almost all tissues in the human organism.It has been widely applied in the form of injectablehydrogels, e.g. for encapsulation and delivery ofLangerhans islets (Johnson et al. 2011), ovarian follicles(Tagler et al. 2012) and stem cells for neural tissueengineering (Banerjee et al. 2009), bone tissueengineering (Zhou and Xu 2011) and skeletal muscleregeneration (Liu et al. 2013). Alginate hydrogel-basedscaffolds were also tested for engineering of cartilage(Wan et al. 2011) and intervertebral discs (Renani et al.2012). In combination with hydroxyapatite, alginatehydrogels were applied for regenerating theosteochondral interface (Khanarian et al. 2012). Alginatehydrogels incorporated with poly(ethylene glycol)molecules and antibodies served for capture ofendothelial progenitor cells from human blood (Hatch etal. 2011).In the form of porous scaffolds, alginate hasbeen used for creating a capillary bed in newlyreconstructed tissues (Yamamoto et al. 2010), and in theform of electrospun nanofibrous scaffolds, forconstructing vascular replacements containing endothelialcells and smooth muscle cells (SMC) (Hajiali et al.2011), and for skin tissue engineering (Jeong et al. 2012).Alginate was also a component of scaffolds for heartvalve engineering (Hockaday et al. 2012), and incombination with gold nanowires, for cardiac tissueengineering (Dvir et al. 2011).Polysaccharides in Tissue EngineeringS33AgarAgar is a polysaccharide in red algae, serving asthe primary structural support for their cell walls. Agar isa mixture of two components: the linear polysaccharideagarose, and a heterogeneous mixture of smallermolecules called agaropectin. Agarose, the predominantcomponent of agar, is a linear polymer, consisting of arepeating monomeric unit of agarobiose. Agarobiose is adisaccharide containing D-galactose and 3,6-anhydro-Lgalactopyranose. Agaropectin is a heterogeneous mixtureof smaller molecules that occur in lesser amounts,and is made up of alternating units of D-galactose andL-galactose, heavily modified with acidic side-groups,such as sulfate and pyruvate (for a review, see McHugh1987).Agar is well-known as growth substrate forbacteria and other microbes, though it has also been usedfor cultivating cells of higher plants in order to developeffective methods for large-scale production of artificialseeds (Al-Hajry et al. 1999). Another well-knownapplication is in testing the migratory potential andinvasiveness of various cell types, including cells fortissue engineering (Ramaswamy et al. 2012), stem cellsfor cell therapies (Sabapathy et al. 2012) and foridentifying cancer cells (for a review, see Discher et al.2005).Other current applications of agar-basedmaterials are in testing the cytotoxicity of various drugs,chemicals and also artificial materials for tissueengineering (Korkmaz et al. 2007, Verma et al. 2009),and in testing the effects of mechanical loading on cellbehavior (Shelton et al. 2003). Foils made of soy agarand collagen gel served as substrates for the deposition ofcells by thermal inkjet printing, i.e. an advancedtechnology developed for tissue engineering (Xu et al.2005). Similarly as alginate, agar can be used for cellencapsulation in tissue engineering applications. Due toits chondrogenic potential, agar was selected to entrapchondrocytes within poly-L-lactide scaffolds (Gong et al.2007). In combination with gelatin, agar has been usedfor engineering nucleus pulposus (Strange and Oyen2012), and in combination with hydroxyapatite andhyaluronic acid, for bone tissue engineering (Wagner etal. 2007). Composite membranes made of agar and type Icollagen proved to be promising wound dressings forhealing burns or ulcers (Bao et al. 2008). Agar gel,attached to the luminal surface of a microporous acrylatetube, supported the formation of in vivo tissue engineeredautologous vascular prosthetic tissues, called “biotubes”

S34Bačáková et al.(Nakayama and Tsujinaka 2014). Agarose was used forneural tissue engineering (Bellamkonda et al. 1995) andas a vocal fold substitute (Choo et al. 2010). Togetherwith hyaluronan and water, agar was used for creating anartificial vitreous humor (Kummer et al. 2007).Dextran and pullulanDextran is a branched glucan (i.e. apolysaccharide composed of many glucose units)containing chains of varying lengths (from 3 to2000 kDa). Dextran is synthesized from sucrose bycertain lactic-acid bacteria (Leuconostoc mesenteroides,Streptococcus mutans, Lactobacillus brevis). In medicalpractice, it is used particularly for its antithromboticproperties. For tissue engineering applications, it has beenused in combination with pullulan, anotherpolysaccharide consisting of maltotriose units andproduced from starch by the fungus Aureobasidiumpullulans. Dextran and pullulan have been appliedparticularly in vascular and bone tissue engineering. Inthe form of electrospun nanofibrous scaffolds, dextranand pullulan promoted the development of a stableconfluent monolayer of endothelial cells, and also thetransition of vascular SMC from proliferative phenotypeto quiescent contractile phenotype in vitro (Shi et al.2012b). In the form of porous scaffolds, dextran andpullulan supported the viability, proliferation,differentiation and function of human endothelialprogenitor cells isolated from cord blood (Lavergneet al. 2012). In bone tissue engineering, porousdextran/pullulan scaffolds, pure or supplemented withnanocrystalline hydroxyapatite, induced osteogenic celldifferentiation in vitro and the formation of mineralizedbone tissue in vivo (Fricain et al. 2013).Gellan and xanthanGellan and xanthan have been shown to beexcellent carriers for growth factors and matrices forseveral tissue engineering applications. These materialsare able to gelify in situ within seconds, to retain largequantities of water, and thus to provide a similarenvironment to that of natural ECM (Khang et al. 2012,Dyondi et al. 2013). In addition, their mechanicalproperties can be fine-tuned to mimic the replaced naturaltissues. Gellan/xanthan gels loaded with chitosannanoparticles, basic fibroblast growth factor (bFGF), andbone morphogenetic protein 7 (BMP-7) promoted thedifferentiation of human fetal osteoblasts. At the sametime, these gels showed antibacterial effects againstVol. 63Pseudomonas aeruginosa, Staphylococcus aureus andStaphylococcus epidermidis, i.e. major pathogens causingthe failure of bone implants (Dyondi et al. 2013). Gellangum was loaded with alkaline phosphatase in order tosupport enzymatic mineralization of scaffolds for bonetissue engineering (Douglas et al. 2012). Gellan gum alsosupported the viability of encapsulated cells of thenucleus pulposus, and thus it holds promise in theconstruction of intervertebral disc replacements (Khanget al. 2012). A xanthan gum derivative was successfullyused for encapsulation and delivery of chondrocytes forpotential cartilage tissue engineering (Mendes et )arelongunbranched polysaccharides consisting of a repeatingdisaccharide unit. The general disaccharide unit consistsof an N-acetyl-hexosamine and a hexose or hexuronicacid, either or both of which may be sulfated. The onlyGAG that is exclusively non-sulfated is hyaluronan, andsulfated GAGs include heparin, chondroitin sulfate,dermatan sulfate, keratan sulfate and heparan sulfate.In addition to sulfating, the members of theglycosaminoglycan family vary in the type ofhexosamine, hexose or hexuronic acid unit that theycontain (e.g. glucuronic acid, iduronic acid, galactose,galactosamine, glucosamine). They also vary in thegeometry of the glycosidic linkage (for a review, seeCollins and Birkinshaw 2013, Schnabelrauch et al. 2013).Hyaluronan (also called hyaluronic acid orhyaluronate) is an anionic, non-sulfated GAG, which isan important component of ECM and synovial fluid. Ashyaluronan is an important component of cartilage, it hasbeen widely used for cartilage tissue engineering. Todate, there are almost 3000 papers in the PubMeddatabase dealing with the role of hyaluronan in cartilagetissue engineering, reconstruction and regeneration.Hyaluronan has been used mainly for engineeringarticular cartilage (Erickson et al. 2012, Kim et al. 2013),but also for meniscus reconstruction (Zellner et al. 2010),osteochondral defects (Filová et al. 2008, Galperin et al.2013), tracheal defects (Hong et al. 2012) andintervertebral discs (Park et al. 2012). For these purposes,hyaluronan has been applied in the form of a hydrogelwith encapsulated chondrocytes (Filová et al. 2008, Parket al. 2012) or mesenchymal stem cells (Zellner et al.2010, Erickson et al. 2012, Kim et al. 2013). It is alsopossible to create hyaluronan fibers of submicron- and

2014nano-size by an electrospinning technique (Kim et al.2013) or hyaluronan-based porous scaffolds (Yan et al.2013). For constructing these scaffolds, hyaluronan hasoften been combined with other natural and syntheticpolymers, such as collagen I and fibrin (Filová et al.2008, Hong et al. 2012), silk (Park et al. 2012), chitosan(Chen et al. 2013) or methacrylates (Galperin et al.2013), and for bone tissue engineering, also with amineral component, e.g. calcium phosphates (Chen et al.2013, Galperin et al. 2013). Hyaluronan is also a majorcomponent of skin, and it has therefore been applied indermal tissue engineering (Mineo et al. 2013, Yan et al.2013). Other tissue engineering applications ofhyaluronan include reconstruction of heart valves (Duanet al. 2013), myocardial tissue (Dahlmann et al. 2013),skeletal muscle tissue (Desiderio et al. 2013), and alsosmall-caliber arteries (diameter 2-4 mm). Hyaluronanbased biodegradable vascular grafts implanted into pigssupported spontaneous development of a neoarterysegment composed of mature smooth muscle cells(SMC), collagen, and elastin fibers organized in layersand completely covered by endothelial cells on theluminal surface (Zavan et al. 2008). Hyaluronan has alsobeen reported to play an important role in the onset anddevelopment of atherosclerosis and other vasculardiseases, namely by its stimulatory effects on migrationand proliferation of vascular SMC (Vigetti et al. 2008).This behavior of SMC can also lead to stenosis andfailure of vascular replacements. However, these negativeeffects are typical for hyaluronan with low molecularweight (less than 500 kDa), while native high molecularweight hyaluronan (more than 1000 kDa) has beenreported to have antimigratory and antiproliferativeeffects on SMC (Kothapalli et al. 2010). Antiproliferativeactivity of hyaluronan can also be induced byO-sulfonation of hyaluronan molecules (Garg et al.1999).The presence of sulfur in the molecules of GAGsis generally associated with antiproliferative effects onSMC. In adult healthy vascular wall, sulfated GAGs keepSMC in quiescent differentiated contractile phenotype(Glukhova and Koteliansky 1995). These effects can alsobe exploited in vascular tissue engineering. For example,SMC in hybrid vascular grafts, constructed on knittedDacron grafts using endothelial cells, SMC, fibroblastsand artificial ECM consisting of type I collagen anddermatan sulfate, were predominantly of contractilephenotype after 12-week-implantation into dogs(Ishibashi and Matsuda 1994). Sulfated GAGs alsoPolysaccharides in Tissue EngineeringS35supported endothelialization of vascular graftsand their antithrombogenic properties. Expandedpolytetrafluoroethylene (ePTFE) vascular grafts coatedwith perlecan, i.e. the major heparan sulfate proteoglycanin cell basement membranes, stimulated the growth ofendothelial cells and suppressed the adhesion of platelets(Lord et al. 2009). The retention of endothelial cells onpoly(carbonate-urea)urethane vascular grafts under shearstress was enhanced by modifying these grafts with amatrix consisting of collagen type IV and dermatansulfate (Salacinski et al. 2001). Heparin in vascular graftsimproved not only their antithrombogenicity, but also theretention of growth factors for endothelial cells, such asvascular endothelial growth factor (VEGF) (Ye et al.2012) and basic fibroblast growth factor (bFGF) (Pitarresiet al. 2013). In addition, heparin enabled stableattachment of stromal cell-derived factor-1α (SDF-1α) tovascular prostheses, which served as a capture moleculefor endothelial and smooth muscle progenitor cells fromblood (Yu et al. 2012).Heparin and other sulfated GAGs have also beenapplied in many other areas of tissue engineering. Forexample, heparin was used for bioactivation ofelectroconductive polypyrrole-based scaffolds for bonetissue engineering (Meng et al. 2013) and for synthesis ofcarbonated apatites, which proved to be more favorable tothe proliferation and differentiation of MC3T3-E1 preosteoblasts than apatites prepared by a traditional method(Deng et al. 2013). Heparin was also a component ofpolymeric scaffolds for neural tissue engineering (Kuoand Wang 2012). In the form of hydrogels, heparin hasbeen used for efficient seeding of chondrocytes onpolymeric scaffolds for cartilage tissue engineering (Kimet al. 2012), for modifying scaffolds for liver tissueengineering (Bao et al. 2011) and for encapsulatingdermal fibroblasts for skin tissue engineering (Choi andYoo 2013). Keratan sul

usually either structure-related or storage-related. The main storage polysaccharides are starch and glycogen, while structural polysaccharides include cellulose, chitin, agar, arabinoxylans and pectins. Some polysaccharides are secreted by bacteria, fungi and algae as a