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Carbohydrate Polymers 180 (2018) 128–144

Contents lists available at ScienceDirect

Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Review

Application of xanthan gum as polysaccharide in tissue engineering: A MARK


review
⁎ ⁎
Anuj Kumara,b, , Kummara Madhusudana Raoa,b, Sung Soo Hana,b,
a
School of Chemical Engineering, Yeungnam University, 280 Daehak-Ro, Gyeongsan 38541, South Korea
b
Department of Nano, Medical and Polymer Materials, Yeungnam University, 280 Daehak-Ro, Gyeongsan 38541, South Korea

A R T I C L E I N F O A B S T R A C T

Keywords: Xanthan gum is a microbial high molecular weight exo-polysaccharide produced by Xanthomonas bacteria (a
Polysaccharide Gram-negative bacteria genus that exhibits several different species) and it has widely been used as an additive
Xanthan gum in various industrial and biomedical applications such as food and food packaging, cosmetics, water-based
Extracellular matrix paints, toiletries, petroleum, oil-recovery, construction and building materials, and drug delivery. Recently, it
Hydrogels
has shown great potential in issue engineering applications and a variety of modification methods have been
Tissue engineering
employed to modify xanthan gum as polysaccharide for this purpose. However, xanthan gum-based biomaterials
need further modification for several targeted applications due to some disadvantages (e.g., processing and
mechanical performance of xanthan gum), where modified xanthan gum will be well suited for tissue en-
gineering products. In this review, the current scenario of the use of xanthan gum for various tissue engineering
applications, including its origin, structure, properties, modification, and processing for the preparation of the
hydrogels and/or the scaffolds is precisely reviewed.

1. Introduction films, foams, hydrogels, depending upon particular tissue application.


In this case hydrogel form may provide most favored 3D micro-
Tissue engineering is a process of the regeneration of damaged tis- environment compared to other forms. For this, natural occurring
sues or the reconstruction of the organs, by adapting appropriate three- polymers show many advantages over synthetic polymers in synthe-
dimensional (3D) microenvironment for proper cell adhesion, pro- sizing appropriate hydrogels for cell fate and proper regeneration of
liferation, followed by the formation of new tissues. This proposed 3D tissues. Natural polymers have high availability, easy processing, ex-
microenvironment can be achieved by the fabrication of artificial bio- cellent biocompatibility, and they mimic biologically to natural ECM of
material structure that may mimic biologically to natural extracellular the tissues (Singh et al., 2016).
matrix (ECM) as supportive architecture (Priya, Jungvid, & Kumar,
2008; Singh, Kaur, Rana, & Kennedy, 2016). Various polymers (natural 1.1. Polysaccharides
or synthetic or their combinations) have extensively been used for this
purpose to produce artificial biomaterial structures in different forms Polysaccharides as natural polymers are highly abundant in the
such as micro-spheres, micro-beads, micro/nano-particles, nanofibrous nature in the form of the renewable resources such as plants, animals,

Abbreviations: ECM, extracellular matrix; 3D, three-dimensional; XG, xanthan gum; USDA, United Sates Department of Agriculture; FDA, United States Food and Drug Administration;
AFM, atomic force microscope; HA, hyaluronic acid; EMGG, enzymatically (α-galactosidase)-modified guar gum; STMP, sodium trimetaphosphate; –OH, hydroxyl group; –CH2COOH,
carboxymethyl group; HEMA, 2-hydroxyethyl methylacrylate; AA, acrylic acid; AAm, acrylamide; MPA, mercaptopropionic acid; TGA, thioglycolic acid; CMX, carboxymethylated-XG;
SAn-XG, succinic anhydride-functionalized XG; PMAO, poly (maleic anhydride/1-octadecene); BIS, N,N’-methylenebisacrylamide; A-CD, cyclodextrin acrylate; XGMNIPAm, XG maleate/
N-isopropylacrylamide; DOPE, 1,2-dioleoyl-sn-glycerophosphoetilamine; MNPs, Magnetic nanoparticles; ESMF, electro-static magnetic field; GNPs, gold nanoparticles; nHAp, nano-
hydroxyapatite; BG, bioactive glass; CNCs, cellulose nanocrystals; SG, silica-based glass; GG, gellan gum; HA, hyaluronic acid; CMC, carboxymethyl cellulose; CS, chondroitin sulfate;
CHT, chitosan; GMA, glycidyl methacrylate; CHX, chlorhexidine; AgNPs, silver nanoparticles; PEHs, polyelectrolyte hydrogels; MMT, montmorillonite; GTM, galactomannan; CC, cur-
cumin; CGN, carrageenan; KG, konjac gum; HNTs, halloysite nanotubes; PPy, polypyrrole; MC, methylcellulose; PLLA, poly-L-lactic acid; OA, osteoarthritis; MMPs, matrix metallo-
proteinases; TIMP-1, tissue inhibitors of metalloproteinase-1; IL-1β, interleukin-1β; LPS, lipopolysaccharide; βlg, β-lactoglobulin; LCST, lower critical solution temperature; BSA, bovine
serum albumin; LYZ, lysozyme; GAGs, glycosaminoglycans; SNPs, sodium nitroprusside; PGE2, prostaglandin E2; GS, gentamicine; HLECs, human lens epithelial cell; DOX, doxorubicin;
bFGF, basic fibroblast growth factor; BMP7, bone morphogenetic protein 7; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; NAG, N-acetyl-D-glucosamine; CAM,
chick embryo chorioallantoic membrane; PLL, poly-L-lysine; IgM, immunoglobulin M; IgG, immunoglobulin G; CHT-NPs, chitosan nanoparticles

Corresponding authors at: School of Chemical Engineering, Yeungnam University, 280 Daehak-Ro, Gyeongsan 38541, South Korea.
E-mail addresses: anuj.budhera@gmail.com (A. Kumar), sshan@yu.ac.kr (S.S. Han).

http://dx.doi.org/10.1016/j.carbpol.2017.10.009
Received 20 August 2017; Received in revised form 20 September 2017; Accepted 2 October 2017
Available online 05 October 2017
0144-8617/ © 2017 Elsevier Ltd. All rights reserved.
A. Kumar et al. Carbohydrate Polymers 180 (2018) 128–144

and microorganisms and have widely been applied in various potential X. campestris, X. fragaria, X. gummisudans, X. juglandis, X. phaseoli, X. vas-
biomedical applications (e.g. drug delivery and tissue engineering) due culorium. Industrially, XG is produced with the fermentation of glucose by X.
to their readily availability, cost-effective, easy processing, and ex- Campestris, a plant-associated bacterium (Becker, Katzen, Pühler, & Ielpi,
cellent biocompatibility nature (Kumbar et al., 2011; Malafaya, 1998; Garcıa-Ochoa, Santos, Casas, & Gomez, 2000; Petri, 2015). The bio-
Silva, & Reis, 2007; Shelke, James, Laurencin, & Kumbar, 2014). Among synthetic pathway of XG synthesis can be categorized in three steps: (1)
polysaccharides, xanthan gum is microbial and an exo-polysaccharide Uptake of simple sugars followed by conversion to nucleotidal derivatives,
that has also been used in biomedical applications due to its excellent (2) Assembly of pentasaccharide subunits associated to an iso-
biocompatibility and gelling property (Petri, 2015). pentylpyrophosphate carrier, and (3) Polymerization of repeat units of
pentasaccharide followed by their secretion (Palaniraj & Jayaraman, 2011;
2. Xanthan gum Rosalam & England, 2006). XG acts as a polyanion at pH > 4.5 because of
the protonation of O-acetyl and pyruvyl residues (Petri, 2015). XG has ex-
2.1. History tensively been used in the food industry because of its efficient thickening
ability, high solution viscosity at very low concentration, pseudoplastic
Xanthan gum (XG) was discovered in 1950s at the National Center behavior in aqueous solution (Faria et al., 2011; Milas, Rinaudo,
for Agricultural Utilization (previously known as Northern Regional Knipper, & Schuppiser, 1990), high stability in broad range of temperature,
Research Center) of the United Sates Department of Agriculture pH, ionic strength, and stability under shear (Hui, 2006; Petri, 2015). XG
(USDA). First industrial production of XG was performed in 1960 fol- chains have the ability to form physical networks with bivalent cations by
lowed by the availability of commercially product in 1964 involving two disaccharide unites in the main chain and O-acetyl and
(Margaritis & Zajic, 1978). This is the second microbial polysaccharide, pyruvyl residues in side chains which lead to intramolecular crosslinking
after dextran (early 1940s), which was industrially commercialized. XG and chains contraction. These acetyl and pyruvyl residues vary with bac-
is non-toxic, non-sensitizing, and does not cause any eye or skin irri- terial species and fermentation conditions to prepare the XG gum. The
tation, and has also been approved by the United States Food and Drug secondary structure of XG undergoes an “order-disorder” transformation
Administration (FDA) in 1969 (Kennedy & Bradshaw, 1984). from helix to coil structure depending on pH, nature of electrolyte, ionic
strength of solutions, and acetyl and pyruvyl contents (Khouryieh, Herald,
2.2. Origin, structure, and chemistry of XG Aramouni, Bean, & Alavi, 2007). The content of acetyl and pyruvyl residues
also affects the behavior of XG aqueous solution. In general, lower pyruvyl
XG has good water-solubility, excellent biocompatibility, and is a high content shows low viscosity and higher pyruvate content promotes gel
molecular weight exo-polysaccharide having branched polymeric chains behavior via improved macromolecular association, whereas higher acetyl
(Le & Turgeon, 2013; Rao, Kumar, Haider, & Han, 2016). The primary content inhibits the gelation behavior of XG aqueous solution (e.g. deace-
structure of XG was established in 1975 and it consists of pentasaccharide tylation leads an increase in viscosity) (Bergmann, Furth, & Mayer, 2008;
subunits. XG comprises of D-glucosyl, D-mannosyl, and D-glucuronyl acid Dário, Hortêncio, Sierakowski, Neto, & Petri, 2011; Petri, 2015; Shatwell,
residues in a 2:2:1 molar ratio with varying proportions of O-acetyl and Sutherland, & Ross-Murphy, 1990; Tako & Nakamura, 1984). This con-
pyruvyl residues. The structure of XG is a linear (1–4)-linked D-glucose si- formational transition to double-helix is a pre-requisite for gel formation
milar to cellulose backbone (Faria et al., 2011; Jansson, Kenne, & Lindberg, that leads to stronger materials. As described elsewhere, XG gels (from
1975). The secondary structure of XG is shown as five-fold right-hand he- 9.1–50.0 wt%) have been prepared by using ionic liquid as green solvent
lical structure with a pitch of 4.7 nm and a diameter of 1.9 nm (Moorhouse, (e.g., 1-butyl-3-methylimidazolium chloride; BMIMCl) via regularly-ordered
Walkinshaw, & Arnott, 1977) and undergoes via a thermally-induced order- interaction of the imidazolium counter cations of XG with other BMIMCl
disorder conformational transition and could be formed by high tempera- molecules. The obtained gels showed thermally-induced shape-memory
tures and low salt concentrations (Cheetham & Mashimba, 1992). Here salt effect and good mechanical performance strongly depending on XG con-
stabilizes the order conformation which is responsible for extraordinary centrations. These gels showed facile conversion into high performance
stability of the XG (Young, Martino, Kienzle-Sterzer, & Torres, 1994), and hydrogels by soaking them in water and further ionically crosslinked with
that evidenced most commonly the dimeric or double-stranded (Hjerde, Na+, Ca2+, and Fe3+ in their corresponding aqueous solutions of 1.0 M
Kristiansen, Stokke, Smidsrød, & Christensen, 1994; Stokke & Christensen, NaCl, 0.2 M CaCl2, and 0.1 M FeCl3, respectively. The obtained ion-ex-
1996). XG is soluble in cold and hot water and requires intensive agitation changed hydrogel with Ca2+ showed better mechanical performance under
when exposed to aqueous medium to avoid the lumps formation. XG so- both tensile and compressive modes compared with ion-exchanged hy-
lutions behave like non-Newtonian fluids and are of highly pseudoplastic drogel with Na+, whereas hydrogel with Fe3+ exhibited hard and brittle
behavior, and the apparent viscosity (conformational status of polymeric nature of the ion-exchanged hydrogel network. Moreover, ion-exchanged
chains) that altered significantly with time and/or shear rate. Generally, the hydrogels with Na+ and Ca2+ showed salt concentration-induced re-
thermal stability of XG against hydrolysis is far better than many other sponsive properties with reversible swelling-shrinking behavior (Izawa,
water-soluble polysaccharides or polymers (Stokke & Christensen, 1996), Kaneko, & Kadokawa, 2009; Izawa & Kadokawa, 2010).
possibly because of the ordered helical structure of XG that protects the
molecules from de-polymerization. Therefore, viscosity of XG solutions is 2.3. Biocompatibility and immunological response
only affected minimally by heat treatment steps (e.g., sterilization) and is
also stable over a wide range of pH values. XG is known as fully biode- XG is well known for its non-toxicity, excellent biocompatibility and
gradable (completely within 2 days) naturally occurring polymer. However, intrinsic ability as immunological agent (Han, Ling, Wang, Wang,
due to the backbone similarity with cellulose, XG is resistant to the attack of & Shao, 2012; Kumar, Rao, Kwon, Lee, & Han, 2017; Le & Turgeon,
the common cellulases and the tri-saccharide side chains of XG are likely to 2013; Rao et al., 2016). As described elsewhere, the biocompatibility of
be a barrier to enzymatic attack. Only in disordered form, it can be cata- the hydrogel prepared by complexation of XG with other poly-
lyzed the cleavage of the main chain when attacked with fungal cellulases, saccharide as chitosan also investigated in vitro and in vivo models with
not in ordered helical form (Rinaudo & Milas, 1980). The molecular weight fibroblast cell line L-929 and showed good biocompatibility analysis
distribution of XG ranges from 2 × 106 to 20 × 106 Da, depending on the (Chellat et al., 2000). Further, to maintain mobility in the joint and
association of chains forming aggregation of several individual chains and reducing the degeneration of cartilage, several glycosaminoglycans
the variation in fermentation conditions. The synthesis of XG is believed to (e.g., chitosan (Chen, Liu, Du, Peng, & Sun, 2006), sodium alginate
be similar to the synthesis of exo-polysaccharide by other Gram-negative (Legendre, Baugé, Roche, Saurel, & Pujol, 2008), hyaluronic acid
bacteria and generally produced by various species of Xanthomonas bac- (Abate, Pulcini, Iorio, & Schiavone, 2010)) have shown chondro-pro-
teria (a Gram-negative bacteria genus), such as X. arboricola, X. axonopodis, tective effects and have been used for the treatment of osteoarthritis

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Fig. 1. Common methods of modification of XG


polymer chains used in tissue engineering.

(OA). Intra-articular injection of hyaluronic acid (HA) is considered as Mishra, & Ramasamy, 2015; Hazirah, Isa, & Sarbon, 2016), cosmetics
an effective treatment for OA. However, HA is unstable and prone to (Garcıa-Ochoa et al., 2000; Palaniraj & Jayaraman, 2011), water-based
degrade quickly by hydrolytic or enzymatic reactions in vivo (Zhong paints (Palaniraj & Jayaraman, 2011), water treatments (Mittal, Parashar,
et al., 1994). For better alternative, intra-articular injection of XG has Mishra, & Mishra, 2014; Mittal, Kumar, & Ray, 2016), jet injection printing
been prepared that could protect the cartilage at joint and reduce the (Faria et al., 2011), petroleum industry (Faria et al., 2011), toiletries
papain-induced osteoarthritis (OA) progression due to its similar (Palaniraj & Jayaraman, 2011), oil recovery (Garcıa-Ochoa et al., 2000),
rheology and viscosity as that of HA (Han, Ling et al., 2012; Han, Shao construction and building materials (Chang, Im, Prasidhi, & Cho, 2015),
et al., 2012). In addition, XG alone showed no adverse effects on rabbit drug delivery (Bhunia, Giri, Nasim, Chattopadhyay, & Bandyopadhyay,
chondrocyte cells viability (Han, Wang et al., 2012). Further, the in- 2013; Jian, Zhu, Zhang, Sun, & Jiang, 2012; Lungan et al., 2015; Shalviri,
trinsic adjuvant property of XG can be used for improving the immune Liu, Abdekhodaie, & Wu, 2010), etc., because of its superior rheological
response of other agents. In this case, Ishizaka, Sugawara, Hasuma, properties like ability to form highly viscous solution at low shear forces,
Morisawa, and Möller (1983) investigated the effect of XG as adjuvant high pseudo-plasticity, and a high viscosity yield value (Rosalam & England,
on murine lymphocytes in vitro. XG successfully influenced both a sig- 2006). The XG solution is stable over a broad range of temperatures (up to
nificant improvement in DNA synthesis in mouse splenic B cells and 90 °C), salt concentrations, and pH (2–11) (Rosalam & England, 2006).
thymocytes, including polyclonal immunoglobulin M (IgM) and im- These superior properties of XG resulted in using for biomedical applica-
munoglobulin G (IgG) antibody responses in B cells. However, XG- tions (e.g., drug delivery and tissue engineering) apart from industrial ap-
modified thymocytes did not show helper or suppressor functions. XG plications (Faria et al., 2011). These superior properties of XG can be un-
showed similar effect on polyclonal antibody responses as lipopoly- derstood in terms of non-Newtonian behavior, high viscosity yield (even at
saccharide (LPS) in murine systems. Further, XG showed weak trigger low concentrations; e.g., 600–2000 ppm), low sensitivity of viscosity to
of Hamster spleen cells for the production of nonspecific antibody, changes in salinity, resistant to mechanical degradation, biodegradable and
while LPS did not trigger spleen cells at all. In addition, immature B environment friendly biomaterial (Rosalam & England, 2006). Only limita-
cells can be stimulated by XG as well by LPS. Spleen cells from C57BL/ tion of XG is slow dissolution rate that can further be improved by some
10 mice were fully responsive to LPS, but not responsive or slightly physical means (Sandford, Baird, & Cottrell, 1981). The dissolution process
responsive only to XG. In other side, XG stimulated spleen cells from of XG involves to steps (Van Krevelen, 1976): (1) solvent penetration to XG
C3H/HeJ mice and other seven mouse strains for the production of for swelling and (2) XG dissolves into the solvent phase. In addition, XG as
polyclonal antibody, but non-responsive to LPS (Ishizaka et al., 1983). hydrocolloids can form ‘Fish-Eyes’ (Sandford et al., 1981) during dissolution
In addition, XG has also been applied for antitumor effects (Takeuchi when XG particles begin to hydrate. In brief, a gelatinous layer of partially
et al., 2009), in bioadhesive compositions for intranasal influenza virus hydrated XG forms on the outside of the XG particle and cannot leave ra-
immunizations (Chiou et al., 2009), and in the preparation of subunit pidly the surface of particle. This prevents water for penetrating to complete
vaccine against leptospirosis (Bacelo et al., 2014). dissolution of particle via complete hydration (Su, Ji, Lan, & Dong, 2003).

2.4. Application of XG in industrial and biomedical area 3. Modification of xanthan gum

XG and XG-based biomaterials have extensively been used in a wide XG has large number of hydroxyl groups and carboxylic groups that
range of applications such as foods and food-packaging (Fareez, Lim, may be modified or functionalized by physical, chemical, chemo-

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enzymatic and plasma-irradiation treatments or their combinations in and radius of gyration). Also, the increase in maximum packing fraction
order to obtain improved properties of the XG as biomaterial. These due to the increase in polydispersity and hydrodynamic radius is the
some common modification treatments can be described as follows (as cause of the decrease in viscoelastic behavior. The changes in the
depicted in Fig. 1). structural and rheological properties of XG were not reversible and
significant recovery was not observed over a period of 11 weeks (Eren,
3.1. Modification by physical method Santos, & Campanella, 2015). Further, the effect of micro-fluidization
on the physical properties of XG was investigated and this treatment
The rheological property of the XG solution is an important factor reduced the intrinsic viscosity, pseudo-plastic behavior and flow bi-
for designing biomaterials that can be used to improve remedial refringence of the treated XG samples. A degradation mechanism of XG
amendment delivery into low-permeable zones in the subsurface het- was proposed by using micro-fluidization (a dynamic high pressure
erogeneous compositions and/or injectable hydrogels for biomedical treatment), where increased micro-fluidization severity resulted a re-
applications, especially tissue engineering. The viscosity of XG solutions latively mild degradation of the main chain via (1) the disruption of
increases strongly with the increasing polymer concentration or mole- aggregates and (2) reversible disruption of the double-stranded con-
cular weight. At low shear rate, the viscosity of XG aqueous solution is formation. In addition, XG treated at high ionic strengths facilitated
higher than the magnitude of the viscosity at high shear rate and at low more mechanical degradation due to the increased ordered stiffer mo-
shear rate, this viscosity further increases linearly with the increase of lecules that experienced effective stresses strongly under high shear and
XG solution concentration within the range 0.3–2.0 g/L, when no salt is cavitation forces exist in the micro-fluidization chamber. This treatment
added to the XG solution (Garcıa-Ochoa et al., 2000; Zhong, Oostrom, can be another way to get a controlled and relatively mild degradation
Truex, Vermeul, & Szecsody, 2013), but this viscosity as well as degree of XG without affecting its chemical composition (Laneuville,
of shear-thinning property of XG solution (< 2.0 g/L) substantially Turgeon, & Paquin, 2013).
decreases when salt is added. Further, for XG concentration above
2.0 g/L, the viscosity as well as degree of shear-thinning of the XG so-
lution increases when salt is added. These changes happen due to the 3.1.2. Using oil and/or W/O emulsions
changes in molecular-conformation levels such as ordered (helix mo- The lubrication behavior of the XG fluid gels and sunflower oil
lecular conformation) and disordered (extended configuration with a modified-XG fluid gels was investigated. XG-based fluid gels were
larger hydrodynamic volume due to strong Columbic repulsions be- formed by applying shear forces during cooling via conformational
tween like charges) states. Actually, at lower concentration (< 2.0 g/L), transition. The addition of 10.0% (w/w) sunflower oil to the continuous
the addition of salt neutralizes the ionic charge of XG molecules and phase of XG fluid gels reduced the friction coefficient. Further, the
therefore, elongated molecules are transformed into helical conforma- addition of 10.0% (w/w) of triglycerides stabilized w/o emulsion to the
tion of the molecules by reducing to lower hydrodynamic volume and continuous phase of XG fluid gels similarly reduced the friction coef-
thereby solution viscosity. While at higher polymer concentration ficient. This was possibly due to the surface-parting (during soft-tri-
(> 2.0 g/L), the effect of local charge inversion and subsequent chain bology experiment) of ridged emulsion particles and limiting de-
expansion due to intermolecular interactions (e.g., entanglements of formation of fluid gel particles. In this study, the addition of only 7%
polymeric molecules) is more effective than that of change in hydro- (w/w) of sunflower oil to the XG fluid gels and triglycerides stabilized
dynamic volumes of molecules. Therefore, XG solution prepared with w/o emulsions showed viable route to obtain lubrication behavior si-
deionized water (no ions) had much higher viscosity compared to tap milar to sunflower oil (Hamilton & Norton, 2016).
and groundwater (presence of ions) possibly due to the composition and
the ionic strength of the water source. Further, higher XG concentration
is required to compensate the viscosity loss caused by the presence of 3.1.3. Blending of proteins and/or other unmodified/modified polymers
ions (e.g., Na+, Ca2+) and remedial amendments (e.g., ethyl lactate, The modification of XG with β-lactoglobulin (βlg) by inducing
phosphate, and sodium lactate) in XG solution. However, the addition electrostatic attraction interaction in network formation was per-
of remedial amendments maintained shear-thinning properties formed. In this study, the gelation mechanism of βlg and XG mixtures
(Bergmann et al., 2008; Zhong et al., 2013). In another study, the effect was monitored by ratio of βlg and XG and their concentrations. With
of salt on the secondary structure (double helical conformation) of XG the initial very weak network of XG, the βlg aggregated along the
was analysed in terms of salt-responsive conformation change after chains of XG by facilitating as a crosslinking agent and 4.8 × 10−3 wt%
denaturation and renaturation of XG solution by heating and gradually as lowest concentration of XG was observed for possible gelation, and
cooling, respectively. In pure water, incomplete renaturation (e.g., more elastic gels were formed at higher XG concentrations. Further, the
single helical structure) of XG was observed; whereas double helical ratio of βlg and XG strongly affects the kinetics of gelation and mainly
conformation (as present is native XG) was observed when salt was controls the gelation process and gel-network. The increasing electro-
added. In addition, less rigid chains were observed when salt was added static attractive interaction between βlg and XG with decreased pH
compared with pure water XG solution (Camesano & Wilkinson, 2001). resulted in the formation inter-polymer complexes via the formation of
By physical means, the rheological behavior of XG solutions can be soluble complexes (sol-gel transition) at the point of gelation
modified and achieved in the following ways: (1) using mechanical (Le & Turgeon, 2013). In another study, a gel composed of en-
treatment, (2) using oil and/or W/O emulsions, (3) the blending of zymatically (α-galactosidase)-modified guar gum was prepared and XG
proteins and/or other unmodified/modified polymers, and (4) using of and investigated the critical gelling behavior and rheological properties
metal ions. of the obtained EMGG/XG gels. The EMGG was prepared with a man-
nose/galactose ratio of 3.02 similar to that of locus bean gum. Due to
3.1.1. Using mechanical effect the removal of galactose residues from EMGG showed improvement in
The rheological and polydispersity aspects of XG by mechanical the interaction between GG and XG in the formation of synergistic gel,
modification (high-pressure homogenization) were investigated as an whereas unmodified-GG did not show such a synergistic effect regard-
alternative treatment to enzymatic and chemical modification. In this less the concentration of salt. This synergistic gelation was successfully
treatment, structured-network of XG solutions was lost gradually de- described by the cascade model. The number of crosslinking sites per
pending on the severity of the high-pressure homogenization. The chain (optimal functionalities of EMGG and XG) were measured to be
viscosity, storage and loss modulus of XG solutions were decreased 300 and 30, respectively. However, the values of optimum functionality
upon high-pressure homogenization due to partial loss of junction zones were best measured from the analysis of modulus data (Mao,
and increased disordered structure (slight decrease in molecular mass Zeng, & Chen, 2012).

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3.1.4. Using metal ions CH2CHCOOH) functionality and prepared XG-g-poly(HEMA-co-AA)


For improvement in rheological properties of XG, several trivalent superporous hydrogel system with sodium bicarbonate as foaming
ions (e.g., Cr3+, Al3+, Fe3+) have also been used for modifying gelation agent and a triblock-copolymer of polyoxyethylene-polyoxypropylene-
behavior of XG (Nolte, John, Smidsrød, & Stokke, 1992). As described polyoxyethylene as foam stabilizer for possible use in various industrial
elsewhere, Lund, Smidsrød, Stokke, and Elgsaeter (1988) investigated and biomedical areas (Gils et al., 2009). Further, the graft-
the effect of chromic ions (Cr3+) on gel formation. The rate of gel copolymerization of XG using acrylamide (AAm, CH2CHCONH2) was
formation was highly dependent on the concentration of Cr3+, but performed and the grafting parameters and copolymer properties of
much smaller extent on the concentration of XG. For 50 mM Cr3+, gel modified XG were investigated (Maia, Silva, Curti, & Balaban, 2012),
formation was observed in less than 1 h, while gel formed in about 40 h whereas Badwaik, Sakure, Alexander, Dhongade, and Tripathi (2016)
for 2 mM Cr3+. The minimum concentration of Cr3+ is 1–2 mM to form performed the graft-copolymerization of carboxymethylated-XG using
gel of 1–7 mg/mL of XG (Lund et al., 1988). acrylamide (AAm, CH2CHCONH2) and prepared CMX-g-PAAm showed
a non-Newtonian flow with one-third intrinsic viscosity and higher
3.2. Modification with chemical method thermal stability, but less crystalline network structure compared to
CMX (Badwaik et al., 2016). The mucoadhesive property after
The dissolution rate of XG into water can be improved by imparting modification of XG as thiol-derivatives (via esterification) using
changes in molecular structure with reduced intermolecular interac- mercaptopropionic acid (MPA) and thioglycolic acid (TGA) was
tions using chemicals, such as formaldehyde (Su et al., 2003). In gen- demonstrated. Metronidazole (as model drug) loaded buccal pellets
eral, the chemical method involves the covalent interactions to modify composed of XG and thiolated-XG using chicken buccal pouch
the gelation behavior of XG. membrane showed high ex vivo bio-adhesion time of thiolated-XG
pallets compared to XG pallets. This is possibly due to disulfide bond
3.2.1. Using crosslinking agent formation between mucus and thiolated-XG. The in vitro sustained
Another way is to crosslink the functional groups of XG chains with release of metronidazole from thiolated-XG pallets compared to XG
chemical agents (e.g., sodium trimetaphosphate (STMP)) via covalent pallets over a prolonged period (Bhatia, Ahuja, & Mehta, 2015). Wang,
network for better hydrogel forming kinetics and mechanical stability Han et al. (2016) prepared succinic anhydride-functionalized XG (SAn-
of crosslinked XG-based hydrogels. The obtained hydrogels showed XG) and investigated the properties of the SAn-modified XG hydrogels.
release controlled properties and more elastic and tougher network The obtained SAn-XG hydrogels showed much higher storage (G’) and
(solid-like gel behavior) compared to physically (hydrogen bonds in- loss (G”) modulus via the increased carboxyl groups (eCOOH) and
teraction) crosslinked XG-based hydrogels (Tao et al., 2016). In another resulted in a more rigid structure than that of XG hydrogels (Wang, Han
study, the effect of XG content on gelatinized starch-XG hydrogel films et al., 2016). Further, Wang, Xin et al. (2016) investigated the
crosslinked with STMP as crosslinker. The swelling ratio of the hydrogel modification of XG by esterification with poly (maleic anhydride/1-
films was observed higher for higher amounts of XG and STMP, espe- octadecene) (PMAO) to improve the shearing and thermal stability.
cially at concentration higher than 10%. Further, gel mesh size in- Modified-XG showed a highly improved viscoelastic behavior, whereas
creased (from 2.84 nm to 6.74 nm at pH 7.4) with increasing swelling similar thermal stability of modified-XG and unmodified-XG was
ratio and anionic drug permeability across hydrogel films was sig- observed. In addition, modified-XG showed better performance on
nificantly lower compared to their neutral form mainly due to elec- temperature-resistance, salt tolerance, and shear endurance due to the
trostatic repulsion between the polymer and negatively charged drugs. crosslinking between XG and PMAO (Wang, Xin et al., 2016).
However, the formed range of mesh size is large enough for the
transport of the most of the drugs varying from small molecules to 3.2.3.2. Modification of the eCOOH groups. The modification of XG by
polypeptide and proteins (Shalviri et al., 2010). the chemical treatment for the buccal drug delivery to treat sialorrhea
was investigated. The polymeric backbone of XG was modified with L-
3.2.2. By the removal of existing functionality cysteine (SH) via amide bond formation (thiolation of XG). The
The addition or removal of chemical functionality to the XG struc- obtained modified-XG showed improvement in mucoadhesive
ture is an efficient way to modify the properties of XG. For this, Pinto, strength and modified swelling properties. In addition, modified-XG
Furlan, and Vendruscolo (2011) investigated the rheological properties especially improved the adhesion to the surfaces of buccal mucosal and
of XG after removal of acetyl groups from XG structure under alkali in refining prolonged and controlled drug-release properties. Further,
medium. With higher alkali concentration, the viscosity of XG solution modified-XG showed an improved saliva uptake capacity and tannic
was observed to increase. However, the concentration of XG (0.5 and acid (causing astringent and drying effect) to reduce excessive saliva
1%) did not show any effect on the viscosity of XG solution. The use of flow (Laffleur & Michalek, 2017).
alkali (NaOH and KOH) in 0.01 mol/L exhibited a viscosity of 410 and
420 mPa s at 10 s−1 and 1.3 and 1.4% deacetylation degree, respec- 3.2.3.3. Modification of both eOH and eCOOH groups. A novel
tively (Pinto et al., 2011). interpenetrated polymer hydrogels composed of XG maleate/N-
isopropylacrylamide (XGMNIPAm) via a grafting-crosslinking with
3.2.3. By the addition of new functionality either N,N’-methylenebisacrylamide (BIS) or cyclodextrin acrylate (A-
3.2.3.1. Modification of the eOH groups. The hydroxyl group (eOH) of CD) as crosslinking agent was prepared. A-CD crosslinked-XG hydrogels
XG has been modified by using various chemical agents, such as an showed improved lower critical solution temperature (LCST) and
unsaturated organic acid (acrylic acid), acid reactive derivatives (e.g., porosity. In addition, A-CD crosslinked- XGMNIPAm hydrogels
maleic anhydride, acryloyl chloride), carboxymethylation, etc. (Gils, showed less sensitivity to electrolytes compared to that of BIS.
Ray, & Sahoo, 2009; Hamcerencu, Desbrieres, Popa, Khoukh, & Riess, Furthermore, having preliminary analysis with progesterone (a
2007; Mendes, Baran, Pereira, Azevedo, & Reis, 2012). In this way, hydrophobic drug), A-CD crosslinked hydrogels may have good
Mendes et al. (2012) modified the XG by incorporating new potential for controlled release polymeric systems (Hamcerencu,
carboxymethyl group (eCH2COOH) onto XG chains (eOH groups) Desbrieres, Popa, & Riess, 2009).
and investigated the encapsulation and survival of chondrocytic
ATDC5 cells. The carboxymethylated-XG showed better perspective 3.3. Modification with chemo-enzymatic method
effect for long-term cell therapies (Mendes et al., 2012). In another
case, XG was graft-copolymerized using 2-hydroxyethyl methyla- In general, phosphorylase has been used in several copolymeriza-
crylate (HEMA, CH3C(CH2)COOCH2CH2OH) and acrylic acid (AA, tion reactions (especially in a chemo-enzymatic approach) in order to

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prepare amylose grafted-polysaccharides (Kadokawa, 2012; Karaki, investigated. The crosslinked XG chains showed disordered conforma-
Aljawish, Humeau, Muniglia, & Jasniewski, 2016). In another study, an tion (coils) exposed with large number of carboxylate groups (negative
amylose grafted-XG by using chemo-enzymatic approach was prepared. charges) compared to un-crosslinked XG chains with ordered con-
Initially, an amine functionalized malto-oligosaccharide was introduced formation (helixes). The loading and delivery of proteins as bovine
chemically into XG via condensation with its carboxylates (eCOO−) to serum albumin (BSA) and lysozyme (LYZ) in the un-crosslinked and
prepare a malto-oligosaccharide-grafted XG and then a phosphorylase- crosslinked XG hydrogels have been analyzed and controlled by hy-
catalyzed enzymatic polymerization of glucose 1-phosphate on malto- drogels and proteins net charges, and can be triggered by pH change.
oligosaccharide-grafted-XG from the graft chain ends to obtain the Moreover, the LYZ loaded XG hydrogels showed substantial bactericidal
product as amylose-grafted XG. This product formed a gel in an ionic activity of LYZ by preserving of LYZ native conformation and presented
liquid and converted into hydrogel by replacing ionic liquid with water. as active wound dressings (Bueno & Petri, 2014). In another study, Han,
The obtained hydrogels showed more elastic property compared to XG Wang et al. (2012) investigated the ability of intra-articular injection of
hydrogel. Further, hydrogel was crosslinked ionically (Fe3+) by soaking XG that could protect the cartilage at joint and reduce the papain-in-
the primary hydrogel system in aqueous solution of FeCl3 (Arimura, duced osteoarthritis (OA) progression due to its similar rheology and
Omagari, Yamamoto, & Kadokawa, 2011). viscosity as that of HA. Therefore, purified XG and IA injection of XG
were analyzed for their qualities for reducing papain-induced OA on
3.4. Modification with plasma irradiation rabbit knee OA model and recorded with morphological and histolo-
gical evaluation of articular cartilage and synovium by evaluating
Plasma chemical technology is a dry chemistry route with high sulphated glycosaminoglycans (GAGs) in cartilage. The obtained IA
energy efficiency and cost-effectiveness. The plasma energy is enough injection of XG was observed as with high transparency, low protein,
to break the bonds in mostly all organic molecules, leading to the for- endotoxin-free, heat-stable (might be sterilized at 121 °C for 15 min),
mation of new active chemical states of carbon atoms (Behnisch, 1997). and could be injected fewer times than IA injection of HA, because no
Generally, plasma technique penetrates about 100 from the material statistical significance on OA was observed for the chondro-protective
surface (Hua, Sitaru, Denes, & Young, 1997) and known as surface effects of intra-articular injection of XG (once in every two weeks for 5
modification technique (Jampala, Manolache, Gunasekaran, & Denes, weeks) and HA (once in every week for 5 weeks). It can be seen clearly
2005). The cold plasma technology is an efficient route for the in- macroscopically (as shown in Fig. 3), surface of articular cartilage was
corporation of appropriate functionality (e.g, amine, carboxyl, and with integrity, smooth and lustrous with a light white color in the sham
hydroxyl groups) in different polysaccharides (Behari, Pandey, group (Fig. 3A and E), while this surface observed to be yellowish-
Kumar, & Taunk, 2001; Hua et al., 1997). The cold plasma technology is white, uneven and damaged (Fig. 3B and F) in the saline group. Further,
an efficient and nonenzymatic route to modify XG. Jampala et al. the cartilage surfaces of two treated groups were lustrous with a white
(2005) investigated the effect of cold-plasma treatment to graft primary color and femoral condylar cartilages as resemble to healthy cartilage
amines (-NH2) followed by the crosslinking by glutaraldehyde on were lesioned slightly (Fig. 3C and D). Tiable plateau cartilages ex-
rheological properties of XG solutions. Prior this, the reactive SiHxCly hibited significant cracks and defects (Fig. 3G and H). However, the
groups under DS-cold-plasma atmosphere were incorporated onto XG. severities were milder as compared to saline group. XG-treated group
By increasing the time and concentration of plasma treatment may be showed lower scores than that of the saline group (Han, Wang et al.,
used to obtain stable XG-based gels (Jampala et al., 2005). 2012).
Further, the effect of XG on rabbit chondrocytes as preliminary
4. Application of XG in tissue engineering cytotoxicity in terms of proliferation and the protein expression of
matrix metalloproteinases (MMPs), and tissue inhibitors of metallo-
XG has recently attracted a great attention as a biomaterial for the proteinase-1 (TIMP-1) in interleukin-1β (IL-1β)-induced chondrocytes
preparation of tissue scaffolds (extracellular matrix) for tissue en- was investigated. With various amount of XG with or without 10 ng/mL
gineering applications. It is well known that for proper regeneration of IL-1β, the obtained results demonstrated that XG alone showed no ad-
damaged tissues, XG-based tissue scaffold should have 3D micro- verse effects on chondrocyte cells viability and reversed IL-1β-reduced
environment providing good cell-matrix interaction with surface chondrocyte cell proliferation significantly in a dose-dependent
chemistry, nanotopology, and appropriate stiffness (Murphy, manner. Further, XG exhibited a dose-dependent increase in TIMP-1
McDevitt, & Engler, 2014) XG itself forms weak gel-like structure via expression while inhibition in IL-1β-induced release of MMPs (Han,
double helical conformations in the presence of specific bivalent cation. Shao et al., 2012). In addition, the protective effect of XG against So-
For this purpose, the stiffness or mechanical stability of XG as well as dium nitroprusside (SNP)-induced rabbit articular chondrocytes apop-
other properties can be manipulated by using other biomaterials or tosis in vitro was investigated. In this study, rabbit articular chon-
reinforcements. Schematic representation of the use of XG with other drocytes were incubated with XG of various concentrations for 24 h
components and/or polymers for tissue engineering applications is prior to incubating with 0.5 mmol/L SNP co-treatment for 24 h and
shown in Fig. 2. supernatants were collected for prostaglandin E2 (PGE2) evaluation.
XG offsets the adverse effects in proliferation of SNP-induced chon-
4.1. Pure XG-based biomaterials drocytes. However, the mechanism of XG blocks SNP-induced chon-
drocytes apoptosis remains unclear. The results showed that XG could
For tissue engineering applications, the structure (in liquid) and reverse SNP-reduced cell proliferation significantly and inhibited early
complex mechanical properties of XG scaffold structures have been cell apoptosis rate in a dose-dependent manner (Chen et al., 2015).
investigated by using atomic force microscope (AFM) and AFM-based
force spectroscopy (FS) and the results revealed the complicated in- 4.2. Modified XG-based biomaterials
termolecular interactions among XG fibrils (self-assembling of the XG
network) (Liang et al., 2014). In addition, the molecular chains ag- Mendes et al. (2012) investigated the properties of modified
gregate and dissociate (in an oscillational mode) with increasing an- (-CH2COOH)-XG (CMX) as artificial matrix (in the form of micro-
nealing time in XG aqueous solution. As analysed by AFM, a homo- capsules) for the encapsulation of chondrocytic ATDC5 cells. The mi-
genous network structure can be obtained by annealing of XG aqueous crocapsules with smooth surface and homogenous size having an
solution at 40 °C for 24 h (Iijima, Shinozaki, Hatakeyama, Takahashi, average diameter of 500 μm were prepared. ATDC5 cells encapsulated-
& Hatakeyama, 2007). Further, the molecular conformation of un- CMX microcapsules showed high viability and proliferated for pro-
crosslinked XG chains and citric acid-crosslinked XG chains was longed culture periods with improved metabolic activity. Further,

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Fig. 2. Schematic representation of the use of XG


with other components and/or polymers for tissue
engineering applications.

ATDC5 cells within CMX microcapsules showed retention of the chon- tissue engineering applications because of their unique properties such
drogenic phenotype. This obtained CMX microcapsules may show a as the excellent biocompatibility and an especial amphiphilicity (Li
potential for long-term cell culture for the applications in cell therapies et al., 2015; Monteiro, Martins, Reis, & Neves, 2014). It is believed that
(Mendes et al., 2012). Further, Wang, Han et al. (2016) prepared suc- phospholipid-based may be used to manipulate the cell and tissue re-
cinic anhydride-functionalized XG (SAn-XG) and investigated the sponses towards biomaterial, facilitating the controlled release of
properties of the SAn-modified XG hydrogels. The obtained SAn-XG bioactive agents and reconstructive surgery (Collier & Messersmith,
hydrogels showed much higher storage (G’) and loss (G”) modulus via 2001). In this case, Mendes, Baran, Reis, and Azevedo (2013) synthe-
the increased carboxyl groups (-COOH) and resulted in a more rigid sized XG-phospholipid (1,2-dioleoyl-sn-glycerophosphoetilamine,
structure than that of XG hydrogels. Gentamicine (GS) drug loaded SAn- DOPE) microcapsules (hollow capsular structures) by combining mi-
XG hydrogels showed prolonged release of GS and good human lens crofluidics with self-assembly for cell encapsulation (survival and pro-
epithelial cell (HLECs) attachment, proliferation and migration when liferation). The supramolecular organization of XG-DOPE under the
GS loading was 1 mg g−1. In addition, SAn-XG/GS hydrogels, compared conditions for cells encapsulation was observed as formation of nano-
to XG-SAn hydrogels, demonstrated a significantly lower degree of in- sized aggregates and the four-step synthesis of microcapsule are shown
fection at 7 day in an in vivo rabbit subcutaneous S. aureus infection in Fig. 4(I) (A and B), respectively. ATDC5 cells were encapsulated
model (Wang, Han et al., 2016). within XG-DOPE microcapsules and demonstrated good cell viability
showing increased cellular metabolic activity over 21 days of incuba-
4.3. XG-phospholipid as bioactive agent tion (as shown in Fig. 4(II)-A). In addition, metabolic activity and
proliferation of encapsulated ATDC5 cells within self-assembled XG-
Phospholipids have widely been used in drug delivery systems and DOPE microcapsules were measured by AlamarBlue assay and DNA

Fig. 3. Gross morphological analysis of femoral condyle and tibial plateau. Femoral condyle: sham group (A: normal cartilage surface); saline group (B: yellowish-white and damaged
cartilage surface); HA-treated group (C: cartilage surface lesioned slightly); and XG-treated group (D: cartilage surface lesioned slightly). Tibial plateau: sham group (E: normal cartilage
surface); saline group (F: yellowish-white, uneven and damaged cartilage surface); HA-treated group (G: defective cartilage surface); and XG-treated group (H: defective cartilage surface).
(Reproduced with the permission from Ref. Han, Wang et al. (2012)).

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Fig. 4. (I) Schematic representation of (A) the amphiphilic XG-DOPE and TEM image to show the morphology of the assemblies in the aqueous solution having electrolytes and (B)
microcapsule synthesis steps: (1) injection of XG-DOPE (dispersed phase) with or without cells into mineral oil phase (continuous phase); (2) polymer microdroplets by the emulsification
at the T-junction in the microfluidic device; (3) wall-solidification of XG-DOPE microcapsules in PBS solution via interfacial ionic-complexation; (4) capsule-wall strengthening by coating
with PLL. (Dashed squares indicate the organization at nanoscale). (II) In vitro ATDC5 cells viability and proliferation in XG-DOPE and XG-DOPE/PLL microcapsules: (A) live/dead assay
of encapsulated cells (green cells as live and red cells as dead by Fluorescence microscopy) that showed maintaining cells viability in the microcapsules for up to 21 days in culture, (B)
metabolic activity of encapsulated cells measured by AlamarBlue assay, and (C) proliferation of encapsulated cells measured by DNA quantification. (Reproduced with the permission
from Ref. Mendes et al. (2013)).

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quantification over 21 days. AlamarBlue assay showed improved me- hard tissues (bone repair, bone augmentation, and coating of implants
tabolic activity for a prolonged time (without needing of any external or as fillers in bone or teeth) especially for bone tissue engineering
coating, as the potentially immunogenic poly-L-lysine (PLL)). However, applications, including a variety of biomedical applications, including
at the end of 21 days of culture, XG-DOPE coated with PLL showed drug delivery systems due to its slow in situ biodegradability, bio-
higher metabolic activity, but not significantly compared to uncoated compatibility, good osteoconductive and osteoinductive properties
XG-DOPE microcapsules (as shown in Fig. 4(II)-B). Further, DNA (Habraken, Wolke, & Jansen, 2007; Legeros, 1993; Weiner & Wagner,
quantification showed a gradual and significant increase in DNA be- 1998; Zhou & Lee, 2011). By taking this opportunity, Izawa et al. (2014)
cause of the good cellular proliferation in both XG-DOPE and PLL reported the mineralization of nHAp upon XG hydrogel via the ionic
coated-XG-DOPE microcapsules (as shown in Fig. 4(II)-C). However, interactions between Ca2+ and –COOH and obtained mechanically
ATDC5 cells within XG-DOPE coated with PLL exhibited significantly improved XG-nHAp porous structure for bone tissue engineering ap-
less cellularity compared to uncoated XG-DOPE microcapsules, despite plications (Izawa et al., 2014). In another study, Bueno, Bentini,
showing similar metabolic activity. Therefore, cells could be en- Catalani, Barbosa, and Petri (2014) prepared XG/nHAp and strontium
capsulated with XG-DOPE microcapsules with enhanced metabolic ac- substituted XG/nHAp (XG/nHAp-Sr) particles. The surface charge
tivity for a prolonged duration without needing any external coating density and colloidal stability of XG/nHAp and XG/nHAp-Sr particles
such as potentially immunogenic PLL and might be a biomimetic carrier were increased because of the surface modifying with XG chains and
for cells grow for cell-based transplantation therapies (Mendes et al., further promoted compatibility between XG chains and XG/nHAp or
2013). XG/nHAp-Sr particles. The XG-XG/nHAp with 10 wt% HAp nano-
composites showed improved mechanical performance while porous
4.4. XG-nanomaterials based biomaterials structure, surface energy, gel content and swelling ratio comparable to
pure XG hydrogels or nHAp nanocomposites. In addition, cellular pro-
4.4.1. XG-magnetic nanoparticles liferation of osteoblastic cells onto XG hydrogels was not significantly
Magnetic nanoparticles (MNPs) show main advantage of remote influenced by the incorporation of XG modified HAp. However, the
controlling when cells functionalized with these nanoparticles. MNPs activity of ALP was substantially favored upon increasing XG/nHAp or
functionalized labeled cells facilitate the manipulation of the cell be- XG/nHAp-Sr particles content (from 10 to 30%) (Bueno et al., 2014).
havior and also control the function of cells using external magnetic
field (Ito & Kamihira, 2010). Compared to pure XG hydrogels, magneto- 4.4.4. XG-bioactive glass/cellulose nanocrystals
responsive and non-toxic XG/MNPs hybrid hydrogels showed more Bioactive glass (BG), similar to hydroxyapatites, has shown a great
pronounced murine fibroblasts (3T3 L1) proliferation, particularly potential in the engineering of new bone tissues, by improving the me-
under applied filed of 0.4T. Topographical analysis showed densely chanical stability and good bioactivity (apatite forming ability) of the BG-
packed tiny particles on the surface along with some large aggregation based polymeric scaffolds (Lei et al., 2012). In addition, cellulose nano-
of small particles and MTT assay showed cell viability with and without crystals (CNCs) have shown a good potential in the tissue engineering areas
electro-static magnetic field (ESMF) (Bueno et al., 2013). Further, XG/ due to their unique properties such as nano-scale size, high aspect ratio and
MNPs-based scaffolds showed successful in vitro neuronal differentia- surface area, good hydrophilicity, biocompatibility, low-density, and high
tion of embryonic stem cells and indicated successful synapse formation strength (7.5 GPa) and modulus (ranging from 110 to 220 GPa) (Habibi,
(increased membrane potential amplitudes upon depolarization with Lucia, & Rojas, 2010; Kumar, Negi, Choudhary, & Bhardwaj, 2014a). These
KCl) (Glaser, Bueno, Cornejo, Petri, & Ulrich, 2015). properties make CNCs ideal nano-reinforcements for preparing tissue en-
gineering biomaterials (Domingues, Gomes, & Reis, 2014; Kumar, Negi,
4.4.2. XG-gold nanoparticles Choudhary, & Bhardwaj, 2014b; Kumar, Negi, Choudhary, & Bhardwaj,
Gold nanoparticles (GNPs) have extensively been used in as ther- 2014c; Kumar, Lee et al., 2017; Kumar, Rao, & Han, 2017a). Therefore, the
apeutic agents (Sun et al., 2014), diagnosis agents (Halo et al., 2014), use of BG sol-gel alongwith CNCs has been found advantageous to improve
and imaging agents (Zerda et al., 2015) because of their easy pre- polymeric-network of XG with mechanical stability and apatite forming
paration, nano-scale size (similar to the dimension of biological com- ability of the hybrid tissue engineering scaffolds. In this way, Kumar, Rao,
pounds), high surface area, and easy functionalization. GNPs also serve Kwon et al. (2017) prepared XG hybrid scaffolds reinforced with CNCs and
as a promising tool for tissue engineering applications due to their silica-based glass (SG) by using freeze-casting and freeze drying process and
ability to deliver bioactive molecules, to diagnose differentiation status demonstrated highly porous structure with improved mechanical stability
of stem cells, to track implanted cells, while improving differentiation (in dry and wet states) when incorporated with SG followed by CNCs.
of cells and intracellular growth factor delivery, cell–cell interactions, Further, good osteoblastic MC3T3-E1 cell adhesion and proliferation was
monitoring in real-time cellular events, adhesive and mechanical per- observed to increase with time and stiffness compared to pure XG scaffold
formance of the scaffolds. These remarkable physicochemical proper- and presents the suitability of as-obtained hybrid scaffolds may possibly
ties make them unique compared to polymeric nanoparticles, protein- have promising application in low-loading bone tissue engineering appli-
based nanoparticles, and liposomes (Vial, Reis, & Oliveira, 2017). cations (Kumar, Rao, Kwon et al., 2017).
Therefore, Pooja, Panyaram, Kulhari, Rachamalla, and Sistla (2014)
synthesized optimized GNPs within XG used as both reducing and sta- 4.5. XG-natural polymer and/or nanomaterials based biomaterials
bilizing or capping agent and doxorubicin (DOX)-loaded GNPs showed
colloidal stability at a pH range between pH 5–9 and NaCl concentra- 4.5.1. XG-chondroitin sulfate
tion up to 0.5 M. In addition, for toxicity evaluation, GNPs were found Chondroitin sulfate (CS) is also a member of GAGs family and is an
non-toxic and biocompatible. However, DOX-loaded XG-GNPs showed important component in cartilage and connective tissues. It is sulfated
3 times more cytotoxicity in A549 cells (human lung cancer cells) and branched polysaccharide and composed of a chain of alternating
compared to DOX only (Pooja et al., 2014). repeat units of glucuronic acid and N-acetyl-glactosamine (GalNac)
with sulfation at either 4- or 6-position (Fan et al., 2017; Lee,
4.4.3. XG-nanohydroxyapatite Huang, & Lee, 2006). CS can have a potential impact on cell attach-
Nano-hydroxyapatite (nHAp) shows excellent biocompatibility and ment, migration, proliferation and differentiation. Oprea, Neamtu,
has close chemical similarity to the inorganic component of the bone Stoica, Petreus, and Vasile (2012) prepared XG/CS hydrogels and in-
that is a natural composite of organic (collagen fibrils) and inorganic vestigated in vitro and in vivo biocompatibility. As-obtained XG/CS
phases, which makes it ideal candidate for orthopedic and dental im- hydrogels showed good in vitro biocompatibility with viability of Sac-
plants. Therefore, synthetic nHAp has widely been used in repairing of charomyces pombe (microorganism) analyzed by using

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chemiluminescent assay. The lethal dose (LD50) of XG/CS-based hy- In addition, chlorhexidine (CHX) is an antimicrobial agent a cationic
drogels is bigger than 3200 mg/kg and hence proved low toxicity. bisbiguanide having wide antimicrobial activity range against Gram-
Further, in vivo evaluation performed by subcutaneous implantation positive and Gram-negative bacteria, dermatophytes, yeast, and some
demonstrated that 50/50 XG/CS composition was absorbed easily selective lipophilic-viruses (Jones, 1997; Kim et al., 2017). However, its
without side-effects. The in vitro and in vivo biocompatibility analyses antimicrobial effect damages the inner membrane (cytoplasmic) and
may possibly present the suitability of XG/CS-based hydrogels for leads to the cell lysis and death (Huynh et al., 2010). Further, it shows
controlled release systems for drug delivery and tissue engineering good affinity to skin and mucous membranes and low toxicity against
applications. The cellular growth of 20–25% was observed with CS mammalian tissue (Jones, 1997; Paolantonio et al., 2008) and has been
increment in composition of the hydrogels compared to positive con- used in dental treatments (Paolantonio et al., 2008). Kim et al. (2017)
trol, whereas further increase of CS content did not show any significant prepared XG/CHT-based polyelectrolyte microspheres via anionic XG
influence. XG hydrogel showed a slight decrease in cell viability (∼4%) and cationic CHT and mixed with CHX dihydrochloride (10 mg/mL)
(Oprea et al., 2012). and 0.5% (v/v) CHX digluconate (0.5% v/v from 20% w/v) to form
injectable antibacterial hydrogels to be used potentially for acute or
4.5.2. XG-chitosan chronic periodontitis. As-obtained hydrogels showed viscoelastic
Chitosan (CHT) is a natural polymer having a linear chain com- behavior, selective antibacterial effects against P. Gingivalis (as
prising of β(1–4) glycosidic bonds linked D-glucosamine residues with determined by blood-agar plate assay), and non-cytotoxicity to
varying number of randomly located N-acetyl-D-glucosamine (NAG) human dermal fibroblast (Kim et al., 2017). In this study, silver
groups. It has large number of eOH groups and is biodegradable, nanoparticles (AgNPs) have been used as antimicrobial agent along
bioactive, biocompatible, and anti-bacterial properties and showed with CHT. Rao et al. (2016) prepared XG-CHT based polyelectrolyte
enhanced effect on cell attachment, proliferation, differentiation and hydrogels (PEHs) with Ag-NPs by green chemistry route without using
mineralization (Di Martino, Sittinger, & Risbud, 2005; Saravanan, any organic solvent and reagents. In this study, highly size-controlled
Leena, & Selvamurugan, 2016). As biomaterial, CHT has also been used and spherical-shaped Ag-NPs of 5–20 nm diameters were obtained as
to prepare weak gel with XG by complexation of these two natural confirmed by characteristic surface plasmon band (398–409 nm) for
polysaccharides via the presence of counter-ions (Martínez-Ruvalcaba, various concentration of silver ions. As-obtained Ag-NPs were dispersed
Chornet, & Rodrigue, 2007). For possible application for the treatment homogenously in XG-CHT-based PEHs (as shown in Fig. 5) and
of skin lesions, Bellini, Pires, Vasconcelos, and Moraes (2012) prepared showed good antibacterial activity by inhibiting both Gram-positive
XG/CHT-based porous lamellar membranes incorporated with Tween (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria.
80 or Pluronic F68 for possible use for dermal dressings and tissue In addition, good in vitro cytocompatibility (cell adhesion and
engineering applications. This study demonstrated that XG/CHT (1/1 proliferation) of PEHs with NIH 3T3 fibroblast cells was observed (as
ratio) with or without Pluronic F68 shows transparency, high absorp- shown in Fig. 5), indicating the promising use in antibacterial wound/
tion capacity of physiological fluids with adequate stability, in vitro low burn dressing and possible application for skin tissue engineering (Rao
cytotoxicity (L929 cells), skin compatible mechanical property (tensile et al., 2016).
strength), whereas membranes (regardless of the mass ratio) with
Tween 80 showed massive cell death (highly toxic to L929 cells), de- 4.5.2.4. XG-CHT/montmorillonite. Montmorillonite (MMT) is a kind of
spite having appropriate mechanical resistance and behavior in phy- layered silicates (clay mineral) and well known as a polymer modifier
siologic solutions (Bellini et al., 2012). In addition, Bellini, Oliva-Neto, because of its high specific surface area (Mishra, Allauddin, Narayan,
and Moraes (2015) prepared XG/CHT composite films from various Aminabhavi, & Raju, 2012). MMT has widely been applied in
sources of XG, where XG type did not affect most of the properties pharmaceutical (Lin et al., 2002) and tissue engineering applications
significantly (Bellini et al., 2015). (Depan, Kumar, & Singh, 2009; Olad & Azhar, 2014). With only a low
amount of MMT, solvent resistance and mechanical performance of the
4.5.2.1. XG-glycidyl methacrylate grafted CHT. CHT has been grafted polymer-based composites can significantly be improved (Zheng et al.,
with GMA to improve the crosslinking ability of the components 2007). By taking these advantages, Liu, Nakagawa, Chaudhary,
because it has oxirane group that can be chemically modified for Asakuma, and Tadé (2011) prepared XG/CHT/MMT-based scaffolds
prospective biological interest (Kuan, Horák, Plichta, & Lee, 2014). from their suspension via freeze drying process and investigated that
Elizalde-Peña et al. (2013) synthesized hybrid hydrogels by mixing slow cooling rate of freezing led to larger pore size with more tilted
the hybrid natural-synthetic CHT-g-glycidyl methacrylate (GMA) and pores horizontally and uniform network as well as higher hardness
XG. The results showed that swelling index of obtained hydrogels values compared to rapid freezing rate. Further, pore size was
decreased as GMA mass percentage increased and behave as physical decreased while mechanical performance was increased with the
hydrogels. Viscoelastic behavior of pure CHT-g-GMA was enhanced on incorporation of MMT (Liu et al., 2011).
increasing of XG as polysaccharide. In addition, synthesized hydrogels
showed good cell viability of fibroblast cells and demonstrated 4.5.3. XG-gellan gum
synthesized hydrogels to be used possibly for skin graft devices in Gellan gum (GG) as bacterial polysaccharide is a linear anionic exo-
biomedical applications (Elizalde-Peña et al., 2013). polysaccharide having repeat unit comprising of α-L-rhamnose, β-D-
glucose, and β-D-glucuronate with molar ratios 1:2:1 (Osmałek,
4.5.2.2. XG-CHT/cellulose nanocrystals. Rao, Kumar, and Han (2017) Froelich, & Tasarek, 2014). It can form physical hydrogel in the pre-
prepared bionanocomposite hydrogels composed of CHT, XG, and CNCs sence of mono-, di-, and tri-valent cations (Maiti, Ranjit, Mondol,
in the presence of green acidifying agent via electrostatic attraction and Ray, & Sa, 2011; Tang, Tung, & Zeng, 1996). In addition, native GG
hydrogel bonding (complexation of polymer chains). The results showed forms soft and easily deformable gels, while deacetylated GG forms
significantly improved mechanical performance with increased CNCs rigid and brittle gels (Jampen, Britt, & Tung, 2000; Osmałek et al.,
content. In addition, 5-Flurouracil drug (as model chemotherapeutic 2014).
agent)-loaded bionanocomposite hydrogels showed excellent releasing
ability and cytocompatibility with NIH3T3 fibroblast cells for possible use 4.5.3.1. XG-GG/CHT nanoparticles. Dyondi, Webster, and Banerjee
in drug delivery as well as tissue engineering applications (Rao et al., 2017). (2013) prepared injectable XG/GG hydrogels with CHT nanoparticles
(CHT-NPs) (297 ± 61 nm), basic fibroblast growth factor (bFGF), and
4.5.2.3. XG-CHT/antibacterial agent. CHT also possesses antimicrobial bone morphogenetic protein 7 (BMP7) were used in a dual growth-
activity (Zheng & Zhu, 2003) and can be used as antimicrobial polymer. factor system to promote the differentiation of human fetal osteoblast

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A. Kumar et al. Carbohydrate Polymers 180 (2018) 128–144

Fig. 5. (I) (A) Digital images of the formation of PEHs: (a) pristine hydrogel, (b-d) PEHs incorporated with AgNPs, and (e) schematic representation of AgNPs formation in PEHs; (B) UV-
spectra of AgNPs for XG-CHT-2 (a), XG-CHT-5 (b), and XG-CHT-10 (c); (C) TGA curves of XG-CHT-0 (a), XG-CHT-2 (b), XG-CHT-5 (c), and XG-CHT-10 (d), respectively. (II) A
Antibacterial activity of AgNPs on E. Coli and S. aureus for (a and a-1) XG-CHT-0, (b and b-1) XG-CHT-2, (c and c-1) XG-CHT-5, and (d and d-1) XG-CHT-10, respectively; (B) SEM images
of fibroblast cells (NIH 3T3) attachment on PEHs for 3 days of incubation for XG-CHT-0 (a), XG-CHT-2 (b), XG-CHT-5 (c), and XG-CHT-10 (d); and (C) MTT assay for 24 h and 48 h.
(Reproduced with the permission from Ref. Rao et al. (2016)).

cells. XG/GG hydrogels incorporated with nanoparticles showed surrounding tissues in case of Seprafilm group and A/B groups (Fig. 6(II)
significant improvement in cell proliferation and differentiation (B–D)), while more intense peritendinous-adhesions (fibrous bundles
because of sustained release of growth factors. Hydrogels loaded with bridging) were observed in C and D groups as compared to Seprafilm
dual growth factor exhibited a higher alkaline phosphatase and (Fig. 6(II) E and F). In addition, some residuals of XG/GG/HA hydrogel
deposition of calcium compared to hydrogels loaded with single were observed because of slow degradation and Seprafilm was degraded
growth factor. In this study, L929 mouse fibroblast cells viability was fully. However, the gross adhesion scores were significantly lower
greater than 95% and human fetal osteoblast cells viability was found compared to the control group. Further, in vivo histological assessments
to be 80% in the presence of XG/GG hydrogels as evaluated by MTT supported the gross observations by indicating irregular-peritendinous
assay for 48 h. In addition, XG/GG hydrogels showed antibacterial surface and massive fibrous adhesions scars on repair site of untreated
effect against common pathogens such as Staphylococcus aureus, tendons (Fig. 6(III) A and B), mild fibrous scars on Seprafilm treated site
Staphylococcus epidermidis, and Pseudomonas aeruginosa in implant (Fig. 6(III) C and D), a clear gap between tendon and epidermis including
failure. Therefore, encapsulation and stabilization of growth factors few loose bundles of fibrous tissue and residual materials (Fig. 6(III) E–H).
within nanoparticles and hydrogels are promising for bone tissue In contrast, massive fibrous tissues and adhesions between repaired tendon
regeneration (Dyondi et al., 2013). treated with XG-GG/HA hydrogel membranes of C and D groups and
epidermis were observed (Fig. 6(III) I–L). In conclusion, XG-GG/HA
4.5.3.2. XG-GG/hyaluronic acid. Hyaluronic acid (HA) is one of the GAGs hydrogel membranes, prepared using formulation A and B, showed an
family members in our body and has widely been used in biomedical significant anti-adhesive effect on repaired tendons without influencing
applications due to the active influence on several cellular functions as well healing of the injured tendons (Kuo et al., 2014).
as cell attachment, migration, and proliferation. HA has most commonly
been used as a hydrogel form, but it lacks some limitations related to 4.5.4. XG-galactomannan/curcumin
inadequate mechanical performance and compromised further by its rapid Galactomannan (GTM) is a plant hetero-polysaccharide comprising
degradation by physiological enzymes in vivo (Garcia-Fuentes, Meinel, of a main chain of D-mannose with D-galactose side groups
Hilbe, Meinel, & Merkle, 2009; Ko, Tien, Wang, & Chen, 2012). The (Scherbukhin & Anulov, 1999). GTM is non-toxic, biocompatible, and
prevention of peritendinous attachment after the repairing of tendon- can make biomaterials with improved cohesive strength (Mikkonen
surgeries poses a main clinical challenge. Kuo, Chang, Wang, Tang, and et al., 2007; Siqueira et al., 2015). Further, curcumin (CC) is a naturally
Yang (2014) prepared hydrogel membranes and demonstrated the possible occurring anti-inflammatory and anti-oxidant agent of turmeric (Cur-
prevention of attachment of surgical tendon and synovial sheath that often cuma longa) associated with NF-kβ inhibition, apoptosis regulation and
cause poor mechanical repairing of the tendon. In this study, XG-GG/HA- antioxidant effects, including neuroprotective activity during treatment
based membranes of various compositions as well as commercially available of spinal cord injuries (Ormond et al., 2014; Jin et al., 2014). However,
Seprafilm (comprised of HA and carboxymethyl cellulose (CMC)) were used the low bio-availability and weak stability limit its applicability for
and wrapped around repaired tendons in a 8-week-old male Sprague- large productive purposes (Metzler, Pfeiffer, Schulz, & Dempe, 2013;
Dawley rat model to examine grossly and histologically after 3 weeks of Requejo-Aguilar et al., 2017). Da-Lozzo et al., 2013 prepared XG-GTM/
healing process. The observation showed that certain compositions of XG- CC-based hydrogel to evaluate viscoelastic behavior and biocompat-
GG/HA hydrogel membranes rapidly swelled and readily and closely ibility from topical application on chick embryo chorioallantoic mem-
blanketed onto tendon tissue, and reduced adhesion with equal ability brane (CAM). The addition of CC (≤0.5 mg/mL) had not any effect on
without affecting the strength of tendon compared to Seprafilm. In addition, the viscoelastic behavior of the XG-GTM hydrogel network and XG-
hydrogel membranes degraded slowly, allowing functioning as a barrier for GTM/CC hydrogel systems showed high biocompatibility. Further, XG-
extended duration. For this evaluation, tests were grouped with four GTM hydrogels did not show any tissue injury of CAM. Actually, foreign
formulations A, B, C, or D for tendon wrapped with Seprafilm or XG-GG/ material can cause tissue injury under the CAM and associated-re-
HA membranes (Fig. 6(I) (A–E)). Fig. 6(II) shows in vivo gross observations sponses of tissues (i.e., inflammation and fibrosis) if inflammatory and
of repaired rat Achilles tendons for 3 weeks of healing process, where wound healing steps occur in an uncontrolled-manner. In this study,
intense adhesions were observed by showing of massive and dense poly-L-lactic acid (PLLA) films (which cause CAM tissue injury) as po-
adhesive-bundles between tendon and controgroup surrounding tissues sitive control promoted neovascularization, opacity, fibrosis, while XG-
(Fig. 6(II) (A)). Loose adhesion bundles were formed bridging tendons and GTM and XG-GTM/CC hydrogel systems were completely absorbed and

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A. Kumar et al. Carbohydrate Polymers 180 (2018) 128–144

Fig. 6. (I) Digital micrographs of animal model demonstrating as; (A) Sharp-dissection of Achilles tendon, (B) Transection of Achilles tendon at a mid-point, (C) Repairing of tendon with
5-0 Prolene sutures, (D) smoothly fit XG-GG/HA hydrogel membrane wrapping of post repaired tendon (as indicated by black arrow), (E) irregularly deformation of fitting Seprafilm on
the tendon (as indicated by white arrow). (II) Gross analysis of repaired Achilles tendon after 3 weeks of healing process in a rat model as control group (A), Seprafilm group (B), group A
(C), group B (D), group C (E), and group D (F), respectively (the black circle shows massive dense adhesive band, while black arrow shows residual membranes). (III) H & E staining and
Masson’s trichrome staining of repaired tendon. (Reproduced with the permission from Ref. Kuo et al. (2014)).

did not provoke injury to tissues similar to control system without cases, i-CGN and k-CGN were fibre reinforcements, while KG and XG
implants. In addition, histological sections confirmed an increase in the were as amorphous matrix for composite hydrogels (Juris et al., 2011).
number of micro-vessels in PLLA exposed-CAM and XG-GTM hydrogels In another study, Almeida, Mueller, Hirschi, and Rakesh (2014) in-
either without or with CC did not stimulate any structural changes in vestigated the rheological and thermal properties of the same four
the CAM (as compared to control systems without implants). Moreover, polysaccharides-based skin hydrogels on considering the mixing of
histological sections supported the behavior of obtained hydrogels in precise polysaccharide composition and storage conditions. The ob-
terms of amount of collagen fibers (Da-Lozzo et al., 2013). tained skin hydrogels were found to be strong gels at all times due to
gradual free water loss. However, the value of elastic (G’) and loss (G”)
modulus of hybrid hydrogels was one to two-folds higher than that of
4.5.5. XG-i-/k-carrageenan-konjac gum magnitude of the hydrogels without the presence of either XG or i-CGN
As anionic polysaccharide, carrageenan (CGN) has widely been used after 12 h of time period in air. All four polysaccharide-based strong
as an emulsifier, thickening, gelling, and stabilizing agent in pharma- hydrogels could be analysed as epidermis skin scaffolds, whereas more
ceutical (Mammarella, & Rubiolo, 2003), industrial, and tissue en- porous weak gels without the presence of either XG or i-CGN for dermis
gineering applications (Kumar, Rao, Haider et al., 2017; Popa, skin scaffolds (Almeida et al., 2014).
Gomes, & Reis, 2011; Santo et al., 2009). CGN resembles to naturally
occurring glycosaminoglycans (GAGs) to some extent due to the pre-
sence of sulphated disaccharides in their backbone composition 4.5.6. XG-SA/CNCs/halloysite nanotubes
(Bhattacharyya et al., 2010). Depending on degree of sulfation (be- SA as polysaccharide (produced from brown sea algae) has broadly
tween 15% and 40%), CGN has been categorized in three ways: (1) been used in biomedical applications due to its inherent hydrophilicity,
kappa-CGN (k-CGN, one sulfate group per disaccharide and forms easy processing, biocompatibility, and its gelling properties under mild
strong and rigid gels), iota-CGN (i-CGN, two sulfate groups and forms conditions (Gerecht-Nir, Cohen, Ziskind, & Itskovitz-Eldor, 2004;
soft gels), and lambda-CGN (λ-CGN, three sulfate groups, known as Kumar et al., 2017a). However, it shows some limitations as un-
highly sulfated, and less likely to form a gel structure, but can form gels controlled degradation, low mechanical property, and lack of cell affi-
when mixed with proteins rather than water). In addition, konjac gum nity (Kumar, Lee et al., 2017). Further, halloysite nanotubes (HNTs) as
(KG) is a type of hydrocolloid-gum (produced from the tubers of Ara- clay material have shown a good potential in developing new organic-
ceae amorphophallus) consisting of 60–70% of konjac glucomannan and inorganic hybrid hydrogels for the applications in biomedical area be-
has extensively been used in the industrial applications (e.g., food in- cause of their biocompatible nature (Joussein et al., 2005; Liu, Wu,
dustry, petroleum drilling) (Dhawan & Kaur, 2007). Due to its high Jiao, Xiong, & Zhou, 2013). On considering these facts, Kumar, Rao,
viscosity, KG is normally used with other hydrocolloids in improving and Han (2017) investigated the synergic effect of CNCs and HNTs on
their viscosity and quality of foods (Yaseen, Herald, Aramouni, & Alavi, the structure-property-relationship of the nanocomposite scaffolds
2005), and can greatly change the specific physicochemical properties composed of XG, SA, CNCs, and HNTs. The results showed good in-
of the blended systems with other components (e.g., XG, CGN, SA, MC, terfacial interactions and homogenous dispersion of the CNCs and HNTs
sodium carboxymethylcellulose, hydroxypropylmethylcellulose) (Liang within the XG-SA polymeric network. In addition, the obtained four
et al., 2011). Juris et al. (2011) investigated a range of mechanical component nanocomposite scaffolds exhibited high porosity with good
properties, degradation duration, and good biocompatibility with pore-interconnectivity and improved mechanical stability, thermal
human fibroblast (MeWo) cells of skin hydrogels composed of four stability, and good cytocompatibility with osteoblastic MC3T3-E1 cells
polysaccharide mixtures (XG with i-CGN, k-CGN, and KG). In these (Kumar et al., 2017b).

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4.6. XG-synthetic polymer-based biomaterials injectable hydrogels and revealed that blend solution recovered its high
viscosity immediately and formed hydrogel rapidly at body tempera-
4.6.1. XG-polypyrrole ture (37 °C). For in vivo analysis for gelation, elimination, and bio-
The electrically conducting polymers (e.g., polypyrrole (PPy)) have compatibility in rat body, The volume of hydrogel increased from day 1
been applied for biomedical engineering applications because of their to day 7 post-injection because of swelling and after that its volume
good electrical conductivity, thermal stability, and ability for copoly- decreased gradually followed by the disappearance completely after
merization without compromising electro-activity. For this reason, PPy 36 days of the injection (much faster than the erosion in vitro). The
showed good potential for biomedical applications, especially for tissue inflammatory cells in the tissues were observed around the implanted
engineering (Zelikin et al., 2002). The cytocompatibility (in vitro and in XG/MC hydrogel and inflammation decreased with the increased im-
vivo) of PPy has been proven with a broad range of cell types (Ateh, plantation time. Further, this cells inflammation almost disappeared on
Navsaria, & Vadgama, 2006; George et al., 2005), but the brittleness the day of 27 post-injection and histology of the surrounding tissues
and inability to manipulate mechanically and processing, it needs was recovered completely on the day of 37 post-injection. In addition,
blending with other materials (Sajesh, Jayakumar, Nair, & Chennazhi, in vitro DOX-loaded XG2/MC10 hydrogels showed sustained release
2013). Moreover, the association of conducting polymer as polypyrrole behavior. XG/MC blend could be a promising injectable hydrogel for
(PPy) with XG hydrogel exhibits high charge density (electroactivity), long-term drug delivery system (Liu & Yao, 2015). The summary of the
good biocompatibility, and forms mechanically stable biomaterial recent studies of the XG-based biomaterials for tissue engineering, as
(Bueno, Takahashi, Catalani, de Torresi, & Petri, 2015). The fibroblast described in previous sections, is given in Table 1.
cells adhesion and proliferation onto XG and XG/PPy scaffolds were
analysed in the absence and the presence of external magnetic field
(0.4 T) for different periods and XG/PPy scaffolds revealed more fa- 5. Conclusion and future perspectives
vored fibroblast adhesion and proliferation due to higher surface
roughness and hydrophobicity and particularly under external mag- Xanthan gum is a microbial exo-polysaccharide produced by
netic field (0.4 T) (Bueno et al., 2015). In this case, XG having high Xanthomonas bacteria and it has good water-solubility, excellent bio-
negative charge density (Bueno & Petri, 2014) might cause electrostatic compatibility, intrinsic immunogenic ability and high molecular weight
repulsion to proteins which are responsible for cell adhesion. The polysaccharide having branched polymeric chains. Further, due to
morphological analysis (as shown in Fig. 7) demonstrates that fibroblast several advantages, such as such as high viscosity at a very low con-
cells hardly observed (only few spherical form; Fig. 7A-a) on XG scaf- centration, physical hydrogel with bivalent cation, shear-thinning
fold in the absence of EMF while many cells having more elongated property, pseudo-plastic behavior, and thermal stability (against hy-
form (Fig. 7A-b) in the presence of EMF. In case of XG/PPy scaffolds, drolysis) that is far better than other polysaccharides or polymers
more cells adhered and proliferated in the absence (Fig. 7A-c) and in (Stokke & Christensen, 1996), but the low mechanical performance and
the presence (Fig. 7A-d) of EMF compared to pure XG scaffold. processing of the xanthan gum limit its applicability for different tissue
Further, this proliferation behavior of fibroblast cells was supported engineering applications. Also, it has large number of hydroxyl groups
by MTT assay (Fig. 7B) in the absence and presence of EMF, providing (eOH) and the free carboxyl groups (eCOO−) that can be used for
same level cells proliferation for pure XG scaffold in the presence of chemical functionalization or modification in order to manipulate or
EMF and XG/PPy scaffolds in the absence of EMF. This explored the optimize its physicochemical and biological properties. As described in
manipulation of cells proliferation under magnetic stimulus and charge this review, XG has been successfully applied in several industrial and
density (Bueno et al., 2015). biomedical applications, such as foods, food-packaging, water-treat-
ments, toiletries, petroleum industry, oil recovery, cosmetics, and drug
delivery systems, but could not be exploited much in tissue engineering
4.6.2. XG-methylcellulose
area. Therefore, the recent trends on the use of XG-based poly-
Methylcellulose (MC) is one of the cellulose derivatives (water so-
saccharide formulations for various tissue engineering applications are
luble) and has been applied to increase the viscosity of foods or paints.
precisely reviewed for better understanding of this biopolymer in this
It has good characteristic in aqueous solution to induce temperature
area. It shows a quite promising future as a biopolymer as demonstrated
dependent sol-gel transition leading by hydrophobic interaction and
in this review by emerging tissue engineering products. Moreover, the
can be manipulated by the various parameters, such as concentration,
shear-thinning and gelling property of XG can be more beneficial in the
additives, degree of substitution, and molecular weight (Altomare,
area of 3D bioprinting of the tissue scaffolds and/or tissue models for
Cochis, Carletta, Rimondini, & Farè, 2016; Park, Kim, Yoon, & Park,
future tissue engineering applications.
2016). Liu and Yao (2015) prepared XG/MC thermo-responsive

Fig. 7. (A) SEM images of fibroblast cells grown onto XG scaffold without (a) and with (b) EMF, XG/PPy scaffolds without (c) and with (d) EMF (Scale bar – 100 μm). (B) MTT assay of
fibroblast cells viability/proliferation onto XG and XG/PPy scaffolds without and with EMF. All values were subtracted from control values. (Reproduced with the permission from Ref.
Bueno et al. (2015)).

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Table 1
XG-based biomaterials for tissue engineering applications.

Formulation Microorganisms/Cells/Tissues used (in vitro/in Targeted application Reference


vivo)
Polymer matrix Other components

XG – Chondrocytes cells Articular cartilage tissue engineering Han, Wang et al. (2012)
XG IL-1β Chondrocytes cells Articular cartilage tissue engineering Han, Shao et al. (2012)
XG CA, BSA and LYZ Not used Wound dressing and skin tissues Bueno and Petri (2014)
XG SNP Chondrocytes cells Articular cartilage tissue engineering Chen et al. (2015)
XG -CH2COOH Chondrocytic ATDC5 cells Cell-therapies Mendes et al. (2012)
XG SAn Human lens epithelial cells (HLECs) and white Cell-therapies and drug delivery systems Wang, Han et al. (2016)
rabbits
XG DOPE Chondrocytic ATDC5 cells Cell-based transplantation therapies Mendes et al. (2013)
XG MNPs Murine fibroblasts (3T3 L1) Cells adhesion and proliferation in tissue Bueno et al. (2013)
engineering
XG MNPs Embryonic stem cells Neuronal tissue engineering Glaser et al. (2015)
XG GNPs, DOX Human lung cancer cells (A549 cells) Cancer-therapies Pooja et al. (2014)
XG nHAp Not used Bone tissue engineering Izawa et al. (2014)
XG Str-nHAp Osteoblast cells (OFCOLL II) Bone tissue engineering Bueno et al. (2014)
XG SG, CNCs Osteoblastic MC3T3-E1 cells Bone tissue engineering Kumar, Rao, Kwon, Lee, and
Han (2017)
XG-CS – Cellular growth of Saccharomyces pombe Drug release and Tissue engineering Oprea et al. (2012)
(microorganism) and mouse
XG-CHT Tween 80 or Pluronic Fibroblast cells (L929 cells) Skin tissue engineering Bellini et al. (2012)
F68
XG-(CHT-g-GMA) – Human primary dermal fibroblasts Skin tissue engineering Elizalde-Peña et al. (2013)
XG-CHT CNCs NIH3T3 fibroblast cells Drug delivery as well as tissue engineering Rao et al. (2017)
applications
XG-CHT Ag-NPs NIH3T3 fibroblast cells Wound/or burn/or skin tissue Rao et al. (2016)
XG-CHT MMT Not used Scaffolding for tissue engineering Liu et al. (2011)
XG-CHT CHX Human dermal fibroblast Acute or chronic periodontitis Kim et al. (2017)
XG-GG CHT-NPs Human fetal osteoblast cells and L929 mouse Bone tissue engineering Dyondi et al. (2013)
fibroblast cells
XG-GG/HA – L929 fibroblast cells Tendon surgeries Kuo et al. (2014)
XG-GTM CC In vivo chick embryo CAM model Wound dressing and skin tissues Da-Lozzo et al. (2013)
XG-i- & k-CGN/KG – Human fibroblast (MeWo) cells Skin tissue engineering Juris et al. (2011)
XG-SA CNCs and HNTs Osteoblastic MC3T3-E1 cells Bone tissue engineering Kumar et al. (2017b)
XG-PPy – Fibroblast cells Cells adhesion and proliferation in tissue Bueno et al. (2015)
engineering
XG-MC DOX Male SD rat tissues (in vivo) Drug release and Tissue engineering Liu and Yao (2015)

Acknowledgements Behari, K., Pandey, P. K., Kumar, R., & Taunk, K. (2001). Graft copolymerization of ac-
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grant and by the Basic Science Research Program through the National dressings and scaffolds for the treatment of skin lesions. Journal of Applied Polymer
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