Carbohydrate Polymers 180 (2018) 128–144

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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.

Tissue engineering is a process of the regeneration of damaged tis-
sues or the reconstruction of the organs, by adapting appropriate three-
dimensional (3D) microenvironment for proper cell adhesion, pro-
liferation, followed by the formation of new tissues. This proposed 3D
microenvironment can be achieved by the fabrication of artificial bio-
material structure that may mimic biologically to natural extracellular
matrix (ECM) as supportive architecture (Priya, Jungvid, & Kumar,
2008; Singh, Kaur, Rana, & Kennedy, 2016). Various polymers (natural
or synthetic or their combinations) have extensively been used for this
purpose to produce artificial biomaterial structures in different forms
such as micro-spheres, micro-beads, micro/nano-particles, nanofibrous
In this case hydrogel form may provide most favored 3D micro-
environment compared to other forms. For this, natural occurring
polymers show many advantages over synthetic polymers in synthe-
sizing appropriate hydrogels for cell fate and proper regeneration of
tissues. Natural polymers have high availability, easy processing, ex-
cellent biocompatibility, and they mimic biologically to natural ECM of
the tissues (Singh et al., 2016).
1.1. Polysaccharides
Polysaccharides as natural polymers are highly abundant in the
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.
and microorganisms and have widely been applied in various potential
biomedical applications (e.g. drug delivery and tissue engineering) due
to their readily availability, cost-effective, easy processing, and ex-
cellent biocompatibility nature (Kumbar et al., 2011; Malafaya,
Silva, & Reis, 2007; Shelke, James, Laurencin, & Kumbar, 2014). Among
polysaccharides, xanthan gum is microbial and an exo-polysaccharide
that has also been used in biomedical applications due to its excellent
biocompatibility and gelling property (Petri, 2015).
2. Xanthan gum
2.1. History
Xanthan gum (XG) was discovered in 1950s at the National Center
for Agricultural Utilization (previously known as Northern Regional
Research Center) of the United Sates Department of Agriculture
(USDA). First industrial production of XG was performed in 1960 fol-
lowed by the availability of commercially product in 1964
(Margaritis & Zajic, 1978). This is the second microbial polysaccharide,
after dextran (early 1940s), which was industrially commercialized. XG
is non-toxic, non-sensitizing, and does not cause any eye or skin irri-
tation, and has also been approved by the United States Food and Drug
Administration (FDA) in 1969 (Kennedy & Bradshaw, 1984).
2.2. Origin, structure, and chemistry of XG
XG has good water-solubility, excellent biocompatibility, and is a high
molecular weight exo-polysaccharide having branched polymeric chains
(Le & Turgeon, 2013; Rao, Kumar, Haider, & Han, 2016). The primary
structure of XG was established in 1975 and it consists of pentasaccharide
subunits. XG comprises of D-glucosyl, D-mannosyl, and D-glucuronyl acid
residues in a 2:2:1 molar ratio with varying proportions of O-acetyl and
pyruvyl residues. The structure of XG is a linear (1–4)-linked D-glucose si-
milar to cellulose backbone (Faria et al., 2011; Jansson, Kenne, & Lindberg,
1975). The secondary structure of XG is shown as five-fold right-hand he-
lical structure with a pitch of 4.7 nm and a diameter of 1.9 nm (Moorhouse,
Walkinshaw, & Arnott, 1977) and undergoes via a thermally-induced order-
disorder conformational transition and could be formed by high tempera-
tures and low salt concentrations (Cheetham & Mashimba, 1992). Here salt
stabilizes the order conformation which is responsible for extraordinary
stability of the XG (Young, Martino, Kienzle-Sterzer, & Torres, 1994), and
that evidenced most commonly the dimeric or double-stranded (Hjerde,
Kristiansen, Stokke, Smidsrød, & Christensen, 1994; Stokke & Christensen,
1996). XG is soluble in cold and hot water and requires intensive agitation
when exposed to aqueous medium to avoid the lumps formation. XG so-
lutions behave like non-Newtonian fluids and are of highly pseudoplastic
behavior, and the apparent viscosity (conformational status of polymeric
chains) that altered significantly with time and/or shear rate. Generally, the
thermal stability of XG against hydrolysis is far better than many other
water-soluble polysaccharides or polymers (Stokke & Christensen, 1996),
possibly because of the ordered helical structure of XG that protects the
molecules from de-polymerization. Therefore, viscosity of XG solutions is
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-
gradable (completely within 2 days) naturally occurring polymer. However,
due to the backbone similarity with cellulose, XG is resistant to the attack of
the common cellulases and the tri-saccharide side chains of XG are likely to
be a barrier to enzymatic attack. Only in disordered form, it can be cata-
lyzed the cleavage of the main chain when attacked with fungal cellulases,
not in ordered helical form (Rinaudo & Milas, 1980). The molecular weight
distribution of XG ranges from 2 × 106 to 20 × 106 Da, depending on the
association of chains forming aggregation of several individual chains and
the variation in fermentation conditions. The synthesis of XG is believed to
be similar to the synthesis of exo-polysaccharide by other Gram-negative
bacteria and generally produced by various species of Xanthomonas bac-
teria (a Gram-negative bacteria genus), such as X. arboricola, X. axonopodis,
129
Carbohydrate Polymers 180 (2018) 128–144
X. campestris, X. fragaria, X. gummisudans, X. juglandis, X. phaseoli, X. vas-
culorium. Industrially, XG is produced with the fermentation of glucose by X.
Campestris, a plant-associated bacterium (Becker, Katzen, Pühler, & Ielpi,
1998; Garcıa-Ochoa, Santos, Casas, & Gomez, 2000; Petri, 2015). The bio-
synthetic pathway of XG synthesis can be categorized in three steps: (1)
Uptake of simple sugars followed by conversion to nucleotidal derivatives,
(2) Assembly of pentasaccharide subunits associated to an iso-
pentylpyrophosphate carrier, and (3) Polymerization of repeat units of
pentasaccharide followed by their secretion (Palaniraj & Jayaraman, 2011;
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-
tensively been used in the food industry because of its efficient thickening
ability, high solution viscosity at very low concentration, pseudoplastic
behavior in aqueous solution (Faria et al., 2011; Milas, Rinaudo,
Knipper, & Schuppiser, 1990), high stability in broad range of temperature,
pH, ionic strength, and stability under shear (Hui, 2006; Petri, 2015). XG
chains have the ability to form physical networks with bivalent cations by
involving two disaccharide unites in the main chain and O-acetyl and
pyruvyl residues in side chains which lead to intramolecular crosslinking
and chains contraction. These acetyl and pyruvyl residues vary with bac-
terial species and fermentation conditions to prepare the XG gum. The
secondary structure of XG undergoes an “order-disorder” transformation
from helix to coil structure depending on pH, nature of electrolyte, ionic
strength of solutions, and acetyl and pyruvyl contents (Khouryieh, Herald,
Aramouni, Bean, & Alavi, 2007). The content of acetyl and pyruvyl residues
also affects the behavior of XG aqueous solution. In general, lower pyruvyl
content shows low viscosity and higher pyruvate content promotes gel
behavior via improved macromolecular association, whereas higher acetyl
content inhibits the gelation behavior of XG aqueous solution (e.g. deace-
tylation leads an increase in viscosity) (Bergmann, Furth, & Mayer, 2008;
Dário, Hortêncio, Sierakowski, Neto, & Petri, 2011; Petri, 2015; Shatwell,
Sutherland, & Ross-Murphy, 1990; Tako & Nakamura, 1984). This con-
formational transition to double-helix is a pre-requisite for gel formation
that leads to stronger materials. As described elsewhere, XG gels (from
9.1–50.0 wt%) have been prepared by using ionic liquid as green solvent
(e.g., 1-butyl-3-methylimidazolium chloride; BMIMCl) via regularly-ordered
interaction of the imidazolium counter cations of XG with other BMIMCl
molecules. The obtained gels showed thermally-induced shape-memory
effect and good mechanical performance strongly depending on XG con-
centrations. These gels showed facile conversion into high performance
hydrogels by soaking them in water and further ionically crosslinked with
Na+, Ca2+, and Fe3+ in their corresponding aqueous solutions of 1.0 M
NaCl, 0.2 M CaCl2, and 0.1 M FeCl3, respectively. The obtained ion-ex-
changed hydrogel with Ca2+ showed better mechanical performance under
both tensile and compressive modes compared with ion-exchanged hy-
drogel with Na+, whereas hydrogel with Fe3+ exhibited hard and brittle
nature of the ion-exchanged hydrogel network. Moreover, ion-exchanged
hydrogels with Na+ and Ca2+ showed salt concentration-induced re-
sponsive properties with reversible swelling-shrinking behavior (Izawa,
Kaneko, & Kadokawa, 2009; Izawa & Kadokawa, 2010).
2.3. Biocompatibility and immunological response
XG is well known for its non-toxicity, excellent biocompatibility and
intrinsic ability as immunological agent (Han, Ling, Wang, Wang,
& Shao, 2012; Kumar, Rao, Kwon, Lee, & Han, 2017; Le & Turgeon,
2013; Rao et al., 2016). As described elsewhere, the biocompatibility of
the hydrogel prepared by complexation of XG with other poly-
saccharide as chitosan also investigated in vitro and in vivo models with
fibroblast cell line L-929 and showed good biocompatibility analysis
(Chellat et al., 2000). Further, to maintain mobility in the joint and
reducing the degeneration of cartilage, several glycosaminoglycans
(e.g., chitosan (Chen, Liu, Du, Peng, & Sun, 2006), sodium alginate
(Legendre, Baugé, Roche, Saurel, & Pujol, 2008), hyaluronic acid
(Abate, Pulcini, Iorio, & Schiavone, 2010)) have shown chondro-pro-
tective effects and have been used for the treatment of osteoarthritis
A. Kumar et al.
(OA). Intra-articular injection of hyaluronic acid (HA) is considered as
an effective treatment for OA. However, HA is unstable and prone to
degrade quickly by hydrolytic or enzymatic reactions in vivo (Zhong
et al., 1994). For better alternative, intra-articular injection of XG has
been prepared that could protect the cartilage at joint and reduce the
papain-induced osteoarthritis (OA) progression due to its similar
rheology and viscosity as that of HA (Han, Ling et al., 2012; Han, Shao
et al., 2012). In addition, XG alone showed no adverse effects on rabbit
chondrocyte cells viability (Han, Wang et al., 2012). Further, the in-
trinsic adjuvant property of XG can be used for improving the immune
response of other agents. In this case, Ishizaka, Sugawara, Hasuma,
Morisawa, and Möller (1983) investigated the effect of XG as adjuvant
on murine lymphocytes in vitro. XG successfully influenced both a sig-
nificant improvement in DNA synthesis in mouse splenic B cells and
thymocytes, including polyclonal immunoglobulin M (IgM) and im-
munoglobulin G (IgG) antibody responses in B cells. However, XG-
modified thymocytes did not show helper or suppressor functions. XG
showed similar effect on polyclonal antibody responses as lipopoly-
saccharide (LPS) in murine systems. Further, XG showed weak trigger
of Hamster spleen cells for the production of nonspecific antibody,
while LPS did not trigger spleen cells at all. In addition, immature B
cells can be stimulated by XG as well by LPS. Spleen cells from C57BL/
10 mice were fully responsive to LPS, but not responsive or slightly
responsive only to XG. In other side, XG stimulated spleen cells from
C3H/HeJ mice and other seven mouse strains for the production of
polyclonal antibody, but non-responsive to LPS (Ishizaka et al., 1983).
In addition, XG has also been applied for antitumor effects (Takeuchi
et al., 2009), in bioadhesive compositions for intranasal influenza virus
immunizations (Chiou et al., 2009), and in the preparation of subunit
vaccine against leptospirosis (Bacelo et al., 2014).
2.4. Application of XG in industrial and biomedical area
XG and XG-based biomaterials have extensively been used in a wide
range of applications such as foods and food-packaging (Fareez, Lim,
130
Carbohydrate Polymers 180 (2018) 128–144
Fig. 1. Common methods of modification of XG
polymer chains used in tissue engineering.
Mishra, & Ramasamy, 2015; Hazirah, Isa, & Sarbon, 2016), cosmetics
(Garcıa-Ochoa et al., 2000; Palaniraj & Jayaraman, 2011), water-based
paints (Palaniraj & Jayaraman, 2011), water treatments (Mittal, Parashar,
Mishra, & Mishra, 2014; Mittal, Kumar, & Ray, 2016), jet injection printing
(Faria et al., 2011), petroleum industry (Faria et al., 2011), toiletries
(Palaniraj & Jayaraman, 2011), oil recovery (Garcıa-Ochoa et al., 2000),
construction and building materials (Chang, Im, Prasidhi, & Cho, 2015),
drug delivery (Bhunia, Giri, Nasim, Chattopadhyay, & Bandyopadhyay,
2013; Jian, Zhu, Zhang, Sun, & Jiang, 2012; Lungan et al., 2015; Shalviri,
Liu, Abdekhodaie, & Wu, 2010), etc., because of its superior rheological
properties like ability to form highly viscous solution at low shear forces,
high pseudo-plasticity, and a high viscosity yield value (Rosalam & England,
2006). The XG solution is stable over a broad range of temperatures (up to
90 °C), salt concentrations, and pH (2–11) (Rosalam & England, 2006).
These superior properties of XG resulted in using for biomedical applica-
tions (e.g., drug delivery and tissue engineering) apart from industrial ap-
plications (Faria et al., 2011). These superior properties of XG can be un-
derstood in terms of non-Newtonian behavior, high viscosity yield (even at
low concentrations; e.g., 600–2000 ppm), low sensitivity of viscosity to
changes in salinity, resistant to mechanical degradation, biodegradable and
environment friendly biomaterial (Rosalam & England, 2006). Only limita-
tion of XG is slow dissolution rate that can further be improved by some
physical means (Sandford, Baird, & Cottrell, 1981). The dissolution process
of XG involves to steps (Van Krevelen, 1976): (1) solvent penetration to XG
for swelling and (2) XG dissolves into the solvent phase. In addition, XG as
hydrocolloids can form ‘Fish-Eyes’ (Sandford et al., 1981) during dissolution
when XG particles begin to hydrate. In brief, a gelatinous layer of partially
hydrated XG forms on the outside of the XG particle and cannot leave ra-
pidly the surface of particle. This prevents water for penetrating to complete
dissolution of particle via complete hydration (Su, Ji, Lan, & Dong, 2003).
3. Modification of xanthan gum
XG has large number of hydroxyl groups and carboxylic groups that
may be modified or functionalized by physical, chemical, chemo-
A. Kumar et al.
enzymatic and plasma-irradiation treatments or their combinations in
order to obtain improved properties of the XG as biomaterial. These
some common modification treatments can be described as follows (as
depicted in Fig. 1).
3.1. Modification by physical method
The rheological property of the XG solution is an important factor
for designing biomaterials that can be used to improve remedial
amendment delivery into low-permeable zones in the subsurface het-
erogeneous compositions and/or injectable hydrogels for biomedical
applications, especially tissue engineering. The viscosity of XG solutions
increases strongly with the increasing polymer concentration or mole-
cular weight. At low shear rate, the viscosity of XG aqueous solution is
higher than the magnitude of the viscosity at high shear rate and at low
shear rate, this viscosity further increases linearly with the increase of
XG solution concentration within the range 0.3–2.0 g/L, when no salt is
added to the XG solution (Garcıa-Ochoa et al., 2000; Zhong, Oostrom,
Truex, Vermeul, & Szecsody, 2013), but this viscosity as well as degree
of shear-thinning property of XG solution (< 2.0 g/L) substantially
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
changes in molecular-conformation levels such as ordered (helix mo-
lecular conformation) and disordered (extended configuration with a
larger hydrodynamic volume due to strong Columbic repulsions be-
tween like charges) states. Actually, at lower concentration (< 2.0 g/L),
the addition of salt neutralizes the ionic charge of XG molecules and
therefore, elongated molecules are transformed into helical conforma-
tion of the molecules by reducing to lower hydrodynamic volume and
thereby solution viscosity. While at higher polymer concentration
(> 2.0 g/L), the effect of local charge inversion and subsequent chain
expansion due to intermolecular interactions (e.g., entanglements of
polymeric molecules) is more effective than that of change in hydro-
dynamic volumes of molecules. Therefore, XG solution prepared with
deionized water (no ions) had much higher viscosity compared to tap
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
ions (e.g., Na+, Ca2+) and remedial amendments (e.g., ethyl lactate,
phosphate, and sodium lactate) in XG solution. However, the addition
of remedial amendments maintained shear-thinning properties
(Bergmann et al., 2008; Zhong et al., 2013). In another study, the effect
of salt on the secondary structure (double helical conformation) of XG
was analysed in terms of salt-responsive conformation change after
denaturation and renaturation of XG solution by heating and gradually
cooling, respectively. In pure water, incomplete renaturation (e.g.,
single helical structure) of XG was observed; whereas double helical
conformation (as present is native XG) was observed when salt was
added. In addition, less rigid chains were observed when salt was added
compared with pure water XG solution (Camesano & Wilkinson, 2001).
By physical means, the rheological behavior of XG solutions can be
modified and achieved in the following ways: (1) using mechanical
treatment, (2) using oil and/or W/O emulsions, (3) the blending of
proteins and/or other unmodified/modified polymers, and (4) using of
metal ions.
3.1.1. Using mechanical effect
The rheological and polydispersity aspects of XG by mechanical
modification (high-pressure homogenization) were investigated as an
alternative treatment to enzymatic and chemical modification. In this
treatment, structured-network of XG solutions was lost gradually de-
pending on the severity of the high-pressure homogenization. The
viscosity, storage and loss modulus of XG solutions were decreased
upon high-pressure homogenization due to partial loss of junction zones
and increased disordered structure (slight decrease in molecular mass
131
Carbohydrate Polymers 180 (2018) 128–144
and radius of gyration). Also, the increase in maximum packing fraction
due to the increase in polydispersity and hydrodynamic radius is the
cause of the decrease in viscoelastic behavior. The changes in the
structural and rheological properties of XG were not reversible and
significant recovery was not observed over a period of 11 weeks (Eren,
Santos, & Campanella, 2015). Further, the effect of micro-fluidization
on the physical properties of XG was investigated and this treatment
reduced the intrinsic viscosity, pseudo-plastic behavior and flow bi-
refringence of the treated XG samples. A degradation mechanism of XG
was proposed by using micro-fluidization (a dynamic high pressure
treatment), where increased micro-fluidization severity resulted a re-
latively mild degradation of the main chain via (1) the disruption of
aggregates and (2) reversible disruption of the double-stranded con-
formation. In addition, XG treated at high ionic strengths facilitated
more mechanical degradation due to the increased ordered stiffer mo-
lecules that experienced effective stresses strongly under high shear and
cavitation forces exist in the micro-fluidization chamber. This treatment
can be another way to get a controlled and relatively mild degradation
of XG without affecting its chemical composition (Laneuville,
Turgeon, & Paquin, 2013).
3.1.2. Using oil and/or W/O emulsions
The lubrication behavior of the XG fluid gels and sunflower oil
modified-XG fluid gels was investigated. XG-based fluid gels were
formed by applying shear forces during cooling via conformational
transition. The addition of 10.0% (w/w) sunflower oil to the continuous
phase of XG fluid gels reduced the friction coefficient. Further, the
addition of 10.0% (w/w) of triglycerides stabilized w/o emulsion to the
continuous phase of XG fluid gels similarly reduced the friction coef-
ficient. This was possibly due to the surface-parting (during soft-tri-
bology experiment) of ridged emulsion particles and limiting de-
formation of fluid gel particles. In this study, the addition of only 7%
(w/w) of sunflower oil to the XG fluid gels and triglycerides stabilized
w/o emulsions showed viable route to obtain lubrication behavior si-
milar to sunflower oil (Hamilton & Norton, 2016).
3.1.3. Blending of proteins and/or other unmodified/modified polymers
The modification of XG with β-lactoglobulin (βlg) by inducing
electrostatic attraction interaction in network formation was per-
formed. In this study, the gelation mechanism of βlg and XG mixtures
was monitored by ratio of βlg and XG and their concentrations. With
the initial very weak network of XG, the βlg aggregated along the
chains of XG by facilitating as a crosslinking agent and 4.8 × 10−3 wt%
as lowest concentration of XG was observed for possible gelation, and
more elastic gels were formed at higher XG concentrations. Further, the
ratio of βlg and XG strongly affects the kinetics of gelation and mainly
controls the gelation process and gel-network. The increasing electro-
static attractive interaction between βlg and XG with decreased pH
resulted in the formation inter-polymer complexes via the formation of
soluble complexes (sol-gel transition) at the point of gelation
(Le & Turgeon, 2013). In another study, a gel composed of en-
zymatically (α-galactosidase)-modified guar gum was prepared and XG
and investigated the critical gelling behavior and rheological properties
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
the removal of galactose residues from EMGG showed improvement in
the interaction between GG and XG in the formation of synergistic gel,
whereas unmodified-GG did not show such a synergistic effect regard-
less the concentration of salt. This synergistic gelation was successfully
described by the cascade model. The number of crosslinking sites per
chain (optimal functionalities of EMGG and XG) were measured to be
300 and 30, respectively. However, the values of optimum functionality
were best measured from the analysis of modulus data (Mao,
Zeng, & Chen, 2012).
A. Kumar et al.
3.1.4. Using metal ions
For improvement in rheological properties of XG, several trivalent
ions (e.g., Cr3+, Al3+, Fe3+) have also been used for modifying gelation
behavior of XG (Nolte, John, Smidsrød, & Stokke, 1992). As described
elsewhere, Lund, Smidsrød, Stokke, and Elgsaeter (1988) investigated
the effect of chromic ions (Cr3+) on gel formation. The rate of gel
formation was highly dependent on the concentration of Cr3+, but
much smaller extent on the concentration of XG. For 50 mM Cr3+, gel
formation was observed in less than 1 h, while gel formed in about 40 h
for 2 mM Cr3+. The minimum concentration of Cr3+ is 1–2 mM to form
gel of 1–7 mg/mL of XG (Lund et al., 1988).
3.2. Modification with chemical method
The dissolution rate of XG into water can be improved by imparting
changes in molecular structure with reduced intermolecular interac-
tions using chemicals, such as formaldehyde (Su et al., 2003). In gen-
eral, the chemical method involves the covalent interactions to modify
the gelation behavior of XG.
3.2.1. Using crosslinking agent
Another way is to crosslink the functional groups of XG chains with
chemical agents (e.g., sodium trimetaphosphate (STMP)) via covalent
network for better hydrogel forming kinetics and mechanical stability
of crosslinked XG-based hydrogels. The obtained hydrogels showed
release controlled properties and more elastic and tougher network
(solid-like gel behavior) compared to physically (hydrogen bonds in-
teraction) crosslinked XG-based hydrogels (Tao et al., 2016). In another
study, the effect of XG content on gelatinized starch-XG hydrogel films
crosslinked with STMP as crosslinker. The swelling ratio of the hydrogel
films was observed higher for higher amounts of XG and STMP, espe-
cially at concentration higher than 10%. Further, gel mesh size in-
creased (from 2.84 nm to 6.74 nm at pH 7.4) with increasing swelling
ratio and anionic drug permeability across hydrogel films was sig-
nificantly lower compared to their neutral form mainly due to elec-
trostatic repulsion between the polymer and negatively charged drugs.
However, the formed range of mesh size is large enough for the
transport of the most of the drugs varying from small molecules to
polypeptide and proteins (Shalviri et al., 2010).
3.2.2. By the removal of existing functionality
The addition or removal of chemical functionality to the XG struc-
ture is an efficient way to modify the properties of XG. For this, Pinto,
Furlan, and Vendruscolo (2011) investigated the rheological properties
of XG after removal of acetyl groups from XG structure under alkali
medium. With higher alkali concentration, the viscosity of XG solution
was observed to increase. However, the concentration of XG (0.5 and
1%) did not show any effect on the viscosity of XG solution. The use of
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-
tively (Pinto et al., 2011).
3.2.3. By the addition of new functionality
3.2.3.1. Modification of the eOH groups. The hydroxyl group (eOH) of
XG has been modified by using various chemical agents, such as an
unsaturated organic acid (acrylic acid), acid reactive derivatives (e.g.,
maleic anhydride, acryloyl chloride), carboxymethylation, etc. (Gils,
Ray, & Sahoo, 2009; Hamcerencu, Desbrieres, Popa, Khoukh, & Riess,
2007; Mendes, Baran, Pereira, Azevedo, & Reis, 2012). In this way,
Mendes et al. (2012) modified the XG by incorporating new
carboxymethyl group (eCH2COOH) onto XG chains (eOH groups)
and investigated the encapsulation and survival of chondrocytic
ATDC5 cells. The carboxymethylated-XG showed better perspective
effect for long-term cell therapies (Mendes et al., 2012). In another
case, XG was graft-copolymerized using 2-hydroxyethyl methyla-
crylate (HEMA, CH3C(CH2)COOCH2CH2OH) and acrylic acid (AA,
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Carbohydrate Polymers 180 (2018) 128–144
CH2CHCOOH) functionality and prepared XG-g-poly(HEMA-co-AA)
superporous hydrogel system with sodium bicarbonate as foaming
agent and a triblock-copolymer of polyoxyethylene-polyoxypropylene-
polyoxyethylene as foam stabilizer for possible use in various industrial
and biomedical areas (Gils et al., 2009). Further, the graft-
copolymerization of XG using acrylamide (AAm, CH2CHCONH2) was
performed and the grafting parameters and copolymer properties of
modified XG were investigated (Maia, Silva, Curti, & Balaban, 2012),
whereas Badwaik, Sakure, Alexander, Dhongade, and Tripathi (2016)
performed the graft-copolymerization of carboxymethylated-XG using
acrylamide (AAm, CH2CHCONH2) and prepared CMX-g-PAAm showed
a non-Newtonian flow with one-third intrinsic viscosity and higher
thermal stability, but less crystalline network structure compared to
CMX (Badwaik et al., 2016). The mucoadhesive property after
modification of XG as thiol-derivatives (via esterification) using
mercaptopropionic acid (MPA) and thioglycolic acid (TGA) was
demonstrated. Metronidazole (as model drug) loaded buccal pellets
composed of XG and thiolated-XG using chicken buccal pouch
membrane showed high ex vivo bio-adhesion time of thiolated-XG
pallets compared to XG pallets. This is possibly due to disulfide bond
formation between mucus and thiolated-XG. The in vitro sustained
release of metronidazole from thiolated-XG pallets compared to XG
pallets over a prolonged period (Bhatia, Ahuja, & Mehta, 2015). Wang,
Han et al. (2016) prepared succinic anhydride-functionalized XG (SAn-
XG) and investigated the properties of the SAn-modified XG hydrogels.
The obtained SAn-XG hydrogels showed much higher storage (G’) and
loss (G”) modulus via the increased carboxyl groups (eCOOH) and
resulted in a more rigid structure than that of XG hydrogels (Wang, Han
et al., 2016). Further, Wang, Xin et al. (2016) investigated the
modification of XG by esterification with poly (maleic anhydride/1-
octadecene) (PMAO) to improve the shearing and thermal stability.
Modified-XG showed a highly improved viscoelastic behavior, whereas
similar thermal stability of modified-XG and unmodified-XG was
observed. In addition, modified-XG showed better performance on
temperature-resistance, salt tolerance, and shear endurance due to the
crosslinking between XG and PMAO (Wang, Xin et al., 2016).
3.2.3.2. Modification of the eCOOH groups. The modification of XG by
the chemical treatment for the buccal drug delivery to treat sialorrhea
was investigated. The polymeric backbone of XG was modified with L-
cysteine (SH) via amide bond formation (thiolation of XG). The
obtained modified-XG showed improvement in mucoadhesive
strength and modified swelling properties. In addition, modified-XG
especially improved the adhesion to the surfaces of buccal mucosal and
in refining prolonged and controlled drug-release properties. Further,
modified-XG showed an improved saliva uptake capacity and tannic
acid (causing astringent and drying effect) to reduce excessive saliva
flow (Laffleur & Michalek, 2017).
3.2.3.3. Modification of both eOH and eCOOH groups. A novel
interpenetrated polymer hydrogels composed of XG maleate/N-
isopropylacrylamide (XGMNIPAm) via a grafting-crosslinking with
either N,N’-methylenebisacrylamide (BIS) or cyclodextrin acrylate (A-
CD) as crosslinking agent was prepared. A-CD crosslinked-XG hydrogels
showed improved lower critical solution temperature (LCST) and
porosity. In addition, A-CD crosslinked- XGMNIPAm hydrogels
showed less sensitivity to electrolytes compared to that of BIS.
Furthermore, having preliminary analysis with progesterone (a
hydrophobic drug), A-CD crosslinked hydrogels may have good
potential for controlled release polymeric systems (Hamcerencu,
Desbrieres, Popa, & Riess, 2009).
3.3. Modification with chemo-enzymatic method
In general, phosphorylase has been used in several copolymeriza-
tion reactions (especially in a chemo-enzymatic approach) in order to
A. Kumar et al.
prepare amylose grafted-polysaccharides (Kadokawa, 2012; Karaki,
Aljawish, Humeau, Muniglia, & Jasniewski, 2016). In another study, an
amylose grafted-XG by using chemo-enzymatic approach was prepared.
Initially, an amine functionalized malto-oligosaccharide was introduced
chemically into XG via condensation with its carboxylates (eCOO−) to
prepare a malto-oligosaccharide-grafted XG and then a phosphorylase-
catalyzed enzymatic polymerization of glucose 1-phosphate on malto-
oligosaccharide-grafted-XG from the graft chain ends to obtain the
product as amylose-grafted XG. This product formed a gel in an ionic
liquid and converted into hydrogel by replacing ionic liquid with water.
The obtained hydrogels showed more elastic property compared to XG
hydrogel. Further, hydrogel was crosslinked ionically (Fe3+) by soaking
the primary hydrogel system in aqueous solution of FeCl3 (Arimura,
Omagari, Yamamoto, & Kadokawa, 2011).
3.4. Modification with plasma irradiation
Plasma chemical technology is a dry chemistry route with high
energy efficiency and cost-effectiveness. The plasma energy is enough
to break the bonds in mostly all organic molecules, leading to the for-
mation of new active chemical states of carbon atoms (Behnisch, 1997).
Generally, plasma technique penetrates about 100 from the material
surface (Hua, Sitaru, Denes, & Young, 1997) and known as surface
modification technique (Jampala, Manolache, Gunasekaran, & Denes,
2005). The cold plasma technology is an efficient route for the in-
corporation of appropriate functionality (e.g, amine, carboxyl, and
hydroxyl groups) in different polysaccharides (Behari, Pandey,
Kumar, & Taunk, 2001; Hua et al., 1997). The cold plasma technology is
an efficient and nonenzymatic route to modify XG. Jampala et al.
(2005) investigated the effect of cold-plasma treatment to graft primary
amines (-NH2) followed by the crosslinking by glutaraldehyde on
rheological properties of XG solutions. Prior this, the reactive SiHxCly
groups under DS-cold-plasma atmosphere were incorporated onto XG.
By increasing the time and concentration of plasma treatment may be
used to obtain stable XG-based gels (Jampala et al., 2005).
4. Application of XG in tissue engineering
XG has recently attracted a great attention as a biomaterial for the
preparation of tissue scaffolds (extracellular matrix) for tissue en-
gineering applications. It is well known that for proper regeneration of
damaged tissues, XG-based tissue scaffold should have 3D micro-
environment providing good cell-matrix interaction with surface
chemistry, nanotopology, and appropriate stiffness (Murphy,
McDevitt, & Engler, 2014) XG itself forms weak gel-like structure via
double helical conformations in the presence of specific bivalent cation.
For this purpose, the stiffness or mechanical stability of XG as well as
other properties can be manipulated by using other biomaterials or
reinforcements. Schematic representation of the use of XG with other
components and/or polymers for tissue engineering applications is
shown in Fig. 2.
4.1. Pure XG-based biomaterials
For tissue engineering applications, the structure (in liquid) and
complex mechanical properties of XG scaffold structures have been
investigated by using atomic force microscope (AFM) and AFM-based
force spectroscopy (FS) and the results revealed the complicated in-
termolecular interactions among XG fibrils (self-assembling of the XG
network) (Liang et al., 2014). In addition, the molecular chains ag-
gregate and dissociate (in an oscillational mode) with increasing an-
nealing time in XG aqueous solution. As analysed by AFM, a homo-
genous network structure can be obtained by annealing of XG aqueous
solution at 40 °C for 24 h (Iijima, Shinozaki, Hatakeyama, Takahashi,
& Hatakeyama, 2007). Further, the molecular conformation of un-
crosslinked XG chains and citric acid-crosslinked XG chains was
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Carbohydrate Polymers 180 (2018) 128–144
investigated. The crosslinked XG chains showed disordered conforma-
tion (coils) exposed with large number of carboxylate groups (negative
charges) compared to un-crosslinked XG chains with ordered con-
formation (helixes). The loading and delivery of proteins as bovine
serum albumin (BSA) and lysozyme (LYZ) in the un-crosslinked and
crosslinked XG hydrogels have been analyzed and controlled by hy-
drogels and proteins net charges, and can be triggered by pH change.
Moreover, the LYZ loaded XG hydrogels showed substantial bactericidal
activity of LYZ by preserving of LYZ native conformation and presented
as active wound dressings (Bueno & Petri, 2014). In another study, Han,
Wang et al. (2012) investigated the ability of intra-articular injection of
XG that could protect the cartilage at joint and reduce the papain-in-
duced osteoarthritis (OA) progression due to its similar rheology and
viscosity as that of HA. Therefore, purified XG and IA injection of XG
were analyzed for their qualities for reducing papain-induced OA on
rabbit knee OA model and recorded with morphological and histolo-
gical evaluation of articular cartilage and synovium by evaluating
sulphated glycosaminoglycans (GAGs) in cartilage. The obtained IA
injection of XG was observed as with high transparency, low protein,
endotoxin-free, heat-stable (might be sterilized at 121 °C for 15 min),
and could be injected fewer times than IA injection of HA, because no
statistical significance on OA was observed for the chondro-protective
effects of intra-articular injection of XG (once in every two weeks for 5
weeks) and HA (once in every week for 5 weeks). It can be seen clearly
macroscopically (as shown in Fig. 3), surface of articular cartilage was
with integrity, smooth and lustrous with a light white color in the sham
group (Fig. 3A and E), while this surface observed to be yellowish-
white, uneven and damaged (Fig. 3B and F) in the saline group. Further,
the cartilage surfaces of two treated groups were lustrous with a white
color and femoral condylar cartilages as resemble to healthy cartilage
were lesioned slightly (Fig. 3C and D). Tiable plateau cartilages ex-
hibited significant cracks and defects (Fig. 3G and H). However, the
severities were milder as compared to saline group. XG-treated group
showed lower scores than that of the saline group (Han, Wang et al.,
2012).
Further, the effect of XG on rabbit chondrocytes as preliminary
cytotoxicity in terms of proliferation and the protein expression of
matrix metalloproteinases (MMPs), and tissue inhibitors of metallo-
proteinase-1 (TIMP-1) in interleukin-1β (IL-1β)-induced chondrocytes
was investigated. With various amount of XG with or without 10 ng/mL
IL-1β, the obtained results demonstrated that XG alone showed no ad-
verse effects on chondrocyte cells viability and reversed IL-1β-reduced
chondrocyte cell proliferation significantly in a dose-dependent
manner. Further, XG exhibited a dose-dependent increase in TIMP-1
expression while inhibition in IL-1β-induced release of MMPs (Han,
Shao et al., 2012). In addition, the protective effect of XG against So-
dium nitroprusside (SNP)-induced rabbit articular chondrocytes apop-
tosis in vitro was investigated. In this study, rabbit articular chon-
drocytes were incubated with XG of various concentrations for 24 h
prior to incubating with 0.5 mmol/L SNP co-treatment for 24 h and
supernatants were collected for prostaglandin E2 (PGE2) evaluation.
XG offsets the adverse effects in proliferation of SNP-induced chon-
drocytes. However, the mechanism of XG blocks SNP-induced chon-
drocytes apoptosis remains unclear. The results showed that XG could
reverse SNP-reduced cell proliferation significantly and inhibited early
cell apoptosis rate in a dose-dependent manner (Chen et al., 2015).
4.2. Modified XG-based biomaterials
Mendes et al. (2012) investigated the properties of modified
(-CH2COOH)-XG (CMX) as artificial matrix (in the form of micro-
capsules) for the encapsulation of chondrocytic ATDC5 cells. The mi-
crocapsules with smooth surface and homogenous size having an
average diameter of 500 μm were prepared. ATDC5 cells encapsulated-
CMX microcapsules showed high viability and proliferated for pro-
longed culture periods with improved metabolic activity. Further,
A. Kumar et al. Carbohydrate Polymers 180 (2018) 128–144

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-
drogenic phenotype. This obtained CMX microcapsules may show a
potential for long-term cell culture for the applications in cell therapies
(Mendes et al., 2012). Further, Wang, Han et al. (2016) prepared suc-
cinic anhydride-functionalized XG (SAn-XG) and investigated the
properties of the SAn-modified XG hydrogels. The obtained SAn-XG
hydrogels showed much higher storage (G’) and loss (G”) modulus via
the increased carboxyl groups (-COOH) and resulted in a more rigid
structure than that of XG hydrogels. Gentamicine (GS) drug loaded SAn-
XG hydrogels showed prolonged release of GS and good human lens
epithelial cell (HLECs) attachment, proliferation and migration when
GS loading was 1 mg g−1. In addition, SAn-XG/GS hydrogels, compared
to XG-SAn hydrogels, demonstrated a significantly lower degree of in-
fection at 7 day in an in vivo rabbit subcutaneous S. aureus infection
model (Wang, Han et al., 2016).
4.3. XG-phospholipid as bioactive agent
Phospholipids have widely been used in drug delivery systems and
tissue engineering applications because of their unique properties such
as the excellent biocompatibility and an especial amphiphilicity (Li
et al., 2015; Monteiro, Martins, Reis, & Neves, 2014). It is believed that
phospholipid-based may be used to manipulate the cell and tissue re-
sponses towards biomaterial, facilitating the controlled release of
bioactive agents and reconstructive surgery (Collier & Messersmith,
2001). In this case, Mendes, Baran, Reis, and Azevedo (2013) synthe-
sized XG-phospholipid (1,2-dioleoyl-sn-glycerophosphoetilamine,
DOPE) microcapsules (hollow capsular structures) by combining mi-
crofluidics with self-assembly for cell encapsulation (survival and pro-
liferation). The supramolecular organization of XG-DOPE under the
conditions for cells encapsulation was observed as formation of nano-
sized aggregates and the four-step synthesis of microcapsule are shown
in Fig. 4(I) (A and B), respectively. ATDC5 cells were encapsulated
within XG-DOPE microcapsules and demonstrated good cell viability
showing increased cellular metabolic activity over 21 days of incuba-
tion (as shown in Fig. 4(II)-A). In addition, metabolic activity and
proliferation of encapsulated ATDC5 cells within self-assembled XG-
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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A. Kumar et al. Carbohydrate Polymers 180 (2018) 128–144

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)).

135
A. Kumar et al.
quantification over 21 days. AlamarBlue assay showed improved me-
tabolic activity for a prolonged time (without needing of any external
coating, as the potentially immunogenic poly-L-lysine (PLL)). However,
at the end of 21 days of culture, XG-DOPE coated with PLL showed
higher metabolic activity, but not significantly compared to uncoated
XG-DOPE microcapsules (as shown in Fig. 4(II)-B). Further, DNA
quantification showed a gradual and significant increase in DNA be-
cause of the good cellular proliferation in both XG-DOPE and PLL
coated-XG-DOPE microcapsules (as shown in Fig. 4(II)-C). However,
ATDC5 cells within XG-DOPE coated with PLL exhibited significantly
less cellularity compared to uncoated XG-DOPE microcapsules, despite
showing similar metabolic activity. Therefore, cells could be en-
capsulated with XG-DOPE microcapsules with enhanced metabolic ac-
tivity for a prolonged duration without needing any external coating
such as potentially immunogenic PLL and might be a biomimetic carrier
for cells grow for cell-based transplantation therapies (Mendes et al.,
2013).
4.4. XG-nanomaterials based biomaterials
4.4.1. XG-magnetic nanoparticles
Magnetic nanoparticles (MNPs) show main advantage of remote
controlling when cells functionalized with these nanoparticles. MNPs
functionalized labeled cells facilitate the manipulation of the cell be-
havior and also control the function of cells using external magnetic
field (Ito & Kamihira, 2010). Compared to pure XG hydrogels, magneto-
responsive and non-toxic XG/MNPs hybrid hydrogels showed more
pronounced murine fibroblasts (3T3 L1) proliferation, particularly
under applied filed of 0.4T. Topographical analysis showed densely
packed tiny particles on the surface along with some large aggregation
of small particles and MTT assay showed cell viability with and without
electro-static magnetic field (ESMF) (Bueno et al., 2013). Further, XG/
MNPs-based scaffolds showed successful in vitro neuronal differentia-
tion of embryonic stem cells and indicated successful synapse formation
(increased membrane potential amplitudes upon depolarization with
KCl) (Glaser, Bueno, Cornejo, Petri, & Ulrich, 2015).
4.4.2. XG-gold nanoparticles
Gold nanoparticles (GNPs) have extensively been used in as ther-
apeutic agents (Sun et al., 2014), diagnosis agents (Halo et al., 2014),
and imaging agents (Zerda et al., 2015) because of their easy pre-
paration, nano-scale size (similar to the dimension of biological com-
pounds), high surface area, and easy functionalization. GNPs also serve
as a promising tool for tissue engineering applications due to their
ability to deliver bioactive molecules, to diagnose differentiation status
of stem cells, to track implanted cells, while improving differentiation
of cells and intracellular growth factor delivery, cell–cell interactions,
monitoring in real-time cellular events, adhesive and mechanical per-
formance of the scaffolds. These remarkable physicochemical proper-
ties make them unique compared to polymeric nanoparticles, protein-
based nanoparticles, and liposomes (Vial, Reis, & Oliveira, 2017).
Therefore, Pooja, Panyaram, Kulhari, Rachamalla, and Sistla (2014)
synthesized optimized GNPs within XG used as both reducing and sta-
bilizing or capping agent and doxorubicin (DOX)-loaded GNPs showed
colloidal stability at a pH range between pH 5–9 and NaCl concentra-
tion up to 0.5 M. In addition, for toxicity evaluation, GNPs were found
non-toxic and biocompatible. However, DOX-loaded XG-GNPs showed
3 times more cytotoxicity in A549 cells (human lung cancer cells)
compared to DOX only (Pooja et al., 2014).
4.4.3. XG-nanohydroxyapatite
Nano-hydroxyapatite (nHAp) shows excellent biocompatibility and
has close chemical similarity to the inorganic component of the bone
that is a natural composite of organic (collagen fibrils) and inorganic
phases, which makes it ideal candidate for orthopedic and dental im-
plants. Therefore, synthetic nHAp has widely been used in repairing of
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Carbohydrate Polymers 180 (2018) 128–144
hard tissues (bone repair, bone augmentation, and coating of implants
or as fillers in bone or teeth) especially for bone tissue engineering
applications, including a variety of biomedical applications, including
drug delivery systems due to its slow in situ biodegradability, bio-
compatibility, good osteoconductive and osteoinductive properties
(Habraken, Wolke, & Jansen, 2007; Legeros, 1993; Weiner & Wagner,
1998; Zhou & Lee, 2011). By taking this opportunity, Izawa et al. (2014)
reported the mineralization of nHAp upon XG hydrogel via the ionic
interactions between Ca2+ and –COOH and obtained mechanically
improved XG-nHAp porous structure for bone tissue engineering ap-
plications (Izawa et al., 2014). In another study, Bueno, Bentini,
Catalani, Barbosa, and Petri (2014) prepared XG/nHAp and strontium
substituted XG/nHAp (XG/nHAp-Sr) particles. The surface charge
density and colloidal stability of XG/nHAp and XG/nHAp-Sr particles
were increased because of the surface modifying with XG chains and
further promoted compatibility between XG chains and XG/nHAp or
XG/nHAp-Sr particles. The XG-XG/nHAp with 10 wt% HAp nano-
composites showed improved mechanical performance while porous
structure, surface energy, gel content and swelling ratio comparable to
pure XG hydrogels or nHAp nanocomposites. In addition, cellular pro-
liferation of osteoblastic cells onto XG hydrogels was not significantly
influenced by the incorporation of XG modified HAp. However, the
activity of ALP was substantially favored upon increasing XG/nHAp or
XG/nHAp-Sr particles content (from 10 to 30%) (Bueno et al., 2014).
4.4.4. XG-bioactive glass/cellulose nanocrystals
Bioactive glass (BG), similar to hydroxyapatites, has shown a great
potential in the engineering of new bone tissues, by improving the me-
chanical stability and good bioactivity (apatite forming ability) of the BG-
based polymeric scaffolds (Lei et al., 2012). In addition, cellulose nano-
crystals (CNCs) have shown a good potential in the tissue engineering areas
due to their unique properties such as nano-scale size, high aspect ratio and
surface area, good hydrophilicity, biocompatibility, low-density, and high
strength (7.5 GPa) and modulus (ranging from 110 to 220 GPa) (Habibi,
Lucia, & Rojas, 2010; Kumar, Negi, Choudhary, & Bhardwaj, 2014a). These
properties make CNCs ideal nano-reinforcements for preparing tissue en-
gineering biomaterials (Domingues, Gomes, & Reis, 2014; Kumar, Negi,
Choudhary, & Bhardwaj, 2014b; Kumar, Negi, Choudhary, & Bhardwaj,
2014c; Kumar, Lee et al., 2017; Kumar, Rao, & Han, 2017a). Therefore, the
use of BG sol-gel alongwith CNCs has been found advantageous to improve
polymeric-network of XG with mechanical stability and apatite forming
ability of the hybrid tissue engineering scaffolds. In this way, Kumar, Rao,
Kwon et al. (2017) prepared XG hybrid scaffolds reinforced with CNCs and
silica-based glass (SG) by using freeze-casting and freeze drying process and
demonstrated highly porous structure with improved mechanical stability
(in dry and wet states) when incorporated with SG followed by CNCs.
Further, good osteoblastic MC3T3-E1 cell adhesion and proliferation was
observed to increase with time and stiffness compared to pure XG scaffold
and presents the suitability of as-obtained hybrid scaffolds may possibly
have promising application in low-loading bone tissue engineering appli-
cations (Kumar, Rao, Kwon et al., 2017).
4.5. XG-natural polymer and/or nanomaterials based biomaterials
4.5.1. XG-chondroitin sulfate
Chondroitin sulfate (CS) is also a member of GAGs family and is an
important component in cartilage and connective tissues. It is sulfated
and branched polysaccharide and composed of a chain of alternating
repeat units of glucuronic acid and N-acetyl-glactosamine (GalNac)
with sulfation at either 4- or 6-position (Fan et al., 2017; Lee,
Huang, & Lee, 2006). CS can have a potential impact on cell attach-
ment, migration, proliferation and differentiation. Oprea, Neamtu,
Stoica, Petreus, and Vasile (2012) prepared XG/CS hydrogels and in-
vestigated in vitro and in vivo biocompatibility. As-obtained XG/CS
hydrogels showed good in vitro biocompatibility with viability of Sac-
charomyces pombe (microorganism) analyzed by using
A. Kumar et al.
chemiluminescent assay. The lethal dose (LD50) of XG/CS-based hy-
drogels is bigger than 3200 mg/kg and hence proved low toxicity.
Further, in vivo evaluation performed by subcutaneous implantation
demonstrated that 50/50 XG/CS composition was absorbed easily
without side-effects. The in vitro and in vivo biocompatibility analyses
may possibly present the suitability of XG/CS-based hydrogels for
controlled release systems for drug delivery and tissue engineering
applications. The cellular growth of 20–25% was observed with CS
increment in composition of the hydrogels compared to positive con-
trol, whereas further increase of CS content did not show any significant
influence. XG hydrogel showed a slight decrease in cell viability (∼4%)
(Oprea et al., 2012).
4.5.2. XG-chitosan
Chitosan (CHT) is a natural polymer having a linear chain com-
prising of β(1–4) glycosidic bonds linked D-glucosamine residues with
varying number of randomly located N-acetyl-D-glucosamine (NAG)
groups. It has large number of eOH groups and is biodegradable,
bioactive, biocompatible, and anti-bacterial properties and showed
enhanced effect on cell attachment, proliferation, differentiation and
mineralization (Di Martino, Sittinger, & Risbud, 2005; Saravanan,
Leena, & Selvamurugan, 2016). As biomaterial, CHT has also been used
to prepare weak gel with XG by complexation of these two natural
polysaccharides via the presence of counter-ions (Martínez-Ruvalcaba,
Chornet, & Rodrigue, 2007). For possible application for the treatment
of skin lesions, Bellini, Pires, Vasconcelos, and Moraes (2012) prepared
XG/CHT-based porous lamellar membranes incorporated with Tween
80 or Pluronic F68 for possible use for dermal dressings and tissue
engineering applications. This study demonstrated that XG/CHT (1/1
ratio) with or without Pluronic F68 shows transparency, high absorp-
tion capacity of physiological fluids with adequate stability, in vitro low
cytotoxicity (L929 cells), skin compatible mechanical property (tensile
strength), whereas membranes (regardless of the mass ratio) with
Tween 80 showed massive cell death (highly toxic to L929 cells), de-
spite having appropriate mechanical resistance and behavior in phy-
siologic solutions (Bellini et al., 2012). In addition, Bellini, Oliva-Neto,
and Moraes (2015) prepared XG/CHT composite films from various
sources of XG, where XG type did not affect most of the properties
significantly (Bellini et al., 2015).
4.5.2.1. XG-glycidyl methacrylate grafted CHT. CHT has been grafted
with GMA to improve the crosslinking ability of the components
because it has oxirane group that can be chemically modified for
prospective biological interest (Kuan, Horák, Plichta, & Lee, 2014).
Elizalde-Peña et al. (2013) synthesized hybrid hydrogels by mixing
the hybrid natural-synthetic CHT-g-glycidyl methacrylate (GMA) and
XG. The results showed that swelling index of obtained hydrogels
decreased as GMA mass percentage increased and behave as physical
hydrogels. Viscoelastic behavior of pure CHT-g-GMA was enhanced on
increasing of XG as polysaccharide. In addition, synthesized hydrogels
showed good cell viability of fibroblast cells and demonstrated
synthesized hydrogels to be used possibly for skin graft devices in
biomedical applications (Elizalde-Peña et al., 2013).
4.5.2.2. XG-CHT/cellulose nanocrystals. Rao, Kumar, and Han (2017)
prepared bionanocomposite hydrogels composed of CHT, XG, and CNCs
in the presence of green acidifying agent via electrostatic attraction and
hydrogel bonding (complexation of polymer chains). The results showed
significantly improved mechanical performance with increased CNCs
content. In addition, 5-Flurouracil drug (as model chemotherapeutic
agent)-loaded bionanocomposite hydrogels showed excellent releasing
ability and cytocompatibility with NIH3T3 fibroblast cells for possible use
in drug delivery as well as tissue engineering applications (Rao et al., 2017).
4.5.2.3. XG-CHT/antibacterial agent. CHT also possesses antimicrobial
activity (Zheng & Zhu, 2003) and can be used as antimicrobial polymer.
137
Carbohydrate Polymers 180 (2018) 128–144
In addition, chlorhexidine (CHX) is an antimicrobial agent a cationic
bisbiguanide having wide antimicrobial activity range against Gram-
positive and Gram-negative bacteria, dermatophytes, yeast, and some
selective lipophilic-viruses (Jones, 1997; Kim et al., 2017). However, its
antimicrobial effect damages the inner membrane (cytoplasmic) and
leads to the cell lysis and death (Huynh et al., 2010). Further, it shows
good affinity to skin and mucous membranes and low toxicity against
mammalian tissue (Jones, 1997; Paolantonio et al., 2008) and has been
used in dental treatments (Paolantonio et al., 2008). Kim et al. (2017)
prepared XG/CHT-based polyelectrolyte microspheres via anionic XG
and cationic CHT and mixed with CHX dihydrochloride (10 mg/mL)
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
chronic periodontitis. As-obtained hydrogels showed viscoelastic
behavior, selective antibacterial effects against P. Gingivalis (as
determined by blood-agar plate assay), and non-cytotoxicity to
human dermal fibroblast (Kim et al., 2017). In this study, silver
nanoparticles (AgNPs) have been used as antimicrobial agent along
with CHT. Rao et al. (2016) prepared XG-CHT based polyelectrolyte
hydrogels (PEHs) with Ag-NPs by green chemistry route without using
any organic solvent and reagents. In this study, highly size-controlled
and spherical-shaped Ag-NPs of 5–20 nm diameters were obtained as
confirmed by characteristic surface plasmon band (398–409 nm) for
various concentration of silver ions. As-obtained Ag-NPs were dispersed
homogenously in XG-CHT-based PEHs (as shown in Fig. 5) and
showed good antibacterial activity by inhibiting both Gram-positive
(Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria.
In addition, good in vitro cytocompatibility (cell adhesion and
proliferation) of PEHs with NIH 3T3 fibroblast cells was observed (as
shown in Fig. 5), indicating the promising use in antibacterial wound/
burn dressing and possible application for skin tissue engineering (Rao
et al., 2016).
4.5.2.4. XG-CHT/montmorillonite. Montmorillonite (MMT) is a kind of
layered silicates (clay mineral) and well known as a polymer modifier
because of its high specific surface area (Mishra, Allauddin, Narayan,
Aminabhavi, & Raju, 2012). MMT has widely been applied in
pharmaceutical (Lin et al., 2002) and tissue engineering applications
(Depan, Kumar, & Singh, 2009; Olad & Azhar, 2014). With only a low
amount of MMT, solvent resistance and mechanical performance of the
polymer-based composites can significantly be improved (Zheng et al.,
2007). By taking these advantages, Liu, Nakagawa, Chaudhary,
Asakuma, and Tadé (2011) prepared XG/CHT/MMT-based scaffolds
from their suspension via freeze drying process and investigated that
slow cooling rate of freezing led to larger pore size with more tilted
pores horizontally and uniform network as well as higher hardness
values compared to rapid freezing rate. Further, pore size was
decreased while mechanical performance was increased with the
incorporation of MMT (Liu et al., 2011).
4.5.3. XG-gellan gum
Gellan gum (GG) as bacterial polysaccharide is a linear anionic exo-
polysaccharide having repeat unit comprising of α-L-rhamnose, β-D-
glucose, and β-D-glucuronate with molar ratios 1:2:1 (Osmałek,
Froelich, & Tasarek, 2014). It can form physical hydrogel in the pre-
sence of mono-, di-, and tri-valent cations (Maiti, Ranjit, Mondol,
Ray, & Sa, 2011; Tang, Tung, & Zeng, 1996). In addition, native GG
forms soft and easily deformable gels, while deacetylated GG forms
rigid and brittle gels (Jampen, Britt, & Tung, 2000; Osmałek et al.,
2014).
4.5.3.1. XG-GG/CHT nanoparticles. Dyondi, Webster, and Banerjee
(2013) prepared injectable XG/GG hydrogels with CHT nanoparticles
(CHT-NPs) (297 ± 61 nm), basic fibroblast growth factor (bFGF), and
bone morphogenetic protein 7 (BMP7) were used in a dual growth-
factor system to promote the differentiation of human fetal osteoblast
A. Kumar et al.
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
significant improvement in cell proliferation and differentiation
because of sustained release of growth factors. Hydrogels loaded with
dual growth factor exhibited a higher alkaline phosphatase and
deposition of calcium compared to hydrogels loaded with single
growth factor. In this study, L929 mouse fibroblast cells viability was
greater than 95% and human fetal osteoblast cells viability was found
to be 80% in the presence of XG/GG hydrogels as evaluated by MTT
assay for 48 h. In addition, XG/GG hydrogels showed antibacterial
effect against common pathogens such as Staphylococcus aureus,
Staphylococcus epidermidis, and Pseudomonas aeruginosa in implant
failure. Therefore, encapsulation and stabilization of growth factors
within nanoparticles and hydrogels are promising for bone tissue
regeneration (Dyondi et al., 2013).
4.5.3.2. XG-GG/hyaluronic acid. Hyaluronic acid (HA) is one of the GAGs
family members in our body and has widely been used in biomedical
applications due to the active influence on several cellular functions as well
as cell attachment, migration, and proliferation. HA has most commonly
been used as a hydrogel form, but it lacks some limitations related to
inadequate mechanical performance and compromised further by its rapid
degradation by physiological enzymes in vivo (Garcia-Fuentes, Meinel,
Hilbe, Meinel, & Merkle, 2009; Ko, Tien, Wang, & Chen, 2012). The
prevention of peritendinous attachment after the repairing of tendon-
surgeries poses a main clinical challenge. Kuo, Chang, Wang, Tang, and
Yang (2014) prepared hydrogel membranes and demonstrated the possible
prevention of attachment of surgical tendon and synovial sheath that often
cause poor mechanical repairing of the tendon. In this study, XG-GG/HA-
based membranes of various compositions as well as commercially available
Seprafilm (comprised of HA and carboxymethyl cellulose (CMC)) were used
and wrapped around repaired tendons in a 8-week-old male Sprague-
Dawley rat model to examine grossly and histologically after 3 weeks of
healing process. The observation showed that certain compositions of XG-
GG/HA hydrogel membranes rapidly swelled and readily and closely
blanketed onto tendon tissue, and reduced adhesion with equal ability
without affecting the strength of tendon compared to Seprafilm. In addition,
hydrogel membranes degraded slowly, allowing functioning as a barrier for
extended duration. For this evaluation, tests were grouped with four
formulations A, B, C, or D for tendon wrapped with Seprafilm or XG-GG/
HA membranes (Fig. 6(I) (A–E)). Fig. 6(II) shows in vivo gross observations
of repaired rat Achilles tendons for 3 weeks of healing process, where
intense adhesions were observed by showing of massive and dense
adhesive-bundles between tendon and controgroup surrounding tissues
(Fig. 6(II) (A)). Loose adhesion bundles were formed bridging tendons and
138
Carbohydrate Polymers 180 (2018) 128–144
surrounding tissues in case of Seprafilm group and A/B groups (Fig. 6(II)
(B–D)), while more intense peritendinous-adhesions (fibrous bundles
bridging) were observed in C and D groups as compared to Seprafilm
(Fig. 6(II) E and F). In addition, some residuals of XG/GG/HA hydrogel
were observed because of slow degradation and Seprafilm was degraded
fully. However, the gross adhesion scores were significantly lower
compared to the control group. Further, in vivo histological assessments
supported the gross observations by indicating irregular-peritendinous
surface and massive fibrous adhesions scars on repair site of untreated
tendons (Fig. 6(III) A and B), mild fibrous scars on Seprafilm treated site
(Fig. 6(III) C and D), a clear gap between tendon and epidermis including
few loose bundles of fibrous tissue and residual materials (Fig. 6(III) E–H).
In contrast, massive fibrous tissues and adhesions between repaired tendon
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
hydrogel membranes, prepared using formulation A and B, showed an
significant anti-adhesive effect on repaired tendons without influencing
healing of the injured tendons (Kuo et al., 2014).
4.5.4. XG-galactomannan/curcumin
Galactomannan (GTM) is a plant hetero-polysaccharide comprising
of a main chain of D-mannose with D-galactose side groups
(Scherbukhin & Anulov, 1999). GTM is non-toxic, biocompatible, and
can make biomaterials with improved cohesive strength (Mikkonen
et al., 2007; Siqueira et al., 2015). Further, curcumin (CC) is a naturally
occurring anti-inflammatory and anti-oxidant agent of turmeric (Cur-
cuma longa) associated with NF-kβ inhibition, apoptosis regulation and
antioxidant effects, including neuroprotective activity during treatment
of spinal cord injuries (Ormond et al., 2014; Jin et al., 2014). However,
the low bio-availability and weak stability limit its applicability for
large productive purposes (Metzler, Pfeiffer, Schulz, & Dempe, 2013;
Requejo-Aguilar et al., 2017). Da-Lozzo et al., 2013 prepared XG-GTM/
CC-based hydrogel to evaluate viscoelastic behavior and biocompat-
ibility from topical application on chick embryo chorioallantoic mem-
brane (CAM). The addition of CC (≤0.5 mg/mL) had not any effect on
the viscoelastic behavior of the XG-GTM hydrogel network and XG-
GTM/CC hydrogel systems showed high biocompatibility. Further, XG-
GTM hydrogels did not show any tissue injury of CAM. Actually, foreign
material can cause tissue injury under the CAM and associated-re-
sponses of tissues (i.e., inflammation and fibrosis) if inflammatory and
wound healing steps occur in an uncontrolled-manner. In this study,
poly-L-lactic acid (PLLA) films (which cause CAM tissue injury) as po-
sitive control promoted neovascularization, opacity, fibrosis, while XG-
GTM and XG-GTM/CC hydrogel systems were completely absorbed and
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
implants. In addition, histological sections confirmed an increase in the
number of micro-vessels in PLLA exposed-CAM and XG-GTM hydrogels
either without or with CC did not stimulate any structural changes in
the CAM (as compared to control systems without implants). Moreover,
histological sections supported the behavior of obtained hydrogels in
terms of amount of collagen fibers (Da-Lozzo et al., 2013).
4.5.5. XG-i-/k-carrageenan-konjac gum
As anionic polysaccharide, carrageenan (CGN) has widely been used
as an emulsifier, thickening, gelling, and stabilizing agent in pharma-
ceutical (Mammarella, & Rubiolo, 2003), industrial, and tissue en-
gineering applications (Kumar, Rao, Haider et al., 2017; Popa,
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
(Bhattacharyya et al., 2010). Depending on degree of sulfation (be-
tween 15% and 40%), CGN has been categorized in three ways: (1)
kappa-CGN (k-CGN, one sulfate group per disaccharide and forms
strong and rigid gels), iota-CGN (i-CGN, two sulfate groups and forms
soft gels), and lambda-CGN (λ-CGN, three sulfate groups, known as
highly sulfated, and less likely to form a gel structure, but can form gels
when mixed with proteins rather than water). In addition, konjac gum
(KG) is a type of hydrocolloid-gum (produced from the tubers of Ara-
ceae amorphophallus) consisting of 60–70% of konjac glucomannan and
has extensively been used in the industrial applications (e.g., food in-
dustry, petroleum drilling) (Dhawan & Kaur, 2007). Due to its high
viscosity, KG is normally used with other hydrocolloids in improving
their viscosity and quality of foods (Yaseen, Herald, Aramouni, & Alavi,
2005), and can greatly change the specific physicochemical properties
of the blended systems with other components (e.g., XG, CGN, SA, MC,
sodium carboxymethylcellulose, hydroxypropylmethylcellulose) (Liang
et al., 2011). Juris et al. (2011) investigated a range of mechanical
properties, degradation duration, and good biocompatibility with
human fibroblast (MeWo) cells of skin hydrogels composed of four
polysaccharide mixtures (XG with i-CGN, k-CGN, and KG). In these
cases, i-CGN and k-CGN were fibre reinforcements, while KG and XG
were as amorphous matrix for composite hydrogels (Juris et al., 2011).
In another study, Almeida, Mueller, Hirschi, and Rakesh (2014) in-
vestigated the rheological and thermal properties of the same four
polysaccharides-based skin hydrogels on considering the mixing of
precise polysaccharide composition and storage conditions. The ob-
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
magnitude of the hydrogels without the presence of either XG or i-CGN
after 12 h of time period in air. All four polysaccharide-based strong
hydrogels could be analysed as epidermis skin scaffolds, whereas more
porous weak gels without the presence of either XG or i-CGN for dermis
skin scaffolds (Almeida et al., 2014).
4.5.6. XG-SA/CNCs/halloysite nanotubes
SA as polysaccharide (produced from brown sea algae) has broadly
been used in biomedical applications due to its inherent hydrophilicity,
easy processing, biocompatibility, and its gelling properties under mild
conditions (Gerecht-Nir, Cohen, Ziskind, & Itskovitz-Eldor, 2004;
Kumar et al., 2017a). However, it shows some limitations as un-
controlled degradation, low mechanical property, and lack of cell affi-
nity (Kumar, Lee et al., 2017). Further, halloysite nanotubes (HNTs) as
clay material have shown a good potential in developing new organic-
inorganic hybrid hydrogels for the applications in biomedical area be-
cause of their biocompatible nature (Joussein et al., 2005; Liu, Wu,
Jiao, Xiong, & Zhou, 2013). On considering these facts, Kumar, Rao,
and Han (2017) investigated the synergic effect of CNCs and HNTs on
the structure-property-relationship of the nanocomposite scaffolds
composed of XG, SA, CNCs, and HNTs. The results showed good in-
terfacial interactions and homogenous dispersion of the CNCs and HNTs
within the XG-SA polymeric network. In addition, the obtained four
component nanocomposite scaffolds exhibited high porosity with good
pore-interconnectivity and improved mechanical stability, thermal
stability, and good cytocompatibility with osteoblastic MC3T3-E1 cells
(Kumar et al., 2017b).

139
A. Kumar et al.
4.6. XG-synthetic polymer-based biomaterials
4.6.1. XG-polypyrrole
The electrically conducting polymers (e.g., polypyrrole (PPy)) have
been applied for biomedical engineering applications because of their
good electrical conductivity, thermal stability, and ability for copoly-
merization without compromising electro-activity. For this reason, PPy
showed good potential for biomedical applications, especially for tissue
engineering (Zelikin et al., 2002). The cytocompatibility (in vitro and in
vivo) of PPy has been proven with a broad range of cell types (Ateh,
Navsaria, & Vadgama, 2006; George et al., 2005), but the brittleness
and inability to manipulate mechanically and processing, it needs
blending with other materials (Sajesh, Jayakumar, Nair, & Chennazhi,
2013). Moreover, the association of conducting polymer as polypyrrole
(PPy) with XG hydrogel exhibits high charge density (electroactivity),
good biocompatibility, and forms mechanically stable biomaterial
(Bueno, Takahashi, Catalani, de Torresi, & Petri, 2015). The fibroblast
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-
vored fibroblast adhesion and proliferation due to higher surface
roughness and hydrophobicity and particularly under external mag-
netic field (0.4 T) (Bueno et al., 2015). In this case, XG having high
negative charge density (Bueno & Petri, 2014) might cause electrostatic
repulsion to proteins which are responsible for cell adhesion. The
morphological analysis (as shown in Fig. 7) demonstrates that fibroblast
cells hardly observed (only few spherical form; Fig. 7A-a) on XG scaf-
fold in the absence of EMF while many cells having more elongated
form (Fig. 7A-b) in the presence of EMF. In case of XG/PPy scaffolds,
more cells adhered and proliferated in the absence (Fig. 7A-c) and in
the presence (Fig. 7A-d) of EMF compared to pure XG scaffold.
Further, this proliferation behavior of fibroblast cells was supported
by MTT assay (Fig. 7B) in the absence and presence of EMF, providing
same level cells proliferation for pure XG scaffold in the presence of
EMF and XG/PPy scaffolds in the absence of EMF. This explored the
manipulation of cells proliferation under magnetic stimulus and charge
density (Bueno et al., 2015).
4.6.2. XG-methylcellulose
Methylcellulose (MC) is one of the cellulose derivatives (water so-
luble) and has been applied to increase the viscosity of foods or paints.
It has good characteristic in aqueous solution to induce temperature
dependent sol-gel transition leading by hydrophobic interaction and
can be manipulated by the various parameters, such as concentration,
additives, degree of substitution, and molecular weight (Altomare,
Cochis, Carletta, Rimondini, & Farè, 2016; Park, Kim, Yoon, & Park,
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)).
140
Carbohydrate Polymers 180 (2018) 128–144
injectable hydrogels and revealed that blend solution recovered its high
viscosity immediately and formed hydrogel rapidly at body tempera-
ture (37 °C). For in vivo analysis for gelation, elimination, and bio-
compatibility in rat body, The volume of hydrogel increased from day 1
to day 7 post-injection because of swelling and after that its volume
decreased gradually followed by the disappearance completely after
36 days of the injection (much faster than the erosion in vitro). The
inflammatory cells in the tissues were observed around the implanted
XG/MC hydrogel and inflammation decreased with the increased im-
plantation time. Further, this cells inflammation almost disappeared on
the day of 27 post-injection and histology of the surrounding tissues
was recovered completely on the day of 37 post-injection. In addition,
in vitro DOX-loaded XG2/MC10 hydrogels showed sustained release
behavior. XG/MC blend could be a promising injectable hydrogel for
long-term drug delivery system (Liu & Yao, 2015). The summary of the
recent studies of the XG-based biomaterials for tissue engineering, as
described in previous sections, is given in Table 1.
5. Conclusion and future perspectives
Xanthan gum is a microbial exo-polysaccharide produced by
Xanthomonas bacteria and it has good water-solubility, excellent bio-
compatibility, intrinsic immunogenic ability and high molecular weight
polysaccharide having branched polymeric chains. Further, due to
several advantages, such as such as high viscosity at a very low con-
centration, physical hydrogel with bivalent cation, shear-thinning
property, pseudo-plastic behavior, and thermal stability (against hy-
drolysis) that is far better than other polysaccharides or polymers
(Stokke & Christensen, 1996), but the low mechanical performance and
processing of the xanthan gum limit its applicability for different tissue
engineering applications. Also, it has large number of hydroxyl groups
(eOH) and the free carboxyl groups (eCOO−) that can be used for
chemical functionalization or modification in order to manipulate or
optimize its physicochemical and biological properties. As described in
this review, XG has been successfully applied in several industrial and
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
area. Therefore, the recent trends on the use of XG-based poly-
saccharide formulations for various tissue engineering applications are
precisely reviewed for better understanding of this biopolymer in this
area. It shows a quite promising future as a biopolymer as demonstrated
in this review by emerging tissue engineering products. Moreover, the
shear-thinning and gelling property of XG can be more beneficial in the
area of 3D bioprinting of the tissue scaffolds and/or tissue models for
future tissue engineering applications.
A. Kumar et al. Carbohydrate Polymers 180 (2018) 128–144

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
Authors would like to thank the authors whose research work and re-
view articles have been cited in this review article. The writing of this re-
view article was fully supported by the 2017 Yeungnam University research
grant and by the Basic Science Research Program through the National
Research Foundation of Korea (NRF) funded by the Ministry of Education,
Science and Technology (Grant No. 2017R1D1A3B03036276).
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