Polysaccharides and Their Derivatives For Versatil

Review
Polysaccharides and Their Derivatives for

Versatile Tissue Engineering Application
Ferdous Khan,* Sheikh Rafi Ahmad
Research and development in the design, synthesis, modification, evaluation, and character-
ization of polysaccharide-based bioactive polymeric materials for guiding and promoting new
tissue in-growth is reviewed. Emphasis is given in this interdisciplinary field of tissue
engineering (TE) with particular reference to bone, cartilage, and skin TE. Current strategies
in scaffold-guided TE approaches using polymers of natural origin and their composites are
elaborated. Innovative modification techniques
in creating functional materials for advanced
TE applications are presented. Challenges and
possible solutions in the technological innovation
in factor molecules incorporation and surface
functionalization for improving the fabrication
of biomaterials scaffolds for cost-effective TE
are also presented.
1. Introduction body or to foster remodeling of tissues in active manner in a

frame of a three-diomensional (3D) scaffold. Fundamental
Tissue engineering (TE) is a branch of biotechnology for requirements for designing polymer-based scaffolds are as
generating complex organisms to repair damaged or follows: a) possession of high porosity with appropriate
malfunctioning tissues or organs as an alternative to whole pore size distribution and high surface area; b) biodegrad-
organ transplantation.[1–3] In the last decade, significant ability with the degradation rate matching the rate of neo-
advancement has been made in the area of TE for tissue formation; and c) possession of the required
therapeutic applications. This field of research and devel- structural integrity of the scaffold to prevent the pores
opment includes a plethora of disciplines, including from collapsing during neo-tissue formation. Additionally,
materials science and engineering, cell biology, and life the scaffold should be nontoxic to cells and be biocompa-
sciences. This research, in turn, involves the use of living tible, positively interacting with the cells to promote cell
cells, manipulated either genetically or through their extra adhesion, proliferation, migration, and differentiated cell
cellular environment. The goal, therefore, is the develop- function. However, any specific requirements mentioned
ment of biological substitutes for implantation into the above will depend on the type of tissue involved in the
regeneration process.
Research on biomaterials has been the subject of interest
Dr. F. Khan[+] both within the academia and in the industry sectors over
School of Chemistry, The University of Edinburgh, West Mains
the past few decades.[4–6] The variety of polymeric
Road, Kings Buildings, EH9 3JJ, UK
materials with specific chemical structures and the ability
E-mail: ferdous.khan0@gmail.com; fkhan@staffmail.ed.ac.uk
Dr. S. R. Ahmad
of precisely controlling the molecular architecture and
Head, Centre for Applied Laser Spectroscopy, CDS, DEAS, Cranfield morphology has allowed numerous uses of polymers in
University, Shrivenham, Swindon, Wiltshire SN6 8LA, UK biomedical fields. For example, biocompatible polymers
[+]
Present address: Ocutech Limited, 3 Clark Way, Bellshill have been used for creating artificial organs and also for
ML4 3NX, UK drug delivery systems.[7,8]
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200409 1
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F. Khan, S. R. Ahmad
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In TE, a common approach is to isolate specific cells Ferdous Khan was a research fellow at the
University of Edinburgh, where he served over
through a small biopsy from a patient for culturing them on
6 years and devoted to research on polymer-
a 3D polymeric scaffold under controlled conditions. The
based materials for the applications of tissue
construct is then delivered to the desired site in the patient’s
engineering and regenerative medicine. Ferdous
body with the aim to promote new tissue formation into has developed polymer hydrogels for cartilage
the scaffold that can be degraded over time. An alternative tissue regeneration, stem cells functioning and
approach is to implant polymer scaffolds ‘‘in vivo’’ for scaffolds for bone tissue engineering, in a cost
tissue ingrowths with the purpose to stimulating and to effective manner. In December 2012, he moved
promoting tissue formation in situ.[9,10] The advantage of to biotech industry as a senior polymer scientist
this approach is the reduction in the number of operations and a team lead (in Ocutech Limited) for the
needed, thus resulting in a shorter recovery time for the development of biomedical devices. Prior to
patient. Edinburgh, he was a PDRA at Carleton University
in association with XRCC, Canada, where he
The human body is made up of complex and
investigated the ‘‘Structure and properties of
sensitive biological systems. There is, therefore, a
polymers, smart materials and composites’’.
stringent requirement for precise and appropriate proper-
He graduated from Dhaka University (Physics)
ties of synthetic materials for tissue regeneration. For in Bangladesh. Afterward, he attended Cranfield
this reason great emphasis has been given for the University and earned his Ph.D. degree in poly-
development of new materials. Recently, polysaccharides mer science in 1999. At Edinburgh, Ferdous
have attracted a great deal of interest in medical and applied his knowledge and ideas for the
scientific communities due to some attractive properties development of 100s of effective biomaterials
such as, biodegradability, low toxicity, low manufacture via a simple blending approach. Recently,
costs, low disposal costs, environmentally friendly he developed polymer blend materials with a
aspects, renewability prospect, etc.[11] A large variety of honeycomb structure for bone tissue engineer-
ing. He applies synthetic polymer chemistry
natural materials have been studied and proposed for
approaches for multicomponent polymers syn-
biotechnological applications some of which offers addi-
thesis, and recently, one of his multi-complex
tional advantages for TE applications such as, biological
polyurethane compounds has been undertaken
signaling, cell adhesion, cell responsive degradation, by FibromEd Ltd. These materials have appli-
re-modeling, and so on. However, the down side of using cations in tissue engineering, drug/gene delivery,
natural polymers is that these may rapidly degrade with and other biotechnology.
the possible loss of biological properties during formulation
and storage, often compromising their use as unique Rafi Ahmad is a founder and lead of the Centre
of Applied Laser Spectroscopy at Cranfield
scaffold materials.
University (Shrivenham). He received his Doctor
Despite the minor down side, current research has been
of Philosophy (D.Phil.) degree from the Univer-
focused on the use of natural polymers, which include
sity of Oxford (UK) in 1972 on the topic of Laser
chitin and chitosan, hyaluronic acid (HA), alginate, starch, Interaction with Materials. The field of his
and cellulose-based polymers and their derivatives, for the research extended to include, among many
development of scaffolds fabrication for bone, cartilage, others, laser ignition of energetic materials,
and skin tissues regeneration. In general, however, this and laser-induced processing of natural and
review will be confined to the topic of the process of synthetic polymer for biomedical applications,
modification of polysaccharides for suitable biomaterials funded by many national and international
generation, their ‘‘in vivo’’ and ‘‘in vitro’’ characteristics and bodies. He was the principle investigator of
future prospects. many EEC funded projects on cellulose research
within the INCO-DC scheme and a key member
of the consortium of six European organizations
for research on an optical technique for plastic
2. Chemistry of Polysaccharides identification under the Brite-Euram Scheme
(BE-7148). He was the UK representative in the
It is well known that some of the structural characteristics management committee of the EEC’s COST-G7
of polysaccharides and their derivatives such as, degree of Action and the EULASNET network.
substitution (DS) and molecular weight (Mw ), etc. largely
govern their properties, such as solubility, physiological
activities, chemical reactivity, and biodegradability.[12] to initiate the reaction (e.g., solvents, initiators, etc.).
Polysaccharides can be modified physically, chemically, Therefore, it is essential to remove such toxic agents by
or biochemically. In some modification processes of purification process, in order to ensure the safety regulation
polysaccharides involved toxic reactive agents or chemicals and the biocompatibility of the final product.
Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200409

2 www.MaterialsViews.com
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Polysaccharides and Their Derivatives for Versatile Tissue Engineering Application
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2.1. Chitin, Chitosan (CS), and Their Modifications with functional materials, can be employed for the
modification of chitin and CS.
Chitin is a natural polysaccharide and second most
abundant polymer after cellulose, produced annually in 2.1.1.1. Carboxymethylation of Chitin and CS
the world. It was first identified in 1884 and is found in the
The introduction of carboxymethyl groups into chitin and
exoskeleton of arthropods (e.g., in the shells of crabs and
CS has been investigated by a number of research
shrimp), the cuticles of insects, and the cell walls of fungi
groups.[13–21] A number of derivatives of chitin and CS
and yeast. It is a polymer with a chemical structure of
can be synthesized (Figure 2). The degree of carboxymethy-
2-acetamido-2-deoxy-D-glucopyranose whereas, CS, how-
lation of chitin contributing to the water solubility is
ever, is the most important (an N-deacetylated) derivative
determined by the concentration of sodium hydroxide
of chitin containing varying fractions of the two residues[12]
present during the preparation of alkaline chitin by freezing
randomly distributed b-(1–4)-linked D-glucosamine (dea-
process.
cetylated unit) and N-acetyl-D-glucosamine (acetylated
Among the water-soluble CS derivatives, O-carboxy-
unit). CS is obtained by (partial) deacetylation of chitin
methyl chitosan (O-CMCS) is an amphiprotic ether deriva-
(Figure 1) in the solid state under alkaline conditions (i.e.,
tive, contains COOH groups and –NH2 groups in the
concentrated NaOH) or by enzymatic hydrolysis in the
molecule. O-CMCS (Figure 2b) can be prepared by reacting
presence of chitin deacetylase.
chitin powder with monochloroacetic acid in isopropyl
The lack of solubility of chitin in common solvents (such
alcohol as a solvent using the condensation reaction.[22] The
as acetone, alcohol, water, etc.) limits the practical
synthesis of N-CMCS (Figure 2c) can be prepared by using CS,
utilization of this material. Although CS can be dissolved
glyoxalic acid with sodium cyanoborohydride solution, and
in aqueous solutions with a pH < 6.5, the required
also by pH adjustment.[23]
neutralization prior to biological applications may cause
The synthesis of N-carboxymethylation of CS is
changes in the shape and size of the host materials.
performed by reacting free amino groups of CS with
Therefore, chemical modification of both chitin and CS has
glyoxylic acid to produce a soluble aldimine. The reduction
been found to be necessary to increase their solubility in
of the aldimine product is carried out with a reducing
common solvents for their maximum utilization. The
agent (e.g., sodium cyanoborohydride). This preparation
modification of chitin and CS can be divided into two
does not require warming or cooling and it produces
categories such as: i) chemical modification via covalent
an N-CMCS, containing acetyl, carboxymethyl, and free
bonding, and ii) physical blending with other materials, as
amino groups in proportions readily controlled through
discussed in the following section.
the choice of the properties of the starting CS (in terms
2.1.1. Chemical Modification of Chitin and CS of degree of deacetylation and molecular weight) and
also by the amount of glyoxylic acid used. Recent studies
Numerous methods, for example carboxymethylation,
have shown that a water-soluble derivative, N,O-CMCS,
graft-copolymerization and crosslinking, and blending
can be prepared by introducing carboxymethyl groups
onto some of the amino and primary hydroxyl sites
of the glucosamine units of the CS structure. This CS
OH H3COC OH derivative is a promising biomaterial.[24–26] N,O-CMCS is a
H H
H CS derivative bearing carboxymethyl substituents at
H NH H
O
H some of both amine and 6-hydroxyl sites of glucosamine
O HO O
units. Synthesis of N,O-CMCS can be performed using CS,
O
HO
NH
O HO sodium hydroxide, isopropanol with chloroacetic acid[27]
H H H NH
H H H as solvent.
H OH H
H3COC H3COC
The synthesis of N-CBCS, on the other hand can be
Chitin performed by using CS, levulinic acid, and sodium
borohydride with water as a solvent (Figure 2d). The
OH OH
H H N,N-dicarboxymethyl chitosan (N,N-DCMCS) (Figure 2e)
H
H NH2 H derivative forms a transparent and mechanically
H
O O HO O resistant film. N,N-DCMCS-calcium phosphate possesses
HO O
O
HO
excellent osteoinductive properties,[28] and can be
H NH2 H H NH2 prepared using CS, water, glacial acetic acid, and glyoxylic
H H H
H H
OH acid with sodium borohydrid. The synthesis of N-CECS
can be performed by using water, 3-halopropionic acids
Chitosan
under mild alkaline media (pH 8–9) with, NaHCO3
Figure 1. Chemical structure of chitin and chitosan. (Figure 2f).[29]

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OH transition occurring at temperatures that

H OCH2COOH
H are similar to that of the human body.
H NaOH,
ClCH2COOH H
O O O This characteristic is an advantage in the
HO Isopropanol H design of drug delivery systems, because
NH 37 °C HO
H
H
H NH the body temperature may change due to
H3COC H H
fever, local infections, or disease.[35]
H3COC H
Chitin a) Carboxymethyl Chitin Grafting of NIPAAm onto CS provides
an increase in the water content on
OCH2COOH exposure to aqueous media and improve-
H OCH2COOH
H ment of the mechanical properties and
H H
O the temperature responsive proper-
O
HO HO
ties.[36] Kim and co-workers[37] synthe-
NaO a cid HO HO
ic sized a CS-graft-NIPAAm copolymer
H NH2 ClC H, lin H
H H HC e vu 4 H using Ce(IV) ammonium nitrate as the
2 O OH L BH H
OH Na
b) O-Carboxymethyl Iso
H NHR initiator, and the copolymer was then
p
Chitosan 50 ropan H R= CHCH2CH2COOH crosslinked with glutaraldehyde. The
°C ol O OH CH3 efficiency and percentage of copolymer-
HO d) N-Carboxybutyl ization increased as the monomer con-
H/ H NH2 CH Chitosan
O O C H H
OH C 60 ° N 3 C O centration (NIPAAm) increased. Such
H CH 3OH, Chitosan G aOH, OH/
Na cid ly H OH findings have been confirmed by Cai
H lic a Na oxali 2O H
a B et al. in their copolymerization of the
3-halopropionic
O ox H c aci
NaHCO3
Gly BH 4 4 d H
same material by gamma-radiation.[30]
HO HO Na O
H HO HO The resulting copolymer exhibited pH-
H H
responsive and temperature responsive
acid
H H
NHCH2COOH H
CH2COOH behaviors, with swelling ratios higher
H OH N
c) N-Carboxymethyl Chitosan CH2COOH at pH 4 than at pH 7. At 35 8C, above the
H
lower critical solution temperature (LCST)
O e) N, N-Dicarboxymethyl
HO HO Chitosan (32 8C), the equilibrium water content
H was lower in comparison to the one at
H H 25 8C.[37] CS-based membranes may be
NHR
R= CH2CH2COOH synthesized for dual effects: to accelerate
wound healing due to the bioactivities of
f) N-Carboxyethyl Chitosan CS and, at the same time, an efficient drug
Figure 2. Various chemical modification pathways of chitin and chitosan are showing to delivery system. Hydroxyethyl metha-
form derivatives. (a) Carboxymethyl chitin, (b) O-carboxymethyl chitosan, (c) N-carbox- crylate and acrylic acid monomers have
ymethyl chitosan, (d) N-carboxythyl chitosan, (e) N,N-carboxymethyl chitosan, and (f) N- been grafted with CS[38,39] and the
carboxyethyl chitosan. authors have found that the grafting of
both monomers showed remarkable
effects on the degree of swelling, cyto-
2.1.1.2. Graft Copolymerization toxicity, thrombogenicity, haemolytic activity, and on
thermal properties.[39]
Chemical modification of CS by grafting of vinyl mono- CS was first reacted with propylene oxide to form
mer(s), and then crosslinking the material, is another hydroxypropylated CS, which was subsequently linked
approach of improving its performance and extending its with ethylene glycol functionalized nano-hydroxyapatite
scope of applications.[30–34] For instance, pure CS hydrogels to form an organic–inorganic hybrid network structure
are known to provide fast degradation and dissolution and (Figure 4a,b).[34] Several research groups have reported
allow limited improvement in mechanical properties. the graft copolymerization of CS-graft-PCL[40] and CS-graft-
Various types of monomers can be grafted onto the CS poly(DL-lactide),[41] and grafted scaffolds (Figure 4e,f) have
backbone using a number of synthetic methods such as shown a significant improvement in TE application.
ceric-ion initiation and radiation-induced methods. An
2.1.2. Modification by Blending
example of ceric-ion initiation of grafting scheme is
presented in Figure 3. One of the intensively investigated To provide a wide range of new physicochemical and
monomers is the N-isopropyl acrylamide (NIPAAm). This is mechanical properties, the blending of chitin and CS with a
due to its ability to form hydrogels with liquid–gel variety of synthetic or natural polymers and ceramic nano

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OH
OH
and phase-inversion process.[57] Hong
H
H H et al.[48] reported on the blending of CS
6 H
O O 4 O
and alginate to form polyelectrolyte
(NH4)2Ce(NO3)6 5 O
2 complex (PEC). Such blended complex
HO 3
NH O 1
was found to be a potential candidate in
H 2
NH
H H H H TE, and delivery of bioactive molecules
H
Chitosan (CS) to stimulate tissue regeneration. Kim
Ce4+ et al.[45] have demonstrated that the
(a) CS-Ceric Ion Complex
blending of CS with bacterial cellulose
3+
(BC) form porous structure with inter-
-H , -
e
+,-C connected network that has a large
+
-H
surface area and useful for vasculariza-
Ce
OH
3+
OH tion.
H H
H H Blending of CS with hydrophobic
O O O polyestyers, such as poly(e-caprolactone)
O
[51,53,56]
H (PCL) and poly(L-lactic acid)
O HO H [57]
NH2 NH (PLLA) was performed in a single
(b) H (c) H
H H phase without any complex chemical
(b, c) CS radicals modification.
H 2C CH
H 2C CH
C O Several other studies[55,58–62] have
+ + C O
shown that the blend of CS can be
NH
NH prepared with ceramic and phosphate-
CH
CH based glass materials. Such bioactive
H 3C CH3 H3C
H OH CH3 implants with bone-like calcium phos-
H OH NIPAAm H
phate minerals may bond to native bone
H O O
O H and enhance bioactivity.[44] Injectable
O
HO H bone substitute material was developed
H NH
O by Liu et al.[59] using blending approach
NH2 H
H H
consisting of CS, citric acid and glucose
H H solution as the liquid phase, and trical-
H2C C H2C C
n n cium phosphate (TCP) powder as the solid
C O C O
phase. Kong et al.[60] have prepared CS/
(d) NH (e) NH hydroxyapatite (HAp) nanocomposite
CH CH scaffolds, and studied the bioactivity of
H3C CH3 H 3C CH3 the composite scaffolds by examining the
(d, e) CS-graf t-PNIPAAm apatite formed on the scaffolds incubated
in synthetic body fluid (SBF), and the
Figure 3. Ceric-ion initiation of graft copolymer synthesis of NIPAAm monomer onto activity of preosteoblasts when cultured
chitosan (CS) shows (a) CS-ceric ion complex formation, (b, c) radical formation onto CS
onto scaffolds. The authors suggested
chain, and (d, e) CS-graft-PNIPAAm.
that compared with pure CS, the compo-
site (CS/HAp) could form apatite more
particles are currently being investigated, as these are readily during the biomimetic process. CS/HAp micro-
unlikely to compromise the biocompatibility and biode- sphere-based scaffolds were also produced by Chesnutt
gradability of the blends.[42–55] For example, CS blended et al.[61] using a co-precipitation method. Chesnutt et al.[61]
with silk fibroin in a solution process and fabricated as a found the surface area and surface roughness of composite
tubular scaffold using freeze drying and crystallization scaffolds were significantly greater than that of scaffolds
process showed a 3D network structure (required for TE composed of only CS. Moreover, composite scaffolds
application) and was analyzed by polarized microscopy.[43] swelled significantly less than CS scaffolds and are,
The blends of chitin and CS form a complex gel,[46,47] therefore, expected to maintain their shape better in vivo.
molded in various shapes and forms with a well-designed Hybrid CS-PLA/HAp ternary system composite materials
porous structure, required for TE application. These are done were also developed.[62] In this system the concentration of
[45,46]
using a variety of techniques such as freeze-drying, PLA plays a significant role on the structure and properties
rapid prototyping and internal bubbling,[48] electrospin- of the scaffold. The CS/HAp composite scaffolds has been
ning,[53,54] precipitation, extrution,[56] and supercritical found to exhibit an excellent bioactivity in vitro and was

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Figure 4. (a) Grafted CS-functionalized nHAp (CS-g-nHAp), (b) fluorescence micrograph of pure CS-g-nHAp scaffold is showing the 3D
network structure, (c, d) fluorescence micrographs illustrating proliferation of pre-osteoblasts (MC3T3-E1 cell line) cultured on CS–g-nHAp
scaffolds, days 7 (c) and 28 (d), respectively. The fluorescence micrographs of cell-scaffold constructs were obtained after staining with
nucleic acid staining dye (Hoechst 33342). (e) Scanning electron microscopic (SEM) image of CS-g-PDLLA fibrous scaffold (fabricated by
electrospining method). (f) SEM image of MC3T3-E1 cells cultured on CS-g-PDLLA scaffold after 7 d, cells were observed spanning the gaps
between electrospun fibers, as indicated by the flattened and spreading morphology covering the surface. (a–d) Reproduced with
permission.[34] Copyright 2011, Elsevier. (e & f) Reproduced with permission.[41] Copyright 2010, Elsevier.
tested by immersion in SBF solution for different time A number of chemical methods[68] can be employed to
periods (i.e., through its ability to promote the deposition modify HA. These methods can take place in three target
of an apatatic layer). Araujo et al.[62] also the observed sites: a) glucuronic acid carboxylic acid (–COOH), b) primary
microstructure of SBF deposition onto the scaffolds and secondary hydroxyl groups (–OH), and c) N-acetyl group
and after 30 d, the composite scaffold was found to be (–NHCOCH3). Some examples of resulting chemical struc-
completely covered with a nanostructured apatitic layer tures of HA derivatives with functional groups are
which could be used as potential material for biomedical presented in Figure 5.
applications.
2.2.1. Modification of the –COOH
Amidation with carbodiimides is one of the most widely
used methods for HA modification.[64,69,70] The reaction is
2.2. Chemistry of Hyaluronic Acid (HA)
carried out by dissolving HA in water or DMSO in the
HA is a linear high molecular weight polysaccharide presence of predominantly 1-ethyl-3-[3-(dimethylamino)-
consisting of alternating disaccharide units of, b-1,4-D- propyl]-carbodiimide (EDC).[64] However, HA first needs to
glucuronic acid and b-1,3-N-acetyl-D-glucosamine[63] as is be converted from its native sodium salt form into its acidic
illustrated in Figure 5. HA is highly hydrated polyanionic form to be soluble in the organic solvent.
macromolecule with molecular weights in the range of 100 Hyaluronan based polymeric networks have been
and 8000 kDa.[64] This and its derivatives have been synthesized by several research groups.[71–73] They have
clinically used as medical products, and recently it has utilized well known crosslinking processes based on
been recognized as an important building block to generate aqueous Ugi condensation reactions. A diamine was used
new biomaterials for TE and regenerative medicine.[65–67] as a crosslinking agent to form diamide linkages between
For diverse biological applications, there is a need for the polysaccharide chains. The reaction is performed in
chemical modification of HA to improve desire properties water at pH 3 formulated with formaldehyde, cyclohexyl
(e.g., the hydrophobicity and biological activity of resulting isocyanide, and the diamine. First, the diamine condenses
materials). with formaldehyde to form a protonated diamine which

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alkylation of the tetra(n-butyl)ammo-

OH OH
H OH OH nium salt of hyaluronan with an alkyl
O H
H O H halide in dimethylformamide (DMF)
O HO O H H
O O O HO O solution and subsequently purified.[76]
O O
HO In particular, HYAFF 11 at 50% of
H OH H NH HO
H H H H H OH H NH esterification is a hydrophilic polymer
H H H H
O CH3 n (water solubility 60 mg ml1), which
D-glucuronic acid O CH3
N-acetyl-D-glucosamine may be processed as a gel. This has been
found to be very similar to native HA,
(a) Repeating disaccharide unit albeit with a higher resistance to the
action of the hyaluronidases. The solubi-
HA lity of hyaluronan can be dramatically
NH2 O reduced so that it is solid even after
HN HO Glycidyl methacrylate remaining for an extended period in
O aqueous environments by benzyl ester-
Adipic dihydrazide O
ification of the free carboxyl groups on
HA-BDDE Crosslinked O
the glucuronic acid component of the
O
hyaluronan chain (HYAFF).[77] By varying
O
O OH the degree of carboxyl modification, the
NH OH
degradation rate can be tailored to meet
HN O H O OH O the requirements of the implantation site
H H O H
O HO O H H and clinical application. At higher per-
O O O HO O
O centages of esterification, the resulting
HO O
H OH H NH HO HYAFF materials became insoluble in
H H H H H OH H NH
H H H H water.[78] Benzyl ester of hyaluronan
O CH3 n
(b) O CH3 (HYAFF) is one of the most promising
materials which can be processed to
Figure 5. Hyaluronic acid (HA) and its chemical modification show (a) chemical structure obtain several types of devices such as
of HA with a repeating disaccharide unit and (b) examples of HA chemical modification tubes, membranes, nonwoven fabrics,
on the target sites of carboxylic acid group (adipic dihydrazide) and hydroxyl group (HA- gauzes, and sponges. All these scaffolds
BDDE crosslinked and glycidyl methacrylate).
are highly biocompatible thus rendering
them potential biomaterials for TE appli-
cation.[79]
then reacts with the cyclohexyl isocyanide. The carboxyl Several authors have described the reaction of HA
group of HA then eliminates the activated cyanide with glycidyl methacrylate to synthesize methacrylated
intermediate to form an (acylamino) amide bond. The HA.[80–82] The reaction is performed in water in the presence
use of formaldehyde, which is known to be carcinogenic, of excess triethylamine as a catalyst. Bencherif et al.[80]
requires specific handling. However, this method leads to suggest that the reaction occurs mainly on the carboxylic
the formation of a secondary amide, adding a second groups of HA and that the secondary trans-esterification on
pending group and in this case, a cyclohexyl. the hydroxyl groups is reversible.
Carboxylic group of HA can be modified by esterification
2.2.2. Modifications of the –OH
using alkyl halides, such as alkyl iodide or alkyl bromide. For
example, synthesis of amphiphilic HA esters was per- The modification of HA on the –OH site was the reported
formed[74] with bromide alkyls in DMSO to convert the case of HA crosslinking by ether formation using epox-
native HA sodium salt into its TBA salt, and the reaction was ides,[83] where 1,2,3,4-diepoxybutane was used as a cross-
performed for 24 h. The HA crosslinking was performed by linking agent. They performed the reaction in strong
esterification using tetraethylene glycol, functionalized by alkaline conditions at pH 13–14 (0.2 M NaOH and 0.1%
two tosylate groups. Another example of the same sodium borohydride) and at 50 8C for 2 h. The crosslinking of
esterification chemistry the use of tosylate as the leaving HA was also performed using butanediol-diglycidyl ether
group, described by Huin-Amargier et al.,[75] in which HA (BDDE)[84] in a 0.25 M NaOH solution. Other bisepoxides
crosslinking was performed by esterification using tetra- have been applied to prepare crosslinked HA gels, such as
ethylene glycol functionalized by two tosylate groups. The ethylene glycol diglycidyl ether and polyglycerol poly-
reaction is performed in DMSO from the TBA salt of HA. glycidyl ether.[85] BDDE (Figure 5b) is used for most
Esterified hyaluronan biomaterials have been prepared by crosslinked HA hydrogels currently available in the market.

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This offers the advantage of ease of synthesis, besides, HA– derivatives at pH values above 8 due to the higher pKa of
BDDE degradation products have not shown any cytotoxi- the amino groups.[98] The in situ generation of potentially
city as is known that the epoxide compounds are toxic degradation hydrazide products may result in side
hydrolyzed into simple diols.[86] effects which need to be evaluated using in vivo
Ester formation is another method of modification of HA methods.[98]
on the –OH site. HA has been modified using alkyl succinic Shu et al. reported[99] the synthesis of hydrogels by
anhydrides such as, octenyl succinic anhydride[87] in which crosslinking HA–disulfide derivatives, and in this case thiol-
the hydroxyl groups of HA react with the anhydride to form modified HA was prepared using a carbodiimide-mediated
ester bonds. A novel method of graft-copolymerization has hydrazide chemical method. Hydrogels were then formed
been described[88] using an acyl-chloride activated carbox- under mild conditions by air oxidation of thiols to
ylate compound onto the hydroxyl groups of HA to form disulfides. This type of reaction is interesting because it
ester bonds. HA esterification with methacrylic anhydride does not rely on synthetic crosslinking agents, and can be
was performed to obtain methacrylated HA.[89,90] The formed in physiological conditions. Kurisawa et al.[100]
presence of methacrylate groups enabled further photo- synthesized in situ crosslinkable hydrogels from tyramine-
crosslinking of the HA derivatives. grafted HA by treatment with horseradish peroxidase
and H2O2. Formation of the hydrogel was performed in situ
2.2.3. Modification of the –NHCOCH3
using two syringes, one containing HA–tyramine and H2O2
Deacetylation of the N-acetyl group of HA recovers an and the second containing horseradish peroxidase to
amino group which can then react with an acid using induce the crosslinking reaction, which proceeds at the
amidation methods.[91] However, deacetylation can cause CC and CO positions between phenols. The in situ
severe chain fragmentation, and therefore milder treat- crosslinked HA hydrogels proved to be biocompatible.
ments have been reported to induce HA chain degradation Bencherif et al.[101] and Leach et al.[102] the synthesis of
via b-elimination of the glucuronic moiety.[91] Oerther HA hydrogels by photoinduced crosslinking method. The
et al.[92] used this method to crosslink HA with the methacrylated-HA products, obtained via glycidyl metha-
carboxylic groups of alginic acid. Several other researchers crylate or via methacrylate anhydride were further cross-
have demonstrated[93] that the deacetylation of HA can be linked by free-radical photoinduced polymerization
performed using enzymes. For obtaining derivatives with method when exposed to under UV-light (365 nm). In this
longer stability times after injection, other techniques are case a photoinitiator [e.g., 2-oxo-ketoglutaric acid or 4-(2-
continuously being explored. Deacetylated HA can be used hydroxyethoxy) phenyl-(2-hydroxy-2-propyl) ketone (Irga-
for further crosslinking where the deacetylated amino cure 2959)] is essential to initiate radicals.
groups react with the carboxylic groups of HA to form an
auto-crosslinked hydrogel.
2.3. Alginates and Its Modification
2.2.4. Crosslinking of HA
Alginates are naturally derived linear unbranched poly-
The crosslinking of polymer is a well-known chemical saccharides which are produced by several brown algae as
modification method to increase network structure and well as some bacteria,[103] consisting b-(1–4)-linked D-
increase in mechanical stability. For clinical applications of mannuronic acid (M block) and a-(1–4)-linked L-guluronic
HA, there is a need to increase stability of HA solutions after acid (G block) units arranged in an irregular block wise
injection. Most commercial HA products for articular pattern of varying proportion of GG, MG, and MM blocks
injections (e.g., Synvisc by Genzyme) and for dermal filling (Figure 6a) that change in composition and sequence along
(Restylane by QMed, Juvederm by Allergan, Teosyal by the polymer chain. The mannuronic acid forms b(1 ! 4)
Teoxane, etc.), therefore, use crosslinking to achieve non- linkages so that M block segments show linear and flexible
soluble hydrogels, providing increasing mechanical stabi- conformation. The guluronic acid, on the other hand, gives
lity. Clinical case studies have reported the filling effect rise to a (1 ! 4) linkage, which serves to introduce a steric
durations of 6–9 months.[94–96] Many commercially avail- hindrance around the carboxyl groups. For this reason the G
able injectable HA hydrogels are crosslinked[97] with BDDE, block segments provide folded and rigid structural con-
divinylsulfone, and glutaraldehyde. formations that are responsible for a pronounced stiffness
Several research groups[98,99] have investigated the of the molecular chains, and widely used as a type of desired
crosslinking of HA–amine or HA–hydrazide derivatives biomaterials for cell immobilization,[104] TE,[105] and drug
obtained using homo- or hetero-functional crosslinking delivery.[106]
agents which include bis(sulfosuccinimidyl) suberate (BS3), Alginate has a number of free hydroxyl and carboxyl
3,3-dithiobis (sulfosuccinimidyl) propionate (DTSSP), or 2- groups distributed along the backbone, and therefore, it is
methylsuberimidate (DMS). They react with HA–hydrazide an ideal candidate for chemical functionalization to
derivatives at pH values of above 5, or HA–amine improve desired properties. A variety of techniques which

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Figure 6. Alginate and its proposed chemical modification on the hydroxyl group via grafting reaction, (a) molecular structure of alginate
(shows GG, MM, and MG blocks) and (b) the synthetic route of alginate-graft-PEG copolymer.
include graft copolymerization, oxidation/reduction, cross- sium peroxydiphosphate/thiourea redox system. The
linking, and sulfation can be employed for the chemical synthesized graft copolymer increased swelling, metal
modification of alginate, as discussed in the following ion uptake, flocculation and resistance to biodegradable
section. properties in comparison to those in un-grafted alginate.
2.3.1. Graft Copolymerization 2.3.2. Oxidation

Graft copolymer reaction can take place at the sites of Oxidation of alginate by periodate ion was for a long time as
hydroxyl group of alginates. Laurienzo et al.[107] have a classical method for modification.[110,111] Oxidation
synthesized a novel alginate–polyethylene glycol (PEG) reactions on –OH groups at C-2 and C-3 positions of the
graft copolymer by reacting a mono-carboxyl terminated uronic units of sodium alginate are performed with sodium
PEG with a sodium alginate, modified by inserting a given periodate (Figure 6b), which leads, by rupture of carbon–
amount of amine functionalities (Figure 6b). The coupling carbon bond, to the formation of two aldehyde groups in
between PEG and alginate was carried out using carbodii- each oxidized monomeric unit. Therefore, larger rotational
mide chemistry in aqueous solutions. The alginate-graft- freedom and new reactive groups along the backbone are
PEG copolymers retain the gelation characteristics of obtained. Exclusion of light during oxidation is essential for
alginate. The presence of grafted PEG molecules inside the limitation of side reaction. It is possible to control the
alginate gels will increase the pore dimensions, and at the alginate degree of oxidation by varying the concentration
same time, will induce improved cell anchorage, and can of the oxidant.
be used as potential candidate materials where higher The oxidized alginate with aldehyde groups on the
biocompatibility and pore dimensions are required, polymer chain gives new reactive groups for chemical
and for gel entrapment devices and for use in micro- modification, especially by reductive-amination. The sub-
encapsulation techniques. sequent reductive amination is performed with alkyl amine
Sen et al. reported[108] the synthesis of various grades by using NaCNBH3 as reducing agent, as it is more reactive
of graft copolymers, based on acrylamide and sodium and selective than the frequently employed sodium
alginate, via microwave irradiation. Results of their hydroborate (NaBH4). A study reported by Bouhadir
research showed that the copolymer having higher degree et al.[112] demonstrated that periodate oxidation can be
of grafting and molecular weight persisted better floccula- utilized to produce biodegradable alginates suitable for TE
tion compared to those of other grades of the grafted application. By limiting the degree of oxidation to 5%, the
polymer and sodium alginate. Alginate has also been ability to form gels with Ca2þ was retained,[113] whereas,
grafted with vinyl sulfonic acid[109] by employing potas- the oxidized (dialdehyde) residues provided degradability

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at physiological conditions. Interestingly, analysis of the ultimately dictates the physical properties of the gels.
degradation kinetics showed an initial phase of rapid With lower concentration of Ca2þ, temporary gels as highly
degradation followed by a slower process. It has been viscose solutions can be obtained. At higher calcium levels,
reported in a separate study[114] that by combining precipitation or gelation results form permanent associa-
periodate oxidation with enzyme alginates gel formation tions of the chains. However, the high level crosslinking and
with Ca2þ, can be tailored. The rate of such gelation, as permanent gels are not suitable for injectable application,
well as the elasticity of the formed gels, has been found but only very mildly crosslinked alginate gels can be
to be strongly depended on the presence of sufficiently injected.[121] The functional properties, such as biocompat-
long G-blocks. However, nongelling sequences were ibility, immunological characteristics, porosity, swelling
reported to influence the system. Oxidized and unoxidized behavior, stability, biodegradability, and the mechanical
alginates having the same average length of G-blocks and properties of the gels are strongly dependent on the
comparable content of G as well as comparable molecular chemical structure, molecular size, gel forming kinetics, and
weights were studied. The results demonstrated a remark- the cation.
able influence of the introduction of flexible (oxidized/
2.3.4. Crosslinking of Alginate with Cationic Polymers
reduced) residues, as the elasticity of the gels dropped
rapidly with increasing extent of oxidation. Indeed, true In recent years, crosslinking of alginate with cationic
gels were only observed for 2 and 4% oxidation. The results polymers has been of significant interest for biomedical
show clearly that the segments between the gelling application. Alginate-CS is known to form a complex
G-blocks play an important role in the formation of elastic networks which can be processed to form gels, 3D porous
Ca-alginate gels, such elastic gels could have potential scaffold, membrane, fibers, etc.[122–126] Several other
application in TE. studies also demonstrated that the PECs of alginate with
peptide[127] and gelatine[128] can be synthesized. The
2.3.3. Crosslinking of Alginate with Polyvalent Cationic
alginate interact with gelatine via ionic and hydrogen
Materials
bonding interactions to provide a complex networks.[128]
Recent research has shown an increasing demand on
2.3.5. Sulfation
crosslinking of alginates using polyvalent cations,[115] to
focus mainly to prepare alginates-based gels for TE When alginate is sulfated it will show high blood
applications. Polyvalent cations have high affinities of compatibility, because the structural similarity to that of
binding to alginates and form gels via crosslinking. The heparin, which has been widely used for anticoagulant
gelation and crosslinking of the polymers are mainly therapy for more than 60 years. Ronghua et al.[129]
achieved by the exchange of ions from the guluronic acids synthesized the alginate sulfates from sodium alginate
with the divalent cations, and the stacking of these through reaction with ClSO3H in formamide. Other
guluronic groups to form the characteristic egg-box methods of chemical modification of alginates are ester-
structure. The cations bind to the a-L-guluronic acid blocks ification, the Ugi reaction, and amidation which have been
in a highly cooperative manner and subsequently become well reviewed by Yang et al.[130]
a gel.[116] Literatures revealed that divalent cations
(Cd2þ, Co2þ, Cu2þ, Mn2þ, Ni2þ, Pb2þ, and Zn2þ) are suitable
2.4. Starch
crosslinking agents but not monovalent cations or
Mg2þ.[117,118] The chelation at the G-residue of the alginate Starch consists of a mixture of a linear poly(1,4-b-D-
molecules results in ionic interaction between the guluro- glucopyranose) (amylose) (Figure 7a) and a branched
nic acid groups while the van der Waal forces between poly(1,4-b-D-glucopyranose) with branches of (1,6-D-gluco-
alginate segments result in a 3D gel network.[119] Divalent pyranose) (amylopectin) (Figure 7b) and occurring nearly
alkaline ions (Ca2þ, Ba2þ, and Sr2þ) bound mainly to GG every 25 glucosidic moieties.[131] It is naturally produced in
segments while trivalent lanthanide ions (La3þ, Pr3þ, and the form of semicrystalline granules with different size and
Nd3þ) showed affinity for both GG and MM segments.[120] composition, depending on the source of origin.[131] Starch
The ultimate affinity of binding will depend on the ionic is a biodegradable and inexpensive natural polymer, and
radius, coordination number of crosslinking ions and the can be processed using various techniques to form shapes
presence of water of hydration surrounding trivalent like 3D porous scaffolds, microparticles, and bone cements.
ions.[120] In order to form gel networks, each alginate chain The above shape of starch-based polymers has shown an
dimerizes to form junctions with many other chains with enormous potential for a wide range of applications in the
ions. For example, the Ca2þ induced alginate gel networks biomedical field and commodity items.[131–133] However,
by inducing dimeric association of the G-block regions. the intrinsic properties such as thermal, mechanical, and
However, the chain association will depend on the biological properties of starch are such that it cannot
concentration of Ca2þ present in the system, which be used directly in most applications. Typically, it must be

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Figure 7. Molecular structure of starch macromolecules comprising anhydroglucose units connected by a-1,4 linkages in amylase chains
(a) which are often branched by connection through a-1,6-linkages (b) (amylopectin branch).
modified or blended with other materials to achieve a 2.4.2. Modification of Starch by Graft Copolymerization
more appropriate property.[134]
The modification of starch by graft copolymerization of
2.4.1. Modification of Starch by Blending
monomers onto its backbone has been carried out by
Starch-based polymeric systems are commonly blended several research groups.[140–145] Graft copolymerization of
with thermoplastic polymers to increase thermal and acrylamide,[140] acrylonitrile,[141] ethyl methacrylate,[142]
mechanical stabilities, and for ease of processing.[135] hydroxyethyl methacrylate,[142] glycidyl methacrylate,[143]
Blends of starch with poly(vinyl alcohol), cellulose acetate, methacrylic acid,[144] and vinyl acetate[145] onto starch
polycaprolactone, and poly(lactic acid) have been proposed backbone has been carried out using radical polymerization
as potential alternative biodegradable materials for a wide technique. In the radical initiation technique, the compet-
range of biomedical applications, including bone cements, ing homopolymerization is one of the main side-reactions
hydrogels for drugs controlled delivery, and bone sub- that accompany radical-induced grafting. The graft copo-
stitutes.[135] The structure and functional properties of lymerization by ceric-ion initiation is typically carried out
these materials depend on blend components and their under acidic conditions which may have adverse effect to
compositions, material processing technique, nature of starch stability, particularly higher process temperatures.
additives, and reinforcement fillers.[136–139] Ceric ion-initiated copolymerization should have the

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Figure 8. A proposed mechanism of graft-copolymerization onto starch by ceric-ion initiation (C1–C2 and C2–C3 linkages) method shows
(a) starch–ceric ion complex formation, (b, c) radical formation, and (d, e) graft copolymer.
advantage that the initiating species are generated directly major pathways of cellulose formation are shown in
on the starch substrate. The proposed mechanism of Figure 9. The chemosynthesis from glucose derivatives
grafting onto starch is illustrated in Figure 8. This pathway can also be a source of cellulose. As can be seen (Figure 9)
will only lead to one graft per starch molecule, and because that the molecular chain of cellulose is very long and
of the hemiacetal linkage, the grafted chain should be consists of one repeating unit (cellobiose).
readily cleaved under acid-catalysis. Grafting, however, The properties of cellulose are very much dependent on
may also be initiated though oxidative cleavage of the 2,3- the characteristics of their sources of origin. For example,
diol linkage (Figure 8). With peroxide initiators in this the properties of BC are quite different from those of plant
grafting process the initial reaction is expected to be celluloses, especially concerning the ultrafine network
hydrogen abstraction from starch. architecture, high hydrophilicity, and moldability.[148] BC
synthesized by the bacterium acetobacter xylinum, has
some desirable properties such as, high purity, nanofibrous
2.5. Cellulose
structure, high crystallinity, high tensile strength, and good
Cellulose is the most abundant, renewable polymer biocompatibility.[149,150] BC is an unbranched polymer,
resource available Worldwide. The primary occurrence of chemically identical to plant cellulose, but different from
cellulose is the existing lignocellulosic materials plants. The the latter in its macromolecular structure and proper-
most popular and commercially important step is the ties.[151] In all forms, cellulose is a very highly crystalline,
isolation of cellulose from plants to remove lignin and high molecular weight polymer, which is infusible and
hemicelluloses.[146] Other pathways of cellulose prepara- insoluble in all but the most aggressive, hydrogen-bond-
tion include biosynthesis of microorganisms (e.g., algae, breaking solvents such as N-methylmorpholine-N-oxide.
fungi, bacteria) and enzymatic in vitro synthesis.[147] The Because of its infusibility and insolubility, cellulose is

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Figure 9. The major pathways of the cellulose formation are demonstrated from (a) plants (b) microorganisms (background: algae, fungi)
and (c) enzymatic synthesis (structure of enzyme is the background).
usually converted into derivatives to make it ease of into the BC were examined by Wan et al.,[166] Grande
processing.[152] et al.,[167] Zimmermann et al.[168] It has been reported that
Modification of cellulose can have a significant influence there are different interaction processes taking place
on biological responses and on materials functionality.[153] between unphosphorylated and phosphorylated BC fibers
Several modification techniques have been proposed to and HAp[166] as illustrated in Figure 10. Jiang et al.[169]
generate cellulose-based biomaterial, including phospor- developed a new type of bone-replacing material composed
ylation, graft copolymerization, blending with other of different weight ratios of nano-HAp and a CS-carbox-
materials to form composites, and chemical derivatiza- ymethyl-cellulose.
tion.[154–160] Rhee and Tanaka[171] studied HAp formation on cellulose
with the aid of citric acid. Citric acid (C6H8O7), which has a
2.5.1. Modification of Cellulose by Phosporylation
hydroxyl and three carboxyl groups in its molecule, may
The attachment of phosphate groups onto cellulose chain adhere to the cellulose surface through hydrogen bonding
offers the possibility of rendering cellulose more suitable between two hydroxyl groups in citric acid and cellulose
for TE applications by enhancing its bioactivity. The cloth, respectively, and it is ionized to the (C6H5O7)3– (cit)
methods of preparation cellulose-phosphate have been form[171] by the loss of hydrogen ions in 1.5 SBF solution (pH
investigated by several research groups,[161–165] and a 7.4). Negatively charged carboxylate head groups will
proposed mechanism of phosphorylation reaction is bind calcium ions in 1.5 SBF solutions and form a Ca-cit
presented in Figure 10. This method may also be applied complex at the cellulose fiber surface. A cluster of critical
to incorporate phosphate group onto other polysaccharides size will then be formed by adsorbing calcium, phosphate
to enhance their bioactivity. ions and other citric acids on the Ca-cit complex 3D and
clusters may act as nuclei for the HA crystal growth.
2.5.2. Cellulose Composites
The observed 3D porous network structure and intercon-
Several research groups have investigated the cellulose nected pores (Figure 10), which can be adjusted over a wide
composites by incorporating inorganic and polymeric range, make the BC/HAp composites promising materials
materials.[166–171] The incorporation of HAp nano-particles in TE.

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Figure 10. The proposed interaction processes of unphosphorylated and phosphorylation BC with HAp. (a) Chemical structure of BC,
(b) mechanism of HAp growth on unphosphorylated BC (pieces of BC were immersed in a 0.1 M CaCl2 solution at 37 8C for 3 d prior to
biomimetic mineralization) and (c) SEM image BC sample (after soaking in 1.5 SBF) showing cross-section view showing HAp particles
entangled with BC fibers. (d) Structure of phosphorylated BC before immersion in the CaCl2, (e) structure of the interaction of SBF with
phosphorylated BC after soaking in 1.5 SBF, and (f) SEM image is showing the microstructure of HAp crystals. (c,f) Reproduced with
permission.[166] Copyright 2007, Elsevier.
3. Polysaccharide Derivatives in TE and heart tissues substitutes. To discuss all these TE

applications is beyond the scope of this review. Thus, we
Polysaccharide-based materials have good biocompatibil- focus on the application of polysaccharide-based scaffolds
ity with acceptable host response, and have the ability to for tissue regeneration, in particular to bone, cartilage, and
promote cell adhesion, proliferation, and differentiation. skin tissues engineering.
These polymers have been used, with little or no fibrous
encapsulation, to create various tissue analogs in the past
decades. 3.1. Bone Tissue
Tissue engineering covers a broad range of applications,
There is a major clinical need for the generation of new bone
in practice the term is closely associated with applications
to replace or restore the function of diseased, damaged, or
that repair or replace portions of or whole tissues such as
lost bones. Although bone formation approaches are very
bone, cartilage, skin, blood vessels, bladder, muscle, nerve
attractive, fully functional and mechanically competent
conduits, liver, and heart. Among these tissues, bone
bone formation have not been achieved yet. The bone
disorders are of significant concern due to increase in
formation strategy combines the progenitor or mature cells
the median age of our population. While cartilage lacks
with biocompatible materials/scaffolds, with or without
regenerative capabilities, and it is essential to develop
growth factors to initiate repair and regeneration, as
approaches for the regeneration of cartilage tissue damaged
demonstrated in Figure 11.
due to disease or trauma. Skin is the largest organ in the
human body. A significant research effort has been
3.1.1. Chitin and CS-Based Materials
translated to actual therapies, particularly in the area of
skin replacement. The effort has also been made for Chitin and CS-based materials enhance bone formation
cartilage and bone tissues repairing. Thoughtful research both in vitro and in vivo,[172,173] but its mechanical
work has also yielded prototypes of nerve conduits, liver weakness and instability, together with its incapacity to

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Figure 11. Regeneration of bone tissue using cell seeded scaffold. (a) Schematic representation of defected bone repairing by implantation of
cells seeded scaffold into the defect region. (b) SEM image of CS-alginate scaffold is showing the porous 3D network structure. Tissue
compatibility of CS–alginate scaffolds infused with bone marrow was assessed in vivo by implanting them into rats and harvesting after 4
and 12 weeks of implantation. All surgical incisions healed without evidence of infection or other complications. (c) After harvest scaffolds
stained with hematoxylin and eosin (H&E) at week 12 showing no evidence of fibrotic layers and well integration with tissue, and
(d) scaffolds stained with Masson’s trichrome at week 4 showing the formation of a large number of blood vessels in red and deposition of
collagen in blue surrounding that the blood vessels (S: scaffold; M: muscle; C: collagen; BV: blood vessels). Starch-based scaffold implanted
rate femora: immunohistological images of (e) starch/(ethylene-vinyl-alcohol) (SEVA) (50/50) blend, (f) SEVA coated with Ca-P, and
(g) starch/cellulose acetate (SCA) blend scaffolds implanted in rat femora after 1 week. H&E sections (e–g) are illustrating the peri-implant
tissue indicating early form of bone formation. (B ¼ bone, C ¼ connective tissue, S ¼ scaffold). (b–d) Reproduced with permission.[184]
Copyright 2005, Elsevier. (e–g) Reproduced with permission.[210] Copyright 2007, Wiley InterScience.
maintain a predefined shape, limits its application.[174] reinforced microstructure has been achieved.[176] The
Therefore, TE researchers has combined CS with a variety of loading of natural coralline into CS microporous scaffolds
materials, such as alginate, HAp, HA, calcium phosphate, was also found to cause an increase in compressive
poly(methyl methacrylate), PLLA for potential application modulus, and to have a positive impact on the adhesion
in orthopedics and cell-based TE applications.[175] Several of MSCs.[177] The composites of CS/HAp have been found to
materials, based on CS and its derivatives, have been used as promote the formation of bone-like apatite on their
osteogenic bone substitutes. By incorporating calcium surfaces after soaking in SBF and to enhance the attach-
phosphate into a CS scaffold both the compressive modulus ment, proliferation and differentiation of osteoblast-like
and yield strength are significantly improved and a cells.[178] Recently, CS hydrogel–HAp composite mem-

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branes were prepared through deposition of HAp on the by many groups of researchers for bone TE. HA has been
surface of the hydrogel by a wet chemical synthesis chemically modified with aldehyde functional groups,[192]
method.[179] These CS–HAp membranes showed high glycidyl methacrylate,[193,194] 2-aminoethyl methacryl-
degree of biocompatibility as evaluated using MG-63 ate,[195] and conjugated with peptides[196] to form stable
osteosarcoma cell line, which suggest that these composite crosslinked networks that can be used for scaffolds. HA
membranes can be useful for bone TE applications.[179] hydrogel scaffolds can also be utilized for the controlled
Several research groups have developed a biomimetic delivery of osteoinductive and angiogenic growth factors
nanocomposite of CS/HAp by in situ co-precipitation with tunable degradation properties, have been applied to
synthetic approach by means of electrospinning,[180] and bone tissue regeneration.[194–196] Scaffolds, delivering bone
lyophilized in a freeze drying process.[181] In vitro cell morphogenetic protein-2 (BMP-2) were evaluated in a rat
culture with human fetal osteoblast cells for up to 15 d calvarial bone critical size defect model.[194,196] BMP-2
demonstrated that the incorporation of HAp nanoparticles delivery from the HA hydrogels had a clear osteoinductive
into fibers led to significant bone formation compared to effect in vivo and, resulted in a significant mineralization
that onto pure electrospun CS scaffolds. Homogeneous CS- compared to control hydrogels. Other research groups[197–199]
PLA/HAp nanocomposites have recently been developed by have also demonstrated that the HA-based hydrogels can be
in situ precipitation. Rod-like inorganic particles, composed utilized as a scaffold for BMP-2 delivery in a controlled
of randomly orientated sub-particles about 10 nm in manner to promote bone regeneration. A more recent
diameter, were homogenously dispersed in the composite study[195] has shown that a photoirradiated HA hydrogel
polymeric matrix. The characterization of the developed loaded with Simvastatin (SIM) can play an important role in
scaffolds indicated that HAp incorporation led to significant producing an effective scaffold for bone tissue regeneration.
improvement in the elastic modulus and compressive SIM is an inhibitor of the competitive 3-hydroxy-3-methyl
strength and that PLA plays a key role in both the control of coenzyme A (HMG-CoA) reductase and is a very efficient drug
inorganic particles structure and the enhancement of for the induction of osteoblastic differentiation.
the mechanical properties.[182] Chemically modified HAp-
3.1.3. Alginate-Based Materials
chitin and HAp-CS composite materials were reported
to be osteoinductive and to exhibit rapid degradation, Several research groups[200–202] investigated the use of
neovascularization, and indication bone tissue formation in alginate hydrogels covalently modified with RGD-contain-
vivo.[183] CS/alginate hybrid scaffolds displayed improved ing peptides to control cell behavior and bone formation.
mechanical strength and structural stability and were shown Simmons et al.[200] reported the delivery of bone marrow
to stimulate new bone formation and rapid vascularization stromal cells (BMSCs) rather than mature osteoblasts
during in vivo experiments (Figure 11b–d).[184] Improved because BMSCs are clinically relevant autologous progeni-
cell attachment ability is achieved by incorporation of tor cell source. Authors also investigated the effects of
collagen into CS scaffold.[185] physiological quantities of BMP2 and TGF-b3 on bone
Porous CS/collagen scaffolds loaded with plasmid and regeneration in the alginate system. The growth factors
adenoviral vector encoding TGF-b showed higher prolifera- were delivered both individually and in combination, and
tion rate of seeded human periodontal ligament cells observe enhanced bone formation with dual delivery.
than unmodified scaffolds and, after implantation in vivo, The different growth factor conditions with both slowly
good integration with the surrounding tissue.[186] In vitro and rapidly degrading scaffolds were also examined. The
studies further showed that CS has good characteristics scaffold degradation rate is a critical design parameter
for promoting differentiation of MSCs.[187,188] Recently, the for bone tissue regeneration because appropriate scaffold
CS-alginate gel/MSCs/BMP-2 systems were found to degradation provides new space for matrix deposition
stimulate new bone formation when injected in vivo.[189] and coalescence, which may ultimately lead to improved
CS gel has been proposed as an in situ-forming scaffold quantity and quality of regenerated bone.[202]
entrapping MSCs. When gel solutions containing MSCs
3.1.4. Starch-Based Materials
were injected into rats, the resulting gel was retained for
28 d without the development of inflammation at the Human osteoblasts culturing experiments involving scaf-
injection site. Moreover, cells survived well on the scaffold folds, based on starch and cellulose acetate (SCA) blend
and showed immuno-suppression effect by decreasing (50:50 wt.-%), processed by extrusion in combination with
macrophage accumulation.[190] blow agents, showed adequate scaffold porosity and
bioactivity for cells adhesion and proliferation. Cells
3.1.2. Hyaluronic Acid-Based Materials
remained viable homogenously on the surface and filled
The polysaccharide of HA has diverse functions in develop- some pores within the scaffolds producing a skeletal
ment, growth, and remodeling in skeletal biology.[191] structure typical of bone ECM.[203] A study conducted on
Different types of hydrogels, based on HA have been used scaffolds obtained from different starch-based materials

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showed the systems posses adequate porous structure, lated BC in SBF, carbonate-containing HAp crystals with
with pores size within a perceived optimal range for bone structural features close to those of biological apatites were
cell culturing and bone tissue ingrowth. Pores structure and uniformly formed on the BC fibers.[166] Moreover, human
interconnectivity, which affect the degradation behavior bone marrow MSCs seeded on the nanocomposites
and mechanical properties, depend on the processing exhibited better adhesion and activity than those seeded
method adopted (e.g., solvent casting and particulate onto the pure BC. The cells proliferated faster on nano-
leaching, compression molding and particulate leaching, composites probably because of the improved pore size and
extrusion with blowing agents, in situ polymerization).[204] of the presence of the inorganic component. In addition, cell
In general, scaffolds produced in this study presented better osteoblastic differentiation was enhanced even without
mechanical properties than those reported in the literature osteogenic reagents.[211]
obtained from other biodegradable polymers and proposed
for bone TE applications. Particles of soluble starch and
3.2. Cartilage Tissue
composite starch/bioactive glass showed promising results
for further studies involving these materials as scaffold for
3.2.1. Chitin/CS-Based Materials for Cartilage
bone regeneration. The particles tested in vitro were shown
to form an apatite-like Ca-P layer at their surfaces, to CS has been investigated as a scaffolding material in
preserve clearly cells viability in short-term tests, and to cartilage engineering.[212,213] Chondrocytes, cultured in
support attachment and growth of undifferentiated cells vitro on CS substrates, maintained round morphology
that expressed osteopontin both in the presence and and preserved synthesis of cell-specific ECM mole-
absence of dexamethasone.[205] Further studies showed cules,[15,213] and CS scaffolds seeded with chondrocytes
that pre-osteoblast cells adhered to the surface of both showed partial repair of cartilage defects in vivo. CS was
polymeric and composite starch-based microparticles, combined with other polymeric materials, like poly(lactic
expressed the typical osteoblastic marker genes and acid), hyaluronan, and poly(ethylenimine), to improve
mineralized the ECM.[206] The presence of HAp as reinforce- chondrocyte attachment and the consequent cell adhesion,
ment in various starch-based materials may be helpful in proliferation, and biosynthetic activity.[46,214,215] When the
the achievement of osteoblast-like cell adhesion, spreading, human skeletal cells, derived from predominantly cartila-
and proliferation. The physico-chemical properties of ginous fetal femora, were cultured within the CS/PEI
starch-based biomaterials influence the cell proliferation hydrogels, the cells maintained chondrocyte-like morphol-
rate and the presence of HAp affects the degradation ogy (Figure 12a–d) and the characteristic functional
behavior and the surface properties.[207,208] features were similar to those of normal cartilage.
The immunogenicity of starch-based scaffolds [starch and Composites of CS–alginate–hyaluronan have been eval-
ethylene vinyl alcohol (SEVA), SCA, starch and polycapro- uated as scaffold for the development of cartilage regenera-
lactone (SPCL)], as well as HAp—reinforced starch-based tion and in vitro experiments showed neocartilage formation
composites, and the host cellular response by subcutaneous while implanted seeded scaffold led to partial repair of
implantation in rats have been investigated.[207] SPCL and cartilage defect in vivo.[216] Recently, a thermosensitive CS–
SCA composites were found to be the materials that pluronic hydrogel was designed as an injectable cell delivery
stimulated the greatest tissue responses and facilitated in carrier for cartilage regeneration. In vitro cell culture using
vivo vascularization.[209] In general, however, the investi- bovine chondrocytes showed a substantial increase in cells
gated materials did not induce a severe reaction at the studied proliferation and GAGs synthesis during 28 d of incuba-
implantation times. The in vivo endosseous response to tion.[217] Furthermore, CS-based scaffolds have also been
scaffolds made of SCA, SEVA, and SEVA and coated with loaded with growth factors to promote cartilage regenera-
biomimetic calcium phosphate (Ca-P) layer, produced by tion: both CS and collagen/CS/GAGs scaffolds loaded with
extrusion with blowing agents, was evaluated through TGF-1 were reported to promote cartilage regenera-
implantation in rat distal femours. All scaffolds exhibited tion.[218,219] CS scaffolds loaded with epidermal growth factor
a favorable bony response and were well-integrated in resulted in the promotion of chondrogenesis.[220] The CS/
the defect site, but only in the case of SCA was the bone (N,N-dicarboxymethyl) scaffolds, loaded with BMPs, have
around the defect in direct contact with the implant[210] been reported to induce the healing of articular cartilage
(Figure 11e–g). lesion in rabbit.[221]
3.1.5. Cellulose-Based Materials 3.2.2. HA-Based Materials for Cartilage
HAp/BC nanocomposites have been developed in order to HA is known to interact with chondrocytes via various
enhance osteoconductivity, mechanical properties, and to surface receptors involved in sophisticated signaling
increase the relatively small pore size of BC which is pathways, which allow chondrocytes to maintain their
composed of dense microfibrils.[211] By soaking phosphory- original phenotype.[222] In addition, HA has been conju-

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Figure 12. Examples of polysaccharide-based materials used as a scaffold for cartilage tissue regeneration. CS-based scaffold: (a) hydrogels
prepared by blending solutions of CS and PEI (gel top left) and SEM image of freeze dried gel showing porous network structure. Human
skeletal cells were cultured up to four weeks in the CS-PEI hydrogel. Micrographs of cells cultured on hydrogels on (b) day 21. (c, d) Analysis of
gene expression (Pcna and Col2a1) by fetal skeletal cells cultured within hydrogels for 28 d with and without TGF-b3 (relative gene
expressions were normalized by b-actin expression (house-keeping gene). HA-based scaffold: (e) histological evaluation of the defect
implanted with chondrocyte-seeded HYAFF 11 at 24 weeks after transplantation. High prevalence of hyaline cartilage with an irregular
surface (toluidine blue staining) is evident. (f) Alcian blue staining of the defect implanted with chondrocyte-seeded HYAFF 11 at 24 weeks
after transplantation. This is evidence for proteoglycans production throughout the thickness of the newly formed cartilage. Alginate-
collagen-elastin scaffold: the immunohistochemically detected (g) fibronectin, and (h) collagen type II in the ECM produced after 4-weeks
transplants of the aggregated porcine chondrocytes suspended in alginate/collagen/k-elastin (magnification 100). Cellulose-based
scaffold for cartilage: (i, j) Macroscopic and histological appearance of defects treated with Si-HPMC alone, (k, l) with Si-HPMC containing
rabbit nasal chondrocytes (RNC) (Si-HPMC/RNC) after 6 weeks implantation indicating tissue formation. Movat’s pentachrome staining was
used (j, l). (m) Immunohistological analysis of articular cartilage defects after 6 weeks of treatment, appeared to be newly formed repair tissue
and the adjacent healthy cartilage. The sections of defects filled with Si-HPMC containing autologous RNC were stained with hematoxylin/
phloxin/safran (HC, healthy cartilage; RT, repaired tissue). (e,f) Reproduced with permission.[231] Copyright 2001, Elsevier. (g,h) Reproduced with
permission.[240] Copyright 1999, John Wiley & Sons, Inc. (i–m) Reproduced with permission.[243] Copyright 2008, Wiley Periodicals, Inc.
gated to alginate, CS, and fibrin gel matrices to provide experiments.[228] The ethyl and the benzyl esters of
artificial ECM environments.[223] However, the hydrophilic, hyaluronan named HYAFF 7 and HYAFF 11, respectively,
polyanionic surfaces of HA biomaterials do not thermo- are two of the most characterized hyaluronan derivative
dynamically favor cell attachment and tissue forma- polymers, from both physicochemical and biological view-
tion.[224] Therefore, to enhance cell interactions, surfaces, points, degrading at predictable rates through hydrolytic
coated with ECM proteins, such as type I collagen and degradation of the ester bonds (around 2 months for
fibronectin, have been developed by creating physically or complete hydrolysis).[87] Human chondrocytes, grown onto
covalently linked functional domains.[225,226] Physical and HYAFF 11 3D scaffolds, are able to re-express in vitro their
biological characteristics of HA in its purified form, such as differentiated phenotype[229] and to reduce the expression
water solubility, rapid resorption, short residence time in and production of molecules involved in cartilage degen-
the tissue, limit its application as biomaterial.[87] Several erative diseases.[230] Histological evaluation of repaired
attempts have been made to modify HA molecular tissue by HYAFF 11 scaffold, employed in chondrocyte
structure and improve its properties. Covalent crosslinking transplantation in vivo, demonstrated a significant
and photocrosslinking have been used to overcome these improvement of the quality of the healing in comparison
limitations and to provide long term stability and increased to defects without grafted chondrocytes (Figure 12e,f).[231]
mechanical strength.[227] For instance, porcine chondro-
3.2.3. Alginate-Based Materials for Cartilage
cytes, encapsulated in photopolymerized HA hydrogels
maintained viability and were able to produce neocartilage Alginate-based hydrogels have been widely studied for its
within the porous networks during 8 weeks in vitro potential application in cartilage tissue regeneration both

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as scaffolds and as matrix for entrapment and delivery of the way for new therapeutic strategies for the treatment of
biologically active molecules or cells.[232–234] In several cartilage defects. Svensson et al.[246] have investigated the
studies, alginates have been combined with chondrocytes native and chemically modified BC materials using bovine
and either injected into the site of interest[235–237] or molded chondrocytes. The results indicate that unmodified BC
and then implanted.[238] Wang et al.[239] have reported on supports chondrocyte proliferation at levels of approxi-
highly-organized 3D alginate scaffold and utilized for mately 50% of the collagen type II substrate while providing
seeding with porcine chondrocytes, then implanted in significant advantages in form of improved mechanical
the dorsal subcutaneous site of SCID mice and demon- properties. Compared with tissue culture plastic and
strated cartilage-like structures were formed after 4 weeks calcium alginate, unmodified BC showed significantly
implantation. Results of in vivo studies indicated that higher levels of chondrocyte growth. Chemical sulfation
after 4 weeks of implantation the chondrocytes were and phosphorylation of the BC, performed to mimic the
had produced ECM proteins consistent with cartilage glucosaminoglycans of native cartilage, did not enhance
(Figure 12g,h).[237–240] chondrocyte growth, while the porosity of the material did
affect chondrocyte viability. Pulkkinen et al.[247] investi-
3.2.4. Starch-Based Materials for Cartilage
gated the suitability of viscose cellulose sponges as a
Starch and PCL blend scaffolds had been fabricated by melt scaffold for cartilage TE. The sponges were tested alone, or
spinning followed by fiber bonding. The processing with recombinant human type II collagen crosslinked
technique involves fiber packing in an appropriate mold inside the material. Cellulose and cellulose/recombinant
with posterior heating below the melting temperature (Tm) type II collagen sponges were biocompatible, and a
for a determined residence period that will allow the fibers significant cell proliferation was observed. However, the
to form a stable fiber mesh structure. The material used was constructs remained soft during the observation period,
a 30/70 (wt.-%) blend of corn starch with polycaprolactone and were devoid of extracellular matrix (ECM) composition,
(SPCL). These scaffolds were tested for cartilage TE. After typical for normal articular cartilage, and is therefore a
6 weeks of cell culture, bovine articular chondrocytes, potential choice for cartilage TE.
seeded on the scaffolds under dynamic conditions,
presented normal morphological features with extensive
3.3. Skin Tissue Engineering
cells presence at the surface of the support structures, and
penetrating the scaffolds pores.[241] It was observed that the Skin is the largest organ in the human body and provides a
fiber mesh network for support of cell growth and protective layer against various environmental insults.
development presents good interconnectivity. The fiber Skin generally consists of two layers: i) a keratinized,
network structure and an extensive porous area (approxi- stratified epidermis, and ii) an underlying thick layer of
mately 75% as estimated by mCT) is an advantage toward collagen-rich dermal connective tissue.[248] The aim of skin
cells penetration into the bulk of the scaffold, while also TE is to rapidly produce a construct that offers the complete
enhancing nutrients diffusion and removal of metabolic regeneration of functional skin, which should allow the
wastes. Sa-Lima et al.[242] evaluated the starch-CS hydrogels skin to fulfill its normal functions: barrier formation,
induced chondrocytic differentiation and cartilage matrix pigmentory defense against UV irradiation, thermoregula-
accumulation and also the influence of starch in the tion, and mechanical and aesthetic functions.[249] Some of
chondrogenesis of encapsulated adipose derived stromal the functions can be restored with existing skin substitutes.
(ADSC) cells. The ADSC were found to be homogeneously Skin substitutes include cultured keratinocyte grafts, a
encapsulated, viable, proliferating, and maintaining the cellular substitutes, and cell matrices. A skin substitute
expression of typical chondrogenic markers genes, and in a 3D structure consist of multiple skin layers,[250,251]
depositing cartilage ECM molecules. and a scaffold requires suitable microstructures such as, a
100–200 mm average pore size and porosity greater than
3.2.5. Cellulose-Based Materials for Cartilage
90%.[252] Li et al.[253] demonstrated the normal human
Vinatier et al.[243–245] recently developed a self-setting skin has an ultimate tensile modulus ranging from 15
cellulose based hydrogels consisting of silanized hydro- to 150 MPa and an ultimate strain of 35–115%. The
xypropyl methylcellulose (Si-HPMC). The authors demon- schematic representation of the requirements to create a
strated that this Si-HPMC hydrogel was a suitable matrix for fully functional skin tissue is presented in Figure 13a.
the in vitro 3D culture of rabbit articular chondrocytes. The In the past decades, many skin substitutes such as
transplantation of Si-HPMC hydrogel containing autolo- xenograft, allografts, and autografts have been employed
gous nasal chondrocytes led to the repair of an articular for wound healing. However, due to the antigenicity or the
cartilage defect in rabbits (Figure 12i–m). Transplanting Si- limitation of donor sites, the skin substitutes cannot
HPMC hydrogel containing autologous cells in an articular accomplish the purpose of the skin recovery and hence
cartilage defect through percutaneous injection could pave not used widely.[254,255] The main role of skin substitutes is

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Figure 13. (a) The schematic representation of the requirements such as cells, growth factors, scaffold, etc. (top left) to create a fully
functional skin tissue (structure of skin, top right). Examples of the polysaccharide-based material for skin tissue: (b) Confocal image of
human dermal fibroblasts cultured over the CS-collagen porous scaffold, the histological response to the CS-collagen porous after
embedded in rabbit ear on 28 d (c) (M: the implanted collagen/CS scaffold, T: the subcutaneous connective). HA-based scaffold: (d, e) in vivo
tissue augmentation by HA hydrogels in ICR hairless mice. Histological analysis of ICR hairless mouse skin treated with HA hydrogel samples
after H&E staining in 12 weeks (d) wrinkled mice without tissue augmentation (untreated) and (e) treated with HA hydrogels crosslinked
with HMDA shows tissue augmentation. (E: epidermis, D: dermis, and H: hypodermis) (scale ¼ 100 mm). (a) Reproduced with permission.[249]
Copyright 2006, The Royal Society. (b,c) Reproduced with permission.[255] Copyright 2003, Elsevier. (d,e) Reproduced with permission.[264]
Copyright 2010, American Chemical Society.
to promote wound healing by simulating the host to It has been demonstrated that CS in the form of CS-cotton
produce various cytokines. These cytokines play an (a superior grade of cotton), accelerate wound healing by
important role not only in preventing dehydration and promoting infiltration of polymorphonuclear cells at the
increasing inflammation, but also in promoting the wound site which is an essential event in rapid wound
formation of granulation tissue in wound healing pro- healing. Recently, Mizuno et al. also reported[259] that CS
cesses. Fibroblast in a dermal equivalent enhances epider- was a good wound healing material and incorporation of
mal differentiation and dermal regeneration by secreting basic fibroblast growth factor (bFGF) into CS accelerated the
cytokines. Therefore, skin substitutes containing rate of healing. Howling et al.[260] demonstrated that highly
cultured fibroblasts can accelerate the healing process deacetylated CS is more biologically active than chitin and
owing to the injection of fibroblasts into the wound tissue, less deacetylated CSs. As mentioned earlier, these results
and promote the synthesis of new tissue in the initial are closely related to the electrostatic interaction of CS with
stage.[256] anionic glycosaminoglycan (GAG), depending on the degree
of deacetylation of CS and pH environment. The GAG,
3.3.1. CS-Based Materials for Skin
distributed widely throughout the body, is known to bind
CS has been extensively used as a skin substitute material and modulate a number of cytokines/growth factors.
due to its many advantages for wound healing such as Further studies emphasized on the combination of CS with
hemostasis (i.e., by accelerating the tissue regeneration and other materials which have a potential way of achieving
stimulating the fibroblast synthesis of collagen).[257,258] rapid wound healing. Yan et al.[261] prepared CS in

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combination with alginate as PEC membranes. These controlled the pore structure of the hydrophilic natural
biodegradable CS–alginate PEC membranes showed greater polymers and manipulated the material properties of the
stability to pH changes and hence more effective as final scaffold. In particular, this alginate has the ability to
controlled-release membranes than either CS or alginate accelerate epithelialization and granulation tissue forma-
itself.[262] The PEC membranes were found to promote tion, which supports a thin sheet of skin epithelium. The cell
accelerated healing of incisional wounds in a rat model. Ma viability was measured using a keratinocyte/fibroblast co-
et al.[255] fabricated porous CS/collagen scaffold by cross- culture for skin tissue regeneration to observe the cellular
linking these with glutaraldehyde and freeze-drying to efficiency of the scaffolds. Finally, the core/shell scaffold
improve biostability and good biocompatibility. They also was implanted in mice as a dermal substitute and
reported that the potential cytotoxicity of glutaraldehyde histological characterization was carried out in vivo that
might be decreased through the presence of CS. This means revealed skin tissue regeneration. Tanihara et al.[268] have
that CS can function as a bridge to increase the crosslinking synthesized an artificial ECM of alginate and demonstrated
efficiency of glutaraldehyde in the collagen-based scaffolds that the matrix stimulated the regeneration of skin, nerve,
owing to the large number of amino groups in its molecular and bone tissues.
chain. The glutaraldehyde-treated CS-collagen scaffold
3.3.4. Cellulose-Based Materials for Skin
retained the original good biocompatibility and could
successfully induce the fibroblasts infiltration from the Microbial cellulose (MC) can be penetrated by skin cells
surrounding tissue (Figure 13b,c). which are able to push away the MC fibrils and migrate deep
into the cellulose net (up to 100 mm).[269] This fact may be
3.3.2. HA-Based Materials for Skin
very important for the treatment of third-degree burns,
The key properties relevant to the production and where new dermis has to be completely replaced and
performance of HA as for skin TE are: the degree of regenerated. With MC, the fibroblasts and keratinocytes
crosslinking, gel hardness, gel consistency, viscosity, would be able to penetrate the microporous net of cellulose,
extrusion force, HA concentration, and hydration are synthesize an ECM, and eventually form dermal tissue.
important.[263] Yeom et al.[264] have developed HA hydro- Despite the fact that the MC is not a biodegradable material,
gels crosslinked with hexamethylenediamine (HMDA), it could stay in the body forever without causing any toxic
biocompatible, and nontoxic for skin tissue augmentation or inflammatory reactions. A novel material which can stay
and regeneration. HA hydrogels were prepared by direct in the body for an extended period of time is highly valued
amide bond formation between the carboxyl groups of HA for the treatment of burned skin since a good substitute for
and HMDA with an optimized carboxyl group modification skin grafting is not currently on the market. The MC has
for effective for skin TE. Wrinkled model mice were proven to be a remarkably versatile biomaterial and can be
prepared for tissue augmentation tests by daily treatment used in wide variety of TE applications which has been well
of the dorsal skins and HA-HMDA hydrogels were injected reviewed.[270]
into the dorsal subcutaneous tissues of wrinkled model It is expected that in the next generation skin substitutes,
mice. The skin tissue generation effect was assessed using a biomaterial scaffolds would be carefully engineered for
surface image analyzer with histological analyses after controlled release of various signal molecules (e.g., growth
hematoxylin-eosin (H&E) and Masson’s trichrome staining factors and proteins) for cell migration, adhesion, prolifera-
(Figure 13d,e). Recently, Hillel et al.[265] developed HA-PEG tion, and differentiation. From the studies conducted so far
bioactive materials for skin tissue replacement which can it may be concluded that the CS-based materials has highest
be injected and then transdermal photocrosslinking. The potential for being used as a clinically acceptable effective
authors confirmed the potential use of these materials by scaffold for skin substitutes.
testing both in animal model and pilot clinical in human
patients, and the feasibility of the transdermal photo-
crosslinking approach for implantation in abdomen skin 4. Conclusion and Outlook
tissue regeneration.[265]
Much progress has been made in the design and
3.3.3. Alginate-Based Materials for Skin
functionalization of natural polysaccharide materials and
The biodegradable and biocompatible alginate structure in processing technologies able to produce porous structure
resembled GAG, which is an important component of the with tailored architecture. This has provided unique means
ECM in human tissue, and it has good tissue compatibility for the development of biodegradable polymeric scaffolds
and mechanical properties and widely used in skin TE.[266] for TE. The studies have revealed that scaffold design must
Kim and co-workers[267] fabricated a 3D structured core meet specific features such as, appropriate porosity and
(alginate)/shell (collagen) scaffold using a core/shell nozzle biodegradability, as well as specific requirements asso-
of a cryogenic co-extrusion process, which precisely ciated with individual defects, such as a custom shape and

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size. From a technological point of view, a great challenge of needed in order to bring MC products to successful
skeletal TE is to design and manufacture a biodegradable commercialization. For example, a wide variety of mam-
scaffold, possessing a required porosity and pore sizes very malian cells need to be seeded onto MC membranes in order
close to the skeletal tissue structure and able to withstand to assess their viability and proliferation. A number of
in vivo loads as well as maintain a suitable structure for a clinical studies will be necessary to prove its usefulness and
sufficient extended time. functionality. If MC proves to be effective in wound repair
Combinations of various cell lines, materials (either and TE, then it will have to be produced on an industrial
natural or synthetic) for the design of optimal bone and scale. Due to its simple fermentation process, large scale MC
cartilage tissue constructs have been reported. In particular, production appears to be quite feasible; however, specific
polysaccharides–inorganic bioactive phase composites engineering details need to be elaborated. Also, more
have been investigated to display bioactive behavior, biochemical and genetic investigations need to be con-
adjustable biodegradation kinetics, and mechanical proper- ducted in order to fully understand and improve the
ties suitable for applications in skeletal tissue regeneration. cellulose production process.
Processing techniques have been adapted and extended for Over past 40 years, a number of biomaterials proposed as
incorporation of inorganic bioactive phases into porous 3D ‘‘ideal’’ scaffolds for cell growth yet few have reached
polymer networks. Hydrogels, especially in injectable form, clinical efficacy. Biomaterials, permanent or biodegradable,
are gaining increasing attention due to their hydrophilicity, naturally occurring or synthetic, need to be biocompatible
suitability for direct cells encapsulation, similarity to with a structural integrity and compatible with native
natural ECM, and the prospect of noninvasive applications. tissue to fulfill their desired role in tissue regeneration.
Moreover, the incorporation of this material into biode- These materials provide cell anchorage sites, mechanical
gradable scaffolds of bioactive molecules to promote stability, and structural guidance and within an in vivo
regeneration of bone, cartilage and skin has been found environment, provide the interface to respond to physio-
to be very promising. logical and biological changes and to remodel the ECM in
A great challenge in materials science and technology order to integrate with the surrounding native tissue.
within the field of TE is the ability to control the accuracy Appropriate matrices for the delivery of stem cells, which
and reproducibility of the manufactured scaffolds. This is to stimulate differentiation and tissue conduction, skeletal
allow standardization of the fabrication process for future tissue as for example have included HAp and calcium
industrial applications. Various scaffold production tech- phosphate and a range of ceramic biomaterials. However,
niques for the processing of a variety of polymeric and HAp and calcium phosphate are not osteoinductive and are
composite materials and for the development of different resorbed relatively slowly. Moreover, there are problems
microstructures are currently in use. However, each associated with biodegradability, inflammatory, and
technique presents unique shortcomings (e.g., lack of immunologic reactions and in disease transmission when
control of scaffold parameters such as porosity, pore size used as carriers for osteoinductive factors.
and distribution, the presence of residual toxic solvent In the case of failure to identify suitable biological
into the scaffold and, most importantly, lack of suitable scaffold to fulfill the desired function of new tissue
mechanical properties). formation that will promote integration and, most
Polysaccharides are proving to be a very versatile class of importantly, significant vascularization has led to an
materials. It can be used in a wide variety of biomedical increased interest in optimizing biomaterials to promote
applications, from topical wound dressings to the durable specific cell–biomaterial interactions. For example, Arg-
scaffolds required for TE. Many scientists are already trying Gly-Asp (RGD) sequence peptides (involved in integrin-
to develop novel biomaterials from synthetic polymers. mediated cell adhesion) can be incorporated onto the
These new materials could be used in many biomedical scaffold surface to enhance cell adhesion and spreading.
and biotechnological applications (e.g., TE, drug delivery, More recently, drug delivery techniques such as entrap-
wound dressings, and medical implants). However, many ment within a hydrogel matrix allowing growth factors to
of these synthetic polymers have their drawbacks. For be released in a controlled fashion from the scaffold to aid
instance, they often do not possess the correct mechanical the regenerating tissue. Such approaches are appealing
properties and are usually not biocompatible. Initial studies as they avoid the use of solvents, and high temperatures
indicate that MC is a better candidate for TE since it is both (and therefore protein degradation) and subsequent release
durable and biocompatible. In fact, MC-based materials of the growth factor is controlled, in response to environ-
are particularly interesting for the development of many mental stimuli. This strategy can be employed in TE, where
different biomedical devices. In some cases, such as wound appropriate growth factor molecules/cytokines can be
healing and organ replacement, a number of clinical studies incorporated into polysaccharide molecules via chemical
have been performed showing its effectiveness in these and/or physical crosslinking prior to in vivo implantation
applications. However, much interdisciplinary research is for desire tissue regeneration.

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Review

Polysaccharides and Their Derivatives for

Ferdous Khan,* Sheikh Rafi Ahmad

1. Introduction body or to foster remodeling of tissues in active manner in a

Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200409

Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200409

OH transition occurring at temperatures that

Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200409

Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200409

Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200409

alkylation of the tetra(n-butyl)ammo-

Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200409

Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200409

2.3.1. Graft Copolymerization 2.3.2. Oxidation

Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200409

Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200409

Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200409

Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200409

Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200409

3. Polysaccharide Derivatives in TE and heart tissues substitutes. To discuss all these TE

Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200409

Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200409

Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200409

Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200409

Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200409

Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200409

Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200409

Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200409

Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200409

Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200409

Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200409

Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200409

Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200409

Macromol. Biosci. 2013, DOI: 10.1002/mabi.201200409

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