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Title
25th anniversary article: Rational design and applications of hydrogels in regenerative
medicine.
Permalink
https://escholarship.org/uc/item/1mc3f01j
Journal
Advanced materials (Deerfield Beach, Fla.), 26(1)
ISSN
0935-9648
Authors
Annabi, Nasim
Tamayol, Ali
Uquillas, Jorge Alfredo
et al.
Publication Date
2014
DOI
10.1002/adma.201303233
Peer reviewed
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University of California
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25th Anniversary Article: Rational Design and Applications
of Hydrogels in Regenerative Medicine
Nasim Annabi , Ali Tamayol , Jorge Alfredo Uquillas , Mohsen Akbari ,
Luiz E. Bertassoni ,
Chaenyung Cha , Gulden Camci-Unal ,
Mehmet R. Dokmeci , Nicholas A. Peppas ,*
and Ali Khademhosseini *
Dr. N. Annabi, Dr. A. Tamayol, Dr. J. A. Uquillas,
Dr. M. Akbari, Dr. C. Cha, Dr. L.E. Bertassoni,
Dr. G. Camci-Unal, Dr. M. R. Dokmeci,
Prof. A. Khademhosseini
Center for Biomedical Engineering
Department of Medicine, Brigham and Women’s Hospital
Harvard Medical School
Boston, MA 02139, USA
Harvard-MIT Division of Health Sciences and Technology
Massachusetts Institute of Technology
Cambridge , MA 02139 , USA
E-mail: alik@rics.bwh.harvard.edu
Dr. N. Annabi, Dr. M. Akbari, Prof. A. Khademhosseini
Wyss Institute for Biologically Inspired Engineering
Harvard University
Boston , MA, 02115 , USA
Prof. N. A. Peppas
Department of Biomedical Engineering
Biomedical Engineering Building 3.110B
The University of Texas at Austin
1 University Station, C0800 ,
Austin , Texas , 78712–1062, USA
E-mail: peppas@che.utexas.edu
DOI: 10.1002/adma.201303233
1 . Introduction
Hydrogels are three-dimensional (3D) net-
works consisting of hydrophilic polymer
chains, which are crosslinked to form
matrices with high water content (up to
thousand of times their dry weight).
[
1
]
Due to their remarkable characteristics,
including tunable physical, chemical, and
biological properties, high biocompat-
ibility, versatility in fabrication, and simi-
larity to native extracellular matrix (ECM),
hydrogels have emerged as promising
materials in the biomedical fi eld.
[
1–3
]
Sig-
nifi cant progress has been made in the
synthesis and fabrication of hydrogels
from both natural and synthetic sources
for various applications; these include
regenerative medicine, drug/gene delivery,
stem cell and cancer research, and cell
therapy.
[
4–6
]
Naturally-derived hydrogels,
such as collagen, chitosan, hyaluronic
acid (HA), alginate, gelatin, elastin, chon-
droitin sulfate, and heparin, are appealing
for biological applications due to their cell
signaling and cell-interactive properties, and biodegradability.
[
7
]
However, their limitations include low mechanical properties,
inability to control their degradation and structure, and poten-
tial immunogenicity. On the other hand, synthetic hydrogels,
such as poly(ethylene glycol) (PEG), poly(vinyl alcohol)(PVA),
poly(2-hydroxyethyl methacrylate) (PHEMA), and polyacryla-
mide (PAM), possess controllable degradation and micro-
structure, generally show high mechanical properties, but lack
biological moieties.
[
3,7
]
Due to the distinct properties of each
of these hydrogel classes, gels that are based on the combina-
tion of natural and synthetic polymers have attracted signifi cant
attention for biological and biomedical applications.
[
8
]
Various crosslinking approaches, including chemical and
physical, have been employed to create polymer networks
and preserve their 3D structures in aqueous environments.
In physically crosslinked gels, physical interactions between
polymer chains prevent dissociation of the hydrogel, while in
chemically crosslinked gels, covalent bonds between polymer
chains create stable hydrogels. Physically crosslinked hydrogels
are formed through changes in environmental conditions (e.g.,
pH, temperature, and ionic interactions), hydrogen bonds,
H y d r o g e l s a r e h y d r o p h i l i c p o l y m e r - b a s e d m a t e r i a l s w i t h h i g h w a t e r c o n -
tent and physical characteristics that resemble the native extracellular
matrix. Because of their remarkable properties, hydrogel systems are used
for a wide range of biomedical applications, such as three-dimensional
(3D) matrices for tissue engineering, drug-delivery vehicles, composite
biomaterials, and as injectable fi llers in minimally invasive surgeries. In
addition, the rational design of hydrogels with controlled physical and
biological properties can be used to modulate cellular functionality and
tissue morphogenesis. Here, the development of advanced hydrogels with
tunable physiochemical properties is highlighted, with particular emphasis
on elastomeric, light-sensitive, composite, and shape-memory hydrogels.
Emerging technologies developed over the past decade to control hydrogel
architecture are also discussed and a number of potential applications
and challenges in the utilization of hydrogels in regenerative medicine are
reviewed. It is anticipated that the continued development of sophisticated
hydrogels will result in clinical applications that will improve patient care
and quality of life.
Adv. Mater. 2014, 26, 85–124
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and protein interactions. There has been a growing interest
in using this class of hydrogels for tissue regeneration as the
gelation often occurs in mild conditions and aqueous solu-
tion in the absence of chemical crosslinkers.
[
9
]
Various inject-
able hydrogels based on alginate, collagen, agarose, HA, and
chitosan have been synthesized by using physical crosslinking
approaches for engineering different tissues.
[
10
]
These gels can
be confi ned in the damaged site and eliminate the need of inva-
sive surgery. However, low mechanical properties of physically
crosslinked hydrogels may limit their tissue engineering appli-
cations, particularly in the regeneration of load bearing tissues.
Chemically crosslinked gels have been obtained by radical poly-
merization, chemical reactions, energy irradiation, and enzy-
matic crosslinking. Some examples of chemically crosslinked
gels for tissue engineering applications include PHEMA,
glutaraldehyde (GA) crosslinked PVA, elastin, and chitosan,
UV crosslinked methacrylated gelatin and elastin, transglut-
aminases crosslinked fi brinogen hydrogels.
[
9,11–13
]
Generally,
chemically crosslinked gels have higher mechanical properties
compared to their physically crosslinked counterparts, but the
residual chemical crosslinkers, organic solvents, and photoini-
tiator may cause cytotoxicity.
Over the past decade, complex hydrogels have been designed
as a result of major breakthroughs in the fi eld of polymer sci-
ence, microscale technologies, and molecular biology.
[
4,6
]
These
advances have set the framework to overcome some of the chal-
lenges in regenerative medicine by rational design of hydrogels
for various medical applications. This review covers the design
principles being applied to synthesize advanced hydrogels with
enhanced mechanical, biological, chemical and electrical prop-
erties. Due to their important biomedical applications, par-
ticular emphasis is given to elastomeric, photo-sensitive, hybrid
and shape-memory hydrogel systems. In addition, emerging
techologies for controlling the micro- and nanoscale architec-
tures of 3D hydrogel constructs and their potential applications
are highlighted.
2 . Advanced Hydrogels with Tunable Properties
2.1 . Elastomeric Materials
Biomaterials have been used as an artifi cial ECM to support
the regeneration of various tissues. Since elasticity is one of
the major mechanical characteristics of soft tissues, signifi -
cant efforts have been made to engineer elastomeric biomate-
rials, which mimic the ability of native tissues to extend under
stress. Mimicking the non-uniform elasticity of innate tissues
including skin, blood vessel, lung, cardiac, and muscle is one
of the major challenges in tissue engineering. Due to the high
stretchability of native tissues, thermoplastic polymers with
elongation break of less than 3% fail to replicate the innate
tissue elasticity, as they undergo plastic deformation under vari-
able loading.
[
14
]
To overcome this limitation, elastomeric hydro-
gels have been developed for biomedical applications.
[
15,16
]
However, one of the challenges associated with these elasto-
meric systems is their inability to mimic non-uniform elasticity
of the native tissue. For example, many of the native tissues
Nicholas A. Peppas is the
Fletcher S. Pratt Chair in
Biomedical Engineering,
Chemical Engineering,
and Pharmacy, and the
Director of the Center for
Biomaterials, Drug Delivery
and Bionanotechnology at the
University of Texas at Austin.
He has been elected a member
of the National Academy
of Engineering (NAE), the
Institute of Medicine (IOM) of the National Academies, the
Royal National Academy of Spain, and the National Academy
of Pharmacy of France. He is the recipient of the 2012 NAE
Founders Award for his contributions to biomaterials and
hydrogels. He received his Diploma in Engineering (D. Eng.)
from NTU of Athens, Greece in 1971 and his Sc.D. from MIT
in 1973, both in chemical engineering.
Ali Khademhosseini i s a n
associate professor at Harvard
University, and holds appoint-
ments at the Harvard-MIT
Division of Health Sciences
Techno logy, Brigham &
Women’s Hospital. In addi-
tion, he is on the faculty of the
Wyss Institute for Biologically
Inspired Engineering at
Harvard University and the
World Premier International-
Advanced Institute for Materials Research (WPI-AIMR) at
Toh oku Un iversi ty. He is a n inte rnatio nally re cognized bio-
engineer regarded for his contributions and research in the
areas of bioengineering, biomaterial synthesis, microscale
technologies, and tissue engineering. His research involves
the development of micro- and nanoscale technologies to
control cellular behavior, fabrication of microscale biomate-
rials and engineering systems for tissue engineering, drug
discovery and cell-based biosensing.
Nasim Annabi is a postdoc-
toral fellow at the Harvard-MIT
Division of Health Sciences
Technology, Brigham and
Women’s Hospital, and the
Wyss Institute for Biologically
Inspired Engineering at Harvard
University. Her research is
based on developing advanced
biomaterials and combining
them with micro- and nanoscale
technologies to control the
cellular microenvironment and
engineer complex tissue constructs.
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decellularization, which removes the cellular component of
explant tissues by detergent, enzymatic digestions, and sol-
vent extraction processes. Due to their stability and durability,
elastin-based tissues preserve their functions and structure
after decellularization. Decellularized elastic scaffolds have
been used as suitable replacements of lung, bladder, artery,
heart valve, skin, and vascular graft.
[
28–30
]
Despite their advan-
tages, decellularized scaffolds have several limitations. For
example, the decellularization process involves harsh reaction
conditions (e.g., enzymatic, chemical, or physical treatments)
that may compromise the biological and mechanical properties
of the constructs, particularly when additional steps of tissue
purifi cation are used.
[
31
]
Other limitations include batch-to-
batch variability, risk of pathogen transfer, inability to obtain
highly purifi ed elastic tissue, and lack of versatility and uni-
formity of decellularized elastic tissues.
[
31
]
Elastin Hydrogels Made from Soluble Elastin : Hydrolyzed
elastin, soluble in aqueous solvents, has been used to engineer
elastic hydrogels. The insolubility of intact elastin fi bers in tis-
sues prevents their processing into elastin-based hydrogels. To
solve this problem, elastic tissues have been treated with oxalic
acid or potassium hydroxide to yield soluble forms of elastin
(e.g.,
α
-elastin and Κ -elastin).
[
32,33
]
These hydrolyzed elastin
molecules have properties similar to the native tropoelastin,
such as ability to coacervate as well as to regulate cell signaling
via the elastin receptors. This demonstrates the potential bio-
logical value of this class of elastin derivatives for biomedical
applications.
Several elastin-based hydrogels have been synthesized from
solubilized elastin for engineering different tissues such as
skin,
[
32,34,35
]
cartilage,
[
36,37
]
and blood vessels.
[
38
]
For example,
α
-elastin hydrogels have been fabricated through chemical
crosslinking approaches using various types of crosslinking
agents.
[
32,34,40
]
Highly porous and elastic hydrogels were also
engineered by crosslinking
α
-elastin with glutaraldehyde
(GA)
[
34
]
and hexamethylene diisocyanate (HMDI)
[
32
]
under high
pressure CO
2
. The fabricated hydrogels facilitated the infi ltra-
tion, attachment, and growth of 3T3 fi broblasts within the 3D
structure of the hydrogels.
[
32,34
]
In addition, the combination
of
α
-elastin with poly caprolactone (PCL) promoted chondro-
cyte adhesion and proliferation.
[
36,39
]
Regeneration of cartilage
tissue has also been achieved by using composite hydrogels
containing Κ -elastin, alginate, and collagen.
[
37
]
Chondrocytes
isolated from porcine and human were embedded inside the
hydrogel composite and subsequently implanted in nude mice.
After 12 weeks of implantation, cartilage-specifi c components
including proteoglycans, collagen, and elastin fi bers were
formed within the engineered tissues which closely mimicked
the native articular cartilage.
[
37
]
Despite its extensive use in
tissue engineering, animal-derived soluble elastin is a hetero-
geneous mixture of peptides which are partially crosslinked and
may not have adequate cell binding sites.
[
41
]
In addition, the
clinical use of animal-derived proteins is often restricted due to
the risk of pathogen transfer and immunological rejection.
[
42
]
Recombinant Elastin-Based Hydrogels : Elastin-based elasto-
mers can be also produced from various recombinant elastin
proteins (e.g., recombinant elastin like polypeptides (ELP)
and recombinant human tropoelastin). These proteins are
obtained via the expression of recombinant DNA in different
display strain stiffening and are responsive to applied strain,
which can not be easily obtained by elastomeric systems.
[
17
]
The use of synthetic elastomers for medical devices dates
back to 1890s when the rubber industry was developed. Since
then, natural and synthetic rubbers, such as silicones, polyole-
fi ns, and polydienes, and polyurethanes have been widely used
as elastomers to engineer various medical devices due to their
biocompatibility, mechanical durability, and low cost.
[
15
]
In the last three decades, the rise of hydrogels as a popular
choice of elastomeric materials for a variety of applications
has been observed.
[
18
]
In this section, we focus on natural- and
synthetic-derived elastomeric hydrogels, which are particularly
useful for soft tissue engineering applications. We also discuss
their limitations and potential applications for engineering bio-
mimetic tissue constructs.
2.1.1 . Naturally-Derived Elastin-Based Elastomers
Elastin is one of the main elastomeric proteins in connective
tissues that are exposed to repetitive strains such as major vas-
cular vessels, aorta, skin, elastic cartilage, tendon, and lung.
Elastin is the essential component that provides elasticity and
resilience needed for the proper function of these tissues. For
example, the presence of elastin in arterial walls facilitates the
blood transfer from the heart, lowers the mechanical work per-
formed by the heart, and preserves the steady fl ow of oxygen
to tissues.
[
19
]
In addition, elastin fi bers allow blood vessels to
withstand continuous cycles of contraction and expansion over
the course of a life time.
[
20
]
Elastin is also known for being the
most persistent and durable protein in the human body, with a
half-life of 70 years.
[
18
]
Elastin plays a critical biological role in regulating cel-
lular functions. Various cell-surface proteins including elastin
binding protein (EBP),
[
21
]
glycosaminoglycans (GAGs),
[
22
]
and
integrin
α
v
β
3
[
23
]
have been identifi ed as receptors for elastin
and its derivatives. Binding with these receptors has been
shown to facilitate various cellular interactions. For example,
it was found that elastin induced the attachment and prolifera-
tion of endothelial cells (ECs) and formation of vascular net-
works.
[
24
]
In addition, elastin derivatives could enhance the in
vitro proliferation of skin fi broblasts.
[
23,25
]
Elastin fi bers in the
skin were also shown to infl uence cellular phenotypes during
wound healing processes by controlling the differentiation of
proliferative dermal fi broblasts into contractile myofi broblasts
to help close the wound.
[
26
]
The presence of various cell-inter-
active segments in elastin and its derivatives enable them to
modulate cellular functions. For example, VGAPG peptide
sequences in elastin facilitate the formation of epidermis layer
by inducing the migration and differentiation of epidermal
keratinocytes.
[
27
]
These unique features demonstrate the poten-
tial value of elastin as a biologically active molecule for engi-
neering elastic hydrogels in tissue engineering.
Various techniques have been developed to synthesize and
purify elastin molecules from natural sources to engineer
elastin-based hydrogels. Elastin can be obtained by partial
hydrolysis of decellularized elastin-rich tissues in animals or by
expression of recombinant protein.
Decellularized Tissues as Elastin-Based Scaffolds : Natural
elastin-containing scaffolds can be generated by tissue
Adv. Mater. 2014, 26, 85–124
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GAGs
[
58
]
and the use of high pressure CO
2
.
[
52
]
In addition, the
BS3 crosslinked rhTE hydrogels, that were implanted subcu-
taneously in guinea pigs, exhibited high biocompatibility and
stability up to 13 weeks of culture (Figure 1 Bii).
[
57
]
A physical
crosslinking approach was also used to generate rhTE hydro-
gels by increasing the pH of protein solutions, which facili-
tated the self-assembly of rhTE spherules in a sol-gel transition
process.
[
55
]
This approach eliminated the use of chemical or
enzyme crosslinkers. The resulting hydrogel was highly fl ex-
ible and elastic with compressive modulus of about 1.7 MPa
over 5 cycles (Figure 1 Ci). These hydrogels also facilitated
the attachment and proliferation of dermal fi broblast in vitro
and were stable for two weeks after intradermal injection into
rats (Figure 1 Cii).
[
55
]
Recently, a highly elastic photocrosslink-
able hydrogel, methacrylated tropoelastin (MeTro), with tun-
able physical properties has been synthesized by functionali-
zation of rhTE with methacrylate groups and subsequent UV
crosslinking.
[
13
]
This approach was used to control the physical
properties of resulting hydrogels including swelling behavior,
porosity, and mechanical properties by altering the methacryla-
tion degree and MeTro concentration. The fabricated MeTro
hydrogels displayed high resilience, reversible deformation
with low energy loss following cyclic compressions, and sub-
stantial extensibility up to 400% before rupture (Figure 1 Di).
In addition, in vitro studies showed that MeTro hydrogels
supported cellular attachment and growth in both 2D and 3D
hosts including plants,
[
43–45
]
yeast,
[
46,47
]
and Escherichia coli
(E. coli).
[
48
]
Recently, human recombinant tropoelastin (rhTE) has been
used to generate elastic rhTE-based hydrogels. Previously, rhTE
was obtained in very low yield by construction of an expression
vector containing the cDNA sequence of an isoform of human
tropoelastin.
[
49
]
To enhance the production yield, Martin and
Weiss developed a 2210-bp synthetic human TEL-encoding
gene (SHEL) which contained codons optimized for maximum
expression of rhTE in commercial yields.
[
50
]
This rhTE has been
processed into a variety of promising hydrogels for tissue engi-
neering applications.
[
27
]
Elastic rhTE-based hydrogels with excellent cell-interactive
properties have been created by using various approaches
including, enzymatic crosslinking using yeast lysyl oxi-
dase (PPLO),
[
51
]
chemical crosslinking,
[
52,53
]
using a fungal
copper amine oxidase,
[
54
]
physical crosslinking,
[
55
]
and
UV crosslinking.
[
13,56
]
For example, rhTE were chemically
crosslinked by GA ( Figure 1 Ai)
[
52
]
or bis(sulfosuccinimidyl)
suberate (BS3) (Figure 1 Bi)
[
57
]
to generate hydrogels in various
forms such as sheets, sponges, and tubes. The fabricated hydro-
gels promoted in vitro attachment, proliferation, and growth
of dermal fi broblast cells (Figure 1 Aii).
[
52
]
Furthermore, cel-
lular penetration within the 3D structures of these hydrogels
was signifi cantly promoted by increasing the level of porosity
and average pore sizes of the gels through the incorporation of
Figure 1. Examples of naturally-derived elastin-based hydrogels. A) GA crosslinked rTE/elastin hydrogels produced under high pressure CO
2
;i) struc-
ture of the hydrogel after swelling, ii) SEM image of dermal fi broblast cells penetrated and attached within the 3D structure of the gel. Reproduced
with permission.
[
52
]
Copyright 2010, Elsevier B.V. B) BS3 crosslinked rTE gel; i) an image from an elastic hydrogel sheet, (ii) hematoxylin and eosin-
stained sample explanted after 13 weeks of implantation (hydrogel is shown in bright red). Reproduced with permission.
[
57
]
Copyright 2004, Elsevier
B.V. C) Physically crosslinked rTE gel; i) representative stress–strain curves over 5 cycles, the resulting gel could be tied in a knot, demonstrating its
high fl exibility, ii) a hematoxylin and eosin-stained explant showing the injection site (the elastic deposit is marked with an E). Reproduced with permis-
sion.
[
55
]
Copyright 2009, Elsevier B.V. D) Methacrylated rTE gel; i) image of an elastic MeTro gel before and after stretching, ii-iv) formation of patterns
with various geometries on MeTro gel by using different microfabrication techniques, v) immunostaining of CM markers on MeTro gel on day 8 of
culture, gel stained for sarcomeric
α
-actinin (green)/connexin-43 (red)/nuclei (blue) (scale bar = 50 µ m), vi) beating behavior of CMs on micropat-
terned MeTro gel. Reproduced with permission
[
13
]
Copyright 2013, Elsevier B.V; Reproduced with permission.
[
56
]
Copyright 2013, John Wiley & Sons, Inc.
Adv. Mater. 2014, 26, 85–124
![Figure 15. Assembly of microgels. A) Schematic diagram of the assembly process; cell-laden modules were immersed inside a hydrophobic oil where they self assembled to minimize the surface energy; Reproduced with permission. [ 388 ] Copyright 2008, National Academy of Sciences. B) Fabricated modules and their assembly; i-iii) lock-and-key constructs loaded with FITC-dextran and Nile red; iv,v) rings containing concentric layers of HUVEC- and SMC-laden hydrogel; these rings were assembled to form a microvessel. Reproduced with permission. [ 384 ] Copyright 2011, John Wiley & Sons, Inc.](/figures/figure-15-assembly-of-microgels-a-schematic-diagram-of-the-1irhdn6a.webp)
![Figure 6. Molecular mechanism of temperature-responsive SMHs. The left panels represent the T T related to the switching of conformational structure. The right panel represents the conformational change of the SMH from shape B to shape A upon cooling, and back from shape A to shape B upon application of heat. The covalent chemical bonds are represented by the red dots and the physical non-covalent bonds by the entangled lines. Reproduced with permission. [ 222 ] Copyright 2013, American Chemical Society.](/figures/figure-6-molecular-mechanism-of-temperature-responsive-smhs-ps4jobyf.webp)