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com

ScienceDirect
Recent advances in hyaluronic acid hydrogels for
biomedical applications
Christopher B Highley1, Glenn D Prestwich2 and
Jason A Burdick1
Hyaluronic acid (HA) is widely used in the design of engineered
hydrogels, due to its biofunctionality, as well as numerous sites
for modification with reactive groups. There are now
widespread examples of modified HA macromers that form
either covalent or physical hydrogels through crosslinking
reactions such as with click chemistry or supramolecular
assemblies of guest-host pairs. HA hydrogels range from
relatively static matrices to those that exhibit spatiotemporally
dynamic properties through external triggers like light. Such
hydrogels are being explored for the culture of cells in vitro, as
carriers for cells in vivo, or to deliver therapeutics, including in
an environmentally responsive manner. The future will bring
new examples of HA hydrogels due to the synthetic diversity of
HA.
Addresses
1
Department of Bioengineering, University of Pennsylvania,
Philadelphia, PA, USA
2
Department of Medicinal Chemistry, University of Utah, Salt Lake City,
UT, USA
Corresponding author: Burdick, Jason A (burdick2@seas.upenn.edu)

Current Opinion in Biotechnology 2016, 40:3540

This review comes from a themed issue on Tissue, cell and pathway
engineering
Edited by April Kloxin and Kyongbum Lee
For a complete overview see the Issue and the Editorial
Available online 27th February 2016
http://dx.doi.org/10.1016/j.copbio.2016.02.008
0958-1669/# 2016 Elsevier Ltd. All rights reserved.

Introduction
Hydrogels are hydrated polymeric networks with diverse
properties that have, in recent years, seen continued
advancement and creativity in their design, fabrication,
and application. Areas of use range from cell and therapeutic delivery to in vitro platforms for creating and
controlling cellular environments. Hyaluronic acid or
hyaluronan (HA) represents one biopolymer that can
be modified and processed to form hydrogels for biomedical applications [1]. Owing to their biocompatibility,
tunable properties, and native biofunctionality, hydrogels
built from HA are increasingly versatile for a myriad of
applications [1]. HA has inherent biological importance
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due to its binding to receptors such as CD44, ability to

degrade via oxidative species and hyaluronidases, and
relevance during development, wound healing, and in the
function and structure of adult tissues [2]. With these
features in mind, several HA hydrogel systems have
already advanced to clinical use in human and veterinary
patients, particularly as dermal fillers, as intra-articular
viscosupplements, for corneal and dermal wound repair,
and for post-surgical adhesion prevention, among other
uses.
HA hydrogels are now evolving in their design to be
responsive to a range of cues, to present dynamic environments, and to possess multiple functionalities such as
sophisticated structures and biochemical signals. Our goal
with this report is to highlight recent advances in the
development of HA hydrogels and their continued application to numerous biomedical conditions. Indeed, since
our review on this topic in 2011 [1], significant new
research advances have occurred. We aim to further
explore the advantages of HA-based hydrogels for continued development of versatile, biocompatible materials
based on a growing diversity of chemical modifications
and processing techniques available.

HA gelation via covalent crosslinking

Scientists and engineers working with HA continue to
expand upon and refine existing chemistries for synthesizing and crosslinking HA macromers (see Figure 1).
Many of the specific, orthogonal click chemistries have
been used to produce hydrogels from HA-derivatives. For
example, strain-promoted [3 + 2] cycloaddition crosslinking between HA modified with either azide or cyclooctyne groups formed biocompatible hydrogels [3].
Selective Diels-Alder reactions have also been used to
crosslink furan-functionalized HA with either maleimidefunctionalized HA [4] or maleimide-poly(ethylene glycol)
(PEG) crosslinkers [5,6] and maleimide-modified HA has
been directly crosslinked via Michael addition reactions
with dithiol crosslinkers, such as peptides containing
cysteines [7]. HA modified with a tetrazine derivative
was crosslinked with bis-trans-cyclooctene molecules [8],
and norbornene-modified HA supports thiol-ene click
crosslinking using spatially controlled photoinitiation
[9]. These crosslinking approaches can provide direct
functionality to the hydrogel (e.g., enzymatically degradable peptide crosslinkers) and specific, orthogonal reactions may enable the engineering of spatiotemporal
control into hydrogel platforms.
Current Opinion in Biotechnology 2016, 40:3540

36 Tissue, cell and pathway engineering

Figure 1

pyridyl disulfide [12]

maleimide [7]
adamantane [15]

lipoic acid [14]

tetrazine [8]
norbornene [9]

-cyclodextrin [15]

phenylalanine [19]

diaminohexane [18]
azide [3]
spermine [18]

cucurbit[6]uril [18]

cyclooctyne [3]

furan [5]

bisphosphonate [20]

cysteine [13]

aldehyde [10]

Hyaluronic acid
(a)

(b)

(c)
selfhealing
(within 5 secs)

100 m

500 m

(d)

(e)

100 m

(f)

1 mm
Before
removal

100 m

200 m

1 mm

After
removal

200 m
Current Opinion in Biotechnology

Chemical modification of hyaluronic acid (HA) has yielded HA-based hydrogels with a variety of chemistries and functionalities. In the top half of
this figure, recent chemical modifications to HA (to either the acid or alcohol groups) are presented. Below this, images illustrating advances in
hydrogel structures and functionalities achieved using HA-based materials are shown. (a) Multilayer microsphere hydrogels through the interfacial
crosslinking of tetrazine-modified HA and an inwardly diffusing crosslinker [8]. (b) Composite hydrogels crosslinked by non-covalent bonds
between bisphosphonate-HA and calcium phosphate nanoparticles that exhibit self-healing behavior [20]. (c) High-resolution 3D structures
fabricated with methacrylated HA according to computer models using two-photon polymerization [33]. (d) Electrospun nanofibers from HAnorbornene and patterned with multiple molecules in independent patterns to allow orthogonal specification of both chemical and
nanotopographical cues [31]. (e) 3D printing of an ink (poly(N-isopropylacrylamide) grafted HA and methacrylated HA) that gels quickly on a
heated print stage and can be covalently crosslinked for longer term stability [25]. (f) Hydrogels crosslinked by non-covalent supramolecular bonds
used as both an ink and support material in 3D printing to allow the printing of material-structure and channel-structure at high resolution at any
location in 3D space [37].

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Advances in hyaluronic acid hydrogels Highley, Prestwich and Burdick 37

In addition to these new modifications to HA, existing

modifications and crosslinking approaches have been
revisited in order to maintain HA integrity or improve
crosslinking conditions. For example, aldehydes have
now been introduced without oxidizing glucuronic acid
residues, helping to maintain HA molecular weight
[10,11]. Disulfide crosslinking has also been achieved
using a pyridyl disulfide modification [12] and free thiols
have been introduced through the cysteine modification
of HA, allowing reaction under mild conditions [13]
Lastly, HA-lipoic acid derivatives allow crosslinking by
photoinitiated opening of the 5-member lipoic acid ring,
resulting in the formation of intermolecular dithiol
bridges [14].

Supramolecular and non-covalent assembly

of HA hydrogels
Non-covalent, supramolecular interactions have also been
advanced for HA hydrogel formation, due to their unique
properties. These interactions are reversible, assembling
and disassembling in response to physical or chemical
perturbations, which allows hydrogels to sense and respond to local cues. For example, HA has been modified
with host-guest molecules such as b-cyclodextrin
(b-CD) (host) and adamantane (guest) to yield a
shear-thinning and self-healing hydrogel due to inclusion
complex formation between b-CD and adamantane. Hydrogel properties were rationally designed based on degree of modification, material concentration, and the ratio
of host-to-guest molecules [15] and could be used as an
injectable formulation since bonds yielded under shear
force to move through a needle and reformed immediately at the site of injection [16].
Azobenzenes have also been grafted to HA to enable
photo-reversible supramolecular crosslinks with b-CDmodified viral particles, due to the cis-trans photoisomerization of azobenzene in response to various wavelengths
of light [17]. Cucurbit[6]uril-modified and diaminohexane-modified HA derivatives also form hydrogels upon
mixing as a result of host-guest interactions [18]. Similarly, HA-modified pendant phenylalanines were mixed
with soluble cucurbit[8]uril (CB[8]) to form a ternary
complex where two phenylalanines are bound together
noncovalently by the CB[8] host [19]. Bisphosphonates,
which can reversibly bind calcium, have also been conjugated to HA and mixed with calcium phosphate nanoparticles to create self-healing, injectable composite
hydrogels [20].
Non-covalent interactions also enable the self-assembly of
HA nanogels, as in the case of hydrophobic molecules that
can be conjugated to HA to render the HA-derivative
amphiphilic. For example, modification of HA with octenyl
succinic anhydride [21], cholesterol [22], or a phospholipid
(1,2-ditetradecanoyl-sn-glycero-3-phosphoethanolamine,
DMPE) [23] yielded macromers that self-assembled into
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nanoscale polymeric micelles when suspended in aqueous

medium. Layers of lipid membranes around nanogels allow
multilamellar peripheral structures that enable further
functionality [23], for example in pharmaceutical applications. Cholesterol-HA nanoparticles have been shown to
assemble and associate in an ionic environment based on
degree of modification, dissociating in the presence of
soluble b-CD [22]. Finally, thermoresponsive HA derivatives have been developed through pendant modification
with a PEG-based copolymer [24] or poly(N-isopropylacrylamide) [25].

Enhanced functionality through composite

hydrogels
In addition to new chemical modifications, new compositions such as colloid mixtures of microparticles and
nanoparticles with HA macromers or dual network hydrogels consisting of HA macromers and other polymers
are also expanding the diversity of HA hydrogels. For
example, hydrogels composed of HA nanoparticles crosslinked using carbodiimide coupling of adipic acid dihydrazide can be combined with methacrylated HA
(MeHA) to create an injectable colloidal hydrogel that
can maintain an extruded shape until crosslinked through
photopolymerization [26]. Another hybrid system consists
of covalently bonding block copolymer micelles to a
MeHA network to deliver drugs. The coupling of the
particles to the network modified the mechanical properties of the material system and allowed accelerated release upon mechanical compression of the hydrogel [27].
Further triggered drug delivery functionality has been
built into composite hydrogels through magnetically responsive particles used as crosslinkers [28].
Double-networks are also of considerable interest owing
to their high mechanical strength and toughness, which
far exceed those of the constituent materials alone or a
simple additive combination of their properties. Biocompatible double-network hydrogels, for example, include
MeHA combined with PEG-diacrylate [29] for enhancing
hydrogel toughness, or HA-tyramine combined with fibrin [30] to resist cell-traction induced network compaction or degradation in the presence of plasmin.

Directed assembly of HA hydrogel structures

Rapidly developing techniques such as electrospinning
[31,32], photopatterned material assembly [33,34], and
3D printing (3DP) [25,3537] are important towards
assembling hydrogel structures that mimic important
biological structures and length-scales (see example in
Figure 1). The use of multiphoton lasers for initiating
photopolymerizations has been applied to numerous HA
systems, enabling the localization of crosslinking with
micron-scale resolution in 3D space. Consequently, HAvinyl ester hydrogels [34] and MeHA hydrogels [33],
potentially with PEG-diacrylate, could be processed into
structures in 3D space with high-resolution.
Current Opinion in Biotechnology 2016, 40:3540

38 Tissue, cell and pathway engineering

3DP is a particularly active area due to its potential to

deposit multiple materials at high resolution in 3D space,
which is difficult to achieve by other means. The materials used in 3DP applications are critically important to
harnessing hardware capabilities, and must be capable of
undergoing some physical assembly and then further
stabilization as a printed structure. Recent examples of
HA-based inks include partially crosslinked MeHA which
is further polymerized upon printing to stabilize the
printed structure [35], which often occurs through a
photocrosslinking process [25,36]. Recently, shear-thinning supramolecular HA-hydrogels were used as both an
ink and support material, allowing high-resolution patterning of arbitrary hydrogel and channel structures anywhere within the 3D space occupied by the support
hydrogel [37]. The ink rapidly gelled upon removal of
shear forces, allowing a hydrogel to be printed within
another hydrogel.

Catechol-modified HA has also been synthesized, and

offers a biocompatible, tissue-adhesive HA-derivative that
can be employed to immobilize hydrogels in vivo [43].

Hydrogels for cell-based therapies and

research

Beyond external control of HA hydrogels, harnessing of

non-covalent associations that define supramolecular assemblies can be used to introduce dynamic features into
hydrogel structures. For example, free polyamine [18] or
diaminohexane guest moieties [45] in HA-hydrogels
crosslinked by host-guest interactions with CB[6]-HA
have been used to sequester CB[6]-conjugated molecules
from solution and enabled control over differentiation of
encapsulated cells [45].

An important application of HA hydrogels is their use in

conjunction with cells, where they can serve as matrices for
studying the effects of environmental cues on cell fate, as
well as a means of delivering cells therapeutically. These
materials may be designed as relatively static environments or as those with tuned properties through external
signals (e.g., application of light). Highly specific, bioorthogonal, click-type chemistries are particularly useful
in directing material properties, where they can offer a
means of modifying hydrogel properties via synthetic cues.
In the simplest approach, hydrogels are crosslinked
around cells, providing relatively static, 3D matrices in
vitro or in vivo. These systems can be powerful, introducing particular biomolecules to a cells environment, such
as a peptide binding motif from N-cadherin that mimics
cell-cell interactions [38] or through simple interactions
with HA via receptors (e.g., CD44). The addition of
peptide sequences for cellmatrix integrin-binding interactions, as well as proteolytic cleaveage of crosslinks, are
appreciated and can be leveraged to control cellular
behavior and fate [39]. HA hydrogels have been further
functionalized to sequester and present growth factors to
facilitate specific cellular functions [40]. These features
have been combined in acrylated-HA hydrogels containing DNA/polyethyleneimine particles and crosslinked
with MMP-sensitive peptides to modulate delivery of
the particles to cells [41].
The delivery of cells in hydrogels that crosslink in situ is
important towards translating cell-based therapies where,
for example, retention and control of cellular fate is important to therapeutic outcomes. HA-derivatives which have
enabled cell injections in vivo include aldehyde-HA [10],
thiolated-HA [42], and acrylated-HA [39], towards
repair of skin, cardiac, and neural tissues, respectively.
Current Opinion in Biotechnology 2016, 40:3540

In contrast to relatively static environments, HA hydrogel

properties can also be externally controlled, most commonly with light. Light-initiated reactions offer temporal
control over hydrogel properties, such as to investigate
how temporal changes in matrix mechanics affect stem
cell differentiation [44] and to elucidate mechanisms
fundamental to this process [7]. The use of light to control
chemical compositions at high spatial resolution is well
established, and photo-patterning of functional groups
into hydrogels without affecting mechanics has likewise
been demonstrated [5,6,9,31]. In fact, through thiol-ene
chemistry, it was possible to use light-based reactions to
independently modify both chemically bound functional
groups and mechanics in space-and-time [9,31].

Acellular hydrogels for regenerative therapies

HA hydrogels can also be designed as cell-free therapies,
such as through the delivery of encapsulated molecules
and proteins and through stimulating natural healing
processes through the recruitment of endogenous cells.
For example, injectable, supramolecular HA-hydrogels
that are stabilized via secondary covalent bonding have
been used to mechanically stabilize cardiac tissue to limit
remodeling after infarction [16]. Injectable, supramolecular hydrogels can also be engineered to control release
kinetics of payloads that have affinities for elements of
the supramolecular system, such as doxycycline and
doxorubicin, which complex with cyclodextrins [46].
The inhibition of proteolytic enzymes, such as matrix
metalloproteinases (MMPs) and aggrecanase, in diseased
and injured tissues can attenuate adverse tissue remodeling. Release of an aggrecanase inhibitor from an HA
hydrogel was used to ameliorate osteoarthritis pathologies
[47]. In an approach to release a molecule in an ondemand fashion, according to therapeutic need in its local
environment, an HA hydrogel was engineered to reduce
MMP activity by delivering an MMP-inhibitor in response to local MMP activity through the use of
MMP-degradable crosslinks [48]. Likewise, the introduction of MMP-sensitive crosslinks into hydrogel nanofibers created by electrospinning offered a means to
support cell-mediated degradation in tissue regeneration
applications [32].
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Advances in hyaluronic acid hydrogels Highley, Prestwich and Burdick 39

Translational potential of HA hydrogels

The rapid progress in the development and application of
HA-based materials in many biomedical areas underlines
the great potential and important role of HA in materials
for regenerative medicine. Promising therapies currently
in the clinic in human or veterinary patients, or in clinical
development currently rely on chemistries from the
19802004 time frame, as clinical translation of new
biomaterials is complex and costly, creating a 1020 year
time lag between laboratory discovery and clinical utility
[49,50]. Many regenerative medicine applications involving cell delivery will require matrices for cell delivery and
retention [51], and only since 2015 has the first in situ
crosslinkable HA hydrogel entered clinical trials for treatment of HIV-induced lipoatrophy with patient-sourced
adipose-derived stromal fraction.
Many of the newer chemistries and processes reviewed
herein will ultimately displace these first generation
living HA hydrogels. Research and development to
create improved, tunable biochemical and biophysical
properties, in combination with technology from other
fields will expand the potential of these next generation
HA hydrogels, some of which will eventually reach the
clinic. Some of the stimuli-responsive HA-materials and
materials programmed to precisely direct therapeutic
outcomes may find future uses in drug delivery, cell
therapies and basic research. In time, some of these more
complex and tunable HA hydrogel systems, much like the
simpler HA material already used in treating patients, will
find their place into common medical practice.

Dedication
We dedicate this article to the memory of Professor Endre
(Bandi) Balazs who recently passed away at age 95 and
dedicated his life to the research of hyaluronan, and
pioneered the development of HA biomaterials for clinical use. He was the father of the International Society for
Hyaluronan Study (ISHAS) and has mentored at least
four generations of HA researchers dedicated to excellence in basic and applied studies of the chemistry and
biology of HA.

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