Sat Haye 2015

Advanced Review
Rheology of peptide- and

protein-based physical hydrogels:
Are everyday measurements just
scratching the surface?
Sameer Sathaye,1 Armstrong Mbi,2 Cem Sonmez,3,4 Yingchao Chen,1
Daniel L. Blair,2 Joel P. Schneider4 and Darrin J. Pochan1
Rheological characterization of physically crosslinked peptide- and protein-based
hydrogels is widely reported in the literature. In this review, we focus on solid
injectable hydrogels, which are commonly referred to as shear-thinning and
rehealing materials. This class of what sometimes also are called yield-stress
materials holds exciting promise for biomedical applications that require
well-dened morphological and mechanical properties after delivery to a desired
site through a shearing process (e.g., syringe or catheter injection). In addition to
the review of recent studies using common rheometric measurements on peptideand protein-based, physically crosslinked hydrogels, we provide experimentally
obtained visual evidence, using a rheo-confocal microscope, of the fracture and
subsequent ow of physically crosslinked -hairpin peptide hydrogels under
steady-state shear mimicking commonly conducted experimental conditions using
bench-top rheometers. The observed fracture demonstrates that the supposed bulk
shear-thinning and rehealing behavior of physical gels can be limited to the yielding of a hydrogel layer close to the shearing surface with the bulk of the hydrogel below experiencing negligible shear. We suggest some measures to be taken
while acquiring and interpreting data using bench-top rheometers with a particular
focus on physical hydrogels. In particular, the use of confocal-rheometer assembly is intended to inspire studies on yielding behavior of hydrogels perceived as
shear-thinning and rehealing materials. A deeper insight into their yielding behavior will lead to the development of yield-stress, injectable, solid biomaterials, and
hopefully inspire the design of new shear-thinning and rehealing hydrogels and
more thorough physical characterization of such systems. Finally, more examples
of bulk fracture in some physical hydrogels based on peptides and proteins are
explored in the light of their behavior as yield-stress materials. 2014 Wiley Periodicals,
Inc.
How to cite this article:
WIREs Nanomed Nanobiotechnol 2015, 7:3468. doi: 10.1002/wnan.1299
Correspondence
1 Department
to: pochan@udel.edu
of Materials Science and Engineering and Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA
2 Department
of Physics, Georgetown University, Washington, DC, USA

of Chemistry, University of Delaware, Newark, DE, USA
4 Chemical Biology Laboratory, National Cancer Institute, Frederick National Laboratory for Cancer Research, Frederick, MD, USA
3 Department
Conflict of interest: The authors have declared no conflicts of interest

for this article.
34
Additional Supporting Information may be found in the online

version of this article.
2014 Wiley Periodicals, Inc.
Volume 7, January/February 2015
WIREs Nanomedicine and Nanobiotechnology
Rheology of peptide- and protein-based physical hydrogels
INTRODUCTION
ydrogel is a general term used for water swollen

and porous materials of polymeric, protein,
peptidic, colloidal, surfactant, or lipid origin. Hydrogels are a mainstay in the food and pharmaceutical
industries but are also increasingly finding applications in areas such as biosensing,1,2 microfluidics,36
drug delivery,710 and tissue engineering.7,9,11,12 A
broad classification of hydrogels based on the type of

crosslinking is commonly made, i.e., chemically (covalently) crosslinked or physically crosslinked (based
on secondary interactions such as hydrogen bonding, hydrophobic and electrostatic interactions). The
highly hydrated and porous nature of hydrogels in
general can be leveraged for their utility as encapsulating agents and delivery devices for therapeutic agents
such as cells,1318 growth factors,1921 DNA,2225
peptides and proteins,2628 and drugs.2933
A very important property of some hydrogels relevant to their applications as drug delivery vectors,34
tissue engineering scaffolds,35 or biomedical implants
and biosealants3638 is that they can be injected using
a simple device such as a syringe or a catheter. A large
number of studies have been focused on injection of
liquid precursors that are injected at a desired site into
the body and then undergo designed sol to gel transition (via physical or chemical mechanisms) at the
site of delivery and, thus, solidify postinjection. These
materials have the capacity to encapsulate therapeutic
agent and cell payloads as a consequence of gelation at
the site of delivery. Although injectable delivery of liquid precursors is a widely studied strategy, the strategy
can suffer from a number of setbacks. Liquid precursors injected at a desired site of delivery in the body
are subject to dilution with bodily fluids or leakage
into neighboring tissue.16,39 Owing to these factors,
mechanical properties of the hydrogels formed from
these precursors ex vivo might be compromised,40,41
and toxicity concerns might arise because of the presence of unreacted initiators, crosslinking agents, and
uncrosslinked polymers. Injection of liquid precursors offers little control over flow properties, spatial
location, and biological response of uncrosslinked liquid precursors immediately after injection. This lack
of control can lead to potential ambiguity in desired
performance of in vivo hydrogels in terms of established ex vivo parameters such as specific morphology,
drug/protein release profile, and encapsulated cell biological behavior.
In this tutorial review, we focus on the solid,
injectable, shear-thinning, and rehealing hydrogels.
Ex vivo, physically crosslinked, solid hydrogels that
are formed prior to injection can undergo flow during
injection and reheal into percolated, solid networks
after injection are of particular interest in biomedical

applications.1618,26,27,30,40,4252 These materials simply will be referred to as solid injectable hydrogels
in this review. Drugs, cell, and growth factor payloads (or combinations thereof) can be encapsulated
in preformed solid hydrogels and injected directly at
a specific desired site owing to the shear-thinning
nature of injectable solid hydrogels. They offer several advantages over injectable liquid precursor materials that are usually crosslinked at the site of delivery and solidify postinjection. Preformed solids can
maintain their structural integrity even immediately
after injection, allowing the encapsulated therapeutics to be localized effectively and largely eliminating limitations posed by injectable liquid precursors.53
Because they are already physically crosslinked, preformed solids eliminate chemical effects of the hydrogel crosslinking process such as UV irradiation and
thermal crosslinking on the underlying body tissue.
The ex vivo crosslinked nature of injectable solid
hydrogels offers reliable predictability of hydrogel
morphology, flow behavior, and encapsulated payload
release profile in vivo as compared with liquid precursors, thus overcoming some of the major limitations of
injectable liquid precursors.16,17 Thus, injectable, preformed solid hydrogels demonstrate great potential in
delivery of encapsulated therapies over injectable precursor liquids. Yield-stress materials exhibit solid-like
response of resistance to flow when subject to smaller
stress values but liquid-like response of yielding and
flowing above a threshold stress value.54 On a commercial scale, these materials are everywhere around
us used in products such as paints, mayonnaise,
cement, toothpaste, and concrete.55 Injectable solid
hydrogels demonstrate a rheological response similar to yield-stress materials, and thus are amenable
as injectable materials that flow when subjected to
syringe-induced shear.
Physically crosslinked, solid injectable hydrogels
can be developed from peptides, proteins, or polymers. With rapid advances in peptide,56,57 protein,58,59
polymer,60,61 and proteinpolymer conjugate synthesis62,63 and materials development, rheological
characterization has become an increasingly important tool to obtain more information about the viscoelastic and flow properties of hydrogels based on
peptides, proteins, and polymers. Common rheological studies conducted on hydrogel materials include
measurement of shear storage modulus (G ) (qualitatively the material stiffness), loss modulus (G ) (qualitatively the material liquid-like flow properties), and
loss factor tan () (the ratio of liquid-like behavior to
solid-like behavior), measured as functions of time,
oscillatory frequency, and oscillatory strain. Such
35
wires.wiley.com/nanomed
Advanced Review
studies can provide insight about gelation kinetics,

linear viscoelastic regions, and relaxation timescales
relevant to the studied hydrogels. A commonly conducted measurement for studying the shear-thinning
and rehealing behavior of physical hydrogels is the
storage modulus evolution of a hydrogel immediately after it has been subject to steady-state shear
of large amplitude by the upper plate of a bench-top
rheometer. Such a measurement often shows a significant reduction in the value of the storage modulus
upon shearing and subsequent gradual storage modulus evolution post-shear cessation.16,26,41,44,6468 Solid
injectable hydrogels are subject to shear treatment
usually using a rheometer and also, but less often, a
syringe or capillary to study their under shear and
post-shear behavior.
Rheological characterization is a widely used
and convenient strategy for investigating shear
response of solid injectable hydrogels. Studying the
shear response of solid injectable hydrogels in terms of
exact flow profile and potential changes in hydrogel
bulk structural characteristics is a step forward in
understanding the shear behavior of solid injectable
hydrogels. Physically crosslinked hydrogels, which
form the hydrogel class representing most solid
injectable hydrogels, have been reported to demonstrate diverse behavior such as bulk fracture,6972
strain stiffening,7376 and shear banding77 when subjected to large amplitude oscillatory or steady-state
shear. Rheological characterization provides information about the changes in mechanical behavior of
physically crosslinked, solid, injectable hydrogels. It
is often not possible to obtain correlations between
mechanical and morphological behavior of physically
crosslinked hydrogels when subject to shear solely by
the use of rheometric experiments. In this review, we
discuss the use of a confocal-rheometer compound
assembly that provides a direct visual evidence of the
flow pattern of a solid injectable peptide hydrogel sample under steady-state shear. The rheometer-induced
shear treatment in the experiment reported herein
is very similar to that reported in citations in the
previous paragraph that discuss shear-thinning and
rehealing behavior of physical hydrogels. Thus, using
a combined assembly of a rheometer and a confocal
microscope, we subject the hydrogel to steady-state
shear as described by reports studying shear-thinning
and rehealing hydrogels with a traditional bench-top
rheometer while being able to simultaneously obtain
a visual understanding of the exact flow pattern of the
hydrogel. Upon being subjected to certain amplitude
of shear, the peptide hydrogel displays bulk fracture a
thin layer away from the shearing upper plate of the
rheometer. Preliminarily, the thickness of the fractured
36
layer depends upon the amplitude of applied shear.

The phenomenon of bulk fracture demonstrates that
the perceived shear-thinning and rehealing behavior
of physical gels might, perhaps, be due to yielding
behavior of a thin layer of the hydrogel very close
to the shearing surface that is the top plate of the
rheometer, with a bulk of the hydrogel below that
layer experiencing negligible shear. Additionally,
upper plate-induced shear produced in standard
rheometry might not be the best representation of the
shear treatment via syringe injection for a variety of
reasons. Unless carefully conducted, a measurement
involving rheometer-induced shear is also prone to
artifacts such as wall slip, gap effects, time effects, and
shear history. Measurements carried out in a manner
similar to the one described above might literally and
figuratively be scratching the surface of what exact
phenomena might be occurring across the entire gap
height in a standard rheometer.
In the next section, we suggest some protocols and precautionary measures to be observed
while acquiring and interpreting data using common bench-top rheometers with a particular focus
on physical hydrogels. To put the phenomenon of
bulk fracture observed by the experiments reported
here into perspective, we review some of the recent
accounts of bulk fracture observed in physically
crosslinked hydrogel systems. Correlation of changes
in rheological behavior to bulk structural changes in
physically crosslinked proteins and peptides hydrogels is a crucial step in understanding the behavior
of these hydrogels in various mechanical and biological environments. The following section also
includes a review of rheological characterization of
peptide- and protein-based physically crosslinked
hydrogels. These studies demonstrate that rheological characterization is critical to understanding of
hydrogel characteristics such as gelation kinetics, gel
stiffness, solid-like character, and yield-stress behavior. The study of these and related hydrogels can
potentially provide interesting correlations between
shear-induced structural changes and rheological
properties.
RHEOLOGICAL MEASUREMENTS OF
PHYSICAL HYDROGELS: PROTOCOLS
AND PRECAUTIONS
Some measurements common to all rheometric experiments, such as oscillatory time sweep, frequency
sweep and strain sweep measurements, are carried
out using common bench-top rheometers while studying rheological properties of physical hydrogels. Some
experimental protocols, for example, the order in
1000
G, G (Pa)
which these measurements should be carried out

along with any specific precautions that should be
taken while studying different physical hydrogels,
are discussed in this section. For detailed explanation of measurements of raw, physical parameters by
rheometers (for example, torque, rotational speed, and
deflection angle) and their conversion to corresponding rheological parameters such as shear stress, shear
rate, and shear deformation, the reader is referred
to Refs 78 and 79 In the case of controlled-strain
rheometers, shear strain is applied to the sample in a
sinusoidal oscillation, (t) = o (sin t), and the measured shear stress is a phase-shifted sine wave with
(t) = o (sin t + ) in which is the applied angular
frequency and is the phase difference between the
two waves. For stress-controlled rheometers, the shear
stress is applied as (t) = o (sin t) and the resulting
shear strain is measured as (t) = o (sin t + ). The
equation relating shear stress and shear strain is given
by (, t) = G . o . (sin t) + G . o . (cos t), with
the coefficients G and G , respectively, indicating
the energy stored elastically and energy lost through
flow per cycle when oscillatory shear is applied to
a viscoelastic material. For a purely elastic material,
the strain and stress waves are in phase ( = 0 ) while
a purely viscous response has the two waves out of
phase by 90 ( = 90 ). Most viscoelastic materials,
if not all, demonstrate an intermediate phase angle
0 < < 90 .80 Since the G and G are ratios of the
oscillatory stress measured in Pascal to the oscillatory
strain which is a dimensionless quantity, G and G
are measured in the unit of stress, which is Pascal
(Pa).78,81
In a common oscillatory rheological measurement, the storage modulus, G , and loss modulus,
G , are the most common parameters that are measured for a hydrogel. G (Pa) and G (Pa) are usually
monitored as a function of time, applied angular frequency, and applied oscillatory strain. In a viscous sol
state, G is greater than G . Therefore, for a solid,
physical gel the storage modulus is greater than the
loss modulus (G G ), particularly at low frequencies as shown in Figure 1. An extensive account of
the theory and practical techniques concerning accurate measurement of a gel point of polymers has
been provided by Winter and Mours.8284 Microrheology has also been used to monitor the gelation
of physical hydrogel systems through use of the
motion of dispersed colloidal probe particles to measure viscoelastic properties.8587 Schultz and Furst88
have described multiple particle-tracking microrheology as a technique useful for precise determination
of hydrogel solgel transitions in a comprehensive
review.
100
10
1
0.1
10
100
1000
Strain %
FIGURE 1 | Oscillatory frequency sweep measurement of a 0.5%

(w/v) hydrogel at pH 9 (125 mM Boric Acid 10 mM NaCl), showing
relative independence of G (Pa) to applied angular frequency
(rad/second) indicating solid-like character of MAX1 0.5% (w/v)
hydrogel. G (Pa) indicated by solid squares, G (Pa) indicated by hollow
circles (G G ). The hydrogel sample shows G G for all
frequencies, particularly the lower frequencies.
Measurement of the moduli as a function of

frequency shows behavior of a hydrogel at short versus long timescales. The frequency dependence of
the moduli is a feature critical to hydrogel characterization. At high frequencies (fast timescales),
a viscoelastic liquid system can appear solid-like
(G G ), while at lower frequencies or longer
timescales, the same material will exhibit liquid-like
responses (G G ) and easily flow. Polymeric solutions with a concentration above the entanglement
concentration, as well as entangled polymeric melts
which are not chemically or physically crosslinked,
show G > G (Pa) with a crossover point reached
owing to increases in frequency after which G > G
(Pa).78,89 However, a solid, physical hydrogel will
exhibit solid-like properties (G G ) at all frequencies and timescales observed.
By monitoring the moduli versus strain, the linear viscoelastic regime (LVR) for a given material
can be determined. The LVR is a window of applied
strain values within which G and G are independent of applied strain. Linear rheological measurements are classified as studies conducted within the
LVR. Conversely, nonlinear rheological measurements
are obtained when experiments are performed outside of the LVR [e.g., when the physical hydrogel is
subjected to a constant shear rate from a spinning
top plate and consequently flows or when the material is subjected to large-amplitude oscillatory strain
(LAOS) treatments]. While steady-state shear is used
to observe the flow of hydrogels, LAOS measurements
37
Advanced Review
are instrumental in characterizing large, rapid material

deformations and help offer a more complete perspective of soft material responses to processing. LAOS
measurements and some typical rheological responses
from complex fluids have been reviewed by Hyun
et al.90 and Deshpande et al.91 In order to ascertain
the strain and frequency windows in which the studied hydrogel behaves like a solid (i.e., measurements
are performed in the LVR), the oscillatory strain sweep
measurement and the frequency sweep measurement
should be the first measurements to be carried out on
a hydrogel during its rheological characterization. A
precise idea of frequency and strain response is important since the values of G and G measured by the
rheometer during an oscillatory time sweep measurement (e.g., during initial gelation or during rehealing
after shear-thin flow) will be at constant frequency and
need to be at strains within the LVR.
Common rheological measurements such as evolution of G and G over time or frequency can
help the researcher gain knowledge about the mechanism and kinetics of physical gel network formation. In particular, various solution stimuli such as
pH, temperature, specific metal ions, enzymes, and
light are instrumental in triggering the assembly of
peptides and polypeptides. Rheology provides a direct
view of the effects of different types and magnitudes of assembly stimuli on peptides designed to
be stimulus-dependent materials. Extensive reviews of
stimuli-dependent (pH, salt, cations/anions, temperature, enzyme) peptide-based, self-assembled materials
have been provided by Lowik et al.92 and Raeburn
et al.93 For example, zinc ion-triggered assembly of
a -hairpin peptide containing a zinc binding nonnatural amino acid residue has been reported to
form hydrogels by Micklitsch et al.94 Cation-induced
hydrogelation of napthylalanine dipeptides at high pH
has been reported by Chen et al.95 Similarly, a series
of functionalized dipeptide systems were reported to
undergo salt-induced gelation in solution to form
shear-thinning and rehealing hydrogels.67 In another
account, cysteine-containing -hairpin peptides undergoing metal-triggered assembly by binding to metals
such as arsenic have been reported by Knerr et al.96
Toledano et al.97 have discussed protease-triggered
self-assembly of peptides into nanofibrous hydrogels
via reversed hydrolysis. In a vast majority of these
reports and the reports reviewed by Lowik et al.,92
the magnitude of triggering stimulus greatly influences peptide and polypeptide self-assembly pathways
and, in some cases, ultimate mechanical properties
of the hydrogels formed. For some peptide-based
gels, the rate of peptide assembly strongly influences bulk mechanical hydrogel properties such as
38
stiffness.40,46,98100 For these systems, the faster the

kinetics of self-assembly, the stiffer the formed hydrogels are. An example is shown in Figure 2, where the
dependence of gel formation over time relative to different solution ionic strengths was demonstrated using
an oscillatory time sweep measurement for G , with
the value of G G for all samples. The assembling
-hairpin peptide samples at weaker stimulus, which is
the lower ionic-strength buffer solution conditions in
this case, show a characteristic lag time while developing into fully percolated gels depending upon the
ionic strength. The sample with 20 mM NaCl concentration shows the highest lag time of about 45 min
and leads to the least stiff hydrogels, while the sample
with 400 mM NaCl concentration assembles fastest
with almost no lag time and leads to the stiffest hydrogels. The hydrogel sample triggered using a buffer with
150 mM NaCl concentration shows an intermediate
lag time of 15 min as compared to the 20 mM and
400 mM NaCl before it shows a sharp increase in G
values. Interestingly, the final hydrogels all have the
same peptide concentration despite having moduli that
differ over an order of magnitude, a clear indication
of differences in network structure due to the different assembly stimuli magnitude and resultant network
structures. A final example of stimuli-triggered assembly is the specially designed -hairpin peptide that contains a photocage that is released from the sequence
when the peptide solution is irradiated by UV radiation as discussed by Haines et al.101 The UV irradiation produced consequent peptide assembly and
hydrogelation as evidenced by oscillatory rheology.
The stimuli-triggered assembly behavior and hydrogel rheological characterization of peptides demonstrate the diversity of the types of stimuli that can be
used to assemble different peptide designs. Many more
examples of peptide and polypeptide physical assembly into hydrogels will be discussed in the remainder
of the paper.
In addition to the study of gelation kinetics
and the effects of different stimuli on assembly, rheological measurements have a particular relevance
in studying mechanical properties of biomaterials
intended as tissue engineering scaffolds and cell
culturing substrates. For example, the mechanical
properties of hydrogels are important in applications as scaffolds until formation of extracellular
matrix (ECM) by cells is complete.102,103 For applications of hydrogels as therapeutic cell delivery
vectors, cellular viability and phenotype depend on
hydrogel mechanical properties.104 When hydrogels
are used as substrates for tissue engineering, cellular differentiation, proliferation, and migration
are influenced by hydrogel mechanical properties
10,000
G (Pa)
1000
100
10
1
0
30
60
90
120
150
Time (min)
FIGURE 2 | Dynamic Time Sweep (1% strain, 6 rad/second) of 2 wt
% MAX1 [(VK)4 -VD PPT-(KV)4 ] solution with 20 mM (G , lled circles),

150 mM (G , lled triangles and 400 mM (G , lled squares) NaCl at
20 C. For all three samples (G G ) indicating gel-like rheological
behavior from all samples at all times. (Reprinted with permission from
Ref 46. Copyright 2004 American Chemical Society)
as discussed by Discher et al.105 A seminal report

from the Discher group106 discusses the influence
of hydrogel matrix elasticity on stem cell lineage
specification. Oda et al.107 have reported dependence
of proliferation rate and cell cycles of pluripotent
stem cells on the storage moduli of the hydrogels
when the cells are encapsulated within chemically
crosslinked hydrogels. The hydrogels were based on
poly(2-methacryloyloxyethyl phosphorylcholine-con-butyl methacrylate-co-p-vinylphenylboronic acid)
(PMBV) and poly(vinyl alcohol) (PVA) polymers with
finely tunable storage modulus values. The conversion of mechanical signals from their environment
to biochemical signals by cells due to cell-surface
receptor-mediated connection between the ECM and
cytoskeleton has also been suggested. These examples
underscore the importance of mechanical properties
of hydrogels that can be potentially used as ECM
mimetic materials.108,109
An important choice relevant to rheological measurement pertains to the instrument geometry used
in the rheological measurement. Different types of
rheometer geometries are available for making rheological measurements namely parallel plate, cone and
plate, concentric cylinder, double walled concentric
cylinder, and combined cone and plate with cylinder
(Mooney/Ewart measuring systems).79 For rheological measurements on physical hydrogels, parallel plate
and cone and plate geometries are most popular. The
cone and plate geometry consists of a flat plate and
a low-angle cone (1 < < 4 ) that rotates against
the flat plate. A truncated cone may also be used to
avoid damage to the apex of the cone and preventing
trapping of sample particles between the apex and the

flat plate. The main advantage of the cone and plate
geometry is that the shear rate does not vary from
the axis of rotation because both the linear velocity
and the gap between the cone and plate increase with
increase of distance from the rotational axis. Parallel plate geometry consists of two parallel plates with
adjustable gap for holding the sample between the
plates. The advantage of this geometry is the variable
gap that can be adjusted to accommodate dispersions
with large particles or domain sizes as well as samples
that have large heterogeneities in structure (e.g., large
variation in pore sizes). For parallel plates, variations
in shear rate across the gap height and instabilities in
shear field at high rates are observed.110 Thus, in case
of parallel plates, calculation of the actual shear rate
from the shear rates measured at different gap must be
carried out. Synthetic limitations often constrain the
amount of peptide or protein available for the formulation of a peptidic or protein-based hydrogel. Given
this constraint, the cone and plate geometry is typically
implemented for rheological studies, as it requires the
least amount of sample volume for a measurement.
Cone and plate geometries are not the best choice for
particulate hydrogels or hydrogels with large domain
sizes like hierarchically assembled fibers, when the particle size can be on the order of the gap size.110 Sample
sedimentation in the case of heterogeneous hydrogels
over time that results in a layer depleted of particles
near the cone has also been reported to skew the rheological measurement.110 Thus, for particulate hydrogels and gels containing large domain sizes, parallel
plate geometries should be used.
Gap effects are commonly observed during rheological characterization of soft, physically crosslinked
hydrogels. A precise calibration of the gap height, i.e.,
the distance between the upper plate and a lower plate
in parallel plate geometries, must be obtained. Before
conducting any measurement, the gap measurement
at which two surfaces are in physical contact with
one another should be calibrated as the zero gap.
This zero gap calibration helps as reference in setting
a desired gap height for the experiments. When measurements of G and G are carried out for the same
sample at different gap heights, different values of the
measured parameters may be obtained because of the
effects of heterogeneity. Thus, multiple measurements
at various gap heights should be carried out and a suitable gap height should be fixed such that values of
measured parameters stay constant at heights above
this gap height.
Similarly, flow-induced heterogeneity with or
without shear and specific physical or chemical interactions between the sample and the plate walls can
39
Advanced Review
lead to wall slippage and distort the measurement.111

Multiple measurements carried out at various gap
heights can also help in analyzing the presence of
potential wall slip, which, if present, can lead to differences in measurements at different gap heights. Wall
slip can be minimized in case of measurements using
parallel plate geometries by roughening of the geometry surface, ensuring adequate momentum exchange
with the gel thereby avoiding slip. A simple solution toward eliminating wall slip is the attachment
of a piece of high grit sand paper to either or both
plates of a bench-top rheometer. Detailed accounts
of attempts made at determination and elimination
of wall slip have been given by Yoshimura et al.112
and Carotenuto et al.,113 who specifically discuss the
implementation of rough tool walls in order to avoid
slip in their rheological measurements. Walls et al.114
reported a significant reduction in wall slip during rheological characterization of fumed silica gels by using
geometries with rough surfaces as opposed to geometries with smooth surfaces. Clasen et al.115 and Kelessidis et al.116 have presented specific examples of the
determination wall slip in yield-stress fluids.
Another important consideration for rheological measurements on peptide- and polypeptide-based
hydrogels is the material used to fabricate the rheometer geometries. Some examples of materials used
to fabricate rheometer geometries include stainless
steel and acrylate resins. Peptides and polypeptides
demonstrate a multitude of secondary interactions
such as hydrophobic interactions, hydrogen bonding,
and electrostatic interactions. Taking such interactions
into account, a careful choice of geometry based on
the material used to fabricate it should be made. For
most experimental setups, the geometries used for
the characterization of hydrogels from these systems
should be, ideally, chemically inert to these systems.
For samples that demonstrate wall slip at the tool surface, it is sometimes beneficial to ensure appropriate
adhesive interactions between the sample and the tool
surface via secondary interactions to avoid wall slip.
Any potential physical bonding interactions between
the materials under study and the rheometer geometry should be carefully accounted for while interpreting rheological data using the concerned geometry.
For example, if hydrophobic interactions dominate
the self-assembly and hydrogel formation from certain peptide molecules and a stainless steel or acrylic
rheometer geometry is used to probe the gelation
kinetics, hydrophobic interactions between the peptide molecules and the geometry must be considered.
Hydrophilic-functionalized geometries might help in
minimization of hydrophobic interactions between
the geometry and the sample in such a case. As
40
an experimental illustration of sampleplate interactions, Walls et al.114 have reported that in case of a
hydrophobic sample, a fumed silica gel, hydrophobic plates undergo hydrophobic interactions leading to
decrease in wall slip. When hydrophilic plates are used
they repel the hydrophobic silica sample leading to a
particle-lean layer near the plate leading to occurrence
of wall slip.
The next section describes solid injectable hydrogels based on -hairpin peptides that undergo hierarchical self-assembly into fibrillar nanostructures. The
rheological characterization of the hydrogels based
on this family of peptides has been conducted taking the above-described protocols and precautions
into consideration. While these hydrogels demonstrate
shear-thinning and rehealing behavior when investigated with a bench-top rheometer, they undergo bulk
fracture under steady-state shear as evidenced by the
confocal-rheometry experimental study described in
the next section.
BIOMEDICALLY PROMISING SOLID

INJECTABLE HYDROGELS FROM
SELF-ASSEMBLING -HAIRPIN
PEPTIDES
Hydrogels from -Hairpin Peptides Based
on the Parent Sequence MAX1
[VKVKVKVK-(VD PPT)-KVKVKVKV-NH2 ]
Solid, injectable hydrogels are formed by the hierarchical self-assembly of -hairpin forming peptides.
The -hairpin peptide assembly process leading to
hydrogel formation, and the importance of rheological characterization in studying hydrogel mechanical
properties, gelation kinetics, and yield-stress material
behavior is discussed in this section. The rheological
characterization of these hydrogels has been carried
out in accordance to the protocols and precautions discussed in Section Rheological Measurements of Physical Hydrogels: Protocols and Precautions.
The Pochan and Schneider groups have studied
extensively the MAX family of peptides, which
is based on a parent sequence MAX1.45,46,117119
MAX1 is a 20 amino acid residue amphiphilic peptide
sequence VKVKVKVK-(VD PPT)-KVKVKVKV-NH2 ,
with alternating hydrophobic valine (V) and
hydrophilic lysine (K) residues.45 The type II turn
sequence -VD PPT-; where D P is the nonnatural,
right-handed enantiomer of proline, P is the natural,
left-handed proline amino acid, and T is threonine; in the center is responsible for chain reversal and
-hairpin formation at elevated pH, ionic strength and
temperature solution conditions from a random coil
(a)
(c)
(b)
(d)
50 nm
(f)
(e)
FIGURE 3 | Schematic of MAX1 self-assembly (a) MAX1 random coil conformation at low to neutral pH and low temperature, the pink side
chains represent lysine side chains and the blue side chains represent valine side chains. (b) -Hairpin conformation induced by rise in the pH,
temperature, and/or ionic strength of the peptide solution. (c) Subsequent to intramolecular folding, facial hydrophobic collapse of two hairpins
leading to formation of a bilayer type structure. (d) Direction of lateral hydrophobic interactions among multiple bilayer type structures (e)
hierarchically assembled branched bril of MAX1. (f) Cryogenic transmission electron microscopy showing brillar structure of MAX1.
conformation in aqueous solution of low to neutral

pH and low ionic strength (Figure 3(b)). An increase
in pH of an acidic/neutral peptide solution serves to
deprotonate some of the lysine side chain primary
amino groups and allows folding and self-assembly
to occur. In addition, increasing the ionic strength of
a neutral peptide solution can also initiate peptide
folding and assembly by screening the positive charges
on the lysine side chains, both leading to -hairpin
formation. The -hairpin conformation has the two
amphiphilic arms of MAX1 parallel to each other
stabilized by intramolecular hydrogen bonding. An
increase in temperature emphasizes hydrophobic interactions primarily between the valine side chains and
serves as another factor promoting the peptide folding
and self-assembly. Facial hydrophobic interactions
(Figure 3(c)) at the valine face between two -hairpins
form the core of the growing nanofibrils.117 Lateral
intermolecular hydrophobic interactions (Figure 3(d))
along the axis of the growing fibrils, along with the
facial hydrophobic interactions, lead to hierarchical
assembly of these -hairpins into uniform fibrils
(Figure 3(e)).118 Lateral intermolecular hydrogen
bonding interactions also play a significant role in the
assembly. Sometimes, during the facial hydrophobic
collapse, two hairpins undergo an incomplete burial
of the hydrophobic face. This incomplete burial manifests itself as a defect that is responsible for nucleation
of two fibrils emanating from the defect junction.
Thus, these junctions of fibrils act as branching points
and contribute to physical crosslinking of the fibrils
in addition to fibrillar entanglement leading to formation of self-standing, solid hydrogels.120 The cryogenic
transmission electron micrograph (Figure 3(f)) shows
the uniform fibrillar structure of the MAX1 network. The fibrils have a uniform thickness 3 nm,
corresponding to the strand length of each -hairpin
of MAX1.
In case of derivatives of MAX1 with slightly
different primary structures, the specific pH, ionic
strength, and temperature conditions, or suitable
combination of these solution parameters, used for
-hairpin formation and consequent intermolecular
assembly and gelation are dependent on the specific
peptide primary sequence. As an example, the peptide
MAX8 is obtained by point substitution of a positively charged lysine residue in MAX1 with a glutamic
acid residue with a negative charge. At the same peptide concentration and solution conditions, MAX8
demonstrates faster assembly kinetics owing to less
overall positive charge (+7 as compared with +9 in
case of MAX1) and additional attractive electrostatic
41
Advanced Review
42
2500
2000
G, G (Pa)
interactions between the added glutamic acid and

lysine residues.40,121 MAX8 peptides can undergo
self-assembly to form hydrogel materials under physiological pH (7.4),121 ionic strength30 (150 mM
salt), and temperature conditions (37 C).30,52,122
MAX8 allows uniform three-dimensional (3D) living
mesenchymal cell or drug payload encapsulation
due to the fast gelation time (<1 min vs 30 min
for MAX1) at physiological solution conditions.122
This expedited gelation leading to ultimately stiffer
hydrogels from MAX8, as compared to those from
MAX1 at the same peptide concentration and solution conditions, has been shown clearly by oscillatory
rheological measurements.123 Thus, MAX8 has
particular relevance to homogeneous 3D cell encapsulation and potential tissue engineering applications.
Leonard et al.124 have provided solid-state NMR
spectroscopy evidence of the -hairpin conformation
being the dominant conformation within assembled
fibrils of MAX8 peptide. MAX8 also has been shown
to have a very similar fibrillar nanostructure as MAX1
in terms of fibril width (3.2 nm).120 Owing to faster
gelation kinetics than MAX1, MAX8 fibrils form
networks with smaller pore sizes indicating more
crosslink density of MAX8 fibrils as compared to
MAX1 as evidenced by cryogenic transmission electron microscopy (cryo-TEM) and small-angle neutron
scattering (SANS).125 Hydrogels based on MAX1
and MAX8 have demonstrated cytocompatibility,126
non-inflammatory properties122 and in some cases
biologically effective properties such as antibacterial activity (MAX1127 and its derivatives47 ). These
peptide systems provide flexibility of peptide structure through introduction of specific biochemical
functionalities such as post-self-assembly chemical
crosslinking to yield stiffer hydrogels.51 Macromolecule self-diffusion and bulk release studies with
MAX1 and MAX8 hydrogels have shown macromolecule mobility within, and release out of, the
gels.27,29 This demonstrates the ability of the porous
-hairpin peptide hydrogels with tunable mesh sizes
as viable candidates for tissue engineering and drug
delivery scaffolds since they allow transport of nutrients and metabolites. Thus, owing to the favorable
therapeutic encapsulation, initial biocompatibility
and, in some cases, bioactivity, MAX1 and MAX8
demonstrate significant potential for their use in
biomedical applications, particularly in light of the
injectable solid attributes displayed as discussed
below.
A well-defined linear viscoelastic region and
yield strain value (1040%) dependent upon the specific peptide sequence and solution conditions used
to trigger self-assembly are demonstrated by these
1500
1000
500
0
0
20
40
60
80
100
Time (min)
120
140
160
180
FIGURE 4 | Oscillatory time sweep measurement (6 rad/second and

1% strain) from 0 to 80 min after mixing peptide solution with buffer
solution to form a 2% (w/v) MAX1 peptide hydrogel at pH 9 (125 mM
Boric Acid 10 mM NaCl) showing evolution of storage modulus of 2%
(w/v) pH 9 (125 mM Boric Acid 10 mM NaCl) to 2200 Pa followed by
steady-state shear of amplitude 1000/second for 2 min indicated by the
dotted line. The shear step is followed by another oscillatory time sweep
measurement probing rehealing of the hydrogel to G value close to the
initial G value. G (Pa) indicated by solid squares, G (Pa) indicated by
hollow circles (G G ), indicating gel-like rheological behavior from
all samples at all times.
hydrogels subject to a strain sweep measurement

at a constant frequency.45,46,53,117,118,120,128 Figure S1
shows an oscillatory strain sweep measurement conducted on a 0.5% (w/v) hydrogel of MAX1 at solution conditions pH 9 (125 mM Boric Acid, 10 mM
NaCl) that demonstrates a well-defined LVR 60%.
During a frequency sweep measurement at constant,
small amplitude strain within the LVR, the hydrogels display an elastic modulus, G , almost independent to applied angular frequency (0.1100 1/second)
over several decades of applied angular frequency
as shown in Figure 1. [0.5% (w/v) MAX1 pH 9
(125 mM Boric Acid 10 mM NaCl)] and discussed in
other reports.30,45,46,53,117,119,120 The solid injectable
gel behavior is characterized by the study of storage
modulus evolution of a fully formed hydrogel after
subjecting it to a steady-state shear using a bench-top
rheometer. Figure 4 shows the shear-thinning and
rehealing behavior of a 2% (w/v) MAX1 hydrogel subject to steady-state shear of 1000/second for
2 min (indicated by dotted line). As discussed by Yan
et al.,53 MAX1 and MAX8 hydrogels, as well as other
MAX1 derivatives, undergo shear-thinning behavior
when subject to rheometer-induced shear and immediately form a solid on cessation of shear leading
to a value of stiffness modulus G 600 Pa, G G
(immediately after the dotted line in Figure 4.). When
allowed to age, these hydrogels heal into networks

with modulus G comparable to that of the network pre-shear (2000 Pa). The significant observation from the results shown in Figure 4 is that the
hydrogels have G (600 Pa) G (20 Pa) immediately after cessation of shear, indicating a solid percolated network nature. This can be considered as the
recovery of the sheared materials under flow to a
solid network immediately after shear cessation. The
increase of the G (Pa) values to pre-shear values over
several hours is indicative of further healing/stiffening
of the hydrogels over time, with a decrease in the
tan and a corresponding increase in elastic nature
of the hydrogels. The rheological characterization of
the MAX1/MAX8 hydrogels demonstrates the utility
of these materials as preformed solid hydrogels that
can retain their solid nature when injected in vivo.
This shear-thinning and rehealing behavior will be
discussed in more detail later in Section Rheological
Characterization of the Shear-Thinning and Rehealing
Behavior of -Hairpin Hydrogels.
Along with MAX1 and MAX8, different primary structures of MAX1 derivative peptides lead
to differences in the assembly stimulus requirement,
assembly kinetics, the nanostructure, and mechanical properties of hydrogels formed from the peptides.
The examples discussed below demonstrate differences in physical properties of hydrogels from peptides with different primary structures based on the
MAX1 peptide sequence. For example, the MAX3
peptide is obtained by substitution of two hydrophobic side chain valine residues with hydrophilic side
chain threonine residues. Owing to these point substitutions, the MAX3 peptide under the same solution
conditions as MAX1 forms hydrogels only at a much
higher temperature (70 C) than MAX1 at the same
solution conditions (30 C). When cooled to temperatures significantly below 70 C (5 C), gelation and
assembly are reversed and the material becomes a low
viscosity solution.117 MAX3 hydrogels can undergo
multiple cycles of sol to gel and gel to sol transitions. [Such reversibility of hydrogel like properties
is observed also in case of MAX1 when temperature
is used as the dominant stimulus for self-assembly
along with appropriate pH/ionic-strength solution
conditions (i.e., low pH, low ionic strength).] These
cyclic changes in MAX3 mechanical properties were
observed using oscillatory rheological measurements.
Other examples of MAX1 derivatives include peptides with varying properties such as twisted fibrils, non-twisted and laminated fibrils all forming
stiff, shear-thinning and rehealing hydrogels.129131
Owing to the designs of the appropriately named
strand swapping peptides, SSP1, SSP2, and SSP3
undergo strand swapping. Hydrophobic association

of strand-swapped dimers drives core fibril structure
formation. But, instead of two hairpins forming a fibril cross section in the case of MAX1, four hairpins
are required to form a fibril structure cross section
by burial of valine side chains in the SSP peptides.
Thus, effectively twice the concentration of peptides
was required to form fibrils and hydrogels with SSP
peptides as compared to MAX1 as shown via rheology. The strand swapping incurred during peptide
folding results in the formation of different nanostructures are formed from SSP1 (singular twisted fibrillar),
SSP2 (singular non-twisted fibrillar), and SSP3 (laminated non-twisted fibrillar).
Rheological measurements also were used
to probe the controlled biodegradation of peptide
hydrogels based on a series of degrading peptides
(DP) through interaction with metalloproteinase-13
(MMP-13).132 Oscillatory rheological characterization was used to measure stiffness values of hydrogels
as they were subject to degradation using MMP-13
and thus helped directly validate that the enzymes
were degrading the peptide fibrils that constituted the
hydrogel. Hydrogel based on enantiomeric mixtures
of self-assembling -hairpins (MAX1 and D-MAX1)
showing non-additive, synergistic, enhancement in
material rigidity, compared to gels prepared from
either pure enantiomer, have been reported by Nagy
et al.133 The fibrillar morphology of the hydrogel
formed from the enantiomeric mixture is negligibly
different from that based on either pure enantiomer.
Results from rheometeric experiments are useful in
demonstrating the non-additive mechanical synergistic effects when peptide enantiomers are mixed.
Similar to -hairpin peptides, hydrogels from
amphiphilic linear peptides that form hydrogels based
on tapes and laminates have been reported.49 The
absence of a -hairpin conformation affects the nanostructure formed from linear peptides, but the hydrogels can be studied rheologically. Many examples of
linear peptides are discussed later in the paper.
Rheological Characterization of the

Shear-Thinning and Rehealing Behavior
of -Hairpin Hydrogels
As discussed above, rheological conditions that
employ small amplitude oscillatory strain conditions
have been used to determine various mechanical
attributes of hydrogels based on MAX1, MAX8, and
derivative peptide sequences. Results from rheological
measurements help in the understanding of various
properties such as hydrogel stiffness, linear viscoelastic region windows, and yield-stress values of these
43
Advanced Review
FIGURE 5 | (a) Flow prole of a cell suspension and cells encapsulated in MAX8 gels. Solid symbols: one-dimensional ow velocity of living MG
63 cells against lateral position when suspended in aqueous buffer (pH 7.4, 25 mM HEPES, 37 C). Open symbols: one-dimensional ow velocity of
living MG 63 cells when encapsulated in 0.75 wt % MAX8 gels, pH 7.4, 25 mM HEPES, 37 C). (b) Three-dimensional confocal microscope images
showing live-dead assays of MG63 cells encapsulated in 0.5 wt % MAX8 hydrogel after injection. (Reprinted with permission from Ref 52. Copyright
2012 American Chemical Society)
hydrogels. These properties help establish the utility

of the hydrogels as biomaterials. An important property of MAX1, MAX8, and derivative peptide-based
hydrogels is their solid injectable behavior. As discussed in Introduction, solid injectable hydrogels of
these peptides offer significant promise as delivery
vehicles for drugs,30 cells, and macromolecules such
as proteins27 and polysaccharides.29
The behavior of MAX1 and MAX8 -hairpin
peptide hydrogels during and after steady-state
shear-induced flow has been explored by Yan et al. in
order to fundamentally understand the mechanisms
of the shear-thinning and rehealing behavior.53 As
shown in Figure 4, a MAX1 hydrogel at 2% (w/v)
concentration exhibits shear-thinning and flows in
the shear field when subject to steady-state shear
(indicated by the dotted line) in Figure 4. In order
to subject the hydrogels to steady-state shear, a
bench-top rheometer equipped with parallel plate
geometry was used. Upon cessation of the shear, the
material immediately rehealed to a solid hydrogel
material as indicated by the data immediately to
the right of the dotted line. The shear-thinning and
rehealing ability of the -hairpin hydrogels is preserved when the hydrogels are used to encapsulate
biologically important materials such as drugs30 and
macromolecules such as proteins27,29 and cells.40,52
Although this experimental setup offers appropriate rheometric characterization of the shear
response of the hydrogel, it cannot offer information
44
about any structural change exhibited by the hydrogel

during simultaneously shear-induced flow. Because
the rheometer measures purely mechanical shear
response of the -hairpin hydrogels, it cannot track
shear-induced flow patterns exhibited by the hydrogels. As an example of investigation of shear-induced
flow patterns by the MAX8 -hairpin hydrogels, Yan
et al.52 reported that the -hairpin hydrogels underwent plug flow upon injection through a capillary in
a scenario mimicking syringe injections. As shown in
the velocity profile in Figure 5, apart from the portion
of the gel directly in contact with the capillary wall
where significant shearing took place, the gel across
the majority of the capillary bore maintained a constant velocity and was subject to negligible shear. This
flow profile result obtained by using a capillary device
would not have been observed if the measurements
were carried out solely using a conventional parallel
plate or cone and plate geometry in a bench-top
rheometer. A negligible gradient in the velocity profile
over the bulk of the hydrogel used to deliver the cells
is observed in comparison to a laminar velocity profile
for cells suspended in buffer solution injected using
the same capillary, Figure 5. The observation of no
shear within the bulk of the gel during capillary flow
led to observations of cells encapsulated within the
hydrogel also experiencing minimal shear during flow
with significant cell viability within the hydrogel network after the shear injection process. The plug flow
and consequent protection of encapsulates from high
Centerline
x
200 m
Capillary wall
0.25
Velocity (mm/s)
0.20
0.15
0.10
Average velocity = 0.151 mm/s
0.05
0.00
0.0
0.1
0.2
0.3
0.4
Radial position (mm)
FIGURE 6 | Visualization of the ow prole in capillary ow of a PC10P hydrogel labeled with uorescent microsphere tracers. Time-dependent
line scans along the diameter of a capillary (1.93 ms/scan) show that the transit time for a particle to cross the plane of the line scan is approximately
the same for all distances from the center of the capillary, indicative of a plug-ow-type prole. This implies yielding in the gel layer adjacent to the
capillary wall, consistent with the shear-banding mechanism. (Reprinted with permission from Ref 16. Copyright 2010 American Chemical Society)
shear rates reinforces the utility of MAX1/MAX8

hydrogels as injectable solid hydrogels.
Two other recent reports in the literature show
the utility of confocal microscopy during hydrogel
capillary flow for insight into the specific plug-flow
behavior of injectable, physically crosslinked gels. A
recent account from Aguado and Heilshorn17 shows
a similar plug-flow regime as essential for protection
of cells from deformation and shear while being
injected as encapsulated within physically crosslinked
alginate hydrogels. Olsen et al.16 report a physically
crosslinked injectable telechelic hydrogel that demonstrates extreme shear-thinning and rapid rehealing
properties. Flow visualization and LAOS results
demonstrate the formation of zones of nonhomogenous shear within the hydrogel. As shown in Figure 6,
visualization of the velocity profile in capillary flow
of a PC10P hydrogel labeled with fluorescent microsphere tracers shows that the portion of the gel closest
to the capillary wall undergoes yielding indicating a
plug-flow profile for the gel in a capillary geometry.16
An important characteristic of the shear response
behavior of the MAX1/MAX8 or any of the -hairpin
peptide hydrogels is that they do not exhibit synerisis, or phase separation of peptide fibrils and aqueous
medium under shear. We have not found in the literature the demonstration of synerisis by other physically
crosslinked peptide/protein hydrogels like the hydrogels mentioned in Section Rheological Characterization of Physically Crosslinked hydrogels.
In order to obtain information of any potential
structural change exhibited by the -hairpin hydrogels
while being subject to steady-state shear-induced flow,
Yan et al.53 have used the combination of rheology
and material flow with the concurrent study of material nanostructure through small-angle scattering. One
combination was the use of rheo-SANS, or rheology
combined with SANS, a concentric cylinder rheometer tool and an incident neutron beam for small-angle
scattering. The other combination technique was to
flow the hydrogel through a capillary while observing
the scattering of an incident synchotron X-ray beam.
In both these techniques, the neutron or X-ray beam
is directed normal to the direction of shear induced
by the rotating cylinder tool/injection through capillary such that the incident neutrons or X-rays can
scan a cross section of a hydrogel sample representing the entire hydrogel sample affected by the shear.
Rheo-SANS is a compound technique that facilitates
the study of structural change of a soft matter system
45
Advanced Review
while simultaneously monitoring changes in rheological properties of the system. An extensive review of
Rheo-SANS study of soft matter systems has been
provided by Eberle et al.134 The construction of a
modified commercial rheometer used to conduct rheometric measurements while simultaneously being able
to monitor structural changes has been discussed by
Porcar et al.135 SANS and small-angle X-ray scattering (SAXS) data from the hydrogels under shear
help to elucidate any changes in hydrogel nanostructure during the process of rheometer-induced or
capillary-induced shear. Results from the combination techniques demonstrate that there is no noticeable
change in hydrogel morphology during rheometer- or
capillary-induced shear. Based on these results and
results from bench-top rheology of -hairpin peptide
(MAX1/MAX8) hydrogels, a model has been proposed to explain how the gel network is fractured
into domains during shear-thinning and flow.53 The
domains can flow during shear but can immediately
re-percolate into a solid hydrogel when the shear
is stopped. Within the fractured domains exists the
same nanostructure, same fibrillar thickness and same
porosity as the original bulk network. The lack of
changes in hydrogel nanostructure during rheometer
or capillary-induced shear may also point to bulk fracture within a certain thickness of the hydrogel sample
away from the shearing surface as explained by Yan
et al.53 Instead of fracturing into domains within the
bulk of the gel thus allowing the material to flow,
a hydrogel layer near the stationary rheometer tool
may fracture from the bulk material and consequently
flow. In this case, the bulk of the hydrogel would
remain stationary and thus would appear as the original bulk network by scattering. Given the possibility of bulk fracture of the hydrogel throughout the
sample cross section or fracture limited to a certain
layer of the hydrogel sample, obtaining visual evidence
of the exact, possible fracture characteristics of the
MAX1/MAX8 hydrogels via imaging techniques such
as confocal microscopy is an important step forward
in understanding the shear response from these solid
injectable hydrogels.
Given the plug-flow behavior of the injectable
solid MAX1 or MAX8 hydrogels, one could expect
fracture of a surface layer next to the top rheometer plate in the hydrogels when subjected to shear
treatment using a rotating upper plate of a bench-top
rheometer. Intuitively, it can be expected that the shear
induced by the rheometer upper plate might be fracturing only a localized layer of the hydrogel very near
the upper plate, while the bulk of the gel might be
negligibly affected by shear. Thus, direct visualization of the flow pattern of the hydrogel when subject
46
to shear using a rheometer upper plate would reveal

the exact flow patterns obtained using a bench-top
rheometer. Rehealing behavior of MAX1 or MAX8
hydrogels after being subject to steady-state shear has
already been observed to be affected by the differences in amplitude and duration of the applied shear.53
For example, a hydrogel subject to 1000/second of
steady-state shear for 2 min recovers to a lower value
of stiffness modulus, G (Pa), as compared to a hydrogel subject to 1000/second of steady-state shear for
5 seconds. Is this difference due to differences in bulk
shear of the gels or to differences in the fractured
layer experiencing shear? Thus, imaging during shear
application would help verify hydrogel flow patterns
and any fracture properties as functions of various
parameters associated with the shear treatment such as
applied stress amplitude, duration, and shear history.
In the experiments reported herein, a confocal
rheometer136 was used to obtain direct visualization
of the structural response of -hairpin hydrogel when
subject to steady-state shear using a bench-top parallel plate rheometer. Experimental details and the
instrumental configuration have been described in
Section Experimental. Carboxyl-functionalized, fluorescent microparticles were encapsulated within the
MAX8 hydrogels used for the study. The fluorescent
microparticles indicated the presence of the hydrogel
across the gap height when imaged using the confocal
microscope in the device. Video data obtained using
the confocal microscope are included in the Supporting Information. These data show a sheared hydrogel sample at various steady-state shear rates while
it is being viewed from the bottom of the hydrogel
sample looking upwards at the rheometer upper plate
as shown in Figure 7. Specifically, each video shows
images of multiple layers of the hydrogels at increasing
heights within the sample away from the rheometer
lower plate. These increasing heights are indicated by
an increasing z value in the videos and the constant x
and y values indicate the position of the layers relative
to the lower plate. The total thickness of the hydrogel
scanned (m), thickness of fractured layers (m), the
corresponding rates of steady shear (second1 ), and
the thickness of the fractured layers as a percentage
of the total hydrogel thickness scanned are reported in
Table 1. The video data obtained on the MAX8 hydrogel at the different steady-state shear rates (5/second,
50/second, 250/second, 400/second, and 1000/second) show fractured layers of different thicknesses.
The total hydrogel thickness between rheometer plates
for the shear rate 5/second was 980 m, while for
the shear rates of 50/second, 250/second, 400/second, and 1000/second the total gel thickness was
790 m. Under steady-state shear of 5/second, the
Shear amplitude
&
thickness of
fractured layer
5/s
T f~
50 m
Rheometer
upper plate
Hydrogel
sample
at rest
Glass window
for confocal
microscope
Fractured
layer
50/s
Tf~
64 m
Incident laser
from confocal
microscope
Stationary
layer
400/s
Tf~
400 m
FIGURE 7 | Schematic showing results obtained from confocal-rheometer compound assembly. The hydrogel undergoes shear rate-dependent
fracture in a layer of certain thickness away from the upper plate of the rheometer geometry, while the rest of the hydrogel undergoes no or negligible
ow. The square schematics indicate volumes across the cross section of the hydrogel sample between the rheometer plates when the sample is
subject to 5/second, 50/second, 250/second, 400/second, and 1000/second rate of steady-state shear. The light blue and dark blue layers indicate the
fractured and consequently owing layers and stationary layers of the MAX8 hydrogel, respectively. T f indicates the % thickness of the fractured layer.
bulk of the encapsulated microparticles, i.e., the bulk

of the MAX8 hydrogel, undergoes zero or negligible flow. Only a layer of average thickness 23 m
near the upper plate (at the top of the bulk hydrogel) shows flow of encapsulated microparticles. This
result clearly shows evidence of bulk fracture of the
physically assembled MAX8 hydrogel. When the same
hydrogel is subject to a steady-state shear of 50/second the layer of microparticles undergoing flow stays
approximately the same at an average of 25 m. When
the rate of steady-state shear is increased to 250/second the fracture between stationary and flowing gel
next to the upper plate occurs at an average thickness of 307 m from the upper plate. At a steady-state
shear of 400/second, the microparticles appear to be
in motion at an average thickness of 625 m. At
a steady-state shear of 1000/second the microparticles appear to be in motion at thickness of 650 m.
These results provide evidence of the bulk fracture
and yielding behavior of the MAX8 hydrogel that is
commonly considered as a shear-thinning and rehealing material. The experimental results acquired using
the confocal-rheomter studying the MAX8 hydrogel
clearly indicate shear rate-dependent fracture of the
hydrogel.
The results from the confocal-rheometer assembly provide important direct visualization of the fracture and flow behavior of the -hairpin hydrogels
under commonly employed shear treatment using a
rheometer. The data clearly show that the hydrogels
demonstrate bulk fracture under steady-state shear in
TABLE 1 Results Obtained from Confocal-Rheometer Assembly

Total Gel
Fractured
Shear
Thickness
Layer
Rate
(Gap Height)
Thickness
% Thickness
(m)
(m)
Fractured
(second1 )
5
980
23 2
2.4 0.2
50
790
25 4
2.8 0.9
250
790
307 9
38.8 1.2
400
790
624.6 11
79.0 1.4
1000
790
652.0 5
82.5 0.7
The thickness of the hydrogel undergoing shear rate-dependent fracture away

from the upper plate of the rheometer geometry is obtained by recording
the z values from the videos at which the encapsulated microparticles
start demonstrating motion. The z values are then converted to thickness
of hydrogel layer in which negligible microparticle motion takes place
by multiplying by the factor (2.37) fixed during the confocal microscopy
measurement. The thickness of the fractured layer is obtained by subtracting
the thickness of the stationary microparticle layer from the total hydrogel
thickness. Measurements performed at each shear rate were performed in
triplicate.
layers with thicknesses dependent on the shear rate. As

shown in Table 1, the thickness of the fractured layer
increases with increased shear rate.
According to the model proposed by Yan
et al.,53 MAX1/MAX8 hydrogels undergo fracture
into smaller domains under shear indicated by yielding and flowing of the hydrogel within the shear field.
Upon termination of rheometer or syringe-induced
shear, these fractured domains re-percolate to form
a solid hydrogel network, indicated by bench-top
47
Advanced Review
rheology with G > G (Pa). The bulk fracture indicated by the confocal rheometer serves to reinforce the
hydrogel fracture model proposed by Yan et al. The
data from the compound assembly prove that yielding
and flow of the MAX1 or MAX8 hydrogels take place
but only within a defined layer parallel to the shearing
plate of the rheometer. These data are also consistent
with the yielding and shearing of MAX8 hydrogels
in a layer close to the walls of a capillary during
capillary flow of MAX8 hydrogels, while the bulk
of the material within the capillary flows as a plug
and experiences no shear. The experimental results
from the capillary geometry and the compound confocal rheometer clearly indicate shear rate-dependent
fracture exhibited by the -hairpin peptide hydrogels. At higher amplitudes of steady-state shear, a
significant majority of the hydrogel bulk away from
the rheometer upper plate undergoes fracture and
shear-thinning. Upon cessation of this shear, it also
demonstrates rehealing behavior into a bulk solid
hydrogel. Importantly, even if one is confined to
use only of a bench-top rheometer, which does not
yield a direct correlation between hydrogel structural
changes under flow and the corresponding rheological
shear response, the rheometer still faithfully represents shear-thinning and rehealing behavior within a
certain layer thickness. As long as a shear-fractured
layer constitutes a significant thickness of a bulk
physical hydrogel, the rheological behavior of the
fractured layer can be considered qualitatively representative of that of the bulk hydrogel sample when
under shear. While difficult to determine what layer
thickness is considered a significant thickness, in all
measurements performed herein, at least 20 m of
hydrogel experienced fracture and shear flow at the
lowest shear rate of 5/second. When subject to high
rates of steady-state shear (1000/second), the hydrogel
exhibits fracture and flow of a layer with thickness
82% of the entire hydrogel sample thickness, a large
majority of the bulk of the hydrogel. Therefore, higher
shear rates are desired if one desires flow of as much
hydrogel volume as possible within the bench-top
rheometer. Importantly, both low and high shear
rate data exhibit qualitatively similar shear-thinning
and immediate rehealing behavior. Almost identical,
immediate rehealing behavior of a bulk hydrogel
when subject to syringe injection-induced shear has
been reported by Yan et al.53 This syringe observation also helps to validate the utility of exclusively
rheometer-induced flow for studying shear-thinning
and rehealing behavior of -hairpin peptide-based
hydrogels and other physical hydrogels that show this
behavior. Therefore, a combination of a visual technique such as a confocal microscope and a rheometer
48
helps elucidate the correlation between the structural

changes and corresponding rheological behavior from
physical hydrogels.
The characteristic of -hairpin peptide-based
hydrogels of undergoing plug flow when injected using
a syringe has been studied in depth by Yan et al.
and the confocal-rheometer assembly. The plug-flow
behavior of the hydrogels can confer a specific benefit
to the use of these hydrogels in therapeutic applications like syringe delivery of pharmaceutical actives
and cells encapsulated in the hydrogels. This specific
benefit is the protection of the encapsulates in the
bulk of the hydrogels from syringe-induced shear. The
encapsulates in the hydrogel bulk undergo negligible
stress as only the hydrogel material at the walls of the
syringe undergoes yielding and syringe-induced shear.
As is inferred from the results of the confocal-rheomter
assembly, higher is the magnitude of shear to which
the hydrogels are subjected, larger is the portion of the
hydrogels that yields and flows. This shear-dependent
fracturing of the hydrogels is an important property
that allows injection of these hydrogels in cavities of
different sizes. The gels can re-percolate immediately
into bulk networks at the injection sites, thus being
particularly viable from a therapeutic standpoint.
Rheological characterization of -hairpin hydrogels as discussed in Section Hydrogels from -Hairpin
Peptides Based on the Parent Sequence MAX1 [VK
VKVKVK-(VDPPT)-KVKVKVKV-NH2] and other
assembled physical hydrogels as discussed in
Section Rheological Characterization of Physically
Crosslinked Hydrogels has been carried out using
a bench-top rheometer using parallel plate or cone
and plate geometries. These oscillatory rheological
measurements have been carried out with applied
strain amplitudes within the LVR of the hydrogels and correctly represent the hydrogel behavior.
These results help in understanding parameters such
as hydrogel stiffness and frequency dependence
behavior from the hydrogels. This utility of a pure
rheometer-induced flow can be reinforced by the
use of simultaneous visual investigation of potential
structural changes of hydrogels under flow. The compound confocal-rheometer assembly is an example
of an instrument with such reinforced utility. Efforts
toward obtaining a complete perspective about the
structural as well as mechanical changes exhibited by
various types of soft materials have been the focus
of research well over a decade now. Visual confirmation of flow behavior of polymers solutions, melts,
and networks has been investigated using particle
tracking in a variety of reports.137140 Wang and
colleagues describe the development and implications of the method of particle tracking velocimetry
Rotating bob
Focusing lenses
Glass cell
Laser
Camera
FIGURE 8 | Schematic representation of a particle-tracking

velocimetry apparatus. (Reprinted with permission from Ref 147.
Copyright 2012 American Chemical Society)
(PTV). This technique has been instrumental in direct

visualization of flow behavior in case of rheological
response from a variety of materials such as DNA
solutions,141144 polymeric melts,142,145 and wormlike
micelles146 from surfactants. A basic setup of a PTV
instrument as reported by Skrzeszewska et al.147 is
shown in Figure 8. The principle of the PTV method
is to optically track the fluorescing or diffracting
particles embedded into the sample under study when
illuminated by a laser. This method provides the
visual proof of phenomena such as wall slip and/or
inhomogeneous shear in case of polymer melts, and
this method has been suggested by the authors as an
indispensable part of nonlinear rheological studies of
highly viscoelastic materials.148
In light of the popularity of oscillatory rheometry as a characterization technique for studying
self-assembly pathways, mechanical properties and
flow behavior of physical hydrogels based on peptides and polypeptides, it is important that there is
awareness regarding these protocols and precautions
discussed in Section Rheological Measurements of
Physical Hydrogels: Protocols and Precautions.
Additionally, combination techniques like the
confocal-rheometer assembly and other visualization
techniques such as PTV would help in elucidation
of the flow behavior of various shear-thinning and
rehealing hydrogels under rheometer-induced shear, a
commonly used technique for characterization of such
gels. It could also help in easy determination of wall
slip at the surface of hydrogels being studied using a
bench-top rheometer which is capable to distorting a
rheological measurement.
An overwhelming majority of rheological studies on physical hydrogels as described later in Sections
Hydrogels Based on -Sheet Structures, Hydrogel

Materials Based on Specific Molecular Recognition, Elastin and Related Hydrogels, Gelatin Gels,
Globular Protein Hydrogels, Fibrous Protein Hydrogels, Hydrogels Based on Peptide Amphiphiles, and
Hydrogel Based on Low Molecular Weight Peptide
Gelators of this review are focused on the linear
rheological properties, i.e., the properties within the
LVR. Representation of shear environments during
flow of physical hydrogels (e.g., syringe injection)
cannot be obtained by restriction to oscillatory
rheological measurements only within small strain
amplitude oscillatory conditions. Exposing hydrogels
to steady-state shear such as injection through a
syringe is a good strategy to understand the ability
of the hydrogel to be processed and/or shaped into a
desired morphology. For example, fully formed solid
MAX8 hydrogels injected using a syringe into a cavity
or crevice can easily adopt the shape of the cavity or
crevice. In case of steady-state shear treatment, information about elastic relaxation of hydrogels subject
to shear cannot be acquired. Sometimes, a thorough
understanding of the nonlinear rheological behavior
of these materials is interesting and beneficial to further explore their use as injectable drug/cell delivery
vehicles and load bearing biomaterials. Reviews of
large amplitude oscillatory shear (LAOS) measurements (i.e., oscillatory application of strain outside of
the LVR) and their utility in probing nonlinear (i.e.,
outside the LVR) viscoelastic properties of complex
fluids such as polymer solutions and melts, emulsions,
biological gels, and micellar solutions has been provided by Hyun et al.,90 and Deshpande.91 Steady-state
shear treatment, the non-oscillatory spinning of the
rheometer top plate at a specified rate, consequently
applying strain that is far outside the LVR, is the other
experimental rheological technique used to subject a
hydrogel to shear treatment. While carrying out SAOS
(small amplitude oscillatory stress) measurements like
the ones being reviewed herein, a linear viscoelastic
response is obtained when oscillatory strain amplitude
of a small value is applied to viscoleastic materials
such as a physically crosslinked hydrogel. A linear
viscoelastic response is characterized by independence
of G (storage modulus) and G (loss modulus) to the
applied strain and amplitude at a constant angular frequency, and the resulting stress is sinusoidal in nature.
In the nonlinear region, however, applied strain is of
a large amplitude and the G (storage modulus) and
G (loss modulus) become dependent on the applied
strain amplitude at a constant angular frequency. The
resulting stress waveform deviates from a sinusoidal
form. On the basis of the characteristic deviations of
different types of complex fluids, nonlinear properties
49
Advanced Review
of different materials can be studied using correlations and comparisons to the already known typical
responses. Thus, steady-state and large amplitude
oscillatory shear measurements can be utilized to
study rheological response of physical hydrogels in
practical environments.
Bulk fracture as observed in case of MAX1 or
MAX8 hydrogels is an example of many important
high steady-state shear responses demonstrated by different physically crosslinked systems. The next section
reviews some of the latest studies of bulk fracture
observed in physically crosslinked materials such as
networks from telechelic polymers and crosslinked
emulsions. Following this section, rheological characterization of physically crosslinked peptide- and
protein-based hydrogels is discussed. These hydrogels
can be used as model systems of diverse molecular origins to gather in-depth understanding of relationships
between shear-induced structural changes and rheological behavior.
ACCOUNTS OF FRACTURING IN
PHYSICALLY CROSSLINKED
HYDROGELS
Solids, when subject to stress undergo a finite deformation while fluids under the same stress, flow,
or continuously deform. Viscoelastic materials such
as physical hydrogels, display properties intermediate to both these classes of materials beyond their
respective LVR by showing shear responses such as
macroscopic fracture,6972 strain stiffening,7376 and
shear banding.77 Macroscopic fracture in physically
crosslinked hydrogels is an effect that can interfere
with rheological measurements carried out using conventional geometries. As discussed in this review, a
physical hydrogel based on self-assembled peptides
can undergo macroscopic fracture at a certain distance
along the gel thickness from the shearing rheometer
plate. Thus, only the portion of the gel within the
fractured volume might yield and flow while a certain layer of the gel might be negligibly affected by
shear. Thus, an advanced study of macroscopic fracture behavior of physical hydrogels can enable deeper
understanding of their deformation behavior under
large amplitude shear. Physical hydrogels are excellent
candidates for potential biomaterial applications such
as developing extracellular matrix mimetic materials.
Thus, such studies are a step in the direction of simulating real-life mechanical and morphological environments that might be encountered by these physical
hydrogels used as biomaterials. An interesting perspective about fracture of elastomers and gels has been provided by Shull.70 A comprehensive review focusing on
50
fracture behavior in transient networks has been provided by Ligoure et al.72 A few studies related closely
to the fracture of physical hydrogels are highlighted
below.
Tabuteau et al.69 have reported a study of fracture nucleation in a model viscoelastic fluid and proposed that like real solids, viscoelastic fluids can fail
in a brittle manner because of lowering of the overall strength by the presence of micro-cracks. These
transient viscoelastic networks are telechelic polymers
connecting oil-in-water droplets in a microemulsion.
The telechelic polymers have a hydrophilic Polyethylene oxide (PEO) backbone with a hydrophobic group
at both ends. These end chains stick reversibly into
the hydrophobic core of the oil droplets and either
loop or link the droplets, leading to a self-assembled
structure. Under shear, these links can escape and
rebound to droplets when shear is removed. The shear
stress increases linearly with shear rate up to a critical
stress after which the flow curve shows a sharp discontinuity and the network fractures instantaneously
with cracks opening up all around the sample that
grow rapidly. The fracture is proposed to take place
because of already present micro-cracks in the material. The tensile stress rather than the shear stress
is reported to cause the fracturing of the networks.
The critical stress for rupture is experimentally and
theoretically verified to be on the order of magnitude of the Youngs modulus of the networks. Tixier et al.149 report on interesting new materials that,
topologically, are self-assembled transient networks
comprised of surfactant micelles linked reversibly by
telechelic polymers. Morphologies of the micelles help
define three distinct domains, a domain of isolated
but not entangled micelles, partially entangled and
fully entangled micelles. These domains correspond
to three distinguishable failure modes in the nonlinear rheology: a brittle mode, an intermediate mode,
and a ductile/shear-banding mode, respectively. These
networks serve to combine the properties of wormlike micelles with those of the bridged micro emulsions discussed earlier. Ramos et al.71 report on tunable transient networks made of telechelic PEO based
polymer linked cetylpyridinuim chloride micelles that
align under shear. The authors have proposed that
fluctuations in the degree of micelle alignment can be a
structural probe of the fracture behavior under shear.
The critical shear rate for fracture was reported to be
either very close to the shear rate at the onset of nonlinear behavior (for brittle samples) or significantly
larger than it (for ductile samples).
Berret et al.150 have reported a study of nonlinear rheological behavior of physical networks from
telechelic polymers with a poly(ethylene oxide) middle
block and semiperfluorinated end caps. This is one

of the first reports on shear-induced brittle solid-like
fracture in a physical network. A flow visualization
technique, very similar to the PTV setup as discussed
in Section Accounts of Fracturing in Physically
Crosslinked Hydrogels in this review, was used. The
flow visualization technique indicates the presence of
a clear fracture zone when the networks are subject
to strains above a certain threshold strain. Another
study conducted by Berret et al.151 using very similar
polymers discussed the existence of a discontinuity
in the stress versus shear rate curve due to inhomogeneous flow conditions created by the rupture of
the network. As the steady shear stress for these networks was studied in response to the shear rate, two
stress regimes stood out with increasing shear rate
separated by a discontinuity. The first regime is shear
thickening while the second is shear-thinning. The
shear-thickening regime was reported to display an
enhancement of the elastic modulus of the networks.
This discontinuous behavior was attributed to rupture or fracture inside the networks. An example of
hydrogels that fracture under shear and reheal upon
removal of shear has been discussed by Skrzeszewska
et al.147 They report the study of macroscopic fracture of physical networks formed by collagen-inspired
telechelic polypeptides under shear by particle image
velocimetry (PIV), which is principally similar to
the PTV technique discussed in Section Accounts
of Fracturing in Physically Crosslinked Hydrogels.
The stress and strain at which failure occurs depend
on the applied shear rate. The width of the fracture
zone (zone in which almost all the physical crosslink
points are ruptured) as studied by PIV was reported
to be much greater than the distance between two
crosslinks and increased with magnitude of applied
shear. Detailed study by the authors of the dependence of the rupturing of the gel at constant stress and
different shear rates has also been reported.
In the next section, we will review recent work
that reports basic rheological characterization on
physically crosslinked hydrogels. The oscillatory rheometric measurements carried out in a majority of
the studies reported herein are similar to the measurements described in Section Rheological Measurements of Physical Hydrogels: Protocols and Precautions, e.g., oscillatory time sweep, frequency sweep,
and strain sweep measurements. In all these accounts
of physically crosslinked hydrogels from various peptides and proteins, rheological characterization was
carried out by conducting time sweep, oscillatory
strain, frequency sweep measurements, or any combination of these measurements, with the applied
shear stress values and resultant strains within the
linear viscoelastic regions of these hydrogels. The

shear-thinning and rehealing behavior of some of the
hydrogels has been studied by applying steady shear,
or oscillatory shear at large strains outside of the
LVR using the rheometer upper plate as described in
Section Rheological Measurements of Physical Hydrogels: Protocols and Precautions.
RHEOLOGICAL CHARACTERIZATION
OF PHYSICALLY CROSSLINKED
HYDROGELS
This section describes rheological characterization of
physically crosslinked hydrogels based on peptide
-sheet structures, proteins, and peptides undergoing
specific molecular interactions, elastin, gelatin, globular proteins, fibrous proteins, peptide amphiphiles
(PAs), and low molecular weight peptide gelators. An
overwhelming majority of measurements carried out
on samples of various hydrogels from these materials have been conducted using a bench-top rheometer using either a parallel plate or cone and plate
geometry. Many of these measurements can be carried out with greater reliability and reproducibility
when precautions and protocols for measurements
as described in Section Rheological Measurements of
Physical Hydrogels: Protocols and Precautions are
observed. These precautions include evaluation of
wall slip potential of hydrogel sample in contact with
geometry, adjustment of appropriate gap height and
study of different gap heights for conducting measurements and an appropriate choice of geometry and
material of fabricating the geometry depending upon
the hydrogel material under study.
Hydrogels Based on -Sheet Structures
Aulisia et al.44 have reported on shear-thinning and

rehealing hydrogels based on multi-domain peptides;
(a) K2 (QL)6 K2 , (b) E(QL)6 E, (c) K2 (SL)6 K2 , (d)
E(SL)6 E, and (e) E(CLSL)3 E. These multi-domain peptides have an ABA structure with the middle B domain
containing alternating hydrophilic and hydrophobic
amino acids. The A block at the end contains charged
amino acids that at neutral pH counters the tendency
of the central block to associate. These peptides are
shown to form nanofibers that, in turn, form viscoelastic hydrogels. These gels display varying abilities of
rehealing on removal of shear treatment. Upon formation, the charged nanofibers are crosslinked ionically
with either phosphate or magnesium ions. Cryogenic
transmission electron microscopy was used to confirm the fibrillar identity of the self-assembled structures. Fibers formed by peptides containing serine as
51
Advanced Review
compared to glutamine in their central blocks were

reported to form much longer and more entangled
fibers leading to stiffer hydrogels. The nanofibers from
peptides containing serines were reported to show a
much larger rise in storage modulus when crosslinked
with an oppositely charged crosslinker (Mg+2 ions) as
compared to those containing glutamines crosslinked
with phosphate ions. The hydrogels from serine
containing peptides were shown to demonstrate an
increased propensity to reheal after being subjected
to shear treatment by flowing through a narrow
bore syringe. When the nanofibers containing cystine residues were oxidized using hydrogen peroxide,
forming covalent disulphide bonds along or normal
to the fiber axis, the stiffness of the formed hydrogels was dramatically increased (60). This increase
was reported to be larger than that obtained when the
nanofibers were crosslinked with Mg+2 ions. As suggested by the authors, this approach shows the versatility of the multi-domain peptide hydrogel mechanical
properties as a function of the peptide sequence.
Further work by Bakota et al.152 employed
extensive rheological strain sweep and time sweep
measurements to demonstrate the enzymatic
crosslinking of nanofibers from multi-domain peptides. This phenomenon of exogenous enzymatic
oxidative crosslinking has been suggested as a facile
strategy for tuning nanofibrillar hydrogel stiffness
from multi-domain peptides. Multi-domain peptides
containing the cell attaching peptide sequence RGD
have also been reported by the same group to form
mechanically stable nanofibrous, shear-thinning,
and rehealing hydrogels, which makes their syringe
delivery possible.26
Bowerman et al.153 have studied the relative
importance of hydrophobicity of the non-aromatic
versus aromatic amino acids in the (XEXE)2 peptides
during amyloid-like self-assembly into helical fibrils.
The authors have correlated the hydrogelation properties of a set of peptides, inferred from the rheological
behavior of their hydrogels, to the hydrophobic character of the individual amino acids (alanine, valine,
leucine, phenylalanine, and cyclohexylalanine) in the
X position of (XEXE)2 . The rheological measurements provide a pivotal piece of information that
indicates an increase in the generic hydrophobicity,
and not aromaticity, contributes to the non-covalent
supramolecular cross-linking of the fibrils. TEM
has played an important role in determining morphology of the fibrils from different peptides. Some
peptides in this series form stable left-handed helices
or uniformly thick fibrils while some form transient
helices that undergo morphological changes into
mature fibrils. TEM provides structural information
52
complementary to rheological characterization to

understand self-assembly processes of these peptides.
The Nilsson group also reports the rheological characterization of -sheet peptide hydrogels based on
a reductive trigger which induces a conformational
change from a peptide macrocycle to a -sheet.154
Ramachandran et al.66,155 report on co-assembly
of amphiphilic peptides into hydrogels with potential
biomedical applications.43,153,155 The authors discuss
peptides KAW10, KAW15, EAW10, and EAW15. Peptide molecules having the same charged residue (lysine
or glutamic acid) do not undergo self-assembly with
each other but do so when blended with molecules
containing an opposite charge (glutamic acid or lysine,
correspondingly). The assembly strategy discussed by
the authors involved making hydrogels by blending
pairs of peptides from the four peptides generating
four pairs, two with sticky ends (with unmatched
lengths of amino acids in peptide pair), and two with
blunt ends (with exactly matched lengths of amino
acids in peptide pair). All four pairs were reported
to undergo co-assembly with very different kinetics
but form shear-thinning and rehealing fibrillar hydrogels, as confirmed by TEM. The KAW:EAW blunt-end
pair was found to form the most stable hydrogels
post-cessation of shear, negating the importance of the
sticky ends for stiffer network formation.
Guilbaud et al.156 have reported on the correlation between enzyme concentration and hydrogel
morphology and properties of enzymatically triggered
-sheet forming tetrapeptide hydrogels. They have
shown formation of heterogeneous hydrogel morphology due to denser hydrogel domains in the vicinity of the enzyme. These denser domains act as a
reinforcement phase for the hydrogels as determined
by rheometeric experiments. Peptide hydrogels based
on mussel inspired iron crosslinking and formation
of shear-thinning and rehealing hydrogels have been
reported by Ceylan et al.157
Hydrogel Materials Based on Specific

Molecular Recognition
Specific interactions between peptide, protein, and
polymer molecules have been used widely for designing smart responsive materials. This section discusses
the use of rheological characterization to probe
various assembly parameters such as optimum gelation concentration, temperature, pH, and solution
ionic strength and their effects on bulk hydrogel
mechanical properties formed as a result of specific
molecular recognition interactions in peptides and
proteins. The Tirrell group put forward one of the first
accounts of materials based on specific interactions,
that of coiled-coil formation.158 These materials are

multi-domain proteins with a water-soluble polyelectrolyte block flanked on both ends by leucine zipper
domains. The zipper domains formed coiled-coil
dimers because of interhelical hydrophobic intreractions. These dimers act as crosslink junctions
that are shown to help in formation of transient
hydrogels.158 The Kopecek group and other groups
have used the strategy of incorporating leucine zipper domains.159163 Susceptibility of these leucine
zipper domains to denaturation at high pH or high
temperature leads to reversal of these gels into solutions. Plateau moduli of the hydrogels formed as
a function of peptide concentration, pH, and ionic
strength were studied using rheological measurements and were attributed to network topology.164
Another study from the same group shows that the
pH-dependent viscosity transition of such networks
can be attributed to the dynamic properties of the
networks.165 Further, disulfide covalent bonding due
to cysteine incorporation in the leucine zipper domain
has been reported to show enhancement of hydrogel
rheological properties.166,167 Xu and Kopecek have
presented coiled-coil-containing block copolypeptides
in which the gelation process was monitored by
microrheology.161 The authors report a reversible
solgel transition observed on addition or removal of
guanidine hydrochloride that denatures the coiled-coil
domains. Hybrid hydrogels based on -sheet peptides
grafted onto HPMA copolymers specifically for the
purpose of nucleation of hydroxyapetite, ultimately
intended for bone regeneration, have been reported
by Wu et al.168 The bulk mechanical properties of
the hybrid gels probed by rheological measurements
were shown to be dependent on the peptide concentration and incubation time. Woolfson and colleagues
discuss development of hybrid peptides by incorporation of -helix and -hairpin characteristics into
one peptide sequence that could transform from a
-helix into a -hairpin upon heating. The ultimately
fibrous gel could be reversed into a solution at lower
pH values.169 The same group has reported on a
-helical, peptide-based, fibrillar hydrogel based on
coiled-coil interactions with the exception of the
amino acids at certain positions on the coiled-coil
surfaces replaced with three alanine residues for
enhanced intermolecular interaction. Mixing of complementary peptides under chilled conditions or room
temperature was reported to form self-supporting,
thermally stable physical gels.170,171 Deming and
colleagues172174 have used bulk rheological measurements to study the extent of hydrogel formation
from polypeptide diblock copolymers comprised of
hydrophilic polyelectrolyte charged segments and a
corresponding hydrophobic segment. The charged

polyelectrolyte block was shown to determine the
solgel transition. The final gel modulus was dependent on the hydrophilic to hydrophobic ratio and
block length. Two factors, the conformational differences in the hydrophobic segment in the diblock
and solution ionic strength, were shown to affect
the critical gelling concentration. These hydrogels
demonstrated shear-thinning and rehealing properties
(8090% of their pre-shear rigidity value after a
large-amplitude oscillatory shear of 1000% strain).
Grove at al.92,93 have described the fabrication of
ionic-strength-responsive hydrogels by using modular,
bottom-up protein design based on the 34 amino
acid tetratricopeptide (TPR) repeat. Specifically,
non-covalent interactions between repeat protein
modules and their partner peptide Polyethylene Glycol (PEG) ligands act as crosslink junctions for these
hydrogels. Both bulk rheology and microrheology
have been used to study the kinetics and viscoelastic
properties of these hydrogels. The authors discuss
the potential of hydrogels based on these modular
protein designs for tissue engineering applications.
Foo et al.65 have reported a similar simple mixing
strategy to induce a solgel phase transition without
any extensive changes in environmental conditions
by using molecular recognition interactions between
proline-rich and di-tryptophan (WW)-rich domains in
engineered protein materials. The transient physical
cross-link junctions in such gels yield relatively weak
(50 Pa) gels (as studied by microrheology), but
provide them a shear-thinning and rehealing nature.
Lu et al.18 report on a Dock and Lock physically
crosslinked hydrogel in which the crosslink junctions
are the interactions between the Docking and Dimerization Domain (DDD) of cAMP-dependent protein
kinase A (PKA) and the anchoring domain (AD) of
A-kinase anchoring proteins (AKAP). These hydrogels
were found to be relatively stiff (1000 Pa) as compared to similar protein-based molecular recognition
gels and to demonstrate shear-thinning and much
faster rehealing properties due to inherently expedited
kinetics of the DDD and AD binding. Extensive
oscillatory time, frequency, and strain sweep measurements were carried out to characterize these
gels rheologically. Liu et al.175 reported on a physically crosslinked hydrogel based on thiol-containing
polypeptide macromer assembled via coiled-coil
interactions. These coiled-coil interactions act as
points of physical crosslinking, imparting a physically
crosslinked nature to the hydrogel network formed
from these macromers. These polypeptide macromers
are covalently bound to vinyl sulfone-containing PEG
molecules through a biologically benign Michael type
53
Advanced Review
addition reaction. These unique gels were also shown

to be benign toward epithelial cells encapsulated
within them. Oscillatory bulk rheology was used to
characterize the mechanical properties of these gels.
Elastin and Related Hydrogels

Elastin is a natural protein found abundantly in
elastic tissues in the human body. Elasticity and the
mechanical reversible character of elastin allow tissues
such as arteries, skin, and vocal folds to extend upon
stress addition and recover upon stress removal. The
elastomeric properties of this protein provide major
motivation for the design of mimetic molecules,
elastin-like polypeptides (ELPs).176 The pentapeptide
repeat unit valine-proline-glycine-X-glycine (where X
is any residue but proline) is the signature sequence
of elastin. The critical temperature of inverse phase
transition, above which ELP solubility decreases and
ELP hydrogelation occurs, can be varied by changing
the X amino acid.177,178 Thermal reversible gelling
hydrogels for cartilage repair were developed using
the thermal reversibility of the elastin molecule.179,180
Cytocompatible ELP coacervates, which mechanically do not measure up to natural connective tissue,
have also been reported.179182 A step up from
these coacervates, crosslinked ELP hydrogels that
displayed enhanced mechanical properties over ELP
coacervates have been reported.182186 ELP hydrogels
with improved mechanical properties, developed by
controlling the phase separation characteristics in
ELP solutions, have also been reported.183,184,187189
These efforts demonstrate control of rheological properties by variation in polypeptide sequence and ionic
strength. Hybrid ELPs obtained by incorporating silk
residue repeat sequences into ELP sequences for drug
and DNA delivery have also been reported.190,191
These proteins, with temperature-controlled kinetics
of gelation, have been shown to be used for in situ
gelation application after syringe injection.192,193 The
rheological characterization of cysteine-containing
elastin-like proteins, which undergo thermal as well as
oxidative gelation, has been reported by the Chilkoti
group.194,195
Gelatin Gels
Gelatin gels find utility in biomedical applications
such as protein delivery due to biocompatibility
and temperature sensitive gelation.196,197 Gelatin
is produced by denaturation of naturally derived
collagen in solution through an acidic or alkaline
process that leads to separation of the triple-helical
tropocollagen into three single-stranded gelatin
molecules. These single-stranded molecules undergo
54
a coilhelix transformation when cooled in aqueous solutions.198,199 Above a critical protein

concentration, thermoreversible gelation takes place
from helix association.198200 Dependence of gel
modulus and gel melting temperature on gelatin concentration and molecular weight, both experimentally
and theoretically, has been reported by Gilsenan
and Ross-Murphy201,202 Creep and creep recovery
measurements used to observe long-term viscoelastic
behavior of gelatin gels were also reported by the same
authors.203 Comparison of creep compliance with the
inverse of the final gel modulus, which provided
potential proof of elastic solid-like behavior of gelatin
gels, has also been reported. According to the authors,
an observed non-recovery of the deformation at the
end of the recovery phase provides an indication of
viscoelastic behavior of gelatin gels.
Globular Protein Hydrogels

Globular proteins is another class of proteins that
demonstrate gel-forming capabilities. Gels from
globular proteins are not only relevant industrially
for commercial applications in foods, but have also
shown great promise in emerging areas such as tissue engineering. Lysozome-based gels204206 are a
good, recent example. Unfolding of globular proteins at higher temperatures allows aggregation of
individual protein molecules above a certain critical
gelling concentration. This aggregation has been
attributed to secondary interactions such as hydrogen bonding and hydrophobic effects. Depending
on whether solution pH of the proteins is closer or
further from the pI, globular proteins can undergo
aggregation to form particulate or fine stranded
hydrogels. Extensive rheological studies focused on
gels from globular proteins such as bovine serum
albumin, -lactoglobulin, and ovalbumin, and the
correlation of their rheological properties with their
structural and molecular characteristics, have been
reported in the literature200,206212 Theoretical modeling has also been reported for better understanding
of the gelation mechanisms. These models attempt to
explain the dependence of gelation time, critical gelation concentration, and equilibrium gel modulus on
variables such as protein concentration, pH, temperature, and ionic strength. In a series of publications,
Ross-Murphy and colleagues190,210214 have reported
on several models to describe the gelling behavior of
fibrillar -lactoglobulin gels formed at both acidic and
basic pH. Van der Linden and colleagues215218 have
described an adjusted random contact model that
correlates the critical percolation concentration and
ionic strength for fine-stranded gels. Studies involving
developments of models that use parameters obtained
from data on the aggregation process, hydrogel rheology, and complementary techniques such as light
scattering have also been reported.207,219223 Miller
et al. have reported on thermoreversible lysozyme
gels formed in mixtures of water and dithiothreitol
as solvents. The lysozyme gelation was reported
to be achieved by heating the protein solution up
to 85 C and then slowly cooling back to room
temperature, denaturing the lysozyme proteins and
forming entangled -sheet-rich fibrils forming a gel
network.204,205 Dynamic oscillatory measurements
were reported to probe the gelation behavior and
mechanical properties.224 The plateau elastic modulus
of the gels was reported to be dependent on lysozyme
concentration in a power law relation.
Rheological characterization of whey protein
gels has been reported by Eissa et al. Heating acidic
whey protein solutions (pH < 4.6) produces weak
and brittle gels. Eissa et al.225 have described an
enzyme-catalyzed two-step process of producing protein gels with substantially higher elastic moduli and
higher yield-stress and strain values. The enzyme treatment was reported to be carried out under alkaline conditions. In these studies, oscillatory rheological characterization was utilized as a critical method
in analyzing materials with significant commercial
potential in the food industry. The enzyme (transglutaminase) treatment allowed for crosslinking of the
proteins. The same authors have also described an
improvement on the above-described method226 by
using neutral solution conditions instead of alkaline
solution conditions for the enzymatic crosslinking of
why proteins. In this report, in addition to stiffness
values of the gels, rheological characterization was
also used to study fractal dimensions of the gels, which
were compared to the fractal dimensions on the same
samples studied using confocal microscopy. As an
alternative to forming whey protein gels at pH < 4.5
which leads to brittle and weak gels, whey protein
gels with strong elastic and textural properties were
reported by Vardhanabhuti et al.227 These gels were
formed at higher pH (7.5) and equilibrated at different acid solutions. The elastic properties of the gels
were reported to be affected by the acid type, acid concentration, equilibration time, and equilibrating solution pH.
Fibrous Protein Hydrogels

Silks form a class of highly studied fibrous proteins
that can form fibers with mechanical properties
(Youngs modulus and tensile strength228,229 ) unsurpassed by almost any other polymer of biological
or synthetic origin. The biocompatible and degradable silk proteins230232 serve as motivation for
various studies to explore silk gels as tissue engineering scaffolds233235 and drug delivery agents when
blended with gelatin.236,237 Silk fibroin is mainly
found in protein from natural silkworm fibers.238
Above a critical concentration silk fibroin assembles in solution leading to a physical hydrogel of
-sheet-rich fibrils. Cytocompatibility and biodegradability of silk fibroin have been established by various
accounts in the literature.233,235,239243 These fibers
are distinct from self-assembled fibrillar nanostructures from -sheet peptides in terms of dimensions
of fibrillar structures formed. The hydrogels formed
from silk proteins are composed of micron-scale
macromolecular clusters of -sheets244 as compared to the well-defined nanosized -sheets fibrillar
structures from synthetic amphiphilic peptides as
discussed in Sections Hydrogels from -Hairpin
Peptides Based on the Parent Sequence MAX1
[VKVKVKVK-(VDPPT)-KVKVKVKV-NH2]
and
Hydrogels Based on Peptide Amphiphiles.
Kang et al.158 and Yoo et al.252 report that the
stability and rigidity of silk fibroin hydrogel were
affected by the amount of an added polymer, poloxamer 407. Wang et al245,246 has reported on expediting gelation kinetics of silk fibroin under physiological conditions facilitating homogeneous 3D encapsulation of living mesenchymal stem cells. Yucel et al.244
report mechanical stimuli (vortexing) as a means of
expediting gelation kinetics of fibroin protein solution.
Moreover, these mechanically stimulated silk hydrogels were also shown to be capable of shear-thinning
by injection and rehealing to pre-shear rigidity immediately after shear cessation. Vollrath et al.247 discuss
pH-dependent gelation of spidroin proteins at lower
pH (5.5) and reversal to solution at pH 7. Fibrillar
networks from synthetic spidroins stabilized by chemical or physical crosslinks have also been reported. The
physical spidroin hydrogels were reported to be easily disrupted because of the purely topological fibrillar
entanglement acting as crosslinks, in contrast to much
stiffer chemically crosslinked gels with elastic moduli
up to around 1000 Pa.248250
Fibrin is another natural, fibrous protein with
potential biomaterial applications for wound healing and growth factor delivery.251 In the event of an
injury fibrinogen is covalently crosslinked by thrombin, thus forming fibrin. When further crosslinked by
factor XIII, fibrin forms a clot and forms a blood
coagulating network.252 Fukada and Kaibara provide early reports on various techniques to study
dynamic rheological properties of fibrin clots along
with mechanism of blood coagulation.253256 Ryan
et al.257,258 have reported a structureproperty relationship study relating rheological behavior of fibrin
55
Advanced Review
clots to their structural characteristics. They suggest a

direct dependence of mechanical rigidity of fibrin clots
on the concentration of thrombin, calcium, and fibrinogen and network cross-link densities.
Hydrogels Based on Peptide Amphiphiles

PAs are an interesting family of functionally diverse
molecules whose assembly properties have been
extensively studied both fundamentally and with
respect to biomedical applications.39,100,259268 Some
applications targeted for PAs are biomineralization, scaffolding for therapeutic delivery and tissue
regeneration.259261,269 Some of the first studies of
PA self-assembly were put forward by Hartgerink
et al.259,269 A PA molecule is a hydrophilic peptide sequence covalently bonded to a hydrophobic
aliphatic segment. PAs are particularly suited to
tissue repair and the controlled therapeutic release
as they offer flexibility of design. For example, a
biological ligand can be used as a head group of the
peptide sequence of an individual PA molecule for
biofunctionalization.39 Additionally, kinetics of PA
self-assembly and the assembled nanostructure can
be controlled by controlling the individual amino
acid sequences in the peptide sequence.268 Molecular amphiphilicity leads PAs to self-assemble into
nanofibers39,259,269 or nanobelts268 and further into
fibrillar networks above certain concentrations. Stupp
and colleagues demonstrated that the solgel transition was triggered upon mixing acidic and basic
PAs at physiological pH.262 With the cell-binding
ligand IKVAV as the head group, a PA solution
mixed with neural cells could form a solid hydrogel
when injected in vivo.39 The authors also discussed
the gelation behavior and viscoelastic properties of
PA-based gels as studied via dynamic oscillatory
measurements. Stendahl et al.100 performed a detailed
investigation on effects of pH, ionic strength, and
type of metal ions on mechanical stiffness of PA
gels. Mechanical stiffness of PA-based hydrogels as
a function of increasing calcium concentration in
the solution environment has been reported by Jun
et al.270 The authors have attributed the decrease
of storage modulus after a threshold value of calcium concentration to possible phase separation
that occurs within the gel. In a separate study, it
was found that stability and mechanical strength of
PA-based hydrogels were dependent on the number
and position of glycine residue that can hydrogen
bond.44 Pashuck et al.271 have reported the control
of mechanical properties of nanofibrillar PA hydrogels based on number and positions of hydrophobic
amino acid residues in the PAs. Greenfield et al.272
56
have reported tuning rheological properties such

as stiffness and strain response of self-assembled
fibrillar PA networks by changing the types of interfibrillar interactions such as hydrogen bonding and
ionic bridges. Toksoz et al.273 report the use of
oscillatory rheological measurements to probe the
self-assembly and gelation properties of PA molecules,
specifically upon isolation and neutralization of
ionic charges on the molecules. Charge neutralization was reported by the use of counter-ions like
calcium and also macromolecules such as heparin
and DNA.
Hydrogels Based on Low Molecular Weight

Peptidic Gelators
In addition to peptides and polypeptides from amino
acids, oligopeptides of low molecular weight, such
as fluorenylmethoxycarbonyl (Fmoc)-protected amino
acids, can self-assemble into supramolecular hydrogels that can be studied using oscillatory rheology measurements. Biological studies have demonstrated potential biomedical applications for these
hydrogels such as tissue regeneration274276 and drug
delivery.277279 These short peptides form hydrogels when using pH and temperature as a gelation
trigger275,277,280282 or in the presence of a natural
enzyme.97,277,279,283286
The Ulijn group has reported hydrogelation
of Fmoc-functionalized amino acids initiated under
physiological conditions. Fmocdiphenylalanine
(FmocF2 ) molecules were reported to undergo
self-assembly into fibrous hydrogels that support 3D
cell culture of living cells.274276 Functionalization
of the network with different chemical moieties has
been explored as an approach to tune mechanical
rigidity of FmocF2 gels. The resulting gel networks
were shown to display optimized compatibility with
various cells.275 Xu and coworkers have presented a
series of Fmocdipeptides, which show a reversible
solgel transition in response to shift in temperature and pH.281,282 In addition, the same group
reported hydrogelation in response to the binding
of vancomycin where gel stiffness was observed to
increase significantly.287 Mahler et al.284 have demonstrated solgel transition from FmocF2 solution in
water at an appropriate concentration, leading to
shear-thinning FmocF2 gels. Adams et al.283 have
reported the gelation of Fmocdipeptides induced by
hydrolysis of glucono--lactone. The resulting hydrogels were homogeneous with reproducible mechanical
properties irrespective of the pre-shear history experienced by the hydrogels. The Adams group has
also reported self-assembly of different dipeptides
triggered by various stimuli such as temperature and

solvent composition68 and salts.67 They have also
provided a report of rheological characterization of
dipeptide-based hydrogels showing differences in
gel properties with different characteristics such as
overall hydrophobicity.288 Several groups have also
studied enzyme-induced gelation of low molecular
weight peptides. Specifically, the Ulijn group has
discussed the hydrogelation of FmoctyrosineOH
triggered under physiological conditions by the presence of alkaline phosphatase. The concentration
of alkaline phosphatase was crucial in determining the gelation kinetics and stiffness of hydrogels
formed.285 Xu and coworkers discuss hydrogelation
of Fmoctyrosine at 37 C at pH 6.0 and pH 9.6281
induced by alkaline phosphate. The final gels were
reversed to a solution with kinase.282 Other enzymes
such as thermolysin,97 -lactamase,289 and MMP-9290
have been used to trigger hydrogelation of various
hydrogelators.
CONCLUSION
Physically crosslinked hydrogels demonstrate significant potential as biomaterials for drug/cell/growth
factor delivery agents and tissue engineering
scaffolds.291 Such hydrogels exhibit a rich diversity in hydrogelation stimuli such as pH, temperature,
ionic strength, enzymes, and metal ions. In particular,
preformed, solid hydrogels, such as the -hairpin
peptide-based hydrogels, can be injected as a solid
hydrogel using a syringe to a desired site in the body
and reheal into solid hydrogels immediately after
injection. These hydrogels, along with some other
similar physical hydrogels, demonstrate a plug-flow
pattern when injected using a syringe that results
from the yielding of a specific layer of hydrogel near
the syringe wall while the bulk of the hydrogel experiences minimal shear. This plug-flow behavior of
the hydrogels offers a particular advantage toward
syringe delivery of cells encapsulated in the hydrogels
by subjecting them to minimal syringe-induced shear.
When subject to steady-state shear using a bench-top
rheometer upper plate, these hydrogels undergo
bulk fracture at a specific distance away from the
shearing upper plate depending on the amplitude of
applied shear. The complex flow patterns observed
in these yield-stress hydrogels under shear, when
studied with various rheological geometries, present
challenges toward physical characterization. Various
other physically crosslinked hydrogels that inherently
behave as yield-stress, shear-thinning materials intuitively can be expected to demonstrate such complex
flow patterns when characterized using an everyday
bench-top rheometer assembly. A large number of

physically crosslinked hydrogel systems demonstrate
the phenomenon of bulk fracture as described in the
review. Thus, this tutorial review attempts to bring
forth a helpful set of precautions and protocols that
warrant careful consideration during rheological
characterization of physically crosslinked yield-stress
hydrogels. A large variety of physically crosslinked
hydrogels based on peptide, protein, PA, and small
molecule peptide hydrogelator systems clearly reinforce the tremendous biomedical potential of such
hydrogels.
EXPERIMENTAL
Confocal-Rheometer Compound Assembly
The assembly is constructed by mounting an MCR
301 Anton Paar rheometer unto a Leica TCS SP5
high-speed confocal microscope. The base plate of the
rheometer is replaced by a homemade cup-like base
that has an aperture to allow light from the objective to reach the sample. A glass coverslip is placed
over the aperture of the cup-like base. In order to
reduce vibrations from the rheometer to the microscope, a plastic brace is used to couple both devices
through the cup-like base. In this configuration, both
devices operate independently of each other. Details
of the device are described by Dutta et al.136 Specifically for this measurement, both the surface of the
rheometer geometry which acts as the upper plate and
the cover glass which functions as the lower plate
in the rheological measurement have been covered
with pieces of 600 grit sand paper (Mc-Master Carr,
Elmhurst, IL, USA) to ensure as much elimination of
wall slip effects as possible. The confocal microscope
is allowed to image the in situ sample between the
rheometer plates via an opening created by cutting out
a little portion of the sandpaper, thus uncovering the
aperture.
Analysis of Video Data Acquired from

Confocal-Rheometer Compound Assembly
Video data obtained using the confocal microscope
are included as Supporting Information. These data
show a sheared MAX8 hydrogel sample at various
steady-state shear rates while it is being viewed from
the bottom of the hydrogel sample looking upwards
at the rheometer upper plate as shown in Figure 7.
Specifically, each video shows images of multiple
layers of the hydrogels at increasing heights within
the sample away from the rheometer lower plate.
These increasing heights are indicated by an increasing z value in the videos. The constant x and y
57
Advanced Review
values that indicate the position of the layers relative to the lower plate remain the same. The thickness of the hydrogel undergoing shear rate-dependent
fracture away from the upper plate of the rheometer geometry is obtained by recording the z values
from the videos at which the encapsulated microparticles start demonstrating motion. The z values are
then converted to thickness of hydrogel layer in which
negligible microparticle motion takes place by multiplying by the factor (2.37) fixed during the confocal
microscopy measurement. The thickness of the fractured layer is obtained by subtracting the thickness
of the stationary microparticle layer from the total
hydrogel thickness. The total thickness of the hydrogel scanned (m), thickness of fractured layers (m),
the corresponding rates of steady-shear (second1 ),
and the thickness of the fractured layers as a percentage of the total hydrogel thickness scanned have
been reported in Table 1. The video data obtained
on the MAX8 hydrogel at the different steady-state
shear rates (5/second, 50/second, 250/second, 400/second, 1000/second) show fractured layers of different thicknesses corresponding to the shear rates. The
range of the z variable for the shear rate 5/second
is 0 < z < 414, corresponding to a minimum thickness
of 0 m and maximum thickness scanned 980 m.
For the shear rates of 50/second, 250/second, 400/second, and 1000/second, the range of the z variable is
0 < z < 333, corresponding to a minimum thickness
of 0 m and maximum thickness scanned 790 m.
Under steady-state shear rate of 5/second, the majority of the encapsulated microparticles, i.e., the bulk
of the MAX8 hydrogel, undergo zero or negligible
flow with only 20 m, or 2.5% of the gel, experiencing fracture and flow. When the same hydrogel is
subject to a steady-state shear rate of 50/second, the
layer of microparticles undergoing flow stays approximately the same at an average of 25 m (z = 325/333).
When the rate of steady-state shear is increased to
250/second, the fracture between stationary and flowing gel next to the upper plate occurs at an average thickness of 307 m (z = 125/333) from the upper
plate. At a steady-state shear rate of 400/second, the
microparticles appear to be in motion at a z value
of 75/333, corresponding to an average thickness of
fractured MAX8 hydrogel 625 m. At a steady-state
shear rate of 1000/second, which is the most widely
used shear rate to apply rheometer-induced shear
in the literature, the microparticles appear to be in
motion at a z value of 53/333, corresponding to
58
an average thickness of fractured MAX8 hydrogel

650 m.
Sample Preparation
The zero gap height calibration for the rheometer
was performed. A 2-mg aqueous solution of MAX8
peptide in 100 L of HEPES (50 mM) is prepared
leading to a 2% (w/v) solution. Fluorescent carboxylfunctionalized polystyrene microspheres (0.50.99 m
in diameter) from Bangs Laboratories (Fishers, IN,
USA) are dispersed in DMEM (Dulbeccos Modified
Eagle Medium, with 25 mM HEPES) An equal volume
of the DMEM is added to the HEPES peptide solution
to give a buffered solution of MAX8. The solution is
quickly transferred to the lower plate of the assembly,
which is the microscopy cover glass. A 25-mm parallel
plate tool is slowly lowered at a speed of 5 m/second
to a measuring gap H = 680 m. The solvent trap is
put in place to prevent drying of the sample. A 10
objective is used on the microscope. The objective is
positioned so that it is at a distance of r/2 of the
radius of the tool. A 488 nm laser is used for imaging.
Using the rheometer a steady-state shear of various
shear rates 5/second, 50/second, and 100/second is
applied to the hydrogel. At the onset of shear, 3D
images of the gel using the confocal microscope are
acquired.
Cryogenic Transmission Electron

Microscopy
Cryo-TEM was performed on a Tecnai G2-12 Twin
Transmission Electron Microscope (FEI Inc., Hillsboro, OR, USA) operating at voltage of 120 kV
located in the W.M.Keck Electron Microscopy Facility
at the University of Delaware. For sample preparation, a copper TEM grid, 300 mesh pre-coated with
lacey carbon film (Ted Pella, Redding, CA, USA). A
droplet of 2 L solution was placed onto the copper
grid loaded in an FEI Vitrobot. Each grid was blotted twice in order to obtain suitable specimen thickness for imaging. The blotting time was set as 2 seconds. The sample was quickly plunged into liquefied
ethane (90 K) cooled by a reservoir of liquid nitrogen to ensure the vitrification of water. The vitrified
samples were transferred to a single tilt cryo transfer holder in a cryo transfer stage immersed in liquid nitrogen. During the imaging, the cryo-holder was
kept below 170 C to prevent sublimation of vitreous
solvent.
ACKNOWLEDGMENTS
This manuscript was supported by the Center for Neutron Science at UD under cooperative agreement
70NANB12H239 from NIST, U.S. Department of Commerce. The statements, findings, conclusions, and
recommendations are those of the author(s) and do not necessarily reflect the view of NIST or the U.S.
Department of Commerce. Armstrong Mbi and Daniel L. Blair wish to thank the NSF for support through
Grant DMR-0847490 and the PRF through grant 49778-DNI7.
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Rheology of peptide- and

How to cite this article:

WIREs Nanomed Nanobiotechnol 2015, 7:3468. doi: 10.1002/wnan.1299

of Physics, Georgetown University, Washington, DC, USA

Conflict of interest: The authors have declared no conflicts of interest

Additional Supporting Information may be found in the online

2014 Wiley Periodicals, Inc.

Volume 7, January/February 2015

WIREs Nanomedicine and Nanobiotechnology

Rheology of peptide- and protein-based physical hydrogels

ydrogel is a general term used for water swollen

broad classification of hydrogels based on the type of

after injection are of particular interest in biomedical

2014 Wiley Periodicals, Inc.

studies can provide insight about gelation kinetics,

layer depends upon the amplitude of applied shear.

2014 Wiley Periodicals, Inc.

Volume 7, January/February 2015

WIREs Nanomedicine and Nanobiotechnology

Rheology of peptide- and protein-based physical hydrogels

Volume 7, January/February 2015

which these measurements should be carried out

FIGURE 1 | Oscillatory frequency sweep measurement of a 0.5%

Measurement of the moduli as a function of

2014 Wiley Periodicals, Inc.

are instrumental in characterizing large, rapid material

stiffness.40,46,98100 For these systems, the faster the

2014 Wiley Periodicals, Inc.

Volume 7, January/February 2015

WIREs Nanomedicine and Nanobiotechnology

Rheology of peptide- and protein-based physical hydrogels

FIGURE 2 | Dynamic Time Sweep (1% strain, 6 rad/second) of 2 wt

% MAX1 [(VK)4 -VD PPT-(KV)4 ] solution with 20 mM (G , lled circles),

as discussed by Discher et al.105 A seminal report

trapping of sample particles between the apex and the

2014 Wiley Periodicals, Inc.

lead to wall slippage and distort the measurement.111

BIOMEDICALLY PROMISING SOLID

2014 Wiley Periodicals, Inc.

Volume 7, January/February 2015

WIREs Nanomedicine and Nanobiotechnology

Rheology of peptide- and protein-based physical hydrogels

conformation in aqueous solution of low to neutral

2014 Wiley Periodicals, Inc.

interactions between the added glutamic acid and

FIGURE 4 | Oscillatory time sweep measurement (6 rad/second and

hydrogels subject to a strain sweep measurement

2014 Wiley Periodicals, Inc.

Volume 7, January/February 2015

WIREs Nanomedicine and Nanobiotechnology

Rheology of peptide- and protein-based physical hydrogels

allowed to age, these hydrogels heal into networks

undergo strand swapping. Hydrophobic association

Rheological Characterization of the

2014 Wiley Periodicals, Inc.

hydrogels. These properties help establish the utility

about any structural change exhibited by the hydrogel

2014 Wiley Periodicals, Inc.

Volume 7, January/February 2015

WIREs Nanomedicine and Nanobiotechnology

Rheology of peptide- and protein-based physical hydrogels

Radial position (mm)

shear rates reinforces the utility of MAX1/MAX8