A protocol for rheological characterization of hydrogels for tissue

engineering strategies

Jonathan M. Zuidema,1 Christopher J. Rivet,1 Ryan J. Gilbert,1 Faith A. Morrison2
1
Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies,
Rensselaer Polytechnic Institute, Troy, New York 12180-3590
2
Department of Chemical Engineering, Michigan Technological University, Houghton, Michigan, 49931

Received 30 July 2013; revised 6 November 2013; accepted 16 November 2013
Published online 6 December 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33088

Abstract: Hydrogels are studied extensively for many tissue Frequency sweep to determine the linear equilibrium modu-
engineering applications, and their mechanical properties lus plateau of the hydrogel. (4) Time sweep with values
influence both cellular and tissue compatibility. However, it is obtained from strain and frequency sweeps to accurately
difficult to compare the mechanical properties of hydrogels report the equilibrium moduli and gelation time. Finally, the
between studies due to a lack of continuity between rheologi- rheological characterization protocol was evaluated using a
cal protocols. This study outlines a straightforward protocol composite MatrigelTM-methylcellulose hydrogel blend whose
to accurately determine hydrogel equilibrium modulus and mechanical properties were previously unknown. The proto-
gelation time using a series of rheological tests. These proto- col described herein provides a standardized approach for
cols are applied to several hydrogel systems used within tis- proper analysis of hydrogel rheological properties. V C 2013

sue engineering applications: agarose, collagen, fibrin, Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater,
MatrigelTM, and methylcellulose. The protocol is outlined in 102B: 1063–1073, 2014.
four steps: (1) Time sweep to determine the gelation time of
the hydrogel. (2) Strain sweep to determine the linear- Key Words: biomaterial, hydrogel, rheology, agarose, colla-
viscoelastic region of the hydrogel with respect to strain. (3) gen, fibrin, MatrigelTM, methylcellulose

How to cite this article: Zuidema JM, Rivet CJ, Gilbert RJ, Morrison FA. 2014. A protocol for rheological characterization of
hydrogels for tissue engineering strategies. J Biomed Mater Res Part B 2014:102B:1063–1073.

INTRODUCTION ties of hydrogels, such as equilibrium modulus and gelation
There is significant interest in the use of hydrogels for tis- time, is of critical importance.
sue engineering applications, as displayed in a recent review Rheology is an appropriate method for characterizing
by Van Vlierberghe et al.1 This interest is driven by the ben- hydrogel mechanical properties since it is quick, sensitive,
eficial characteristics that hydrogels present for both in vitro requires small sample sizes, and is revealing of differences
and in vivo applications. In vitro, hydrogels may serve as in architecture such as degree of crosslinking, proximity of
either a two-dimensional (2D) growth-supporting substrate2 the glass transition, structural homogeneity/heterogeneity,
or as a three-dimensional (3D) microenvironment.3 Recent and molecular weight. There are a number of reviews of the
reviews4,5 describe the complex, mechanically driven com- rheological properties of hydrogels composed of proteins,11
munications that occur between cells and their environment polysaccharides,12 or both.13 The main rheological technique
in vitro. Hydrogels are important for studying mechanical to characterize hydrogels is small-amplitude oscillatory
interactions because their mechanical properties are tunable shear (SAOS). In SAOS, a small amount of sample (<1 g) is
through the use of cross-linking agents6 or by altering the placed between parallel disks or a cone and a disk, and a
physical2 and chemical7 composition of the formulation. small-amplitude torsional oscillation generates shear flow in
These findings may then be translated to in vivo and clinical the sample.14 In the linear-viscoelastic regime (LVE), the
applications, as many hydrogels are injectable and conform frequency-dependence of the dynamic moduli G0 (x) and
to their implantation site geometry. Hydrogels are also capa- G00 (x) may be used to deduce important aspects of gel
ble of sustained release for drug8 or gene therapies9 and structure and mechanical behavior. For example, with fre-
can provide an injection vehicle/temporary scaffold for cell- quency sweeps we can detect gel character (degree of cross
based therapies.10 For all these applications, understanding linking), entanglement, and the glass transition, and some
and properly quantifying the underlying mechanical proper- details of chain architecture.15 Essential to the use of SAOS

Correspondence to: F.A. Morrison (e-mail: fmorriso@mtu.edu)
Contract grant sponsor: NIH; contract grant numbers: HD061096, NS062392
Contract grant sponsor: NSF; contract grant number: NSF CAREER: 1150125

C 2013 WILEY PERIODICALS, INC.
V 1063

a mechanical property not related to the time to gelation of a MatrigelTM-methylcellulose hydrogel shear modulus. The protocol reported ensures that the assumptions sweep used to measure Ge also allows us to identify x1. sample was removed and dispensed onto the rheometer because the measurement requires a mechanical perturba. To do this. strains and stresses need to be kept low during rheological testing. enzymatic cross-linking. ples. and thus the choice of imposed strain amplitude trogen (Grand Island.. deformation (LVE limit).e. To not destroy or modify hydrogel sam. and methylcellulose.g. distilled water and 0. Fibrinogen and thrombin were purchased 1064 ZUIDEMA ET AL A PROTOCOL FOR RHEOLOGICAL CHARACTERIZATION OF HYDROGELS . so 300 lL of solu- one moves from one hydrogel system to another (e.5 mM HEPES buffer) using a the methods established by Chambon and coworkers.12 for gels that are not too gelation profiles. and enzymatic).2 The hydrogel was fabricated In those tests. Collagen. The protocol presented herein pro- gels include the creep experiment11 and the unconstrained vides a clear method for accurately determining the rheologi- tensile test.is that the moduli be independent of the amplitude of the chosen examples are in widespread use: agarose. cellulose are polysaccharide hydrogels that exhibit opposing rium shear modulus of a gel. tion and a 1000-lm gap were chosen for collagen instead of tein hydrogels to polysaccharide hydrogels). and gen. G0 and the time to gelation. the 250 lL hydrogel volume and 500-lm gap height used The goal of this work is to develop and evaluate a proto./wt.18 composite whose mechanical properties were unknown. col to provide the equilibrium strength. The SAOS frequency sweep. temperature. the of SAOS are respected and permits meaningful comparisons lower frequency limit at which solid (gel. In brief. Type I. We evaluated the ability of weak gel. A 300 observed. pH. MD) and used to prepare a 2% (wt. Once x1 is known. or a proscribed amount of Agarose.11 Note that the confined elongation test16.. The test geometry was lowered to tion of the system. Attention to the solution (5 mg/mL in acetic acid) was diluted to 4 mg/mL effect of frequency is important as well.) structural reformation with SAOS. The enzyme thrombin. The same configura- tion may be used to monitor structural breakdown in METHODS hydrogels by imposing a nonlinear strain (large-amplitude Hydrogel fabrication oscillatory shear.23 The fibrin to display the utility of the protocol across multiple gelation hydrogel was created by crosslinking fibrinogen using the mechanisms (e. A panel of hydrogels was chosen version of a previously published procedure.7 Detailed characterization hydrogel in saline solution (0. relaxation) behavior. the desired gap height and excess hydrogel was discarded. Collagen was get all the details of a rheological test right. plate preheated to 37 C. as well as the time to gelation for hydrogels frequently used in tissue Fibrin. SeaPrepTM Agarose was purchased from Lonza preshear19) on the gel and subsequently monitoring the (Walkersville. and it is recommended that that are effective on stronger gels. following deionized. Agarose and methyl- In particular. To determine the equilibrium modulus of a gel. Colla- and G00 (x) are measured in the linear-viscoelastic limit. NY) and was prepared according to must be a compromise between generating adequate signal the manufacturer’s protocol. agarose gelation occurs upon cooling stiff. rat-tail collagen was purchased from Invi- however. The SAOS measurement thus both important first steps towards decoding other rheologi- tracks the growth of the G0 value from zero (sol) to the cal signatures present in SAOS. the evolution of a egies. particularly as easier to manipulate at larger volumes. followed by a second time can be monitored as a function of time. as a function of lasticity and the frequency dependence of the moduli are time or extent of reaction. constant modulus) among studies in hydrogel-based tissue-engineering strat- behavior is observed. MatrigelTM. fibrin. Rheometers have a finite sensitivity. collagen. Our SAOS is ideal for monitoring changing systems (such as suggested protocol is a cycle of the three tests: time sweep. for example. shear modulus. and MatrigelTM are protein-based hydrogels that the limiting value of G0 at low frequency is identified as the undergo gelation by pH changes. both frequency and time are varied on a gel. the creep test is used investigators wanting to assess these properties follow the when a precise measurement of Ge is desired on a very thoroughness of this protocol. moment when G0 5 G00 for all frequencies. The hydrogel was kept at this temperature until a 250 lL Using SAOS on fragile hydrogels presents a challenge.g. frequency sweep. and the gelation point is recognized as the quently heating to 60 C for 15 min under constant stirring. Ge. pro. fibrin. Other methods of measuring Ge for hydro. A fibrin hydrogel was prepared by using a modified engineering applications. Ge.15 SAOS and the tensile test are both quick tests cal characteristics of hydrogels. for the other hydrogels. gelation of a hydrogel) since the measurement is quick and strain sweep.17 meas.85% sodium chloride in of the gelation point is also possible with SAOS. by first dispersing agarose in cold saline solution and subse- ling system. SAOS is effective in identifying the equilib.. the protocol to measure the equilibrium strength and the ures bulk modulus. since the choice of with a neutralization buffer [1N sodium hydroxide in frequency determines the relaxation processes that are phosphate-buffered saline (PBS)] while kept on ice. Although we limit our discussion to obtaining Ge and sol to a gel may be monitored with SAOS. respectively. and heating. the stock collagen and preserving the integrity of the sample. It can be a challenge to geometry was lowered to the desired height. value of the equilibrium modulus Ge. determining the limits of linear viscoe- G00 are measured at a frequency below x1. G0 (x) whereas methylcellulose gelation occurs upon heating.20–22 previously published protocol. Always using the same frequency across many lL sample of the neutralized solution was immediately dis- types of hydrogels is not correct since each gel has its own pensed on the rheometer plate chilled to 4 C and the test frequency (i.

Using a previously pub. The test geometry was a 20 mm diameter plate. Each sample the methylcellulose. and percent strains are presented for all tests ran in the study. MatrigelTM was thawed overnight ated a hydrogel that had the composition of 80% (vol. MI). To fabricate the com- formation while confined between the plate and the test posite hydrogel.) methylcellulose. from Sigma Aldrich. dispensed on the rheometer plate cooled to 4 C. MatrigelTM-methylcellulose hydrogel blend. equilibrium times. geometry was lowered to the desired gap height and excess gen and thrombin were dissolved in DPBS at concentrations solution was discarded. A 250 lL sample was removed and while in liquid state.1–100 5 5 MatrigelTM-MC Temperature ( C) 37 37 37 Gap Size (lm) 500 500 500 Equilibrium Time (min) 30 30 – Frequency (Hz) 1 0. to 60 C.1–100 10 10 Fibrin Temperature ( C) 37 37 37 Gap Size (lm) 500 500 500 Equilibrium Time (min) 30 30 – Frequency (Hz) 1 0. ice at 4 C. MatrigelTM was first thawed overnight on geometry.01–100 1 % Strain 0.01–100 1 % Strain 0. Parameter Table./wt. The solution was was dispensed on the preheated/cooled rheometer plate kept on ice until use.01–100 1 % Strain 0. Louis.2 a 6% (wt.1–100 10 10 Methylcellulose Temperature ( C) 37 37 37 Gap Size (lm) 500 500 500 Equilibrium Time (min) 30 30 – Frequency (Hz) 1 0. The test geometry was lowered to the JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | JUL 2014 VOL 102B. of 10 mg/mL and 2 U/mL. NJ) and prepared according to the manufac./wt. forming a clear liquid. MatrigelTM and 1% (wt.1–100 5 5 MatrigelTM Temperature ( C) 37 37 37 Gap Size (lm) 500 500 500 Equilibrium Time (min) 30 30 – Frequency (Hz) 1 0.1–100 10 10 The temperatures.001–100 1 % Strain 0. Methylcellulose. The sample was removed and dispensed on the rheometer plate test geometry was lowered to the desired gap height and cooled to 4 C./ (Franklin Lakes. DE)./vol. The test geometry was lowered to the desired excess solution was discarded.1–100 10 10 Collagen Temperature ( C) 37 37 37 Gap Size (lm) 1000 1000 1000 Equilibrium Time (min) 45 45 – Frequency (Hz) 1 0.) on ice at 4 C and a 250 lL sample of the solution was dis. ensuring gel mechanical properties of MatrigelTM. Fibrino. This cre- turer’s protocol. ORIGINAL RESEARCH REPORT TABLE I. respectively. Gelation occurs rapidly so and methylcellulose were combined to investigate the ability the test geometry was lowered to the desired gap height of methylcellulose to alter the gelation characteristics and immediately after the thrombin was dispensed. (St. MethocelTM methylcellulose was provided Rheological characterization by Dow Chemical (Midland. Hydrogel Parameter Strain Sweep Frequency Sweep Time Sweep  Agarose Temperature ( C) 10 10 10 Gap Size (lm) 500 500 500 Equilibrium Time (min) 30 30 – Frequency (Hz) 1 0.) of MatrigelTM to 5% methylcellulose solution. MatrigelTM pensed followed by the thrombin. In brief. vol. gap sizes. The dispersion was then placed on ice to dissolve and Table I outlines the testing parameters.01–100 1 % Strain 0. ISSUE 5 1065 . 5% methylcellulose (wt./wt.01–100 1 % Strain 0. The test co’s phosphate-buffered saline (DPBS) with calcium. gap height and excess solution was discarded. Rheological characterization was performed on all hydrogel lished protocol.) hydrogel was prepared by samples using a TA Instruments AR-G2 rheometer (New first dispersing the methylcellulose in saline solution heated Castle. MatrigelTM was then mixed MatrigelTM. MatrigelTM was purchased from BD Biosciences with methylcellulose on ice at the proportions of 4:1 (vol. The rheometer plate was chilled to 4 C and the fibrinogen solution was first dis. MO) and dissolved in Dulbec. A 250 lL pensed on the rheometer plate that was chilled to 4 C. frequencies.) in saline solution was created as described above.

FIGURE 1. Strain sweeps. The testing time to determine produce larger torques. G0 was determined from 0. The linear-viscoelastic limit was determined with respect to strain.1 to 100% strain for aga- rose (A). The testing sequence was then The accuracy of SAOS moduli measurements depends on applied to the sample. Each hydrogel sample was used for the magnitude of the torque generated by the deformation only one test. we found that the accuracy of G00 1066 ZUIDEMA ET AL A PROTOCOL FOR RHEOLOGICAL CHARACTERIZATION OF HYDROGELS . fibrin (C). The AR-G2 is rated to measure a lower limit of 0. No values of G00 were obtained due to reasons stated in the methods. for experiments that seek long-time was discarded.003 therefore no need to use a humidified chamber or trap for mNm of torque. collagen (B). larger plates produce larger tor- gelation time and modulus was very short and there was ques. Each test was performed in triplicate and the and by the instrument’s ability to resolve the phase angle d data represents the average of the three tests with corre. desired gap height (500 or 1000 lm). Smaller gaps sponding standard deviation. between the strain wave and the stress wave. plate initiated gelation. and excess hydrogel these experiments. MatrigelTM (D). Rapidly heating or cooling the rheometer behavior of gels such precautions are essential. and methylcellulose (E).

We recommend the appropriate amount of time as determined by the pre- that the first step in the process be to produce a gel while ceding time sweep step in the absence of rheological moni- monitoring with SAOS at an arbitrary strain and frequency. The second recommended test is to check for the LVE limit ues that are referenced in the literature for gels that are simi.] measurements were compromised when low torque signals in vivo at or near this temperature. of G00 correspond to a minimum value of torque of at least whether it is generated by chemical crosslinking or by phys- 0. val. if possible. minimum torque of at least 0. rheometer plate and the test geometry.g. A strain sweep from 0.e. which is available at wileyonlinelibrary. the gelation times are either 30 or 45 min (Table I). The gel was allowed to reach equilibrium for according to appropriate rheological criteria. formed prior to dispensing on the rheometer plate and sub- sequently compressed by lowering the test geometry to the RESULTS desired height. G00 was too small to measure for all hydrogels. ical electrostatic associations.3 rad s21) (cho- tial data point for the study—providing a general indication of sen arbitrarily) on the fully formed gel. Plate diame. strain sweep and frequency sweep) are per. ORIGINAL RESEARCH REPORT FIGURE 2. toring. the reported values This ensures that the physical structure of the hydrogel. likewise. If a hydrogel is straints formed by the amount of sample available. occurs confined between the ter was chosen to be as large as possible within the con. to know what SAOS settings to use for initial characterization [e.com. At low frequencies. lower temperatures are accuracy of G0 measurements were compromised when low required (i. sion of this below). For the hydrogels tested in expected for fully formed hydrogels (Figure 2. strain sweeps for the gels studied here are shown in Figure quent tests (e.01. G00 goes like x through the region of the glass transition as shown. At high frequencies.15 [Color figure can be viewed in the online issue.003 mNm. onto the rheometer plate as a liquid and the test geometry The reported values of G0 in this article correspond to a is lowered to the desired height while still a liquid sample. This is especially critical to ensure that subse. ISSUE 5 1067 . A new lar to the one being tested (we chose 5–10% strain and 1 Hz hydrogel solution was placed on the rheometer plates and to start). conducted at a frequency of x0 5 1. and a strain of 10% was selected for JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | JUL 2014 VOL 102B. then the resulting data is indicative of a Gelation time deformed hydrogel network and not the naturally forming When working with a new hydrogel formulation.14.010 mNm and G00 /G0 5 tan d of at least 0. The transition of G0 between these two plateaus characterizes the glass transition of the polymer. The loss modulus G00 for a gel is very low and cannot be measured.g. Typical linear-viscoelastic behavior of a crosslinked gel. by performing a strain sweep on a fully formed gel. The results of the gelation time. each sample is loaded values of torque are combined with phase angles near 90 . as is to be formed on a fully gelled sample. The values Strain sweeps used for the first gelation time test should be... Furthermore. combined with phase angles of close to 0 . In cases such as agarose. strain amplitude (c0) and the frequency (x0)]. in the glassy region G00 goes through a maximum. it is difficult network. Agarose showed linear behavior (con- Each test should be conducted at 37 C if possible. the glassy behavior of polymers is observed (G0 5 109 Pa). G0 approaches a plateau.. since stant values as strain is varied) of G0 up to 15 % strain in tissue engineering applications the gel will be forming [Figure 1(A)].1 to 100% strain was then this time (or extent of reaction) sweep then serves as an ini. more discus- this study. For a gel about which little is known. the corresponding thermal conditions were applied to initi- ble to choose the strain amplitude (c0) and the frequency (x0) ate gelation. 1. it is not possi. the which forms a gel by cooling. the gel shear modulus.0 Hz (6. 10 C).

and 10% strain was In the initial time sweep conducted at the beginning of this selected for subsequent sweeps. ear strain region up to 10 % strain [Figure 1(E)]. protocol. Values of G00 were removed if the data were not reliable as discussed in the report. Fibrin exhibited a linear plateau of the SAOS frequency sweep (Figure 2). MatrigelTM had a linear frequency must correspond to this low-frequency plateau. and 5% strain was SAOS to monitor gel formation or reformation. MatrigelTM (D). we arbitrarily chose the value of frequency to use. region up to 13% strain [Figure 1(D)]. Collagen had a linear strain region also Frequency sweeps up to 15% strain [Figure 1(B)]. The linear equilibrium modulus plateau (Ge) with respect to frequency was determined. and 5% the results of the frequency sweep using the appropriate strain was chosen for subsequent sweeps. fibrin (C). subsequent sweeps. strain (as determined in the preceding section) allow us to 1068 ZUIDEMA ET AL A PROTOCOL FOR RHEOLOGICAL CHARACTERIZATION OF HYDROGELS .01 to 100 Hz for agarose (A). the testing selected for subsequent sweeps. collagen (B). To use region up to 10% strain [Figure 1(C)]. Frequency sweeps. and 10% strain was The presence of a gel is associated with the low frequency selected for subsequent sweeps. G0 and G00 were deter- mined from 0. Methylcellulose had a lin.FIGURE 3. and methylcellulose (E).

ORIGINAL RESEARCH REPORT FIGURE 4. collagen (B).01 to 100 Hz were conducted at The purpose of the third recommended test is to find an the LVE strain amplitude determined in the preceding sec- appropriate value of frequency to employ in a second time tion. ISSUE 5 1069 . for frequency ior and a frequency of 1 Hz was selected for future tests sweeps a new hydrogel solution was placed on the rheome. based on good torque signal at that frequency. while G00 is only presented for methylcellulose (E) due to limits on instrument sensitivity. As in the case of the strain sweeps. all frequencies measured reflected gel behav- time. For agarose. rigorously choose a frequency to ensure that the time ter plate and the appropriate amount of time elapsed for sweeps we perform reflect the formation of the gel network the gel to reach equilibrium (no rheological monitoring). MatrigelTM (D). and not some other structural change. Gel formation was monitored with a time sweep for 30 min for agarose (A). and methylcellulose (E). fibrin (C). JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | JUL 2014 VOL 102B. Frequency sweeps from 0. Final time sweeps. G0 is presented for all tests. For collagen. The results of frequency sweeps are shown in Figure sweep that defines the equilibrium strength and gelation 3.

so we utilized a 30 min time sweeps. Fibrin time sweeps were conducted at 5% strain and 1 Hz at 37 C. it was unknown if strain was small enough to be in the LVE and if the frequency was a value in the low- frequency gel plateau. Collagen time sweeps were conducted at 10% strain and 1 Hz at 37 C. For methylcellulose the influence of the glass transition extended to approxi- mately 5–10 Hz.1 to 100% strain The MatrigelTM-methylcellulose blend formed a stable gel in was then conducted at a frequency of x0 5 1. This second time sweeps was performed in order to obtain the hydrogel equilibrium modulus. Therefore. The hydrogel strain [10% strain (Figure 1(D)] and frequency [1 Hz (Fig. blend exhibited LVE behavior up to 9 Hz [Figure 5(B)]. reaching stability at 120 Pa in 3 min [Figure 4(B)]. Fibrin formed a stabilized gel in 10 min near 160 Pa [Figure 4(C)]. The parameters for strain determine the frequency domain within the LVE for the and frequency were taken from the results of the MatrigelTM MatrigelTM-methylcellulose hydrogel blend. Methylcellulose then continued to increase the G0 modulus until stabilizing at 25 min near 340 Pa [Figure 4(E)]. Once these limits were established. because it is not possible to know 1070 ZUIDEMA ET AL A PROTOCOL FOR RHEOLOGICAL CHARACTERIZATION OF HYDROGELS . The gelation kinetics of methylcellulose were slower than the other hydrogels. The first four hydrogels formed rapidly and the cross- over of G0 and G00 was not observed (G00 was too low to mea- sure for the extent of the tests). and ure 3(D)] sweeps. FIGURE 5. For methylcellulose G0 crossed G00 at 5 min. hydrogel blend. a a frequency of 1 Hz was chosen to perform subsequent stable gel formed in under 10 min. fully formed gel.01 to 100 Hz were conducted at 10% strain to trial-and-error approach. the gel region was observed below 20 Hz. Time sweeps were conducted on MatrigelTM- preincubation time at 37 C before running the strain and methylcellulose blends using 10% strain and 1 Hz at 37 C. After running the time sweep at 37 C. reaching stability in 20 min near 1200 Pa [Figure 4(A)]. that is. frequency sweeps. 1 Hz was therefore also an acceptable fre- quency choice for this system. and the crossover of G0 and G00 was captured for this material. 1 Hz was selected for future testing. The final step of the protocol is a second time sweep at the now verified strain and frequency values. we were able to select appropriate values for future tests. Agarose slowly formed a gel. A strain sweep from 0.0 Hz on the 5 min near 30 Pa [Figure 5(C)]. showing when the material changed from more of a liquid state to being dominated by the gel state [Figure 4(E)]. Collagen quickly formed a gel. MatrigelTM-Methylcellulose hydrogel characterization. MatrigelTM formed a stable gel in 5 min near 90 Pa [Figure 4(D)]. a time test was first used to determine the time of gelation (data not shown). The linear-viscoelastic limit was determined with respect to strain (A). MatrigelTM time sweeps were conducted at 10% strain and 1 Hz at 37 C.fibrin and MatrigelTM. we chose a strain of 10% DISCUSSION for the subsequent frequency sweep. It was found that the MatrigelTM -methyl- cellulose blend was in the LVE range up to nearly 20% strain [Figure 5(A)]. for the intent of the first time sweep was solely to identify gelation time. and gel formation and equilib- To start the analysis for this previously uncharacterized rium modulus (Ge) was monitored by time sweeps for 30 min (C). Time sweeps The time sweep performed as step one of this protocol employed values of strain and frequency that were unveri- fied. Methylcellulose time sweeps were conducted at 5% strain and 1 Hz at 37 C. Agarose time sweeps were conducted at 10% strain and 1 Hz at 10 C. the linear equilibrium modulus plateau (Ge) with respect to frequency was MatrigelTM-methylcellulose hydrogel blend determined using frequency sweeps (B). indicating a transition from a primarily liquid sample to a predominately solid material. Frequency sweeps Characterization of a new hydrogel with SAOS requires a from 0.

which were conducted at 10 C (Figure 4). determine the time to gelation. Because the sample starts as a low viscosity liquid. For our forms. however. hydrogel mismatch. how the hydrogel conforms to implantation site geometry est. however. and this applied SAOS test. cially important parameter to consider when delivering surement is interpretable. tool for monitoring this change. which greater than G00 (glassy solid behavior). The chosen parameters must also comply One goal of SAOS characterization of gels in tissue engi- with the material. because it impacts the moduli measured to the material characteristics of inter. and (2) the angle d. because higher frequencies generate large changes in G0 and G00 during gelation. the tor- featured in Figure 2).e. for example. towards glassy behavior at high frequency. these features can be convenient technique. At lower frequencies of testing. causing the dynamic instrument calculates G0 and G00 14: moduli to cross. potentially modifying therapeutic delivery. exhibiting an entanglement pla. which combined with geometry information gives a mer gel at high frequency exhibits G0 5 109 Pa and G0 value of complex modulus (G*). gel formation). ORIGINAL RESEARCH REPORT a priori the appropriate values of strain. Slow gelation times may permit hydrogel leakage from the Characterization begins with an initial time sweep to delivery site. and the initial phase angle is quency at low frequency (see. which must not be destroyed by the neering is to monitor gelation (i. the polymer near 90 (sin d 5 0). SAOS is an ideal higher torques. From these two measurements the with G0 dropping more rapidly than G00 . A poly. genes. even when equipment and is provided by the time sweep test. ISSUE 5 1071 . G00 does not decrease as the first power of fre. the viscosity of the sample (related to G00 ) grows. The issue of strain is very important in SAOS measure. frequency. For these materials G0 is strongly que increases and G00 becomes measureable. Furthermore. such as the frequency sweep where strain is cient time for the gels tested to reach their final moduli val- held constant. In all which presents limitations in terms of strain and phase. terized by very low values of both G0 and G00 . The strain needs to produce a sufficiently high signal perform once the appropriate values of strain and frequency without destroying the gel. G0 exceeds G00 . that is. The storage affected by entanglement. the large changes however. value chosen within the LVE from the strain sweep in sub. the equilibrium strength (Ge) of the gel. is the measured phase difference between the oscillating cies are tested. angle sensitivity. care must be taken to verify that an observed plateau in G0 and thus G00 5 G*sin90 5 G* and G0 5 G*cos90 is zero. The fre. we must be able to relate drugs. or cells to an injury site. torque. Time sweeps of a forming gel are straightforward to ments. and fre- must comply with the capabilities of the SAOS instrument. Time sweeps were con- strain sweeps to determine the LVE limit and employed a ducted for 30 min at 37 C for all gels except for agarose. strain maxima and minima to control the sample deforma. When the gelling of a sample is observed with the SAOS tion and signal strength. because of the intrinsic nature of liquids. it is important that the mea. The probed physics change with frequency. the instrument features that allow users to choose stress and tests can be extended as necessary. strain and stress waves. In this work we performed are identified. For a liquid. Because of the affects signal strength. their own measurement issues. When working with gels that take longer to form. G00 5G sin d with G0 tending at low frequencies towards a plateau. Because of this. This value is then employed while rapid gelation times may inhibit the hydrogel from in a subsequent strain sweep to determine the maximum conforming to the lesion geometry. While the sample is a sol. as mentioned above. and thus to interpret the SAOS response the mea. a frequency of 1 Hz was within that plateau. cases studied. Until JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | JUL 2014 VOL 102B. When the gel forms. and may influence the outcomes of tissue regeneration. The choices for these parameters studied here there was no entanglement plateau.15 before progressing stages. This was a suffi- sequent tests. between 5 does the elastic behavior (G0 ). Gelation time is an espe- material issues are addressed. the elastic modulus G0 levels off at the value of the equilib- Frequency is another parameter of SAOS tests that rium modulus. As lower frequen. In this work.. and large relative values of the two dynamic moduli cause surement frequency must be chosen with care. and reflects the equilibrium modulus of the crosslinked gel gelation time to employ towards obtaining a value for the rather than the entanglement plateau. the strain and the stress are 90 out of phase. An alternative choice is to use automatic ues. the sample begins as a pure liquid and is charac- and may be used when users are accustomed to the per. initial signal of torque is small. modulus G0 is not capable of being measured at these early teau at intermediate frequencies14. as we now discuss. and in the same region G0 5G cos d G00 decreases proportional to the frequency as lower and G00 =G0 5tan d lower frequencies are tested. For the hydrogels equilibrium gel strength. As the gelation reaction occurs. quency tests revealed the location of the gel plateau. Note that for polymeric gels that have entangled strands or other sources of strong elas. as instrument and for the hydrogels studied herein. As the gel formance of their instrument and test materials. the elastic response grows in importance and G0 and G00 cross again. creating a tissue- strain that delivers a material response within the LVE limit. G0 becomes equal to while keeping the material response within the LVE limit G00 20 and as the reaction proceeds. the tic behavior. quency behavior of long-chain polymeric gels has been There are two raw signals in an SAOS rheometer: (1) discussed at length in the literature15 (see Figure 2). G00 and 10% strain was sufficient to generate appropriate signal is greater than G0 . and G00 approaches zero. ately characterizes hydrogels through SAOS. both G0 and G00 drop (the glass transition). we illustrate such a process that appropri. Ultimately (Table I). Ge.

different studies.19 large-amplitude oscillatory strates (80 kPa) resulting in increased gene expression of shear (LAOS). and we compared our shear modulus results from a 2% agarose gel SUMMARY to published results for the shear modulus determined by This study outlines a protocol to assess the linear- unconfined compression tests. the type of agarose used.12 and superposed osteogenic markers. G0 rose quickly above G00 for the centage than the hydrogel tested here (2%) in order to cre- other gels tested. cous signal are incorporated into the purely elastic gel.12 steady shear. methylcellulose the tissue of interest interact favorably with the implanted 340 Pa. adipose 2. Agarose.24 The shear modulus that viscoelastic mechanical properties of hydrogels using small- Dekosky et al. reported was 8. gives the same information as small-amplitude testing in a shear mode (when divided by a factor of three). The methylcellulose time sweep spinal cord. modulus range of brain tissue.73 6 1. but value of 30 Pa (Figure 5). with the stiffer sub- ture strength. Com. but hydrogel obtained if there is sufficient torque [Figure 4(E)]. showing that the gel state dominates at an ate a hydrogel that is similar to the mechanical properties early time for these hydrogels. low If agarose is pursued for central nervous system repair. MatrigelTM 90 Pa. when the ratio of G0 to G00 (or the 100% MatrigelTM formulation (90 Pa) (Figure 4). The MatrigelTM-methylcellulose hydrogel formed a measure. and G0 becomes measurable. For The protocol outlined here was used on a gel formulation the ripening gel G0 is large and G00 is small.5–15 kPa. When divided by amplitude oscillatory shear. shows that before 5 min the material is mostly a liquid. this gel could have some utility in applications to avoid assigning meaning to signals that reflect noise that require hydrogels with very low moduli.sufficient elasticity develops in the sample. but draws and G00 are obtained through the gel point.2 kPa low to measure. The G00 to G0 ) is larger than 10 the phase angle reliability of reduction in the percentages of the hydrogel materials. This material of choice for central nervous system regeneration. When the phase angle is too small to record. As the preparation. Cells in different tissue Figure 2 is expected of all gels. and testing parame- sample progresses towards the gel point.14. entanglement plateau is observed between Ge and the glass cartilage 20–25 kPa.26 Smooth muscle cells have also been shear and SAOS. mal cell attachment and differentiation. As a rule of thumb. and bone 25–45 kPa. strong and weak. compared with stiffer gels (9000 Pa). G0 grows strongly and G00 decreases as the loose directly compare hydrogel mechanical properties between chains and dangling ends that are responsible for the vis. is about 200 Pa above the gives an example of a gelation crossover point [Figure 4(E)]. G00 was too small to measure of central nervous system tissue. so it may not be the ideal G0 crosses over and rises above G00 at about 5 min.27 cussion of the relative strengths of different gels. collagen mechanical properties of the hydrogel to ensure that cells of 120 Pa. Soft gels (200 Pa) pro- gel. G0 remains too three. one viscosity material. Regardless of issue.2–1 kPa.3 kPa. For gels microenvironments are presented with different elasticities: with entangled strands between crosslinks. the possibility of reducing methylcellulose chain entangle- plex moduli ratios of greater than 102 or less than 1022 are ment and MatrigelTM gelation by the competition between unlikely to be accurate for any instrument due to inherent the two different hydrogels are the two most likely causes noise in detecting weak phase-angle signals. Our average of 1. Ge. The final values of equilibrium modulus. the gel in less than 5 min and reached an equilibrium modulus measurement of G0 is still quite accurate (cos d 5 1).5 kPa. fibrin 160 Pa. The gels presented in this study are mostly weak gels. although accurate values of G00 may be is slightly less than the average of 2. Small amplitude testing in a compression mode material.28 Whenever a new hydro- accurately throughout the time sweeps for all but the meth.73 kPa. and then after 5 min the material forms may fabricate an agarose hydrogel with a lower weight per- a gel as G0 rises above G00 .25 Substrates with sue engineering applications and may be pursued after LVE mechanical property gradients affected bone marrow stro- characterization is complete. an intermediate brain 0.12 Results from such tests would permit dis. The similarity grows. The difference in the mechanical properties of tissues is The experiments discussed here are all in the LVE an important parameter that needs to be taken into account regime.1 kPa. Because of this for the reduction in the equilibrium modulus. care should be taken in reporting values of G0 and G00 the cause. leading to a very not evaluated previously in the literature in order to show small phase angle that eventually becomes too small to its utility. and rheological instruments are approaching their limits. which results from a deformation that is outside the moted enhanced neurite growth of mixed cortical cultures LVE limit. the phase angle d ters can account for these differences. and The time sweep results for the five systems studied here would be possible tissue engineering substrates for brain or are shown in Figure 4.15 The division of gels into strong and have been conducted detailing the effects of substrate stiff- weak12 is a distinction based on the fracture strength of the ness on cell responses in vitro. gel material is developed for a specific tissue engineering ylcellulose.5–3. Nonlinear tests include frac. shown to respond to differences in substrate stiffness.2 6 3. start-up of steady shearing. however. muscle 8. Within this regime the SAOS behavior discussed in when developing new hydrogels. but after the gel awareness to the need for a standardized approach to point. This value is much lower than the accurate values of G00 may no longer be obtained [Figure 6% methylcellulose hydrogel (340 Pa) and is also less than 4(A–D)]. the values are 2. The first step of the protocol is 1072 ZUIDEMA ET AL A PROTOCOL FOR RHEOLOGICAL CHARACTERIZATION OF HYDROGELS . it is important to accurately characterize the the five gels were found to be: agarose 1200 Pa.5 Several studies transition behavior. Tests that disrupt a gel may be of interest in tis. for situation. Good values of both G0 between the two results validates both methods. rather than material behavior.

98:281–294. gel modulus). 17. Roatch CH.123:297–308. Pochan DJ. Buxboim A.4: neering as the implanted material must not only interface 17–25. Biomacromolecules 2011. hydrogel delivery system for osteosarcoma gene therapy with Biomaterials 2001. Wang DA. Kim Y. Scientific World J 2003. release of neurotrophin-3 from fibrin gels for spinal cord injury. Winter HH. Mikos The outcome of this protocol presents a robust procedure AG. 8. this first test provides an idea of the gelation kinetics for 10. ery. Rev 2010. Ivanovska IL. New York. Snedeker JG. Laurencin SJ. Shatwell KP. Macromolecules 1986. Dunstan DE. limit and a frequency sweep to determine a frequency that 39:3528–3540. increase efficiency and ultimately implant effectiveness. Dubruel P. Acta Biomater 2008. Therapeutic cell delivery and the system and ensures that subsequent tests will be per. Hydrogel nanoparticles in drug deliv. Volume 1. Morrison FA.32: lage tissue engineering. Sakiyama-Elbert SE. Rheology of biopolymer solutions and gels. Biomaterials 2008. rices with compliance comparable to that of brain tissue select 5. Guan Z. Rafiei P. of smooth muscle cells in a 3-D biosynthetic hydrogel system. The initial time sweep is fol.29:2597–2607. 3. Sawyer ES.7:1634–1643. Adv Drug Deliv Rev 2008. Biomaterials 2011. Gehrke SH. ately reflects the growth of the gel’s strength and structure 14. hydrogels as scaffolds for tissue engineering applications: A 31:683–697. Ross-Murphy SB. Hennink WE. Rheology of model polyurethanes at the gel point. 12. Maher SA. NY: Oxford in the course of gel formation. Selinger E. Characterisa- manner (gelation time). Science 2005. Seliktar D. ISSUE 5 1073 . Varshney RR. Morrison FA.12:1387–1408. Azadi A. toward tendon and bone. Hassager O. Acta Biomater 2011.19: 2146–2149. New York.60:1638–1649. and micro- Following the outlined procedure ensures accurate results computer tomography. Biochemical and biomechanical gra- 6. Petrovic ZS.30:217–227. Sharma RI. Ingavle GC. MacKnight WJ. 13. Controlled 2. Ross-Murphy SB. and fracture strength. 1980. Polysaccharide strong and weak Finally.415: 95–101. Researchers in tissue engineering tion of the interactive properties of microcrystailline cellulose- may find this protocol useful as a first step in developing carboxymethyl cellulose hydrogels. Ananthanarayanan B. 24. 26. Charlton D. Tissue Eng Part C Methods 2010. Viscoelastic Properties of Polymers. McDonald JW III. Szymanski A. Balgude AP. Novel crosslinking methods to dients for directed bone marrow stromal cell differentiation design hydrogels. DesKosky BJ. may be performed to probe samples’ nonlinear behavior 15. 18. Ferry JD. Biomaterials 2009. Analysis of linear viscoelasticity of a for desirable characteristics prior to implantation that will crosslinking polymer at the gel point. Dormer NH. blends for neural repair. Yu TB. van Nostrum CF.22:1077–1084. but it must also do so in a clinically relevant 19.62:699–710. Elucidating the mecha. Wang YL. Discher DE. review. Taylor SJ. Biophys J keletal forces and physics of the nucleus: how deeply do cells 2006. Zhao GH. Adv Drug Deliv formed on fully gelled samples. J cation and characterization of tunable polysaccharide hydrogel Control Release 2004.310:1139–1143. Heirarchically designed agarose and nobiology of malignant brain tumors using a brain matrix. Gilbert RJ. REFERENCES 22. Matrix elasticity. Understanding Rheology. 1542. Liebschner MAK. fate control in hydrogels and hydrogel hybrids. when reporting equilibrium gel modulus and gelation time of Superporous hydrogels for cartilage repair: Evaluation of the a hydrogel. 16. Meaney DF. Int J Pharmaceutics 2011. Guthrie JT. Lowman AM. 2001. Dynamics of Polymeric mechanically well with the surrounding tissue (equilibrium Liquids.30:367–382. Discher DE. hydrogel-based strategies.3:105–121. Yu X.131:17638–17646. Biopolymer-based crosslinking PDMS with imbalanced stoichiometry. New York. Lomakin J. Chambon F. J Am effects of matrix stiffness and RhoA on the phenotypic plasticity Chem Soc 2009. Linear viscoelasticity at the gel point of a 1. Biomacromolecules 2002. Winter HH. Tissue cells feel and respond to 25. Dass CR. Agarose gel 9.90:3012–3018. Miller WJ. ’feel’ outside and in? J Cell Sci 2010. Jaroch DB. The final time sweep thus appropri. Peyton SR. providing a screening mechanism 20. Zuidema JM. De novo design of saccharide-peptide 27. 28. morphological and mechanical properties. lowed by a strain sweep to determine the linear-viscoelastic 11. gels using gravimetry. Choong PF. 23. These two features are integral to tissue engi. Janmey PA. Putnam AJ. Biomaterials 2010.30:4815–4823. Schacht E. Picout DR. Kapur N. Adv Drug Deliv Rev 2002. Kim PD.54:13–36. the time sweep is repeated with appropriate values gels. A chitosan stiffness determines rate of DRG neurite extension in 3D cultures. Janmey P.31:7695–7704. Pap MM. pigment epithelium-derived factor combined with chemotherapy. confined compression testing. Behravesh E. Georges PC. Subsequent nonlinear tests University Press. Mat- the stiffness of their substrate. 1987. Hamidi M. Winter HH. Chambon F. Ta HT. appropriately reflects the equilibrium gel modulus Ge. Chem Soc Rev 2010. Liao SW. poly(ethylene glycol) interpenetrating network hydrogels for carti- mimetic hyaluronic acid hydrogel platform. 4. Carlin B. of strain and frequency. 2nd ed. 7. The hydrogels as synthetic scaffolds for tailored cell responses. Chambon F. NY: Wiley. J Rheol 1986.16:1533– 7913–7923. Ghajar CM. JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | JUL 2014 VOL 102B.3:1263–1270. cytos. Spiller KL. Rheological properties of peptide-based hydro- gels for biomedical and other applications. Bird RB. Kumar S. 21. Bellamkonda RV. Biorheology 1993. Yan C. Lemoine JJ. neuronal over glial growth in mixed cortical cultures. Wang C. Timmer MD. Armstrong RC. Detamore MS. Fabri. 3rd ed. Larson I. NY: Wiley. Van Vlierberghe S. Evaluation of the in vitro degradation of macroporous hydro- that is not often conducted in the field of tissue engineering. J Rheol 1987. ORIGINAL RESEARCH REPORT a time sweep with arbitrary strain and frequency choices.