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Research Papers

Mechanical Characterization of a Dynamic and Tunable Methacrylated Hyaluronic Acid Hydrogel

[+] Author and Article Information
Matthew G. Ondeck

Material Science Program,
UC San Diego,
La Jolla, CA 92093

Adam J. Engler

Material Science Program,
UC San Diego,
La Jolla, CA 92093;
Department of Bioengineering,
UC San Diego,
La Jolla, CA 92093;
Sanford Consortium for Regenerative Medicine,
La Jolla, CA 92037
e-mail: aengler@ucsd.edu

1Corresponding author.

Manuscript received November 8, 2015; final manuscript received December 30, 2015; published online January 27, 2016. Editor: Victor H. Barocas.

J Biomech Eng 138(2), 021003 (Jan 27, 2016) (6 pages) Paper No: BIO-15-1566; doi: 10.1115/1.4032429 History: Received November 08, 2015; Revised December 30, 2015

Hyaluronic acid (HA) is a commonly used natural polymer for cell scaffolding. Modification by methacrylate allows it to be polymerized by free radicals via addition of an initiator, e.g., light-sensitive Irgacure, to form a methacrylated hyaluronic acid (MeHA) hydrogel. Light-activated crosslinking can be used to control the degree of polymerization, and sequential polymerization steps allow cells plated onto or in the hydrogel to initially feel a soft and then a stiff matrix. Here, the elastic modulus of MeHA hydrogels was systematically analyzed by atomic force microscopy (AFM) for a number of variables including duration of UV exposure, monomer concentration, and methacrylate functionalization. To determine how cells would respond to a specific two-step polymerization, NIH 3T3 fibroblasts were cultured on the stiffening MeHA hydrogels and found to reorganize their cytoskeleton and spread area upon hydrogel stiffening, consistent with cells originally cultured on substrates of the final elastic modulus.

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Figures

Grahic Jump Location
Fig. 1

Functionalization and characterization of MeHA hydrogels. (a) NMR spectrum of 50 kDa HA with methacrylate functionalization (∼65% methacrylate functionalized). (b) Elastic modulus of 1% and 3% w/v of MeHA polymerized for 1, 2, and 3 min with 350 nm UV light. All samples are statistically different from one another based on one-way ANOVA with p < 10−4. (c) A 10 μm × 10 μm elastic modulus map for 1% and 3% MeHA gels UV polymerized for 1 min.

Grahic Jump Location
Fig. 2

Comparison of on-demand versus continuous stiffening. (a) Elastic modulus measured for 1% MeHA gels polymerized for 1, 2, and 4 min and gels polymerized for 1 and 2 min, stiffened additionally with 1 and 2 min of UV light exposure, respectively. Using nonparametric t-tests: *p < 10−12 and **p < 10−8. (b) Elastic modulus measured for 3% MeHA gels polymerized for 1, 2, and 4 min and gels polymerized for 1 and 2 min, stiffened additionally with 1 and 2 min of UV light exposure, respectively. Using nonparametric t-tests: *p < 10−7 and **p < 10−6. (c) A 10 μm × 10 μm elastic modulus map for 1% MeHA gel UV polymerized for 1 min then stiffened with an additional 1 min of UV light.

Grahic Jump Location
Fig. 3

Impact of degree of methacrylation on stiffness. (a) NMR spectrum of MeHA with 38% (black arrow) and 65% functionalization (gray arrow), with peaks representing the methacrylate group and HA indicated. (b) Elastic modulus of 38% and 65% methacrylate functionalized 3% MeHA polymerized for 1, 2, and 3 min with 350 nm UV light. One-way ANOVA indicated that conditions were statistically different with p < 10−4 for UV exposure time within each methacrylation percentage, although post hoc Tukey analysis did not find a difference between 2 and 3 min exposure time for 1% MeHA.

Grahic Jump Location
Fig. 4

Dynamic stiffening affects cell spreading. (a) NIH 3T3 fibroblasts were cultured separately on 1% MeHA gels UV polymerized for 1 and 2 min, and dynamic MeHA gels polymerized for 1 min and then stiffened on for 1 additional minute. Cultures were stiffened on day 1 and fixed on day 3. Arrowheads indicate stress fibers. (b) Fibroblast cell area was measured at day 3 for cells cultured on 1% and 3% MeHA gels polymerized for 1 and 2 min and 1 + 1 min stiffened gels. The gray background is the range of cell areas for fibroblasts cultured on tissue culture glass as a comparison. One-way ANOVA indicated that only the 1% MeHA conditions were statistically different with p < 0.1 for UV exposure time.

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