(33h) Environmentally Responsive Methacrylated Alginate Hydrogel Gradients for Studying NIH/3T3 Fibroblasts
Environmentally responsive methacrylated alginate hydrogel
gradients for studying NIH/3T3 fibroblasts
Boddupalli1, Kaitlin M. Bratlie1, 2, 3
1Department of Chemical & Biological Engineering, Iowa State
University, Ames, Iowa 50011
2Department of Materials Science & Engineering, Iowa State
University, Ames, Iowa 50011
3Ames National Laboratory, Ames, Iowa 50011
Alginate is natural biomaterial that has been used for
diverse tissue engineering applications. Covalently crosslinked methacrylated
alginate (ALGMA) can increase the stability of the gels, along with mechanical
strength. Step growth, chain growth, and a mixture of the two, termed mixed
mode, crosslinking mechanisms were examined here. The effect of these
crosslinking mechanisms on compressive modulus, degradation, swelling, and
cellular responses of NIH/3T3 fibroblasts was measured. The heterogeneity of
natural tissue can be mimicked here using photomasks to tune the mechanical
properties ALGMA hydrogels. Comparing the differentiation of fibroblasts to
myofibroblasts these patterned alginate substrates can better understand how
mechanical properties influence cell responses.
Materials and Methods: ALGMA hydrogels were fabricated by methacrylating medium
viscosity alginic acid. The pre-gel solution was photo-crosslinked using
Irgacure 2959 and degassed with and without dithiothreitol (DTT) to obtain step
and chain growth polymerization respectively. Combining Irgacure 2959, DTT, and
degassing the pre-gel solution resulted in mixed mode polymerization. Dually
crosslinked ALGMA hydrogels were fabricated by adding strontium chloride to the
three covalently crosslinked gels to obtain a total of six hydrogel formulations.
Compressive moduli for the hydrogels were evaluated under step-load conditions.
The swelling behavior of these hydrogels was measured after incubating them in
sodium acetate buffers of varied pH. NIH/3T3 fibroblasts were encapsulated in
the different hydrogels as well as seeded on the alginate gradient, with their proliferation
response evaluated through live/dead assays. Immunohistochemical staining of
the fibroblasts seeded on the photopatterned alginate was conducted to observe
the differentiation to myofibroblasts.
Results and Discussion: The compressive moduli for the hydrogels was in the
range of 9.3 ± 0.2 to 22.6 ± 0.3 kPa with
the softest being step growth and the stiffest being mixed mode hydrogel with
strontium ionic crosslinks. The hydrogels fabricated here swelled the most (up
to 17.1 ± 0.6 times their original dry weight for the step growth gels) under
basic pH conditions. All six hydrogel formulations had half-lives around 25
days under accelerated degradation conditions in 0.1 mM sodium hydroxide.
NIH/3T3 cells encapsulated in these hydrogels showed no observable cell death. The
highest cell proliferation was on the stiffest ionically crosslinked mixed mode
hydrogels (Figure 1). Patterned alginate hydrogels were prepared masking
exposure to UV light on the ionically crosslinked chain growth hydrogels. These
fabricated hydrogels had a compressive modulus range of 6.9 ± 0.4 to
20.2 ± 0.4 kPa using varying opacities of the photomask. Fibroblasts seeded on
the photopatterned alginate proliferated better when the substrate was
functionalized with fibronectin and there were clear distinctions in
differentiation to myofibroblasts.
1. Proliferation of NIH/3T3 cells encapsulated
in alginate hydrogels. NIH/3T3 fibroblasts were mixed with alginate and
crosslinked. Cells seeded on tissue culture plastic served as the controls.
Representative micrographs of live (green) and dead (red) cells cultured for 48
h. Scale bar is 100 mm.
stiffness alters swelling responses and cell proliferation of encapsulated
cells. Surface seeded fibroblasts patterned substrates of ionically crosslinked
chain growth polymerized ALGMA hydrogels resulted in higher cell proliferation
on stiffer substrates. The
significantly higher swelling response to basic pH environments as well as the
instigation of myofibroblast differentiation suggests the potential of these
hydrogel formulations responsive microenvironments for studying how fibroblasts
would respond in chronic wound environments.
Acknowledgements: This work was supported by Roy J. Carver Charitable Trust Grant No.
13-4265 and the Mike and Denise Mack faculty fellowship to Kaitlin Bratlie.