Biophysically defined and cytocompatible covalently adaptable networks as viscoelastic 3D cell culture systems - PubMed (original) (raw)

Biophysically defined and cytocompatible covalently adaptable networks as viscoelastic 3D cell culture systems

Daniel D McKinnon et al. Adv Mater. 2014.

Abstract

Presented here is a cytocompatible covalently adaptable hydrogel uniquely capable of mimicking the complex biophysical properties of native tissue and enabling natural cell functions without matrix degradation. Demonstrated is both the ability to control elastic modulus and stress relaxation time constants by more than an order of magnitude while predicting these values based on fundamental theoretical understanding and the simulation of muscle tissue and the encapsulation of myoblasts.

Keywords: dynamic materials; hydrogels; polymeric materials; self-healing materials; supramolecular chemistry.

© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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Figures

Figure 1

Figure 1

a) Table showing the relevant kinetic and thermodynamic information for reactions between N-methyl hydrazine and butyraldehyde or 4-nitrobenzaldehyde at 25 °C in pH 7.4 buffer containing 1% DMSO. b) Chemical structures of 4-H and 4-AA showing reversible gelation. c) Rheological time sweep plot showing rates of gelation. Evolution of 90% of G∞ occurs in 5 minutes for 4-H:4-AA (thick solid line, G’; thick dashed line G”) and ca. 1 hour for 4-H:4-BA (thin solid line, G’; thin dashed line G”).

Figure 2

Figure 2

a) Hydrogel modulus can be controlled by incorporating crosslinkers of different functionalities or varying the stoichiometry of functional groups (dark bars, on stoichiometry; light bars 50% excess hydrazine, Student's t-test, p < 0.05). b) 4-H:4-AA shows frequency-dependent crossover below 0.03 rad/s, which indicates that it behaves as a Maxwellian viscoelastic fluid (filled squares, G’; empty squares, G”; solid line, G’ Maxwell fit; dashed line, G” Maxwell fit).[43] c) Stress relaxation is strongly dependent on the ratio of aliphatic to aryl aldehyde crosslinker, with relaxation times ranging from tens of seconds to tens of hours (bars, 100:0 4-AA:4-BA; triangles, 80:20; circles, 0:100). When the mole fraction of 4-BA crosslinker crosses the percolation threshold of the system, the stress relaxation behavior collapses to that of 0:100. Stress relaxation of 4-H:4-AA and 4-H:4-BA are in good agreement with the Maxwell model (dashed lines). Covalent adaptability can be abolished upon treatment by sodium cyanoborohydride, which reduces the hydrazone bond to the corresponding secondary hydrazine (asterisks). d) Stress relaxation can be shown macroscopically through molding. 4-H:4-AA and 4-H:4-BA were pressed into square and triangular molds for 60 s, with 4-H:4-AA adopting the shape of the mold as a viscoelastic fluid and 4-H:4-BA retaining its shape as a more elastic solid (scale bar = 3 mm).

Figure 3

Figure 3

The rheological properties of complex tissue can be emulated using covalently adaptable hydrogels. a) The elastic modulus of mouse muscle (thin solid line, G′; thin dashed line, G″) begins to sharply decrease below 1 rad/s, possibly due to the fibrous nature of the tissue, while the 8-H:8-AA (thick solid line, G′; thick dashed line, G″) exhibits this property below 0.05 rad/s. However, G∞ of the mouse muscle can be exactly matched. b) The mouse muscle (triangles) demonstrates significant stress relaxation over 10 minutes and fits a two-mode Maxwell model (solid line). 8-H:8-AA (diamonds) comes close to reproducing the initial and final stress with just one mode of relaxation. 8-H:8-BA (squares) relaxes much more slowly.

Figure 4

Figure 4

a) Images of encapsulated C2C12 myoblasts 72 hours post-encapsulation stained with calcium-AM (green, live) and ethidium homodimer (red, dead; scale bar 200 μm). b) Cell viability quantified at 24 hours (dark bars), 72 hours (medium bars), and 240 hours (light bars). c) Representative images of cells stained for f-actin (red) and the nucleus (blue) are shown (scale bar 20 μm). After 10 days in culture, the cells encapsulated in 8-H:8-AA exhibit significant spreading and robust actin fiber formation. Cells encapsulated in 8-H:(80% 8-AA,20% 8-BA) extend lamellipodia and filopodia and show actin filaments but do not deviate as far from rounded. Cells encapsulated in 8-H:8-BA remain rounded. d) Some cells in the 8-H:8-AA gels fuse into multinuclear structures. e) Quantification of cell spreading (dark bars, left axis) and fraction of cells extending processes (light bars, right axis) by 10 days in culture. Cells in 8-H:8-AA and 8-H:(80% 8-AA,20% 8-BA) both displayed significantly more spreading, as measured by projected cell area and process extension, than those in 8-H:8-BA (Student's t-test, p<0.05).

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