Hydrogel substrate stress-relaxation regulates the spreading and proliferation of mouse myoblasts

Abstract

Mechanical properties of the extracellular microenvironment are known to alter cellular behavior, such as spreading, proliferation or differentiation. Previous studies have primarily focused on studying the effect of matrix stiffness on cells using hydrogel substrates that exhibit purely elastic behavior. However, these studies have neglected a key property exhibited by the extracellular matrix (ECM) and various tissues; viscoelasticity and subsequent stress-relaxation. As muscle exhibits viscoelasticity, stress-relaxation could regulate myoblast behavior such as spreading and proliferation, but this has not been previously studied. In order to test the impact of stress relaxation on myoblasts, we created a set of two-dimensional RGD-modified alginate hydrogel substrates with varying initial elastic moduli and rates of relaxation. The spreading of myoblasts cultured on soft stress-relaxing substrates was found to be greater than cells on purely elastic substrates of the same initial elastic modulus. Additionally, the proliferation of myoblasts was greater on hydrogels that exhibited stress-relaxation, as compared to cells on elastic hydrogels of the same modulus. These findings highlight stress-relaxation as an important mechanical property in the design of a biomaterial system for the culture of myoblasts.

Statement of significance: This article investigates the effect of matrix stress-relaxation on spreading and proliferation of myoblasts by using tunable elastic and stress-relaxing alginate hydrogels substrates with different initial elastic moduli. Many past studies investigating the effect of mechanical properties on cell fate have neglected the viscoelastic behavior of extracellular matrices and various tissues and used hydrogels exhibiting purely elastic behavior. Muscle tissue is viscoelastic and exhibits stress-relaxation. Therefore, stress-relaxation could regulate myoblast behavior if it were to be incorporated into the design of hydrogel substrates. Altogether, we showed that stress-relaxation impacts myoblasts spreading and proliferation. These findings enable a better understanding of myoblast behavior on viscoelastic substrates and could lead to the design of more suitable substrates for myoblast expansion in vitro.

Keywords: Alginate; Hydrogel; Myoblast; Stress-relaxation; Viscoelasticity.

Figure 1

Summary of the experimental approach and muscle stress-relaxation. A) C2C12 cells and primary myoblasts were cultured on elastic and stress-relaxing alginate hydrogels and their spreading and proliferation were assessed. B) Explanted muscle from a rat hindlimb was subjected to a constant 15 % strain compression, and the stress required to maintain the strain was monitored.

Figure 2

Alginate hydrogels used for culture studies. A) Chemical structure of the ionically crosslinked and covalently crosslinked alginate gels. Calcium was utilized for ionic crosslinking, while adipic acid dihydrazide (AAD) was utilized to form covalent linkages. B) Stress-relaxation behavior of ionically and covalently crosslinked alginate hydrogels subjected to constant 15 % strain compression. C) Initial elastic modulus of elastic (covalent) and stress-relaxing (ionic) hydrogels. Gels were crosslinked to different levels to allow the initial moduli to be matched. Moduli were measured from compression tests.

Figure 3

Spreading and proliferation of C2C12 cells on elastic and stress-relaxing hydrogels of various initial elastic moduli. A) Representative images of C2C12 cells cultured for 22 h on elastic and stress-relaxing hydrogels with initial moduli of 2.8, 12.2, 49.5 kPa. Actin is represented in green and the nucleus is represented in blue. Scale bar: 20 µm. B) Quantification of the spreading area of single C2C12 cells cultured on the different hydrogel conditions. C) Cell circularity was quantified. A value of 1 indicates a perfect circle and as the value approaches 0, the shape is increasingly elongated. D) The Aspect Ratio (ratio between the major and minor axis) was quantified. B,C,D) The area and the shape factors were quantified using ImageJ and the shape factor plug-in. For each condition, the values of cell area, circularity or aspect ratio were pooled from four gels, resulting in n = 434–737 cells analyzed per condition. E) Representative images of C2C12 cells stained for EdU, as a metric of cell proliferation. EdU positive nuclei are represented in red and non-EdU nuclei are represented in blue. Scale bar: 200 µm F) Quantification of the proliferation of C2C12 cells cultured on the different hydrogel conditions, as indicated by the percentage of EdU positive cells versus the total number of cells. Values represent the mean and the standard deviation (SD) of minimum three replicates. All data were compared using a two-tailed unpaired Student’s t-test (* p < 0.05, ** p < 0.01, *** p < 0.001). a.u. : arbitrary units.

Figure 4

Proliferation of primary myoblasts on elastic and stress-relaxing hydrogels of 12.2 kPa stiffness. A) Representative phase contrast image of primary myoblasts cultured on collagen-coated TCP at day 6. Scale bar; 1000 µm. B) Representative FACS plot demonstrating M-Cadherin and Syndecan-4 staining of primary myoblasts, and table with the percentage of cells staining positive for each marker. C–D) Representative CFSE staining profiles (C) and quantification of mean CFSE intensity (D) of primary myoblasts on elastic or stress-relaxing substrates, both with initial elastic modulus of 12.2 kPa. (E) Quantification of mean CFSE intensity of M-Cadherin positive primary myoblasts on elastic or stress-relaxing substrates of 12.2 kPa D–E) Values represent the mean and the standard deviation (SD) of minimum three replicates. Data were compared using a two-tailed unpaired Student’s t-test (** p < 0.01, ** p < 0.001).