Visible-Light Stiffness Patterning of GelMA Hydrogels Towards In Vitro Scar Tissue Models

Abstract

Variations in mechanical properties of the extracellular matrix occurs in various processes, such as tissue fibrosis. The impact of changes in tissue stiffness on cell behaviour are studied in vitro using various types of biomaterials and methods. Stiffness patterning of hydrogel scaffolds, through the use of stiffness gradients for instance, allows the modelling and studying of cellular responses to fibrotic mechanisms. Gelatine methacryloyl (GelMA) has been used extensively in tissue engineering for its inherent biocompatibility and the ability to precisely tune its mechanical properties. Visible light is now increasingly employed for crosslinking GelMA hydrogels as it enables improved cell survival when performing cell encapsulation. We report here, the photopatterning of mechanical properties of GelMA hydrogels with visible light and eosin Y as the photoinitiator using physical photomasks and projection with a digital micromirror device. Using both methods, binary hydrogels with areas of different stiffnesses and hydrogels with stiffness gradients were fabricated. Their mechanical properties were characterised using force indentation with atomic force microscopy, which showed the efficiency of both methods to spatially pattern the elastic modulus of GelMA according to the photomask or the projected pattern. Crosslinking through projection was also used to build constructs with complex shapes. Overall, this work shows the feasibility of patterning the stiffness of GelMA scaffolds, in the range from healthy to pathological stiffness, with visible light. Consequently, this method could be used to build in vitro models of healthy and fibrotic tissue and study the cellular behaviours involved at the interface between the two.

Keywords: GelMA; digital micromirror device; force indentation; hydrogel; mechanical properties; photopatterning; projection; visible light crosslinking.

FIGURE 1

Scheme of the reactions for the synthesis of GelMA (A) and for the formation of GelMA hydrogels (B).

FIGURE 2

AFM force indentation measurements of the average elastic moduli of 10 wt% (black diamond) and 15 wt% (red circle) GelMA hydrogels as a function of their exposure time to the green LED. The error bars represent the standard error (10 wt%: between 15 and 21 force maps over four separate gels for each exposure time; 15 wt%: between 27 and 31 force maps over seven separate gels for each exposure time).

FIGURE 3

Stiffness patterning of 10 wt% GelMA hydrogels crosslinked for 4 min under the green LED with two binary photomasks: “33–0” (A) and “50–0” (B) masks. The elastic modulus of the hydrogels was significantly reduced by the opaque area of the photomask, which modulated the green LED irradiance (error bars are the standard error over at least six force maps on two samples per photomask, p < 0.0005). Photograph of a “50–0” binary photomask placed on top of a pre-gel solution into a mould (C).

FIGURE 4

Gradient of stiffness on a 10 wt% GelMA hydrogel crosslinked for 4 min under the green LED (A) (error bars = standard deviation of the force maps). Photograph of the gradient filter (0.04–4.0 OD) used for the patterning of hydrogels (B). The red circle represents the approximate position of the sample under the mask.

FIGURE 5

Crosslinking of GelMA via the projector system (A). With this system, a digital image is directly projected at the surface of the pre-gel solution to create patterns of stiffness in the GelMA (B). The resulting elastic modulus of 10 wt% GelMA hydrogels according to their exposure time under the projector has been measured by force indentation measurements with AFM (C) (errors bars: standard error over five force maps per exposure time).

FIGURE 6

Relationship between the projected irradiance and the 10 wt% GelMA gels’ elastic moduli after exposure of 4 min (A). The average elastic moduli have been assessed by force indentation measurements with AFM (error bars: standard error over at least four force maps per area). Images of the gels taken with a USB microscope show that the projector system does not produce sub-patterning over the masked area (B) compared to patterned gels prepared with the printed photomask (C) (scale bars = 2 mm). The numbers on (B,C) are the greyscale values that were applied to the gels. The boundary between the two areas of a 1/0.5 binary gel crosslinked for 3 min was probed with AFM and showed a height gradient (D) as well as a stiffness gradient on the force map (E) (scale bars: 20 μm). The numbers above maps (D,E) represent the orientation of the boundary in terms of greyscale values. Three lines on the force map (E) were averaged (lines identified with *) and plotted against the distance to show the strength of the gradient (F) (error bars: standard error of the mean over the three lines).

FIGURE 7

Stiffness gradients with the projector set-up. 10 wt% GelMA hydrogels were crosslinked for 4 min with a gradient ranging from greyscale values of 1 to 0.5 over a distance of approximately 6 mm (A) or 2 mm (B). Another method to produce a stiffness gradient with a sliding mask was also used (C). Error bars: standard deviation of the elastic modulus over the force maps.

FIGURE 8

Complex shapes such as a star (A) or the University of Auckland logo (B) were projected for 4 min at the surface of a 10 wt% GelMA pre-gel (scale bars: 1 mm). It resulted in hydrogels with specific designs (the gels were dried for 1 h at RT before the pictures were taken).