Dual Crosslinked Gelatin Methacryloyl Hydrogels for Photolithography and 3D Printing

Abstract

Gelatin methacryloyl (GelMA) hydrogels have been used in tissue engineering and regenerative medicine because of their biocompatibility, photopatternability, printability, and tunable mechanical and rheological properties. However, low mechanical strength limits their applications in controlled drug release, non-viral gene therapy, and tissue and disease modeling. In this work, a dual crosslinking method for GelMA is introduced. First, photolithography was used to pattern the gels through the crosslinking of methacrylate incorporated amine groups of GelMA. Second, a microbial transglutaminase (mTGase) solution was introduced in order to enzymatically crosslink the photopatterned gels by initiating a chemical reaction between the glutamine and lysine groups of the GelMA hydrogel. The results showed that dual crosslinking improved the stiffness and rheological properties of the hydrogels without affecting cell viability, when compared to single crosslinking with either ultraviolet (UV) exposure or mTGase treatment. Our results also demonstrate that when treated with mTGase, hydrogels show decreased swelling properties and better preservation of photolithographically patterned shapes. Similar effects were observed when three dimensional (3D) printed and photocrosslinked substrates were treated with mTGase. Such dual crosslinking methods can be used to improve the mechanical properties and pattern fidelity of GelMA gels, as well as dynamic control of the stiffness of tissue engineered constructs.

Keywords: 3D printing; dual crosslinking; enzymatic crosslinking; microbial transglutaminase; photocrosslinking; photolithography.

Figure 1

Schematic elucidating the crosslinking mechanism of GelMA (a) Modification of gelatin to GelMA (b) Photocrosslinking of GelMA hydrogel (c) Enzymatic crosslinking of GelMA hydrogel.

Figure 2

Viscoelastic properties of hydrogels crosslinked with 30 s and 60 s of UV exposure with or without mTGase treatment (a) time sweep results (b) frequency sweep results for 30 s of UV exposure condition (c) frequency sweep results for 60 s of UV exposure condition. (w/: with; w/o: without; mTGase: microbial transglutaminase).

Figure 3

Swelling and mechanical characteristics of the gels (a) Swelling properties (b) Stiffness analysis using nanoindentation.

Figure 4

Enzymatic degradation of GelMA gels with or without mTGase treatment (mTGase: microbial transglutaminase).

Figure 5

3D printed constructs made of 2 layers, prepared using (a,b,e) 30 s of UV crosslinking without mTGase treatment, (f) 30 s of UV crosslinking with 30 min mTGase treatment, (c,d,g) 60 s of UV crosslinking without mTGase treatment, (h) 60 s of UV crosslinking with 30 min mTGase treatment. The treatment with mTGase reduced the swelling and helped the structure to preserve its shape. (Scale shows 2000 µm)

Figure 6

3D printed constructs made of 6 layers, prepared using (a,b,e) 30 s of UV crosslinking without mTGase treatment, (f) 30 s of UV crosslinking with 30 min mTGase treatment, (c,d,g) 60 of s UV crosslinking without mTGase treatment, (h) 60 s of UV crosslinking with 30 min mTGase treatment. (Scale shows 2000 µm for upper row and 200 µm for lower row)

Figure 7

Cell viability analysis of cell-laden micropatterned gels prepared using (a) 30 s of UV treatment without mTGase treatment (b) 30 s of UV treatment with 30 min mTGase treatment, (c) 60 s of UV treatment without mTGase treatment, (d) 60 s of UV treatment with 30 min mTGase treatment, (e) live cell percentage comparison for 30 s of UV treatment with or without mTGase treatment and (f) live cell percentage comparison for 60 s of UV treatment with or without mTGase treatment.

Figure 8

Micropatterned cell-laden gels created using photolithography (a) dumbbell patterns, (b) zoomed in view of dumbbell pattern, (c) triangular pattern, (d) dumbbell patterns, (e) zoomed in view of dumbbell pattern, (f) triangular pattern.