Rheological Properties of Coordinated Physical Gelation and Chemical Crosslinking in Gelatin Methacryloyl (GelMA) Hydrogels

Abstract

Synthetically modified proteins, such as gelatin methacryloyl (GelMA), are growing in popularity for bioprinting and biofabrication. GelMA is a photocurable macromer that can rapidly form hydrogels, while also presenting bioactive peptide sequences for cellular adhesion and proliferation. The mechanical properties of GelMA are highly tunable by modifying the degree of substitution via synthesis conditions, though the effects of source material and thermal gelation have not been comprehensively characterized for lower concentration gels. Herein, the effects of animal source and processing sequence are investigated on scaffold mechanical properties. Hydrogels of 4-6 wt% are characterized. Depending on the temperature at crosslinking, the storage moduli for GelMA derived from pigs, cows, and cold-water fish range from 723 to 7340 Pa, 516 to 3484 Pa, and 294 to 464 Pa, respectively. The maximum storage moduli are achieved only by coordinated physical gelation and chemical crosslinking. In this method, the classic thermo-reversible gelation of gelatin occurs when GelMA is cooled below a thermal transition temperature, which is subsequently "locked in" by chemical crosslinking via photocuring. The effects of coordinated physical gelation and chemical crosslinking are demonstrated by precise photopatterning of cell-laden microstructures, inducing different cellular behavior depending on the selected mechanical properties of GelMA.

Keywords: GelMA; animal source; gelatin; gelatin methacryloyl; gelation; mechanical properties; photopolymerization.

Figure 1.

GelMA undergoes (a) only chemical gelation via crosslinking in the presence of Irgacure 2959 photoinitiator and UV light above the thermal gel point, (b) only physical gelation when cooled below the gel point, and (c) coordinated physical gelation and chemical gelation via crosslinking when cooled below the gel point and exposed to UV light in the presence of Irgacure 2959 photoinitiator. (d) Adherent cells undergo morphological changes depending on the GelMA crosslinking method and subsequent scaffold stiffness.

Figure 2.

GelMA synthesized from (a) porcine gelatin, (b) bovine gelatin, and (c) cold-water fish gelatin was photo-cross-linked (λ =365 nm) on a parallel plate rheometer at room temperature with 5mW·cm⁻² intensity for 5 minutes after incubation at 37°C to determine crosslinking kinetics. GelMA synthesized from (d) porcine gelatin, (e) bovine gelatin, and (f) cold-water fish gelatin was photo-crosslinked (λ =365 nm) with the same testing parameters after incubation at 4°C to determine crosslinking kinetics of materials with both physical and chemical crosslinks. GelMA samples were tested at high, mid, and low DS. Data represents the average Storage modulus (G’) across three replicates.

Figure 3.

Cure points for GelMA synthesized from different animal sources and (a) low, (b) mid, and (c) high degrees of substitution were determined based on crosslinking kinetics. Cure points were used to determine polymerization times with different UV intensities for all conditions, useful for defining printability of each material. The green region highlights cure points that require less than 30 seconds of UV Exposure, a metric important for ‘bioprintability’ and micropatterning. Error bars are plotted as the standard deviation (n≥3).

Figure 4.

Storage modulus (G’) was compared for samples polymerized after incubation at 37 °C and 4°C for (a) porcine GelMA (b) bovine GelMA and (c) cold-water fish GelMA. Generally, storage modulus (G’) increases with increasing DS. Porcine GelMA had higher storage moduli compared to samples synthesized from other sources and cold-water fish GelMA had the lowest. These measurements correlated to Bloom strength of unmodified gelatin. The trends noted for chemical crosslinking alone were conserved for dual crosslinked GelMA. Except for cold-water fish gelatin, which does not undergo physical gelation, GelMA samples demonstrated much higher storage moduli when dual crosslinked compared to chemical crosslinking alone. Storage modulus (G’) measurements of physical crosslinks alone were compared to that of coordinated physical and chemical crosslinks for (d) porcine GelMA, (e) bovine GelMA, and (f) piscine GelMA. Statistical analysis included t-test comparisons with a p-value of 0.05 (n=9). Error bars are plotted as the standard deviation.

Figure 5.

Rheological measurements were collected sweeping from 4-50 °C for porcine samples at (a) low, (b) mid, and (c) high degrees of substitution. GelMA polymerized at 4 °C is shown in blue and GelMA polymerized at 37 °C is shown in red. GelMA with physical crosslinking alone and the lowest DS showed the highest change in storage modulus (G’) between set temperatures, while the GelMA with the highest DS showed the lowest change in storage modulus (G’). GelMA crosslinked after incubation at 4°C showed less change in storage moduli with differing temperature compared to GelMA crosslinked at a warmer temperature. Data represents the average Storage modulus (G’) across three replicates.

Figure 6.

Human dermal fibroblasts adopted different morphologies depending on matrix stiffness. (a) HDFns grown on porcine GelMA polymerized at 4°C showed a longer more fibril morphology, while (b) HDFns cultured on top of porcine GelMA polymerized at 50°C showed a rounder morphology and cytoskeletal organization. (c) HDFns grown on GelMA polymerized at 4°C had a significantly higher aspect ratio compared to those from softer porcine GelMA scaffolds polymerized at 50°C. Cells were analyzed at n ≥ 55, imaged from 40X magnification. Statistical analysis included t-test comparisons with a p-value of 0.05. Error bars are plotted as the standard deviation.

Figure 7.

(a) Longer and thinner HDFn morphologies on stiffer gels and (b) rounder HDFn morphologies on softer gels were observed in (c) porcine GelMA scaffolds patterned using lithography and temperature differences. (d) Brightfield images show clear lines between stiff GelMA polymerized at 4°C and soft GelMA polymerized at 50°C.

Figure 8.

MDAMB231 human mammary spheroids were cultured within GelMA scaffolds with different animal sources, degrees of substitution, and polymerization conditions. Phalloidin and DAPI stains were used to determine cell invasion into surrounding matrix. Stiffer matrices resulted in minimal spheroid outgrowth. Matrices with storage moduli between 400 and 1100 Pa demonstrated invasion into the surrounding matrix. The GelMA with the highest and lowest storage modulus (G’) demonstrated minimal invasion into the surrounding matrix. Optimal cell proliferation was realized in hydrogels will storage moduli between 400 and 800 Pa.