Fig 1.
Synthesis of fish gelatin methacryloyl (GelMA) and fabrication of photocrosslinked GelMA hydrogel.
(A) Gelatin was reacted with methacrylic anhydride (MA) to introduce a methacryloyl substitution group on the reactive amine and hydroxyl groups of the amino acid residues. (B) GelMA photocrosslinking to form A hydrogel matrix under UV irradiation. The free radicals generated by the photoinitiator initiated chain polymerization with methacryloyl substitution. (C) Schematic of formation of patterned hydrogels using photolithography.
Fig 2.
Degree of methacrylation as determined by TNBS assay.
(A) Various volume percentages of methacrylic anhydride (0.25%, 1.25% and 20%) were analyzed to investigate the degree of methacrylation of the synthesized fish GelMA. (B) Comparison of a high degree of methacrylation (20% MA) according to the origin of gelatin (fish GelMA vs. porcine GelMA); there was no significant difference. The percentage of incorporated substitution was calculated by comparing the amount of remaining amino groups (-NH2) in GelMA to that in pristine gelatin. Error bars represent standard deviations (SDs) of measurements performed on six samples.
Fig 3.
Mechanical properties of fish GelMA hydrogels with various degrees of methacrylation and gel percentages.
(A) Compressive modulus for 5%, 10% and 15% (w/v) fish GelMA at low, medium and high degree of methacrylation, with the exception of low degree, 5% and 10% (w/v) GelMA which formed gels too weak to be handled for testing. (B) Representative stress-strain curve of a 15% (w/v) GelMA according to degree of methacrylation. (C) Comparison of fish and porcine GelMA hydrogels with a high degree of methacrylation. (D) Representative curve of 15% (w/v) GelMA for fish and porcine GelMA comparison. Conditions of 10% and 15% (w/v) GelMA were significantly different (*p<0.05, **p<0.01, ***p<0.001). Error bars represent SDs of measurements performed on 3 samples.
Fig 4.
Equilibrium swelling properties of GelMA hydrogels.
(A) The mass swelling ratios of fish GelMA hydrogels containing 5%, 10% and 15% (w/v) GelMA and low, medium and high degrees of methacrylation were significantly different (*p<0.05, **p<0.01, ***p<0.001). GelMA (5% and 10% (w/v)) with low methacrylation produced hydrogels which were too weak to be handled and therefore were not tested. (B) Comparison of fish and porcine GelMA hydrogels with a high degree of methacrylation. Error bars represent SDs of measurements of five samples.
Fig 5.
Degradation characteristics of fish GelMA hydrogels.
(A) Degradation profiles of fish GelMA hydrogels with various degrees of methacrylation (low, medium and high) and GelMA concentrations (5%, 10% and 15%) upon exposure to collagenase type II. (B) Comparison of fish and porcine GelMA hydrogels with high degree of methacrylation and 10% gel concentration. Error bars represent SDs of measurements performed on three samples. Representative cross-sectional SEM images of fish GelMA (C~F) and porcine GelMA (G~J) hydrogels reveal different gel morphologies after degradation with collagenase type II. (K) Pore size distribution of GelMA hydrogels (Pore size frequency obtained from 5 SEM images per condition).
Fig 6.
Fabrication of micropatterned fish GelMA hydrogel and viability of cells on micropatterned gel surfaces.
NIH3T3 cells readily adhered to fish GelMA surfaces irrespective of macromer concentration. (A-C) Pattern fidelity of fish GelMA using 5%, 10% and 15% macromer (scale bar = 800 μm). (D-F) LIVE/DEAD assay at 24 h after adhesion (scale bar = 200 μm). (G) Quantification of cell viability demonstrated high cell survival under all conditions and there was no significant difference between GelMA conditions. Error bars represent SDs of averages obtained from five images per condition.
Fig 7.
Cell adhesion and proliferation on fish GelMA surfaces.
NIH3T3 cells on 5%, 10% and 15% (w/v) GelMA with a medium and high degree of methacrylation adhered and proliferated (day 5). (A-I) Representative images of NIH3T3 cells on fish GelMA surfaces stained with phalloidin (green)/DAPI (blue) on day 1 and day 3 of culture (scale bar = 100 μm). (J-L) MTS assay of cells on GelMA hydrogels after 1, 3 and 5 days. Error bars represent SDs of averages of three samples per condition. There were no significant differences between fish and porcine GelMA.
Fig 8.
Behavior of cells encapsulated in fish GelMA hydrogels.
(A-F) NIH3T3 cells embedded in medium and high degree of methacrylation fish GelMA containing 5% (w/v) GelMA were stained using calcein-AM/ethidium homodimer at 3 h and 24 h after encapsulation to evaluate the cell viability compared to high degree of methacrylation porcine GelMA. (G-I) After 2 days of culture, cells proliferated and elongated in fish and porcine GelMA hydrogel. (J-L) Representative images of the cells stained with phalloidin for the actin filaments (green) and nuclei counterstained with DAPI (blue) at 72 h. (P) Viability of the encapsulated cells. (Q) Quantification of cell proliferation in GelMA hydrogel until 5 days. Cell proliferation rate was not significantly different at all condition.