Characterizing the Effects of Synergistic Thermal and Photo-Cross-Linking during Biofabrication on the Structural and Functional Properties of Gelatin Methacryloyl (GelMA) Hydrogels

Abstract

Gelatin methacryloyl (GelMA) hydrogels have emerged as promising and versatile biomaterial matrices with applications spanning drug delivery, disease modeling, and tissue engineering and regenerative medicine. GelMA exhibits reversible thermal cross-linking at temperatures below 37 °C due to the entanglement of constitutive polymeric chains, and subsequent ultraviolet (UV) photo-cross-linking can covalently bind neighboring chains to create irreversibly crosslinked hydrogels. However, how these cross-linking modalities interact and can be modulated during biofabrication to control the structural and functional characteristics of this versatile biomaterial is not well explored yet. Accordingly, this work characterizes the effects of synergistic thermal and photo-cross-linking as a function of GelMA solution temperature and UV photo-cross-linking duration during biofabrication on the hydrogels' stiffness, microstructure, proteolytic degradation, and responses of NIH 3T3 and human adiposederived stem cells (hASC). Smaller pore size, lower degradation rate, and increased stiffness are reported in hydrogels processed at lower temperature or prolonged UV exposure. In hydrogels with low stiffness, the cells were found to shear the matrix and cluster into microspheroids, while poor cell attachment was noted in high stiffness hydrogels. In hydrogels with moderate stiffness, ones processed at lower temperature demonstrated better shape fidelity and cell proliferation over time. Analysis of gene expression of hASC encapsulated within the hydrogels showed that, while the GelMA matrix assisted in maintenance of stem cell phenotype (CD44), a higher matrix stiffness resulted in higher pro-inflammatory marker (ICAM1) and markers for cell-matrix interaction (ITGA1 and ITGA10). Analysis of constructs with ultrasonically patterned hASC showed that hydrogels processed at higher temperature possessed lower structural fidelity but resulted in more cell elongation and greater anisotropy over time. These findings demonstrate the significant impact of GelMA material formulation and processing conditions on the structural and functional properties of the hydrogels. The understanding of these material-process-structure−function interactions is critical toward optimizing the functional properties of GelMA hydrogels for different targeted applications.

Multiscale Anisotropic Tissue Biofabrication via Bulk Acoustic Patterning of Cells and Functional Additives in Hybrid Bioinks

“…The pore sizes were determined from CryoSEM images (3500X magnification) using a custom algorithm in MATLAB that we developed in our previous work. [43] Assessment of Viability and Elongation of Cells in Pure GelMA Constructs under Different Thermo-Reversible and Photocrosslinking Conditions: For the study shown in Figure 2, the media above the constructs in the 24-well plates was extracted and 1 mL of PBS containing Live/Dead assay reagents (L3224, Thermo Fisher) at a concentration of 0.25 μL mL −1 for calcein-AM and 1 μL mL −1 of ethidium homodimer-1 added. The constructs were then washed and imaged using fluorescence microscopy (SP8, Leica Microsystems).…”

“…The GelMA used in this study was purchased in a lyophilized form (80% degree of substitution (DOS), 900 496, Millipore Sigma). The consistency of DOS across different purchased batches was verified using 1 HNMR spectroscopy [ 43 ] to be within ±7.5%. The lyophilized GelMA was constituted at 5% w/v in phosphate buffered saline (PBS) and the photoinitiator Lithium phenyl‐2,4,6‐trimethylbenzoylphosphinate (LAP) added at a concentration of 0.25% w/v to formulate the pure GelMA photoink.…”

“…temperatures induces entanglement of polypeptide networks, and subsequent UV crosslinking creates covalent bonds between the entangled networks to prevent their de-crosslinking when the temperature is increased, [43] which is evident in the CryoSEM images (Figure 2A).…”

“…Figure 2. Investigating the effects of dual-crosslinking property of GelMA photoinks and synthesis of hybrid GelMA formulations containing CM or PM.A) Below 37 °C, polypeptide chains of GelMA undergo tight interlinking leading to thermo-reversible gelation, and subsequent photocrosslinking leads to formation of nonreversible covalent crosslinking of the GelMA matrix [43]. Based on the pre-photocrosslinking solution temperature and subsequent UV exposure duration, varying degree of crosslinking can be achieved within the GelMA matrix.…”

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

“…However, the liquid can cool down very rapidly with such a small volume. Several studies found that cooling down a GelMA pre-gel solution to temperatures below 25 °C before crosslinking actually increased the elastic modulus of the crosslinked hydrogel compared to crosslinking the pre-gel solution directly at RT (Rizwan et al, 2017;Young et al, 2020;Chansoria et al, 2021). This is due to the physical gelation of the gelatine before light-curing, which induces a better organisation of the gelatine chains resulting in a higher crosslinking density.…”