Poster M16 - Role of Photoinitiator in Regulating Cell Response for Cell-Laden Gelatin Methacryloyl Bioinks

Introduction:

Introduction: Cell-laden hydrogel systems crosslinked by light energy have been widely used for tissue engineering and in vitro disease modeling [1]. The biocompatibility issues of the photoinitiators (PIs) associated with reactive species (i.e., free radicals, cations, or anions) have limited their applications of PIs [2]. For example, PI can lead to cytotoxicity, genotoxicity, and oxidative stress. Reactive oxygen species (ROS) created by free radicals are crucial in cellular signaling and play a pivotal role in wound healing and tissue regeneration. Their overproduction can lead to oxidative stress, potentially damaging cells [3]. This dual nature of ROS underscores the importance of carefully balancing their levels in tissue engineering. We studied how ROS in mesenchymal stem cells (MSCs) in gelatin-based hydrogels is correlated with three classes of PIs well used in the literature: Eosin Y, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and Irgacure 2959. We measured cellular oxidative stress, a critical factor influencing cell viability, stem cell fate, and function in tissue engineering applications. Our results help bioengineers better design cell-laden hydrogel systems for creating biomimetic scaffolds and disease models.

Materials and

Methods:

Methods: We synthesized gelatin methacryloyl (GelMA) [4] to prepare a 5% w/v GelMA hydrogel precursor with varying concentrations of LAP, Irgacure, and Eosin Y (0.01 to 1% w/v). Crosslinking times were optimized for LAP and Irgacure samples under 365 nm light at 10 mW/cm², and for Eosin Y under 512 nm light at 12 mW/cm². Hydrogels were subjected to swelling and biodegradation tests based on a well-established protocol [4]. Standard compression testing was conducted to determine the stiffness of the material under physical load. Bioink was prepared with bone marrow human mesenchymal stem cells (PT-2501, Lonza) with varying PI types and concentrations to investigate the role of PI type and concentration on stem cell fate. We conducted Live/Dead, CCK-8, and DCFDA/H2DCFDA- Cellular ROS assays. Focal adhesion properties were investigated via actin cytoskeleton and focal adhesion staining according to the manufacturer's protocol (FAK100, Millipore Sigma, Millipore). Statistical analysis was performed using the GraphPad Prism statistical tool. A one-way analysis of variance (One-way ANOVA) test was used for data analysis.

Results, Conclusions, and Discussions:

Results: Increasing PI concentrations in GelMA hydrogels led to higher crosslinking density, resulting in a higher compressive Young’s modulus, and reducing the swelling/degradation rates. Conversely, samples subjected to collagenase degradation with the lowest concentration of PIs showed the highest mass loss, suggesting a less cross-linked network that was more susceptible to enzymatic cleavage. In contrast, hydrogels with the highest PI concentrations maintained a significant percentage of their mass up to Day 5, demonstrating a more densely cross-linked matrix that resisted collagenase activity. CCK-8 and Live/Dead assays indicated that cell viability within the GelMA hydrogels was influenced by the concentration and the type of photoinitiator, with LAP and Eosin Y samples showing higher proliferative and viability rates than those with Irgacure. ROS generation showed a parallel trend with viability and proliferation, where decreased PI concentration and the use of LAP and Eosin Y led to lower ROS generation. Morphologically, MSCs exhibited a more branched distribution in GelMA with lower PI ratios, whereas a round and compact morphology was observed in GelMA with higher PI concentrations. These observations suggest that increased PI concentration leads to a denser hydrogel network (stiffer matrix) and higher ROS generation, which synergistically influencing stem cell fate. Additionally, MSCs exhibited increased actin filament formation at lower concentrations of LAP compared to Irgacure samples, indicating that both the type and concentration of PI significantly influence focal adhesion properties and, ultimately, stem cell fate.

Conclusion: The concentrations and types of PIs significantly impact the biophysical properties of GelMA hydrogels and, consequently, the cellular mechanisms in MSCs. In future studies, we will examine how ROS generation affects these cellular mechanisms and stem cell fate, specifically analyzing related genes such as p21, p53, Ki-67, and CASP3 using RT-qPCR.

Acknowledgements (Optional): References

[1] Goodarzi H. et al. ACS Biomater. Sci. Eng, 2022.
[2] Chen M. et al. Chemical Reviews, 2016.
[3] Hu Q. et al. Oncotarget, 2018.
[4] Dogan E. et al. Acta Biomaterialia, 2024 (submitted).