Poster R2 - Synthesis and Characterization of Gelatin Methacryloyl: Introducing Chemistry Students to the Applications of Hydrogels in Medicine

Introduction: Given that a significant number of chemistry and biochemistry majors at our primarily undergraduate institution (PUI) in Central Texas aspire to careers in medicine, we believe it is essential to familiarize our students with biopolymers they might encounter in the development of cutting-edge biomaterial therapeutics. Therefore, we created an experiential laboratory for synthesizing, refining, and characterizing gelatin methacryloyl (GelMA). GelMA is an appropriate material for an experiential undergraduate laboratory because the biopolymer is widespread in biomedical engineering literature and relatively facile to synthesize. We were particularly inspired by the recent efforts of the Lee, Cho, and Tan groups to develop refined quantitative protocols to develop GelMA with minimal batch-to-batch variation. In adapting these efforts to the undergraduate laboratory, we had several objectives. First, we wanted to expose upper-level undergraduate students to the interdisciplinary disciplines of tissue engineering and regenerative medicine. Second, we sought to illustrate the tangible real-world implications of the students’ synthesis efforts. Third, we aimed to provide students with a comprehensive production pipeline, from synthesis to purification to quantification, enabling students to gain experience with each material production stage. We hypothesized that students would strengthen their understanding of macromolecular chemistry, polymerization reactions, and purification techniques. In addition, we aimed to reinforce students’ ability to use a micropipette, a nearly ubiquitous tool in the molecular sciences, and Microsoft Excel. In summary, this laboratory experiment nurtures a holistic approach to scientific inquiry and encourages students to envision the transformative potential of chemistry in addressing real-world challenges.

Materials and

Methods: Groups of two or three undergraduate students carried out the procedures conducted in this experiential laboratory. Type A gelatin was added slowly to a 0.25 M CB buffer solution heated to 50 C and stirred until dissolved. The pH was then adjusted to 9.4 with NaOH. After adding methacrylic anhydride (MAAnh), the reaction proceeded for one hour at 50 C and was stopped by adjusting the pH to 7.4 with HCl. The solution was transferred into dialysis tubing and dialyzed against distilled water for at least 48 hours. After dialysis, the GelMA was frozen, lyophilized, and stored in a desiccator until use.

The final molecular structure of the modified polymer was determined via 1-H NMR. A TNBS assay was conducted to analyze the number of unmodified amine groups. A Fe(III)-hydroxamic acid-based assay test was performed to test for the presence of unmodified hydroxyl (-OH) groups. 10 wt%/v GelMA and 0.05 wt%/v of LAP were added to 5 mL DI H20 in a 20 mL scintillation vial and stirred at 37 C overnight until fully dissolved. The solution was cross-linked in the mold in an ultraviolet cross-linking cabinet (UVP CL-1000, ~365 nm) until cross-linked (about 3 J). The hydrogels were weighed regularly, and the swelling degree was calculated. A stress-strain curve was generated on a CellScale UniVert by compressing the sample at 1 mm/min for one minute with a pre-load set to 0.1 N.

Results, Conclusions, and Discussions: Since Southwestern University is a small undergraduate-only institution, class sizes are small to maximize instructor-student interaction; in the study described here, we supervised 14 students. Most students in the class section successfully synthesized GelMA with a high degree of modification, confirmed primarily by the two downfield methacryloyl peaks from 5.3-6.2 ppm. The TNBS assay adds a quantitative measurement of on-target modification of the free amino groups on the gelatin backbone. Some student groups successfully produced data that validated their 1-H NMR spectra results. Still, most absorbance readings of the gelatin sample were higher than the upper limit of the calibration curve. The Fe(III)-hydroxamic acid-based assay performed by the students generally yielded appropriate results, indicating minimal hydroxyl group modification. We compared two batches of GelMA: one with 0.317 mL MAAnh (LoGelMA) and 1 mL of MAAnh (HiGelMA). We observed, as expected, that the cross-linked HiGelMA maintained a compressive modulus about triple that of the LoGelMA and that the LoGelMA swelled significantly more than the LoGelMA.

An IRB-approved Google Form was used to evaluate student learning across the laboratory and determine if our protocols and instructions were easy to follow. From the responses, the students did the suggested pre-laboratory reading and gained familiarity with tissue engineering, hydrogels, and GelMA. The maturity and depth of the student responses were notably improved at the end. The students indicated that the laboratory was well-designed and either reinforced or enhanced their skills. Most students found the lab to be structured logically and that the resources provided them provided the necessary tools that may have been lacking from prior experience.

In conclusion, we demonstrated that GelMA, a well-established biomacromolecule in the biomaterials discipline, is particularly well-suited to be adapted to the undergraduate chemistry laboratory. The students at Southwestern University were able to follow the provided instructions by synthesizing and purifying this modified polymer. In future laboratories, we would like the students to fabricate bulk tissue constructs via 3D bioprinting and eventually incorporate mammalian cell culture to investigate cell-material interactions at the bulk and microscopic scale.

Acknowledgements (Optional): The authors would like to acknowledge the help of Andrew Vergote in refining the GelMA synthesis and purification protocol. The authors would also like to acknowledge funding support from the Welch Foundation (AF-0005), the Sam Taylor Fellowship, and internal funding from Southwestern University via a generous gift from Bob and Annie Graham.