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Biomechanics

Biomechanics - Poster Session D

Poster E3 - Multilayered Hydrogel Stiffness Evaluation for Engineering Cell Environments

Friday, October 25, 2024
3:30 PM - 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction: Recreating complex cell environments with varying structures require complex materials that contain differing architectures such as layers. However, many in vitro models of cell behavior utilize homogenous biomaterials as platforms for cell cultures. For the past decade, studies have committed to understanding cell models through experimentation on variations of structures such as hydrogels. Throughout the literature, cells are characterized by their mechanical environments in particular. One factor of their environment that can be manipulated is stiffness. Our group’s focus is on connective tissues with complex structures such as the intestinal wall and heart valves. Heart valve interstitial cells are responsive to their mechanical environment, but there is still limited research measuring their growth on multilayered hydrogels. With an aim to characterize and optimize the appropriate hydrogel formulation for intestinal epithelium, the project focuses on the gel and cell interactions building upon previous research methods published from the Grande-Allen Lab. This study fabricates hydrogels with latex fluorescent beads utilizing a 3D printed spin coater designed to spread the bead layers providing a vivid illustration of tissue and organ replication. Through porcine aortic valve interstitial cells (PAVICs) and green fluorescent protein (GFP), the cells will operate using the gene replicator to display coloring simultaneously of the fluorescent beaded gels allowing for analysis and modification through microscopy.

Materials and

Methods: To fabricate gels of different PEGDA percentages, silicone molds were prepared and added to glass sides treated with a commercial silicon solution. The first procedure was making the prepolymer solution added with the photoinitiator made with varying weight-per-volume percentages ranging from 2-5%. For 1mL of solution, the following were combined in a sterile and low-light environment: 1mL of Thioxoborane (HBS) +1.5% Triethanolamine (TEAO) solution; 10uL of 1mM tetrabromofluorescein (Eosin Y) solution; 3.5 uL of N-Vinyl-2-Pyrrolidone (NVP). After the solution was combined, the PEGDA weight was added to the solution, and vortexed for 30 seconds. Solution was later pipetted into the silicon molds to crosslink under white light for 8 minutes, then were carefully removed from slides with PBS and placed into petri dishes filled with PBS to allow the gels to hydrate and swell overnight. After 24hrs, gels were characterized for their mechanical properties. The process remained mostly unchanged when it came to the fabrication of the bilayer hydrogels, the main change being crosslinking time. The first, stiffer, gels were partially crosslinked for 4.5 minutes, then the second gels were added for an additional 10 min. This procedure would change to all layers crosslinking for 4.5 minutes then varying PEGDA layers crosslinked with molded plates coated with silicone solutionwhere the gel would rest to avoid gel damage and encourage layered crosslinking. In addition to standard gels, gels containing latex fluorescent beads were prepared using a custom built spin coater.

Results, Conclusions, and Discussions: Gels made from 2.5% PEGDA were very weak and broke easily; their stiffness was 3.5kPa. Hydrogels with 5% PEGDA yielded stiffness of 7-12 kPa. 7.5% PEGDA gels displayed a stiffness of 26-33 kPa. The single layer hydrogels compression tested granted stiffer curves with higher percentages of PEGDA. As for the bilayered hydrogels, they experienced often delamination between halves and tearing during removal from the molds. The difference in stiffness and swelling in the layers and the possibly insufficient application of the silicon solution may have been the cause of these issues. Additionally, the higher the PEGDA fraction, the more often the gels attached to the used glass slide, probably also caused by the extended time cross linking. In addition to the single layered gels, the bilayered gels of both 5% PEGDA displayed stiffness records of 34kPa. Addition of the fluorescent beads was challenging and the beads solution would clump unless centrifugal force was applied to spread the beads around the gel solution.
Preparing the layered hydrogels with varying stiffnesses in the different layers was challenging. Future procedures would benefit from crosslinking all layers for 4.5min instead of just the top gel layer, as we found with the bilayer 5%/5% gel.
Filling the gel molds and removing the crosslinked gels was also tricky. It was often necessary to overfill by 30% to ensure gels were made without air pockets. It was noted that higher PEGDA gels were more adhesive to the glass slides underneath the mold. Applying a silicon solution to the glass slide, which helped with the gel removal.
After optimizing gel formation, applying the fluorescence beads to display the gel layers was improved by using a spin coater. The beads applied without a spin coater clumped into large masses appearing as one large volume.
The work represented an initial study to use different PEGDA percentages for bilayering hydrogels to replicate differing mechanical environments of cells. Future work will apply GFP transfected cells to the bilayered gel surface and track the cell behavior and fluorescent bead deformation allowing for analysis and modification through microscopy.

Acknowledgements (Optional): Rice University Take Flight Program