(469f) Impact of Microgel Aspect Ratio on Granular Hydrogel Porosity and Cell Infiltration for Cardiac Application

Introduction: Injured cardiac tissue has limited capacity for self-repair, mainly due to the non-proliferative nature of adult cardiomyocytes. This continues to be one of the main challenges for developing therapeutics to treat cardiac injury following myocardial infarction (MI). Without medical intervention after MI, the cascade of deleterious remodeling significantly increases the likelihood of fatal heart failure. This work focuses on the development of injectable acellular granular hyaluronic acid (HA) hydrogels to provide cues to 1) limit the damage to the infarcted area and 2) allow revascularization and repair during ventricle remodeling. We hypothesize that these acellular granular hydrogels will allow for the recruitment of endogenous cells to mechanically bulk the cardiac tissue and limit adverse ventricular remodeling to improve overall cardiac function. Prior work with injectable acellular HA hydrogels for cardiac repair demonstrated improved angiogenesis and cardiac function, even without potent angiogenic cues(1); however, the lack of macroporosity of bulk hydrogels structures previously implemented limited rapid cellular infiltration. To address this limitation, we employed granular hydrogels, which exhibit an inherent porosity to support cell infiltration and activity while providing mechanical support (Fig. 1A).

Most granular hydrogels used for biomedical applications consist of spherical particles; however, we recently reported that granular hydrogels from rod-like microgels of aspect ratio 2.2 improved cellular invasion in both in vitro and in vivo studies(2). However, there is a limited understanding of how increased aspect ratios affect granular hydrogel packing, injectability, cell invasion, and tissue reconstruction. To address this, we fabricated rod-like microgels with increased aspect ratios of 3 to 5 (Fig. 1B) and compared these to control spherical microgels that matched the volumes of rods (Fig. 1C).

Methods and results: Microgels were fabricated using norbornene-modified hyaluronic acid (NorHA) macromers and using a PDMS-based single-channel microfluidic device. HA is a naturally-derived polymer that can interact with cells via the CD44 receptor and degrade in the presence of hyaluronidases and prior work showed that HA microparticles attenuate the fibrotic response after MI compared to PEG gels(3, 4). For this study, NorHA (3 wt%), dithiothreitol (0.08wt%), and a photoinitiator (Lithium phenyl 2,4,6 trimethylbenzoylphosphinate -LAP) (0.1wt%) were dissolved in PBS, to form the aqueous phase droplets in the microfluidic device. FITC-dextran (0.2wt%) was added into the aqueous phase to image the particles in the fluorescence microscope. A low viscosity perfluorinated oil HEF-7500 (3M) with 2% v/v of the surfactant Krytox-157 FSH (Dupont) was used to fabricate the rod-like particles. The average length of the rod-like particles varies from 255 to 355 m, and the aspect ratio ranges from 2.70 to 5.00. To produce spherical particles, mineral oil with Span 80 (2 wt%) was used as the continuous phase. The spheres were fabricated to match the rod-like particle volume. The average diameter of the spheres ranges from 129 to 172 m. Particles were washed multiple times and suspended in PBS as previously described (2).

Microgels were jammed using centrifugation to form granular hydrogels. The jammed granular hydrogels were transferred to a glass bottom 96-well plate for imaging. Granular hydrogel porosity was assessed by analyzing z-stacks of confocal images, which showed a higher porosity for rods (Fig. 1D). This trend was confirmed by performing rigid body dynamic simulations in Cinema4D from sphere and rod microgels that matched experimental microgel measurements (Fig. 1F). For simulations, the porosity increased with the particle dimensions and the porosity was larger for rods than spheres of equivalent volume. In addition, the z-stacks of granular hydrogels were analyzed using BoneJ, a FIJI plugin, to skeletonize the interstitial space in 3D and determine branch lengths between pores as a representation of mean free migratory pathways (Fig. 1F). The analysis demonstrated longer uninterrupted pathways in granular hydrogels from rods when compared to their volume-matched sphere counterparts. To monitor cellular invasion, spheroids of endothelial/mesenchymal cells (2:1 ratio, ~1000 cells/spheroid, prepared with AggreWell 400 templated agarose wells) were introduced to the granular materials and cultured for 3 days to assess cells sprouting in the granular material using confocal microscopy (Fig. 1G). Sprout displacements were larger when the spheroids were placed in the granular hydrogels from microgels with high aspect ratio rods when compared to spheres of similar volume (Fig. 1H). These results demonstrate that rods of higher aspect ratios have higher porosity and cell sprouting, providing key engineering design aspects for granular material in biomedical applications.

Towards biomedical applications, injectability is an important design feature. Rheological analyses demonstrate the shear-yielding behavior of the granular hydrogels across formulations (Fig. 1I). For cardiac applications; we previously showed the injectability of granular hydrogels into cardiac tissue through these features (5).

Future Work: Ongoing work includes the assessment of granular hydrogels in a rat model of MI. The knowledge gained from these studies will help in the design of new therapeutics for treating MI. The findings could also be applied to other microgel formulations and applications in which porosity and pore sizes could be adjusted by changing the particle aspect ratio to promote cell infiltration.

References
1. S. J. Yoon et al., Regeneration of ischemic heart using hyaluronic acid-based injectable hydrogel. Journal of Biomedical Materials Research Part B: Applied Biomaterials 91B, 163-171 (2009).
2. T. H. Qazi et al., Anisotropic Rod-Shaped Particles Influence Injectable Granular Hydrogel Properties and Cell Invasion. Advanced Materials 34, 2109194 (2022).
3. L. V. Le et al., Injectable hyaluronic acid based microrods provide local micromechanical and biochemical cues to attenuate cardiac fibrosis after myocardial infarction. Biomaterials 169, 11-21 (2018).
4. Microtopographical Cues in 3D Attenuate Fibrotic Phenotype and Extracellular Matrix Deposition: Implications for Tissue Regeneration. Tissue Engineering Part A 16, 2519-2527 (2010).
5. J. E. Mealy et al., Injectable Granular Hydrogels with Multifunctional Properties for Biomedical Applications. Advanced Materials 30, 1705912 (2018).