Impact of microgel aspect ratio on granular hydrogel porosity and cell infiltration for cardiac applications

Introduction:: Injured cardiac tissue has a 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). 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 and compared these to control spherical microgels that matched the volumes of rods.


Materials and Methods:: Microgels were fabricated using norbornene-modified hyaluronic acid (NorHA) macromers and using a PDMS-based single-channel microfluidic device. 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. To produce spherical particles, mineral oil with Span 80 (2 wt%) was used as the continuous phase. 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. We performed rigid body dynamic simulations in Cinema4D from spheres and rod-like microgels that matched experimental measurements to assess the theoretical porosity. 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.

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.


Results, Conclusions, and Discussions:: The average length of the rod-like particles varied from 255 to 355 mm, and the aspect ratio ranged from 2.70 to 5.00 (Fig. 1B). Various spheres batches were fabricated to match the rod-like particle volume (Fig. 1C). The average diameter of the selected spheres batches ranged from 129 to 172 mm, with a volume of approximately 1 to 3 nL. The experimental porosity after jamming the microgels was higher for rod-like particles (Fig. 1D). This trend was confirmed by the rigid body dynamic simulations in Cinema4D of the microgels that matched the experimental measurements (Fig. 1E). For the simulation results, the porosity increased with the particle dimensions, and the porosity was larger for rods than spheres of equivalent volume. The skeletonize analysis further quantified the interstitial space between microgels (Fig. 1G). The analysis demonstrated longer uninterrupted pathways in granular hydrogels from rods when compared to their volume-matched sphere counterparts.

The in vitro model for cell invasion demonstrated significant sprouting in all groups after 3 days (Fig. 1G). One key finding of this work was that the 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 less tortuosity to enable cell sprouting, providing key engineering design aspects for granular materials in biomedical applications.

Towards biomedical applications, injectability is an important design feature. Rheological analyses demonstrated 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). 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.


Acknowledgements (Optional): :

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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).