Modulating the Porosity and Granular Diameter of Brain-mimetic Hydrogels for Cell Delivery.

Saturday, October 11, 2025

9:10 AM - 9:15 AM PDT

Location: Room 32B

Presenting Author(s)

Esmeralda Castaneda

Undergraduate Researcher
University of Oklahoma
Oklahoma City, Oklahoma, United States

Co-Author(s)

Christopher Pierce

Graduate Research Assistant
University of Oklahoma
Norman, Oklahoma, United States

Last Author(s)

John R. Clegg

Assistant Professor
The University of Oklahoma
Norman, Oklahoma, United States

Primary Investigator(s)

John R. Clegg

Assistant Professor
The University of Oklahoma
Norman, Oklahoma, United States

Introduction: : Injectable hydrogel scaffolds are emerging as a therapeutic for brain injuries and neural regeneration [1]. This could be particularly useful in the context of regulating immune responses following brain injury, to prevent chronic inflammation, which is characterized by excessive activation of microglia and macrophages to a classical by “M1” phenotype in the peri-injury tissue [4]. If unresolved, chronic inflammation can progress to neurodegenerative disease [4]. However, “M2” macrophages are mediators of the immune system that repair surrounding tissue and suppress inflammation. Our long-term goal is to deliver adoptive M2 macrophages to the peri-injury tissue as a therapeutic strategy, potentially reversing inflammation through the release of anti-inflammatory cytokines like IL-4 and IL-10. However, cellular therapies are limited by poor retention locally and low cell survival. These limitations of cell delivery could be overcome by using a granular hydrogel scaffold, a class of injectable and porous biomaterials, because of their interparticle porosity. The size of microspheres affects the degree of packing, with smaller microspheres leading to tighter packing and larger microspheres enhancing interstitial space, leading to nutrient transport and promotion of regenerative phenotypes [6]. An average diameter of ~200 µm allows for cell adherence and injectability. To increase viability and M2 cell phenotype, we hypothesize that granular hydrogels with microsphere diameters designed to achieve porosity in the 30-400 µm range will support a more “M2” rich environment [2]. The purpose of this study was to systematically optimize fabrication parameters for brain-inspired hydrogel microspheres, enabling their future use as scaffolds for macrophage delivery.

Materials and

Methods: : To fabricate the hydrogel precursor, hyaluronic acid methacrylate (HAMA), polyethylene glycol diacrylate (PEGDA), and lithium phenyl-2,5,6-triethylbenzoylphosphinate (LAP) were chemically crosslinked via UV exposure. Granular hydrogels were formed through a batch emulsion process, producing injectable microspheres [3]. To control microsphere diameter, hydrogel precursors were added dropwise into a stirring oil bath at different stirring speeds: 250 rpm, 450 rpm, and 650 rpm. Nitrogen purging was performed for 10 minutes to ensure microsphere formation before UV exposure to eliminate oxygen, which can inhibit crosslinking. UV exposure was set for 20 minutes at 200-Watt Mercury Arc to finalize the crosslinking. The microsphere diameter was imaged using an EVOS M7000 at 10x magnification (Figure 1a) and analyzed in ImageJ. To modulate porosity, microspheres were centrifuged at three stirring speeds: 250 rcf, 1000 rcf, and 1800 rcf, allowing controlled packing density and pore size formulation. Porosity of the assembled granular hydrogel was visualized using a Leica SP8 confocal microscope with a 20x oil immersion objective lens. Image analysis was performed using ImageJ to quantify pore architecture. Cells were blended with granular hydrogel group and bulk hydrogel group at different compositions, such as pure HAMA and HAMA blended with GelMA, in a 96-well plate to determine cell viability across different biomaterial structures. GraphPad Prism was used to conduct statistical analysis of microsphere diameter, granular hydrogel porosity, and cell viability.

Results, Conclusions, and Discussions:: The microsphere experimental run evaluated the impact of stirring speed on microsphere diameter. As shown in Figure 1, microspheres consistently formed at approximately 200 µm in diameter across all stirring conditions, regardless of stirring speed. However, the 650-rpm stirring speed produced the most uniform size distribution and was therefore selected for subsequent porosity modulation experiments. Porosity was controlled by varying centrifugation speeds during microsphere packing. Both the lowest (250 rcf) and highest packing (1800 rcf) resulted in greater individual pore size as well as porosity percentage, shown in Figure 2. Packing at 1000 rcf resulted in the smallest average pore size at ~100 µm2 and the lowest porosity percentage at ~13%. A higher cell viability was shown with the granular hydrogel compared to the bulk hydrogel at around 60%, being ~10% more than the bulk hydrogel, as shown in Figure 3. Our study successfully demonstrated the reproducible formation of microspheres with ~200 µm diameter and identified a centrifugation rate (1000 rcf) that yields a starting point for suitable pore sizes and optimal porosity. These findings will allow for future research, where primary macrophages are encapsulated by similar granular hydrogel biomaterials with varying porosity, such that we can determine the impact of specific physical scaffold parameters on macrophage cell activation.

Acknowledgements and/or References (Optional): : 1. Howard, D., Buttery, L. D., Shakesheff, K. M., & Roberts, S. J. (2008). Tissue engineering: strategies, stem cells and scaffolds. Journal of Anatomy, 213(1), 66–72. https://doi.org/10.1111/j.1469-7580.2008.00878.x
2. Yang, X., Gao, J., Yang, S., Wu, Y., Liu, H., Su, D., & Li, D. (2023). Pore size-mediated macrophage M1 to M2 transition affects osseointegration of 3D-printed PEEK scaffolds. International journal of bioprinting, 9(5), 755. https://doi.org/10.18063/ijb.755
3. Qazi, T. H., Wu, J., Muir, V. G., Weintraub, S., Gullbrand, S. E., Lee, D., Issadore, D., & Burdick, J. A. (2022). Anisotropic Rod-Shaped Particles Influence Injectable Granular Hydrogel Properties and Cell Invasion. Advanced materials (Deerfield Beach, Fla.), 34(12), e2109194. https://doi.org/10.1002/adma.202109194
4. Rotaru-Zăvăleanu, A. D., Dinescu, V. C., Aldea, M., & Gresita, A. (2024). Hydrogel-Based Therapies for Ischemic and Hemorrhagic Stroke: A Comprehensive Review. Gels (Basel, Switzerland), 10(7), 476. https://doi.org/10.3390/gels10070476
5. Feigin, V. L., Brainin, M., Norrving, B., Martins, S., Sacco, R. L., Hacke, W., Fisher, M., Pandian, J., & Lindsay, P. (2022). World Stroke Organization (WSO): Global Stroke Fact Sheet 2022. International journal of stroke: official journal of the International Stroke Society, 17(1), 18–29. https://doi.org/10.1177/17474930211065917
6. Annabi, N., Nichol, J. W., Zhong, X., Ji, C., Koshy, S., Khademhosseini, A., & Dehghani, F. (2010). Controlling the porosity and microarchitecture of hydrogels for tissue engineering. Tissue engineering. Part B, Reviews, 16(4), 371–383. https://doi.org/10.1089/ten.TEB.2009.0639