(A-27) Capturing Cell-Mediated Remodeling and Describing Granular Hydrogel Behavior Using a Yield-Stress Fluid Model

Introduction:: Granular hydrogels comprising packed hydrogel microparticles, or microgels, have emerged as a novel class of biomaterial with distinct advantages over traditional bulk hydrogels. These include injectability, heterogeneity, porosity, and enhanced biotransport. As granular hydrogels see more use, novel ways to characterize these constructs and cell-matrix interactions will become increasingly important. Such tools will be crucial to describe increasingly complex granular hydrogel models of natural tissues.

Granular hydrogels are commonly defined in terms of average rheological behavior, capturing quantitative metrics such as storage modulus, loss modulus, viscosity, etc. While storage modulus may provide information on cell-mediated remodeling, transient large amplitude oscillatory shear (LAOS) behavior may prove more sensitive to these processes. We aim to describe cell-mediated remodeling using brittility, a fitting parameter that measures mechanical heterogeneity, within a proposed yield-stress fluid model. To do this, we will encapsulate cells within microgels and culture for 1-4 weeks, taking weekly measurements. We hypothesize that brittility will increase as cells remodel their local environments over time. We also anticipate that granular mixtures composed of 50% acellular microgels and 50% cell-laden microgels may enhance the sensitivity of brittility as a measuring tool, as only half of the granular gel is subject to remodeling. We aim to use this yield-stress fluid model to describe granular hydrogel rheology broadly, utilizing it to detect cell-mediated matrix changes by proxy of mechanical properties.

Materials and Methods:: Microgels are formed using a flow-focusing microfluidic device. Hydrogel precursor – either gelatin maleimide (GelMAL) or PEG 4-arm maleimide (PEG-4MAL) is dissolved and pumped into the device. Microgels are formed by the impingement of a continuous oil phase containing emulsified dithiothreitol (DTT, crosslinker), and crosslinking begins immediately as emulsified DTT diffuses into microgels. Microgels are washed by centrifugation to remove residual oil, and granular hydrogels are finally formed by centrifuging and removing the supernatant. Granular hydrogels are formed from 4wt% GelMAL microgels (166.2 ± 8.4 µm diameter) or 5wt% PEG-4MAL microgels (156.2 ± 7.0 µm diameter). Future work will examine granular mixtures (1:2 and 2:1 GelMAL:PEG-4MAL) using the proposed yield-stress fluid model.

C57Bl/6 mouse bone marrow mesenchymal stem cells (mMSC, Cyagen) are cultured in Mouse Bone Marrow MSC Complete Medium (Cyagen). To encapsulate mMSCs, cells are detached from culture flasks, washed to remove serum proteins, and suspended in GelMAL precursor prior to microgel fabrication as described above. mMSCs are encapsulated at p9 at concentrations of 2 x 106 cell/mL and 4 x 106 cell/mL.

Rheological tests are performed on an Anton-Paar MCR 302e rheometer using a 20mm parallel plate at 22°C or 37°C. Amplitude sweeps and LAOS were carried out at a frequency of 1 rad/s. Tests were conducted with enough sample volume to ensure the granular gel height is at least 10x the diameter of individual microgels.

Results, Conclusions, and Discussions:: A comparison of the experimental data and model predictions for amplitude sweeps are shown in Fig 1, for acellular granular hydrogels PEG-4MAL and GelMAL. An overshoot in loss modulus is observed for both hydrogels at intermediate amplitudes, followed by decrease in both moduli. The model accurately captures this behavior. The stricter test for model validity can be conducted by comparing transient responses, as shown in Fig 2. The model captures all the features observed experimentally, including the overall shapes and the instantaneous slopes of the curves in different regions. Both hydrogels have brittility factor of greater than one, indicating that local structure is relatively stiffer than the bulk. We found the GelMAL granular gel more difficult to pack into a material that constitutes a yield-stress fluid, compared to the PEG-4MAL granular gel.

These data provide a proof-of-concept for treating granular hydrogel as a yield-stress fluid. We anticipate our yield-stress fluid model will be able to explain granular hydrogel behavior at a level deeper than average rheological techniques. Work in the coming months will seek to encapsulate mMSCs within GelMAL microgels and mix these into a 50:50 granular hydrogel comprising acellular and cell-laden microgels. This is due to large sample volumes required for shear rheology; however, we also anticipate the sensitivity of brittility to be enhanced in a granular gel with non-uniform remodeling potential. This approach is relevant to anticipated future work in which we aim to co-culture distinct cell-laden microgel populations. This work will supplement future efforts in granular hydrogel culture by establishing brittility that may be connected to other metrics of remodeling (gene expression, proteomics, nascent protein deposition) to build a holistic profile of these processes.

We will seek to describe acellular granular mixtures of 4wt% GelMAL and 5wt% PEG-4MAL, demonstrating the power of this yield-stress model to describe complex granular mixtures broadly. This work has significant implications for 3D printing, for which granular hydrogels are an excellent candidate biomaterial. In particular, this model describes yield-stress fluid behavior in with continuity rather than piecewise, and may smooth the process of 3D printing optimization for granular hydrogels and other bioinks.

Acknowledgements (Optional): :

References (Optional): : Daly, A.C., Riley, L., Segura, T. et al. Hydrogel microparticles for biomedical applications. Nat Rev Mater 5, 20–43 (2020). https://doi.org/10.1038/s41578-019-0148-6

Krutarth Kamani, Gavin J. Donley, and Simon A. Rogers. Unification of the Rheological Physics of Yield Stress Fluids.
Phys. Rev. Lett. 126, 218002 (2021). https://doi.org/10.1103/PhysRevLett.126.218002