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Biomaterials

Biomaterials - Poster Session E

P29 - Tuning Cell Behavior Using MMP-Degradable RGD Peptides

Saturday, October 11, 2025
10:00 AM - 11:00 AM PDT
Location: Exhibit Hall F, G & H

Introduction: : RGD peptides are commonly used to promote cell adhesion due to their ability to bind integrin receptors on the cell surface [1] However, cell-matrix interactions in tissues are highly dynamic and integrins are frequently co-localized with proteases on the cell surface. Additionally, ligand-bound integrins can be internalized and integrins are recycled to the cell surface. While the RGD sequence effectively binds integrins, its non-degradable nature fails to mimic aspects of cell-matrix interactions, which could influence cellular processes such as migration, spreading, and proliferation [2] . To overcome this limitation, we have developed a hydrogel system that allows cells to both attach and dynamically remodel their surroundings by incorporating protease-sensitive ligands.
We hypothesize that incorporating MMP-degradable sequences into the hydrogel system will enable internalization of ligand-bound integrins through protease-mediated cleavage, thereby promoting more favorable cell behaviors such as spreading and viability. Understanding how MMP-degradable motifs modulate these processes will inform the design of more effective biomaterials that support cell-driven matrix remodeling and dynamic cellular responses.

Materials and

Methods: : We used a 3.5% 20kDa 8-arm PEG dibenzocyclooctyne (DBCO) crosslinked with a protease-degradable N₃-KGPQGIWGQK-N₃ peptide for all gels. MMP-sensitive peptides were incorporated in the gels at a total concentration of 1.5 mM in a 20 µL gel, using one of three sequences: N₃-GRGDS, N₃-KLVADLMASAE-GRGDS, or N₃-KGPQGIWGQK-GRGDS. Human mesenchymal stem cells (hMSCs) were seeded into the gels cell viability was measured using the Alamar Blue assay. The cells were stained after seven days with rhodamine-phalloidin and DAPI. Hydrogels were imaged using a confocal microscope using a confocal microscope (Nikon C2+ laser scanning). Quantitative image analysis, including average cell area and relative viability, was performed using ImageJ (Fiji). Data was analyzed using a one-way ANOVA followed by a Tukey post-hoc test in R.

Results, Conclusions, and Discussions:: Results and

Discussion: To evaluate how MMP specificity influences cell behavior, we incorporated two MMP-degradable RGD peptides into a PEG-based hydrogel system: GPQGIWGQ, a broadly cleavable sequence recognized by multiple MMPs [3], and KLVADMASAE, a sequence preferentially cleaved by membrane-specific MMPs [4]. Both were tethered to the matrix via the integrin-binding RGD motif. A non-degradable peptide, N₃-GRGDS, served as a control that allows integrin attachment but restricts protease-mediated remodeling.
Gels functionalized with the broadly cleavable sequence supported the highest cell viability (118%), while those with the membrane-associated peptide showed a moderate improvement (104%) from the non-degradable control (95%) (Fig 1. A). These results suggest that incorporating protease-sensitive linkages into the hydrogel creates a more permissive microenvironment that supports cell survival.
Differences in cell viability were accompanied by notable changes in cell morphology across conditions. hMSCs in the GPQGIWGQ condition exhibited significantly greater spreading (1027.5 µm²), as measured by projected cell area, and appeared more elongated in morphology (Fig 1. C). In contrast, cells in the KLVADMASAE (789.5 µm²) and N₃-GRGDS (799.8 µm²) conditions remained more compact, with smaller projected areas (Fig 1. B).
Our results demonstrate that incorporating MMP-degradable linkers into PEG-based hydrogels improves hMSC viability and morphology, underscoring the importance of matrix degradability in designing cell-instructive environments. This supports our hypothesis that protease-mediated cleavage of tethered adhesive ligands facilitates complex cell-matrix interactions, including internalization of ligand-bound integrins, which promotes spreading and survival [5]. In the GPQGIWGQ condition, cell-secreted proteases efficiently cleave the MMP-sensitive sequence at adhesion sites, releasing RGD ligands from the matrix. In contrast, the non-degradable N₃-GRGDS prevents integrin-ligand internalization, limiting integrin recycling and adhesion formation. The membrane-specific condition showed only moderate improvements compared to the non-degradable control, suggesting that both cleavage specificity and efficiency critically regulate integrin recycling and cell behavior. Efficient ligand release supports integrin internalization cycles that underlie effective cell spreading and proliferation.

Conclusions: We developed a hydrogel platform functionalized with MMP-degradable RGD peptides, which significantly improves upon non-degradable systems by enabling protease-mediated matrix remodeling. This degradability allows for internalization and recycling of ligand-bound integrins, supporting dynamic adhesion turnover that enhances cell viability and spreading.

Acknowledgements and/or References (Optional): : Acknowledgements:
We would like to acknowledge our funding sources, the NIH (1R21GM143593–01) and NSF (Award 2138723). We are grateful to the lab of Lesley Chow for the use of their preparative high performance liquid chromatography.

References:
1. Bellis, S. L. (2011). Advantages of RGD peptides for directing cell association with biomaterials. Biomaterials, 32(18), 4205–4210. https://doi.org/10.1016/j.biomaterials.2011.02.029
2. Moghaddam, A. S., Dunne, K., Breyer, W., Wu, Y., & Pashuck, E. T. (2025). Hydrogels with multiple RGD presentations increase cell adhesion and spreading. Acta Biomaterialia, 199, 142–153. https://doi.org/10.1016/j.actbio.2025.04.037
3. Patterson, J., & Hubbell, J. A. (2010). Enhanced proteolytic degradation of molecularly engineered PEG hydrogels in response to MMP-1 and MMP-2. Biomaterials, 31(30), 7836–7845. https://doi.org/10.1016/j.biomaterials.2010.06.061
4. Wu, Y., Rozans, S. J., Moghaddam, A. S., & Pashuck, E. T. (2024). Optimizing peptide crosslinks for cell-responsive hydrogels. bioRxiv. https://doi.org/10.1101/2024.05.16.594348
5. Paul, N. R., Jacquemet, G., & Caswell, P. T. (2015). Endocytic trafficking of integrins in cell migration. Current Biology, 25(22), R1092–R1105. https://doi.org/10.1016/j.cub.2015.09.049