(L-476) Affibody-Conjugated Polyethylene Glycol Hydrogels Tailor Release of Human Mesenchymal Stromal Cell Secretome

Introduction:: High cell donor variability poses a significant challenge in the use of cells as regenerative therapies for musculoskeletal diseases. Prior research revealed that several proteins secreted by mesenchymal stromal cells (MSCs)—including granulocyte-macrophage colony-stimulating factor (GM-CSF), bone morphogenetic protein 2 (BMP-2), and vascular endothelial growth factor (VEGF)—were key biomarkers for high therapeutic efficacy. Further research then looked at developing a statistical model to correlate protein secretion from each donor to therapeutic efficacy. Validation of donors that were found to be therapeutic was assessed in-vivo. Donors that showed the highest potential were also correlated with high GM-CSF and VEGF protein production. Biomaterials-based approaches have the potential to mimic hMSC spatiotemporal secretion profiles and be an alternative cell-free hydrogel platform. Through the controlled release of proteins released into media, biomaterials can mimic natural MSC secretion profiles. The goal of this work was to engineer a cell-free biomaterial platform to mimic the spatiotemporal secretion of key therapeutic factors from MSCs using affibody-functionalized hydrogels. We hypothesized that proteins from MSC-conditioned media would be sequestered by these hydrogels and slowly released over 24hrs.

Materials and Methods:: Human MSCs were stimulated with 20ng/mL interleukin-1β (IL-1β) to secrete proteins in response to an inflammatory stimulus. After 24hrs, Conditioned media was collected after 24 hrs and analyzed using multiplexed ELISA analysis, demonstrating that, following IL-1β stimulation, MSCs increased production of granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 6 (IL-6), interleukin 8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), and vascular endothelial growth factor (VEGF). Affibodies with high affinities to either GM-CSF (KD = 441.4nM) , BMP2 (KD = 10.7nM) , or VEGF (58.3nM) were identified and used. Affibodies were conjugated to 5 w/v% PEG-Maleimide hydrogels (PEG-4Mal) at 500x molar excess through a thiol click chemistry reaction. After synthesis, hydrogels were incubated with 1mL of conditioned cell media for 24hrs. Hydrogels were then incubated with 1mL of PBS and samples of the supernatant were collected at 1, 3, 6, and 24hrs. Cytokines were analyzed by multiplexed ELISA using a Luminex 200 system.

Results, Conclusions, and Discussions:: We observed significant sequestration of several cytokines, including GM-CSF, IL-6, IL-8, and MCP-1, from the media incubated with each of the hydrogels. Hydrogels with affibodies demonstrated a burst release of proteins at 1 hour, while PEG hydrogels without affibodies released more proteins at the later time points (3,6, and 24 hours) compared to those with affibodies. The affibody-functionalized hydrogels sequestered and released key MSC-secreted cytokines, mimicking the hMSC secretion profile. This platform has the potential to move towards an acellular and more reproducible therapeutic approach mimicking hMSC therapies.

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