Assistant Professor University of Virginia, United States
Introduction: Over 20 million people in the United States have a biomaterial device implant. A critical obstacle to implant success is overcoming the foreign body response and fibrotic encapsulation, which lead to implant failure. The medical and surgical cost of treating certain device failures averages up to $50000 per patient. Implant device failure can severely compromise both the implant’s function and the recipient’s health. A key contributor to this failure is the foreign body response (FBR), which involves a complex cascade of immune modulators, various cell types, soluble mediators, and cellular interaction. When foreign materials, such as implants, medical devices, or scaffolds, are introduced into the body, they often trigger complex biological responses. This leads to an inflammatory reaction that eventually results in fibrotic encapsulation, which compromises the functionality of the implanted biomaterial, making it a critical challenge to address in biomaterials. Recent studies have shown that Thy-1, a cell surface glycoprotein, modulates this fibrotic response. Specifically, Thy-1 loss has been associated with increased cell contractility-driven stiffening of the extracellular matrix, elevated αvβ3 integrin activation, and heightened fibrosis. In other fibrotic disorders, it has been shown that restoring Thy-1 expression is able to restore homeostatic fibroblast function and inhibit fibrosis. Therefore, we hypothesize that using a hydrogel system to deliver Thy-1 can modulate integrin activity and fibroblast phenotype, potentially reducing the fibrotic response and improving the performance of biomaterial implants.
Materials and
Methods: The microporous Annealed Particle (MAP) hydrogel system features annealed microgel particles composed of PEG-maleimide (PEG-MAL), methacrylamide (Meth-MAL), RGD peptides, and matrix metalloproteinase (MMP-2)-degradable crosslinkers, which were processed via microfluidic devices to form microgel particles. The resulting microgels were purified using a series of oil, PBS, and hexane. Thy-1 Fc fusion protein or the control Fc protein were covalently attached to the MAP hydrogel. Each condition was tested in triplicate using three wells per condition: Thy-1-MAP, control protein-MAP, and MAP.Thy-1 Knockout (thy-1 KO) cells were seeded on our different MAP gel conditions, and after 72 hours, cells were harvested using liberase. To evaluate integrin activity and Thy-1 presence, Cells were stained with WOW-1 antibody for active αvβ3 and for Thy-1 and measured expression via flow cytometry.
Results, Conclusions, and Discussions: We first confirmed that we were able to tether Thy-1 to MAP gel; this process involved conjugating Thy-1 to the microgel particle, introducing thiol groups to the Thy-1, which enables covalent linking to the maleimide present on MAP microgel. We performed an OPA protein Assay to confirm successful tethering, which showed an elevation in protein content compared to MAP alone. When Thy-1 KO cells were cultured on these MAP gels to determine whether Thy-1 can be transferred, they did not uptake Thy-1 from MAP gels. Therefore, we wanted to answer the question of whether Thy-1 can regulate integrins via trans interactions from MAP gels. Using a WOW-1 antibody for active avb3 integrins, we did not see a change in αvβ3 activity when Thy-1 KO cells were seeded on Thy-1 tethered MAP gels. Moving forward, our ongoing work will be assessing changes in fibroblast activation in response to Thy-1 delivery and integrin activity in other integrin species.
Conclusion: Tethering of Thy-1 on hydrogel strategy highlights the potential of using biomaterial modifications to control cellular behavior, improve the performance and longevity of implantable devices, and help mitigate fibrosis.
.jpg "Mathew K. Kibet photo")