Host responses to microgel-based biomaterial interfaces

Although medical devices and biomaterial implants are used clinically in a variety of applications, the process of implanting them damages local tissue and initiates a localized non-specific inflammatory response that is detrimental to device performance. Extensive research efforts have focused on developing material surface treatments and systems to deliver anti-inflammatory agents to prevent biofouling and abrogate biomaterial-mediated inflammation. Traditional surface modification strategies are capable of reducing protein adsorption and cell adhesion in vitro; however, their use as long-term implant coatings is limited due to reduced non-fouling behavior, continued inflammation and fibrous encapsulation. This work aims to address these limitations by developing a novel and versatile implant coating with non-fouling properties using a system based on hydrogel microparticles (i.e. microgels), which offers many material advantages over current methods. The overall objective of this project was to evaluate host responses to these microgel coatings. Using the rationale that macrophages are the key mediators of inflammatory and regenerative responses our central hypothesis was that macrophage adhesion and subsequent activities can be modulated, and the intensity of the foreign body response to biomaterials can be reduced using these novel coatings.;As a first step toward testing this hypothesis, we characterized the surface properties of the microgel coatings using multiple techniques. Microgel coatings were synthesized from poly(N-isopropyl acrylamide)- co-acrylic acid covalently crosslinked with poly(ethylene glycol)-diacrylate and deposited onto a clinically relevant substrate, poly(ethylene terephthalate). We found that microgel coatings can be successfully deposited using a simple spin coating technique, and the incorporation of a photoaffinity label enables covalent attachment to the substrate and enhances long-term stability. We have confirmed the presence of nitrogen-rich pNIPAm microgel particles on the surface of PET and generated a homogeneous monolayer coating. Microgel particles effectively covered material defects commonly present on the surface of the underlying PET substrate. The ability to generate conformal and complete microgel coatings on heterogeneous/rough, biomedically relevant materials is a major advantage of this strategy over existing polymer chain grafting approaches. Importantly, using radiolabeled protein assays, we determined that microgel-coated samples also adsorbed significantly lower levels of human fibrinogen compared to unmodified PET controls.;Further characterization of these materials involved the evaluation of cellular responses using an in vitro culture system to model acute leukocyte interactions with biomaterial surfaces. Macrophages were cultured for 48 h on biomaterials, and adherent cells were imaged and scored for viability, adherent density, and spreading area. We demonstrated that microgel coatings significantly reduced the adhesion and spreading of macrophages compared to PET controls using a murine macrophage cell line, as well as primary human blood-derived monocytes. The low levels of in vitro cell adhesion and spreading combined with the protein adsorption-resistance characteristics on the microgels provide indirect evidence that these coatings impart non-fouling properties to biomaterial supports.;Implanted materials were then evaluated for early cellular responses in the intraperitoneal cavity of mice, a rigorous model of acute inflammation. Analyses of explanted biomaterials using immunofluorescence staining techniques revealed that microgel-coated samples significantly reduced the density of surface-adherent cells; additionally, fewer CD68+ macrophages were observed on these samples. Moreover, adherent cells were harvested and immunostained intracellularly for a panel of inflammatory cytokines (TNF-alpha, MCP-1, IL-1beta, and IL-10), and were then analyzed by flow cytometry to quantify relative cytokine levels. We demonstrated that microgel-coated samples exhibited significantly lower levels of pro-inflammatory cytokines in adherent leukocytes compared to unmodified PET, indicating that these coatings modulate cellular pro-inflammatory activities. Microgel-coated samples did not elicit pro-inflammatory cytokine expression beyond levels associated with the surgical procedure (sham group); therefore, increased cytokine expression was associated with leukocyte adhesion to the implanted PET biomaterial. These reductions of in vivo leukocyte adhesion and pro-inflammatory cytokine expression associated with the microgel coating contrasts with results of other non-fouling surface treatments.;Finally, we used an established model of chronic inflammation to evaluate these coatings for their efficacy at longer implantation time points. Unmodified PET controls, microgel-coated samples, and EG3 SAMs were implanted subcutaneously for 4 wk. Explants were processed histologically and stained for various markers. Collagen staining demonstrated that the microgel coatings significantly reduced fibrous capsule thickness, and those capsules appeared less compact and structurally ordered than PET controls. Microgel-coated samples also contained significantly fewer total cells within the capsule. Additional sections were stained for the macrophage marker CD68 using immunohistochemical methods to determine the inflammatory cellular profile at the cell-material interface. Unexpectedly, microgel-coated samples contained proportionately more CD68+ cells (relative to total cell numbers) than PET controls. Sections were also scored for multinucleated FBGC, but no significant differences were found among treatment groups.;In summary, this research has established a simple and reproducible method of surface functionalization to create effective coatings that resist protein adsorption and leukocyte adhesion. Collectively, these results demonstrate that microgel particles can be applied as relatively stable implant coatings to modulate inflammation and achieve more desirable host responses in vivo with the potential to extend implant lifetime. This work is innovative because it applies hydrogel particles to the development of a novel microscale implant coating. Our strategy offers unique control over synthesis parameters with the possibility of generating complex coatings onto a variety of biomedically-relevant materials. Furthermore, this research provides the foundation for developing a hydrogel-based coating system incorporating various bioactive signaling agents within a low-fouling background. Such a system will support controlled interactions with inflammatory cells, which will enable unprecedented regulation of host responses to implanted biomaterials.