Poster L25 - HemRGB Trichrome Staining for Assessment of Encapsulation of a Novel IL-4-Eluting Polypropylene Mesh in a Mouse Model

Friday, October 25, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Hallie Maytin (she/her/hers)

Student
University of Pittsburgh
Pittsburgh, Pennsylvania, United States

Co-Author(s)

AN

Alexis L. Nolfi

Graduate Student
University of Pittsburgh, Pennsylvania, United States
MK

Mangesh Kulkarni

Research Assistant Professor
University of Pittsburgh, United States
CS

Clint Skillen

Laboratory Manager
University of Pittsburgh, United States

Last Author(s)

BB

Bryan Brown

Associate Professor
University of Pittsburgh, United States

Introduction: Pelvic organ prolapse (POP), the descent of the pelvic organs into the vagina, is a debilitating disorder that can lead to significant pain and interrupted quality of life. 13% of women undergo surgical treatment to repair POP by age 80. Surgical treatment commonly includes implantation of synthetic polypropylene mesh (PPM) to improve anatomical outcomes. However, high rates of complications such as mesh exposure (>10%) are observed, and often fail to resolve, even with mesh removal. Recent research suggests that these complications may arise due to the macrophage response to PPM. Macrophages are highly plastic and have phenotypes ranging from M1 pro-inflammatory to M2 anti-inflammatory extremes. Ideally, after PPM implantation, the M1 macrophages transition to M2 in a timely manner driving mesh integration with the host. However, we have observed that M1 macrophages persist chronically, suggesting that the transition does not happen with PPM, driving downstream complications.
Recently, we demonstrated that M1 macrophages can be polarized to the M2 phenotype using the cytokine interleukin-4 (IL-4), and we have developed a surface coating that captures IL-4 and releases it following implantation to drive local polarization to the M2 phenotype. Here, we examine the effects of changes in IL-4 dosing achieved by changing the number of IL-4 containing layers and/or the concentration of IL-4 within the PPM coating upon long-term integration of the mesh in a mouse subcutaneous implantation model.

Materials and

Methods: A nanometer thickness, layer-by-layer coating was developed with the ability to release IL-4 with the amount and timing of release based on the number of coating layers, as previously described (Hachim, Biomaterials, 2017). This tunability was tested and variable release of IL-4 confirmed utilizing ELISA assays. Coated mesh was implanted into a subcutaneous pocket on the abdomen of 8–10-week-old female C57BL/6 mice. Flow cytometry was used to assess the degree to which variable IL-4 released polarized macrophages towards the M2 phenotype at 7 and 14 days.
Additional implantations were performed, and tissue samples were harvested at 90 and 180 days to assess the impacts of IL-4 release pattern and degree of macrophage polarization upon long-term mesh integration with the surrounding tissues. Formalin-fixed paraffin embedded tissues were sectioned and stained utilizing a recently developed RGB-Trichrome protocol (picrosirius red, alcian blue, and fast green, (Gaytan et al., Sci Rep., 2020)) or using picrosirius red staining alone.
RGB-trichrome samples were imaged with brightfield microscopy (Motic Easy Scan) to visualize collagen content. These images were analyzed in QuPath by measuring the average thickness of bands of collagen between two mesh knots (i.e., tissue formed within the mesh pores). Slides were additionally imaged under polarized light to understand the composition and orientation of these same bands of collagen. In QuPath, each section was analyzed by determining the percentage of collagen that appears red, green, and yellow, indicating its size/maturity. Results obtained using the RGB-trichrome stain were compared to those using traditional picrosirius red labeling.

Results, Conclusions, and Discussions: ELISA results showed that the mesh coating released IL-4 depending on the amount of IL-4-containing-layers and the IL-4 concentration within the polymer solution during the application process. As layers were added, total IL-4 released increased; similarly, as concentration within each layer increased, there was a proportional increase in IL-4 released compared to groups with the same number of layers but lesser concentrations of IL-4.
90-day RGB-trichrome stained tissues were imaged and thickness of the collagen bands between mesh areas was assessed (n≥6 animals/group, Fig. 1). For age-matched-controls and sham-operated animals, the thickness of the band of collagen immediately between the skin and abdominal muscle was assessed. We observed that uncoated mesh produced bands of collagen that were not significantly different than groups that had polymer coating only or a polymer coating with IL-4. Compared to groups without mesh, mesh-containing groups had a significantly higher average width of collagen bands at 90 days (p < 0.05) (Fig. 2a). At 180-days, results were similar to what was observed in 90-day samples (n≥4 animals/group, except for the uncoated mesh group, in progress at the time of abstract submission) (Fig. 2b).
Polarized light revealed that, in 180-day tissues, there is an abundance of type I collagen, aligning with our expectation of mature collagen deposition at this timepoint. Additionally, in comparison to groups without mesh, tissues with mesh had a more consistent alignment of collagen between areas of mesh fibers, indicating better organization of these tissues (Fig. 3). Groups implanted with mesh containing 40 and 60 layers of IL-4 coating appeared more aligned than other mesh groups.
These results demonstrate the ability to control IL-4 release at the surface of mesh implantation, resulting in M2 macrophage polarization. Collagen thickness was similar across groups; however, organization of the collagen in the 40- and 60-layer coated mesh groups appeared more aligned, suggesting better integration. While these results suggest that mesh integration can be controlled by modulating the early macrophage response, additional studies are needed to quantitatively assess collagen fiber content and alignment and their impact upon biomechanical integration in a clinically relevant model of POP repair.

Acknowledgements (Optional): This work was funded by the University of Pittsburgh Swanson School of Engineering, the Office of the Provost, and the Department of Bioengineering.