(H-320) The Role of Macrophage Scavenger Receptors in Engineered Biomaterial Scaffold Remodeling

Introduction:: : CD206, also known as mannose receptor C type 1, is frequently used as a marker to identify M2 polarized macrophages. M2 macrophages aid in tissue repair, inflammation reduction, immune tolerance development, and defense against excessive inflammation. In the context of biomaterials, CD206 is found on the surface of M2-like macrophages and dendritic cells and has been correlated with positive outcomes in biological scaffold remodeling. Furthermore, previous research has suggested that CD206 plays a role in the internalization of collagen fragments in tumors. The goal of this study is to see if CD206 is more than just an M2 marker in response to biomaterials.

Materials and Methods:: We evaluated CD206 on macrophages in vitro cell assays and in vivo traumatic injury model Our injury model is a volumetric muscle loss (VML). To generate the injury, we surgically remove a 3mm area of the quadriceps muscle which results in a permanent defect. The biomaterial treatments for VML are the extracellular matrix (ECM) of the porcine small intestinal submucosa, saline (control), polyethylene powder (PE), and uninjury (muscle without injury). Muscles are analyzed using FFPE immunohistochemistry of CD206 and flow cytometry with a panel of 22 myeloid markers. We performed surgery on WT, Batf3 (lack of cross-presenting dendritic cells) Rag^-/- (lack adaptive immune cells) mice, at different time points (7 days & 21 days post-injury) to evaluate the myeloid profile. In vitro, we evaluate the direct interactions of macrophages with ECM through immunocytochemistry and microscopy. To create an ECM deposit in vitro, we'll use WI38 fibroblast-secreted matrix decellularized with Triton/NH₄OH. We'll extract the femur and tibia of B6 mice to isolate macrophages, which will then be co-cultured with the ECM under three polarization stimuli on glass chamber slides. Three stimuli include M0 media (DMEM medium + M-CSF), M1 media (DMEM medium + lipopolysaccharide (LPS)), and M2 media (DMEM medium + IL-4). ECM deposition will be evaluated with Picrosirius Red (PSR) along with two stimulants, Fibronectin, and L-ascorbic acid (Ascorbate). Finally, Collagens of the ECM and morphology of macrophages are detected by staining with Collagen 1 (AF488), ActinRed (red), and DAPI (blue).


Results, Conclusions, and Discussions:: Results & Discussion: To compare the expression of CD206 in different mice using in vivo methods, we began with wild-type mice. It was observed that the levels of CD206 expression were high in macrophages and dendritic cells of wild-type mice treated with ECM scaffolds. Expression of CD206 on macrophages and dendritic cells in Rag1^-/- mice, who lack adaptive immune cells, is lower than in WT mice at day 7 post-injury (Fig. 1A). The CD206 expression in mice without cross-presenting dendritic cells (Batf3^-/- mice) is decreased at 7- and 21-days post-injury compared to WT (Figure 1B). Histologically, there is a significant amount of CD206 expression (brown DAB staining) on the capsular surface of the ECM as opposed to the muscle interface (Fig.1C). For a better understanding of the interaction of macrophages with ECM, in vitro testing was used produce more precise results in a less complicated model. Two stimulants, fibronectin, and ascorbate, were utilized to determine if they altered the ECM production in the WI38 fibroblast culture. After 5 days of culture, slides were decellularized and stained with PSR (Fig. 1D). We found that both fibronectin and ascorbate together yield an optimal ECM deposition and adherence to chamber slides. After decellularization of the resulting fibroblast-secreted matrix and seeding macrophages on ECM, the cells begin to elongate after 24 hrs. and are associated along collagen fibrils (green) at 42 hrs. (Fig. 1E).

Conclusion: Overall, the results showed that CD206 expression is upregulated in response to ECM scaffolds and dependent upon both adaptive immune cells and cross-presenting dendritic cells. In vitro, we successfully generated a decellularized extracellular matrix through the culture of WI38 cells on fibronectin-coated glass slides in the presence of supplemental ascorbate. Macrophages cultured on top of ECM adopted a more elongated morphology in comparison to glass slides alone, and cells were associated with collagen fibrils. Future studies will involve evaluating other scavenger receptors and how the absence of CD206 affects macrophage behavior.


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