(402b) Macrophages Exhibit Stable Phenotypic Markers in Extended Culture on Model Biomaterial Surfaces

Introduction. All biomaterial implants cause chronic and acute host inflammatory responses followed by atypical fibrosis at the wound site. The molecular events that orchestrate this process, known as the foreign body reaction (FBR), remain poorly understood. As key cellular players in this response and in both natural and specific immunity, macrophages both react to and secrete cytokine signals that direct complex interactions among immune and wound healing cells. Upon adhesion to implanted materials, macrophages secrete important chemical signals (chemokines, cytokines) involved in the immune response, recruiting additional cells of numerous lineages to the wound site, and promoting chronic inflammation of the implant site, often resulting in implant failure. Understanding the mechanisms by which macrophages interrogate, activate, adhere to, and differentiate on implant surfaces is an important step towards understanding the FBR and improving implant biocompatibility. This work explores an in vitro approach of determining some initial molecular events involved in macrophage adhesion to medically relevant biomaterial surfaces, as well as events resulting from long-term adhesion of macrophage lineage cells to these surfaces.

Materials and Methods. Primary-derived bone marrow macrophages (BMMO) were harvested from C57/BL-6 mice (Jackson Labs). Murine monocyte/macrophage cell lines (IC-21, J774A.1, and RAW 264.7) were purchased from the ATCC (Manassas, VA) and were cultured under standard conditions. Model surfaces were prepared as previously described.^1-3 These cells were then cultured on control and model biomaterial surfaces continuously for 21 days, a period of time equivalent to the completion of FBR mediated fibrosis in vivo. Media was changed daily, and cell-enriched media was analyzed for expression of many cytokines involved in inflammation and the FBR (GM-CSF, IFN-γ, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, MCP-1, and TNF). Cells were also imaged daily to observe morphology. At day 21 cells were probed for an assortment of surface markers that serve as macrophage markers, adhesion molecules, indicators of activation, and molecules implicated in macrophage fusion into foreign body giant cells (F4/80, CD14, CD40, CD11b, CD11c, CD18, CD54, Fc Receptor, Macrophage Mannose Receptor, CD40, and TLR-4).

Results and Discussion. Microscopic evaluation of each cell type on each surface over time showed an overall change in morphology among immortalized cell lines to a more rounded phenotype, whereas BMMO cells showed a more consistent morphology over time, keeping an astral shape despite crowding on the surface. Interestingly, RAW 264.7 cells exhibited fusion into multi-nuclear foreign body giant cells, indicating that certain macrophage lineage cells are capable of fusion without cytokine signaling from other inflammatory cells. Our previous work has involved the phenotyping of these cell types using flow cytometry to assay cell-surface protein expression.³ Compared to previous expression levels, very little change occurred after 21 days of continuous culture for any of the markers studied with the exception of the macrophage mannose receptor (MMR). MMR expression increased for all cell types on all surfaces, as MMR has been implicated in the fusion of macrophages this result indicates a greater potential for fusion of these cells after extended culture on biomaterial surfaces.

References. 1. Godek ML, Sampson JS, Duchsherer NL, McElwee Q, Grainger DW. J. Biomat. Sci. Polym. Ed., 17, 1141, (2006); 2. Godek ML, Michel R, Chamberlain LM, Castner DC, Grainger DW. (submitted J. Biomed. Mater. Res., 2006); 3. Chamberlain LM, Godek ML, Gonzalez-Juarrero M, Grainger DW. (submitted J. Biomed. Mater. Res., 2007).