(229a) Investigating Macrophage Plasticity and Migration in a 3D Wound Healing Model

Cellular interactions with neighboring cells and their microenvironments are critical to wound healing. Specifically, in a 3D environment matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) enable cellular migration and remodeling of the extracellular matrix (ECM), while cytokine secretions modulate phenotypic functions. Despite the critical roles played by macrophages and fibroblasts during wound healing, changes in macrophage activation, protein expression and the corresponding changes in fibroblast function are not fully understood. We have designed 3D and 2D macrophage-fibroblast co-culture systems that mimic different phases of the wound healing process. The ratios of macrophages to fibroblasts were varied to simulate early and late stages of wound healing. We investigate the effects of matrix elasticity, EC composition and cellular interactions in 3D and 2D system on macrophage plasticity, migratory behavior, cell-cell adhesion molecules, and the secretion of MMPs and TIMPs.

Collagen gels exhibiting Youngâ??s modulus values of 6 or 36kPa were assembled to mimic early and late-stage wound environments. Co-cultures of RAW 264.7 macrophages and BALB/c 3T3 fibroblasts were cultured within (3D) or on the surface (2D) of these gels. Time-lapse images obtained on a confocal microscope were used to track cells in 3D. Immunofluorescence stainings were conducted to detect cell-cell adhesion molecules and ELISA assays were conducted to measure the concentration of pro- and anti-inflammatory cytokines, MMPs and TIMPs.

Macrophages co-cultured with fibroblasts showed a strong tendency to converge on and adhere to fibroblasts in both 2D and 3D systems. In 2D cultures, cells migrated independently or in aggregates of cells. Migratory speeds increased 28.5% for individual macrophages and 101.2% for cell aggregates at the 1:1 ratio when compared to the macrophage monocultures on 6kPa gels. This trend held for cells on 36kPa gels, however migratory speeds were reduced. Surprisingly, macrophages in 3D monocultures often migrated at speeds up to 30μm/h, similar to their 2D counterparts. We are currently analyzing migration in 3D co-cultures as well as investigating the signaling molecules responsible for attractions between fibroblasts and macrophages. As we have observed strong adhesion between macrophages and fibroblasts, we have examined the expression of intercellular adhesion molecule 1 (ICAM-1), a mediator of inflammatory cell recruitment. Fibroblasts expressed ICAM-1 across all conditions. In macrophage monocultures on 6kPa gels, almost all macrophages expressed ICAM-1 at the cell periphery and points of contact between neighboring cells. Macrophages cultured on the stiffer 36kPa gels showed decreased expression compared to those on the 6kPa gels. The expression of ICAM-1 by macrophages was significantly reduced when fibroblasts were added at a 1:1 ratio. These trends suggest that the presence of fibroblasts may be inhibiting the inflammatory profile of macrophages. Interestingly, decreases in ICAM-1 expression in macrophages could be correlated to a reduction in the concentration of tumor necrosis factor alpha (TNF-a). The reduction of this inflammatory cytokine between mono- and co-cultures of macrophages with fibroblasts (1:1 ratio) was approximately 50%.

We have developed a platform to study the complex interplay between two cell types that play critical roles in wound healing. Significant changes were observed between 2D and 3D migration of co-cultures of these cell types. The co-cultures can be expanded to include different cells types and tissue environments enabling the investigations into cell-cell interaction in 3D migration.