(9e) Macrophages Affect and Are Affected by Cells Encapsulated in PEG-Based Hydrogels: An in Vitro Co-Culture Study

Photopolymerized poly(ethylene glycol) (PEG) based hydrogels hold promise as in situ forming cell carriers for tissue engineering. The in vivo success of PEG-based hydrogels will depend largely on how the host responds to the implanted material. The early and late stages involving inflammation and resolution of the foreign body reaction may have significant adverse consequences on the performance and integration of tissue engineering constructs. Recent studies from our group demonstrated that PEG-based hydrogels promote macrophage attachment and adhesion in vitro and elicit a strong inflammatory reaction in vivo (Lynn et al. J Biomed Mater Res, 20(5): 738-747 (2010)). In an effort to elucidate the effects of the FBR on cell-laden synthetic scaffolds, we have developed an in vitro testing platform for cell-laden hydrogels aimed at mimicking macrophage interrogation and attack of the in vivo reaction.

Specifically, a co-culture system was developed consisting of RAW264.7 macrophages seeded directly onto a model cell-laden PEG-based hydrogel comprised of encapsulated NIH/3T3 fibroblast cells and cultured for up to 3 days. The cell adhesion ligand, RGD, was incorporated into the PEG hydrogel to permit fibroblast attachment within the hydrogel and to enhance macrophage adhesion to the surface of the hydrogel. The impact of each cell type on the other was investigated by gene expression within the co-culture and analyzed for genes associated with inflammation, tissue production and wound healing.

In the co-culture system, the presence of macrophages negatively impacted gene expression for extracellular matrix molecule by down-regulating collagen I and III expressions by 6-fold and 3-fold, respectively, but interestingly stimulating vascular endothelial growth factor A expression by 12-fold after 48 hours of culture when compared to fibroblast monocultures. All genes remained up-regulated after 72 hours, but not to the same level. Similarly, the fibroblasts had a dramatic impact on the phenotype of the macrophages, spanning among classically activated, wound healing, and regulatory. After 48 hours, macrophages expressed 1000-fold, 200-fold, and 800-fold greater expressions for inducible nitric oxide (indicative of classical activation), arginase type I (indicative of wound healing), and interleukin-10 (indicative of a regulatory phenotype), respectively, when compared to macrophage monocultures. However, by 72 hours, only iNOS was up-regulated in the co-culture system suggesting that a classically activated phenotype was more profound throughout the study. These findings indicate that the crosstalk between macrophages and fibroblasts, likely through paracrine signaling of secreted cytokines and other molecules, dramatically impacts the phenotypic response of both cell types, at least at the gene level. Taken together, our work highlights the negative impact that macrophages may have on cell-laden synthetic scaffolds and the need for intelligently designing scaffolds that are capable of attenuating this potential deleterious host response.