In vitro two-step granuloma formation model for testing innate immune response to implants and coatings

The extensive innate immune response to implanted biomaterials contributes significantly to their sub-par performance and failure. Granuloma formation is one of such reactions which results in multi-cell type clusters in line with the immune reaction to implanted materials. However, currently no in vitro model of granuloma formation exists that takes into account the arrival of multiple cell types (immune cells and connective tissue cells) to the implant insertion site. In this study, we developed a two-step model based on stimulated macrophage seeding followed by fibroblast introduction after a physiologically relevant time period for mimicking initial steps of immune reaction to biomaterials and inducing granuloma like behavior. Both LPS and TNF-α induction resulted in granuloma like formations which persisted longer than the control conditions. Introduction of human fibroblasts resulted in the colonization of the surfaces where the cell numbers and the collagen secretion were dependent on the microenvironment. In order to demonstrate the capacity of our model system to monitor the reaction to a given coating, a validated antimicrobial coating (Polyarginine (PAR)/Hyaluronic acid (HA)) was used as a testing bed. The coating prevented the adhesion of macrophages while allowing the adhesion of the fibroblast at the time of their arrival. Similar to its antimicrobial activity, macrophage metabolic activity and M2 differentiation in the presence of PAR was dependent to its chain length. The incorporation of fibroblasts resulted in decreased TNF-α and increased IL-1RA secretion especially in stimulation conditions. The pro- and anti-inflammatory cytokine secretions were low for PAR/HA coatings in line with the decreased number of macrophage presence. In the presence of complex PBMC population, the coating resulted in slightly less cellular attachment, without any significant cytokine secretion; the absence of inflammatory reaction was also demonstrated in vivo in a mouse model. The described in vitro granuloma testing system can control the macrophage reaction as a function of stimulation. It can also be used for testing new biomaterials for the potential innate immune responses and also for validation of implant coatings beyond their primary function from the immune response point of view.