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.

Corrigendum: Immune Assisted Tissue Engineering via Incorporation of Macrophages in Cell-Laden Hydrogels Under Cytokine Stimulation.

[This corrects the article DOI: 10.3389/fbioe.2018.00108.].

Immune Assisted Tissue Engineering via Incorporation of Macrophages in Cell-Laden Hydrogels Under Cytokine Stimulation.

The function of soft tissues is intricately linked to their connections with the other systems of the body such as circulation, nervous system, and immune system. The presence of resident macrophages in tissues provides a means to control tissue homeostasis and also a way to react to the physical/biological insults and tissue damage. Thus, incorporation of resident macrophage like phenotype-controlled macrophages in engineered tissues can improve their fidelity as model tissues and also improve their rate of integration and facilitate the resolution of inflammation for regenerative medicine applications. Herein, we demonstrate two potential ways to immunoassist the remodeling process of engineered soft tissues in three-dimensional (3-D) gelatin based hydrogels containing fibroblasts and/or endothelial cells: (i) with supplementation of interleukin-4 (IL-4) in the presence of macrophages and (ii) in tri-culture via naive monocytes or differentiated macrophages. The presence of IL-4 had a proliferative effect on fibroblasts, with a significant boosting effect on proliferation and cytokine secretion in the presence of differentiated macrophages with an upregulation of activin, interleukin-1 receptor antagonist (IL-1RA), tumor necrosis factor alpha (TNF-α), and interleukin-1 beta (IL-1ß), creating a more stimulating microenvironment. The addition of IL-4 in endothelial cell/macrophage co-culture configuration improved the organization of the sprout-like structures, with a boost in proliferation at day 1 and with an upregulation of IL-6 and IL-1RA at the earliest stage in the presence of differentiated macrophages creating a favorable microenvironment for angiogenesis. In tri-culture conditions, the presence of monocytes or macrophages resulted in a denser tissue-like structure with highly remodeled hydrogels. The presence of differentiated macrophages had a boosting effect on the angiogenic secretory microenvironment, such as IL-6 and IL-8, without any additional cytokine supplementation. The presence of fibroblasts in combination with endothelial cells also had a significant effect on the secretion of angiopoietin. Our results demonstrate that incorporation of macrophages in a resident macrophage function and their phenotype control have significant effects on the maturation and cytokine microenvironment of 3-D multiple cell type-laden hydrogels, which can be harnessed for better integration of implantable systems and for more physiologically relevant in vitro tissue models with an immune component.