Introduction: Protein adsorption to biomaterials is a well-known phenomenon thought to contribute to the progression of the foreign body reaction (FBR). While the contribution of serum proteins has been studied extensively, those derived from tissues damaged during implantation have received little attention. Toll-like receptors (TLRs) may mount an inflammatory response against these damage-associated molecular patterns (DAMPs) through their activation of NF-κB and AP-1 transcription factors[1]. The purpose of this study was to determine whether TLR-mediated macrophage recognition of DAMPs adsorbed to biomaterial surfaces contributes to the progression of the FBR.
Methods: Fetal bovine serum (FBS) and 3T3 fibroblast lysate were used as sources of serum proteins and DAMPs, respectively. Total protein concentrations were determined by microBCA assay. Tissue culture polystyrene (TCPS) was incubated in lysate and/or FBS, then rinsed with phosphate-buffered saline (PBS). Activation of NF-κB/AP-1 in RAW-Blue macrophages induces expression of secreted embryonic alkaline phosphatase (SEAP), the enzymatic activity of which can be quantified with the QUANTI-Blue assay. These cells, cultured in 10% heat-inactivated FBS, were exposed to TCPS alone (negative control), lipopolysaccharide (LPS; positive control), and lysate/FBS-adsorbed TCPS. Culture was ended after 24hrs. Morphology of May-Grünwald-Giemsa –stained cells was evaluated. IL-4 and IFN-γ concentrations were determined by ELISAs. NF-κB/AP-1 –dependent SEAP activity was quantified. All experiments were run in triplicate, ANOVAs with Tukey’s post-hoc analyses used, and p<0.05 accepted as statistically significant. All values reported as mean±SD.
Results and Discussion: Lysate induced significantly greater macrophage fusion than TCPS (39.2±13.9% vs 0.5±0.3%, respectively; p<0.05), yielding multinucleated cells (27.0±19.9 nuclei/cell; 9068.0±6548.0µm2) of irregular morphology and random nuclear arrangement (Fig 1). Previous models have relied on IL-4 and IFN-γ to induce fusion[2], but neither was detected suggesting an alternative mechanism. Lysate induced NF-κB/AP-1 activation to the same extent as known TLR-agonist LPS (11.4±0.1 vs 11.9±1.1 fold greater than TCPS, respectively), which was significantly greater than that induced by FBS (0.3±0.1 fold; p<0.001). While this suggests that TLRs mount an inflammatory response against DAMPs but not serum proteins, both compete for adsorption as evidenced by the dose-dependent suppression of NF-κB/AP-1 activity with dilution of lysate in FBS but not PBS (p<0.0001; Fig 2). Importantly, lysate continued to elicit significant activation compared to FBS when comprising as little as 0.06% of total protein added (p<0.05), supporting the relevance of DAMPs to the FBR in vivo.
Conclusion: These findings indicate that DAMPs-adsorbed biomaterials elicit an inflammatory response in macrophages and promote their fusion to yield foreign body giant cells, the hallmark of the FBR.
Canada Foundation for Innovation’s (CFI) John R. Evans Leaders Fund (JELF); Queen’s University Senate Advisory Research Committee (SARC) Grant
References:
[1] Yu M, Wang H, Ding A, Golenbock DT, Latz E, Czura CJ, Fenton MJ, Tracey KJ, Yang H. HMGB1 signals through Toll-like receptor (TLR) 4 and TLR2. Shock 26: 174 - 179, 2006.
[2] McNally AK, Anderson JM. Interleukin-4 induces foreign body giant cells from human monocytes/macrophages: differential lymphokine regulation of macrophage fusion leads to morphological variants of multinucleated giant cells. American Journal of Pathology 147: 1487 - 1499, 1995.