Titanium Surface Characteristics Induce the Specific Reprogramming of Toll-like Receptor Signaling in Macrophages

Abstract

Most of the research on titanium-based dental implants (Ti-discs) is focused on how they are able to stimulate the formation of new tissue and/or cytotoxic studies, with very scarce data on their effects on functional responses by immunocompetent cells. In particular, the link between the rewiring of innate immune responses and surface biomaterials properties is poorly understood. To address this, we characterize the functional response of macrophage cultures to four different dental titanium surfaces (MA: mechanical abrasion; SB + AE: sandblasting plus etching; SB: sandblasting; AE: acid etching). We use different Toll-like receptor (TLR) ligands towards cell surface receptors (bacterial lipopolysaccharide LPS for TLR4; imiquimod for TLR7; synthetic bacterial triacylated lipoprotein for TLR2/TLR1) and endosomal membrane receptor (poly I:C for TLR3) to simulate bacterial (cell wall bacterial components) or viral infections (dsRNA and ssRNA). The extracellular and total LDH levels indicate that exposure to the different Ti-surfaces is not cytotoxic for macrophages under resting or TLR-stimulated conditions, although there is a tendency towards an impairment in macrophage proliferation, viability or adhesion under TLR4, TLR3 and TLR2/1 stimulations in SB discs cultures. The secreted IL-6 and IL-10 levels are not modified upon resting macrophage exposure to the Ti-surfaces studied as well as steady state levels of iNos or ArgI mRNA. However, macrophage exposure to MA Ti-surface do display an enhanced immune response to TLR4, TLR7 or TLR2/1 compared to other Ti-surfaces in terms of soluble immune mediators secreted and M1/M2 gene expression profiling. This change of characteristics in cellular phenotype might be related to changes in cellular morphology. Remarkably, the gene expression of Tlr3 is the only TLR that is differentially affected by distinct Ti-surface exposure. These results highlight the relevance of patterned substrates in dental implants to achieve a smart manipulation of the immune responses in the context of personalized medicine, cell-based therapies, preferential lineage commitment of precursor cells or control of tissue architecture in oral biology.

Keywords: Toll-like receptors; dental implants; immune regulation; macrophage cells; surfaces; titanium discs.

Figure 1

Composition of the different titanium discs by XR microfluorescence. The surface treatments were: mechanical abrasion (MA), sandblasting plus acid etching (SB + AE), sandblasting (SB) and acid etching (AE).

Figure 2

Ti-disc analysis (A) by FESEM and confocal scanning of the titanium discs with different surface treatment: mechanical abrasion (MA), sandblasting plus acid etching (SB + AE), sandblasting (SB) and acid etching (AE). Below, 3D representations of the surface analysis by optical profiling (B).

Figure 3

LDH extracellular (A) and total (B) quantification of RAW 264.7 cells cultured over the Ti-discs with different surfaces: mechanical abrasion (MA), sandblasting plus etching (SB + AE), sandblasting (SB) and acid etching (AE) and with or without the TLR ligands LPS (TLR4), imiquimod (TLR7), poly(I:C) (TLR3) and Pam3Csk4 (TLR2/1) (n = 6).

Figure 4

Cell morphology of RAW 264.7 cells cultured over the different Ti-discs by fluorescence microscopy. Surface treatment: mechanical abrasion (MA), sandblasting plus etching (SB + AE), sandblasting (SB) and acid etching (AE). In blue, nuclei with DAPI; in green, actin filaments with phalloidin 488. Scale bar: 25 µm. (n = 3).

Figure 5

Quantitative determination by ELISA of IL-6 (A) and IL-10 (B) cytokine levels in supernatants from RAW 264.7 cells cultured over Ti-discs with different surfaces: mechanical abrasion (MA), sandblasting plus etching (SB + AE), sandblasting (SB) and acid etching (AE) and with or without the TLR ligands LPS (TLR4), imiquimod (TLR7), poly(I:C) (TLR3) and Pam3Csk4 (TLR2/1) (n = 3). **, p < 0.0021; ***, p < 0.0002; ****, p < 0.0001.

Figure 6

Relative gene expression of iNos (A) and ArgI (B) mRNAs by qRT-PCR in RAW 264.7 cells cultured over Ti-discs with different surfaces: mechanical abrasion (MA), sandblasting plus etching (SB + AE), sandblasting (SB) and acid etching (AE) and with or without the TLR ligands LPS (TLR4), Imiquimod (TLR7), Poly(I:C) (TLR3) and Pam3Csk4 (TLR2/1). * p < 0.00332; **** p < 0.0001.

Figure 7

Relative gene expression of Tlr 4 (A), Tlr7 (B), Tlr3 (C), Tlr2 (D) and Tlr1 (E) mRNAs by qRT-PCR in RAW 264.7 cells cultured over the Ti-discs with different surfaces: mechanical abrasion (MA), sandblasting plus etching (SB + AE), sandblasting (SB) and acid etching (AE) and with or without the TLR ligands LPS (TLR4), Imiquimod (TLR7), Poly(I:C) (TLR3) and Pam3Csk4 (TLR2/1). *, p < 0.00332; ****, p < 0.0001.

Figure 8

Potential mechanistic insights into Ti-discs caused the reprogramming of macrophages. TLR signaling imprinting of the MA-exposed macrophages displays differential functional responses compared to SB + AE, SB and AE titanium-based materials after membrane or endosomal TLRs elicited by specific ligands (LPS for TLR4, synthetic bacterial triacylated lipoprotein for TLR2/1, poly I:C for TLR3 and imiquimod for TLR7). TLR downstream signaling regulated by titanium-based dental materials affects both the MyD88-dependent and MyD88-independent pathways, suggesting a potential role in IRF3-, IRF7- and p50/p65-mediated control of the transcriptional activity. Abbreviations: MyD88, MyD88 innate immune signal adaptor protein; TRAF6, TNF receptor-associated factor 6; TRIF, TIR-domain-containing adapter-inducing interferon-β; IRF3, interferon regulatory factor 3; IRF7, interferon regulatory factor 7; p50/p65, subunits of NF-kappa B nuclear transcription factor; MA, mechanical abrasion; SB, sandblasting plus etching; SB, sandblasting; AE, acid etching.