Adherent Endotoxin on Dental Implant Surfaces: A Reappraisal
Osteoimmunology is the crosstalk between cells from the immune and skeletal systems, suggesting a role of pro-inflammatory cytokines in the stimulation of osteoclast activity. Endotoxin or bacterial challenges to inflammatory cells are directly relevant to dental implant pathologies involving bone resorption, such as osseointegration failure and peri-implantitis. While the endotoxin amount on implant devices is regulated by standards, it is unknown whether commercially available dental implants elicit different levels of adherent-endotoxin stimulated cytokines. The objective of this work is to develop a model system and evaluate endotoxin-induced expression of pro-inflammatory cytokine genes relevant to osteoclast activation on commercially available dental implants. Murine J774-A1 macrophages were cultured on Ti disks with different level of lipopolysaccharide (LPS) contamination to define the time-course of the inflammatory response to endotoxin, as evaluated by reverse transcription polymerase chain reaction analysis. The developed protocol was then used to measure adherent endotoxin on commercially available packaged and sterile dental implants in the “as-implanted” condition. Results show that tested dental implants induce variable expression of endotoxin-stimulated genes, sometimes above the level expected to promote bone resorption in vivo. Results are unaffected by the specific surface treatment; rather, they likely reflect care in cleaning and packaging protocols. In conclusion, expression of genes that enhance osteoclast activity through endotoxin stimulation of inflammatory cells is widely different on commercially available dental implants. A reappraisal of the clinical impact of adherent endotoxins on dental (and bone) implant devices is required in light of increasing knowledge on crosstalk between cells from the immune and skeletal systems.
Expression of interleukin-1 gene by J774A-1 macrophages after 1-, 4-, and 24-hour culturing on endotoxin-free (control) and lipopolysaccharide (LPS)-contaminated Ti samples. Data are expressed as fold expression over the value of the control sample at 1 hour; (a) full y-axis scale, (b) reduced y-axis scale.
Expression of interleukin-6 gene by J774A-1 macrophages after 1-, 4- and 24-hour culturing on endotoxin-free (control) and lipopolysaccharide (LPS)-contaminated Ti samples. Data are expressed as fold expression over the value of the control sample at 1 hour; (a) full y-axis scale, (b) reduced y-axis scale.
Dependence of the expression of interleukin-1 (IL-1) and IL-6 genes by J774A-1 macrophages after 4-hour culturing on the concentration of lipopolysaccharide (LPS) in the solution used to prepare LPS-contaminated Ti samples. Data are expressed as fold expression over expression of the control sample.
Expression of interleukin-6 (IL-6) and IL-1 genes by J774A-1 macrophages after 4-hour culturing on solvent-cleaned machined (Mach), Ti-blasted, and double acid etched (DAE) implants and on the same implants after a complete endotoxin removal cycle (shown by +). Data are expressed as fold expression over the value of the Mach +; (a) full y-axis scale, (b) reduced y-axis scale.
Expression of interleukin-6 (IL-6) gene by J774A-1 macrophages after 4-hour culturing on 22 different commercially available dental implants. Data are expressed as fold expression over the value of an endotoxin-free control dental implant. The horizontal LPS1, LPS5, and LPS10 lines show the reference values of IL-6 fold expression obtained on purposely contaminated samples, already shown in Figure 3; (a) full y-axis scale, (b) reduced y-axis scale. Measurements performed on a single specimen for each sample, the error bar shows intra-experiment accuracy through the standard deviation obtained from triplicate aliquots of cDNA.
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