## 3 Results  
The expressions of genes that code for interleukin IL-1\(\beta\), IL-6, TNF-\(\alpha\), NOS 2, MMP-8, MMP-9 and TIMP-3 have been evaluated through quantitative PCR, preceded by reverse transcription (RT-qPCR) at 4 h of incubation of murine macrophage cells J774a.1 in direct contact with different implant types. The results of the analysis of gene TIMP-3 expression showed very low levels of mRNA, with amplification curves that amounted to >35 Ct. As a consequence, they were excluded from the final analysis. The outcomes of gene expression are reported in Figure 1. Figure 2 shows the statistically significant differences among the samples.  
Figure 1: The graph shows the expression of the genes that code for IL-6, TNF-\(\alpha\), NOS 2, MMP-8, and MMP-9 by J774-A1 macrophage cells placed in contact with the implant samples as well as with the negative control (polystyrene for cell cultures) for a duration of 4 h. The data are presented as fold expression of the gene with respect to the control, which is the ratio between gene expression in the sample under examination and gene expression in the control (i.e., if the measured value is 1, it means that the inflammatory cells in contact with the sample under examination express the same quantity of the gene as the inflammatory cells in contact with the negative control; if the value is 2, it means that they express double, and so on). Results are expressed as Mean (SD), \(n=3\).  
Figure 2: Outcomes of the statistical analysis of gene expression data for each sample for each gene analyzed. Samples presenting a statistically significant difference in gene expression are indicated with *. * \(p<0.05\), ** \(p<0.01\). Results are expressed as Mean (SD), \(n=3\).  
RTqPCR analysis results showed a non-inflammation pattern of gene expression for all the tested implants, except for a mild pro-inflammatory effect induced by 3 iT3 hybrid implants. In particular, in macrophage cells in contact with 3 iT3 implants, a statistically significant increase in transcripts for IL-1\(\upbeta\) (1.47-fold) in comparison with polystyrene and Nobel Pearl implants was found with \(p<0.05\), and compared with the other implant types with \(p<0.01\).  
Regarding the expression of IL-6 (Figure 2), the response was very low in all the samples analyzed. Only the 3 iT3 sample promoted an expression that was roughly 1.5 times greater than that of the negative control without a statistically significant difference, while the difference (1.63-fold) was significant compared to Shard and Syra (\(p<0.05\)), which are the two implants that induced the lower response, even lower than that of the negative control (albeit not statistically significantly).  
Prama implant showed a significantly greater expression of MMP-9 (\(p<0.05\)), while Shard had a significantly lower expression of NOS-2 (\(p<0.05\)) compared to the negative control.  
On the contrary, no statistically significant differences were observed for any of the implants in the expression of TNF-\(\alpha\) and of MMP-8 genes in comparison with polystyrene nor between the individual implant types.  
The low levels of mRNAs of IL-1\(\upbeta\) and IL-6, that are the main pro-inflammatory cytokines involved in foreign body reactions and in pathogen response, show that 3 iT3 implants induced a mild inflammatory response in macrophages at 4 h of incubation, differently from the other implants. In any case, the response was very low, and all the implants tested might be considered devoid of pro-inflammatory effects.