*3.1. Cytokine Profile Analysis*

#### 3.1.1. Analysis of Cytokine Levels in Periimplant Tissue

Cytokine levels were measured in periimplant tissue obtained from revision or primary surgery to identify a potential local immune response (Figure 1).

**Figure 1.** Cytokine profiles of periimplant tissue. Cytokines are shown in graph (**a**) and (**b**) with different concentration scales. Except from IL-15 and IL-17, patients with aseptic loosening AL (+) showed a statistically significant increase in the cytokine levels when compared with the control group. Out of the statistically significant cytokines, IL-4 and TNF-α did not show any statistical significance (NS) when comparing the two revision groups. IL-8 was found to be highly increased in patients with AL. Results are expressed as the mean (±SEM). The Mann-Whitney U test was used for the statistical analysis with a significance level of 0.05. *p* values are given by \* *p* < 0.05, \*\* *p* ≤ 0.01, \*\*\* *p* ≤ 0.001.

Altogether, 10 cytokines (IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-15, IL-17A, IFN-γ and TNF-α) and growth factor GM-CSF were analyzed (Figure 1a/b). We found a highly increased cytokine profile in patients with AL, with a statistical significant increase of IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF, IFN-γ and TNF-α when compared to the AL (+) and the control group. When compared to the AL (−) group we found a statistically significant increase for all cytokines except from IL-4, IL-15, GM-CSF, and TNF-α. Of note, IL-8 was highly increased and the most strongly associated cytokine with AL.

#### 3.1.2. Analysis of Cytokine Levels in Serum

An identical cytokine profile analysis was performed in serum to investigate a corresponding systemic response (Figure 2). Cytokine levels in serum appeared 10–100 fold lower and although IL-8 and IFN-γ seemed increased in the AL (+) group, no statistical differences could be established. Together these results show a general increase of the investigated cytokine profile, in periimplant tissue obtained from patients with AL, but also that cytokine levels in periimplant tissue are not necessarily reflected in blood serum. Among other increased cytokines, IL-8 was established as the most potent marker of AL.

**Figure 2.** Cytokine profiles in serum. Patients with aseptic loosening are represented as AL (+), patients with dislocations are represented as AL (−) and the controls. Increased IL-8 and IFN-γ levels appeared for the AL (+) group. Results are expressed as the mean concentration (±SEM). No statistically significant differences could be established between the groups using the Mann-Whitney U test with a significance level of 0.05.

#### *3.2. Patch Test*

All patient groups were subjected to a comprehensive patch test containing orthopedically relevant metals and methyl methacrylate, the monomer of poly (methyl methacrylate) (PMMA) used as bone cement in THR (Table 2). Positive and doubtful reactions to these metals are summarized in Table 2. No statistical significant differences between either of the groups could be established. Few positive test reaction were observed even for the metals used in the standard series (Cr, Co and Ni), only one reaction to Ni and one to Cr were observed in all three groups. However, three positive reactions for Ti and two positive skin reactions to V were observed in the (AL+) group. In fact, the two positive reactions to V were observed in the same patient who had a positive reaction to Cr (Figure 3).

**Table 2.** Skin reactions. Positive (+) and doubtful (+?) skin reactions to different metals and methyl methacrylate. Patch test reactions were scored using the International Contact Dermatitis Research Group's (ICDRG) criteria [30]. Only definite +1, +2 and +3 reactions were regarded as positive. No reactions were categorized as +2 and +3 reactions in this study and only compounds with either positive (+1) or doubtful (+?) reactions are listed in the table. Prevalence of positive reactions was tested against the control group using Fisher's exact test with two tailed *p* values. No statistical significant differences were found.


**Figure 3.** Patch test. (**A**) Example of a positive (+) and a doubtful skin reactions (+?) to vanadium and chromium in a patient from the AL (+) group. (**B**) Enlarged photograph of the skin reaction to vanadium.
