Biocompatibility of silver ions and silver containing alloy

5.1.1 Effect of AgNO3 and MgAg1% sticks on the viability of different cells Ag⁺ salts have been shown to exert an inhibitory effect on proliferation and differentiation of several cell lines: bone marrow cells and keratinocytes (HOLLINGER, 1996), hepatocytes (LIU et al., 1991), lymphocytes (HUSSAIN et al., 1992) or leukocytes (JANSSON and HARMS-RINGDAHL, 1993). Our experiments provided detailed information about the biocompatibility of AgNO₃ and degradation medium of MgAg1% sticks on primary cell cultures (fibroblasts and epithelial cells), murine fibroblasts and macrophages. AgNO3 is known to have a good antimicrobial action with low toxicity (BRUTEL DE LA RIVIERA et al., 2000; OLSON et al., 2002;

KLASEN, 2000; HOLLINGER, 1996). Antiseptics such as AgNO₃, which are potent broad-spectrum antimicrobial agents, are administered until there is closure of the epithelium to minimize the risk of infection. COOPER et al. (1991) stated that cytotoxicity of Ag⁺ is considered to be low while the bactericidal power is high and that is one of the reasons why we choose Ag⁺ for our alloy, in addition to BOSWALD et al. (1999), GREIT et al. (1999), OLOFFS (1994) who stated that there is no cytotoxic effect of Ag in addition to its good biocompatibility.

In the present study, several end-point parameters (MTS, Neutral red) were used to indicate the cytotoxic action of AgNO₃ and degradation medium of MgAg1% sticks.

Protein content was used as an indirect method for assessing cell number revealed that AgNO3 produces an evident loss of cell protein content depending on the concentration of AgNO₃. Our cell viability results classify AgNO₃ as low cytotoxic, since there was an affect on the viability of different cells either primary or cell lines, but only at high concentrations. When MTS and neutral red tests were conducted, AgNO₃ solution had a slight influence on the viability of primary mammary epithelium cells at high concentrations, while MgAg1% degradation medium did not show an influence on on the viability of primary mammary epithelium. AgNO₃ showed a weak influence on the viability of murine fibroblasts at 0.3 mmol/l, but primary mammary fibroblasts were more resistant as AgNO3 affected its viability at 1 mmol/l. MgAg1%

degradation medium did not show an influence on the viability of both murine and

Discussion

mammary fibroblasts cell. Murine and raw macrophages viability was not affected by MgAg1% degradation medium, while they showed a decrease in the viability at 0.3 mmol/l of AgNO₃. If the results obtained in our study are compared with those presented by TEEPE et al. (1993) in similar conditions (24-hour incubation with AgNO₃ in 10 % FCS), but performed in human keratinocytes, they found that AgNO₃ concentrations which are totally toxic for fibroblasts are approximately 40-50 times lower than those found by those authors for keratinocytes. In addition, when FCS 10

% is added to the fibroblast culture, AgNO₃ toxicity decreased markedly and only began to appear at 6.5 mmol/l. However, this AgNO3 concentration with FCS 5 % or 2 % produced a total loss of protein content (TEEPE et al., 1993)

This would indicate that fibroblasts are far more susceptible to AgNO₃ than keratinocytes. We can conclude that degradation medium of MgAg1% did not have an influence on the viability of our cell cultures (mammary epithelium and fibroblasts, murine fibroblasts and murine and raw macrophages), while AgNO₃ solution had a slight affect on our cell cultures at high concentrations, as silver is well known to be dose dependent (PERRELLI and PIOLATTO 1992; BRUTEL DE LA RIVIERA et al., 2000).

5.1.2 Effect of AgNO3 and MgAg1% sticks on the metabolic activities

Ag ions are also actively involved in the cellular energy metabolism for the most part by influencing many enzymes participating in glycolysis, the citric acid cycle and the respiratory chain (WOLF and TRAPANI, 2008; COWAN, 2002). Pyruvate kinase and succinate dehydrogenase were chosen as marker enzymes for mitochondria and cytosol. Inhibition by Ag ions of succinate uptake into membrane vesicles of E. coli has been noted (RAYMAN et al., 1972).

Quite surprisingly, our results indicate an enhanced activity of SDH and PK activity in a tissue in contact with MgAg1% sticks implant as well as the different cell cultures treated with AgNO₃ solution and degradation medium of MgAg1% sticks. SDH and PK activity was found to be significantly increased in case of higher concentrations of AgNO₃ solution and degradation medium of MgAg1% sticks in the culture medium.

The reason for this outcome is yet unknown.

Discussion

Our results indicate a higher activity by mitochondrial dehydrogenase. To our best knowledge, these findings are described here for the first time and refer to a basic aspect of cell physiology. SCHREURS and ROSENBERG (1981) stated that if Ag⁺ damaged the cell membrane, making it freely permeable esterified phosphate should appear outside the cell after Ag⁺ treatment and that probability could be applied on succinate and pyruvate enzymes, but we found that there was no decrease in SDH and PK activity after treatment with either AgNO3 solution or degradation medium of MgAg1%. In contrast to this, increased amounts of AgNO₃ concentrations showed a stimulation of SDH and PK activity, but unfortunately the reason is still unknown.

The results showed that SDH and PK activity of primary mammary epithelium and fibroblasts was high after treatment with either AgNO₃ solution at 1 mmol/l or degradation medium of MgAg1% at 1 ml. similary, primary mammary epithelium cells showed a high SDH activity under light microscope too, when treated with AgNO₃ solution at 1 mmol/l , furthermore, histologically, bovine teats treated with MgAg1%

sticks showed a high SDH activity in comparison to the untreated bovine teats, while murine fibroblasts showed a higher SDH and PK activity than primary mammary cell cultures at 0.3 mmol/l, when treated with AgNO₃ solution.

Interestingly, the histochemical SDH activity showed the same results for the bovine teats tissues after treatment with MgAg1% sticks in comparison to the untreated teats. It showed a higher SDH activity represented in a dark blue colour in comparison to the control untreated teat. The definite explanation for this point still can not be stated, but whatever the explanation for this increase is, we did not have any decrease in the metabolic activity which means that the mitochondrial functions and lysosomes were not affected by our MgAg1% alloys or with different concentration of AgNO₃. HIDALGO and DOMINGUEZ (1998) demonstrated that Ag ions impaired mitochondrial activity in human fibroblasts assessed by succinate dehydrogenase activity.

Discussion

5.1.3 Effect of AgNO3 and MgAg1% sticks on the biomarkers of inflammatory reactions

The aim was to measure the inflammatory reaction in ex vivo and in vitro experiments, as potential inflammation is a major concern in the context of biomaterials and its constitutes are an important element of biocompatibility (WITTE et al., 2008; PURNAMA et al., 2010).

PGE₂ is an important indicator for measuring the inflammatory reactions in vitro as well as ex vivo; BÄUMER and KIETZMANN (2000) showed that isolated udder is a suitable model for inflammatory skin reactions. In our experiments, teat tissues from isolated udder showed no enhanced production of PGE₂ in reaction to MgAg1%

sticks regarding that PGE₂ in the treated teat was nearly of the amount in comparison to the untreated teat for 6 experiments. On the other hand, it was determined in one of the isolated udder experiments an enhanced production of PGE₂, but it might be related to an already persisting infection in the udder, while there was no enhanced production of PGE2 in other isolated udder experiments, that might be referred to short duration of the experiments which trigger PGE₂ production, but the absence of other increased inflammatory markers released from the other isolated udders would increase the probability towards the first reason.

We found that primary mammary epithelium cells and primary mammary fibroblasts also did not show an enhanced production of TNF-alpha in comparison to untreated control, while they were stimulated with 100 μg/ml LPS as a positive control. LPS is a part of the bacterial membrane of gram-negative bacteria and reacts as a bacterial endotoxin. This endotoxin, like other microbial components mediates its pro-inflammatory effects through binding to toll-like receptors (TLRs). LPS is recognized by the TLR4 (SONG et al. 2001), which has been detected in epithelial cells and keratocytes, but not in endothelial cells (SONG et al., 2001; JOHNSON et al. 2005;

KUMAGAI et al. 2005). We did not measure the other cytokines compared to the other cell lines, as TNF-alpha was the only cytokine available for measurement for bovine cell line. In addition, neither murine fibroblasts nor murine macrophages showed an enhanced production of IL-1beta, IL-6 and TNF-alpha, although these cells were stimulated with 100 μg/ml LPS as a positive control. LPS treated cells

Discussion

showed a significant increase of TNF-alpha, IL-1beta and IL-6 compared to the treated control. The fact that none of these markers were found after the incubation with degradation medium of MgAg1% or different concentrations of AgNO3 confirms our last step towards the biocompatibility experiments.