EFFECT OF BIOFILMS AND AGGRESSIVE IONS ON THE CORROSION OF DENTAL ALLOYS

M. Fernndez Lorenzo de Mele, M.C. Cortizo

1. INTRODUCTION Different metallic alloys are employed in dental works according to the requirements of the treatment. The noble metal alloys are extra-hard and thus suitable for metal-ceramic restorations but they are very expensive, have a low elastic module and their density is high (1). Due to their lower cost, low density and high elastic module, the nickel alloys and cobalt-chromium alloys are also frequently used instead of noble metal alloys when functionality and aesthetics allow it. The addition of Mo and Mn to nickel-chromium alloys has improved their corrosion resistance. However, nickel can cause allergic problems in sensitive patients (4-6). Silver and titanium as well as Ti-6Al-4V alloy are employed as filling materials of the root canals while stainless steel wires are generally used in orthodontic treatments (2-7). In bridges and crowns hard alloys with low content of gold and those of type Pd-Ag and most recently titanium alloys are employed (2,3). Ternary and quaternary alloys with gold or palladium base employed in dental restorations and intracanal implants usually contain silver. As a common feature they are darkened and corroded after the immersion in the biological medium (1). Tarnish was detected in those alloys with higher silver content (2,8). Silver cones used to be utilised as filling material of the dental root. They were specially employed in the filling of thin and curved canals because they are easily handled and allow a definite radiographic image. However, it has been reported that when silver is in contact with the tissue fluids, it deteriorates and forms citotoxic products, inflammation reactions and a defective filling (9). Besides, traces of silver and sulphur were detected in the biopsy of the surrounding tissues. The sulphur may arrive at the interface canal-cone through the organic fluids. This is in good agreement with the fact that the sulphur concentration detected through EDAX analysis of cones after an endodontical treatment decreases with the distance from the apex. X-Ray diffraction analysis shows the formation of Ag2S among the compounds formed (10). Corrosion of dental materials can also be induced by microbial activity because, like teeth, foreign materials surfaces are usually covered by a dental plaque. The dental plaque includes bacteria, their extracellular product, glucoproteins and inorganic deposits. The orthodontic materials attached in the oral cavity as well as the restorations are covered by the dental plaque. Besides, the endodontic materials as well as the implants are also often colonised by bacteria (11-13). Prophylactic products like NaF are frequently used to prevent tooth decay. The mechanisms of its action is not exactly understood but it is known that fluoride modulates the demineralisationremineralisation process of the hydroxiapatite (14,15). Besides fluoride could inhibit the production of acid enzymes by the microorganisms (16-18). However, this prophylactic action may be accompanied by the corrosion of the metallic dental materials by this aggressive anion.

The aim of this work is to study the biodeterioration of several metallic biomaterials used in dental works. Synthetic saliva and physiological solutions of different compositions were assayed to investigate the effect of chloride, sulphide and fluoride on the biomaterials performance. In addition, the effect of bacterial biofilm formation on the corrosion of these metals was also analysed as well as the influence of prophylactic products in the prevention of the biodeterioration process. 2. EXPERIMENTAL Experimental conditions are similar to those described elsewhere (19). Titanium, silver, chromiumnickel (Cr-Ni), cobalt-chromium (Co-Cr) silver- palladium (Ag-Pd) and copper-aluminium alloys (Cu-Al) provided with an electric contact and embedded in a resin were used as electrodes. Prior to each run, pre-treatment of samples was made by polishing up to alumina 1 m size. In all cases coupons were degreased with acetone, followed by rinsing in distilled water. Optical and scanning microscopic observations of the samples were made after each run. Electrochemical measurements were performed in a double wall electrochemical cell using a Pt counterelectrode and a saturated calomel electrode (SCE) as reference. Synthetic salivas of different compositions (19-20) were used as electrolytes. Occasionally, human plasma was also used. Polarisation curves were made at a constant scan rate of 0.01 V/min from -1.0 V up to different anodic limits. SEM observations of the films formed in the synthetic salivas sterile and inoculated with microorganisms were made. Microorganisms were isolated from the dental plaque of several patients. After exposure, and in order to preserve biological material, metallic specimens were fixed with 2 % glutaraldehyde in sterile saliva, washed with distilled water, dehydrated through an acetone series to 100 % and critical point dried. Estimation of total bacterial numbers was carried out through a direct cell count method using epifluorescent microscopy. After exposure the samples were removed, stained with 0.01 % acridine orange for 5 minutes, rinsed in sterile saliva to remove loose cells, and examined under ultraviolet light through epifluorescent microscopy at 400X. Cells were counted in 10 randomly chosen fields (field area: 0.0464 cm2) per sample. 3. RESULTS AND DISCUSSION 3.1 Electrochemical behaviour of dental metallic materials Fig. 1 shows the open circuit potentials (Ecorr) vs. time curves corresponding to titanium, Cr-Ni, AgPd, Cu-Al alloys immersed in the sterile synthetic saliva. Ecorr. did not exhibit important changes, except for titanium. In the case of Cu-Al alloy Ecorr records showed scattered values. Ecorr values were very close to those corresponding to high dissolution of copper as Cu (II) ions and/or dealloying. Polarisation curves made with titanium and Co-Cr coupons exhibit a passive region which extends up to high potential values (above 0.7 V) (20). Conversely, passivity breakdown was reported for CrNi and Cu-Al alloys. Selective corrosion occurs by dissolution of Ni-rich interdendritic regions formed during solidification of Cr-Ni alloys. The breakdown potentials were found above 0.07 V, depending on the alloy and saliva composition and oral cavity diseases (20,23).

The addition of fluoride did not alter the passivity of Ni-Cr, Ag-Pd, Co-Cr alloys under open circuit conditions. Higher dissolution was observed in Cu-Al alloys in agreement with previous results (2325). The behaviour of titanium coupons was very complex in this medium. Titanium owes its high corrosion resistance to the presence of a thin oxide layer. Immediately after the immersion the Ecorr increases attesting the oxide formation and the passivating processes occurring on the surface. It has been reported that passivity breakdown in fluoride media depends on the fluoride concentration (18). Titanium passivity was not altered in 0.02 M NaF in the potential range analysed (up to 0.3 V (SCE)) but it can be broken in 0.2 M solutions.

20 0 -20 Potential/ mV(SCE) -40 -60 -80 -100 -120 -140 -160 0 400 800 1200 Time (min) Fig. 1. Open circuit potential vs time plot of several dental materials. NiCr Ti CuAl AgPd

The action of fluoride in 0.2 M solutions is also dependent on the stage of the titanium oxide formation. When fluoride anions were added a day after the immersion the oxide was completely formed and no effect could be detected. Conversely, when the addition was made before the beginning of the record or after the 0.0 V value was reached, Ecorr decreases abruptly indicating the dissolution of the oxide layer. When a stable passive layer is formed the fluoride action is hindered and passivity is not modified even at high fluoride concentrations. However if the oxide film is formed under high fluoride concentrations the stability and homogeneity of the oxide could be altered. This could explain the contradictory results reported recently (25-27).

100 0 Potential/ mV(SCE) -100 -200 -300 -400 -500 0 200 400 600 800 1000 1200 1400 Time/min without NaF with NaF addition (see arrow)

Fig. 2. Effect of fluoride addition on the open circuit potential of titanium. Voltammograms made using silver and Ag-Pd alloy showed similar features. Current-voltage relationships obtained using chloride solutions and human plasma showed lower current values in the latter case. SEM observations revealed the formation of a mixed films on the electrode surface with different structural characteristics (21). Experiments carried out using the rotating ring-disk electrode technique lead to the detection of silver soluble species (silver chloride complexes). In the case of metals immersed in organic solutions containing chlorides, protein adsorption occurs preceeding the adhesion of the blood cells (22). Probably plasmatic proteins also adsorb on the silver surface hindering the chloride adsorption, and changing the morphology of the film. This justifies the current decrease observed when plasma is employed as electrolyte, with respect to that obtained in NaCl solutions. When Na2S is added to the chloride solution a current increase at very cathodic potential values (0.75 V) is recorded. It is associated to the formation of a Ag2S monolayer which is bright and compact and exhibits interference colours. Microscopic observations made after the removal of the film showed irregular micropits on the metal surface. Current transients depict the formation of a Ag2S monolayer on silver electrodes followed by the massive growth of a mixed film. EDX analysis of this layer detected sulphur and chlorine signals. At open circuit the Ag2S formation occurs when the electrode is immersed. The mixed film, Ag2S and AgCl, is formed at higher potentials. In all cases, the metal tarnishes losing its original brightness. Experiments made with Ag-Pd alloy showed similar tarnish processes when synthetic saliva of different compositions were used as electrolytes.

According to the results tarnish and pigmentation of silver and silver alloys under the experimental conditions assayed is related to the formation of AgCl, Ag2S and reduced Ag on the surface. The formation of micropits and corrosion products could induce failures in endodontic therapy and in dental restorations. 3.2 Bacterial adherence on dental materials With the aim of studying the influence of biofilm formation on the corrosion processes occurring in the oral cavity metal coupons were immersed in synthetic saliva inoculated with bacteria. These bacteria had been isolated from the dental plaque of several patients. No significant differences in the Ecorr values were detected in the case of titanium coupons immersed in synthetic saliva inoculated with microorganisms with respect to those immersed in sterile solutions (Table 1). Accordingly, previous results did not show any change in polarisation curves (18). However, variations of Ecorr up to 0.1 V could be observed for Ag-Pd, Ni-Cr, Cu-Al and CoCr alloys (Table 1). Particularly, the corrosion products structure of CuAl alloy was modified. The inner copper (I) and the outer copper (II) corrosion layers structural characteristics change in the presence of biofilms (28). To investigate the degree of microbial adhesion to different dental metallic material several dental alloys (Co-Cr, Ni-Cr, Cu-Al) and titanium microbial biofilms were analysed through epifluorescent microscopy. It could be observed that the highest bacterial number corresponded to titanium coupons followed in decreasing order by Co-Cr, Ni-Cr and Cu-Al alloys. The rate of adhesion of microorganisms to metallic surface is influenced by several factors such as the presence of biocides and the flow conditions. Besides, the rate of microorganisms attachment varies according to the nature of the substrate (29). Results show that the highest rate was found for titanium coupons on which the largest number of microorganisms were counted in the shortest period. The lowest rate was detected for Cu-Al alloys. There, a long induction period was needed to form a conditioned surface for bacterial adhesion, due to the toxic characteristics of this substrate. Bacterial cells were not uniformly distributed on the metal surface. Little holes and ditches in which diffusion was restricted were preferentially occupied by sessile bacteria. The rate of plaque maturation also seems to be positively correlated with surface roughness (30). Adsorbed and attached cells may be protected against shear forces and abrupt changes taking place in the bulk by the shelters and local irregularities provided by the rough surface.

Table 1. Corrosion potential (Ecorr/V (SCE) of several dental biomaterials after 20 h exposure period in a synthetic saliva, in the presence and in the absence of microorganisms. Metal/ Electrolyte solution Sterile saliva Sterile saliva inoculated with microorganisms Cr-Ni(A) -0.087 -0.155 Cu-Al -0.108 -0.160 Co-Cr -0.232 -0.126 Ag-Pd -0.150 -0.091 Ti 0.010 0.005

Formation of carious lesions is frequently associated to the attachment of Streptococcus mutans followed by Lactobacillus (31). A Streptococcus chains attached on titanium surface in contact with synthetic saliva could be seen by SEM. Oral microorganisms show sensitivity to fluoride (16-18). A rapid decrease in the planktonic population was found when fluoride was added to the synthetic saliva. However, biofilms formed on titanium surface seem to be less sensitive to this biocide. Larger amounts of extracellular polymeric substances were found close to bacteria in this case and lower reduction in the number of microorganisms than in the case of planktonic bacteria was detected. This indicates that sessile microorganisms have higher resistance to biocide action probably related to polymeric substances production. During the early stages of biofilm formation exopolysaccharides are overproduced and act as cements, reinforcing the adhesion mechanisms. Then, they may restrict the diffusion of agents from the surrounding medium. Such effects seem to be more pronounced for the activity of biocides (32, 33). Besides, high density microbial aggregates could be formed within the biofilm making the penetration of the biocide difficult (34). Copper and silver cause tarnish phenomena and are the preferentially corroded elements of noble metal alloys (1). The increase in the gold and palladium levels often decreases the rate of these processes. However, it must be kept in mind that corrosion, pigmentation and tarnish are not only related to the concentration of each element but to the relationship existing between these concentrations. 4. CONCLUSIONS Fluoride action did not significantly alter the passivity of the dental metallic biomaterials assayed except for copper alloys which corrosion products changed their structural characteristics. Besides, titanium dissolution could occur at high fluoride concentrations (0.2M) during the oxide layer formation. The antimicrobial effect of fluoride was more significant against planktonic than against sessile microorganisms. The diversity, abundance and rate of attachment of sessile microorganisms varied according to the nature of the metallic substrate. The highest rate was found for titanium and the lowest for Cu-Al alloy. Tarnish processes on silver and silver alloys under the experimental conditions assayed were related to the formation of AgCl, Ag2S and Ag deposits on the metal surface. The formation of micropits and corrosion products could induce failures, imperfect fillings and aesthetic problems in endodontic therapy and in dental restorations. Biofilms formation did not produce important changes in the corrosion potential values with respect to sterile conditions. However, the structural characteristics of the corrosion layers formed on copper and silver alloys changed in the presence of the biofilm. 5. ACKNOWLEDGEMENTS Authors wish to acknowledge to CONICET (PIP 4377/96) and the National University of La Plata for the financial support of this research project.

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Biography of the presenting author: Dr. Mnica Fernndez Lorenzo de Mele is a researcher of the CONICET (National Council of Technical and Scientific Research of Argentina). She belongs to INIFTA, Pure and Applied Physicochemical Research Institute of La Plata, Argentina. Her doctoral Thesis was about Metal/biofilm/solution interactions. She is Professor of the Engineering Faculty of the National University of La Plata. Author of 70 papers related to Biocorrosion and Biofouling, Corrosion of Biomaterials and Marine Corrosion which has been published in international journals. She has been the argentinian delegate of the BIOCORR network of CYTED (Iberoamerican Program of Science and Technology), which deals with Biocorrosion and Biofouling in Industrial Systems