Journal of Clinical Feriodontology: 1977: 4: 231-239 Key words: Denial plaque - copper amalgam - bacteriological e.'lect. Accepted for publication: November 24, 1976.
The plaque-inhibiting effect of copper amalgam TuuLA HYYPPA AND KEIJO PAUNIO Department of Periodontology, Institute of Dentistry, University of Turku, Turku, Finland Abstract. In order to assess whether copper amalgam accumulates less bacterial plaque than silver amalgam, 14 copper amalgam cervical restorations were placed in 10 patients. Control silver amalgam fillings were placed in homologous teeth of the same jaw. To evaluate the amount of plaque, the stained areas on the amalgams were analyzed from photographs with a planimetric method. Further quantitative analyses of plaque growth on copper amalgam were carried out with microbiological methods, for which a new group of 10 patients was selected. Cervical cavities were prepared on both sides of the jaw. Specimens of copper amalgam and silver amalgam restorations were placed on opposite sides. The plaque was collected after 2 and 3 days and cultivated under aerobic and anaerobic conditions on blood agar plates and on Mitis Salivarius Agar. The results of both methods show a very significant inhibition of plaque growth on copper amalgam. These results were confirmed by scanning electron micrographs which showed that copper amalgam only had plaque on small areas. The plaque appeared in band formations arranged irregularly on the surface. Higher magnifications revealed a filamentous structure with predominating coccoid organisms. Scanning electron micrographs also showed that pellicle formation, which is considered to be necessary for baeterial adherence to the surface, took place on both amalgams.
It is generally accepted that bacterial plaque plays a decisive role in the initiation of gingivitis. It has been shown that in the absence of oral hygiene, plaque accumulates at the gingivai margin, and in the course of 10 to 21 days, gingivitis can be observed (Lde et al. 1965). Plaque accumulation is promoted by overhanging filling margins and rough surfaces of restorations (Waerhaug 1965, Bjoi'n et al. 1969, Karlsen 1970). It has been shown that even well-adapted and polished restorations with subgingival extension cause gingival inflammation (Mormann et al. 1974). In light of this fact, some degree of gingival inflammation seems unavoidable adjacent to subgingival restorations unless
the material itself eould influence the bacterial plaque. Studies have been conducted to determine the antibacterial properties of restorative materials. TUrkheini (1953) observed in in vitro experiments that copper amalgam had a very strong antibacterial effect. Shay et al. (1956) also found copper amalgam to be the most efficacious of the tested dental materials in vitro. Lind et al. (] 964) found that copper amalgam caused a lower rate of contact caries and had consequently a better cariostatic effect than silver amalgam. Their study was carried out on systematically treated school children. In the present study, planimetric analysis,
232
HYYPPA AND PAUNIO
scanning electron microscopy and microbiologic methods are used to assess the amount of plaque on cervical copper amalgam restorations. Material and Methods
Planimetric Analysis For planimetric analysis of the amount of plaque on cervical restorations, 10 patients (seven male and three female) aged between 14 and 58 years (mean age 35) were included in the study. All patients had primary carious lesions on bucco-gingival surfaces which required class V restorations. A total of 14 restorations of copper amalgam (Cupro-Muc®, Mertz Co.) were made in either a first molar, premolar or canine. Restorations of silver amalgam (PresiDent® alloy, Presi-Dent AB) in the contralateral tooth of the same jaw served as controls. In five cases concerning the premolars the control restorations were made in the adjacent premolar. In order to test the plaque deposition on the fillings, the teeth were cleaned with a paste of pumice and a rotating rubber cup. Interdental cleaning was carried out with reciprocating interproximal tips (Eva Prophylaxis System, Dentatus). The patients were asked to refrain from all oral hygiene procedures for 3 days. The accumulated plaque was stained with 0.1 % basic fuchsin (in 10 % ethanol). Colour photographs, magnification 2 x, were taken of the test and the control tooth. The photographs were analyzed through planimetry, carried out with a digitizer (Hewlett-Packard Digitizer, model 9864A) entering Cartesian coordinate data from the above graphic records into a calculator (Howlett-Packard 9821 A Series calculator) according to the method of Rekola & Scheinin (1977). For each observed tooth the following data was entered for planimetric analysis: a) the entire surface of the cervical restoration, b) the areas of the fill-
ing covered by plaque, both lightly and heavily stained. The age of the restorations at the plaque registration varied from 10 days to 101 days, the average being 47 days. Microbiological Studies For further quantitative estimation of the amounts of plaque on copper and silver amalgam, another group of 10 patients was selected, one female and nine male. The age ratige of the patients was 17-65 years, mean age 40. Cervical class V cavity preparations were made in homologous teeth of tbe same Jaw. Cylindrical specimens of copper and silver amalgam were prepared as follows: a fairly rigid plastic tube, inner diameter 1.9 mm was cut in slices of equal width. The slice served as a matrix and was filled with amalgam. The specimens were fitted into tbe cavities and embedded in wax (Astynax®, Associated Dental Products, Ltd.) leaving only the outer surface free for plaque accumulation. No oral hygiene was practiced for 2 or 3 days. The teeth were first gently rinsed with water to remove saliva and food debris after which the pieces were dislodged with a sterile carver and dropped into a test tube containing 1.5 ml nutrient broth (Difco) with 0.1 % cysteine. The sample was cooled in an ice bath to 4°C and the plaque was dispersed by sotiication for 30 s with an amplitude of 7 ^m. The sample was serially diluted in the above medium and plated on blood agar and Mitis Salivarius Agar (Difco). Four sets of blood plates were ineubated aerobically at 37°C. Triplicate sets of blood agar plates were incubated anaerobically in jars with 80 % Ns, 10 % CO2 and 10 % H, for 3 to 5 days at 37°C. Triplicate plates of Mitis Salivarius Agar were incubated anaerobically for 24 h at 31°C and kept at room temperature for another 24 h. The number
THE PLAQUE-INHIBITING EFFECT OF COPPER AMALGAM of colonies on all plates was determined; the final count represents a mean of 3 or 4 counts of plates with 0-500 colony forming units. For statistical analysis of the colony counts the Mann-Whitney U-test was used. Scanning Electron Microscopy Copper and silver amalgam specimens were placed in the mouth as described above and kept in place for 20 and 60 min for pellicle formation and 2 days for plaque formation. Samples were taken from three individuals. The pieces were removed, washed with distilled de-ionized water and rapidly frozen in methanol and freeze-dried. Approximately 200 A of gold-palladium was evaporated onto the surface. The specimens were examined in a ISM U3 scanning electron microscope at an accelerating voltage of 15 kV.
233
vation of microorganisms grown on silver and copper amalgam restorations for 3 days gave the following mean counts on blood agar plates: silver amalgam 1.5 X lO*" and copper amalgam 2.0 X 10^ microorganisms/ surfaces. The difference between the silver and copper amalgam counts is statistically highly significant.
Results Planimetric Analysis Copper amalgam restorations had much less plaque than silver restorations (Figs. lA and IB). On copper amalgam small amounts of plaque could be observed in two cases, while on 12 copper restorations there was no visible plaque. The mean distribution of plaque on copper amalgam expressed as percentage of the totai filling area was 1.93 ± 1.36 (mean ± S.E.). In contrast, the silver amalgam restorations accumulated a considerable amount of plaque; the percentage distribution of plaque on these fillings was 58.07 ± 9.0 (mean ± S.E.). The difference between the two groups is statistically highly significant. Microbiological Studies Viable total counts on blood agar incubated aerobically and anaerobically and viable streptococcal counts on Mitis Salivarius Agar are shown in Table 1. Aerobic culti-
B Fig. 1. The effect of copper amalgam restoration (A) on plaque growth when oral hygiene procedures were suspended for 3 days. The control silver amalgam restoration (B) was made in the contra-lateral tooth of the same jaw. Die Wirkung der KupferamalgamfUllung (A) aiif das Plaquewachstum bei 3-tagigem Einstellen oraler Hygienemassnahmen. Die KonlroHfiiUung in Silberamalgam (B) stammt von dein kontralateralen Zahn des gleichen Kiefers. Effet dc I'obturation a i'amalgame de ciiivre (A) siir le developpement de la plaque lorsque les soins d'liygiene bucco-dentaire soni suspendus pendant 3 jours. L'obturation tenioin d'amalgame d'argent (B) avail e!e faite sur la dent controlaterale de la meme mdchoire.
HYYPPA AND PAUNIO
234
Table 1. Plaque on copper and silver amalgam specimens. Mean counts of microorganisms on blood agar and Mitis Salivarius Agar Plaque auf Kupfer- und Silberamalgamproben. Mittelwerte der Anzahl von Mikroorganismen auf Blut- und Mitis Salivarius Agar. Plaque sur les specimens d'amalgame de cuivre et d'amalgame d'argent. Nombres moyens de microorganismes sur agar au sang et sur "Mitis Salivarius Agar" Subjeet
Age of plaque
Aerobic count
nX:
Anaerobic count nXlO'
Mitis Salivarius Agar
silver
copper"
silver
copper"
silver
copper"
1 2
2 days
116 414
9.7 10.3
44 220
1.6 4.9
20 36
0.6 0.6
mean
(n = 2)
265
10
132
3.3
28
0.6
3 days
1700 3500
15 1.8 1.4 O.I 1.9 49 11 15 20
330
2 1 0.2 0.2 2 29 66 10 14
21 780 25 210 130 140 200 180 210
no growth
880 310
3000 800
1400 10 Mean (n
690
1500
3300 74 270
3700 560 810 510
1200
0.7 0.2
no growth 1.5 3.9 74 39 15
"There is a highly significant difference between the silver amalgam and copper amalgam groups (Mann-Whitney U-test) ^^Zwischen den bei Silber- und Kupferamalgamgruppcn gefundenen Werten liegen stark signifikante Unterschiede vor (Mann-Whitney U-Test). "II existe une difference hautement significative entre le groupe de I'amalgaine d'argent et le groupe de I'amalganie de cuivre Age of plaque {Alter der Plaque, age de la plaque), aerobic count {Anzahl Aerober, nombre d'aerobies), anaerobic count {Anzahl Anaerober, nombre d'anaerobies).
Under anaerobic conditions the following counts were obtained: on silver amalgam 1.2 X 100 and on copper amalgam 1.0 X 10^ microorganisms/surface. The difference between the silver and copper amalgam counts is statistically highly significant. Viable streptococcal counts on Mitis Saiivarius Agar yielded the following mean counts: on silver amalgam 2.1 X 105 and on copper amalgam 1.5 X 10^ microorganisms per surface. Comparison of mean microbial counts on blood agar with the counts on Mitis Salivarius Agar shows that streptococci comprised 14 to 18 % of the total microflora of 3-day-old plaque on silver
amalgam. On copper amalgam the percentage of streptococci varied from 75 to 100 % of the total viable counts. Scanning Electron Microscopy Scanning electron microscopy revealed that pellicle formation occurred on both amalgams studied. It was seen as an organic film covering the surface structure of the amalgam in as little as 20 min after exposure to the oral environment. After 2 days heavy plaque formation was obtained on most silver amalgam restorations (Fig. 3 A and B). The outlines of individual cells could often not be seen
THE PLAQUE-INfflBIlTNG EFFECT OF COPPER AMALGAM
SILVER
AMALGAM
60 COPPER
AMALGAM
'.O
20
Fig. 2. Percentage distribution of stained plaque in 10 test subjects on 14 copper and silver amalgam restorations after 3 days of refraining from oral hygiene procedures. Prozentuelle Verteilung eingefdrbler Plague bei 10 Probanden, bezogen auf 10 Kupfer- und Silberamalgamfullungen nach dreitdgigem Eitistellen aller oraler Hygienemassnahmen. Repartition de la plaque (exprimee en pourcentage) apres coloration chez 10 sujets sur 14 obturations a I'amalgame d'argent et a I'amalgame de cuivre aprcs 3 jours d'abstention des soins d'hygiene bucco-dentaire.
because of the amorphous organic material covering the bacteria. High magnifications revealed a granular matrix between the organisms. Areas with thin layers of plaque made it possible to observe that the plaque was composed of filamentous organisms and cocci. On copper amalgam specimens bands of plaque were spread irregularly over the surface leaving large areas free of plaque (Fig. 3C). An organic film covering the surface of the amalgam was also observed. A higher magnification of the plaque covered area showed a network of filamentous organisms with intermingled cocci dominating. Attachment between the organisms could also be seen (Fig. 3 D). Discussion
The antibacterial effect of copper amalgam in vivo has been confirmed using three different methods. First, analysis with planimetry showed that plaque accumulation on
235
cervical copper amalgam restorations was markedly reduced when compared with silver amalgam restorations. This observation was confirmed by microbial counts and scanning electron micrographs of plaque grown on copper amalgam specimens. Planimetric analysis for evaluating the plaquecovered areas of the fillings was chosen because reproducible scoring can be carried out with considerable accuracy (Rekola & Scheinin 1977). The results show that there is a striking difference in the amounts of stained plaque on these two amalgams. In the five cases concerning premolars where the test and control restorations were situated in adjacent teeth, however, it was observed that the plaque on silver amalgam was slightly less stained than on the other control restorations. This indicates that dissolved copper ions may be transmitted to the surface of the silver amalgam restorations and inhibit plaque growth there as well. The microbiologic investigations of 2and 3-day-oId plaque from 10 different subjects verify the above results of reduced plaque growth on copper amalgam. The total amounts of microorganisms per sample are comparable since the surface used for collecting the plaque was of standardized size. From the 3-day-old plaque grown on silver amalgam only a small percentage of streptococci was recovered. On the other hand, plaque from copper amalgam specimens seemed to be made up predominantly of streptococci. The high proportion of streptococci on copper amalgam could be explained by the toxic effect of copper ions on bacterial growth. Previous reports (e.g. Theilade & Theilade 1970) have shown that coccoid organisms dominate in early plaque formation, and since the plaque is not able to mature because of constantly dying organisms, this accounts for the high proportion of streptococci in th^'e viable counts.
236
HYYPPA AND PAUNIO
The formation of dental plaque involves an initial attachment of bacteria to the acquired pellicle (Gibbons & van Houte 1973). To observe whether pellicle formation occurs on copper amalgam, specimens were placed in the mouth in a cervical cavity for 20 and 60 min. The scanning micrographs show that there is an organic film covering both the copper and silver amalgam surfaces. Thus it seems that cop-
per ions do not prevent pellicle formation even though the growth of microorganisms is inhibited. On the other hand, the structure of the pellicle formed on the copper amalgam restoration may be so changed, that it is unlikely that bacteria will attach to it. The 2-day-old plaque formed on copper amalgam appeared as bands arranged irregularly on the surface, showing that there
THE PLAQUE-INHIBITING EFFECT OF COPPER AMALGAM were large areas free of plaque accumulation. This explains why the viable counts obtained from the copper amalgam pieces were so small. The filamentous structure of the plaque with predominantly coccoid organisms does not differ from similar structures found in scanning electron micrographs of plaque grown on enamel (Newman 1972). There were no epithelial cells on the surface of the copper amalgams or in the plaque formed on them as was seen in plaque grown on enamel (Tinanoff et al. 1976). Gingival restorations which extend below the gingival margin are likely to accumulate plaque and it is concluded by Waerhaug (1975) that these restorations are involved in the etiology of destructive periodontai disease. Therefore, it is important to consider the plaque-inhibiting effect of the restorative material chosen. The present study indieates that copper amalgam is less likely to accumulate plaque than silver
237
amalgam. However, more research in this field is necessary in order to elucidate the therapeutic action of copper amalgam in restorations and its long-term effect on the gingiva. Zusammenfassung
Plaqueinhibierender Effekt von Kupferamalgam Um das Vermogen von Kupferamalgam zu studieren Plaqueansammlungen zu reduzieren, wurden bei 10 Patienten vierzehn zervikale Kupferamalgarafiillungen gelegt. Kontrollfiillungen in Silberamalgam wurden an homologen Zabnen des gleichen Kiefers angebracbt. Um die Piaquemenge zu schatzen, warden die eingefarbten Abschnitte der Test- und KontroHfiJllungen nach fotografischer Registrierung mit planimetrischer Methode analysiert. Weiterhin wurden quantitative Analysen des Plaquewaehstums auf Kupferamalgam mit mikrobiologischen Methoden vorgenommen. Hierfur wurde eine iieue Groppe von 10 Patienten ausgewahlt. Zervikale Kavitaten wurden in Zahne beider Kieferseiten prapariert. Test und Kontrollflillungen in Kupfer- bzw. Silberamal-
Eig. 3. Scanning electron micrographs from the surface of silver and copper amalgam pieces after 2 days in tooth cavities. The test subjects refrained from oral hygiene procedures during this time. A) shows a 740 X magnification and B) a 7400 X magnification of the silver amalgam restorations. A heavy amorphous organic material covers the plaque surface (A). In the bottom of the crevice microorganisms are seen arranged parallel to each other (B). C) shows a 740 X magnification and D) 7400 X magnification of the piece of copper amalgam. Note the plaque-free areas on the copper amalgam surface. The plaque is composed of filamentous organisms with intermingled cocci. (D) Scanning-elektronische Mikrographien der Oberfldche von Silber- und Kupferamalgamfiillungen nach 2-tagiger Applikation in Zahnkavitdten. Die Probanden stellten wahrend dieser Zeit alle orale Hygienemassnahmen ein. A) zeigt die SilheramalgamfUUungen in 740-facher und B) in 7400-facher Vergrosserung. Die Plaqueoberfldche wird von einer Menge amorphen organischen Materials bedeckt (A). Auf dem Boden des Spaltes sind paralell liegende Mikroorganismen sichthar (B). (C) zeigt die Kupferamalgamprobe in 740-facher und (D) in 7400'facher Vergrosserung. Bitte beachten Sie die plaquefreien Stellen auf der Oberfldche des Kupferamalgams. Die Plaque besteht aus fadenforniigen Organismen, vermischt mit Kokken (D). Cliches au microscope electroniquc a balayage: surface de specimens d'amalgame d'argent et d'amalgame de cuivre places depuis 2 jours dans les cavites. Les sujets s'etaient pendant cette periode abstenus de soins d'hygiene bucco-dentaire. A) montre un grossissement a 740 X et B) un grossissement a 7400 X des obturations d'amalgame d'argent. Une couche compacte de substance organique amorphe couvre la surface de la plaque (A). Au fond de Vanfractuosite, on volt des micro-organismes disposes en direction parallele les uns aux autres (B). C) montre un grossissement a 740 X et D) un grossissement a 7400 X du specimen d'amalgame de cuivre. La plaque est constitute d'organismes filamenteux entremeles de cocci (D).
HYYPPA AND PAUNIO
238
gam wurden an einander gegeniiberliegenden Seitea gelegt. Nach 2 imd 3 Tagen wurde die Plaque gesammelt und unter aeroben und anaeroben Verhaltnlssen auf Blut-Agarplatten und Mitis Salivarius Agar kultiviert. Die Resuitate beider Untersuchungsmethoden zeigen signifikante Inhibition von Piaquewachstum auf Kupferamalgam. Diese Resultate wurden durch Scanningelektronische Mikrographie bestatigt die zeigte, dass sich auf Kupferamalgam nur kleine Plaqueareale befanden. Die Plaque hatte irregulare Bandformation. Bei hoherer Vergrbsserung wurden fadenformige Stnikturen mit uberwiegenden kokkoiden Organismen gesehen. Die Scanning-elektronische Mikrographie zeigte weiterhin, dass eine PelUkelbildung, die als Voraussetzung fiir die Haftung von Bakterien angesehen wird, an beiden Araalgamen stattfand. Resume
Inhibition de la plaque par i'amalgame de cuivre Aiin d'etabiir si i'amalgame de cuivre accumule moins de plaque microbienne que l'amalgame d'argent, on a place chez 10 patients des obturations du coliet en amalgame de cuivre, et, a la meme machoire, dans les dents homologues, on a fait des obturations temoins en amalgame d'argent. Pour evaluer !a quantite de plaque, on a, apres application de colorant, fait une analyse de la surface des amalgames sur photographies par une methode planimetrique. On a de plus effectue par des methodes bacteriologiques une analyse quantitative du developpement de la plaque sur I'amalgame de cuivre. Pour cette analyse, un nouveau groupe de 10 patients a ete selectionne. Des cavites ont ete preparees au niveau du collet sur des dents des deux cotes de la machoire. Des specimens d'obturations a I'amalgame d'argent et a I'amalgame de cuivre ont ete places sur les cotes opposes. La plaque a ete recueillie au bout de 2 jours et au bout de 3 jours et cultivee dans des conditions aerobies et anaerobies sur des plaques d'agar au sang et sur milieu "Mitis Salivarius Agar", Les resultats obtenus par les deux methodes montraient une inhibition tres significative du developpement de la plaque par I'amalgame de cuivre. Ces resultats ont ete confirmes par des cliches au microscope electronique a balayage qui montraient que I'amalgame de cuivre ne presentait que de petites zones couvertes de
plaque. La plaque se presentait sous forme de bandelettes placees sur la surface de fagon irregulifire. A plus fort grossissement, on constatait une structure filamenteuse avec predominance d'organismes semblables a des cocci. Les cliches au microscope eiectronique a balayage revelaient qu'il se produisait sur les deux sortes d'amalgame une formation de peilicule, ce qui est considere comme etant necessaire pour l'adhesion microbienne a la surface. References
Bjorn, A.-L., Bjom, H. & Grkovic, B. (1969) Marginal fit of restorations and its reiation to bone level. I. Metal fillings. Odontologisk Revy 20, 311-321. Gibbons, R. J. & van Houte, J. (1973) On the formation of dental plaque. Journal of Periodontology 44, 347-359. Karlsen, K. (1970) Gingiva! reactions to dental restorations. Ada Odontologica Scandinavica 28, 895-904. Lind, V., Wennerhoim, G. & Nystrom, S. (1964) Contact caries in connection with silver amalgam, copper amalgam and silicate fillings. Acta Odontologica Scandinavica 22, 333-341. Loe, H., Theilade, E. & Jensen, S. B. (1965) Experimental gingivitis in man. Journal of Periodontology 36, 177-187. Morman, W., Regolati, B. & Renggli, H. (1974) Gingival reaction to well-fitted subgingival proximal gold inlays. Journal of Clinical Periodonlology 1, 120-125. Newman, H. N. (1972) Structure of approximal human dental plaque as observed by scanning electron microscopy. Archives of Oral Biology, 17, 1445-1453. Rekola, M. & Scheinin, A. (1977) Quantification of dental plaque through planimetric analysis. Scandinavian Journal of Dental Research 85, 51-55. Shay, D. E., Allen, T. J. & Mantz, R. F. (1956) The antibacterial effects of some dental restorative materials. Journal of Dental Research 35, 25-32. Theilade, E. & Theilade, J. (1970) Bacteriological and ultrastnictura! studies of developing dental plaque. In Dental Plaque, ed. McHugh, W. D., pp. 27-40. Edinburgh: E. & S. Livingstone Ltd. Tinanoff, N., Gross, A. & Brady, J. M. (1976) Development of plaque on enamel. Parallel investigations. Journal of Periodontal Research 11, 197-209.
THE PLAQUE-INHIBITING EFFECT OF COPPER AMALGAM Tiirkheim, H. I. (1953) Bacteriological investigations on dental filling materials. BrUish Dental Journal 95, 1-7. Waerhaug, J. (1956) Effect of rough surfaces upon gingival tissue. Journal of Dental Research 35, 323-325. Waerhaug, J. (1975) Presence or ahsence of plaque on suhgingival restorations. Scandinavian Journal of Dental Research, 83, 193201.
Address: Tuula Hyyppd Department of Periodontology Institute of Dentistry University of Turku Lemminkdisenkatu 2 SF~20520 Turku 52 Finland
239
The plaque-inhibiting effect of copper amalgam TuuLA HYYPPA AND KEIJO PAUNIO Department of Periodontology, Institute of Dentistry, University of Turku, Turku, Finland Abstract. In order to assess whether copper amalgam accumulates less bacterial plaque than silver amalgam, 14 copper amalgam cervical restorations were placed in 10 patients. Control silver amalgam fillings were placed in homologous teeth of the same jaw. To evaluate the amount of plaque, the stained areas on the amalgams were analyzed from photographs with a planimetric method. Further quantitative analyses of plaque growth on copper amalgam were carried out with microbiological methods, for which a new group of 10 patients was selected. Cervical cavities were prepared on both sides of the jaw. Specimens of copper amalgam and silver amalgam restorations were placed on opposite sides. The plaque was collected after 2 and 3 days and cultivated under aerobic and anaerobic conditions on blood agar plates and on Mitis Salivarius Agar. The results of both methods show a very significant inhibition of plaque growth on copper amalgam. These results were confirmed by scanning electron micrographs which showed that copper amalgam only had plaque on small areas. The plaque appeared in band formations arranged irregularly on the surface. Higher magnifications revealed a filamentous structure with predominating coccoid organisms. Scanning electron micrographs also showed that pellicle formation, which is considered to be necessary for baeterial adherence to the surface, took place on both amalgams.
It is generally accepted that bacterial plaque plays a decisive role in the initiation of gingivitis. It has been shown that in the absence of oral hygiene, plaque accumulates at the gingivai margin, and in the course of 10 to 21 days, gingivitis can be observed (Lde et al. 1965). Plaque accumulation is promoted by overhanging filling margins and rough surfaces of restorations (Waerhaug 1965, Bjoi'n et al. 1969, Karlsen 1970). It has been shown that even well-adapted and polished restorations with subgingival extension cause gingival inflammation (Mormann et al. 1974). In light of this fact, some degree of gingival inflammation seems unavoidable adjacent to subgingival restorations unless
the material itself eould influence the bacterial plaque. Studies have been conducted to determine the antibacterial properties of restorative materials. TUrkheini (1953) observed in in vitro experiments that copper amalgam had a very strong antibacterial effect. Shay et al. (1956) also found copper amalgam to be the most efficacious of the tested dental materials in vitro. Lind et al. (] 964) found that copper amalgam caused a lower rate of contact caries and had consequently a better cariostatic effect than silver amalgam. Their study was carried out on systematically treated school children. In the present study, planimetric analysis,
232
HYYPPA AND PAUNIO
scanning electron microscopy and microbiologic methods are used to assess the amount of plaque on cervical copper amalgam restorations. Material and Methods
Planimetric Analysis For planimetric analysis of the amount of plaque on cervical restorations, 10 patients (seven male and three female) aged between 14 and 58 years (mean age 35) were included in the study. All patients had primary carious lesions on bucco-gingival surfaces which required class V restorations. A total of 14 restorations of copper amalgam (Cupro-Muc®, Mertz Co.) were made in either a first molar, premolar or canine. Restorations of silver amalgam (PresiDent® alloy, Presi-Dent AB) in the contralateral tooth of the same jaw served as controls. In five cases concerning the premolars the control restorations were made in the adjacent premolar. In order to test the plaque deposition on the fillings, the teeth were cleaned with a paste of pumice and a rotating rubber cup. Interdental cleaning was carried out with reciprocating interproximal tips (Eva Prophylaxis System, Dentatus). The patients were asked to refrain from all oral hygiene procedures for 3 days. The accumulated plaque was stained with 0.1 % basic fuchsin (in 10 % ethanol). Colour photographs, magnification 2 x, were taken of the test and the control tooth. The photographs were analyzed through planimetry, carried out with a digitizer (Hewlett-Packard Digitizer, model 9864A) entering Cartesian coordinate data from the above graphic records into a calculator (Howlett-Packard 9821 A Series calculator) according to the method of Rekola & Scheinin (1977). For each observed tooth the following data was entered for planimetric analysis: a) the entire surface of the cervical restoration, b) the areas of the fill-
ing covered by plaque, both lightly and heavily stained. The age of the restorations at the plaque registration varied from 10 days to 101 days, the average being 47 days. Microbiological Studies For further quantitative estimation of the amounts of plaque on copper and silver amalgam, another group of 10 patients was selected, one female and nine male. The age ratige of the patients was 17-65 years, mean age 40. Cervical class V cavity preparations were made in homologous teeth of tbe same Jaw. Cylindrical specimens of copper and silver amalgam were prepared as follows: a fairly rigid plastic tube, inner diameter 1.9 mm was cut in slices of equal width. The slice served as a matrix and was filled with amalgam. The specimens were fitted into tbe cavities and embedded in wax (Astynax®, Associated Dental Products, Ltd.) leaving only the outer surface free for plaque accumulation. No oral hygiene was practiced for 2 or 3 days. The teeth were first gently rinsed with water to remove saliva and food debris after which the pieces were dislodged with a sterile carver and dropped into a test tube containing 1.5 ml nutrient broth (Difco) with 0.1 % cysteine. The sample was cooled in an ice bath to 4°C and the plaque was dispersed by sotiication for 30 s with an amplitude of 7 ^m. The sample was serially diluted in the above medium and plated on blood agar and Mitis Salivarius Agar (Difco). Four sets of blood plates were ineubated aerobically at 37°C. Triplicate sets of blood agar plates were incubated anaerobically in jars with 80 % Ns, 10 % CO2 and 10 % H, for 3 to 5 days at 37°C. Triplicate plates of Mitis Salivarius Agar were incubated anaerobically for 24 h at 31°C and kept at room temperature for another 24 h. The number
THE PLAQUE-INHIBITING EFFECT OF COPPER AMALGAM of colonies on all plates was determined; the final count represents a mean of 3 or 4 counts of plates with 0-500 colony forming units. For statistical analysis of the colony counts the Mann-Whitney U-test was used. Scanning Electron Microscopy Copper and silver amalgam specimens were placed in the mouth as described above and kept in place for 20 and 60 min for pellicle formation and 2 days for plaque formation. Samples were taken from three individuals. The pieces were removed, washed with distilled de-ionized water and rapidly frozen in methanol and freeze-dried. Approximately 200 A of gold-palladium was evaporated onto the surface. The specimens were examined in a ISM U3 scanning electron microscope at an accelerating voltage of 15 kV.
233
vation of microorganisms grown on silver and copper amalgam restorations for 3 days gave the following mean counts on blood agar plates: silver amalgam 1.5 X lO*" and copper amalgam 2.0 X 10^ microorganisms/ surfaces. The difference between the silver and copper amalgam counts is statistically highly significant.
Results Planimetric Analysis Copper amalgam restorations had much less plaque than silver restorations (Figs. lA and IB). On copper amalgam small amounts of plaque could be observed in two cases, while on 12 copper restorations there was no visible plaque. The mean distribution of plaque on copper amalgam expressed as percentage of the totai filling area was 1.93 ± 1.36 (mean ± S.E.). In contrast, the silver amalgam restorations accumulated a considerable amount of plaque; the percentage distribution of plaque on these fillings was 58.07 ± 9.0 (mean ± S.E.). The difference between the two groups is statistically highly significant. Microbiological Studies Viable total counts on blood agar incubated aerobically and anaerobically and viable streptococcal counts on Mitis Salivarius Agar are shown in Table 1. Aerobic culti-
B Fig. 1. The effect of copper amalgam restoration (A) on plaque growth when oral hygiene procedures were suspended for 3 days. The control silver amalgam restoration (B) was made in the contra-lateral tooth of the same jaw. Die Wirkung der KupferamalgamfUllung (A) aiif das Plaquewachstum bei 3-tagigem Einstellen oraler Hygienemassnahmen. Die KonlroHfiiUung in Silberamalgam (B) stammt von dein kontralateralen Zahn des gleichen Kiefers. Effet dc I'obturation a i'amalgame de ciiivre (A) siir le developpement de la plaque lorsque les soins d'liygiene bucco-dentaire soni suspendus pendant 3 jours. L'obturation tenioin d'amalgame d'argent (B) avail e!e faite sur la dent controlaterale de la meme mdchoire.
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Table 1. Plaque on copper and silver amalgam specimens. Mean counts of microorganisms on blood agar and Mitis Salivarius Agar Plaque auf Kupfer- und Silberamalgamproben. Mittelwerte der Anzahl von Mikroorganismen auf Blut- und Mitis Salivarius Agar. Plaque sur les specimens d'amalgame de cuivre et d'amalgame d'argent. Nombres moyens de microorganismes sur agar au sang et sur "Mitis Salivarius Agar" Subjeet
Age of plaque
Aerobic count
nX:
Anaerobic count nXlO'
Mitis Salivarius Agar
silver
copper"
silver
copper"
silver
copper"
1 2
2 days
116 414
9.7 10.3
44 220
1.6 4.9
20 36
0.6 0.6
mean
(n = 2)
265
10
132
3.3
28
0.6
3 days
1700 3500
15 1.8 1.4 O.I 1.9 49 11 15 20
330
2 1 0.2 0.2 2 29 66 10 14
21 780 25 210 130 140 200 180 210
no growth
880 310
3000 800
1400 10 Mean (n
690
1500
3300 74 270
3700 560 810 510
1200
0.7 0.2
no growth 1.5 3.9 74 39 15
"There is a highly significant difference between the silver amalgam and copper amalgam groups (Mann-Whitney U-test) ^^Zwischen den bei Silber- und Kupferamalgamgruppcn gefundenen Werten liegen stark signifikante Unterschiede vor (Mann-Whitney U-Test). "II existe une difference hautement significative entre le groupe de I'amalgaine d'argent et le groupe de I'amalganie de cuivre Age of plaque {Alter der Plaque, age de la plaque), aerobic count {Anzahl Aerober, nombre d'aerobies), anaerobic count {Anzahl Anaerober, nombre d'anaerobies).
Under anaerobic conditions the following counts were obtained: on silver amalgam 1.2 X 100 and on copper amalgam 1.0 X 10^ microorganisms/surface. The difference between the silver and copper amalgam counts is statistically highly significant. Viable streptococcal counts on Mitis Saiivarius Agar yielded the following mean counts: on silver amalgam 2.1 X 105 and on copper amalgam 1.5 X 10^ microorganisms per surface. Comparison of mean microbial counts on blood agar with the counts on Mitis Salivarius Agar shows that streptococci comprised 14 to 18 % of the total microflora of 3-day-old plaque on silver
amalgam. On copper amalgam the percentage of streptococci varied from 75 to 100 % of the total viable counts. Scanning Electron Microscopy Scanning electron microscopy revealed that pellicle formation occurred on both amalgams studied. It was seen as an organic film covering the surface structure of the amalgam in as little as 20 min after exposure to the oral environment. After 2 days heavy plaque formation was obtained on most silver amalgam restorations (Fig. 3 A and B). The outlines of individual cells could often not be seen
THE PLAQUE-INfflBIlTNG EFFECT OF COPPER AMALGAM
SILVER
AMALGAM
60 COPPER
AMALGAM
'.O
20
Fig. 2. Percentage distribution of stained plaque in 10 test subjects on 14 copper and silver amalgam restorations after 3 days of refraining from oral hygiene procedures. Prozentuelle Verteilung eingefdrbler Plague bei 10 Probanden, bezogen auf 10 Kupfer- und Silberamalgamfullungen nach dreitdgigem Eitistellen aller oraler Hygienemassnahmen. Repartition de la plaque (exprimee en pourcentage) apres coloration chez 10 sujets sur 14 obturations a I'amalgame d'argent et a I'amalgame de cuivre aprcs 3 jours d'abstention des soins d'hygiene bucco-dentaire.
because of the amorphous organic material covering the bacteria. High magnifications revealed a granular matrix between the organisms. Areas with thin layers of plaque made it possible to observe that the plaque was composed of filamentous organisms and cocci. On copper amalgam specimens bands of plaque were spread irregularly over the surface leaving large areas free of plaque (Fig. 3C). An organic film covering the surface of the amalgam was also observed. A higher magnification of the plaque covered area showed a network of filamentous organisms with intermingled cocci dominating. Attachment between the organisms could also be seen (Fig. 3 D). Discussion
The antibacterial effect of copper amalgam in vivo has been confirmed using three different methods. First, analysis with planimetry showed that plaque accumulation on
235
cervical copper amalgam restorations was markedly reduced when compared with silver amalgam restorations. This observation was confirmed by microbial counts and scanning electron micrographs of plaque grown on copper amalgam specimens. Planimetric analysis for evaluating the plaquecovered areas of the fillings was chosen because reproducible scoring can be carried out with considerable accuracy (Rekola & Scheinin 1977). The results show that there is a striking difference in the amounts of stained plaque on these two amalgams. In the five cases concerning premolars where the test and control restorations were situated in adjacent teeth, however, it was observed that the plaque on silver amalgam was slightly less stained than on the other control restorations. This indicates that dissolved copper ions may be transmitted to the surface of the silver amalgam restorations and inhibit plaque growth there as well. The microbiologic investigations of 2and 3-day-oId plaque from 10 different subjects verify the above results of reduced plaque growth on copper amalgam. The total amounts of microorganisms per sample are comparable since the surface used for collecting the plaque was of standardized size. From the 3-day-old plaque grown on silver amalgam only a small percentage of streptococci was recovered. On the other hand, plaque from copper amalgam specimens seemed to be made up predominantly of streptococci. The high proportion of streptococci on copper amalgam could be explained by the toxic effect of copper ions on bacterial growth. Previous reports (e.g. Theilade & Theilade 1970) have shown that coccoid organisms dominate in early plaque formation, and since the plaque is not able to mature because of constantly dying organisms, this accounts for the high proportion of streptococci in th^'e viable counts.
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HYYPPA AND PAUNIO
The formation of dental plaque involves an initial attachment of bacteria to the acquired pellicle (Gibbons & van Houte 1973). To observe whether pellicle formation occurs on copper amalgam, specimens were placed in the mouth in a cervical cavity for 20 and 60 min. The scanning micrographs show that there is an organic film covering both the copper and silver amalgam surfaces. Thus it seems that cop-
per ions do not prevent pellicle formation even though the growth of microorganisms is inhibited. On the other hand, the structure of the pellicle formed on the copper amalgam restoration may be so changed, that it is unlikely that bacteria will attach to it. The 2-day-old plaque formed on copper amalgam appeared as bands arranged irregularly on the surface, showing that there
THE PLAQUE-INHIBITING EFFECT OF COPPER AMALGAM were large areas free of plaque accumulation. This explains why the viable counts obtained from the copper amalgam pieces were so small. The filamentous structure of the plaque with predominantly coccoid organisms does not differ from similar structures found in scanning electron micrographs of plaque grown on enamel (Newman 1972). There were no epithelial cells on the surface of the copper amalgams or in the plaque formed on them as was seen in plaque grown on enamel (Tinanoff et al. 1976). Gingival restorations which extend below the gingival margin are likely to accumulate plaque and it is concluded by Waerhaug (1975) that these restorations are involved in the etiology of destructive periodontai disease. Therefore, it is important to consider the plaque-inhibiting effect of the restorative material chosen. The present study indieates that copper amalgam is less likely to accumulate plaque than silver
237
amalgam. However, more research in this field is necessary in order to elucidate the therapeutic action of copper amalgam in restorations and its long-term effect on the gingiva. Zusammenfassung
Plaqueinhibierender Effekt von Kupferamalgam Um das Vermogen von Kupferamalgam zu studieren Plaqueansammlungen zu reduzieren, wurden bei 10 Patienten vierzehn zervikale Kupferamalgarafiillungen gelegt. Kontrollfiillungen in Silberamalgam wurden an homologen Zabnen des gleichen Kiefers angebracbt. Um die Piaquemenge zu schatzen, warden die eingefarbten Abschnitte der Test- und KontroHfiJllungen nach fotografischer Registrierung mit planimetrischer Methode analysiert. Weiterhin wurden quantitative Analysen des Plaquewaehstums auf Kupferamalgam mit mikrobiologischen Methoden vorgenommen. Hierfur wurde eine iieue Groppe von 10 Patienten ausgewahlt. Zervikale Kavitaten wurden in Zahne beider Kieferseiten prapariert. Test und Kontrollflillungen in Kupfer- bzw. Silberamal-
Eig. 3. Scanning electron micrographs from the surface of silver and copper amalgam pieces after 2 days in tooth cavities. The test subjects refrained from oral hygiene procedures during this time. A) shows a 740 X magnification and B) a 7400 X magnification of the silver amalgam restorations. A heavy amorphous organic material covers the plaque surface (A). In the bottom of the crevice microorganisms are seen arranged parallel to each other (B). C) shows a 740 X magnification and D) 7400 X magnification of the piece of copper amalgam. Note the plaque-free areas on the copper amalgam surface. The plaque is composed of filamentous organisms with intermingled cocci. (D) Scanning-elektronische Mikrographien der Oberfldche von Silber- und Kupferamalgamfiillungen nach 2-tagiger Applikation in Zahnkavitdten. Die Probanden stellten wahrend dieser Zeit alle orale Hygienemassnahmen ein. A) zeigt die SilheramalgamfUUungen in 740-facher und B) in 7400-facher Vergrosserung. Die Plaqueoberfldche wird von einer Menge amorphen organischen Materials bedeckt (A). Auf dem Boden des Spaltes sind paralell liegende Mikroorganismen sichthar (B). (C) zeigt die Kupferamalgamprobe in 740-facher und (D) in 7400'facher Vergrosserung. Bitte beachten Sie die plaquefreien Stellen auf der Oberfldche des Kupferamalgams. Die Plaque besteht aus fadenforniigen Organismen, vermischt mit Kokken (D). Cliches au microscope electroniquc a balayage: surface de specimens d'amalgame d'argent et d'amalgame de cuivre places depuis 2 jours dans les cavites. Les sujets s'etaient pendant cette periode abstenus de soins d'hygiene bucco-dentaire. A) montre un grossissement a 740 X et B) un grossissement a 7400 X des obturations d'amalgame d'argent. Une couche compacte de substance organique amorphe couvre la surface de la plaque (A). Au fond de Vanfractuosite, on volt des micro-organismes disposes en direction parallele les uns aux autres (B). C) montre un grossissement a 740 X et D) un grossissement a 7400 X du specimen d'amalgame de cuivre. La plaque est constitute d'organismes filamenteux entremeles de cocci (D).
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238
gam wurden an einander gegeniiberliegenden Seitea gelegt. Nach 2 imd 3 Tagen wurde die Plaque gesammelt und unter aeroben und anaeroben Verhaltnlssen auf Blut-Agarplatten und Mitis Salivarius Agar kultiviert. Die Resuitate beider Untersuchungsmethoden zeigen signifikante Inhibition von Piaquewachstum auf Kupferamalgam. Diese Resultate wurden durch Scanningelektronische Mikrographie bestatigt die zeigte, dass sich auf Kupferamalgam nur kleine Plaqueareale befanden. Die Plaque hatte irregulare Bandformation. Bei hoherer Vergrbsserung wurden fadenformige Stnikturen mit uberwiegenden kokkoiden Organismen gesehen. Die Scanning-elektronische Mikrographie zeigte weiterhin, dass eine PelUkelbildung, die als Voraussetzung fiir die Haftung von Bakterien angesehen wird, an beiden Araalgamen stattfand. Resume
Inhibition de la plaque par i'amalgame de cuivre Aiin d'etabiir si i'amalgame de cuivre accumule moins de plaque microbienne que l'amalgame d'argent, on a place chez 10 patients des obturations du coliet en amalgame de cuivre, et, a la meme machoire, dans les dents homologues, on a fait des obturations temoins en amalgame d'argent. Pour evaluer !a quantite de plaque, on a, apres application de colorant, fait une analyse de la surface des amalgames sur photographies par une methode planimetrique. On a de plus effectue par des methodes bacteriologiques une analyse quantitative du developpement de la plaque sur I'amalgame de cuivre. Pour cette analyse, un nouveau groupe de 10 patients a ete selectionne. Des cavites ont ete preparees au niveau du collet sur des dents des deux cotes de la machoire. Des specimens d'obturations a I'amalgame d'argent et a I'amalgame de cuivre ont ete places sur les cotes opposes. La plaque a ete recueillie au bout de 2 jours et au bout de 3 jours et cultivee dans des conditions aerobies et anaerobies sur des plaques d'agar au sang et sur milieu "Mitis Salivarius Agar", Les resultats obtenus par les deux methodes montraient une inhibition tres significative du developpement de la plaque par I'amalgame de cuivre. Ces resultats ont ete confirmes par des cliches au microscope electronique a balayage qui montraient que I'amalgame de cuivre ne presentait que de petites zones couvertes de
plaque. La plaque se presentait sous forme de bandelettes placees sur la surface de fagon irregulifire. A plus fort grossissement, on constatait une structure filamenteuse avec predominance d'organismes semblables a des cocci. Les cliches au microscope eiectronique a balayage revelaient qu'il se produisait sur les deux sortes d'amalgame une formation de peilicule, ce qui est considere comme etant necessaire pour l'adhesion microbienne a la surface. References
Bjorn, A.-L., Bjom, H. & Grkovic, B. (1969) Marginal fit of restorations and its reiation to bone level. I. Metal fillings. Odontologisk Revy 20, 311-321. Gibbons, R. J. & van Houte, J. (1973) On the formation of dental plaque. Journal of Periodontology 44, 347-359. Karlsen, K. (1970) Gingiva! reactions to dental restorations. Ada Odontologica Scandinavica 28, 895-904. Lind, V., Wennerhoim, G. & Nystrom, S. (1964) Contact caries in connection with silver amalgam, copper amalgam and silicate fillings. Acta Odontologica Scandinavica 22, 333-341. Loe, H., Theilade, E. & Jensen, S. B. (1965) Experimental gingivitis in man. Journal of Periodontology 36, 177-187. Morman, W., Regolati, B. & Renggli, H. (1974) Gingival reaction to well-fitted subgingival proximal gold inlays. Journal of Clinical Periodonlology 1, 120-125. Newman, H. N. (1972) Structure of approximal human dental plaque as observed by scanning electron microscopy. Archives of Oral Biology, 17, 1445-1453. Rekola, M. & Scheinin, A. (1977) Quantification of dental plaque through planimetric analysis. Scandinavian Journal of Dental Research 85, 51-55. Shay, D. E., Allen, T. J. & Mantz, R. F. (1956) The antibacterial effects of some dental restorative materials. Journal of Dental Research 35, 25-32. Theilade, E. & Theilade, J. (1970) Bacteriological and ultrastnictura! studies of developing dental plaque. In Dental Plaque, ed. McHugh, W. D., pp. 27-40. Edinburgh: E. & S. Livingstone Ltd. Tinanoff, N., Gross, A. & Brady, J. M. (1976) Development of plaque on enamel. Parallel investigations. Journal of Periodontal Research 11, 197-209.
THE PLAQUE-INHIBITING EFFECT OF COPPER AMALGAM Tiirkheim, H. I. (1953) Bacteriological investigations on dental filling materials. BrUish Dental Journal 95, 1-7. Waerhaug, J. (1956) Effect of rough surfaces upon gingival tissue. Journal of Dental Research 35, 323-325. Waerhaug, J. (1975) Presence or ahsence of plaque on suhgingival restorations. Scandinavian Journal of Dental Research, 83, 193201.
Address: Tuula Hyyppd Department of Periodontology Institute of Dentistry University of Turku Lemminkdisenkatu 2 SF~20520 Turku 52 Finland
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