Journal of Clinical Periodontology: 1979; 6: 115-130 Key words: Ptaque composition ~ gingivitis - hydrogen peroxide Accepted for ptiblicaJion: June 20, 1978.
momhwasii.
Effect of hydrogen peroxide on developing piaque and gingivitis in man JAN WENNSTROM AND JAN LINDHE
Department of Periodontology, Faculty of Odontology, University of Gothenburg, Gothenburg, Sweden Abstract. The present experiment was undertaken to assess the effect of hydrogen peroxide release during mouth rinsings on the composition of the microbiota of developing plaque in humans and the amount and pathogenecity of the piaque formed. The trial was designed as a double-blind crossover study of the effect of a mouthwash (Amosan®) and a placebo rinse on the development of plaque and gingivitis in young adults. The active compound was available as a powder; the rinse consisted of 1.7 g powder dissolved in 30 ml hot tap water. Fourteen dental students participated in the trial. The students were examined during two consecutive periods, each consisting of one preparatory (during which active tooth cleaning measures were carefully practiced) and one main test period (during which mouth rinsings were the only plaque control measure). Each of the two test periods was initiated by a baseline examination following which the participants rinsed either with the active or the placebo mouthwash. The rinsings were performed immediately after breakfast, after lunch and after dinner. Measurements of Piaque and Gingival Index scores were perfornied 4, 7 and 14 days after the start of the no-toothbrushing period. Bacteria were sampled and examined after 7 and 14 days of trial. The results demonstrated that a mouthwash which released hydrogen peroxide effectively prevented the colonization of filametits, fusiforms, motile and curved rods as well as spirochetes hi developing plaque. The mouthwash which was used as the only oral hygiene measure during a 2-week period furthermore markedly reduced the amount of plaque formed and significantly retarded gingivitis development. It is suggested that HgO, released by mouthwashes during rinsing may prevent or retard the colonization and multiplication of anaerobic bacteria.
Findings reported by Loe et al. (1965) and Theilade et al. (1966) from experitnents in young adults have demonstrated that the abolishment of mechanical tooth cleaning measures results in a rapid accumulation of plaque and in generalized gingivitis after 9-21 days. During the no-toothbrushing period there was not only an increase in the numher of microorganisms in the plaque, but in addition a change in the composition
of the plaque flora. Thus, at the start of the experiments, i.e. when the gingivae were normal, the plaque flora was predominated by Gram-positive cocci and rods. Later the microflora changed into a complex population in which filaments, fusobacteria and eventually vibrios and spirochetes made up approximately 30 % of all cells present. The authors concluded that gingivitis is associated with an increase in number of bacteria
0303-6979/79/020115-16$02.50/0© 1979 Munksgaard, Copenhagen
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WENNSTROM AND LINDHE
and a change in the flora towards a larger representation of Gram-negative organisms. Other methods have also been used to study the mieroflora associated with periodontal health and disease, such as "direct microscopic examination", "cultivation in nonselective media", "electron microscopic assessments of plaque in situ" and assessments of the "predominant cultivable organisms" from pocket samples (for review see Socransky 1977). Slots (1977) and Slots et al. (1978) studied the predominant cultivable microbiola of healthy sulci and inflamed gingival pockets in man. They observed that a Gram-positive flora predominated O> 75 % of ail isolates) the microbiota of healthy sulci, whereas close to 50 % of the microorganisms in gingivitis pockets were obligate anaerobes. In addition, Gram-negative anaerobic rods made up approximately 25 % oi the isolates. Listgarten & Hellden (1978) examined the subgingival microfiora of healthy and diseased sites in humans by darkfield microscopy. They reported that coccoid cells were predominant (75 %) in healthy sites while at diseased sites fusiforms and motile bacteria including spirochetes made up approximately 60 % of the bacterial cells. In attempts to further correlate the colonization of certain types of plaque bacteria with the initiation of gingival inflammation, trials have been performed in which the bacterial composition of the bacterial composition of the developing plaque was altered via the use of antibiotics administered either via the systemic route or applied topically in the oral cavity (for review see Loesche 1976). Also in this context, the gingivitis model by Loe et al. (1965) has frequently been used to evaluate antimicrobial agents for their ability to prevent disease development. For instance, Lbe et al. (1967) and Jensen et al. (1968) showed that topical application of antibiotics such as Tetracycline, Vaneomycin and Polymyxin B re-
sulted in a pronounced change of the plaque flora. As a consequence of this change, varying degrees of inhibition of subclinical and clinical signs of gingivitis were noted. In a similar manner different substances such as antiseptics, enzymes, tensioactive agents, etc., have been employed in experiments to prevent or reduce plaque formation and gingivitis development (for review see, e.g. Gjermo 1974, Loesche 1976). During recent years data has accumulated demonstrating the importance of the obligate anaerobic microorganisms in plaques associated with gingivitis and periodontitis (for review see Socramky 1977). Since the obligate anaerobes are sensitive towards oxygen, microbiologists have been prompted to develop elaborate techniques to ascertain proper living conditions for the anaerobic plaque microorganisms during sampling and cultivation. This knowledge has been utilized to some extent in the formulation of certain types of mouthwashes which operate by the release of hydrogen peroxide or oxygen (Cobe 1960, Stripman et al. 1971, Ericsson & Lundbeck 1955). The aim of the present experiment was to study the effect of a hydrogen peroxide releasing mouthwash on (1) the composition of the microbiota of the developing plaque in humans and (2) the amount and pathogenecity of the plaque formed. Material and Methods The trial was designed as a double-blind crossover study of the effect of a mouthwash (Amosan®) releasing hydrogen peroxide and a placebo drug on the development of plaque and gingivitis in young adults. The active compound (Amosan, Cooper Lab. Inc., Cedar Knolls, New Jersey, USA) was available as a powder and stored in disposable airtight envelopes. Each envelope contained 1.7 g of the powder which immediately prior to use was dissolved in 30 ml
EFFECT OE H^O^ ON PLAQUE AND GINGIVITIS
117
Baseline enamination
Initial examination Preparatory
Main te5t phase
Group A "Activp compound"
Group A "Placebo" Sroup B "Active compound" L..
I- Group E "°lacebo" Day
Day
0
4
7
1:1
ClI
Bacterial sample
Preparatory pnase: 2-3 «ee(;5
Fig. 1. Outline of the experiment. Fourteen dental students participated in the trial. During a preparatory period of 2-3 weeks prior to the test periods, careful mechanical toothcleaning was practiced. Clinical examinations were performed on days 0, 4, 7 and 14 during the main test periods. Bacterial plaque was sampled on days 0, 7 and 14. Die Organisation des Versuches: Vierzehn Studenten der Zahnmedizin nahinen an dem Test teil. Wahrend eines vorbereitenden Versuchsabschnittes von 2~3 Wochen wurde eine sorgfdltige mechanische Zahnreinigung durchgefiihrt. Die klinischen Untersuchungen wurden an den Tagen 0, 4, 7 und 14 wahrend des eigentlichen Versuchsabschnittes vorgenommen. Abstriche bakterieller Plaque wurden an den Tagen 0, 7 und 14 entnommen. Plan de l'experience. Quatorze etudiants en dentisterie participaient a cet essai. Pendant une phase preparatoire de 2-3 semaines pre,cedant chacune des phases experimentales (test phase), le nettoyage mecaniqtie des dents etait pratique d'une fa(;on meticuleuse. Les examens clinigues ont ete effeetues aux Jours 0, 4, 7 et 14 pendant les phases experimentales principales. Des prelevements de plaque microbienne (bacterial sample) ont ete faits aux Jours 0, 7 et 14.
hot tap water. The composition of the preparation was 68.6 % sodiumi perborate, 29.4 % sodium bitartrate and 1.9 % flavoring substances such as menthol and peppermint. The active ingredient is, according to the manufacturer, hydrogen peroxide. Fourteen dental students volunteered to participate in the dinieal investigation. They had at least 24 teeth and showed no sign of active periodontai tissue breakdown. All the students were examined during two consecutive periods (Fig. 1), each consisting of one preparatory period (during which active, mechanical, oral hygiene measures were carefully practiced) and one main test period (during which no mechanical tooth eleaning measures were performed).
At the start of the two preparatory periods the teeth of all the participants were carefully scaled and polished. Detailed instruction in proper oral hygiene measures was given to eaeh individual. At the end of each of the two preparatory periods, the oral hygiene status and the gingival conditions of the perticipants were examined. The oral hygiene status was assessed using the Plaque Index system (Silness & Loe 1964). The amount of plaque was determined on all surfaces of all teeth in the dentition except 18, 28, 38 and 48. The frequency distribution of seores 0, 1, 2 and 3 was calculated for each individual. The gingival conditions were examined using the Gingival Index system (Loe & Sil-
118
WENNSTROM AND LINDHE
ness 1963). The frequency distribution of scores 0, 1, 2 and 3 was calculated for each individual. Following the clinical examination, supragingival plaque was collected from the buccal surface of tooth 14. The composition of the plaque sample was analyzed by darkfield microscopy according to a method recently described by Listgarten & Heliden (1978). Thus, the frequency distribution of coccoid cells, straight rods, fusitorms, filaments, motile and curved rods and spirochetes was calculated. Following the baseline examination, the participants were asked to abstain from all mechanical tooth cleaning measures during a 2-week period. They were instructed to rinse their mouth, three times a day (2 min-
utes each time) with 30 ml of the active mouthwash (1.7 gm Amosan dissolved in 30 ml warm-hot tap water) or a placebo mouthwash. The rinsings were performed immediately after breakfast, after lunch and after dinner. AU participants were given careful instruction in the use of the mouthrinse. During this first test period subjects 3, 5, 6, 7, 10, 13 and 14 used the active compound and subjects 1, 2, 4, 8, 9, 11 and 12 used the placebo control. Measurements of Plaque and Gingival Index scores were performed 4, 7 and 14 days after the start of the no-toothbrushing period. Bacterial samples were collected from the buccal surfaces of tooth 14 on days 0 and 7 and from tooth 24 on day 14. At the end of the first main test period,
COMPOSITION OF PLAOUE SAMPLES
•
BASELINE-NORMAL
^
TEST PERIOD
Q
CONTROL PERIOP
^
M O COCCOID CELLS STRAIGHT ROOS
7
14
FUSIFORMS
0 MOTILE AND
7
14
SPIROCHETES
RODS
Fig. 2. Diagram showing the composition of the developing plaque on the buccal tooth surfaces examined during the test and control periods. Mean (x) percentage and standard error (S.E.). Das Diagramm veranschaulicht die Zusammensetzung der Plaque an den bukkalen Flachen der wdhrend der Test- und Kontroltperioden untersuchten Zahne. Mittlere Prozentwerte (x) und Standardirrtum (SE). Diagramme motitrant la composition de la plaque se formant sur les faces vestibulaires examinees pendant les periodes experimentales (test) et temoins (eontrol), Pourcentage moyen (x) et erreur-type (S.E.).
EFFECT OF
ON PLAQUE AND GINGIVITIS
119
Table 1. The frequency distribution of different bacteria of developing plaque during the test (Amosan) and control (Placebo) periods. Mean (x) and standard error (S.E.) Die Frequenzverteilung verschiedener Bakterien in der sich entwiekelnden Plaque wahrend der Test- (Amosan) und Kontrollperioden (Plazebo). Mittelwerte (x) und Standardirrtum (SE) Distribution de frequence des differentes bacteries de la plaque se formant pendant les periodes experimentales avec Ainosati (test) et temoins avec Placebo (control). Moyenne (x) et erreurtype (S.E.) Coccoid cells -!Straight rods
Filaments
0 7 14
84,2 ± 3.9 51.7 ± 4.8a 40.4 ± 6.0a
7.0 ± 1.8 20.3 ± 2.1a 17.7 ± I,8b
4.3 ± 2.0 16.3 ± 2.6b
Test
0 7 14
88.2 ± 4.6 88.0 ± 3.5 84.9 ± 4.7
4.7 ± 1.8 4.6 ± 1.4 6.9 ± 2.0
Test vs Control
0 7 14
NS P < 0.001 P < 0.001
NS F < 0.001 P < 0.001
Period
Control
Days
Motile Fusiforms
+
Spirochetes
Curved rods
14.9 ± 1.815
4.5 + 1.3 11.6 ± 2.9f 22.1 ± 4.0b
0,1 ± 0.1 0.1 ± 0.1 4.9 ± 1.80
4.0 ± 1 . 3 5.6 ± 1.9 5.4 ± 1.8
3.0 ± 1.2 1.4 ± 0.5 2.6 ± 1.8
0,1 ± 0.1 0.4 ± 0.6 0.1 ± 0.1
NS P < 0.01 P < 0.001
NS NS P < 0.05
NS P < 0.01 P < 0.01
" Significantly different from day 0: P < 0.001 il Significantly different from day 0: P < 0.01 c Significantly different from day 0; P < 0.05 Coccoid celis -I- Straight rods (Kokkoide Zellen + gerade Stdbchen, cellules coccoides + batonnets droits), filaments (Filarien, filametits), fusiforms (Fusobakterien, fusiformes). Motile -j- curved rods (Bewegliche + gekrUmmte Stabchen, bdtontiets tnobiles -\- iticurves), spirochetes (Spirochaten, spirochetes), a, -|- b 4- c Significantly different from day 0: (a -f b -]- c Signifikant unterschiedlieh vom Tage 0.', a -j- b -|- i; difference significative par rapport au jour 0:).
the teeth of the participants were again carefully scaled and polished. They were instructed to cleati their teeth regularly and in a meticulous manner during the subsequent 2—3 weeks. Professional tooth cleaning (Lindhe & Axelsson 1973) was performed twice during this second preparatory period. Towards the end ot" the period a new baseline examination was performed following whicli the second main test period was initiated (Fig. I). During this period subjects 1, 2, 4, 8, 9, 11, 12 used the active compound and subjects 3, 5, 6, 7, 10, 13, 14 the placebo mouthrinse. The clinical and bacteriological examinations were repeated after 4, 7 and 14 days of no mechanical tooth cleaning. Differences regarding PU and GI scores as well as frequency distri-
bution of bacteria between test and control periods were analyzed using Student's t-test. Differences between days were assessed by analysis of variance. Results
Microbiology At the start of the two main test periods, i.e. when the Plaque and Gingival Index scores were zero or close to zero for all buccal, lingual and approximal tooth surfaces and gingival units, coccoid cells and straight rods accounted for almost 90 % of the bacterial population in the plaque samples (Fig. 2). The remaining bacteria were identified as filaments («* 6 %; Table 1 and Fig. 2), fusiforms ( ^ 4 %) and motile rods
120
WENNSTROM AND LINDHE
( ~ 4 % ) . Spirochetes were absent in most of the samples and accounted on the average for only 0.1 % of the total flora. During the "placebo period" (control period) of no mechanical tooth cleaning there was a marked change in the hacterial composition of the developing plaque on the buccal tooth surfaces examined. Hence, after 7 and 14 days of experiment the percentage of coccoid cells and straight rods had decreased (52 % on day 7 and 40 % on day 14; Fig. 2) and the frequency distribution of filaments, fusiforms, motile and curved rods increased (Table 1). On day 7 spirochetes were observed in samples from two individuals and on day 14 plaque samples collected from eight individuals harbored a significant number of spirochetes {x = AB %\ Fig. 2). Consequently, during the control period the microbiota of the supragingival plaque shifted from a predominantly coccoid cell + straight rod flora to a complex flora containing large numbers of filaments -1- fusiforms O 30 %),
curved -f motile rods ( > 20 %) and spirochetes («^5 %). During the test period, when the participants regularly rinsed with the hydrogen peroxide mouthwash, the supragingival plaque maintained its characteristics of day 0, i.e. the flora on days 7 and 14 of notoothbrushing was dominated by coccoid cells and straight rods ( > 80 % of ail bacteria counted; Fig. 2). Furthermore, during the test period the frequency distribution of filaments, fusiforms, motile + curved rods and spirochetes did not change (Table 1, Fig. 2). Clinical Parameters At the start of the two no-toothbrushing periods practically all tooth surfaces were without clinically detectable plaque, i.e. the frequency distribution of Plaque Index score 0 was close to 100 % (Table 2, Fig. 3). The examination of the gingival conditions revealed that most gingival units were healthy and that the percentage of units
Table 2. Frequency distribution of Plaque Index scores 0, 1, 2 and 3 during the test and control periods. Mean (x) and standard error (S.E.) Die Frequenzverteilung der Plaqueindex-Bewertungseinheiten (scores) 0, 1, 2 und 3 wahrend der Test- und Kontrollperioden. Mittelwerte (x) utid Standardirrtum (SE) Distribution de frequence des scores 0, 1,2 et 3 pour l'indice de Plaque pendant les periodes experimentaies et temoins. Moyenne (x) et erreur-type (S.E.) Period
Days
Control
0 4 7 14
98.7 41.4 28.0 17.2
± ± ± ±
0.5 4.0 4.6 2.9
1.3 51.0 45.4 37.0
± ± ± ±
0.5 3.0 4.5 4.8
Test
0 4 7 14
98.9 73.2 64.6 46.4
± ± ± ±
0.5 4.9 5.5 4.3
1.0 24.4 31.9 45.2
± ± ± ±
0.4 3.6 4.2 3.6
Test vs Control
0 4 7 14
NS P < 0.001 P < 0.001 P < 0.001
0
1
NS P < 0.001 NS NS
2
3
0 8.4 ± 2.8 26,6 ± 6.3 44.4 ± 6.2
0 0 0 1.4 ± 0.9
0.1 3.1 3.4 8,7
± ± ± ±
0.1 2.0 1.9 3.6
NS NS P < 0.001 P < 0.001
0 0 0 0
M' NS NS
EFFECT OF
ON PLAQUE AND GINGIVITIS
121
PLAQUE INDEX SCORE 0
^
I
TEST PERIOD
I CONTROL PEfllOD
.
SCORE 2
DAY 0
DA* 4
DAV 7
DAY 4
DftY 7
I
i
Fig. 3. The mean ("xj percentage of tooth surfaces with Plaque Index scores 0 and 2 during the test and control periods. The top diagram presents the data for all surfaces combined. The bottom diagram presents the data for buceal, lingual and approximal surfaces separately. Das Vorkotnmen (tnittlere Prozentwerte, x) der Bewertungseinheiten 0 und 2 des Plaqueindexes wahrend der Test- und Kontrollperioden. Das obere Diagramm enthalt die kombinierten Werte aller ZahnoberflUchen. Das untere Diagramm enthdlt die separaten Werte der bukkalen, lingualen und approxitnalen Zahtioberflachen. Pourcentage moyeti (x) des faces dentaires ayant des scores 0 et 2 pour I'indice de Plaque pendant les periodes experimentales et temoins. Le diagramme du hatit represente les donttees pour I'ensemble des faces. Le diagramtne du bas represente separetnent les donnees eoncernant les faces vestibulaires (buccal), linguales et proximales.
WENNSTROM AND LINDHE
GINGIVAL INDEX SCORE O
Ir
P^ j
TEST PERIOD [ CONTROL PERIOD
SCORE 2
APPROXIUAL
^
DA* 0
DAY a
I r OaV 7
DBI 14
DAY D
DAY 4
DA* 7
DA* 14
DAY 0
DAY i
DAY 7
DAY 14
F/^. •#. The mean (x) percentage of gingival units with GJ scores 0 and 2 during the test and control periods. The top diagram presents the data for all gingival units combined. The bottom diagram presents the data for buccal, lingual and approximal units separately. Das Vorkommen (mittlere Prozentwerte, x) gingivaler Abschnitte tnit den Bewertungseinheiten 0 und 2 des Gingivalindexes wdhrend der Test- und Kontrollperioden. Das obere Diagramm enthdlt die kombinlerten Werte aller Gingivalabschnitte. Das untere Diagramm enthalt die separaten Werte der bukkalen, lingualen und approximalen Abschnitte. Poureentage moyen (x) des unites gingivales presentant les scores 0 et 2 pour I'indice Gingival pendant les periodes experimentales et temoins. En haut, donnees pour I'ensemble des faces; en bas, donnees pour les faces vestibulaires, linguales et proximales, separement.
EFFECT OF
ON PLAQUE AND GINGIVITIS
Table 3. Frequency distribution of Gingival Index scores 0, 1, 2 and 3 during the test and control periods. Mean (x) and standard error (S.E.) Die Frequenzverteilung der Gingivalindex-Bewertungseinheiten (scores) 0, I, 2 und 3 wdhrend der Test- und Kontrollperioden. Mittelwerte (x) und Standardirrtum (SE) Distribution de frequence des scores 0, I, 2 et 3 pour l'indice Gingival pendant les periodes experimentales et temoins. Moyenne (x) et erreur-type (S.E.) Period
Days
Control
0 4 7 14
74.0 57.4 31.4 13.0
±2.3 ± 2.0 ± 3.0 ± 2.3
25.4 37.4 55.8 54.9
± ± ± ±
2.2 2.4 2.5 3.4
n.9 ± 2.5
0 0 0
31.6 ± 4.6
0
Test
0 4 7 14
72.1 66.9 52.4 27.4
±1.8 ± 1.6 ± 2.7 ± 7.3
26.4 29.3 42.2 53.3
± ± ± ±
1.7 1.8 2.4 1.4
1.5 3.2 5.4 19.4
0 0 0 0
Test vs Control
0 4 7 14
NS P < 0.001 P < O.OOJ P < 0.001
0
1
2
NS P < 0.05 P < 0.01 NS
receiving a GI score of 2 was very low (start of control period: 0.6 %; start of test period: 1.5 %; Table 3, Fig. 4). During the control period the number of plaque-free tooth surfaces rapidly deereased (Fig. 3). Thus, already after 4 days of no toothbrushing only 40 % of the surfaces were clean and after 7 and 14 days the corresponding figures were 28 and 17 %. During the 2 weeks of observation there was a corresponding increase of Plaque Index seores 1 and 2 (Tables 2 and 4, Fig. 3). At the reexaminations on days 4, 7 and 14, the frequeney of Piaque Index score 2 was 8.4, 26.6 and 44.4 %, respeetively. The most marked inerease of the plaque scores occurred on the buccal and approximal tooth surfaees (Fig. 3, Table 4). Also during the test period the frequency of Plaque Index seore 0 decreased. This decrease was, however, moderate in eomparison to the ehange observed during the control period (Fig. 3). Thus, after 4, 7 and 14 days of no toothbrushing but rinsing with Amosan, the frequency of Pll = 0 was 73.2 % (control = 41.4 %), 64.6 % (control
3
0.6 ± 0.3 3.9 ± 0.8
± ± ± ±
0.6 1.0 0.8 2.4
NS NS P < 0.01 F < 0.01
NS NS NS NS
^ 2 8 % ) and 46.4% (eontrol = 17.2 %). The differenee wilh regard to seore PH = 0 between the test and eontrol periods was at eaeh reexamination highly significant (P < 0.003; Table 2). Only towards the end of the test period did the frequency of Plaque Index score 2 differ significantly from the day 0 value. Also with regard to the PII seore 2 eould a highly signifieant differenee be calculated between the test and eontrol regimen (days 7 and 14; P < 0.001, Table 2). Fig. 3 and Table 4 illustrate the Plaque Index alterations on different tooth surfaees during the two no-toothbrushing periods. It is obvious that the aetive mouthwash was more effective on buccal and lingual than on appriximal tooth surfaces. However, also on the approximal surfaces the differenee regarding PU seores 0 and 2 between the test and eontrol period was marked (Table 4). During both periods of no toothbrushing the gingival conditions gradually deteriorated (Table 3, Fig. 4). This is illustrated by the increase in the frequency of Gingival
124
WENNSTROM AND LINDHE
Table 4, Frequency distribution of Plaque Index scores 0, 1, 2 and 3 for the buccal, lingual and approximal tooth surfaces during the test (Amosan) and control (Plaeebo) periods. Mean (x) and standard error (S.E.) Die Frequenzverteilung der Plaqueindex-Bewertungseinheiten (scores) 0, 1, 2 und 3 an den bukkalen, lingualen und approximalen Zahnoberfldchen wahrend der Test- (Amosan) und Kontrollperioden (Plazebo). Mittelwerte (x) und Standardirrtum (SE) Distribution de frequence des scores 0, 1, 2 et 3 pour I'indice de Plaque au niveau des faces vestibulaires, linguales et proximales pendant les periodes experimetitales avec Amosan (test) et temoins avec Placebo (control). Moyenne (x) et erreur-type (S.E.) 0
Buccal surfaces „ „ Amosan ^^^^ Placebo
99.7 ± 0.3 100.0 ± 0
1
0.3 ± 0.3 0± 0
••
2
3
z
—
Day 4
A P
80,1 + 6.2*** 44.9 ± 6.4
16.1 + 4.3*** 47.1 ± 5.1
4.1 ± 2.5 7.6 ± 2.7
Day 7
A P
68.7 + 5.7*** 23,3 ± 7.7
27.3 ± 5.9** 49.6 + 4.6
4.7 + 2.4** 27.4 ± 6.1
—
Day 14
A P
48.1 + 6.7*** 8.8 ± 4.0
41.1 ± 4.8 39.5 + 6.6
10.8 + 3,9** 50.4 ± 8,5
0 ±0 1.4 ± 1.1
1.1 ± 0.5 0.6 ± 0.3
—
—
—
Ling It al surfaces DayO
^
98.9 + 0,5 99.4 ± 0.4
Day 4
A P
86.9 ± 6.3 80.6 + 3.6
6,9 ± 1.6*** 18.0 ± 2.8
0.6 ± 0,4 1.4 ± 1.1
—
Day 7
A P
91.9 + 2.0*** 65.9 ± 4.5
6.7 ± 1.6*** 29.4 ± 3.9
.1.4 + 1.1* 4.8 ± 1.8
—
Day 14
A P
83,1 50.9 ± 5.2
13.4 + 2.4*** 39.6 ± 4.5
2.4 ± 1,3 9.7 ± 3,4
Approximal sttrfaces DayO
p
98.6 + 0.9 97.7 ± 1.0
Day 4
A P
59.4 ± 6.7*** 19.1 ± 4.8
36.9 ± 5.2*** 69.4 ± 4.1
3,8 ± 2.5** 11.1 ± 3.6
—
Day 7
A P
48.9 + 6.9*** 11.2 ± 4.5
46.8 ± 5.5 51.9 ± 7.0
4.4 + 2.4*** 36.9 ± 8,8
—
Day 14
A P
26.7 + 5.4*** 4.9 + 2.7
62,4 + 4.4* 34,8 ± 6.1
10.9 + 4 9*** 58.4 ± 7.3
0± 0 1.9 ± 1.4
1.3 ± 0.8 2.3 ± 1.0
—
* Significantly different from Placebo: P < 0.05 ** Significantly different from Placebo: P < 0.01 *** Significantiy different from Placebo: P < 0.001 Buccal surface (Bukkale Oberflache, faces vestibttlaires), lingual surfaces (Linguale Oberflachen, faces linguales), approximal surfaces (Approximale Oberflachen, faces proximales).
EFFECT OF H^O^ ON PLAQUE AND GINGIVITIS
125
Table 5. Frequency distribution of Gingival Index scores 0, 1 and 2 for the buccai, lingual and approxitnal tooth surfaces during the test (Amosan) and control (Placebo) periods. Mean (x) and standard error (S.E.) Die Frequenzverteilung der Gingivalindex-Bewertungseinheiten (scores) 0, 1, 2 und 3 an den bukkalen, lingualen utid approximalen Gingivaabschnitten wdhrend der Test- (Amosan) und Kontrollperioden (Plazebo). Mittelwerte (x) und Standardirrtum (SE) Distribtition de frequence des scores 0, 1 et 2 potir I'Indice Gingival au niveati des faces vestibulaires, linguales et proximales pendant les periodes experimentales avec Amosan (test) et temoins avec Placebo (control). Moyenne (x) et erreur-type (S.E.) 0 Buccai surfaces Amosan °^^" Placebo
92.1 ± 1.7 93.7 ± 1.2
Day 4
2
6.5 ± 1.4 5.6 ± 1.1
1.3 ± 0.9 0.8 ± 0.6
91.6 ± 1.9**''^ 79.6 ± 2.5
5.8 ± 1.4** 15.7 ± 2.2
3.1 ± 1.3 4.1 ± 1.1
82.1 ± 2.5*** 54.1 ± 4.7
15.2 ± 2.6*** 38.0 ± 4.3
2.7 ± 0.7* 7.9 ± 1.8
45.4 ± 4.]** 21.3 ± 4.7
41.4 ± 2.9* 57.8 ± 4.1
p
69.1 ± 3.2 74.9 ± 2.6
29.1 ± 2.8 24.8 ± 2.8
0.9 X 0.4
A
67.3 ± 3 . 7 62.6 + 2.5
28.9 ± 3.6 32.9 ± 2.1
3.9 ± 1.6 4.5 ± 1 . 2
A
38.2 + 2.9** 47.4 ± 4.4
36.8 ± 2.5 39.7 ± 2.8
5.0 ± 0.9** 12.9 ± 2.4
A
44.4 ± 3.7** 28.5 ±3.0
36.6 ± 3.4 43.9 ± 3.4
18.6 ± 3.2* 27.4 ± 3.7
63.8 ± 2.9 65.0 ± 2.8
34.4 ± 3.1 34.3 ± 2.7
1.8 ± 0.7 0.7 ± 0.3
A
54.6 ± 2.5** 41.1 ± 3.4
41.3 ± 2.7*** 55.7 ± 3.2
4.1 ± 1.2 3.2 ± 0.8
A
35.4 ± 3.9*** 12.7 ± 2.8
57.4 ± 3.7** 72.4 ± 3 . 3
7.2 ± 1.6* 14.9 ± 3.6
A P
9.6 ± 1.1*** 2.6 ± 1.4
67.4 ± 3.0 58.5 ± 5.3
22.3 ± 2.9* 38.9 ± 5.9
p
Day 7 Day 14
1
A
13.2 ± 2.8* 20.9 ± 4.3
Lingual surf aces
Day 0 Day 4 Day 7 Day 14
1.4 ± 0.6
Approximal surfaces DayO Day 4 Day 7 Day 14
p
Significantly different from Placebo: P < 0.05 Significantly different from Placebo: F < 0.01 Significantly different from Placebo: P < 0.001
126
WENNSTROM AND LINDHE
Index scores > 0. The degree of deterioration, however, was more marked during the control than during the test period (Tables 3 and 5). Thus during the 2 weeks when the hydrogen peroxide mouthwash was used as the only oral hygiene measure, the percentage gingival units scored GI = 2 (Fig. 4) increased from 1.5 % (day 0) to 3.2 % (day 4), 5.4 % (day 7) and 19.4 % (day 14). The corresponding figures for the control period, when the participants used a placebo mouthrinse, were 0.6 % (day 0), 3.9 % (day 4), 12.9 % (day 7) and 31.6 % (day 14). At the reexamination on days 7 and 14 the difference between the test and control periods regarding the percentage of GI score 2 was found to be statistically significant (P < 00.1). The difference was significant for buccal, lingual as well as for interproximal units.
Discussion The present experiment clearly demonstrated that a mouthwash which releases hydrogen peroxide effectively prevents the colonization of filaments, fusobacteria, motile and curved rods and spirochetes in developing plaque. The mouthwash which was used as the only oral hygiene measure during a 2-week period (three times daily), in addition, markedly reduced the amount of plaque formed and significantly retarded gingivitis development. Since it is well known that most obligate anaerobes are sensitive to exposure of hydrogen peroxide, there are reasons to believe that H^Og released by the wouthwash during rinsing prevented or retarded the colonization and multiplication of preferably anaerobic bacteria. The trial reported was designed as a double-blind crossover study of the effect of Amosan and a placebo mouthwash on the development of plaque and gingivitis in young adults. This means that intraindivid-
ual comparisons were made from test and control periods. Attempts were also made not only to assess the effect of Amosan on gross plaque formation (Plaque Index scorings) but also to analyze the frequency distribution of various microorganisms occurring in the plaques formed. This analysis was made by darkfield microscopy, a technique which earlier was proved to be of value in distinguishing between plaques associated with healthy gingivae and diseased sites in humans (Listgarten & Hellden 1978). Darkfield microscopy has also been used to investigate the effect of different modes of periodontal treatment in humans (Listgarten et al. 1978, Lindhe et al. 1979) and dogs (Listgarten et al. 1979, Heijl & Lindhe 1979). Plaques sampled at the two baseline examinations, i.e. when the participants had clinically healthy gingivae, were dominated by coccoid cells and straight rods with filaments, fusiforms, motile and curved rods being present in low numbers only. This finding agrees with results by Listgarten & Hellden (1978) who reported that plaque samples from the gingiva! sulcus region of "relatively healthy" gingival sites were characterized by their preponderance of coccoid cells and straight rods. The findings also corroborate data by, e.g. Loe et aL (1965), Theilade et al. (1966), Jensen et al. (1968) showing that Gram-positive cocci and small rods characterized the plaque sampled from individuals with healthy gingivae. After 7 days of no toothbrushing the frequency distribution of filaments and fusiforms had undergone a threefold increase (from 31.3 to 36.6%; Table 1). A similar change in the composition of piaque was observed by Loesche & Syed (1975) and Syed et al. (1975) who reported that the developing microbial plaque was characterized mainly by the influx of Actinomyces species. Between days 7 and 14 in the present experiment, the percentage of coccoid
EFFECT OF H^O^ ON PLAQUE AND GINGIVITIS
127
Table 6. The composition of tbe bacterial plaque in relation to the gingivai status at the sampling sites. Mean percentage (x) and standard error (S.E.) Die Zusatnmensetzung der bakteriellen Plaque in Bezug auf den Gesundheitszustand der Gingiva der Regionen, an denen die Abstriche entnommen wurde Composition de la plaque microbienne et etat de la gencive au niveau des preievements. Pourcentage moyen (x) et erreur-type (S.E.) Coccoid cells Gingival Index
Ovsl
Motile
+
+
Straight rods
Filaments
Fusiforms
Curved rods
Spirochetes
86.5 ± 2.5 60.2 ± 4,4 55.2 ± 9.1
6.3 ± 1.2 15.0 ± 1.5 12.4 ± 2.4
4,3 ± 0.9 13.0 ± 1.7 12.0 ± 2.8
2.6 ± 0.7 11.1 ± 2,4 16.1 ± 4.0
0.3 ± 0.2 0,7 ± 0.4 4,2 ± 2.1
P < 0.001 P < 0,001 NS
P < 0.001 P < 0.05
P < 0.001 P < 0.001 NS
P < 0,001 P < O.OOI NS
P < 0.01 P < 0.05
NS
cells and straight rods further decreased while the number of motile -F curved rods increased and spirochetes became more frequently represetited in the samples. Concomitant with this change of the microbiota, the amount of plaque markedly increased and gingivitis became established at the plaque sampling sites. The frequency distribution of various bacteria in the plaque samples has, in Table 6, been compared to the Gingival Index scores of the plaque areas, i.e. the buccal surface of teeth 14 or 24. It is obvious that, irrespective of experimental time, the relative number of coccoid cells and straight rods decreases and the number of filaments, fusiforms, motile and curved rods increases with increasing GI scores. It is also interesting to note that plaques sampled from areas with a GI score of 2, in comparison to GI score 1, have a significantly larger relative content of spirochetes, whereas the difference regarding motile + cui-ved rods, filaments and fusobacteria is insignificant. This observation is in accordance with findings from studies in humans and dogs by Listgarten & Hellden (1978) and Listgarten et al. (1979) who reported on a decreasing percentage of coc-
NS
coid cells and straight rods with increasing GI scores and an increasing tendency of motile rods and spirochetes. It should be observed, however, that whereas Listgarten & coworkers studied the suicus flora, the present data are derived from examinations of supragingivally located plaques. In most respects the observed shift of the microbiota during plaque and gingivitis development agrees with Theilade et ai. (1966) who stated that approximately at the same time as the complex microflora was established the first chnically detectable signs of localized gingivitis could be recognized. During the 2-week period when the participants in the present trial rinsed with the hydrogen peroxide releasing mouthwash, the microbiota of the plaque samples from the buccal surfaces of 14 and 24 maintained its characteristics of day 0, i.e. the gradual influx of fusobacteria, motile + curved rods and spirochetes was obviously prevented. During the same period, plaque formation (PU score 2) on the buccal surfaces (Table 4) was markedly reduced (10.8 % versus 50.4 % of the control period) and gingivitis development obviously retarded. If it is assumed that a regular use of a HgO^-releasing
WENNSTROM AND LINDHE
tnouthwash interferes with the anaerobic bacteria, the findings reported here demotistrate the importance of the anaerobes for plaque growth and gingivitis development and therefore confirm observations by Listgarten et al (1978, 1979) and Heijl & Lindhe (1979) showing that administration, via the systemic route, of antimicrobial compounds known to eliminate anaerobic microorganisms decreases signs of plaque formation and gingivitis. The observations made in this study imply that this change of the microhiota can be accomplished by HgOg release from a mouthwash. Whether this method can be used for extended periods to control dental plaque and prevent or cure gingivitis must await farther research. The finding that Amosan effectively influences the composition of a microbial plaque agrees to large extent with data reported hy Wade et al. (1961, 1963), BundgaardJorgensen & Clausen (1962) and Wade & Mirza (1964). They studied the effectiveness of different drugs in overcoming the acute phase of ulcerative gingivitis (Vincent's type) and reported that penicillin administered as capsules - and peroxyhorate monohydrate - used as a mouthwash three times daily - yielded satisfactory results. Both clinical and microbiologic parameters were examined. In this context it should be realized that in the Vincent's type ulcerative gingivitis obligate anaerobes such as fusobacteria and spirochetes are considered to be important etiological factors. The finding that an H^O^-releasing mouthwash has an effect also in plaques associated with developing gingivitis accords with results reported by, e.g. Johansen et al. (1970). They evaluated the effect of Ascoxal T® in dentai students and demonstrated a significant inhibition of plaque formation after 3 and 8 days of no mechanical toothcieaning. Similar findings regarding Ascoxal T were reported by Bergenholtz et al. (1969) who were unable to find a similar effect of Amo-
san (Bocosept®). Rundegren et al. (1973) studied the effect of a hydrogen peroxide toothpaste for its (1) bactericidal effects in vitro and (2) effect on gingivitis and plaque in dental students. Significant effects were observed from both types of experiment. Zusammenfassung
Der Effekt von Wasserstoffsuperoxyd auf die Entwicklung von Plaque und Gingivitis beim Menschen Die vorliegende Stndie wurde untemommen um den Effekt von naszierendem Wasserstoff auf die Zusammensetzung, den Gehait an Mikroorganismen und die krankheitserregenden Eigenschaften der sich entwiekelnden Plaque bei menschlichen Probanden zu beobachten. Die Versuehe wurden als doppelte Elindtests nach dem Kreuzmodell vorgenommen und soil ten die Wirkung einer aktiven Mundsp iii losung (Amosan©) auf die Entwicklnng von Plaque nnd Gingivitis mit dem Effekt von Spulungen mit Piazebofllissigkeiten vergleichen, Der aktive Bestandteil wurde als Pulver geliefert. Fiir eine Mundspliiung wurde 1,7 g Pulver in 30 ml heissem Wasserleitungswasser aufgelost. Vierzehn Studenten der Zahnmedizin hatten sich als Prohanden zur Verfiigung gestellt. Die Probanden wurden wahrend zweier aufeinander folgenden Testperioden untersucht. Jede dieser Testperioden bestand aus einem vorbereilenden (aktive Mundhygiene wurde genauestens durchgefiihrt} und einem eigentiichen Versuchsabschnitt (Mundspulungen als einzige Methode zur Plaquekontrolle). Jeder der eigentiichen Versuchsahschnitte beider Testperioden wurde mit einer Ausgangsuntersuchung eingeleitet. Dann spulten die Probanden entweder mit der aktiven oder der Piazebofilissigkeit. Die Mundsplilungen viTirden direkt nach dem Frlihstuck, nach dem Mittagessen und nach dem Abendbrot vorgenommen. Die Bewertungseinheiten (scores) der Plaque- und Gingivalindizes wurden 4, 7 und 14 Tage nach dem Beginn des eigentiichen Versuchsabschnittes (kein Zahneputzen gestattet) festgestellt. Bakterielle Abstriche wurden nach 7 und 14-tagiger Versuchsdauer untersucht. Die Resultate zeigten, dass eine Mundsplilfltissigkeit die naszierenden Wasserstoff enthalt eine Besieddung der sich entwickelnden Plaque mit sowohl Filarien,, Fusobakterien, bewegliChen und gekriimmten Stabchen als auch Spiro-
EFFECT OF H.O^ ON PLAQUE AND GINGIVITIS chaten effektiv verhinderte. Die Mundspijlungen, die wahrend einer Periode von zwei Wochen als einziges Hilfsmittel zur oralen Hygiene zur Anwendung kamen, verringerten weiterhin deutlich die Menge sich neubildender Plaque und verzogerten signifikant die Entwicklung der Gingivitis. Es wird vermutet, dass wahrend der MundspUlung naszierendes H^O^ die Fahigkeit anaerober Bakterien zur Besiedelung und Vermehrung verhindern oder verzogern kann.
biles et incurves, ainsi que Ie developpement de colonies de spirochetes dans la plaque en formation. Ce bain de bouche, utilise comme unique soin d'hygiene bucco-dentaire pendant une periode de deux semaines, reduisait de plus de fa?on marquee la quantite de plaque qui se formait et retardait I'apparition d'une gingivite. II semblerait que l'eau oxygenee liberee par les bains de bouche pendant les rincages puisse empecher ou retarder le developpement de; colonies de bacteries anaerobies et leur multiplication.
Resumi Action du peroxyde d'hydrogene chez l'homme sur la formation de plaque et l'apparition de gingivite Cette etude experimentale a ete entreprise pour evaluer Taction de rincages de bouche produisant du peroxyde d'hydrogene sur la composition mierobienne de la plaque en formation et sur la quantite et le caractere pathogene de Ia plaque formee. Les essais ont ete concus comme une etude a double insu par methode croisee concernant J'action d'un bain de bouche (Amosan®) et d'un rincage placebo sur Ia formation de piaque et l'apparition de gingivite chez de jeunes adultes. Le produit actif se presentait sous forme de poudre; Ie rincage se faisait avec 1,7 g de poudre en solution dans 30 ml d'eau chaude du robinet. Quatorze etudiants en dentisterie ont participe a cette etude. Les etudiants ont etc examines pendant 2 periodes consecutives, consistant ehacune en une phase preparatoire (pendant laquelie les procedes actifs de nettoyage des dents etaient pratiques d'une fa^cn meticuleuse) et une phase experimentale principale (pendant laquelle les rin^ages de bouche etaient les seules mesures destinees a combattre la plaque). Chacune des deux phases experimentales debutaient par un examen de reference apres lequel les participants commencaient les rincages, soit avec le bain de bouche actif, soit avec le bain de bouche placebo. Les rincages avaient lieu immediatement apres le petit dejeuner, apres le dejeuner et apres le diner. Les scores de l'indice de Plaque et de rindice Gingival ont ete etablis 4, 7 et 14 jours apres le debut de la periode sans brossage des dents. Des prelevements bacteriologiques ont ete faits et examines apres 7 jours d'experience et apres 14 jours d'experience. Les resultats ont montre que le bain de bouche degageant du peroxyde d'hydrogene previent de maniere efficace le developpement de colonies de filaments, de fusiformes, de batonnets mo-
References
Bergenholtz, A., Hugoson, A., Lundgren, D. & Ostgren, A. (1969) The plaque-inhibiting property of some mouthwashes and their effecl on the oral mucosa. Svensk Tandlakare Tidskrift 62, 7-14. Bundgaard-Jorgensen, F. &. Clausen, F. P. (1962) Kliniske erfaringer vid behandling af parodontale lidelser med Bocosept®. Tandlaegebladet 66, 691-696. Cobe, H. M. (1960) Investigations of a new dental chemotherapeutic agent in the presence of blood. Oral Surgery 13, 678-685. Ericsson, Y. & Lundbeck, H. (1955) Antimicro-' bical effect in vitro of the ascorbic acid oxidation. Acta Patologica et Microbiologica Scandinavica 37, 493-506. Gjermo, P. (1974) Chlorhexidine in dental practice. Journal of Clitiical Periodontology 1, 143-152. Heiji, L. & Lindhe, J. (1979) The effect of metronidazole on the development of plaque and gingivitis in the beagle dog. Journal of Clinical Periodotitology, in press. Jensen, S. B., Loe, H., Schiott, C. R. & Theilade, E. (1968) Experimental gingivitis in man. IV. Vancomycin-induced changes in bacterial plaque composition as related to development of gingiva] inflammation. Journat of Periodontat Research 3, 284-293. Johansen, J. R., Flotra, L. & Gjermo, P. (1970) A clinical evaluation of the effect of Ascoxal T® on plaque formation and gingivitis. Acta Odontologica Scandinavica 28, 661-677. Lindhe, J. & Axelsson, P. (1973) The effect of controlled oral hygiene and topical fluoride application on caries and gingivitis in Swedish schoolchildren. Community Dentistry and Oral Epidemiology 1. 9-16. Lindhe, J., Heijl, L., Goodson, I. M. & Socransky, S. S. (1979) Local tetracycline delivery using hollow fiber devices in periodontal
13d
WENNSTROM AND LINDHE
therapy. Journal of Clinical Periodontology, in press. Listgarten, M. A. & Heilden, L. (1978) Relative distribution of bacteria at clinically healthy and periodontally diseased sites in humans. Journal of Clinical Periodontology 5, 115132. Listgarten, M. A., Lindhe, J. & Hellden, L. (1978) The effect of tetracycline and/or scaling on human periodontal disease - clinical, microbiological and histological observations. Journal of Clinical Periodontology 5, 246-271. Listgarten, M. A., Lindhe, J. & Parodi, R. (1979) The effect of systemic antimicrobial therapy on plaque and gingivitis in dogs. Jourtial of Periodontal Research. In press. Loesehe, W. J. (1976) Chemotherapy of dental plaque infections. In: Preventive dentistry: Nature, pathogenicity and clinical control of plaque. Oral Sciences Reviews 9, 65-107. Loesche, W. J. & Syed, S. A. (1975) Bacteriology of dental plaque in experimental gingivitis. 1. Relationship between gingivitis and flora. Journal of Dental Research 54, (Special Issue A) 71, Abstr. No. 108. Loe, H. Silness, J, (1963) Periodontal disease in pregnancy. I. Prevalence and severity. Acta Odontologiea Scandinavica 21, 533—551. Loe, H., Theilade, E. & Jensen, S. B. (1965) Experimental gingivitis in man. Journat of Perlodontology 36, 177-187. Loe, H., Theilade, E., Jensen, S. B. & Schiott, C. R. (1967) Experimental gingivitis in man. III. The influence of antibiotics on gingival plaque development. Journal of Periodontal Research 2, 282-289. Rundegren, J., Fomeli, J. & Ericsson, T. (1973) In vivo and in vitro studies on a new peroxide-containing toothpaste. Scandinavian Journal of Dental Research SI, 543-547. Shipman, B., Cohen, E. & Kaslick, R. S. (1971) The effect of a urea peroxide ge! on plaque deposits and gingival status. Journal of Periodontology 42, 283-285. Silness, J. & Loe, H. (1964) Periodontal disease in pregnancy. II. Correlation between oral
hygiene and periodontal condition. Acta Odontologiea Scandinavica 22, 121-135. Slots, J. (1977) Microflora in the healthy gingival sulcus in man. Scandinavian Journal of Dental Research 85, 247-254. Slots, J., Moenbo, D., Langebaek, J. & Frandsen, A. (1978) Microbiota of gingivitis in man. Scandinavian Journal of Dental Research 86, 174-181. Socransky, S. S. (1977) Microbiology of periodontai disease - Present status and future considerations. Journat of Periodontology 48, 497-504. Syed, S. A,, Loesche, W. J. & Loe, H. (1975) Bacteriology of dental plaque in experimental gingivitis. 2. Relationship between time, plaque score and flora. Journal of Dental Research 54 (Special Issue A) Abstr. No. 109. Theilade, E., Wright, W. H., Jensen, S. B. & Loe, H. (1966) Expermiental gingivitis in man, II. A longitudinal and bacteriological investigation. Journal of Periodontal Research 1, 1-13. Wade, A. B. & Mirza, R. B. (1964) The relative effectiveness of sodium peroxyborate and hydrogen peroxide in treating acute ulcerative gingivitis. Dental Practitiotier 14, 185187. Wade, A. B., Blake, G. C, Manson, J. D., Anderson, E. G. B. & Arnold, M. E. (1961) A comparative assessment of three medicaments for the treatment of acute ulcerative gingivitis of the Vincent's type. British Dental Journal 111, 280-285. Wade, A. B,, Blake, G. C, Manson, J. D., Berdon, J. K., Mathieson, F. & Bate, D. M. (1963) Treatment of the acute phase of ulcerative gingivitis (Vincent's type). British Dental Journal 115, 372-375. Address: Department of Periodontology Faculty of Odontology University of Gothenburg Gothenburg Sweden
momhwasii.
Effect of hydrogen peroxide on developing piaque and gingivitis in man JAN WENNSTROM AND JAN LINDHE
Department of Periodontology, Faculty of Odontology, University of Gothenburg, Gothenburg, Sweden Abstract. The present experiment was undertaken to assess the effect of hydrogen peroxide release during mouth rinsings on the composition of the microbiota of developing plaque in humans and the amount and pathogenecity of the piaque formed. The trial was designed as a double-blind crossover study of the effect of a mouthwash (Amosan®) and a placebo rinse on the development of plaque and gingivitis in young adults. The active compound was available as a powder; the rinse consisted of 1.7 g powder dissolved in 30 ml hot tap water. Fourteen dental students participated in the trial. The students were examined during two consecutive periods, each consisting of one preparatory (during which active tooth cleaning measures were carefully practiced) and one main test period (during which mouth rinsings were the only plaque control measure). Each of the two test periods was initiated by a baseline examination following which the participants rinsed either with the active or the placebo mouthwash. The rinsings were performed immediately after breakfast, after lunch and after dinner. Measurements of Piaque and Gingival Index scores were perfornied 4, 7 and 14 days after the start of the no-toothbrushing period. Bacteria were sampled and examined after 7 and 14 days of trial. The results demonstrated that a mouthwash which released hydrogen peroxide effectively prevented the colonization of filametits, fusiforms, motile and curved rods as well as spirochetes hi developing plaque. The mouthwash which was used as the only oral hygiene measure during a 2-week period furthermore markedly reduced the amount of plaque formed and significantly retarded gingivitis development. It is suggested that HgO, released by mouthwashes during rinsing may prevent or retard the colonization and multiplication of anaerobic bacteria.
Findings reported by Loe et al. (1965) and Theilade et al. (1966) from experitnents in young adults have demonstrated that the abolishment of mechanical tooth cleaning measures results in a rapid accumulation of plaque and in generalized gingivitis after 9-21 days. During the no-toothbrushing period there was not only an increase in the numher of microorganisms in the plaque, but in addition a change in the composition
of the plaque flora. Thus, at the start of the experiments, i.e. when the gingivae were normal, the plaque flora was predominated by Gram-positive cocci and rods. Later the microflora changed into a complex population in which filaments, fusobacteria and eventually vibrios and spirochetes made up approximately 30 % of all cells present. The authors concluded that gingivitis is associated with an increase in number of bacteria
0303-6979/79/020115-16$02.50/0© 1979 Munksgaard, Copenhagen
116
WENNSTROM AND LINDHE
and a change in the flora towards a larger representation of Gram-negative organisms. Other methods have also been used to study the mieroflora associated with periodontal health and disease, such as "direct microscopic examination", "cultivation in nonselective media", "electron microscopic assessments of plaque in situ" and assessments of the "predominant cultivable organisms" from pocket samples (for review see Socransky 1977). Slots (1977) and Slots et al. (1978) studied the predominant cultivable microbiola of healthy sulci and inflamed gingival pockets in man. They observed that a Gram-positive flora predominated O> 75 % of ail isolates) the microbiota of healthy sulci, whereas close to 50 % of the microorganisms in gingivitis pockets were obligate anaerobes. In addition, Gram-negative anaerobic rods made up approximately 25 % oi the isolates. Listgarten & Hellden (1978) examined the subgingival microfiora of healthy and diseased sites in humans by darkfield microscopy. They reported that coccoid cells were predominant (75 %) in healthy sites while at diseased sites fusiforms and motile bacteria including spirochetes made up approximately 60 % of the bacterial cells. In attempts to further correlate the colonization of certain types of plaque bacteria with the initiation of gingival inflammation, trials have been performed in which the bacterial composition of the bacterial composition of the developing plaque was altered via the use of antibiotics administered either via the systemic route or applied topically in the oral cavity (for review see Loesche 1976). Also in this context, the gingivitis model by Loe et al. (1965) has frequently been used to evaluate antimicrobial agents for their ability to prevent disease development. For instance, Lbe et al. (1967) and Jensen et al. (1968) showed that topical application of antibiotics such as Tetracycline, Vaneomycin and Polymyxin B re-
sulted in a pronounced change of the plaque flora. As a consequence of this change, varying degrees of inhibition of subclinical and clinical signs of gingivitis were noted. In a similar manner different substances such as antiseptics, enzymes, tensioactive agents, etc., have been employed in experiments to prevent or reduce plaque formation and gingivitis development (for review see, e.g. Gjermo 1974, Loesche 1976). During recent years data has accumulated demonstrating the importance of the obligate anaerobic microorganisms in plaques associated with gingivitis and periodontitis (for review see Socramky 1977). Since the obligate anaerobes are sensitive towards oxygen, microbiologists have been prompted to develop elaborate techniques to ascertain proper living conditions for the anaerobic plaque microorganisms during sampling and cultivation. This knowledge has been utilized to some extent in the formulation of certain types of mouthwashes which operate by the release of hydrogen peroxide or oxygen (Cobe 1960, Stripman et al. 1971, Ericsson & Lundbeck 1955). The aim of the present experiment was to study the effect of a hydrogen peroxide releasing mouthwash on (1) the composition of the microbiota of the developing plaque in humans and (2) the amount and pathogenecity of the plaque formed. Material and Methods The trial was designed as a double-blind crossover study of the effect of a mouthwash (Amosan®) releasing hydrogen peroxide and a placebo drug on the development of plaque and gingivitis in young adults. The active compound (Amosan, Cooper Lab. Inc., Cedar Knolls, New Jersey, USA) was available as a powder and stored in disposable airtight envelopes. Each envelope contained 1.7 g of the powder which immediately prior to use was dissolved in 30 ml
EFFECT OE H^O^ ON PLAQUE AND GINGIVITIS
117
Baseline enamination
Initial examination Preparatory
Main te5t phase
Group A "Activp compound"
Group A "Placebo" Sroup B "Active compound" L..
I- Group E "°lacebo" Day
Day
0
4
7
1:1
ClI
Bacterial sample
Preparatory pnase: 2-3 «ee(;5
Fig. 1. Outline of the experiment. Fourteen dental students participated in the trial. During a preparatory period of 2-3 weeks prior to the test periods, careful mechanical toothcleaning was practiced. Clinical examinations were performed on days 0, 4, 7 and 14 during the main test periods. Bacterial plaque was sampled on days 0, 7 and 14. Die Organisation des Versuches: Vierzehn Studenten der Zahnmedizin nahinen an dem Test teil. Wahrend eines vorbereitenden Versuchsabschnittes von 2~3 Wochen wurde eine sorgfdltige mechanische Zahnreinigung durchgefiihrt. Die klinischen Untersuchungen wurden an den Tagen 0, 4, 7 und 14 wahrend des eigentlichen Versuchsabschnittes vorgenommen. Abstriche bakterieller Plaque wurden an den Tagen 0, 7 und 14 entnommen. Plan de l'experience. Quatorze etudiants en dentisterie participaient a cet essai. Pendant une phase preparatoire de 2-3 semaines pre,cedant chacune des phases experimentales (test phase), le nettoyage mecaniqtie des dents etait pratique d'une fa(;on meticuleuse. Les examens clinigues ont ete effeetues aux Jours 0, 4, 7 et 14 pendant les phases experimentales principales. Des prelevements de plaque microbienne (bacterial sample) ont ete faits aux Jours 0, 7 et 14.
hot tap water. The composition of the preparation was 68.6 % sodiumi perborate, 29.4 % sodium bitartrate and 1.9 % flavoring substances such as menthol and peppermint. The active ingredient is, according to the manufacturer, hydrogen peroxide. Fourteen dental students volunteered to participate in the dinieal investigation. They had at least 24 teeth and showed no sign of active periodontai tissue breakdown. All the students were examined during two consecutive periods (Fig. 1), each consisting of one preparatory period (during which active, mechanical, oral hygiene measures were carefully practiced) and one main test period (during which no mechanical tooth eleaning measures were performed).
At the start of the two preparatory periods the teeth of all the participants were carefully scaled and polished. Detailed instruction in proper oral hygiene measures was given to eaeh individual. At the end of each of the two preparatory periods, the oral hygiene status and the gingival conditions of the perticipants were examined. The oral hygiene status was assessed using the Plaque Index system (Silness & Loe 1964). The amount of plaque was determined on all surfaces of all teeth in the dentition except 18, 28, 38 and 48. The frequency distribution of seores 0, 1, 2 and 3 was calculated for each individual. The gingival conditions were examined using the Gingival Index system (Loe & Sil-
118
WENNSTROM AND LINDHE
ness 1963). The frequency distribution of scores 0, 1, 2 and 3 was calculated for each individual. Following the clinical examination, supragingival plaque was collected from the buccal surface of tooth 14. The composition of the plaque sample was analyzed by darkfield microscopy according to a method recently described by Listgarten & Heliden (1978). Thus, the frequency distribution of coccoid cells, straight rods, fusitorms, filaments, motile and curved rods and spirochetes was calculated. Following the baseline examination, the participants were asked to abstain from all mechanical tooth cleaning measures during a 2-week period. They were instructed to rinse their mouth, three times a day (2 min-
utes each time) with 30 ml of the active mouthwash (1.7 gm Amosan dissolved in 30 ml warm-hot tap water) or a placebo mouthwash. The rinsings were performed immediately after breakfast, after lunch and after dinner. AU participants were given careful instruction in the use of the mouthrinse. During this first test period subjects 3, 5, 6, 7, 10, 13 and 14 used the active compound and subjects 1, 2, 4, 8, 9, 11 and 12 used the placebo control. Measurements of Plaque and Gingival Index scores were performed 4, 7 and 14 days after the start of the no-toothbrushing period. Bacterial samples were collected from the buccal surfaces of tooth 14 on days 0 and 7 and from tooth 24 on day 14. At the end of the first main test period,
COMPOSITION OF PLAOUE SAMPLES
•
BASELINE-NORMAL
^
TEST PERIOD
Q
CONTROL PERIOP
^
M O COCCOID CELLS STRAIGHT ROOS
7
14
FUSIFORMS
0 MOTILE AND
7
14
SPIROCHETES
RODS
Fig. 2. Diagram showing the composition of the developing plaque on the buccal tooth surfaces examined during the test and control periods. Mean (x) percentage and standard error (S.E.). Das Diagramm veranschaulicht die Zusammensetzung der Plaque an den bukkalen Flachen der wdhrend der Test- und Kontroltperioden untersuchten Zahne. Mittlere Prozentwerte (x) und Standardirrtum (SE). Diagramme motitrant la composition de la plaque se formant sur les faces vestibulaires examinees pendant les periodes experimentales (test) et temoins (eontrol), Pourcentage moyen (x) et erreur-type (S.E.).
EFFECT OF
ON PLAQUE AND GINGIVITIS
119
Table 1. The frequency distribution of different bacteria of developing plaque during the test (Amosan) and control (Placebo) periods. Mean (x) and standard error (S.E.) Die Frequenzverteilung verschiedener Bakterien in der sich entwiekelnden Plaque wahrend der Test- (Amosan) und Kontrollperioden (Plazebo). Mittelwerte (x) und Standardirrtum (SE) Distribution de frequence des differentes bacteries de la plaque se formant pendant les periodes experimentales avec Ainosati (test) et temoins avec Placebo (control). Moyenne (x) et erreurtype (S.E.) Coccoid cells -!Straight rods
Filaments
0 7 14
84,2 ± 3.9 51.7 ± 4.8a 40.4 ± 6.0a
7.0 ± 1.8 20.3 ± 2.1a 17.7 ± I,8b
4.3 ± 2.0 16.3 ± 2.6b
Test
0 7 14
88.2 ± 4.6 88.0 ± 3.5 84.9 ± 4.7
4.7 ± 1.8 4.6 ± 1.4 6.9 ± 2.0
Test vs Control
0 7 14
NS P < 0.001 P < 0.001
NS F < 0.001 P < 0.001
Period
Control
Days
Motile Fusiforms
+
Spirochetes
Curved rods
14.9 ± 1.815
4.5 + 1.3 11.6 ± 2.9f 22.1 ± 4.0b
0,1 ± 0.1 0.1 ± 0.1 4.9 ± 1.80
4.0 ± 1 . 3 5.6 ± 1.9 5.4 ± 1.8
3.0 ± 1.2 1.4 ± 0.5 2.6 ± 1.8
0,1 ± 0.1 0.4 ± 0.6 0.1 ± 0.1
NS P < 0.01 P < 0.001
NS NS P < 0.05
NS P < 0.01 P < 0.01
" Significantly different from day 0: P < 0.001 il Significantly different from day 0: P < 0.01 c Significantly different from day 0; P < 0.05 Coccoid celis -I- Straight rods (Kokkoide Zellen + gerade Stdbchen, cellules coccoides + batonnets droits), filaments (Filarien, filametits), fusiforms (Fusobakterien, fusiformes). Motile -j- curved rods (Bewegliche + gekrUmmte Stabchen, bdtontiets tnobiles -\- iticurves), spirochetes (Spirochaten, spirochetes), a, -|- b 4- c Significantly different from day 0: (a -f b -]- c Signifikant unterschiedlieh vom Tage 0.', a -j- b -|- i; difference significative par rapport au jour 0:).
the teeth of the participants were again carefully scaled and polished. They were instructed to cleati their teeth regularly and in a meticulous manner during the subsequent 2—3 weeks. Professional tooth cleaning (Lindhe & Axelsson 1973) was performed twice during this second preparatory period. Towards the end ot" the period a new baseline examination was performed following whicli the second main test period was initiated (Fig. I). During this period subjects 1, 2, 4, 8, 9, 11, 12 used the active compound and subjects 3, 5, 6, 7, 10, 13, 14 the placebo mouthrinse. The clinical and bacteriological examinations were repeated after 4, 7 and 14 days of no mechanical tooth cleaning. Differences regarding PU and GI scores as well as frequency distri-
bution of bacteria between test and control periods were analyzed using Student's t-test. Differences between days were assessed by analysis of variance. Results
Microbiology At the start of the two main test periods, i.e. when the Plaque and Gingival Index scores were zero or close to zero for all buccal, lingual and approximal tooth surfaces and gingival units, coccoid cells and straight rods accounted for almost 90 % of the bacterial population in the plaque samples (Fig. 2). The remaining bacteria were identified as filaments («* 6 %; Table 1 and Fig. 2), fusiforms ( ^ 4 %) and motile rods
120
WENNSTROM AND LINDHE
( ~ 4 % ) . Spirochetes were absent in most of the samples and accounted on the average for only 0.1 % of the total flora. During the "placebo period" (control period) of no mechanical tooth cleaning there was a marked change in the hacterial composition of the developing plaque on the buccal tooth surfaces examined. Hence, after 7 and 14 days of experiment the percentage of coccoid cells and straight rods had decreased (52 % on day 7 and 40 % on day 14; Fig. 2) and the frequency distribution of filaments, fusiforms, motile and curved rods increased (Table 1). On day 7 spirochetes were observed in samples from two individuals and on day 14 plaque samples collected from eight individuals harbored a significant number of spirochetes {x = AB %\ Fig. 2). Consequently, during the control period the microbiota of the supragingival plaque shifted from a predominantly coccoid cell + straight rod flora to a complex flora containing large numbers of filaments -1- fusiforms O 30 %),
curved -f motile rods ( > 20 %) and spirochetes («^5 %). During the test period, when the participants regularly rinsed with the hydrogen peroxide mouthwash, the supragingival plaque maintained its characteristics of day 0, i.e. the flora on days 7 and 14 of notoothbrushing was dominated by coccoid cells and straight rods ( > 80 % of ail bacteria counted; Fig. 2). Furthermore, during the test period the frequency distribution of filaments, fusiforms, motile + curved rods and spirochetes did not change (Table 1, Fig. 2). Clinical Parameters At the start of the two no-toothbrushing periods practically all tooth surfaces were without clinically detectable plaque, i.e. the frequency distribution of Plaque Index score 0 was close to 100 % (Table 2, Fig. 3). The examination of the gingival conditions revealed that most gingival units were healthy and that the percentage of units
Table 2. Frequency distribution of Plaque Index scores 0, 1, 2 and 3 during the test and control periods. Mean (x) and standard error (S.E.) Die Frequenzverteilung der Plaqueindex-Bewertungseinheiten (scores) 0, 1, 2 und 3 wahrend der Test- und Kontrollperioden. Mittelwerte (x) utid Standardirrtum (SE) Distribution de frequence des scores 0, 1,2 et 3 pour l'indice de Plaque pendant les periodes experimentaies et temoins. Moyenne (x) et erreur-type (S.E.) Period
Days
Control
0 4 7 14
98.7 41.4 28.0 17.2
± ± ± ±
0.5 4.0 4.6 2.9
1.3 51.0 45.4 37.0
± ± ± ±
0.5 3.0 4.5 4.8
Test
0 4 7 14
98.9 73.2 64.6 46.4
± ± ± ±
0.5 4.9 5.5 4.3
1.0 24.4 31.9 45.2
± ± ± ±
0.4 3.6 4.2 3.6
Test vs Control
0 4 7 14
NS P < 0.001 P < 0.001 P < 0.001
0
1
NS P < 0.001 NS NS
2
3
0 8.4 ± 2.8 26,6 ± 6.3 44.4 ± 6.2
0 0 0 1.4 ± 0.9
0.1 3.1 3.4 8,7
± ± ± ±
0.1 2.0 1.9 3.6
NS NS P < 0.001 P < 0.001
0 0 0 0
M' NS NS
EFFECT OF
ON PLAQUE AND GINGIVITIS
121
PLAQUE INDEX SCORE 0
^
I
TEST PERIOD
I CONTROL PEfllOD
.
SCORE 2
DAY 0
DA* 4
DAV 7
DAY 4
DftY 7
I
i
Fig. 3. The mean ("xj percentage of tooth surfaces with Plaque Index scores 0 and 2 during the test and control periods. The top diagram presents the data for all surfaces combined. The bottom diagram presents the data for buceal, lingual and approximal surfaces separately. Das Vorkotnmen (tnittlere Prozentwerte, x) der Bewertungseinheiten 0 und 2 des Plaqueindexes wahrend der Test- und Kontrollperioden. Das obere Diagramm enthalt die kombinierten Werte aller ZahnoberflUchen. Das untere Diagramm enthdlt die separaten Werte der bukkalen, lingualen und approxitnalen Zahtioberflachen. Pourcentage moyeti (x) des faces dentaires ayant des scores 0 et 2 pour I'indice de Plaque pendant les periodes experimentales et temoins. Le diagramme du hatit represente les donttees pour I'ensemble des faces. Le diagramtne du bas represente separetnent les donnees eoncernant les faces vestibulaires (buccal), linguales et proximales.
WENNSTROM AND LINDHE
GINGIVAL INDEX SCORE O
Ir
P^ j
TEST PERIOD [ CONTROL PERIOD
SCORE 2
APPROXIUAL
^
DA* 0
DAY a
I r OaV 7
DBI 14
DAY D
DAY 4
DA* 7
DA* 14
DAY 0
DAY i
DAY 7
DAY 14
F/^. •#. The mean (x) percentage of gingival units with GJ scores 0 and 2 during the test and control periods. The top diagram presents the data for all gingival units combined. The bottom diagram presents the data for buccal, lingual and approximal units separately. Das Vorkommen (mittlere Prozentwerte, x) gingivaler Abschnitte tnit den Bewertungseinheiten 0 und 2 des Gingivalindexes wdhrend der Test- und Kontrollperioden. Das obere Diagramm enthdlt die kombinlerten Werte aller Gingivalabschnitte. Das untere Diagramm enthalt die separaten Werte der bukkalen, lingualen und approximalen Abschnitte. Poureentage moyen (x) des unites gingivales presentant les scores 0 et 2 pour I'indice Gingival pendant les periodes experimentales et temoins. En haut, donnees pour I'ensemble des faces; en bas, donnees pour les faces vestibulaires, linguales et proximales, separement.
EFFECT OF
ON PLAQUE AND GINGIVITIS
Table 3. Frequency distribution of Gingival Index scores 0, 1, 2 and 3 during the test and control periods. Mean (x) and standard error (S.E.) Die Frequenzverteilung der Gingivalindex-Bewertungseinheiten (scores) 0, I, 2 und 3 wdhrend der Test- und Kontrollperioden. Mittelwerte (x) und Standardirrtum (SE) Distribution de frequence des scores 0, I, 2 et 3 pour l'indice Gingival pendant les periodes experimentales et temoins. Moyenne (x) et erreur-type (S.E.) Period
Days
Control
0 4 7 14
74.0 57.4 31.4 13.0
±2.3 ± 2.0 ± 3.0 ± 2.3
25.4 37.4 55.8 54.9
± ± ± ±
2.2 2.4 2.5 3.4
n.9 ± 2.5
0 0 0
31.6 ± 4.6
0
Test
0 4 7 14
72.1 66.9 52.4 27.4
±1.8 ± 1.6 ± 2.7 ± 7.3
26.4 29.3 42.2 53.3
± ± ± ±
1.7 1.8 2.4 1.4
1.5 3.2 5.4 19.4
0 0 0 0
Test vs Control
0 4 7 14
NS P < 0.001 P < O.OOJ P < 0.001
0
1
2
NS P < 0.05 P < 0.01 NS
receiving a GI score of 2 was very low (start of control period: 0.6 %; start of test period: 1.5 %; Table 3, Fig. 4). During the control period the number of plaque-free tooth surfaces rapidly deereased (Fig. 3). Thus, already after 4 days of no toothbrushing only 40 % of the surfaces were clean and after 7 and 14 days the corresponding figures were 28 and 17 %. During the 2 weeks of observation there was a corresponding increase of Plaque Index seores 1 and 2 (Tables 2 and 4, Fig. 3). At the reexaminations on days 4, 7 and 14, the frequeney of Piaque Index score 2 was 8.4, 26.6 and 44.4 %, respeetively. The most marked inerease of the plaque scores occurred on the buccal and approximal tooth surfaees (Fig. 3, Table 4). Also during the test period the frequency of Plaque Index seore 0 decreased. This decrease was, however, moderate in eomparison to the ehange observed during the control period (Fig. 3). Thus, after 4, 7 and 14 days of no toothbrushing but rinsing with Amosan, the frequency of Pll = 0 was 73.2 % (control = 41.4 %), 64.6 % (control
3
0.6 ± 0.3 3.9 ± 0.8
± ± ± ±
0.6 1.0 0.8 2.4
NS NS P < 0.01 F < 0.01
NS NS NS NS
^ 2 8 % ) and 46.4% (eontrol = 17.2 %). The differenee wilh regard to seore PH = 0 between the test and eontrol periods was at eaeh reexamination highly significant (P < 0.003; Table 2). Only towards the end of the test period did the frequency of Plaque Index score 2 differ significantly from the day 0 value. Also with regard to the PII seore 2 eould a highly signifieant differenee be calculated between the test and eontrol regimen (days 7 and 14; P < 0.001, Table 2). Fig. 3 and Table 4 illustrate the Plaque Index alterations on different tooth surfaees during the two no-toothbrushing periods. It is obvious that the aetive mouthwash was more effective on buccal and lingual than on appriximal tooth surfaces. However, also on the approximal surfaces the differenee regarding PU seores 0 and 2 between the test and eontrol period was marked (Table 4). During both periods of no toothbrushing the gingival conditions gradually deteriorated (Table 3, Fig. 4). This is illustrated by the increase in the frequency of Gingival
124
WENNSTROM AND LINDHE
Table 4, Frequency distribution of Plaque Index scores 0, 1, 2 and 3 for the buccal, lingual and approximal tooth surfaces during the test (Amosan) and control (Plaeebo) periods. Mean (x) and standard error (S.E.) Die Frequenzverteilung der Plaqueindex-Bewertungseinheiten (scores) 0, 1, 2 und 3 an den bukkalen, lingualen und approximalen Zahnoberfldchen wahrend der Test- (Amosan) und Kontrollperioden (Plazebo). Mittelwerte (x) und Standardirrtum (SE) Distribution de frequence des scores 0, 1, 2 et 3 pour I'indice de Plaque au niveau des faces vestibulaires, linguales et proximales pendant les periodes experimetitales avec Amosan (test) et temoins avec Placebo (control). Moyenne (x) et erreur-type (S.E.) 0
Buccal surfaces „ „ Amosan ^^^^ Placebo
99.7 ± 0.3 100.0 ± 0
1
0.3 ± 0.3 0± 0
••
2
3
z
—
Day 4
A P
80,1 + 6.2*** 44.9 ± 6.4
16.1 + 4.3*** 47.1 ± 5.1
4.1 ± 2.5 7.6 ± 2.7
Day 7
A P
68.7 + 5.7*** 23,3 ± 7.7
27.3 ± 5.9** 49.6 + 4.6
4.7 + 2.4** 27.4 ± 6.1
—
Day 14
A P
48.1 + 6.7*** 8.8 ± 4.0
41.1 ± 4.8 39.5 + 6.6
10.8 + 3,9** 50.4 ± 8,5
0 ±0 1.4 ± 1.1
1.1 ± 0.5 0.6 ± 0.3
—
—
—
Ling It al surfaces DayO
^
98.9 + 0,5 99.4 ± 0.4
Day 4
A P
86.9 ± 6.3 80.6 + 3.6
6,9 ± 1.6*** 18.0 ± 2.8
0.6 ± 0,4 1.4 ± 1.1
—
Day 7
A P
91.9 + 2.0*** 65.9 ± 4.5
6.7 ± 1.6*** 29.4 ± 3.9
.1.4 + 1.1* 4.8 ± 1.8
—
Day 14
A P
83,1 50.9 ± 5.2
13.4 + 2.4*** 39.6 ± 4.5
2.4 ± 1,3 9.7 ± 3,4
Approximal sttrfaces DayO
p
98.6 + 0.9 97.7 ± 1.0
Day 4
A P
59.4 ± 6.7*** 19.1 ± 4.8
36.9 ± 5.2*** 69.4 ± 4.1
3,8 ± 2.5** 11.1 ± 3.6
—
Day 7
A P
48.9 + 6.9*** 11.2 ± 4.5
46.8 ± 5.5 51.9 ± 7.0
4.4 + 2.4*** 36.9 ± 8,8
—
Day 14
A P
26.7 + 5.4*** 4.9 + 2.7
62,4 + 4.4* 34,8 ± 6.1
10.9 + 4 9*** 58.4 ± 7.3
0± 0 1.9 ± 1.4
1.3 ± 0.8 2.3 ± 1.0
—
* Significantly different from Placebo: P < 0.05 ** Significantly different from Placebo: P < 0.01 *** Significantiy different from Placebo: P < 0.001 Buccal surface (Bukkale Oberflache, faces vestibttlaires), lingual surfaces (Linguale Oberflachen, faces linguales), approximal surfaces (Approximale Oberflachen, faces proximales).
EFFECT OF H^O^ ON PLAQUE AND GINGIVITIS
125
Table 5. Frequency distribution of Gingival Index scores 0, 1 and 2 for the buccai, lingual and approxitnal tooth surfaces during the test (Amosan) and control (Placebo) periods. Mean (x) and standard error (S.E.) Die Frequenzverteilung der Gingivalindex-Bewertungseinheiten (scores) 0, 1, 2 und 3 an den bukkalen, lingualen utid approximalen Gingivaabschnitten wdhrend der Test- (Amosan) und Kontrollperioden (Plazebo). Mittelwerte (x) und Standardirrtum (SE) Distribtition de frequence des scores 0, 1 et 2 potir I'Indice Gingival au niveati des faces vestibulaires, linguales et proximales pendant les periodes experimentales avec Amosan (test) et temoins avec Placebo (control). Moyenne (x) et erreur-type (S.E.) 0 Buccai surfaces Amosan °^^" Placebo
92.1 ± 1.7 93.7 ± 1.2
Day 4
2
6.5 ± 1.4 5.6 ± 1.1
1.3 ± 0.9 0.8 ± 0.6
91.6 ± 1.9**''^ 79.6 ± 2.5
5.8 ± 1.4** 15.7 ± 2.2
3.1 ± 1.3 4.1 ± 1.1
82.1 ± 2.5*** 54.1 ± 4.7
15.2 ± 2.6*** 38.0 ± 4.3
2.7 ± 0.7* 7.9 ± 1.8
45.4 ± 4.]** 21.3 ± 4.7
41.4 ± 2.9* 57.8 ± 4.1
p
69.1 ± 3.2 74.9 ± 2.6
29.1 ± 2.8 24.8 ± 2.8
0.9 X 0.4
A
67.3 ± 3 . 7 62.6 + 2.5
28.9 ± 3.6 32.9 ± 2.1
3.9 ± 1.6 4.5 ± 1 . 2
A
38.2 + 2.9** 47.4 ± 4.4
36.8 ± 2.5 39.7 ± 2.8
5.0 ± 0.9** 12.9 ± 2.4
A
44.4 ± 3.7** 28.5 ±3.0
36.6 ± 3.4 43.9 ± 3.4
18.6 ± 3.2* 27.4 ± 3.7
63.8 ± 2.9 65.0 ± 2.8
34.4 ± 3.1 34.3 ± 2.7
1.8 ± 0.7 0.7 ± 0.3
A
54.6 ± 2.5** 41.1 ± 3.4
41.3 ± 2.7*** 55.7 ± 3.2
4.1 ± 1.2 3.2 ± 0.8
A
35.4 ± 3.9*** 12.7 ± 2.8
57.4 ± 3.7** 72.4 ± 3 . 3
7.2 ± 1.6* 14.9 ± 3.6
A P
9.6 ± 1.1*** 2.6 ± 1.4
67.4 ± 3.0 58.5 ± 5.3
22.3 ± 2.9* 38.9 ± 5.9
p
Day 7 Day 14
1
A
13.2 ± 2.8* 20.9 ± 4.3
Lingual surf aces
Day 0 Day 4 Day 7 Day 14
1.4 ± 0.6
Approximal surfaces DayO Day 4 Day 7 Day 14
p
Significantly different from Placebo: P < 0.05 Significantly different from Placebo: F < 0.01 Significantly different from Placebo: P < 0.001
126
WENNSTROM AND LINDHE
Index scores > 0. The degree of deterioration, however, was more marked during the control than during the test period (Tables 3 and 5). Thus during the 2 weeks when the hydrogen peroxide mouthwash was used as the only oral hygiene measure, the percentage gingival units scored GI = 2 (Fig. 4) increased from 1.5 % (day 0) to 3.2 % (day 4), 5.4 % (day 7) and 19.4 % (day 14). The corresponding figures for the control period, when the participants used a placebo mouthrinse, were 0.6 % (day 0), 3.9 % (day 4), 12.9 % (day 7) and 31.6 % (day 14). At the reexamination on days 7 and 14 the difference between the test and control periods regarding the percentage of GI score 2 was found to be statistically significant (P < 00.1). The difference was significant for buccal, lingual as well as for interproximal units.
Discussion The present experiment clearly demonstrated that a mouthwash which releases hydrogen peroxide effectively prevents the colonization of filaments, fusobacteria, motile and curved rods and spirochetes in developing plaque. The mouthwash which was used as the only oral hygiene measure during a 2-week period (three times daily), in addition, markedly reduced the amount of plaque formed and significantly retarded gingivitis development. Since it is well known that most obligate anaerobes are sensitive to exposure of hydrogen peroxide, there are reasons to believe that H^Og released by the wouthwash during rinsing prevented or retarded the colonization and multiplication of preferably anaerobic bacteria. The trial reported was designed as a double-blind crossover study of the effect of Amosan and a placebo mouthwash on the development of plaque and gingivitis in young adults. This means that intraindivid-
ual comparisons were made from test and control periods. Attempts were also made not only to assess the effect of Amosan on gross plaque formation (Plaque Index scorings) but also to analyze the frequency distribution of various microorganisms occurring in the plaques formed. This analysis was made by darkfield microscopy, a technique which earlier was proved to be of value in distinguishing between plaques associated with healthy gingivae and diseased sites in humans (Listgarten & Hellden 1978). Darkfield microscopy has also been used to investigate the effect of different modes of periodontal treatment in humans (Listgarten et al. 1978, Lindhe et al. 1979) and dogs (Listgarten et al. 1979, Heijl & Lindhe 1979). Plaques sampled at the two baseline examinations, i.e. when the participants had clinically healthy gingivae, were dominated by coccoid cells and straight rods with filaments, fusiforms, motile and curved rods being present in low numbers only. This finding agrees with results by Listgarten & Hellden (1978) who reported that plaque samples from the gingiva! sulcus region of "relatively healthy" gingival sites were characterized by their preponderance of coccoid cells and straight rods. The findings also corroborate data by, e.g. Loe et aL (1965), Theilade et al. (1966), Jensen et al. (1968) showing that Gram-positive cocci and small rods characterized the plaque sampled from individuals with healthy gingivae. After 7 days of no toothbrushing the frequency distribution of filaments and fusiforms had undergone a threefold increase (from 31.3 to 36.6%; Table 1). A similar change in the composition of piaque was observed by Loesche & Syed (1975) and Syed et al. (1975) who reported that the developing microbial plaque was characterized mainly by the influx of Actinomyces species. Between days 7 and 14 in the present experiment, the percentage of coccoid
EFFECT OF H^O^ ON PLAQUE AND GINGIVITIS
127
Table 6. The composition of tbe bacterial plaque in relation to the gingivai status at the sampling sites. Mean percentage (x) and standard error (S.E.) Die Zusatnmensetzung der bakteriellen Plaque in Bezug auf den Gesundheitszustand der Gingiva der Regionen, an denen die Abstriche entnommen wurde Composition de la plaque microbienne et etat de la gencive au niveau des preievements. Pourcentage moyen (x) et erreur-type (S.E.) Coccoid cells Gingival Index
Ovsl
Motile
+
+
Straight rods
Filaments
Fusiforms
Curved rods
Spirochetes
86.5 ± 2.5 60.2 ± 4,4 55.2 ± 9.1
6.3 ± 1.2 15.0 ± 1.5 12.4 ± 2.4
4,3 ± 0.9 13.0 ± 1.7 12.0 ± 2.8
2.6 ± 0.7 11.1 ± 2,4 16.1 ± 4.0
0.3 ± 0.2 0,7 ± 0.4 4,2 ± 2.1
P < 0.001 P < 0,001 NS
P < 0.001 P < 0.05
P < 0.001 P < 0.001 NS
P < 0,001 P < O.OOI NS
P < 0.01 P < 0.05
NS
cells and straight rods further decreased while the number of motile -F curved rods increased and spirochetes became more frequently represetited in the samples. Concomitant with this change of the microbiota, the amount of plaque markedly increased and gingivitis became established at the plaque sampling sites. The frequency distribution of various bacteria in the plaque samples has, in Table 6, been compared to the Gingival Index scores of the plaque areas, i.e. the buccal surface of teeth 14 or 24. It is obvious that, irrespective of experimental time, the relative number of coccoid cells and straight rods decreases and the number of filaments, fusiforms, motile and curved rods increases with increasing GI scores. It is also interesting to note that plaques sampled from areas with a GI score of 2, in comparison to GI score 1, have a significantly larger relative content of spirochetes, whereas the difference regarding motile + cui-ved rods, filaments and fusobacteria is insignificant. This observation is in accordance with findings from studies in humans and dogs by Listgarten & Hellden (1978) and Listgarten et al. (1979) who reported on a decreasing percentage of coc-
NS
coid cells and straight rods with increasing GI scores and an increasing tendency of motile rods and spirochetes. It should be observed, however, that whereas Listgarten & coworkers studied the suicus flora, the present data are derived from examinations of supragingivally located plaques. In most respects the observed shift of the microbiota during plaque and gingivitis development agrees with Theilade et ai. (1966) who stated that approximately at the same time as the complex microflora was established the first chnically detectable signs of localized gingivitis could be recognized. During the 2-week period when the participants in the present trial rinsed with the hydrogen peroxide releasing mouthwash, the microbiota of the plaque samples from the buccal surfaces of 14 and 24 maintained its characteristics of day 0, i.e. the gradual influx of fusobacteria, motile + curved rods and spirochetes was obviously prevented. During the same period, plaque formation (PU score 2) on the buccal surfaces (Table 4) was markedly reduced (10.8 % versus 50.4 % of the control period) and gingivitis development obviously retarded. If it is assumed that a regular use of a HgO^-releasing
WENNSTROM AND LINDHE
tnouthwash interferes with the anaerobic bacteria, the findings reported here demotistrate the importance of the anaerobes for plaque growth and gingivitis development and therefore confirm observations by Listgarten et al (1978, 1979) and Heijl & Lindhe (1979) showing that administration, via the systemic route, of antimicrobial compounds known to eliminate anaerobic microorganisms decreases signs of plaque formation and gingivitis. The observations made in this study imply that this change of the microhiota can be accomplished by HgOg release from a mouthwash. Whether this method can be used for extended periods to control dental plaque and prevent or cure gingivitis must await farther research. The finding that Amosan effectively influences the composition of a microbial plaque agrees to large extent with data reported hy Wade et al. (1961, 1963), BundgaardJorgensen & Clausen (1962) and Wade & Mirza (1964). They studied the effectiveness of different drugs in overcoming the acute phase of ulcerative gingivitis (Vincent's type) and reported that penicillin administered as capsules - and peroxyhorate monohydrate - used as a mouthwash three times daily - yielded satisfactory results. Both clinical and microbiologic parameters were examined. In this context it should be realized that in the Vincent's type ulcerative gingivitis obligate anaerobes such as fusobacteria and spirochetes are considered to be important etiological factors. The finding that an H^O^-releasing mouthwash has an effect also in plaques associated with developing gingivitis accords with results reported by, e.g. Johansen et al. (1970). They evaluated the effect of Ascoxal T® in dentai students and demonstrated a significant inhibition of plaque formation after 3 and 8 days of no mechanical toothcieaning. Similar findings regarding Ascoxal T were reported by Bergenholtz et al. (1969) who were unable to find a similar effect of Amo-
san (Bocosept®). Rundegren et al. (1973) studied the effect of a hydrogen peroxide toothpaste for its (1) bactericidal effects in vitro and (2) effect on gingivitis and plaque in dental students. Significant effects were observed from both types of experiment. Zusammenfassung
Der Effekt von Wasserstoffsuperoxyd auf die Entwicklung von Plaque und Gingivitis beim Menschen Die vorliegende Stndie wurde untemommen um den Effekt von naszierendem Wasserstoff auf die Zusammensetzung, den Gehait an Mikroorganismen und die krankheitserregenden Eigenschaften der sich entwiekelnden Plaque bei menschlichen Probanden zu beobachten. Die Versuehe wurden als doppelte Elindtests nach dem Kreuzmodell vorgenommen und soil ten die Wirkung einer aktiven Mundsp iii losung (Amosan©) auf die Entwicklnng von Plaque nnd Gingivitis mit dem Effekt von Spulungen mit Piazebofllissigkeiten vergleichen, Der aktive Bestandteil wurde als Pulver geliefert. Fiir eine Mundspliiung wurde 1,7 g Pulver in 30 ml heissem Wasserleitungswasser aufgelost. Vierzehn Studenten der Zahnmedizin hatten sich als Prohanden zur Verfiigung gestellt. Die Probanden wurden wahrend zweier aufeinander folgenden Testperioden untersucht. Jede dieser Testperioden bestand aus einem vorbereilenden (aktive Mundhygiene wurde genauestens durchgefiihrt} und einem eigentiichen Versuchsabschnitt (Mundspulungen als einzige Methode zur Plaquekontrolle). Jeder der eigentiichen Versuchsahschnitte beider Testperioden wurde mit einer Ausgangsuntersuchung eingeleitet. Dann spulten die Probanden entweder mit der aktiven oder der Piazebofilissigkeit. Die Mundsplilungen viTirden direkt nach dem Frlihstuck, nach dem Mittagessen und nach dem Abendbrot vorgenommen. Die Bewertungseinheiten (scores) der Plaque- und Gingivalindizes wurden 4, 7 und 14 Tage nach dem Beginn des eigentiichen Versuchsabschnittes (kein Zahneputzen gestattet) festgestellt. Bakterielle Abstriche wurden nach 7 und 14-tagiger Versuchsdauer untersucht. Die Resultate zeigten, dass eine Mundsplilfltissigkeit die naszierenden Wasserstoff enthalt eine Besieddung der sich entwickelnden Plaque mit sowohl Filarien,, Fusobakterien, bewegliChen und gekriimmten Stabchen als auch Spiro-
EFFECT OF H.O^ ON PLAQUE AND GINGIVITIS chaten effektiv verhinderte. Die Mundspijlungen, die wahrend einer Periode von zwei Wochen als einziges Hilfsmittel zur oralen Hygiene zur Anwendung kamen, verringerten weiterhin deutlich die Menge sich neubildender Plaque und verzogerten signifikant die Entwicklung der Gingivitis. Es wird vermutet, dass wahrend der MundspUlung naszierendes H^O^ die Fahigkeit anaerober Bakterien zur Besiedelung und Vermehrung verhindern oder verzogern kann.
biles et incurves, ainsi que Ie developpement de colonies de spirochetes dans la plaque en formation. Ce bain de bouche, utilise comme unique soin d'hygiene bucco-dentaire pendant une periode de deux semaines, reduisait de plus de fa?on marquee la quantite de plaque qui se formait et retardait I'apparition d'une gingivite. II semblerait que l'eau oxygenee liberee par les bains de bouche pendant les rincages puisse empecher ou retarder le developpement de; colonies de bacteries anaerobies et leur multiplication.
Resumi Action du peroxyde d'hydrogene chez l'homme sur la formation de plaque et l'apparition de gingivite Cette etude experimentale a ete entreprise pour evaluer Taction de rincages de bouche produisant du peroxyde d'hydrogene sur la composition mierobienne de la plaque en formation et sur la quantite et le caractere pathogene de Ia plaque formee. Les essais ont ete concus comme une etude a double insu par methode croisee concernant J'action d'un bain de bouche (Amosan®) et d'un rincage placebo sur Ia formation de piaque et l'apparition de gingivite chez de jeunes adultes. Le produit actif se presentait sous forme de poudre; Ie rincage se faisait avec 1,7 g de poudre en solution dans 30 ml d'eau chaude du robinet. Quatorze etudiants en dentisterie ont participe a cette etude. Les etudiants ont etc examines pendant 2 periodes consecutives, consistant ehacune en une phase preparatoire (pendant laquelie les procedes actifs de nettoyage des dents etaient pratiques d'une fa^cn meticuleuse) et une phase experimentale principale (pendant laquelle les rin^ages de bouche etaient les seules mesures destinees a combattre la plaque). Chacune des deux phases experimentales debutaient par un examen de reference apres lequel les participants commencaient les rincages, soit avec le bain de bouche actif, soit avec le bain de bouche placebo. Les rincages avaient lieu immediatement apres le petit dejeuner, apres le dejeuner et apres le diner. Les scores de l'indice de Plaque et de rindice Gingival ont ete etablis 4, 7 et 14 jours apres le debut de la periode sans brossage des dents. Des prelevements bacteriologiques ont ete faits et examines apres 7 jours d'experience et apres 14 jours d'experience. Les resultats ont montre que le bain de bouche degageant du peroxyde d'hydrogene previent de maniere efficace le developpement de colonies de filaments, de fusiformes, de batonnets mo-
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