Clin Oral Invest DOI 10.1007/s00784-013-1160-7
ORIGINAL ARTICLE
Treatment effect of ozone and fluoride varnish application on occlusal caries in primary molars: a 12-month study E. Johansson & J. W. V. van Dijken & L. Karlsson & I. Andersson-Wenckert
Received: 30 August 2013 / Accepted: 26 November 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Aim The aim of this study is to evaluate the effect of ozone and fluoride varnish on occlusal caries in primary molars in a split-mouth study. Materials and methods Caries risk was estimated by treating Public Dental Health Service dentists. Children with occlusal caries with Ekstrand index scores ≤3 (VI ≤3) were included. Selection of caries lesions was discontinued for ethical reasons due to non-acceptable clinical results during the followup. In the continued evaluation pairs of teeth with noncavitated caries lesions, Ekstrand score ≤2a (VI ≤2) were selected. Fifty pairs of carious primary molars were included, 18 boys and 15 girls (mean 4.7 years, range 3–8). At baseline, the lesions were assessed by visual inspection (VI) and laserinduced fluorescence (LF), in each pair to treatment with 40 s ozone (HealOzone TM , 2,100 ppm) or fluoride varnish Duraphat®. The treatments and evaluations were repeated at 3, 6 9 months and evaluations only at 12 months. Results Medium-high caries risk was observed in VI ≤3 children and low-medium risk in VI ≤2a children. In the 15 pairs VI ≤3 lesions, 8 treated with ozone and 9 with fluoride progressed to failure. In the 35 pairs VI ≤2a lesions, one lesion failed. Median baseline LF values in the VI ≤3 group were 76 and 69, for ozone and fluoride lesions, respectively, and 21 and 19 in the VI ≤2a group. At 12 months, LF values in the VI E. Johansson : J. W. V. van Dijken : I. Andersson-Wenckert Department of Odontology, Faculty of Medicine, Umea University, Umea, Sweden L. Karlsson Division of Cariology, Department of Dental Medicine, Karolinska Institutet, Stockholm, Sweden E. Johansson (*) Dental Hygienist Education, Department of Odontology, Dental School, Umea University, SE-901 87 Umea, Sweden e-mail: [email protected]
≤2a group were 15 and 18. No improvement or difference in LF values was found over time between the caries lesions treated with ozone or fluoride. Conclusions Neither ozone nor fluoride varnish treatments stopped the progression of caries in cavitated lesions. In low and medium caries risk children, non-cavitated lesions following both treatments showed slight or no progression. The use of ozone or fluoride varnish treatments in this regime as caries preventive method, added to the daily use of fluoridated toothpaste, to arrest caries progression in primary molars must therefore be questioned. Keywords Caries . Clinical . Fluoride . Ozone
Introduction Despite the dramatic decline in dental caries over the past few decades, particularly in industrialised countries, the disease persists, however, with a skewed distribution [1–3]. The polarisation of the caries disease in child and youth populations has been confirmed in several studies [2, 4]. Early identification of children with caries activity as a means of preventing and arresting the disease has been shown to be more efficacious than restorative treatments later in life [5, 6]. In Sweden, a decline of caries prevalence in 4 year olds has been observed since 1967 [7–9]. The main decline occurred from 1967 to 1980, after which a levelling out was seen. In 2007, still 38 % of the 4-year olds had cavitated and noncavitated caries lesions, in spite of a further significant decrease in the prevalence and distribution of the dmfs values between 2002 and 2007 [9]. Today, fluoride regimes and fissure sealants are commonly used treatments of early caries lesions in children [10, 11]. Fluoride varnish application aims to prevent tissue loss and to reverse or arrest lesions and thereby avoid restorations [12,
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13]. However, a recent review failed to present sufficient evidence for most preventive measures and the need for new approaches and further well-designed clinical trials was emphasised [11]. During the last decade, ozone was suggested as a new caries preventive method [14–16]. Ozone is an unstable gas containing three oxygen atoms and is a powerful oxidant and highly potent antimicrobial agent [17]. Ozone applications have been shown to reduce caries-associated bacteria in primary root caries lesions in vitro [14, 15] and in vivo [16]. However, conflicting results regarding the effect of ozone on bacteria have been obtained in the biofilm and in caries lesions [18–20]. There are only a few longitudinal studies published investigating the effect of ozone treatment in non-invasive management of early caries lesions. Two studies on root caries lesions, with a follow-up time of 6 and 18 months, respectively, showed root lesions to be arrested non-operatively with ozone [21, 22]. Three studies on occlusal caries in children and adolescents with a follow up time of 3, 8 and 26 months, respectively, showed conflicting results regarding the ability of ozone to arrest or prevent caries [23–25]. Only one of these studies evaluated ozone treatment in the primary dentition [24]. The aim of this study was to evaluate in an intra-individual comparison the effect of ozone and fluoride varnish treatments on occlusal caries in primary molars during 1 year, monitored with visual inspection (VI) and with laser-induced fluorescence (LF). The null hypothesis was that there was no difference between the two treatments concerning the effectiveness to arrest occlusal caries progression.
Material and methods Study design This split mouth study received ethical approval from the Regional Ethical Review Board, Umea University, §563/03, dnr 03-486 and dnr 07-189 M. Preschool children were recruited from public dental health clinics in Umea and Nordmaling, County of Västerbotten, Sweden. All children attending their dental clinics for regular check-ups and meeting the inclusion criteria, occlusal caries in one or two bilateral matched pairs of first and/or second primary molars, were invited to take part in the study. Sample collection was performed between December 2003 and May 2008. Exclusion criteria were health problems such as severe asthma, failure to cooperate and teeth with obvious signs of enamel hypomineralisation or hypoplasia in the fissure. Information on the purpose and procedures of the study was given to the parents or guardians and written informed consent was obtained before starting the treatments. The power calculation was based on a clinical difference of 10 % dentin caries (VI
score 4) between the treatment groups. To detect a significant difference between the two treatments with a two-sided significance level of 0.05 and a power of 80 %, it was estimated that 40 bilaterally matched pairs of first and/or second primary molars were needed. The study was conducted in two parts. A total of 50 pairs of primary molars with caries lesions were included. In the first part of the study, children with at least two occlusal caries lesions (one pair) with Ekstrand ≤3 scores (VI ≤3), according to visual inspection criteria described by Ekstrand et al. (Table 1) [26], were included. Four boys and seven girls (mean age 4.8 year, range 3–6 years) took part in the study. Four children had two pairs and seven children one pair. Selection of children in the VI ≤3 group was discontinued for ethical reasons during the course of the study due to the high number of non-acceptable clinical results. All failed lesions during the 1 year follow-up were cavitated ones (see Results). In order to further evaluate the effectiveness of ozone and fluoride varnish, the threshold for inclusion of the pair’s caries lesions was therefore decreased to non-cavitated lesions only. A continued selection of children with bilateral occlusal caries lesions with Ekstrand score ≤2a, (VI ≤2a) was performed. In this VI ≤2a group, 35 pairs of primary molars were evaluated in 14 boys and 8 girls (mean age 4.5 years, range 4– 8 years), resulting in a power of 75 %. Fourteen children had 2 pairs and 7 children had 1 pair. Test (ozone) and positive control treatment (fluoride varnish) Ozone gas, generated by the device HealOzoneTM (KaVo, Biberach, Germany) was used and a silicon cup was applied over the occlusal surface of the fissure with the lesion to be treated. The device produces ozone at a fixed concentration of 2,100 ppm and at a flow rate of 615 cm3 min−1 as confirmed by the manufacturer’s service protocol before the start of the study. When an intact seal around the lesion was obtained with the silicone cup, ozone was delivered under vacuum for 40 s. Table 1 Criteria used at the visual inspection (VI), according to Ekstrand et al. [26] Score Criteria 0 1 1a 2 2a 3 4
No or slight change in enamel translucency after prolonged air drying (>5 s) Opacity (white) hardly visible on the wet surface, but distinctly visible after air drying Opacity (brown) hardly visible on the wet surface, but distinctly visible after air drying Opacity (white) distinctly visible without air drying Opacity (brown) distinctly visible without air drying Localised enamel breakdown in opaque or discoloured enamel and/or greyish discolouration from the underlying dentine Cavitation in opaque or discoloured enamel exposing the dentine beneath
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The occlusal surface of the positive control tooth was air dried for 5 s and topical fluoride varnish Duraphat® (Colgate Oral Pharmaceuticals, New York, NY, USA), 22,600 ppm NaF, was applied in a thin layer with a micro-brush. After the applications, the children were asked to refrain from eating and drinking for the next hour and to avoid brushing their teeth until the next morning. In order to evaluate the effectiveness of ozone only, neither additional fluoridated or antimicrobial mouth rinses or medications, nor the additional remineralisation solution (containing xylitol, fluoride, calcium, phosphate and zinc) as suggested by the manufacturer, were used during the study period [24]. The parents and/or the guardians were recommended to continue helping to brush their children’s teeth twice daily with fluoride toothpaste (in Sweden, commonly a concentration of 1,000 ppm) during the study period. Evaluation At baseline, anamnestic data were noted. Caries risk of the children, on a three-grade scale (low, medium or high caries risk), was assessed by the general dentists at the Public Dental Health Service clinics by means of clinical and sociodemographic information routinely available at the clinical examinations, e.g. incipient caries lesions and former caries histories. The caries lesions were detected, scored and measured by VI and LF. Before the assessments, the lesions were cleaned with a saline-wetted rotating brush. VI was scored with the visual ranked caries scoring system according to Ekstrand et al. [26]. The LF measurements were carried out with the chair side laser device DIAGNOdentTM (KaVo DIAGNOdent 2095, KaVo, Biberach, Germany), which allows reading values from 0 to 99 [27]. The same device and A-tip were used throughout the study. Before every treatment session, the instrument was calibrated using a ceramic standard provided by the manufacturer, and measurements were performed after the lesion was air dried for 5 s using an air syringe. At each measurement, the standard value was first calibrated by measuring a sound enamel reference point. When measuring, the tip was slightly tilted and rotated along its own axis to record the peak value of the area according to the manufacturer’s instructions. The selected measurement points in the lesions, including the sound reference point, were noted graphically in a protocol to be able to repeat the calibrations and measurements during the study, both for the VI scoring and LF readings. The included lesions were then randomly assigned within each pair to test (ozone) or positive control (fluoride varnish) treatment by throwing a die. An odd number meant that the lesion assigned to treatment with ozone was to be on the right side of the jaw and the lesion with fluoride varnish treatment on the left, and an even number the reverse. If two pairs of molars with lesions were present, the second test lesion was always on the same side as the first.
Two operators were trained prior to the study start in VI scoring and LF measurements with in vitro exercises until good agreement was achieved. VI scoring, LF measurements, ozone and fluoride varnish applications were conducted at baseline, 3, 6 and 9 months or until failure, i.e. VI score 4 (Table 1), indicating necessity of an operative treatment. At 12 months, only VI scoring and LF measurements were performed. At each visit, assignment and previous registrations were concealed until the time of treatment. All baseline VI scoring and LF measurements were performed by both investigators, while further assessments in the VI ≤3 group were performed by operator IAW and in the VI ≤2a group by operator EJ. Statistical methods The data were analysed using the Statistical Package for the Social Sciences, SPSS15 and PASW 18, (SPSS Inc., Chicago, Il, USA). Descriptive statistics were used to describe the study groups concerning background data, VI and LF values. Intraoperator agreement was assessed in the in vitro exercises and inter-operator agreement was assessed at the baseline visit for VI and LF using Spearman’s rank order correlation tests. A Bland Altman plot was used to identify systemic differences of LF values. For paired readings (inter-examiner), the difference between the measurements was plotted against their means (mean difference + 1.96 SD). Wilcoxon signed-rank test was used to analyse differences between the study groups at baseline and, in the VI ≤2a group, changes from baseline to 12 months in LF values. The level of statistical significance was set at p 0.05, Table 3). During the evaluation period, eight lesions treated with ozone and nine treated with fluoride varnish progressed to failure, i.e. VI score 4 (Table 2). All of the lesions in the VI ≤3 group with VI score 3 at baseline failed during the 1-year period. Two children were assessed as medium caries risk and nine children were assessed as high caries risk. In the high-risk children, only one lesion pair remained during the study time. Since only five pairs remained at the Table 2 Study flow in the VI ≤3 and VI ≤2a groups, n =number of tooth pairs, Failure=score 4 according to VI criteria by Ekstrand et al. [26] operative treatment necessary. Dropout or withdrawn was due to lack of cooperation, relocation, other tooth in pair failed or repair of the proximal surface of the tooth
a
Two children with a total of three pairs failed to show up at 9 months, but were assessed at 12 months
Time: months
VI ≤3 group 0 (n =15) 3 (n =15) 6 (n =11) 9 (n =4a) 12 (n =5) Total VI ≤2a group 0 (n =35) 3 (n =33) 6 (n =33) 9 (n =33) 12 (n =32) Total
VI scores
LF values
O
F
O
F
VI ≤3 group 0 (n =15) 3 (n =15) 6 (n =11) 9 (n =4) 12 (n =5)
2a (1–3) 2a (1a–4) 2a (2–4) 2a (2a–4) 3 (2–4)
2a (1–3) 2 (1a–4) 3 (1a–4) 3 (1a–4) 2a (2–4)
76 (0–99) 63 (18–99) 50 (32–99) 51 (44–99) 57 (33–90)
69 (0–99) 60 (20–99) 75 (16–99) 66 (41–99) 58 (20–78)
VI ≤2a group 0 (n =35) 3 (n =33) 6 (n =33) 9 (n =33) 12 (n =32)
2a (1–2a) 2a (1–2a) 2a (1–2a) 2a (1–3) 2a (1–4)
2a (1–2a) 2a (1–2a) 2a (1–2a) 2a (1–3) 2a (1–3)
21 (2–68) 20 (2–54) 15 (4–70) 15 (2–98) 15 (4–99)
19 (2–69) 17 (2–69) 15 (2–40) 14 (2–96) 18 (5–99)
O treatment with ozone, F treatment with fluoride varnish
12 months follow up, no further statistical analyses were performed. At baseline, no VI score higher than 2a were registered in the VI ≤2a group. The median (min–max) LF values were 21 (2–66) and 19 (2–69) for lesions to be treated with ozone and fluoride, respectively. The baseline values of VI and LF did not differ significantly (p >0.05, Table 3). After 12 months, a tendency towards increased VI scores was recorded both for lesions treated with ozone and fluoride (Fig. 2). One of the lesions treated with ozone failed at 12 months, reaching VI score 4. At 12 months, median (min–max) LF values for
Ozone
Fluoride varnish
Failure
Drop out/withdrawn
Failure
Drop out/withdrawn
3 2 1 2 8
1 2 1 0 4
3 3 2 1 9
1 1 0 0 2
0 0 0 1 1
2 0 1 0 3
0 0 0 0 0
2 0 1 0 3
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Fig. 2 Distribution of visual inspection (VI) scores at baseline and at 12 months after treatment every third month with ozone or fluoride. Score criteria see Table 1. Count number of lesions with respective scores
ozone and fluoride varnish-treated lesions were 15 (4–99) and 17.5 (2–85), respectively. No significant improvement or difference in LF values was found over time between the lesions treated with ozone or fluoride (p >0.05). Sixteen out of 22 children were assessed as low caries risk and 6 as medium. The failed lesion was found in a child with low caries risk.
Discussion Finding alternative non-invasive treatment strategies to stop and reverse caries lesion progression is desirable in order to avoid operative therapy. In this study, we investigated the effect of repeated 40 s ozone applications to arrest occlusal caries lesions in primary molars. In the split-mouth model, Duraphat® applications were used as a positive control. In earlier studies of the effect of ozone on occlusal caries lesions, the intervention group’s ozone (10 s) plus reductant group and reductant group only were compared [28–30]. In order to evaluate the effect of ozone application alone, the remineralisation solution (reductant and patient kit) recommended by the manufacturer, containing high concentrations of fluoride, was omitted as also suggested in a recent systematic review of the effectiveness of HealOzone for the treatment of caries [30]. Duggal et al. concluded recently that ozone had no additional effect on the inhibition of enamel and dentin demineralisation compared to when the reductant/patient kits were used on their own, under a cariogenic challenge in situ [31]. Neither ozone nor Duraphat® treatments could prevent
progression of cavitated lesions in the VI ≤3 group during the 1-year follow-up. The selection of cavitated caries lesions was therefore discontinued for ethical reasons and the ozone effectiveness evaluation was continued with VI ≤2a lesions only. In this non-cavitated lesions group, a tendency towards increased VI scores was recorded during the follow up. There were no significant differences observed between the treatment groups and the null hypothesis was therefore accepted. Ozone in the gaseous or aqueous phase has been shown to be a powerful antimicrobial agent [32]. In vitro, ozone showed an antibacterial effect on cariogenic microorganisms. However, the presence of saliva hampered the bacterial killing [33]. In vivo, the ozone application on the exposing dental surfaces will encounter an environment of both saliva and biofilm. Baysan and Beighton [18] found no antibacterial effect of ozone treatment when they studied its effect on the dentinal microflora of non-cavitated occlusal lesions. Freshly extracted molar teeth with occlusal surface LF values of between 11 and 30 and Ekstrand score of 3 were exposed for 40 s. No significant difference was observed in total amount of bacteria recovered from dentine of the ozone-treated and non-treated groups. The results are contradictory compared with reported effect of ozone treatments on bacteria in root caries lesions [18]. A long lasting antibacterial effect of ozone on the dental surface is limited because of its properties and no long-term antibacterial effect of ozone has been demonstrated. To improve the results, an increase of the number of applications per time period and/or longer exposure periods may be discussed, but from a cost-effectiveness view, this seems unrealistic to suggest for a general practise [30]. In our study, the more superficial caries lesions selected in the second part of the study and the lower caries risk of the children in the VI ≤2a group may explain the difference in outcome compared with the VI ≤3 group rather than a lasting antibacterial effect of the treatments. The present study was carried out as split-mouth design, which makes it possible to decide if one treatment is more effective than another, if carry-across effects are under control [34]. This design minimises the subject effect, for instance differences in eating habits, salivary factors and oral hygiene including use of fluoridated toothpaste and possible changes of these factors during the study period [34]. Biased treatment efficacy is one of the risks with this study design and might occur when the active substance of an intervention can carry over to the other side of the mouth. In order to minimise this potential risk, only two types of treatments were evaluated in this study, and bilateral lesions in the fissures of the primary molars were used. The ozone treatment is a local application regime that covers the lesion to be treated by using a silicon cup, and after treatment, no further ozone is applied until next visit. In order to contribute fluoride and at the same time minimise potential carry across effects, the varnish was only applied on the elected positive control lesion [35, 36].
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Caries diagnosis is a complicated process and combining two methods is supposed to improve the accuracy [10, 27]. This was the incitement of using both VI and LF to detect and indirectly monitor changes. The visual ranked caries scoring system with six stages was used to enable detection and registration of small changes in caries progression. Scores 1 up to 2a represent enamel lesion without tissue breakdown, whereas score 3 and 4 indicates presence of cavity formation. In this study, score 4 was chosen as outcome variable, i.e. failure and necessary operative treatment. Ekstrand et al. [26] investigated the ability of the VI system to detect and assess the depth of caries lesions and found a strong relationship between visual, electronic caries monitoring and radiographic assessments. The use of LF has been suggested to be a useful adjunct to conventional methods for the detection of occlusal caries. Anttonen et al. showed increased LF values with the increase of visual score [27]. The authors used a modified version of VI criteria with only three steps on the scale, compared to the six steps used in this study. They found that the higher the visual score, the higher the mean LF values, but in each visual category, large variations in LF values were found. Three studies evaluating ozone treatment of caries lesions used LF readings in order to indirectly monitor caries arrest, reversal or progression [23–25]. In a study by Dähnhardt et al. [24], soft dentin was excavated in open single surface lesions in primary teeth. The test teeth were then treated with ozone for 20 s every second month, while the controls were left untreated. After 8 months, they found significantly increased hardness values and nonsignificantly lower LF values for the ozone-treated teeth compared with the control. They concluded ozone treatment to be suitable for uncooperative and anxious children [24]. Kronenberg et al. [25] concluded that LF readings were not appropriate to use for caries measurements contiguous orthodontic brackets. In our study, the readings of LF showed a large variation both within each visual category at baseline and for the individual lesions at the recall visits. The LF method has shown good validity and reliability in vitro [37]. Contradictory results have been observed in vivo in both dentitions and a recent published systematic review of caries detection methods confirmed the limited evidence for LF method in vivo for both primary and permanent teeth [38, 39]. The body of evidence for the LF method was from the beginning based primarily on in vitro studies, while extrapolation to clinical setting still remains uncertain. The relative poor performance and large variations by LF under clinical conditions, both in our study and other clinical trials, may be attributed to several factors. The presence of saliva, plaque and stain and limited access to test site influence the quality of the measurements. The LF method is based on bacterial metabolites and measures changes in organic material in comparison to tooth mineral content. The method may therefore be less appropriate for in vivo monitoring of small changes in enamel and more useful for detection of dentinal caries with high bacterial metabolites. In the VI ≤3
group lesions in general, high LF readings were observed with median baseline values for ozone and fluoride of 76 and 69, respectively. No difference was observed between the ozone and fluoride treatment in the ability to stop progression of the lesions in the primary molars during the follow-up period. The lesions progressed to dentin despite treatment every third month. The median baseline LF values in the VI ≤2a group were for ozone 21 and for fluoride 19, far lower than for the VI ≤3 group lesions. Slightly increased VI scores were seen from baseline to 12 months follow up, i.e. low detectable progression of the lesions. The median LF readings, for both ozone and fluoride, showed a non-statistically significant decrease from baseline to 12 months. Only one caries lesion needed operative treatment. A low caries progression was observed in the non-cavitated lesions group which contained a high frequency of low caries risk children. It has been shown that in low caries risk adults using fluoridated dentifrices, proximal caries lesions showed a slow progression [40]. All children in the present study used fluoridated toothpaste and it may be possible that there was no or only a low additional effect of the treatments evaluated. Inclusion of a negative control tooth without additional treatment could have given an answer, but due to ethical reasons, it was no option to leave caries lesions untreated. As earlier mentioned, Duggal et al. showed recently that ozone had no additional effect in combination with adjunct remineralisation products on inhibition of demineralisation of the dental hard tissues in situ [31]. In another split mouth study of treatment of non-cavitated occlusal caries in newly erupted permanent molars, the effect of one ozone treatment for 40 s was compared with untreated contra-lateral control lesions [23]. After 3 months, they found that the ozone-treated lesions in the high risk caries group showed significantly more caries reversal or reduced progression than the control lesions. These changes were based on LF difference values from baseline to 3 months with start values no higher than 30. These results were not confirmed in our study, where we found large variations in LF values irrespective of LF start values and estimated caries risk. The use of ozone in the perspective of primary prevention was evaluated by Kronenberg et al. [25]. Its caries protective effect on white spot formation was studied in high-risk patients during multibracket therapy. Ozone treatment for 30 s was compared with Cervitec/Fluor Protector® application and assessments and treatments were performed every third month. After 26 months, the fluoride varnish application therapy was significantly superior to ozone treatment. They reported that ozone application was well accepted by children as a quick, taste free and easy treatment [24]. In conclusion, despite ozone treatment every third month, cavitated lesions failed during the 1 year follow up as did the local fluoride varnish treated lesions. In the VI ≤2a group with
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non-cavitated lesions only, a tendency to caries progression was observed for both treatments despite the lower caries risk of the children. The use of ozone or fluoride varnish treatments in this regime as caries preventive method, added to the daily use of fluoridated toothpaste, to arrest caries progression in primary molars must therefore be questioned.
Acknowledgments We acknowledge all children for their participation, personnel at the Public Dental Health clinics in Umea, Nordmaling and the Department of Odontology/Paediatric dentistry in Umea. We are grateful for statistical analyses by Marie Eriksson at the Department of Statistics, Umea University. The Healozone® (KaVo, Biberach, Germany) equipment was provided by KaVo. Conflict of interest The authors declare that they have no conflict of interest.
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39. Twetman S, Axelsson S, Dahlén G, Espelid I, Mejáre I, Norlund A, Traeneus S (2013) Adjunct methods for caries detection: a systematic review of literature. Acta Odontol Scand 71:388–397 40. Mejàre I, Stenlund H, Zelezny-Holmlund C (2004) Caries incidence and lesion progression from adolescence to young adulthood: a prospective 15-year cohort study in Sweden. Caries Res 38:130–141
ORIGINAL ARTICLE
Treatment effect of ozone and fluoride varnish application on occlusal caries in primary molars: a 12-month study E. Johansson & J. W. V. van Dijken & L. Karlsson & I. Andersson-Wenckert
Received: 30 August 2013 / Accepted: 26 November 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Aim The aim of this study is to evaluate the effect of ozone and fluoride varnish on occlusal caries in primary molars in a split-mouth study. Materials and methods Caries risk was estimated by treating Public Dental Health Service dentists. Children with occlusal caries with Ekstrand index scores ≤3 (VI ≤3) were included. Selection of caries lesions was discontinued for ethical reasons due to non-acceptable clinical results during the followup. In the continued evaluation pairs of teeth with noncavitated caries lesions, Ekstrand score ≤2a (VI ≤2) were selected. Fifty pairs of carious primary molars were included, 18 boys and 15 girls (mean 4.7 years, range 3–8). At baseline, the lesions were assessed by visual inspection (VI) and laserinduced fluorescence (LF), in each pair to treatment with 40 s ozone (HealOzone TM , 2,100 ppm) or fluoride varnish Duraphat®. The treatments and evaluations were repeated at 3, 6 9 months and evaluations only at 12 months. Results Medium-high caries risk was observed in VI ≤3 children and low-medium risk in VI ≤2a children. In the 15 pairs VI ≤3 lesions, 8 treated with ozone and 9 with fluoride progressed to failure. In the 35 pairs VI ≤2a lesions, one lesion failed. Median baseline LF values in the VI ≤3 group were 76 and 69, for ozone and fluoride lesions, respectively, and 21 and 19 in the VI ≤2a group. At 12 months, LF values in the VI E. Johansson : J. W. V. van Dijken : I. Andersson-Wenckert Department of Odontology, Faculty of Medicine, Umea University, Umea, Sweden L. Karlsson Division of Cariology, Department of Dental Medicine, Karolinska Institutet, Stockholm, Sweden E. Johansson (*) Dental Hygienist Education, Department of Odontology, Dental School, Umea University, SE-901 87 Umea, Sweden e-mail: [email protected]
≤2a group were 15 and 18. No improvement or difference in LF values was found over time between the caries lesions treated with ozone or fluoride. Conclusions Neither ozone nor fluoride varnish treatments stopped the progression of caries in cavitated lesions. In low and medium caries risk children, non-cavitated lesions following both treatments showed slight or no progression. The use of ozone or fluoride varnish treatments in this regime as caries preventive method, added to the daily use of fluoridated toothpaste, to arrest caries progression in primary molars must therefore be questioned. Keywords Caries . Clinical . Fluoride . Ozone
Introduction Despite the dramatic decline in dental caries over the past few decades, particularly in industrialised countries, the disease persists, however, with a skewed distribution [1–3]. The polarisation of the caries disease in child and youth populations has been confirmed in several studies [2, 4]. Early identification of children with caries activity as a means of preventing and arresting the disease has been shown to be more efficacious than restorative treatments later in life [5, 6]. In Sweden, a decline of caries prevalence in 4 year olds has been observed since 1967 [7–9]. The main decline occurred from 1967 to 1980, after which a levelling out was seen. In 2007, still 38 % of the 4-year olds had cavitated and noncavitated caries lesions, in spite of a further significant decrease in the prevalence and distribution of the dmfs values between 2002 and 2007 [9]. Today, fluoride regimes and fissure sealants are commonly used treatments of early caries lesions in children [10, 11]. Fluoride varnish application aims to prevent tissue loss and to reverse or arrest lesions and thereby avoid restorations [12,
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13]. However, a recent review failed to present sufficient evidence for most preventive measures and the need for new approaches and further well-designed clinical trials was emphasised [11]. During the last decade, ozone was suggested as a new caries preventive method [14–16]. Ozone is an unstable gas containing three oxygen atoms and is a powerful oxidant and highly potent antimicrobial agent [17]. Ozone applications have been shown to reduce caries-associated bacteria in primary root caries lesions in vitro [14, 15] and in vivo [16]. However, conflicting results regarding the effect of ozone on bacteria have been obtained in the biofilm and in caries lesions [18–20]. There are only a few longitudinal studies published investigating the effect of ozone treatment in non-invasive management of early caries lesions. Two studies on root caries lesions, with a follow-up time of 6 and 18 months, respectively, showed root lesions to be arrested non-operatively with ozone [21, 22]. Three studies on occlusal caries in children and adolescents with a follow up time of 3, 8 and 26 months, respectively, showed conflicting results regarding the ability of ozone to arrest or prevent caries [23–25]. Only one of these studies evaluated ozone treatment in the primary dentition [24]. The aim of this study was to evaluate in an intra-individual comparison the effect of ozone and fluoride varnish treatments on occlusal caries in primary molars during 1 year, monitored with visual inspection (VI) and with laser-induced fluorescence (LF). The null hypothesis was that there was no difference between the two treatments concerning the effectiveness to arrest occlusal caries progression.
Material and methods Study design This split mouth study received ethical approval from the Regional Ethical Review Board, Umea University, §563/03, dnr 03-486 and dnr 07-189 M. Preschool children were recruited from public dental health clinics in Umea and Nordmaling, County of Västerbotten, Sweden. All children attending their dental clinics for regular check-ups and meeting the inclusion criteria, occlusal caries in one or two bilateral matched pairs of first and/or second primary molars, were invited to take part in the study. Sample collection was performed between December 2003 and May 2008. Exclusion criteria were health problems such as severe asthma, failure to cooperate and teeth with obvious signs of enamel hypomineralisation or hypoplasia in the fissure. Information on the purpose and procedures of the study was given to the parents or guardians and written informed consent was obtained before starting the treatments. The power calculation was based on a clinical difference of 10 % dentin caries (VI
score 4) between the treatment groups. To detect a significant difference between the two treatments with a two-sided significance level of 0.05 and a power of 80 %, it was estimated that 40 bilaterally matched pairs of first and/or second primary molars were needed. The study was conducted in two parts. A total of 50 pairs of primary molars with caries lesions were included. In the first part of the study, children with at least two occlusal caries lesions (one pair) with Ekstrand ≤3 scores (VI ≤3), according to visual inspection criteria described by Ekstrand et al. (Table 1) [26], were included. Four boys and seven girls (mean age 4.8 year, range 3–6 years) took part in the study. Four children had two pairs and seven children one pair. Selection of children in the VI ≤3 group was discontinued for ethical reasons during the course of the study due to the high number of non-acceptable clinical results. All failed lesions during the 1 year follow-up were cavitated ones (see Results). In order to further evaluate the effectiveness of ozone and fluoride varnish, the threshold for inclusion of the pair’s caries lesions was therefore decreased to non-cavitated lesions only. A continued selection of children with bilateral occlusal caries lesions with Ekstrand score ≤2a, (VI ≤2a) was performed. In this VI ≤2a group, 35 pairs of primary molars were evaluated in 14 boys and 8 girls (mean age 4.5 years, range 4– 8 years), resulting in a power of 75 %. Fourteen children had 2 pairs and 7 children had 1 pair. Test (ozone) and positive control treatment (fluoride varnish) Ozone gas, generated by the device HealOzoneTM (KaVo, Biberach, Germany) was used and a silicon cup was applied over the occlusal surface of the fissure with the lesion to be treated. The device produces ozone at a fixed concentration of 2,100 ppm and at a flow rate of 615 cm3 min−1 as confirmed by the manufacturer’s service protocol before the start of the study. When an intact seal around the lesion was obtained with the silicone cup, ozone was delivered under vacuum for 40 s. Table 1 Criteria used at the visual inspection (VI), according to Ekstrand et al. [26] Score Criteria 0 1 1a 2 2a 3 4
No or slight change in enamel translucency after prolonged air drying (>5 s) Opacity (white) hardly visible on the wet surface, but distinctly visible after air drying Opacity (brown) hardly visible on the wet surface, but distinctly visible after air drying Opacity (white) distinctly visible without air drying Opacity (brown) distinctly visible without air drying Localised enamel breakdown in opaque or discoloured enamel and/or greyish discolouration from the underlying dentine Cavitation in opaque or discoloured enamel exposing the dentine beneath
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The occlusal surface of the positive control tooth was air dried for 5 s and topical fluoride varnish Duraphat® (Colgate Oral Pharmaceuticals, New York, NY, USA), 22,600 ppm NaF, was applied in a thin layer with a micro-brush. After the applications, the children were asked to refrain from eating and drinking for the next hour and to avoid brushing their teeth until the next morning. In order to evaluate the effectiveness of ozone only, neither additional fluoridated or antimicrobial mouth rinses or medications, nor the additional remineralisation solution (containing xylitol, fluoride, calcium, phosphate and zinc) as suggested by the manufacturer, were used during the study period [24]. The parents and/or the guardians were recommended to continue helping to brush their children’s teeth twice daily with fluoride toothpaste (in Sweden, commonly a concentration of 1,000 ppm) during the study period. Evaluation At baseline, anamnestic data were noted. Caries risk of the children, on a three-grade scale (low, medium or high caries risk), was assessed by the general dentists at the Public Dental Health Service clinics by means of clinical and sociodemographic information routinely available at the clinical examinations, e.g. incipient caries lesions and former caries histories. The caries lesions were detected, scored and measured by VI and LF. Before the assessments, the lesions were cleaned with a saline-wetted rotating brush. VI was scored with the visual ranked caries scoring system according to Ekstrand et al. [26]. The LF measurements were carried out with the chair side laser device DIAGNOdentTM (KaVo DIAGNOdent 2095, KaVo, Biberach, Germany), which allows reading values from 0 to 99 [27]. The same device and A-tip were used throughout the study. Before every treatment session, the instrument was calibrated using a ceramic standard provided by the manufacturer, and measurements were performed after the lesion was air dried for 5 s using an air syringe. At each measurement, the standard value was first calibrated by measuring a sound enamel reference point. When measuring, the tip was slightly tilted and rotated along its own axis to record the peak value of the area according to the manufacturer’s instructions. The selected measurement points in the lesions, including the sound reference point, were noted graphically in a protocol to be able to repeat the calibrations and measurements during the study, both for the VI scoring and LF readings. The included lesions were then randomly assigned within each pair to test (ozone) or positive control (fluoride varnish) treatment by throwing a die. An odd number meant that the lesion assigned to treatment with ozone was to be on the right side of the jaw and the lesion with fluoride varnish treatment on the left, and an even number the reverse. If two pairs of molars with lesions were present, the second test lesion was always on the same side as the first.
Two operators were trained prior to the study start in VI scoring and LF measurements with in vitro exercises until good agreement was achieved. VI scoring, LF measurements, ozone and fluoride varnish applications were conducted at baseline, 3, 6 and 9 months or until failure, i.e. VI score 4 (Table 1), indicating necessity of an operative treatment. At 12 months, only VI scoring and LF measurements were performed. At each visit, assignment and previous registrations were concealed until the time of treatment. All baseline VI scoring and LF measurements were performed by both investigators, while further assessments in the VI ≤3 group were performed by operator IAW and in the VI ≤2a group by operator EJ. Statistical methods The data were analysed using the Statistical Package for the Social Sciences, SPSS15 and PASW 18, (SPSS Inc., Chicago, Il, USA). Descriptive statistics were used to describe the study groups concerning background data, VI and LF values. Intraoperator agreement was assessed in the in vitro exercises and inter-operator agreement was assessed at the baseline visit for VI and LF using Spearman’s rank order correlation tests. A Bland Altman plot was used to identify systemic differences of LF values. For paired readings (inter-examiner), the difference between the measurements was plotted against their means (mean difference + 1.96 SD). Wilcoxon signed-rank test was used to analyse differences between the study groups at baseline and, in the VI ≤2a group, changes from baseline to 12 months in LF values. The level of statistical significance was set at p 0.05, Table 3). During the evaluation period, eight lesions treated with ozone and nine treated with fluoride varnish progressed to failure, i.e. VI score 4 (Table 2). All of the lesions in the VI ≤3 group with VI score 3 at baseline failed during the 1-year period. Two children were assessed as medium caries risk and nine children were assessed as high caries risk. In the high-risk children, only one lesion pair remained during the study time. Since only five pairs remained at the Table 2 Study flow in the VI ≤3 and VI ≤2a groups, n =number of tooth pairs, Failure=score 4 according to VI criteria by Ekstrand et al. [26] operative treatment necessary. Dropout or withdrawn was due to lack of cooperation, relocation, other tooth in pair failed or repair of the proximal surface of the tooth
a
Two children with a total of three pairs failed to show up at 9 months, but were assessed at 12 months
Time: months
VI ≤3 group 0 (n =15) 3 (n =15) 6 (n =11) 9 (n =4a) 12 (n =5) Total VI ≤2a group 0 (n =35) 3 (n =33) 6 (n =33) 9 (n =33) 12 (n =32) Total
VI scores
LF values
O
F
O
F
VI ≤3 group 0 (n =15) 3 (n =15) 6 (n =11) 9 (n =4) 12 (n =5)
2a (1–3) 2a (1a–4) 2a (2–4) 2a (2a–4) 3 (2–4)
2a (1–3) 2 (1a–4) 3 (1a–4) 3 (1a–4) 2a (2–4)
76 (0–99) 63 (18–99) 50 (32–99) 51 (44–99) 57 (33–90)
69 (0–99) 60 (20–99) 75 (16–99) 66 (41–99) 58 (20–78)
VI ≤2a group 0 (n =35) 3 (n =33) 6 (n =33) 9 (n =33) 12 (n =32)
2a (1–2a) 2a (1–2a) 2a (1–2a) 2a (1–3) 2a (1–4)
2a (1–2a) 2a (1–2a) 2a (1–2a) 2a (1–3) 2a (1–3)
21 (2–68) 20 (2–54) 15 (4–70) 15 (2–98) 15 (4–99)
19 (2–69) 17 (2–69) 15 (2–40) 14 (2–96) 18 (5–99)
O treatment with ozone, F treatment with fluoride varnish
12 months follow up, no further statistical analyses were performed. At baseline, no VI score higher than 2a were registered in the VI ≤2a group. The median (min–max) LF values were 21 (2–66) and 19 (2–69) for lesions to be treated with ozone and fluoride, respectively. The baseline values of VI and LF did not differ significantly (p >0.05, Table 3). After 12 months, a tendency towards increased VI scores was recorded both for lesions treated with ozone and fluoride (Fig. 2). One of the lesions treated with ozone failed at 12 months, reaching VI score 4. At 12 months, median (min–max) LF values for
Ozone
Fluoride varnish
Failure
Drop out/withdrawn
Failure
Drop out/withdrawn
3 2 1 2 8
1 2 1 0 4
3 3 2 1 9
1 1 0 0 2
0 0 0 1 1
2 0 1 0 3
0 0 0 0 0
2 0 1 0 3
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Fig. 2 Distribution of visual inspection (VI) scores at baseline and at 12 months after treatment every third month with ozone or fluoride. Score criteria see Table 1. Count number of lesions with respective scores
ozone and fluoride varnish-treated lesions were 15 (4–99) and 17.5 (2–85), respectively. No significant improvement or difference in LF values was found over time between the lesions treated with ozone or fluoride (p >0.05). Sixteen out of 22 children were assessed as low caries risk and 6 as medium. The failed lesion was found in a child with low caries risk.
Discussion Finding alternative non-invasive treatment strategies to stop and reverse caries lesion progression is desirable in order to avoid operative therapy. In this study, we investigated the effect of repeated 40 s ozone applications to arrest occlusal caries lesions in primary molars. In the split-mouth model, Duraphat® applications were used as a positive control. In earlier studies of the effect of ozone on occlusal caries lesions, the intervention group’s ozone (10 s) plus reductant group and reductant group only were compared [28–30]. In order to evaluate the effect of ozone application alone, the remineralisation solution (reductant and patient kit) recommended by the manufacturer, containing high concentrations of fluoride, was omitted as also suggested in a recent systematic review of the effectiveness of HealOzone for the treatment of caries [30]. Duggal et al. concluded recently that ozone had no additional effect on the inhibition of enamel and dentin demineralisation compared to when the reductant/patient kits were used on their own, under a cariogenic challenge in situ [31]. Neither ozone nor Duraphat® treatments could prevent
progression of cavitated lesions in the VI ≤3 group during the 1-year follow-up. The selection of cavitated caries lesions was therefore discontinued for ethical reasons and the ozone effectiveness evaluation was continued with VI ≤2a lesions only. In this non-cavitated lesions group, a tendency towards increased VI scores was recorded during the follow up. There were no significant differences observed between the treatment groups and the null hypothesis was therefore accepted. Ozone in the gaseous or aqueous phase has been shown to be a powerful antimicrobial agent [32]. In vitro, ozone showed an antibacterial effect on cariogenic microorganisms. However, the presence of saliva hampered the bacterial killing [33]. In vivo, the ozone application on the exposing dental surfaces will encounter an environment of both saliva and biofilm. Baysan and Beighton [18] found no antibacterial effect of ozone treatment when they studied its effect on the dentinal microflora of non-cavitated occlusal lesions. Freshly extracted molar teeth with occlusal surface LF values of between 11 and 30 and Ekstrand score of 3 were exposed for 40 s. No significant difference was observed in total amount of bacteria recovered from dentine of the ozone-treated and non-treated groups. The results are contradictory compared with reported effect of ozone treatments on bacteria in root caries lesions [18]. A long lasting antibacterial effect of ozone on the dental surface is limited because of its properties and no long-term antibacterial effect of ozone has been demonstrated. To improve the results, an increase of the number of applications per time period and/or longer exposure periods may be discussed, but from a cost-effectiveness view, this seems unrealistic to suggest for a general practise [30]. In our study, the more superficial caries lesions selected in the second part of the study and the lower caries risk of the children in the VI ≤2a group may explain the difference in outcome compared with the VI ≤3 group rather than a lasting antibacterial effect of the treatments. The present study was carried out as split-mouth design, which makes it possible to decide if one treatment is more effective than another, if carry-across effects are under control [34]. This design minimises the subject effect, for instance differences in eating habits, salivary factors and oral hygiene including use of fluoridated toothpaste and possible changes of these factors during the study period [34]. Biased treatment efficacy is one of the risks with this study design and might occur when the active substance of an intervention can carry over to the other side of the mouth. In order to minimise this potential risk, only two types of treatments were evaluated in this study, and bilateral lesions in the fissures of the primary molars were used. The ozone treatment is a local application regime that covers the lesion to be treated by using a silicon cup, and after treatment, no further ozone is applied until next visit. In order to contribute fluoride and at the same time minimise potential carry across effects, the varnish was only applied on the elected positive control lesion [35, 36].
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Caries diagnosis is a complicated process and combining two methods is supposed to improve the accuracy [10, 27]. This was the incitement of using both VI and LF to detect and indirectly monitor changes. The visual ranked caries scoring system with six stages was used to enable detection and registration of small changes in caries progression. Scores 1 up to 2a represent enamel lesion without tissue breakdown, whereas score 3 and 4 indicates presence of cavity formation. In this study, score 4 was chosen as outcome variable, i.e. failure and necessary operative treatment. Ekstrand et al. [26] investigated the ability of the VI system to detect and assess the depth of caries lesions and found a strong relationship between visual, electronic caries monitoring and radiographic assessments. The use of LF has been suggested to be a useful adjunct to conventional methods for the detection of occlusal caries. Anttonen et al. showed increased LF values with the increase of visual score [27]. The authors used a modified version of VI criteria with only three steps on the scale, compared to the six steps used in this study. They found that the higher the visual score, the higher the mean LF values, but in each visual category, large variations in LF values were found. Three studies evaluating ozone treatment of caries lesions used LF readings in order to indirectly monitor caries arrest, reversal or progression [23–25]. In a study by Dähnhardt et al. [24], soft dentin was excavated in open single surface lesions in primary teeth. The test teeth were then treated with ozone for 20 s every second month, while the controls were left untreated. After 8 months, they found significantly increased hardness values and nonsignificantly lower LF values for the ozone-treated teeth compared with the control. They concluded ozone treatment to be suitable for uncooperative and anxious children [24]. Kronenberg et al. [25] concluded that LF readings were not appropriate to use for caries measurements contiguous orthodontic brackets. In our study, the readings of LF showed a large variation both within each visual category at baseline and for the individual lesions at the recall visits. The LF method has shown good validity and reliability in vitro [37]. Contradictory results have been observed in vivo in both dentitions and a recent published systematic review of caries detection methods confirmed the limited evidence for LF method in vivo for both primary and permanent teeth [38, 39]. The body of evidence for the LF method was from the beginning based primarily on in vitro studies, while extrapolation to clinical setting still remains uncertain. The relative poor performance and large variations by LF under clinical conditions, both in our study and other clinical trials, may be attributed to several factors. The presence of saliva, plaque and stain and limited access to test site influence the quality of the measurements. The LF method is based on bacterial metabolites and measures changes in organic material in comparison to tooth mineral content. The method may therefore be less appropriate for in vivo monitoring of small changes in enamel and more useful for detection of dentinal caries with high bacterial metabolites. In the VI ≤3
group lesions in general, high LF readings were observed with median baseline values for ozone and fluoride of 76 and 69, respectively. No difference was observed between the ozone and fluoride treatment in the ability to stop progression of the lesions in the primary molars during the follow-up period. The lesions progressed to dentin despite treatment every third month. The median baseline LF values in the VI ≤2a group were for ozone 21 and for fluoride 19, far lower than for the VI ≤3 group lesions. Slightly increased VI scores were seen from baseline to 12 months follow up, i.e. low detectable progression of the lesions. The median LF readings, for both ozone and fluoride, showed a non-statistically significant decrease from baseline to 12 months. Only one caries lesion needed operative treatment. A low caries progression was observed in the non-cavitated lesions group which contained a high frequency of low caries risk children. It has been shown that in low caries risk adults using fluoridated dentifrices, proximal caries lesions showed a slow progression [40]. All children in the present study used fluoridated toothpaste and it may be possible that there was no or only a low additional effect of the treatments evaluated. Inclusion of a negative control tooth without additional treatment could have given an answer, but due to ethical reasons, it was no option to leave caries lesions untreated. As earlier mentioned, Duggal et al. showed recently that ozone had no additional effect in combination with adjunct remineralisation products on inhibition of demineralisation of the dental hard tissues in situ [31]. In another split mouth study of treatment of non-cavitated occlusal caries in newly erupted permanent molars, the effect of one ozone treatment for 40 s was compared with untreated contra-lateral control lesions [23]. After 3 months, they found that the ozone-treated lesions in the high risk caries group showed significantly more caries reversal or reduced progression than the control lesions. These changes were based on LF difference values from baseline to 3 months with start values no higher than 30. These results were not confirmed in our study, where we found large variations in LF values irrespective of LF start values and estimated caries risk. The use of ozone in the perspective of primary prevention was evaluated by Kronenberg et al. [25]. Its caries protective effect on white spot formation was studied in high-risk patients during multibracket therapy. Ozone treatment for 30 s was compared with Cervitec/Fluor Protector® application and assessments and treatments were performed every third month. After 26 months, the fluoride varnish application therapy was significantly superior to ozone treatment. They reported that ozone application was well accepted by children as a quick, taste free and easy treatment [24]. In conclusion, despite ozone treatment every third month, cavitated lesions failed during the 1 year follow up as did the local fluoride varnish treated lesions. In the VI ≤2a group with
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non-cavitated lesions only, a tendency to caries progression was observed for both treatments despite the lower caries risk of the children. The use of ozone or fluoride varnish treatments in this regime as caries preventive method, added to the daily use of fluoridated toothpaste, to arrest caries progression in primary molars must therefore be questioned.
Acknowledgments We acknowledge all children for their participation, personnel at the Public Dental Health clinics in Umea, Nordmaling and the Department of Odontology/Paediatric dentistry in Umea. We are grateful for statistical analyses by Marie Eriksson at the Department of Statistics, Umea University. The Healozone® (KaVo, Biberach, Germany) equipment was provided by KaVo. Conflict of interest The authors declare that they have no conflict of interest.
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