A study to determine the effects of calcium based toothpastes in orthodontic patients

Aims To measure demineralisation/remineralisation of sub-surface caries-like lesions placed in situ on an orthodontic appliance and treated with Tooth Mousse™ (GC Corporation, Tokyo, Japan). Objectives To assess the degree of change in mineralisation of subsurface lesions following the application of Tooth Mousse™ with Transverse Microradiography (TMR) as volume mineral loss (ΔZ), lesion depth and lesion width. Null Hypothesis This study will test the null hypothesis that there is no difference between the remineralising abilities of Tooth Mousse™ used alongside normal fluoride toothpastes in orthodontic patients. Design This was a randomised cross-over in situ study. Ethical Approval Ethical approval was sought and obtained from the National Research Ethics service (NRES) and the NHS Research and Development Offices. This study was given the REC reference number: 10/H1005/85. Setting The Oral Health Research laboratories and The Orthodontic Clinic. Liverpool University Dental Hospital. Sample Size The sample size was calculated on the primary outcome of mineral loss (vol%μm). Data were used from a previous study (Benson 2009) using the Transverse Microradiography (TMR) technique, the residual standard deviation was estimated at 189 vol%μm . Four Latin square blocks, with a total of 12 participants, would allow detection of pairwise differences between groups of 300 vol%μm, with 90% power, at the 5% confidence level. To allow for possible dropout, a fifth block was added giving a total of 15 participants. Participant selection Inclusion criteria; a. Age 12 to 16 years. b. Had adequate space between the lower 2nd premolar region and lower 1st molar region in order to provide space to place the carrier on the archwire. c. Subjects were in good health. Exclusion criteria; a. The subjects were allergic to milk products. b. The subjects had taken antibiotics in the last 2 months. Randomisation Randomisation was generated in blocks of 3, from random number tables by a statistician who was not involved in the recruitment or allocation of the interventions. The method of randomisation was by envelope concealment. The patients were randomly allocated to one of three possible orders of intervention; A, B, C; B, C, A or C, A, B. Interventions Each participant received the three pastes in random order determined by a Latin square design in blocks of 3. A. A normal fluoride toothpaste (1450ppm). B. A normal fluoride toothpaste (1450ppm) and topically applied Tooth Mousse™ Plus toothpaste, which incorporates CPP-ACP and fluoride (900ppm) to the carrier only. C. A normal fluoride toothpaste and topically applied Tooth Mousse™ to be applied to the carrier only. Blinding Blinding of the participants was only possible with the Tooth Mousse™ and the Tooth Mousse™ Plus. The tubes were covered in insulation tape. The samples were recoded prior to being sectioned and analysed so the principal investigator was blind to the subject and intervention. Following TMR analysis the coding was revealed. Samples Following consent a previously prepared demineralised human premolar enamel sample that had been sterilised was placed in a carrier, which was then attached to the archwire. The carrier was a modified version of a carrier used in previous clinical studies (Benson 2000) and consisted of a stainless steel attachment. Base-line TMR analysis had been carried out to determine the degree of demineralisation of the enamel samples. Method Each participant was provided with the intervention and a toothbrush and was asked to brush their teeth for two minutes twice a day with a pea sized amount for a 4-week period. The participants were asked not to use any other toothpaste or mouth rinses during the 4-week period. For example; Treatment A paste is given to the subject 1 and instructed to use it for 4 weeks. At the four-week appointment a routine orthodontic appointment was carried out and the carrier with the enamel sample was removed from the mouth and taken to the laboratory for TMR analysis. Subject 1 was then instructed to continue with their normal oral hygiene procedures in the following 4 weeks. This was the washout period. Subject 1 then returned for another routine orthodontic appointment and insertion of a new enamel sample in a carrier. The subject was given treatment B paste to use for 4 weeks. This continued until the subject had used all the treatment pastes. The minimum length of time each participant was involved in the study was 20 weeks, including the washout periods. Outcome measures The main outcome measure was the difference in mineral loss/gain between the control specimens and the specimens used in the participants. Analysis of mineral content profiles, three parameters were obtained; mineral loss ΔZ (vol%μm) lesion depth Ld (μm) and lesion width Lw (μm). From these, the data were normalised and a percentage changes in mineral loss, lesion depth and lesion width were calculated. This was completed by dividing the sample value by the control value and multiplying by 100 (Strang et al 1987). Statistical analysis The data from TMR analyses were entered into the Statistical Package for Social Sciences (SPSS v. 20, Chicago, Illinois, USA) and tested for normality using frequency histograms. Hypothesis testing was carried out using a 3-way ANOVA. A Pearson correlation coefficient was calculated for the quantity of Tooth Mousse™ versus the percentage change in mineralisation. Results 1. TMR analysis revealed that there was no significant effect on the percentage change in mineral loss for the intervention (p=0.278), the order in which they received the intervention (p=0.625) or for each subject (p=0.66). There was no significant effect on the lesion depth for the intervention (p=0.184), the order (p=0.474) or the subject (p=0.957). There was also no significant effect on the lesion depth for the intervention (p=0.553), the order (p=0.582) or the subject (p=0.977). 2. There was large individual variability, with large ranges and no association between intervention and degree of mineral loss. 3. There was no correlation between the amount of mineral loss and the amount of Tooth Mousse™ or Tooth Mousse™ Plus used (r=0.144). 4. 22% of the samples were lost. Conclusion There were large intra-subject and inter-subject ranges with no association between intervention and degree of mineral loss or gain. There was no significant difference between the three interventions on mineral gain or loss. A large number of samples were lost to analysis and this could have had a large effect on the results. In this study the null hypothesis that there was no difference between the remineralising abilities of Tooth Mousse™ with normal fluoride toothpastes in orthodontic patients could not be rejected. Based on the results of this study, orthodontic patients are at risk of enamel demineralisation during treatment and attentiveness by the clinician should be maintained at all times. The aims of this study have been met, however, further improvements of the in situ method are required and a movement towards in vivo method is needed in order to advance our knowledge of the remineralising potential of this group of agents. Implications Following this study, future research should concentrate on developing the in situ model such that it is as representative as far as possible to the in vivo method. The work completed in this study should form the basis for subsequent investigation. This in situ model is as close a representation as possible to the in vivo model, however it cannot fully replicate the in vivo model, which could also have had an impact on the results. In order to assess the full remineralising efficacy of casein phosphopeptide, development of the in situ model is recommended. The other possibility is to move to an in vivo model, which would require a non-destructive method of measuring demineralisation such as QLF. A randomised controlled clinical trial is an ideal method to answer this question