Microcrystalline hydroxyapatite is not inferior to fluorides in clinical caries prevention: a randomized, double-blind, non-inferiority trial

Recent evidence for a significant interference of microcrystalline hydroxapatite (HAP) particles with re- and demineralisation processes at the tooth-biofilm interface suggested, that they may be promising candidates for efficacious caries prevention. This multicenter randomized controlled non-inferiority trial evaluated the impact of the 2 x daily use of a HAP dentifrice without fluoride on the progression of enamel caries in adolescent caries-risk patients subjected to orthodontic therapy, with a fluoridated AmF/SnF dentifrice serving as a positive control. Primary study outcome was the occurrence of enamel caries lesions ≥ ICDAS (International Caries Detection and Assessment System) code 1 around orthodontic brackets on the vestibular surfaces of teeth 15-25 within the 168 days observation period. Secondary study outcomes were the occurrence of enamel caries lesion ≥ ICDAS code 2, Plaque Index (PlI) and Gingival Index (GI). Out of 150 recruited patients, 147 were included in the intent to treat analysis (ITT); 133 finished the study per protocol (PP). PP data analysis revealed the occurrence of enamel caries ≥ ICDAS code 1 in 54.7% of the HAP group patients compared to 60.9% of the fluoride control. In the ITT analysis the corresponding numbers were 56.8% (HAP) and 61.6% (control). Non-inferiority testing of the ITT as well as the PP data set proved that the caries preventive efficacy of the HAP dentifrice was not inferior to the protection provided by the fluoridated AmF/SnF control. Regarding all assessed secondary outcomes (enamel caries ≥ ICDAS code 2, GI, PlI) no significant differences between both experimental groups were observed. Within the restraints set by design and study population of this trial microcrystalline HAP as ingredient of toothpaste may thus be regarded a promising supplement to fluorides in clinical caries prevention (ClinicalTrials.gov: NCT02705456).


Introduction 60
In recent years findings, mostly derived from in vitro studies, suggested, that micro-61 crystalline hydroxyapatite (HAP) particles may be promising candidates for the 62 prevention of cariogenic demineralization and the stimulation of remineralization 63 processes on enamel and dentine surfaces [1][2][3]. Huang et al. (2011) reported a 64 regain of mineral content and an increase in microhardness on demineralized bovine 65 enamel slabs that had subsequently been exposed to microcrystalline HAP particles 66 where enamel dissolution usually is initiated [4]. In an in situ -study the use of a zinc-71 carbonate HAP microcluster-containing mouth rinse significantly reduced bacterial 72 colonization on bovine enamel slabs worn intraorally by healthy volunteers [5]. 73 Hannig and Hannig (2010) put these in situ and in vitro findings into a more compre-74 hensive perspective by stating that already physiological tooth wear constantly 75 releases HAP particles into the oral environment, which may subsequently interfere 76 with de-and remineralisation processes as well as with the metabolism of the oral 77 microbiota at the tooth-bacterial biofilm interface [6]. The impact of microcrystalline 78 HAP as an ingredient of dentifrices has been positively evaluated in controlled clinical 79 trials regarding dentinal hypersensitivity [7-10] and parameters of periodontal health 80 [11]. Up to date, however, comparable data regarding the caries-preventive 81 properties of HAP toothpastes are mostly missing. They are limited to positive 82 findings from in situ studies on extracted teeth or standardized enamel and dentine 83 specimen, being subjected to different toothpaste slurries and worn in between by 84 the cleaning/disinfection procedures as well as the return/handing over of the 136 toothpaste supply performed as described before. At day 84 (visit 5) the recording of 137 GI, PlI and ICDAS II scores as well as cleaning and disinfection were repeated as 138 described before. Next to a new supply of toothpaste also a new Pulsar 35 electric 139 toothbrush was handed over. At day 112 (visit 6) and at day 140 (visit 7) procedures 140 performed were identical to those at day 56 (visit 4). At day 168 (visit 8) the final 141 assessment of PlI, GI and ICDAS II scores as well as the return of the study 142 dentifrices was conducted as described before. Furthermore, at each study visit 143 patients were asked about the occurrence of important harms or unintended effects 144 related or unrelated to the use of the study dentifrices.   premolars, canines and incisors with surface integrities ICDAS codes 0-3. 50% of 210 the pictures of a given sample were randomly presented in duplicates to evaluate the 211 ability of the examiners to reproduce their own assessments. 212 Interrater reliability analysis revealed a mean weighted kappa = 0.75 for the first 213 assessment run, which increased to kappa = 0.80 for the final calibration, indicating 214 "substantial agreement" among the different examiners throughout the study [25]. streptococci was extrapolated to be p=80% for the control group using the fluoridated 234 toothpaste. The difference between both experimental groups not be regarded 235 clinically relevant was set to Δ ≤ 20%. A sample size of 2 x 74 study patients was 236 calculated to be sufficient to reject the null hypothesis, that the test dentifrice is 237 inferior to the control dentifrice, using a non-inferiority margin of Δ = 20% for the 238 primary outcome measure and one-sided, exact Fisher Test (α = 5%, power = 80%). 239 240 Blinding, randomisation 241 The trial was designed to blind study patients and examiners to the group assign-242 ment. For this purpose, both study dentifrices (test/control) were filled into neutral 243 plastic tubes of identical shape and color by an independent, GMP certified 244 laboratory for cosmetics. Using block randomization with a block size of 4 a random 245 list was generated to code-label test and control tubes with consecutive unique 246 identification numbers. Randomization of dentifrice assignment was stratified by 247 study center. Handing out of the experimental dentifrices to the study patients 248 followed the sequence of the identification numbers and was performed by trained 249 study nurses not involved in the examination of the study participants. To maintain 250 blinding of examiners and study patients, the study patients were instructed not to 251 discuss toothpaste-related issues with the examiners but with the study nurses only, 252 who were also responsible for instructing the patients in efficacious oral hygiene and 253 taking back the empty or unused dentifrice tubes at the subsequent visits. The 254 number of study nurses varied between a minimum of one and a maximum of four 255 per study center. 256 257 Statistical analysis 258 The primary outcome measure was analysed primarily for the PP population and 259 repeated for sensitivity reasons, for the ITT population. The exact confidence limits 260 (Clopper-Pearson) were computed to test non-inferiority (cp. [28]). For the primary 261 outcome measure, non-inferiority was claimed, if the upper limit of the one-sided 95% 262 confidence for the corresponding difference between test and control dentifrice was 263 less than Δ ≤ 20%. 264 In addition, two-sided Wilcoxon-Mann-Whitney tests were used for between group Six patients of the test group and 4 patients of the control group terminated study 284 participation prematurely due to lack of interest or not keeping the follow-up appoint-285 ments. Further 6 patients completed the study but were excluded from the PP 286 analysis due to insufficient dosing of the assigned dentifrice, calculated from the 287 residual weight of the returned dentifrice tubes. All but one patient of the test group 288 and all patients of the control group received at least one dose of the assigned 289 dentifrice (n=149) and were thus primarily included in the ITT analysis set. As two 290 study patients left the trial already before the first reevaluation at week 4, the total 291 number of study individuals suitable for an inclusion in the ITT analysis of caries 292 development further decreased to n=147. No important harms or unintended effects 293 related or unrelated to the use of the study dentifrices were reported. Finally, a total 294 of 133 study patients (64 test / 69 control) was included in the PP analysis set (Fig 2).  Blinded change of the primary outcome 305 A blinded analysis of the ICDAS data at the end of the study revealed, that the 306 overall observed occurrence of ICDAS lesions ≥ code 2 in the study population was 307 29.3% and therefore considerably lower than the anticipated value (p = 80%) used 308 for the sample size calculation. As the difference between the groups not be 309 regarded clinically relevant had been set in the study protocol to ∆ ≤ 20% a clinically 310 meaningful verification of non-inferiority was no longer warranted. Thus, the primary 311 endpoint was changed to the more frequent overall occurrence of ICDAS lesions  Non-inferiority analysis 338 Table 2 displays the difference between both experimental groups regarding the 339 percentage of study subjects experiencing the new occurrence of at least one ICDAS 340 lesion ≥ code 1 (primary outcome) or at least one ICDAS lesion ≥ code 2 (secondary 341 outcome) including the corresponding one-sided 95% confidence intervals. As the 342 upper limits of the 95% confidence intervals for the primary outcome are well below 343 the given non-inferiority margin of ∆ ≤ 20% for both analysis sets (PP: 8%; ITT: 9%) 344 the HAP group has to be considered as non-inferior to the fluoride control. 345 Also regarding the secondary outcome (ICDAS lesion ≥ code 2) the upper limits of 346 the 95% confidence intervals are substantially below the given non-inferiority margin 347 of 20% for both analysis sets (PP: 3%, ITT: 7%), indicating that again the HAP test 348 group has to be considered being non-inferior to the fluoridated control.

Plaque Index (PlI), Gingival Index (GI)
390 The results of the ITT analysis of the PlI and the GI data are shown in Table 4. Mean 391 PlI as well as mean GI scores increased significantly (p < 0.0001) between baseline 392 and day 168 in both groups. Neither at baseline nor at day 168 differences between 393 the experimental groups were statistically significant.   age 12-16 subjected to orthodontic therapy with fixed appliances, the regular use of a 438 highly concentrated 5000 ppm fluoride dentifrice was accompanied by a significantly 439 lower incidence of white spot enamel lesions when compared to the regular use of a 440 standard 1450 ppm fluoride control dentifrice [30]. While this suggests, that 1450 441 ppm may not be the optimal fluoride concentration for a dentifrice to be used in unblinded PP data set however revealed, that the actual difference between both 477 experimental groups was 6.2% in favour of the HAP test dentifrice with an exact 478 upper one-sided 95% confidence limit of 8.3%, i.e. substantially lower than the preset 479 non-inferiority margin of Δ = 20%. 480

Secondary Outcomes 481
The data for the secondary outcomes Plaque Index (PlI) and Gingival Index (GI) 482 furthermore confirmed the findings of preceding studies, reporting a significant 483 increase of gingival inflammation and bacterial plaque mass after the onset of 484 orthodontic therapy with fixed appliances [18,29]. Differences between both 485 experimental groups regarding the recorded PlI and GI data could not be verified 486 statistically for any of the evaluated time points, which is also in good agreement with 487 the results of a previous trial comparing the plaque-and gingivitis-reducing properties 488 of a fluoride-free HAP test dentifrice and a fluoridated AmF/SnF control in a study 489 cohort of periodontitis patients [11].  Settings and locations where the data were collected 11 Interventions 5 The interventions for each group with sufficient details to allow replication, including how and when they were actually administered

5-6
Outcomes 6a Completely defined pre-specified primary and secondary outcome measures, including how and when they were assessed 8-9 6b Any changes to trial outcomes after the trial commenced, with reasons 12 Sample size 7a How sample size was determined 9 7b When applicable, explanation of any interim analyses and stopping guidelines -Randomisation: Sequence generation 8a Method used to generate the random allocation sequence 10 8b Type of randomisation; details of any restriction (such as blocking and block size) 10 Allocation concealment mechanism 9 Mechanism used to implement the random allocation sequence (such as sequentially numbered containers), describing any steps taken to conceal the sequence until interventions were assigned 10 Implementation 10 Who generated the random allocation sequence, who enrolled participants, and who assigned participants to interventions 10 Blinding 11a If done, who was blinded after assignment to interventions (for example, participants, care providers, those 10 assessing outcomes) and how 11b If relevant, description of the similarity of interventions 5-6 Statistical methods 12a Statistical methods used to compare groups for primary and secondary outcomes 10 12b Methods for additional analyses, such as subgroup analyses and adjusted analyses 10

Results
Participant flow (a diagram is strongly recommended) 13a For each group, the numbers of participants who were randomly assigned, received intended treatment, and were analysed for the primary outcome 11 13b For each group, losses and exclusions after randomisation, together with reasons 11 Recruitment 14a Dates defining the periods of recruitment and follow-up 11 14b Why the trial ended or was stopped -Baseline data 15 A Sources of funding and other support (such as supply of drugs), role of funders 7 *We strongly recommend reading this statement in conjunction with the CONSORT 2010 Explanation and Elaboration for important clarifications on all the items. If relevant, we also recommend reading CONSORT extensions for cluster randomised trials, non-inferiority and equivalence trials, non-pharmacological treatments, herbal interventions, and pragmatic trials.
Additional extensions are forthcoming: for those and for up to date references relevant to this checklist, see www.consort-statement.org.