Effect of self-assembling peptide P11-4 on orthodontic treatment-induced carious lesions.

This study aimed to evaluate the effect of self-assembling peptide P11-4 (SAP) in the therapy of initial smooth surface caries (white spot lesions, WSL) following orthodontic multibracket treatment. Twenty-three patients (13f/10m; average age 15.4 years) with at least two teeth with WSL were recruited for the randomised controlled clinical trial with split-mouth design. In opposite to the control teeth, the test teeth were treated with SAP on Day 0. The primary endpoint was the impedance measurement of WSL using customised tray to ensure reproducibility of the measurement location. The secondary endpoint was the morphometric measurement of WSL using a semi-automated approach to determine the WSL size in mm2. Treatment effects were adjusted for site-specific baseline values using mixed models adapted from the cross-over design. Test WSL showed a mean baseline impedance value of 46.7, which decreased to 21.1, 18.4, and 19.7 after 45, 90, and 180 days, respectively. Control WSL showed a mean baseline value of 42.0, which decreased to 35.0, 29.5, and 33.7, respectively. The overall treatment contrast was -13.7 (95% CI: -19.6 - -7.7; p < 0.001). For the secondary endpoint, the test WSL size decreased from 8.8 at baseline to 6.5 after 180 days. The control WSL decreased from 6.8 to 5.7, respectively. The related treatment contrast was -1.0 in favour of test WSL (95% CI: -1.6 - -0.5; p = 0.004). The treatment of initial carious lesions with self-assembling peptide P11-4 leads to superior remineralisation of the subsurface lesions compared with the control teeth.


Results
Baseline characteristics. The mean duration of the orthodontic treatment with fixed appliances was 27 months (min/max 13/39). Of the 23 recruited patients (13f 10 m; mean age 15.4 years), 21 could be analysed in mixed models (12f/9 m; mean age 15.3 years). One patient showed established dentin caries (primary endpoint = 100) on both teeth after 30 days and cavitated lesions after 90 days. The related four values of the primary endpoint after 90 and 180 days were set to 100. No further cavitated lesions were observed. The QHI at t0 was 2.2/2.2 (test/control teeth).

Impedance measurement of white spot lesion (primary endpoint). Both test and control teeth
exhibited a substantial decrease of impedance readings throughout the study period (Table 1). Control teeth showed a mean baseline value of 42.0 at day 0, which decreased to 35.0 at day 45,29.5 at day 90, and 33.7 at day 180. Test teeth showed a mean baseline value of 46.7 and exhibited a markedly larger decrease to 21.1 (day 45), 18.4 (day 90), and 19.7 (day 180).
The treatment effect was statistically significant (overall treatment contrast, which is the mean of the three single contrasts in Table 1: -13.7, 95% CI: −19.6 -−7.7; p < 0.001). More importantly, the global F test of no treatment effect was rejected (p = 0.003 for the test including one term for treatment and two terms for the interaction between treatment and time). After treatment, the difference between test and control tooth changed only slightly 2), respectively; for the "missing not at random" scenario by adding 50 to the treatment site of the three dropouts: −9.7 (95% CI: −18.9 -−0.5)).

Morphometric measurement of white spot lesion (secondary endpoint).
After treatment, the decrease in size according to semi-automated morphometric measurements was more pronounced in test teeth (p = 0.030 for the interaction between treatment and time; Fig. 2). After 180 days, the difference between test and control group was statistically significant in mixed model analysis although the 95% CI was also consistent with a weak to moderate effect ( Table 2). The global test of no treatment effect was rejected (p = 0.013).
Missing values were also dealt with by multiple imputation, including sensitivity analysis for missing data when true values are systematically lower or higher (nonignorable non-response). Both WSL of one patient were too small to be measured semi-automatically. The two cavitated lesions were not measurable after 90 and 180 days. These paired nonignorable non-responses are levelled off by the split-mouth design. Missing values, for which ignorable non-responses were assumed, occurred for a total of four subjects. One of the two patients who were excluded in mixed models for missing baseline values could be re-included in robust analysis, resulting in 15 patients available for complete case analysis. Robust analysis of contrasts between the first and third time points (-t 1 + t 3 to model the interaction) confirmed the result of the mixed model by using complete and imputed Figure 1. Impedance measurement of White Spot Lesion at different time points (black: test tooth/grey: control tooth). As "temporally and logically, a baseline cannot be a response to treatment, so baseline and response cannot be modeled in an integrated framework" 43 , baseline and response were graphed differently. Consequently, the response and the 95% CI are adjusted for baseline values 43 . p = 0.001, p = 0.005, and p = 0.007 for treatment differences after 45, 90, and 180 days, respectively.

Figure 2.
Morphometric measurement of White Spot Lesion Size in mm 2 at different time points (black: test tooth/grey: control tooth). As "temporally and logically, a baseline cannot be a response to treatment, so baseline and response cannot be modeled in an integrated framework" 43 , baseline and response were graphed differently. Consequently, the response and the 95% CI are adjusted for baseline values 43 . p = 0.969, p = 0.137, and p = 0.004 for treatment differences after 45, 90, and 180 days, respectively. www.nature.com/scientificreports www.nature.com/scientificreports/ data without dropouts (15 and 21 subjects, respectively). The treatment effect became uncertain if "ignorable non-response" did not hold in sensitivity analyses after multiple imputation and by including dropouts (23 subjects).

Discussion
Our trial raised comparable parameters as the previously reported trials [25][26][27]32 but differs from the other trials on buccal caries, as active carious lesions (clinically visible as rough, chalky and matte surface) were treated, and not predominantly inactive ones (clinically visible as a white spot under a hard, shiny surface) [25][26][27] . Both the impedance values and morphometric measurements showed favourable WSL reduction results for the test group.
The greater difference between test and control lesions, however, occurred in the impedance values compared with that in the morphometric measurements, thereby confirming that SAP P 11 -4 acts predominantly by regenerating the mineral structure of the enamel of the lesion body and not just the surface 23,24 . Moreover, at the last study visit at day 180, the impedance values for test lesions indicated regression of caries into very outer enamel, whereas the impedance values for the control lesions indicated still caries throughout the whole enamel layer 31,33 , which agrees with previous data that fluoride (in the present study as fluoride-based professional prophylaxis paste and home care toothpaste) only acts in the top 30-50 µm of the enamel surface 13,14,34 .
In recent years, caries activity has become a central topic within cariology, as activity defines the clinical need and opportunity to intervene in the decay process. Active carious lesions, as treated in the present study, have open pores allowing communication between the subsurface carious lesion body and the oral cavity. From the lesion body, calcium and phosphate ions can diffuse out (demineralisation) or in (remineralisation). In a similar fashion, the open pores allow SAP P 11 -4 to diffuse into the lesion body to support remineralisation and thus regression of the tooth decay.
The active carious lesions in the conventional treatment control exhibited spontaneous regression of the measured WSL size over the study period until day 180 3,8 , which is comparable to literature data 27,29 . However, SAP P 11 -4 enhanced the remineralisation to a higher decrease of the measured WSL size.
The present clinical trial is not without shortcomings. The possibility of random error because of the relatively small sample size. Second, the trial was run within a university setting and the patients were Caucasians, thereby limiting the generalizability of the findings. Third, information bias could have influenced our results. The morphometric assessment represents only a part of the clinical situation because it is related only to the visible tooth surface. Critical clinical assessment factors such as the hardness and gloss of the lesion could not be taken into account as they could not be measured reliably within an in vivo study. It would be interesting to measure the enamel hardness induced by SAP P 11 -4, and compare those to the previously reported in vitro microhardness results 34,35 .

Baseline vs
Day 45 Day 90 Day 180  www.nature.com/scientificreports www.nature.com/scientificreports/ from one site to another 39 . Note that the theory of causality 37 has been rapidly developed since the split-mouth design was criticized for statistical reasons in 1990 40 . Third, the split-mouth design was very efficient because the within-patient correlation was clearly different from zero (p = 0.61 for the primary endpoint, p = 0.72 for the secondary endpoint). For the primary and secondary endpoint, the sample sizes of n = 21 and n = 17 in our  www.nature.com/scientificreports www.nature.com/scientificreports/ split-mouth design correspond to total sample sizes of 108 and 118 patients in a parallel group design, respectively 39 . Fourth, three measurements of the primary endpoint were taken to increase reliability. Fifth, the duration of the trial worked for both endpoints. It was long enough to gain important insights into the mechanism of the treatment, especially for the secondary endpoint. Finally, the statistical analysis is state-of-the-art 39,41-43 , follows rigorously the intention-to-treat principle by accounting for the uncertainty due to dropouts, and supports confidence intervals 44 .
Overall, our results not only agree with other clinical trials on the effectiveness of SAP P 11 -4 20,25-27 but also show that SAP P 11 -4 is effective in the treatment of active WSL 26,27 supporting as already mentioned the proposed mechanism of action within the subsurface lesion body 23,24 . Thus, SAP P 11 -4 treatment gets one step closer to a regeneration ad integrum, that would be the ultimate goal in healing carious enamel.
One of the reasons that the treated enamel does not return to full translucency is seen in the fact that the SAP P 11 -4-induced fibres support the formation of de novo hydroxyapatite crystals in a fan-type non-prismatic arrangement around the fibres 24,45 , which have another refractive index than the prismatic hydroxyapatite crystals produced by the Tomes processes from ameloblasts in the final stage of enamel deposition.
Although buccal carious lesions chosen in order to quantitatively assess the effect of the treatment are rare outside the orthodontics, the clinical significance of the results relates to the treatment of caries in general. Thus, the aesthetic shortcomings of the SAP P 11 -4 treatment 46 are neglectable for almost all of the initial carious lesions as most clinically relevant caries develops in proximal or occlusal sites and if buccal lesions occur, they are mostly positioned on premolars and molars.
Professionals are trained to identify initial caries in any location, either visually, with caries diagnostics or on x-rays. Based on those assessments the clinician will decide on whether invasive restorative treatments are needed. Regenerative procedures should be considered whenever possible, in order to conserve natural tooth structure and function and to avoid or at least to delay as far as possible the entry into the conventional filling approach leading to ever larger fillings 47 . Bröseler et al. have shown that the SAP P 11 -4 treatment can be transferred into a practice setting 27 .

Conclusion
The treatment of initial carious lesions with self-assembling peptide P 11 -4 leads to superior remineralisation of the subsurface lesions compared with the control teeth.

Material and Methods
Study design. The clinical trial was designed and performed as a split-mouth conventional treatment-controlled trial investigating the effect of the treatment of buccal carious lesions following orthodontic treatment by using SAP P 11 -4 as add-on with the conventional treatment control. Clinical study procedures were performed parallel to regular appointments at the Orthodontic Department of the University Medicine Greifswald (Fig. 3) according to the Declaration of Helsinki and in compliance with ISO 14155:2012. Approval for all clinical procedures and the trial was obtained by the ethical committee of the University of Greifswald (Code BB 99/12, date of approval: 28 th August 2012).
The clinical trial was registered in the German Database for clinical trials (DRKS00016501, date of registry: 30 th January 2019).
Patient and study tooth selection. Twenty-three patients (13f/10 m; average age 15.4 years) with at least two WSL after removal of fixed orthodontic appliances could be recruited into the trials from the Orthodontic Department of the University Medicine Greifswald and treated by the Restorative Department. The written informed consent was obtained from every participance or from a parent of every participance under 18 years prior to any study-related procedures two days before baseline (Fig. 3).
Patients had to fulfil all the following selection criteria: Inclusion www.nature.com/scientificreports www.nature.com/scientificreports/ • At least two active carious lesions around the bracket area with a rough, chalky and matte surface; • Size and form of the active carious lesion: The carious lesion must be fully visible and assessable and accessible; • Able and willing to observe good oral hygiene throughout the study; • ≥20 teeth and a BP score of <3; • Age ≥12 years and ≤18 years; • Willing and able to attend the on-study visits; • Willing and able to understand all study-related procedures; • Written informed consent before participation in the study. Exclusion • Pre-treated WSL; • Tooth with another carious lesion apart from the ones developed during orthodontic treatment; • Evidence of tooth erosion; History of head and neck illnesses; • Any pathology or concomitant medication affecting salivary flow; • Concurrent participation in another clinical trial.

Randomisation and blinding.
Within each subject, the treatment was randomly allocated to a site by flipping a coin. For three subjects having had test and control teeth in the same quadrant, the more anterior tooth was assigned to the opposite site. Based on our add-on study design, the investigator and patient were not blinded during applying SAP on the tooth, however, outcome assessor and statistician (except for sensitivity analysis after multiple imputation described below) were blinded.
Study treatment procedures (besides the regular orthodontic treatment). At the Day 0 visit, all teeth were cleaned with a non-fluoridated tooth cleaning paste (Clean Polish, Kerr, Germany), (Fig. 3).
Further, the patients received an electrical tooth brush (Oral-B Pro 1000 Precision Clean, P&G, Germany) and an instruction in it. After that, general patient assessment was recorded prior to allocation of the treatment.
"Each participant acts as their own control" 36 . In opposite to the control teeth, which got no further treatment on Day 0, the test teeth were prepared according to the instruction of the use for the SAP P 11 -4 product (Curodont Repair, Credentis, Windisch, Switzerland). The test teeth were cleaned with 2-3% NaOCl, rinse with H 2 O; etched with 35% Phosphoric acid for 20 sec (Ultra-Etch, Ultradent Products Inc., USA), rinsed with H 2 O and air-dried. After cleaning, the SAP P 11 -4 solution was applied to the WSL and left in place for 5 minutes.
The patients were strongly encouraged to use fluoride toothpaste (at least 1450 ppm Fluoride) twice daily. At every follow-up visits (Day 45, Day 90 and Day 180) parallel to regular orthodontic control appointments, all teeth (including test tooth and control tooth) were cleaned with a fluoridated prophylaxis paste (Flairesse 12.300 ppm Fluoride, DMG, Germany) as at 2 days before baseline (Day 0). Oral hygiene instructions were provided at every study visit (Fig. 3).
Outcomes. Primary endpoint of the study was initial carious lesions as measured by the impedance (CarieScan, Orange Dental, Biberach, Germany) [29][30][31] . Before measurements, the device was checked and handled according to the manufacturer's instructions. The area was kept dry by optragate (Ivoclar Vivadent, Ellwangen, Germany) and the tooth surface air-dried for 6 seconds. The impedance measurements were taken three times and the internal CarieScan values were averaged for each time point. To ensure reproducibility of the location of the WSL throughout the study, customised trays were prepared, and holes were drilled at the place of the WSL fitted to the nozzle of the diagnostic. The measurements were performed with the tray in place to ensure that readings were taken at the same place (Fig. 4).
The secondary endpoint (WSL size) throughout the study were evaluated morphometrically via Shadepilot (DeguDent, Hanau, Germany) (Fig. 5). In order to ensure reproducibility of the measurement, a representative and isolated area of the WSL on the study tooth was selected and its size semi-automatically determined as follows: A buccal tooth area was selected on the measuring device, the WSL area within the area calculated by the device, and refined, if necessary, by the blinded assessor. For clarity: Not necessarily the whole affected area of the study tooth was taken into account but only clearly definable WSL (Fig. 6).

Sample size estimation.
At the time of designing the study previous clinical data were not available whereas the SAP treatment was new. The experience over the last years supports that "In practice, for many trials, it is unlikely that there will be data to support a realistic estimate" of the within-subject correlation 36 . This is especially true in dentistry, where data from at least three levels (subject, jaw, and tooth) are required to account for within-subject variability. Thus, "it is a more sensible approach to sample-size determination to have a look at what sort of trial has been run in the past in a particular area and see what sort of inferences were possible rather than going through some complicated power calculation: often this is no more than a ritual" 48 . This is what was done; for the primary endpoint, which is continuous, at least 22 participants were aimed for 49,50 . Note that inferences from confidence intervals as presented herein are more appropriate than inferences from p-values 44,51 . Statistical analysis. Replacing "period" by "site", the split-mouth design was analysed as cross-over design 39 .
The repeated measurements were examined in mixed models for random subject effects and in robust analyses for the two-treatment design 52 . Treatment, time, the interaction between treatment and time, site-specific baseline values using restricted cubic splines with 3 knots, site, and the interaction between site and time were fixed factors. In addition, subject and site were included as hierarchical random factors, and time was modeled as repeated factor by three variance and three unstructured covariance terms within site, yielding a total of seven random factors 52 . The Kenward-Roger method 41 using the observed information matrix was applied Scientific RepoRtS | (2020) 10:6819 | https://doi.org/10.1038/s41598-020-63633-0 www.nature.com/scientificreports www.nature.com/scientificreports/ to correct for small-sample inference. Mixed models can deal with imbalanced or missing data if the "missing at random" assumption holds 43 . The complex mixed models were analysed using Stata software (release 14.2, Stata Corporation, College Station, TX, USA) and checked using SAS software (release 9.4, Cary, NC, USA). In robust analyses of the direct treatment effect (not including baseline values), the two-sample t-test was used for differences in linear combinations of repeated measurements between groups defined by the site of treatment 52 . To cover the uncertainty due to dropouts, multiple imputations were generated by fully conditional specification using R software 43 . Scenarios other than "missing at random" were examined in sensitivity analysis after multiple imputation.

Data availability
The datasets of the current study are available from the corresponding author on request.