Randomized Clinical Trial investigating Self-Assembling Peptide P11-4 for Treatment of Early Occlusal Caries

Non-invasive caries treatment is a major focus in modern dentistry. The present study was designed to evaluate the effectiveness of monomeric self-assembling peptide P11-4 (SAP P11-4) in combination with fluoride varnish or polymeric self-assembling peptide matrix (SAPM) in treatment of non-cavitated occlusal caries. Ninety children and adolescents were included in this randomized, gold-standard-controlled clinical trial. Test Group 1 received SAP P11-4 and twice fluoride varnish at baseline and Day 180, Test Group 2 received SAP P11-4 on baseline and twice weekly SAPM (home-application), and Control Group received fluoride varnish on baseline and Day 180. Caries progression was measured by laser fluorescence, Nyvad Caries Activity, ICDAS-II-codes, and investigator assessments. Laser fluorescence changes demonstrated superior results for Test Group 1 and 2, as values decreased compared to an increase for the Control Group (p < 0.0005). ICDAS-II codes at Day 360 showed partial regression for Test Group 1 (6.7%) and Test Group 2 (20.0%) and partial progression for Control Group (23.3%) (p < 0.01). Nyvad Caries Activity yielded superior caries inactivation for Test Groups, compared to Control Group (p = 0.002). This trial showed that SAP P11-4, applied either in combination with fluoride varnish or twice weekly SAPM, was a superior treatment for early caries compared to fluoride varnish alone.

a function of the enamel lesion depth have shown that remineralization after SAP P 11 -4 application occurs deep into the lesion body. In contrast, mineralization with fluoride is restricted to the outer enamel layer 15 .
For SAP P 11 -4 to be translated into daily clinical practice, it is essential that clinicians can visualize the treatment effect with available diagnostics. Both ex vivo and in vivo studies have reported the successful use of laser fluorescence, PTR-LUM, Impedance, QLF and radiography in monitoring remineralization after SAP P 11 -4 application in a time-dependent manner 12,13,[16][17][18][19][20] .
Yet, the ultimate test for any device is the clinical efficacy within daily clinical practice. Early clinical trials have proven the safety of SAP P 11 -4 and have indicated the efficacy of the product 19,21 . Recent reports on randomized controlled clinical trials demonstrated superior efficacy compared to fluoride varnish, placebo or saliva in the treatment of initial caries -both in combination with fluoride varnish or with normal oral hygiene, including fluoride toothpaste 16,18,22,23 .
Two clinical trials are of particular interest. The first investigated a combination of SAP P 11 -4 and fluoride varnish in remineralisation of early carious lesions, with results showing superior remineralisation, caries inactivation and regression of SAP P 11 -4 and fluoride varnish compared to fluoride varnish alone, yet raising several clinically relevant question 16 : Is there an additive, synergistic, or inhibiting effect between SAP P 11 -4 used alongside fluoride varnish? Could fluoride varnish be substituted with a home care solution avoiding risks and logistic difficulties associated with high fluoride application? Is the efficacy of SAP P 11 -4 and fluoride varnish in fully erupted molars and premolars different to that of erupting molars (as only erupting molars were treated)? Is the regenerative process supported by SAP P 11 -4 fully completed at the 6 month recall -the last recall of the study?
A second publication reported an in situ trial investigating the use of a gel including SAP P 11 -4 in its matrix form (SAPM) in the prevention of demineralisation and treatment of early demineralised lesions. The results suggested that SAPM gel with 900ppm fluoride and twice a week application showed equal or superior efficacy in high risk caries patients compared to fluoride varnish 24 . P 11 -4 within the SAPM gel was developed for home treatment of caries and prevention thereof, primarily by inhibiting demineralisation, whereas the monomeric SAP P 11 -4 product for professional use was specifically developed for treatment of initial carious lesions. SAPM and SAP P 11 -4 differ in their primary mode of action, formulation and concentration of P 11 -4. Whereas SAP P 11 -4 diffuses into the lesion body upon application on the tooth surface of active carious lesions, the location of action of SAPM is primarily the tooth surface protecting the tooth minerals, as the fibres are too long to diffuse into the lesion body.
In the present clinical study, we investigated for the first time the possibility of substituting the use of high concentration fluoride (i.e. fluoride varnish) with an SAPM gel including toothpaste levels of fluoride (900 ppm Fluoride) suitable for home use. Furthermore, this is the first clinical study investigating the combinational of SAPM and SAP P 11 -4. We based the design on a previous clinical trial, which studied a combination of SAP P 11 -4 and fluoride varnish (SAP P 11 -4+Fluoride Varnish; Test Group 1) and compared it to fluoride varnish only (Fluoride Varnish; Control) in the treatment of children and adolescents. Yet, we extended the study design, using the same control group, and the previous test group 1 as a positive control, adding a third treatment group of SAP P 11 -4 and twice weekly home application of an SAPM (SAP P 11 -4+SAPM; Test Group 2) and a longer follow-up period, and a wider patient selection. We could thus address the clinically relevant questions raised by the previous trial: The efficacy of SAP P 11 -4 without simultaneous application of fluoride varnish. The efficacy of SAP P 11 -4 in treatment of initial occlusal caries of fully erupted molars and premolars, and determine the time in which the SAP P 11 -4 supported remineralisation is completed and whether it is stable over 12 months.
We tested the hypothesis that SAP P 11 -4+Fluoride Varnish and SAP P 11 -4+SAPM would show no difference in clinical success in the treatment of early occlusal caries and that both would be superior to the current clinical gold-standard of fluoride varnish alone -corroborating data from the previous clinical trial 16 and providing an alternative for the clinician to substitute semi-annual high fluoride varnish application with a twice weekly application of an SAPM gel.

Results
The present study was approved by the University Dentistry  Ninety children and adolescents were recruited into the trial and equally assigned at random to the three study groups (Fig. S1). None of the baseline values showed statistical differences among the groups (Table 1). Throughout the study, 7 patients in the Control Group were lost. One patient was lost after baseline, two patients were lost due to clinically indicated placement of a filling at the study lesion after completion of the Day 90 visit, and four more were lost after the Day 180 visit. An additional 4 lesions in the Control Group required a filling after completion of the study visit at Day 360. For those patients, all assessments are available, including Day 360 assessments. Test Group 1 lost 3 patients throughout the study, one each after Baseline, Day 90 and Day 180. Test Group 2 lost 3 patients throughout the study; two patients were lost after Day 90, and one lesion required filling after Day 180 (Fig. S1).
Laser fluorescence. Change in the laser fluorescence values was the primary parameter ( www.nature.com/scientificreports www.nature.com/scientificreports/ follow-up compared to the Control Group (p < 0.0005) but there were no statistical differences between Test Group 1 and Test Group 2 (p = 0.595).

ICDAS-II code and lesions requiring restorative intervention.
All three study groups had distributions between ICDAS-II codes 2 and 3 (Table 1) at baseline. The changes in ICDAS-II Code of the study lesions are shown in Table 3. Throughout the study period, none of the control lesions regressed into a lower caries class; however, until Day 360, 9 (30%) lesions increased by at least one class (Day 360: 7 increases and 2 lesions that needed filling after Day 90). None of the Test Group 1 or 2 lesions moved into a higher ICDAS-II Code, including the lesion in Test Group 2, which was restored after the Day 180 visit upon request of the guardian. In Test Group 1, two lesions (6.7%) regressed into a smaller ICDAS-II code by Day 360; in Test Group 2, six lesions (20%) regressed into a smaller ICDAS-II code. There was no statistical significant difference between Test Group 1 & 2 (p = 0.26), but both Test Groups 1 & 2 performed superior to the control (p < 0.05). Throughout the study, 6 (20%) of the lesions in the Control Group required restorative intervention (2 after Day 90 and 4 after Day 360); no lesions were restored in Test Group 1, and one lesion was restored in Test Group 2 after Day 180.
Nyvad Activity Criteria. All three study groups had a comparable distribution of active and inactive caries lesions at baseline (Table 1). Changes in the Nyvad Activity Criteria throughout the study are described in Table 4. In the Control Group, the inactive/active lesions changed from 4/26 at baseline to 13/10 at Day 360. Test Group 1 showed superior caries inactivation to the Control Group, as the inactive/active lesions increased from 1/29 (Day 0) 27/0 (Day 360) (p < 0.0005). Test Group 2 showed superior caries inactivation to the Control Group, as the inactive/active lesions increased from 3/27 (Day 0) to 27/0 (Day 360) (p = 0.002). There were no statistical significant differences between the two Test Groups (p = 0.205).
Visual Analogue Scale for surface texture. All   Global Impression of Change questionnaire. The Global Impression of Change (GOIC) questionnaires at the three follow-up visits showed an overall worsening of the caries in the Control Group and a clear superior improvement of the carious enamel of Test Groups 1 & 2 (p < 0.0005) (Fig. 1). There was no statistical difference observed between Test Groups 1 & 2 (p > 0.05).

Adverse events.
No adverse events or safety issues were observed throughout the study in any study group.

Discussion
In the present study, both Test Groups, including SAP P 11 -4 either with fluoride varnish or with SAPM, showed superior results in terms of enamel remineralisation compared to the Control Group treated with fluoride varnish. As would be expected, the Control and Test Group 1 show similar results as in the previous clinical study with a similar design including those two treatment groups 16,22,23 . In addition, it also showed similar results as other randomized controlled trials investigating SAP P 11 -4 or SAPM.
Differences to Alkilzy et al. 2017 could be seen in the magnitude of the laser fluorescence signal decrease for both Test Groups compared to the previous study 16 , yet this might have been due to the overall lower starting values in the present study. The relative decreases of laser fluorescence among the SAP P 11 -4 groups in the studies were similar in the two studies. The corroborating ICDAS-II code data were also similar, showing between 6.7-20% regression into lower ICDAS-II codes in the present study and similar numbers in the previous study 16 . The laser fluorescence readings of the Control Group in the present study increased slightly (non-significant) and remained unchanged in the previous study 16 .
The present trial addressed critical questions raised by the previous trials on SAP P11-4. As there was no statistically significant difference between the two Test Groups, it suggests that the effect of Fluoride Vanish 2x per year leads to similar results as SAPM 2x per week, if used in combination with SAP P 11 -4, and thus SAPM could be substituted for fluoride varnish treatment without loosing effectiveness. Secondly, erupted molars and premolars benefit similarly from the treatment as did erupting molars. Finally the regenerative process supported by SAP P 11 -4 mostly occurs within the first 3 months as can be seen by the inactivation of the caries and the laser fluorescence values, but regression of the carious enamel could be observed throughout the year, especially changes in ICDAS-II codes.
The underlying rationally designed biomimetic technology of SAP P 11 -4 has gone through rational development and clinical testing. It was first investigated within the basic sciences 25 , then entered oral biology 11 and afterwards translated into clinical practice in a combined effort of academia and industry 16,18,19,21,23 . In recent years, several clinical studies, both in university clinic and private practice settings, have been reported, all supporting the effectiveness and safety of SAP P 11 -4 and its superiority over other conventional remineralizing agents, such as Fluoride 16,18,19,21,23 .
To adapt our clinical practice and use SAP P 11 -4 as a routine prevention measure, the clinical relevance of the superiority seen in SAP P 11 -4 over fluoride varnish should be discussed. The main advantage shown in the clinical trial -and in this one in particular -is that SAP P 11 -4 can initiate a regression for the caries and not just an arresting/inactivation of the lesions, as observed in many fluoride studies. To preserve the natural tooth structure  www.nature.com/scientificreports www.nature.com/scientificreports/ as long as possible and delay a restorative intervention, a regression of the caries, by a biomimetic agent such as SAP P 11 -4, is a significant clinical benefit. For the first time, such a guided enamel regeneration enables the tooth to get closer to its healthy state and further from the disease state 3,26 . Arresting a lesion with fluoride stops the imminent clinical issue at hand, but does little to alleviate the thread of a lesion becoming active again and progressing to a cavity 27 .
Based on the clinical benefit provided by, and the bulk of scientific and clinical data available for SAP P 11 -4 in the treatment of carious lesions, a translation into every day practice is imminent and should be welcomed by the profession as a new effective tool to decrease caries rates.
The present clinical study is not without limitations. The main limitation is the blinding within the study, which was limited to Control Group and Test Group 1 patients. This might have affected the Nyvad Activity Criteria and ICDAS-II assessments and could have led to a bias for the subjective assessments, VAS Scores, and GOIC. However, as these assessments corroborate the primary parameter of laser fluorescence, obtained with a validated device, there seems to be robustness for the data as a whole.
Scientifically, it would have been interesting to provide SAPM (Test Group 2) without the 900-ppm fluoride. However, as fluoride toothpaste was used throughout the study, the investigators felt that the additional 900 ppm fluoride twice a week supplied by SAPM had little effect on the overall outcome of the clinical trial.

conclusion
The data obtained from the clinical trial showed SAP P 11 -4, applied either in combination with fluoride varnish or twice weekly SAPM, to be a superior treatment for early caries lesions compared to fluoride varnish alone. The efficacy of the two groups including SAP P 11 -4 did not show differences in the treatment of occlusal caries as in the present study no statistically significant differences were observed. These findings are based on laser fluorescence, ICDAS-II Code, Nyvad Caries Activity Criteria and investigator assessments. www.nature.com/scientificreports www.nature.com/scientificreports/

Materials and Methods
Study design. The present study was designed as a randomized, gold-standard controlled, single blinded clinical trial comparing the combined treatment of a single application of SAP P 11 -4 and fluoride varnish (Test Group 1) and the combined treatment of a single application of SAP P 11 -4 and a twice weekly self-application of SAPM (Test Group 2) against the gold standard fluoride varnish applied twice per year (Control).
All assessments were performed in accordance with ISO 14155:2011 Clinical investigation of medical devices for human subjects -Good clinical practice and the national regulation for clinical trials in the Republic of Kosovo. Subjects were recruited from the daily clinical practice of the paediatric dental department of the University Dentistry Clinical Center of Kosovo and fulfilled the following selection criteria: Inclusion criteria.
• Present early occlusal carious lesions (ICDAS-II codes 2 or 3) that do not require invasive treatment • Age ≥ 6 years and ≤ 15 years • Size and form of the carious lesions must both be fully visible, assessable, and accessible • Willing and able to attend the on-study visits and to observe good oral hygiene throughout the study • Provide written informed consent before participation in the study As subjects were minors, informed consent was provided by the parent and/or legal guardian.
• Evidence of tooth erosion • Fluoride varnish application <3 months prior to study treatment • History of head and neck illnesses (e.g., head/neck cancer) • Any pathology or concomitant medication affecting salivary flow or dry mouth • Any metabolic disorders affecting bone turnover • Concurrent participation in another clinical trial To ensure similar fluoride levels among subjects, all subjects were asked whether they use fluoride tablets or drops in addition to their fluoride toothpaste, which all subjects declined. There is no water-, milk-, or salt-fluoridation anywhere in Kosovo. If a subject presented more than one early carious lesion, then all lesions where treated according to the study allocation, but only one lesion was included in the assessment. Within the study, lesions on molars surpassed lesions on pre-molars.
Randomization and blinding. Subjects were randomly assigned to either of the 3 study groups by drawing consecutively numbered envelopes providing the study group assignment. The randomization envelopes were provided by a third party. As a single investigator performed the treatment and all assessments, blinding of the investigator was not possible. Patients were only blinded with regards to Test Group 1 and Control Group, as Test Group 2 patients were given home applications of SAPM.
Treatment. Subjects were treated according to their study group allocation: Control Group lesions were treated with fluoride varnish (Fluor Protector S (7′700ppm Fluoride, Ivoclar Vivadent, Schaan, Lichtenstein) at baseline and Day 180; Test Group 1 lesions were treated once with SAP P 11 -4 at baseline (Curodont Repair, credentis, Windisch, Switzerland), including pretreatment according to the instructions for use: Fluoride-free prophypaste (Proxypaste RDA 36 fluoride free, Ivoclar Vivadent, Schaan, Lichtenstein), 2% NaOCl (Chloraxid, Cerkamed, Poland), and 35% Etch Gel (Voco, Cuxhaven, Germany), followed by fluoride varnish application. Test Group 1 received fluoride varnish application at Day 180 as the control group did. Test Group 2 lesions were treated once with SAP P 11 -4 at baseline identical to Test Group 1 but without fluoride varnish application. Instead, SAPM (Curodont Protect, credentis, Windisch, Switzerland) was provided, and study patients were instructed on the application. SAPM was to be applied twice per week with a finger. Subjects of Test Group 2 were provided with a calendar to monitor SAPM application.
All study materials used in the present study, especially SAP P 11 -4 and SAPM are suitable and recommended by the manufacturer for the use in children and adolescents.
Study patients were asked to attend follow-up visits at Day 90, Day 180 and Day 360; Control Group and Test Group 1 received an additional fluoride varnish application at Day 180.
Study visits and assessments. Baseline assessments were performed after written informed consent for study participation was provided by all study patients prior to study group assignment (i.e., randomization) and first study treatments.
All assessments at all study visits were performed by a single trained investigator as no alternative clinical investigator trained in both GCP and study procedures was available at the study site. The following assessments were performed only at baseline: Simplified Oral Hygiene Index (OHI-S) 28 , Plaque Index, Caries Risk Assessment 29 , DMFT. The following assessments were performed at each study visit: Laser Fluorescence (DiagnoDent Pen, KaVo, Biberach, Germany) -Triple measurements were performed, and the average was taken for statistical analysis; ICDAS-II code of the study lesion (not including activity of the carious lesion) 4 ; Nyvad Activity Criteria assessment of the study lesion 30  www.nature.com/scientificreports www.nature.com/scientificreports/ lesions, and positive values soft(er)/rough(er) carious lesions 21,31 ; Global Impression of Change Questionnaire (GIOC) was assessed by the investigator at all follow-up visits 21 . The GIOC is a seven-point scale with answers ranging from: "very much worse", "worse", "a little worse", "unchanged", "a little better", "better", and "very much better". To explore if the development of the examined parameters during the 1-year study period was different between the treatment groups, tests of interaction of time and group were performed in the framework of multilevel models.

Statistical analysis.
For metric parameters -laser fluorescence, VAS texture assessment -these multilevel models were linear mixed models, while ICDAS-II class, Nyvad Activity Status and GOIC were analysed in the framework of generalized estimation equation (GEE) for ordinal outcome variables; all were adjusted for their corresponding baseline values, sex, age, and plaque index.
In addition to these global tests, group differences in progression in the tested parameters and changes from baseline to day 90, day 180 and day 360 were assessed. Pairwise comparisons between the treatment groups with respect to the changes from baseline to day 90, day 180 and day 360 were performed by means of ANCOVA or ordinal logistic regressions, depending on the scale of the outcome variables. Again, the models were adjusted for their corresponding baseline values, sex, age, and plaque index.
Knowledge transfer statement. The results of this study should aid clinicians in their decision on whether non-invasive early caries treatment might be suitable for a patient. In addition, it educates the clinician on how to quantify the success of a non-invasive treatment and what changes to expect. This can lead to a more prevention-orientated treatment decision and thus less invasive dentistry, enabling the patient to keep their natural teeth longer.

Data availability
The datasets generated during and analysed during the current study are available from the corresponding author on request.