In-Vivo Study on the Efficacy of a Photodynamic System with Riboflavin (Vitamin B2) and Polychromatic LED Light Source on Gum Health Measured by Bleeding-on-Probing (BoP).   Client: Mana Health Technologies GmbH 1. Objective of the Study The objective of the study was to measure and evaluate the efficacy of photodynamic care using a novel system in the oral cavity. The product system "manadental" to be tested was conceived, developed, and brought to market readiness by Mana Health Technologies GmbH. 28 subjects participated. The study, led by principal investigator Dr. med. dent. Andreas Inthal, was conducted on behalf of Mana Health Technologies GmbH. Dr. med. dent. Andreas Inthal is the study director of Mana Health Technologies GmbH, involved as an investor, and significantly involved in the product development of the photodynamic care system "manadental". The product "manadental" to be tested is not subject to the German Medicinal Products Act or the Medical Devices Act. The electronic device (measuring glass, charging station, emitter) is a consumer product within the meaning of the German Product Safety Act. The photodynamic liquid is classified as a cosmetic product. In accordance with Regulation (EC) No. 1223/2009 and Regulation (EU) No. 655/2013, ethical and safety-relevant requirements were considered by the principal investigator in advance of the study, and this study was conducted in accordance with "good scientific practice."   In a group of subjects, clinically objective parameters of efficacy on the gingiva were investigated, color matching was performed, and the subjects' subjective perception was recorded. 2. Subjects, Materials, and Methods 28 subjects participated in the study, which was conducted in the practice of the principal investigator Dr. med. dent. Andreas Inthal. The devices and the photodynamic liquid were provided by Mana Health Technologies GmbH. Participation was limited to 3 weeks for each subject. The subject group consisted of healthy individuals in good general condition between 20 and 75 years of age. Participation in the study was voluntary and could be terminated at any time without giving reasons. The manadental product system was handed over to the subjects, including its packaging. A detailed user manual was enclosed with the product packaging. In addition, the subjects received thorough training on product application at the practice. The product was applied every second evening before brushing teeth during the 3-week observation period.   2.1 Endpoints The primary endpoint of the study was to assess the product's efficacy on gum health using Bleeding-on-Probing (BoP). Secondary endpoints included assessing the product's influence on tooth color and analyzing product perception through an anonymized questionnaire.   2.2 Bleeding-on-Probing To assess the condition of the gums, Bleeding-on-Probing (BoP) according to Ainamo and Bay (1975) was used. A blunt-tipped probe is pressed against defined measuring points at the gingival margin. It is dichotomously recorded (yes/no) whether the probe manipulation causes bleeding or not. Six sites, distributed over the entire sulcular circumference of the tooth (which corresponds to 32 x 6 = 192 measuring points in a fully developed dentition), are recorded dichotomously (yes/no). The unmodified BoP according to Ainamo and Bay (1975) represents the total number of all bleeding probing points as a percentage of the total number of all probing points [4], [5]. In the present study, a common modification of BoP according to Ainamo and Bay was used. The result given in this modification expresses the total number of bleeding teeth in relation to the total number of probed teeth. This contrasts with the conventional calculation, in which the number of bleeding sites is expressed in relation to the total number of all probed sites. Accordingly, in the present modification, a tooth in which the inducible bleeding has been reduced, for example, from 5 sites to one site, is still recorded as "bleeding." This further increases the robustness of the parameter. To further reduce the risk of bias by the investigator, a periodontic probe (WHO probe) calibrated to 0.2N was used in accordance with "good scientific practice." 2.3 Tooth Shade Matching To determine tooth shade, the "Classical" shade guide from VITA® manufacturer was used. The shades include ranks A1-D4®. The three-dimensional tooth replicas with corresponding shading from A1-D4 are held directly against the tooth and matched [6]. 2.4 Questionnaire The evaluation of subjective perception was carried out using open questions, single-choice questions, scaled questions, and polarity profiles. Quantifiable question models were evaluated with percentages and/or absolute numbers in pie or bar charts. 3. Results of Bleeding-on-Probing Bleeding-on-Probing was tested on an average of 26.6 (median 28, minimum 21, maximum 30) teeth per subject. Before the intervention, Bleeding-on-Probing was found on an average of 18.4 (20; 2 – 28) teeth; after the intervention, it was only on 11.5 (10.5; 0-22) teeth. This reduction was highly significant (Wilcoxon test for paired samples: z = 4.627, p < 0.0001; Fig. 1; Fig. 2). Figure 1: Number of teeth with Bleeding-on-Probing before (BoP T0) and after a three-week intervention (BoP T3W). Figure 2: Wilcoxon test for paired samples for the number of teeth with Bleeding-on-Probing before (BoP T0) and after a three-week intervention (BoP T3W; z = 4.627, p < 0.0001).   The number of bleeding teeth decreased by an average of 43.1%. The tendency to bleed was reduced in all subjects over the observation period.   4. Results of Tooth Color Matching Tooth discoloration, determined by the defined tooth shade guides, showed no color change in 23 subjects (82.1%) and a lightening in 5 subjects (17.9%). A darker discoloration after the intervention did not occur in any person (Fig. 3). Figure 3: Frequency of tooth color change after 3-week intervention 5. Questionnaire Evaluation The following presents some aspects from the questionnaire evaluation. The anonymized questionnaire was completed by the subjects after the 3-week application period and before the final examination, so as not to overlie the subjective impression.     5.1 Mouthfeel: "Please rate the mouthfeel directly after application" This question served to assess the "subjective mouthfeel" of the subjects after the 3-week application. The following answer options were available: Mouth feels less clean. Mouth feels the same as before. Mouth feels cleaner. (Fig. 4) Figure 4: Evaluation of the question "Mouthfeel" as a pie chart. 5.2. Ergonomics: "Please rate the irradiation application in the mouth from unpleasant (1) to pleasant (5):" The question served to gather subjects' subjective assessment of the product's ergonomics after 3 weeks of "manadental" use. The rating was determined using a polarity profile, ranging from unpleasant (1) to pleasant (5). On average, ergonomics were rated with 4.36 points out of 5, underscoring the pleasant application of the "manadental" product system. (Fig. 5) Figure 5: Evaluation of the question "Ergonomics" as a scale. 5.3 Effect: Your subjective assessment of the effect of "manadental" after 3 weeks The question was intended to gauge the subjects' subjective assessment of the effect of "manadental" after 3 weeks of use. The following answer options were available: Oral health has worsened. Oral health has remained the same. Oral health has improved. (Fig. 6) Figure 6: Evaluation of the question "Effect" as a pie chart.   5.4. Usage: Would you like to continue using the product in your daily life? This question served to assess the subjective potential for product adoption after 3 weeks of use. The following answer options were available: Yes. No. Undecided. (Fig. 7) Figure 7: Evaluation of the question "Usage" as a pie chart.   6. Interpretation and Summary The in-vitro studies conducted so far demonstrate an impressive effect of the photodynamic care system on the pathogenic oral bacterium Streptococcus mutans. The method achieves a germ reduction of approx. 5.5 log stages and is superior to conventional mouthwashes by approx. 3 log stages (99.9%). The results of the in-vivo efficacy of the "manadental" photodynamic care system, measured by the BoP parameter, show a highly significant improvement in the condition of the gingiva (Wilcoxon test for paired samples: z = 4.627, p < 0.0001, Fig. 1, Fig. 2). The average improvement of the BoP parameter over the observation period is 43.1%. Overall, an improvement of the parameter was observed in all subjects; no case of stagnation or deterioration of the gingival condition was recorded. The clarity of the present results, considering the robust BoP parameter, is remarkable. The investigators and professionals of the conducting institution also evaluate the observed improvement during the observation period as a success, based on clinical experience. The results of the tooth shade matching indicate that the application of the photodynamic care system does not cause tooth discoloration. In addition to the tooth shade matching, no partial discolorations of the enamel or gingiva were observed by the investigators during the second examination. The slight lightening of tooth color, which was observed in 5 individuals, is not considered significant by the investigators and professionals of the conducting institution over the observation period. The scale jumps were small (0.5 – 1 VITA® scale units) and were observed with predominantly lighter baseline tooth color (AX), which is empirically more easily influenced by ambient light fluctuations and investigator bias. Within the present study, no side effects of the application of the photodynamic care system were discernible based on the measured parameters, the general assessment of oral health, and the follow-up discussion at the final examination. This study shows a highly significant improvement in the gingival condition of the subjects after a 3-week application of the "manadental" photodynamic care system. No immediate side effects of the photodynamic care system were observed.   References   [1] Carrera, E., Dias, H. B., Corbi, S. C. T., Marcantonio, R. A. C., Bernardi, A. C. A., Bagnato, V. S., Hamblin, M. R. & De Souza Rastelli, A. N. (2016). The application of antimicrobial photodynamic therapy (aPDT) in dentistry: a critical review. 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