Client: Mana Health Technologies GmbH

1. Aim of the tests

The aim of these tests was to investigate the antibacterial effect of the “manadental” oral photodynamic cleaning system developed by Mana Health Technologies GmbH on the pathogenic oral bacteria Streptococcus mutans. As a comparison, two conventional, commercially available mouthwashes were tested for their antibacterial effectiveness.

The test method was to use the indicator bacteria Streptococcus mutans as an example to investigate in vitro the extent to which the process can reducing the number of colony forming units (CFU/m
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2. Materials and methods

Before illumination, the pathogenic oral bacteria Streptococcus mutans was incubated for 48 hours on COL-S agar plates at 37°C with 5% CO2 concentration, then suspended in 0.9% NaCl and set to a defined starting bacteria number (107 – 108) using OD600 measurement. For the test, the bacteria suspension was mixed with the solutions to be tested (riboflavin rinse, conventional mouthwash 1, conventional mouthwash 2).

The LED light source used for the testing has a polychromatic emission spectrum which includes the wavelength at the required energy level necessary for photophysical stimulation of the riboflavin. The illumination was applied in 5-minute (300-second) cycles.

The photodynamic rinse was a water-based solution with a special vitamin B2 (riboflavin) derivative as a photosensitiser. The formula was designed to be a mouthwash and therefore contains flavourings and additional active ingredients, which have a synergistic relationship with the primary photodynamic effect.

After manipulation and incubation with the mouthwashes, the solutions were mixed again with the pipette and diluted and plated in a 1:10 dilution series up to 107. Each test was evaluated twice in the form of a technical replicate. The photodynamic illumination was also designed as a biological replicate.

The following test methods were compared with each other:

• Control
• LED illumination
• Riboflavin rinse
• Riboflavin rinse + LED illumination
• Conventional mouthwash 1
• Conventional mouthwash 2

3. Results

The results of the test methods can be found in the following table:

The results of the testing prove that neither illumination alone (BK) nor the isolated riboflavin rinse (RK) have demonstrable bactericidal effects compared to the growth control (WK1 and WK2). By contrast, the combination of illumination and riboflavin rinse (BES I, BES II) demonstrates a strong antibacterial effect against Streptococcus mutans (bacteria reduction of ~5.5 log levels) in the test.

The relative difference in effectiveness between the conventional mouthwashes (SP1, SP2) and the photosensitising illuminated riboflavin rinse (BES I, BES II) is ~3.0 log levels in the test. As three logarithmic units (3.0 log levels) mean a percentage reduction of 99.9%, it can be assumed that the manadental photodynamic cleaning system is 99.9% more effective against Streptococcus mutans than the tested conventional mouthwashes.

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4. Interpretation and summary

The results of this testing indicate that the antimicrobial effect of the photodynamic system under test is much greater than the conventional mouthwashes with incubation as specified by the manufacturer

(Fig. 1).

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Responsible Investigators: Werner Voss, M.D., Frauke Behrens M.D., Sigird Brune M.D, Vanessa Gomes M.D.

1. Aim of the study 

Aim of the study was to evaluate the effectiveness of a new photodynamic system on gingival health. The product in question, „manadental“, has been designed, developed and brought to market maturity by Mana Health Technologies GmbH Inc.

The present study (Nr: 2310251797) was carried out by the German testing institute Dermatest GmbH. The product „manadental“ is neither amenable to the Medicines Law nor to the Medical Devices Law. The multi-component system (measuring glass, charging station, toothlight) is a consumer product and as such subject to the Product Safety Act.The liquid used for photosensitization has been rated as cosmetic product.

The study was based on a group of test persons and included clinical objective parameters such as Bleeding-on-Probing (BoP), Plaque Index (PI) and tooth discolouration. During the four weeks testing period care was taken to document intolerance, irritation or other local side effects.

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2. Test persons, materials and methods 

50 test persons participated in the study. Both the product system and the photosensitizer liquid were provided free of charge by Mana Health Technologies GmbH Inc. Participation in the study was limited to 4 weeks and could be terminated at any timepoint without specifiying the reasons.

The test persons were healthy volunteers in fair general condition, aged between 18 and 75 years. All of them were told to keep up their usual oral hygiene. In addition to their usual oral hygiene, the persons in the application group used the test product “manadental” once every other day (A2G).

The study design was 2-arm, the test persons were randomised into two groups:
• 25 test persons were assigned to the control group (KG)
• 25 test persons were assigned to the application group (A2G)

The product system „manadental“ was handed out to the test persons in the application group in the original package with the detailed manual included. In addition, the appliation group had a meticulous instruction concerning the handling of the product in the dentists´ office.

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‍2.1  Measurement parameters

In each participant the measurement parameters were documented on the first day (T=0) and after four weeks (T=28). For Bleeding-on-Probing (BoP) and Plaque-Index (PI) a statistical difference of p less than 0.05 in the two-sample T-test was considered significant.

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2.1.1 Bleeding-on-Probing (BoP)

‍In order to evaluate the condition of the gingiva the Bleeding-On-Probing test (BoP; bleeding induced by pressure of a probe) first described by Ainamo u. Bay 1975 was used. For testing a blunt-tipped probe is pressed towards previously defined measurement points at the gingival margin. The result (whether bleeding is induced or not) is recorded in a dichotomous way (yes/no). Since six measurement points along the dental circumference of each tooth are defined, a total of 192 measurement points (32 x 6) result in a complete set of teeth. The original BoP by Ainamo and Bay describes the cumulative number of bleeding points as a percentage of all points that underwent probing [4],[5].

In the present study a common modification of the original BoP which increases its informative value has been applied. This modification describes the number of teeth at the gingival margins of which bleeding has been induced in relation to the total number of teeth that underwent probing. By using this method e.g. a tooth at the circumference of which bleeding points have been reduced from 5 to 1 would still be counted as “bleeding”. In order to reduce an inter-investigator bias and according to good clinical practice a paradontal probe calibrated to a pressure of 0.2N (WHO-probe) was used.

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2.1.2 Plaque Index (PI)

Dental plaque consists of multiple layers with a complex structure containing proteins, carbohydrates, phosphates and microorganisms. It forms especially on dental surfaces that cannot be kept clean by natural or artificial means and can cause dental caries, parotontitis and gingivitis. To assess both presence and degree of dental plaque formation the Plaque Index (PI) has been developed as a quantification tool after staining of the plaque. In the present study the PI according to Quigley and Hein was used [4].

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The plaque was stained with the Relevator Mira-2-Ton® (Hager & Werken GmbH & Co. KG; Färbetabletten). After meticulous rinsing with water the degree of plaque formation on the buccal surface (surface facing the cheek) of each tooth was evaluated by a qualified study nurse according to the scale depicted in figure 1. The sum of all degrees of plaque formation divided by the number of teeth per test person corresponds to the PI.

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2.1.3 Tooth shades

To assess the tooth shade the colour scale „Classical” (VITA®) was used. The shades cover the ranks A1-D4®. The three-dimensional replicas of dental surfaces with shades corresponding to A1 till D4 were aligned to the teeth of the test persons for comparison, to detect post-treatment changes. [6]

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2.1.4 Clinical assessment by dental specialists

‍In addition to the quantitative and qualitative evaluations clinical assessment criteria were applied by specialists in dental medicine. All test persons underwent meticulous examination for pathological changes of teeth, gingiva and oral mucosa before, during and after the application study.

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2.2 Endpoints of the study

‍Endpoints of the present study were the evaluation of the effectiveness of the new photodynamic product system on gingival health measured by Bleeding-on-Probing (BoP) and its influence on the Plaque Index (PI) as well as on tooth shade.

In addition, clinical findings in teeth, gingiva and oral mucosa were documented.

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‍4. Results

4. 1 Results of Bleeding-on-Probing (BoP)

Within the observation period a reduction in bleeding of a mean of 19.5% was found in the control group (KG). In comparison the reduction in the application group (A2G) that had used the product system every other day was at a mean of 49,39%.

The reduction of bleeding in the application group using the product system every other day (A2G) in comparison to the controls (KG) was highly significant (two-sample t-Test, p = 0.0053).

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4. 2 Results of Plaque-Index (PI) 

During the observation period the mean reduction of Plaque-Index (PI) in the controls (KG) was 52,07%.  In the application group that used the product system every other day (A2G) the mean reduction of PI was 63,79% .

Der reduction of the Plaque-Index in the application group using the product system every other day (A2G) compared with the controls (KG) was not statistically significant (two-sample t-Test, p = 0.454).

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4.3 Results for tooth shades

‍In none of the participants in either group any changes of tooth shade were found.

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‍4.4 Results of the clinical assessment by dental specialists

‍Before, during and after the study none of the participants showed any evidence of gingival disease or oral mucosal changes. There was no need for dental-medical treatment in any case, likewise it was not necessary to discontinue the study. In summary the product system was very well tolerated and did not cause any detectable pathological changes to the gingiva or the oral mucosa.

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‍5. Interpretation and conclusion

The present study in 50 test persons demonstrated that bleeding induced by probing was reduced in both controls (KG) and in the application group (A2G) that had used the photodynamic product system every other day. After 4 weeks the reduction of Bleeding on Probing (BoP) in the controls was 19,5% compared with 49,39% in the application group, the difference is statistically highly significant (p=0,0053). Thereby the present study confirms the results of a previous in-vivo study which had equally shown significant reduction of BoP.

Also the formation of dental plaque measured by the Plaque-Index showed reduction in both controls and application group after 4 weeks, but though the reduction in the latter was more pronounced, the difference was not statistically significant (KG = 52,07%, A2G = 63,79% ; p = 0.454).

The results of the Plaque Index (PI) suggest that a better basic dental hygiene throughout the observation period caused plaque reduction in both groups. However, considering the highly significant reduction of bleeding in the application group mere better oral hygiene alone does not seem to exert effect the same effect on the gingival health as the photodynamic product system does.

The tooth shade remained unchanged throughout the observation period.

In conclusion, a significant positive effect of the photodynamic care system „manadental“ on gingival health, as measured by Bleeding on Probing (BoP) can be stated. The clincial assessment by dental specialists documented that the application was well tolerated and did not cause any side effects on teeth, gingiva or the oral mucosa.

Client: Mana Health Technologies GmbH

1. Aim of the study

The aim of the study was to measure and evaluate the effectiveness of oral photodynamic cleaning using a new system. The “manadental” product system to be tested was designed, developed and introduced to the market by Mana Health Technologies GmbH. 28 test subjects took part. The study was conducted under the leadership of principal investigator Dr Andreas Inthal on behalf of Mana Health Technologies GmbH. Dr Inthal is the principal investigator at Mana Health Technologies GmbH, is involved as an investor, and played a significant role in developing the “manadental” photo dynamic cleaning product.

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The “manadental” product to be tested is not subject to the Austrian Medicinal Products Act (Arzneimittelgesetz) or the Austrian Medical Devices Act (Medizinproduktegesetz). The electronic device (measuring glass, charging point, emitter) is a consumer product for the purposes of the Product Safety Act (Produktsicherheitsgesetz). The photodynamic liquid is classified as a cosmetic product. In accordance with EC Regulation No 1223/2009 and EU Regulation No 655/2013, the principal investigator took the necessary ethical and safety considerations into account before the study began, and the study was conducted in accordance with good scientific practice.

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A group of test subjects was studied using objective clinical parameters for effectiveness on gums and a colour comparison, and the subjective opinions of the test subjects were also obtained.

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2. Test subjects, materials and methods

28 test subjects took part in the study, which was conducted at the medical practice of principal investigator Dr Andreas Inthal. The devices and the photodynamic liquid were supplied by Mana Health Technologies GmbH. Participation for each test subject was limited to 3 weeks.

The test subject group was composed of healthy individuals in good general condition between the ages of 20 and 75. Participation in the study was voluntary, and the test subjects could withdraw at any time without providing reasons.

The “manadental” product was given to the test subjects with all its packaging, which included detailed instructions. The test subjects were also given detailed training in how to use the product at the medical practice. During the 3-week observation period, the test subjects used the product every other evening before brushing their teeth.

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2.1 Endpoints

The primary endpoint of the study was to test the effectiveness of the product on gum health using bleeding on probing (BoP). The secondary endpoints were to test the impact of the product on tooth colour and to analyse the perception of the product using an anonymous questionnaire.

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2.2 Bleeding on probing

To evaluate the condition of the gums, bleeding on probing (BoP) in accordance with Ainamo & Bay 1975 was used. A blunt probe is pressed against defined measuring points on the gingival margin, with a dichotomous yes/no answer to whether pressing the probe produces bleeding. There are six measuring points distributed across the sulcular circumference of the tooth (in fully mature teeth, this results in 32 x 6 = 192 measuring points) where a yes/no answer is recorded. If BoP follows the exact Ainamo & Bay 1975 method, the total number of bleeding measuring points is expressed as a percentage of the total number of all measuring points. [4],[5]

In the present study, a common modification of the Ainamo & Bay BoP method was used. The result given using this modification expresses the total number of bleeding teeth in relation to the total number of probed teeth. This is in contrast to the conventional calculation, where the number of bleeding measuring points is expressed in relation to the total number of all measuring points. Using this modification, a tooth where bleeding caused by probing has been reduced from 5 points to 1 point (for example) is still recorded as “bleeding”. This makes the parameter even more robust. To further reduce the risk of bias on the part of the principal investigator, and in accordance with good scientific practice, a periodontal probe (WHO probe) pressure-calibrated to 0.2N was used.

2.3 Tooth colour card comparison

To record the tooth colour, the “Classical” colour scale from VITA® was used. The shades cover the A1-D4® shade range. The three-dimensional replica teeth in the corresponding A1-D4 shades are held up next to each tooth and compared. [6]

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2.4 Questionnaire

A questionnaire including open questions, multiple choice questions, scale questions and polarity scales was used to evaluate subjective opinions. Quantifiable question models with percentage and/or absolute numbers in pie charts or bar charts were used to evaluate the results.

3. Results of bleeding on probing

Bleeding on probing was tested on an average of 26.6 teeth (median 28, minimum 21, maximum 30) per test subject. Before the intervention, bleeding on probing was found in an average of 18.4 (20; 2-28) teeth; after the intervention, it was only found on 11.5 (10.5; 0-22) teeth. This decrease was highly significant (Wilcoxon test for paired samples: z = 4.627, p < 0.0001; Fig. 1; Fig. 2).

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The number of bleeding teeth decreased by an average of 43.1%. In all test subjects, the tendency to bleed was reduced during the observation period.

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4. Results of the tooth colour comparison

As determined by the defined tooth colour cards, the tooth colour comparison showed no change in colour for 23 test subjects (82.1%) and a brightening in 5 test subjects (17.9%). Darker colours were not observed in any of the test subjects after the intervention (Fig. 3).

5. Analysing the questionnaire

The following section explains several aspects of the questionnaire analysis. The test subjects filled out the anonymous questionnaire after using the product for 3 weeks and before the subsequent examination, to avoid influencing their subjective opinions.

5.1 Mouthfeel: “Please evaluate your mouthfeel immediately after using the product”

This question served to evaluate “subjective mouthfeel” in the test subjects after using the product for 3 weeks. They could choose from the following potential answers: My mouth feels less clean. My mouth feels as clean as before. My mouth feels cleaner. (Fig. 4)

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5.2.  Ergonomics: “Please evaluate the illumination in your mouth from unpleasant (1) to pleasant (5):”

This question allowed the test subjects to give their subjective opinion of the ergonomics of the “manadental” product after using it for 3 weeks. The answers were evaluated using a polarity scale ranging from unpleasant (1) to pleasant (5). On average, the product was rated 4.36 out of 5 in terms of ergonomics, which demonstrates that the “manadental” product is pleasant to use. (Fig. 5)

5.3   Effectiveness: Your subjective opinion of the effectiveness of “manadental” after 3 weeks

This question allowed the test subjects to give their subjective opinion of the effectiveness of the “manadental” product after using it for 3 weeks. They could choose from the following potential answers: My oral health has got worse. My oral health has remained the same. My oral health has improved. (Fig. 6)

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5.4.  Benefit: Would you be happy to continue using this product in your everyday life?

This question allowed the test subjects to give their subjective opinion of the possibility of continuing to use the “manadental” product after using it for 3 weeks. They could choose from the following potential answers: Yes. No. Unsure. (Fig. 7)

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6. Interpretation and summary

Previous in vitro studies have demonstrated that the photodynamic cleaning system has an impressive effect on the pathogenic oral bacteria Streptococcus mutans. This method reduces bacteria by approx. 5.5 log levels and is approx. 3 log levels (99.9%) superior to conventional mouthwashes.

The results of the in vivo effectiveness of the “manadental” photodynamic cleaning system, measured using the BoP parameter, show a highly significant improvement in the condition of the gums. (Wilcoxon test for paired samples: z = 4.627, p < 0.0001; Fig. 1; Fig. 2).

The average improvement in the BoP parameter during the observation period was 43.1%. Overall, an improvement in this parameter was identified in all test subjects, and there was no case of stagnation or of deterioration in the condition of their gums.

In consideration of the robust BoP parameter, it is noteworthy that these results are extremely clear.  Based on their clinical experience, the principal investigators and the specialists conducting the study consider the objective improvement during the observation period to be a success.

The results of the tooth colour card comparison suggest that using the photodynamic cleaning system does not lead to discolouration of teeth. In addition to the tooth colour card comparison, the principal investigators did not identify any partial discolouration of the enamel or the gums in the second examination. The principal investigators and the specialists conducting the study consider the slight brightening in tooth colour identified in 5 test subjects during the observation period to be insignificant. The steps in the scale were small (0.5-1 VITA® scale units) and were overwhelmingly identified with a brighter basic tooth shade (AX), which experience has shown can more easily be influenced by variable light in the room and test subject bias.

In this study, no side-effects were observed from use of the photodynamic cleaning system on the basis of the measured parameters, general observation of oral health and in the follow-up consultation in the final examination.

This study shows a highly significant improvement in gum condition in the test subjects after using the “manadental” photodynamic cleaning system for 3 weeks. No immediate side-effects of the photodynamic cleaning system were observed.