Abstract
Objectives
Intra-oral pH plays an important role in the pathogenesis of tooth erosion and decay, but there is limited information about its variation in real life settings. The aims of this research were to: 1) develop a wireless device, which can be used to continuously monitor intra-oral pH and temperature in real-time; 2) test and validate the device under controlled laboratory conditions; and 3) collect data in a natural environment in a sample of healthy volunteers.
Methods
A wireless device for measuring pH and temperature simultaneously was developed, calibrated and validated against the gold standard glass electrode pH meter. A smart phone was used as data logger. The wireless device was embedded in an oral appliance and worn by eleven participants (mean age 31.1 ± 6.9 years) for 24 h, while conducting standardised drinking tasks and regular daily activities.
Results
The wireless device could accurately measure pH and temperature both in vitro and in vivo . The recovery time following the swallow of a standard acidic drink varied markedly among individuals (mean = 1.3 ± 0.9 min). The intra-oral pH and temperature recorded in the natural environment also showed a large inter- and intra-individual variability. The average intra-oral pH when asleep (6.7 ± 0.5) was lower (p < 0.001) than when awake (7.2 ± 0.5). The average intra-oral temperature during sleep (35.6 ± 0.5 °C) was higher (p < 0.001) than when awake (34.5 ± 0.7 °C).
Conclusions
Intra-oral pH and temperature can be continuously and wirelessly assessed in real-life settings, and show individual-specific patterns with circadian variations. Intra-oral pH becomes slightly acidic during sleep while intra-oral temperature increases and fluctuates less.
Clinical significance
We propose a wireless device that is capable of measuring intra-oral pH over a 24-h period. We found marked inter-individual variation after acidic stimuli, and day to sleep time variation of both intra-oral temperature and pH. Our approach may provide new insight into the relationship between oral pH, tooth wear and decay.
1
Introduction
Chemical erosion of dental hard tissues can occur in almost one third of the general population . Its prevalence varies with age and is steadily increasing over time . The aetiology of dental erosion is multifactorial and results from an interaction between biological, chemical and behavioural risk factors . A prerequisite for the dissolution of tooth structure, however, is the lowering of intra-oral pH due to exposure of the oral cavity to acids of extrinsic and/or intrinsic origin . Extrinsic acids are mostly from dietary sources, but can also be found in the environment. Intrinsic acids are related to disorders of the gastro-oesophageal tract where gastric acids are regurgitated to the oral cavity . Intra-oral pH also plays a significant role in the pathogenesis of dental caries, with a critical pH (5.5⿿5.7), in which pH levels below this threshold will initiate the dissolution of enamel . In erosion, there is no defined critical pH as it is the dissolution of dental hard tissues without plaque. It has been suggested that erosion depends on the concentration of calcium, phosphate and fluoride in the solution and that dissolution of tooth mineral is initiated at pH levels below 4.5 with the calcium concentration normally present in saliva.
Erosive tooth wear is time dependent and the prolonged exposure to a lower intra-oral pH can greatly increase the risk of enamel dissolution . Despite the major role played by intra-oral pH in oral health, there is currently very little information about its normal and pathological variation over time . Indeed, conventional assessment of oral pH consists of collecting samples of un-stimulated or stimulated saliva over a limited period of time and measuring the pH. These assessments are not representative of in vivo values; there is loss of carbon dioxide from samples when they are removed from the mouth which results in an inaccurate (more alkaline) pH reading . pH values also depend on temperature, and this further complicates their measurement in the oral environment, as intra-oral temperature can vary markedly during the day . Intra-oral pH and temperature should therefore be assessed simultaneously over an extended period of time in a natural environment, where participants carry out normal daily activities. This setup poses significant technical challenges. With recent technological advances, however, small wireless biosensors can be attached to, or implanted in, the body so that physiological and pathological processes can be continuously assessed non-invasively and unobtrusively .
The first aim of this study was to develop a small intra-oral wireless device capable of assessing and transmitting pH and temperature values continuously and in real-time to a smart phone acting as a data logger. The second aim was to test and validate the wireless device under controlled laboratory conditions using a glass electrode pH meter as gold standard. The third aim was to collect real-time pH and temperature data in a group of healthy volunteers over an extended time period, by embedding the wireless device in an oral appliance. Data were collected in real-life settings, while participants carried out their normal daily activities such as drinking and sleeping. We hypothesise that intraoral pH and temperature are different when individuals are awake compared with when they are asleep.
2
Material and methods
2.1
Development of the wireless device
The wireless device ( Fig. 1 a) included a power supply printed circuit board (PCB; L ÿ W ÿ H: 14.0 ÿ 10.0 ÿ 0.8 mm) and a main PCB (L ÿ W ÿ H: 16.0 ÿ 10.5 ÿ 0.8 mm; Kamahi Electronics, Dunedin, New Zealand). The power supply PCB hosted a lithium magnesium di-oxide battery (CR1025, Renata, Switzerland), which provided sufficient power to collect data for >2 weeks. The main PCB included: an amplifier (Zerø-Crossover Rail-to-rail I/O operational amplifier, Texas Instruments, Dallas, Texas, USA); a microcontroller (PIC16FLF1825, Microchip Technology Incorporation, USA); a linear active thermistor (MCP9700AT, Microchip Technology Incorporation, Minnesota, USA); and an ANT + ultra-low power single chip (NRF24AP2, Dynastream Innovation, Trondheim, Norway). The pH and temperature measurements were transmitted to an ANT + -supported smartphone (Xperia M2, Sony, Tokyo, Japan) once every two seconds (0.5 Hz) using the Industrial, Scientific and Medical band (2.4 GHz). During each transmission, the radiofrequency transmitter only operated for 0.2 ms and otherwise remained off. This device fully complies with the New Zealand Standard for radiofrequency field exposure NZS 2772.1-1999. A small (ÿ = 2.4 mm) pH antimony electrode (Primus, Restech, San Diego, California, USA) was connected to the main PCB. The antimony electrode detects pH in aerosolised droplets without requiring full immersion or contact with fluid for electrical continuity .
2.2
Software application
An Android software application was developed for the calibration, monitoring and recording of pH and temperature by the smart phone. The application was designed to be user-friendly, showing real-time dynamic charts of pH and temperature values (Appendix B). The graphing library ⿿achartengine⿿ ( www.achartengine.org ) was used to create the graphs. On-phone calibration was based on the standard two-point temperature and pH calibration process. The application saved individual log files for each recording session, which included basic demographic data.
2.3
Device testing and validation
In vitro experiments were conducted to test the accuracy and precision of the device. These experiments were conducted with the wireless unit not incorporated in an oral appliance. The accuracy of the thermistor was tested against a laboratory thermometer (Brannan thermometers, Cleator Moor, Cumbria, England), while heating the wireless unit with a laboratory incubator (Laboratory equipment Ptw, Marrickville, New South Wales, Australia).
The effect of temperature on the antimony pH electrode was tested using pH 7.4 phosphate buffered saline solution (Sigma-Aldrich ® , St. Louis, Missouri, USA), in water baths set at 25, 35, or 45 ° C. The reading stability was tested by continuously measuring pH with the antimony electrode at a constant temperature (23.5 ° C) and pH (7.0) for 24 h.
The accuracy of the antimony electrode was tested against a standard glass pH electrode (PL-700 PVS, GOnDO Electronic, Taipei, Taiwan). The antimony and glass electrodes were firstly calibrated and then immersed in a pH 7.0 buffer solution. The buffer solution was then slowly titrated with either NaOH or HCl. At each titration step, the pH readings from the glass and antimony electrodes were recorded.
The effect of prolonged exposure of the electrode tip to desiccation and oxygen were also tested and reported elsewhere .
2.4
Study participants
A pilot study was used to estimate the required sample size. We aimed to detect a pH difference 0.5 between awake and asleep readings using a repeated measurements study design. Setting both α-error and β-error (95% power) to 0.05 (one-tailed test), we estimated that eleven participants were needed.
Participants (N = 11; mean age = 31.1 ± 6.9 years) were recruited among University of Otago staff and students. The following exclusion criteria were applied: smoking, gastro-oesophageal reflux disease, tooth wear, active caries, alcoholism, sleep disordered breathing, eating disorders, and intake of medication at enrolment. The study protocol was approved by the local Ethics Committee (H13/014) and a written informed consent was collected from all participants.
2.5
Oral appliance
The wireless device was embedded in a vacuum-formed oral appliance within two layers of hard-elastic polyethylene terephthalate glycol (PET-G; Duran ® 0.5 ÿ 125 mm, Scheu Dental GmbH, Iserlohn, Germany). The device was secured to the dental cast using soft silicone (Sil-Kitt Clear, Scheu-Dental GmbH, Iserlohn, Germany). The pH probes were positioned close to the palatal surface of the upper central incisors in eight participants and more distally in three participants. These sites were chosen because they are most frequently affected by erosive tooth wear . The two PET-G foils were sealed using two sealants (Duralay, Reliance Dental, IL, USA; and 215-CTH-UR-SR, Dymax, Torrington, Connecticut, USA). The oral appliance with the embedded PCBs was then smoothed to optimise fitting and improve comfort ( Fig. 1 b).
2.6
Procedure
The experiment consisted of a clinical and an experimental session separated by at least one week. During the clinical session, the subjects were informed about the procedures and clinically evaluated to assess their eligibility for inclusion in the study. Alginate dental impressions (Kromopan, Lascod, Florence, Italy) of the maxillary arch were taken and poured with stone (Vel-Mix Stone type IV, Kerr, USA).
Prior to commencement of the 24-h study, the wireless units were embedded in the oral appliance and then calibrated for pH and temperature measurements using the custom-made software application. The temperature measurements were calibrated by immersing the appliance firstly into cold water and then warm tap water. Then the pH measurements were calibrated using a pH 7.0 (±0.02) and then a pH 4.01 (±0.02) buffer solution (Restech, San Diego, California, USA).
Once the device was calibrated, the participants were required to perform two standardised tasks that included: 1) three swallows of 10 ml of still water (NZ Natural Spring, Frucor, Kaiapoi, New Zealand; pH 7.2), one swallow every 1 min; and 2) three swallows of 10 ml of orange juice (McCoy, Frucor; pH 4.4), one swallow every 5 min. Both beverages were at room temperature (21 °C).
After completion of the standardised tasks, participants were free to continue with their daily activities and were asked to wear the intra-oral appliance for a period of 24 h. They were also given instructions on how to use the smartphone software. The participants were requested to keep the smartphone within approximately 2 m of the wireless device at all times to ensure a stable connection. The smartphone vibrated in the event of signal loss thus alerting the participant to re-establish connection.
Beverage intake and the main activities performed were recorded via smartphone software as well as in a logbook. The participants entered the time when the appliance was removed and replaced back into the mouth, as well as the sleep time, which was defined as the time the bedroom light was switched off until the participants first opened their eyes in the morning. The software also allowed participants to record the start and stop time of other casual activities, such as drinking, showering or exercise. When the ⿿activity stop⿿ button was pressed, participants were prompted to enter the type of activity that had been done.
Participants were requested to wear the intra-oral appliance at all times except during chewing and tooth-brushing. When not worn, the appliance was stored in a plastic container and kept wet using gauze moistened in distilled water.
2.7
Data analysis
The wireless appliance transmitted (0.5 Hz) the following information to the smart phone: unit ID, a data stamp, raw pH, temperature, and remaining battery voltage. Signals were further processed on-phone by adding a time stamp, the calibrated pH and temperature signals, and four activity markers (sleep start/stop, device in/out, drinking, other activity), which were stored as binary data. Data collected were stored on phone as comma separated values (CSV) files for subsequent off-line analyses. These analyses included a data quality check with removal of pH values outside the optimal range of the antimony electrode, as determined from laboratory experiments (Appendix A). The activity markers were used to cut the signal portions where the intra-oral appliance was removed and to identify tasks, such as drinking, sleeping or other activities.
The mean individual intra-oral pH and temperature while awake and asleep were calculated by averaging the corresponding signals delimited by the sleep markers. The standardised swallowing tasks were assessed by calculating the minimum intra-oral pH value and the recovery time. This was defined as the time needed for the intra-oral pH to return to its baseline value after a swallow of a drink.
The data collected were analysed using conventional descriptive statistics, linear regression, and a linear mixed-effects model. The response variables of the mixed model analysis were ⿿pH⿿ and ⿿temperature⿿, the variables ⿿time⿿ (either asleep or awake) and ⿿participant⿿ was entered as fixed and random factor, respectively. All analyses were performed using SPSS (20.0, IBM, Chicago, Illinois, USA). Statistical significance was accepted at P < 0.05.
2
Material and methods
2.1
Development of the wireless device
The wireless device ( Fig. 1 a) included a power supply printed circuit board (PCB; L ÿ W ÿ H: 14.0 ÿ 10.0 ÿ 0.8 mm) and a main PCB (L ÿ W ÿ H: 16.0 ÿ 10.5 ÿ 0.8 mm; Kamahi Electronics, Dunedin, New Zealand). The power supply PCB hosted a lithium magnesium di-oxide battery (CR1025, Renata, Switzerland), which provided sufficient power to collect data for >2 weeks. The main PCB included: an amplifier (Zerø-Crossover Rail-to-rail I/O operational amplifier, Texas Instruments, Dallas, Texas, USA); a microcontroller (PIC16FLF1825, Microchip Technology Incorporation, USA); a linear active thermistor (MCP9700AT, Microchip Technology Incorporation, Minnesota, USA); and an ANT + ultra-low power single chip (NRF24AP2, Dynastream Innovation, Trondheim, Norway). The pH and temperature measurements were transmitted to an ANT + -supported smartphone (Xperia M2, Sony, Tokyo, Japan) once every two seconds (0.5 Hz) using the Industrial, Scientific and Medical band (2.4 GHz). During each transmission, the radiofrequency transmitter only operated for 0.2 ms and otherwise remained off. This device fully complies with the New Zealand Standard for radiofrequency field exposure NZS 2772.1-1999. A small (ÿ = 2.4 mm) pH antimony electrode (Primus, Restech, San Diego, California, USA) was connected to the main PCB. The antimony electrode detects pH in aerosolised droplets without requiring full immersion or contact with fluid for electrical continuity .
2.2
Software application
An Android software application was developed for the calibration, monitoring and recording of pH and temperature by the smart phone. The application was designed to be user-friendly, showing real-time dynamic charts of pH and temperature values (Appendix B). The graphing library ⿿achartengine⿿ ( www.achartengine.org ) was used to create the graphs. On-phone calibration was based on the standard two-point temperature and pH calibration process. The application saved individual log files for each recording session, which included basic demographic data.
2.3
Device testing and validation
In vitro experiments were conducted to test the accuracy and precision of the device. These experiments were conducted with the wireless unit not incorporated in an oral appliance. The accuracy of the thermistor was tested against a laboratory thermometer (Brannan thermometers, Cleator Moor, Cumbria, England), while heating the wireless unit with a laboratory incubator (Laboratory equipment Ptw, Marrickville, New South Wales, Australia).
The effect of temperature on the antimony pH electrode was tested using pH 7.4 phosphate buffered saline solution (Sigma-Aldrich ® , St. Louis, Missouri, USA), in water baths set at 25, 35, or 45 ° C. The reading stability was tested by continuously measuring pH with the antimony electrode at a constant temperature (23.5 ° C) and pH (7.0) for 24 h.
The accuracy of the antimony electrode was tested against a standard glass pH electrode (PL-700 PVS, GOnDO Electronic, Taipei, Taiwan). The antimony and glass electrodes were firstly calibrated and then immersed in a pH 7.0 buffer solution. The buffer solution was then slowly titrated with either NaOH or HCl. At each titration step, the pH readings from the glass and antimony electrodes were recorded.
The effect of prolonged exposure of the electrode tip to desiccation and oxygen were also tested and reported elsewhere .
2.4
Study participants
A pilot study was used to estimate the required sample size. We aimed to detect a pH difference 0.5 between awake and asleep readings using a repeated measurements study design. Setting both α-error and β-error (95% power) to 0.05 (one-tailed test), we estimated that eleven participants were needed.
Participants (N = 11; mean age = 31.1 ± 6.9 years) were recruited among University of Otago staff and students. The following exclusion criteria were applied: smoking, gastro-oesophageal reflux disease, tooth wear, active caries, alcoholism, sleep disordered breathing, eating disorders, and intake of medication at enrolment. The study protocol was approved by the local Ethics Committee (H13/014) and a written informed consent was collected from all participants.
2.5
Oral appliance
The wireless device was embedded in a vacuum-formed oral appliance within two layers of hard-elastic polyethylene terephthalate glycol (PET-G; Duran ® 0.5 ÿ 125 mm, Scheu Dental GmbH, Iserlohn, Germany). The device was secured to the dental cast using soft silicone (Sil-Kitt Clear, Scheu-Dental GmbH, Iserlohn, Germany). The pH probes were positioned close to the palatal surface of the upper central incisors in eight participants and more distally in three participants. These sites were chosen because they are most frequently affected by erosive tooth wear . The two PET-G foils were sealed using two sealants (Duralay, Reliance Dental, IL, USA; and 215-CTH-UR-SR, Dymax, Torrington, Connecticut, USA). The oral appliance with the embedded PCBs was then smoothed to optimise fitting and improve comfort ( Fig. 1 b).
2.6
Procedure
The experiment consisted of a clinical and an experimental session separated by at least one week. During the clinical session, the subjects were informed about the procedures and clinically evaluated to assess their eligibility for inclusion in the study. Alginate dental impressions (Kromopan, Lascod, Florence, Italy) of the maxillary arch were taken and poured with stone (Vel-Mix Stone type IV, Kerr, USA).
Prior to commencement of the 24-h study, the wireless units were embedded in the oral appliance and then calibrated for pH and temperature measurements using the custom-made software application. The temperature measurements were calibrated by immersing the appliance firstly into cold water and then warm tap water. Then the pH measurements were calibrated using a pH 7.0 (±0.02) and then a pH 4.01 (±0.02) buffer solution (Restech, San Diego, California, USA).
Once the device was calibrated, the participants were required to perform two standardised tasks that included: 1) three swallows of 10 ml of still water (NZ Natural Spring, Frucor, Kaiapoi, New Zealand; pH 7.2), one swallow every 1 min; and 2) three swallows of 10 ml of orange juice (McCoy, Frucor; pH 4.4), one swallow every 5 min. Both beverages were at room temperature (21 °C).
After completion of the standardised tasks, participants were free to continue with their daily activities and were asked to wear the intra-oral appliance for a period of 24 h. They were also given instructions on how to use the smartphone software. The participants were requested to keep the smartphone within approximately 2 m of the wireless device at all times to ensure a stable connection. The smartphone vibrated in the event of signal loss thus alerting the participant to re-establish connection.
Beverage intake and the main activities performed were recorded via smartphone software as well as in a logbook. The participants entered the time when the appliance was removed and replaced back into the mouth, as well as the sleep time, which was defined as the time the bedroom light was switched off until the participants first opened their eyes in the morning. The software also allowed participants to record the start and stop time of other casual activities, such as drinking, showering or exercise. When the ⿿activity stop⿿ button was pressed, participants were prompted to enter the type of activity that had been done.
Participants were requested to wear the intra-oral appliance at all times except during chewing and tooth-brushing. When not worn, the appliance was stored in a plastic container and kept wet using gauze moistened in distilled water.
2.7
Data analysis
The wireless appliance transmitted (0.5 Hz) the following information to the smart phone: unit ID, a data stamp, raw pH, temperature, and remaining battery voltage. Signals were further processed on-phone by adding a time stamp, the calibrated pH and temperature signals, and four activity markers (sleep start/stop, device in/out, drinking, other activity), which were stored as binary data. Data collected were stored on phone as comma separated values (CSV) files for subsequent off-line analyses. These analyses included a data quality check with removal of pH values outside the optimal range of the antimony electrode, as determined from laboratory experiments (Appendix A). The activity markers were used to cut the signal portions where the intra-oral appliance was removed and to identify tasks, such as drinking, sleeping or other activities.
The mean individual intra-oral pH and temperature while awake and asleep were calculated by averaging the corresponding signals delimited by the sleep markers. The standardised swallowing tasks were assessed by calculating the minimum intra-oral pH value and the recovery time. This was defined as the time needed for the intra-oral pH to return to its baseline value after a swallow of a drink.
The data collected were analysed using conventional descriptive statistics, linear regression, and a linear mixed-effects model. The response variables of the mixed model analysis were ⿿pH⿿ and ⿿temperature⿿, the variables ⿿time⿿ (either asleep or awake) and ⿿participant⿿ was entered as fixed and random factor, respectively. All analyses were performed using SPSS (20.0, IBM, Chicago, Illinois, USA). Statistical significance was accepted at P < 0.05.
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