Materials and Methods
Approval from Research Ethics Committee
This study protocol was approved by the Kocaeli University Ethical
Committee of Clinical Research, Non-Invasive Clinical Research Ethical
Committee (KOU GOKAEK 2019/10.34) and was conducted following the
Declaration of Helsinki of 1975, as revised in 2000. All participants
signed an informed consent form for being included in the study. This
clinical trial is registered at ClinicalTrials.gov as NCT04853745.
Sample selection and processing of teeth
The patients were recruited from individuals seeking periodontal and/or
dental treatment at the Faculty of Dentistry, Kocaeli University,
Kocaeli, Turkey. The teeth samples were collected from the participants
who met the following criteria: being older than the age of 18, having
no history of any infectious disease such as hepatitis and/or HIV (+),
being willing to participate in the study, having a single-rooted tooth
with a gingival recession on all surfaces and need to be extracted, the
tooth must be free of caries, defects, and restorations.
All patients who met the criteria were informed about the study.
Patients who gave written informed consent were included in the study.
Before the teeth extractions, root surfaces were marked all around with
a bur at the gingival margin. Extractions were performed under local
anesthesia with articaine hydrochloride and epinephrine 1:200,000
(Ultracain® D-S, Sanofi Aventis, Istanbul, Turkey).
Following the extractions, tooth surfaces were cleaned with an
ultrasonic scaler (Woodpecker® Medical Instrument Co.
Ltd., Guilin, China) to remove deposits and soft tissue remnants and
were inspected under 2.7X magnification
(Orangedental®, Biberach, Germany). They were
sectioned using a metal disc attached to the handpiece by gingival
margin level. Extracted teeth were collected and were randomly divided
into 3 groups as follows: sodium bicarbonate group,glycine group, and erythritol group. The samples were stored in 1%
thymol.
Evaluation with Micro-CT scanning
Samples were scanned before and after polishing powder application with
a high-resolution micro-CT device (Bruker SkyScan 1275, Kontich,
Belgium). The scanning conditions were 100 kVp; 100-mA, 0.5-mm Al/Cu
filter; 13.1-μm pixel size; and rotation at 0.2 steps. According to the
manufacturer’s instructions, each sample was rotated 360°. The mean
scanning time was around 1h. Also, to measure tissue mineral density
(TMD), two different mineral concentrations of conical MD phantoms
(rods) of 0.25 and 0.75 gHAp cm3 were placed next to
the samples, and the scan was repeated at the same conditions at
baseline and after the polishing procedure.
Tooth surfaces instrumentation
The same air-polishing device (AIR-FLOW® Master Piezon, EMS SA, Nyon,
Switzerland) was used for all powder instrumentations. Three types of
air-polishing powders were used: sodium bicarbonate-based particles
(CLASSIC®, EMS SA, Nyon, Switzerland ), glycine-based
particles (PERIO®, EMS SA, Nyon, Switzerland ), and
erythritol-based particles (PLUS®, EMS SA, Nyon,
Switzerland). All surfaces were numbered, and application was made to
the mesial and distal surfaces of the root, buccal and lingual surfaces
of the crown to avoid repeated instrumentations. After the device and
samples were fixed, a metal plate with a 5 mm diameter hole was placed
on the sample to limit the application area. Surfaces one and three were
air-polished at medium power setting (9 LED power setting) and surfaces
two and four were air-polished at maximum power setting (17 LED power
setting). The distance between the handpiece and the tooth surface was
kept constant at 5 mm, and the treatment angulation was adjusted to
600. In all applications, the application time was 5
seconds, and the water setting was medium (6 LED). The powder chambers
of the device were filled to the maximum level in each application.
Micro-CT image analysis
The special software (NRecon ver. 1.7.4.2, Skyscan, Kontich, Belgium)
was used for reconstructions. For the reconstruction parameters, ring
artifact correction and smoothing were fixed at 7 and 3 in the order,
and the beam artifact correction was set at 40%. 16-bit
gray-value images were obtained. Reconstructed images were superimposed
using another software (DataViewer ver. 1.5.6.2, Skyscan,
Kontich, Belgium). The pre-application images and the incisal edges and
buccal cusps of the teeth in this image were chosen as a landmark to
align the pre-application and post-application images. The reference and
target images were superimposed, and a different image was obtained from
the different areas of the two images. This image represented the change
in the area after the air-polishing. After reconstruction, the
interpolated regions of interest (ROI) were drawn using the software
(CTAn ver. 1.16.1.0, Skyscan, Aartselaar, Belgium ). Using these ROIs,
defect depth, defect volume, and demineralization depth were measured.
Thresholding was applied to each of the images. The threshold was set as
follows: the lower limit was set between 20 and 255 (in grayscale
values), and the upper limit was set at the top end of the brightness
spectrum. A multilevel Otsu threshold was used.
To evaluate TMD, ROIs were manually drawn to include the enamel and
exposed root surface which is cementum. Other regions were excluded from
the ROI selection. The ROIs were subtracted from the original image by
software (CTAn ver. 1.16.1.0, Skyscan, Aartselaar, Belgium ) to allow
average gray value and density analysis, and TMD was calculated. To aid
TMD calculations, grayscale values were converted into MD values (gHAp
cm3) with a linear calibration curve based on
grayscale values obtained from two mineral concentration conical
phantoms of 0.25 and 0.75 gHAp cm3.
Statistical analysis
Power analysis was performed using software (G*Power Software version
3.1.9.2, Düsseldorf, Germany) to estimate the minimum required sample
size. Sample counts were calculated at 12 for each group. Type I error
and test power was set to 5% and 90% respectively. Considering the
losses that may arise during sample preparation, the sample size was
determined to be 48.
The data were analyzed using commercially available statistical analysis
software (MedCalc Statistical Software version 12.7.7, MedCalc Software
bvba, Ostend, Belgium).
Descriptive statistics that were used to analyze the results included
the mean, standard deviation, median, minimum, and maximum. Kruskal
Wallis test was used to compare differences in more than two independent
variables. Mann-Whitney U test was performed to compare differences
between two independent variables. Wilcoxon test was used to compare
differences between two dependent variables. P -values less than
0.05 were considered statistically significant.