This clinical pre-post study with a follow-up of 12 months was registered at clinical trials (NCT03198832-02/06/2017). All procedures received approval from the Human Research Ethics Committee of Institute of Science and Technology (ICT) UNESP in São José dos Campos– São Paulo, Brazil (n01.504.963).
For this study, 356 diabetic patients were screened from May 2016 until August 2017. Forty-eight were included in the pre-established criteria (inclusion and exclusion), of which 24 patients Group 1 had Periodontitis stage III and IV, grade modifiers B and C and 24 patients Group 2 had Gingivitis -dental biofilm - induced mediated by systemic or local risk factors. Considering our previous results11(Monteiro et al.), a sample size of 22 patients per group would be necessary to detect variations of 0.12 units of moLDL between the groups, with standard deviation 0.14. By using a two-tailed test of variance we have type I error α = 0.05, type II error β = 0.2, and a power of 80%. Twenty-four patients per group were, then, included in the study.
These patients came from the ICT– UNESP (State University of São Paulo Júlio de Mesquita Filho) dentistry school in São José dos Campos – São Paulo, Brazil.
Included in the study were individuals older than 35 years who had DM2 diagnoses for more than five years; had A1c levels between 7% and 11%; and had been diagnosed with periodontitis or gingivitis. They must have presented with the loss of clinical insertions >3 mm in two non-adjacent teeth and the loss of clinical insertions ≥5 mm in 30% or more of the teeth present20, and they must have had at least 20 teeth present. They had to agree to participate in the study and sign the informed consent form. Patients with cardiovascular disease, cancer, gastrointestinal disorders, skin diseases, pregnancy, lactation, smoking habit, arthritis, lupus; those who had undergone periodontal treatment within the past 12 months; those who had made use of antioxidant supplements, anti-inflammatories, or antibiotics within the previous three months; those who had changed their glycemic control medication during the past three months; and those with any other diseases of inflammatory origin were excluded.
All of the participants received oral hygiene instructions (Bass brushing technique and the use of dental floss or interproximal brushes), and an experienced clinician (JFP) obtained periodontal parameters. The diabetic patients with periodontitis received full-mouth scaling and root planning with an ultrasonic device (Prof Neo, Dabi Atlante-BR, TipPerio Sub-EVMWQHED3 Ribeirão Preto, SP, Brazil) and manual curettes (Millennium, Golgran, SP Brazil). These procedures were performed under infiltrative local anesthesia with mepivacaine with epinephrine (Mepivalem AD Dentsply Catanduva, SP, Brazil40/0.005 mg/mL) in a single session21. Diabetic patients with gingivitis received supra-gingival scaling and prophylaxis in a single session. Every three months, patients from both groups received maintenance therapy through prophylaxis.
Clinical parameters measured were: probing depth (PD), clinical attachment level (CAL), gingival resection (GR), bleeding on probing index (BOP) and plaque index (IPL). A trained examiner (JFP) performed the clinical measurements (Kappa 0.86) using a 15-mm standardized manual periodontal probe (UNC® Hu-Friedy, Jacarepaguá - Rio de Janeiro, Brazil). The examiner's calibration was done as follows: the examiner measured the PS and the NIC of 10 patients twice in a 24-hour interval. Then the measurements were submitted to the intra class correlation test and the examiner was judged calibrated if a 90% agreement in the measurements was reached. This procedure was repeated until the examiner reached this index.
After anamnesis and the collection of clinical measurements, all patients were referred to the clinical analysis laboratory for the initial blood collection process. Blood was collected into Vacutainer tubes (Vacutainer, BD do Brasil, Curitiba, PR, Brazil) containing ethylenediaminetetraacetic acid from each individual after 12 h of fasting at baseline and then six months and 12 months after the treatment, and the laboratory was blinded to study groups. The first baseline blood collection for laboratory analysis was performed prior to periodontal treatment. The following blood collections (6 and 12 months) were performed 6 and 12 months after the date of periodontal treatment.
The following analysis were: overnight fasting glucose, high-sensitivity C-reactive protein (hs-CRP), glycated hemoglobin (A1c), total cholesterol, HDL-c, LDL-c, triglycerides (TG) and oxidized low-density lipoprotein (oxLDL) level. Plasma was obtained following the centrifugation of the blood at 1000 g for 10 min at 4 °C and was stored at −80 °C. Details about the detection of the oxLDL in the plasma may be encountered in Monteiro et al.11
Samples preparationBlood from patients were centrifuged at 103 g and 4 °C for 15 minutes to isolate the plasma. Benzamidine (2 mM), gentamicin (0.5%) and chloramphenicol (0.25%), PMSF (phenyl–methyl–sulfonyl–fluoride) (0.5 mM) and aprotinin (0.1 unit/mL) were added. The LDL-c (hereafter called the "LDL solution sample" for the optical experiments – ZS and absorbance measurements) was isolated from the infranatant (that contains HDL cholesterol, albumin and other proteins present in the plasma) via sequential preparative ultra-centrifugation22 at 105 g and 4 °C using a Hitachi Ultracentrifuge. The protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA) with bovine serum albumin as the standard.
Z-Scan techniqueThe LDL-c sample is illuminated by a laser beam (wavelength λ = 532 nm). Periodic on and off states of about 30 milliseconds of light irradiation are employed. The pulsed-light beam is focused at the position of z = 0 via a lens. All Z-scan experiments were performed at physiological temperature (37 °C). The sample moves along the z-axis, from a position before the focus until a position after the focus. The accuracy of ZS measurements depends on the intensity stability of the laser, the Gaussian profile of the beam, the setup optical alignment and the uniformity of the sample23. To avoid spurious effects in our measurements, the laser beam intensity was monitored splitting the beam in two. One of them goes directly to a detector to measure the laser intensity and the other goes to the ZS setup. Eventual fluctuations on the intensity during the experiments are corrected by using the information from the first beam. The second beam enters in the ZS setup and passes th rough the sample. The transmitted beam is detected by a detector with an iris positioned at its front. At least 10–15 independent measurements (at fixed z position) are made to achieve good data statistics. The reproducibility of our results were checked by measuring the same sample in different days, obtaining results within the experimental errors. More details about the setup, data acquisition and data treatment can be found in our previous works13,24. The typical result in the ZS experiment is a peak to valley (or valley to peak) curve of the normalized transmittance as a function of the sample z position. The peak to valley amplitude is proportional to the phase shift (θ) of the thermal lens formed. This parameter depends on the sample absorption coefficient (α) (which is proportional to the concentration of light absorbers), thermo-optical coefficient (dn/dT), laser power (P) and sample thickness (L), and it is inversely proportional to the thermal conductivity (k) of the sample:
$$\theta =\frac{0.24P}{k\lambda }\frac{dn}{dT}(1-{e}^{-\alpha L})\cdot $$
(1)
In this study, each patient and, correspondingly, each sample had different LDL-c concentrations, so we normalized the values of θ to compare the results from various patients. The parameters of α, P, L and dn/dT were measured independently. Therefore, the ZS allows for the measurement of the sample thermal conductivity, which is a characteristic of the LDL solution.
UV-vis spectroscopyThe linear-absorbance spectra were obtained via a Uv-vis spectrophotometer with a wavelength range from 200 nm to 1100 nm. Samples were placed into a quartz cuvette with an optical-path length of 1 cm. All experiments were performed at physiological temperature (37 °C). The spectrophotometer measures the extinction spectrum, which is the sum of both the Rayleigh scattering and the absorbance. The Rayleigh scattering is proportional to λ−4, and its intensity is estimated for each one of the samples25. The absorbance is then calculated removing the scattering contribution from the extinction spectra. As mentioned before, the LDL-c contains various molecules, and each of them has different absorption spectra. For instance, the maximum light absorption of the APOB-100 is at λApo ≈ 280 nm26,27, the cholesterol at λchol < 200 nm28, the α-Tocopherol at λα-Toc ≈ 210 nm and the carotenoids at λβ-Car ≈ λα-Car ≈ 440 and 480 nm16,17. I n this study, we specifically investigated the absorbance values measured at λ = 480 nm (one of the wavelength at the maximum of absorbance of Carotenoids), at various times of blood extraction, for example, baseline and after six and 12 months.
Statistical analysisThe data were consolidated and made available on the mean value ± standard deviation (SD) in the case of normal distribution or on the median ± interquartile range otherwise. The evaluation of the normality of the data was made by using the Shapiro-Wilk test. The values of the clinical and laboratorial parameters were compared depending on the case by using: t-test, χ2 test, Mann-Whitney Rank Sum Test, one-way repeated measures ANOVA/Tukey Test and Friedman test.
Ethical approvalAll procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Informed consentInformed consent was obtained from all individual participants included in the study.
