Laser Doppler Flowmeter as a Periodontal Evaluation Method: A Clinical Pilot Study

Background and objectives: Periodontal disease, as an inﬂ ammatory pathology, induces hemodynamic changes that can be evaluated by diﬀ erent unbiased methods such as laser Doppler ﬂ owmetry. This clinical investigation assesses laser Doppler as a non-invasive procedure to monitor gingival vascularization and its potential relationship with the response to treatment of periodontal disease. Materials & methods: 45 sites of white Spanish patients with active periodontitis undertake a complete periodontal analysis. This included periodontal pathogens identiﬁ cation along with the monitoring of the gingival margin microvascularization using a Doppler laser at the points exhibiting the most periodontal damage. All assessments were performed before and after periodontal combined treatment PCT (scaling, root planing, and antibiotic therapy prescription) ( n = 45 sites). Results: Parameters of periodontal disease showed a positive correlation with pathogen levels. Blood ﬂ ow readings decreased signiﬁ cantly after PCT ( p < 0,05), although this parameter was not statistically correlated with periodontal nor microbial assessments in a signiﬁ cant range. Conclusion: Laser Doppler is a complementary method of monitoring periodontal inﬂ ammation to traditional techniques of clinical periodontal evaluation. Further studies are necessary to determine its usefulness as a predictive method of periodontal disease evolution.


Introduction
Periodontitis is the main cause of dental loss in the adult population.However, diagnosis of periodontal disease is still based upon subjective clinical examination procedures, which are time-consuming and poorly implemented in general dental practice [1].Research into periodontal disease is one of the leading topics in dental knowledge.Special attention has been focused on new innovative methods in periodontal diagnosis, speci ically built on the momentum of the genomic and proteomic era, as well as advances in cell biology and cell signaling upon periodontal diagnosis and therapy.Considering the essential need for translation of basic research into of ice practice, and the thorny issue of how cost-effective periodontal therapy is, new approaches in parameters for

Abstract
Background and objectives: Periodontal disease, as an infl ammatory pathology, induces hemodynamic changes that can be evaluated by diff erent unbiased methods such as laser Doppler fl owmetry.This clinical investigation assesses laser Doppler as a non-invasive procedure to monitor gingival vascularization and its potential relationship with the response to treatment of periodontal disease.
Materials & methods: 45 sites of white Spanish patients with active periodontitis undertake a complete periodontal analysis.This included periodontal pathogens identifi cation along with the monitoring of the gingival margin microvascularization using a Doppler laser at the points exhibiting the most periodontal damage.All assessments were performed before and after periodontal combined treatment PCT (scaling, root planing, and antibiotic therapy prescription) (n = 45 sites).
Results: Parameters of periodontal disease showed a positive correlation with pathogen levels.Blood fl ow readings decreased signifi cantly after PCT (p < 0,05), although this parameter was not statistically correlated with periodontal nor microbial assessments in a signifi cant range.

Conclusion:
Laser Doppler is a complementary method of monitoring periodontal infl ammation to traditional techniques of clinical periodontal evaluation.Further studies are necessary to determine its usefulness as a predictive method of periodontal disease evolution.
periodontal diagnosis and treatment monitoring are of paramount relevance.
Periodontal health is de ined as the absence of in lammation even in a reduced periodontium.In lammation constitutes a paramount sign of periodontal disease and the irst step in the cellular and humoral immune response [2].Periodontal pathogens and their invasion of gingival epithelium cells, alter periodontum in two ways: A.-causing tissue destruction; and B.-producing harmful substances that, during tissue damage, act as proin lammatory cytokines that stimulate the in lammatory response [3][4][5][6].Early changes of this defensive reaction occur at a microvascular level as a form of angiogenesis, due to dilatation of the capillaries and an increase in the number of them [7,8].This can be transferred to both the initial gingivitis and chronic in lammatory periodontal processes.
Several studies employ laser Doppler lowmetry (LDF) as a non-invasive method to assess tissue vascularity.This technique can be used in different microvascular systems such as skin, colonic, muscular, gingival, pulpal, and oral mucosal tissues [9][10][11][12][13][14][15][16][17][18][19][20][21][22][23].An animal study measured the in luence of irradiation on decreased jaw-bone vascularization, validating LDF to monitor alveolar bone vascularity [24].The establishment of a normal values range for bone micro-vascularization may reduce the risk of osteoradionecrosis in implant treatment of irradiated patients [25].Normal blood low velocity values could be calculated relative to periodontal tissues, as recently reported for eye microvasculature [26].In Periodontics, LDF appraisals of tissue response to either basic periodontal treatment (scaling and root planning) [27,28] or surgical approach [18,19] have proved laser Doppler readings to be positively correlated to gingival in lammation reduction [28][29][30].Other studies that focus on gingival micro-vascularization differences between smokers and non-smokers failed to ind a lower gingival blood low in non-smoking patients with the same degree of periodontitis.Tobacco has not proved to be capable of causing vasoconstriction in oral tissues, contrary to clinical observations in which there is a lower tendency to bleeding on probing in this group of patients [18,21].
Eventually, it could be possible to establish a threshold in periodontal vascularity value beyond which periodontal bone resorption could be triggered.As a matter of fact, impartial clinical decisions in periodontal diagnosis and treatment outcomes might be in luenced by assessing micro-vascularity in periodontal tissues.Hence, clinical implementation of laser Doppler readings may increase the predictability of periodontal treatments.However, to prove this method to be useful in human beings, normal values of human periodontal vascularity measured by LDF constitute a pre-requisite.As far as we know, no study has considered how the presence of periodontal pathogens in luences gingival blood low/gingival in lammation.This study tackles the evolution of the gingival blood low after combined therapy with basic periodontal treatment and antibiotics.

Study design and population
This pilot study includes a total of 9 white Spanish patients (4 women and 5 men) between 26 and 71 years old (mean 44.67, standard deviation SD 13.17).All participants sought periodontal treatment in private practice and exhibited periodontal biotype medium [31] and disease stage III and IV, grade C [32].It was distinguished between smokers and nonsmokers.
The following exclusion criteria were considered: patients with a history of excessive consumption of drugs and/ or alcohol, pregnant or breastfeeding, infectious diseases, intravenous bisphosphonates treatment, antibiotic treatment for less than 2 months before starting with the irst part of the study, diabetics, chemotherapy, head or neck irradiation, hematologic disorders, as well as in psychiatric patients.
The protocol study complies with the ethical precepts formulated in the Declaration of Helsinki of the World Medical Association on the ethical principles for medical research in human beings and in their subsequent revisions, as well as those requirements for the applicable legal regulations.It was approved by the clinical research ethics committee of the San Carlos Clinical Hospital in Madrid (C.I. 17/129C).All patients signed an informed consent of the medical process and its inclusion in the research study.
The timeline of the experiment (Figure 1) had three checkpoints: 1. Periodontal study which included periodontogram, periodontal pathogens identi ication, and pre-treatment blood low laser Doppler readings at the 5 sites exhibiting the greatest periodontal disease activity; 2. One-stage scaling and root planning treatment followed by antibiotic prescription (Metronidazole 500 mg / 12h / 7 days [33,34]), 3. Eight weeks later, all parameters were re-evaluated to assess the correlation between clinical intervention, microbial counts, and LDF readings.

Periodontal evaluation
Clinical data from patients included an exhaustive anamnesis form, orthopantomography (OPG), and standardized periapical radiographic series [35].The periodontal chart was ful illed using a CP-12 periodontal probe, and included assessments of probing depth (PD), bleeding on probing (BOP) scored according to the Löe and Silness criteria, plaque index (PI) recorded by the O'Leary plaque index, clinical attachment loss (CAL) and recessions (REC).All these measurements involved six tooth aspects (mesiobuccal, buccal, distobuccal, mesio-lingual, lingual, and disto-lingual) [36][37][38][39][40]. Periodontogram also came with tooth mobility, sulcular suppuration, and furcation defects.blood low with a sampling frequency of 40 Hz and a depth of 1 mm.Doppler effect consists of the apparent frequency change of a wave produced by the relative movement of a source with respect to an observer.In the blood low, by striking a beam of light on any human tissue, it is dispersed both by the static structures and by the red blood cells.Those beams returned by the red blood cells suffer a deviation in their frequency that is enlarged, however, those that affect the static structures are not modi ied.Both light fractions are captured by a photodetector and processed to determine blood low [24].

Microbiological testing of periodontal microorganisms
The ive sites with higher PD values from periodontal evaluation were allocated for bacterial sample collection.Protocol for sulcular crevicular luid bacterial samples included supragingival extensive drying and plaque removal with sterile cotton pellets.
The processing of the samples was carried out in the Microbiology and Molecular Virology Unit of the Analysis Laboratories Dr. Echevarne (Barcelona, Spain) within 24 hours.For the identi ication of periodontal pathogens, in samples of crevicular luid, a conventional polymerase chain reaction (PCR) was performed followed by identi ication by reverse hybridization on a colorimetric strip.The microIDent® kit (Hain Lifescience, GmbH, Nehren, Germany) was used, based on DNA • STRIP® technology, which allows the semi-quantitative detection of Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf), Treponema denticola (Td) and Prevotella intermedia (Pi).
For this, a multiplex PCR was carried out, using complementary oligonucleotides of fraction 16 of the rDNA corresponding to these microorganisms, followed by simultaneous reverse hybridization on a nylon strip with colorimetric labeling.Multiplex ampli ication was accomplished with biotin-labeled primers.The reverse hybridization procedure was performed according to the instructions of the manufacturer of the microIDent® kit (Hain Lifescience, Nehren, Germany).Each strip supplied by the manufacturer of the microIDent® test has a total of 7 reaction zones: 2 corresponding to quality control (Conjugate Control (CC) and Ampli ication Control (AC)), and 5 speci ic bands for each bacterial species (Aa, Pg, Tf, Td and Pi) (Figure 2A).The results were determined according to the intensity of staining of each line in the speci ic bands, corresponding to each bacterium (Figure 2B).The degree of staining of each band was represented by crosses (Figure 2C), following a scale from lower to higher: (+), +, ++, +++, which the commercial company makes correspond with a speci ic concentration for each bacterium (Figure 2D).The absence of staining was considered negative, indicating that the sample contained less than the detectable level of nucleic acid for the target microorganism.The detection limit was determined by the manufacturer and corresponded to 10 3 genomes, in the case of Aa, and 10 4 genomes for Pg, Pi, Tf, and Td.

Micro-vascular fl ow assessments
Laser Doppler Flowmeter Figure 3 (Moor VMS-PC®, Moor Instruments Limited UK) surveyed blood low at the periodontal gingival margin.The probe used in this experiment displayed a 1.5 mm diameter and a iber distance of 0.5 mm (VP3 needle-shaped probe) Figure 4.This lowmeter is a semiconductor diode laser with a wavelength of 780 nm, based on the Doppler effect that provides continuous monitoring of   Flowmeters measure blood low in perfusion units (PU) as the product of the average speed and concentration of blood cells in a single volume of tissue."Standard Motility" consisting in a low concentration of polystyrene microspheres in water submitted along thermal movement (brownian movement) calibrates the probe.A memory chip in the probe stores igures from the calibration process.Probe positioning on the gingival margin was stabilized by means of a customized splint in order to sustain measurement reproductivity.Splint is important to minimize scattering from structures other than blood cells that could generate Doppler changes and produce an indistinguishable signal from that caused by blood low itself.LDF readings were recorded during an interval of 20 seconds (Figure 5).Blood low assessments were performed at each site independently so that positioning in the split did not scatter readings during probe recording.
Factors such as room temperature and brightness are confounding factors for LDF readings and may cause bias in results.Since low temperatures (< 15 ºC) reduces signi icantly Doppler laser lowmeter signal, the room was maintained at all times in a 23 ºC -26 ºC range during procedures.The dental chair was switched off and exposure from outside light was controlled to minimize probe scattering from external light energy sources.

Statistical analysis
Statistics software SPSS® was used to perform data analysis.All measurements were described by means and SD, statistical signi icance was pointed for a p < 0.05.Blood low was considered a dependent variable and compared with indirect variables such as gender, age, toxic habits (smoke), PD, BOP, PI, CAL, and periodontal pathogens presence.Measurement values at different timeline points were analyzed by the T-Student test for paired samples, using the range with Wilcoxon signs when appropriate.Pearson correlation coef icient was used to determine the correlation between blood low (PU) and the rest of the parameters.Differences between subjects (smoker or not) were compared by means of the Mann-Whitney U test.
Data analysis revealed signi icantly lower PU in female patients compared with male patients at T1(week -1) and at T2 (week 8).T-Student Test showed also a decrease in PU values statistically signi icant in female patients (p < 0.05) after PCT.
Pearson's correlation test indicates a statistical signi ication comparing microvascular readings with age, younger patients exhibited bigger difference in LDF readings after PTC than older ones (p = 0.016).
No statistical differences were found between smokers or not in microvascular readings previous or after periodontal treatment (Mann-Withney U test p > 0.05) (Table 1).

Periodontal parameters and blood fl ow readings (PU)
Men had worse indicators of periodontal health than women, even this was not statistically signi icant.Eight weeks after PCT, all patients had improvements in all periodontal parameters, in terms of pocket reduction, gain of clinical attachment level and reduction in full mouth bleeding and plaque scores.Probing depth decreased mean -2.08, SD 1.51 p < 0.05.The full mouth bleeding on probing percentage was 65% in week -1 and after PCT (week 8) was 27.44%.In addition, the plaque index was signi icantly reduced and the mean percentage of plaque in the whole mouth went from 79% to 48% after PCT.Regarding CAL, it experienced an average improvement of -2.08 SD 1.51.Pearson's correlation tests did not show a signi icant correlation with the decrease in LDF (p > 0.05) (Table 2).

Periodontal pathogens count and periodontal and microvascular parameters
Pearson's correlation test showed a signi icant reduction in all periodontal pathogens (p = 0.000) after PCT, except for the reduction in Td, which was not statistically signi icant.
A positive relationship could be established between certain pathogen reductions and PU levels, as well as plaque index and bleeding on the probing index (Table 3).

Discussion
Until today, the most used methods for evaluation and monitoring periodontal disease are indirect procedures based on clinical evaluations and determinations of the subgingival micro lora.The direct methods used previously required invasive techniques such as biopsies, which cause an irreversible change in the tissues studied [41].
Several studies in animals have shown that blood low is greater in an in lamed gingiva than in a healthy one due to blood stasis [42,43].Likewise, when gingival in lammation occurs in humans, changes are detected in the marginal gingiva, consisting of an increase in the number of visible vessels, among others [44].These changes never reverted back to the original blood low healthy pattern even when in lammation had resolved [45].Therefore, the use of blood low as a predictive indicator of future healing and prognosis of periodontal disease is a concept worth pursuing [46].
In the present experiment, the blood low in the marginal gingiva was notably greater before PCT than 8 weeks after it, having produced both a clinical and statistical reduction in the evaluation parameters of gingival in lammation and periodontal status, considering in some cases the site studied without gingival in lammation (no BOP, PD£3mm).The classic diagnostic parameters of PD, BOP, and CAL, as well as their effectiveness in the monitoring of periodontal disease, have been widely described.It was possible to establish a positive correlation between these parameters and gingival blood low in the marginal and intracellular gingiva at the disease sites compared to healthy ones.This relationship is also directly proportional to the severity of periodontitis, increasing with greater severity [27,47].In this study, we also have found a positive relationship, although not statistically signi icant, between the classic parameters to evaluate periodontal disease and blood low, the analysis of the data shows a signi icant decrease after treatment in all parameters, both periodontal and low of blood.
A possible limitation in the data collection process was the size of the probe.The gingival LDF signal is dominated by the low mainly from the super icial vessels, so with the current wavelength of the laser light and the construction of the probe, it did not allow us to obtain records of the blood low within the periodontal pocket, where the irst in lammatory changes occur and the largest number of pathogens are present.
All patients included had established stage III and IV and grade C periodontitis, but it would be interesting to focus future research on the evolution of blood low in relation to in lammation development since greater readings have been described in chronic periodontitis than in the initial gingivitis [28].
Probe splinting or manual blood low measurement was a controversial issue in previous studies [28,48].It has been reported higher measure values when performed by hand than splinting, which may be due to the pressure of the splinting material that compresses the gingiva.For this reason, in this study, the measurements were always carried out by the same operator with a customized splint, seeking not to interfere with the natural vascularization conditions by not contacting the gingival tissue and avoiding previously performed injections of anesthesia with a vasoconstrictor Figure 6.
Comparison with previous studies was hampered by the variety of Doppler lasers used with different calibration constants and units of measurement, as well as by the different study protocols.Even so, the minimum time required to measure blood low was established, in line with these studies, at 20 consecutive seconds per monitored site [21,[49][50][51].Other studies based on microvascular low assessments, employed different methods such as Optical Coherence Technology Angiography (OCTA) [52].This technique, although non-invasive, is also limited by its expensiveness, slow acquisition time, and small ield of imaging among others [53].Fluorescein angiography (FA) has considerable shortcomings, such as being invasive, having a long image acquisition time, and not providing quantitative data.Also, near-infrared spectroscopy provides deeper tissue information on blood low but it's not vessel-speci ic [54].
LDF is a proven monitoring tool of vascularization in other human tissues as alveolar bone [25].The main application will be evaluating the osseointegration of dental implants, making microvascularization a crucial factor in implant stability.Human besides animal studies showed that LDF is an adequate method for bone microvascularity evaluation and might determine future implant success [55].Therefore, laser Doppler lowmetry seemed to be a valid option in the search for new direct and non-invasive methods of microvascular low measurements.
One of the factors that in luence the progression of periodontal disease is the combined action of related pathogens, capable of causing changes in the total oral microbiota, altering the homeostatic balance of the tissue [55][56][57][58].Above all, the presence of the Socransky red complex bacteria (dominant in the late stages of plaque development and mainly found in cases of periodontitis in adults) is strongly associated with parameters of periodontal in lammation, including PD and BOP [56,59].The presence of these pathogenic species has also been detected in healthy individuals, so for periodontal disease to manifest itself clinically, in addition to a subgingival microbiota shift (favoring excessive growth of pathogenic species), a suitable environment and susceptible host are essentiall [57,60].
Recent studies have focused on the role of innate immunity in the pathogenesis of periodontitis [61].The inal immune response occurs at the local level, being essential for microbiota regulation, by stimulating microenvironmental changes and limiting the increase in periodontal pathogens that help the most protective bacteria to predominate [62].Despite the fact that, in this study, the relationship between the decrease in periodontopathogenic species in the studied sites and the evolution of blood low and in lammation parameters appears to be positive, it lacks suf icient statistical signi icance.This supports the theory that, although the role of periodontopathogenic bacteria in the development of periodontal disease is undeniable, it is not exclusively an infectious but an in lammatory pathology, with an essential role in the host's immune system.
Since the exact mechanisms of the pathogenesis of periodontitis remain unclear, and multiple factors can destabilize this balance contributing to the pathogenesis of periodontitis, the counterbalance of different diagnostic parameters results in challenging for such a multifactorial disease.

Conclusion
In light of the indings of this study, LDF is a feasible method for gingival vascularization assessments, which in combination with traditional periodontal parameters, provides useful information for periodontal disease diagnosis and treatment monitoring.
Especially in the most clinically affected sites, a reduction in the means of blood low readings was detected, as well as the correlation between the parameters of the evaluation of periodontal disease and the number of pathogens found.
Further studies are necessary to focus on its usefulness as a predictive method of the evolution of periodontal disease with a larger sample size and an increased follow-up time.

Figure 1 :
Figure 1: Timeline of the experiment.Week 1: Diagnostic phase with a periodontal chart fulfi lled, microbiological testing of periodontal microorganism, and microvascular blood fl ow readings using laser Doppler fl owmetry.Week 0: Periodontal treatment (scaling, root planing, and antibiotic prescription).Week 8: Periodontal reevaluation (periodontal chart, periodontal pathogen identifi cation, and blood fl ow readings using laser Doppler fl owmetry).

Figure 2 :
Figure 2: PCR results interpretation accord to the microIDent® kit manufacturer (Hain Lifescience, GmbH, Nehren, Germany) A-Reaction zones (sites 1-7).B-Staining of line in the specifi c bands, corresponding to each bacterium.C-Degree of staining of each band represented by crosses.D-Explanation of pathogens concentrations.

Figure 5 :
Figure 5: Blood fl ow measurements in PU with the Moor-VMS software.20 seconds selected.

Table 1 :
Demographic characteristics and blood fl ow readings were measured in perfusion units.Means, standard deviations, and p values.

Table 2 :
Periodontal parameters correlate with blood fl ow readings measured in perfusion units.Means, standard deviations, and p values.