**Surface Alterations Induced on Endodontic Instruments by Sterilization Processes, Analyzed with Atomic Force Microscopy: A Systematic Review**

**Mario Dioguardi 1,\*, Vito Crincoli 2, Luigi Laino 3, Mario Alovisi 4, Enrica Laneve 1, Diego Sovereto 1, Bruna Raddato 1, Khrystyna Zhurakivska 1, Filiberto Mastrangelo 1, Domenico Ciavarella 1, Lucio Lo Russo <sup>1</sup> and Lorenzo Lo Muzio <sup>1</sup>**


Received: 29 October 2019; Accepted: 14 November 2019; Published: 17 November 2019

**Abstract:** Endodontic canal disinfection procedures that use sodium hypochlorite, and subsequently, heat sterilization procedures can alter the surface of endodontic instruments, described as corrosion and micropitting. These phenomena can be visualized on the surface of the instruments by SEM and atomic force microscopy analyses. The endodontic instruments used in probing, pre-enlargement, and shaping phases are made of steel alloy or nickel-titanium alloy (NiTi) and are subject to torsional, flexor, and cyclic fatigue; indeed, reuse of these instruments must be done with the knowledge that these instruments are subject to fracture following stress caused during their use. Fracture of the instrument within the canal is an eventuality that can lead to failure of the treatment, and therefore it is important to try to reduce situations that can contribute to the fracture. This review was performed based on the PRISMA protocol. Studies were identified through bibliographic research using electronic databases. A total of 1036 records were identified on the PubMed and Scopus databases. After screening the articles, restricted by year of publication (1979 to 2019), there were 946 records. With the application of the eligibility criteria (all the articles pertaining to the issue of sterilization in endodontics), there were 228 articles. There were 104 articles after eliminating overlaps. There were 50 articles that discussed the influence of sterilization procedures on the surface characteristics of endodontic instruments, and 26 articles that measured parameters on surface alteration. Applying the inclusion and exclusion criteria resulted in a total of eleven articles for quantitative analysis. Four articles were in reference to the primary outcome, eight articles to secondary outcome, and five articles to tertiary outcome. The meta-analysis showed a statistically significant surface alteration effect after five autoclaves and after immersion in the canal irrigants after 10 min.

**Keywords:** autoclave; endodontic sterilization; atomic force microscopy; NiTi alloy; endodontics; corrosion

#### **1. Introduction**

Endodontic instruments are commonly used in dental practice to perform endodontic treatments of vital and necrotic teeth, endodontic retreatments, pulpotomies, pulpectomy and specification

procedures. Depending on the phase of treatment, endodontic instruments are divided into instruments for probing the endodontic canal for the pre-enlargement, and glidepath for shaping the canal or instruments for the closure and three-dimensional sealing of endodontic canals [1,2]. Many of these tools are reusable after performing cleaning, disinfection, and sterilization procedures by autoclaving [3].

Endodontic canal disinfection procedures that use sodium hypochlorite [4], and subsequently, heat sterilization procedures, can alter the surface of endodontic instruments, described as corrosion and micropitting, phenomena [5] that can be visualized by SEM and atomic force microscopy analyses.

The endodontic instruments used in the probing, pre-enlargement, and shaping phases are made of steel alloy or nickel-titanium alloy (NiTi) and are subject to torsional, flexor, and cyclic fatigue, indeed the reuse of these instruments must be done with the knowledge that these tools are subject to fracture following stress caused during their use [6]. Fracture of the instrument within the canal is an eventuality that can lead to failure of the treatment, and therefore it is important to try to reduce situations that can contribute the fracture.

On the surface subject to fatigue, surface alterations can give rise to microcrack which can lead to fracture of the instrument, and also reduce the cutting capacity of the blades on the endodontic files [7]. Therefore, in order to maintain the same cutting efficiency, the endodontist has to exert greater pressure on the instrument with an increase in torsional fatigue stress [6].

An atomic force microscope is an instrument capable of analyzing the surface of instruments. It consists of a cantilever with a pointed tip (tip) mounted on the end, typically composed of silicon or silicon nitride and having a radius of curvature of the "order of nanometers". The sample to be scanned, through the Vand Der Waals forces, interacts with the tip of the detector by flexing it. There are several methods to detect any cantilever movement. The majority of atomic force microscopy (AFM) systems use laser beam detection, which is an optical system with position sensitive detectors called photodiodes. The laser light is reflected by the cantilever on the position-sensitive photodiode. Very small forces are produced between the probe and the surface to be scanned, and these are the forces that allow the AFM system to record the deflection of the cantilever. The cantilever deflection is called "cantilever rigidity". This rigidity can be measured by Hooke's law. Rigidity is recorded visually and can be viewed on the computer in real time. The surface scan of endodontic instruments is used both in non-contact mode and in contact mode and, in general, the scanned surfaces start at 3 mm from the tip of the instrument up to 6 mm. The parameters which are considered with AFM for the analysis of a surface are the arithmetic mean roughness(AMR) of the maximum height (MH) and root mean square (RMS). The atomic force microscopy, therefore, provides detailed information with measurable parameters of possible alterations and irregularities present on the surface of an instrument [8].

Surface alterations can represent a problem in the use of endodontic instruments. A study by Ylmaz, in 2018, identified surface alterations described as surface roughness with statistically significant results for instruments constructed with new M-wire and EDM alloys [9].

One problem of reusing endodontic instruments that are subject to fatigue is the deterioration they suffer that results from their use in the dental canal for the removal of dentin, as well as the corrosive action by the root canal irrigants such as sodium hypochlorite, and subsequently, the action of the temperature and steam induced by the autoclave sterilization process. The surface alterations are well described in a study by Inan, in 2007, on the universal ProTaper, after clinical use and sterilization [10].

Fayyad and Mahran, in a 2013 study, demonstrated by AFM analysis that the alterations on Twisted Files [11], Hero Shaper, RaCe, and GTX instruments were statistically significant after immersion in 5% sodium hypochlorite, however, the alterations were not statistically significant after EDTA immersion. In contrast, Ametrano et al., in 2010, reported significant results for instruments immersed in EDTA [12]. Other studies have report conflicting data, such as the study conducted by Casella, in 2011, in which there was no variation in corrosion resistance for some instruments (K FILE and GT-rotary) unlike the K3 knife immersed in 5% sodium hypochlorite [13]. In addition, studies conducted at the Sem da Razavianet, in 2015, reported an increase in roughness directly related to the number of sterilization cycles performed on endodontic instruments [14].

In contrast, there is debate within the scientific community regarding whether there are statistically significant surface alterations induced by the autoclave or the canal irrigants. This review aims to try to clarify this aspect by investigating the literature to extrapolate the data on surface alterations in endodontic instruments in order to statistically analyze them in a meta-analysis.

Previous systematic reviews on this topic have not included the effect of surface alterations of endodontic instruments subjected to heat sterilization. There is only one systematic review that analyzes the variations in torsional properties subjected to autoclave sterilization.

This review could help endodontists who perform endodontic therapy and reuse endodontic instruments daily. Awareness of the greater or lesser risk of potential fracture triggered by surface variations due to heat or use of canal irrigants on the instruments could be helpful.

#### **2. Materials and Methods**

This systematic review was conducted based on the Prisma protocol.

The study was constructed using the following PICO elements for questions: Population (endodontic instruments); intervention (surface alterations induced by sterilization processes and root canal irrigation); control (new endodontic instruments not subject to sterilization; and outcome (surface alterations induced by the sterilization process by autoclave, and by root canal irrigants such as sodium hypochlorite and EDTA).

The following PICO question was formulated: To what extent, statistically significant, the sterilization processes and the used canal irrigants alter the surface of the rotating endodontic instruments with respect to the control?

After an initial selection phase of article identification in the databases, the potentially eligible articles were qualitatively evaluated in order to investigate the surface alterations of endodontic instruments resulting from the sterilization of instruments and disinfection of endodontic canals.

#### *2.1. Eligibility Criteria*

This literature review took into consideration in vitro and clinical studies that concerned the subject of sterilization and the influence of the latter on the physical and chemical properties of endodontic instruments. In particular, articles that dealt with the corrosive phenomena and surface alterations considered by microscopy methods (atomic force microscopy), conducted in recent years, and published with abstracts in English, were considered potentially eligible.

Articles from the last 40 years were chosen, because disinfection and sterilization procedures have changed in light of new discovered infectious contaminants, such as HIV and HCV viruses and the prong of spongiform encephalopathy. Furthermore, the methods used to manufacture the instruments have changed with the introduction of new alloys and new instruments. Therefore, in summary, potentially eligible articles included studies that investigated the influence of sterilization and disinfection procedures on endodontic canals, as well as on the physical and chemical characteristics of endodontic instruments, however, articles published more than 40 years ago and those that did not present an abstract in English were excluded.

Finally, the articles that were potentially eligible were subjected to a full text analysis to verify their use for a qualitative and quantitative analysis.

The inclusion and exclusion criteria applied in the full text analysis are the following:


#### *2.2. Research Methodology*

The studies were identified through a bibliographic research on electronic databases.

The literature search was conducted using the search engines "PubMed" and "Scopus". The search on the providers was conducted between 12 September 2019 and 18 September 2019 and the last search for a partial update of the literature was conducted on 1 October 2019.

The following search terms were used on PubMed and Scopus: "Endodontic sterilization" PubMed 333 and Scopus 269; "endodontic autoclave" PubMed 38 and Scopus 52; "atomic force microscopy" AND "endodontic" PubMed 21 and Scopus 33; "roughness" AND "endodontic" Pub Med 42 and Scopus 67; "roughness" AND "ethylenediaminetetraacetic acid" PubMed 15 and Scopus 40; "roughness" AND "sodium hypochlorite" PubMed 1 and Scopus 1; "sodium hypochlorite" AND "atomic force microscopy" PubMed 40 and Scopus 80; "atomic force microscopy" AND "NiTi rotary instruments" PubMed 1 and Scopus 2 (Table 1).

#### *2.3. Screening Methodology*

The records obtained were, subsequently, examined by two independent reviewers (M.D. and S.D), and a third reviewer (E.L.) acted as a decision maker in situations of doubt. The screening included the analysis of the title and the abstract to eliminate the recordings not related to the topics of the review. After the screening phase, the overlaps were removed and the complete texts of the articles were analyzed, from which the ones eligible for the qualitative analysis and the inclusion in the meta-analysis for the three results were identified. The results sought by the two reviewers were:


The fourth reviewer, with supervisory duties, was L.Lo.M. The K agreement between the two screening reviewers was 0.8464 (Table 2). The K agreement was based on the formulas of the *Cochrane Handbook for Systematic Reviews* [15].

The Newcastle–Ottawa scale for case-control studies was used to assess the risk of bias in the included studies. The quantitative analysis was performed with the Rev Manager software 5.3 (Cochrane Collaboration, Copenhagen, Denmark [16].


**1.**Completeoverviewofthesearchmethodology.

**Table**

**Table 2.** K agreement calculation, Po = 0.94 (proportion of agreement), Pe = 0.6092 (agreement expected), K agreement = 0.8464 (<0 no agreement, 0.0 to 0.20 slight agreement, 0.21 to 0.40 fair agreement, 0.41 to 0.60 moderate agreement, 0.61 to 0.80 substantial agreement, and 0.81 to 1.00 almost perfect agreement). The K agreement was calculated from the 50 articles and included eleven articles with the application of the inclusion and exclusion criteria.


#### **3. Results**

A total of 1036 records were identified on the PubMed and Scopus databases (Table 1). After screening the articles, with the restriction by year of publication (1979 to 2019), there were 946 records. With the application of the eligibility criteria (all the articles pertaining to the issue of sterilization in endodontics), there were 228 articles. There were 104 articles after eliminating overlaps. There were 50 articles that discussed the influence of sterilization procedures on the surface characteristics of endodontic instruments, and 26 that measured parameters on surface alteration.

Applying the inclusion and exclusion criteria resulted in a total of eleven articles for quantitative analysis.

Four articles were in reference to the primary outcome, eight to the secondary outcome, and five to the tertiary outcome. The entire selection and screening procedures are described in the flow chart (Figure 1).

#### *3.1. Study Characteristics and Data Extraction*

The studies included for quantitative analysis were:


The extracted data included the magazine (author, data, and journal); the endodontic instrumentation object of measurement (name, taper, and diameter at tip); the method of sterilization by heat (temperature, pressure, and time); the number autoclave cycles or irrigants; the number of instruments (control and experimental); the number of surfaces scanned by the instrument; the number of total scans; the size of the scanning surface; and the data concerning the root mean square (RMS) ± standard deviation.

The data extracted for the tree outcomes are shown in Tables 3 and 4.

*3.2. Risk of Bias*

The risk of bias was assessed through the Newcastle–Ottawa case-control scale. The results are reported in detail in Table 5. For each category, a value of one to three was assigned (one = low and three = high).

**Figure 1.** Flow chart of the different phases of the systematic review.


**Table 3.** Primary outcome (extraction of data relating to the root mean square detected on the surface of the endodontic instruments subjected to atomic

 force

Inan et al., 2007, J. Endod. [10]

Autoclave 134 ◦C for 18 min

ProTaper F2 (25/08)

0

1

 1

 1

 11

 11

 1 × 1 μm

 7.29 ± 0.88 nm

 11

 11

 1 × 1 μm

 1.46 ± 0.45 nm



The risk of bias within the individual studies was low enough that the methods of investigation adopted for the controls were identical to the cases included in the meta-analysis. The Prasad study [23] was the only study of the exposure time of endodontic instruments to canal irrigants that was not well defined, exposing the study to a bias.

The risk of bias between the various studies was considered high, and therefore partly limited the importance of the results. The heterogeneity of the studies depended mainly on the diversity of the instruments, which were similar, in some cases, only in terms of tip diameter, taper, and type of metal alloy.

The heterogeneity of the studies was represented by funnel plots of the four outcomes, as shown in Figure 2.

**Figure 2.** Funnel plots of the evaluation of heterogeneity for the (**A**) first, (**B**) second, (**C**) third outcomes.

#### *3.3. Data Analysis*

The statistical analysis of the data was performed using the Rev Manager 5.3 software (Copenhagen, 153 Denmark, The Nordic Cochrane Centre, The Nordic Cochrane Collaboration, 2014) and the results were represented by forest plots for each of the outcomes.

For the primary outcome, variations of the root mean square root (RMS) of endodontic instruments subjected to five autoclave cycles as compared with the non-autoclaved control, the comparison showed high heterogeneity of the studies, with an I2 equal to 100%. For this reason, a random effects model was used. Overall, for the primary outcome, meta-analysis was favorable for the control group. The studies that present data with a statistically significant difference are Inan et al., 2007 [10] and Spagnuolo et al., 20012 [17]. The studies by Ylmaz et al., 2018 [9] and Can Saglam et al., 2015 [22] are exactly at the center of the line of no effect. The studies by Ylzam and Can Saglam are exactly at the center of the line of no effect, however, the remaining two studies are favorable for the group subjected to control, their confidence intervals do not intercept the line of no effect (Figure 3).


**Figure 3.** Forest plot of the random effects model of the meta-analysis of the primary outcome.

For the secondary outcome, variations of the root mean square (RMS) of endodontic instruments exposed to sodium hypochlorite 5% as compared with the control group, the comparison showed high heterogeneity among the studies, with an I2 equal to 98%. For this reason, for the second outcome, a random effects model was applied to avoid minimizing the roles of smaller-dimension studies. For the second outcome, the forest plot is in favor of the subject group control.

The studies that reported statistically significant data in favor of the control group are Ametrano, 2011; Prasad, 2014; Topuz, 2008; and Uslu, 2018. The Cai's study was the only study that was in favor of the group subjected to sodium hypochlorite, even though its confidence interval crosses the line of no effect. The other studies report statistically insignificant data (Figure 4).

**Figure 4.** Forest plot of the random effects model of the meta-analysis of the secondary outcome.

For the tertiary outcome, variations of the root mean square (RMS) of the endodontic instruments exposed to EDTA 10% as compared with the control group, the comparison showed high heterogeneity between the studies, with an I2 of 99%, and therefore a random effects model was applied. For the tertiary outcome, the forest plot is in favor of the control group except for the study by Fayyad which is positioned in the line of no effect (Figure 5).


#### **4. Discussion**

The results of the meta-analysis for the three outcomes are in agreement in establishing that the superficial alterations induced by autoclave, from the sodium hypochlorite and from the EDTA, are statistically significant surface alterations that represent points where instrument fractures can be triggered. In addition, the alterations induced on the surface analyzed by SEM and AFM show that the alterations can also be expressed on the cutting surface, altering, in a pejorative sense, the cutting efficacy.

For the first outcome, the studies, in the literature, that supported a statistically significant alteration are:


Sodium hypochlorite certainly alters the surface of NiTi instruments and innumerable studies are in agreement such as Uslu et al., 2018 [19]; Ametrano et al., 2010 [12]; Topuz et al., 2008 [20]; and Prasad et al., 2014 [23]. Furthermore, a study conducted by Yokoyama et al., 2004 [24] stated that the action of sodium hypochlorite causes a worsening of the surface in endodontic instruments that facilitates their rupture following flexor and torsional stress.

The statistical analysis, in a similar way but with fewer studies, also confirms that the EDTA determines an increase in surface irregularities in a statistically significant way, and studies that confirm it after 10 min of exposure are well highlighted in the forest plot (Figure 5). Studies that are in contrast to the present meta-analysis report conflicting data regarding the action of EDTA on the surface. It seems that for exposures less than 5 min they do not alter the surface, however, according to Bonaccorsa et al. [25], a passivation phenomenon could lead to the creation of a complex between the metallic ions and the EDTA at a PH lower than four which renders the instrument resistant.

#### **5. Conclusions**

In conclusion, based on the present systematic analysis we affirm that autoclave induces a statistically significant corrosive phenomena, called micropitting, after five cycles of autoclave and determined by the heat, and comparatively, hypochlorite determines corrosion after only 5 min of exposure and EDTA after 10 min of exposure.

Superficial alterations, which are widely discussed in the literature, can determine the triggering of fractures in instruments subjected to cyclic fatigue and torsional fatigue. Therefore, it is important for endodontist to have knowledge of such corrosive phenomena, induced by irrigants such as sodium hypochlorite and EDTA, on instruments that can be reused and autoclaved.

**Author Contributions:** Conceptualization, M.D., D.S., and B.R.; methodology, K.Z. and M.D.; software, M.A. validation, M.D., and L.L.M.; formal analysis, D.S., M.D., and V.C.; investigation, L.L. and M.D.; resources, L.L.M.; data curation, E.L. and M.D.; writing—original draft preparation, M.D.; writing—review and editing, M.D., L.L.M., L.L.R., D.C., and F.M.

**Funding:** This research received no external funding.

**Acknowledgments:** All the acknowledgements go to Lorenzo Lo Muzio, Director of the Dental Clinic and President of the Department of Clinical and Experimental Medicine of the University of Foggia, who gave fundamental technical support in the writing of this article.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Influence of Myeloperoxidase Levels on Periodontal Disease: An Applied Clinical Study**

#### **Alessandro Polizzi \*, Salvatore Torrisi, Simona Santonocito, Mattia Di Stefano, Francesco Indelicato and Antonino Lo Giudice**

Department of General Surgery and Surgical-Medical Specialties, School of Dentistry, University of Catania, AOU Policlinico—P.O. Vittorio Emanuele, Via Plebiscito 628, 95124 Catania, Italy; ture\_torrisi@hotmail.it (S.T.); simonasantonocito.93@gmail.com (S.S.); mattiadistefano@live.it (M.D.S.); indelicato@policlinico.unict.it (F.I.); nino.logiudice@gmail.com (A.L.G.)

**\*** Correspondence: alexpoli345@gmail.com; Tel./Fax: +39-0957435359

Received: 26 January 2020; Accepted: 3 February 2020; Published: 4 February 2020

**Abstract:** In this trial, we evaluated the influence on plasma and salivary myeloperoxidase (MPO) levels of periodontal health, coronary heart disease (CHD), periodontitis, or both periodontitis and CHD. Clinical and periodontal parameters were collected from periodontitis patients (n = 31), CHD patients (n = 31), patients with both periodontitis and CHD (n = 31), and from healthy patients (n = 31) together with saliva and plasma samples. The median concentrations of salivary and plasma MPO were statistically higher in the CHD patients [plasma: 26.2 (18.2–34.4) ng/mg; saliva 83.2 (77.4–101.5) ng/mL, p < 0.01] and in the periodontitis plus CHD patients [plasma: 27.8 (22.5–35.7) ng/mg; saliva 85.6 (76.5–106.7) ng/mL, p < 0.001] with respect to periodontitis and control patients. Through a univariate regression analysis, c-reactive protein (CRP) and CHD (both p < 0.001) and periodontitis (p = 0.024) were statistically correlated with MPO in plasma. The multivariate regression analysis demonstrated that only CRP was statistically the predictor of MPO in plasma (p < 0.001). The multivariate regression analysis in saliva demonstrated that, regarding MPO levels the only predictors were CRP (p < 0.001) and total cholesterol (p = 0.035). The present study evidenced that subjects with CHD and periodontitis plus CHD had higher plasma and salivary levels of MPO compared to subjects with periodontitis and healthy controls.

**Keywords:** myeloperoxidase; periodontitis; cardiovascular disease; applied model

#### **1. Introduction**

Periodontal disease is a common oral inflammatory multifactorial disease that causes the disruption of the periodontium and the tissues that support the tooth, such as bone and cementum main caused by oral bacteria, that ultimately leads to the loss of the tooth [1]. Almost all adults in the USA present periodontal disease forms and nearly ten percent of the population worldwide express severe type of periodontal disease [2,3].

More recently, observational reports have shown a correlation between periodontal disease and cardiovascular disease, such as stroke, heart disease and endothelial dysfunction [4,5]. Moreover, some studies demonstrated a specific correlation among periodontal disease and an augmented risk of stroke [6] and, coronary heart disease (CHD) [7,8].

The pathogenesis of periodontal disease includes inflammatory and bacteria responses which may determine an increased host response subsequent to the presence of pathogenic oral biofilm in gingival tissues [9]. More specifically, periodontal disease has been correlated with an increase of levels of some systemic inflammatory mediators in serum, such as prostaglandin, interleukin 1 (IL-1), IL-6, and C-reactive protein (CRP) [10].

Myeloperoxidase (MPO) is one of the more mediators expressed within tissues during the progression of inflammation [11]. It was demonstrated that MPO, secreted by endothelial cells after exposure to pathogenic bacteria, represents a potent mediator of vascular inflammation and a vasoconstrictor [12].

In this regard, it has been shown that several proinflammatory cytokines, including IL-1, -6, and -8, have been reported to upregulate the secretion of MPO [13]. The expression of MPO was strongly associated in the gingival tissue and endothelial cells during periodontitis [14]. More specifically, a clinical study found that, in gingival crevicular fluid, MPO increased with the progression of the periodontitis, and also that MPO was involved in the regulation of IL-1b expression in gingival tissues [15,16].

During the last few decades, several studies have analyzed the association between periodontal disease endothelial dysfunction, and increased risk of CHD and cardiovascular disease (CVD) [17,18]. More specifically, it has been supposed that the inflammatory mediators that are present and released during the active phase of periodontal disease such as CRP, interleukins, prostaglandins, and metalloproteases, can negatively influence the release of nitric oxide (NO) [19]. The altered release of NO can affect the endothelium which in turn regulates vascular tone, and, finally, dysfunction of the endothelium and enhanced risk of CVD [20,21]. For these causes, there is growing interest to investigate some other oral mediators that can regulate and impact the subclinical endothelial dysfunctions as an early sign of augmented risk of CHD and CVD. In this regard, a correlation between high proportion of MPO, CRP, and endothelial dysfunction was recently reported [22,23].

The production of NO at local level has been shown to be fundamental in the aetiology and progression of periodontitis. The increment and reduction of NO metabolites in saliva production in periodontal tissue against periodontopathogenic microbiota during periodontitis have been demonstrated to be correlated endothelial dysfunction [24,25]. Furthermore, it has been reported that MPO plays an important role in the reduction of NO synthase especially during periodontitis [26].

Based on these findings, the aims of this trial were to consider a possible association of periodontitis, CVD, or both periodontitis plus CVD on serum and salivary MPO. Futhermore, we analyzed the possible correlation between MPO in serum and saliva and if serum CRP mediated the association between salivary or serum MPO levels.

#### **2. Materials and Methods**

#### *2.1. Study Design*

For the present study, 311 healthy controls and patients with periodontitis or CHD were chosen at the School of Odontostomatology of the University of Catania, Italy, from October 2018 to December 2019. Patients were chosen in a specific range of age (35–65 years old) and on gender in order to have similar proportion of patients in each category characterized by the selection variable. 50% of the patients and controls were males with 45–54 year of age.

The study was performed following the 2016 revision of the Helsinki declaration on medical research. Ethical approval was obtained by the local International Review Board (IRB) (#18-18). The study was registered at clinicaltrials.gov (NCT04152023). An informed written consent was obtained from each enrolled patient. The trial was performed in accordance to the guidelines for the strengthening of reporting of observational studies (STROBE) [27].

The inclusion criteria for the subjects enrolled in the periodontitis group were: (1) at least 16 teeth, (2) at least of 40% of periodontal sites with clinical attachment level (CAL) ≥2 mm and probing depth (PD) ≥4 mm [28]; (3) at least one periodontal site with ≥2 mm of crestal alveolar bone loss confirmed on digital periapical x-rays; (4) at least ≥40% sites with bleeding on probing (BOP) [29]. Healthy controls had any systemic disorder, at least ≤10% sites with BOP, and no periodontal sites with PD or CAL ≥4 mm, or x-ray signs of bone loss.

For the CVD group, the inclusion criteria were: at least ≥18 years; a diagnosis of CVD with ≥50% of stenosis of at least one coronary artery verified by coronary angiography, or past or current percutaneous coronary intervention [30]. Each type of previous disease, taking drugs, or previous CVD exams (e.g., electrocardiography, etc.) were recorded. In all patients, the diagnosis of CVD was performed by the same operator from medical record information. For the periodontitis plus CVD subjects the inclusion criteria were the same of the single disease (periodontitis and CVD).

The exclusion criteria of all subjects, were (1) consumption of contraceptive drugs; (2) consumption of antibiotics, anti-inflammatory or immunosuppressive drugs during the three months previous the trial; (3) presence of gestation or suction; (4) intake of alcohol; (5) anesthetic allergy; (6) intake of nifedipine, hydantoin or cyclosporin a drugs; (7) any type of periodontal treatment in the three months before baseline.

Then, 187 subjects were left out from the study because they did not meet the inclusion criteria (n = 129), failed to join in the study (n = 37), or were lost at the first assessment (n = 21). For these reasons, for the present study, 31 healthy subjects, 31 periodontitis patients, 31 CHD patients, and 31 patients with both diseases (periodontitis plus CHD) were enrolled in the end (Figure 1).

**Figure 1.** Flowchart of the study.

In each patient, every demographic characteristic (such as educational level) and demographic indices such as age, gender, body mass index (BMI), diabetes and other systemic events were recorded together with the type of drug taken. Diabetes was recorded on the patient's medical story or on fasting blood glucose ≥125 mg/dL. The BMI was recorded by calculating the patient's weight divided by the square of his height in kg/m2. All enrolled subjects were also classified on their smoking history, such as normal smokers, ex-smokers (subjects who have not smoked for ≥5 years), and non-smokers.

The periodontal evaluation comprised clinical attachment loss (CAL), probing depth (PD), bleeding on probing (BOP), and plaque score (PI) [31]. CAL was verified, such as PD plus gingival recession using the cementoenamel junction as a reference. All periodontal indexes were registered, in all patients, by two independent calibrated examiners (a principal examiner and a control examiner), exonerated in the subsequent study steps, using a periodontal probe (UNC-15, Hu-Friedy, Chicago, IL, USA).

It was assessed the inter- and intra-examiner reliability for PD and CAL through the Intraclass Correlation Coefficient (ICC) analysis. The obtained inter-examiner reliability was in agreement for PD (ICC= 0.819) and CAL (ICC = 0.832) with a good degree of reliability. The intra-examiner reliability of PD and CAL was done only on 24 subjects (six random subjects per group) for both examiners. For the first examiner, the intra-examiner reliability presented an agreement for PD (ICC = 0.819) and CAL (ICC = 0.808); for the second examiner, the intra-examiner reliability was good for both PD (ICC = 0.818) and CAL (ICC = 0.801). All periodontal indexes were registered, in each enrolled subject, at six sites in each tooth.

A power analysis was executed in order to evaluate the sample size needed for the study. The sample size was determined considering four groups: an effect size of 0.29 for MPO (primary outcome chosen), a two-sided significance level of 0.05, a standard deviation of 1.5 [23], and a power

level of 80%. It was established that would be required around 28 patients per group, with a total number of 114 patients required to obtain a power level of 80%. a total of 124 patients were enrolled, so the study power was 81%. Power and sample size calculation was performed with statistical software (G\*Power version 3.1.9.4, Universitat Dusseldorf, Germany).

#### *2.2. Evaluation of Salivary and Serum MPO*

All serum and saliva samples were collected on an in all patients between 8:00 and 10:00 a.m., before the periodontal examination, on the same day by the same examiner. All enrolled subjects were requested to refrain from drinking, eating, chewing, brushing their teeth, or other oral hygiene maneuvers in the 12 h preceding the sampling of serum and saliva.

For the collection of serum, a venous blood sample was taken which, after the collection, was immediately cooled with ice and centrifuged at 4 ◦C (800× *g* for 10 min). For the collection of saliva samples, the enrolled patients were asked to moisten by chewing a cotton roll for two minutes using the salivette method (Sarsted, Verona, Italy). Subsequently, the saliva sample in each patient was instantaneously centrifuged at 4 ◦C (1000× *g* for 2 min). Both saliva and serum samples were stored at −20 ◦C.

Magnetic bead-based luminex assay (R&D Systems, Minneapolis, MN, and Sigma-Aldrich, Saint Louis, MO, USA) were used to detect serum and salivary concentrations of MPO, following the manufacturers' instructions. Levels of hs-CRP were calculated using a nephelometric assay kit. Levels of hs-CRP >3 mg/L were related to an augmented CVD risk. Routine methods were applied to assess glucose and plasma lipids levels.

#### *2.3. Statistical Analysis*

Median, 25%, and 75% percentile were used to express numerical variables while number and % were used to express categorical variables. Nearly all of the variables analyzed (e.g., fasting glucose, triglycerides, all periodontal index) did not have normal distribution, as confirmed by Kolmogorov–Smirnov test. Only age, BMI, and salivary and serum MPO were normally distributed; for this reason, nonparametric tests were used to analyze all data in the present analysis [32]. More specifically, to confront all numerical variables in the 4 groups of patients, was applied the Kruskal Wallis test while the Mann Whitney test was applied to obtain the two-by-two comparisons. Bonferroni's correction was applied for numerous evaluations; the α level of 0.050 was split by the potential comparisons (n = 6), and the adjusted significance level equalled 0.008 (0.050/6).

The *p*-trend analysis for salivary and serum and MPO levels was obtained using the Jonckheere–Terpstra Test to evaluate whether MPO levels were statistically augmented in the four analyzed groups. To asses any significant interdependence between MPO in saliva and serum and hs-CRP, the Spearman correlation test was used.

Moreover, a univariate and multivariable linear regression analysis were applied in all enrolled patients to evaluate the dependence of MPO levels in serum and saliva (which resulted normally distributed) on possibly explicative outcomes such as sex, education, age, socioeconomic status (SES), triglycerides, total cholesterol, BMI, CRP, and CVD drugs (yes/no). In the multivariate final model, sex, age, and education SES were incorporated such as possible confounders, and tested to analyze if CHD, periodontitis, and hs-CRP influenced MPO in serum. For the evaluation of MPO in saliva, the same analysis was performed using salivary MPO levels as an outcome. All statistical analyses were executed using statistical software (SPSS 22.0 for Windows package, SPS srl, Bologna, Italy). a *p*-value < 0.05 was set such as significant.

#### **3. Results**

The demographic and serological characteristics of the enrolled patients are shown in Table 1. All groups were matched for age and sex, and they did not presented any statistically significant differences regarding education levels, smoking, BMI, and serological features (Table 1).

**Table 1.** Sample characteristics of enrolled patients. Data are represented as median (25th; 75th percentiles) or number with percentage. \* *p* < 0.001 and \*\* *p* < 0.001 significant differences vs. healthy subjects computed by the Mann Whitney test. §§ *p* < 0.001 significant differences vs. periodontitis patients calculated by the Mann Whitney test. # *p* < 0.008 significant differences vs. coronary heart disease (CHD) patients calculated by the Mann Whitney test.


Compared to healthy controls, patients with periodontitis, CDH and a combination of periodontitis and CVD presented a higher value of hs-CRP (*p* < 0.001). Moreover, patients with CHD and periodontitis plus CHD presented no significant differences regarding past CVD events.

In Table 2 are represented dental characteristics of all enrolled patients. Compared with CHD and control patients, subjects with periodontitis and periodontitis plus CVD showed higher periodontal parameters (CAL, PD, BOP, PI) and smaller number of teeth (*p* < 0.001) (Table 2).



Figure 2 represents median (25th; 75th percentile) values of MPO levels in saliva and serum of all enrolled patients. Compared to control subjects, patients with CVD (*p* < 0.01) and with periodontitis plus CVD (*p* < 0.001) had higher median concentrations of MPO in saliva and serum. More specifically, in comparison with periodontitis subjects, patients with periodontitis plus CVD presented increased salivary and serum concentrations of MPO (*p* < 0.01) (Figure 2).

**Figure 2.** Median values (25%; 75% percentiles) of coronary heart disease (MPO) in saliva and serum. \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001 significant differences vs. control subjects (derived by the Kruskal–Wallis test). §§ *p* < 0.01 significant differences vs. periodontitis patients. *p* < 0.001.

Moreover, the p-for trend analysis test evidenced that MPO in serum increased gradually in subjects with periodontitis, CVD, and with periodontitis plus CVD (*p*-trend <0.001) (Figure 3). No statistically significant associations were found in MPO levels between serum and saliva (rs = 0.213, *p* = 0.098). Moreover, in all enrolled patients presented a positive correlation between serum/salivary MPO and hs-CRP levels (rs = 0.341, *p* < 0.001)/(rs = 0.609, *p* < 0.001) (Figure 3).

**Figure 3.** Analysis of correlation of serum and salivary MPO levels with c-reactive protein (CRP) in all patients.

The univariate regression analysis evidenced that there was a significant direct impact of hs-CRP on serum and salivary MPO (both *p* < 0.001). Furthermore, the adjusted multivariate linear regression analysis evidenced that hs-CRP variable was the only significant predictor for serum MPO (*p* < 0.001).

Moreover, hs-CRP (*p* < 0.001) and total cholesterol (*p* = 0.035) were the statistically significant predictor variables for salivary MPO (Table 3).

**Table 3.** Uni- and multivariate linear regression analysis for MPO levels in serum and saliva in all patients. Age was included as continuous variable. For periodontitis and cardiovascular disease (CVD), controls served as reference. For gender, male served as reference.


#### **4. Discussion**

This trial was aimed at evaluating the impact of conditions such as periodontal disease, CVD, or periodontitis plus CVD on MPO levels in serum and saliva. The present trial evidenced that the occurrence of CVD caused increased levels of MPO and hs-CRP in serum and saliva. Nevertheless, in comparison with periodontitis and healthy controls, only the group of subjects with CVD and periodontitis plus CVD had significantly elevated MPO levels in serum and saliva, endorsing the suggestion that CVD influenced the increment of MPO levels in serum and saliva. Furthermore, results of the present study show that the simultaneous presence of periodontitis in patients with CVD can determine an increased activation of MPO and therefore represent a subclinical stimulus for the purpose of an increase in CVD development.

In accordance with the results of the present study, some reports have shown that high levels of MPO in serum represent real independent risk factors of CVD development and increased mortality index, possibly by inactivating NO signaling [33]. Specifically, it has also been shown that, in patients with atherosclerosis, high systemic levels of MPO are associated with significant carotid epithelial dysfunctions, underlining the fundamental inhibitory role of NO exercised by MPO [34]. Therefore, the simultaneous presence of CVD on the one hand and periodontitis on the other hand can be a real explanation for the deterioration of endothelial function due to high levels of MPO. In this regard, recent research has shown that the treatment of periodontitis has significantly reduced the systemic levels of MPO in patients with coronary disease [35].

Moreover, several reports demonstrated that increased hs-CRP levels in serum can facilitate the increment of MPO levels in serum in several diseases in humans [23,35–38]. In accordance with the results of our study, several studies have been demonstrated that situations which may cause an increase of oxidative stress, such as CVD and periodontal disease, cause the high release of CRP, which in turn, can arouse the production of MPO in saliva and serum in order to defend tissue damage determined by oxidative stress condition [36]. In agreement with the results of the present study, Magan-Fernandez et al. [39] demonstrated that CRP and MPO levels were higher during active phases of periodontal disease.

However, while evidence has previously been demonstrated regarding high serum MPO levels as primary mediators of endothelial dysfunction or in the development of cardiovascular risk, from the authors' knowledge, there is no specific evidence to determine MPO levels in saliva in order to evaluate whether the increased expression of salivary MPO levels determines, by reflection, an increase in MPO in serum and then analyzes the salivary levels of MPO as an index of endothelial dysfunction. In this regard, however, it should be noted that this study did not reveal a significant correlation between serum and saliva MPO levels, as salivary MPO levels are influenced in patients enrolled independently only of hs-CRP and total cholesterol. This explanation can be determined by the way that MPO salivary levels may be due to an exclusive local oral production of MPO.

In this regard, it should be noted that, from the studies currently present in the literature, while the effect of MPO at a systemic level mediated by the reduction of NO on endothelial damage has been previously highlighted, the impact of MPO activation orally (e.g., in saliva) is less clear. However, there are studies that show that periodontal disease is positively correlated with high levels of NO and therefore with related stress-oxidative damage [19,24]. The presence of high levels of NO at salivary level can be explained as NO is produced orally in response by the host as a specific salivary defense in the presence of periodontal pathogenic bacteria that are exacerbated during periodontitis [12,24,40]. Furthermore, some studies suggested that decreasing activities of NO and some other enzymatic antioxidants, such as superoxide dismutase catalase, were associated with periodontitis and high levels of MPO, whereas others claim that antioxidants function as protective agents against free radicals during CP progression [19,24,41].

However, there is no unanimous consensus in the literature on the effects of NO levels on tissue damage during periodontitis. Some reports have shown high levels of NO in periodontal tissue in the active periodontal period [39,42] while, on the other hand, other authors have shown lower levels of NO in saliva of subjects with periodontal disease [25,43]. However, results in the literature may have been determined by the different homogeneity of the patients enrolled in the studies, by the different age ranges of the patients analyzed, or by the excessive presence of patients who smoke; in fact it has been shown that smoking can cause a high increase in salivary NO salivary levels [44–46]. Another explanation for the different results found in the literature can be determined by the different salivary sampling method performed in the different studies. Furthermore, the cause of the different expression of MPO at the salivary and serum level may be due to a different production of NO at the oral level which may be different from the serum one.

As an explanation of the results of the present study, it should be highlighted that the dysfunctional damage at the endothelium level found in patients with periodontitis and with CVD can be determined by a specific inflammatory and immune pathway in which MPO modulates a response towards pathogenic bacteria of the oral biofilm which are exacerbated during the active phases of periodontal damage. It has also been shown that MPO, during periodontal disease, mediates the immune response at the endothelial level through specific heat shock proteins which has been shown to be useful for stimulating the production of cross-reactive T cells [47–52]. In this regard, this process which sees MPO as a key modulator [53–58], has also been shown to influence the host defense mechanism that determines a subsequent activation of endothelial cell production [55,58–63] which leads to an increased risk of future tissue damage effects due to periodontal pathogens bacteria in several oral diseases [43,64–68]. Moreover, the oral microbiota is a key factor in the protection against the colonization of extrinsic pathogens that could impact systemic health [68]. However, the imbalance of the ecosystem together with high levels of MPO, which can be caused by a weak immune system, lead to a challenge for oral and systemic health [68]. The ecological conditions of these habitats are constantly changing, so ecosystems are subject to frequent variations [68,69].

However, the present trial has some limitations. Among the main limitations there is the type of study, which makes it difficult to analyze the cause and effect on a temporal level of MPO. The small sample size, due to high inclusion and exclusion levels, and to the important excluded confounders, also represents a limitation of the present preliminary study. However, the exclusion of several confounders represents a positive and rigorous aspect for the clear evaluation of these confounders on the concentration of serum and salivary levels of MPO.

Recently, different approaches have been developed with the aim of easily evaluating innovative salivary markers useful for early and subclinically validating the development of different diseases. This study indicates that patients suffering from periodontitis and CVD have higher serum and salivary levels of MPO than subjects with periodontitis and healthy subjects.

The results of this study propose that mostly CVD is a stimulus to the increased serum MPO levels which may be beyond a pathway intermediated by hs-CRP. Therefore, these results are promising but at the same time require further studies with a larger sample of analysis in order to better comprehend the function of MPO during periodontitis.

**Author Contributions:** Conceptualization, A.P.; methodology, S.T.; validation, S.S., formal analysis, M.D.S.; writing—original draft preparation, F.I.; A.L.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


molars: a randomized, controlled clinical trial. *Int. J. Oral Maxillofac. Surg.* **2019**, *48*, 1348–1354. [CrossRef] [PubMed]


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