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Salivary glucose concentration and daily variation in the oral fluid of healthy patients



 Polimeni A., Tremolati M., Falciola L., Pifferi V., Ierardo G., Farronato G. 
Original Article, 1-6
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Aim. The aim of this study was to determine the concentration and the variations of salivary glucose in healthy patients who were sampled at five different intervals during the day. Materials and methods. Samples of un-stimulated saliva have been collected from 21 healthy patients using the drooling technique and they were divided into two categories. In the first category, patients were asked not to toothbrush their teeth in the 8 hours prior to collection of the salivary sample. Patients in the second category were instead asked to toothbrush 90 minutes before the collection of the first sample of saliva. The glucose concentration was measured in all patients via an enzymatic spectrophotometry. Patients have been selected following a strict inclusion criteria, which included&lt;5% of plaque presence e according to the plaque Index and a total absence of oral disease that could possibly interfere with sample taking or oral fluid analysis. The average age of patients was 22,4±2,6 years old of which 45% were female and 55% were male. Samples were collected five times between 8 am and 12 pm, before and after breakfast. Data was statistically analyzed using the Skewness/Kurtosis Test, Shapiro-Wilk Test, Kruskal Wallis Test and Linear Regression Model, considering values of p&lt;0,05 to be significant. Results. The average rate of un-stimulated salivary flow was 0,53±0,21 ml/min. There were no significant differences between salivary glucose values and salivary flow in female and male patients. However, we observed a typical trend which recurred for each patient and resembled a “pseudo-glycemic curve”. Conclusions. The collected data suggests that glucose concentration results did not show statistically significant differences (p&gt;0,078) which could however be due to the little number of patients assessed, nonetheless there is a trend, recalling a “pseudo-glycemic curve” not referable to changes in flow rates, probably due to glucose catabolism and shunt.




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Annali di stomatologia
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Salivary glucose concentration and daily variation in the oral fluid of healthy patients
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Salivary glucose concentration and daily variation in the oral fluid of healthy patients
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                                                                                                     Original article



    Salivary glucose concentration and daily variation
    in the oral fluid of healthy patients




                                                                                                                      li
                                                                   na
    Antonella Polimeni, MD, DDS1                              Results. The average rate of un-stimulated sali-
    Marco Tremolati, DDS, MS, PhDC2                           vary flow was 0,53±0,21 ml/min. There were no




                                                                 io
    Luigi Falciola, MD, PhD3                                  significant differences between salivary glucose
    Valentina Pifferi, MD3                                    values and salivary flow in female and male pa-
    Gaetano Ierardo, DDS1                                     tients. However, we observed a typical trend
    Giampietro Farronato, MD, DDS2                            which recurred for each patient and resembled a




                                                              az
                                                              “pseudo-glycemic curve”.
    1 Department of Oral and Maxillo-facial Sciences,         Conclusions. The collected data suggests that
      “Sapienza” University of Rome, Italy                    glucose concentration results did not show sta-
    2 Department of Biomedical, Surgical and Dental           tistically significant differences (p>0,078) which




                                                           rn
      Sciences, University of Milan, Italy                    could however be due to the little number of pa-
    3 Department of Chemistry, University of Milan and        tients assessed, nonetheless there is a trend, re-
      Sensors Group of the Italian Chemical Society, Italy    calling a “pseudo-glycemic curve” not referable
                                                              to changes in flow rates, probably due to glucose




                                                        te
                                                              catabolism and shunt.
    Corresponding author:
    Giampietro Farronato                                      Key words: salivary glucose, saliva, oral hygiene,
                                                     In
    Department of Biomedical, Surgical and Dental Sci-
    ences, University of Milan
                                                              oral fluid, spectrophotometry.

    Via Commenda, 10
    20122 Milano, Italy                                       Introduction
    E-mail: giampietro.farronato@unimi.it
                                              ni
                                                              Oral fluid is an organic fluid, easy to collect and to
                                                              preserve, which may give us plenty of information
    Summary                                                   about the presence of systemic and local diseases
                                                              via non-invasive sampling methods. It is constituted
                               o

    Aim. The aim of this study was to determine the           by a complex chemical milieu of teeth and oral soft
    concentration and the variations of salivary glu-         tissues, consisting mainly on water, essential elec-
                           izi


    cose in healthy patients who were sampled at five         trolytes, glycoproteins, antimicrobial enzymes and nu-
    different intervals during the day.                       merous other important constituents like glucose (1,
    Materials and methods. Samples of un-stimulated           2). In the last decade the concentration of many bio-
    saliva have been collected from 21 healthy patients       markers present in saliva has acquired increasing im-
            Ed




    using the drooling technique and they were divid-         portance possibly showing the presence of systemic
    ed into two categories. In the first category, pa-        illness and reflecting the metabolic, nutritional, im-
    tients were asked not to toothbrush their teeth in        munological, hormonal, emotional and neurological
    the 8 hours prior to collection of the salivary sam-      state of the patient (3-5).
    ple. Patients in the second category were instead         Many articles have been written regarding the role
    asked to toothbrush 90 minutes before the collec-         and presence of glucose in oral fluids such as saliva
    C




    tion of the first sample of saliva. The glucose con-      and gingival crevicular fluid (GCF) in diabetic pa-
    centration was measured in all patients via an en-        tients. However, little efforts have been made to as-
CI




    zymatic spectrophotometry. Patients have been se-         sess healthy patients. Above all, there are no articles
    lected following a strict inclusion criteria, which in-   that describe repeated sample taking at different in-
    cluded <5% of plaque presence e according to the          tervals of the day (10-14).
    plaque Index and a total absence of oral disease          Glucose is a small molecule (180 DA) that diffuses
    that could possibly interfere with sample taking or       through blood vessel membranes and passes through
    oral fluid analysis. The average age of patients was      blood serum into GCF and salivary ducts to finally
©




    22,4±2,6 years old of which 45% were female and           reach the oral cavity (15). Several studies assert that
    55% were male. Samples were collected five times          salivary glucose levels are not related to glycaemia,
    between 8 am and 12 pm, before and after break-           even if the oral fluid of diabetic patients presents higher
    fast. Data was statistically analyzed using the           levels of glucose than those in healthy controls (14-17).
    Skewness/Kurtosis Test, Shapiro-Wilk Test,                Sampling oral fluid is easy, non-invasive for patients
    Kruskal Wallis Test and Linear Regression Model,          and safe for clinicians, However, several authors do
    considering values of p<0,05 to be significant.           not agree on the results obtained from such analyses,

    Annali di Stomatologia 2014; V (1): 1-6                                                                            1
    A. Polimeni et al.


    due to the different available protocols in sampling         them their toothbrush and toothpaste in order to
    procedures and the different analytical techniques.          demonstrate and explain to them the correct oral hy-
    Ramsaier et al. and Kinney et al. report using one of        giene techniques.
    the most repeatable and simple procedures to sam-            Salivary samples were always collected between 8
    ple oral fluid: the drooling technique (2, 18).              am and 12 pm. Un-stimulated whole saliva from each




                                                                                                                             li
    Many authors sampled oral fluid in a specific range of       participant was collected at each study visit via pas-
    time and patients were instructed not to brush their         sive drooling into a sterile plastic tube.




                                                                     na
    teeth, eat, drink or smoke 2-3 hours before the time of      The first sample was taken after 8 hours from tooth
    saliva sample collection, but none of them have as-          brushing (from 8:00 am to 8:30 am) or after 90 minutes
    sessed a plaque index score and/or oral health evalua-       (following two different Protocols). After the first oral flu-
    tion. This is essential as plaque residuals may play an      id sample collection, patients were asked to eat a stan-




                                                                   io
    important role in salivary glucose concentration (19-21).    dardized breakfast (selected by us). After breakfast, pa-
    Based on these premises, the goal of the present study       tients were asked to brush their teeth as they would
    is to evaluate the presence and therefore compare sali-      normally. One hour after tooth brushing the second oral




                                                                az
    vary glucose concentration in healthy young individuals      fluid sample was collected. Other three samples were
    with excellent oral care. We also intend to analyze un-      taken respectively after 90, 120 and 180 minutes.
    stimulated oral fluid samples taken at different times       One hundred and twenty five total samples were col-
    before and after a standardized breakfast, comparing         lected and statistically normalized using the Skew-




                                                             rn
    two different protocols; with tooth brushing (90 min be-     ness/Kurtosis and Shapiro-Wilk Normality Tests, and
    fore first sample taking) and without tooth brushing (8      then analyzed with the Kruskal Wallis Test and Linear
    hours from the last domiciliary oral hygiene care).          Regression Model. Values where p<0,05 were con-
                                                                 sidered to be significant.




                                                          te
    Materials and methods
                                                                 Reagents and solutions
    Thirty-seven patients who mantained all permanent
                                                       In
    teeth and had no salivary dysfunctions were included         Glucose oxidase/peroxidase, o-dianisidine dihy-
    in the study. Patients were trained for 6 months on          drochloride and sodium fluoride were purchased by
    how to carry out effective oral domiciliary hygiene          Aldrich; Sodium azide, D(+)-glucose monohydrate
    care. They were monitored until they reached a               and sulphuric acid (95-97%) were Merck reagents.
    Plaque Index score, which was less than 5% accord-           Artificial saliva was prepared, according to the litera-
                                          ni

    ing to the Silness and Loe index. Of the thirty-seven        ture (22) by dissolving in water the following
    patients assessed, only twenty-one of them have              reagents: 0.9 gL -1 KCl (Aldrich), 0.66 gL -1 KH 2PO 4
    managed to reach a 5% plaque level within the 6              (Baker), 0.08 gL-1 MgCl2 6H2O (Carlo Erba), 0.49 gL-1
                             o

    months. Hence, these patients have been included in          KHCO3 (Merck), 0.37 gL-1 NaNO3 (Merck), 0.12 gL-1
    the study only one diabetic patient was included in          CaCO3 (Merck), 0.64 gL-1 NH4Cl (Merck), 0.20 gL-1
                         izi


    the study under his specific request.                        Urea (Merck), 2.00 gL-1 Mucin (Aldrich). All the solu-
    The patients selected were between 20 to 25 years            tions were prepared with Millipore Milli-Q ultrapure
    old (average 22,4 ± 2,6 years). Of these, 45% were           water (resistivity > 18 MΩ cm-1).
    female and 55% were males. All patients were in              Standard solutions of D(+)-glucose monohydrate
           Ed




    good systemic and periodontal health, as assessed            were prepared for the calibration plot, diluting a 100
    from both their medical history and oral and periodon-       ppm solution.
    tal examination.                                             5 mg of o-dianisidine dihydrochloride were dissolved in
    Exclusion criteria for the study were: familiarity with or   1 mL of water and 0.8 mL of this solution were added to
    suspected diabetes, presence of metabolic diseases,          a Glucose oxidase/peroxidase solution, prepared dis-
    use of pharmacological drugs during the month prior to       solving the content of a capsule in 39.2 mL of water.
    C




    sample collection, presence of gingivitis or periodontal     Solutions of 0.01 M NaF and 0.01 M NaN3 were used
    disease, incorrect or unusual nutritional habits, pres-      as glycostatic and bacteriostatic agents respectively,
CI




    ence of xerostomia or reduced salivary flow.                 and a 12 N H2SO4 solution was used to stop the en-
    A single clinician (M.T.) evaluated all patients, using      zymatic reaction.
    a recent medical history and clinical findings. M.T
    was also responsible for instructing the patients on
    correct oral hygiene care and collecting repeated            Determination method
    samples. The information recorded was: age, gender,
©




    number of teeth and the plaque Index score accord-           The method used for the determination of glucose in
    ing to Silness and Loe.                                      saliva samples is an optimization of the GAGO-20
    After an additional evaluation, patients received de-        Sigma Aldrich Technical Bulletin. It is a spectrophoto-
    tailed information about the study procedures. All pa-       metric enzymatic technique based on the transforma-
    tients have been asked not to brush their teeth, drink       tion of glucose to gluconic acid mediated by glucose
    or eat within 8 hours prior to the first sample collec-      oxidase and the consequent production of H2O2. Hy-
    tion (at 8 am). They were also asked to take with            drogen peroxide reacts with o-dianisidine in the pres-

    2                                                                                    Annali di Stomatologia 2014; V (1): 1-6
    Salivary glucose concentration and daily variation in the oral fluid of healthy patients


    ence of peroxidase, producing a colored product,                 For both protocols the standardized breakfast con-
    whose absorbance, proportional to glucose concen-                sisted in a sweetened coffee or tea (with 2 tea-
    tration, is measured at 427 nm.                                  spoons of sugar corresponding to 8,4 g) and an
    2 mL of the solution containing glucose oxidase/per-             empty croissant.
    oxidase and o-dianisidine were added to each sam-                The collection procedure of saliva involved taking




                                                                                                                              li
    ple to be analyzed; after 30 min at 37° C in a thermo-           two samples of 0.5 mL to which 0.25 mL of 0.01 M
    stating bath and 2 mL of 12 N H2SO4 were used to                 NaF and 0.25 mL of 0.01 M NaN 3 were added as




                                                                       na
    stop the enzymatic reaction. The absorbance spec-                glycostatic and bacteriostatic agents. The first sam-
    trum was measured in the range 380-800 nm, mea-                  ple was mixed with 2 mL of the solution containing
    suring the maximum at 427 nm.                                    glucose oxidase/peroxidase and o-dianisidine whilst
                                                                     the second sample was mixed with 2 mL of water




                                                                     io
                                                                     (blank). Both samples were treated as described in
    Calibration plot                                                 the method section. The measurements of glucose
                                                                     concentration in the samples were obtained sub-
    The calibration plot was obtained using calibration              tracting the blank absorbance and using the calibra-




                                                                  az
    solutions gathered by mixing 0.25 mL of 0.01 M NaF,              tion plot. The blank subtraction is essential to par-
    0.25 mL of 0.01 M NaN3, 0.25 mL of artificial saliva             tially eliminate the contribution to the absorbance
    and 0.25 mL of standard glucose solutions in the con-            caused by the different opacity of the saliva sam-
    centration range of 0 to 40 ppm. Each of these sam-              ples, which comes from different patients. In such




                                                               rn
    ples was treated with the procedure explained before,            way, the measurement can only be referred to the
    measuring and correcting the absorbance with the                 glucose content.
    blank value.




                                                            te
                                                                     Results
    Protocols
                                                                     This study involved 21 non-diabetic patients; 11
    Two protocols were established to evaluate the glu-
    cose measurements and behavior in human saliva:
                                                         In          (52,4%) male and 10 (47,6%) female patients aged
                                                                     between 20 to 25 years. The average age was 22,4 ±
    • Protocol 1 (12 healthy patients): the first sample             2,6 years. For the whole oral fluid sample the aver-
       was collected after 8 hours from tooth brushing,              age flow rate was 0,53 ± 0,21 ml/min. A statistically
       before breakfast (shown by the red continuous                 significant correlation between salivary glucose and
                                              ni
       vertical line in Fig. 1) and subsequent morning               un-stimulated salivary flow rate was found (P=0,001).
       tooth brushing (shown by the green dashed verti-
       cal line in Fig. 1). Saliva was then collected at 60,
       90, 120, 180 minutes from breakfast and morning               Calibration plot
                               o

       tooth brushing.
    • Protocol 2 (9 healthy patients + 1 diabetic pa-                The calibration plot (Fig. 2) of the analytical method-
                           izi


       tient): the first sample was collected in the mor-            ology chosen for this study was obtained in the range
       ning after 8 hours from tooth brushing, (shown by             of 0-4 mg dL-1 with a positive correlation. The limit of
       the green dashed vertical line in Fig. 2). The pa-            detection was calculated at 0.011 mg dL-1 of glucose
       tients were then asked to have breakfast (shown               and the limit of quantification at 0.0325 mg dL -1 of
            Ed




       by the red continuous vertical line in Fig. 2). Sali-         glucose. These results show that the method used is
       va was then collected at 60, 90, 120, 180 minutes             applicable to analyze the glucose content in human
       from breakfast.                                               saliva.
    C




                                                                               Figure 1. General glucose concentration trend
                                                                               (“pseudo-glycemic curve”) for the two Protocols
                                                                               (two examples for each protocol: continuous lines
CI




                                                                               from data in bold in Tables 1 and 2). The dashed
                                                                               line represents the trend of the diabetic patient.
©




    Annali di Stomatologia 2014; V (1): 1-6                                                                                    3
    A. Polimeni et al.


    Discussion                                                       Nevertheless, a particular trend, showing a maximum
                                                                     value around 90 min from breakfast has been ob-
    Many authors have proved the efficiency and impor-               served. Figure 1 shows two examples for each proto-
    tance of saliva as diagnostic fluid. Salivary tests to           col (continuous lines, data in bold in Tab. 1 and 2)
    detect both periodontal and systemic illnesses are               representing this trend. This data suggests a correla-




                                                                                                                                  li
    now an established possibility. In fact, in literature           tion between food intake and salivary glucose. How-
    there are plenty of articles available which discuss             ever, such correlation has to be further assessed due




                                                                       na
    the applicability of oral fluid sample analyses (from            to the small number of samples analyzed.
    the simplest one to the most complex) as the integrat-           The difference between the two Protocols is particu-
    ed microfluidic platform for oral diagnostics, usually           larly evident in the first collected samples.
    known as Point-of-care (POC) salivary tests (23-26).             Protocol 1, in which the first sample is collected in the




                                                                     io
    Several studies evaluate the concentration of glucose            morning before tooth brushing (8 hours after the last
    in the saliva of diabetic patients. However, only a mi-          tooth brushing), demonstrates that during the night,
    nority of them focuses on healthy patients assessed              when the salivary flow rate is reduced, the levels of
                                                                     glucose in oral fluids increases and probably accumu-




                                                                  az
    at different times during the day with the purpose of
    investigating whether their glucose levels vary ac-              lates on oral hard and soft tissues.
    cording to several factors such as tooth-brushing and            Protocol 2, which involves tooth brushing before the
    nutritional habits.                                              first sample collection (precisely 90 minutes before),
                                                                     demonstrates the importance of effective oral hygiene




                                                               rn
    Table 1 and Table 2 show the results of the glucose
    determination according to both Protocols.                       care which reduces the level of glucose in the oral
    The data collected varies significantly form patient to          cavity (compared to Protocol 1) up to a measurement
    patient (as expected), probably due to the different             of 0 in half of the cases. Moreover, salivary flux acti-




                                                            te
    metabolism of each individual.                                   vation in these patients, due to tooth brushing, proba-

                                                         In                                   Figure 2. Calibration plot for glu-
                                                                                              cose determination via enzymatic
                                                                                              spectrophotometric method.
                             o              ni
                         izi
           Ed




    Table 1. Glucose concentration data for all the samples collected in Protocol 1.

    P
    C
CI




    Table 2. Glucose concentration data for all the samples collected in Protocol 2; a diabetic patient has also been recorded.
©




    P




    4                                                                                        Annali di Stomatologia 2014; V (1): 1-6
    Salivary glucose concentration and daily variation in the oral fluid of healthy patients


    bly plays a role in the salivary glucose concentration                  Paludetti G, Scarano E. Potential applications of human sali-
    observed.                                                               va as diagnostic fluid. Acta Otorhinolaryngol Ital.
    The dashed line in Figure 2 - Protocol 2, shows the                     2011;31:347-357.
                                                                       2.   Kinney JS, Morelli T, Braun T, Ramseier CA, Herr AE, Sug-
    behavior of glucose in the diabetic patient. The trend
                                                                            ai JV, Shelburne CE, Rayburn LA, Singh AK, Giannobile WV.
    of the “pseudo-glycemic curve” is the same already




                                                                                                                                       li
                                                                            Saliva/pathogen biomarker signatures and periodontal dis-
    discussed, but shifted at higher values, demonstrat-                    ease progression. J Dent Res. 2011; 90:752-758.
    ing how it may be possible to use salivary glucose




                                                                          na
                                                                       3.   Pohjamo L, Knuuttila M, Tervonen T, Haukipuro K. Caries
    concentration in the monitoring of diabetic patients.                   prevalence related to the control of diabetes. Proc Finn Dent
    Regarding the concentration of salivary glucose, the                    Soc. 1988;84:247-52.
    average collected from all patients involved in this               4.   Arcella D, Ottolenghi L, Polimeni A, Leclercq C. The rela-
    study was lower than the measurement obtained by                        tionship between frequency of carbohydrates intake and den-




                                                                        io
    Soares et al. (27), which was 5,94 mg/dL and Di Gioia                   tal caries: a cross-sectional study in Italian teenagers.
    et al. (28) which was 5,57 mg/dL. Agha-hosseini et al.                  Public Health Nutr. 2002;5(4):553-60.
                                                                       5.   Farronato G. Odontostomatologia per l’Igienista Dentale, Basi
    observed an average salivary glucose measurement of
                                                                            anatomo-cliniche e protocolli operativi. Piccin-Nuova Libraria




                                                                     az
    13,6 mg/dL. As proposed by Soares et al. (27), one                      2007.
    possible explanation of salivary glucose differences ob-           6.   Farronato G, Regaldo G, Mascardi A. Azione enzimatica
    tained in these studies may be the various study de-                    dell’Alfa-Amilasi sulla placca batterica. Dent Cadm.
    signs as well as the diversity of the methods used and                  1987;7:59-63.




                                                                  rn
    selection criteria of the sample collection (13, 29). This         7.   Farronato G, Venerando B, de Colle C, Tonon M. Azione di
    data suggests that domiciliary oral hygiene techniques                  una destranasi sulla placca batterica in soggetti sottoposti
    of each patient should always be assessed before oral                   a chirutgia ortognatodontica. Prevenzione e Assistenza Den-
    fluid sample taking or, if possible, previously set.                    tale 1992: 5.
                                                                       8.   Farronato G, Venerando B, Biagi R, De Colle C, Passuello




                                                               te
    In accordance to the results obtained from this study, the
                                                                            F, Tonon M. Azione di una destranasi sulla placca batteri-
    majority of authors observe a statistically significant cor-
                                                                            ca in soggetti handicappati: sperimentazione clinica. Pre-
    relation between flow rate and salivary glucose concen-                 venzione e Assistenza Dentale. 1992:3.
    tration. These suggest a pivotal role of flow rate, not only       9.   Polimeni A., Gallottini L., Dolci M. La secrezione salivare e
    for the assessment of glucose concentration but also for
                                                            In              le sue alterazioni. Roma. Euroma, Editrice Univ. La Goliardica
    the identification of biomarkers levels in the diagnosis or             1995.
    screening of systemic and local diseases (27-29).                 10.   Twetman S, Johansson I, Birkhed D, Nederfors T. Caries in-
                                                                            cidence in young type 1 diabetes mellitus patients in rela-
                                                                            tion to metabolic control and caries-associated risk factors.
                                              ni
    Conclusions                                                             Caries Res. 2002;36:31-5.
                                                                      11.   Karjalainen KM, Knuuttila ML, Käär ML. Salivary factors in
                                                                            children and adolescents with insulin-dependent diabetes mel-
    Based on the results obtained from the samples stud-                    litus. Pediatr Dent. 1996;18:306-11.
                               o

    ied, it is possible to determine that salivary glucose is         12.   Ben-Aryeh H, Serouya R, Kanter Y, Szargel R, Laufer D. Oral
    present even when plaque levels and oral health sta-                    health and salivary composition in diabetic patients. J Dia-
    tus are strictly controlled. However, further studies
                           izi


                                                                            betes Complicat. 1993;7:57-62.
    are required to prove a correlation between glucose               13.   Moore PA, Guggenheimer J, Etzel KR, Weyant RJ, Orchard
    intake and salivary glucose as the number of patients                   T. Type 1 diabetes mellitus, xerostomia, and salivary flow
    and samples collected in our study were limited.                        rates. Oral Surg Oral Med Oral Pathol Oral Radiol Endod.
                                                                            2001;92:281-91.
            Ed




    Moreover, an alternative, complementary, more rapid,
                                                                      14.   Jurysta C, Bulur N, Oguzhan B, Satman I, Yilmaz TM,
    precise and accurate analytical methodology (capa-
                                                                            Malaisse WJ, Sener A. Salivary glucose concentration and
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                                                                            excretion in normal and diabetic subjects. J Biomed
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    analytical techniques, capable of monitoring small                      doi:10.1155/2009/430426
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    C




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                                                                            1988; 2: 96-99.
                                                                      16.   Amer S, Yousuf M, Siddqiui PQ, Alam J. Salivary glucose
CI




    Acknowledgements                                                        concentrations in patients with diabetes mellitus-a minimally
                                                                            invasive technique for monitoring blood glucose levels. Pak
                                                                            J Pharm Sci. 2001;14:33-37.
    Authors thank Colgate-Palmolive Company for finan-
                                                                      17.   Chávez EM, Borrell LN, Taylor GW, Ship JA. A longitudinal
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                                                                            with type 2 diabetes. Oral Surg Oral Med Oral Pathol Oral
©




                                                                            Radiol Endod. 2001 Feb;91(2):166-73.
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    6                                                                                            Annali di Stomatologia 2014; V (1): 1-6
