10.59987/ads/2016.3.38-45
Aim. The aim of the present in vitro study was to evaluate the protective effects of a zinc-hydroxyapatite toothpaste against an erosive challenge produced by a soft drink (Coca-Cola) using Scanning Electron Microscopy (SEM).
Methods. Forty specimens were assigned to 4 groups of 10 specimens each (group 1: no erosive challenge, no toothpaste treatment, group 2: erosive challenge, no toothpaste treatment, group 3: erosive challenge, fluoride toothpaste treatment, group 4: erosive challenge, zinc-hydroxyapatite toothpaste treatment). The surface of each specimen was imaged by SEM. A visual rating system was used to evaluate the condition of the enamel surface; results were analyzed by nonparametric statistical methods.
Results. Statistically significant differences were found between the samples untreated and those immersed in Coca-Cola (group 1, 2); the highest grade of damage was found in group 2, while the lowest grade was recorded in the samples of group 4. Comparing the groups, the two analyzed toothpaste tended to protect in different extend.
Conclusions. In this study treatment of erosively challenged enamel with Zn-Hap toothpaste showed a clear protective effect. This was greater than the effect observed for a normal fluoride toothpaste and confirmed the potential benefit the Zn-HAP technology can provide in protecting enamel from erosive acid challenges.
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Annali di stomatologia
toothpastes
Protective effects of a zinc-hydroxyapatite toothpaste on enamel erosion: SEM study
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Original Article
2022-08-10
2022-08-10
2016-09-01
2023-12-04
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Ann Stomatol (Roma)
C. Poggio
2016
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toothpastes
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Protective effects of a zinc-hydroxyapatite toothpaste on enamel erosion: SEM study
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                                                                                                 Original article



    Protective effects of a zinc-hydroxyapatite
    toothpaste on enamel erosion: SEM study




                                                                                                              i
                                                                                                            al
    Marco Colombo                                         Zn-HAP technology can provide in protecting ena-
    Riccardo Beltrami                                     mel from erosive acid challenges.




                                                                                              n
    Davide Rattalino
    Maria Mirando                                         Key words: enamel erosion, fluoride, hydroxyap-




                                                                                           io
    Marco Chiesa                                          atite, SEM, surface analysis, toothpastes.
    Claudio Poggio

                                                          Introduction




                                                              az
    Department of Clinical, Surgical, Diagnostic and
    Pediatric Sciences, Section of Dentistry,             The prevalence of dental erosion (erosive tooth wear)
    University of Pavia, Italy                            is thought to be increasing, due to the wide availability,




                                                            rn
                                                          and frequent consumption of acidic drinks such as soft
                                                          drinks, sports drinks and fruit juices (1). The develop-
    Corresponding author:                                 ment of erosion involves a chemical process in which
    Claudio Poggio                                        the inorganic phase of the tooth is demineralized,




                                                          te
    Department of Clinical, Surgical, Diagnostic and      thereby reducing the hardness of tooth substrates (2).
    Pediatric Sciences                                    To prevent dental erosion many strategies have been
    Section of Dentistry, Policlinico “San Matteo”        developed (3). To improve enamel and dentin resis-
    Piazzale Golgi 3
                                                       In tance, toothpastes were considered effective and af-
    27100 Pavia, Italy                                    fordable vehicles (4). The incorporation of protective
    E-mail: claudio.poggio@unipv.it                       agents in toothpastes has become increasingly com-
                                                          mon; also because dental sensitivity, a problem often
                                                          related to acid erosion, is a common complaint
                                          ni

    Summary                                               among patients. Currently, conventional fluoride-
                                                          based toothpastes do not seem to be able to effec-
    Aim. The aim of the present in vitro study was to     tively protect teeth against erosion (5). Recently, new
                                  io


    evaluate the protective effects of a zinc-hydroxya-   toothpastes formulations have been introduced to
    patite toothpaste against an erosive challenge        contrast enamel and dentin erosion. Among the large
    produced by a soft drink (Coca-Cola) using Scan-      amount of commercially available products, several
                          iz



    ning Electron Microscopy (SEM).                       new toothpaste technologies were subject of our pre-
    Methods. Forty specimens were assigned to 4           vious studies (6, 7). Changes in tooth structure due to
    groups of 10 specimens each (group 1: no ero-         extrinsic factors, such as acidic challenges from food
             Ed




    sive challenge, no toothpaste treatment, group 2:     and drink, have been widely investigated through
    erosive challenge, no toothpaste treatment, group     Scanning Electron Microscopy (SEM). The aim of the
    3: erosive challenge, fluoride toothpaste treat-      present study was to test the impact of toothpaste
    ment, group 4: erosive challenge, zinc-hydroxyap-     with Zinc-Hydroxyapatite (Zn-HAP) on preventing
    atite toothpaste treatment). The surface of each      enamel erosion compared to toothpaste with fluoride
    specimen was imaged by SEM. A visual rating           by using Scanning Electron Microscopy (SEM).
 IC




    system was used to evaluate the condition of the
    enamel surface; results were analyzed by non-
    parametric statistical methods.                       Materials and methods
    Results. Statistically significant differences were
C




    found between the samples untreated and those         The test toothpaste was: Zn-HAP (Microrepair®)
    immersed in Coca-Cola (group 1, 2); the highest       toothpaste, without fluoride (Biorepair; Coswell
    grade of damage was found in group 2, while the       S.P.A., 40050 Funo, Italy). The control toothpaste
    lowest grade was recorded in the samples of           was: fluoride toothpaste, 1450 ppm F- as NaF (Eu-
@




    group 4. Comparing the groups, the two analyzed       fresh; CIO Farmaceutici s.r.l., 81100 Caserta, Italy).
    toothpaste tended to protect in different extend.     The study involved four different treatment groups:
    Conclusions. In this study treatment of erosively     group 1: no erosive challenge, no toothpaste treat-
    challenged enamel with Zn-Hap toothpaste              ment;
    showed a clear protective effect. This was greater    group 2: erosive challenge, no toothpaste treatment;
    than the effect observed for a normal fluoride        group 3: erosive challenge, fluoride toothpaste treat-
    toothpaste and confirmed the potential benefit the    ment;

    38                                                                        Annali di Stomatologia 2016;VII (3):38-45
    Protective effects of a zinc-hydroxyapatite toothpaste on enamel erosion: SEM study


    group 4: erosive challenge, zinc-hydroxyapatite tooth-           36 h; during these intervals the specimens were kept
    paste treatment.                                                 in artificial saliva. The specimens of group 2, 3 and 4
                                                                     were immersed in 6 ml of the soft drink for 2 min at
    Specimen preparation                                             room temperature before rinsing with deionized wa-
                                                                     ter. Four consecutive intervals of the immersion pro-
    Specimens were prepared from 40 human incisors,                  cedure were carried out (8). The immersions in the
    extracted for orthodontic and periodontal reasons.               soft drink were repeated as described above at 0, 8,




                                                                                                                      i
    Debris and soft tissue were eliminated and teeth were            24 and 36 h.




                                                                                                                    al
    inspected for cracks, hypoplasia, white spot lesions
    and reconstruction. Teeth were cleaned to remove                 Scanning Electron Microscopy (SEM)
    soft tissue and stored in a solution of 0.1% (wt/vol)




                                                                                                        n
    thymol. The enamel specimens were cut at the enam-               The specimens were gently air dried, dehydrated with
    el-dentin junction with a high-speed diamond rotary              alcohol, sputter‐coated with gold. Enamel and dentin




                                                                                                     io
    bur with a water-air spray. The samples were placed              were characterized using a field emission scanning
    into Teflon molds measuring 10 x 8 x 2 mm and em-                electron microscope (FE‐SEM, MIRA3, TESCAN).
    bedded in self-curing, fast-setting acrylic resin (Rapid         Serial SEM microphotographs of the surfaces of each




                                                                         az
    Repair, DeguDent GmbH, Hanau, Germany) in such                   specimen at 2.50 KX and 5.00 KX original magnifica-
    a way that the exposed buccal surface was plano-                 tions were obtained (9). A systematic assessment
    parallel to the bottom of the mold.                              method was adopted for grading the SEM images.
                                                                     SEM images recorded were evaluated in terms of




                                                                       rn
    Demineralization and remineralization                            enamel damages by three experienced assessors
                                                                     who randomly examined the samples twice in a blind
    A soft drink (Coca Cola, Coca Cola Company, Milano,              manner. A scoring scale (Tab. 2) was adopted to de-
    Italy) was chosen for the demineralization process               scribe the enamel surface (10).




                                                                     te
    (6). The pH at 20˚C, buffering capacity, and concen-
    tration of calcium and phosphate of the beverage                 Statistical Analysis
    were measured by standard chemical methods. The
    pH of soft drink was measured with a pH meter (Acc-
                                                            In       Descriptive statistics for the scores of the morphologi-
    umet AB15, Fisher Scientific, Pittsburgh, PA). Ca 2-             cal analysis were calculated. Data were analyzed
    and PO43- were determined by flame atomic absorp-                with the Kruskal-Wallis test. The Mann-Whitney U
    tion (Perkin Elmer 1100 B spectrophotometer). Mea-               test was performed for post hoc comparisons. Signifi-
    surements were performed in triplicate and average               cance was set at a P value <0.05. In order to check
                                                 ni

    values calculated (Tab. 1).                                      the intra - and inter-observer reliability the Intraclass
    The samples were then assigned to the four treat-                Correlation Coefficient was calculated; it was greater
    ment groups with 10 specimens per group.                         than 0.9.
                                       io


    The toothpastes were applied neat onto the surface
    of the specimens to cover the enamel surface without
    brushing and then wiped off with distilled water wash-           Results
                              iz



    ing after every treatment to remove residual tooth-
    paste; the control specimens (group 1) were taken on             The mean amounts of scores for the morphological
    storage for the whole experimentation and they did               analysis of the images are reported in Table 3 and in
               Ed




    not receive any treatment. The toothpastes were ap-              Figure 1. The Kruskal-Wallis test showed the pres-
    plied to the enamel surfaces for 3 min at 0, 8, 24 and           ence of significant differences among the different


    Table 1. Chemical properties of the soft drink used in the study.
 IC




    Beverage                 Ph                 Buffering Capacity         Po4 (Mg/L)             Ca (Mg/L)

    Coca Cola                2,44               0,0056                     175,7                  20,83
C




    Table 2. Scoring criteria used for the evaluation of SEM images.

    Grade     Status
@




    0         Enamel surface remained perfectly intact with no grooves, pits, and porosity

    1         Presence of surface irregularities on enamel surface, without demineralization of prismatic and/or

              interprismatic enamel

    2         Presence of wrinkles and demineralization of prismatic/interprismatic enamel

    3         Diffuse demineralization involved the rod core, with decomposition of morphology of prism


    Annali di Stomatologia 2016;VII (3):38-45                                                                              39
    M. Colombo et al.


    Table 3. Means and standard deviations of the morphological SEM scores provided by the three observers and overall.
    Different superscript letters indicate significant differences (p <0.05).

                         Observer 1             Observer 2            Observer 3              Overall

    Group 1              0.53 ± 0.52            0.68 ± 0. 44          0.63 ± 0.61             0.61 ± 0.52      a


    Group 2              2.73 ± 0.46            2.44 ± 0.66           2.89 ± 0.34             2.69 ± 0.49




                                                                                                                     i
                                                                                                               b




                                                                                                                   al
    Group 3              2.2 ± 0.41             2.11 ± 0.57           1.97 ± 0.44             2.1 ± 0.47   c


    Group 4              1.27 ± 0.46            1.18 ± 0.35           1.45 ± 0.33             1.3 ± 0.38   d




                                                                                                    n
                                                                                                 io
    groups (p<0.05). On enamel surfaces not exposed to          Discussion and conclusions
    the erosive challenge by the soft drink (group 1), the
    typical structures of sound enamel such as grooves          In this study the morphological analysis of enamel




                                                                    az
    and perichimata lines were apparent; also small de-         surfaces after an erosive acid challenge from a soft
    pressions or ditches or grinding marks were found in-       drink followed by treatment with Zn-Hap toothpaste
    dicative of the cumulative mechanical effects the           showed a clear protective effect. This was greater
    teeth have experienced were observed (Figs. 2, 3).          than the effect observed for a normal fluoride tooth-




                                                                  rn
    The enamel surface of teeth exposed to the acidic           paste and confirmed the potential benefit the Zn-HAP
    challenge by the soft drink clearly demonstrated deep       technology can provide in protecting enamel from
    changes in enamel structure (Figs. 4, 5). After 32 min      erosive acid challenges.
    eosure to the acidic challenge (four immersions of 8        The morphological analysis of enamel was based on




                                                                te
    min each) an irregular pattern of surface erosion           images taken by scanning electron microscopy
    could be observed and the presence of honeycomb             (SEM), a technique that is suitable for use with native
    structures suggests demineralization of enamel              unpolished surface samples and enamel having been
    prisms. The morphological scores for the acid chal-
                                                        In      exposed to acidic challenge or toothpaste treatment.
    lenged specimens were significantly higher than the         In the present in vitro study, SEM was used to verify
    scores for the specimens not exposed to acid chal-          the protective effect of the two toothpastes on enamel
    lenge (Mann-Whitney U test, p<0.05).                        exposed to erosive action of a soft drink. The SEM
                                            ni
    The acid-challenged specimens treated with the con-         study allowed to understand qualitatively the process-
    trol and test toothpastes demonstrated a lower de-          es of demineralization of the enamel surface through
    gree of demineralization on the enamel surface as           the observation of specific morphological and struc-
                                      io


    evident in the SEM images, Figures 6-9 respectively.        tural features that characterize the enamel itself.
    This is reflected in the lower morphological scores for     A classification scale was used in order to help quan-
    these two groups compared to the acid-challenged            tifying and describe the damage grade on enamel.
                           iz



    samples not treated with toothpaste. In the SEM im-         Scoring criteria modification of demineralization eval-
    ages (Figs. 6, 7) of the specimens treated with fluo-       uation (11) was followed, as reported in Table 2: a
    ride toothpaste (group 3) honeycomb structures that         score of zero was assigned to enamel surface per-
              Ed




    were typical of the demineralization enamel were still      fectly intact with no grooves, pits and porosity, while
    visible, and a slight irregular pattern of erosion could    a score of three to those where diffuse demineraliza-
    be observed. The average morphological score of             tion involved the rod core, resulting in a lesion form-
    this group was significantly lower than the scores for      ing the “keyhole” like structure.
    the acid challenged specimens (p<0.05).                     The experimental protocol of the present study was
    The specimens treated with the Zn-HAP toothpaste            conducted in attempt to better simulate the daily
 IC




    (group 4) showed evidence of deposited material in          habits of soft drink consumption. To predict the ero-
    the SEM images (Figs. 8, 9), with little evidence of        sive potential of a soft drink, the method used should
    erosive damage to the tooth surface.                        simulate what happens in vivo when the drink enters
    Specimens of group 4 showed the lowest morphologi-          the mouth. For this reason, the method used in the
C




    cal SEM scores of even if not as similar as intact          present study (four consecutive intervals of 2 minutes
    enamel (P<0.05). Overall mean and standard devia-           for four times, at 0, 8, 24 and 36 hours) was consid-
    tions of the morphological SEM scores confirmed that        ered to mimic, as closely as possible, the natural con-
    Zn-HAP toothpaste provided the lowest evidence of           sumption of cola drink during the main daily meals.
@




    erosive damage to the tooth surface (p < 0.001).            During the entire experimental protocol, the speci-
    Thus, if comparing the action of Zn-HAP (group 4)           mens were maintained in fresh artificial saliva until
    and fluoride (group 3) toothpastes against an eroded        the next time of application of pastes. This means
    enamel surface (group 2), it resulted that enamel           that the specimens were in contact with the bioactive
    specimens of group 4 tended to be significantly more        agent for 12 min without suffering a demineralizing
    protected after the treatment.                              acid attack and then stayed in remineralizing solution.



    40                                                                              Annali di Stomatologia 2016;VII (3):38-45
    Protective effects of a zinc-hydroxyapatite toothpaste on enamel erosion: SEM study


                                                                                  Figure 1. Means and standard deviations
                                                                                  of the morphological SEM scores provided
                                                                                  by the three observers.




                                                                                                                    i
                                                                                                      n           al
                                                                                                   io
                                                                       az
                                                                     rn
                                                                                  Figure 2. SEM image at 5.00 KX magnifi-
                                                                                  cation of intact enamel surface (Group 1).




                                                                   te
                                                          In
                                                ni
                                       io
                              iz
               Ed




                                                                                  Figure 3. SEM image at 2.50 KX magnifi-
                                                                                  cation of intact enamel surface (Group 1).
 IC
C
@




    Annali di Stomatologia 2016;VII (3):38-45                                                                            41
    M. Colombo et al.


    Figure 4. SEM image at 5.00 KX magnifi-
    cation of enamel exposed to Coca-Cola
    (Group 2).




                                                                                        i
                                                                        n             al
                                                                     io
                                                       az
                                                     rn
    Figure 5. SEM image at 2.50 KX magnifi-
    cation of enamel exposed to Coca-Cola
    (Group 2).




                                                   te
                                                   In
                                               ni
                                     io
                             iz
              Ed




    Figure 6. SEM image at 5.00 KX magnifi-
    cation of intact enamel surface treated with
    Eufresh (Group 3).
 IC
C
@




    42                                                  Annali di Stomatologia 2016;VII (3):38-45
    Protective effects of a zinc-hydroxyapatite toothpaste on enamel erosion: SEM study


                                                                                  Figure 7. SEM image at 2.50 KX magnifi-
                                                                                  cation of intact enamel surface treated with
                                                                                  Eufresh (Group 3).




                                                                                                                     i
                                                                                                      n            al
                                                                                                   io
                                                                       az
                                                                     rn
                                                                                  Figure 8. SEM image at 5.00 KX magnifi-
                                                                                  cation of intact enamel surface treated with
                                                                                  Biorepair (Group 4).




                                                                   te
                                                          In
                                                ni
                                       io
                              iz
               Ed




                                                                                  Figure 9. SEM image at 2.50 KX magnifi-
                                                                                  cation of intact enamel surface treated with
                                                                                  Biorepair (Group 4).
 IC
C
@




    Annali di Stomatologia 2016;VII (3):38-45                                                                              43
    M. Colombo et al.


    In the oral environment, host factors (such as the        agents since they are used routinely as an oral hy-
    mineral concentration of the tooth, and the pellicle      giene measure. Jager et al. (21) showed that different
    and plaque formation) can influence the progression       exposure times to acid beverages also result in very
    of demineralization (12-14).                              different estimates of erosive potential, and that effect
    Salivary factors, such as the salivary flow rate, com-    of the choice of study methodology may affect the re-
    position and buffering capacity, might exert protective   sults of the study.
    action on dental surface (12, 15, 16). For this reason,   For this reason, the effects of dentifrices on dental




                                                                                                                        i
                                                              erosion have been exhaustively studied (12, 22-24).




                                                                                                                      al
    a further step was taken in this study to enhance the
    relevance of the model by storing specimens in artifi-    While some studies have shown that products with
    cial saliva between the experimental procedures.          bioactive agents such as fluoride, CPP-ACP, and cal-
    Among soft drinks, Cola drink has the highest erosive     cium sodium phosphosilicate have the potential to




                                                                                                       n
    potential (17, 18) and this was the rationale for using   prevent enamel demineralization (20, 23, 24), other
    it in the present study.                                  studies have shown no favorable effects of these




                                                                                                    io
    The effect of demineralizing, acidic drinks such as       agents (12, 22).
    Coca Cola was assessed in this study by comparing
    the SEM images of enamel treated with the drink with




                                                                  az
    those of the unchallenged samples, shown in Figure        Acknowledgements
    2 and 3. As expected, the surface of enamel treated
    with an acidic drink shows the presence of honey-         We are grateful to Clara Cassinelli (Nobil Bio Ricerche
    comb structures, which suggests demineralization of       S.r.l., Portacomaro, Asti, Italy) for providing the SEM




                                                                rn
    enamel prisms. Diffuse demineralization involved the      images and technical assistance.
    rod core, with decomposition of morphology of prims:
    they were severely affected and a greater prism-core
                                                              Conflict of interest statement




                                                              te
    dissolution compared with that in the interprismatic
    areas gave the enamel a “keyhole pattern” or “honey-      The Authors of this study have no conflict of interest
    comb pattern” of demineralization (Figs. 4, 5).           to disclose.
    The main aim of the present study was to evaluate,
                                                       In
    by SEM analysis, the protective efficacy of a Zn-HaP
    toothpaste on enamel after acidic challenge, com-         References
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    Annali di Stomatologia 2016;VII (3):38-45                                                                                            45
