10.59987/ads/2014.3.98-102
Aim. The aim of the present in vitro study was the evaluation of two products: a CPP-ACP paste (GC Tooth Mousse, GC Corp.) and a desensitizing toothpaste (Colgate Sensitive Pro Relief, Colgate-Palmolive) on preventing enamel erosion produced by a soft drink (Coca Cola) by using Atomic Force Microscopy (AFM).
Methods. Thirty enamel specimens were assigned to 6 groups of 5 specimens each. 1: intact enamel, 2: enamel + soft drink, 3: intact enamel + Colgate Sensitive Pro Relief, 4: enamel + soft drink + Colgate Sensitive Pro Relief, 5: intact enamel + GC Tooth Mousse, 6: enamel + soft drink + GC Tooth Mousse. The surface of each specimen was imaged by AFM. The root mean-square roughness (Rrms) was obtained from the AFM images and the differences in the averaged values among the groups were analyzed by ANOVA test.
Results. Comparing groups 4 and 6 (soft drink + toothpastes) with group 2 (eroded enamel) a statistical difference (P&lt;0.05) was registered, suggesting effectiveness in protecting enamel against erosion of the products investigated. Conclusions. The use of new formulation toothpastes can prevent enamel demineralization.
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Annali di stomatologia
surface roughness
Atomic force microscopy study of enamel remineralization
Text.Serial.Journal
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Original Article
2022-08-12
2022-08-12
2014-09-01
2023-12-04
98-102
Ann Stomatol (Roma)
M. Colombo
2014
10.59987/ads/2014.3.98-102
98
1971-1441
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surface roughness
en
102
Atomic force microscopy study of enamel remineralization
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                                                                                                     Original article



    Atomic force microscopy study of enamel
    remineralization




                                                                                                        i
                                                                                                      al
                                                                                                   on
    Claudio Poggio, MD, DDS1                                  Key words: AFM, enamel, remineralization, SEM,
    Matteo Ceci, DMD, PhD1                                    soft drinks, surface roughness.
    Riccardo Beltrami, DMD, PhD2
    Marco Lombardini, DMD, PhD1
    Marco Colombo, DMD, PhD1                                  Introduction




                                                                                         zi
                                                              Dental erosion has been defined commonly as the
    1 Department of Clinical, Surgical, Diagnostic and Pe-    chemical dissolution of the hard tissues of teeth by




                                                                           na
      diatric Sciences, Section of Dentistry, University of   acids of non-microbiological origin (1). Dental erosion
      Pavia, Italy                                            is a common problem in modern societies, owing to
    2 Department of Brain and Behavioural Sciences,           the increased consumption of acid drinks such as
      Section of Statistics, University of Pavia, Italy       soft drinks, sport drinks, fruit juices (2). The develop-
                                                              ment of erosion involves a chemical process in which




                                                                er
                                                              the inorganic phase of the tooth is demineralized,
    Corresponding author:                                     thereby reducing the hardness of the tooth sub-
    Claudio Poggio                                            strates. Subsequent abrasive challenges through
    Department of Clinical, Surgical, Diagnostic


                                                        t     brushing increase the loss of the tooth substrates (3).
    and Pediatric Sciences, Section of Dentistry,
    University of Pavia
                                                     In       The early stages of dental erosion are characterized
                                                              by a softening of the enamel surface to a depth of the
    Piazzale Golgi 3                                          order of 1-10 microns. Many studies have been car-
    27100 Pavia, Italy                                        ried out to understand the process of enamel dem-
    Phone: +39 0382 516257, +39 3398124925                    ineralization at the early stages, but there are still lit-
                                         ni
    Fax: +39 0382 516224                                      tle known about if these early stages are reversible
    E-mail: claudio.poggio@unipv.it                           (4). Biological and chemical factors in the oral envi-
                                                              ronment influence the progress of dental erosion.
                                io

                                                              Saliva provides protective effects by neutralizing and
    Summary                                                   clearing the acids; it is also a source of inorganic
                                                              ions necessary for the remineralization process (5).
    Aim. The aim of the present in vitro study was the        Enamel has no spontaneous capability to repair
                        iz



    evaluation of two products: a CPP-ACP paste (GC           when affected by specific dental pathologies such as
    Tooth Mousse, GC Corp.) and a desensitizing               caries, abrasions or fractures because it contains no
    toothpaste (Colgate Sensitive Pro Relief, Colgate-        cells (6). Therefore, when enamel is exposed to oral
          Ed




    Palmolive) on preventing enamel erosion pro-              environment the only possibility to be reconstructed
    duced by a soft drink (Coca Cola) by using Atom-          depends on the application of alloplastic materials.
    ic Force Microscopy (AFM).                                Toothpastes have been considered effective and ac-
    Methods. Thirty enamel specimens were assigned            cessible vehicles to improve enamel resistance to
    to 6 groups of 5 specimens each. 1: intact enam-          erosive attacks (7). The incorporation of protecting
    el, 2: enamel + soft drink, 3: intact enamel + Col-       agents in toothpastes has become increasingly more
IC




    gate Sensitive Pro Relief, 4: enamel + soft drink +       common, because sensitivity is a common complaint
    Colgate Sensitive Pro Relief, 5: intact enamel +          among patients. Many types of toothpaste recently
    GC Tooth Mousse, 6: enamel + soft drink + GC              introduced are claimed to prevent erosion. Fluoride
    Tooth Mousse. The surface of each specimen was            dentifrices have some protective effect on enamel
C




    imaged by AFM. The root mean-square roughness             eroded to brushing abrasion when immersed in vitro
    (Rrms) was obtained from the AFM images and the           in a cola drink (8). Currently, conventional fluoride-
    differences in the averaged values among the              containing toothpastes do not appear to be able to
    groups were analyzed by ANOVA test.                       protect efficiently against erosion (9). Colgate Sensi-
©




    Results. Comparing groups 4 and 6 (soft drink +           tive Pro Relief (Colgate-Palmolive, New York, NY,
    toothpastes) with group 2 (eroded enamel) a sta-          USA) is based on arginine 8% (Pro-Argin™). Pro-Ar-
    tistical difference (P<0.05) was registered, sug-         gin ™ technology contains arginine, an amino acid
    gesting effectiveness in protecting enamel                naturally found in saliva, and a compound of insolu-
    against erosion of the products investigated.             ble calcium in the form of calcium carbonate. Argi-
    Conclusions. The use of new formulation tooth-            nine and calcium carbonate bind to the dentin-enam-
    pastes can prevent enamel demineralization.               el negatively charged surface, creating a protective

    98                                                                           Annali di Stomatologia 2014; V (3): 98-102
    AFM study of enamel remineralization


    layer, resistant to acid stimuli, that covers the surface   tion (19). The specimens were cut at the enamel-
    defects and occludes the exposed dentinal tubules           dentin junction, with a high-speed diamond rotary bur
    with a consequently reduced sensitivity (10). GC            with a water-air spray. The labial surfaces near the
    Tooth Mousse (GC Corp., Tokio, Japan) is based on           enamel dentin junction were ground using silicon car-
    CPP-ACP. Casein phosphopeptides (CPP) contain-              bide papers (grades 600 to 1200) under water irriga-




                                                                                                       i
    ing the cluster sequence – Ser(P)-Ser(P)- Ser(P)-           tion to produce flat enamel surfaces. Samples were




                                                                                                     al
    Glu-Glu – have a remarkable ability to stabilize calci-     placed into Teflon moulds measuring 10 x 8 x 2 mm,
    um phosphate (ACP) in metastable solution. Through          embedded in flowable composite resin and polymer-
    the multiple phosphoseryl residues, the CPP binds to        ized. The baseline root mean-square roughness,




                                                                                                  on
    forming nanoclusters of ACP, preventing their growth        R rms , was measured for all the specimens before
    to the critical size required for nucleation and phase      starting experimentation. No statistical difference in
    transformation (11). CPP-ACP has been demonstrat-           R rms values (150±5) was recorded, suggesting that
    ed to have anticariogenic activity in laboratory, ani-      the specimens may be comparable.
    mal and human in situ experiments (12). The CPP-




                                                                                          zi
    ACP solutions have shown to significantly remineral-
    ize enamel subsurface lesions in vitro (13). CPP-ACP        Demineralization and remineralization
    has been successfully incorporated into oral health




                                                                             na
    products such as a mouth rinses, chewing gums and           A soft drink (Coca Cola, Coca Cola Company, Milano,
    sports drink to reduce enamel erosion (14). Changes         Italy) was chosen for the demineralization process.
    in tooth structure due to extrinsic factors have been       The pH at 20˚C, buffering capacity, concentration of
    widely investigated. Whereas the material losses (ab-       calcium and phosphate of the beverage were mea-




                                                                  er
    solute erosions) have been carefully characterized,         sured (20). Measurements were performed in tripli-
    only minor attention has been devoted to the investi-       cate and average values calculated. Two remineral-
    gation of tooth surface change during erosion and           izating agents were used: GC Tooth Mousse and, as
    demineralization (15). The kinetic roughening of a          reference, Colgate Sensitive Pro Relief, a desensitiz-
    surface is the process that takes place when material


                                                          t     ing toothpaste. The samples were then assigned to 6

    and experimental studies showed that this process
                                                       In
    is removed from or added to the surface. Theoretical        groups, each made of 5 teeth:
                                                                - group 1: intact enamel
    might be often interpreted in terms of self-affinity        - group 2: enamel + soft drink
    concepts and simple scaling laws. However, whereas          - group 3: enamel + Colgate Sensitive Pro Relief
    many works have been devoted to the investigation           - group 4: enamel + soft drink + Colgate Sensitive
                                                 ni
    of film growth by chemical or physical vapor deposi-            Pro Relief
    tion methods, less attention has been reserved to the       - group 5: enamel + GC Tooth Mousse.
    inverse problem of surface etching, polishing, or ero-      - group 6: enamel + soft drink + GC Tooth Mousse.
                                     io

    sion (16). Atomic Force Microscopy (AFM) is capable         The control specimens (group 1) were taken on stor-
    of giving images with atomic resolution with minimal        age for the whole experimentation and they did not
    sample preparation. This technique has been widely          receive any treatment. The specimens of groups 2, 4
    used to characterize the erosion of enamel and den-         and 6 were immersed in 6mL of the soft drink for 2
                           iz



    tine (17). More recently, also AFM nanoindentation          min at room temperature before rinsing with deion-
    has been applied to the study of enamel erosion (18).       ized water. Four consecutive intervals of the immer-
    The aim of the present in vitro study was the evalua-       sion procedure were carried out at 0, 8, 24 and 36 h
          Ed




    tion of a CPP-ACP paste and of a desensitizing              for a total of 8 minutes (21). The toothpastes were
    toothpaste (Colgate Sensitive Pro Relief, Colgate-          applied neat onto the surface of the specimens of
    Palmolive) on preventing enamel erosion produced            groups 3, 4, 5 and 6 without brushing for 3 min at 0,
    by a soft drink (Coca Cola) by using Atomic Force           8, 24 and 36 h and then wiped off with distilled water
    Microscopy (AFM).                                           washing. In groups 4 and 6 the toothpastes were ap-
                                                                plied after demineralization with Coca Cola.
IC




    Materials and methods
                                                                Atomic Force Microscopy (AFM) observations
    Specimens’ preparation
C




                                                                After 24 hours from the last procedure of demineral-
    Specimens were prepared from 30 human incisors              ization and remineralization (15), specimens were ob-
    free of caries and defects, extracted for periodontal       served with an atomic force microscopy AutoProbe
    reasons. After the extraction, the teeth were cleansed      CP 100 (Thermomicroscopes, Veeco, Plainview, NY,
©




    of soft tissue debris and inspected for cracks, hy-         USA), equipped with a piezoelectric scanner, which
    poplasia and white spot lesions; they were disinfected      can cover an area of 100 x 100 μm2 with a range of 7
    in 5.25% sodium hypochlorite solution for one hour          μm in the z-direction. The most common topographi-
    and stored in artificial saliva (pH 7.0, 14.4 mM NaCl;      cal parameters were determined, such as the surface
    16.1 mM KCl; 0.3m mM Cl2.6H20; 2.9 mM K2HPO4;               roughness (Rrms). Rrms is given by the standard devia-
    1.0 mM CaCl2.2H2O; 0.10 g/100 ml sodium car-                tion of the heights, obtained from the AFM images by
    boxymethylcellulose) during the whole experimenta-          testing, for each sample, at least 10 different film ar-

    Annali di Stomatologia 2014; V (3): 98-102                                                                       99
    C. Poggio et al.


    eas of 30 × 30 μm2 with a resolution of 256 x 256 pix-           Discussion and conclusion
    els. From the analyses of the AFM height profiles, it
    was also possible to estimate the erosion cavities               In the present in vitro study, AFM was used to verify
    depth of the enamel surface. The data were obtained              the protective effect of a CPP-ACP paste on enamel
    by averaging on at least 20 selected lines of the im-            exposed to the erosive action of a soft-drink. AFM




                                                                                                              i
    age. Measurements were performed on the treatment                was used to study tooth surfaces in order to compare




                                                                                                            al
    specimens and on the matching controls.                          the pattern of particle distribution in the outermost
                                                                     layer of the tooth surfaces (19). It was found that
                                                                     AFM gives high-contrast, high-resolution images and




                                                                                                         on
    Statistical analysis                                             is an important tool as a source of new structural in-
                                                                     formation: tapping mode AFM (TM-AFM) images are
    Differences in the averaged values among the groups              able to show net differences between exposed and
    were analyzed by ANOVA test. Statistical difference              unexposed enamel areas (4). There is a clear rela-
    was set at P<0.05. Post hoc Bonferroni test was per-             tionship between erosion and temperature of the bev-




                                                                                               zi
    formed to assess the differences between the differ-             erages (2). In this study, the beverage was kept at a
    ent groups.                                                      constant temperature of 20° C. Although erosion pro-
                                                                     ceeds more slowly in vivo than in vitro owing to the




                                                                                  na
                                                                     protective effect of saliva and acquired pellicle, the
    Results                                                          effect of temperature can be expected to be signifi-
                                                                     cant (22). In order to stress their demineralizing po-
    The surface roughness (Rrms) is an index of the sur-             tential, the soft drink was replenished every 2 min to




                                                                       er
    face quality. The mean Rrms values recorded before               ensure that it was carbonated and to reduce the
    experimentation was not statistically different when             buffering effect from ions dissolved from the enamel
    compared with the mean Rrms value of group 1 (intact             surface (21). Enamel surface is often aprismatic and
    enamel) after immersion in artificial saliva. Table 1 re-        more highly mineralized than enamel subsurface.
    ports mean Rrms values with related standard devia-


                                                               t     This prismless enamel arises at the end of amelogen-
    tions obtained in the six groups. Comparing untreated
    specimens (group 1) with demineralized specimens
                                                            In       esis. Although this layer of enamel is more frequent
                                                                     on the surface of deciduous teeth, it can also be
    (group 2), a statistically significant difference                found on the surface of permanent teeth. It is known
    (P<0.05) in R rms values was registered, with an in-             that the prism-free enamel is gradually worn off dur-
    crease of surface roughness passing from intact                  ing mastication but it is retained in protected areas.
                                              ni
    enamel to enamel exposed to Coca Cola. A signifi-                Flat and polished specimens were used in the pre-
    cant difference (P < 0.05) was registered when com-              sent study in an attempt to standardize specimens
    paring the R rms values of group 3 (enamel treated               and remove natural variations in surface enamel be-
                                     io

    with Colgate Sensitive Pro Relief) with intact enamel            tween teeth and between different tooth sites and
    (group 1). Significant higher R rms values are regis-            types, which may result in different responses to acid
    tered with GC Tooth Mousse (P < 0.05). Comparing                 dissolution (1). However, it should be noted that nat-
    the Rrms values of groups 4 and 6 (soft drink + tooth-
                          iz


                                                                     ural tooth surfaces erode more slowly than polished
    pastes) with group 2 (enamel only demineralized) a               surfaces (22). The specimens were cleaned with 5%
    statistical difference (P<0.05) was registered, sug-             NaOCl for 1 hour, which could not alter enamel sur-
    gesting a protective effect of the toothpastes investi-          face. The erosive potential of orange juice modified
          Ed




    gated against enamel demineralization.                           by food additives in enamel and dentine was evaluat-
                                                                     ed; it was concluded that only the combination of cal-
                                                                     cium lactate pentahydrate and sodium linear
    Table 1. Mean roughness values (Rrms) ± standard devia-
                                                                     polyphosphate reduced erosion (23).
    tion obtained in the six groups. Different superscript letters
                                                                     CPP-ACP clusters seem to be anticariogenic in labo-
    means significant differences between the groups (P <
                                                                     ratory animal in situ experiments (24) and on human
IC




    0.05).
                                                                     caries in vitro (13). A CPP-ACP crème was effective
    Groups                                    Mean roughness         in remineralizing early enamel lesions of the primary
                                              values (Rrms)          teeth (25), while recently a CCP-ACP paste was
                                                                     found to be effective in promoting enamel remineral-
                                              148 ± 19a
C




    group 1: intact enamel
                                                                     ization (20). Finally CPP-ACP complex has been suc-
    group 2: enamel + soft drink              261 ± 28b              cessfully added to chewing-gums: the efficacy of
    group 3: intact enamel +                                         three commercially available sugar-free chewing
    Colgate Sensitive Pro Relief              59 ± 5c                gums was compared, concluding that the superior
©




    group 4: enamel + soft drink                                     remineralization activity of the gum was attributed to
    + Colgate Sensitive Pro Relief            93 ± 7d                the presence of casein phosphopeptide-amorphous
    group 5: intact enamel +                                         calcium phosphate nanocomplexes (CCP-ACP) (26).
    GC Tooth Mousse                           155 ± 14a              Que et al., (27) study demonstrated that the new Pro-
                                                                     Argin formula toothpaste provided a significant reduc-
    group 6: enamel + soft drink +
                                                                     tion in dentin hypersensitivity. The reduced sensitivity
    GC Tooth Mousse                           167 ± 12a
                                                                     recorded after a single application can be maintained

    100                                                                                Annali di Stomatologia 2014; V (3): 98-102
    AFM study of enamel remineralization


    over time with daily use of the product for a period of
    24 weeks (28). In addition the desensitizing pastes
    based on argine and calcium carbonate have proven
    to be useful in reducing dentinal sensitivity if applied
    before a session of professional oral hygiene (29).




                                                                                                           i
    The Atomic Force Microscopy could analyze the topo-




                                                                                                         al
    graphical aspects of enamel samples and the effects
    of the demineralization/remineralization processes on
    the enamel morphology, in the presence of the studied




                                                                                                      on
    toothpastes. The most common topographical parame-
    ter was therefore determined, such as the surface
    roughness (Rrms), to quantitatively evaluate the surface
    aspect. After demineralization with an acidic substance
    such as Coca Cola, the surface should appear much




                                                                                             zi
    rougher. The process of erosion causes, in fact, an in-
    crease of Rrms. The demineralizing process was inves-
    tigated by comparing the fraction of intact enamel (Fig.




                                                                               na
    1) with the one treated with the soft drink (Fig. 2). After
    the demineralization process, cavities of diameter be-
    tween 4 and 7 microns were formed. This is reflected
    in the values of Rrms, which increased from 148 to 261




                                                                    er
    nm. As regards the effect of the toothpastes in the
    process of enamel remineralization, several considera-
    tions can be made. Comparing the R rms values of
    groups 3 (enamel + Colgate Sensitive Pro Relief) with
    intact enamel (group 1) a statistical significant (P <


                                                            t
    0.05) decrease of surface roughness suggests a rem-
    ineralizating effect on intact enamel of the product
                                                         In
    used. In groups 3, in fact, enamel shows the typical          Figure 2. 2D and 3D AFM images: enamel exposed to soft
    aprismatic surface layer, on which it is possible to          drink (A) and enamel demineralised and then reminer-
                                                                  alised with Colgate Sensitive Pro Relief (B) and with GC
                                                 ni
                                                                  Tooth Mousse (C).

                                                                  identify the precipitated crystals of the remineralizating
                                     io

                                                                  agents (Fig. 1). Instead significant higher Rrms values
                                                                  are registered in group 5 (enamel + GC Tooth Mousse)
                                                                  (P < 0.05). However, in group 5, AFM images showed
                                                                  a random distribution along the surface of the protec-
                           iz



                                                                  tive agent to form globular aggregates and/or precipi-
                                                                  tates of mineral substances, more or less abundant (as
                                                                  seen in the lighter areas of images). This may justify
          Ed




                                                                  roughness values, significantly higher than those
                                                                  recorded for the other product tested (Fig. 1). Compar-
                                                                  ing the Rrms values of groups 4 and 6 (enamel + soft
                                                                  drink + toothpastes) with group 2 (eroded enamel) a
                                                                  statistical difference (P<0.05) was registered, suggest-
                                                                  ing a remineralizing power on eroded enamel of the
IC




                                                                  two products. In group 4 the prismatic structure of hy-
                                                                  droxyapatite, which typically becomes evident as a re-
                                                                  sult of erosion, was not observed. Therefore Colgate
                                                                  Sensitive Pro Relief seems to able to regenerate a ho-
C




                                                                  mogeneous, very compact, thick and uniform surface
                                                                  layer (Fig. 2). As regards the effects of GC Tooth
                                                                  Mousse on eroded enamel (group 6), the prismatic
                                                                  structure is slightly evident but the cavities seems to
©




                                                                  be well-filled. In this case the superficial layer of the
                                                                  remineralizing agent is probably constituted by a glob-
                                                                  ular arrangement of the mineral substances (Fig. 2).
                                                                  Under the limitations of the present in vitro study, the
    Figure 1. 2D and 3D AFM images: intact enamel (A) and         application of the tested toothpastes can be consid-
    enamel treated with Colgate Sensitive Pro Relief (B) and      ered effective on preventing enamel erosion pro-
    with GC Tooth Mousse (C).                                     duced by a soft drink.

    Annali di Stomatologia 2014; V (3): 98-102                                                                          101
    C. Poggio et al.


    Acknowledgments                                                              bleaching and casein phosphopeptide-amorphous calcium
                                                                                 phosphate (CPP-ACP) treatment. J Dent. 2007;37:297-306.
    Nothing to declare.                                                    15.   Bertassoni LE, Habelitz S, Pugach M, et al. Evaluation of sur-
                                                                                 face structural and mechanical changes following reminer-
                                                                                 alization of dentin. Scanning. 2010;32:312-19.




                                                                                                                           i
                                                                           16.   Barbour ME, Rees JS. The laboratory assessment of
    Conflict of interest statement




                                                                                                                         al
                                                                                 enamel erosion: a review. J Dent. 2004;32:591-602.
                                                                           17.   De-Deus G, Paciornik S, Pinho Mauricio MH, Prioli R. Real-
    The authors of this study have no conflict of interest                       time atomic force microscopy of root dentin during dem-
    to disclose.                                                                 ineralization when subjected to chelating agents. Int Endod




                                                                                                                      on
                                                                                 J. 2000;39:683-92.
                                                                           18.   Lippert F, Parker DM, Jandt KD. In vitro demineralization/rem-
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        conditioned and bonded enamel following carbamide peroxide               Argin™ Special Issue.
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