Odontology https://doi.org/10.1007/s10266-022-00688-7 ORIGINAL ARTICLE The power of weak ion‑exchange resins assisted by amelogenin for natural remineralization of dental enamel: an in vitro study Sandra Diez‑García1 · María‑Jesús Sánchez‑Martín1 · Manuel Valiente1 Received: 6 July 2021 / Accepted: 20 January 2022 © The Author(s) 2022 Abstract This study aims to develop an innovative dental product to remineralize dental enamel by a proper combination of ion- exchange resins as controlled release of mineral ions that form dental enamel, in the presence of amelogenin to guide the appropriate crystal growth. The novel product proposed consists of a combination of ion-exchange resins (weak acid and weak base) individually loaded with the remineralizing ions: C ­ a2+, ­PO43− and F ­ −, also including Z ­ n2+ in a minor amount as antibacterial, together with the protein amelogenin. Such cocktail provides onsite controlled release of the ions necessary for enamel remineralization due to the weak character of the resins and at the same time, a guiding tool for related crystal growth by the indicated protein. Amelogenin protein is involved in the structural development of natural enamel and takes a key role in controlling the crystal growth morphology and alignment at the enamel surface. Bovine teeth were treated by applying the resins and protein together with artificial saliva. Treated teeth were evaluated with nanoindentation, scanning electron microscopy and energy-dispersive X-ray spectroscopy. The innovative material induces the dental remineralization creating a fluorapatite layer with a hardness equivalent to sound enamel, with the appropriate alignment of corresponding nanocrystals, being the fluorapatite more acid resistant than the original mineral. Our results suggest that the new product shows potential for promoting long-term remineralization leading to the inhibition of caries and protection of dental structures. Keywords Controlled release system · Amelogenin · Enamel remineralization · Dental caries · Fluorapatite Introduction from biofilms of dental plaque on dietary fermentable car- bohydrates, diffuse into the tooth and dissolve the mineral Dental caries is a complex disease that afflicts a large pro- [6–13]. Dental caries is a process, which can be stopped portion of the world’s population, regardless of gender, age and reversed in its initial stages. If left untreated, the early and ethnicity, although it is more likely to be present in reversible lesion can progress to the point of being irrevers- individuals with a low socioeconomic status [1–4]. Despite ible [14, 15]. Another factor that puts oral health at risk and the fact that the prevalence of dental caries has descended makes it necessary to find a solution is the popularity of over the last decades, the disease is still a major problem the whitening systems and their side effects, such as dental for adults and children, and an improved approach to pre- erosion and hypersensitivity [16–19]. The mature enamel vention and therapy is currently needed [5]. Dental caries has no residual cellular components that can carry out the progresses when organic acids, produced by bacterial action repair when it is damaged, so the restoration depends on physicochemical events at the tooth surface [1, 2]; here are protective factors that can prevent or reverse it, such as sali- Sandra Diez-García and María-Jesús Sánchez-Martín shared the co-first authorship. vary proteins, calcium, phosphate, salivary flow and fluoride in saliva [5, 20, 21]. María-Jesús Sánchez-Martín is the co-first author. Enamel is the outer layer of the teeth and is the hardest and most mineralized tissue in vertebrates [22–25]. It is an * María‑Jesús Sánchez‑Martín mariajesus.sanchez@uab.cat acellular mineralized tissue comprised of densely packed crystallites of calcium hydroxyapatite organized into rod and 1 GTS Research Group, Department of Chemistry, interrod structures. Tooth enamel rods seem to extend from Faculty of Science, Universitat Autònoma de Barcelona, the dentino-enamel junction to the tooth surface and they are 08193 Bellaterra, Spain 13 Vol.:(0123456789) Odontology remarkably longer than wide. Interrods are interprismatic oral application time of many fluoride products, fluoride ions crystallites oriented in different directions [26]. are released rapidly producing high concentrations in a short During tooth germination, the enamel mineral is a car- time [21]. There has been an increase in the exposure to fluo- bonated apatite, not pure hydroxyapatite (HA). The min- ride ingestion in children increasing the risk of toxicity and eral is related to hydroxyapatite but is much more soluble dental fluorosis due to the high levels of fluoride released in acid, as well as calcium deficient (calcium is replaced into biological fluids [35]. by sodium, magnesium and zinc) and contains between 3 A different approach to induce remineralization is used and 6% of carbonate ions replacing phosphate ions in the in the present study to avoid high concentrations of fluoride crystal lattice. Demineralization and remineralization is a with its side effects and prolong the contact time between dynamic process. The carbonate is preferentially lost during fluoride ions and teeth. The fluorapatite formation could be the demineralization process, and it is avoided during remin- guided controlling the release speed of fluoride in the oral eralization. Thus, the calcium-deficient and carbonate-rich environment, in conjunction with the release of calcium regions of the crystal are markedly susceptible to be attacked and phosphate ions to induce the remineralization. For this during demineralization [5]. When the O ­ H− groups in pure purpose, a product (called NMTD [36], new material for hydroxyapatite ­(Ca10(PO4)6(OH)2) are completely replaced dental treatment, from its Spanish acronym Nuevo Material by fluoride ions (­ F−), it results in mineral fluorapatite (FA) de Tratamiento Dental) that provides a controlled release ­(Ca10(PO4)6F2), which is really resistant to dissolution by system for the anticaries treatment of dental tissues is used. acid. Mature enamel is mostly a mixture of hydroxyapatite NMTD is a combination of ion-exchange resins of weak and fluorapatite and for this reason, it is much less soluble acid and weak base ion-exchangers composition, loaded with than the original mineral [5, 21, 27]. calcium, fluoride, phosphate and zinc. The molar ratio of Fluoride is widely used in dental products because pri- the ions has to be close to that of the organomineral tissue marily via topical mechanisms, it has a cariostatic effect to be remineralized; in the case of the teeth, the approximate inhibiting the demineralization at the crystal surfaces, molar ratio between C ­ O43−, is 2:1:1, respec- ­ a2+, ­F− and P enhancing the remineralization, interfering with plaque tively. In contact with NMTD, the organomineral tissues are formation and inhibiting bacterial metabolism [5, 10, 21, remineralized in an effective way, especially in the presence 28–32]. The presence of fluoride ions restricts the forma- of ­Zn2+ ions. Zinc has two effects: it helps to combat the tion of acidic, more soluble calcium phosphates as dicalcium micro-organisms which cause caries due to its bactericidal phosphate dihydrate or octacalcium phosphate, and facili- properties against oral bacteria [37], and it is an initiator of tates the creation of more acid-resistant fluorapatite or fluor- the ionic release of the other structural ions. A toothpaste hydroxyapatite (partial substitution of fluoride by hydroxyl containing NMTD was proven to be effective in limiting groups). Fluorapatite or fluorhydroxyapatite are formed in vitro enamel demineralization and in enhancing remin- when low levels of fluoride ions are present, and calcium flu- eralization [38]. The application of ion-exchange materials oride will be created with high levels of fluoride ions. This has advantages compared to conventional chemical reagents, calcium fluoride will hydrolyze to fluorhydroxyapatite in the since the release of ions is only due to the ion-exchange presence of acid phosphate or phosphate ions. Moreover, mechanism and they do not introduce undesirable ions into fluoride is more effective in inhibiting hydroxyapatite dis- the solution, they are characterized by practically neutral pH solution when calcium and phosphate ions are also present in values and they can also adsorb bacteria on their surfaces. the solution [2]. With fluoride availability, demineralization Most of these resins are non-toxic and are used in the phar- is reduced because part of the calcium and phosphate lost maceutical industry, in medical applications and also in the by the dissolution of hydroxyapatite returns to the enamel food industry [39]. as more acid-resistant fluorapatite [33]. On the other hand, the organic matrix composed of proteins Even though systemic fluoride by means of water fluori- secreted by the ameloblasts provides a scaffold for the enamel dation has been promoted in the past for the decline in dental minerals to grow. But following this stage of development, the caries, it has been found that the systemic benefits of fluoride enamel enters a maturation phase in which most of the organic are minimal and the primary reduction in dental caries is due matter is degraded [23, 24, 40–42]. The protein amelogenin, to the topical effect of water fluoridation and the availabil- that constitutes the 90% of the protein matrix, plays a central ity of fluoridated toothpastes. In several European countries role in guiding the hierarchical organization of apatite crystals without water fluoridation, a caries reduction has been seen observed in mature enamel [22, 43–46]. It is known that the after the introduction of fluoridated toothpastes [3]. Tooth- crystal morphology and alignment of enamel, as well as the pastes are probably the most widespread products in health correct enamel thickness, are the result of a protein-guided care and are one of the most effective ways to deliver free or uniaxial growth process. The exact mechanism guiding this soluble fluoride [34]. Nevertheless, high concentrations of process remains undetermined due to the rapid degradation fluoride can cause numerous side effects and due to the short of the underlying protein matrix during tissue maturation. 13 Odontology However, the role of self-assembly of enamel matrix proteins, calcium fluoride (95%) and tricalcium phosphate (35–40% particularly amelogenin, has been widely recognized as a (Ca)) at the ratio of 1.67 Ca/P. Subsequently, the reagents key factor in controlling enamel structural development [22, were placed in the muffle furnace (Selecta 366 PE, Barce- 43, 47]. Therefore, there is considerable interest in the use lona, Spain) and heated to 1200 °C for 2 h. The solid FA was of amelogenin and other similar self-assembling peptides for then grounded during 15 min into powder. dental remineralization [48–54]. The aim of this study is to assess the NMTD remineralization capacity in the presence Mineral growth in solution and absence of amelogenin to understand the influence of this protein on the crystal morphology and alignment during the To form FA in solution, 0.6 g of NMTD product was mixed remineralization process. with 1 ml of artificial saliva (KCl 0.24 g/l, ­CaCl2∙2H2O 0.078 g/l, ­KH2PO4 0.544 g/l, M­ gCl2∙6H2O 0.041 g/l, and Materials and methods HEPES 4.77 g/l; adjusted to pH 7.1 ± 0.4 with KOH pellets) and placed in a closed container inside an incubator at 37 °C Materials during 24 h. The resultant paste is filtered with the aid of a funnel and a Kitasato flask under vacuum and dried at room Hydrochloric acid (37%) and potassium hydroxide pel- temperature. lets (85%) were purchased from Panreac (Barcelona, Spain). Chloramine T trihydrate (98–103%), HEPES Amelogenin production (4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid, 99.5%), magnesium chloride hexahydrate (99%), and cal- A human 175 amino acid amelogenin (Swissprot Q99217, cium fluoride (95%) were purchased from Sigma-Aldrich isoform 1, excluding the signal peptide) was expressed in (Steinheim, Germany); calcium chloride dihydrate the Escherichia coli strain BL21 (DE3) and purified with (74–78%), potassium dihydrogen phosphate (99.5%), and an acid/heat treatment as described previously Svensson tricalcium phosphate [35–40% (Ca)] from Panreac (Bar- Bonde and Bulow [56]. The proteins were analyzed by celona, Spain); potassium chloride (99–100.5%) from J. T. matrix‐assisted laser desorption/ionization, sodium dodecyl Baker (Deventer, Holland), all in powder form. Deionized sulfate polyacrylamide gel electrophoresis and western blot water was purified through a Millipore purification system to confirm the obtainment of amelogenin. The recombinant from Millipore (Milford, MA, USA). Reference sample of amelogenin was quantified with a nanodrop. the hydroxyapatite was of analytical grade and was used Protein purification has been performed by the ICTS as received without any further purification (> 90%, Fluka, “NANBIOSIS”, more specifically by the Protein Produc- Sigma-Aldrich, Steinheim, Germany). tion Platform of CIBER in Bioengineering, Biomaterials and The base of the weak acid ion-exchange resins is a Nanomedicine (CIBER-BBN)/IBB, at the UAB SePBioEs copolymer from acrylic acid, divinylbenzene, and aliphatic scientific-technical service (http://​www.​nanbi​osis.​es/​portf​ diene with carboxylic acid functional groups (Lewatit S olio/​u1-​prote​in-​produ​ction-​platf​orm-​ppp/). 8528, Lanxess, Leverkusen, Germany); and the base of the weak base ion-exchange resins is a styrene–divinylbenzene- Specimen preparation copolymer with tertiary amine functional groups (Lewatit S 4528, Lanxess, Leverkusen, Germany). These food grade Bovine mandibular incisors have a great macroscopic and ion-exchange resins already charged with different ions microscopic similarity to human teeth, so they have been ­(Zn2+, ­Ca2+, ­F− and ­PO43−) were purchased from MionTec commonly used as a model in dental studies [57–59]. Human (Leverkusen, Germany). They were ground to below 50 µm and bovine incisors have certain small differences that are particle size and mixed to form the NMTD product, in which important to consider when conducting physical or chemical the corresponding molar ratio between ­Ca2+, ­F− and ­PO43−, studies of dental specimens. For example, bovine enamel is 2:1:1, respectively. Moreover, ­Zn2+ ions-charged resin is crystals present larger diameter than human enamel crys- added, representing the 0.2% of the dry weight of the related tals and bovine prisms differ in shape compared to human resin. prisms. In addition, calcium distribution is more homogene- ous in bovine enamel than in human enamel. However, there Methods are no significant differences in fluoride uptake or in carbon- ate content between human and bovine enamel. Moreover, Reference fluorapatite sample both kinds of teeth have also similar hardness, porosity and amount of interprismatic enamel [60–63]. The fluorapatite reference powder was synthetized follow- Specimens of deciduous bovine incisors were cleaned of ing a solid phase reaction [55] mixing in the agate miller gross debris and the root was sectioned using a diamond 13 Odontology saw (South Bay Technology, San Clemente, CA, USA). Samples were embedded in a Fastray acrylic resin (Harry J. Bosworth, Skokie, IL, USA) to close the root hole. Then, teeth were etched with 1 M HCl for 30 s [64] to simulate the early stage of dental erosion and cleaned by rinsing with MilliQ water while brushing for 20 s with an electric tooth- brush Vitality (Braun Oral-B) with brush head EB20 (Braun Oral-B). In vitro remineralizing treatment Groups of six teeth were prepared as explained in “Specimen preparation” for each time and treatment: NMTD, NMTD with human amelogenin, and blank samples. Blank samples were immersed in the artificial saliva solu- tion (prepared as explained in “Mineral growth in solution”) at 37 °C during the different times of the experiment and cleaned with MilliQ water while brushing with the electric Fig. 1  Bovine teeth with NMTD treatment placed in the container toothbrush for 20 s every 24 h. with artificial saliva on the bottom prior to sealing For both remineralizing treatments, artificial saliva (in the presence or absence of 100 µg/ml of human amelogenin) was added to NMTD until obtaining the optimal consistence of thick gel for its application onto the tooth (about twice as much artificial saliva is added as NMTD resin). The mixture was applied directly on the enamel surface of the etched teeth (Fig. 1). The samples were placed in a closed container with artificial saliva on the bottom and inside an incubator to maintain the humidity and the natural temperature of the oral cavity (37 °C). Every 24 h, each treatment was renewed by carefully washing the specimens with MilliQ water and brushing with the electric toothbrush for 20 s before apply- ing a fresh portion of treatment. The different groups of samples were treated between 4 and 20 days (Fig. 2). Finally, teeth were cleaned by brushing with MilliQ water for 20 s using the electric toothbrush and stored in a 0.5% chloramine T solution until their analysis. Moreover, some samples that were treated during 15 days with NMTD were brushed continuously for 15 min with the electric toothbrush Vitality (Braun Oral-B) with brush head EB20 (Braun Oral-B) maintaining constant force to study the strength of the remineralization. Fig. 2  Scheme of the teeth with the different treatments indicating To study the remineralized layer, selected samples were sample collection times longitudinally cut in two halves along the central lobe with a Struers Minitom precision diamond saw (Copenhagen, Denmark) and polished with a Struers LaboPol-25 polisher After each polishing, the samples were sonicated for 1 min (Copenhagen, Denmark). A sequence of silicon carbide to clean the polishing residues. paper was used to polish the longitudinal side, starting at grit size P1000 and sequentially increasing to P4000, under SEM–EDX measurements a constant flow of water. Then, a water-based diamond sus- pension containing monocrystalline diamonds and cooling The structure and elemental composition of the samples lubricant with a mean particle size of 1 µm (DiaDuo-2, Stru- were analyzed by scanning electron microscopy (SEM) and ers, Copenhagen, Denmark) was used to finish the polishing. energy-dispersive X-ray spectroscopy (EDX) with a Zeiss 13 Odontology Merlin field emission SEM equipped with an EDX Oxford formed in the presence of the ions released from the NMTD. INCA X-Max detector. This microscope has a unique charge The basic parameters that are necessary for the calculation compensation system that allows the high-resolution imag- of distribution diagrams, including equilibrium constants, ing of non-conductive samples, and electrons which accu- are included in the program database. mulate on the sample surface are swept away by a fine jet The SEM images treatment was done using ImageJ [69, of nitrogen. The images were taken on the surface and the 70], a public domain image processing and analysis program longitudinal section of the teeth with the secondary electron developed at the US National Institutes of Health. detector with a low voltage of 1–3 kV and the EDX meas- GraphPad Prism 9 (GraphPad Software, San Diego, CA, urements were taken at 10 kV. The powder from the min- USA) was used for statistical analysis. eral growth experiment and the HA and FA references were characterized under the same conditions as described above. All the experiments were performed at room temperature. Results Nanoindentation Mineral growth in solution The hardness of the teeth was measured using an MTS Nano Theoretically, the predominant species formed in the pres- Indenter XP with a Berkovich tip that provides a fast and ence of the ions released from the NMTD at pH 7.1 would reliable way to acquire mechanical data on the submicron be calcium fluoride and fluorapatite, as it can be seen in the scale. The continuous stiffness measurements (CSM) were chemical species distribution diagram of Fig. 3. performed in the longitudinal section of the tooth with a Figure 4 shows the SEM image of the elongated crystals depth limit of 1000 nm and a Poisson’s ratio of 0.25 [65, obtained after the incubation of NMTD in artificial saliva 66]. Continuous stiffness measurement with depth, in con- solution for 24 h and the EDX results of one crystal and the junction with the known indenter tip area function, allows fluorapatite reference powder. continuous hardness monitoring [67]. All the experiments were performed at room temperature. Before every measure- In vitro experiment ment, the Berkovich diamond indenter was calibrated on a standard fused silica specimen. SEM–EDX measurements Software Teeth were lengthwise cut and the remineralized layer thick- ness after each treatment time was measured using ImageJ MEDUSA program (Stockholm, Sweden) [68] was used software. Figure 5 shows that the layer grows gradually until to create the chemical equilibrium diagram of the species day 15, when it reaches a plateau around 23 ± 1 µm. Fig. 3  Theoretical distribution diagram of the species formed in the presence of the released ions from NMTD as function of medium pH (artificial saliva solution in our case) 13 Odontology Fig. 4  SEM image of the crystals obtained after the incubation of NMTD in artificial saliva solution during 24 h with 5000X magnification (a), EDX spectrum of one of the crystals (b), EDX spectrum of the fluorapatite reference powder (c) To study the formed layer bonding strength to the enamel surface, teeth were brushed continuously for 15 min after 15 days of treatment. Figure 6 represents a longitudinal section of a tooth showing the remineralized layer after brushing. The difference between the treatment in the presence and absence of amelogenin was studied. In Fig. 7, it can be appre- ciated that the crystals are bigger in the presence of amelo- genin. The diameter of the crystals for the 4-day treatment is 0.11 ± 0.02 µm for NMTD and 0.14 ± 0.02 µm for NMTD in the presence of amelogenin. In the case of the 15-day treat- ment, the diameter of the crystals is 0.13 ± 0.02 µm for the treatment without protein and 0.17 ± 0.03 µm with amelo- genin. The 15-day treatment with protein shows significant differences in crystal size versus NMTD alone, according to the one-way ANOVA (analysis of variance) test performed Fig. 5  Evolution of the remineralized layer thickness with the time of with GraphPad Prism 9. NMTD treatment To study the composition of the remineralized layer, an EDX study was performed by measuring blank and treated samples after 4 and 15 days of treatment and comparing them to the references of fluorapatite and hydroxyapatite powder. EDX results are shown in Table 1. were taken in longitudinally cut samples of 15 days To study the evolution of the formed layer across its through the formed layer until the enamel. Figure 8 shows outer part to the enamel, different EDX measurements the variations of each element when moving across the new layer to the natural bovine enamel. 13 Odontology Fig. 6  Longitudinal section SEM images of a sample treated with NMTD for 15 days and brushed for 15 min with × 250 magnification (a) and × 1000 magnification of the square area (b) Fig. 7  SEM images at × 20,000 magnification level of the tooth surface after 4 and 15 days of treatment: blank (a), NMTD (b), NMTD and amelogenin (c) Table 1  Surface EDX measurements of the different treatments at dif- sample after 15 days (NMTD15), NMTD-amelogenin-treated sam- ferent times: blank sample after 4 days (BL4), NMTD-treated sam- ple after 15 days (AH15), fluorapatite powder reference (FA), ple after 4 days (NMTD4), NMTD+amelogenin-treated sample after hydroxyapatite powder reference (HA) 4 days (AH4), blank sample after 15 days (BL15), NMTD+treated BL4 NMTD4 AH4 BL15 NMTD15 AH15 FA HA Ca (atomic %) 18 ± 5 14 ± 1 20.7 ± 0.1 20 ± 4 14.78 ± 0.01 20.6 ± 0.1 25 ± 4 21 ± 3 P (atomic %) 15 ± 3 10.5 ± 0.5 14.68 ± 0.08 19 ± 2 10.9 ± 0.1 14.53 ± 0.04 14.3 ± 0.8 14.7 ± 0.9 O (atomic %) 66 ± 8 64.8 ± 1.0 56.5 ± 0.4 61 ± 5 63.3 ± 0.5 56.4 ± 0.5 56 ± 2 65 ± 3 F (atomic %) 0.3 ± 0.1 10.8 ± 0.5 8.1 ± 0.2 0.1 ± 0.1 11.0 ± 0.4 8.5 ± 0.4 4.9 ± 0.7 0.00 ± 0.01 13 Odontology Fig. 8  Longitudinal section SEM–EDX analysis of blank sample after 15 days (a), NMTD-treated sample after 15 days (b), NMTD+amelogenin-treated sample after 15 days (c) Nanoindentation the EDX spectrum of the crystals matches the fluorapatite reference spectrum. To assess the hardness of both kinds of remineralized layer The remineralization of the tooth samples has been suc- with respect to the enamel, CSM measurements were taken cessful, as it can be seen in Fig. 5, the thickness of the rem- in the layer and the enamel of longitudinally cut samples; ineralized layer progressively increases until day 15, when it the measurements are shown in Fig. 9. reaches its natural maximum at around 23 ± 1 µm. Figure 6 shows the remineralized layer of the 15 days treatment to remain intact after brushing for 15 min, dem- onstrating that it is attached to the tooth surface strongly Discussion enough to resist the brushing of the daily hygiene. The increase in crystal size with amelogenin for both The results of the solution experiment confirm the expected treatment times (4 and 15 days) seen in Fig. 7 suggests formation of fluorapatite. It can be observed in Fig. 4 that amelogenin protein to accelerate the crystallization rate [75]. the shape of the crystals formed with the NMTD in artifi- We can observe in Table 1 that the presence of amelo- cial saliva solution after 24 h resembles the first phase of genin during the remineralization process produces a layer the formation of fluorapatite spherulites [71–74]. Moreover, more similar in composition to pure fluorapatite, with more Fig. 9  Longitudinal section continuous stiffness measurements of a 15-day NMTD-treated sample (a) and a 15-days NMTD+amelogenin-treated sample (b) 13 Odontology Ca and P but less O and F than the layer formed only with most supporting evidence to sustain their use among those NMTD that also shows the formation of other minerals like commercially available for dental remineralization [80]. In calcium fluoride. This calcium fluoride is a precursor that the case of the NMTD product, fluorapatite is also formed will derive in fluorapatite in the presence of phosphate [2, since these same ions are supplied. The ions are delivered in 76]. The composition of the blank samples treated with a controlled manner by means of weak ion-exchange resins, artificial saliva alone is similar to that of hydroxyapatite as avoiding the precipitation of unwanted compounds by the expected. The results obtained from the 4-day and 15-day massive encounter of ions outside the enamel surface, and treatments are similar, which means that extending the treat- allowing remineralization with fluorapatite by delivering ment time increases the thickness of the layer, as observed the ions slowly to the enamel surface. The drawback of this previously, but does not alter its composition. necessary controlled release would be the long application In Fig. 8, comparing the remineralized layer (first 23 µm) time of the NMTD product required to achieve adequate to natural enamel (beneath this 23 µm), fluorine concentra- remineralization, but it avoids high fluoride concentrations tion is higher and the oxygen concentration is lower due to in the oral cavity unlike non-controlled-release products. the expected production of fluorapatite in the layer (increase In conclusion, the NMTD product is effective and induces of fluoride content) replacing corresponding hydroxyapatite the remineralization after an acid attack creating a fluorapa- (decrease of the ­OH− content). In the presence of amelo- tite layer of around 23 µm after 15 days of treatment, over- genin, calcium and phosphorus remain largely similar along coming the brushing process of the daily hygiene. The the new layer to the original enamel since fluorapatite and presence of amelogenin protein during the remineralization hydroxyapatite contain the same amount of calcium and process improves the layer hardness and the crystal mor- phosphorus, thus supporting our expected mineral forma- phology, also accelerating the crystallization rate. Further- tion. On the contrary and in the absence of amelogenin, more, amelogenin seems to induce the composition of the the observed decrease of oxygen, calcium, and phospho- layer closer to that of pure fluorapatite. The novel product of rus in the formed layer against the original enamel may be NMTD with amelogenin induces complete remineralization interpreted by the formation of a considerable proportion with a hardness that reaches the levels observed in natural of ­CaF2 instead of just fluorapatite. In the blank samples, healthy enamel. Therefore, the novel product is promising these changes are not observed due to the absence of the to provide long-term remineralization to inhibit caries and remineralized layer. protect tooth structures. Nevertheless, further studies would We can observe in Fig. 9 the remineralized layer without be necessary to evaluate the clinical applicability of this bio- protein to be slightly harder than enamel during the first mimetic material. 500 nm although it loses hardness from there unlike enamel. However, in the presence of amelogenin, the hardness of the Acknowledgements The Spanish Ministerio de Economía y Competi- tividad is acknowledged for the financial support provided (Project: remineralized layer is maintained at values similar to the CTM2015-65414-C2-1-R). Sandra Diez-García acknowledges the enamel. These results may be due to the role of amelogenin FI-2017 fellowship from Agència de Gestió d’Ajuts Universitaris i de in guiding the morphology and alignment of crystals forma- Recerca (Generalitat de Catalunya). tion [22, 43]. The development of technologies to rebuild tooth enamel Funding Open Access Funding provided by Universitat Autonoma de Barcelona. This work was supported by the Agència de Gestió d’Ajuts and preserve tooth structure is of great interest due to the Universitaris i de Recerca (Generalitat de Catalunya) [Grant number inability of the mature tooth enamel to regenerate itself after FI-2017]; and the Spanish Ministerio de Economía y Competitividad substantial mineral loss [1, 2, 25, 40, 47, 77–79]. A widely [Grant number CTM2015-65414-C2-1-R]. The funders had no role studied remineralizing system uses nanocomplexes of casein in study design, data collection and analysis, decision to publish, or preparation of the manuscript. phosphopeptide-amorphous calcium phosphate (CPP-ACP) to stabilize and deliver bioavailable ions [80]. CPP-ACPs have been introduced in toothpastes, mouthwashes, lozenges, Declarations chewing gums and even in bovine milk [81–83]. Another Conflict of interest The authors declare that they have no conflicts of option is the combination of CPP-ACP with fluoride or the interest. application of casein phosphopeptide-amorphous calcium fluoride phosphate (CPP-ACFP) to form fluorapatite. CPPs Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. prevent the promotion of dental calculus, which consists of dental plaque mineralization, by stabilizing the calcium, phosphate, and fluoride ions. Yet, the ions are freely bioa- Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- vailable to induce remineralization by their diffusion through tion, distribution and reproduction in any medium or format, as long concentration gradients into enamel subsurface lesions [83, as you give appropriate credit to the original author(s) and the source, 84]. CPP-ACP and CPP-ACFP are the technologies with provide a link to the Creative Commons licence, and indicate if changes 13 Odontology were made. The images or other third party material in this article are 15. Fan M, Yang J, Xu HHK, Weir MD, Tao S, Yu Z, et al. Rem- included in the article's Creative Commons licence, unless indicated ineralization effectiveness of adhesive containing amorphous otherwise in a credit line to the material. 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