pubs.acs.org/ac Article Combination of Two Synchrotron Radiation-Based Techniques and Chemometrics to Study an Enhanced Natural Remineralization of Enamel Sandra Diez-García,§ María-Jesús Sánchez-Martín,*,§ José Manuel Amigo, and Manuel Valiente Cite This: Anal. Chem. 2022, 94, 5359−5366 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: The limitations to assess dental enamel remineraliza- tion have been overcome by a methodology resulting from the appropriate combination of synchrotron radiation-based techniques on both, infrared microspectroscopy and micro X-ray diffraction, with the help of specific data mining. Since amelogenin plays a key role in modulating the mineralization of tooth enamel, we propose a controlled ion release for fluorapatite structural ions (Ca2+, PO43−, and F−, also including Zn2+) by using weak acid and weak base ion- exchange resins in the presence of amelogenin to remineralize the surface of etched teeth. This combination provides the necessary ions for enamel remineralization and a guide for crystal growth due to the protein. Remineralized tooth samples were analyzed by applying the indicated methodology. The synchrotron data were treated using principal component analysis and multivariate curve resolution to analyze the mineral layer formed in the presence and absence of amelogenin. The remineralizing treatment created a fluorapatite layer free of carbonate impurities and with a similar orientation to that of the natural enamel thanks to amelogenin contribution. ■ INTRODUCTION Tooth enamel is the most mineralized and hardest tissue in the the material properties of mineralized tissues such as dental enamel, including mineralization, crystallinity, or carbonate human body. The enamel is constituted by multiple rodlike substitution.10 The incorporation of a microscope in FTIR microspectroscopy (μFTIR) has introduced the possibility of apatite crystals, which are arranged in ordered prisms.1,2 combining biochemical and spatial information. Coupling this Mineral composition of mature enamel is a mixture of technique to a synchrotron radiation light source, synchrotron compounds, primarily hydroxyapatite (HA), which is crystal- radiation-based FTIR microspectroscopy (SR-μFTIR), allows line calcium phosphate (Ca10(PO4)6(OH)2) that has a a much better signal-to-noise ratio and the use of a smaller hexagonal crystal system with the P63/m space group.3,4 This beam size without losing signal efficiency, since one of the enamel HA is not stable and suffers changes due to chemical most outstanding properties of a synchrotron radiation source interactions. The major change is produced when phosphate is is its high brightness compared with a traditional globar substituted by carbonate because this substitution increases the source.11 solubility of the enamel. However, if the hydroxyl ions of HA On the other hand, as apatites have a well-defined hexagonal are replaced by fluoride ions, fluorapatite (FA), a more acid- crystal structure, diffraction methods can be used to analyze resistant compound, is generated.5 the crystal structure in teeth.1 The crystallographic properties Several techniques have been used to assess tooth of enamel and fluoridated enamel have been investigated using remineralization, such as scanning electron microscopy, atomic X-ray diffraction (XRD). Incorporating fluoride into the force microscopy, or indentation. However, these techniques enamel structure may lead to crystallographic changes in have certain limitations regarding the analysis of the enamel (contraction of the a,b-axis, increase in apatite crystal composition of enamel remineralization and crystal orienta- tion.6,7 Therefore, synchrotron radiation-based infrared micro- spectroscopy and microdiffraction were used for this study to Received: December 20, 2021 overcome these constraints. Accepted: March 11, 2022 Fourier transform infrared (FTIR) spectroscopy is a well- Published: March 23, 2022 recognized molecular vibrational technique that has been widely used to investigate the chemical structural properties of natural materials.8,9 It has been extensively used for mapping © 2022 The Authors. Published by American Chemical Society https://doi.org/10.1021/acs.analchem.1c05498 5359 Anal. Chem. 2022, 94, 5359−5366 Analytical Chemistry pubs.acs.org/ac Article size and reduction of crystal defects). Nevertheless, enamel enamel demineralization, and zinc also has antibacterial and crystallinity varies at different layers. If the enamel is ground malodor control properties.21,30 into powder mixing them, it may affect the accuracy of the Furthermore, enamel matrix proteins play a vital part during measurement.12 The micro X-ray diffraction (μXRD) analysis the development of enamel in the regulation of mineralization overcomes the limitations of conventional powder XRD and and crystal organization. Then, the protein matrix is can obtain site-specific data directly from the tooth allowing proteolytically degraded during the enamel maturation stage. the study of the enamel crystallinity at different levels.13 The Amelogenin constitutes more than 90% of the organic matrix, synchrotron through-the-substrate micro X-ray diffraction (tts- being the most abundant protein in the forming enamel. The μXRD) used in the present work allows performing punctual importance of the amelogenin protein is well-known because analysis in thin sections with a spot size of few micrometers. amelogenin self-assembly controls the morphology, size, and Therefore, this technique enables data collection directly on orientation of the growing crystals.23 For this reason, thin tooth sections preserving the textural context and allowing amelogenin in conjunction with NMTD is used in this local identification of mineral phases.14 research to achieve a remineralization similar to that of natural There are several factors that put oral health at risk, such as enamel. the popularity of whitening systems with their side effects or This study aims to evaluate the efficiency of the the excessive consumption of acidic foods and beverages.15−18 remineralization after the treatment with NMTD and Demineralization caused by regular exposure of the tooth amelogenin and to determine the protein influence on the enamel to acids, such as those produced within accumulations morphological changes of the remineralized enamel. Synchro- of bacterial plaque from dietary carbohydrates, removes tron infrared microspectroscopy and microdiffraction, joined mineral ions from HA crystals and may cause a caries lesion. with the proper chemometric method, allow us to study the Demineralization in early caries lesions can be reversed by evolution of the structure of apatites and their distribution after calcium and phosphate in saliva.19−22 However, mature tooth the remineralization process. The chemometric methods used enamel is acellular and does not regenerate itself after are principal component analysis (PCA) and multivariate curve substantial loss.23,24 Mechanical properties of the macroscopic resolution (MCR). PCA is a variable reduction tool to identify enamel tissue are highly dependent on the alignment and trends and patterns in an extremely efficient manner by orientation of HA crystals. Therefore, changes in surface analyzing the variance of the data.31 On the contrary, MCR structure as a result of caries or microscopic damage can cause allows us to understand the physical or chemical differences of significant impairment of the dental function and, ultimately, the mineral formed in the presence and absence of tooth loss.1 More than 12 million dental implants are needed amelogenin.32 annually worldwide as part of routine oral rehabilitation.25 Fluoride is the most popular agent for enhancing remineralization. Besides the antibacterial properties at low ■ EXPERIMENTAL SECTION In Vitro Dental Remineralization. All the reagents used concentrations, fluoride stops the demineralization and favors are detailed in Supporting Information section 1.1. Bovine the opposite process of remineralization on the tooth surface.20 teeth are used as a model, given their similarity to human During remineralization processes, fluoride ions promote the teeth.33,34 Bovine tooth specimens were cleaned of gross debris formation of FA in the presence of calcium and phosphate before removing the root with a diamond saw (South Bay ions. At higher concentrations, it creates a calcium fluoride Technology, San Clemente, CA). The resulting tooth samples layer that acts as a reservoir source for fluoride and protects the were embedded in a Paladur clear autopolymerizing acrylic enamel from the formation of caries.26 Therefore, fluoride is resin (Heraeus Kulzer, Hanau, Germany), closing the root added to toothpastes, mouthwashes, and drinking water as an aperture and leaving the front surface exposed. The embedded anticaries agent.20 Despite its benefits, high concentrations of teeth were then etched with hydrochloric acid 1 M for 30 s to fluoride can cause undesirable side effects and unfortunately, in mimic the early stage of dental erosion and immediately many dental products, fluoride ions are released too rapidly, afterward cleaned by rinsing with Milli-Q water while brushing producing high concentrations in a brief period due to the for 20 s with an electric toothbrush. short oral application time.27,28 Artificial saliva was prepared by mixing the following This research employs an innovative approach against dental compounds in the indicated concentrations: potassium demineralization to avoid the side effects of high fluoride chloride 0.24 g/L, calcium chloride dihydrate 0.078 g/L, concentrations in the oral environment and extend the contact potassium dihydrogen phosphate 0.544 g/L, magnesium time between the fluoride ions and the tooth surface, enabling chloride hexahydrate 0.041 g/L, HEPES 4.77 g/L. After successful remineralization. The product called NMTD29 (new complete dissolution of the saliva components, the pH was material for dental treatment, from its Spanish acronym Nuevo adjusted to 7.1 ± 0.4 with potassium hydroxide pellets. Material de Tratamiento Dental) provides a controlled release In order to perform the remineralizing treatments, artificial system for the anticaries treatment and it is composed of a saliva was added to NMTD until the consistency of dense gel combination of weak acid and weak base ion-exchange resins was reached for its application to bovine teeth. In the case of loaded with calcium, fluoride, phosphate, and zinc. This agent the treatment with NMTD and amelogenin (synthesis detailed allows the formation of FA by controlling the rate of fluoride in Supporting Information section 1.3), 100 μg/mL of human release into the oral environment, in conjunction with the amelogenin was added to the saliva before mixing with release of calcium and phosphate ions to induce remineraliza- NMTD. In both cases the mixture of NMTD with the tion. The molar ratio of the calcium, fluoride, and phosphate corresponding saliva (with or without amelogenin) was ions has to be close to that of the organomineral tissue to be distributed on the enamel surface of acid-etched teeth, and remineralized. Zinc ions act as initiators of the ionic release of then the samples were placed in a sealed vessel. The base of the structural ions. In addition, zinc incorporation into enamel this container was filled with saliva to maintain the humidity of may accelerate its remineralization and reduce the rate of the environment and the recipient was placed inside an 5360 https://doi.org/10.1021/acs.analchem.1c05498 Anal. Chem. 2022, 94, 5359−5366 Analytical Chemistry pubs.acs.org/ac Article Figure 1. (a) Average of the specular reflectance FTIR spectra of enamel and band assignment numbers (n = 11). (b) Average of spectra corresponding to enamel, NMTD layer with amelogenin, and NMTD layer without amelogenin normalized to a maximum (n = 11). incubator at the normal temperature of the oral cavity (37 °C). More details of data acquisition and data treatment are shown Every 24 h for 15 days, each treatment was renewed by in Supporting Information section 1.5. washing the samples carefully with Milli-Q water and brushing for 20 s before applying a fresh treatment portion. Finally, all teeth were cleaned by brushing with Milli-Q water for 20 s and ■ RESULTS AND DISCUSSION Specular Reflectance SR-μFTIR. After 15 days of the stored in a 0.5% chloramine T solution until the preparation remineralizing treatments, teeth had a new mineral layer for the synchrotron experiments. between 20 and 30 μm of thickness measurable with the Specular Reflectance SR-μFTIR Experiment. Two microscope of MIRAS beamline. Measurements were taken on samples of each treatment were completely embedded in the a frame including points of the enamel and of this new layer to same Paladur acrylic resin and longitudinally cut in two halves study the similarities and differences of the crystals of the with a Struers Minitom precision diamond saw (Copenhagen, newly formed layers for the different treatments (NMTD with Denmark) to obtain a mesial view from the inside. A sequence amelogenin or NMTD alone) with the underlying enamel. of silicon carbide paper was used to polish the exposed tooth Infrared spectroscopy of apatites generally provides two area with a Struers LaboPol-25 polishing machine (Copenha- different kinds of information. The crystalline quality is gen, Denmark), starting at grit size P2500 and increasing to evaluated from the width of the absorption bands due to the P4000, under a constant flow of tap water. A sequence of phosphate vibrational modes. The other type of information is diamond suspensions with a mean particle size of 3 and 1 μm based on the presence of molecular species, like carbonate was used to finish the polishing and obtain the appropriate groups, which are detected by specific vibrational bands.37 reflection properties for the measurements. A sample image Bands corresponding to carbonates and phosphates in the can be observed in the Supporting Information Figure S1a. enamel HA are identified in Figure 1a. The bands associated Tooth samples were analyzed by μFTIR coupled to with the ν3PO4 vibrations (antisymmetric stretching) are synchrotron radiation (SR-μFTIR) in reflectance mode. The observed between 1170 and 965 cm−1.38,39 In the literature, experiment was carried out at MIRAS beamline of ALBA the deconvolution of the ν3PO4 bands in stoichiometric apatite Synchrotron (Cerdanyola del Vallès, Spain)35 using a Hyper- is described as secondary phase vibrations of Ca−O−P at 1103 ion 3000 microscope coupled to a Vertex 70 spectrometer cm−1, P−O at 1091 cm−1, Ca−O at 1047 cm−1, and O−Ca−O (Bruker, Ettlingen, Germany) and equipped with a mercury- at 1031 cm−1.40 The weak ν1PO4 band (symmetric stretching) cadmium-telluride (MCT) detector. More details of data is present at 966 cm−1.37 The region between 1580 and 1320 acquisition and data treatment are shown in Supporting cm−1 corresponds to ν3CO3 antisymmetric stretching, where Information section 1.4. two maximums at 1442 and 1401 cm−1 can be appreciated.8 Synchrotron tts-μXRD Experiment. A couple of samples An additional small band related to structural carbonates of each treatment were released from the Paladur acrylic resin (ν2CO3 symmetric angular deformation) is present at 867 and embedded in Epofix resin (Struers, Copenhagen, Den- cm−1.8,37,39 Carbonate can substitute into two anionic sites of mark). The tooth samples were longitudinally cut in half, fixed the HA structure. In carbonated apatite type B, which is the on a glass substrate of 1.5 mm thickness, and polished to predominant type in biological apatite, it is located at reduce the sample thickness down to 30 μm and achieve a flat phosphate group sites, while in carbonated apatite type A, it surface. A tooth thin section image can be seen in the is found at hydroxyl group sites.41 According to the peak Supporting Information Figure S1b. These sections were deconvolution from the literature, the ν3CO3 vibration in analyzed by synchrotron tts-μXRD. The experiments were carbonated apatite type A splits into two peaks at 1530 and performed at the Materials Science and Powder Diffraction 1465 cm−1. In contrast, type B can be characterized by peaks at (MSPD) beamline of ALBA Synchrotron (Cerdanyola del 1456 and 1423 cm−1 in the IR spectrum.41,42 In the case of the Vallès, Spain).36 The MSPD beamline is equipped with ν2CO3 out-of-plane bending mode, a peak at 879 cm−1 is Kirkpatrick−Baez mirrors and a Rayonix SX165 CCD detector. reported to belong to type A and another at 872 cm−1 to type 5361 https://doi.org/10.1021/acs.analchem.1c05498 Anal. Chem. 2022, 94, 5359−5366 Analytical Chemistry pubs.acs.org/ac Article Figure 2. (a) PCA scores graph of the FTIR spectra from the enamel and layer of two samples with amelogenin and two samples without amelogenin. (b) PC 1 and PC 2 loadings of the PCA. B.41,43 The combined presence of A and B carbonates affects the carbonate peak positions compared to apatite with only A or only B type.41 Meaning that the carbonate peaks detected in the regions 1580−1320 cm−1 and 880−830 cm−1 of the enamel in reflection mode show a mixture of type A and B carbonates. Both types of layers (Figure 1b) present the characteristic peak of apatites due to the phosphate group near 1050 cm−1, indicating a high crystallinity.44 In both layers, there is an absence of the two small peaks near 1400 cm−1 and the small peak at 867 cm−1 that are present in the enamel due to the carbonate group.45 The lack of the carbonate substitution group of high solubility and the narrowing of the phosphate peak in both layers indicate conversion to a more highly crystalline apatite compared to enamel. In addition, the layers appear to have a more evident vibration at 1103 cm−1 than the enamel, corresponding to the Ca−O−P secondary phase vibration. This could be due to a higher amount of calcium interacting with phosphate than in enamel since there are no carbonates in the layer, and therefore there is no calcium Figure 3. Diffractograms obtained by synchrotron tts-μXRD for HA bound to carbonate as in enamel. Further evidence is included reference, FA reference, enamel, NMTD layer with amelogenin, and in the Supporting Information Figure S2. NMTD layer without amelogenin, normalized to maximum and In Figure 2a, the PCA of spectra corresponding to enamel showing reflections. and both layers shows that principal component 1 (PC 1) separates the two treatments (NMTD with and without observed in the (211), (112), and (300) reflections of the amelogenin) from each other and principal component 2 (PC external layer and the FA reference (synthesis explained in 2) separates the enamel from the treatments as it separates Supporting Information section 1.2), compared to the enamel according to the depth of the measurement. PC 1 represents and the HA reference, as expected from the slight reduction in 59.95% of the variation, while PC 2 accounts for the 33.10%. the a,b-axis dimension when F− ions replaces OH− ions.47,48 In Figure 2b, the loadings for PC1 and PC2 show the main This shift confirmed the substitution of fluoride ions into the peaks in the regions 1580−1320 cm−1 and 1170−965 cm−1, apatite lattice, therefore the remineralized layer formed is corresponding to the ν3CO3 and the ν3PO4 vibrations. composed of FA, more resistant than enamel HA. Therefore, these vibrations are responsible for the scores Synchrotron tts-μXRD was also used to investigate the separation in Figure 2a. The points in the PCA belonging to texture (or preferred orientation) of enamel crystallites in the the layer with protein are statistically closer to the enamel tooth sections. The preferred orientation refers to the degree points than those belonging to the layer with NMTD alone, of alignment of the crystallites. The intensities of different which may be due to the 1103 cm−1 shoulder being more XRD peaks can be employed to estimate the macroscopic pronounced in the NMTD layer (Figure 1b). level-specific orientation of crystals. Sharp and intense (002) Synchrotron tts-μXRD. A typical diffraction pattern of HA and (004) peaks in the apatite, as can be seen for the new was found inside the enamel, the main (hkl) Miller indices4,46 layers in Figure 3, indicate that crystals prefer to be aligned (002), (211), (112), (300), (202), (222), (213), and (004) are along the c-crystallographic axis as in real enamel.49,50 The indicated in Figure 3. A shift to a slightly higher angle was lattice planes (002) and (004) along the c-axis of the enamel 5362 https://doi.org/10.1021/acs.analchem.1c05498 Anal. Chem. 2022, 94, 5359−5366 Analytical Chemistry pubs.acs.org/ac Article Figure 4. 2D μXRD images from (a) a sample with amelogenin and (b) a sample without amelogenin (enamel point on the top and layer point on the bottom). Representation of the azimuthal plots from a line of (c) a sample with amelogenin and (d) a sample without amelogenin. crystals are oriented perpendicular to the tooth surface, both kinds of samples (NMTD treatment with or without following the direction of the enamel prism arrangement.1,51 amelogenin) was found in the reflection (002), as can be Perpendicularity to the enamel surface maximizes the strength appreciated in Figure 4a,b. The lattice plane reflection (002) and bending capability and enhances the wear resistance does not overlap with other reflections and has the greatest capacity.52 intensity variation with maxima normal to the c-axis.52 A high degree of crystalline anisotropy, as in dental enamel, Moreover, in the diffractograms shown in Figure 3 a shift produces a change in the intensity around the Debye ring of was not observed in the (002) peak, since the contribution is Bragg reflections in two dimensions that correlates with the due to the c-axis in this reflection and the c-axis dimension degree of crystallite alignment or ordering.53 The intensity does not change from HA to FA.3 To analyze the different variations around the diffraction rings are indicative of tooth behavior of crystal orientation along the lines of points that enamel texture.51 Diffraction spots in Figure 4a,b are enter into the teeth, the evolution of the azimuthal intensity of concentrated in distinct arcs, both in the enamel and in the the reflection (002) has been plotted. This gives a linear layers, which means that the crystals are ordered.54 The representation of the evolution of the pixel intensity along the strongest texture (the most extreme intensity variation) for ellipse specified by the angle 2θ (Debye ring).55 Figure 4c,d 5363 https://doi.org/10.1021/acs.analchem.1c05498 Anal. Chem. 2022, 94, 5359−5366 Analytical Chemistry pubs.acs.org/ac Article Figure 5. Examples of MCR analysis of the azimuthal plots from a line of (a) the sample with amelogenin and (b) the sample without amelogenin. (c) Loadings for each component of both samples, with amelogenin and without amelogenin. shows a typical example of the azimuthal plot for all the points by imposing non-negativity in both the intensities and of one line from both samples where there are two pronounced azimuthal profiles. The orientation of component 1 (blue) is peaks separated by approximately 180°. The two pronounced mostly present in the layer, while component 2 (orange) peaks represent the opposing (002) reflection maxima that can appears in the outer enamel and component 3 (yellow) be observed in the 2D μXRD images of Figure 4a,b. Sharp becomes more important in the deeper enamel where new intense peaks are evidence of a strong preferred orientation, preferred orientations appear. Component 4 (purple) belongs while broad peaks would indicate a more random orientation to the points outside before reaching the samples. The of the crystallites.56 It can be seen in Figure 4c,d that both evolution of the different components confirms that the kinds of layers have a strong preferred orientation with intense orientation of the layer with NMTD and amelogenin (Figure peaks, even exceeding the intensity of the underlying enamel. 5a) follows the enamel underneath better than the layer with Moreover, the treatment with NMTD and amelogenin (Figure only NMTD (Figure 5b). In the NMTD layer with 4a,c) produces a layer that follows the preferred orientation of amelogenin, the change of the components 1 and 2 between the surface enamel better than the treatment with only NMTD the layer and the enamel is more gradual than in the one with (Figure 4b,d), since the positions of the arcs and the peaks for NMTD alone, which is consistent with the movement of the the reflection (002) match better with enamel ones. The presence of another population of crystallites with distinct peaks in Figure 4c,d. The changes in the intensities of the preferred orientation can be appreciated deeper in the enamel components as they enter deeper into the tooth (Figure 5a,b) of the sample with protein where there are two additional can also be correlated with the intensities of the peaks in the peaks, also separated by approximately 180° (Figure 4c). This azimuthal plots at different depths. The intensity in the layers effect of four peaks due to two crystal populations with of the azimuthal plots in Figure 4c,d is also higher than in the different preferred orientation is due to the natural structure of enamel. More examples are included in the Supporting the enamel and can also be seen in the azimuthal plots of the Information Figure S3. enamel of both sample types in the Supporting Information The loadings in Figure 5c show peaks separated by 180°, as Figure S3a,b. the peaks of the original azimuthal plots. The peaks for In Figure 5, MCR analysis of the azimuthal plots of the components 1 and 2 are in similar positions, yet component 3 reflection (002) from both treatments (NMTD with has them shifted to lower angles. This is consistent with amelogenin and NMTD alone) is shown. MCR was developed component 3 being predominant in the inner enamel (Figure 5364 https://doi.org/10.1021/acs.analchem.1c05498 Anal. Chem. 2022, 94, 5359−5366 Analytical Chemistry pubs.acs.org/ac Article 5a,b) where azimuthal peaks appear at lower angles (Figure Notes 4c,d). The authors declare no competing financial interest. ■ CONCLUSIONS The combination of synchrotron infrared microspectroscopy ■ ACKNOWLEDGMENTS This work was supported by the Spanish Ministerio de and micro X-ray diffraction have shown to conform a useful Economía y Competitividad (Project CTM2015-65414-C2-1- methodology, together with the proper data treatment, to R). Sandra Diez-García acknowledges the FI-2017 fellowship analyze the structure of apatites in samples of hard dental from Agència de Gestió d’Ajuts Universitaris i de Recerca tissues. Infrared microspectroscopy provides information on (Generalitat de Catalunya). The SR-μFTIR experiment was chemical structural properties such as carbonate substitution, performed at MIRAS beamline and the tts-μXRD experiment while micro X-ray diffraction allows to study the crystal at MSPD beamline at ALBA Synchrotron (Cerdanyola del structure and texture distribution on dental specimens. Vallès, Spain), with the collaboration of ALBA staff (Dr. Synchrotron radiation allows one to detect small changes Ibraheem Yousef and Dr. Oriol Vallcorba). This work has been with better resolution and to analyze microzones with high funded by ALBA Synchrotron through granted proposals signal intensity. (Grant References 2018093150 and 2020024308). The Since mature tooth enamel does not regenerate after authors acknowledge Dr. Roberto Boada and Mr. Jorge substantial loss, finding a suitable solution for the dental Rodríguez-Martínez from Universitat Autònoma de Barcelona problems is essential.23,24,56 The NMTD resin with or without for their support. amelogenin protein creates a remineralized layer on the surface of acid-etched teeth composed of carbonate free FA that has higher physicochemical stability than tooth HA. Nevertheless, ■ REFERENCES (1) Fujisaki, K.; Todoh, M.; Niida, A.; Shibuya, R.; Kitami, S.; amelogenin is a crucial component of this remineralizing Tadano, S. J. Mech. Behav. Biomed. Mater. 2012, 10, 176−182. product since it plays a critical role in controlling the preferred (2) Kis, V. K.; Sulyok, A.; Hegedus, M.; Kovács, I.; Rózsa, N.; orientation of the growing crystal to resemble dental enamel. Kovács, Z. Acta Biomater 2021, 120, 104−115. 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