Anal Bioanal Chem DOI 10.1007/s00216-015-8742-y
Raman spectroscopic characterisation of resin-infiltrated hypomineralised enamel Arun K. Natarajan 1 & Sara J. Fraser 3,4 & Michael V. Swain 2 & Bernadette K. Drummond 1 & Keith C. Gordon 3
Received: 5 February 2015 / Revised: 7 April 2015 / Accepted: 27 April 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Raman spectroscopy was used to investigate how the effect of pre-treatment protocols, with combinations of hydrochloric acid (HCl), sodium hypochlorite (NaOCl) and hydrogen peroxide (H 2 O 2 ), for molar–incisor hypomineralisation (MIH) altered the penetration depth of polymer infiltrants (ICON, DMG, Hamburg, Germany). Furthermore, the effect on the structure of the MIH portions of the teeth with treatment is examined using multivariate analysis of spectra. It was found that pre-treatment protocols improved penetration depths. The structure of the MIH portion post-treatment appeared much closer to that of normal enamel suggesting a diminution of protein in the MIH region with treatment.
Keywords Raman spectroscopy . Resin infiltration . Hypomineralisation . Enamel
Electronic supplementary material The online version of this article (doi:10.1007/s00216-015-8742-y) contains supplementary material, which is available to authorized users. * Sara J. Fraser sara[email protected]
* Keith C. Gordon [email protected]
Department of Oral Sciences, Faculty of Dentistry, University of Otago, PO Box 647, Dunedin 9054, New Zealand
Biomaterials Science Research Unit, Faculty of Dentistry, University of Sydney, Sydney Dental Hospital, Surry Hills, NSW 2010, Australia
MacDiarmid Institute of Advanced Materials and Nanotechnology, Department of Chemistry, University of Otago, PO Box 56, Dunedin 9054, New Zealand
Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland
Introduction The outermost layer of human tooth crowns in the oral cavity is enamel, which is the hardest tissue consisting of 95 % mineral, 1 % organic matter and 4 % water by weight . Disturbance during tooth development can lead to enamel hypomineralisation where regions of the enamel contain lesions with lower levels of the mineral component . The hypomineralisation defects can be further classified based on changes in colour, pattern of occurrence and on the distribution of the defects in the mouth. Molar–incisor hypomineralisation (MIH) is a condition where abnormal opaque-coloured lesions (white, yellow and brown) with distinct margins are found on first permanent molars, with or without the involvement of incisor teeth and sometimes second primary molar teeth. Previous epidemiological studies have reported prevalence of MIH ranging between 2.4 and 40.2 % in different populations [3–11]. Investigators have used a range of methods to study the structure of MIH enamel. The affected enamel is typically characterised by poor biophysical properties. This can lead to fracture during normal function and poor aesthetics. The structure of MIH enamel is more porous  with decreased mineral and increased protein/peptide content [13–15]. The mean mineral density (MD) has been shown to be significantly lower for MIH enamel (19 % lower) compared with normal enamel . Mahoney et al. reported that the hardness and modulus of elasticity of MIH-affected enamel were significantly lower when compared with that of normal enamel . Jalevik et al., using secondary ion mass spectrometry and X-ray microanalysis, found that the median Ca/P ratios in the hypomineralised areas were significantly lower (1.4) when compared to those in the adjacent normal enamel (1.8) . MIH teeth have an increased protein content, both retained developmental proteins and serum proteins that appear to have been incorporated during the defective development . Several other studies have documented the presence of increased organic content in MIH-affected
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enamel [17, 20, 14]. In general, brown-discoloured enamel showed a 15- to 21-fold higher protein/peptide content, whereas yellow and white chalky enamel showed an approximately 8fold higher protein/peptide content than sound enamel [14, 20]. Xie et al. suggested that improved clinical outcomes are likely to depend on removal or alteration of the protein prior to etching for adhesive bonding . The high protein content inhibits adequate etch patterns and associated poor bonding of dental materials to these teeth . Attempts have been made to remove protein/peptide from the enamel surface to facilitate further mineralisation and increase bonding of resin materials to hypomineralised enamel [22–25]. Raman spectroscopy enables identification and characterisation of a variety of materials, cells and tissues including human dental enamel, dentine and cementum [26–37]. The technique has been applied to teeth [38–41, 35, 36, 42, 43], bone [44–49] and dental materials [50–55]. The ability to couple the technique to a microscope has made it possible to look at the chemical composition of teeth over a spatially resolved area. This technique has been used to detect faults in enamel, investigate the dentine–enamel interface and observe dental caries at a microscopic length scale [42, 36, 35, 39, 15]. In addition, this tool has also been used for assessment of bone healing around dental implants [56, 57]. Recent attempts have been made to find a suitable resin infiltrant that can improve the mechanical properties, improve appearance and decrease sensitivity [24, 58, 25]. A major difficulty is that etching has not allowed effective infiltration beyond a thin surface layer due to the increased organic content [14, 20, 59]. Resin infiltrants have been used to successfully treat MIH enamel. Recently, Crombie and co-workers also attempted to investigate the effectiveness of resin infiltration [24, 25]. Their study using polarised light microscopy reported inconsistent results with respect to penetration of the resin infiltrant. This study, for the first time, uses Raman spectroscopy in combination with principal component analysis (PCA), a nondestructive analytical tool, to investigate the penetrability of a low-viscosity resin infiltrant into enamel affected by MIH with various different pre-treatment agents. Raman spectroscopy combined with PCA was also used to investigate how the different pre-treatments affect the chemical composition of the treated lesions; this highlighted that the additional dental pretreatment procedures prior to resin infiltration into the hypomineralised lesions cause the treated enamel to appear chemically closer to that of normal enamel.
Experimental Specimen preparation Ethics approval was obtained from the University of Otago Human Ethics Committee (Ref no: MEC/11/11/087).
Following consent, first permanent molars removed as part of needed dental treatment were donated for the study. Seven molar teeth showing brown-demarcated opacities without surface enamel breakdown were selected. Only enamel samples with brown hypomineralisation were selected to have consistency in the severity of the defects. Teeth were cleaned using pumice to remove any debris and stored in Hank's balanced salt solution (HBSS) and thymol crystals at 4° C until use. At the time of investigation, each tooth was sectioned perpendicular to the surface across an MIH defect (giving two halves of each lesion) using a water-cooled diamond grit-embedded saw fitted on a precise sectioning machine (Accutom-50, Struers A/S, Ballerup, Denmark). The sectioned teeth were then polished using 1,200- and 4,000-grit grinding paper followed by 6-, 3and 1-μm diamond suspensions. A total of eight lesion halves were obtained. The lesion halves were allocated to four different infiltration groups (n=2 per group), and infiltration was carried out according to the protocols described in supplementary information section (cf. Electronic Supplementary Material Fig. S1). Treatment R had no pre-treatment and used 15 % hydrochloric acid (HCl) etching followed by resin infiltration (standard protocol). Pre-treatment A used 15 % HCl followed by 5 % sodium hypochlorite (NaOCl) and resin infiltration. Pre-treatment B used 5 % NaOCl followed by 15 % HCl and resin infiltration. Pre-treatment C used 30 % hydrogen peroxide (H2O2) followed by 15 % HCl and resin infiltration. To ensure that resin infiltrated the enamel from the outer surface and not through the cut surface, the cut surface was coated with nail varnish . The resin infiltrant used in this study was ICON caries infiltrant (DMG, Hamburg, Germany). This consists of 90 % triethylene glycol dimethacrylate (TEGDMA), 9.9 % ethanol, 0.5 % camphorquinone and 0.5 % ethyl 4-(dimethylamino)benzoate. The resin was polymerised using light curing with an output of 450 nm and an intensity of 800 mW/cm2. After the hypomineralised lesions were infiltrated, the nail varnish on the cut surface was removed by polishing. Raman microscopy The spectra were obtained with a Senterra Raman microscope (Bruker Optics, Ettlingen, Germany) equipped with 532- and 785-nm lasers. This instrument uses OPUS 6.5 (Bruker Optics, Ettlingen, Germany) for spectral acquisition and settings control. The 785-nm laser was used to avoid fluorescence of organic components of teeth. Spectra were collected using both 3–5- and 9–18-cm−1 resolution. The higher resolution spectra were collected over a narrower spectral region to better inspect differences in the bioapate. The signal has a band reproducibility within 0.5 cm−1 using OPUS SureCal with backgrounds collected every 1,000 s. Initially, line measurements with ×50 objective were collected on samples TR1 and TR2 after treatment via resin
Raman spectroscopic characterisation of enamel 960
Dentine Enamel Hypomineralised enamel Resin
Relative intensity / a.u.
1639 1721 1607 1668
Raman shift / cm
Fig. 1 Typical Raman spectra of dentine (red), enamel (blue), hypomineralised enamel (purple) and resin (green)
infiltration to detect resin infiltration into the sample. Each spectrum is a composite of 30 spectra taken for 10 s each with 50-mW incident laser power. Line measurements were collected across the cut section from the tooth surface towards
the dentine–enamel junction (DEJ), with additional spectra collected across the very outer tooth surface. Investigation of resin infiltration after pre-treatment of the enamel with the different protocols also used line measurements. Measurements were collected prior to and post-infiltration. The Raman spectra of each sectioned tooth (samples TA1, TA2, TB1, TB2, TC1 and TC2) were collected over a series of five line measurements. Each line measurement began from the outer tooth surface and continued across the enamel to the DEJ with incremental measurements taken at set distances. Lines 1, 4 and 5 involved measurements every 100 μm, and lines 2 and 3 involved measurements every 25 μm. Lines 2 and 3 were taken at closer distances to monitor depth of resin infiltration. An example of the line positions and distance between points on a sample is shown in Fig. 2b. All samples followed the same sampling pattern. The ×20 objective was used. Each spectrum is a composite of 30 spectra taken for 10 s with 50mW incident laser power.
Table 1 A summary of the representative wavenumber and associated assignment of key bands from enamel, dentine and hypomineralised enamel from human teeth spectra Enamel ṽ / cm−1
Dentine ṽ / cm−1
Hypomineralised ṽ / cm−1
Resin ṽ / cm−1
Assignment v2(PO43−) v4(PO43−)
602 v4(PO43−) v(CC) skeletal backbone 838 δ(CCH) aromatic, v(CC) (proline)
855 860 960
v(CC) aromatic ring breathing, ν1(HPO42−)
1,004 1,039 1,045 1,070
v3(PO43−) out of phase, v(CO32−) b-type ν1(CO32−)
1,045 1,069 1,127 1,190
1,246 1,284 1,405 1,452 1,608 1,639
δ(CH2) scissors Phenyl ring v(C=C) v(C=O) amide I v(C=O)
Tooth component assignments are made based on studies by Awonusi et al., Kirchner et al. and Penel et al. [54, 43, 51]. The resin assignments are based on Shin et al. 
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Fig. 2 Overview photos, Raman measurement positions and representative Raman spectra from sample TB1. (a) Overview image and measurement positions prior to treatment, (b) overview image and Raman measurement positions after treatment with details on measurement positioning parameters and (c) example spectra taken
across a series of points along line 1 of sample TB1. In (c), black indicates an example resin spectrum, red indicates spectra containing resin signals, blue indicates spectra collected from the hypomineralised enamel region and green indicates spectra collected from the dentine region of the tooth
Principal component analysis
outlying spectra, such as those focused slightly off the sample, were removed from the calculation.
The PCA and associated pre-processing of the spectra were calculated using the Unscrambler-X 10.3 software (CAMO, Norway). All the high-resolution spectra were analysed together after linear baseline correction (LBC) and standard normal variate (SNV) pre-processing of the spectral region 200 to 1,100 cm−1. PCA was calculated over this same spectral region using random cross-validation with 20 segments. Due to the high number of spectra and limited spectral region studied, the PCA analysis was also done on the low-resolution spectra with the three treatment groups analysed separately. These spectra were also pre-processed using LBC and SNV but over the larger spectral region of 200 to 1,800 cm−1 followed by PCA analysis with full cross-validation. In these analyses, any Table 2
Results and discussion The typical spectra of enamel, dentine, hypomineralised enamel and the resin used in this study are shown in Fig. 1. The normal enamel contains the strongest mineral features, in particular the ν1(PO43−) band at 960 cm−1. Both the dentine and hypomineralised enamel spectra have a less intense ν1(PO43−) band compared with normal enamel; the dentine also visibly contains bands associated with its higher organic content. A summary of the band wavenumber and
The approximate resin infiltration depth into samples with and without additional pre-treatments
Approximate resin infiltration depth (μm)
Pattern of infiltration
Resin Resin Type A + resin Type A + resin Type B + resin Type B + resin Type C + resin Type C + resin
TR1 TR2 TA1 TA2 TB1 TB2 TC1 TC2
0–300 0 500 700 800 (900 along line A) 0 500 480 (600 along line A)
Irregular pattern of infiltration No infiltration occurred but resin deposition on surface enamel noted Inconsistent pattern of infiltration with varying degrees of resin penetration Inconsistent pattern of infiltration with varying degrees of resin penetration Inconsistent pattern of infiltration with varying degrees of resin penetration No infiltration occurred but resin deposition on surface enamel noted Relatively consistent infiltration across the tooth Relatively consistent infiltration across the tooth
Raman spectroscopic characterisation of enamel
assignments from the average spectra of enamel, dentine, hypomineralised enamel and the resin used for treatment in this study is given in Table 1. The resin spectrum is distinctive from that of each of the components of the tooth (Fig. 1). It is thus possible to identify it, and this has been done for the samples both with and without the series of pre-treatment protocols. An example of the line mapping of resin infiltration is shown in Fig. 2, and the data for the series of teeth are presented in Table 2. From these data, it appears that infiltration is poor without pre-treatment. Optical images suggest the resin is deposited to the surface in the untreated samples, and this is corroborated with Raman microscopy. Line analysis shows that infiltration is sporadic in one of the untreated samples and does not appear to reach depths beyond 300 μm. Any of the three pre-treatment protocols yields more effective infiltration. In the case of treatment C, this appears to give consistent infiltration results. A key question is what are the effects of these pre-treatments on the teeth structure that lead to this more effective infiltration? This may be gleaned by examining the spectra taken across each of the teeth before and after the treatment protocols. These data are examined with PCA, and from these analyses, it is possible to group the spectra from the differing components. The high-resolution spectra from untreated and treated teeth were subjected to PCA to assess how the spectra, and hence chemical composition and/or structure of the tooth, changed on treatment. PCA of all spectra from samples before and after treatment showed major chemical changes after treatment. The first two principal components (PCs) account for 79 % of the total spectral variance, and the associated score plot for these PCs is given in Fig. 3a. The normal enamel
clusters in slightly negative PC1 and negative PC2 space, the dentine clusters in positive PC1, negative PC2 space and the hypomineralised enamel form the greatest variance across all of PC1 space and in neutral through to positive PC2 space as observed in Fig. 3a. Whilst the normal enamel and dentine from all the samples appear to cluster in similar regions in PC space indicating close chemical composition, the hypomineralised lesions vary significantly between samples despite trying to use only brown severity lesions, indicating high person-to-person variability in the hypomineralised dental tissue. The spectral features which are separating these samples can be found by inspection of the loading plots (Fig. 3c). Both PC1 and PC2 appear to be separating based on the mineral component of the teeth (hydroxyapatite), with more phosphate-type signals such as ν1(PO43−) at 960 cm−1 observed in negative PC1 and PC2 loadings. For the normal enamel and dentine, this separation pattern along PC1 is consistent with what is already known on the chemical makeup of enamel and dentine with enamel having higher levels of the mineral component than the dentine allowing it to have a harder structure [16, 13, 17, 19, 1]. The second PC also follows what would be expected, with positive samples (hypomineralised lesions) having lower mineral content than the negative samples (enamel and dentine). The phosphate features around 430, 585 and 609 cm−1 appear distorted, likely indicating changes in the phosphate structure. A previous study by the authors suggests that this is due to increased disorder in the bioapatite . Interestingly, some of sample A1 enamel is clustering away from all the other sample enamel spectra around A1-
Fig. 3 PCA analysis from spectra of samples before and after the three treatment regimes. Score plot of PC1 versus PC2 accounts for 79 % of the overall spectral variance with (a) highlighting spectra taken before treatment and (b) highlight spectra taken after treatment. The associated
loading plots are given in (c), and the scores of PC3 versus PC4 are shown in (d) where some separation due to the resin signal becomes more apparent
A.K. Natarajan et al.
Fig. 4 PCA analysis of low resolution spectra from samples with treatment A; 15 % HCl followed by 5 % sodium hypochlorite (NaOCl) and resin infiltration. Score plot of PC1 versus PC2 is shown with (a)
initial spectra highlighted and (b) post-treatment spectra highlighted. Samples before and after treatments are shown in (c) sample A1 and (d) sample A2. The associated first two loadings are given in (e)
Fig. 5 PCA analysis of low-resolution spectra from samples with treatment A; 15 % HCl followed by 5 % sodium hypochlorite (NaOCl) and resin infiltration. Signal due to the resin itself is not observed until PCs 4 and 5 within (a) the score plot and (b) the associated loading plots
Raman spectroscopic characterisation of enamel
hypomineralised enamel spectra. This suggests that whilst visually it may have looked like normal enamel, it is in fact hypomineralised. After treatment, the position of the majority of the spectra from hypomineralised regions moved from their starting position in positive PC2 space towards the normal enamel and dentine in neutral PC2 space (Fig. 3b). The signal due to the resin itself does not appear until the third and fourth PCs as seen in the scores (Fig. 3d) and associated loadings (Fig. 3c). This suggests that the resin itself cannot be the cause of these shifts in PC1 and PC2 space. Most likely the demineralisation and deproteination pre-treatment steps have caused changes to the bioapatite. It is theorised that the demineralisation step preferentially removes the more disordered apatite first, leaving behind the more ordered and hence more like more normal tooth bioapatite behind. The deproteination step is believed to aid this by removing the excess protein that prevents remineralisation. The removal of protein and disordered apatite is likely the cause of the treated samples appearing more
like normal enamel. An important thing to consider is that despite the treated hypomineralised lesion being more spectroscopically and hence chemically similar to normal enamel after treatment, the lesion may still be porous. In theory, the cap of resin on the outer tooth surface may block any stimulus hopefully preventing pain and sensitivity. Upon treatment, the spectral features are modified such that the hypomineralised cluster shifts towards an apatite-like spectrum. This suggests diminution of the protein bands and, in the absence of any mineral source, would lead to a more open normal enamel structure. This would be consistent with the observation of more favourable infiltration. These samples are infiltrated after treatment, yet the signature from the resin is in fact only a minor component of the overall spectral population with resin signature appearing in the loading plots of PC3 (7 %) and PC4 (5 % variance). In other words, the effect of the treatment and infiltration is to modify the enamel structure in the hypomineralised region so that is has less protein and appears spectrally to be more like normal enamel.
Fig. 6 PCA analysis of low-resolution spectra from samples with treatment B; 5 % NaOCl followed by 15 % HCl and resin infiltration. Score plot of PC1 versus PC2 is shown with (a) initial spectra highlighted and
(b) post-treatment spectra highlighted. Samples before and after treatments are shown in (c) sample B1 and (d) sample B2. The associated first two loadings are given in (e)
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The effects of the individual treatment procedures were analysed more closely using separate PCA analyses for each group. PCA analysis of the A-treatment method highlighted that the dominant spectral variances are associated with bioapatite to organic constituents (PC1, 64 % variance) and differences in the bioapatite structure and organic constituents (PC2, a further 19 % variance) as seen in the loadings in Fig. 4e. Sample A2 shows major changes in the composition of the hypomineralised lesion after treatment with movement in PC space towards normal enamel (Fig. 4a, b). Interestingly, sample A1 showed minimal movement in PC space, and the corresponding visual images (Fig. 4c, d) showed resin infiltration into A1 was less consistent than that of A2 across the lesion surface. These changes are believed to be due to the pretreatments and not the resin itself, as the resin signal is not observed until PCs 4 and 5, accounting for only 2 % of the overall spectral variance each (Fig. 5). PCA analysis of type B treatment shows similar results with the dominant spectral differences attributed to protein
to apatite content (PCA, 46 % variance) and the apatite form or disturbances to the apatite (PC2, 23 % variance) as seen in the loading plots (Fig. 6e). Following treatment, both B1 and B2 hypomineralised lesions constrict (become more uniform) and move towards normal enamel in PC space (Fig. 6a, b). Despite these changes, the resin infiltration only occurs on sample B1, not B2 (Fig. 6c, d). It is unknown if the deproteination and demineralisation steps have made this lesion more susceptible to stimuli in the absence of the resin cap or if the removal of the protein will enable the lesion to demineralise more efficiently. PCA analysis of the third treatment showed the most dramatic changes with the hypomineralised spectra from both C1 and C2 constricting significantly and moving very close to normal enamel (Fig. 7a, b). The first principal component is dominated by the relative apatite to proteinaceous content (PC1, 65 % variance). However, for these samples, the resin signal contribution is greater than that for A and B treatments with resin signal portioning from the apatite signal in PC2 (PC2, 18 % variance)
Fig. 7 PCA analysis of low resolution spectra from samples with treatment C; 30 % hydrogen peroxide (H2O2) followed by 15 % HCl and resin infiltration. Score plot of PC1 versus PC2 is shown with (a)
initial spectra highlighted and (b) post-treatment spectra highlighted. Samples before and after treatments are shown in (c) sample B1 and (d) sample B2. The associated first two loadings are given in (e)
Raman spectroscopic characterisation of enamel
as seen in the loadings in Fig. 7e. The infiltration of these samples appeared to be the most consistent across the whole lesion surface (Fig. 7c, d). This earlier contribution of the resin signal and similar infiltration patterns may be indicative of the two C samples being closer in chemical composition compared to the other tooth pairs. Many more samples would be required to better understand if these differences are related to the treatment methods or due to the underlying lesions itself. It is understood that defects seen in MIH enamel is a result of disruption to hydroxyapatite crystal mineralisation during the maturation phase of tooth development [61, 19]. This disruption to the enamel mineralisation process prevents the proteases to remove the excess proteins/peptides [62, 20, 14] and subsequently the crystal expansion resulting in apatite crystals that are less thicker/wider and loosely packed [63, 19]. The closeness of the cluster size proximity to the dentine spectra suggests that the properties of the treated tooth will be variable, somewhere in between that of normal enamel and dentine. This may arise because the thickness/width of the apatite crystals in the MIH enamel is smaller, and the composition is more dominated by the internal defective structure than for sound enamel [64, 19]. It appears that the use of HCl preferentially demineralises the disordered hydroxyapatite in the hypomineralised lesions, leaving behind the more ordered hydroxyapatite structure closer to that of normal enamel and dentine. The NaOCl and H2O2 are helpful in removing some excess proteins and peptides which prevent further crystal growth of hydroxyapatite in MIH enamel [14, 65, 66, 62]. It is possible that just the removal of some excess protein without resin infiltration may potentially allow more continuing mineralisation to occur . If the resin has infiltrated the hypomineralised enamel, it may prevent ingress of acids or bacteria in the mouth. Raman spectroscopy was used to both monitor the resin infiltration into hypomineralised lesions and the effects of the pre-treatment conditions on the tooth structure itself. The use of deproteination and demineralisation appeared to enhance resin infiltration. Whilst further studies are required to mitigate the effects of sample-to-sample variation in these results as the variation between lesions was very high, this study indicates the potential for Raman spectroscopy to be used to monitor tooth treatments and their effects on the tooth structure.
Conclusions Raman spectroscopy was used to detect the depth of resin infiltration into hypomineralised lesions to monitor how various different pre-treatments affect this resin infiltration depth. Raman spectroscopy also showed insight into how the dental tissue was altered with the various pre-treatment methods. The results indicate that resin infiltration is inconsistent and variable depending partly on pre-treatment protocols. It appears
that without some form of pre-treatment, little or no infiltration will occur. This analysis of resin infiltration into teeth with hypomineralised areas demonstrates that zones of resin infiltration in the hypomineralised enamel were not homogeneous in the samples. This has also been shown in early caries lesions; however, the penetration was deeper in caries lesions . Our findings corroborate those reported in a previous optical microscopic study that showed variable resin infiltration in MIH-affected enamel [24, 58]. Raman spectroscopy was able to show the change in the apatite crystalline structure with different pre-treatment agents used prior to resin infiltration. PCA analysis showed hypomineralised lesions became more like normal enamel and dentine after deproteination and demineralisation preceding resin infiltration. It appears that the dissolution of disordered hydroxyapatite occurs more readily than well-ordered crystalline hydroxyapatite after the excess protein is reduced. This may offer a path to improve further mineralisation in hypomineralised enamel. Raman spectroscopy shows promise for inspecting how new dental treatments interact with bioapatite tissue. Acknowledgments Arun Natarajan and Sara Fraser would like to acknowledge the University of Otago for postgraduate publishing bursaries.
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