University of Birmingham Disruption of enamel crystal formation quantified by synchrotron microdiffraction

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Introduction
Diseases associated with mineralization defects are frequently investigated using structural characterisation of affected hard tissues to complement an existing understanding of disease pathogenesis informed by cellular and molecular studies. A wide variety of techniques used to study bone and dental hard tissues include light and electron microscopy 1 ; Methods: Two-dimensional synchrotron X-ray diffraction has been utilized as a sophisticated and useful technique to spatially quantify preferred orientation in mineralized healthy deciduous dental enamel, and the disrupted crystallite organization in enamel affected by a systemic disease affecting bone and dental mineralization (mucopolysaccharidosis Type IVA and Type II are used as examples). The lattice spacing of the hydroxyapatite phase, the crystallite size and aspect ratio, and the quantified preferred orientation of crystallites across whole intact tooth sections, have been determined using synchrotron microdiffraction.
Results: Significant differences in mineral crystallite orientation distribution of affected enamel have been observed compared to healthy mineralized tissue. The gradation of enamel crystal orientation seen in healthy tissue is absent in the affected enamel, indicating a continual disruption in the crystallite alignment during mineral formation.
Conclusions: This state of the art technique has the potential to provide a unique insight into the mechanisms leading to deranged enamel formation in a wide range of disease states.
Clinical relevance: Characterising crystal orientation patterns and geometry in health and following disruption can be a powerful tool in advancing our overall understanding of mechanisms leading to the tissue phenotypes seen clinically. Findings can be used to inform the appropriate dental management of these tissues and/or to investigate the influence of therapeutic interventions or external stressors which may impact on amelogenesis.
atomic force microscopy 2,3 ; X-ray microtomography 4,5 ; X-ray diffractometry [6][7][8] ; and increasingly synchrotron X-ray scattering. [9][10][11] In particular electron microscopy studies can show qualitatively hierarchical features on the microscopic lengthscale such as prismatic and interprismatic structures of enamel revealing the variation in prismatic structure between species showing that enamel ultrastructure and function are closely linked to evolutionary development. 12 In particular, recent evaluation of Hunter-Schreger bands in human enamel reveal that these specific prismatic orientations have evolved to optimise resistance to fracture and wear over the lifetime of an individual. 13,14 Uniquely, synchrotron X-ray microdiffraction can determine spatial distributions of basic crystallographic parameters of the hydroxyapatite (HA) phase within mineralized tissues. Characterising crystal orientation patterns and geometry in health and following disruption can therefore become a powerful tool advancing our overall understanding of mechanisms leading to phenotypic expression. Dental hard tissues are unique in terms of their accessibility for such analyses. Deciduous teeth exfoliate naturally and permanent teeth are frequently available following routine extraction. More importantly dental enamel is highly mineralized and it's unique hierarchical structure forms incrementally over extended time periods with individual teeth mineralizing in 4-5 years. Accordingly disruption in crystallographic features of dental enamel due to disease progression or therapeutic intervention can be closely correlated with event time points.
The spectrum metabolic disorders known as mucopolysaccharidosis (MPS) diseases have incidences reported to range from 1:50,000 to 1:250,000 births 15 and will be used as a case study to highlight the capabilities of the technique. In particular mucopolysaccharidosis Type IVA (MPS IVA), or Morquio Syndrome, has manifestations in primary and secondary dentition. MPS IVA, an autosomal recessive lysosomal storage disease, 16,17 is characterised by reduced activity of enzyme N-acetylgalactosamine 6-sulphatase (GALNS) encoded by a gene on human chromosome 16q24. 3 18,19 which leads to intracellular accumulation of partially degraded glycosaminoglycans (GAGs) keratan sulphate and chondroitin 6-sulphate in connective tissue, the skeletal system and teeth. 20,21 Clinically it manifests after infancy and is associated with severe skeletal abnormalities, restrictive lung disease, impaired endurance, hearing impairment, and aortic valvular disease. 22 Enzyme therapies developed for MPS IVA are currently being investigated through clinical trials (NCT ID: NCT01242111 and NCT01275066), with the potential to revolutionise treatment for patients.
Basic dental histological investigations have demonstrated that MPS IVA enamel is abnormally thin and pitted 23 with increased porosity correlating to the striae of Retzius. 24 Electron microscopy has revealed an interstitial layer of amorphous material 3-4 mm thick at the amelodentinal junction (ADJ). 25 In MPS IVA it has been suggested that pathological accumulation of GAGs occurs in the lysosomes of secretory stage ameloblasts. 21 However, there is no consensus on whether the effects of impaired lysosomal pathway function result in disturbances in protein secretion; matrix mineralization; degradation processes of amelogenins; or a combination which lead to the enamel structural changes. 2D synchrotron X-ray diffraction across whole intact sections of dental enamel can provide important insights into the spatial distribution of HA crystallite orientation. 9,26 The aim of this study was to ally detailed analysis of physical characteristics of affected enamel in a system with a known, precise, underlying genetic lesion. We aim to demonstrate that the technique can not only give novel insight into the mechanistic understanding of the disease pathogenesis (in MPS), but also provide better understanding of basic processes of enamel biomineralization in health by relating known genetic defects to measured changes in crystallographic parameters.

2.
Materials and methods

Specimen preparation
Tooth specimens were collected following ethical approval (UK National Research Ethics Service Reference 08/H1202/119) and consent at Birmingham Children's Hospital NHS Foundation Trust. Two deciduous maxillary incisors from different patients affected by MPS IVA; one affected by MPS II (with no previous reported effect on enamel formation); and one healthy control deciduous maxillary incisor were used. Each extracted tooth, stored in thymol-saline solution, was serially sectioned bucco-lingually using a 0.3 mm diamond blade cutter (Met Prep, Coventry, United Kingdom) then polished to 100 mm thick. A spatially equivalent 0.4 mm Â 0.2 mm area, located 1 mm superior to the enamel-cementum junction was identified as a representative and comparable region of interest on each tooth section. An illustration highlighting the equivalent region of interest on each tooth section is given in Fig. 1. For synchrotron studies tooth sections were kept hydrated during measurement using a reservoir of thymolsaline solution. For scanning electron microscopy the sections were dehydrated in ethanol, etched with 35% orthophosphoric acid for 15 s, and stored in a desiccator prior to use.

Experimental procedure
Synchrotron X-ray diffraction was used to explore the texture (or preferred orientation) of enamel crystallites in intact tooth sections. Preferred orientation refers to the degree of crystallite alignment. For a polycrystalline, isotropic material, there is random orientation of crystallites averaging the Bragg scattering intensity uniformly around the Debye rings of 2D X-ray diffraction patterns. However, a high degree of crystalline anisotropy, such as in dental enamel, produces a change in intensity around the Debye ring of Bragg reflections in twodimensions correlating to the degree of crystallite alignment or ordering (Fig. 2, inset A). This change in intensity around Debye rings from different regions of enamel was measured to quantify the spatial texture distribution, and therefore the enamel crystallite organisation as a function of position. 2D synchrotron X-ray diffraction experiments were carried out on the XMaS beamline (BM28) at the European Synchrotron Radiation Facility (ESRF). Performing experiments at central synchrotron radiation facilities through a peerreviewed beamtime application process, means there are strict time constraints such that carrying out repeatability studies is not straight forward. For this study (the first of its kind to explore texture variation in MPS affected tissue) it was decided to prioritise the collection of high quality, high resolution images instead of sampling many repeated specimens. Therefore the X-ray beamspot was defined as 20 Â 20 mm using vacuum tube slits, and diffraction images were collected every 20 s. An X-ray energy of 15.0 keV was used -corresponding to a wavelength of 0.82 Å . Each specimen was mounted onto a goniometer on an XY-motorized travelling sample platform and aligned to the centre of rotation. A co-ordinate system for the region of interest on the tooth was established using a calibrated telescope. 2D Xray diffraction images were collected using a 2048 Â 2048 pixel CCD camera mounted perpendicular to the X-ray beam 170 mm from the sample. 2D maps were collected by translating the specimen relative to the beam in horizontal and vertical directions. Subsequently, scanning electron microscopy was used to image the same regions of interest on the enamel.

Data analysis
A total of eight hundred 2D diffraction images of enamel were processed with the Fit2d software. 27 Intensity patterns around the Debye ring of the (0 0 2) Bragg reflection were used to quantify the texture parameter (or preferred orientation) since this reflection is normal to the c-axis of enamel HA crystallites. The intensity was integrated over 3608 in a narrow band containing the (0 0 2) reflection and plotted versus the azimuthal angle. The peaks were fitted to a Gaussian peakshape and the full width half maximum (FWHM) value determined. By applying this procedure to each of the diffraction images, a spatial map of texture distribution in the (0 0 2) direction in the enamel was constructed.
The diffraction data of ten patterns close to the surface of the healthy enamel, and MPS IVA affected enamel respectively were summed and analysed by Rietveld refinement 28 using GSAS software. 29 The instrument parameters were calibrated using a LaB 6 standard sample. The two summed patterns were fitted with a HA phase with hexagonal space group P6 3 /m. Lattice parameters, Lorentzian line broadening and spherical harmonic texture parameters were refined. Microstrain, although a refinable parameter within GSAS, was found to be negligible in these enamel specimens therefore was kept fixed.

Results
A typical diffraction pattern fitted to the calculated HA phase using Rietveld refinement is demonstrated in Fig. 2 together  with its 2D X-ray diffraction image (Fig. 2. inset). The tabled results of the refinement are given in Table 1. Lattice parameters (a and c), Lorentzian line broadening ( p jj and p ? ), and spherical harmonic texture parameters (0 0 2 (SH) ) were refined producing a good fit to the HA phase. Lorentzian line broadening was refined to find the anisotropic particle sizes from the Scherrer equation. The parallel ( p k ) and perpendicular ( p ? ) components to the anisotropic particle size effects were given by: where l is the X-ray wavelength; X is Lorentzian particle size broadening; and X e is the anisotropic coefficient of Lorentzian broadening. 29 An example of the typical variations in Intensity around the (0 0 2) Debye ring for healthy and MPS IVA affected enamel are plotted in Fig. 3 with a Gaussian peak shape fitted to determine the FWHM. It was observed that the FWHM of affected enamel is over double that of healthy enamel, indicating less orientation of crystallites in affected enamel.
The results of the texture distribution analysis are shown in Fig. 4a-d. The x-axis represents distance from the surface enamel towards the ADJ and the y-axis represents vertical distance. In healthy enamel (Fig. 4a) the FWHM values decrease as a function of distance from the enamel surface. In contrast the FWHM values of the MPS II and MPS IVA affected enamel (Fig. 4b-d) do not vary significantly as a function of distance. Fig. 5 shows four typical scanning electron micrographs for the healthy deciduous enamel section (Fig. 5a-d), and for MPS IVA affected enamel (Fig. 5e-h). In Fig. 5d (healthy enamel close to ADJ) clear scalloping (Fig. 5d, arrow 1) and close integration of dentine and enamel can be seen with a small region of closely packed enamel crystallites at the ADJ (Fig. 5d, arrow 2), with prismatic order emerging $5 mm from the ADJ. In contrast in Fig. 5h (MPS IVA affected enamel close to ADJ) there is no evidence of scalloping, instead a microgap between dentine and enamel is observed (Fig. 5h, arrow 3). Compared to the healthy ADJ, there is a larger region of tightly packed enamel crystallites (Fig. 5h, arrow 4), and a less well defined prismatic order emerges. In the healthy enamel closer to the surface (Fig. 5a-c) there is good differentiation between prisms and interprismatic enamel (Fig. 5c, arrow 5); sharp, well defined crystallite boundaries; and a uniform prismatic structure. Whereas in the MPS IVA affected enamel ( Fig. 5eg) a non-uniform prismatic structure emerges with atypical prism shapes (Fig. 5f, arrow 6); larger regions of aprismatic enamel; and the individual crystallites are less well defined.

Discussion
There is a 0.2% variation in lattice parameter between healthy and affected enamel. Enamel is not pure hydroxyapatite, rather a carbonate substitute apatite, with CO 3 2À concentrations ranging from 1-5% from surface to tissue interior. 30 The 0.2% variation lies within the intratooth variations we have reported previously of 0.1-0.6% 9 therefore is likely to be attributed to slight variations in chemical composition rather than related to the metabolic disorder. The anisotropic particle size broadening varies between healthy and MPS IVA affected enamel, with the crystallite Table 1 -Refined structural parameters for surface enamel diffraction patterns fitted with HA phase. The low x 2 value (<1.0) is likely due to low-noise summed area detector data which has been converted into 1D underestimating the collection statistics.

Parameter
Healthy enamel MPS IVA affected enamel   Results presented in Fig. 4 indicate that texture distributions in MPS II and IVA enamel are substantially different to that of healthy enamel. In healthy deciduous enamel (Fig. 4a) there is a steady decrease in ordering (crystallite alignment) as a function of depth into the tooth. We have reported a similar trend in the past in the permanent dentition, where we observed that HA crystallites are most aligned in the cuspal   (5), non-uniform prism structure (6). The magnification is the same for each image and the scale bar is given.
regions, whilst along sides of the tooth away from the cusps and deeper into the enamel, crystallites are less ordered, likely due to the convolution of prism directions which occurs towards the ADJ. 9,26 In contrast, the plots shown in Fig. 4b-d indicate that the texture in MPS II and IVA affected enamel does not vary as a function of depth into the enamel. Instead the degree of crystallite ordering remains constant throughout the tooth thickness. This trend is seen also on the prismatic length scale from the SEM images shown in Fig. 5.
In MPS IVA the deficiency of N-acetylgalactosamine 6-sulphatase (GALNS) manifests clinically and radiographically in enamel which is hypoplastic and can detach easily from the underlying dentine. Generally a thin enamel layer is indicative of a developmental disturbance during the secretory stage of amelogenesis. Through in situ hybridization of a day 1 mouse incisor it has been shown that GALNS mRNA is most abundant in secretory ameloblasts. 21 Therefore it is likely that the disruption in the texture gradation and reduced ordering in the prismatic structure we observed in MPS IVA affected enamel is likely to start in the secretory stage of amelogenesis. However, although mineralization starts in the secretory stage, the thickening of enamel crystallites, where the degree of preferred orientation is defined continues through the maturation stage, therefore it is likely that the texture distribution is determined by disruption of cellular and matrix-mediated events at both stages. At the earliest stages of amelogenesis, it has been suggested that GAGs may serve as a matrix for anchoring amelogenin at the ADJ so that a close bond is established between enamel and dentine. 31 The absence of good integration between enamel and dentine at the ADJ of the MPS IVA enamel (Fig. 5h) may be indicative of the lack of the specific sulphatase which should remove the GAGs from the dentine tubule sites to allow proper intergration at the ADJ.
In addition, normal enamel matrix mineralization has been demonstrated to be disrupted in animal models by suppression of Ca 2+ ATPase activity and ATP-dependent calcium pumps are observed ultracytochemically at specific regions of the Tomes process of ameloblasts. 32 Moreover, the potential role of Ca 2+ trafficking has been reported in many lysosomal storage disorders including evidence for a potential link with mitochondrial dysfunction. 33 This was not predicted to be present in patients affected by MPS IVA but these findings in enamel may provide some evidence that other tissues like bone and muscle should be investigated in this cohort.
To the authors' knowledge there have been no reports of enamel defects associated with Hunter syndrome (MPS II), however in this study we clearly see disturbances in MPS II enamel crystal organisation. In fact, disruption in enamel texture appears more severe in MPS II than in MPS IVA. Accumulation of GAGs in the dental follicle of developing teeth in other MPS diseases, such as Hurler syndrome (MPS I) has been reported in the past, 23 but dental abnormalities are not always clinically obvious. It may be in MPS II the disruption affects the crystallography and nanostructure of enamel but has not yet been detected clinically on the macroscale.
We have shown that the use of 2D synchrotron microdiffraction, a state of the art technique, has the potential to provide unique insights into the mechanisms leading to deranged enamel formation in a disease state. The detailed characterisation of apatite crystallite orientation in dental enamel can provide an accessible and minimally invasive route to improve our understanding of the biomineralization process in enamel and our understanding of pathogenesis of systemic disturbances affecting mineralization. The structural information provided may have direct relevance for clinicians managing disease prevention or restoration of these tissues. For example, in the scenario provided, the lack of regular HA crystal orientation throughout the enamel thickness in MPS II and IVA may explain the poor success of adhesive restorations bonded to etched-enamel which is observed clinically. 15

Conclusions
We have used 2D synchrotron X-ray diffraction to study the texture distribution in enamel affected by MPS IVA and II. Significant differences were observed in the texture distribution of the MPS IVA and II affected enamel as compared to healthy tissue, characterised by less gradation of enamel crystal orientation in MPS-affected enamel compared to healthy tissue. The use of this state of the art technique has the potential to provide a unique insight into the mechanisms leading to deranged enamel formation in a wide range of disease states.