Development of a new approach to diagnosis of the early fluorosis forms by means of FTIR and Raman microspectroscopy

www.nature.com/scientificreports OPEN Development of a new approach to diagnosis of the early fluorosis forms by means of FTIR and Raman microspectroscopy Pavel Seredin1,2*, Dmitry Goloshchapov1, Yuri Ippolitov3 & Jitraporn Vongsvivut4 This study is aimed at investigating the features of mineralization of the enamel apatite at initial stages of fluorosis development. Samples of teeth with intact and fluorotic enamel in an early stage of the disease development (Thylstrup–Fejerskov Index = 1–3) were studied by Raman scattering and FTIR using Infrared Microspectroscopy beamline at Australian Synchrotron equipment. Based on the data obtained by optical microspectroscopy and calculation of the coefficient R [A-type/B-type], which represents the ratio of carbonation fraction of CO32−, replacing phosphate or hydroxyl radicals in the enamel apatite lattice, the features of mineralization of enamel apatite in the initial stages of development of the pathology caused by an increased content of fluorine in the oral cavity were established. Statistical analysis of the data showed significant differences in the mean values of R [A-type/B-type] ratio between the control and experimental groups for surface layers (p < 0.01). The data obtained are potentially significant as benchmarks in the development of a new approach to preventive diagnostics of the development of initial and clinically unregistered stages of human teeth fluorosis, as well as personalized control of the use of fluoride-containing caries-preventive agents. It has been established that caries prevalence is associated with the intake of fluorine c ompounds1,2, that is, the less fluoride, the higher is caries frequency3,4. The program to introduce water fluorination in several European countries has led to a reduction in the number of carious pathologies in the p opulation1. At the same time, it was shown that fluorination is not a panacea1,2, especially under excessive fluoride intake in cases of medium severity leading to osteosclerosis and osteoporosis2. According to WHO, redundant fluoride in drinking water, as well as the irrational use of fluoride toothpaste, leads to the development of fl uorosis2,5,6. The excess of fluorine affects millions of people globally, although it is most often manifested in light and medium-light f orms2,7. Enamel fluorination is one of the methods to combat initial c aries5,8, stabilising the inorganic part of the dental matrix, nanocrystalline defective calcium carbonate substituted hydroxyapatite (CHAP), which in case of biogenic apatite can be described by the structural formula Ca8.8Mg0.1(PO4)4.9(HPO4)0.6(CO3)0.5(OH)0.99 or more precisely (Ca)5.x(Mg)q(Na)u(HPO4)v(CO3)w(PO4)3.y(OH,F)x_z where x, q, u, v, w, y, and z are the stoichiometry coefficients10. Fluorine ions replace hydroxyl groups and vacancies in the apatite, forming a more stable crystal lattice, calcium fluorapatite (CFA)8,10–13, which is less susceptible to caries. However, the formation of FA, given its physical and chemical properties different from those ones of native apatite, leads to a disrupted mineralization and transport functions of e namel5,8,14. Prolonged exposure of teeth to substances containing high levels of fluoride can lead to the formation of cracks and chips due to the formation of calcium fluoride (CaF2) enamel in the subsurface layers5,12,14. Consequently, many individuals, due to excess exposure to toothpaste and other fluorine-containing components, have an increased fluorosis5–7. The indicators of prevalence and severity of this pathology in developed countries have increased not only in time but by orders of magnitude. As a result of fluoride accumulation, fluorosis does not manifest clinically until the colour and morphology of the teeth change, which can be observed during the visual examination of the patient14–16. This requires the development of a diagnostic technique for the enamel condition to register changes in the enamel matrix during the development of fluorosis17–19. Early diagnosis of dental diseases using precision monitoring methods is 1 Department of Solid State Physics and Nanostructures, Voronezh State University, University Sq. 1, Voronezh, Russia 394018. 2Ural Federal University, 19 Mira Street, Ekaterinburg, Russia 620002. 3Department of Pediatric Dentistry With Orthodontia, Voronezh State Medical University, Studentcheskaya St. 11, Voronezh, Russia 394006. 4Australian Synchrotron (Synchrotron Light Source Australia Pty LTD), 800 Blackburn Rd, Clayton, VIC 3168, Australia. *email: Scientific Reports | (2020) 10:20891 | https://doi.org/10.1038/s41598-020-78078-8 1 Vol.:(0123456789) www.nature.com/scientificreports/ a key paradigm for ensuring p ublic15,20–24, hence the diagnosis of this disease at different levels of development is a priority scientific direction in therapeutic dentistry in the developed countries. Since the condition of human teeth is determined by changes occurring in the phase composition of the tissue at the micro and nano level, the use of spectroscopic methods of molecular identification is the most promising and sensitive tool to precisely assess such changes in the dental e namel24–26. Raman spectroscopy is most often used for diagnostics of dental diseases as a non-destructive method of analysis, which allows to obtain direct data regarding the local atomic structure, chemical and molecular composition of biological o bjects18,20,24,27–29. Also, the hard tissue of a human tooth can be investigated by infrared spectroscopy methods30–34, which due to minimal external influences, has not been modified by the m ethod35. At the same time using the IR-microspectroscopy with synchrotron radiation as a source allows one to analyze biological specimens (human tooth tissues) and pathology processes in them with a greater lateral resolution during data acquisition and high signal-to-noise ratio without the long-term accumulation of the desired signal that is important for the study of biological specimens36–38. The use of infrared microspectroscopy with synchrotron radiation makes it possible to clarify the molecular composition and study local changes in the mineral-organic matrix of dental enamel from the areas less than 10 µm239. Moreover, in any case the applicability of synchrotron IR-microspectroscopy to the dental tissue analysis is more convenient than Raman microspectroscopy, even though the Raman microscopy is also an excellent approach to vibrational spectroscopy, with an equivalent, or often a better, spatial resolution than synchrotron infrared m icroscopy31,33,37,40. It should be noted that the correlation between the data obtained by Raman methods and infrared (IR) microspectroscopy for the samples of dental hard tissue affected by fluorosis, taking into account the thickness of the enamel, was not made. However, separate studies of the molecular structure of teeth with fluorosis by methods of optical microspectroscopy have b (...truncated)