Rapid Deposition of the Biomimetic Hydroxyapatite-Polydopamine-Amino Acid Composite Layers onto the Natural Enamel.

In this work, we developed a technology that enables rapid deposition of biomimetic composite films onto natural enamel slices (known as biotemplates). These films are composed of polydopamine (PDA) and nanocrystalline carbonate-substituted hydroxyapatite (nano-cHAp) that have been functionalized with amino acid l-Arginine. We utilized atomic force microscopy (AFM) and scattering scanning near-field optical microscopy (s-SNOM) combined with infrared (IR) synchrotron to achieve nanoscale spatial resolution for both IR absorption and topography analyses. This combined analytical modality allowed us to understand how morphology connects to local changes in the chemical environment on the biotemplate surface during the deposition of the bioinspired coating. Our findings revealed that when using the proposed technology and after the deposition of the first PDA layer, the film formed on the enamel surface nearly covers the entire surface of the specimen whose thickness is larger on the surface of the emerging enamel prisms. Calculation of the crystallinity index for the biomimetic layer showed a multiple increase compared with natural enamel. This indicates regular and dense aggregation of nano-cHAp into larger crystals, imitating the morphology of natural enamel rods. The microhardness of the formed PDA-based biomimetic layer mineralized with nano-cHAp functionalized with amino acid l-Arginine deposited on natural enamel was practically the same as that of natural enamel. The characterization of nano-cHAp-amino acid-PDA layers using IR and Raman microspectroscopy showed that l-arginine acts as a conjunction agent in the formation of mineralized biomimetic composite coatings. The uniformity of the mechanisms of PDA layer formation under different deposition conditions and substrate types allows for the formation of coatings regardless of the macro- and micromorphology of the template. Therefore, the results obtained in this work have a high potential for future clinical applications in dental practice.

Changes in Dental Biofilm Proteins' Secondary Structure in Groups of People with Different Cariogenic Situations in the Oral Cavity and Using Medications by Means of Synchrotron FTIR-Microspectroscopy.

This work unveils the idea that the cariogenic status of the oral cavity (the presence of active caries lesions) can be predicted via a lineshape analysis of the infrared spectral signatures of the secondary structure of proteins in dental biofilms. These spectral signatures that work as natural markers also show strong sensitivity to the application in patients of a so-called modulator-a medicinal agent (a pelleted mineral complex with calcium glycerophosphate). For the first time, according to our knowledge, in terms of deconvolution of the complete spectral profile of the amide I and amide II bands, significant intra- and intergroup differences were determined in the secondary structure of proteins in the dental biofilm of patients with a healthy oral cavity and with a carious pathology. This allowed to conduct a mathematical assessment of the spectral shifts in proteins' secondary structure in connection with the cariogenic situation in the oral cavity and with an external modulation. It was shown that only for the component parallel ß-strands in the amide profile of the biofilm, a statistically significant (p < 0.05) change in its percentage weight (composition) was registered in a cariogenic situation (presence of active caries lesions). Note that no significant differences were detected in a normal situation (control) and in the presence of a carious pathology before and after the application of the modulator. The change in the frequency and percentage weight of parallel ß-strands in the spectra of dental biofilms proved to be the result of the presence of cariogenic mutans streptococci in the film as well as of the products of their metabolism-glucan polymers. We foresee that the results presented here can inherently provide the basis for the infrared spectral diagnosis of changes (shifts) in the oral microbiome driven by the development of the carious process in the oral cavity as well as for the choice of optimal therapeutic treatments of caries based on microbiome-directed prevention measures.

A Study of the Peculiarities of the Formation of a Hybrid Interface Based on Polydopamine between Dental Tissues and Dental Composites, Using IR and Raman Microspectroscopy, at the Submicron Level.

The creation of buffer (hybrid) layers that provide improved adhesion to two heterogeneous materials is a promising and high-priority research area in the field of dental materials science. In our work, using FTIR and Raman microspectroscopy at the submicron level in a system of dental composites/intact dental enamel, we assessed the molecular features of formation and chemically visualized the hybrid interface formed on the basis of a nature-like adhesive, polydopamine (PDA). It is shown that a homogeneous bioinspired PDA-hybrid interface with an increased content of O-Ca-O bonds can be created using traditional methods of dental tissue pretreatment (diamond micro drilling, acid etching), as well as the subsequent alkalinization procedure and the developed synthesis technology. The development of the proposed technology for accelerated deposition of PDA-hybrid layers, as well as the creation of self-assembled biomimetic nanocomposites with antibacterial properties, may in the future find clinical application for minimally invasive dental restoration procedures.

Compositional Analysis of the Dental Biomimetic Hybrid Nanomaterials Based on Bioinspired Nonstoichiometric Hydroxyapatite with Small Deviations in the Carbonate Incorporation.

In our paper, we discuss the results of a comprehensive structural-spectroscopic and microscopic analysis of non-stoichiometric nanocrystalline hydroxyapatite (CHAp) with low carbonate anion content and biomimetic hybrid nanomaterials produced on its basis. It was shown that hydroxyapatite nanocrystals synthesized by chemical precipitation and biogenic calcium source mimic the properties of biogenic apatite and also have a morphological organization of "core-shell" type. The "core" of the CHAp nanocrystal is characterized by an overabundance of calcium Ca/P~1.9. Thus "a shell" with thickness of ~3-5 nm is formed from intermediate apatite-like phases where the most probable are octocalcium phosphate, dicalcium phosphate dihydrate and tricalcium phosphate. The multimode model of the Raman profile of samples CHAp and biomimetic composites for spectral region 900-1100 cm-1 proposed in our work has allowed to allocate precise contribution of B-type carbonate substitution, taking into account the presence on a surface of "core" HAp nanocrystal of various third-party intermediate apatite-like phases. The calibration function constructed on the basis of the described model makes it possible to reliably determine small concentrations of carbonate in the structure of hydroxyapatite with the application of Raman express method of diagnostics. The results of our work can inspire researchers to study the processes of induced biomineralization in mineralized tissues of the human body, using non-destructive methods of control with simultaneous analysis of chemical bonding, as well as determining the role of impurity atoms in the functions exhibited by biotissue.

Development of a Hybrid Biomimetic Enamel-Biocomposite Interface and a Study of Its Molecular Features Using Synchrotron Submicron ATR-FTIR Microspectroscopy and Multivariate Analysis Techniques.

Using a biomimetic strategy and bioinspired materials, our work proposed a new technological approach to create a hybrid transitional layer between enamel and dental biocomposite. For this purpose, an amino acid booster conditioner based on a set of polar amino acids (lysine, arginine, hyaluronic acid), calcium alkali, and a modified adhesive based on BisGMA and nanocrystalline carbonate-substituted hydroxyapatite are used during dental enamel restoration. The molecular properties of the hybrid interface formed using the proposed strategy were understood using methods of multivariate statistical analysis of spectral information collected using the technique of synchrotron infrared microspectroscopy. The results obtained indicate the possibility of forming a bonding that mimics the properties of natural tissue with controlled molecular properties in the hybrid layer. The diffusion of the amino acid booster conditioner component, the calcium alkali, and the modified adhesive with nanocrystalline carbonate-substituted hydroxyapatite in the hybrid interface region creates a structure that should stabilize the reconstituted crystalline enamel layer. The developed technology can form the basis for an individualized, personalized approach to dental enamel restorations.

Biomimetic Mineralization of Tooth Enamel Using Nanocrystalline Hydroxyapatite under Various Dental Surface Pretreatment Conditions.

In this report, we demonstrated the formation of a biomimetic mineralizing layer obtained on the surface of dental enamel (biotemplate) using bioinspired nanocrystalline carbonate-substituted calcium hydroxyapatite (ncHAp), whose physical and chemical properties are closest to the natural apatite dental matrix, together with a complex of polyfunctional organic and polar amino acids. Using a set of structural, spectroscopy, and advanced microscopy techniques, we confirmed the formation of a nanosized ncHAp-based mineralized layer, as well as studying its chemical, substructural, and morphological features by means of various methods for the pretreatment of dental enamel. The pretreatment of a biotemplate in an alkaline solution of Ca(OH)2 and an amino acid booster, together with the executed subsequent mineralization with ncHAp, led to the formation of a mineralized layer with homogeneous micromorphology and the preferential orientation of the ncHAp nanocrystals. It was shown that the homogeneous crystallization of hydroxyapatite on the biotemplate surface and binding of individual nanocrystals and agglomerates into a single complex by an amino acid booster resulted in an increase (~15%) in the nanohardness value in the enamel rods area, compared to that of healthy natural enamel. Obtaining a similar hierarchy and cleavage characteristics as natural enamel in the mineralized layer, taking into account the micromorphological features of dental tissue, is an urgent problem for future research.

Effect of Exo/Endogenous Prophylaxis Dentifrice/Drug and Cariogenic Conditions of Patient on Molecular Property of Dental Biofilm: Synchrotron FTIR Spectroscopic Study.

(1) Objectives: This study is the first one to investigate the molecular composition of the dental biofilm during the exogenous and endogenous prophylaxis stages (use of dentifrice/drug) of individuals with different cariogenic conditions using molecular spectroscopy methods. (2) Materials and Methods: The study involved 100 participants (50 males and 50 females), aged 18-25 years with different caries conditions. Biofilm samples were collected from the teeth surface of all participants. The molecular composition of biofilms was investigated using synchrotron infrared microspectroscopy. Changes in the molecular composition were studied through calculation and analysis of ratios between organic and mineral components of biofilm samples. (3) Results: Based on the data obtained by synchrotron FTIR, calculations of organic and mineral component ratios, and statistical analysis of the data, we were able to assess changes occurring in the molecular composition of the dental biofilm. Variations in the phosphate/protein/lipid, phosphate/mineral, and phospholipid/lipid ratios and the presence of statistically significant intra- and inter-group differences in these ratios indicate that the mechanisms of ion adsorption, compounds and complexes arriving from oral fluid into dental biofilm during exo/endogenous prophylaxis, differ for patients in norm and caries development. (4) Conclusions: The conformational environment and charge interaction in the microbiota and the electrostatic state of the biofilm protein network in patients with different cariogenic conditions play an important role. (5) Clinical Significance: Understanding the changes that occur in the molecular composition of the dental biofilm in different oral homeostasis conditions will enable successful transition to a personalised approach in dentistry and high-tech healthcare.

Development of a Visualisation Approach for Analysing Incipient and Clinically Unrecorded Enamel Fissure Caries Using Laser-Induced Contrast Imaging, MicroRaman Spectroscopy and Biomimetic Composites: A Pilot Study.

This pilot study presents a practical approach to detecting and visualising the initial forms of caries that are not clinically registered. The use of a laser-induced contrast visualisation (LICV) technique was shown to provide detection of the originating caries based on the separation of emissions from sound tissue, areas with destroyed tissue and regions of bacterial invasion. Adding microRaman spectroscopy to the measuring system enables reliable detection of the transformation of the organic-mineral component in the dental tissue and the spread of bacterial microflora in the affected region. Further laboratory and clinical studies of the comprehensive use of LICV and microRaman spectroscopy enable data extension on the application of this approach for accurate determination of the boundaries in the changed dental tissue as a result of initial caries. The obtained data has the potential to develop an effective preventive medical diagnostic approach and as a result, further personalised medical treatment can be specified.

The Molecular and Mechanical Characteristics of Biomimetic Composite Dental Materials Composed of Nanocrystalline Hydroxyapatite and Light-Cured Adhesive.

The application of biomimetic strategies and nanotechnologies (nanodentology) has led to numerous innovations and provided a considerable impetus by creating a new class of modern adhesion restoration materials, including different nanofillers. An analysis of the molecular properties of biomimetic adhesives was performed in this work to find the optimal composition that provides high polymerisation and mechanical hardness. Nanocrystalline carbonate-substituted calcium hydroxyapatite (nano-cHAp) was used as the filler of the light-cured adhesive Bis-GMA (bisphenol A-glycidyl methacrylate). The characteristics of this substance correspond to the apatite of human enamel and dentin, as well as to the biogenic source of calcium: avian eggshells. The introduction and distribution of nano-cHAp fillers in the adhesive matrix resulted in changes in chemical bonding, which were observed using Fourier transform infrared (FTIR) spectroscopy. As a result of the chemical bonding, the Vickers hardness (VH) and the degree of conversion under photopolymerisation of the nano-cHAp/Bis-GMA adhesive increased for the specified concentration of nanofiller. This result could contribute to the application of the developed biomimetic adhesives and the clinical success of restorations.

Investigation of the Effect of Nanocrystalline Calcium Carbonate-Substituted Hydroxyapatite and L-Lysine and L-Arginine Surface Interactions on the Molecular Properties of Dental Biomimetic Composites.

Differences in the surface interactions of non-stoichiometric nanocrystalline B-type carbonate-substituted hydroxyapatite (n-cHAp) with the amino acids L-Lysine hydrochloride (L-LysHCl) and L-Arginine hydrochloride (L-ArgHCl) in acidic and alkaline media were determined using structural and spectroscopic analysis methods. The obtained data confirm that hydroxyapatite synthesized using our technique, which was used to develop the n-cHAp/L-LysHCl and n-cHAp/L-ArgHCl composites, is nanocrystalline. Studies of molecular composition of the samples by Fourier transform infrared spectroscopy under the change in the charge state of L-Lysine in environments with different alkalinity are consistent with the results of X-ray diffraction analysis, as evidenced by the redistribution of the modes' intensities in the spectra that is correlated with the side chains, i.e., amide and carboxyl groups, of the amino acid. During the formation of a biomimetic composite containing L-Lysine hydrochloride and n-cHAp, the interaction occurred through bonding of the L-Lysine side chain and the hydroxyl groups of hydroxyapatite, which created an anionic form of L-Lysine at pH ≤ 5. In contrast, in biocomposites based on L-Arginine and n-cHAp, the interaction only slightly depends on pH value, and it proceeds by molecular orientation mechanisms. The X-ray diffraction and infrared spectroscopy results confirm that changes in the molecular composition of n-cHAp/L-ArgHCl biomimetic composites are caused by the electrostatic interaction between the L-ArgHCl molecule and the carbonate-substituted calcium hydroxyapatite. In this case, the bond formation was detected by Fourier transform infrared (FTIR) spectroscopy; the vibrational modes attributed to the main carbon chain and the guanidine group of L-Arginine are shifted during the interaction. The discovered interaction mechanisms between nanocrystalline carbonate-substituted hydroxyapatite that has physicochemical properties characteristic of the apatite in human dental enamel and specific amino acids are important for selecting the formation conditions of biomimetic composites and their integration with the natural dental tissue.

Raman and XANES Spectroscopic Study of the Influence of Coordination Atomic and Molecular Environments in Biomimetic Composite Materials Integrated with Dental Tissue.

In this work, for the first time, the influence of the coordination environment as well as Ca and P atomic states on biomimetic composites integrated with dental tissue was investigated. Bioinspired dental composites were synthesised based on nanocrystalline calcium carbonate-substituted hydroxyapatite Ca4ICa6IIPO46-xCO3x+yOH2-y (nano-cHAp) obtained from a biogenic source and a set of polar amino acids that modelled the organic matrix. Biomimetic composites, as well as natural dental tissue samples, were investigated using Raman spectromicroscopy and synchrotron X-ray absorption near edge structure (XANES) spectroscopy. Molecular structure and energy structure studies revealed several important features related to the different calcium atomic environments. It was shown that biomimetic composites created in order to reproduce the physicochemical properties of dental tissue provide good imitation of molecular and electron energetic properties, including the carbonate anion CO32- and the atomic Ca/P ratio in nanocrystals. The features of the molecular structure of biomimetic composites are inherited from the nano-cHAp (to a greater extent) and the amino acid cocktail used for their creation, and are caused by the ratio between the mineral and organic components, which is similar to the composition of natural enamel and dentine. In this case, violation of the nano-cHAp stoichiometry, which is the mineral basis of the natural and bioinspired composites, as well as the inclusion of different molecular groups in the nano-cHAp lattice, do not affect the coordination environment of phosphorus atoms. The differences observed in the molecular and electron energetic structures of the natural enamel and dentine and the imitation of their properties by biomimetic materials are caused by rearrangement in the local environment of the calcium atoms in the HAp crystal lattice. The surface of the nano-cHAp crystals in the natural enamel and dentine involved in the formation of bonds with the organic matrix is characterised by the coordination environment of the calcium atom, corresponding to its location in the CaI position-that is, bound through common oxygen atoms with PO4 tetrahedrons. At the same time, on the surface of nano-cHAp crystals in bioinspired dental materials, the calcium atom is characteristically located in the CaII position, bound to the hydroxyl OH group. The features detected in the atomic and molecular coordination environment in nano-cHAp play a fundamental role in recreating a biomimetic dental composite of the natural organomineral interaction in mineralised tissue and will help to find an optimal way to integrate the dental biocomposite with natural tissue.

To the Question on the Use of Multivariate Analysis and 2D Visualisation of Synchrotron ATR-FTIR Chemical Imaging Spectral Data in the Diagnostics of Biomimetic Sound Dentin/Dental Composite Interface.

In this short communication, we provide information on the use of the hierarchical cluster analysis of synchrotron ATR-FTIR 2D chemical imaging spectral data as a useful and powerful approach to the microspectroscopic diagnostics of molecular composition in the hybrid sound dentin/dental composite interfaces and materials, including ones developed with the use of biomimetic strategies. The described diagnostic approach can be successfully transferred to the analysis and visualisation of 2D spectral data, collected using laboratory Raman and FTIR microspectroscopy techniques.

Engineering of a Biomimetic Interface between a Native Dental Tissue and Restorative Composite and Its Study Using Synchrotron FTIR Microscopic Mapping.

The aim of this work is to develop a biomimetic interface between the natural tooth tissue and the restorative composite and to study it on the basis of synchrotron micro-FTIR mapping and multidimensional processing of the spectral data array. Using hierarchical cluster analysis of 3D FTIR data revealed marked improvements in the formation of the dentine/adhesive/dental hybrid interface using a biomimetic approach. The use of a biomimetic strategy (application of an amino acid-modified primer, alkaline calcium and a nano-c-HAp-modified adhesive) allowed the formation of a matrix that can be structurally integrated with natural dentine and dental composite. The biomimetic hybrid layer was characterised by homogeneous chemical composition and a higher degree of conversion of the adhesive during polymerisation, which should provide optimal integration of the dental composite with the dentine.

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

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.

Spectroscopic signature of the pathological processes of carious dentine based on FTIR investigations of the oral biological fluids.

The aim of our work is to find a spectroscopic signature of the pathological processes of carious dentine based on the investigations of the molecular composition of the oral biological fluids with the use of FTIR synchrotron techniques. This complex analysis of the obtained data shows that a number of signatures are present only in the spectra of dentine and gingival fluids from the patients developing caries of the deep dentine tissues. The detected features and complex analysis of the quantitative and qualitative data representing signatures of the development of oral cavity pathologies can enhance the quality of dental screening.

Pathology-specific molecular profiles of saliva in patients with multiple dental caries-potential application for predictive, preventive and personalised medical services.

BACKGROUND: Improving the quality of life is part of the global agenda. The focus is predominantly on prevention of socially significant diseases. Combating dental caries-related diseases is a top priority as it has a huge impact on people's social lives. Therefore, the purpose of the work was to study the changes in the molecular composition of saliva from subjects with multiple caries lesions using spectroscopic methods of analysis to identify potential tissue markers of caries development for predictive, preventive and personalised medical services. OBJECTIVES AND METHODS: The molecular composition of mixed saliva (oral fluid) from subjects with and without multiple caries was analysed with the use of spectroscopic techniques, FTIR with synchrotron radiation for the excitation. The IR spectra of the oral fluid as well as the calculated mineral-organic, carbon-phosphate, Amide II/Amide I and protein/thiocyanate ratios were compared between subjects with and without multiple caries. RESULTS: This complex analysis of the obtained experimental data determined that the molecular composition of the oral fluid from those with multiple caries differed from those without caries; the organic-mineral balance in the oral fluid of those with multiple caries shifted towards a reduction in the mineral complexes, accompanied by an increase in the organic component. The thiocyanate content increased more than twofold, accompanied by increased carboxyl groups of esters, lipids and carbohydrates. CONCLUSION: The detected features in the IR spectra of mixed saliva as well as the calculated changes in the ratios between organic and inorganic components can be used as biomarkers of cariogenesis in the oral cavity, as a diagnostic criterion in the analysis of the oral fluid samples.

Phase transformations in a human tooth tissue at the initial stage of caries.

The aim of the paper is to study phase transformations in solid tissues of the human teeth during the development of fissure caries by Raman and fluorescence microspectroscopy. The study of the areas with fissure caries confirmed the assumption of the formation of a weak interaction between phosphate apatite enamel and organic acids (products of microorganisms). The experimental results obtained with by Raman microspectroscopy showed the formation of dicalcium phosphate dihydrate - CaHPO4-2H2O in the area of mural demineralization of carious fissure. A comparative analysis of structural and spectroscopic data for the intact and carious enamel shows that emergence of a more soluble phase - carbonate-substituted hydroxyapatite - is typical for the initial stage of caries. It is shown that microareas of dental hard tissues in the carious fissure due to an emerging misorientation of apatite crystals have a higher fluorescence yield than the area of the intact enamel. These areas can be easily detected even prior to a deep demineralization (white spot stage) for the case of irreversibly changed organomineral complex and intensive removal of the mineral component.