Relationship between Changes in Chemical Composition of Enamel Subsurface Lesions and the Emitted Nonlinear Optical Signals: An in vitro Study.

The development of new diagnostic technologies based on the light scattering and autofluorescence properties of dental tissues is required to improve the diagnostic ability of initial caries lesions earlier than previously done and promoting the potential of treatment without surgical intervention. The aim of this study is to correlate fluorescence-based results provided by multiphoton microscopy (MPM) with confocal Raman microscopy records using phosphate level at 960 cm-1 and the organic matrix at ∼2,931 cm-1 in healthy and demineralized human enamel. Measurements on 14 teeth were made using two incident lights of different wavelengths, released by confocal Raman microscopy and MPM. Raman phosphate peak intensity at 960 cm-1 along with organic to mineral ratio at (2,931/430 cm-1) and nonlinear optical signals (second harmonic generation [SHG] and intrinsic two-photon excited fluorescence [I2PEF]) were recorded from the demineralized and healthy enamel sites. Raman spectral maps showed that the higher the organic/mineral ratio in the demineralized enamel, the lower the intensity of mineral component in the same zone. MPM revealed new optical indicators of carious lesion as shown by the presence of a red-shifted fluorescence peak in the 650- to 750-nm area of the fluorescence spectrum of demineralized enamel. Moreover, on sample regions with insignificant autofluorescence, the emergence of the SHG signal could be noted. By comparing I2PEF images with the structural motifs observed by the confocal Raman imaging system, the morphological similarity of the acquired images was quite evident. Any change in the I2PEF spectra reflects alterations in the chemical composition of enamel. These findings may provide an important basis for potentially valuable applications of photonic tools in the clinical diagnosis of tooth pathological conditions, besides exposing the fundamental role of organic matrix in enamel integrity and reparation.

In vitro investigation of fluorescence of carious dentin observed with a Soprolife® camera.

OBJECTIVES: Our aim was to determine the origin of the red fluorescence of carious dentine observed with the Soprolife® camera. METHODS: We conducted in vitro studies to evaluate the origin of the red fluorescence using acids and matrix metalloproteinase (MMP) to mimic caries and methylglycoxal (MGO) to evaluate the effect of glycation reactions on the red fluorescence. In every step of these models, we detected the changes of dentin photonic response with Soprolife® in daylight mode and in treatment mode. A Raman spectroscopy analysis was performed to determine the variations of the dentin organic during the in vitro caries processes. Raman microscopy was performed to identify change in the collagen matrix of dentine. RESULTS: The red fluorescence observed in carious dentine using a Soprolife® camera corresponds to the brownish color observed using daylight. Demineralization using nitric acid induces a loss of the green fluorescence of dentine. The red fluorescence of carious dentine is resistant to acid treatment. Immersion of demineralized dentine in MGO induces a change of color from white to orange-red. This indicates that the Maillard reaction contributes to lesion coloration. Immersion of demineralized dentine in an MMP-1 solution followed by MGO treatment results in a similar red fluorescence. Raman microspectroscopy analysis reveals accumulation of AGE's product in red-colored dentine. CONCLUSIONS: Our results provide important information on the origin of the fluorescence variation of dentine observed with the Soprolife® camera. We demonstrate that the red fluorescence of carious dentine is linked to the accumulation of Advanced Glycation End products (AGE). CLINICAL RELEVANCE: The study provides a new biological basis for the red fluorescence of carious dentine and reinforces the importance of the Soprolife® camera in caries diagnostics.

Properties of dentin, enamel and their junction, studied with Brillouin scattering and compared to Raman microscopy.

OBJECTIVE: Dentin, enamel and the transition zone, called the dentin-enamel junction (DEJ), have an organization and properties that play a critical role in tooth resilience and in stopping the propagation of cracks. Understanding their chemical and micro-biomechanical properties is then of foremost importance. The aim of this study is to apply Brillouin microscopy on a complex biological structure, that is, the DEJ, and to compare these results with those obtained with Raman microscopy. DESIGN: Both techniques allow noncontact measurements at the microscopic scale. Brillouin microscopy is based on the interaction between acoustic phonons and laser photons and gives a relation between the frequency shift of the scattered light and the stiffness of the sample. Raman spectra contain peaks related to specific chemical bonds. RESULTS: Comparison of the Brillouin and Raman cartographies reveals correlations between mechanical and chemical properties. Indeed, the shapes of the phosphate content and stiffness curves are similar. The two spectroscopies give compatible values for the mean distance between two tubules, i.e., 4-6 µm. Moreover, for the first time, the daily cross striations of enamel could be studied, indicating a relationship between the variation in the phosphate concentration and the variation in the rigidity within the enamel prisms. CONCLUSIONS: We demonstrate here the possibility of using Brillouin scattering microscopy to both study complex biological materials such as the enamel-dentin junction and visualize secondary structures. Correlations between the chemical composition and mechanical properties could help in better understanding the tissue histology.

Nanostructured Porous Silicon for Bone Tissue Engineering: Kinetics of Particle Degradation and Si-Controlled Release.

Nanostructured porous silicon (pSi) is a synthetic silicon-based material. Its biocompatibility and bioresorbability in body fluids make pSi an appealing biomaterial for tissue engineering, with surfaces characteristics facilitating human cell adhesion and differentiation. The resorption kinetics of such porous biomaterials is crucial for in vivo bone regeneration, in order to adapt biomaterial resorption to tissue formation, and to control the release of loaded bioactive molecules. We investigated pSi as a bioactive scaffold for bone tissue engineering, with an emphasis on kinetics of pSi resorption and silicon release. PSi particles and chips were fabricated from crystalline silicon, and functionalized by oxidation and chemical grafting of amine groups to mimic biological structures. Materials resorption over time was investigated with Raman spectroscopy, infrared spectroscopy, and Scanning Electron Microscopy. Silicon release was followed by mass spectrometry. Particle degradation and inclusion in newly formed bone were studied in vivo. The in vitro experiments revealed that non-oxidized pSi had an accelerated initial dissolution in ddH2O and an inhibition of initial Si release in SBF. This high reactivity also led to transformation towards amorphous non-resorbable silica when incubated in SBF. PSi resorption started immediately with a maximal dissolution in the first 24 h. Later, the dissolution rate decreased over time. In comparison, the resorption process of oxidized pSi seemed delayed, but more continuous. This delayed dissolution increased the bioactivity and stability, leading to enhanced bone formation in vivo. Delayed pSi degradation provided a constant surge of silicic acid over time and promoted bone regeneration, demonstrating the high potential of pSi for bone tissue engineering: Oxidized pSi were almost completely resorbed after 2 months of healing, with remaining partially dissolved particles surrounded by newly formed bone. On the contrary, non-oxidized particles were still obviously present after 2 months with limited bone regeneration. This delayed resorption is consistent with the in vitro observations in SBF, and particles' transformation towards silica.

Molecular structural analysis of carious lesions using micro-Raman spectroscopy.

In clinical situations carious dentine tissues can be discriminated by most caries fluorescence detection tools, including a new fluorescence intra-oral camera. The objectives of this study were: (i) to analyze the Raman spectra of sound, carious, and demineralized dentine, (ii) to compare this spectral analysis with the fluorescence variation observed when using a fluorescence camera, and (iii) to evaluate the involvement of the Maillard reaction in the fluorescence variations. The first positive hypothesis tested was that the fluorescence of carious dentine obtained using a fluorescence camera and the Raman spectra variation were closely related. The second was that the variation of fluorescence could be linked with the Maillard reaction. Sound dentine, sound dentine demineralized in aqueous nitric acid solution, carious soft dentine, sound dentine demineralized in lactic acid solution, sound dentine demineralized in aqueous nitric acid solution and immersed in methylglycoxal solution, and sound dentine demineralized in aqueous nitric acid solution and immersed in methylglycoxal and glucose solutions, were studied using micro-Raman spectroscopy. Modifications in the band ratio of amide, phosphate, and carbonate were observed in the decayed and demineralized groups compared with the sound dentine group. The results indicate that a close relationship exists between the Maillard reaction and fluorescence variation.

Confocal Raman microscopy and SEM/EDS investigations of the interface between the zirconia core and veneering ceramic: the influence of a liner and regeneration firing.

The aim of this study was to evaluate the changes in the transition layer at the interface between yttria partial stabilized tetragonal zirconia polycrystalline (Y-TZP) core and veneering feldspathic ceramic (VITA VM(®)9), under different manufacturing methods. Confocal Raman microscopy and energy dispersive X-ray spectroscopy (EDS) analyses were carried out on tapered veneered cross sections of the interface. For some samples, an additional firing of the core was used, as the application of an optional liner material between the core and veneer. Single Raman spectra were distinguishable between Y-TZP and the veneering materials. VM(®)9 and liner spectra were broadly superimposable. No substantial differences appeared in their chemical elemental composition. 2D Raman images and EDS analysis emphasized changes in the interdiffusion thickness; the additional firing of the core decreased the interdiffusion zone, and the highest firing temperature of the liner increased the interdiffusion zone. These results, which will help us understand the changes in this transition layer, are discussed.

Specific intracellular signature of SARS-CoV-2 infection using confocal Raman microscopy.

SARS-CoV-2 infection remains spread worldwide and requires a better understanding of virus-host interactions. Here, we analyzed biochemical modifications due to SARS-CoV-2 infection in cells by confocal Raman microscopy. Obtained results were compared with the infection with another RNA virus, the measles virus. Our results have demonstrated a virus-specific Raman molecular signature, reflecting intracellular modification during each infection. Advanced data analysis has been used to distinguish non-infected versus infected cells for two RNA viruses. Further, classification between non-infected and SARS-CoV-2 and measles virus-infected cells yielded an accuracy of 98.9 and 97.2 respectively, with a significant increase of the essential amino-acid tryptophan in SARS-CoV-2-infected cells. These results present proof of concept for the application of Raman spectroscopy to study virus-host interaction and to identify factors that contribute to the efficient SARS-CoV-2 infection and may thus provide novel insights on viral pathogenesis, targets of therapeutic intervention and development of new COVID-19 biomarkers.

Glucose Starvation or Pyruvate Dehydrogenase Activation Induce a Broad, ERK5-Mediated, Metabolic Remodeling Leading to Fatty Acid Oxidation.

Cells have metabolic flexibility that allows them to adapt to changes in substrate availability. Two highly relevant metabolites are glucose and fatty acids (FA), and hence, glycolysis and fatty acid oxidation (FAO) are key metabolic pathways leading to energy production. Both pathways affect each other, and in the absence of one substrate, metabolic flexibility allows cells to maintain sufficient energy production. Here, we show that glucose starvation or sustained pyruvate dehydrogenase (PDH) activation by dichloroacetate (DCA) induce large genetic remodeling to propel FAO. The extracellular signal-regulated kinase 5 (ERK5) is a key effector of this multistep metabolic remodeling. First, there is an increase in the lipid transport by expression of low-density lipoprotein receptor-related proteins (LRP), e.g., CD36, LRP1 and others. Second, an increase in the expression of members of the acyl-CoA synthetase long-chain (ACSL) family activates FA. Finally, the expression of the enzymes that catalyze the initial step in each cycle of FAO, i.e., the acyl-CoA dehydrogenases (ACADs), is induced. All of these pathways lead to enhanced cellular FAO. In summary, we show here that different families of enzymes, which are essential to perform FAO, are regulated by the signaling pathway, i.e., MEK5/ERK5, which transduces changes from the environment to genetic adaptations.

Adsorption of proteins on TiO2 particles influences their aggregation and cell penetration.

TiO2 nanoparticles known as E171 are one controversial food additive due to its potential toxicity. In this work, the main hypothesis is that the proteins adsorbed on the TiO2 nanoparticles prevent their aggregation and favor the cell penetration. To do so, the TiO2 nanoparticles were coated with gelatin and ß-lactoglobulin to reach interfacial concentrations about 0.25 mg/mg and 0.32 mg/mg, respectively. The measurement of NP size showed that the protein coating improve the colloidal stability of TiO2 nanoparticles. The FTIR analysis suggests that the ß-lactoglobulin structure is modified after adsorption. The penetration of TiO2 penetration inside human intestinal epithelial cells was shown and quantify by using confocal Raman microscopy. The promoting role of the protein coating on the cell penetration was demonstrated for both the gelatin and ß-lactoglobulin. Finally, the results allow establishing a correlation between the ability of proteins to prevent NP aggregation and the cell penetration.

Confocal Raman data analysis of tufts and spindles at the human dentin-enamel junction.

OBJECTIVE: The aim of this article is to analyze the chemical mapping of tufts and spindles of the human dental enamel using confocal Raman microscopy measuring length, structuration and composition of spindles and tufts. DESIGN: we used Raman diffusion, based on the interaction between photons and optic phonons, to reveal chemical bound. Adult molars were selected and longitudinally sectioned. Areas of 120 * 120 µm were scanned near the dentin-enamel junction and grooves. Spectra were collected and phosphate and proteins peak intensities images were reconstructed, related to HPA concentration. Images of Phosphate (PO43-, 960 cm-1) and protein (CH, 2800/3000 cm-1) intensities have been reconstructed. K-mean cluster has been calculated to compare centroid spectra from enamel, dentin and tuft or spindle. RESULTS: intensity profile revealed spindles as less mineralized areas than enamel, from 5 to 10 µm large. In the groove of molar, long tufts were found, more than 150 µm. CONCLUSIONS: Confocal Raman microscopy is a very interesting tool to characterize chemically secondary structure of enamel. The size of a tuft in the groove allows us make the hypothesis that they could play a role in long term resilience of mechanical stress.

Assessing Cobalt Metal Nanoparticles Uptake by Cancer Cells Using Live Raman Spectroscopy.

PURPOSE: Nanotechnology applied to cancer treatment is a growing area of research in nanomedicine with magnetic nanoparticle-mediated anti-cancer drug delivery systems offering least possible side effects. To that end, both structural and chemical properties of commercial cobalt metal nanoparticles were studied using label-free confocal Raman spectroscopy. MATERIALS AND METHODS: Crystal structure and morphology of cobalt nanoparticles were studied by XRD and TEM. Magnetic properties were studied with SQUID and PPMS. Confocal Raman microscopy has high spatial resolution and compositional sensitivity. It, therefore, serves as a label-free tool to trace nanoparticles within cells and investigate the interaction between coating-free cobalt metal nanoparticles and cancer cells. The toxicity of cobalt nanoparticles against human cells was assessed by MTT assay. RESULTS: Superparamagnetic Co metal nanoparticle uptake by MCF7 and HCT116 cancer cells and DPSC mesenchymal stem cells was investigated by confocal Raman microscopy. The Raman nanoparticle signature also allowed accurate detection of the nanoparticle within the cell without labelling. A rapid uptake of the cobalt nanoparticles followed by rapid apoptosis was observed. Their low cytotoxicity, assessed by means of MTT assay against human embryonic kidney (HEK) cells, makes them promising candidates for the development of targeted therapies. Moreover, under a laser irradiation of 20mW with a wavelength of 532nm, it is possible to bring about local heating leading to combustion of the cobalt metal nanoparticles within cells, whereupon opening new routes for cancer phototherapy. CONCLUSION: Label-free confocal Raman spectroscopy enables accurately localizing the Co metal nanoparticles in cellular environments. The interaction between the surfactant-free cobalt metal nanoparticles and cancer cells was investigated. The facile endocytosis in cancer cells shows that these nanoparticles have potential in engendering their apoptosis. This preliminary study demonstrates the feasibility and relevance of cobalt nanomaterials for applications in nanomedicine such as phototherapy, hyperthermia or stem cell delivery.

Simultaneous label-free live imaging of cell nucleus and luminescent nanodiamonds.

In recent years, fluorescent nanodiamond (fND) particles containing nitrogen-vacancy (NV) centers gained recognition as an attractive probe for nanoscale cellular imaging and quantum sensing. For these applications, precise localization of fNDs inside of a living cell is essential. Here we propose such a method by simultaneous detection of the signal from the NV centers and the spectroscopic Raman signal from the cells to visualize the nucleus of living cells. However, we show that the commonly used Raman cell signal from the fingerprint region is not suitable for organelle imaging in this case. Therefore, we develop a method for nucleus visualization exploiting the region-specific shape of C-H stretching mode and further use k-means cluster analysis to chemically distinguish the vicinity of fNDs. Our technique enables, within a single scan, to detect fNDs, distinguish by chemical localization whether they have been internalized into cell and simultaneously visualize cell nucleus without any labeling or cell-fixation. We show for the first time spectral colocalization of unmodified high-pressure high-temperature fND probes with the cell nucleus. Our methodology can be, in principle, extended to any red- and near-infrared-luminescent cell-probes and is fully compatible with quantum sensing measurements in living cells.

Formation and assessment of enamel subsurface lesions in vitro.

The present study compared two pH-cycling models designed to induce subsurface lesions (SLs) with a less demineralized surface layer on teeth, with the aim of developing new technologies for assessment of such lesions by examining the performance of confocal Raman microscopy for detection of white spot lesions (WSLs). Twelve sound premolars were exposed to two sets of model conditions (A, B) designed to induce SLs. Teeth on which white lesions had formed in vivo were used as positive controls. All specimens were inspected using an intraoral camera and Raman microscopy to detect small changes in the appearance and structure of the enamel. Changes in the natural color of the teeth during the treatment were recorded via the camera. Phosphate maps with their spectra were constructed from the phosphate peak at 960 cm-1. The depth of lesions was measured on the basis of variations in phosphate peak intensity. Protocol B was reliable for reproducing SLs in a relatively short period. Both protocols had intrinsic limitations in not completely simulating the complex intraoral conditions leading to WSL formation with respect to lesion depth and preservation of an intact surface layer. Raman microscopy can be considered the gold standard for analysis of hard tissue mineralization.

Reparative Mineralized Tissue Characterization after Direct Pulp Capping with Calcium-Silicate-Based Cements.

Nowadays, the preservation of dental pulp vitality is an integral part of our daily therapies. The success of these treatments depends on the clinical situation as well as the biomaterials used. Mineral Trioxide aggregate and BiodentineTM are commonly used as pulp capping materials. One objective of vital pulp therapy is the repair/regeneration of the pulp. In addition to the initial inflammatory status of the pulp, the nature and quality of the new mineralized tissue obtained after pulp capping directly influence the success of the treatment. In order to characterize the reparative dentin, in the current study, the chemical composition and microstructure of the dentin bridge after direct pulp capping using Biodentine™ and mineral trioxide aggregate (MTA) was studied by using Raman microspectroscopy and scanning electron microscopy, respectively. The results showed that the reparative dentin bridge observed in both groups presented dentin tubules and chemical composition similar to primary dentin. With the limitations of this study, the calcium-silicate-based cements used as pulp capping materials provide an optimal environment for pulp healing, resulting in a reparative dentin resembling on certain points of the primary dentin and the regeneration of the pulp.

Dental pulp stem cells used to deliver the anticancer drug paclitaxel.

BACKGROUND: Understanding stem cell behavior as a delivery tool in cancer therapy is essential for evaluating their future clinical potential. Previous in-vivo studies proved the use of mesenchymal stem cells (MSCs) for local delivery of the commonest anticancer drug, paclitaxel (PTX). Dental pulp is a relatively abundant noninvasive source of MSCs. We assess dental pulp stem cells (DPSCs), for the first time, as anticancer drug carriers. Confocal Raman microscopy is a unique tool to trace drug and cell viability without labeling. METHODS: Drug uptake and cell apoptosis are identified through confocal Raman microscope. We traced translocation of cytochrome c enzyme from the mitochondria, as a biomarker for apoptosis, after testing both cancer and stem cells. The viability of stem cells was checked by means of confocal Raman microscope and by cytotoxicity assays. RESULTS: In this study, we prove that DPSCs can be loaded in vitro with the anticancerous drug without affecting their viability, which is later released in the culture medium of breast cancer cells (MCF-7 cells) in a time-dependent fashion. The induced cytotoxic damage in MCF-7 cells was observed consequently after PTX release by DPSCs. Additionally, quantitative Raman images of intracellular drug uptake in DPSCs and MCF-7 cells were obtained. Cytotoxic assays prove the DPSCs to be more resistant to PTX as compared to bone marrow-derived MSCs, provided similar conditions. CONCLUSIONS: Applications of dental stem cells for targeted treatment of cancer could be a revolution to reduce morbidity due to chemotherapy, and to increase the efficacy of systemic cancer treatment.

Confocal Raman mapping of collagen cross-link and crystallinity of human dentin-enamel junction.

The separation zone between enamel and dentin [dentin-enamel junction (DEJ)] with different properties in biomechanical composition has an important role in preventing crack propagation from enamel to dentin. The understanding of the chemical structure (inorganic and organic components), physical properties, and chemical composition of the human DEJ could benefit biomimetic materials in dentistry. Spatial distribution of calcium phosphate crystallinity and the collagen crosslinks near DEJ were studied using confocal Raman microscopy and calculated by different methods. To obtain collagen crosslinking, the ratio of two peaks 1660 cm-1 over 1690 cm-1 (amide I bands) is calculated. For crystallinity, the inverse full-width at half maximum of phosphate peak at 960 cm-1, and the ratio of two Raman peaks of phosphate at 960/950 cm-1 is provided. In conclusion, the study of chemical and physical properties of DEJ provides many benefits in the biomaterial field to improve the synthesis of dental materials in respect to the natural properties of human teeth. Confocal Raman microscopy as a powerful tool provides the molecular structure to identify the changes along DEJ and can be expanded for other mineralized tissues.

Apatite precipitation on a novel fast-setting calcium silicate cement containing fluoride.

Aim: Calcium silicate cements are widely used in endodontics. Novel fast-setting calcium silicate cement with fluoride (Protooth) has been developed for potential applications in teeth crowns including cavity lining and cementation. Objective: To evaluate the surface apatite-forming ability of Protooth compositions as a function of fluoride content and immersion time in phosphate-buffered saline (PBS). Material and methods: Three cement compositions were tested: Protooth (3.5% fluoride and 10% radiocontrast), ultrafast Protooth (3.5% fluoride and 20% radiocontrast), and high fluoride Protooth (15% fluoride and 25% radiocontrast). Powders were cap-mixed with liquid, filled to the molds and immersed in PBS. Scanning electron microscopy, energy dispersive X-ray analysis, and Raman spectroscopy were used to characterize the precipitations morphology and composition after 1, 7, 28, and 56 days. Apatite/belite Raman peak height indicated the apatite thickness. Results: Spherical calcium phosphate precipitations with acicular crystallites were formed after 1-day immersion in PBS and Raman spectra disclosed the phosphate band at 965 cm-1, supporting the apatite formation over Protooth compositions. The apatite deposition continued and more voluminous precipitations were observed after 56 days over the surface of all cements. Raman bands suggested the formation of ß-type carbonated apatite over Protooth compositions. High fluoride Protooth showed the most compact deposition with significantly higher apatite/belite ratio compared to Protooth and ultrafast Protooth after 28 and 56 days. Conclusions: Calcium phosphate precipitations (apatite) were formed over Protooth compositions after immersion in PBS with increasing apatite formation as a function of time. High fluoride Protooth exhibited thicker apatite deposition.

Confocal Raman microscopy to monitor extracellular matrix during dental pulp stem cells differentiation.

Regenerative medicine brings promising applications for mesenchymal stem cells, such as dental pulp stem cells (DPSCs). Confocal Raman microscopy, a noninvasive technique, is used to study osteogenic differentiation of DPSCs. Integrated Raman intensities in the 2800 to 3000 cm⁻¹ region (C-H stretching) and the 960 cm⁻¹ peak (ν1 PO4³â») were collected (to image cells and phosphate, respectively), and the ratio of two peaks 1660 over 1690 cm⁻¹ (amide I bands) to measure the collagen cross-linking has been calculated. Raman spectra of DPSCs after 21 days differentiation reveal several phosphate peaks: ν1 (first stretching mode) at 960 cm⁻¹, ν2 at 430 cm⁻¹, and ν4 at 585 cm⁻¹ and collagen cross-linking can also be calculated. Confocal Raman microscopy enables monitoring osteogenic differentiation in vitro and can be a credible tool for clinical stem cell based research.

Sprayed cells and polyelectrolyte films for biomaterial functionalization: the influence of physical PLL-PGA film treatments on dental pulp cell behavior.

Further development of biomaterials is expected as advanced therapeutic products must be compliant to good manufacturing practice regulations. A spraying method for building-up polyelectrolyte films followed by the deposition of dental pulp cells by spraying is presented. Physical treatments of UV irradiation and a drying/wetting process are applied to the system. Structural changes and elasticity modifications of the obtained coatings are revealed by atomic force microscopy and by Raman spectroscopy. This procedure results in thicker, rougher and stiffer film. The initially ordered structure composed of mainly α helices is transformed into random/ß-structures. The treatment enhanced dental pulp cell adhesion and proliferation, suggesting that this system is promising for medical applications.