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.
The European Union (EU) directive 2010/63/EU on the protection of animals used for scientific purpose focused on reducing the number of animals and refinement of breeding. Animal studies are necessary to protect human health. Lots of animal models exist to study bone regeneration, but a reliable, well reproducible, and relatively inexpensive model with the possibility for multiple testing in the same animal is still missing. Rats may serve as good models for this due to the small animal size and good cost/benefit ratio. The present study aimed to develop a novel rat caudal vertebrae critical size defect model for bone regeneration and implant osseointegration studies The study was performed using Wistar rats with weight from 380 to 450 g. An incision was made on the dorsal side of the tail. After skin and muscles retractions, the vertebrae were exposed. Critical size defects for bone tissue engineering or implant placements for titanium body experiments were possible in each of the first four caudal vertebrae. Micro-computed tomography (CT) and histology were used to detect bone growth. There was no bone formation in the defects after 1 or 2 months of healing. When a calcium phosphate biomaterial was used (Bio-Oss; Geistlich Pharma AG, Wolhusen, Switzerland), a good stability of the material in the defect was noted and bone growth was visible after 1 or 2 months. Results based on implant placement showed good primary stability after 3 months of healing. MicroCT showed integrated implant position through the vertebra. These results suggest that the rat caudal vertebrae may serve as a good new model for studying bone regeneration and implant osseointegration with the possibility of multiple testing within the same experimental animal and the potential to decrease number of experimental animals.
Dental enamel is the most mineralized tissue in the human body and has a very complex organization. The aim of the present study was to evaluate chemical composition and orientation of crystals in human enamel cross striation using confocal Raman microscope. Slices of teeth were prepared and scanned using a confocal Raman microscope. Cross striation spacing was calculated according to the variation of PO43− peak intensity. The measurements showed a characteristic length between two cross striations of 2.70 (SD 0.43) μm for permanent teeth and 1.75 (SD 0.37) for deciduous teeth. Ratios between OH and PO43− peaks, for 0 and 90 degrees angulation, are, respectively, 9.18 (SD 0.80) and 44.74 (SD 3.12) for decidual teeth, and 11.72 (SD 1.46) and 22.12 (SD 3.15) for adult teeth. This confirmed that the hydroxyapatite crystal is well oriented along the enamel prism, as reported in the literature, but with a significant increase in deciduous teeth. K‐mean cluster binary images were calculated to compare the ratio of the centroid peaks of the cross striation zone and inter cross striation material. The regions corresponding to inner prism, included cross striations, are more mineralized than the outer part of enamel prism. In the prism, the Pearson correlation test was performed based on the intensity of hydroxyapatite characteristic peaks (ν1 PO43−, ν1 CO32−, and OH). The variations were well correlated: These results indicated that cross striations corresponded to the variation of hydroxyapatite concentration and not to a variation of crystals orientation.
White lesions represent an early phase of caries formation. 20 human sound premolars were subjected to pH cycling procedure to induce subsurface lesions (SLs) in vitro. In addition, 2 teeth with naturally developed white spot lesions (WSLs) were used as references. All specimens characterized by confocal Raman microscopy being used for the first time in examining white & subsurface lesions and providing a high resolution chemical and morphological map based on phosphate peak intensity alterations at 960 cm−1. Nanoindentation technique was used to measure Hardness (H) and Young’s modulus (E) of enamel. Phosphate map of examined samples exhibited presence of intact surface layer (ISL) followed by severe depletion in (PO43−) peak in the area corresponding to the body of the lesion. In all examined groups, the mechanical properties of enamel were decreased in lesion area and found to be inversely related to penetration depth of indenter owing to enamel hierarchical structure. By combining the above two techniques, we linked mechanical properties of enamel to its chemical composition and ensured that the two methods are highly sensitive to detect small changes in enamel composition. Further work is required to bring these two excellent tools to clinical application to perceive carious lesions at an early stage of development.
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.
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