Frederico Barbosa de Sousa

Effect of cinnamomum zeylanicum blume essential oil on the rowth and morphogenesis of some potentially pathogenic Aspergillus species

Cinnamomum zeylanicum Blume is known for a wide range of medicinal properties. This study aimed to assess the interference of C. zeylanicum essential oil on the growth and morphogenesis of some potentially pathogenic Aspergillus species. The essential oil presented strong antifungal effect causing the growth inhibition of the assayed strains and development of large growth inhibition zones. MIC50 and MIC90 values were 40 and 80 µL/mL, respectively. 80, 40 and 20 µL/mL of the oil strongly inhibited the radial mycelial growth of A. niger, A. flavus and A. fumigatus along 14 days. 80 and 40 µL/mL of the oil caused a 100% inhibition of the fungal spore germination. Main morphological changes observed under light microscopy provided by the essential oil in the fungal strains were decreased conidiation, leakage of cytoplasm, loss of pigmentation and disrupted cell structure indicating fungal wall degeneration. It is concluded that C. zeylanicum essential oil could be known as potential antifungal compound, particularly, to protect against the growth of Aspergillus species.

Enamel mineralization in the absence of maturation stage ameloblasts

The role of maturation stage ameloblasts is not clear yet. The aim of this study was to verify to which extent enamel mineralizes in the absence of these cells. Maturation stage ameloblasts and adjacent dental follicle cells from rat lower incisors were surgically removed and the limits of this removal were marked by notches made in the enamel. Histological analysis confirmed that the ameloblasts had been removed within the limits of the notches. The teeth erupted and when the notches appeared in the mouth, the enamel in the experimental teeth was hard but whitish compared to the yellowish colour of the contralateral incisors used as control. SEM images revealed similar enamel rod arrangement in both groups. Decreased mineral content was observed in some specimens by polarized light microscopy, and microhardness values were much lower in the experimental teeth. FTIR analysis showed that higher amounts of protein were found in most experimental teeth, compared with the control teeth. Enamel proteins could not be resolved on 15% SDS-PAGE gels, suggesting that most of them were below 5kDa. These results suggest that the enamel matured in the absence of ameloblasts has increased protein content and a much lower mineral content, suggesting that maturation stage ameloblasts are essential for proper enamel mineralization.

Exposure to lead exacerbates dental fluorosis

AIM Our aim was to test the hypothesis that co-exposure to lead and fluoride alter the severity of enamel fluorosis. MATERIALS AND METHODS Wistar rats were allocated in four groups: control, and 3 groups that received water containing 100 ppm of fluoride (F), 30 ppm of lead (Pb), or 100 ppm of F and 30 ppm of Pb (F+Pb) from the beginning of gestation. Enamel analysis and F and Pb determinations in enamel, dentine, and bone were performed in 81-day-old animals. Fluorosis was quantified using a new fluorosis index based on the identification of incisor enamel defects (white bands and white islets, representing hypomineralization, and cavities) weighted according to their severity and quantity. Hypomineralization was validated histopathologically by polarizing microscopy and microradiography. Scores were given by two blinded calibrated examiners (intra and interexaminer kappa values were 0.8 and 0.86, respectively). RESULTS The control and the Pb groups presented normal enamel. The F+Pb group presented more severe enamel defects compared with the F group (P<0.0001). CONCLUSIONS This study shows that lead exacerbates dental fluorosis in rodents, suggesting that co-exposure to lead may affect the degree of fluorosis.

A new approach for improving the birefringence analysis of dental enamel mineral content using polarizing microscopy

The main problem in interpreting birefringence of dental enamel under polarizing microscopy is the lack of physical constants able to allow the Wiener equation to be applied directly to the composition of such tissue. The present study introduces a new approach to circumvent this constraint. Because the nonmineral phase of enamel is heterogeneous, its refractive index can be computed in terms of its components (namely, water, which is partially replaced by the immersion medium, and organic matter), thereby providing a more acceptable refractive index to be used in the Wiener equation. Furthermore, the enamel mineral volume is ordinarily calculated on the basis of the density 3.15 g cm−3. The density 2.99 g cm−3 has been, however, reported to be more accurate for enamel hydroxyapatite, so enamel mineral volumes from selected published data were converted using such a density. The birefringence of mature enamel computed by the Wiener equation, taking into account the above refinements, matched, for the first time, published experimental birefringence values. The theoretical water and organic contents were also consistent with published experimental data. Thus, a direct application of the Wiener equation to the enamel composition has now been achieved. It is speculated that quantitative data on the mineral, the water and the organic contents of mature dental enamel can be derived from interpretation of birefringence in two immersion media (obtained before and after extraction of the organic matter) with this new approach.

Recovery and identification of mature enamel proteins in ancient teeth.

Proteins in mineralized tissues provide a window to the past, and dental enamel is peculiar in being highly resistant to diagenesis and providing information on a very narrow window of time, such as the developing period; however, to date, complete proteins have not been extracted successfully from ancient teeth. In this work we tested the ability of a whole-crown micro-etch technique to obtain enamel protein samples from mature enamel of recently extracted (n = 2) and ancient (n = 2; ad 800 to 1100) third molars. Samples were analyzed using matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry, and the resulting spectra were searched against the Swiss-Prot protein database using the Mascot software for protein identification. In our protocol, the separation of proteins in gel is not necessary. Successful identification of specific enamel proteins was obtained after whole-crown superficial enamel etching with 10% HCl. Most protein fragments recovered from dry teeth and mummy teeth contained amino-terminal amelogenin peptides. Only one peptide specific for the amelogenin X-isoform was identified. In conclusion, the reported techniques allowed the successful recovery of proteins specific to dental enamel from samples obtained in a very conservative manner, which may also be important in forensic and/or archeological science.

Morphological characterization of rat incisor fluorotic lesions

The morphological characterization of fluorotic rat incisor enamel was carried out. Experimental adult animals received drinking water with 45 mg F/L of fluoride, and the control group received distilled water. Fluoride concentrations found in the control and fluorosis groups were 0.04 and 0.09 microg/mL (plasma), 0.26 and 0.66 microg/mg (whole tibia), and 0.24 and 2.3 microg/mg (tibia surface), with P < or = 0.001 for all comparisons between the groups. A succession of white and pigmented bands was observed in the fluorotic rat incisors. Under polarizing light microscopy, cross-sections of superficial areas corresponding to the white bands (from the surface to approximately 20 microm) showed high positive birefringence. These fluorotic lesions also exhibited the lowest resistance to superficial acid etching. No morphological differences in inner enamel were seen under scanning electron microscopy. In fluorotic enamel, only the surface layer related to the white areas presented lower birefringence compared with the enamel of control teeth and the surface layer of the pigmented areas (normal ones) of fluorotic teeth. In conclusion, the white bands of fluorotic rat enamel represent hypomineralized superficial areas and are not subsurface lesions. The detailed description of these lesions is important to understand dental fluorosis.

A comparative study on component volumes from outer to inner dental enamel in relation to enamel tufts.

BACKGROUND AND AIM Dental enamel presents marked mechanical properties gradients from outer to inner enamel, a region lacking component volumes profiles. Tufts, structures of inner enamel, have been shown to play a role in enamel resilience. We aimed at comparing component volumes from inner to outer enamel in relation to enamel tufts. MATERIALS AND METHODS Transversal ground sections from the cervical half of unerupted human third molars (n=10) were prepared and histological points were selected along transversal lines (extending from innermost to outer enamel) traced across tufts and adjacent control areas without tufts. Component volumes were measured at each histological point. RESULTS Component volumes ranges were: 70.6-98.5% (mineral), 0.02-20.78% (organic), 3.8-9.8% (total water), 3-9% (firmly bound water), and 0.02-3.3% (loosely bound water). Inner enamel presented the lowest mineral volumes and the highest non-mineral volumes. Mineral, water and organic contents differed as a function of the distance from innermost enamel but not between the tuft and control lines. Tufts presented opaqueness in polarizing microscopy (feature of fracture lines). Organic volume gradient correlated with a relatively flat profile of loosely bound water. Inner, but not outer enamel, rehydrated after air-dried enamel was heated to 50°C and re-exposed to room conditions, as predicted by the organic/water gradient profiles. CONCLUSIONS Component volumes vary markedly from outer to inner enamel, but not between areas with or without tufts (that behave like fracture lines under polarizing microscopy).

Growth inhibition and morphological alterations of Trichophyton rubrum induced by essential oil from Cymbopogon winterianus Jowitt ex Bor

Trichophyton rubrum is one of the most common fungi causer of dermatophytosis, mycosis that affect humans and animals around the world. Researches aiming new products with antifungal activity become necessary to overcome difficulties on treatment of these infections. Accordingly, this study aimed to investigate the antifungal activity of essential oil from Cymbopogon winterianus against the dermatophyte T. rubrum. The antifungal screening was performed by solid medium diffusion method with 16 T. rubrum strains, minimum inhibitory concentration (MIC) and minimum fungicide concentration (MFC) were determined using the microdilution method. The effects on mycelial dry weight and morphology were also observed. Screening showed essential oil in natura inhibited all the tested strains, with inhibition zones between 24-28 mm diameter. MIC50 and MIC90 values of the essential oil were 312 µg/mL for nearly all the essayed strains (93.75 %) while the MFC50 and MFC90 values were about eight times higher than MIC for all tested strains. All tested essential oil concentrations managed to inhibit strongly the mycelium development. Main morphological changes on the fungal strains observed under light microscopy, which were provided by the essential oil include loss of conidiation, alterations concerning form and pigmentation of hyphae. In the oil presence, colonies showed folds, cream color and slightly darker than the control, pigment production was absent on the reverse and with evident folds. It is concluded that C. winterianus essential oil showed activity against T. rubrum. Therefore, it could be known as potential antifungal compound especially for protection against dermatophytosis.Trichophyton rubrum is one of the most common fungi causer of dermatophytosis, mycosis that affect humans and animals around the world. Researches aiming new products with antifungal activity become necessary to overcome difficulties on treatment of these infections. Accordingly, this study aimed to investigate the antifungal activity of essential oil from Cymbopogon winterianus against the dermatophyte T. rubrum. The antifungal screening was performed by solid medium diffusion method with 16 T. rubrum strains, minimum inhibitory concentration (MIC) and minimum fungicide concentration (MFC) were determined using the microdilution method. The effects on mycelial dry weight and morphology were also observed. Screening showed essential oil in natura inhibited all the tested strains, with inhibition zones between 24-28 mm diameter. MIC50 and MIC90 values of the essential oil were 312 μg/mL for nearly all the essayed strains (93.75 %) while the MFC50 and MFC90 values were about eight times higher than MIC for all tested strains. All tested essential oil concentrations managed to inhibit strongly the mycelium development. Main morphological changes on the fungal strains observed under light microscopy, which were provided by the essential oil include loss of conidiation, alterations concerning form and pigmentation of hyphae. In the oil presence, colonies showed folds, cream color and slightly darker than the control, pigment production was absent on the reverse and with evident folds. It is concluded that C. winterianus essential oil showed activity against T. rubrum. Therefore, it could be known as potential antifungal compound especially for protection against dermatophytosis.

Dental enamel birefringence for a wide mineral content range and for different immersion media's refractive indexes: an improved mathematical interpretation

The aim of this study is to extend, for a larger range of mineral volume (V1) and different aqueous immersion media, the model for calculating the refractive index of the non‐mineral phase of the human dental enamel proposed by Sousa et al. Published experimental birefringence data of carious and developing human enamel in air and aqueous immersion media with different refractive indexes were interpreted. For 48%≤V1≤ 95%, quantitative estimates on the non‐mineral contents consistent with the values described in the literature were derived. New polynomial relationships of the non‐mineral contents as a function of the mineral content (48%≤V1≤ 95%) are introduced, which allows computing the theoretical mean enamel birefringence in water and air for most of the points of developing, carious and mature enamel with a difference of one order of magnitude in relation to the classical model for the non‐mineral contents refractive index and closer to the experimental data. For enamel birefringence in immersion media other than water and air, a less expressive improvement was obtained. Perspectives for future studies are discussed.

Anatomical variations in primary teeth microelements with known differences in lead content by micro-Synchrotron Radiation X-Ray Fluorescence (μ-SRXRF) – A preliminary study

Shed teeth have been proposed as trace element biomarkers. This study determined variations in the spatial distribution of Ca, K, Zn, Pb, Mn, Cu, and Sr in four anatomical locations: superficial enamel (SE, 0-10μm), subsuperficial enamel (SSE, 10-30μm), primary dentin (PD), and secondary dentin (SD). Five primary incisors were analyzed by micro Synchrotron Radiation X-Ray Fluorescence (μ-SRXRF). Two teeth had low concentrations of lead in the SE (<250μg/g), while three contained very high lead concentrations in the SE (>2000μg/g). Teeth were sliced, and five spot measurements (20μm beam diameter) were accomplished in each location. The data are shown as absolute values and as the ratio between the different elements and Ca. The distribution of K was close to that of Ca. Zn was the third most abundant element, with the highest levels being found in the SE and SD and low levels detected in the PD. Increasing Sr levels were found progressing from the enamel to the dentin, with the highest levels being found in the SD, a distribution that was unique. Pb, Mn, and Cu exhibited a similar trend, with higher signals for these elements detected in the SE. This study provides preliminary data on the heterogeneous distribution of different elements in the tooth, highlighting the importance of the first 10μm of the SE for determination of some elements, such as Zn, Pb, Mn, and Cu.