PEDRO ARTHUR AUGUSTO DE CASTRO

PEDRO ARTHUR AUGUSTO DE CASTRO

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Q-switched Nd:YAG laser on dental enamel with photoabsorber
2022 - CASTRO, PEDRO; PEREIRA, DAISA; ANA, PATRICIA; MATOS, CHRISTIANO; ZEZELL, DENISE
Nd:YAG lasers emitting λ=1064 nm at microsecond and nanosecond pulses are alternatives to prevent dental caries and erosion in clinics.This wavelength allows most of photons to penetrate deep in the hard tissue due to low absorption of hydroxyapatite in the region. It is necessary to use photoabsorbers so most of photons are absorbed in the surface of the tissue preventing dental pulp necrosis. Currently the coal paste is used as a photoabsorber but the irradiated tissue turns darker what implies in the patients low adherence to the treatment due to aesthetic reasons. [1,2]. Confocal Raman spectroscopy is a non-destructive optical method to obtain detailed information about molecular composition of biological structures in depth. The most prominent feature of Confocal Raman spectroscopy is the reliable capability to provide the biomolecular data with no use of ionizing radiation to penetrate in the sample. This work aims to characterize the dental enamel irradiated with Nd:YAG laser with nanoseconds pulses, in order to describe the depth related changes promoted in the enamel, by the heat generated due to laser irradiation. For these measurements, 30bovine enamel blocks of 8 mm2, were randomized into 3 groups: G1 – enamel untreated; G2 – enamel irradiated with Nd:YAG nanopulsed laser (1064 nm, 4 W; 1,05 J/cm2; 5 ns 20 Hz, Brilliant, Quantel Laser) using a coal paste as photoabsorber; G3 -enamel irradiated with Nd:YAG nanopulsed laser ( same parameters as G2) using squid ink as photoabsorber. The assessments of three different depth regions of the cubic shaped samples were: region A- left corner above of the sample, region B- middle of the sample and Region C- right corner below of the sample. The intensity map of phosphate (950 cm-1)regarding the position, were calculated [3,4] as shown in the Figure 01. The comparative results in the Fig.1 demonstrated that application of coal paste associated with Nd:YAG (G2) can preserve the inorganic content better than the squid ink group (G3). These findings have crucial clinical implications in the laser protocol development and itwas possible to correlate the heat penetration depth of the laser irradiation with photoabsorber using the images obtained bythe confocal Raman.
FTIR imaging on glass substrates evaluation of histological skin burn injuries specimens treated by femtosecond laser pulses
2022 - ZEZELL, DENISE; CASTRO, PEDRO; DEL-VALLE, MATHEUS; CAMILLO-SILVA, CARLOS; SAMAD, RICARDO; DE ROSSI, WAGNER; SANTOS, MOISES
Burn injuries continue to be one of the leading causes of unintentional death and injury in low- and middle-income countries [1]. Burns are considered an important public health problem, because in addition to physical problems that can lead the patient to death, they cause psychological and social damage. An estimated 180,000 deaths every year are caused by burns [2]. The use of infrared (IR) spectroscopy for studying biological specimens is nowadays a wide and active area of research. The IR microspectroscopy has proved to be an ideal tool for investigating the biochemical composition of biological samples at the microscopic scale, as well as its fast, sensitive, and label-free nature [3]. IR image spectral histopathology has shown great promise as an important diagnostic tool, with the potential to complement current pathological methods, reducing subjectivity in biopsy samples analysis. However, the use of IR transmissive substrates which are both fragile and prohibitively very expensive, hinder the clinical translation. The goal of this study is to evaluate the potential of discriminating healing process, in burned skin specimens treated with ultrashort pulses laser 3 days after the burn. This study is considering a previous paper [4], in which it analyzed only micro-ATR-FTIR spectra of a frozen sample point. The specimens were obtained from third degree burn wound. The wounds treatment were performed three days after the burn, and the animals were sacrificed 3 and 14 days post-treatment. Using coverslipped H&E stained tissue on glass from previous histopathological analysis and applying the analytical techniques PCA and K-means on N−H, O−H, and C−H stretching regions occurring at 2500−3800 cm−1 (high wavenumber region), were possible to discriminate burned epidermal and dermal regions from irradiated in same regions on sample. In the figures is shown the average spectrum at (a) day 3 and (b) day 14. , in both there were increase of burned+laser treated bands. The great potential of this study was to analyse coverslipped H&E stained tissue on glass, without compromising the histopathologist practices and contribute for clinical translation.
Assessment of burn wounds status using mid-infrared spectroscopy
2019 - CASTRO, PEDRO; LIMA, CASSIO; ZORN, TELMA; ZEZELL, DENISE M.
Burns are one of the major causes of morbidity and the most costly traumatic injuries worldwide. Better understanding of the molecular mechanisms associated with wound healing might provide improved clinical strategies to speed up the tissue repair process and reduce the global impact of burns on public health services. The traditional techniques used to assess the biochemical events related to wound repair are laborious, time-consuming and require multiple staining. Thus, the present study aims to evaluate the feasibility of Fourier transform infrared (FTIR) spectroscopy in order to monitor the progress and healing status of burn wounds. Third-degree burn injuries were induced on Wistar rats by water vapor exposure. Afterwards, biopsies specimen was extracted for further histopathological examination and spectroscopic evaluation at 4 time-points (3, 7, 14 and 21 days). Raw spectral data were offset-corrected and normalized by amide I band area. The second derivatives were compared by the Principal Component Analysis (PCA). On days 3 and 7, when compared to healthy group, biomolecules bands were most prominent. However, on days 14 and 21, these molecular bands decreased. Therefore, our pairwise comparison revealed that metabolic activity induced by thermal injury decreases as the healing process progresses. Our findings show that FTIR spectroscopy can monitor the biochemical development induced by burn injury and detect the status of wound repair.
Semi quantitative evaluation of skin burn wound healing
2019 - CASTRO, PEDRO A.A. de; ZEZELL, DENISE M.; MORAIS, MYCHEL R.P.T.; ZORN, TELMA M.
The role of skin wound healing the biochemical mechanisms are still not fully understood and spectroscopy may shine some light on this information. The Fourier Transform Infrared spectroscopy coupled with Attenuated Total Re°ectance (ATR-FTIR) has been demonstrated potential to monitor the biomolecule activity upon biological samples. This work aims to evaluate the feasibility of using ATR-FTIR to discriminate burned skin throughout wound stages. Water vapor at 90±C was applied to the dorsum of Wistar rats resulting in standardized third-degree burns. Tissue samples were collected after 3±, 7±, 14± and 21 days post burn injury. For the acquisition of the spectra, 150 scans were averaged with a resolution of 4 cm¡1 and wavenumbers ranging from 4000 to 400 cm¡1. Analysis of the spectra was calculated using MATLABR°R2015a (MathWorks, Natick, MA) software. Fingerprint region (900 - 1800 cm¡1) and high wavenumber (2800 - 3000 cm¡1) of spectra were o®set-corrected and vector normalized. Area under the curve (AUC) of collagen (1236 cm¡1), amide II (1540 cm¡1), amide I (1632 cm¡1) and lipid (2852 cm¡1) of each group were performed by integration method. The FTIR results exhibit the high biochemical activity of the tissue in the 14o post burn injury when it is compared with control group which correspond to the stage when the new tissue formation is still ongoing. The lipid content decrease in the 21 day, which suggest that the metabolic activity and structural reorganization decrease as the wound healing progress. For future works, micro-FTIR imaging will be used spatially discriminate the bands.
Heat-induced depth of Nd:YAG laser irradiation in biological hard tissues
2019 - PEREIRA, DAISA L.; CASTRO, PEDRO A.A.; GOMES, GABRIELA V.; ZAMATARO, CLAUDIA B.; ZEZELL, DENISE M.; RIBEIRO, HENRIQUE B.; MATOS, CHRISTIANO J.S.; ANA, PATRICIA A.
Confocal Raman spectroscopy is a non-destructive and non-invasive technique which provides surface Raman spectra and depth images of biological structures contactless with the sample, with no use of ionizing radiation to penetrate in the sample under analysis. These characteristics allow its experimental use without any side e®ects to the sample. The depth images are obtained by Raman microscopy and are related to the characteristics of the tissues. This study aims to characterize irradiated hard tissues and correlate the depth reached by the heat of the laser irradiation with the obtained images. For this, thirty 8 mm2 blocks of bovine enamel and bovine root dentin, were randomized into 6 groups: G1- enamel untreated; G2- enamel irradiated with Nd:YAG micropulsed laser (1064 nm, 10 Hz- Lares Research R°) using a coal paste as photoabsorber; G3- enamel irradiated with Nd:YAG nanopulsed laser (1064 nm, 20 Hz, Brilliant, Quantel Laser) using a coal paste as photoabsorber; G4-G6 (bovine root dentin in the same conditions of treatment of G1-G3). The measurements were performed in three di®erent depth regions of the cubic shaped samples: region A- left corner above of the sample, region B- middle of the sample and Region C- right corner below of the sample. The area under the phosphate, carbonate, amide I, II, and III bands were calculated. The Raman spectra of the Nd:YAG irradiated samples detected a reduction in all the organic components of the enamel after laser irradiation. Previous studies of our group demonstrated that di®erences in carbonate substitution in the apatite lattice are related to the apatite instability and demineralization susceptibility. Considering that carbonate free apatite is less susceptible to acid attack, the results of this study suggest that Nd:YAG lased enamel can be more resistant to caries, in a direct correlation to the thickness of the treated area. It was found that for micropulsed Nd:YAG laser, the heat induced depth was 10 § 2 ¹m and for nanopulsed laser the heat induced depth was 8 § 3 ¹m. So, it is possible to correlate the heat penetration depth of the laser irradiation with the images obtained by the confocal Raman.
Calcium analysis from gamma sterilized human dentin and enamel
2019 - ZAMATARO, CLAUDIA B.; KUCHAR, NIELSEN G.; SCAPIN, MARCOS A.; ZANINI, NATHALIA; CASTRO, PEDRO; RABELO, THAIS F.; JUVINO, AMANDA C.; ZEZELL, DENISE
Gamma radiation changes the patients0 oral cavity undergoing radiotherapy. Alterations cause an unsaturated environment of calcium and phosphate into the oral cavity. After approval of the Ethics Committee, 20 hu- man teeth were sectioned to obtain 20 human enamel and 20 dentin samples, polished plane. Samples were randomized in the irradiated group and control group (untreated). Then, the treatment group was irradiated with 25:0 kGy at the 60Co multipurpose irradiator. After the gamma irradiation, Fourier Transformed Infrared Spectroscopy (FTIR), percentage of surface microhardness loss (%SMHL) and Scanning Electron Microscopy (SEM) were performed. At the end, acidic biopsies were performed to quantify the concentration of calcium present in the samples. FTIR showed that the molecular structure of HA of the enamel is similar to the non- irradiated, with no formation or loss of molecular compounds occurring. X-ray °uorescence at enamel samples was performed. Microscopic morphological analysis did not shown signi¯cant di®erences. Surface microhardness is an indirect indicator of the mineral content of the samples. The mean obtained was 258:2 (38:8) KHN within the hardness spectrum of the healthy natural enamel. The compounds present in the samples and the values of the ratios of Calcium and Phosphate oxides and relation between the elements Calcium and Phosphorus. The ratio of the most stable oxides shows a variation with linear correlation. In the enamel, the ratio (Ca/P) shows a change in the elemental content with linear correlation (R2 = 1). These ¯ndings lead us to a new hypothesis of behaviour of the HA crystal versus gamma irradiation. On the other hand for the irradiated dentin, the Knoop hardness number was within the range of the spectrum similar to that of natural dentin of human origin. X-ray °uorescence shows that irradiated dentin has great similarity with natural dentin from the point of view of chemical composition. SEM analyses showed that there was no thermal damage or interprismatic morpho- logical changes in the hydroxyapatite structure of human dental dentin outside the buccal environment when using doses of gamma irradiation up to 25 kGy.
ATR-FTIR spectral monitoring of burn wound healing in skin by hierarchical cluster
2018 - CASTRO, PEDRO A.A. de; ZEZELL, DENISE M.; MORAIS, MYCHEL R.P.T.; ZORN, TELMA M.T.
Efficient biochemical characterization of burn wound healing stages can improve clinical routine to adjust the patient treatment. The Fourier Transform Infrared spectroscopy coupled with Attenuated Total Reflectance (ATR-FTIR) is an analytical technique that has potential capability to provide spectral biomarkers in biological material. This study aims to evaluate the feasibility of using ATR-FTIR to classify burned skin, to be able, in the future, to follow the regenerative process in patients. Wistar rat burn tissues were evaluated by ATRFTIR spectroscopy at 3, 7, 14, 21 days after burn and compared with the healthy group samples (H). For the acquisition of one spectrum, 150 scans were averaged with a resolution of 4 cm-1 and wavenumbers ranging from 4000 to 400 cm-1. Analysis of the spectra was performed using MatLab R2017a (MathWorks, EUA) software. The fingerprint region between 900 to 1800 cm-1 was separated and normalized by amide I band area. For smoothing purpose, spectra were submitted to Savitzky-Golay filter with a polynomial of second order in a fifteen points window. All spectra data were submitted to the hierarchical cluster using the single method and standardized Euclidean distance. The classification results demonstrated separation to non-wounded groups with an accuracy of 91,8% (H vs. 3D), 87,83% (H vs. 7D) and 97,96% (H vs. 14D). Interestingly, when the results from the healthy group were compared to the 21D group, the accuracy dissimilarity was 26%, which suggests that they are chemically similar implying that after 21 days the burned lesions are entirely recovered. These findings indicate that when an adequate multivariate method is applied, ATR-FTIR is suitable to detect the wounded and non-wounded skin as well as its healing stages. In this way, it is possible to conclude that ATR-FTIR can be an auxiliary analysis for the clinical routine for skin burn wound healing.
FTIR spectroscopy
2018 - CASTRO, PEDRO; LIMA, CASSIO; ZORN, TELMA M.T.; ZEZELL, DENISE M.
In this study, we investigated the ability of Fourier transform infrared spectroscopy to discriminate healthy tissue and thermal injury, aiming the development of an optical method to evaluate the wound healing process.
Discrimination of ionizing radiation effects on bone using Fourier Transform Infrared Spectroscopy using K-means
2018 - CASTRO, PEDRO A.A. de; ZEZELL, DENISE M.
We demonstrated the feasibility of using ATR-FTIR spectroscopy associated with k-means clustering to evaluate the recognition of different doses. Our results open up new possibilities for protein monitoring relating to dose responses.
Biochemical evaluation of bone submitted to ionizing radiation by ATR-FTIR spectroscopy
2017 - CASTRO, PEDRO A.A. de; DIAS, DERLY A.; VELOSO, MARCELO N.; ZEZELL, DENISE M.
FTIR spectroscopy associated with PC-LDA was able to discriminate bone samples receiving different ionizing radiation doses (0,01 kGy, 1 kGy, 15 kGy), showing potential to the use of phosphate vibrational modes as a dose marker. OCIS codes: (170.4580) Medical optics and biotechnology – Optical diagnostics for medicine; (170.6510) Medical optics and biotechnology – Spectroscopy, tissue diagnostics; (170.1580) Medical optics and biotechnology – Chemometrics.