PEDRO ARTHUR AUGUSTO DE CASTRO
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Tese IPEN-doc 29223 Chemometrics of biological tissues using infrared spectroscopy2022 - CASTRO, PEDRO A.A. deVibrational spectroscopy techniques have been considered for the diagnosis of diseases. In particular, Fourier transforms infrared spectroscopy (FTIR) is an optical method widely adopted in biomedical research. This is because FTIR images allow the collection of various molecular information enabling a correlation between spectral characteristics and structural changes. Incorporating qualitative and quantitative aspects is essential to implementing a chemometric procedure that transcribes spectral variance into meaningful information. In the case of burns, the degree of tissue damage will strongly depend on several factors, such as severity, depth, and size, which influence the healing process. Laser scar treatment has been used for decades for post-healing remodeling. However, conventional laser techniques may not wholly normalize mature scar tissue, and a preventive approach to laser exposure during wound healing has emerged. Early intervention has already been investigated in several clinical studies, but there is no consensus. Therefore, the objectives of this study were; to investigate FTIR in biological tissue analysis to provide better chemometric strategies; to study the feasibility of FTIR to establish a profile of the skin and bone tissue, and to evaluate the effect of laser on wound healing and the development of a new chemometric methodology for automatic digital dewaxing. To this end, thermal injuries were performed on the back of Wistar rats by applying water vapor at 90ºC for 12s. Subsequently, on day three post-burn, laser irradiation was performed. In the laser procedure, 3 protocols were adopted: 1) Er:glass (SellasEvo®) with 3025FPA, 100mJ, 3s; 2) LLT with red laser with = 660nm, power 40mW; 3) Combination of the two lasers with the previous parameters. Then, a histological and chemometric analysis was performed. For the chemometric evaluation of the data, an analysis protocol was developed based on a pre-processing using OCTAVVS followed by implementing a baseline, threshold, and application of PCA. From that point, the images are classified, they can be normalized and go through the Fuzzy C-means (FCM) technique to determine the spectral differences. All-laser groups demonstrate a shrinkage of the wound region in the histopathological findings. Er:Glass, showed a smaller retraction area and less epithelium recovery. The PBM group has a dense crust. Both treatments demonstrate intense shrinkage and more consistent recovery of the epidermis. In the FCM, three trends occur 1) in the range of 900-950 cm-1 it has a significantly reduced amplitude in the control group; 2) in the range of 1200-1250 cm-1 the control group has a similar amplitude demonstrating bands of sensitive markers; and 3) the amide regions provide various spectral markers. The control group, as expected, remains with minimal changes in this FTIR region. On the other hand, in the PBM group, there is a shift from 1660 to 1679 cm-1, which may correspond to changes in the molecular structure of a protein in the context of type I collagen. FTIR is a valuable alternative for tissue research on a biochemical level through fingerprints that can be translated into morphological features.Resumo IPEN-doc 26009 Semi quantitative evaluation of skin burn wound healing2019 - 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.Resumo IPEN-doc 26006 Heat-induced depth of Nd:YAG laser irradiation in biological hard tissues2019 - 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.Resumo IPEN-doc 26005 Calcium analysis from gamma sterilized human dentin and enamel2019 - ZAMATARO, CLAUDIA B.; KUCHAR, NIELSEN G.; SCAPIN, MARCOS A.; ZANINI, NATHALIA; CASTRO, PEDRO; RABELO, THAIS F.; JUVINO, AMANDA C.; ZEZELL, DENISEGamma 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.Resumo IPEN-doc 25613 ATR-FTIR spectral monitoring of burn wound healing in skin by hierarchical cluster2018 - 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.Dissertação IPEN-doc 24678 Espectroscopia de absorção no infravermelho em pele queimada: avaliação de potenciais biomarcadores para o reparo tecidual2018 - CASTRO, PEDRO A.A. deUma caracterização bioquímica eficiente da cicatrização das feridas por queimadura pode melhorar a rotina clínica para a ajustar os tratamento dos pacientes. Considerando a dificuldade de monitoramento de cicatrização, a espectroscopia no infravermelho por transformada de Fourier acoplado com o acessório de Reflexão Total Atenuada (ATR-FTIR) é uma técnica analítica a qual tem demonstrado capacidade de prover biomarcadores espectrais no materiais biológicos. Esse estudo avaliou a capacidade de usar ATR-FTIR para classificar a pele queimada com o intuito de monitorar o reparo tecidual em pacientes. Ratos Wistar com pele queimada foram avaliados pela espectroscopia ATR-FTIR nos dias 3, 7, 14 e 21 após a queimadura e comparados com amostras de grupos sadios. Os espectros obtidos foram separados nas regiões entre 900 a 1800 cm-1 e entre 2800 a 3000 cm-1 para posterior adequação quimiométrica. Avaliou-se as amplitudes das derivadas de segunda ordem dos espectros como parâmetro de discriminação, nos quais demonstraram diferenças significativas para os grupos não queimados com controle e entre grupos de queimadura usando Glicogênio (1030 cm-1), Amida I (1628 cm-1), Amida II (1514 cm-1), Colágeno em 1281 cm-1 e Colágeno em 1336 cm-1. Esses resultados indicam que ATR-FTIR é sensível para detectar os estágios de reparo tecidual e pode futuramente ser um instrumento auxiliar para a rotina clínica.