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.
Bone as a biomarker for detecting low dose of ionizing radiation
2022 - CASTRO, PEDRO A.A. de; DIAS, DERLY A.; VELOSO, MARCELO N.; ZEZELL, DENISE M.
FTIR spectroscopy was able to discriminate bone samples receiving low-dose ionizing radiation doses (0.002 kGy, 0.004 kGy, 0.007 kGy), offering valuable insights in view of the understanding of radiation dose response in biological systems.
Chemometrics of biological tissues using infrared spectroscopy
2022 - CASTRO, PEDRO A.A. de
Vibrational 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.
Assessment of bone dose response using ATR-FTIR spectroscopy
2022 - CASTRO, PEDRO A.A. de; DIAS, DERLY A.; DEL-VALLE, MATHEUS; VELOSO, MARCELO N.; SOMESSARI, ELIZABETH S.R.; ZEZELL, DENISE M.
The health care application of ionizing radiation has expanded worldwide during the last several decades. While the health impacts of ionizing radiation improved patient care, inaccurate handling of radiation technology is more prone to potential health risks. Therefore, the present study characterizes the bone dose response using bovine femurs from a slaughterhouse. The gamma irradiation was designed into low-doses (0.002, 0.004 and 0.007 kGy) and high-doses (1, 10, 15, 25, 35, 50 and 60 kGy), all samples received independent doses. The combination of FTIR spectroscopy and PLS-DA allows the detection of differences in the control group and the ionizing dose, as well as distinguishing between high and low radiation doses. In this way, our findings contribute to future studies of the dose response to track ionizing radiation effects on biological systems.
The impact of scan number and its preprocessing in micro-FTIR imaging when applying machine learning for breast cancer subtypes classification
2021 - DEL-VALLE, MATHEUS; SANTOS, MOISES O. dos; SANTOS, SOFIA N. dos; CASTRO, PEDRO A.A. de; BERNARDES, EMERSON S.; ZEZELL, DENISE M.
The breast cancer molecular subtype is an important classification to outline the prognostic. Gold-standard assessing using immunohistochemistry adds subjectivity due to interlaboratory and interobserver variations. In order to increase the diagnosis confidence, other techniques need to be examined, where the FTIR spectroscopy imaging allied with machine learning techniques may provide additional and quantitative information regarding the molecular composition. However, the impact of co-added scans acquisition parameter into machine learning classifications still needs better evaluation. In this study, FTIR images of Luminal B and HER2 subtypes were acquired varying the scan number and preprocessing techniques. It was demonstrated a spectral quality improvement when the scan number was increased, decreasing the standard deviation and outliers. Six machine learning models were used to classify the subtypes: Linear Discriminant Analysis, Partial Least Squares Discriminant Analysis, K-Nearest Neighbors, Support Vector Machine, Random Forest and Extreme Gradient Boosting. Best mean accuracy of 0.995 was achieved by Extreme Gradient Boosting model. It was found that all models achieved similar high accuracies with groups b256_064 (256 background and 064 scans), b256_128 and b128_128. Besides assessing the performance of different models, the b256_064 was established as the optimal group due to the minimum acquisition time. Therefore, this work indicates b256_064 for breast cancer subtype classification and also as a basis for other studies using machine learning for cancer evaluation.
Machine Learning methods for micro-FTIR imaging classification of human skin tumors
2021 - DEL VALLE, MATHEUS; STANCARI, KLEBER; CASTRO, PEDRO A.A. de; SANTOS, MOISES O. dos; ZEZELL, DENISE M.
This review presents some methods applied to micro-FTIR imaging for classification of human skin tumors. It is a collection of the pre-processing pipeline and machine learning classification models. The aim of this review is to update and summaiize the current methods which an applied in our skin tumor research.
Healing status of bum wound healing
2021 - CASTRO, PEDRO; ZORN, TELMA; ZEZELL, DENISE
The purpose of this study is to use infrared spectroscopy (FTIR) for monitoring biological changes in burned skin. Wistar rats dorsum samples were compared to healthy group samples at 7, 14, 21 days after burn. Proteins changes of burn wounds were monitored by area under the curve (AUC) of bands at 1630 cm 1 , 1543 cm 1 and 1743 cm 1 . Kruskal-Wallis normality tests, unpaired t test with Welch's correction were used to evaluate the differences between AUC. These bands suggest association between collagen activity during wound healing stages. Our result indicates progressive recovery of 7,14 and 21 days tissues when compared with the healthy group.
Assessment of the preventive effects of Nd:YAG laser associated with fluoride on enamel caries using optical coherence tomography and FTIR spectroscopy
2021 - DIAS-MORAES, MARCIA C.; CASTRO, PEDRO A.A.; PEREIRA, DAISA L.; ANA, PATRICIA A.; FREITAS, ANDERSON Z.; ZEZELL, DENISE M.
Objective This in vitro study characterized and monitored, by Optical Coherence Tomography (OCT) and Fourier Transformed Infrared Spectroscopy (FTIR), the effects of the association of acidulated phosphate fluoride gel (APF-gel) and Nd:YAG (neodymiun:yttrium-aluminum-garnet) laser, as sequencial treatments, in the prevention of incipient enamel caries lesions. Methods 120 human enamel samples were randomized into 3 groups (n = 40): APF-gel (1.23% F-, 4 min.); Laser+APF (Nd:YAG laser irradiation—0.6W, 84.9J/cm2, 10Hz, followed by APF-gel); and APF+Laser (APF-gel followed by laser irradiation). The samples were subjected to a 15-day pH-cycling, evaluated by OCT (quantification of optical attenuation coefficient–OAC) and FTIR (analysis of carbonate and phosphate content) before treatments, after treatments, and on the 5th, 10th and 15th days of pH-cycling. The statistical analysis was performed (α = 5%). Results The Optical Attenuation Coefficient (OAC) assessed by OCT increases with the progression of demineralization, and the Laser+APF presented the highest values of OAC in 10th and 15th days of pH-cycling. Nd:YAG decreased the carbonate content after treatment regardless of the application order of the APF-gel, while APF-gel did not interfere in the composition of enamel. The carbonate content was also changed in the first 5 days of the pH-cycling in all groups. Conclusion Nd:YAG laser irradiation before or after the application of APF-gel did not influence the appearance of incipient caries lesions, showing no synergistic effect. Regardless of the application order of the APF-gel, laser irradiation reduces the carbonate content of the enamel, which also changes during the demineralization process. However, irradiation before the application of APF-gel increased the speed of progression of the lesions, which positively impacts public health as it can prevent caries disease, even in high risk individuals. OCT and FTIR are suitable for assessing this effect.
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.