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.
Burn wound healing by infrared spectroscopy imaging
2022 - CASTRO, P.; SILVA, C.; ZEZELL, D.
Aim: Burns are one of the major causes of morbidity and the most costly traumatic injuries worldwide1. 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. Methods : Third-degree burn injuries were induced on Wistar rats by water vapor exposure. Afterwards, biopsies specimen was extracted for further histopathological examination and IR imaging evaluation at 7 days. The spectral imaging was performed using a micro- FTIR equipment in transflection mode (MirrIR, Kevley Technologies), with a 32 x 32 FPA of 5.5 μm pixel size. All spectra data were baseline corrected and vector normalized. The preprocessing and image comparison was performed using Cytospec software (Cytospec, version 2.00.5)2. Results : The pairwise analysis was evaluated in the wavenumber region maps of 1200-1300 cm-1. The biomolecule band associated to collagen content (1200 – 1300 cm-1) was most prominent3 on the 7th day image in the post-burn that in healthy group. Conclusions : Therefore, our pairwise comparison revealed that metabolic activity induced by thermal injury increases the chemical activity associated to the healing progresses. Our findings show that FTIR spectroscopy has potential to identify the biochemical signatures induced by burn injury.
Assessment of bound water of saliva samples by using FT-IR spectroscopy
2022 - FERREIRA, MARIA C.C.; MONTEIRO, GABRIELA R.; PERALTA, FELIPE; CASTRO, PEDRO A.A.; ZEZELL, DENISE; NOGUEIRA, MARCELO S.; CARVALHO, LUIS F.C.S.
The objective of the present work is to show the relationship of the high-wavenumber spectral region concern OH vibrations, which show in a way how bound water can be altered in different sample groups.
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.
Nd:YAG laser on dental enamel in the reduction of artificial caries demineralization
2019 - ZEZELL, DENISE M.; SILVA, MATEUS R.; CASTRO, PEDRO A.A.; SILVA, TANIA M.; GONCALVES, SERGIO E.P.
Nd:YAG-laser associated to a photoabsorber, in the reduction of artificial caries in enamel was evaluated. Eighty bovine specimens with 6mm diameter and 2mm high were obtained and a half of the surface of each was protected as a control. Microdurometer and FTIR were performed initially and 8 groups (n=10) were obtained according to treatments: G1(- control): no-treatment; G2(+control): fluorophosphate; G3(Nd:YAG 60mJ/pulse, 10Hz, 48J/cm2, non-contact); G4(photoabsorber + Nd:YAG 60mJ); G5(Nd:YAG 80mJ/pulse, 10Hz, 64J/cm2); G6(photoabsorber + Nd:YAG 80mJ); G7(Nd:YAG 100mJ/pulse, 10Hz, 80J/cm2); G8(photoabsorber + Nd:YAG 100mJ). De-remineralization cycle were performed for induction of artificial caries and to interferometer, microdurometer and FTIR. Microhardness data were submitted to 2-way ANOVA and Tukey/Dunnett tests 5%. Statistically differences were obtained in the photoabsorberfactor individually and in the interaction between laser and photoabsorber. There was a lower percentage of microhardness loss in the groups with photoabsorber; G8 presented microhardness similar to G2. FTIR data were submitted to T-test 5%. Compared with G2, higher concentrations of carbonate were found in G4, G6 and G8; phosphate in G8; lower Amide-I concentration at G8 and higher Carbonate/Phosphate ratio at G4 and G6. The interferometry results were submitted to 3-way ANOVA of repeated measures 5%. There were statistically differences in the photoabsorber-factor individually and in the time-factor. Photoabsorber decreased the demineralization; Nd:YAG-laser without photoabsorber were less effective than fluoride; Nd:YAG-laser 100mJ with photoabsorber was as effective as fluoride and; the Nd:YAG-laser, associated or not to the photoabsorber, was no more effective than fluoride in the reduction of artificial decay.
Chemometric analysis of bone dose response
2022 - CASTRO, PEDRO A.A. de; DIAS, DERLY A.; ZEZELL, DENISE M.
The use of ionizing radiation for treatment and diagnosis of many diseases has been well documented. The health effects of ionizing radiation improved patient care, however, incorrect handling of radiation technology may pose potential health risks. In light of this, Fourier-transform infrared spectroscopy combined with attenuated total reflectance (ATR-FTIR) offers a nondestructive, label-free technique for identifying functional groups in biological samples. In this work, fragments of bone were collected from bovine femur diaphysis. Samples were cut and polished until 1 cm x 1 cm x 1 mm, which were then stored properly in the refrigerated environment. Samples irradiation was performed with a multipurpose irradiator of Cobalt-60 at doses of 15 kGy and 25 kGy.Spectral data was submitted for the second derivative. Thereafter, the amplitude of the second derivative was organized for hierarchical clustering in conjunction with a heatmap. In order to carry out the chemometric analyses, all procedures were performed using MATLAB (The Mathworks Inc., Natick, MA, USA). Based on these results, an examination of the cluster heatmap reveals that the unique behavior for each group which may be correspond to the ionizing radiation change in the spectra. Hence, these findings suggest new possibilities for spectral monitoring of dose responses.
Q-switched Nd:YAG laser on dental enamel with photoabsorber
2020 - CASTRO, P.; PEREIRA, D.; ANA, P.; MATOS, C.; ZEZELL, D.
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, 30 bovine 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 it was possible to correlate the heat penetration depth of the laser irradiation with photoabsorber using the images obtained by the confocal Raman.
Biochemical characterization of saliva of smoking and non-smoking patients by using Fourier-transform infrared spectroscopy
2022 - FERREIRA, MARIA C. de M.S.C.; LEAL, LEONARDO B.; NOGUEIRA, MARCELO S.; CASTRO, PEDRO A.A.; PERALTA, FELIPE; ZEZELL, DENISE M.; CARVALHO, LUIS F. das C. e S. de
More than 90% of oral malignant neoplasms consist of squamous cell carcinoma originated from extrinsic and/or intrinsic multifactorial etiology on the epithelium of the oral mucosa. This multifactorial etiology makes early-stage cancer detection challenging due to the lack of associated oral-tissue clinical features and absence of changes on conventional cellular-imaging, serological and histopathological exams. By using a molecular-sensitive optical technique such as Fourier-transform infrared (FT-IR) spectroscopy, disease-specific biochemical changes can be detected non-destructively, non-invasively and with small sample volumes. In this study, we have used FT-IR spectroscopy to analyze saliva samples of control, smoker, and occasional smoker groups and determine their intrinsic molecular changes as well as the performance of sample differentiation by using a neural network classifier. Saliva samples were collected by spitting, homogenized and stored at -20ºC until analysis. Spectral data was collected in the fingerprint region (900cm-1 to 1800cm- 1) by using a Thermo Scientific Nicolet 6700 ATR FT-IR Spectrometer, in which 1-μl samples were placed on the crystal without additives and left to dry completely by an average time of 5 minutes. Data pre-processing and analysis was performed on the OriginPro8.5 and Orange software. Saliva-sample classification was performed with leave-one-out validation. Correctly classified instances were 72.7% for the control group, 65.5% for occasional smokers and 75% for smokers. Sample differences were observed in the peaks at 1076cm-1 (skeletal cis conformation of DNA and symmetric stretching of phosphate [PO2], 1403cm-1 symmetric CH3 modes of protein methyl groups and δsCH3 of collagen, 1451cm- 1 asymmetrical CH3 bending modes of the protein methyl groups, 1547cm-1 of protein band, amide II, peptide and proteins amide II, and 1646cm-1 amide I, C5 methylated cytosine, C==O bond, C==C stretching uracil and NH2 guanine.