Antimicrobial Photodynamic Therapy (aPDT) for periodontal disease (PD) treatment has been studied for more than three decades, but there is no consensus among researchers about its role on PD control. PD is an oral infection linked with a variate of systemic diseases affecting mainly immunocompromised and elderly patients. The aim of this study is to evaluate aPDT for PD control and to analyze the periodontal microbiological profile regarding healthcare-associated infections (HAI). Six patients were enrolled in this study and the microbiological profile of 36 sites were evaluated in different moments: initial, after scaling and root planing, after aPDT and one-week post-treatment. aPDT was performed with 100μM water solution of methylene blue (MB), and a diode laser (100mW, 660nm, 90s per point, 9J). After periodontal therapy the pockets were irrigated with hydrogen peroxide (3%), washed with water, and MB was applied when no bleeding was detected. To collect the periodontal pocket content, a sterilized paper point was placed inside the pocket for 30s. The material was transferred to a vial with transport culture medium and sent to a microbiological facility to be analyzed blindly. The results showed the presence of pathogens linked with HAI as Acinetobacter baumannii, Candida albicans e Serratia marcescens immediately after therapy, but one week after treatment, none of them were detected and the periodontal index of all patients improved. This preliminary result point out that aPDT may not only improve periodontal clinical conditions, but also can diminish the systemic-health threat represented by this disease.
Cryo-electron tomography (cryo-ET) is an emerging technology that enables thin samples, including small intact prokaryotic cells, to be imaged in three dimensions in a near-native 'frozenhydrated' state to a resolution sufficient to recognize very large macromolecular complexes in situ. This methodology has been fundamental to provide information about cellular ultrastructure. This study used cryo-ET to evaluate the photodynamic effect on the viability and envelope architecture of a Gram-negative bacteria. Bacterial suspension of E. coli minicells were submitted to photodynamic treatment with methylene blue solution (100μM) and a 100mW low power diode laser emitting at 660nm with 6 and 18J of energy. As a control group, a suspension of minicells were submitted to 462 IU/mL penicillin G for 60 min at 30 °C, to compare the damage in cell wall structure. After treatment bacteria were immediately plunge-frozen across EM grids and standard cryo-ET tilt series were collected, 3D images reconstructions were calculated and recorded. The imagens showed detachment of the bacterial cell walls and mesosome-like structures. In addition, some sites showed interrupted stretches in both inner and outer membranes and cell wall degradation, indicating bacterial envelope damage. Cryo-electron tomography revealed that the effects of photodynamic therapy on Gram negative bacteria was based on damage to the outer membrane, cell wall and inner membrane and occurs in an energy-dependent manner.
Antimicrobial photodynamic therapy (APDT) combines the use of light with a photosensitizer (PS) and oxygen to kill microbial cells. Even though this technique was first reported in the beginning of the 20th century, APDT never took off as antimicrobial chemotherapy did. However, microbial resistance to chemotherapy is currently expanding in faster rates than drug discovery. Therefore, introduction of therapeutic alternatives that bypass mechanisms of drug resistance now presents an urgent status. Fortunately, the scientific and technological development related to APDT made it far more feasible for mainstream clinical applications. Our research group has been working on mechanisms and applications of APDT for almost 20 years. We have already reported that successful APDT results depend on a number of factors, such as PS and light parameters, cell type, and oxygen abundance, among others. We have also demonstrated that APDT is an effective adjuvant in endodontics and periodontics and can be a non-invasive treatment for caries, candidiasis and cutaneous leishmaniasis. In Veterinary Medicine, we have reported effective treatment for penguin pododermatitis, snake stomatitis and dog otitis. This presentation will give an integrated perspective from the basic APDT mechanisms, preclinical and clinical trials to protocol optimization and future perspectives.
Photodynamic inactivation (PDI) has been reported to be effective to eradicate a wide variety of pathogens, including antimicrobial-resistant microorganisms. However, there are conflicting reports in the literature about the effect of growth phase on the susceptibility to PDI. The aim of this study was to identify the potential molecular targets of PDI on Candida albicans in exponential growth phase after PDI mediated by methylene blue (50μM) and exposure to a 660nm-LED (P=360mW). For this task, scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FT-IR) techniques were employed. Pre-irradiation time was set at 10min and exposure time was 15 min delivering a radiant exposure of 162 J/cm2 on a 24-well plate of about 2 cm2. Morphological analysis revealed cell damage after PDI. FT-IR predominantly showed degradation of functional groups related to C-O of deoxyribose; C-C of DNA; C-O stretching vibration of C-OH group of ribose-RNA; P-O stretching modes from the phosphodiester groups of nucleic acids; C=C, C=N, C=O, N=H proteins and amides. Previous studies from our group had demonstrated different targets on the same cells but in stationary growth phase. Therefore, we can conclude that PDI promoted damage to intracellular structures in fungal cells at exponential-phase growth and information on the susceptibility of different growth phases to PDI can be of great importance for the development of treatment strategies that would lead to inactivation of fungal cells in all possible phases of growth in a way that would turn the clinical PDI treatment effective and predictable.
Laser Induced Breakdown Spectroscopy (LIBS) is a technique capable to perform elemental analyses of a variety of samples, independent of matter state. Other spectroscopy techniques may require a destructive and time-consuming sample preparation. On the other hand, LIBS is a less destructive technique with no (or considerably less) sample preparation, using a relatively simple experimental setup. LIBS also provides a multielement analysis into one single spectrum acquisition, applying a Nd:YAG short-pulsed laser to ensure the stoichiometry between the sample and the generated plasma. LIBS have been applied on the study of carious lesions using a Nd:YAG into its fundamental emission at 1064 nm. It was shown that ratio of P/Ca and Zn/Ca can be used to monitor the cariogenic process. Another minor elements, e.g. C and Cu, associated with bacteria biofilm were also measured with the Nd:YAG laser. The fundamental wavelength emission (1064 nm) of Nd:YAG is coincident with a hydroxyapatite transmission window and it may affect the result. In order to address this issue a study used the second harmonic of the Nd:YAG laser at 532 nm. It was show that it is also possible perform LIBS on carious lesion using the Nd:YAG at 532 nm. However, there is not a work direct comparing the LIBS at 532 nm and 1064 nm for carious lesion detection. So, the aim of this work was to investigate the influence of laser wavelength on the LIBS performance for carious lesion detection. In both cases the carious lesion was detected with the advantage of no interference with hydroxyapatite at 532 nm.
An endotracheal tube (ETT) is required for the management of critically ill, mechanically ventilated patients. Ventilatorassociated pneumonia (VAP) affects patients hospitalized in intensive care units; its risk of occurrence is 1% to up 3% for each day of mechanical ventilation. The polymicrobial nature of VAP is established with mixed bacterial-fungal biofilms colonizing the ETT. The microbial interaction enhances the microbial pathogenesis contributing to high indexes of morbidity/mortality. Antimicrobial Photodynamic Therapy (aPDT) could be a suitable therapy for decontamination of oral cavity and ETT at the same time, but the use of a fiber optics inside the ETT seems to not be appropriated since a cannula for secretion aspiration has to be introduced into the ETT to keep it´s lumen. The aim of this study is to proof the concept that an external light source from a LED is capable of reach all areas of the ETT. We use a commercial ETT, 60μM methylene blue (MB), and a 660nm diode laser and calculated the transmission coefficient of light in different situations as only tube, tube with biofilm and biofilm+MB. The results prove that is possible to transmit light through the tube even in the presence of MB and biofilm although a high attenuation of about 60% was measured depending on the tested condition.
The objective of this work was to develop a LIDAR-like equation model to analyze the measured Optical
coherence tomography (OCT) signal and determine the total extinction coefficient of a scattering sample.
OCT is an interferometric technique that explore sample backscattering feature to acquire in depth cross-section
images using a low coherence light source. Although, almost of the OCT applications are intended
to generate images for diagnostic, similar to histological images, but the backscattering signal carries much
more information. The backscattering problem is similar to those found on LIDAR (Light Detection And
Ranging) problem, this similar situation indicate a path that should be followed to solve the OCT problem.
To determine the total extinction coefficient three inversion methods was used: the slope, boundary point
and optical depth methods solutions. These algorithms were used to analyze the OCT signal of a single and
double layer dentist resin polymer. The total extinction coefficient variations along the optical path were
obtained in order to evaluate the potential of this technique to differentiate structures with different optical
properties. The sample optical characteristics extracted from OCT signal can be use as an additional
quantitative method to help clinical diagnoses when applied on biological tissues among others.
Photodynamic antimicrobial therapy (PAT) may become a useful clinical tool to treat microbial infections, overcoming
microbial resistance that is a major problem nowadays. The aim of our work was to verify the damage caused by
photosensitization over a Escherichia col) via atomic force microscopy (AFM), looking for structural changes that might
occur in cells after PAT. Cells culture were grown until a stationary phase to reach a concentration of approximately 108
cells/mL allowing the production of extracellular slime in a
biofilm-like structure. The cells including the extracellular
matrix were put in a slide and its structure was observed using AFM; subsequently a water solution of methylene blue at
60μM was applied over the cells and a pre-irradiation time of 3 minutes was waited and followed by illumination with a
diode laser (λ=660nm, power 40mW, 3min, fluence 180J/cm2, beam diameter 0.04cm2). The same cells were observed
and the images stored. A second set of experiments was performed with a smaller number of cells/area and without
extracellular slime, using the parameters abovementioned. The results showed alterations on cellular scaffold markedly
dependent on the number of cells and the presence of extracellular slime. The slime is targeted by the photosensitizer,
and after irradiation a destruction of the matrix was observed; when fewer cells were evaluated the destruction is much
more evident. The images suggested rupture of the cellular membrane and cellular fragments were observed. Our
findings indicate that AFM seems is a useful tool to investigate parameters linked with photodestruction of
microorganisms.
This study compares the antimicrobial effect of photodynamic therapy (PDT) combined to endodontic treatment
with conventional endodontic treatment alone in patients with necrotic pulp and has a 6-month radiographic follow up
comparing the healing of periapical lesions. Fifteen patients with periapical lesion and requiring root canal treatment
were selected. Microbiological samples were taken after accessing the root canal, conventional manual endodontic
therapy (group 1 n=5) and after accessing the canal, endodontic therapy and PDT (group 2 n=10). All the root canals
were filled with a calcium hydroxide paste for 1 week. Radiographs were taken after obturation and following 6 months.
Endodontic therapy alone presented an 87% reduction in microorganisms while the combination with PDT had a 95%
reduction. Radiographic follow up showed 32% higher reduction in the lesion area in PDT group. Results suggest that
the use of PDT added to conventional endodontic treatment leads to a further major reduction of microbial load. PDT is
an efficient alternative to chemical antimicrobial agents. It is a non-cumulative local treatment, which may be an
appropriate approach for the treatment of infections in the oral cavity.
Photodymanic antimicrobial therapy (PAT) has been proposed to treat oral infections and the phenothiazinic compound
Methylene Blue (MB) has been considered as a suitable photosensitizer for this application. MB is a known
methachromatic compound and the dimerization process may lead to different photochemical reactions, into the oral
cavity, where the complete isolation of the saliva may not be possible. The aim of this study is to monitor the
dimerization process of MB in the presence of human saliva through absorption spectroscopy. Absorption spectra of
30μM MB solutions in water and in human saliva were recorded in the wavelengths ranging from λ=400nm to λ=700nm.
The spectra were recorded immediately after mixture and 1 min, and 5 minutes after blending. The results were evaluated
by spectral analyses and through the calculus of the dimer/monomer ratio. The results demonstrated that immediately
after mixture a hypochromic effect characterized by the diminishing on the total absorption in the visible range of the
spectrum (λ= 400nm - 700nm) is observed, but the aggregation process is not detectable. After 1 minute the ratio between
dimer and monomer absorption increase and this increase became higher upon increasing the contact time. The results
indicate that the addition of saliva into the mixture leads to and hypochromic effect follow by the dye aggregation.
Aggregation is probably an important variable to be analyzed when choosing the pre-irradiation time in oral cavity
application, because it may lead to different photochemical routes.
Severe burns cause extensive damage and are complicated by loss of body fluids, injury in the cutaneous vasculature and delayed wound healing. Low-intensity laser therapy (LILT) has been studied as an alternative method to accelerate wound healing. This study was carried out to evaluate LILT effects (λ= 660 nm) in rat burned skin with two different dose regimens. Thirty-six male adult Wistar rats with two burns created on their back using steam water were divided into 3 groups. In the fractioned dose laser group (FG), the lesions were irradiated with 1J/cm2 on days 1, 3, 8 and 10; in the single dose laser group (SG), the lesions were irradiated with 4J/cm2 on day 1. On control group (CG), lesions were not irradiated. Three animals per group were sacrificed on days 1, 3, 8, 10, 15 and 21 post-wounding and skin specimens were collected and processed to histomorphometry. At days 1, 3 and 8, statistical significant differences were not observed among groups. On the 10th day, mean values of the number of blood vessels for FG was significantly higher than CG. Irradiated groups showed a peak of new blood vessels formation at day 15 while for CG the peak was at day 21. The number of vessels in CG was significantly higher than FG and SG at day 21. These findings suggest that LILT may accelerate angiogenesis compared to control group, however, no significant differences were observed between laser groups with fractioned or single dose during all experiment.
We use the optical path difference (OPD) technique to quantify the organization of collagen fibers during skin repair of full-thickness burns following low-intensity polarized laser therapy with two different polarization incidence vectors. Three burns are cryogenerated on the back of rats. Lesion L is irradiated using the electric field vector of the polarized laser radiation aligned in parallel with the rat's occipital-caudal direction. Lesion L is irradiated using the electric field vector of the polarized laser radiation aligned perpendicularly to the aforementioned orientation. Lesion C is untreated. A healthy area labeled H is also evaluated. The tissue samples are collected and processed for polarized light microscopy. The overall finding is that the OPD for collagen fibers depends on the electric field vector of the incident polarized laser radiation. No significant differences in OPDs are observed between L and H in the center, sides, and edges of the lesion. Lesions irradiated using the electric field vector of the polarized laser radiation aligned in parallel with the rat's occipital-caudal direction show higher birefringence, indicating that collagen bundles in these lesions are more organized.
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