Overall Results for a National Program of Photodynamic Therapy for Basal Cell Carcinoma: A Multicenter Clinical Study to Bring New Techniques to Social Health Care.

Along the past years, a national program to implement photodynamic therapy (PDT) for nonmelanoma skin cancer (NMSC) was performed over the Brazilian territory. Using a strategy involving companies, national bank, and medical partners, equipment, medication, and protocols were tested in a multicenter study. With results collected over 6 years, we could reach a great deal of advances concerning the use of PDT for skin cancer. We present the overall reached results of the program and discuss several aspects about it, including public politics of treatment. A discussion about advantages of this technique within conditions of health care is placed, comparing PDT with surgery, including an analysis about the implementation of PDT in countries in development as Brazil, considering not only technical but social aspects, as the distribution of medical doctor in the Brazilian territory. The program resulted in a huge dissemination of PDT in Brazil and many countries in Latin America, in a partnership among public politics, universities, companies, and hospitals and clinics and in the insertion of national technologies as option to treat NMSC. Consequence of the program is mainly the continuation of the use of PDT in Brazil and many countries in Latin America.

Vascular Effects of Photodynamic Therapy with Curcumin in a Chorioallantoic Membrane Model.

Photodynamic Therapy (PDT) is a treatment that requires light, a photosensitizing agent, and molecular oxygen. The photosensitizer is activated by light and it interacts with the oxygen that is present in the cellular microenvironment. The molecular oxygen is transformed into singlet oxygen, which is highly reactive and responsible for the cell death. Therefore, PS is an important element for the therapy happens, including its concentration. Curcumin is a natural photosensitizer and it has demonstrated its anti-inflammatory and anti-oxidant effects that inhibit several signal transduction pathways. PDT vascular effects of curcumin at concentrations varying from 0.1 to 10 mM/cm² and topical administration were investigated in a chick Chorioallantoic Membrane (CAM) model. The irradiation was performed at 450 nm, irradiance of 50 mW/cm² during 10 min, delivering a total fluence of 30 J/cm². The vascular effect was followed after the application of curcumin, with images being obtained each 30 min in the first 3 h, 12 h, and 24 h. Those images were qualitatively and quantitatively analyzed with a MatLAB®. Curcumin was expected to exhibit a vascular effect due to its angio-inhibitory effect. Using curcumin as photosensitizer, PDT induced a higher and faster vascular effect when compared to the use of this compound alone.

Necrosis response to photodynamic therapy using light pulses in the femtosecond regime.

One of the clinical limitations of the photodynamic therapy (PDT) is the reduced light penetration into biological tissues. Pulsed lasers may present advantages concerning photodynamic response when compared to continuous wave (CW) lasers operating under the same average power conditions. The aim of this study was to investigate PDT-induced response when using femtosecond laser (FSL) and a first-generation photosensitizer (Photogem) to evaluate the induced depth of necrosis. The in vitro photodegradation of the sensitizer was monitored during illumination either with CW or an FSL as an indirect measurement of the PDT response. Healthy liver of Wistar rats was used to evaluate the tissue response. The photosensitizer was endovenously injected and 30 min after, an energy dose of 150 J cm(-2) was delivered to the liver surface. We observed that the photodegradation rate evaluated via fluorescence spectroscopy was higher for the FSL illumination. The FSL-PDT produced a necrosis nearly twice as deep when compared to the CW-PDT. An increase of the tissue temperature during the application was measured and was not higher than 2.5 °C for the CW laser and not higher than 4.5 °C for the pulsed laser. FSL should be considered as an alternative in PDT applications for improving the results in the treatment of bulky tumors where higher light penetration is required.

Correlation between light transmission and permeability of human dentin.

The influence of dentin permeability on transdentinal LED light propagation should be evaluated since this kind of phototherapy may further be clinically used to stimulate the metabolism of pulp cells, improving the healing of damaged pulps. This study evaluated the influence of the dentin permeability on the transdentinal LED light (630 nm) transmission. Forty-five 0.5-mm-thick dentin disks were prepared from the coronal dentin of extracted sound human molars. An initial measurement of transdentinal LED light transmission was carried out by illuminating the discs in the occlusal-to-pulpal direction onto a light power sensor to determine light attenuation. The discs were treated with EDTA for smear layer removal, subjected to analysis of hydraulic conductance, and a new measurement of transdentinal LED light transmission was taken. Spearman's correlation coefficient was used for analysis of data and showed a weak correlation between dentin permeability and light attenuation (coefficient = 0.19). This result indicates that higher or lower dentin permeability does not reflect the transdentinal propagation of LED light. Significantly greater transdentinal propagation of light was observed after treatment of dentin surface with EDTA (Wilcoxon test, p < 0.05). According to the experimental conditions of this in vitro study, it may be concluded that dentin permeability does not interfere in the transdentinal LED light transmission, and that smear layer removal facilitates this propagation.

Photodynamic therapy of adenoid hypertrophy in acute rhinosinusitis.

A first step for microorganisms to reach the respiratory system and cause infectious disease is colonization in the nasopharynx. Humans inhale a bacterial load of up to 106 per cubic meter of air per day [1], including transient microorganisms between the upper and lower airways. This can lead to lung infections, amounting to billions of dollars in annual direct treatment costs in the United States, depending on the etiologic agent [2,3]. Curcumin has been described as a photosensitizer (PS) that, activated at 450 nm, is efficient against planktonic bacteria [4] and biofilms [5]. At the same time, effects on microbial interactions are commonly detected in the upper respiratory tract and should be considered for the treatment of adenoids [6]. We, therefore, propose in this study to optimize photodynamic therapy (PDT) conditions in vitro by simulating bacterial coinfection conditions in nasal cavities evaluated by the response surface method, where we can evaluate interactions of treatment variables. From this, the clinical case of the treatment of rhinosinusitis was carried out using PDT with nasal lighting. The absence of symptoms that characterize the disease was monitored and evaluated by the Kepler Meyer method. The study points out considerations that can be evaluated for the treatment to be a possibility of clinical indication in the control of rhinosinusitis.

A new photodynamic therapy protocol for nodular basal cell carcinoma treatment: Effectiveness and long-term follow-up.

BACKGROUND: Photodynamic therapy (PDT) has been reported as an excellent option for the treatment of small nodular basal cell carcinomas (nBCC). The standard protocol consists of two sessions, one week apart. Sometimes, returning to the hospital after one week can be impractical for elderly patients, due to comorbidities and mobility issues. Therefore, a new technique performed in one day could be superior for those patients. OBJECTIVE: Evaluate the effectiveness of a PDT Single-visit protocol comparing to the standard protocol, as well as pain and long-term recurrence-free follow-up for nBCC. METHODS: A total of 120 nBCC were treated through a Standard PDT protocol(two sessions, one week apart), and 120 nBCC were treated through a Single-visit PDT(two sessions in one day). A 30-day-after biopsy was performed in order to evaluate the results after the treatment. The lesions that had successful treatment were clinically and dermoscopically evaluated every 6 months up to 60 months. The pain score was compared between the groups(assessed every 3 min during PDT). RESULTS: A complete response at 30-days-after PDT biopsy was observed in 85% of Standard PDT and in 93.3% of Single-visit PDT. Regarding the pain during the illumination, less pain was observed during the second session of the Single-visit PDT. The recurrence-free follow up showed, after 60 months, an 69.0% cumulative probability of recurrence-free for Standard PDT and 80.6% for Single-visit PDT. CONCLUSIONS: The suggested Single-visit PDT protocol resulted in better outcomes at 30-day-after PDT biopsy and in lower recurrence rates than the Standard PDT protocol. A more comfortable and more efficient treatment was offered for the patients, with lower pain.

Temperature effect on the PpIX production during the use of topical precursors.

Decreasing incubation time, as well as enhanced PpIX production, are present challenges for topical photodynamic therapy (PDT). There are reports concerning the existence of a strong relationship between site temperature and porphyrin synthesis in biological tissue, that suggest temperature increase in the tissue can improve the formation of PpIX. The main objective of this study is to determine whether the temperature change of the tissue favors the production of PpIX. Creams containing aminolevulinic acid (ALA) and methyl aminolevulinate (MAL) were topically administered for 30 min on healthy skin of rats' back and the formation of PpIX was evaluated for 180 min. The animals were divided into 5 groups: cooling tissue to 20 °C or heating tissue to 40 °C (either before or after incubation of the cream) and control group (unchanged temperature). The tissue temperature was evaluated by thermography. The influence of temperature was evaluated both concerning cream penetration and the production of PpIX. It was found that both ALA and MAL led to an increase of about 20% PpIX production when the tissue was warmed before incubation of the cream, suggesting that the penetration improved. When the thermal change was promoted after incubation of the cream, the production of PpIX decreased both by heating and cooling, probably related to enzyme modification. The results found in this study suggest that the increase of tissue temperature before the cream incubation can improve the clinical protocols of topical PDT using ALA or MAL, improving the efficiency of the procedure by increasing the production of PpIX and allowing the decrease of the incubation period.

Optical clearing agent increases effectiveness of photodynamic therapy in a mouse model of cutaneous melanoma: an analysis by Raman microspectroscopy.

Melanoma is the most aggressive type of skin cancer and a relevant health problem due to its poor treatment response with high morbidity and mortality rates. This study, aimed to investigate the tissue changes of an improved photodynamic therapy (PDT) response when combined with optical clearing agent (OCA) in the treatment of cutaneous melanoma in mice. Photodithazine (PDZ) was administered intraperitoneally and a solution of OCA was topically applied before PDT irradiation. Due to a resultant refractive index matching, OCA-treated tumors are more optically homogenous, improving the PDT response. Raman analysis revealed, when combined with OCA, the PDT response was more homogenous down to 725 µm-depth in thickness.

Energy analysis of PDT using thermography during the treatment of basal cell carcinoma.

The changes in tissue temperature of basal cell carcinoma lesions were investigated during photodynamic therapy in order to better understand the effects and mechanisms of PDT in tissue. In this study, the monitoring of 40 lesions of basal cell carcinoma was performed during photodynamic therapy. The lesion region becomes thermally evident throughout the procedure, and there is an improved contrast of the lesion edges after the end of the irradiation. The comparison between thermal and fluorescence images showed a correlation between the PpIX evidenced through widefield fluorescence and the temperature gradient of the thermal images after the procedure, indicating that thermography is a potential diagnostic tool to evaluate the selective response of PDT. A model was created to calculate the amount of light energy converted to heat, tissue damage, and other energy transfer processes involved in the PDT. Using this model, it was shown that most of the energy conversion was in photodynamic action (48.7% and 48.3%, in first and second session, respectively), followed by the energy ratio attributable to blood perfusion (37.2%). This is evidence that photodynamic therapy does not generate a significant thermal component, an important aspect of the study of its mechanisms.

Photodynamic therapy dosimetry using multiexcitation multiemission wavelength: toward real-time prediction of treatment outcome.

Evaluating the optical properties of biological tissues is needed to achieve accurate dosimetry during photodynamic therapy (PDT). Currently, accurate assessment of the photosensitizer (PS) concentration by fluorescence measurements during PDT is typically hindered by the lack of information about tissue optical properties. In the present work, a hand-held fiber-optic probe instrument monitoring fluorescence and reflectance is used for assessing blood volume, reduced scattering coefficient, and PS concentration facilitating accurate dosimetry for PDT. System validation was carried out on tissue phantoms using nonlinear least squares support machine regression analysis. It showed a high correlation coefficient (>0.99) in the prediction of the PS concentration upon a large variety of phantom optical properties. In vivo measurements were conducted in a PDT chlorine e6 dose escalating trial involving 36 male Swiss mice with Ehrlich solid tumors in which fluences of 5, 15, and 40 J cm - 2 were delivered at two fluence rates (100 and 40 mW cm - 2). Remarkably, quantitative measurement of fluorophore concentration was achieved in the in vivo experiment. Diffuse reflectance spectroscopy (DRS) system was also used to independently measure the physiological properties of the target tissues for result comparisons. Then, blood volume and scattering coefficient measured by the fiber-optic probe system were compared with the corresponding result measured by DRS and showed agreement. Additionally, tumor hemoglobin oxygen saturation was measured using the DRS system. Overall, the system is capable of assessing the implicit photodynamic dose to predict the PDT outcome.

Development of a system to treat and online monitor photodynamic therapy of skin cancer using PpIX near-infrared fluorescence.

The limited adoption of photodynamic therapy (PDT) around the medical field may be tied to the unpredicted treatment response that an unmonitored therapy could deliver. Given the high variability in the lesions optical and physiological parameters, it is of fundamental importance to monitor PDT, since different lesions require different therapeutic parameters. We developed a system to treat and online monitor PDT of skin cancer, using protoporphyrin-IX (PpIX) near-infrared fluorescence imaging. The system can be operated up to 150 mW/cm2 at 633 nm, with real-time fluorescence monitoring around 700 nm, using the treatment light itself for fluorescence excitation. This technology allows system portability, simplicity, and low cost. This study describes the system development and its comparison with a 400-450 nm commercial system to detect the PpIX fluorescence during a PDT in murine skin cancer model. The developed device was able to acquire considerably more fluorescence signal from deeper regions when compared to the violet excitation device.

Single visit PDT for basal cell carcinoma - A new therapeutic protocol.

Non-melanoma skin cancer is the most prevalent type of cancer in Brazil and worldwide. Topical Photodynamic Therapy is a technique that offers advantages as: excellent aesthetic result, possibility of application for outpatients in ambulatory setting, and presenting a minimum functional impact of the treated anatomic site. Fractionated Photodynamic Therapy is a modification of the usual technique in which the full dose of light is delivered in steps separated by a periods of time ("dark intervals"). In Brazil, no studies using this technique for treatment of BCC have been published. Thus, we proposed to evaluate the complete and partial response to the four different protocols of fractional Photodynamic Therapy, when evaluated 30 days after treatment. The study showed a complete response of 65.8%, 67.6%, 72.7% and 95.4% in the groups 1, 2, 3 and 4, respectively. We observed that the dark interval and the irradiated light dose are parameters of great importance for the final response to the treatment. Our results suggest that Fractionated Photodynamic Therapy is a technique with excellent aesthetic result and complete response when evaluated 30 days after treatment. However, a longer follow-up will be necessary for better understanding of the behavior of the lesions treated.

Fluorescence spectroscopy in the visible range for the assessment of UVB radiation effects in hairless mice skin.

Ultraviolet (UV) radiation may induce skin alterations as observed in photoaging. Some recognized modifications are epidermal hyperplasia, amorphous deposition of degraded elastic fibers and reduction in the number of collagen fibers. They alter the tissue biochemical properties that can be interrogated by steady state fluorescence spectroscopy (SSFS). In this study, we monitored the changes in endogenous fluorescence emission from hairless mice skin during a protocol of photoaging using UVB irradiation. To perform the fluorescence spectroscopy, it was used a violet laser (408nm) to induce the native fluorescence that is emitted in the visible range. Under 408nm excitation, the emission spectrum showed bands with peaks centered around 510, 633 and 668nm for irradiated and control groups. A relative increase of the fluorescence at 633nm emission on the flank was observed with time when compared to the ventral skin at the same animal and the non-irradiated control group. We correlated the emission at 633nm with protoporphyrin IX (PpIX), and our hypothesis is that the PpIX metabolism in the photoaged and aged skin are different. PpIX fluorescence intensity in the photoaged skin is higher and more heterogeneous than in the aged skin. Notwithstanding, more spectroscopic and biochemistry studies investigating the 510 and 633nm emission are needed to confirm this hypothesis.

Temperature analysis during bonding of brackets using LED or halogen light base units.

The purpose of our investigation is to compare the intrapulpal temperature changes following blue LED system and halogen lamp irradiation at the enamel surface of permanent teeth. The fixation of brackets using composite resin is more comfortable and faster when using a photo-curable composite. Several light sources can be used: halogens, arc plasma, lasers, and recently blue LED systems. An important aspect to be observed during such a procedures is the temperature change. In this study, we have used nine human extracted permanent teeth: three central incisors, three lateral incisors, and three canines. Teeth were exposed to two light sources: blue LED system (preliminary commercial model LEC 470-II) and halogen lamp (conventional photo-cure equipment). The surface of teeth was exposed for 20, 40, and 60 sec at the buccal and lingual enamel surface with an angle of 45 degrees. Temperature values measured by a thermistor placed at pulpar chamber were read in time intervals of 1 sec. We obtained plots showing the temperature evolution as a function of time for each experiment. There is a correlation between heating quantity and exposition time of light source: with increasing exposition time, heating increases into the pulpal chamber. The halogen lamp showed higher heating than the LED system, which showed a shorter time of cooling than halogen lamp. The blue LED system seems like the indicated light source for photo-cure of composite resin during the bonding of brackets. The fixation of brackets using composite resin is more comfortable and faster when using a photo-curable composite. Blue LED equipment did not heat during its use. This could permit a shorter clinical time of operation and better performance.

Time-resolved fluorescence lifetime for cutaneous melanoma detection.

Melanoma is the most aggressive skin cancer type. It is characterized by pigmented lesions with high tissue invasion and metastatic potential. The early detection of melanoma is extremely important to improve patient prognosis and survival rate, since it can progress to the deadly metastatic stage. Presently, the melanoma diagnosis is based on the clinical analysis of the macroscopic lesion characteristics such as shape, color, borders following the ABCD rules. The aim of this study is to evaluate the time-resolved fluorescence lifetime of NADH and FAD molecules to detect cutaneous melanoma in an experimental in vivo model. Forty-two lesions were analyzed and the data was classified using linear discriminant analysis, a sensitivity of 99.4%, specificity of 97.4% and accuracy of 98.4% were achieved. These results show the potential of this fluorescence spectroscopy for melanoma detection.

Relationship between the chemical and morphological characteristics of human dentin after Er:YAG laser irradiation.

The effects of laser etching on dentin are studied by microenergy-dispersive x-ray fluorescence spectrometry (µ-EDXRF) and scanning electron microscopy (SEM) to establish the correlation of data obtained. Fifteen human third molars are prepared, baseline µ-EDXRF mappings are performed, and ten specimens are selected. Each specimen received four treatments: acid etching (control-CG) or erbium:yttrium-aluminum-garnet (Er:YAG) laser irradiation (I-100 mJ, II-160 mJ, and III-220 mJ), and maps are done again. The Ca and P content are significantly reduced after acid etching (p<0.0001) and increased after laser irradiation with 220 mJ (Ca: p<0.0153 and P: p=0.0005). The Ca/P ratio increased and decreased after CG (p=0.0052) and GI (p=0.0003) treatments, respectively. CG treatment resulted in lower inorganic content (GI: p<0.05, GII: p<0.01, and GIII: p<0.01) and higher Ca/P ratios than laser etching (GI: p<0.001, GII: p<0.01, and GIII: p<0.01). The SEM photomicrographies revealed open (CG) and partially open dentin tubules (GI, GII, and GIII). µ-EDXRF mappings illustrated that acid etching created homogeneous distribution of inorganic content over dentin. Er:YAG laser etching (220 mJ) produced irregular elemental distribution and changed the stoichiometric proportions of hydroxyapatite, as showed by an increase of mineral content. Decreases and increases of mineral content in the µ-EDXRF images are correlated to holes and mounds, respectively, as found in SEM images.

Apical sealing quality of in vitro apicectomy procedures after using both Er:YAG and Nd:YAG.

The aim of this study was to evaluate the apical sealing of dentinal tubules after root-end surface cutting by using Er:YAG and Nd:YAG lasers. After root-canal instrumentation and filling, apices of 50 extracted maxillary canine human teeth were resected by Er:YAG with 400 mJ, 10 Hz, for 30 sec. The samples were randomly assigned to five groups (n = 10): (GI) treated without root-end cavity, but with Nd:YAG (1.0 W, 10 Hz, 20 sec) for dentinal tubules sealing; (GII) treated with root-end cavity without the use of Nd:YAG; (GIII) treated with root-end cavity and Nd:YAG application; (GIV) treated with root-end cavity made by Er:YAG with no focus and without Nd:YAG application; and (GV) treated without root-end cavity and without Nd:YAG application. The root-end cavities were performed by using Er:YAG at 300 mJ, 10 Hz, for 20 sec. Subsequently, all teeth were waterproofed and immersed in 2% methylene blue for 48 h in a vacuum environment. The samples were longitudinally sectioned, and microleakage was measured. ANOVA and the Fisher LSD test showed that GIV was less susceptible to microleakage than were the other groups (p < 0.05). Interestingly, the use of the Er:YAG with no focus showed superior dentinal tubule sealing in comparison with the other groups, even with or without root-end cavity and Nd:YAG application.