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A clean smile

Studies on using LEDs against bacteria and fungi are advancing

EDUARDO CESARAbove, LED-equipped rod for treatment of the inside of the mouth. Below, dishes with microorganisms for studying under the lightEDUARDO CESAR

LEDs may soon be used in Brazil to destroy bacteria and fungi that harm our oral health. A study involving a number of institutions in the country, led by the physicist Vanderlei Salvador Bagnato, a professor at the University of São Paulo”s Physics Institute at São Carlos (IFSC-USP), is being finalized jointly with the company Gnatus, from the city of Ribeirão Preto. This company produces medical and dental equipment. Since 2009, Bagnato”s team has been conducting studies to create and test a mouth decontamination treatment using a technique called photodynamic therapy (PDT). The same procedure is already being used experimentally for other purposes, such as lesions on external parts of the body in the treatment of diseases such as skin cancer and leishmaniasis, or to treat burns.

Until the beginning of the last century, man had few weapons with which to fight fungi and bacteria, other than his own immune system, which was often defeated. The situation started changing in 1928, when the Scottish bacteriologist Alexander Fleming discovered penicillin, the first antibiotic, which became a medical drug as from 1941. It then looked as if humankind was the winner, but this turned out to be a mistaken assumption. The bacteria revealed themselves to be a more powerful enemy than people had imagined. With each new antibiotic created, they develop resistance. Today, there are superbacteria that are immune to the most powerful drugs. This is where photodynamic therapy comes into the picture. It consists of using light from lasers or light emitting diodes (LEDs) to kill microorganisms. In the case of Bagnato”s team, the dental studies involve LEDs.

The technique is relatively simple. The first step is to apply on the infected region a photosensitizing substance, normally in liquid form, allowing it to act for a few minutes. During this time, it is absorbed by the microorganisms or adheres to their external membrane. Then, a colored light (blue or red, for instance), with the precise electromagnetic wavelength that is most appropriate for each case, is directed at the treated area. This excites the molecules of the photosensitizer and reacts with the oxygen found in that environment or in the microorganism. In this reaction, this element loses electrons while highly reactive free radicals are formed. “These, in turn, oxidize the area where they are, leading the microorganisms” membrane to rupture, which causes them to die,” explains the dentist Cristina Kurachi, from IFSC-USP, who is on Bagnato”s team. The study also includes researchers from the Dental School of Paulista State University (Unesp) at Araraquara, the Dental School of the Federal University of Bahia (UFBA), the State University of Ponta Grossa (UEPG), in Paraná state, the Federal University of São Carlos (UFSCcar), the School of Veterinary Medicine and Zootechnology of Unesp at Botucatu, and the Sírio-Libanês Hospital in the São Paulo state capital.

EDUARDO CESARUltrasound device combined with LED for dental hygieneEDUARDO CESAR

For this therapy to be used by physicians and dentists, first a safe protocol that determines the treatment parameters must be established. Thus, one must know which photosensitizing substance is the most effective against the microorganism, the light of what color should be applied for what length of time. Bagnato”s team has progressed in the sense of finalizing the protocol. They tested three photosensitizers: porphyrin, a drug made from a substance also found in blood; methylene blue; and curcuminoid salt, made from curcumin extracted from saffron. The studies of this last photosensitizer included Professor Ana Claudia Pavarina, from the Unesp Dental School at Araraquara. The first two photosensitizers are activated with a red light of a wavelength of 630 to 660 nanometers (nm), and the third, with a blue light of 450 nm.

According to Cristina, these photosensitizers, lit by the right light for different lengths of time, were tested on microorganisms such as the bacteria Porfiromonas gingivalis that cause gum or periodontal diseases, Streptococcus mutans and Lactobacillus casei, responsible for caries, and Staphylococcus aureus, which causes hospital infections. “We also tested this against the fungus Candida albicans, which causes the prosthetic candidiasis that can affect those who wear dental prostheses,” she explains. “We got our best results with porphyrin and the curcuminoid salt.” For her, one of the treatments nearest to being developed is the treatment for periodontal diseases, for which the clinical protocol is close to being finalized.

However, treatments and protocols are not the only outcome of the project, which also yielded technological progress. “We developed several instruments and pieces of equipment during the course of the research,” says Bagnato. “Many of them are to be released onto the market.” The researchers explained what they needed and Gnatus, along with the university, made the tools. One of these is a mouth decontamination kit that comprises several metal rods, each of which has an LED at its end. What differentiates them is the position of this light emitter, which is determined by the area of the mouth on which the light is to shine. Thus, the one that aims at the inner cheek has its LED on the side of the rod”s end. The one that shines on the tongue is shaped like a small racket, while the one used to access the entire mouth cavity has a rounded point. The team has also developed an ultrasound device for periodontal treatment, with an LED attached to it. “While dental plaque is removed, the device conducts photodynamic therapy,” explains Cristina. This equipment should get to the market within one year.

EDUARDO CESARDish for the evaluation of the efficiency of photodynamic therapyEDUARDO CESAR

Light therapy
Even though the concept is an old one, having been around for more than 40 years, PDT research is relatively recent throughout the world, which is why there is no such treatment being used routinely to date. All PDT treatments are experimental. Even in developed countries, PDT is still at the stage of study and determination of protocols. “The earliest works in literature on the effects of photodynamic therapy upon oral bacteria date from 1992 and were conducted by Professor Brian Wilson, from the Ontario Cancer Institute in Canada,” he explains. “They tested the bactericidal potential of several photosensitizing agents.” One year later, new studies were published showing that PDT”s antimicrobial action was efficient against the bacteria that cause caries, such as Streptococcus mutans, Lactobacillus casei and Actinomyces viscosus, which are found in human dentin.

The rising interest in PDT is no accident. “Given forecasts concerning the end of the era of antibiotics, due to resistance to treatment developed by microorganisms, photodynamic therapy for microbe control has become highly important,” says Bagnato. “Additionally, it might provide several advantages relative to the traditional antimicrobial agents. One of them is that the death of the bacteria is quick, reducing the need to maintain high concentrations of chemical substances for a long time, as is the case with antibiotics and antiseptics. Furthermore, photodynamic therapy conserves the healthy tissue.” Secondly, Bagnato mentioned that PDT does not allow microorganisms to develop resistance. “As the death of the bacteria is not linked to the mediation of chemical radicals, resistance is unlikely to develop,” he explains. “Another advantage is that neither the photosensitizer nor the light used are bactericides when applied independently, so that the death of bacteria can be controlled, being limited only to the irradiated area, which avoids the destruction of the microbiota in other areas. Moreover, this technique allows countless applications, with no associated side effects provided suitable protocols are used.”

The low cost of the treatment is yet another advantage of PDT. Until recently, lasers were the most often used PDT light source. Though efficient, they are expensive. “Now, thanks to the development of LEDs, studies have arisen using this source of light in PDT,” Bagnato tells us. “Comparing the efficacy of LEDs and of lasers, it became apparent that LED emitting devices are far less expensive but yield a similar photodynamic response. Thus, with inexpensive sources of light and of photosensitizers, this type of treatment becomes financially viable.” So much so that the Brazilian team is already thinking about new photodynamic therapy applications. They have already requested patents for applications such as body decontamination and the treatment of mycoses, seborrhea and pneumonia. In the case of the latter, the lighting would come from outside the body toward its inside and the photosensitizing substance would be inhaled by the patient. The treatment might turn out to be faster than the traditional antibiotics one. The research is unlikely to stop at this. “The field is still new and is developing fast, with a lot of room for contributions from other groups,” says Bagnato.

To conduct the project that is under way which is expected to take three years, the team received funding in the amount of R$1.5 million from Finep (the Studies and Projects Finance Agency), R$1 million from Gnatus, R$300 thousand from the São Carlos Center of Optics and Photonics Research, coordinated by Bagnato, and R$300 thousand from the National Institute of Optics and Photonics. In addition to the progress of the development of equipment for photodynamic therapy, the research studies, to date, have yielded about twenty scientific articles and five patents.

The Project
Technological innovation program of the São Carlos Center of Optics and Photonics Research (Cepof) (nº 98/14270-8); Type Centers for Research, Innovation and Dissemination (Cepids); Coordinator Vanderlei Salvador Bagnato – USP; Investment R$ 300,000.00 for the dentistry LEDs/PDT project (FAPESP)

Scientific articles
MIMA, E. G. O. et al. Susceptibility of Candida albicans to photodynamic therapy in a murine model of oral candidosis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. v. 109, n. 3, p. 392-401. 2010.
GOIS, M. M. et al. Susceptibility of Staphylococcus aureus to porphyrin-mediated photodynamic antimicrobial chemotherapy: an in vitro study. Lasers Medical Science. v. 25, n. 3, p. 391-35. 2010.