Transcrição reversa e reação em cadeia da polimerase: princípios e aplicações em odontologia

Various molecular biology techniques have become available in the last few years. One of the most revolutionary of these techniques regarding nucleic acid analysis is the polymerase chain reaction (PCR), which was first described in 1985. This method relies on the exponential amplification of specific DNA fragments, resulting in millions of copies that can serve as templates for different kinds of analyses. PCR can be preceded by a reverse transcription (RT) reaction in order to produce cDNA from RNA (RT-PCR). RT-PCR provides the possibility to assess gene transcription in cells or tissues. PCR and RT-PCR techniques have been instrumental in dental research, and show potential to be used for diagnosis as well as for treatment and prevention of many diseases (dental caries, periodontal disease, endodontic infections and oral cancer). Compared to other traditional methodologies, PCR and RT-PCR show many advantages including high specificity, sensitivity, and speed. Since PCR and RT-PCR are relatively new techniques and are not available to most students and professionals involved with dentistry, the aim of this work is to present the details of these techniques as well as dental literature reports in which they were used.Várias técnicas de biologia molecular têm sido disponibilizadas nos últimos anos. Uma que revolucionou a análise de ácidos nucléicos foi a reação em cadeia da polimerase (PCR), descrita pela primeira vez em 1985. Esta técnica baseia-se na possibilidade de amplificação exponencial de fragmentos específicos de DNA, com a criação de milhões de cópias que servirão como matéria-prima para diferentes tipos de análises. A PCR pode ser precedida por uma reação de transcrição reversa (RT) para a obtenção de cDNA a partir de RNA (RT-PCR), representando, por exemplo, uma possibilidade de análise de expressão gênica em células ou tecidos. As técnicas de PCR e RT-PCR têm sido utilizadas em pesquisas odontológicas como ferramentas auxiliares para nortear não só o diagnóstico como também o tratamento e prevenção de várias doenças (cárie, doença periodontal, lesões endodônticas e câncer), uma vez que se mostram vantajosas em comparação a outras técnicas tradicionais pela sua alta especificidade, alta sensibilidade e rapidez. Por se tratar de técnicas relativamente novas e não disponíveis para a maioria dos profissionais e estudantes de Odontologia, o objetivo do presente trabalho é apresentar detalhes da PCR e da RT-PCR, bem como trabalhos da literatura em que ambas foram utilizadas

Oral antibacterial effect of chlorhexidine treatments and professional prophylaxis in children

Aim: The primary aim of this longitudinal study was to evaluate additional effects of 4-week chlorhexidine digluconate (CHX) gel treatments to control Aggregatibacter actinomycetemcomitans counts in children after professional dental prophylaxis. Porphyromonas gingivalis and Streptococcus mutans counts were also determined to evaluate the secondary effects of anti-plaque treatments on microbial shifts. Methods: Twenty-six children with A. actinomycetemcomitans counts >4 log10/ mL of saliva and/or Quigley-Hein plaque index >3.0 were enrolled in this study. Patients were randomly assigned to groups GI (placebo gel), GII (0.5% CHX gel), GIII (1% CHX gel), and GIV (2% CHX gel). Four sessions of treatment were performed during 4 weeks after a session of professional dental prophylaxis. Real-Time polymerase chain reaction (PCR) was used to determine viable microorganism counts in non-stimulated whole saliva samples collected at baseline, one week, one month and three months after interruption of treatments. Results: A reduction of all bacterial counts was detected after the 3-month follow-up in all groups. Lower counts of P. gingivalis were achieved from 1 week on after treatments. The 2% CHX concentration seemed to contribute to lower A. actinomycetemcomitans levels and increase S. mutans levels. Conclusions: Professional dental prophylaxis was effective to control salivary levels of A. actinomycetemcomitans, P. gingivalis and S. mutans. Additional antimicrobial effects, however, were not observed by the combination of professional dental prophylaxis and 4-week chlorhexidine gel treatments

Perfil de tratamento de urgência de crianças de 0 a 15 anos atendidas no serviço de urgência odontológica da Faculdade de Odontologia de Bauru, Universidade de São Paulo

Dental recordings of 0 to 15-year-old patients assisted at Urgency Dental Service (UDS) from Bauru Dental School, University of São Paulo, in 2001 and 2002, were assessed in order to quantify the number of patients that used the service, to determine attendance patterns, and to record the frequency of different types of dental emergencies and their performed treatment. Data were plotted and submitted to a descriptive statistical analysis. Among the total of patients attended at UDS (6020), 1166 (19.37%) were children, with mean age of 9.24 years. Trauma was the cause for 199 (17.06%) of the recorded urgency visits. It occurred more frequently in children between 0 and 3 years of age (34.42%), and between 7 and 12 years of age (18.12%). The main treatments performed were temporary restoration (33.33%) for coronal fracture, and orientation (24.44%) for luxation. Nontraumatic events were the etiology for 967 (82.92%) of the total urgency diagnosis. The most commonly found nontraumatic diagnosis was dental caries lesions (61.75%), followed by problems of eruption or root resorption (14.27%) and bone or soft tissue lesions (6.51%), among others (17.47%). The most frequent treatments performed for caries lesions were: excavation and temporary restoration (39.39%) when there was no abscess, and coronal opening and dressing (40.95%) for caries lesions with abscess. There was an increasing trend in caries lesions prevalence according to the rising of the age, in contrast to trauma prevalence. Treatment for both situations was done according to the indicated protocol for each case.Prontuários odontológicos de crianças de 0 a 15 anos de idade atendidas no Setor de Urgência Odontológica (SUO) da Faculdade de Odontologia de Bauru, Universidade de São Paulo, em 2001 e 2002, foram avaliados a fim de se quantificar o número de pacientes que utilizaram o serviço, determinar os padrões de atendimento e relatar a freqüência de diferentes tipos de emergências odontológicas e dos tratamentos realizados. Os dados foram tabulados e submetidos a uma análise estatística descritiva. Do total de pacientes atendidos no SUO (6020), 1166 (19,37%) eram crianças, com média de idade de 9,24 anos. Lesões traumáticas foram responsáveis por 199 (17,06%) do total de visitas de emergência. Isso ocorreu mais freqüentemente em crianças entre 0 e 3 anos (34,42%), e entre 7 e 12 anos (18,12%). Os principais tratamentos realizados foram restauração temporária (33,33%) para fratura coronária, e orientação (24,44%) para luxação. Eventos não-traumáticos foram responsáveis por 967 (82,92%) do total de diagnósticos de emergência. O diagnóstico mais comumente encontrado foi cárie dentária (61,75%), seguida por problemas de irrupção e reabsorção óssea (14,27%) e lesões em tecido duro ou mole (6,51%), entre outros (17,47%). Os tratamentos realizados com maior freqüência para os casos de cárie foram: escavação e restauração temporária (39,39%) quando não havia abscesso, e abertura coronária e curativo (40,95%) para cáries com abscesso. Houve uma tendência para o aumento na prevalência de cárie com a idade. O oposto foi observado para traumatismo dentário. O tratamento para ambos estava de acordo com o preconizado para este tipo de serviço

A randomized clinical trial of cavity liners after selective caries removal: one-year follow-up

Alternatives for the treatment of caries disease, such as minimally invasive approaches, have been developed in recent years. Objective: To carry out clinical and radiographic evaluations of three cavity liners after selective caries removal. Methodology: Thirty-six primary molars with deep occlusal caries lesions without pulp involvement (from children of both genders, aged between 5 and 8 years) were randomly divided into the following groups: calcium hydroxide cement (CHC) group; mineral trioxide aggregate (MTA) group and Portland cement with added zirconium oxide (PCZ) group. The following-up period was 6- and 12-month. The clinical and radiographic success rates were evaluated through chi-square test. The radiographic measurements were compared by ANOVA followed by Tukey's test (p<0.05). Results: Thirty-six patients were included, but thirty-four returned for 12-month follow-up. The overall success rate of the therapy for the three groups was 94.11% and no statistically significant differences occurred in the comparison among groups (p>0.05). Nineteen radiographs were selected to measure the dentin barrier thickness. The intragroup comparison presented a statistically significant increase of the dentin barrier for all groups, at 12-month follow-up. However, the MTA group showed increase of the dentin barrier, over time, 6- to 12-month follow-up. The intergroup comparison revealed no statistically significant differences (p>0.05). Conclusion: The clinical and radiographic data showed that all cavity liners provided effective treatment of primary teeth after selective caries removal

Comparison between the rotary (Hyflex EDM®) and manual (k-file) technique for instrumentation of primary molars: a 12-month randomized clinical follow-up study

Rotational instrumentation is an alternative for the clinical practice of pediatric dentists. However, there are few records in the literature on the clinical and radiographic aspects of treated teeth over time. Objectives: Compare instrumentation time and filling quality between manual (k-file) and rotary (Hyflex EDM®) files, and clinically and radiographically follow-up the treated teeth for 12 months. Moreover, the characteristics of glass ionomer restorations and their interference in the treatment prognosis over time were evaluated. Methodology: In total, 40 children with pulp involvement in primary molars received treatment with Hyflex EDM® or manual rotary files, performed by an operator. Clinical and radiographic aspects were observed at different times to determine the effectiveness of each technique. Results: The rotary system reduced instrumentation time when compared to the use of manual files (p≤0.05), but there was no difference in filling quality between the groups (p≥0.05). Moreover, both types of instrumentation were effective for 12 months (p≥0.05), and restoration retention influenced the emergence of periapical lesions (p≤0.05). Conclusion: Although rotary files reduce clinical time, the clinical and radiographic aspects of both techniques were similar over 12 months. Moreover, restoration retention has been shown to be related to treatment prognosis

Effects of low-level laser therapy on stem cells from human exfoliated deciduous teeth

Low-Level Laser Therapy stimulates the proliferation of a variety of types of cells. However, very little is known about its effect on stem cells from human exfoliated deciduous teeth (SHED). Objective This study aimed to evaluate the influence of different laser therapy energy densities on SHED viability and proliferation. Material and Methods SHED were irradiated according to the groups: I (1.2 J/cm2 - 0.5 mW – 10 s), II (2.5 J/cm2 – 10 mW – 10 s), III (3.7 J/cm2 – 15 mW – 10 s), IV (5.0 J/cm2 – 20 mW – 10 s), V (6.2 J/cm2 – 25 mW – 10 s), and VI (not irradiated – control group). Cell viability was assessed 6 and 24 h after irradiation measuring the mitochondrial activity and using the Crystal Violet assay. Cell proliferation was assessed after 24, 48, and 72 h of irradiation by SRB assay. Results MTT assay demonstrated differences from 6 to 24 hours after irradiation. After 24 h, groups I and IV showed higher absorbance values than those of control group. Crystal Violet assay showed statistically differences in the absorbance rate from 6 to 24 h after irradiation for groups III and VI. At 24 h after irradiation, Group III absorbance rate was greater than that of groups I, II, and IV. Group VI absorbance rate was greater than that of groups I and IV. SRB assay showed that the group I had higher rates than those of groups II, III, V, and VI, at 24 h after irradiation. After 48 h, group I exhibited the greatest cell proliferation rate followed by groups III, V, and VI. After 72 h, group III exhibited the lowest cell proliferation rate than those of groups II, IV, and V. Conclusions The Low-Level Laser Therapy energy densities used in this study did not cause loss of cell viability and stimulated SHED proliferation within the parameters described in this study

Surgical techniques for the treatment of ankyloglossia in children: a case series

This paper reports a series of clinical cases of ankyloglossia in children, which were approached by different techniques: frenotomy and frenectomy with the use of one hemostat, two hemostats, a groove director or laser. Information on the indications, contraindications, advantages and disadvantages of the techniques was also presented. Children diagnosed with ankyloglossia were subjected to different surgical procedures. The choice of the techniques was based on the age of the patient, length of the frenulum and availability of the instruments and equipment. All the techniques presented are successful for the treatment of ankyloglossia and require a skilled professional. Laser may be considered a simple and safe alternative for children while reducing the amount of local anesthetics needed, the bleeding and the chances of infection, swelling and discomfort

Pulp therapy in deciduous teeth: therapeutic possibilities based on evidences

Introduction: Pulpotomy of primary teeth has been the treatment of choice in cases of inflammation of the coronal pulp caused by caries or trauma with no involvement of the radicular pulp, thus avoiding the premature loss of the teeth. Not only is the diagnosis of the inflamed dental pulp required for this therapy to succeed, but also the selection of an effective and biocompatible medicament. However, none of the drugs available and recommended for pulpotomy therapy present all the requirements of an ideal pulp capping agent. Objective: Through a systematic literature review, between the years of 2000 and 2011, focusing in randomized clinical trials, systematic reviews and meta-analysis, aiming to analyze and discuss based on alternatives for the treatment of pulpotomy in human deciduous teeth. Conclusion: Scientific evidences showed that the medicaments and techniques influence were few. Thus, further and well-designed studies are suggested for clarification. The information generated in such experiments may contribute to a better understanding of the mechanisms of pulp therapy to new therapeutic approaches aimed at improving pulp therapy in deciduous teeth.Introdução: A pulpotomia em dentes decíduos é uma técnica conservadora de terapia pulpar amplamente utilizada \ud em Odontopediatria, sendo de fundamental importância para evitar a perda prematura desses dentes, quer seja \ud por alterações provocadas pela cárie dentária ou traumatismo dentário. Apesar de ser uma técnica estudada há \ud muitos anos, causa muitas controvérsias e discussões, principalmente em termos de biocompatibilidade dos \ud medicamentos empregados e pelas dificuldades e falhas no diagnóstico da condição pulpar. Objetivo: Por meio \ud de uma revisão sistemática da literatura, no período compreendido entre 2000 e 2011, e com enfoque em estudos \ud clínicos randomizados, revisões sistemáticas e meta-análises, este trabalho teve como objetivo discutir, com base em \ud evidências científicas, alternativas para o tratamento de pulpotomia em dentes decíduos humanos. Conclusão: As \ud evidências científicas fidedignas com o uso de materiais capeadores pulpares e as técnicas de Eletrocirurgia e Laser \ud de Baixa Potência foram escassas. Desta forma, sugere-se a realização de estudos complementares bem delineados \ud estatisticamente para maiores esclarecimentos. As informações geradas em tais experimentos poderão contribuir \ud para um melhor entendimento dos mecanismos da terapia pulpar, podendo gerar o desenvolvimento de protocolos \ud com novas formas terapêuticas, que visam a melhorar a terapia pulpar em dentes decíduo

Pulpotomies with portland cement in human primary molars

Two clinical cases in which Portland cement (PC) was applied as a medicament after pulpotomy of mandibular primary molars in children are presented. Pulpotomy using PC was carried out in two mandibular first molars and one mandibular second molar, which were further followed-up. At the 3, 6 and 12-month follow-up appointments, clinical and radiographic examinations of the pulpotomized teeth and their periradicular area revealed that the treatments were successful in maintaining the teeth asymptomatic and preserving pulpal vitality. Additionally, the formation of a dentin bridge immediately below the PC could be observed in the three molars treated. PC may be considered as an effective alternative for primary molar pulpotomies, at least in a short-term period. Randomized clinical trials with human teeth are required in order to determine the suitability of PC before unlimited clinical use can be recommended

Initial inflammatory response after the pulpotomy of rat molars with MTA or ferric sulfate

Purpose: To compare, both qualitatively and quantitatively, the inflammatory cells, vascular density and IL-6 immunolabeled cells present in the pulp after pulpotomy with white MTA versus 15.5% ferric sulfate (FS). Methodology: Forty-eight mandibular first molars from 24 Wistar rats were divided into MTA or FS groups and subdivided according to the period after pulpotomy procedure (24, 48 and 72 hours). Four teeth (sound and untreated) were used as controls. Histological sections were obtained and assessed through the descriptive analysis of morphological aspects of pulp tissue and the quantification of inflammatory cells, vascular density and interleukin-6 (IL-6) expression. Data were statistically analyzed (p<0.05). Results: The number of inflammatory cells was similar in both groups, being predominantly localized at the cervical radicular third. In the MTA group, increased inflammation was observed at 48 hours. Vascular density was similar in both groups and over time, being predominant in the medium radicular third. No correlation was found between the number of inflammatory cells and the vascular density. Pulp tissue was more organized in MTA-treated teeth. In both groups, a weak to moderate IL-6 expression was detected in odontoblasts and inflammatory cells. Comparing both groups, there was a greater IL-6 expression in the cervical radicular third of teeth treated with MTA at 24 hours and in the medium and apical thirds at 72 hours, while in the FS group a greater IL-6 expression was found in the apical third at 24 hours. Conclusion: The MTA group presented better histological features and greater IL-6 expression than the FS group. However, no difference was observed between the groups regarding the inflammatory status and vascularization, suggesting the usefulness of FS as a low-cost alternative to MTA