Avaliou-se a proliferação das células tronco da polpa de dentes decíduos esfoliados humanos (SHED) após aplicação única do laser de baixa potência. Foi realizada a análise da viabilidade das SHED cultivadas sob déficit nutricional e em condições ideais após irradiação com o laser de baixa potência vermelho de Indio Gálio Alumínio e Fósforo - InGaAlP (660nm, 40mW e 10J/cm2) e infravermelho (780nm, 40mW e 10J/cm2) durante 4 e 8 segundos, nos períodos de 24, 48 e 72 horas através dos ensaios de redução do MTT e do ensaio colorimétrico de Busatti e Gomes. Para análise estatística utilizou-se o teste ANOVA complementado pelo teste de Tukey com nível de significância de 5% (p< 0,05). Observou-se tanto com o MTT quanto com o ensaio colorimétrico de Busatti e Gomes uma tendência de aumento da proliferação celular diretamente relacionada à dose do LBP, estatisticamente significante nos períodos de 24, 48 e 72 horas. Ao analisar os resultados e considerando os parâmetros utilizados e o protocolo de LBP, pode-se concluir que o LBP promoveu a proliferação das SHED tanto a 660 nm quanto a 780nm, pode influenciar a viabilidade e a proliferação das SHED nas doses e comprimentos de onda utilizados e os ensaios do MTT e colorimétrico de Busati e Gomes demonstraram dentro de suas limitações ser eficientes para determinar a viabilidade e proliferação das SHED. / It was evaluated the proliferation of stem cells from human exfoliated deciduous teeth (SHED) after a single application of low power laser. The viability of SHED grown under ideal conditions and under nutritional deficit after irradiation with red laser (660/780nm, 10J/cm2 and 40mW) during periods of 4 and 8 seconds was analyzed through the MTT reduction assays and rapid colorimetric assay of Busatti and Gomes. Statistical analysis was performed using the ANOVA and Tukey´s multiple comparisons test with a significance level of 5% (p < 0.05). It was observed with the MTT assay and Busatti and Gomes assay a trend of cell proliferation increase directly releated to the irradiation dose, statistically significant. After 24, 48 and 72 hours, all the groups showed higher cell proliferation when compared to control. Analyzing the results and considering the used parameters and LBP protocol, it can be concluded that LBP promoted the proliferation of SHED both 660nm and 780nm according to the dosage and wavelengths used, and MTT assay and colorimetric Busatti and Gomes demonstrated within their limitations to be effective in determining the viability and proliferation of SHED.

Abstract Dentin forms the hard tissue portion of the dentin-pulp complex, while the dental pulp is soft connective tissue that retains the vitality of the dentin. Odontoblasts form the outermost cell layer of pulp and play a central role during dentin formation by producing and mineralizing the dentin matrix. The understanding of the defensive reactions in the dentin-pulp complex is limited. Information about the transcriptome and proteome of pulp tissue and odontoblasts would facilitate understanding of their functions during health and disease. The aim of this study was to investigate the expression profiles of human pulp tissue and odontoblasts in vivo and in vitro using large-scale expression analysis methods. Also, the suitability of these methods in pulp biological research in vivo and in vitro was evaluated. cDNA microarray revealed only minor variation and 2-D electrophoresis combined with mass spectrometry revealed no differences between healthy and carious teeth pulp tissue in vivo. The effect of transforming growth factor β1 (TGF-β1) on pulp and odontoblasts was studied in vitro using oligonucleotide-based microarrays, and marked changes in the transcriptome were revealed, especially in the expression of chemokine- and cytokine-related genes. Transiently increased interleukin expression was confirmed at the protein level by antibody array. DNA microarray analysis of native pulp tissue and odontoblasts was used to search for potential odontoblast markers. Only one gene related to extracellular matrix organization and biogenesis, matrilin 4, and two expressed sequence tags (ESTs), which represent transcribed sequences encoding possibly unknown genes, were identified in odontoblasts but not in pulp. Analysis of mature native odontoblasts and cultured odontoblast-like cells by DNA microarray revealed a high similarity (84%) between native and cultured cells. Also, differential expression levels of selected neuronal proteins were observed and confirmed at the mRNA and protein levels. In conclusion, microarray is a powerful tool for pulp biology, especially for in vitro studies. TGF-β1 was revealed as a potent regulator of proinflammatory responses in the dentin–pulp complex. In addition, several potential odontoblast markers were identified by microarray, and the similarity of cultured odontoblast-like cells used in the study with native odontoblasts was confirmed.

The cornea is the transparent, avascular and highly innervated outer anterior layer of the eye. The cornea is a very delicate structure and any traumatic insult may lead to damage and limbal stem cell deficiency (LSCD), leading to chronic discomfort, visual impairment and ultimately blindness. The resultant issues can have a significant effect on patients and reduce their quality of life. Whilst conservative and therapeutic management of these problems play a part in the treatment of corneal injuries often surgery is indicated. However, surgical repair of damaged corneas may be limited by the availability of suitable donor tissue and donor site morbidity. Corneal grafts or penetrating keratoplasty (PK) or donor limbal grafts may lead to surgical complications such as corneal scarring, infection and graft rejection. First described in 1908 by A. Maximow, stem cells offer the opportunity to produce functional cell specific tissues from undifferentiated “primordial” cells. By using stem cells from human adult or deciduous tooth pulp, repair and regeneration of the cornea may be possible. Furthermore, it may lead to development of new and innovative treatments of other corneal disorders and injuries. The aim of the investigations detailed in this thesis was to characterize dental pulp stem cells (DPSC), help establish their use in regenerative medicine and help enhance the repair and regeneration of damaged corneal epithelium. Using various laboratory techniques including PCR, western blot and immunostaining it was determined that DPSC possess adequate potency and plasticity to be differentiated into a number of cell-lines. Co-culture of DPSC with human cornea demonstrated that stem cells were attracted to the tissue and migrate towards it and attach to the surface of the limbal explant. Additionally, using soft contact lenses it has been shown that DPSC can be successfully transferred from culture to human cornea in vitro. Expression of terminally differentiated corneal epithelium markers such as cytokeratin 3 & 12 further supports the concept that DPSC were transdifferentiated into epithelial progenitor cells. Once transferred onto the corneal surface, DPSC supported corneal epithelium regeneration, allowed corneal epithelial like cells to grow and avert conjunctivalisation and thus maintained cornea transparency. Further studies are needed to provide a better understanding of the DPSC’s role in corneal regeneration, but it is clear that DPSC are promising candidates for this novel and non-invasive method of corneal epithelium regeneration.

Magister Scientiae Dentium - MSc(Dent) / The purpose of this study was to determine duration time, breakage and apical displacement, whilst using six different endodontic filing systems to prepare molar teeth. A total of 96 molar teeth were used in the study, divided equally, ie 16 teeth per system selected randomly, totalling 48 canals per system. A standardised access cavity was prepared for all the teeth before selection. The canals were filed according to the manufacturers’ guidelines. The result showed that PROTAPER®, K3™ and the combination of: HERO Shaper®, HERO Apical® and Endoflare® (Referred from hereon as HERO System for convenience) were statistically faster than PROFILE® and FlexMaster®, which were in turn faster than AETTM. Although breakage did occur in K3™ and HERO System this was not deemed statistically significant. Apical displacement occurred in the form of Type 1 in the AETTM, PROFILE® and HERO System, but once again this was not statistically significant. It was concluded that more aggressive cutting features such as a positive rake angle, pyramidal shaped tip, progressive taper and absence of radial lands, if present, could have enabled K3™, HERO System and PROTAPER® to have faster times, and in addition these features did not compromise these systems with regard to apical foramina transportation and breakage. / South Africa

Orientador: Milton Carlos Kuga / Resumo: Os objetivos do presente estudo foram avaliar os efeitos dos solventes para remoção de resíduos de cimento endodôntico à base de resina epóxi (Sealer Plus) da dentina da câmara pulpar e a repercussão sobre a interface adesiva, com o sistema adesivo Universal (Single Bond Universal e Ambar Universal). Foram selecionados 90 incisivos bovinos com coroa dental similar, os quais foram seccionados, obtendo-se fragmentos da câmara pulpar, na proporção de 5x5x5mm para avaliação da microscopia eletrônica de varredura (MEV), e seus efeitos sobre a dentina da câmara pulpar, por meio de microscopia EDX e mensuração da microdureza dentinária, após o uso dos protocolos de limpeza contendo acetato de amila (AA), acetona pura (AC) ou etanol a 95% (ET) e combinações entre cada um destes protocolos (soluções experimentais AA-AC, AA-ET, AC-ET e AA-AC-ET), além dos controles positivo (PC) e negativo (NC). Além disso, quarenta fragmentos de dentina foram impregnados com cimento endodôntico e removidos com os protocolos de limpeza: etanol a 95% (ET), xilol (XI), acetato de amila (AA) ou ação mecânica (PC) e dez espécimes não receberam tratamento (NC) para avaliação também da persistência de resíduos e a repercussão sobre a interface adesiva. A avaliação da persistência de resíduos foi realizada em MEV (500x) e após, foram obtidos 100 espécimes, onde 80 foram impregnados e submetidos aos protocolos de limpeza e 20, não. Em cada protocolo, os espécimes foram divididos em 2 subgrupos (n=10), de acord... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Indiana University-Purdue University Indianapolis (IUPUI) / Background: Root canal disinfection is an essential requirement for the success of regenerative endodontics. Currently, the so-called triple antibiotic paste (TAP) is considered the standard of care. Notwithstanding the good antimicrobial capacity, the high concentration of TAP has shown significant toxicity to human cells, especially dental pulp stem cells. A novel drug release system, i.e., a triple antibiotic paste-mimic electrospun scaffold containing low concentrations of the antibiotics present in the TAP, has emerged as an effective and reliable alternative to fight root canal infections without potential toxic effects on dental stem cells, which are an integral part of the regenerative treatment. Objectives: The aim of this study was to determine the antimicrobial efficacy of an innovative three-dimensional (3D) triple antibiotic paste-mimic tubular scaffold against Actinomyces naeslundii biofilm formed inside human root canal dentinal tubules. Materials and methods: Pure polydioxanone (PDS) polymer solution and PDS loaded with metronidazole, ciprofloxacin and minocycline (35 wt.% of each antibiotic, 3D-TAP-mimic scaffold) were spun into 3D fibrous scaffolds. A. naeslundii (ATCC 43146) was centrifuged to induce biofilm formation inside human root canal dentinal tubules using a dentin slice model (1 mm thickness and 2.5 mm canal diameter). The infected dentin slices were exposed to the 3D-TAP-mimic scaffold, TAP solution (50 mg/mL of each antibiotic), and antibiotic-free PDS. Biofilm elimination was quantitatively and qualitatively analyzed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM), respectively. Results: A dense penetration of A. naeslundii biofilm was observed by CLSM throughout the dentinal tubules. 3D-TAP-mimic scaffold significantly reduced the percentage of viable bacteria compared with PDS (p <.05). TAP solution completely eliminated viable bacteria without differing from 3D-TAP-mimic scaffolds. SEM images showed results similar to CLSM. Conclusion: Collectively, the proposed tubular 3D-TAP-mimic scaffold holds significant clinical potential for root canal disinfection strategy prior to regenerative endodontics.

Yes / Implementing the principles of tissue engineering within the clinical management of non-vital immature permanent teeth is of clinical interest. However, the ideal scaffold remains elusive. The aim of this work was to assess the feasibility of decellularising rat dental pulp tissue and evaluate the ability of such scaffold to support stem cell repopulation. Rat dental pulps were retrieved and divided into control and decellularised groups. The decellularisation protocol incorporated a low detergent concentration and hypotonic buffers. After decellularisation, the scaffolds were characterised histologically, immunohistochemistry and the residual DNA content quantified. Surface topography was also viewed under scanning electron microscopy. Biocompatibility was evaluated using cytotoxicity assays utilising L-929 cell line. Decellularised scaffolds were recellularised with human dental pulp stem cells up to 14 days in vitro. Cellular viability was assessed using LIVE/DEAD stain kit and the recellularised scaffolds were further assessed histologically and immunolabelled using makers for odontoblastic differentiation, cytoskeleton components and growth factors. Analysis of the decellularised scaffolds revealed an acellular matrix with histological preservation of structural components. Decellularised scaffolds were biocompatible and able to support stem cell survival following recellularisation. Immunolabelling of the recellularised scaffolds demonstrated positive cellular expression against the tested markers in culture. This study has demonstrated the feasibility of developing a biocompatible decellularised dental pulp scaffold, which is able to support dental pulp stem cell repopulation. Clinically, decellularised pulp tissue could possibly be a suitable scaffold for use within regenerative (reparative) endodontic techniques.

Thesis (M.S.)--University of Michigan, 1993.

Thesis (M.S.D.)--Indiana University School of Dentistry, 2009. / Title from PDF t. p. (viewed Aug. 21, 2009) Advisor(s): Mychel Vail, Acting Chair of the Research Committee, Joseph Legan, Kenneth Spolnik, Susan L. Zunt, Edwin Parks. Curriculum vitae. Includes abstract. Includes bibliographical references (leaves 109-120).

Cavalcanti, BN. Resposta de fibroblastos de polpa humanos frente a substâncias liberadas por capeadores pulpares diretos [Tese de Doutorado]. São Paulo: Faculdade de Odontologia da USP; 2003. RESUMO O objetivo do presente estudo foi o de avaliar os efeitos citotóxicos de substâncias liberadas durante a aplicação de materiais utilizados em capeamento pulpar direto, sobre fibroblastos de polpa dentária humana. Utilizou-se para o experimento meios condicionados pelas substâncias a serem testadas, divididas nos grupos a seguir: grupo I: controle (meio de cultivo sem condicionamento); grupo II: cimento de hidróxido de cálcio; grupo III: adesivo dentinário; grupo IV: ácido ortofosfórico a 37%. O condicionamento foi realizado, colocando-se meio de cultivo fresco sobre os materiais de modo que a presa (grupo II), polimerização (grupo III) ou o contato direto (grupo IV) liberassem substâncias para esse meio de cultivo. Esse meio era colocado sobre as células durante todo o experimento, excetuando-se o grupo IV, onde o contato foi feito por um período de 15 segundos, conforme recomendações clínicas. Posteriormente foram realizadas contagens em hemocitômetro pelo método de exclusão por azul de Trypan, que cora somente as células mortas. As contagens foram realizadas em períodos de 0, 6, 12 e 24 horas para o experimento de viabilidade celular (curto prazo), onde se avaliou o percentual de células vivas sobre o total de células, e em períodos de 1, 3, 5 e 7 dias para o experimento de sobrevivência celular, no qual se avaliou o número absoluto de células vivas. Observou-se que as substâncias liberadas pelo adesivo dentinário são citotóxicas em qualquer período, diminuindo consideravelmente a viabilidade celular e afetando suas curvas de crescimento. Aquelas liberadas pelo ácido ortofosfórico a 37% provocam diminuição da viabilidade somente nos primeiros momentos do contato com as células, enquanto as substâncias liberadas durante a presa do hidróxido de cálcio não são citotóxicas em nenhum momento. / The purpose of the present study was to evaluate the cytotoxic effects of substances leached during the use of direct pulp capping materials, on human pulp fibroblasts. There were used cell culture mediums conditioned by the test materials, as follows: group I: control (fresh medium without conditioning); group II: calcium hydroxide cement; group III: bonding system; group IV: 37% orthophosphoric acid. The medium conditioning was made, pouring the fresh conditioning medium on the materials, in order that its setting (group II), polymerization (group III) or the direct contact (group IV) would be able to leach substances to this culture medium. These conditioned mediums were put on the cells for the entire experiment, excepting the group IV, in which the mediums were put in contact with the cells for 15 seconds, following clinical recommendations. Cell counting was performed in hemocytometer, using the Trypan blue exclusion method, which mark only the dead cells. These counting was made at experimental times of 0, 6, 12 and 24 hours for the cell viability assay (short term), where it is evaluated the percentage of live cells on the total number of cells, and at experimental times of 1, 3, 5 and 7 days for the survival assay, in which is evaluated the absolute number of live cells. It was observed that the substances leached by the bonding system are cytotoxic at all experimental times, decreasing significantly the cell viability and affecting its growing rate. Those leached by the 37% orthophosphoric acid decreased the cell viability only at the first contact with the cells, and the substances leached during the setting of the calcium hydroxide cement are not cytotoxic.