A Standardized Novel Method to Measure Radiographic Root Changes after Endodontic Therapy in Immature Teeth

Outcome studies of endodontic treatment of necrotic immature permanent teeth rely on radiographic measures as surrogates of whether the treatment achieved regeneration/revascularization/revitalization. An increase in radiographic root length and/or width is thought to result in a better long-term prognosis for the tooth. In this study a method to measure radiographic outcomes of endodontic therapies on immature teeth was developed and validated

Stem cells from human apical papilla decrease neuro-inflammation and stimulate oligodendrocyte progenitor differentiation via activin-A secretion

Secondary damage following spinal cord injury leads to non-reversible lesions and hampering of the reparative process. The local production of pro-inflammatory cytokines such as TNF-α can exacerbate these events. Oligodendrocyte death also occurs, followed by progressive demyelination leading to significant tissue degeneration. Dental stem cells from human apical papilla (SCAP) can be easily obtained at the removal of an adult immature tooth. This offers a minimally invasive approach to re-use this tissue as a source of stem cells, as compared to biopsying neural tissue from a patient with a spinal cord injury. We assessed the potential of SCAP to exert neuroprotective effects by investigating two possible modes of action: modulation of neuro-inflammation and oligodendrocyte progenitor cell (OPC) differentiation. SCAP were co-cultured with LPS-activated microglia, LPS-activated rat spinal cord organotypic sections (SCOS), and LPS-activated co-cultures of SCOS and spinal cord adult OPC. We showed for the first time that SCAP can induce a reduction of TNF-α expression and secretion in inflamed spinal cord tissues and can stimulate OPC differentiation via activin-A secretion. This work underlines the potential therapeutic benefits of SCAP for spinal cord injury repair

Treatment of trigeminal ganglion neurons in vitro with NGF, GDNF or BDNF: effects on neuronal survival, neurochemical properties and TRPV1-mediated neuropeptide secretion

BACKGROUND: Nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) all play important roles in the development of the peripheral sensory nervous system. Additionally, these growth factors are proposed to modulate the properties of the sensory system in the adult under pathological conditions brought about by nerve injury or inflammation. We have examined the effects of NGF, GDNF and BDNF on adult rat trigeminal ganglion (TG) neurons in culture to gain a better understanding of how these growth factors alter the cytochemical and functional phenotype of these neurons, with special attention to properties associated with nociception. RESULTS: Compared with no growth factor controls, GDNF, at 1 and 100 ng/ml, significantly increased by nearly 100% the number of neurons in culture at 5 days post-plating. A significant, positive, linear trend of increasing neuron number as a function of BDNF concentration was observed, also peaking at nearly 100%. NGF treatment was without effect. Chronic treatment with NGF and GDNF significantly and concentration-dependently increased 100 nM capsaicin (CAP)-evoked calcitonin gene-related peptide (CGRP) release, reaching approximately 300% at the highest concentration tested (100 ng/ml). Also, NGF and GDNF each augmented anandamide (AEA)- and arachidonyl-2-chloroethylamide (ACEA)-evoked CGRP release, while BDNF was without effect. Utilizing immunohistochemistry to account for the proportions of TRPV1- or CGRP-positive neurons under each growth factor treatment condition and then standardizing evoked CGRP release to these proportions, we observed that NGF was much more effective in enhancing CAP- and 50 mM K(+)-evoked CGRP release than was GDNF. Furthermore, NGF and GDNF each altered the concentration-response function for CAP- and AEA-evoked CGRP release, increasing the E(max )without altering the EC(50 )for either compound. CONCLUSIONS: Taken together, our results illustrate that NGF, GDNF and BDNF differentially alter TG sensory neuron survival, neurochemical properties and TRPV1-mediated neuropeptide release in culture. In particular, our findings suggest that GDNF and NGF differentially modulate TRPV1-mediated neuropeptide secretion sensitivity, with NGF having a much greater effect on a per neuron basis than GDNF. These findings are discussed in relation to possible therapeutic roles for growth factors or their modulators in pathological pain states, especially as these relate to the trigeminal system

O ensino da leitura e da escrita no 1º ano de escolaridade: os resultados dos alunos em leitura

Tese de Doutoramento em Psicologia Aplicada, Área de especialidade Psicologia EducacionalO presente estudo teve como objetivo caracterizar as práticas de ensino da linguagem escrita em Portugal e perceber o seu impacto na aquisição da leitura no final do 1º ano de escolaridade. A primeira etapa deste estudo correspondeu a um estudo descritivo e comparativo com o realizado por Fijalkow (2003) em França. A partir da resposta a um questionário de 883 professores foi possível verificar que os professores dos dois países se aproximam no que diz respeito aos aspetos metodológicos do ensino da leitura e à avaliação da leitura e da escrita dos alunos. Contudo, os professores portugueses dizem utilizar com maior frequência do que os professores franceses, atividades a partir de livros infantis, outros materiais para além do manual e diferentes propostas de escrita. Na segunda etapa deste estudo, o questionário utilizado foi validado através da sua estrutura fatorial e foram utilizados os fatores revelados para a realização de uma análise hierárquica de clusters. Esta análise revelou três grupos de professores: a) um grupo de professores que centra as suas práticas no ensino do código da linguagem escrita (unidades curtas); b) um grupo que realça a construção de significado na interação com o material escrito (unidades longas); c) e um grupo de professores que reúne nas suas práticas características de ambos os grupos referidos anteriormente (unidades diversificadas). A terceira etapa realizada diz respeito à validação das respostas do questionário através da observação de sala de aula. Foram selecionados 5% dos professores de cada um dos três grupos constituídos (N=42). Cada professor foi observado duas vezes durante 60 minutos em situações escolhidas por si como situações privilegiadas para o ensino da linguagem escrita. Através da utilização de uma grelha de observação que divide as atividades desenvolvidas em atividades que remetem para o código ou para o significado, foi possível confirmar as respostas ao questionário para 55% dos professores participantes. No sentido de perceber o impacto das práticas dos professores na aquisição da leitura dos alunos foram selecionados os professores cujas observações foram coincidentes com as respostas ao questionário e avaliados os seus alunos com recurso a três provas de leitura: leitura de palavras isoladas, decisão lexical e compreensão. Participaram 461 alunos distribuídos pelos três grupos referidos anteriormente. Através da realização de uma MANCOVA, controlando a escolaridade das mães dos alunos, verificou-se que os alunos do grupo unidades diversificadas obtêm melhores resultados em todas as provas do que os alunos dos outros dois grupos. Foi ainda possível constatar que não existiram diferenças de desempenho entre os alunos do grupo unidades curtas e os alunos do grupo unidades longas.ABSTRACT: The present study aimed to characterize the teaching practices of written language and understand its impact on reading acquisition by the end of 1st grade. The first step of this study was a descriptive and comparative study to the one conducted by Fijalkow (2003) in France. Based on questionnaire answers of 883 teachers, we found that teachers from both countries have similar approaches regarding methodological aspects of teaching of reading and students’ assessment of reading and writing abilities. However, Portuguese teachers stated that they use more frequently activities from children's literature, other materials besides the manual and different writing proposals than French teachers. In the second step of the study, the questionnaire was validated through its factorial structure and the factors disclosed were used to perform a hierarchical cluster analysis. This analysis revealed three groups of teachers: a) a group of teachers who focus its practices in teaching the code of written language (short units), b) a group that emphasizes the construction of meaning in interaction with the written material (long units), c) and a group of teachers who gathered in their practices characteristics of both groups mentioned above (diversified units). The third step concerns the validation of the questionnaire answers through classroom observation. 5% of the teachers in each of the three groups were selected (N=42). Each teacher was observed twice during 60 minutes in situations identified by them as privileged situations for teaching written language. Using an observation grid which divides the activities conducted in code-activities or the meaning-activities it was possible to confirm questionnaire answers for 55% of the participating teachers. In order to understand the impact of teachers’ practices in students reading acquisition, teachers whose observations were coincident with the questionnaire answers were selected and their students were evaluated using three reading tests: single words reading, lexical decision task and comprehension task. Participants were 461 students distributed through the three groups previously mentioned. By conducting a MANCOVA, controlling mothers’ education level, we concluded that students in the diversified unit group obtain better results in all reading tests than students from the other groups. It was also possible to confirm that there were no differences in performance between students of the short units group and students of the long units group.Tese de Doutoramento em Psicologia Aplicada, Área de especialidade Psicologia EducacionalApoio financeiro da Fundação para a Ciência e Tecnologia (SFRH/BD/64630/2009

Direct and Indirect Effect of Chlorhexidine on Survival of Stem Cells from the Apical Papilla and Its Neutralization

Introduction: Several irrigants have been used for disinfection in regenerative endodontic procedures including chlorhexidine (CHX). In this context, the antibacterial properties of disinfectants are mainly in focus of research even though they may have an undesirable impact on the fate of stem cells. In this study, we hypothesized that CHX has both a direct effect when applied to stem cells of the apical papilla (SCAPs) and an indirect effect when SCAPs are exposed to dentin previously conditioned with CHX. Methods: Cell toxicity was evaluated in vitro using the CellTox green fluorescence assay (Promega, Madison, WI) and CellTiter-Glo (Promega) after SCAPs were exposed directly to a dynamic concentration range of CHX; apical papilla explant cultures were stained with ApopTag (Merck Millipore, Billerica, MA) after culture with CHX. Furthermore, standardized slabs from human dentin were treated with CHX and consecutively rinsed in EDTA, L-alpha-lecithin (Sigma-Aldrich, St Louis, MO), or L-alpha-lecithin followed by EDTA. After that, SCAPs were cultured on the slabs for 5 days, and cellular viability was determined (indirect effect). Data were treated nonparametrically and analyzed using the Krukal-Wallis test (P <=.05). Results: Direct exposure of SCAPs to CHX highly affected cell viability at concentrations above 10(-3)%, whereas lower concentrations had no adverse effect. During the initial 60 minutes, concentrations of 10(-2)% CHX or higher resulted in early pronounced toxicity with a maximum effect within 15 minutes after exposure. Likewise, CHX-conditioned dentin slabs were detrimental to SCAP survival; however, the deleterious effects were completely reversed by neutralization with L-alpha-lecithin. Conclusions: Chlorhexidine is toxic to SCAPs when applied directly or indirectly via conditioned dentin. If applied for a short time and neutralized by L-alpha-lecithin, it can be a gentle and cell-preserving disinfectant before endodontic regeneration

Expression of neurotrophic factors in human dentin and their regulation of trigeminal neurite outgrowth

Introduction: Neurotrophic factors play a significant role in the innervation of the pulp-dentin complex during and after organogenesis. There have been numerous bioactive molecules identified in the dentin extracellular matrix; however, the expression of neurotrophic factors in the dentin matrix and their biological activity are largely unknown. The purpose of this study was to characterize the relative expression of neurotrophic factors in human dentin matrix proteins (DMPs) and their effect on neurite outgrowth of trigeminal (TG) neurons. Methods: Dentin was powdered in liquid nitrogen from noncarious human third molar teeth. DMPs were solubilized through an EDTA extraction method, dialyzed, and lyophilized until use. The relative expression of nerve growth factor, brain-derived neurotrophic factor, glial cell-line derived neurotrophic factor, neurotrophin 3, and neurotrophin 4/5 was determined by the enzyme-linked immunosorbent assay. Rat TG neurons were cultured and exposed to different concentrations of DMPs (1-10(5) ng/mL) or vehicle, and a quantitative neurite outgrowth assay was performed. Results: Human DMPs contained all of the tested neurotrophic factors, with glial cell-line derived neurotrophic factor and neurotrophin 4/5 found at the highest levels. DMPs were able to promote the neurite outgrowth of rat TG neurons at an optimum concentration of 10-10(2) ng/mL, whereas the effect was partially inhibited at higher concentrations (>10(3) ng/mL). Conclusions: The human dentin extracellular matrix is a rich reservoir for neurotrophic factors that are key components for neuronal homeostasis, differentiation, and regeneration. These data suggest that neurotrophins in DMPs could play an important role as signaling molecules for the innervation of the pulp-dentin complex during the processes of tooth formation, repair, and regeneration

Neurotrophic Proteins in Dentin and Their Effect on Trigeminal Sensory Neurons

Introduction: A plethora of bioactive molecules present during tooth formation become sequestered in the mineralized dentin matrix and can be released into the pulp tissue after demineralization from carious lesions. However, neurotrophic factors are differentially expressed and secreted during various stages of odontogenesis. Thus, the aims of this study were (1) to investigate their presence and relative abundance in crown and root dentin and (2) to evaluate the bioactivity of dentin-derived proteins on neuronal cells. Methods: Dentin matrix proteins (DMPs) were isolated from matched roots and crowns of extracted healthy human third molars. The total protein amount as well as the concentration of growth factors and neurotrophic proteins were quantified. The impact on neuritogenesis was determined with mouse trigeminal neurons in vitro and by a hydrogel implant model in vivo. Transient receptor potential cation channel subfamily V member 1 (TRPV1) sensitization of DMP-conditioned neurons was evaluated by single-cell calcium imaging. Results: The relative concentration of neurotrophic molecules revealed that nerve growth factor is the most abundant neurotrophin with 3-fold increased expression in radicular dentin. Similarly, brain-derived neurotrophic factor and neurotrophin 3 are more abundant in radicular than corona! dentin. Conversely, glial cell line- derived neurotrophic factor is more abundant in corona! dentin, whereas neurotrophin 4 is equally distributed. Dentin matrix proteins promoted neurite outgrowth in vitro and axonal targeting in vivo, with a greater effect observed by radicular dentin extracts. Furthermore, DMPs sensitized TRPV1 responses in mouse trigeminal neurons with greater activity seen with extracts from root dentin. Conclusions: Neurotrophic factors are differentially distributed between corona! and radicular dentin with different effects of dentin-derived proteins on axonal growth and targeting as well as the sensitization of TRPV1. Thus, extracellular proteins from the dentin matrix are likely involved in neurogenic responses to caries and could be exploited in clinical regenerative endodontics to promote reinnervation and enhance tissue regeneration

Mixed and Purified SCAPs: Similar Growth, Phenotypic and Differentiation Characteristics

Objective: To compare the phenotype, the growth curve and the osteo- and chondrogenic differentiation potential of mixed vs purified stem cells populations from the apical papilla (SCAPs); gingival fibroblasts (GFs) were used as control. Method: Four cell populations were considered: 1)mixed SCAPs (M-SCAPs); 2&3)SCAPs purified by immunomagnetic separation based either on Stro-1 (P-SCAPs1), or on a combination of CD73, CD90 and CD105 (P-SCAPs2); 4)gingival fibroblasts (GFs). Each population was subcultured, and their growth curve determined. Their phenotype was characterized by flow cytometry using antibodies against CD29, CD31, CD34, CD90, CD105 and Stro-1. The cells were then cultured in osteo- and chondrogenic media during 25 days. Osteogenic and chondrogenic differentiation were evaluated respectively by alizarin red S and alcian blue staining. Result: All four cell populations had similar growth curves, were homogeneously positive to CD29, CD90 and CD105 (percentage of positive cells \u3e97% in all cases but P-SCAPs1 for CD90 – 91.5%), and negative to CD31, CD34 and Stro-1 (\u3c3% in all cases but GFs for CD34 – 7.5%). All cell populations were positive for alizarin red S, and all but GFs were positive for alcian blue. Conclusion: The present results question the specificity of certain markers considered to identify dental mesenchymal stem cells. The expression of Stro-1 was notably lost in P-SCAPs1, which however conserved their multi-differentiation potential. The rationale of sorting cells based on these markers is therefore called into question vs using a mixed population. Finally, the results suggest that GFs either contain a certain proportion of osteogenic progenitor cells, or that they intrinsically possess an osteogenic differentiation potential

Mixed and Purified SCAPs: Similar Growth, Phenotypic and Differentiation Characteristics

Objective: To compare the phenotype, the growth curve and the osteo- and chondrogenic differentiation potential of mixed vs purified stem cells populations from the apical papilla (SCAPs); gingival fibroblasts (GFs) were used as control. Method: Four cell populations were considered: 1)mixed SCAPs (M-SCAPs); 2&3)SCAPs purified by immunomagnetic separation based either on Stro-1 (P-SCAPs1), or on a combination of CD73, CD90 and CD105 (P-SCAPs2); 4)gingival fibroblasts (GFs). Each population was subcultured, and their growth curve determined. Their phenotype was characterized by flow cytometry using antibodies against CD29, CD31, CD34, CD90, CD105 and Stro-1. The cells were then cultured in osteo- and chondrogenic media during 25 days. Osteogenic and chondrogenic differentiation were evaluated respectively by alizarin red S and alcian blue staining. Result: All four cell populations had similar growth curves, were homogeneously positive to CD29, CD90 and CD105 (percentage of positive cells \u3e97% in all cases but P-SCAPs1 for CD90 – 91.5%), and negative to CD31, CD34 and Stro-1 (\u3c3% in all cases but GFs for CD34 – 7.5%). All cell populations were positive for alizarin red S, and all but GFs were positive for alcian blue. Conclusion: The present results question the specificity of certain markers considered to identify dental mesenchymal stem cells. The expression of Stro-1 was notably lost in P-SCAPs1, which however conserved their multi-differentiation potential. The rationale of sorting cells based on these markers is therefore called into question vs using a mixed population. Finally, the results suggest that GFs either contain a certain proportion of osteogenic progenitor cells, or that they intrinsically possess an osteogenic differentiation potential

Advances in endodontics: Potential applications in clinical practice

Contemporary endodontics has seen an unprecedented advance in technology and materials. This article aimed to review some of the challenges and advances in the following sections: (1) endodontic imaging, (2) root canal preparation, (3) root canal disinfection, (4) root canal filling, and (4) regenerative endodontic procedures (REPs). Jointly, these advances are aimed at improving the state of the art and science of root canal treatment