60 % of school children have some form of untreated tooth decay or have suffered trauma to the front teeth which results in pulp damage. If left untreated, these teeth are susceptible to premature fracture/loss under daily stresses. In cases of adolescent tooth loss, teenagers cannot get dental implants until after the growth spurts; their only option is using removable dentures which lowers their quality of life. Conventional endodontic treatment (root canal treatment) is used in cases of pulp necrosis, but cannot be performed in immature permanent teeth due to major differences in tooth anatomy. Currently the American Dental Academy has approved a procedure called Regenerative Endodontic Treatment (RET) for such cases, but the outcomes are still unpredictable and the method is largely unreliable. One issue that we are trying to address in this work is the regeneration of the pulp-dentin complex (PDC), specifically the interface. Endeavors in regenerating either pulp or dentin have been successful individually, but the interface region is the anatomical and physiologic hallmark of the PDC and has not been addressed. We have proposed a biomimetic scaffold to facilitate early stage stratification of these different tissues and allow recapitulation of their interface. Tissue engineering principles and biomaterial processing techniques were used simultaneously to encourage dental pulp stem cells into mineralize selectively only on one side. This effectively allows the scaffold to serve as the interface region between the hard dentin and the soft vascular pulp. / Bioengineering

Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: Regenerative endodontic procedures are an effective treatment option for immature teeth with infected necrotic pulps to allow for healing and potential continued root development, yet challenges to ideal treatment outcomes remain. Consistent development of root length and width of dentin remains a challenge, as does development of the pulp-dentin complex. Previous in vitro studies have assessed the role of different growth factors and bioactive molecules in combination with scaffolds to potentially facilitate continued development of the pulp-dentin complex using dental pulp stem cells (DPSCs). The proline-rich tyrosine kinase 2 (Pyk2) is linked with osteoblast activity and the regulation of bone mass. Further, the Pyk2 inhibitor PF-4618433 (PF-46) has been shown in previous studies to enhance osteoblast activity and mineral deposition in vitro. However, whether Pyk2 targeting promotes the osteogenic differentiation of DPSCs remains unknown. Objective: The purpose of this study was to investigate the effect of a Pyk2 inhibitor, PF-46, on the proliferation, differentiation, and mineralization of human DPSCs. Materials and Methods: Human DPSCs were cultured in 24-well plates with α-MEM with 10% FBS, and containing 0 μM (vehicle control) or 0.1 μM, 0.3 μM, or 0.6 μM PF-46. Fresh media and treatments were replaced every 2-3 days. After 1 day incubation, cytotoxic effects were evaluated by using an MTS proliferation assay. After 4 days of treatment, direct cell counting was performed. To induce osteogenic differentiation, ascorbic acid and β-glycerol phosphate were added to the culture media and the DPSCs were cultured with PF-46 for 14 days. Then, an alkaline phosphatase (ALP) assay and mineral deposition assay were performed. Differences between treatment groups were analyzed by a one-way ANOVA followed by pair-wise tests conducted using Tukey’s multiple comparisons procedure with a 5% significance level. Results: The 0.6 μM PF-46 group had a significantly higher cell count, ALP activity and mineral deposition when compared to 0 μM PF-46. The 0.1 and 0.3 μM PF-46 groups also had significantly higher ALP activity compared to the 0 μM PF-46 group after 14 days of incubation. There was a general trend of increased differentiation and mineral deposition as the concentration of PF-46 increased from 0.1 μM to 0.6 μM. Conclusion: There was a general concentration-dependent increase in cell count, differentiation, and mineral deposition by human DPSCs as the concentration of PF-46 increased from 0 μM up to 0.6 μM, with the highest activity observed with 0.6 μM PF-46. Although further research is needed, these results suggest that strategies that target Pyk2 may potentially be used to improve the osteogenic differentiation of DPSCs to aid endodontic regeneration.

Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: Regenerative endodontic therapy in immature teeth promotes continuation of root development and likely increases the prognosis of these teeth. The use of double antibiotic paste (DAP), equal parts of ciprofloxacin and metronidazole, followed by the dentin conditioner, ethylenediaminetetraacetic acid (EDTA), has been suggested for canal disinfection and facilitation of stem cell attachment/proliferation, respectively. However, the effect is unknown when all these agents are used on on radicular dentin surfaces to facilitate the level of stem cell proliferation. Objectives: The aim of this in-vitro study is to compare the proliferation of human dental pulp stem cells (hDPSCs) on human radicular dentin treated with two different concentrations of DAP followed by EDTA. Materials and Methods: Human premolars and incisors were prepared into standardized polished 4 mm x4 mm radicular dentin specimens. Groups of specimens were treated with DAP 500 mg/mL, DAP 1 mg/mL, DAP 500 mg/mL followed by 17-percent EDTA, DAP 1 mg/mL followed by 17-percent EDTA; 17% EDTA, or no treatment. All groups treated with antibiotics were incubated with DAP at 37°C for one week. All specimens were washed with distilled water. The hDPSCs were seeded across all specimens and unattached cells were collected after 24 hours. LDH assay was completed on unattached cells for quantification. Three days after attachment, WST viability and LDH cytotoxicity assays were performed. Hypothesis: There is no significant difference in hDPSC viability, unattachment, and cytotoxicity on dentin specimens treated with DAP and 17-percent EDTA. Clinical Significance: These results can be used to help identify the best treatment concentrations when using DAP and/or EDTA to promote endodontic regeneration. Results: The results demonstrated significantly less viability of hDPSCs on specimens treated with 500 mg/mL DAP with and without 17-percent EDTA. Groups treated with 1 mg/mL DAP, 1 mg/mL DAP and 17-percent EDTA, and 17-percent EDTA alone had no statistically significant difference in viability compared with control untreated dentin. The results of the unattached cells from the LDH demonstrated that cells from the specimens treated with solely 500 mg/mL and 1 mg/mL DAP had significantly higher levels of unattached cells when compared with all other groups. The LDH assays in summation with the WST assays showed a trend of a lack of proliferation on groups treated with 500 mg/mL DAP with and without 17-percent EDTA. Conclusions: Paste-like concentrations (500 mg/mL) of DAP are detrimental to hDPSC viability, whereas the present study supports the use of low-concentration antibiotics consistent with current recommendations for intracanal medicaments used during endodontic regenerative procedures.

Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: During root canal treatment (RCT), an opening is made through the crown of the tooth to access and to disinfect the root canal system (RCS). Traditional endodontic access (TEA) may sacrifice tooth structure and weaken the tooth. Cone beam computed tomography (CBCT) provides information about the exact location of the root canals. This information can be used for the design of a canal-directed endodontic access (CDEA). It may also be used for the 3D printing of an acrylic endodontic stent that could help to create a conservative CDEA. Objective: 1) Evaluate the ability of the Dolphin 3D imaging software to assist in creating a CDEA; 2) Compare tooth structure loss in a CDEA to that in a TEA by measuring the volume of remaining tooth structure, surface area of the access opening at the occlusal, and remaining dentin thickness at the CEJ. Materials and Methods: Thirty extracted human mandibular premolars were used. Teeth with large, wide canals were excluded. CBCT images will be taken for all teeth using Kodak 9000. Fifteen teeth were randomly assigned to the TEA group and 15 teeth were assigned to the CDEA group. The CDEA path was mapped using Dolphin 3D imaging software. Acrylic access stents were designed using Rhino 3D software and printed using a 3D printer. The teeth were accessed through the corresponding stents. The 15 teeth that are part of the traditional access group were accessed without a stent. A CBCT scan was taken post-access for all 30 teeth. Wilcoxon Rank Sum Tests were performed to compare the following outcomes for the two groups: the volume of remaining tooth structure, the surface area of the access opening at the occlusal, and remaining dentin thickness at the CEJ. Results: The remaining dentin thickness (percent loss) was not significantly larger for TEA than for CDEA. The surface area (post-treatment) was significantly larger for TEA than for CDEA, and volume (percent loss) was significantly larger for TEA than for CDEA. Conclusion: The use of the CBCT and Dolphin 3D imaging provided an accurate and more conservative CDEA with the guide of an acrylic stent.

Indiana University-Purdue University Indianapolis (IUPUI) / Accurate determination of working length during endodontic therapy is critical in achieving a predictable and successful outcome. Working length is determined by the use of electronic apex locators, tactile perception, knowledge of average tooth lengths and dental radiography. Due to the increasing use of digital radiography in clinical practice, a comparison with conventional film in working length determination is justified. The purpose of this study is to determine if there is a difference between Schick digital radiography and Kodak Ultra-speed film in the accurate determination of working lengths when vertical angulation of the object is variable. Twelve teeth with #15 K-flex files at varying known lengths from the anatomical apex were mounted in a resin-plaster mix to simulate bone density. A mounting jig for the standardization of projection geometries allowed for exact changes in vertical angulation as it related to the object (tooth) and the film/sensor. Each tooth was imaged using Schick CDR and Kodak Ultra-speed film at varying angles with a consistent source-film distance and exposure time. Four dental professionals examined the images and films independently and measured the distance from the tip of the file to radiographic apex and recorded their results. The error in working length was calculated as the observed value minus the known working length for each tooth type. A mixed-effects, full-factorial analysis of variance (ANOVA) model was used to model the error in working length. Included in the ANOVA model were fixed effects for type of image, vertical angulation, and the interaction of angle and film type. Tooth type and examiner were included in the model as random effects assuming a compound symmetry covariance structure. The repeatability of each examiner, for each film type, was assessed by estimating the intra-class correlation coefficient (ICC). The ICC was determined when 12 randomly selected images and radiographs were reevaluated 10 days after initial measurements. The repeatability of each examiner for Schick CDR was good with ICCs ranging from 0.67 to 1.0. Repeatability for the conventional film was poor with ICCs varying from -0.29 to 0.55. We found the error in the working length was not significantly different between film types (p = 0.402). After adjusting for angle, we found that error in the working length from the digital image was only 0.02 mm greater (95-percent CI: -0.03, 0.06) than the conventional film. Furthermore, there was not a significant difference among the angles (p = 0.246) nor in the interaction of image type with angle (p = 0.149). Based on the results of our study, we conclude that there is not a statistically significant difference in determining working length between Schick CDR and Kodak Ektaspeed film when vertical angulation is modified.

La revue de la littérature montre que la pulpe dentaire est une source utile pour le diagnostic des bactériémies, y compris en paléomicrobiologie. Les précédents travaux en paléomicrobiologie réalisés dans le laboratoire ont tous mis en évidence une amplification possible de courts ou très courts fragments d'ADN bactérien partant de l'hypothèse que l'ADN ancien est fragmenté. Dans le premier travail, le modèle expérimental de dégradation de l'ADN des macrophages murins J774 et de Mycobacterium smegmatis par la chaleur sèche à 90°C a montré une différence statistiquement significative (p < 0.05) entre la vitesse de fragmentation de l'ADN bactérien et eucaryote. Ces résultats suggèrent que les diagnostics paléomicrobiologiques peuvent détecter des fragments plus longs d'ADN bactérien à partir des échantillons anciens. Dans un deuxième travail, un système de détection rapide de 7 agents pathogènes par PCR multiplex en temps réel a été utilisé pour détecter ces pathogènes suspectés à partir de la pulpe dentaire de 1192 dents anciennes collectées dans 12 charniers dont un localisé à Douai (1710 – 1712). Après la détection de Bartonella quintana dans ce charnier, les PCRs en temps réel emboîtées ultra-sensibles et la «PCR suicide» ont été utilisées pour confirmer la présence de Rickettsia prowazekii souche Madrid E génotype B dans 6/55 pulpes dentaires (11%) collectées de 6/21 squelettes (28.6%) de soldats à Douai. Ces résultats supportent l'hypothèse que le typhus a été introduit en Europe par les soldats espagnols au retour des conquêtes en Amérique. / Reviewing the literature shows that dental pulp is a useful source for bacteremic and paleomicrobiological diagnoses. In the first work, an experimental model of DNA degradation of the murine macrophage cell line J774 and Mycobacterium smegmatis by exposure to 90°C dry heat showed a statistically significant difference (p < 0.05) of fragmentation level between bacterial and eukaryotic DNA. These results suggest that paleomicrobiological diagnosis can detect more large fragments of bacterial DNA from ancient buried specimens. In the second work, a system of rapid detection of seven pathogens by multiplex real-time PCR was used for detecting suspected pathogens from dental pulp of 1192 ancient teeth collected from 12 multiple burials including a mass grave in Douai, 1710 – 1712. After the Bartonella quintana detection in this site, real-time nested PCR and ultra-sensitive “suicide PCR” were used to confirm the presence of Rickettsia prowazekii strain Madrid E genotype B in 6/55 dental pulp specimens collected from 6/21 (29%) skeletons of soldiers buried in Douai.These results support the hypothesis that typhus was imported into Europe by Spanish soldiers from America. In the last work, DNA extracted from 5 dental pulp specimens collected from multiple burials at Issoudun, 17th – 18th centuries, was analyzed by pyrosequencing which detected Yersinia pestis sequences in the metagenome. For paleomicrobiology, pyrosequencing is a sensitive technic which can be used as baseline test to detect both suspected and unexpected pathogens from ancient specimens. We named this new approach «paleometagenomics».

Foram analisados dentes decíduos com cárie dental profunda de 110 crianças, sendo coletadas 103 amostras de polpa necrosada e 7 de fístulas gengivais. Morfotipos bacterianos foram visualizados pelas colorações de Gram e Brenn-Brown, e os DNA foram obtidos e usados na detecção bacteriana por PCR. A predominância de cocos Gram positivos (81,8%) e cocobacilos Gram negativos (49,1%) foram observadas. Em 88 amostras de polpas, microrganismos com maior ocorrência foram: Enterococcus spp. (50%), P. gingivalis (49%), F. nucleatum (25%) e P. nigrescens (11,4%). Foram detectados em fístulas: P. gingivalis (43%), Enterococcus spp. (28,6%), F. nucleatum (14,3%), P. nigrescens (14,3%), e D. pneumosintes (14,3%). Os nossos resultados permitem concluir que a microbiota envolvida nas infecções pulpares em dentes decíduos é similar em termos qualitativos àquela observada em dentes permanentes. Entretanto, a predominância de Enterococcus spp. e P. gingivalis deve ser levado em consideração pelos clínicos em casos necessários de tratamento endodôntico em crianças com dentição decídua. / In this study, deciduous teeth with deep caries from 110 children, with 103 pulp necrosis and 7 gingival fistula samples were evaluated. Bacterial morphotypes were visualized by Gram staining and Brown-Brenn. DNA were obtained and used in bacterial detection by using PCR. The predominance of Gram-positive cocci (81.8%) and Gram-negative coccobacilli (49.1%) were observed. In 88 pulp samples a high frequency of microrganisms were observed: Enterococcus spp. (50%), P. gingivalis (49%), F. nucleatum (25%) and P. nigrescens (11.4%). In fistulas were detected: P. gingivalis (43%), Enterococcus spp. (28.6%), F. nucleatum (14.3%), P. nigrescens (14.3%), and Dialister pneumosintes (14.3%). Our results, suggest that the involved microbiota in pulp infections of deciduous teeth are qualitatively similar than those permanent teeth. However, a predominance of Enterococcus spp. and P. gingivalis was observed, and it must be considered in the endodontic treatment in children with primary dentition.

A translação da regeneração pulpar com células tronco para a clinica requerirá o uso de scaffolds injetáveis. O objetivo foi estudar o comportamento de células tronco obtidas de dentes decíduos exfoliados (SHED) injetados em canais radiculares de pré-molares humanos com ápice aberto com scaffold de colágeno recombinante humano tipo I (rhC-I) e a base de nanofibras auto-organizáveis (SA). Para determinar a viabilidade e potencial de diferenciação de SHED in vitro, raízes nao instrumentadas foram posicionadas com o ápice em meio de cultura. SHED ressuspendida em rhC e SA foram injetadas nos canais (n=24, 5X105 células/mL). Os controles foram SHED e scaffolds sozinhos. Marcadores para diferenciação odontoblastica (DSPP, DMP-1 e MEPE) foram avaliados semanalmente por RT-PCR por 28 dias. Para avaliar a diferenciação odontoblástica e formação de tecido in vivo, SHED transuzida com GFP foram injetadas em canais radiculares (n=8, 106 célulass/mL) utilizando os mesmos grupos e implantadas subcutaneamente em camundongos imunodeprimidos. O controle (C+) foi um pré-molar humano extraído. Analise estatística foi feita com ANOVA (=0.05). Os marcadores de diferenciação odontoblástica aumentaram para SA e rhC-I mas nao nos controles. Crescimento de tecido pulpar-símile ( do que 60% do comprimento da raiz) foi observado em 75% dos implantes para SA e rhC-I e 0% nos controles. Analise de imunohistoquimica para GFP confirmou a origem tecidual a partir de SHED. PCNA e ensaio de TUNEL mostraram alta ativiade proliferativa e poucas células apoptóticas. Injeções de tetraciclina evidenciaram neoformação de dentina. A densidade microvascular e nomero de odontoblastos delineando a dentinta foi similar em rhC-I, SA e C+. A associação de SHED com scaffolds injetáveis foi capaz de originar um tecido pulpar capaz de produzir dentina e constitui um passo a mais frente ao objetivo de regeneração pulpar em pacientes humanos. / The translation of dental pulp regeneration with stem cells to the clinic will require the use of injectable scaffolds. The aim was to study the behavior of stem cells from exfoliated deciduous teeth (SHED) injected in the root canal of opened-apex human premolars with either recombinant human collagen I (rhC-I) or selfassembling nanofiber (SA) scaffolds. To assess in vitro SHED viability and differentiative potential, non-instrumented roots were set with the apex in culture media. SHED were mixed in rhC-I or SA and injected into canals (n=24, 5X105 cells/mL). Controls were SHED or scaffolds alone. Odontoblastic differentiation markers (DSPP, DMP-1 and MEPE) were assessed weekly by RT-PCR for 28 days. To evaluate odontoblast differentiation and tissue formation in vivo, SHED transduced with GFP were injected in canals (n=8, 106 cells/mL) using same groups and implanted subcutaneously in immunodeficient mice. Positive control (C+) was extracted premolar. Statistic was done with ANOVA (=0.05). Odontoblastic differentiation markers increased in SA and rhC-I but not in controls. Pulp-like tissue growth ( than 60% of root length) was observed in 75% of implants for SA and rhC-I and 0% in controls. GFP staining confirmed SHEDs tissue origin. PCNA staining and TUNEL assay showed high proliferative activity and few apoptotic cells. Tetracycline injections showed newly formed dentin. Microvessel density and odontoblastic-like cell number lining dentin were similar in rhC-I, SA and C+. Injectable scaffolds and SHED allowed for the engineering of a pulp-like tissue and constitute one step forward towards the goal of dental p ulp regeneration in human patients.

Há um grande interesse no estudo de células-tronco em função de sua capacidade de auto-renovação e plasticidade. Estas características capacitam as células-tronco a produzirem células de diferentes linhagens que participam ativamente do processo de homeostase, da resposta à injúria e da regeneração e reparação tecidual. A polpa dental é o tecido mais estudado na Odontologia em relação a células-tronco, mas diversos estudos já mostraram a presença dessas células também na região periodontal. É importante salientar, que dependendo de sua origem, as células-tronco apresentam comportamentos diversos, especialmente no que tange ao transplante in vivo. Além disso, os microambientes onde as células-tronco residem (nichos), têm um papel fundamental no comportamento das mesmas, pois controlam aspectos essenciais como o estado de indiferenciação e a auto-renovação. Esse projeto de pesquisa teve como objetivos duas análises distintas. Na primeira, verificar se existem células-tronco mesenquimais derivadas da curetagem do alvéolo dental humano após extrações dentais. Na segunda, analisar o nicho de células-tronco nas polpas dentais de camundongos. Em comum, as duas análises se basearam no uso de marcadores previamente utilizados na literatura para o estudo de células-tronco. Como não existem marcadores únicos e específicos para a identificação dessas células, diferentes combinações desses marcadores entre si e de técnicas laboratoriais foram empregadas. No primeiro estudo, após o isolamento das células, deu-se especial atenção à sua caracterização, que foi realizada através de ensaios que avaliaram propriedades e comportamentos que sabidamente ocorrem em células-tronco. Já na segunda parte desse estudo, utilizamos a polpa dental de camundongos para a análise in vivo de nichos. Em camundongos, a localização do nicho responsável pelo crescimento continuo dos incisivos é conhecida. De uma maneira geral, nossos resultados demonstram que: 1) É possível isolar células-tronco/progenitoras a partir de tecidos curetados de alvéolo dental após extrações dentárias. Esse fato é inédito e abre novas perspectivas em termos de oportunidade de coleta e futura aplicação clínica. 2) É possível observar a diversidade populacional nas células que formam o nicho de células-tronco em polpa dental de camundongos, e através de uma organização hierárquica, propusemos um modelo para este nicho. Esse modelo servirá de base para estudos subsequentes que visem avaliar o comportamento das células que formam o nicho, quando isoladas das demais, e abrirá possibilidade para análises em outros tecidos dentais e não dentais. / There is great interest in the study of stem cells due to their ability to produce mature cells of different lineages that participate actively in the process of homeostasis, injury response, regeneration and tissue repair. The dental pulp is the most studied tissue in dentistry, but several studies have shown the presence of these cells in the periodontal tissue region. It is important to note that depending on their origin, these cells exhibit different behaviours, especially in regard to in vivo transplantation. Furthermore, the microenvironment in which these cells are found (niches) have an essential role in their behaviour as they control important aspects such as differentiation and self-renewal. This research project aimed for two distinct analyses. The first was to verify if there are mesenchymal stem cells derived from human dental socket curettage after dental extractions. The second, to analyse the stem cell niche in mouse dental pulp. In common, the two analyses were based on the use of markers previously used in the literature for the study of stem cells. As there are no specific and unique markers to identify these cells, an extensive combination of these markers among themselves, and laboratory techniques were performed. In the first study, after the isolation of the cells, special attention was given to their characterization by combining assays to evaluate stem cells properties and behaviours. In the second part of this study, we used the mouse dental pulp model for in vivo analysis of stem cell niches. In mice, the location of the niche responsible for the incisors continuous growth is known. In general our results showed that: 1) It is possible to isolate progenitor/stem cells from tissue curettage of the alveolus after tooth extractions. This fact is unprecedented and opens new perspectives for the obtention of stem cells for future clinical applications. 2) It is possible to observe the cells population diversity of the mouse dental pulp niche and through a hierarchical organization, we proposed a model for this niche. This model will provide basis for further studies aimed at evaluating the behavior of cells that form the niche, when isolated from the others, and opens the possibility for the analysis of the niche structure in other dental and non-dental tissues.

O hidróxido de cálcio (HCa), o agregado de trióxido mineral (MTA) e o óleo-resina de copaíba (COP) isoladamente apresentam características biológicas do material de capeamento pulpar direto mais apropriado. Com o pressuposto que associados poderiam originar materiais mais apropriados para serem aplicados em capeamento pulpar direto, este estudo objetivou analisar in vitro proliferação, diferenciação e migração de células-tronco de polpa de dente decíduo humano esfoliado (SHEDs) em resposta a substâncias liberadas pelo COP isolado ou associado ao HCa ou ao MTA. Proliferação, diferenciação e migração de SHEDs (Linhagem PDH3) foram analisadas através do ensaio de redução do MTT; da atividade de fosfatase alcalina (ALP), formação de nódulos mineralizados pelo ensaio de Vermelho de Alizarina e expressão dos genes (BGLAP, DSPP, DMP1 e HSP-27) pelo qRT-PCR; e, do ensaio do Scratch, respectivamente. As células foram submetidas à ação de meios condicionados pelos biomateriais, de acordo com os seguintes grupos experimentais: COP isolado (COP); HCa isolado (HCa); HCa associado ao COP (HCa+COP); MTA isolado (MTA) e MTA associado ao COP (MTA+COP). Células crescidas em meio de cultura fresco serviram de controle. Os dados foram comparados utilizando ANOVA complementado pelo teste de Tukey (p 0,05). O grupo HCa apresentou número de células viáveis significativamente menor que o dos demais grupos, inclusive o do grupo HCa+COP (p<0,01) em todos os tempos experimentais. A atividade de ALP em 14 dias foi similar em todos os grupos experimentais. Em 21 dias, o grupo COP apresentou quantidade maior de mineralização que os demais grupos (p<0,01) e o grupo HCa+COP maior que o grupo HCa (p<0,01). O gene DMP1 não foi expresso pelas células em nenhum grupo experimental. O grupo COP apresentou as menores expressões dos genes BGLAP, DSPP e HSP-27. As SHEDs do grupo MTA+COP apresentaram superexpressão dos genes BGLAP, DSPP e HSP-27, e as do grupo HCa+COP superexpressão dos genes BGLAP e HSP-27. O grupo HCa foi o único que não apresentou células em migração em todos os tempos experimentais. Os demais grupos apresentaram migração similar à do grupo Controle, exceto o grupo MTA em 12 e 24 horas. As SHEDs dos grupos HCa+COP e MTA+COP migraram significativamente mais que as dos grupos HCa e MTA, respectivamente. O COP, isolado ou em associação aos demais biomateriais, não interfere na proliferação de SHEDs e quando associado ao HCa é capaz de anular a citotoxicidade deste biomaterial isolado. O COP, isoladamente ou em associação, não interfere na atividade de fosfatase alcalina. Isoladamente, o COP induz a maior diferenciação funcional e quando associado ao HCa melhora substancialmente a diferenciação induzida por este biomaterial isolado. Os biomateriais isolados ou associados não são capazes de induzir expressão de DMP1. O COP associado aos biomateriais induz superexpressão de genes relacionados à formação de matriz extracelular e de diferenciação odontoblástica. O COP isoladamente não interfere na migração celular e, quando associado ao HCa, anula por completo a inibição de migração causada por esse biomaterial isolado. Quando associado ao MTA, o COP aumenta a velocidade de migração das células em relação ao MTA isolado. Óleo-resina de copaíba (COP) promove proliferação, diferenciação e migração de células-tronco de polpa dental sendo uma proposta de um biomaterial para capeamento pulpar. / The calcium hydroxide (CaH), the mineral trioxide aggregate (MTA) and the oil-resin copaiba (COP) by themselves have biological characteristics of the ideal direct pulp capping material. With the assumption that when associated they could originate materials more appropriated for direct pulp capping, this study aimed to analyze the in vitro proliferation, differentiation and migration of stem cells from human exfoliated deciduous teeth (SHEDs) in response to substances leached from COP alone or associated with CaH or MTA. Proliferation, differentiation and migration of SHEDs (PDH3 lineage) were analyzed through the MTT reduction assay; alkaline phosphatase (ALP) activity, mineralized nodules formation using the Alizarin Red assay and gene expression (BGLAP, DSPP, DMP1 e HSP-27) using qRT-PCR; and the Scratch assay, respectively. The cells were submitted to the culture medium conditioned by the biomaterials according to the following experimental groups: COP alone (COP); CaH alone (CaH); CaH associated to COP (CaH+COP); MTA alone (MTA) and MTA associated to COP (MTA+COP). Cells grown in fresh culture medium served as control. Data were compared by ANOVA complemented by the Tukey´s test (p 0.05). The CaH group presented number of viable cells significantly smaller (p<0.01) than those of all other experimental groups, including the CaH+COP, during whole experimental time. The ALP activity in 14 days was similar in all experimental groups. In 21 days, the COP group presented the amount of mineralization higher (p<0.01) than those of all other groups. The gene DMP1 was not expressed by the cells in all experimental groups. The COP group presented the smallest expression (p<0.01) of BGLAP, DSPP and HSP-27 genes. The SHEDs of the MTA+COP group presented superexpression of the BGLAP, DSPP and HSP-27 genes, and in the CaH+COP group the cells superexpressed the BGLAP and HSP-27 genes. The CaH group was the only one where there was no cell migration during whole experiment. Migration of all other groups was similar to that of the control group, except by the MTA group in 12 and 24 hours. The CaH+COP and MTA+COP migrated significantly more than the CaH and MTA groups, respectively. COP, alone or in association to the other biomaterials, does not interfere with the proliferation of the SHEDs, and when associated to CaH is able to annul the cytotoxicity of the CaH alone. The COP, alone or in association to the other biomaterials, does not interfere with the ALP activity. The COP alone induces the highest functional differentiation of the SHEDs and when associated to the CaH substantively improves this differentiation. The biomaterials, alone or in association, are not able to induce the expression of the DMP1 gene. The COP associated to the biomaterials induces superexpression of the genes related to the extracellular matrix formation and odontoblastic differentiation. The COP alone does not interfere with the cell migration and, when associate to CaH, completely annuls the inhibition of migration caused by this material alone. When associated to MTA the COP increases the cell migration speed compared to the effect of the MTA alone. Oil-resin copaiba (COP) promotes proliferation, differentiation and migration of stem cells from dental pulp with a proposal for a biomaterial for pulp capping.