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Background: Disinfection and dentin conditioning promote a favorable scenario for regenerative endodontic treatment. Clinical reports have confirmed periapical normality with high variability in disinfection protocols; nevertheless, the nature of neoformed tissue varied between them. Thus, this study aimed to present the impact of disinfection protocols on the clinical, histological, and molecular outcomes of regenerative endodontics procedures in permanent teeth with incomplete root formation. Materials and Methods: Eighteen teeth with incomplete root formation which required endodontic regenerative treatment were treated with different disinfection and conditioning agents and followed under clinical control. One case was evaluated under histological and immunohistochemical analyses. Results: Clinical outcomes revealed periapical repair in 17/18 cases. Histological and immunohistochemical analyses confirmed the neoformation of the dentinal matrix and its mineralization. Conclusions: Chemical conditioning could impact the outcome of regenerative endodontic procedures. The histological and immunohistochemical analysis showed the nature of the newly formed tissue that correlates with the clinical outcome.
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Abstract This study aimed to evaluate the microstructure formed after the chemical treatment of teeth, for the development of autogenous grafts from the demineralized dentin matrix (DDM) technique, in order to identify the most efficient demineralizing solution. The specimens, originating from the root and coronal portion, were submitted to ultrasonic cleaning and drying in an oven for 1h at 100 ºC. Then, the density was determined by Archimedes' principle for each specimen, using distilled water as immersion liquid. The samples were separated into five groups: Control group: negative control, Distilled water;EDTA group: positive control, trisodium EDTA; NaOCl group: 2.5% sodium hypochlorite; HCl-0.6M group: 0.6M hydrochloric acid; and H2O2/H2SO4 group: hydrogen peroxide and sulfuric acid. Each specimen was immersed for 1h in the corresponding group descaling solution at 60 ºC. Subsequently, the mass loss and density of the treated specimens were determined by Archimedes' principle. Ultimately, the specimens of each group were characterized by microtomography, Scanning Electron Microscopy, and Energy Dispersive Spectrometry X-ray (SEM-EDS). The results demonstrated that the H2O2/H2SO4 solution allowed the formation of interconnected micropores, suggesting better pore structures for application in scaffolds, when compared to the other studied solutions.
Resumo Este estudo teve como objetivo avaliar a microestrutura formada após o tratamento químico em dentes, para o desenvolvimento de enxertos autógenos a partir da técnica de matriz de dentina desmineralizada (DDM), a fim de identificar a solução desmineralizante mais eficiente. Os espécimes, provenientes da raiz e porção coronal, foram submetidos à limpeza ultrassônica e secagem em estufa por 1h a 100 ºC. Em seguida, a densidade foi determinada pelo princípio de Arquimedes para cada espécime, utilizando água destilada como líquido de imersão. As amostras foram separadas em cinco grupos: Controle: controle negativo, Água destilada; EDTA: controle positivo, EDTA trissódico; NaOCl: hipoclorito de sódio 2,5%; HCl-0.6M: ácido clorídrico 0,6M; e H2O2/H2SO4: peróxido de hidrogênio e ácido sulfúrico. Cada espécime foi imerso por 1h na solução descalcificante de grupo correspondente a 60 ºC. Posteriormente, a perda de massa e a densidade dos espécimes tratados foram determinadas pelo princípio de Arquimedes. Por fim, os espécimes de cada grupo foram caracterizados por microtomografia, microscopia eletrônica de varredura e espectrometria de energia dispersiva de raios-X (SEM-EDS). Os resultados demonstraram que a solução H2O2/H2SO4 permitiu a formação de microporos interligados, sugerindo melhores estruturas de poros para aplicação em scaffolds, quando comparada às demais soluções estudadas.
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OBJECTIVES: To develop a 3D-printed, microparticulate hydrogel supplemented with dentin matrix molecules (DMM) as a novel regenerative strategy for dental pulp capping. MATERIALS AND METHODS: Gelatin methacryloyl microgels (7% w/v) mixed with varying concentrations of DMM were printed using a digital light projection 3D printer and lyophilized for 2 days. The release profile of the DMM-loaded microgels was measured using a bicinchoninic acid assay. Next, dental pulp exposure defects were created in maxillary first molars of Wistar rats. The exposures were randomly capped with (1) inert material - negative control, (2) microgels, (3) microgels + DMM 500 µg/ml, (4) microgels + DMM 1000 µg/ml, (5) microgels + platelet-derived growth factor (PDGF 10 ng/ml), or (6) MTA (n = 15/group). After 4 weeks, animals were euthanized, and treated molars were harvested and then processed to evaluate hard tissue deposition, pulp tissue organization, and blood vessel density. RESULTS: All the specimens from groups treated with microgel + 500 µg/ml, microgel + 1000 µg/ml, microgel + PDGF, and MTA showed the formation of organized pulp tissue, tertiary dentin, newly formed tubular and atubular dentin, and new blood vessel formation. Dentin bridge formation was greater and pulp necrosis was less in the microgel + DMM groups compared to MTA. CONCLUSIONS: The 3D-printed photocurable microgels doped with DMM exhibited favorable cellular and inflammatory pulp responses, and significantly more tertiary dentin deposition. CLINICAL RELEVANCE: 3D-printed microgel with DMM is a promising biomaterial for dentin and dental pulp regeneration in pulp capping procedures.
Assuntos
Dentina Secundária , Microgéis , Agentes de Capeamento da Polpa Dentária e Pulpectomia , Ratos , Animais , Polpa Dentária , Compostos de Cálcio/uso terapêutico , Capeamento da Polpa Dentária/métodos , Materiais Biocompatíveis , Silicatos/uso terapêutico , Ratos Wistar , Regeneração , Impressão Tridimensional , Combinação de Medicamentos , Óxidos/uso terapêuticoRESUMO
Abstract The aim of this study was to evaluate a Demineralized Human Dentine Matrix (DHDM) as viable biomaterial for alveolar ridge preservation in a rat model. Wistar rats were submitted to the extraction of maxillary first molars bilaterally. Sockets were filled with biomaterials and divided into 4 experimental groups (n=5): blood clot, autogenous bone, bovine-derived xenograft (BDX) and DHDM. Animals were sacrificed at 7, 14 e 28 days. Microtomography (uCT) volumetric evaluation and qualitative histological analyses were performed. Results obtained through the uCT showed similar values between the DHDM and the other experimental groups. The histological evaluation demonstrated DHDM with an unspecific inflammatory process and bone neoformation with slow reabsorption of the material. This result indicates that DHDM implanted in rat sockets is biocompatible and reduces the alveolar ridge volume loss after tooth extraction.
Resumo O objetivo deste estudo foi avaliar a Matriz Dentinária Humana Desmineralizada (MDHD) como biomaterial viável para preservação do rebordo alveolar, no modelo em rato. Ratos Wistar foram submetidos à exodontias dos primeiros molares superiores bilateralmente. Os alvéolos foram preenchidos com biomateriais e divididos em 4 grupos experimentais (n=5): coágulo sanguíneo, osso autógeno, osso xenógeno de origem bovina e MDHD. Os animais foram sacrificados aos 7, 14 e 28 dias. Foram realizadas avaliações volumétricas por microtomografia (uCT) e análises histológicas qualitativas. Os resultados obtidos por meio do uCT mostraram valores semelhantes entre o MDHD e os demais grupos experimentais. A avaliação histológica demonstrou MDHD com processo inflamatório inespecífico e neoformação óssea com lenta reabsorção do material. Esse resultado indica que a MDHD implantada em alvéolo de rato é biocompatível e reduz a perda de volume do rebordo alveolar após extração dentária.
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The aim of this study was to analyze the suitability of pluripotent stem cells derived from the amnion (hAECs) as a potential cell source for revitalization in vitro. hAECs were isolated from human placentas, and dental pulp stem cells (hDPSCs) and dentin matrix proteins (eDMPs) were obtained from human teeth. Both hAECs and hDPSCs were cultured with 10% FBS, eDMPs and an osteogenic differentiation medium (StemPro). Viability was assessed by MTT and cell adherence to dentin was evaluated by scanning electron microscopy. Furthermore, the expression of mineralization-, odontogenic differentiation- and epithelial-mesenchymal transition-associated genes was analyzed by quantitative real-time PCR, and mineralization was evaluated through Alizarin Red staining. The viability of hAECs was significantly lower compared with hDPSCs in all groups and at all time points. Both hAECs and hDPSCs adhered to dentin and were homogeneously distributed. The regulation of odontoblast differentiation- and mineralization-associated genes showed the lack of transition of hAECs into an odontoblastic phenotype; however, genes associated with epithelial-mesenchymal transition were significantly upregulated in hAECs. hAECs showed small amounts of calcium deposition after osteogenic differentiation with StemPro. Pluripotent hAECs adhere on dentin and possess the capacity to mineralize. However, they presented an unfavorable proliferation behavior and failed to undergo odontoblastic transition.
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Polpa Dentária , Osteogênese , Âmnio , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Células Epiteliais , Humanos , Osteogênese/genética , Regeneração , Células-Tronco/metabolismoRESUMO
AIM: Lyophilized demineralized dentin matrix (LDDM) consists of a type 1 collagen complex matrix containing growth factors and no mineral crystals. Although the efficacy of LDDM for bone grafting is well known, there is limited evidence on the biological response to human lyophilized DDM. Therefore, the aim of this study was to evaluate the biological response of subcutaneous tissues in rats to powdered LDDM, mineral trioxide aggregate (MTA), and Biodentine implanted using polyethylene tubes. METHODS: Forty Wistar rats were divided into four groups (n = 10 each) depending on the experimental time intervals and were placed in polyethylene tubes containing LDDM, MTA, biodentine, and one empty control. After 3, 7, 15, and 30 observation days, the animals were sacrificed and quantitative and qualitative analysis of the subcutaneous tissue samples was carried out. The intensity of the inflammatory response was scored from 0 (no response) to 3 (severe response), and the data were statistically analyzed using ANOVA and Bonferroni tests (p < 0.05). RESULTS: All groups exhibited moderate inflammation after 3 and 7 days of observation, with presence of inflammatory infiltrate predominantly consisting of macrophages and angioblastic proliferation being observed. After 15 observation days, the control group exhibited mild inflammation and a predominance of fibroblasts, and this differed significantly from the remaining cement groups that exhibited moderate inflammation. After 30 days of observation, all groups exhibited a mild inflammatory response, predominance of fibroblasts, and a greater amount of collagen fibers. CONCLUSION: Within the limitations of this study, it can be concluded that LDDM exhibited an acceptable biological response similar to MTA and Biodentine in the subcutaneous tissues of rats.
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OBJECTIVES: This study aimed to revisit benign odontogenic ghost cell lesions (BOGCL) by hematoxylin and eosin staining and immunohistochemistry. MATERIALS AND METHODS: Thirty cases of calcifying odontogenic cyst (COC) and 6 cases of dentinogenic ghost cell tumor (DGCT) were selected for histopathological and immunohistochemical analysis. Sections stained for cytokeratin (K) 14, K-19, amelogenin, collagen type 1 (COL-1), and dentin matrix acidic phosphoprotein 1 (DMP-1) were evaluated using qualitative analysis. Sections stained for Ki-67 and minichromosome maintenance protein-2 (MCM-2) were evaluated using semi-quantitative analysis. RESULTS: A morphologic overlap was noticed in all BOGCL. Moreover, no differences were detected in the expression of K-14 and K-19. The expression of proliferative markers Ki-67 and MCM-2 was similar between cystic and tumor lesions (p > .05). The presence of COL-1 and absence of amelogenin in the so-called dysplastic dentin, associated with its histologic pattern, suggest that this is in fact an enameloid-like tissue. CONCLUSIONS: The dysplastic dentin should be considered an enameloid-like tissue in these lesions. CLINICAL RELEVANCE: The similarity in histology, protein expression, and proliferative marker indices between COC and DGCT suggest that they are a sole entity and likely represent types of the same neoplasia.
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Dentina , Cisto Odontogênico Calcificante , Tumores Odontogênicos , Colágeno Tipo I , Humanos , QueratinasRESUMO
Apesar de sua capacidade de reparo, o tecido ósseo pode ser submetido a alguns tipos de fraturas, cirurgias ou patologias que podem levar a grandes defeitos ósseos. As principais estratégias de tratamento de defeitos ósseos são baseados em osteoindução ou osteocondução. Matriz dentinária desmineralizada humana (MDDH) é uma alternativa biocompatível para preencher defeitos ósseos, melhorando a qualidade e quantidade de osso produzido. 24 Ratos Wistar foram selecionados, submetidos à extração de ambos os segundos molares superiores (direito e esquerdo). Os alvéolos foram separados em dois grupos: controle (direita) preenchido com coágulo sanguíneo e experimental (esquerda) preenchido com MDDH. Os animais foram sacrificados aos 5, 10 e 21 dias. Foram realizadas análises histológicas, histomorfométricas (análise de variância - ANOVA e teste de Tukey) e imunohistoquímica para osteopontina (OPN) como indicador de osteogênese. Aos 5 dias MDDH foi incorporada pelas novas trabéculas ósseas. Aos 10 dias observou-se organização do tecido conjuntivo e trabéculas no grupo experimental. Detectou-se coloração intensa para OPN em área adjacente à MDDH no grupo experimental. Aos 21 dias no grupo experimental verificou-se trabéculas maduras. Houve diferença estatísticamente significatva (p <0,05). Maior número de trabéculas em grupos experimentais que nos grupos controle em todos os períodos de análise. MDDH implantadas em alvéolos de ratos, induz a aceleração da osteogênese. Presença OPN observada mais intensamente aos 10 dias próximo à MDDH
BACKGROUND - Despite its good capacity for regeneration, bone tissue subjected to some types of fractures or surgery that can lead to large bone defects. The major bone defects treatment strategies are based in osteoinduction or osteoconduction. Demineralized human dentin matrix (DHDM) is a biocompatible alternative to fill bone defect, improving the quantity and quality of bone produced. METHODS - Wistar rats were selected, submitted to the extraction of both second molars (right and left). Alveoli were separated into two groups: control (right) filled with blood clot and experimental (left) filled with DHDM. Animals were sacrificed at 5, 10 and 21 days. Histological and histoquantitative analyzes (analysis of variance - ANOVA, and Tukey's test) were performed and immunostaining for osteopontin (OPN) as osteogenesis indicator. RESULTS - 5 days - DHDM incorporated by new trabeculae. 10 days - connective tissue organization and new trabeculae in the experimental group. Intense staining for OPN close to DHDM in the experimental group. 21 days - experimental group showing mature trabeculae. Statistical difference observed (p<0.05). Higher number of trabeculae in experimental groups in all periods of analysis. CONCLUSIONS - DHDM implanted in alveoli induces the acceleration of osteogenesis. Presence of OPN observed more intensely at 10 days close to DHDM
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Animais , Ratos , Processo Alveolar/anormalidades , Matriz Óssea/anormalidades , Regeneração Óssea/fisiologia , Dente Molar/anatomia & histologia , Osteopontina/análise , Osteopontina/genética , Análise de Variância , Interpretação Estatística de DadosRESUMO
Dentin matrix protein 1 (DMP1) is an acidic phosphoprotein that plays an important role in mineralized tissue formation by initiation of nucleation and modulation of mineral phase morphology. The purpose of the present study was to examine the immunoexpression of DMP1 in tooth germs of 7 human fetuses at different gestational ages (14, 16, 19, 20, 21, 23 and 24 weeks) comparing with completed tooth formation erupted teeth. The results showed the presence of DMP1 in the dental lamina, as well as in the cells of the external epithelium, stellate reticulum and stratum intermedium of the enamel organ. However, in the internal dental epithelium, cervical loop region and dental papilla some cells have not labeled for DMP1. In the crown stage, DMP1 was expressed in the ameloblast and odontoblast layer, as well as in the dentinal tubules of coronal dentin near the odontoblast area. Erupted teeth with complete tooth formation exhibited immunolabeling for DMP1 only in the dentinal tubules mainly close to the dental pulp. No staining was observed in the enamel, predentin or dental pulp matrix. DMP1 is present in all developing dental structures (dental lamina, enamel organ, dental papilla) presenting few immunoexpression variations, with no staining in mineralized enamel and dentin.
A proteína da matriz dentinária 1 (DMP1) é uma fosfoproteína ácida que tem sido relacionada diretamente ao processo de mineralização dos tecidos em formação sendo iniciadora do processo de nucleação e modulação da fase mineral. O objetivo desse trabalho foi avaliar a imunoexpressão da DMP1 em germes dentários em diferentes fases da odontogênese, obtidos de 7 fetos humanos em diversos estágios gestacionais (14, 16, 19, 20, 21, 23 e 24 semanas), comparando-se com dentes com rizogênese completa. Os resultados mostraram que a DMP1 esteve expressa na lâmina dentária, bem como, nas células do epitélio externo, retículo estrelado e estrato intermediário do órgão do esmalte. Diferentemente, no epitélio interno do órgão do esmalte, alça cervical e papila dentária algumas células não apresentaram a DMP1. Nas fases de coroa, os ameloblastos e odontoblastos apresentaram marcação positiva para a DMP1, bem como os túbulos dentinários da dentina coronária próximos à região odontoblástica. Os dentes com rizogênese completa exibiram marcação para a DMP1 apenas nos túbulos dentinários principalmente próximos à polpa dentária. Nenhuma marcação foi observada na matriz de esmalte ou pré-dentina, nem na polpa dentária. Concluímos que a DMP1 está presente em todas as fases da odontogênese, tanto na lâmina dentária, órgão do esmalte, bem como na papila dentária, com pequenas variações de nuances de expressão, estando ausente na dentina e esmalte mineralizados.