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Mesenchymal stem-cell-derived extracellular vesicles (MSC-EVs) have been increasingly investigated for cancer therapy and drug delivery, and they offer an advanced cell-free therapeutic option. However, their overall effects and efficacy depend on various factors, including the MSC source and cargo content. In this study, we isolated EVs from the conditioned medium of human immature dental pulp stem cells (hIDPSC-EVs) and investigated their effects on two papillary thyroid cancer (PTC) cell lines (BCPAP and TPC1). We observed efficient uptake of hIDPSC-EVs by both PTC cell lines, with a notable impact on gene regulation, particularly in the Wnt signaling pathway in BCPAP cells. However, no significant effects on cell proliferation were observed. Conversely, hIDPSC-EVs significantly reduced the invasive capacity of both PTC cell lines after 120 h of treatment. These in vitro findings suggest the therapeutic potential of hIDPSC-EVs in cancer management and emphasize the need for further research to develop novel and effective treatment strategies. Furthermore, the successful internalization of hIDPSC-EVs by PTC cell lines underscores their potential use as nanocarriers for anti-cancer agents.

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BACKGROUND: Histological techniques are essential for the microscopic study and investigation of the human dental pulp. The aim of this study was to evaluate the impact of decalcification-free technique by examining dental pulp morphology by histological staining with haematoxylin and eosin and immunohistochemistry. MATERIALS AND METHODS: The sample consisted of 30 healthy third molars extracted for orthodontic indication, the pulp tissue was obtained by removing the mineralized tissues, separating the enamel and dentine and by marking with a flexible diamond disc on the coronal surface and longitudinal axis of the root. These guides made it possible to separate the fragments and obtain the pulp tissue for fixation and staining with H&E and subsequent immunohistochemistry with CD34 and S-100 antibodies. RESULTS: The technique showed preservation of pulp morphology with adequate preservation of microscopic structures. No alterations in tissue viability were observed. The staining allowed an accurate assessment of vascular and nervous components by means of CD34 and S-100 markers, respectively. CONCLUSIONS: This technique allows preservation of pulp tissue, maintaining viable tissue for histological analysis and immunohistochemistry tests, as well as reducing sample processing time.

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BACKGROUND: Validation of the reference gene (RG) stability during experimental analyses is essential for correct quantitative real-time polymerase chain reaction (RT-qPCR) data normalisation. Commonly, in an unreliable way, several studies use genes involved in essential cellular functions [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S rRNA, and ß-actin] without paying attention to whether they are suitable for such experimental conditions or the reason for choosing such genes. Furthermore, such studies use only one gene when Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines recommend two or more genes. It impacts the credibility of these studies and causes distortions in the gene expression findings. For tissue engineering, the accuracy of gene expression drives the best experimental or therapeutical approaches. AIM: To verify the most stable RG during osteogenic differentiation of human dental pulp stem cells (DPSCs) by RT-qPCR. METHODS: We cultivated DPSCs under two conditions: Undifferentiated and osteogenic differentiation, both for 35 d. We evaluated the gene expression of 10 candidates for RGs [ribosomal protein, large, P0 (RPLP0), TATA-binding protein (TBP), GAPDH, actin beta (ACTB), tubulin (TUB), aminolevulinic acid synthase 1 (ALAS1), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta (YWHAZ), eukaryotic translational elongation factor 1 alpha (EF1a), succinate dehydrogenase complex, subunit A, flavoprotein (SDHA), and beta-2-microglobulin (B2M)] every 7 d (1, 7, 14, 21, 28, and 35 d) by RT-qPCR. The data were analysed by the four main algorithms, ΔCt method, geNorm, NormFinder, and BestKeeper and ranked by the RefFinder method. We subdivided the samples into eight subgroups. RESULTS: All of the data sets from clonogenic and osteogenic samples were analysed using the RefFinder algorithm. The final ranking showed RPLP0/TBP as the two most stable RGs and TUB/B2M as the two least stable RGs. Either the ΔCt method or NormFinder analysis showed TBP/RPLP0 as the two most stable genes. However, geNorm analysis showed RPLP0/EF1α in the first place. These algorithms' two least stable RGs were B2M/GAPDH. For BestKeeper, ALAS1 was ranked as the most stable RG, and SDHA as the least stable RG. The pair RPLP0/TBP was detected in most subgroups as the most stable RGs, following the RefFinfer ranking. CONCLUSION: For the first time, we show that RPLP0/TBP are the most stable RGs, whereas TUB/B2M are unstable RGs for long-term osteogenic differentiation of human DPSCs in traditional monolayers.

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Background: Antibiotic pastes used as intracanal medication in cases of revascularization therapy might cause negative effects on tooth properties, such as a reduction in dentin microhardness. This in vitro study investigated dentin microhardness in three different locations distancing from the canal lumen after 20 days of treatment with a tri-antibiotic paste (ciprofloxacin, metronidazole, and minocycline), and with a double-antibiotic paste (ciprofloxacin and metronidazole), with calcium hydroxide [Ca(OH)2] UltracalTM XS-treated dentin as comparison. Material and Methods: Human mandibular premolars (n = 48) had the root canals cleaned and shaped and were used to produce dentin slices. Dentin slices remained immersed in the medications for 20 days. The Knoop microhardness (KHN) test was performed before (baseline/Day-0) and after treatment (Day-20) with the medications. Indentations were made at 25 µm, 50 µm, and 100 µm distances from the root canal lumen. The KHN was compared intra-group using Wilcoxon's test. Independent groups were compared using Mann-Whitney's and Kruskal-Wallis' tests, at α = 5%. Results: The microhardness in all the tested groups was reduced at Day-20 in comparison with Day-0 (p < 0.001) (intra-group comparison/same distances). The Day-0 values were similar, and the Day-20 values were higher for the Ca(OH)2 group (p < 0.05) (comparison between groups/same distances). Conclusions: Calcium hydroxide for 20 days would be preferred rather than antibiotic pastes to minimize the expected reduction in dentin microhardness during regenerative procedures.

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Aims: This ex vivo study aimed to assess the dissolving capacity of 2.5% sodium hypochlorite using eight agitation protocols within swine pulp tissue. Subjects and Methods: Twelve lower first premolars were prepared and split into the fragments with a groove housing porcine dental pulp. Groups were assigned based on agitation systems: manual, passive ultrasonic, Easy Clean and XP-Endo Finisher. Two agitation time protocols were applied: One min (3 s × 20 s cycles) and 2 min (6 s × 20 s cycles). Wilcoxon Mann-Whitney U test was used to compare the groups. Results: Both time frames demonstrated superior results compared to manual group (P > 0.5). However, in the two min groups, no significant differences were observed among the other protocols (P < 0.5). Intriguingly, increasing cycle numbers significantly improved results within each group (P > 0.5). Conclusion: Extending the chemical agitation time during final irrigation enhances tissue removal, regardless of the irrigation protocol employed.

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The objective of this study was to create injectable photo-crosslinkable biomaterials, using gelatin methacryloyl (GelMA) hydrogel, combined with a decellularized bone matrix (BMdc) and a deproteinized (BMdp) bovine bone matrix. These were intended to serve as bioactive scaffolds for dentin regeneration. The parameters for GelMA hydrogel fabrication were initially selected, followed by the incorporation of BMdc and BMdp at a 1% (w/v) ratio. Nano-hydroxyapatite (nHA) was also included as a control. A physicochemical characterization was conducted, with FTIR analysis indicating that the mineral phase was complexed with GelMA, and BMdc was chemically bonded to the amide groups of gelatin. The porous structure was preserved post-BMdc incorporation, with bone particles incorporated alongside the pores. Conversely, the mineral phase was situated inside the pore opening, affecting the degree of porosity. The mineral phase did not modify the degradability of GelMA, even under conditions of type I collagenase-mediated enzymatic challenge, allowing hydrogel injection and increased mechanical strength. Subsequently, human dental pulp cells (HDPCs) were seeded onto the hydrogels. The cells remained viable and proliferative, irrespective of the GelMA composition. All mineral phases resulted in a significant increase in alkaline phosphatase activity and mineralized matrix deposition. However, GelMA-BMdc exhibited higher cell expression values, significantly surpassing those of all other formulations. In conclusion, our results showed that GelMA-BMdc produced a porous and stable hydrogel, capable of enhancing odontoblastic differentiation and mineral deposition when in contact with HDPCs, thereby showing potential for dentin regeneration.

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INTRODUCTION: Pulp stones (PS) are calcifications commonly found in the pulp tissue that may be associated with systemic diseases. OBJECTIVE: To evaluate the association between PS and systemic diseases. METHODS: A case-control study with the inclusion of individuals from 18 to 65 years of age, of both sexes. Analysis was made of 1047 digital panoramic radiographs. The controls could not have any teeth with PS; the cases were the contrary. A questionnaire comprising demographic, habit, and general health (diabetes, problems with blood vessels, altered cholesterol level, heart attack, kidney or gallbladder stone, arthritis, or autoimmune disease, and for women, endometriosis, and ovarian cyst). Data were submitted to the Student's t-test to identify differences between groups about sex and age. The Chi-square test was applied to the cross-tabulation. The analyses were performed using SPSS®, version 25.0, with a 5% significance level. RESULTS: 490 patients participated (242 cases and 248 controls). There was no difference between groups for the sex (p=0.966) and age (p=0.186). Only "kidney stone" was associated with the case group (p=0.001), being almost three times higher when compared to the control group. No significant differences were found in females about the presence or absence of PS (p>0.05). CONCLUSÃO: In this research, it is suggested the existence of an association between kidney stones and the presence of pulp stones.

INTRODUÇÃO: Nódulos pulpares (NP) são calcificações comumente encontradas no tecido pulpar que podem estar associadas a doenças sistêmicas. OBJETIVO: Avaliar a associação entre NP e doenças sistêmicas. MÉTODOS: Estudo caso-controle com inclusão de indivíduos de 18 a 65 anos de idade, de ambos os sexos. Foram analisadas 1047 radiografias panorâmicas digitais. Os controles não poderiam ter dentes com NP; os casos foram o contrário. Foi aplicado um questionário aos participantes, contendo variáveis demográficas, de hábitos e de saúde geral (diabetes, problemas com vasos sanguíneos, nível de colesterol alterado, ataque cardíaco, cálculo renal ou biliar, artrite ou doença autoimune, e para as mulheres, endometrioses e cisto no ovário). Os dados foram submetidos ao teste t de Student para identificar diferenças entre os grupos em relação ao sexo e à idade. O teste Qui-quadrado foi aplicado para a tabulação cruzada. As análises foram realizadas no SPSS®, versão 25.0, com nível de significância de 5%. RESULTADOS: Participaram 490 pacientes (242 casos e 248 controles). Não houve diferença entre os grupos para sexo (p=0,966) e idade (p=0,186). Apenas "cálculo renal" associou-se ao grupo caso (p=0,001), sendo quase três vezes maior quando comparado ao grupo controle. Não foram encontradas diferenças significativas no sexo feminino em relação à presença ou ausência de PS (p>0,05). CONCLUSÃO: Nesta pesquisa, sugere-se a existência de uma associação entre cálculos renais e presença de Nódulos pulpares.

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BACKGROUND: Amelogenesis imperfecta is a hereditary disorder affecting dental enamel. Among its phenotypes, hypocalcified AI is characterized by mineral deficiency, leading to tissue wear and, consequently, dental sensitivity. Excessive fluoride intake (through drinking water, fluoride supplements, toothpaste, or by ingesting products such as pesticides or insecticides) can lead to a condition known as dental fluorosis, which manifests as stains and teeth discoloration affecting their structure. Our recent studies have shown that extracts from Colombian native plants, Ilex guayusa and Piper marginatum, deposit mineral ions such as phosphate and orthophosphate into the dental enamel structure; however, it is unknown whether these extracts produce toxic effects on the dental pulp. OBJECTIVE: To assess cytotoxicity effects on human dental pulp stem cells (hDPSCs) exposed to extracts isolated from I. guayusa and P. marginatum and, hence, their safety for clinical use. METHODS: Raman spectroscopy, fluorescence microscopy, and flow cytometry techniques were employed. For Raman spectroscopy, hDPSCs were seeded onto nanobiochips designed to provide surface-enhanced Raman spectroscopy (SERS effect), which enhances their Raman signal by several orders of magnitude. After eight days in culture, I. guayusa and P. marginatum extracts at different concentrations (10, 50, and 100 ppm) were added. Raman measurements were performed at 0, 12, and 24 h following extract application. Fluorescence microscopy was conducted using an OLIMPUS fv1000 microscope, a live-dead assay was performed using a kit employing a BD FACS Canto TM II flow cytometer, and data analysis was determined using a FlowJo program. RESULTS: The Raman spectroscopy results showed spectra consistent with viable cells. These findings were corroborated using fluorescence microscopy and flow cytometry techniques, confirming high cellular viability. CONCLUSIONS: The analyzed extracts exhibited low cytotoxicity, suggesting that they could be safely applied on enamel for remineralization purposes. The use of nanobiochips for SERS effect improved the cell viability assessment.

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O objetivo do presente estudo foi identificar microrganismos das espécies Enterococcus spp e Enterobacteriaceae em dentes com canais radiculares infectados portadores de infecção primária e/ou secundária/persistente. Métodos: A amostra do presente estudo foi de 23 pacientes que apresentaram necessidade de tratamento ou retratamento endodôntico. Foram coletadas amostras de 28 dentes infectados usando pontas de papel absorventes estéreis, transportadas em solução salina, diluídas, plaqueadas e incubadas em estufa de cultura bacteriológica. Para o crescimento de microrganismos foram utilizados jarros com gerador de atmosfera de anaerobiose. Colônias microbianas foram isoladas, caracterizadas e identificadas. Os dados coletados foram estatisticamente analisados com a utilização do software SPSS for Windows 10.0 (SPSS Inc., USA). Resultados: Foi isolada somente uma cepa do gênero Enterococcus spp, e nenhuma espécie do gênero Enterobacteriaceae. Das coletas microbiológicas realizadas em 28 canais radiculares, todas apresentaram crescimento microbiano em anaerobiose. Dezoito dentes apresentavam necrose pulpar e lesão periapical. Os outros 10 dentes já haviam recebido tratamento endodôntico prévio e em 6 destes houve constatação de lesão periapical, sendo que nos outros 4, não. Conclusão: Nas condições experimentais do presente estudo, pode-se concluir que não houve correlação da presença de espécies microbianas das famílias Enterococcus spp e/ou Enterobacteriaceae com infecção primária ou secundária do canal radicular.

The objective of the present study was to identify microorganisms of the Enterococcus spp and Enterobacteriaceae species in teeth with infected root canals with primary and/or secondary/persistent infection. Methods: The sample of the present study consisted of 23 patients who required endodontic treatment or retreatment. Samples of 28 infected teeth were collected using sterile absorbent paper points, transported in saline solution, diluted, plated and incubated in a bacteriological culture oven. For the growth of microorganisms, jars with an anaerobic atmosphere generator were used. Microbial colonies were isolated, characterized and identified. The collected data were statistically analyzed using the SPSS for Windows 10.0 software (SPSS Inc., USA). Results: Only one strain of the genus Enterococcus spp was isolated, and no species of the genus Enterobacteriaceae. From the microbiological collections carried out in 28 root canals, all showed microbial growth in anaerobic conditions. Eighteen teeth had pulp necrosis and periapical lesion. The other 10 teeth had already received previous endodontic treatment and in 6 of them there was a periapical lesion, and in the other 4, no. Conclusion: Under the experimental conditions of the present study, it can be concluded that there was no correlation between the presence of microbial species of the Enterococcus spp and/or Enterobacteriaceae families with primary or secondary root canal infection.

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BACKGROUND: In recent years, dental pulp stromal cells (DPSCs) have emerged as a promising therapeutic approach for Parkinson's disease (PD), owing to their inherent neurogenic potential and the lack of neuroprotective treatments for this condition. However, uncertainties persist regarding the efficacy of these cells in an undifferentiated state versus a neuronally-induced state. This study aims to delineate the distinct therapeutic potential of uninduced and neuronally-induced DPSCs in a rodent model of PD induced by 6-Hydroxydopamine (6-OHDA). METHODS: DPSCs were isolated from human teeth, characterized as mesenchymal stromal cells, and induced to neuronal differentiation. Neuronal markers were assessed before and after induction. DPSCs were transplanted into the substantia nigra pars compacta (SNpc) of rats 7 days following the 6-OHDA lesion. In vivo tracking of the cells, evaluation of locomotor behavior, dopaminergic neuron survival, and the expression of essential proteins within the dopaminergic system were conducted 7 days postgrafting. RESULTS: Isolated DPSCs exhibited typical characteristics of mesenchymal stromal cells and maintained a normal karyotype. DPSCs consistently expressed neuronal markers, exhibiting elevated expression of ßIII-tubulin following neuronal induction. Results from the animal model showed that both DPSC types promoted substantial recovery in dopaminergic neurons, correlating with enhanced locomotion. Additionally, neuronally-induced DPSCs prevented GFAP elevation, while altering DARPP-32 phosphorylation states. Conversely, uninduced DPSCs reduced JUN levels. Both DPSC types mitigated the elevation of glycosylated DAT. CONCLUSIONS: Our results suggested that uninduced DPSCs and neuronally-induced DPSCs exhibit potential in reducing dopaminergic neuron loss and improving locomotor behavior, but their underlying mechanisms differ.

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