Effect of a novel bioceramic root canal sealer on the angiogenesis-enhancing potential of assorted human odontogenic stem cells compared with principal tricalcium silicate-based cements

Abstract Objective: This study evaluated the angiogenesis-enhancing potential of a tricalcium silicate-based mineral trioxide aggregate (ProRoot MTA), Biodentine, and a novel bioceramic root canal sealer (Well-Root ST) in human dental pulp stem cells (hDPSCs), human periodontal ligament stem cells (hPLSCs), and human tooth germ stem cells (hTGSCs). Methodology: Dulbecco's modified Eagle's medium was conditioned for 24 h by exposure to ProRoot MTA, Biodentine, or Well-Root ST specimens (prepared according to the manufacturers' instructions). The cells were cultured in these conditioned media and their viability was assessed with 3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxy-methoxy-phenyl)-2-(4-sulfo-phenyl)-2H tetrazolium (MTS) on days 1, 3, 7, 10, and 14. Angiogenic growth factors [platelet-derived growth factor (PDGF), basic fibroblast growth factor (FGF-2), and vascular endothelial growth factor (VEGF)] were assayed by sandwich enzyme-linked immunosorbent assay (ELISA) on days 1, 7, and 14. Human umbilical vein endothelial cell (HUVEC) migration assays were used to evaluate the vascular effects of the tested materials at 6-8 h. Statistical analyses included Kruskal-Wallis, Mann-Whitney U, and Friedman and Wilcoxon signed rank tests. Results: None of tricalcium silicate-based materials were cytotoxic and all induced a similar release of angiogenic growth factors (PDGF, FGF-2, and VEGF) (p>0.05). The best cell viability was observed for hDPSCs (p<0.05) with all tricalcium silicate-based materials at day 14. Tube formation by HUVECs showed a significant increase with all tested materials (p<0.05). Conclusion: The tricalcium silicate-based materials showed potential for angiogenic stimulation of all stem cell types and significantly enhanced tube formation by HUVECs.

Avaliação da expressão apical de fatores pró-angiogênicos e citocinas em reposta à infecção endodôntica / Evaluation of apical expression of pro-angiogenic factors and cytokines in response to endodontic infection

A progressão e desenvolvimento de patologias pulpares e periapicais estão intimamente relacionados à presença de microrganismos e seus subprodutos nos sistema de canais radiculares (SCR), que induzem uma resposta de defesa adjacente ao ápice radicular. A angiogênese é apontada como fator essencial na patogênese das alterações periapicais crônicas, estando relacionada ao seu estabelecimento e manutenção, por ser fonte constante de citocinas, quimiocinas e proteases. A angiogênse também está relacionada ao reparo tecidual que segue à resolução das alterações perirradiculares após a realização da terapia endodôntica. Neste estudo, avaliou-se a expressão de fatores pró-angiogêncios e citocinas relacionadas, em amostras coletadas de pacientes (n=20) com dentes portadores de Periodontite Apical Crônica imediatamente após a instrumentação dos SCR e 7 dias após os procedimentos de desinfecção. As amostras foram analisadas por meio de reação em cadeia da polimerase em tempo real (PCR-RT). Verificou-se a expressão gênica de fatores pró-angiogênicos e citocinas Angiopoetina-1 (AGT1), Fator de crescimento endotelial vascular-A (VEGF-A), Fator de crescimento fibroblástico básico (FGF-ß), Proteína quimiotática de monócitos (CCL2/MCP-1), Proteína inflamatória de macrófagos-1ß (CCL4), C-X-C Receptor de quimiocina tipo 4 (CXCR4), C-C Receptor de quimiocina tipo 6 (CCR6), TNF-α, IFN-γ, IL-17, IL-10, IL6, RANK-L e MMP-9. A expressão do mRNA dos mediadores avaliados revelou aumento significativo nos níveis de AGT1, CCL2/MCP-1, CCL4, CCR6, TNF-α, IFN- γ, IL-10, RANK-L e MMP-9 no dia 7 quando comparado com o dia 0 (P <0,05). Para VEGF-A, FGF-ß, CXCR4, IL-17 e IL-6 as expressões de mRNA foram semelhantes em ambos tempos mensurados (P> 0,05). Pode-se concluir que, após desinfecção do SCR, houve aumento nos níveis de expressão de mRNA de importantes mediadores envolvidos nos fenômenos angioproliferativos e osteogênicos.(AU)

The progression and development of pulpal and periapical pathologies are closely related to the presence of microorganisms and their by-products in the infected root canal system (RCS), which induces a defense response adjacent to the root apex. Angiogenesis has been identified as an essential factor in the pathogenesis of chronic periapical alterations, being related to its establishment and maintenance, being a constant source of cytokines, chemokines and proteases. Angiogenesis is also related to the tissue repair that follows the resolution of the periradicular alterations after the implementation of endodontic therapy. In this study, was evaluated the expression of pro-angiogenic factors and correlated cytokines in samples collected from patients (n = 20) on teeth with Chronic Apical Periodontitis immediately after RCS instrumentation and 7 days after disinfection procedures. Samples were analyzed by real-time polymerase chain reaction (RT-PCR). The gene expression of pro-angiogenic factors and cytokines Angiopoetin-1 (AGT1), Vascular endothelial growth factor-A (VEGF-A), Basic fibroblast growth factor (FGF-ß), Monocyte Chemoattractant Protein-1 (CCL2/MCP-1), Macrophage inflammatory protein-1ß (CCL4), C-X-C chemokine receptor motif 4 (CXCR4), C-C chemokine receptor motif 6 (CCR6), TNF-α, IFN-γ, IL-17, IL-10, IL-6, RANK-L and MMP-9. The mRNA expression of the mediators evaluated revealed a significant increase in levels of AGT1, CCL2/MCP-1, CCL4, CCR6, TNF-α, IFN-γ, IL-10, RANK-L and MMP-9 on day 7 when compared to day 0 (P <0.05). As for VEGF-A, FGF-ß, CXCR4, IL-17 and IL-6 their mRNA expressions was similar at both observed times (P> 0.05). In conclusion, after cleaning and shaping procedures of the RCS, there was an increase in mRNA expression levels of important mediators involved in angioproliferative and osteogenic phenomenon.(AU)

Estudo da imunoexpressão do VEGF165 e do VEGF165B em lesões orais de líquen plano e pênfigo vulgar / Study of VEGF165 and VEGF165B immunoexpression in oral lesions of lichen planus and pemphigus vulgaris

O Líquen Plano Oral (LPO) é uma doença mucocutânea mediada imunologicamente, de etiologia desconhecida, relativamente comum, com prevalências, na população mundial, que variam de 0,22 a 5%. O pênfigo vulgar é uma doença autoimue crônica que pode acometer a mucosa oral sendo o mais comum dos tipos de pênfigo. Entretanto, sua ocorrência é rara, com incidência estimada na população geral de um a cinco casos por milhão de pessoas diagnosticadas a cada ano. O VEGF-A é a proteína angiogênica mais potente tanto na angiogênese normal quanto na patológica. O splicing alternativo do éxon 8 do gene do VEGFA dá origem a duas famílias conhecidas de proteínas isofórmicas, uma desempenhando papel angiogênico, VEGFxxxa, e outra um papel antiangiogênico, VEGFxxxb. Este trabalho se propôs a avaliar a expressão imunoistoquímica do VEGF165 (angiogênico), do VEGF165b (antiangiogênico) em 46 casos de LPO reticular, 23 casos de LPO erosivo e 12 casos de PV, usando como controle 11 casos de hiperplasia fibrosa. Todos os espécimes das lesões e os casos controle foram divididos em e zonas para a análise das marcações, em zona superficial (Z1), média (Z2) e profunda (Z3). Os resultados deste experimento foram submetidos a testes estatístico não-paramétricos com nível de significância de 5%. Comparando apenas as lesões para o marcador anti-VEGF165 foram observadas diferenças significativas apenas nas zonas mais profundas entre as lesões de LPO reticular e PV, e entre as lesões de LPO erosivo e PV. Para o marcador anti-VEGF165b diferenças significativas foram observadas nas zonas médias entre as lesões de LPO reticular e PV; e nas zonas profundas entre LPO erosivo e PV e entre LPO reticular e PV. Avaliando o marcador VEGF165b nos espécimes sem categorizá-los por zonas foram observadas diferenças significativas entre as lesões de LPO reticular e PV. Na análise da correlação entre ambos os marcadores em cada lesão foram observadas correlação positiva fraca e significativa nas zonas média e profunda do LPO reticular e na zona superficial do LPO erosivo. Os resultados do presente estudo sugerem a participação do processo angiogênico na patogênese do LPO e na progressão das lesões de líquen plano oral e pênfigo vulgar, porém outros estudos devem ser realizados a fim de que esses achados, principalmente em relação ao pênfigo vulgar seja fundamentado, uma vez que a presente pesquisa é uma das primeiras que avalia a angiogênese na lesão já estabelecida dessa doença (AU).

Oral Lichen Planus is an immunologically mediated mucocutaneous disease of relatively unknown etiology with prevalences in the world population varying from 0.22 to 5%. Pemphigus vulgaris is a chronic autoimmune disease that may affect the oral mucosa being the most common type of pemphigus. However, its occurrence is rare, with an estimated incidence in the general population of one to five cases per million people diagnosed each year. Angiogenesis plays an important role in tumor growth and in the progression of chronic inflammatory diseases. VEGF-A is the most potent angiogenic protein in both normal and pathological angiogenesis. The alternative splicing of exon 8 VEGF-A gene gives rise to two known families of isoform proteins, one playing angiogenic role, VEGFxxxa, and another an antiangiogenic role, VEGFxxxb. The aim of this study was to evaluate the immunohistochemical expression of VEGF165 (angiogenic), VEGF165b (antiangiogenic) in 46 cases of reticular OLP, 23 cases of erosive OLP and 12 cases of PV, using as control 11 cases of fibrous hyperplasia. All specimens of the lesions and the control cases were divided into zones for the analysis of the immunohistochemical stains, in superficial (Z1), medium (Z2) and deep zones (Z3). The results of this experiment were submitted to non-parametric statistical tests with significance level of 5%. For all immunohistochemical stains the comparison between the lesions with the control group (HF) showed significant differences. Comparing only the lesions to the anti-VEGF165 stains, significant differences were observed only in the deeper zones between the reticular LPO lesions X PV; and between erosive LPO lesions X PV. For the anti-VEGF165b stains, significant differences were observed in medium zones between reticular OLP X PV lesions; and in deep zones between erosive LPO X PV and between reticular LPO and PV. And evaluating VEGF165b stains in specimens without categorizing them by zones was observed a significant difference between reticular LPO and PV lesions. In the analysis of the correlation between both markers in each lesion, a weak and significant positive correlation was observed in medium and deep zones of reticular OLP; and a weak positive correlation in superficial zone of erosive LPO. The present study results suggest angiogenic process participation in the pathogenesis and progression of lesions of oral lichen planus and pemphigus vulgaris. However other studies must be carried out in order that this implication, mainly in relation to pemphigus vulgaris be based once this is one of the first studies to evaluate angiogenesis in the already established lesion of this disease (AU).