Abstract Protease-activated receptor-2 (PAR2) is associated with the pathogenesis of many chronic diseases with inflammatory characteristics, including periodontitis. This study aimed to evaluate how the activation of PAR2 can affect the osteogenic activity of human periodontal ligament stem cells (PDLSCs) in vitro. PDLSCs collected from three subjects were treated in osteogenic medium for 2, 7, 14, and 21 days with trypsin (0.1 U/mL), PAR2 specific agonist peptide (SLIGRL-NH2) (100 nM), and PAR2 antagonist peptide (FSLLRY-NH2) (100 nM). Gene (RT-qPCR) expression and protein expression (ELISA) of osteogenic factors, bone metabolism, and inflammatory cytokines, cell proliferation, alkaline phosphatase (ALP) activity, alizarin red S staining, and supernatant concentration were assessed. Statistical analysis of the results with a significance level of 5% was performed. Activation of PAR2 led to decreases in cell proliferation and calcium deposition (p < 0.05), calcium concentration (p < 0.05), and ALP activity (p < 0.05). Additionally, PAR2 activation increased gene and protein expression of receptor activator of nuclear factor kappa-Β ligand (RANKL) (p < 0.05) and significantly decreased the gene and protein expression of osteoprotegerin (p <0. 05). Considering the findings, the present study demonstrated PAR2 activation was able to decrease cell proliferation, decreased osteogenic activity of PDLSCs, and upregulated conditions for bone resorption. PAR2 may be considered a promising target in periodontal regenerative procedures.

Abstract: PAR1 is a G-coupled protein receptor that regulates several cellular metabolism processes, including differentiation and proliferation of osteogenic and cementogenic related cells and our group previously demonstrated the regenerative potential of PAR1 in human periodontal ligament stem cells (hPDLSCs). In this study, we hypothesized that PAR1 regulates the cementogenic differentiation of hPDLSCs. Our goal was to identify the intracellular signaling pathway underlying PAR1 activation in hPDSLC differentiation. hPDLSCs were isolated using the explant technique. Cells were cultured in an osteogenic medium (OST) (α-MEM, 15% fetal bovine serum, L-glutamine, penicillin, streptomycin, amphotericin B, dexamethasone, and beta-glycerophosphate). The hPDLSCs were treated with a specific activator of PAR1 (PAR1 agonist) and blockers of the MAPK/ERK and PI3K pathways for 2 and 7 days. The gene expression of CEMP1 was assessed by RT-qPCR. The activation of PAR1 by its agonist peptide led to an increase in CEMP1 gene expression when compared with OST control. MAPK/ERK blockage abrogated the upregulation of CEMP1 gene expression induced by PAR1 agonist (p < 0.05). PI3K blockage did not affect the gene expression of CEMP1 at any experimental time (p > 0.05). We concluded that CEMP1 gene expression increased by PAR1 activation is MAPK/ERK-dependent and PI3K independent, suggesting that PAR1 may regulate cementogenetic differentiation of hPDLSCs.

Resumo Este estudo investigou o efeito de três materiais comerciais à base de silicato de cálcio (CSBM) na citotoxicidade e na produção de citocinas pró e antiinflamatórias em células-tronco do ligamento periodontal humano (hPDLSCs). Cultura de hPDLSCs foi estabelecida e caracterizada. Extratos de Bio-C Sealer (Angelus, Londrina, PR, Brasil), MTA Fillapex (Angelus, Londrina, PR, Brasil) e PBS Cimmo HP® (Cimmo Soluções em Saúde, Pouso Alegre, MG, Brasil) foram preparados com a colocação de espécimes dos cimentos (5 x 3 mm) em meio de cultura. Em seguida, os extratos foram diluídos (1, 1: 2, 1: 4, 1: 8, 1:16) e inseridos nos poços semeados de células para ensaio de citotoxicidade por meio de MTT por 24, 48 e 72 h. As citocinas TNF-α e IL-10 foram quantificadas por ELISA em sobrenadantes de células de 24 h. Os dados foram analisados por ANOVA e teste de Tukey (α = 0,05). Todos os CSBM exibiram alguma citotoxicidade que variou de acordo com a concentração do extrato e o tempo de avaliação. O MTA Fillapex apresentou os maiores efeitos citotóxicos com redução significativa da atividade metabólica / viabilidade celular quando comparado ao Bio-C Sealer e Cimmo HP®. O TNF-α foi regulado positivamente pelos três cimentos testados (p <0,05), enquanto apenas o MTA Fillapex regulou positivamente a liberação de IL-10 em comparação com o controle. Tomados em conjunto, os resultados mostraram que PBS Cimmo HP®, Bio-C Sealer e MTA Fillapex apresentam citotoxicidade leve e transitória e induziram a produção de TNF-α. O MTA Fillapex regulou positivamente a liberação de IL-10 por hPDLSCs.

Abstract This study investigated the effect of three commercial calcium silicate-based materials (CSBM) on cytotoxicity and pro-and anti-inflammatory cytokines production in cultured human periodontal ligament stem cells (hPDLSCs). Culture of hPDLSCs was established and characterized. Extracts of Bio-C Sealer (Angelus, Londrina, PR, Brazil), MTA Fillapex (Angelus, Londrina, PR, Brazil) and PBS Cimmo HP (Cimmo Soluções em Saúde, Pouso Alegre, MG, Brazil) were prepared by placing cement specimens (5 x 3 mm) in culture medium. Then, the extracts were serially two-fold diluted (1, 1:2, 1:4, 1:8, 1:16) and inserted into the cell-seeded wells for 24, 48 and 72 h for MTT assays. TNF-α and IL-10 cytokines were quantified by ELISA at 24h-cell supernatants. Data were analyzed by ANOVA and Tukey’s test (α = 0.05). All CSBM exhibited some cytotoxicity that varied according to extract concentration and time of evaluation. MTA Fillapex presented the highest cytotoxic effects with significant reduction of metabolic activity/cell viability when compared to Bio-C Sealer and Cimmo HP®. TNF-α was significantly upregulated by the three tested cements (p < 0.05) while only MTA Fillapex significantly upregulated IL-10 in comparison to control. Taken collectively, the results showed that PBS Cimmo HP®, Bio-C Sealer and MTA Fillapex present mild and transient cytotoxicity and slightly induced TNF-α production. MTA Fillapex upregulated IL-10 release by hPDLSCs.

Resumo No presente estudo nós comparamos os efeitos de um inibidor seletivo da COX-2 (etoricoxibe) com um anti-inflamatório não esteroidal não seletivo (AINE) (diclofenaco de potássio) no processo inflamatório e perda óssea alveolar em modelo de periodontite experimental. Noventa ratos Holtzman machos (250 g) foram randomizados em quatro grupos experimentais: Sham+CMC e Ligadura+CMC (controle) que receberam solução de carboximetilcelulose de sódio (CMC) a 0,5%; Ligadura+Diclofenaco e Ligadura+Etoricoxibe que receberam diclofenaco de potássio e etoricoxibe, respectivamente, suspensos em CMC 0,5% (10 mg/kg/dia). 7, 14 e 21 dias após colocação de ligadura bilateral na região cevical dos primeiros molares inferiores, os animais foram submetidos à eutanásia. No fim de cada período, as mandíbulas foram coletadas para exame radiográfico de perda óssea alveolar. Em adição, osso alveolar e ligamento periodontal foram coletados para determinar a expressão da enzima COX-2, e o tecido gengival foi coletado para determinar a expressão de PGE2. Animais submetidos à indução da doença periodontal pela ligadura (Grupo Ligadura) apresentaram um aumento significativo da expressão gênica de COX-2 nos dias 7, 14 e 21 (p<0,05). Todavia, ambos os tratamentos resultaram em uma significativa redução da perda óssea alveolar em comparação ao grupo Ligadura (p<0,05). Esse estudo mostrou que ambos os fármacos foram capazes de reduzir a perda óssea alveolar após indução da doença periodontal.

Abstract In the present study we compared the effects of the selective COX-2 inhibitor etoricoxib with those of the classical non-selective NSAID diclofenac on the inflammatory process and alveolar bone loss in an experimental model of periodontitis in rats. Ninety male Holtzman rats (250 g) were randomly sorted into four experimental groups: Sham+CMC and Ligature+CMC (control) groups which received 0.5% carboxymethylcellulose sodium (CMC) solution; Ligature+Diclofenac and Ligature+Etoricoxib groups which received Potassium Diclofenac and Etoricoxib, respectively, suspended in 0.5% CMC (10 mg/kg/day). At 7, 14 and 21 days after placing ligatures in the cervical region of both the lower right and left first molars, the animals were euthanized. At the end of each period, the mandibles were collected for radiographic examination of alveolar bone loss. In addition, alveolar bone and periodontal ligament tissue samples were collected for COX-2 expression analysis and gingival tissues were collected for measurement of PGE2 contents. Animals with ligature-induced periodontal disease showed significant increased COX-2 gene expression at days 7, 14 and 21 (p<0.05) on alveolar bone and periodontal ligament. However, both treatments resulted in significantly reduced alveolar bone loss when compared to the untreated Ligature group (p<0.05), with no statistical difference between Etoricoxib and Diclofenac Potassium groups. This study shows that both drugs were able to reduce alveolar bone loss after periodontal disease induction.

Abstract Endodontic revascularization is based on cell recruitment into the necrotic root canal of immature teeth after chemical disinfection. The clinical outcome depends on the ability of surviving cells from the apical tissue to differentiate and promote hard tissue deposition inside the dentinal walls. Objective To investigate the effect of calcium hydroxide (CH) and modified triple antibiotic paste (mTAP – ciprofloxacin, metronidazole and cefaclor) on the viability and mineralization potential of apical papilla cells (APC) in vitro . Material and Methods APC cultures were kept in contact with CH or mTAP (250-1000 µg/mL) for 5 days, after which cell viability was assessed using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Next, APCs were subjected to CH or mTAP at 250 µg/mL for 5 days before inducing the differentiation assay. After 14 and 21 days, calcium deposition was assessed by the Alizarin Red S staining method, followed by elution and quantification using spectrophotometry. Data were analyzed using ANOVA followed by Tukey post hoc test. Results CH induced cell proliferation, whereas mTAP showed significant cytotoxicity at all concentrations tested. APC treated with CH demonstrated improved mineralization capacity at 14 days, while, for mTAP, significant reduction on the mineralization rate was observed for both experimental periods (14 and 21 days). Conclusion Our findings showed that CH induces cell proliferation and improves early mineralization, whereas mTAP was found cytotoxic and reduced the mineralization potential in vitro of APCs.

Abstract The aim of this longitudinal prospective study was to evaluate the effects of periodontal treatment on the clinical, microbiological and immunological periodontal parameters, and on the systemic activity (ESSDAI) and subjective (ESSPRI) indexes in patients with primary Sjögren’s Syndrome (pSS). Twenty-eight female patients were divided into four groups: pSS patients with or without chronic periodontitis (SCP, SC, respectively), and systemically healthy patients with or without chronic periodontitis (CP, C, respectively). Periodontal clinical examination and immunological and microbiological sample collection were performed at baseline, 30 and 90 days after nonsurgical periodontal treatment (NSPT). Levels of interleukin IL-1β, IL-8 and IL-10 in saliva and gingival crevicular fluid (GCF) were evaluated by ELISA, as well as the expression of Porphyromonas gingivalis (Pg), Aggregatibacter actinomycetemcomitans, (Aa) Tannerella forsythia (Tf), and Treponema denticola (Td), by qPCR. Systemic activity and pSS symptoms were evaluated by ESSDAI and ESSPRI. NSPT resulted in improved periodontal clinical parameters in both SCP and CP groups (p>0.05). Pg, Aa, and Tf levels decreased after NSPT only in CP patients (p<0.05). Significantly greater levels of IL-10 in GCF were verified in both SCP and CP groups (p<0.05). SCP patients showed increased salivary flow rates and decreased ESSPRI scores after NSPT. In conclusion, NSPT in pSS patients resulted in improved clinical and immunological parameters, with no significant effects on microbiological status. pSS patients also showed increased salivary flow and lower ESSPRI scores after therapy. Therefore, it can be suggested that NSPT may improve the quality of life of pSS patients.

Abstract Recent studies investigating protease-activated receptor type 2 (PAR-2) suggest an association between the receptor and periodontal inflammation. It is known that gingipain, a bacterial protease secreted by the important periodontopathogen Porphyromonas gingivalis can activate PAR-2. Previous studies by our group found that PAR-2 is overexpressed in the gingival crevicular fluid (GCF) of patients with moderate chronic periodontitis (MP). The present study aimed at evaluating whether PAR-2 expression is associated with chronic periodontitis severity. GCF samples and clinical parameters, including plaque and bleeding on probing indices, probing pocket depth and clinical attachment level, were collected from the control group (n = 19) at baseline, and from MP patients (n = 19) and severe chronic periodontitis (SP) (n = 19) patients before and 6 weeks after periodontal non-surgical treatment. PAR-2 and gingipain messenger RNA (mRNA) in the GCF of 4 periodontal sites per patient were evaluated by Reverse Transcription Polymerase Chain Reaction (RT-qPCR). PAR-2 and gingipain expressions were greater in periodontitis patients than in control group patients. In addition, the SP group presented increased PAR-2 and gingipain mRNA levels, compared with the MP group. Furthermore, periodontal treatment significantly reduced (p <0.05) PAR-2 expression in patients with periodontitis. In conclusion, PAR-2 is associated with chronic periodontitis severity and with gingipain levels in the periodontal pocket, thus suggesting that PAR-2 expression in the GCF reflects the severity of destruction during periodontal infection.

Knowledge about the presence of some important oral pathogens is an important step in better identifying children at risk for periodontal and/or caries diseases in later life. The purpose of this study was to detect the presence of Streptococcus mutans (Sm), Aggregatibacter actinomycetemcomitans (Aa), Campylobacter rectus (Cr), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), and Tannerella forsythia (Tf) in gingival biofilm samples from 196 children, and to assess whether any of these pathogens are more associated with gingival inflammation extension and the Decayed/Missing/Filled teeth (DMFT/dmft) index. The subjects presented plaque index greater than 80% and were divided in 3 groups according to the bleeding index (BI): I) Low bleeding (< 30%), II) Medium bleeding (31 - 59%) and III) High bleeding (> 60%). The presence of each pathogen was determined by PCR. The prevalence of Sm was 71.9% and the mean dmft/DMFT was 6.68. The prevalence in low, medium and high bleeding groups was 43.5%, 34.5% and 46.7% for Aa; 43.5%, 37.9%, and 36.7% for Cr; 99.1%, 100%, and 96.7% for Pg; 56.5%, 56.9%, and 66.7% for Pi; and 58.3%, 60.3%, and 56.7% for Tf, respectively. Pg (99.0%) was the most prevalent periodontal pathogen detected followed by Tf (58.7%), Pi (58.2%), Aa (41.3%) and Cr (40.8%). Our study indicated that in this high plaque index population studied, a high prevalence of Sm and high mean DMFT were observed. In addition, the presence of Pi was associated with the presence of inflammation (P < 0.05) whereas Cr was associated with periodontal health (P < 0.05).

Ciclosporina A (CsA) é um potente imunossupressor usado no transplante de órgãos e no tratamento de várias doenças auto-imunes. Recentes estudos têm demonstrado que a CsA estimula a deposição de cemento na superfície radicular. O objetivo deste estudo foi de avaliar a espessura do cemento periapical e largura do forame apical em ratos tratados com CsA. Os ratos pesavam 50 g e foram tratados com doses diárias de 10 mg/kg de peso corporal de CsA no período de 60 dias. O cemento do primeiro molar inferior foi examinado histologicamente e morfometricamente por análises de cortes em parafina com 5µm de espessura no sentido vestíbulo-lingual e corados com hematoxilina e eosina. As análises histométricas e estereológicas revelaram a presença de largos depósitos de cemento em todas as superfícies radiculares, particularmente maior na região periapical e obliterando o forame. A densidade volumétrica dos cementoblastos não foi aumentada. No período de 5 a 90 dias após o término da terapia com CsA, não houve redução na espessura do cemento. Estes resultados sugerem que o depósito de cemento não é reversível após o tratamento com CsA ser cessado.

Cyclosporin A (CsA) is a potent immunosuppressor used in organ transplantation and in the management of various autoimmune diseases. Recent studies have shown that CsA stimulates deposition of cementum on root surfaces. The aim of this study was to evaluate the periapical cementum thickness and the apical foramen width in CsA-treated rats. Rats weighing 50 g were treated with a daily injection of 10 mg/kg body weight of CsA in the chow for 60 days. The cementum of the mandibular 1st molars was histologically and morphometricaly examined by analysis of 5-µm-thick serial buccolingual paraffin sections stained with hematoxylin and eosin. Histometric and stereologic analyses revealed the presence of large amounts of cementum in all root surfaces, particularly abundant in the periapical region and obliterating the foramen. The volume density of cementoblasts did not increase. Five to 90 days after the termination of CsA therapy, there was no reduction of cementum thickness. These results suggest that cementum deposition is not reversible after cessation of CsA treatment.

A ciclosporina A (CsA) é usada como um agente imunossupressor e promove efeitos colaterais como o crescimento gengival que permanece um problema significante. Alguns fatores de risco podem aumentar esses efeitos, como a duração do tratamento. Ainda não há estudos estereológicos e bioquímicos explorando os efeitos de um longo período de terapia com CsA no tecido gengival. O objetivo do presente estudo foi investigar o nível de TGF-beta1 na saliva e descobrir a densidade de fibroblastos e fibras colágenas no tecido gengival de ratos tratados por um longo período com CsA. Os ratos foram tratados por 60, 120, 180 e 240 dias com injeções subcutâneas diárias de 10 mg/kg de peso corporal de CsA. Ao final dos períodos experimentais, a saliva era coletada para determinação do TGF-beta1. Após o processamento histológico, o epitélio oral e a área do tecido conjuntivo foram medidos, bem como a densidade volumétrica dos fibroblastos (Vf) e das fibras colágenas (Vcf). Após 60 e 120 dias de tratamento com CsA ocorreu um significante aumento de Vf e Vcf, bem como aumento significante de TGF-beta1. Após 180 e 240 dias, houve redução do crescimento gengival associada com decréscimo significante do nível de TGF-beta1, e também diminuição observada de Vf e Vcf. Os dados presentes neste estudo sugerem que, após longo período de terapia, ocorre uma diminuição do nível de TGF-beta1, o que deve contribuir para um aumento na atividade proteolítica dos fibroblastos na gengiva, favorecendo a normalidade da síntese da matriz extracelular.

Cyclosporin A (CsA) is used as an immunosuppressive agent and its prominent side effect is the induction of gingival overgrowth, which remains a significant problem. The risk factors appraised include the duration of treatment. However, there are no stereological and biochemical studies exploring the effects of long-term CsA therapy on gingival tissue. The purpose of the present study was to investigate the level of TGF-beta1 in saliva and describe the densities of fibroblasts and collagen fibers in the gingival tissue of rats treated with CsA for long periods. Rats were treated for 60, 120, 180 and 240 days with a daily subcutaneous injection of 10 mg/kg of body weight of CsA. At the end of the experimental periods, saliva was collected for the determination of TGF-beta1 levels. After histological processing, the oral epithelium and the connective tissue area were measured as well as the volume densities of fibroblasts (Vf) and collagen fibers (Vcf). After 60 and 120 days of CsA treatment, there was a significant increase in Vf and Vcf as well as a significant increase in TGF-beta1 levels. After 180 and 240 days, reduction in the gingival overgrowth associated with significant decreases in the level of TGF-beta1, and also decreased Vf and Vcf, were observed. The data presented here suggest that after long-term therapy, a decrease in TGF-beta1 levels occurs, which might contribute to an increase in the proteolytic activity of fibroblasts in the gingiva, favoring the normality of extracellular matrix synthesis.