Abstract Periodontal regeneration is a challenge, and tissue engineering based on periodontal ligament stem cells (PDLSCs) has been shown to be a promising alternative to this process. However, the need for scaffolds has limited the therapeutic use of PDLSCs. In this context, scaffold-free tissue engineering using the cell sheet (CS) technique has been developed as an alternative approach to improve tissue regeneration. Previously, we showed that Protease-activated receptor-1 (PAR1) can regulate PDLSCs. Herein, we evaluate whether PAR1 influences osteogenesis in CSs produced from PDLSCs, without the use of scaffolds. PDLSCs were isolated and immunophenotyped. Then, CSs were obtained by supplementing the culture medium with ascorbic acid (50 µg/mL), and PAR1 was activated through its agonist peptide (100 nM). Scaffold-free 3D CSs were successfully produced from PDLSCs, and they showed higher proliferation potential than isolated PDLSCs. Also, PAR1 activation decreased senescence and improved osteogenic differentiation of CSs by increasing mineralized nodule deposition and alkaline phosphatase concentration; PAR1 also modulated osteogenic markers at the gene and protein levels. We further demonstrated that this effect was regulated by Wnt, TGF-βI, MEK, p38 MAPK, and FGF/VEGF signaling pathways in PDLSCs (p < 0.05%). Overall, PAR1 activation increased osteogenic activity in CSs, emerging as a promising scaffold-free therapeutic approach for periodontal regeneration. challenge (PDLSCs process However context scaffoldfree scaffold free CS (CS Previously Proteaseactivated Protease receptor1 receptor 1 receptor- PAR (PAR1 Herein immunophenotyped Then 50 (5 µg/mL, µgmL µg/mL , µg mL µg/mL) 100 (10 nM. nM . nM) Scaffoldfree Scaffold D Also concentration levels Wnt TGFβI, TGFβI TGF βI, βI TGF-βI MEK p p3 MAPK FGFVEGF FGF VEGF 0.05%. 005 0.05% 0 05 0.05%) Overall (PAR 5 ( 10 (1 00 0.05 0.0 0.

Resumo Defeitos ósseos craniofaciais causados por tumores, traumas, perda dentária prolongada ou doenças periodontais representam um grande desafio no campo da engenharia tecidual. Em casos de periodontite e peri-implantite, a terapia reconstrutiva também representa um grande desafio para o cirurgião-dentista. Lipoxinas, resolvinas, protectinas e maresinas, conhecidas como mediadores lipídicos especializados pró-resolutivos (SPMs - Specialized Pro-resolving Lipid Mediators), têm sido amplamente estudadas no campo da pesquisa odontológica para a regeneração óssea devido às suas ações na restauração da homeostase tecidual e na promoção da cicatrização e regeneração tecidual. Portanto, este estudo foca em uma revisão sobre o uso dos SPMs para a regeneração óssea craniofacial e alveolar. Assim, foram realizadas buscas eletrônicas em cinco bases de dados para identificar estudos pré-clínicos que avaliaram as ações dos SPMs na regeneração óssea craniofacial e alveolar. Dos 523 artigos recuperados das bases de dados eletrônicas, 19 foram incluídos na análise. A resolvina (Rv) E1 foi o SPM mais avaliado (n=8), seguida pelas maresinas (Ma) R1 (n=3), lipoxinas (Lx) A4 (n=3), RvD1 (n=3), RvD2 (n=1), LxB4 (n=1) e maresina (M)-CTR3 (n=1). A meta-análise mostrou que os SPMs aumentaram o osso recém-formado em 14,85% em comparação ao grupo controle (p<0,00001), diminuíram a área do defeito remanescente em 0,35 mm² (p<0,00001) e reduziram a distância linear entre o defeito e a crista óssea em 0,53 mm (p<0,00001). A RvE1 reduziu a reabsorção óssea inflamatória em defeitos periodontais e osteólise em calvárias e aumentou a regeneração óssea quando combinada com um enxerto ósseo bovino. A RvD2 induziu a resolução ativa da inflamação e a regeneração tecidual em lesões periapicais, enquanto a RvD1 controlou o microambiente inflamatório em defeitos em calvárias em ratos, promovendo a cicatrização óssea e a angiogênese. A MaR1 induziu a proliferação e migração de células-tronco mesenquimais, osteogênese e angiogênese em defeitos em calvárias, e a benzo (b)-LxA4 e a LxA4 promoveram a regeneração óssea em defeitos craniofaciais e alveolares em ratos, induzindo a regeneração sob condições inflamatórias. Em resumo, os SPMs emergiram como contribuintes fundamentais para a resolução da inflamação e a facilitação da neoformação óssea em defeitos craniofaciais e alveolares. Esses resultados são baseados em estudos pré-clínicos, in vivo e in vitro, e fornecem uma revisão atualizada sobre o impacto dos SPMs na engenharia tecidual. tumores traumas periimplantite, periimplantite peri implantite, implantite peri-implantite cirurgiãodentista. cirurgiãodentista cirurgião dentista. dentista cirurgião-dentista Lipoxinas resolvinas próresolutivos pró resolutivos Proresolving Pro resolving Mediators, Mediators , Mediators) Portanto alveolar Assim préclínicos pré clínicos 52 1 análise Rv (Rv E n=8, n8 n n=8 8 (n=8) Ma (Ma R n=3, n3 n=3 3 (n=3) Lx (Lx RvD n=1, n1 n=1 LxB (n=1 MCTR3 MCTR M CTR3 CTR (M)-CTR n=1. . metaanálise meta recémformado recém formado 1485 14 85 14,85 p<0,00001, p000001 p p<0,00001 0 00001 035 35 0,3 (p<0,00001 053 53 0,5 p<0,00001. RvE bovino periapicais ratos MaR célulastronco células tronco mesenquimais bLxA4 bLxA b LxA (b)-LxA inflamatórias resumo préclínicos, clínicos, vitro 5 n= (n=8 (n=3 (n= 148 14,8 p00000 p<0,0000 0000 03 0, (p<0,0000 05 (n 14, p0000 p<0,000 000 (p<0,000 p000 p<0,00 00 (p<0,00 p00 p<0,0 (p<0,0 p0 p<0, (p<0, p<0 (p<0 p< (p< (p

Abstract Craniofacial bone defects caused by tumors, trauma, long-term tooth loss, or periodontal disease are a major challenge in the field of tissue engineering. In periodontitis and peri-implantitis, reconstructive therapy is also a major challenge for the dental surgeon. Lipoxins, resolvins, protectins, and maresins, known as specialized pro-resolving lipid mediators (SPMs), have been widely studied in the field of dental, oral, and craniofacial research for bone regeneration for their actions in restoring tissue homeostasis and promoting tissue healing and regeneration. Therefore, this study focuses on a survey of the use of SPMs for craniofacial and alveolar bone regeneration. Thus, electronic searches of five databases were performed to identify pre-clinical studies that evaluated the actions of SMPs on craniofacial and alveolar bone regeneration. Of the 523 articles retrieved from the electronic databases, 19 were included in the analysis. Resolvin (Rv) E1 was the mostly assessed SPM (n=8), followed by maresins (Ma) R1 (n=3), lipoxins (Lx) A4 (n=3), RvD1 (n=3), RvD2 (n=1), LxB4 (n=1), and maresin (M)-CTR3 (n=1). Meta-analysis showed that SPMs increased the newly formed bone by 14.85% compared to the control group (p<0.00001), decreased the area of the remaining defect by 0.35 mm2 (p<0.00001), and decreased the linear distance between the defect to the bone crest by 0.53 mm (p<0.00001). RvE1 reduced inflammatory bone resorption in periodontal defects and calvarial osteolysis and enhanced bone regeneration when RvE1 was combined with a bovine bone graft. RvD2 induced active resolution of inflammation and tissue regeneration in periapical lesions, while RvD1 controlled the inflammatory microenvironment in calvarial defects in rats, promoting bone healing and angiogenesis. MaR1 induced the proliferation and migration of mesenchymal stem cells, osteogenesis, and angiogenesis in calvarial defects, and benzo (b)-LxA4 and LxA4 promoted bone regeneration calvarial and alveolar bone defects in rats, inducing regeneration under inflammatory conditions. In summary, SPMs have emerged as pivotal contributors to the resolution of inflammation and the facilitation of bone neoformation within craniofacial and alveolar bone defects. These results are based on pre-clinical studies, in vivo and in vitro, and provide an updated review regarding the impact of SPMs in tissue engineering. tumors trauma longterm long term loss engineering periimplantitis, periimplantitis peri implantitis, implantitis peri-implantitis surgeon Lipoxins resolvins protectins proresolving pro resolving SPMs, , (SPMs) oral Therefore Thus preclinical pre clinical 52 1 analysis Rv (Rv E n=8, n8 n n=8 8 (n=8) Ma (Ma R n=3, n3 n=3 3 (n=3) Lx (Lx A RvD n=1, n1 n=1 (n=1) LxB MCTR3 MCTR M CTR3 CTR (M)-CTR n=1. . Metaanalysis Meta 1485 14 85 14.85 p<0.00001, p000001 p p<0.00001 0 00001 (p<0.00001) 035 35 0.3 053 53 0.5 p<0.00001. RvE graft lesions rats MaR cells osteogenesis bLxA4 bLxA b LxA (b)-LxA conditions summary vitro (SPMs 5 n= (n=8 (n=3 (n=1 148 14.8 p00000 p<0.0000 0000 (p<0.00001 03 0. 05 (n= 14. p0000 p<0.000 000 (p<0.0000 (n p000 p<0.00 00 (p<0.000 p00 p<0.0 (p<0.00 p0 p<0. (p<0.0 p<0 (p<0. p< (p<0 (p< (p

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. Proteaseactivated Protease activated receptor2 2 receptor- PAR (PAR2 characteristics periodontitis (PDLSCs vitro 7 14 0.1 01 0 1 (0. U/mL, UmL U/mL , U mL U/mL) SLIGRLNH2 SLIGRLNH SLIGRL NH2 NH (SLIGRL-NH2 100 (10 nM, nM nM) FSLLRYNH2 FSLLRYNH FSLLRY (FSLLRY-NH2 nM. . RTqPCR RT qPCR (RT-qPCR ELISA (ELISA factors metabolism cytokines (ALP staining assessed 5 performed p 0.05, 005 0.05 05 0.05. Additionally kappaΒ kappa Β RANKL (RANKL <0 05. 05) findings resorption procedures (PAR 0. (0 (SLIGRL-NH 10 (1 (FSLLRY-NH 00 0.0 (

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.

Abstract Objective Obesity is a chronic disease that negatively affects an individual’s general and oral health. The present study aimed to compare the clinical and microbiological effects of non-surgical periodontal therapy with the full mouth disinfection (FMD) protocol on obese and non-obese individuals at 9 months post-therapy. Methodology This clinical study was first submitted and approved by the Ethics Committee. Fifty-five obese patients and 39 non-obese patients with periodontitis were evaluated. The full-mouth periodontal clinical parameters, clinical attachment level (CAL), probing depth (PD), gingival index (GI), and plaque index (PI), were monitored at baseline, 3, 6, and 9 months after periodontal treatment with full mouth disinfection (FMD) protocol. The mean count of Tannerella forsythia , Porphyromonas gingivalis , Treponema Denticola , and Aggregatibacter actinomycetemcomitans was determined by quantitative polymerase chain reaction on subgingival biofilm samples. Demographic data were assessed by Chi-square test. For clinical and microbiological parameters, two-factor repeated-measures ANOVA was used. Results In both groups, periodontal therapy using the one-stage full-mouth disinfection protocol significantly improved CAL, PD, GI, and PI (p<0.05). Obese and non-obese patients equally responded to non-surgical periodontal therapy (p>0.05). Microbial count found no major differences (p>0.05) between obese and non-obese individuals who had undergone non-surgical periodontal therapy. Conclusions Obesity did not affect the clinical and microbiological outcomes of non-surgical periodontal therapy.

Resumo Este estudo foi realizado para investigar o perfil microbiano e os níveis de endotoxina de lesões endoperiodontais de origem periodontal. Amostras periodontais e endodônticas foram obtidas de bolsas periodontais e canais radiculares necróticos de 10 dentes com lesões endoperiodontais. A investigação de 40 espécies bacterianas diferentes foram determinadas em cada amostra endodôntica e periodontal usando o método de hibridização de DNA-DNA (checkerboard) e o ensaio cinético cromogênico LAL foi usado para quantificação de endotoxinas. O teste exato de Fisher correlacionou as espécies bacterianas com a microbiota endodôntica ou periodontal. Os níveis de endotoxina (EU/mL) encontrados nas amostras do canal radicular e na bolsa periodontal foram comparados pelo teste de Wilcoxon (p<0,05). Bactérias e unidades de LPS foram encontradas em 100% das amostras endodônticas e periodontais. As espécies E. faecium, P. acnes, G. morbillorum, C. sputigena e L. buccalis foram fortemente correlacionadas com a microbiota endodôntica e P. nigrescens com a microbiota periodontal. P. intermedia, P. endodontalis e V. parvula foram mais prevalentes em ambas microbiotas endodôntica e periodontal. Os níveis de endotoxina na bolsa periodontal (89600 EU/mL) foram significativamente maiores do que no canal radicular (2310 EU/mL). Concluiu-se que a microbiota presente nos tecidos periodontal e endodôntico é semelhante, com maior prevalência de espécies do complexo laranja e maior nível de endotoxina nas bolsas periodontais.

Abstract This study was carried out to investigate the microbial profile and endotoxin levels of endodontic-periodontal lesions of periodontal origin. Periodontal and endodontic samples were taken from periodontal pockets and necrotic root canals of 10 teeth with endodontic-periodontal lesions. Evidencing of 40 different bacterial species were determined in each endodontic and periodontal sample using the checkerboard DNA-DNA hybridization method and Kinetic chromogenic LAL assay was used for quantification of endotoxins. Fisher’s exact test correlated the bacterial species with the endodontic or periodontal microbiota. The endotoxin levels (EU/mL) found in samples of the root canal and periodontal pocket were compared by the Wilcoxon test (p<0.05). Bacteria and LPS units were found in 100% of the endodontic and periodontal samples. The species E. faecium, P. acnes, G. morbillorum, C. sputigena and L. buccalis were strongly correlated with the endodontic microbiota and P. nigrescens with the periodontal microbiota. P. intermedia, P. endodontalis and V. parvula were more prevalent in both endodontic and periodontal microbiots. The endotoxin levels in the periodontal pocket (89600 EU/mL) were significantly higher than in the root canal (2310 EU/mL). It was concluded that the microbiota present in the periodontal and endodontic tissues is similar, with a higher prevalence of species of the orange complex and a higher level of endotoxin in the periodontal pockets.

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.