Cytotoxicity and cytokine production by calcium silicate-based materials on periodontal ligament stem cells

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 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.

Long non-coding RNA potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 regulates the proliferation and osteogenic differentiation of human periodontal ligament stem cells by targeting miR-24-3p / 华西口腔医学杂志

OBJECTIVES@#This study aims to explore the effect and molecular mechanism of long non-coding RNA (lncRNA) potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 (KCNQ1OT1) on proliferation and osteogenic differentiation in human periodontal ligament stem cells (hPDLSCs).@*METHODS@#The hPDLSCs of normal periodontal tissues were isolated and cultured. The mineralized solution induced the osteoblast differentiation of hPDLSCs. The down-regulation of lncRNA KCNQ1OT1, the overexpression of anti-miR-24-3p on the proliferation and the levels of osteocalcin (OCN), osteopontin (OPN) and alkaline phosphatase (ALP) of hPDLSCs were investigated. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the levels of lncRNA KCNQ1OT1, miR-24-3p, OCN, OPN, and ALP. Methyl thiazolyl tetrazolium (MTT) method was used to detect cell viability and activity. Cell proliferation was evaluated by MTT. Western blot was used to detect protein expression. The targeted relationship between lncRNA KCNQ1OT1 and miR-24-3p was detected by double-luciferase experiment.@*RESULTS@#The expression level of lncRNA KCNQ1OT1 increased, and that of miR-24-3p decreased during the osteogenesis of hPDLSCs (@*CONCLUSIONS@#Down-regulation of lncRNA KCNQ1OT1 inhibited the proliferation and osteogenic differentiation of hPDLSCs by targeting the up-regulated expression of miR-24-3p.

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.

Effect of Milk Renewal on Cell Viability In Vitro at Different Time Frames

Abstract The purpose of this study was to evaluate if the renewal of milk as a storage medium, every 12, 24 and 48 h, is able to increase its ability to maintain human periodontal ligament fibroblasts (PDLF) viability over time. PDLF were soaked in Minimum Essential Medium at 37 °C (MEM-37) (positive control), tap water (Water) (negative control) and in skimmed milk (44 wells) at 5 °C and 20 °C. The skimmed milk was renewed every 12 h (Milk-12), 24 h (Milk-24) and 48 h (Milk-48) in 11 wells of each plate, and the milk in the remaining 11 wells of each plate was maintained in situ (not renewed milk) (NRM). After 24, 48, 72, 96 and 120 h, cell viability was determined by the tetrazolium salt-based colorimetric (MTT) assay. Data were statistically analyzed by Kruskal-Wallis, Scheffé and Mann-Whitney tests (a=5%). At 5 °C, only Milk-48 was significantly better than NRM. At 20 °C, NRM was more effective than Milk-12 and Milk-24 in all time periods. In relation to the temperature (5 °C or 20 °C), renewal of milk at 5 °C was better in maintaining cell viability than the renewal at 20 °C. In conclusion, the renewal of milk was able to increase its ability to maintain cell viability only when performed every 48 h in milk maintained at 5 °C.

Resumo O objetivo deste estudo foi avaliar se a renovação do leite, a cada 12, 24 e 48 h, é capaz de aumentar sua capacidade de manter a viabilidade de fibroblastos do ligamento periodontal humano (FLPH) ao longo do tempo. FLPH foram conservados em Meio Essencial Mínimo a 37 °C (MEM-37) (controle positivo), água da torneira (água) (controle negativo) e em leite desnatado (44 poços) a 5 °C e 20 °C. O leite desnatado foi renovado a cada 12 h (leite-12), 24 h (leite-24) e 48 h (leite-48) em 11 poços de cada placa, e em outros 11 poços de cada placa o leite foi deixado in situ (leite não renovado) (LNR). Depois de 24, 48, 72, 96 e 120 h, a viabilidade celular foi determinada pelo ensaio colorimétrico à base de sal tetrazólio (MTT). Os dados foram analisados estatisticamente pelos testes de Kruskal-Wallis, Scheffé e Mann-Whitney (α=5%). A 5 °C, somente o leite-48 foi significantemente melhor do que o LNR. A 20 °C, LNR foi mais efetivo do que o leite-12 e leite-24 em todos os períodos de tempo. Em relação à temperatura (5 °C ou 20 °C), a renovação do leite a 5 °C foi melhor na manutenção da viabilidade celular do que a renovação a 20 °C. Concluindo, a renovação do leite foi capaz de aumentar sua habilidade em manter a viabilidade celular apenas quando realizada a cada 48 h no leite mantido a 5 °C.

Epithelial rests of Malassez: from latent cells to active participation in orthodontic movement

ABSTRACT Introduction: The epithelial rests of Malassez (ERM) represent a group of cells in the periodontal ligament classically consisting of latent or quiescent structures associated with pathological processes. However, recent evidence shows that these structures cannot be considered only as cellular debris. The ERM is a major tissue structure, with functions in maintaining the homeostasis of periodontal tissue, including the maintenance of orthodontic movement. Objective: The present literature review aims at presenting the potential functions of ERM, with emphasis on orthodontic movement and the functional structure of the periodontium. Conclusion: ERM cells have a functional activity in modulation of orthodontic movement, trough their potential for differentiation, maintenance functions and the capacity of repairing periodontium.

RESUMO Introdução: os remanescentes epiteliais de Malassez (REM) se configuram como um grupo de células epiteliais presentes no ligamento periodontal, classicamente consideradas estruturas latentes ou quiescentes, associadas a processos patológicos. Entretanto, ao longo dos anos, esse paradigma vem sendo rompido e hoje não mais são consideradas apenas como restos celulares, mas sim uma importante estrutura tecidual, com funções na manutenção da homeostase do periodonto, inclusive durante a movimentação ortodôntica. Objetivo: na presente revisão da literatura, buscou-se apresentar as funções dessas estruturas, com ênfase nelas durante a movimentação ortodôntica, rompendo o conceito errôneo de que são meros restos celulares, e defendendo sua compreensão como uma estrutura funcional do periodonto. Conclusão: os REM possuem uma atividade funcional na modulação da movimentação ortodôntica, por meio de seu potencial para diferenciação, de suas funções de manutenção e de sua capacidade de reparação periodontal.

Osteogenic potential of periodontal ligament stem cells are unaffected after exposure to lipopolysaccharides

Abstract Periodontitis develops as a result of a continuous interaction between host cells and subgingival pathogenic bacteria. The periodontium has a limited capacity for regeneration, probably due to changes in periodontal ligament stem cells (PDLSCs) phenotype. The aim of this study was to evaluate the effects of lipopolysaccharides from Porphyromonas gingivalis (PgLPS) on mesenchymal phenotype and osteoblast/cementoblast (O/C) potential of PDLSCs. PDLSCs were assessed for Toll-like receptor 2 (TLR2) expression by immunostaining technique. After, cells were exposed to PgLPS, and the following assays were carried out: (i) cell metabolic activity using MTS; (ii) gene expression for IL-1β, TNF-α and OCT-4 by real-time polymerase chain reaction (RT-qPCR); (iii) flow cytometry for STRO-1 and CD105, and (iv) osteogenic differentiation. PDLSCs were positive for TLR2. PgLPS promoted cell proliferation, produced IL-1β and TNF-α, and did not affect the expression of stem cell markers, STRO-1, CD105 and OCT-4. Under osteogenic condition, PDLSCs exposed to PgLPS showed a similar potential to differentiate toward osteoblast/cementoblast phenotype compared to control group as revealed by mineralized matrix deposition and levels of transcripts for RUNX2, ALP and OCN. These results provide evidence that PgLPS induces pro-inflammatory cytokines, but does not change the mesenchymal phenotype and osteoblast/cementoblast differentiation potential of PDLSCs.

In Vitro Periodontal Ligament Cell Viability in Different Storage Media

Abstract The aim of this study was to evaluate the viability of periodontal ligament cells of avulsed teeth in three different storage media. Forty-five mature premolars extracted for orthodontic therapeutic purposes were randomly and equally divided into three groups according to the storage medium: milk (control), rice water and egg white. After placing extracted teeth for 30 min in storage media, the scrapings of the periodontal ligament (PDL) were collected in Falcon tubes containing collagenase in 2.5 mL of phosphate buffer saline and were incubated for 30 min and centrifuged for 5 min at 800 rpm. Cell viability was analyzed by Trypan blue exclusion. Rice water had a significantly higher number of viable cells compared to egg white and milk. There was no statistically significant difference between egg white and milk. Rice water may be able to maintain PDL cell viability of avulsed teeth better than egg white or milk.

Resumo O objetivo deste estudo foi avaliar a viabilidade das células do ligamento periodontal de dentes avulsionados armazenados em três diferentes meios. Quarenta e cinco pré-molares com formação radicular completa extraídos por razões terapêuticas foram aleatoriamente distribuídos em três grupos, de acordo com o meio de armazenagem: leite (controle), água de arroz e clara de ovo). Após armazenar os dentes avulsionados por 30 min no meio, raspas do ligamento periodontal (LPD) foram coletadas em tubos Falcon contendo 2,5 mL de solução tamponada de soro fosfatado, incubadas por 30 min e a seguir centrifugadas por 5 min a 800 rpm. A viabilidade celular foi analisada pelo método de exclusão do Azul de Trypan. A água de arroz teve um número significativamente maior de células viáveis em comparação com o leite e a clara de ovo. Não houve diferença estatisticamente significativa entre o leite e a clara de ovo. A água de arroz pode ser capaz de manter a viabilidade das células do PDL de dentes avulsionados, melhor que o leite ou a clara de ovo.

Avaliação dos eixos ECA / ANG II / AT1 e ECA-2 / ANG (1-7) / MAS em fibroblastos de gengiva e ligamento periodontal humanos estimulados por IL-1ß e sua contribuição na produção e regulação de citocinas e quimiocinas / Evaluation ACE /ANG II /AT1 and ACE-2 /ANG (1-7) /MAS axis in gingival and periodontal ligament human fibroblasts stimulated with IL-1ß and its contribution in the production of cytokines and chemokines

O Sistema renina-angiotensina (SRA) tem sido relatado como um importante modulador de processos inflamatórios e imunológicos, incluindo a doença periodontal (DP). Estudos sugerem neste sistema um eixo alternativo (ECA-2 /ANG(1-7) /MAS) que atuaria como um contra-regulador de efeitos mediados pelo clássico eixo (ECA /ANGII /AT1). Sabe-se que bactérias periodontopatogênicas, como a Porphyromonas gingivalis (Pg), possuem componentes bioativos de membrana (ex. lipopolissacarídeos-LPS) capazes de induzir uma forte resposta imune no hospedeiro devido à liberação de citocinas nas células, entre elas Interleucina (IL)- 1ß. Neste contexto, fibroblastos são as células mais abundantes nos tecidos periodontais e possuem em sua superfície celular receptores necessários para o reconhecimento da invasão bacteriana, ativando cascatas intracelulares, que levam à produção de citocinas. O objetivo deste estudo foi verificar se os eixos ECA/ ANGII/ AT1 e ECA-2/ ANG(1-7)/ MAS contribuem para a produção e/ ou regulação de citocinas inflamatórias (CI) por fibroblastos de gengiva humana (HGF) e ligamento periodontal humano (HPLF) estimulados por IL-1ß. Após o pré-tratamento com Losartan e Ang (1-7) ou silenciamento mediado por RNA de interferência (RNAi) de AT1, HGF e HPLF foram estimulados por IL-1ß por 3 horas (RNAm) ou 24 horas (proteína). Expressão de RNAm para AT1, MAS, ECA, ECA-2, IL-1ß, TNF-α, IL-6, IL-8, IL-10, TGF-ß, CXCL12, RANK-L e OPG foram avaliados por RT-qPCR e das proteínas IL-6, IL-8, ECA e ECA-2 por ELISA. Foi realizado também Western Blot para detecção de AT1 e ECA nos extratos celulares e dosagem de nitrito no sobrenadante das culturas. Ambos os subtipos de fibroblastos mostraram aumento da expressão de RNAm para AT1, IL-1ß, IL-6, IL-8, TNF-α e OPG, quando estimulados por IL-1ß. No entanto, apenas em HPLF foi observado aumento para MAS, ECA e TGF-ß. Losartan e Ang (1-7) não modularam o transcrito, a secreção de CI e nem a produção de nitrito no sobrenadante das culturas, tanto em HGF como em HPLF. O silenciamento do receptor AT1 reduziu a secreção de IL-6 e IL-8 induzida por IL-1ß em cultura de HGF e HPLF e aumentou a expressão gênica de OPG somente em HGF. Estes resultados sugerem que o silenciamento de AT1, mas não o bloqueio farmacológico deste receptor pelo antagonista Losartan, em HGF e HPLF, pode controlar a produção de IL-6 e IL-8, que por sua vez contribuem para a patogênese periodontal.(AU)

The renin-angiotensin system (RAS) has been reported as an important modulator of inflammatory and immune responses, including periodontal disease (PD). Studies suggest an alternative axis as part of this system (ACE-2 / ANG (1-7) / MAS) that would act as counter-regulatory to the classical axis (ECA / ANGII / AT1). It is known that periodontal bacteria such as Porphyromonas gingivalis (Pg) have bioactive components in their membrane (such as lipopolysaccharide-LPS) capable of inducing a strong immune response in the host due to the release of cytokines in cells, including interleukin (IL) - 1ß. In this regard, fibroblasts are the most abundant cells in periodontal tissues and receptors needed for the recognition of bacterial invasion by activating intracellular cascades that lead to cytokine production. The aim of this study was to determine whether the axes ACE / ANGII / AT1 and ACE-2 / ANG (1-7) / MAS contribute to the production and / or regulation of inflammatory cytokines (IC) by fibroblasts of human gingiva (HGF) and human periodontal ligament (HPLF) stimulated IL-1ß. After pre-treatment with Losartan, Ang (1-7) or silencing mediated by RNA interference (RNAi) of AT1, HGF and HPLF were stimulated by IL-1ß for 3 hours (RNAm) or 24 hours (protein). Expression mRNA for AT1, MAS, ACE, ACE-2, IL-1ß, TNF-α, IL-6, IL-8, IL-10, TGF-ß, CXCL12, RANK-L and OPG was assessed by RT- qPCR and proteins IL-6, IL-8, ACE and ACE-2 by ELISA. Western Blot for the detection of AT1 and ECA and dosage of nitrite was also performed. Experiments stimulated by IL-1ß showed a positive control for gene expression AT1, IL-1ß, IL-6, IL-8, TNF-α and OPG in HGF and HPLF and MAS, ACE and TGF-ß only HPLF. Losartan and Ang (1-7) did not modulate the transcription and secretion of IC and no nitrite production in the culture supernatant of HGF and HPLF. The silencing AT1 reduced IL-6 secretion and IL-8 induced by IL- ß in cultured HGF and HPLF and increased OPG gene expression only HGF. These results suggest that silencing AT1, but not pharmacological blockade of this receptor by Losartan in HPLF and HGF, can control the production of IL-6 and IL-8, which in turn contribute to the pathogenesis of periodontal disease.(AU)

Effects of IL-10 and glucose on expression of OPG and RANKL in human periodontal ligament fibroblasts

The effects of interleukin-10 (IL-10) and glucose on mRNA and protein expression of osteoprotegerin (OPG), and its ligand, receptor activator of nuclear factor-κB ligand (RANKL), were investigated in human periodontal ligament fibroblasts (HPDLFs). Primary HPDLFs were treated with different concentrations of IL-10 (0, 1, 10, 25, 50, and 100 ng/mL) or glucose (0, 5.5, 10, 20, 30, and 40 mmol/L). Changes in mRNA and protein expression were examined using the reverse-transcription polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. After IL-10 treatment, mRNA and protein levels of OPG were increased, while mRNA and protein levels of RANKL were decreased (P<0.05), both in a concentration-dependent manner. Glucose stimulation had the opposite concentration-dependent effect to that of IL-10 on OPG and RANKL expression. IL-10 upregulated OPG expression and downregulated RANKL expression, whereas high glucose upregulated RANKL and downregulated OPG in HDPLFs. Abnormal levels of IL-10 and glucose may contribute to the pathogenesis of periodontal disease.

Effects of fluoride on proliferation and mineralization in periodontal ligament cells in vitro

Fluoride, which is often added to toothpaste or mouthwash in order to protect teeth from decay, may be a novel therapeutic approach for acceleration of periodontal regeneration. Therefore, we investigated the effects of fluoride on proliferation and mineralization in human periodontal ligament cells in vitro. The periodontal ligament cells were stimulated with various concentrations of NaF added into osteogenic inductive medium. Immunohistochemistry of cell identification, cell proliferation, alkaline phosphatase (ALP) activity assay, Alizarin red S staining and quantitative real-time-polymerase chain reaction (RT-PCR) were performed. Moderate concentrations of NaF (50-500 μmol/L) had pro-proliferation effects, while 500 μmol/L had the best effects. ALP activity and calcium content were significantly enhanced by 10 μmol/L NaF with osteogenic inductive medium. Quantitative RT-PCR data varied in genes as a result of different NaF concentrations and treatment periods. We conclude that moderate concentrations of NaF can stimulate proliferation and mineralization in periodontal ligament cells. These in vitro findings may provide a novel therapeutic approach for acceleration of periodontal regeneration by addition of suitable concentrations of NaF into the medication for periodontitis treatment, i.e., into periodontal packs and tissue patches.

Static compression regulates OPG expression in periodontal ligament cells via the CAMK II pathway

ABSTRACT Objective This study aimed to investigate the potential role of CAMK II pathway in the compression-regulated OPG expression in periodontal ligament cells (PDLCs). Material and Methods The PDL tissue model was developed by 3-D culturing human PDLCs in a thin sheet of poly lactic-co-glycolic acid (PLGA) scaffolds, which was subjected to static compression of 25 g/cm2 for 3, 6 and 12 h, with or without treatment of KN-93. After that, the expression of OPG, RANKL and NFATC2 was investigated through real-time PCR and western blot analysis. Results After static compression, the NFATC2 and RANKL expression was significantly up-regulated, while partially suppressed by KN-93 for 6 and 12 h respectively. The OPG expression was significantly down-regulated by compression in 3 h, started to elevate in 6 h, and significantly up-regulated in 12 h. The up-regulation after 12 h was significantly suppressed by KN-93. Conclusions Long-term static compression increases OPG expression in PDLCs, at least partially, via the CAMK II pathway.

Advanced glycation end products (AGEs) and their receptor (RAGE) induce apoptosis of periodontal ligament fibroblasts

Diabetics have an increased prevalence of periodontitis, and diabetes is one of the causative factors of severe periodontitis. Apoptosis is thought to be involved in this pathogenic relationship. The aim of this study was to investigate apoptosis in human periodontal ligament (PDL) fibroblasts induced by advanced glycation end products (AGEs) and their receptor (RAGE). We examined the roles of apoptosis, AGEs, and RAGE during periodontitis in diabetes mellitus using cultured PDL fibroblasts that were treated by AGE-modified bovine serum albumin (AGE-BSA), bovine serum albumin (BSA) alone, or given no treatment (control). Microscopy and real-time quantitative PCR indicated that PDL fibroblasts treated with AGE-BSA were deformed and expressed higher levels of RAGE and caspase 3. Cell viability assays and flow cytometry indicated that AGE-BSA reduced cell viability (69.80±5.50%, P<0.01) and increased apoptosis (11.31±1.73%, P<0.05). Hoechst 33258 staining and terminal-deoxynucleotidyl transferase-mediated nick-end labeling revealed that AGE-BSA significantly increased apoptosis of PDL fibroblasts. The results showed that the changes in PDL fibroblasts induced by AGE-BSA may explain how AGE-RAGE participates in and exacerbates periodontium destruction.

Chronic stress enhances progression of periodontitis via alpha1-adrenergic signaling: a potential target for periodontal disease therapy

This study assessed the roles of chronic stress (CS) in the stimulation of the sympathetic nervous system and explored the underlying mechanisms of periodontitis. Using an animal model of periodontitis and CS, the expression of tyrosine hydroxylase (TH) and the protein levels of the alpha1-adrenergic receptor (alpha1-AR) and beta2-adrenergic receptor (beta2-AR) were assessed. Furthermore, human periodontal ligament fibroblasts (HPDLFs) were stimulated with lipopolysaccharide (LPS) to mimic the process of inflammation. The proliferation of the HPDLFs and the expression of alpha1-AR and beta2-AR were assessed. The inflammatory-related cytokines interleukin (IL)-1beta, IL-6 and IL-8 were detected after pretreatment with the alpha1/beta2-AR blockers phentolamine/propranolol, both in vitro and in vivo. Results show that periodontitis under CS conditions enhanced the expression of TH, alpha1-AR and beta2-AR. Phentolamine significantly reduced the inflammatory cytokine levels. Furthermore, we observed a marked decrease in HPDLF proliferation and the increased expression of alpha1-ARfollowing LPS pretreatment. Pretreatment with phentolamine dramatically ameliorated LPS-inhibited cell proliferation. In addition, the blocking of alpha1-ARsignaling also hindered the upregulation of the inflammatory-related cytokines IL-1beta, IL-6 and IL-8. These results suggest that CS can significantly enhance the pathological progression of periodontitis by an alpha1-adrenergic signaling-mediated inflammatory response. We have identified a potential therapeutic target for the treatment of periodontal disease, particularly in those patients suffering from concurrent CS.

In Vitro Cytotoxicity of White MTA, MTA Fillapex® and Portland Cement on Human Periodontal Ligament Fibroblasts

The aim of this study was to compare the in vitro cytotoxicity of white mineral trioxide aggregate (MTA), MTA Fillapex® and Portland cement (PC) on human cultured periodontal ligament fibroblasts. Periodontal ligament fibroblast culture was established and the cells were used for cytotoxic tests after the fourth passage. Cell density was set at 1.25 X10 4 cells/well in 96-well plates. Endodontic material extracts were prepared by placing sealer/cement specimens (5X3mm) in 1mL of culture medium for 72 h. The extracts were then serially two-fold diluted and inserted into the cell-seeded wells for 24, 48 and 72 h. MTT assay was employed for analysis of cell viability. Cell supernatants were tested for nitric oxide using the Griess reagent system. MTA presented cytotoxic effect in undiluted extracts at 24 and 72 h. MTA Fillapex® presented the highest cytotoxic levels with important cell viability reduction for pure extracts and at ½ and » dilutions. In this study, PC did not induce alterations in fibroblast viability. Nitric oxide was detected in extract-treated cell supernatants and also in the extracts only, suggesting presence of nitrite in the soluble content of the tested materials. In the present study, MTA Fillapex displayed the highest cytotoxic effect on periodontal ligament fibroblasts followed by white MTA and PC.

Resumo O objetivo deste estudo foi comparar a citotoxicidade in vitro de agregado trióxido mineral (MTA) branco, MTA Fillapex® e cimento Portland (PC) em cultura de fibroblastos de ligamento periodontal humano. A cultura de fibroblastos de ligamento periodontal foi estabelecida e as células foram utilizadas para os testes citotóxicos após a quarta passagem. A densidade celular foi ajustada em 1,25X10 4 células/poço em placas de 96 poços. Extratos dos materiais endodônticos foram preparados por meio da inserção de corpos de prova dos cimentos (5 X 3 mm) em 1 mL de meio de cultura durante 72 h. Os extratos foram diluídos serialmente na razão de ½ e inseridos aos poços contendo as células por 24, 48 e 72 h. Ensaio de MTT foi realizado para a avaliação da viabilidade celular. O sobrenadante das células foi testado em relação à presença de óxido nítrico utilizando o sistema de reagentes de Griess. O MTA apresentou efeito citotóxico quando o extrato era aplicado sem diluição durante 24 e 72 h. O MTA Fillapex apresentou os maiores níveis de citotoxicidade com importante redução da viabilidade celular quando o extrato foi aplicado puro e em diluições de ½ e ». Neste estudo, PC não induziu alterações na viabilidade de fibroblastos. Óxido nítrico foi detectado no sobrenadante de células tratadas com os extratos e ainda nos extratos somente, o que sugere a presença de nitrito no conteúdo solúvel dos materiais testados. No presente estudo, MTA Fillapex foi o material que demonstrou o maior efeito citotóxico sobre fibroblastos de ligamento periodontal seguido do MTA branco e do PC. .

Zimography is an effective method for detection of matrix metalloproteinase 2 (MMP-2) activity in cultured human fibroblasts

PURPOSE: To describe a method to characterize the gelatinase activity of cultured human periodontal fibroblasts stimulated with Pam3Cys and E. coli LPS, ligands of TLR2 and TLR4 respectively, and by centrifugation of the cultures, simulating an orthodontic force. METHODS: To study MMP-2 activity, primary cultures of human periodontal fibroblasts were stimulated with the addition of TLRs 2 and 4 ligands and the application of mechanical force by centrifugation at 141 x g for 30 min. Supernatant media was collected 24 hours later to perform protein quantification and zymography. RESULTS: MMP-2 activity suffered an increase in cultures co-stimulated with TLRs 2 and 4 ligands alone or with the presence of mechanical force application compared to basal levels. CONCLUSION: Zymography, one of the several methods to study MMPs activities, is a simple, qualitative and efficient method based on electrophoresis of bis-acrylamide gels copolymerized with a protein substrate.

Evaluation of two protocols for low-level laser application in patients submitted to orthodontic treatment

INTRODUCTION: Different low-level laser (LLL) irradiation protocols have been tested to accelerate orthodontic tooth movement (OTM). Nevertheless, divergent results have been obtained. It was suggested that the stimulatory action of low level laser irradiation occurs during the proliferation and differentiation stages of bone cellular precursors, but not during later stages. OBJECTIVE: The purpose of this study was to determine the effect of two protocols of LLL irradiation on experimental tooth movement: One with daily irradiations and another with irradiations during the early stages. METHODS: Thirty-six rats were divided into control groups (CG1, CG2, CG3) and irradiated groups (IrG1, IrG2, IrG3) according to the presence of: experimental tooth movement, laser irradiation, type of laser irradiation protocol and date of euthanasia (3th or 8th day of experiment). At the end of experimental periods, a quantitative evaluation of the amount of OTM was made and the reactions of the periodontium were analyzed by describing cellular and tissue reactions and by counting blood vessels. RESULTS: The amount of OTM revealed no significant differences between groups in the same experimental period (p < 0.05). Qualitative analysis revealed the strongest resorption activity in irradiated groups after seven days, especially when using the daily irradiation protocol. There was a higher number of blood vessels in irradiated animals than in animals without orthodontic devices and without laser irradiation (p < 0.05). CONCLUSION: Moreover, angiogenesis was verified in some of the irradiated groups. The irradiation protocols tested were not able to accelerate OTM and root resorption was observed while they were applied.

INTRODUÇÃO: diferentes protocolos de irradiação por laser de baixa potência (LBP) têm sido testados para potencializar o movimento ortodôntico; entretanto, há resultados divergentes. Foi sugerido que seu efeito bioestimulador ocorre nas fases de proliferação e diferenciação celular, não agindo em estágios tardios. OBJETIVO: avaliar o efeito de dois protocolos de irradiação do LBP na movimentação ortodôntica: um com irradiações diárias e outro em que irradiações foram realizadas apenas nos períodos iniciais. MÉTODOS: trinta e seis ratos Wistar foram divididos em grupos controles (GC1, GC2 e GC3) e irradiados (GIr1, GIr2 e GIr3), de acordo com a presença de dispositivo ortodôntico, a presença de irradiação, o tipo de protocolo de irradiação e a data de eutanásia (3º ou 8º dia de experimento). Ao final dos períodos experimentais, foram realizadas mensurações da movimentação dentária, análise qualitativa das reações celulares e teciduais do periodonto e contagem de vasos sanguíneos no ligamento periodontal. RESULTADOS: a quantidade de movimentação não diferiu entre os grupos num mesmo tempo experimental (p < 0,05). A análise qualitativa revelou maior atividade absortiva nos grupos irradiados ao final de 7 dias, especialmente quando as irradiações foram diárias. Nos grupos irradiados diariamente, a contagem de vasos foi aumentada em relação aos animais isentos de dispositivo ortodôntico e de aplicações de LBP (p < 0,05). CONCLUSÃO: apesar de verificada angiogênese em certos grupos irradiados, os protocolos de irradiação testados não foram capazes de acelerar a movimentação dentária, e foi possível verificarem-se absorções radiculares.

Soy milk as a storage medium to preserve human fibroblast cell viability: an in vitro study

Soy milk (SM) is widely consumed worldwide as a substitute for cow milk. It is a source of vitamins, carbohydrates and sugars, but its capacity to preserve cell viability has not been evaluated. The purpose of the present study was to investigate the efficacy of SM to maintain the viability of human fibroblasts at short periods compared with different cow milks. Human mouth fibroblasts were cultured and stored in the following media at room temperature: 10% Dulbecco's Modified Eagle Medium (DMEM) (positive control group); long shelf-life ultra-high temperature whole cow milk (WM); long shelf-life ultra-high temperature skim cow milk (SKM); powdered cow milk (PM); and soy milk (SM). After 5, 15, 30 and 45 min, cell viability was analyzed using the MTT assay. Data were analyzed statistically by the Kruskal-Wallis test with post-analysis using the Dunn's method (α=0.05). SKM showed the lowest capacity to maintain cell viability in all analyzed times (p<0.05). At 30 and 45 min, the absorbance levels in control group (DMEM) and SM were significantly higher than in SKM (p<0.05). Cell viability decreased along the time (5-45 min). The results indicate that SM can be used as a more adequate storage medium for avulsed teeth. SKM was not as effective in preserving cell viability as the cell culture medium and SM.

O leite de soja (LS) é largamente consumido em todo o mundo como substituto para o leite bovino. Este é uma fonte de vitaminas, carboidratos e açúcares, mas a sua capacidade para preservar a viabilidade celular não foi avaliada. A finalidade do estudo foi investigar a eficácia do LS em manter a viabilidade de fibroblastos humanos em períodos curtos em comparação com diferentes leites bovinos. Fibroblastos de boca humanos foram cultivados e armazenados nos seguintes meios à temperatura ambiente: 10% de meio Dulbecco's Modified Eagle (DMEM) (grupo controle positivo); leite bovino integral longa vida (LI); leite bovino desnatado longa vida - LD; leite em pó - LP; leite de soja - LS. Depois de 5, 15, 30 e 45 min, a viabilidade celular foi analisada usando o método de MTT. Os dados foram analisados estatisticamente pelo teste de Kruskal-Wallis e posteriormente usando o método de Dunn (α=0,05). O grupo LD apresentou a menor capacidade para manter a viabilidade celular em todos os tempos analisados (p<0,05). Aos 30 e aos 45 min, os níveis de absorbância no grupo controle (DMEM) e LS foram significativamente maiores que no grupo LD (p<0,05). A viabilidade celular diminuiu ao longo do tempo (5-45 min). Os resultados indicaram que LS pode ser usado como meio de armazenamento mais adequado para dentes avulsionados. LD não foi eficaz na preservação da viabilidade das células como o meio de cultura de células e o LS.

El rol de los restos epiteliales de Malassez en el ligamento periodontal / The role of Malassez epithelial rests in the periodontal ligament

El periodonto de inserción es definido como aquellos tejidos que soportan y rodean a la raíz dentaria y está compuesto opr tres estructuras que constituyen una unidad topográfica y funcional: el cemento radicular, el ligamento periodontal y el hueso alveolar. Estas estructuras evolucionan interrelacionada y coordinadamente durante la vida del diente, adaptándose a las modificaciones que surgen en el medio bucal. El objetivo de este trabajo es transmitir los nuevos conceptos emergentes vinculados al periodonto de inserción, centrándonos fundamentalmente en la estructura y función de los restos epiteliales de Malassez (REM), células presentes en el ligamento periodontal. Si bien es conocida la participación de estas células en procesos patológicos, entre ellos, quistes inflamatorios y tumores odontogénicos, poco se sabe de los mismos. Por ello, para poder llevar a cabo esta actualización sobre los REM, se consideraron las últimas publicaciones al respecto que figuran en la base de datos de Pubmed. A través de esta búsqueda, se obtuvo información referida a su descubrimiento, origen embriológico, características histológicas, productos elaborados por los REM y posibles funciones por ellos desempeñadas. Se puede decir entonces, que los REM desempeñarían un papel importante dentro del ligamento periodontal normal y, posiblemente, durante las terapias periodontales regenerativas. Si bien se requieren más estudios al respecto, es posible por lo tanto, reivindicar a estas células en su función y considerarlas mucho más que simples vestigios de la odontogénesis, presentes en el ligamento periodontal.

Dental pulp tissue engineering

Dental pulp is a highly specialized mesenchymal tissue that has a limited regeneration capacity due to anatomical arrangement and post-mitotic nature of odontoblastic cells. Entire pulp amputation followed by pulp space disinfection and filling with an artificial material cause loss of a significant amount of dentin leaving as life-lasting sequelae a non-vital and weakened tooth. However, regenerative endodontics is an emerging field of modern tissue engineering that has demonstrated promising results using stem cells associated with scaffolds and responsive molecules. Thereby, this article reviews the most recent endeavors to regenerate pulp tissue based on tissue engineering principles and provides insightful information to readers about the different aspects involved in tissue engineering. Here, we speculate that the search for the ideal combination of cells, scaffolds, and morphogenic factors for dental pulp tissue engineering may be extended over future years and result in significant advances in other areas of dental and craniofacial research. The findings collected in this literature review show that we are now at a stage in which engineering a complex tissue, such as the dental pulp, is no longer an unachievable goal and the next decade will certainly be an exciting time for dental and craniofacial research.

A polpa dental é um tecido conjuntivo altamente especializado que possui uma restrita capacidade de regeneração, devido à sua disposição anatômica e à natureza pós-mitótica das células odontoblásticas. A remoção total da polpa, seguida da desinfecção do canal radicular e seu preenchimento com material artificial proporciona a perda de uma significante quantidade de dentina deixando como sequela um dente não vital e enfraquecido. Entretanto, a endodontia regenerativa é um campo emergente da engenharia tecidual, que demonstrou resultados promissores utilizando células-tronco associadas à scaffolds e moléculas bioativas. Desta forma, esse artigo revisa os recentes avanços obtidos na regeneração do tecido pulpar baseado nos princípios da engenharia tecidual e fornece aos leitores informações compreensivas sobre os diferentes aspectos envolvidos na engenharia tecidual. Assim, nós especulamos que a combinação ideal de células, scaffolds e moléculas bioativas pode resultar em significantes avanços em outras áreas da pesquisa odontológica. Os dados levantados em nossa revisão demonstraram que estamos em um estágio no qual, o desenvolvimento de tecidos complexos, tais como a polpa dental, não é mais inatingível e que a próxima década será um período extremamente interessante para a pesquisa odontológica.