Potential of Mesenchymal Stem Cell Sheets on Periodontal Regeneration: A Systematic Review of Pre-Clinical Studies.

BACKGROUND: Cell sheet technique using mesenchymal stem cells is a high-level strategy in periodontal regenerative medicine. Although recent studies have shown the role of MSCSs in increased dental supporting tissues and bone, there is no systematic review focused specifically on assessing periodontal regeneration in orthotopic animal models. OBJECTIVE: To evaluate the potential of mesenchymal stem cell sheets (MSCSs) on periodontal regeneration, compared to control, in experimental animal models Methods: Pre-clinical studies in periodontal defects of animal models were considered eligible. The electronic search included the MEDLINE, Web of Science, EMBASE and LILACS databases. The review was conducted according to the Preferred Reporting Item for Systematic Reviews and Meta-Analyses statement guidelines. RESULTS: A total of 17 of the 3989 studies obtained from the electronic database search were included. MSCSs included dental follicle (DF) MSCSs, periodontal ligament (PL) MSCSs, dental pulp (DP) MSCSs, bone marrow (BM) MSCSs, alveolar periosteal (AP) MSCSs and gingival (G) MSCSs. Regarding cell sheet inducing protocol, most of the studies used ascorbic acid (52.94%). Others used culture dishes grafted with a temperature-responsive polymer (47.06%). Adverse effects were not identified in the majority of studies. Meta-analysis was not considered because of methodological heterogeneities. PDL-MSCSs were superior for periodontal regeneration enhancement compared to the control, but in an induced inflammatory microenvironment, DF-MSCSs were better. Moreover, DF-MSCSs, DP-MSCSs, and BM-MSCSs showed improved results compared to the control. CONCLUSION: MSCSs can improve periodontal regeneration in animal periodontal defect models.

Oral biofilm dysbiosis during experimental periodontitis.

OBJECTIVES: We have previously characterized the main osteoimmunological events that occur during ligature periodontitis. This study aims to determine the polymicrobial community shifts that occur during disease development. METHODS: Periodontitis was induced in C57BL/6 mice using the ligature-induced periodontitis model. Healthy oral mucosa swabs and ligatures were collected every 3 days from 0 to 18 days post-ligature placement. Biofilm samples were evaluated by 16SrRNA gene sequencing (Illumina MiSeq) and QIIME. Time-course changes were determined by relative abundance, diversity, and rank analyses (PERMANOVA, Bonferroni-adjusted). RESULTS: Microbial differences between health and periodontal inflammation were observed at all phylogenic levels. An evident microbial community shift occurred in 25 genera during the advancement of "gingivitis" (3-6 days) to periodontitis (9-18 days). From day 0 to 18, dramatic changes were identified in Streptococcus levels, with an overall decrease (54.04%-0.02%) as well an overall increase of Enterococcus and Lactobacillus (23.7%-73.1% and 10.1%-70.2%, respectively). Alpha-diversity decreased to its lowest at 3 days, followed by an increase in diversity as disease advancement. Beta-diversity increased after ligature placement, indicating that bone loss develops in response to a greater microbial variability (p = 0.001). Levels of facultative and strict anaerobic bacteria augmented over the course of disease progression, with a total of eight species significantly different during the 18-day period. CONCLUSION: The data supports that murine gingival inflammation and alveolar bone loss develop in response to microbiome shifts. Bacterial diversity increased during progression to bone loss. These findings further support the utilization of the periodontitis ligature model for microbial shift analysis under different experimental conditions.

Osteogenesis in human periodontal ligament stem cell sheets is enhanced by the protease-activated receptor 1 (PAR1) in vivo.

Human periodontal ligament stem cells (PDLSCs) have been studied as a promising strategy in regenerative approaches. The protease-activated receptor 1 (PAR1) plays a key role in osteogenesis and has been shown to induce osteogenesis and increase bone formation in PDLSCs. However, little is known about its effects when activated in PDLSCs as a cell sheet construct and how it would impact bone formation as a graft in vivo. Here, PDLSCs were obtained from 3 patients. Groups were divided into control, osteogenic medium and osteogenic medium + PAR1 activation by TFLLR-NH2 peptide. Cell phenotype was determined by flow cytometry and immunofluorescence. Calcium deposition was quantified by Alizarin Red Staining. Cell sheet microstructure was analyzed through light, scanning electron microscopy and histology and transplanted to Balb/c nude mice. Immunohistochemistry for bone sialoprotein (BSP), integrin ß1 and collagen type 1 and histological stains (H&E, Van Giesson, Masson's Trichrome and Von Kossa) were performed on the ex-vivo mineralized tissue after 60 days of implantation in vivo. Ectopic bone formation was evaluated through micro-CT. PAR1 activation increased calcium deposition in vitro as well as BSP, collagen type 1 and integrin ß1 protein expression and higher ectopic bone formation (micro-CT) in vivo.

Microenvironmental Factors that Shape Bacterial Metabolites in Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is a significant global health problem that involves chronic intestinal inflammation and can involve severe comorbidities, including intestinal fibrosis and inflammation-associated colorectal cancer (CRC). Disease-associated alterations to the intestinal microbiota often include fecal enrichment of Enterobacteriaceae, which are strongly implicated in IBD development. This dysbiosis of intestinal flora accompanies changes in microbial metabolites, shaping host:microbe interactions and disease risk. While there have been numerous studies linking specific bacterial taxa with IBD development, our understanding of microbial function in the context of IBD is limited. Several classes of microbial metabolites have been directly implicated in IBD disease progression, including bacterial siderophores and genotoxins. Yet, our microbiota still harbors thousands of uncharacterized microbial products. In-depth discovery and characterization of disease-associated microbial metabolites is necessary to target these products in IBD treatment strategies. Towards improving our understanding of microbiota metabolites in IBD, it is important to recognize how host relevant factors influence microbiota function. For example, changes in host inflammation status, metal availability, interbacterial community structure, and xenobiotics all play an important role in shaping gut microbial ecology. In this minireview, we outline how each of these factors influences gut microbial function, with a specific focus on IBD-associated Enterobacteriaceae metabolites. Importantly, we discuss how altering the intestinal microenvironment could improve the treatment of intestinal inflammation and associated disorders, like intestinal fibrosis and CRC.

Effect of alpha-humulene incorporation on the properties of experimental light-cured periodontal dressings.

The objective of this study was to formulate an experimental light-cured periodontal dressing containing alpha-humulene and to compare its physical, antimicrobial, and cytotoxicity properties with commercial gold standards (Barricaid® and Periobond®). Two periodontal dressing formulations were developed (a and b). The formulations were divided into 5 groups according to the alpha-humulene concentration as follows: Ea - control group, Ea1 - 1%, Ea5 - 5%, Ea10 - 10%, and Ea20 - 20%; Eb - control group, Eb1 - 1%, Eb5 - 5%, Eb10 - 10%, and Eb20 - 20%. Materials characterization was performed using the degree of conversion, cohesive strength, sorption, and solubility assays. Antimicrobial assay was performed using the modified direct contact test against E. faecalis and S. aureus. Cytotoxicity was assessed by the cell viability experiment using L929 fibroblasts. In general, the cohesive strength values of materials decreased as the alpha-humulene concentration increased. All the experimental dressings showed antimicrobial activity against both bacteria tested. Cell viability results for the Ea, Ea1, Eb, and Eb1 groups showed moderate cytotoxic effect. The formulations containing alpha-humulene showed similar behavior to the commercial references. Thus, formulations containing alpha-humulene have potential to be used as periodontal dressing.

Protease-activated receptor type 1 (PAR1) increases CEMP1 gene expression through MAPK/ERK pathway.

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.

Interferon activated gene 204 protects against bone loss in experimental periodontitis.

BACKGROUND: Periodontal destruction can be the result of different known and yet-to-be-discovered biological pathways. Recent human genetic association studies have implicated interferon-gamma inducible protein 16 (IFI16) and absent in melanoma 2 (AIM2) with high periodontal interleukin (IL)-1ß levels and more destructive disease, but mechanistic evidence is lacking. Here, we sought to experimentally validate these observational associations and better understand IFI16 and AIM2's roles in periodontitis. METHODS: Periodontitis was induced in Ifi204-/- (IFI16 murine homolog) and Aim2-/- mice using the ligature model. Chimeric mice were created to identify the main source cells of Ifi204 in the periodontium. IFI16-silenced human endothelial cells were treated with periodontal pathogens in vitro. Periodontal tissues from Ifi204-/- mice were evaluated for alveolar bone (micro-CT), cell inflammatory infiltration (MPO+ staining), Il1b (qRT-PCR), and osteoclast numbers (cathepsin K+ staining). RESULTS: Ifi204-deficient mice> exhibited >20% higher alveolar bone loss than wild-type (WT) (P < 0.05), while no significant difference was found in Aim2-/- mice. Ifi204's effect on bone loss was primarily mediated by a nonbone marrow source and was independent of Aim2. Ifi204-deficient mice had greater neutrophil/macrophage trafficking into gingival tissues regardless of periodontitis development compared to WT. In human endothelial cells, IFI16 decreased the chemokine response to periodontal pathogens. In murine periodontitis, Ifi204 depletion elevated gingival Il1b and increased osteoclast numbers at diseased sites (P < 0.05). CONCLUSIONS: These findings support IFI16's role as a novel regulator of inflammatory cell trafficking to the periodontium that protects against bone loss and offers potential targets for the development of new periodontal disease biomarkers and therapeutics.

Encephalitis and cytokine storm secondary to respiratory viruses in children: Two case reports.

Introduction: Encephalitis is a syndrome characterized by brain damage secondary to an inflammatory process that is manifested by cognitive impairment and altered cerebral spinal fluid analysis; it may evolve with seizures and coma. Despite viral infections representing the main cause of encephalitis in children, respiratory syncytial virus (RSV) and parainfluenza virus are mostly associated with respiratory presentations. Uncommonly, the inflammatory phenomena from encephalitis secondary to viral agents may present with an exacerbated host response, the so-called cytokine storm. The link between these infectious agents and neurologic syndromes resulting in a cytokine storm is rare, and the underlying pathophysiology is still poorly understood. Case presentation: A 5-year-old girl and a 2-year-old boy infected with parainfluenza and RSV, respectively, were identified through nasopharyngeal polymerase chain reaction. They were admitted into the pediatric intensive care unit due to encephalitis and multiple organ dysfunction manifested with seizures and hemodynamic instability. Magnetic resonance imaging findings from the first patient revealed a bilateral hypersignal on fluid-attenuated inversion recovery in the cerebral hemispheres, especially in the posterior parietal and occipital regions. The girl also had elevated IL-6 levels during the acute phase and evolved with a fast recovery of the clinical presentations. The second patient progressed with general systemic complications followed by cerebral edema and death. Conclusion: Encephalitis secondary to respiratory viral infection might evolve with cytokine storm and multiorgan inflammatory response in children.

Protease-activated receptor type 1 (PAR1) increases CEMP1 gene expression through MAPK/ERK pathway

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.

Effect of alpha-humulene incorporation on the properties of experimental light-cured periodontal dressings

Abstract The objective of this study was to formulate an experimental light-cured periodontal dressing containing alpha-humulene and to compare its physical, antimicrobial, and cytotoxicity properties with commercial gold standards (Barricaid® and Periobond®). Two periodontal dressing formulations were developed (a and b). The formulations were divided into 5 groups according to the alpha-humulene concentration as follows: Ea - control group, Ea1 - 1%, Ea5 - 5%, Ea10 - 10%, and Ea20 - 20%; Eb - control group, Eb1 - 1%, Eb5 - 5%, Eb10 - 10%, and Eb20 - 20%. Materials characterization was performed using the degree of conversion, cohesive strength, sorption, and solubility assays. Antimicrobial assay was performed using the modified direct contact test against E. faecalis and S. aureus. Cytotoxicity was assessed by the cell viability experiment using L929 fibroblasts. In general, the cohesive strength values of materials decreased as the alpha-humulene concentration increased. All the experimental dressings showed antimicrobial activity against both bacteria tested. Cell viability results for the Ea, Ea1, Eb, and Eb1 groups showed moderate cytotoxic effect. The formulations containing alpha-humulene showed similar behavior to the commercial references. Thus, formulations containing alpha-humulene have potential to be used as periodontal dressing.

Cytotoxicity of Reparative Endodontic Cements on Human Periodontal Ligament Stem Cells

ABSTRACT Objective To compare the cytotoxicity of commercial reparative endodontic cements on human periodontal ligament stem cells (hPDLSCs). Material and Methods The culture of hPDLSCs was established. Cell density was set at 2 × 104 cells/well in 96-well plates. Extracts of Biodentine, Bio-C Repair, Cimmo HD, MTA Repair HP and White MTA were prepared. Then, the extracts were diluted (pure, 1:4 and 1:16) and inserted into cell-seeded wells for 24, 48, and 72 h to assess cell viability through MTT assay. hPDLSCs incubated with culture medium alone served as a negative control group. Data were analyzed by Two-Way ANOVA and Tukey's test (α=0.05). Results At 24 h, pure extract of MTA Repair HP and Biodentine 1:16 presented higher cell viability compared to control. Lower cell viability was found for pure extract of Cimmo HD, MTA Repair HP 1:4 and 1:16, and White MTA 1:16. At 48 h, pure extract of Bio-C Repair and MTA Repair HP presented higher cell viability compared to control. At 72 h, only the pure extract of MTA Repair HP led to higher cell proliferation compared to control. Conclusion Biodentine, Bio-C Repair and MTA Repair HP were able to induce hPDLSCs proliferation. Cimmo HD and White MTA were found to be mostly cytotoxic in hPDLSCs.

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

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.

Antimicrobial and cytotoxic capacity of pyroligneous extracts films of Eucalyptus grandis and chitosan for oral applications.

Chitosan films containing distilled pyroligneous extracts of Eucalyptus grandis (DPEC), characterized and developed by Brazilian Agricultural Research Corporation-Embrapa Temperate Agriculture (EMBRAPA-CPACT), were evaluated for antimicrobial activity against Candida albicans, Streptococcus mutans, and Lactobacillus acidophilus by direct contact test. Further, their capacity for the prevention of teeth enamel demineralization and cytotoxicity in vitro were also determined. The natural polymers were tested at different concentrations (1500-7500 µg mL-1) and the formulation of an experimental fluoride varnish with antimicrobial activity was evaluated by direct contact test, whereas cytotoxicity was analyzed through the colorimetric MTT assay. Preliminary data showed no statistically significant differences in cytotoxicity to NIH/3T3 cell line when DPEC is compared to the control group. On the other hand, the antimicrobial capacity and demineralization effects were found between the test groups at the different concentrations tested. Chitosan films containing distilled pyroligneous extracts of E. grandis may be an effective control strategy to prevent biofilm formation related to dental caries when applied as a protective varnish. They may inhibit the colonization of oral microorganisms and possibly control dental caries through a decrease in pH and impairment of enamel demineralization.

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.

Protease-activated receptor 1 as a potential therapeutic target for COVID-19.

Acute respiratory disease caused by a novel coronavirus (SARS-CoV-2) has spread all over the world, since its discovery in 2019, Wuhan, China. This disease is called COVID-19 and already killed over 1 million people worldwide. The clinical symptoms include fever, dry cough, dyspnea, headache, dizziness, generalized weakness, vomiting, and diarrhea. Unfortunately, so far, there is no validated vaccine, and its management consists mainly of supportive care. Venous thrombosis and pulmonary embolism are highly prevalent in patients suffering from severe COVID-19. In fact, a prothrombotic state seems to be present in most fatal cases of the disease. SARS-CoV-2 leads to the production of proinflammatory cytokines, causing immune-mediated tissue damage, disruption of the endothelial barrier, and uncontrolled thrombogenesis. Thrombin is the key regulator of coagulation and fibrin formation. In severe COVID-19, a dysfunctional of physiological anticoagulant mechanisms leads to a progressive increase of thrombin activity, which is associated with acute respiratory distress syndrome development and a poor prognosis. Protease-activated receptor type 1 (PAR1) is the main thrombin receptor and may represent an essential link between coagulation and inflammation in the pathophysiology of COVID-19. In this review, we discuss the potential role of PAR1 inhibition and regulation in COVID-19 treatment.