Cell sheet produced from periodontal ligament stem cells activated by PAR1 improves osteogenic differentiation.

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-ßI, 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.

Fusobacterium nucleatum triggers senescence phenotype in gingival epithelial cells.

The prevalence of periodontitis increases with physiological aging. However, whether bacteria associated with periodontal diseases foster aging and the mechanisms by which they may do so are unknown. Herein, we hypothesize that Fusobacterium nucleatum, a microorganism associated with periodontitis and several other age-related disorders, triggers senescence, a chief hallmark of aging responsible to reduce tissue repair capacity. Our study analyzed the senescence response of gingival epithelial cells and their reparative capacity upon long-term exposure to F. nucleatum. Specifically, we assessed (a) cell cycle arrest by analyzing the cyclin-dependent kinase inhibitors p16INK4a and p14ARF and their downstream cascade (pRb, p53, and p21) at both gene and protein levels, (b) lysosomal mediated dysfunction by using assays targeting the expression and activity of the senescence-associated ß-galactosidase (SA-ß-Gal) enzyme, and (c) nuclear envelope breakdown by assessing the expression of Lamin-B1. The consequences of the senescence phenotype mediated by F. nucleatum were further assessed using wound healing assays. Our results revealed that prolonged exposure to F. nucleatum promotes an aging-like phenotype as evidenced by the increased expression of pro-senescence markers (p16INK4a , p21, and pRb) and SA-ß-Gal activity and reduced expression of the counter-balancing cascade (p14ARF and p53) and Lamin-B1. Furthermore, we also noted impaired wound healing capacity of gingival epithelial cells upon prolong bacterial exposure, which was consistent with the senescence-induced phenotype. Together, our findings provide a proof-of-concept evidence that F. nucleatum triggers a pro-senescence response in gingival epithelial cells. This might affect periodontal tissue homeostasis by reducing its repair capacity and, consequently, increasing susceptibility to periodontitis during aging.

Cell sheet produced from periodontal ligament stem cells activated by PAR1 improves osteogenic differentiation

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.

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.

Periodontitis, chronic liver diseases, and the emerging oral-gut-liver axis.

The liver carries out a wide range of functions ranging from the control of metabolites, nutrient storage, and detoxification to immunosurveillance. While inflammation is essential for the tissue remodeling and maintenance of homeostasis and normal liver physiology, constant exposure to dietary and microbial products creates a niche for potentially prolonged immune activation and unresolved inflammation in susceptible host. Failure to restrain inflammation can lead to development of chronic liver diseases characterized by fibrosis, cirrhosis and eventually liver failure. The liver maintains close interactions with numerous organs which can influence its metabolism and physiology. It is also known that oral cavity microenvironment can influence the physiological conditions of other organs and emerging evidence implicates that this could be true for the liver as well. Presence of chronic inflammation and dysbiotic microbiota is a common feature leading to clinical pathology both in periodontitis and chronic liver diseases (CLDs). In fact, known CLDs appear to have some relationship with periodontitis, which impacts the onset or progression of these conditions in a bidirectional crosstalk. In this review, we explore the emerging association between oral-gut-liver axis focusing on periodontitis and common CLDs including nonalcoholic fatty liver disease, chronic viral hepatitis, liver cirrhosis, and hepatocellular cancer. We highlight the immune pathways and oral microbiome interactions which can link oral cavity and liver health and offer perspectives for future research.

Bifidobacterium Strains Present Distinct Effects on the Control of Alveolar Bone Loss in a Periodontitis Experimental Model.

Periodontitis is an inflammatory disease induced by a dysbiotic oral microbiome. Probiotics of the genus Bifidobacterium may restore the symbiotic microbiome and modulate the immune response, leading to periodontitis control. We evaluated the effect of two strains of Bifidobacterium able to inhibit Porphyromonas gingivalis interaction with host cells and biofilm formation, but with distinct immunomodulatory properties, in a mice periodontitis model. Experimental periodontitis (P+) was induced in C57Bl/6 mice by a microbial consortium of human oral organisms. B. bifidum 1622A [B+ (1622)] and B. breve 1101A [B+ (1101)] were orally inoculated for 45 days. Alveolar bone loss and inflammatory response in gingival tissues were determined. The microbial consortium induced alveolar bone loss in positive control (P + B-), as demonstrated by microtomography analysis, although P. gingivalis was undetected in oral biofilms at the end of the experimental period. TNF-α and IL-10 serum levels, and Treg and Th17 populations in gingiva of SHAM and P + B- groups did not differ. B. bifidum 1622A, but not B. breve 1101A, controlled bone destruction in P+ mice. B. breve 1101A upregulated transcription of Il-1ß, Tnf-α, Tlr2, Tlr4, and Nlrp3 in P-B+(1101), which was attenuated by the microbial consortium [P + B+(1101)]. All treatments downregulated transcription of Il-17, although treatment with B. breve 1101A did not yield such low levels of transcripts as seen for the other groups. B. breve 1101A increased Th17 population in gingival tissues [P-B+ (1101) and P + B+ (1101)] compared to SHAM and P + B-. Administration of both bifidobacteria resulted in serum IL-10 decreased levels. Our data indicated that the beneficial effect of Bifidobacterium is not a common trait of this genus, since B. breve 1101A induced an inflammatory profile in gingival tissues and did not prevent alveolar bone loss. However, the properties of B. bifidum 1622A suggest its potential to control periodontitis.

Probiotics improve re-epithelialization of scratches infected by Porphyromonas gingivalis through up-regulating CXCL8-CXCR1/CXCR2 axis.

Porphyromonas gingivalis inhibits the release of CXCL8 by gingival epithelial cells and reduces their proliferation. We previously reported that Bifidocaterium sp. and Lactobacillus sp. immunomodulate gingival epithelial cells response to this periodontal pathogen, but their effects on re-epithelialization properties are still unknown. Herein we explored these activities of potential probiotics on gingival epithelial cells and clarified their mechanisms. The immortalized OBA-9 lineage was used to perform in vitro scratches. Twelve clinical isolates and commercially available strains of Bifidobacterium sp. and Lactobacillus sp. were screened. L. casei 324 m and B. pseudolongum 1191A were selected to perform mechanistic assays with P. gingivalis W83 infection and the following parameters were measured: percentage of re-epithelialization by DAPI immunofluorescence area measurement; cell number by Trypan Blue exclusion assay; CXCL8 regulation by ELISA and RT-qPCR; and expression of CXCL8 cognate receptors-CXCR1 and CXCR2 by Flow Cytometry. Complementary mechanistic assays were performed with CXCL8, in the presence or absence of the CXCR1/CXCR2 inhibitor-reparixin. L. casei 324 m and B. pseudolongum 1191A enhanced re-epithelialization/cell proliferation as well as inhibited the harmful effects of P. gingivalis W83 on these activities through an increase in the expression and release of CXCL8 and in the number of cells positive for CXCR1/CXCR2. Further, we revealed that the beneficial effects of these potential probiotics were dependent on activation of the CXCL8-CXCR1/CXCR2 axis. The current findings indicate that these potential probiotics strains may improve wound healing in the context of the periodontal tissues by a CXCL8 dependent mechanism.

Therapeutic potential of periodontal ligament stem cells.

Inflammatory periodontal disease known as periodontitis is one of the most common conditions that affect human teeth and often leads to tooth loss. Due to the complexity of the periodontium, which is composed of several tissues, its regeneration and subsequent return to a homeostatic state is challenging with the therapies currently available. Cellular therapy is increasingly becoming an alternative in regenerative medicine/dentistry, especially therapies using mesenchymal stem cells, as they can be isolated from a myriad of tissues. Periodontal ligament stem cells (PDLSCs) are probably the most adequate to be used as a cell source with the aim of regenerating the periodontium. Biological insights have also highlighted PDLSCs as promising immunomodulator agents. In this review, we explore the state of knowledge regarding the properties of PDLSCs, as well as their therapeutic potential, describing current and future clinical applications based on tissue engineering techniques.

Maresin-1 and Resolvin E1 Promote Regenerative Properties of Periodontal Ligament Stem Cells Under Inflammatory Conditions.

Maresin-1 (MaR1) and Resolvin E1 (RvE1) are specialized pro-resolving lipid mediators (SPMs) that regulate inflammatory processes. We have previously demonstrated the hard and soft tissue regenerative capacity of RvE1 in an in vivo model of the periodontal disease characterized by inflammatory tissue destruction. Regeneration of periodontal tissues requires a well-orchestrated process mediated by periodontal ligament stem cells. However, limited data are available on how SPMs can regulate the regenerative properties of human periodontal ligament stem cells (hPDLSCs) under inflammatory conditions. Thus, we measured the impact of MaR1 and RvE1 in an in vitro model of hPDLSC under stimulation with IL-1ß and TNF-α by evaluating pluripotency, migration, viability/cell death, periodontal ligament markers (α-smooth muscle actin, tenomodulin, and periostin), cementogenic-osteogenic differentiation, and phosphoproteomic perturbations. The data showed that the pro-inflammatory milieu suppresses pluripotency, viability, and migration of hPDLSCs; MaR1 and RvE1 both restored regenerative capacity by increasing hPDLSC viability, accelerating wound healing/migration, and up-regulating periodontal ligament markers and cementogenic-osteogenic differentiation. Protein phosphorylation perturbations were associated with the SPM-induced regenerative capacity of hPDLSCs. Together, these results demonstrate that MaR1 and RvE1 restore or improve the regenerative properties of highly specialized stem cells when inflammation is present and offer opportunities for direct pharmacologic treatment of lost tissue integrity.

Oral and Fecal Microbiome in Molar-Incisor Pattern Periodontitis.

In order to improve our understanding on the microbial complexity associated with Grade C/molar-incisor pattern periodontitis (GC/MIP), we surveyed the oral and fecal microbiomes of GC/MIP and compared to non-affected individuals (Control). Seven Afro-descendants with GC/MIP and seven age/race/gender-matched controls were evaluated. Biofilms from supra/subgingival sites (OB) and feces were collected and submitted to 16S rRNA sequencing. Aggregatibacter actinomycetemcomitans (Aa) JP2 clone genotyping and salivary nitrite levels were determined. Supragingival biofilm of GC/MIP presented greater abundance of opportunistic bacteria. Selenomonas was increased in subgingival healthy sites of GC/MIP compared to Control. Synergistetes and Spirochaetae were more abundant whereas Actinobacteria was reduced in OB of GC/MIP compared to controls. Aa abundance was 50 times higher in periodontal sites with PD≥ 4 mm of GC/MIP than in controls. GC/MIP oral microbiome was characterized by a reduction in commensals such as Kingella, Granulicatella, Haemophilus, Bergeyella, and Streptococcus and enrichment in periodontopathogens, especially Aa and sulfate reducing Deltaproteobacteria. The oral microbiome of the Aa JP2-like+ patient was phylogenetically distant from other GC/MIP individuals. GC/MIP presented a higher abundance of sulfidogenic bacteria in the feces, such as Desulfovibrio fairfieldensis, Erysipelothrix tonsillarum, and Peptostreptococcus anaerobius than controls. These preliminary data show that the dysbiosis of the microbiome in Afro-descendants with GC/MIP was not restricted to affected sites, but was also observed in supragingival and subgingival healthy sites, as well as in the feces. The understanding on differences of the microbiome between healthy and GC/MIP patients will help in developing strategies to improve and monitor periodontal treatment.

Probiotics alter biofilm formation and the transcription of Porphyromonas gingivalis virulence-associated genes.

BACKGROUND AND OBJECTIVE: The potential of probiotics on the prevention and control of periodontitis and other chronic inflammatory conditions has been suggested. Lactobacillus and Bifidobacterium species influence P. gingivalis interaction with gingival epithelial cells (GECs) but may not act in a unique way. In order to select the most appropriate probiotic against P. gingivalis, we aimed to evaluate the effect of several strains on Porphyromonas gingivalis biofilm formation and transcription virulence-associated factors (PgVAFs). METHODS: Cell-free pH neutralized supernatants (CFS) and living Lactobacillus spp. and Bifidobacterium spp. were tested against P. gingivalis ATCC 33277 and W83, in mono- and multi-species (with Streptococcus oralis and S. gordonii) biofilms. Relative transcription of P. gingivalis genes (fimA, mfa1, kgp, rgp, ftsH and luxS) was determined in biofilms and under GECs co-infection. RESULTS: Probiotics CFS reduced P. gingivalis ATCC 33277 levels in mono-species biofilms and living probiotics reduced P. gingivalis abundance in multi-species biofilms. L. acidophilus LA5 down-regulated transcription of most PgVAFs in biofilms and GECs. CONCLUSIONS: Probiotics affect P. gingivalis biofilm formation by down-regulating overall PgVAFs with the most pronounced effect observed for L. acidophilus LA5.

Chemokines and cytokines profile in whole saliva of patients with periodontitis.

Clinical features suggest differences in immune response among periodontitis forms, albeit a large number of cytokines and chemokines remain to be evaluated. The saliva is an available source of mediators and its analysis would be valuable in order to understand pathophysiological differences. The objective of this study was analyze chemokines/cytokines profile in whole saliva of individuals with severe periodontitis (Stage III) presenting moderate [Grade B; GB] or rapid progression rate with a localized incisor-molar pattern [Grade C; GC/IMP]. A case-control study was designed for each periodontitis group. GB (n = 9) and GC/IMP (n = 7) patients and their healthy controls (C-GB, n = 9 and C-GC, n = 7) were evaluated. Non-stimulated saliva samples were assessed by a multiplex assay for a total of 40 cytokines, C-C and C-X-C motif chemokines. GC/IMP group presented higher levels of CCL17 and CCL27 (p = 0.04, FDR > 0.05), and lower levels of CCL2 (p = 0.04, FDR > 0.05) and CCL25 (p = 0.006, FDR < 0.05) when compared to its control. GB patients had higher levels of IL-6, IL-1ß (p = 0.04, FDR > 0.05), and elevated pro-inflammatory (TNF-α,IL-1ß,INF-γ,IL-6, IL-16): anti-inflammatory (IL-2, IL-4, IL-10) ratio (p = 0.01, FDR < 0.05) compared to its control [p-values by Mann-Whitney test, and False Discovery Rate (FDR) by Benjamini-Hochburg corrections]. CCL-chemokines and cytokines contributed to differences between GC/C-GC and GB/C-GB, respectively (p < 0.05, PERMANOVA test). These preliminary data revealed that each periodontitis phenotype presented distinct immune profiles differentially expressed in saliva compared to their related controls, suggesting differences in the etiopathogenesis of GB and GC/IMP.

Effect of periodontal treatment on Aggregatibacter actinomycetemcomitans colonization and serum IgG levels against A. actinomycetemcomitans serotypes and Omp29 of aggressive periodontitis patients.

OBJECTIVE: To evaluate the effect of the periodontal treatment on Aggregatibacter actinomycetemcomitans JP2 clone, and the IgG serum levels against its outer membrane protein (Omp29) and A. actinomycetemcomitans serotypes in aggressive periodontitis (AgP). SUBJECTS AND METHODS: Seventeen patients with generalized (GAgP), 10 with localized (LAgP), and 10 healthy controls were included. AgP participants were submitted to periodontal treatment-scaling and root planing plus antibiotics (SRP+A). Periodontal parameters, for example, probing depth (PD) and clinical attachment loss (CAL), were evaluated at baseline and at 1-year. Serum IgG against Omp29 and serotypes a, b, and c were determined by ELISA. The levels of A. actinomycetemcomitans JP2 clone were determined in subgingival biofilm samples by qPCR. RESULTS: Periodontal treatment resulted in significant reductions of PD, CAL, and IgG levels against Omp29, serotypes b, and c. After therapy, IgG levels against A. actinomycetemcomitans serotypes, as well as the levels of the JP2 clone in AgP, became similar to controls. The reduction in JP2 clone count was correlated with a reduction of PD and IgG response against Omp29. CONCLUSION: Scaling and root planing plus antibiotics decreased IgG levels response against Omp29 and A. actinomycetemcomitans serotypes involved in the disease (b and c), while the serum response increased against tne commensal serotype (a), similar to what occurs in periodontally healthy individuals.

Probiotics alter the immune response of gingival epithelial cells challenged by Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: Although previous studies revealed the potential use of probiotics in the control of periodontitis, little is known about their interactions with gingival epithelial cells (GECs). Since GECs comprise the first defense in the subgingival microenvironment, the aim of this study was to evaluate the effect of probiotic lactobacilli and bifidobacteria strains on OBA-9 cells challenged with Porphyromonas gingivalis. METHODS: Immortalized human GECs (OBA-9) were challenged with live P. gingivalis (strains W83 and ATCC33277) and co-infected with one of 12 tested probiotic strains at a multiplicity of infection (MOI) of 1:1000 for 2 hours. Bacterial adhesion and invasion were determined by antibiotic exclusion analysis and CFU counting. OBA-9 viability was assessed by MTT assay, and levels of inflammatory mediators (TNF-α, IL-1ß, and CXCL8) in the supernatants were determined by ELISA. The expression of genes encoding Toll-like receptors (TLR2, TLR4) was evaluated by RT-qPCR. RESULTS: Both strains of P. gingivalis were able to adhere and invade OBA-9 cells, with significant loss in cell viability, increase in the levels of TNF-α and IL-1ß, and upregulation of TLR4. However, co-infection with probiotics attenuated these effects in P. gingivalis challenged GECs. Most probiotics maintained OBA-9 viability and reduced pathogens adhesion and invasion. Furthermore, probiotics were able to adhere to GECs, which was enhanced for most strains in the presence of P. gingivalis. The synthesis of IL-1ß and TNF-α by P. gingivalis in challenged GECs was reduced in co-culture with most of the tested probiotics, whereas the secretion of CXCL8 increased, and TLR4 was downregulated. CONCLUSION: Probiotics can alter the interaction of GECs with P. gingivalis by modulating the pathogen's ability to adhere and invade these cells, as well as by regulating the innate immune response. Such properties are strain-specific and may indicate the most efficient probiotics to control periodontitis.

A systematic review and meta-analysis on the effects of crown lengthening on adjacent and non-adjacent sites.

OBJECTIVES: The objective of the study was to assess the impact of periodontal crown lengthening surgery on clinical parameters at adjacent and non-adjacent sites compared to treated sites. MATERIAL AND METHODS: An electronic search was carried out on MEDLINE-PubMed, The Cochrane Library, and ISI Web of Science databases between 1978 and 2015. Methodological quality assessment was based on Cochrane recommendations. Meta-analyses were assessed with RevMan 5.0 and heterogeneity between studies by the Higgin test (I 2). Clinical attachment level (CAL) and probing depth (PD) were the primary outcome variables. Four case series studies were included and three in the meta-analysis. All studies showed high risk of bias. RESULTS: The surgery promoted significant changes in treated, adjacent, and non-adjacent sites. There were greater changes in PD (mean difference -0.14, 95 % CI -0.18 to -0.10, p < 0.00001) and CAL (mean difference 0.16, 95 % CI 0.13 to 0.20, p < 0.00001) in treated sites when compared to adjacent and non-adjacent sites for PD (mean difference -0.09, 95 % CI -0.12 to -0.05, p < 0.00001) and CAL (mean difference 0.91, 95 % CI 0.87 to 0.94, p < 0.00001). CONCLUSION: Crown lengthening surgery results in changes of clinical parameters in treated, adjacent, and non-adjacent sites. CLINICAL RELEVANCE: Clinical and esthetic alterations on the adjacent/non-adjacent teeth can lead to clinical and esthetic alterations, which must be considered in surgical planning.