Synthesis of multifunctional chlorhexidine-doped thin films for titanium-based implant materials.

Our goal was to create bio-functional chlorhexidine (CHX)-doped thin films on commercially pure titanium (cpTi) discs using the glow discharge plasma approach. Different plasma deposition times (50, 35 and 20 min) were used to create bio-functional surfaces based on silicon films with CHX that were compared to the control groups [no CHX and bulk cpTi surface (machined)]. Physico-chemical and biological characterizations included: 1. Morphology, roughness, elemental chemical composition, film thickness, contact angle and surface free energy; 2. CHX-release rate; 3. Antibacterial effect on Streptococcus sanguinis biofilms at 24, 48 and 72 h; 4. Cytotoxicity and metabolic activity using fibroblasts cell culture (NIH-F3T3 cells) at 1, 2, 3 and 4 days; 5. Protein expression by NIH-F3T3 cells at 1, 2, 3 and 4 days; and 6. Co-culture assay of fibroblasts cells and S. sanguinis to assess live and dead cells on the confocal laser scanning microscopy, mitochondrial activity (XTT), membrane leakage (LDH release), and metabolic activity (WST-1 assay) at 1, 2 and 3 days of co-incubation. Data analysis showed that silicon films, with or without CHX coated cpTi discs, increased surface wettability and free energy (p < 0.05) without affecting surface roughness. CHX release was maintained over a 22-day period and resulted in a significant inhibition of biofilm growth (p < 0.05) at 48 and 72 h of biofilm formation for 50 min and 20 min of plasma deposition time groups, respectively. In general, CHX treatment did not significantly affect NIH-F3T3 cell viability (p > 0.05), whereas cell metabolism (MTT assay) was affected by CHX, with the 35 min of plasma deposition time group displaying the lowest values as compared to bulk cpTi (p < 0.05). Moreover, data analysis showed that films, with or without CHX, significantly affected the expression profile of inflammatory cytokines, including IL-4, IL-6, IL-17, IFN-y and TNF-α by NIH-F3T3 cells (p < 0.05). Co-culture demonstrated that CHX-doped film did not affect the metabolic activity, cytotoxicity and viability of fibroblasts cells (p > 0.05). Altogether, the findings of the current study support the conclusion that silicon films added with CHX can be successfully created on titanium discs and have the potential to affect bacterial growth and inflammatory markers without affecting cell viability/proliferation rates.

TiO2 nanotubes improve physico-mechanical properties of glass ionomer cement.

OBJECTIVES: The aim of this study was to determine the physico-mechanical properties of a high viscosity glass ionomer cement (GIC) reinforced with TiO2 nanotubes (TiO2-nt). METHODS: TiO2-nt was incorporated into the GIC powder components (Ketac Molar EasyMix™) in concentrations of 0% (control group), 3%, 5%, 7% by weight. Compressive strength (n = 10/group), three point bending for flexural strength (n = 18/group), microshear bond strength to dentin and failure mode (n = 20/group), and surface roughness and weight loss before and after brushing simulation (30,000 cycles) (n = 8/group) were evaluated. Data were submitted to Shapiro-Wilk, ANOVA, Tukey and Chi-square tests (α ≤ 0.05). RESULTS: Addition of 5% of TiO2-nt into GIC presented the highest values for compressive strength and differed from the control, 3% and 7% groups (p = 0.023). There were no significant differences in flexural strength (p = 0.107) and surface roughness before and after the dental brushing (p = 0.287) among the groups. GIC added with 5% TiO2-nt showed the lowest weight loss values (p = 0.01), whereas the control, 3% or 5% TiO2-nt groups presented similar microshear bond strength values (p ≥ 0.05). The 5% TiO2-nt group featured higher microshear bond strength than the 7% TiO2-nt group (p = 0.034). Cohesive in material was the most representative failure mode for all groups. SIGNIFICANCE: The incorporation of TiO2-nt did not affect GIC's adhesiveness to dentin, but improved its compressive strength at 5%. Furthermore, TiO2-nt decreased the percentage of weight loss after GIC's surface wear.

Novel LRAP-binding partner revealing the plasminogen activation system as a regulator of cementoblast differentiation and mineral nodule formation in vitro.

Amelogenin isoforms, including full-length amelogenin (AMEL) and leucine-rich amelogenin peptide (LRAP), are major components of the enamel matrix, and are considered as signaling molecules in epithelial-mesenchymal interactions regulating tooth development and periodontal regeneration. Nevertheless, the molecular mechanisms involved are still poorly understood. The aim of the present study was to identify novel binding partners for amelogenin isoforms in the cementoblast (OCCM-30), using an affinity purification assay (GST pull-down) followed by mass spectrometry and immunoblotting. Protein-protein interaction analysis for AMEL and LRAP evidenced the plasminogen activation system (PAS) as a potential player regulating OCCM-30 response to amelogenin isoforms. For functional assays, PAS was either activated (plasmin) or inhibited (ε-aminocaproic acid [aminocaproic]) in OCCM-30 cells and the cell morphology, mineral nodule formation, and gene expression were assessed. PAS inhibition (EACA 100 mM) dramatically decreased mineral nodule formation and expression of OCCM-30 differentiation markers, including osteocalcin (Bglap), bone sialoprotein (Ibsp), osteopontin (Spp1), tissue-nonspecific alkaline phosphatase (Alpl) and collagen type I (Col1a1), and had no effect on runt-related transcription factor 2 (Runx2) and Osterix (Osx) mRNA levels. PAS activation (plasmin 5 µg/µl) significantly increased Col1a1 and decreased Bglap mRNA levels (p < .05). Together, our findings shed new light on the potential role of plasminogen signaling pathway in the control of the amelogenin isoform-mediated response in cementoblasts and provide new insights into the development of targeted therapies.

Treatment of dehiscence-type defects with collagen matrix and/or enamel matrix derivative: Histomorphometric study in minipigs.

BACKGROUND: This study aimed to evaluate, histomorphometrically, the use of collagen matrix (CM) and/or enamel matrix derivative (EMD) for the treatment of dehiscence-type recession defects in minipigs. METHODS: Eight healthy, male, young BR-1 minipigs, with no periodontal disease were treated. Bilateral dehiscence-type defects were surgically created on the buccal of the mandibular premolars (PI and PII). After 30 days, the defects were randomly assigned to four groups: coronally advanced flap (CAF); CAF + CM; CAF + EMD; and CAF + CM + EMD (split-mouth design). The evaluated parameters (mm): total defect length; new cementum (NC); new bone (NB); gingival margin position; total epithelium length; epithelium on the root; connective tissue adaptation; and soft tissue thickness (STT). RESULTS: The EMD-treated groups showed a superior length of NC [4.13 ± 1.22 (CAF + EMD); 3.95 ± 1.11 (CAF + CM + EMD); 2.94 ± 0.77 (CAF + CM); 2.72 ± 0.81 (CAF), P = 0.02] and NB [3.21 ± 0.68 (CAF + CM + EMD); 3.01 ± 0.56 (CAF + EMD); 2.15 ± 0.47 (CAF + CM); 2.29 ± 0.82 (CAF), P = 0.005]. The CAF and CAF + CM groups showed a superior epithelial length when compared to EMD-treated groups after 3 months. A superior STT was observed for CAF + CM + EMD group (1.5 ± 0.33) when compared with the other groups [1.09 ± 0.26 (CAF + EMD); 1.04 ± 0.34 (CAF + CM); and 1.14 ± 0.29 (CAF), P = 0.03]. CONCLUSION(S): The results of the present study indicate that EMD application, irrespective of the combination with CM, may improve the periodontal regeneration of dehiscence-type defects in this animal model.

Periodontal ligament-derived mesenchymal stem cells modulate neutrophil responses via paracrine mechanisms.

BACKGROUND: Mesenchymal stem cells differentiate into distinct mesenchymal cell lineages and regulate the immune response. The aim of this study was to determine whether periodontal ligament-derived mesenchymal stem cells (PDLSCs) have the ability to modulate neutrophil responses via paracrine mechanisms. METHODS: CD105-enriched PDLSCs were seeded for 24 h and challenged with Porphyromonas gingivalis total protein extract (PgPE) (0 or 2 ug/mL) for 3 h. Cells were then washed and further cultured for 18 h and the supernatants were collected and stored. Next, neutrophil-differentiated human promyelocytic leukemia HL-60 cells (HL60D) were treated with PDLSCs supernatants and HL-60D activation and functional responses were determined. RESULTS: PgPE treatment induced higher secretion of inflammatory markers and chemokines by PDLSCs, including RANTES, eotaxin, interferon (IFN)-γ- inducible protein 10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), IFN-γ, interleukin (IL)-6, IL-8 and IL-1ra (P < 0.05). HL-60D recruitment rate was increased by 4.7 ± 1.09-fold when exposed to PgPE-treated PDLSCs supernatants. PgPE-treated PDLSCs supernatants promoted a 1.78 ± 1.04-fold increase in the production of intracellular reactive oxygen species (ROS) by PMA-stimulated HL-60D, whereas PgPE-untreated PDLSCs supernatants led to a 16% reduction in intracellular ROS. In sharp contrast, neither PgPE-untreated nor PgPE-treated PDLSCs supernatants altered tumor necrosis factor (TNF)-α and IL-1ß secretion by HL-60D cells. CONCLUSION: Together, these findings suggest an important role of PDLSCs in the recognition of P. gingivalis, paracrine recruitment and activation of antimicrobial mechanisms in innate immune cells, without interfering in cytokine responses.

Does enamel matrix derivative application improve clinical outcomes after semilunar flap surgery? A randomized clinical trial.

OBJECTIVES: To evaluate the treatment of gingival recessions by semilunar coronally positioned flap plus enamel matrix derivative (SCPF + EMD). MATERIALS AND METHODS: Thirty patients with class I localized gingival recession were included. They were randomly allocated in two groups: SCPF + EMD and SCPF. Recession height (RH), recession width (RW), width of keratinized tissue (WKT), thickness of keratinized tissue (TKT), probing depth (PD), and clinical attachment level (CAL) were measured at baseline, 6 and 12 months post-surgery. Patient/professional evaluation of esthetics and root sensitivity was performed. RESULTS: After 12 months, mean root coverage was 1.98 ± 0.33 mm for SCPF + EMD (90.86 ± 14.69%) and 1.85 ± 0.41 mm (79.76 ± 17.44%) for SCPF (p > 0.05). The esthetic evaluation by the patient showed preference for SCPF + EMD. According to the professional evaluation (QCE), the use of EMD decreases the appearance of postoperative scar tissue line. There was a significant reduction in root hypersensitivity with no further complaints by the patients. CONCLUSIONS: The addition of EMD provides significantly better esthetics to SCPF, according to patient and professional assessments. SCPF + EMD is effective but not superior to SCPF for root coverage, after 12 months. CLINICAL RELEVANCE: Previous clinical trials showed that the combination of EMD with coronally advanced flaps may enhance the outcome of root coverage. There is a lack of studies testing the combination of EMD with SCPF. The combination SCPF + EMD provides better esthetics when compared to the SCPF and is effective, but not superior, to SCPF for root coverage, after 12 months. TRIAL REGISTRATION: NCT02459704.

Transcriptome of healthy gingival tissue from edentulous sites in patients with a history of generalized aggressive periodontitis.

BACKGROUND: This study evaluates the transcriptome of healthy gingival tissue from edentulous sites in patients with a history of generalized aggressive periodontitis (GAgP), chronic periodontitis (CP), and in patients with no history of periodontitis (H), using microarray and quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. METHODS: Healthy gingival tissue from edentulous sites was taken from patients from the GAgP (n = 12), CP (n = 12), and H (n = 12) groups. Initially, total RNA from four tissue samples per group was used in transcriptomic microarray analysis. Differential gene expression (fold-change), gene ontology (GO; biologic process), and pathway analyses were performed. Genes that were differentially expressed and showing a significant role on altered pathways were validated by qRT-PCR analysis on 12 samples per group. RESULTS: In total, 270 probe sets and 50 GO groups were differentially expressed (upregulated or downregulated) between GAgP and H. Natural killer cell receptors and other genes related to the immune system were upregulated in GAgP, whereas genes with functions in neural processes and in proliferation/differentiation of keratinocytes were underexpressed. There were 220 probe sets and 75 GO groups that were differentially expressed when comparing CP and GAgP. CP was characterized by increased expression of genes related to responses to external stimuli and an underexpression of immune system-related genes. qRT-PCR analysis confirmed microarray results, that killer cell immunoglobulin (Ig)-like receptor, two Ig domains and long cytoplasmic tail 4; interleukin-6; and selectin E were more highly expressed in patients from the GAgP group compared with the CP and H groups. CONCLUSION: This study demonstrates differences in the transcriptome of healthy gingival tissue from edentulous sites in patients with GAgP compared with patients from H or CP groups.

Computational analysis for GNAQ mutations: New insights on the molecular etiology of Sturge-Weber syndrome.

Somatic activating mutations in the GNAQ have been recently associated with several congenital genetic disorders and tumors; however, the molecular mechanism/etiology that leads to GNAQ somatic mosaic mutation are unknown. Here, we reported a case of Sturge-Weber Syndrome (SWS) manifesting cutaneous vascular malformations (hemifacial Port-wine stain), cerebral and ocular vascular abnormalities (including epilepsy and glaucoma) and harboring a c.548G>A (p.R183Q) somatic mosaic mutation in GNAQ. Computational modeling studies were performed to assistant with the comprehension of the functional impact of p.R183Q and p.Q209L mutations in GNAQ, which encodes a G protein subunit alpha q (Gαq). The p.R183Q mutation was predicted to abolish hydrogen bonds between R183 residue and GDP molecule, destabilizing the inactive GDP-bound conformation of the Gαq mutants. Furthermore, replacement of R183 by Q183 residue was predicted to promote conformation changes in protein surface features affecting the switch I region, a key region that undergoes conformational changes triggered by receptor binding during signal transduction. In addition, replacement of Q209 by L209 residue was predicted to affect the molecular interaction between Gαq and Gß subunit, impairing formation of the inactive heterotrimeric complex. These findings, in association with PPI network analysis, indicate that p.R183Q and p.Q209L mutations result in the over-activation of different downstream effectors, which in turn will determine the distinct cell responses and phenotype. These findings bring new insights on molecular etiology of vascular malformations associated to SWS and on different mechanisms underlying hyperactivation of downstream pathways to Gαq.

Xenogenous Collagen Matrix and/or Enamel Matrix Derivative for Treatment of Localized Gingival Recessions: A Randomized Clinical Trial. Part II: Patient-Reported Outcomes.

BACKGROUND: Gingival recession (GR) might be associated with patient discomfort due to cervical dentin hypersensitivity (CDH) and esthetic dissatisfaction. The aim is to evaluate the effect of root coverage procedure with a xenogenous collagen matrix (CM) and/or enamel matrix derivative (EMD) in combination with a coronally advanced flap (CAF) on CDH, esthetics, and oral health-related quality of life (OHRQoL) of patients with GR. METHODS: Sixty-eight participants with single Miller Class I/II GRs were treated with CAF (n = 17), CAF + CM (n = 17), CAF + EMD (n = 17), and CAF + CM + EMD (n = 17). CDH was assessed by evaporative stimuli using a visual analog scale (VAS) and a Schiff scale. Esthetics outcome was assessed with VAS and the Questionnaire of Oral Esthetic Satisfaction. Oral Health Impact Profile-14 (OHIP-14) questionnaire was used to assess OHRQoL. All parameters were evaluated at baseline and after 6 months. RESULTS: Intragroup analysis showed statistically significant reduction in CDH and esthetic dissatisfaction with no intergroup significant differences (P >0.05). The impact of oral health on QoL after 6 months was significant for CAF + CM, CAF + EMD, and CAF + CM + EMD (P <0.05). Total OHIP-14 score and psychologic discomfort, psychologic disability, social disability, and handicap dimensions showed negative correlation with esthetics. OHIP-14 physical pain dimension had positive correlation with CDH (P <0.05). OHIP-14 showed no correlation with percentage of root coverage, keratinized tissue width, or keratinized tissue thickness (P >0.05). CONCLUSION: Root coverage procedures improve patient OHRQoL by impacting on a wide range of dimensions, perceived after reduction of CDH and esthetic dissatisfaction of patients with GRs treated with CAF + CM, CAF + EMD, and CAF + CM + EMD.

Viability and Osteogenic Differentiation of Human Periodontal Ligament Progenitor Cells Are Maintained After Incubation With Porphyromonas gingivalis Protein Extract.

BACKGROUND: Porphyromonas gingivalis (Pg) is a major periodontal pathogen that contains immunostimulatory components. Periodontal ligament mesenchymal stem cells (PDLMSCs) are responsible for regeneration of the periodontium that is lost due to periodontitis. Pathologic factors within the microenvironment that impair resident PDLMSCs are not well understood. The present study investigates in vitro the effects of Pg protein extract (PgPE) on biologic properties of CD105-enriched PDL progenitor cell populations (PDL-CD105+). METHODS: Five populations of PDL-CD105+ cells were exposed to PgPE and assessed for cell viability, apoptosis, and proinflammatory gene expression (interleukin-1ß [IL-1ß], tumor necrosis factor-alpha [TNF-α], and IL-6) by quantitative reverse transcription polymerase chain reaction, IL-6 immunostaining, activation of IL-6/signal transducer and activator of transcription (STAT) 3 signaling pathway, and osteogenic differentiation potential. RESULTS: PgPE treatment (2 µg/mL) did not affect cell viability or survival but induced a significant increase in IL-1ß, TNF-α, and IL-6 messenger RNA (mRNA) expression and positive staining for IL-6. A total of 29 genes from the IL-6/STAT3 pathway were upregulated on PgPE stimulation. These genes are related to biologic processes involved in the control of cell survival (B-cell lymphoma 2 [BCL2]), cell proliferation (hepatocytehepatocyte growth factor), cytokine-mediated signaling pathway (suppressor of cytokine signaling 3, C-X-C ligand 8 [CXCL8]), and response to stress (CXCL8, mitogen-activated protein kinase 3, BCL2-associated X protein, and BCL2). Additionally, PgPE treatment caused an increase in alkaline phosphatase mRNA expression in PDL-CD105+ cells after 7 days of osteogenic induction, although mineral nodule formation was comparable to the control group. CONCLUSIONS: These results suggest that the inflammatory profile induced by PgPE treatment in PDL-CD105+ cells did not affect cell viability, apoptosis, or osteogenic differentiation, perhaps due to increased expression of genes involved in the control of cell proliferation and protection against cell death.

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

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-1ß, 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-1ß 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.

Leucine-Rich Amelogenin Peptide (LRAP) Uptake by Cementoblast Requires Flotillin-1 Mediated Endocytosis.

Basic, pre-clinical, and clinical studies have documented the potential of amelogenin, and its variants, to affect cell response and tissue regeneration. However, the mechanisms are unclear. Thus, the aim of the present study was to identify, in cementoblasts, novel binding partners for an alternatively spliced amelogenin form (Leucine-Rich Amelogenin Peptide-LRAP), which is supposed to act as a signaling molecule in epithelial-mesenchymal interactions. LRAP-binding protein complexes from immortalized murine cementoblasts (OCCM-30) were achieved by capture affinity assay (GST pull down) and proteins present in these complexes were identified by mass spectrometry and immunoblotting. Flotillin-1, which functions as a platform for signal transduction, vesicle trafficking, endocytosis, and exocytosis, was identified and confirmed by co-precipitation and co-localization assays as a protein-binding partner for LRAP in OCCM-30 cells. In addition, we found that exogenously added GST-LRAP recombinant protein was internalized by OCCM-30 cells, predominantly localized in the perinuclear region and, that inhibition of flotillin1-dependent functions by small interference RNA (siRNA) methodology significantly affected LRAP uptake and its biological properties on OCCM-30 cells, including LRAP effect on the expression of genes encoding osteocalcin (Ocn), bone sialoprotein (Bsp), and runt-related transcription factor 2 (RunX2). In conclusion, LRAP uptake by cementoblast involves flotillin-assisted endocytosis, which suggests an involvement of LRAP in lipid-raft-dependent signaling pathways which are mediated by flotillin-1. J. Cell. Physiol. 232: 556-565, 2017. © 2016 Wiley Periodicals, Inc.

Full-mouth ultrasonic debridement associated with povidone iodine rinsing in GAgP treatment: a randomised clinical trial.

OBJECTIVE: This study evaluated the clinical, immunological and microbiological results of full-mouth ultrasonic debridement (FMUD) with 10 % povidone iodine (PVPI) as the cooling liquid in the treatment of generalised aggressive periodontitis (GAgP). MATERIAL AND METHODS: Twenty-eight patients presenting GAgP were randomly assigned to one of the following groups for evaluation: FMUD + SS (n = 14)--single session of FMUD with 0.9 % saline solution as cooling agent and FMUD + PVPI (n = 14)--single session of FMUD with PVPI solution as cooling agent. Probing depth (PD), relative clinical attachment level (RCAL), relative position of gingival margin, plaque index (FMPI) and bleeding score (FMBS), immunological (interleukin-10 and interleukin-1ß concentrations in gingival crevicular fluid) and microbiological (Aa and Pg amounts) parameters were evaluated at baseline, first, third and sixth months after treatment. RESULTS: The two groups presented reduction of FMPI and FMBS and had statistically significant PD reductions, RCAL gains and gingival recession (p < 0.05). Both therapies reduced Pg levels in deep and in moderate pockets (p < 0.05). FMUD + PVPI reduced Aa levels in deep pockets. However, no inter-group differences in clinical, immunological and microbiological parameters were observed (p > 0.05). CONCLUSIONS: It could be concluded that 10 % PVPI used as an irrigant solution in FMUD decreased Aa levels in deep pockets but had no additional benefits when compared with saline solution irrigation in terms of clinical, microbiological and immunological results. CLINICAL RELEVANCE: The FMUD is a valid option for the treatment of GAgP, but the use of 10 % PVPI did not improve the results of the periodontal therapy.

Penetration of Filled and Unfilled Resin Sealants on Different Enamel Substrates.

PURPOSE: To evaluate the penetration ratio of filled and unfilled resin-based sealants on different enamel substrates and pit and fissure morphologies. METHODS: Forty-eight occlusal enamel blocks obtained from impacted human third molars were randomly divided (n equals eight) according to enamel substrates (sound; caries-like lesion; caries-like lesion plus topical fluoride application) and sealant material (FluroShield; Helioseal Clear Chroma). Sealants were applied on the enamel surface. The specimens were stored in 100 percent humidity for 24 hours at 37 degrees Celsius, sectioned in a buccal-lingual direction (at approximately 50 µm), and examined to determine the sealant penetration ratio (b x 100/a; a equals total fissure length and b equals sealant penetration length) and pit and fissure morphology (V-, U-, or Y-shaped). Statistical analysis was performed using Friedman and Kruskal-Wallis tests (P<0.05). RESULTS: Enamel substrate and sealant material did not affect the sealant penetration ratio, with no interactions between these factors. Moreover, the morphology significantly affected the sealant penetration, with the "Y"-shaped fissures presenting the lowest sealant penetration ratio compared to "U-"shaped (P=0.0001) and "V-" shaped fissures (P=0.0018). CONCLUSIONS: Pit and fissure morphology was a critical factor on sealant's penetration capacity; however, enamel substrate and sealant type did not affect sealant's penetration ratio.

Periodontal clinical and microbiological characteristics in healthy versus generalized aggressive periodontitis families.

AIM: Generalized aggressive periodontitis (GAP) is a severe and multifactorial disease in which a familial aggregation and a specific microbiological profile have been suggested. Thus, this case-control study evaluated the clinical and subgingival microbial profile of GAP subjects and their families compared to healthy families. METHODS: Fifteen families with parents presenting periodontal health and 15 with parents with a history of GAP were selected. Each family should have at least one child between 6 and 12 years old. Plaque index (PI), gingival index (GI), and periodontal probing depth (PPD), as well as Porphyromonas gingivalis, Tannerella forsythia, and Aggregatibacter actinomycetemcomitans (Aa) amounts (by qPCR), were assessed from all subjects. RESULTS: Children of GAP families showed a higher PI, GI, and PPD when compared to children of healthy families (p ≤ 0.05). A higher frequency of detection and amounts of Aa was observed in GAP children compared to children of healthy families (p ≤ 0.05). Moreover, a significant association between Aa amounts and gingival bleeding was observed in children (p ≤ 0.05, r = 0.37). CONCLUSION: Children from GAP families have worst clinical conditions, i.e. higher levels of PI, GI, and PPD, a more pathogenic microbiological profile, and the amount of Aa are associated with a higher marginal inflammation.

Exposure of periodontal ligament progenitor cells to lipopolysaccharide from Escherichia coli changes osteoblast differentiation pattern.

UNLABELLED: Periodontal ligament mesenchymal stem cells (PDLMSCs) are an important alternative source of adult stem cells and may be applied for periodontal tissue regeneration, neuroregenerative medicine, and heart valve tissue engineering. However, little is known about the impact of bacterial toxins on the biological properties of PDLSMSCs, including self-renewal, differentiation, and synthesis of extracellular matrix. OBJECTIVE: This study investigated whether proliferation, expression of pro-inflammatory cytokines, and osteogenic differentiation of CD105-enriched PDL progenitor cell populations (PDL-CD105(+) cells) would be affected by exposure to bacterial lipopolysaccharide from Escherichia coli (EcLPS). MATERIAL AND METHODS: Toll-like receptor 4 (TLR4) expression was assessed in PDL-CD105(+) cells by the immunostaining technique and confirmed using Western blotting assay. Afterwards, these cells were exposed to EcLPS, and the following assays were carried out: (i) cell viability using MTS; (ii) expression of the interleukin-1 beta (IL-1ß), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor alpha (TNF-α) genes; (iii) osteoblast differentiation assessed by mineralization in vitro, and by mRNA levels of run-related transcription factor-2 (RUNX2), alkaline phosphatase (ALP) and osteocalcin (OCN) determined by quantitative PCR. RESULTS: PDL-CD105+ cells were identified as positive for TLR4. EcLPS did not affect cell viability, but induced a significant increase of transcripts for IL-6 and IL-8. Under osteogenic condition, PDL-CD105+ cells exposed to EcLPS presented an increase of mineralized matrix deposition and higher RUNX2 and ALP mRNA levels when compared to the control group. CONCLUSIONS: These results provide evidence that CD105-enriched PDL progenitor cells are able to adapt to continuous Escherichia coli endotoxin challenge, leading to an upregulation of osteogenic activities.

Tooth-derived stem cells: Update and perspectives.

Tissue engineering is an emerging field of science that focuses on creating suitable conditions for the regeneration of tissues. The basic components for tissue engineering involve an interactive triad of scaffolds, signaling molecules, and cells. In this context, stem cells (SCs) present the characteristics of self-renewal and differentiation capacity, which make them promising candidates for tissue engineering. Although they present some common markers, such as cluster of differentiation (CD)105, CD146 and STRO-1, SCs derived from various tissues have different patterns in relation to proliferation, clonogenicity, and differentiation abilities in vitro and in vivo. Tooth-derived tissues have been proposed as an accessible source to obtain SCs with limited morbidity, and various tooth-derived SCs (TDSCs) have been isolated and characterized, such as dental pulp SCs, SCs from human exfoliated deciduous teeth, periodontal ligament SCs, dental follicle progenitor cells, SCs from apical papilla, and periodontal ligament of deciduous teeth SCs. However, heterogeneity among these populations has been observed, and the best method to select the most appropriate TDSCs for regeneration approaches has not yet been established. The objective of this review is to outline the current knowledge concerning the various types of TDSCs, and discuss the perspectives for their use in regenerative approaches.

Skin fibroblasts from individuals with Chediak-Higashi Syndrome (CHS) exhibit hyposensitive immunogenic response.

BACKGROUND: Chediak-Higashi Syndrome (CHS) is a rare autosomal recessive disease characterized by immunodeficiency, oculocutaneous albinism, neurological dysfunction, and early death. Individuals with CHS present with increased susceptibility to infections of the skin, upper-respiratory tract, gastrointestinal tract, and oral tissues. Classical CHS is caused by mutations in the gene encoding lysosomal trafficking regulator (LYST). Although defects in cytotoxic T cell lytic secretory granule secretion and neutrophil phagocytosis are suggested to contribute to the immunodeficiency in CHS, the underlying molecular mechanisms are unknown. We hypothesized that skin fibroblasts from CHS subjects exhibit impaired immune response due to defective trafficking of inflammatory factors. METHODS AND RESULTS: Primary skin fibroblasts from CHS subjects or healthy controls were assessed for genes encoding inflammatory response factors using PCR array. At baseline, we found CD14, IL1R1 and TLR-1 were down-regulated significantly (≥2 fold change) and the genes encoding TLR-3, IL-1ß and IL-6 were up-regulated in CHS cells compared to control cells. When challenged with E. coli lipopolysaccharide (LPS), CHS cells were less responsive than control cells, with only 8 genes significantly up-regulated (3-68 fold change) compared to baseline values, whereas 28 genes in control cells were significantly up-regulated at a much higher magnitude (3-4,629 fold change). In addition, 50% of the genes significantly up-regulated in LPS-treated control cells were significantly lower in LPS-treated CHS cells. IL-6, a fibroblast-derived proinflammatory cytokine essential for fighting infections was significantly lower in culture media of CHS cells with or without LPS. Furthermore, Western blot and immunofluorescent staining revealed that TLR-2 and TLR-4 were diminished on cell membranes of CHS cells and dissociated from Rab11a. CONCLUSIONS: For the first time, results from our study indicate defective trafficking of TLR-2 and TLR-4 contributes to the hyposensitive response of CHS skin fibroblasts to immunogenic challenge, providing a potential therapeutic target for clinical intervention in CHS.

Salivary carriage of periodontal pathogens in generalized aggressive periodontitis families.

BACKGROUND: Generalized aggressive periodontitis (GAP) is a multifactorial disease that shows a specific microbial profile and a familial aggregation. AIM: This study evaluated the salivary microbial profile of families with a history of GAP and compared them with healthy families. DESIGN: Fifteen families with parents presenting periodontal health and 15 with parents with a history of GAP were selected. Each family had a child aged 6-12 years. Stimulated saliva was collected from all subjects, and Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf), and Aggregatibacter actinomycetemcomitans (Aa) amounts were determined. RESULTS: Children of GAP families showed higher detection of Aa (90%) than children of healthy families (45%) (P < 0.05). Parents with GAP showed a Pg salivary concentration statistically higher than that of healthy parents (P < 0.05).Children of GAP families, however, exhibited similar Pg concentration than healthy children (P > 0.05). Tf amounts did not differ either in parents or in children (P > 0.05) The infection risk calculation indicates that children who have one parent who is positive for Aa have 16.3 times (95% CI 3.1-87.2) more risk of being infected with Aa (P < 0.05) than children from an Aa-negative family. CONCLUSION: It may be concluded that children of parents with aggressive periodontitis have higher levels and higher risk of Aa infection.

Intermittent parathyroid hormone administration improves periodontal healing in rats.

BACKGROUND: Intermittent administration of parathyroid hormone (PTH) promotes new bone formation in patients with osteoporosis and bone fractures. It was shown previously that PTH also reduces periodontitis-related bone loss. The aim of this study is to evaluate the effect of treatment with PTH on periodontal healing in rats. METHODS: Fenestration defects were created at the buccal surface of the distal root of the mandibular first molars, and both periodontal ligament (PDL) and cementum were removed. Animals were then assigned to two groups (eight animals per group): group 1: control, placebo administration; and group 2: test, human PTH (hPTH) 1-34 administration at a concentration of 40 µg/kg. For both groups, the animals were injected every 2 days, and the animals were sacrificed at 14 and 21 days after surgery. Specimens were harvested and processed for routine decalcified histologic sections. The following parameters were assessed: 1) remaining bone defect extension (RBDE); 2) newly formed bone density (NFBD); 3) total callus area (TCA); 4) osteoclast number (ON) in the callus region; and 5) newly formed dental cementum-like tissue (NFC). Birefringence of root PDL reattachment was also evaluated. RESULTS: Birefringence analysis showed root PDL reattachment for both groups 21 days after treatment. Intermittent hPTH 1-34 administration decreased RBDE (P <0.01) and increased NFBD (P <0.01), TCA (P <0.01), area of NFC (P <0.01), and ON in the callus region (P <0.01). CONCLUSION: Within the limits of the present study, intermittent administration of hPTH 1-34 led to an enhanced periodontal healing process compared with non-treated animals.