BMP-2 and asporin expression regulate 5-aza-dC-mediated osteoblast/cementoblast differentiation of periodontal dental ligament mesenchymal progenitor cells.

Periodontal dental ligament (PDL) is composed of heterogeneous population of mesenchymal progenitor cells. The mechanisms that regulate the differentiation of these cells towards osteoblast/cementoblast phenotype are not fully understood. Some studies have demonstrated that is possible to change the pattern of cell differentiation via epigenetic mechanisms. The proposal of this study was to investigate whether 5-aza-2'-deoxycytidine (5-aza-dC) treatment would stimulate the osteoblast/cementoblast differentiation of periodontal ligament mesenchymal progenitor cells (PDL-CD105+ enriched cells), characterized as low osteoblast potential, through bone morphogenetic protein-2 (BMP-2) modulation. PDL-CD105+ cells from a single donor were cloned and characterized in two populations as high osteoblast/cementoblast potential (HOP) and low osteoblast/cementoblast potential (LOP) by mineralization in vitro and expression of osteogenic gene markers, such as runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteocalcin (OCN), bone morphogenetic protein 2 (BMP-2) and asporin (ASPN). Next, two LOP clones (L1 and L2) were pretreated with 5-aza-dC (10 µM) for 48 h, cultured under osteogenic condition and evaluated for mineralized matrix in vitro, transcription modulation of osteogenic gene markers, methylated and hydroxymethylated DNA levels of BMP-2 and ASPN and intracellular/extracellular expression of BMP-2 protein. LOP clones showed high expression of ASPN transcripts associated with low mRNA levels of BMP-2, RUNX2, ALP, and OCN. 5-aza-dC treatment raised hydroxymethylated DNA levels of BMP-2 and increased the expression of BMP-2 transcripts in both LOP clones. However, BMP-2 protein (intracellular and secreted forms) was detected only in L1 cell clones, in which it was observed an increased expression of osteoblast/cementoblast markers (RUNX2, ALP, OCN) associated with higher mineralization in vitro. In L2 cell clones, 5-aza-dC increased gene expression of ASPN, with no great change in for osteoblast/cementoblast differentiation potential. These data show that 5-aza-dC improves osteoblast/cementoblast differentiation of PDL-CD105+ cells via BMP-2 secretion, and this effect depends on low levels of ASPN expression.

Local IL-10 level as a predictive factor in generalized aggressive periodontitis treatment response.

Generalized aggressive periodontitis (GAgP) presents a reduced response to non-surgical therapy. However, it is not clear if the initial clinical, microbiological or immunological characteristics are impacting the worse response to treatment. This study aimed to identify the predictive value of clinical, microbiological and immunological patterns on the clinical response to therapy in GAgP patients. Twenty-four GAgP patients were selected, and gingival crevicular fluid (GCF) and subgingival biofilm were collected. Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythia levels were evaluated by qPCR, and IL-1ß and IL-10 concentration by ELISA. Twelve patients were treated with SRP (scaling and root planning), and twelve with SRP plus 375 mg amoxicillin and 250 mg metronidazole (8/8 hours, 7 days) (SRP + AM). The clinical changes (Probing Pocket Depth [PPD] reduction and Clinical Attachment Level [CAL] gain) 6 months post-treatment were correlated to the initial clinical, inflammatory and microbiological variables using stepwise logistic regression (α = 5%). CAL gain at 6 months was 1.16 ± 0.77 for SRP and 1.74 ± 0.57 mm for SRP + AM (P > .05). PPD reduction was 1.96 ± 0.82 for SRP and 2.45 ± 0.77 mm for SRP + AM (P < .05). In the SRP group, IL-10 showed a predictive value for clinical response. The higher the IL-10 concentration at baseline, the higher the reduction in PPD at 6 months (P = .01, r = .68). However, when antimicrobials were administered, no significant influence was detected (P > .05). It can be concluded that the IL-10 levels in GFC act as a predictor of clinical response to GAgP. Moreover, the intake of antimicrobials appears to overlap the influence of the inflammatory response on clinical response to treatment. Clinical trial registration number: NCT03933501.

Experimental and clinical studies on regenerative periodontal therapy.

The recognition of a periodontal therapy as a regenerative procedure requires the demonstration of new cementum, periodontal ligament, and bone coronal to the base of the defect. A diversity of regenerative strategies has been evaluated, including root surface conditioning, bone grafts and bone substitute materials, guided tissue regeneration, enamel matrix proteins, growth/differentiation factors, combined therapies and, more recently, tissue-engineering approaches. The aim of this chapter of Periodontology 2000 is to review the research carried out in Latin America in the field of periodontal regeneration, focusing mainly on studies using preclinical models (animal models) and randomized controlled clinical trials. This review may help clinicians and researchers to evaluate the current status of the therapies available and to discuss the challenges that must be faced in order to achieve predictable periodontal regeneration in clinical practice.

Evaluation of peri-implant marginal tissues around tissue-level and bone-level implants in patients with a history of chronic periodontitis.

OBJECTIVE: To evaluate clinical and radiographic characteristics in peri-implant marginal tissues in patients with a history of chronic periodontitis, rehabilitated using tissue-level or bone-level implants. MATERIAL AND METHODS: Using a split-mouth design, 20 patients with a history of chronic periodontitis were selected and received two different implants, tissue-level group (n = 20) and the bone-level group (n = 20). Peri-implant probing depth, relative peri-implant mucosal margin position, relative peri-implant clinical attachment level, peri-implant plaque index and peri-implant bleeding on probing were evaluated at prosthesis installation, 1, 3, 6, 12 and 24 months after implant loading. Radiographic marginal bone level was evaluated at implant insertion, prosthesis installation, 6 and 24 months after implant loading. RESULTS: The mean difference of peri-implant marginal bone resorption from implant installation to 24 months in function was 0.75 ± 1.12 mm for the tissue-level group and 0.70 ± 0.72 mm for the bone-level group. No statistically significant difference was found between groups at all assessment periods for clinical and radiographic peri-implant evaluation. CONCLUSION: Under a rigid supportive therapy, both approaches performed likewise regarding clinical and radiographic parameters for rehabilitation of patients with a history of chronic periodontitis.

Enamel matrix protein derivative and/or synthetic bone substitute for the treatment of mandibular class II buccal furcation defects. A 12-month randomized clinical trial.

OBJECTIVE: This study aims to clinically evaluate the treatment of mandibular class II furcation defects with enamel matrix derivative (EMD) and/or a bone substitute graft made of ß-tricalcium phosphate/hydroxyapatite (ßTCP/HA). MATERIALS AND METHODS: Forty-one patients, presenting a mandibular class II buccal furcation defect, probing pocket depth (PPD) ≥4 mm and bleeding on probing, were included. They were randomly assigned to the groups: 1-EMD (n = 13); 2-ßTCP/HA (n = 14); 3-EMD + ßTCP/HA (n = 14). Plaque index (PI), gingival index (GI), relative gingival margin position (RGMP), relative vertical and horizontal attachment level (RVCAL and RHCAL), and PPD were evaluated at baseline and 6 and 12 months. The mean horizontal clinical attachment level gain was considered the primary outcome variable. RESULTS: No significant intragroup differences were observed for RGMP, but significant changes were observed for RVCAL, RHCAL, and PPD for all groups (p < 0.05). After 12 months, the mean horizontal clinical attachment level gain was 2.77 ± 0.93 mm for EMD, 2.64 ± 0.93 mm for ßTCP/HA, and 2.93 ± 0.83 mm for EMD + ßTCP/HA, with no significant differences among the groups. At the end of the study, 85.3 % of the sites were partially closed; however, no complete closure was observed. CONCLUSION: EMD + ßTCP/HA does not provide a significant advantage when compared to the isolated approaches. All three tested treatments promote significant improvements and partial closure of class II buccal furcation defects. Based on its potential to induce periodontal regeneration, EMD may be considered an attractive option for this type of defect, but complete closure remains an unrealistic goal. CLINICAL RELEVANCE: The partial closure of buccal furcation defects can be achieved after the three tested approaches. However, the combined treatment does not provide a significant benefit when compared to the isolated approaches.

DNA methylation analysis of SOCS1, SOCS3, and LINE-1 in microdissected gingival tissue.

OBJECTIVES: DNA methylation plays a critical role in the regulation of the transcription of the suppressors of cytokine signaling (SOCS) 1 and SOCS3, which are modulators in the inflammation. We hypothesized that the methylation status of SOCS1, SOCS3, and long interspersed nuclear element (LINE)-1 in gingival tissues previously inflamed would be similar to that found in gingival tissues without clinical inflammation in the period studied. MATERIALS AND METHODS: Laser capture microdissection was performed to isolate epithelial and connective gingival tissues. The groups were comprised by ten patients without history of periodontitis and absence of clinical signs of inflammation in the gingiva during the study (healthy group) and ten patients with history of periodontitis, presenting inflammation in the gingival tissue at the first examination of the study (controlled chronic periodontitis group). The gingival biopsies from the controlled chronic periodontitis group were collected after controlling the inflammation. DNA methylation patterns were analyzed using methylation-specific high-resolution melting and combined bisulfite restriction analysis. RESULTS: DNA methylation levels for SOCS1 and SOCS3 did not differ between groups or tissues; likewise, no differences were observed in total LINE-1 methylation or at specific loci. CONCLUSION: At 3 months following control of inflammation in gingival tissues, the methylation profile of SOCS1, SOCS3, and LINE-1 is similar between connective and epithelial tissues from patients that were previously affected or not by chronic inflammation. CLINICAL RELEVANCE: Clinical results of a successful treatment are observed after inflammation control and the molecular findings illustrate local and general methylation patterns in recovering tissues toward health conditions and might help to understand events that are occurring in oral cells.

Photodynamic therapy in the treatment of class II furcation: a randomized controlled clinical trial.

AIM: To investigate the effect of photodynamic therapy (PDT) as adjunct to mechanical therapy in furcations. MATERIALS AND METHODS: A double-blind, parallel, randomized controlled clinical trial was conducted in subjects presenting class II furcations. The subjects were randomly allocated to a test (PDT; n = 16) or control group (non-activated laser/only photosensitizer; n = 21). At baseline, 3 and 6 months, clinical, microbiological and cytokine pattern evaluation was performed. Clinical attachment level was defined as the primary outcome variable. RESULTS: Clinical parameters improved after both therapies (p < 0.05) with no differences between groups at any time point (p > 0.05). At 6 months, real-time PCR evaluation showed a decrease in Porphyromonas gingivalis and Tannerella forsythia only in the PDT group (p < 0.05) with no inter-group differences. Regarding cytokines, IL-4 and IL-10 levels increased in both groups at 6 months. GM-CSF, IL-8, IL-1ß and IL-6 levels decreased only in the PDT group after 3 months (p < 0.05). At 3 months, inter-group analyses showed that GM-CSF, IFN-γ, IL-6 and IL-8 levels were lower in the PDT group. At 6 months, lower IL-1ß levels were also observed in the PDT group (p < 0.05). CONCLUSION: Photodynamic therapy did not promote clinical benefits for class II furcations; however, advantages in local levels of cytokines and a reduction in periodontopathogens were demonstrated.

Hydroxyapatite/ß-tricalcium phosphate and enamel matrix derivative for treatment of proximal class II furcation defects: a randomized clinical trial.

OBJECTIVE: To clinically evaluate proximal furcations treated with hydroxyapatite/ß-tricalcium phosphate (HA/ß-TCP) isolated or combined with enamel matrix derivative (EMD). MATERIAL AND METHODS: Thirty patients, presenting at least one proximal class II furcation defect, probing pocket depth (PPD) ≥5 mm and bleeding on probing, were included. The defects were assigned to the HA/ß-TCP group (n = 15); open-flap debridement (OFD) + HA/ß-TCP filling, or, HA/ß-TCP-EMD group (n = 15); OFD + HA/ß-TCP + EMD filling. Plaque (PI) and gingival index (GI), PPD, relative gingival margin position (RGMP), vertical and horizontal attachment level (RVAL and RHAL), vertical and horizontal bone level (RVBL and RHBL), and furcation diagnosis were evaluated at baseline and at 6 months. RESULTS: Both groups presented improvements after therapies (p < 0.05); however, no inter-group differences could be seen in any single parameter (p > 0.05). At 6 months, the gains in rVCAL in the HA/ß-TCP and HA/ß-TCP-EMD groups were 1.47 ± 0.99 and 2.10 ± 0.87 mm, while the RHCAL gains were 1.47 ± 1.46 and 1.57 ± 1.58 mm (p > 0.05). The RVBL and RHBL gains for the HA/ß-TCP and HA/ß-TCP-EMD group were 1.47 ± 1.13 and 1.70 ± 1.26 mm, and 1.90 ± 1.11 and 1.70 ± 1.37 mm respectively (p > 0.05). The HA/ß-TCP-EMD group showed seven closed furcations versus four in the HA/ß-TCP group (p > 0.05). CONCLUSION: Both treatments lead to improvements in all clinical variables studied in the present trial. However, the closure of proximal class II furcation defects is still unpredictable.

Clinical and microbiological changes after minimally invasive therapeutic approaches in intrabony defects: a 12-month follow-up.

OBJECTIVES: This 12-month randomized, controlled trial evaluated the clinical effects and microbiological changes of minimally invasive nonsurgical and surgical approaches for the therapy of intrabony defects. MATERIALS AND METHODS: Twenty-nine subjects with intrabony defects in single-rooted tooth were randomly assigned to; (1) minimally invasive nonsurgical technique (MINST) or (2) minimally invasive surgical technique (MIST). Quantities of Aggregatibacter actinomycetemcomitans, Tannerella forsythia, and Porphyromonas gingivalis, determined by using real-time PCR, were evaluated at baseline, 3, 6, and 12 months after the treatments. Clinical recordings--probing depth (PD), position of the gingival margin (PGM), and relative clinical attachment level (RCAL)--were obtained at baseline and 12 months post-therapy. The primary outcome variable of the study was RCAL. RESULTS: Both treatment modalities resulted in an improvement in all clinical recordings, with significant PD reductions (p < 0.05), RCAL gains (p < 0.05), and no change in the PGM (p > 0.05) after 12 months in both MINST and MIST groups. No clinical differences were observed between groups (p > 0.05). Regarding the microbiological outcomes, at the re-examinations, a significant decrease was observed for T. forsythia and P. gingivalis when compared with baseline (p < 0.05) for both treatments. The amount of A. actinomycetemcomitans did not reduced decrease throughout the study (p > 0.05). Intergroup differences in the microbiological assay were not found at any time point (p > 0.05). CONCLUSIONS: Both MINST and MIST provided comparable clinical results and microbiological changes in the treatment of intrabony defects over 12 months follow-up. CLINICAL RELEVANCE: This randomized, controlled, parallel trial revealed that both therapeutic modalities may promote clinical and microbiological benefits at 12 months post-therapy.

Cementocyte alterations associated with experimentally induced cellular cementum apposition in Hyp mice.

BACKGROUND: Cellular cementum, a mineralized tissue covering apical tooth roots, grows by apposition to maintain the tooth in its occlusal position. We hypothesized that resident cementocytes would show morphological changes in response to cementum apposition, possibly implicating a role in cementum biology. METHODS: Mandibular first molars were induced to super-erupt (EIA) by extraction of maxillary molars, promoting rapid new cementum formation. Tissue and cell responses were analyzed at 6 and/or 21 days post-procedure (dpp). RESULTS: High-resolution micro-computed tomography (micro-CT) and confocal laser scanning microscopy showed increased cellular cementum by 21 dpp. Transmission electron microscopy (TEM) revealed that cementocytes under EIA were 50% larger than control cells, supported by larger pore sizes detected by micro-CT. Cementocytes under EIA displayed ultrastructural changes consistent with increased activity, including increased cytoplasm and nuclear size. We applied EIA to Hyp mutant mice, where cementocytes have perilacunar hypomineralization defects, to test cell and tissue responses in an altered mechanoresponsive milieu. Hyp and WT molars displayed similar super-eruption, with Hyp molars exhibiting 28% increased cellular cementum area versus 22% in WT mice at 21 dpp. Compared to control, Hyp cementocytes featured well-defined, disperse euchromatin and a thick layer of peripherally condensed heterochromatin in nuclei, indicating cellular activity. Immunohistochemistry (IHC) for cementum markers revealed intense dentin matrix protein-1 expression and abnormal osteopontin deposition in Hyp mice. Both WT and Hyp cementocytes expressed gap junction protein, connexin 43. CONCLUSION: This study provides new insights into the EIA model and cementocyte activity in association with new cementum formation.

Novel rare frameshift variation in aggressive periodontitis: Exomic and familial-screening analysis.

BACKGROUND: Aggressive periodontitis (AgP), currently periodontitis grade C, presents early onset, rapid progression, and a poorly established genetic association. Thus, this study aimed to identify genetic variants associated with AgP via whole exome sequencing (WES) through a familial screening approach. METHODS: WES was performed in two nuclear families, including a proband and a parent affected by AgP and an unaffected parent and sibling. Common variants among affected individuals, excluding those common to healthy people, from each family, composed the data set associated with AgP. In silico analysis evaluated the impact of each variant on protein structure and protein-protein interactions. Moreover, identified deleterious variants were validated in a populational analysis (n = 96). RESULTS: The missense single nucleotide variations (SNVs) rs142548867 in EEFSEC (c.668C>T), rs574301770 in ZNF136 (c.466C>G), and rs72821893 in KRT25 (c.800G>A) and the frameshift indels rs37146475 in GPRC6A (c.2323-2324insT) and c.1366_1372insGGAGCAG in ELN were identified in AgP and have a predicted functional impact on proteins. In silico analysis indicated that the indel in GPRC6A generates a loss of the C-terminal tail of the Gprca protein. Furthermore, this SNV was significantly associated with AgP in a population-based investigation. CONCLUSION: Novel frameshift variation in GPRC6A (c.2323-2324insT) was identified as a potential genetic alteration associated with AgP occurrence.

Histometric evaluation of bone around titanium implants with different surface treatments in rats exposed to cigarette smoke inhalation.

OBJECTIVES: There is a lack of histological information about the influence of cigarette smoke on bone around surface-treated implants. The aim of the present study was to test the influence of titanium surface treatment on osseointegration in animals that were exposed to intermittent cigarette smoke inhalation. MATERIAL AND METHODS: Twenty-two male Wistar rats were used. One tibia, chosen at random, received a machined titanium implant (MI) while the other received an aluminum oxide-blasted surface implant (ABI). The animals were randomly assigned to one of the following groups: Group 1 - control (n=11) and Group 2 - intermittent cigarette smoke inhalation (n=11). Sixty days after surgery, the animals were sacrificed. The degree of bone-to-implant contact (BIC), bone filling (BF) within the limits of the threads of the implants and bone density (proportion of mineralized bone in a 500-mum-wide zone lateral to the implant - BD) were measured in the cortical (zone A) and cancellous bone (zone B) areas. RESULTS: Data analysis showed significant differences when comparing the groups and implant surfaces in both zones for BIC (two-way ANOVA -P<0.05). The two groups presented higher BIC mean values for ABI, when compared with MI (P<0.05). In group 2, cigarette smoke inhalation negatively affected BF in both zones (P<0.05). Group 2 presented a significantly decreased BD in both zones (P<0.05). No statistically significant differences were observed between surfaces in any of the groups for BD. CONCLUSION: Within the limits of the present study, it can be concluded that the aluminum oxide blast surface treatment may increase the degree of BIC but cannot overcome the detrimental effect of tobacco smoke on bone around titanium implants.

Semilunar coronally positioned flap or subepithelial connective tissue graft for the treatment of gingival recession: a 30-month follow-up study.

BACKGROUND: The objective of this prospective, controlled clinical trial was to evaluate the long-term outcomes of subepithelial connective tissue graft (SCTG) or semilunar coronally positioned flap (SCPF) for the treatment of Miller Class I gingival recession defects. METHODS: Seventeen patients with bilateral Miller Class I gingival recessions (< or =4.0 mm) in maxillary canines or premolars were selected. The recessions were randomly assigned to receive SCPF or SCTG. 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 and at 6 and 30 months post-surgery. Patient satisfaction with esthetics and root sensitivity was also evaluated. RESULTS: The root-coverage outcomes obtained at 6 months were maintained throughout the study. At the 30-month examination, the average percentage of root coverage was 89.25% for SCPF and 96.83% for SCTG (P >0.05); complete root coverage was observed in 58.82% and 88.24% of patients, respectively. SCTG maintained a statistically significant increase in TKT (P <0.05) at 30 months. At this time, there were no significant differences between the two groups with regard to RH, RW, WKT, PD, and CAL. The evaluation of the esthetic outcome by the patient showed a preference for the SCTG treatment. Furthermore, in this group, no patient complained of residual or additional root hypersensitivity. In the SCPF group, three patients had this complaint at 30 months. CONCLUSIONS: SCPF and SCTG can be successfully used to treat Class I gingival recession, presenting outcomes with long-term stability. However, patient-oriented outcomes, such as esthetics and root sensitivity, favor SCTG therapy.

Full-mouth ultrasonic debridement associated with amoxicillin and metronidazole in the treatment of severe chronic periodontitis.

BACKGROUND: The purpose of this study was to evaluate the adjunctive clinical, microbiologic, and immunologic effects of the systemic administration of amoxicillin and metronidazole in the full-mouth ultrasonic debridement of patients with severe chronic periodontitis. METHODS: Twenty-five patients presenting at least eight teeth with probing depth (PD) > or =5 mm and bleeding on probing (BOP) were selected and randomly assigned to full-mouth ultrasonic debridement + placebo (control group) or full-mouth ultrasonic debridement + amoxicillin and metronidazole (test group). The clinical outcomes evaluated were visible plaque index, BOP, position of the gingival margin, relative attachment level (RAL), and PD. Real-time polymerase chain reaction (PCR) was used for quantitative analysis of Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans), Porphyromonas gingivalis, and Tannerella forsythia (previously T. forsythensis). The enzyme-linked immunosorbent assay (ELISA) technique permitted the detection of prostaglandin E(2,) interleukin-1beta, and interferon-gamma levels in gingival crevicular fluid. All parameters were evaluated at baseline and at 3 and 6 months post-treatment. RESULTS: At 6 months, the test treatment resulted in lower BOP and an additional reduction (0.83 mm) in PD (P <0.05). Data also showed RAL gain > or =2 mm at 43.52% of sites in control patients compared to 58.03% of sites in test patients (P <0.05). However, both groups had similar mean RAL gain (1.68 and 1.88 mm for the control and test groups, respectively). Real-time PCR and ELISA failed to identify significant differences between the groups. CONCLUSIONS: Both treatments resulted in significant clinical improvements; however, there was a slight, but significantly greater, improvement in BOP and the percentage of sites with PD > or =5 mm exhibiting RAL gain > or =2 mm in the test group. Nevertheless, no improvement in the microbiologic or immunologic outcome was observed with the adjunctive use of systemic amoxicillin and metronidazole.

Periodontal debridement as a therapeutic approach for severe chronic periodontitis: a clinical, microbiological and immunological study.

AIM: To clinically, microbiologically and immunologically characterize periodontal debridement as a therapeutic approach for severe chronic periodontitis. MATERIAL AND METHODS: Twenty-five patients presenting at least eight teeth with a probing pocket depth (PPD) of >or=5 mm and bleeding on probing (BOP) were selected and randomly assigned to quadrant-wise scaling and root planing or one session of full-mouth periodontal debridement. The following clinical outcomes were assessed: plaque index, BOP, position of gingival margin, relative attachment level (RAL) and PPD. Real-time PCR was used for quantitative analysis of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythia. The enzyme-linked immunosorbent assay permitted the detection of IL-1beta, prostaglandin E(2), INF-gamma and IL-10 in gingival crevicular fluid (GCF). All the parameters were evaluated at baseline, and at 3 and 6 months after treatment. RESULTS: Both the groups had similar means of PPD reduction and attachment gain over time. Besides a significant reduction in the bacterial level after treatment in both groups, microbiological analysis failed to demonstrate significant differences between them. Finally, no difference was observed between groups with respect to the levels of inflammatory mediators in GCF. CONCLUSION: Periodontal debridement resulted in a similar clinical, microbiological and immunological outcome when compared with standard scaling and root planing and therefore may be a viable approach to deal with severe chronic periodontitis.

Root cementum may modulate gene expression during periodontal regeneration: a preliminary study in humans.

BACKGROUND: Previous data demonstrated that root cementum may affect periodontal regeneration. As such, this study aimed to explore further possible mechanisms involved in this process by investigating in humans whether root cementum modulates gene expression in the regenerating tissue formed under membrane-protected intrabony defects. METHODS: Thirty subjects with deep intrabony defects (> or =5 mm; 2- or 3-wall) were selected and assigned to the control or test group. The control group received scaling and root planing with the removal of granulation tissue and root cementum; the test group underwent removal of granulation tissue and soft microbial deposits by cleaning the root surface with a microbrush and saline solution, aiming at cementum preservation. Guided tissue regeneration (GTR) was applied to both groups. Twenty-one days later, the newly formed tissue under the membrane was assessed for the expression of the following genes: alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (OCN), platelet-derived growth factor-alpha (PDGFA), bone sialoprotein (BSP), and basic fibroblast growth factor (bFGF). RESULTS: Data analysis demonstrated that mRNA levels for PDGFA, BSP, and bFGF were higher in the sites where root cementum was kept in place compared to the sites where root cementum was removed completely as part of the periodontal therapy (P <0.05); in contrast, OCN levels were lower (P <0.05). No difference for ALP or OPN was observed between the control and test groups (P >0.05). CONCLUSION: Root cementum may modulate the expression of growth and mineral-associated factors during periodontal regeneration.

Root coverage outcome may be affected by heavy smoking: a 2-year follow-up study.

BACKGROUND: Although subepithelial connective tissue graft (CTG) has been reported to be a predictable procedure for root coverage, the impact of smoking on the long-term outcome of periodontal plastic surgery is unclear. Hence, the aim of this study was to evaluate the effect of smoking, on a long-term basis, on the stability of gingival tissue following CTG treatment of gingival recession. METHODS: Twenty-two defects were treated by CTG in canine and premolar Miller Class I and II gingival recessions (11 smokers and 11 non-smokers). The following clinical measurements were obtained at baseline and at 1, 2, 3, 4, 6, 12, 18, and 24 months after surgery: plaque and gingival indexes, extension of gingival recession (GR), probing depth (PD), clinical attachment level (CAL), and gingival thickness. Individuals smoking > or =20 cigarettes/day for > or =5 years were considered smokers. RESULTS: Data analysis demonstrated that both groups presented similar plaque and gingival indexes (P >0.05), and an intragroup analysis showed that CTG was able to promote root coverage and increase gingival thickness in both groups over time (P <0.05). However, at 24 months postoperatively, statistical analysis showed that smokers presented poorer outcomes with regard to PD, GR, and CAL (P <0.05); in addition, a more satisfactory stabilization of the gingival tissue was found in the non-smoker group. CONCLUSION: Smoking may represent a challenge to root coverage outcome for CTG because smoking significantly affected the stability of gingival tissue over time.

Chronic stress may modulate periodontal disease: a study in rats.

BACKGROUND: The present study aimed to evaluate whether chronic stress (CS) affects ligature-induced periodontal disease and to investigate the impact of CS on the mRNA levels of interleukin (IL)-1beta, -1 receptor antagonist, -6, and -10, interferon-gamma (IFN-gamma), receptor activator of nuclear factor-kappa B ligand (RANKL), and osteoprotegerin in the gingival tissues of rats. METHODS: Sixty male Wistar rats were assigned randomly to three groups: G1 (control; non-ligated sites), G2 (periodontal disease), and G3 (periodontal disease associated with restraint stress for 12 hours/day for the entire study). After 30 days, all animals were sacrificed by decapitation. Blood samples were taken, and the concentrations of corticosterone and catecholamines were measured as biomarkers of CS. Marginal tissues around ligated and non-ligated teeth were harvested, and gene expression was assessed by quantitative polymerase chain reaction. Moreover, the area of bone loss (ABL) was determined histometrically. RESULTS: Data analysis showed that CS increased serum levels of stress biomarkers (P <0.05), ligature placement resulted in a significant ABL compared to non-ligated sites, CS significantly increased the amount of ABL in inflamed sites (P <0.001), and CS significantly increased mRNA levels of proinflammatory (IL-1beta and -6 and IFN-gamma) and anti-inflammatory (IL-10) cytokines and proresorptive factor (RANKL) in ligated sites (P <0.05). CONCLUSION: CS significantly increased bone loss resulting from ligature-induced periodontitis by a local increase in proinflammatory and proresorptive factors.

RG108 increases NANOG and OCT4 in bone marrow-derived mesenchymal cells through global changes in DNA modifications and epigenetic activation.

Human bone marrow-derived mesenchymal stem cells (hBMSCs) are important for tissue regeneration but their epigenetic regulation is not well understood. Here we investigate the ability of a non-nucleoside DNA methylation inhibitor, RG108 to induce epigenetic changes at both global and gene-specific levels in order to enhance mesenchymal cell markers, in hBMSCs. hBMSCs were treated with complete culture medium, 50 µM RG108 and DMSO for three days and subjected to viability and apoptosis assays, global and gene-specific methylation/hydroxymethylation, transcript levels' analysis of epigenetic machinery enzymes and multipotency markers, protein activities of DNMTs and TETs, immunofluorescence staining and western blot analysis for NANOG and OCT4 and flow cytometry for CD105. The RG108, when used at 50 µM, did not affect the viability, apoptosis and proliferation rates of hBMSCs or hydroxymethylation global levels while leading to 75% decrease in DNMTs activity and 42% loss of global DNA methylation levels. In addition, DNMT1 was significantly downregulated while TET1 was upregulated, potentially contributing to the substantial loss of methylation observed. Most importantly, the mesenchymal cell markers CD105, NANOG and OCT4 were upregulated being NANOG and OCT4 epigenetically modulated by RG108, at their gene promoters. We propose that RG108 could be used for epigenetic modulation, promoting epigenetic activation of NANOG and OCT4, without affecting the viability of hBMSCs. DMSO can be considered a modulator of epigenetic machinery enzymes, although with milder effect compared to RG108.

Comparative study of ultrasonic instrumentation for the non-surgical treatment of interproximal and non-interproximal furcation involvements.

BACKGROUND: The aim of this clinical trial was to compare the outcome of non-surgical treatment of interproximal and non-interproximal Class II furcation involvements. METHODS: Thirty-eight patients presenting at least one Class II furcation involvement that bled on probing with a probing depth (PD) > or = 5 mm were recruited. Furcation involvements were grouped as either buccal and lingual furcation involvements (BLFI) or interproximal furcation involvements (IFI). The following clinical outcomes were evaluated: visible plaque index, bleeding on probing (BOP), position of the gingival margin, relative attachment level (RAL), PD, and relative horizontal attachment level (RHAL). N-benzoyl-l-arginine-p-nitroanilide (BAPNA) testing was used to analyze trypsin-like activity in dental biofilm. All parameters were evaluated at baseline and 1, 3, and 6 months after non-surgical subgingival instrumentation. RESULTS: Six months after treatment, both groups had similar means of RAL and RHAL gain (P >0.05). These variables were 1.22 and 1.07 mm in the IFI group and 1.38 and 1.20 mm in the BLFI group, respectively. The PD reduction was significantly greater in the BLFI group than in the IFI group (2.59 and 2.11 mm, respectively; P <0.05). The BLFI group presented fewer sites with PD > or = 5 mm than the IFI group at all post-treatment periods. At 6 months, the BAPNA test showed that only the BLFI group had values significantly different from baseline. This means that the BLFI group had significantly lower BAPNA values compared to the IFI group at 6 months. CONCLUSION: Buccal and lingual Class II furcation involvements respond better to non-surgical therapy compared to interproximal Class II furcation involvements.