Phytocystatin CsinCPI-2 Reduces Osteoclastogenesis and Alveolar Bone Loss.

Periodontal disease (PD) is a polymicrobial chronic inflammatory condition of the supporting tissues around the teeth, leading to the destruction of surrounding connective tissue. During the progression of PD, osteoclasts play a crucial role in the resorption of alveolar bone that eventually leads to the loss of teeth if the PD is left untreated. Therefore, the development of antiresorptive therapies targeting bone-resorbing cells will significantly benefit the treatment of PD. Here, we demonstrate the inhibitory effect of CsinCPI-2, a novel cysteine peptidase inhibitor from the orange tree, on periodontitis-induced inflammation, alveolar bone loss, and osteoclast differentiation. Using the ligature-induced periodontitis model in mice, we show that treatment with CsinCPI-2 (0.8 µg/g of body weight) significantly reduced inflammatory cell infiltrate in the connective tissue and prevented the loss of alveolar bone mass (BV/TV) caused by PD, effects associated with diminished numbers of TRAP-positive multinucleated cells. Furthermore, CsinCPI-2 significantly downregulated the numbers of inflammatory cells expressing CD3, CD45, MAC387, and IL-1ß. In vitro, CsinCPI-2 inhibited RANKL-induced TRAP+ multinucleated osteoclast formation in mouse bone marrow macrophage cultures in a concentration-dependent manner. This effect was not due to cytotoxicity, as demonstrated by the MTT assay. CsinCPI-2 inhibited RANKL-induced mRNA expression of Acp5, Calcr, and Ctsk, as well as the RANKL-induced upregulation of Nfatc1, a crucial transcription factor for osteoclast differentiation. Based on our findings, CsinCPI-2 prevents bone loss induced by PD by controlling the inflammatory process and acting directly on osteoclastogenesis, suggesting an interesting potential for CsinCPI-2 in the strategy for PD treatment.

Behavior of rat bone marrow stem cells on titanium surfaces modified by laser-beam and deposition of calcium phosphate.

OBJECTIVES: The aim of this study was to evaluate the behavior of rat bone marrow stem cells seeded on a Ti-15Mo alloy surface modified by laser-beam irradiation followed by calcium phosphate deposition. MATERIALS AND METHODS: A total of four groups were evaluated: polished commercially pure titanium (cpTi): Ti-P; laser irradiation + calcium phosphate deposition on cpTi: Ti-LCP; polished Ti-15Mo alloy: Ti15Mo-P; and laser irradiation + calcium phosphate deposition on Ti-15Mo alloy: Ti15Mo-LCP. Before and after laser irradiation and calcium phosphate deposition on the surfaces, physicochemical and morphological analyses were performed: Scanning Electron Microscopy (SEM) and Energy Dispersive Spectroscopy (EDX). The wettability of the samples was evaluated by contact angle measurement. In addition, the behavior of osteoblast-like cells to these surfaces was evaluated for cell morphology, adhesion, proliferation and viability, evaluation of alkaline phosphatase formation and gene expression of osteogenesis markers. RESULTS: Surfaces wet-abrade with grit paper (P) showed oriented groves, while the laser irradiation and calcium phosphate deposition (LCP) produced porosity on both cpTi and Ti15Mo alloy groups with deposits of hydroxyapatite (HA) crystals (SEM). EDX showed no contamination after surface modification in both metal samples. A complete wetting was observed for both LCP groups, whereas P surfaces exhibited high degree of hydrophobicity. There was a statistical difference in the intragroup comparison of proliferation and viability (p < 0.05). The ALP activity showed higher values in the Ti15Mo alloy at 10 days of culture. The gene expression of bone related molecules did not present significant differences at 7 and 14 days among different metals and surface treatments. CONCLUSION: Ti15-Mo seems to be an alternative alloy to cpTi for dental implants. Surface treatment by laser irradiation followed by phosphate deposition seems to positively interact with bone cells. CLINICAL RELEVANCE: Ti-15Mo alloy surface modified by laser-beam irradiation followed by calcium phosphate deposition may improve and accelerate the osseointegration process of dental implants.

Regulation of the autophagy-marker Sequestosome 1 in periodontal cells and tissues by biomechanical loading.

PURPOSE: Orthodontic treatment is based on the principle of force application to teeth and subsequently to the surrounding tissues and periodontal cells. Sequestosome 1 (SQSTM1) is a well-known marker for autophagy, which is an important cellular mechanism of adaptation to stress. The aim of this study was to analyze whether biomechanical loading conditions regulate SQSTM1 in periodontal cells and tissues, thereby providing further information on the role of autophagy in orthodontic tooth movement. METHODS: Periodontal ligament (PDL) fibroblasts were exposed to cyclic tensile strain of low magnitude (3%, CTSL), and the regulation of autophagy-associated targets was determined with an array-based approach. SQSTM1 was selected for further biomechanical loading experiments with dynamic and static tensile strain and assessed via real-time polymerase chain reaction (RT-PCR) and immunoblotting. Signaling pathways involved in SQSTM1 activation were analyzed by using specific inhibitors, including an autophagy inhibitor. Finally, SQSTM1 expression was analyzed in gingival biopsies and histological sections of rats in presence and absence of orthodontic forces. RESULTS: Multiple autophagy-associated targets were regulated by CTSL in PDL fibroblasts. All biomechanical loading conditions tested increased the SQSTM1 expression significantly. Stimulatory effects of CTSL on SQSTM1 expression were diminished by inhibition of the c­Jun N­terminal kinase (JNK) pathway and of autophagy. Increased SQSTM1 levels after CTSL were confirmed by immunoblotting. Orthodontic force application also led to significantly elevated SQTSM1 levels in the gingiva and PDL of treated animals as compared to control. CONCLUSIONS: Our in vitro and in vivo findings provide evidence of a role of SQSTM1 and thereby autophagy in orthodontic tooth movement.

Immune response mediated by Th1 / IL-17 / caspase-9 promotes evolution of periodontal disease.

INTRODUCTION: Periodontitis is characterized by inflammatory mediators beyond T lymphocyte function and phenotype (Th1/Th2/Th17). The clinical diversity in periodontitis makes it difficult to characterize the immune response in patients. This study evaluated the profile of the adaptive immune response in the periodontal disease model. METHODS: 72 rats (Wistar) were divided into a control group (CTL/day 0) and periodontitis (PD15/15 days and PD60/60 days). In the PD15 and PD60 groups, periodontal disease was induced by ligature with a silk thread placed in the cervical region of the upper first molar. After euthanasia, the periodontal tissue was analyzed by flow cytometry (CD4, CD8, CD25, CD44), semi-quantitative RT-PCR (T-bet, GATA-3, RORγt), semi-quantitative RT-PCR and ELISA IFN-γ, TNF-α, IFN-γ, IL-4, IL-6, IL-10, IL-17) and by Western blotting (Caspase-9, PCNA). RESULTS: The number of CD4+CD25+, CD4+CD44+, CD8+CD25+ and CD8+CD44+ cells and expression levels of T-bet and GATA-3 are increased in the PD60 group compared to PD15 and CTL. The RORγ-t gene transcript increased in the PD15 group in relation to PD60 and CTL. The cytokines IFN-γ, TNF-α and IL-17 increased in the PD60 group in relation to PD15. The expression of Caspase-9 was higher in the PD60 group than in PD15. CONCLUSIONS: The results suggest that the evolution of gingivitis to periodontitis is related to the accumulation of activated Th1 cells (IFN-γ and TNF-α) associated with the presence of increased IL-17. Studies with inhibitors of these cytokines in periodontal disease may lead to therapy directed at blocking the inflammatory process in this pathology, interrupting bone loss.

Damage-regulated autophagy modulator 1 in oral inflammation and infection.

OBJECTIVES: Damage-regulated autophagy modulator (DRAM) 1 is a p53 target gene with possible involvement in oral inflammation and infection. This study sought to examine the presence and regulation of DRAM1 in periodontal diseases. MATERIAL AND METHODS: In vitro, human periodontal ligament fibroblasts were exposed to interleukin (IL)-1ß and Fusobacterium nucleatum for up to 2 days. The DRAM1 synthesis and its regulation were analyzed by real-time PCR, immunocytochemistry, and ELISA. Expressions of other autophagy-associated genes were also studied by real-time PCR. In vivo, synthesis of DRAM1 in gingival biopsies from rats and patients with and without periodontal disease was examined by real-time PCR and immunohistochemistry. For statistics, ANOVA and post-hoc tests were applied (p < 0.05). RESULTS: In vitro, DRAM1 was significantly upregulated by IL-1ß and F. nucleatum over 2 days and a wide range of concentrations. Additionally, increased DRAM1 protein levels in response to both stimulants were observed. Autophagy-associated genes ATG3, BAK1, HDAC6, and IRGM were also upregulated under inflammatory or infectious conditions. In vivo, the DRAM1 gene expression was significantly enhanced in rat gingival biopsies with induced periodontitis as compared to control. Significantly increased DRAM1 levels were also detected in human gingival biopsies from sites of periodontitis as compared to healthy sites. CONCLUSION: Our data provide novel evidence that DRAM1 is increased under inflammatory and infectious conditions in periodontal cells and tissues, suggesting a pivotal role of DRAM1 in oral inflammation and infection. CLINICAL RELEVANCE: DRAM1 might be a promising target in future diagnostic and treatment strategies for periodontitis.

Role of Cathepsin S in Periodontal Inflammation and Infection.

Cathepsin S is a cysteine protease and regulator of autophagy with possible involvement in periodontitis. The objective of this study was to investigate whether cathepsin S is involved in the pathogenesis of periodontal diseases. Human periodontal fibroblasts were cultured under inflammatory and infectious conditions elicited by interleukin-1ß and Fusobacterium nucleatum, respectively. An array-based approach was used to analyze differential expression of autophagy-associated genes. Cathepsin S was upregulated most strongly and thus further studied in vitro at gene and protein levels. In vivo, gingival tissue biopsies from rats with ligature-induced periodontitis and from periodontitis patients were also analyzed at transcriptional and protein levels. Multiple gene expression changes due to interleukin-1ß and F. nucleatum were observed in vitro. Both stimulants caused a significant cathepsin S upregulation. A significantly elevated cathepsin S expression in gingival biopsies from rats with experimental periodontitis was found in vivo, as compared to that from control. Gingival biopsies from periodontitis patients showed a significantly higher cathepsin S expression than those from healthy gingiva. Our findings provide original evidence that cathepsin S is increased in periodontal cells and tissues under inflammatory and infectious conditions, suggesting a critical role of this autophagy-associated molecule in the pathogenesis of periodontitis.

Clinical outcomes of periodontal therapy are not influenced by the ATC/TTC haplotype in the IL8 gene.

BACKGROUND AND OBJECTIVE: Although chronic periodontitis (CP) is a multifactorial condition, few studies have investigated the potential association of gene variants with the outcome of periodontal therapy. In a previous study, we reported that variants in the interleukin-8 (IL8) gene were associated with CP in a Brazilian population. The aim of this nonrandomized study was to investigate whether genetic susceptibility to CP, conferred by the presence of the IL8 ATC/TTC haplotype, influences the clinical outcomes of nonsurgical periodontal therapy and the IL-8 protein levels in the gingival crevicular fluid. MATERIAL AND METHODS: Forty-one individuals were grouped according to the presence (susceptible to CP; n = 21) or absence (not susceptible to CP; n = 20) of the IL8 ATC/TTC haplotype. These individuals received nonsurgical periodontal therapy from one periodontist, who was blinded to the genetic status of each patient, and follow up continued for 45 d. The clinical parameters and gingival crevicular fluid samples were collected at baseline and on day 45. The IL-8 levels were determined by an ELISA. The data were subjected to the Mann-Whitney U-test, Wilcoxon and Spearman tests and to multiple logistic-regression analysis. RESULTS: No significant differences between patients with or without the IL8 ATC/TTC haplotype were found for the outcome of nonsurgical periodontal therapy and IL-8 levels. The multiple logistic-regression analysis did not show a statistically significant association between the IL8 haplotype and the variables studied. CONCLUSION: In this longitudinal clinical study, we observed that neither the outcome of nonsurgical periodontal therapy nor the IL-8 levels were influenced by the IL8 ATC/TTC CP-susceptibility haplotype. Additional studies of CP patients from other ethnic populations are necessary to confirm these results.

Periodontopathogens levels and clinical response to periodontal therapy in individuals with the interleukin-4 haplotype associated with susceptibility to chronic periodontitis.

Periodontitis is an inflammatory disease that results from an interaction between dental biofilm agents and the host immune-inflammatory response. Periodontopathogenic organisms, such as Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola, as well as the host's susceptibility, represented by the host's genetic makeup, are the key factors that influence this complex disease. Recently, we identified haplotypes in the IL4 gene that were associated with chronic periodontitis (CP). This study aimed to evaluate whether subjects with different IL4 haplotypes (TCI/CCI and TTD/CTI) would be differentially colonized by periodontopathogens and whether they would respond differently to non-surgical periodontal therapy. Thirty-nine patients carrying the IL4 haplotype of genetic susceptibility to CP (IL4+) or protection against CP (IL4-) were evaluated. Those groups were further subdivided into individuals with CP (CP IL4+ or CP IL4-) and those that were periodontally healthy (H) (H IL4+ or H IL4-). CP patients were submitted to non-surgical periodontal therapy. Clinical and microbiological analyses were performed considering the data at baseline and 45 and 90 days after periodontal therapy. Periodontopathogens levels were evaluated by absolute quantitative polymerase chain reaction (qPCR). The baseline data revealed that the total levels of periodontopathogens were higher in the CP IL4+ than in the CP IL4- groups. Clinical analyses revealed that the periodontal therapy was equally effective, independent of the subject's IL4 genetic load. The TCI/CCI IL4 haplotype, previously associated with genetic susceptibility to CP, was also associated with increased levels of periodontopathogenic bacteria, but this genetic background did not influence the response to non-surgical periodontal treatment.

Association between IL8 haplotypes and pathogen levels in chronic periodontitis.

Chronic periodontitis (CP) is considered to be a multifactorial disease influenced by microbial and genetic factors. The aim of the present study was to investigate whether the genetic susceptibility to CP in individuals with the IL8 ATC/TTC haplotype is associated with subgingival levels of periodontopathogens. Sixty-five individuals, grouped according to the presence (n = 28) or absence (n = 37) of the IL8 haplotype, were evaluated. After clinical periodontal evaluation, each group was subdivided according to the presence (CP) or absence (H) of periodontitis. Four subgingival samples were obtained from CP and two samples per subject from H patients. The levels and proportions of Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola were analyzed using quantitative real-time polymerase chain reaction (q-PCR). No differences were found in the proportion of periodontopathogenic bacteria between groups with the presence or absence of the IL8 haplotype. However, in the CP groups, the levels of periodontopathogens were significantly higher in the individuals without the IL8 haplotype than in the individuals with the IL8 haplotype. These results suggest that periodontal destruction may occur in patients who are considered to be genetically susceptible to CP with a lower microbial challenge because of the presence of the IL8 ATC/TTC haplotype than in patients without this haplotype.

Effect of periodontal treatment on the incidence of preterm delivery: a systematic review.

Preterm birth is a major problem in public health in developed and developing countries and the search for risk factors of this event is important. The aim of this study was to review the effect of periodontal treatment on the incidence of preterm delivery. A wide research was executed considering an evaluation period between November of 1998 and October of 2009 at MEDLINE/PUBMED databases. The selection strategy consisted of the search for the following key-words: "periodontal therapy" or "periodontal disease" and "pregnancy outcome" or "preterm birth". The search was limited for articles written in English. The randomized clinical trials that evaluated the effect of the non-surgical periodontal treatment on the incidence of Preterm Low Birth Weight (PLBW) were selected. In a total of 7 papers selected, the incidence of PLBW was lower in groups of women who were submitted to periodontal treatment. Reductions of Preterm Birth (PTB) ranged from 0.8% to 28.01%, while reduction of Low Birth Weight (LBW) ranged from 0.44% to 33%. In studies that analyzed these two variables together, there was variation between 4.57% to 71.5% in rates reduction. Due to heterogeneity of the data, the meta-analysis was not applied. The majority of the studies concluded that non-surgical periodontal treatment in pregnant women reduces incidence of preterm babies with low weight.

PDGF-B gene therapy accelerates bone engineering and oral implant osseointegration.

Platelet-derived growth factor-BB (PDGF-BB) stimulates repair of healing-impaired chronic wounds such as diabetic ulcers and periodontal lesions. However, limitations in predictability of tissue regeneration occur due, in part, to transient growth factor bioavailability in vivo. Here, we report that gene delivery of PDGF-B stimulates repair of oral implant extraction socket defects. Alveolar ridge defects were created in rats and were treated at the time of titanium implant installation with a collagen matrix containing an adenoviral (Ad) vector encoding PDGF-B (5.5 x 10(8) or 5.5 x 10(9) pfu ml(-1)), Ad encoding luciferase (Ad-Luc; 5.5 x 10(9) pfu ml(-1); control) or recombinant human PDGF-BB protein (rhPDGF-BB, 0.3 mg ml(-1)). Bone repair and osseointegration were measured through backscattered scanning electron microscopy, histomorphometry, micro-computed tomography and biomechanical assessments. Furthermore, a panel of local and systemic safety assessments was performed. Results indicated that bone repair was accelerated by Ad-PDGF-B and rhPDGF-BB delivery compared with Ad-Luc, with the high dose of Ad-PDGF-B more effective than the low dose. No significant dissemination of the vector construct or alteration of systemic parameters was noted. In summary, gene delivery of Ad-PDGF-B shows regenerative and safety capabilities for bone tissue engineering and osseointegration in alveolar bone defects comparable with rhPDGF-BB protein delivery in vivo.

Tumor necrosis factor alpha and interleukin-1beta modulate calcium and nitric oxide signaling in mechanically stimulated osteocytes.

OBJECTIVE: Inflammatory diseases often coincide with reduced bone mass. Mechanoresponsive osteocytes regulate bone mass by maintaining the balance between bone formation and resorption. Despite its biologic significance, the effect of inflammation on osteocyte mechanoresponsiveness is not understood. To fill this gap, we investigated whether the inflammatory cytokines tumor necrosis factor alpha (TNFalpha) and interleukin-1beta (IL-1beta) modulate the osteocyte response to mechanical loading. METHODS: MLO-Y4 osteocytes were incubated with TNFalpha (0.5-30 ng/ml) or IL-1beta (0.1-10 ng/ml) for 30 minutes or 24 hours, or with calcium inhibitors for 30 minutes. Cells were subjected to mechanical loading by pulsatile fluid flow (mean +/- amplitude 0.7 +/- 0.3 Pa, 5 Hz), and the response was quantified by measuring nitric oxide (NO) production using Griess reagent and by measuring intracellular calcium concentration ([Ca(2+)](i)) using Fluo-4/AM. Focal adhesions and filamentous actin (F-actin) were visualized by immunostaining, and apoptosis was quantified by measuring caspase 3/7 activity. Cell-generated tractions were quantified using traction force microscopy, and cytoskeletal stiffness was quantified using optical magnetic twisting cytometry. RESULTS: Pulsatile fluid flow increased [Ca(2+)](i) within seconds (in 13% of cells) and NO production within 5 minutes (4.7-fold). TNFalpha and IL-1beta inhibited these responses. Calcium inhibitors decreased pulsatile fluid flow-induced NO production. TNFalpha and IL-1beta affected cytoskeletal stiffness, likely because 24 hours of incubation with TNFalpha and IL-1beta decreased the amount of F-actin. Incubation with IL-1beta for 24 hours stimulated osteocyte apoptosis. CONCLUSION: Our results suggest that TNFalpha and IL-1beta inhibit mechanical loading-induced NO production by osteocytes via abrogation of pulsatile fluid flow-stimulated [Ca(2+)](i), and that IL-1beta stimulates osteocyte apoptosis. Since both NO and osteocyte apoptosis affect osteoclasts, these findings provide a mechanism by which inflammatory cytokines might contribute to bone loss and consequently affect bone mass in rheumatoid arthritis.

AAV2/1-TNFR:Fc gene delivery prevents periodontal disease progression.

Periodontal disease is a chronic inflammatory condition induced by tooth-associated microbial biofilms that induce a host immune response. Therapeutic control of progressive tissue destruction in high-risk patients is a significant challenge in therapy. Soluble protein delivery of antagonists to tumor necrosis factor-alpha (TNF-alpha) inhibits alveolar bone resorption due to periodontitis. However, protein therapy raises several concerns, such as recurrence of disease activity after treatment cessation and repeated dosing regimens. In this study, we used pseudotyped adeno-associated virus vector based on serotype 1 (AAV2/1) to deliver the TNF receptor-immunoglobulin Fc (TNFR:Fc) fusion gene to rats subjected to experimental Porphyromonas gingivalis (Pg)-lipopolysaccharide (LPS)-mediated bone loss. Animals received Pg-LPS delivered to the gingivae thrice weekly for 8 weeks, vehicle alone, Pg-LPS and intramuscular delivery of pseudotyped AAV2/1-TNFR:Fc vector (1 x 10(11) DNase I-resistant particles) or AAV2/1-TNFR:Fc vector delivered to naive animals. AAV2/1-TNFR:Fc therapy led to sustained therapeutic levels of serum TNFR protein and protected against Pg-LPS-mediated loss of bone volume and density. Furthermore, AAV2/1-TNFR:Fc administration reduced local levels of multiple proinflammatory cytokines and osteoclast-like cells at the periodontal lesions. These findings suggest that delivery of AAV2/1-TNFR:Fc may be a viable approach to modulate periodontal disease progression.

Comparison of manual versus sonic and ultrasonic toothbrushes: a review.

PURPOSE: This review of the literature intends to evaluate the effect of brushes with high frequency motion when compared with manual toothbrushes regarding the indices of plaque and gingival bleeding. METHODS: Patients presenting gingivitis and/or chronic periodontitis were evaluated in addition to patients having osseointegrated implants and fixed orthodontic appliances. Pertinent literature was reviewed to select articles according to previously defined inclusion criteria. RESULTS: In the assessed studies results showed significant decreases in plaque and gingival indices by utilization of both types of brushes. However, in the selected studies where sonic brushes were tested in orthodontic and dental implant patients there was a more significant decrease in the indices. Furthermore, there was no indication of gingival recession attributed to product use. CONCLUSION: Future studies with a more homogeneous methodology and better experiment designs will be needed.

Regeneration of Class III furcation defects with basic fibroblast growth factor (b-FGF) associated with GTR. A descriptive and histometric study in dogs.

BACKGROUND: The poor predictability of periodontal regenerative treatment of Class III furcation defects stimulates the study of alternatives to improve its results, such as the use of polypeptide growth factors. The objective of this study was to evaluate, both histologically and histometrically, the effects of topical application of basic fibroblast growth factor (b-FGF) associated with guided tissue regeneration (GTR) in the treatment of Class III defects surgically induced in dogs. METHODS: All second and fourth premolars of 5 mongrel dogs were used and randomly assigned to one of three treatment groups: group 1 (control), treated with scaling and root planing, tetracycline hydrochloride (125 mg/ml) conditioning, and GTR with a collagen membrane; group 2, same treatment as group 1 plus 0.5 mg of b-FGF; group 3, same treatment as group 1 plus 1.0 mg of b-FGF. After a 90-day healing period, routine histologic processing and staining with hematoxylin and eosin and Masson trichrome were performed. RESULTS: The descriptive analysis indicated better regenerative results in both groups treated with b-FGF while the histometric data, analyzed by means of analysis of variance (ANOVA), showed greater filling of the defects in group 2 in comparison to the defects in groups 3 and 1, respectively, which was represented by a smaller area of plaque-occupied space (P = 0.004) as well as a greater amount of newly formed cementum (P = 0.002). CONCLUSIONS: These results indicate that b-FGF, especially in smaller doses, may enhance the regenerative results in Class III furcation lesions, leading to greater filling of these defects with both mineralized and non-mineralized tissues.

Evaluation of anionic collagen membranes in the treatment of class II furcation lesions: an histometric analysis in dogs.

This study aims to evaluate the effect of using anionic collagen membranes in guided tissue regeneration treatment of Class II furcation lesions in dogs. The defects were created in the buccal furcation of 16 mandibular premolars of four dogs. After 56 days without plaque control, the sites were scaled and divided into two groups according to the treatment applied: control sites, open flap debridement; and test sites, guided tissue regeneration treatment. The animals were killed after 3 months. Histological and histometrical analyses showed that the collagen membrane was better than open flap debridement in terms of newly formed cementum and epithelial migration prevention. It provided effective blockade of epithelial tissue and promoted regeneration of lost periodontal tissues, suggesting that the membrane warrants further study.