Francisco Humberto Nociti

Impact of smoking on inflammation: overview of molecular mechanisms

BackgroundInflammation is a critical component of normal tissue repair, as well as being fundamental to the body’s defense against infection. Environmental factors, such as smoking, have been reported to modify the host response and hence modify inflammation progression, severity and outcome. Therefore, a comprehensive understanding of the molecular mechanisms by which smoking affects inflammation is vital for preventive and therapeutic strategies on a clinical level.AimThe purpose of the present article is to review the potential biological mechanisms by which smoking affects inflammation, emphasizing recent developments.ResultsSmoking is reported to effect a number of biological mediators of inflammation through its effect on immune-inflammatory cells, leading to an immunosuppressant state. Recent evidence strongly suggests that the molecular mechanisms behind the modulation of inflammation by smoking mainly involve the nuclear factor-kappa B (NF-kB) family, through the activation of both an inhibitor of IkB kinase (IKK)-dependent and -independent pathway. In addition to NF-kB activation, a number of transcriptional factors including GATA, PAX5 and Smad 3/4, have also been implicated.ConclusionMultiple mechanisms may be responsible for the association of smoking and inflammation, and the identification of potential therapeutic targets should guide future research.

Cementum: A Phosphate-sensitive Tissue

Ectopic calcification within joints has been reported in humans and rodents exhibiting mutations in genes that regulate the level of extracellular pyrophosphate, e.g., ank and PC-1; however, periodontal effects of these mutations have not previously been examined. These initial studies using ank and PC-1 mutant mice were done to see if such mineral deposition and resulting ankylosis were occurring in the periodontium as well. Surprisingly, results indicated the absence of ankylosis; however, a marked increase in cementum formation on the root surfaces of fully developed teeth of these mutant mice was noted. Examination of ank mutant mice at earlier ages of tooth root formation indicated that this striking observation is apparent from the onset of cementogenesis. These findings suggest that cells within the periodontal region are highly responsive to changes in phosphate metabolism. This information may prove valuable in attempts to design successful therapies for regenerating periodontal tissues.

Mesenchymal Stem Cell Properties of Periodontal Ligament Cells From Deciduous and Permanent Teeth

BACKGROUND Human postnatal stem cells have been identified in periodontal ligaments (PDLs). In this study, the in vitro biologic properties of CD105(+) enriched cell subsets from PDLs harvested from deciduous (DePDL) and permanent (PePDL) teeth are comparatively assessed. METHODS PDL tissue was obtained from 12 teeth (six primary and six permanent) from which CD105(+) CD34(-) CD45(-) cells were isolated by magnetic cell sorting. To identify and quantitatively compare the stem cell markers, DePDL and PePDL cells were assessed for CD166 surface antigen expression by flow cytometry, real-time polymerase chain reaction, and immunostaining for Stro-1 and Oct-4, osteogenic and adipogenic differentiation, and proliferation rate by trypan blue method. RESULTS Magnetic cell sorting isolated cell populations containing 23.87% (+/- 11.98%) and 11.68% (+/- 6.27%) of CD105(+) expressing cells from PePDL and DePDL, respectively. Flow cytometric analysis demonstrated a higher proportion of CD105(+) cells coexpressing CD166 surface antigen in PePDL, whereas immunostaining and real-time polymerase chain reaction analysis demonstrated that both cell subsets expressed Stro-1 and Oct-4. DePDL-CD105(+) subsets were more proliferative compared to PePDL subsets, and both cell populations showed multipotential capabilities to differentiate in vitro to osteoblast/cementoblast- and adipocyte-like cells. However, a higher expression of adipogenic-related genes was observed in DePDL cells, whereas PePDL-CD105(+) cell subset presented a more homogeneous osteoblast/cementoblast response. CONCLUSION These findings demonstrate that highly purified mesenchymal progenitor cell subsets can be obtained from the PDLs of both deciduous and permanent teeth, and further indicate phenotype dissimilarities that may have an impact on their clinical applications.

Levels of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, inflammatory cytokines and species-specific immunoglobulin G in generalized aggressive and chronic periodontitis.

BACKGROUND AND OBJECTIVE Aggressive periodontitis pathogenesis still is not completely understood in the literature regarding the relationship between microbial and inflammatory aspects. So this study aimed to compare microbial and inflammatory patterns in the gingival crevicular fluid of generalized aggressive and chronic periodontitis patients. MATERIAL AND METHODS Forty aggressive and 28 chronic periodontitis patients were selected. Biofilm and gingival crevicular fluid were collected from a deep pocket (periodontal probing depth >7 mm) and a moderate pocket (periodontal probing depth = 5 mm) of each patient, and microbiological and immunoenzymatic assays were performed. Real-time PCR was used to determine quantities of Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis. Enzyme-linked immunosorbent assay (ELISA) was employed to determine gingival crevicular fluid levels of interleukin-1beta, interferon-gamma, prostaglandin E(2) and interleukin-10. In addition, immunoglobulin G (IgG) levels against A. actinomycetemcomitans and P. gingivalis lipopolysaccharide were also determined by ELISA. Analysis of variance/Tukey test, Mann-Whitney U-test and the Pearson correlation test were used to determine differences and correlations between variables analysed (alpha = 5%). RESULTS Patients suffering from generalized aggressive periodontitis had their mouth colonized by higher amounts of A. actinomycetemcomitans and P. gingivalis than chronic periodontitis patients. Conversely, the gingival crevicular fluid levels of IgG against both pathogens were statistically inferior in aggressive periodontitis patients (p < 0.05). With regard to gingival crevicular fluid levels of cytokines, aggressive periodontitis patients presented reduced levels of interleukin-10 (p < 0.05). CONCLUSION In comparison to chronic periodontitis, generalized aggressive periodontitis patients have an imbalance in the host response, with reduced levels of interleukin-10 and IgG, and increased periodontal pathogens.

Subgingival biodiversity in subjects with uncontrolled type‐2 diabetes and chronic periodontitis

BACKGROUND AND OBJECTIVE There is a bidirectional relationship between periodontal disease and type-2 diabetes mellitus (DM). Inflammatory mediators may negatively affect glycemic control, and increased glucose levels and resultant glycation end-products may alter the host response against bacterial infection. However, no agreement has been reached regarding the effect of DM on periodontal subgingival microbiota. Therefore, the purpose of the present study was to compare the subgingival biodiversity in deep periodontal pockets of subjects with chronic periodontitis and either uncontrolled type-2 diabetes or no diabetes using 16S rRNA gene cloning and sequencing. MATERIAL AND METHODS Twelve subjects with uncontrolled type-2 diabetes (glycated hemoglobin > 8%) and eleven nondiabetic subjects presenting severe and generalized chronic periodontitis were selected. Subgingival biofilm from periodontal pockets > 5 mm were assessed using the 16S rRNA gene cloning and sequencing technique. RESULTS Significant differences were observed in subgingival microbiota between diabetic and nondiabetic subjects. Diabetic subjects presented higher percentages of total clones of TM7, Aggregatibacter, Neisseria, Gemella, Eikenella, Selenomonas, Actinomyces, Capnocytophaga, Fusobacterium, Veillonella and Streptococcus genera, and lower percentages of Porphyromonas, Filifactor, Eubacterium, Synergistetes, Tannerella and Treponema genera than nondiabetic individuals (p < 0.05). Moreover, some phylotypes, such as Fusobacterium nucleatum, Veillonella parvula, V. dispar and Eikenella corrodens were detected significantly more often in diabetic subjects than in nondiabetic subjects (p < 0.05). CONCLUSION Subjects with uncontrolled type-2 diabetes and chronic periodontitis presented significant dissimilarities in subgingival biodiversity compared with nondiabetic subjects.

Tooth root dentin mineralization defects in a mouse model of hypophosphatasia.

Tissue‐nonspecific alkaline phosphatase (TNAP) is expressed in mineralizing tissues and functions to reduce pyrophosphate (PPi), a potent inhibitor of mineralization. Loss of TNAP function causes hypophosphatasia (HPP), a heritable disorder marked by increased PPi, resulting in rickets and osteomalacia. Tooth root cementum defects are well described in both HPP patients and in Alpl−/− mice, a model for infantile HPP. In Alpl−/− mice, dentin mineralization is specifically delayed in the root; however, reports from human HPP patients are variable and inconsistent regarding dentin defects. In the current study, we aimed to define the molecular basis for changes in dentinogenesis observed in Alpl−/− mice. TNAP was found to be highly expressed by mature odontoblasts, and Alpl−/− molar and incisor roots featured defective dentin mineralization, ranging from a mild delay to severely disturbed root dentinogenesis. Lack of mantle dentin mineralization was associated with disordered and dysmorphic odontoblasts having disrupted expression of marker genes osteocalcin and dentin sialophosphoprotein. The formation of, initiation of mineralization within, and rupture of matrix vesicles in Alpl−/− dentin matrix was not affected. Osteopontin (OPN), an inhibitor of mineralization that contributes to the skeletal pathology in Alpl−/− mice, was present in the generally unmineralized Alpl−/− mantle dentin at ruptured mineralizing matrix vesicles, as detected by immunohistochemistry and by immunogold labeling. However, ablating the OPN‐encoding Spp1 gene in Alpl−/− mice was insufficient to rescue the dentin mineralization defect. Administration of bioengineered mineral‐targeting human TNAP (ENB‐0040) to Alpl−/− mice corrected defective dentin mineralization in the molar roots. These studies reveal that TNAP participates in root dentin formation and confirm that reduction of PPi during dentinogenesis is necessary for odontoblast differentiation, dentin matrix secretion, and mineralization. Furthermore, these results elucidate developmental mechanisms underlying dentin pathology in HPP patients, and begin to explain the reported variability in the dentin/pulp complex pathology in these patients.

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.

Deficiency in Acellular Cementum and Periodontal Attachment in Bsp Null Mice

Bone sialoprotein (BSP) is an extracellular matrix protein found in mineralized tissues of the skeleton and dentition. BSP is multifunctional, affecting cell attachment and signaling through an RGD integrin-binding region, and acting as a positive regulator for mineral precipitation by nucleating hydroxyapatite crystals. BSP is present in cementum, the hard tissue covering the tooth root that anchors periodontal ligament (PDL) attachment. To test our hypothesis that BSP plays an important role in cementogenesis, we analyzed tooth development in a Bsp null (-/-) mouse model. Developmental analysis by histology, histochemistry, and SEM revealed a significant reduction in acellular cementum formation on Bsp-/- mouse molar and incisor roots, and the cementum deposited appeared hypomineralized. Structural defects in cementum-PDL interfaces in Bsp-/- mice caused PDL detachment, likely contributing to the high incidence of incisor malocclusion. Loss of BSP caused progressively disorganized PDL and significantly increased epithelial down-growth with aging. Bsp-/- mice displayed extensive root and alveolar bone resorption, mediated by increased RANKL and the presence of osteoclasts. Results collected here suggest that BSP plays a non-redundant role in acellular cementum formation, likely involved in initiating mineralization on the root surface. Through its importance to cementum integrity, BSP is essential for periodontal function.

Current perspective of the impact of smoking on the progression and treatment of periodontitis

This literature review provides an overview of the current scenario regarding the impact of smoking on the progression and treatment of periodontitis; clinical, microbiological and immunological data from studies from our and other groups are presented. In general, preclinical and clinical data are unanimous in demonstrating that smokers present increased susceptibility, greater severity and faster progression of periodontal disease compared with nonsmokers. The evidence further demonstrates that smokers lose more teeth and have a less favorable response to therapy than do nonsmokers. Although it is well established that smoking significantly impacts on the onset, progression and outcome of periodontal disease, the mechanisms involved remain unclear. More importantly, some of the reported deleterious effects of smoking on periodontal tissues have been reported to be reversible upon participation in smoking-cessation programs. Therefore, clinicians should strongly advise smokers to enroll in cessation strategies, even temporarily, in order to improve the overall outcome.

Wnt/β‐catenin pathway regulates bone morphogenetic protein (BMP2)‐mediated differentiation of dental follicle cells

BACKGROUND AND OBJECTIVE Bone morphogenetic protein 2 (BMP2)-induced osteogenic differentiation has been shown to occur through the canonical Wnt/βcatenin pathway, whereas factors promoting canonical Wnt signaling in cementoblasts inhibit cell differentiation and promote cell proliferation in vitro. The aim of this study was to investigate whether putative precursor cells of cementoblasts, dental follicle cells (murine SVF4 cells), when stimulated with BMP2, would exhibit changes in genes/proteins associated with the Wnt/β-catenin pathway. MATERIAL AND METHODS SVF4 cells were stimulated with BMP2, and the following assays were carried out: (i) Wnt/β-catenin pathway activation assessed by western blotting, β-catenin/transcription factor (TCF) reporter assays and expression of the lymphoid enhancer-binding factor-1 (Lef1), transcription factor 7 (Tcf7), Wnt inhibitor factor 1 (Wif1) and Axin2 (Axin2) genes; and (ii) cementoblast/osteoblast differentiation assessed by mineralization in vitro, and by the mRNA levels of runt-related transcription factor 2 (Runx2), osterix (Osx), alkaline phosphatase (Alp), osteocalcin (Ocn) and bone sialoprotein (Bsp), determined by quantitative PCR after treatment with wingless-type MMTV integration site family, member 3A (WNT3A) and knockdown of β-catenin. RESULTS WNT3A induced β-catenin nuclear translocation and up-regulated the transcriptional activity of a canonical Wnt-responsive reporter, suggesting that the Wnt/β-catenin pathway functions in SVF4 cells. Activation of Wnt signaling with WNT3A suppressed BMP2-mediated induction of cementoblast/osteoblast maturation of SVF4 cells. However, β-catenin knockdown showed that the BMP2-induced expression of cementoblast/osteoblast differentiation markers requires endogenous β-catenin. WNT3A down-regulated transcripts for Runx2, Alp and Ocn in SVF4 cells compared with untreated cells. In contrast, BMP2 induction of Bsp transcripts occurred independently of Wnt/β-catenin signaling. CONCLUSION These data suggest that stabilization of β-catenin by WNT3A inhibits BMP2-mediated induction of cementoblast/osteoblast differentiation in SVF4 cells, although BMP2 requires endogenous Wnt/β-catenin signaling to promote cell maturation.