The Roles of FOXO1 in Periodontal Homeostasis and Disease.

Periodontitis is an oral chronic inflammatory disease that is initiated by periodontal microbial communities and requires disruption of the homeostatic responses. The prevalence of periodontal disease increases with age; more than 70% of adults 65 years and older have periodontal disease. A pathogenic microbial community is required for initiating periodontal disease. Dysbiotic immune-inflammatory response and bone remodeling are characteristics of periodontitis. The transcription factor forkhead box protein O1 (FOXO1) is a key regulator of a number of cellular processes, including cell survival and differentiation, immune status, reactive oxygen species (ROS) scavenging, and apoptosis. Although accumulating evidence indicates that FOXO1 activity can be induced by periodontal pathogens, the roles of FOXO1 in periodontal homeostasis and disease have not been well documented. The present review summarizes how the FOXO1 signaling axis can regulate periodontal bacteria-epithelial interactions, immune-inflammatory response, bone remodeling, and wound healing.

Adhesion of anaerobic periodontal pathogens to extracellular matrix proteins.

Extracellular matrix (ECM) proteins are highly abundant in the human body and can be found in various tissues, most prominently in connective tissue and basement membrane. For invasive bacterial pathogens, these structures function as physical barriers that block access to underlying tissues. The ability to bind and degrade these barriers is important for the establishment of infections and migration to other body sites. In the oral cavity, the ECM and the basement membrane (BM) are important components of the Junctional epithelium (JE) that closes the gap between the teeth surface and the mucosa. In periodontitis, the JE is breached by invading pathogenic bacteria, particularly strict anaerobic species. In periodontitis, invading microorganisms induce an unregulated and destructive host response through polymicrobial synergism and dysbiosis that attracts immune cells and contributes to the destruction of connective tissue and bone in the periodontal pocket. Colonization of the periodontal pocket is the first step to establish this infection, and binding to ECM is a major advantage in this site. Several species of strict anaerobic bacteria are implicated in acute and chronic periodontitis, and although binding to ECM proteins was studied in these species, few adhesins were identified so far, and the mechanisms involved in adhesion are largely unidentified. This review summarizes the data available on the interaction of strict anaerobic bacteria and components of the ECM.

Selective bacterial degradation of the extracellular matrix attaching the gingiva to the tooth.

The junctional epithelium (JE) is a specialized portion of the gingiva that seals off the tooth-supporting tissues from the oral environment. This relationship is achieved via a unique adhesive extracellular matrix that is, in fact, a specialized basal lamina (sBL). Three unique proteins - amelotin (AMTN), odontogenic ameloblast-associated (ODAM), and secretory calcium-binding phosphoprotein proline-glutamine rich 1 (SCPPPQ1) - together with laminin-332 structure the supramolecular organization of this sBL and determine its adhesive capacity. Despite the constant challenge of the JE by the oral microbiome, little is known of the susceptibility of the sBL to bacterial degradation. Assays with trypsin-like proteases, as well as incubation with Porphyromonas gingivalis, Prevotella intermedia, and Treponema denticola, revealed that all constituents, except SCPPPQ1, were rapidly degraded. Porphyromonas gingivalis was also shown to alter the supramolecular network of reconstituted and native sBLs. These results provide evidence that proteolytic enzymes and selected gram-negative periodontopathogenic bacteria can attack this adhesive extracellular matrix, intimating that its degradation could contribute to progression of periodontal diseases.

Impact of Oral Commensal Bacteria on Degradation of Periodontal Connective Tissue in Mice.

BACKGROUND: Innate and adaptive immunosurveillance mechanisms in response to the normal commensal bacteria can affect periodontal innate defense status. However, it is still unclear how commensal bacteria contribute to the inflammatory responses of junctional epithelium (JE) and periodontal connective tissue (PCT). The aim of the present study is to investigate the contribution of commensal bacteria on inflammatory responses in JE and PCT in mice. METHODS: The periodontal tissue of germ-free (GF) and specific-pathogen-free (SPF) mice were compared at age 11 to 12 weeks (n = 6 per group). In this study, the number of neutrophils and expression of intercellular adhesion molecule (ICAM)-1, fibroblast growth factor receptor (FGFR)-1, matrix metalloproteinase (MMP)-1, and MMP-8 within the JE and the PCT are evaluated. The collagen density was also determined in PCT stained with picrosirius red (PSR). PSR staining combined with or without polarized light microscopy has been used to assess the organization and maturation of collagen matrix. RESULTS: In the present findings, the area of JE in SPF mice was significantly greater than that in GF mice (P <0.05). In addition, the JE and PCT in SPF mice showed greater migration of neutrophils and higher expression of ICAM-1, FGFR-1, MMP-1, and MMP-8 than those in GF mice (P <0.05). Furthermore, the density of collagen in PCT in SPF mice was lower compared to GF mice (P <0.05). CONCLUSION: These results indicate that commensal bacteria induced a low-grade inflammatory state in JE and that such conditions may contribute to degradation of collagen in PCT in mice.

Smad2 is involved in Aggregatibacter actinomycetemcomitans-induced apoptosis.

Apoptosis is thought to contribute to the progression of periodontitis. It has been suggested that the apoptosis of epithelial cells may contribute to the loss of epithelial barrier function. Smad2, a downstream signaling molecule of TGF-ß receptors (TGF-ßRs), is critically involved in apoptosis in several cell types. However, the relationship between smad2 and bacteria-induced apoptosis has not yet been elucidated. It is possible that the regulation of apoptosis induced by periodontopathic bacteria may lead to novel preventive therapies for periodontitis. Therefore, in the present study, we investigated the involvement of smad2 phosphorylation in apoptosis of human gingival epithelial cells induced by Aggregatibacter actinomycetemcomitans (Aa). Aa apparently induced the phosphorylation of smad2 in primary human gingival epithelial cells (HGECs) or the human gingival epithelial cell line, OBA9 cells. In addition, Aa induced phosphorylation of the serine residue of the TGF-ß type I receptor (TGF-ßRI) in OBA9 cells. SB431542 (a TGF-ßRI inhibitor) and siRNA transfection for TGF-ßRI, which reduced both TGF-ßRI mRNA and protein levels, markedly attenuated the Aa-induced phosphorylation of smad2. Furthermore, the disruption of TGF-ßRI signaling cascade by SB431542 and siRNA transfection for TGF-ßRI abrogated the activation of cleaved caspase-3 expression and repressed apoptosis in OBA9 cells treated with Aa. Thus, Aa induced apoptosis in gingival epithelial cells by activating the TGF-ßRI-smad2-caspase-3 signaling pathway. The results of the present study may suggest that the periodontopathic bacteria, Aa, activates the TGF-ßR/smad2 signaling pathway in human gingival epithelial cells and induces apoptosis in epithelial cells, which may lead to new therapeutic strategies that modulate the initiation of periodontitis.

Increased bacterial invasion and differential expression of tight-junction proteins, growth factors, and growth factor receptors in periodontal lesions.

BACKGROUND: Many pathogens are known to modulate epithelial physical barriers, particularly tight-junction (TJ) proteins, to enter host cells and/or tissues. Growth factors have been implicated in the regulation of TJ proteins. The aim of this study is to determine differences in the levels of TJ proteins, growth factors, and their receptors in relation to bacterial invasion in diseased gingival tissues obtained from patients with periodontitis. METHODS: The presence of bacteria and expression of junctional adhesion molecule (JAM)-A, occludin, epidermal growth factor (EGF), keratinocyte growth factor (KGF), insulin-like growth factor-I (IGF-I), EGF receptor, KGF receptor, and IGF-1 receptor (IGF-1R) were evaluated in gingival tissues from healthy (n = 10) and diseased (n = 10) sites in patients with periodontitis by in situ hybridization and immunohistochemistry. RESULTS: The bacterial invasion of gingival tissue was increased in periodontal lesions compared with healthy sites. Although the levels of JAM-A and occludin were not significantly different between the healthy and diseased sites, aberrant cytoplasmic expression of JAM-A and occluding was often observed in the lesions. In addition, more leukocytes expressing JAM-A or occludin were observed within the disease-associated epithelia. Compared with the healthy sites, the differential expression of KGF, IGF-I, and IGF-1R was observed in the periodontal lesions. The levels of TJ proteins showed positive correlations with those of growth factors. CONCLUSION: The aberrant expression of growth factors and TJ proteins may contribute to increased bacterial invasion and disease progression in periodontal lesions.

Gram-positive bacteria as an antigen topically applied into gingival sulcus of immunized rat accelerates periodontal destruction.

BACKGROUND AND OBJECTIVE: Periodontitis is generally accepted to relate to gram-negative bacteria, and the host defense system influences its onset and progression. However, little is known about the relation between gram-positive bacteria and periodontitis. In this study, we topically applied gram-positive and gram-negative bacterial suspensions to the gingival sulcus in rats after immunization, and then histopathologically examined their influence on periodontal destruction. MATERIALS AND METHODS: Rats previously immunized with heat-treated and sonicated Staphylococcus aureus or Aggregatibacter actinomycetemcomitans were used as immunized groups. The non-immunized group received only sterile phosphate-buffered saline. In each animal, S. aureus or A. actinomycetemcomitans suspension was applied topically to the palatal gingival sulcus of first molars every 24 h for 10 d. Blood samples were collected and the serum level of anti-S. aureus or anti-A. actinomycetemcomitans immunoglobulin G (IgG) antibodies was determined by enzyme-linked immunosorbent assay. The first molar regions were resected and observed histopathologically. Osteoclasts were stained with tartrate-resistant acid phosphatase (TRAP). The formation of immune complexes was confirmed by immunohistological staining of C1qB. RESULTS: Serum levels of anti-S. aureus and anti-A. actinomycetemcomitans IgG antibodies in the immunized groups were significantly higher than those in the non-immunized groups were. The loss of attachment, increase in apical migration of the junctional epithelium, and decreases in alveolar bone level and number of TRAP-positive multinuclear cells in each immunized group were significantly greater than in each non-immunized group. The presence of C1qB was observed in the junctional epithelium and adjacent connective tissue in the immunized groups. CONCLUSIONS: Heat-treated and sonicated S. aureus and A. actinomycetemcomitans induced attachment loss in rats immunized with their suspensions. Our results suggest that not only gram-negative but also gram-positive bacteria are able to induce periodontal destruction.

Fusobacterium nucleatum biofilm induces epithelial migration in an organotypic model of dento-gingival junction.

BACKGROUND: Effects of Fusobacterium nucleatum (F. nucleatum) biofilm on epithelial cell proliferation, apoptotic cell death, and basement membrane constituent collagen IV production were examined in an organotypic dento-epithelial (OD-E) model. METHODS: The OD-E model was constructed by seeding keratinocytes on fibroblast-containing collagen gels and by placing tooth pieces on top. A 3-day-old biofilm either a laboratory strain (American Type Culture Collection [ATCC] 25586) or a clinical strain (Anaerobe Helsinki Negative [AHN] 9508) of F. nucleatum was placed on the top of the model. The coculture was incubated ≤24 hours. The expression and localization of Ki-67, caspase-3, and collagen IV were examined by immunohistochemistry. RESULTS: Hematoxylin and eosin staining showed epithelial migration and lateral sprouting into the connective tissue matrix in F. nucleatum OD-E cocultures. The proliferation pattern of the in vitro dento-epithelial junction was changed. In controls without bacterial challenge, the Ki-67 expression was abundant in the cells attached to the tooth, whereas in F. nucleatum biofilm-treated cultures, the Ki-67-expressing cells were more often in the connective tissue-facing side of the epithelium. An apoptotic marker caspase-3 was expressed in controls and in F. nucleatum laboratory strain ATCC cocultures throughout the epithelium, in contrast to cultures treated with F. nucleatum clinical strain AHN, in which caspase-3 was absent. Collagen IV stainings were negative in both controls and F. nucleatum cocultures. CONCLUSION: F. nucleatum biofilm coculture with OD-E model causes lateral sprouting of the epithelium with an altered epithelial proliferation pattern, resembling the histologic changes seen in vivo in the early pathogenesis of periodontal disease.

Interaction of oral bacteria with gingival epithelial cell multilayers.

Primary gingival epithelial cells were cultured in multilayers as a model for the study of interactions with oral bacteria associated with health and periodontal disease. Multilayers maintained at an air-liquid interface in low-calcium medium displayed differentiation and cytokeratin properties characteristic of junctional epithelium. Multilayers were infected with fluorescently labeled Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum or Streptococcus gordonii, and bacterial association was determined by confocal microscopy and quantitative image analysis. Porphyromonas gingivalis invaded intracellularly and spread from cell to cell; A. actinomycetemcomitans and F. nucleatum remained extracellular and showed intercellular movement through the multilayer; whereas S. gordonii remained extracellular and predominantly associated with the superficial cell layer. None of the bacterial species disrupted barrier function as measured by transepithelial electrical resistance. P. gingivalis did not elicit secretion of proinflammatory cytokines. However, A. actinomycetemcomitans and S. gordonii induced interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), IL-6 and IL-8 secretion; and F. nucleatum stimulated production of IL-1ß and TNF-α. Aggregatibacter actinomycetemcomitans, F. nucleatum and S. gordonii, but not P. gingivalis, increased levels of apoptosis after 24 h infection. The results indicate that the organisms with pathogenic potential were able to traverse the epithelium, whereas the commensal bacteria did not. In addition, distinct host responses characterized the interaction between the junctional epithelium and oral bacteria.

Virulence of major periodontal pathogens and lack of humoral immune protection in a rat model of periodontal disease.

OBJECTIVE: This study was designed to test the hypothesis that periodontal pathogens Tannerella forsythia and Porphyromonas gingivalis are synergistic in terms of virulence potential using a model of mixed-microbial infection in rats. MATERIALS AND METHODS: Three groups of rats were infected orally with either T. forsythia or P. gingivalis in mono-bacterial infections or as mixed-microbial infections for 12 weeks and a sham-infected group were used as a control. This study examined bacterial infection, inflammation, immunity, and alveolar bone loss changes with disease progression. RESULTS: Tannerella forsythia and P. gingivalis genomic DNA was detected in microbial samples from infected rats by PCR indicating their colonization in the rat oral cavity. Primary infection induced significantly high IgG, IgG2b, IgG1, and IgG2a antibody levels indicating activation of mixed Th1 and Th2 immune responses. Rats infected with the mixed-microbial consortium exhibited significantly increased palatal horizontal and interproximal alveolar bone loss. Histological examinations indicated significant hyperplasia of the gingival epithelium with moderate inflammatory infiltration and apical migration of junctional epithelium. The results observed differ compared to uninfected controls. CONCLUSION: Our results indicated that T. forsythia and P. gingivalis exhibit virulence, but not virulence synergy, resulting in the immuno-inflammatory responses and lack of humoral immune protection during periodontitis in rats.

The relationship between physiologic halitosis and periodontopathic bacteria of the tongue and gingival sulcus.

To determine the influence of oral status on halitosis, the relationship between halitosis and periodontopathic bacteria present in plaque on the tongue and the subgingival sulcus was examined in 62 periodontally healthy adults. Halitosis indicators used were the organoleptic score; gas chromatography results [total volatile sulfur compounds (VSCs) = H(2)S + CH(3)SH + (CH(3))(2)S]; Halimeter values; and the results of three clinical tests, plaque control record (PlCR), plaque index (PlI), and tongue coat status. Significant correlations with organoleptic scores was observed for PlCR, PlI, tongue coat status, VSC amounts, and Halimeter values, indicating that halitosis in periodontally healthy subjects tended to originate from tongue plaque deposits. Polymerase chain reaction analysis was used to detect six periodontopathic bacteria (Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythensis, and Treponema denticola) from the tongue and subgingival plaque. Significant effects on the organoleptic scores, tongue coat status, total VSC, H(2)S and CH(3)SH amounts, and Halimeter values were observed only for T. denticola and F. nucleatum and only in the tongue plaque, not in the subgingival plaque. Thus, therapies developed to inhibit the growth of these bacteria may lead to future treatments of halitosis.

Expression of beta-defensin-2 in human gingival epithelial cells in response to challenge with Porphyromonas gingivalis in vitro.

BACKGROUND AND OBJECTIVE: Human beta-defensin-2 (hBD-2) is an antimicrobial peptide that is produced by epithelial cells after stimulation with microorganisms and inflammatory mediators. Compared with gram-positive bacteria, gram-negative bacteria, which are typically detected in the periodontal pockets in periodontitis, elicit a stronger antibacterial peptide response of hBD-2 by epithelial cells. The purpose of this study was to investigate the expression of hBD-2 and relationships between it and inflammatory mediators in human gingival epithelial cells (HGEC) in response to challenge with Porphyromonas gingivalis in vitro. MATERIAL AND METHODS: mRNA expression of hBD-2 in HGEC stimulated with or without P. gingivalis was assessed using a semiquantitative reverse transcription-polymerase chain reaction. Primary cultured HGEC were activated by live P. gingivalis, and inflammatory cytokine production was examined using an enzyme-linked immunosorbent assay. RESULTS: The level of hBD-2 mRNA in HGEC treated with P. gingivalis increased with exposure time. After 48 h, the mRNA in P. gingivalis was significantly increased compared with that in control HGEC. The interleukin-8 production rate was much greater in stimulated HGEC than in the control HGEC, almost always showing a significant difference after 3 h. The production of interleukin-1beta was not increased as much as that of interleukin-8. CONCLUSION: These findings suggest that the expression of hBD-2 in HGEC is P. gingivalis-dependently induced and is likely to be connected with the initial stage of the inflammatory response.

Surface morphology of pocket epithelium.

The pocket epithelium in periodontitis differs from the clinically healthy epithelium in its increase in sulcular depth. However, closer surface morphological distinctions have not been described. To study the surface characteristics of pocket gingiva, the authors analyzed pocket and sulcular epithelium biopsies by scanning and transmission electron microscopy using cytochemical staining for visualization of bacterial adhesion. The clinically healthy and the marginal pocket epithelium were characterized by squamous epithelial cells joined by tight junctions and an inconspicuous surface lacking a distinctive papillary formation. The large quantity of bacteria that adhered to the clinically healthy and marginal pocket epithelium did not appear to elicit any significant defense response. The deeper part of the pocket epithelium revealed a wrinkled papillary relief, increased exfoliation of epithelial cells, leukocyte transmigration, and bacterial internalization, as well as internalization-induced epithelial apoptosis. The alteration of the deep pocket epithelium surface might be either genuine or due to environmental changes of the crevice, or both. Therefore, the periodontitis recovery after removing the deep pocket epithelium might now be related to the pathological alterations of the deep pocket epithelium.

The junctional epithelium: from health to disease.

The junctional epithelium is located at a strategically important interface between the gingival sulcus, populated with bacteria, and the periodontal soft and mineralized connective tissues that need protection from becoming exposed to bacteria and their products. Its unique structural and functional adaptation enables the junctional epithelium to control the constant microbiological challenge. The antimicrobial defense mechanisms of the junctional epithelium, however, do not preclude the development of gingival and periodontal lesions. The conversion of the junctional to pocket epithelium, which is regarded as a hallmark in disease initiation, has been the focus of intense research in recent years. Research has shown that the junctional epithelial cells may play a much more active role in the innate defense mechanisms than previously assumed. They synthesize a variety of molecules directly involved in the combat against bacteria and their products. In addition, they express molecules that mediate the migration of polymorphonuclear leukocytes toward the bottom of the gingival sulcus. Periodontopathogens-such as Actinobacillus actinomycetemcomitans or, in particular, Porphyromonas gingivalis-have developed sophisticated methods to perturb the structural and functional integrity of the junctional epithelium. Research has focused on the direct effects of gingipains, cysteine proteinases produced by Porphyromonas gingivalis, on junctional epithelial cells. These virulence factors may specifically degrade components of the cell-to-cell contacts. This review will focus on the unique structural organization of the junctional epithelium, on the nature and functions of the various molecules expressed by its cells, and on how gingipains may attenuate the junctional epithelium's structural and functional integrity.

Adhesion of Porphyromonas gingivalis strains to cultured epithelial cells from patients with a history of chronic adult periodontitis or from patients less susceptible to periodontitis.

BACKGROUND: The present study aimed to explain the interindividual variation in periodontitis susceptibility by differences in the initial adhesion rate of Porphyromonas gingivalis to the pocket epithelium of these individuals, and/or by inter-P. gingivalis strain differences in association capacity (adhesion and internalization). METHODS: Adhesion assays were performed on epithelial monolayers (cultured in vitro from pocket epithelium belonging to patients who were less or more susceptible to chronic adult periodontitis) using 11 genetically different clinical strains of P. gingivalis. RESULTS: Both the disease category (less susceptible versus susceptible) and the interstrain variation were found to have a significant effect (both P <0.05) on the initial bacterial association. The chronic adult periodontitis group showed significantly more association of P. gingivalis when compared to less susceptible patients (4.2 x 10(6) versus 3.5 x 10(6)). Also, the interstrain variation was significant, with strains Pg 4 and 5 representing the least and best associating bacteria (1.8 x 10(6) colony forming units for Pg 4, 9 x 10(6) for Pg 5). CONCLUSIONS: These results indicate that periodontitis susceptibility is influenced by both the interindividual differences in pocket epithelium (allowing more adhesion of P. gingivalis) or by the strain type by which the patient is infected (intra-species differences in adhesion capacity).

The characteristic cellular organization and CEACAM1 expression in the junctional epithelium of rats and mice are genetically programmed and not influenced by the bacterial microflora.

BACKGROUND: The epithelial cell adhesion molecule CEACAM1 exhibits an interesting dynamic expression during tooth development. It is first expressed in the reduced enamel epithelium, its expression then increases in the orally faced reduced epithelium and the overlying oral epithelium that then fuse to give rise to the junctional epithelium. The expression of CEACAM1 remains at high levels in the junctional epithelium, in contrast to the surrounding oral sulcular epithelium which shows much lower expression levels. We investigated if the high expression levels of CEACAM1 and the loosely organized cells characteristic of the junctional epithelium are genetically programmed or result from bacterial infiltration. METHODS: Oral tissues from germ-free rats and mice and animals with conventional bacterial flora were analyzed by transmission electron microscopy and immunohistochemical staining for CEACAM1. RESULTS: The junctional epithelium of both germ-free and conventional animals was identical with respect to both CEACAM1 expression and morphology. Also the presence of leukocytes was the same in both types of animals. CONCLUSIONS: The results indicate that the characteristic morphology and the high expression levels of CEACAM1 in the junctional epithelium are genetically programmed and not a result of bacterial infiltration. This suggests that CEACAM1 has an important role for the structural integrity of the junctional epithelium. This conclusion was supported by the observation that the junctional epithelium does not express any E-cadherin, which is another abundant epithelial cell adhesion molecule.

Accessing restoration margins--a multidisciplinary approach.

Dentists frequently balance periodontal, restorative, and esthetic concerns that will provide adequate tooth length, ensure periodontal health, and provide biological predictability. This report discusses the current and classical literature and provides a multidisciplinary approach of predictably "accessing" the restoration margins to facilitate long-term gingival health and tooth survival.

A correlation study of inflammatory cell mobilization in response to subgingival microbial colonization.

This study evaluated site-by-site the relations between subgingival microbial colonization and gingival tissue reactions. Experimental, deep periodontal defects were established at buccal surfaces of mandibular and maxillary canine teeth in 5 beagle dogs. The root surfaces were instrumented by a flame-shaped, fine-grained, rotating diamond point, or by a sharp curet. Following a 10-day postsurgical healing period, the dogs were fed a plaque-inducing diet for 70 days. The animals were then sacrificed and tissue blocks of the experimental sites including teeth and periodontal tissues were secured. The buccal gingiva was removed and processed for histomorphometric analysis while the teeth were prepared for scanning electron microscopic evaluation of the extent of subgingival microbial colonization. The results revealed that inflammatory cell density in the junctional epithelium and in the connective tissue were positively correlated to subgingival microbial colonization (P < 0.01). Furthermore, the degree of significance decreased with increasing distance from the plaque. The present study demonstrates that a close relation may exist between the extent of subgingival microbial colonization and inflammatory gingival tissue reactions.