Quantifying oral inflammatory load: oral neutrophil counts in periodontal health and disease.

BACKGROUND AND OBJECTIVE: Neutrophils are the primary white blood cells that are recruited to fight the initial phases of microbial infections. While healthy norms have been determined for circulating blood neutrophil counts in order to identify patients with suspected systemic infections, the levels of oral neutrophils (oPMNs) in oral health and in the presence of periodontal diseases have not been described. It is important to address this deficiency in our knowledge as neutrophils are the primary immune cell present in the crevicular fluid and oral environment and previous work has suggested that they may be good indicators of overall oral inflammation and periodontal disease severity. The objective of this study was to measure oPMN counts obtained in a standardized oral rinse from healthy patients and from those with chronic periodontal disease in order to determine if oPMN levels have clinical relevance as markers of periodontal inflammation. A parallel goal of this investigation was to introduce the concept of 'oral inflammatory load', which constitutes the inflammatory burden experienced by the body as a consequence of oral inflammatory disease. MATERIAL AND METHODS: Periodontal examinations of patients with a healthy periodontium and chronic periodontal disease were performed (n = 124). Two standardized consecutive saline rinses of 30 s each were collected before patient examination and instrumentation. Neutrophils were quantified in the rinse samples and correlated with the clinical parameters and periodontal diagnosis. RESULTS: Average oPMN counts were determined for healthy patients and for those with mild, moderate and severe chronic periodontal diseases. A statistically significant correlation was found between oPMN counts and deep periodontal probing, sites with bleeding on probing and overall severity of periodontal disease. CONCLUSIONS: oPMN counts obtained through a 30-s oral rinse are a good marker of oral inflammatory load and correlate with measures of periodontal disease severity.

Periodontal Inflammation Primes the Systemic Innate Immune Response.

The presence of periodontal diseases (PDs) often strongly correlates with other severe chronic inflammatory conditions, including cardiovascular disease, diabetes, and arthritis. However, the mechanisms through which these diseases interact are unclear. In PD, tissue and bone destruction in the mouth is driven by elevated recruitment of polymorphonuclear neutrophils (PMNs), which are primed and recruited from the circulation to sites of inflammation. We predicted that systemic effects on PMN mobilization or priming could account for the interaction between PD and other inflammatory conditions. We tested this using a mouse model of ligature-induced PD and found elevated PMN counts specifically in bone marrow, supporting a systemic effect of periodontal tissue inflammation on PMN production. In contrast, mice with induced peritonitis had elevated PMN counts in the blood, peritoneum, and colon. These elevated counts were further significantly increased when acute peritonitis was induced after ligature-induced PD in mice, revealing a synergistic effect of multiple inflammatory events on PMN levels. Flow cytometric analysis of CD marker expression revealed enhanced priming of PMNs from mice with both PD and peritonitis compared to mice with peritonitis alone. Thus, systemic factors associated with PD produce hyperinflammatory PMN responses during a secondary infection. To analyze this systemic effect in humans, we induced gingival inflammation in volunteers and also found significantly increased activation of blood PMNs in response to ex vivo stimulation, which reverted to normal following resolution of gingivitis. Together, these results demonstrate that periodontal tissue inflammation has systemic effects that predispose toward an exacerbated innate immune response. This indicates that peripheral PMNs can respond synergistically to simultaneous and remote inflammatory triggers and therefore contribute to the interaction between PD and other inflammatory conditions. This suggests larger implications of PD beyond oral health and reveals potential new approaches for treating systemic inflammatory diseases that interact with PD.

Oral and Blood Neutrophil Activation States during Experimental Gingivitis.

Polymorphonuclear neutrophils (PMNs) are the primary leukocytes present in the healthy and inflamed oral cavity. While unique PMN activation states have been shown to differentiate health and periodontitis, little is known about the changes in PMN activation states that occur during the transition from periodontal health to gingivitis. The objective of this study was to characterize oral and circulatory PMNs during induction and resolution of experimental gingivitis. Healthy volunteers were recruited to undergo experimental gingivitis. Clinical assessment of pocket depths, bleeding on probing, gingival index, and plaque index, as well as flow cytometric analysis of CD (cluster of differentiation) activation markers on blood and oral PMNs, was performed weekly. All clinical parameters increased significantly during the induction period and returned to baseline levels during the resolution phase. During the induction phase, while oral PMN counts increased, oral PMN activation state based on surface expression of CD63, CD11b, CD16, and CD14 was diminished compared to those seen in health and during the resolution phase. PMNs in circulation during onset showed increased activation based on CD55, CD63, CD11b, and CD66a. Using clinical parameters and oral PMN counts assessed at day 21, we noted 2 unique disease patterns where one-third of subjects displayed an exaggerated influx of oral PMNs with severe inflammation compared to the majority of the population who experienced a moderate level of inflammation and PMN influx. This supports the notion that PMN influx and severe inflammatory changes during gingivitis could identify subjects at risk for the development of severe gingival inflammation and progression toward destructive periodontitis. This study demonstrates that oral PMN activation states are reduced in gingivitis and suggest that only in periodontitis do PMNs become hyperactivated and tissue damaging. Knowledge Transfer Statement: Our article creates a paradigm for future studies of the evolution of essential oral and circulatory biomarkers to identify individuals at risk to develop periodontitis at an early stage of periodontal disease, which is reversible upon proper oral hygiene practices and dental treatments.

The relationship between mouthrinse matrix metalloproteinases (MMP-1, 8, 13) and albumin levels with the degree of oral mucositis in allogeneic stem cell transplant patients.

Our aim was to examine the relationship between mouthrinse matrix metalloproteinases (MMPs) and whole albumin levels (AL) relative to oral mucositis (OM) in allogeneic stem cell transplant (alloSCT) patients. Mouthrinse vertebrate collagenase levels are positively correlated with connective tissue destruction (CTD) in periodontitis and may also be involved in CTD associated with OM. Increases in salivary AL have been noted prior to OM onset and may serve as a predictive tool for OM and as a positive control in this study. A total of 23 alloSCT patients were visited eight times over 4 weeks following the transplant. OM was scored via a previously validated examiner-based ordinal system. Mouthrinse samples were collected and analyzed for MMP-1, 8, 13 (members of the vertebrate collagenase group) and AL. No significant correlation was found for MMP levels relative to OM scores. AL were positively and significantly associated with OM scores (P<0.001). MMP levels may not be an important factor in OM development and severity; however, mouthrinse AL may serve as a more objective measure of OM development and severity.

Factitious injury of the oral mucosa: a case series.

Gingival and oral mucosal tissues can be the site of a number of mucocutaneous and ulcerative conditions. Generally, these are not difficult to identify on the basis of clinical characteristics, and diagnosis can be aided by the use of routine histopathological and immunopathological techniques as well as other laboratory investigations. Self-induced or factitious injury (FI) of the oral mucosal tissues may present a confusing clinical picture, and be diagnosed erroneously as a mucocutaneous disorder in spite of the absence of appropriate pathological and immunopathological findings, or a failure to respond to routine treatment. A case series is presented here outlining 4 cases of FI which presented initially as mucocutaneous disease. These cases were investigated to rule out systemic or local causes, in order to establish a diagnosis of FI. Treatment of these conditions was facilitated with placebo or sham procedures which were designed primarily to cover the lesions. In most cases, the self injurious behavior could be linked to secondary gain.

Effects of Porphyromonas gingivalis 2561 extracts on osteogenic and osteoclastic cell function in co-culture.

This study was undertaken to determine the direct effects of extracts derived from Porphyromonas gingivalis on bone formation and mineral resorption in an osteogenic/osteoclastic cell in vitro co-culture model. Osteogenic bone marrow derived stromal cells were isolated from 18-day old embryonic chickens, while osteoclastic cells were isolated from laying white Leghorn hens on calcium deficient diets. Osteoclastic cells (5 x 10(5)) were seeded onto mineral thin films and suspended above osteogenic cells (1 x 10(4)) already plated on the bottoms of tissue culture plate wells. Sonicated P. gingivalis 2561 extracts were prepared from whole bacterial cells and added in varying proportions (0 to 2 microg/ml) to the co-culture growth medium. These co-cultures, and appropriate mono-culture controls, were incubated for a further 4 days. Parameters of bone forming cell activity including alkaline phosphatase activity, calcium and inorganic phosphate accumulation were performed on the osteogenic cells. Mineral substrate resorption by osteoclastic cells was assessed morphometrically. In their respective mono-cultures, the addition of P. gingivalis sonicate to the culture medium had no effect on osteoclastic mineral resorption, but significantly inhibited osteogenesis (up to 45%; P <0.05). In co-cultures, however, the sonicate induced significant increases in mineral resorption (up to 70%; P <0.05), whereas bone forming cell activity was still inhibited, although to a significantly lesser extent than in mono-cultures (up to 25%; P <0.05). These results suggest that P. gingivalis sonicate induced up-regulation of mineral resorption may be mediated via osteogenic cells.

Management of gingival vitiligo with the use of a tattoo technique.

The purpose of this report is to describe the management of gingival vitiligo in a black female. This patient was referred by her psychiatrist to the Mount Sinai Hospital Dental Department (Toronto) for assessment of progressive loss in gingival pigmentation. According to her psychiatrist this loss of pigmentation, which the patient indicated may be considered in Africa as a hallmark of HIV infection, was a significant exogenous factor in relation to the patient's ongoing clinical depression. After obtaining informed consent, a modification of a tattooing method used for skin was applied to the patient's attached gingival tissues. Test sites were tattooed prior to performing full gingival tattooing under local anaesthesia. The results demonstrate that it was possible to restore this patient's gingival pigmentation in a highly esthetically acceptable manner. The resulting coloration was reminiscent of the patient's natural pigmentation that had been lost ostensibly because of her systemic disorder. Our findings also showed that the artificial pigmentation established via the tattoo method was stable 4 months postoperatively and continues to be stable, as expected. A profound improvement in the patient's mood was noted.

Inhibition of osteogenesis in vitro by a cigarette smoke-associated hydrocarbon combined with Porphyromonas gingivalis lipopolysaccharide: reversal by resveratrol.

BACKGROUND: Smoking and infection with Gram-negative bacterial pathogens are risk factors for alveolar bone loss. The aims of this study were: 1) to examine the combined effects of an aryl hydrocarbon, benzo[a]pyrene (BaP), that is concentrated in cigarette smoke, and lipopolysaccharide (LPS) extracted from Porphyromonas gingivalis on osteogenesis in a rat bone marrow cell (RBMC) model of osteogenesis; and 2) to determine whether resveratrol (Res), an aryl hydrocarbon receptor antagonist, could reverse the putative inhibitory effects of BaP + LPS on osteogenesis. METHODS: LPS of P. gingivalis strain 2561 was introduced in various concentrations to the RBMC in 96-well plates and kept in culture for 8 to 12 days. The same protocol was used for studying BaP and LPS + BaP combinations. Following the incubation periods, parameters of osteogenesis were measured, including formation of mineralized bone nodules, alkaline phosphatase activity, and total cell protein. Transcription of the pro-inflammatory cytokine interleukin (IL)-1beta in the cultures was determined by reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Bone nodule formation generally decreased significantly with increasing LPS concentrations (P<0.05), whereas total cell protein decreased only slightly (P>0.05). BaP in previously high concentrations alone also caused a significant dose-dependent decrease in bone nodule formation (P<0.05) but when half maximal doses were used, significant decreases were most often seen when LPS was added. Hence, in combination, the inhibitory effects of LPS + BaP on osteogenesis were additive, inhibiting bone nodule formation up to 9-fold. Resveratrol partially reversed the inhibitory effects of low concentrations of LPS alone, and completely reversed the inhibition of nodule formation when low concentrations of LPS were combined with BaP. IL-1beta expression generally fluctuated inversely to the inhibitory activity of LPS, LPS + BaP, and LPS + BaP + Res combinations. CONCLUSIONS: Smoke-derived aryl hydrocarbons and bacterial LPS may act additively to inhibit bone formation. The findings may explain, in part, why net periodontal bone loss is greater and bone healing is less successful in smokers than non-smokers with periodontal infections. Reversal of the inhibitory effects in vitro by resveratrol suggests that this phytoalexin should be studied further for its potential therapeutic value, given its aryl hydrocarbon receptor antagonism and apparent anti-inflammatory activity.

Reduced neuropsychologic measures as predictors of treatment outcome in patients with temporomandibular disorders.

AIMS: To determine via a prospective investigation whether the presence of neuropsychologic or cognitive deficiencies could be identified in patients with temporomandibular disorders (TMD) and used to predict treatment outcome. This was based on the theory that measurable reductions in neuropsychologic and cognitive function might have a negative impact on treatment outcome in patients with essentially nontraumatic TMD, as has been shown for patients with posttraumatic TMD. METHODS: Various neuropsychologic, psychosocial, and clinical parameters (including but not limited to the Peterson-Peterson Consonant Trigram Test and the California Verbal Learning Test) were used to pretest patients suffering from TMD prior to treatment. Patients were then entered into treatment, after which determination of treatment success was made both by the use of visual analog scales for pain and global transitional outcome measures (e.g., "better," responders versus "same/worse," nonresponders). After determination of treatment success was made, treatment response was correlated with the various clinical, cognitive, and neuropsychologic test scores. RESULTS: Overall, the nonresponders did worse in both the neuropsychologic and psychosocial assessments, with greater memory deficits, sleep disturbances, depression, and fatigue and lower energy levels as compared to responders. Among the best predictors of treatment outcome were the Peterson-Peterson Consonant Trigram Test scores, as well as the scores on the California Verbal Learning Test (i.e., poorer test outcomes predicted nonresponse). Neither responders nor nonresponders could be distinguished from one another based on clinical parameters of maximum interincisal opening or muscle tenderness. Three psychosocial variables were also found to be predictors of poor outcome: sleep disturbance, fatigue, and income. Pretreatment pain on chewing was also found to be a reliable predictor of poor treatment outcome. CONCLUSION: We conclude that various neuropsychologic, psychosocial, and some clinical parameters may provide pretreatment prediction of treatment outcome in an idiopathic TMD population.

Differentiation between musculoligamentous, dentoalveolar, and neurologically based craniofacial pain with a diagnostic questionnaire.

A self-administered questionnaire consisting of 21 questions, diagrams for chief pain location, and a digital pain scale was used prospectively to sort 92 patients with orofacial pain into three categories: (1) musculoligamentous (ie, temporomandibular disorders); (2) neurologically based (ie, migraine, trigeminal neuralgia, tension-type headache, cluster headache, and atypical facial pain); and (3) dentoalveolar pain. Sensitivity, specificity, as well as negative and positive predictive values suggest that this questionnaire may be used reliably to identify patients with orofacial pain that fits the above-described pain categories without prior knowledge of the clinical diagnosis. Digital pain scale findings indicated that on presentation, pain level could not be correlated with any particular pain category, but when using this scale to describe past pain experience, patients with neurologically based pain selected the highest digital pain scale values up to six times more frequently than patients with musculoligamentous or dentoalveolar pain. Patients with musculoligamentous or dentoalveolar pain selected the lowest digital pain scale values up to 15 times more frequently than those with neurologically based pain. Although this questionnaire may be used for initial categorization of pain, there is still no substitute for a thorough history and clinical examination.

Evaluation of temporomandibular joint internal derangement.

The incidence of internal derangement of the temporomandibular joint has been documented in patients with temporomandibular disorders. However, the detection and diagnosis of a displacement of the temporomandibular joint disc in relation to internal derangement is not always accurate, and it varies according to the method of examination. A prospective clinical investigation of 26 patients (45 temporomandibular joints) with signs and symptoms of temporomandibular joint pain and dysfunction was completed to examine the accuracy of clinical examination, sagittal recording device tracings, arthrography, and magnetic resonance imaging in detecting internal derangement in the temporomandibular joint. A group of 16 asymptomatic control subjects (32 temporomandibular joints) was examined for the presence of internal derangement by the methods under consideration. Incidence of bilateral internal derangement in the temporomandibular joints of the symptomatic patients was also assessed. Findings obtained through clinical examination and sagittal recording device tracings agreed most often with the arthrographic findings of internal derangement. Magnetic resonance imaging often failed to detect the presence of arthrographically detected internal derangement. Internal derangement was identified bilaterally in a significant number of patients, despite the absence of bilateral symptoms. This incidence varied according to the technique used. In the control group, 9% of the temporomandibular joints that had been assessed as normal according to clinical examination and sagittal recording device tracings were found to have internal derangement according to magnetic resonance imaging.

Clinical management of avulsed permanent incisors using Emdogain: initial report of an investigation.

The enamel matrix derivative Emdogain was recently approved for clinical use in a number of countries, including Canada. It has been shown to stimulate regeneration of periodontal ligament following periodontal surgery in adults. This paper reviews pertinent clinical and laboratory studies of Emdogain and describes the protocol and methods used for a longitudinal outcome study of replantation of avulsed permanent incisors in children and adolescents. Application of these methods is described in an illustrative case report of Emdogain use. This paper is meant to inform clinicians and guide those who are instituting similar investigations.

Periodontitis as an early presentation of HIV infection.

OBJECTIVE: To determine whether the presence of rapidly progressive periodontitis (RPP) in people at high risk for acquired immunodeficiency syndrome (AIDS) may be the first symptom of previously unrecognized human immunodeficiency virus (HIV) infection. DESIGN: Case series. SETTING: Dental clinic. PATIENTS: Twenty patients who presented or were referred to the dental clinic over 6 months for the treatment of unexplained RPP and were at high risk for AIDS. OUTCOME MEASURES: Diagnosis of HIV infection: identification of candidal organisms in cytologic smears, determination of complete and differential blood counts and of ratio between T4 (helper) and T8 (suppressor) lymphocytes, and performance of HIV antibody assays. MAIN RESULTS: All of the patients were men, although sex was not an inclusion criterion. Sixteen (80%) of the 20 patients were found to have HIV infection. Four had been aware that they were HIV positive: two admitted it only when their T4:T8 ratio was known and the other two when the T4:T8 test was explained or requested. Fifteen of the patients were homosexual, three came from AIDS-endemic areas, and two had hemophilia. The RPP was responsible for alveolar bone loss in all of the patients. One patient lost bone in one site because of localized osteomyelitis. Only five patients had concurrent candidal overgrowth, and three had Kaposi's sarcoma. The mean T4:T8 ratio was 0.57 (standard deviation 0.52). CONCLUSIONS: These findings suggest that periodontal disease may be one of the first clinical presentations of previously undiagnosed HIV infection. Thus, patients at high risk for AIDS who present with aggressive periodontal disease should be investigated for possible HIV infection. However, further, prospective studies are required to confirm the contention that RPP is one of the first signs of HIV infection or AIDS.

Characterization of inhibitory effects of suspected periodontopathogens on osteogenesis in vitro.

By using an in vitro bone-forming culture system, the chick periosteal osteogenesis (CPO) model, the direct effects on osteogenesis of sonicated extracts derived from oral bacteria were examined. Both extracts from bacterial species having strong associations with periodontal diseases (Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, and Prevotella intermedia, hereinafter referred to as suspected periodontopathogens) and extracts from species not correlated with periodontal disease (Streptococcus sanguis, Veillonella atypica, and Prevotella denticola, hereinafter referred to as nonpathogenic bacteria) were tested. All bacterial cultures were grown under standard anaerobic culture conditions. Sonicated bacterial extracts were prepared from the bacterial pellet. These were added in various proportions to the CPO cultures. Parameters of osteogenesis, including alkaline phosphatase activity, calcium and P(i) accumulation, and collagen synthesis, were measured in 6-day-old cultures. Compared with controls grown in the absence of bacterial products, osteogenesis was inhibited significantly in cultures treated with extracts derived from the suspected periodontopathogens. No osteogenic inhibition was observed in cultures treated with extracts from the nonpathogenic bacteria. These results suggest that the ability to inhibit osteogenesis in vitro may be a pathogenic property shared by a limited group of species. Further characterization of the P. gingivalis extracts revealed that both proteinaceous and nonproteinaceous products, including lipopolysaccharide, were able to inhibit osteogenesis. P. gingivalis extract-mediated inhibition of osteogenesis in CPO cultures was blocked by indomethacin, implicating prostaglandins in the regulation of the bacterial effects. The bacterial extracts had either reversible or irreversible inhibitory effects on osteogenesis when added after differentiation or before/during differentiation of bone cells, respectively.

Direct effects of metabolic products and sonicated extracts of Porphyromonas gingivalis 2561 on osteogenesis in vitro.

It is well documented that oral microorganisms play a significant role in the initiation and progression of periodontal disease. By using various in vitro models, it has been shown that some bacteria considered periodontal pathogens or their products can stimulate bone resorption and some other parameters of osteoblast-like cell activity. However, the effects of these organisms and their products on osteogenesis itself are not known. This study was undertaken to determine the direct effects of metabolic products and sonicated extracts of Porphyromonas gingivalis on bone formation in the chick periosteal osteogenesis model. Cultures of P. gingivalis 2561 were grown under standard anaerobic culture conditions. The spent medium was collected, and following centrifugation, sonicated bacterial extracts were prepared from the bacterial pellet. These were added in various proportions to the chick periosteal osteogenesis cultures. Sonicated extracts were further fractionated into five molecular-size ranges and similarly tested. Parameters of osteogenesis, including alkaline phosphatase activity, calcium and Pi accumulation, and collagen synthesis, were measured on 6-day-old cultures. Compared with controls devoid of bacterial products, osteogenesis was inhibited significantly in cultures treated with either conditioned medium or extracts obtained from P. gingivalis. Various amounts of inhibitory activity were observed in the different ultrafiltration molecular-size fractions, with very profound inhibitory effects observed in the < 5-kDa range. Histological observations indicated the presence of cells, some bone, and/or new fibrous connective tissue at all concentrations, indicating that toxicity was not a factor. These results suggest that periodontal pathogens such as P. gingivalis might contribute to the bone loss in periodontal diseases not only by stimulating resorption but, possibly, by inhibiting bone formation directly.

Prosol-chlorhexidine irrigation reduces the incidence of bacteremia during ultrasonic scaling with the Cavi-Med: a pilot investigation.

The purpose of this pilot investigation was to determine whether the incidence of bacteremia following subgingival ultrasonic scaling and root planing could be reduced by the use, pre- and intraoperatively, of an irrigant containing 0.12 per cent chlorhexidine (CHX); Prosol. Individuals having evidence of significant periodontal disease (minimum of seven sites per quadrant 4.0 mm and bleeding on probing) were entered into this study. By use of a random number table, patients were assigned to either the experimental or control groups. The procedures, as described below, were carried out in a double blind fashion so that neither the investigator nor the patient was aware of whether Prosol or placebo was being used. The placebo solution was flavored to make it indistinguishable from Prosol. Patients were first anesthetized. Their gingival crevices were then irrigated using the Cavi-Med ultrasonic scaler. At this point, the ultrasonic action was not activated. Ten minutes later, ultrasonic scaling and root planing with the Cavi-Med unit were begun with a continuous flow of either the placebo or control solutions. Blood samples were taken preoperatively, while postoperative samples were taken one minute after completing the scaling of each quadrant and then 10 minutes after scaling the second quadrant. Routine aerobic and anaerobic bacterial culture methods were used to identify viable blood-borne bacteria. The results show that there was no difference in the distribution or presentation of periodontal disease between the experimental and control quadrants.(ABSTRACT TRUNCATED AT 250 WORDS)