Identification of Genes Differentially Expressed in Simvastatin-Induced Alveolar Bone Formation.

Local delivery of simvastatin (SIM) has exhibited potential in preventing inflammation and limiting bone loss associated with experimental periodontitis. The primary aim of this study was to analyze transcriptome changes that may contribute to SIM's reduction of periodontal inflammation and bone loss. We evaluate the global genetic profile and signaling mechanisms induced by SIM on experimental periodontitis bone loss and inflammation. Twenty mature female Sprague Dawley rats were subjected to ligature-induced experimental periodontitis around maxillary second molars (M2) either unilaterally (one side untreated, n = 10) or bilaterally (n = 10). After the ligature removal at day 7, sites were injected with either carrier, pyrophosphate (PPi ×3), 1.5-mg SIM-dose equivalent SIM-pyrophosphate prodrug, or no injection. Three days after ligature removal, animals were euthanized; the M1-M2 interproximal was evaluated with µCT, histology, and protein expression. M2 palatal gingiva was harvested for RNA sequencing. Although ligature alone caused upregulation of proinflammatory and bone catabolic genes and proteins, seen in human periodontitis, SIM-PPi upregulated anti-inflammatory (IL-10, IL-1 receptor-like 1) and bone anabolic (insulin-like growth factor, osteocrin, fibroblast growth factor, and Wnt/ ß-catenin) genes. The PPi carrier alone did not have these effects. Genetic profile and signaling mechanism data may help identify enhanced pharmacotherapeutic approaches to limit or regenerate periodontitis bone loss. © 2018 The Authors. JBMR Plus Published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

Impact of Modifiable Risk Factors on Bone Loss During Periodontal Maintenance.

OBJECTIVES: The aim of this study was to analyze modifiable patient risk factors from dental chart histories and radiographs for progressive mild-moderate periodontitis during periodontal maintenance (PM). METHODS AND MATERIALS: Bitewing radiographs of 442 elderly periodontal maintenance patients were taken before and after two years of periodontal maintenance. Each progressive periodontitis (PP) patient (with at least one site of posterior interproximal bone loss of ≥2 mm, n=71) was matched to a periodontitis stable (PS) patient (no sites with bone loss, n=71) of the same gender and age (±five years) to control for these variables and was compared for measurements of general patient (medical history, smoking, hygiene and compliance habits) and tooth-related (bone loss, overhangs, interproximal dimensions) factors at baseline. Fisher exact and t-tests were used to compare groups. RESULTS: While the elderly PM patients with mild-moderate periodontitis were generally stable, 71 of 442 were PP patients. No significant differences from PS patients were observed at baseline with regard to the systemic factors measured. However, the PP group had less cementoenamel junction to bone length (bone loss p<0.0001) and more interproximal width (2.3±1.0 mm) than did the PS group (1.7±0.6 mm, p=0.0016). This was reflected in more open sites without adjacent tooth contact in PP (42% vs 15%, p=0.0006). CONCLUSIONS: In the short term, systemic and behavior factors are of limited value in identifying mild-moderate periodontitis patients on PM at increased risk of bone loss. However, interproximal width and lack of adjacent tooth contacts are related to the likelihood of losing interproximal bone during periodontal maintenance, suggesting the need for restorative therapy.

Effects of local simvastatin-alendronate conjugate in preventing periodontitis bone loss.

BACKGROUND AND OBJECTIVE: Local host-modulation therapy is an emerging approach to prevent disease progression in sites with moderate periodontitis. The combination of simvastatin and alendronate would be an intriguing host-modulatory strategy because of the bone-anabolic properties of simvastatin and the antiresorptive/bone-targeting characteristics of alendronate. The objective of this study was to evaluate the effects of local administration of a simvastatin-alendronate-ß-cyclodextrin (SIM-ALN-CD) conjugate for preventing experimental periodontitis bone loss. MATERIAL AND METHODS: Twenty-four mature female Sprague-Dawley rats were treated with three, 12 µL injections, administered one week apart, bilaterally into the palatal/interproximal gingiva. The injections contained: (i) a conjugate of 0.5 mg of SIM and 3.75 mg of ALN-CD in H2 O; (ii) H2 O alone; or (iii) no treatment. One week later, the same sites were subjected to induction of experimental periodontitis by three injections (i.e. one injection administered every other day for five d) of 0.01 mg of Escherichia coli endotoxin [lipopolysaccharide (LPS)] in phosphate-buffered saline (PBS) or PBS alone. After an additional week, the rats were killed, the palates were harvested and interproximal bone volume and adjacent thickness were calculated using microcomputed tomography. Subsequently, specimens were decalcified, and interproximal histologic sections were stained with hematoxylin and eosin for evaluation of alveolar crest osteoclasts and surrounding inflammation. Values were compared among treatment groups using analysis of variance and the Kruskal-Wallis test. RESULTS: Interproximal bone volume was reduced by LPS injections (p ≤ 0.04), yet when experimental periodontitis was preceded by treatment with SIM-ALN-CD, more bone was preserved than after treatment with carrier alone (p = 0.007). While LPS caused a significant loss in bone thickness over the palatal roots (p ≤ 0.04), the injection protocol (PBS) also caused a significant loss of palatal bone thickness (p ≤ 0.03). However, prophylactic SIM-ALN-CD injections resulted in no further loss of bone thickness during experimental periodontitis. LPS injections gave histologic evidence of increased osteoclasts and subsulcular inflammation, both of which were reduced when preceded by treatment with SIM-ALN-CD (p ≤ 0.0002). CONCLUSION: The primary conclusion of this study was that locally applied SIM-ALN-CD has the potential to prevent episodes of periodontitis bone loss.

Serum bone biomarkers and oral/systemic bone loss in humans.

UNLABELLED: We recently reported that subantimicrobial-dose doxycycline (SDD) significantly reduced serum bone-resorption biomarkers in subgroups of post-menopausal women. We hypothesize that changes in serum bone biomarkers are associated not only with systemic bone mineral density (BMD) changes, but also with alveolar bone changes over time. One hundred twenty-eight eligible post-menopausal women with periodontitis and systemic osteopenia were randomly assigned to receive SDD or placebo tablets twice daily for two years, adjunctive to periodontal maintenance. Sera were analyzed for bone biomarkers. As expected, two-year changes in a serum bone biomarker were significantly associated with systemic BMD loss at the lumbar spine (osteocalcin, bone-turnover biomarker, p = 0.0002) and femoral neck (osteocalcin p = 0.0025). Two-year changes in serum osteocalcin and serum pyridinoline-crosslink fragment of type I collagen (ICTP; bone-resorption biomarker) were also significantly associated with alveolar bone density loss (p < 0.0001) and alveolar bone height loss (p = 0.0008), respectively. Thus, we have shown that serum bone biomarkers are associated with not only systemic BMD loss, but with alveolar bone loss as well. CLINICAL TRIAL REGISTRATION INFORMATION: Protocol registered at ClinicalTrials.gov, NCT00066027.

Doxycycline effects on serum bone biomarkers in post-menopausal women.

We previously demonstrated that subantimicrobial-dose-doxycycline (SDD) treatment of post-menopausal osteopenic women significantly reduced periodontal disease progression, and biomarkers of collagen destruction and bone resorption locally in periodontal pockets, in a double-blind placebo-controlled clinical trial. We now hypothesize that SDD may also improve biomarkers of bone loss systemically in the same women, consistent with previous studies on tetracyclines (e.g., doxycycline) in organ culture and animal models of bone-deficiency disease. 128 post-menopausal osteopenic women with chronic periodontitis randomly received SDD or placebo tablets daily for 2 years adjunctive to periodontal maintenance therapy every 3-4 months. Blood was collected at baseline and at one- and two-year appointments, and sera were analyzed for bone resorption and bone formation/turnover biomarkers. In subsets of the study population, adjunctive SDD significantly reduced serum biomarkers of bone resorption (biomarkers of bone formation were unaffected), consistent with reduced risk of future systemic bone loss in these post-menopausal women not yet on anti-osteoporotic drugs.

Cyclooxygenase-2 inhibitor reduces simvastatin-induced bone morphogenetic protein-2 and bone formation in vivo.

BACKGROUND AND OBJECTIVE: Simvastatin, a cholesterol-lowering drug, also stimulates oral bone growth when applied topically, without systemic side-effects. However, the mechanisms involved in vivo are not known. We hypothesized that bone morphogenetic protein-2, nitric oxide synthase, and cyclooxygenase-2 are involved, based on prior in vitro evidence. MATERIAL AND METHODS: A rat bilateral mandible model, where 0.5 mg of simvastatin in methylcellulose gel was placed on one side and gel alone on the other, was used to quantify nitric oxide, cyclooxygenase-2 and bone morphogenetic protein-2 (via tissue extraction, enzyme activity or immunoassay), and to analyze the bone formation rate (via undecalcified histomorphometry). Cyclooxygenase-2 and nitric oxide synthase inhibitors (NS-398 and L-NAME, respectively) were administered intraperitoneally. RESULTS: Simvastatin was found to stimulate local bone morphogenetic protein-2, nitric oxide and the regional bone formation rate (p < 0.05), whereas NS-398 inhibited bone morphogenetic protein-2 and reduced the bone formation rate (p < 0.05). CONCLUSION: These data suggest an association between simvastatin-induced bone morphogenetic protein-2 and bone formation in the mandibular microenvironment, and the negative effect of cyclooxygenase-2 inhibitors on bone growth.

Bone turnover markers in serum and periodontal microenvironments.

BACKGROUND: Periodontitis is characterized by altered bone turnover, but local measurements are difficult. OBJECTIVES: The objective of this study was to develop a method to measure multiple markers of bone turnover from single samples collected at various bone surfaces of the periodontium, and to test the ratios of these markers against more traditional serum and gingival crevicular fluid (GCF) samples. MATERIALS AND METHODS: Fourteen subjects with untreated periodontitis were recruited for sampling serum, GCF (from sites > or = 5 mm probing depth that bled on probing) and washes of periodontal bone surfaces (adjacent interproximal, vestibular cortical and trabecular bone) with a novel irrigating device. All samples were analyzed for osteocalcin (OC, bone turnover marker; RIA), cross-linked N-telopeptide of type I collagen (NTx, bone resorption marker; ELISA) and albumin (Alb, serum protein; ELISA). Results were reported as ratios: OC/NTx to determine relative bone turnover, and OC/Alb or NTx/Alb to determine local OC or NTx production. RESULTS: The OC/NTx ratio was significantly higher (p < or = 0.05) in serum vs. GCF (OC undetectable), interproximal bone and cortical vestibular bone, but significantly lower than in trabecular vestibular bone. The OC/Alb ratio for serum was also statistically lower than for vestibular trabecular bone. The NTx/Alb ratio for serum was statistically lower than for GCF and all the bone wash test sites. The results indicated considerable local production of both OC and NTx. CONCLUSIONS: This system demonstrated that multiple markers of bone turnover can be harvested by irrigation from periodontal bone microenvironments. Bone turnover profiles from periodontal bone surfaces and GCF differed from systemic bone turnover profiles (serum) and may be valuable in tracking site-specific responses to disease or treatment.

Effects of locally-delivered human macrophage products and estrogen on murine inflammatory bone resorption.

The objective of this study was to use an in vivo model of periodontitis (mouse calvaria) to quantify the effects of local release of secreted human macrophage products, 17beta-estradiol (E2), and proinflammatory lipopolysaccharide (LPS) on histologic bone resorption. Human THP-1 monocytes (106) were converted to macrophage phenotype by 500 ng/ml phorbol 12-myristate- 13-acetate (PMA) and treated as follows: no stimulation or Escherichia coli LPS (10 microg/ml) alone or in combination with a physiologic dose of E2 (100 pg/ml) for 24 h in RPMI/10% FBS, washed extensively, then incubated for 24 h in serum-free media. Supernatant products were concentrated and incorporated into a 4% (w/v) methylcellulose gel. Separate gels were incorporated with the following: LPS (500 microg/animal) alone, high dose of E2 (10 ng/animal) alone, a combination of LPS + E2, or gel only (controls). Loaded or control gels were placed into a polylactic acid occlusive dome, inserted subcutaneously over the calvaria of mature ovariectomized ICR Swiss mice (8 mice x 7 groups x 2 times [5/14 days] = 112 animals), then calvaria were evaluated histologically. Macrophage stimulation with LPS alone, but not LPS in combination with E2, produced supernatants which upregulated osteoclast numbers in the suture area compared to gel controls at 5 days (p = 0.009). The addition of LPS directly to the local delivery gels significantly upregulated osteoclasts in endosteal surfaces compared to gel controls at 5 days (p = 0.024) and at 14 days (p = 0.025). The addition of E2 to LPS down-regulated resorption to a level not different from gel controls at 14 days. This in vivo model appears effective in studying inflammatory bone resorption, which may be inhibited by E2 directly or through its influence on secreted macrophage products.

Outcomes associated with supportive periodontal therapy in smokers and nonsmokers.

PURPOSE: This study evaluated the effects of smoking status on retrospective clinical and radiographic measures of periodontal disease and compared these to prospective changes in digital radiographic bone height. METHODS: Clinical data on moderate (4 to 6 mm) and severe (> 6 mm) periodontal pocket depths, and bleeding on probing, were obtained from 95 subjects on suggested three-month supportive periodontal therapy (SPT) for AAP Class III/IV periodontitis. Standardized radiographic data were obtained concerning posterior interproximal alveolar bone height from 36 of the 95 subjects using computer-assisted digital technology at baseline and one year later. The subjects were divided into groups by smoking status: current, former, and never. Data were evaluated using a general linear statistical model. RESULTS: Evaluation of clinical data showed that current smokers exhibited a significantly higher percentage of moderate (18%) and severe (1%) periodontal pockets than nonsmokers (10% and 0%, respectively; p < 0.002). Baseline radiographic interproximal bone height loss also was greater in current smokers (5.75 +/- 1.07 v. 4.64 +/- 1.16 mm). Bone loss over one year occurred in 5% of the sites, but was not significantly different among groups. CONCLUSION: Clinical periodontal pockets and bone loss accumulated more rapidly in smokers, even though they submitted to regular supportive periodontal therapy. Although this population was clinically compliant over a one year period, digital radiography showed a high incidence of detectable bone loss. The impact of smoking, however, may require longer than one year to show longitudinal changes. It is recommended that a periodic radiographic analysis on bone height be considered during SPT, and longer term studies be conducted in order to accurately identify the outcome of smoking status on this variable.

Human interleukin-1 beta and interleukin-1 receptor antagonist secretion and velocity of tooth movement.

The cytokines interleukin-1 beta (IL-1 beta) and IL-1 receptor antagonist (IL-1RA) probably play a part in orthodontic tooth movement. Here, the force magnitudes and the area of force application in the compressed periodontal ligament (PDL) were controlled and the velocity of tooth movement correlated with concentrations of IL-1 beta and IL-1RA in the gingival crevicular fluid (GCF). Seven individuals undergoing orthodontic treatment involving maxillary first premolar extractions and distal movement (bodily retraction) of the maxillary canines participated in the 84-day study. For each participant, continuous retraction forces were applied so that they received equivalent PDL stresses of 13 kPa for one canine and 4 kPa for the other. GCF cytokine concentrations from experimental and control teeth were expressed relative to total protein in the GCF and compared using an 'Activity Index' (AI)=Experimental (IL-1 beta/IL-1RA)/Control (IL-1 beta/IL-1RA). The results showed that the velocity of tooth movement in an individual was related to their AI. The correlation between AI and tooth movement was stronger from the distal (R(d)=0.78) than from the mesial (R(m)=0.65) of retracted teeth. The results demonstrate that equivalent force systems produce individual differences in cytokine production, which correlate with interindividual differences in the velocity of canine retraction.

Comparison of methods for measuring root and mucogingival sensitivity.

OBJECTIVE: The aim of this study was to compare the variability of measurements of root and mucogingival sensitivity over a 24-hour period. STUDY DESIGN: Sixteen individuals (46.8 +/- 3.2 years old) were randomly tested for pain thresholds with calibrated electrical stimulation of the root and adjacent mucosa (electric pulp tester), pressure on mucosa (pressure-sensitive probe), and cold on the root (experimental thermocoupler probe) at baseline and after 4, 8, and 24 hours. Variability between and within subjects was estimated by using analysis of variance for random effects. RESULTS: Intrasubject variability was highest for electric testing of the root and lowest for cold testing of the root across time. Of all subjects, 93% fell within 5 degrees C at all periods for the cold stimulation/moderate pain threshold. CONCLUSIONS: Calibrated cold stimulation of root areas appears to provide the most sensitive measure to assess therapeutic interventions to control cervical dental pain because of low intrasubject variability in untreated patients.

The association of cigarette smoking with alveolar bone loss in postmenopausal females.

BACKGROUND, AIMS: The purpose of this 2-year longitudinal clinical study was to determine the impact of smoking on alveolar bone height and density changes in postmenopausal females. METHODS: 59 postmenopausal women completed this study, including 38 non-smokers and 21 smokers. All subjects had a history of periodontitis, participated in 3- to 4-month periodontal maintenance programs and were within 5 years of menopause at the study outset. 4 vertical bite-wing radiographs of posterior sextants were taken at baseline and 2-year visits. Radiographs were evaluated using computer-assisted densitometric image analysis (CADIA); changes in interproximal alveolar bone density and changes in alveolar bone height were determined. Relative clinical attachment levels (RCAL) and presence/absence of plaque and bleeding on probing were recorded. RESULTS: Smokers exhibited a higher frequency of alveolar bone height loss (p<0.05) and crestal (p<0.03) and subcrestal (p<0.02) density loss relative to non-smokers. Smokers exhibited a trend (p<0.08) toward a higher frequency of > or =2.0 mm RCAL loss over the 2-year period. Plaque and bleeding on probing did not differ between smokers and non-smokers. A significant interaction, determined by repeated measures ANOVA, was noted between systemic bone mineral density (BMD) at the lumbar spine and smoking on alveolar bone density change (p<0.05). Only non-smoking patients with normal BMD realized a mean net gain in alveolar bone density; osteoporotic/osteopenic subjects (n=25) and smokers lost alveolar bone density. CONCLUSION: Postmenopausal female smokers were more likely to lose alveolar bone height and density than non-smokers with a similar periodontitis, plaque and gingival bleeding experience. In addition, both smoking and osteoporosis/osteopenia provided a negative influence on alveolar bone.

C5a modulation of interleukin-1 beta-induced interleukin-6 production by human osteoblast-like cells.

Periodontal bone resorption is controlled by osteoblast products, including interleukin (IL)-6, which are stimulated by other cytokines and complement components in the pro-inflammatory milieu. This study demonstrated that human osteoblast-like osteosarcoma cells (MG-63) responded to human recombinant (hr) C5a by releasing significant amounts of the bone-resorbing cytokine IL-6. C5a-induced release of IL-6 was enhanced 330% when cells were exposed to IL-1beta prior to C5a challenge at optimal concentrations (1.0 microg/ml C5a, 0.1 ng/ml IL-1beta). Cells simultaneously challenged with these concentrations of C5a and IL-1beta produced a 700% increase in IL-6 release relative to cells challenged with IL-1beta alone. Incubation of IL-1beta-treated cells with anti-human C5a receptor (C5aR) Ab resulted in a 78% suppression of the C5a-induced release of IL-6, but C5aR neutralization did not affect C5a/IL-1beta co-stimulation of IL-6. In addition, neither IL-1beta nor C5a significantly altered the other's cell-surface receptor relative to binding affinity or density. These results indicate that while MG-63 cells express functional C5aRs, the synergistic effect of C5a and IL-1beta on osteoblast IL-6 production is probably controlled by post-receptor signaling events. C5a agonists and antagonist used to alter critical C5a concentrations may present a new point of therapeutic intervention for the treatment of inflammatory bone resorption such as is found in periodontitis.

Longitudinal alveolar bone loss in postmenopausal osteoporotic/osteopenic women.

The purpose of this 2-year longitudinal clinical study was to investigate alveolar (oral) bone height and density changes in osteoporotic/osteopenic women compared with women with normal lumbar spine bone mineral density (BMD). Thirty-eight postmenopausal women completed this study; 21 women had normal BMD of the lumbar spine, while 17 women had osteoporosis or osteopenia of the lumbar spine at baseline. All subjects had a history of periodontitis and participated in 3- to 4-month periodontal maintenance programs. No subjects were current smokers. All patients were within 5 years of menopause at the start of the study. Four vertical bitewing radiographs of posterior sextants were taken at baseline and 2-year visits. Radiographs were examined using computer-assisted densitometric image analysis (CADIA) for changes in bone density at the crestal and subcrestal regions of interproximal bone. Changes in alveolar bone height were also measured. Radiographic data were analyzed by the t-test for two independent samples. Osteoporotic/osteopenic women exhibited a higher frequency of alveolar bone height loss (p<0.05) and crestal (p<0.025) and subcrestal (p<0.03) density loss relative to women with normal BMD. Estrogen deficiency was associated with increased frequency of alveolar bone crestal density loss in the osteoporotic/osteopenic women and in the overall study population (p<0.05). These data suggest that osteoporosis/osteopenia and estrogen deficiency are risk factors for alveolar bone density loss in postmenopausal women with a history of periodontitis.

Influence of estrogen and osteopenia/osteoporosis on clinical periodontitis in postmenopausal women.

BACKGROUND: In Western societies, more than one-third of the female population above age 65 suffers from signs and symptoms of osteoporosis, a disorder characterized by low bone mass. Estrogen deficiency is the dominant pathogenic factor for osteoporosis in women. The impact of estrogen deficiency and osteopenia/osteoporosis on periodontitis is unclear, partially due to the lack of longitudinal studies evaluating clinical signs of gingival inflammation and periodontitis progression. The purpose of this investigation was to analyze prospectively the influence of serum estradiol levels and osteopenia/osteoporosis on common clinical measurements of periodontal disease over a 2-year period. METHODS: Fifty-nine moderate/advanced adult periodontitis patients and 16 non-periodontitis subjects, all within 5 years after menopause at baseline, completed the study. Serum estradiol levels (E2) were measured yearly by 125I radioimmunoassay, and osteopenia/osteoporosis was determined by dual energy x-ray absorptiometry of the lumbar spine. Posterior interproximal clinical measurements were obtained every 6 months for the periodontitis patients, including explorer-detectable supragingival plaque, bleeding on probing (BOP) and relative clinical attachment level (RCAL). Baseline probing depths, smoking history, and demographic data also were collected. RESULTS: Data indicated that baseline demographic measurements and bone mineral density (BMD) of the lumbar spine were not different between E2-deficient and E2-sufficient subjects. Smoking activity (packs smoked/day, years smoked) was higher in periodontitis patients (P=0.0001). E2-sufficient periodontitis subjects had a higher frequency of supragingival plaque without increasing gingival inflammation. E2 status did not influence the percentage of sites losing RCAL for either periodontitis or non-periodontitis groups, but when non-smoking osteopenic/osteoporotic periodontitis patients were evaluated, E2-deficient subjects had more BOP (43.8% versus 24.4%, P<0.04) and a trend toward a higher frequency of > or =2.0 mm RCAL loss (3.8% versus 1.2%, P<0.1) than E2-sufficient subjects. CONCLUSIONS: These data suggest that E2 supplementation (serum E2>40 pg/ml) is associated with reduced gingival inflammation and a reduced frequency of clinical attachment loss in osteopenic/osteoporotic women in early menopause.

Nickel release from orthodontic arch wires and cellular immune response to various nickel concentrations.

AIMS: Results from two previous clinical studies suggested that exposure to high nickel-containing orthodontic arch wires may induce hypersensitivity in certain individuals. The purpose of this study was to measure the amount of nickel released from three types of nickel-containing arch wires into a synthetic saliva in vitro, and determine if the concentrations were sufficient to elicit either cytotoxic (trypan blue exclusion test) or stimulatory (MTT test) responses in human peripheral blood mononuclear cells (PBMCs) derived from nickel-sensitive and nickel-nonsensitive individuals. PBMCs were exposed to five concentrations of nickel sulfate solutions ranging from 0-29 ppm, and results were compared, particularly at concentrations obtained from nickel release experiments. FINDINGS: The amount of nickel released into synthetic saliva ranged from 0.4-4.1 ppb. Wires subjected to a combination of soaking and cyclic straining released significantly more nickel than those that were soaked only (p

Gingival fluid IL-1beta in postmenopausal females on supportive periodontal therapy. A longitudinal 2-year study.

Posterior interproximal alveolar bone in 59 women, within 5 years after menopause, was assessed at baseline and after 2 years of supportive periodontal therapy (history of moderate/advanced periodontitis) using digitized image analysis. Baseline lumbar spine bone mineral density, smoking status, and yearly serum estradiol (E2) levels also were obtained to group subjects. An additional 16 non-periodontitis postmenopausal women were followed 2 years for clinical and estrogen status. 2-min GCF IL-1beta levels averaged from 2 baseline periodontal pockets (in periodontitis subjects) and 2 non-periodontitis sites (in non-periodontitis and periodontitis subjects) were determined with an enzyme immunoassay. A progressive and stable site were also monitored every 6 months for GCF IL-1beta in 15 patients. Results after 2 years indicated that 17 subjects had no posterior interproximal sites losing > or =0.4 mm of alveolar crest bone height, while 13 subjects had > or =3 such sites. Using analysis of variance, none of the above clinical groupings resulted in a significant difference in mean baseline or longitudinal GCF IL-1beta levels. However, when subjects who lost alveolar crest bone height were considered, E2-sufficient subjects had significantly depressed baseline GCF IL-1beta (in past-periodontitis sites) compared to E2-deficient patients (9.1+/-2.1 versus 31.7+/-10.2 pg/2-min sample, p<0.05), suggesting E2 influences gingival IL-1beta production in progressive periodontitis patients.

Histological alterations following short-term smokeless tobacco exposure in humans.

The purpose of this study was to investigate the histological changes following short-term smokeless tobacco application in humans. Sixteen smokeless tobacco-using subjects participated in this trial. Each subject had used at least 3 cans of snuff per week for the previous 2 yr and had an existing lesion at the site of habitual snuff placement. The experimental design included subject placement of moist snuff (University of Kentucky reference tobacco brand 1S3) at a new site in the mandibular arch. At either 2 or 7 d, biopsies were taken from the new lesions and from a non-placement site in the opposing arch. The volume density of inflammatory cells was determined by point counting. Keratin and epithelial thickness were evaluated by digitizing morphometry. Data were analyzed by repeated measures analysis of variance. In 7-d lesions, increased keratin thickness was observed at the new sites compared to the non-placement sites (p = 0.05). Increased volume density of fibroblasts (p = 0.027) and decreased volume densities of macrophages (p = 0.0083) and mast cells (p = 0.05) were observed at 2 d in new versus non-placement sites. Clinically, the new sites showed erythema, erythema plus ulceration, or white striations. This study demonstrated histological and clinical changes at new snuff placement sites in as few as 2-7 d, underscoring the rapidity of tissue alterations following snuff use.

Development of smokeless tobacco-induced oral mucosal lesions.

This study examined clinical and inflammatory mediator parameters during the development of snuff-induced mucosal lesions. Nineteen smokeless tobacco (ST) users placed moist snuff at designated new placement sites over either a 2- or 7-day period. By day 2, the predominant clinical alteration was an erythematous reaction, and one-third of the subjects demonstrated white striations in combination with erythema or ulceration. By 7 days, 56% of the subjects displayed white striated lesions. Tissue concentrations (pg/mg) of IL-1beta were 4.6+/-1.6 at new placement sites as compared with 0.7+/-0.4 at control sites (P<0.05). PGE2 and IL-1alpha concentrations also were significantly higher (P<0.05) at new placement sites as compared with control sites (9.4+/-2.2 vs 4.1+/-0.7 and 6.2+/-1.3 vs 3.2+/-0.7, respectively). In view of the recognized role of PGE2 and IL-1 in immune and inflammatory functions, these mediators may play a role in the pathogenesis of clinical alterations at ST placement sites.