Exercise-driven metabolic pathways in healthy cartilage.

OBJECTIVE: Exercise is vital for maintaining cartilage integrity in healthy joints. Here we examined the exercise-driven transcriptional regulation of genes in healthy rat articular cartilage to dissect the metabolic pathways responsible for the potential benefits of exercise. METHODS: Transcriptome-wide gene expression in the articular cartilage of healthy Sprague-Dawley female rats exercised daily (low intensity treadmill walking) for 2, 5, or 15 days was compared to that of non-exercised rats, using Affymetrix GeneChip arrays. Database for Annotation, Visualization and Integrated Discovery (DAVID) was used for Gene Ontology (GO)-term enrichment and Functional Annotation analysis of differentially expressed genes (DEGs). Kyoto Encyclopedia of Genes and Genome (KEGG) pathway mapper was used to identify the metabolic pathways regulated by exercise. RESULTS: Microarray analysis revealed that exercise-induced 644 DEGs in healthy articular cartilage. The DAVID bioinformatics tool demonstrated high prevalence of functional annotation clusters with greater enrichment scores and GO-terms associated with extracellular matrix (ECM) biosynthesis/remodeling and inflammation/immune response. The KEGG database revealed that exercise regulates 147 metabolic pathways representing molecular interaction networks for Metabolism, Genetic Information Processing, Environmental Information Processing, Cellular Processes, Organismal Systems, and Diseases. These pathways collectively supported the complex regulation of the beneficial effects of exercise on the cartilage. CONCLUSIONS: Overall, the findings highlight that exercise is a robust transcriptional regulator of a wide array of metabolic pathways in healthy cartilage. The major actions of exercise involve ECM biosynthesis/cartilage strengthening and attenuation of inflammatory pathways to provide prophylaxis against onset of arthritic diseases in healthy cartilage.

Azithromycin decreases crevicular fluid volume and mediator content.

Azithromycin enhances the response to root planing and produces anti-inflammatory effects in treating chronic lung disease. This led us to hypothesize that azithromycin inhibits inflammatory mediator production in gingiva, leading to decreased gingival crevicular fluid (GCF) volume. To test this hypothesis, ten periodontally healthy volunteers received azithromycin every 24 hours for 48 hours. GCF samples were collected from 12 maxillary interproximal sites prior to azithromycin (baseline) and 2, 4, 7, and 14 days later. Samples were assayed for IL-1beta, IL-8, TNF-alpha, VEGF, IL-6, and IL-10. With azithromycin treatment, GCF volume decreased significantly on days 2 through 7 (P < 0.05), but increased toward baseline levels on day 14. This was accompanied by a transient decrease in the content of IL-1beta, IL-8, TNF-alpha, and VEGF (P < 0.05). IL-6 and IL-10 were not detected. Since plaque was absent throughout the study, the findings suggest that azithromycin produces anti-inflammatory effects in gingiva.

Salivary detection of periodontopathic bacteria in periodontally healthy children.

BACKGROUND: Salivary occurrence of periodontopathic bacteria is of interest especially in children as a risk indicator for the transmission, development and control of periodontal disease. We assessed the prevalence of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, Prevotella nigrescens and Treponema denticola as microbial complexes in the saliva of children with mixed dentition and healthy gingiva. MATERIALS AND METHODS: Paraffin-stimulated saliva samples were collected from 41 children (22 boys and 19 girls), aged 6-13 years old. Gingival health was determined during the initial screening exam. The test bacteria were identified using a 16S rRNA-based PCR analysis. RESULTS: P. nigrescens was the most frequent species (80%), followed by T. denticola (32%), A. actinomycetemcomitans (24%) and P. gingivalis (12%). P. intermedia and T. forsythia were not detected. P. nigrescens was also common species in combinations. Paired and triple bacterial combinations were found in 24% and 20% of all children, respectively. There was no positive association between bacterial combinations in colonization and subject's gender (P>0.05, Fisher exact test). CONCLUSION: The salivary presence of P. nigrescens, T. denticola, A. actinomycetemcomitans and P. gingivalis but not P. intermedia and T. forsythia can occur in childhood without clinical signs of gingival disease. Thus, the possible risk of bacterial transmissions through saliva and, the need to screen for periodontal pathogens should be considered before mixed dentition.

Resonance frequency analysis of one-stage dental implant stability during the osseointegration period.

BACKGROUND: Currently, there is no available clinical tool to evaluate the amount of osseointegration and stability around dental implants. It has been recently suggested that changes in the stiffness of an implant in bone during healing may be monitored by using resonance frequency analysis (RFA). The aim of this study was to determine whether RFA can be integrated into the routine clinical evaluation of initial healing of dental implants. METHODS: Thirty-one patients (18 female and 13 male; mean age of 51.7 years) were included into this study. A total of 122 implants and three different, but comparable, implant designs were evaluated by using RFA. The specific transducer for each implant system was used. ISQ (implant stability quotient) readings were obtained for each implant at the time of surgery, 3 and 6 weeks postoperatively, and at the time of loading (3 or 6 months following surgery). Data were analyzed for different healing times, various anatomical locations, implant length, and type. Average time in function was 12 months. RESULTS: Two implants failed during healing. Implant stability was higher on the mandible compared to the maxilla for each implant system studied (Mann-Whitney test, P <0.01). ISQ readings decreased significantly at 3 and 6 weeks post-surgery compared to readings obtained at surgery (Wilcoxon matched pairs sign-rank tests, P <0.01). A recovery to the initial ISQ levels was noted at the time of implant loading. The possible effects of different types and lengths of implants to ISQ readings were examined. CONCLUSIONS: Results of this study support the need for a clinical tool to evaluate dental implant stability prior to loading, especially for implants placed in the maxilla. It appears that implant stability is weakest at 3 to 6 weeks in one-stage non-loaded dental implants. ISQ readings can be used to determine different healing phases and the stability of dental implants. However, it is difficult to define a general standardized range of ISQ readings for successful implant integration for various implant systems. Thus, RFA values/ISQ levels should be calibrated for each implant system separately. Further studies are needed to compare the early changes seen in immediately loaded dental implants and to determine whether there is any time in which the total recovery in ISQ levels may occur.

Radiographic evaluation of dental implants placed using an osteotome technique.

BACKGROUND: The osteotome technique has been successfully used for implant placement when a limited vertical height is available at posterior maxilla. However, it is not clear if new bone is formed at the apical portion of the implant placed by this technique without any bone graft. The aim of this study was to radiographically evaluate bone formation around dental implant surfaces exposed to the space created at the sinus floor without the presence of any graft material. METHODS: Forty patients (21 male, 19 female; mean age 46.7 years) who received a total of 75 dental implants together with indirect sinus lifting procedure were included. Initial and 6-month postoperative panoramic films were scanned and analyzed using a commercially available software program. Implants were divided into two groups: initial alveolar bone height <9 mm or > or =9 mm. This helped determine the effect of available bone and exposed implant surface on bone formation in a system where the shortest implant was 8 mm. RESULTS: The mean implant length placed at locations with <9 mm initial bone height (mean 7 +/- 1.3 mm, N = 29 implants) was 11 +/- 1.7 mm; gain in bone height was 3.9 +/- 1.9 mm. At locations where minimum bone height was 9 mm (mean 10.4 +/- 0.7 mm), 44 implants were placed with a 13.5 +/- 1.06 mm mean length. Mean gain in bone height was 2.9 +/- 1.2 mm at these sites. Two implants were lost at stage 2 surgery. The success rate after 25 months of loading was 97.3%. CONCLUSIONS: It is possible to radiographically observe a gain of approximately 3 to 4 mm of bone from the sinus floor to the implant apex. The amount of initial alveolar bone height, presence of sinus membrane perforation, and the amount of exposed implant surface appear to play a role in the presence or absence of radiopacity within the elevated sinus floor, following 6 months of healing.

Histologic analysis of alveolar bone following guided bone regeneration.

BACKGROUND: Questions on new bone quality following guided bone regeneration (GBR) with various graft materials and its importance in osseointegration have been raised. This study reports histologic analysis of bone sections from future implant sites at upper and lower jaws that were augmented with bovine porous bone mineral graft material plus a porcine collagen membrane. METHODS: Due to severe atrophy of the alveolar crest, GBR prior to implant placement was indicated in 11 patients (mean age 45.5 years). Following an average of 7 months of healing, implant placement surgery was performed. Bone sections from implant beds were fixed in formalin, decalcified in sodium citrate and formic acid, and placed in paraffin. Sections 5 to 7 microm thick were prepared, stained with hematoxylin and eosin, and analyzed under light microscopy. Results for 27 implant sites are presented. RESULTS: Compared to the lower jaw, segments from the upper jaw had a lower percentage of bone and higher percentage of residual material and vascularization. CONCLUSIONS: Within the limits of this study, we concluded that osteogenesis is completed and the rate of vascularization and osteoclastic activity was reduced by 7 months. Also, the upper jaw significantly differed from the lower jaw in bone formation, vascularization, and the amount of residual material. Thus, the anatomical location of the defect may be as important as the properties of the graft material used in obtaining successful osteogenesis using guided bone regeneration techniques.

A randomized trial of amifostine as radioprotector in the radiotherapy of head and neck cancer.

PURPOSE: Radiotherapy (RT) for head and neck cancer typically involves the major salivary glands bilaterally and can cause acute and chronic xerostomia and mucositis. The degree of xerostomia has been reported to depend on the radiation dose and the salivary gland volume irradiated. In this study, we evaluated the efficacy of the radioprotector amifostine to improve xerostomia and mucositis in head and neck cancer patients who received RT. PATIENTS AND METHODS: A total of 53 patients with head and neck cancer entered this prospective randomized study. Patients were randomly assigned to undergo RT or RT plus short intravenous (i.v.) infusion of amifostine 210 mg/m(2) before each RT fraction. RESULTS: No statistically significant difference was seen between the 2 arms in terms of mucositis. Acute xerostomia occurred in 31 (93.9%) patients in the amifostine arm and all of the patients in the RT-alone arm (p <0.05). Grade 3 acute xerostomia occurred in 13 (39.3%) patients in the amifostine arm, and in 9 (45%) patients in the RT-alone arm (p=0.04). Late xerostomia occurred in 19 (57.5%) patients in the amifostine arm, and in 14 (70%) patients in RT-alone arm (p=0.03). CONCLUSION: The administration of amifostine in head and neck cancer patients receiving RT improved significantly acute and late xerostomia, while did not offer protection in the prevention of mucositis. Further prospective studies are needed in order to better define the role of this agent.

Short-chain carboxylic acids produced by gram-negative anaerobic bacteria can accelerate or delay polymorphonuclear leukocyte apoptosis in vitro.

BACKGROUND: Short-chain carboxylic acids (SCCA) are metabolic byproducts of anaerobic subgingival bacteria associated with human periodontal disease. We examined the effect of 4 SCCA (butyric, propionic, succinic, and lactic acids) on human polymorphonuclear leukocyte (PMN) apoptosis over the range of concentrations (1 to 30 mM) found in the diseased periodontium. METHODS: PMN suspensions were incubated at 37 degrees C with medium alone (control) or one of the 4 SCCA at concentrations of 1, 5, or 30 mM. Aliquots were withdrawn hourly to assess apoptosis and viability by fluorescence microscopy. RESULTS: Relative to untreated controls, PMN incubated for at least 5 hours with 1 mM butyric or propionic acids exhibited significant delays in apoptosis (P<0.05), while those incubated with succinic or lactic acids exhibited no significant differences from controls (P>0.05). At a concentration of 5 mM, propionic, succinic, and lactic acids had little effect on apoptosis (P>0.05), but butyric acid significantly accelerated apoptotic changes (P<0.05). At 30 mM, all SCCA except lactic acid significantly accelerated apoptosis (P<0.05). Incubation with SCCA did not adversely affect cell viability (typically >98%). Lysates from PMN incubated 6 hours with 30 mM butyric or propionic acids contained significantly more caspase-3 activity than lysates from untreated control PMN (P<0.05). Moreover, pretreatment with a specific inhibitor of caspase-3 blocked acceleration of PMN apoptosis by butyric or propionic acids (P<0.05). CONCLUSION: Low concentrations of butyric or propionic acids delay PMN apoptosis and extend their functional lifespan, while higher concentrations accelerate apoptosis through a mechanism that appears to involve caspase-3.

Polyamines found in the inflamed periodontium inhibit priming and apoptosis in human polymorphonuclear leukocytes.

BACKGROUND: Polymorphonuclear leukocytes (PMNs) are exposed to high concentrations of polyamines in the inflamed periodontium and possess a transport system for taking up these compounds. Previous studies suggest that polyamines are involved in priming of the PMN respiratory burst by tumor necrosis factor-alpha (TNF-alpha) and can stabilize DNA against degradation. The purpose of this study was to determine whether exogenous polyamines can modulate priming by TNF-alpha or delay nuclear changes associated with PMN apoptosis (programmed cell death). METHODS: Isolated human PMNs were incubated with putrescine or spermidine in vitro. Superoxide generation was measured with a cytochrome C reduction assay, and apoptotic changes were assessed by fluorescence microscopy (after cell staining with acridine orange and ethidium bromide). RESULTS: Incubation with 1 mM putrescine for 1 hour inhibited superoxide production by TNF-primed PMNs by 20%, but enhanced the production of superoxide by unprimed cells by 38%. Both effects were dose dependent and statistically significant (P <0.03, repeated measures ANOVA and Dunnett's test). Spermidine had no significant effects on PMN oxidative function. With regard to apoptosis, 1 mM putrescine or spermidine produced a statistically significant reduction in the proportion of apoptotic PMNs within 6 to 9 hours (P <0.05). In cells incubated for 7 hours with 300 microM putrescine or spermidine, the proportion of apoptotic cells was approximately 30% lower than in untreated controls (P <0.05, Dunnett's test). The delay of apoptosis by spermidine was less profound than that produced by TNF-alpha and was not additive to the effects of this cytokine. CONCLUSIONS: Polyamines could potentially impair the priming of PMN oxidative function by TNF-alpha at sites where this cytokine is present. In the absence of TNF-alpha, polyamines could enhance PMN superoxide release and enhance the maintenance of PMN function in the periodontal pocket.

Alkaline conditions accelerate polymorphonuclear leukocyte apoptosis in vitro.

Apoptosis was monitored in polymorphonuclear leukocytes (PMNs) cultured under mildly acidic, neutral, and alkaline conditions. Within 3 h, 9.0% of the PMNs underwent apoptosis at pH 6.7, as did 12% at pH 7.2, 38% at pH 7.7, and 60% at pH 8.2. Inhibitors of serine proteases, caspase-1, or caspase-3 significantly inhibited PMN apoptosis at pH 8.2, suggesting an involvement by these enzymes.

pH changes observed in the inflamed gingival crevice modulate human polymorphonuclear leukocyte activation in vitro.

Previous studies have noted a positive correlation between gingival inflammation and crevicular pH, which reportedly varies from 6.5 to 8.5. In the present study, we characterized the manner in which deviation from the "physiological" pH of blood (7.2) influences activation of chemotaxis, phagocytosis, superoxide generation, and degranulation by human polymorphonuclear leukocytes (PMNs). Purified PMNs were suspended in HEPES-buffered balanced salts solutions adjusted to pH 6.7, 7.2, 7.7, or 8.2. In a modified Boyden chamber, the chemotactic response to fMet-Leu-Phe was maximal at pH 7.2. In comparison, chemotaxis was significantly depressed at pH 7.7 and pH 8.2 (P < 0.05), but was not significantly different at pH 6.7. Activation of the respiratory burst by fMet-Leu-Phe was optimal at pH 7.2, but was significantly depressed at pH 6.7 and 8.2 (P < 0.05). pH had little effect on N-acetyl-beta-glucosaminidase release from primary granules. However, lactoferrin release from the secondary granules of fMet-Leu-Phe-activated PMNs was significantly lower at pH 7.2 than at pH 6.7 or 8.2 (P < 0.05). Moreover, phagocytosis of opsonized bacteria was significantly lower at pH 7.2 than at pH 7.7. In addition to these effects on functional activation, extracellular pH influenced the magnitude of intracellular Ca2+ mobilization. Peak fMet-Leu-Phe-induced Ca2+ levels were significantly higher at pH 8.2 than at pH 7.2 (P < 0.01). These findings suggest that the pH of the periodontal environment can selectively influence PMN activation, thereby altering the balance between bacteria and the host response.

An inhibitor of polyamine biosynthesis impairs human polymorphonuclear leukocyte priming by tumor necrosis factor alpha.

TNF primes polymorphonuclear leukocytes (PMNs) for enhanced oxidative and secretory activity and directly induces adhesion and IL-1 beta expression. Previous reports suggest that polyamine biosynthesis by ornithine decarboxylase (ODC) has an essential role in macrophage activation by TNF. In the current study, TNF induced rapid increases in the putrescine and spermine content of PMNs. Difluoromethylornithine (DFMO), a selective inhibitor of ODC, inhibited these increases and blunted the enhancement of superoxide generation and secondary granule release associated with priming by TNF. DFMO did not affect the expression of TNF receptors or block receptor-independent activation of the respiratory burst by phorbol esters. Moreover, DFMO did not antagonize induction of adhesion or IL-1 beta mRNA expression by TNF. Thus, polyamine biosynthesis plays an important role in priming by TNF, but is not involved in all PMN responses to this cytokine. This suggests that ODC is a potential target for selective chemotherapeutic modulation of the inflammatory response.