Milan Petelin

Periodontal Disease Burden and Pathological Changes in Organs of Dogs

Bacterial plaque associated periodontal disease is the most common chronic infection in man and dogs. In man, there is an association between periodontal disease and myocardial infarction and stroke, while in dogs it has also been associated with changes in internal organs. Inflamed periodontal tissues present a ‘periodontal disease burden’ to the host and the extent of this inflammatory disease burden is likely to affect the degree of associated pathological change in distant organs. This hypothesis was investigated in dogs with naturally occurring periodontal disease. Post-mortem investigations including periodontal assessment, standard necropsy, and organ histology were performed on 44 mature toy and miniature Poodles (related, periodontitis predisposed breeds) that died naturally or were euthanized based on clinical disease. Animals with gross primary organ pathology were excluded. The periodontal disease burden was estimated from the total surface area of periodontal pocket epithelium using six measurements of probing depth for each tooth and the tooth circumferences. Ordinal logistic regression (OR) analysis established that for each square centimeter of periodontal disease burden there was a 1.4-times higher likelihood of greater changes being present in the left atrio-ventricular valves (OR = 1.43), plus 1.2 and 1.4 times higher likelihood for greater liver and kidney pathology (OR = 1.21; OR = 1.42), respectively. The results show that there is a link between the estimated ‘periodontal disease burden’ resulting from plaque-bacteria associated periodontal disease and the level of internal pathology in this population, implying that periodontitis might contribute to the development of systemic pathology in dogs.

EPR study of mucoadhesive ointments for delivery of liposomes into the oral mucosa

Local drug delivery to the oral cavity tissues has been used for treatment of periodontal disease, aphthosus stomatitis, lichen planus, bacterial and fungal infections. The liposome stability in different mucoadhesive ointments, their transport into dogs oral mucosa and gingiva and washing out of ointments from tissue surface was investigated by electron paramagnetic resonance (EPR). Liposomes were composed of soya lecithin, cholesterol and lipoaminosalt (55:35:10 w/w) and encapsulated with spin labeled water soluble spin probe ASL (N-1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl-N-dimethyl-N-hydroxyethylammonium iodide). Polymethyl methacrylate, Carbopol 934P and Orabase as the bioadhesive ointments were used. The stability of liposomes did not change significantly after mixing with polymethyl methacrylate, but decreases in Carbopol and even more in Orabase. Washing out experiment shows that all three ointments adhere well to oral mucosa and gingiva. After 10 min of washing, approximately 30% of hydrogels were washed out. The transport experiment showed that liposomes limit the transport of hydrophilic substance to the superficial layer of epithelium. In oral mucosa, as opposed to gingiva, hydrogels enhance the transport as compared to solution of ASL or liposomes. Among the examined ointments, polymethyl methacrylate proved to be the most appropriate for local application of liposome entrapped drug to oral mucosa or gingiva.

Oral health of the elderly living in residential homes in Slovenia

OBJECTIVES To evaluate oral health status of the elderly, living in eight randomly selected residential homes for senior citizens across the country. BACKGROUND The percentage of the elderly is growing worldwide. With ageing, risks of various oral diseases, including dental caries and periodontal disease, are growing. METHODS Altogether 296 elderly people (88 men, 208 women) of average age 79.89 ± 7.4 years were questioned about their medical condition and oral health practice and examined orally. Evaluation of clinical examination was carried out by DMFT, plaque index (Silness and Löe, 1964) and Community Periodontal Index of Treatment Need (CPITN). RESULTS Of 296 participants, 106 (35.8%) were edentulous, 95 (32.1%) had one to nine teeth and 95 persons (32.1%) had 10 or more teeth. The average number of teeth in an individual was small: 6.76 ± 7.47. The average number of teeth with caries lesions was 3.59 ± 4.70, filled teeth 1.94 ± 3.63 and teeth without caries or fillings 1.19 ± 2.41. The average DMFT value was 30.75. In 69.5% of participants, dental plaque was visible with the naked eye. Of 171 subjects, in whom CPITN index was appraised, 81.9% would need oral hygiene education, 56.7% would need scaling and root planning and 21.6% would need periodontal surgical treatment. CONCLUSIONS The results of this study indicate poor oral health of the elderly living in residential homes situated in different towns in Slovenia. It is of utmost importance to highlight the necessity of improving oral health care of this population.

THE PERMEABILITY OF HUMAN CEMENTUM IN VITRO MEASURED BY ELECTRON PARAMAGNETIC RESONANCE

The structure and permeability of cementum are changed during the course of periodontal disease. In this study, the transport of water-soluble, spin-labelled molecules through cementum was studied by electron paramagnetic resonance (EPR). Cementum samples cut from different parts of the root were classified into four different groups: (A) samples exposed to the oral environment, (B) samples exposed to the periodontal-pocket environment; (C) samples cut from periodontally involved teeth but not exposed to saliva or periodontal pocket and (D) samples from sound young teeth extracted for orthodontic reasons. In order to obtain undamaged cementum, a dentine layer was left on each sample. Two methods were used to measure the diffusion coefficients of spin-labelled molecules in cementum dentine samples. First, the method of one-dimensional EPR imaging (EPRI) was used to evaluate the penetration of spin-labelled molecules into the cementum/dentine structure. Second, the diaphragm-cell method was used to determine the diffusion coefficients of the labelled molecules through the cementum under steady-state conditions. The results indicate that the interface between cementum and dentine is a barrier to diffusion. A set of diffusion (D) and partition (K) coefficients to describe the molecular transport in cementum, barrier and dentine was generated from the experimental data of both methods. For cementum (c), the barrier (b) and dentine (d) these coefficients were: Dc= 10(-8)cm2/s, Db= 10(-10)cm2/s, Dd= 10(-6)cm2/s and K=0.1. For the particular periodontally involved and uninvolved teeth the value of the rate-limiting barrier was DbA= 0.3 +/- 0.03 x 10(-10)cm2/s, DbB= 1 +/-0.3 x 10(-10)cm2/s, DbC= 0.3 +/- 0.03 x 10(-10)cm2/s, DbD= 0.4 +/- 0.05 x 10(-10)cm2/s. The largest diffusion flux across the dental hard tissue was found in the samples that had been exposed to the pocket environment (3.1 +/- 0.2) x 10(-9)cm2/s (p < 0.01), which coincided with the permeability calculated from the data evaluated by EPRI. The transport of the labelled molecules into and through the cementum dentine samples depends on the structure of the dental hard tissues, which changes during the course of periodontal disease. Knowledge of molecular diffusion across the tooth cementum/dentine structure is likely to be important for planning new treatments for periodontal disease.

Systemic use of selective iNOS inhibitor 1400W or non-selective NOS inhibitor L-NAME differently affects systemic nitric oxide formation after oral Porphyromonas gingivalis inoculation in mice

OBJECTIVE Nitric oxide synthase (NOS) inhibitors are reported to protect against the local tissue damage in gingivitis and periodontal disease by reducing nitroxidative stress during inflammation, but their systemic effects are not well investigated. DESIGN NOS inhibitors systemic effects were investigated in a murine chronic oral inoculation model using live Porphyromonas gingivalis ATCC 33277 (0.3 ml; 10(9)cfu/ml) or sterile broth (0.3 ml). Organ nitric oxide (NO) and plasma nitrite/nitrate (NOx) were determined in mice treated with non-selective NOS inhibitor l-NAME (50mg/kg/24h i.p.) or selective iNOS inhibitor 1400W (10mg/kg/6h i.p.) for the last 5 days of the experiment. Differences between groups were evaluated by nonparametric Wilcoxons rank-sum one-sided two-sample test and the results compared to those obtained from sham-treated (sterile broth) sham-inoculated animals (water for injection i.p./6h). RESULTS Repeated ingestion of P. gingivalis resulted in generalized production of NO in organs and NOx in plasma, the levels of both typically being reduced in P. gingivalis-inoculated-1400W-treated mice, whilst the use of l-NAME was largerly ineffective, even promoting NO/NOx formation. Application of either inhibitor to sham-inoculated animals enhanced NO/NOx formation, due only in part to the repeated i.p. injections. CONCLUSIONS The systemic use of 1400W or l-NAME differently affects systemic nitric oxide formation in mice orally challenged with P. gingivalis, but the sequelae of such an intervention should be evaluated further.

Chronic ingestion of Porphyromonas gingivalis induces systemic nitric oxide response in mice

INTRODUCTION Porphyromonas gingivalis induces nitric oxide (NO) production in various cells, systemic NO elevation being expected in chronic oral challenge. METHODS Groups of BALB/c mice were inoculated orally with either live P. gingivalis ATCC 33277 or sterile broth on days 0, 2 and 4, with or without later administration of the inducible nitric oxide synthase (iNOS) inhibitor 1400W. Plasma and tissues were harvested on day 42 for assays of tumor necrosis factor-alpha (TNF-alpha), nitrite and nitrate (NOx) and tissue NO, or histology and iNOS immunohistochemistry. RESULTS No signs of gingivitis were observed, but plasma NOx was significantly elevated (P = 0.028) as was TNF-alpha (P = 0.079) in P. gingivalis-inoculated animals compared with controls, NOx being reduced when 1400W was used. NO production in organs showed a similar trend, with significant elevation in liver (P = 0.017) and kidneys (P = 0.027), whereas concomitant treatment of inoculated animals with 1400W caused significant reductions in NO in aorta (P = 0.008) and kidneys (P = 0.046). Sham-inoculated 1400W-treated animals had significantly increased plasma NOx (P = 0.004) and liver NO (P = 0.04). NOx in plasma correlated significantly with NO production in lungs (0.35, P = 0.032) and kidneys (0.47, P = 0.003). Immunohistochemistry demonstrated iNOS activity in many tissues in all groups. CONCLUSION Repeated oral administration of P. gingivalis induced systemic NO and NOx production in mice, probably by activating iNOS as suggested by the response to 1400W.

Aerosolized clindamycin is superior to aerosolized dexamethasone or clindamycin-dexamethasone combination in the treatment of severe Porphyromonas gingivalis aspiration pneumonia in an experimental murine model

ABSTRACT Adjunctive corticosteroid treatment to reduce excessive local inflammatory response in pneumonia is controversial. To study the effects of an early local adjunct dexamethasone treatment on the course of pneumonia and inflammatory/cytokine response, mice were intratracheally inoculated with live Porphyromonas gingivalis and treated with either clindamycin (C), dexamethasone (D), C+D combination, or were not treated (Pg). Six mice from each group were euthanized at 6, 24, 72, and 168 hours after inoculation. Levels of tumor necrosis factor (TNF)-α, soluble TNF-α receptors (sTNFR1 and sTNFR2), interleukin (IL)-1β, and IL-6 in the serum and lung-homogenate supernatant were determined. Lung samples were histopathologically assessed and all findings compared to those found in 24 sham-inoculated mice (phosphate-buffered saline [PBS]). Severe P. gingivalis–induced bronchopneumonia progressed from 24 hours, peaked at 72 hours, and resolved after 168 hours with changes in local and systemic cytokine levels. Clindamycin-treated mice developed only mild bronchopneumonia that resolved fast (72 hours) with an early (6–24 hours) normalization of local and systemic cytokine levels. Similar course of pneumonia and cytokine level changes were observed in mice treated with C+D, but later. Early (6–24 hours) local elevation of sTNFRs was observed in C and C+D groups of mice, whereas nontreated (Pg) mice had increased systemic sTNFRs. Severe bronchopneumonia with delayed resolution was observed in D-group mice, with an early local and systemic decrease in sTNFR1 and persistent elevation of local TNF-α. Clindamycin or a clindamycin-dexamethasone combination treatment significantly improves the course of P. gingivalis-aspiration pneumonia, but more so if clindamycin alone is used. A favorable course of pneumonia seems to be associated with an early elevation of sTNFRs and normalization of TNF-α.

Periodontal inflammatory burden correlates with C-reactive protein serum level.

Abstract Objective. The aim of study was to present a new method for evaluation of the periodontal inflammatory burden, to apply the method to the adult population and to correlate it with serum levels of C-reactive protein (CRP). Materials and methods. On 515 extracted teeth was measured the neck circumferences (NC). The average values of the NC were obtained for 16 male and 16 female individual tooth types. In the clinical part of this study 238 dentate subjects were included. The subgingival area, inflamed area and periodontal wound size were calculated from NC, probing depth and BOP. The sum of the inflamed and ulcerated subgingival areas of all teeth represented the total periodontal inflammatory burden of an individual. Serum levels of CRP were measured by immunochemical method. Results. The average subgingival area in 238 subjects was calculated to be 13.11 ± 6.35 cm2 and inflammatory burden area 9.25 ± 5.57 cm2. The periodontal bleeding wound (p < 0.05) was significantly larger in men. The increased serum levels of CRP correlated with periodontal inflammatory burden (p < 0.05). Conclusions. This new method quantifies the inflammatory burden caused by periodontal disease. The size of the inflammatory burden is correlated with increased serum levels of CRP.

LACK OF SOLUBLE TUMOR NECROSIS FACTOR ALPHA RECEPTOR 1 AND 2 AND INTERLEUKIN-1β COMPARTMENTALIZATION IN LUNGS OF MICE AFTER A SINGLE INTRATRACHEAL INOCULATION WITH LIVE PORPHYROMONAS GINGIVALIS

Porphyromonas gingivalis aspiration pneumonia induces local and systemic cytokine responses, but the dynamic of the immune response following lung exposure to live P. gingivalis is poorly understood. Groups of 50 12-week-old male BALB/c mice were inoculated intratracheally with live P. gingivalis ATCC 33277 using low dose (2 × 105 colony-forming units [CFU]), high dose (2.9 × 109 CFU), or phosphate-buffered saline (PBS; sham-inoculated), and the 3 groups were sacrificed at 2, 6, 24, 72, 168 hours. Lung and serum samples were collected for tumor necrosis factor alpha (TNF-α), soluble TNF-α receptors (sTNFRs), interleukin (IL)-1β, and IL-6 analysis and lung histology. Pneumonia, only observed in the high-dose group, was associated with an early increase in lung TNF-α, IL-1β, and IL-6, whereas no significant changes were observed in lung sTNFRs. Serum sTNFRs were significantly increased in high-dose animals at all times. IL-1β elevation occurred earlier in serum than in lungs. IL-1β was also significantly elevated in serum from low-dose animals at 6 hours. Serum IL-6 and sTNFRs remained raised at 7 days, whereas all other measured cytokines returned to basal levels with resolution of pneumonia. Development of pneumonia is dependent on the P. gingivalis dose; however, part of the cytokine response is unique to the systemic compartment, even in animals that do not develop pneumonia.

Single gavage with Porphyromonas gingivalis reduces acute systemic nitric oxide response in mice

INTRODUCTION Porphyromonas gingivalis, an important periodontal pathogen, can also induce host responses in distant tissues. P. gingivalis induces nitric oxide (NO) production in immune system cells and non-immune system cells, therefore NO might be involved in an acute systemic host response. METHODS Eighteen female BALB/c mice were perorally inoculated with 10(8) colony-forming units live P. gingivalis ATCC 33277. Plasma nitrite and nitrate (NOx) and NO production in lungs, aorta, heart, liver, spleen, kidneys, and brain were measured at intervals after inoculation and compared with levels in 11 control animals. RESULTS NOx levels were significantly (P = 0.017) lower at 7, 13, and 25 h after P. gingivalis inoculation. A similar trend in NO production occurred in most tested organs, but never reached statistical significance. The correlation between NOx in plasma and NO in liver was positive (Spearman correlation coefficient = 0.81, P = 0.0025) and marginal for kidney (0.58, P = 0.059). CONCLUSION Single peroral inoculation of mice with P. gingivalis reduces the acute systemic NO response. As NO is important for host defense, the reduction of NO levels after exposure is likely to delay the host response, increasing the chances that infection with P. gingivalis will become established.