Periodontal disease, periodontal treatment and systemic nitric oxide in dogs.

Thirty-two client-owned dogs treated for periodontal disease were divided in group 1 if no periodontitis, group 2 if ≤25%, and group 3 if >25% of the teeth present were affected with periodontitis. Blood was tested before and 2 weeks after periodontal therapy for nitrosyl hemoglobin (HbNO), plasma nitrite/nitrate (NOx) and 3-nitrotyrosine (NT) levels. No HbNO was detected in any of the animals tested. There was no significant difference in the NOx plasma levels within each group or across the groups before and after the treatment, but a noticeable increase in NOx plasma levels was observed in group 3 after the treatment. Plasma NT was detected in only one third of the animals. NO levels varied greatly across individual dogs. The data are suggestive of an overall increase in systemic NO response 2 weeks after periodontal treatment in dogs with advanced periodontal disease, but the response is greatly individually-dependent.

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.

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.

The temperature effect during pulse application on cell membrane fluidity and permeabilization.

Cell membrane permeabilization is caused by the application of high intensity electric pulses of short duration. The extent of cell membrane permeabilization depends on electric pulse parameters, characteristics of the electropermeabilization media and properties of cells exposed to electric pulses. In the present study, the temperature effect during pulse application on cell membrane fluidity and permeabilization was determined in two different cell lines: V-79 and B16F-1. While cell membrane fluidity was determined by electron paramagnetic resonance (EPR) method, the cell membrane electropermeabilization was determined by uptake of bleomycin and clonogenic assay. A train of eight rectangular pulses with the amplitude of 500 V/cm, 700 V/cm and 900 V/cm in the duration of 100 micros and with repetition frequency 1 Hz was applied. Immediately after the pulse application, 50 microl droplet of cell suspension was maintained at room temperature in order to allow cell membrane resealing. The cells were then plated for clonogenic assay. The main finding of this study is that the chilling of cell suspension from physiological temperature (of 37 degrees C) to 4 degrees C has significant effect on cell membrane electropermeabilization, leading to lower percent of cell membrane permeabilization. The differences are most pronounced when cells are exposed to electric pulse amplitude of 900 V/cm. At the same time with the decreasing of temperature, the cell membranes become less fluid, with higher order parameters in all three types of domains and higher proportion of domain with highest order parameter. Our results indicate that cell membrane fluidity and domain structure influence the electropermeabilization of cells, however it seems that some other factors may have contributing role.

Vascular disrupting action of electroporation and electrochemotherapy with bleomycin in murine sarcoma.

Electrochemotherapy has a direct cytotoxic effect on tumour cells, and presumably, a vascular disrupting effect. In this study, on the basis of the prediction of the mathematical model, histological evaluation and physiological measurements of the tumours were carried out to confirm that electroporation and electrochemotherapy of tumours have a vascular disrupting action. In the study, SA-1 solid subcutaneous sarcoma tumours in A/J mice were treated by bleomycin (BLM) given intravenously (1 mg kg(-1)), application of electric pulses (8 pulses, 1040 V, 100 micros, 1 Hz) or a combination of both - electrochemotherapy. The vascular effect was determined by laser Doppler flowmetry, power Doppler ultrasonographic imaging and Patent blue staining. The extent of tumour hypoxia was determined immunohistochemically by hypoxia marker pimonidazole and partial pressure of oxygen (pO(2)) in tumours by electron paramagnetic resonance oximetry. Electrochemotherapy with BLM induced good antitumour effect with 22 days, tumour growth delay and 38% tumour cures. The application of electric pulses to the tumours induced instant but transient tumour blood flow reduction (for 70%) that was recovered in 24 h. During this tumour blood flow reduction, we determined an increase in hypoxic tumour area for up to 30%, which was also reflected in reduced tumour oxygenation (for 70%). According to the described mathematical model, endothelial cells lining in tumour blood vessels are exposed to a approximately 40% higher electric field than the surrounding tumour cells, and therefore easily electroporated, allowing access of high BLM concentration to the cytosol. Consequently, electrochemotherapy has, besides the immediate vascular disrupting action, also a delayed one (after 24 h), as a consequence of endothelial cell swelling and apoptosis demonstrated by extensive tumour necrosis, tumour hypoxia, prolonged reduction of tumour blood flow and significant tumour growth delay, and tumour cures. Our results demonstrate that in addition to the well-established direct cytotoxic effect on tumour cells, electrochemotherapy also has an indirect vascular disrupting action resulting altogether in extensive tumour cell necrosis leading to complete regression of tumours.

Influence of different anesthetics on skin oxygenation studied by electron paramagnetic resonance in vivo.

The effects of two general anesthetics on skin oxygenation in mice are evaluated by electron paramagnetic resonance oximetry. Up to now no data on the effects of different anesthetics on skin oxygenation could be found. In this study animals were anesthetized with ketamine/xylazine or isoflurane, and partial pressure of oxygen (pO(2)) in the skin, heart rate and hemoglobin oxygen saturation were followed as a function of time and inhaled oxygen concentration. The skin pO(2) significantly increased continuously for about 60 min in mice anesthetized with isoflurane and remained constant after that. During ketamine/xylazine anesthesia, the pO(2) in the skin only slightly decreased. The skin pO(2) increased with higher inspired oxygen concentrations for both anesthetics groups. When breathing 21% oxygen, mice anesthetized with isoflurane had two-fold higher pO(2) in the skin compared to mice anesthetized with ketamine/xylazine. The heart rate was significantly lower in animals anesthetized with ketamine/xylazine, while hemoglobin saturation was almost the same in both groups at all inhaled oxygen concentrations. These results show that the type of anesthesia is an important parameter that needs to be considered in experiments where skin pO(2) is followed.

Free radical scavenging activities of yellow gentian (Gentiana lutea L.) measured by electron spin resonance.

Yellow gentian (Gentiana lutea L.) is a herbal species with a long-term use in traditional medicine due to its digestive and stomachic properties. This paper presents an investigation of the free radical scavenging activity of methanolic extracts of yellow gentian leaves and roots in two different systems using electron spin resonance (ESR) spectrometry. Assays were based on the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) and the superoxide radicals (O2*-) generated by the xanthine/xanthine oxidase (X/XO) system. The results of gentian methanolic extracts were compared with the antioxidant capacity of synthetic antioxidant butylated hydroxyanisole (BHA). This study proves that yellow gentian leaves and roots exhibit considerable antioxidant properties, expressed either by their capability to scavenge DPPH or superoxide radicals.

In vivo study of liposomes as drug carriers to oral mucosa using EPR oximetry.

The purpose of this study was to select the best types of liposomes for use as drug carriers for topical treatment of oral mucosal lesions. Electron paramagnetic resonance (EPR) oximetry, using the paramagnetic probe lithium phthalocyanine, was used in vivo to measure the effects of a hyperemic drug, benzyl nicotinate (BN) which was incorporated into liposomes of varying size and composition. The liposomes were made from either hydrogenated or non-hydrogenated soy lecithin and mixed with polymethyl methacrylate ointment for application. EPR oximetry was used to measure the partial pressure of oxygen (pO2) in the oral mucosa before and after application of liposomes. It was found that the most pronounced changes of pO2 in oral mucosa and also the longest action of the drug occurred after the topical application of BN in multi-lamellar liposomes made from hydrogenated soy lecithin (p<0.0001). When these liposomes were applied to oral mucosa over 3 successive days it was found that pO2 increased the most on the first day, the effect gradually decreased following application on the second and third days. The duration of the resulting hyperemia was the longest on the second day (p<0.01). Among the examined carriers, multi-lamellar liposomes made from hydrogenated soy lecithin appear to be the most appropriate for local drug delivery to oral mucosa.

Hydrophobic barrier in liposomes studied by FT-ESEEM.

An improvement of the electron spin echo envelope modulation technique (ESEEM) for studying the hydrophobic barrier in lipid membranes was proposed. Water penetration depth into the lipid bilayer composed of egg-phosphatidylcholine and dicetylphosphate was studied. For this purpose the stearic acid spin probes with nitroxide moiety at different positions on the acyl chain were dissolved in the bilayer of liposomes prepared in buffer solution with D2O. The ESEEM method is based on the deuterium effect to the 14N-hyperfine splitting constant of the spin probe. For the analysis of the spectra we suggested the frequency domain approach (Fourier transform-ESEEM) instead of the time domain. In order to test this approach, the influence of cholesterol and octanol on the water penetration depth into liposomes was measured. FT-ESEEM appeared to be a more sensitive method for the quantitative measurements of the water penetration depth than the time domain. This approach can become one of the most effective methods for studying the influence of surfactants and other biologically active molecules on water penetration depth into membranes.

Interactions of solid lipid nanoparticles with model membranes and leukocytes studied by EPR.

Solid lipid nanoparticles (SLN) are colloidal systems which have been proposed for several administration routes. Only limited data are available about the mechanism and rate of interaction of SLN with cells and tissues. The aim of our study was to investigate interactions of SLN with model membranes (liposomes) and cells (leukocytes). SLN dispersions composed of glyceryl tripalmitate, phosphatidylcholine, water, and poloxamer 188 or Tween 20 were prepared by the melt-emulsification process. Spin-labeled phosphatidylcholine (PC(10,3)) and the methylester of doxyl palmitic acid (MeFASL(10,3)) were incorporated into SLN as spin probes (SPs) in order to determine the rate and mechanism of cell interaction by electron paramagnetic resonance (EPR) spectroscopy. Our results indicate that the exchange of SP between SLN and liposomes is much faster for MeFASL(10,3) than for PC(10,3), probably due to the smaller size of the former. In contrast to liposomes, in leukocytes no significant difference in the transfer rates of the two SP was observed after incubation, suggesting that there is an uptake of SLN to leukocytes (endocytosis) although simultaneous SP diffusion is not excluded. The interaction of SLN with leukocytes appears to depend significantly on the stabilizer used. Transfer of PC(10,3) from SLN coated with poloxamer 188 is much faster than from SLN coated with Tween 20.

Reduced blood flow and oxygenation in SA-1 tumours after electrochemotherapy with cisplatin.

Electrochemotherapy is an antitumour treatment that utilises locally delivered electric pulses to increase cytotoxicity of chemotherapeutic drugs. Besides increased drug delivery, application of electric pulses affects tumour blood flow. The aim of this study was to determine tumour blood flow modifying effects of electrochemotherapy with cisplatin, its effects on tumour oxygenation and to determine their relation to antitumour effectiveness. Electrochemotherapy of SA-1 subcutaneous tumours was performed by application of electric pulses to the tumours, following administration of cisplatin. Tumour blood flow modifying effects of electrochemotherapy were determined by measurement of tumour perfusion using the Patent blue staining technique, determination of tumour blood volume, and microvascular permeability using contrast enhanced magnetic resonance imaging, and tumour oxygenation using electron paramagnetic resonance oximetry. Antitumour effectiveness was determined by tumour growth delay and the extent of tumour necrosis and apoptosis. Tumour treatment by electrochemotherapy induced 9.4 days tumour growth delay. Tumour blood flow was reduced instantaneously and persisted for several days. This reduction in tumour blood flow was reflected in reduced tumour oxygenation. The maximal reduction in partial oxygen pressure (pO2) levels was observed at 2 h after the treatment, with steady recovery to the pretreatment level within 48 h. The reduced tumour blood flow and oxygenation correlated well with the extent of tumour necrosis and tumour cells apoptosis induced by electrochemotherapy with cisplatin. Therefore, the data indicate that antitumour effectiveness of electrochemotherapy is not only due to increased cytotoxicity of cisplatin due to electroporation of tumour cells, but also due to anti-vascular effect of electrochemotherapy, which resulted in reduced tumour blood flow and oxygenation.

The influence of cholesterol and charge on the membrane domains of alkylphospholipid liposomes as studied by EPR.

Alkylphospholipids are physiologically active derivatives of lipids effective in the treatment of breast cancer. Among them, octadecyl-(1,1-dimethyl-4-piperidino-4-yl)-phosphate (OPP) was demonstrated recently to have the strongest antitumor effect in micellar as well as in sterically stabilised liposome suspension with a low cholesterol content. In this work electron paramagnetic resonance (EPR) was used to study the influence of cholesterol, charge, and sterical stabilisation by PEG2000DSPE on the domain structure and fluidity characteristics of the membrane of OPP liposomes. As a spin probe 5-doxylpalmitoyl methyl ester was used. By computer simulation of the EPR spectra it was found that the experimental spectra are composed of three spectral components, which were attributed to three types of domains with different fluidity characteristics. The EPR parameters as well as the proportions of the individual domains were found to be mainly dependent on the amount of cholesterol, and only to a minor degree on charge and sterical stabilisation. There was a pronounced increase in the proportion of membrane domains with low order parameter, when the molar ratio of cholesterol to OPP was decreased below 1. At the same time the order parameters of all domains decreased, pointing to a transition from a less to a more fluid membrane organisation. These results coincide with an improved therapeutic activity of formulations with a low molar ratio of cholesterol to OPP and indicates that the fluidity characteristics of the membrane may be important for the effectiveness of liposomal alkylphospholipids against breast cancer cells.

Reduced tumor oxygenation by treatment with vinblastine.

Vinblastine (VLB) previously has been shown to perturb tumor blood flow, but the effect of these perturbations on tissue oxygenation is not known. The recent development of electron paramagnetic resonance (EPR) oximetry now has made it feasible to measure the effects of changes of perfusion on the pO(2) in tumors and normal tissues as a function of time and dose. We measured changes in tumor perfusion by Patent blue staining, tumor blood volume and microvascular permeability by contrast-enhanced magnetic resonance imaging, and tumor oxygenation by EPR in s.c. SA-1 murine tumors. We found that treatment with VLB induced dose-dependent reduction in tumor perfusion. One hour after i.p. treatment of mice with 2.5 mg/kg VLB, tumor perfusion was reduced to 20% of the pretreatment value and returned to close to original values within 48 h. A transient tumor blood flow-modifying effect of VLB was demonstrated also by contrast-enhanced magnetic resonance imaging; reduction of tumor blood volume and microvascular permeability was found. Reduced tumor oxygenation was found as measured by EPR oximetry, with the same time course of changes in tumor blood flow. Tumor oxygenation was reduced to 50% of pretreatment value 1 h after the treatment with 2.5 mg/kg VLB and returned to pretreatment levels within 24 h after the treatment. Although the directions of the changes in perfusion and oxygenation were similar, they were quantitatively different. Reduction in oxygenation of normal tissues, muscle, and subcutis also occurred but was smaller and returned to pretreatment values more quickly compared to the changes induced in the tumors. In conclusion, the present study demonstrates that VLB causes a profound reduction in tumor blood flow and oxygenation, which may have implications in controlling side effects of therapy and the planning of combined treatment with VLB, either with other chemotherapeutic drugs or with radiotherapy.

Improved skin oxygenation after benzyl nicotinate application in different carriers as measured by EPR oximetry in vivo.

The development of formulations, which increase skin oxygenation and of methods for measuring oxygen levels in skin are important for dealing with processes affected by the level of oxygen, e.g., rate of healing and efficiency of radiation oncology. In this study we have investigated the role of carriers on the efficacy of benzyl nicotinate (BN) action in skin after dermal application in different formulations by EPR oximetry in vivo. The time course of pO2 in the skin after application of rubefacient is followed directly for the first time. The results obtained proved the applicability of in vivo EPR oximetry as a sensitive method by which small alterations in pO2 can be detected. We have found that the type of vehicle significantly influences the time when BN starts to act, the duration of its action, and the maximal increase in pO2. The ranking of vehicle efficiency was: lipid nanoparticles in hydrophilic gel>liposomes in hydrophilic gel>hydrophilic gel>hydrophobic ointment>hydrophobic cream. Primarily the semi-solid vehicle determines the lag-time of action, but the maximal oxygen level is influenced decisively by the particulate carrier systems. BN effectiveness was dose dependent. 2.5% w/w concentration of BN appears to be the most appropriate for therapeutic application. After repeated application a successive increase of pO2 base line in skin and of the maximal pO2 was noticed.

Development of liposome encapsulated clindamycin for treatment of acne vulgaris.

The enhancement of topical delivery of hydrophilic substances by use of multilammelar liposomes was measured ex vivo on pig ear skin and in vivo on hairless mice by electron paramagnetic resonance method (EPR). Multilamellar liposomes with different lipid composition (final concentration of membrane components is 48 mg/ml) were loaded with a hydrophilic spin probe GluSL, which does not penetrate the liposome membrane easily. They were characterized with respect to their stability, entrapped volume and enhancement characteristics. We observed significant differences in the properties of different types of liposomes with respect to their stability when in contact with the skin and their penetration into the skin. The results measured in vivo are consistent with those obtained ex vivo. On the basis of these findings the liposomes with appropriate stability and intradermal penetration characteristics were chosen for the development of liposome-encapsulated 1% clindamycin preparation for therapy of acne vulgaris. A double-blind clinical study was conducted to assess the safety and efficiency of liposome-encapsulated 1% clindamycin solution versus 1% clindamycin solution (Klimicin T, Lek). On the basis of the clinical trial it may be concluded that liposome-encapsulated 1% clindamycin solution was therapeutically superior over conventional 1% clindamycin solution in the treatment of acne vulgaris.

In vivo EPR of topical delivery of a hydrophilic substance encapsulated in multilamellar liposomes applied to the skin of hairless and normal mice.

In vivo low frequency EPR was used to measure the enhancement of topical delivery of hydrophilic substances by use of multilamellar liposomes. The contribution of transepidermal or/and transfollicular routes of transport was investigated using hairless and normal mice. Two liposome dispersions that previously had been shown to have different enhancement properties on ex vivo skin were used. The kinetics of the reduction of hydrophilic spin probe GluSL (N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-2,3,4,5, 6-pentahydroxy-hexaneamide) applied to the skin encapsulated into the liposomes was measured. To distinguish the reduction of GluSL on the skin surface from its reduction inside the skin, the oxidizing agent potassium ferricyanide (KFeCN) was used. This does not penetrate into the skin and therefore it oxidizes hydroxylamines back to nitroxide only on the surface of the skin. We observed significant differences in the properties of the two types of liposomes with respect to their stability when in contact with skin and their transport characteristics. The results measured in vivo are consistent with those obtained ex vivo, indicating that in vivo L-band EPR is a powerful technique for following pharmacokinetics in the skin of live animals. The results also show that clearance by blood flow and possible alterations of skin after sacrifice of animal do not influence the results of penetration of liposome entrapped substances into the skin during the time of our experiment (typically around 60 min). The reduction of GluSL in the skin of hairless vs. normal mice was similar, indicating that the transfollicular penetration was not of major importance in vivo in this experimental model.

Influence of spin probe structure on its distribution in SLN dispersions.

Solid lipid nanoparticles (SLN) are drug carrier system composed of biodegradable substances, which are solid at room temperature. The physico-chemical properties and structure of the incorporated compounds can affect their partitioning in SLN dispersions. In this work the influence of lipophilicity and structure of different SP on its location in SLN were studied. By electron paramagnetic resonance (EPR) measurements it was found that lipophilic SP distribute between a solid glyceride core and a soft phospholipid layer, with the more polar part (piperidine ring or methylcarboxylic groups) oriented toward the water-lipid interface. The majority of SP is located in the phospholipid layer, but the portion in the solid lipid core increases with SP lipophilicity. The hydrophilic Tempol does not incorporate into SLN.

Fast and accurate characterization of biological membranes by EPR spectral simulations of nitroxides.

A method by which it is possible to characterize the membranes of biological samples on the basis of the EPR spectral lineshape simulation of membrane-dissolved nitroxide spin probes is described. The presented simulation procedure allows the determination of the heterogeneous structure of biological membranes and fluidity characteristics of individual membrane domains. The method can deal with isotropic and anisotropic orientations of nitroxides introduced into the biological samples described by restricted fast motion with a correlation time between 0.01 and 10 ns. The linewidths of the Lorentzian lineshapes are calculated in a restricted fast-motion approximation. In the special case of samples with high concentrations of nitroxides or in the presence of paramagnetic ions, the lineshapes are calculated directly from the exchange-coupled Bloch equations. The parameters describing ordering, relaxation, polarity, and the portions of the individual spectral components are extracted by optimizing the simulated spectra to the experimental spectrum with either a Simplex or a Monte Carlo algorithm. To improve the algorithm's efficiency, a new way of characterizing the goodness of fits is introduced. The new criterion is based on the standard least-squares function, but with special weighting of the partial sums. Its benefits are confirmed with membrane spectral simulation. Two classes of examples-simulation and optimizations of synthetic spectra to evaluate the accuracy of the optimization algorithms and simulation and optimization of EPR spectra of nitroxides in liposome suspensions in the presence of a broadening agent and in human leukocytes are shown.

Local delivery of liposome-encapsulated superoxide dismutase and catalase suppress periodontal inflammation in beagles.

BACKGROUND, AIMS: The purpose of this study was to evaluate the influence of oxygen free radical scavengers on periodontal inflammation and healing process. METHOD: Experimental periodontitis was induced by elastic ligatures around premolars (P2, P3, P4) and 1st molars (M1) in the upper and lower jaws of 15 beagle dogs. 9 months after the beginning of the experiment, the ligatures were removed. After 3 weeks of stabilization period, all teeth were supragingivally scaled. The animals were divided into 3 groups of 5 dogs. The 1st group received a liposome-encapsulated superoxide dismutase (SOD), the 2nd group a liposome-encapsulated catalase (CAT) and the 3rd group received both enzymes encapsulated in liposomes. 4 treatment modalities were tested in each group; i.e., supragingival scaling only (1), supragingival scaling and enzymes (2), supra- and sub-gingival scaling and root planing (3) and supra- and sub-gingival scaling and root planing with subgingival application of enzymes (4). Enzymes were delivered subgingivally on a daily basis for a period of 6 weeks. Gingival index (GI), probing depth (PD), clinical attachment level (CAL), radiographic analysis and the histological evaluation were performed. RESULTS: Around the teeth with scaling and root planing followed by the application of SOD, the greatest suppression of gingival inflammation (GI = 1.8 +/- 0.1 before versus GI = 1.2 +/- 0.2 after treatment) (p<0.003), the smallest size of connective tissue infiltrate (5.5 +/- 4.3%) (p<0.01), the greatest reduction of PD (PD= 3.2 +/- 1.0 mm before versus 2.00.7 mm after treatment) (p<0.001), and gain of CAL (CAL=3.0 +/- 1.7 mm before versus CAL=2.4 +/- 1.1 mm after treatment) (p<0.001) were observed. In addition, radiographic analysis showed the greatest alveolar bone apposition in the group of teeth treated with scaling and root planing followed by subgingival application of SOD or both enzymes (p<0.001). CONCLUSION: In conclusion, we demonstrated that scaling and root planing with subgingival application of liposome-encapsulated SOD suppress peridontal inflammation on experimentally induced periodontitis in beagle dogs.