| BackgroundPeriodontitis is one of the common diseases of mankind, and the pathogenic factors of this chronic disease are always complicated. Periodontitis can cause gingival inflammation, periodontal pocket formation and alveolar bone resorption, as a result the affected teeth become mobility and eventually fall off. According to WHO investigations, the overall morbidity of periodontal disease is 75%. In some parts of China, this rate can be as high as 93%. It has been proved that bacterium and its products are the causes of parodontopathy in bacteria plaque, and subgingival plaque is closely related to the destruction of periodontal tissues. Control of specific bacteria relating to the occurring and developing of parodontopathy is the key approach to effective treatment. Medication is one of the important adjutant therapies for parodontopathy, and local delivery of medicines in periodontal tissues is widely used in clinical applications because it can avoid the side effects of general medications. Local delivery of medicines has a variety of patterns and can be divided into supragingival medications and subgingival medications, and the latter include rinsing the periodontal pockets and sustained-release medications. In recent years, the development and application of local sustained-release medicines has greatly improved the effectiveness of clinical treatment. Sustained-release medicines can maintain a stable blood concentration, and thus prolong the effective treatment time, decreasing the medication frequency, improve complicance of patients, reduce the total medication dose and lower the risks of side effect. Currently the substances of subgingival medications include fibers, powders, medication strips, pastes, glues and ointments.ObjectiveThis study is to obtain an optimized drug concentration by comparing the bacteriostasis effects of 5 kinds of ordinary suppositories of different concentrations (without sustained-release constituents) in bacteriostasis experiments in vitro, and thus to provide theoretical support for the preparation of the compound ornidazole and pefloxacinmesylate sustained-release gum suppositories. After preparation, the compound sustained-release gum suppositories will be used in the sustained-release bacteriostasis experiment, bacterial sensitivity experiment and ground substance effect experiment in vitro. Then the anti-inflammatory experiment will be carried out to verify the anti-inflammation and detumescence effectiveness of the prepared compound sustained-release gum suppository. Ultimately an experimental animal model of periodontitis will be established to study the treatment effect of the compound sustained-release gum suppository on experimental rats, and to form the basis of clinical experiments.Materials and methods1. 5 kinds of compound ornidazole and pefloxacinmesylate gum suppository (without sustained-release constituent) of different concentrations (1%,2%,5%,10%,20%) were prepared on the drug-sensitive papers plates of 6 mm in diameter(50μl in total medicine dose), then K-B method was used to test the bacteriostasis effect on the 4 strains of anaerobic bacteria (Pg, Fn, Pi, Ps), and 3 strains of anaerobic bacteria (Ms, Sa, Se). By comparing the size of bacteriostasis annuluses to choosing optimized drug concentration, it provides vital theoretical evidence for the preparation of compound sustained-release gum.2. 5 of the compound ornidazole and pefloxacinmesylate sustained-release gum suppositories were randomly selected and placed in a beaker containning 4 ml pure water. 5 of the ordinary compound ornidazole and pefloxacinmesylate gum suppositories (without sustained-release ground substance) were also randomly selected and placed in another beaker containning 4 ml pure water. 1 ml samples were obtained from each of the beakers at 30 minutes,1,2,3,4,5,6,7,8,9,10,11,12.16,20,24 hours respectively. From each of the obtained 1ml liquid samples, a micro pipette was used to draw 5 ul liquid to the filter paper (6 mm )to perform bacteriostasis test, and to compare bacteriostasis effects of the 2 groups of compound gum suppositories. The compound gum suppositories were then stored in a refrigerator at 4oC for 6 months to repeat the bacteriostasis tests, and to compare the bacteriostasis annulus diameters with those of 6 months before. The basic medicines (ornidazole and pefloxacinmesylate) were weighed out according to the prescribed dosage to perform bacteriostasis tests (with no ground substance added to it), and then compare the bacteriostasis annulus diameters with those of the sustained-release compound gum suppositories containing the ground substance.3. With mice fixed stably, xylene (100%) was used to smear the left auricles of the selected mice on both sides to induce inflammation, 0.02 ml for each mice. 30 minutes later, experimental medicines were used to smear the left auricles of the 4 groups of mice. The smearing was performed at intervals of one hour, and 4 times in total. 30 minutes later, the mice were sacrificed, and the left auricles and the right auricles were cut off, and a hole puncher was used on the same location of the left auricles and the right auricles to make auricle slices (6 mm each), then their weights were obtained to calculate the swelling levels of the left auricles and the right auricles.4. The experimental SD rats of each group were fed with 5~10ml water before medication, 30 minutes later, a micrometer was used to measure the thickness of the their paws, then corresponding medicines were used to smear the left paws of each rat of the 4 groups every 30 minutes, 4 times in total. 10 minutes later, white egg (10%) was used to perform hypodermic injection into the left paws of each rat to induce inflammation, 0.1 ml for each one. Then the corresponding medicines were used to smear the left paws at every 30 minutes for 4 times in total. 30 minutes after the inflammation was induced, a micrometer was used to measure the thickness at the same location of both the left paws and the right paws at 0.5,1,2,3,4 hours respectively to calculate the swelling levels.5. Dental cervix ligation was used with the healthy SD rats (half male and half female, 180~220g each) to perform modeling. 8 weeks later, 10 modeling rats and 10 normal rats were randomly selected to test the periodontal specifications to conform whether the modeling was successful. Then 48 rats of successful modeling and 12 normal rats were divided into 5 groups to receive experimental medication. 24 hours later they were sacrificed to test the periodontal specifications and histopathological change of the 5 groups of rats, and to verify the treatment effects of the compound sustained-release suppositories.Results1. The experimental results showed that all the 5 kinds of ordinary compound ornidazole and pefloxacinmesylate gum suppository of different concentrations (1%,2%,5%,10%,20%) had a strong bacteriostasis effect on the 7 major suspected pathogenic bacteria. The sizes of bacteriostasis annuluses of the 7 different bacteria varied, and the sizes of bacteriostasis annuluses of the 5 different concentrations varied, and there were mutual effects with the different concentrations and the different kinds of bacteria. The results showed that the concentration of 20% had the strongest effect on Sa (the bacteriostasis annulus diameter was 27 mm), and the concentration of 1% had the weakest effect on Ms (the bacteriostasis annulus diameter was 7.9 mm). Compared with the annulus diameters of the other 4 concentrations, the bacteriostasis annulus diameter of the concentration of 20% is the largest one, showing that the concentration of 20% has the best and strongest bacteriostasis effect.2. The liquid samples (obtained at 0.5,1,2,3,4,5,6,7,8,9,10,11,12,16,20,24 hours respectively) from the sustained-release gum suppository group had corresponding bacteriostasis effects on the suspected pathogenic bacteria. Especially for the sensitive bacteria such as Pg,Pi,Sa,Se, the sustained-release effect was sustained for 24 hours. For the non-sensitive bacteria such as Ps,Ms, the sustained-release effect was sustained for 10 hours, showing an obvious advantage over the ordinary suppository. As for the ordinary gum suppository group (without sustained-release substance), their bacteriostasis effect on the sensitive bacteria such as Pg,Fn,Pi,Sa,Se was sustained for 9 hours, and the effect on Ps and Ms bacteria could only be sustained for 6 hours, which means it could not meet the sustained-release demand. The bacteriostasis annuluses of the compound sustained-release gum suppositories did not show any significant difference after they were stored in a refrigerator at 4oC for 6 months. The bacteriostasis effect of the 2 pure medicines (ornidazole and pefloxacin mesylate, without any ground substance) was similar with that of the compound sustained-release gum suppositories. As for the compound sustained-release gum suppositories (containing ground substance), the bacteriostasis effect on Pg,Fn,Ps,Sa was relatively weak (p<0.05). However, the bacteriostasis effect on Pi,Ms,Se did not show any significant difference.3. In experiment of ear swelling of mices, the swelling levels of the auricles of the 4 groups of mice varied. The high-dosage group of compound sustained-release gum suppository had the lowest swelling level (1.09 mg), while the blank control group had the highest swelling level (7.61 mg). The comparison of the high-dosage group and the low-dosage group with the blank control group showed a distinct difference (p<0.001), and the comparison of the high-dosage group with the positive control group also showed a distinct difference (p<0.05).4. In voix pedis swelling experiment, the swelling levels of the 4 groups of rat paws varied, and the swelling levels at different time points varied, and there were mutual relationships between different groups and the different time points. The high-dosage group of the compound sustained-release gum suppository had the lowest swelling level (1.134 mm) at 4 hours, while the blank control group had the highest swelling level (5.672 mm) at 1 hour. The comparison of the high-dosage group of the compound sustained-release gum suppository with the positive control group showed no difference(P>0.05) at 0.5,1,2 ,4 hours. However it showed significant difference (P>0.001) when compared with the blank control group at the same time points.5. 8 weeks after the modeling, all the periodontal specifications showed significant changes, and there was a distinctive difference (p<0.001) in comparison with the normal group, suggesting a successful modeling of the SD rats. The rats were sacrificed after the final dosage was given at the end of 5 group of medical test. Among the various evaluations, it was discovered that the following groups were improved: periodontal specification of compound sustained-release gum suppository group, metronidazole stilus group and ornidazole intragastric adiminstration group. In microscopical study, the group of compound sustained-release gum suppository was similar to the positive control group (metronidazole stilus). In comparison with the periodontitis group, the cellular infiltration of soft tissues in the group of compound sustained-release gum suppository was greatly alleviated, periodontal pockets became much shallower, and alveolar resorption ceased.Conclusions1. All the 5 kinds of compound gum suppositories of different concentrations have strong bacteriostasis effect. Having referred to relative documents, we finally selected the concentration of 10% as the optimized concentration for medication.The compound ornidazole and pefloxacinmesylate sustained-release gum suppository has sustained-release effect and a stable bacteriostasis effect on the suspected pathogenic bacteria in periodontal tissues. The ground substance does not affect the effectiveness of the sustained-release compound gum suppository.2. The anti-inflammation experiment shows that the compound ornidazole and pefloxacinmesylate sustained-release gum suppository has a strong inhibitory effect on the swelling of mice auricles (which was induced by xylene) and on the swelling of the rat paws (which was induced by egg white).3. The compound ornidazole and pefloxacinmesylate sustained-release gum suppository is effective in treating periodontitis, especially in reducing the inflammation and destruction of periodontal tissues.
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