| BACKGROUDPeriodontitis is one of the most common infections and is a major cause of tooth loss.It is a polymicrobial infection, and thus, the complex interaction among microorganismsmakes the disease a challenging one to understand and treat. These pathogens includeanaerobes, such as Porphyromonas gingivalis, Prevotella intermedia, Fusobacteriumnudeatum and Peptostreptococcus anaerobiu, as well as facultative anaerobes, includingStaphylococcus aureus and Staphylococcus epidermidis, among others. Bacterial plaque isrecognized as the primary agent for the initiation and progression of periodontitis. Plaquemicroorganisms can damage the periodontium by releasing their proteolytic and noxiouswaste products and by stimulating the host cells to produce pro-inflammatory cytokines,inducing connective tissue and alveolar bone destruction.Therefore, a successful treatment depends on the elimination or control of thepathogens together with a microbial shift toward a microbial population, which is typicallypresent in healthy individuals. Treatments include mechanical and drug therapy. Mechanicaltherapy involves nonsurgical scaling and root planing (SRP) and drug therapy is adjunctiveto SRP.Drug therapy includes local and systemic administration. Although systemicadministration has some benefits, an inadequate concentration in the periodontium and ahigh plasma concentration may be associated with bacterial resistance and side effects. Animportant advantage of local administration is the higher therapeutic concentration in thelesion location, leaving residual parts unaffected.Ornidazole is a member of the nitromidazoles and is widely used in the treatment ofthe oral disease, which have better activity against anaerobes than quinolones. Pefloxacinmesylate is a member of the quinolones, and these have better activity against facultative anaerobes than nitromidazoles. As previously mentioned, anaerobe is dominant microbialpopulation, but facultative anaerobe also plays a part in break down of deep periodontaltissues.The use of PLGA as a biodegradable polymer in microsphere production is common inthe literature due to its attractive properties, including the availability of variousco-polymer compositions and molecular weights, which makes the manufacture ofmicrospheres with tailored characteristics accessible.So, the aim of this study can be summarized as preparation of ornidazole/pefloxacin-loaded PLGA microspheres with optimized characteristics and in vivo evaluation of t thepharmacological effect on chronic periodontitis.MATERIALS AND METHODS1. Preliminary preparation of compound ornidazole/pefloxacin PLGA sustained releasemicrospheresDouble emulsion→solvent evaporation method was used to prepare the microspheres,high performance liquid chromatographic analysis (HPLC) was used to measure thedrug content. With the encapsulation efficiency as the main investigation index,preparation device, organic solvents combination, PVA concentration in external waterphase was preliminary optimized.2. Prescription and process optimization of compound ornidazole/pefloxacin PLGAsustained release microspheresOn the basis of preliminary improvement, with the encapsulation efficiency as themain investigation index, preparation device, the molecular weight and concentration ofPLGA, proportion of PLA/PGA, the proportion of drugs/PLGA, the proportion ofmethylene chloride/ethyl acetate in oil phase, stirring speed and time of high-speed shearmachine were optimized through single factor experiment. And then, the concentration ofPLGA, the proportion of methylene chloride/ethyl acetate in oil phase, and the stirring timeof high-speed shear machine were futher optimized through orthogonal test on3factors3levels.3. Drying and sterilizationPowder traits, surface morphology, encapsulation efficiency and burst release effect ofthe microspheres were investigated and compared under freezing drying and room temperature vacuum drying methods. Through the microbial inspection, the best radiationsterilization dose of60Co was screened from2,5,10,15kGy.4. Performance testing of compound ornidazole/pefloxacin PLGA sustained releasemicrospheres after optimizationIn order to determinate wheather pharmaceutical preparation is suitable for localadministration for periodontitis, the powder traits, surface morphology, encapsulationefficiency, pH value of suspension liquid drugs-release trait in vitro of the microsphereswere investigated after optimization.5. Impacts of the microspheres in vitro on proliferation, mineralization of periodontalligamnent fibroblastsPeriodontal ligamnent fibroblasts were cultured by tissue culture method,cell wasidentificated through morphology and immunohistochemistry. In order to judge cell safetyof the Pharmaceutical preparation, proliferation activity, and cell toxicity andmineralization activity in50mg/ml microspheres, blank microspheres, and5mg/ml ofornidazole/pefloxacin in1~7d were investigated through MTT method and alkalinephosphatase activity determination method.6. Antibacterial experiment of the microspheres in vitroAnaerobes (Porphyromonas gingivalis, Prevotella intermedia, Fusobacteriumnudeatum and Peptostreptococcus anaerobiu), afacultative anaerobes (Staphylococcusaureus and Staphylococcus epidermidis) were cultured in vitro. Drugs released fromsustained release microspheres were collected at1d,2d,3d,4d,5d,6d,8d,10d,12d,14d,16d,18d,20d,22d and24d. K-B method was used to evaluate the antibacterial effect atdifferent time points.7. Effects of the microspheres on experimental periodontitis in ratsPeriodontitis was induced in rats by placing a thin-steel ligature around the upper firstmolars and inoculating them with Porphyromonas gingivalis381. After8weeks, SBI, PI,PPD, ABL, AST-GCF and histopathology were used to evaluate the success of the rat ECP.After the successful induction, effects of systemic ornidazole, systemic compoundornidazole, local sustained release microspheres and local YAKANG (metronidazole) onexperimental periodontitis in rats were evaluated. RESULTS1. In HPLC, the retention time of ornidazole and pefloxacin respectively is10.299minand13.107min, the separation in good condition. The linear relation between peak area andconcentration of drug was good in1~30μ g/ml range.2. Optimization results: the container was beaker, the stirring device was high-speedshear machine, the stirring time was1.5min, methylene chloride/ethyl acetate was1:2,PLGA molecular weight was25000, the concentration of PLGA in organic solvent was200mg/ml, LA/GA was75:25, drug/PLGA was1:10, the concentration of PVA in externalwater phase was3%.3. After freeze-drying processing the powder of microspheres was in good liquiditywithout adhesion, however, after room temperature vacuum drying processing the powderappeared agglomerate and adhesion phenomenon. After room temperature vacuum dryingprocessing, the burst release effect increased obviously and the encapsulation efficiency islow.2kGy60Co could not achieve sterilization thoroughly,5,10,15kGy60Co could allachieve sterilization thoroughly.4. After optimization, color of powder was white, roundness degrees of powder were35.99±1.03°, repose angle of powder was36.1±2.20°, and bulk density of powder was0.3804±0.0311g/ml. Under Light and scanning electron microscopy, the microspheres weresmooth, in good balling index and in lesss adhesion state. After screening throughmolecular sieve, the particle sizes of microspheres were in normal distribution (14~30μm); the mean value was21.60±2.41μm. the encapsulation efficiency of ornidazole andpefloxacin was68.15±0.40%and64.07±0.37%respectively, drug loadings was6.18±0.15%and3.95±0.21%respectively. pH value of Microspheres in1%physiological saline,5%glucose and pure water was7.01±0.03,4.91±0.27,5.57±0.16respectively.At24h,ornidazole in vitro released about25%by degrees, pefloxacin released about35%. Theburst release effects existed. Over time, the drug release rate remained stable.At20d,ornidazole in vitro released about95%by degrees, pefloxacin released above97%.5. Periodontal ligamnent fibroblasts cultured in vitro were from mesoderm and were ingood condition. At different time point, there was no obvious change between differenttreatment groups on cell proliferation, there was no cell toxicity in microspheres, and there wasno obvious change between different treatment groups on cell alkaline phosphatase activity. 6. The antibacterial experiment of sustained release microspheres in vitro showed thatduring the whole experiment time, the pharmaceutical preparation could have effectiveantibacterial activity; at the fist day, the bacteriostatic effect was the strongest; during2~20d,the bacteriostatic effect maintain stable; after20d the bacteriostatic effect decreasedobviously.7. After8weeks' induction, the periodontal health indicators in periodontitis rats, suchas SBI, PI and PPD, GCF-AST, ABL showed statistically significant differences comparedwith normal rats.Histopathology observation in ECP rats revealed inflammatory cellinfiltration, apical migration of the junctional epithelium, severe cementum destructionresembling a periodontitis lesion. The disorders of these indicators indicated the success ofperiodontitis mode. After20days treatment, compared with ECP, all the combinedtreatments produced beneficial changes in the clinical indicators.The histopathology of theperiodontium revealed a significant reduction in the numbers of inflammatory cellinfiltration as well as in the inhibition of destruction of cementum. Among these treatmentgroups, the Local sustained release microspheres gruop is most effective.CONCLUSIONS1. After optimization, the microspheres were smooth, in good balling index and inlesss adhesion state. The encapsulation efficiency, drug loadings and release activity in vitroof the microspheres were in good condition. The pharmaceutical preparation was suitablefor local injection treatment of periodontitis.2. The microspheres exerted no obvious influence on the proliferation andmineralization of periodontal ligamnent fibroblasts. The pharmaceutical preparation exertedno toxicity on periodontal ligamnent fibroblasts.3. The pharmaceutical preparation had obvious bacteriostatic effect to the mainpathogenic peiodontitis bacteria in20days in vitro.4. A rat model of peiodontiti, which can provide an important basis for follow-uptreatment experiments, was successfully established. In the treatment of chronicperiodontitis, the pharmaceutical preparation (microspheres) was adjuvant to periodontalinstruments, has certain advantages.
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