Development Of HPDLF And HGF Drug Transport Models And Preliminary Study On The Associated Mechanism

Some periodontitis pathogen can invade oral epithelial cells and gingival fibroblasts,evading the host response.Some antibiotics can attain higher levels in the cells described above and in gingival crevice fluid than that in blood,while some antibiotics couldn't be accumulated in cells.The study on the uptake mechanism of antimicrobials by cells will provide a guide to clinical appilication of antimicrobials and improve the therapeutic effect of antimicrobials.Uptake and transcellular transport of agents by human periodontal ligaments fibroblasts(HPDLF) and human gingival fibroblasts(HGF) are theoretical basis of dental implant administration system and dental root canal administration system. To explore the feasibility of local drug administration routes described above,we studied the uptake mechanism of antibiotics by HPDLF and HGF preliminarily, and developed HPDLF and HGF drug transport models.The study includes two parts.Part 1 Preliminary Study on the Uptake Mechanism of Antibiotics by HPDLF and HGFExperiment 1 Study on the Uptake Mechanisms of Metronidazole and Tinidazole by HPDLF and HGFObjective:To investigate the uptake of metronidazole and tinidazole byHPDLF and HGF.Methods:After HPDLF and HGF incubated with metronidazole and tinidazole respectively,the intracellular agents concentrations were measured at different times with high performance liquid chromatography(HPLC).The relationship between the intracellular agents concentrations and extracellurlar agents concentrations,and the effect of temperature,pH and transport inhibitors on the uptake of agents by HPDLF and HGF were studied.Results:The intracellular metronidazole and tinidazole concentrations reached the extracellular levels in 3 minutes after incubation;The intracellular metronidazole and tinidazole concentrations increased linearly with the extracellular level; Temperature,pH and transport inhibitors had no significant effect on the uptake of agents by HPDLF and HGF.Conclusions:HPDLF and HGF can take up metronidazole and tinidazole rapidly via simple diffusion,which proves the feasibility of drug delivery through the dental implant administration system and root canal.Experiment 2 Expression of Organic Ion Transporters mRNA in Human Gingival FibroblastsObjective:To detect the expression of organic cation transporters(OCTs) and organic anion transporters(OATs) mRNA in human gingival fibroblasts(HGF) and.Methods:RT-PCR was used to detect the expression of OCTs and OATs mRNA in HGF.RESULTS:The expression of OCT1,OCT2,OCTN1 and OAT1-4 mRNA in HGF was found.CONCLUSION:The findings of the test verify the speculation that there exist OCTs and OATs in the HGF.Part 2 Development of HPDLF and HGF Drug Transport Cell Models and Transcellular Transport of AntibioticsExperiment 1 Development and Validation of HPDLF and HGF Drug Transport Cell ModelsObjective:To develop HPDLF and HGF drug transport cell models.Methods:HPDLF and HGF were cultivated on polycarbonate filter membrane of transwell respectively.After1,2,3 and 4 weeks of culture,transepithelia electrical resistance(TEER) was detected and the growth of HPDLF and HGF were observed by light microscope.After 2 weeks of culture,the models were observed by the scanning electron microscope(SEM),transmission electron microscope (TEM),and the permeability was measured with fluorescein sodium.RESULTS: HPDLF and HGF cells connected each other tightly and completely after 2 weeks. Observation of section of filter membrane revealed a stratified cell growth of HPDLF and HGF 2 weeks after inoculation,and TEERs of HPDLF and HGF were 56.14±7.43 and 57.34±7.62Ω·cm^-2 respectively.Being incubated for 30 minutes, less than 1%of fluorescein sodium reached the inferior filter membrane 2 week after inoculation.CONCLUSION:The tests of permeability and TEER were consistent with the demands of development of drug transport cell models. HPDLF and HGF drug transport cell models could be used to study drug transport by HPDLF and HGF in vitro. Experiment 2 Transcellular Transport of Metronidazole,Tinidazole, Minocycline and Cefotaxime Sodium in HPDLF and HGF Drug Transport Cell ModelsObjective:To investigate the transcellular transport of metronidazole,tinidazole, minocycline and cefotaxime sodium in drug transport cell models of HPDLF and HGF.Methods:The transcellular transport of metronidazole,tinidazole, minocycline and cefotaxime were measured with HPLC.Results: Metronidazole,tinidazole and minocycline could be transported through the HPDLF and HGF cell layers completely in 7-8min.Cefotaxime sodium could be transported through the HPDLF and HGF cell layers completely in 50 and 46 min..Conclusion:The transcellular transport of agents by HPDLF and HGF were observed in our study,which proves the feasibility of drug delivery through the root canal.