Biological Transport Of Antibiotics By Human Periodontal Ligament Fibroblasts

The invasion of bacteria to host cells made it more difficult to deal with the periodontal dieases. The concentrations of some medicine in gingival crevicular fluid were higher than those in serum. To verify the hypothesis of delivering medicine to the periodontium and whole body through the root canal, we carried out experiments to study the biological transport of 8 kinds of antibiotics by human periodontal ligaments fibroblasts (HPDLF). There are:Part 1: the biological transport of metronidazole by HPDLF Objective: To investigate biological transport of metronidazole by HPDLF. Methods: Premolars extracted for orthodontic reasons were used in this study. HPDLF were primarily cultured by tissue explant. HPDLF were incubated in metronidazole solutions. After disrupting cells by supersonic wave, the intracellular antibiotics contents were measured by HPLC and the cell total protein was measured by Bradford protein assay. Results: HPDLF were primarily cultured successfully and passaged stably. The intracellular metronidazole content of HPDLF couldn't be detected, incubated in 20 μg/ml antibiotics solution. The MC3T3-E1 cells intracellular contents were 0.165±0.034 ng/μg, 0.206±0.065 ng/μg at 5 min, 10 min respectively. Conclusion: HPLC is an accurate, sensitive method for measurement of the intracellular antibiotics. Metronidazole can't be transported by HPDLF. The transport is cell specific.Part 2: the biological transport of tinidazole by HPDLFObjective: To investigate biological transport of tinidazole by HPDLF. Methods: HPDLF and MC3T3-E1 cells were incubated in antibiotics solutions. The intracellular antibiotics contents were measured by HPLC and the cell total protein was measured by Bradford protein assay. Results: The HPDLF intracellular contents were 0.067±0.049 ng/μg, 0.099±0.024 ng/μg, 0.078±0.015 ng/μg at 1min, 5 min, 10 min respectively, incubated in 100 μg/ml antibiotics solution. The HPDLF intracellular content was 0.142±0.011 ng/μg at 5 min, incubated in 200 μg/ml antibiotics solution. The MC3T3-E1 intracellular content was 0.082±0.027 ng/μg at 5 min, incubated in 100 μg/ml tinidazole solution. Conclusion: HPLC is an accurate, sensitive method for measurement of the intracellular antibiotics. Tinidazole can be transported by HPDLF. The transport is time-dependent, concentration-dependent and cell specific.Part 3: the biological transport of tetracycline hydrochloride by HPDLF Objective: To investigate biological transport of tetracycline hydrochloride by HPDLF. Methods: HPDLF and MC3T3-E1 cells were incubated in antibiotics solutions. The intracellular antibiotics contents were measured by HPLC and the cell total protein was measured by Bradford protein assay. Results: The HPDLF intracellular contents were 0.192±0.008 ng/μg, 0.212±0.082 ng/μg, 0.620±0.075 ng/μg at 1min, 5 min, 10 min respectively, incubated in 10 μg/ml antibiotics solution (p<0.01). The HPDLF intracellular content was 0.503±0.056 ng/μg at 5 min , incubated in 20 μg/ml antibiotics solution and was siginificantly higher than that of 10 μg/ml group (p<0.05). The MC3T3-E1 intracellular content was 0.666±0.560 ng/μg at 5 min, incubated in 10 μg/ml antibiotics solution. Conclusion: HPLC is an accurate, sensitive method for measurement of the intracellular antibiotics. Tetracycline hydrochloride can be transported by HPDLF. The transport is time-dependent, concentration-dependent and cell specific.Part 4: the biological transport of minocycline by HPDLF Objective: To investigate biological transport of minocycline by HPDLF. Methods: HPDLF and MC3T3-E1 cells were incubated in minocycline solutions. The intracellular antibiotics contents were measured by HPLC and the cell total protein was measured by Bradford protein assay. Results: The HPDLF intracellular contents were 1.661±0.520 ng/μg, 1.949± 0.415 ng/μg, 2.154± 0.728 ng/μg at 1min, 5 min, 10 min respectively, incubated in 20 μg/ml antibiotics solution (p<0.05). The HPDLF intracellular content was 2.155±0.728 ng/μg at 5 min, incubated in 20 μg/ml antibiotics solution. The MC3T3-E1 intracellular contents were 1.532±0.396 ng/μg, 3.331±0.538 ng/μg, 2.811±0.798 ng/μg at 1min, 5 min, 10 min respectively, incubated in 20 μg/ml antibiotics solution (p<0.05). The MC3T3-E1 intracellular contents were 0.751±0.265 ng/μg, 3.188±0.636 ng/μg at 5 min respectively, incubated in 10 μg/ml and 40μg/ml antibiotics solution (p<0.01). Conclusion: HPLC is an accurate, sensitive method for measurement of the intracellular antibiotics. Minocycline can be transported by HPDLF. The transport is time-dependent, concentration-dependent and cell specific.Part 5: the biological transport of roxithromycin by HPDLF Objective: To investigate biological transport of roxithromycin by HPDLF. Methods: HPDLF and MC3T3-E1 cells were incubated in antibiotics solutions. The intracellular antibiotics contents were measured by HPLC and the cell total protein was measured by Bradford protein assay. Results: The intracellular antibiotics content of HPDLF and MC3T3-E1 cells couldn't be detected at 1min, 5 min, 10 min, incubated in 20 μg/ml and 40 μg/ml antibiotics solutions. Conclusion: HPLC is an accurate, sensitive method for measurement of roxithromycin. With the limitations, roxithromycin can't be detected of transport by HPDLF.Part 6: the biological transport of norfloxacin by HPDLF Objective: To investigate biological transport of norfloxacin by HPDLF. Methods: HPDLF and MC3T3-E1 cells were incubated in norfloxacin solutions. The intracellular antibiotics contents were measured by HPLC and the cell total protein was measured by Bradford protein assay. Results: The HPDLF intracellular contents were 0.095±0.032 ng/μg, 0.096±0.052 ng/μg, 0.104±0.078 ng/μg at 1min, 5 min, 10 min respectively, incubated in 10 μg/ml antibiotics solution. The MC3T3-E1 intracellular contents were 0.033±0.018 ng/μg, 0.034±0.013 ng/μg, 0.082±0.029 ng/μg at 1min, 5 min, 10 min respectively, incubated in 10 μg/ml antibiotics solution (p<0.05). The MC3T3-E1 intracellular contents were 0.070±0.057 ng/μg at 5 min , incubated in 20 μg/ml antibiotics solution. Conclusion: HPLC is an accurate, sensitive method for measurement of the intracellular antibiotics. Norfloxacin can be transported by HPDLF. The transport is time-dependent, concentration-dependent and cell specific.Part 7: the biological transport of lincomycin hydrochloride by HPDLF Objective: To investigate biological transport of lincomycin hydrochloride by HPDLF. Methods: HPDLF and MC3T3-E1 cells were incubated in antibiotics solutions. The intracellular antibiotics contents were measured by HPLC and the cell total protein was measured by Bradford protein assay. Results: The intracellular antibiotics content of HPDLF and MC3T3-E1 cells couldn't be detected at 1min, 5 min, 10 min , incubated in 10 μg/ml and 20 μg/ml antibiotics solutions. Conclusion: HPLC is an accurate, sensitive method for measurement of lincomycin hydrochloride. With the limitations, lincomycin hydrochloride can't be detected of transport by HPDLF.Part 8: the biological transport of cefotaxime sodium by HPDLF Objective: To investigate biological transport of cefotaxime sodium by HPDLF. Methods: HPDLF and MC3T3-E1 cells were incubated in antibiotics solutions.The intracellular antibiotics contents were measured by HPLC and the cell total protein was measured by Bradford protein assay. Results: The HPDLF intracellular contents were 0.104±0.030 ng/μg, 0.151±0.007 ng/μg, 0.161±0.046 ng/μg at 1min, 5min, 10min respectively, incubated in 100 μg/ml antibiotics solution. The MC3T3-E1 intracellular content was 0.096±0.027 ng/μg at 5 min, incubated in 100 μg/ml antibiotics solution. Conclusion: HPLC is an accurate, sensitive method for measurement of the intracellular antibiotics. Cefotaxime sodium can be transported by HPDLF. The transport was time-dependent, cell specific.