The Transmembrane Transport Of Antibiotics By Human Gingival Fibroblasts

Some periodontitis pathogen can invade oral epithelial cells and gingival fibroblasts, evading the host response,which results in progress and recurrence of periodontitis. The majority systemic antibiotics can attain higher levels in gingival fluid and gingival tissure than in blood, the mechanism of which is the host cells can take up, accumulate, and redistribute these antibiotics.The foundation of dental implant administration system and dental root canal administration system is that the host cells in periodontium can transmembrane transport the agents. So, the present study is to investigate transmembrane transport of seven agents by human gingival fibroblasts to explore the feasibility of local drug delivery in periodontium and the possibility of the design of dental implant administration system and dental root canal administration system.The study includes two parts.Part 1 Comparison of Primary Culture Methods of Human Gingival Fibroblast in VitroObjective: To establish and evaluate the primary culture methods of human gingival fibroblast (HGF) cell lines and to investigate their biological features. Methods: The primary cells were cultured with tissue explants method, and two improved enzymatic digestion -explants methods respectively. The cells were identified by means of morphological analysis and immunofluorescence.The biological features were investigated with inverted phase contrast microscope and MTT colorimetric assay. Compare the effectiveness of the three methods in primary culture of HGF. Results: Cultured cells were spindle-shaped, and had a positive reaction to antibodies against vimentin and a negative reaction to antibodies against keratin. Their morphological and biological characteristicswere similar to those of typical HGF. The success rate of primary cells culture were 26.7% (tissue explants method), 54%(improved enzymatic digestion -explants method[invert flask], 60%(improved enzymatic digestion -explants method[coverslip] respectively(P<0.01). There was no statistically significant difference between the two enzymatic digestion-explants methods (P>0.05). Conclusions: The cell lines established in this study are proved to be human gingival fibroblasts.The two improved enzymatic digestion- explants methods can noticeably increase the success rate of primary culture of human gingival fibroblasts.Part 2 The Transmembrane Transport of Antibiotics by Human Gingival FibroblastsObjective: To investigate transmembrane transport of seven antibiotics by human gingival fibroblasts(HGF), and explore the effect of agent concentration and incubating time on transport to verify the feasibility of local drug delivery in periodontium and possibility of the design of dental implant administration system and dental root canal administration system. Methods: 1 Co- incubated the HGF cells and agent solutions at 37℃, then after incubation for indicated times(1,5, 10, 15(min), the agent solutions were quickly removed. The cells were collected and disrupted by supersonic wave. The lysate agent concentration were measured by high performance liquid chromatography (HPLC) and the cell total protein were measured by Bradford assay. 2 Monitor the transport of tetracycline hydrochloride and minocycline hydrochloride by HGF using Laser Scanning Confocal M icroscope. Results: 1 At steady state (agent concentration: 40ug/ml, incubation time: 10min), the HGF intracellular agent contents were 1.340±0.288 ng/μg (metronidazol), 2.071±1.028 ng/μg(tinidazole), 2.117±0.272 ng/μg (tetracycline hydrochloride), 7.039±0.937ng/μg(minocycline hydrochloride),0.570±0.042 ng/μg (cefotaxime sodium),1.172±0.391 ng/μg (norfloxacin),1.448±0.178ng/μg(levofloxacin) respectively.The transport ability of these agents are as follow:minocycline hydrochloride> tetracycline hydrochloride>tinidazole> levofloxacin>metronidazol>norfloxacin> cefotaxime sodium. 2 There were statistically significant differences in intracellular agent contents between different agent concentrations and between different co-incubating time (P<0.01). 3 There were statistically significant differences in co-incubating time needed to a achieve steady state among different agent concentrations(P<0.01).4 Fluorescence intensity emitted by tetracycline hydrochloride and minocycline hydrochloride enhanced with agent concentration and co-incubating time in Laser Scanning Confocal Microscope.The fluorescence of tetracycline hydrochloride and minocycline hydrochloride is located only in cytoplasm in 15 min.Conclusions: 1 HGF can transport all these antibiotics into cells, and there are differences in the transport ability among the antibiotics, which demonstrates the feasibility of local drug delivery in periodontium and the the possibility of the design of dental implant administration system and dental root canal administration system .2 The intracellular antibiotics contents are dependent on the extracellular agent concentration and also related with co-incubating time. 3 HPLC is an sensitive, accurate, precise method of the measurement of the intracellular agents .4 Laser Scanning Confocal Microscope can be used for monitoring the transport of agents with Fluorescence by living cells.