| In clinic, periodontitis and peri-implant infection caused by bacteria are very difficult to cure. It may relate with the theory that some bacteria are capable of invading host cells and escape immunity response and some antibacterial that can not be accumulated in cells. Staphylococcus aureus (SA) is a common pathogenic bacterium in infective diseases. Many studies showed that SA could invade osteoblasts or be phagocytized by phagocyte, which caused the persistence and relapse of the infection. It is very critical to choose the antibacterials that can penetrate osteoblasts and have no effect on cell function. Our previous studies showed that minocycline (MINO) could penetrate osteoblasts, but there have been no studies to determine whether MINO is capable of playing antibacterial effect in osteoblasts. Our experiments were carried out to investigate this problem and understand initially intracellular activity of MINO. It will benefit to choose antibiotics reasonably in treatment of SA invasive infection and provide experimental evidence for the hypothesis of administration through artificial tooth implant.In this study, mature rat alveolar osteoblast (MRAOBs) were cultured in vitro.Then the influences of minocycline on the proliferation, protein synthesis and alkaline phosphatase (ALP) activity of MRAOBs were investigated in order to confirm the proper minocyline concentration to kill the bacteria in osteoblasts. The intracellular and extracelluar antibacterial activity of MINO were observed in infected osteoblasts by SA. Fistly, various concentrations of minocycline were added to the medium of cultured MRAOBs respectively. After 2 days, the effect of minocycline on cell proliferation and protein synthesis were assayed. After 4 days, the ALP activity was measured. Results showed that the presence of minocycline (1～200mg/L) in the medium resulted in no morphological change of MRAOBs.Significantly enhanced the proliferative activity and protein synthesis of MRAOBs (P<0.01). Moreover, as the increase of minocycline concentration within 1～100mg/L, the effects of promoting proliferative activity and protein synthesis were enhanced. The maximum promotion was obtained at 100mg/L. However, at a concentration range of 1 to 200mg/L, minocycline significantly inhibited cellular ALP activity. And the inhibition was ehanced with increasing the concentration of minocycline gradually. So the concentration of 100mg/L minocyline is chosen to observe the amount of minocycline uptake by MRAOBs during 15 to 60 minutes. Result indicated that the intracellular accumulation of minocycline during 15 to 60 minutes was no significant difference (P>>0.05), maybe reach saturation of cytosis, which prove MINO was capable of maintaining higher content for longer in osteoblast. Meanwhile after MRAOBs were infected by SA, intracellular bacterium colony forming units (CFU) were calculated and the status of invision was observed through transmission electron microscope (TEM) examination. Result showed that SA were internalized at latter three time point with SA being ingested more at 60min and followed at 45min and 30min. Finally, the samples of MRAOBs invaded by SA strain ATCC25923 for 60min were divided into two group. After killing extracellular SA with gentamicin, 100mg/L minocycline were added in one group. Intracellular antibacterial activity was observed exclusively. Result showed that the intracellular bacteria were reduced gradually with the prolongation of minocycline effect during 1 to 24 hours and were almost killed completely for 24 hours. While 100mg/L minocycline were added directly in another group, the intracellular and extracellular bacteria were reduced simultaneously. And extracellular bacteria were almost killed for 3 hours, almost all intracellular bacteria were not present for 24 hours.In conclusion, a certain range concentration of minocycline could promote osteoblast proliferation and protein synthesis, inhibit osteoblast differentiation. So the concentration of 100mg/L minocyline may be chosen as the properconcentration for killing the bacteria intra-osteoblasts and extra-osteoblasts. MINO possesses the character of antibacterial effect on SA in osteoblasts.
|
PDF Full Text Request