The Transmembrane Transport Of Metformin And Pravastatin By Osteoblasts From Rat Mandible

Diabetes and osteoporosis are main systemic diseases that affect the dental implant treatment.They impact the restoration and reconstruction on bone tissue by inducing a series of pathological changes around the dental implant and decrease the success ratio of dental implant.Metformin,an anti-hyperglucose drug,can not only lower blood glucose,but also promote osseointegration around the implant;pravastatin,an anti-hyperlipidemic drug,can treat osteoporosis efficiently,and it is the only one that has the ability to promot the bone formation and inhibite the bone resorption.Therefore,topical administration around dental implant become the focus of the study in present.In particular,Liu Hong-chen proposed dental implant drug administration system—through the contact between implant and bone tissue,drugs can be transported to the local and the whole body so as to achieve the better therapeutic effects.So,we need to understand the capacity of transporting drugs by the bone tissue cells.In this study, we explored the possible transport mechanism through observing the transmembrane transport of metformin and pravastatin by osteoblasts from rat's mandible to provide the experimental basis for drug administration by dental implant.PartⅠThe establishment of the transmembrane transport transwell model of rat mandible osteoblastsObsjective:To built the transmembrane transport transwell model of rat mandible osteoblasts. Methods:Primary osteoblasts were isolated,cultured,identified and inoculated in the filter membrane of polycarbonate at the bottom of the upper well.Light microscope was utilized to observe the cells growth,scanning and transmission electron microscope was applied to observe the cell junction,membrane resistance and Fluorescein Sodium permeation experiment were detected the barrier function of osteoblasts.Results:Osteoblasts were cultured and inoculated in the filter membrane of polycarbonate successfully;cells(1×10~5/ml)converged completely,connected closely and displayed ambi-layer generation 2 weeks after inoculation;tight junction between each cell was observed by scanning and transmission electron microscope;cells barrier function shown that TER of cells was increasing with time adding and reach the maximum(88.65±7.33)Ω/cm~2 which maintained till the 3rd week after inocubation;Fluorescein Sodium permeation experiment presented that after incubated for 30 minutes,only 0.17%of Fluorescein Sodium reached the inferior filter membrane.Conclusions:The transwell can be used to set up the model of osteoblasts with growth of ambi-layer and investigate the mechanic of the transmembrane transport of drugs.PartⅡThe transmembrane transport of metformin by osteoblasts from rat mandibleObjective:To detect the transmembrane transport of metformin by osteoblasts, investigate the possible mechanic of transport and explore the condition and feasibility of dental implant drug delivery.Methods:HPLC(High Performance Liquid Chromatography)was utilized to detect the transmembrane transport of metformin by osteoblasts.The effect of time,drug concentration,temperature,pH,cimetidine(an inhibitor of organic cation transporter),and verapamil(an inhibito of P-protein)on the transport of metformin were studied.Octs(Organic cation transporters)expression were characterized by Immunocytochemistry and RT-PCR.Western blotting was ulilized to detect the effect ofmetformin on the expression of Oct1.Results:Osteoblasts has the ability of transmembrane transport of metformin. The transport of metformin could be saturable with time and drug concentration adding;a certain range of temperature and pH can promote the transport of metformin;The transport of metformin was inhibited by cimetidine and phenformin,but promoted by verapamil;Oct1 but no Oct2 was expressed extensively in cell membrane and cytoplasm of osteoblasts;The protein level of Oct1 can be up-regulated by metformin with different drug concentrations and times,but no obvious differentces from each group.Conclusions:Osteoblasts from rat mandible have the ability of transmembrane transport of metformin.,and the transport of metformin in osteoblasts was facilitated diffusion mediated by Oct1 and an active transportation mediated by P-gp which proves the possibility of the local drug delivery around dental implant in diabetes patients.PartⅢThe transmembrane transport of pravastatin by osteoblasts from rat mandibleObjective:To detect the transmembrane transport of pravastatin by osteoblasts, investigate the possible mechanic of transport and explore the condition and feasibility of dental implant drug delivery.Methods:HPLC(High Performance Liquid Chromatography)was utilized to detect the transmembrane transport of pravastatin by osteoblasts.The effect of time,drug concentration,temperature,pH,simvastain and lovastatin(the other stins)and verapamil on the transport of pravastatin were studied.Oatp1(Organic anion transporting polypeptide 1)expression were characterized by Immunocytochemistry.Western blotting was ulilized to detect the effect of pravastatin on the expression of Oatp1.Results:Osteoblasts has the ability of transmembrane transport of metformin. The transport of metformin could be saturable with time and drug concentration adding;a certain range of temperature and pH can promote the transport of metformin;The transport of metformin was inhibited by simvastain and lovastatin, but not effected by verapamil;Oatp1 was expressed extensively in cell membrane and cytoplasm of osteoblasts;The protein level of Oatp1 can be up-regulated by pravastatin with different drug concentrations,but no obvious differentces from each group.Conclusions:Osteoblasts from rat mandible have the ability of transmembrane transport of pravastatin,and the transport of pravastatin in osteoblasts was facilitated diffusion mediated by Oatp1 and an active transportation which proves the possibility of the loca1 drug delivery around dental implant in osteoporosis patients.