Biological Transport Of Glybenclamide And Insulin By Rat Mandibular Osteoblasts

Choosing rational form of medication is given extensive interest in medication of diabetic in recent years. Local drug delivery is one of the efficacious way to lessen the side effects of systemic administration. Prof. Liu have proposed the hypothesis on delivering medicine to the whole body through the implant and root canal. To verify the hypothesis, The experiments were carried out of biological transport of glybenclamide and insulin in osteoblast. The whole study included 2 parts. There are: Part 1:Modified primary culture of mandibular osteoblasts from rat mandible Objective: To investigate an efficient method of primary culture of osteoblasts from rat mandible, and to establish a foundation for further experimental study. Methods: The mandible harvested from 4-6 weeks SD rats was stripped off all soft tissues including the periosteum, rinsed and cut to trivial bone block under sterile condition. Then, the bone block was subcultured in culture flask after digested with modified enzymatic digestion-explants method, the acquired cells were identified with invert microscope, alkaline phosphorase stain, immunohistochemistry stain of osteocalcin and stain of calcified nodules. Results: The cells obtained from rat's alveolar bone were identified to be osteoblasts by invert microscope, alkaline phosphorase stain, immunohistochemistry stain of osteocalcin and stain of calcified nodule. Conclusion: The modified enzymatic digestion-explants method is an ideal method to obtain and culture mandibular osteoblasts having typical characteristics.Part 2: Biological Transport of glybenclamide and insulin by osteoblas Objective: To investigate transmembrane transport of glybenclamide and insulin by osteoblasts, verifying the feasibility of local drug delivery of the design of dental implant administration system. Methods: 1. Incubated the MC3T3-E1 and rat MO with agent solutions, then after incubation for indicated times( 1, 5, 10, 15, 20, 30, 45min), the agent solutions were quickly removed . The cells were collected andwere disrupted by ultrasonic wave. The lysate agent concentration was measured by high performance liquid chromatography (HPLC) and the cell total protein was measured by Bradford assay. Results: Glybenclamide could be transported by both kinds of cells. The intracellular contents were dependent on the extracellular concentration and incubation time. The intracellular concentration in 100ug/ml was even bigger than it in 50ug/ml at identical time. The intracellular contents increased rapidly with incubation time in 3 minutes and decreased after 20 minutes. The intracellular contents of osteoblasts were significantly lower than that of MC3T3-E1 cells ( P<0.01) . The intracellular insulin of MC3T3-E1 couldn't be detected during the total observation time. Conclusion: 1. HPLC is a sensitive, accurate, precise method of the measurement of the intracellular agents. 2. Rat mandibular osteoblast and MC3T3-E1 cell can transport glybenclamide into cells. 3. The intracellular drug contents are dependent on the extracellular agent concentration and also related with co-incubating time. 4. Insulin can not be transport into MC3T3-E1 cell on the extracellular concentration.