| Background and Objective:Oral implanting has gradually become one of the main ways to patients who lackof tooth and needs to repair, implant osseointegration and long-term stability mainlydepends on sufficient implant-bone contact ratio and the bone around theimplant.Implant osteolysis leads to aseptic loosening is the most common cause ofimplant failure. Osteoporosis (OP) is a disease characterized with mineral bonedensity, damage of microstructure, increasing porosity. OP patient’sskeletonmetabolism and bone quality have so many pathological changes that theimplant are difficult to obtain best retention. That greatly reduces the success ofosteoporosis patients. Therefore, how to enhance body retention in osteoporosis sitehas became a problem to be solved.Parathyroid hormone (PTH) is secreted by the parathyroid gland containing84amino acids of single polypeptide protein, bioactive fragments is PTH1-34which canmediated osteoblast’s synthesis metabolismã€stimulate the secretion of bone matrixand inhibit osteoblast apoptosis. It is the most important factor which directly effectson bone and calcium homeostasis regulation of kidney.In2002, the reorganizationparathyroid hormone (PTH1-34) is approved by the US.Food and DrugAdministration (FDA)as the treatment of osteoporosis and promote skeleton syntheticdrugs.Currently,PTH1-34has still deliveried by subcutaneous injection, which is moreexpensive, shorter half-life variability, poorer targeting, with painful andinconvenience to patients. Controlled release technology make the release of bioactiveagents in specific time possible. The biological surfactant coating into biodegradablepolymer material, that can protect effective components from the enzymaticdegradation in body and can control or extend its release concentration and duration.The technology can be used to deliver drugs in vitro, hormones, growth factors, etc. Poly glycolide-lactide (PLGA) because of its good biocompatibility, degradability anddosage controlling become the ideal drug delivery system and approved by the FDAas the security biological materials used in clinical. Our experiment prepared PLGAmicrospheres loaded with PTH1-34by double emulsion, realizing controlled releaseof PTH1-34, with the rat pre-ossification cells cultivation, through cytology andmolecular biology experiment in vitro to make evaluations for effects of the drugdelivery system.Method：Biodegradable polylactic acid microspheres loaded with parathyroid relatedpeptide on PTH (1-34) was measured by NaOH-SDS method to determine the drugloadings.Configured to release with physiological saline solution concentration ofdrug controlled release and co-cultured with MC3T3-E1cell that has already bydifferention,72h after planking ALP (alkaline phosphatase) staining.28daysplanking alizarin red staining to observe the degree of mineralization in vitro, and insetting time (24h,72h), to detect the microspheres controlling release solution’sinfluence of RUNX2, ALP and VEGF mRNA expression of MC3T3-E1cell.Results：1. The NaOH-SDS method measured PLGA microspheres of drug-loading rate was0.78%, microspheres with high drug-loading rate. It provides the basis for cellularand molecular biology experiments next step.2. ALP staining results of induced liquid culture after72h, ALP activity sites aredark or brown, some gathered together, ALP staining degree of experimentalgroup and positive control group are higher than control group.3. When osteoblast was cultivated by induced liquid to28d,alizarin red staining, thecell growthed stratified and have a calcium salt deposition.Alizarin red stainingshowed that the intermittent group (group b) and the experimental group’s (groupe) calcium nodule diameter were larger significantly compared with control group(group a)(P <0.05), group of microspheres without drug (group d) has fewcalcium nodules, continuing group (group c) did not see calcium nodules.4. Every group was cultivated by induced liquid after24h and72h, we examined detection of osteogenesis markers RUNX2, ALP and VEGF gene.RUNX2andALP expression trend high overall from the first day on the third day, includingintermittent dosing group (group b) and experiment group (group e) wassignificantly higher than the control group (group a)(P <0.05), the expression ofALP trend is consistent with the ALP staining results; VEGF expression is higher,on the first day and on the third day declined, but the experimental group (group e)expression quantity is always higher than control group which consisit with thepositive control group (group b).Conclusion：1. PLGA microspheres loaded with the parathyroid related peptide has high deliveryrate, which provides a basis for the preparation of the final concentration in celland molecular experiments.2. PLGA microspheres loaded with the parathyroid related peptide can obviouslypromote the ALP activity of MC3T3-E1cells and reveals the microspheres hadsignificant effect on the osteogenetic activity.3. PLGA microspheres loaded with the parathyroid related peptide can promoteMC3T3-E1cell terminal matrix calcification.4. PLGA microspheres loaded with the parathyroid related peptide promotesskeleton anabolic and blood vessel related genes mRNA expression of MC3T3-E1cells,that proved polylactic acid microspheres loaded drugs significantlypromoting MC3T3-E1cells’s osteogenesis differentiation in vitro from themolecular level once again.It predicts the control-release system in oralimplanting has a potential prospect in the field of clinic application and provides anew possible solution to solve such problems as insufficient bone aroundperi-implant in clinical... |
