| Objectives: Prostaglandin E2(PGE2)is a lipid compound with high biological activity and a complex multifunctional bone metabolism regulator.It is involved in angiogenesis,bone repair and cartilage metabolism.PGE2 is now recognized as an important target molecule for tissue engineered bone and cartilage tissue regeneration solutions.As a signal molecule in periodontal remodeling in oral field,Acceleration of orthodontic tooth movement on PGE2 is already demonstrated in a large number of researches.In bone tissue,PGE2 is mainly synthesized by osteoblasts.It can directly interacts with osteoblasts and osteoclasts,on the other hand with bone marrow cells indirectly.Mediate bone is remodelled by increasing the recruitment of osteoclasts and osteogenic precursor cells.The effect of PGE2 on bone remodeling is biphasic.Replace "on low concentrations or in the presence of glucocorticoids,PGE2 promotes differentiation of osteoblasts and bone formation;but on high concentrations or with insulin-like growth factors-1 in the presence of PGE2,it can inhibit bone formation by narrow collagen synthesis.PGE2 plays a key regulatory role in orthodontic treatment and remodels alveolar bone directly or indirectly to regulate bone formation and absorption.However,PGE2 is a local hormone,local synthesis,and local inactivation,the half-life period is very short(usually 1-2 minutes),which Impose restrictions on its application.Therefore,in the field of orthodontic study,it is very important to construct PGE2 controlled-release system sustained-release PGE2.PLGA is a biodegradable polymeric release sustained-release material that is prepared from polylactic acid and glycolic acid in different proportions.It is one of the few carrier permitted by the US FDA and has been used clinically.It shows low toxicity,high safety,good stability,and good biocompatibility.It is often used as a carrier for peptides,proteins and anti-tumor drugs.Through its own degradation,it can release the drug at a more stable rate in a few weeks or months,maintain effective blood drug concentration,reduce the number of drug half-life administration,and it is suitable for drugs with short half-life but for a long-term use.It has been widely used nowadays in tissue engineering scaffolds and drug carriers.Research show that nano-hydroxyapatite particles(nano-HAP)can promote osteoblast proliferation,adhesion,and mineralization-related protein expression.The mineral matrix in bone matrix accounts is about 35%,mainly composed of calcium and phosphoric acid hydroxyapatite(HA).The formation of bone matrix is the result of differentiation and mineralization of osteoblasts and issential for bone formation.When osteoblasts enter into the differentiation stage,they secrete large amounts of extracellular matrix,including type I collagen,non-collagen and proteoglycans,and under the regulation of osteoblasts and various ions,proteins,lipids and enzymes,calcium and phosphate.After the ions reach a certain concentration,hydroxyapatite is mineralized in the matrix vesicles,and then deposited on the type I collagen fibers to continue mineralization to generate more hydroxyapatite.Synthetic hydroxyapatite is very similar to human hard tissue in both composition and structure.Due to its better biocompatibility and osteoinductivity,it has been widely used as the biomedical material and the drug carrier.The Ca2+ and PO43-ions released by HA degradation can promote bone mineralization and accelerate new bone formation.Studies have confirmed that the combination of HA and PLGA can significantly increase the strength of the base material,slow down the degradation rate,and its basic product can neutralize some of the acidic products produced by polylactic acid.The increase of surface roughness and porosity can promote Bone cell adhesion and drug adsorption.Research and development of good carrier systems can overcome the weaknesses of drugs and different carrier materials,these are very important to the medical field.Within this study,the PGE2-loaded PLGA microspheres(PGE2-PLGA-MSs)drug carrier is constructed to solve the problem of short half-life of PGE2.The physicochemical properties and sustained-release properties of the carrier are determined through experimental studies of drug encapsulation efficiency and sustained-release curves.Effects of Drugs and Vectors on the Biological Properties and Osteogenic Gene Expression of MC3T3-E1 Cells determines the effect of low concentration of PGE2 on the osteoblasts and the sustained-release effect of the carrier,and the effect on the proliferation and differentiation of osteoblasts;Bone cells simultaneously applies to PGE2 and hydroxyapatite,and two kinds of osteogenic inducing factors to observe whether the growth and differentiation of osteoblasts are positively promoted;the searchis for the excellent drug carrier system and its exploration by using the HAP-PLGA-PGE2 hybridization system Preparation methods and conditions.We hope that through the above series of studies,we can provide research directions and quantitative indicators for the development and utilization of sustained release drug carriers.Methods: PGE2-PLGA-MSs with uniform particle size is prepared by membrane emulsification method.The drug loading of microspheres and drug release in vitro were determined by enzyme-linked immunosorbent assay.Using no additive medium,blank microspheres,and hydroxyapatite(HAP)as control group,PGE2,PGE2+HAP and PGE2-PLGA-MSs(0.0125,0.05,0.2,0.8,3.2,12.8 μmol/L)as experimental group,incubates with the osteoblast-like cell line MC3T3-E1,detecting the proliferative activity of the osteoblast-like cell line MC3T3-E1 at different times by using the MTT assay,the A value of the sample,the alkaline phosphatase kit,and the microscope counts each field.Under the number of mineralized nodules,flow cytometry was used to detect cell proliferation cycle and apoptosis,RT-PCR was used to detect the expression level of osteogenic related genes and transcription factors.Results: 1.PLGA loaded PGE2,prepare the GE2-PLGA-MSs experimentally with particle size of about 5 μm,and a narrow particle size distribution;the microsphere drug loading is 2.5% when PLGA(IV=0.28)is as the carrier material,cumulative release of48 h drug is about 80%.2.Sustained release of PGE2,PGE2 + HAP and PGE2 has the bidirectional regulation of MC3T3-E1 cell proliferation,0.0125 μmol/L concentration promoted cell proliferation,and 0.2,0.8,3.2,and 12.8 μmol/L concentrations inhibits cell proliferation in a dose-dependent manner.The inhibition of PGE2 and PGE2+HAP at 24 h is lower than the PGE2 at 24 h,but there is no significant difference among the three groups of 48 h and 72 h.The concentration of 0.05 μmol/L has no effect on cell proliferation.3.PGE2,PGE2 + HAP,PGE2-PLGA-MSs have bothway affection of MC3T3-E1 cell cycle;0125μmol/L concentration promotes cell proliferation;0.2,0.8,3.2,12.8μmol/L concentration inhibits cell proliferation,PGE2-The inhibitory effect of PLGA-MSs is significantly lower than that of equal concentrations of PGE2,HAP+PGE2,PI index is higher than that of PGE2,HAP+PGE2 and also dose-dependent;0.05 μmol/L concentration has no effect on cell cycle.4.PGE2,PGE2 + HAP,PGE2-PLGA-MSs have bidirectional affection of MC3T3-E1 cell apoptosis,0.0125μmol/L concentration inhibits apoptosis,0.2,0.8,3.2,12.8μmol/L concentration promotes apoptosis.,And time-dependent,PGE2-PLGA-MSs to promote apoptosis is lower than the equivalent concentration of PGE2,PGE2 + HAP;0.05 mol / L concentration has no effect on cell apoptosis.HAP has a certain role in promoting the apoptosis of MC3T3-E1 cells(P<0.05)and time-dependent.5.Each concentration of PGE2,PGE2+HAP and PGE2-PLGA-MSs promotes the secretion of alkaline phosphatase(ALP)in MC3T3-E1 cells in a dose-dependent manner.The peak promoting take effect on the 5th day and began to decline on the 7th day.The prolonged release of PGE2 and PGE2+HAP is lower than the equal concentration of PGE2 on the 3rd day,and higher than that on the equal concentration of the PGE2 during the 5th and 7th days.6.All The concentrations of PGE2,PGE2+HAP and PGE2-PLGA-MSs promote the mineralization of MC3T3-E1 cells in a dose-dependent manner.The PGE2-PLGA-MSs and PGE2+HAP promotes the mineralization of the cytoskeletal matrix on the 9th day,which is lower than that of the equal concentration of PGE2,and it is higher than the equal concentration of PGE2 around the 13 th and 15 th days.7.On the 5th,7th,9th and11 th day,With real-Time PCR detection of osteogenic genes and transcription factor expression levels,PGE2,PGE2-HAP,PGE2-PLGA-MSs all promote ALP,OPN,OCN,COL-1;RUNX2,Oxteix;c-fos,c-fra1,c-The expression of fra2,jun-D and c-jun m RNA inhibites the expression of BSP.By compared with the control group,PGE2-PLGA-MSs prolong the gene expression time,and the concentration of PGE2 at 0.05 μmol/L enhance the expression of gene regulatory factors.Conclusions: 1.PLGA can be loaded with PGE2,and loaded and sustained release effect of low molecular weight PLGA(IV=0.28)microspheres drug are better than high molecular weight.2.PGE2-PLGA-MSs has sustained release effect under the in-vitro culture conditions.It can prolong the action time of PGE2 on the proliferation,differentiation,osteogenic and related osteogenic gene expression of MC3T3-E1 cells.3.PGE2 has a bidirectional adjusting function on the proliferation of MC3T3-E1 cells,and the low concentration of PGE2 promoted cell proliferation in a periodically and closely ependent manner.PGE2 at a concentration of 0.05 μmol/L in this experiment is a critical concentration that promotes proliferation of MC3T3-E1 cells.4.In this research,concentration PGE2,PGE2-HAP and PGE2-MSs can accelerate the biological performance of osteoblast-like cells MC3T3-E1 and related osteogenic gene expression,to inhibit the expression of bone sialoprotein gene.5.PGE2-HAP positively promotes the biological performance of osteoblast-like cells MC3T3-E1 and related osteogenic gene expression. |
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