| BackgroundIn recent years,with its unique and irreplaceable advantages,more and more patients with dentition defect prefer oral implant therapy.The implants achieve the retention through the formation of osseointegration between the implants and the alveolar bone.However,there is often insufficient bone mass at implanted area caused by some possible reasons.Insufficient bone mass not only limits implanting,but also seriously affects the aesthetic effect and service life of implant prostheses.How to better promote bone regeneration and improve bone mass of implanted area is a recent research focus of dental implantology.Through biochemical methods adding osteogenic bioactive molecules in bone substitutes is an effective way to promote bone regeneration.20(S)-Hydroxycholesterol(20(S)-OXY)belongs to this class of bioactive molecules,and is one of the intermediates of cholesterol metabolism pathway.It can induce osteogenic differentiation of mesenchymal stem cells,simultaneously inhibit adipogenic differentiation.But the 20(S)-OXY has some shortcomings.First,its hydrophobicity is not conducive to be attached to the surface of hydrophilic materials.Second,its in vivo half-life is short because it is easily metabolized by the liver.Currently the researches are focused on changing the chemical structure of 20(S)-OXY in order to improve its osteogenic ability,however no drug delivery system has been used to encapsulating 20(S)-OXY,and to observe the efficacy of this drug delivery system in vitro and in vivo.Objectives1.To prepare 20(S)-hydroxycholesterol loaded mPEG-PLA polymeric micelles(20(S)-OXY-PM)and study on its physical characteristics and sustained release effect in vitro.2.To investigate the osteogenic efficiency of 20(S)-OXY-PM in vitro and in vivo and the possible mechanisms.Methods1.The polymeric micelle systerm containing 20(S)-OXY(20(S)-OXY-PM)was constructed by the nano precipitation method using poly(ethylene glycol)-polylactide amphiphilic block polymer as the carrier.In order to select the synthetic conditions suitable for this study,the experimental conditions were optimized by single factor experiments in three aspects,organic solvent type,the stirring speed,the first addition ratio of 20(S)-OXY.The particle size,particle size distribution and Zeta potential were measured by dynamic light scattering(DLS).The morphology of micelle particles was observed by transmission electron microscopy(TEM).Entrapment efficiency and drug loading capacity of 20(S)-OXY-PM and its sustained release capacity in vitro were measured by high performance liquid chromatography(HPLC).2.Using MTS assay,ALP activity determination by stainning and colorimetric method,inverted phase contrast microscope,alizarin red staining,qRT-PCR,immunocytofluorescent and Western blot to detect cell proliferation,ALP activity,morphologic changes,calcification deposition,Runx2,OSX,ALP,OCN osteogenic related gene expression,and OCN protein expression of the mouse bone marrow mesenchymal stem cell line M2-10B4 affected by 20(S)-OXY-PM.By using the Hedgehog signal pathway inhibitor cyclopamine,the expression of Glil transcription factor was detected by Western blot to investigate the effects of 20(S)-OXY-PM on Hedgehog signaling pathway.3.With gelatin sponge as a carrier,20(S)-OXY,20(S)-OXY-PM and empty micelles were implanted into rabbit skull bone defects.Rabbits were sacrificed at 3 and 6 weeks after operation.Using Micro-CT to three-dimensional reconstruct the bone defects and measure the new bone mineral density(BMD)and volume fraction(BV/TV).Fluorescence staining labeled the newly formed bone tissue.Toluidine blue staining were performed to observe the histomorphology of new bone formation and measure the new bone area.Results1.Through the single factor experiments to determine the synthesis conditions for this study:organic solvent by DMSO,organic phase and water stirring speed was 500rpm,the content ratio of 20(S)-OXY:mPEG-PLA in 2:10.The results of DLS showed that the average size of 20(S)-OXY-PM was 58.94nm,polydispersity index was 0.11,Zeta potential was-9.95mV.The TEM image showed that 20(S)-OXY-PM particals were spherical.The drug loading capacity of 20(S)-OXY-PM was 10.79%,the encapsulation efficiency was 60.46%and in vitro release time was 120 hours which extended than the same content of 20(S)-OXY.2.The results of MTS showed that there was no significant effect of empty micelles on cell proliferation and 20(S)-OXY dose dependently reduced cell proliferation.20(S)-OXY-PM increased the ALP secretion and the mineralization capacity more than 20(S)-OXY at the same concentration,the effects of high concentration 20(S)-OXY-PM groups were the most obvious.After induction culture,the morphology of mesenchymal stem cells changed into osteoblasts'.At the same concentration,the morphological changes were more obvious in the 20(S)-OXY-PM groups.High concentration 20(S)-OXY-PM and 20(S)-OXY could increase Runx2,OSX,ALP,OCN gene expression.Compared to 20(S)-OXY,20(S)-OXY-PM increased gene expression more obviously.The immunocytofluorescent and Western blot results showed that more OCN protein expressed in the 20(S)-OXY-PM groups compared to in the 20(S)-OXY groups at the same concentration and the effects of high concentration 20(S)-OXY-PM groups were the most obvious.Western blot detecting the expression of Gli1 found that the expression was higher in 20(S)-OXY-PM groups than in 20(S)-OXY groups,adding cyclopamine could inhibit the upregulation of Glil expression in both groups.3.The values of BV/TV and BMD measured by Micro-CT software at 3 and 6 weeks were higher in the 20(S)-OXY-PM and 20(S)-OXY groups compared to the gelatin sponge and empty micelles groups,and they were most obvious in the 20(S)-OXY-PM groups.The bone defects were filled with new bone only in the 20(S)-OXY-PM groups at 6 week shown by 3D reconstruction.The results of fluorescence staining showed that closer to the center of the bone defects,the fluorescence intensity was stronger indicating that the new bone usually growed from the edge of bone defects to the center.There were some elliptical fluorophores in the center of the bone defects which were isolated from the new bone at the edge in the 20(S)-OXY-PM groups indicating that the osteogenic induction ability of 20(S)-OXY-PM in situ is stronger.The new bone morphology of toluidine blue staining was consistent with fluorescence staining.The results of quantitative analysis about new bone area were similar with BV/TV results measured by Micro-CT.Conclusions1.20(S)-OXY_PM can be prepared by nanoprecipitation method with suitable experimental conditions.The synthesized 20(S)-OXY-PM with narrow particle size distribution could obviously prolong the release time of 20(S)-OXY in vitro.2.In vitro cell experiments showed that mPEG-PLA nanomicelles encapsulating 20(S)-OXY could improve the osteogenic induction ability.On the one hand,this phenomenon may be due to 20(S)-OXY-PM can promote the activation of Hedgehog signaling pathway,on the other hand,it may be related to the drug sustained release.3.The experimental results of bone regeneration by skull defects in rabbits showed that the in situ bone activation of 20(S)-OXY-PM was stronger than that of 20(S)-OXY,which could better promote the formation of new bone. |
PDF Full Text Request