| Patients with osteoporosis are prone to suffering from decreased primary stability and delay bone bonding during implant treatment,which may impair the successful rate in implant surgery.Icariin?ICA?has the osteoinductivity,could promote osteogenesis and angiogenesis,inhibit bone absorption,and is effective on curing osteoporosis,without adverse effects.Loaded ICA on the surface of the titanium?Ti?–based implants can promote the bone formation around the implant and realize the early osseointegration.Objective:The present research was to study the effects of different concentrations of ICA on the MC3T3-E1 preosteoblast cells.On this basis,two kinds of samples with different surface modification methods were employed.The first surface modification method was titanium dioxide?TiO2?nanotube?NT?loaded with ICA and covered with poly?lactic-co-glycolic acid?,abbreviated as PLGA.The second method was the establishment of a local ICA delivery system via a layer-by-layer self-assembly?LbL?system on phase-transited lysozyme?PTL?primed Ti surface according to the principle of green and environmental protection.The two kinds of modified samples were characterized by scanning electron microscopy?SEM?,X-ray photoelectron spectroscopy?XPS?and contact angle test.Then the drug release test was performed to observe the drug release profiles of different modified samples.In addition,the effects of the different samples on MC3T3-E1 cells'biological behaviors were observed by in vitro experiments.The effects of TiO2 nanotube loaded ICA covered with PLGA coating on normal rats's osseointegration were studied by in vivo experiments.Morevevr,the effects of ICA coating loaded via LbL system on PTL primed Ti surface samples on bone formation were observed using both normal and osteoporotic animal models.Methods:1.MC3T3-E1 cells were cultured in mediums containing different concentrations of ICA.The cell morphology was observed by microscope.Cell proliferation,differentiation and calcification were evaluated by cell counting kit-8?CCK-8?,alkaline phosphatase?ALP?and alizarin red staining,respectively.2.Two kinds of surface modification methods were used.One is soaking the TiO2nanotubes surface in the ICA solution to prepare NT-ICA sample and further cover the NT-ICA surface with PLGA coating to establish the NT-ICA-PLGA sample.Another method is applying the ICA loaded in chitosan?CS?layers which adsorb with hyaluronic acid?HA?layers to form multilayers via the LbL system on the PTL nanofilm primed Ti substrate to construct the Ti-PTL-[HA/CS?ICA?]3 surface according to the principle of green and environmental protection.3.The surface morphology,composition and wettablility of different samples were characterized by SEM,XPS and contact angle test respectively to evaluate the two kinds of implant surface modification methods.Drug release profiles of ICA were detected by High Performance Liquid Chromatography.In addition,the effects of the ICA sustained-release system were investigated using in vitro MC3T3-E1 cell culture tests including CCK-8,ALP activity,acridine orange/ethidium bromide?AO/EB?,actin staining,real-time polymerase chain reaction?PCR?and Western Blot.Morever,an in vivo rat model was used to evaluate the bone formation effects of different substrates by histological staining.4.Ti and Ti-PTL-[HA/CS?ICA?]3 samples were implanted into the femus of the rats of osteoporosis and sham operation groups.Histological analyses were used to observe effect of Ti-PTL-[HA/CS?ICA?]3 sample for promoting osteogenesis.Results:1.Different concentrations of ICA could promote differentiation and proliferation of MC3T3-E1 cells.The results showed that low concentrations(10-7-10-9mol/L)of ICA promoted proliferation while high concentrations(10-5-10-6mol/L)of ICA promoted differentiation.2.For the modification samples of TiO2 nanotube loaded ICA covered with PLGA,SEM showed that ICA and PLGA were successfully loaded on nanotube structure.The contact angle test displayed that the wettability of NT-ICA-PLGA increased.The drug release experiment indicated that the NT-ICA-PLGA extended the drug release span to 2 weeks.MC3T3-E1 cells grown on coatings showed higher adhesion,proliferation and differentiation ability than others.Histological staining revealed significant bone formation in the NT-ICA-PLGA group.3.For modification samples of ICA coating loaded via LbL system on PTL primed Ti surface,SEM and XPS demonstrated successful fabrication of the Ti-PTL-[HA/CS?ICA?]3 surface.The surfaces with different ICA concentrations revealed controlled release profiles of ICA during a 2-week span.MC3T3-E1 grown on the coated substrates displayed higher adhesion,proliferation and differentiation than the Ti group.Both decalcified and undecalcified sections revealed much obvious bone formation in the Ti-PTL-[HA/CS?ICA?]3 group than Ti group in normal rat animal model.4.In rat osteoporosis model,histological staining of decalcified sections showed that the volume of new bone increased around the implant-tissue interface of Ti-PTL-[HA/CS?ICA?]3 group,suggesting higher bone bonding ability for the ICA modified group.Conclusion:Our study shows that different concentrations of ICA have different effects on MC3T3-E1 cells.The two kinds of titanium surface modification methods can both achieve a sustained release system of ICA during early bone formation span.The modified surfaces can promote the adhesion,proliferation and differentiation of MC3T3-E1 preosteoblast cells in vitro.NT-ICA-PLGA samles can accelerate the new bone formation in vivo.Morever,Ti-PTL-[HA/CS?ICA?]3 samples displayed superior bone formation ability in normal and osteoporosis rat models. |
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