| 【Background】Maxillofacial bone defect caused by various reasons is a common clinical problem.At present,the most commonly used clinical treatment method is xenogeneic bone powder or autologous bone transplantation,which is mainly used in orthopedics and oral and maxillofacial surgery.However,xenogeneic bone meal can be used only for small bone increment,and the source of autogenous bone is limited.The incidence rate of complications is relatively high,including the incidence,pain,infection,nerve and vascular injury of donors.The direct biological basis of bone regeneration is the differentiation of osteoblasts,so how to induce bone matrix mineralization and calcium deposition ability of osteoblasts is the core problem to solve bone regeneration.Nano hydroxyapatite(nHA)is a widely studied bioactive mineral additive and synthetic bone biomaterial,which can enHAnce the ability of stem cells to differentiate into osteoblasts.It is an ideal nano material to induce osteoblast differentiation.However,its surface drug loading capacity is limited,and it is difficult to obtain satisfactory bone regeneration and repair in complex bone defects.Mesoporous silica(MS)is a kind of porous degradable material.It is widely used in drug delivery because of its unique regular hexagonal nano porous structure,which is easy to carry small molecule drugs.However,the holes of MS coating prepared by conventional sol-gel method are parallel to the substrate,which can not achieve high-efficiency drug loading;the newly reported "oil-water two-phase method" can be very convenient to prepare MS coating on the vertical substrate surface.Therefore,in this study,a new "water oil two-phase method" was used to coat mesoporous silica(MS)on the surface of nHA to achieve efficient drug delivery on the surface of nHA,and further enHAnce the promotion of osteoblast differentiation.【Objective】To construct nHA-MS complex,the endogenous osteogenic activity of nHA combined with the drug loading characteristics of MS can promote osteoblast differentiation and promote bone defect regeneration.【Method】(1)nHA,MSN,nHA-MS and other materials were prepared according to the literature method.The materials were characterized by SEM,TEM and FTIR.(2)The physical adsorption method was used to load DEX,and the absorbance test was used for quantitative analysis of DEX.The loading efficiency and release characteristics of different nanoparticles on DEX were detected.(3)MC3T3-E1 cells were treated with multiple dilution of nanoparticles.The cell viability was analyzed by MTT method,and the safe concentration range was selected.After long-term culture,apoptosis and cell cycle were detected to evaluate the long-term cytotoxicity of nanoparticles.(4)After the cells were labeled with FITC,the uptake of the nanoparticles was observed by confocal laser scanning,and the intracellular status of the nanoparticles was observed by transmission electron microscopy.(5)Osteogenic differentiation was induced by different nanoparticles.The expression of osteogenic related genes and proteins was detected by real-time PCR and Western blot.The mineralization ability of cells was analyzed by alizarin red staining.(6)MC3T3-E1 cells were seeded on the surface of sponge and induced with osteogenic induction solution.The osteogenic efficiency of MC3T3-E1 cells was analyzed in vitro.(7)The changes of bone tissue were observed by Micro CT and HE staining,and the promoting effect of the scaffold on the repair of the ultimate skull defect was studied.【Result】(1)The results show that nHA has a typical solid rod structure with an average size of about 50 nm to 100 nm;MSN is spherical and rich in nano channels,with a particle size of 60-120 nm and an average particle size of 90 nm,while the channel diameter is about2-6 nm,with an average of about 3 nm.After DEX adsorption,the shape and size of MSN particles do not change significantly,while the channels and pore sizes become blurred.The outer layer of nHA-ms is porous coating,the pore size is about 4nm and the distribution is uniform.(2)The results showed that the average drug loading and entrapment efficiency of MSN were about 5.32% and 54.5%,respectively,which could release DEX continuously in vitro;the average drug loading amount of Dex in nHA-ms was about 7.18%,the entrapment efficiency was about 45.61%,and the drug loading rate of DEX was 59.12 μg / mg,which was significantly higher than that of nHA,and the release rate was slowed down.(3)After MC3T3-E1 cells were treated with nHA concentration below 10 μ g / ml,the cytotoxicity of nHA MS was similar to that of MSN and nHA,the safe concentration range was less than 10 μ g / ml,and there was no obvious cell apoptosis and cycle change after long-term cell culture;there was no significant difference between nHA-MS and nHA on cell viability.(4)Result was observed by confocal laser scanning and transmission electron microscopy,it shows that nHA,MSN and nHA-MS could be absorbed by cells,and almost all of them had uptake behavior and degradation in cells.(5)Both nHA and nHA-MS could promote the osteogenic differentiation of MC3T3-E1 cells.When DEX was loaded with nHA-MS,the osteogenic differentiation ability of MC3T3-E1 cells was significantly improved.(6)The results show that the nanoparticles could still be absorbed by cells after they were encapsulated in chitosan sponge,which showed the similar osteogenic activity as the cells treated with nanoparticles alone.(7)The model of skull defect in vivo confirmed that chitosan sponge containing nanoparticles could significantly promote bone repair.Group DEX@nHA-MS showed the best repair effect.【Conclusion】The results show that the vertical MS coating on the surface of nHA can significantly improve the drug loading performance,and greatly enHAnce the osteoinductive activity of nHA after loading DEX.It shows excellent osteogenic activity in vitro and in vivo.It is a new type of nano material for repairing bone defects. |
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