| Critical bone defect and nonunion are still great challenges in the field of clinical bone repair,and the main problem of bone implants are lack of bioactivity at present.The establishment of bioactive bone implants usually need to combine with biodegradable biomaterials,seed cells and growth factors in bone tissue engineering.Because of the excellent mechanical property,degradability and biocompatibility,the composite materials consist of composite of synthetic polymer materials poly lactic-co-glycolic acid?PLGA?and inorganic nanoparticles hydroxyapatite?HA?are the most widely used for bone repair materials.Although the composie material can promote the repair of bone defect,it doesn't have bioactivity.Therefore,the technology of modifing the surface of implants utilizing bioactive molecule such as growth factors is of great significance for bone regeneration and repair.Bone morphogenetic protein 2?BMP2?plays an important role in bone healing and reestablishment.But the effect of BMP2 is limited because of unsteadiness in vivo,short half-life and easily degraded and inactivated.Therefore,it has been a crucial research topic to immobilize the growth factors onto the surface of bone implants to lengthen or maintain the bioactivity.The underwater adhesion protein derived from sea mussels has extensive adhesion ability,which can adhere to the surface of many kinds of materials sucn as polymers,metals and ceramics and so on.It has been kown that the bases of the protein is 3,4-dihydroxylphenylalanine?DOPA?.Therefore,inspired by this kind of adhesion DOPA,to intergrate the active site of waterborne adhesive DOPA molecule into BMP2 to produce a kind of waterborne adhesive growth factor DOPA-BMP2 like mussels,which can modify the surface of bone implants materials and produce composite materials with excellent bioactivity and bone induction function.RhBMP2 and rhBMP2-LPETG with the recognition sequence of protein ligase-transpeptidase A were respectively expressed in E.coli in this study.After the transformation was successfully verified by sequencing,the Ni column affinity chromatography was used for purification.PLGA/HA composite membrane was prepared by coating method,and the PLGA/HA composite membrane modified by DOPA-BMP2 was obtained by hydroxylation of DOPA molecular precursor YKYKY-GGG,which was converted into DOPA-GGG and fixed on PLGA/HA composite membrane surface.And rhBMP2-LPETG was connected with DOPA-GGG by Sortase A protein ligase.The molecular weight,concentration and secondary structure of rhBMP2 and rhBMP2-LPETG were determined by polyacrylamide gel electrophoresis and circular dichroism.The adhesion of membrane surface was detected by FTIR.The hydrophilicity of PLGA/HA composite membrane was measured by water contact Angle method.The ability of each protein binding to PLGA/HA composite membrane was analyzed by ELISA.Inoculated MC3T3-E1 cells to the PLGA/HA composite membrane modified by different proteins,cultured at 37°C,5%CO2 environment,detected the proliferation,alkaline phosphatase and the effects of cell adhesion of MC3T3-E1 cells using the method of CCK-8,ALP staining method,FITC staining method respectively.The expression levels of osteogenic genes and proteins of MC3T3-E1 cells were detected by RT-PCR and cellular immunofluorescence.The calcium deposition on PLGA/HA composite membrane was analyzed by alizarin red staining.The results showed that rhBMP2 and rhBMP2-LPETG gene vectors were successfully constructed by E.coli expression system,and were obtained after induced by IPTG,expression,purification and renaturation.The result of circular dichroism showed that the addition of LPETG sequence didn't affect the basic structure of BMP2,and the secondary structure of rhBMP2-LPETG was basically consistent with rhBMP2.The PLGA membrane was prepared by solution replacement method,and the PLGA/HA composite membrane was prepared by coating method,both of the surface was modified by DOPA-BMP2.The result of PLGA membrane detected by FTIR showed that rhBMP2,rhBMP2-LPETG and YKYKY-GGG could be adsorbed onto the surface by physical adsorption,and DOPA-BMP2 was also successfully adsorbed onto the film surface after hydroxylation reaction.The result of water contact angle test showed that the water contact angle of PLGA/HA composite film modified by DOPA-BMP2?47.5°±0.57?is significantly lower than the groups of PLGA/HA?83.1°±1.52?,BMP2?63.7°±0.85?,YKYKY-GGG?64.7°±0.76?and BMP2-LPETG?50.6°±0.42??P<0.05?,indicating that PLGA/HA composite film modified by DOPA-BMP2 had the best hydrophilicity.The result of ELISA showed that the absorbance of DOPA-BMP2 group?0.1144±0.0044?at 562 nm was significantly higher than the grouop of PLGA/HA?0.1024±0.0021?,BMP2?0.1039±0.005?,YKYKY-GGG?0.0981±0.0037?and BMP2-LPETG?0.1011±0.0056??P<0.05?,indicating that PLGA/HA composite film modified by had the largest amount of adhesion.The result of CCK-8showed that both dissociative rhBMP2 and rhBMP2-LPETG had no significant effect on the proliferation of MC3T3-E1 cells.the result of ALP staining showed that compared with the group of control?704.253±226.5?,PLGA/HA?6279.157±1462.4?,50 ng BMP2?18523.81±3518.6?,1 ng DOPA-BMP2?10195.3±2159.0?,10 ng DOPA-BMP2?13881.96±1725.3?the total area of ALP in 50 ng DOPA-BMP2 group?24745.8±12491.9?was significantly increas?P<0.05?,indicating that rhBMP2 and DOPA-BMP2 could promote the early osteoblastic differentiation of MC3T3-E1 cells to some extent,and the promoting effect of 50 ng DOPA-BMP2 was most obvious,therefore 50 ng is the best optimal dosage.FITC staining and quantitative analysis results showed that the average area of single cell in DOPA-BMP2 group?3511.051±608.698?was siginificantly higher than the group of control?1068.382±178.735?,PLGA/HA?686.487±239.805?,BMP2?854.449±392.417?,YKYKY-GGG?780.808±201.99?and BMP2-LPETG?1202.999±363.201??P<0.05?,at the same time,compared with the group of control?12±1?,PLGA/HA?14.7±2.08?,BMP2?19±2.08?,YKYKY-GGG?19±1.73?and BMP2-LPETG?19±2.65?the average number of cells in the group of DOPA-BMP2?26±2?was significantly increased?P<0.05?,indicating that the proliferation and growth of the cells in DOPA-BMP2 group were the best,and the PLGA/HA composite membrane modified by DOPA-BMP2 could promote the adhesion of MC3T3-E1 cells.The results of qRT-PCR and cellular immunofluorescence showed that DOPA-BMP2 could significantly promote the expression of RUNX2,OPN and OCN?P<0.05?,as well as the osteogenic related proteins RUNX2 and OPN in MC3T3-E1 cells.The results of alibi red staining showed that both the red spots of BMP2 group and BMP2-LPETG group increased compared with the control group,while the DOPA-BMP2 group showed a more obviously increase and a deeper color,indicating that PLGA/HA composite film modified by DOPA-BMP2 could more effectively promote the accumulation of calcium elements on the surface of PLGA/HA composite film.In summary,PLGA/HA composite membrane surface was modified by DOPA in this study,and BMP2 was fixed to the material surface by Sortase A protein ligase to obtain the PLGA/HA composite membrane modified by DOPA-BMP2.The results showed that the material modified by DOPA had better adhesion ability and was more favorable for the adhesion of BMP2,which not only provided a good regeneration environment for bone repair,but also obtained a bone graft material with strong osteogenic induction ability. |
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