Effect Of TiO₂/SrHA Composite Loaded Hippocampus Extract On Proliferation And Differentiation Of Osteoblasts

Related bone diseases caused by bone defects seriously affect the normal life of humans.Typically,small bone defects would healing via the host tissue repair mechanism,while the implantation of biomaterials is required when the size of bone defects reach or exceed the critical size defect.Titanium alloy is a metal implant material widely used in clinical practice,but due to its biological inertness,the surface is easily covered by fibrous tissue and separated from the surrounding host bone tissue,which cannot meet the desired effect of bone adhesion.A large number of researchers have improved the biological activity of implant materials by modifying the surface,the basic composition of bone tissue and introducing bioactive substances.In this research,three-dimensional bionic composite-stents of titanium dioxide/strontium doped hydroxyapatite?TiO₂/SrHA?were constructed based on the three-dimensional multistage structure of human bone and the self-healing process of bone trauma.And the scaffolds were loaded with phenolic extracts from hippocampus,and the effects of the composite nanomaterials on extracts release kinetics and cell proliferation and differentiation were studied by using the mouse osteoblast model.The main contents and results are as follows:1.TiO₂ nanotubes were prepared by anodization and annealed at 400°C,500°C,600°C and 700°C.The structural characterization is obtained and characterized by scanning electron microscopy?SEM?and X-ray diffraction?XRD?.The phenolic substance 1-?5-bromo-2-hydroxy-4-meth-oxyphenyl?ethanone?BHM?extracted from the Hippocampus kuda Bleeler was coated on the surface of TiO₂ nanotubes and then was co-cultured with MC3T3-E1 cells.The effects of TiO₂/BHM composite scaffolds on proliferation,differentiation and mineralization of preosteoblasts were studied by MTT assay,alkaline phosphatase activity?ALP?and alizarin red staining?ARS Staining?.The pre-osteoblast MC3T3-E1 was implanted on the surface of TiO₂ nanotubes at different annealing temperatures.The optimal annealing temperature was 500°C by MTT and ALP activity detection.When the concentration of BHM was 20?mol/L,the viability of co-cultured MC3T3-E1 cells was the highest.And the microstructure and morphology of TiO₂ nanotubes promoted the proliferation and differentiation of the cells to a certain extent when the main crystalline form of TiO₂ nanotubes was anatase.When BHM was loaded on TiO₂ nanotubes,the proliferation,differentiation and mineralization of the cells were improved significantly.2.Strontium hydroxyapatite microspheres were successfully prepared by replacing the Ca element of hydroxyapatite with metal element Sr under hydrothermal reaction.Scanning electron microscopy?SEM?,X-ray diffraction?XRD?,and X-ray photoelectron spectroscopy?XPS?were applied to characterize SrHA?strontium-doped hydroxyapatite?microspheres.The hippocampal active compound BHM was loaded by physical adsorption,and the osteogenic ability of BHM,SrHA and BHM/SrHA composite microspheres were evaluated by MTT assay and alkaline phosphatase activity test.The results showed that the diameter of the prepared SrHA microspheres was about 11?m,and the co-cultured MC3T3-E1 cells had the highest viability when the concentration of SrHA microspheres was 20?mol/L.Compared with pure SrHA,BHM,BHM/SrHA composite microspheres had more significant effect on MC3T3-E1 osteogenesis of osteoblasts.3.The TiO₂/SrHA composite scaffolds were prepared by hydrothermal self-assembly.The hippocampal active compound BHM was loaded into the scaffolds.The release kinetics of the scaffolds was studied by fluorescence spectrophotometer.Cell compatibility and osteogenic properties of the scaffolds were evaluated by in vitro cell assay?MTT,ALP,ARS staining and real-time quantitative PCR?.The results showed that the prepared TiO₂/SrHA composite scaffold can successfully load compound BHM.In the vitro simulation release experiments,the release concentration of BHM showed a good linear relationship within the first 5hours,which has a correlation coefficient reached 0.98.Given a BHM concentration at 20?mol/L,TiO₂/SrHA/BHM composite scaffolds exhibited the most significant cellular compatibility,osteogenesis and mineralization,compared with pure SrHA,TiO₂ nanotubes and TiO₂/SrHA.Real-time quantitative PCR assay showed that the scaffold could up-regulate the expression of bone marker genes such as alkaline phosphatase?ALP?,osteopontin?OPN?,bone-specific transcription factor?RunX2?,osteocalcin?Osteocalcin?and collagen I?COL1a1?in varying degrees.