Preparation And Application Of PEEK Functional Microspheres Loaded With Nano-materials In Bone Repair

Objective: In order to explore the application of PEEK composite microspheres as filling materials in bone repair,the novel strategy of biofunctionalization of PEEK materials was developed.Materials and Methods: 1.In this study,cerium sulfate（Ce（SO₄）₂）was introduced into PEEK,and Ce（SO₄）₂ loaded PEEK composite microspheres were prepared by gas driving method.Ce（SO₄）₂ in composite microspheres was completely converted into ceria nano-particles（CeO₂NPs）by hydrothermal assisted hydrolysis for the first time,and PEEK composite microspheres loaded with CeO₂ NPs were prepared.The micromorphology of the microspheres was observed by SEM,and the components of the microspheres were qualitatively and quantitatively analyzed by XRD,XPS,FT-IR,TGA and ICP-OES.Finally,the protein adsorption capacity and antioxidant capacity of the composite microspheres were studied by protein adsorption test and DPPH method.2.The prepared Ce（SO₄）₂/PEEK microspheres and CeO₂NPs/PEEK microspheres were tested in vitro and in vivo,in order to explore the biocompatibility and osteogenic properties of the microspheres.The effects of composite microspheres on the adhesion and proliferation of MC3T3-E1 cells were evaluated by CCK-8 assay,Calcein AM/PI and Phalloidin/DAPI fluorescence staining.The bone induction properties of the microspheres were evaluated by alkaline phosphatase staining and activity detection,alizarin red staining and calcium quantitative detection,and the mechanism of osteogenic differentiation of the microspheres was preliminatively investigated by qRT-PCR.The oxidative stress model was constructed with H₂O₂ to explore the protective effect of microspheres against oxidative stress and cells.Finally,a rat skull defect model was established,and the effect of composite microspheres on bone repair in vivo was further evaluated by Micro-CT and histological section staining.3.In order to explore the effective integration of PEEK and n-HA,PEEK microspheres with porous structure were obtained by hydroxylation treatment.Porous PEEK microspheres loaded with n-HA were prepared by hydrothermal in situ coprecipitate method using porous PEEK microspheres as carrier.The microstructure of the composite microspheres was observed by SEM,and the composition of the composite microspheres was qualitatively and quantitatively analyzed by XRD,FT-IR and TGA.The effect of microspheres on proliferation and adhesion of MC3T3-E1 cells was evaluated by Phalloidin/DAPI fluorescence staining and CCK-8 assay.The bone induction performance of the microspheres was evaluated by alkaline phosphatase staining and activity detection,alizarin red staining and calcium quantitative detection,and the expression level of osteogenic genes.Finally,the bone repair effect of the microspheres was further evaluated by Micro-CT and histological section staining through the establishment of a rat skull defect model.Results: 1.The CeO₂NPs/PEEK microspheres prepared by hydrothermal assisted hydrolysis have a unique "topology" structure,and the loaded CeO₂ NPs particles are uniform and can be uniformly distributed in the microspheres.The characterization results of XRD,XPS and FT-IR confirmed that Ce（SO₄）₂ in the composite microspheres could be completely converted into CeO₂ NPs.TGA and ICP-EOS results showed that the loading capacity of CeO₂ NPs in composite microspheres was 3.33 ± 1.81%,and the integration efficiency of CeO₂ NPs in the microsphere was 57.91 ± 8.79%.The results of protein adsorption experiment and DPPH free radical scavenging experiment showed that compared with PEEK microspheres,the protein adsorption capacity and free radical scavenging capacity of CeO₂NPs/PEEK microspheres were significantly improved.2.The prepared composite microspheres were co-cultured with MC3T3-E1 cells,and the results of CCK-8 assay,Calcein AM/PI and Phalloidin/DAPI fluorescence staining showed that compared with PEEK microspheres, CeO₂NPs/PEEK microspheres not only had good cell compatibility,but also could promote cell adhesion and proliferation.Through alkaline phosphatase staining and activity detection,alizarin red staining and calcium quantitative detection,it was confirmed that CeO₂NPs/PEEK microspheres could promote extracellular matrix mineralization and alkaline phosphatase expression.In oxidative stress model,CCK8 assay and DCFH-DA and FITC/DAPI fluorescence staining confirmed that CeO₂NPs/PEEK microspheres could effectively protect cells from oxidative stress damage.In addition,qRT-PCR technique was used to study the mechanism of osteogenesis promoted by composite microspheres,and found that CeO₂ NPs in composite microspheres can promote the expression of Smp/Fam53 B protein,activate the Wnt/β-catenin signaling pathway,and activate the transcription of downstream target genes.Through the construction of rat skull defect model,MicroCT bone reconstruction showed that CeO₂NPs/PEEK microspheres were implanted into the skull defect area,and the new bone in the central area could be connected with the edge area of the bone defect.The staining results of histological sections showed that in the CeO₂NPs/PEEK microsphere group,more new bone and mature bone surrounded the microspheres at both ends of the bone defect area,and the autogenous bone coincided with the new bone,and the bone remodeling was obvious.3.The porous n-HA/ PEEK microspheres prepared in this chapter have unique porous structure and uniform n-HA “coating”.XRD and FT-IR results confirm that n-HA deposited in porous PEEK microspheres has a good crystallinity,with no other impurity phases exist.The quantitative analysis of the composite microspheres by TGA method showed that the actual load of n-HA in the microspheres was 11.5 ± 1.71%.The co-culture of the composite microspheres with MC3T3-E1 cells,CCK-8 assay and Phalloidin/DAPI fluorescence staining proved that the composite microspheres had good cytocompatibility and could promote the adhesion and proliferation of osteoblasts.Through alkaline phosphatase staining,alizarin red staining and osteogenesis-related gene expression detection,it was found that the staining of porous n-HA/PEEK microspheres was obvious,and the alkaline phosphatase activity,extracellular matrix calcium deposition and osteogenesisrelated gene expression level were significantly higher than those of PEEK microspheres group.Micro-CT bone reconstruction and quantitative data analysis confirmed the osteogenic properties of porous n-HA/PEEK microspheres from multiple angles.Histological section staining results showed that compared with PEEK microspheres,porous n-HA/PEEK microspheres further increased the amount and thickness of new bone in the bone defect area,and the distribution was more uniform,and the new bone tissue spanning the microspheres could be seen in the central area of the outer plate.The results indicated that porous n-HA/PEEK microspheres had excellent bone repair ability.Conclusion: 1.The hydrothermal-assisted hydrolysis not only effectively removed the residual sulfuric acid solvent in the microspheres,but also induced the forced hydrolysis of Ce（SO₄）₂ loaded in PEEK composite microspheres to CeO₂ NPs in the weak acidic environment.The diameter of CeO₂ NPs is uniform and distributed uniformly in the composite microspheres.2.CeO₂NPs/PEEK microspheres can not only promote cell adhesion and proliferation,improve extracellular matrix mineralization and alkaline phosphatase expression,but also activate Wnt/β-catenin signaling pathway and promote osteoblast differentiation by promoting Smp/Fam53 B protein expression.at the same time,in the oxidative stress model constructed in vitro,CeO₂ NPs loaded with composite microspheres can protect cells from intracellular ROS accumulation caused by oxidative stress and make cells have a better spreading morphology.3.In the rat skull defect model,PEEK composite microspheres loaded with CeO₂ NPs not only showed better bone defect repair effect,but also occupied iNOS substrate by up-regulating the expression of Arg1 protein,thus inhibiting the production of ROS in local tissues and creating a more favorable microenvironment for bone repair.4.The acicular n-HA was loaded onto the interior and surface of the hydroxylated porous PEEK microspheres by hydrothermal in situ coprecipitation.n-HA loaded by composite microspheres has a good crystallinity,no other impurity phase.5.The unique three-dimensional network structure of porous PEEK composite microspheres loaded with n-HA not only facilitates cell migration and adhesion,but also enhances the expression levels of alkaline phosphatase,extracellular matrix calcium deposition and osteogenesis related genes by uniform n-HA "coating" of composite microspheres,and has excellent performance in rat skull defect models.