The Effect Of The Bionic Porous Structure And Piezoelectric Properties Of HA/BT On Osteogenesis

Background:The existing bone scaffolds are still limited by its poor bio-activity to induce osteogenesis,and absence of suitable porous structures to facilitate cell migration,bone tissue and blood vessels in-growth to the deep part of the scaffold.So,addressing bone regeneration in the deeper layers of bone scaffolds come into be a hot topic in the field of tissue engineering at present.Bio-piezoelectric ceramic materials have piezoelectric properties similar to those of human bone tissue,which can generate charge signals under stress on its surface,while electrical stimulation is believed to be capable to promote bone growth.In this study,hydroxyapatite/barium titanate（HA/BT）composite ceramics with porous structure,similar to the natural bone Haver’s system,was prepared using a modified ice mold method.This unique porous structure consisted of a central tubules and radial arrangement,which formulated a fully open laminate structure.The omnidirectional porous structure can ensure the oxygen and nutrient supply and provide channels for bone tissue to grow into the scaffolds,while the piezoelectric charges was used to promote the osteogenesis.The combined bionics between structure and function is desired to enhance the bone formation in bone scaffolds.Objective:In this paper,we obtained HA/BT bone scaffolds by the modified ice mold method,on the basis of the study on the influence of process parameters on the porous structure,mechanical properties and piezoelectric properties.In order to explore the effects of the bionic porous structure and piezoelectric property of HA/BT on osteogenesis,the effects of piezoelectric property on osteoblast function were investigated following the study for the effects of bionic porous HA/BT porosity and chemical composition of on osteoblast function and optimization of structural factors;subsequently,the effects of piezoelectric property of HA/BT on macrophage polarization conversion and the possible mechanisms were further investigated,as well as the influence of macrophage polarization conversion on osteoblast and vascular endothelial cell functions;finally,the impacts of the bionic porous structure and piezoelectric properties on bone growth inside the scaffold structure and osseointegration were examined in vivo.Method:（1）By investigating the relationships between component particle collocation,solid volume fraction,freezing rate and slurry viscosity coefficient,porous morphology,mechanical properties,piezoelectric properties,and porosity of sample,the influence of process parameters on porous structure and properties was disclosed and the bionic porous HA/BT bone scaffolds with porous structure and piezoelectric properties similar to natural cortical bone were prepared.（2）MTT method was used to detect the cytotoxicity of bionic porous HA/BT composite ceramics with different BT contents on L929cells.（3）To explore the effect of different porosity bionic porous HA/BT composite ceramics on the function of co-cultured osteoblasts and optimize the porosity of samples,CCK-8 method was used to detect proliferation,scanning electron microscope was used to observe morphology and ELISA was implored to detect the secretion of cytokines related to osteogenic differentiation.（4）Bionic porous HA,BT,and HA/BT ceramics with similar surface roughness and sample porosity were prepared by changing particle collocation.The effects of chemical components of scaffold materials on osteoblast function were investigated by using CCK-8method to detect cell proliferation,scanning electron microscopy to observe cell morphology,and ELISA to detect the secretion levels of cytokines related to osteogenic differentiation.（5）On the basis of optimization of scaffold porosity,polarized bionic porous HA/BT composite ceramics were co-cultured with osteoblasts under 1 HZ,5 HZ,10 HZ and 15 HZ stress.To study the effects of piezoelectric signals with different frequency on the adhesion,proliferation and differentiation of osteoblasts,the osteogenic differentiation-related genes and cytokines secretion were examined by RT-PCR and ELISA,respectively;cell proliferation was determined by CCK-8 method;cell morphology was observed by scanning electron microscope.（6）The polarized porous HA/BT composite ceramics with optimized porosity were co-cultured with macrophages under stress loading,and the cell proliferation was detected by CCK-8 method,the cell morphology was observed by scanning electron microscopy,the expression levels of polarization-related genes were detected by RT-PCR,the expression levels of membrane surface proteins and secretory functional proteins were determined by immuno-fluorescence staining and Western Blot,the expression levels of pro-/anti-activated proteins were detected by ELISA.The effects of electrical signals generated by porous HA/BT composite ceramics under the stress on the proliferation and polarization of macrophages were investigated at the cellular,protein and molecular levels,and the possible signaling mechanisms were initially explored.（7）The expression levels of pro-osteogenic and pro-angiogenic related genes and cytokines in macrophages co-cultured with polarized HA/BT under stress were detected by RT-PCR and ELISA,respectively.The cells were inoculated in conditioned medium containing macrophage supernatant,and an indirect co-culture system of macrophages,osteoblasts,and vascular endothelial cells was established to simulate the immune microenvironment around the scaffolds.To study the effect of the polarization transformation of macrophages induced by piezoelectric stimulation on the osteogenic function of osteoblasts,migration and tube formation of vascular endothelial cells,RT-PCR and ELISA were used to detect osteogenic gene expression and ALP activity of osteoblast,respectively;CCK-8 method was used to detect the proliferation rate of osteoblasts at different time points;Alizarin red staining was used to observe and quantify the formation of mineralized matrix.Transwell cell migration Experiment and tube formation test to evaluate the migration and tube formation ability of human umbilical vein endothelial cells（HUVECs）.（8）The polarized and non-polarized biomimetic porous HA/BT composite ceramics were implanted into the distal end of the New Zealand rabbit femur,and the femoral specimens containing the implanted samples were obtained at 1 w,4 w,and 8 w after implantation,respectively.X-ray examination,analysis on fluorescently labeled sections and non-decalcified hard tissue sections with Golder’s staining,scaffold compressive strength detection,and ELISA to determine the contents of ALP and OC in scaffolds,were used to study the effect of biomimetic porous structure and piezoelectric properties on bone growth in porous structures and osseointegration.Results:（1）Under the conditions of the same solid volume fraction and freezing speed,the samples prepared by the slurry with 0.2μm HA/1.49μm BT particle collocation has denser laminate structures,smoother surface,and the highest piezoelectric coefficient(d₃₃);With the increase of the solid volume fraction and the freezing speed,the porosity and the laminar spacing decreased,and piezoelectrical coefficient and compressive strength increased;solid volume fraction and freezing gradient temperature had similar control efficiency on porosity and laminar spacing,but solid volume fraction had higher control efficiency than freezing speed on compressive strength and piezoelectric coefficient(d₃₃).（2）HA/BT with different BT contents had no obvious cytotoxicity to L929;the biomimetic porous HA/BT composite ceramic with a porosity of 50%had the best porous structure and was more conducive to the adhesion,proliferation,and differentiation of osteoblasts.HA,BT and HA/BT bionic porous ceramics with similar porosity and laminate surface roughness were co-cultured with MC3T3-E1 cells,and the cell proliferation and ALP concentration in the HA/BT group were higher than those in the BT group at 7 d.（3）The piezoelectric signal generated by polarized bionic HA/BT composite ceramics under stress can increase the protein adsorption and promote the proliferation and differentiation of MC3T3-E1 cells,and the cells change from polygonal to shuttle shape under the influence of piezoelectric signal on the scaffold surface;the amplitude of electrical stimulation response of MC3T3-E1 cells to piezoelectricity is the largest when the frequency of stress action is 10 HZ.（4）The electrical signals generated by polarized biomimetic porous HA/BT composite ceramics under stress can promote the proliferation of macrophage RAW264.7,and also promotes the transformation of macrophages from pro-inflammatory M1 type to anti-inflammatory repair type M2,characterized by the increase of the m RNA,protein expression of M2 macrophage markers CD206,Arg-1 and repair cytokines such as-10,IL-10,TGF-β1.JAK2/STAT6 signaling pathway partially mediates piezoelectric signaling Intracellular signal transduction,inhibition of macrophage JAK2 can reduce the protein expression of Arg-1,CD206,which were anti-inflammatory markers.（5）The electrical signal generated by polarized bionic HA/BT under stress promoted the m RNA and protein expression of TGF-β1,BMP2,and PDGF in macrophages;the altered macrophage function brought about by piezoelectric signaling promoted the proliferation of osteoblasts and the expression of osteogenic genes BMP2,Runx2,and ALP,enhanced the mineralization of matrix and facilitated vascular endothelial cell migration and tube formation.（6）4 weeks post implantation,polarized HA/BT interlocked with the host bone,with blue-stained bone in the laminar structure and central canal,while only orange collagen tissue was visible in the laminar structure and central canal of unpolarized HA/BT;after 8 w of implantation,blue bone tissue appeared in the deep laminar structure and central canal of both polarized HA/BT and unpolarized HA/BT;the compressive strength at 4 w and 8 w,and the ALP and OC content at 4w of polarized HA/BT were higher than those of unpolarized HA/BT.Conclusion:（1）By constructing a radially distributed temperature gradient field and regulating the process parameters,biomimetic porous HA/BT composite ceramics with a central tubule and a radially distributed laminate structure similar to the natural bone can be obtained by using the freeze casting method.（2）The bionic porous HA/BT composite ceramic with 50%porosity has the best porous structure;the addition of HA increases the bioactivity of BT.（3）The piezoelectric stimulation generated by the biomimetic porous HA/BT composite ceramics under stress can promote the adhesion,proliferation,and differentiation of osteoblasts,while the frequency is most pronounced at 10 Hz.（4）Piezoelectric stimulation generated by polarized biomimetic porous HA/BT composite ceramics promotes the transformation of macrophages from pro-inflammatory M1 type to repairing anti-inflammatory M2 type,and the JAK2/STAT6 pathway is partially involved in the intracellular conduction of piezoelectric signals;The polarization transformation of macrophages induced by piezoelectric stimulation can promote the adhesion,proliferation,differentiation of osteoblasts and tube formation of vascular endothelial cells.（5）The biomimetic porous structure can provide structural conditions for the in-growth of bone tissue,and the piezoelectric effect can accelerate bone regeneration;the biomimetic HA/BT composite ceramic realizes the bone regeneration in the center of the scaffold material through the combination between the biomimetic porous structure and piezoelectric properties.