| PART 1 THE CONSTRUCTION,CHARACTERIZATION AND IN VITRO BIOCOMPATIBILITY OF THE STRUCTURE METAL BONE TRABECULAE LOADED WITH ESTRIOL NANOPARTICLESObject : Eq-PLGA nanoparticles were constructed by two-step phacoemulsification,and PDA coating was prepared on porous tantalum metal bone trabecular materials by PDA bionic method.A large number of EQ PLGA nanoparticles were adsorbed on the surface of the coating to prepare a structural metal bone trabecular Eq-PDA-Tan.And its physical and chemical properties and biocompatibility were tested,providing a basis for further research.Method : Eq-PLGA nanoparticles were prepared by two-step phacoemulsification.The physical and chemical properties of Eq-PLGA were evaluated by observing the morphological characteristics.The particle size potential,encapsulation efficiency,loading capacity and in vitro drug release of Eq were tested.The cell viability of C3H10 T1/2 and RAW264.7incubated with Eq-PLGA was tested and cell phagocytosis of fluorescently labeled nanoparticles was observed to evaluate in vitro biocompatibility.PDA coating was prepared on porous tantalum metal bone trabeculae by PDA bionic method.Eq-PLGA nanoparticles were compounded on the surface of PDA-Tan by vacuum freeze-drying,and Eq-PDA-Tan metal bone trabeculae was successfully constructed.The surface morphology and hydrophilicity of Eq-PDA-Tan were examined.After incubation with the cells,the adhesion,expansion and proliferation of the cells on the materials and inside the pores of the materials were observed to evaluate the biocompatibility of Eq-PDA-Tan metal bone trabecula.Results:The scanning electron microscope and transmission electron microscopy showed that the Eq-PLGA had uniform size and good dispersion,with a particle size of(291.5±29.54)nm,zeta potential of(-22.3±1.98)m V and a dispersion index of 0.149.The encapsulation efficiency of Equol was(74.39±4.18)% and loading capacity was(6.86±0.54)%.In vitro drug release experiments showed that EQ-PLGA nanoparticles continuously released Eq for 30 days,with a total release accumulation rate of(72.44±1.70)%.The cell viability after incubation with nanoparticles was higher than 90%.C3H10 T1/2 cells gobbled Eq-PLGA nanoparticles into the cytoplasm and nucleus with good cytocompatibility.General observation and SEM confirmed that PDA coating and EQ-PLGA nanoparticles were successfully prepared on the Tan surface,and PDA coating could increase the surface hydrophilicity of the material.CCK-8,SEM and CLSM found that Eq-PDA-Tan promoted cell proliferation(P<0.05),improved cell adhesion and extension on the surface of the material,and promoted cell growth into the pores of the material..Conclusion : Eq-PDA-Tan structural metal bone trabeculae was constructed successfully.The material shows good release performance in vitro,good surface hydrophilicity,promotes cell proliferation and guides cell growth,and has good biocompatibility in vitro,indicating that it is a safe and promising bone implant material.PART 2 IN VITRO STUDY ON OSTEOGENIC DIFFERENTIATION AND OSTEOCLASTIC DIFFERENTIATION OF EQ-PDA-TAN STRUCTURAL METAL BONE TRABECULAEObject : To explore the influence of Eq-PDA-Tan structural metal bone trabeculae on the biological effects of cell osteoblast differentiation and osteoclast differentiation in vitro,so as to provide a premise for the treatment of osteoporic bone defect in vivo.Method : The morphological changes of Eq-PDA-Tan metal bone trabecula incubated with C3H10T1 / 2 cells and RAW264.7 cells in the presence of inducible factors were observed by light microscopy.The effects of metal bone trabeculae Tan,PDA-Tan and Eq-PDA-Tan on cell osteogenic differentiation and osteoclast differentiation were analyzed by ALP staining and activity detection,alisarin red staining,Western-blot,q RT-PCR,ELISA and TRAP activity detection.Results : Eq-PDA-Tan metal bone trabecula were incubated with C3H10 T1/2 cells and RAW264.7 cells,respectively.After inducible factor was added,C3H10 T1/2 cells gradually differentiated into osteoblasts,calcium nodules appeared,cells became oblate,and cytoplasm appeared cavities.The black pseudopodia of RAW264.7 cells were appeared to connect each other to form multinucleated osteoclasts.ALP staining and activity showed that after 7 and 14 days of osteogenesis induction,the purple color of Eq-PDA-Tan group was deeper,and the activity of ALP was significantly higher than that of other groups(P<0.05).Alizarin red staining showed that after 21 days of osteogenesis induction,red-stained calcium nodules were deep red in Eq-PDA-Tan group were all over the whole field of vision.IOD value was significantly higher than that in other groups(P<0.01).For osteoblasts,COL-1,RUNX2 and Osterix were significantly higher in Eq-PDA-Tan group than that in other groups(P<0.01)after 7days of induction,while OCN expression was not significantly different in each group(P>0.05).After 21 days of induction,COL-1 expression in Eq-PDA-Tan group was still higher than that in other groups(P<0.01),and COL-1 expression in PDA-TAN group and Tan group was higher than that in the control group(P<0.01).For OCN,Runx2 and Osterix,the Eq-PDA-Tan group was significantly higher than the other groups(P<0.01),the PDA-Tan group was higher than the Tan group(P<0.01),and the Tan group was higher than the blank control group(P<0.01).For osteoclast-associated proteins NFATc1,c-Fos and CTSK,the expression levels were the lowest in the Eq-PDA-Tan group(P<0.01)after 7 days of induction,and there was no difference between the other groups(P>0.05).Detection of genes related to osteogenesis and osteoclast differentiation showed the changes were similar to those of proteins.The ratio of OPG/RANKL in the supernatant of the Eq-PDA-Tan group was the highest(P<0.05),and the TRAP content was the lowest in the Eq-PDA-Tan group(P<0.05).Conclusion : Eq-PDA-Tan metal trabecular bone shows excellent osteogenic differentiation in vitro,and Eq-PDA-Tan metal trabecular bone inhibits the osteoclast differentiation of cells through the release of Eq,which is expected to be an ideal bone implant for patients with osteoporosis and bone defect.PART 3 IN VIVO STUDY ON THE TREATMENT OF OSTEOPOROTIC BONE DEFECTS IN RATS WITH EQ-PDA-TAN METAL TRABECULAR BONEObject:The critical bone defect model of femoral condyle in rats with osteoporosis was established.The Eq-PDA-Tan metal trabecular bone repair ability,bone integration effect and biocompatibility in vivo were evaluated by imaging and histology methods,so as to provide a basis for clinical application and transformation.Method:1.SD female rats were divided into OVX(ovariectomy,40rats)group and Sham(Sham operation,10 rats)group.Bone metabolism markers and bone density were detected,micro-CT bone scan and histological observation were used to evaluate the success of osteoporosis model.2.OVX rats were randomly divided into 4 groups.A circular defect with a diameter of 3.5mm and a depth of 4mm was made in the femoral condyle.3.X-ray was used at 0W,4W,8W and 16 W to observe the metal trabecular bone was successfully implanted into the femoral condyle defect and the repair of bone defect in the blank control group.4.Samples were taken out at 4W,8W and 16 W after surgery,and bone defect repair and surrounding tissues were observed in general.The bone penetration inside the metal trabeculae and the bone-material interface were observed by Magenta-methylene blue staining and toluidine blue staining,and the bone area penetration percentage in the pore space of the metal trabeculae and the implant contact rate(BIC)were calculated.The implant bone growth and the interface between bone and material were observed by SEM,and the elements of new bone tissue were analyzed by EDS.5.The cerebral cortex,heart,liver,kidney and spleen tissues were histological observed at16 W after surgery to evaluate their biocompatibility in vivo.Results:1.According to the detection of bone metabolism markers,after 12 W of surgery,serum osteocalcin level of OVX group was significantly lower than that of Sham group(P<0.01),while TRACP-5b level was significantly higher than that of Sham group(P<0.01)and bone mineral density was significantly lower than that of Sham group(P<0.01).Mirco-CT showed that BV/TV,Conn.D,Tb.N and Tb.Th levels in OVX group were significantly lower than those in Sham group(P<0.01),while Tb.Sp level was significantly higher than that in Sham group(P<0.01).Histological observation showed that the bone trabeculae in OVX group became thinner,the space between trabeculae widened,the bone trabeculae were fractured,the bone collagen decreased,and the number of osteoclasts increased compared with Sham.2.X-ray observation: On the day after the operation,there was a rounded shadow of the bone defect at the femoral condyle in the blank control group,the implant placement was good in each group,and there were subtle gaps between the material and the bone defect interface.After 4,8,16 w,the bone defect was smaller with the extension of time in the blank control group,but the defects of bone are still clearly visible,the bone sclerosis appeared.The implant position of the other groups did not change or become loose with the extension of time.Callus was formed around the implant,and the gap between the implant and the bone defect interface became smaller and stronger.At 16 W after surgery,gaps between implant and bone interface were still visible in Tan group,but the gaps between implant and bone interface were invisible to the naked eye in PDA-Tan group and Eq-PDA-Tan group.3.General observation:Infection and rejection did not occur in each group,and the implants in each group were in good position and secure.With the extension of the implantation time,the materials in each group were covered with fibrous tissue and callus.In the blank group,the bone defect was gradually filled with fibrous tissue,but the segmental defect was still visible.4.Observation of magenta-methylene blue staining and toluidine blue staining in hard tissue sections: Eq-PDA-Tan group had more new bone growth than the other two groups(P<0.05),and PDA-Tan group also had more bone growth than the Tan group(P<0.05).The BIC values of the Eq-PDA-Tan group and the PDA-Tan group were significantly higher than those of the Tan group at all time points(P<0.05),but there was no statistical difference between the two groups.5.SEM and EDS: With the extension of time,Eq-PDA-Tan group showed that a large number of new bone was generated and grew into pores.The interface between new bone and material was closely jointed,and the new bone transitioned from braided bone to lamellar bone,and finally became mature lamellar bone.The ratio of calcium to phosphorus in the new bone of each group decreased with the extension of time,and the ratio of calcium to phosphorus in the Eq-PDA-Tan group was significantly lower than that in the other groups(P<0.05),which was 1.686±0.029.6.Histological observation: Heart,liver,spleen,cerebral cortex and kidney tissues showed no obvious inflammatory cell infiltration and no pathological changes.Conclusion:Eq-PDA-Tan metal bone trabeculae effectively promotes the formation of new bone under the micro-environment of osteoporosis,improves the bone integration effect of bone-material interface,and has good biocompatibility and bone inductivity in vivo. |
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