Preparation Of Silk Fibroin/Calcium Phosphate Cement (SF/CPC) Composite And Experimental Study On Augmenting Sheep Vertebral Body Defect Model By The Composite

Partâ… :Preparation of Silk Fibroin/Calcium Phosphate Cement(SF/CPC) Composite and Study on the Physicochemical Properties【Objective】To prepare a novel bone substitute of silk fibroin/calcium phosphate cement(SF/CPC) composite for bone substitute,and to explore the influences of silk fibroin on the compressive strength,setting time and injectability and select a cement composite with a proper ratio of silk fibroin for further clinical experiments.【Methods】The loosened refined silk fiber were dissolved in the CaCl₂·CH₃CH₂OH·H₂O ternary solution at a mole ratio of 1:2:8 to get a pure SF solution then prepared into SF powder using spray dryer.Tetracalcium phosphate(TTCP) was prepared with liquid phase precipitation method,then mixed with dicalcium phosphate anhydrous(DCPA) at a mole ratio of 1:1,hydroxyapatite(HA) was also added with 4 wt%. Six groups were set according to the concentrations of SF powder in the solid phase of CPC:0.5 wt%,1.0 wt%,1.5 wt%,2.0 wt%,2.5 wt%,3.0 wt%respectively.Then the solid phase blended with the liquid phase with a ratio of 0.4ml/g to prepare the cement composite.Compressive strength,setting time and injectability were measured among the six groups of cement composite as well as CPC group.The structural characteristics of all groups were observed under scanning electron microscope(SEM),final products were analyzed using x-ray diffraction analyzer(XRD).【Results】The compressive strength increased along with the mass fraction of SF powder in the solid phase,while the trend of setting time was opposite.Injectability was also impaired by SF powder;however,it was still higher than 85%with a 2.5 wt%SF/CPC. SEM observation showed SF penetrated throughout CPC crystals,which were tightly connected.The XRD displayed that hydration reaction of SF/CPC almost ended after 24 hours and the final product of SF/CPC was poor crystalline hydroxyapatite.【Conclusions】SF/CPC with a SF mass fraction of 2.5 wt%in its solid phase have a high compressive strength and low setting time,together with an acceptable injectability of more than 85%.The final products of the cement composite were unchanged.It could be suitable for bone substitute application.Thus,we will choose this kind of SF/CPC for the next cytocompatibility study.Partâ…¡:Studies on Cellular Compatibility of the SF/CPC【Objective】To evaluate SF/CPC cellular compatibility by using co-culturing of bone marrow stromal cells(BMSCs) from rabbit's marrow and SF/CPC.【Methods】Primarily cultured BMSCs were isolate from a rabbit's bone marrow by density gradient centrifugation.The third passage BMSCs were seeded into CPC and SF/CPC for 3 to 5 days.BMSCs alone were cultured at the same condition to act as controls.The cellular morphology and function(attachment,proliferation and differentiation) were assessed separately by means of phase contrast microscope,SEM and MTT assay.The material leaching liquor was used to test cell toxicity.【Results】BMSCs could adhere to CPC and SF/CPC,and proliferate and grow on the surface of the composites normally.The cellular activity and function were not affected by the materials,and no statistical difference was found between the two groups and the control group(P＞0.05).Cell toxicity test discovered these materials had no toxic effect on BMSCs.【Conclusions】BMSCs could be isolated,cultured and proliferated with active function in vitro.CPC and SF/CPC have a good cellular biocompatibility and can be used as a bone substitute.Partâ…¢:Study on sheep vertebral body defect model augmentation with SF/CPC in vitro【Objective】To develop a sheep vertebral body defect model for screening vertebral augmentation filler materials and to evaluate the augmentation effect of SF/CPC in this model in vitro. 【Methods】Forty-eight lumbar vertebrae harvested from ten fresh skeletally mature female sheep cadavers were evenly assigned to eight groups,6 vertebrae each.Bone mineral density(BMD) was examined each group.Four groups of vertebrae were drilled in the perpendicular direction to center of the sagittal plane of the vertebra,with the different drills,φ2.0mm,φ4.0mm,φ6.0mm,φ8.0mm to form a cylindrical bone defect.The depth was 10.0mm,keeping the contralateral cortex intact.The fifth group served as control.All vertebrae were compressed in a material testing system to determine the strength and stiffness.Based on the above test,a certain defect was selected as the vertebral body defect model.The three left groups were prepared into this model,and then augmented by CPC,SF/CPC,PMMA.The strength and stiffness of the three groups were evaluated after setting for 24h in simulated body fluid.【Results】There was no difference in BMD among the eight groups of vertebrae.It was found that the strength and stiffness of the vertebrae with bone defect decreased as the diameter of the bone defect increasing.The compressive strength and stiffness of the vertebrae withφ6.0mm defect was significantly lower in comparison with the control group(P＜0.05).After augmentation,there was no difference among SF/CPC,PMMA and intact groups(P＞0.05),while CPC group had a lower value compared with intact group in compressive strength and stiffness(P＜0.05).【Conclusion】The sheep vertebrae with aφ6.0mm and 10.0mm in depth defect is a suitable model to screen vertebral augmentation filler materials.SF/CPC has a prompt augmentation effect in this model.This model keeps the vertebral figuration intact,so it can be used in further studies in vivo.Partâ…£:Studies on sheep vertebral defect model augmentation with SF/CPC in vivo【Objective】To determine the feasibility of SF/CPC composites as an injectable bone subsitute and investigate the possibility on clinical use as a new vertebral augmentation filler material.【Methods】Twelve adult sheep were operated on left retroperitoneum approach and cavities were made on L3,L4 or L5.CPC,SF/CPC,and PMMA was transplanted to the cavities at random order.Six sheep were killed at 1- and 6-month-time point postoperation, respectively.Two specimens were subjected to un-decalcified sections and stained with...