Experimental Evaluation Of Silk Fibroin/Calcium Phosphate Cement (SF/CPC) Composite In Animal Pulmonary Embolism Model And Alginate Gel Intervertebral Disc Culture System

PartⅠ: Preparation of Silk Fibroin/Calcium Phosphate Cement (SF/CPC) Composite, Study of Washout resistance and coagulation test in vitro【Objective】To prepare a novel bone substitute of silk fibroin/calcium phosphate cement (SF/CPC) composite, and to explore the influences of silk fibroin and its fraction on the washout resistance of CPC in fluids. The effects of cements including PMMA, CPC and SF/CPC on the plasmatic phase of coagulation were assayed for further animal experiments.【Methods】The loosened refined silk fiber were dissolved, dialyzed and spray dried to extract SF powder. 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. CPC free from SF served as control. Washout behaviour was observed and percent remaining cement was measured to determine the stability of samples in the wet environment. After cements curing, particles were contacted with the fresh porcine plasma, after which the prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen quantitation (Fig) and D-dipolymer were assayed.【Results】With the addition of SF, the washout resistance of CPC in fluids were highly developed. Percentage of remaining cement increased with the mass fraction of SF and reached approximately 100% in SF/CPC composites with 2.5 wt% and 3.0 wt% of SF. Only a slight decrease of PT value in CPC group occurred after the plasma in contact with three kinds of cements, without any significant change in other coagulation parameters.【Conclusions】The incorporation of SF remarkably improved the anti-decay properties of CPC in an aqueous environment. The washout resistance ability increased with the mass fraction of SF. In consideration of both stability and injectability, SF/CPC containing 2.5 wt% SF served as SF/CPC object in the following research. PMMA, CPC and SF/CPC barely influence the coagulation parameters in vitro. Only a slight decrease of PT value was observed in CPC group compared to control. The incorporation of SF further develops the blood compatibility of CPC.PartⅡ: Test of SF/CPC using an animal pulmonary cement embolism model【Objective】To develop an animal pulmonary cement embolism model, by which the effect of cements on the hemodynamic change, respiratory function and the antithrombin activity were measured after the pulmonary embolism caused by cement injection. The efficiency of SF reducing the risk of cardiovascular complication was evaluated.【Methods】After general anesthesia, animals were endotracheal intubated and mechanically ventilated by a respirator. A normal tidal volume and a normal arterial carbon dioxide were maintained during the experiment. Fluid-filled catheter was inserted into abdominal aorta via the left femoral artery for the monitoring of arterial blood pressure and blood sampling. A pulmonary catheter was placed into the pulmonary artery through the right jugular vein for the measurement of mean pulmonary arterial pressure and central venous pressure. The pulmonary trunk was exposed via a median sternotomy. Cement was injected into the main pulmonary artery via a venous catheter. The following hemodynamics were measured and analyzed: mean arterial blood pressure (MABP, mmHg), mean central venous pressure (MCVP, mmHg), mean pulmonary arterial pressure (MPVP, mmHg) and heart rate (HR, beats/min). Blood samples were drawn at each time point for blood gases analyzing, and pre- and post-injection for measurement of AT III. Postmortem lungs were subject to computer tomography (CT) examination along with three-dimensional reconstructions of cement casts.【Results】CPC was showed to have the most severe influence upon cardiovascular system, with a maximum elevation of 21.33±5.44mmHg in PAP, a maximum decrease of -27.15±11.79mmHg in MABP. At the 10 min after injection, a transient decrease (-27.7±6.17 beats/min) of HR was observed in CPC group. The changes in PMMA and SF/CPC group were relatively slight with a statistically significant difference from CPC groups. Blood gases revealed significant hypercarbia, acidemia and hypoxemia in CPC group after injection, while these impairments of respiratory function were mild in PMMA and SF/CPC group. There were a largest change of -0.13±0.02 in PH value (60 min after injection), 15.9±7.67mmHg in partial pressure of arterial carbon dioxide (60 min after injection) and -23±1.26mmHg in arterial oxygen tension (10 min after injection) in CPC group. Although all dropped, the decline degree of AT III activity level was the most in CPC group with statistically significant (p<0.05) difference between other two groups.【Conclusions】The pulmonary cement embolism model in animals were successfully developed by directly injecting cements into the pulmonary arterial trunk. The results indicated that, the washout resistance properties of cements significantly influenced the severity of cardiovascular complications post pulmonary embolism. The incorporation of SF evidently improved the stability of CPC that effectively reduced the formation of embolus especially the micro-embolus. The effect on hemodynamic changes and respiratory function were well reduced by the addition of SF, as well as a decreased risk of stimulation of clotting system. The rate of acute cardiovascular deterioration and mortality were therefore decreased.PartⅢ: The influence of SF/CPC on cell viability, metabolism of proteoglycan and collagen of the intervertebral disc cultured in an alginate gel system【Objective】To investigate the influence of SF/CPC, CPC and PMMA on cell viability, metabolism of proteoglycan and collagen of the intervertebral disc cultured in the alginate gel system.【Methods】The discs were taken from rabbits and cultured in alginate gel system with either ordinary complete medium (control group) or complete medium in which cements samples had been extracted. At 1w, 2w and 3w, cell viability was measured by Hoechst 33342–PI cocktail nuclear staining; the expressions of type I, II collagen were detected using immunohistochemistry; proteoglycan contant measurement were done using Safranin O staining. After Hoechst 33342–PI cocktail nuclear staining, the number of cells with both bright blue and bright red were counted under fluorescence microscope in separate fields of view per slide and expressed as percentage of the total cells counted. Integrated option density (IOD) of brown DAB deposits staining type I, II collagen were measured quantitatively using Image Pro Plus software; Integrated option density of orange-red part in safranin O stained sections were also analyzed.【Results】The percentage of dead cells in PMMA group and CPC group were significantly higher than that of control and SF/CPC group. The population of dead cells in SF/CPC was lower than that either in PMMA on in CPC group, which was statistically significant at 1w (week). The difference was not significant on the population of dead cells between SF/CPC group and PMMA group, but between SF/CPC group and CPC group at 3w (P<0.05). The percentage of dead cells was and 2w (P<0.05). The results of the IOD measurement of type I collagen staining in the outer layer of the anulus fibrosus showed that, the value in PMMA group at 1w was prominently lower that other groups; at 2w and 3w, the values in CPC and SF/CPC group were higher than control. The results of the IOD measurement of type II collagen staining in the inner layer of the anulus fibrosus and nucleus pulposus field showed that, the values of PMMA and CPC group were markedly lower than SF/CPC group; at 3w, there was some restore in CPC group, the value of SF/CPC group was still equal to the control. The results of the IOD measurement of Safranin O staining showed that, even in the control group the IOD value kept declining, the decline rate was the most in PMMA group.【Conclusion】PMMA significantly inhibits the cell viability, metabolism of proteoglycan and collagen of the intervertebral disc cultured in the alginate gel system. Compared to CPC, SF/CPC presents a better biocompatibility with intervertebral disc. The bone cements used in vertebroplasty might accelerate the degeneration of disc with different degree depending on the cement type. Appropriate material should be selected to eliminate the incidence of this complication.PartⅣ: Influence of SF/CPC on the genes expression of intervertebral disc【Objective】To study the influence of SF/CPC, CPC and PMMA on the degeneration related genes expression of intervertebral disc. To explore the mechanism of the degeneration of intervertebral disc after vertebroplasty at a gene level, which will apply the theoretical basis on target genes and interaction mechanisms for the prevention and treatment.【Methods】The discs were taken from rabbits and cultured in alginate gel system with complete medium in which cements samples had been extracted. At 1w and 2w, the mRNA were isolated and a reverse transcription–competitive polymerase chain reaction (RT–PCR) technique was used to measure expression of products, along with the quantitative real-time RT-PCR using SYBR? Green I fluorescent dye to quantitatively detect and compare the expression of AGC1,CollagenⅡ,CollagenⅠ,BMP-2,IL-1β,TIMP-1,MMP-3,MMP-2,SOX-9,TGF-β1,Cox-2 between groups. The housekeeper gene GAPDH was used to calibrate the target gene and the relative expression of genes were calculated using 2-△△CT method.【Results】At 1w, the expression of AGC1 in PMMA group was lower than control while were higher in CPC and SF/CPC group. The expression of AGC1 kept elevated in SF/CPC group at 2w. At 1w, the expression of COL1 in all cements group increased reactively. At 2w all felt back except which in SF/CPC group still keeping similar to control. COL2 did not express in detectable amounts in all cements group at 1w and although detectable at 2w was extremely low. At 1w, the expression of MMP-2 and MMP-3 in PMMA and CPC groups were both lower than control and at 2w were expressed in reduced amounts in CPC group, while was expressed in lower amount in SF/CPC group. TIMP-1 expression was stimulated at 1w and declined at 2w in all cements groups. IL-1βoverexpressed in all cements group all the time while the value of SF/CPC group was lower than others at 2w. CPC group showed a higher expression of COX-2 Compared with others. The expression of TGF-β1, BMP-2 and SOX9 were higher than control at 1w and decline back in different degree with the expression of TGF-β1 and SOX9 being lower than control.【Conclusion】The bone cements materials have effect on the degeneration related genes expression of intervertebral disc. The effect may accelerate the degeneration process of disc cultured in vitro, while also stimulates the self protective and self healing function. The cements differ from each other on the expression of certain gene. The expression of COL1 drop after a transient reactive increase in PMMA and CPC groups while keep at a fair level in SF/CPC group. AGC1 is sensitive to PMMA characterizing with a down regulation. The expression of matrix degradation relative genes such as MMP-2 and MMP-3 are not elevated by SF/CPC, which also has a less influence on the same elevating of proinflammatory genes with PMMA and CPC group. At the gene level, SF/CPC has the lowest effect on the degeneration of disc.