Experimental Study Of Bionic Porous Silk Fibroin/biphasic Calcium Phosphate Bone Scaffold Preparation And The Ectopic Bone Formation Ability

PartⅠ:preparation and physical and chemical property analysis of biomimeticporous silk fibroin(SF)/biphasic calcium phosphate(BCP) bone scaffoldObjcective: To prepare biomimetic silk fibroin/biphasic calcium phosphate(SF/BCP)bone scaffold via improved gas foaming-phase separation method, according to thedifferent constituent of natural bone matrix; to study the influence of different contents ofSF on the pore diameter, porosity, the degradable variation and the ultimate resistancestrength,and select a scaffold as an excellent carrier on which the seed cell can grow andproliferate by comparing the comprehensive performance.Methods: Firstly,we we prepare the SF solution, hydroxyapatite(HA) and the β-tricalcium phosphate (β-TCP) powder,and then mix the HA and β-TCP intoBCP,according to the ratio of m (HA)/m(β-TCP)=6:4just like the bone matrix, thematerial composition was researched by FT-IR and SEM method.Secondly, we chose7%wt SF solution as the curing liquid referring to the results of preliminary experiments,and divided the scaffolds into0%SF,20%SF,40%SF,60%SF groups. After adding the gasporogen, the materials were stirred to foam thoroughly within60℃Water bath pot,thenwe use the improved gas foaming-phase separation method to get the scaffold after thematerials were transferred into a homemade mold. Finally, the composite, pore diameter,porosity, the degradable variation, mechanical strength were researched by XRD,Micro-CT, SEM, SBF and biomechanical tester respectively.Result:1: Pore size: the0%SF scaffold was (40.43±1.09)μm;20%SF:(64.77±1.90)μm;40%SF:(150.35±1.21)μm;60%SF:(200.93±0.81)μm. The40%SF and60%SF ones hadmore appropriate pore size which was good for cell growth. 2:The porosity rate(%) was30.48±1.20,63.75±1.22,85.67±1.06,90.75±1.33from0%SFto60%SF respectively. Compared with the BCP materials, the porosity rate of biomimeticscaffold was more suitable for the needs of connections between cells(P <0.01).3. The maximum compressive strength was (7.57±0.56)MPa；(22.37±0.61)MPa；(40.70±0.39)MPa；(18.75±0.82)MPa respectively;Of all the bionic scaffolds, the40%SFone can withstand the maximum pressure significantly(P<0.05).4.As the rise of the SF content,the degradation rate of the SF/BCP biomimeticscaffold accelerated (42.21%,51.81%,60.43%respectively),while the BCP material wasonly28.12%on the56th day.Conclusion: The pore size and the porosity rate were proportional to the SF content,and the40%SF and60%SF ones had more appropriate pore size and porosity rate whichwere good for proliferation of osteoblast.The compressive strength values of threebiomimetic groups were greater than BCP ones, and showed elevated levels after the firstincreased(40%SF) and then decreased(60%SF),the compression performance of40%SFscaffold is significantly better than the other three groups, with considerable intension ofcancellous bone.The degradation rate of biomimetic scaffold is consistent with the rate ofbone formation, In summary,the40%SF scaffold can meet the needs of bone tissueengineering． Part Ⅱ: Experimental study of biocompatibility and ectopic boneformation ability of the biomimetic porous scaffoldObjective: Different scaffolds were co-cultured with osteoblasts to analyze thebiocompatibility, then the scaffold that loaded with osteoblasts was implanted in musclebag of the rats to observe ectopic bone formation ability.Methods: Firstly, osteoblasts were cultured within the leachate from four differentscaffolds, then acridine orange and MTT methods were used to test the biocompatibility atdifferent time.After co-cultured with biomimetic scaffold for7days, we researched theproliferation of the cells by SEM. Finally, three groups(SF/BCP/MC3T3-E1; SF/BCP;MC3T3-E1) were implanted in the muscle bag of rats to test the ectopic bone formationability by X-ray and the content of ALP.Result: the leachate from biomimetic scaffold promoted the proliferation ofMC3T3-E1apparently, compared with BCP materials, cells can cling on biomimeticmaterials successfully, extending pseudopodia and secreting bone matrix. According toradiology and ALP test, the form that SF/BCP scaffold loaded with osteoblasts hasexcellent ability of ectopic bone.Conclusion: Biomimetic SF/BCP scaffold can mimic the natural bone matrix and theosteogenic microenvironment, it can facilitate the adhesion, proliferation of osteoblastswith excellent biocompatibility, it has strong ectopic bone formation ability and wouldhave broad application prospects of spinal fusion in the future.