Preparation And Optimization Of SF/PCL Composite Fibrous Scaffold For Bone Tissue Engineering

Bone tissue engineering is an interdisciplinary research field,which strives to repair and regenerate damaged tissue using scaffolds.In this field,a desired scaffold is similar to nature ECM and could serve as a 3D template to support cell attachment and proliferation.Electrospinning is an economical and simple technique for the fabrication of fibers with diameters ranging from nanometer to micron scale which has been widely used in bone tissue engineering.In this study,SF and PCL were used as materials for the preparation of SF/PCL nanofibrous scaffold which applied to bone tissue engineering.In the process of electrospinning,we found that the phase separation phenomenon could be obviously observed in the SF/PCL solution after being left for 3 hours when using FA as the solvent.As far as we know,no research has mentioned the phase separation phenomenon when using FA as the solvent for preparing the SF/PCL electrospinning solution.In this study,we deeply investigated the causes of phase separation phenomenon and its influences on the electrospinning progress.The morphology and composition of the SF/PCL nanofibrous scaffolds were examined by scanning electron microscopy,Fourier transform infrared spectroscopy and thermal gravimetric analyzer.Attachment and proliferation of mouse osteoblast MC3T3-E1 cells were tested by scanning electron microscopy and cytotoxity assay.The results indicated that the phase separation of SF/PCL solution might led to inhomogeneous morphology and composition of the composite scaffolds,there appeared to be many fused fibrous structure on the fibers collected at 6-7 h,and the content of SF in the electrospun SF/PCL fibers had very significant differences with the increase of electrospinning time.The content of SF in the composite nanofibers collected at 0-1 h was about 50%.When increased the electrospinning time to 2-3 h,the contentof SF fell to its lowest level(10%).The content of SF started to increase during the rest of the electrospinning time and achieved its maximum value(65%)at 6 h.The inhomogeneity of the SF/PCL scaffolds with formic acid as solvent had a remarkable difference on cell adhesion and proliferation.To address the phase separation,acetic acid,small molecule nonsolvent for silk fibroin,was first introduced to SF/PCL/FA solution,a homogeneous solution without separation for over several days was achieved after mixing for 5 h.The morphology and composition of the acetic acid-modified SF/PCL fibrous scaffolds were examined by scanning electron microscopy,Fourier transform infrared spectroscopy and thermal gravimetric analyzer.Finally,the biological responses of SF/PCL nanofibrous scaffolds have also been studied by scanning electron microscopy(SEM)and cytotoxicity assay.The results indicated that the introduction of appropriate amount of HAc to the mixed solution contributes to the homogeneity in the SF/PCL blended system,which was not going to separate into two phases for over several days.Nanofibers obtained also appeared to be smooth and homogeneous,which are desired for uniform SF/PCL nanofibers.And there are no significant differences for cell adhesion and proliferation on the resultant homogeneous nanofibrous scaffolds collected at different time periods.The modified SF/PCL nanofibrous scaffolds with only small pore size,which may limit the usefulness of electrospinning in tissue engineering strategies.Therefore,it's necessary to improve the performance of the SF/PCL scaffold by optimizing the pore size of the membrane.In this work,several methods had been developed for the optimization of pore size.Firstly,microfibers with diameter range from 5-10 ?m and large enough pore size had been prepared by improved electrospinning device.After that,dual-component electrospinning was used to obtain SF and PCL micro/nanofibrous composite scaffolds.The introduction of microfibers can significantly increase the pore size of composite scaffold and nanofibers can provide an ECM-like environment,these give composite scaffolds many excellent properties.Finally,for the first time,the SF/PCL scaffolds with various ratios of microfiber to nanofiber was obtained by one step,the ratios were tuned by adjusting the collection position of electrospun fibers.The morphology,composition,pore properties of the composite scaffolds was investigated with field-emission scanning electron microscopy,Fourier transform infrared and pore measurement.To assay cell proliferation,cell viability,and infiltration ability,mouse osteoblast MC3T3-E1 cells were seeded on the SF and PCL micro/nanofibrous composite scaffolds.The results showed that,fibers with diameters of up to 10 ?m were obtained by improved electrospinning device,the pore size of scaffolds was positively linked to fibers diameter.The ratios of microfiber to nanofiber have approximate gradient change,and the changes of pore size(2 ?m-30 ?m)were same as the change of ratios.The scaffold with a ratio of 83:17 for microfibers to nanofibers and pore size of 7.6 ?m was proved to be better for cells adhesion,proliferation and infiltration.The micro/nanofibrous composite scaffold prepared by the improved method will be better applied to bone tissue engineering.