| Silk fibroin(SF)and sericin(SS)are natural animal proteins approved by the US Food and Drug Administration(FDA).They have excellent biocompatibility and good physical and chemical properties,and are widely used in the biomedical field.The study of silk fibroin as a scaffold material for bone tissue engineering has also received widespread attention,but the mechanical properties of regenerated silk fibroin materials are difficult to meet the requirements of bone replacement.Previous studies have pointed out that different silk degumming methods have different effects on the destruction of silk fibroin.In this paper,a mild hot water method without adding any chemical or biological reagents is used to degumming.On the other hand,sericin has been proven to provide an organic template for biomineralization.Therefore,in this paper,by adjusting the degree of degumming,different proportions of silk fibroin/sericin(SF/SS)blend materials were prepared,and the structural properties of the blend materials were studied.In this paper,silk fibers with different sericin content were prepared by degumming with boiling water and controlling different degumming times(1h,2h,3h,and 5h).They were dissolved with lithium bromide and dialysis desalted to prepare silk protein solutions with different sericin content.The amino acid composition of the silk protein solution was determined.The SF/SS blended smooth film and porous scaffold were prepared by ethanol treatment and PEG-DE cross-linking.The secondary structure and crystal structure of the SF/SS blended material were analyzed.The tensile properties of the SF/SS blended smooth film and the compressive properties of the porous scaffold were tested.And the thermal stability of the blended materials were studied.The chemical composition of the surface of the SF/SS blended smooth film after ethanol treatment was studied,and the chemical state of each element was determined.Finally,the in-situ precipitation method was used to investigate the mineralization properties of SF/SS blends.Conventional Na2CO3 degummed silk fibers and regenerated silk fibroin materials were used as controlsSEM observation of the silk fiber after degumming showed that compared with the degumming method of boiling water and Na2CO3 degumming,no grooves appeared on the longitudinal surface of the silk fibroin at different degumming times,and the cross section was not split.Amino acid composition analysis results showed that the boiling time of degumming in water was shortened,and the content of sericin in the SF/SS blend solution increased,showing that the aspartic acid,serine,and threonine increased correspondingly.SDS-PAGE electrophoresis revealed that the molecular weight of silk proteins in the boiling water degumming group was mainly above 150kDa,and the molecular weight distribution of Na2O3 degumming silk protein was scattered,with deep bands between 100-25kDa,and accompanied by a large number of protein molecules less than 25kDa.The viscosity test results of the SF/SS blend solution showed that the extreme values of the shear viscosity of the silk protein solution for boiling water for 1h,3h,and 5h were 10.51Pa.s,9.24Pa.s,and 5.38Pa.s,respectively,and were far greater than the extreme viscosity of Na2CO3 degummed SF solution is 0.40Pa.s.Fourier transform infrared(FTIR)and X-ray diffraction(XRD)were used to study the structure of SF/SS blended materials treated with ethanol and PEG-DE.The FTIR chart compared with the untreated blended material showed that the characteristic peak of amide? of the SF/SS blended smooth film cross-linked by ethanol treatment and PEG-DE shifted from 1637cm-1 to 1618cm-1,and changed from the original random coil conformation to ?-sheet.The amide ? characteristic peaks of SF/SS blended porous scaffolds treated with ethanol and PEG-DE were shifted from 1534cm-1 to 1516cm-1 and 1517cm-1,and the amide? characteristic peaks were moved from 1238cm-1 to 1234cm-1 and 1232cm-1,the molecular conformation changed from random coil to ?-sheet.The infrared spectrum peak fitting results showed that the ?-sheet structure content of SF/SS blended materials debonded for 3h and 5h in boiling water was significantly higher than that of SF materials debonded with Na2CO3.The XRD pattern showed that the untreated SF/SS blended smooth film and porous X-ray diffraction peak showed a broad "biaotou peak" near 19.9°.SF/SS blended smooth films and porous scaffolds treated with ethanol and PEG-DE cross-linked diffraction peaks that are characteristic of Silk ? type crystals around 9,1°,20.7°,and 24.1°.The tensile test results show that compared with the Na2CO3 degumming group,the stress and strain of the SF/SS blended smooth film treated with ethanol are significantly improved.For example,the tensile strength(8.83±1.25MPa)of the ethanol treated for 3h is significantly greater than that of the Na2CO3 degummed(5.36±0.53MPa).The SF/SS blended smooth film in the PEG-DE crosslinked group had a slightly lower breaking strength than that of the control group except that the sample was debonded for 1 h in boiling water,and the stress and strain properties of the other samples were improved.The SF/SS blended smooth film in the PEG-DE crosslinked group had a slightly lower breaking strength than that of the control group except that the sample was debonded for 1 h in boiling water,and the stress and strain properties of the other samples were improved.The mechanical properties of SF/SS blended smooth films treated with ethanol were better than those of PEG-DE crosslinked group.The compression test results of SF/SS blended porous scaffolds show that in the ethanol treatment group,the order of compressive strength and compression modulus is 5h>3h>Na2CO3>1h,such as the compression strength of ethanol for 5h(51.60±9.12MPa)much larger than Na2CO3 degumming(33.69±7.35MPa).In the PEG-DE crosslinked group,the order of compressive strength and compressive modulus was Na2CO3>5h>3h>1h,and the compression modulus of the ethanol-treated group was significantly larger than that of the PEG-DE crosslinked group.Thermogravimetric-microentropy thermogravimetry(TG-DTG)results show that the order of the fastest temperature of the decomposition rate of the ethanol-treated SF/SS blended smooth film is:SS>1h>3h=5h>Na2CO3,respectively 327.35?,311.20?,310.80?,310.80? and 308.30?.The order of the fastest temperature of the decomposition rate of the SF/SS blended porous scaffolds treated with ethanol is:SS>lh=3h>5h>Na2CO3,respectively 323.89? ?,313.50?,313.50?,310.67? and 309.17?.The XPS results showed that the O concentration in the boiling water degumming 1h group was higher than that in the boiling water degumming 5h group,and the C=O corresponding to C1s and the C-O content corresponding to O1s were greater than the boiling water degumming 5h group with lower sericin content.Mineralization of SF/SS blends treated with ethanol in a simulated body fluid environment.The SEM image shows that after 1 day of mineralization,there are more nano-scale crystals than the SF smooth film degummed by Na2CO3.After 3 days of mineralization,the crystals grow on the surface of the SF/SS blended smooth film to form a dense and rough mineral layer.After 7 days of mineralization,the SF smooth film was completely covered by nanocrystals,grown and stacked on each other.Similarly,the growth trend of crystals on SF/SS blended porous scaffolds is similar to that of SF/SS blended smooth films.After 7 days of mineralization,the crystal growth volume on the pore walls of SF/SS blended porous scaffolds is larger.Energy dispersive X-ray spectroscopy(EDX)detection of SF/SS blended materials after mineralization shows that the content of Ca and P deposited on the SF/SS blended smooth film and porous scaffold increased in a time-dependent manner with the extension of mineralization time.After 7 days of mineralization,the Ca/P value of the mineral deposited on the surface of the SF/SS blended smooth film was close to that of natural bone at about 1.75,and the Ca/P value of the mineral deposited on the SF/SS blended porous scaffold was about 2.10.FTIR,XRD and TG-DTG were used to analyze the chemical structure and crystal structure of ethanol-treated SF/SS blended materials after mineralization.The characteristic peaks of protein amide ? and amide ? of mineralized SF/SS blended smooth membrane and porous scaffold were greatly weakened,and the characteristic peak of amide ?disappeared.Strong spikes of hydroxyapatite(HAp)appeared near 1016cm-1,598cm-1,and 554cm-1.And after mineralization for 1 day,the HAp characteristic peak of the boiling water degumming 1h group was stronger than other groups.The XRD pattern shows that the intensity of the diffraction peak of Silk ? crystal structure of the mineralized SF/SS blend material near 20.50 is obviously weakened,and the diffraction peaks corresponding to the(002)and(211)crystal planes of HAp appear,and with the increase of mineralization time,the characteristic diffraction peak intensity of HAp gradually increased.The results of thermal analysis showed that the residual rates of SF/SS blended smooth films demineralized in boiling water for 1h,5h and Na2CO3 after 7 days of mineralization increased from the initial 34.69%,33.65%and 24.83%to 58.22%,52.41%and 53.90%.The results show that the SF/SS blend material prepared by boiling water degumming not only has a denser structure,better mechanical properties and thermal stability,but also can effectively regulate the nucleation,growth and aggregation of minerals,which is expected to provide a possible alternative graft for efficient regeneration of bone tissue. |
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