| Silk fibroin materials as tissue engineering scaffold has a wide prospect of development and application prospect because of its excellent biocompatibility,low immunogenicity and biodegradation.However,on the one hand,when silk fibroin three-dimensional scaffolds are used for tissues regeneration of dermis and blood vessels,the three-dimensional structures will collapse prematurely due to rapid local degradation and fragmentation,the degradation rate needs to be delayed when they are used for repair of hard tissues especially.On the other hand,the ability of Bombyx mori silk fibroin materials to guide tissue regeneration needs to be strengthened because the activity of promoting cell adhesion and proliferation is not as good as that of natural protein materials such as collagen and fibrin.How to prevent the scaffolds from being prematurely biodegraded and collapse as well as how to make the scaffolds guide tissue regeneration faster in order to make the biodegradation speed match the tissue regeneration speed is an important subject in the field of silk-based tissue engineering scaffolds.In view of the above problems,this study adopted two ways that delayed the biodegradation rate of regenerated silk fibroin three-dimensional scaffolds by improving the molecular weight of regenerated silk fibroin and promoted cell adhesion and proliferation by reversing its surface charge from negative to positive so that the biodegradation rate of silk fibroin three-dimensional scaffold could match the rate of tissue regeneration and could further effectively guide the tissue regeneration.To this end,papain,which is specific to sericin,was used as degumming agent to avoid the large-scale hydrolysis and destruction of silk fibroin during the degumming process,and high molecular weight silk fibroin(S-1)was obtained;low-molecular-weight polyethyleneimine was grafted on the side chain of silk fibroin to prepare cationized high molecular weight silk fibroin.Based on the self-assembly behavior of silk fibroin in water environment,the effects of increasing molecular weight and cationic modification on self-assembly behavior of silk fibroin was systematically studied.Then the effects of increasing molecular weight and cationic modification of silk fibroin on the self assembled structure,molecular conformation,pors structure,mechanical properties,biodegradation properties,cell adhesion and proliferation properties and guiding dermal tissue regeneration properties of three-dimensional scaffolds were studied in vitro and in vivo.Firstly,the sericin of raw silk was removed by degumming with 3.0 g/L papain,8 mol/L urea,0.5 g/L sodium carbonate and 5.0 g/L sodium carbonate solution,respectively,to explore the effect of degumming method on the molecular weight and composition of silk fibroin.The results showed that the degumming rate of 3.0 g/L papain degumming method was similar to that by 0.5 g/L sodium carbonate.The surface of silk fibroin was smooth and flat,the molecular weight and intrinsic viscosity of silk fibroin obtained by 3.0 g/L papain degumming were higher than that obtained by 8 mol/L urea degumming,significantly higher than that of 0.5 g/L sodium carbonate degumming and extremely significantly higher than that of 5.0 g/L sodium carbonate degumming.The average molecular weights were 142.47,126.94,105.93 and 81.19 kDa.Secondly,the high molecular weight silk fibroin obtained by papain was incubated in aqueous solution to explore the changes of silk fibroin in morphology,Zeta potential,molecular conformation,and shear viscosity over time.In aqueous solution,the initial state of low molecular weight silk fibroin(S-3)is nanoparticles with a diameter of about 230 nm,while high molecular weight silk fibroin(S-1)is about 30 nm in diameter short fiber connected about 270 nm in diameter particles to form beads,the short fibers not only accelerate the process that silk fibroin nanoparticles transformed from globular/elliptic ball to spindle and further assembled to nanofiber,but also effected the the arrangement and distribution of nanofibers.Unlike low molecular weight silk fibroin that eventually assembles into parallel-arranged nanofibers,nanofibers assembled from the high molecular weight silk fibroin were crisscrossed without obvious parallel arrangement tendency.During the assembly process of high molecular weight silk fibroin,the Zeta potential of silk fibroin aggregates decreased,the conformation of silk fibroin gradually changed from random curly structure to β-sheet structure,and the apparent viscosity of solution increased.The β-sheet structure content and apparent viscosity of high molecular weight silk fibroin aggregates were higher than those of low molecular weight silk fibroin.Thirdly,the silk fibroin three dimensional scaffolds were prepared by freeze-drying technology to explore the structure and physicochemical properties of high molecular weight silk fibroin materials.The results showed that compared with low molecular weight silk fibroin which prepared by 0.5 g/L sodium carbonate degumming method,the pore size of the high molecular weight porous scaffolds decreased,while the thickness and roughness of pore wall,the particle size of pore wall surface,the porosity and the proportion of β-sheet structure all increased.Correspondingly,the particle size of the pore wall of the uncrosslinked porous scaffold increased,and the compressive modulus and compressive strength of the uncrosslinked porous scaffold increased by about 4 times and 2 times,respectively.The enzymatic degradation rate of crosslinked porous scaffolds delayed significantly.Especially,the uncrosslinked high molecular weiht silk fibroin scaffold was mainly random coil structure,but its dissolve-loss rate after immersing in water for 24 h was close to that of the chemically crosslinked scaffolds,which was mainly due to the S-1 assemblied into nanofiber networks during the freeze-forming process.Meanwhile,there were a large number of nano-scale protrusions on the surface of the pore wall surface of S-1 crosslinked porous scaffold,which provided surface for cells to adhere and spread,therefore,the rate of cell adhesion and proliferation was significantly accelerated.Subsequently,under the mediation of carbodiimide,low molecular weight polyethyleneimine(PEI,1.8 kDa)was grafted on the side chain of S-1 to obtain cationized high molecular weight silk fibroin(CS-1).When PEI accounted for 6%of S-1,the grafting efficiency of PEI was about 27.57%,the Zeta potential of the modified silk fibroin reached+14.28 mV.The CS-1 solution was incubated at 37℃ to explore the changes of cationized high molecular weight silk fibroin on the morphology,Zeta potential and molecular conformation.Compared with negatively charged silk fibroin,the self-assembly rate of cationized silk fibroin was accelerated,the size of silk fibroin aggregates was increased,but the spherical CS-1 particles assembled into short fibers instead of spindle deformation during the transformation from spherical particles to nanofibers.Furthermore,CS-1 scaffolds were prepared by freeze-drying technology to study the the structure and properties of cationized high molecular weight porous scaffolds.Compared with negatively charged silk fibroin scaffolds,the particle size of the pore wall of the uncrosslinked CS-1 scaffold was increased significantly.Meanwhile,crosslinked CS-1 scaffolds can further significantly promote the adhesion and proliferation of human vascular endothelial(HUVECs)cells,and inhibited the growth of escherichia coli and staphylococcus aureus.It was worth noticed that the β-sheet structure content of CS-1 scaffold was slower than that of negatively charged scaffold,but the degradation rate was slower,indicating that cationic modification of silk fibroin could not only bestow the silk fibroin scaffold the antibacterial function and enhance the adhesion ability of the scaffold to cells,but also delay the biodegradation of the scaffold by shielding protease attack or assembling into micron size aggregates.Finally,the cationized high molecular weight silk fibroin three dimensional scaffolds were implanted into SD rat full-thickness skin defect wounds.The in vivo results demonstrated that increasing the molecular weight of silk fibroin and modifying cationically not only significantly delayed the degradation rate of the scaffold in vivo,but also promoted the tissue growth,collagen deposition and blood vessel formation,which allowed a better match between the degradation rate of the scaffold and the regeneration rate of tissue.It was found that the biodegradation rate of the cationized high molecular weight silk fibroin three-dimensional scaffold in vivo was basically consistent with the tissue regeneration rate,showing a strong mutual match between the biodegradation and the tissue regeneration rate.In summary,this paper explored the effects of increasing molecular weight and cationic modification on the self-assembly behavior of silk fibroin,providing an experimental basis for understanding the reasons of microself-assembly supramolecular structure of silk fibroin materials.The porous scaffold prepared by cationized high molecular weight silk fibroin has slow biodegradation rate and strong cell adhesion.The biodegradation rate is basically the same as the tissue regeneration rate and can effectively guide dermal tissue regeneration.The results provide reliable experimental basis for the optimization and improvement of silk fibroin based tissue engineering scaffolds as well as the development and clinical application of new generation of tissue repair products. |
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