| Objective:To prepare a novel silk fibroin/nano hydroxyapatite/graphene oxide(SF/nHAp/GO)hybrid porous scaffold with oriented channel-like structure.Characterized its physical and chemical properties and studied in vitro cell response and osteogenesis ability to preliminarily evaluate the feasibility of the hybrid porous scaffold as a bone repair scaffold material.Methods:In this study,conventional spongy(unoriented)SF,SF/nHAp,SF/nHAp/GO porous scaffolds and SF/nHAp/GO porous scaffold with oriented channel-like structure were prepared by freeze-drying technology and directed temperature field freeze-drying technology.These scaffolds were characterized using scanning electron microscopy(SEM),X-ray Diffraction(XRD),Fourier Transform infrared spectroscopy(FT-IR)and universal materials tester to assess the effects of nHAp and GO on scaffolds’ properties.In order to estimate cytocompatibility of these scaffolds,BMSCs were seeded on them,the viability and morphology of the BMSCs were assessed by CCK-8 assay,LSCM and SEM.In addition,HUVECs were cultivated onto the unoriented and oriented SF/nHAp/GO porous scaffold to evaluate the effect of oriented channel-like structure on vascularization and the viability and morphology were observed the same as above.For further test in vitro,osteogenic induction was carried out on BMSCs inoculated on the four groups of scaffolds,then alkaline phosphatase(ALP)activity was tested and cell mineralization was observed to dectect the ability of promoting osteogenic differentiation.Results:1.SEM results showed that all the scaffolds presented interconnected porous structure and the pore size gradually decreased with the addition of nHAp and GO.The porosity of unoriented SF,SF/nHAp,SF/nHAp/GO scaffolds was around 80 ~ 83% while the oriented SF/nHAp/GO scaffold was above 90%.The four scaffolds showed a typical Silk II structure according to XRD and FT-IR.Moreover,scaffolds exhibited both of favorable compression strength(P)and elastic modulus(E)significantly improved with the addition of nHAp and GO.Otherwise,the oriented SF/nHAp/GO porous scaffold exhibited anisotropic mechanical properties,and their compression strength and elastic modulus in axial direction were as much 3 times as those in radial direction.2.LSCM images and CCK-8 assays showed that all the four groups of scaffold materials had good cytocompatibility,and the addition of nHAp significantly enhanced cell proliferation.In the unoriented groups,compared with SF and SF/nHAp groups,the pore size of SF/nHAp/GO group decreased sharply,and the cell proliferation also slowed down significantly.Compared with the unoriented groups,the oriented SF/nHAp/GO porous scaffold exhibited better ability to promote cell proliferation.Otherwise,it was found that HUVECs in the oriented SF/nHAp/GO porous scaffold grew faster and deeper than those in the unoriented SF/nHAp/GO group.The cells migrated,colonized and proliferated in the unobstructed cannels,forming dense vascular-like cell bands from the top to the bottom.3.In the osteoblast differentiation of BMSCs,at each time point,the oriented channel-like SF/nHAp/GO composite stimulated alkaline phosphatase secretion,and mineral deposition more strongly than the traditional spongy SF,SF/nHAp and SF/nHAp/GO scaffolds.Conclusion:A novel oriented channel-like SF/nHAp/GO porous scaffold was successfully fabricated by directional temperature field freeze-drying technology.The nHAp and GO incorporation enhanced the mechanical properties but decreased the pore size to some extent.Cell culture and CCK8 assay indicated that the oriented channel-like SF/nHAp/GO hybrid scaffold had good cell affinity and cytocompatibility.The oriented pore channel with appropriate diameter,high interconnectivity and anisotropic mechanical propertieswere beneficial for cell prolongation and proliferation,and promoted the vascularization and osteogenic differentiation.In conclusion,the SF/nHAp/GO hybrid porous scaffold with oriented channel-like structure may prove to be a promising scaffold for bone tissue engineering. |
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