| Objective:As an important carrier of cells and growth factors,scaffold materials have attracted wide attention in the field of bone tissue engineering,but how to construct scaffolds with both composition and structure is still a challenge.In this study,silk fibroin(Silk Fibroin,SF)/ nano-hydroxyapatite(nano-Hydroxyapatite,nHAp)/ graphene oxide(Graphene Oxide,GO)porous scaffolds were prepared by directional temperature field freeze-drying technique.The characterization,morphology,degradation rate,biocompatibility and biological activity of directional porous scaffolds were systematically studied and compared with traditional disordered sponge SF porous scaffolds,SF/nHAp porous scaffolds and SF/nHAp/GO porous scaffolds.It provides a meaningful reference for the further study of directional porous scaffolds in dentistry and orthopedics department.Methods:(1)Preparation and Physicochemical Properties of SF/nHAp/GO porous scaffolds based on directional pore structureIn this study,traditional disordered sponge SF,SF/nHAp,SF/nHAp/GO porous scaffolds and SF/nHAp/GO porous scaffolds based on directional pore structure were prepared by freeze-drying technology and directional temperature field freeze-drying technology.The morphology,pore diameter and porosity of each scaffold were characterized and compared by field emission scanning electron microscope(Scanning Electron Microscope,SEM).In order to evaluate the in vitro mineralization ability of the scaffolds,the PBS solution containing 1U/ml protease XIV was used to evaluate the mineralization ability of the scaffolds.The surface morphology and element composition of the scaffolds were observed by scanning electron microscope(SEM)and Fourier transform infrared spectroscopy(Fourier transform infrared spectroscopy,FT-IR).(2)Study on cytotoxicity,cytocompatibility and osteogenic induction of SF/nHAp/GO porous scaffolds based on directional pore structure in vitroIn order to evaluate the cytocompatibility,cytotoxicity and osteogenic induction ability of porous scaffolds in each group,resuscitated mouse bone marrow mesenchymal stem cells(murine Bone marrow mesenchymal stem cells,m BMSCs)were cultured on porous scaffolds and cultured for 1,3 and 7 days.The cytotoxicity was detected by living / dead cell staining(living/dead staining)and rhodamine labeled cyclopeptide-DAPI staining.The viability,proliferation and adhesion of cells were observed by laser confocal microscope(LSCM).Real-time fluorescence quantitative PCR(RT-q PCR)was used to detect the expression of osteogenesis-related genes in porous scaffolds,including ALP,Runx-2,OC and Col-I.(3)The experimental study of SF/nHAp/GO porous scaffold based on directional pore structure on the repair of rat skull defect.Twenty-four male and healthy Sprague Dawley(nominal SD)rats were randomly selected and divided into two groups.Round defect models were established on bilateral skulls of rats.Ⅰ group of rats were implanted with SF and SF/nHAp stents,and Ⅱ groups of rats were implanted with SF/nHAp/GO and directional pore SF/nHAp/GO stents.12 rats were killed at 6 and 12 weeks after operation.The rats killed at 6 weeks were injected with fluorescent labeling at 3 weeks,4 weeks and 5 weeks after operation,and the rats killed at12 weeks were injected with fluorescent markers at 6 weeks,8 weeks and 10 weeks.X-ray photographs(X-ray)were taken to observe the bone repair in the bone defect area of rats in different time points.Then the non-decalcified but dehydrated skull tissue sections were made,and the fluorescence staining was observed under laser confocal microscope,and the bone tissue sections were made by using a series of tissue section equipment,and the osteocytes and inflammatory cells were observed under light microscope by HE and Masson staining.After the rats were killed,the viscera such as heart,liver,spleen and kidney were dissected,stained with HE,the cell morphology of each organ was observed,and the biological safety of the experimental materials was evaluated.Results:1.Preparation and Physicochemical Properties of SF/nHAp/GO porous scaffolds based on directional pore structureThe characterization results show that there is no obvious phase separation of SF,nHAp and GO in the porous scaffolds,which indicates that the three components have good compatibility.Although the addition of nHAp and GO reduces the pore diameter and porosity of the material to a certain extent,the directional temperature field freeze-drying technology can improve the pore diameter and porosity of the scaffold,which makes the directional channel SF/nHAp/GO scaffold have better pore connectivity and more suitable porosity.Degradation experiments showed that nHAp and GO enhanced the ability of scaffolds to resist enzyme degradation to a certain extent,so that SF/nHAp/GO scaffolds and SF/nHAp/GO scaffolds with directional pores had a corresponding degradation rate of matching bone tissue.The results of mineralization experiment in vitro show that the introduction of nHAp and GO can increase the ability of scaffold materials to induce bonelike apatite and effectively improve the ability of promoting osteogenesis.2.Study on cytotoxicity,cytocompatibility and osteogenic induction of SF/nHAp/GO porous scaffolds based on directional pore structure in vitroThe results of cytocompatibility experiment showed that the scaffolds of each group had almost no adverse effect on cells for a period of time,and the cell survival rate and cell morphology of nHAp and GO scaffolds were better than those of pure SF scaffolds,indicating that both nHAp and GO could promote cell survival,and nHAp and GO had stronger synergistic effect on the proliferation of m BMSCs and better cytocompatibility.The SF/nHAp/GO scaffold with directional channels benefits from its unique pore structure,which makes the adhesion and proliferation of bone marrow mesenchymal stem cells more favorable.RT-PCR results showed that the expression of osteogenic genes in each group of scaffolds was up-regulated,and the ability of osteogenic differentiation induced by SF/nHAp/GO scaffolds with oriented pores was more prominent.3.The experimental study of SF/nHAp/GO porous scaffold based on directional pore structure on the repair of rat skull defect.All the rats recovered smoothly in the experiment.The skull specimens were obtained at 6 and 12 weeks after operation,and the experimental materials were sutured after the bone defect was placed,and there was no exfoliation and bleeding.The structure of in vivo toxicity test showed that there was no significant difference in the morphology of heart,liver,spleen and kidney in each group,and no abnormal changes were found,such as cell degeneration,hyperemia,edema,etc.,indicating that the scaffold material would not destroy the important organs of rats in a short time,and the imaging examination showed that the ossification gray value of SF/nHAp/GO stent group and SF/nHAp/GO stent group was the highest,and that of directional orifice stent group was better.The results of sequence fluorescence labeling experiment showed that after 8-10 weeks of implantation,the bone mineralization deposition rate of SF/nHAp/GO stent with directional pore was the highest,followed by non-directional SF/nHAp/GO stent,and the bone mineralization deposition rate of SF/nHAp stent and SF stent decreased as a whole.Histological observation,the effect of angiogenesis and osteogenesis of SF/nHAp/GO stent with directional pore was the most obvious.Therefore,the oriented pore SF/nHAp/GO porous scaffold has stronger biological activity and bone tissue integration ability.Conclusion:In this study,a new type of directional porous SF/nHAP/GO scaffold was successfully prepared by directional temperature field freezing technology.And the research results show that the directional freezing technology can increase the pore diameter and porosity of the scaffold and provide a better microenvironment.Cell adhesion and proliferation experiments in vitro showed that the oriented pore-like SF/nHAp/GO scaffold had good cell affinity and was beneficial to cell growth and proliferation.The experiments of biomineralization in vitro,osteogenic gene expression and skull defect repair in vivo show that SF/nHAp/GO scaffolds with directional pores can effectively increase apatite deposition,up-regulate osteogenic gene expression and induce new bone formation.Therefore,directional pore SF/nHAp/GO scaffolds have good bioactivity and good biocompatibility in vivo and in vitro. |
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