背景:生物活性物质Biodentine具有良好的抗压强度、粘接强度及较少的微渗漏等诸多优点,已被成功应用于多种临床适应证,然而有关Biodentine是否可促进成骨细胞成骨的研究罕见。目的:研究不同浓度Biodentine对人骨肉瘤细胞MG-63增殖及成...背景:生物活性物质Biodentine具有良好的抗压强度、粘接强度及较少的微渗漏等诸多优点,已被成功应用于多种临床适应证,然而有关Biodentine是否可促进成骨细胞成骨的研究罕见。目的:研究不同浓度Biodentine对人骨肉瘤细胞MG-63增殖及成骨作用的影响。方法:制备不同浓度梯度(1、1/2、1/4、1/8和1/16)的Biodentine浸提液。将人骨肉瘤细胞MG-63分6组培养,分别加入MEM培养液(对照组)及5种浓度的Biodentine浸提液,采用CCK-8法检测培养第1,3,5,7天的细胞增殖,筛选Biodentine浸提液最佳浓度。将人骨肉瘤细胞MG-63分2组培养,分别加入MEM培养液(空白对照组)及最佳浓度的Biodentine材料浸提液(实验组),培养第1,3,5,7天,采用Real-time PCR法检测细胞中成骨因子Runx2 m RNA表达;培养第10,14天行茜素红染色,观察细胞钙化结节形成情况。结果与结论:(1)培养第1,3天时,不同浓度Biodentine材料浸提液组活细胞数量与对照组相比无差异;培养第5天,1浓度材料浸提液组活细胞数量明显低于对照组(P<0.05),1/2、1/4、1/8浓度材料浸提液组活细胞数量与对照组比较无差异,1/16浓度材料浸提液组活细胞数量明显高于对照组(P<0.05),因此实验选择1/16浓度材料浸提液进行后续实验;(2)实验组培养第1,3,5,7天的Runx2 m RNA表达量分别为空白对照组的1.14,5.29,1.08,2.11倍(P均<0.05);(3)培养第10天时,实验组与空白对照组均未见矿化结节;培养第14天,两组均可见矿化结节,实验组的面积更大、颜色更深;(4)结果表明在一定浓度下,Biodentine可促进人骨肉瘤细胞MG-63增殖及成骨活性。展开更多
Background:Mesenchymal stem or stromal cells(MSCs)derived from the induced pluripotent stem cells(iPSCs)have uniform biological activity,which makes the clinical application of MSCs in bone repair possible.Culturing t...Background:Mesenchymal stem or stromal cells(MSCs)derived from the induced pluripotent stem cells(iPSCs)have uniform biological activity,which makes the clinical application of MSCs in bone repair possible.Culturing the iPSC-MSCs onto osteoconductive materials is a promising tissue engineering-based strategy in bone regeneration.The aim of this work was to evaluate the effects of semaphorin 3A(Sema3A)and hypoxia inducible factor 1 subunit alpha(HIF1α)co-overexpression on the survival and osteogenic differentiation of iPSC-MSCs.Methods:Sema3A and HIF1αwere linked together with the three(GGGGS;G,glycine;S,serine)peptide fragment,and their co-expression in iPSC-MSCs was mediated by a lentiviral vector.The fusion protein retained the immune reactivity for both Sema3A and HIF1αas determined with Western blotting.iPSC-MSCs were infected with overexpression lentivirus(oeLenti)as negative control,oeLenti-Sema3A,oeLenti-HIF1αor oeLenti-Sema3A-HIF1αlentiviruses.Results:Sema3A overexpression alone promoted the osteogenic differentiation of iPSC-MSCs(the activity and/or expression of osteoblast markers,such as alkaline phosphatase,osteopontin,and osteocalcin,were upregulated),and suppressed cell survival.The Sema3A-HIF1αfusion protein showed a comparable osteoconductive effect to that of Sema3A without reducing cell survival.We further seeded iPSC-MSCs modified by SemaA-HIF1αoverexpression onto hydroxyapatite(HA)scaffolds,and evaluated their growth and differentiation on this three-dimensional material.Additional data indicated that,as compared to iPSC-MSCs cultured in ordinary two-dimensional dishes,cells cultured in HA scaffolds grew(blank vs.HA scaffolds:0.83 vs.1.39 for survival)and differentiated better(blank vs.HA scaffolds:11.29 vs.16.62 for alkaline phosphatase activity).Conclusion:Modifying iPSC-MSCs with pro-osteogenic(Sema3A)and pro-survival(HIF1α)factors may represent a promising strategy to optimize tissue engineering-based strategy in bone repair.展开更多
文摘背景:生物活性物质Biodentine具有良好的抗压强度、粘接强度及较少的微渗漏等诸多优点,已被成功应用于多种临床适应证,然而有关Biodentine是否可促进成骨细胞成骨的研究罕见。目的:研究不同浓度Biodentine对人骨肉瘤细胞MG-63增殖及成骨作用的影响。方法:制备不同浓度梯度(1、1/2、1/4、1/8和1/16)的Biodentine浸提液。将人骨肉瘤细胞MG-63分6组培养,分别加入MEM培养液(对照组)及5种浓度的Biodentine浸提液,采用CCK-8法检测培养第1,3,5,7天的细胞增殖,筛选Biodentine浸提液最佳浓度。将人骨肉瘤细胞MG-63分2组培养,分别加入MEM培养液(空白对照组)及最佳浓度的Biodentine材料浸提液(实验组),培养第1,3,5,7天,采用Real-time PCR法检测细胞中成骨因子Runx2 m RNA表达;培养第10,14天行茜素红染色,观察细胞钙化结节形成情况。结果与结论:(1)培养第1,3天时,不同浓度Biodentine材料浸提液组活细胞数量与对照组相比无差异;培养第5天,1浓度材料浸提液组活细胞数量明显低于对照组(P<0.05),1/2、1/4、1/8浓度材料浸提液组活细胞数量与对照组比较无差异,1/16浓度材料浸提液组活细胞数量明显高于对照组(P<0.05),因此实验选择1/16浓度材料浸提液进行后续实验;(2)实验组培养第1,3,5,7天的Runx2 m RNA表达量分别为空白对照组的1.14,5.29,1.08,2.11倍(P均<0.05);(3)培养第10天时,实验组与空白对照组均未见矿化结节;培养第14天,两组均可见矿化结节,实验组的面积更大、颜色更深;(4)结果表明在一定浓度下,Biodentine可促进人骨肉瘤细胞MG-63增殖及成骨活性。
基金This work was supported by a grant from the National Natural Science Foundation of China(No.81601726).
文摘Background:Mesenchymal stem or stromal cells(MSCs)derived from the induced pluripotent stem cells(iPSCs)have uniform biological activity,which makes the clinical application of MSCs in bone repair possible.Culturing the iPSC-MSCs onto osteoconductive materials is a promising tissue engineering-based strategy in bone regeneration.The aim of this work was to evaluate the effects of semaphorin 3A(Sema3A)and hypoxia inducible factor 1 subunit alpha(HIF1α)co-overexpression on the survival and osteogenic differentiation of iPSC-MSCs.Methods:Sema3A and HIF1αwere linked together with the three(GGGGS;G,glycine;S,serine)peptide fragment,and their co-expression in iPSC-MSCs was mediated by a lentiviral vector.The fusion protein retained the immune reactivity for both Sema3A and HIF1αas determined with Western blotting.iPSC-MSCs were infected with overexpression lentivirus(oeLenti)as negative control,oeLenti-Sema3A,oeLenti-HIF1αor oeLenti-Sema3A-HIF1αlentiviruses.Results:Sema3A overexpression alone promoted the osteogenic differentiation of iPSC-MSCs(the activity and/or expression of osteoblast markers,such as alkaline phosphatase,osteopontin,and osteocalcin,were upregulated),and suppressed cell survival.The Sema3A-HIF1αfusion protein showed a comparable osteoconductive effect to that of Sema3A without reducing cell survival.We further seeded iPSC-MSCs modified by SemaA-HIF1αoverexpression onto hydroxyapatite(HA)scaffolds,and evaluated their growth and differentiation on this three-dimensional material.Additional data indicated that,as compared to iPSC-MSCs cultured in ordinary two-dimensional dishes,cells cultured in HA scaffolds grew(blank vs.HA scaffolds:0.83 vs.1.39 for survival)and differentiated better(blank vs.HA scaffolds:11.29 vs.16.62 for alkaline phosphatase activity).Conclusion:Modifying iPSC-MSCs with pro-osteogenic(Sema3A)and pro-survival(HIF1α)factors may represent a promising strategy to optimize tissue engineering-based strategy in bone repair.
