负载γ⁃Fe2O3的壳聚糖多孔海绵对大鼠骨髓间质干细胞增殖和成骨分化的影响
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国家自然科学基金(81870807);江苏省高校优势学科建设工程资助项目(2018-87)


Effects of γ ⁃ Fe2O3 ⁃ loaded chitosan porous sponge on proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells
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    摘要:

    目的:研究负载γ-Fe2O3的壳聚糖多孔海绵对大鼠骨髓间质干细胞(rat bone marrow mesenchymal stem cell,rBMSC)增殖和成骨分化的影响。方法:使用冻干-交联法制备负载γ-Fe2O3浓度分别为1%、5%、10%和20%的壳聚糖海绵,并制备空白对照组。将rBMSC培养于海绵上,通过扫描电镜和共聚焦显微镜观察细胞黏附及增殖情况,通过CCK-8法检测细胞第1、3、5、7天的增殖情况,通过碱性磷酸酶(alkaline phosphatases,ALP)染色及活性检测和荧光定量PCR检测第7、14天ALP活性和成骨指标 ALP、骨形态发生蛋白(bone morphogenetic protein 2,Bmp2)、胶原蛋白(collagen Ⅰ,Col1)和Runt相关转录因子2(Runt-related transcription factor 2,Runx2)的表达;使用茜素红定量法评估第21、28天细胞外基质矿化情况。结果:CCK-8结果显示rBMSC均能在材料上持续增殖,添加γ-Fe2O3对rBMSC增殖有促进作用;ALP染色及活性检测结果和PCR结果显示添加γ-Fe2O3能提高ALP活性并促进成骨指标表达;茜素红定量结果显示添加浓度为5%和10%时,矿化物形成量高于对照组(P < 0.05)。结论:负载γ- Fe2O3的壳聚糖海绵能够促进 rBMSC 的增殖和早期成骨分化,浓度为 5%和 10%时对 rBMSC 成骨分化晚期矿化物形成有促进作用。

    Abstract:

    Objective:This study aims to observe the effects of γ-Fe2O3-loaded chitosan porous sponge on the proliferation and early osteogenic differentiation of rat bone marrow mesenchymal stem cells(rBMSC). Methods:Chitosan sponges loaded with γ- Fe2O3 at concentrations of 1%,5%,10% and 20% were prepared by freeze -drying and cross -linking,and blank control group was prepared. Scanning electron microscopy and confocal microscopy were used to evaluate the adhesion and proliferation of rBMSC on the sponges. Cell proliferation at 1,3,5 and 7 days was detected by CCK -8. The early osteogenic differentiation of rBMSC at 7 and 14 days was evaluated by alkaline phosphatases(ALP)staining and activity detection. Real - time fluorescence quantitative reverse transcription PCR was used to detect the expression of ALP,bone morphogenetic protein 2(Bmp2),collagen I(Col1)and Runt-related transcription factor 2(Runx2)after 7 and 14 days of osteogenic induction. The mineralization of extracellular matrix at 21 and 28 days was assessed by alizarin red staining quantitative method. Results:CCK-8 results showed each group added with γ-Fe2O3 promoted the proliferation of rBMSC. ALP staining and activity detection results showed the addition of γ-Fe2O3 can improve the activity of ALP. The results of real - time fluorescence quantitative reverse transcription PCR showed that the addition of γ - Fe2O3 can promote the expression of osteogenic indexes(ALP,Bmp2,Col1 and Runx2). The quantitative detection results of alizarin red staining showed that the amount of mineralization in the groups with γ-Fe2O3 added concentration of 5% and 10% was higher than that in the control group(P < 0.05).Conclusion:The chitosan porous sponge loaded with γ - Fe2O3 promoted the proliferation and the early indicators of osteogenic differentiation of rBMSC,and loading γ-Fe2O3 at concentration of 5% and 10% can promote the formation of mineralization in the late stage of osteogenic differentiation of rBMSC.

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孙上雯,陈汉帮,胡姝颖,章非敏.负载γ⁃Fe2O3的壳聚糖多孔海绵对大鼠骨髓间质干细胞增殖和成骨分化的影响[J].南京医科大学学报(自然科学版),2023,(3):326-333

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  • 在线发布日期: 2023-03-11
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