负载γ-Fe_(2)O_(3)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  

Effects of γ-Fe_(2)O_(3)-loaded chitosan porous sponge on proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells

作　　者：孙上雯 陈汉帮 胡姝颖[1,2] 章非敏[1,2] SUN Shangwen;CHEN Hanbang;HU Shuying;ZHANG Feimin(Jiangsu Province Key Laboratory of Oral Diseases,Nanjing Medical University,Nanjing 210029,china;Department of Prosthodontics,the Affiliated Stomatological Hospital of Nanjing Medical University,Nanjing 210029,china)

机构地区：[1]南京医科大学口腔疾病研究江苏省重点实验室,江苏南京210029 [2]南京医科大学附属口腔医院修复科,江苏南京210029

出　　处：《南京医科大学学报（自然科学版）》2023年第3期326-333,共8页Journal of Nanjing Medical University(Natural Sciences)

基　　金：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)。

摘　　要：目的:研究负载γ-Fe_(2)O_(3)的壳聚糖多孔海绵对大鼠骨髓间充质干细胞(rat bone marrow mesenchymal stem cell,r BMSC)增殖和成骨分化的影响。方法:使用冻干-交联法制备负载γ-Fe_(2)O_(3)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(collagenⅠ,Col1)和Runt相关转录因子2(core binding factor alphal 1,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-Fe_(2)O_(3)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-Fe_(2)O_(3)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-Fe_(2)O_(3)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。Objective:This study aims to observe the effects of γ-Fe_(2)O_(3)-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-Fe_(2)O_(3)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-Fe_(2)O_(3)promoted the proliferation of r BMSC. ALP staining and activity detection results showed the addition of γ-Fe_(2)O_(3)can improve the activity of ALP. The results of real-time fluorescence quantitative reverse transcription PCR showed that the addition of γ-Fe_(2)O_(3)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-Fe_(2)O_(3)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-Fe_(2)O_(3)promoted the proliferation and the early indicators of osteogenic differentiation of rBMSC,and loading γ-Fe_(2)O_(3)at concentration of 5% and 10% can promote the formation of mineralization in the late stage of osteogenic differentiation of r BMSC.

关 键 词：γ-Fe_(2)O_(3)磁性纳米颗粒 壳聚糖多孔海绵 骨组织工程 成骨分化 

分 类 号：R336[医药卫生—人体生理学]