收稿日期: 2022-10-12
网络出版日期: 2023-01-31
Effect of pH on the chelation between strontium ions and decellularized small intestinal submucosal sponge scaffolds
Received date: 2022-10-12
Online published: 2023-01-31
目的: 设计不同pH值下螯合锶(strontium, Sr)的脱细胞小肠黏膜下层(decellularized small intestinal submucosa, dSIS)海绵支架(Sr/dSIS), 以该支架的理化性能和生物相容性为评价指标, 为制备Sr/dSIS选择合适的pH值。方法: (1) Sr/dSIS制备及分组: 将dSIS溶液与氯化锶溶液等体积混合, 调节溶液pH分别为3、5、7、9, 于37℃下充分反应后经冷冻干燥制得多孔支架, 分别命名为Sr/dSIS-3、-5、-7、-9, 以dSIS支架为对照; (2)理化性能评价: 观察支架大体形貌, 采用扫描电镜分析微观形貌并测定孔隙率和孔径, 能谱分析表面元素, 红外光谱分析官能团结构, 原子吸收分光光度法测定螯合率, 比重法检测吸水率, 万能力学测试机评价压缩强度; (3)生物相容性评价: 采用Calcein-AM/PI活细胞/死细胞染色法评价各组支架的毒性和促骨髓间充质干细胞(bone mesenchymal stem cells, BMSCs)增殖效果。结果: 扫描电镜下各组支架具有三维多孔网络结构, 孔径和孔隙率差异无统计学意义; 能谱分析中Sr/dSIS-5、-7、-9组检测出锶元素的特征峰, 且锶元素均匀分布于支架; 官能团分析验证了Sr/dSIS-5、-7、-9组有螯合物形成; 螯合率分析显示Sr/dSIS-7组锶离子螯合率最高, 与其他组的差异有统计学意义(P < 0.05);各组支架吸水性良好; Sr/dSIS-5、-7、-9组的压缩强度显著高于对照组(P < 0.05);各组支架的生物相容性良好, Sr/dSIS-7组展现出最佳的促细胞增殖能力。结论: pH为7时, Sr/dSIS支架具有高锶离子螯合率以及更好的促BMSCs增殖效果, 是制备Sr/dSIS支架的理想pH值。
李雨柯 , 王梅 , 唐琳 , 刘玉华 , 陈晓颖 . 不同pH值对脱细胞小肠黏膜下层海绵支架螯合锶离子的影响[J]. 北京大学学报（医学版）, 2023 , 55(1) : 44 -51 . DOI: 10.19723/j.issn.1671-167X.2023.01.007
Objective: To investigate the preparation of decellularized small intestinal submucosa (dSIS) sponge scaffolds with chelated strontium (Sr) ions at different pH values, and to select the appropriate pH values for synthesizing Sr/dSIS scaffolds using the physicochemical properties and biocompatibility of the scaffolds as evaluation indexes. Methods: (1) Sr/dSIS scaffolds preparation and grouping: After mixing dSIS solution and strontium chloride solution in equal volumes, adjusting pH of the solution to 3, 5, 7, and 9 respectively, porous scaffolds were prepared by freeze-drying method after full reaction at 37℃, which were named Sr/dSIS-3, -5, -7, and -9 respectively, and the dSIS scaffolds were used as the control group. (2) Physicochemical property evaluation: The bulk morphology of the scaffolds was observed in each group, the microscopic morphology analyzed by scanning electron microscopy, and the porosity and pore size determined, the surface elements analyzed by energy spectroscopy, the structure of functional groups analyzed by infrared spectroscopy, the chelation rate determined by atomic spectrophotometry, the water absorption rate detected by using specific gravity method, and the compression strength evaluated by universal mechanical testing machine.(3) Biocompatibility evaluation: The cytotoxicity and proliferative effect to bone mesenchymal stem cells (BMSCs) of each group were evaluated by Calcein-AM/PI double staining method. Results: Scanning electron microscopy showed that the scaffolds of each group had an interconnected three-dimensional porous structure with no statistical difference in pore size and porosity. Energy spectrum analysis showed that strontium could be detected in Sr/dSIS-5, -7 and -9 groups, and strontium was uniformly distributed in the scaffolds. Functional group analysis further supported the formation of chelates in the Sr/dSIS-5, -7 and -9 groups. Chelation rate analysis showed that the Sr/dSIS-7 group had the highest strontium chelation rate, which was statistically different from the other groups (P < 0.05). The scaffolds in all the groups had good water absorption. The scaffolds in Sr/dSIS-5, -7 and -9 groups showed significantly improved mechanical properties compared with the control group (P < 0.05). The scaffolds in all the groups had good biocompatibility, and the Sr/dSIS-7 group showed the best proliferation of BMSCs. Conclusion: When pH was 7, the Sr/dSIS scaffolds showed the highest strontium chelation rate and the best proliferation effect of BMSCs, which was the ideal pH value for the preparation of the Sr/dSIS scaffolds.
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