熔盐法制备含钴氯磷灰石及其抗氧化性能和细胞相容性研究

doi:10.15541/jim20220040

无机材料学报 ›› 2022, Vol. 37 ›› Issue (11): 1225-1235.DOI: 10.15541/jim20220040

所属专题：【生物材料】骨骼与齿类组织修复

熔盐法制备含钴氯磷灰石及其抗氧化性能和细胞相容性研究

舒朝琴¹^,²(), 朱敏¹(), 朱钰方²()

1.上海理工大学材料与化学学院, 上海 200093
2.中国科学院上海硅酸盐研究所高性能陶瓷与超微结构国家重点实验室, 上海 200050

收稿日期:2022-01-21 修回日期:2022-02-18 出版日期:2022-03-10 网络出版日期:2022-03-10
通讯作者: 朱敏, 副教授. E-mail: mzhu@usst.edu.cn;
朱钰方, 教授. E-mail: zjf2412@163.com
作者简介:舒朝琴(1995-), 女, 硕士研究生. E-mail: 1573738940@qq.com
基金资助:
国家自然科学基金(51872185);国家自然科学基金(52072246)

Cobalt-incorporated Chlorapatite: Preparation by Molten Salt Method, Anti-oxidation and Cytocompatibility

SHU Chaoqin¹^,²(), ZHU Min¹(), ZHU Yufang²()

1. School of Materials and Chemistry, University of Shanghai for Science and Technology, Shanghai 200093, China
2. State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, China

Received:2022-01-21 Revised:2022-02-18 Published:2022-03-10 Online:2022-03-10
Contact: ZHU Min, associate professor. E-mail: mzhu@usst.edu.cn;
ZHU Yufang, professor. E-mail: zjf2412@163.com
About author:SHU Chaoqin (1995-), female, Master candidate. E-mail: 1573738940@qq.com
Supported by:
National Natural Science Foundation of China(51872185);National Natural Science Foundation of China(52072246)

摘要/Abstract

摘要：

生物活性陶瓷骨修复材料虽然具有优异的成骨性能, 但缺乏抗氧化应激的能力, 妨碍骨修复进程。本研究以β相磷酸三钙(β-TCP)粉体为原料, 采用LiCl-KCl熔盐体系, 以六水合氯化钴(CoCl₂·6H₂O)为钴源, 利用熔盐法制备出含钴氯磷灰石(Co-MS-TCP)。通过Co-MS-TCP粉体清除过氧化氢(H₂O₂)分析了含钴氯磷灰石的抗氧化能力; 通过细胞活性、胞内活性氧(ROS)含量变化评价了材料的细胞相容性和细胞水平抗氧化性能。结果表明, 熔盐处理β-TCP粉体能够制备含钴氯磷灰石, 钴含量随CoCl₂·6H₂O加入量增加而增大; H₂O₂清除能力随氯磷灰石中钴含量的增加而增强, 6 h内对H₂O₂的清除率可达90%以上。细胞实验证实, 含钴氯磷灰石具有良好的细胞相容性和抗氧化性能, 1.5 mg·mL^-1加3% Co盐的MS-TCP (3%Co-MS-TCP)即可保证软骨细胞和骨髓间充质干细胞存活率大于85%, 并且3% Co-MS-TCP可有效清除H₂O₂, 使得细胞内ROS含量显著降低。因此, 通过熔盐法制备含钴生物活性陶瓷是实现抗氧化应激的一种有效途径, 这也为开发催化活性高、生物相容好的功能化生物活性陶瓷提供了新的策略。

关键词: 生物活性陶瓷, 熔盐法, 钴, 抗氧化

Abstract:

Orthopedic surgery and postoperative inflammation are easy to induce oxidative stress, which hinders the process of bone repair. Bioactive ceramics have excellent osteogenic properties, but lack the ability to resist oxidative stress. Therefore, it is of great significance to develop a bioactive ceramic material with antioxidant function. Here, cobalt-incorporated chloroapatite (Co-MS-TCP) was prepared by a molten salt method, in which the mixture of lithium chloride and potassium chloride was used as a molten salt system, and β-phase tricalcium phosphate (TCP) and cobalt chloride hexahydrate (CoCl₂∙6H₂O) were used as raw material and cobalt source, respectively. The antioxidant ability of Co-MS-TCP was determined by catalyzing H₂O₂ clearance. The cytocompatibility and anti-oxidation of Co-MS-TCP were further evaluated by analyzing the changes of cell viability and intracellular reactive oxygen species (ROS). Results showed that Co-MS-TCP with controllable cobalt content can be prepared by a molten salt method with changing the addition amount of CoCl₂∙6H₂O source. The scavenging capacity of H₂O₂ increased with the increase of cobalt content in chlorapatite, and more than 90% of H₂O₂ could be scavenged within 6 h due to the catalytic activity of Co-MS-TCP. Furthermore, cell experiments confirmed the cytocompatibility and antioxidative property of Co-MS-TCP. 3% Co-MS-TCP at a concentration of 1.5 mg·mL^-1 could still ensure the survival rate of bone marrow mesenchymal stem cells and chondrocytes to be higher than 85%, and 3% Co-MS-TCP can also significantly reduce the content of intracellular ROS for the H₂O₂-stimulated cells. Therefore, molten salt method is an effective way to prepare cobalt-incorporated bioactive ceramics with antioxidative property, which also provides a promising strategy for the development of functional bioactive ceramics with high catalytic activity and biocompatibility.

Key words: bioactive ceramics, molten salt method, cobalt, anti-oxidation

中图分类号:

TQ174

舒朝琴, 朱敏, 朱钰方. 熔盐法制备含钴氯磷灰石及其抗氧化性能和细胞相容性研究[J]. 无机材料学报, 2022, 37(11): 1225-1235.

SHU Chaoqin, ZHU Min, ZHU Yufang. Cobalt-incorporated Chlorapatite: Preparation by Molten Salt Method, Anti-oxidation and Cytocompatibility[J]. Journal of Inorganic Materials, 2022, 37(11): 1225-1235.

图/表 13

图1 熔盐处理保温不同时间制备的Co-MS-TCP粉体的XRD图谱

Fig. 1 XRD patterns of Co-MS-TCP powders prepared by molten salt method with different holding time The color figure can be obtained from online edition

图2 β-TCP粉体经熔盐处理保温不同时间前后样品的微观形貌SEM照片

Fig. 2 SEM images of β-TCP powders before and after molten salt treatment with different holding time (A1-A2) β-TCP; (B1-B2) 0 h Co-MS-TCP; (C1-C2) 0.5 h Co-MS-TCP; (D1-D2) 1 h Co-MS-TCP; (E1-E2) 2 h Co-MS-TCP; (F1-F2) 3 h Co-MS-TCP

图3 熔盐处理保温不同时间制备的(A)MS-TCP和(B) Co-MS-TCP粉体的清除H2O2情况

Fig. 3 H2O2 scavenging effect of (A) MS-TCP and (B) Co-MS-TCP powders prepared by molten salt method with different holding time *** p<0.001

图4 加入不同钴盐量所获熔盐处理制备的Co-MS-TCP粉体的XRD图谱

Fig. 4 XRD patterns of Co-MS-TCP powders prepared by molten salt method with different cobalt salt additions

图5 不同钴盐加入量所获熔盐处理制备的Co-MS-TCP粉体的SEM照片

Fig. 5 SEM images of Co-MS-TCP powders prepared by molten salt method with different cobalt salt additions (A1, A2) MS-TCP; (B1, B2) 1% Co-MS-TCP; (C1, C2) 3% Co-MS-TCP; (D1, D2) 5% Co-MS-TCP

图6 钴含量及其价态在含钴氯磷灰石催化清除H2O2中的作用

Fig. 6 Contents and valence state of cobalt in Co-MS-TCP on H2O2 scavenging ability (A) H2O2 scavenging effect of Co-MS-TCP powders with different cobalt contents; (B) XPS spectra of cobalt in 3% Co-MS-TCP powder; (C, D) Cobalt contents in MS-TCP, 1% Co-MS-TCP, 3% Co-MS-TCP, and 5% Co-MS-TCP powders determined by (C) ICP-AES and (D) XRF methods. *p<0.05, **p<0.01, ***p<0.001. The color figures can be obtained from online edition

图7 β-TCP、MS-TCP和3%Co-MS-TCP粉体在Tris-HCl缓冲液中(A) Ca、(B) P、(C) Co离子的释放情况

Fig. 7 Releases of (A) Ca, (B) P and (C) Co ions from β-TCP, MS-TCP and 3% Co-MS-TCP powders in Tris-HCl buffer The color figures can be obtained from online edition

图8 SBF溶液中浸泡7 d后的β-TCP (A1, A2)、MS-TCP (B1, B2)和3% Co-MS-TCP (C1, C2)粉体的SEM照片和(D)红外光谱图

Fig. 8 SEM images of (A1, A2) β-TCP, (B1, B2) MS-TCP and (C1, C2) 3% Co-MS-TCP after soaking in SBF for 7 d and (D) their corresponding FT-IR spectra The color figure can be obtained from online edition

图9 不同浓度MS-TCP和3% Co-MS-TCP 粉体对(A)软骨细胞和(B)骨髓间充质干细胞的细胞毒性评估

Fig. 9 Cell viabilities of MS-TCP and 3% Co-MS-TCP powders with different concentrations on (A) cartilage cells and (B) bone marrow mesenchymal stem cells **p<0.01, *** p<0.001; The color figures can be obtained from online edition

图10 (A)空白组和(B) β-TCP, (C) MS-TCP, (D) 3% Co-MS-TCP粉体处理H2O2刺激的软骨细胞内ROS染色荧光显微镜照片和(E)对应的荧光强度统计

Fig. 10 Images of intracellular ROS staining fluorescence in cartilage cells stimulated by H2O2 treatment with powders (A) Blank, (B) β-TCP, (C) MS-TCP, (D) 3% Co-MS-TCP and (E) corresponding fluorescence intensity statistics ***p<0.001; The color figures can be obtained from online edition

图11 (A)空白组和(B) β-TCP, (C) MS-TCP, (D) 3% Co-MS-TCP粉体处理H2O2刺激的骨髓间充质干细胞后的细胞内ROS染色荧光显微镜照片和(E)对应的荧光强度统计

Fig. 11 Images of intracellular ROS staining fluorescence in bone marrow mesenchymal stem cells stimulated by H2O2 treatment with powders (A) Blank, (B) β-TCP, (C) MS-TCP, (D) 3% Co-MS-TCP and (E) Corresponding fluorescence intensity statistics. * p<0.05, ** p<0.01 The color figures can be obtained from online edition

图12 (A)空白组和(B) β-TCP, (C) MS-TCP, (D) 3% Co-MS-TCP粉体处理(A1-D1) 无H2O2刺激与(A2-D2) H2O2刺激的软骨细胞后的细胞活(绿色)/死(红色)Calcein-AM-PI染色荧光显微镜照片

Fig. 12 Calcein AM and PI fluorescence images of cartilage cells treated with different powders((A) Blank; (B) β-TCP; (C) MS-TCP; (D) 3% Co-MS-TCP; (A1-D1) Cells without H2O2 stimulation; (A2-D2) Cells stimulated with H2O2 showing live (green) and death (red) cells) The color figures can be obtained from online edition

图13 (A)空白组和(B) β-TCP, (C) MS-TCP, (D) 3% Co-MS-TCP粉体处理(A1-D1)无H2O2刺激与(A2-D2) H2O2刺激的骨髓间充质干细胞的活/死Calcein-AM-PI染色荧光显微镜照片

Fig. 13 Calcein AM and PI fluorescence images of bone marrow mesenchymal stem cells treated with powders (A) Blank; (B) β-TCP; (C) MS-TCP; (D) 3% Co-MS-TCP; (A1-D1) Cells without H2O2 stimulation; (A2-D2) Cells stimulated with H2O2 showing live (green) and death (red) cells The color figures can be obtained from online edition

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熔盐法制备含钴氯磷灰石及其抗氧化性能和细胞相容性研究

Cobalt-incorporated Chlorapatite: Preparation by Molten Salt Method, Anti-oxidation and Cytocompatibility

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