含钼的硅酸盐生物陶瓷释放的化学离子对肌腱-骨相关的多细胞调控★

doi:10.6023/A23040120

摘要/Abstract

研究生物材料所产生的化学信号与细胞行为之间的作用关系, 对于高性能组织工程材料的设计与制备具有重要的指导意义. 本工作中, 采用化学共沉淀法成功地合成了含钼(Mo)的硅酸盐(MS)生物活性陶瓷粉体, 并探究其降解产生的活性离子化学信号对肌腱-骨相关细胞(肌腱干/祖细胞(TSPCs)和骨髓间充质干细胞(BMSCs))的调控作用. 所合成的MS生物陶瓷颗粒大小均一, 其降解后的浸提液中含有Mo、钙(Ca)、硅(Si)三种生物活性离子. 特定浓度范围的MS浸提液可以同时促进TSPCs和BMSCs的增殖和迁移活性. 更重要的是, MS浸提液可以同时刺激TSPCs和BMSCs的特异性分化, 说明MS生物陶瓷释放的化学离子所构建的微环境, 能够同时协调TSPCs和BMSCs的细胞行为. 因此, MS生物陶瓷有望作为一种有效的“生物活性因子”用于肌腱-骨损伤修复.

关键词: 含钼的硅酸盐, 化学信号, 多细胞, 细胞行为

Understanding the relationship between the chemical signals generated by biological materials and cellular behaviors has great significance for the design and preparation of high-performance tissue engineering biomaterials. In the past several decades, silicate bioceramics have been widely used in tissue engineering. Bioactive ions released from silicate bioceramics can act as chemical signals to regulate cellular behaviors and promote the tissue regeneration. Moreover, by regulating the components of silicate bioceramics, silicate bioceramics can generate specific chemical signals to regulate cellular behaviors of multiple cells. Here, by introducing molybdenum (Mo) element into silicate bioceramics, we have successfully developed Mo-containing silicate (MS) bioceramics which are able to regulate cellular behaviors of tendon stem/progenitor cells (TSPCs) and bone marrow mesenchymal stem cells (BMSCs) simultaneously. Using ammonium molybdate as a source of Mo element, MS bioceramics were prepared by chemical coprecipitation method. The synthesized MS bioceramics were mostly below 10 μm in size and had uniform distribution of Mo elements. Moreover, MS bioceramics were composed of high-purity CaMoO₄ and CaSiO₃. To explore the effect of chemical signals generated from MS bioceramics on TSPCs and BMSCs, we prepared MS extracts for cell culture. MS bioceramics supported the survival of TSPCs and BMSCs and maintained a better cellular state during the culture period of 5 d. Due to the released Ca, Si and Mo ions from MS bioceramics, TSPCs and BMSCs cultured with MS extracts exhibited excellent proliferation and migration activities. Interestingly, after cultured with MS extracts in the appropriate concentration, the expression of osteogenic genes and protein of BMSCs and the expression of tenogenic genes and protein of TSPCs were significantly enhanced, suggesting that chemical signals generated by MS bioceramics simultaneously promoted the specific differentiation of TSPCs and BMSCs. Such MS bioceramics are believed to be an effective “bioactive factor” for repairing injury at the tendon-bone interfaces.

Key words: Mo-containing silicate, chemical signals, multiple cells, cellular behaviors

引用此文

杜琳, 薛健民, 郇志广, 吴成铁. 含钼的硅酸盐生物陶瓷释放的化学离子对肌腱-骨相关的多细胞调控^★[J]. 化学学报, 2023, 81(10): 1334-1340.

Lin Du, Jianmin Xue, Zhiguang Huan, Chengtie Wu. Regulation of Tendon-bone Related Multiple Cells by Chemical Ions Released from Mo-containing Silicate Bioceramics^★[J]. Acta Chimica Sinica, 2023, 81(10): 1334-1340.

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图/表 7

图1. 含钼(Mo)的硅酸盐(MS)生物陶瓷粉体的表征. (A) MS颗粒的X射线衍射(XRD)图谱. (B) MS颗粒的扫描电镜图(SEM). (C) MS颗粒的粒径分布图. (D) MS颗粒的元素分布图(EDS). 具有均匀粒度尺寸和均匀Mo元素分布的MS生物陶瓷颗粒被成功制备

Figure 1. Characterization of Mo-containing silicate (MS) bioceramic powders. (A) XRD pattern of MS particles. (B) SEM image of MS particles. (C) Particle size distribution of MS particles. (D) EDS elemental mapping of MS particles. MS bioceramics were successfully prepared with uniform particle size and homogeneous distribution of Mo element

Ionic conc./(mg•L^－1)	Ca	Si	Mo
Control	71.85±2.58	2.45±1.13	0±0
1/2	83.77±4.34	136.06±7.61	14.29±0.94
1/4	96.18±6.39	73.19±4.80	7.67±0.62
1/8	85.99±2.43	39.29±0.85	3.92±0.31
1/16	79.29±4.85	21.09±0.89	1.95±0.07
1/32	76.39±2.83	11.75±0.20	1.27±0.08
1/64	70.53±1.67	6.96±0.35	0.43±0.26
1/128	72.99±0.36	4.86±0.21	0.37±0.13
1/256	70.92±1.82	3.40±0.94	0.18±0.22

表1. 不同稀释倍数的MS浸提液中的化学信号含量

Table 1. The concentration of chemical signals generated from different concentrations of MS extracts

图2. 用不同稀释倍数的MS浸提液培养后的肌腱干/祖细胞(TSPCs)和骨髓间充质干细胞(BMSCs)的增殖活性及存活情况. TSPCs (A)和BMSCs (B)用不同稀释倍数的MS浸提液培养后的增殖活性(n=3) *p<0.05, **p<0.01, ***p<0.001. 用MS浸提液培养1 d和5 d后的TSPCs (C)和BMSCs (D)的live/dead染色图

Figure 2. Proliferation and live/dead assay of TSPCs (tendon stem/progenitor cells) and BMSCs (bone marrow mesenchymal stem cells) cultured with different concentrations of MS extracts. Proliferative activity of (A) TSPCs and (B) BMSCs after cultured with different dilution of MS extracts (n=3) *p<0.05, **p<0.01, ***p<0.001. Live/dead assay of TSPCs (C) and BMSCs (D) after cultured with MS extracts for 1 and 5 d

图3. 不同稀释倍数的MS浸提液对TSPCs和BMSCs的迁移影响. MS浸提液诱导后发生迁移的TSPCs (A)和BMSCs (B)的结晶紫染色图. 发生迁移的TSPCs (C)和BMSCs (D)的细胞数目统计(n=4). *p<0.05, **p<0.01, ***p<0.001

Figure 3. The effect of different dilution of MS extracts on migration of TSPCs and BMSCs. After induced with MS extracts, crystalline violet staining images of the migration of TSPCs (A) and BMSCs (B). Quantification of TSPCs (C) and BMSCs (D) induced to migrate (n=4). *p<0.05, **p<0.01, ***p<0.001

图4. MS浸提液培养后的TSPCs的成肌腱相关基因和Tenomodulin (TNMD)蛋白表达情况. 不同稀释倍数的MS浸提液培养后的TSPCs的BGN (A)、DCN (B)和TNC (C)的表达情况(n=6). *p<0.05, **p<0.01, ***p<0.001. (D)不同稀释倍数的MS浸提液培养后的TSPCs的TNMD蛋白的免疫荧光染色图片

Figure 4. The expression of tenogenic differentiation-related genes and Tenomodulin (TNMD) of TSPCs cultured with different dilutions of MS extracts (1/16 and 1/32). The expression of (A) BGN, (B) DCN and (C) TNC of TSPCs incubated with different dilutions of MS extracts after 5 days of culture (n=6). *p<0.05, **p<0.01, ***p<0.001. (D) Immunofluorescence staining images of TNMD protein of TSPCs cultured with different concentrations MS extracts

图5. MS浸提液培养后的BMSCs的成骨相关基因和Bone sialoprotein (BSP)蛋白表达情况. 不同稀释倍数的MS浸提液培养后的BMSCs的ALP (A)、BMP2 (B)、Run×2 (C)和OPN (D)的表达情况 (n=6). *p<0.05, **p<0.01, ***p<0.001. (E)不同浓度的MS浸提液培养后的BMSCs表达的BSP蛋白的免疫荧光染色图片

Figure 5. The expression of osteogenic differentiation-related genes and Bone sialoprotein (BSP) of BMSCs cultured with different dilutions of MS extracts. (A) The expression of ALP, (B) BMP2, (C) Run×2 and (D) OPN of BMSCs incubated with different dilution of MS extracts after 5 days of culture (n=6). *p<0.05, **p<0.01, ***p<0.001. (E) Immunofluorescence staining images of BSP protein of BMSCs cultured with different concentrations of MS extracts

图6. TNMD (A)和BSP (B)免疫荧光染色图的半定量分析(n=3). *p<0.05, **p<0.01, ***p<0.001

Figure 6. Semi-quantitative analysis of immunofluorescence staining images of TNMD (A) and BSP (B) expression (n=3). *p<0.05, **p<0.01, ***p<0.001

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