钙磷涂层的JDBM镁合金多孔支架促进血管新生及骨缺损修复的效果评估

doi:10.3969/j.issn.1674-8115.2021.06.005

摘要/Abstract

摘要：

目的·研究含有钙磷涂层（CaHPO₄·2H₂O，DCPD）的JDBM（Mg-Nd-Zn-Zr）镁合金支架体内外促血管新生和骨缺损修复的生物学效应。方法·应用模板复制法和化学沉积法构建JDBM-DCPD和JDBM-MgF₂支架，使用微型CT（micro-CT）和扫描电子显微镜检测2种支架的表征。在支架表面种植骨髓间充质干细胞（bone marrow mesenchymal stem cell， BMSC）并通过CCK-8实验及细胞黏附实验观察支架对细胞的生物相容性。通过Transwell细胞迁移实验和成管实验检测支架浸提液对内皮细胞系Ea.hy926细胞迁移和成管能力的影响，并通过免疫荧光染色进一步观察血管内皮生长因子（vascular endothelial growth factor，VEGF）的分泌情况；通过碱性磷酸酶和茜素红染色检测浸提液对BMSC成骨能力的影响。构建SD大鼠股骨髁临界性骨缺损模型并植入支架，术后8周通过Microfil血管灌注、micro-CT扫描、组织切片染色等方法评估JDBM-DCPD支架的促血管新生及促成骨能力。结果·JDBM-DCPD支架的主孔径为400~450 μm，钙磷颗粒均匀分布在孔壁上，大小为15~25 μm。BMSC能够在JDBM-DCPD支架表面黏附且生长良好。与对照组和JDBM-MgF₂支架浸提液相比，JDBM-DCPD支架浸提液在体外能够显著促进Ea.hy926内皮细胞迁移、成管以及VEGF的表达，同时可显著增强BMSC早期和晚期成骨分化。体内植入8周后，JDBM-DCPD支架促进缺损区血管和新骨再生作用显著优于JDBM-MgF₂支架。结论·JDBM-DCPD支架在体内外实验中展现了优良的成血管效应，特别是在体内植入后可以实现早期血管化，从而更加有效地促进骨再生。

关键词: Mg-Nd-Zn-Zr合金, DCPD涂层, 血管新生, 成骨分化, 骨再生, 骨组织工程

Abstract:

Objective·To evaluate the biological effects of JDBM (Mg-Nd-Zn-Zr) scaffold coated with DCPD (CaHPO₄·2H₂O) on angiogenesis and repairing bone defects in vivo and in vitro.

Methods·The JDBM-DCPD and JDBM-MgF₂ scaffolds were constructed by using template replication method and chemical deposition method, and the characteristics of the scaffolds were observed by micro-CT and scan electron microscope. The bone marrow mesenchymal stem cells (BMSCs) were seeded on the scaffolds and the biocompatibility of scaffolds was evaluated by CCK-8 experiment and cell adhesion experiment. Transwell cell migration experiment and tube formation experiment were used to detect the effects of scaffold extracts on the migration and tube-forming ability of endothelial cell line Ea.hy926 cells, and immunofluorescence was used to further observe the secretion of vascular endothelial growth factor (VEGF). Alkaline phosphatase staining and alizarin red staining were used to detect the effect of the extracts on the osteogenic ability of BMSCs. Furthermore, the model of critical bone defect of femoral condyle was constructed in SD rats. The JDBM-DCPD and JDBM-MgF₂ scaffolds were implanted into the defects, respectively, and the effects of scaffolds on the osteogenesis and vascularization were assessed by Microfil vascular perfusion, micro-CT scanning, and tissue section staining after 8 weeks of operation.

Results·The main spherical pore size of JDBM-DCPD scaffold was 400?450 μm and the calcium and phosphorus particles were evenly distributed on the pore wall with the size of 15?25 μm. BMSCs adhered and grew well on the surface of JDBM-DCPD scaffold. Compared with the control group and the JDBM-MgF₂ scaffold extract, the JDBM-DCPD scaffold extract could significantly promote the migration, tube formation and VEGF expression of Ea.hy926 cells, and significantly enhance the early and late osteogenic differentiation of BMSCs in vitro. After 8 weeks of implantation, the JDBM-DCPD scaffold dramatically facilitated the regeneration of new bone and new vessels in the defect area compared with the JDBM-MgF₂ scaffold.

Conclusion·The JDBM-DCPD scaffold exhibits excellent vascularization effects both in vivo and in vitro, especially early vascularization effect after implantation and bone regeneration promotion in vivo.

Key words: Mg-Nd-Zn-Zr alloy (JDBM), DCPD coat, angiogenesis, osteogenic differentiation, bone regeneration, bone tissue engineering

中图分类号:

R318.08

王青, 王伟, 姜达君, 贾伟涛. 钙磷涂层的JDBM镁合金多孔支架促进血管新生及骨缺损修复的效果评估[J]. 上海交通大学学报(医学版), 2021, 41(6): 732-740.

Qing WANG, Wei WANG, Da-jun JIANG, Wei-tao JIA. Evaluation of JDBM porous scaffold coated with DCPD in promoting angiogenesis and repairing bone defects[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(6): 732-740.

图/表 7

图1 JDBM-DCPD与JDBM支架表征的比较Note: A. 3D reconstruction images of JDBM-MgF2 and JDBM-DCPD porous scaffolds. B. SEM to observe the pore size and surface structure of the scaffolds (above ×100, below ×5 000). C. The porosities of JDBM-MgF2 and JDBM-DCPD porous scaffolds. ① P=0.000， compared with the JDBM-MgF2 group.

Fig 1 Comparison of JDBM-DCPD and JDBM-MgF2 porous scaffolds in characteristics

图2 JDBM-DCPD与JDBM-MgF2支架生物相容性的比较Note: A. Cell proliferation activity of BMSCs cultured on the scaffolds for 7 d detected by CCK8 kit. ①P=0.000, compared with the JDBM-MgF2 group. B. Cell adhesion of BMSCs cultured on the scaffolds for 7 d observed by SEM (above ×250, below ×1 000).

Fig 2 Comparison of JDBM-DCPD and JDBM-MgF2 in biocompatibility

图3 JDBM-DCPD与JDBM-MgF2支架浸提液对Ea.hy926细胞迁移能力和成管情况影响的比较Note: A. Representative photographs showing the effect of the extracts of JDBM-DCPD and JDBM-MgF2 on the migration of Ea.hy926 cells after incubation for 24 h detected by Transwell migration assay (crystal violet staining, ×100). B. Quantitation of migrating Ea.hy926 cells. C. Representative photographs showing the effect of the extracts of JDBM-DCPD and JDBM-MgF2 on the tubule formation of Ea.hy926 cells after incubation for 3 h and 6 h (×100). D. Quantitation of tubule formation of Ea.hy926 cells. ①P=0.000, compared with the control group; ②P=0.007, ③P=0.000, compared with the JDBM-MgF2 group. HPF—high power field.

Fig 3 Comparison of the effects of JDBM-DCPD and JDBM-MgF2 scaffold extracts on Ea.hy926 cell migration and tube formation

图4 JDBM-DCPD与JDBM-MgF2支架浸提液对Ea.hy926细胞VEGF表达水平影响的比较Note:A. Representative photographs showing the effect of the extracts of JDBM-DCPD and JDBM-MgF2 on the expression of VEGF in Ea.hy926 cells after incubation for 3 d detected by immunofluorescent (×200). B. Semi-quantitative analysis of fluorescence intensity. ①P=0.001, ②P=0.000, compared with the control group; ③P=0.004, compared with the JDBM-MgF2 group.

Fig 4 Comparison of the effects of JDBM-DCPD and JDBM-MgF2 scaffold extracts on the expression of VEGF in Ea.hy926 cells

图5 JDBM-DCPD与JDBM-MgF2支架浸提液对BMSC成骨分化影响的比较Note:A. ALP staining of BMSCs cultured in the scaffold extracts for 7 d and 14 d, respectively (×100). B. ARS staining of BMSCs cultured in the scaffold extracts for 14 d (×100).

Fig 5 Comparison of effects of JDBM-DCPD and JDBM-MgF2 scaffold extracts on osteogenic differentiation of BMSCs

图6 植入8周后影像学检测JDBM-DCPD与JDBM-MgF2支架的体内成骨、成血管作用Note: A. X-ray photos. B. Three-dimensional reconstruction and sagittal images by CT. C. Microfil perfusion to observe the formation of new blood vessels in the defect area. The white circles indicate the bone defect area.

Fig 6 Osteogenesis and angiogenesis of JDBM-DCPD and JDBM-MgF2 scaffolds in vivo detected by radiography 8 weeks after implantation

图7 植入8周后JDBM-DCPD组与JDBM-MgF2组大鼠骨缺损区的组织学观察Note:A. H-E staining of the bone defect areas (above×200, below×400). B. Expression of OCN in the defect areas (immunohistochemical staining, ×400). C. Expression of CD31 in the defect areas (immunohistochemical staining, ×400). The white arrows represent the new bone tissues and the red arrows represent the new blood vessels.

Fig 7 Histological observation of the bone defect areas in JDBM-DCPD group and JDBM-MgF2 group rats 8 weeks after implantation

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