A 3D-printed calcium polyphosphate/icariin bone scaffold induces osteogenic differentiation of bone marrow mesenchymal stem cells for treating bone defects

doi:10.3969/j.issn.2095-4344.1758

Abstract

Abstract:

BACKGROUND: With the rapid development of tissue engineering, seeking for optimal scaffold materials, osteogenic factors and abundant stem cells with strong differentiation and proliferation potential has been an issue of concern in the field of orthopedics.
OBJECTIVE: To explore the effect of calcium polyphosphate/icariin bone scaffold to induce osteogenic differentiation of bone marrow mesenchymal stem cells for the treatment of bone defects.
METHODS: The CPP/ICA composite bone scaffold was prepared using 3D printing technology. The rabbit bone marrow mesenchymal stem cells were isolated and cultured. Bone defect models were made in 36 New Zealand white rabbits, and equally randomized into control group and experimental group, followed by implantation of calcium polyphosphate/icariin composite bone scaffold. Afterwards, in the experimental group, 1.5 mL of bone marrow mesenchymal stem cells (1×10⁹/L) was injected through the femoral vein into the rabbits, while the rabbits in the control group were given the same amount of normal saline via the femoral vein. At 4, 8 and 12 weeks postoperatively, six rabbits from each group were taken to isolate and culture bone marrow mesenchymal stem cells. Transwell chamber assay was used to measure the migration ability of bone marrow mesenchymal stem cells. Fluorescence quantification-reverse transcriptase polymerase chain reaction was used to determine the type I collagen and CD44 mRNA levels in the cells. Rabbit femoral condyle specimens were taken, and X-ray films were used to observe the formation of epiphyses around the composite scaffold. Van-Gieson staining was used to observe the histological changes of bone tissues. Immunohistochemical staining was used to determine the level of epiphyseal nerve growth factors.
RESULTS AND CONCLUSION: The number of migrated bone marrow mesenchymal stem cells was significantly higher in the experimental group than the control group (P < 0.05). The type I collagen and CD44 mRNA levels in the cells were also higher in the experimental group than the control group (P < 0.05). Findings from X-ray examination and histological observation showed more osteophytes in the experimental group than the control group. The level of epiphyseal nerve growth factors in the experimental group was significantly higher than that in the control group (P < 0.05). To conclude, the calcium polyphosphate/icariin bone scaffold prepared by 3D printing technology can induce the osteogenic differentiation of bone marrow mesenchymal stem cells, and has a good effect in the treatment of bone defects.

Key words: femoral condyle, bone defect, calcium polyphosphate, icariin, composite bone scaffold, 3D printing technology, bone marrow mesenchymal stem cells, osteogenic differentiation

CLC Number:

Shi Yongxin, Pang Zengjin, Yang Mingzhi, Peng Lijun, Li Fuqin, Song Hui, Luo Fuwei. A 3D-printed calcium polyphosphate/icariin bone scaffold induces osteogenic differentiation of bone marrow mesenchymal stem cells for treating bone defects[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(21): 3309-3315.

Figures/Tables 2

2.1 骨髓间充质干细胞形态培养24 h见细胞贴壁生长，培养第3天细胞呈短梭形，散在分布，见图1A，培养第5天细胞呈集落样生长，数量明显增多，见图1B，培养第10天细胞呈明显的长梭形，集落面积增加，集落之间相互融合，见图1C，培养第14天时细胞达80%以上融合，单个细胞呈狭长梭形，有一定方向性，呈漩涡状排列，见图1D。第3代细胞呈梭形，呈漩涡状排列，见图1E。 2.2 骨髓间充质干细胞鉴定结果免疫组化染色显示，细胞阳性表达CD44，阴性表达CD34，表明培养细胞为骨髓间充质干细胞，见图2。 2.3 聚磷酸钙/淫羊藿苷复合骨支架的超微结构扫描电镜下可见淫羊藿苷均匀分布在聚磷酸钙支架表面，见图3。 2.4 两组大白兔X射线检查结果第4周，对照组复合骨支架和两端股骨界面清晰，无骨痂包绕，实验组复合骨支架和两端股骨界面模糊，骨支架旁侧有少量骨痂生长；第8周，对照组复合骨支架和两端股骨界面模糊，骨支架旁侧有少量骨痂生成，实验组复合骨支架和两端股骨界面模糊，骨支架旁侧有较多骨痂生长；第12周，对照组复合骨支架和两端股骨界面处有大量骨痂包裹，实验组复合骨支架周围完全被新生的骨痂组织包裹，见图4。 2.5 两组大白兔组织学观察(Van-Gieson染色)结果第4周，实验组骨支架和新生骨结合较紧密，骨支架边缘部分降解，对照组骨支架和新生骨界限明显，骨支架无明显降解；第8周，实验组骨支架和新生骨交界被新生骨小梁包绕，骨支架进一步降解，新生骨小梁沿骨支架降解形成的空隙向骨支架内部生长，对照组骨支架边缘部分降解，有少量新生骨小梁；第12周，实验组骨支架中央降解形成空隙，新生骨小梁沿骨支架降解空隙继续长入，对照组新生骨小梁少，骨支架降解缓慢，骨小梁长入骨支架空隙的范围比较局限，见图5。 2.6 两组大白兔骨髓间充质干细胞迁移数实验组大白兔骨髓间充质干细胞迁移数高于对照组(P < 0.05)，见表1和图6。 2.7 两组大白兔骨髓间充质干细胞Ⅰ型胶原蛋白、CD44 mRNA表达比较第4，8，12周时，实验组大白兔骨髓间充质干细胞Ⅰ型胶原蛋白、CD44 mRNA表达均高于对照组(P < 0.05)，见表2。 2.8 免疫组化染色检测骨痂神经生长因子水平第4，8，12周时，实验组大白兔骨痂神经生长因子水平均高于对照组(P < 0.05)，见表3和图7。"

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