Objective: To investigate the effects of three-dimensional (3D) printed Ti6Al4V-4Cu alloy on inflammation and osteogenic gene expression in mouse bone marrow mesenchymal stem cells (BMSCs) and mouse mononuclear macrophage line RAW264.7.
Methods: Ti6Al4V and Ti6Al4V-4Cu alloys were prepared by selective laser melting, and the extracts of the two materials were prepared according to the biological evaluation standard of medical devices. The effects of two kinds of extracts on the proliferation of mouse BMSCs and mouse RAW264.7 cells were detected by cell counting kit 8 method. After co-cultured with mouse BMSCs for 3 days, the expression of osteogenesis- related genes [collagen type Ⅰ (Col-Ⅰ), alkaline phosphatase (ALP), Runx family transcription factor 2 (Runx-2), osteoprotegerin (OPG), and osteopontin (OPN)] were detected by real-time fluorescence quantitative PCR. After co-cultured with mouse RAW264.7 cells for 1 day, the expressions of inflammation-related genes [interleukin 4 (IL-4) and nitric oxide synthase 2 (iNOS)] were detected by real-time fluorescence quantitative PCR, and the supernatants of the two groups were collected to detect the secretion of vascular endothelial growth factor a (VEGF-a) and bone morphogenetic protein 2 (BMP-2) by ELISA. The osteogenic conditioned medium were prepared with the supernatants of the two groups and co-cultured with BMSCs for 3 days. The expressions of osteogenesis-related genes (Col-Ⅰ, ALP, Runx-2, OPG, and OPN) were detected by real-time fluorescence quantitative PCR.
Results: Compared with Ti6Al4V alloy extract, Ti6Al4V-4Cu alloy extract had no obvious effect on the proliferation of BMSCs and RAW264.7 cells, but it could promote the expression of OPG mRNA in BMSCs, reduce the expression of iNOS mRNA in RAW264.7 cells, and promote the expression of IL-4 mRNA. It could also promote the secretions of VEGF-a and BMP-2 in RAW264.7 cells. Ti6Al4V-4Cu osteogenic conditioned medium could promote the expressions of Col-Ⅰ, ALP, Runx-2, OPG, and OPN mRNAs in BMSCs. The differences were all significant ( P<0.05).
Conclusion: 3D printed Ti6Al4V-4Cu alloy can promote RAW264.7 cells to secret VEGF-a and BMP-2 by releasing copper ions, thus promoting osteogenesis through bone immune regulation, which lays a theoretical foundation for the application of metal prosthesis.
目的: 探究 3D 打印 Ti6Al4V-4Cu 合金对小鼠 BMSCs 及小鼠单核巨噬细胞系 RAW264.7 炎症及成骨基因表达的影响。.
方法: 通过选择性激光熔融技术制备 Ti6Al4V 与 Ti6Al4V-4Cu 合金，依据医疗器械生物学评价标准制备两种材料浸提液。通过细胞计数试剂盒 8 法检测两种浸提液对小鼠 BMSCs、小鼠 RAW264.7 细胞增殖的影响。两种浸提液与小鼠 BMSCs 共培养 3 d 后，通过实时荧光定量 PCR 法检测成骨相关基因Ⅰ型胶原（collagen type Ⅰ，Col-Ⅰ）、ALP、Runx 家族转录因子 2（Runx family transcription factor 2，Runx-2）、骨保护素（osteoprotegerin，OPG）、骨桥蛋白（osteopontin，OPN）mRNA 的表达；与小鼠 RAW264.7 共培养 1 d 后，通过实时荧光定量 PCR 法检测炎症相关基因 IL-4、一氧化氮合成酶 2（nitric oxide synthase 2，iNOS）mRNA 的表达，并收集两组上清液通过 ELISA 法检测其中 VEGF-a、BMP-2 的分泌量。将两组上清液制备成成骨条件培养基，与 BMSCs 共培养 3 d，实时荧光定量 PCR 法检测成骨相关基因 Col-Ⅰ、ALP、Runx-2、OPG、OPN mRNA 的表达。.
结果: 与 Ti6Al4V 合金浸提液比较，Ti6Al4V-4Cu 合金浸提液对 BMSCs 及小鼠 RAW264.7 细胞的增殖影响不明显；但是可促进 BMSCs OPG mRNA 的表达，减少小鼠 RAW264.7 细胞炎症相关基因 iNOS mRNA 的表达、促进抑炎因子 IL-4 mRNA 的表达，还可促进小鼠 RAW264.7 细胞分泌 VEGF-a 及 BMP-2，而 Ti6Al4V-4Cu 成骨条件培养基又可促进 BMSCs 成骨相关基因 Col-Ⅰ、ALP、Runx-2、OPG、OPN mRNA 的表达，上述指标差异均有统计学意义（ P<0.05）。.
结论: 3D 打印 Ti6Al4V-4Cu 合金通过释放铜离子，促进 RAW264.7 细胞分泌 VEGF-a 及 BMP-2，从而通过骨免疫调节促进成骨发生，为金属假体的应用奠定了理论基础。.
Keywords: Three-dimensional printing; Ti6Al4V-4Cu alloy; bone immune regulation; osteogenic microenvironment.