Three-dimensional printed Ti6Al4V-4Cu alloy promotes osteogenic gene expression through bone immune regulation_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHANG Chenke ^1,2 , LU Yanjin ³ , GUO Yupeng ² , CHEN Wan ² , TANG Hong ² , LI Huaisheng ² , TANG Kanglai ² ,  HE Qingyi ¹

1. Department of Orthopedics, the First Affiliated Hospital of Army Medical University of Chinese PLA, Chongqing, 400038, P.R.China;
2. Department of Orthopedics/Sports Medicine Center, the First Affiliated Hospital of Army Medical University of Chinese PLA, Chongqing, 400038, P.R.China;
3. Fujian Institute of Research on the Structure of Matter, Fuzhou Fujian, 350002, P.R.China;

Corresponding author：

HE Qingyi, Email: qingyihe18625@163.com

Keywords：

Three-dimensional printing; Ti6Al4V-4Cu alloy; osteogenic microenvironment; bone immune regulation

DOI：

10.7507/1002-1892.201912139

Video：

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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.

Citation： ZHANG Chenke, LU Yanjin, GUO Yupeng, CHEN Wan, TANG Hong, LI Huaisheng, TANG Kanglai, HE Qingyi. Three-dimensional printed Ti6Al4V-4Cu alloy promotes osteogenic gene expression through bone immune regulation. Chinese Journal of Reparative and Reconstructive Surgery, 2020, 34(9): 1170-1176. doi: 10.7507/1002-1892.201912139 Copy

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2.	Dimitriou R, Mataliotakis GI, Angoules AG, et al. Complications following autologous bone graft harvesting from the iliac crest and using the RIA: a systematic review. Injury, 2011, Suppl 2: S3-S15.
3.	万志鹏, 蒋文涛, 王宠, 等. 三维打印 Ti-6Al-4V 合金孔洞几何特征与空间分布研究. 生物医学工程学杂志, 2017, 34(6): 876-882.
4.	Hanawa T. Titanium-tissue interface reaction and its control with surface treatment. Front Bioeng Biotechnol, 2019, 7: 170.
5.	Li Y, Ding Y, Munir K, et al. Novel β-Ti35Zr28Nb alloy scaffolds manufactured using selective laser melting for bone implant applications. Acta Biomater, 2019, 87: 273-284.
6.	Wang CC, Hu HX, Li ZP, et al. Enhanced osseointegration of titanium alloy implants with laser microgrooved surfaces and graphene oxide coating. ACS Appl Mater Interfaces, 2019, 11(43): 39470-39483.
7.	Bosshardt DD, Chappuis V, Buser D. Osseointegration of titanium, titanium alloy and zirconia dental implants: current knowledge and open questions. Periodontol, 2000, 2017, 73(1): 2-40.
8.	Li RC, Deng CJ, Li XX, et al. Copper-incorporated bioactive glass-ceramics inducing anti-inflammatory phenotype and regeneration of cartilage/bone interface. Theranostics, 2019, 9(21): 6300-6313.
9.	彭建强, 易志新, 武明鑫, 等. 氧化应激活化 RAW264.7 巨噬细胞对 MC3T3-E1 成骨细胞迁移、增殖及成骨基因表达影响的实验研究. 中国修复重建外科杂志, 2016, 30(9): 1146-1152.
10.	Chen XN, Wang ML, Chen FY, et al. Correlations between macrophage polarization and osteoinduction of porous calcium phosphate ceramics. Acta Biomater, 2020, 103: 318-332.
11.	Julier Z, Park AJ, Briquez PS, et al. Promoting tissue regeneration by modulating the immune system. Acta Biomater, 2017, 53: 13-28.
12.	Chen Z, Klein T, Murray RZ, et al. Osteoimmunomodulation for the development of advanced bone biomaterials. Materials Today, 2016, 19(6): 304-321.
13.	Fraga CG. Relevance, essentiality and toxicity of trace elements in human health. Mol Aspects Med, 2005, 26(4-5): 235-244.
14.	李永乐, 何强, 范先东, 等. 人脐血对兔骨折愈合期间全血微量元素含量的影响. 中国修复重建外科杂志, 2013, 27(6): 673-679.
15.	Mir E, Hossein-Nezhad A, Bahrami A, et al. Adequate serum copper concentration could improve bone density, postpone bone loss and protect osteoporosis in women. Iranian Journal of Public Health, 2007, 36(2): 24-29.
16.	Djoko KY, Ong CI, Walker MJ, et al. The role of copper and zinc toxicity in innate immune defense against bacterial pathogens. J Biol Chem, 2015, 290(31): 18954-18961.
17.	Tapiero H, Townsend DM, Tew KD. Trace elements in human physiology and pathology. Copper. Biomed Pharmacother, 2003, 57(9): 386-398.
18.	Ren L, Wong HM, Yan CH, et al. Osteogenic ability of Cu-bearing stainless steel. J Biomed Mater Res B Appl Biomater, 2015, 103(7): 1433-1444.
19.	Liu F, Wang F, Shimizu T, et al. Hydroxyapatite formation on oxide films containing Ca and P by hydrothermal treatment. Ceramics International, 2006, 32(5): 527-531.
20.	Guo S, Lu YJ, Wu SQ, et al. Preliminary study on the corrosion resistance, antibacterial activity and cytotoxicity of selective-laser-melted Ti6Al4V-xCu alloys. Mater Sci Eng C Mater Biol Appl, 2017, 72: 631-640.
21.	Bohner M, Miron RJ. A proposed mechanism for material-induced heterotopic ossification. Materials Today, 2019, 22: 132-141.
22.	康明, 黄杰华, 张理选, 等. 壳聚糖/胡须/磷酸钙骨水泥复合生物材料的力学性能及对诱导多能干细胞成骨潜能的影响. 中国修复重建外科杂志, 2018, 32(7): 959-967.
23.	邓廉夫, 燕宇飞. 骨修复材料的研究现状与进展. 中国修复重建外科杂志, 2018, 32(7): 815-820.
24.	满星云, 索海瑞, 刘家利, 等. 基于三维打印的磷酸三钙骨组织工程支架烧结工艺研究. 生物医学工程学杂志, 2020, 37(1): 112-118.
25.	Chen XN, Wang J, Chen Y, et al. Roles of calcium phosphate-mediated integrin expression and MAPK signaling pathways in the osteoblastic differentiation of mesenchymal stem cells. J Mater Chem B, 2016, 4(13): 2280-2289.
26.	Pajarinen J, Lin T, Gibon E, et al. Mesenchymal stem cell-macrophage crosstalk and bone healing. Biomaterials, 2019, 196: 80-89.
27.	Hotchkiss KM, Reddy GB, Hyzy SL, et al. Titanium surface characteristics, including topography and wettability, alter macrophage activation. Acta Biomater, 2016, 31: 425-434.
28.	Luo J, Ding X, Song W, et al. Inducing macrophages M2 polarization by dexamethasone laden mesoporous silica nanoparticles from titanium implant surface for enhanced osteogenesis. Acta Metallurgica Sinica (English Letters), 2019, 10: 1253-1260.
29.	Liu W, Li J, Cheng M, et al. Zinc-modified sulfonated polyetheretherketone surface with immunomodulatory function for guiding cell fate and bone regeneration. Adv Sci (Weinh), 2018, 5(10): 1800749.
30.	Shi M, Chen Z, Farnaghi S, et al. Copper-doped mesoporous silica nanospheres, a promising immunomodulatory agent for inducing osteogenesis. Acta Biomater, 2016, 30: 334-344.
31.	Zhang RR, Liu XJ, Xiong ZY, et al. The immunomodulatory effects of Zn-incorporated micro/nanostructured coating in inducing osteogenesis. Artif Cells Nanomed Biotechnol, 2018, 46(sup1): 1123-1130.
32.	Zhang XF, Chen QP, Mao XL. Magnesium enhances osteogenesis of BMSCs by tuning osteoimmunomodulation. Biomed Res Int, 2019, 2019: 7908205.
33.	Honda Y, Anada T, Kamakura S, et al. Elevated extracellular calcium stimulates secretion of bone morphogenetic protein 2 by a macrophage cell line. Biochem Biophys Res Commun, 2006, 345(3): 1155-1160.
34.	Chen ZT, Wu CT, Gu WY, et al. Osteogenic differentiation of bone marrow MSCs by β-tricalcium phosphate stimulating macrophages via BMP2 signalling pathway. Biomaterials, 2014, 35(5): 1507-1518.
35.	Champagne CM, Takebe J, Offenbacher S, et al. Macrophage cell lines produce osteoinductive signals that include bone morphogenetic protein-2. Bone, 2002, 30(1): 26-31.
36.	Freytes DO, Kang JW, Marcos-Campos I, et al. Macrophages modulate the viability and growth of human mesenchymal stem cells. J Cell Biochem, 2013, 114(1): 220-229.
37.	Zhang J, Wu H, He F, et al. Concentration-dependent osteogenic and angiogenic biological performances of calcium phosphate cement modified with copper ions. Mater Sci Eng C Mater Biol Appl, 2019, 99: 1199-1212.
38.	Weng L, Boda SK, Teusink MJ, et al. Binary doping of strontium and copper enhancing osteogenesis and angiogenesis of bioactive glass nanofibers while suppressing osteoclast activity. ACS Appl Mater Interfaces, 2017, 9(29): 24484-24496.
39.	Li H, Li J, Jiang J, et al. An osteogenesis/angiogenesis-stimulation artificial ligament for anterior cruciate ligament reconstruction. Acta Biomaterialia, 2017, 54: 399-410.
40.	Rigiracciolo DC, Scarpelli A, Lappano R, et al. Copper activates HIF-1alpha/GPER/VEGF signalling in cancer cells. Oncotarget, 2015, 6(33): 34158-34177.

1. Wu XC, Stroll SI, Lantigua D, et al. Eggshell particle-reinforced hydrogels for bone tissue engineering: an orthogonal approach. Biomater Sci, 2019, 7(7): 2675-2685.
2. Dimitriou R, Mataliotakis GI, Angoules AG, et al. Complications following autologous bone graft harvesting from the iliac crest and using the RIA: a systematic review. Injury, 2011, Suppl 2: S3-S15.
3. 万志鹏, 蒋文涛, 王宠, 等. 三维打印 Ti-6Al-4V 合金孔洞几何特征与空间分布研究. 生物医学工程学杂志, 2017, 34(6): 876-882.
4. Hanawa T. Titanium-tissue interface reaction and its control with surface treatment. Front Bioeng Biotechnol, 2019, 7: 170.
5. Li Y, Ding Y, Munir K, et al. Novel β-Ti35Zr28Nb alloy scaffolds manufactured using selective laser melting for bone implant applications. Acta Biomater, 2019, 87: 273-284.
6. Wang CC, Hu HX, Li ZP, et al. Enhanced osseointegration of titanium alloy implants with laser microgrooved surfaces and graphene oxide coating. ACS Appl Mater Interfaces, 2019, 11(43): 39470-39483.
7. Bosshardt DD, Chappuis V, Buser D. Osseointegration of titanium, titanium alloy and zirconia dental implants: current knowledge and open questions. Periodontol, 2000, 2017, 73(1): 2-40.
8. Li RC, Deng CJ, Li XX, et al. Copper-incorporated bioactive glass-ceramics inducing anti-inflammatory phenotype and regeneration of cartilage/bone interface. Theranostics, 2019, 9(21): 6300-6313.
9. 彭建强, 易志新, 武明鑫, 等. 氧化应激活化 RAW264.7 巨噬细胞对 MC3T3-E1 成骨细胞迁移、增殖及成骨基因表达影响的实验研究. 中国修复重建外科杂志, 2016, 30(9): 1146-1152.
10. Chen XN, Wang ML, Chen FY, et al. Correlations between macrophage polarization and osteoinduction of porous calcium phosphate ceramics. Acta Biomater, 2020, 103: 318-332.
11. Julier Z, Park AJ, Briquez PS, et al. Promoting tissue regeneration by modulating the immune system. Acta Biomater, 2017, 53: 13-28.
12. Chen Z, Klein T, Murray RZ, et al. Osteoimmunomodulation for the development of advanced bone biomaterials. Materials Today, 2016, 19(6): 304-321.
13. Fraga CG. Relevance, essentiality and toxicity of trace elements in human health. Mol Aspects Med, 2005, 26(4-5): 235-244.
14. 李永乐, 何强, 范先东, 等. 人脐血对兔骨折愈合期间全血微量元素含量的影响. 中国修复重建外科杂志, 2013, 27(6): 673-679.
15. Mir E, Hossein-Nezhad A, Bahrami A, et al. Adequate serum copper concentration could improve bone density, postpone bone loss and protect osteoporosis in women. Iranian Journal of Public Health, 2007, 36(2): 24-29.
16. Djoko KY, Ong CI, Walker MJ, et al. The role of copper and zinc toxicity in innate immune defense against bacterial pathogens. J Biol Chem, 2015, 290(31): 18954-18961.
17. Tapiero H, Townsend DM, Tew KD. Trace elements in human physiology and pathology. Copper. Biomed Pharmacother, 2003, 57(9): 386-398.
18. Ren L, Wong HM, Yan CH, et al. Osteogenic ability of Cu-bearing stainless steel. J Biomed Mater Res B Appl Biomater, 2015, 103(7): 1433-1444.
19. Liu F, Wang F, Shimizu T, et al. Hydroxyapatite formation on oxide films containing Ca and P by hydrothermal treatment. Ceramics International, 2006, 32(5): 527-531.
20. Guo S, Lu YJ, Wu SQ, et al. Preliminary study on the corrosion resistance, antibacterial activity and cytotoxicity of selective-laser-melted Ti6Al4V-xCu alloys. Mater Sci Eng C Mater Biol Appl, 2017, 72: 631-640.
21. Bohner M, Miron RJ. A proposed mechanism for material-induced heterotopic ossification. Materials Today, 2019, 22: 132-141.
22. 康明, 黄杰华, 张理选, 等. 壳聚糖/胡须/磷酸钙骨水泥复合生物材料的力学性能及对诱导多能干细胞成骨潜能的影响. 中国修复重建外科杂志, 2018, 32(7): 959-967.
23. 邓廉夫, 燕宇飞. 骨修复材料的研究现状与进展. 中国修复重建外科杂志, 2018, 32(7): 815-820.
24. 满星云, 索海瑞, 刘家利, 等. 基于三维打印的磷酸三钙骨组织工程支架烧结工艺研究. 生物医学工程学杂志, 2020, 37(1): 112-118.
25. Chen XN, Wang J, Chen Y, et al. Roles of calcium phosphate-mediated integrin expression and MAPK signaling pathways in the osteoblastic differentiation of mesenchymal stem cells. J Mater Chem B, 2016, 4(13): 2280-2289.
26. Pajarinen J, Lin T, Gibon E, et al. Mesenchymal stem cell-macrophage crosstalk and bone healing. Biomaterials, 2019, 196: 80-89.
27. Hotchkiss KM, Reddy GB, Hyzy SL, et al. Titanium surface characteristics, including topography and wettability, alter macrophage activation. Acta Biomater, 2016, 31: 425-434.
28. Luo J, Ding X, Song W, et al. Inducing macrophages M2 polarization by dexamethasone laden mesoporous silica nanoparticles from titanium implant surface for enhanced osteogenesis. Acta Metallurgica Sinica (English Letters), 2019, 10: 1253-1260.
29. Liu W, Li J, Cheng M, et al. Zinc-modified sulfonated polyetheretherketone surface with immunomodulatory function for guiding cell fate and bone regeneration. Adv Sci (Weinh), 2018, 5(10): 1800749.
30. Shi M, Chen Z, Farnaghi S, et al. Copper-doped mesoporous silica nanospheres, a promising immunomodulatory agent for inducing osteogenesis. Acta Biomater, 2016, 30: 334-344.
31. Zhang RR, Liu XJ, Xiong ZY, et al. The immunomodulatory effects of Zn-incorporated micro/nanostructured coating in inducing osteogenesis. Artif Cells Nanomed Biotechnol, 2018, 46(sup1): 1123-1130.
32. Zhang XF, Chen QP, Mao XL. Magnesium enhances osteogenesis of BMSCs by tuning osteoimmunomodulation. Biomed Res Int, 2019, 2019: 7908205.
33. Honda Y, Anada T, Kamakura S, et al. Elevated extracellular calcium stimulates secretion of bone morphogenetic protein 2 by a macrophage cell line. Biochem Biophys Res Commun, 2006, 345(3): 1155-1160.
34. Chen ZT, Wu CT, Gu WY, et al. Osteogenic differentiation of bone marrow MSCs by β-tricalcium phosphate stimulating macrophages via BMP2 signalling pathway. Biomaterials, 2014, 35(5): 1507-1518.
35. Champagne CM, Takebe J, Offenbacher S, et al. Macrophage cell lines produce osteoinductive signals that include bone morphogenetic protein-2. Bone, 2002, 30(1): 26-31.
36. Freytes DO, Kang JW, Marcos-Campos I, et al. Macrophages modulate the viability and growth of human mesenchymal stem cells. J Cell Biochem, 2013, 114(1): 220-229.
37. Zhang J, Wu H, He F, et al. Concentration-dependent osteogenic and angiogenic biological performances of calcium phosphate cement modified with copper ions. Mater Sci Eng C Mater Biol Appl, 2019, 99: 1199-1212.
38. Weng L, Boda SK, Teusink MJ, et al. Binary doping of strontium and copper enhancing osteogenesis and angiogenesis of bioactive glass nanofibers while suppressing osteoclast activity. ACS Appl Mater Interfaces, 2017, 9(29): 24484-24496.
39. Li H, Li J, Jiang J, et al. An osteogenesis/angiogenesis-stimulation artificial ligament for anterior cruciate ligament reconstruction. Acta Biomaterialia, 2017, 54: 399-410.
40. Rigiracciolo DC, Scarpelli A, Lappano R, et al. Copper activates HIF-1alpha/GPER/VEGF signalling in cancer cells. Oncotarget, 2015, 6(33): 34158-34177.

Chinese Journal of Reparative and Reconstructive Surgery

Three-dimensional printed Ti6Al4V-4Cu alloy promotes osteogenic gene expression through bone immune regulation

Abstract Full text Figures/Tables Video References Cited by

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