Microstructure, properties and cytocompatibility evaluation of hydroxyapatite/Ti-13Nb-13Zr composites
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摘要: 为了改善Ti-13Nb-13Zr医用钛合金的生物活性与细胞相容性，利用放电等离子烧结(SPS)技术制备了Ti-13Nb-13Zr合金和羟基磷灰石(HA)含量5wt%的5HA/Ti-13Nb-13Zr复合材料并进行退火处理，研究了两种材料的显微组织、力学性能、表面润湿性、体外矿化行为及细胞增殖与凋亡等生物学性能。结果表明：合金主要由β-Ti和α-Ti相组成，复合材料由β-Ti、α-Ti、HA相及少量陶瓷反应相(Ca3(PO4)2、CaZrO3、CaO)组成，退火后部分初生α-Ti转变为β-Ti且组织更均匀，HA的加入会使得晶粒细化；退火后两种材料抗压强度、屈服强度、屈强比和弹性模量均略微下降；HA的加入提高了复合材料亲水性、类骨磷灰石形成能力、细胞增殖率并降低了细胞凋亡率；综合分析，退火后的5HA / Ti-13Nb-13Zr复合材料抗压强度、屈服强度和弹性模量分别为(1744±9) MPa、(1493±12) MPa和(43±1.6) GPa，具有优异的类骨磷灰石形成能力，同时细胞增殖率达到99.1%±0.8%，表明HA的加入明显提高了Ti-13Nb-13Zr合金的生物活性与细胞相容性。Abstract: In order to improve the bioactivity and cytocompatibility of Ti-13Nb-13Zr biomedical titanium alloy, Ti-13Nb-13Zr alloy and 5HA/Ti-13Nb-13Zr composite with hydroxyapatite (HA) content 5wt% were fabricated by spark plasma sintering (SPS) technique and then annealed. The microstructure, mechanical properties, surface wettability, in vitro mineralization behavior, cell proliferation and apoptosis of the two materials were investigated. The results show that the alloy is mainly composed of β-Ti and α-Ti phases, and the composite is mainly composed of β-Ti, α-Ti, HA phases and a small amount of ceramic reaction phases (Ca3(PO4)2, CaZrO3, CaO). After annealing, the part of α-Ti transforms to β-Ti and the microstructure is more uniform. The addition of HA can refine the grains to a certain extent. The compressive strength, yield strength, yield ratio and elastic modulus of the two materials decrease slightly after annealing. The HA not only strengthens the hydrophilicity and osteoapatite-like formation ability, but also increases cell viability rate and cell apoptosis rate of the composites. Comprehensive analysis, the compressive strength, yield strength and elastic modulus of the 5HA/Ti-13Nb-13Zr are (1744±9) MPa, (1493±12) MPa and (43±1.6) GPa, respectively. Moreover, the 5HA/Ti-13Nb-13Zr composite have excellent bone-like apatite formation ability and the cell viability reaches 99.1%±0.8%. It indicated that the addition of HA significantly improves the bioactivity and cytocompatibility of Ti-13Nb-13Zr alloy.
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图 1 Ti-13Nb-13Zr合金和5-羟基磷灰石(HA)/Ti-13Nb-13Zr复合材料退火前后的物相分析
Figure 1. Phase analysis of Ti-13Nb-13Zr alloy and 5-hydroxyapatite (HA)/Ti-13Nb-13Zr composite before and after annealing
TCP—Ca3(PO4)2
图 2 退火前后Ti-13Nb-13Zr合金和5HA/Ti-13Nb-13Zr复合材料的微观形貌：((a)、(b)) 烧结态合金；((c)、(d)) 退火态合金；((e)、(f)) 烧结态复合材料；((g)、(h)) 退火态复合材料
Figure 2. Microstructure of Ti-13Nb-13Zr alloy and 5HA/Ti-13Nb-13Zr composite before and after annealing: ((a), (b)) Sintered alloy; ((c), (d)) Annealed alloy; ((e), (f)) Sintered composite; ((g), (h)) Annealed composite
图 3 退火前后Ti-13Nb-13Zr合金和5HA/Ti-13Nb-13Zr复合材料的力学性能
Figure 3. Mechanical properties of Ti-13Nb-13Zr alloy and 5HA/Ti-13Nb-13Zr composite before and after annealing
Rm—Compressive strength; RP0.2—Yield strength
图 4 纯H2O和NaCl溶液在Ti-13Nb-13Zr合金和5HA/Ti-13Nb-13Zr复合材料的表面接触角
Figure 4. Contact angle of pure water and NaCl solution on the surface of Ti-13Nb-13Zr alloy and 5HA/Ti-13Nb-13Zr composite
图 5 退火态Ti-13Nb-13Zr合金和5HA/Ti-13Nb-13Zr复合材料在模拟人工体液(SBF)溶液中浸泡7天的微观形貌图：(a) 合金；(b) 复合材料
Figure 5. Microstructure of Ti-13Nb-13Zr alloy and 5HA/Ti-13Nb-13Zr composite after annealing immersed in simulated artificial body fluid (SBF) solution for 7 days: (a) Alloy; (b) Composite
图 6 退火态Ti-13Nb-13Zr合金和5HA/Ti-13Nb-13Zr复合材料的光密度(OD)值(a)和细胞增殖率(b) (*p< 0.05；**p< 0.01)
Figure 6. Optical density (OD) values (a) and cell viabilities (b) of Ti-13Nb-13Zr alloy and 5HA/Ti-13Nb-13Zr composite after annealing (*p< 0.05; **p< 0.01)
图 7 退火态Ti-13Nb-13Zr合金和5HA/Ti-13Nb-13Zr复合材料浸提液诱导人成骨肉瘤细胞(MG63 细胞)凋亡实验：(a)合金；(b)复合材料 (X轴、Y轴分别表示PI 和 Annexin V，一般认为PI单染的细胞是已经死亡的细胞，Annexin V单染的细胞是凋亡早期的细胞，而双染是凋亡中期的细胞；Q1区表示死细胞；Q2表示凋亡晚期细胞；Q3表示正常细胞；Q4表示凋亡早期细胞)
Figure 7. Apoptosis experiment of Human osteosarcoma cells (MG63 cells) induced by extraction of Ti-13Nb-13Zr alloy and 5HA/Ti-13Nb-13Zr composite after annealing: (a) Alloy; (b) Composite (X and Y axes indicate PI and annexin V respectively. It is generally accepted that the cells with PI mono-staining are dead cells, the cells with annexin V mono-staining are cells in the early apoptotic stage, and the cells with double staining are cells in the middle apoptotic stage; Q1 represents dead cells; Q2 represents late apoptotic cells; Q3 represents normal cells; Q4 represents early apoptotic cells)
图 8 Ti-13Nb-Zr合金和5HA/Ti-13Nb-13Zr复合材料诱导MG63细胞凋亡率统计图 (*p< 0.05；**p< 0.01)
Figure 8. Cell apoptosis rate statistical graph of MG63 cells induced by extraction of Ti-13Nb-Zr alloy and 5HA/Ti-13Nb-13Zr composite (*p< 0.05; **p< 0.01)
表 1 两组材料在SBF溶液浸泡后的表面成分分析
Table 1. Surface composition analysis of two groups of materials soaked in SBF solution
Material Element/at% Ti O Na Ca P Cl K Point A 1.1 19.6 39.7 — — 38.3 1.1 Point B 0.6 46.6 15.7 10.1 6.8 13.3 7.5 
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