紫外线照射对两种纳米形貌钛种植体表面巨噬细胞生物学行为及炎症细胞因子分泌的影响

Effects of two nanotopographies of ultraviolet-treated titanium implant surface on macrophage behaviour and inflammatory cytokines secretion

目的研究紫外线照射对两种纳米形貌钛种植体表面巨噬细胞生物学行为及炎症细胞因子分泌的影响,为紫外线照射在种植体改性方面的临床应用提供依据。方法钛试件经氢氟酸酸蚀或氢氟酸酸蚀+阳极氧化分别制备酸蚀组和氧化组钛试件各27个,场发射扫描电镜观察两组各1个试件的表面形貌。试件避光保存8周后,两组各取13个试件用紫外线照射48 h(即酸蚀+紫外线组和氧化+紫外线组),以未经紫外线照射的酸蚀组和氧化组钛试件作为对照。检测各组钛试件表面接触角;各组钛试件与小鼠巨噬细胞共培养4、24和72 h后,检测各组细胞黏附和增殖情况,扫描电镜观察细胞形态;液相芯片技术检测培养24和72 h时细胞上清液中肿瘤坏死因子α(tumor necrosis factor α,TNF-α)、单核细胞趋化蛋白1(monocyte chemotactic protein-1,MCP-1)和巨噬细胞炎症蛋白1α(macrophage inflammatory protein-1α,MIP-1α)的质量浓度。结果酸蚀组钛试件表面可见微米凹坑和纳米柱,氧化组钛试件表面可见微米凹坑和TiO2纳米管结构。酸蚀+紫外线组和氧化+紫外线组表面接触角(分别为20.2°±2.8°和0.0°±0.0°)均显著小于酸蚀组和氧化组(P<0.05)。培养4和24 h时各组巨噬细胞黏附和增殖差异均无统计学意义(P>0.05),培养72 h时酸蚀+紫外线组和氧化+紫外线组细胞增殖[A值分别为(0.92±0.13)和(1.10±0.08)]均分别显著大于酸蚀组和氧化组(P<0.05);两组TNF-α质量浓度[分别为(1.03±0.11)和(0.87±0.10)ng/L]、MCP-1质量浓度[分别为(301.7±50.3)和(240.8±18.7)ng/L]、MIP-1α质量浓度[分别为(224.9±30.6)和(233.9±14.9)ng/L]均分别显著小于酸蚀组和氧化组(P<0.05)。结论紫外线照射能增加两种纳米形貌钛试件亲水性,尤其是氧化+紫外线组。紫外线照射可促进两种纳米形貌钛表面巨噬细胞增殖、减轻炎症反应。

Objective To investigate the effects of two nanotopographies of ultraviolet (UV)-treated titanium surface on macrophage biological behaviour and inflammatory cytokines secretion, and to provide basis for clinical application of UV-treatment in dental implant modification. Methods Titanium disks were allocated into two groups. Samples in one group were acid-etched in hydrofluoric acid (Acid Ti group), and those in the other group were acid-etched and anodized (Anodization group) to form two nanotopographies respectively. The surface morphology was evaluated by field-emission scanning electron microscopy (FE-SEM). The samples were stored in the dark for 8 weeks. Thirteen samples from each group were exposed to UV-irradiation for 48 h (Acid Ti+UV group and Anodization+UV group), UV-untreated samples from Acid Ti and Anodization groups served as control. Hydrophilicity of samples was measured using contact angle measuring device. After 4, 24 and 72 h of incubation, macrophage cell adhesion and proliferation were conducted using cell counting kit-8. Cytokine/chemokine secretions [tumor necrosis factor-α(TNF-α), monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-1α(MIP-1α)] were measured from cell culture supernatants at 24 and 72 h using magnetic luminex assay. Cell morphology was examined using FE-SEM after 2 h of incubation. Results Micropitted/nanopillar and micropitted/nanotubular topographies were observed in Acid Ti group and Anodization group respectively. Contact angles in Acid Ti+UV and Anodization+UV groups (20.2°±2.8° and 0.0°±0.0°) were significantly smaller than those in the Acid Ti and Anodization groups (P<0.05). Cell adhesion and proliferation in all groups at 4 and 24 h showed no difference (P>0.05). Cell proliferation in Acid Ti+UV and Anodization+UV groups at 72 h were (0.92±0.13) and (1.10±0.08) respectively, which were significantly higher than those in Acid Ti and Anodization groups. TNF-α concentration in Acid Ti+UV and Anodization+UV groups at 72 h were (1.03±0.11) and (0.87±0.10) ng/L, MCP-1 were (301.7±50.3) and (240.8±18.7) ng/L, MIP-1α were (224.9±30.6) and (233.9±14.9) ng/L respectively, which were significantly lower than those in Acid Ti and Anodization groups (P<0.05). Conclusions UV treatment can increase hydrophilicity of two titanium surface topographies, especially of Anodization+UV group. UV-treated titanium surfaces can promote macrophage proliferation and reduce the inflammatory response in vitro.