[Influence of antimicrobial peptide biofunctionalized TiO2 nanotubes on the biological behavior of human keratinocytes and its antibacterial effect].

Objective: To fabricate TiO2 nanotube material functionalized by antimicrobial peptide LL-37, and to explore its effects on biological behaviors such as adhesion and migration of human keratinocytes (HaCaT) and its antibacterial properties. Methods: The TiO2 nanotube array (NT) was constructed on the surface of polished titanium (PT) by anodization, and the antimicrobial peptide LL-37 was loaded on the surface of TiO2 nanotube (LL-37/NT) by physical adsorption. Three samples were selected by simple random sampling in each group. Surface morphology, roughness, hydrophilicity and release characteristics of LL-37 of the samples were analyzed with a field emission scanning electron microscope, an atomic force microscope, a contact angle measuring device and a microplate absorbance reader. HaCaT cells were respectively cultured on the surface of three groups of titanium samples. Each group had 3 replicates. The morphology of cell was observed by field emission scanning electron microscope. The number of cell adhesion was observed by cellular immunofluorescence staining. Cell counting kit-8 (CCK-8) assay was used to detect cell proliferation. Wound scratch assay was used to observe the migration of HaCaT. The above experiments were used to evaluate the effect of each group on the biological behavior of HaCaT cells. To evaluate their antibacterial effects, Porphyromonas gingivalis (Pg) was respectively inoculated on the surface of three groups of titanium samples. Each group had 3 replicates. The morphology of bacteria was observed by field emission scanning electron microscope. Bacterial viability was determined by live/dead bacterial staining. Results: A uniform array of nanotubes could be seen on the surface of titanium samples in LL-37/NT group, and the top of the tube was covered with granular LL-37. Compared with PT group [the roughness was (2.30±0.18) nm, the contact angle was 71.8°±1.7°], the roughness [(20.40±3.10) and (19.10±4.11) nm] and hydrophilicity (the contact angles were 22.4°±3.1° and 25.3°±2.2°, respectively) of titanium samples increased in NT and LL-37/NT group (P<0.001). The results of in vitro release test showed that the release of antimicrobial peptide LL-37 was characterized by early sudden release (1-4 h) and long-term (1-7 d) slow release. With the immunofluorescence, more cell attachment was found on NT and LL-37/NT than that on PT at the first 0.5 and 2.0 h of culture (P<0.05). The results of CCK-8 showed that there was no significant difference in the proliferation of cells among groups at 1, 3 and 5 days after culture. Wound scratch assay showed that compared with PT and NT group, the cell moved fastest on the surface of titanium samples in LL-37/NT group at 24 h of culture [(96.4±4.9)%] (F=35.55, P<0.001). A monolayer cells could be formed and filled with the scratch in 24 h at LL-37/NT group. The results of bacterial test in vitro showed that compared with the PT group, the bacterial morphology in the NT and LL-37/NT groups was significantly wrinkled, and obvious bacterial rupture could be seen on the surface of titanium samples in LL-37/NT group. The results of bacteria staining showed that the green fluorescence intensity of titanium samples in LL-37/NT group was the lowest in all groups (F=66.54,P<0.001). Conclusions: LL-37/NT is beneficial to the adhesion and migration of HaCaT cells and has excellent antibacterial properties, this provides a new strategy for the optimal design of implant neck materials.目的： 探讨抗菌肽生物功能化TiO2纳米管的抗菌性能及其对人角质形成细胞黏附、迁移等生物学行为的影响。 方法： 采用阳极氧化法在光滑钛片（光滑钛组）表面构建TiO2纳米管阵列（纳米管组），通过物理吸附方式将抗菌肽（LL-37）加载至TiO2纳米管表面（抗菌肽组）。每组采用简单随机抽样法抽取3个试样，借助场发射扫描电镜、原子力显微镜、接触角测量仪、荧光标记和荧光酶标仪观察各组试样表面形貌、粗糙度、亲水性以及抗菌肽释放特点。将人角质形成细胞分别培养于3组钛试样表面，每组设置3个重复，场发射扫描电镜观察细胞黏附形态，细胞免疫荧光染色观察黏附数量；细胞计数试剂盒检测细胞增殖能力；划痕实验分析细胞迁移特点，评价各组钛试样对HaCaT细胞生物学行为的影响。将牙龈卟啉单胞菌（Porphyromonas gingivalis，Pg）接种至3组钛试样表面，每组设置3个重复，场发射扫描电镜观察细菌形态，活死细菌染色法测定细菌活力，评价各组钛试样对Pg的抑制作用。 结果： 抗菌肽组试样表面可见均匀排列的纳米管阵列，管口处有颗粒状抗菌肽覆盖。纳米管组和抗菌肽组钛试样表面粗糙度［分别为（20.40±3.10）和（19.10±4.11）nm］和亲水性（接触角分别为22.4°±3.1°和25.3°±2.2°）均比光滑钛组［粗糙度为（2.30±0.18）nm，接触角为71.8°±1.7°］显著增加（P<0.05）。抗菌肽的释放表现为早期的突释（1~4 h）和长期（1~7 d）的缓释过程。免疫荧光显示，HaCaT细胞接种至钛试样表面0.5和2.0 h后，纳米管和抗菌肽组钛试样表面细胞黏附数量均比光滑钛组显著增加（P<0.05）；细胞计数结果显示，接种1、3及5 d后各组细胞增殖活性差异均无统计学意义（P>0.05）；划痕实验显示，与光滑钛和纳米管组相比，划痕后24 h时抗菌肽组钛试样表面愈合率最高［（96.4±4.9）%］（F=35.55，P<0.001），抗菌肽组钛试样24 h即可形成单层细胞并填充划痕。体外细菌共培养实验显示，相比于光滑钛组，纳米管和抗菌肽组钛试样表面细菌皱缩明显，抗菌肽组钛试样表面可见明显的菌体破裂；活死细菌染色显示，抗菌肽组钛试样表面绿色荧光强度为各组最低（F=66.54，P<0.001）。 结论： 抗菌肽生物功能化TiO2纳米管材料具备良好的抗菌性能，有利于人角质形成细胞的黏附及迁移。.