目的:本研究的目的是研究AgNPs掺杂Ti6Al4V表面的生物相容性,通过评估人牙龈成纤维细胞(HGF)的活力和增殖率,作为种植在修饰表面上的种植体周围软组织的优势细胞。
方法:通过电化学沉积将8和30nm尺寸分布的AgNPs掺入到Ti6Al4V样品表面上,使用具有增加的AgNP浓度为100、200和300ppm的胶体银分散体。研究中包括一个对照和六个实验组:(i)对照(Ti6Al4V),(ii)8nm/100ppm,(iii)8nm/200ppm,(iv)8nm/300ppm,(v)30nm/100ppm,(vi)30nm/200ppm,(vii)30nm/300ppm。从牙周健康供体患者中分离人牙龈成纤维细胞(HGF)细胞原代培养物。AgNP掺杂的Ti-6Al-4V样品对HGF的细胞毒性通过MTT和BrdU分析测试进行评估。使用钙黄绿素AM和埃塞俄比亚同二聚体-EthD1荧光染色来确定活细胞和死细胞。通过SEM测定HGFs的形态和附着特性。
结果:EDX分析证实了样品上存在AgNP。MTT显示,与接种在对照圆盘上的细胞相比,接种在测试大小和所有浓度的AgNP样品圆盘上的细胞均表现出降低的细胞代谢活性。与对照组相比,两种大小的AgNPs的所有浓度均影响细胞增殖率,正如BrdU分析所揭示的。总的来说,改性Ti6Al4V表面的细胞毒性取决于细胞暴露时间。通过共聚焦显微镜观察证实了MTT和BrdU测定测试的结果。具体来说,大多数细胞在72h培养期间保持存活。SEM显微照片显示,相邻的细胞彼此形成键,创建共轭细胞的汇合层。
结论:本研究的结果表明,在100ppm浓度下用8nm和30nmAgNPs改性的Ti6Al4V表面,200ppm,和300ppm对HGF不产生任何严重的细胞毒性。HGFs增殖速率的初始阻滞在72小时恢复。这些结果,结合同一研究小组的研究,关于对常见牙周病原体的抗菌活性,建议AgNP掺杂的Ti6Al4V表面是用于植入物基台以预防植入物周围疾病的潜在候选者。
OBJECTIVE: To investigate the biocompatibility of silver nanoparticle (AgNP)-doped Ti-6Al-4V surfaces by evaluating the viability and proliferation rate of human gingival fibroblasts (HGFs)-as the dominant cells of peri-implant soft tissues-seeded on the modified surfaces.
METHODS: AgNPs (sizes 8 nm and 30 nm) were incorporated onto Ti-6Al-4V specimen surfaces via electrochemical deposition, using colloid silver dispersions with increasing AgNP concentrations of 100 ppm, 200 ppm, and 300 ppm. One control and six experimental groups were included in the study: (1) control (Ti-6Al-4V), (2) 8 nm/100 ppm, (3) 8 nm/200 ppm, (4) 8 nm/300 ppm, (5) 30 nm/100 ppm, (6) 30 nm/200 ppm, and (7) 30 nm/300 ppm. HGF cell primary cultures were isolated from periodontally healthy donor patients and cultured in direct contact with the group specimens for 24 and 72 hours. The cytotoxicity of AgNP-doped Ti-6Al-4V specimens toward HGF was assessed by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and BrdU (5-bromo-2\'-deoxyuridine) assay tests. Calcein AM and ethidium homodimer (EthD-1) fluorescent stains were used to determine the live and dead cells. The morphology and attachment properties of the HGFs were determined via scanning electron microscopy (SEM).
RESULTS: Energy dispersive x-ray (EDX) analysis confirmed the presence of AgNPs on the specimens. The MTT test revealed that AgNPs of both sizes and all concentrations presented a decreased cellular metabolic activity compared to the control discs. All concentrations of both sizes of AgNPs affected the cell proliferation rate compared to the control group, as revealed by the BrdU assay. Overall, cytotoxicity of the modified Ti-6Al-4V surfaces depended on cell exposure time. Observation via confocal microscopy confirmed the results of the MTT and BrdU assay tests. Specifically, most cells remained alive throughout the 72-hour culture period. SEM images revealed that adjacent cells form bonds with each other, creating confluent layers of conjugated cells.
CONCLUSIONS: The findings of the present study indicate that Ti-6Al-4V surfaces modified with 8 nm and 30 nm AgNPs at concentrations of 100 ppm, 200 ppm, and 300 ppm do not produce any serious cytotoxicity toward HGFs. The initial arrest of the HGF proliferation rate recovered at 72 hours. These results on the antibacterial activity against common periodontal pathogens, in combination with the results found in a previous study by the same research group, suggest that AgNP-doped Ti-6Al-4V surfaces are potential candidates for use in implant abutments for preventing peri-implant diseases.