使用微流控装置实时检测兔骨膜细胞中的抗生素细胞毒性，并与传统培养方法进行比较。
Real-time detection of antibiotics cytotoxicity in rabbit periosteal cells using microfluidic devices with comparison to conventional culture assays.
机构信息
Bone and Joint Research Center, Department of Orthopedic Surgery, Chang Gung Memorial Hospital-Linkou and University College of Medicine, 5th, Fu-Shin Street, Kweishan Dist, Taoyuan, 333, Taiwan, Republic of China.
Graduate Institute of Medical Mechatronics, Chang Gung University, Taiwan, Republic of China.
出版信息
BMC Musculoskelet Disord. 2019 Jul 26;20(1):339. doi: 10.1186/s12891-019-2705-y.
BACKGROUND
Local antibiotic application has been widely used in orthopedic surgery. The dose-related toxicity of antibiotics towards periosteal tissues and resulting effects on osteogenic expression are yet to be studied.
METHODS
Periosteal cells harvested from the medial tibia of New Zealand White rabbits were used. A seeding density of 5 × 10 cells/cm was determined to be optimal for testing in the pilot study; the cells were cultured in xCELLigence 96-well plates. Microfluidic impedance analyzers were used to monitor cellular proliferation in microfluidic culture systems with exposure to three different concentrations (10 μg/mL, 100 μg/mL, and 1000 μg/mL) of cefazolin, ciprofloxacin, and vancomycin, respectively. The correlation of cell index at day 7 with optical density values from WST-1 assays using conventional cultures was evaluated by calculating the Pearson's coefficient. RNA analysis was performed to investigate the expression of osteogenic markers in the cultured cells, including core-binding factor alpha 1 (Cbfa1), osteopontin (OPN), and osteopontin promoter (OPNp), relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the endogenous control.
RESULTS
A significant dose-related inhibition of cell index was found for all the 3 antibiotics, whereas the WST-1 assays showed a significant dose-related inhibition of cellular proliferation only at a high dose of cefazolin (1000 μg/mL) and medium-to-high dose of ciprofloxacin (100 μg/mL and 1000 μg/mL). Pearson's coefficient analysis indicated a high correlation between the cell index and optical density values of WST-1 assays only for medium and high doses of ciprofloxacin (100 μg/mL and 1000 μg/mL); a moderate correlation was seen for cefazolin, and a low dose of ciprofloxacin (10 μg/mL). RNA analysis confirmed significant dose-related inhibition of cfba1, OPN, and OPNp expression by all three antibiotics.
CONCLUSION
With optimal seeding amounts, rabbit periosteal cells can be dynamically monitored in the xCELLigence microfluidic system. Dose-related inhibition of cellular proliferation and osteogenic expression was found after exposure to cefazolin and ciprofloxacin. By providing real-time detection and exhibiting comparable correlation, microfluidic impedance-based analyzer is a feasible alternative to the conventional WST-1 assays.
背景
局部应用抗生素在骨科手术中得到了广泛应用。然而，抗生素对骨膜组织的剂量相关毒性及其对成骨表达的影响仍有待研究。
方法
从新西兰白兔的胫骨内侧采集骨膜细胞。在初步研究中确定最佳的接种密度为 5×10 个细胞/cm，将细胞接种于 xCELLigence 96 孔板中。使用微流控阻抗分析仪监测在三种不同浓度（10μg/mL、100μg/mL 和 1000μg/mL）头孢唑林、环丙沙星和万古霉素暴露下的细胞在微流控培养系统中的增殖情况。通过计算 Pearson 系数，评估微流控培养系统中细胞指数与传统培养中 WST-1 检测的光密度值之间的相关性。通过 RNA 分析，检测培养细胞中成骨标志物核心结合因子α 1（Cbfa1）、骨桥蛋白（OPN）和骨桥蛋白启动子（OPNp）的表达，以甘油醛-3-磷酸脱氢酶（GAPDH）作为内参。
结果
所有三种抗生素均显示出显著的剂量相关的细胞指数抑制，而 WST-1 检测仅在头孢唑林高剂量（1000μg/mL）和中高剂量（100μg/mL 和 1000μg/mL）时显示出显著的剂量相关的细胞增殖抑制。Pearson 系数分析表明，只有中高剂量（100μg/mL 和 1000μg/mL）的环丙沙星与 WST-1 检测的光密度值具有高度相关性，头孢唑林具有中度相关性，低剂量（10μg/mL）的环丙沙星具有低度相关性。RNA 分析证实，三种抗生素均显著抑制 Cbfα1、OPN 和 OPNp 的表达。
结论
在最佳接种量下，兔骨膜细胞可在 xCELLigence 微流控系统中进行动态监测。头孢唑林和环丙沙星暴露后，发现细胞增殖和成骨表达呈剂量相关抑制。微流控阻抗分析仪通过提供实时检测并表现出可比较的相关性，是一种可行的替代传统 WST-1 检测的方法。
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