Real-time detection of antibiotics cytotoxicity in rabbit periosteal cells using microfluidic devices with comparison to conventional culture assays.

Abstract:

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 × 103 cells/cm2 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.

authors

Chiu CH,Lei KF,Chan YS,Ueng SWN,Chen AC

doi

10.1186/s12891-019-2705-y

subject

Has Abstract

pub_date

2019-07-26 00:00:00

pages

339

issue

1

issn

1471-2474

pii

10.1186/s12891-019-2705-y

journal_volume

20

pub_type

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