Protein array method for assessing in vitro biomaterial-induced cytokine expression.

Abstract

This study demonstrates the feasibility of a cytokine-based in vitro test for biomaterials. The combination of monocyte culture and protein array technology tested in this study permitted the detection of subtle changes in cytokine expression following an exposure to titanium (Ti) particles. However, a broader range of materials and sample configurations must be examined before these promising results can be translated into a reliable and predictive in vitro biomaterials testing protocol. Modified glass slides were robotically printed with eight identical arrays consisting of capture antibodies against four mouse cytokines [IL-6, TNF-alpha, MIP-2, TGF-beta1] and two positive and two negative detection controls. RAW 264.7 mouse monocytes seeded into six-well plates at 10(5)cells/well were treated with either sterilized Ti particles (test biomaterial), or lipopolysaccharide (LPS; positive control), or untreated (negative control). Aliquots (80 microl) of culture medium collected at 1, 6, 24, 48, and 72 h were applied to the protein arrays for simultaneous sandwich fluoroimmunoassay, followed by imaging the fluorescent intensities on a conventional microarray scanner. LPS induced the release of all four cytokines between 1 and 6h treatment periods, whereas Ti induction of cytokines showed a gradual and subtle increase in cytokine expression for >24 h. Among the four cytokines assayed, TNF-alpha and MIP-2 were most prominently expressed, while IL-6 was slightly elevated and TGF-beta1 was undetected above background.