The Quantification of Inflammatory Cellular Responses Using Real-Time Polymerase Chain Reaction (RT-PCR)

LeeAnn O. Bailey, Newell R. Washburn, Carl G. Simon Jr., Edward S. Chan, 

Francis W. Wang

Biomaterials Group, Polymers Division, National Institute of Standards and Technology, Gaithersburg, MD. 20899-8545

The introduction of tissue engineering approaches for the repair and replacement of human body components extends the application and importance of synthetic materials (biomaterials). Implanted biomaterials frequently evoke inflammatory responses, which are quite complex and not well defined at present. The goals of this work are to develop improved measurement methods for the quantification of cellular inflammatory responses to biomaterials and to obtain data that culminates in an enhanced understanding of the ways in which the body responds to the introduction of biomaterials. To evaluate the biocompatibility of materials, we have established a system that allows for the analysis and quantitation of cellular inflammatory responses in vitro. Elevated cytokine production serves as an indicator of inflammatory responses, thus we monitored the levels of tumor necrosis factor alpha and interleukin-1b produced by cells. In this study, the inflammatory responses of non-transformed murine macrophages (Raw 264.7) were analyzed. The cells were incubated with polymethylmethacrylate (PMMA) microspheres in the presence and absence of lipopolysaccharide (LPS) at 8 h and 18 h. The analysis of the genetic material obtained from the cells was quantitated using Real-Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). The cell populations treated with LPS alone or PMMA microspheres alone resulted in an elevation of cytokine levels, while cells incubated with LPS and PMMA particles demonstrated a synerginistic effect by producing a marked increase in the level of cytokine expression, 336 fold greater than that of the untreated control. Florescence microscopy studies that assessed cellular viability were also performed and are consistent with the RT-PCR results. Thus, we have developed an in vitro assay for quantifying cellular inflammatory responses. Furthermore, we have demonstrated that the presence of a bacterial component and wear debris particles can work in concert to elevate cytokine production.