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THE IN VITRO REGULATION OF OSTEOGENIC DIFFERENTIATION BY INFLAMMATORY CYTOKINES
Presenter: Lauren L Zammerilla, BS
Co-Authors: Shakir S; Naran S; Cray JJ; Smith DM; Wang D; Losee JE; Cooper GM
University of Pittsburgh

Background: Morselized bone components have been shown to induce an inflammatory response after long bone fracture. Given that similar components are often utilized in calvarial reconstruction, we hypothesized that a connection exists between the inflammatory response generated by these morselized bone components and subsequent bone healing. In the current study, we sought to determine the effects of specific inflammatory mediators on osteogenic differentiation.

Methods: A mouse myoblast cell line (C2C12) with an established response to BMP2 stimulation was utilized and their response to BMP2 alone, and BMP2 in combination with specific inflammatory mediators was quantified. Treatment groups included proliferation medium (negative control), BMP2, and BMP2 in combination with TGF≤?1, TGF≤?2, TGF≤?3, TNF, IL-10, or HMGB1. After 24 hours, cells underwent alkaline phosphatase (ALP) staining, quantitative ALP activity assay, and MTS cell proliferation assay. Quantitative PCR was used to analyze gene expression of the bone-related genes RUNX2, OSX, TGF≤?1, 2 & 3, and FGFR2. One-way ANOVA analysis was used, with p<0.05 being significant.

Results: BMP2-TGF≤?1, BMP2-TGF≤?2, BMP2-TGF≤?3, and BMP2-TNF groups showed reduced ALP staining when compared to BMP2-alone. ALP activity was significantly reduced in the BMP2-TGF≤?1 (p=0.011), BMP2-TGF≤?3 (p=0.004), and BMP2-TNF (p=0.039) groups compared to BMP2-alone. MTS assay revealed increased proliferation in the BMP2-TGF≤?1, BMP2-TGF≤?2, BMP2-TGF≤?3, and BMP2-TNF groups. Quantitative PCR revealed a two-fold or greater decrease in FGFR2 expression in cells treated with BMP2-TGF≤?1, BMP2-TGF≤?2, BMP2-TGF≤?3, and BMP2-TNF.

Conclusions: These data suggest a role for inflammation-related proteins in bone healing as TGF≤?1, TGF≤?3, and TNF inhibited BMP2-induced ALP expression in C2C12 cells. Interestingly, these same factors promoted cell proliferation. The down-regulation of FGFR2 expression in cells treated with TGF≤?1, TGF≤?2, TGF≤?3, and TNF may play a role in this response.


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