Polymethylmethacrylate (PMMA) particles have been shown to inhibit the differentiation of osteoprogenitor cells, but the mechanism of this inhibitory effect has not been investigated. We hypothesize that the inhibitory effects of PMMA particles involve impairment of osteoprogenitor viability and direct inhibition of transcription factors that regulate osteogenesis. We challenged MC3T3-E1 osteoprogenitors with PMMA particles and examined the effects of these materials on osteoprogenitor viability and expression of transcription factors Runx2, osterix, Dlx5, and Msx2. MC3T3-E1 cells treated with PMMA particles over a 72-h period showed a significant reduction in cell viability and proliferation as indicated by a dose-and time-dependent increase in supernatant levels of lactate dehydrogenase, an intracellular enzyme released from dead cells, a dose-dependent decrease in cell number and BrdU uptake, and the presence of large numbers of positively labeled Annexin V-stained cells. The absence of apoptotic cells on TUNEL assay indicated that cell death occurred by necrosis, not apoptosis. MC3T3-E1 cells challenged with PMMA particles during the first 6 days of differentiation in osteogenic medium showed a significant dose-dependent decrease in the RNA expression of Runx2, osterix, and Dlx5 on all days of measurement, while the RNA expression of Msx2, an antagonist of Dlx5-induced osteogenesis, remained relatively unaffected. These results indicate that PMMA particles impair osteoprogenitor viability and inhibit the expression of transcription factors that promote osteoprogenitor differentiation. 7,8 and osteogenic differentiation of human mesenchymal stem cells. 9,10 Given that osteoprogenitors are the precursors to osteoblasts, the survival and differentiation of these cells is crucial to the process of bone formation in the implant bed during exposure to wear particles.Osteoprogenitor differentiation is regulated by the transcription factors Runx2, osterix, Dlx5, and Msx2, and the transcriptional activator b-catenin. 11 Runx2 dictates the expression of key osteoblast proteins such as alkaline phosphatase, collagen, and osteocalcin, and is expressed in bone cells throughout the osteogenic lineage. 11 Osterix lies downstream of Runx2 and is required for the differentiation of pre-osteoblasts into mature osteoblasts. 11,12 Dlx5 is a homeobox domain transcription factor that also regulates osteogenesis [13][14][15] and is involved in the response of osteogenic cells to BMP-2. [16][17][18] Msx2, another homeodomain transcription factor, is a functional antagonist and repressor of Dlx5-induced osteogenesis. 19-23 b-Catenin, a transcriptional activator of the canonical Wnt signaling pathway, associates with the DNA-binding proteins TCF and LEF, and stimulates Runx2 transcription. 11,24,25 Expression of these transcription factors is normally required for the proper differentiation of osteoprogenitor cells.Our group has previously shown that PMMA particles inhibit the osteogenic differentiation of murine MC3T3-E1 ...

In order to optimize and modulate bone formation it is essential to understand the expression patterns of key bone-specific growth factors, as osteoprogenitor cells undergo the processes of proliferation, differentiation and maturation. This study reports the sequential expression of bone-related growth and transcription factors when bone marrow-derived osteoprogenitor cells from C57BL mice were cultured on allograft bone discs. Mineralization and osteocalcin protein levels were used to track osteogenic differentiation and maturation. Bone-related growth factors, such as Bmp-2, Bmp-7, Ctnnb-1, Fgf-2, Igf-1, Vegf-a and Tgf-β1, and transcription factors, such as Runx-2 and osteocalcin, were examined by enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR). Total density of mineralized bone was significantly increased 7.6 ± 0.7% in allografts cultured with cells, compared with a 0.5 ± 2.0% increase in the controls without cells (p < 0.01). Osteocalcin protein levels peaked at day 4. Protein expression showed peaks of BMP-2 and TGF-β1 on day 2, with VEGF peaking on day 8, and IGF-1 decreasing on day 2. mRNA for Pdgf-a peaked on day 2; Bmp-2 on days 4 and 16; Ctnnb-1 on days 8 and 20; Vegf-a, Fgf-2, Runx-2 and Igf-1 on day 12; Tgf-β1 on day 16; and Pdgf-b on day 20. Osteogenic growth factors correlated with Runx-2 and Ctnnb-1, whereas a predominant vascular growth factor, Vegf-a, did not follow this pattern. Specific bone-related genes and proteins were expressed in a time-dependent manner when osteoprogenitor cells were cultured on cortico-cancellous bone discs in vitro.

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