Plastic Surgery Research Council
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PSRC 60th Annual Meeting

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TGF Beta is a Potent Inhibitor of BMP2 Mediated Osteogenic Differentiation in a Primary Murine Muscle Cell in vitro Model of Heterotopic Ossification
S. Alex Rottgers, M.D., Laurie B. Meszaros, PhD, Anand R. Kumar, M.D..
University of Pittsburgh, Pittsburgh, PA, USA.

Introduction: Heterotopic ossification (HO) is pathologic bone formation in extremity muscles. Alterations in inflammatory mediators and bone morphogenetic proteins acting on muscle derived progenitor cells (MDCs) are thought to be critical for HO formation. The aim of this study is to evaluate the effects of bone morphogenetic protein (BMP2) and inflammatory mediators (TNFα, TGFβ1) on MDC osteogenic differentiation.
Methods: Primary mouse muscle cells were isolated from 8-week-old C57B/6J mice. Hindlimb muscles were sterilely processed, followed by pre-plating on collagen-coated flasks for 2 hours to remove fibroblast. 100,000 cells per well were cultured in DMEM-based proliferation medium (PM) alone or with 50ng/mL of BMP2, with 1 ng/ml TNFα , with 5 ng/ml of TGFβ1 alone or in various combinations.
Samples were collected after 3 days. RNA was isolated and reverse transcribed to cDNA. Quantitative PCR for Osx, Alp and Runx2 was performed. Changes in target gene expression were expressed relative to untreated MDCs with expression normalized to GAPDH.
Results: FACS analysis revealed a mixed population of cells, with high Sca-1 and CD34 expression and low CD31, CD 56, CD144 and CD146 expression. Muscle cells cultured with BMP2 demonstrated increased expression of Osx (36.6±25.8), Alp (19.0±4.16) and Runx2 (2.26±0.61) relative to untreated cells. TNFα decreased expression of Osx (0.19±0.002), Alp (0.360±0.15) and Runx2 (0.75±0.18 fold). TGFβ1 demonstrated decreased expression of Osx (0.01±0.07), Alp (0.004±0.0005) and Runx2 (0.59±0.01).
Cells cultured with BMP2/TNFα demonstrated increased expression of Osx (19.1±4.39) relative to untreated cells but significantly less than BMP alone (p=0.001). BMP-2 mediated expression of Alp (15.9±2.13) and Runx2 (1.64±0.10) was maintained. BMP2/TGFβ1 treatment demonstrated decreased expression of Osx (0.003±0.001), Alp (0.004±0.0005) and Runx2 (0.56±0.05) from baseline. Combined BMP2/TNFα/TGFβ1 treatment also decreased expression of Osx (1.58±0.52), Alp (0.021±0.006) and Runx2 (0.92±0.10).
Discussion: Using a mixed population of primary MDCs we have demonstrated in vitro osteogenic differentiation following application of BMP2. TNFα significantly decreased BMP2 mediated osteogenic changes. We have demonstrated a more robust osteogenic inhibition of BMP2 mediated changes using TGFβ1. TGFβ1 and TNFα agonist therapy may represent a novel therapy for HO. Future studies evaluating changes within the intracellular SMAD pathway may elucidate the mechanism of inhibition.


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