3D Constructs of Human Skeletal Muscle-Derived Cells as a Model of Bone Formation: Osteogenic Differentiation in Response to BMP2

In plastic and reconstructive surgery, surgeons are often challenged with a shortage of bone tissue at the site of bone defect. Human skeletal muscle-derived cells (skMDCs) undergo osteogenic differentiation in response to bone morphogenetic proteins (BMP) and have been explored for use in bone reconstructions.^1,2 Before cell therapy can be considered for clinical use, delivery of cells and control of bone growth must be optimized. The aim of this study was to determine the effect of BMP2 on differentiation of skMDCs fabricated into 3D constructs, thereby validating this novel model of bone formation. 3D constructs provide a more accurate representation of in vivo setting than monolayer cultures and may be used in bone grafting without additional structural support.

skMDCs isolated from 20, 25, and 32 year-old male donors  were purchased from Cook MyoSite and cultured in growth media. 1x10^5 skMDCs were fabricated into 3D constructs with extracellular matrix components using Flexcell Tissue Train System then cultured in osteogenic media with or without BMP2 (50ng/mL) for 3 days. RNA isolation and reverse transcription were then performed. Expression of marker genes of osteogenic and myogenic differentiation, Osx and MyoD, respectively, were evaluated via qPCR. They were normalized to GAPDH, and fold differences of BMP2 groups over corresponding controls were computed. The data was analyzed using ANOVA and Tukey's test with P<0.05 considered statistically significant.

All skMDCs groups treated with BMP2 showed statistically significant difference in both Osx and MyoD expression compared to the corresponding groups not treated with BMP2. Osx expression was increased with BMP2 treatment with mean fold difference over control ± standard deviation of 5.12±1.01 for 20 yo, 2.85±0.10 for 25 yo, and 3.67±1.09 for 32 yo (Figure 1). MyoD expression was decreased with BMP2 treatment with mean fold difference over control ± standard deviation of 0.61±0.06 for 20 yo, 0.51±0.10 for 25 yo, and 0.25±0.11 for 32 yo (Figure 2).

The results indicate that BMP2 promotes osteogenic differentiation of skMDCs in 3D constructs as evidenced by increased Osx expression and decreased MyoD expression in groups treated with BMP2. Human skMDCs fabricated into 3D constructs showed an osteogenic response to BMP2 comparable to other models and can therefore serve as an excellent in vitro model of bone formation to identify factors that promote osteogenesis.