Mechanical and Vascular Cues Synergistically Enhance Osteogenesis in Human Mesenchymal Stem Cells

Abstract

Development and maintenance of a vascular network are critical for bone growth and homeostasis; strategies that promote vascular function are critical for clinical success of tissue-engineered bone constructs. Co-culture of endothelial cells (ECs) with mesenchymal stem cells (MSCs) and exposure to 10% cyclic tensile strain have both been shown to regulate osteogenesis in isolation, but potential synergistic effects have yet to be explored. The objective of this study was to expose an MSC-EC co-culture to 10% cyclic tensile strain to examine the role of this mechanical stimulus on MSC-EC behavior. We hypothesized that paracrine signaling from ECs would stimulate osteogenesis of MSCs, and exposure to 10% cyclic tensile strain would enhance this anabolic signal. Human umbilical vein ECs and human bone marrow-derived MSCs were either monocultured or co-cultured at a 1:1 ratio in a mixed osteo/angiogenic medium, exposed to 10% cyclic tensile strain at 1 Hz for 4 h/day for 2 weeks, and biochemically and histologically analyzed for endothelial and osteogenic markers. While neither 10% cyclic tensile strain nor co-culture alone had a significant effect on osteogenesis, the concurrent application of strain to an MSC-EC co-culture resulted in a significant increase in calcium accretion and mineral deposition, suggesting that co-culture and strain synergistically enhance osteogenesis. Neither co-culture, 10% cyclic tensile strain, nor a combination of these stimuli affected endothelial markers, indicating that the endothelial phenotype remained stable, but unresponsive to the stimuli evaluated in this study. This study is the first to investigate the role of cyclic tensile strain on the complex interplay between ECs and MSCs in co-culture. The results of this study provide key insights into the synergistic effects of 10% cyclic tensile strain and co-culture on osteogenesis. Understanding mechanobiological factors affecting MSC-EC crosstalk will help enhance strategies for creating vascularized tissues in tissue engineering and regenerative medicine.

FIG. 1.

To enhance EC attachment, ECs were cultured in EGM for 3 days before co-culture with MSCs or exposure to 10% cyclic tensile strain. From days 0 to 14, ECs, MSCs, and MSC-EC co-cultures were all cultured in OEM and either exposed to 10% cyclic tensile strain (1 Hz for 4 h/day) or left in static conditions. EC, endothelial cell; EGM, endothelial growth medium; MSC, mesenchymal stem cell; OEM, osteogenic-endothelial medium. Color images available online at www.liebertpub.com/tea

FIG. 2.

(A) Osteogenic endothelial growth medium (OEM) was found to enhance MSC viability relative to EGM, while maintaining viability of ECs and MSC-EC co-cultures. While a laminin coating slightly increased EC viability (B) and endothelial markers CD31 and VE-Cdh staining (D), a collagen type I coating greatly enhanced MSC and MSC-EC viability (B) and osteogenesis, (C) and thus was chosen for all experiments. Scale bars, 100 μm. *p < 0.05 for black versus white bars. Color images available online at www.liebertpub.com/tea

FIG. 3.

Representative images demonstrating that ECs elongated in response to 10% cyclic tensile strain, while MSCs and MSC-EC co-cultures elongated and aligned perpendicular to the direction of strain. Scale bars, 100 μm (200 μm for inserts). Color images available online at www.liebertpub.com/tea

FIG. 4.

Representative images demonstrating that ECs maintained expression of CD31 and VE-Cdh in co-cultures with MSCs and in response to 10% cyclic tensile strain, indicating that EC phenotypic stability was preserved. Scale bars, 100 μm. Color images available online at www.liebertpub.com/tea

FIG. 5.

(A) Calcium accretion normalized to DNA and (B) mineral deposition were synergistically enhanced by co-culture and application of 10% cyclic tensile strain. Scale bars, 100 μm. *p < 0.05. Color images available online at www.liebertpub.com/tea