Development of physicochemical co-culture models for engineering prevascularized bone tissue.

摘要

An in vitro co-culture system consisting of mesenchymal stem cells (MSCs) and endothelial cells (HUVE-12 cell line) was developed to observe biochemical and physical cues in a pre-vascularized, osteo-differentiating engineered-tissue. Many co-culture models simplify the influence of physical cues in their models. This work attempted to reduce or eliminate a set of chemical inductive reagents within the co-culture model to allow for further optimization of other influences in the model, such as physical cues. When cultured alone in osteogenic media, endothelial cells displayed signs of hypertrophy, low cell yield and a significant increase in calcium content in comparison to HUVE-12 cells in different chemical media as well as compared against cell type. When cultured in angiogenic media, hMSCs did not express PECAM-1, a marker displayed by endothelial cells. While chemical induction can osteo-differentiate hMSCs, endothelial cells did not behave optimally in osteogenic media. Substrate elasticity was explored as a means to osteo-differentiate hMSCs and was categorized in terms of Alizarin Red S staining, osteogenic gene regulation from PCR experiments, and values of elastic moduli using AFM force-deflection curves fit to the Hertz Cone model. HUVE-12 invasive behavior through a collagen gel was also investigated: the addition of exogenous growth factors compared to hMSC-conditioned media did not increase the depth of EC invasions. Knowing this, a monolayer of HUVE-12 was seeded atop a collagen gel, atop hMSCs on a PDMS substrate (E = 2 MPa) in diluted angiogenic media. By exploiting the secretion of growth factors (e.g., VEGF) by hMSCs, we promoted angiogenesis in a virtually exogenous-free model, allowing for further study of the effects of physical cues on a co-culture.