(11e) Controlled Exploration of Synergistic Effects of Heterotypic Cell-Cell Interactions and Mechanical Stimulation On Blood Vessel Formation

Small diameter blood vessel replacements are necessary in the treatment of advanced atherosclerosis, aneurysmal and peripheral vascular disease, and trauma. Although initial results with many of the tissue engineered vascular grafts (TEVG) constructed to date are encouraging, potential for mechanical failure has limited the general success of these grafts. The disparity in mechanical properties between TEVG and native vessels is due in large measure to differences in the amount, composition, and microarchitecture of the extracellular matrix (ECM) produced by associated smooth muscle cells (SMC). To improve SMC ECM production and phenotype in TEVG, we need to understand the manner in which SMC respond to various stimuli to which they are exposed during development and homeostasis. Embryological data suggest that endothelial cells (EC) direct the recruitment and differentiation of mesenchymal stem cells into SMC. In addition, physiological pulsation has been shown to enhance SMC differentiation. We therefore reasoned that concurrent EC exposure and mechanical stimulation would further enhance mesenchymal stem cell progression toward an SMC phenotype. To enable systematic study of potential synergistic effects between EC presence and pulsatile flow on mesenchymal stem cell differentiation, poly(ethylene glycol) diacrylate (PEGDA) hydrogels were employed. PEGDA has several material properties which make it desirable for studies focusing on elucidating the effects of specific stimuli on cell behavior, including its biological ?blank slate? character and its tunable mechanical properties. In addition, PEGDA hydrogels are photopolymerizable in the presence of cells, allowing fabrication of the seamless multi-layered tubular constructs required for the proposed studies.

In the present study, bilayered PEGDA constructs were exposed to ~7% strain at 160 bpm pulsatile flow and a mean shear stress of ~5 dynes/cm2. Constructs were cultured in DMEM supplemented with 10% heat inactivated FBS and antibiotics/anti-mycotics. Following 21 days total culture time, samples were harvested and the differentiation state of the mesenchymal stem cells in both the EC+ and EC- constructs was analyzed biochemically and histologically. Deposition of three ECM molecules key to vessel mechanics, namely collagen type I, collagen type III, and elastin, was examined. Collagen type I expression was lower in EC+ constructs than in EC- constructs (p < 0.05). In contrast, elastin levels were higher in EC+ constructs than in EC- constructs. To assess 10T½ differentiation, expression of SMC marker calponin h1 was examined and found to be increased in EC- constructs relative to EC+ constructs (p < 0.05)These variations in 10T½ ECM production and phenotype were reflected in differences in internal cellular signaling in EC+ and EC- constructs. SRF and myocardin, two transcription factors that directly regulate calponin h1 expression, were each decreased in EC+ constructs relative to EC- constructs (p < 0.05). Similarly, SMAD3/4, which enhances SRF transcription, was increased in EC- constructs (p < 0.05).

Contrary to expectation, combined EC presence and pulsatile flow stimulation appeared to drive mesenchymal stem cells away from a differentiated SMC phenotype. These results suggest that these two stimuli, each of which is separately known to stimulate SMC differentiation, may need to be applied to mesenchymal stem cells in a sequential manner to achieve desired differentiation.