The Effects of Mechanical Stimulation on Controlling and Maintaining Marrow Stromal Cell Differentiation Into Vascular Smooth Muscle Cells

CHD is the leading cause of morbidity and mortality in the U.S. [1]. For patients suffering from severe CHD, plaque build-up in the wall of the coronary arteries can cause inadequate oxygen supply to the heart, leading to cardiac ischemia and eventually death [2]. In order to restore adequate blood circulation, coronary artery bypass grafts (CABG) are performed. CABG procedures are highly invasive and involve the harvesting of a vessel at a secondary site [3,4]. Although synthetic materials such as Dacron and polytetrafluoroethylene are often used for vascular grafts, they can lead to thrombosis or neointimal hyperplasia [5]. More importantly, while synthetic grafts work well for arteries bigger than 12 mm, they are not suitable for the geometry of coronary arteries [6,7]. A cell-based TEBV has great potential to overcome the morbidity of autografts and the issues with synthetic materials. In order to attain the proper organization and mechanical properties found in native vessels, VSMCs are used as a crucial component of TEBVs [8–11]. Physiological conditions expose native vessels to a constant cyclic mechanical strain of 9–12% strain at roughly 1 Hz [12]. Under pulsatile flow, VSMCs in culture have been shown to express a more organized and layered structure in addition to a higher level of extracellular matrix (ECM) reorganization, resulting in significantly higher Young's modulus and burst strength [13,14]. VSMCs play a crucial role in TEBVs and it is evident that the application of cyclic strain can help to duplicate their organization and mechanical properties found in native vessels. Nevertheless, isolation of patient VSMCs for graft construction is a highly invasive procedure and may not be the most efficient TEBV cell source. With growing interest in stem cells, research has shown that MSCs, or mesenchymal stem cells, are a potential cell source for VSMCs. Compared to VSMCs, MSCs are more easily harvested and isolated, resulting in a process that is less invasive and leads to less morbidity than a vessel harvest for VSMCs [15]. In addition, MSCs have a much higher proliferative capacity and can potentially generate more tissue in less time than VSMCs [16,17]. The differentiation capacity of MSCs spans the broadest spectrum of terminally differentiated cell types among adult stem cells; the documented differentiation capacity of MSCs includes bone, cartilage, muscle, marrow stroma, tendon/ligaments, adipose tissue, connective tissues, and smooth muscle [17–22]. These combined factors make MSCs a potential cell source for vascular regeneration and a great candidate for the fabrication of tissue engineered vascular constructs. Studies in recent years have shown bone marrow-derived MSCs to differentiate into VSMCs in response to transforming growth factor β-1 and ascorbic acid [23–26]. Despite success in static conditions, MSCs differentiated into VSMCs must retain their cell type under dynamic conditions to be useful in a functional TEBV. To this end, several studies have shown that mechanical stimulation involving cyclic strain was capable of inducing MSC differentiation into the VSMC lineage in as short as 24 h [27–30]. Higher strain frequencies were observed to yield more occurrences of VSMC commitment, while lower strain frequencies were often observed to induce osteogenesis [31,32]. The distinction between strain conditions suggests that there may be two distinct mechanical conditions capable of directing MSC differentiation, one leading to VSMCs and the other to osteoblasts. In order to implement MSCs in TEBV designs, it is essential to elucidate the stability of these lineage commitments. In this study, we focused on the stability of MSC differentiation into VSMCs and osteoblasts when differentiation was induced solely by cyclic mechanical stimulation, thus mimicking the strain a TEBV would be exposed to in vivo. We apply conditions centering on the physiological conditions of 10% strain at 1 Hz [11] experienced by native VSMCs to study the effective frequency range of using mechanical conditioning for directed differentiation. By using single- and dual-stage conditioning, we investigated the stability of mechanically induced MSC differentiation into VSMCs, and in so doing determined whether any fluctuation in stretch frequency pre- or postimplantation could potentially compromise a constructed graft.