Increased Cell Traction-Induced Prestress in Dynamically Cultured Microtissues.

Prestress is a phenomenon present in many cardiovascular tissues and has profound implications on their in vivo functionality. For instance, the in vivo mechanical properties are altered by the presence of prestress, and prestress also influences tissue growth and remodeling processes. The development of tissue prestress typically originates from complex growth and remodeling phenomena which yet remain to be elucidated. One particularly interesting mechanism in which prestress develops is by active traction forces generated by cells embedded in the tissue by means of their actin stress fibers. In order to understand how these traction forces influence tissue prestress, many have used microfabricated, high-throughput, micrometer scale setups to culture microtissues which actively generate prestress to specially designed cantilevers. By measuring the displacement of these cantilevers, the prestress response to all kinds of perturbations can be monitored. In the present study, such a microfabricated tissue gauge platform was combined with the commercially available Flexcell system to facilitate dynamic cyclic stretching of microtissues. First, the setup was validated to quantify the dynamic microtissue stretch applied during the experiments. Next, the microtissues were subjected to a dynamic loading regime for 24 h. After this interval, the prestress increased to levels over twice as high compared to static controls. The prestress in these tissues was completely abated when a ROCK-inhibitor was added, showing that the development of this prestress can be completely attributed to the cell-generated traction forces. Finally, after switching the microtissues back to static loading conditions, or when removing the ROCK-inhibitor, prestress magnitudes were restored to original values. These findings show that intrinsic cell-generated prestress is a highly controlled parameter, where the actin stress fibers serve as a mechanostat to regulate this prestress. Since almost all cardiovascular tissues are exposed to a dynamic loading regime, these findings have important implications for the mechanical testing of these tissues, or when designing cardiovascular tissue engineering therapies.

Decoupling the Effect of Shear Stress and Stretch on Tissue Growth and Remodeling in a Vascular Graft.

The success of cardiovascular tissue engineering (TE) strategies largely depends on the mechanical environment in which cells develop a neotissue through growth and remodeling processes. This mechanical environment is defined by the local scaffold architecture to which cells adhere, that is, the microenvironment, and by external mechanical cues to which cells respond, that is, hemodynamic loading. The hemodynamic environment of early developing blood vessels consists of both shear stress (due to blood flow) and circumferential stretch (due to blood pressure). Experimental platforms that recapitulate this mechanical environment in a controlled and tunable manner are thus critical for investigating cardiovascular TE. In traditional perfusion bioreactors, however, shear stress and stretch are coupled, hampering a clear delineation of their effects on cell and tissue response. In this study, we uniquely designed a bioreactor that independently combines these two types of mechanical cues in eight parallel vascular grafts. The system is computationally and experimentally validated, through finite element analysis and culture of tissue constructs, respectively, to distinguish various levels of shear stress (up to 5 Pa) and cyclic stretch (up to 1.10). To illustrate the usefulness of the system, we investigated the relative contribution of cyclic stretch (1.05 at 0.5 Hz) and shear stress (1 Pa) to tissue development. Both types of hemodynamic loading contributed to cell alignment, but the contribution of shear stress overruled stretch-induced cell proliferation and matrix (i.e., collagen and glycosaminoglycan) production. At a macroscopic level, cyclic stretching led to the most linear stress-stretch response, which was not related to the presence of shear stress. In conclusion, we have developed a bioreactor that is particularly suited to further unravel the interplay between hemodynamics and in situ TE processes. Using the new system, this work highlights the importance of hemodynamic loading to the study of developing vascular tissues.

A Bioreactor to Identify the Driving Mechanical Stimuli of Tissue Growth and Remodeling.

Tissue growth and remodeling are essential processes that should ensure long-term functionality of tissue-engineered (TE) constructs. Even though it is widely recognized that these processes strongly depend on mechanical stimuli, the underlying mechanisms of mechanically induced growth and remodeling are only partially understood. It is generally accepted that cells sense mechanical changes and respond by altering their surroundings, by means of extracellular matrix growth and remodeling, in an attempt to return to a certain preferred mechanical homeostatic state. However, the exact mechanical cues that trigger cells to synthesize and remodel their environment remain unclear. To identify the driving mechanical stimuli of these processes, it is critical to be able to temporarily follow the mechanical state of developing tissues under physiological loading conditions. Therefore, a novel "versatile tissue growth and remodeling" (Vertigro) bioreactor was developed that is capable of tissue culture and mechanical stimulation for a prolonged time period, while simultaneously performing mechanical testing. The Vertigro's unique two-chamber design allows easy, sterile handling of circular 3D TE constructs in a dedicated culture chamber, while a separate pressure chamber facilitates a pressure-driven dynamic loading regime during culture. As a proof-of-concept, temporal changes in the mechanical state of cultured tissues were quantified using nondestructive mechanical testing by means of a classical bulge test, in which the tissue displacement was tracked using ultrasound imaging. To demonstrate the successful development of the bioreactor system, compositional, structural, and geometrical changes were qualitatively and quantitatively assessed using a series of standard analysis techniques. With this bioreactor and associated mechanical analysis technique, a powerful toolbox has been developed to quantitatively study and identify the driving mechanical stimuli of engineered tissue growth and remodeling.