Creating complex, controllable environments for scaffold-free tissue engineering using acoustic levitation

Tissue engineering has been limited by the complex structure of biological tissue. Methods have therefore been devised using scaffolds made from synthetic materials (polymers) or natural materials (extracellular matrix from cadavers or animal tissues). These scaffolds are artificially introduced materials with potentially adverse side effects. This project’s objective was to determine the parameters necessary to create an environment to employ a scaffold-free method for building tissue. This method uses ultrasonic standing waves in fluid to generate complex patterns acting as a template to organize suspended cells into tissue structures. Parameters investigated included ultrasonic waveform shape, frequency, and intensity; water density and temperature; tissue cell size; transducer properties; and container configuration for standing-waves. Computer models were used to evaluate various container designs. Ultrasonic transducers from 0.20 to 1.0 MHz were coupled to containers and driven using an arbitrary waveform generator and RF amplifier. Polyethylene microspheres of 10-50 micrometer diameter were used to simulate biological cells. Experiments with cylindrical containers demonstrated that microspheres can be levitated in water to form layers, and revealed that the use of a square-wave modulated waveform generated more distinct layers. Experiments were also conducted with square containers using two transducers. These experiments produced a lattice-type structure of microspheres.