Intermittent Applications of Continuous Ultrasound on the Viability, Proliferation, Morphology, and Matrix Production of Chondrocytes in 3D Matrices

Abstract

Chondrocytes, the cellular component of the articular cartilage, have long been recognized as strain-sensitive cells, and have the ability to sense mechanical stimulation through surface receptors and intracellular signaling pathways. This strain-induced biological response of chondrocytes has been exploited to facilitate chondrocyte culture in in vitro systems; examples include the application of hydrostatic pressure, dynamic compression, hydrodynamic shear (i.e., rotating bioreactors), and low-intensity pulsed ultrasound (US). While the ability of US to influence chondrogenesis has been documented, the precise mechanisms of US-induced stimulation continue to be investigated. There remains a critical need to evaluate the impact of US on chondrocytes in 3D culture, which is a necessary microenvironment for maintaining the chondrocyte phenotype. In this study, a continuous US wave for predetermined time intervals was employed, as opposed to pulsed US used in previous studies, to stimulate chondrocytes seeded in 3D scaffolds. The chondrocytes (n = 6) were subjected to US stimulation as follows: 1.5 MHz for 161 seconds, 5.0 MHz for 51 seconds, and 8.5 MHz for 24 seconds, and the US signal was applied twice in a 24-hour period. Scaffolds that are not stimulated by US served as the control. Both the control and the US-stimulated groups were maintained in culture for 10 days, and at the conclusion of the culture period, chondrocytes were assayed for total DNA content, morphology, and cartilage-specific gene expression by reverse transcriptase polymerase chain reaction. Our results show that chondrocytes when stimulated with continuous US for predetermined time intervals possessed higher cellular viability (1.2 to 1.4 times) and higher levels of type II collagen and aggrecan mRNA expression when compared to controls.