Relation of low-intensity pulsed ultrasound to the cell density of scaffold-free cartilage in a high-density static semi-open culture system

A scaffold-free cartilage construct, analogous to those found during embryonic precartilage condensation, has received much attention as a novel modality for tissue-engineered cartilage. In the present study, we developed an uncomplicated culture system by which scaffold-free cartilage-like tissues are produced using cell-cell interactions. With this system, we attempted to prevent dedifferentiation and reverse the phenotypic modulations by adjusting the cell density. We investigated whether low-intensity pulsed ultrasound (LIPUS) enhances matrix synthesis of the scaffold-free cartilage construct. Rat articular chondrocytes multiplied in monolayers were seeded onto the synthetic porous membrane at stepwise cell densities (i.e., 1.0, 2.0, and 4.0 x 10(7) cells/cm(2)) to allow formation of a scaffold-free cartilage construct via cell-cell interaction. The cartilage constructs were then stimulated by LIPUS for 20 min/day. To investigate the effect of LIPUS stimulation on matrix synthesis, expression of mRNA for cartilage matrix molecules was quantified by a real-time reverse transcription-polymerase chain reaction. Synthesis of type II collagen, type I collagen, and proteoglycan was also assessed histologically. Only the chondrocytes cultured at high cell densities in the 2.0 x 10(7)cells/cm(2) group became concentrated and formed a plate-like construct similar to native articular cartilage by macroscopic and histological assessments. Statistical analysis on the matrix gene expression demonstrated that the levels of type II collagen and aggrecan mRNA of the 2.0 x 10(7)cells/cm(2) group were significantly higher than with the other two cell-density groups. Interestingly, the LIPUS application led to a statistically significant enhancement of aggrecan gene expression only in the 2.0 x 10(7) cells/cm(2) group. The current study presents a semi-open static culture system that facilitates production of the scaffold-free constructs from monolayer-cultured chondrocytes. It suggests that the LIPUS application enhances matrix production in the construct, and its combination with the scaffold-free construct might become a feasible tool for production of implantable constructs of better quality.