Effect of Rock Inhibition on Mass Transport in Mesenchymal Stem Cell Aggregates Undergoing Chondrogenesis

The objective of the research was to manipulate biomolecular signaling inside differentiating human mesenchymal stem cells (hMSCs) to improve properties of tissue engineered cartilage. Enhanced transport conditions formed by manipulating the biophysical mechanisms activated during hMSC aggregation upon chondrogenic differentiation were investigated. Manipulation was achieved through Rho/ROCK signaling, a key biophysical signaling pathway that interferes with actin-myosin mediated contractile force generation and subsequent aggregation (Kim & Sheetz, 2012). The effect of the Rho/ROCK inhibition has not been investigated yet in hMSCs, which highlights the importance of this study.

Chondrogenesis is the process by which chondrocytes are formed from progenitor cells. Currently, small aggregates of scaffold-free cartilage constructs can be formed by subjecting hMSCs to culture medium containing growth factors such as TGF-β1. Within 24 hours after chondrogenesis initiation, the cells aggregate spontaneously to form a spherically-shaped pellet. Aggregates have been used primarily as an investigative tool and not as a therapeutic tool due to their inferior properties compared to the native tissue.

While properties of hMSCs undergoing Rho/Rock inhibition were not previously investigated, hMSCs have been observed to form a â??loosely packed pelletâ?. A more loosely packed pellet of hMSCs can be linked to greater transport properties allowing for signaling molecules to permeate aggregates and therefore improving conditions for cartilage tissue culture. While pellets being formed during the first few days will not have the structure necessary for transport properties to be observed, they could be fully observed after seven days. As the extracellular matrix accumulates during chondrogenesis, the transport properties will be inhibited, which occurs beginning day 7. Rho/Rock inhibition was able to significantly enhance the transport properties due a less compact pellet. The central hypothesis is that modulation of transport properties via ROCK inhibition during chondrogenesis will lead to improved cartilage tissue. The resulting construct due to the Rho/ROCK signaling will lead to larger pellets, allowing the aggregates to be used as a more practical tool for clinical applications.

The goal of this study was focused on the effects of the inhibition of Rho/ROCK and its effect on hMSCs towards chondrogenesis and enhancing the process. This was evidenced by the increased mass transport of tracer dextran molecules of different sizes. Fully formed ROCK inhibited pellets were noted to have improved transport properties than those without the inhibition. Biochemical property data supports this above conclusion.