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Effects of simulated microgravity on nitric oxide production and proteoglycan synthesis by chondrocytes encapsulated in 3D PEG hydrogels.   I. Villanueva¹, B. Klement², D. von Deutsch³, and S.J. Bryant¹.  ¹Dept. of Chemical and Biological Engineering, University of Colorado ²Dept. of Anatomy and Neurobiology, and ³Dept. of Pharmacology and Toxicology, Morehouse School of Medicine,.

   Overproduction of nitric oxide (NO) due to shear stress and disruptive loading has been implicated in cartilage degeneration within the articulating joints.  Rotating wall vessel (RWV) bioreactors provide tissues with a rotation about a horizontal axis, which is depicted by extremely low fluid shear stresses and turbulence.  The constructs placed in the RWV remain suspended within a constantly moving body of nutrient medium.  In this study, neutral polyethylene glycol (PEG) hydrogels with varying crosslinking densities (ρ_x) were used as 3D scaffolds for chondrocyte (cartilage forming cells) culture.  These cartilage models were used to study chondrocyte activity in the absence of loading and high shear stresses. A spinner flask was used as the control.  Chondrocyte response was measured by NO production and proteoglycan production, both normalized to total DNA content. PEG gels were fabricated with two _x’s, 0.1 and 0.5 mol/L, by varying the PEG concentration to yield gels with the same chemistry but different network structures. Chondrocytes were encapsulated in the gels 24 hours prior to loading in the RWV. Gel constructs were cultured in the RWV bioreactors for 2 and 5 days with an average rotational speed of 22rpm.  Mean proteoglycan content increased and mean NO production  decreased compared to controls at each  time point for each _x.  However,  signficance was only observed at day 2 in the low ρ_x gel with NO inhibition in the RWV compared to controls. Crosslinking density did not affect chondrocyte response over the 5 days of culture. Overall, an inhibition in NO was correlated with upregulation in PG synthesis (r= 0.52 ,  p<0.001).This study demonstrates that  PEG hydrogel constructs cultured within the RWV culture provide a 3D environment that inhibits NO production and has positive effects on the formation of matrix components, which is essential for healthy cartilage maintenance..  

(Supported by NASA NAG2-1274 and NIH:  Grant # K22 DE 016608)