(L-451) Investigating the effects of 3D Printed Scaffolds on reducing Nitric Oxide in chondrocyte cells under Hypoxic Conditions

Osteoarthritis (OA) is the degeneration of the joint that affects articular cartilage (AC) and the underlying bone. OA is the most common form of arthritis, affecting 36.8% of adults in the United States alone. AC is an avascular tissue made up of chondrocytes that live in low-oxygen environments consequently. Under normal conditions (1% - 6%), chondrocytes produce low concentrations of reactive oxygen species (ROS). ROS are free radicals essential for maintaining cellular homeostasis and function, but in hypoxia (< 1%), ROS is produced excessively. Excessive amounts of ROS promote cartilage degradation by inducing matrix-degrading proteases, reducing extracellular matrix (ECM) synthesis, and inducing chondrocyte apoptosis. Antioxidants are deployed to reduce ROS, but when there's an insufficient ratio of antioxidants to oxidants, oxidative stress (OS) occurs. Cells counteract OS via radical scavenging of excessive free radicals that restores healthy cellular redox balance. Direct implications of ROS in some joint diseases have been accumulated in the cartilage of arthritis patients, specifically derivatives of nitric oxide (NO^-). Cerium oxide (CeO₂) is a potent ROS scavenger due to its ability to co-exist between trivalent (Ce³⁺) and tetravalent (Ce⁴⁺) states in a redox reaction. This study aims to investigate the effects of 3D-printed scaffolds on reducing NO^- under hypoxic conditions using the Griess Assay. To assess CeO₂ reducing capabilities, scaffolds prepared with CeO₂ are compared to scaffolds prepared with poly(N-vinyl caprolactam) (PVCL). We hypothesize that the 3D-printed scaffolds prepared with CeO₂ will be efficient in reducing NO^-  in chondrocytes.

The Griess Assay is conducted using the Biotic Citation 1 Plate Reader with glass chamber. 1mL of conditioned media is removed from C28/I2 Human Chondrocyte Cell Line (cells resuspended in 100µL of culture media) and pipetted into a 12 Well-plate with pieces of scaffold samples evenly distributed among them. The 12 Well-plate is then incubated in the PHCbi CO₂ Incubator for 1 hour at 37°C and 5.0% CO₂. 150µL of media from each well is removed from 12 Well-plate and transferred to wells in 96 Well-plate. 20µL of Griess reagent is added to each well along with 130µL of deionized (DI) water. Griess reagent is made by mixing 10µL of N-(1-naphthyl)ethylenediamine (Component A) and 10µL of sulfanilic acid (Component B). Incubate 96 Well-plate for 30 minutes at room temperature in a dark environment. Measure absorbance in Cytation 1 Plate Reader at 548 nm. Absorbance readings are converted to nitrite concentrations via calibration.  To calibrate, prepare sodium nitrite (NaNO₂) solution with concentrations between 1100µM by diluting the nitrite standard solution (Component C) with DI water. Samples are prepared and measured the same as described previously but using standard nitrite solutions in place of the experimental samples. A standard curve is plotted of nitrite concentrations (x-axis) versus absorbance (y-axis). Nitrite concentrations are read corresponding to the absorbance of experimental samples from the standard plot.

The figure represents a bar graph of data collected after the performance of the Griess Assay at distinct oxygen levels. The Griess assay is a colorimetric method used to quantify derivatives of NO^-. NO^- has two oxidative states of nitrite (NO₂) and nitrate (NO₃), but in this study nitrite concentrations is the only derivative observed. Previous reports have demonstrated that nitrite concentrations in hypoxia remained higher than concentrations in hyperoxia (20%), so it was expected that concentrations measured at 5% O₂ would remain higher than concentrations measured at 20% O₂. Cell culture media served as the negative control of the experiment, so the minimum concentration accumulated allowed us to gauge the additional amount of NO^- that would be added to the samples with scaffolds present. It was unexpected that samples prepared with CeO₂ (MPV2010 and M2050) would exhibit higher nitrite concentrations than samples prepare with PVCL (ML0050 and MLPV0050). CeO₂ has emerged as a powerful antioxidant therapeutic tool in the prevention and treatment of oxidative-stress-related diseases. Cerium (Ce) redox capability to co-exist as both Ce³⁺ and Ce⁴⁺, allows oxygen to be stored and released when present in the form of CeO₂. Under physiological conditions, the oxygen storage capabilities of Ce can scavenge free radicals as soon as they are produced during metabolic imbalance. The antioxidant properties of CeO₂ have displayed minimum toxicity to normal tissue and provide cellular protection from varying ROS derivatives. Although the hypothesis was falsified, CeO₂ may not be efficient in reducing NO^- but may be efficient in reducing other ROS derivatives.