Poster AA5 - Cell Surface Hydrophobicity in Osteoarthritis versus Healthy Donor Cells over 21 days of Culture through Contact Angle Measurements

Introduction: Osteoarthritis (OA) is a leading cause of disability, affecting over half a billion individuals globally, with an occurrence that has significantly increased over recent decades. This condition not only leads to joint pain and stiffness but also brings economic costs due to direct healthcare expenses and indirect costs such as decreased productivity and increased caregiving needs. The pathogenesis of OA involves the breakdown of articular cartilage (AC), which is critical for joint function. Despite considerable research, OA's exact mechanisms remain partially understood, specifically at the cellular level. Chondrocytes, the main cells of AC, play a crucial role in the maintenance and repair of AC tissue. However, in OA, these cells fail to properly repair and maintain the AC matrix upon injury, leading to progressive deterioration. With this in mind, developing disease models of OA that rely on cells extracted from OA donors is challenging. Researchers in turn try to utilize cells extracted from healthy donors to study OA disease mechanisms and potential treatments. However, prior to doing that, fundamental understanding of the differences in properties between chondrocytes isolated from healthy and OA donors should be gained to assess the feasibility of this approach. In a step towards that, hydrophobicities of chondrocytes isolated from both healthy and osteoarthritic donors were assessed utilizing water contact angles (WCAs) and compared over 21 days of monoculture. By identifying the similarities and differences in cellular hydrophobicities of variable phenotypic chondrocytes, representative models of OA can be developed and effective treatment interventions can be tested.

Materials and

Methods: Chondrocytes were harvested from six males (62 years, P=0.17), three healthy and three with osteoarthritis (OA). Upon revival, cells underwent a thawing process, and centrifugation, and were then cultured for 21 days in DMEM/F-12 with supplements at 37°C, 5% CO2, and 20% oxygen. At days 0, 1, 7, 14, and 21 days, cultures were digested with collagenase type 1 (0.1% w/v) for approximately 30 mins at 37 ᵒC, 5% CO2, 95% relative humidity, and 20% oxygen. Cells were then isolated via repetitive washing and centrifugation steps following established protocols. Pellets of cells were then resuspended in water at a calculated cell density (1.2×106 cells/ml) required to cover a filter membrane (Whatman 4-micron grade 589/3) with 5 monolayers of cells. Filters were kept on a gelatin layer to ensure water equilibrium is achieved. Contact angles on monolayers of cells at different points in culture were then measured using the sessile drop method with a drop shape analyzer DSA100 (KRŰSS Hamburg Germany). Various solvents (deionized water, formamide, and diiodomethane) with different dielectric constants were used to measure the contact angles. For each time point, at least three filters were prepared and 5-9 measurements per filter were performed. Additionally, colorimetric assays to quantify DNA, collagen (COL), and glycosaminoglycans (GAG) of the engineered AC tissues were carried out according to standard methods. Statistical analyses, including ANOVA and Welch’s t-test, were applied to interpret the data and assess significant differences between different cell lines.

Results, Conclusions, and Discussions: Results and

Discussion: This study revealed that chondrocytes from OA donors consistently demonstrated higher WCAs compared to chondrocytes from healthy donors, with statistically significant differences on days 0, 7, and 14 (Figure 1). Further, all measured WCAs for cells were above 80o at all time points of culture indicating that all chondrocytes are hydrophobic (Figure 1). It is well established in the literature that WCAs that are below 65o reflect hydrophilic surfaces. When donor to donor variability in WCAs was assessed, our data suggested that a triplicate is sufficient for WCA measurements as there were time points during culture in which all WCAs were constant (Figure 2). Variations in WCAs amongst donors may be influenced by several physiological and lifestyle factors beyond OA. For instance, factors such as a high Body Mass Index or low physical activity levels contribute to the production of inflammatory cytokines and exert mechanical stress on joints. Such stress can lead to lipid peroxidation, wherein free radicals damage the lipid bilayer, creating hydrophobic patches. This in part explains why cells isolated from OA donors are more hydrophobic than cells isolated from healthy donors. The differences in the hydrophobicities of cells of healthy and OA phenotypes suggest that hydrophobicities can be potentially used to sort cells. By day 21, the WCAs of cells of OA and healthy donors were statistically similar, suggesting the potential to restore an OA cell phenotype to a healthy phenotype over 21 days of culture. Further, total GAG/DNA and total COL/DNA were significantly higher by 6- and 8- fold on average at all points of culture for OA donor cells compared to healthy donor cells (P < 0.0001). The higher contents of COL/DNA and GAG/DNA for OA cultures reflect the need of these tissues to form mature extracellular matrices to support them.

Conclusions: These findings highlight the complexity of OA pathophysiology at the cellular level. Our findings suggest that modifying the cellular microenvironment could offer means to restore the regenerative capabilities of chondrocytes within 21 days of culture; offering means for enhanced tissue regeneration and design of representative TE OA models.

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