Poster Q11 - Heterogeneities in the Nanomechanical Properties of Chondrocytes Isolated from Healthy and Diseased Humans Assessed by Atomic Force Microscopy

Introduction: Osteoarthritis (OA) is a degenerative condition impacting over 32 million adults in the U.S., characterized by damage to articular cartilage (AC) which induces joint inflammation. This condition progresses irreversibly, leading to severe pain, diminished mobility, and a reduction in quality of life, thereby accentuating the urgent need for novel interventions. Despite the significant burden of OA, there are currently no cures for its reversal; underscoring a pressing need for new therapeutic approaches. The development of viable interventions is hindered by the lack of model systems that accurately represent the complexities of human OA. However, in vitro tissue-engineered models emerge as a promising avenue, offering possibilities to replicate healthy AC and restore functionality to damaged tissues. Nonetheless, research and therapeutic development is complicated by the complex microenvironment in which chondrocytes, the primary cells in AC, exist. The limited availability of chondrocytes and their propensity to dedifferentiate in culture add further complications. To overcome these limitations, OA models are often developed using chondrocytes isolated from healthy donors or from animal species. However, the feasibility of using engineered tissues derived from chondrocytes of healthy donors to accurately replicate OA's tissue and microenvironment is to be tested. Investigating the physiochemical properties of chondrocytes from healthy and diseased donors is essential to develop accurate OA models. As a step toward this goal, atomic force microscopy (AFM) was used to assess variations in the elasticities of chondrocytes obtained from healthy and diseased male donors of similar ages.

Materials and

Methods: Chondrocytes were isolated from three male healthy and OA donors, each (age 62, P=0.17). Cells were seeded at 25,000 cells/cm² in tissue culture dishes. Cells were grown under standard conditions (37°C, 90% humidity, 5% CO2, 20% O2) in DMEM supplemented with 10% FBS and 1% antibiotics, with bi-daily media changes up to 21 days. Prior to AFM measurements, cells underwent three PBS washes and immobilization with Poly-L-Lysine. AFM measurements were conducted using the NanoWizard IV AFM, equipped with non-conductive silicon nitride tips and the CellHesion module, in PBS at 37°C. The QITM Imaging mode was used to scan cell surfaces, and an 8×8 force map was collected per cell. AFM measurements were performed on days 0, 1, 7, 14, and 21 on 3-5 cells per culture and for three cultures per time point. The force-indentation curves collected were fitted to the Hertz model of contact mechanics using a conical indenter geometry to estimate the Young's modulus of elasticity with the JPK data processing software. Finally, cultures were colorimetrically assessed for the production of key AC’s extracellular matrix (ECM) markers including glycosaminoglycans (GAGs), total Collagen (COL) and DNA. Statistical analysis was performed using ANOVA to compare elasticities across groups and results were presented as mean ± SEM.

Results, Conclusions, and Discussions: Results and discussion: As expected, as the culture progressed, chondrocytes from both healthy and diseased origins expanded in length and width and flattened in height (Table 1). When compared to each other, the chondrocytes of OA and healthy origins were morphologically similar. With exception to day 7, the elasticities of chondrocytes of OA’s origin were slightly higher than these of healthy origin (Table 1). During the culture period, chondrocytes of healthy origin reached a maximum Young’s modulus at day 7, which was 17% higher than the mean values quantified at days 0, 1, 14, and 21. In comparison, chondrocytes of OA’s origin gradually kept increasing their Young’s modulus from day 0 to day 21 with a linear slope of 2.2 Pa (R2=0.95); suggesting continued need to enhance their mechanics. Further, chondrocytes of OA’s origin were characterized by a higher Young’s moduli at all time points investigated than chondrocytes of healthy origin except at day 7. The higher Young’s moduli of OA cells may help them withstand mechanical stresses in a microenvironment that is less supportive due to degraded ECM. Supporting this observation is the fact that the total collagen per DNA for the OA cultures was significantly lower than that of healthy cultures at all time points investigated (Table 1). Interestingly, the GAG/DNA for the OA cultures was lower at days 0 and 1 compared to healthy cultures but was larger at days 7, 14, and 21 with a maximum ratio of 1.33 at day 14. The increased GAG content in the OA culture compared to the healthy cultures can be attributed to the need of OA cells to attract more medium to enable appropriate growth.

Conclusions: The observed differences in the nanomechanical properties between chondrocytes of healthy and osteoarthritic origins highlight the need for development of in vitro models that closely simulate the osteoarthritic microenvironment. Such models are critical for creating innovative therapies that may slow or reverse the progression of OA, thereby enhancing the quality of life for millions affected.

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