Heterogeneities in the adhesion energies of chondrocytes isolated from healthy and osteoarthritic donors to a model surface evaluated over 21 days by atomic force microscopy

Introduction:

Introduction: Articular cartilage (AC) covers moving joints. Osteoarthritis (OA) marks the degradation of AC and is associated with severe pain and reduction of mobility. Due to hypoxia and lack of vascularity, repairing AC once injured is challenging. OA has no current treatments; calling for OA disease models to test for effective interventions or for engineered tissues to replace damaged ones. To address the above, tissue engineering (TE) offers a promising approach. Chondrocytes, the primary cell type in AC, are preferred over other cells for TE. However, chondrocytes make up only 2% of AC’s volume, making harvesting a reliable number of cells to work with extremely hard. To compound the problem further, the number of cells obtained from an OA donor is 30-fold less than those obtained from a healthy one. With the above in mind, designing OA TE models that can rely on cells taken from healthy donors or even from large animal species can overcome the limitations imposed by the limited number of chondrocytes isolated from OA donors. However, to enable this approach, a comprehensive fundamental understanding of how chondrocytes isolated from healthy and OA donors vary in their physiochemical properties such as dimensions, hydrophobicities, adhesion, elasticities, and chemical composition of surface molecules is necessary. With such knowledge, altering environmental conditions can guide means through which cells of healthy donors can be manipulated to capture the OA diseased phenotypes of cells.

Materials and

Methods: In a step towards addressing the gap defined above, differences in the adhesion energies of chondrocytes to a model of silicon nitride were quantified using atomic force microscopy (AFM) over 21 days of monoculture under PBS. Human chondrocytes (HCs) were donated from three healthy and three OA donors; both groups were similar in age (62 years, P = 0.1687). Cells were thawed, centrifuged, and cultured in DMEM/F-12 at 37°C, 5% CO2, and 20% oxygen. AFM imaging and force measurements were conducted at different time points during the cell culture process (days 0, 1, 7, 14, and 21), utilizing the Nano-Wizard IV AFM (JPK Instruments, Billerica, MA) coupled with the Cell-Hesion module at 37 oC. Once imaged, a force-volume map of 8×8 was created and 64 retraction force curves were collected per cell. At least 3-5 cells per culture for three different cultures per donor were investigated. The area under the force-distance curve was calculated as the adhesion energy (Figure 1). Energies were compared at the cellular levels across the 64 points mapped on the surface, culture level across three different cultures per culture time point per donor, at the donor level for all measurements taken from the three cultures per donor, and at the phenotype level for all measurements done for healthy or OA donors. Statistical standard methods were used to compare the differences amongst groups of interest.

Results, Conclusions, and Discussions: Results and discussion: At the cellular level, data suggested that individual cells of healthy donors varied in their adhesion energies by up to 5.8, 4.0, 5.5, 8.3, and 8.4 folds on days 0, 1, 7, 14, and 21, respectively. In comparison, variation in adhesion energies of cells extracted from OA donors differed by up to 2.6 folds at different culture time points. Cell-to-cell variability in adhesion is to be expected. Cells express many molecules on their surfaces; allowing the AFM cantilever to contact different molecules each time. These molecules will vary in their composition and makeup; leading to changes in quantified adhesion. At the culture level, results indicated that cultures of cells of healthy donors are more heterogeneous than those of diseased donors. Literature suggests that cells residing in healthier environments with reduced stress levels produce a wider variety of molecules on their surfaces; enabling them to adjust their metabolic behavior and control their phenotype features. This results in more heterogeneous surfaces compared to cells residing in stressed environments such as those isolated from OA donors. At the donor’s level, data suggested that cells of diseased donors are more heterogenous compared to cells of healthy donors (Figure 2). This can possibly be attributed to variable medical history amongst the OA donors. Finally, at the phenotypic level, the ratios of the mean of the adhesion energies quantified for all cells of healthy donors to these of cells of diseased donors varied significantly by up to 6.3, 1.4, 1.1, 1.6, and 2.3 folds on days 0, 1, 7, 14, and 21, respectively. This finding suggests that the adhesion strength measured for cells isolated from healthy donors always exceeded these measurements compared to the cells separated from diseased donors.

Conclusions: Our data suggested that chondrocytes isolated from healthy and OA donors significantly vary in their adhesion energies at all investigated levels. This advocates for caution to be experienced when designing TE models that utilize cells from healthy donors to optimize treatments for OA. Further, results suggested that three cultures may not be sufficient to represent the biological heterogeneity inherited in chondrocytes.

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