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Introduction: Chondrocytes beneath the joint surface display a distinct superficial chondrocyte spatial organization (SCSO), which is a marker of tissue ultrastructure and function that undergoes a proliferative remodeling in early osteoarthritis [1,2]. Cellular filamentous actin (F-actin) and cell volume, two proliferation cofactors, change with osteoarthritis and might contribute to proliferation in early osteoarthritis. We asked whether chondrocyte proliferation rate (PR) can be controlled by controlling cell shape, and whether cell shape, the macroscopic tissue disease state (MTDS), or cytoskeletal F-actin intensity, density, or distribution has the strongest impact on the PR. To answer this question, we established a quantitative biology approach, combining single cell analyses with partial least square (PLS) analyses and random forest (RF) classification and regression predictive modeling.
Methodology: Chondrocytes were isolated from two MTDSs, macroscopically intact (MIA) or osteoarthritic-lesional areas (MOA), and cultured on custom-designed micro-patterned adhesion sites (MPs) for 1 or 7 days. Fixed chondrocytes were stained with phalloidin iFluor488 and DAPI and imaged using intensity-calibrated fluorescent beads for calculating F-actin intensity per cell and F-actin density. Fiji was used for textural analyses to quantify F-actin distribution, cell shape, and PR. PLS and RF were used to identify the most relevant factors for controlling and predicting PR.
Results: The PR of chondrocytes varied on different MP designs from 0.25 to 8.37, demonstrating that MP geometry allows minimizing and maximizing chondrocyte proliferation. PLS revealed that the highest impact on PR (in descending order) had cell shape (day_7), cell shape (day_1), and MP shape. The PR correlated negatively with cell solidity and roundness (day_7) and positively with cell area, length and aspect ratio (day_7), cell length being the most crucial factor. Interestingly, the PR of MOA chondrocytes was always higher than that of MIA chondrocytes. Using circular and H-shaped MPs in two sizes, we characterized single cell F-actin content, density, and distribution over time, revealing that F-actin density on day 1 was largely determined by MP size but not geometry or MTDS, whereas at day 7 F-actin density was determined by MTDS but not by MP size, geometry, or cell shape. PLS revealed that F-actin density (day_7), MTDS, and F-actin amount (day_7) had the highest impact on the PR, whereas other factors were relatively unimportant for controlling PR. Using RF regression with cell shape and actin parameters as input, the MTDS was predicted with an accuracy of 84.27 %, confirming the relation of these parameters to the PR of human chondrocytes.
Conclusion: The PR of chondrocytes is not only controlled by MP or cell shape, but to a greater extent by F-actin density and MTDS. In cell culture, cytoskeletal F-actin density was initially dependent on MP size but this was superseded over time by F-actin density being determined by MTDS. Thus, these data suggest that in situ proliferation leading to loss of SCSO in early osteoarthritis appears to be more controlled by MTDS-associated changes in F-actin than MTDS-associated changes in cell shape.
References: 1:Rolauffs et al. Arthritis Rheum. 2010 62(2):489-98; 2:Felka et al. Osteoarthritis Cartilage. 2016 Jul;24(7):1200-9.
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