Large number tracking of depth and mechanics dependent calcium signaling in chondrocytes of articular cartilage

Abstract

Purpose: Moderate loading of cartilage stimulates chondrocytes to synthetize matrix proteins, but excessive loading can lead to apoptosis. It is well known that macroscale strains affect cell health, but the mechanism behind how tissue level strain causes cellular and subcellular biological response is poorly understood. Previous work done by our group showed impact-induced tissue strain resulting in mitochondrial depolarization in chondrocytes, with correlation between strain and chondrocyte death, demonstrating the importance of microscale mechanics driving chondrocyte fate after impact. The correlation between strain and mitochondrial dysfunction indicates the mitochondria’s role in mechanosensing. However, the mechanotransduction and cell signaling pathway driving this process has not been fully characterized. In traumatic brain injury, Ca2+ signaling is known to produce mitochondrial swelling, while in other tissues Ca2+ is buffered by mitochondria. However, if too much Ca2+ is taken up, the mitochondrial permeability transition pore will open, leading to depolarization and cell death. In brain injury, this process can be averted if the mitochondrial swelling is addressed immediately. This indicates an opportunity to introduce mitoprotective therapies in a similar vein in articular cartilage. To better understand chondrocyte fate and investigate the process of mitochondrial depolarization after strain, an understanding of the Ca2+ signaling during mechanical stimulation is required. We predict that chondrocytes in different regions of cartilage will show different Ca2+ signal responses, in relation to the depth dependent compressive and shear properties of the tissue. In order to test this prediction, we compare changes in Ca2+ signaling under various magnitudes of compressive and shear strain, and compare the signaling response with previous work that determined impact-induced strain thresholds for mitochondrial dysfunction and chondrocyte death. Methods: Explants of articular cartilage were sterilely dissected from the femoral condyles of neonatal calves. The explants were cultured in a solution of DMEM and Antibiotic-Antimycotic for 24 hours. After removal from culture, the explants were vertically bisected into hemicylinders. They were stained for Ca2+ ions in 5μM Fluo-8 AM for one hour at 37° C, and rinsed in pure DMEM three times for ten minutes each to remove excess stain. The bisected samples were affixed to a custom-built Tissue Deformation Imaging Stage (TDIS) that can apply compressive and shear strain while directly imaging the tissue on a Zeiss LSM 510 inverted confocal microscope [Fig. 1]. Each sample was subjected to compression and shear. Recordings were made for 1000s while uncompressed, immediately following compression, post-relaxation, and immediately following shear. From these videos we were able to extract cell locations as a function of time through a centroid tracking algorithm [Fig. 2]. We extracted location and intensity information for all cells framed within a 636× 636μm frame (∼1000 cells per video), and identified cells showing Ca2+ transients. Local strain was calculated using a 2D digital image correlation program and the intensity curve of each cell was matched with the corresponding local strain. Results: Cell tracking showed different families of curves in Ca2+ signal intensity for cells located in the superficial (0 to 200μm depth) and middle (> 200μm depth) regions [Fig. 3], as well as multiple different families of cell signals present for uncompressed, compressed, and sheared samples [Fig. 4]. Increased proportion of cells showing Ca2+signaling was observed at greater compressive strain and in comparison with unstrained samples, indicating a threshold at which abnormal Ca2+ signaling occurs. Conclusions: The development of a technique to track a large number of cells in a non-equilibrium state is an improvement upon previous methods. This method allows for the study of transient signals despite changes in location and brightness, allows for large-scale tracking of cells for population statistics and provides the basis to directly study the relationship between Ca2+ signaling and mitochondrial response. The observation of differences in cell Ca2+ signaling between the surface and middle zones before any tissue strain was applied reinforces the idea that the superficial layer has a protective role for articular cartilage. This is in agreement with previous work showing that removal of the superficial layer allowed strain to penetrate deeper into the tissue. The identification of strain thresholds for increased Ca2+ signaling aligns with previous work showing strain thresholds for mitochondrial depolarization and cell death, further promoting the role that Ca2+ has in the cellular response pathway to strain.View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)