Murine chondrocytes within intact medial femoral condyles have dysregulated calcium responses to mechanical loading after dmm surgery

Abstract

Purpose: Intracellular calcium mobilization is known to be a downstream signaling response to different types of mechanical loading in a variety of cells, including chondrocytes. Osteoarthritis involves aberrant loading, however, the calcium responses of chondrocytes to the mechanical loading changes induced by osteoarthritis have not been examined. Therefore, the purpose of this study was to generate novel methods to begin to address this question using the destabilization of the medial meniscus (DMM) mouse model of osteoarthritis. By better understanding how chondrocyte response to loading is altered in osteoarthritis, novel targets for therapies may be uncovered. Methods: We created a novel line of mice by crossing Collagen II cre mice with GCaMP6s loxp mice. GCaMP6s is an ultra-sensitive fluorescent calcium reporter that can be genetically expressed in specific cell types using cre-loxp recombination (in this case, chondrocytes). In these Collagen II-GCaMP6s mice, when intracellular calcium increases in chondrocytes, an increase in green fluorescence can be measured. DMM surgery in these mice causes joint damage similar to wild-type mice. To apply mechanical loading to mouse femoral condyle cartilage while performing calcium imaging, we built a system using a Zeiss Axio Observer inverted fluorescent microscope and an Aurora Scientific dual-mode lever system (300C) in order to apply compressive strain with 1 μm resolution (Fig 1). This setup enables us to compress murine medial femoral cartilage against a glass coverslip while imaging from below. We verified that our applied displacement resulted in accurate movement of our sample by an Eddy sensor (Micro-Epsilon ES04) with a resolution of 0.04 μm. DMM (n=4) or sham (n=3) surgery was performed in the right knee of 10-week old male Collagen II-GCaMP6 mice. Sixteen weeks after surgery, each femur was freshly dissected and prepared for calcium imaging. Calcium Imaging and Data Analysis: A defined sodium-calcium buffer, commonly used for calcium imaging, was used for all imaging and was slowly perfused through the chamber during each experiment. Images were taken at a frequency of 1 Hz, with an exposure time of 20 ms, using a GFP filter set. For each femur, baseline images were acquired without loading for 5 min, loading using a square waveform with amplitude 7 μm and frequency 0.05 Hz was applied for 5 min, 5 min of imaging was acquired post loading with the femur out of contact, and 8 min of imaging was performed while a 50% hypo-osmotic buffer was perfused past the femur. Images were processed using custom macros in Fiji to correct for minor movement as well as to calculate (F-Fo)/Fo, where Fo = the first 2-20 frames imaged. Responses in individual chondrocytes during the different imaging phases were compared between sham and DMM using unpaired, two-tailed t-tests (GraphPad Prism 6), where p<0.05 was considered statistically significant. Results: Seven microns of compression (∼18% strain; our sham mouse medial condyle cartilage was on average 39 μm thick) in medial femoral condyles resulted in greater numbers of chondrocytes generating intracellular calcium responses in sham mice compared to DMM mice 16 weeks after surgery (3.8-fold higher; p=0.03) (Fig 2). However, on average, DMM chondrocyte calcium responses were greater in amplitude and duration than sham chondrocyte responses (2.5-fold higher; p=0.03) (Fig 2). The average peak forces observed during compression were similar for sham (13.4±2.6 mN) and DMM mice (16.3±1.6 mN) (p = 0.35). Exposure to hypo-osmotic salt solution has been used previously as a positive control for inducing intracellular calcium responses in chondrocytes as it causes cells to stretch. Here, we found that 50% hypo-osmotic solution induced similar numbers of chondrocyte responses in both sham (54±10) and DMM (42±11; p=0.46) mice 16 weeks after surgery, and similar amplitude and duration of responses (sham: 6.9±0.2 ΔF/Fo x seconds; DMM: 9.7±2.6 ΔF/Fo x seconds; p=0.36). Conclusions: Here, we have generated a novel strain of mice and a novel mechanical stimulation and imaging system in order to image real-time responses of chondrocytes to mechanical loading within intact mouse femoral condyles after DMM surgery. Our results suggest that after DMM surgery, femoral condyle chondrocytes are still able to generate intracellular calcium responses to changes in osmolarity, but they have become dysregulated in response to mild levels of cartilage compression. Future work will seek to understand which calcium signaling pathways may be mediating these changes and how these changes may contribute to other pathological cartilage changes in osteoarthritis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)