Effect of Osmotic Stress on Intracellular Calcium Signaling of In Situ Juvenile and Mature Chondrocytes

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1 Effect of Osmotic Stress on Intracellular Calcium Signaling of In Situ Juvenile and Mature Chondrocytes Yilu Zhou, Michael A. David, Jie Ma, Liyun Wang, X. Lucas Lu. University of Delaware, Newark, DE, USA. Disclosures: Y. Zhou: None. M. David: None. J. Ma: None. L. Wang: None. X. Lu: None. Introduction: Natural aging has been shown to alter the phenotype and metabolic activity of articular cartilage chondrocytes [1] and may also alter their mechanotransduction processes. Intracellular calcium ([Ca 2+ ] i) signaling is among one of earliest mechanotransduction responses of chondrocytes when exposed to mechanical, fluid, electric and osmotic stresses. Studies have observed spontaneous [Ca 2+ ] i signaling in mature chondrocytes when exposed to various osmotic stresses in vitro [2-4] ; however, it is not clear whether the age-related differences in chondrocytes can ultimately affect their mechanotransduction behaviors and associated [Ca 2+ ] i signaling. In this study, we will investigate the effect of different osmotic stresses (hypertonic, hypotonic, and isotonic) on calf (juvenile) and adult bovine (mature) chondrocytes spontaneous [Ca 2+ ] i signaling in situ. It is hypothesized that mature chondrocytes will demonstrate altered spontaneous [Ca 2+ ] i signaling when exposed to hypo/hypertonic stress compared to juvenile chondrocytes. Methods: Cartilage explant isolation: 3 mm diameter cartilage explants were isolated from juvenile (calf; ~3 months) and mature (bovine: ~18 months) fresh bovine knee joints within 24 hours of slaughter (Green Village, NJ). Explants were cultured in chemically defined medium [5] until osmotic loading. Osmotic loading: Prior to confocal imaging, cylindrical explants (juvenile n = 6; mature n = 7) were cut into two halves, one for each osmotic condition, and incubated with Fluo-8 AM for 40 minutes. After incubation, explants were placed in an imaging chamber and positioned on a confocal microscope (Zeiss LSM 510). Samples were allowed to equilibrate for 15 minutes to eliminate any stress from experimental handling, and then middle-to-superficial zone chondrocytes spontaneous [Ca 2+ ] i signaling responses were captured under isotonic stress (DMEM medium; 310 mosm) and then immediately followed under either hypertonic (NaCl addition; 600 mosm) or hypotonic (distilled water addition; 165 mosm) stress. Each osmotic stress was imaged for 16 minutes (960s, 2s per frame). Data Analysis and Statistics: [Ca 2+ ] i intensity of each cell was recorded and analyzed to obtain the percentage of responsive cells, defined as [Ca 2+ ] i intensity increase more than four times of the baseline standard deviation, and the spatiotemporal parameters of the [Ca 2+ ] i peaks. Chi-square analysis was performed to detect the difference of responsive percentage between the two groups, and Mann-Whitney U test was performed to detect the differences of spatial-temporal parameters from [Ca 2+ ] i signaling. Results: Spontaneous [Ca 2+ ] i signaling responses were successfully captured from both juvenile (Figure 1A) and mature (Figure 1B) chondrocytes. Typical [Ca 2+ ] i intensity oscillation curves under all osmotic conditions were shown in Figure 1C. Under all osmotic stresses, the responsive percentages were significantly higher in mature chondrocytes than the corresponding percentage in juvenile group (p<.001). In addition, for both mature and juvenile chondrocytes, hypotonic stress increased (p<.001) while hypertonic stress decreased the response percentages (p<.001) compared to isotonic stress (Figure 2). When juvenile chondrocytes were under hypotonic stress, the number of [Ca 2+ ] i peaks

2 (p<.01), magnitude of peaks (p<.01), time to reach a peak (p<.001), peak relaxation time (p<.001) were increased, while the time between two neighboring peaks was decreased (p<.001) compared to isotonic stress (Figure 3A-E). When hypertonic stress was applied, there was an increase in time to reach a peak (p<.001) and peak relaxation time (p<.001) while a decrease in the number (p<.001) and magnitude of peaks (p<.05) compared to isotonic stress. Regarding mature chondrocytes, under hypotonic stress there was an increase in the number of peaks (p<.001) while a decrease in all other parameters (p<.001) compared to isotonic stress. Additionally, hypertonic stress caused an increase in the peak relaxation time (p<.001) and time between two peaks (p<.001) while decreasing the number of peaks (p<.001) compared to isotonic stress (Figure 3F-J). The magnitude of peaks in juvenile group is significantly higher (p<.05) than that in mature group under all osmotic conditions. Under isotonic condition, time to reach a peak, peak relaxation time and time between peaks in juvenile group are shorter (p<.001) than those in mature group. Discussion: In the present study, cartilage explants allowed for a more appropriate in situ environment for chondrocytes to reside in for the analysis of mechanotransduction response compared to previous monolayer tests. Age-related differences were revealed in the mechanotransduction behaviors of chondrocytes via spontaneous [Ca 2+ ] i signaling under osmotic stress. A higher responsive percentage in mature chondrocytes under all osmotic conditions compared to juvenile suggests that mature chondrocytes might be more sensitive to physical signals experienced in vivo. A variety of differences exist in the spatiotemporal features of [Ca 2+ ] i peaks for both juvenile and mature chondrocytes under different osmotic conditions. In general, the hypotonic condition generated higher frequency of [Ca 2+ ] i peaks, and the peaks are more spike-like with shorter rising and relaxation time compared to iso- and hypertonic conditions. Because negatively charged proteoglycans attract more anions into cartilage and induce an osmotic pressure within the tissue, change of proteoglycan content due to aging or disease would inevitably cause fluctuations in osmotic environments that chondrocytes experience. Our results demonstrated that chondrocytes are capable of sensing their osmotic environment in terms of the frequency and spatiotemporal features of spontaneous [Ca 2+ ] i signaling, which may play important roles for chondrocytes to adapt their phenotype and metabolic activity according to environmental cues. Significance: The [Ca 2+ ] i signaling of in situ chondrocytes under all osmotic conditions appears to be altered in an age-dependent manner. Osmotic environments may be a target to modulate chondrocytes function and to overcome the adverse effects of aging on cartilage properties.

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5 ORS 2015 Annual Meeting Poster No: 0434