Viability and volume of in situ bovine articular chondrocytes—changes following a single impact and effects of medium osmolarity  Dr Peter G. Bush, Ph.D., Peter D. Hodkinson, M.Sc., Georgina L. Hamilton, B.Sc., Dr Andrew C. Hall, Ph.D.  Osteoarthritis and Cartilage  Volume 13, Issue 1, Pages 54-65 (January 2005) DOI: 10.1016/j.joca.2004.10.007 Copyright © 2004 OsteoArthritis Research Society International Terms and Conditions

Fig. 1 An overview of the pattern of chondrocyte death arising from the application of a single injurious impact to bovine articular cartilage. Explants of cartilage were incubated with calcein-AM and PI as described (see Materials and Methods) and then subjected to (a) no impact, or (b) a single impact of 100g from 10cm. The cartilage was then imaged by CLSM after 60min, and the projected image viewed perpendicular to the cartilage surface is shown (see Materials and Methods). Intracellular calcein or PI fluorescence appears green or red, respectively. Confocal images were acquired at 10μm z-step intervals using a 5× dry objective lens to a depth of ∼60μm. Bar=100μm. Osteoarthritis and Cartilage 2005 13, 54-65DOI: (10.1016/j.joca.2004.10.007) Copyright © 2004 OsteoArthritis Research Society International Terms and Conditions

Fig. 2 Chondrocyte death following a single impact. Explants of bovine articular cartilage were subjected to (a) no impact, (b) 100g from 10cm, or (c) 200g from 10cm and then maintained in culture. Maximum projected confocal images of calcein (live cells; green) and PI (dead cell nuclei; red) were taken of transverse sections 24h after impact. Confocal images were acquired at 10μm z-step intervals using a 10× dry objective lens. For the experimental protocols for this and other figures, see Materials and Methods. Bar=100μm. Osteoarthritis and Cartilage 2005 13, 54-65DOI: (10.1016/j.joca.2004.10.007) Copyright © 2004 OsteoArthritis Research Society International Terms and Conditions

Fig. 3 The time course of chondrocyte death resulting from a single impact load. Bovine articular cartilage incubated with calcein-AM and PI, was positioned with the synovial surface uppermost, and subjected to a single impact load of 100g from 10cm, and then viewed by CLSM. Upper panels show the maximum projected confocal images of calcein (live cells; green) and PI (dead cell nuclei; red) (a) 5min, (b) 15min, (c) 25min, (d) 35min, and (e) 45min after impact. To visualise the time course of cell death more easily, the lower panels indicate the additional cell death observed in sequential images over the following periods (0–5min, 5–15min, 15–25min, 25–35min, and 35–45min). Confocal images were acquired at 10μm z-step intervals using a 5× dry objective lens. Bar=200μm. Osteoarthritis and Cartilage 2005 13, 54-65DOI: (10.1016/j.joca.2004.10.007) Copyright © 2004 OsteoArthritis Research Society International Terms and Conditions

Fig. 4 Changes to the viability of SZ chondrocytes near the injured edge resulting from a single impact load. Bovine articular cartilage was subjected to a single impact load (100g from 10cm) and viewed from the synovial surface. Panels show maximum projected confocal images of calcein (live cells; green) and PI (dead cell nuclei; red): (a) 3min, (b) 6min, (c) 12min, and (d) 20min after impact. The chondrocytes used for volume determinations in this experiment are identified individually using a white spot [panel (d)]. Confocal images were acquired at 1μm z-step intervals using a 63× water-immersion objective lens. Bar=25μm; image approximately 150μm×150μm. Osteoarthritis and Cartilage 2005 13, 54-65DOI: (10.1016/j.joca.2004.10.007) Copyright © 2004 OsteoArthritis Research Society International Terms and Conditions

Fig. 5 The decrease in chondrocyte viability after two single impacts of different magnitude. The proportion of viable cells as a percentage of the total cell number studied was determined over 20min for (1) control cartilage explants (i.e., not exposed to impact), and for explants subjected to (2) 100g from 5cm and (3) 100g from 10cm. The data are shown at the time points corresponding to initiation of the scan, although note that the total scanning time required for each measurement was ∼90s. At the first time point studied, there was a significant (P<0.001, indicated by †) decrease in the proportion of viable cells. Although the subsequent decrease in viability appeared greater for 100g from 10cm compared to the same load from 5cm, this difference was only significant at 20min (P<0.01, indicated by *). Over the entire time course, the rate of cell death was not significantly different (P=0.718; two-way ANOVA; see Results). Data (means±s.e.m.) are from at least five experiments under each condition. Osteoarthritis and Cartilage 2005 13, 54-65DOI: (10.1016/j.joca.2004.10.007) Copyright © 2004 OsteoArthritis Research Society International Terms and Conditions

Fig. 6 The change in cell volume of in situ chondrocytes following a single impact. Cartilage explants were positioned with the synovial surface uppermost, and then impacted. The edge of the injury was located quickly as described and the volume of viable chondrocytes near the edge determined over 20min (see Materials and Methods, and Figs. 1 and 4). The control cartilage was not subjected to impact and the volume of in situ chondrocytes determined at the time points indicated, with the mean initial value taken as 100%. For both impact loads, chondrocyte shrinkage was significantly different (P<0.006, indicated by *) but the rate of shrinkage was not different between the two loads (P=0.089; two-way ANOVA; see Results). Data (n[N]) are given as means±s.e.m. for (4[18]), (3[26]) and (5[17]), respectively. Osteoarthritis and Cartilage 2005 13, 54-65DOI: (10.1016/j.joca.2004.10.007) Copyright © 2004 OsteoArthritis Research Society International Terms and Conditions