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. 2016 Aug 1;126(8):2893-902.
doi: 10.1172/JCI83676. Epub 2016 Jul 18.

Induced superficial chondrocyte death reduces catabolic cartilage damage in murine posttraumatic osteoarthritis

Minjie Zhang, Sriniwasan B Mani, Yao He, Amber M Hall, Lin Xu, Yefu Li, David Zurakowski, Gregory D Jay, Matthew L Warman

Induced superficial chondrocyte death reduces catabolic cartilage damage in murine posttraumatic osteoarthritis

Minjie Zhang et al. J Clin Invest. .

Abstract

Joints that have degenerated as a result of aging or injury contain dead chondrocytes and damaged cartilage. Some studies have suggested that chondrocyte death precedes cartilage damage, but how the loss of chondrocytes affects cartilage integrity is not clear. In this study, we examined whether chondrocyte death undermines cartilage integrity in aging and injury using a rapid 3D confocal cartilage imaging technique coupled with standard histology. We induced autonomous expression of diphtheria toxin to kill articular surface chondrocytes in mice and determined that chondrocyte death did not lead to cartilage damage. Moreover, cartilage damage after surgical destabilization of the medial meniscus of the knee was increased in mice with intact chondrocytes compared with animals whose chondrocytes had been killed, suggesting that chondrocyte death does not drive cartilage damage in response to injury. These data imply that chondrocyte catabolism, not death, contributes to articular cartilage damage following injury VSports手机版. Therefore, therapies targeted at reducing the catabolic phenotype may protect against degenerative joint disease. .

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Figures

Figure 1
Figure 1. Measuring chondrocyte density and number.
(A–F) 3D reconstructed images representing a 760 μm × 760 μm × 50 μm volume containing a DAPI-stained, wild-type femoral head. The centroids of individual chondrocyte nuclei in the entire cartilage volume (A) and in a volume extending to a depth of 10 μm below the surface (C) are indicated by yellow dots. The cartilage volumes across the entire thickness (B) and within the most superficial 10-μm region below the surface (D) are colored yellow; the volume covered by the yellow volume in D is colored blue (inset, D). (E and F) Centroids of chondrocyte nuclei in 100 μm × 100 μm areas (boxes) at depths of 10 μm (E) or 20 μm (F) are indicated by yellow dots. (G) Bland-Altman plots comparing measures of cell density (number of nuclei/0.01 mm3 cartilage) across the entire cartilage volume (global cell density = chondrocyte nuclei counted in A divided by cartilage volume as calculated in B), the surface volume extending to a depth of 10 μm (superficial cell density = chondrocyte nuclei counted in C divided by surface volume as calculated in D), and cell numbers in the center 100 μm × 100 μm areas at 10 μm (E) and 20 μm (F) depths for the femoral head. Each circle represents a comparison between contralateral joint surfaces (n = 10 mice and 3 comparisons per mouse). Note the circles from an individual mouse cluster near one another. Numbers indicate the median mice with regard to average cell numbers. Blue lines indicate the mean ± 1.96 SD. (H) Graphs depicting the mean (+ SD) values of the 4 primary measures for the femoral head obtained after storage in fixative for 3 days, 10 days, and 17 days. No significant differences are attributable to the length of time in fixative.
Figure 2
Figure 2. DTA-induced killing of surface chondrocytes.
(A) Schematic depicting the experimental timeline for inducing DTA-mediated cell death by administering tamoxifen from 21 to 30 days of age and then monitoring chondrocyte division by administering EdU from 31 to 40 days of age. Animals were analyzed after the final EdU dose. (B) Confocal images showing EdU-positive chondrocyte nuclei in a plane 20 μm below the surface of DAPI-stained femoral heads of a control mouse (Prg4CreERt2+R26mTmG/+ mouse) (left) and a DTA-ablated mouse (Prg4CreERt2/+R26mTmG/DTA mouse) (right). Note the decreased numbers of total (blue) and dividing (pink) chondrocyte nuclei in the DTA-ablated mouse. (C and D) Confocal femoral head images taken at different planar depths (10, 20, 30, and 40 μm) in control mice (C) and DTA-ablated mice (D) identify DAPI-stained chondrocyte nuclei (blue), nonrecombined membrane–anchored dTomato-expressing chondrocytes (red), and Cre-recombined membrane-anchored eGFP-expressing chondrocytes (green). Identical thresholds were employed for all images. Insets indicate DAPI windows (pseudocolored white) from a 100 μm × 100 μm area in the center of the image. (E and F) Bar graphs depicting the mean (± SD) values for chondrocyte density and chondrocyte number for the femoral (E) and humeral (F) heads of control (blue bars) and DTA-ablated (red bars) mice (n = 5). *P < 0.05, between control and DTA-ablated mice using Student’s t test. Scale bar: 100 μm.
Figure 3
Figure 3. Preservation of cartilage surfaces despite DTA-induced killing of surface chondrocytes.
(A) Schematic depicting the experimental timeline for inducing cell death with tamoxifen-mediated induction of DTA expression from 21 to 30 days of age. Subsequent cell division was monitored by administering EdU from 31 to 40 days of age or from 2 to 3 months of age. (B, D, and F) Representative H&E-stained sagittal sections through the tibial-femoral joint of 40-day-old (B), 3-month-old (D), and 9-month-old (F) control mice (left) and DTA-ablated mice (right) (n = 5/time point). (C, E, and G) Representative Safranin O– and Fast Green stained sagittal sections through the tibial-femoral joint of 40-day-old (C), 3-month-old (E), and 9-month-old (G) control mice (left) and DTA-ablated mice (right) (n = 5/time point). Scale bars: 50 μm. (H and I) Bar graphs depicting the mean (± SD) global cell densities and superficial cell densities in the lateral condyle (H) and femoral head (I) of control (blue bars) and DTA-ablated (red bars) mice at different time points (n = 5/time point). Note chondrocyte density measured globally and in the superficial zone decreased significantly in 40-day-old and 3-month-old DTA-ablated mice compared with controls, with the greatest decrease occurring in the superficial zone. However, this decrease disappeared at 9 months of age. *P < 0.05, Student’s t test. (J) Representative histograms depicting the distribution of nearest neighbor distances for DAPI-stained nuclei in the femoral heads of 9-month-old control (upper) and DTA-ablated (lower) mice. Note the significantly increased mean distance in the DTA-ablated mice (P < 0.001, Student’s t test, n = 5) and the significant difference in the histogram shape (P = 0.023, Levene’s test) between control and DTA-ablated mice. (K) Bar graphs depicting mean (± SD) percentages of DAPI-stained chondrocyte nuclei that are also EdU positive (n = 5). Note the DTA-ablated mice (red bars) exhibit significantly lower percentages of EdU-positive cells at 40 days of age compared with control mice (blue bars), but significantly higher percentages of EdU-positive cells at 3 months of age. *P < 0.05, Student’s t test.
Figure 4
Figure 4. Chondrocyte death following mechanical destabilization in the knee.
(A) Representative H&E-stained coronal sections (left) and confocal images of DAPI-stained nuclei at depths of 20 μm (right, pseudocolored white) in medial and lateral condyles of 14-week-old mice that had undergone sham (upper) or DMM (lower) surgery at 10 weeks of age (n = 6). (B) Bar graphs depicting the mean (± SD) values for global cell density, superficial cell density, and cartilage thickness in condyles from 14-week-old mice that had undergone DMM surgery (red bars) or sham surgery (blue bars) 4 weeks earlier (n = 6). Note only the superficial cell density on the medial side of the medial condyle is lower in the DMM compared to shame treated mice. *P < 0.05 Student’s t test. (C) Representative H&E-stained coronal sections (left) and confocal images of DAPI-stained nuclei at depths of 20 μm (right, pseudocolored white) in medial and lateral condyles of 22-week-old mice that had undergone sham (upper) or DMM (lower) surgery at 10 weeks of age (n = 9). Note the appearance of surface roughening in both condyles and the reduction in the number of DAPI-stained nuclei in the medial, but not in the lateral, condyle of the DMM mice compared with sham-treated mice. (D) Bar graphs depicting the mean (± SD) values for global cell density, superficial cell density, and cartilage thickness in condyles from 22-week-old mice that had undergone DMM surgery (red bars) or sham surgery (blue bars) 12 weeks earlier (n = 9). Note the significant decrease in chondrocyte density in medial surface of the medial condyle. *P < 0.01, Student’s t test. The medial surface of the medial condyle is normally protected when the meniscus is intact. Scale bar: 50 μm.
Figure 5
Figure 5. Chondrocyte loss prevents cartilage from being damaged by DMM surgery.
(A) Representative confocal images of DAPI-stained nuclei at depths of 20 μm (pseudocolored white) in medial and lateral femoral condyles of mice that had undergone sham (upper) or DMM (lower) surgery 12 weeks earlier (n = 6). Scale bar: 100 μm. (B and C) Bar graphs depicting the mean (± SD) values for global cell density (B) and superficial density (C) in condyles from mice that had undergone DMM surgery (red bars) or sham surgery (blue bars) 12 weeks earlier (n = 6). Note the significant decrease in chondrocyte density in all condyles of the DTA mice compared with control mice. Also note that DMM surgery did not further affect chondrocyte density in the DTA-ablated mice, whereas cell density decreased significantly in the medial condyles of nonablated mice that underwent DMM versus sham surgery. *P < 0.05, control vs. DTA, Student’s t test. #P < 0.05, sham vs. DMM, Student’s t test. (D) H&E-stained coronal sections in medial femoral condyles (left) and medial tibial plateau (right) of 22-week-old mice that had undergone sham (upper) or DMM (lower) surgery 12 weeks earlier. Images are from the mouse with the median OARSI score in each group (n = 6). Scale bar: 50 μm. (E) OARSI scores of control or DTA-ablated mice that underwent sham or DMM surgery 12 weeks earlier (n = 6/group). Note the significantly higher OARSI score in DMM joints in both control and DTA mice versus sham joints and significantly lower OARSI score in DMM joints in DTA mice versus DMM joints in controls. *P < 0.05, control vs. DTA, nonparametric Wilcoxon rank-sum test. #P < 0.05, sham vs. DMM, nonparametric Wilcoxon rank-sum test.
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