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. 2020 Oct 16;9:e55212. doi: 10.7554/eLife.55212

Figure 5. The SOC protects epiphyseal chondrocytes from apoptosis induced by mechanical stress.

(A–D) Quantification of propidium iodide- (PI) (A–B) and TUNEL- (C–D) positive cells in the growth plates of the tibia with and without SOCs loaded vertically (A, C) or at an angle (B, D). (E–F) Representative images and quantification of TUNEL staining (green) (E) and cleaved caspase-3 staining (red) (F) in the growth plates of loaded tibias with and without SOCs. DAPI was used for counterstaining (blue). Data are means ± SD, two-tailed Student’s t-test. In (A–F), n = 3. The control and loaded tibia were from the same animal. VL, vertical load, AL, angled load. RZ and PZ, resting zone and proliferative zone combined, HZ, hypertrophic zone. ‘tibia with SOC’ and ‘tibia without SOC’ in the figure refer to tibias from 30-day-old mice and 10-day-old rats, respectively (see Figure 5—figure supplement 1A–B).

Figure 5.

Figure 5—figure supplement 1. Establishing and characterizing SOC± model in relation to stifness and cell death.

Figure 5—figure supplement 1.

(A) Mouse (30-day-old) and rat (10-day-old) tibia with and without a SOC, respectively, have similar physical dimensions of the epiphyses (d1, d2, h). (B) Representative images and quantification of the nanoindentation test of 30-day-old mice and 10-day-old rats. (C–D) The stiffness of bones with fully developed SOCs (tibia with SOC) and with immature SOCs (tibia without SOC) was measured at different loads and in both the vertical (C) and angled (D) direction. (E–J) Representative load/deformation (E–F) and stress/strain (G–H) curves of vertically (VL) (E, G) and angularly (AL) (F, H) loaded tibias with and without SOCs. (I–J) The elasticity of tibias with and without SOCs under vertical (I) and angled load (J). (K–L) The QPCR analysis of Indian hedgehog (Ihh) (K) and parathyroid hormone-related peptide (PTHrP) (L) levels in the epiphyseal end of tibia with and without SOCs. (M) Representative images of growth plates of tibias with and without SOCs fixed after dissection and prior loading. TUNEL staining (green) and nucleus counterstaining with DAPI (blue). (N) Fluorescent in situ hybridization of collagen type X (ColX) in the loaded growth plates of tibias with and without SOCs. VL, vertical load, AL, angled load. RZ and PZ, resting zone and proliferative zones, HZ, hypertrophic zone. (O) QPCR analysis of ColX level in the epiphyseal end of tibia with and without SOCs exposed to different loading. Data are means ± SD, two-tailed Student’s t-test, except in (B) where Mann-Whitney U-test was used. In (A), n = 23 for ‘Mouse’ and n = 15 for ‘Rat’. In (B), n = 5 for ‘Mouse’ and n = 4 for ‘Rat’. In (C), n = 10 for ‘tibia with SOC’ at all loads; n = 9 for ‘tibia without SOC’ at 0–1N and 1–4N, n = 4 for>4N. In (D), n = 11 for ‘tibia with SOC’ at all loads; n = 8 for ‘tibia without SOC’ at all loads. In (I–L, O), n = 3. ns, not significant.
Figure 5—figure supplement 2. Death of the growth plate chondrocytes upon various loading in the presence and absence of the SOC.

Figure 5—figure supplement 2.

(A–B) Representative images and quantification of the distribution of propidium iodide (PI)-positive cells in the growth plates of tibia with (A) and without a SOC (B) loaded vertically or angularly. During the end of culture period, bones were incubated with PI, which penetrates only into dead cells (red). Representative pictures (images) and quantifications (graphs) are shown. DAPI (blue) counterstaining of all nucleus was done on the fixed tissue sections. RZ and PZ, resting zone and proliferative zones, HZ, hypertrophic zone. ‘tibia with SOC’ and ‘tibia without SOC’ refer to tibias from 30-day-old mice and 10-day-old rats, respectively (see Figure 5—figure supplement 1A–B). Data are means ± SD, two-tailed Student’s t-test. In (A–B), n = 3.
Figure 5—figure supplement 3. YAP/p73 activation the growth plate chondrocytes upon loading.

Figure 5—figure supplement 3.

(A–C) Immunostaining and quantification of YAP (A), p73 (B), and YAP + p73 double-positive cells (C) in the growth plates of loaded tibias with and without SOCs loaded vertically (VL) and angularly (AL). RZ and HZ, resting zone and proliferative zone, HZ, hypertrophic zone. ‘tibia with SOC’ and ‘tibia without SOC’ refer to tibias from 30-day-old mice and 10-day-old rats, respectively (see Figure 5—figure supplement 1A–B). Data are means ± SD, two-tailed Student’s t-test. In (A–C), n = 3.