3D profiling of mouse epiphyses across ages reveals new potential imaging biomarkers of early spontaneous osteoarthritis

Abstract

Worldwide research groups and funding bodies have highlighted the need for imaging biomarkers to predict osteoarthritis (OA) progression and treatment effectiveness. Changes in trabecular architecture, which can be detected with non-destructive high-resolution CT imaging, may reveal OA progression before apparent articular surface damage. Here, we analysed the tibial epiphyses of STR/Ort (OA-prone) and CBA (healthy, parental control) mice at different ages to characterise the effects of mouse age and strain on multiple bony parameters. We isolated epiphyseal components using a semi-automated method, and measured the total epiphyseal volume; cortical bone, trabecular bone and marrow space volumes; mean trabecular and cortical bone thicknesses; trabecular volume relative to cortical volume; trabecular volume relative to epiphyseal interior (trabecular BV/TV); and the trabecular degree of anisotropy. Using two-way ANOVA (significance level ≤0.05), we confirmed that all of these parameters change significantly with age, and that the two strains were significantly different in cortical and trabecular bone volumes, and trabecular degree of anisotropy. STR/Ort mice had higher cortical and trabecular volumes and a lower degree of anisotropy. As the two mouse strains reflect markedly divergent OA predispositions, these parameters have potential as bioimaging markers to monitor OA susceptibility and progression. Additionally, significant age/strain interaction effects were identified for total epiphyseal volume, marrow space volume and trabecular BV/TV. These interactions confirm that the two mouse strains have different epiphyseal growth patterns throughout life, some of which emerge prior to OA onset. Our findings not only propose valuable imaging biomarkers of OA, but also provide insight into ageing 3D epiphyseal architecture bone profiles and skeletal biology underlying the onset and development of age-related OA in STR/Ort mice.

Keywords: biomarkers; cortical bone; epiphysis; imaging; micro-CT; mouse model; osteoarthritis; trabecular bone.

FIGURE 1

Trabecular segmentations of mouse tibia epiphyses. Red, CBA (healthy) and blue, STR/Ort (OA) trabeculae. Left‐hand side images (a, c, e, g, i, k) show central slices from reconstructed μCT scans viewed in coronal plane, with segmented trabeculae highlighted (blue or red). Right‐hand side images (b, d, f, h, j, l) show corresponding trabeculae rendered as 3D volume, viewed from above. (a/b) 8–11 week CBA; (c/d) 8–11 week STR/Ort; (e/f) 18–20 week CBA; (g/h) 18–20 week STR/Ort; (i/j) 40+ week CBA; (k/l) 40+ week STR/Ort. Age groups are indicated on right‐hand side. Scale bars = 1 mm. c.b., cortical bone, m.s., marrow space; t.b., trabecular bone.

FIGURE 2

Parameters affected by mouse age but with no significant difference between strains at any of the time points. (a) Epiphyseal trabecular volume relative to cortical volume and (b) trabecular thickness was significantly affected by mouse age in younger CBA mice, but with no significant difference identified between the two strains at any given age. Lines on plot connect mean values distinguishing between STR/Ort (OA, blue) and CBA (healthy, red) mice. (a) Two‐way ANOVA with Tukey's post hoc test. (b) Non‐parametric independent‐samples Kruskal–Wallis test. Significance levels p < 0.05 are indicated by *, p < 0.01 by **, and p < 0.001 by ***. Purple dotted lines denote a significant difference between age groups when strains are combined (Table 5). Trabecular/cortical volume was significantly smaller in 18–20 and 40+ week mice compared to 8‐ to 11‐week‐old mice (p < 0.001). Trabecular thickness in CBA mice was significantly larger at 18–20 weeks versus 8‐ to11‐week‐old mice (p < 0.001).

FIGURE 3

Cortical thickness is affected by mouse strain and age, but exhibits overlap between strains, showing poor promise as a biomarker. STR/Ort mice (blue) have significantly higher cortical thickness (p = 0.040) than CBA mice (red). Eight‐ to eleven‐week‐old mice have significantly lower cortical thickness than 18–20‐week‐old mice (p < 0.001) and than 40‐week‐old mice (p < 0.001). Significance levels p < 0.05 are indicated by *, p < 0.01 by ** and p < 0.001 by ***. Blue indicates STR/Ort mice (OA) and red indicates CBA mice (healthy). Purple dotted lines denote a significant difference between age groups when strains are combined (Table 5). Black dashed lines denote a significant difference between the two strains. Two‐way ANOVA with Tukey's post hoc test, significance level p < 0.05.

FIGURE 4

Cortical volume, trabecular volume and trabecular anisotropy differ between STR/Ort and CBA mice. These parameters also change with age, but there is no significant strain‐age interaction, meaning strains are differentiable throughout life and follow similar ageing trajectories. (a) STR/Ort mice have significantly greater cortical bone volume than CBA (p < 0.001), a feature that also increases significantly for both strains with age between 8–11 and 18–20 weeks (p = 0.001). (b) STR/Ort mice have significantly greater trabecular bone volume than CBA (p = 0.001), a feature which also decreases with age (p = 0.007 between 8–11 and 18–20 weeks, and p = 0.052 between 18–20 weeks and 40+ weeks). (c) STR/Ort mice have significantly lower trabecular anisotropy than CBA (p = 0.002), and trabecular anisotropy decreases significantly between 18–20 and 40 + weeks for both strains (p = 0.016). (a–c) Two‐way ANOVA with Tukey's post hoc test. * denotes p < 0.05, ** p < 0.01 and *** p < 0.001 by. STR/Ort (blue, OA) and CBA mice (red, healthy). Purple dotted lines denote a significant difference between age groups when strains are combined (Table 5). Black dashed lines denote a significant difference between the two strains.

FIGURE 5

Total epiphyseal volume and marrows space volume (MSV) depend on strain, and strains show different ageing effects. (a) STR/Ort (OA, blue) and CBA (healthy, red) mice have no significant difference between epiphyseal volumes at 8–11 weeks (p = 0.841) and 40+ weeks (p = 0.416) of age, but their growth patterns are divergent, such that STR/Ort mice have a significantly greater epiphyseal volume than CBA at 18–20 weeks (p < 0.001), which represents early‐stage OA. Both mouse strains have significantly greater epiphyseal volumes at 40+ weeks than at 8–11 weeks of age (p < 0.006), and STR/Ort mice also have significantly greater total epiphyseal volume at 18–20 weeks versus 8–11 weeks (p = 0.002). (b) STR/Ort (OA, blue) mice reveal a significantly lower marrow space volume value than CBA mice at 8–11 weeks of age (p = 0.001), but this is reversed by 18–20 weeks, when STR/Ort MSV is significantly greater (p = 0.009) than that of their CBA (healthy, red) counterparts. There are no significant MSV differences detectable at 40+ weeks between the strains (p = 0.083). Eight‐ to eleven‐week‐old CBA mice have significantly higher marrow space volume than 18–20 week mice, and 18–20 week mice have significantly lower marrow space volume than 40+ week mice (both p‐values <0.001). For STR/Orts, only 40+ week mice are significantly different from 8‐ to 11‐week‐old mice (p = 0.016). Two‐way ANOVA with Tukey's post hoc test, significance levels p < 0.05 are indicated by *, p < 0.01 by ** and p < 0.001 by ***. Blue indicates STR/Ort mice (OA) and red indicates CBA mice (healthy). Black dashed lines denote a significant difference between the two strains.

FIGURE 6

Trabecular BV/TV depends on strain, and strains show disparate ageing phenotypes. Blue indicates STR/Ort mice (OA) and red indicates CBA mice (healthy). There is no significant difference between the two strains at 18–20 weeks (p = 0.1.0) or 40+ weeks of age (0.092), but CBA have a significantly lower relative trabecular volume than STR/Ort at 8–11 weeks old (p < 0.001). Two‐way ANOVA with post hoc pairwise comparison test, significance levels p < 0.05 are indicated by *, p < 0.01 by ** and p < 0.001 by ***. Black dashed lines denote a significant difference between the two strains.