Effect of rapamycin on bone mass and strength in the α2(I)-G610C mouse model of osteogenesis imperfecta

Abstract

Osteogenesis imperfecta (OI) is commonly caused by heterozygous type I collagen structural mutations that disturb triple helix folding and integrity. This mutant-containing misfolded collagen accumulates in the endoplasmic reticulum (ER) and induces a form of ER stress associated with negative effects on osteoblast differentiation and maturation. Therapeutic induction of autophagy to degrade the mutant collagens could therefore be useful in ameliorating the ER stress and deleterious downstream consequences. To test this, we treated a mouse model of mild to moderate OI (α2(I) G610C) with dietary rapamycin from 3 to 8 weeks of age and effects on bone mass and mechanical properties were determined. OI bone mass and mechanics were, as previously reported, compromised compared to WT. While rapamycin treatment improved the trabecular parameters of WT and OI bones, the biomechanical deficits of OI bones were not rescued. Importantly, we show that rapamycin treatment suppressed the longitudinal and transverse growth of OI, but not WT, long bones. Our work demonstrates that dietary rapamycin offers no clinical benefit in this OI model and furthermore, the impact of rapamycin on OI bone growth could exacerbate the clinical consequences during periods of active bone growth in patients with OI caused by collagen misfolding mutations.

Keywords: autophagy stimulation; biomechanics; diseases and disorders of bone; micro-computed tomography; osteogenesis imperfecta; rapamycin treatment.

Figure 1

Effect of rapamycin treatment on mouse growth and long bone length. WT and OI male mice were treated with dietary rapamycin (Rapa) or control diet for 5 weeks from 3 weeks to 8 weeks of age. N = 8 mice per genotype and treatment. (A) Mouse body weight (g) at start of treatment and at end of 5 weeks treatment (+Rapa). Mean with 95% CI. (B) Tibial length at end of the treatment. Control and rapamycin (Rapa) treatment groups are indicated, ● WT, ■ OI. All values are the mean ± SD; Two‐way ANOVA with Tukey's post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001. All WT and OI body weight data were significantly different (P < 0.0001) between 3 week and 8 week samples. For clarity in the figure, significance bars are not in shown between these samples

Figure 2

Micro‐CT of tibial trabecular bone. (A) BV/TV = bone volume fraction (%); (B) Tb.Th = trabecular thickness (μm); (C) Tb.N = trabecular number (mm⁻¹); (D) Tb.Sp = trabecular separation (μm). Control and rapamycin (Rapa) treatment groups are indicated, ● WT, ■ OI. All values are the mean ± SD; Two‐way ANOVA with Tukey's post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Figure 3

Micro‐CT of tibial cortical bone. (A) Ma.Ar = medullary area (mm²); (B) Ct.Ar = cortical bone area (mm²); (C) Endocortical perimeter (mm); (D) Periosteal perimeter (mm); (E) Cortical thickness (μm); (F) pMOI = polar moment of inertia (mm⁴). Control and rapamycin (Rapa) treatment groups are indicated, ● WT, ■ OI. All values are the mean ± SD; Two‐way ANOVA with Tukey's post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Figure 4

Four‐point bend testing of long bones. (A) Femora—Maximum load to failure (N); (B) Femora—Stiffness (N/mm); (C) Femora—Energy to maximum load (mJ); (D) Femora—Energy after maximum load (mJ); (E) Tibiae—Maximum load to failure (N); (F) Tibiae—Stiffness (N/mm); (G) Tibiae—Energy to maximum load (mJ); (H) Tibiae—Energy after maximum load (mJ). Control and rapamycin (Rapa) treatment groups are indicated; ● WT, ■ OI. Data were analysed using a two‐way ANOVA with Tukey's post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Figure 5

Compression testing of L4 vertebrae. (A) Maximum load to failure (N); (B) Stiffness (N/mm); (C) Energy to maximum load (mJ). Control and rapamycin (Rapa) treatment groups are indicated, ● WT, ■ OI. Data were analysed using a two‐way ANOVA with Tukey's post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Figure 6

OI mechanical data corrected for body size and bone length. To correct for the reduced body weight and bone length of rapamycin‐treated OI mice the data were plotted against individual mouse weight × bone length previously described.28 For each plot, the significance (P‐value) of the difference between the slopes of untreated OI (■, OI Control) and rapamycin‐treated OI (●, OI Rapa) bones are indicated (ANCOVA). The r ² value and P‐value for each plot is given below to describe if the slope is significantly non‐zero. (A) Femora—Maximum load to failure (N); OI Control r ² = 0.01558, P = 0.7684; OI Rapa r ² = 0.07437, P = 0.5540. (B) Femora—Stiffness (N/mm); OI Control r ² = 0.00668, P = 0.8475; OI Rapa r ² = 0.1436, P = 0.4019. (C) Tibiae—Maximum load to failure (N); OI Control r ² = 0.3598, P = 0.1160; OI Rapa r ² = 0.00185, P = 0.9271. (D) Tibiae—Stiffness (N/mm): OI Control r ² = 0.2116, P = 0.2514; OI Rapa r ² = 0.00069, P = 0.9554. Compression testing of L4 vertebrae. (E) Maximum load to failure (N); OI Control r ² = 0.365, P = 0.1127; OI Rapa r ² = 0.1602, P = 0.3259. (F) Stiffness (N/mm); OI Control r ² = 0.08933, P = 0.4721; OI Rapa r ² = 0.00005, P = 0.9870