Distinct cyclosporin a doses are required to enhance bone formation induced by cyclic and rest-inserted loading in the senescent skeleton

Abstract

Age-related decline in periosteal adaptation negatively impacts the ability to utilize exercise to enhance bone mass and strength in the elderly. We recently observed that in senescent animals subject to cyclically applied loading, supplementation with Cyclosporin A (CsA) substantially enhanced the periosteal bone formation rates to levels observed in young animals. We therefore speculated that if the CsA supplement could enhance bone response to a variety of types of mechanical stimuli, this approach could readily provide the means to expand the range of mild stimuli that are robustly osteogenic at senescence. Here, we specifically hypothesized that a given CsA supplement would enhance bone formation induced in the senescent skeleton by both cyclic (1-Hz) and rest-inserted loading (wherein a 10-s unloaded rest interval is inserted between each load cycle). To examine this hypothesis, the right tibiae of senescent female C57BL/6 mice (22 Mo) were subjected to cyclic or rest-inserted loading supplemented with CsA at 3.0 mg/kg. As previously, we initially found that while the periosteal bone formation rate (p.BFR) induced by cyclic loading was enhanced when supplemented with 3.0 mg/kg CsA (by 140%), the response to rest-inserted loading was not augmented at this CsA dosage. In follow-up experiments, we observed that while a 30-fold lower CsA dosage (0.1 mg/kg) significantly enhanced p.BFR induced by rest-inserted loading (by 102%), it was ineffective as a supplement with cyclic loading. Additional experiments and statistical analysis confirmed that the dose-response relations were significantly different for cyclic versus rest-inserted loading, only because the two stimuli required distinct CsA dosages for efficacy. While not anticipated a priori, clarifying the complexity underlying the observed interaction between CsA dosage and loading type holds potential for insight into how bone response to a broad range of mechanical stimuli may be substantially enhanced in the senescent skeleton.

Figure 1. Bone response at the mid-shaft tibiae from animals subject to loading with or without CsA supplementation.

Illustrative fluorescent images from animals subject to cyclic loading (a) and supplemented with 3.0 mg/kg CsA (b), or subject to rest-inserted loading (c) and supplemented with 0.1 mg/kg CsA (d).

Figure 2. CsA supplementation significantly amplified bone response to cyclic loading.

Periosteal MS/BS (a), MAR (b), and BFR/BS (c; mean+s.e.) were significantly increased in experimentally loaded (X) versus contralateral controls (C; ‘+’). Supplementing cyclic loading with CsA (at 3.0 mg/kg) significantly enhanced MAR and BFR/BS compared with that induced by loading alone (‘#’).

Figure 3. Lower-dose CsA supplementation was required to amplify bone response to rest-inserted loading.

Periosteal MS/BS (a), MAR (b), and BFR/BS (c; mean+s.e.) were significantly increased in experimentally loaded (X) versus contralateral controls (C; ‘+’). Supplementing rest-inserted loading, but not cyclic loading, with low-dose CsA (at 0.1 mg/kg) significantly enhanced MAR and BFR/BS compared with that induced by rest-inserted loading alone (‘#’). Additionally, CsA supplementation enhanced MAR (at 0.1, 0.3 mg/kg) and BFR/BS (at 0.1 mg/kg) in animals subject to rest-inserted versus cyclic loading (‘$’).

Figure 4. Dose-response relations simulated by the implemented non-linear models.

A single model, which assumed that there is no adaptive benefit of loading supplemented with CsA, could simulate the rp.MS/BS data (a; mean ± s.e.). In contrast, two separate models were required to simulate rp.MAR (b) and rp.BFR/BS (c) induced by cyclic and rest-inserted loading supplemented with CsA. Please note that CsA dosage is plotted on a log-scale.