Microdamage induced by in vivo Reference Point Indentation in mice is repaired by osteocyte-apoptosis mediated remodeling.

Reference Point Indentation (RPI) is a technology that is designed to measure mechanical properties that relate to bone toughness, or its ability to resist crack growth, in vivo. Independent of the mechanical parameters generated by RPI, its ability to initiate and propagate microcracks in bone is itself an interesting issue. Microcracks have a crucial biological relevance in bone, are central to its ability to maintain homeostasis. In healthy tissues, a process of targeted remodeling routinely repairs microcracks in a process mediated by osteocyte apoptosis. However, in diseases such as osteoporosis this process becomes deficient and microcracks can accumulate. Small animal models such are crucial for the study of such diseases, but it is technically challenging to create microcracks in these animals without causing outright failure. Therefore we sought to use RPI as a focal microdamage placement tool, to introduce microcracks to mouse long bones and investigate whether the same pathway mediates their repair as that described in other microdamage systems. We first used SEM to confirm that microdamage is formed RPI in mouse bone. Then, since RPI is carried out transdermally, we sought to confirm that no periosteal response occurred at the indented region. We then used a pan-caspase inhibitor (QVD) to determine whether osteocyte apoptosis plays the same pivotal role in microdamage repair in this model, as has been demonstrated in others. In conclusion, we validated that the microdamage-apoptosis-remodeling pathway is maintained with this method of microdamage induction in mice. We show that RPI can be used as a reliable and reproducible microdamage placement tool in living mouse long bones without inducing a periosteal response. We also used a caspase inhibitor, to block osteocyte apoptosis and thus abrogate the remodeling response to microdamage. This demonstrates that the well described microdamage repair system, involving targeted remodeling mediated by osteocyte apoptosis, is conserved in this novel mouse model using an in vivo RPI loading system.

Temporal effect of in vivo tendon fatigue loading on the apoptotic response explained in the context of number of fatigue loading cycles and initial damage parameters.

Accumulation of damage is a leading factor in the development of tendinopathy. Apoptosis has been implicated in tendinopathy, but the biological mechanisms responsible for initiation and progression of these injuries are poorly understood. We assessed the relationship between initial induced damage and apoptotic activity 3 and 7 days after fatigue loading. We hypothesized that greater apoptotic activity (i) will be associated with greater induced damage and higher number of fatigue loading cycles, and (ii) will be higher at 7 than at 3 days after loading. Left patellar tendons were fatigue loaded for either 100 or 7,200 cycles. Diagnostic tests were applied before and after fatigue loading to determine the effect of fatigue loading on hysteresis, elongation, and loading and unloading stiffness (damage parameters). Cleaved Caspase-3 staining was used to identify and calculate the percent apoptosis in the patellar tendon. While no difference in apoptotic activity occurred between the 100 and 7,200 cycle groups, greater apoptotic activity was associated with greater induced damage. Apoptotic activity was higher at 7 than 3 days after loading. We expect that the decreasing number of healthy cells that can repair the induced damage in the tendon predispose it to further injury.