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

Abstract

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.

Osteocyte death and bone overgrowth in mice lacking Fibroblast Growth Factor Receptors 1 and 2 in mature osteoblasts and osteocytes

“…This possibility could be tested by pharmacologically inhibiting osteocyte apoptosis with the pan-caspase inhibitor QVD, which has been shown to block osteocyte apoptosis and prevent pathological bone remodeling. (17,(63)(64)(65) At both 3 and 12 weeks of age, we observed increased Fgf23 mRNA expression in cortical bone. Given that the number of viable osteocytes is in decline, this suggests that the increased Fgf23 expression likely occurs in osteoblasts or pre-osteocytes.…”

Osteocyte Death and Bone Overgrowth in Mice Lacking Fibroblast Growth Factor Receptors 1 and 2 in Mature Osteoblasts and Osteocytes

“…This possibility could be tested by pharmacologically inhibiting osteocyte apoptosis with the pan-caspase inhibitor QVD, which has been shown to block osteocyte apoptosis and prevent pathological bone remodeling. (17,(67)(68)(69) At both 3 and 12 weeks of age, we observed increased Fgf23 mRNA expression in cortical bone of OC-Fgfr1/2 DCKO mice. Given that the number of viable osteocytes is in decline, this suggests that the increased Fgf23 expression likely occurs in osteoblasts or pre-osteocytes.…”

“…The increase in bone mass in OC‐Fgfr1/2 DCKO is preceded by the initial loss of osteocytes, suggesting that increased bone accrual is secondary to osteocyte loss. This possibility could be tested by pharmacologically inhibiting osteocyte apoptosis with the pan‐caspase inhibitor QVD, which has been shown to block osteocyte apoptosis and prevent pathological bone remodeling …”

“…At both 3 and 12 weeks of age, we observed increased Fgf23 mRNA expression in cortical bone of OC‐Fgfr1/2 DCKO mice. Given that the number of viable osteocytes is in decline, this suggests that the increased Fgf23 expression likely occurs in osteoblasts or pre‐osteocytes . Interestingly, it has been shown that viable osteocytes, within 100 to 300 µm from dying osteocytes, have increased expression of VEGF and RANKL .…”

“…Given that the number of viable osteocytes is in decline, this suggests that the increased Fgf23 expression likely occurs in osteoblasts or pre-osteocytes. (69) Interestingly, it has been shown that viable osteocytes, within 100 to 300 µm from dying osteocytes, have increased expression of VEGF and RANKL. (66,71) We posit that Fgf23 expression may be similarly increased in nearby viable osteocytes or pre-osteocytes in response to their dying neighbors.…”