Abstract / Summary
Abstract Quantifying how local mechanical strain around individual osteocytes relates to Wnt-catenin signaling in situ remains technically challenging. Load-induced activation of the Wnt/β-catenin signaling pathway in osteocytes is an important step leading to anabolic bone formation, and neighboring osteocytes in murine cortical bone exhibit differential levels of Wnt/β-catenin signaling following cyclic axial compression loading. To better understand the mechano-responsiveness of individual osteocytes, we developed a protocol to quantify in situ relative Wnt/β-catenin reporter activity in individual osteocytes using the TOPGAL model and to relate this readout to local axial strain in the perilacunar bone matrix. In this proof-of-concept experiment, the right forelimb of a 5-mo-old male TOPGAL Wnt/β-catenin reporter mouse was subjected to cyclic axial compression loading at 2.25 N and 2 Hz for 100 cycles. One hour after loading, the non-loaded left and loaded right ulnae were co-stained with two dyes: DDAO to indicate the level of Wnt/β-catenin signaling in osteocytes, and FITC to label the lacunocanalicular structure. Micro-CT scans were used to model the ulnae, and three-dimensional confocal images were collected from slices in compressed and tensive regions. Image processing and 3D modeling software were used to evaluate DDAO signal intensity as a TOPGAL reporter readout of Wnt/β-catenin signaling for individual osteocytes and develop finite element models for strain analysis in the bone matrix. In non-loaded bone, every observed osteocyte showed a low, basal level of DDAO signal. Relative DDAO signal was approximately 25- to 50-fold higher in loaded than non-loaded bone and was heterogeneous among osteocytes following loading. Robust linear regression using an exploratory high-strain summary metric showed a positive association between Wnt/β-catenin activation and the predicted longitudinal strain magnitude in bone regions immediately surrounding each osteocyte. This approach provides a workflow for correlating single-osteocyte Wnt-catenin activation with local lacunar wall strain and can be extended in future studies to larger cohorts and additional signaling pathways.