Document Type

Article

Publication Date

11-2026

Identifier

DOI: 10.1093/jbmrpl/ziag162; PMCID: PMC13695144

Abstract

Quantifying how local mechanical strain around individual osteocytes relates to Wnt/β-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 (TCF Optimal Promoter-β-galactosidase) Wnt/β-catenin reporter mouse 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 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 2 dyes: DDAO (7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one)) to indicate the level of Wnt/β-catenin signaling in osteocytes, and FITC (Fluorescein isothiocyanate isomer I) 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/β-catenin activation with local lacunar wall strain and can be extended in future studies to larger cohorts and additional signaling pathways.

Journal Title

JBMR Plus

Volume

10

Issue

11

First Page

162

Last Page

162

PubMed ID

42927309

Keywords

DDAO; Wnt/β-catenin; analysis/quantitation of bone; confocal microscopy; finite element analysis; mechanotransduction; multiplexed imaging; osteocyte; strain

Comments

Grants and funding

This article is a work of the United States government. Such works are not entitled to domestic copyright protection under U.S. law and are therefore in the public domain.

Publisher's Link: https://academic.oup.com/jbmrplus/article/10/11/ziag162/8869159?login=false

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