RESEARCH SUMMARY
This study investigated whether partial Beclin-1 deficiency alters bone mechanical quality and the osteocyte lacunar-canalicular microenvironment independently of changes in bone mass or geometry. Fourteen-week-old male Becn1+/− mice were compared with age-matched wild-type controls using micro-CT, whole-bone mechanical testing, tissue-level microindentation, histology, osteocyte lacunar-canalicular imaging, mineral analysis, and molecular assays. Despite reduced Beclin-1 expression, the mutant mice showed no significant differences in trabecular bone volume, trabecular architecture, cortical thickness, cortical area, minimum moment of inertia, or cortical tissue mineral density. Mechanical performance, however, was substantially impaired. Whole-femur three-point bending showed 23.7% lower maximum load, 22.3% lower stiffness, 31.9% lower work to fracture, 22.9% lower apparent bending modulus, and 23.6% lower ultimate bending stress in Becn1+/− mice. Separate cortical microindentation also showed reduced tissue-level Young’s modulus and yield stress. Osteocyte lacunar-canalicular architecture was altered, with canalicular length reduced by 44.2%, canalicular width increased by 34.3%, and pericanalicular thickness increased by 27.7%. The lacunar-wall calcium-to-phosphorus ratio was reduced while interstitial cortical matrix Ca/P remained unchanged, and osteocyte lacunae showed increased relative acidification. Expression of the acidification- and remodeling-associated markers Atp6v0b, Ctsk, and Acp5 increased, whereas Mmp13 was unchanged. Similar Ctsk and Acp5 responses were reproduced after Becn1 knockdown in MLO-Y4 osteocyte-like cells. Overall, the results demonstrate that reduced Beclin-1 expression can impair cortical bone mechanical competence despite preserved bone mass and geometry and suggest that alterations in osteocyte canalicular architecture, pericellular geometry, mineral composition, and remodeling activity may contribute to reduced bone quality.
A CellScale UniVert mechanical test system was used to perform three-point bending to failure on femurs from 14-week-old male Becn1+/− mice and age-matched wild-type controls. Before testing, the same femurs had undergone micro-CT analysis for measurement of bone geometry. Each femur was positioned in a consistent anatomical orientation on two supports separated by a 7 mm span. A 0.1 N preload was applied to establish contact, after which the UniVert loaded the femoral mid-diaphysis at a displacement rate of 0.05 mm/s until failure. Force-displacement data were recorded at 0.2-second intervals and analyzed with the authors’ Python-based 3PB-Analyzer software. The UniVert data provided maximum load, stiffness, yield load, post-yield displacement, and work to fracture. When combined with mid-diaphyseal geometry measured by micro-CT, the same force-displacement results were also used to calculate apparent bending modulus and ultimate bending stress. Compared with wild-type controls, Becn1+/− femurs showed a 23.7% reduction in maximum load, 22.3% lower stiffness, 31.9% lower work to fracture, 22.9% lower apparent bending modulus, and 23.6% lower ultimate bending stress. These UniVert measurements demonstrated a clear loss of whole-bone mechanical competence despite the absence of significant genotype-related differences in cortical geometry or tissue mineral density, supporting the study’s central conclusion that Beclin-1 insufficiency impairs bone quality rather than simply reducing bone quantity.
