Gestational Protein Restriction Promotes Osteoporotic Phenotype During Aging

Abstract Osteoporosis results from impaired bone remodeling and deterioration of bone microarchitecture, ultimately increasing fracture risk. Although early-life nutritional stress has been implicated in long-term health outcomes, its impact on skeletal remodeling mechanisms remains unclear. We investigated whether gestational protein restriction induces persistent alterations in the skeletal immune microenvironment that contribute to age-related bone fragility. Pregnant C57BL/6J mice were fed either a normal-protein or low-protein (LP) diet during gestation. Male offspring were evaluated at 12 and 18 months of age. Microcomputed tomography revealed significant reductions in trabecular bone volume fraction, bone mineral density, and trabecular number in 18-month-old LP offspring, along with reduced cortical thickness at both ages. Three-point bending tests demonstrated reduced maximal load in LP-18 M femora, indicating compromised mechanical strength. Ultrastructural analyses revealed increased cortical porosity and disrupted collagen fiber organization in aged LP bone. RNA sequencing identified differentially expressed genes enriched in pathways related to macrophage recruitment, complement signaling, and osteoclast differentiation. RT-qPCR confirmed increased expression of C5ar1 , Ccl3 , Ccr2 , Lilrb4a , and Trem2 . Immunohistochemical analysis demonstrated increased CD68 + cells in aged LP animals, indicating expansion of the skeletal myeloid compartment. These findings demonstrate that gestational protein restriction induces long-term reprogramming of the skeletal immune microenvironment, associated with enhanced osteoclast-related signaling and impaired bone mechanical competence during aging.