Aging-Induced Discrepant Response of Fracture Healing is Initiated from the Organization and Mineralization of Collagen Fibrils in Callus

Using a murine tibial osteotomy model, this study investigated how aging alters fracture-healing structure–function outcomes and how mechanical immobilization differentially modulates healing in young versus aged animals. Young (5-week) and aged (68-week) mice underwent internal fixation with an intramedullary steel needle and were assigned to free-movement controls or hindlimb tail-suspension immobilization. At day 21 post-fracture, the callus microstructure (micro-CT), mineral/organic composition (TGA, EDS, Raman, XRD), collagen fibril organization (AFM), and multiscale mechanics (nanoindentation and whole-bone bending) were quantified. Aging delayed healing and reduced callus mineralization and mechanics (lower BV/TMD, lower mineral-to-organic ratio, shorter mineral crystals, reduced modulus/hardness and bending strength). Immobilization produced age-dependent effects: in aged mice it increased mineral crystal length and mineral-to-matrix ratios and improved callus quality, whereas in young mice it disordered collagen fibrils, increased carbonate substitution, and reduced mineral content/crystal length, leading to inferior mechanical recovery. The authors conclude that age-specific mechanobiological interventions may be required because collagen organization and mineralization pathways respond differently to immobilization across age groups.