Ex vivo validation of osteogenic efficacy in a bioceramic dual drug-delivery system

Alendronate (ALN) is widely employed against skeletal disorders, but its low oral bioavailability and prolonged skeletal retention necessitate high systemic doses that carry significant risk. The research team previously developed a novel bioceramic drug-delivery system (DDS) that sequentially releases gentamicin during the first two weeks post-implantation (with reported therapeutic ceoncentrations released daily) and ALN thereafter to to be used as a bone substitute. The main objective of this study was to validate the DDS’s osteogenic capacity ex vivo , once gentamicin had been depleted and only ALN is released. Before ex vivo testing,there was the need to confirm, via in vitro biological assessment, if the release profile of ALN achieved the therapeutic values needed. For that purpose, human bone-marrow stromal cells (hBMSCs) were exposed to ALN concentrations between 10⁻⁴ and 10⁻¹¹ M for 20 days. Concentrations from 10⁻⁶ to 10⁻¹⁰ M enhanced metabolic activity, alkaline-phosphatase activity, matrix mineralisation (Alizarin Red, von Kossa), and early- to mid-stage osteogenic gene expression (RUNX2, SP7, COL1A1, ALP, BMP-2), while late marker BGLAP remained suppressed, indicating ongoing differentiation. Having confirmed that the ALN release profile of the DDS met the therapeutic need, the DDS was then validated, as an osteogenic bone sustitute system, in the ex vivo model. For validation of the DDS, embryonic chick femurs were cultured ex vivo for 11 days with DDS preconditioned by release assay at day 17—representing the alendronate release phase, only. Treated femurs exhibited a significant increase in bone volume fraction, accompanied by enhanced deposition of a collagenrich extracellular matrix and a more highly organized trabecular network, while total femoral volume remained constant. Histological staining and polarizedlight microscopy further revealed that the extracellular matrix in the sample group was denser, more mature and organized than in controls. These findings verify that the sequential DDS releases ALN at osteogenically effective concentrations precisely when antibiotic coverage ends, affirming its capacity to couple early infection control with subsequent, localised stimulation of bone formation while avoiding systemic exposure.