Osteogenic Potential of Tetracyclines: Insights From a Femoral Organotypic Model

Objectives: To evaluate the biological activity of four tetracyclines (TCs) from distinct generations, in bone structure and function, using a translational ex vivo femoral organotypic system (FOS).
Methods: FOS was established using dissected femora from 11-day-old chick embryos (Gallus domesticus) cultured at the air/liquid interface, 37°C, 5% CO₂ in air. FOS was maintained for 11 days in basal medium (αMEM containing 1% ascorbic acid) as controls, while experimental groups received basal medium supplemented with tetracycline (Tetra), doxycycline (Doxy), minocycline (Mino), or sarecycline (Sare) at 1 or 10µg/mL. FOS underwent histomorphometric analyses (n=9) through micro-computed tomography (Bruker Skyscan-1276), or histological evaluation upon staining with Alcian blue/Sirius red. The assessment of gene expression (n=5) was carried out by qRT-PCR. Statistical analysis was assessed using one-way ANOVA, followed by Tukey’s post-hoc test, p<0.05.
Results: Histomorphometric analyses of microtomographic and histological samples showed that TCs at 1µg/mL (excluding Tetra) significantly increased bone volume compared to control (p<0.045). Similarly, these experimental conditions promoted a thicker and more mature collagen outer layer at the diaphyseal area, while no significant effects were observed at 10µg/mL for the tested TCs. qPCR assay revealed that key osteogenic-related genes (RUNX2 and COL1A2) were upregulated by the incubation with Doxy, Mino and Sare (p<0.012). SOST expression was increased by Tetra, Mino and Sare (p<0.04), while osteochondrogenic genes such as SOX9 and ACAN were upregulated mostly by Doxy (p<0.01).
Conclusions: These findings suggest that low concentrations of second- and third-generation TCs, particularly Doxy, hold promise for enhancing bone regeneration. Doxy may be uniquely suited for applications requiring osteochondrogenic induction.