Abstract
BACKGROUND: Besides its antimicrobial action, doxycycline (DX) has lately been repurposed as a small-molecule drug for osteogenic purposes. However, osteogenic DX application is impeded by its dose-dependent cytotoxicity. Further, high-dose DX impairs cell differentiation and mineralization. PURPOSE: Integrating DX into a biomaterial-based delivery system that can control its release would not only ameliorate its cytotoxic actions but also augment its osteogenic activity. In this work, we managed to engineer novel composite DX-hydroxyapatite-polycaprolactone nanoparticles (DX/HAp/PCL) to modify DX osteogenic potential. METHODS: Employing a 2(3)-factorial design, we first optimized HApN for surface-area attributes to maximize DX loading. Composite DX/HAp/PCL were then realized using a simple emulsification technique, characterized using various in vitro methods, and evaluated for in vitro osteogenesis. RESULTS: The developed HApN exhibited a favorable crystalline structure, Ca:P elemental ratio (1.67), mesoporous nature, and large surface area. DX/HAp/PCL achieved the highest reported entrapment efficiency (94.77%±1.23%) of DX in PCL-based particles. The developed composite system achieved controlled release of the water-soluble DX over 24 days. Moreover, the novel composite nanosystem managed to significantly ameliorate DX cytotoxicity on bone-marrow stem cells, as well as enhance its overall proliferation potential. Alkaline phosphatase and mineralization assays revealed superior osteodifferentiation potential of the composite system. Quantification of gene expression demonstrated that while DX solution was able to drive bone-marrow stem cells down the osteogenic lineage into immature osteoblasts after 10-day culture, the innovative composite system allowed maturation of osteodifferentiated cells. To the best of our knowledge, this is the first work to elaborate the impact of DX on the expression of osteogenic genes: RUNX2, OSP, and BSP. Further, the osteogenicity of a DX-loaded particulate-delivery system has not been previously investigated. CONCLUSION: Our findings indicate that repurposing low-dose DX in complementary biomaterial-based nanosystems can offer a prominent osteogenic candidate for bone-regeneration purposes.