Biomaterial-Based Nanocomposite for Osteogenic Repurposing of Doxycycline

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.

Preparation and Characterization of Doxycycline-Loaded Electrospun PLA/HAP Nanofibers as a Drug Delivery System

“…Doxy inhibits the production of protein by bacteria, which reduces their activity or leads to their complete death [ 27 , 28 ]. Due to the limited direct cell–drug contact, in the case of Doxy-loaded polymer-based systems, the dose-dependent cytotoxicity of the cells is eliminated [ 29 ]. Thus, the design and investigation of polymer-Doxy-based systems are primarily important in research.…”

Translating Material Science into Bone Regenerative Medicine Applications: State-of-The Art Methods and Protocols

“…The relative number of metabolically active cells seeded on the tested biomaterial is usually evaluated by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) colorimetric assay, based on the reduction of the yellow tetrazolium salt MTT to purple formazan crystals by NAD(P)H-dependent oxidoreductase enzymes present in metabolically active cells [ 14 , 56 , 60 , 61 , 65 , 68 , 82 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 , 100 ]. The enzyme lactate dehydrogenase (LDH) cytotoxicity assay is another widely used colorimetric assay that allows one to quantify the extracellular concentration of the LDH secreted upon cell damage [ 55 , 83 , 92 , 98 ].…”

“…They can proliferate and differentiate into osteoblasts upon appropriate stimuli. MSCs isolated from bone marrow are regarded as the gold standard [ 14 , 53 , 55 , 57 , 67 , 68 , 73 , 76 , 77 , 79 , 83 , 91 , 94 , 98 , 107 , 108 , 109 ]. MSC-like cells isolated from the stromal vascular fraction of the adipose tissue, namely, adipose-derived stem cells, ASC, are seldom used in selected applications [ 2 , 65 , 72 , 82 , 89 , 104 , 109 , 110 , 111 , 112 ].…”

“…In the vast majority of papers in the literature, the expression levels of one or more genes associated with osteoblast differentiation are usually evaluated by quantitative real-time PCR (qRT-PCR) and/or Western blot and/or immunofluorescence analysis and/or ELISA assay [ 14 , 55 , 57 , 58 , 60 , 62 , 64 , 65 , 68 , 69 , 70 , 72 , 73 , 76 , 77 , 79 , 80 , 82 , 83 , 84 , 86 , 87 , 89 , 90 , 91 , 94 , 96 , 98 , 100 , 108 , 109 , 113 ]. Table 3 reports the most representative osteogenic genes usually analyzed.…”

“…The analysis of alkaline phosphatase (ALPL) activity is one of the most common tests used to evaluate the osteogenic process and numerous easy-to-use and highly sensitive assays to measure ALPL activity are available on the market [ 14 , 53 , 54 , 55 , 58 , 61 , 65 , 66 , 67 , 68 , 70 , 71 , 73 , 76 , 79 , 80 , 83 , 85 , 86 , 88 , 90 , 91 , 93 , 94 , 95 , 96 , 97 , 98 , 99 , 107 , 114 , 115 ].…”