Accelerated Bone Regeneration by Nitrogen-Doped Carbon Dots Functionalized with Hydroxyapatite Nanoparticles

We investigated the osteogenic potential of nitrogen-doped carbon dots (NCDs) conjugated with hydroxyapatite (HA) nanoparticles on the MC3T3-E1 osteoblast cell functions and in a zebrafish (ZF) jawbone regeneration (JBR) model. The NCDs-HA nanoparticles were fabricated by a hydrothermal cum co-precipitation technique. The surface structures of NCDs-HA nanoparticles were characterized by X-ray diffraction; Fourier transform infrared (FTIR), UV-vis, and laser fluorescence spectroscopies; and scanning electron microscopy, transmission electron microscopy (TEM), energy-dispersive spectrometry (EDS), and NMR analyses. The TEM data confirmed that the NCDs are well conjugated on the HA nanoparticle surfaces. The fluorescent spectroscopy results indicated that the NCDs-HA exhibited promising luminescent emission in vitro. Finally, we validated the chemical structure of NCDs-HA nanoparticles on the basis of FTIR, EDS, and P-31 NMR analysis and observed that NCDs are bound with HA by electrostatic interaction and H-bonding. Cell proliferation assay, alkaline phosphatase, and Alizarin red staining were used to confirm the effect of NCDs-HA nanoparticles on MC3T3-E1 osteoblast proliferation, differentiation, and mineralization, respectively. Reverse transcriptase polymerase chain reaction was used to measure the expression of the osteogenic genes like runt-related transcription factor 2, alkaline phosphatase, and osteocalcin. ZF-JBR model was used to confirm the effect of NCDs-HA nanoparticles on bone regeneration. NCDs-HA nanoparticles demonstrated cell imaging ability, enhanced alkaline phosphatase activity, mineralization, and expression of the osteogenic genes in osteoblast cells, indicating possible theranostic function. Further, NCDs-HA nanoparticles significantly enhanced ZF bone regeneration and mineral density compared to HA nanoparticles, indicating a therapeutic potential of NCDs-HA nanoparticles in bone regeneration and fracture healing.