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 31 P 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.