Developing a biodegradable scaffold remains a major challenge in bone tissue engineering. This study was aimed at developing novel alginate-chitosan-collagen (SA-CS-Col)-based composite scaffolds consisting of graphene oxide (GO) to enrich porous structures, elicited by the freeze-drying technique. To characterize porosity, water absorption, and compressive modulus, GO scaffolds (SA-CS-Col-GO) were prepared with and without Ca-mediated crosslinking (chemical crosslinking) and analyzed using Raman, Fourier transform infrared (FTIR), X-ray diffraction (XRD), and scanning electron microscopy techniques. The incorporation of GO into the SA-CS-Col matrix increased both crosslinking density as indicated by the reduction of crystalline peaks in the XRD patterns and polyelectrolyte ion complex as confirmed by FTIR. GO scaffolds showed increased mechanical properties which were further increased for chemically crosslinked scaffolds. All scaffolds exhibited interconnected pores of 10-250 μm range. By increasing the crosslinking density with Ca, a decrease in the porosity/swelling ratio was observed. Moreover, the SA-CS-Col-GO scaffold with or without chemical crosslinking was more stable as compared to SA-CS or SA-CS-Col scaffolds when placed in aqueous solution. To perform in vitro biochemical studies, mouse osteoblast cells were grown on various scaffolds and evaluated for cell proliferation by using MTT assay and mineralization and differentiation by alizarin red S staining. These measurements showed a significant increase for cells attached to the SA-CS-Col-GO scaffold compared to SA-CS or SA-CS-Col composites. However, chemical crosslinking of SA-CS-Col-GO showed no effect on the osteogenic ability of osteoblasts. These studies indicate the potential use of GO to prepare free SA-CS-Col scaffolds with preserved porous structure with elongated Col fibrils and that these composites, which are biocompatible and stable in a biological medium, could be used for application in engineering bone tissues.

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 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.

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Carbon quantum dots (CDs) are widely investigated because of their low toxicity, outstanding water solubility, and high biocompatibility. Specifically, fluorescent CDs have attracted ever-increasing interest. However, so far, only a few studies have focused on assessing the fluorescence of nitrogen-doped CDs (N@CDs) during in vivo exposure. Here, we describe a strategy for low-cost, one-pot synthesis of N@CDs. The low toxicity and suitability of the N@CDs for fluorescence imaging are validated using zebrafish (ZF) as a model. Strong fluorescence emission from ZF embryos and larvae confirms the distribution of N@CDs in ZF. The retention of N@CDs is very stable, long lasting, and with no detectable toxicity. The presence of a strong fluorescence at the yolk sac, especially in the vicinity of the intestine, suggests that a high content of N@CDs entered the digestive system. This indicates that N@CDs may have potential imaging applications in elucidating different aspects of lipoprotein and nutritional biology, in a ZF yolk lipid transport and metabolism model. On the other hand, the presence of a strong selective fluorescence at the eyes and melanophore strips at the trunk and tail region of ZF larvae suggests that N@CDs has a high melanin-binding affinity. These observations support a novel and revolutionary use of N@CDs as highly specific bioagents for eye and skin imaging and diagnosis of defects in them. N@CDs are known for their multifunctional applications as highly specific bioagents for various biomedical applications because of their exceptional biocompatibility, photostability, and selective affinity. These characteristics were validated in the developmental ZF model.

The aim of this study was to develop a chitosan-based risedronate/zinc-hydroxyapatite intrapocket dental film (CRZHDF) for applications in the treatment of alveolar bone loss in an animal model of periodontitis. The physical characteristics (folding endurance, pH, mucoadhesive strength, risedronate content and release) of CRZHDF, exhibited results within the limit. X-ray diffraction analysis indicates reduced or disappeared crystallinity of risedronate and zinc-hydroxyapatite in presence of chitosan. Further, FTIR studies revealed stability of CRZHDF and compatibility between risedronate, zinc-hydroxyapatite and chitosan. Periodontitis was induced by Porphyromonas gingivalis-lipopolysaccharide injections around the mandibular first molar. We divided rats into 5 groups (12 rats/group): healthy, untreated periodontitis; periodontitis plus CRZHDF-A, periodontitis plus CRZHDF-B, and periodontitis plus chitosan film. After four weeks, blood samples and mandibles were obtained for biochemical, radiographic and histological analysis. Bone specific alkaline phosphatise activity and tartrate resistant acid phosphatase 5b was statistically lower in CRZHDF-A and CRZHDF-B groups as compared to the untreated periodontitis group (p < 0.0001). The expression of osteocalcin was statistically higher in CRZHDF-A and CRZHDF-B groups as compared to the untreated periodontitis group (p < 0.0001). Alveolar bone was intact in the healthy group. Local administration of CRZHDF resulted in significant improvements in the mesial and distal periodontal bone support (MPBS and DPBS, respectively) proportions (%), bone mineral density, and also reversed alveolar bone resorption when compared to the untreated periodontitis group (p < 0.001). The study reported here reveals that novel CRZHDF treatment effectively reduced alveolar bone destruction and contributes to periodontal healing in a rat model of experimental periodontitis.

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