| Hydroxyapatite(HAP) is the main inorganic component of bones and teeth in vertebrates. It has been reported that the biocompatibility of HAP depend on their morphology and size. HAP with suitable morphology and size can promote osteoblasts(OBs) proliferation and differentiation. Therefore, HAP has broad application prospects for bone tissue engineering. Nano-sized HAP could cross cell membrane and enter cells, however the pathway of HAP enters cells and the intracellular distribution of HAP after exposure is still unclear, and the effect on osteogenic differentiation of OBs needs further studies. This dissertation focuses on the research team synthesized defect-related luminescent nano- and microstructured HAP, the physicochemical properties characterize, biocompatibility evaluation, uptake and subcellular localization, and the effect on the differentiation of OBs. The main contents are as follows:1. Synthesis and characterization of defect-related luminescent nano- and microstructured HAP. In this paper, three types of defect-related luminescent HAP are synthesized by hydrothermal synthesis: short nanorod(S1), long nanorod(S2), and bur-like microsphere(S3) which self-assembled from nanorods. Chemical composition and the functional groups of the materials are confirmed by XRD and FTIR analysis. Fluorescence spectroscopy shows excitation wavelength falls in the ultraviolet region, the emission wavelength falls in the blue-violet region. DLS and the Zeta potential results showed that the nanorod HAP(S1, S2) particles were agglomerated in Dulbeccol’s Modified Eagle’s Medium(DMEM), and had a good stability in DMEM. There is not any other luminescent groups added during the synthesis process of defect-related luminescent HAP, and the synthesized HAP has the same chemical composition similar to natural HAP. Therefore, studies on the biological effects of defect-related luminescent HAP are analogy to the real effects of natural HAP in bone tissue.2. Biocompatibility evaluation of defect-related luminescent HAP. Biocompatibilities of three defect-related luminescent HAP particles are studied in five areas including material corrosion resistance, cytotoxicity, inflammatory, genotoxicity and blood compatibility. All of the HAP showed non-inflammatory, good corrosion resistance and blood compatibility. Cytotoxicity results showed sample S1 and S3 inhibited the activity of OBs, causing the OBs shrinkage and increased intracellular ROS production. Comet assay also confirmed a certain genetic toxicity of sample S1 and S3. Sample S2 shows neither cytotoxicity nor genotoxicity, and it has good biocompatibility.3. The mechanism of cellular uptake and subcellular localization of defect-related luminescent HAP in OBs. It has been reported that nano-sized HAP can enter cells, but the mechanism of cellular uptake and subcellular localization has not been described clearly. In this paper, it is confirmed that HAP entered into OBs mainly through macropinocytosis-dependent and caveolae-mediated internalization. Confocal microscopy revealed that nanorod S1 and S2 entered into OBs and located in the lysosomes, but unable to enter the nucleus. Bur-like microsphere S3 did not enter OBs.4. The effect of defect-related luminescent HAP on OBs osteogenic differentiation function. Defect-related luminescent HAP particles inhibit the activity of ALP which was an early osteogenic differentiation of OBs, but HAP promotes the expression of collagen and the formation of mineralization nodules. By real-time quantitative PCR analysis, S2 promotes the expression of osteogenic genes OCN, while S1 and S3 suppressed the expression of OCN for up to 14 days of culture. Then the mineralization nodules were observed by fluorescence microscopy, we can found that the centre of the mineralized nodules emitted blue fluorescence, which came from defect-related luminescent HAP particles. The results suggested that HAP has a certain nucleation role in promoting the formation of mineralized nodules.In summary, S1 and S2 enter OBs through the pathways of macropinocytosis and caveolae-mediated endocytosis. Intracellular S1 promotes the increase of intracellular ROS level, which resulted in DNA damage. Internalized S1 and S2 are located in lysosome and release Ca2+ in the acidic environment of lysosomes, which provides Ca2+ source for cell mineralization. S1, S2 and S3 outside OBs promote mineralization due to the nucleation effect. Nano-HAP promotes the formation of mineralization nodules through both intracellular and extracellular pathway. |
