Nanohydroxyapatite shape and its potential role in bone formation: an analytical study

Bone cells (osteoblasts) produce a collagen-rich matrix called osteoid, which is mineralized extracellularly by nanosized calcium phosphate (CaP). Synthetically produced CaP nanoparticles (NPs) have great potential for clinical application. However few studies have compared the effect of CaP NPs with different properties, such as shape and aspect ratio, on the survival and behaviour of active bone-producing cells, such as primary human osteoblasts (HOBs). This study aimed to investigate the biocompatibility and ultrastructural effects of two differently shaped hydroxyapatite [Ca10(PO4)6(OH)2] nanoparticles (HA NPs), round- (aspect ratio 2.12, AR2) and rice-shaped (aspect ratio 3.79, AR4). The ultrastructural response and initial extracellular matrix (ECM) formation of HOBs to HA NPs were observed, as well as matrix vesicle release. A transmission electron microscopy (TEM)-based X-ray microanalytical technique was used to measure cytoplasmic ion levels, including calcium (Ca), phosphorus (P), sodium (Na) and potassium (K). K/Na ratios were used as a measure of cell viability. Following HA NP stimulation, all measured cytoplasmic ion levels increased. AR2 NPs had a greater osteogenic effect on osteoblasts compared with AR4 NPs, including alkaline phosphatase activity and matrix vesicle release. However, they produced only a moderate increase in intracellular Ca and P levels compared with AR4. This suggests that particular Ca and P concentrations may be required for, or indicative of, optimal osteoblast activity. Cell viability, as measured by Na and K microanalysis, was best maintained in AR2. Initial formation of osteoblast ECM was altered in the presence of either HA NP, and immuno-TEM identified fibronectin and matrilin-3 as two ECM proteins affected. Matrilin-3 is here described for the first time as being expressed by cultured osteoblasts. In summary, this novel and in-depth study has demonstrated that HA NP shape can influence a range of different parameters related to osteoblast viability and activity.