No abstract

The biomedical materials research community frequently accepts that amorphous calcium phosphate (ACP) can be adsorbed and assimilated more readily by living organisms to produce new bone tissue than crystallized calcium phosphates such as hydroxyapatite (HAP). Previous studies also confirm that ACP has improved bioactivity compared to HAP since more adhesion and proliferation of osteogenic cells are observed on the ACP substrates. However, we note that the different size -effects of calcium phosphates are not taken into account in these studies and the used ACP are always smaller than the HAP. Our recent study reveals that the dimensions of nanoparticles are directly related to the bioactivities of calcium phosphates, e.g. the smaller nanocrystallites have a greater promotion effect on the proliferation of bone marrow mesenchymal stem cells (BMSCs). In order to understand the influence of crystallinity of calcium phosphate on the osteogenic cells correctly, it is critical to use ACP and HAP nanoparticles which have the same size distribution in such comparisons. In the present work, y20 nm ACP and HAP particles are synthesized and the effects of crystallinity of calcium phosphates are studied. The adhesion, proliferation, and differentiation of BMSCs are measured on ACP and HAP films, which are compared at the same size scale. It is surprising that more cells adsorb and proliferate on the film of well crystallized HAP than those on the ACP film. Alkaline phosphatase (ALP) activity assay and reverse transcription-polymerase chain reaction (RT-PCR) assay are also used to evaluate the differentiation of BMSCs. The results show that the differentiation of BMSCs to osteoblasts is promoted significantly by NanoHAP. The current experimental phenomena clearly demonstrate that the crystallized phase of calcium phosphate, HAP, provides a better substrate for BMSCs than the amorphous one, ACP, when the factor of size effect is removed. A new view on the relationship between the crystallinity of calcium phosphate and the responses of BMSCs indicates the importance of size and phase controls in the application of biomedical materials.

Coculturing scaffolds with seeded cells in vitro is an indispensable process for construction of engineered tissues. It is essential to understand effects of the constituent particles of scaffold on seeded cells. In this study, we investigated the influence of nano-sized hydroxyapatite (nHAP) particles on the proliferation and osteogenic differentiation of bone marrow-derived mesenchymal stem cells (MSCs). nHAP particles were cocultured with MSCs separated from rabbit. Cellular effects of particles were determined by cell counts, Vonkossa stains, and reverse transcription-polymerase chain reaction (RT-PCR) examinations. Results showed that nHAP particles could promote the MSCs growth when particle concentrations were lower than 20 microg/10(4) cells. This effect disappeared when the particles and the cells were cocultured in serum-free media. Higher particle concentrations could significantly inhibit the cell growth. Under the standard culture condition, the only effect of nHAP particles on osteogenic differentiation of MSCs was to enhance the expression of collagen I. Under the osteogenic-inductive culture condition, nHAP particles could inhibit mineralization of cells but promote their osteogenic differentiation. These cellular effects of particles still existed when the particles and the cells were cultured in indirect coculture system. nHAP particles could decrease calcium and phosphate concentrations of culture media, which possibly contributed to the cellular effects of nHAP particles.

As the main inorganic component of biological bone and tooth enamel, hydroxyapatite (HAP) is highly biocompatible and bioactive. Our recent work has shown that 20 nm HAP can be readily internalized by living cells. However, HAP nanoparticles are not luminescent so that they are difficult to measure in living cells during in vitro experiments. A novel inorganic biological probe was suggested by doping 20 nm HAP with terbium. The calcium ions on the HAP particle surface could be partially replaced by Tb, which resulted in the 20 nm Tb-HAP particles. The Tb-doped HAP became luminescent and could be observed under a visible excitation of 488 nm. Compared with the usual probes, the modified HAP nanoparticles were more photostable and were almost nontoxic. It was important that the HAP characteristics remained after the surface modification with a small amount of Tb. After incubating with rabbit bone marrow mesenchymal stem cells (MSCs) in culture, the luminescence of the internalized HAP in the living cells was clearly observed under a fluorescent microscope. Transmission electron microscopy analysis also confirmed the uptake of the particles by MSCs. It was demonstrated that the modified HAP was a stable biological probe for cellular research. We suggested that the Tb-doped HAP should have a great potential to trace the evolvement of nanoparticles, which is a key topic in nanobiotechnology.