Particulate beta-tricalcium phosphate and hydroxyapatite doped with silver promote in vitro osteoblast differentiation in MC3T3-E1 cells.

BACKGROUND: Calcium phosphates including ß-tricalcium phosphate (ß-TCP) and hydroxyapatite (HAp) have been widely used for bone regeneration application because of their high osteoconductive activities. In addition, various kinds of inorganic ions enhance differentiation, proliferation, and mineralization of osteoblasts. However, information about the effects of silver-doped ß-TCP [ß-TCP (Ag)] and HAp [HAp (Ag)] particles on osteogenic differentiation is not available yet. OBJECTIVE: We focused on the impact of ß-TCP (Ag) and HAp (Ag) particles on the osteogenic differentiation of MC3T3-E1 osteoblast precursor cells. METHODS: MC3T3-E1 osteoblast precursor cells were pre-treated by ß-TCP (Ag) or HAp (Ag). And then the medium was changed to differentiation medium. Subsequently, osteoblast differentiation-related markers were determined. RESULTS: We found that treatment with ß-TCP (Ag) or HAp (Ag) particles increased alkaline phosphatase activity in MC3T3-E1 cells. Expression of osteoblast differentiation-related genes also increased after treatment with ß-TCP (Ag) or HAp (Ag) particles, a response thought to be regulated by zinc finger-containing transcription factor osterix. The ratio of the receptor activator of nuclear factor kappa-B ligand (RANKL) to osteoprotegerin (OPG) was decreased by ß-TCP (Ag) and HAp (Ag) particles. CONCLUSION: Silver doping of ß-TCP and HAp particles is effective for bone regeneration.

In vivo bioresorbability and bone formation ability of sintered highly pure calcium carbonate granules.

Calcium carbonate-based bone substitutes derived from natural coral exoskeleton (aragonite) are resorbed and remodeled faster than calcium phosphate-based substitutes. However, coral species with structures appropriate for use as bone substitutes are very limited. Therefore, it is important to evaluate potential of artificial calcium carbonate ceramics as a bone substitute. In this study, calcium carbonate granules with various porosities and pore sizes were prepared by sintering a highly pure (>99.98%) calcium carbonate powder (calcite), and their resorption properties and bone formation abilities were examined in vivo for the first time. The sintered calcium carbonate was resorbed faster than ß-tricalcium phosphate, which has a similar structure. However, sintered calcium carbonate did not promote new bone formation during long-term implantation. Furthermore, both resorption and new bone formation were affected by the pore structure. The optimal structures of the artificially sintered calcium carbonate bone substitute were also discussed.

Comparison of proinflammatory potential of needle-shaped materials: aragonite and potassium titanate whisker.

Among the crystal forms of calcium carbonate, aragonite has needle-like shape. Although materials with needle-shaped crystals are associated with pulmonary toxicity, the toxic activity of aragonite is unclear. Therefore, proinflammatory potential of aragonite, neutralized aragonite and potassium titanate whisker was evaluated. The cellular effects of aragonite were weaker than those of potassium titanate whisker. Aragonite treatment induced the expression of chemokines in A549 cells and macrophages. Although aragonite exhibited proinflammatory effects in vitro, pulmonary inflammation was not observed in vivo after intratracheal administration of aragonite in mice. We did not observe the induction of inflammatory cytokine secretion or tissue lesion in the lungs of mice after administration of aragonite. Potassium titanate whisker treatment induced chemokine secretion in vitro. An increase in the number of neutrophils was observed in the mice lung tissue after administration of potassium titanate whisker. Aragonite and neutralized aragonite both induced an increase in the levels of intracellular calcium, but the levels were significantly higher in cells treated with aragonite than in cells treated with neutralized aragonite. These results suggested that intracellular calcium release mediates the cellular effects of aragonite. The toxicity of aragonite based on its needle-like structure was also not observed.

Effect of calcium carbonate particle shape on phagocytosis and pro-inflammatory response in differentiated THP-1 macrophages.

Phagocytosis is a physiological process used by immune cells such as macrophages to actively ingest and destroy foreign pathogens and particles. It is the cellular process that leads to the failure of drug delivery carriers because the drug carriers are cleared by immune cells before reaching their target. Therefore, clarifying the mechanism of particle phagocytosis would have a significant implication for both fundamental understanding and biomedical engineering. As far as we know, the effect of particle shape on biological response has not been fully investigated. In the present study, we investigated the particle shape-dependent cellular uptake and biological response of differentiated THP-1 macrophages by using calcium carbonate (CaCO3)-based particles as a model. Transmission electron microscopy analysis revealed that the high uptake of needle-shaped CaCO3 particles by THP-1 macrophages because of their high phagocytic activity. In addition, the THP-1 macrophages exposed to needle-shaped CaCO3 accumulated a large amount of calcium in the intracellular matrix. The enhanced release of interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α) by the THP-1 macrophages suggested that the needle-shaped CaCO3 particles trigger a pro-inflammatory response. In contrast, no pro-inflammatory response was induced in undifferentiated THP-1 monocytes exposed to either needle- or cuboidal-shaped CaCO3 particles, probably because of their low phagocytic activity. We also found that phosphate-coated particles efficiently repressed cellular uptake and the resulting pro-inflammatory response in both THP-1 macrophages and primary peritoneal macrophages. Our results indicate that the pro-inflammatory response of macrophages upon exposure to CaCO3 particles is shape- and surface property-dependent, and is mediated by the intracellular accumulation of calcium ions released from phagocytosed CaCO3 particles.

Evolution of Calcite Nanocrystals through Oriented Attachment and Fragmentation: Multistep Pathway Involving Bottom-Up and Break-Down Stages.

A nonclassical multistep pathway involving bottom-up and break-down stages for the evolution of calcite nanograins ∼50 nm in size was demonstrated in a basic aqueous system. Calcite nanofibrils ∼10 nm wide were produced as the initial crystalline phase via amorphous calcium carbonate through ion-by-ion assembly by the carbonation of Ca(OH)2 at a high pH of ∼13. Bundles ∼50 nm in diameter were then formed by the subsequent oriented attachment of the nanofibrils. Monodispersed calcite nanograins were finally obtained through spontaneous fragmentation of the fibrous forms via a decrease in pH by further carbonation.