Toshiisa Konishi

Development of a,b-plane-oriented hydroxyapatite ceramics as models for living bones and their cell adhesion behavior

In vertebrate bones and tooth enamel surfaces, the respective a,b-planes and c-planes of hydroxyapatite (HAp) crystals are preferentially exposed. However, the reason why the HAp crystals show different orientations depending on the type of hard tissues is not yet understood. To clarify this question, appropriate ceramic models with highly preferred orientation are necessary. In the present study, dense HAp ceramic models which have the same orientation as living bones were fabricated using composite powders of c-axis-oriented single-crystal apatite fibers (AF) and wet-synthesized apatite gels (AG). The results of crystalline identification and ultrastructural observation showed that the resulting HAp ceramics maintained the c-axis orientation of the AF particles, and their high a,b-plane orientation degrees could be maintained with small additive amounts of AG; however, when the AG content was over 30 mass%, this value decreased. The influence of orientation degree on the surface characteristics was investigated by evaluating the surface zeta-potential and wettability. These results show that increasing the a,b-plane orientation degree shifted the surface charge from negative to positive, and decreased the surface wettability. Initial cell-attachment assays were performed on these resulting ceramics using MC3T3-E1 cells as models of osteoblasts. The results show that the cell-attachment efficiency decreased with increasing a,b-plane orientation degree.

Enhanced early osteogenic differentiation by silicon-substituted hydroxyapatite ceramics fabricated via ultrasonic spray pyrolysis route

The influence of silicon-substituted hydroxyapatite (Si-HAp) on osteogenic differentiation was assessed by biological analysis. Si-HAp was prepared by ultrasonic spray pyrolysis (USSP) technique using various amounts of Si (0, 0.8, and 1.6 mass%). Chemical analysis revealed that Si was incorporated into the hydroxyapatite (HAp) lattice with no other crystalline phase and which caused the change of crystal structure. Biological analyses showed that the Si contents affected the cell proliferation and morphology, suggesting that there is an optimal Si content for cell culture. As for differentiation, alkaline phosphatase activity and osteocalcin production of Si-HAp were higher than those of HAp. Gene expression profiles also revealed that substitution of Si (0.8 mass%) up-regulated the expression levels of osteocalcin and especially Runx2, a master gene for osteoblast development. These results suggest that incorporating Si into the HAp lattice may enhance the bioactivity, particularly during early osteoblast development.

In vitro and in vivo antimicrobial properties of silver-containing hydroxyapatite prepared via ultrasonic spray pyrolysis route

Hydroxyapatite (HAp), with its high biocompatibility and osteoconductivity, readily absorbs proteins, amino acids and other substances, which in turn favor the adsorption and colonization of bacteria. To prevent bacterial growth and biofilm formation on HAp discs, silver-containing (1-20 mol%) HAp (Ag-HAp) powders were synthesized using an ultrasonic spray pyrolysis (USSP) technique. The X-ray diffraction (XRD) peaks were very broad, indicating low crystallinity, and this induced the release of Ag(+) ions from Ag-HAp powders. In addition, a gradual increase in Ca(2+) ion release was observed. These results suggest that dissolution of Ca(2+) ion in Ag-HAp triggered the release of Ag(+) ions. The antimicrobial efficacy of Ag-HAp disc was tested against Staphylococcus aureus. Samples with Ag contents of more than 5 mol% were found to be highly effective against bacterial colonization and biofilm formation in vitro. In vivo antibacterial tests using bioluminescent strains also showed reductions in the viability of bacteria with Ag-HAp (5 mol%) discs. Biocompatibility tests using a modified Transwell® insert method showed that Ag-HAp (5 mol%) discs have negative effects on osteoblast proliferation. These results indicate that Ag-HAp (5 mol%) has effective antibacterial activity and good biocompatibility both in vitro and in vivo together with good biocompatibility, thus confirming its utility as a bactericidal material.

Fabrication of novel biodegradable α-tricalcium phosphate cement set by chelating capability of inositol phosphate and its biocompatibility

Biodegradable α-tricalcium phosphate (α-TCP) cement based on the chelate-setting mechanism of inositol phosphate (IP6) was developed. This paper examined the effect of the milling time of α-TCP powder on the material properties of the cement. In addition, biocompatibility of the result cement in vitro using osteoblasts and in vivo using rabbit models will be studied as well. The α-TCP powders were ballmilled using ZrO2 beads in pure water for various durations up to 270 minutes, with a single-phase α-TCP obtained at ballmilling for 120 minutes. The resulting cement was mostly composed of α-TCP phase, and the compressive strength of the cement was 8.5±1.1MPa, which suggested that the cements set with keeping the crystallite phase of starting cement powder. Thecell-culture test indicated that the resulting cementswere biocompatiblematerials. In vivo studies showed that the newly formed bones increased with milling time at a slight distance fromthe cement specimens and grewmature at 24weeks, and the surface of the cementwas resorbed by tartrate-resistant acid phosphatase-(TRAP-)positive osteoclast-like cells until 24weeks of implantation. The present α-TCP cement is promising for application as a novel paste-like artificial bone with biodegradability and osteoconductivity.

Development of Bioresorbable Calcium-Phosphate Cements Hybridized with Gelatin Particles and their In Vivo Evaluation Using Pig’s Tibia Model

Novel bioresorbable calcium-phosphate cement (CPC) with anti-washout property was developed by adding thermally cross-linked gelatin particles as pore generator into a CPC. The CPC was composed of α-tricalcium phosphate (α-TCP) and surface-modified hydroxyapatite (HAp) with inositol phosphate as a chelating agent (IP6-HAp). The bioresorbable CPC hybridized with gelatin particles was successfully fabricated by mixing the aqueous sodium chondroitin sulfate solution including Na2HPO4 and the pre-mixed powders composed of α-TCP (72 mass%), IP6-HAp (18 mass%), and the gelatin particles (10 mass%). The hybridized CPC paste showed initial setting time (IST) of 5 minutes and exhibited anti-washout property. Compressive strength after setting for 24 h reached to 4.2 MPa. An in vivo preliminary study using pig’s tibia model demonstrated that the hybridized CPC could be easily injected and set promptly without washout. In addition, no fragmentation in the specimens was observed after 8 weeks implantation. Moreover, a histological observation (Villanueva bone stain) revealed that almost 80% of the hybridized CPC specimens were resorbed and that immature bones were formed inside the specimens.

In Vitro Biological Evaluation of Anti-Tumor Effect of the Chelate-Setting Hydroxyapatite Cement

Cancers frequently metastasize to bone, where it leads to secondary tumor growth, and osteolytic bone degradation. Bone metastases are often associated with fractures and severe pain resulting in decreased quality of life. Accordingly, effective therapies to inhibit the development or progression of bone metastases will have important clinical benefits. Bone cement, one of the powerful tools as bone substitutes, is used to fill the resection voids. The aim of this study was to develop a local drug delivery system using HAp cement as a carrier of chemotherapeutic agents. In the present study, we have fabricated chelate-setting apatite cements (IP6-HAp cements) using HAp particles surface-modified with inositol hexaphosphate (IP6) and evaluated their anti-tumor effect. Human osteosarcoma (HOS) cultured on IP6-HAp cements (over 3000 ppm IP6) resulted in inhibition of cell growth. DNA microarray analysis indicated changes in the expression of apoptosis-related genes on IP6-HAp cement surface-modified with 5000 ppm IP6 compared with HAp cement, suggesting activation of apoptosis machinery by IP6-HAp cement. To clarify the mechanism of anti-tumor effect of IP6-HAp cement, the properties of cement were investigated. The release kinetics of IP6 from IP6-HAp cement showed that the level of released IP6 was insufficient to induce anti-tumor activity. These results led us to consider that locally high concentration of IP6 which was released from cement acts on the cells directly as anti-tumor agent and induces the apoptosis. Consequently, IP6-HAp cement might gain the anti-tumor effect and act as a carrier for local drug delivery system.

Preparation of Silver-Containing Hydroxyapatite Powder by Ultrasonic Spray-Pyrolysis Technique and its Antibacterial Property

We prepared silver-containing hydroxyapatite (Ag-HAp) powders by an ultrasonic spray-pyrolysis (USSP) technique and evaluated their antibacterial activity.　　Ag-HAp powders with Ag contents of 0, 1, 2, 5, and 20 mol% as nominal composition were prepared by the above USSP technique. The droplets of starting solutions were dried at 300 °C and then pyrolyzed at 850 °C to prepare the Ag-HAp powders. Ag-HAp powders showed a HAp single phase and were composed of spherical particles with a diameter of 0.5 - 3.0 μm. To evaluate the antibacterial activity of resulting powders, colony count method was performed using Staphylococcus aureus. Ag-HAp powders with the contents of Ag more than 5 mol% showed good antibacterial activity.

Effects of Adding Polysaccharides and Citric Acid into Sodium Dihydrogen Phosphate Mixing Solution on the Material Properties of Gelatin-Hybridized Calcium-Phosphate Cement

We have succeeded in improving the material properties of a chelate-setting calcium-phosphate cement (CPC), which is composed of hydroxyapatite (HAp) the surface of which has been modified with inositol hexaphosphate (IP6) by adding α-tricalcium phosphate (α-TCP) powder. In order to create a novel chelate-setting CPC with sufficient bioresorbability, gelatin particles were added into the IP6-HAp/α-TCP cement system to modify the material properties. The effects of adding polysaccharides (chitosan, chondroitin sulfate, and sodium alginate) into the sodium dihydrogen phosphate mixing solution on the material properties of the gelatin-hybridized CPC were evaluated. The results of mechanical testing revealed that chondroitin sulfate would be the most suitable for fabricating the hybridized CPC with higher compressive strength. Moreover, further addition of an appropriate amount of citric acid could improve the anti-washout capability of the cement paste. In summary, a gelatin-hybridized IP6-HAp/α-TCP cement system prepared with a mixing solution containing chondroitin sulfate and citric acid is expected to be a beneficial CPC, with sufficient bioresorbability and material properties.

Preparation and Characterization of β-Tricalcium Phosphate Powders with High Solubility for Chelate-Setting Calcium-Phosphate Cements

We have previously developed a novel chelate-setting β-tricalcium phosphate (β-TCP) cement with non-fragmentation property in vivo. This novel cement has been set on the basis of chelate-setting mechanism of inositol phosphate (IP6). In this study, β-TCP powders were synthesized by mechanochemical method, and the as-prepared powders were heated at 600-1300°C for 1 h. Some properties of the resulting powders were examined. The crystalline phase of the resulting powders in the range of 600-1100°C was of β-TCP single phase. In the cases at 1200°C and 1300°C, the resulting powders were composed of β-TCP and α-TCP. Median particle sizes of the resulting powders increased with heating temperature from 5.35 μm up to 47.7 μm. Dissolution rate of Ca2+ ions from the β-TCP powders was measured by Japanese Industrial Standard T 0330-3. When the heating temperature was at 700°C, the Ca2+ ions solubility was highest among examined ones. The β-TCP powder heated at 700°C for 1 h will be expected as the starting powder for paste-like artificial bone filler with excellent bioresorbability.

Fabrication and characterization of chelate-setting β-tricalcium phosphate cements with enhanced bioresorbability

A novel chelate-setting β-tricalcium phosphate (β-TCP) cement with anti-washout properties have been fabricated previously. This cement has been set on the basis of chelating ability of inositol phosphate (IP6). In this study, the ball-milling and surface-modification conditions of starting β-TCP cement powders were optimized in terms of bioresorbability. Starting powders were prepared by simultaneously ball-milling at 300 rpm for 3 h with 1 mm diameter ZrO2 beads and surface-modifying with 40 cm3 of 3000 ppm IP6 solution. The resulting starting powder was consisted of β-TCP single phase, and had high specific surface area of 48.3 m2∙g-1. Cement pastes were prepared by mixing the starting powder and the aqueous solution composed of 2.5 mass% sodium hydrogen phosphate, 1.5 mass% citric acid and 1.0 mass% sodium alginate at a powder/liquid ratio of 1/0.90 [g∙cm-3] for 2 min. After setting in pure water for 72 h, compressive strength of the cement specimens was higher than that of human cancellous bone. Dissolution rate of Ca2+ ions was measured by according to Japanese Industrial Standard T 0330-3. The results of Ca2+ ions dissolution rate test demonstrated that the cement specimens derived from the above starting powder were the highest dissolution rate among examined ones. This cement would be expected as bone fillers with high bioresobability.