Minori Mizumoto

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.

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.

Adsorption Behavior of Sodium Inositol Hexaphosphate on the Surface of Hydroxyapatite

We have previously developed hydroxyapatite (HAp) cement based on the chelate-setting mechanism of sodium inositol hexaphosphate (IP6), in which HAp powder was prepared by surface-modification with IP6 after ball-milling of the HAp powder (conventional process). Meanwhile, we have recently established novel powder preparation process (modified process). In the present study, the adsorption behavior of IP6 on the surface of HAp at both the processes was circumstantially examined to clarify the chelating mechanism of IP6. The adsorbed amount of IP6 increased with the IP6 concentration in both the processes; however, the adsorbed amount of IP6 at the modified process was lower than that at the conventional process. X-ray photoelectron spectroscopic study revealed that the IP6 adsorbed on the surface of HAp powders. The degree in dispersion of the HAp particles at the modified process was higher than that at conventional process. Furthermore, the elution of IP6 from the powders prepared at the novel process was lower than that of the powders at the conventional process.

Comparative study on bioresorbability of chelate-setting cements with various calcium-phosphate phase using rabbit model

We have developed novel calcium-phosphate cements (CPCs) based on the chelate-setting mechanism of inositol phosphate (IP6) using hydroxyapatite (HAp), β-tricalcium phosphate (β-TCP) and α-TCP as starting materials. These cements (IP6-HAp, IP6-β-TCP and IP6-α-TCP cements) have different bioresorbability due to the chemical composition of starting materials. In the present study, biocompatibility and bioresorbability of the above three cements and commercially available cement (Biopex®-R) was histologically evaluated in vivo using rabbit model for 4, 8, and 24 weeks, in addition to their dissolution in vitro. The dissolution of these cements increased in the order of IP6-HAp, IP6-β-TCP and IP6-α-TCP cements. The newly-formed bones were directly in contact with both the IP6-HAp and Biopex®-R cement specimens. As for the IP6-β-TCP and IP6-α-TCP cements, newly-formed bones were formed time-dependently slightly apart from the cement specimens. Resorption rate for Biopex®-R, IP6-HAp, IP6-β-TCP, and IP6-α-TCP cements after 24 weeks implantation were of 7.2, 5.0, 13.7, and 16.2%, respectively, compared to original cements. The present chelate-setting cements with different bioresorbability are promising candidates for application as the novel CPCs.

In Vitro Evaluation of Chelate-Setting Cements Fabricated from Silicon-Containing Apatite Powder Using Osteoblastic Cells

In our previous study, silicon-containing hydroxyapatite (Si-HAp) powder was prepared via an aqueous precipitation reaction. The Si-HAp powders were synthesized with desired Si contents (0, 0.4, 0.8, 1.6, and 2.4 mass%) as a nominal composition. Another previous study in our group demonstrated surface-modification of HAp powder with inositol phosphate (IP6) enhanced the compressive strength of apatite cement. Thus, to fabricate the cements with higher bioactivity, the above Si-HAp powders were surface-modified with IP6 (IP6-Si-HAp). The IP6-Si-HAp cements with various Si contents were fabricated by mixing with pure water at the powder/liquid ratio of 1/0.4 [w/v]. In order to clarify biocompatibility of the IP6-Si-HAP cements in the present work, MC3T3-E1 cells as a model of osteoblast were seeded on the cement specimens. As for the numbers of cells cultured on the IP6-Si-HAp cements, the substitution of lower levels of Si into HAp lattice did not greatly influence the cell proliferation. However, the substitution of Si amount over 0.8 mass% enhanced the cell proliferation. Especially, the IP6-Si-HAp cement with the Si content of 2.4 mass% showed excellent cell proliferation among examined specimens. Therefore, to fabricate the cements with higher bioactivity, it is necessary to control the amount of Si in the IP6-Si-HAp cements. The usage of these IP6-Si-HAp cements may make it possible to fabricate the cements with higher bioactivity, compare to conventional pure HAp cements.

Fabrication of Chelate-Setting Cement Using Silicon-Substituted Hydroxyapatite and its Property

We have developed novel hydroxyapatite (HAp) cement, “chelate-setting HAp cement” on the basis of chelate bonding of inositol hexaphosphate (IP6). In order to create the cement with enhanced bioactivity, we used a silicon-substituted hydroxyapatite (Si-HAp) as a starting material instead of pure HAp powder. The Si-HAp powders were prepared via an aqueous precipitation reaction and surface-modified with IP6 (IP6-Si-HAp). The Si-HAp were synthesized with desired Si contents (0, 0.4, 0.8, 1.6, and 2.4 mass%). Regardless of the amount of Si, the XRD patterns and FT-IR spectra of the powders were similar to each other. Additionally, chemical composition of Si-HAp powders were the almost same as the nominal one. The substitution of Si resulted in a decrease in the a-axis and increase in the c-axis of unit cell of HAp. While, the compressive strength of IP6-Si-HAp cements was higher than that of Si-HAp cements. These results suggest that surface-modification of any powders with IP6 is effective for enhancement of mechanical property. Comparison of mechanical property between HAp and Si-HAp cement specimens revealed that the substitution of lower levels of Si into0 HAp lattice did not greatly influence compressive strength. However, the substitution of high dose of Si (over 2.4 mass%) reduced the compressive strength. Therefore, to fabricate the chelate-setting cements with enhanced bioactivity, it is necessary to control the amount of Si.