Kazuo Isama

Synthesis of a novel b-tricalcium phosphate/hydroxyapatite biphasic calcium phosphate containing niobium ions and evaluation of its osteogenic properties

To promote the osteogenic properties of osteoblasts, we synthesized a hydroxyapatite (HAp) with β-tricalcium phosphate (β-TCP) biphasic calcium phosphate containing Nb ions (NbTCP/HAp). NbTCP/HAp was prepared by annealing precipitates obtained by coprecipitation of an aqueous solution of Ca(NO3)2 and a mixture of (NH4)2HPO4 and aqueous Nb solution. The precipitates can be regarded as a calcium-deficient HAp, the PO4 sites of which are partly occupied by Nb ions. NbTCP/HAp was successfully synthesized by thermal decomposition of the precipitates. NbTCP/HAp enhanced the calcification of normal human osteoblasts (NHOst), and the amount of calcified tissue increased in proportion to the Nb ion concentration in the NbTCP/HAp. The alkaline phosphatase (ALP) activity of NHOst was also enhanced by NbTCP/HAp. Because Nb ions significantly enhance the ALP activity of NHOst, calcification by NbTCP/HAp is considered to be due to enhancement of ALP activity induced by Nb ions dissolved from NbTCP/HAp. These results indicate that NbTCP/HAp can be an effective bone repair material.

Enhancing effect of poly(L-lactide) on the differentiation of mouse osteoblast-like MC3T3-E1 cells.

Poly(L-lactide) (PLLA) has bioabsorbability and biocompatibility, and it is used as biodegradable screws, pins and plates for internal bone fixation. The purpose of this study was to clarify the effects of low molecular weight (Mw) PLLA on the proliferation and differentiation of mouse osteoblast-like MC3T3-E1 cells. MC3T3-E1 cells were cultured with the concentration of 5-50 microg/ml of PLLA with weight average Mw of 5000 (PLLA-5k) and 10,000 (PLLA-10k) for 2 weeks using the micromass culture. Both PLLAs did not affect the proliferation of MC3T3-E1 cells. However, the calcifications of MC3T3-E1 cells were stimulated with increasing the concentration of the PLLAs. Then PLLA-5k increased the calcification of MC3T3-E1 cells more than PLLA-10k. Additionally, both PLLAs increased the alkaline phosphatase (ALP) activity and calcium content of MC3T3-E1 cells up to the similar level to the calcification. These results indicated that low Mw PLLA enhanced the differentiation of MC3T3-E1 cells with no effect on the proliferation. Moreover, it was suggested that the increase of the ALP activity was a key step to stimulate the calcification of MC3T3-E1 cells. The osteoconductivity of implanted PLLA would be based on the enhancing effect of low Mw PLLA on the differentiation of the osteoblasts.

In vivo tissue/biomaterials toxic responses: Correlation with cytotoxic potential but not cell attachment

Abstract The relationships between cell attachment, cytotoxicity and in vivo tissue response such as muscle inflammation and eye irritation were investigated using lens materials, radiation vulcanized natural rubber latex (RVNRL) and standard reference materials (SRM). Five RVNRL materials, which induced muscle tissue inflammation, were cytotoxic and also significantly decreased cell attachment activities. The cytotoxic potential of a SRM made of segmented polyurethane correlated well with the decrease in cell attachment activity. However, among lens materials, cytotoxicity was detected in fluorosilicone acrylate but not in poly(methylmethacrylate) (PMMA), although the cell attachment activity of PMMA was the lowest. Eye irritation induced by wearing various kinds of lenses correlated with cytotoxic potential (r = −0·90) but not with cell attachment activities. In conclusion, in vivo toxic tissue responses correlated well with cytotoxic potentials but not always with cell attachment activity. Our experimental results also showed that direct contact colony formation was mainly inhibited by cytotoxicity, but not the low activity of cell attachment.

Novel Calcium Phosphate Ceramics: The Remarkable Promoting Action on the Differentiation of the Normal Human Osteoblasts

To promote the activity of normal human osteoblasts (NHOst), the novel HAp ceramics containing Nb ions (NbHAp) were synthesized by wet chemical process, which reacting aqueous solution containing a mixture of Ca(NO3)2, (NH4)2HPO4, and the Nb aqueous solution. X-ray diffraction patterns indicated that NbHAp had a monolithic apatitic structure, although crystallite decreased as Nb content increased. From inductively coupled plasma analysis, maximum amount of Nb ions in the sample was almost 8.2atom% of P ions. The NbHAps were presented as aggregates and composed of fine crystal of <1µm in diameter. Nb ions in NbHAp were uniformly distributed in the aggregates. Furthermore, high-resolution XPS spectra of Nb 3d5/2 indicated that Nb ions in the HAp were presented as Nb5+. These results suggested that Nb ions were at PO4 site in crystal structure of HAp. When NHOst were cultured with the NbHAp, their ALP activity were twice as much as that of NHOst cultured with HAp without Nb ions.

Self-organization of the compositional gradient structure in hyaluronic acid and poly(N-isopropylacrylamide) blend film.

A compositional gradient structure in hyaluronic acid (HA) and poly(N-isopropylacrylamide) (PIPAAm) blend film was self-organized from a homogeneous aqueous solution in a plasma-treated polystyrene dish (PTPSD), and the formation mechanisms of the gradient structure were studied by casting the same solution on PTPSD and a non-treated polystyrene dish (NTPSD) under ambient and vacuum conditions. The formation of a compositional gradient structure in HA/PIPAAm blend film was confirmed by scanning electron microscopy, energy dispersive X-ray (EDX) mapping analysis and step-scan photoacoustic Fourier transformed infrared spectroscopy (PAS–FT-IR) measurements. The EDX mapping measurements for Na element revealed that the HA component gradually decreases from the dish-side to the air-side of the film cast on PTPSD, while for the film cast on NTPSD no such obvious change was observed on the cross-section. Further studies on the films prepared on PTPSD and NPTPSD under ambient and vacuum conditions demonstrated that the hydrophilic interaction and the solvent evaporation rate were the most significant factors leading to the formation of a compositional gradient structure in the HA/PIPAAm blend system.

Osteoblast Differentiation and Apatite Formation on Gamma-Irradiated PLLA Sheets

The effects of the γ-irradiatted PLLA on the osteoblasts and apatite formation were investigated in vitro. The PLLA sheet was γ-ray irradiated at the dose of 10, 25 or 50 kGy. The mouse osteoblast-like MC3T3-E1 cells and normal human osteoblast NHOst cells were micromass cultured on the PLLA sheet for 2 weeks, and then the proliferation and differentiation of the cells were determined. The proliferations of MC3T3-E1 and NHOst cells hardly changed with increasing irradiation dose. However, the differentiations of MC3T3-E1 and NHOst cells increased with irradiation dose. On the other hand, the surface of the PLLA sheet after soaking in the medium without the cells was characterized by SEM, EDX, FT-IR and XPS. The hydroxyapatite was formed on the surface of the PLLA sheet after soaking, and the amount of hydroxyapatite increased with irradiation dose. In summary, the γ-irradiated PLLA increased the differentiation of osteoblasts and also increased apatite-forming ability even without the osteoblasts. The osteoblast differentiation was enhanced well in the apatite formation on the surface of PLLA after the γ-irradiation.