Yoshiyuki Yokogawa

Porous calcium phosphate coating over phosphorylated chitosan film by a biomimetic method.

A porous calcium phosphate coating deposited on chitosan films was studied using scanning electron microscopy, energy-dispersive X-ray analysis, micro-Fourier transform infrared spectroscopy (micro-FTIR) and thin-film X-ray diffractometry (XRD). Chitosan films were first prepared by dissolving chitosan powder in dilute acetic acid and drying in a flat petri dish. The films were phosphorylated using urea and H3PO4 with the P content being 0.1-0.2 wt%. Phosphorylated films soaked in saturated Ca(OH)2 solution for 8 days led to the formation of a calcium phosphate precursor phase over the entire surface. This precursor phase stimulated the growth of a porous coating of calcium-deficient hydroxy apatite when immersed in 1.5 x SBF for more than 20 days. Phosphorylated films not treated with Ca(OH)2 did not show any calcium phosphate growth upon immersion in SBF solution. The precursor phase is thought to be octacalcium phosphate, which nucleates a HAP phase during SBF treatment. Initially, this treatment in SBF results in the formation of a single-layer calcium phosphate particles over the film surface. As immersion time in SBF increases, further nucleation and growth produce a porous HAP coating. The Ca/P ratio of the HAP coating is a function of SBF immersion time.

Surface instability of calcium phosphate ceramics in tissue culture medium and the effect on adhesion and growth of anchorage-dependent animal cells

The surface of biocompatible ceramics made of synthesized hydroxyapatite (HAP) and beta-tricalcium phosphate (TCP) was found to be extremely active in tissue culture medium. Using mixed ceramics of HAP and TCP which had been prepared with different Ca/P molar ratios adjusted in stepwise fashion to values of 1.50, 1.55, 1.60, 1.64 and 1.67, the characteristics of the surface were investigated. The time-dependent variation of zeta potential of the TCP-HAP ceramics immersed in distilled water and in culture medium with and without addition of fetal bovine serum showed that the surface was unstable with significant changes in the charge being observed. Dry TCP powder had a zeta potential of -19 mV, which shifted to -7 mV after soaking in water and to -26 mV in culture medium. In contrast, HAP had a zeta potential of -11 mV in a dry state, -9 mV in water and -29 mV in culture medium. Concentrations of calcium and phosphate dissolved in distilled water showed the solubility was higher for TCP than for HAP. In comparison, it was found that dissolved calcium and phosphate in the medium were removed from the solution by deposition on immersed TCP-HAP ceramics. These results suggested that the stability of the surface was closely related to both reactions of association and dissociation of calcium and phosphate in tissue culture medium. The zeta potential analysis also suggested that Ca-deficient HAP, which has a similar crystal structure to HAP with a Ca/P ratio less than 1.67, was generated by degradation and reforming of the surface layer. The most stable structure which was the most suitable for adhesion of L-929 cells was obtained by the mixture of 20% TCP and 80% HAP ceramics. In conclusion, the stability of the surface structure was considered to be the dominant factor for the enhancement of the adhesiveness of cells on TCP-HAP ceramics.

Growth of calcium phosphate on phosphorylated chitin fibres.

Calcium phosphate growth on chitin phosphorylated fibres was studied using scanning electron microscopy and energy dispersive X-ray analysis (SEM, EDX), micro-Fourier transform infrared spectroscopy (FTIR), and solid state magic angle spinning nuclear magnetic resonance (MAS NMR) techniques. The C6 chemical shift positions of 13C MAS NMR in the chitin fibres phosphorylated using urea and H3PO4 are obvious indicating that phosphorylation takes place not in the C1 but in the C6 region. Micro-FTIR and 31P MAS NMR suggested that ammonium hydrogen phosphate formed during the phosphorylation procedure. Chitin fibres phosphorylated using urea and H3PO4 and then soaked in saturated Ca(OH)2 solution at ambient temperature, which lead to the formation of thin coatings formed by partial hydrolysis of the PO4 functionalities, were found to stimulate the growth of a calcium phosphate coating on their surfaces after soaking in 1.5×SBF solution for as little as one day. The thin layer after Ca(OH)2 treatment functioned as a nucleation layer for further calcium phosphate deposition after soaking in 1.5×SBF solution. EDX-measured Ca : P ratios of the coatings of Ca(OH)2-treated phosphorylated chitin in 1.5×SBF solution suggested that calcium-deficient apatite was formed.

ζ-Potentials of planar silicon plates covered with alkyl- and fluoroalkylsilane self-assembled monolayers

Abstract ζ-potentials of planar Si plates covered with an alkyl- or a fluoroalkyl organosilane self-assembled monolayer (SAM) were measured over a pH range of 3–11 by means of an electrophoretic light scattering spectrophotometer, and were compared with those of Si plates covered with native oxide (SiO 2 ), polyethylene (PE) and polytetrafluoroethylene (PTFE). The SAMs were prepared on SiO 2 /Si substrates by a chemical vapor deposition (CVD) method in which an alkylsilane, that is, octadecyltrimethoxysilane [CH 3 (CH 2 ) 17 Si(OCH 3 ) 3 , i.e. ODS] or a fluoroalkylsilane, that is, (heptadecafluoro-1,1,2,2-tetrahydrodecyl) trimethoxysilane [CF 3 (CF 2 ) 7 CH 2 CH 2 Si(OCH 3 ) 3 , i.e. FAS] was used as a precursor. The SAM-covered Si plates were found to be 35–65% less charged than the SiO 2 /Si plate. Furthermore, SAM formation reduced surface acidity. The isoelectric points (IEPs) of the ODS- and FAS-SAM surfaces observed at pH 3.5–4 were higher than that of SiO 2 /Si (∼pH 2.0). These results are attributable to a reduction in the density of surface silanol (Si–OH) groups on the SiO 2 /Si substrate. Si–OH groups were consumed due to the formation of siloxane bondings with the organosilane molecules which compose the SAMs. Finally, the IEPs of the ODS- and FAS-SAM covered surfaces were found to be almost identical to those of the PE and PTFE bulk surfaces.

Growth of calcium phosphate on surface-modified cotton

A study of the growth of amorphous calcium phosphate on surface-modified cotton fibres by a combination of scanning electron microscopy/electron diffraction X-ray analysis, micro-FTIR and X-ray photoelectron spectroscopy is reported. Cotton fibres phosphorylated by the urea/phosphorous acid method and then soaked in saturated Ca(OH)2 for approximately one week were found to stimulate the growth of a calcium phosphate coating on their surfaces after soaking in 1.5×SBF for as little as 1 day. Ca(OH)2 soaking of the fibres is found to produce highly crystalline clusters lodged in the fibres which were confirmed by micro-FTIR to be calcium phosphite monohydrate (CaHPO3·H2O). In contrast, phosphorylated fibres not subjected to the Ca(OH)2 treatment did not exhibit calcium phosphate growth upon immersion in 1.5×SBF solution. Soaking of the Ca(OH)2-treated fibres with time in the 1.5×SBF solution produced progressively thicker layers of calcium phosphate on the fibres as confirmed by scanning electron microscopy and X-ray photoelectron spectroscopy. In general, calcium phosphate coatings formed over 1 1–5 day period soaking in 1.5×SBF solution appeared to consist of agglomerations of a large number of small spherical particles, while coatings formed after 17 days of soaking were distinctly chunky, thick and non-uniform in appearance. Micro-FTIR indicated that CaHPO3·H2O clusters were still present in cotton samples even after 4 days of soaking, while after 17 days, only the infrared spectrum typical of calcium phosphate was observed. EDX-measured Ca:P ratios of the coatings, although variable, suggested amorphous calcium phosphate. The mechanism of formation of the coating is believed to involve dissolution of the CaHPO3.H2O clusters upon introduction of the Ca(OH)2-treated phosphorylated cotton into the 1.5×SBF solution which elevates the Ca2+ ion concentration in the vicinity of the fibres so stimulating calcium phosphate formation. It is postulated that phosphite groups chemically bound to the cotton fibre surface or a calcium phosphite coating on the fibres act as nucleation sites for calcium phosphate growth in 1.5×SBF solution.

Further studies of calcium phosphate growth on phosphorylated cotton fibres

Further studies using scanning electron microscopy/energy dispersive X-ray analysis (SEM/EDX), micro-Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and solid state magic angle spinning nuclear magnetic resonance (MAS NMR) techniques of calcium phosphate growth on Ca(OH)2-treated urea/H3PO3- and urea/H3PO4-modified cotton fibres are reported. In the case of the Ca(OH)2-treated urea/H3PO3-modified fibres which have been reported in an earlier paper, further experiments subjecting the urea/H3PO3-modified cotton to alternative soaking treatment procedures to Ca(OH)2 as well as different calcium phosphate growth media such as the alkaline phosphatase-catalysed hydrolysis of disodium p-nitrophenylphosphate to free phosphate have reaffirmed the importance of the Ca(OH)2 treatment step for the stimulus and growth of calcium phosphate growth on the fibres. Studies on cotton phosphorylated by a slightly different method using urea/H3PO4 instead of urea/H3PO3 show that a phosphorylated cotton with similar properties to the urea/H3PO3-modified fibres can be produced. Soaking of these fibres in saturated Ca(OH)2 solution leads to cotton coated with thin layers of calcium phosphate formed by partial hydrolysis of the PO4 functionalities in the phosphorylated cotton which are believed to act as nucleation layers for further calcium phosphate deposition when the fibres are subsequently soaked in 1.5×SBF solution. SEM/EDX studies of the calcium phosphate coatings formed on the Ca(OH)2-treated urea-H3PO4 fibres as a function of soaking time in 1.5 × SBF show that coatings deposit and become noticeably thick after approximately 9 days. XPS studies indicated the presence of carbonate species in the calcium phosphate coating deposited. In common with the calcium phosphate coated Ca(OH)2-treated urea/H3PO3-modified fibres studied earlier, the average EDX-measured Ca: P ratios of the coatings formed on the Ca(OH)2-treated urea/H3PO4 fibres are ∼ 1.60 and give very similar micro-FTIR spectra with evidence of carbonate which suggests that amorphous calcium deficient apatite has deposited.

Initial anchoring and proliferation of fibroblast L-929 cells on unstable surface of calcium phosphate ceramics.

Calcium phosphate ceramics constructed from beta-tricalcium phosphate (TCP) and hydroxyapatite (HAP) have been successfully used as implant materials. However, there is a possibility that these materials are responsible for an unwanted inflammatory response during wound healing. Since TCP is soluble in the body, the instability of this material may contribute to this inflammatory response. Using composite ceramics of TCP and HAP that possessed Ca/P molar ratios of 1.50, 1.55, 1.60, 1.64, and 1.67, the effect of surface instability on fibroblast L-929 cells was investigated. The time-dependent variation of the initial anchoring ratio, cell density, and cell viability were measured. In general, the cells were severely damaged and ruptured on the highly soluble thin surface layer of the TCP-HAP ceramics. The initial anchoring ratio for TCP-HAP ceramics was as high as that for the polystyrene dish (Lux, control). However, viability at 6 h decreased to less than 50% of the initial cell density on ceramics with a Ca/P molar ratio of 1.64 (20% TCP-80% HAP), while 85% of the cells were viable on Lux. The viability on 100% TCP, whose surface is the most highly soluble among the TCP-HAP ceramics used in this study, was reduced to 20%. Morphological observation showed that the anchored cells were ruptured when grown in culture medium on the 100% TCP. Although the high solubility of the thin surface layer on the TCP-HAP ceramics of the carrier was found to be the dominant factor in the decreasing cell viability, the initial viability was enhanced by the stabilization of the surface of the TCP-HAP ceramics by pre-incubating the scaffolds in a culture medium containing 10% fetal bovine serum for 3 d.

Growth and Adhesion of Osteoblast-Like Cells Derived from Neonatal Rat Calvaria on Calcium Phosphate Ceramics

The effects of biocompatible ceramics on the growth and adhesion of osteoblast-rich rat calvarial cell cultures were investigated. Osteoblast-like cells and mouse fibroblast-like L-929 cells were cultured on composite sinters of hydroxyapatite (HAP) and beta-tricalcium phosphate (TCP) culture carriers, whose Ca/P molar ratios were adjusted to values of 1.50, 1.55, 1.60, 1.64 and 1.67. The growth rates of both cell types were accelerated on the TCP-HAP ceramics as compared to those on polystyrene plastic (LUX) or bioinert zirconia ceramics. The population of osteoblast-like cells reached a density of 2.28 x 10(5) cells/cm2 on 100% HAP (Ca/P ratio 1.67) at 9 d of culture, while the corresponding cell density was 1.66 x 10(5) cells/cm2 on LUX and 1.26 x 10(5) cells/cm2 on zirconia. Adhesion of the osteoblast-like cells on TCP-HAP ceramics was similarly increased as compared with that on LUX or zirconia ceramics. The adhesion of L-929 cells on TCP-HAP ceramics was found to be weaker than that on cultures on LUX or zirconia ceramics. The time-dependent variations in the alkaline phosphatase activity of the osteoblast-like cells showed that the osteoblastic phenotype was potentiated by culturing the cells in calcium-rich media. The surface analyses of the Ca/P ratio and the microstructure by XRD and FTIR suggest that the Ca-rich surface was newly formed by recombination on the surface layer in the culture medium containing fetal bovine serum. These results suggest that the surface of TCP-HAP ceramics, especially that of 100% HAP ceramics, are effective for accelerating growth and differentiation of osteoblast-like cells. This is most probably due to the chemical and physical instability and composition of 100% HAP, which promote the formation of a Ca-rich layer at the cell-material interface and provision of Ca ions to the osteoblast-like cells.

Time-dependent variation of the surface structure of bioceramics in tissue culture medium and the effect on adhesiveness of cells

Abstract Biocompatible ceramics made of β-tricalcium phosphate (TCP) were found to be actively changing their surface characteristics in tissue culture medium. The time-dependent variation of the zeta potential of TCP ceramics immersed in distilled water and in culture medium with and without addition of fetal bovine serum showed that the surface was unstable with significant changes in the charge being measured. Dry TCP had a zeta potential of −9.3 mV, which shifted to −1.8 mV after soaking in water and to −27.6 mV in culture medium with serum. To clarify the effect of the time-dependent variation of the surface structure on growth and adhesion of anchorage-dependent animal cells, the zeta potential of ceramics in dry state was regulated from −11.5 mV to +27.2 mV by means of silane coupling modification. After soaking in distilled water for six days, zeta potential of the modified TCPs shifted to between +7.5 mV and −7.6 mV, while they were between −9.9 mV and −23.7 mV in culture medium with serum. Concentrations of calcium and phosphate dissolved in distilled water and in culture medium showed the solubility of the ceramics was considerably high and depended on the pH of the surface layer. The suitable surface property for adhesion of L-929 cells was obtained by the most stable ceramics in the culture condition. In conclusion, the solubility of the thin surface layer of the carrier was considered to be the dominant factor in decreasing the adhesiveness of cells on TCP ceramics.

Effects of the surface wettability and zeta potential of bioceramics on the adhesiveness of anchorage-dependent animal cells

Abstract Using calcium phosphate ceramics that have high biocompatibility with the living body, the effects of the surface characteristics of the bioceramics on cell adhesiveness were investigated. In the case of carriers with contact angles from 35° to 60°, the cell adhesiveness increased according to the increase in the wettability. Measurement of the zeta potentials of HAP-TCP sinters showed that these bioceramics had negative potentials from −2 mV to −6 mV. Electrochemical analysis suggested that the initial cell anchoring ratio (Ria) and adhesive strength (Fa,enz) were affected by the surface ionic condition of the ceramic material. To clarify the effects of the surface potential of the ceramics on cell adhesiveness, the ceramic surface was modified chemically by means of various silane coupling reagents. The surface potential was regulated from −20 mV to +24 mV. Using these ceramics, the affinity and adhesiveness of the cells to the ceramics were found to be dominantly regulated by the surface potential. A negative potential was effective in increasing the adhesiveness, even though living cells have negative charges.