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.     

致密磷酸钙陶瓷在动态SBF中类骨磷灰石层形成研究

磷酸钙陶瓷植入体内后其表面类骨磷灰石层的形成是诱导成骨的先决条件．本实验在模拟体液(ｓｉｍｕｌａｔｅｄ ｂｏｄｙ ｆｌｕｉｄ,ＳＢＦ)以人体骨骼肌组织内体液的正常生理流率(２ｍＬ/１００ｍＬ·ｍｉｎ)和偏离正常生理流率流动的动态条件下,研究在动态ＳＢＦ中影响致密磷酸钙陶瓷表面类骨磷灰石层形成的因素．结果表明:在生理流率条件下,材料的粗糙表面有利于类骨磷灰石的形成,加大ＳＢＦ中Ｃａ^２＋、ＨＰＯ_４^２－离子浓度,类骨磷灰石层的形成速度加快．比起通常使用的静态浸泡试验,ＳＢＦ以生理流率流动的动态试验能够更好地模拟类骨磷灰石生长的体内环境．动态ＳＢＦ对了解类骨磷灰石形成,进而了解磷酸钙陶瓷在体内诱导成骨机理是十分有用的．     

Dynamic study of calcium phosphate formation on porous HA/TCP ceramics

Bone-like apatite formation on porous calcium phosphate ceramics was investigated in static simulated body fluid (SBF) and dynamic SBF at different flowing rates. The results of a 14-day immersion in static SBF showed that the formation of bone-like apatite occurred both on the surface and in the pores of the samples. When SBF flowed at the physiological flow rate in muscle (2 ml/100 ml⋅min), bone-like apatite could be detected only in internal surface of the pores of samples. The result that bone-like apatite formation could only be found in the pores when SBF flowed at physiological flow rate was consistent with that of porous calcium phosphate ceramics implanted in vivo: osteoinduction was only detected inside the pores of the porous calcium phosphate ceramics. This result implicates that the bone-like apatite may play an important role in the osteoinduction of Ca-P materials. The dynamic model used in this study may be better than usually used static immersion model in imitating the physiological condition of bone-like apatite formation. Dynamic SBF method is very useful to understand bone-like apatite formation in vivo and the mechanism of ectopic bone formation in calcium phosphate ceramics.     

Effect of fluoride on apatite formation from Ca4(PO4)2O in 0.1 mol L−1 KH2PO4

The effect of fluoride on the hydrolysis of tetracalcium phosphate (TTCP; Ca4(PO4)2O) was investigated in 0.1 mol l–1KH2PO4 containing 0–83 mol l–1 KF. Characterization of the final apatite phase formed by the hydrolysis was made with X-ray diffraction and SEM. The initial pH was between 4.5 and 5.4, depending on the solutions, and the pH rapidly increased and was kept constant between 7.3 and 6.5. An increase in KF concentration tended to lower the pH in the final stage of hydrolysis. The calcium concentration was considerably lower than the phosphorus concentration throughout the reaction. The fluoride concentration decreased shortly after the start of hydrolysis. The hydrolysis of TTCP in 0.1 mol l–1 KH2PO4 proceeded to form hydroxyapatite via DCPD when the KF concentration was low. The hydrolysis product was a calcium-deficient non-stoichiometric hydroxyapatite with a Ca/P ratio of about 1.5. With an increase in the KF concentration in the 0.1 mol l–1 KH2PO4 solution, TTCP directly transformed into hydroxyapatite containing F- ions or fluorapatite and with improved crystallinity. The addition of fluoride in the solution initially accelerated the formation of apatite. However, the layer of newly formed apatite adhering to the TTCP particles retarded TTCP dissolution; as a result, hydrolysis was delayed. IR analysis showed that the apatite phase contained HPO2–4 ions in the structure. The formula for the hydrolysis product of TTCP in the presence of fluoride can be expressed as follows: Ca10–x(HPO4)x(PO4)10–x(OH)2–x–yFy. © 1998 Chapman & Hall.     

Formation mechanism of biomedical apatite coatings on porous titania layer

A titania containing calcium and phosphate with rough and porous structure was prepared by microarc oxidation. The in vitro bioactivity was examined by immersing the samples into the simulated body fluid (SBF). And the mechanism was also discussed. The results show that only 3 days of immersion in SBF, apatite was formed on the surface, and after 6 days, nearly all the surface covered by apatite. This indicates that the layer can induce the formation of apatite in simulated body fluid. It is analyzed that the key factors of the apatite formation are the hydrolysis of the CaTiO₃ and special structure.     

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.     

Dynamic study of calcium phosphate formation on porous HA/TCP ceramics

Bone-like apatite formation on porous calcium phosphate ceramics was investigated in static simulated body fluid (SBF) and dynamic SBF at different flowing rates. The results of a 14-day immersion in static SBF showed that the formation of bone-like apatite occurred both on the surface and in the pores of the samples. When SBF flow at the physiological flow rate in muscle (2 ml/100 ml min1), bone-like apatite could be detected only in internal surface of the pores of samples. The result that bone-like apatite formation could only be found in the pores when SBF flown at physiological flow rate was consistent with that of porous calcium phosphate ceramics implanted in vivo: osteoinduction was only detected inside the pores of the porous calcium phosphate ceramics. This result implicates that the bone-like apatite may play an important role in the osteoinduction of Ca-P materials. The dynamic model used in this study may be better than usually used static immersion model in imitating the physiological condition of bone-like apatite formation. Dynamic SBF method is very useful to understand bone-like apatite formation in vivo and the mechanism of ectopic bone formation in calcium phosphate ceramics.     

Apatite-forming ability of glass-ceramic apatite–wollastonite – polyethylene composites: effect of filler content

The bioactivity of a range of glass-ceramic apatite–wollastonite (A–W) – polyethylene composites (AWPEXs) with glass-ceramic A–W volume percentages ranging from 10 to 50, has been investigated in an acellular simulated body fluid (SBF) with ion concentrations similar to those of human blood plasma. The formation of a biologically active apatite layer on the composite surface after immersion in SBF was demonstrated by thin-film X-ray diffraction (TF-XRD) and field-emission scanning electron microscopy (FE-SEM). An apatite layer was formed on all the composites, with the rate of formation increasing with an increase in glass-ceramic A–W percentage. For composites with glass-ceramic A–W filler contents 30 vol %, the apatite layer was formed within 12 h of immersion, which is a comparable time for apatite formation on monolithic glass-ceramic A–W. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) demonstrated that the apatite formation on AWPEX samples with 50 vol % filler content occurred in a manner similar to that seen on pure glass-ceramic A–W, in that the calcium, silicon, and magnesium ion concentrations increased and, conversely, a decrease was observed in the phosphate ion concentration. These results indicate that a suitable in vitro response was achieved on a composite incorporating particulate glass-ceramic A–W with a particularly favorable response being observed on the AWPEX sample with 50 vol % filler content.     

Process of formation of bone-like apatite layer on silica gel

It has been proposed that a hydrated silica plays an important role in forming a biologically active apatite layer on the surfaces of bioactive glasses and glass-ceramics in the body. Recent experiments have shown that a silica hydrogel actually induces apatite formation on its surface in a simulated body fluid (SBF). In the present study the process of apatite formation on silica gel was investigated by means of thin-film X-ray diffraction, Fourier-transformed infrared reflection spectroscopy and scanning electron microscopic observation of the surface of the silica gel, as well as the measurement of changes in the ion concentration of the fluid. It was found that the induction period for the apatite nucleation on the surface of the silica gel was about 6 days. Once the apatite nuclei were formed they grew, taking a spherulitic form by consuming the calcium and phosphate ions from the surrounding fluid. Each spherulite consisted of a lot of flake that clustered into a petal-like morphology. The flake was carbonate-containing hydroxyapatite of small-crystallites and/or defective structure. The Ca/P ratio of the apatite was estimated as 1.5–1.6. Thus, the apatite formed was able to induce secondary nucleation of the apatite.     

XPS study of the process of apatite formation on bioactive Ti—6Al—4V alloy in simulated body fluid

Bioactive Ti—6Al—4V alloy, which spontaneously forms a bonelike apatite layer on its surface in the body and bonds to living bone through this apatite layer, can be prepared by producing an amorphous sodium titanate on its surface by NaOH and heat treatments. In this study, the process of apatite formation on the bioactive Ti—6Al—4V alloy was investigated in vitro, by analyzing its surface with X-ray photoelectron spectroscopy as a function of soaking time in a simulated body fluid 4SBF). Thin-film X-ray diffractometry of the alloy surface and atomic emission spectroscopy of the fluid were also performed complementarily. It was found that immediately after immersion in the SBF,the alloy exchanged Na¹ ions from the surface sodium titanate with H₃O¹ ions in the fluid to form Ti-OH groups on its surface. The Ti-OH groups, immediately after their formation,incorporated the calcium ions in the fluid to form calcium titanate. The calcium titanate thereafter incorporated the phosphate ions in the fluid to form an amorphous calcium phosphate, which was later crystallized into bonelike apatite. This process of apatite formation on the alloy was the same as on the pure titanium metal, because the alloy formed the sodium titanate free of Al and V by the NaOH and heat treatments. The initial formation of the calcium titanate is proposed to be a consequence of the electrostatic interaction of negatively charged units of titania dissociated from the Ti-OH groups with the positively charged calcium ions in the fluid. The calcium titanate is postulated to gain a positive charge and interact with the negatively charged phosphate ions in the fluid to form amorphous calcium phosphate, which eventually stabilizes into crystalline apatite.     

XPS study of the process of apatite formation on bioactive Ti–6Al–4V alloy in simulated body fluid

Bioactive Ti–6Al–4V alloy, which spontaneously forms a bonelike apatite layer on its surface in the body and bonds to living bone through this apatite layer, can be prepared by producing an amorphous sodium titanate on its surface by NaOH and heat treatments. In this study, the process of apatite formation on the bioactive Ti–6Al–4V alloy was investigated in vitro, by analyzing its surface with X-ray photoelectron spectroscopy as a function of soaking time in a simulated body fluid (SBF). Thin-film X-ray diffractometry of the alloy surface and atomic emission spectroscopy of the fluid were also performed complementarily. It was found that immediately after immersion in the SBF, the alloy exchanged Na⁺ ions from the surface sodium titanate with H₃O⁺ ions in the fluid to form Ti-OH groups on its surface. The Ti-OH groups, immediately after their formation, incorporated the calcium ions in the fluid to form calcium titanate. The calcium titanate thereafter incorporated the phosphate ions in the fluid to form an amorphous calcium phosphate, which was later crystallized into bonelike apatite. This process of apatite formation on the alloy was the same as on the pure titanium metal, because the alloy formed the sodium titanate free of Al and V by the NaOH and heat treatments. The initial formation of the calcium titanate is proposed to be a consequence of the electrostatic interaction of negatively charged units of titania dissociated from the Ti-OH groups with the positively charged calcium ions in the fluid. The calcium titanate is postulated to gain a positive charge and interact with the negatively charged phosphate ions in the fluid to form amorphous calcium phosphate, which eventually stabilizes into crystalline apatite.     

Chemical surface treatment of silicone for inducing its bioactivity

It has been confirmed that the apatite nucleation is induced by silanol (Si–OH) groups formed on the surfaces of materials and/or silicate ions adsorbed on them.It was previously shown that apatite nuclei are formed on organic polymers when the polymers are placed on CaO, SiO2-based glass particles soaked in a simulated body fluid (SBF) with ion concentrations nearly equal to those of human blood plasma, and that they grow spontaneously to form a dense and uniform apatite layer together with high adhesive strength to the substrates when the polymers are soaked in another solution with ion concentrations 1.5 times the SBF. In the present study, silanol groups bonded covalently to the surface of the silicone substrate were formed and its apatite-forming ability was examined. When silicone substrates were treated with 5 or 10 M NaOH with pH 7.25 at 36.5°C for more than 3 h, silanol groups were formed on the surfaces of the substrates. When thus NaOH-treated substrates were soaked in 1.5SBF at 36.5°C, a bone-like apatite was formed on the substrates in a short period. © 1998 Chapman & Hall.     

Comparative study of apatite formation on CaSiO<Subscript>3</Subscript> ceramics in simulated body fluids with different carbonate concentrations

Apatite formation on CaSiO₃ ceramics was investigated using two different simulated body fluids (SBF) proposed by Kokubo (1990) and Tas (2000) and three sample/SBF (S/S) ratios (1.0, 2.5 and 8.3 mg/ml) at 36.5°C for 1–25 days. The CaSiO₃ ceramic was prepared by firing coprecipitated gel with Ca/Si = 0.91 at 1400°C. The bulk density was 2.14 g/cm³ and the relative density about 76%. The two SBF solutions contain different concentrations of HCO₃^– and Cl^– ions, the concentrations of which are closer to human blood plasma in the Tas SBF formulation than in the Kokubo formulation. The pH values in the former solution are also more realistic. The CaSiO₃ ceramics show apatite formation in SBF (Kokubo) after soaking for only 1 day at all S/S ratios whereas different phases were formed at each S/S ratio in SBF (Tas). The crystalline phases formed were mainly apatite at S/S = 1.0 mg/ml, carbonate-type apatite at 2.5 mg/ml and calcite at 8.3 mg/ml. At higher S/S ratios the increase in the Ca concentration became higher while the P concentration became lower in the reacted SBF. These changes in SBF concentrations and increasing pH occurred at higher S/S ratios, producing more favorable conditions in the SBF for the formation of carbonate bearing phases, finally leading to the formation of calcite instead of apatite in the higher HCO₃^– ion concentration SBF (Tas). Apatite is, however, formed in the lower HCO₃^– ion concentration SBF (Kokubo) even though the Ca and P concentrations change in a similar manner to SBF (Tas).     

NaOH treatment of vacuum-plasma-sprayed titanium on carbon fibre-reinforced poly(etheretherketone)

Carbon fibre-reinforced polyetheretherketone (CF-PEEK) substrates were coated with titanium by vacuum-plasma-spraying and chemically treated in 10 M sodium hydroxide (NaOH) solution. After NaOH treatment, the specimens were immersed in simulated body fluid (SBF) containing ions in concentrations similar to those of human blood plasma. Scanning electron microscopy, energy-dispersive X-ray analysis and diffuse reflectance Fourier transformed–infrared spectroscopy were used to analyse the NaOH-treated VPS-Ti surface and the calcium phosphate layer formed during immersion in SBF. It was observed that a carbonate-containing calcium phosphate layer was formed on the NaOH-treated VPS-Ti surface during immersion in SBF, whereas no calcium phosphate precipitation occurred on the untreated surfaces. It is therefore concluded that vacuum-plasma-spraying with titanium and subsequent chemical modification in 10 M NaOH solution at 60°C for 2 h is a suitable method for the preparation of bioactive coatings for bone ongrowth on CF-PEEK.     

通过仿生法在硅橡胶表面制备磷灰石薄膜的研究

用CaCl2的乙醇溶液和K2HPO4溶液对硅橡胶进行预处理,将处理过的硅橡胶分别浸渍于模拟体液和钙磷饱和溶液中来制备磷灰石薄膜.利用薄膜X射线衍射、红外吸收光谱和扫描电子显微镜对形成的薄膜进行了表征.结果表明,分别在模拟体液中7d和在钙磷饱和溶液中5d后,硅橡胶表面形成了一层磷灰石薄膜;在模拟体液中的薄膜表面呈网状并分布有许多球状晶粒,在钙磷饱和溶液中的薄膜为结晶良好的片状晶体.     

Apatite deposition on polyamide films containing carboxyl group in a biomimetic solution

The development of organic–inorganic hybrids composed of hydroxyapatite and organic polymers is attractive because of their novelty in being materials that show a bone-bonding ability, i.e. bioactivity, and because they have mechanical properties similar to those of natural bone. The biomimetic process has received much attention for fabricating such a hybrid, where bone-like apatite is deposited under ambient conditions on polymer substrates in a simulated body fluid (SBF) having ion concentrations nearly equal to those of human extracellular fluid or related solutions. It has been shown that the carboxyl group is effective for inducing heterogeneous nucleation of apatite in the body. In the present study, apatite deposition on polyamide films containing various numbers of carboxyl groups was investigated in 1.5 SBF, which had ion concentrations 1.5 times those of a normal SBF. The effect of incorporation of calcium chloride on the formation of apatite was examined. Polyamide films containing 33 mol % CaCl₂ did not form apatite, even after soaking in 1.5 SBF for 7 days, and even when the polymer film contained 50 mol % carboxyl group. On the other hand, those modified with 40 mass % CaCl₂ formed apatite on their surfaces in 1.5 SBF. The ability of the modified film to form an apatite layer increased, and the adhesion of the apatite layer bonded to the film improved, with increasing carboxyl group content. It is concluded that novel apatite–polyamide hybrids can be prepared by a biomimetic process.     

Development of bioactive zirconium–tin alloy by combination of micropores formation and apatite nuclei deposition

In previous studies, Zr gained apatite‐forming ability by various methods; however, it took more than 7 days in simulated body fluid (SBF) to gain apatite‐forming ability. In this study, the authors developed the method to achieve apatite‐forming ability in Zr alloy within 1 day in SBF by a combination with apatite nuclei that promote apatite formation in SBF. First, Zr–Sn alloy was soaked in concentrated sulphuric acid, and pores in micro‐level were formed on the surface of Zr–Sn alloy. To attain apatite forming ability in Zr–Sn alloy, second, apatite nuclei were formed in the micropores. To evaluate apatite‐forming ability, thus‐obtained Zr–Sn alloy with apatite nuclei was soaked in SBF; hydroxyapatite formation was observed on the whole surface of the Zr–Sn alloy plates. From this result, it was clarified that higher apatite‐forming ability was attained on the apatite nuclei‐treated Zr–Sn alloy with micropores in comparison with that without micropores. When adhesive strength of formed hydroxyapatite film with respect to Zr–Sn alloy plates was measured, high‐adhesive strength of the formed apatite film was attained by forming micropores and subsequently precipitating apatite nuclei in the fabrication process because of an interlocking effect caused by hydroxyapatite formed in the micropores.Inspec keywords: precipitation, zirconium alloys, calcium compounds, bioceramics, tin alloys, adhesion, thin filmsOther keywords: apatite forming ability, micropore formation, hydroxyapatite film, bioactive zirconium‐tin alloy, apatite nuclei‐treated zirconium‐tin alloy, zirconium‐tin alloy plates, simulated body fluid, concentrated sulphuric acid, hydroxyapatite formation, adhesive strength, precipitating apatite nuclei, time 1.0 d, ZrSn, Ca₁₀ (PO₄)₆ (OH)₂      

Biological control of apatite growth in simulated body fluid and human blood serum

The surface transformation reactions of bioactive ceramics were studied in vitro in standard K9-SBF solution and in human blood serum (HBS)—containing simulated body fluid (SBF). The calcium phosphate ceramics used for this study were stoichiometric hydroxyapatite (HA), β-tricalcium phosphate (β-TCP) and brushite. Immersion of each calcium phosphate tested in this study, in simulated body fluid, led to immediate surface precipitation of apatite. The use of HBS resulted in a delay in the onset of precipitation and a significant inhibition of the dissolution reaction normally observed for brushite in solution. However, apatite formation still occurred. The use of HBS and SBF in this investigation, which has shown the ability to induce similar crystal growth as that observed in vivo, suggests that there is scope for the use of serum proteins in simulated body fluid in order to create a protein-rich surface coating on biomedical substrates.     

Bioactive coating on a cobalt base alloy by heat treatment

A method to promote a bioactive surface on the cobalt base alloy ASTM F-75 was tested. A set of cylindrical samples was obtained using the investment casting technique and packed in a mixture composed of 70% β-tricalcium phosphate and 30% bioactive glass and then heat treated for 1 h at 1220 °C. To characterize the in vitro bioactivity, a set of heat treated metallic specimens was immersed in a simulated body fluid with an ionic concentration nearly equal to that of human blood plasma (SBF) for 7, 10 and 21 days and in a more concentrated solution (1.5 SBF). After heat treatment, fine agglomerates homogeneously distributed, containing Ca, P, O and Na were observed on the metallic surface. After immersion of the samples in simulated body fluids, a thicker layer identified as apatite was formed on the samples immersed in SBF for 21 days and on all samples immersed in 1.5 SBF.     

磷酸钙陶瓷在不同动物的模拟体液中类骨磷灰石的形成研究

根据人、狗、猪、猴和兔五种动物体液的钙离子浓度和pH值的差异,配制了不同组分的模拟体液,将孔壁致密和有微孔的多孔磷酸钙陶瓷分别浸泡在这些模拟体液中,研究陶瓷孔隙表面类骨磷灰石的形成情况.结果表明:在模拟体液中浸泡14天后,孔壁致密的材料未见有类骨磷灰石层形成;有微孔的多孔磷酸钙陶瓷,材料孔壁表面(包括陶瓷表面较深孔隙)有类骨磷灰石层的形成,这与体内植入实验观察到的类骨磷灰石层形成和诱导成骨情况相似,可以推论类骨磷灰石层的形成的确是骨诱导的先决条件.随着钙离子浓度的增加,其孔壁表面类骨磷灰石层的形成也更为均匀,但类骨磷灰石生长快慢顺序与动物组织学观察到的骨诱导性高低的次序不完全一致.