Low temperature fabrication of spherical brushite granules by cement paste emulsion

Secondary protonated calcium phosphates such as brushite (CaHPO₄·2H₂O) or monetite (CaHPO₄) have a higher resorption potential in bone defects than sintered ceramics, e.g. tricalcium phosphate or hydroxyapatite. However, processing of these phosphates to monolithic blocks or granules is not possible by sintering due to thermal decomposition of protonated phosphates at higher temperatures. In this study a low temperature technique for the preparation of spherical brushite granules in a cement setting reaction is presented. These granules were synthesized by dispersing a calcium phosphate cement paste composed of β-tricalcium phosphate and monocalcium phosphate together with a surfactant to an oil/water emulsion. The reaction products were characterized regarding their size distribution, morphology, and phase composition. Clinically relevant granule sizes ranging from 200?μm to 1?mm were obtained, whereas generally smaller granules were received with higher oil viscosity, increasing temperature or higher powder to liquid ratios of the cement paste. The hardened granules were microporous with a specific surface area of 0.7?m²/g and consisted of plate-like brushite (>95?% according to XRD) crystals of 0.5–7?μm size. Furthermore it was shown that the granules may be also used for drug delivery applications. This was demonstrated by adsorption of vancomycin from an aqueous solution, where a load of 1.45–1.88?mg drug per g granules and an almost complete release within 2?h was obtained.     

Enzyme-catalysed synthesis of calcium phosphates

A biomimetic method is described for the precipitation of nanosized calcium phosphates using the alkaline phosphatase (EC 3.1.3.1), which is responsible for hydrolysis of organic and inorganic phosphates in vivo. Buffered solutions containing glycerol-2-phosphate and CaCl₂ in addition to MgCl₂ and the respective enzyme were prepared for calcium phosphate precipitation. The phosphate group of glycerol-2-phosphate was cleaved through enzymatic hydrolysis. The local inorganic phosphate concentration increased resulting in the precipitation of nanosized calcium phosphates phases (Ca–P phase) composed of calcium deficient hydroxyapatite (CDHA) and hydroxyapatite (HA). At high Ca²⁺-concentration and large enzyme amounts mixed phases of HA/CDHA with an increasing quantity of HA were favoured. Under basic conditions (pH > 9) formation of HA was observed, whereas at neutral pH of 7.5 CDHA was primarily formed. The assignment of Ca–P phases was accomplished by FT-IR and Raman-spectroscopy in addition to X-ray diffractometry. The Ca–P materials exhibited BET surface areas of 173 m²/g. SEM-micrographs of the Ca–P powders showed globular-shaped agglomerates of Ca–P particles. The size of the Ca–P crystallite ranged from 9 nm to 25 nm according to transmission electron microscopy (TEM), where round-shaped, platelike and fibrelike crystallites were found. All crystallites showed diffuse ring patterns in electron diffraction confirming the nanosize of the precipitate. Using the developed technique, it was possible to synthesise 100 g of bonelike Ca–P materials in 1 day using 15 L batches with optimised parameters.     

Doped nanocrystalline calcium carbonate phosphates

The formation of nanocrystalline calcium carbonate phosphates doped with Fe²⁺, Mg², Zn²⁺, K⁺, Si⁴⁺, and Mn²⁺ has been studied by X-ray diffraction, IR spectroscopy, differential thermal analysis, and energy dispersive X-ray fluorescence analysis. The results indicate that the synthesis involves the formation of hydroxy carbonate complexes from the three calcium carbonate polymorphs (calcite, vaterite, and aragonite) in a solution of ammonium chloride and ammonium carbonate, followed by reaction with orthophosphoric acid. This ensures the preparation of a bioactive material based on chlorophosphates, octacalcium hydrogen phosphate, and calcium chloride hydroxide phosphates containing cation vacancies. Particle-size analysis data show that the materials contain nanoparticles down to 10 nm in size. Heat treatment of the doped calcium carbonate phosphates produces calcium hydroxyapatite containing cation vacancies, which can be used as a bioactive ceramic.     

Sorption of strontium ions from solutions onto calcium and magnesium phosphates

A comparative study of the uptake of Sr ions from aqueous solutions with calcium and magnesium phosphates of various chemical compositions was made. Calcium and magnesium phosphates in the concentration range 10–5000 mg L^?1 absorb up to 280 mg g^?1 Sr, with the ability of magnesium phosphates to take up Sr ions exceeding that of calcium phosphates by an order of magnitude. The degree of decontamination of solutions from ⁹⁰Sr with the magnesium-containing samples exceeds 80%, and the distribution coefficient is (4.7–5.4) × 10³ cm³ g^?1.     

Preparation of high specific surface area hydroxyapatite for environmental applications

We report the preparation of hydroxyapatite in powdered form by aqueous reaction of calcium nitrate or hydroxide with phosphate ion at room temperature. With a slow maturation step of 48 h avoiding heat, the resulting products show large specific surface areas above 150 m²/g. The specific surface areas also depend on stirring speed with a maximum observed with gentle mixing. Ageing causes a decrease in specific surface area which tends to stabilize near 100 m²/g following a 1 year period. The hydroxyapatite may be spray dried and conserve interesting physical properties for environmental applications.     

Using a synthetic body fluid (SBF) solution of 27 mM HCO3− to make bone substitutes more osteointegrative

A Tris–HCl-buffered synthetic body fluid (SBF) solution, mimicking the human blood plasma, with the following ion concentrations of 27 mM HCO₃⁻, 2.5 mM Ca²⁺, 1.0 mM HPO₄²⁻, 142 mM Na⁺, 125 mM Cl⁻, 5 mM K⁺, 1.5 mM Mg²⁺, and 0.5 mM SO₄²⁻ was used as an aqueous medium to process a number of bone substitute materials under the so-called biomimetic conditions of 37 °C and pH 7.4. This solution was named as Tris–SBF-27 mM. Firstly, collagen sponges were soaked in Tris–SBF-27 mM solution at 37 °C and were found to be fully covered with nanoporous apatitic calcium phosphate (Ap-CaP). The composites of collagen–Ap-CaP biomaterials are expected to be used in orthopedic and dental surgery. Secondly, Ap-CaP short whiskers or microrods with a novel nanotexture and surface areas higher than 45 m²/g were synthesized in Tris–SBF-27 mM solution. Thirdly, calcium sulfate cements doped with CaHPO₄ (monetite), were shown to have apatite-inducing ability upon ageing in Tris–SBF-27 mM. CaHPO₄ addition in calcium sulfate was found to improve its mechanical strength, measured after cement setting reaction. Pure calcium sulfate cement pellets were not stable in Tris–SBF-27 mM solutions and crumbled into a powder. All the samples were characterized by SEM, XRD, FTIR, surface area and mechanical strength measurements.     

Ca3(PO4)2:Er3+,Yb3+: An upconversion phosphor for in vivo imaging

Using a sol-gel process, we have synthesized compounds isostructural with the biogenic mineral whitlockite and containing calcium phosphate, β-Ca₃(PO₄)₂, codoped with Er³⁺ and Yb³⁺ in various concentrations and ratios. Their particle size was determined to be ～42–57 nm by atomic force microscopy and ～93 nm by dynamic light scattering. The particles were shown to be weak anion exchangers (their zeta potential is ?13.3 mV). The observed luminescence of the β-Ca₃(PO₄)₂:Er³⁺,Yb³⁺ phosphates in the visible spectral region (λ = 0.525, 0.550, and 0.650 μm) under IR excitation (λ = 0.98 μm) is due to the upconversion mechanism and is acceptable for in vivo imaging in terms of safety (green emission) and intensity. Biocompatibility testing results demonstrate that the β-Ca₃(PO₄)₂:Er³⁺,Yb³⁺ phosphates meet the relevant biosafety and nontoxicity criteria.     

Comparison of failure mechanisms for cements used in skeletal luting applications

Glass Polyalkenoate Cements (GPCs) based on strontium calcium zinc silicate (Sr–Ca–Zn–SiO₂) glasses and low molecular weight poly(acrylic acid) (PAA) have been shown to exhibit suitable compressive strength (65 MPa) and flexural strength (14 MPa) for orthopaedic luting applications. In this study, two such GPC formulations, alongside two commercial cements (Simplex^® P and Hydroset?) were examined. Fracture toughness and tensile bond strength to sintered hydroxyapatite and a biomedical titanium alloy were examined. Fracture toughness of the commercial Poly(methyl methacrylate) cement, Simplex^® P, (3.02 MPa m^1/2) was superior to that of the novel GPC (0.36 MPa m^1/2) and the commercial calcium phosphate cement, Hydroset?, for which no significant fracture toughness was obtained. However, tensile bond strengths of the novel GPCs (0.38 MPa), after a prolonged period (30 days), were observed to be superior to commercial controls (Simplex? P: 0.07 MPa, Hydroset?: 0.16 MPa).     

Effects of cooling conditions and chitosan coating on the properties of porous calcium phosphate granules produced from hard clam shells

The main inorganic components of clam shells are calcium carbonate and trace elements of magnesium, strontium, and zinc. Clam shells can be used as a calcium source to synthesize calcium phosphates, and these trace elements promote the growth of bone tissue and improve the performance of bioceramics. In this study, hydroxyapatite (HA) powders were synthesized from clam shells, and porous calcium phosphate granules were prepared through the gas foaming technique. In addition, the effects of a chitosan coating and cooling conditions on the strength of the porous granules were investigated. The results indicated that the samples produced under the furnace cooling condition were biphasic hydroxyapatite/β-tricalcium phosphate granules (HA/β-TCP), whereas the samples produced under the air-cooling condition were triphasic hydroxyapatite/β-tricalcium phosphate/α-tricalcium phosphate granules (HA/β-TCP/α-TCP). The compressive strength of the porous granules prepared through air cooling was 79% higher than that of the granules produced through furnace cooling. The compressive strength of the air-cooled sample after the subsequent application of the chitosan coating further increased by 21%. A degradability test revealed that the weight loss rate of the air-cooled samples was greater than that of the furnace-cooled samples, which was due to the presence of high-solubility α-TCP in the air-cooled samples.     

Block-copolymer-assisted synthesis of hydroxyapatite nanoparticles with high surface area and uniform size

Abstract

We report the synthesis of hydroxyapatite nanoparticles (HANPs) by the coprecipitation method using calcium D-gluconate and potassium hydrogen phosphate as the sources of calcium and phosphate ions, respectively, and the triblock copolymer F127 as a stabilizer. The HANPs were characterized using scanning electron microscopy, x-ray diffraction, and nitrogen adsorption/desorption isotherms. Removal of F127 by solvent extraction or calcination alters the structure of HANPs. The solvent-extracted HANPs were single crystals with their 〈001〉 axis oriented along the rod axis of the HANP, whereas the calcined HANPs contained two crystal phases that resulted in a spherical morphology. The calcined HANPs had much higher surface area (127 m² g^?1) than the solvent-extracted HANPs (44 m² g^?1).     

Catalytic performance of ceria fibers with phosphatase-like activity and their application as protein carriers

Ceria (CeO₂) synthesized by cerium nitrate hexahydrate in alkaline solution under hydrothermal treatment produces a fiber structure that allows high O-P bond cleavage activity. Brunauer–Emmett–Teller (BET) analysis revealed that fiber-morphological CeO₂ with high surface area (73.9 m²/g) and pore volume (0.42 cm³/g) showed better hydrolytic activity than nanopolyhedral and cubic morphologies. The CeO₂ fiber displayed hydrolytic activity in a tris(hydroxymethyl)aminomethane (Tris) buffer; however, no reaction occurred in a phosphate buffer. From analysis by based on scanning transmission electron microscopy (STEM) and energy-dispersive X-ray spectroscopy (EDX), the phosphate group in the buffer was seen to be immediately adsorbed on the surface of CeO₂ particles; therefore, the CeO₂ catalysts could not attack the phosphoric esters as a substrate. In addition, the CeO₂ fiber showed hydrolytic activity for deoxyribonucleic acid (DNA). Moreover, enzymes loaded on the CeO₂ fiber particles retained activity levels equivalent to free-solution enzymes. It is thought that the findings of the present study regarding the properties of CeO₂ fiber will have a significant impact in the fields of not only antibacterial and antimicrobial reagents, but also biosensors and biocatalysts.     

Hydrolyses of calcium phosphates-allografts composite in physiological solutions

Hydrolysis of calcium phosphates cement- allografts composite in calf serum and that in saline were examined in comparison with those of the calcium phosphates cement in both the solutions. The calcium phosphates cement consists of α-tricalcium phosphate (α-TCP), tetracalcium phosphate (TetCP), dicalcium phosphate dihydrate (DCPD), and hydroxyapatite (HAP), which is clinically used as Biopex. In the hydrolyses of Biopex-allografts composite in both the solutions, the calcium phosphates cement was transformed into HAP. On the other hand, in the hydrolyses of Biopex, HAP was formed after 1 day and octacalcium phosphate (OCP) was gradually formed after 7 days. In the presence of allografts, plate-like crystals were deposited and in the absence of allografts, needle-like crystals were deposited in both the solutions. By the addition of allografts, the hydrolysis process of the calcium phosphates cement was significantly changed.     

Chemical transformation of some biologically relevant calcium phosphates in aqueous media during a steam sterilization

The purpose of this study was to investigate the effect of steam sterilization on some biologically relevant calcium phosphates: CaHPO₄· 2H₂O (DCPD), calcium deficient apatite (CDA) and biphasic calcium phosphate (BCP). Suspensions of 0.2 g of each calcium phosphate compound with 5.0 ml of deionized water were prepared and steam sterilized in an autoclave (20 min at 121 °C). After sterilization the suspensions were filtered and the dried solids characterized with scanning electron microscopy, IR-spectroscopy and X-ray diffraction. The pH and calcium concentrations of the filtrates were determined with ion selective electrodes. Similar measurements were made with the same samples which were not sterilized. The sterilization procedure was found to result in the dehydration of DCPD and hydration of calcium oxide incorporated into the BCP. Solution pH was observed to change from 7.3 to 5.5 for the solutions in equilibrium with DCPD and from 8.5 to 10.6 for those in equilibrium with BCP. Minor changes both with the solid and liquid phases were found to occur during the steam sterilization of CDA. These results indicate that steam sterilization may have different effects on different calcium phosphate suspensions: it can result in dehydration of DCPD, fast hydration for CaO in BCP, but no significant effect on CDA.     

Synthesis of lamellar mesostructured calcium phosphates using n-alkylamines as structure-directing agents in alcohol/water mixed solvent systems

Lamellar mesostructured calcium phosphates constructed by ionic bonds were prepared by using n-alkylamines (n-C _n H_2n+1NH₂, n = 8–18) at room temperature in the mixed solvent systems of aliphatic alcohol (C _n H_2n+1OH, n = 1–4) and water, and the synthetic conditions were investigated in detail. The mixed solvent systems suppressed the formation of crystalline calcium phosphates like brushite (CaHPO₄·2H₂O) and monetite (CaHPO₄) at low temperatures, successfully affording pure lamellar mesostructured calcium phosphates. Other crystalline phases such as hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂) were not formed under the conditions with the Ca/P molar ratios in the range of 0.7–1.0 in the starting mixtures. The Ca/P molar ratio of the lamellar mesostructured calcium phosphates was ca. 1.0, calculated by ICP and ³¹P MAS NMR data. Interestingly, the kind of alcohols strongly influenced the solubilities of calcium phosphate species and n-alkylamines, and then lamellar mesostructured phases were obtained with some morphological variation.     

Mineral phase deposition on pectin microspheres

The nucleation of apatite and calcium phosphates onto natural polysaccharides containing carboxyl groups can be obtained by immersion in biomimetic solutions. The deposition of hydroxyapatite has been recently described also on pectin, and pectin–apatite hybrid gels were proposed as scaffolds for bone tissue regeneration. In this work, injectable calcium phosphate/pectin microspheres were prepared to promote both the natural process of biomineralization and the controlled release of encapsulated cells, genes, enzymes, proteins or drugs in the pathological situ. Pectin microspheres were prepared with different formulations from aqueous solutions using an extrusion system, and incubated in H₂O or SBF for 14 days. The presence of an excess of CaCl₂, deriving from the preparation process, was determinant in promoting the deposition of calcium phosphates from SBF, whereas no mineral deposition was detected on pectin microspheres extensively purified after preparation. The nucleation of calcium phosphates occurred on pectin microspheres incubated in SBF for 14 days and was evaluated through ESEM–EDS analysis and FT-IR spectroscopy.     

Bioactivity of calcium phosphate coatings prepared by electrodeposition in a modified simulated body fluid

The purpose of this study was to investigate bioactivity of calcium phosphate coatings prepared by electrodeposition in a modified simulated body fluid (SBF). Calcium phosphates were electrodeposited on commercially pure titanium substrates in the modified SBF at 60 °C for 1 h maintaining the cathodic potentials of − 1.5 V, − 2 V, and − 2.5 V (vs. SCE). Subsequently, the calcium phosphate coatings were transformed into apatites during immersion in the SBF at 36.5 °C for 5 days. The apatites consisted of needle-shaped crystallites distributed irregularly with different grain sizes. As the coatings were electrodeposited at higher cathodic potential, the crystallite of the apatites got denser and the grain sizes of the apatites became bigger during subsequent immersion in the SBF. However, as the coatings were electrodeposited at higher cathodic potential, the coatings were transformed into apatites with lower crystallinity and the Ca/P atomic ratio of the apatites got higher than 1.67, that of stoichiometric hydroxyapatite, after subsequent immersion in the SBF. In addition, CO₃²⁻ ions contained in the modified SBF were incorporated in the calcium phosphate coating during electrodeposition and had an influence on transforming the calcium phosphate into bonelike apatite during subsequent immersion in the SBF showing that CO₃²⁻ incorporated in the apatites disturbed crystallization of the apatites. These results revealed that the coating electrodeposited at − 2.0 V (vs. SCE) in the modified SBF containing CO₃²⁻ ions was the most bioactive showing transformation into carbonate apatite similar to bone apatite.     

Zinc phosphate as versatile material for potential biomedical applications Part 1

Synthetic α - and β -Hopeite, two polymorphs of zinc phosphate tetrahydrates (ZPT) have been synthesized by hydrothermal crystallization from aqueous solution at 20 ^∘C and 90 ^∘C respectively. Aside from their subtitle crystallographic differences originating from a unique hydrogen bonding pattern, their thermodynamic interrelation has been thoroughfully investigated by means of X-Ray diffraction (XRD) and differential scanning calorimetry (DSC), combined with thermogravimetry (TGA-MS). Using a new heterogeneous step-reaction approach, the kinetics of dehydration of the two forms of ZPT was studied and their corresponding transition temperature determined. Low temperature DRIFT, FT-Raman and ¹H, ³¹P MAS-NMR reveal an oriented distortion of the zinc phosphate tetrahedra, due to a characteristic hydrogen bonding pattern and in accordance with the molecular tetrahedral linkage scheme of the phosphate groups. Biogenic Hydroxyapatite (HAP) and one of its metastable precursors, a calcium dihydrogen phosphate dihydrate (DCPD) or Brushite were also obtained and used to underline the resulting variations of chemical reactivity in zinc phosphates.     

An electrodeposition method of calcium phosphate coatings on titanium alloy

Calcium phosphates coatings were deposited onto titanium alloy discs via en electrodeposition method. Titanium alloy discs were blasted with calcium phosphate particles, then etched in a mixture of nitric and fluoric acids and rinsed in demineralized water. The titanium alloy disc (cathode) and platinum mesh (anode) were immersed in a supersaturated calcium phosphate electrolyte buffered at pH 7.4 and connected to a current generator. The microstructure, chemical composition and crystallinity of the electrodeposited coatings were studied as function of time 10–120 min, temperature 25–80°C, current density 8–120 mA/cm², magnesium and hydrogen carbonate amounts (0.1–1 mM). Uniform calcium phosphate coatings were obtained in 30 min but coating thickness increased with deposition time. Raising the temperature of electrolyte resulted in more uniform coatings as ionic mobility increased. Low current density was preferable due to hydrogen gas evolving at the cathode, which disturbed the deposition of calcium phosphate crystals on titanium. The amounts of magnesium and hydrogen carbonate ions affected both the homogeneity and morphology of the coatings. This study showed that the electrodeposition method is efficient for coating titanium with osteoconductive calcium phosphate layers.     

Biomimetic mineralisation of apatites on Ca2+ activated cellulose templates

We investigated the activation of regenerated cellulose 2D model thin films and 3D fabric templates with calcium dihydroxide. The Langmuir–Blodgett (LB) film technique was applied for manufacturing of the model thin films using a trimethylsilyl derivative of cellulose (TMS-cellulose). Regenerated cellulose films were obtained by treating the TMS-cellulose LB-films with hydrochloric acid vapours. For 3D templates, regenerated cellulose fabrics (Lyocell®) were used. The regenerated cellulose templates were activated with a Ca(OH)₂-suspension and subsequently exposed to 1.5 × SBF to induce the in situ formation of biomimetic calcium phosphate phases. FTIR and Raman spectroscopy showed that the Ca(OH)₂ and calcite present from reaction with HCO₃⁻ on the template surface were dissolved in the initial stage of exposure to the 1.5 × SBF. After 1 day, the formation of apatitic phases in 1.5 × SBF was observed. According to detailed calculations, high supersaturation levels S in close vicinity to the template surface (S > 16) resulting from the Ca²⁺ diffusion induced the formation of biomimetic calcium phosphate. The biomimetic calcium phosphates were identified by FTIR and Raman spectroscopy as highly carbonated apatites (HCA) lacking hydroxyl ions. 3D fabric templates of regenerated cellulose covered with a biomimetic coating of apatite might be of particular interest for novel scaffold architectures in bone repair and tissue engineering.     

Seeded growth of hydroxyapatite in simulated body fluid

The precipitation of calcium phosphates was investigated, in simulated body fluid (SBF), pH 7.40 and 37°C. The kinetics of the mineral phase forming in the SBF was measured using the constant supersaturation method. The approach provides a detailed investigation in the processes taking place in the SBF which is widely used for the study of biomineralization. The pH adjustment was done by a pH-stat instead of Tris-Buffer [Tris (hydroxymethyl) Aminomethane] to avoid the presence of organic soluble compounds. The stability of SBF was investigated and the stable supersaturated solutions were seeded. The technique of seeded precipitation was employed for the achievement of accurate and reproducible kinetics measurements. The crystal growth experiments in which SBF solutions of variable supersaturations were seeded with hydroxyapatite [Ca₅(PO₄)₃OH, HAP] crystals showed that the precipitation of calcium phosphates took place exclusively on specific active sites provided on the surface of the synthetic seed crystals. The crystal growth mechanism showed that the process was controlled by surface diffusion. The phase formed was HAP in the lattice of which CO₃²⁻ and Mg²⁺ ions were incorporated. SBF was the source of these ions. Moreover it was found that the less stable calcium phosphate dihydrate (CaHPO₄·2H₂O, DCPD) may form as a transient phase hydrolyzing rapidly into the more stable HAP. Morphological examination of the carbonated apatites formed in the SBF showed appreciable aggregation.