Calcium Phosphate Films Research Articles

Bioinspired Calcium Phosphate Coated Mica Sheets by Vapor Diffusion and Its Effects on Lysozyme Assembly and Crystallization

We propose for the first time the vapor diffusion method to deposit bioinspired calcium phosphate films on mineral substrates, that is, delaminated mica muscovite sheets and to assess the capability of these films to affect the nucleation and growth of lysozyme crystals. Deposited calcium phosphate layers were composed of octacalcium phosphate (OCP) and apatite (Ap) nanocrystals, with increased amount of OCP at higher crystallization times. In the presence of poly(acrylic acid) (PAA) deposited layers were composed of amorphous calcium phosphate (ACP). Results of lysozyme crystallization showed that OCP/Ap-coated mica sheets slowed down the nucleation process without altering significantly the number of nucleated crystals per droplet respect to the uncoated control. ACP-coated mica sheets acted as an inhibitor, thus delaying the nucleation and reducing in addition the number of crystals. These results contrasted with the nucleation induction effect observed when calcium phosphate nanopowders were added to ...

Construction of different morphology of calcium phosphate film on titanium base oxidation layer superhydrophobic-superhydrophilic pattern

Commercial ink-jet printer was applied to print defined pattern on the titanium base oxidation layer which was covered with a superhydrophobic coating. Then the superhydrophobic molecules in some regions were degraded under ultraviolet radiation to get a superhydrophobic/ superhydrophilic micro-pattern. Afterwards, CaP film with different morphologies was deposited onto the superhydrophobic and the superhydrophilic regions by electrochemical methods. The plate-like CaP films which formed on superhydrophilic zones had a more porous surface than those on superhydrophobic zones. In vitro cell culture experiment results showed that flat CaP coatings in superhydrophobic zones were better for MG63 osteoblast-like cells adhesion than the CaP film coating in superhydrophilic zone.

Alternate Calcification in Microcapillaries for the Fabrication of Hydroxyapatite Films without Light Exposure, Calcination, or Applied Voltage.

We discuss an alternate method for calcification in microcapillaries for fabricating calcium phosphate films using silicone molds and calcifying solutions. Calcium phosphate films with a line/space of 5-50 µm were fabricated by controlling the concentrations of calcium chloride and sodium phosphate solutions. Plate-type crystals of hydroxyapatite were grown when the calcium phosphate films were immersed in hydroxyapatite precursors. In the initial stage of hydroxyapatite crystal growth, the c-plane of the crystals was grown parallel to the substrates, and subsequently the growth followed with the c-plane growing perpendicular to the substrates. In narrow capillaries, dendritic structures were formed with a tendency to grow in a direction parallel to the direction of the microcapillaries. This technique is useful in the micropatterning of biocompatible ceramics with a minimized material consumption and a short fabrication time.

Calcium phosphate film on niobium substrate obtained by two-step oxidation

Calcium phosphate film on niobium substrate obtained by two-step oxidation

Nanoscale analysis of calcium phosphate films obtained by RF magnetron sputtering during the initial stages of deposition

In the present work, we investigated the nanostructure of calcium phosphate films formed during the initial stages of deposition (between 30 and 240s; deposition rate of 3.7nm/min) by radio-frequency magnetron sputtering. The sputtering deposition parameters and the plasma energy were previously adjusted to produce nearly stoichiometric nanocrystalline hydroxyapatite films after 180min. Grazing-incidence X-ray diffraction and X-ray photoelectron spectroscopy analyses revealed that the films formed in the initial stages were composed of an amorphous calcium phosphate phase with a Ca/P ratio Ca/P=1.60±0.12, which is close to that of hydroxyapatite. Using high-resolution transmission electron microscopy, we demonstrated that the films were not a unique amorphous phase. An additional phase composed of dense calcium phosphate nanospheres was found in all periods and investigated. The nanospheres grew in size as the deposition time increased, and small nanospheres were always observed. This observation suggests the continuous formation of nanospheres at the surface of the film. Nanocrystalline regions were revealed by electron diffraction and high-resolution electron microscopy after a deposition of 60s with a corresponding film thickness of 3.7nm. Electron energy-loss spectroscopy analysis comparing individual nanospheres to the amorphous calcium phosphate layer showed differences in the oxygen K edge, suggesting a difference in the chemical bonding of the oxygen species. Nanospheres were observed in association with isolated crystals, suggesting that the nanospheres may participate in the crystallization of hydroxyapatite according to a heterogeneous nucleation process.

Morphological, chemical and kinetic characterisation of zein protein-induced biomimetic calcium phosphate films.

This study examines zein protein-induced growth of calcium phosphate (CaP) thin films at air-liquid interfaces. The results demonstrate that zein protein films in contact with simulated body fluids (SBF) promote the growth of hemispherical CaP particles of 150 nm radius, which combine to form a film. The CaP films formed on zein were less continuous than those obtained using a hexadecanoic acid monolayer self-assembled at the air-liquid interface. In situ ellipsometry measurements were used to follow the nucleation and growth of CaP in these systems. The kinetic parameters extracted from the ellipsometry data were dependent on the organic layer and also the SBF concentration. The mineralisation process was slower and final CaP film thicknesses smaller when zein films were used. XPS analyses revealed the presence of zein protein only on the air-facing side of the CaP films, confirming that the zein protein is involved in nucleating the unusual hemispherical CaP morphology. The zein protein-induced CaP thin films showed iridescence with a distinct range of colours as a function of CaP film thickness. Templated CaP mineralisation with zein protein is a simple and chemically facile method for synthesising CaP films with novel morphologies and controllable thicknesses.

Nucleation, growth and evolution of calcium phosphate films on calcite

Marble, a stone composed of the mineral calcite, is subject to chemically induced weathering in nature due to its relatively high dissolution rate in acid rain. To protect monuments and sculpture from corrosion, we are investigating the application of thin layers of hydroxyapatite (HAP) onto marble. The motivation for using HAP is its low dissolution rate and crystal and lattice compatibility with calcite. A mild, wet chemical synthesis route, in which diammonium hydrogen phosphate salt was reacted with marble, alone and with cationic and anionic precursors under different reaction conditions, was used to produce inorganic HAP layers on marble. Nucleation and growth on the calcite substrate was studied, as well as metastable phase evolution, using scanning electron microscopy, grazing incidence X-ray diffraction, and atomic force microscopy. Film nucleation was enhanced by surface roughness. The rate of nucleation and the growth rate of the film increased with cationic (calcium) and anionic (carbonate) precursor additions. Calcium additions also influenced phase formation, introducing a metastable phase (octacalcium phosphate) and a different phase evolution sequence.

Effect of Strontium Enhanced Calcium Phosphate Coating on <i>In Vitro</i> Behavior of Human Mesenchymal Stem Cell (hMSC)

Calcium phosphate is a widely used material as coating for metallic implants. This research describes a biomimetic coating techniques based on deposition of calcium phosphate films on a Ti6Al4V plates that was used to study the effect of strontium additive on the behavior of hMSCs. In this study, strontium additive was homogenously deposited onto calcium phosphate films on a Ti6AlV plates by using a biomimetic techniques. Strontium affected composition and morphology of calcium phosphate deposited on a Ti6Al4V plates to a varying degree, according to concentration of solutions used. The effect of strontium additive on proliferation and differentiation of hMSCs depended on the solution and concentration tested. In general, all individual three coatings showed decreased hMSCs proliferation. Strontium additive demonstrated a significant increase in differentiation into osteogenic lineage when compared with the control and calcium phosphate films without strontium additive. However, no cytotoxic effect of strontium additive in the concentrations tested was detected. The Fourier transform infrared spectra showed that this new coating closely resembles bone mineral. The techniques illustrated in this study mimics bone mineral containing strontium additive, making it constructive for studying basic processes of in vitro bone formation. The results showed in this study can be used for changing bone graft substitutes by addition of strontium additive on implants in order to affect their performance in bone repair and regeneration. Also, the system can aid rapid bone formation around the implant, reducing therewith the patient’s recovery time after surgery.

The potential-assisted deposition as valuable tool for producing functional apatite coatings on metallic materials

Nitinol was coated with hydroxyapatite (HA) by the two-step method that includes electrodeposition of the calcium phosphate (brushite) film, as a precursor for the HA deposition, which is the second reaction step.This work concerns direct comparison of structural and barrier properties of the HA coatings produced by the phase transformation of brushite to HA in (i) an alkaline solution (NaOH) and (ii) a simulated body fluid solution (SBF).Chemical composition and morphology of the surface films were characterized using field emission scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), X-ray diffraction analysis (XRD) and X-ray photoelectron spectroscopy (XPS).The corrosion resistance was determined using in situ method of electrochemical impedance spectroscopy (EIS), under in vitro conditions, in order to optimize the production of bioactive HA coatings on metallic (Nitinol) implants for medical applications.The combination of advantages of the electrodeposition of the HA precursor film on the Nitinol surface and the treatment in an SBF solution, under in vitro conditions, has the potential to provide the HA coatings with the structural and microstructural homogeneity, excellent barrier properties (corrosion resistance) as well as bioactivity.

Bioinspired Crystallization of Continuous Calcium Phosphate Films on a Langmuir Monolayer of Zein Protein: Their Mechanical Performance, Hydrophilicity, and Biocompatibility

In this article, zein, a major protein of corn, has been used as biomimetic mineralization template to synthesize calcium phosphate by a bioinspired approach. A zein Langmuir monolayer, formed by self-assembling nanoglobules, is used to induce the mineralization of calcium phosphate at the air–liquid interface of the 10 times concentrated simulated body fluid. After biomimetic mineralization, zein films are covered with continuous minerals, grown from a small, curved morphology to straight, flake-like morphology, which are a mixture of dicalcium phosphate dehydrate and hydroxyapatite. With the continuous calcium phosphate incorporation, the modulus and hardness of mineralized zein films are enhanced to 11.6 and 0.32 GPa, higher than the values of pure zein films. The hybridization of inorganic minerals decreases the water contact angles from about 63° to 20°. The higher mechanical properties and hydrophilic properties could endow a friendly environment for fibroblast cells attachment, spread, and proliferation. These results suggested that the biomimetic mineralized zein film could serve as a new biomimetic scaffolds for bone tissue engineering.

Biocompatibility Research of the Biphasic Calcium Phosphate Composite Ceramic Film Obtained through Micro-Arc Oxidation on the Medical Titanium Surfaces

To obtain optimal medical ceramic film with bioactivity, Microarc oxidized TiO2-based films containing Ca and P on titanium alloy were formed in electrolytes containing hydroxyapatite and β-tricalcium phosphate salts. After that the biocompatibility tests including invitro soaking test and invivo implant experiment were carried out, and component and phases of films were analysed by energy dispersive spectrometer and X-ray diffraction, respectively.The obtained result shows the porosity biomedical ceramic films with different proportional of Hydroxyapatite/β-tricalcium are fabricated through changing the electrolyte composions,respectively.The bioactivity of biphasic calcium phosphate is enhanced comparing to the pure HA ones.So the biphasic calcium phosphate film material can be satified with medical application.

Using synchrotron radiation to study the structure of calcium-phosphate films on the surfaces of medical implants

In situ X-ray diffractometry with synchrotron radiation (SR) is used to study structural transformations in thin films of calcium-phosphate on the surfaces of medical implants made from alumina ceramics and oxidized titanium. The temperature limits of crystallization and structural composition of films on various substrates are found.

A study on the interfacial chemistry of magnesium hydroxide surfaces in aqueous phosphate solutions: Influence of Ca2+, Cl− and protein

In recent years, magnesium based materials have been proposed as a potential biodegradable metallic implant material for orthopedic applications. Magnesium alloys are an excellent material for this purpose because they have mechanical properties that are similar to bone, have been shown to dissolve in biological fluids and are non-toxic. However, there is still relatively little information on the surface chemistry of these materials in physiological solutions. The interaction of phosphates with magnesium alloys is of particular interest because the deposition of calcium phosphate at implant surfaces is critical to the healing process in orthopedic applications. In the present work, the chemistry at the magnesium hydroxide/solution interface for model solutions containing physiologically relevant ions and protein was investigated using in situ ATR-FTIR. These studies are complemented by ex situ analysis of magnesium alloy coupons exposed to similar solutions. Our results demonstrate that precipitation of phosphate minerals at the solid/liquid interface dominates the observed changes in surface chemistry. The mineralization process was further observed to be strongly affected by the presence of chloride salts and protein in solution.

Modification of silica surface with nonstoichiometric calcium phosphate and its effects on protein adsorption and osteoblast response

Surface modification of silica particle by calcium phosphate (CaP) film using a heterogeneous precipitation method is reported in this study. Surface potential of silica particle was actively changing at 50min of soaking at 37°C. The surface morphological change observed using field emission scanning electron microscopy (FE-SEM) revealed the highly porous structure of the film and formation pattern was well agreed with that of zeta potential. Fourier transform infrared spectroscopy (FTIR) was used to evaluate the chemical composition of the surface of the CaP film. Chemical composition acquired from energy dispersive X-ray spectroscopy (EDX) illustrated that the CaP films consist of calcium and phosphorus and provided the atomic molar ratio of calcium and phosphorus indicating that the CaP films are non-stoichiometric apatites. The specific surface area of the silica particles was increased by ~2.5-fold when the CaP film was formed on the silica particles. Protein adsorption assays demonstrated that the CaP film had a significant effect on interaction with protein characterized in terms of adsorbed mass and surface charge. The adsorption of model proteins, albumin and lysozyme, was reduced in the presence of CaP film. Lysozyme adsorption appeared to be governed by electrostatic attraction, whereas albumin caused gradual decrease in the negativity of surface potential with increase of lysozyme solution concentration. Cell viability assays using human osteoblast MG63 cells confirmed that the film is cytocompatible. This study strengthened our understanding of interaction between protein and CaP and can be applied to surface modification of bone substitutes in granular shapes for the promotion of bone regeneration.

Surface properties of Al2O3-YSZ ceramic composites modified by a combination of biomimetic coatings and electric polarization

Alumina-containing yttria-stabilized zirconia composite (Al-YSZ) has a higher flexural strength than YSZ, although Al-YSZ cannot directly bond with bone tissues. To expand the biomedical applications of Al-YSZ, new surface modifications were investigated to improve the bioactivity in the present study. The combination of a biomimetic coatings method using an alternate soaking technique and electric polarization treatment was performed on Al-YSZ. The crystalline phases of the starting Al-YSZ sintered disks were assigned to tetragonal ZrO2 and α-Al2O3 phases. The analyses of the electrical properties were carried out using complex impedance and thermally stimulated depolarized current measurements for polarization treatments. Our results indicate that the stored charges in Al-YSZ via polarization decreased in YSZ, because of the added alumina, while the calcium phosphate film deposited by the biomimetic coatings method on polarized Al-YSZ was observed to be thicker than the non-polarized film. The coating method would be suitable for the complex geometry of biomaterials. As a result, polarized Al-YSZ fabricated using an alternate soaking process is expected to be useful as a biomaterial under load-bearing conditions.

The incorporation of a zone of calcified cartilage improves the interfacial shear strength between in vitro-formed cartilage and the underlying substrate

A major challenge for cartilage tissue engineering remains the proper integration of constructs with surrounding tissues in the joint. Biphasic osteochondral constructs that can be anchored in a joint through bone ingrowth partially address this requirement. In this study, a methodology was devised to generate a cell-mediated zone of calcified cartilage (ZCC) between the in vitro-formed cartilage and a porous calcium polyphosphate (CPP) bone substitute in an attempt to improve the mechanical integrity of that interface. To do so, a calcium phosphate (CaP) film was deposited on CPP by a sol–gel process to prevent the accumulation of polyphosphates and associated inhibition of mineralization as the substrate degrades. Cartilage formed in vitro on the top surface of CaP-coated CPP by deep-zone chondrocytes was histologically and biochemically comparable to that formed on uncoated CPP. Furthermore, the mineral in the ZCC was similar in crystal structure, morphology and length to that formed on uncoated CPP and native articular cartilage. The generation of a ZCC at the cartilage–CPP interface led to a 3.3-fold increase in the interfacial shear strength of biphasic constructs. Improved interfacial strength of these constructs may be critical to their clinical success for the repair of large cartilage defects.

Primary human bone marrow adipocytes support TNF-α-induced osteoclast differentiation and function through RANKL expression

PurposeIn previous reports, it was demonstrated that bone marrow adipocytes were related to steroid osteoporosis through osteoclastogenesis induced by Receptor Activator of Nuclear factor κ-B Ligand (RANKL) expression. The purpose of this study was to evaluate the effect of Tumor necrosis factor-alpha (TNF-α) on RANKL expression in bone marrow adipocytes, and osteoclast differentiation supported by human bone marrow adipocytes. MethodsRANKL, osteoprotegerin (OPG), and macrophage-colony stimulating factor (M-CSF) mRNA expression in bone marrow adipocytes and their regulation by TNF-α treatment were measured by real-time RT-PCR. Co-cultures of bone marrow adipocytes and osteoclast precursors were performed with or without TNF-α, and osteoclast differentiation was evaluated morphologically and functionally. ResultsRANKL expression and an increase in the RANKL/OPG ratio in bone marrow adipocytes were stimulated by TNF-α treatment. In co-culture of bone marrow adipocytes and osteoclast precursors with TNF-α, the number of TRAP-positive multinuclear cells and resorption cavity formations of calcium phosphate film were increased. Osteoclast differentiation was suppressed by anti-RANKL antibody treatment. In co-culture with non-cell-contact conditions, no TRAP-positive cells or resorption cavity formations were observed. ConclusionsTNF-α increased RANKL expression in primary human bone marrow adipocytes. TNF-α induced the ability of bone marrow adipocytes to promote osteoclast differentiation and activity in a manner directly related to RANKL expression.

Biocompatibility and osteoconduction of active porous calcium–phosphate films on a novel Ti–3Zr–2Sn–3Mo–25Nb biomedical alloy

The purpose of this study is to investigate the biocompatibility and osteoconduction of active porous calcium-phosphate films on the novel Ti-3Zr-2Sn-3Mo-25Nb biomedical alloy. The active porous calcium-phosphate films were prepared by the micro-arc oxidation method on the surface of a near β biomedical Ti-3Zr-2Sn-3Mo-25Nb alloy, and then activated in a hydroxyl solution followed by an aminated solution. The phase composition, surface micro-topography and elemental characteristics of the active porous calcium-phosphate films were investigated with XRD, SEM, EDS and XPS. The biocompatibility was assessed using corrosion testing, the in vitro osteoblast cultivation test and implantation in soft tissue (subcutaneous and musculature). The osteoconduction was evaluated using the simulated body fluid test and by implantation in hard tissue. The results show that the active porous films are mainly composed of TiO(2) anatase and rutile. The oxide layer is a kind of porous ceramic intermixture containing Ca and P. Immersion in simulated body fluid can induce apatite formation on the porous calcium-phosphate films resulting in excellent bioactivity. Cell cultures revealed that MC3T3-E1 cells grew on the surface exhibiting favorable morphologies. These results indicate that the Ti-3Zr-2Sn-3Mo-25Nb biomedical alloy coated with an active porous calcium-phosphate film has been shown to have excellent corrosion resistance, good biocompatibility and osteoconduction, which can promote cell proliferation and bone formation.

Preparation and osteoinduction of active micro-arc oxidation films on Ti-3Zr-2Sn-3Mo-25Nb alloy

A layer of porous film containing Ca and P was prepared by the micro-arc oxidation method on the surface of a novel near β biomedical Ti-3Zr-2Sn-3Mo-25Nb alloy, and then NH 2 − active group was introduced to the films by activation treatment. The phase composition, surface micro-topography and elemental characteristics of the micro-arc oxidation films were investigated with XRD, SEM, EDS and XPS, and the osteoinduction of the micro-arc oxidation films was tested using the simulated body fluid immersion, the in-vitro osteoblast cultivation test and animal experiment. The results show that the oxide layer is a kind of porous ceramic intermixture and contains Ca and P. The films in the simulated body fluid can induce apatite formation, resulting in excellent bioactivity. The cell test discovers that osteoblasts can grow well on the surface of micro-arc oxidation films. And the Ti-3Zr-2Sn-3Mo-25Nb biomedical alloy coated with active porous calcium-phosphate films shows better osteoinduction in vivo.

Human bone marrow adipocytes support dexamethasone-induced osteoclast differentiation and function through RANKL expression

The TNF-family molecule, Receptor Activator of Nuclear factor κ B Ligand (RANKL) is known as a key regulator for bone remodeling, and is essential for the development and activation of osteoclasts. In this study, we examined the regulation of RANKL in primary human bone marrow adipocytes and the relationship between bone marrow adipocytes and bone metabolism. RANKL expression and the RANKL/osteoprotegerin (OPG) mRNA ratio in marrow adipocytes increased following dexamethasone treatment. In co-cultures of human osteoclast precursors and bone marrow adipocytes with dexamethasone, osteoclast precursors differentiated to TRAP-positive multinuclear cells. Moreover, the ability of bone resorption was confirmed in co-culture in flasks coated with calcium phosphate film. Osteoclast precursor differentiation and bone resorption were blocked by RANKL antibody pretreatment. TRAP-positive multinuclear cells did not form in coculture without cell-to-cell contact conditions. We conclude that primary human bone marrow adipocytes have the ability to promote osteoclast differentiation and activities, similar to osteoblasts and other RANKL-expressing cells.