Calcium Phosphate Films Research Articles

Effect of calcium phosphate coating composition and crystallinity on the response of osteogenic cells in vitro.

The aim of this study was to investigate the effect of calcium phosphate coating crystallinity and composition on the proliferation and differentiation of rat bone marrow (RBM) cells. Grit-blasted titanium substrates were provided with thin sputter-coated calcium phosphate (Ca-P) films of different composition. The Ca-P-coated substrates were used as-sputtered or were heat-treated. XRD measurements showed that the as-sputtered coatings had an amorphous structure, whereas the heat-treated substrates showed an amorphous-crystalline structure. RBM cells were cultured on these substrates and on noncoated titanium substrates. After specific culture times, the expression of osteogenic markers by the cells was studied. On the amorphous-crystalline coatings as well as on titanium substrates, RBM cells proliferated, expressed alkaline phosphatase and showed mineralization. More mineralization was found on the amorphous-crystalline coatings than on the titanium substrates. Some precipitation was also found on substrates that were incubated in complete culture medium without cells. This precipitate disappeared after prolonged incubation. Alkaline phosphatase expression differed on the various amorphous-crystalline Ca-P-coated substrates, but no difference was found in the mineralization on these substrates. The amorphous Ca-P coatings showed extensive dissolution and some signs of precipitation after longer culture periods. Proliferation and differentiation of RBM cells was not seen on the amorphous coatings, regardless of Ca-P composition. We conclude that amorphous-crystalline Ca-P coatings stimulate differentiation of RBM cells, with only limited differences between coatings of various composition. In contrast, Ca-P coatings with an amorphous structure inhibit the growth and differentiation of RBM cells. This effect was found on all amorphous substrates, regardless of Ca-P composition.

Routes to calcified porous silicon: implications for drug delivery and biosensing

This work describes methods for the fabrication of calcium phosphate films on crystalline silicon substrates, with the long-term goal of employing such materials in either drug delivery or sensing applications. The emphasis of this particular account encompasses both film fabrication as well as surface modification strategies relevant to the sensing of bio-molecular species. For the former, a new process for calcium phosphate film formation is described entailing the use of a high energy DC spark onto silicon surfaces in the presence of Ca 10 (PO 4 ) 6 (OH) 2 . For sensing purposes, routes to modification of the calcium phosphate/porous silicon surface have been achieved via the use of bifunctional bisphosphonate derivatives, with proof-of-concept demonstrated by the covalent attachment of fluorescent fluorescein tags.

Variation in Biological Properties of Thin Calcium Phosphate Films Formed by e-Beam Evaporation

Variation in Biological Properties of Thin Calcium Phosphate Films Formed by e-Beam Evaporation

Plasma processing of materials for medical applications

The thin and defect-free calcium phosphate coating layer deposited to a thickness of 1 μm by electron beam evaporation was characterized in vivo. Calcium phosphate film deposited implants made of commercially pure Ti were inserted in rabbit tibiae. The bone forming ability was compared with the as-machined and as-blasted implants. Eighteen white rabbits were ovariectomized 2 months before insertion for osteoporic condition. The calcium phosphate coated implants had the most bone-to-metal contact and required the highest removal torque force in ovariectomized rabbit. The removal torque force was increased to slightly more than 50% by calcium phosphate coating for the same surface finished condition. The sacral nerve, S3, stimulation of a 38-year-old male patient with C4 and C5 tetraplegia by iridium coated electrode increased the reservoir volume and decreased the bladder pressure at the same time.

Casein and soybean protein-based thermoplastics and composites as alternative biodegradable polymers for biomedical applications.

This work reports on the development and characterization of novel meltable polymers and composites based on casein and soybean proteins. The effects of inert (Al(2)O(3)) and bioactive (tricalcium phosphate) ceramic reinforcements over the mechanical performance, water absorption, and bioactivity behavior of the injection-molded thermoplastics were examined. It was possible to obtain materials and composites with a range of mechanical properties, which might allow for their application in the biomedical field. The incorporation of tricalcium phosphate into the soybean thermoplastic decreased its mechanical properties but lead to the nucleation of a bioactive calcium-phosphate film on their surface when immersed in a simulated body fluid solution. When compounded with 1% of a zirconate coupling agent, the nucleation and growth of the bioactive films on the surface of the referred to composites was accelerated. The materials degradation was studied for ageing periods up to 60 days in an isotonic saline solution. Both water uptake and weight loss were monitored as a function of the immersion time. After 1 month of immersion, the materials showed signal of chemical degradation, presenting weight losses up to 30%. However, further improvement on the mechanical performance and the enhancement of the hydrolytic stability of those materials will be highly necessary for applications in the biomedical field.

Effects of ion beam assist on the formation of calcium phosphate film

Calcium phosphate films were formed by electron beam evaporation of β-TCP with and without simultaneous Ar ion beam bombardments. The Ca/P ratio was 0.76 without bombardments, but it was increased to 1.80 by bombarding of Ar ion beam extracted from the ion gun set to 0.8 A. Hydroxyapatite peaks were developed and became stronger with increases in beam current. The variations in chemical composition had an effect on the dissolution behaviors in isotonic saline solution showing two distinct features, pinhole and flat region. The dissolved thickness measured in the flat region of all calcium phosphate coating layers after 10 h immersion was similar with the range of 5450 and 6100 Å. However, the size of pinhole was larger for the calcium phosphate coating layer with the lower Ca/P ratio indicating more active dissolution.

Growth of calcium phosphate thin films by in situ assisted ultraviolet pulsed laser deposition

Calcium phosphate (CaP) thin films including hydroxyapatite were intensively studied in order to optimize the technology of the bone prostheses manufacturing. A drawback in the CaP films processing is the poor mechanical characteristics, especially hardness, tensile strength and adherence to the metallic substrate. We report a new method for the growth of high quality CaP films with substantial improvement of the mechanical properties: pulsed laser deposition (PLD) assisted by in situ ultraviolet (UV) radiation emitted by a low pressure Hg lamp. The depositions were made on Si and Ti-5Al-2.5Fe alloys in very low ambient oxygen at pressures of 10 −2 to 10 −1 Pa with the substrates maintained at 500–600 °C temperature. The films were analyzed by electron microscopy, white light confocal microscopy (WLCM), grazing incidence X-ray diffraction and Berkovich nanoindentation. The films were crystalline and exhibited remarkable mechanical characteristics with values of hardness and Young modulus of 6–8 and 150–170 GPa, respectively, which are uncommonly high for the CaP ceramics. The UV lamp radiation enhanced the gas reactivity and atoms mobility during processing, while the tensile strength between the film’s grains and the bonding strength at the CaP film-substrate interface were increased.

Properties of Ruthenium Complex-Doped Calcium Phosphate/Porous Silicon Nanostructures

In this work, we focus on the optical properties of the ruthenium complexes [Ru(phen)3]Cl2 and [Ru(bpy)3]Cl2 incorporated into biocompatible calcium phosphate films on porous Si substrates to both characterize the distribution of such species in the film as well as analyze their diffusion from the porous matrix upon aqueous exposure. For the latter measurements, photoluminescence spectroscopy is employed to monitor the changes in emission intensity for the both the calcium phosphate surface and surrounding aqueous environment as a consequence of diffusion from the film. In selected cases, visible absorption spectroscopy was also utilized to measure the appearance of a given complex in the surrounding solution. The effects of heating and anodic bias on the calcium phosphate/porous Si/Si substrate on the kinetics and available equilibrium concentrations of a given Ru complex have also been evaluated.

Effect of pH on Deposition of Calcium Phosphate Films from Electrolytic Solution

Effect of pH on Deposition of Calcium Phosphate Films from Electrolytic Solution

An innovative auto-catalytic deposition route to produce calcium-phosphate coatings on polymeric biomaterials.

The aim of this research is to develop a new methodology to obtain bioactive coatings on bioinert and biodegradable polymers that are not intrinsically bioactive. In this study three types of materials were used as substrates: (i) high molecular weight polyethylene (HMWPE) and two different types of starch based blends (ii) starch/ethylene vinyl alcohol blends, SEVA-C and (iii) starch/cellulose acetate blends, SCA. Two types of baths were originally proposed and studied to produce novel auto-catalytic calcium-phosphate (Ca-P) coatings. Then, the coated surfaces were analyzed by scanning electron microscopy and energy dispersive spectroscopy (SEM/EDS), as produced, and after different immersion periods in SBF. The evolution of Ca and P concentrations was determined by induced-coupled plasma emission (ICP) spectroscopy. The crystalline phases present on the films formed on the different material surfaces, after a certain soaking time, were identified by thin-film X-ray diffraction (TF-XRD). The obtained results indicated that it was possible to coat the materials surfaces with a Ca-P layer with only 60 min of immersion in both types of auto-catalytic solutions. Furthermore, it was possible to observe the clear bioactive nature of the Ca-P coatings after different immersion periods in a simulated body fluid (SBF). The results from TF-XRD confirmed the presence of partially amorphous Ca-P films with clearly noticeable hydroxylapatite peaks. These new methodologies allow for the production of an adherent bioactive film on the polymeric surfaces prior to implantation, which may allow for the development of bone-bonding, bioabsorbable implants and fixation devices.

Various Ca/P ratios of thin calcium phosphate films

Various Ca/P ratios of calcium phosphate films were formed by electron beam evaporation with and without simultaneous Ar ion bombardment. The Ca/P ratio of film was controlled by either using the evaporants having the different ratio of Ca/P with addition of CaO or increases in ion beam current. Without ion beam bombardments, the Ca/P ratio of film increased from 1.12 to 1.71 with addition of 25 wt.% CaO. The effect of ion beam assistance was also observed, and the ratio increased with ion beam current. The Ca/P ratio of the film was intimately related to the structure formed after heat treatment and the dissolution behavior. The films had the average bonding strength of 64.8 MPa that was much higher than the plasma sprayed hydroxyapatite coating layer. Calcium phosphate film showed beneficial effects on osteoblast cell attachment and proliferation.

In vitro mineralization of a glass‐ceramic of the MgO‐3CaO · P2O5‐SiO2 system: Wettability studies

The calcium phosphate deposition on the surface of a bioactive glass-ceramic of the MgO-3CaO · P2O5-SiO2 system during a 1-week immersion in biological model fluids, was investigated through wettability studies. Two model fluids with chemical composition similar to the human blood plasma were tested: Hanks’ balanced salt solution (HBSS) and a simulated body fluid (SBF) with a higher calcium content. The effect of the presence of albumin, both in solution or previously adsorbed to the surface, was assessed. The behavior observed using two SBF solutions prepared with and without TRIS buffer was compared. The surface free energy of the glass-ceramic samples was determined, before and after immersion, and dynamic contact angle analysis was performed using the biological model fluids as scanning liquids. Scanning electron microscope observations and x-ray photoelectron spectroscopy (XPS) analysis were performed to complement the wettability studies. The experimental results led to the conclusion that the precipitation of a calcium phosphate film in HBSS occurred mainly when the immersion solution was renewed daily or in the presence of adsorbed albumin. In SBF, the addition of TRIS buffer seemed to inhibit the formation of the calcium phosphate film. © 2002 Wiley Periodicals, Inc. J Biomed Mater Res 61: 99–108, 2002

X-ray microanalysis of multi-elements coatingsusing Auger formalism: application to biomaterials

For the thickness determination of thin coatings on substrate in scanning electron microscopy using x-ray energy dispersive spectrometry, a method called Auger formalism has been established in our laboratory. It consists of applying a formalism derived from that in the Auger spectroscopy which assumes a linear relationship between the signal intensity emitted by the coating and its thickness.In this paper, a development of the Auger formalism in the case of heterogeneous coatings is presented. Using x-ray energy dispersive spectrometry, this method allows one to determine the atomic concentrations of the elements which composed the coating. The capability of the proposed method is illustrated by two different applications: a Ni-Cu film on titanium alloy substrate (academic specimen) and calcium phosphate film on titanium alloy substrate (prosthetic coating). An iterative procedure established for the proposed method is also presented. The results show that the Auger formalism may now be applied not only to simple coatings for thickness determination but also to the complex ones for concentration determination. And its use does not require the local knowledge of the substrate composition which is not accessible in the Auger spectroscopy.

C-myc is required for osteoclast differentiation.

The role of the receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL)-a tumor necrosis factor (TNF)-related cytokine-in osteoclast formation has been established clearly. However, the downstream signaling pathways activated by this cytokine remain largely unknown. To identify genes that play a role in osteoclastogenesis, we used RAW 264.7 mouse monocytes as a model system for the differentiation of multinucleated osteoclasts from mononucleated precursors. RAW 264.7 cells were induced with RANKL to form multinucleated giant osteoclast-like cells (OCLs) that expressed a number of osteoclast-specific markers and were able to form resorption pits on both calcium phosphate films and bone slices. This system was used to identify genes that are regulated by RANKL and may play a role in osteoclast differentiation. The proto-oncogene c-myc was strongly up-regulated in RANKL-induced OCLs but was absent in undifferentiated cells. Expression of Myc partners Max and Mad, on the other hand, was constant during OCL differentiation. We expressed a dominant negative Myc in RAW 264.7 cells and were able to block RANKL-induced OCL formation. Northern Blot analysis revealed a delay and a significant reduction in the level of messenger RNA (mRNA) for tartrate-resistant acid phosphatase (TRAP) and cathepsin K. We conclude that c-myc is a downstream target of RANKL and its expression is required for RANKL-induced osteoclastogenesis.

In vitro mineralization of a glass-ceramic of the MgO-3CaO x P2O5-SiO2 system: wettability studies.

The calcium phosphate deposition on the surface of a bioactive glass-ceramic of the MgO-3CaO. P(2)O(5)-SiO(2) system during a 1-week immersion in biological model fluids, was investigated through wettability studies. Two model fluids with chemical composition similar to the human blood plasma were tested: Hanks' balanced salt solution (HBSS) and a simulated body fluid (SBF) with a higher calcium content. The effect of the presence of albumin, both in solution or previously adsorbed to the surface, was assessed. The behavior observed using two SBF solutions prepared with and without TRIS buffer was compared. The surface free energy of the glass-ceramic samples was determined, before and after immersion, and dynamic contact angle analysis was performed using the biological model fluids as scanning liquids. Scanning electron microscope observations and x-ray photoelectron spectroscopy (XPS) analysis were performed to complement the wettability studies. The experimental results led to the conclusion that the precipitation of a calcium phosphate film in HBSS occurred mainly when the immersion solution was renewed daily or in the presence of adsorbed albumin. In SBF, the addition of TRIS buffer seemed to inhibit the formation of the calcium phosphate film.

Generation and activity of equine osteoclasts in vitro: effects of the bisphosphonate pamidronate (APD)

Equine osteoclast-like cells (ocl s) were generated from the bone marrow (bm) of two ponies and one horse in the presence of rankl, the receptor activator of NFκB ligand and macrophage colony-stimulating factor (m-csf). The phenotype of these cells was confirmed by demonstration of characteristics typical of osteoclasts (oc s) including: the expression of tartrate-resistant acid phosphatase (trap), the vitronectin receptor (vnr) and the calcitonin receptor (ctr), the demonstration of responsiveness to calcitonin (ct) and the ability to form resorption lacunae on ivory slices and calcium phosphate films. The bisphosphonate pamidronate (apd) dose-dependently inhibited resorption of calcium phosphate films by equine ocl s with an IC50 of 5·8×10−7M in one horse. apd also dose-dependently inhibited the number ofocl s present in bm cultures after 7 days. However, this effect is most likely attributable to increasedocl death rather than decreased ocl formation. Paradoxically, apd appeared to cause an early, transient, increase in ocl formation in bm cultures, however, this effect was reversed after 7 days. These preliminary in vitro data support the potential use of apd in clinical conditions characterised by increased bone turnover such as osteomyelitis, osteitis, septic osteoarthritis, navicular disease, cystic bone lesions and immobilisation-induced osteoporosis and provide useful information for future pharmacokinetic studies and clinical trials in vivo.

The Use of Spark Ablation to Produce Calcium Phosphate Films on Silicon

A method for fabrication of calcium phosphate films on crystalline silicon substrates which employs a high energy dc spark on silicon surfaces in the presence of Ca 1 0 (PO 4 ) 6 (OH) 2 is described. Such a process kinetically traps the desired calcium phosphate on the substrate with concomitant formation of a porous layer. Scanning electron micrographs of the silicon substrate show a nanoporous surface morphology for those areas exposed to spark processing. Energy dispersive X-ray analysis and infrared vibrational spectroscopy of the spark-processed regions of the Si substrate are clearly consistent with the presence of the desired biologically relevant material. In conjunction with an automated stage, facile formation of deliberate micrometer-size patterns is demonstrated.

Biological performance of calcium phosphate films formed on commercially pure Ti by electron-beam evaporation

Thin and defect-free calcium phosphate films with a Ca/P ratio of 1.62 were formed by electron-beam evaporation. The as-deposited films had average bonding strengths to the metal implants of 64.8 MPa and the dissolution rates of 47.5 nm/h in isotonic saline solutions. The interface mechanical characteristics and histology of the as-machined, as-blasted, and calcium phosphate coating on the machined surfaces of commercially pure titanium were investigated. After a healing period of 12 weeks, the implants were unscrewed with a torque gauge instrument at the day of sacrifice. The coated sample showed a removal torque of 48.5 Ncm (SD 5.4) compared to 32.3 Ncm (SD 2.91) for the uncoated implant with the same surface roughness, and 47.3 Ncm (SD 5.8) for the grit blasted screw. The histomorphometric analyses of the calcium-phosphate-coated implants revealed a mean of 52.4% (SD 6.3) as the highest bone to implant contact.

In-Situ AFM Study of the Mechanisms Involved on Calcium-Phosphate Film Formation on Partially Crystallized Bioglass<sub>Ⓡ</sub> and on Hydroxylapatite Immersed in a Simulated Body Fluid

In-Situ AFM Study of the Mechanisms Involved on Calcium-Phosphate Film Formation on Partially Crystallized Bioglass<sub>Ⓡ</sub> and on Hydroxylapatite Immersed in a Simulated Body Fluid

Calcium Phosphate Coating on Titanium Plates by Electrodeposition

ABSTRACTCalcium phosphate films were deposited on titanium electrodes cathodically from CaCl2·2H2O and Ca(H2PO4)2 · H2O aqueous solutions. In this study, H2O2 addition into electrolytes was applied to enhance the electrochemical process at the solution/electrode at a smaller cathodic potential than no H2O2 addition. Deposited films were analyzed by scanning electron microscopy (SEM) observation and X-ray diffraction (XRD). Cathodic current of the Ti electrode decreased once and increased in the solution with H2O2. It shows a cathodic current peak at c.a. 25 min in the case of potentiostatic condition at - 0.756 V (vs. Ag - AgCl, sat. KCl). The calcium phosphate film grows mainly with the decrease in current after the cathodic peak. The characteristics for the electrodeposited film such as crystal morphology depends on cathodic potential, solution pH, deposition temperature and amount of H2O2 addition. Dense calcium phosphate film composed of relatively good crystalline was obtained at pH 5.5 and – 0.756 V. Film adhesion on Ti appeared to be strong by peeling test. At larger cathodic potential of – 1.156 V, the film coverage on titanium plates was smaller and film adhesion worsened. Larger cathodic polarization of more than – 1.556 V was necessary to reduce water in case without H2O2 addition.