Cytotoxicity investigations of plasma sprayed calcium phosphate coatings

One potential alternative material to replace hydroxyapatite (HAp) as a coating material for plasma-sprayed coatings on implants for hip replacement is fluorapatite (FAp). FAp has advantages over HAp regarding the capability of being chemically stable during the coating process. This leads to surface coatings containing high apatite rates with a mechanical stability (bond strength, microhardness) comparable to HAp. From the technical point of view the production of FAp coatings is well investigated, although studies on biocompatibility of FAp coatings are fewer. This paper reports the production of HAp and FAp coatings with varying solubilities by plasma spraying and their in vitro cytotoxicity. Varying solubilities were realized by using modified plasma-spray parameters in common with suitable apatite powders with different crystallinities. Coating solubilities were evaluated by immersing the plasma-sprayed coatings in deionized water and electrolyte solution. Afterwards, cytotoxicity tests were performed using a modified half-slide technique. Cell attachment and cell morphology were evaluated. Neither HAp nor FAp coatings exhibited cytoxic influence on cells in culture. Results suggest that HAp coatings stimulate cell growth and FAp coatings do not. This could be explained by a negative effect on cell growth of the dissolved fluoride ions.     

Properties of calcium phosphate coatings deposited and modified with lasers

Physical, chemical and biological properties of calcium phosphate coatings fabricated by a pulse laser deposition method at room temperature (RT PLD) have been studied. In vitro evaluation of RT PLD coatings on bioresorbable polymers (Poly--caprolactone and Poly-L-lactide) have been carried out. It was shown that both polymers support osteoblast growth, with increased cell activity, alkaline phosphatase activity and total protein content on those surfaces that have been coated. The advantages of RT PLD coatings in biomaterials surface optimization are discussed.     

Characterisation of calcium phosphate/titanium dioxide hybrid coatings

The role of titanium dioxide (TiO₂) as a means to engender enhanced stability into calcium phosphate (Ca-P) coatings has been well recognised. Several different methods have been used to create such Ca-P/TiO₂ hybrid layers on a range of substrates. This paper reports the properties of a Ca-P/TiO₂ system created by the sputter deposition of hydroxyapatite onto a titanium surface and the subsequent thermal diffusion of TiO₂ through the porous Ca-P layer. The role of temperature in determining the surface contribution from TiO₂ has been determined. Coatings annealed up to 600 °C did not exhibit any hybrid nature in the uppermost surface, however the coatings annealed to 700 °C did show the presence of both HA and rutile TiO₂. The surfaces annealed to 800 °C were predominantly rutile TiO₂. It was also observed that the Ca/P ratio decreased with increasing annealing temperature and that the coating annealed to 700 °C had a value of 1.82 ± 0.07, which was closest to stoichiometric HA. Furthermore, the coatings that were annealed to 700 °C displayed a Ca-P/TiO₂ hybrid nature, specifically in their uppermost surface and supported the growth and proliferation of osteoblast-like cells more readily when compared to the HA coatings or the rutile TiO₂ surfaces.     

In vitro testing of Nd:YAG laser processed calcium phosphate coatings

Nd:YAG laser cladding is a new method for deposition of a calcium phosphate onto metallic surfaces of interest in implantology. The aim of this study was to compare the biologic response of MG-63 human osteoblast-like cells grown on Ti-6Al-4V substrates coated with a calcium phosphate layer applied using different methods: plasma spraying as reference material and Nd:YAG laser cladding as test material. Tissue culture polystyrene was used as negative control. The Nd:YAG laser clad material showed a behaviour similar to the reference material, plasma spray, respective to cell morphology (SEM observations), cell proliferation (AlamarBlue assay) and cytotoxicity of extracts (MTT assay). Proliferation, as measured by the AlamarBlue assay, showed little difference in the metabolic activity of the cells on the materials over an 18 day culture period. There were no significant differences in the cellular growth response on the test material when compared to the ones exhibited by the reference material. In the solvent extraction test all the extracts had some detrimental effect on cellular activity at 100% concentration, although cells incubated in the test material extract showed a proliferation rate similar to that of the reference material. To better understand the scope of these results it should be taken into account that the Nd:YAG clad coating has recently been developed. The fact that its in vitro performance is comparable to that produced by plasma spray, a material commercially available for more than ten years, indicates that this new laser based method could be of commercial interest in the near future.     

Dissolution behavior of simultaneous vapor deposited calcium phosphate coatings in vitro

Solubility is one of the most important properties in the field of biomaterial. The present paper evaluated the dissolution behavior of simultaneous vapor deposited calcium phosphate coatings in vitro. The coatings were immersed in calcium-free Hank's solution at different periods of time. Characterization of the coatings was performed using scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy and Rutherford backscattering spectroscopy, prior to and after immersion. Amorphous coatings showed complete dissolution. Crystalline coatings showed that alpha tricalcium phosphate (α-TCP) phase dissolved steadily throughout the testing time leaving the stable hydroxyapatite phase undegraded. The increased in calcium and phosphate ions due to dissolution of α-TCP provided the means for reprecipitation of apatite on the coating, which became apparent after 7 days of immersion.     

Production of thin calcium phosphate coatings from glass source materials

Calcium phosphate (CaP) coatings, from 40 000 to 200 000 nm thick, on titanium and titanium alloy substrates, were produced using radio frequency (RF) sputtering. Such coatings on dental implants have the potential for improving initial bone ingrowth rates. The success of these coatings may allow the movement from two stage implant systems to single stage implant systems, significantly reducing the time required for healing and fixture placement. Glass source materials were developed for the RF sputtering facility and the resultant coatings were characterized and compared to coatings sputtered from a conventional plasma sprayed hydroxyapatite (HA) source material. The coatings were characterized according to their chemistry, crystalline orientation, and residual strain.     

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.     

Phase evolution in calcium phosphate coatings obtained by in situ laser cladding

Calcium phosphate coating was fabricated by in situ laser cladding using mixed powders of CaCO₃ and CaHPO₄, which presented a complex phase constitution since the reactions between CaCO₃ and CaHPO₄ would produce not only hydroxyapatite (HA) in the coating, but also other phases, such as Ca₄(PO₄)₂O (TTCP) and α-Ca₃(PO₄)₂ (α-TCP). In order to realize the control of the phase constitution, the effects of the Ca/P molar ratio of mixed powders, laser power, scanning velocity and heat treatment on the phase constitution of the coatings were investigated through X-ray diffraction analysis. It is found that the variation of the Ca/P molar ratio of the mixed powders, laser power and scanning velocity can adjust, to a certain extent, the proportion of HA, α-TCP, and TTCP in the coating. However, the α-TCP and TTCP cannot be eliminated from the coating due to the intrinsic high cooling rate of the laser melt pool during laser cladding. By suitable post heat treatment, the TTCP and α-TCP in the coating can be partially or completely transformed into HA. Therefore, HA coating or coatings with desirable proportion of HA, α-TCP and TTCP can be obtained by in situ laser cladding plus post heat treatment.     

Formation of carbonate apatite on calcium phosphate coatings containing silver ions

The prerequisite for bioactive materials to bond to living bone is the formation of biologically equivalent carbonate apatite on their surfaces in the body. Calcium phosphate ceramic surfaces can be transformed to a biological apatite through a series of surface reactions including dissolution–precipitation and ion exchange. In the present work, apatite coatings with different crystallinity, compositions and crystal sizes, including a well-crystallized hydroxyapatite coating, were synthesized electrochemically and doped with silver ions in silver nitrate solution at room temperature. The formation of a new carbonate apatite on the surface of these coatings was investigated in an acellular simulated body fluid with ion concentrations comparable with those of human blood plasma, using scanning electron microscopy and Fourier transform-infrared spectroscopy. The results show that small quantities of silver ions incorporated into apatite coatings may have a strong stimulatory effect on the formation of carbonate apatite without adversely affecting the chemical stability of these coatings.     

Development of a novel calcium phosphate cement composed mainly of calcium sodium phosphate with high osteoconductivity

Two novel calcium phosphate cements (CPC) have been developed using calcium sodium phosphate (CSP) as the main ingredient. The first of these cements, labeled CAC, contained CSP, α-tricalcium phosphate (TCP), and anhydrous citric acid, whereas the second, labeled CABC, contained CSP, α-TCP, β-TCP, and anhydrous citric acid. Biopex^®-R (PENTAX, Tokyo, Japan), which is a commercially available CPC (Com-CPC), and OSferion^® (Olympus Terumo Biomaterials Corp., Tokyo, Japan), which is a commercially available porous β-TCP, were used as reference controls for analysis. In vitro analysis showed that CABC set in 5.7 ± 0.3 min at 22 °C and had a compressive strength of 86.0 ± 9.7 MPa after 5 days. Furthermore, this material had a compressive strength of 26.7 ± 3.7 MPa after 2 h in physiologic saline. CAC showed a statistically significantly lower compressive strength in the presence of physiologic saline and statistically significantly longer setting times than those of CABC. CABC and CAC exhibited apatite-forming abilities in simulated body fluid that were faster than that of Com-CPC. Samples of the materials were implanted into the femoral condyles of rabbits for in vivo analysis, and subsequent histological examinations revealed that CABC exhibited superior osteoconductivity and equivalent bioresorbability compared with Com-CPC, as well as superior osteoconductivity and bioresorbability compared with CAC. CABC could therefore be used as an alternative bone substitute material.     

Immobilization of calcium phosphate nano-clusters into alkoxy-derived porous TiO2 coatings

Alkoxy-derived porous coatings of titanium oxide were fabricated on commercially pure titanium substrates by an electrochemical method in methanolic electrolytes. Nano-clusters of brushite (CaHPO4 · 2H2O) were immobilized into the pores of the oxide network by reacting these coatings in acidic calcium phosphate solutions at 50°C. The acid-base reaction between calcium phosphate solutions and the hydroxyl groups of the oxide network resulted in the formation of nano-clusters of brushite crystals immobilized inside the oxide pores. This treatment resulted in the conversion of the porous oxide network into a coherent mass with improved physical integrity. Nano-clusters of brushite crystals immobilized in the oxide matrix were converted into amorphous calcium phosphate (ACP) and poorly crystallized hydroxyapatite (HA) by further treatment of the oxide in alkaline solutions. The porous oxide coating also reacted strongly with concentrated phosphoric acid. The phosphate-modified oxide resulting from this reaction was further treated in calcium hydroxide solution to form nano-clusters of poorly crystallized HA within the oxide network.     

Cytocompatibility of calcium phosphate coatings deposited by an ArF pulsed laser

In the current studies, we deposited ultra-thin hydroxyapatite films on a pure titanium substrate by pulsed laser deposition, and we examined the effects of these surfaces on rat bone marrow (RBM) cells. This method allowed deposition of 500-, 2,000-, and 5,000-A-thick hydroxyapatite films. X-ray diffraction showed that the amorphous films recrystallized to a hydroxyapatite crystal structure after annealing. The proliferation of RBM cells was unaffected by the hydroxyapatite films, but osteocalsin and alkaline phosphatase mRNA and protein levels were elevated in cells grown on 2,000- and 5,000-A-thick films. These results indicate that ultra-thin hydroxyapatite films generated by pulsed laser deposition are better at promoting osteogenesis than pure titanium surfaces.     

Cytocompatibility of calcium phosphate coatings deposited by an ArF pulsed laser

In the current studies, we deposited ultra-thin hydroxyapatite films on a pure titanium substrate by pulsed laser deposition, and we examined the effects of these surfaces on rat bone marrow (RBM) cells. This method allowed deposition of 500-, 2000-, and 5000-Angstrom-thick hydroxyapatite films. X-ray diffraction showed that the amorphous films recrystallized to a hydroxyapatite crystal structure after annealing. The proliferation of RBM cells was unaffected by the hydroxyapatite films, but osteocalsin and alkaline phosphatase mRNA and protein levels were elevated in cells grown on 2000- and 5000-Angstrom-thick films. These results indicate that ultra-thin hydroxyapatite films generated by pulsed laser deposition are better at promoting osteogenesis than pure titanium surfaces.     

Release behaviors of drug loaded chitosan/calcium phosphate coatings on titanium

Implants with antibiotic drug loaded bioactive coatings have been increasingly applied in orthopedic operations. Here we report the drug release behavior of gentamycin loaded chitosan/calcium phosphate coatings on titanium. Chitosan/calcium phosphate coatings with different component ratios and surface topographies were prepared by electrochemical deposition method. Our results showed that the drug release from these coatings was controlled by their component ratio and surface topography, and the former ratio played a more significant role. The present coatings could provide an effective way to create both good bioactivity and antibacterial activity.     

A new evaporation-based method for the preparation of biomimetic calcium phosphate coatings on metals

This study reports a new method to prepare biomimetic calcium phosphate coatings on titanium, stainless steel, CoCrMo, and tantalum. The method does not require surface etching, high supersaturation, or tight control of solution conditions. Metallic samples were dipped into a supersaturated calcium phosphate solution, withdrawn, and left to dry at room temperature. Calcium phosphate crystallites formed on and completely covered the surfaces by repeating the dip-and-dry treatment. The crystallite-covered surfaces readily grew to calcium phosphate coatings when immersed in the supersaturated solution. The mechanism of the treatment was suggested to be an evaporation-induced surface crystallization process.     

Pulsed electrodeposition for the synthesis of strontium-substituted calcium phosphate coatings with improved dissolution properties

Strontium-substituted calcium phosphate coatings are synthesized by pulsed electrodeposition on titanium alloy (Ti6Al4V) substrates. Experimental conditions of the process are optimized in order to obtain a coating with a 5% atomic substitution of calcium by strontium which corresponds to the best observations on the osteoblast cells activity and on the osteoclast cells proliferation. The physical and chemical characterizations of the obtained coating are carried out by scanning electron microscopy associated to energy dispersive X-ray spectroscopy (EDXS) for X-ray microanalysis and the structural characterization of the coating is carried out by X-ray diffraction. The in vitro dissolution/precipitation properties of the coated substrates are investigated by immersion into Dulbecco's Modified Eagle Medium (DMEM) from 1 h to 14 days. The calcium, phosphorus and strontium concentrations variations in the biological liquid are assessed by Induced Coupled Plasma - Atomic Emission Spectroscopy for each immersion time. The results show that under specific experimental conditions, the electrodeposition process is suitable to synthesize strontium-substituted calcium phosphate coatings. Moreover, the addition of hydrogen peroxide (H₂O₂) into the electrolytic solution used in the process allows us to observe a control of the strontium release during the immersion of the prosthetic materials into DMEM.     

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.     

Increased osteoblast density in the presence of novel calcium phosphate coated magnetic nanoparticles

Bone diseases (including osteoporosis, osteoarthritis and bone cancer) are of great concern to the medical world. Drugs are available to treat such diseases, but often these drugs are not specifically targeted to the site of the disease and, thus, lack an immediate directed therapeutic effect. The optimal drug delivery system should enhance healthy bone growth with high specificity to the site of bone disease. It has been previously shown that magnetic nanoparticles can be directed in the presence of a magnetic field to any part of the body, allowing for site-specific drug delivery and possibly an immediate increase in bone density. The objective of the present study was to build off of this evidence and determine the density of osteoblasts (bone forming cells) in the presence of various uncoated and coated magnetic nanoparticles that could eventually be used in drug delivery applications. Results showed that some magnetic nanoparticles (specifically, γ-Fe(2)O(3)) significantly promoted osteoblast density (that is, cells per well) after 5 and 8 days of culture compared to controls (no particles). These magnetic nanoparticles were further coated with calcium phosphate (CaP; the main inorganic component of bone) to tailor them for treating various bone diseases. The coatings were conducted in the presence of either bovine serum albumin (BSA) or citric acid (CA) to reduce magnetic nanoparticle agglomeration, a common problem resulting from the use of nanoparticles which decreases their effectiveness. Results with these coatings showed that magnetic nanoparticles, specifically (γ-Fe(2)O(3)), coated in the presence of BSA significantly increased osteoblast density compared to controls after 1 day. In this manner, this study provided unexpected evidence that CaP-coated γ-Fe(2)O(3) magnetic nanoparticles increased osteoblast density (compared to no particles) and, thus, should be further studied to treat numerous bone diseases.