Thin calcium phosphate coatings on titanium by electrochemical deposition in modified simulated body fluid

Adherent and optically semitransparent thin calcium phosphate (CaP) films were electrochemically deposited on titanium substrates in a modified simulated body fluid at 37 degrees C. Coatings deposited by using periodic pulsed potentials showed better adhesion and better mechanical properties than coatings deposited with use of a constant potential. Scanning electron microscopy was used to study the morphology of the coatings. The coatings displayed a polydispersed porous structure with pores in the range of a few nanometers to 1 mum. Furthermore, X-ray diffractometry and the O(1s) satellite peaks in X-ray photoelectron spectroscopy indicated that the coatings possessed a similar surface chemistry to that of natural bone minerals. These results were confirmed by inductively coupled plasma optical emission spectrometry, which yielded a Ca:P ratio of 1.65, close to that of hydroxyapatite. Contact mode atomic force microscopy (AFM) showed the average thickness of the coatings was in the order of 200 nm. Root-mean-square (RMS) roughness values, also derived by AFM, were shown to be much higher on the titanium-CaP surfaces in comparison with untreated titanium substrates, with RMS values of about 300 and 110 nm, respectively. Cell culture experiments showed that the CaP surfaces are nontoxic to MG63 osteoblastic cells in vitro and were able to support cell growth for up to 4 days, outperforming the untreated titanium surface in a direct comparison. These easily prepared coatings show promise for hard-tissue biomaterials.     

Mechanical properties of calcium phosphate coatings deposited by laser ablation

Amorphous calcium phosphate and crystalline hydroxyapatite coatings with different morphologies were deposited onto Ti-6Al-4V substrates by means of the laser ablation technique. The strength of adhesion of the coatings to the substrate and their mode of fracture were evaluated through the scratch test technique and scanning electron microscopy. The effect of wet immersion on the adhesion was also assessed. The mechanisms of failure and the critical load of delamination differ significantly depending on the phase and structure of the coatings. The HA coatings with granular morphology have higher resistance to delamination as compared to HA coatings with columnar morphology. This fact has been related to the absence of stresses for the granular morphology.     

Interface potential of calcium phosphate in simulated body fluid

Calcium phosphate ceramics are used in the substitution of injured or damaged bones. Nevertheless, the behaviour of these materials, and in particular, the mechanisms guiding their interface response in physiological environment is still unknown. This work describes the construction of hydroxyapatite and tricalcium phosphate electrodes used to determine the interface potential behaviour of these materials in a simulated body fluid, in a pH range corresponding to the variation observed in human body injuries, at ambient and physiological temperatures. These measurements are associated with the adsorption/desorption of ions from the materials. The results show that hydroxyapatite and tricalcium phosphate have similar behaviour in that they reach an interface potential equilibrium state faster when the solution pH is decreased and the temperature increased. This behaviour may be attributed to their ability to form a calcium-rich layer and is relevant to their quality as implantable materials.     

Theoretical analysis of calcium phosphate precipitation in simulated body fluid

The driving force and nucleation rate of calcium phosphate (Ca-P) precipitation in simulated body fluid (SBF) were analyzed based on the classical crystallization theory. SBF supersaturation with respect to hydroxyapatite (HA), octacalcium phosphate (OCP) and dicalcium phosphate (DCPD) was carefully calculated, considering all the association/dissociation reactions of related ion groups in SBF. The nucleation rates of Ca-P were calculated based on a kinetics model of heterogeneous nucleation. The analysis indicates that the nucleation rate of OCP is substantially higher than that of HA, while HA is most thermodynamically stable in SBF. The difference in nucleation rates between HA and OCP reduces with increasing pH in SBF. The HA nucleation rate is comparable with that of OCP when the pH value approaches 10. DCPD precipitation is thermodynamically impossible in normal SBF, unless calcium and phosphate ion concentrations of SBF increase. In such case, DCPD precipitation is the most likely because of its highest nucleation rates among Ca-P phases. We examined the influences of different SBF recipes, interfacial energies, contact angle and molecular volumes, and found that the parameter variations do not have significant impacts on analysis results. The effects of carbonate incorporation and calcium deficiency in HA were also estimated with available data. Generally, such apatite precipitations are more kinetically favorable than HA.     

Bone growth on and resorption of calcium phosphate coatings obtained by pulsed laser deposition

Three different calcium phosphate coatings of crystalline hydroxyapatite (HA), alpha- and beta-tricalcium phosphate (alpha+beta-TCP), or amorphous calcium phosphate (ACP) obtained by pulsed laser deposition on Ti-6Al-4V were incubated in a potentially osteogenic primary cell culture (rat bone marrow) in order to evaluate the amount and mode of mineralized bone matrix formation after 2 weeks with special emphasis on the type of interfacial structure that was created. Evaluation techniques included fluorescence labeling and scanning electron microscopy. The resistance to cellular resorption by osteoclasts was also studied. Bone matrix delaminated from the ACP coatings, while it remained on the HA and the alpha+beta-TCP coatings even after fracturing. A cementlike line was seen as the immediate contiguous interface with the nondegrading dense HA surface and with the surface of the remaining porous beta-TCP coating. Highly dense and crystalline HA coatings do not dissolve but are capable of establishing a strong bond with the bone matrix grown on top. Chemical and mechanical bonding were considered in this case. Cellular resorption was practically not observed on the HA coatings, but it was observed on the alpha+beta-TCP coatings. Resorption took place as dissolution that was due to the acidic microenvironment.     

Dissolution properties of calcium phosphate granules with different compositions in simulated body fluid

Calcium phosphate granules were produced through the calcination of three hydroxyapatites (HAs) at 1150 degrees C: nearly-stoichiometric (NS-HA), calcium-deficient (CD-HA), and carbonated (Carb-HA). The characterization using scanning electron microscope (SEM), X-ray diffraction (XRD), and Fourier-transformed infrared spectroscopy (FT-IR) showed that those granules exhibit strong differences in chemical composition, surface texture, and dissolution behavior. Sample dissolution in a simulated body fluid (SBF) and precipitation of a calcium phosphate layer on the granule surface were followed up for 7 days by chemical analysis of calcium content in SBF. After 80-min reaction, precipitation of new calcium phosphate phases predominates over the dissolution of original granules. Comparison between SEM images and XRD/FT-IR analysis revealed that the composition of the calcium phosphate layer and its precipitation rate depend on the HA composition and its heat treatment. Calcined carbonated apatite induces the precipitation of an octacalcium phosphate (OPC) layer, whereas a carbonated apatite layer is formed from calcined-deficient HA. The calcined nearly-stoichiometric HA exhibits low efficiency in producing carbonated apatite layer.     

Characterization of calcium phosphate coatings deposited by Nd:YAG laser ablation at 355 nm: influence of thickness

Calcium phosphate coatings were deposited by pulsed laser ablation with a radiation of 355 nm from a Nd:YAG laser. All the coatings were obtained at the same conditions, but deposition was stopped after different number of pulses to get coatings with different thickness. The influence of thickness in the structural and mechanical properties of the coatings was investigated. Coatings structure was characterised by scanning electron microscopy, grazing incidence X-ray diffractometry and Raman spectroscopy. The mechanical properties were evaluated by scratch test. The morphology of the coatings is dominated by the presence of droplets. The coatings are composed mainly of hydroxyapatite, alpha tricalcium phosphate and amorphous calcium phosphate. Thinner coatings withstand higher loads of failure in the scratch test.     

Evaluation of apatite ceramics containing alpha-tricalcium phosphate by immersion in simulated body fluid

The purpose of this study was to estimate the availability of alpha-tricalcium phosphate (alpha-TCP) on/in hydroxyapatite (HAP) ceramics for bioactivity as bone-substitute materials by immersion in a simulated body fluid (SBF; Hanks' solution) containing ion concentrations similar to those in human blood plasma. Two alpha-TCP-surface-modified HAP and alpha-TCP-HAP composite materials were prepared by orthophosphoric acid treatment of sintered HAP and controlling the crystal phases of calcium phosphate cement, respectively. After immersion in SBF, the sintered HAP modified on the surface in an approximately 0.2 microm alpha-TCP layer was more effective for the precipitation of carbonated apatites than an approximately 2 microm alpha-TCP layer and HAP-only layer. In the calcium phosphate cements consisting of HAP and alpha-TCP phases, after immersion for 1 week, the specimens precipitated large amounts of apatites having alpha-TCP contents of approximately 25% and 50% in the cement. The results of immersion tests imply the possibility that the alpha-TCP on/in HAP ceramics may be a bioactive agent for bone-substituting HAP materials.     

Micro- and nano-testing of calcium phosphate coatings produced by pulsed laser deposition

Micro- and nano-testing methods have been explored to study the thin calcium phosphate coatings with high adhesive strength. The pulsed laser deposition (PLD) technique was utilised to produce calcium phosphate coatings on metal substrates, because this type of coatings exhibit much higher adhesive strength with substrates than conventional plasma-sprayed coatings. Due to the limitations of the conventional techniques to evaluate the mechanical properties of these thin coatings (1 microm thick), micro-scratch testing has been applied to evaluate the coating-to-substrate adhesion, and nano-indentation to determine the coating hardness and elastic modulus. The test results showed that the PLD produced amorphous and crystalline HA coatings are more ductile than titanium substrates, and the PLD coatings are not delaminated from the substrates by scratch. Also, the results showed that the crystalline HA coating is superior in internal cohesion to the amorphous one, even though the lower elastic modulus of amorphous coating could be more mechanically compatible with natural bone.     

Human osteoblast response to pulsed laser deposited calcium phosphate coatings

Octacalcium phosphate (OCP) and Mn(2+)-doped carbonate hydroxyapatite (Mn-CHA) thin films were deposited on pure, highly polished and chemically etched Ti substrates with pulsed laser deposition. The coatings exhibit different composition, crystallinity and morphology that might affect their osteoconductivity. Human osteoblasts were cultured on the surfaces of OCP and Mn-CHA thin films, and the cell attachment, proliferation and differentiation were evaluated up to 21 days. The cells showed a normal morphology and a very good rate of proliferation and viability in every experimental time. Alkaline phosphatase activity was always higher than the control and Ti groups. From days 7 to 21 collagen type I production was higher in comparison with control and Ti groups. The level of transforming growth factor beta 1 (TGF-beta1) was lower at 3 and 7 days, but reached the highest values during following experimental times (14 and 21 days). Our data demonstrate that both calcium phosphate coatings favour osteoblasts proliferation, activation of their metabolism and differentiation.     

Behavior of human osteoblast-like cells in contact with electrodeposited calcium phosphate coatings

Calcium-deficient hydroxyapatite (Ca-def-HAP) thin films were elaborated on Ti6Al4V substrates by electrodeposition. The coatings exhibit two different morphologies and crystallinities. Human osteoblast-like cells (MG-63) were cultured on the surfaces of these materials; the cell content and viability were evaluated up to 28 days. The scanning electron microscopy and biological investigations showed cells with a normal morphology, good proliferation, and viability from 7 to 21 days. But after 28 days, the number of live cells decreases in both cases; however, this decrease is less important in the case of calcium phosphate (CaP) coating surface when compared with the control (cell culture plastic). The cells cultured on Ca-def-HAP coating exhibit more cellular extensions and extracellular matrix. RT-PCR for type I collagen, alkaline phosphatase, and osteocalcin studies were also carried out, and was found that the CaP enhances gene expression of ALP and OC and thus the differentiation of osteoblast-like cells. Moreover, this study shows that the difference in the morphology of CaP coatings has no effect on the biocompatibility.     

Calcium phosphate coatings obtained by Nd:YAG laser cladding: physicochemical and biologic properties

The plasma spray (PS) technique is the most popular method commercially in use to produce calcium phosphate (CaP) coatings to promote fixation and osteointegration of the cementless prosthesis. Nevertheless, PS has some disadvantages, such as the poor coating-to-substrate adhesion, low mechanical strength, and brittleness of the coating. In order to overcome the drawbacks of plasma spraying, we introduce in this work a new method to apply a CaP coating on a Ti alloy using a well-known technique in the metallurgical field: laser surface cladding. The physicochemical characterization of the coatings has been carried out by means of X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy dispersive X-ray analysis (EDX). The biologic properties of the coatings have been assessed in vitro with human osteoblast-like MG-63 cells. The overall results of this study affirm that the Nd:YAG laser cladding technique is a promising method in the biomedical field.     

Calcium phosphate and fluorinated calcium phosphate coatings on titanium deposited by Nd:YAG laser at a high fluence

Calcium phosphate coatings are known to enhance long-term fixation, reliability and promote osteointegration of cementless titanium-based implant devices. This study was aimed at the pulsed laser deposition of calcium phosphate coatings onto titanium using hydroxyapatite and hydroxyapatite-fluorapatite targets. The deposition was carried out at the high laser beam fluence conditions, about 12 J/cm(2). The coatings were characterized with respect to their morphology, phase composition and hardness. X-ray energy dispersive analysis revealed the coatings retain their elemental composition, and fluoride content within the film is the same as in the initial target. However, unlike sintered targets, the deposited films contain no apatite-like phases. The hardness of the films, about 18 GPa, is surprisingly high compared to that of hydroxyapatite and hydroxyapatite-fluorapatite ceramic targets. The deposited coatings of 2.7-2.9 microm thickness have uniform and dense microstructure, containing the solidified droplets of the expulsed from the target phase. The uncommon structure and hardness of the films can be attributed to the melting and phase decomposition of the initial material in the laser plasma.     

Immersion behavior of RF magnetron-assisted sputtered hydroxyapatite/titanium coatings in simulated body fluid.

The focus of the present study was on the dissolution/degradation behavior of a series of magnetron-sputtered, single-layered HA/Ti coatings on Ti-6Al-4V substrate immersed in SBF. Changes in coating morphology, crystal structure, and adhesion strength with immersion time are characterized. XRD, FTIR, and LVSEM results consistently indicate that highly crystalline monolithic HA coating is very dissolvable in SBF. The monolithic HA coating is largely delaminated in 3 weeks and entirely peeled off the substrate in 7 weeks. The dissolution is even greater for 95HA/5Ti coating, which severely disintegrated in only 1 week. The amorphous-like coatings sputtered from targets comprising 10 vol % or more Ti, however, appeared almost intact, and their adhesion strengths, which were all higher than 60 MPa, did not change much (within 10%) even after 14 weeks of immersion. The coatings from targets comprising roughly 10-50 vol % Ti combine advantages of high and nondeclining adhesion strength, high resistance to SBF attack, and possibly much higher bioactivity (with large amounts of Ca, P, etc., dissolved in the coatings) than that of Ti.     

Corrosion of anodic TiO coatings on Ti-6Al-4V in simulated body fluid

Anodic TiO coatings were produced on Ti-6Al-4V substrates using aqueous electrolytes containing dissolved calcium and phosphorus. Two baths containing Ca and P compounds, in the molar ratios 5 and 15 were used. Different coatings were produced by electrolysis for 3 and 10 h at a constant current density of 10 mA/cm(2). X-ray diffraction and X-ray fluorescence were used to identify the phases and chemical composition respectively. Thickness of the coatings was measured using ellipsometry. Electrochemical polarization and AC impedance studies were performed on the coatings by exposing them to simulated body fluid (SBF) for a period of 1 week. The coating produced by 10-h electrolysis from bath of Ca/P ratio 15 showed low corrosion current and high impedance to the week-long attack of SBF.     

The modulation of osteogenesis in vitro by calcium titanium phosphate coatings

Calcium phosphate coated titanium and titanium alloy are widely used as dental and orthopaedic implants. This study examines the effect of novel calcium titanium and calcium titanium zirconium phosphates suitable for plasma-spraying onto titanium substrata on the expression of bone-related genes and proteins by human bone-derived cells (HBDC) and compares this behavior to that on native titanium and hydroxyapatite-coated titanium. Test materials were an acid etched and sand-blasted titanium surface (Ti-DPS), a plasma-sprayed hydroxyapatite coating (HA), and five materials which were created from CaTi(4)(PO(4))(6) (CTP) and CaZr(4)(PO(4))(6) (CZP): sintered CaTi(4)(PO(4))(6) (CTP-S1), sintered 46CaO.23TiO(2).31P(2)O(5) (CTP-S2), sintered CaTiZr(3)(PO(4))(6), (CTZP-S1), sintered 46CaO.23ZrO(2).31P(2)O(5) (CTZP-S2) and sintered 55CaO.20TiO(2).31P(2)O(5) (CTP-S3). HBDC were grown on the substrata for 3, 7, 14 and 21 d, counted and probed for various mRNAs and proteins (type I collagen, osteocalcin, osteopontin, osteonectin, alkaline phosphatase and bone sialoprotein). All substrates significantly affected cellular growth and the temporal expression of an array of bone-related genes and proteins. At 14 and 21 d, cells on CTP-S3 displayed significantly enhanced expression of all osteogenic mRNAs. Surfaces of CTP-S1 and CTP-S3 had the most effect on osteoblastic differentiation inducing a greater expression of an array of osteogenic markers than recorded for cells grown on Ti-DPS and HA, suggesting that these novel materials may possess a higher potency to enhance osteogenesis.     

Apatite formation on the surface of wollastonite/tricalcium phosphate composite immersed in simulated body fluid

Wollastonite/tricalcium phosphate composites were prepared and immersed in SBF for various periods to investigate the apatite-formation mechanism on their surfaces. Surface morphologies and composition before and after immersion were analyzed by SEM and EDS technologies. The concentration changes of calcium, silicon, and phosphorus in SBF due to the immersion of the samples were measured with inductively coupled plasma atomic emission spectroscopy, and the corresponding pH values in SBF were recorded. XRD and IR were used to examine the changes in crystalline phases and functional groups. It was found that, after immersion for 1 day, the samples were covered by pom-pom-like apatite granules, resulting from the dissolution of wollastonite and the simultaneous transformation of TCP. This quickly led to the subsequent precipitation of another apatite layer, which covered the earlier-formed surface completely. These results indicate that the wollastonite/tricalcium phosphate composite is a potential biomaterial candidate because of its high bioactivity and in situ porous structure.     

Low-temperature crystallization of calcium phosphate coatings synthesized by ion-beam-assisted deposition.

Crystallization temperature of the amorphous calcium phosphate coating synthesized by ion-beam-assisted deposition successfully was decreased to 400 degrees C for the making of orthopedic implants with better qualities. Transmission electron microscopy (TEM) and X-ray diffraction (XRD) were used to investigate the morphological and structural evolution of the crystals in the coating during post-heat treatment. The crystallization of calcium phosphate coating is a hydroxyl-diffusion-controlled process, which is thought to be the mechanism responsible for the decrease of the crystallization temperature. In addition, the detailed crystallization process of calcium phosphate coating is elaborated in the present paper. The results show that the crystallinity of the hydroxyapatite coating can be well controlled by adjusting the post-heat-treatment time.     

Osteoclastic resorption of biomimetic calcium phosphate coatings in vitro

A new biomimetic method for coating metal implants enables the fast formation of dense and homogeneous calcium phosphate coatings. Titanium alloy (Ti6Al4V) disks were coated with a thin, carbonated, amorphous calcium phosphate (ACP) by immersion in a saturated solution of calcium, phosphate, magnesium, and carbonate. The ACP-coated disks then were processed further by incubation in calcium phosphate solutions to produce either crystalline carbonated apatite (CA) or octacalcium phosphate (OCP). The resorption behavior of these three biomimetic coatings was studied using osteoclast-enriched mouse bone-marrow cell cultures for 7 days. Cell-mediated degradation was observed for both carbonated apatite and octacalcium phosphate coatings. Numerous resorption lacunae characteristic of osteoclastic resorption were found on carbonated apatite after cell culture. The results showed that carbonated apatite coatings are resorbed by osteoclasts in a manner consistent with normal osteoclastic resorption. Osteoclasts also degraded the octacalcium phosphate coatings but not by classical pit formation.     

Mechanism of calcium disilicide-induced calcification of crystalline silicon surfaces in simulated body fluid under zero bias

A dry-etch spark ablation method was used to produce calcium disilicide (CaSi2/Si) layers on silicon surfaces, and their biomineralization under zero bias was followed by means of scanning electron microscopy, X-ray energy dispersive analysis, and Raman spectroscopy. CaSi2/Si wafers are bioinert at 25 degrees C and bioactive at 37 degrees C. Mechanistic insights regarding biomineralization were derived from an analysis of film growth morphology and chemical composition after various soaking periods in standard simulated body fluid (SBF). Changes in CaSi2/Si calcification behavior as a function of reaction temperature and pH, SBF concentration, and various surface modification processes were also employed for this purpose. During CaSi2/Si calcification under zero bias, calcium phosphate (CaP) growth is strongly dependent on the structural degradation of CaSi2/Si grains. Surface silanol groups, initially present on the as-prepared material, cannot induce CaP nucleation, which begins only upon delamination of CaSi2/Si layers. The calcium phosphate phases, which are present during various growth stages, possibly include a combination of Mg-substituted whitlockite, monetite, and tricalcium phosphate.