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.     

The study of surface transformation of pulsed laser deposited hydroxyapatite coatings

Hydroxyapatite (HA) coatings generally exhibit very good biocompatibility owing to their compositional resemblance to the natural hard tissue and to bioactive properties that are directly related to surface transformations in physiological fluids. In this study, two types of porous HA coatings produced with pulsed laser deposition were tested with respect to their dissolution/reprecipitation in a semidynamic simulated physiological solution. Coatings with higher porosity produced with a 355-nm wavelength laser exhibited significant reprecipitation earlier than those produced with a 266-nm wavelength laser. The dissolution of the non-HA phases played a major role in the reprecipitation of HA-like material as indicated by X-ray diffraction (XRD). The coatings' Ca/P ratio became closer to the theoretical value of HA. The newly formed HA had imperfect crystal structure and/or small crystal size as suggested by XRD. The reprecipitation resulted in a very dense morphology as shown by scanning electron microscopy, suggesting a mechanically strong structure after reprecipitation. Despite undergoing dissolution and reprecipitation, the coatings showed sufficient stability in the solution, as XRD and energy-dispersive X-ray studies indicated no significant loss of the coatings. The stability of these HA coatings and their ability to cause reprecipitation of HA in the simulated physiological solution showed the potential of these coatings for clinical applications.     

Plasma sprayed coatings of hydroxylapatite

The technique of plasma spraying has been applied to deposit a thin, dense layer of hydroxylapatite onto a titanium substrate. Bond strength of such apatite coatings with the substrate have been measured, as well as the (absence of) influence of the coating process on fatigue properties of the substrate. Animal studies showed similar histological reactions to apatite coatings as to (well documented) apatite bulk materials.     

Structural and morphological study of pulsed laser deposited calcium phosphate bioceramic coatings: influence of deposition conditions, laser parameters, and target properties

Calcium phosphate (CaP) bioceramics, especially hydroxyapatite (HA), have been used as coatings on implants owing to their biocompatible properties. The commercial practice for applying HA coating, plasma spraying, has some disadvantages which limit the long-term stability of the implants. Pulsed laser deposition (PLD) is being investigated as an alternative technique. The purpose of this research was to systematically study the effect of various parameters of the PLD process on the properties of CaP coatings. In this study, three types of HA targets and two laser wavelengths were used to make six categories of coatings. Predominantly crystalline HA coatings were produced under all six categories at optimum conditions, although small amounts of minor phases sometimes were found. Sufficient coating/substrate bond strength was also obtained. A wide variety of coating morphologies was obtained, from rather dense and uniform to rough and porous. The important factors that affected the morphology included target properties, vacuum level, deposition temperature, and laser wavelength and energy density. PLD's ability to produce both amorphous and crystalline, and both smooth/dense and rough/porous coatings may be a unique advantage.     

XPS, EDX and FTIR analysis of pulsed laser deposited calcium phosphate bioceramic coatings: the effects of various process parameters

Many techniques have been used to produce calcium phosphate, especially hydroxyapatite (HA), coatings on metallic implant surfaces for improved biocompatibility. Although some techniques have produced coatings used clinically, the long-term stability of the coating/implant is still questionable. As a new technique for making HA coatings, pulsed laser deposition (PLD) shows some advantages in controlling the coatings' crystal structure and composition. In this study, three types of HA target and two wavelengths of laser were used to produce calcium phosphate coatings. Despite PLDs ability to improve the crystal structure by incorporating water vapor into the deposition process, the characterization with EDX and XPS showed that coatings had different Ca/P ratios from that of the pure HA targets, which almost assured the presence of non-HA phases. FTIR spectra also showed differences in phosphate bands of coatings and targets although the difference in data collecting modes might have been a factor. The observed differences might be related to the differences between the surface and bulk chemistries of the coatings. Nevertheless, when evaluating the suitability of the PLD technique for making HA coatings, the possibility of the formation of non-HA phases cannot be excluded, although it may not necessarily be a negative factor.     

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.     

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.     

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.     

Influence of thickness on the properties of hydroxyapatite coatings deposited by KrF laser ablation.

The growth of hydroxyapatite coatings obtained by KrF excimer laser ablation and their adhesion to a titanium alloy substrate were studied by producing coatings with thicknesses ranging from 170 nm up to 1.5 microm, as a result of different deposition times. The morphology of the coatings consists of grain-like particles and also droplets. During growth the grain-like particles grow in size, partially masking the droplets, and a columnar structure is developed. The thinnest film is mainly composed of amorphous calcium phosphate. The coating 350nm thick already contains hydroxyapatite, whereas thicker coatings present some alpha tricalcium phosphate in addition to hydroxyapatite. The resulting coating to substrate adhesion was evaluated through the scratch test technique. Coatings fail under the scratch test by spallating laterally from the diamond tip and the failure load increases as thickness decreases, until not adhesive but cohesive failure for the thinnest coating is observed.     

Degradation of hydroxylapatite, fluorapatite, and fluorhydroxyapatite coatings of dental implants in dogs.

Calcium phosphate coatings on dental implants enhance integration of the material. Resorption of the ceramic coatings has raised some concern about the behavior of the bone-implant interfaces after the coating disappearance. Substitution of the OH- ions by fluoride in the hydroxylapatite (HA) lattice makes the calcium phosphate more stable. We investigated the degradation rate of dental implants with 50- and 100-microm coatings of HA, fluorapatite (FA), or fluorhydroxylapatite (FHA). The implants were inserted in dog jaws and retrieved for histological analysis after 3, 6, and 12 months. The thickness of the calcium phosphate coatings was evaluated using an image analysis device. A relative resorption index and its standard deviation were studied. HA and FA coatings (even at 100-microm thickness) were almost totally degraded within the implantation period. In contrast, the FHA coatings did not show significant degradation during the same period. The standard deviation showed that the resorption process for FHA with thicknesses of 50 or 100 microm was the same. Such a difference was not observed between the 50- and 100-microm thick coatings of FA and HA. In conclusion, the FHA coatings showed good integration in the bone tissue and lasted much longer than classic calcium phosphate coatings.     

A mechanical investigation of fluorapatite, magnesiumwhitlockite, and hydroxylapatite plasma-sprayed coatings in goats.

Ceramic coatings of fluorapatite (FA), magnesiumwhitlockite (MW), and hydroxylapatite (HA), and noncoated Ti-6Al-4V alloy (Ti) implants were evaluated before and after implantation in an animal study. Cylindrical plugs were coated by plasma-spraying with FA, MW, and HA. X-ray-diffraction patterns showed for FA and HA a crystalline and for MW an amorphous-crystalline coating structure. The plugs were implanted into the right femora and left humeri of 16 adult goats. Follow-up periods were 12 and 25 weeks. The in vivo results were evaluated using push-out tests and scanning electron microscopy. There were significant differences in push-out strengths between femur and humerus. The FA and HA implants showed significantly higher push-out strengths than the MW and Ti alloy implants, especially for the 12 week follow-up period. Furthermore, at 12 weeks, MW showed significantly lower push-out strengths than Ti alloy. SEM-investigation of the interfaces revealed that FA did not degrade while both MW and HA showed extensive degradation at 12 and 25 weeks. In addition, the interface after push-out testing showed for the MW, HA, and Ti alloy implants to be fractured at the implant-tissue interface and for the FA to be fractured at the coating-titanium interface.     

Different patterns of bone fixation with hydroxyapatite and resorbable CaP coatings in the rabbit tibia at 6, 12, and 52 weeks

Applying bioactive coatings on orthopedic implants can increase the fixation and long-term implant survival. In our study, we compared a resorbable electrochemically deposited calcium phosphate coating (Bonit?) to a thin (40 μm) plasma-sprayed hydroxyapatite (HA) coating, applied on grit-blasted screw-shaped Ti-6Al-4V implants in the cortical region of rabbit tibia, implanted for 6, 12, and 52 weeks. The removal torque results demonstrated stronger bone-to-implant fixation for the HA than Bonit-coated screws at 6 and 12 weeks. After 52 weeks, the fixation was in favor of the Bonit-coated screws, but the difference was statistically insignificant. Coat flaking and delamination of the HA with multinucleated giant cell activity and bone resorption observed histologically seemed to preclude any significant increase in fixation comparing the HA implants at 6 versus 12 weeks and 12 versus 52 weeks. The Bonit-coated implants exhibited increasing fixation from 6 to 12 weeks and from 12 to 52 weeks, and the coat was resorbed within 6 weeks, with minimal activity of multinucleated giant cells or bone resorption. A different fixation pattern was observed for the two coatings with a sharper but time limited increase in fixation for the HA-coated screws, and a slower but more steadily increasing fixation pattern for the Bonit-coated screws. The side effects were more serious for the HA coating and limiting the expected increase in fixation with time.     

The effect of sol-gel-formed calcium phosphate coatings on bone ingrowth and osteoconductivity of porous-surfaced Ti alloy implants

Ti-6Al-4V implants formed with a sintered porous surface for implant fixation by bone ingrowth were prepared with or without the addition of a thin surface layer of calcium phosphate (Ca-P) formed using a sol-gel coating technique over the porous surface. The implants were placed transversely across the tibiae of 17 rabbits. Implanted sites were allowed to heal for 2 weeks, after which specimens were retrieved for morphometric assessment using backscattered scanning electron microscopy and quantitative image analysis. Bone formation along the porous-structured implant surface, was measured in relation to the medial and lateral cortices as an indication of implant surface osteoconductivity. The Absolute Contact Length measurements of endosteal bone growth along the porous-surfaced zone were greater with the Ca-P-coated implants compared to the non-Ca-P-coated implants. The Ca-P-coated implants also displayed a trend towards a significant increase in the area of bone ingrowth (Bone Ingrowth Fraction). Finally, there was significantly greater bone-to-implant contact within the sinter neck regions of the Ca-P-coated implants.     

Physical, chemical, and biological characterization of pulsed laser deposited and plasma sputtered hydroxyapatite thin films on titanium alloy

The physical, chemical, and biological properties of pulsed laser deposited (PLD) and plasma sputtered (PS) hydroxyapatite (HA) coatings were compared. Human osteoblast-like cell responses to these coatings in vitro were assayed for proliferation and phenotypic expression. PS coatings formed smooth and continuous thin films that followed the contours of the substrate surface. PLD coatings consisted of numerous spheroidal micro- and macroparticles. The crystallinity of all coatings was quantified by comparison with the HA target used for both the PS and PLD processes. The XRD and FTIR results indicated that unannealed PLD coatings deposited at room temperature had X-ray spectra consistent with an amorphous structure and were found to dissolve after only a few hours in saline solution. Annealing at 400 degrees C increased the crystallinity (87-98%), which resulted in improved stability and cell activity. The PS coatings showed greater chemical stability than the unannealed PLD coatings and contained an approximate 15% crystalline phase, increasing to 65% postannealing. Cell proliferation and alkaline phosphatase production were significantly higher on unannealed PS specimens than the other coating treatments. There may be benefits in engineering the presence of a minor percentage of a microcrystalline phase in an amorphous or nanometer scale polycrystalline HA structure.     

Effects of cyclic bending and physiological solution on plasma-sprayed hydroxylapatite coatings of varying crystallinity.

This study investigated the effects of cyclic bending stress levels and testing in simulated physiological solutions or air on the integrity of plasma-sprayed hydroxylapatite (HA) coatings of two different crystallinities. Hydroxylapatite-coated commercially pure (CP) Ti rods were evaluated by immersion testing in Hank's Balanced Salt Solution (HBSS) and by rotating bending in air and HBSS. Static immersion testing of nonstressed specimens resulted in significant microcracking of coating surfaces after 42 days. Specimens cyclically tested at bending stresses above the yield strength of Ti experienced low cycle fatigue failure of the Ti substrates prior to spallation of the HA coatings. Coatings tested at 1 x 10(6) cycles with interface bending stresses of 180 MPa displayed increased surface microcracking, but no bulk coating spallation. Coatings cycled in HBSS displayed greater amounts of microcracking and surface alteration than samples cycled in air. There was no apparent relation between HA crystallinity and mechanical integrity under cyclic bending stresses.     

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.     

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.     

Local release of magnesium from mesoporous TiO2 coatings stimulates the peri-implant expression of osteogenic markers and improves osteoconductivity in vivo

Local release of Mg ions from titanium implant surfaces has been shown to enhance implant retention and integration. To clarify the biological events that lead to this positive outcome, threaded implants coated with mesoporous TiO₂ thin films were loaded with Mg-ions and placed in the tibia of rabbits for 3 weeks, after surface characterization. Non-loaded mesoporous coated implants were used as controls. Peri-implant gene expression of a set of osteogenic and inflammatory assays was quantified by means of real-time quantitative polymerase chain reaction. The expression of three osteogenic markers (OC, RUNX-2 and IGF-1) was significantly more pronounced in the test specimens, suggesting that the release of Mg ions directly at the implant sites may stimulate an osteogenic environment. Furthermore, bone healing around implants was evaluated on histological slides and by diffraction-enhanced imaging (DEI), using synchrotron radiation. The histological analysis demonstrated new bone formation around all implants, without negative responses, with a significant increase in the number of threads filled with new bone for test surfaces. DEI analysis attested the high mineral content of the newly formed bone. Improved surface osteoconductivity and increased expression of genes involved in the bone regeneration were found for magnesium-incorporation of mesoporous TiO₂ coatings.     

A removal torque of the laser-treated titanium implants in rabbit tibia

The purpose of the present study is to evaluate the significance of different surface textures by comparison of the removal forces for laser-treated and machined titanium screw 8 weeks after the installation in rabbit tibia. A total of 14 screw shaped, commercially pure titanium implants with a length of 5 mm, a diameter of 3.75 mm were grouped as follows: Group A: seven implants left as-machined; Group B: seven implants treated with laser method (CSM implant, CSM company, Daegu, Korea) Topographic evaluation was performed with scanning electron microscope (Hitachi S-4200, Japan) to compare the surface structure of laser-treated and machined ones. Installation procedures were done according to Branemark protocol after pre-threading, machined implants were inserted in right tibia metaphysics and laser-treated surface implants were inserted in left ones. Eight weeks post surgically seven rabbits were sacrificed. The implant sites were exposed, and the bone and soft tissues that had formed on top of the implants were carefully removed. Subsequently, the force needed to unscrew the implants (n=14) was measured using a digital torque gauge (Mark-10 corporation, USA). Scanning electron micrographs of the laser-treated and machined control groups demonstrated created a deep and regular honey-comb pattern with small pore, while machined treatment created the typical microscopically grooved and relatively smooth surface characteristic. Eight weeks after implant placement, the average removal torque was 23.58+/-3.71 N cm for the machined implants, 62.57+/-10.44 N cm for the laser-treated implants. The torque measurements yielded statistically significant differences between the machined group and the laser-etched group (p=0.00055) (Wilcoxon's signed-rank test). The laser-treated group achieved higher removal torque values compared to the machined control group.