Bioceramic coatings like hydroxyapatite (HA) have shown promising bioactive properties in load-bearing implant applications. The two-year project is to deposit functionally graded HA/Ti layers consisting of an underlying Ti bond coat, the alternating layer, and an HA top-layer on Ti6Al4V substrates using plasma spray to improve the coating-substrate interface properties. The alternating layers were created by means of changing the feeding rate and input power of Ti and HA powders, which gradually decreasing Ti content with increasing depth from the Ti bond-coat. After which, characterization of RGD peptide-grafted coatings following heat treatment will be emphasized. The presently first-year results indicated that surface chemistry and morphology of the graded coatings were similar to those of monolithic HA coatings. The bond strength values of the as-sprayed graded coatings were much superior to those of monolithic HA coatings. The cyclic fatigue did have a statistically significant effect on bond strength of monolithic HA coatings, with a decrease of 23%.
However, the graded coatings were able to survive 1 million cycles of loading in air without significantly reduced bond strength. The in vitro electrochemical measurement results also indicated that the graded coatings had a more beneficial and desired behavior than monolithic HA coatings after fatigue. In the secondary year, the major consideration was to examine the corrosion resistance and bond strength of graded coatings after various postheat treatment temperatures ranging from 400oC to 700oC. Gentamicin loading onto the plasma-sprayed coating was used to improve its antibacterial activity. RGD grafting is also another focus. Our results indicate that an appropriate heat treatment resulted in recrystallization of amorphous calcium phosphate of the as-sprayed coatings. The enhancement in corrosion resistance took place after heat treatment. Gentamicin loading might provide effective release at early time points (up to 1 h). As for RGD grafting on coating surface, the MTT assay indicated there are no significant differences between all samples with or without RGDC peptide. The heat treatment at 600oC for 1 h in air, leading to increased crystallinity and fewer defects, as well as enhanced bond strength, may be a suitable post-deposition treatment method to promote the characteristics of graded HA/Ti coatings that also serve as a drug carrier.
Keywords: Titanium, hydroxyapatite, graded coating, implant, plasma spray, peptide
3. 緣由與目的
HA-coated implants have shown promising bioactive properties in orthorpaedic and dental surgery [1-2]. A variety of coating techniques, such as plasma spraying [1], magnetron sputtering [3], and sol-gel processing [4], have been explored to coat HA onto metals. Among these techniques, plasma spraying is perhaps most popularly used due to its process feasibility such as ease of operation. However, the plasma-sprayed HA coatings generally contained cracks, pores, second phases, and residual stresses that can reduce their durability and lead to partial or complete delamination of the coatings in body fluids [5]. In addition, the major causes of failure of HA-coated implants appear to reside in the coating-substrate interface [6,7]. In a study by Kangasniemi et al. [7] the failure mode of bone-HA interfacial tensile testing was observed to occur consistently at the HA-Ti interface, indicating clearly that it was difficult to develop reliable HA/Ti alloy bonding.
The adhesion of the HA coating to its Ti substrate and the integrity of the substrate/coating interface are always concerned in determining the performance and reliability of any HA-coated devices. Short-term clinical trials have shown HA coatings to enhance bone apposition, fixation, and reduce healing time. Nevertheless, the long-term stability of such coatings was the predominate factor causing the success of the implant. For improving the coating-substrate bond strength and other properties, many attempts have been made to develop biocoatings with higher strength. In this case, double layer or multiplayer coatings have been considered recently [6,8]. The deposition of a composite coating, wherein a metallic phase is introduced to serve as either an intermediate layer (bond coat) or a second (continuous or dispersed) phase in the HA matrix was pursued to enhance the interface properties. For example, Gu et al. [6] used a approach of ceramic slurry mixing to prepare composite powders with an inner core of Ti6Al4V particles swathed by a layer of mixed HA/
yttria stabilized zirconia powders for plasma spraying onto Ti6Al4V substrates.
Another more effective approach in the design of eliminating material-property discontinuities to significantly increase adhesion strength is by means of grading the material composition near the interfaces or through the coatings, so-called functionally graded materials. The top layer of functionally graded coatings may provide good bioactive properties to accelerate bone healing and the underlying bond coat may be designed to achieve maximum adhesion strength. Between these two layers is a transition layer with intermediate properties. Gruner developed a multi-layered coating composed of a Ti precoat, a Ti/HA composite layer and an HA top-layer by plasma deposition [9]. This implant study showed a fast and stable fusion between the coated implant and the bone. Inagaki et al. [10] plasma-sprayed gradient-like HA/Ti composite coatings at different RF power and suggested that the bond strength increased with increasing power of plasma due to the increase in density of the coatings. However, their work did not provide the results of cyclic fatigue and corrosion behaviors of the potent graded coatings, which are of paramount importance, when implanted in body [9,10].
It is believed that HA coatings with higher crystallinity always yield a decreased dissolution rate [11] and enhanced rate of cell proliferation [12]. In order to improve the quality of plasma-sprayed coatings further than optimizing plasma spray processing and altering material composition [13], heat treatment is sometimes used to increase coating crystallinity and reduce the residual stress of HA-based coatings [14–16]. In a recent paper [17], we found that treatment at 600oC could play a predominant role in enhancing the characteristics of plasma-sprayed HA coatings.
Bacterial infection of bone is still a non-negligible concern in dental and orthopedic surgery. In the treatment of carious teeth, bacteria left in the cavity of infected dentin are one of the factors leading to secondary caries or pulpal injury after restoration [18]. Antibacterial
treatment of the cavity is thus recommended before restoration is completed. Systems of controlled release can target delivery of biologically active agents to specific bone tissue areas for enhanced healing and repair. Local delivery can yield higher concentrations in the relevant tissues, improving their efficacy and possibly reducing the necessary duration of treatment [19].
Extracellular matrix (ECM) proteins contain signaling domains which can be synthesized as bioactive peptide fragments. Many biomaterials have been modified with short peptides in an effort to enhance cell-material interactions, and mimic the role of ECM proteins [20,21]. The best studied of these peptides is the RGD sequence, a motif that binds to integrin cell adhesion receptors and thereby promotes cell attachment to the material surface.
The use of RGD-peptide may be a future way of improving biomaterial surface. According to the literature [22,23], the autologous human bone marrow stroma cells may be successfully associated to RGD-grafted biomaterials Ti to promote cell colonization.
In the present project, the graded coatings were plasma-sprayed onto Ti6Al4V substrates.
By independently changing the feeding rate and plasma power of the Ti and HA powders, compositional control of the coating was achieved. It was attempted to combine the advantages of both the high chemical affinity between the underlying coating containing Ti and Ti6Al4V substrate and functionally graded coating concepts. The focus was to investigate the cyclic fatigue resistance of plasma-sprayed coatings using three-point bending test. The coating morphology, crystal structure and bond strength were also characterized. Next, to further enhance the quality of the potential HA/Ti graded coatings by means of post-deposition heat treatment was achieved. Additionally, combining osteoconductive properties of bioactive coatings with an antibiotic release could produce a two-fold beneficial effect.
Finally, the evaluation of RGD grafting on coatings was another focus.
4. 實驗方法
4.1. Fabrication of graded coatings
Commercial HA powder (AMDRY 6021, Plasma Technik A.G., Wohlen, Switzerland) with particle size between 44 and 149 μm was used in this study. Pure Ti particles of 100 μm were purchased from Alloy Metals Inc (AMDRY 918, Troy, MI). Commercially available plates of Ti6Al4V alloy (100 × 10 × 3 thick) were used as the substrate material. Prior to plasma spray, the substrate surface was mechanically polished to #1200 grit level, cleaned and sandblasted with 450 μm SiC particles. To obtain a uniform coating, the substrate was mounted on a disk that could rotate during spraying in air using a Plasma-Technik A-3000 system (Plasma Technik A.G., Wohlen, Switzerland).
Functionally graded HA/Ti coatings consisting of an underlying Ti bond coat, the alternating layer, and an HA top-layer were deposited onto Ti6Al4V substrates, as shown in Figure 1. A Ti layer (about 30 μm) was first deposited as an initial bond coat (precoat) onto the substrates. After that, alternating deposition was performed using two torches that allowed for the use of independent processing parameters and divided into three steps. For example, the plasma power, flow rate of plasma gas (Ar/H2), and powder feeding rate (Ti/HA) were changed from the parameters for Ti of 27.5 kW, 60/3 L min-1, and 20/0 g min-1, respectively, to 29.8 kW, 56/5 L min-1, and 15/5 g min-1 so that Ti gradually decreased and HA simultaneously increased during deposition. Accordingly, the next step was to increase input power levels and to decrease the feeding rate ratio of Ti and HA powders, in addition to change the flow rate of plasma gas mixtures (Ar/H2). Finally a 30 μm thick HA top-layer was plasma-sprayed onto the alternating layer. The plasma spray parameters for graded coatings were listed in Table I. Monolithic HA coating was also fabricated for comparison using the parameters for HA top-layer. Coating thickness for the two specimens was about 100 μm.
4.2. Heat treatment
In order to study the effect of heat treatment, the as-sprayed coatings were heat-treated at 400, 500, 600 and 700oC for 1 h each at a heating rate of 5oC/min in an air-circulated furnace.
After treatment, the furnace was allowed to cool to room temperature. The optimal temperature was used for the coating loading drug or gafting RGD peptide.
4.3. Phase composition and morphology
Phases of the two types of the as-sprayed coatings were analyzed by an X-ray diffractometer (XRD, Shimadzu XD-D1, Kyoto, Japan) operated at 30 kV and 30 mA. A Fourier transform infrared (FTIR) spectroscopy (Bomem DA8.3, Hartman & Braun, Canada) in reflection absorption mode with a spectral resolution of 1 cm-1, was used to characterize the various functional groups on the coating surface. The microstructure/microchemistry was characterized under field emission scanning electron microscope (FESEM) (Hitachi S-4200, Hitachi, Tokyo, Japan) equipped with an energy dispersive spectrometer (EDS, Noran Instrument Inc., Middleton, WI, USA). The Ca, P, and Ti concentration profiles of the graded coatings were measured using point analysis of EDS. Specimens for cross-sectional microscopy were prepared by mounting the coated specimens in epoxy resin, followed by polishing through 1 μm alumina.
4.4. Bond strength measurement
The plasma sprayed coating for load-bearing applications must be evaluated by fatigue ability due to the cyclic nature of in vivo loading. For the investigation of the fatigue resistance, the coated samples were fatigued under cyclic three-point bending test in air at room temperature using Shimadzu servopulser 48000 system (Kyoto, Japan). The fatigued samples were 50 mm in length, 10 mm in width, 3 mm in thickness with a gauge length of 40 mm. A cyclic loading of 280 MPa, which corresponds with the reported maximum bending stress of a clinically implant subjected to in vitro circumstances, with a stress ratio of Smin/Smax
= zero was imposed at 5 Hz up to 1 million cycles, and then performing tensile bond strength and corrosion tests. Bond strength (or adhesive strength) of the two coatings before and after cyclic fatigue was used to represent the present pull-out test results using an EZ-Test machine (Shimadzu, Kyoto, Japan) at a loading rate of 0.5 mm/min. In doing the testing, a 2.7 mm dia.
aluminum pull stud (Quad Group, Spokane, WA) was bonded to the coated surface with a solid epoxy and then cured at 150oC for 1 h in oven.3 After the coated specimen/stud assembly was gripped on a platen, the stud was then pulled down against the platen until failure. The maximum fracture force can be recorded and averaged to obtain the mean value and standard derivation. The number of measurements is twelve for each group.
4.5. Electrochemical testing
After fatigue and heat treatment, the coatings were electrochemically measured, in addition to as-sprayed coatings. The electrochemical measurements carried out on the samples included open circuit potential (OCP)-time measurements and potentiodynamic polarization in Hank’s Balanced Salt Solution (HBSS), using a CHI 660A electrochemical system (CH Instrument, Austin, Texas). For OCP measurement, only two electrodes (working electrode and reference electrode) were involved, while for potentiodynamic polarization method a conventional three-electrode cell was used. A saturated calomel reference electrode (SCE) and a platinum counter electrode were employed. The sample surface was cleaned by distilled water. Deaerated conditions under N2 gas purging and a temperature of 37°C were used for in vitro experiments. The evaluation of potentiodynamic polarization was started after immersion
in HBSS for 1 h. The scanned potential range varied from -1 V up to 2 V toward the anodic direction at a sweep rate of 0.5 mV/s in the Tafel mode. Six measurements were performed for each group. Corrosion potential and corrosion current were provided after being analyzed by the software.
4.6. In vitro release profile
Gentamicin is chosen as the drug model for its broad spectrum, effectiveness in treating osteomyelitis, and capacity for preventing osseous staphylococcal infections. Before loading the drug into the implant, the used drug was dissolved into pH 7.0 Gibco Dulbecco’s phosphate-buffered saline (Invitrogen, Carlsbad, CA) without calcium and magnesium ions.
Then, each sample was coated separately with 3 mL of drug solution, continually removed, and replaced into fresh immersion solution at regular intervals. The amount of gentamicin released was determined at 332 nm using a UV/VIS spectrophotometer (V-630, JASCO, Tokyo, Japan). For each antibiotics/implant combination, at least three parallel experiments were tested. In addition, SEM was also used to characterize the coated samples before and after release studies.
4.7. Evaluation of RGD grafting
Before RGDC grafting, all specimens were ultrasonically cleaned with dry ethanol for 10 min and dried at 60 . Then, metal samples were sterilized vi℃ a autoclave at 120oC for 30 min). For RGDC immobilization, 100 μl of 1 mM RGDC solution in water was spin-coated onto the coating surfaces and air dried. MG63 human osteoblast-like cells were suspended in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Langley, OK) supplemented with 10% fetal bovine serum (Gibco, Langley, OK) and antibiotics (1% of a penicillin (10,000 U/ml)/streptomycin (10,000 mg/ml) solution (Gibco, Langley, OK). The cells were plated at a density of 2 × 104 cells/mL in 24-well plates containing various steriled samples. Plates were cultured in standard conditions at 37oC and 95% humidity, and 5% CO2.
The quantification of the adhered cells was performed by measuring their mitochondrial dehydrogenase activity using MTT assay. After 1, 4 and 24 h, the cells were treated with 5 mg/mL of thiazolyl blue tetrazolium bromide (Sigma, St. Louis, MO) in PBS (Gibco, Langley, OK) and incubated in the humidified atmosphere of 5% CO2. After culture for 4 h, dimethyl sulfoxide (J.T. Baker, Phillipsburg, NJ) was used and the optical density was measured at the absorbance of 550 nm. The morphology of the cells adhering on coating surface was observed by SEM.
4.8. Statistical analysis
One-way analysis of variance (ANOVA) was used to evaluate the significant differences between the means in bond strength and electrochemical data. Scheffe’s multiple comparison test was used to determine the significance of the standard deviations in the data of the various coated samples. In all cases, the results were considered statistically different at p <
0.05.
5. 結果與討論
5.1. Characterization of as-spayed coatings
In Fig. 1 the plasma-sprayed coatings generally have lower apatite crystallinity than HA powders used for plasma spray. Besides the broadening of apatite peaks and increased intensity of CaO phase, TCP phases were observed in monolithic HA and graded coatings.
The high temperature involved in plasma spray process had obviously enhanced the decomposition of apatite as well as chemical reactions within HA phase. Since its top-layer
comprised HA phase of roughly 30 μm, it is reasonable that surface phase composition of the graded coatings detected by XRD was almost same as that of monolithic HA coatings.
Similarly, both monolithic HA coating and graded coating should consistently exhibit many absorption bands of apatite structure in its FTIR reflection spectrum (Fig. 2). The broad band at 980-1120 cm-1 in FTIR reflection spectra of the two coatings, probably resulted from overlapping apatite and TCP signals, were attributed to HPO4/PO4 functional groups. The bands between 600 and 560 cm-1 were suggested to result from the vibrational mode of PO4
groups. In contrast to those of HA powder, the decrease of the stretching mode at 3570 cm-1 and the librational mode at 630 cm-1 of OH groups, suggested the OH release of a hydroxylated HA structure that occurred in the two coatings. Additionally, the split sharp bands at 570, 600, 1050, and 1110 cm-1 indicated a well-crystallized apatite powder used for plasma spray.
Despite their different deposition processing, the two coatings had a similar morphology that had patches of smooth and shiny glassy films and irregularly-shaped particles (Fig. 3 (a)).
Randomly distributed pores of different sizes and microcracks were also observed. The surface morphology depended strongly on the sizes and shapes of the feeding powder particles.
During plasma spraying, small size powder was completely melted and formed a glassy phase during fast cooling, while larger size powder, such as the one used in the present study, was only partially melted resulting in a coating morphology comprising the observed irregularly-shaped particles. This observation is consistent with the earlier-discussed XRD results (e.g.
broadening in apatite peaks).
The cross-sectional SEM micrographs showed that the layer defects between the splats within coatings are observed to run roughly parallel to the coating surface, as shown in Fig. 3 (b). Besides, there were a lot of perpendicular defects inside the coatings, e.g., pores and cracks. These plasma spray-induced layer defects were frequently observed in plasma sprayed HA and other coatings. It was found that the microstructure of graded coatings gradually transferred from Ti bond coat to HA topcoat accompanied with the change in composition distribution through coating thickness.
Chemical distribution of three major elements, Ca, P and Ti, in the graded coatings cross-section was studied using SEM-EDS technique, as also shown in Figure 3 (c). In doing through-thickness chemical analysis of the coatings, a series of point analyses were made on the polished cross section of the coating. These elemental point analysis illustrated the graded coatings were with a controlled compositional gradient. The alternating layer within the coating had a gradually decreasing concentration of the Ti toward the outer surface with the exception of initial bond coat, along with a gradually increasing Ca and P contents up to a bioactive top-layer composed of an apatite. The composition changes were so gradual that the interface between the substrate and the coating layer as well as the boundaries among the phases within the coating layer should seem to be tightly bonded, which was the main purpose to be achieved. The measured Ca/P ratio on HA top layer was in the range of 1.9-2.1, higher than the stoichiometric Ca/P ratio (1.67) of HA, and also observed in other plasma-sprayed
Chemical distribution of three major elements, Ca, P and Ti, in the graded coatings cross-section was studied using SEM-EDS technique, as also shown in Figure 3 (c). In doing through-thickness chemical analysis of the coatings, a series of point analyses were made on the polished cross section of the coating. These elemental point analysis illustrated the graded coatings were with a controlled compositional gradient. The alternating layer within the coating had a gradually decreasing concentration of the Ti toward the outer surface with the exception of initial bond coat, along with a gradually increasing Ca and P contents up to a bioactive top-layer composed of an apatite. The composition changes were so gradual that the interface between the substrate and the coating layer as well as the boundaries among the phases within the coating layer should seem to be tightly bonded, which was the main purpose to be achieved. The measured Ca/P ratio on HA top layer was in the range of 1.9-2.1, higher than the stoichiometric Ca/P ratio (1.67) of HA, and also observed in other plasma-sprayed