Effects of MgO and SiO2 on Plasma-Sprayed Hydroxyapatite Coating

Jul 28, 2017 - Effects of MgO and SiO2 on Plasma-Sprayed Hydroxyapatite Coating: An in Vivo Study in Rat Distal Femoral Defects ... *Phone: 509-335-74...
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Effects of MgO and SiO2 on Plasma-Sprayed Hydroxyapatite Coating: An in Vivo Study in Rat Distal Femoral Defects Dongxu Ke,† Samuel F. Robertson, William S. Dernell,‡ Amit Bandyopadhyay,† and Susmita Bose*,† †

W. M. Keck Biomedical Materials Research Laboratory, School of Mechanical and Materials Engineering, Washington State University, Pullman, Washington 99164-2920, United States ‡ Veterinary Teaching Hospital, Washington State University, Pullman, Washington 99164, United States S Supporting Information *

ABSTRACT: Plasma-sprayed hydroxyapatite (HA)-coated titanium implants have been widely used in orthopedic applications due to their inheritance of an excellent mechanical property from titanium and great osteoconductivity from HA. However, the lack of osteoinductivity limits their further applications. In this study, 1 wt % MgO and 0.5 wt % SiO2 were mixed with HA for making plasma-sprayed coatings on titanium implants. Plasma-sprayed HA- and MgO/SiO2−HA-coated titanium implants showed adhesive bond strengths of 25.73 ± 1.92 and 23.44 ± 2.89 MPa, respectively. The presence of MgO and SiO2 significantly increased the osteogenesis, osseointegration, and bone mineralization of HA-coated titanium implants by the evaluation of their histomorphology after 6, 10, and 14 weeks of implantation in rat distal femoral defects. Implant pushout tests also showed a shear modulus of 149.83 ± 3.69 MPa for MgO/ SiO2−HA-coated implants after 14 weeks of implantation, compared to 52.68 ± 10.41 MPa for uncoated implants and 83.92 ± 3.68 MPa for pure HA-coated implants; These are differences in the shear modulus of 96% and 56.4%, respectively. This study assesses for the first time the quality of the bone−implant interface of induction plasma-sprayed MgO and SiO2 binary-doped HA coatings on load-bearing implants compared to bare titanium and pure HA coatings in a quantitative manner. Relating the osseointegration and interface shear modulus to the quality of implant fixation is critical to the advancement and implementation of HA-coated orthopedic implants. KEYWORDS: plasma-sprayed hydroxyapatite coating, titanium implant, MgO and SiO2, in vivo histomorphology, implant pushout test, osseointegration, osteogenesis implant interface.3−5 Long-term clinical trials also indicated that HA-coated femoral stems had a high survival rate in young and active patients.6,7 Coating techniques, such as electrophoretic deposition, dip coating, and plasma-sprayed coating were reported to deposit HA coatings on metallic substrates.8−10 Plasma-sprayed coating has the advantages of a high deposition rate, low cost, and low contamination and is the only widely used coating method applied commercially for orthopedic implants.11 Therefore, plasma-sprayed HA coating should be an excellent solution for improving the osteoconductivity of Ti implants. Even though HA is an osteoconductive material, it lacks osteoinductivity to enhance bone growth. Additives, such as MgO and SiO2, were reported to improve the osteoinductivity of β-tricalcium phosphate (β-TCP) in vitro and in vivo.12,13 MgO was reported to increase the osteoinductivity of β-TCP by improving the osteoblastic attachment, proliferation, and alkaline phosphatase production in vitro.14 An in vivo study also showed that the presence of MgO in HA enhanced osteogenesis in rabbit femoral defects.15 The presence of SiO2

1. INTRODUCTION Hip implants have become much more common over the past few decades. The need for total hip replacement has drastically increased from 138700 in 2000 to 310800 in 2010 from the last reported study.1 As one of the most successful surgeries, the medical practices of total hip replacement surgery have matured and streamlined. The academic community is now focusing on improving the mechanical and biological properties of total hip implants to increase their survival rate and lifespan, expedite patients’ recovery, and reduce patients’ pain after surgery. The hip stem is one of the most important parts of the total hip implant, which is inserted into the femur and anchors the implant in a fixed position.2 The hip stem is commonly made of titanium due to its light weight and excellent mechanical properties. Since the hip stem is usually fully covered by host tissue, its limited osteoconductivity and osseointegration become disadvantageous. One possible solution is to apply osteoconductive coatings on the Ti hip stem to improve its osteoconductivity and osseointegration. Hydroxyapatite (HA) is known as a stable coating material because of its excellent biocompatibility and low degradation rate. In addition, HA coatings can reduce the implant fixation time, enhance bonding between implants and host tissues, and create uniform bone growth near the bone− © XXXX American Chemical Society

Received: April 21, 2017 Accepted: June 2, 2017

A

DOI: 10.1021/acsami.7b05574 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces Scheme 1. Concepts of the Experimental Design for This Study

was reported to enhance the osteogenesis of HA in vivo compared to that of pure HA by showing an increased amount of bone growth.16,17 Besides the improvement of osteogenic properties, SiO2 was found to decrease osteoclastogenesis and increase angiogenesis in vitro as well.18,19 These studies were performed using granulated HA and porous HA scaffolds; however, scientific research focusing on these binary dopants in plasma-sprayed coatings to enhance the healing rate as well as the long-term success of metallic implants is still lacking. In this study, MgO and SiO2 were added to HA for preparing plasma-sprayed coatings on Ti substrates. The objective of this study is to investigate the effects of MgO and SiO2 on the biological properties of plasma-sprayed HA coatings in vivo. It is hypothesized that MgO and SiO2 can enhance the osteogenesis and osseointegration of plasma-sprayed HA coating in vivo. To test the hypothesis, plasma-sprayed HA and MgO/SiO2−HA coatings were prepared and characterized by X-ray diffraction (XRD), Fourier-transformed infrared (FTIR) spectroscopy, the interface microstructure, and the adhesive bond strength for evaluating their physical and mechanical properties. Then uncoated Ti, HA-coated Ti, and MgO/SiO2−HA-coated Ti implants were inserted into rat distal femoral defects for in vivo characterizations using histomorphometric staining and implant pushout tests after implantation for 6, 10, and 14 weeks. The whole design of the experiments is shown in Scheme 1.

characterizations. The Ti rods had a diameter of 3 mm and were cut to 30 mm in length. The substrates and rods were sandblasted and then ultrasonicated two times with alternating deionized (DI) water and acetone. 2.2. Coating Powder Preparation. The HA powder used in this study was commercial grade HA with a particle size between 150 and 212 μm (Monsanto, St. Louis, MO). Silica (SiO2) and magnesium oxide (MgO) were obtained from Sigma-Aldrich (St. Louis, MO). MgO/SiO2−HA powder was prepared by making a 50 g batch including 0.25 g of SiO2, 0.5 g of MgO, and HA mixed with 75 mL of anhydrous ethanol and 100 g of milling media at 70 rpm for 6 h. The same amount of HA powder was also mixed with the same amount of ethanol and milling media as the control. The mixtures were dried at 60 °C for 72 h as the final powders for plasma coatings. 2.3. Plasma Coating Preparation. A 30 kW induction-coupled radio frequency (rf) plasma spray system (Tekna, Sherbrooke, QC, Canada) was used to deposit the coatings on substrates with an axial powder feeding system and supersonic nozzle. In this study, 25 kW and a 100 mm working coordinate were used for the preparation of HA and MgO/SiO2−HA coatings on the basis of previous optimizations.10 2.4. Phase and Chemical Group Identifications. An X-ray diffractometer using Cu Kα radiation at 35 kV and 30 mA was used to identify the phase formation of HA and MgO/SiO2−HA coatings. A range between 20° and 45° was scanned for the coatings with a dwelling of 0.5 s and a step size of 0.05°. The quantitative crystallinity of the coatings

2. MATERIALS AND METHODS

was calculated, where Ac and Aa are the integrated intensities of crystalline HA peaks and amorphous HA humps, respectively, between 25° and 37°.5 Ac was calculated by multiplying the area of the 100% HA (211) peak by 3.23, which is the ratio of the total intensity of all crystalline HA peaks in JCPDS no. 9-432 to the intensity of the (211)

crystallinity (%) = Ac /(Ac + A a ) × 100

2.1. Ti Substrate and Rod Preparation. Titanium plates and rods were purchased from President Titanium (Hanson, MA). The plates were cut by a water jet cutter to have a diameter of 2.54 cm for the adhesive bond strength test and a diameter of 1.22 cm for other B

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based on three samples and is reported as the mean ± standard deviation (n = 3).

peak in the range between 25° and 37°. Ac + Aa was calculated by integrating the area of all HA peaks within 25° and 37°. Plasma-sprayed HA and MgO/SiO2−HA coatings were scanned by FTIR to identify their chemical groups. The coatings were placed on an attenuated total reflection (ATR) diamond crystal followed by scanning with an FTIR spectrometer in the range from 400 to 1200 cm−1. The final graph was achieved by plotting the transmittance against the wavenumber. 2.5. Coating Interface Microstructure. Samples with HA and MgO/SiO2−HA coatings were mounted with polymer resins followed by sectioning using a diamond saw at a speed of 300 rpm. After sectioning, the samples were etched followed by the observation of coating interfaces using field emission scanning electron microscopy (FESEM). 2.6. Adhesive Bond Strength Test. The adhesive bond strength was tested on HA-coated and MgO/SiO2−HA-coated Ti substrates. All test parameters were based on ASTM C633.20 Briefly, samples for adhesive bond strength tests were glued to two sandblasted counterparts using the protocol-recommended glue Armstrong A-12 (Resin Technology Group, LLC, Easton, MA). Then they were cured at 200 °F for 30 min followed by the test using a standard tensile test setup at a constant crosshead speed of 0.0013 cm/s until failure. The adhesive bond strength was calculated as the maximum force divided by the sample area. The data are reported as the mean ± standard deviation (n = 5). 2.7. In Vivo Surgery in Rat Distal Femoral Defects. All implants (⌀ 3 × 30 mm) were sterilized by an autoclave before the surgeries. Twenty-seven male Sprague−Dawley rats (Simonsen Laboratories, Gilroy, CA) with an average body mass of 300 g were used in this study. Before the surgeries, buprenorphine was injected into the rats as the premedication for their pain relief. Anesthesia was conducted using isoflurane (Abbott Laboratories, North Chicago, IL) coupled with oxygen prior to the surgery. The rats’ legs were shaved and disinfected by alternately being rubbed with chlorohexane and 70% ethanol. Then longitudinal incisions were made in the distal femur regions. Distal femoral defects were created in the distal femurs using a drill at a slow rotation speed. The remaining bone fragments were washed away by physiological saline. Ti, HA-coated Ti, and MgO/SiO2−HA-coated Ti rods were pressed into the defects. Three rats were used for each composition at each time point. After implantation, the muscles and skin were sutured separately using MONOCRYL (poliglecaprone 25) synthetic absorbable surgical sutures (Ethicon Inc., Somerville, NJ). Then the wounds were rubbed with 5% povidone iodine disinfectant to prevent infections. Meloxicam was injected in the next 3 days after surgery as postmedication for their pain relief. After 6, 10, and 14 weeks, the rats were euthanized by overdosing CO2 in a sealed box followed by creating a bilateral pneumothorax to ensure their fatality. The bone implants were taken out for in vivo characterizations by an implant pushout test and Masson Goldner’s trichrome staining. All surgery procedures can be found in the approved protocol of the Institutional Animal Care and Use Committee (IACUC) at Washington State University. 2.8. Histomorphometric Analysis. The bone implants were fixed in 10% buffered formalin solution for 72 h after the surgeries. Then they were dehydrated in 70% ethanol, 95% ethanol, 100% ethanol, ethanol−acetone (1:1), and 100% acetone. After dehydration, the samples were embedded in Spurr’s resin. The embedded implant blocks were sectioned parallel to the circle cross-section of the implant using a low-speed diamond saw at a speed of 300 rpm. The sections were glued to glass slides followed by polishing to obtain thin sections. Finally, they were stained by Masson Goldner’s trichrome stain and observed under a light microscope. The osseointegration was characterized using the mineralized bone area within the coatings divided by the total coating area (p < 0.05, where n = 3). 2.9. Implant Pushout Test. Implant pushout tests were performed to determine the interfacial shear modulus between the tissue and implant using a universal material testing machine (Instron, Grove City, PA) in compression with a 3000 kg load cell. The maximum shear modulus was calculated from the stress−strain plots of the implant pushout test experiments. Each implant pushout result was

3. RESULTS 3.1. Phase and Chemical Identifications. In both the plasma-sprayed HA and MgO/SiO2−HA coatings, the FTIR spectra contained bands associated with the phosphate group’s symmetric P−O stretching mode ν1 (962 cm−1), antisymmetric P−O stretching modes ν3 (1030−1092 cm−1), and antisymmetric P−O bending modes ν4 (565−604 cm−1) and the O−H stretching mode of the hydroxide group (632 cm−1).21 These characteristic peaks suggest the composition of both coatings to be HA rather than other orthophosphates formed by thermal decomposition, such as β-TCP and α-TCP, which exhibit ν4 mode bands at 555 and 609 cm−1 and at 551, 563, 585, 597, and 613 cm−1.22 It was noted that the peak sharpness of MgO/ SiO2−HA was much higher than that of pure HA, as shown in Figure 1.

Figure 1. FTIR spectra of plasma-sprayed HA (black) and MgO/ SiO2−HA (red) coatings on Ti substrates.

Diffraction spectra revealed the phase composition of both coatings was nearly 100% HA, as shown in Figure 2, according to the standard HA phase (JCPDS file no. 09-0432). This was in good agreement with the FTIR spectra. Surprisingly, the crystallinity of MgO/SiO2−HA was much higher than that of pure HA. 3.2. Interface Microstructure and Adhesive Bond Strength. Plasma-sprayed HA and MgO/SiO2−HA coatings resulted in gap-free coating interfaces, as indicated by FESEM and shown in Figure 3. The thickness of both coatings was around 50 μm. Adhesive bond strengths of 25.73 ± 1.92 and 23.44 ± 2.89 MPa were achieved for HA- and MgO/SiO2−HAcoated Ti substrates, respectively. 3.3. Histomorphometric Characterization. Week 6 histological staining showed that osteoid formation was only observed around uncoated Ti implants while a wide gap existed between the osteoid and implant. For HA-coated Ti, mineralized bone (MB) formation was observed around the HA coating, and the gap between the MB and implant was much smaller compared to that for uncoated Ti. MgO/SiO2− HA-coated Ti showed a gap-free interface and a 45.63% ± 4.14% mineralization within the coating, as shown in Figures 4c and 5. At week 10, MB formation was observed around uncoated Ti, while the gap between the MB and implant, although still present, decreased in comparison to that at week C

DOI: 10.1021/acsami.7b05574 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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week 14, where the shear modulus of HA-coated Ti samples increased to 83.92 ± 3.68 MPa and the shear modulus of MgO/SiO2−HA-coated Ti samples was recorded at 149.83 ± 3.69 MPa, a significant increase and above the average values of uncoated and HA-coated Ti by 184.4% and 78.54%, respectively. In addition, two different pushout failure modes were noted for the coated and uncoated samples. Pushout tests of uncoated Ti resulted in intact bone structure near the pushout site, while coated Ti displayed considerable bone fractures as shown in Table 1.

4. DISCUSSION The primary goal of this study is to understand the effects of MgO and SiO2 on osteogenesis and osseointegration of plasmasprayed HA coating in vivo. However, to study the in vivo effects of these two additives, the prerequisite is to prepare high-quality plasma coatings. Phase purity, phase crystallinity, and adhesive bond strength are three crucial factors affecting the overall quality of plasma-sprayed coatings. In this study, the FTIR spectra shown in Figure 1 suggested an unaltered HA chemistry in both coatings as well as better crystallinity of the plasma-sprayed MgO/SiO2−HA compared to plasma-sprayed HA as indicated by sharper spectral peaks for plasma-sprayed MgO/SiO2−HA. XRD further confirmed this conclusion for both HA and MgO/SiO2−HA compositions as the spectra showed almost 100% HA phase. This can be attributed to the placement of the supersonic plasma nozzle in the lower region of the plasma torch, where the temperature is much lower than that at the center of the plasma torch. Hence, this prevents the HA decomposition into α-TCP and β-TCP during the plasmaspraying process. In addition, an analysis of the percent crystallinity using the XRD spectra gave a much higher calculated crystallinity for MgO/SiO2−HA coatings than for pure HA, as shown in Figure 2. There are two possible explanations for this phenomenon: First, Mg2+ and Si4+ substitution for Ca2+ into the HA crystal structure during the plasma-spraying process led to a more stable structure, which can decrease the nucleation energy and increase the crystallinity of the coating. Second, the temperature of the plasma zone can be above 10000 °C, where HA, MgO, and SiO2 will reach their melting points and then impinge onto the substrates to form coatings. The melting points of HA, MgO, and SiO2 are 1100, 2852, and 1713 °C, respectively.23 Therefore, the average melting temperature of MgO/SiO2−HA should be higher than that of pure HA when the particles reach the substrates. Thus, MgO/SiO2−HA should take a longer time to crystallize because of its higher temperature, which results in its higher crystallinity than that of pure HA. The adhesive bond strengths for plasma-sprayed HA and MgO/SiO2−HA are 25.73 ± 1.92 and 23.44 ± 2.89 MPa, respectively, which are much higher than the clinically required minimum of 15 MPa.10,24 In addition, the adhesive bond strength of the plasma-sprayed HA coating is not significantly affected by the presence of additives, which is also shown in other studies.25−27 In addition, gap-free coating interfaces were observed between the coatings and implants, as shown in Figure 3, confirming that the plasmasprayed coatings were strongly bonded with the substrate. After high-quality coatings were made, further study was conducted on the effects of MgO and SiO2 on the biological properties of the plasma-sprayed HA coating in vivo using the rat distal femoral model. During bone regeneration, osteoid is secreted by osteoblasts followed by mineralization to form MB. At week 6, osteoid formation was observed around uncoated Ti

Figure 2. X-ray diffraction plots of plasma-sprayed HA and MgO/ SiO2−HA coatings on Ti substrates and their crystallinities.

Figure 3. Interface microstructure of plasma-sprayed HA (a) and MgO/SiO2−HA (b) coatings on Ti substrates showing crack-free interfaces.

6. Both HA-coated Ti and MgO/SiO2−HA-coated Ti showed gap-free interfaces, but the mineralization within the MgO/ SiO2−HA coating was significantly higher than that within the HA coating, as shown in Figures 4e,f and 5. At week 14, the gap between uncoated Ti and MB continued to decrease from that at week 10, but bone approximation to the implant surface remained incomplete. Bone−implant interfaces of the undoped and doped coatings remained gap-free; however, mineralization within the MgO/SiO2−HA coating was still significantly higher than that within the HA coating, as shown in Figures 4h,I and 5. 3.4. Implant Pushout Test. The pushout test results showed that, for all samples, the tissue−implant interface shear modulus increased with respect to time. For uncoated Ti samples, the calculated shear modulus was 19.43 ± 2.50 MPa at week 6. At weeks 10 and 14, the shear modulus for uncoated Ti had continued to increase to 33.56 ± 2.50 and 52.68 ± 10.41 MPa, respectively. In contrast, week 6 pushout tests showed HA-coated Ti samples had a shear modulus of 62.34 ± 1.99 MPa. Similarly, MgO/SiO2−HA-coated Ti samples had a shear modulus of 64.14 ± 9.07 MPa. This trend continued into the week 10 results, where HA-coated and MgO/SiO2−HA-coated Ti results showed shear moduli of 70.89 ± 4.07 and 73.92 ± 3.68 MPa, respectively. A distinct difference was apparent at D

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Figure 4. From left to right, light microscope images of uncoated Ti, HA-coated Ti, and MgO/SiO2−HA-coated Ti sections stained by Masson Goldner’s trichrome stain. Images were taken at 6 weeks (a−c), 10 weeks (d−f), and 14 weeks (g−i) postimplantation. Note the good osseointegration of doped samples for all time points. Black arrows indicate coatings (light blue), white arrows indicate osteoid formation (orange), and dark blue-green areas represent mineralized bone.

Table 1. Maximum Implant Pushout Moduli of Uncoated Ti, HA-Coated Ti, and MgO/SiO2−HA-Coated Ti after Implantation in Rat Distal Femurs for 6, 10, and 14 Weeks (Left) and Two Post-Pushout Failure Modes, Intact Bone for Uncoated Ti and Fractured Bone for Coated Ti (Right)a

Figure 5. Mineralization within HA and MgO/SiO2−HA coatings after implantation in rat distal femurs for 6, 10, and 14 weeks. It is calculated using the mineralized bone area within the coatings divided by the total coating area. The middle and late time points show statistically a significant increase in bone integration for MgO/SiO2− HA (*, p < 0.05, where n = 3).

implants, which indicated that Ti was biocompatible and did not hinder new bone formation. Despite this, a large gap was observed between the uncoated Ti and osteoid since Ti was not an osteoconductive material. Compared to uncoated Ti at week 6, HA-coated Ti had a large amount of MB formation instead of osteoid with smaller gaps between the bone and implants, which indicated that plasma-sprayed HA coatings expedited the bone regeneration process and improved the osseointegration of Ti implants. Similar results were reported by Vahabzadeh et al. showing smaller gaps and more MB formation when comparing HA-coated Ti to uncoated Ti.28 In this study, the implant pushout results also confirm the better osseointegration of HA-coated Ti by showing a higher maximum implant pushout modulus compared to that of uncoated Ti, as shown in

maximum pushout moduli (MPa)

time points

compositions

6 weeks

uncoated Ti HA-coated Ti MgO/SiO2−HA-coated Ti uncoated Ti HA-coated Ti MgO/SiO2−HA-coated Ti uncoated Ti HA coated Ti MgO/SiO2−HA-coated Ti

10 weeks

14 weeks

19.43 62.34 64.14 33.56 70.89 73.92 52.68 83.92 149.83

± ± ± ± ± ± ± ± ±

2.50 1.99 9.07 2.50 4.07 3.68 10.41 3.68 3.69

The data suggest superior bioactive fixation of MgO/SiO2−HA compared to pure HA or bare Ti. *Only coated Ti at 10 and 14 weeks show fractured bone after pushout test.

a

Table 1. Interestingly, a near-gap-free interface was observed between the coating surface and host bone tissue in the MgO/ E

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ACS Applied Materials & Interfaces SiO2−HA-coated Ti samples. In addition, plasma-sprayed HA coatings are porous coatings, which can allow bone ingrowth toward the coatings. Hence, it is reasonable to observe osseointegration within HA coatings. The percentage of MB formation within the doped coating was 45.63% ± 4.14%, whereas HA-coated samples had no observable MB formation within the coating, as shown in Figures 4 and 5. It was reported that the addition of MgO could enhance the osteogenesis of tricalcium phosphate by accelerating the bone mineralization in vivo.12,29 SiO2 was reported to enhance the bone remodeling and biomineralization process.30,31 Therefore, the gap-free interface and mineralization within the MgO/SiO2−HA coating at week 6 might be the result of expedited mineralization and enhanced osteogenesis due to the presence of MgO and SiO2. At the week 10 time point, histological images of uncoated Ti implants began to show MB formation, which had an implant apposition similar to that of HA-coated and MgO/SiO2−HAcoated Ti implants at week 6. The gap between MB and the uncoated Ti decreased significantly compared to that at week 6, further elucidating that although Ti was not osteoconductive, it is still a biocompatible material, and bone mineralization should occur around uncoated Ti. In addition, the osseointegration of uncoated Ti was improved with the implantation time as indicated by the decreasing gap distance between MB and the implant. A decrease in the tissue−implant gap for uncoated Ti from 4 weeks to 10 weeks was similarly reported by Bandyopadhyay et al.32 For both HA-coated and MgO/ SiO2−HA-coated Ti, histological images indicate no gaps in the interface between the coatings and MB at 10 weeks. Notably, the calculated mineralization within the MgO/SiO2− HA coating is significantly higher than that within the HA coating, as shown in Figures 4 and 5. In a previous study, Mastrogiacomo et al. reported that Si4+ had a special coupling between scaffold resorption and new bone formation, where Si−TCP was progressively resorbed and replaced by new bone formation.33 In this study, the faster mineralization within the MgO/SiO2−HA coating might be caused by the presence of SiO2 as well. In addition, the lower contact angles (Table S1, Supporting Information), higher pushout shear modulus, and fractured bone pushout failure mode for coated Ti at week 10 onward confirm the better osseointegration of MgO/SiO2− HA-coated Ti compared to uncoated Ti and even HA-coated Ti. At week 14, there is still a noticeable gap between MB and the uncoated Ti, which indicates that the uncoated Ti is still not well integrated with the host tissue. The mineralization within the MgO/SiO2−HA coating is still significantly higher than that within the HA coating, as shown in Figures 4a−c and 5, which might still be caused by the presence of SiO2. In addition, the MgO/SiO2−HA-coated samples show a maximum implant pushout modulus of 149.83 ± 3.69 MPa, which is significantly higher than that of the HA-coated samples, 83.92 ± 3.68 MPa, and that of the uncoated samples, 52.68 ± 10.41 MPa, as shown in Table 1. This result also supports the histomorphometric data that MgO/SiO2 −HA-coated Ti has an enhanced osseointegration. Results show that the presence of MgO and SiO2 enhances the crystallinity, in vivo osseointegration, and osteogenesis of the plasma-sprayed HA coating, which is promising to the development of future load-bearing implants.

bone tissue engineering applications, a knowledge gap exists in quantifying the quality of the interface and implant fixation of this coating system on load-bearing implants. This study reports a novel plasma-sprayed MgO/SiO2−HA coating system for improving the biological properties of Ti implants using a rat distal femoral model for in vivo characterizations. XRD and adhesive bond strength tests showed that the presence of MgO and SiO2 increases the crystallinity and has no significant deleterious effects on the adhesive bond strength of the plasmasprayed HA coating. Plasma-sprayed HA coatings expedite the early stage of bone regeneration in vivo by directly showing well-approximated MB at week 6. Simultaneously, the addition of MgO and SiO2 enhances osteogenesis and osseointegration of the plasma-sprayed HA coating by showing significantly enhanced mineralization within the coating and a maximum implant pushout modulus of 149.83 ± 3.69 MPa at week 14, which is much higher compared to 83.92 ± 3.68 MPa for HAcoated Ti and 52.68 ± 10.41 MPa for bare Ti. In summary, this study successfully quantifies improved physical and biological properties of the plasma-sprayed HA coating, which is promising for use in improving load-bearing orthopedic implants.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b05574. Description of the contact angle measurement procedure and data as shown in Table S1 and additional information on the histomorphometric analysis procedure and included image, Figure S1 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: 509-335-7461. Fax: 509-335-4662. E-mail: sbose@ wsu.edu. ORCID

Samuel F. Robertson: 0000-0002-8433-7393 Funding

We acknowledge the funding from the National Institutes of Health under Grant No. 1R01AR066361. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Number of Hip Replacements Has Skyrocketed, U.S. Report Shows. Consumer HealthDay, Feb 12, 2015. https://consumer. healthday.com/senior-citizen-information-31/demographic-arthritisnews-37/number-of-hip-replacements-has-skyrocketed-u-s-reportshows-696419.html (accessed Aug 26, 2016). (2) Hip Implant Basics. https://www.hipreplacement.com/ technology/implants/basics (accessed Aug 26, 2016). (3) Lind, M.; Overgaard, S.; Bünger, C.; Søballe, K. Improved Bone Anchorage of Hydroxyapatite Coated Implants Compared with Tricalcium-Phosphate Coated Implants in Trabecular Bone in Dogs. Biomaterials 1999, 20, 803−808. (4) Dumbleton, J.; Manley, M. T. Hydroxyapatite-Coated Prostheses in Total Hip and Knee Arthroplasty. J. Bone Jt. Surg., Am. Vol. 2004, 86A, 2526−2540. (5) Sun, L.; Berndt, C. C.; Khor, K. A.; Cheang, H. N.; Gross, K. A. Surface Characteristics and Dissolution Behavior of Plasma Sprayed Hydroxyapatite Coating. J. Biomed. Mater. Res. 2002, 62, 228−236.

5. CONCLUSION Despite previous studies using SiO2 and MgO as viable dopants for increasing the biological properties of HA and β-TCP for F

DOI: 10.1021/acsami.7b05574 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces (6) Gabbar, O. A.; Rajan, R. A.; Londhe, S.; Hyde, I. D. Ten- to Twelve-Year Follow-Up of the Furlong Hydroxyapatite-Coated Femoral Stem and Threaded Acetabular Cup in Patients Younger than 65 Years. J. Arthroplasty. 2008, 23, 413−417. (7) Shah, N. N.; Edge, A. J.; Clark, D. W. Hydroxyapatite-CeramicCoated Femoral Components in Young Patients Followed-Up for 16 to 19 Years: An Update of A Previous Report. J. Bone Jt. Surg., Br. Vol. 2009, 91B, 865−869. (8) Sridhar, T. M.; Mudali, U. K. Development of Bioactive Hydroxyapatite Coatings on Type 316L Stainless Steel by Electrophoretic Deposition for Orthopaedic Applications. Trans. IIM 2003, 56, 221−230. (9) Shi, D.; Jiang, G.; Bauer, J. The Effect of Structural Characteristics on the In Vitro Bioactivity of Hydroxyapatite. J. Biomed. Mater. Res. 2002, 63, 71−78. (10) Roy, M.; Bandyopadhyay, A.; Bose, S. Induction Plasma Sprayed Nano Hydroxyapatite Coatings on Titanium for Orthopaedic and Dental Implants. Surf. Coat. Technol. 2011, 205, 2785−2792. (11) Sun, L.; Berndt, C. C.; Grey, C. P. Phase, Structural and Microstructural Investigations of Plasma Sprayed Hydroxyapatite Coatings. Mater. Sci. Eng., A 2003, 360, 70−84. (12) Bose, S.; Tarafder, S.; Banerjee, S. S.; Davies, N. M.; Bandyopadhyay, A. Understanding In Vivo Response and Mechanical Property Variation in MgO, SrO and SiO2 Doped β-TCP. Bone. 2011, 48, 1282−1290. (13) Bandyopadhyay, A.; Bernard, S.; Xue, W.; Bose, S. Calcium Phosphate-Based Resorbable Ceramics: Influence of MgO, ZnO, and SiO2 Dopants. J. Am. Ceram. Soc. 2006, 89, 2675−2688. (14) Xue, W.; Dahlquist, K.; Banerjee, A.; Bandyopadhyay, A.; Bose, S. Synthesis and Characterization of Tricalcium Phosphate with Zn and Mg Based Dopants. J. Mater. Sci.: Mater. Med. 2008, 19, 2669− 2677. (15) Landi, E.; Logroscino, G.; Proietti, L.; Tampieri, A.; Sandri, M.; Sprio, S. Biomimetic Mg-Substituted Hydroxyapatite: from Synthesis to In Vivo Behaviour. J. Mater. Sci.: Mater. Med. 2008, 19, 239−247. (16) Hing, K. A.; Revell, P. A.; Smith, N.; Buckland, T. Effect of Silicon Level on Rate, Quality and Progression of Bone Healing within Silicate-Substituted Porous Hydroxyapatite Scaffolds. Biomaterials 2006, 27, 5014−5026. (17) Patel, N.; Best, S. M.; Bonfield, W.; Gibson, I. R.; Hing, K. A.; Damien, E.; Revell, P. A. A Comparative Study on the In Vivo Behavior of Hydroxyapatite and Silicon Substituted Hydroxyapatite Granules. J. Mater. Sci.: Mater. Med. 2002, 13, 1199−1206. (18) Mladenović, Ž .; Johansson, A.; Willman, B.; Shahabi, K.; Björn, E.; Ransjö, M. Soluble Silica Inhibits Osteoclast Formation and Bone Resorption In Vitro. Acta Biomater. 2014, 10, 406−418. (19) Li, H.; Chang, J. Bioactive Silicate Materials Stimulate Angiogenesis in Fibroblast and Endothelial Cell Co-Culture System through Paracrine Effect. Acta Biomater. 2013, 9, 6981−6991. (20) ASTM C633-13: Standard Test Method for Adhesion or Cohesion Strength of Thermal Spray Coatings, 2013. http://dx.doi. org/10.1520/C0633 (accessed Jan 26, 2016). (21) Panda, R. N.; Hsieh, M. F.; Chung, R. J.; Chin, T. S. FTIR, XRD, SEM and Solid State NMR Investigations of CarbonateContaining Hydroxyapatite Nano-Particles Synthesized by HydroxideGel Technique. J. Phys. Chem. Solids 2003, 64, 193−199. (22) Malakauskaite-Petruleviciene, M.; Kareiva, A.; Beganskiene, A.; Garskaite, E.; Niaura, G.; Stankeviciute, Z. Characterization of Sol-Gel Processing of Calcium Phosphate Thin Films on Silicon Substrate by FTIR Spectroscopy. Vib. Spectrosc. 2016, 85, 16−21. (23) 1306-06-5 (Hydroxyapatite) Product Description. http://us. chemicalbook.com/ChemicalProductProperty_US_CB2131094.aspx (accessed July 13, 2016). (24) Tsui, Y. C.; Doyle, C.; Clyne, T. W. Plasma Sprayed Hydroxyapatite Coatings on Titanium Substrates. Part 1: Mechanical Properties and Residual Stress Levels. Biomaterials 1998, 19, 2015− 2029.

(25) Roy, M.; Bandyopadhyay, A.; Bose, S. Induction Plasma Sprayed Sr and Mg Doped Nano Hydroxyapatite Coatings on Ti for Bone Implant. J. Biomed. Mater. Res., Part B 2011, 99B, 258−265. (26) Roy, M.; Fielding, G. A.; Beyenal, H.; Bandyopadhyay, A.; Bose, S. Mechanical, In Vitro Antimicrobial, and Biological Properties of Plasma-Sprayed Silver-Doped Hydroxyapatite Coating. ACS Appl. Mater. Interfaces 2012, 4, 1341−1349. (27) Fielding, G. A.; Roy, M.; Bandyopadhyay, A.; Bose, S. Antibacterial and Biological Characteristics of Silver Containing and Strontium Doped Plasma Sprayed Hydroxyapatite Coatings. Acta Biomater. 2012, 8, 3144−3152. (28) Vahabzadeh, S.; Roy, M.; Bandyopadhyay, A.; Bose, S. Phase Stability and Biological Property Evaluation of Plasma Sprayed Hydroxyapatite Coatings for Orthopedic and Dental Applications. Acta Biomater. 2015, 17, 47−55. (29) Tarafder, S.; Davies, N. M.; Bandyopadhyay, A.; Bose, S. 3D Printed Tricalcium Phosphate Scaffolds: Effect of SrO and MgO Doping on In Vivo Osteogenesis in A Rat Distal Femoral Defect Model. Biomater. Sci. 2013, 1, 1250−1259. (30) Pietak, A. M.; Reid, J. W.; Stott, M. J.; Sayer, M. Silicon Substitution in the Calcium Phosphate Bioceramics. Biomaterials 2007, 28, 4023−4032. (31) Tanizawa, Y.; Suzuki, T. Effects of Silicate Ions on the Formation and Transformation of Calcium Phosphates in Neutral Aqueous Solutions. J. Chem. Soc., Faraday Trans. 1995, 91, 3499− 3503. (32) Bandyopadhyay, A.; Shivaram, A.; Tarafder, S.; Sahasrabudhe, H.; Banerjee, D.; Bose, S. In vivo Response of Laser Processed Porous Titanium Implants for Load-Bearing Implants. Ann. Biomed. Eng. 2017, 45, 249−260. (33) Mastrogiacomo, M.; Papadimitropoulos, A.; Cedola, A.; Peyrin, F.; Giannoni, P.; Pearce, S. G.; Alini, M.; Giannini, C.; Guagliardi, A.; Cancedda, R. Engineering of Bone Using Bone Marrow Stromal Cells and A Silicon-Stabilized Tricalcium Phosphate Bioceramic: Evidence for A Coupling Between Bone Formation and Scaffold Resorption. Biomaterials 2007, 28, 1376−1384.

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DOI: 10.1021/acsami.7b05574 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX