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Surface characterization and in vivo performance of plasma-sprayed hydroxyapatite-coated porous Ti6Al4V implants generated by electron beam melting
Surface characterization and in vivo performance of plasma-sprayed hydroxyapatite-coated porous Ti6Al4V implants generated by electron beam melting Hai Huang, Ping-Heng Lan, Yong-Quan Zhang, Xiao-Kang Li, Xing Zhang, Chao-Fan Yuan, Xue-Bin Zheng, Zheng Guo PII: DOI: Reference:
S0257-8972(15)30342-X doi: 10.1016/j.surfcoat.2015.10.047 SCT 20666
To appear in:
Surface & Coatings Technology
Received date: Revised date: Accepted date:
7 June 2015 21 October 2015 22 October 2015
Please cite this article as: Hai Huang, Ping-Heng Lan, Yong-Quan Zhang, Xiao-Kang Li, Xing Zhang, Chao-Fan Yuan, Xue-Bin Zheng, Zheng Guo, Surface characterization and in vivo performance of plasma-sprayed hydroxyapatite-coated porous Ti6Al4V implants generated by electron beam melting, Surface & Coatings Technology (2015), doi: 10.1016/j.surfcoat.2015.10.047
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ACCEPTED MANUSCRIPT Surface
characterization
and
in
vivo
performance
of
plasma-sprayed
hydroxyapatite-coated porous Ti6Al4V implants generated by electron beam
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melting
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Hai Huang1☆, Ping-Heng Lan1☆，Yong-Quan Zhang1, Xiao-Kang Li1, Xing Zhang2, Chao-Fan Yuan1, Xue-Bin Zheng3 ，Zheng Guo1*
1 Department of Orthopedics, Xijing Hospital, Fourth Military Medical University,
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Xi’an, Shaanxi, China, 2 Shenyang National Laboratory for Materials Science,
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Institute of Metal Research, Chinese Academy of Sciences, Shenyang, Liaoning, China, 3 Key laboratory of Inorganic Coating Materials, Chinese Academy of
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Sciences, Shanghai, China
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Funding: This project was supported by the National Natural Science Foundation of
and
the
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China(NO.51271199), National Natural Science Foundation of China(NO.81171773) National
High
Technology
Research
and
Development
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Program("863"Program) of China(NO.2015AA033702). The funder had no role in study design, data collection, decision to publish, or preparation of the manuscript. Corresponding Author: Zheng Guo Email: [email protected]: +8602984773411 ☆ These authors contributed equally to this work. Abstract Porous titanium with high strength and a low elastic modulus has received attention as an excellent orthopedic implant; however, the fabrication and biological performance of this material needs to be improved. A porous Ti6Al4V implant(TI) was prepared 1
ACCEPTED MANUSCRIPT using the electron beam melting method, and some samples underwent surface modification with a hydroxyapatite coating(HA-TI) by plasma-spraying. After
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characterization of their surfaces, the TI and HA-TI materials were implanted into
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distal femur bone defects of sheep, and bone formation and osteointegration were evaluated at 2 and 4 months post-implantation. A micro computed tomography analysis indicated that the porous Ti6Al4V implant with interconnected pores had a
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high porosity~69±5%and large pore size~514±35μm. Scanning electron microscopy,
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energy dispersive spectrometry, and X-ray diffraction analyses revealed that the HA coating was successfully deposited on the exterior surface of the implants; the
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elemental composition of the internal surface of the HA-TI material included calcium
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and phosphorus, as well as titanium and aluminum. Confocal laser scanning
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microscopy revealed that the surface roughness of the HA-TI group was significantly higher than that of the TI group. Micro computed tomography and histological
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analyses indicated that the bone formation of the HA-TI group was superior to that of the TI group at both 2 and 4 months post-implantation. In the HA-TI group, the new bone contacted the coating of the implant directly, and no fibrous tissue or gaps were observed at the bone-implant interface. This study demonstrates hydroxyapatite coating of porous Ti6Al4V implants by plasma-spraying is suitable to improve the bone formation and osteointegration capabilities. In addition, the results demonstrate that HA-coated porous Ti6Al4V implants generated by electron beam melting have excellent prospects in orthopedic applications. Key word: Porous titanium; Electron beam melting; Bone ingrowth; Hydroxyapatite; 2
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Osteointegration; Surface treatment
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ACCEPTED MANUSCRIPT Introduction Titanium and its alloys are used extensively as orthopedic and dental implants
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due to their high resistance to corrosion, excellent biocompatibility, high
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strength-to-weight ratio, and low elastic modulus. However, a number of studies reported that the biomechanical mismatch between the elastic moduli of metallic implants and the surrounding bone tissue may lead to stress-shielding phenomena,
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which induces an inappropriate stress distribution at the bone-implant interface
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resulting in bone resorption, implant loosening and failure of prosthesis fixation[1-3]. To overcome these drawbacks, porous titanium structures with reduced elastic moduli
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are currently being developed; these materials avoid the generation of stress-shielding
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implants [4,5].
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effects and are favorable for new bone ingrowth, which increases fixation of the
Porous titanium can be manufactured using many different techniques, such as
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the powder sintering, solid-state foaming, and polymeric foam replication methods [6,7]. However, these traditional fabrication methods have a number of limitations regarding the control of the internal pore structure and the outer shape of the implants. Several studies demonstrated that pore structure, connectivity, and diameter affect the mechanical properties and biological performance of porous implants [8-10].In addition, their regular internal structures of porous implants fabricated by conventional methods hinder stress transfer and lead to implant fractures caused by local stress concentration[11]. In addition, the low porosity and poor connectivity of these porous titanium materials can disrupt body fluid circulation and thereby affect 4
ACCEPTED MANUSCRIPT new bone ingrowth. It is difficult to control the structures and parameters of porous titanium alloy
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sprepared using traditional methods; hence, the mechanical properties and biological
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performance of the materials can fail to meet the implant requirements. Recently, advanced rapid prototyping technologies, such as electron beam melting (EBM), became a better choice for the preparation of porous metal implants. EBM is capable
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of manufacturing porous titanium implants with controlled pore shapes, sizes, and
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distributions. The porous metal implants fabricated by EBM not only decrease stress-shielding effect but also allow the tissue ingrowth, thus improve long-term
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stability of implants. Using this technique, Parthasarathyet al.[12]created porous
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titanium with fully connected internal pores and a porosity of 49–70%, and Murret
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al.[13,14] fabricated porous titanium alloys with a porosity of 60–80% and an elastic modulus of the material
was only 3–6 GPa, which is close to that of cortical bone.
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In addition to the internal structure, the surface physicochemical properties of biological implants play an important role in the bone ingrowth process. Because titanium is a bioinert material, titanium alloy implants are often surrounded by fiber tissue rather than newly formed bone, which can disrupt osteointegration[15,16].To improve bone ingrowth and osteointegration, various surface modification methods have been used to enhance the bioactivity of titanium alloy implants [17].HA can adhere to soft and osseous tissue directly without an intermediate layer. Plasma spray is by far most-established commercial technique for HA coating, owing to the thick deposit, high deposition rate and low operation cost. HA coating has been widely used 5
ACCEPTED MANUSCRIPT for clinical implants and prosthesis, which is able to bind to bone directly and promotes bone ingrowth and osteointegration[18].Due to the complex internal
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structure of porous implants, a blind territory can be generated inside implants for HA
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coating using plasma-spraying. However, few studies reported HA coating for surface modification of EBM-generated porous titanium alloy implants with high porosity and connectivity using a plasma-spraying method.
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The purpose of this study was to evaluate the internal coating and in vivo
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biological performance of porous uncoated or HA-coated titanium alloy(Ti6Al4V) implants with well-controlled porous structures generated by EBM. The physical
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properties of porous implants with or without the HA coating were analyzed by
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scanning electron microscopy (SEM),micro computed tomography(micro-CT), energy
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dispersive spectrometry (EDS), confocal laser scanning microscopy (CLSM), and X-ray diffraction (XRD) analyses. The coated and uncoated samples were implanted
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into cylindrical bone defects in the bilateral distal femur of sheep, and bone ingrowth and osteointegration were evaluated by X-ray, micro-CT, and histological analyses.
Materials and Methods
Fabrication of porous Ti6Al4V implants Cylindrical porous implants with a length of 20mm and a diameterof10mm were fabricated using EBM (EBM S12;Arcam AB, Mölndal, Sweden). Three-dimensional (3D) models of the samples were generated using computer-aided design 6
ACCEPTED MANUSCRIPT software(Materialise/MagicsTM).To provide layer information, the 3D models were sliced into layers with a thickness of 100μm.Then,thedata were imported into the
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EBM device, and the implants were produced. The implants with and without the HA
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coating had similar structures; however, the HA-coated implants possessed a solid end (length =8mm)that enabled them to be clamped duringthe plasma-spraying process.
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Surface modification
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So far, atmospherical plasma spray(APS) was the conventional method for HA coating. However, Zheng et al.[19] reported that higher crystallinity has been obtained
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while the HA coating was deposited by VPS as compared with APS and the in vitro
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tests show that both APS and VPS HA coating posses good bioactivity.
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In this study, VPS method was applied for HA coating preparation. Prior to plasma-spraying, the substrates were ultrasonically cleaned in acetone and then grit
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blasted using corundum sand (particle size 250-300μm). A vacuum plasma-spray （VPS） system (F4-VB;Sulzer-Metco, Winterthur, Switzerland) was used to apply the HA coating, using the modified spray parameters listed in Table 1. Commercially available HA powder (AlfaAesar, Ward Hill, MA) was used as feedstock. The thickness of the HA coating was approximately 150μm. Characterization To determine the mean pore sizes and porosities, the porous implants were examined by SEM(SSX-550; Shimadzu, Kyoto, Japan) and micro-CT(Inveon MM Gantry; Siemens, Munich, Germany).The surface topographies of the samples with or 7
ACCEPTED MANUSCRIPT without coatings were observed by CLSM(LEXT OLS4000; Olympus, Tokyo, Japan),and the average roughness values(Ra)were determined.To confirm the coating
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composition, an XRD measurement was conducted to analyze the surface elemental
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compositions of the two types of implants. In addition, to investigate the internal coating of the implants, a sample was cut from the middle of the transverse axis, and
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the EDS analysis was performed.
The
uncoated
and
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Implantation assay coated
materials
were
implanted
into
six
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20–30-month-oldsmall-tail Han sheep weighing 35–55kg. To ensure their good health,
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all of the sheep underwent a general physical examination and were quarantined for
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approximately 2 weeks prior to the implantation operation. The study protocol was approved by the Fourth Military Medical University Committee on Animal
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Care(Permit Number:14015). The sheep were housed at the Department of Orthopedics of Xijing Hospital Animal Resources Center in two shacks with an average acreage of 18 m2. All animal experiments were conducted in accordance with the Guidelines for the Use of Laboratory Animals of the National Institutes of Health. All surgical procedures were performed under sterile conditions. The sheep underwent food and water restriction for 24 h prior to surgery, and were then placed under general anesthesia and fixed on the operation table. The distal femur areas of the two hind limbs were shaved and disinfected with iodine solution. A straight skin incision was made at the lateral femoral condyle, and the distal lateral aspect of the femur was 8
ACCEPTED MANUSCRIPT exposed. A cylindrical bone defect(20 mm in depth and 10 mm in diameter) was created at the lateral femoral condyle. The defect was washed with hydrogen peroxide and
then
the
porous
uncoated
Ti6Al4V
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saline,
implants(TI)
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and
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andHA-coatedTi6Al4V implants(HA-TI) were randomly placed into the right or left hind limb bone defects. Finally, the wounds were washed and closed in layers. After the operation, each sheep received intramuscular injections of an antibiotic for 3days
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to prevent infection. At 2 or 4 months post-implantation, the sheep were euthanized
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by an intravenous overdose of pentobarbital sodium, and the specimens were
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collected.
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Imaging
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X-ray imaging was performed immediately after the implantation procedure. After sacrifice of the animals 2 or 4 months later, the collected specimens were
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immersed in 10% formalin, and further X-ray images were obtained. To quantify new bone ingrowth into the porous implants, the collected specimens were scanned by micro-CT(Explore Locus SP Micro-CT;GE Healthcare, Wauwatosa, WI) with arotationor360°, a resolution of 14μm, a voltage of 80kV, and a current of 80μA.The 3D structure inside the defect was rebuilt using Explore Reconstruction UtilityTMsoftware (GE Healthcare),and the amount of newly formed bone in the porous implants was measured.
Histological analysis 9
ACCEPTED MANUSCRIPT The specimens were fixed in 10% formalin for 1week, and then dehydrated in graded concentrations of ethanol and embedded in methyl methacrylate. After
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polymerization, the samples were cut into sections with a thickness of approximately
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250μm using a histotome, and these sections were sanded to 80μm thick. After polishing and cleaning, the sections were stained with 1.2% trinitrophenol and 1% acid fuchsin(VanGieson staining), and observed under a light microscope(Leica
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DMLA, Bensheim, Germany). To generate an overall image, regional micrographs at
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low magnification were joined together using Photoshop6.0 software (Adobe,San Jose, CA). The new bone volume in the defect was measured, and the percentage of bone
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volume in the total available pore space was calculated using Image-Pro Plus
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Statistics
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implant was evaluated.
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6.0(Media Cybernetics,Rockville, MD). In addition, the integration of bone into the
One-way analysis of variance (ANOVA) was performed using SPSS 19.0 software (IBM, Armonk,NY). Data are represented as the mean ±standard deviation. p<0.05 was considered statistically significant.
Results
Characterization Figure 1A shows the appearance of the porous Ti6Al4V implants fabricated by 10
ACCEPTED MANUSCRIPT EBM. The pore structure was examined by SEM, and micro-CT reconstructed 3D images revealed that the samples displayed full connectivity(Figure 1B). The
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structural properties of the implants are listed in Table 2.The SEM analyses revealed
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that, unlike the uncoated implant(TI),the HA-coated implant (HA-TI) had a granular substance layer distributed on its curved face(Figure 2). Surface morphology 3D images were generated by CLSM and used to determine the roughness (Ra values)of
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the two types of implant (Figure 3). As shown in Figure 4, the average Ra value of the
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HA-TI group (8.45±0.80μm) was significantly higher than that of the TI group(4.95±1.26μm).Next, the surface elemental compositions of the TI and HA-TI
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materials were examined by EDS. Titanium, vanadium and aluminum were present on
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the surface of the TI material, whereas calcium, oxygen and phosphorus were the
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main elements on the surface of the HA-TI material (Figure 5).An XRD analysis confirmed that the phase layer on the HA-coated implant surface was
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hydroxyapatite(Figure 6).
To evaluate the internal coating of the implants, samples were cut from the middle of the transverse axis, and EDS was performed. Both coating elements (calcium, oxygen and phosphorus) and alloy elements (titanium, vanadium and aluminum) were detected on the internal surface of the HA-TI material (Figure 7B), indicating that the HA coating was deposited on the internal surface and the vacuum plasma-spray method is suitable for modification of porous implant surface. Imaging X-ray images were taken immediately after implantation and 2 or 4 months 11
ACCEPTED MANUSCRIPT post-implantation. The images taken after the surgical procedure revealed that all of the samples were implanted in the correct location (Figures 8A and 8B).The X-ray
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images collected at 2 months post-implantation showed that a widespread low-density
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shadow surrounded the implants (Figures 8C and 8D);however, at 4 months post-implantation, this area of low-density shadow had reduced (Figures 8E and 8F).Micro-CT analyses performed at 2 and 4 months post-implantation confirmed
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these findings (Figure 9). A quantitative analysis revealed that the bone volume
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fractions of the HA-TI and TI groups were higher at 4 months post-implantation than 2 months post-implantation, and the bone volume fraction of the HA-TI group was
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significantly higher (p=0.018 for 2 months, p=0.008 for 4 months)than that of the TI
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group at both time points (Figure 10).In addition, at 2 months post-implantation, the
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new bone tissue formed mainly around rather than inside the implants. By contrast, at 4 months post-implantation, the new bone tissue in the HA-TI group was found not
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only at the edge of the implants, but also in the internal region, whereas osteosis in the TI group was still concentrated at the margin of the implants. Histological analyses Observations revealed no signs of inflammation or foreign body reactions in the samples collected from the implanted sheep. Low magnification imaging revealed a similar pattern of healing in the TI and HA-TI groups; specifically, new bone tissue generated from the margin of the implant migrated into the internal region of the defect by implant attachment and creeping growth. At 2 months post-implantation, a small amount of newly generated immature woven bone was present in the rims of the 12
ACCEPTED MANUSCRIPT implants, but new bone formation was absent from the core regions of both types of implant(Figures 11A and 11B). At 4 months post-implantation, the amount of new
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bone tissue increased in the HA-TI group, almost half of the pores were filled with
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bone tissue, and osteosis was detected in the core region of the implant; however, at the same time point, new bone formation barely increased in the TI group, and the internal area of the implants was occupied by fibrous tissue rather than new bone
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(Figures 11C and 11D).Histometric measurements revealed similar results; at both2
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(p=0.03)and 4 (p<0.01)months post-implantation, the mineralized bone fraction of the HA-TI group was significantly higher than that of the TI group (Figure 12).The
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HA-TI group displayed excellent osteogenesis performance; the newly formed
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immature bone attached to the HA coating of the implant tightly, and a large number
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of osteoblasts were distributed linearly on the other side (Figure 13), indicating a huge potential for bone growth in the HA-TI group.
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To evaluate the osteointegration of both types of implants, the bone-implant interfaces were examined at high magnification(Figure 14).Gaps and fibrous tissue were located between the bone and the TI surface at 2 and 4 months post-implantation. By contrast, the new bone was closely connected to the HA coating on the HA-TI surface at the same time points, indicating that the HA-TI material promoted better osteointegration than the TI material.
Discussion The usage of porous titanium alloy implants with 3D pore structures and low 13
ACCEPTED MANUSCRIPT elastic moduli reduce stress shielding and improve both bone ingrowth and fixation of the implant. Previous research indicated that implants with high porosity and
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connectivity promote better gas and nutrient exchange between the interior and
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exterior regions of the implant[20,21], which can promote cell migration and tissue growth into the implant. In addition, pore size is important for bone ingrowth. Frosh et al.[22] compared the growth behaviors of osteoblasts in porous implants with pore
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sizes of 300,400,500,600, and 1000μm; the 600μm implant displayed the best
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migration and proliferation performance, and the implants with pore sizes of 400–600μm displayed a higher proportion of mineralized tissue. Unfortunately,
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conventional manufacturing methods are unable to guarantee the production of
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implants with the desired pore architecture; however, the advantage of EBM is that it
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enables accurate control of a sample’s structural parameters based on 3D model creation and additive manufacturing. Based on these findings, implants with pore
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diameters of approximately 500μm and high porosity and connectivity were designed and fabricated by EBM. A micro-CT analysis confirmed that the architectural parameters of the samples(pore size:514±35μm;porosity: 69±5%; interconnected in all dimensions) met the design requirements. Although porous titanium alloy implants enable surrounding bone ingrowth, new bone formation and osteointegration still have shortcomings because titanium is a bioinert material. Branemark[23]was the first to describe osteointegration as “contact established between normal and remodeled bone and an implant surface without the interposition of non-bone or connective tissue, at the light microscopic level”. 14
ACCEPTED MANUSCRIPT However, in most cases, fibrous tissue and gaps occur between the titanium implant and the bone tissue[24,25], and these defects can lead to a reduction in bone ingrowth
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or implant fixation weakness. HA has great biocompatibility and outstanding
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osteoconduction; hence, it has been used as an artificial bone graft material for decades. Ripamontiet al.[26] fabricated a plasma-sprayed HA-coated Ti6Al4V implant with 36 concavities measuring 1600μm in diameter and 800μm in depth, and
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implanted the material into the rectus abdominis muscle of Chacma baboons. After 31
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months, the geometrically-constructed plasma-sprayed titanium implants exhibited osteoinduction, which could initiate bone differentiation. Based on this finding, HA
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has been widely used as a coating on orthopedic implants to facilitate bone repair and
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improve implant-bone interface responses. Other bioactive materials, such as
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bioglasses, have also shown good biocompatibility and osteoconductivity. However, the weak interfacial bonding between titanium alloy and bioglass, as well as the fast
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degradation in vivo, limits usage of these bioglasses as implant coatings[27]. Here, the porous Ti6Al4V implant was surface modified with HA by plasma-spraying to improve its bioactivity.SEM and EDS analyses revealed that a uniform layer of the HA coating was deposited on the exterior surface of the implant; however, a scattered coating was observed on the internal surface, and some interior regions were not covered with HA. In addition, a CLSM analysis revealed that the roughness of the surface of the implant increased after HA coating. Several previous studies demonstrated that rough surface facilitate osteoblast proliferation and bone formation more efficiently than smooth surfaces [28,29]. 15
ACCEPTED MANUSCRIPT In the in vivo experiment, the micro-CT analysis revealed that bone formation in the HA-TI group was superior to that in the TI group at 2 and 4 months
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post-implantation. Reconstructed 3D images showed that the newly formed bone was
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distributed in the peripheral region of the TI material at both time points; however, the new bone tissue penetrated into the core area of the HA-TI material. These imaging results were also confirmed by histological analyses. Low magnification images
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showed that, although no bone tissue located in the core region of either type of
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implant at 2 months post-implantation, the core region of the HA-coated implant was colonized by newly formed immature woven bone at 4 months post-implantation. At
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the same time point, the interior of the uncoated implant was filled with fibrous tissue,
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which may impede new bone ingrowth. The implant-bone interfaces were also
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observed under high magnification. In the TI group, newly generated bone did not make direct contact with the uncoated implant, and fibrous tissue and gaps were
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identified between the implant and bone. By contrast, immature bone was associated tightly with the HA coating of the HA-TI material “without the interposition of non-bone or connective tissue”, as defined by Branemark[23].Based on these findings, we concluded that the HA-coated implant exhibited better osteointegration and bone ingrowth than the uncoated Ti6Al4V implant. A unique phenomenon was identified by high magnification imaging of the HA-TI group; specifically, numerous osteoblasts were linearly distributed along the exterior surface of the immature bone, which is favorable to new bone formation. This behavior of osteoblasts is a possible explanation for the results of the quantitative 16
ACCEPTED MANUSCRIPT analyses based on micro-CT and histometric measurements; these analyses revealed that the amount of new bone generated in the HA-coated implant was significantly
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higher than the amount generated in the uncoated implant at both 2 and 4 months
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post-implantation, and that the bone formation rate increased more rapidly in the HA-TI group than the TI group.
Recently, several researchers examined the in vitro and in vivo bioactivities of
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porous titanium implants with different biomimetic coatings[30,31];however, few
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details of the coatings on the internal surfaces were reported. Li et al.[32] evaluated Ti6Al4V implants with and without a biomimetic apatite coating and found that the
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rates of bone ingrowth and bone formation were similar in the two groups in vivo.
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Here, the internal surface of the HA-coated implant was examined by cutting a sample
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from the middle of the transverse axis. Although the internal surface of the Ti6Al4V implant was not completely coated with HA, it still had higher osteogenic and
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osteointegration capabilities than the uncoated implant. This finding indicates that the HA coatingimproves the bone formation, bone ingrowth, and bone-implant integration abilities of Ti6Al4V implants. Based on the results described above, we observed that new bone tissue grew inside of HA coated porous implant and helped anchor the implant in the implantation site. It was suggested that such “anchoring effect” could improve the fixation strength of the implant, which may reduce this risk of surface coating delamination. The mechanical strength and possibility of delamination of this HA coating will be further investigated in future work. 17
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Conclusion
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Here, a porous Ti6Al4V implant with accurately controlled pore structures and
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architectural parameters(porosity ~69±5%and pore size ~514±35μm)was generated using the EBM technique. Surface characterization demonstrated that the HA coating by plasma spraying was successfully deposited on the exterior surface of the implant;
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however, the coating on the internal surface was distributed non-uniformly.
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Histometric measurements revealed that the mineralized bone fraction of the HA-TI group was significantly higher than that of the TI group at both 2 and 4 months
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post-implantation. The HA-TI group displayed excellent osteogenesis performance;
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the newly formed immature bone attached to the HA coating of the implant tightly,
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and a large number of osteoblasts were distributed linearly on the other side. These results indicated the HA-coated implant exhibited remarkable bone ingrowth and
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osteointegration capabilities in vivo. To further evaluate the bonding strength of the bone-implant interface, the biomechanics testing should be conducted in future work. Thus, our results suggest that HA coating by plasma-spraying is likely suitable for biological modification of porous Ti6Al4V implants, which has a great potential for clinical applications.
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[1]. Gefen A (2002) Computational simulations of stress shielding and bone resorption around existing and computer-designed orthopaedic screws. Medical and Biological Engineering and Computing 40: 311-322. [2]. Engh Jr CA, Young AM, Engh Sr CA, Hopper Jr RH (2003) Clinical consequences of stress shielding after porous-coated total hip arthroplasty. Clinical orthopaedics and related research 417: 157-163. [3]. Oh I-H, Nomura N, Masahashi N, Hanada S (2003) Mechanical properties of porous titanium compacts prepared by powder sintering. Scripta Materialia 49: 1197-1202. [4]. Kujala S, Ryhänen J, Danilov A, Tuukkanen J (2003) Effect of porosity on the osteointegration and bone ingrowth of a weight-bearing nickel–titanium bone graft substitute. Biomaterials 24: 4691-4697. [5]. Ryan G, Pandit A, Apatsidis DP (2006) Fabrication methods of porous metals for use in orthopaedic applications. Biomaterials 27: 2651-2670. [6]. Singh R, Lee P, Dashwood R, Lindley T (2010) Titanium foams for biomedical applications: a review. Materials Science and Technology 25: 127-136. [7]. Dunand DC (2004) Processing of titanium foams. Advanced Engineering Materials 6: 369-376. [8]. Habibovic P, Yuan H, van der Valk CM, Meijer G, van Blitterswijk CA, et al. (2005) 3D microenvironment as essential element for osteoinduction by biomaterials. Biomaterials 26: 3565-3575. [9]. Otsuki B, Takemoto M, Fujibayashi S, Neo M, Kokubo T, et al. (2006) Pore throat size and connectivity determine bone and tissue ingrowth into porous implants: three-dimensional micro-CT based structural analyses of porous bioactive titanium implants. Biomaterials 27: 5892-5900. [10]. Mastrogiacomo M, Scaglione S, Martinetti R, Dolcini L, Beltrame F, et al. (2006) Role of scaffold internal structure on in vivo bone formation in macroporous calcium phosphate bioceramics. Biomaterials 27: 3230-3237. [11]. Liu Y, Lim J, Teoh S-H (2013) Review: Development of clinically relevant scaffolds for vascularised bone tissue engineering. Biotechnology advances 31: 688-705. [12]. Parthasarathy J, Starly B, Raman S, Christensen A (2010) Mechanical evaluation of porous titanium (Ti6Al4V) structures with electron beam melting (EBM). Journal of the mechanical behavior of biomedical materials 3: 249-259. [13]. Murr L, Gaytan S, Medina F, Lopez H, Martinez E, et al. (2010) Next-generation biomedical implants using additive manufacturing of complex, cellular and functional mesh arrays. Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences 368: 1999-2032. [14]. Murr L, Amato K, Li S, Tian Y, Cheng X, et al. (2011) Microstructure and mechanical properties of open-cellular biomaterials prototypes for total knee replacement implants fabricated by electron beam melting. Journal of the mechanical behavior of biomedical materials 4: 1396-1411. 19
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[15]. Heinl P, Müller L, Körner C, Singer RF, Müller FA (2008) Cellular Ti–6Al–4V structures with interconnected macro porosity for bone implants fabricated by selective electron beam melting. Acta biomaterialia 4: 1536-1544. [16]. Göransson A, Jansson E, Tengvall P, Wennerberg A (2003) Bone formation after 4 weeks around blood-plasma-modified titanium implants with varying surface topographies: an in vivo study. Biomaterials 24: 197-205. [17]. Liu X, Chu PK, Ding C (2004) Surface modification of titanium, titanium alloys, and related materials for biomedical applications. Materials Science and Engineering: R: Reports 47: 49-121. [18]. Sun L, Berndt CC, Gross KA, Kucuk A (2001) Material fundamentals and clinical performance of plasma‐sprayed hydroxyapatite coatings: A review. Journal of biomedical materials research 58: 570-592. [19]. Zheng XB, Ji H, Huang JQ, Ding CX (2005) PLASMA SPRAYED Ti AND HA COATINGS： A COMPARATIVE STUDY BETWEEN APS AND VPS. Acta metallurgica sinica(English letters)18: 339-344. [20]. Karageorgiou V, Kaplan D (2005) Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials 26: 5474-5491. [21]. Hutmacher DW (2000) Scaffolds in tissue engineering bone and cartilage. Biomaterials 21: 2529-2543. [22]. Frosch KH, Barvencik F, Viereck V, Lohmann CH, Dresing K, et al. (2004) Growth behavior, matrix production, and gene expression of human osteoblasts in defined cylindrical titanium channels. Journal of Biomedical Materials Research Part A 68: 325-334. [23]. Branemark P-I (1983) Osseointegration and its experimental background. The Journal of prosthetic dentistry 50: 399-410. [24]. Ponader S, Von Wilmowsky C, Widenmayer M, Lutz R, Heinl P, et al. (2010) In vivo performance of selective electron beam‐melted Ti‐6Al‐4V structures. Journal of biomedical materials research Part A 92: 56-62. [25]. Søballe K, Hansen ES, B‐Rasmussen H, Jørgensen PH, Bünger C (1992) Tissue ingrowth into titanium and hydroxyapatite‐coated implants during stable and unstable mechanical conditions. Journal of Orthopaedic Research 10: 285-299. [26]. Ripamonti U, Roden LC, Renton LF (2012) Osteoinductive hydroxyapatite-coated titanium implants. Biomaterials 33: 3813-3823. [27]. Wang G, Lu Z, Liu X, Zhou X, Ding C, et al. (2011) Nanostructured glass-ceramic coatings for orthopaedic applications. J R Soc Interface 8: 1192-1203. [28]. Rosales-Leal J, Rodríguez-Valverde M, Mazzaglia G, Ramon-Torregrosa P, Diaz-Rodriguez L, et al. (2010) Effect of roughness, wettability and morphology of engineered titanium surfaces on osteoblast-like cell adhesion. Colloids and surfaces A: Physicochemical and Engineering aspects 365: 222-229. [29]. Martin J, Schwartz Z, Hummert T, Schraub D, Simpson J, et al. (1995) Effect of titanium surface roughness on proliferation, differentiation, and protein synthesis of human osteoblast‐like cells (MG63). Journal of biomedical 20
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materials research 29: 389-401. [30]. Takemoto M, Fujibayashi S, Neo M, Suzuki J, Kokubo T, et al. (2005) Mechanical properties and osteoconductivity of porous bioactive titanium. Biomaterials 26: 6014-6023. [31]. Fujibayashi S, Takemoto M, Neo M, Matsushita T, Kokubo T, et al. (2011) A novel synthetic material for spinal fusion: a prospective clinical trial of porous bioactive titanium metal for lumbar interbody fusion. European spine journal 20: 1486-1495. [32]. Li X, Feng Y-F, Wang C-T, Li G-C, Lei W, et al. (2012) Evaluation of biological properties of electron beam melted Ti6Al4V implant with biomimetic coating in vitro and in vivo. PloS one 7: e52049.
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Figure Legends
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Figure 1.Appearance and pore structure of the porous Ti6Al4V implants. (A) Overall appearance of the porous Ti6Al4V implant fabricated using the EBM
Figure
of
images
the
surface
morphologies
of
the
porous
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2.SEM
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interconnected pore structure.
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method.(B) Micro-CT reconstructed 3D image of the porous implant showing the
Ti6Al4Vimplants. (A) The TI group. (B) The HA-TI group. The red arrows indicate
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that a granular layer was distributed on the surface of the HA-TI but not the TI. Figure 3.Reconstructed 3D topographic image of the porous Ti6Al4V implants. The images were used to determine the Ra values for the TI and HA-TI groups. Figure 4. Surface roughness of the implants. The mean Ra values were determined based on the images shown in Figure 3 (*p <0.05). Figure 5.EDS analyses of the elemental compositions of the exterior surfaces of the porous implants. The surface elements of the TI group comprised metallic elements(titanium, vanadium and aluminum), whereas those of the HA-TI group comprised coating elements(calcium, oxygen and phosphorus). 21
ACCEPTED MANUSCRIPT Figure 6.XRDanalysis of the coated porous implant. The coating phase was identified as hydroxyapatite.
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Figure 7.Internal surface elemental composition of the HA-TI.(A) The overall
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appearance of a section of the HA-TI material cut from the middle of the transverse axis. (B) EDS analysis of the elemental composition of the interior of the coated implants. Both metallic and coating elements were detected on the internal surface of
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the HA-TI.
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Figure 8.Radiographs taken after implantation.(A) Lateral radiograph taken
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immediately after implantation. (B) Anteroposterior radiograph taken immediately
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after implantation.(C) The TI group at 2 months post-implantation. (D) The HA-TI group at 2 months post-implantation. (E) The TI group at 4 months post-implantation.
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(F) The HA-TI group at 4 months post-implantation.
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Figure 9.Reconstructed 3D images of the implants. New bone (khaki) formation around the implant (gray) in the TI group at 2 months post-implantation (A), the HA-TI group at 2 months post-implantation (B),the TI group at 4 months post-implantation (C),and the HA-TI group at 4 months post-implantation (D).
Figure 10.Bone volume fractions of the TI and HA-TI groups at 2 and 4 months post-implantation.*p < 0.05.
Figure 11.Histological observations of the TI and HA-TI groups at low 22
ACCEPTED MANUSCRIPT magnification(Van Gieson stain,16×).The distribution of new bone (red) and fibrous tissue (dark blue) in the TI group at 2 months post-implantation (A),the HA-TI group
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the HA-TI group at 4 months post-operation (D).
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months post-implantation. *p<0.05.
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Figure 12. Mineralized bone fractions of the TI and HA-TI groups at 2 and 4
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Figure 13. Osteoblast behavior in the HA-coated implant(Van Gieson stain,200×).
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The greenarrowindicates severalosteoblasts adhered tothe new bone, which contains a
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lot of osteocytes. The yellow arrow indicates the HA coating between the metallic
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matrix and the newly formed bone tissue.
Figure 14.Osteointegration of the porous implant(Van Gieson stain, 400×).(A)
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The TI group. The green arrow indicates the fibrous tissue locatedbetween the implant and bone tissue.(B) The HA-TI group. The yellow arrow indicatesthe HA coating connecting the implant to the new bone.
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ACCEPTED MANUSCRIPT Table 1.Spray parameters. Value
Argon plasma gas flow rate (SLPM)
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Parameter
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Hydrogen plasma gas flow rate (SLPM) Spray distance (mm) Argon powder carrier gas (SLPM)
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Current (A)
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Voltage (V) Powder feed rate (g/min)
100 3.5 650 68 25 400
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Pressure (mbar)
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SLPM, standard liters per minute.
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ACCEPTED MANUSCRIPT Table 2.Structural properties of theporous Ti6Al4V implant fabricated by EBM. Pore size(μm)
Trabecular diameter(μm)
Porosity(%)
Φ10×20
514±35
238±23
69±5
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ACCEPTED MANUSCRIPT Highlights The porous Ti6Al4V implant was prepared using the electron beam melting method.
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The HA coating deposited on the surface of the porous implants by plasma-spraying
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The porous implants were implanted into distal femur bone defects of sheep.
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Plasma-spraying HA improved bone formation and osteointegration of porous implants.
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S0257-8972(15)30342-X doi: 10.1016/j.surfcoat.2015.10.047 SCT 20666
To appear in:
Surface & Coatings Technology
Received date: Revised date: Accepted date:
7 June 2015 21 October 2015 22 October 2015
Please cite this article as: Hai Huang, Ping-Heng Lan, Yong-Quan Zhang, Xiao-Kang Li, Xing Zhang, Chao-Fan Yuan, Xue-Bin Zheng, Zheng Guo, Surface characterization and in vivo performance of plasma-sprayed hydroxyapatite-coated porous Ti6Al4V implants generated by electron beam melting, Surface & Coatings Technology (2015), doi: 10.1016/j.surfcoat.2015.10.047
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Surface
characterization
and
in
vivo
performance
of
plasma-sprayed
hydroxyapatite-coated porous Ti6Al4V implants generated by electron beam
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melting
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Hai Huang1☆, Ping-Heng Lan1☆，Yong-Quan Zhang1, Xiao-Kang Li1, Xing Zhang2, Chao-Fan Yuan1, Xue-Bin Zheng3 ，Zheng Guo1*
1 Department of Orthopedics, Xijing Hospital, Fourth Military Medical University,
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Xi’an, Shaanxi, China, 2 Shenyang National Laboratory for Materials Science,
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Institute of Metal Research, Chinese Academy of Sciences, Shenyang, Liaoning, China, 3 Key laboratory of Inorganic Coating Materials, Chinese Academy of
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Sciences, Shanghai, China
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Funding: This project was supported by the National Natural Science Foundation of
and
the
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China(NO.51271199), National Natural Science Foundation of China(NO.81171773) National
High
Technology
Research
and
Development
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Program("863"Program) of China(NO.2015AA033702). The funder had no role in study design, data collection, decision to publish, or preparation of the manuscript. Corresponding Author: Zheng Guo Email: [email protected]: +8602984773411 ☆ These authors contributed equally to this work. Abstract Porous titanium with high strength and a low elastic modulus has received attention as an excellent orthopedic implant; however, the fabrication and biological performance of this material needs to be improved. A porous Ti6Al4V implant(TI) was prepared 1
ACCEPTED MANUSCRIPT using the electron beam melting method, and some samples underwent surface modification with a hydroxyapatite coating(HA-TI) by plasma-spraying. After
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characterization of their surfaces, the TI and HA-TI materials were implanted into
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distal femur bone defects of sheep, and bone formation and osteointegration were evaluated at 2 and 4 months post-implantation. A micro computed tomography analysis indicated that the porous Ti6Al4V implant with interconnected pores had a
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high porosity~69±5%and large pore size~514±35μm. Scanning electron microscopy,
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energy dispersive spectrometry, and X-ray diffraction analyses revealed that the HA coating was successfully deposited on the exterior surface of the implants; the
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elemental composition of the internal surface of the HA-TI material included calcium
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and phosphorus, as well as titanium and aluminum. Confocal laser scanning
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microscopy revealed that the surface roughness of the HA-TI group was significantly higher than that of the TI group. Micro computed tomography and histological
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analyses indicated that the bone formation of the HA-TI group was superior to that of the TI group at both 2 and 4 months post-implantation. In the HA-TI group, the new bone contacted the coating of the implant directly, and no fibrous tissue or gaps were observed at the bone-implant interface. This study demonstrates hydroxyapatite coating of porous Ti6Al4V implants by plasma-spraying is suitable to improve the bone formation and osteointegration capabilities. In addition, the results demonstrate that HA-coated porous Ti6Al4V implants generated by electron beam melting have excellent prospects in orthopedic applications. Key word: Porous titanium; Electron beam melting; Bone ingrowth; Hydroxyapatite; 2
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Osteointegration; Surface treatment
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ACCEPTED MANUSCRIPT Introduction Titanium and its alloys are used extensively as orthopedic and dental implants
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due to their high resistance to corrosion, excellent biocompatibility, high
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strength-to-weight ratio, and low elastic modulus. However, a number of studies reported that the biomechanical mismatch between the elastic moduli of metallic implants and the surrounding bone tissue may lead to stress-shielding phenomena,
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which induces an inappropriate stress distribution at the bone-implant interface
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resulting in bone resorption, implant loosening and failure of prosthesis fixation[1-3]. To overcome these drawbacks, porous titanium structures with reduced elastic moduli
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are currently being developed; these materials avoid the generation of stress-shielding
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implants [4,5].
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effects and are favorable for new bone ingrowth, which increases fixation of the
Porous titanium can be manufactured using many different techniques, such as
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the powder sintering, solid-state foaming, and polymeric foam replication methods [6,7]. However, these traditional fabrication methods have a number of limitations regarding the control of the internal pore structure and the outer shape of the implants. Several studies demonstrated that pore structure, connectivity, and diameter affect the mechanical properties and biological performance of porous implants [8-10].In addition, their regular internal structures of porous implants fabricated by conventional methods hinder stress transfer and lead to implant fractures caused by local stress concentration[11]. In addition, the low porosity and poor connectivity of these porous titanium materials can disrupt body fluid circulation and thereby affect 4
ACCEPTED MANUSCRIPT new bone ingrowth. It is difficult to control the structures and parameters of porous titanium alloy
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sprepared using traditional methods; hence, the mechanical properties and biological
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performance of the materials can fail to meet the implant requirements. Recently, advanced rapid prototyping technologies, such as electron beam melting (EBM), became a better choice for the preparation of porous metal implants. EBM is capable
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of manufacturing porous titanium implants with controlled pore shapes, sizes, and
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distributions. The porous metal implants fabricated by EBM not only decrease stress-shielding effect but also allow the tissue ingrowth, thus improve long-term
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stability of implants. Using this technique, Parthasarathyet al.[12]created porous
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titanium with fully connected internal pores and a porosity of 49–70%, and Murret
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al.[13,14] fabricated porous titanium alloys with a porosity of 60–80% and an elastic modulus of the material
was only 3–6 GPa, which is close to that of cortical bone.
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In addition to the internal structure, the surface physicochemical properties of biological implants play an important role in the bone ingrowth process. Because titanium is a bioinert material, titanium alloy implants are often surrounded by fiber tissue rather than newly formed bone, which can disrupt osteointegration[15,16].To improve bone ingrowth and osteointegration, various surface modification methods have been used to enhance the bioactivity of titanium alloy implants [17].HA can adhere to soft and osseous tissue directly without an intermediate layer. Plasma spray is by far most-established commercial technique for HA coating, owing to the thick deposit, high deposition rate and low operation cost. HA coating has been widely used 5
ACCEPTED MANUSCRIPT for clinical implants and prosthesis, which is able to bind to bone directly and promotes bone ingrowth and osteointegration[18].Due to the complex internal
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structure of porous implants, a blind territory can be generated inside implants for HA
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coating using plasma-spraying. However, few studies reported HA coating for surface modification of EBM-generated porous titanium alloy implants with high porosity and connectivity using a plasma-spraying method.
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The purpose of this study was to evaluate the internal coating and in vivo
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biological performance of porous uncoated or HA-coated titanium alloy(Ti6Al4V) implants with well-controlled porous structures generated by EBM. The physical
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properties of porous implants with or without the HA coating were analyzed by
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scanning electron microscopy (SEM),micro computed tomography(micro-CT), energy
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dispersive spectrometry (EDS), confocal laser scanning microscopy (CLSM), and X-ray diffraction (XRD) analyses. The coated and uncoated samples were implanted
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into cylindrical bone defects in the bilateral distal femur of sheep, and bone ingrowth and osteointegration were evaluated by X-ray, micro-CT, and histological analyses.
Materials and Methods
Fabrication of porous Ti6Al4V implants Cylindrical porous implants with a length of 20mm and a diameterof10mm were fabricated using EBM (EBM S12;Arcam AB, Mölndal, Sweden). Three-dimensional (3D) models of the samples were generated using computer-aided design 6
ACCEPTED MANUSCRIPT software(Materialise/MagicsTM).To provide layer information, the 3D models were sliced into layers with a thickness of 100μm.Then,thedata were imported into the
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EBM device, and the implants were produced. The implants with and without the HA
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coating had similar structures; however, the HA-coated implants possessed a solid end (length =8mm)that enabled them to be clamped duringthe plasma-spraying process.
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Surface modification
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So far, atmospherical plasma spray(APS) was the conventional method for HA coating. However, Zheng et al.[19] reported that higher crystallinity has been obtained
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while the HA coating was deposited by VPS as compared with APS and the in vitro
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tests show that both APS and VPS HA coating posses good bioactivity.
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In this study, VPS method was applied for HA coating preparation. Prior to plasma-spraying, the substrates were ultrasonically cleaned in acetone and then grit
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blasted using corundum sand (particle size 250-300μm). A vacuum plasma-spray （VPS） system (F4-VB;Sulzer-Metco, Winterthur, Switzerland) was used to apply the HA coating, using the modified spray parameters listed in Table 1. Commercially available HA powder (AlfaAesar, Ward Hill, MA) was used as feedstock. The thickness of the HA coating was approximately 150μm. Characterization To determine the mean pore sizes and porosities, the porous implants were examined by SEM(SSX-550; Shimadzu, Kyoto, Japan) and micro-CT(Inveon MM Gantry; Siemens, Munich, Germany).The surface topographies of the samples with or 7
ACCEPTED MANUSCRIPT without coatings were observed by CLSM(LEXT OLS4000; Olympus, Tokyo, Japan),and the average roughness values(Ra)were determined.To confirm the coating
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composition, an XRD measurement was conducted to analyze the surface elemental
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compositions of the two types of implants. In addition, to investigate the internal coating of the implants, a sample was cut from the middle of the transverse axis, and
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the EDS analysis was performed.
The
uncoated
and
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Implantation assay coated
materials
were
implanted
into
six
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20–30-month-oldsmall-tail Han sheep weighing 35–55kg. To ensure their good health,
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all of the sheep underwent a general physical examination and were quarantined for
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approximately 2 weeks prior to the implantation operation. The study protocol was approved by the Fourth Military Medical University Committee on Animal
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Care(Permit Number:14015). The sheep were housed at the Department of Orthopedics of Xijing Hospital Animal Resources Center in two shacks with an average acreage of 18 m2. All animal experiments were conducted in accordance with the Guidelines for the Use of Laboratory Animals of the National Institutes of Health. All surgical procedures were performed under sterile conditions. The sheep underwent food and water restriction for 24 h prior to surgery, and were then placed under general anesthesia and fixed on the operation table. The distal femur areas of the two hind limbs were shaved and disinfected with iodine solution. A straight skin incision was made at the lateral femoral condyle, and the distal lateral aspect of the femur was 8
ACCEPTED MANUSCRIPT exposed. A cylindrical bone defect(20 mm in depth and 10 mm in diameter) was created at the lateral femoral condyle. The defect was washed with hydrogen peroxide and
then
the
porous
uncoated
Ti6Al4V
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saline,
implants(TI)
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and
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andHA-coatedTi6Al4V implants(HA-TI) were randomly placed into the right or left hind limb bone defects. Finally, the wounds were washed and closed in layers. After the operation, each sheep received intramuscular injections of an antibiotic for 3days
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to prevent infection. At 2 or 4 months post-implantation, the sheep were euthanized
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by an intravenous overdose of pentobarbital sodium, and the specimens were
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collected.
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Imaging
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X-ray imaging was performed immediately after the implantation procedure. After sacrifice of the animals 2 or 4 months later, the collected specimens were
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immersed in 10% formalin, and further X-ray images were obtained. To quantify new bone ingrowth into the porous implants, the collected specimens were scanned by micro-CT(Explore Locus SP Micro-CT;GE Healthcare, Wauwatosa, WI) with arotationor360°, a resolution of 14μm, a voltage of 80kV, and a current of 80μA.The 3D structure inside the defect was rebuilt using Explore Reconstruction UtilityTMsoftware (GE Healthcare),and the amount of newly formed bone in the porous implants was measured.
Histological analysis 9
ACCEPTED MANUSCRIPT The specimens were fixed in 10% formalin for 1week, and then dehydrated in graded concentrations of ethanol and embedded in methyl methacrylate. After
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polymerization, the samples were cut into sections with a thickness of approximately
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250μm using a histotome, and these sections were sanded to 80μm thick. After polishing and cleaning, the sections were stained with 1.2% trinitrophenol and 1% acid fuchsin(VanGieson staining), and observed under a light microscope(Leica
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DMLA, Bensheim, Germany). To generate an overall image, regional micrographs at
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low magnification were joined together using Photoshop6.0 software (Adobe,San Jose, CA). The new bone volume in the defect was measured, and the percentage of bone
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volume in the total available pore space was calculated using Image-Pro Plus
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Statistics
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implant was evaluated.
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6.0(Media Cybernetics,Rockville, MD). In addition, the integration of bone into the
One-way analysis of variance (ANOVA) was performed using SPSS 19.0 software (IBM, Armonk,NY). Data are represented as the mean ±standard deviation. p<0.05 was considered statistically significant.
Results
Characterization Figure 1A shows the appearance of the porous Ti6Al4V implants fabricated by 10
ACCEPTED MANUSCRIPT EBM. The pore structure was examined by SEM, and micro-CT reconstructed 3D images revealed that the samples displayed full connectivity(Figure 1B). The
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structural properties of the implants are listed in Table 2.The SEM analyses revealed
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that, unlike the uncoated implant(TI),the HA-coated implant (HA-TI) had a granular substance layer distributed on its curved face(Figure 2). Surface morphology 3D images were generated by CLSM and used to determine the roughness (Ra values)of
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the two types of implant (Figure 3). As shown in Figure 4, the average Ra value of the
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HA-TI group (8.45±0.80μm) was significantly higher than that of the TI group(4.95±1.26μm).Next, the surface elemental compositions of the TI and HA-TI
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materials were examined by EDS. Titanium, vanadium and aluminum were present on
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the surface of the TI material, whereas calcium, oxygen and phosphorus were the
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main elements on the surface of the HA-TI material (Figure 5).An XRD analysis confirmed that the phase layer on the HA-coated implant surface was
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hydroxyapatite(Figure 6).
To evaluate the internal coating of the implants, samples were cut from the middle of the transverse axis, and EDS was performed. Both coating elements (calcium, oxygen and phosphorus) and alloy elements (titanium, vanadium and aluminum) were detected on the internal surface of the HA-TI material (Figure 7B), indicating that the HA coating was deposited on the internal surface and the vacuum plasma-spray method is suitable for modification of porous implant surface. Imaging X-ray images were taken immediately after implantation and 2 or 4 months 11
ACCEPTED MANUSCRIPT post-implantation. The images taken after the surgical procedure revealed that all of the samples were implanted in the correct location (Figures 8A and 8B).The X-ray
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images collected at 2 months post-implantation showed that a widespread low-density
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shadow surrounded the implants (Figures 8C and 8D);however, at 4 months post-implantation, this area of low-density shadow had reduced (Figures 8E and 8F).Micro-CT analyses performed at 2 and 4 months post-implantation confirmed
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these findings (Figure 9). A quantitative analysis revealed that the bone volume
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fractions of the HA-TI and TI groups were higher at 4 months post-implantation than 2 months post-implantation, and the bone volume fraction of the HA-TI group was
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significantly higher (p=0.018 for 2 months, p=0.008 for 4 months)than that of the TI
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group at both time points (Figure 10).In addition, at 2 months post-implantation, the
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new bone tissue formed mainly around rather than inside the implants. By contrast, at 4 months post-implantation, the new bone tissue in the HA-TI group was found not
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only at the edge of the implants, but also in the internal region, whereas osteosis in the TI group was still concentrated at the margin of the implants. Histological analyses Observations revealed no signs of inflammation or foreign body reactions in the samples collected from the implanted sheep. Low magnification imaging revealed a similar pattern of healing in the TI and HA-TI groups; specifically, new bone tissue generated from the margin of the implant migrated into the internal region of the defect by implant attachment and creeping growth. At 2 months post-implantation, a small amount of newly generated immature woven bone was present in the rims of the 12
ACCEPTED MANUSCRIPT implants, but new bone formation was absent from the core regions of both types of implant(Figures 11A and 11B). At 4 months post-implantation, the amount of new
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bone tissue increased in the HA-TI group, almost half of the pores were filled with
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bone tissue, and osteosis was detected in the core region of the implant; however, at the same time point, new bone formation barely increased in the TI group, and the internal area of the implants was occupied by fibrous tissue rather than new bone
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(Figures 11C and 11D).Histometric measurements revealed similar results; at both2
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(p=0.03)and 4 (p<0.01)months post-implantation, the mineralized bone fraction of the HA-TI group was significantly higher than that of the TI group (Figure 12).The
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HA-TI group displayed excellent osteogenesis performance; the newly formed
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immature bone attached to the HA coating of the implant tightly, and a large number
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of osteoblasts were distributed linearly on the other side (Figure 13), indicating a huge potential for bone growth in the HA-TI group.
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To evaluate the osteointegration of both types of implants, the bone-implant interfaces were examined at high magnification(Figure 14).Gaps and fibrous tissue were located between the bone and the TI surface at 2 and 4 months post-implantation. By contrast, the new bone was closely connected to the HA coating on the HA-TI surface at the same time points, indicating that the HA-TI material promoted better osteointegration than the TI material.
Discussion The usage of porous titanium alloy implants with 3D pore structures and low 13
ACCEPTED MANUSCRIPT elastic moduli reduce stress shielding and improve both bone ingrowth and fixation of the implant. Previous research indicated that implants with high porosity and
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connectivity promote better gas and nutrient exchange between the interior and
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exterior regions of the implant[20,21], which can promote cell migration and tissue growth into the implant. In addition, pore size is important for bone ingrowth. Frosh et al.[22] compared the growth behaviors of osteoblasts in porous implants with pore
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sizes of 300,400,500,600, and 1000μm; the 600μm implant displayed the best
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migration and proliferation performance, and the implants with pore sizes of 400–600μm displayed a higher proportion of mineralized tissue. Unfortunately,
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conventional manufacturing methods are unable to guarantee the production of
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implants with the desired pore architecture; however, the advantage of EBM is that it
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enables accurate control of a sample’s structural parameters based on 3D model creation and additive manufacturing. Based on these findings, implants with pore
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diameters of approximately 500μm and high porosity and connectivity were designed and fabricated by EBM. A micro-CT analysis confirmed that the architectural parameters of the samples(pore size:514±35μm;porosity: 69±5%; interconnected in all dimensions) met the design requirements. Although porous titanium alloy implants enable surrounding bone ingrowth, new bone formation and osteointegration still have shortcomings because titanium is a bioinert material. Branemark[23]was the first to describe osteointegration as “contact established between normal and remodeled bone and an implant surface without the interposition of non-bone or connective tissue, at the light microscopic level”. 14
ACCEPTED MANUSCRIPT However, in most cases, fibrous tissue and gaps occur between the titanium implant and the bone tissue[24,25], and these defects can lead to a reduction in bone ingrowth
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or implant fixation weakness. HA has great biocompatibility and outstanding
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osteoconduction; hence, it has been used as an artificial bone graft material for decades. Ripamontiet al.[26] fabricated a plasma-sprayed HA-coated Ti6Al4V implant with 36 concavities measuring 1600μm in diameter and 800μm in depth, and
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implanted the material into the rectus abdominis muscle of Chacma baboons. After 31
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months, the geometrically-constructed plasma-sprayed titanium implants exhibited osteoinduction, which could initiate bone differentiation. Based on this finding, HA
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has been widely used as a coating on orthopedic implants to facilitate bone repair and
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improve implant-bone interface responses. Other bioactive materials, such as
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bioglasses, have also shown good biocompatibility and osteoconductivity. However, the weak interfacial bonding between titanium alloy and bioglass, as well as the fast
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degradation in vivo, limits usage of these bioglasses as implant coatings[27]. Here, the porous Ti6Al4V implant was surface modified with HA by plasma-spraying to improve its bioactivity.SEM and EDS analyses revealed that a uniform layer of the HA coating was deposited on the exterior surface of the implant; however, a scattered coating was observed on the internal surface, and some interior regions were not covered with HA. In addition, a CLSM analysis revealed that the roughness of the surface of the implant increased after HA coating. Several previous studies demonstrated that rough surface facilitate osteoblast proliferation and bone formation more efficiently than smooth surfaces [28,29]. 15
ACCEPTED MANUSCRIPT In the in vivo experiment, the micro-CT analysis revealed that bone formation in the HA-TI group was superior to that in the TI group at 2 and 4 months
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post-implantation. Reconstructed 3D images showed that the newly formed bone was
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distributed in the peripheral region of the TI material at both time points; however, the new bone tissue penetrated into the core area of the HA-TI material. These imaging results were also confirmed by histological analyses. Low magnification images
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showed that, although no bone tissue located in the core region of either type of
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implant at 2 months post-implantation, the core region of the HA-coated implant was colonized by newly formed immature woven bone at 4 months post-implantation. At
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which may impede new bone ingrowth. The implant-bone interfaces were also
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observed under high magnification. In the TI group, newly generated bone did not make direct contact with the uncoated implant, and fibrous tissue and gaps were
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identified between the implant and bone. By contrast, immature bone was associated tightly with the HA coating of the HA-TI material “without the interposition of non-bone or connective tissue”, as defined by Branemark[23].Based on these findings, we concluded that the HA-coated implant exhibited better osteointegration and bone ingrowth than the uncoated Ti6Al4V implant. A unique phenomenon was identified by high magnification imaging of the HA-TI group; specifically, numerous osteoblasts were linearly distributed along the exterior surface of the immature bone, which is favorable to new bone formation. This behavior of osteoblasts is a possible explanation for the results of the quantitative 16
ACCEPTED MANUSCRIPT analyses based on micro-CT and histometric measurements; these analyses revealed that the amount of new bone generated in the HA-coated implant was significantly
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Recently, several researchers examined the in vitro and in vivo bioactivities of
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porous titanium implants with different biomimetic coatings[30,31];however, few
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details of the coatings on the internal surfaces were reported. Li et al.[32] evaluated Ti6Al4V implants with and without a biomimetic apatite coating and found that the
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osteointegration capabilities than the uncoated implant. This finding indicates that the HA coatingimproves the bone formation, bone ingrowth, and bone-implant integration abilities of Ti6Al4V implants. Based on the results described above, we observed that new bone tissue grew inside of HA coated porous implant and helped anchor the implant in the implantation site. It was suggested that such “anchoring effect” could improve the fixation strength of the implant, which may reduce this risk of surface coating delamination. The mechanical strength and possibility of delamination of this HA coating will be further investigated in future work. 17
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Conclusion
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Here, a porous Ti6Al4V implant with accurately controlled pore structures and
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architectural parameters(porosity ~69±5%and pore size ~514±35μm)was generated using the EBM technique. Surface characterization demonstrated that the HA coating by plasma spraying was successfully deposited on the exterior surface of the implant;
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however, the coating on the internal surface was distributed non-uniformly.
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Histometric measurements revealed that the mineralized bone fraction of the HA-TI group was significantly higher than that of the TI group at both 2 and 4 months
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the newly formed immature bone attached to the HA coating of the implant tightly,
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and a large number of osteoblasts were distributed linearly on the other side. These results indicated the HA-coated implant exhibited remarkable bone ingrowth and
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osteointegration capabilities in vivo. To further evaluate the bonding strength of the bone-implant interface, the biomechanics testing should be conducted in future work. Thus, our results suggest that HA coating by plasma-spraying is likely suitable for biological modification of porous Ti6Al4V implants, which has a great potential for clinical applications.
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ACCEPTED MANUSCRIPT References
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[1]. Gefen A (2002) Computational simulations of stress shielding and bone resorption around existing and computer-designed orthopaedic screws. Medical and Biological Engineering and Computing 40: 311-322. [2]. Engh Jr CA, Young AM, Engh Sr CA, Hopper Jr RH (2003) Clinical consequences of stress shielding after porous-coated total hip arthroplasty. Clinical orthopaedics and related research 417: 157-163. [3]. Oh I-H, Nomura N, Masahashi N, Hanada S (2003) Mechanical properties of porous titanium compacts prepared by powder sintering. Scripta Materialia 49: 1197-1202. [4]. Kujala S, Ryhänen J, Danilov A, Tuukkanen J (2003) Effect of porosity on the osteointegration and bone ingrowth of a weight-bearing nickel–titanium bone graft substitute. Biomaterials 24: 4691-4697. [5]. Ryan G, Pandit A, Apatsidis DP (2006) Fabrication methods of porous metals for use in orthopaedic applications. Biomaterials 27: 2651-2670. [6]. Singh R, Lee P, Dashwood R, Lindley T (2010) Titanium foams for biomedical applications: a review. Materials Science and Technology 25: 127-136. [7]. Dunand DC (2004) Processing of titanium foams. Advanced Engineering Materials 6: 369-376. [8]. Habibovic P, Yuan H, van der Valk CM, Meijer G, van Blitterswijk CA, et al. (2005) 3D microenvironment as essential element for osteoinduction by biomaterials. Biomaterials 26: 3565-3575. [9]. Otsuki B, Takemoto M, Fujibayashi S, Neo M, Kokubo T, et al. (2006) Pore throat size and connectivity determine bone and tissue ingrowth into porous implants: three-dimensional micro-CT based structural analyses of porous bioactive titanium implants. Biomaterials 27: 5892-5900. [10]. Mastrogiacomo M, Scaglione S, Martinetti R, Dolcini L, Beltrame F, et al. (2006) Role of scaffold internal structure on in vivo bone formation in macroporous calcium phosphate bioceramics. Biomaterials 27: 3230-3237. [11]. Liu Y, Lim J, Teoh S-H (2013) Review: Development of clinically relevant scaffolds for vascularised bone tissue engineering. Biotechnology advances 31: 688-705. [12]. Parthasarathy J, Starly B, Raman S, Christensen A (2010) Mechanical evaluation of porous titanium (Ti6Al4V) structures with electron beam melting (EBM). Journal of the mechanical behavior of biomedical materials 3: 249-259. [13]. Murr L, Gaytan S, Medina F, Lopez H, Martinez E, et al. (2010) Next-generation biomedical implants using additive manufacturing of complex, cellular and functional mesh arrays. Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences 368: 1999-2032. [14]. Murr L, Amato K, Li S, Tian Y, Cheng X, et al. (2011) Microstructure and mechanical properties of open-cellular biomaterials prototypes for total knee replacement implants fabricated by electron beam melting. Journal of the mechanical behavior of biomedical materials 4: 1396-1411. 19
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[15]. Heinl P, Müller L, Körner C, Singer RF, Müller FA (2008) Cellular Ti–6Al–4V structures with interconnected macro porosity for bone implants fabricated by selective electron beam melting. Acta biomaterialia 4: 1536-1544. [16]. Göransson A, Jansson E, Tengvall P, Wennerberg A (2003) Bone formation after 4 weeks around blood-plasma-modified titanium implants with varying surface topographies: an in vivo study. Biomaterials 24: 197-205. [17]. Liu X, Chu PK, Ding C (2004) Surface modification of titanium, titanium alloys, and related materials for biomedical applications. Materials Science and Engineering: R: Reports 47: 49-121. [18]. Sun L, Berndt CC, Gross KA, Kucuk A (2001) Material fundamentals and clinical performance of plasma‐sprayed hydroxyapatite coatings: A review. Journal of biomedical materials research 58: 570-592. [19]. Zheng XB, Ji H, Huang JQ, Ding CX (2005) PLASMA SPRAYED Ti AND HA COATINGS： A COMPARATIVE STUDY BETWEEN APS AND VPS. Acta metallurgica sinica(English letters)18: 339-344. [20]. Karageorgiou V, Kaplan D (2005) Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials 26: 5474-5491. [21]. Hutmacher DW (2000) Scaffolds in tissue engineering bone and cartilage. Biomaterials 21: 2529-2543. [22]. Frosch KH, Barvencik F, Viereck V, Lohmann CH, Dresing K, et al. (2004) Growth behavior, matrix production, and gene expression of human osteoblasts in defined cylindrical titanium channels. Journal of Biomedical Materials Research Part A 68: 325-334. [23]. Branemark P-I (1983) Osseointegration and its experimental background. The Journal of prosthetic dentistry 50: 399-410. [24]. Ponader S, Von Wilmowsky C, Widenmayer M, Lutz R, Heinl P, et al. (2010) In vivo performance of selective electron beam‐melted Ti‐6Al‐4V structures. Journal of biomedical materials research Part A 92: 56-62. [25]. Søballe K, Hansen ES, B‐Rasmussen H, Jørgensen PH, Bünger C (1992) Tissue ingrowth into titanium and hydroxyapatite‐coated implants during stable and unstable mechanical conditions. Journal of Orthopaedic Research 10: 285-299. [26]. Ripamonti U, Roden LC, Renton LF (2012) Osteoinductive hydroxyapatite-coated titanium implants. Biomaterials 33: 3813-3823. [27]. Wang G, Lu Z, Liu X, Zhou X, Ding C, et al. (2011) Nanostructured glass-ceramic coatings for orthopaedic applications. J R Soc Interface 8: 1192-1203. [28]. Rosales-Leal J, Rodríguez-Valverde M, Mazzaglia G, Ramon-Torregrosa P, Diaz-Rodriguez L, et al. (2010) Effect of roughness, wettability and morphology of engineered titanium surfaces on osteoblast-like cell adhesion. Colloids and surfaces A: Physicochemical and Engineering aspects 365: 222-229. [29]. Martin J, Schwartz Z, Hummert T, Schraub D, Simpson J, et al. (1995) Effect of titanium surface roughness on proliferation, differentiation, and protein synthesis of human osteoblast‐like cells (MG63). Journal of biomedical 20
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materials research 29: 389-401. [30]. Takemoto M, Fujibayashi S, Neo M, Suzuki J, Kokubo T, et al. (2005) Mechanical properties and osteoconductivity of porous bioactive titanium. Biomaterials 26: 6014-6023. [31]. Fujibayashi S, Takemoto M, Neo M, Matsushita T, Kokubo T, et al. (2011) A novel synthetic material for spinal fusion: a prospective clinical trial of porous bioactive titanium metal for lumbar interbody fusion. European spine journal 20: 1486-1495. [32]. Li X, Feng Y-F, Wang C-T, Li G-C, Lei W, et al. (2012) Evaluation of biological properties of electron beam melted Ti6Al4V implant with biomimetic coating in vitro and in vivo. PloS one 7: e52049.
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Figure Legends
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Figure 1.Appearance and pore structure of the porous Ti6Al4V implants. (A) Overall appearance of the porous Ti6Al4V implant fabricated using the EBM
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2.SEM
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interconnected pore structure.
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method.(B) Micro-CT reconstructed 3D image of the porous implant showing the
Ti6Al4Vimplants. (A) The TI group. (B) The HA-TI group. The red arrows indicate
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that a granular layer was distributed on the surface of the HA-TI but not the TI. Figure 3.Reconstructed 3D topographic image of the porous Ti6Al4V implants. The images were used to determine the Ra values for the TI and HA-TI groups. Figure 4. Surface roughness of the implants. The mean Ra values were determined based on the images shown in Figure 3 (*p <0.05). Figure 5.EDS analyses of the elemental compositions of the exterior surfaces of the porous implants. The surface elements of the TI group comprised metallic elements(titanium, vanadium and aluminum), whereas those of the HA-TI group comprised coating elements(calcium, oxygen and phosphorus). 21
ACCEPTED MANUSCRIPT Figure 6.XRDanalysis of the coated porous implant. The coating phase was identified as hydroxyapatite.
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Figure 7.Internal surface elemental composition of the HA-TI.(A) The overall
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appearance of a section of the HA-TI material cut from the middle of the transverse axis. (B) EDS analysis of the elemental composition of the interior of the coated implants. Both metallic and coating elements were detected on the internal surface of
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the HA-TI.
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Figure 8.Radiographs taken after implantation.(A) Lateral radiograph taken
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immediately after implantation. (B) Anteroposterior radiograph taken immediately
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after implantation.(C) The TI group at 2 months post-implantation. (D) The HA-TI group at 2 months post-implantation. (E) The TI group at 4 months post-implantation.
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(F) The HA-TI group at 4 months post-implantation.
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Figure 9.Reconstructed 3D images of the implants. New bone (khaki) formation around the implant (gray) in the TI group at 2 months post-implantation (A), the HA-TI group at 2 months post-implantation (B),the TI group at 4 months post-implantation (C),and the HA-TI group at 4 months post-implantation (D).
Figure 10.Bone volume fractions of the TI and HA-TI groups at 2 and 4 months post-implantation.*p < 0.05.
Figure 11.Histological observations of the TI and HA-TI groups at low 22
ACCEPTED MANUSCRIPT magnification(Van Gieson stain,16×).The distribution of new bone (red) and fibrous tissue (dark blue) in the TI group at 2 months post-implantation (A),the HA-TI group
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the HA-TI group at 4 months post-operation (D).
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at 2 months post-implantation (B), the TI group at 4 months post-operation (C), and
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Figure 12. Mineralized bone fractions of the TI and HA-TI groups at 2 and 4
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Figure 13. Osteoblast behavior in the HA-coated implant(Van Gieson stain,200×).
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The greenarrowindicates severalosteoblasts adhered tothe new bone, which contains a
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lot of osteocytes. The yellow arrow indicates the HA coating between the metallic
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matrix and the newly formed bone tissue.
Figure 14.Osteointegration of the porous implant(Van Gieson stain, 400×).(A)
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The TI group. The green arrow indicates the fibrous tissue locatedbetween the implant and bone tissue.(B) The HA-TI group. The yellow arrow indicatesthe HA coating connecting the implant to the new bone.
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Argon plasma gas flow rate (SLPM)
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Hydrogen plasma gas flow rate (SLPM) Spray distance (mm) Argon powder carrier gas (SLPM)
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Voltage (V) Powder feed rate (g/min)
100 3.5 650 68 25 400
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Pressure (mbar)
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SLPM, standard liters per minute.
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ACCEPTED MANUSCRIPT Table 2.Structural properties of theporous Ti6Al4V implant fabricated by EBM. Pore size(μm)
Trabecular diameter(μm)
Porosity(%)
Φ10×20
514±35
238±23
69±5
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ACCEPTED MANUSCRIPT Highlights The porous Ti6Al4V implant was prepared using the electron beam melting method.
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The HA coating deposited on the surface of the porous implants by plasma-spraying
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The porous implants were implanted into distal femur bone defects of sheep.
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Plasma-spraying HA improved bone formation and osteointegration of porous implants.
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