fabrication and characterization of selective laser melting printed ti 6al 4v alloys subjected to heat treatment for customized implants design

Progress in Natural Science: Materials International (xxxx) xxxx–xxxx Contents lists available at ScienceDirect HOSTED BY Progress in Natural Science: Materials International journal homepage: www.elsevier.com/locate/pnsmi Original Research Fabrication and characterization of selective laser melting printed Ti–6Al– 4V alloys subjected to heat treatment for customized implants design☆ Mengke Wanga,b,1, Yuwei Wua,b,1, Songhe Lua,b, Tong Chena,b, Yijiao Zhaob, Hu Chenb, ⁎ Zhihui Tanga,b, a 2nd Dental Center, School and Hospital of Stomatology, Peking University, Beijing 100081, China National Engineering Laboratory for Digital and Material Technology of Stomatology, School and Hospital of Stomatology, Peking University, Beijing 100081, China b A R T I C L E I N F O A BS T RAC T Keywords: Selective laser melting (SLM) Titanium alloy Heat treatment Physiochemical properties Cytocompatibility Selective laser melting (SLM) is a promising technique capable of rapidly fabricating customized implants having desired macro- and micro-structures by using computer-aided design models However, the SLM-based products often have non-equilibrium microstructures and partial surface defects because of the steep thermal gradients and high solidification rates that occur during the laser melting To meet clinical requirements, a heat treatment was used to tailor the physiochemical properties, homogenize the metallic microstructures, and eliminate surface defects, expecting to improve the cytocompatibility in vitro Compared with the as-printed Ti– 6Al–4V substrate, the heat-treated substrate had a more hydrophilic, rougher and more homogeneous surface, which should promote the early cell attachment, proliferation and osseointegration More importantly, a crystalline rutile TiO2 layer formed during the heat treatment, which should greatly promote the biocompatibility and corrosion resistance of the implant Compared to the untreated surfaces, the adhesion and proliferation of human bone mesenchymal stem cells (hBMSCs) on heat-treated substrates were significantly enhanced, implying an excellent cytocompatibility after annealing Therefore, these findings provide an alternative to biofunctionalized SLM-based Ti–6Al–4V implants with optimized physiochemical properties and biocompatibility for orthopedic and dental applications Introduction Medical titanium alloys, especially the Ti–6Al–4V alloys, are characterized by excellent osseointegration, superior corrosion resistance and favorable mechanical properties over their counterparts such as cobalt alloys and stainless steel They have been successfully used in the orthopedic and dental fields for long-term and load-bearing bone implants [1,2] Conventional processing technologies used for manufacturing implants include casting and forging, which are time- and material-consuming and not allow the realization of customized implants having complex geometries [3] Recently, additive manufacturing (AM) has emerged as a revolutionary technique for the one-step fabrication of near-net-shaped implants The method uses virtual three-dimensional (3D) model data, has high material utilization rates, requires only short lead times and has reduced tooling costs [4] Among the available AM techniques, selective laser melting (SLM) is a superior candidate for meeting the anatomical and functional requirements at the recipient site of implantation because of its high precision and excellent performance [5,6] Additionally, in contrast to electron beam melting, no preheating of the powder and no complicated vacuum equipment are required during the entire SLM manufacturing process [7,8] However, steep thermal gradients and high solidification rates occur because of the rapidly-moving intense laser beam; SLM-based samples have been reported to have non-equilibrium microstructures and partial surface defects as a consequence [9,10] Therefore, to meet current clinical requirements, a heat treatment was identified that would tailor the physiochemical properties [1,11], eliminate surface defects and homogenize the metallic microstructures, looking forward to improving the in vitro cytocompatibility eventually Heating process is also a cost-effective way to transform superficial amorphous titania layers into the rutile crystalline structure, which could increase the biocompatibility and corrosion resistance of the implants [12] As reported, annealing below 550 °C could result in the increase of age Peer review under responsibility of Chinese Materials Research Society ⁎ Corresponding author at: 2nd Dental Center, School and Hospital of Stomatology, Peking University, Beijing 100081, China E-mail address: zhihui_tang@126.com (Z Tang) These authors equally contributed http://dx.doi.org/10.1016/j.pnsc.2016.12.006 Received 10 January 2015; Accepted August 2015 1002-0071/ © 2016 Published by Elsevier B.V on behalf of Chinese Materials Research Society This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/) Please cite this article as: Wang, M., Progress in Natural Science: Materials International (2016), http://dx.doi.org/10.1016/j.pnsc.2016.12.006 Progress in Natural Science: Materials International (xxxx) xxxx–xxxx M Wang et al hardening and embrittlement, while annealing above the β-transus temperature (approximately 1000 °C) could lead to excessive grain growth of the β phase [13] Proved by the previous studies, sub-βtransus annealing treatments in the range 800–850 °C not only could relieve residual stress, but also could generate the preferred lamellar α+β equilibrium structure with desirable mechanical properties, which were recommended for the versatile post-treatment of SLM-based titanium alloys [13–15] Since surface physiochemical characteristics strongly influence osseointegration, there have been extensive researches on modifying the implant surface to obtain a homogenous microstructure having excellent hydrophilicity, appropriate roughness and compatible cellular compatibility [16–18] The studies focusing on the thermal treatment of SLM-fabricated Ti–6Al–4V alloys are available [10,15,19], but a systematic analysis of its efficiencies on the surface properties and biocompatibility is still lacking This article reports the surface alteration of the heat-treated Ti–6Al–4V substrate, including the superficial wettability, morphology, roughness, crystalline structure and mechanical properties In addition, the attachment and proliferation behaviors of human bone mesenchymal stem cells (hBMSCs) on these SLMprinted substrates were further evaluated The crystalline structures of the Ti–6Al–4V disks with and without annealing were examined and compared by X-ray diffraction (XRD6100, SHIMADZU Corp., Kyoto, Japan) using a Cu target as the radiation source at 40 kV and 100 mA The diffraction angles (2θ) were set at 20–60°, with a step size of 0.02° 2θ and a scan speed of 4° 2θ/ Superficial mechanical properties were measured in-situ by nanoindentation using a nanomechanical test system (TI–900 TriboIndenter, HYSITRON, Minneapolis, USA) Five indentations were made perpendicularly to different flat regions using a pyramidal diamond Berkovich indenter (with a total included angle of 142.31°), operating at a constant load of mN In all cases, a trapezoidal loading–unloading profile was used, with s loading and unloading segments, including a s holding segment Before performing each array of indents, the stability of the nano-indentation instrument was checked by measuring a fused-quartz reference sample with known properties (E*=71.0 GPa) The elastic modulus (E*) and nano-hardness (H) were calculated from the experimental unload–displacement curves using the Oliver and Pharr model The surface hydrophilicity was evaluated by measuring the contact angles of untreated and heat-treated Ti–6Al–4V surfaces (OCA20, DATAPHYSICS, Filderstadt, Germany) The contact angles, using the sessile drop method, were measured at ambient temperature Six specimens were used to provide an average and standard deviation Material and methods 2.1 Materials and samples preparation 2.3 Cell culture Gas-atomized Ti–6Al–4V ELI powder (Grade 5, DENTAURUM, Ispringen, Germany) with a particle size of 15–45 µm was used as the base material All of the samples evaluated in this study were manufactured by an SLM machine (Mlab Cusing R, Concept Laser GmbH, Lichtenfels, Germany) at a scanning speed of 900 mm/s, laser power of 95 W, spot size of 40 µm and layer thickness of 30 µm Layers were scanned using the continuous laser mode in a zig-zag pattern, which was rotated 90° between each layer The whole process was performed in an argon atmosphere and the samples were built upon a solid titanium substrate After production, the samples were removed from the substrate using wire electro-discharge machining and were processed into cylinders with an approximate diameter of 15 mm and a height of mm Half of the samples were annealed at 820 °C (10 °C/min) for h in a furnace under argon shield and then were gradually cooled down to room temperature The untreated samples were used as controls All specimens were degreased ultrasonically in baths of acetone and anhydrous ethanol for h, respectively, with de-ionized water rinsing for h after applications of each solvent For metallographic analysis each sample was wet ground in a round device with silicon carbide sandpaper of decreasing grit size and then polished with alumina suspensions, to obtain a flat and homogeneous surface The metallographic features of the specimens were disclosed by etching them at room temperature for 15–20 s with Kroll's reagent (2 mL hydrofluoric acid, mL nitric acid and 100 mL distilled water) The human bone mesenchymal stem cells (hBMSCs) purchased from ScienCell (California, America) were chosen to evaluate the cytocompatibility in the present study They were cultured in standard tissue culture dishes using α-minimal essential medium (a-MEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco), and 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA) The cultures were maintained at 37 °C in a humidified 5% CO2 incubator (MCO-18AIC, Japan) Cells were fed every days and passaged at 1:3 splitting ratio upon 90% confluent by exposure to 0.25% trypsin-EDTA solution (Gibco) for 30 s All the Ti–6Al–4V disks were autoclaved, rinsed with sterile PBS and transferred into 12-well tissue culture plates Prior to cell seeding, the specimens were equilibrated in culture medium for 10 Subsequently, the hBMSCs were drop-seeded on the substrates at a density of 5×104 cells/mL and incubated statically for at least h to allow cell attachment 2.4 Immunofluorescence After 12 h of seeding, the hBMSCs were separately fixed with 4% paraformaldehyde (PFA) for 30 and permeabilized with 0.1% Triton X-100 (Solarbio, Beijing, China) for at room temperature (RT) Subsequently, samples were washed with PBS three times and then incubated with fluorescein isothiocyanate (FITC)-phalloidin (10 µg/mL, Sigma-Aldrich, America) for 40 at RT in order for visualization of filamentous actin (F-actin) Finally, cell nuclei were counterstained with DAPI (1 µg/mL, Sigma-Aldrich) for at RT and visualized immediately by a laser confocal microscopy (LSM5, Carl Zeiss, Germany) 2.2 Microstructure and surface feature analysis The microstructural analysis was then conducted using an incident light microscope (BX51M, OLYMPUS, Tokyo, Japan) The surface morphology of the origenal and heat-treated Ti–6Al–4V substrates were characterized by a field-emission scanning electron microscope (FE-SEM, JSM-6701F, JEOL, Tokyo, Japan) at an accelerating voltage of 20 kV Heated and unheated specimens were separately observed under a 3D laser scanning microscope (VK-X100K, KEYENCE, Osaka, Japan); 10 different areas of each sample were chosen and scanned to obtain the 3D surface profile These images were analyzed with the VK-H1XP software to obtain the average roughness (Ra), peak-to-valley roughness (Rz) and root-mean-square roughness (Rq) 2.5 Cell proliferation assay At 1, 4, and days of incubation, the disks were transferred into new 12-well dishes and evaluate the cell proliferation using cell count kit-8 assay (CCK-8, Dojindo, Japan) Briefly, at desired time intervals of cultivation, CCK-8 solution was added into each well at a proportion of 1:10 (v/v) for h incubation in dark Then 100 μl supernatant from each well was transferred to new 96-well cell culture plates The absorbance value of the supernatant optical density (OD value) for Progress in Natural Science: Materials International (xxxx) xxxx–xxxx M Wang et al topographical features are thought to promote osteogenesis and implant stabilization, perhaps because the larger surface area improves bone-to-implant contact and mechanical interlocking between regenerated bone and the implants [20,26,27] Additionally, in terms of biological activity, nano-topography may enhance the adsorption of ECM proteins, improve cell proliferation and stimulate cell differentiation towards osteogenic lineage [20] To further characterize the surface topography after the annealing treatment, the surface roughness was evaluated using a 3D laser scanning microscope The control disk (Fig 3a) exhibited a heterogeneous and irregular surface topography, with high peaks (orangered) and low valleys (blue) random distributing in view In contrast, symmetrical distribution of peaks and valleys were seen on the heattreated surface, displaying a relatively homogeneous surface texture Furthermore, some objective parameters (Fig 3c–e) were introduced to quantify the roughness differences, i.e., average roughness (Ra), peak-to-valley roughness (Rz) and root-mean-square roughness (Rq) Consistent with 3D surface reconstructions, the heat-treated sample had a Ra value of 7.59 ± 0.39 µm, which is slightly higher and more stable than the control (6.31 ± 0.72 µm) The Rz and Rq parameters were chosen to reflect local height fluctuations in a given area For the untreated disk, the Rz and Rq values were 61.72 ± 9.16 and 9.60 ± 1.14 µm, respectively After heat treatment, the surface became more homogeneous, which was reflected in the lower means and standard deviations of Rz (42.66 ± 1.43 µm) and Rq (7.31 ± 0.52 µm) It has been suggested that a rough surface can provide anchors for protein adsorption and cell adhesion, as well as regulate osteoblast differentiation and matrix production, and thereby accelerate the osteogenetic process [28,29] However, the standard ISO 20160 requires that dental implants have a homogeneous surface microstructure to ensure material integrity and mechanical properties [30] The above results proved that heat-treated sample had a rougher but more homogeneous surface morphology than the untreated case, which could assist early fixation and long-term mechanical stability of an implanted prosthesis Fig Illustration of the selective laser melting manufacturing process of the printed Ti–6Al–4V substrates and the post-annealing treatment each group was measured with a microplate reader (Model 680, BioRad, Canada) at 450 nm Six parallel specimens were used to provide an average and standard deviation 2.6 Statistical analysis All data were expressed as means ± standard deviation One-way analysis of variance (ANOVA) with Tukey's post-hoc test applied was used to assess differences among the groups; p < 0.05 was considered statistically significant Results and discussion The Ti–6Al–4V substrates in this study were manufactured using the SLM technique (Fig 1) This method can produce preciselycontrolled micro/nano-architectures, which facilitate cell attachment and proliferation and accelerate the osseointegration rate [10,20] Heat treatments are typically used to homogenize microstructures and optimize mechanical properties [21–23] According to previous studies, an overly low or overly high annealing temperature might have a negative effect [13,14] In order to match with the selected machine type and laser processing parameters, the optimum heat-treated parameter in this study was recommended as 820 °C for h, which was repeatedly verified by the Concept Laser Company Compared with the as-received substrates, the effectiveness of this mild annealing treatment was carefully assessed by surface characterization and cellular compatibility in vitro 3.2 Microstructural analysis Metallographic analysis was conducted to study the microstructural changes that occurred during the annealing process Fig 4a–b shows that the control group had the typical α′-martensitic structure with a very fine acicular morphology, which was reported previously [7,22,31] In comparison, heating at 820 °C for h (Fig 4c–d) transformed the α'-martensite into the coarser lamellar (α+β) structure with white platelets (α-phase) and dark regions (β-phase), in which the dominant α-phase was present as coarser laths and separated by small amounts of narrow interphase regions (β-phase) [1] The small amount of the β-phase is ascribed to the presence of α-stabilizers (such as oxygen) that were incorporated during manufacturing [32] As indicated previously, a coarser lamellar microstructure has high fracture toughness, which implies superior resistance to creep and fatigue crack growth [1] The large temperature gradients that occurred during the SLM process enabled diffusion-less transformation of the high-temperature β-phase to the low-temperature α-phase, which resulted in the α′martensitic microstructure [32] The α′-martensite results from rapid solidification and its features correlate with the direction of heat conduction [23] Heating above the martensite start temperature (Ms, 650 °C), followed by slow cooling in the furnace, transformed the as-fabricated α′-phase into the more stable lamellar α+β phase [10], which is advised to provide implants with a more desirable combination of strength and toughness [33] 3.1 Surface morphology and roughness analysis Low-magnification images (Fig 2a and c) showed that aggregates of non-melted Ti–6Al–4V globules were present on the as-printed samples, which were only loosely associated on the surface Heat treatment (Fig 2b and d) caused some of the aggregated metal particles to become fused and bonded to the surface Those remaining looselybonded globules can be deleterious to the mechanical properties and lead to inflammation of surrounding tissues [24,25] Therefore, future work must include removal of the weakly-bonded particles to further improve the surface quality High-magnification images (Fig 2e and f) revealed a distinct difference between the two surfaces: the heat-treated surface had a rough granulated debris pattern compared with the smoother surface of the control specimen A close-up image of the granulated surface (Fig 2g and h) further reveals the difference observed at the nanometer scale: a closely-spaced lattice layer formed on the heat-treated samples, which resulted in nano-elevation of the surface roughness The modified surface of a heat-treated sample had a hierarchical structure consisting of micro-scale features (partially-melted microglobules) and nano-scale features (closely-spaced nano-lattices) Nano- 3.3 Surface crystalline structure analysis Glancing-angle XRD patterns were analyzed to further study the Progress in Natural Science: Materials International (xxxx) xxxx–xxxx M Wang et al Fig Scanning electron microscope images of untreated and heat-treated Ti–6Al–4V substrates under different magnifications (a, b) 100×; (c, d) 500×; (e, f) 2000×; (g, h) 30,000× Fig Quantitative measurement of the surface roughness of untreated and heat-treated Ti–6Al–4V substrates using the three-dimensional (3D) laser scanning microscope 3D surface reconstructions of untreated (a) and heat-treated (b) Ti–6Al–4V substrates are presented at a vertical scale of 80 µm These images were analyzed with the VK-H1XP software to obtain the values of (c) average roughness, (d) peak-to-valley roughness and (e) root-mean-square roughness (n=10, *, p < 0.05; **, p < 0.01) the preferred crystallographic orientation of the hexagonal Ti phase was the (101) plane After annealing, some new peaks appeared at 2θ values of 27.4, 54.2 and 56.6°, which were attributed to the (110), (301) and (112) planes, respectively, of tetragonal rutile TiO2 The tiny peak that appeared at 2θ=39.2° was attributed to the (101) plane of the β-Ti phase, in accordance with the metallographic results Although titanium alloys are prone to form TiO2 layers at room temperature, no oxide peaks were detected in the untreated sample and changes in the crystal structure that occurred because of the heat treatment (Fig 5) The relative intensity (counts per second) is graphed as a function of the diffraction angle (2θ) The Bragg diffraction peaks of the untreated sample at 2θ values of 35.5, 38.6, 40.8 and 53.1° indexed to the (100), (002), (101) and (102) planes, respectively These peaks are consistent with those of hexagonal α-Ti The α′-martensite is assumed to have a similar crystal structure to α-Ti because only peaks corresponding to α-Ti were observed [1,3] The data also revealed that Progress in Natural Science: Materials International (xxxx) xxxx–xxxx M Wang et al Fig Metallographic images of the untreated and heat-treated specimens the superficial non-uniformity In contrast, the heat-treated substrates had a more stable E*(113.21 ± 10.47 GPa) and H (4.11 ± 0.92 GPa), implying that annealing made the entire disks more homogeneous More specifically, the thermal treatment increased the H value 2.36fold, indicating a harder surface that would be more able to resist cracking and fatigue Annealing provided a more statistically stable E* value, which was matched with the normal range (101–125 GPa) of machined Ti–6Al– 4V alloy [36,37] To obtain effective osseointegration, implants must have an elastic modulus that is comparable with that of normal bone (30 GPa) in order to enable better load transfer and minimize the stress-shielding phenomenon [38] As a consequence, additional research is warranted to study the reduction of mechanical mismatch and ultimately improve the long-term fixation by establishing the porous Ti–6Al–4V alloy having interconnected pores and porosity that are similar to natural bone The evenly-distributed lamellar (α+β) microstructure was formed when sufficiently low cooling rates were used following heat treatment below the β transus This improves superficial hardness through the solid-solution strengthening effect of vanadium [33] Additionally, the formation of protective titania layers is also reported to increase the hardness and the fatigue resistance of the superficial layers, and improve the corrosion behavior and reduce the friction coefficient [39] The above results indicate that the annealed Ti–6Al–4V substrate had improved mechanical properties that meet the requirements for dental or bone implants Fig X-ray diffraction patterns of untreated and heat-treated Ti–6Al–4V substrates # and & represent the α-Ti and β-Ti phases, respectively; * represents the rutile phase all of the diffraction peaks were related to hexagonal close-packed α-Ti, suggesting that the naturally-formed titania layers were amorphous [34] Fig reveals that the heat treatment caused the oxide crystallinity to change, i.e., the amorphous oxide converted to the crystalline rutile TiO2 Rutile is the most thermodynamically stable phase of crystalline TiO2, and a coating of crystalline rutile instead of amorphous TiO2 would endow implants with better corrosion resistance and biocompatibility [35] 3.5 Surface wettability analysis Fig 7a shows that a pristine SLM-printed Ti–6Al–4V disk was relatively hydrophobic with a contact angle of ca 79.25° Heat treatment of the disk resulted in a substantial and statistically significant increase in surface wettability, which was manifested by a dramatic reduction of the contact angle to ca 37.54° (Fig 7b) 3.4 Surface mechanical properties The Young's modulus (E*) and nano-hardness (H) values obtained by the nano-indentation tests are presented in Fig The control group had greater variation for both E* and H, which might be originate from Progress in Natural Science: Materials International (xxxx) xxxx–xxxx M Wang et al Fig Mechanical properties obtained from the nano-indentation tests (*, p < 0.05; **, p < 0.01) water [35] It is the hydrogen bonding of water to the surface functional groups that exerts the greatest influence on wettability The micro- or nano-topographical character could also affect the apparent surface hydrophilicity Several articles have reported a positive correlation between surface topography and wettability [45,46], but the detailed mechanism is still uncertain and warrants further study 3.6 Cell morphology and proliferation analysis Assisted by facile heat treatment, an optimal surface with more hydrophilic, rougher and homogeneous texture had been established; its in vitro cellular compatibility was further evaluated by investigating the adhesion and proliferation of hBMSCs After 12 h of seeding, most adherent cells exhibited an elongated or polygonal morphology, and contained expansive networks of actin filaments on both surfaces (Fig 8a), which consistent with the healthy shape of hBMSCs Compared with the untreated group, more amounts of hBMSCs with uniform distribution and well spreading attached on the heat-treated specimens, perhaps owing to the optimized biocompatibility and homogeneity after heating treatment [47] Furthermore, as cell adhesion was enhanced, hBMSCs cultured on the heat-treated substrates spread well with visible presentation of more mature F-actin intracellular stress fibers, indicating an excellent cytocompatibility of heattreated disk As depicted in the Fig 8b, the OD450 value increased as the extension of incubation time, indicating that both groups could facilitate the normal proliferation of hBMSCs However, when the substrates were subjected to heat treatment, the cell viability was statistically higher than that of the pristine samples during the whole culture time (p < 0.01) Furthermore, at day and 4, hBMSCs attached on the heat-treated surface showed a proliferation rate of 175% and 169% of that on untreated surface, respectively, indicating that heating process could promote the cytocompatibility of the SLM-based disks Previous studies showed that surface wettability and roughness were crucially important for cellular responses to the substrates [20,42] As displayed in Figs and 7, a more rougher and hydrophilic surface was observed after the heat treatment, which would provide anchors for the surrounding ions and proteins adsorption, facilitating the cell attachment and proliferation In conclusion, these findings proved that the post-heating treatment could endow SLM-based Ti–6Al–4V substrates with enhanced physiochemical properties and biological characteristics for orthopedic and dental applications Fig Wettability measurements for the untreated and heat-treated Ti–6Al–4V substrates by contact angle goniometry The images show the water attached to the untreated (a) and heat-treated (b) Ti–6Al–4V substrates Graph (c) gives the measured contact angles for the specimens Data are displayed as means ± standard deviations (n=6, p < 0.01) It has been well-proven that surface hydrophilicity plays an advantageous role during the early phase of osseointegration, which is a requirement for the initial fixation and long-term mechanical stability of an implanted prosthesis [40,41] Theoretically, a hydrophilic surface enhances the surface reactivity with the surrounding ions, amino acids and proteins in the tissue fluid and then facilitates osteoblast attachment and proliferation to obtain effect osseointegration [42,43] The improved surface wettability observed after the heat treatment signifies that a more hydrophilic surface was established [28,40] As previously suggested, the improved hydrophilicity after annealing is probably related to the enhanced thickness and crystallinity of the surface rutile TiO2 layer [44] The OH– groups of this layer easily associate with the surface of the implants, leading to an increased concentration of Ti–OH groups, which could hydrogen-bond with Conclusion Summarizing, an optimal surface with more hydrophilic, rougher Progress in Natural Science: Materials International (xxxx) xxxx–xxxx M Wang et al Fig Cell adhesion and proliferation of the hBMSCs cultured on the untreated and heat-treated Ti-6Al-4V surfaces (a) Visualization of the cytoskeleton (green, labeled with FITCphalloidin) and cell nuclei (blue, counterstained with DAPI) after 12 h of seeding Scale bar: 50 µm (b) The cell proliferation was evaluated by CCK-8 assay after 1, 4, and days incubation (n=3, **, p < 0.01) [17] K Lin, L Xia, J Gan, Z Zhang, H Chen, X Jiang, J Chang, ACS 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3674–3683 [47] S Amin Yavari, Y.C Chai, A.J Böttger, R Wauthle, J Schrooten, H Weinans, A.A Zadpoor, Mater Sci Eng C: Mater Biol Appl 51 (2015) 132–138 and homogeneous texture had been established via annealing treatment in order to enhance the biocompability and osseointegration of SLM-based substrates, which was a simple, cost-effective and efficient method Our report has demonstrated that SLM printing followed by a suitable heat treatment can successfully be used to fabricate the customized implants with optimal physiochemical properties and in vitro cytocompatibility, which will accelerate the application of SLM technique in the orthopedic and dental fields Acknowledgements This work was supported by the National Natural Science Foundation of China, Grant number 81300851, which was awarded to Yu-wei Wu, and the Beijing Municipal Natural Science Foundation, Grant number Z151100003715007, which was awarded to Zhi-hui Tang We want to thank Changhui Song for his assistance with 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implants Fig X-ray diffraction patterns of untreated and heat- treated Ti? ? ?6Al? ? ?4V substrates # and & represent the α -Ti and β -Ti phases, respectively; * represents the rutile... Quantitative measurement of the surface roughness of untreated and heat- treated Ti? ? ?6Al? ? ?4V substrates using the three-dimensional (3D) laser scanning microscope 3D surface reconstructions of untreated