To meet clinical requirements, a heat treatment was used to tailor the physiochemical properties, homogenize the metallic microstructures, and eliminate surface defects, expecting to imp
Trang 1H O S T E D B Y Contents lists available atScienceDirect
Progress in Natural Science: Materials International
journal homepage:www.elsevier.com/locate/pnsmi Original Research
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
b National Engineering Laboratory for Digital and Material Technology of Stomatology, School and Hospital of Stomatology, Peking University, Beijing
100081, China
A R T I C L E I N F O
Keywords:
Selective laser melting (SLM)
Titanium alloy
Heat treatment
Physiochemical properties
Cytocompatibility
A B S T R A C T
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 TiO2layer formed during the heat treatment, which should greatly promote the biocompat-ibility 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
1 Introduction
Medical titanium alloys, especially the Ti–6Al–4V alloys, are
characterized by excellent osseointegration, superior corrosion
resis-tance and favorable mechanical properties over their counterparts such
as cobalt alloys and stainless steel They have been successfully used in
the orthopedic and dentalfields for long-term and load-bearing bone
implants[1,2] Conventional processing technologies used for
manu-facturing implants include casting and forging, which are time- and
material-consuming and do not allow the realization of customized
implants having complex geometries[3]
Recently, additive manufacturing (AM) has emerged as a
revolu-tionary 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
http://dx.doi.org/10.1016/j.pnsc.2016.12.006
Received 10 January 2015; Accepted 5 August 2015
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.
1 These authors equally contributed.
E-mail address: zhihui_tang@126.com (Z Tang).
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/).
Trang 2hardening 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-
β-transus 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
altera-tion of the heat-treated Ti–6Al–4V substrate, including the superficial
wettability, morphology, roughness, crystalline structure and
mechan-ical properties In addition, the attachment and proliferation behaviors
of human bone mesenchymal stem cells (hBMSCs) on these
SLM-printed substrates were further evaluated
2 Material and methods
2.1 Materials and samples preparation
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 2 mm
Half of the samples were annealed at 820 °C (10 °C/min) for 4 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 1 h, respectively, with de-ionized water rinsing
for 1 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 aflat 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, 5 mL nitric acid and 100 mL distilled water)
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
rough-ness (Rz) and root-mean-square roughrough-ness (Rq)
The crystalline structures of the Ti–6Al–4V disks with and without annealing were examined and compared by X-ray diffraction
(XRD-6100, 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/ min
Superficial mechanical properties were measured in-situ by nano-indentation 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 5 mN In all cases, a trapezoidal loading–unloading profile was used, with 5 s loading and unloading segments, including a 2 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-hard-ness (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 2.3 Cell culture
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 2 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 min Subsequently, the hBMSCs were drop-seeded on the substrates at a density of 5×104cells/mL and incubated statically for at least 1 h to allow cell attachment
2.4 Immunofluorescence After 12 h of seeding, the hBMSCs were separatelyfixed with 4% paraformaldehyde (PFA) for 30 min and permeabilized with 0.1% Triton X-100 (Solarbio, Beijing, China) for 5 min 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 min at RT in order for visualization of filamentous actin (F-actin) Finally, cell nuclei were counterstained with DAPI (1 µg/mL, Sigma-Aldrich) for 5 min at RT and visualized immediately by a laser confocal microscopy (LSM5, Carl Zeiss, Germany)
2.5 Cell proliferation assay
At 1, 4, and 7 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 2 h incubation in dark Then 100μl supernatant from each well was transferred to new 96-well cell culture plates The absorbance value of the supernatant optical density (OD value) for
Trang 3each group was measured with a microplate reader (Model 680,
Bio-Rad, 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
3 Results and discussion
The Ti–6Al–4V substrates in this study were manufactured using
the SLM technique (Fig 1) This method can produce
precisely-controlled 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
stu-dies, 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 4 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
3.1 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
loosely-bonded 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 scale features (partially-melted
micro-globules) and nano-scale features (closely-spaced nano-lattices)
Nano-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 regen-erated 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 di fferentia-tion 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 hetero-geneous and irregular surface topography, with high peaks (orange-red) and low valleys (blue) random distributing in view In contrast, symmetrical distribution of peaks and valleys were seen on the heat-treated surface, displaying a relatively homogeneous surface texture Furthermore, some objective parameters (Fig 3c–e) were intro-duced 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 para-meters 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 microstruc-ture 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
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 typicalα′-martensitic structure with a very fine acicular morphology, which was reported previously [7,22,31] In comparison, heating at 820 °C for 4 h (Fig 4c–d) transformed theα'-martensite into the coarser lamellar (α+β) structure with white platelets (α-phase) and dark regions (β-phase), in which the dominantα-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 indi-cated 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] Theα′-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-fabricatedα′-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]
3.3 Surface crystalline structure analysis Glancing-angle XRD patterns were analyzed to further study the
Fig 1 Illustration of the selective laser melting manufacturing process of the printed
Ti–6Al–4V substrates and the post-annealing treatment.
Trang 4changes 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 hexagonalα-Ti The α′-martensite is
assumed to have a similar crystal structure toα-Ti because only peaks
corresponding toα-Ti were observed[1,3] The data also revealed that
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
Fig 2 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 3 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).
Trang 5all of the diffraction peaks were related to hexagonal close-packed α-Ti,
suggesting that the naturally-formed titania layers were amorphous
[34].Fig 5reveals that the heat treatment caused the oxide
crystal-linity 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
amor-phous TiO2would endow implants with better corrosion resistance and
biocompatibility[35]
3.4 Surface mechanical properties
The Young's modulus (E*) and nano-hardness (H) values obtained
by the nano-indentation tests are presented inFig 6 The control group
had greater variation for both E* and H, which might be originate from
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 2.36-fold, 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 theβ 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
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)
Fig 4 Metallographic images of the untreated and heat-treated specimens.
Fig 5 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.
Trang 6It 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
hydro-philic surface enhances the surface reactivity with the surrounding
ions, amino acids and proteins in the tissuefluid and then facilitates
osteoblast attachment and proliferation to obtain effect
osseointegra-tion[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
anneal-ing is probably related to the enhanced thickness and crystallinity of
the surface rutile TiO2layer[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
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 adhe-sion was enhanced, hBMSCs cultured on the heat-treated substrates spread well with visible presentation of more mature F-actin intracel-lular stressfibers, indicating an excellent cytocompatibility of heat-treated 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 1 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 inFigs 3 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 attach-ment and proliferation In conclusion, thesefindings 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
4 Conclusion Summarizing, an optimal surface with more hydrophilic, rougher
Fig 6 Mechanical properties obtained from the nano-indentation tests (*, p < 0.05; **, p < 0.01).
Fig 7 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).
Trang 7and homogeneous texture had been established via annealing
treat-ment 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 dentalfields
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
preparation of this manuscript
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Fig 8 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 FITC-phalloidin) 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 7 days incubation (n=3, **, p < 0.01).