Cytocompatibility of Ti3AlC2, Ti3SiC2, and Ti2AlN: In Vitro Tests and

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Cytocompatibility of TiAlC, TiSiC, and TiAlN: In vitro Tests and First-principles Calculations Ke Chen, Nianxiang Qiu, Qihuang Deng, Min-Ho Kang, Hui Yang, JaeUk Baek, Young-Hag Koh, Shiyu Du, Qing Huang, and Hyoun-Ee Kim ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00432 • Publication Date (Web): 10 Aug 2017 Downloaded from http://pubs.acs.org on August 12, 2017

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ACS Biomaterials Science & Engineering

Cytocompatibility of Ti3AlC2, Ti3SiC2, and Ti2AlN: In vitro Tests and First-principles Calculations Ke Chen

a,‡

, Nianxiang Qiu

b,‡

, Qihuang Deng b, Min-Ho Kang a, Hui Yang b, Jae-Uk Baek a,

Young-Hag Koh c, Shiyu Du b, Qing Huang b,*, Hyoun-Ee Kim a,* a.

Department of Materials Science and Engineering, Seoul National University, 1 Gwanak-ro,

Gwanak-gu, Seoul 151-744, Republic of Korea. b.

Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences,

1219 Zhongguan West Road, Ningbo 315201, China. c.

School of Biomedical Engineering, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 136-

703, Republic of Korea. *Corresponding authors: a,*

Department of Materials Science and Engineering, Seoul National University, 1 Gwanak-ro,

Gwanak-gu, Seoul 151-744, Republic of Korea. E-mail address: [email protected] (Hyoun-Ee Kim); b,*

Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences,

1219 Zhongguan West Road, Ningbo 315201, China. E-mail address: [email protected] (Qing Huang). ‡

Ke Chen and Nianxiang Qiu contributed equally.

ACS Paragon Plus Environment

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ABSTRACT: Herein, the cytocompatibility of selected MAX phases, Ti3AlC2, Ti3SiC2, and Ti2AlN, were systematically evaluated using in vitro tests for the first time. These phases were anoxic to preosteoblasts and fibroblasts. Compared with the strong viable fibroblasts, the different cellular responses of these materials were clearly distinguishable for the preosteoblasts. Under the osteoblastic environment, Ti2AlN exhibited better cell proliferation and differentiation performance than the Ti3AlC2 and Ti3SiC2. Moreover, the performance was superior to that of a commercial Ti–6Al–4V alloy and comparable to that of pure Ti. A possible mechanism was suggested based on the different surface oxidation products, which were determined from the binding energy of adsorbed Ca2+ ions using first-principles calculations. Compared with the partially oxidized TiCxOy layer on Ti3AlC2 and Ti3SiC2, the partially oxidized TiNxOy layer on the Ti2AlN had a stronger affinity to the Ca2+ ions, which indicated the good cytocompatibility of Ti2AlN.

KEYWORDS: MAX phase, Ti2AlN, cytocompatibility, in vitro test, first-principles calculations

1. Introduction Traditional Ti-based materials such as pure Ti and Ti–6Al–4V alloy have been widely applied as dental materials.

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However, their poor resistance to physical abrasion and fatigue

fracture require enhancement. 4-6 Metal carbide or nitride coatings (such as TiC, TiN, TiON and TiAlN) are used to strengthen the surface hardness and wear resistance; however, the bonding strength of the interface poses a new challenge.

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Tin+1AXn phases, which are MAX phases,


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exhibit good tribological properties, damage tolerance, and oxidation and corrosion resistance because of their unique structure, in which alternate near-close-packed layers of covalent M6X octahedrons are interleafed with metal-like A atom layers.

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Their flexural strength and

fracture toughness (Ti3AlC2, 340–450 MPa 16, 4.6–9.1 MPa·m1/2 15; Ti3SiC2, 720 MPa 17, 4.5–16 MPa·m1/2 15; Ti2AlN, 350–371 MPa 18, 8.2 MPa·m1/2 19) are much greater than those of common bioceramics (such as hydroxyapatite, 65–85 MPa

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, 0.65–0.70 MPa·m1/2

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). Thus, these

machinable ceramics have been used as concrete dry drills, gas burner nozzles, and heat exchangers. 12, 15 Recently, the MAX phase was also introduced to the biomedical field as a structural material or secondary phase to strengthen and toughen hydroxyapatite.

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However, studies

have specifically focused on Ti3SiC2, which has been confirmed to be bio-inert and will not result in adverse foreign body reaction.

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The SiC-reinforced Ti3SiC2 composite induces a few

foreign-body granulomas and eosinophilic leukocytes.

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The reason for this behavior remains

unclear, and verification of the cytocompatibility of MAX phases, especially for the other phases in this large family, remains insufficient. The biological response to host tissue cells must be considered in the development of synthetic biomaterials.

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In this study, we investigated the cytocompatibility of selected MAX

phases (Ti3AlC2, Ti3SiC2, and Ti2AlN) through cell adhesion, proliferation, and differentiation with preosteoblasts and fibroblasts and compared the results with those for traditional Ti-based materials (pure Ti and Ti–6Al–4V alloy). Moreover, first-principles calculations were employed to further reveal the mechanism. The aim of this work was to explore the factors that affect the cytocompatibility of MAX phases from the direction of different chemical compositions and to establish a reasonable mechanism to infer the cytocompatibility of other MAX phases.


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2. Methods 2.1 Fabrication of Ti3AlC2, Ti3SiC2, Ti2AlN, Ti–6Al–4V alloy, and pure Ti slices The Ti3AlC2 and Ti3SiC2 bulks were prepared from commercial powders (98%, 300 mesh, Beijing Jinhezhi Materials Co., Ltd., China) using hot pressing (ZT-60-24Y, Chenhua Co., Ltd., China) in an Ar atmosphere with a maximum pressure of 20 MPa. The heating rate was 10°C/min, and the temperature was held for 2 h at 1350°C for Ti3AlC2 and at 1400°C for Ti3SiC2. The Ti2AlN bulks were synthesized in-situ using a pulse-electric-current-aided sintering device (HP D 25/3; FCT Group, Germany) with a Ti–1.1Al–TiN ground powder mixture (Ti and Al, 99.5%, 300 mesh; TiN, 300 mesh, 99.9%; Targets Research Center of General Research Institute for Nonferrous Metals, China) in an Ar atmosphere. The green pellet was calcined in a graphite mold at 1350°C for 1 h with a maximum pressure of 30 MPa. 28 The commercial pure Ti (ASTM Grade 4) and Ti–6Al–4V alloy (ASTM Grade 5) were purchased from Seoul Titanium Co., Ltd., Korea. All the specimens were processed into 10 mm × 10 mm slices (for cell adhesion and proliferation) and 20 mm × 20 mm slices (for cell differentiation) using wire cut electric discharge machining. Before the in vitro tests, the slices were sequentially polished using 400, 800, and 1500 grit SiC paper. Then, the as-rinsed slices were disinfected in ethanol for 30 min and ultraviolet rays for 3 days in a clean bench. 2.2 Characterization of the self-made MAX phase bulks The chemical phase-analysis of the synthesized MAX phases were evaluated using X-ray Diffraction (XRD, Bruker AXS D8 Advance, Germany) with Cu Kα radiation and their spectra were collected at a step scan of 0.02° 2θ and a step time of 0.2 s. The microstructure was observed using field emission scanning electron microscope (SEM, Merlin Compact, ZEISS,


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Germany) equipped with energy dispersive spectroscopy (EDS, Thermo Scientific, USA). The surface composition was detected using X-Ray Photoelectron Spectroscopy (XPS, AXIS-His, Kratos, Japan). The hydrophilicity was evaluated based on the contact angle with approximately 10 µL of deionized water using a goniometer (Phoenix 300, Korea) at ambient atmosphere. 2.3 In vitro tests A mouse preosteoblast cell line (MC3T3-E1; ATCC, CRL-2593, USA) and mouse fibroblast cell line (L929; derivative of strain L) were employed in the in vitro test. All the cells were cultivated in alpha-minimum essential medium (α-MEM, Welgene Co., Ltd., Korea) with fetal bovine serum (FBS; Cellgro, USA) (10% FBS for preosteoblasts, 5% FBS for fibroblasts) and 1% penicillin streptomycin (Pen Strep; Life Technologies Co., Ltd., USA) in a humidified incubator (Sanyo Co., Ltd., Japan) with 5% CO2 at 37°C. A dye-exclusion assay (Trypan blue) was used to count the cells. The cell adhesion was determined based on the preosteoblasts or fibroblasts (1 mL, 1×104 cells/mL) after cultured on the surface of the Ti3AlC2, Ti3SiC2, Ti2AlN, Ti–6Al–4V alloy and pure Ti for 3 h. The cells were then stained using phalloidin (Life Technology Co., Ltd., USA) and 4', 6-diamidino-2-phenylindole (DAPI, Life Technology Co., Ltd., USA). The morphology and distribution were recorded using the confocal laser scanning microscopy (CLSM; FluoView FV1000, Olympus Co., Ltd., Japan). The spreading area of the preosteoblasts was calculated using ImageTool 3.0 from fifty CLSM images for each material at ×20 magnification. The MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium) assay was used to evaluate the cell proliferation. After being cultured for 3, 5 or 7 days, the optical density (OD) of the formazan product was detected using a microplate reader


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(EZ Read 400, Biochrom Ltd. UK) at 490 nm. CLSM was also used to examine the distributions of the preosteoblasts after incubation for 7 days for verification. The alkaline phosphatase (ALP) assay was applied to determine the cell differentiation. After incubation for 12 days, the total protein of the cells was determined using the Bio-Rad protein assay kit (Bio-Rad Co., Ltd., USA) based on a series of bovine serum albumin (BSA; Sigma) standards. Then, the ALP activity was predicted based on the color change from pnitrophenyl phosphate (pNPP, Sigma) to p-nitrophenol (pNP). The amount of ALP was calculated based on the absorbance value at 405 nm (OD value) per milligram of total cellular protein. 2.4 Statistical analysis All the experiments were conducted with at least three parallel samples. The results are expressed as the mean ± standard deviation. A value of p < 0.05 was considered statistically significant according to the Student's t-test method using Statistical Product and Service Solutions software. 2.5 Computational details First-principles calculations were preformed to investigate the phenomenon of the Ca2+ ion bound on these selected MAX phases with the CASTEP package. pseudopotentials

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The ultrasoft

and the plane-wave basis were adopted for the spin-polarized density

functional theory computation. A kinetic-energy cutoff for plane-wave expansion was set to 420 eV in our calculations. The generalized gradient approximation (GGA) was expressed by the Perdew–Burke–Ernzerhof (PBE) functional for the exchange-correlation potential.

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The


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effect of van der Waals (vdW) interactions based on the PBE functional was included explicitly using the empirical correction scheme of Grimme. 33 The vacuum distance was set to 16 Å along the z direction to avoid the artificial interlayer interaction caused by periodic boundary condition. All the atoms in the unit cell were fully relaxed until the convergence criterion on each atom was less than 10–5 eV in energy and 0.03 eV Å–1 in force. The Brillouin zones were sampled with a 9×9×1 k-point grid using Monkhorst–Pack scheme. 34

3. Results and discussion 3.1 Characterization of the self-made MAX phase bulks


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Figure 1. Characterization of the self-prepared MAX phases used for in vitro tests: (A) XRD spectra of Ti3AlC2 (black line), Ti3SiC2 (red line), and Ti2AlN (blue line) bulks; (B-D) SEM surface morphology; (E-G) XPS spectra of Ti 2p region. The phase composition of each self-made MAX phase bulk was determined using XRD. The spectra in Figure 1A indicated that all the MAX phase bulks possessed a high purity. In particular, for Ti3SiC2 and Ti2AlN, no apparent impurity peaks were observed. For the Ti3AlC2 bulk, the amount of remaining intermetallic TiAl3 was approximately 2 wt.% according to the supplier. However, this intermetallic phase commonly exists in Ti–6Al–4V alloy,

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which is

widely used as a commercial biomaterial. To the best of our knowledge, the related works did not note that TiAl3 is harmful to the human body. Hence, we believe that these self-prepared bulks could be used as platforms to evaluate the cytocompatibility of the corresponding MAX phases. The SEM images showed that these platforms used for in vitro tests had dense body and similar morphology (Figure 1B-D). The XPS spectra exhibited that besides the Ti-C bond or TiN bond belonged to the MAX phases, the Ti-O bond and Al-O bond existed on the surfaces of the Ti3AlC2 and Ti2AlN, and the Ti-O bond and Si-O bond existed on that of Ti3SiC2 (Figure 1E-G and Figure S1-S3). The presence of oxygen on the surfaces of MAX phases was also detected by the analysis of EDS (Table S1). These results were consistent with the previous works, 37-38 which attested that the MAX phases were covered by partially oxidized layer like the partially oxidized layer on the TiC and TiN. 39-40

3.2 Biological behaviors of preosteoblasts and fibroblasts on the MAX phase


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To evaluate the adhesive behavior of the preosteoblast on the surface of Ti3AlC2, Ti3SiC2, Ti2AlN, Ti–6Al–4V alloy, and pure Ti, CLSM was employed. As observed in Figure 2A, the cells attached on all these materials very well. Compared with the Ti alloy and pure Ti, the spreading of preosteoblast on the MAX phase, particularly on the Ti2AlN was promoted in the i

n

i

t

i

a

l

Figure 2. (A) Typical CLSM images used to determine the morphology of MC3T3-E1 cells attached on the surfaces of Ti3AlC2, Ti3SiC2, Ti2AlN, Ti–6Al–4V alloy, and pure Ti specimens after incubation for 3 h. Phalloidin and DAPI were used to stain the filamentous actin (red) and nucleus (blue), respectively. The seeding cell density was 1×104 cells/mL. (B) Spreading areas of the attached preosteoblasts calculated using ImageTool 3.0 based on fifty CLSM images (shown


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in Figure S4) for each material at ×20 magnification. (**p < 0.01) (C) Hydrophilicity determined by the contact angle with deionized water.

adhesion stage. Based on the fifty CLSM images (shown in Figure S4), the cell spreading area on the Ti2AlN calculated using the ImageTool 3.0 was 2901 µm2/cell, which was larger than that of the Ti alloy (2372 µm2/cell) or pure Ti (2450 µm2/cell) (Figure 2B). Because the hydrophilic materials induced a higher level of the adhesion and spreading of the MC3T3-E1 cells,

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the

contact angle results with deionized water corroborated these findings. The more hydrophilic Ti2AlN (37.0°) exhibited better initial cell attachment compared with the commercial Ti–6Al–4V alloy and pure Ti (52.9° and 51.6° respectively, as shown in Figure 2C).


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Figure 3. Proliferation and differentiation of MC3T3-E1 cells on the Ti3AlC2, Ti3SiC2, Ti2AlN, Ti–6Al–4V alloy, and pure Ti specimens. (A) MTS assay for determination of the cell viability after 5-day and 7-day incubation; (B) typical CLSM image used to determine the corresponding distribution of MC3T3-E1 cells grown after 7 days (DAPI stained the nucleus with blue color); (C) ALP activity used to determine the differentiation of MC3T3-E1 cells after 12-day incubation. The seeding cell density was 1×104 cells/mL. (*p < 0.05, ***p < 0.001) The viability of the preosteoblasts was then characterized by MTS assay and direct CLSM observation. Based on Figure 3A, the MAX phase exhibited non-toxicity on the preosteoblast, and the cells actively proliferated on all the materials. Among the selected MAX phases, Ti2AlN exhibited the best performance based on the optical density value. Moreover, the cell viability in the Ti2AlN group was even higher than that in the commercial Ti–6Al–4V alloy and was comparable to that in the pure Ti after 7 days of incubation. This phenomenon was also supported by the distribution of the preosteoblasts directly observed using CLSM (Figure 3B). In contrast to Ti3AlC2, Ti3SiC2, and Ti–6Al–4V alloy, a larger number of preosteoblasts was universally distributed on the Ti2AlN. During the osteoblastic differentiation, ALP was formed to promote the hydrolysis of phosphate esters, which was used as an early marker to determine the differentiation degree of preosteoblasts.

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In this study, the ALP activity of the MC3T3-E1 cells cultured on the

surfaces of Ti3AlC2, Ti3SiC2, Ti2AlN, Ti–6Al–4V alloy, and pure Ti for 12 days was recorded (Figure 3C). Among the selected MAX phases, the ALP activity in the Ti2AlN group was significantly higher than that in the other two phases. The ALP activity of Ti2AlN was almost twofold and fourfold of that of Ti3AlC2 and Ti3SiC2, respectively. Although a noticeable


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difference in ALP activity between Ti2AlN and pure Ti was hardly detected, the osteoblastic differentiation on the Ti2AlN was superior to that on commercial Ti–6Al–4V alloy. Fibroblasts were also employed to evaluate the cytocompatibility of the Ti3AlC2, Ti3SiC2, Ti2AlN, Ti–6Al–4V alloy, and pure Ti specimens. Similar cell adhesive behavior after seeding for 3 h was detected using CLSM (Figure S5A). The cell proliferation determined from the MTS assay and CLSM images also revealed little difference (Figure S5B-C). The mean values of each group obtained from the MTS assay at 5 days were at similar levels and the error bars overlapped. However, during the initial incubation stage (approximately 3 days), the fibroblasts proliferated faster on Ti2AlN than on the other phases, possibly because of the strong viability of the fibroblasts, whose proliferation rate was almost three times that of the osteoblasts.

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It was

difficult to distinguish slight differences between these materials. In other words, the cytocompatibility of these MAX phases was as good as the reference Ti–6Al–4V alloy and pure Ti under fibroblastic condition.

3.3 Theoretical calculation of Ca2+ ions absorbed on the MAX phase The Ca2+ ion plays a prominent role bridging a net negative substrate and anionic protein (integrin) for cell attachment. 45 The deposition of calcium is the first step of calcium phosphate nucleation, which is critical for implants in contact with bone tissue. 40, 46 Hence, the simulation of the process of Ca2+ adsorbed on the substrate can be used to evaluate the cytocompatibility of the biomaterials. For instance, the negative charge is calculated to prove this process in TiO2 and TiN’s works. 40, 46 Herein, a new quantitative indicator (binding energy of the Ca2+ ions absorbed on the substrate) is used to directly evaluate this absorption process since the surfaces of MAX


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phases (Ti3AlC2, Ti3SiC2, Ti2AlN) are proven negative charged in the normal pH range of body fluids according to the analysis of zeta potential 47-50. In the light of the layered hexagonal crystal, the (100) and (001) surfaces were selected as representative surfaces based on the experimental XRD data (Figure 1A). To assess the stability of the naked surface slab, we first computed the cohesive energy (Ec) of the (100) and (001) surface slabs, which was defined as: = − −

/ / − / / ⁄ , where , , / , and / are the total energies of a surface slab, single Ti atom, single Al or Si atom and single N or C atom, respectively, and , ,

/ , and / are the total number of atoms and the respective number of Ti, Al (or Si) and N (or C) atoms in the supercell. To further evaluate the stability of the formation of the naked surface slabs relative to the simple stable substances, we defined the formation energy as: = − −

/ / − / / ⁄ , where the chemical potentials , / , and / were defined as the energies per atom in bulk Ti, bulk Al or Si, and a nitrogen molecule or graphite, respectively. Ec and Ef of these surface slabs are listed in Table 1. All the bare (001) surfaces were the most stable and the easiest to be observe experimentally because they had the largest Ec and Ef. However, for the bare (100) surface, there were three possible configurations for the MAX phase. As shown in Figure 4 and Table 1, the (100)-3 configuration could be transformed into the (100)-1 configuration for Ti2AlN because the single-coordinated Ti atom was unstable and readily reacted with water or oxygen. However, the single-coordinated Ti atom also tended to be removed from the surface of


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the (100)-2 configuration to form a planar TiC surface, which was similar to the structure of the (100)-1 configuration for the Ti3AlC2 and Ti3SiC2 after exposure to oxygen or water. Thus, the (100)-1 and (100)-2 configurations of Ti2AlN and the (100)-1 and (100)-3 configurations of both Ti3AlC2 and Ti3SiC2 were selected to investigate the following adsorption of the Ca2+ ion on the (100) surface.

Table 1. Ec, Ef, and ∆Ebind of the absorbed Ca2+ ions for each configuration shown in Figure 4 and Figure 5.

Ec

Ef

∆Ebind

Ec

Ef

∆Ebind

eV/atom

eV/atom

eV/atom

eV/atom

eV/atom

eV/atom

TAC001

–7.31

–0.71

–19.14

TSC100-3

–6.78

–0.15

–15.63

TAC100-1

–6.58

–0.26

–16.28

TAN001

–6.45

–1.17

–24.71

TAC100-2

–6.55

–0.20

-

TAN100-1

–5.95

–0.79

–18.53

TAC100-3

–6.63

–0.15

–15.90

TAN100-2

–5.99

–0.88

–15.74

TSC001

–7.47

–0.75

–19.96

TAN100-3

–5.85

–0.63

-

TSC100-1

–6.66

–0.19

–16.94

rTiO2110

-

-

–21.83

TSC100-2

–6.62

–0.026

-

surface

surface

TAC, TSC and TAN represent Ti3AlC2, Ti3SiC2 and Ti2AlN, respectively, in this table.


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Figure 4. Different configurations of the (100) and (001) surfaces of Ti3AlC2, Ti3SiC2, and Ti2AlN.

Figure 5. Optimized configurations for the Ca2+ ion bound on the (001), (100)-1, (100)-2, and (100)-3 surfaces of the Ti3AlC2, Ti3SiC2, and Ti2AlN and the (110) surface of rTiO2.


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The MAX phase should be passivated by an oxidation reaction on the surface in ambient atmosphere.

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Piscanec et al. reported that a partially oxidized TiN surface can be used as a

preferential site for the deposition of Ca2+ ion.

40

We tested various possible partially oxidized

configurations followed by atomic relaxations. The optimized stable configurations of Ti3AlC2, Ti3SiC2, and Ti2AlN are presented in Figure 5. The O atom was bound to three Ti atoms were located above the N/C atom in the (001) configuration. Herein, the O atom was probably bound with the H atom to form a hydroxyl group (OH). However, this process was energetically unfavorable according to the formation energy ((F = − − ) > 0), especially under alkaline conditions. For the (100)-1 configuration of Ti2AlN, the bridging OH group (F= –0.43 eV) was coordinated by two Ti atoms bound to two N atoms, whereas the threefold-coordinated O atom was coordinated by one Al atom and two Ti atoms bound to three N atoms. Rodriguez et al. have shown that the CTiTi hollow is the most stable site for the adsorption of O atoms on the TiC (001) surface.

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In addition, for the (100)-1 configurations of

Ti3AlC2 and Ti3SiC2, one O atom was coordinated by two Ti atoms bound to three C atoms and by one C atom bound to five Ti atoms on the planar TiC surface, which is consistent with reports of Rodriguez et al. The other free surficial Ti and Al atoms were bound to the threefoldcoordinated O atoms. The oxidation behaviors of the (100)-2 configuration for Ti2AlN and the (100)-3 configuration for Ti3AlC2 and Ti3SiC2 were similar to that of the (100)-1 configuration, in which the O atom was also bound to two Ti atoms and an Al atom for Ti2AlN and the CTiTi hollow for Ti3AlC2 and Ti3SiC2, respectively (as observed in Figure 5). We also tested the possible adsorption sites of Ca2+ ions on the surface of partially oxidized configurations of Ti3AlC2, Ti3SiC2, and Ti2AlN (shown in Table S2 and Figure S6-S7). The


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preferred adsorption site of Ca2+ ions on the surface and the corresponding binding energy are shown in Figure 5 and Table 1. The binding energy was defined as: ∆ $ = %& '() − %& ' − *() /* where () , %& ' , and %& '() represent the total energies of a single Ca2+ ion, the specific partially oxidized surface of the MAX phase, and the corresponding configuration after Ca2+ ions absorbed on this surface, respectively. m is the number of absorbed Ca2+ ions. In the (001) configuration, the Ca2+ ion was coordinated by three O atoms (Figure 5). The binding energies of the Ca2+ ion on the (001) configurations of Ti3AlC2, Ti3SiC2, and Ti2AlN were – 19.14, –19.96, and –24.71 eV/atom, respectively. In addition, the Ca2+ ion was coordinated by four O atoms in the (100)-1 configuration (Figure 5), and the binding energies were –16.28, – 16.94, and –18.53 eV/atom for Ti3AlC2, Ti3SiC2, and Ti2AlN, respectively. For the (100)-2 configuration of Ti2AlN, the Ca2+ ion tended to bind to two threefold-coordinated N atoms with a binding energy of –15.74 eV/atom, whereas the Ca2+ ion preferred to bind to two C atoms and one O atom in the (100)-3 configuration with binding energies of –15.90 (–15.63) eV/atom for Ti3AlC2 (Ti3SiC2), respectively (Figure 5). For comparison, we studied the adsorption of Ca2+ ions on a rutile TiO2 (110) surface, which was the most stable representative surface of TiO2 46. The Ca2+ ion was coordinated by two bridging O atoms and two terminal O atoms of the OH group, which was consistent with the result of Svetina’s work 46. The binding energy was –21.83 eV, which was considered the source of the good cytocompatibility of the TiO2. The partially oxidized (001) surface of the Ti2AlN exhibited a stronger affinity to the Ca2+ ion, in contrast with that of TiO2. However, the binding strength of the Ca2+ ion on the partially oxidized (100) surfaces of Ti2AlN and the (001) and (100) surfaces of both Ti3AlC2 and Ti3SiC2 were weaker


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than that on TiO2. These results indicate that the partially oxidized Ti2AlN exhibited improved cytocompatibility compared with the partially oxidized Ti3AlC2 and Ti3SiC2 and comparable cytocompatibility as the pure Ti, which is in excellent agreement with the experimental observations.

3.4 Mechanism for cytocompatibility of the MAX phase The cell affinity is known to be affected by the surface hydrophilicity, chemical composition, net charge, topography, and other biochemical-physical properties. 27, 53 Herein, we observed that not only the hydrophilicity but also the intrinsic features of the chemical elements played important roles in the behavior of these selected MAX phases. Because the real compositions of Ti3AlC2, Ti3SiC2 and Ti2AlN surface are composed of Al2O3 and TiCxOy, SiO2 and TiCxOy, and Al2O3 and TiNxOy, respectively

37, 39-40

(Figure 1E-G and Figure S1-S3), and

the surface of pure Ti and Ti–6Al–4V alloy are protected by TiO2, and TiO2 and Al2O3, 54-55 the different cytocompatibility of these materials can be explained based on the different biological properties of these oxides, carbides and nitrides. It is unquestionable that the TiO2 has the best cytocompatibility among these oxides.

56-58

However, TiC layer and TiN layer were shown to

enhance the cytocompatibility of Ti–6Al–4V alloy

59

, NiTi alloy

60-61

, and Co–Cr alloy

62

. In

particular, the TiN layer even improved the cytocompatibility of pure Ti 63 because the TiN layer not only physically prevented the release of toxic ions, such as vanadium and nickel ions, but also improved the cell functions, including promoting the adsorption of proteins, increasing the metabolism of energy and amino acids, and enhancing the regulation of inflammation. 59-61, 64


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To determine the source of the cytocompatibility, the adsorption of Ca2+ ions on the surface of the materials is commonly used in theoretical calculation. 40, 46 Stefano et al. demonstrated that the TiNxOy surface could initiate spontaneous nucleation of calcium phosphate because of the presence of a higher surface negative charge (TiO2, –0.45; the partially oxidized TiN, –0.80).

40

In our experiment, the binding energy between the absorbed Ca2+ ion and the surface was first calculated using first-principles theory for direct evaluation. Compared with Ti3AlC2 and Ti3SiC2, Ti2AlN had a notable stronger binding energy for the partially oxidized (001) and (100) surfaces. The above analyses were consistent with our in vitro tests, in which Ti2AlN exhibited preferable osteoblastic proliferation and differentiation compared with Ti3AlC2 and Ti3SiC2; the performance was even superior to that of the commercial Ti–6Al–4V alloy and comparable to that of pure Ti. This discovery is interesting, as previous studies on the MAX phase for biomedical applications have only focused on Ti3SiC2, 23-24 which is considered to have the best resistance to oxidation and corrosion of the traditional MAX phases.65-66 However, this study revealed that the inertness was not the dominant factor determining the cytocompatibility of the MAX phase. The Ti2AlN exhibited outstanding cytocompatibility because of the TiNxOy surface. The above analyses illustrated that the chemical composition of the MX layer in MAX phase played a more critical role than the A site atom. This mechanism also provided a direction to select a MAX phase for biomedical applications, which is valuable for such a large family. Based on this work, we speculate that the Tin+1ANn group (such as Ti4AlN3) has excellent cytocompatibility. It is also notable that the two-dimensional early transition metal carbides or nitrides (MXenes), mainly consisting of these (001) surfaces,67-69 probably exhibit remarkable cytocompatibility due to the


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strongest binding energy of the Ca2+ ions on the (001) surface compared with the other surfaces (Table 1).

4. Conclusion This study used self-made pure MAX phase slices as platforms to evaluate the cytocompatibility of Ti3AlC2, Ti3SiC2, and Ti2AlN in in vitro tests. All the phases were determined to be cytocompatible with respect to the commercial Ti–6Al–4V alloy and pure Ti with preosteoblasts and fibroblasts. The cell proliferation and differentiation examination under an osteoblastic environment revealed that Ti2AlN exhibited the optimal performance among these MAX phases; its performance was even superior to that of the commercial Ti–6Al–4V alloy and comparable to that of pure Ti. The mechanism for this behavior was interpreted for the different protective layers. First-principles calculations revealed that Ca2+ ions had a strong affinity for adsorption on the TiNxOy layer, which indicated the good cytocompatibility of Ti2AlN. Our discovery provides a new route for the selection of specific MAX phases for biomedical applications.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.xxxxxxx. The SI contains XPS spectra (Figure S1-S3), EDS data (Table S1), CLSM images for calculating spreading area of


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preosteoblast (Figure S4), adhesion and proliferation of fibroblasts (Figure S5), and the detailed results of the optimization process in first-principles calculation (Table S2 and Figure S6-S7).

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China [Grant Nos. 91226202 and 91426304], CAS Interdisciplinary Innovation Team Project and Ningbo Natural Science Foundation [Grant No. 2016A610273]. Ke Chen acknowledges financial support received from the China Scholarship Council (CSC) [Grant No. 201608260009]. We thank Dr. Cheol-Min Han from KOREA Institute of Science and Technology for revising our manuscript.

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For Table of Contents Use Only Cytocompatibility of Ti3AlC2, Ti3SiC2, and Ti2AlN: In vitro Tests and Firstprinciples Calculations

Ke Chen

a,‡

, Nianxiang Qiu

b,‡

, Qihuang Deng b, Min-Ho Kang a, Hui Yang b, Jae-Uk Baek a,

Young-Hag Koh c, Shiyu Du b, Qing Huang b,*, Hyoun-Ee Kim a,*


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The cytocompatibility of MAX phases (Ti3AlC2, Ti3SiC2, and Ti2AlN) in direction of different compositions were systematically compared. The first-principles calculations attributed the good cytocompatibility of Ti2AlN to the original TiNxOy layer, which had a strong binding energy with Ca2+ ion.


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Cytocompatibility of Ti3AlC2, Ti3SiC2, and Ti2AlN: In Vitro Tests and

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