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Surface Mechanoengineering of a Zr-based Bulk Metallic Glass via Ar-Nanobubble Doping to Probe Cell Sensitivity to Rigid Materials Lu Huang, Mengkun Tian, Dong Wu, Gerd Duscher, Peter K. Liaw, and Wei He ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b12663 • Publication Date (Web): 24 Nov 2017 Downloaded from http://pubs.acs.org on November 29, 2017
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ACS Applied Materials & Interfaces
Surface Mechanoengineering of a Zr-based Bulk Metallic Glass via ArNanobubble Doping to Probe Cell Sensitivity to Rigid Materials Lu Huang1, Mengkun Tian2*, Dong Wu1, Gerd Duscher1,3, Peter K. Liaw 1*, and Wei He1 1
Department of Materials Science and Engineering, University of Tennessee, Knoxville, TN37996, USA 2 Department of Chemical and Biomaterials Engineering, University of Tennessee, Knoxville, TN37996, USA 3 Materials Science and Technology Division, Oak Ridge National Laboratory, Oak Ridge, TN37831, USA
Abstract: In this study, a new materials platform, utilizing the amorphous microstructure of bulk metallic glasses (BMGs) and the versatility of ion implantation, was developed for the fundamental investigation of cell responses to substrate-rigidity variations in the gigapascal modulus range, which was previously unattainable with polymeric materials. The surface rigidity of a Zr-AlNi-Cu-Y BMG was modulated with low-energy Ar-ion implantation owing to the impartment of Ar nanobubbles into the amorphous matrix. Surface softening was achieved due to the formation of nanobubble-doped transitional zones in the Zrbased BMG substrate. Bone-forming cell studies on this newly designed platform demonstrated that mechanical cues, accompanied with the potential effects of other surface properties (i.e. roughness, morphology, and chemistry), contributed to modulating cell behaviors. Cell adhesion and actin filaments were found to be less established on less stiff surfaces, especially on the surface with an elastic modulus of 51 GPa. Cell growth appeared to be affected by surface mechanical properties. A lower stiffness was generally related to a higher growth rate. Findings in this study broadened our fundamental understanding concerning the mechanosensing of bone cells on stiff substrates. It also suggests that surface mechano-engineering of metallic materials could be a potential strategy to promote osseointegration of such materials for bone-implant applications. Further investigations are proposed to fine tune the ion implantation variables in order to further distinguish the surface-mechanical effect on bone-forming cell activities from the contributions of other surface properties. 1 ACS Paragon Plus Environment
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Keywords: Bulk metallic glass; Ion implantation; Microstructure; Mechanical behavior; Cell rigidity sensing.
1. Introduction Cell interactions with implantable biomaterials are essential to the ultimate fates of the implants. Proper cell behaviors on biomaterial surfaces are desired in order to warrant successful implant integrations. To modulate cell behavior on the surface of a biomaterial, surface-modifications in chemistry topography
3-7
1, 2
and
have been extensively researched and validated. In recent
decades, regulating cell behavior with mechanical cues has emerged as a subject of great research interest.
8-10
An increasing number of studies have
revealed that cells can sense the rigidity of their growth substrates, and transduce the effects of the mechanical microenvironment to change adhesion, proliferation, migration, and differentiation behaviors.11, 12 In the majority of these studies on rigidity sensing, cells were interacting with “soft” polymeric materials, such
as
agarose
and
alginate
hydrogels,
polyacrylamide-based
gels,
poly(ethylene glycol)-based hydrogels, and polydimethylsiloxane, with relatively low elastic moduli ranging from tens of pascal (Pa) to several megapascals.
8-17
Recently, Smith et al. used methacrylate-based networks to extend the moduli range to cover 60 to 850 MPa and studied cell responses.
18
Despite the
progress, little has been reported to date on cell responses to substrate rigidity within the gigapascal modulus range. Considering hard tissues, such as mineralized bone with high elastic modulus (~ 20 GPa), 19, 20 and the prevalent use of rigid materials, such as Ti and its alloys (moduli ~ 50-100 GPa) in implantable orthopedic prostheses, it is of significant value to improve our understanding on cell behavior on highly rigid materials. In the current work, we were interested in two initial questions: (1) will cells sense substrate-rigidity change in the gigapascal range? (2) If so, what responses will cells exhibit? To address these two questions, we employed ion implantation on bulk metallic glasses (BMGs) to modulate substrate rigidity in the gigapascal modulus range and investigated cell responses. This approach not only provides opportunities to
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fundamentally comprehend cell mechanosensing on rigid metallic surfaces, but also can be beneficial to guide the engineering of metallic implants for enhanced tissue integrations. The rationale of selecting BMGs over common biomedical alloys as our test substrates to probe cell mechanosensing of high stiffness materials is based on the homogeneous microstructures of BMGs. Traditional crystalline biomedical alloys, such as Ti alloys, consist of different phases, grain boundaries, and segregations, all of which can exhibit distinctive mechanical properties.
21, 22
This
inhomogeneity in the mechanical microenvironments could confound the interpretation of cell responses to the overall mechanical variations among substrates. In contrast, the amorphous microstructure of BMGs overcomes the above issues, and thus allows cell-mechanosensitivity studies conducted on surfaces exhibiting uniform mechanical properties. Although initially developed as a new family of engineering materials, BMGs have attracted great attentions from the biomaterials field because of their bulk mechanical properties, chemical stability, and polymer-like thermal processability23-26, which further makes BMGs a relevant model material for the present work. Among many different types of BMGs available, a Zr-based (Zr0.55Al0.10Ni0.05Cu0.30)99Y1 (at.%) BMG was used. Previous work has demonstrated that this Zr-based BMG is cytocompatible with MC3T3-E1 preosteoblasts,
27, 28
a cell line commonly used for bone-implant
related studies, including in the context of mechanosensing, and is the subject of investigations herein. To vary the surface rigidity of the Zr-based BMG, we opted for a versatile yet facile surface-modification method, namely ion implantation. The effects of ion implantation to alter mechanical properties of BMGs were previously reported. 29-33
By adjusting implantation parameters (i.e., ion-beam energy, fluence, and
flux), it is feasible to fine-tune the surface-mechanical properties of BMGs over a broad span and allow the subsequent study of cell responses to such changes. 2. Results and Discussion In the present study, the Zr-based BMG substrate was modified by low energy implantations with inert Ar ions at different implantation energies and 3 ACS Paragon Plus Environment
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fluences in order to confine the effects to the near-surface region. For denotation purposes, samples were labelled as Zr-AC, Zr-50L, Zr-50H, Zr-100H, and Zr200H for as-cast and various ion-implanted Zr-based BMG surfaces, as summarized in Figure 1a. Mechanical modifications to the Zr-based BMG as a result
of
Ar-ion
implantations
were
characterized
by
nanoindentation.
Representative load-displacement curves (Figure 1b) showed that for all the ionimplanted samples, the maximum loads at the maximum indentation depth of 35 nm were lower than that of the Zr-AC sample. From these curves, elastic modulus (Em) of the respective sample plotted in Figure 1c was derived according to Oliver-Pharr method:34 E = (1-ν2)S/(2γa) Where S is the stiffness, a is the contact radius of the indentation, ν is the Poisson’s ratio of the sample, and it is about 0.362 for Zr-based BMG, 35 γ is the correction factor. 36 Generally, the Zr-based BMG surfaces after ion implantation were “softer” than the Zr-AC sample, and the extent of softening was dependent on the implantation energy and fluence. Specifically, the Em of the BMG surface decreased as the implantation energy decreased or the fluence increased. Collectively, these samples provide a modulus range spanning from 51 to 86 GPa. We also obtained stiffness and nanohardness from nanoindentation tests. The effect of ion implantation on both properties shared a similar trend with that for nanohardness (Figure S1). It is noted that the obtained values based on the nanoindentation technique in the present study are considered semi-quantitative. First, the differences in the modified layer thicknesses and radiation-damage distribution amongst different specimens could result in different “substrate effects”, as explained in greater detail in the supporting information. Second, the potential alterations in other properties (i.e., surface roughness, morphology, etc.) due to the ion-implantation treatment to the Zr-based BMG surfaces could affect the elastic-plastic contact between the indenter and the test sample, leading to discrepancy in the measured results. Nevertheless, the relative comparisons within the test group remained legitimate, which evidenced that ion implantation can be used to successfully adjust the mechanical properties of the Zr-based 4 ACS Paragon Plus Environment
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BMG surfaces. In previous studies of the effect of room temperature Ar ion implantation on various Zr-based BMGs, both surface hardening softening
33
30-32
and
have been observed under different irradiation conditions. The
underlying mechanism governing the ion implantation induced mechanical modifications was commonly suggested as the irradiation-induced microstructural alteration (i.e., structural relaxation or formation of nanocrystals). 30-33
Figure 1 (a) Summary on ion implantation parameters and denotation of tested samples; and (b) representative load-displacement curves and (c) elastic moduli of indicated substrates measured using nanoindentation (groups with statistically-significant differences at p < 0.05 were separated by different symbols).
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To elucidate the specific softening mechanism associated with the Ar-ion implantation conditions used in the current study, we analyzed the microstructure of the surface regions on the Zr-based BMGs using transmission electron microscopy (TEM). Cross-sectional TEM Z-contrast images for the near-surface regions (Figure 2a) revealed the formation of nanosized fine features after ion implantation. Depending on the implantation conditions, the size of those features and their depth profiles varied. For the Zr-50L and Zr-200H specimens, these nanofeatures were limited within a ~ 55 nm-thick and a ~ 250 nm layer depths, respectively. The sizes of these nanofeatures were relatively small with diameters below 10 nm. In contrast, a drastically wider size distribution was noted for the other two implanted samples. For Zr-100H, larger features (40 - 80 nm in diameter) were seen distributed along the mid-section of the implanted zone, and were surrounded by much smaller nanofeatures. As for Zr-50H, features greater than 100 nm were formed when some of the round features merged to each other, resulting in protrusions or pockets on the surface. To further determine whether these implantation-induced nanosized fine features are voids or Ar bubbles, chemical analyses were carried out. The results of energy-dispersive X-ray spectroscopy (EDS) obtained from the implanted Zrbased BMG samples first confirmed the presence of Ar within the near-surface regions after implantation (Figure S2). We then used spatially resolved (~ 0.5 nm) electron energy loss spectroscopy (EELS) to reveal Ar distributions in the implanted layers by continuously recording spectra along the cross-section of the implanted samples. The intensities of the Ar-L2,3 ionization-edge were determined after background subtraction with a combination of a power law and a polynomial: E−r + aE2 + bE + c. Here, a, b, c, and r are fitting parameters, E is the energy.
37, 38
These intensities I of the Ar-L2,3 ionization edge are directly
related to the areal density N of Ar: I(β, ∆) = N(β, ∆) × σ(β, ∆) × IT. 39 Here, N is the absolute number of atoms per unit area, σ is the inelastic scattering crosssection, IT is the total transmitted intensity, β and ∆ represent the dependence of integration energy window and collection angle respectively. Since IT, β, and ∆ was kept constant in this experiment, the relative amount of Ar and Ar 6 ACS Paragon Plus Environment
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distributions could be directly obtained from EELS spectrum images. As shown in Figure 2b and Figure S3, EELS-based Ar mappings clearly suggest the nature of those small nanofeatures (diameter < 20 nm) observed in the Z-contrast images being Ar bubbles formed by the segregation of implanted Ar ions. Despite the absence of Ar signals in the larger diameter features, we rationalize that these apparent voids were originally filled with Ar, considering that the TEM samples were prepared as thin lamella of approximately 50 nm in thickness that inevitably led to the discharge of Ar gas from Ar bubbles with diameters above 50 nm.
Figure 2 Microstructure and Ar distribution analyses of the Zr-based BMG surfaces after ion implantation: (a) Z-contrast images; (b) magnified Z-contrast images and corresponding EELS mappings; as well as SRIM simulated (dashed red lines) and experimentally measured (solid blue lines) Ar-distribution profiles.
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Based on the EELS measurements, Ar depth profiles were also extracted (Figure 2b). The measured profiles exhibited distorted bell shapes, and the depth varied with the implantation energy, a higher value of which allowed deeper ion penetration. Peak concentrations of Ar were mostly located close to the midline of the implantation-modified layer, although disruptions occurred in the experimental measurements due to the artificial voids for Zr-50H and Zr-100H samples. The relative peak Ar concentrations determined by the highest numbers of Ar atoms were found to be dependent on both implantation energy and fluence. To complement the experimental observations, computational simulations using the Monte-Carlo algorithm with the Stopping and Range of Ions in Solids (SRIM) software 40 was further employed to estimate the potential ion- implantation effect on the Zr-based BMG substrates. The simulation results (Figure 2b and Figure S4) showed that the Ar profiles evolved with both the implantation energy and fluence, which are consistent with the experimental findings. In addition to the estimation of the Ar distribution, SRIM simulations can also be used to assess the implantation-induced damages (i.e., displacement per atom, dpa), which were found to be proportional to the applied fluence and decreased with the implantation dpa. With such a high disorder, the microstructure of the Zr-based BMG remained amorphous, which was verified by the selected area diffraction (SAD) and nano-beam electron diffraction (NBED) patterns41-43 in Figure 3b. The SAD patterns for the surface regions including the modified layer and for the substrate region without ion implantation influences were acquired for samples treated under indicated implantation conditions. These diffraction patterns indicate the amorphous (or highly-defective crystalline) microstructure of BMG, throughout the surface region for each substrate. The two reflection rings with the sharp intensity represent the short-range ordering (SRO) of the BMG. The most intense reflection ring represented the SRO of approximately 0.5 nm periodicity, and the second one represented the SRO of approximately 0.3 nm periodicity. In the meantime, these two rings were significantly broadened, implying the deviations of SRO periodicity. The SAD patterns for the surface and bulk-matrix regions after implantation were virtually 8 ACS Paragon Plus Environment
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identical, suggesting that no distinct structural changes were introduced to the implanted surface region of the Zr-based BMG after Ar-ion irradiation. NBED was used to further study the microstructure after implantation. Four different sites were inspected, as indicated in Figure 3b, including locations that were (A) near surface, (B) close to the middle of the implanted layer, (C) farther from surface but still within the implanted layer, and (D) away from the implanted layer. The NBED patterns further validated that no significant structural changes were introduced to the implanted surface region of the Zr-based BMG after Ar-ion irradiation, although the local structural distortion may have been introduced as indicated by the variation in the SRO measurement, which requires further investigations. Overall, it confirmed that no distinctive differences were found between the diffraction from the modified layer and that from the bulk matrix of the substrate. Collectively, these results demonstrate that the implantation processes used in the current study enabled the formation of Ar-nanobubbles doped within the surface layers with a graded distribution, while retaining the amorphous nature of the BMG substrate. The size, distribution, and concentration of these nanobubbles can be tuned by altering implantation parameters. The presence of these Ar-nanobubbles accounts for rigidity alterations on the Zr-based BMG surfaces. The degree of surface softening was directly related to the concentration of Ar within the modified layer. Specifically, a higher concentration of Ar in the surface layer was associated with a lower measured surface rigidity. The lowest Em of 51 GPa of the Zr-50H sample corresponds to the highest calculated Ar concentration of approximately 42 at.%. With the generation of the Ar nanobubble-doped layer on Zr-based BMG surfaces, mechanical variations at high rigidity levels (Em > 50 GPa) were readily achieved to study the mechanoresponses of bone-forming cells. For cell culture studies, we have confined our comparisons amongst Ar-ion-implanted specimens, owing to the significant difference on surface wetting properties of Zr-based BMGs before and after Ar-ion-implantation, which was demonstrated in our previous study. 30 The exclusion of as-cast sample will help to delineate the effect 9 ACS Paragon Plus Environment
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of substrate rigidity and reduce the effect of other surface properties, which will be discussed in more details in a later section.
Figure 3 Effects of Ar-ion implantations on the microstructure of (Zr0.55Al0.10Ni0.05Cu0.30)99Y1 BMG: (a) SRIM simulations on the distribution of disorders; (b) TEM observations on the surface crosssections (SAD images were taken from both ion-implanted and matrix regions; NBED were taken at indicated spots, as marked in the Z-contrast images).
As bone-forming cells are anchorage dependent, cell adhesion to substrates precedes any other cell functions and bears a significant role for cell growth. Therefore, adhesion of MC3T3-E1 cells on the Ar-ion-implanted Zr-based BMG 10 ACS Paragon Plus Environment
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samples was first characterized. All the ion-implanted samples supported the initial cell adhesion and maintained the cell viability (Figure S5). We focused on the adherent cell morphology via fluorescent staining of focal adhesion (FA) plaques and cytoskeletal actin filaments. FAs are important in maintaining cell adhesion to the surface and serve as physical linkages connecting the cytoskeletal system and the microenvironment.
44, 45
Regardless of the rigidity of
the ion-implanted BMG substrates, cells were able to develop a great number of FAs, which are seen primarily concentrated at the periphery of the cells (Figure 4a). However, quantitative analyses of FAs revealed that the average number of FAs per cell was influenced by substrate rigidity, with the least rigid Zr-50H surface (Em = 51 GPa) supporting the lowest amount of FA development (Figure 4b). The density of FAs and cell circularity were also found to be dependent on substrate rigidity (Figure S6). Staining of actin filaments revealed the formation of actin filament bundles running across the cell body and terminating at FAs (Figure 4a). These bundles, also known as stress fibers, are crucial in the formation of the cytoskeletal system and transduction of mechanical indications. 46, 47
Qualitatively speaking, the actin filament bundles also appeared to be
affected by substrate rigidity, where more pronounced stress fiber formation was observed for cells on Zr-50L (Em = 86 GPa) and Zr-200H (Em = 76 GPa) than those on Zr-50H (Em = 51 GPa) and Zr-100H (Em = 66 GPa). These results from the cell adhesion behavior study imply that variations in the substrate rigidity on the gigapascal scale can be sensed by bone-forming cells and contribute to the concurrent changes in cell functions, even though such high elastic moduli are not physiologically attainable. In comparison with previous findings obtained from MC3T3-E1 preosteoblast cells cultured on substrates with elastic moduli spanning kilopascal to megapascal rigidity, although with distinct materials chemistry amongst different studies,
15, 17, 48
the
general effect of substrate rigidity is similar. Cells respond to a stiffer surface by forming a higher density of FAs and exhibiting more developed cytoskeletal structures.
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Figure 4 Adhesion and proliferation of MC3T3-E1 bone-forming cells on indicated Zr-based BMG substrates: (a) representative immunofluorescent images of cells (green: vinculin; red: F-actin; blue: nuclei); (b) number of focal adhesion (FA) sites per cell and (c) Proliferation of MC3T3-E1 bone-forming cells from day 1 to 5 quantified with WST-1 assay (inset: exponential trend lines of the proliferation data to comparatively show the growth rates of cells on the substrates). * denotes statistically significant differences at p < 0.05.
The growth of bone-forming cells into larger population prepares themselves for later biological activities (i.e., differentiation and mineralization). The MC3T3E1 cell growth from days 1 to 5 on different surfaces was examined using the WST-1 assay (Figure 4c). This assay utilizes the mitochondrial dehydrogenation of terazolium salt to formazan, which can be quantified spectroscopically. 12 ACS Paragon Plus Environment
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Therefore, the results can be directly correlated with the metabolic activities of cells, which served as an indicator of cell population. Higher growth rates were detected on Zr-50H, Zr-100H, and Zr-200H surfaces with relatively lower Em, in contrast to the Zr-50L surface with the highest Em in the group. Cell proliferation on indicated surfaces at each time point was also visualized using SEM (Figure 5). The MC3T3-E1 cells grew to larger populations on all surfaces after 5 d of incubation. Cells were spaced and exhibited polygonal shapes at day 1, which confirmed the observations from the fluorescent images in Figure 3a. At day 3, cells number increased with enhanced cell-cell interactions. After 5 d growth, the cell layer reached near-confluency on the Zr-50L surface, while confluent cell layers were formed on Ar-implanted samples treated at the high fluence. The SEM observations were in agreement with the WST-1 results, which further evidenced the higher propagation rates of cells on high fluence Ar-ion implanted samples. Overall, the dependence of cell growth rates on surface stiffness was not as significant in the present study, as compared with the previous reports, which suggested a generally higher growth rate of bone-forming cells on the less stiff surfaces. Interestingly, this observation may have deviated from the results reported by others using the same cell type on chemically-distinct and mechanically-softer surfaces. For example, Kong et al. found that the cellproliferation rate increased from 0.4 to 0.7 days-1 as the stiffness of the alginate hydrogel was raised from 20 to 60 and 110 kPa.
17
Khatiwala et al. compared the
proliferative capacity of MC3T3-E1 cells on type-II collagen functionalized polyacrylamide gels with stiffness of 11.78, 21.6, and 38.98 kPa, and noticed greater cell proliferation on stiffer gels.
48
It is apparent that the material
chemistry of the substrates (i.e., alginate, collagen, etc.) in previous studies was significantly different from the alloy surface in this study, which could contribute to different protein adsorption and lead to diverse cell activities. [17, 45] On top of that, it is postulated that the stiffness level of the investigated BMG substrates encompassed in the current work, being six orders of magnitude higher than those prior studies, may be counted as one of the sources of the different observations concerning the effect of substrate rigidity on cell growth. 13 ACS Paragon Plus Environment
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Additionally, the proliferation behavior of cells on different substrates has been found to be regulated by the signaling through cytoskeleton linkages and cell adhesion.
44
To shed light on such correlation, future studies can be directed
towards specific signaling pathways of different receptors or regulators. Moreover, other surface factors could exist to interfere with the surface-stiffness effect, which will be discussed in the following section.
Figure 5 Cell growth from days 1 to 5: cell morphology on indicated substrates after 1, 3, and 5 days of growth (scale bar: 100 µm)
Based on the experimental results above, it was registered that mechanical cue on the gigapascal level could direct the behavior of bone-forming cells. In the meantime, it is necessary to take into consideration the alterations originated from the concurrent variation of other surface properties after ion implantation. These potential influences include the surface morphology, roughness, chemistry, and wettability, which were widely reported to play imperative roles to regulate protein adsorption and cell responses. 5, 49, 50 The surface morphology of the as-cast and ion-implanted Zr-based BMG samples was visualized under SEM (Figure 6a). While there were no apparent
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morphological variations on Zr-AC, Zr-50L, and Zr-200H samples within the resolution limit, distinct features were detected for the Zr-50H sample characterized by large-sized Ar bubbles or holes formed within the near-surface region. In the meantime, small patch-like features can be identified for the Zr100H specimen showing the unevenness of the surface. Based on the TEM observations discussed above, these distinct surface features were resulted from the protrusions caused by the Ar segregation or formation of air pockets due to the burst of the bubbles. Atomic force microscopy (AFM) images were collected to show the surface topography and roughness of each sample, as presented in Figure 6b. In consistency with SEM observations, the roughest surface was observed on the Zr-50H sample (Ra = 7.74 nm), followed by Zr-100H sample (Ra = 2.13 nm). Zr-AC, Zr-50L, and Zr-200H were found relatively smooth with roughness values of approximately 0.5 nm. Our scrutiny on surface roughness of the Zr-based BMGs before and after ion bombardment indicated that there could have presented a coordination of surface roughness and mechanical properties to modify MC3T3-E1 bone-forming cell behavior on the Ar-ion implanted Zrbased BMGs in this study. The reported effects of surface roughness on cell behavior differed, depending on the scale of roughness (i.e., micron-, submicron, and nano-sized) and topography of the surface (i.e., randomly distributed or patterned). 5, 49-55 Previous observations were made on modified Ti-alloy surfaces with the random distribution of surface features and roughness ranging from tens of nanometers to several microns, which suggested the slower MC3T3-E1 cell growth on rougher surfaces.
51, 53, 54
Therefore, we postulated that the rougher
surface and evenly distributed Ar-bubbles on Zr-50H and Zr-100H specimens may have discouraged cell growth. The surface-roughness effect could counteract the effect of surface stiffness/modulus, resulting in a less apparent liaison between the cell-growth rate and surface stiffness. This potential interaction
between
roughness
and
mechanical
properties
necessitates
additional research to better distinguish the role of surface-mechanical properties. Future studies are desired to assure minimum modification on surface
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roughness by altering the ion-implantation parameters to impinge ions to larger depths.
Figure 6 Morphological and topographical analyses of the Zr-based BMG surfaces before and after Ar-ion implantation using (a) SEM and (b) AFM.
Surface chemistry at the material-cell interface is another important mediator of cell behaviors.
49, 53, 56
In the present study, we have adopted the
implantation of inert Ar ions, aiming at constraining the chemical changes to the Zr-based BMG surface. In our previous study, surface chemistry of the Zr-based BMG after the low-energy Ar-ion implantation was analyzed, using X-ray photoelectron spectroscopy (XPS), which revealed the depletion of Cu and Al, along with the enrichment of ZrO2 on the ion-implanted Zr-based BMG surfaces. 30
Regardless, chemical variations were not significant between the two Ar-ion-
implanted groups in that study, implying a relatively consistent surface chemistry amongst ion-implanted groups. 30 Therefore, the surface-chemistry influence was considered to impose secondary effects to cell responses in the present work, demanding detailed chemical analysis in our follow-up work for specimens with different Ar-ion implantation treatments to validate the assumption or further elaborate the interactions of surface chemistry and mechanical effects on boneforming cell behavior. Wettability and surface free energy (SFE) are commonly used as a predicative index for the potential influence of surface properties on cell response.
50, 57
It has been recognized that modifications of different surface
properties usually concomitantly modulate SFE, which was strongly correlated with the protein-adsorption and the downstream cell activities. 50, 58-60 The trends 16 ACS Paragon Plus Environment
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of protein and cell behavior changes with SFE are more complex than a simple rule of thumb, which are dependent on the constituents in the serum, substrate materials, types of cells, etc.61,
62
To account for the effects from the surface
wettability in this study, we characterized the wetting behavior of both polar (deionized water, DI water) and nonpolar (diiodomethane, DII) liquids on the sample surface, and calculated the SFE of each specimen’63 As shown in Figure 7, the SFE of the as-cast sample
Figure 7 Surface free energy (red: dispersive part, blue: polar part; green: overall) of indicated substrates. The surface free energy and its components for each ion-implanted sample were found significantly different from those of the Zr-AC sample at p < 0.05.
was significantly higher than any of the ion-implanted samples, whereas no statistical differences in SFE were detected among the ion-implanted samples. The considerable difference in SFE of the as-cast specimen from ion-implanted ones justified our exclusion of Zr-AC in the cell culture study, which is to diminish the contributions of other surface properties affecting MC3T3-E1 cell behavior. SFE results provided a first line to acknowledge the interference of other surface properties on cell responses. In this study, the variations in wettability and SFE were found minimal amongst different implanted BMG surfaces. However, sessile drop measurements depicted the universal surface properties over a relatively large area under the coverage of the droplet, which limited the 17 ACS Paragon Plus Environment
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sensitivity and resolution of the test. Studies on surface properties and protein adsorption on much smaller scales are necessary to deepen our understanding on the synergistic effect of mechanical cues and other surface properties. Meanwhile, a comprehensive study should be practiced, based on the findings in this study to optimize ion-implantation parameters in order to achieve substrates with distinct mechanical properties and yet similar surface roughness, morphology, and chemistry. This process will be helpful to isolate the mechanical effect and mechenosensing from other contributing aspects. 3. Conclusions In conclusion, low energy Ar-ion implantation was utilized to modulate the surface rigidity of the Zr-based BMG substrate in the present study. The Zrbased BMG surfaces were softened due to the impartment of Ar nanobubbles into the amorphous matrix. By generating a nanobubble-doped transitional zone between the BMG substrate and bone-forming cells, it was shown for the first time that bone-forming cell adhesion could sense and react to mechanical cues of modulus changes on the gigapascal level at the BMG surfaces. The lower stiffness of the surfaces were found to relate to less established cell adhesion and actin filaments, especially on the surface with an elastic modulus of 51 GPa. Cell growth was found to be affected by surface stiffness, with a general trend of faster growth on the relatively less stiff BMG surfaces. In addition to the mechanical cues induced by Ar-ion implantation, the potential effects on cell activities as a result of concurrent modifications on other surface properties were discussed. Results from the current study enriched our knowledge concerning the cell mechanosensing of the substrate rigidity on metallic materials with elastic moduli higher than 50 GPa. Furthermore, this study introduced a new platform for fundamental investigation of cell responses to substrate rigidity previously unattainable
with
synthetic
materials.
The
homogeneous
amorphous
microstructure of BMGs and the versatility of the ion-implantation technique allows for the preparation of surfaces with tunable mechanical properties to enhance the interaction with cells. By advancing our understanding of bone-cell mechanosensing in a broader range of substrate stiffness, it also makes surface 18 ACS Paragon Plus Environment
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mechano-engineering of metallic orthopedic implants a potential strategy to consider for promoting osseointegration. Based on the findings in the present work, future studies are desired to further distinguish the surface-mechanical effect from the contributions of other surface properties by fine-tuning the ionimplantation parameters. METHODS Material preparation. The Zr-based BMGs with the nominal composition of (Zr0.55Al0.10Ni0.05Cu0.30)99Y1 (at.%) were fabricated using the arc-melting and copper mold casting techniques to form plate samples. Testing samples (5 × 5 × 2 mm3) were cut from the plates and polished to mirror-like surface finish. Ion-Implantation. Ion implantation was conducted in the Michigan Ion Beam Laboratory at the University of Michigan with a 400 kV ion implanter. Ar ions were implanted at different energies and fluences, as summarized in Figure 1a. Nanoindentation. Nanoindentation tests were performed in a displacementcontrol mode using the Hysitron Triboindenter at room temperature. The loading function is composed of a 2 s loading segment, a 1 s holding at the maximum penetration depth, followed by unloading in 2 s. A blunt Berkovich diamond tip was used with an effective tip radius of 759 nm, and a spherical tip geometry was applied at such shallow penetration. The elastic modulus, stiffness, and hardness of each sample were determined from the load-displacement curves using the Oliver-Pharr method. 34 Microstructural and Ar-distribution Analysis. The ion-implanted surfaces were sectioned, using the focused ion beam (FIB) with Zeiss Auriga Crossbeam. Characterization of the BMG was performed with a Zeiss Libra 200 MC TEM at an acceleration voltage of 200 kV. High angle annular dark field (HAADF) images in the scanning transmission electron microscope (STEM) mode (Z-contrast image) were captured to show the microstructure and Ar bubbles in the BMG. The advantage of high contrast in the Z-contrast image is derived from the large atomic number (Z) difference between the Ar and the major element, Zr, in the BMG. Selected area electron diffraction (SAD) taken from a round illuminated area (around 150 nm in diameter) was used to determine the phase(s) of the 19 ACS Paragon Plus Environment
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BMG under TEM. To investigate the possible local structural change of BMG after implanting Ar ions, we utilized the nano-beam electron diffraction (NBED) by converging the electron beam onto a small area (around 0.5 nm) with a convergent angle 2.5 mrad. This technique allows for the detection of the structure of the BMG in the ultra-small area with the same size as the beam probe dimension. Monochromated EELS was employed to determine Ar distributions within the implanted region. Detailed descriptions are included in the supporting information. Surface Morphology and Roughness. Surface morphology of Zr-based BMGs before and after Ar-ion implantation was viewed, using SEM at an acceleration energy of 20 kV. Secondary electron micrographs were collected. Surface topography and roughness of each sample were measured using AFM. Measurements were conducted on an area of 10 × 10 µm2 under non-contact mode in air. Surface Wettability and Free Energy. Wetting behavior of polar (deionized water, DI water) and nonpolar (diiodomethane, DII) liquids on the samples was determined by measuring their contact angles on the surfaces using a CAM-Plus contact angle goniometer (Cheminstruments, USA). About 5 µL of liquid droplet was placed on the samples. Contact angles were measured, using the half-angle sessile-drop method (US Patent 5268733) at room temperature. Repetitive measurements (n = 5) were carried out. Surface free energy (SFE) for the tested samples was calculated, based on contact angle measurements using the Owens-Wendt’s equation: 63 1 + cosθ = 2(γsd)1/2[(γld)1/2／γl] + 2(γsp)1/2[(γlp)1/2／γl] where θ is the contact angle; γl is SFE of measured liquid; γs is SFE of tested sample; the superscripts, d and p, denote dispersive and polar components of SFE. The SFE for each sample can be calculated by γs = γsd + γsp. Simulation. The damage and Ar-ion distribution on the BMG surfaces were simulated, using the SRIM package developed by Ziegler et al.
40
with a full-
cascade mode. The density of the Zr-based BMG applied in the simulation was
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6.65 g/cm3.
30
The experimental fluence and energy were plugged in to calculate
the depth profiles of disorder and Ar-ion concentration. Cell Adhesion. Murine-derived bone-forming MC3T3-E1 pre-osteoblasts (ATCC, USA) were seeded on the ion-implanted surfaces at a density of 1 × 104 cells/cm2. After 24 h of incubation, cells were fixed with paraformaldehyde and stained following a previously described protocol.
27
Vinculin and α-actin were
labeled with Alexa Fluor 488 and 594 fluorophores (Invitrogen, USA) to respectively present cell adhesion and cytoskeleton. Cell nuclei were dyed, using DAPI (Chemicon, CA). Cells were subsequently viewed under a Zeiss Axio Observer A1 inverted fluorescent microscope. ImageJ was used to analyze more than 50 images for each group. Cell Growth. The growth of MC3T3-E1 cells from days 1, 3, and 5 was monitored using a water-soluble tetrazolium salt (WST-1, Roche applied science, USA) assay. Cells at a density of 1 × 104 cells/cm2 were seeded to the Zr-based BMG surfaces in a 48-well plate. At each time point, samples were transferred to a new 48-well plate containing the 150 µl fresh growth medium supplemented with 15 µl WST-1 reagent in each well. The plate was incubated at 37 °C for 2 h to allow reaction, followed by brief mixing on a plate shaker for 1 min, at the end of the incubation. After cooling to room temperature, 100 µl of each supernatant was transferred to a 96-well plate. The absorbance was read against a blank control at 460 nm.
27
The absorbance of the sample was read against a blank
control at 460 nm. The cell population and morphology at different incubation time points from days 1-5 was further observed, using SEM at an acceleration energy of 3 or 5 keV. After rinsing in PBS, the cell layers were fixed with 3% glutaradehyde at 4
o
C for 1 day and washed in PBS for dehydration, as
previously described. [12] Samples were immersed in ascending grades of ethanol (25%, 50%, 75%, 100%, and dry) for 10 min. each, followed by chemical dehydration with hexamethyldisilazane (HMDS). To ensure a complete removal of water, samples were immersed in the mixtures of dry ethanol and HMDS at volume ratios of 2:1, 1:1, and 1:2, followed by the 100% HMDS treatment. HMDS was allowed to evaporate in a vacuum desiccator, where samples were stored 21 ACS Paragon Plus Environment
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prior to imaging. Dried specimens were coated with gold using an SPI sputtering device for 20 s at 20 mA for SEM imaging. SEM secondary electron images were collected.
ASSOCIATED CONTENT Supporting Information Supporting Information is available free of charge on the ACS Publications website at DOI: Experimental details of substrate preparation, nanoidentation, ArDistribution
Analysis, SRIM Simulation, Cell Culture, Cell Viability and
Cell Adhesion
AUTHOR INFORMATION Corresponding Authors *Email:
[email protected] *Email:
[email protected]
ORCID Mengkun Tian: 0000-0003-2790-7799 Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS The authors thank Prof. T. Zhang (Beihang University, China) for the BMG materials and the Joint Institute of Advanced Materials at The University of Tennessee for microscopy access. WH, PKL, and LH acknowledge the financial support from National Science Foundations under grant # CMMI-1100080. PKL very much appreciates the support from the Department of Energy (DOE), Office of Nuclear Energy’s Nuclear Energy University Program (NEUP, grant # 00119262), the DOE, Office of Fossil Energy, National Energy Technology Laboratory (DE-FE-0008855, DE-FE-0011194, and DE-FE-0024054), Innovative Processing and Technologies Program of the National Energy Technology Laboratory's (NETL) Strategic Center for Coal under the RES contract (DE-FE0004000), and the Army Research Office (W911NF-13-1-0438).
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