Developing biomedical nano-grained β-type titanium alloys using high pressure torsion for improved cell adherence

Abstract

Proper surface characteristics for a titanium implant are crucial for the formation of different cellular protrusions known as filopodia and lamellipodia, both of which have a significant impact on cell attachment, spreading, migration, and proliferation. Microstructural features such as grain boundaries and defects of implant surface can modulate the cellular components and structure at the leading edge of cells. Here, a nano-grained Ti–29Nb–13Ta–4.6Zr (NG TNTZ) substrate was produced by high-pressure torsion (HPT) for improved biofunctionality. Cellular response of human osteoblast cells on nano-grained TNTZ substrates is evaluated and compared with the cellular response of those on coarse-grained TNTZ. High wettability, which depends on high internal energy due to the nano-sized grains that are full of boundaries, interfaces, and high dislocation density, influenced the hOBs cells on NG TNTZ to form highly developed cellular protrusions. Large number of filopodia protrusions resulted in excellent cell attachment as consistent with high level of vinculin and superior cell proliferation. This study demonstrates the advantages of nanocrystalline surface modification using HPT for processing metallic biomaterials that are proper for orthopedic implants.

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Introduction

Microstructural features such as grain structure, grain boundaries, and defects are key factors in influencing the majority of all characteristics of titanium as a polycrystalline material.¹ In recent years, the role of grain size in controlling surface bio-functionality has been investigated on a range of metallic biomaterials.^2,3 In the last decade, the development of ultrafine- and nano-grained (UFG/NG) materials has attracted particular attention because of improved mechanical and surface properties compared to those of conventional coarse-grained (CG) materials.⁴ Severe plastic deformation (SPD) is an efficient process for extreme grain refinement, yielding UFG/NG materials.^5,6 For metallic materials, the numerous dislocations introduced by severe deformation gradually become arranged and annihilated into high-angle boundaries, resulting in an UFG/NG microstructure, both on the surface and in the body.⁷ Such UFG/NG materials exhibit outstanding mechanical properties such as high strength and hardness, and good ductility.^6,8 The aim of this study is to produce a titanium implant exhibiting an excellent surface bio-functionality that favors cell attachment, spreading, and proliferation⁹ in addition to outstanding mechanical properties¹⁰ to allow long-term usage. However, although the surface bio-functionality of UFG/NG metallic biomaterials has been reported,^3,11 these materials are mostly fragmented, and the mechanism of enhanced cellular behavior on implant have not been addressed in terms of the cell cytoskeleton.

Developing nano-grained β-type titanium alloy

Herein, the use of a β-type Ti–29Nb–13Ta–4.6Zr (TNTZ) alloy¹² composed of non-toxic and non-allergenic elements is reported for the preparation of substrates bearing CG and UFG/NG microstructures via conventional processes such as solution and aging treatments and high-pressure torsion,⁷ which is one of the SPD processes. Compared to widely used implant material Ti-6A-4V ELI, which has a high Young's modulus (110 GPA), TNTZ exhibits a lower value (60–80 GPa) close to that of bone (10–30 GPa), thus preventing stress shielding effects that can result in osteoporosis or poor osteointegration (also see the section for experiments in the ESI†).¹³ The microstructural characterizations of our materials were studied by optical microscopy (OM), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and high-resolution TEM (HRTEM). Surface characteristics were studied by atomic force microscopy (AFM) and contact angle measurements. Surface wettability was determined by evaluating surface free energy (SFE) using contact angle measurement (also see the section for experiments in the ESI†). The cellular activity of human osteoblast cells (hOBs) cultured on CG and UFG/NG TNTZ alloys was studied with respect to cell attachment and proliferation. The morphology of the attached hOBs were investigated using immunofluorescence (IF) microscopy, by labeling the nuclei, actin and vinculin proteins in the attached hOBs with blue, red, and green colors, respectively, using a Focal Adhesion Kit (also see the section for experiments in the ESI†).

Material characterization

Microstructural features of TNTZ have been summarized in Fig. 1 (please see the ESI section† for the details of the microstructural characterization). The larger images to clearly understand the microstructures were added in the ESI.† Previously reported XRD analysis (Fig. S1†)¹⁴ clearly confirmed this BCC structure has showed a precipitated hexagonal closed packed (HCP) α phase following aging treatment (TNTZ_AT). The OM micrograph in Fig. 1a shows that the solutionized TNTZ (TNTZ_ST) possesses a single body-centered cubic (BCC) equiaxed β grains (∼40 μm diameter) microstructure (Fig. S2a†). The SEM observation (Fig. 1b) shows these α phases are homogeneously distributed in the β grains and exhibit a needle-like morphology (∼750 nm × 25 nm) (Fig. S2b†).

Fig. 1 (a) Optical micrograph of TNTZ_ST (b) SEM images of TNTZ_AT (c) TEM bright image of TNTZ_AHPT with selected area electron diffraction pattern (d) HRTEM bright field image with nano-beam diffraction patterns. (e) An illustration of dislocation generations and microstructural refinement mechanism. The precipitated α phases having needle-like morphology were shown with magnified SEM image. Precipitated α phases were marked with red line. The strain fields between β matrix and α phases were marked with yellow dashed line in the HRTEM image.

The microstructure of TNTZ_AT subjected to HPT (TNTZ_AHPT) was investigated by TEM (Topcon EM-002B, Japan) due to the limitations of OM and SEM (Fig. 1c and S3a†). TEM bright field images (Fig. 1c) show that TNTZ_AHPT exhibits a microstructure consisting of elongated grains (∼285 nm × 35 nm). In addition, a relatively homogeneous microstructure was revealed by a hardness distribution (Fig. S5†). The complete Debye rings in the SAED pattern of TNTZ_AHPT also indicate highly polycrystalline microstructure (Fig. 1c and S3a†). Unfortunately, the α phases were difficult to distinguish in the conventional TEM bright field image. Furthermore, HRTEM (FEI Titan 80-300, Japan) revealed a precipitated α phase (red lines) with a circular morphology (∼12 nm diameter) in the β grains (Fig. 1d and 3b). In contrast, large blurred and wavy contrast variations (yellow dashed lines), which indicate strain fields, surrounding the α phases in the β matrix were also observed.¹⁴ Furthermore, the elongated grains contain nanostructured subgrains with non-uniform morphology. In addition, the grains and subgrains of TNTZ_AHPT surrounding large contrasting and blurred and wavy boundaries with remained dislocations remained during annihilation of arranged dislocations. Such a refining mechanism along with the formation of such non-equilibrium boundaries,^6,15 have been previously reported¹⁴ and are illustrated in Fig. 1e (Fig. S4†).

Surface bio-functionality

Mirror-finish polishing was then performed to ensure identical surface topography for all substrates in order to investigate microstructural effect on surface bio-functionality. AFM (AFM, Nanocute, Seiko, Japan) topographical images of all polished substrates in a 10 μm × 10 μm scan area are shown in Fig. S6 and mean roughness values are listed in Table S1.† AFM analysis reveals that all substrates exhibit identical surface roughness and topography. Surface free energy (SFE) parameters for all substrates were then determined using the contact angle measurements with distilled water and further diiodomethane (DMs-401, Kyowa, Japan). Fig. S7† shows the average contact angles and SFE values for all substrates using drops of distilled water. Although no statistically significant differences were observed among coarse grained substrates, TNTZ_AT gives a slightly smaller contact angle among CG TNTZ_ST, TNTZ_AT, and Ti64 ELI substrates with values of 53.80° ± 2.13°, 51.78° ± 2.87°, and 64.83° ± 2.40°, respectively. On the other hand, the water drops on TNTZ_AHPT substrates show quite smaller contact angle (35.15° ± 2.49°) than those of the CG substrates (Fig. S7b†).

Cell attachment and proliferation

The cellular behavior of hOBs has been quantitatively revealed for attachment studies on titanium substrates (Fig. 2) (please see the ESI section† for the details of the cell culture). Cell numbers were counted using a disposable hemocytometer (C-Chip, NanoEnTek Inc., USA). The number of hOBs attached to TNTZ_AHPT was significantly higher than that on CG TNTZ_ST and TNTZ_AT substrates (Fig. 2) after 6 h incubation. In addition, although the number of hOBs attached to the TNTZ_AT was slightly higher than that on TNTZ_ST, no statistical difference was observed. Individual actin proteins, which are major determinants of shape, migration, and attachment of the hOBs, were then analyzed quantitatively using IF microscopy (Fig. 3) (please see the ESI section† for the details of immunocytochemical staining of actin filaments). The attached hOBs exhibit well-developed and dense actin stress fibers (ASFs) on TNTZ_AHPT whereas ASFs were not observed in all hOBs attached to TNTZ_ST and TNTZ_AT. The magnified IF micrographs (Fig. 3a) show that the hOBs on titanium substrates exhibit lamellipodia and filopodia structures at their leading edges. These protrusions coexist at the protruding front of a migrating cell, with the filopodia tending to extend by the highly dense organization of actin filaments from bundles formed inside the lamellipodia. The titanium implant surface has to present an excellent surface characteristic for improved cell migration for ensuring implant-body biocompatibility in bone formation. The hOBs exhibit larger lamellipodia structures along with significantly higher numbers of filopodia structures on TNTZ_AHPT, than those of TNTZ_ST and TNTZ_AT (Fig. 3b). The magnified IF micrographs also show that although filopodia structures are formed only at the leading edge of the hOBs attached on TNTZ_ST and TNTZ_AT, they surround the majority of hOBs attached to TNTZ_AHPT (Fig. 3a).

Fig. 2 Average numbers of the attached hOBs on the substrates of TNTZ_ST, TNTZ_AT, TNTZ_AHPT, and Ti64 ELI after incubation for 6 h at 307 K.

Fig. 3 (a) Fluorescence micrographs of actin filaments and stress fibers, lamellipodia, and filopodia structures in hOBs attached on TNTZ_ST, TNTZ_AT, and TNTZ_AHPT substrates and (b) number of filopodia spikes of hOBs attached on TNTZ_ST, TNTZ_AT, and TNTZ_AHPT, and Ti64 ELI substrates.

To better understand the substrate–hOBs interactions and the organization of actin filaments, vinculin proteins were also visualized by IF microscopy (Olympus IX71 inverted microscopy, Japan) (Fig. S8a†) (please see the ESI section† for the details of immunocytochemical staining of vinculin). IF micrographs of hOBs attached to TNTZ_AHPT show significantly stronger vinculin signals than those on TNTZ_ST and TNTZ_AT, which was verified by the quantitative data of the vinculin area/cell area in Fig. S8b.† In the case of hOB growth, cell proliferation on TNTZ and Ti64 ELI substrates following 6 d incubation was assessed and is illustrated in Fig. S9.† Although the number of hOBs was slightly higher on TNTZ_AT than those on TNTZ_ST and Ti64 ELI, no significant difference was observed. Furthermore, TNTZ_AHPT gave the highest cell number of all substrates.

Discussion

Since a relatively uniform surface roughness was maintained throughout our study, the observed cell–substrate interactions and their responses were directly related to factors arising from differences in microstructural features, such as dislocations. The energy stored in various dislocation configurations provide the dislocations provide the driving force for the observed recovery and recrystallization phenomena during the HPT.^16–18 Consequently, the exterior dislocations (Fig. 1 and S4†), which contribute the majority of stored energy in NG TNTZ, are integrated mainly into grain boundaries, cell boundaries, or complex entanglements. Therefore, the high surface energy of NG TNTZ (Fig. S7†) due to the large volume fraction of grain boundaries and interfaces exhibiting exterior dislocation. Reports suggest that materials possessing the same composition and surface topography, while having smaller grain diameter, exhibit higher surface energies than their CG counterparts.^19,20 Furthermore, in the case of NG TNTZ, a higher surface energy resulted in a higher wettability, which correlates with the ability of the solid surface to reduce surface tension, and thus reduce the contact angle of the liquid²¹ when compared to CG TNTZ. Wei and his coworkers²² showed that high wettability increases the cell attachment due to high fibronectin absorption to a substrate surface. A similar result regarding hOBs was demonstrated on a graphene substrate by Shi and his coworkers.²³ Therefore, wettability is known to affect surface bio-functionality. This surface functionality is the basis for the osteointegration assessment, for the stimulation of hOB functions such as adhesion, spreading, and proliferation at the bone-implant interface.²⁴ Furthermore, HPT causes a high volume fraction of discontinuities related to the above-mentioned boundaries, interfaces, and junctions in the surface microstructure. These features can lead to the formation of focal adhesion points, which may help signaling among cells during cell attachment, along with migration and proliferation along the NG TNTZ.²⁵ Furthermore, it was reported that cytoskeleton dynamics have been implied by stimulated mechano-receptive responses in fibroblast, related to such discontinuities.²⁶

Examining individual proteins linked to hOBs activity also indicated that NG TNTZ produced more promising results. One of the major structures of actin cytoskeleton, ASFs are formed by bundles of actin filaments in the cytoplasm stretching across the entire hOB length.^27,28 These ASFs play a central role in the attachment, migration and morphology of hOBs.²⁸ IF micrographs of the ASFs show that compared to CG TNTZs, the hOBs on the NG TNTZ exhibit enhanced outstretched morphologies with well-organized and elongated ASFs. In addition, lamellipodia and filopodia aid in actin network structure formation to define the leading edge for migrations.^27,28 In particular, filopodia is responsible for exploring the environment during motility.^29,30 Furthermore, the cytoplasmic actin-binding protein vinculin is mainly found in focal adhesions, and so these junctions were immunocytochemically imaged using IF microscopy (Fig. S6†) to gain a better understanding of the nature of the interactions of between hOBs with different substrates. The highest amount vinculin was found along the extended actin microfilaments of cells seeded on NG TNTZ (Fig. S8a†), resulting in focal adhesion on the surface of these substrates.

Conclusions

In this study, the HPT process enabled us to control the grain size of a new type of alloy TNTZ to modulate both mechanical characteristics and wettability of substrates, which is highly dependent on the grain size and imperfections such as grain and subgrain boundaries and dislocations. High wettability of NG TNTZ substrates led to larger lamellipodia structures along with significantly higher numbers of filopodia protrusions all around the cells and hereby an excellent hOB cell attachment performance. Data regarding difference between cell projections with respect to different alloys observed in this study might lead to better understanding of cell–implant surface interaction for designing better implant materials. Furthermore, evidence of high level of vinculin expression and superior cell proliferation of hOB cells attached to NG TNTZ, suggests that an NG surface promotes cellular activity.

Acknowledgements

This work was performed in part on commission from the New Energy and Industrial Technology Development Organization (NEDO) and supported in part by Grants-in-Aid for Scientific Research (A), Young Scientists (A), Challenging Exploratory Research from the Japan Society for the Promotion of Science (JSPS), and Inter-University Cooperative Research Program “Innovation Research for Biosis-Abiosis Intelligent Interface” from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. Authors kindly thank Dr Xuetao Shi (Tohoku University, Japan) for providing the hOB cells used in this study.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra23454a

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