Surface Severe Plastic Deformation of an Orthopedic TiâNbâSn Alloy

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Characterization, Synthesis, and Modifications

Surface severe plastic deformation of an orthopedic TiNb-Sn alloy induces unusual precipitate remodeling and supports stem cell osteogenesis through Akt signaling Sumit Bahl, Sai R K Meka, Satyam Suwas, and Kaushik Chatterjee ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00406 • Publication Date (Web): 09 Jul 2018 Downloaded from http://pubs.acs.org on July 10, 2018

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ACS Biomaterials Science & Engineering

Surface severe plastic deformation of an orthopedic Ti-Nb-Sn alloy induces unusual precipitate remodeling and supports stem cell osteogenesis through Akt signaling

Sumit Bahl, Sai Rama Krishna Meka, Satyam Suwas, Kaushik Chatterjee* Department of Materials Engineering Indian Institute of Science, Bangalore, India 560012

Author to whom all correspondence should be addressed: *[email protected]; +91-80-22933408

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Abstract This work presents a strategy to augment the bioactivity of a new-generation metastable β TiNb-Sn alloy through surface severe plastic deformation. Foremost, the alloy was strengthened by precipitation of α phase using a well-designed thermo-mechanical processing route. Subsequently, the surface of the aged alloy was subjected to severe plastic deformation via surface mechanical attrition treatment (SMAT). Upon SMAT, a unique phenomenon of strain-induced precipitate coarsening was observed. A possible mechanism is proposed wherein the precipitates first dissolve due to significant slip transfer across the α/β interface followed by reprecipitation along the other precipitates thereby leading to coarsening. Coarsening of the precipitates abrogated the strengthening caused by plastic deformation as a result of which the hardness did not increase significantly after SMAT in sharp contrast to other alloys. SMAT led to a decrease in the attachment of human mesenchymal stem cells due to an increase in the roughness-mediated surface hydrophobicity. On the other hand, an increase in the roughness led to the formation of more number of focal adhesions. This in turn enhanced the proliferation rate and more importantly, osteogenic differentiation of stem cells. Detailed investigation into the underlying mechanism revealed that an increase in focal adhesions activated the Akt mediated mechano-transduction signaling pathway that enhanced the osteogenic differentiation. In summary, the potential of surface severe plastic deformation to impart bioactivity to the next-generation of orthopedic β Ti alloys is underscored in this work. Keywords: β Titanium alloys; Strain-induced coarsening; Orthopedic implants; Severe plastic deformation; Osteogenesis; Akt pathway


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1. Introduction Ti-Nb-Sn based metastable β Ti alloys are promising materials for use in orthopedic applications. A combination of properties such as high strength, low elastic modulus, nontoxic elemental constituents and high corrosion resistance make these alloys the preferable choice for implants 1. Akin to any other metallic system for structural applications, these β alloys ought to be thermo-mechanically processed for enhancement of their mechanical properties. Typically, metastable β Ti alloys can be strengthened by precipitation of α phase using an aging treatment thereby advancing their capabilities for load bearing orthopedic applications

2-3

. Recently, we have developed high strength Ti-Nb-Sn based β Ti alloys by

controlling the nanoscale precipitation of α phase during aging treatment

2-3

. Aside from

mechanical properties, a highly bioactive surface of the material is equally essential for the long-term clinical success of the orthopedic implants. Although the surfaces of these alloys are biocompatible they are essentially bio-inert in nature resulting in poor osseointegration 4. The bioactivity of metallic biomaterials can be improved either by modifying the chemical composition or topography of a surface 5. Chemical modification usually involves coating the surfaces with bioactive ceramic or polymeric materials. Surface topography is modified by a broad spectrum of sophisticated etching techniques on one hand or relatively facile acid etching techniques on the other hand

4, 6

. Although, bioactivity improves

effectively, these techniques suffer from several limitations. For example, the durability of the modified surfaces vis-à-vis the interface strength with the substrate metal is a significant concern. The potential detrimental impact of rough surface topographies on the fatigue life can outweigh the anticipated benefits on the cell growth. Notably, the bioactivity of metallic surfaces can also be improved by surface severe plastic deformation

7-12

. The ultrafine or

nanocrystalline microstructure thereby produced has shown to improve the attachment, proliferation and osteogenic differentiation of cells in vitro. In contrast to chemical and 3 ACS Paragon Plus Environment


topographical modifications, surface severe plastic deformation will not degrade the fatigue strength of a material. Rather, the fatigue strength can potentially be improved due to the surface hardening and generation of compressive residual stresses induced by severe plastic deformation

7, 12-14

. Although fine grain size produced by severe plastic deformation

techniques is known to enhance cell responses, its mechanism remains elusive 15. The effect of other interfering factors such as varied materials classes, surface topography, cell lines and animal models make it further difficult to elucidate the sole effect of grain size on the cell response 15. Even as the new-generation low modulus, high strength β Ti alloys attract attention for engineering biomedical devices, there is poor understanding of the effect of surface severe plastic deformation on the biological response. Furthermore, the microstructural evolution in β Ti alloys during severe plastic deformation also requires detailed investigation with limited current understanding. It is important to investigate the microstructural evolution during deformation as this underlying microstructure will have consequences on the mechanical and biological performances of the devices. Surface mechanical attrition treatment (SMAT) is a surface severe plastic deformation process that has been demonstrated to successfully produce a nanocrystalline surface in a wide variety of alloys 12, 16-19. In SMAT, the surface of a material is severely deformed by the impacts of hard metallic or ceramic balls moving in random directions with high speed. A few studies have revealed preliminary observation on the effect of SMAT on cellular response with minimal understanding of the underlying molecular mechanisms 20-22. In this investigation, the surface of a dual phase β Ti alloy prepared using an earlier reported thermo-mechanical processing route 3 was modified by SMAT. The high strength of a dual phase microstructure in β Ti alloy makes its suitable for orthopedic application. Subsequently, the microstructural evolution at the surface after SMAT was characterized. 4 ACS Paragon Plus Environment

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The influence of SMAT on modulating the response of human mesenchymal stem cells (hMSCs) in terms of attachment, proliferation and osteogenic differentiation was evaluated. The role of intracellular signaling in mediating the observed response was elucidated. The corrosion behavior was also assessed in simulated body fluid (SBF). The beneficial effects of surface treatment of this alloy by SMAT are highlighted here for its use in the orthopedic applications. 2. Materials and method 2.1.

Materials and processing

The alloy with composition Ti-32Nb-2Sn in wt.% was prepared by non-consumable arc melting in the shape of a pan cake using high purity (>99.9 %) constituent elements Ti, Nb and Sn. The solidified microstructure of the pan-cake was homogenized by hot rolling at 950° C followed by solution treatment at 950° C for 0.5 h and subsequently water quenching. In the next step, the material was subjected to aging for 6 h at 500 °C by inserting in a preheated furnace. These samples will be hereafter referred to as A500. The surfaces of aged samples were prepared using standard metallographic techniques with the final polishing performed using 0.3 µm alumina paste. The aged samples were then subjected to SMAT using hardened steel balls with 4.75 mm diameter for 30 min. These samples will be hereafter referred to as A500-S. 2.2.

Microstructural characterization and X-Ray diffraction

The microstructural characterization before and after SMAT was performed by scanning electron microscope (SEM, Gemini, Zeiss) and transmission electron microscope (TEM, Tecnai T20). Samples for SEM were prepared by standard metallography procedures with the last step performed using electropolishing. The samples were etched in Kroll’s reagent prior to microstructural characterization. The microstructures before and after SMAT were taken at the top surfaces rather than the cross-sections. There were no additional grinding and 5 ACS Paragon Plus Environment


polishing steps performed after SMAT. The increase in roughness after SMAT did not impede high resolution imaging of the microstructure using SEM. Hence the possibility of missing the SMAT affected layer for microstructural characterization is eliminated. Samples for TEM were prepared using ion milling (PIPS, Gatan Inc.). The TEM sample from the surface after SMAT was prepared such that only the non-processed side was grinded and milled during sample preparation. X-Ray diffraction (XRD) was performed using Cu-Kα radiation (PANalytical X’pert Pro) operated 40 kV voltage and 30 mA current with a step size of 0.033°. 2.3.

Mechanical and surface characterization

Hardness at the surface before and after SMAT was measured using micro-Vickers indentation using a 25-gf load and 10 s dwell time. Surface roughness measurements were done using optical profilometer (Talysurf CCI, Hobson) and atomic force microscopy (AFM, Bruker Park systems NX-10). The scan area for AFM was 2.5 µm × 2.5 µm while for the optical profilometer was 840 µm × 840 µm. AFM scans were performed in the non-contact mode using an ACTA cantilever of a spring constant of 40 N/m. The optical profilometer scans were recorded at 20 x magnification. Surface wettability was evaluated by measuring the sessile contact angle of ultra pure water (Sartorius) using a contact angle goniometer (OCA 15EC Dataphysics). The contact angle of A500 and A500-S was measured on their as prepared surfaces (final polishing with 0.3 µm alumina paste). The surfaces of A500 and A500-S were also ground using P3000 grit paper to achieve similar surface roughness which was followed by measuring their water contact angles. The passive oxide layer was characterized using X-ray photoelectron spectroscopy (XPS, Kratos Analytical) using a monochromatic Al source with 1.486 KeV.


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2.4.

Electrochemical behavior

The corrosion behavior before and after SMAT was evaluated in SBF. The composition of the SBF used is reported elsewhere 3. A standard three electrode system with Pt as the counter electrode and standard calomel electrode (SCE) as the reference electrode was used. A total of 3 h were given for the potential to stabilize, which is referred to as open circuit potential (OCP). Potentiodynamic polarization curves were measured to calculate the corrosion rate by Tafel extrapolation method. A scan rate of 2 x 10-4 V/s was used to measure the polarization curves from -0.6 V to 0.4 V. The conductivity of the surface oxide layer was characterized using Mott-Schottky plots. The potentials of the samples were stabilized in SBF for 3 h. A potential sweep with a step size of 50 mV was performed from 1.0 V to -1.0 V and the corresponding impedance was measured. A sinusoidal wave form with an amplitude of 5 mV and a frequency 1 kHz was used for scanning. 2.5.

Biological response

Primary bone marrow-derived hMSCs from a 25-year-old male donor were cultured in the growth medium supplemented with 15 % fetal bovine serum (FBS), 1 % glutamax (Sigma), and 1 % penicillin-streptomycin antibiotic mixture (Sigma). All cell studies were done with due approval of the Institutional Committee on Stem Cell Research of the Indian Institute of Science. Square specimens with an edge length of 7 mm were cut to fit in the wells of 48 well plate. The samples were sterilized by ethanol and UV prior to cell seeding. The samples were incubated in growth medium 24 h prior to seeding cells. 5 x103 cells suspended in 400 µl of medium were seeded per well. The cells were cultured in a conventional 5% CO2 incubator and the medium was refreshed every 3 days. 2.5.1. Cell attachment and proliferation Cell attachment on the surfaces was measured at 1 day after seeding cells and proliferation was measured at 3 days after seeding cells. The quantification was done by measuring total 7 ACS Paragon Plus Environment


DNA content using Picogreen dsDNA kit (Invitrogen), as reported earlier 3. The focal adhesions of the cell were evaluated by staining for Paxillin. The cells were fixed in 3.7 % formaldehyde for 15 min and subsequently permeabilized with 0.2 % Trition (Sigma). The samples were then incubated in the blocking solution (5% BSA) for 45 min. The cells were then incubated overnight at 4° C in the primary anti-human antibody raised in rabbit (Abcam) prepared in the blocking solution with a 1:200 dilution ratio. Next, the cells were incubated with the secondary anti-rabbit antibody raised in goat conjugated with Alexa Fluor 488 (Invitrogen) at 1:500 dilution ratio for 45 min at 25° C. Further, the cells were stained for actin fibers and nuclei. The actin fibers were stained with Alexa Fluor 546 (Invitrogen) with a working concentration of 25 µg/ml and nuclei were strained with DAPI (Invitrogen) with a working concentration of 0.2 µg/ml. The cells were imaged using Zeiss LSM 880 Airyscan Superresolution confocal fluorescence microscope. 2.5.2. Osteogenic differentiation Osteogenic differentiation of hMSCs was evaluated at 14 days after seeding cells. The cells on the sample surfaces were cultured in growth medium supplemented with osteoinductive factors (20 mM β glycerophosphate, 50µM ascorbic acid and 10 nM dexamethasone). The mineral deposited by 14 days was quantified by staining with Alizarin Red S dye (Sigma). The cells were fixed in 3.7 % formaldehyde for 30 min followed by staining with the dye for 30 min. The unbound dye was removed by giving several washes with DI water. The dye bound to the surface was solubilized in a solution of 0.5 N HCl + 5% SDS and the absorbance was measured at 405 nm using a microplate reader (Biotek). The expression of osteogenesis makers namely, bone morphogenetic protein (BMP-2) and runt-related transcription factor 2 (Runx2) at 14 days was evaluated using western blot with GAPDH as the internal loading control. Commercially available primary anti-human antibodies raised in rabbits and secondary antibodies raised in goat were used (Abcam). The 8 ACS Paragon Plus Environment

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details of the protocol are reported elsewhere 23. The blots were analyzed using the myECL imager (Thermo Scientific). The role of Akt mediated pathway in governing the osteogenic differentiation of hMSCs cultured on the alloy surfaces was analyzed using its inhibitor LY294002. The inhibitor was first dissolved in DMSO and further added to the osteogenic medium at a final working concentration of 20 µM. Osteogenic medium with only DMSO was used as the control. The effect of inhibiting the Akt pathway on the osteogenic differentiation of stem cells was confirmed by determining the expression of BMP-2 and Runx2 proteins at 14 days using western blot, as described above. 3. Results 3.1.

Microstructural characterization

Fig. 1a shows the initial microstructure of A500 before SMAT. The microstructure consists of uniformly distributed nanoscale α precipitates in the β matrix. The microstructure of the surface of A500-S after deformation by SMAT also consists of nanoscale α precipitates (Fig. 1b). However, the size of the precipitates is larger compared to the initial microstructure. The size of precipitate increased from 100 ± 40 nm length and 40 ± 10 nm width in A500 to 190 ± 80 nm length and 40 ± 10 nm width in A500-S. This is indicative of a deformation-induced microstructural coarsening in this alloy.



Figure 1 SEM micrograph of the surface of (a) A500 and (b) A500-S

The TEM micrographs of A500 and the surface of A500-S are shown in Fig. 2. The bright field TEM micrograph of A500 (Fig. 2a) confirms the uniform distribution of nanoscale precipitates in the β matrix. This micrograph further displays a uniform distribution of strain contrast in the A500 condition. The dark field TEM micrograph of a different region of the same specimen is shown in Fig 2b. The important feature to be noted here is that the 10 ACS Paragon Plus Environment

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contrast is uniform across the width of the precipitates such as the region showed by the red arrow. The bright field micrograph of A500-S shown in Fig. 2c reveals the uneven distribution of strain in the microstructure, as evident by the dark and bright regions in the upper and lower locations of the micrograph, respectively. This contrasts with the uniform distribution of strain in A500 (Fig. 2a). The bright field and dark field micrographs of the precipitates in A500-S are shown in Fig. 2(d, e). In contrast to the ideal morphology of α precipitates observed in A500, the precipitates in A500-S have an irregular morphology consisting of non-uniform width across their length and steps as marked by yellow arrows in Fig. 2d. Apart from the large sized irregular shaped precipitates, some of the precipitates (encircled in Fig. 2d) are significantly smaller in dimensions and have an irregular morphology. Such precipitates are absent in A500 (Fig. 2a). As opposed to the uniform contrast across the width of a precipitate in A500 (Fig.2b), the precipitates after deformation have a non-uniform contrast across their width (marked by red arrows, Fig. 2(e, f)). A brighter contrast can be observed at the edges as compared to the core regions of the precipitates. Fig. 3a presents the XRD patterns of A500 and A500-S that confirm the presence of α and β phases before and after SMAT, respectively. Fig. 3(b, c) display the full width at half maximum (FWHM) of the peaks of α and β phases in A500 and A500-S, respectively. FWHM of majority of the peaks corresponding to the β phase increased after SMAT. On the contrary, FWHM of majority of the peaks corresponding to the α phase did not change after SMAT. The hardness that was measured at the surface revealed a marginal increase from 2.9 ± 0.1 GPa in A500 to 3.2 ± 0.1 GPa in A500-S.



Figure 2 (a) Bright field and (b) dark field TEM micrographs of A500; (c) and (d) bright field TEM micrographs of the surface of A500-S; (e) Dark field TEM micrograph of the region corresponding to (d); (f) dark field TEM micrograph of a different surface region of A500-S


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110

♦•

220

♦

11.0

10.2 200

♦

10.1

10.0

Intensity (a.u.)

•

−♦ α −♦ β−•

•

♦

310

(a)

•

• A500-S

A500 30

40

50

60

70

80

90

100

2θ θ° (b) 1.4

(c)

A500 A500-S

1.2

1.0 0.9

A500 A500-S

0.8

FWHM°°

1.0

FWHM°°

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


211

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0.8 0.6

0.7 0.6 0.5 0.4

0.4

0.3

0.2

0.2 10.0

10.1

10.2

11.0

110

hk.l

200

211

310

hkl

Figure 3 (a) XRD patterns of A500 and A500-S; FWHM of peaks of (b) α and (c) β phases shown in (a) 3.2.

Surface characterization

The surface topography was characterized using AFM and optical profilometer. Although the vertical resolution (z height) of the optical profilometer is extremely high in the range of subnanometer, the lateral resolution is poor in the range of sub-micron. As a result, topographical features that are smaller than the lateral resolution of the optical profilometer can be mapped only using higher resolution scanning probe techniques such as AFM

24

. The optical

profilometer is more efficient for analyzing large surface areas than AFM. Furthermore, characterization of surfaces with high undulations at the macroscale such as craters produced after SMAT can damage the AFM tips. Therefore, a comprehensive topographical 13 ACS Paragon Plus Environment


characterization of the surface was performed using a combination of optical profilometer and AFM. The 3D surface profiles are shown in Fig. 4. The values of various roughness parameters derived from optical profilometer and AFM are compiled in Table 1. The values of surface roughness characterized by AFM and optical profilometer are different due to different lateral resolutions. This further supports the argument of the need to characterize the topography using multiple techniques. Nevertheless, both the techniques prove that the SMAT led to an increase in the surface roughness. Thus, both nano-topography (AFM) and micro-topography (OP) of the surface changed after SMAT. Surface wettability was evaluated by measuring the water contact angle. The water contact angle increased significantly after SMAT from 33° ± 5° in A500 to 61° ± 2° in A500-S. In other words, the surface became more hydrophobic after SMAT. The surface of A500 and A500-S were ground slightly to deconvolute the role of roughness and microstructure on wettability. The water contact angle of the ground samples was similar for A500 (48 ± 3°) and A500-S (50 ± 4°) indicating that the wettability is governed by roughness rather than microstructure in the present case. The composition of the surface oxide layer was determined using XPS. The wide-angle spectra of A500 and A500-S are compiled in Fig. 5. The two XPS spectra nearly overlap with each other implying that SMAT did not lead to significant changes in the composition of the passively formed oxide layer on the alloy surface. Table1 Values of the surface roughness parameters obtained from AFM and optical profilometer (OP) Ra AFM OP (nm) (nm) A500 1.2 ± 0.1 27 ± 4 A500-S 2.2 ± 0.2 46 ± 18

Sample

Rq AFM OP (nm) (nm) 1.5 ± 0.1 40 ± 6 2.9 ± 0.1 70 ± 20


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Figure 4 AFM image of (a) A500 and (b) A500-S; Optical profilometer image of (c) A500 and (d) A500-S

O

Intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Ti A500→ → Sn

Nb

C

0

200

A500-S→ →

400

600

800

1000

1200

Binding energy (eV) Figure 5 XPS spectra showing similar oxide compositions on the surfaces of A500 and A500S 15 ACS Paragon Plus Environment


3.3.

Corrosion behavior

The Tafel plots of A500 and A500-S measured in SBF are shown in Fig. 6a. The passivation behavior typical of Ti alloys was shown by both A500 and A500-S. The corrosion rate (ICORR) increased marginally after SMAT from (1.8 ± 0.1) x 10-8 A/cm2 in the A500 to (6.3 ± 2.4) x 10-8 A/cm2 in the A500-S (Table 2). The passivation current (IPASS) was also higher for A500-S as compared to A500 (Table 2). Charge carrier density of the oxide layer was calculated from the Mott-Schottky plots shown in Fig. 6b. The oxide layer formed on the surface of the alloy is n-type in nature, as evident by the positive slope of the curve. The conductivity of the oxide layer can be calculated from the Mott-Schottky plots

12

and is inversely proportion to slope of the linear

region in the curve. The linear region in the curve is marked in Fig. 6b. The values of charge carrier density are noted in Table 2. The conductivity of the oxide layer formed on the alloy did not change after SMAT.

(a)

0.4

(b)

A500 A500-S

(F-2cm4

)

0.2

10

A500 A500-S

8 6

10

0.0

1/C2 x 10

Voltage (V vs. SCE)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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-0.2 -0.4 -0.6 -10

-9

-8

-7

-6

-5

4 2

0 -1.0

-0.5

Log I (A/cm2)

0.0

0.5

1.0

Voltage (V vs. SCE)

Figure 6 (a)Tafel plot (b) Mott-Schottky plot of A500 and A500-S measured in SBF

Table 2 Values of corrosion current density (ICORR), corrosion potential ECORR, passivation current (IPASS) and charge carrier density (Nd) Property

A500

A500-S

ICORR (x 10-8 A cm-2) ECORR (V vs. SCE)

1.8 ± 0.1 -0.43 ± 0.05

6.3 ± 2.4 -0.32 ± 0.02


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IPASS (x 10-6 A cm-2) Nd (x 1019 cm-3)

3.4.

0.7 ± 0.1 2.5 ± 0.9

2.4 ± 1.5 3.2 ± 1.7

Biological response

Fig. 7a shows the DNA content measured at 1 day and 3 days after seeding cells, as a quantitative indicator of the attachment and proliferation of hMSCs, respectively. DNA content is assumed to be proportional to the number of cells. The cells attached to the surface at 1 day were fewer on A500-S as compared to A500. However, the cells present at 3 days were similar on both A500 and A500-S surfaces. It implies that although the attachment of hMSCs is lower, the proliferation rate is higher on the surface of A500-S compared to A500. The proliferation rate calculated as the ratio of DNA content at 3 days vs. 1 day is ~ 2.9 for A500 and ~ 4.1 for A500-S. Fig. 7b shows the absorbance values of the solubilized mineral deposited by hMSCs at the end of 14 days. A higher mineral content is observed on the surface of A500-S as compared to A500. In other words, the osteogenic differentiation of hMSCs improved after processing the surface with SMAT. The improvement in osteogenic differentiation was further confirmed using western blot analysis as shown in Fig. 8a. The osteogenic markers assessed include BMP-2 and Runx2 23. Both these are early stage protein markers for osteogenic differentiation

23

. Runx2 is a target gene for BMP-2 signalling that

downstream leads to the osteogenic differentiation of mesenchymal stem cells

25

. The

expression of these proteins was up-regulated in the cells cultured on the surface of A500-S compared to A500, confirming the improved osteogenic differentiation after SMAT. Additionally, the expression of phosphorylated Akt (pAkt) was found to be up-regulated in the cells cultured on A500-S as compared to A500. pAkt was inhibited and the expression of osteogenic markers was determined in the cells cultured on A500-S to determine its role in osteogenic differentiation. The western blot in Fig. 8b shows that the drug successfully inhibited pAkt whereas its expression was similar 17 ACS Paragon Plus Environment


in cells cultured in medium without drug in medium containing DMSO. The expression of BMP-2 and Runx2 was completely suppressed in cells cultured with the drug (dissolved in DMSO). On the other hand, these proteins were expressed in cells cultured in medium with or without DMSO alone. These results indicate that the enhanced osteogenic differentiation in hMSCs cultured on the alloy surfaces subjected to SMAT, in the present study, was mediated by an Akt dependent pathway.

(a) 400

1 day 3 days

350 300

(b)

250 200 150

0.18

∗

0.15

Absorbance

DNA (ng/ml)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.12 0.09 0.06

∗

100

0.03

50 0

0.00 A500

A500-S

A500

Microstructure

A500-S

Microstructure

Figure 7 (a) DNA content at 1 days and 3 days; (b) absorbance reading of mineral deposited at 14 days. * represents statistically significant difference at p

Surface Severe Plastic Deformation of an Orthopedic TiâNbâSn Alloy

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Surface Severe Plastic Deformation of an Orthopedic TiâNbâSn Alloy