Enhanced Osseointegrative Properties of Ultra ... - ACS Publications

Article Cite This: ACS Biomater. Sci. Eng. 2018, 4, 3268−3281

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Enhanced Osseointegrative Properties of Ultra-Fine-Grained Titanium Implants Modified by Chemical Etching and Atomic Layer Deposition Denis V. Nazarov,*,†,‡ Vladimir M. Smirnov,† Elena G. Zemtsova,† Natalia M. Yudintceva,§ Maxim A. Shevtsov,§,∥,⊥ and Ruslan Z. Valiev† †

Saint Petersburg State University, 7/9 Universitetskaya nab., Saint Petersburg 199034, Russia National Technology Initiative Center of Excellence in Advanced Manufacturing Technologies at Peter the Great St. Petersburg Polytechnic University, Politekhnicheskaya 29/1 str., Saint Petersburg 195251, Russia § Institute of Cytology of the Russian Academy of Sciences (RAS), Tikhoretsky ave. 4, Saint Petersburg 194064, Russia ∥ First Pavlov State Medical University of St. Petersburg, Lva Tolstogo str. 6-8, Saint Petersburg 197022, Russia ⊥ Department of Radiation Oncology, Klinikum rechts der Isar, Technische Universität München, Ismaniger Str. 22, 81675 Munich, Germany

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S Supporting Information *

ABSTRACT: An integrated approach combining severe plastic deformation (SPD), chemical etching (CE), and atomic layer deposition (ALD) was used to produce titanium implants with enhanced osseointegration. The relationship between morphology, topography, surface composition, and bioactivity of ultra-fine-grained (UFG) titanium modified by CE and ALD was studied in detail. The topography and morphology have been studied by means of atomic force microscopy, scanning electron microscopy, and the spectral ellipsometry. The composition and structure have been determined by X-ray fluorescence analysis, X-ray diffraction, and X-ray photoelectron spectroscopy. The wettability of the surfaces was examined by the contact angle measurement. The bioactivity and biocompatibility of the samples were studied in vitro and in vivo. CE of UFG titanium in basic (NH4OH/H2O2) or acidic (H2SO4/H2O2) piranha solution significantly enhances the surface roughness and leads to microstructures, nanostructures, and hierarchical micro-/nanostructures on the surfaces. In vitro results demonstrate deterioration of adhesion, proliferation, and differentiation of MC3T3-E1 osteoblasts cell for CE samples as compared to the non-treated ones. Atomic layer deposition of crystalline titanium oxide onto the CE samples increased hydrophilicity, changed the surface composition, and enhanced significantly in vitro characteristics. In vivo experiments demonstrated non-toxicity of the implants. Etching in basic piranha solution with subsequent ALD significantly improved implant osseointegration as compared with the non-modified samples. KEYWORDS: chemical etching, UFG titanium, atomic layer deposition, osteoblast response, osseointegration

1. INTRODUCTION The development of new materials for the dental and orthopedic implants is the important task of the material science for many years.1,2 A large variety of materials has been designed for implantation, but numerous problems have not been solved so far. For example, usually implants’ osseointegration is too long, and reliability is not sufficiently good. Moreover, the steadily increasing average human life duration requires increasing also the life of implants.3 To solve these problems, different approaches were tried. Now it is evident that the solution may only be found by using the synergetic effect arising from the different combinations of surface modification techniques.4−11 Suitable material for the orthopedic implant must combine the various properties, but the implementation of them is complicated. Mechanical properties play a significant role in the © 2018 American Chemical Society

implant’s success. The necessary set of mechanical properties depends on the intended medical application.1,2 High hardness, tensile strength, and fatigue strength are of the utmost importance. In addition, Young’s modulus of the implant should be close to Young’s modulus of bone tissue.1,2 In addition to bulk mechanical properties even more important for implants are the surface properties, since the surface is in direct contact with the living organism. The surface parameters that influence on the host tissue are chemical composition, wettability, electrical charge, topography, and crystal structure.1,12 Received: March 19, 2018 Accepted: July 26, 2018 Published: July 26, 2018 3268

DOI: 10.1021/acsbiomaterials.8b00342 ACS Biomater. Sci. Eng. 2018, 4, 3268−3281

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ACS Biomaterials Science & Engineering

Figure 1. Scheme of integrated approach.

electronics,27 solar cells,28 lithium-ion batteries,29,30 catalysis,31 etc. In recent years, ALD has been actively used to modify the surface of biomaterials,32,33 but there are practically no studies on applying ALD coatings for medical implants. In the current study, we explored integrated approach (Figure 1) to produce high-performance material for orthopedic implants. The approach combines advantages of three complementary methods: severe plastic deformation (SPD), chemical etching (CE), and atomic layer deposition (ALD). First, SPD improves mechanical properties by conversion of titanium into nanostructured UFG form. Next, CE produces a topography and morphology of the surface that is necessary for the rapid and successful implant osseointegration. Finally, ALD serves for deposition of bioactive and biocompatible crystalline TiO2 coating that protects implant from biological corrosion, preserves topography of the etched surface, and favors the growth of new bone tissue.34 Subsequent in vitro investigations provided the relation between the composition, structure, topography, and morphology of the UFG-Ti surface on the one hand and the cytological response on the other hand. Based on in vitro and in vivo data, we made conclusions about the synergetic effects of combination the SPD, CE, and ALD and prospects for an integrated approach.

To date, titanium and its alloys are known as the most successful materials for the fabrication of orthopedic and dental implants.1−3 Their advantages are high strength, durability, plasticity, and chemical stability.1,2 Alloys could additionally enhance mechanical properties of titanium.2,13 However, many of alloys’ components are toxic and can be dangerous while dissolving.14 The most suitable alternative is the use of pure titanium in the nanostructured or ultra-fine-grained (UFG) forms.15−17 UFG-Ti has more implant-suitable mechanical properties (high tensile and fatigue strength, low Young’s modulus) as compared to coarse-grained (CG) Ti.15,17 As a result, UFG-Ti provides better reliability and durability as the implant material. Moreover, UFG structure can promote adhesion, spreading, proliferation, and differentiation of cells, and also can accelerate bone tissue mineralization,18 which eventually promotes the implants’ osseointegration. Unfortunately, nanostructuring cannot provide an ideal osseointegration, so additional modification of surface composition, structure, and topography is required. Commonly surface topography engineering includes the techniques of electrochemical anodization,19,20 sandblasting,12,20 chemical etching.20,21 Surface composition is modified by deposition of bioactive coatings using physical vapor deposition (PVD),20 chemical vapor deposition (CVD),20 sol−gel techniques,20,22 and ionic implantation.23 Among the above-mentioned methods, chemical etching (CE) is currently the most promising technique due to wide possibilities to vary both relief and surface composition combined with its simplicity.20,21 We have recently demonstrated that the variation of the etching medium (NH4OH/H2O2 or H2SO4/H2O2) and time leads to various micro-, nano-, and hierarchical micro-/nanostructures on the UFG or CG titanium surface.24 Surface topography of the obtained structures is already very promising but the surface composition is still not quite suitable. Nevertheless, the disadvantages of the surface composition produced by CE can be compensated by additional surface modification, e.g., coating. Atomic layer deposition (ALD) is applicable as the only method that provides coatings with completely preserved surface topography. ALD is based on the cyclic self-limiting gas−solid chemical reactions on the support surface whereby the coating is grown layer by layer.25 While increasing number of chemical reactions (number of ALD cycles), the coating thickness increases as well. ALD provides two main advantages: high precision of the film thickness and high uniformity of the coating even on the substrates with high aspect ratio and porous substrates.25,26 In addition, the technological features of the ALD make it possible to obtain coatings of high purity, as well as coatings of complex composition.25 Due to these features, ALD is widely applied as the technique for the production of materials for micro-

2. EXPERIMENTAL SECTION 2.1. Mechanical Treatment: SPD, Cutting, Polishing, and Cleaning. The titanium rods (Grade 4) of 1 m length and 12 mm in diameter were used as billets for SPD. The rods were subjected to Equal-Channel Angular Pressing (ECAP-Conform processing at 400 °C) in “Nanomet” LLC, Ufa, Russia. Five passes were used, and the resulting value of total accumulated true strain was equal to 3.5.34 After ECAP-Conform the rods were subjected to drawing at 200 °C that resulted in formation of finite nanostructured (UFG) rods with diameter of 6 mm. The average grain size of UFG titanium was ∼100 nm according to analysis of XRD data by Rietveld method (Figure S1). Before etching rods were treated by machining as previously described.34 First, UFG-titanium rods were cut into discs (thickness of 2−3 mm) with the Buehler IsoMet 1000. Then, the discs were ground and polished with a semiautomatic Buehler MiniMet 1000 to the mirror-like surface (roughness less than 10 nm) using 600, 800, and 1200 grit sandpapers and suspension of silicon dioxide nanoparticles (20 nm). Finally, the samples were cleaned repeatedly with acetone and deionized water in an ultrasonic bath for 15 min and dried in a desiccator.34 2.2. Chemical Etching. The polished UFG-Ti discs were placed into Pyrex glass containers with 40 mL of basic piranha solution (BPS, NH4OH/H2O2) or acidic piranha solution (APS, H2SO4/H2O2). The volume ratio of the reagents for both types of solutions was 7/3. The temperature 20 °C was maintained with thermostat (Elmi TW-2.03). APS and BPS were prepared using ammonium hydroxide (50% NH4OH; Vecton, Russia), sulfuric acid (36 mol/L H2SO4; Vecton, Russia), and aqueous hydrogen peroxide (30% v/v H2O2; Vecton, Russia). The discs were kept in piranha solutions during 5 and 15 min, 3269


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2.5.3. Cell Viability and Proliferation. MC3T3-E1 cells were incubated with PBS (control sample) for 1, 6, 12, 24, and 48 h in a CO2 incubator. After incubation, cells were washed, and 0.4% Trypan blue exclusion test was used for assessment of viability. Additionally, the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to estimate the cytotoxicity of the samples. We applied the Vybrant MTT Cell Proliferation assay kit according to the manufacturer’s protocol (Life Technologies, USA) using Bio-Rad 680 photometer (Bio-Rad Laboratories Inc., USA). Cell proliferation was analyzed using crystal violet assay after 1, 6, and 12 h and 1, 2, 3, and 7 days of co-incubation on the samples. 2.5.4. Cells’ Osteogenic Differentiation Analysis. For evaluation of the cells’ osteogenic differentiation, we analyzed early marker alkaline phosphatase (ALP) and late marker osteopontin (OP).35−37 The assessment was performed after 1 h and 1, 2, 7, 14, 21, and 28 days of cells’ co-incubation in a CO 2 incubator. According to the manufacturer’s protocol, we analyzed culture medium for the concentration of the proteins employing Alkaline Phosphatase Assay Kit (colorimetric, Abcam, USA) and Osteopontin N-Half ELISA Kit (Clon Tech, USA). 2.6. In Vivo Analysis of the Nanocoated Titanium Implants. 2.6.1. Animals. New Zealand male rabbits (weight 3.1 ± 0.25 kg) were obtained from the animal nursery “Rappolovo” RAMN (St. Petersburg, Russia). The experimental protocol was approved by the Animal Ethics Committee at the First Pavlov State Medical University of St. Petersburg (Saint Petersburg, Russia). 2.6.2. Surgical Procedure. Each rabbit received titanium implant in the femur close to the joint. After the below-knee amputation the animals were divided as follows (three rabbits per group): (1) insertion of the non-etched and non-coated titanium implant (control group); (2) insertion of the implant etched in NH4OH/H2O2 for 2 h (BPS2h): (3) insertion of the implant etched in NH4OH/H2O2 for 2 h and coated with ALD (BPS-2h+ALD). For anesthesia, fentanyl and fluanisone at 0.5 mL/kg were intramuscularly injected with intraperitoneal injections of diazepam (2.5 mg per animal, valium, Roche, France). The implants were press-fit into the bone canal of the femur. Immediately after surgery, they were allowed full weight-bearing. During the next 60 days the rabbits were kept in separate cages. After a follow-up period, animals were sacrificed by intravenous injections of Pentobarbital. In the end the implants were removed using the removal torque (RTQ) method. Additionally, the surface of the ejected implants was analyzed using SEM JSM-35.7 (Tokyo, Japan). 2.6.3. X-ray Analysis. X-ray analysis was carried out prior to the surgery and 2 and 8 weeks after the insertion of the intraosseous implants. The rabbits were sedated by an intramuscular injection of xylazine (1−3 mg/kg) and ketamine (10−50 mg/kg) mixture prior to the X-ray. Radiographs (46 kV, 200 mA, 32 ms, Trophy N800 HF, Fujifilm 24 ×30 cm2 IP cassette type C, 1 m film-focus distance) were taken for evaluation of the position of the implants and cortical layer thickness in the bone−implant interface. 2.6.4. Removal Torque Measurements. The removal torque measurements were used for evaluation of the interfacial shear strength between the implant surface and the bone tissue. The static torque was applied to the implant at a linearly increasing rate (9.5 N·cm/s). 2.7. Statistical Analysis. Five samples of each type were used for in vitro studies. Three implants of each type were used for in vivo studies. The error bars in figures represent standard deviation. One- or twotailed Student’s t tests were used to evaluate the differences between the experimental and control groups. All data processing was run using Statistica Version 9.2 for Windows (StatSoft, Inc., Tulsa, OK, USA). Pvalues of 99%). Impurity contents were oxygen 0.10−0.24, iron 0.22−0.25, and copper 0.09−0.15 wt%. The XPS study of the surface of CE and ALD-treated samples indicated the presence of Ti, O, and C as the only common surface contaminants. No other elements were detected. After surface ion etching, carbon completely disappeared (Figure 5). Thus, the presence of carbon on samples surface is caused only by adventitious atmospheric hydrocarbon contamination. All high-resolution Ti 2p XPS spectra contained the Ti 2p1/2 and Ti 2p3/2 peaks with maxima at 464.9 and 459.2 eV (Figure 6). The peaks are attributed to Ti4+.38 No Ti3+ or Ti2+ shoulders at lower binding energies were detected, suggesting that all samples have a predominant TiO2 surface layer. Metallic Ti0 peak was present in the spectrum of non-modified titanium surface (Figure 7). This peak disappeared after APS etching (probably due to the surface oxidation)24 but still presented after BPS etching. The complete disappearance of the Ti0 peaks was observed for ALD-coated samples. This observation confirms the full coverage of the surface of the ALD samples by TiO2 layer. Non-treated, CE-treated, and CE+ALD-treated UFG-Ti each have an intensive O 1s peak corresponded to Ti−O bonds at 530.5 eV (Figure 8) and another peak at a higher energy which can be attributed to −OH and H2O surface species.38 Its intensity is higher for BPS than for APS samples, but after ALD intensity decreases. Variation of relative intensities can be caused either by the difference of surface species concentration or by variation of specific surface area. According to XRD, the ALD coatings are polycrystalline and characterized by (101), (200), (105), (211), and (204) structure reflections of TiO2 anatase (Figure 9). 3.4. Wettability of the Surface. Non-treated titanium surface is hydrophilic (the contact angle is 79 ± 3°); see Figure 10 and Table 1. APS etching does not cause the significant change of the contact angle. However, this leads to the increase of the values’ deviation (APS-24h). After BPS etching the

Figure 2. SEM of four structure types prepared on the UFG-Ti surfaces by CE. Adapted from ref 34.

(1) BPS-15 min, i.e., the samples etched in NH4OH/H2O2 during 15 min. The nanostructures are present in these samples, but the micrometer-sized structures are not. (2) BPS-2h, i.e., the samples etched in NH4OH/H2O2 during 2 h. The nanostructures are present in these samples together with the pits with the diameter of 1−2 μm. (3) APS-15 min, i.e., the samples etched in H2SO4/H2O2 during 15 min. These samples contain sponge-like nanostructures, but they have no microstructures. (4) APS-24h, i.e., the samples etched in H2SO4/H2O2 during 24 h. Well-developed nano- and microtopography is present here. 3.2. Atomic Layer Deposition. Titanium oxide layers have been successfully deposited by ALD on the surfaces of four types CE samples under study. A total of 400 ALD cycles led to final thickness of 20 nm that corresponds to growth rate of 0.05 nm per cycle. The SEM data suggest that nanoscale structures were changed for all samples (Figure 3).34 This effect is quite expected, as the size of the nanostructures prepared by CE did not exceed a few tens of nanometers. So after the ALD, the nanostructures were overgrown by titanium oxide coatings. All samples after ALD demonstrated the presence of the grains of 30−100 nm in diameter. Note that the ALD coating did not destroy initial etched titanium topography at the micrometer scale (Figure 3). However, we observed some healing of microscale holes that is clearly revealed for the sample BPS-2h.34 In order to analyze the changes of the samples topography after CE+ALD, we measured the set of scans 1 × 1 (nanoscale) and 10 × 10 μm (microscale) with AFM and calculated parameters of topography: vertical range, the average mean value of surface roughness (Ra), root-mean-square roughness (RMS), and specific surface area (Ssurf). Representative 3271


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Figure 4. AFM 3d surface topographies of the chemically etched UFG-Ti before and after ALD.

cellular multilayers formation in the ALD-coated samples but not in the CE samples (data not shown). 3.5.2. Cellular Cytotoxicity and Proliferation. After coincubation of the MC3T3-E1 osteoblasts we measured the cytotoxicity after 1, 6, 12, 24, and 48 h employing MTT assay. All samples did not cause toxic activity for the whole 48 h period of observation (Figure 13). Intriguingly, in the ALD samples one could notice the increased cell viability as compared to the control or CE-modified samples. MC3T3-E1 osteoblasts proliferation of ALD samples (Figure 14) was increased as compared to non-coated titanium. The difference in the proliferation of ALD-coated samples with ones subjected to CE only could cause the difference in viability results. In fact, the MTT test demonstrates the total number of living cells. Since proliferation of cells occurs during the coincubation, the viability values at later periods are significantly larger than in the early stages for the same samples. Similarly, the differences in proliferation of various samples cause the differences in their viability. 3.5.3. Osteogenic Cell Differentiation. Alkaline phosphatase (ALP)the early marker of osteogenic differentiationwas analyzed after 1 h and 1, 2, 7, 14, and 28 days of co-incubation with MC3T3-E1 osteoblasts. After 1 day of co-incubation we detected an elevation of ALP in the culture medium (Figure 15). The ALP content was further increased up to 7−14 days of coincubation with subsequent decrease to the day 28. The faster ALP reaches its maximum, the faster the processes of early differentiation of osteoblasts take place, and one can expect an earlier osseointegration of the implant. Based on these data, it can be concluded that the ALP maxima are observed earlier for samples coated with ALD (2−7 days) than for non-treated, control, and CE samples (7−14 days). The comparative analysis also demonstrated that ALD samples induced statistically more significant increase in the absolute value of ALP as compared to control and CE samples (Figure 15). The highest ALP

Figure 5. C 1s XPS spectra of ALD samples before and after ion etching.

contact angle is significantly increased (>90°); as a result, surface becomes hydrophobic. The hydrophobicity of these samples can be related also to the surface composition features. The contact angle for the etched samples is significantly decreased after ALD. The minimal contact angle values were found for the samples APS-15min+ALD (62 ± 2°) and BPS-2h +ALD (68 ± 7°). 3.5. In Vitro Analysis of the Cellular Interactions. 3.5.1. Osteoblasts’ Adhesion and Spreading. After coincubation (for 24 h, 7 days, and 14 days) of the MC3T3-E1 osteoblasts onto modified UFG titanium samples, the latter ones were washed with PBS and fixed for the subsequent SEM analysis. The cells were fusiform (Figure 11). SEM images of the surface of samples coincubated for 7 days with osteoblasts MC3T3-E1 are depicted in Figure 12. CE samples showed the reduced adhesive properties of the MC3T3E1 osteoblasts as compared to the non-treated titanium and ALD-modified samples. Intriguingly, on the ALD-coated samples we observed the formation of the cellular monolayer starting from day 7 of co-incubation. On day 14 we detected the 3272


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Figure 6. High-resolution XPS Ti 2p spectra of the CE- and CE+ALD-modified UFG-Ti samples.

Figure 7. High-resolution XPS Ti 2p spectra of CE- and CE+ALD-modified samples in Ti0 peak region.

activity of OP, and there were no abrupt changes up to a maximum observation time (28 days). Thus, the differentiation process passes to the final stage after 14 days for ALD-coated samples and continues up to 28 days or more for non-coated ones. ALD-modified samples demonstrate better in vitro results than the ones after CE treatment only; however, it is difficult to select the best sample among them. Maximal ALP activity is achieved for the samples that treated in BPS and APS for 15 min, whereas OP accumulation is the best for the BPS-2h+ALD sample. For the in vivo studies, the sample BPS-2h+ALD was selected as it has a complex topography due to the presence of micro- and nanostructures and low contact angle. In addition,

production was observed for UFG-Ti+ALD and APS-24h+ALD samples. Analysis of late osteogenic differentiation marker, osteopontin (OP), demonstrated the gradual increase in the protein production starting from day 1 of co-incubation in all assessed samples (Figure 16). The level of the OP production did not markedly change between the CE-modified samples and nontreated titanium. But samples coated with ALD demonstrated a greater intensity of OP analogously to the case of ALP. In addition, there is a sharp increase before 14 days and a decrease in OP values after 14 days for the ALD samples. This feature may indicate the completion of late differentiation in this period (14−28 days). Non-coated samples showed relatively low 3273


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Figure 8. High-resolution XPS O 1s spectra of the CE- and CE+ALD-modified UFG-Ti samples.

3.6. In Vivo Study of the Implants with Micro-/ Nanotopographic Surface. Non-treated screws, screws etched in BPS, and screws etched in BPS with subsequent ALD were studied by SEM. Images are shown in Figure S2. The morphology of the implants surface is similar to that observed for titanium discs used by us for in vitro studies. After below-knee amputation, the non-treated and modified UFG titanium screws were positioned into the bone. All animals recovered from the operation without complications. We did not observe any complications in the tibia bone (i.e., bone thinning, bone ulceration, etc.) nor any axial displacement of the titanium screws in the follow-up period of 60 days as was shown by the radiographs (Figure 17A). There was no observed any axial displacement of the titanium screws, which indicates that the implant inside tibia was well fixated by the osseointegration process. The data of cortical layer thickness in the bone−implant interface clearly demonstrated the increased new bone formation in the rabbits with CE and CE+ALD screws which constituted 1.29 ± 0.27 and 2.91 ± 0.75 mm, respectively (Figure 17B). Good fixation and high level of osseointegration were further proved with removal torque test that demonstrates the enhanced osseointegration strength for CE and CE+ALD samples: 23.17 ± 5.49 and 38.83 ± 1.17 N·cm, respectively (Figure 17C). Intriguingly, for CE+ALD-modified screws the cortical bone formation is increased and osseointegration is enhanced as compared to the CE-modified implants. Further SEM analysis of the titanium surface clearly demonstrates the presence of the cellular monolayer on the modified screws as compared to the non-modified control implants (Figure 17A).

Figure 9. GIXRD pattern of the ALD titania film deposited on the UFG-Ti surface.

4. DISCUSSION UFG-titanium etching in Piranha solutions produced the surfaces with microstructures, nanostructures, and hybrid micro-/nanostructures. ALD-modification of the etched surfaces led to the formation of crystalline titanium oxide nanocoating. The parameters of the morphology, topography, wettability, surface structure along with the in vitro results are summarized in the Table 1. These parameters are strongly different for various samples. Nevertheless, in vitro tests

Figure 10. Microphotographs of the drops on the surface: UFG titanium, BPS-2h, and BPS-2h+ALD.

for in vivo studies we used an analogous sample without ALDcoating and also a non-modified sample for the control. 3274


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ACS Biomaterials Science & Engineering Table 1. Summary of Characteristics of the Samples topography (Rmax, RMS, surface areaa) contact angles (deg)

adhesion/viabilityb (at 2nd day)

proliferationb/ differentiationb (AF-OP)

smooth/native titania

79 ± 3

good/1.40

0.84/1.53−1.45

19.4 ± 3.7 2.31 ± 0.22 3.6 ± 0.4

smooth/titania, 20 nm (anatase)

80 ± 2

good/1.41

1.24/1.86−1.91

125 ± 13 6.18 ± 0.76 1.0 ± 0.2

17.8 ± 3.4 1.51 ± 0.16 4.0 ± 0.4

nanosponge, microsmooth/titania (amorphous)

75 ± 3

bad/1.06

0.90/1.23−1.04

APS-24h

601 ± 48 53.1 ± 6.5 6.5 ± 0.6

49.3 ± 7.8 5.41 ± 0.85 6.0 ± 0.6

nanostructures, micropits/titania (amorphous)

71 ± 10

bad/1.05

0.99/1.07−0.96

BPS-15min

476 ± 40 52.6 ± 5.2 15.1 ± 1.3

78.1 ± 3.9 9.68 ± 0.23 40.2 ± 1.1

nanonets, micropits/thick titania (amorphous)

100 ± 2

bad/1.05

0.89/1.05−1.03

BPS-2h

912 ± 37 80.2 ± 5.2 35.0 ± 2.1

97.7 ± 5.2 14.3 ± 0.5 41.9 ± 1.9

nanograins, microsmooth/titania (anatase)

120 ± 5

bad/1.03

0.77/1.05−0.98

APS-15min+ALD

135 ± 24 7.75 ± 0.46 0.8 ± 0.1

22.9 ± 3.3 3.20 ± 0.42 4.2 ± 0.7

nanograins, microsmooth/titania (anatase)

62 ± 2

good/1.41

1.28/1.75−1.89

APS-24h+ALD

501 ± 63 37.9 ± 7.4 7.1 ± 2.1

64.6 ± 4.4 8.43 ± 1.38 12.2 ± 3.9

nanograins, micropits/titania (anatase)

72 ± 9

good/1.38

1.11/1.83−2.03

BPS-15min+ALD

402 ± 173 57.9 ± 19.1 4.2 ± 1.4

76.9 ± 6.6 10.2 ± 1.2 7.4 ± 1.9

nanograins, micropits/titania (anatase)

74 ± 4

good/1.39

1.29/1.39−1.85

BPS-2h+ALD

418 ± 27 42.1 ± 1.4 9.4 ± 2.1

56.9 ± 16.7 6.80 ± 1.91 4.2 ± 2.3

nanograins micropits/titania (anatase)

68 ± 7

good/1.38

1.00/1.52−2.28

1 × 1 μm

10 × 10 μm

UFG

126 ± 11 6.54 ± 0.79 1.3 ± 0.3

17.1 ± 3.1 1.91 ± 0.19 2.3 ± 0.4

UFG+ALD

127 ± 13 13,4 ± 0.87 1.5 ± 0.3

APS-15min

sample

surface morphology/composition

a

In percent. bIn comparison to control sample.

Figure 11. Morphology of MC3T3-E1 cells co-incubated on BPS-2h +ALD sample. Live cell imaging after (a) 1 day and (b) 7 days of cultivation. Scale bar = 100 μm.

Figure 12. Representative SEM images for the samples after 7 days of co-incubation with MC3T3-E1 osteoblasts.

It is known that the implant topography significantly affects the cytological response of cells.1,12,20 It is generally believed that microscale topography significantly improves cell adhesion

demonstrate that all the samples are non-cytotoxic and lead to the production of the markers of early and late differentiation in the osteogenic direction. 3275


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Figure 13. MC3T3-E1 osteoblast viability after co-incubation. Data are presented as mean ± SD from five independent series of experiments.

Figure 14. MC3T3-E1 osteoblast proliferation activity after co-incubation. Data are presented as mean ± SD from five independent series of experiments.

Figure 15. Alkaline phosphatase production by MC3T3-E1 osteoblasts. Each value represents mean ± SD from five independent experiments.

interactions of implant surface with host tissue.12,40 However, the influence of values of topography parameters on the biomedical properties is rather complicated and often has an extremal dependence.41

and can promote bone-to-implant contact via mechanical interlocking mechanisms.12,39,40 Nanoscale topography affects chemical reactivity, wettability, and adsorption rate of biomolecules and leads to improvement of biomedical 3276


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Figure 16. Osteopontin production by MC3T3-E1 osteoblasts. Each value represents mean ± SD from five independent experiments.

does not influence on the adhesion, viability, and proliferation of the osteoblasts (CAL-72). Solar et al. showed the deterioration of adhesion and spreading of osteoblasts (MG-63) with an increase in the roughness (RMS) higher than 30 nm.47 In our samples RMS reached 80 nm, whereas for non-etched Ti this parameter was only 6 nm. The worst characteristics of proliferation and differentiation were found for the samples with hierarchical micro-/ nanostructures (BPS-2h, BPS-15min). Note that due to the biomimetic nature, such structures are expected to be the most promising, and they attract special interest of researchers. Most researchers suggest a significant enhancement of the bioactivity and appearance of synergetic effect in the hierarchical micro-/ nanostructures.4−7 Deterioration of the in vitro characteristics for our samples is evidently related to the surface hydrophobicity,48 as well as to the features of surface composition and thickness of the surface oxide layer.49,50 These disadvantages were easily eliminated by the ALD coating. A definite improvement of the cytological response was found for all ALD-modified samples. This effect is observed for all the in vitro parameters (viability, proliferation, ALP, OP). The performance improvement can be caused by a change in the surface composition and by the anatase crystalline structure of the coating. It is known that the crystallinity of the surface oxide layer significantly affects the osseointegration of the bone tissue.51 The higher crystallinity favors the growth of hydroxyapatite, the main inorganic component of bone tissue.52 Rutile is the most suitable crystalline structure for bone tissue mineralization; however, rutile does not demonstrate advantages at in vitro experiments in comparison to anatase.53 On the contrary, anatase can enhance osteoblast adhesion and proliferation by affecting wettability.52 Our coatings have the anatase structure, and experiments really demonstrated the significant decrease of the wetting angles after ALD modification (Table 1). Taking our experimental data into the account, we can compare the in vitro parameters of the samples of the series CE, ALD, and combined CE+ALD. Due to the fact that CE leads to worse in vitro characteristics, whereas ALD to better ones, so ALD-treated sample was expected to demonstrate better

Figure 17. In vivo evaluation of UFG-Ti implants. Data are presented as mean ± SD. (A) Radiographs of the control, BPS-2h, and BPS-2h+ALD implants at 8 weeks after surgery and representative SEM images of the extracted implants’ surface are presented. Scale bar = 30 μm. (B) Cortical layer thickness (mm) in the bone−implant interface. (C) Osseointegration strength (removal torque, N·cm).

From our results, the chemical etching in acidic and basic piranha solutions led to some deterioration of in vitro parameters for the samples with the developed relief as compared to the relatively smooth non-etched titanium. Note that decrease of cytological response is typical both for the samples with micrometer-sized relief (APS-24h) and for ones with nanosized relief (APS-15min). Despite this rather unexpected conclusion, our results are not exceptional. The previous experimental results show that in some cases the microtopography depress osteoblast proliferation,42,43 differentiation, and mineralization.44 Influence of nanotopography can also be controversial.45 So, Cai et al.46 showed that variation of the titanium roughness (RMS) in the range from 2 to 21 nm (scans 50 × 50 μm) almost 3277


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ACS Biomaterials Science & Engineering Table 2. Results of in Vitro Studies of the Hybrid Nano-/Microstructures on the Titanium Surface characteristic

viability (MTT test)

proliferation

differentiation

type of cells

ref

nano-/microstructures + TiO2 (BPS2h+ALD)

1.41 (2 days)a

1.29 (3 days)a

ALP-1.83 (2 days)a ALP-1.4 (7 days)a ALP-1.45 (2 days)b OP-2.84 (14 days)a

MC3T3-E1

this work

nanostructured CG-Ti

0.7b (BPS etching)

1.2b (4 days) (APS etching)

OCb very good (APS etching)

MC3T3-E1

50

hierarchical micro-/nanostructures

∼1.8b (4 days)

MC3T3-E1

4

ALP ∼1.1b (4 days)

BMSCs

5

∼1.2b (4 days)

hierarchical micro-/nanostructures + Sr hierarchical micro-/nanostructures

∼1.2−1.3b (3 days)


BMSCs

6

micro-/nanostructures

∼1.1−1.2


MC3T3-E1

7

UFG-Ti hierarchical porous surface

≥0.8a (3 days) 1−1.1b

MG63

54

ALP ∼2−3 (7 days)c OC ∼3−4 (7 days)c

rat bone marrow cells

8

ALP (7 days) 0.75−1.25 (depends on nanotube size) OC, remarkably worseb

rat calvarial osteoblasts

9

MSCs

10

MC3T3-E1

11

1.15−1.6 (2 days)c

micropits and TiO2 nanonodules

hybrid micropitted/nanotubular

MTT test, no differenceb

hybrid micropitted/nanotubular

1.2 (4 days)b

nanoneedle and nanoporous/ micropits

1.2 (3 days) 1.4 (5 days) 1.6 (7 days)

ALP 3−4 (7 days) + 1.5 (14 days)b

a Compared to control sample. bCompared to untreated Ti. cCompared to micrometer-scale Ti. Abbreviations: OC, osteocalcin; BMSCs, bone marrow stromal cells; MSCs, mesenchymal stem cells.

spark plasma sintering (see the summary in the Table 2). Resulted structures differ in morphology and topography and also have various in vitro characteristics. In common, the viability and differentiation of our CE+ALD samples are equal or surpass these parameters of the majority of studies. But our results are not maximal ones. Viability values for our samples are less than the ones of hierarchal structures prepared by Li.4 Differentiation of our samples is better compared to the results of Li,4 Xu,7 Jiang,6 and Zhao9,10 but worse than the results of Kubo8 and Zhuang.11 In vivo studies in the model of the below-knee amputation in New Zealand rabbits clearly demonstrated that all the samples are non-toxic and well-attached into the intramedullary canal. CE+ALD combination (samples BPS-2h+ALD) demonstrated the best parameters for the formation of the strong bonds with the surrounding tissues. The nontoxicity and well-attachment of the CE- and/or ALD-modified implants is in line with previously reported results of the biocompatibility for implant with surfaces modified by CE, sandblasting, and anodization.55−57 The greatest cortical layer thickening and force of the removal from the bone were observed for the samples with hierarchical micro-/nanostructures (BPS-2h+ALD). This can indicate the accelerated osteogenesis in the area of the contact of the bone with the implant surface. Osseointegration strength (that was measured using removal torque (RTQ) method) could be comparable to that of published previously by Sul58 and more

properties than CE+ALD samples. However, the results showed that the difference is either minor for CE+ALD-modified samples or even they have advantagesynergetic effect of combination of CE and ALD is acted. Probably, the combination of developed topography, the surface composition, and smaller contact angles for CE+ALD samples comparing to these parameters for CE samples determine their high in vitro results. We compared our in vitro results with published data. Results of viability, proliferation differentiation of our samples, and literature data are presented in Table 2. From all the variety of published data, we selected the papers where the authors studied hierarchical micro-/nanostructures. Note that these results are complicated for the comparison because various authors use different cells and control samples. The samples preparation methods along with in vitro tests techniques are also different. Vetrone et al. used APS and BPS chemical etching for titanium modification.50 Authors found that APS-etching enhances adhesion and proliferation, whereas BPS etching deteriorates viability and mineralization. The samples surface morphology was similar to one of our samples; however, the data about topography parameters were not described in detail. In addition, Vetrone et al. used CG titanium, whereas we used UFG titanium. The difference of in vitro characteristics of CG and UFG titanium samples can be significant.18 In other works, various techniques were used to fabricate hierarchical nano/ microstructures, including anodization, chemical etching, and 3278


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ACS Biomaterials Science & Engineering recently by Zemtsova et al.59 Implants with fluorinated TiO2 nanotubes demonstrated significantly increased removal torque strengths (41 vs 29 N·cm; P = 0.008) in rabbit femurs and new bone formation (57.5% vs 65.5%; P = 0.008) as compared to the non-coated implants.48 Microtopographic/nanotopographic surfaces of Ti implants, as shown by Zemtsova et al., increased new bone formation and osseointegration strength up to 41.97 ± 2.54 N·cm.59 Presumably coating of the implant surface by cells prior to the intraosseous implantation could further promote osseointegration. Thus, seeding of the bone-integrated pylon with autologous fibroblasts induced into osteoblast differentiation provided 1.5-fold higher osteogenesis than in control group (as shown by three-phase scintigraphy and histological analysis).60 Surprising is the fact that the sample BPS-2h with the worst in vitro parameters showed higher cortical layer thickness, amount of the cell contacts, and force necessary for the removal compared to the non-modified titanium. Thus, in vivo experiments showed positive effect of hierarchical micro-/nanostructures on osseointegration. Summarizing, we note that the integrated approach suggested by us includes three complementary modification techniques (SPD, CE, and ALD). This approach is promising for the development of orthopedic implants with the high osseointegration ability. SPD significantly improves mechanical properties of the implants,16,61,62 CE forms necessary surface topography and morphology, and ALD enhances wettability as well as chemical and phase composition of the surface. The presented in vivo studies emphasize the influence of the hierarchical micro-/nanotopography formed by CE+ALD combination on osseointegration of the implants.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +7-812-428-4033. ORCID

Denis V. Nazarov: 0000-0002-7230-2070 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was conducted using the equipment of the resource centers of the Research Park of the St. Petersburg State University, “Innovative Technologies of Composite Nanomaterials”, “Physical Methods of Surface Investigation”, “X-ray Diffraction Studies”, “Nanotechnology”, and “Nanophotonics”. This work was supported in part by grants from the StPetersburg State University, No. 6.37.204.2016, grant from the St-Petersburg State University and Event 3-2018. The animal experiments were in part supported by grants from the Russian Science Foundation, No. 14-50-00068, and by the Federal Agency of Scientific Organizations, Russia, State Grant of the Ministry of Health of the Russian Federation No. 32.

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REFERENCES

(1) Brunette, D. M.; Tengvall, P.; Textor, M.; Thomsen, P. Titanium in Medicine; Springer-Verlag: Berlin/Heidelberg, 2001; http://www. springer.com/us/book/9783642631191. (2) Geetha, M.; et al. Ti Based Biomaterials, the Ultimate Choice for Orthopaedic ImplantsA Review. Prog. Mater. Sci. 2009, 54, 397−425. (3) Von der Mark, K.; Park, J. Engineering Biocompatible Implant Surfaces Part II: Cellular Recognition of Biomaterial Surfaces: Lessons From Cell−Matrix Interactions. Prog. Mater. Sci. 2013, 58, 327−381. (4) Li, B. E.; Li, Y.; Min, Y.; Hao, J. Z.; Liang, C. Y.; Li, H. P.; Wang, G. C.; Liu, S. M.; Wang, H. S. Synergistic Effects of Hierarchical Hybrid Micro/Nanostructures on the Biological Properties of Titanium Orthopaedic Implants. RSC Adv. 2015, 5, 49552−49558. (5) Li, Y.; Fu, Q.; Qi, Y.; Shen, M.; Niu, Q.; Hu, K.; Kong, L. Effect of a Hierarchical Hybrid Micro/Nanorough Strontium-Loaded Surface on Osseointegration in Osteoporosis. RSC Adv. 2015, 5, 52296−52306. (6) Jiang, N.; Zhu, S.; Li, J.; Zhang, L.; Liao, Y.; Hu, J. Development of a Novel Biomimetic Micro/Nanohierarchical Interface for Enhancement of Osseointegration. RSC Adv. 2016, 6, 49954−49965. (7) Xu, J.; Chen, X.-s.; Zhang, C.-y.; Liu, Y.; Wang, J.; Deng, F.-l. Improved Bioactivity of Selective Laser Melting Titanium: Surface Modification with Micro-/Nano-Textured Hierarchical Topography and Bone Regeneration Performance Evaluation. Mater. Sci. Eng., C 2016, 68, 229−240. (8) Kubo, K.; Tsukimura, N.; Iwasa, F.; Ueno, T.; Saruwatari, L.; Aita, H.; Chiou, W.-A.; Ogawa, T. Cellular Behavior on TiO2 Nanonodular Structures in a Micro-to-Nanoscale Hierarchy Model. Biomaterials 2009, 30 (29), 5319−5329. (9) Zhao, L.; Mei, S.; Chu, P. K.; Zhang, Y.; Wu, Z. The Influence of Hierarchical Hybrid Micro/Nano-Textured Titanium Surface with Titania Nanotubes on Osteoblast Functions. Biomaterials 2010, 31 (19), 5072−5082. (10) Zhao, L.; Liu, L.; Wu, Z.; Zhang, Y.; Chu, P. K. Effects of Micropitted/Nanotubular Titania Topographies on Bone Mesenchymal Stem Cell Osteogenic Differentiation. Biomaterials 2012, 33 (9), 2629−2641. (11) Zhuang, X. M.; Zhou, B.; Ouyang, J. L.; Sun, H. P.; Wu, Y. L.; Liu, Q.; Deng, F. L. Enhanced MC3T3-E1 Preosteoblast Response and Bone Formation on the Addition of Nanoneedle and Nano-Porous

5. CONCLUSIONS In this work, we studied the advantages and drawbacks of the integrated approach that is directed onto the development of the bioactive material for the medical implants. The approach successfully combined advantages of the severe plastic deformation (SPD), chemical etching (CE), and atomic layer deposition (ALD). Based on in vitro and in vivo tests, we showed that the samples BPS-2h+ALD with hierarchical micro-/ nanostructures are non-toxic and have the most favorable effect on the proliferation and differentiation of cells in osteogenic direction, osseointegration, and reliability of the implants’ osseointegration. We studied the relation between morphology, topography, composition, and wettability of CE- and ALD-modified UFG titanium surface on the one hand and cytological in vitro response on the other hand. Significant effect of the surface treatment type is found on the adhesion, proliferation, and differentiation of cells of MC3T3-E1 osteoblasts. It was shown that CE deteriorates in vitro parameters, whereas ALD coatings enhance these parameters. CE+ALD combination leads to the effect that is either equal or even greater than the case of ALD treatment only. This fact indicates a synergetic effect of CE +ALD combination.

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Figure S1, XRD pattern of the UFG-Ti; Figure S2, SEM images of the UFG-Ti screws prepared for in vivo study (PDF)

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.8b00342. 3279


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