Enhanced osseointegrative properties of the ultrafine-grained titanium

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Tissue Engineering and Regenerative Medicine

Enhanced osseointegrative properties of the ultrafine-grained titanium implants modified by the chemical etching and atomic layer deposition. Denis Vasilievich Nazarov, Vladimir M Smirnov, Elena Zemtsova, Natalia M Yudintceva, Maxim A Shevtsov, and Ruslan Z Valiev ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00342 • Publication Date (Web): 26 Jul 2018 Downloaded from http://pubs.acs.org on July 27, 2018

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ACS Biomaterials Science & Engineering

Enhanced osseointegrative properties of the ultrafine-grained titanium implants modified by the chemical etching and atomic layer deposition. Denis V. Nazarov1,2*, Vladimir M. Smirnov1, Elena G. Zemtsova1, Natalia M. Yudintceva3, Maxim A. Shevtsov,3,4,5 and Ruslan Z. Valiev1 1

Saint Petersburg State University, 7/9 Universitetskaya nab., Saint Petersburg 199034,

Russia 2

National Technology Initiative Center of Excellence in Advanced Manufacturing Technologies at Peter the Great St. Petersburg Polytechnic University, Saint Petersburg, 195251 Politekhnicheskaya 29/1 str., Russia.

3

Institute of Cytology of the Russian Academy of Sciences (RAS), Saint Petersburg, 194064 Tikhoretsky ave., 4, Russia. 4

First Pavlov State Medical University of St.Petersburg, Saint Petersburg, 197022 Lva Tolstogo str. 6-8, Russia 5

Department of Radiation Oncology, Klinikum rechts der Isar, Technische Universität München, 81675 Ismaniger Str. 22, Munich, Germany Keywords: Chemical etching; UFG titanium; Atomic Layer Deposition, Osteoblast response;

osseointegration.

Abstract:

The integrated approach which combined the 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

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composition and bioactivity of ultrafine grained (UFG) titanium modified by CE and ALD is studied in detail. The topography and morphology have been studied by means of atomic force microscopy (AFM), scanning electron microscopy (SEM), and the spectral ellipsometry (SE). The composition and structure have been determined by X-Ray fluorescence analysis (XRF), X-ray diffraction (XRD), and X-Ray Photoelectron Spectroscopy (XPS). 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 the basic (NH4OH/H2O2) or acidic (H2SO4/H2O2) Piranha solutions significantly enhances the surface roughness and leads to micro-, nano- and hierarchical micro/nano structures 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 increase hydrophilicity, change the surface composition and enhanced significantly in vitro characteristics. In vivo experiments demonstrate non-toxicity of the implants. Etching in basic piranha solution with the subsequent ALD significantly improve implant osseointegration as compared with the non-modified samples.

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].


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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 implant’s success. The necessary set of mechanical properties depends on the intended medical application [1,2]. High hardness, tensile and fatigue strength are of the superior 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]. To the moment, 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 ultrafine grained (UFG) forms [15-17]. UFG-Ti has more implant-suitable mechanical properties (high tensile and fatigue strength, low Young 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, 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, the chemical etching (CE) is currently the most promising technique due to wide possibilities to variate 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 films 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 microelectronics [27], solar cells [28], lithiumion 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 highperformance material for orthopedic implants. The approach combines advantages of three


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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. Then, 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, ALD and prospects for an integrated approach.

Figure 1. Scheme of integrated approach 2. Experimental section. 2.1. Mechanical treatment (SPD, cutting, polishing, 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. The 5 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]. Firstly, UFGtitanium 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 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, 15 minutes, 1, 2, 6, and 24 hours. Immediately after etching, the samples were taken out of the etchant and thoroughly washed in distilled water using an ultrasonic bath [34]. 2.3. Atomic Layer Deposition Titanium oxide was coated by ALD on the surface of the polished, etched UFG-Ti and monocrystalline silicon (100) plates (2x2 cm). Silicon plates act as a witness (control sample) for ellipsometry and X-ray reflectometry (XRR) measurements [34]. The deposition was performed in the hot-wall, flow-type reactor (Nanoserf) having slot-type geometry (Nanoengineering Ltd, Russia). The temperature of the reactor was maintained at 250°C.


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Nitrogen (99.9999%) was used as a carrier and purging gas. Total reactor gas flow rate at purge pulse was 300 sccm (standard cubic centimeters per minute). Titanium isopropoxide (Ti(OiPr)4, Sigma Aldrich 99.999%) as a solution in isooctane (99.99%) was supplied into reactor by the injection valve. Isooctane acted as a carrier gas, which evaporates quickly after injection into the Nanoserf reactor. The pulse duration was 20 ms that corresponds to dosage of about 4 µmol Ti(OiPr)4 per cycle. The volume ratio titanium isopropoxide/isooctane was 1/20. The water co-reactant was delivered by vapor (pulse - 500 ms) with the vessel being held at 27°C. Total number of ALD cycles was 400. More detailed description of deposition is given in [34]. 2.4. Morphology, topography and composition of surface. The topography of the samples surfaces was studied by atomic force microscope (AFM) Solver P47 Pro (NT-MDT, Moscow, Russia) using the tapping mode. The measurements were conducted in air atmosphere with scan areas of 1×1, 10×10, 50×50and µm2. A total of 5-6 random positions on the different samples surface were measured. Four parameters including the vertical range, the average mean value of surface roughness (Ra), root mean square roughness (RMS) and surface area difference (the increase of 3D surface area over 2D surface area, in percent) were calculated by the Gwyddion 2.37 software [24]. The morphology of samples was studied with scanning electron microscope (Zeiss Merlin) operated at 10–15 kV in In-lens, SE2 and EDS modes. The magnifications from 300 up to 600 000 (spatial resolution 1 nm) were used. 3-4 random positions on the each sample surface were scanned. The chemical composition of the UFG-Ti before and after the modification was studied by energy dispersive X-ray fluorescence spectrometer EDX Series 800 HS (Shimadzu). The composition of the samples surfaces was studied by X-ray photoelectron spectroscopy (XPS) with “Thermo Fisher Scientific Escalab 250Xi” spectrometer. All samples were excited by Al



Kα (1486.7 eV) X-rays at a base pressure of 7 × 10−8 Pa. High-resolution spectra were automatically charge compensated by setting the binding energy of C1s carbon line to 284.8 eV [34]. The thickness of TiO2 coatings deposited on the polished UFG-Ti and silicone witness was estimated by spectral ellipsometry (350–1000 nm) using Ellips-1891 SAG instrument (CNT, Novosibirsk, Russia). The accuracy of the thickness measurement was estimated as 0.5-1 nm. Wettability was evaluated by measuring the static contact angle (Biolin Scientific Theta Lite). Deionized water (5 µl) was dropped onto each specimen with an auto-pipette at 20°C. The average contact angles were measured from 3 specimens of each group at 4-5 surface positions at 10 seconds after the dropping. 2.5. In vitro assessment of the cellular interactions 2.5.1. Cell Culture. MC3T3-E1 mice osteoblasts (ATCC® CRL-2593TM) were obtained from the Russian Cell Culture Collection at the Institute of Cytology of the Russian Academy of Sciences (RAS) (Saint Petersburg, Russia). MC3T3-E1 cells were harvested in CO2-incubator (37°C, 6% CO2) in DMEM medium (Sigma-Aldrich, USA) supplemented with 2 mM L-glutamine, 10% fetal bovine serum (FBS) and antibiotics (100 units/ml Penicillin G and 100 µg/ml Streptomycin). 2.5.2. Cell Adhesion. MC3T3-E1 cells were co-incubated on the surface of the samples for 24 hours, 7 and 14 days in a CO2-incubator. After incubation, cells were washed with Dulbecco’s Phosphate Buffer Saline (PBS) (Sigma-Aldrich, USA) and fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (Sigma-Aldrich, USA). Evaluation of the cells adhesion and morphology was performed using SEM JSM-35.7 (Tokyo, Japan). 2.5.3. Cell viability and proliferation.


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MC3T3-E1 cells were incubated with phosphate buffered saline (PBS; control sample) for 1, 6, 12, 24 and 48 hours 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-(4,5dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium 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, 12 hours, 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 hour, 1, 2, 7, 14, 21 and 28 days of cells co-incubation in a CO2-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 (3 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 hours (BPS-2h): (3) insertion of the implant etched in NH4OH/H2O2 for 2 hours and coated with ALD (BPS2h+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 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, 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 Ncm/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 two-


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tailed 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). P-values of 99%). Impurities content was oxygen 0.10–0.24, iron 0.22–0.25, copper 0.09–0.15 (wt%). The XPS study of the surface of CE and ALD-treated samples indicated the presence of Ti, O as well as C only as the common surface contaminant. 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.

Figure 5. C1s XPS spectra of ALD samples before and after ion etching


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All high-resolution Ti2p XPS spectra contained the Ti2p1/2 and Ti 2p3/2 peaks with maxima at 464.9 eV 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.

Figure 6. High-resolution XPS Ti2p spectra of the CE and CE+ALD modified UFG-Ti samples. 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.



Figure 7. High-resolution XPS Ti2p spectra of CE and CE+ALD modified samples in Ti0 peak region. Non-treated, CE treated, and CE+ALD treated UFG-Ti have an intensive O1s 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.

Figure 8. High-resolution XPS O1s spectra of the CE and CE+ALD modified UFG-Ti samples. According to XRD, the ALD coatings are polycrystalline and characterized by (101), (200), (105), (211) and (204) structure reflections of TiO2 anatase (Figure 9).


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Figure 9. GIXRD pattern of the ALD titania film deposited on the UFG-Ti surface. 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 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 BPS2h+ALD (68±7°).

Figure 10. Microphotographs of the drops on the surface: UFG titanium, BPS-2h, BPS2h+ALD. Table 1. Summary of characteristics of the samples



Sample

UFG

UFG+ALD

APS-15 min

APS-24h

BPS-15min

BPS-2h

APS-15 min+ALD APS24h+ALD

BPS15min+ALD BPS2h+ALD

Topography (Rmax, RMS, surface area#) 1x1 mkm 10x10mkm 17.1±3.1 126±11 1.91±0.19 6.54±0.79 2.3±0.4 1.3±0.3 127±13 13,4±0.87 1.5±0.3

19.4±3.7 2.31±0.22 3.6±0.4

125±13 6.18±0.76 1.0±0.2

17.8±3.4 1.51±0.16 4.0 ± 0.4

601±48 53.1±6.5 6.5±0.6 476±40 52.6±5.2 15.1±1.3

49.3±7.8 5.41±0.85 6.0±0.6 78.1±3.9 9.68±0.23 40.2±1.1

912±37 80.2±5.2 35.0±2.1 135±24 7.75±0.46 0.8±0.1 501±63 37.9±7.4 7.1±2.1 402±173 57.9±19.1 4.2±1.4 418±27 42.1±1.4 9.4±2.1

97.7±5.2 14.3±0.5 41.9±1.9 22.9±3.3 3.20±0.42 4.2±0.7 64.6±4.4 8.43±1.38 12.2±3.9 76.9±6.6 10.2±1.2 7.4±1.9 56.9±16.7 6.80±1.91 4.2±2.3

Surface morphology/ composition

Contact angles

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Adhesion/ Viability*(at 2nd day)

Proliferation*/ Differentiation * (AF-OP)

Smooth/native titania

79±3

Good/ 1.40

0.84/ 1.53-1.45

Smooth/titania 20 nm (anatase)

80±2

Good/ 1.41

1.24/ 1.86-1.91

75±3

Bad/ 1.06

0.90/ 1.23-1.04

71±10

Bad/ 1.05

0.99/ 1.07-0.96

100±2

Bad/ 1.05

0.89/ 1.05-1.03

120±5

Bad/ 1.03

0.77/ 1.05-0.98

62±2

Good/ 1.41

1.28/ 1.75-1.89

72±9

Good/ 1.38

1.11/ 1.83-2.03

74±4

Good/ 1.39

1.29/ 1.39-1.85

68±7

Good/ 1.38

1.00/ 1.52-2.28

Nanosponge, microsmooth/ titania (amorphous) Nanostructures, micropits/titania (amorphous) Nano-nets, micropits/thick titania (amorphous) Nanograins, microsmooth /titania (anatase) Nanograins, microsmooth /titania (anatase) Nanograins, micropits/titania (anatase) Nanograins, micropits/titania (anatase) Nanograins micropits/titania (anatase)

#

in percent *-in comparison to control sample

3.5. In vitro analysis of the cellular interactions 3.5.1. Osteoblasts adhesion and spreading After co-incubation (for 24 hours, 7 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).


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Figure 11. The 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. SEM images of the surface of samples co-incubated for 7 days with osteoblasts МC3T3-E1 are depicted in the Figure 12. CE samples showed the reduced adhesive properties of the MC3T3-E1 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 cellular multilayers formation in the ALD-coated samples but not in the CE samples (data not shown).

Figure 12. Representative SEM images for the samples after 7 days co-incubation with MC3T3-E1 osteoblasts. 3.5.2. Cellular cytotoxicity and proliferation



After co-incubation of the MC3T3-E1 osteoblasts we measured the cytotoxicity after 1, 6 and 12 hours, 1 and 2 days 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.

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


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Figure 14. MC3T3-E1 osteoblast proliferation activity after co-incubation. Data are presented as mean±S.D. from five independent series of experiments. 3.5.3. Osteogenic cell differentiation Alkaline phosphatase (ALP) – the early marker of osteogenic differentiation – was analyzed after 1 hour, 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 co-incubation 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 this 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 (Fig. 15). The highest ALP production was observed for UFG-Ti+ALD and APS24h+ALD samples.

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



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 non-treated 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 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 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 noncoated ones.

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


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was selected as it has a complex topography due to the presence of micro and nanostructures and low contact angle. In addition, for in vivo studies we used analogous sample without ALD-coating and also non-modified sample for the control. 3.6. In vitro 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.) as well as no axial displacement of the titanium screws in the follow-up period of 60 days as was shown by the radiographs (Fig. 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 boneimplant 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). Well fixation and high level of osseintegration 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 Ncm, 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 17. In vivo evaluation of UFG-Ti implants. Data are presented as mean ± S.D. (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, Ncm). 4.

Discussion

UFG-titanium etching in Piranha solutions produced the surfaces with micro-, nano- 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 demonstrate that all the samples are noncytotoxic and lead to the production of the markers of early and late differentiation in the osteogenic direction. 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


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adhesion and can promote bone-to-implant contact via mechanical interlocking mechanisms [12,39,40]. Nanoscale topography affects chemical reactivity, wettability, adsorption rate of biomolecules and leads to improvement of biomedical interactions of implant surface with host tissue [12,40]. However, influence of values of topography parameters on the biomedical properties is rather complicated and often has an extremal dependence [41]. 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 micron-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 50x50 µm) almost 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 the structures are expected to be the most promising and they attract special interest of researchers. The most of researchers suggest the 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 the surface compositions and the



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 reason for 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 on 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 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


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of published data, we selected the papers where the authors studied hierarchical micronanostructures. 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 spark plasma sintering (see the summary in the Table 2). Resulted structures differ in morphology, 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 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], Zhao [9,10] but worse than the results of Kubo [8] and Zhuang [11].

Table 2. Results of in vitro studies of the hybrid nano-/micro-structures on the titanium surface. Characteristic

Viability (MTT-test)

Proliferation

Differentiation

Type of cells

Ref

Nano/microstructures+TiO

1.41 (2 days)*

1.29 (3 days)*

ALP-1,83 (2 days)* ALP-1.4 (7 days)* ALP-1.45 (2 days)+ OP-2.84 (14 days)*

МC3T3-E1

This work

2

(BPS-2h+ALD)



Nanostructured CG-Ti

0.7+ (BPSetching)

1.2+ (4 days) (APS-etching)

Hierarchical micro/nano structures Hierarchical micro/nano structures+Sr

~1.8+ (4 days)

Hierarchical micro/nano structures micro/nano structures UFG-Ti hierarchical porous surface Micropits and TiO2 nanonodules hybrid micropitted/ nanotubular

(MTT-test) No difference+

OC+-very good (APS-etching)

Page 28 of 42

МC3T3-E1

50

МC3T3-E1

4

ALP~1.1+ (4 days)

BMSCs

5

~1.2-1.3+ (3 days)

ALP~1.4+ (7 days)

BMSCs

6

~1.1-1.2

ALP~1.1+ (7 days)

МC3T3-E1

7

MG63

54

ALP~2-3 (7 days)# OC~3-4 (7 days)#

Rat bone marrow cells

8

ALP (7days) 0.75-1.25 (depends on nanotube size) OC remarkably worse+

rat calvarial osteoblasts

9

Mesenchymal stem cell (MSC) MC3T3-E1

10

~1,2+ (4 days)

≥0.8*(3 days) 1-1.1+

1.15-1.6 (2 days)#

hybrid micropitted/ nanotubular

1.2 (4day)+

nanoneedle and nanoporous/microp its *compared to control sample + compared to untreated Ti #compared to micron-scale Ti OC - osteocalcin BMSCs – bone marrow stromal cells

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

ALP 3-4 (7 day)+ 1.5 (14 day)+

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 wellattachment 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


11

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surface. Osseointegration strength (that was measured using removal torque (RTQ) method) could be comparable to that of published previously by Sul [58] and more recently by Zemtsova et al. [59]. Implants with fluorinated TiO2 nanotubes demonstrated significantly increased removal torque strengths (41 vs 29 Ncm; 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 Ncm [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 the force necessary for the removal comparing 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.

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 nontoxic and have the most favorable effect on the proliferation and differentiation of cells in osteogenic direction, osseointegration and reliability of the implant osseointegration. We studied the relation between morphology, topography, composition, 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 МC3T3-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 ALDtreatment only. This fact indicates a synergetic effect of CE+ALD combination. Supporting Information Figure S1, XRD pattern of the UFG-Ti. Figure S2. SEM images of the UFG-Ti screws prepared for in vivo study. Author information * E-mail: [email protected] Phone: +7-812-428-4033 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


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For Table of Contents Use Only Enhanced osseointegrative properties of the ultrafine-grained titanium implants modified by the 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



Graphic abstract 83x35mm (300 x 300 DPI)


Page 42 of 42

Enhanced osseointegrative properties of the ultrafine-grained titanium

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