New Zr-Ti-Nb Alloy for Medical Application: Development, Chemical and Mechanical Properties, and Biocompatibility

materials

Article New Zr-Ti-Nb Alloy for Medical Application: Development, Chemical and Mechanical Properties, and Biocompatibility

Oleg Mishchenko 1,2, Oleksandr Ovchynnykov 3, Oleksii Kapustian 3 and Maksym Pogorielov 1,4,* 1 NanoPrime, 25 Metalowcow Str., Dedice 39-200, Poland; [email protected] 2 Department of Surgical and Propaedeutic Dentistry, Zaporizhzhia State Medical University, 26, Prosp.Mayakovskogo, Zaporizhzhia 69035, Ukraine 3 Department of Physics and Engineering, Zaporizhzhia Polytechnic National University, 64 Zhukovsky Str, Zaporizhzhia 69063, Ukraine; [email protected] (O.O.); [email protected] (O.K.) 4 Centre of Collective Use of Scientiﬁc Equipment, Sumy State University, 2 R-Korsakova Str, Sumy 40007, Ukraine * Correspondence: [email protected] or [email protected]; Tel.: +38-066-900-5448

Received: 11 February 2020; Accepted: 27 February 2020; Published: 13 March 2020

Abstract: The concept of mechanical biocompatibilities is considered an important factor for orthopedics and dental implants. The high Young modulus of traditional Ti-based alloys can lead to stress-shielding syndrome and late postoperative complications. The development of new Al- and V-free Ti alloys with a low elastic modulus is a critical task for implantology. Despite the relatively low Young modulus and appropriate biological response of metastable beta-Ti alloys, their production requires complex metallurgical solutions and a high final cost that limit commercial application. The current research aimed to develop a Zr-Ti-Nb system with a low Young modulus suitable for biomedical application, including orthopedics and dental implantology. Two different charges were used for new alloy production with melting in a vacuum-arc furnace VDP-1 under atmospheric control (argon + helium) with a non-consumable tungsten electrode and a water-cooled copper crystallizer. Post-treatment included a forging-rolling process to produce a bar suitable for implant production. SEM with EDX and the mechanical parameters of the new alloy were evaluated, and a cell culture experiment provided a biocompatibility assessment. The chemical composition of the new alloy can be represented as 59.57-19.02-21.41 mass% of Zr-Ti-Nb. The mechanical properties are characterized by an extremely low Young modulus—27,27 GPa for the alloy and 34.85 GPa for the bar. The different master alloys used for Zr-Ti-Nb production did not affect the chemical compound and mechanical parameters so it was possible to use affordable raw materials to decrease the final price of the new product. The cell culture experiment demonstrated a full biocompatibility, indicating that this new alloy can be used for dental and orthopedics implant production.

Keywords: Zr-Ti-Nb alloy; Young modulus; mechanical properties; biocompatibility

1. Introduction A high strength, low density, excellent corrosion resistance, high biocompatibility, and the ability of osseointegration are critical properties for metal alloys used for the production of dental and orthopedics implants [1]. Ti alloys have been used for this purpose for many years, with high clinical success [2], and due to their higher life expectancy, implants are now expected to serve for much longer periods. Additionally, the concept of mechanical biocompatibilities is considered an important factor [3,4]. Commercially pure titanium and the Ti–6Al–4V alloy are the most used metals for implant

Materials 2020, 13, 1306; doi:10.3390/ma13061306 www.mdpi.com/journal/materials Materials 2020, 13, 1306 2 of 11 production, with a higher elastic stiffness and a relatively high elastic modulus (110 GPa) in comparison with cortical bone (from 15 to 30 GPa) [5]. However, toxic metal release (Al and V) from Ti–6Al–4V implants has been reported after long-term implantation [6]. A low modulus in dental implants can induce better stress transfer along the bone–implant interface and limit crestal bone loss due to excessive load transmitted to the bone through the crest module [7]. Some research has shown that decreasing the elastic modulus can minimize the bone atrophy due to the stress shielding effect, and increase the durability of orthopedic implants [8,9]. Based on these suggestions, the development of new Al- and V-free Ti alloys with a low elastic modulus is a critical task for implantology [10]. Metastable beta Ti-based alloys with nontoxic metals such as Nb and Zr displayed a reversible stress-induced martensitic transformation that led to a superelastic effect (high recoverable strain) and a very significant reduction of the apparent elastic modulus [11,12]. Some research has shown successful solutions for low-modulus alloy development. Liang S.X. and co-authors developed a β Ti alloy (Ti–31Nb–6Zr–5Mo, wt.%) using hot rolling followed by aging treatments, with a final Young modulus of around 48 GPa [13]. The Ti–24Nb–4Zr–8Sn alloy with a Young modulus of 49 GPa developed by Nune K.S. showed an additional stimulation of osteoblast proliferation [14]. The Ti-24Nb-4Zr-7.9Sn alloy developed by the Institute of Metal Research Chinese Academy of Sciences (elastic modulus—42 GPa) showed a good biocompatibility in both cell culture and animal models [15]. Despite their relatively low Young modulus and appropriate biological response, all mentioned alloys require complex metallurgical solutions and have a high final cost that limit their clinical application. The current research aimed to develop a Zr-Ti-Nb system with a low Young modulus suitable for biomedical material application, including orthopedics and dental practice. To achieve all requirements for medical implants, we expected the development of a Zr-Ti-Nb system with 59.57 mass% (Zr), 19.02 mass% (Ti), and 21.41 mass% (Nb). The predicted mechanical properties expected were a tensile strength of 800–1300 MPa and elastic modulus of 40–70 GPa. Dental implant production requires the development of cylindrical bars with the following characteristics: 6.0 mm in diameter and 1.5–3.0 m in length, with Ra 1.6–3.2.

2. Materials and Methods

2.1. Materials For the development of a Zr-Ti-Nb alloy with the predicted properties, we used two different charges with the following composition: Charge 1—zirconium iodide (Zr-I), Niobium (NbSh), and Titanium Sponge (TiG-120); Charge 2—zirconium CTZ-110, Niobium (NbSh) and Titanium Sponge (TiG-90). Niobium (NbSh) was purchased from PJSC (Titanium Institute, Zaporizhzhia, Ukraine) and manufactured by metallothermal technology. Titanium Sponge (TiG-120) was obtained from Zaporizhzhia Titanium & Magnesium Combine Ltd. (Zaporizhzhia, Ukraine); Zirconium iodide and CTZ were purchased from SSE (Zirconium, Zaporizhzhia, Ukraine). Zirconium iodide has an extremely high price and zirconium CTZ-110 was used in charge 2 to decrease the final price of the Zr-Ti-Nb alloy. Taking into account that zirconium CTZ-110 has a higher oxygen concentration, Titanium Sponge TG-90 was used in charge 2 to equalize the oxygen amount in the final alloy. The element composition of feedstock is listed in Tables1–3.

Table 1. The composition of the zirconium master alloys (main alloying elements).

Mass Fraction of Impurities,% Type of Zr N O C Fe S Ni Cl Al Ca Mn Ti Cr Zr-I 0.005 0.05 0.008 0.03 0.008 0.02 - 0.005 0.02 0.001 0.005 0.02 CTZ-110 0.006 0.14 0.02 0.03 0.01 0.01 0.003 0.005 0.01 0.001 0.007 0.005 Materials 2020, 13, 1306 3 of 11

Table 2. The composition of the titanium master alloys.

Mass Fraction of Impurities,% Type of Ti N O C Fe Si Ni Cl TG-90 0.02 0.04 0.02 0.05 0.01 0.04 0.08 TG-120 0.02 0.06 0.03 0.11 0.02 0.04 0.08

Table 3. The composition of the niobium master alloy.

Mass Fraction of Impurities,% N O C Fe Si Ta Ti Niobium (NbSh) 0.02 0.06 0.03 0.11 0.02 0.04 0.07

2.2. Melting Process Melting was carried out in a vacuum-arc furnace VDP-1 with an atmospheric controller (argon + helium), with a non-consumable tungsten electrode and a water-cooled copper crystallizer. The VDP-1 furnace was invented and produced by PJSC (Titanium Institute, Zaporizhzhia, Ukraine).

2.3. SEM and EDX The structure and composition, as well as the distribution quality, of the main alloying elements, were evaluated using a JSM-IT300LV scanning microscope (Jeol, Tokyo, Japan) equipped with X-Max 80 X-ray energy dispersive microanalysis (Oxford Intruments, Abingdon, UK). The investigation was performed at an accelerating voltage of 15 kV and an electron probe diameter of 4 nm, while the diameter of the x-ray excitation zone was about 2 µm. The chemical composition was determined by the non-standard method of calculating fundamental parameters: by calculating the correction reflection coefficients of the electrons of the probe, the absorption of characteristic x-ray radiation, and fluorescence. Three test specimens were used for each measurement method.

2.4. X-ray Phase Analysis The X-ray powder diffraction technique was used to characterize the phase composition and structural parameters. X-ray diffraction patterns were recorded using a standard powder diffractometer DRON-3 (Burevestnik, Moscow, Russia) with a Bragg-Brentano geometry and filtered Cu Kα radiation. Data were collected in the 2θ range from 20◦ to 120◦, with a step interval of 0.05◦ and a count time of 5 sec per step.

2.5. Metallographic Investigations Specimens were polished using a grinding wheel (grits 240–400–600 µm), with final polishing being conducted by a felt circle with a 3 µm diamond emulsion. Metallographic studies of the macro- and microstructure of the non-etched and etched state and analysis of fractographs of cast and deformed alloys were performed on the optical microscope "NEOPHOT-32" (Carl Zeiss Jena, Jena, Germany), as well as on the scanning electron microscope JSM-IT300LV (Jeol, Tokyo, Japan) at a magnification of X500—5000. For the analysis of the macrostructure, the grinders were etched in solution: 20% hydrofluoric and 20% nitric acids, with a glycerin base. Grinds for microstructure analysis were etched in a solution of hydrofluoric (20%) and nitric (20%) acids, with a glycerol base. Three test specimens were used for each measurement method.

2.6. Mechanical Parameters The mechanical parameters of the developed alloy and ﬁnal bar were investigated using the INSTRON 8801 machine (Instron, Norwood, MA, USA). To determine the mechanical properties, Materials 2020, 13, 1306 4 of 11

the following parameters were evaluated: tensile strength (σB, MPa), yield strength (σO2, MPa), elastic modulus (E, GPa), elongation (δ, %), and transverse narrowing (ψ, %). All of the experiments were Materialsconducted 2020, 13 in, x triplicate. FOR PEER REVIEW 4 of 12

124 2.7.2.7. Biocompatibility Biocompatibility StudyStudy 125 TheThe cell cell culture culture technique technique was was used used to to assess assess the the biocompatibility biocompatibility of of the the final final Zr-Ti-Nb Zr-Ti-Nb bar bar using using 126 a astandard standard biocompatibility biocompatibility test test [16]. [16 ].Cylindrical Cylindrical specimens specimens that that were were 6 6mm mm in in diameter diameter and and 5 5mm mm 127 highhigh were were prepared prepared for for the the cell cell culture culture experiment. experiment. Samples Samples were were sterilized sterilized by by 70% 70% ethanol ethanol for for 3 3h hat at 128 roomroom temperature, temperature, washed washed in PBSPBS twice,twice, and and then then placed placed in 24-wellin 24-well plates. plates. Dulbecco’s Dulbecco’s Modified Modified Eagle 129 EagleMedium Medium/Nutrient/Nutrient Mixture Mixture F-12 (DMEMF-12 (DMEM/F-12)/F-12) with L-glutamine, with L-glutamine, containing containing 100 units 100/mL units/mL penicillin, 130 penicillin,100 µg/mL 100 streptomycin, μg/mL streptomycin, 2.5 µg/mL 2.5 amphotericin μg/mL amphotericin B, 10% Fetal B, Bovine10% Fetal Serum, Bovine and Serum, 1.0 ng/ mLand bFGF, 1.0 131 ng/mLwas added bFGF, to was each added well. to It each was thenwell. incubatedIt was then at incubated 37 ◦C in a humidifiedat 37 °C in a environment humidified environment with 5 % CO 2. 132 withAfter 5 % 24 CO2. h, the After human 24 osteoblasth, the human cells osteoblast (U2OS cells cells obtained (U2OS fromcells obtained Umeo University, from Umeo Umeo, University, Sweden) 4 133 Umeo,were seededSweden) at were 10 cells seeded per at sample 104 cells in 2per mL sample of DMEM in 2 /mLF-12. of TestDMEM/F-12. specimens Test with specimens cells were with incubated cells 134 wereat 37 incubated◦C with 5%at 37 CO °C2, with and media5% CO2, was and changed media everywas changed two days every during two a days seven-day during culture a seven-day period. 135 cultureAll of theperiod. experiments All of the were experiments conducted were in triplicate.conducted in triplicate. 136 TheThe Alamar Alamar Blue Blue (AB) (AB) assay assay was used toto assessassess the the cell cell viability viability on on day da one,y one, three, three, and and seven seven after 137 afterseeding. seeding. The The media media was was removed removed from from each each well well and and washed washed with with PBS. PBS. One One milliliter milliliter of Alamar of Alamar Blue™ 138 Blue™solution solution was added was added to each to well each and well then and incubated then incubated for two for hours. two hours. Two aliquots Two aliquots of 200 ofµL 200 of AlamarμL of 139 AlamarBlue™ Blue™solution solution were collected were collected from each from sca eachffold andscaffold the absorbanceand the absorbance was read was on the read absorbance on the 140 absorbancereader using reader 570 andusing 600 570 nm and wave 600 lengths.nm wave lengths.

2.8. Statistics 141 2.8. Statistics 142 One-wayOne-way ANOVA ANOVA with with multiple multiple comparisons comparisons was was us useded to to assess assess the the difference difference between between groups groups 143 usingusing GraphPad GraphPad Prism Prism 8.0 8.0 software software (V (V 8.0, 8.0, San San Diego, Diego, CA, CA, USA). USA). Statistical Statistical significance significance was was assumed assumed p 144 atat a aconfidence confidence level level of of 95% 95% (p ( < <0.05).0.05). 3. Results and Discussion 145 3. Results and Discussion 3.1. Melting and Bar Preparation 146 3.1. Melting and Bar Preparation Master alloys were tabbed to the furnace simultaneously with tight packing. The mass of the 147 chargeMaster was alloys calculated were fortabbed melting to the the furnace alloy atsimult the amountaneously of with 1.6 kg,tight which packing. is necessary The mass to of obtain the 148 chargecylindrical was calculated billets with for a melting diameter the of alloy 6 mm at andthe amount a length of of 1.6 up kg, to 3000which mm is necessary (taking into to accountobtain 149 cylindricalprocessing billets losses). with For a uniformdiameter distribution of 6 mm and of alloying a length elements of up to and 3000 impurities mm (taking throughout into account the ingot 150 processingvolume, triple losses). remelting For a uniform of each ingotdistribution was performed. of alloying The elements melting and process impurities was performed throughout with the the 151 ingot volume, triple remelting of each ingot was performed. The melting process4 was performed with following parameters: I = 400–600 A; U = 62–64 V; pre-vacuum = 1 10− mbar; and melting medium × −4 152 the= followingargon (99.999 parameters: Ar) at pressure I = 400– P600= А0.5; U kgf= 62/–cm642 V;. After pre-vacuum triple re-melting, = 1×10 mbar; we obtained and melting the ingotmedium used − 2 153 = forargon bar (99.999 preparation Ar) at and pressure the investigation P = −0.5 kgf/cm of the. chemicalAfter triple and re-melting, mechanical we parameters obtained the (Figure ingot1 ).used 154 for bar preparation and the investigation of the chemical and mechanical parameters (Figure 1).

155 156 FigureFigure 1. 1.Zr-Ti-NbZr-Ti-Nb ingot ingot after after arc arc melting melting—upper—upper (A (),A ),lower lower (B (),B ),and and lateral lateral (C ()C view.) view.

157 For the production of the final bar for dental implant manufacturing, obtained ingots were 158 turned onto their side surface, bottom, and top with the base diameter of 72 mm, the top diameter of 159 64 mm, height of 37 mm, and weight of 790 g (Figure 2A). Subsequent forging of ingots (Figure 2B) 160 was carried out on a hammer through a square profile with heating in an oven to 800 °C. Rolling to 161 a diameter of 18 mm (Figure 2 C) was carried out on a three-roll 10-30 cross-helical rolling mill in 6

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For the production of the ﬁnal bar for dental implant manufacturing, obtained ingots were turned onto their side surface, bottom, and top with the base diameter of 72 mm, the top diameter of 64 mm, height of 37 mm, and weight of 790 g (Figure2A). Subsequent forging of ingots (Figure2B) was carried out on a hammer through a square proﬁle with heating in an oven to 800 ◦C. Rolling to a diameter of Materials18 mm 2020 (Figure, 13, x FOR2 C) PEER was REVIEW carried out on a three-roll 10-30 cross-helical rolling mill in 6 passes5 heatedof 12 to 800 ◦C. The rolling in the longitudinal rolling mill was heated to 400–600 ◦C, according to the 162 passesscheme: heated diameter to 800 18 °C. mm The squarerolling 16in the16 longitudinal mm oval 12.7rolling mm mill squarewas heated 12 12 to mm 400–600oval °C, 8 163 according to the scheme: diameter→ 18 mm× → square→ 16 × 16 mm → →oval 12.7 mm× → square→ 12 × 12 × 6 mm diameter 7 mm. After longitudinal rolling, heat treatment was carried out at 600 ◦C to align → → → 164 mmthe bar oval (Figure 8 × 6 mm2D). Heat diameter treatment 7 mm. was After carried longitudinal out in order rolling, to "soften" heat treatment the sample was to carried align theout rod,at 165 600 °C to align the bar (Figure 2D). Heat treatment was carried out in order to "soften" the sample to and the temperature 600 ◦C was chosen to relieve stress. In this way, both processes—alignment and 166 alignannealing—were the rod, and the provided. temperature 600 °C was chosen to relieve stress. In this way, both processes— 167 alignment and annealing—were provided.

168 169 FigureFigure 2. Process 2. Process of Zr-Ti-Nb of Zr-Ti-Nb bar bar manufacturing: manufacturing: ingot ingot after after mechanical mechanical turning turning (A), (A after), after the the forging forging 170 (B),(B cross-helical), cross-helical rolling rolling (C), (C and), and final ﬁnal longitudinal longitudinal rolling rolling (C). (D ).

171 AsAs a final a final result result of ofthe the post-cast post-cast treatment, treatment, we we ob obtainedtained a longitudinal a longitudinal bar bar with with a length a length of of1.5 1.5 m m 172 andand diameter diameter of of6.2 6.2 mm. mm. The The curvature curvature of ofthe the bar bar did did not not exceed exceed 0.5% 0.5% of ofits itslength. length. 3.2. Chemical and Mechanical Parameters 173 3.2. Chemical and Mechanical Parameters The chemical composition and mechanical properties of the new alloy (made from different master 174 The chemical composition and mechanical properties of the new alloy (made from different alloys—charge 1 and charge 2) and obtained bar are shown in Table4. It should be noted that using 175 master alloys—charge 1 and charge 2) and obtained bar are shown in Table 4. It should be noted that different master alloys resulted in the same chemical composition and mechanical properties in the final 176 using different master alloys resulted in the same chemical composition and mechanical properties Zr-Ti-Nb alloy, so CTZ-110 can be the alloy of choice for Zr-Ti-Nb alloy production, which can decrease 177 in the final Zr-Ti-Nb alloy, so CTZ-110 can be the alloy of choice for Zr-Ti-Nb alloy production, which the final price. The chemical composition fully meets the planned expectations and determines the 178 can decrease the final price. The chemical composition fully meets the planned expectations and mechanical properties. The tensile strength of the new alloy was significantly lower than a Grade 5 179 determines the mechanical properties. The tensile strength of the new alloy was significantly lower Ti alloy, but after post-casting treatment, it rose to 900.1 MPa, which is the same as that for standard 180 than a Grade 5 Ti alloy, but after post-casting treatment, it rose to 900.1 MPa, which is the same as materials used for dental implant production. The yield strength of the new materials was significantly 181 that for standard materials used for dental implant production. The yield strength of the new lower than that of Ti, but it should predict reversible deformation in the final dental implant. 182 materials was significantly lower than that of Ti, but it should predict reversible deformation in the 183 final dental implant.

184 Table 4. Chemical composition (EDX) and mechanical properties of the Zr-Ti-Nb alloy and resulting 185 bar.

Chemical composition, mass% Mechanical properties Samples Ti Zr Nb σB, MPa σО2, МPa E, GPa δ, % ψ,% Charge 1 19.0 59.5 21.4 687.5 648.9 28.3 13,2 40,8 Charge 2 19.0 59.5 21.4 568.1 552.7 27.2 12,0 53,1 Bar 19.0 59.5 21.4 850.0-900.1 695,0 37.6 10.5 36,0 186 The elastic modulus of the new alloy varied between 27.27 and 28.32 GPa, which is the lowest 187 known Young modulus used for dental and orthopedic implant production [17]. Alloy post-

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Table 4. Chemical composition (EDX) and mechanical properties of the Zr-Ti-Nb alloy and resulting bar.

Chemical Composition, Mass% Mechanical Properties Samples Ti Zr Nb σB, MPa σO2, MPa E, GPa δ,% ψ,% Charge 1 19.0 59.5 21.4 687.5 648.9 28.3 13,2 40,8 Charge 2 19.0 59.5 21.4 568.1 552.7 27.2 12,0 53,1 Bar 19.0 59.5 21.4 850.0-900.1 695,0 37.6 10.5 36,0

The elastic modulus of the new alloy varied between 27.27 and 28.32 GPa, which is the lowest knownMaterials Young 2020, 13 modulus, x FOR PEER used REVIEW for dental and orthopedic implant production [17]. Alloy post-treatment6 of 12 led to a slightly increasing elastic modulus up to 37.67 GPa, but is still near the mechanical parameter 188 treatment led to a slightly increasing elastic modulus up to 37.67 GPa, but is still near the mechanical of natural cortical bone. Post-casting treatment led to decreasing metal elongation, but was not 189 parameter of natural cortical bone. Post-casting treatment led to decreasing metal elongation, but was significantly different from that of standard Ti alloys [18]. Observing the data of X-ray diffraction 190 not significantly different from that of standard Ti alloys [18]. Observing the data of X-ray diffraction shown in Figure3 and using the Wegard rule, it was found that about 65%-66% of the alloy was a 191 shown in Figure 3 and using the Wegard rule, it was found that about 65%-66% of the alloy was a volume-centered zirconium phase. Therefore, the test alloy can be attributed to β-zircon. There are a 192 volume-centered zirconium phase. Therefore, the test alloy can be attributed to β-zircon. There are a lot of data about positive bone responses and the long-term integration of β-Ti alloys with no toxic 193 lot of data about positive bone responses and the long-term integration of β-Ti alloys with no toxic metals (Al or V), which would allow these implants to be used in clinical cases [19]. Some research has 194 metals (Al or V), which would allow these implants to be used in clinical cases [19]. Some research demonstrated better late postoperative responses to β-Ti implants due to stress shielding reduction [20]. 195 has demonstrated better late postoperative responses to β-Ti implants due to stress shielding Finite element analysis of a one-piece zirconia implant was shown to be less prone to peri-implant bone 196 reduction [20]. Finite element analysis of a one-piece zirconia implant was shown to be less prone to overloading and subsequent bone loss in high-stress areas, especially in the labial-cervical region of 197 peri-implant bone overloading and subsequent bone loss in high-stress areas, especially in the labial- the cortical bone [21]. However, additional experimental and clinical evidence and more investigations 198 cervical region of the cortical bone [21]. However, additional experimental and clinical evidence and of β-zircon alloys for dental implant manufacturing are still required. 199 more investigations of β-zircon alloys for dental implant manufacturing are still required.

200 Figure 3. X-Ray diﬀraction of the Zr-Ti-Nb alloy. 201 Figure 3. X-Ray diffraction of the Zr-Ti-Nb alloy. It should be noted that the existing mechanical properties of the Zr-Ti-Nb alloy in the cast state 202 open perspectivesIt should be noted for implant that the manufacturing existing mechanical using simplerproperties methods, of the Zr-Ti-Nb without thealloy use in of the additional cast state 203 complexopen perspectives and expensive for implant deformation manufacturing processing. using The simpler existing methods, experience without of powder the use produced of additional from 204 ingotscomplex for and additive expensive technologies deformation [22] suggestsprocessing. the The technical existing feasibility experience of producingof powder powdersproduced from from β 205 newingots experimental for additive -zirconiumtechnologies alloys. [22] suggests Taking intothe technical account thefeasibility active developmentof producing ofpowders bioinspired from 206 dentalnew experimental and orthopedics β-zirconium implants alloys. with mechanical Taking into properties account closethe active to those development of human bones,of bioinspired we can β 207 recommenddental and neworthopedics the Zr-Ti-Nb implants alloy with as a materialmechanical for pr additiveoperties technologies. close to those-alloys of human can providebones, we better can 208 bonerecommend responses new compared the Zr-Ti-Nb to conventional alloy as a Ti-alloysmaterial for after additive 3D scaﬀ technologies.old implantation β-alloys [23]. can The provide initial 209 lowbetter Young bone modulus responses of thecompared Zr-Ti-Nb to alloy,conventional in combination Ti-alloys with after additive 3D scaffold manufacturing implantation techniques, [23]. The 210 caninitial provide low anYoung ideal modulus solution forof largethe Zr-Ti-Nb implant manufacturingalloy, in combination for orthopedics. with additive manufacturing 211 techniques,SEM with can EDX provide showed an ideal a uniform solution distribution for large implant of Zr, Ti, manufacturing and Nb within for the orthopedics. new alloy that made 212 tripleSEM re-melting with EDX and furthershowed post-cast a uniform treatment distribution possible of Zr (Figure, Ti, and4). Nb within the new alloy that made 213 triple re-melting and further post-cast treatment possible (Figure 4).

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214 Figure 4. SEM image of the Zr-Ti-Nb ingot (A) and spectral distribution of Ti (B), Zr (C), and Nb (D). 215 Figure 4. SEM image of the Zr-Ti-Nb ingot (A) and spectral distribution of Ti (B), Zr (C), and Nb 216 (DThe). results of metallographic analysis and fractograms are shown in Figures5–7. The microstructure of the Zr-Nb-Ti cast alloy is represented by β-grains that are 150–200 µm in 217 sizeThe (Figure results5A). of Forging metallographic and rolling analysis deformation and fractograms led to a change are shown in the in size figures of the 5, grains, 6, and up 7. toThe the 218 microstructurecomplete absence of the of theirZr-Nb-Ti boundaries cast alloy (Figure is represented5B). An analysis by β of-grains the samples’ that are microstructure 150–200 μm (within size the 219 (Figureinitial cast5A). andForging in a deformedand rolling state) deformation showed that, led afterto a deformation,change in the the size original of the grains grains, were up bentto the and 220 completestretched absence in the metalof their flow boundaries direction. (Figure The grains 5B). An were analysis parallel of the to this samples’ direction, microstructure and the structural (with 221 thechanges initial cast occurred and in in a thedeformed entire volume state) showed of the workpiece that, after (Figuredeformation,5B). the original grains were bent 222 and stretchedThe fracture in the surfacesmetal flow of Zr-Ti-Nbdirection. ingot The grains specimens were afterparallel the to tensile this direction, test had aand cup-shaped the structural shape 223 changesthat is typicaloccurred for in ductile the entire fracture, volume with of a the fibrous workpiece middle (Figure (bottom) 5B). part and a smoother conical surface (bevels) (Figure6). The bottom part had a rounded shape and occupied an area of 1.5 2.0 mm. × The width of the conical surface formed by the mechanism of the viscous destruction characterized the ability of the material for plastic deformation to be around 0.5-1.0 mm. Kim K.M. have shown that increasing the Zr in a Ti-Zr-Nb alloy leads to the formation of a β-phase and Ti-14Nb-18Zr combination with a low modulus and sufficient plastic deformation [24]. Low-modulus alloys have shown better osseointegration in the early postoperative period and a sufficient bone quality in late regeneration terms during in-vivo experiments [25]. The fractographic analysis of fractured surfaces revealed that the fracture of Zr-Ti-Nb alloy specimens was ductile, both in the initial state (cast) and after deformation (Figure7). Our analysis did not show areas of brittle fractures, pores, and other defects, which indicates the high quality of deformation processing. The destruction of the Zr-Ti-Nb alloy occurred by the mechanism of micropore fusion. Previous research has shown that such a mechanism can provide superior mechanical properties during implant exploitation and prevent late postoperative complications, such as the stress shielding effect [26].

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224 Materials 2020, 13, x FOR PEER REVIEW 9 of 12 Figure 5. Microscopic images of the ingot (A) and bar (B) after longitudinal rolling. 225 Figure 5. Microscopic images of the ingot (A) and bar (B) after longitudinal rolling.

226 The fracture surfaces of Zr-Ti-Nb ingot specimens after the tensile test had a cup-shaped shape 227 that is typical for ductile fracture, with a fibrous middle (bottom) part and a smoother conical surface 228 (bevels) (Figure 6). The bottom part had a rounded shape and occupied an area of 1.5 × 2.0 mm. The 229 width of the conical surface formed by the mechanism of the viscous destruction characterized the 230 ability of the material for plastic deformation to be around 0.5-1.0 mm. Kim K.M. have shown that 231 increasing the Zr in a Ti-Zr-Nb alloy leads to the formation of a β-phase and Ti-14Nb-18Zr 232 combination with a low modulus and sufficient plastic deformation [24]. Low-modulus alloys have 233 shown better osseointegration in the early postoperative period and a sufficient bone quality in late 234 regeneration terms during in-vivo experiments [25]. 235 The fractographic analysis of fractured surfaces revealed that the fracture of Zr-Ti-Nb alloy 236 specimens was ductile, both in the initial state (cast) and after deformation (Figure 7). Our analysis 237 did not show areas of brittle fractures, pores, and other defects, which indicates the high quality of 242 Figure 6. Fractographs of the fracture surface of the Zr-Ti-Nb alloy cut from the ingot. 238 deformation processing. The destruction of the Zr-Ti-Nb alloy occurred by the mechanism of 239243 micropore fusion.Figure Previous6. Fractographs research of the has fracture shown surface that of suchthe Zr -Ti-Nba mechanism alloy cut fromcan theprovide ingot. superior 240 mechanical properties during implant exploitation and prevent late postoperative complications, 241 such as the stress shielding effect [26].

244 245 Figure 7. Fractographs of the fracture surface of the Zr-Ti-Nb alloy cut from the bar after final 246 longitudinal rolling.

247 3.3. Biocompatibility Test 248 Commercially pure Ti (Cp-Ti) was used as known biocompatible material (reference). The cell 249 culture experiment demonstrated adequate osteoblast adhesion on day one on both Cp-Ti and Zr-Ti- 250 Nb alloys, with later proliferation on day 3 and 7. These data suggest biocompatibility and the 251 absence of cell toxicity. SEM on day 7 demonstrated that cells adhered to the polished surface by long 252 and short processes and cell-cell communication (Figure 8). The addition of Zr and Nb in the Ti alloy 253 showing a high biocompatibility as a cast and after sandblasting has demonstrated a good 254 biocompatibility in a previous study [27,28]. As mentioned above, toxic Al and V metal can prevent 255 late material toxicity after implantation.

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242 243 Materials 2020, 13Figure, 1306 6. Fractographs of the fracture surface of the Zr-Ti-Nb alloy cut from the ingot. 9 of 11

244 Figure 7. Fractographs of the fracture surface of the Zr-Ti-Nb alloy cut from the bar after ﬁnal 245 longitudinalFigure 7. Fractographs rolling. of the fracture surface of the Zr-Ti-Nb alloy cut from the bar after final 246 longitudinal rolling. 3.3. Biocompatibility Test 247 3.3. CommerciallyBiocompatibility pure Test Ti (Cp-Ti) was used as known biocompatible material (reference). The cell 248 cultureCommercially experiment demonstrated pure Ti (Cp-Ti) adequate was used osteoblast as known adhesion biocompatible on day one material on both Cp-Ti(reference). and Zr-Ti-Nb The cell 249 alloys,culture with experiment later proliferation demonstrated on day adequate 3 and 7. osteobla These datast adhesion suggest on biocompatibility day one on both and Cp-Ti the absence and Zr-Ti- of 250 cellNb toxicity. alloys, SEMwith onlater day proliferation 7 demonstrated on day that 3 cells and adhered 7. These to data the polished suggest surfacebiocompatibility by long and and short the 251 processesabsence of and cell cell-cell toxicity. communication SEM on day 7 (Figuredemonstrated8). The that addition cells ofadhered Zr and to Nb the in polished the Ti alloy surface showing by long a 252 highand biocompatibilityshort processes and as a cell-cell cast and communication after sandblasting (Figure has demonstrated8). The addition a goodof Zr biocompatibilityand Nb in the Ti alloy in a 253 previousshowing study a high [27 ,28biocompatibility]. As mentioned as above, a cast toxic and Al after and Vsandblasting metal can prevent has demonstrated late material toxicitya good Materials 2020, 13, x FOR PEER REVIEW 10 of 12 254 afterbiocompatibility implantation. in a previous study [27,28]. As mentioned above, toxic Al and V metal can prevent 255 late material toxicity after implantation.

256

257 FigureFigure 8. 8. ResazurinResazurin reduction reduction assay assay after after osteoblast osteoblast seeding seeding on on the the Cp-Ti Cp-Ti and and Zr-Ti-Nb Zr-Ti-Nb alloy alloy (A (A) )and and 258 anan SEM SEM image image of of osteoblasts osteoblasts on on the the Zr-Ti-Nb Zr-Ti-Nb alloy alloy on on day day 7 7 ( (BB).). 4. Conclusions 259 4. Conclusions The new Zr-Ti-Nb alloy developed using the vacuum-arc melting process with further 260 The new Zr-Ti-Nb alloy developed using the vacuum-arc melting process with further forging- forging-rolling treatment demonstrated an extremely low Young modulus with adequate tensile 261 rolling treatment demonstrated an extremely low Young modulus with adequate tensile and yield and yield strength and elongation parameters. The size reduction of structural components as a result 262 strength and elongation parameters. The size reduction of structural components as a result of the of the deformation processing of the cast material provided an increase of mechanical properties up to 263 deformation processing of the cast material provided an increase of mechanical properties up to 26%- 26%-60%. The mechanical properties of the new alloy in the deformed state correspond to grade-5 Ti, 264 60%. The mechanical properties of the new alloy in the deformed state correspond to grade-5 Ti, but but the elastic modulus is much closer to that of natural bone. The different master alloys used for 265 the elastic modulus is much closer to that of natural bone. The different master alloys used for Zr-Ti- Zr-Ti-Nb production did not affect the chemical compound and mechanical parameters, so it would be 266 Nb production did not affect the chemical compound and mechanical parameters, so it would be possible use affordable raw materials that decrease the final price of the new product. The cell culture 267 possible use affordable raw materials that decrease the final price of the new product. The cell culture 268 experiment demonstrated full biocompatibility, indicating that the new alloy could be used for dental 269 and orthopedics implant production.

270 Author Contributions: Conceptualization, O.M. and M.P.; methodology, O.O., O.K., and M.P.; formal analysis, 271 O.M. and M.P.; investigation, O.K., O.O., and M.P.; data curation, M.P.; writing—original draft preparation, M.P. 272 and O.O.; writing—review and editing, M.P. and O.M.; supervision, M.P. All authors have read and agreed to 273 the published version of the manuscript.

274 Funding: This research was funded by EU-H2020-MSCA-RISE, grant no 777926 NanoSurf.

275 Acknowledgments: The authors acknowledge the grant from the Ministry of Education and Science of Ukraine 276 (0119U100823) for support in cell culture research.

277 Conflicts of Interest: The authors declare no conflicts of interest.

278 References

279 1. Bédouin, Y.; Gordin, D.-M.; Pellen-Mussi, P.; Pérez, F.; Tricot-Doleux, S.; Vasilescu, C.; Drob, S.I.; Chauvel- 280 Lebret, D.; Gloriant, T. Enhancement of the biocompatibility by surface nitriding of a low-modulus titanium 281 alloy for dental implant applications. J. Biomed. Mater. Res. B Part B 2019, 107, 1483–1490, 282 doi:10.1002/jbm.b.34240. 283 2. Branemark, R.; Branemark, P.I.; Rydevik, B.; Myers, R.R. Osseointegration in skeletal reconstruction and 284 rehabilitation: A review. J. Rehabil. Res. Dev. 2001, 38, 175–181. 285 3. Oleshko, O.; Deineka, V.V.; Husak, Y.; Korniienko, V.; Mishchenko, O.; Holubnycha, V.; Pisarek, M.; 286 Michalska, J.; Kazek-Kesik, A.; Jakóbik-Kolon, A.; et al. Ag nanoparticle-decorated oxide coatings formed 287 via plasma electrolytic oxidation on ZrNb alloy. Materials 2019, 12, 3742, doi:10.3390/ma12223742. 288 4. Abdel-Hady Gepreel, M.; Niinomi, M. Biocompatibility of Ti-alloys for long-term implantation. J. Mech. 289 Behav. Biomed. Mater. 2013, 20, 407–415, doi:10.1016/j.jmbbm.2012.11.014. 290 5. Blanquer, A.; Musilkova, J.; Barrios, L.; Ibáñez, E.; Vandrovcova, M.; Pellicer, E.; Sort, J.; Bacakova, L.; 291 Nogués, C. Cytocompatibility assessment of Ti-Zr-Pd-Si-(Nb) alloys with low Young's modulus, increased

Materials 2020, 13, 1306 10 of 11 experiment demonstrated full biocompatibility, indicating that the new alloy could be used for dental and orthopedics implant production.

Author Contributions: Conceptualization, O.M. and M.P.; methodology, O.O., O.K., and M.P.; formal analysis, O.M. and M.P.; investigation, O.K., O.O., and M.P.; data curation, M.P.; writing—original draft preparation, M.P. and O.O.; writing—review and editing, M.P. and O.M.; supervision, M.P. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by EU-H2020-MSCA-RISE, grant no 777926 NanoSurf. Acknowledgments: The authors acknowledge the grant from the Ministry of Education and Science of Ukraine (0119U100823) for support in cell culture research. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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