Additive Manufacturing

Beta titanium alloys, the third generation of implant dentistry biomaterials: analysis of
physical, chemical, mechanical, and microbiological properties of Ti-35Nb-7Zr-5Ta
and Ti-6Al-4V discs obtained by machining and additive manufacturing
--Manuscript Draft--

Manuscript Number: ADDMA-D-22-04033

Article Type: Research Paper

Keywords: Dental Implants; Ti-6Al-4V; TNZT; Additive Manufacturing; bacteria

Corresponding Author: Andréa Reis

BRAZIL

First Author: Juliana Dias Corpa Tardelli

Order of Authors: Juliana Dias Corpa Tardelli

Mariana Valente

Lucas Otani

Rodolfo Batalha

Fernanda Alves

Marcelo A. Pereira-da-Silva

Vanderlei Bagnato

Regina Dibb

Piter Gargarella

Claudemiro Bolfarini

Andréa Reis

Abstract: The objective was to compare the physical, chemical, mechanical, and microbiological
properties between disks (n=10) of Ti-6Al-4V and Ti-35Nb-7Zr-5Ta (TNZT) obtained by
Machining (M) and additive manufacturing (AM) by Laser Powder Bed Fusion
technique to identify the influence of processing technique and chemical composition
on material properties. Using scanning electron microscopy (SEM), energy dispersive
x-ray spectroscopy (EDX), x-ray diffraction (XRD), wettability, surface free energy,
roughness by confocal laser microscopy, Vickers microhardness (VM), and colony
forming units (CFU) against S. aureus. Two-way ANOVA (p<0.05) was applied. Higher
roughness and irregularity were observed in the MA disks. The chemical composition
of the EDX alloys was compatible with the concentrations expected and available in
the literature. For TNZT the manufacturing technique interfered in the present phases
(α and β). The wettability and surface free energy of TNZT was higher than that of Ti-
6Al-4V and for the manufacturing techniques, there was no significant difference. Ti-
6Al-4V showed greater hardness than TNZT and the U technique greater than AM.
There was no difference in S. aureus CFU between the groups. It was concluded that
the AM technique generated more irregular and rough surfaces, and lower hardness,
without significant changes concerning M in terms of chemical composition, wettability,
surface free energy, and bacterial formation. The TNZT alloy showed higher
hydrophilicity, surface free energy, roughness, lower hardness, manufacturing
techniques that interfered in its phases, and no differences for CFU compared to Ti-
6Al-4V.

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Carta de apresentação

UNIVERSIDADE DE SÃO PAULO

FACULDADE DE ODONTOLOGIA DE RIBEIRÃO PRETO

November 18th, 2022.

Dear Editor,

We would like to submit the attached Original Article entitled “Beta titanium alloys, the

third generation of implant dentistry biomaterials: analysis of physical, chemical,

mechanical, and microbiological properties of Ti-35Nb-7Zr-5Ta and Ti-6Al-4V discs

obtained by machining and additive manufacturing” to be considered for publication in

Additive Manufacturing.

We the undersigned declare that this manuscript is original, has not been published
before and is not currently being considered for publication elsewhere.

We wish to confirm that there are no known conflicts of interest associated with this
publication and there has been no significant financial support for this work that could
have influenced its outcome.

We confirm that the manuscript has been read and approved by all authors, that should
have made substantial contributions.

We further confirm that the order of authors listed in the manuscript has been approved

by all of us.

We acknolowledge the Foundation for Research Support of the State of São Paulo

(FAPESP) for research financial [grant number 2020/05272-2].

Andréa Cândido dos Reis

AVENIDA DO CAFÉ S/Nº - TEL: (0XX16) 602-3952 – FAX: (0XX16) 633-0999

14040-904 – RIBEIRÃO PRETO – S.P. - BRASIL

 UNIVERSIDADE DE SÃO PAULO
FACULDADE DE ODONTOLOGIA DE RIBEIRÃO PRETO

Beta titanium alloys, the third generation of implant dentistry biomaterials: analysis
of physical, chemical, mechanical, and microbiological properties of Ti-35Nb-7Zr-
5Ta and Ti-6Al-4V discs obtained by machining and additive manufacturing

Juliana Dias Corpa Tardellia, Mariana Lima da Costa Valentea, Lucas Barcelos Otanib,

Rodolfo Lisboa Batalhab, Fernanda Alvesc, Marcelo A. Pereira-da-Sivac, Vanderlei

Salvador Bagnatoc, Regina Guenka Palma Dibba, Piter Gargarellab, Claudemiro

Bolfarinib, Andréa Cândido dos Reisa

a
Department of Dental Materials and Prosthesis; School of Dentistry of Ribeirão Preto,

University of São Paulo (USP), 14040-904, Ribeirão Preto, SP, Brazil.

b
Department of Materials Engineering, Federal University of São Carlos (UFSCAR),

13565-905, São Carlos, SP, Brazil.

c
Optics Group from São Carlos Institute of Physics (IFSC), University of São Paulo

(USP), 13560‐ 970, São Carlos, SP Brazil

Corresponding Author:

Dra. Andréa Cândido dos Reis

Departament of Dental Materials and Prosthesis, School of Dentistry of Ribeirão Preto,

University of São Paulo (USP)

Av. do Café, s/n, 14040-904, Ribeirão Preto – SP, Brazil

Telephone: +55-16-3315-4790

Email: andreare73@yahoo.com.br

AVENIDA DO CAFÉ S/Nº - TEL: (0XX16) 602-3952 – FAX: (0XX16) 633-0999

14040-904 – RIBEIRÃO PRETO – S.P. - BRASIL

Destaques

HIGHLIGHTS
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2 The manufacturings did not interfere with the chemical composition of the alloys;
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5 The processing route altered phases in TNZT different from Ti-6Al-4V;
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TNZT has a higher roughness, surface free energy, and wettability than Ti-6Al-4V;
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10 The Manufacturing Technique did not interfere in the wettability;
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SLM surfaces were more rougher, but they did not interfere with CFU results.
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Manuscript File Click here to view linked References

Beta titanium alloys, the third generation of implant dentistry biomaterials:

1
2 analysis of physical, chemical, mechanical, and microbiological properties of
3
4
Ti-35Nb-7Zr-5Ta and Ti-6Al-4V discs obtained by machining and additive
5 manufacturing
6
7
8 ABSTRACT
9
10 The objective was to compare the physical, chemical, mechanical, and microbiological
11
12 properties between disks (n=10) of Ti-6Al-4V and Ti-35Nb-7Zr-5Ta (TNZT) obtained
13
14 by Machining (M) and additive manufacturing (AM) by Laser Powder Bed Fusion
15
16 technique to identify the influence of processing technique and chemical composition on
17
18 material properties. Using scanning electron microscopy (SEM), energy dispersive x-ray
19
20 spectroscopy (EDX), x-ray diffraction (XRD), wettability, surface free energy, roughness
21 by confocal laser microscopy, Vickers microhardness (VM), and colony forming units
22
23 (CFU) against S. aureus. Two-way ANOVA (p<0.05) was applied. Higher roughness and
24
25 irregularity were observed in the MA disks. The chemical composition of the EDX alloys
26
27 was compatible with the concentrations expected and available in the literature. For
28
29 TNZT the manufacturing technique interfered in the present phases (α and β). The
30
31
wettability and surface free energy of TNZT was higher than that of Ti-6Al-4V and for
32 the manufacturing techniques, there was no significant difference. Ti-6Al-4V showed
33
34 greater hardness than TNZT and the U technique greater than AM. There was no
35
36 difference in S. aureus CFU between the groups. It was concluded that the AM technique
37
38 generated more irregular and rough surfaces, and lower hardness, without significant
39
40 changes concerning M in terms of chemical composition, wettability, surface free energy,
41
42
and bacterial formation. The TNZT alloy showed higher hydrophilicity, surface free
43 energy, roughness, lower hardness, manufacturing techniques that interfered in its phases,
44
45 and no differences for CFU compared to Ti-6Al-4V.
46
47
48 Keywords: Dental Implants; Ti-6Al-4V; TNZT; Additive Manufacturing; SLM;
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bacteria.
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 1. INTRODUCTION
1
2 As a raw material for biomedical implants, titanium alloys are most suitable.
3
4 They can be manufactured by subtractive manufacturing, casting followed by machining,
5
6 and additive manufacturing, including Electron Beam Melting (EBM) and Selective
7
8 Laser Melting (SLM), which are most commonly used [1,2]. Understanding the physical,
9
10 chemical, mechanical, and biological properties of titanium alloys depending on the
11
12
chosen manufacturing technique is necessary, as the choice of the ideal biomedical device
13 for the case depending on the local and systemic condition of the patient acts as a
14
15 predictive factor for the rehabilitation success.
16
17 The most used material for biomedical applications is the Ti-6Al-4V alloy as it
18
19 presents the ideal combination of mechanical properties and corrosion resistance [3–6].
20
21 However, this has become the subject of discussion in recent years, as its chemical
22
23
elements aluminum (Al) and vanadium (V) are associated with neurological disorders,
24 allergies and toxicity depending on their concentration and exposure time. To overcome
25
26 this biological disadvantage, beta titanium (Ti-β) alloys are developed with non-toxic
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28 elements such as tantalum (Ta), zirconium (Zr), molybdenum (Mo), and tin (Sn) and are
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30 promising for long-lasting implants for having high mechanical compatibility, that is,
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32 high wear resistance and low modulus of elasticity, in addition to being categorized by
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Abd-Elrhman et al. as the third generation of biomaterials [1,7–10].
35 Among the manufacturing techniques, subtractive manufacturing has the
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37 disadvantages of high material waste (around 80%) and high production cost, so additive
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39 manufacturing techniques are advantageous because they can simplify and accelerate the
40
41 manufacturing process, but they have as a disadvantage the high cost of equipment and
42
43 raw material, in addition to the difficulty of establishing the ideal parameters to obtain
44
the proposed structure [1,2,11,12]. For metals, the Laser Powder Bed Fusion (LPBF)
45
46 technique is the most consolidated, it uses a laser beam to fuse successive layers of
47
48 powder into the desired geometry in a protective atmosphere of argon (Ar) or nitrogen
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50 (N2) to produce structures with a relative density of 99% [1,13–15].
51
52 The study of the properties of the alloys, and means of obtaining, among other
53
54 factors, are important to understand the means of reducing the main cause of the failure
55 of implant-supported rehabilitations, which is the peri-implant infection, the main reason
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57 for patient dissatisfaction with expenses with treatments to contain there [16–25]. In the
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59 biomedical field, the gram-positive bacterium Staphylococcus aureus (S. aureus) has a
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 high affinity for Ti surfaces and has been implicated in infections of metallic implants
1
2 due to its ability to form biofilms resistant to antibiotics and patient defenses [16,18,26].
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Therefore, the search for understanding the factors that determine bacterial adhesion to
5 implant surfaces is the way to determine strategies to prevent such contamination and
6
7 thus increase the survival of implant-supported oral rehabilitations.
8
9 In this article, Ti-35Nb-7Zr-5Ta (TNZT) alloy, promising for combining low
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11 elastic modulus (50-70GPa), high strength, and biocompatibility [1,14,15], as compared
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13 to the most used for biomedical applications, Ti-6Al-4V, to infer the influence of the alloy
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and manufacturing technique on the physical, chemical, mechanical, and biological
16 behavior of these surfaces against S. aureus through CFU analysis.
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18 2. MATERIALS AND METHODS
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21 2.1 Sample preparation
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23 2.1.1Machining samples
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26 The Ti-6Al-4V and TNZT cylindrical bars (ERCATA GmbH, Development Co., Ltd.,
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28 Germany) were sliced out in disk-shaped samples of 5mm in diameter by 1 mm thick
29 using wire electro-discharge machining. The suffix “M” is used for the materials in as
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31 received condition.
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34 2.1.2 SLM samples
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36 The gas-atomized powder of the Ti-6Al-4V and TNZT (ERCATA GmbH, Development
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38 Co., Ltd., Germany) as a spherical powder, and particles with a diameter <63 µm, were
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40 used to produce the disk-shaped samples by SLM in a Realizer SLM 50 with a fiber laser
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42 in the continuous mode (λ=1070 nm), spot size≈60 µm, 129 W applied on a thin layer of
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44 powder with 40 µm thickness, 110 μm of hatching distance, and scanning speed of 0.57
45
m s−1 built in a vertical direction. These optimized L-PBF parameters were established in
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47 previous work [14]. The atmosphere was filled with argon, and the oxygen content was
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49 kept at 0.1 wt% in the chamber. The suffix "SLM" is used to distinguish the samples
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51 produced by such a processing technique.
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2.1.3 Post-processing of the samples
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56 The samples were wet ground with a sequence of #320, #400, and #600 grit sandpaper
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58 (Minimet 1000, Buehler, Lake Bluff, IL, USA). The cleaning process was conducted in
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60 distilled water and acetone for 7 min in an ultrasonic bath and dried with warm air.
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 2.2 Surface characterization
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2 The surface morphologies of the samples were characterized by a scanning electron
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4 microscope (SEM, JSM-6610LV, JEOL, Japan) equipped with an Energy Dispersive X-
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6 ray (EDX) microanalysis system (IXRF Systems 500 Digital Processing). The 3D laser
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8 scanning microscope (LEXT 4000; Olympus, Hamburg, Germany) measured the
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10 roughness parameters (average roughness, Ra; average maximum height of the profile,
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Rz; and average surface roughness, Sa) in 10 different areas per sample. The roughness
13 measurements obtained by atomic force microscopy (Bruker model Dimension ICON)
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15 through the probe model SCM-PIT-V2 (Bruker) of silicon doped with antimony of length
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17 225 micrometers and spring constant 3N/m. The analysis was performed in intermittent
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19 contact (tapping) to obtain the surface profile.
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21 Wettability and surface free energy of the surfaces were assessed using a sessile drop
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23 contact angle goniometer (KSV CAM200) and software (CAM 200 Contact Angle
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25 Measurement System). A drop of 4 μl distilled water, formamide, and diodomethane was
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27 deposited on the surface under constant temperature. Measurements were done after 60 s
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29 for partial stabilization of the drop. Three measures were done per sample; before each
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measure, the discs were dried with a 1 min nitrogen flow. The average of the left and right
32 contact angles was used for the analysis.
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35 2.3 Microstructure Characterization
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37 X-ray diffraction (XRD) was used for structural analysis by a Bruker AXS D2
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39 diffractometer with Cu Kα radiation (λ = 1.54 Å); in the range of 5–50◦, with steps of
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41 0.05◦, with 0.5 s for each step
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44 2.4 Mechanical analysis
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46 Vickers microhardness measurements were performed in a microdurometer (Shimadzu,
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48 HMV-2 Micro Hardness Tester, Shimadzu Corporation, Kyoto, Japan) by applying a load
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50 of 300 g for 15 s. The analysis was performed at three randomly distributed points. The
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52 mean value was calculated for each sample and then for each group to obtain the
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microhardness.
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56 2.5 In vitro biological evaluation
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2.5.1 Experiments with Staphylococcus aureus
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 The bacterial strain used in this study was Staphylococcus aureus (ATC25923). The
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2 bacterium that was storage in Tryptic Soy Broth (TSB) at -80°C was cultured on brain
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heart infusion agar (BHI) (KASVI, São José dos Pinhais, PR, Brazil) plates in an
5 incubator at 37 °C for 24 hours. After incubation, 7 colonies were resuspended in a tube
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7 containing 10 mL of TSB, and the bacterium was incubated at 37 °C for 18 hours. An
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9 aliquot of 500 μL of the suspension was diluted in 9.5 mL of fresh TSB and incubated for
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11 4 hours until reached the mid-log growth phase. Then, the cell concentration was adjusted
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13 to 108 cells/mL in a spectrophotometer (600 nm) (Cary 60 UV-Vis Spectrophotometer –
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Agilent), which corresponds to an optical density of 0.2 arbitrary units (a.u.). After
16 standardization of the bacterial suspension, 500 μL were transferred to the wells of a 24-
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18 wells plate where the specimens were, and incubated at 37 °C under agitation (75 rpm)
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20 for 90 min, for bacterial adhesion on the surface of the material samples. After the
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22 adhesion period, samples were removed from the wells and were carefully washed twice
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24 with 500 μL of saline solution to remove non-adhered cells, buffer the medium and
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remove metabolites. Then, the specimens were inserted into the wells of a 24-wells plate
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27 with 500 μL of TSB for biofilm formation. The plate was kept at 37 °C under agitation
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29 for 48 h, completing the maturation phase of the biofilm. After this period, samples were
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31 removed from the plate and washed twice with 500 μL of saline.
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2.5.2 Biofilm quantification
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36 Biofilm quantification on the surface of the specimens was determined using the CFU/mL
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38 test. For this, the specimens were transferred individually into microcentrifuge tubes
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40 containing 500 μL of saline solution. The biofilms were completely detached from the
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specimens by vigorously vortexing the tubes for 60 seconds. Then, serial dilutions (100
43 to 10-5) were performed and plated in duplicate on BHI Agar. Plates were incubated at 37
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45 °C for 24 h.
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48 After incubation, the bacterial colonies were counted and the CFU/mL values were
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obtained and transformed into logarithms (log10) for statistical analysis purposes.
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55 2.6 Statistical analysis
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 For quantitative data, a two-way ANOVA (factor 1: alloy and factor 2: Manufacturing
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2 Method) was used and the analysis was performed in the SPSS 20.0 software (SPSS,
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Chicago, IL), considering P<0.05 as the level of significance.
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8 3. RESULTS AND DISCUSSION
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10 3.1 Surface characterization
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12 Figure 1 obtained by scanning electron microscopy and Figure 2 obtained by
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14 confocal laser microscopy demonstrate that the surfaces of the Ti-6Al-4V M and TNZT
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16 M samples are completely fused and have less roughness than the Ti-6Al- 4V SLM and
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18 TNZT SLM. The morphological difference of greater roughness and presence of spherical
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20 particles fused, partially fused, and weakly adhered to the surfaces obtained by SLM is
21 attributed to the energy density not being sufficient to melt the powder completely
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23 because the laser beam follows the Gaussian energy distribution, suggesting energy tends
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25 to decrease toward the edges of the laser [14,15,27]. Thus, the surfaces obtained by SLM
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27 in this study through the parameters used, suggest the need for post-processing for the
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29 manufacture of biomedical devices to remove poorly adhered dust particles that can
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promote peri-implant inflammatory processes [13,28,37–41,29–36].
32 Figure 1. Scanning electron microscopy of Ti-6Al-4V M, TNZT M, Ti-6Al-4V SLM, and
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34 TNZT SLM disks A) X 50; B) X1000.
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50 Ti-6Al-4V M X50 Ti-6Al-4V M X1000
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14 TNZT M X50 TNZT M X1000
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30 Ti-6Al-4V SLM X50 Ti-6Al-4V SLM X1000
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46 TNZT SLM X50 TNZT SLM X1000
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 Figure 2. Confocal laser microscopy of Ti-6Al-4V M, TNZT M, Ti-6Al-4V SLM, and
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2 TNZT SLM discs.
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22 Ti-6Al-4V M TNZT M
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41 Ti-6Al-4V SLM TNZT SLM
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3.2 Microstructure Characterization
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47 Table 1 shows that the Ti-6Al-4V M and Ti-6Al-4V SLM samples present the
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49 elements Ti, Al, and V close to the nominal and specific composition for the alloy, as
50
51 well as the presence of only the α phase (hexagonal close-packed, HCP) (Figure 3) for
52
53 both conditions. Thus, it can be inferred that the processing route did not provide
54 significant changes in the chemical composition and final structure of the samples.
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57 Table 1 shows that the TNZT M and TNZT SLM samples present the elements
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59 Ti, Nb, Zr, and Ta close to the nominal and specific composition for the alloy, as well as
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 the α (HCP) and β (BCC) phases, respectively (Figure 3). Thus, the results suggest that
1
2 the processing route alters the phases present so that the machined discs come from forged
3
4
billets and are subsequently machined, thus enabling the most thermodynamically stable
5 structure at room temperature to be completely formed (phase α), while the rapid cooling
6
7 provided by the SLM technique allowed the maintenance of the β phase at room
8
9 temperature.
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12
It is noteworthy that the understanding of the phase formation of the alloys is
13 important, as it directly affects the properties obtained, so the SLM technique for the
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15 TNZT alloy, by providing the maintenance of the β phase in the microstructure, caused a
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17 decrease in the mechanical strength of the alloy, a result also observed by other authors
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19 [42–44] corroborating the results observed concerning microhardness (Table 2).
20
21 Table 1. Chemical composition of the discs, percentage by weight (%).
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23 Weight percentage (%)
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25 Samples Ti Al V Nb Zr Ta
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27 Ti-6Al-4V M 89.7 6.3 3.9 - - -
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29 TNZT M 54.4 - - 33.3 6.4 5.8
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31 Ti-6Al-4V SLM 89.1 7.2 3.7 - - -
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33 TNZT SLM 54.6 - - 33.1 6.7 5.5
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37 Figure 3. X-ray diffractograms (XRD) of Ti-6Al-4V M, TNZT M, Ti-6Al-4V SLM, and
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39 TNZT SLM disks.
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 3.3 Mechanical analysis
1
2 For microhardness, the Manufacturing Technique provided a statistically
3
4 significant difference, as observed by the Ti-6Al-4V M and TNZT M groups presenting
5
6 a higher average when compared to the Ti-6Al-4V SLM and TNZT SLM groups (Table
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8 2). The statistical difference can be explained by the greater relative density of machined
9
10 samples in comparison to SLM due to the differences in processing routes so that most
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12
forged and machined samples result in solid structures with less than 0.01% porosity,
13 while SLM samples have around 1% porosity [14,45]. Future studies that correlate the
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15 porosity of samples by micrographic or tomography analysis are necessary to infer the
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17 influence of porosity on mechanical performance.
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20 The chemical composition of the evaluated alloys, Ti-6Al-4V and TNZT, also
21 provided a statistically significant difference for microhardness, which can be explained
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23 by the effect of the solid solution of the Ti-6Al-4V alloy, since the chemical elements Al
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25 and V cause much more stresses in the alpha phase structure due to the greater size
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27 difference when compared to Ti, which results in a greater hardness of the alloy, to the
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29 TNZT alloy in which the elements Ti, Nb, Zr, and Ta are very similar elements and
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31
consequently do not produce such a strong solid solution effect. Thus, the Ti-6Al-4V
32 alloy has a higher solid solution hardening capacity than TNZT.
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35 When reflecting on the possible clinical implications, the results found for surface
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37 hardness inspire the need to analyze the correlation of the surface of these devices, in this
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39
case, the dental implants, with the surfaces to which they will be related. To contemplate
40 osseointegration, you must consider the implant's external surface and its physical,
41
42 chemical, and biological properties that will work synergistically. While the internal
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44 surface and/or prosthetic platform of the implant will relate to prosthetic components of
45
46 surface hardness dependent on the manufacturing technique and alloy, which may be
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48 compatible or incompatible.
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50 Table 2. Mean and standard deviation of microhardness values for alloys (Ti-6Al-4V
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52
and TNZT) and techniques (Machining and SLM).
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54 P value
Techniques
55 Alloy
56 Assay Alloys Machined SLM
57 Ti-6Al-4V 384.04±11.36 363.95±40.22
Vickers Microhardness
58 TNZT 324.42±83.11 249.43±33.10 <0.001
59 P value
0.018
60 Technique
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 The vertical p-value is related to the comparison between alloys and the horizontal p-
1
2 value is related to the comparison between manufacturing techniques.
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4 3.4 In vitro biological evaluation
5
6
7 SANTHOS KUMAR et al., 2020 [46] and VADILLO-RODRÍGUEZ et al., 2018
8
9 [47] report the bacterial preference for irregular and rough surfaces. The results found in
10 this study for CFU Table 3 disagree with the literature because, despite the greater
11
12 roughness of the surfaces obtained by the SLM technique as expressed in Table 4, the
13
14 Manufacturing Technique was not significant (p=0.057) for the formation of bacterial
15
16 colonies. It is noteworthy that the roughness found in the evaluated groups was higher
17
18 than the minimum value of 0.20 μm, which is the general size of bacteria, and considered
19
20 the minimum size of roughness necessary for bacterial cell walls to detect areas for
21 adhesion.
22
23
24 Table 3. Mean and standard deviation of CFU values for alloys (Ti-6Al-4V and TNZT)
25
26 and techniques (Machining and SLM). The vertical p-value is related to the alloy and the
27
28 horizontal p-value is related to the manufacturing technique.
29
30 P value
31 Techniques
Alloy
32 Assay Alloys Machined SLM
33 Ti-6Al-4V 6.62±0.03 6.65±0.07
34 CFU
TNZT 6.27±0.38 7.23±0.35 0.578
35 P value
36 0.057
Technique
37
38 The vertical p-value is related to the comparison between alloys and the horizontal p-
39 value is related to the comparison between manufacturing techniques.
40
41 Table 4. Mean and standard deviation of roughness values (Ra, Rz, Sa) for alloys (Ti-
42 6Al-4V and TNZT) and techniques (Machining and SLM).
43
44
Techniques P Value
45
Assay Parameters Alloys Machined SLM Alloy
46
47 Ra 0.10±0.007 2.34±1.38 0.204
48 Rz Ti-6Al-4V 0.59±0.04 12.04±7.43 0.096
49 Sa 0.12±0.01 3.27±1.62 0.353
Roughness
50 Ra 0.37±0.21 3.28±2.02 0.204
51 Rz TNZT 1.77±1.10 24.96±20.19 0.096
52 Sa 0.15±0.04 4.25±2.31 0.353
53 P Value
<0.001
54 Technique
55 The vertical p-value is related to the comparison between alloys and the horizontal p-
56
57 value is related to the comparison between manufacturing techniques.
58
59
60
61
62
63
64
65
 In this study, the findings for roughness and CFU allow us to suggest that despite
1
2 the greater roughness of the surfaces obtained by SLM, the applied polishing may have
3
4
interfered by making the roughness peaks rounded (Figure 4), and thus, have led to a
5 decrease in the number of focal points favorable to bacterial adhesion and spread, as
6
7 occurred in the studies by MANGANO et al., 2009 [36] and TSUKANAKA et al., 2016
8
9 [32] corroborating GE et al., 2019 [48], which infers that bacterial adhesion in
10
11 biomaterials is dependent on the available area for adhesion and morphology of the space
12
13 for bacterial confinement. In addition to the observation of Figures 1 and 2 and the
14
15
numerical data expressed in Figure 4, it is possible to infer that the chemical interaction
16 of Ti, Nb, Zr, and Ta produces rougher surfaces than Ti, Al, and V, since the machined
17
18 and SLM surfaces of TNZT showed higher roughness values than those of Ti-6Al-4V.
19
20
21 Figure 4. Topographic analysis of roughness by AFM of Ti-6Al-4V M, TNZT M,
22
23
Ti-6Al-4V SLM, and TNZT SLM discs.
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53 According to the review by GITTENS et al., 2014 [49] bacteria show a preference
54
55 for adhesion to surfaces of the same hydrophilicity, that is, hydrophobic bacteria prefer
56
57 hydrophobic surfaces and hydrophilic bacteria prefer hydrophilic surfaces. The bacterium
58
evaluated in this study (S. aureus) is considered by the literature to be hydrophobic [49–
59
60 52]. However, despite the evaluated groups being considered hydrophilic, with water
61
62
63
64
65
 contact angle concerning the surface lower than 90◦, there was indeed the formation of
1
2 bacterial colonies as shown in Table 3. This fact was attributed to the development of
3
4
obscure adaptation mechanisms in this work because biomolecular assays were not
5 performed to better understand the kinetics of bacterial colonization on the surfaces
6
7 evaluated.
8
9
10 Considering wettability (Table 5), there was no statistically significant difference
11
12
for the Manufacturing Technique (p>0.619), which can be attributed to the fact that the
13 pores on the surface of the samples manufactured by the SLM technique were previously
14
15 filled with air [13,53]. Therefore, they were not being filled with water, which provides
16
17 an increase in the contact angle despite the greater surface area of the SLM samples when
18
19 compared to the machined ones. Meanwhile, for Alloy, there was a statistically significant
20
21 difference (p<0.041), possibly due to the surface oxides formed by the elements Ti, Nb,
22
23
Ta, and Zr being more hydrophilic than the ones composed by Ti, Al, and V, because
24 according to VLCAK et al., 2021[54] the valence state of metal ions influences the
25
26 wettability of oxidized titanium surfaces.
27
28
29 According to the literature, the higher the surface free energy, the greater the
30
31
bioactivity of a surface by favoring the adsorption of proteins and bone cells, and thus,
32 reducing bacterial adhesion by reducing the number of sites for their colonization [55–
33
34 57]. In this in vitro study, the surface free energy was significantly influenced by the type
35
36 of alloy (p=0.025) and allowed us to infer that the TNZT alloy has a higher surface free
37
38 energy, independent of the Manufacturing Technique, corroborating the results for
39
40 wettability for presenting the best wetting ability (Table 5).
41
42 Table 5. Mean and standard deviation of wettability and surface free energy values for
43
44
alloys (Ti-6Al-4V and TNZT) and techniques (Machining and SLM).
45
46 P Value
Techniques
47 Assays Alloys Alloy
48 Machined SLM
49 Ti-6Al-4V 59.49±4.55 63.81±7.55
Wettability
50 TNZT 54.48±7.90 53.69±14.24 0.041
51 P value
0.619
52 Technique
53 Ti-6Al-4V 42.60±2.80 45.36±3.80
Surface Free Energy
54 TNZT 47.96±3.58 45.99±2.94
0.025
55 P Value
0.755
56 Technique
57 The vertical p-value is related to the comparison between alloys and the horizontal p-
58
59 value is related to the comparison between manufacturing techniques.
60
61
62
63
64
65
 3.5 Clinical significance, limitations, and future scope
1
2 In this study, the physical, chemical, mechanical and biological properties of Ti-
3
4 6Al-4V and TNZT alloys obtained by machining and SLM were evaluated to understand
5
6 the behavior of these alloys concerning their possible use in dental implants. It was
7
8 observed that the properties of TNZT were superior to Ti-6Al-4V for wettability and
9
10 surface free energy, which favor wetting by blood, adsorption of proteins and osteoblastic
11
12
cells for the phenomenon of osseointegration, and higher roughness that may favor the
13 mechanical imbrication with the bone tissue, and, therefore, less hardness, which in vivo
14
15 will depend on its relationship with the structure that will be related, as well as if the
16
17 internal surface of the implant has a relationship with the prosthetic component and if the
18
19 external surface of the peri-implant bone tissue. Regarding its behavior against S. aureus,
20
21 its biological behavior did not differ from Ti-6Al-4V in the formation of bacterial
22
23
colonies, but it emphasizes the need for future time-dependent studies for a better
24 evaluation of bacterial cell viability on these surfaces.
25
26
27 As for the manufacturing techniques, the SLM technique provides greater
28
29 roughness, although, for primary stability, rough implants provide greater surface area for
30
31
better mechanical imbrication [58]. For secondary stability, roughness can affect
32 negatively, depending on its influence on bacterial and bone cell adhesion, so future
33
34 studies, in vivo, that correlate roughness with the biological behavior of these groups are
35
36 necessary for better inference of the possible clinical implication of the roughness. In
37
38 addition to the lack of difference in wettability and surface free energy between machined
39
40 surfaces and SLM in vitro, they motivate in vivo studies to assess whether this result is
41
42
maintained and the actual stimulation of bone cells by SLM surfaces.
43
44 This in vitro experimental study indicates satisfactory results for the TNZT alloy
45
46 and motivates the conduct of in vivo studies for validation and verification by in vitro and
47
48 experimental randomized in vivo biocompatibility studies with animals to determine
49
50
whether this alloy is safe for use in human experiments. In addition to the limitation of
51 this study not having evaluated bone cells and other bacterial species or even a
52
53 multispecies biofilm, in future studies to be developed the biological behavior of these
54
55 surfaces against bacterial and bone cells will be clarified and the results will be correlated
56
57 with the properties evaluated in this study.
58
59 4. CONCLUSIONS
60
61
62
63
64
65
 Based on the results obtained, it was possible to conclude that:
1
2 • The morphology observed on the surface of the evaluated groups is consistent with the
3
4 Manufacturing Technique used, being rougher for the SLM;
5
6
7 • The Manufacturing, Machining, and SLM Techniques did not interfere with the
8
9 chemical compositions of Ti-6Al-4V and TNZT alloys;
10
11 • The processing route is the main reason for the variation of phases present in the TNZT
12
13 M and TNZT SLM disks; In addition to Ti-6Al-4V the processing route is not making the
14
15 production of implants with this material unfeasible.
16
17 • TNZT alloy has a rougher surface than Ti-6Al-4V regardless of the Manufacturing
18
19 Technique, subtractive or additive; In addition to post-processing, polishing may have
20
21 interfered with the CFU results in the Ti-6Al-4V groups SLM and TNZT SLM for having
22
23 reduced the focal points favorable to adherence to the make them rounded.
24
25 • The wettability of SLM surfaces is similar to Machined when considering
26
27 Manufacturing Technique, possibly due to air filling the pores which water would wet.
28
29 What's more, the TNZT alloy has a higher surface free energy and wettability than Ti-
30
31 6Al-4V, possibly due to its oxides being more hydrophilic;
32
33 • Special attention should be paid to Alloy and Manufacturing Technique when producing
34
35 a dental implant, as these are factors that can interfere in a significant impact on
36
37 microhardness and the consequent survival of oral rehabilitation. In the way that implants
38
39 are produced by alloys of greater rigidity and by Machining have greater wear resistance
40
41 than SLM implants, depending on the print parameters;
42
43 • The Alloy and Manufacturing Technique did not significantly interfere with the CFU
44
45 results against S. aureus. The results found in this study demonstrate the ability of this
46
47 microorganism to adapt, as it produced several colonies statistically similar between
48
49 polished surfaces (machined) and rough (SLM). In addition to colonization on
50
51
hydrophilic surfaces, although this is hydrophobic.
52
53
54
55
56 ACKNOWLEDGEMENTS
57
58 The authors acknolowledge the Foundation for Research Support of the State of São Paulo
59
60 (FAPESP) for research financial [grant number 2020/05272-2].
61
62
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65
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Figura Click here to access/download;Figure;figure 1.jpg
Figura Click here to access/download;Figure;Figure 2.jpg
Figura Click here to access/download;Figure;Figure 3.jpg
Figura Click here to access/download;Figure;Figure 4.jpg
Table (Editable version) Click here to access/download;Table (Editable
version);TABLES.docx

TABLES
1
2 Table 1. Chemical composition of the discs, percentage by weight (%)
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4 Weight percentage (%)
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6 Samples Ti Al V Nb Zr Ta
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8 Ti-6Al-4V M 89.7 6.3 3.9 - - -
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TNZT M 54.4 - - 33.3 6.4 5.8
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11 Ti-6Al-4V SLM 89.1 7.2 3.7 - - -
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13 TNZT SLM 54.6 - - 33.1 6.7 5.5
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 Table 2. Mean and standard deviation of microhardness values for alloys (Ti-6Al-4V
1 and TNZT) and techniques (Machining and SLM).
2
3 P value
4 Techniques
Alloy
5 Assay Alloys Machined SLM
6 Ti-6Al-4V 384.04±11.36 363.95±40.22
7 Vickers Microhardness
TNZT 324.42±83.11 249.43±33.10 <0.001
8
P value
9 0.018
Technique
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11 The vertical p-value is related to the comparison between alloys and the horizontal p-
12 value is related to the comparison between manufacturing techniques.
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 Table 3. Mean and standard deviation of CFU values for alloys (Ti-6Al-4V and TNZT)
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2 and techniques (Machining and SLM). The vertical p-value is related to the alloy and the
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4
horizontal p-value is related to the manufacturing technique.
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6 P value
7 Techniques
Alloy
8 Assay Alloys Machined SLM
9 Ti-6Al-4V 6.62±0.03 6.65±0.07
10 CFU
TNZT 6.27±0.38 7.23±0.35 0.578
11 P value
12 0.057
Technique
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14 The vertical p-value is related to the comparison between alloys and the horizontal p-
15 value is related to the comparison between manufacturing techniques.
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 Table 4. Mean and standard deviation of roughness values (Ra, Rz, Sa) for alloys (Ti-
1 6Al-4V and TNZT) and techniques (Machining and SLM).
2
3 Techniques P Value
4 Assay Parameters Alloys Machined SLM Alloy
5 Ra 0.10±0.007 2.34±1.38 0.204
6 Rz Ti-6Al-4V 0.59±0.04 12.04±7.43 0.096
7 Sa 0.12±0.01 3.27±1.62 0.353
8 Roughness
Ra 0.37±0.21 3.28±2.02 0.204
9
Rz TNZT 1.77±1.10 24.96±20.19 0.096
10
Sa 0.15±0.04 4.25±2.31 0.353
11
12 P Value
<0.001
13 Technique
14 The vertical p-value is related to the comparison between alloys and the horizontal p-
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16 value is related to the comparison between manufacturing techniques.
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 Table 5. Mean and standard deviation of wettability and surface free energy values for
1 alloys (Ti-6Al-4V and TNZT) and techniques (Machining and SLM).
2
3 P Value
4 Techniques
Assays Alloys Alloy
5 Machined SLM
6 Ti-6Al-4V 59.49±4.55 63.81±7.55
7 Wettability
TNZT 54.48±7.90 53.69±14.24 0.041
8 P value
9 0.619
Technique
10
Ti-6Al-4V 42.60±2.80 45.36±3.80
11 Surface Free Energy
12 TNZT 47.96±3.58 45.99±2.94
0.025
13 P Value
0.755
14 Technique
15 The vertical p-value is related to the comparison between alloys and the horizontal p-
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17 value is related to the comparison between manufacturing techniques.
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Conflito de interesses

Declaration of interests

☒The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: