139
Journal of Oral Science, Vol. 64, No. 2, 139-144, 2022
Original article
Biofilm accumulation on additive manufactured Ti-6Al-4V alloy surfaces
Mari Koike1), Richard J. Mitchell2), Tetsuro Horie3), Susan K. Hummel4), and Toru Okabe5)
1)
The Nippon Dental University College at Tokyo, The Nippon Dental University, Tokyo, Japan
2)
Department of Biomaterials Science, University of Kentucky College of Dentistry, Lexington, KY, USA
3)
Department of Oral Health, The Nippon Dental University, Tokyo, Japan
4)
Harry S Truman Memorial Veterans’ Hospital, Columbia, MO, USA
5)
Department of Biomaterials Science, Texas A & M University, Baylor College of Dentistry, Dallas, TX, USA
Abstract
Purpose: This study investigated whether additive manufactured (AM)
surfaces inhibit accumulation of bacterial biofilm on the surfaces of
Ti-6Al-4V alloy dental implants. Bacterial biofilms are thought to cause
peri-implant disease, which develops in mucosa surrounding titanium (Ti)
and Ti alloy dental implants and can lead to bone loss and implant failure.
Methods: Accumulation of a Streptococcus mutans (ATCC 25175) biofilm
on Ti-6Al-4V alloy was compared in relation to fabrication method, ie,
AM using electron beam melting (EBM) or laser beam melting (LBM).
Conventional lost-wax casting was used as positive control, and Teflon was
used as negative control. Biofilm accumulation on the alloys and negative
control (each n = 10) was conducted at 37°C under anaerobic conditions.
After 4 h, the number of metabolically active S. mutans bacteria adhering
to the alloy was determined with a bioluminescence assay.
Results: The quantitative roughness values of the specimens, before expo-
sure to bacteria, ranked EBM > LBM > cast > Teflon.
Conclusion: The amount of biofilm accumulation on the investigated AM
metals and cast metal controls did not significantly differ.
Keywords: additive manufacturing, biofilm, bioluminescence assay,
dental implants, titanium alloy
Introduction
Because of improvements in lost-wax casting, the dental alloys that can
be successfully cast now includes the more biocompatible titanium (Ti)
alloys. However, the casting process for such alloys consists of multiple
steps and is extremely demanding. Progress in digital technology since
1980 has enabled a new type of solid form fabrication. Additive manufac-
turing (AM) was a significant breakthrough in using digital processes in
product manufacturing. Unlike earlier material manufacturing processes, a
part is fabricated incrementally in layers by additive processes. In 2007, an
article may have been the first to describe the three AM processes used to
make orthopedic implants, namely, electron beam melting (EBM), direct
metal laser sintering (DMLS), and selective laser melting (SLM) or laser
beam melting (LBM). Solid material gradually accumulates by selective
melting and subsequent solidification of powder layers, thereby forming
profiles dictated by computer models. Because this method requires no
fixtures or tooling, cost and lead time are considerably reduced [1]. Prod-
ucts can be directly fabricated with intricate external and internal features,
including lattice and porous features. Most subsequent reports focused on
explaining AM technologies but said little about the success and failure of
AM implants and other devices. Many emphasized orthopedic implants
[2-4] rather than dental implants.
In dentistry, use of techniques such as x-ray tomography and digital
impressions allow for creation of computer-aided design (CAD) files
Correspondence to Dr. Mari Koike, The Nippon Dental University College at Tokyo, The Nippon
Dental University 1-9-20 Fujimi, Chiyoda-ku, Tokyo 112-0071, Japan
Fax: +81-3-3261-8928 E-mail: mkoike@tky.ndu.ac.jp
Color figures can be viewed in the online issue at J-STAGE.
doi.org/10.2334/josnusd.21-0521
DN/JST.JSTAGE/josnusd/21-0521
(Received December 5, 2021; Accepted December 28, 2021)
that drive AM production of parts (e.g., prostheses) that accurately repair
defects in bony or dental structures [5]. This drew the attention of clini-
cians and researchers in medicine and dentistry. It would be beneficial if
such custom-designed replacement parts could be made using biocompat-
ible Ti alloys. Although most of these implants are made of commercially
pure Ti (cp Ti), a smaller, unknown number of implants are made of Ti
alloys. Use of zirconia ceramic implants is growing, but Ti has recently
come to dominate [5]. No other metal is used with any frequency. Specifi-
cally, most implants are made from cp Ti; fewer are made from Ti alloys.
The most widely used Ti dental implant alloy [6] has the same composition
as the most widely used Ti orthopedic implant alloy, namely, Ti-6Al-4V
alloy, which has superior strength. Ti implants replace single teeth, serve
as abutments for fixed partial dentures, and are increasingly used to support
overdentures. The present researchers were among the early evaluators of
AM products and previously published studies comparing 1) the mechani-
cal properties of Ti-6Al-4V alloy processed by EBM compared with those
of wrought Ti-6Al-4V alloy [1], 2) the mechanical properties of Ti-6Al-4V
alloy processed by EBM for dental applications [7], 3) the fatigue resis-
tance of Ti-6Al-4V alloy processed by EBM with the beam aligned parallel
and perpendicular to the added layers [8], 4) osseointegration in rabbits of
Ti-6Al-4V alloy processed by EBM versus osseointegration of a porous-
surface commercial Ti-6Al-4V implant [9], and 5) the surface roughness of
Ti-6Al-4V alloy processed by EBM and SLM [Koike M et al., TMS 2019
148th Annual Meeting & Exhibition. Supplemental Proceedings, Springer,
827-36, 2019] and biological properties using stem cells [10].
A recent review [3] discusses the use of DMLS AM and EBM AM
to make highly porous orthopedic implants that mimic cancellous human
bone. Porosity has two advantages. First, in an orthopedic implant, poros-
ity corrects for the high elastic modulus of the Ti, thus yielding an implant
with stiffness close to that of bone. Second, porosity provides spaces into
which bone can grow, thereby locking the implant into place. Laser and
electron beams can both be finely focused to achieve the required dimen-
sional accuracy and have sufficient energy to quickly sinter or completely
melt metals with high melting points, such as Ti. In addition, technology
has matured to the point that a commercial AM Ti-6Al-4V alloy porous-
surface dental implant was evaluated in a clinical trial. Each implant had
a porous-surface screw-shaped root, and 110 implants were placed, each
of which supported a single crown. Tunchel et al. [11] report that after 3
years in service, 94.3% of the implants survived. Clinical trials suggest that
dental implants are very successful in these applications. A comprehensive
review of 23 studies with a minimum of 10 years’ follow-up reported a
mean survival of 94.6% [12]. However, this success does not fully account
for the prevalence of peri-implant disease, inflammation of the mucosa,
and subsequent bone loss around the margins where the implant emerges
from the mucosa. A review of 11 studies revealed that, in most of the world,
peri-mucositis, a reversible condition, was highly prevalent; it was present
in 43% of implants examined [13]. In the same studies, peri-implantitis,
a more severe, irreversible, condition, was observed in 22% of examined
implants. These infections usually do not end with loss of the implant,
although that is possible. Another systematic review of nine clinical stud-
ies found that 9.6% of the implants were associated with implantitis [14].
Peri-implantitis is associated with a high percentage of late failures of
implanted Ti [15]. Overall, the results vary considerably, in part because of
imprecise definitions of peri-implant disease [16].
In 1994, Pontoriero et al. [17] reported that when bacterial plaque was
140
Table 1   Chemical composition of the alloys given by the manufacturers (wt.%)
Alloy Chemical composition (wt.%)
Ti-6Al-4V ELI powder Al: 6.00, V: 4.00, C: 0.03, Fe: 0.10,
(ASTM grade 23) O: 0.10, N: 0.01, H: <0.003, Ti: balance
Ti-6Al-4V powder Al: 5.50-6.75, V: 3.50-4.50, C: <0.8, Fe: <3.0, O: <2.0,
(ASTM grade 5) N: <0.5, H: <0.15, Ti: balance
Ti-6Al-4V ELI Al: 6.06, V: 4.07, C: 0.013, Fe: 0.18, O: 0.11, N: 0.004,
wrought pieces H: 0.024, Ti: balance
(ASTM grade 23)
Table 2   Printing parameters as indicated by the manufacturers
EBM
Environment vacuum
Temperature >700°C
Power 4,000 W
Powder 40-110 mm
Layer thickness 0.1 mm
allowed to accumulate in humans, gingivitis develops around teeth and
mucositis develops around Ti dental implants. In both cases, inflammation
was reduced when biofilms were removed or suppressed. This and subse-
quent reports suggested a causal relationship between bacterial biofilms
and implantitis. In both periodontal disease and implantitis, inflammation
was reduced when biofilm was removed, or its growth prevented or slowed.
In 2015, a systematic review by Renvert and Polysois confirmed a connec-
tion between biofilms and peri-implant disease [18]. A more recent review
of treatment planning confirmed that biofilms caused peri-implant disease
and cautioned that successful treatment of peri-implantitis was difficult and
depended on many factors, including implant site and patient motivation.
The literature on peri-implant disease continues to evolve. Kotsakis and
Olmedo [19] cautioned against viewing peri-implant disease as equivalent
to periodontal disease. They report that dissolution of Ti alters the micro-
biome around the implant and consequently that the biofilm is different,
which might explain why successful treatment appears to be more difficult
than for periodontal disease [20].
Although there is a substantial need for information on the biocom-
patibility of alloys fabricated by AM, few studies have examined the
relationship between peri-implant disease and surface morphology of
implants made by AM. Koike and colleagues [Koike M et al., TMS 2019
Proceedings, Springer, 827-36, 2019.] have published studies of biofilm
formation on various alloys, including Ti alloys made by EBM equipment.
The present study focused on accumulation of Streptococcus mutans (S.
mutans) biofilm on Ti-6Al-4V alloy and compared biofilm adhering to
Ti-6Al-4V alloy plates fabricated with EBM, LBM, and conventional lost-
wax casting.
Materials and Methods
Materials used
The Ti alloy most widely used for biomedical applications, Ti-6Al-4V alloy,
was employed to make specimens for examining biofilm accumulation.
The spherical alloy powders Ti-6Al-4V ELI (extra-low interstitial, ASTM
Grade 23, Arcam AB, Gothenburg, Sweden) and Ti-6Al-4V (ASTM Grade
5, EOS GmbH, Krailling, Germany) were fabricated by EBM and LBM,
respectively. The chemical compositions of the powders and wrought
alloys are summarized in Table 1. Note that the main difference is the lower
levels of interstitial elements in the powder alloys: the percentages of H, N,
and O in Ti-6Al-4V ELI are much lower than in Ti-6Al-4V. Although tita-
nium alloys with lower interstitial content are superior in some mechanical
characteristics, such a minimal difference in composition should not affect
their physical properties. Table 1 details the chemical composition of the
alloys, as detailed by the manufacturers (wt.%).
Particle and surface characterization
A scanning electron microscope (SEM; S-4000, Hitachi High-Technolo-
gies Corp., Tokyo, Japan) was used to compare the sizes of assemblies of
particles and to characterize the microstructure of sintered surfaces before
and after coverage by biofilm. The particles were compared and catego-
rized by size and the microstructure of sintered powders examined and
Manufacturer
ArcamAB
Gothenburg, Sweden
EOS GmbH, Krailling,
Germany
Titanium Industry, Hillsboro,
TX, USA
LBM
gas
Room
100-1,000 W
20-40 mm
0.02-0.06 mm
described. A Multisizer 4e Coulter Counter (Beckman Coulter Life Sci-
ences, Brea, CA, USA) was used to determine the number and volume of
particles as a function of particle diameter. Before the biofilm experiments,
surface roughness (Sa: µm) was evaluated in randomly chosen areas (1.112
× 1.116 mm) on two disks from each group of disks. The roughness of
cast and sintered surfaces was determined with a coherence scanning inter-
ferometer (CSI: VS 1550, Hitachi High-Technologies Corp.). Specimens
were ranked from most to least rough based on Sa values.
Fabricating AM and cast specimens
AM specimens
Two different AM machines were used to fabricate the two groups of AM
alloy specimens. EBM specimens were fabricated with the Arcam AB
machine (Arcam A2, Arcam AB), and LBM specimens were fabricated
with the EOS machine (Eosint 270, EOS GmbH). For each of these groups,
10 disk-shaped specimens (10 mm in diameter × 2 mm in thickness) were
fabricated. All disks were built up in the direction parallel to top of the disk
surface. The printing parameters for each AM machine are shown in Table
2. As for the AM specimens, no acid or solvent cleaning nor fine abrasion
was used to treat the as-fabricated surfaces.
Cast specimens
Pieces of alloy that were to be melted for casting were cut from a wrought
Ti-6Al-4V ELI (ASTM 23) alloy cylindrical rod (Titanium Industry, Hill-
sboro, TX, USA). A third group of 10 disks with the same dimensions was
made by using a centrifugal casting machine (Ticast Super R, Selec, Co.,
Ltd., Osaka, Japan). These disks were cast in an MgO mold (Selevest CB,
Selec, Co., Ltd.). All specimens were tested in the as-fabricated condition,
i.e., no acid or solvent cleaning nor fine abrasion was used to treat the
as-fabricated surfaces.
Controls
Ten Teflon disks of the same dimensions were cut and polished with 1,500-
grit silicon carbide paper and used as a negative control.
Preparation of bacterial suspensions
S. mutans (ATCC25175) was pre-cultured in 5 mL of brain-heart infusion
medium (BHI, Becton, Dickinson and Company, Tokyo, Japan) overnight
at 37°C under aerobic conditions. After pre-culture, the number of bacteria
in the pre-cultured bacterial suspension was approximately 1.8 × 109 CFU/
mL.
Bacterial adherence and biofilm formation on alloy and Teflon disks
All specimens were tested in the as-fabricated condition. Each specimen
was rinsed in ethanol and then in deionized water. After drying completely,
each disk was UV sterilized and placed in a well with 1 mL of BHI solu-
tion. The BHI solution also contained 5% sucrose (1st grade, Waco Pure
Chemical Industries Ltd., Osaka, Japan) and 5% S. mutans. Each well was
incubated for 4 h at 37°C, under anerobic conditions. These conditions
were produced with an oxygen absorber-CO2 generator (AnaeroPack-
Anaero, Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan).
 Figure 1

141

A B

Figure 2

Fig. 1 SEM images of a typical Ti-6Al-4V ELI alloy (A) and Ti-6Al-4V alloy (B) powder
Ti-6Al-4V ELI for EBM Ti-6Al-4V for LBM

A B
Count

Ti-6Al-4V ELI Ti-6Al-4V

Diameter of the powder particles used (m)

C D
Volume (%)

Ti-6Al-4V ELI Ti-6Al-4V

Diameter of the powder particles used (m)
Fig. 2 Frequency of number (A and B) and volume percent (C and D) of alloy particles as a function of alloy diameter

Evaluation of biological activity on alloy disks and controls
Each disk was washed twice in distilled water by using sonication to
remove non-adhered and/or floating S. mutans. The number of viable S.
mutans cells on each disk was determined by using luminescence signals
(BacTiter-Glo Microbial Cell Viability Assay, Promega KK, Madison, WI,
USA). The assay quantifies ATP (adenosine triphosphate) present. ATP (an
indicator of metabolically active cells) was measured with a microplate-
type luminometer (AB-2350 Phelios, ATTO Corp., Tokyo, Japan).
Preparation of biofilm-covered specimens for SEM observation
Biofilm was prepared for SEM examination by using previously described
methods [21]. Each disk surface was examined with SEM after biofilm
accumulation.
Statistical analysis
The Levene test was performed for data on homoscedasticity, followed
by the Shapiro-Wilk test for ATP values (P > 0.05). Because the analysis
showed that the data were homoscedastic (P > 0.05), numeral data were
analyzed by using one-way ANOVA followed by the Tukey post hoc test
(α = 0.05, IBM SPSS Statistics ver. 23, Chicago, IL, USA).
Results
Surface morphology
The range of particle sizes in these powders is illustrated by the scan-
ning electron micrographs in Fig. 1. The frequency number and volume
of particles as a function of particle diameter are shown in Fig. 2. The
distribution of particle sizes might have affected surface porosity, a feature
we need to measure in future research, tweaking sintering variables might
have corrected for this, for example, adjusting power levels during sinter-
 Figure 3
142
A E
EBM Sa = 36.80 m EBM (×50)
B F
LBM Sa = 6.48 m LBM (×50)
C G
Cast Sa = 2.30 m Cast (×150)
D H
Teflon (×300)
Teflon Sa = 0.03 m
Fig. 3   Topographic images of disk surfaces before biofilm accumulation. Sa is the roughness (μm) of the surface area (1.115 mm × 1.112 mm) as determined by coherence scanning
interferometry (CSI) (A, B, C, and D). SEM images show the surface of the disks before (E, F, G, and H) and after (I, J, K, and L) biofilm accumulation
ing might have been a simple way to eliminate unwanted porosity.
The differences in surface roughness are most vividly seen in CSI
topographic images (which emphasize the z-axis) of the three types of
specimens and the control (Fig. 3A-D). Figure 3 (E, F, and G) shows typi-
cal SEM images of the surface of the Ti-6Al-4V alloy specimens obtained
with EBM, LBM, and lost-wax casting. The figure also shows a surface
image of the Teflon negative control (Fig. 3H). These results are not what
would expect. The expectation is that larger amounts of biofilm will be
I
Biofilm on EBM (×100)
J
Biofilm on LBM (×100)
K
Biofilm on cast (×100)
L
Biofilm on teflon (×100)
attached to rougher surfaces. Also surprising is the observation that the
smooth and highly hydrophobic Teflon holds nearly as much biofilm as
the alloys. Importantly, there is no evidence to support the hypothesis
that roughness inherent to a surface produced by build up of consecutive
micro-step layers will better support attachment of the biofilm. Among the
four surfaces, the EBM specimen is exceptionally rough and has a more
rippled look than the other three specimen types. Alloy particles, some
half melted, are present on the surface; some appear welded to the surface
 Figure 4
140
120
(% relative to the control)
Mean ATP luminescence
100
80
60
40
20
0 0
EBM
Alloys and controls used
Fig. 4   Biofilm accumulation and surface roughness of the alloys and control substrates
below, while others appear to be physically locked to the surface by other
particles. Although not immediately obvious in the SEM image, close
inspection of the surfaces of the LBM specimen, cast specimen, and Teflon
control revealed that all were clearly smoother than the EBM surfaces.
The “rippled look” seen in the SEM images of the EBM specimen is also
evident in CSI images (Fig. 3A). The quantitative roughness value (Sa)
for the EBM specimen was 36.80 µm, whereas the Sa values for the LBM
specimen, cast specimen, and Teflon negative control were 6.48 µm, 2.30
µm, and 0.03 µm, respectively. The Sa values are thus ranked as EBM >
LBM > cast > Teflon.
Biological activity on alloys
The bar graph in Fig. 4 shows the quantitative expression of biofilm accu-
mulation, i.e., mean ATP luminescence values, expressed as a percentage
of the Teflon negative control. ATP values are proportional to the number
of biologically active biofilm cells in the accumulated biofilm. There was
no significant difference in the amount of accumulated biofilm in relation
to specimen type (P > 0.05). In Fig. 4, the surface roughness value, Sa, for
each specimen type is plotted in a line graph. Surface roughness and the
amount of biofilm accumulation were not correlated.
Discussion
To be successful, a dental implant must simultaneously become osseoin-
tegrated to bone as rapidly as possible and resist formation and growth
of oral biofilms that lead to peri-implant disease. If rigorous procedures
are followed, untreated Ti implants will osseointegrate [22]. However, in
the oral environment, biofilm comprises multiple species [23]. Therefore,
biofilms have displaced bacteria as mediators of oral disease on implant
surfaces. Within biofilm, several bacteria species work together to opti-
mize their survival in niches in the oral environment. At start of the 21st
century, researchers came to widely recognize that biofilms evolved to be
successful in niches within oral spaces that lacked free swimming bacte-
ria. An example of this is the strong resistance of biofilms to antibiotics.
For two decades, scientists have been aware that biofilms seldom form
on clean surfaces. Within seconds, an acquired pellicle of proteins from
saliva covers any surface that is exposed to the oral environment [24,25].
However, the potential roles for this pellicle in inhibiting biofilm have not
been clarified.
As awareness increased that metal implants were being colonized by
disease-causing biofilms, researchers attempted to identify which surfaces
resisted attachment and which did not. For example, hydrophilic surfaces
resisted attachment, while rough surfaces did not [26-29]. Implant design-
ers were faced with the problem that a surface suited for one purpose was
ill-suited for another. Roughness and nano-topologies designed to promote
osteogenesis simultaneously produce surfaces prone to attachment by bio-
films. Chemically changing an alloy (by adding Ag or Zn, for example) can
make them bactericidal but may inhibit cells (e.g., osteoblasts) that need to
be active for osseointegration. Schemes that deliver antibiotics, chlorhexi-
dine, and antimicrobial peptides to the surface are also being explored [30].
However, because such bactericidal agents are gradually depleted, they
become less effective with time. Finally, biomimetic approaches have been
143
40
ATP assey Roughness
Roughness (Sa:m)
30
20
10
LBM Cast Teflon
suggested [31,32]. One such idea is to change only the surface topography,
to make it “anti-fouling,” that is, to create a surface that is so hydrophobic
that water soluble bacteria will run off before they can attach.
In the present study, a monoculture of S. mutans was used as the oral
bacteria in biofilm testing. An acid-loving, acid-producing bacteria, S.
mutans has long been regarded as the best candidate in human cariogen-
esis. S. mutans is also well known to form biofilms [33]. Few studies have
examined the interaction between oral bacteria and implant materials. The
most recent [34] used a method similar to the present study: the research-
ers placed cultures of nine species of oral bacteria, each separately as a
monoculture, on Ti thin film substrates or on amorphous carbon thin film
substrates. They incubated the bacteria in human saliva and mycoplasma
and found that, depending on the bacteria species used, adhesion was
affected by the chemistry of the implant material, its roughness, and the
media in which it was incubated. That study [34] and the present study are
the only attempts to directly investigate the effect of Ti alloy on attachment
and growth.
The present study showed the specific results for adhesion of S. mutans
biofilm on Ti-6Al-4V alloy. Instead of a mixture of many bacteria spe-
cies, the tested biofilm consisted of a single bacterial species. S. mutans
was selected because it is an important causative agent within dental
biofilm. A mixed-species biofilm may behave very differently in vivo,
and interactions within a biofilm produced by multiple bacterial species
may be complicated. Interestingly, a previous study of the effect of alloy
constituents on single-species biofilm accumulation [Koike M et al., TMS
2019 Proceedings, Springer, 827-36, 2019] reported that the amount of
biofilm formed was reduced when the substrate alloy contained a cytotoxic
element like palladium (Pd). This suggests that biofilm accumulation can
be controlled or minimized by appropriate alloy selection. To extend the
present study, the interaction between various dental materials and biofilms
produced by mixed species of bacteria should be investigated.
The present results describe biofilm accumulation on Ti-6Al-4V alloy
substrates over a rather short period of time: 4 h. Future tests should include
longer durations of biofilm growth, to account for the fact that bacteria in
a biofilm have different effects than the same bacteria in isolation. In the
presence of a biofilm overlay, the effect of a substrate material on biofilm
accumulation may be significantly reduced.
In biofilm formation, microorganisms such as S. mutans are initially in
planktonic mode in saliva. Once a device is seated intraorally it quickly
becomes coated with mucins and/or other proteins. This coated surface
facilitates adhesion of microorganisms by cell-surface interaction, which
is followed by development of biofilm by cell-to-cell interaction [35,36].
Biofilm accumulation of S. mutans on Ti-6Al-4V alloy fabricated by EBM
or LBM did not significantly differ from that on lost-wax casting; neverthe-
less, the alloy specimens had different surface roughness values. The Sa
value of the Ti-6Al-4V alloy specimen by EBM was greater than 35 µm,
whereas the values for the other alloys and the Teflon control were less
than 3 µm. Although the surfaces of the additive manufactured Ti-6Al-4V
alloy EBM specimen were rougher than those of the other cast alloys, this
roughness, unexpectedly, did not increase biofilm accumulation. This is
because that the surface of the EBM specimens was rougher, but more cur-
vaceous and smoother configuration than that of LBM and cast specimens
144
with less rough but more coarser configurations. In addition, the amount
of biofilm formed on these specimens after 4 h of accumulation did not
significantly differ. However, the results might be different for longer
experiments. In addition, examination of biofilm-covered EBM specimens
with the roughest surfaces—those where the biofilm appeared to attach
itself mechanically, especially by penetrating microscopic crevices—
indicated that biofilm removal would be exceedingly difficult. Lastly, the
bulky biofilm formed on the EBM specimen is a concern. Removing such
biofilm from the EBM surfaces could prove difficult. Therefore, because
the surface properties were affected by the type of machine, the parameters
used, and the materials, among other factors, surface treatments should
be selected after considering the dental applications. Further research on
EBM applications is needed to reduce the surface roughness of devices
produced by this technique.
Acknowledgments
This study was partially supported by a Grant-in Aid for Scientific Research
(C) from the Japan Society for the Promotion of Science (19K10235).
Conflict of interest
The authors declare no potential conflicts of interest with respect to the
research, authorship, or publication of this article.
References
  1. Koike M, Greer P, Owen K, Lilly G, Murr LE, Gaytan SM et al. (2011) Evaluation of
titanium alloys fabricated using rapid prototyping technologies-electron beam melting and
laser beam melting. Materials (Basel) 4, 1776-1792.
  2. Murr LE (2018) A metallographic review of 3D printing/additive manufacturing of metal
and alloy products and components. Metallogr Microst Anal 7, 103-132.
 3. Yuan L, Ding S, Wen C (2018) Additive manufacturing technology for porous metal
implant applications and triple minimal surface structures: a review. Bioact Mater 4, 56-70.
  4. Sarker A, Leary M, Fox K (2020) Metallic additive manufacturing for bone-interfacing
implants. Biointerphases 15, 050801.
  5. Nicholson JW (2020) Titanium alloys for dental implants: a review. Prosthesis 2, 100-116.
  6. Kaur M, Singh K (2019) Review on titanium and titanium based alloys as biomaterials for
orthopaedic applications. Mater Sci Eng C Mater Biol Appl 102, 844-862.
  7. Koike M, Martinez K, Guo L, Chahine G, Kovacevic R, Okabe T (2011) Evaluation of
titanium alloy fabricated using electron beam melting system for dental applications. J
Mater Process Tech 211, 1400-1408.
  8. Joshi GV, Duan Y, Neidigh J, Koike M, Chahine G, Kovacevic R et al. (2013) Fatigue
testing of electron beam‐melted Ti‐6Al‐4V ELI alloy for dental implants. J Biomed Mater
Res B Appl Biomater 101, 124-130.
  9. Chu TG, Khouja N, Chahine G, Kovacevic R, Koike M, Okabe T (2016) In vivo evaluation
of a novel custom-made press-fit dental implant through electron beam melting® (EBM®).
Int J Dent Oral Sci 3, 358-365.
10. Kim JH, Kim MY, Knowles JC, Choi S, Kang H, Park SH et al. (2020) Mechanophysical
and biological properties of a 3D-printed titanium alloy for dental applications. Dent Mater
36, 945-958.
11. Tunchel S, Blay A, Kolerman R, Mijiritsky E, Shibli JA (2016) 3D printing/additive manu-
facturing single titanium dental implants: a prospective multicenter study with 3 years of
follow-up. Int J Dent 2016, 8590971.
12. Moraschini V, Poubel LA, Ferreira VF, Barboza Edos S (2015) Evaluation of survival and
success rates of dental implants reported in longitudinal studies with a follow-up period of
at least 10 years: a systematic review. Int J Oral Maxillofac Surg 44, 377-388.
13. Derks J, Tomasi C (2015) Peri‐implant health and disease. A systematic review of current
epidemiology. J Clin Periodontol 42, S158-171.
14. Atieh MA, Alsabeeha NH, Faggion CM Jr, Duncan WJ (2013) The frequency of peri-
implant diseases: a systematic review and meta-analysis. J Periodontol 84, 1586-1598.
15. Derks J, Håkansson J, Wennström JL, Tomasi C, Larsson M, Berglundh T (2015) Effective-
ness of implant therapy analyzed in a swedish population: early and late implant loss. J
Dent Res 94, 44S-51S.
16. Fu JH, Wang HL (2020) Breaking the wave of peri-implantitis. Periodontol 2000 84, 145-
160.
17. Pontoriero R, Tonelli MP, Carnevale G, Mombelli A, Nyman SR, Lang NP (1994) Experi-
mentally induced peri‐implant mucositis. a clinical study in humans. Clin Oral Implants
Res 5, 254-259.
18. Renvert S, Polyzois I (2015) Risk indicators for peri‐implant mucositis: a systematic litera-
ture review. J Clin Periodontol 42, S172-S186.
19. Kotsakis GA, Olmedo DG (2021) Peri-implantitis is not periodontitis: scientific discoveries
shed light on microbiome-biomaterial interactions that may determine disease phenotype.
Periodontol 2000 86, 231-240.
20. Daubert D, Pozhitkov A, McLean J, Kotsakis G (2018) Titanium as a modifier of the peri-
implant microbiome structure. Clin Implant Dent Relat Res 20, 945-953.
21. Kinden DA, Brown MF (1975) Technique for scanning electron microscopy of fungal
structures within plant cells. Phytopathology 65, 74-76.
22. Brånemark P, Adell R, Albrektsson T, Lekholm U, Lundkvist S, Rockler B (1983) Osseoin-
tegrated titanium fixtures in the treatment of edentulousness. Biomaterials 4, 25-28.
23. Fröjd V, Chávez de Paz L, Andersson M, Wennerberg A, Davies JR, Svensäter G (2011) In
situ analysis of multispecies biofilm formation on customized titanium surfaces. Mol Oral
Microbiol 26, 241-252.
24. Teughels W, Van Assche N, Sliepen I, Quirynen M (2006) Effect of material characteristics
and/or surface topography on biofilm development. Clin Oral Implant Res 17, 68-81.
25. Sterzenbach T, Helbig R, Hannig C, Hannig M (2020) Bioadhesion in the oral cavity and
approaches for biofilm management by surface modifications. Clin Oral Investing 24,
4237-4260.
26. Alghamdi HS, Jansen JA (2020) The development and future of dental implants. Dent
Mater J 39, 167-172.
27. Wang Q, Zhou P, Liu S, Attarilar S, Ma RL, Zhong Y et al. (2020) Multi-scale surface
treatments of titanium implants for rapid osseointegration: a review. Nanomaterials (Basel)
10, 1244-1271.
28. Linklater DP, Baulin VA, Juodkazis S, Crawford RJ, Stoodley P, Ivanova EP (2021)
Mechano-bactericidal actions of nanostructured surfaces. Nat Rev Microbiol 19, 8-22.
29. Matos GRM (2021) Surface roughness of dental implant and osseointegration. J Maxillofac
Oral Surg 20, 1-4.
30. Mas-Moruno C, Su B, Dalby MJ (2019) Multifunctional coatings and nanotopographies:
toward cell instructive and antibacterial implants. Adv Healthc Mater 8, e1801103.
31. Albertini M, Fernandez-Yague M, Lázaro P, Herrero-Climent M, Rios-Santos JV, Bullon
P et al. (2015) Advances in surfaces and osseointegration in implantology. Biomimetic
surfaces. Med Oral Patol Oral Cir Bucal 20, E316-E325.
32. Al-Zubaidi SM, Madfa AA, Mufadhal AA, Aldawla MA, Hameed OS, Yue XG (2020)
Improvements in clinical durability from functional biomimetic metallic dental implants.
Front Mater 7, 106.
33. Deng DM, Hoogenkamp MA, Exterkate RA, Jiang LM, van der Sluis LW, Ten Cate JM et
al. (2009) Influence of streptococcus mutans on enterococcus faecalis biofilm formation. J
Endod 35, 1249-1252.
34. Almaguer-Flores A, Ximénez-Fyvie LA, Rodil SE (2010) Oral bacterial adhesion on amor-
phous carbon and titanium films: effect of surface roughness and culture media. J Biomed
Mater Res B Appl Biomater 92, 196-204.
35. Palmer RJ Jr, White DC (1997) Developmental biology of biofilms: implications for treat-
ment and control. Trends Microbiol 5, 435-440.
36. O’Toole G, Kaplan HB, Kolter R (2000) Biofilm formation as microbial development.
Annu Rev Microbiol 54, 49-79.