UNIVERSIDADE ESTADUAL PAULISTA
“JÚLIO DE MESQUITA FILHO”
Instituto de Ciência e Tecnologia
Campus de São José dos Campos
ORIGINAL ARTICLE DOI: https://doi.org/10.4322/bds.2025.e4663
1
Braz Dent Sci 2025 Jan/Mar;28 (1): e4663
This is an Open Access article distributed under the terms of the Creative Commons Attribution license (https://creativecommons.org/licenses/by/4.0/), which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
In vitro investigation of flower-like micro-nano topography
modifications to improve titanium implant surface properties
Investigação in vitro de modificações em formato de flor de micro-nano topografia para melhorar as propriedades da
superfície de implantes de titânio
Nadia KARTIKASARI1 , Ratri Maya SITALAKSMI1 , Karina MUNDIRATRI , Salsabilla Eliya ANDARU1 ,
Aisyah Rachmadani Putri GOFUR1 , Harry LAKSONO1
1 - Universitas Airlangga, Faculty of Dental Medicine, Department of Prosthodontics, Surabaya, Jawa Timur, Indonesia.
How to cite: Kartikasari N, Sitalaksmi RM, Mundiratri K, Andaru SE, Gofur ARP, Laksono H. In vitro investigation of ower-
like micro-nano topography modications to improve titanium implant surface properties. Braz Dent Sci. 2025;28(1):e4663.
https://doi.org/10.4322/bds.2025.e4663
ABSTRACT
Objective: The surface modication of titanium implants has demonstrated signicant potential in enhancing
the surface characteristics of the implants, improving their performance, and minimizing the risk of infection.
This study examines the advantages of combining acid etching and alkaline heat treatment to enhance titanium
implant surfaces and evaluate their antibacterial effectiveness. Material and Methods: Titanium was divided
into four groups: machine surface (MA), samples subjected to a single acid etching (MR), and samples that
experienced both a single acid etching and two distinct alkaline heat treatment protocols (MN1 and MN2). The
surface properties (topography, chemical composition, surface roughness, and wettability) and antibacterial
evaluation against Porphyromonas gingivalis were evaluated. The statistical signicance of differences among the
groups was determined using ANOVA and Tukey’s Post-Hoc Test (P<0.05). Results: The MN group signicantly
transformed titanium surfaces into a micro-nano ower-like topography with hydroxyl and sodium titanate
structures. The highest surface roughness was shown in MN1 (Sa= 7,58 ± 0,64; Sq 9,78 ± 0,8) (P<0.05) and
the lowest wettability were shown in MN2 (25,75 ± 8,2) (P<0.05). Conversely, MN2 exhibited the lowest
wettability, measured at 25.75 ± 8.2 (P < 0.05). Antibacterial assessments revealed a notable reduction in the
growth of Porphyromonas gingivalis on the MN2 modied surfaces at 27.43 ± 10.43% (P < 0.05). Conclusion:
The combination of acid etching and alkaline heat treatment has shown the ability to create a micro-nano
surface with outstanding properties and signicant antibacterial effects. This advancing titanium dental implant
technology presents a valuable alternative for improving clinical outcomes in dental applications.
KEYWORDS
Antibacterial agents; Dental implant; Medicine; Surface properties; Titanium.
RESUMO
Objetivo: A modicação da superfície dos implantes de titânio tem demonstrado potencial signicativo na
melhoria das características da superfície dos implantes, melhorando seu desempenho e minimizando o risco de
infecção. Este estudo examina as vantagens da combinação de ataque ácido e tratamento térmico alcalino para
melhorar as superfícies de implantes de titânio e avaliar sua ecácia antibacteriana. Materiais e métodos: O
titânio foi dividido em quatro grupos: superfície de máquina (MA), amostras submetidas a um único ataque ácido
(MR) e amostras submetidas a um único ataque ácido e dois protocolos distintos de tratamento térmico alcalino
(MN1 e MN2). Foram avaliadas as propriedades da superfície (topograa, composição química, rugosidade e
molhabilidade) e a avaliação antibacteriana contra Porphyromonas gingivalis. As diferenças estatísticas signicantes
entre os grupos foram determinadas por meio de ANOVA e teste post-hoc de Tukey (p<0,05). Resultados: O
grupo MN transformou signicativamente as superfícies de titânio em uma micro-nanotopograa em forma de
2
Braz Dent Sci 2025 Jan/Mar;28 (1): e4663
Kartikasari N et al.
In vitro investigation of flower-like micro-nano topography modifications to improve titanium implant surface properties
Kartikasari N et al. In vitro investigation of flower-like micro-nano topography
modifications to improve titanium implant surface properties
INTRODUCTION
Titanium implants are widely recognized
as the gold standard in dental implantology
due to their exceptional biomechanical and
biocompatible properties [1-4]. However, the
peri-implantitis poses a significant challenge
in the application of titanium implants [1]. As
titanium is classied as a bioinert material, it
possesses no intrinsic antimicrobial properties.
Consequently, this can facilitate bacterial
colonization, which may obstruct the integration
with adjacent tissues, prolong the postoperative
recovery period, and compromise the overall
health, aesthetics, and functionality of dental
implant restorations [2].
One of the key factors contributing to peri-
implantitis associated with titanium implants is the
colonization of bacteria. Microorganisms such as
Prevotella intermedia
,
Fusobacterium nucleatum
,
and
Porphyromonas gingivalis
play an essential
roles in its development and progression through
biolm formation [5]. Once the titanium surface
is exposed to the oral environment, bacteria
begin to adhere to the titanium surface and form
a biolm. These biolms are characterized by
complex three-dimensional structures composed
of extracellular polymeric substances that
effectively protect bacterial clusters, enabling
their long-term persistence [6]. This protective
layer shields bacteria from antibacterial agents,
antibiotics, nutrient deprivation, and immune
responses, making them more resistant to
treatment [1]. The use of antibacterial agents can
further exacerbate bacterial resistance, creating
a signicant challenge in effectively managing
peri-implant infections.
Recent advancements in the surface
modication of titanium implants at the micro-
nanometer scale topograhy level have gained
signicant attention due to their ability to alter both
surface topography and chemical composition. The
alteration of the surface topography, roughness,
chemical composition, wettability, and electric
charge, play a crucial role in bacterial adhesion
and biolm formation on implant surfaces [6-10].
Consequently, optimizing these parameters
may improve the antibacterial efficacy of the
implants [4]. Although these properties can
be manipulated to mitigate biofilm formation,
it remains challenging to control external
environmental factors such as pH, temperature,
bacterial species, morphology, and size [7]
The effectiveness of bactericidal surfaces is
inuenced by several factors, including topography,
structural height, radius, and spacing. Surfaces
designed to prevent bacterial adhesion can be
categorized as either bactericidal or anti-biofouling.
Bactericidal surfaces compromise the integrity of the
bacterial cell wall, ultimately resulting in cell death.
In contrast, anti-biofouling surfaces inhibit and deter
bacterial attachment through specic topographical
or chemical properties [1]. Micro-topographies
typically possess antifouling characteristics by
inhibiting bacterial adhesion, whereas nano-
topographies demonstrate antibacterial capabilities
by directly rupturing bacterial membranes and
resulting in cell death [11,12]. Implant surface
modification significantly enhance the contact
adhesion area, thereby improving the bactericidal
properties of surfaces compare to the smooth
or no treatment surface [13]. This micro-nano
topography leads to mechanical damage on
bacterial membranes through direct contact,
generating physical stress that contributes to
enhanced bactericidal effects [1].
Acid etching represents a surface modication
technique wherein titanium implants are
immersed in strong acidic solutions, including
hydrochloric acid (HCl), nitric acid (HNO3),
sulfuric acid (H2SO4), or hydrouoric acid (HF),
under controlled conditions [2,8]. This procedure
or, com estruturas de hidroxila e titanato de sódio. A maior rugosidade supercial foi observada em MN1 (Sa
= 7,58 ± 0,64; Sq = 9,78 ± 0,8) (P < 0,05) e a menor molhabilidade foi observada em MN2 (25,75 ± 8,2)
(P < 0,05). Por outro lado, MN2 apresentou a menor molhabilidade medida em 25,75 ± 8,2 (P < 0,05). As
avaliações antibacterianas revelaram uma redução notável no crescimento de Porphyromonas gingivalis nas
superfícies modicadas com MN2 em 27,43 ± 10,43% (P < 0,05). Conclusão: A combinação de ataque ácido
e tratamento térmico alcalino demonstrou a capacidade de criar uma micro-nano superfície com propriedades
excepcionais e efeitos antibacterianos signicativos. Esta tecnologia avançada de implantes dentários de titânio
apresenta uma alternativa valiosa para melhorar os resultados clínicos em aplicações odontológicas.
PALAVRAS-CHAVE
Agentes antibacterianos; Implante dentário; Medicina; Propriedades de superfície; Titânio.
3
Braz Dent Sci 2025 Jan/Mar;28 (1): e4663
Kartikasari N et al.
In vitro investigation of flower-like micro-nano topography modifications to improve titanium implant surface properties
Kartikasari N et al. In vitro investigation of flower-like micro-nano topography
modifications to improve titanium implant surface properties
yields a micro-rough topography characterized
by uniformly distributed ridges and pits [13-20].
Although these micron-scale topographies do not
possess inherent bactericidal properties, they
inuence the interactions between bacteria and
surfaces, thereby reducing bacterial adhesion and
obstructing biolm formation [8]. Conversely,
alkali-heat treatment is a method in which
titanium is immersed in strong alkaline solutions,
such as sodium hydroxide (NaOH) or potassium
hydroxide (KOH), followed by exposure to
elevated temperatures in a furnace for a specied
duration [2,8,13-16]. This treatment facilitates
the development of titanium oxide nanospikes,
which exhibit bactericidal potential through
the mechanical disruption of bacterial cell
membranes [21]. Furthermore, titanium nano
surfaces characterized by dense, anisotropically
patterned nano spikes demonstrate antimicrobial
activity by repelling Gram-positive cocci and
effectively eliminating Gram-negative bacilli [18].
Implant surface modifications were used
to achieve micro- and nano-scale topography
in order to increase biological efficacy. Prior
research indicates that nano-scale topographies
can address the limitations associated with micro-
scale structures by facilitating cell spreading and
signicantly promoting osteoblast differentiation
and osseointegration [22]. In addition, micro-
nano hierarchical surfaces promote improved
osteoblast adhesion and proliferation by closely
resembling the topography natural bone [23].
Micro-nano hierarchical surfaces signicantly
enhance the adhesion and proliferation of
osteoblasts by effectively mimicking the
topographical characteristics of natural bone [24].
Their results indicated that the incorporation of
nano-topography to micro-topography led to
enhanced cell proliferation while preserving
mechanical interlocking and promoting
differentiation [2]. In light of these results, we
would like to integrate the micro topography
produced by acid etching and the alkaline heat
treatment’s nano topography. This study aims to
assess the efcacy of the combined application of
acid etching and alkali-heat treatment in terms
of its surface properties (topography, chemical
composition, surface roughness, and wettability)
and antibacterial effectiveness. Our hypothesis is
that the combination of acid etching and alkaline
heat treatment may induce suitable titanium
surface properties and enhance antibacterial
effectiveness.
MATERIALS AND METHODS
This laboratory experiment was an in vitro
study employing a post-test-only control group
design. Ethical approval for the research was
obtained from the Health Research Ethical
Clearance Committee of the Faculty of Dental
Medicine at Universitas Airlangga (No. 0870/
HRECC.FODM/VIII/2024). The study focused
on examining the topography, surface roughness,
and chemical composition that dene the surface
characteristics at the Department of Materials and
Metallurgical Engineering, Institute of Technology
Sepuluh Nopember (ITS). Additionally, the
wettability and antimicrobial activity of the
materials were assessed against
Porphyromonas
gingivalis
at the Research Center of the Faculty
of Dental Medicine, Universitas Airlangga
(Figure 1). Each sample were evaluated triplicate
and the number of samples of each group is three.
Preparation of titanium surface
This study employed commercially pure-
grade I titanium plates, which were purchased
from PT Special Metals, Jakarta, Indonesia. The
titanium plates were subjected to laser cutting to
create square-shaped samples measuring 10 mm
x 10 mm x 5 mm. Titanium were divided into four
main group: machine surface (MA), Ti samples
underwent a single acid etching (MR), Ti sample
underwent single acid etched and two alkaline
heat treatment protocols (MN1 and MN2). The
machine titanium (MA) underwent a thorough
cleansing process utilizing a series of ethanol
and distilled water, followed by ultrasonication.
The acid-etched surfaces were prepared by
immersed the MA titanium samples in a 67%
(w/w) sulfuric acid solution (Merck, Darmstadt,
Germany) at a temperature of 120°C for a
duration of 75 seconds. Upon completion of the
etching procedure, the titanium surfaces were
allowed to dry at room temperature and were
subsequently designated as micro-roughened
(MR) surfaces.
The combination of acid etching and alkaline
heat treatment were expected to produce micro-
nano roughened (MN). Two distinct alkaline heat
treatments were administered to the MR samples,
according to the previous protocol [13-16]. In
summary, the MR samples were boiled in a sodium
hydroxide (NaOH) solution (Merck, Darmstadt,
Germany) for a period of 24 hours. The alkaline
4
Braz Dent Sci 2025 Jan/Mar;28 (1): e4663
Kartikasari N et al.
In vitro investigation of flower-like micro-nano topography modifications to improve titanium implant surface properties
Kartikasari N et al. In vitro investigation of flower-like micro-nano topography
modifications to improve titanium implant surface properties
heat treatments were categorized as follows: the rst
treatment utilized a 5M sodium hydroxide solution
at 60°C, designated as micro-nano roughened
surface 1 (MN1), while the second treatment
involved a 10M solution at 90°C, designated as
micro-nano roughened surface 2 (MN2). Following
the boiling process, the titanium samples underwent
rinsing with distilled water, were air-dried overnight,
and then subjected to sintering in a furnace at 600°C
for one hour before being allowed to cool to ambient
temperature.
Scanning Electron Microscope-Energy Dis-
persive X-Ray (SEM-EDX)
The topography of the titanium samples
was assessed utilizing scanning electron
microscopy (SEM) with an FEI Inspect-S50
system (Oregon, USA). The SEM images of the
titanium samples were taken at magnications
of 10000x, 15000x, and 50000x to enable a
comprehensive examination of the surface
morphology. Furthermore, elemental analysis
of the titanium surfaces was carried out using
energy-dispersive X-ray spectroscopy (EDX),
which is incorporated into the SEM system.
Fourier transform infrared (FTIR) spectroscopy
Functional groups on the titanium surface
were analyzed using Fourier transform infrared
(FTIR) spectroscopy Thermo Scientic Nicolet
iS10 (Wisconsin, USA). The spectra were recorded
in the range of 4000–2000 cm-1. Background
correction was performed based on the surface
spectrum of MA.
X-ray diffraction (XRD)
The crystalline phase of each titanium
surface was analyzed using MiniFlex 600C
X-ray diffractometer (Tokyo, Japan). Titanium
discs were analyzed with Cu Kα radiation (λ =
1.5406 Å, 40 kV, 40 mA). Diffraction patterns
were collected in the 2θ range of 10° to 90°
Atomic Force Microscopy (AFM)
The surface roughness of the titanium
samples was assessed using an Atomic Force
Microscopy (AFM) device (Bruker-Nano N8
NEOS, Bruker Corp., Billerica, MA, USA). The
surface roughness parameters that were measured
included the arithmetical mean height (Sa), root
mean squared height (Sq), maximum height (Sz),
and maximum surface amplitude (St).
Wettability
The wettability of the titanium surface was
assessed by single-drop of 10 μL of distilled
water onto the titanium surface. The images of
the contact angle were obtained using a camera
Figure 1 - Research Diagram. Machine titanium grade 1 (MA) was prepared (10 mm x 10 mm x 5 mm). The treated titanium group was Ti samples
that underwent a single acid etching (MR), Ti samples underwent single acid etching and two alkaline heat treatment protocols (MN1 and
MN2). For the acid etched treatment, MA titanium samples were placed in a 67% (w/w) sulfuric acid solution at a temperature of 120°C for 75
seconds. Alkaline heat treatment was conducted using 5M sodium hydroxide solution at 60°C, designated as micro-nano roughened surface
1 (MN1), while the second treatment involved a 10M solution at 90°C, designated as micro-nano roughened surface 2 (MN2). The surface
properties and antibacterial effect were evaluated after the Ti sample preparation.
5
Braz Dent Sci 2025 Jan/Mar;28 (1): e4663
Kartikasari N et al.
In vitro investigation of flower-like micro-nano topography modifications to improve titanium implant surface properties
Kartikasari N et al. In vitro investigation of flower-like micro-nano topography
modifications to improve titanium implant surface properties
(Fujifilm, Tokyo, Japan). The contact angle
measurements were subsequently analyzed and
calculated using ImageJ 1.54g software developed
by National Institutes of Health and the Laboratory
for Optical and Computational Instrumentation
(LOCI, University of Wisconsin), USA).
Porphyromonas gingivalis
culture
Porphyromonas gingivalis
(ATCC 33277,
United Kingdom) was obtained from the culture
stock. The bacteria were rst grown in Tryptone
Soya Broth (TSB) and incubated anaerobically
at 37°C for 18–24 hours. Bacterial colonies were
then collected using a sterile loop and transferred
to 3 mL of Brain Heart Infusion (BHI) liquid
media, followed by incubation at 37°C for 18
hours. The bacterial suspension was standardized
to match a McFarland standard of 0.5 (1.5 ×
108 CFU/mL). The standardized suspension was
carefully pipetted and evenly spread onto the
surface of nutrient agar media.
Antibacterial activity
Titanium samples were incubated in
centrifuge tubes containing 5 mL of
Porphyromonas
gingivalis
suspension for 24 hours. Following the
incubation, the tubes were vortexed to dislodge
adherent bacteria from the titanium surface.
Subsequently, 0.1 mL of the resulting suspension
was plated onto Mueller Hinton Agar (MHA)
medium in petri dishes and incubated at 37°C
for 48 hours under anaerobic conditions. After
the incubation period, bacterial colonies were
enumerated to determine the bacterial load.
The bactericidal ratio was calculated using the
following formula:
(1) ( 2 )
(1)
% 100
CFUs CFUs
BR
CFUs
−
= ⋅
(1)
Where CFU(1) represents the colony-forming units
in the control group, and CFU(2) represents the
colony-forming units in the experimental group.
After completing the data collection, a
statistical analysis was conducted using SPSS
software 27 (IBM, Tokyo, Japan). The normality
of the data was assessed using the Kolmogorov-
Smirnov test (P>0.05), and homogeneity was
evaluated using Levene’s test (P>0.05). When
appropriate, One-Way ANOVA (P<0.05) was
used to assess signicant differences among the
groups. Following this, the Post Hoc Tukey’s
Honestly Significant Difference (HSD) test
(P<0.05) was performed to enable specific
comparisons between the groups.
RESULTS
The surface treatment applied to the titanium
modified its appearance, changing from the
original silver metallic hue to a dark grey following
acid etching. Conversely, the combination of acid
and alkaline heat treatments resulted in a light
brown-yellowish tint for the group combination
acid etching and alkaline heat treatment with
5M NaOH (MN1) and a dark brown color for the
group combination acid etching and alkaline heat
treatment with 10M NaOH (MN2) (Figure 2A).
The scanning electron microscopy (SEM) images
of the machine titanium treatment (MA) group
revealed a at surface characterized by a broad
groove (Figure 2B a-a’-a”). In contrast, the group
with acid etching treatment (MR) exhibited
titanium surfaces displaying a relatively uniform
distribution of numerous sharp ridges and pits,
which formed outer honeycomb-like grooves of
varying sizes (Figure 2B b-b’-b”). Both MN1 and
MN2 exhibited a nanoflower-like structure,
however, MN1 displayed additional lopodia-like
petal extensions radiating outward (Figure 2B
c-c’-c”), while MN2 presented a more compact
nanoower characterized by thicker petal-like
formations that were tightly clustered around the
center, featuring overlapping layers (Figure 2B
d-d’-d”). Elemental analysis indicated that both
the machine titanium (MA), titanium with acid
etching (MR), and MN1 groups were composed
of titanium (Ti) and oxygen (O), whereas MN2
contained titanium, oxygen, and sodium (Na).
In all groups, titanium was the predominant
component (Figure 2C).
The FTIR spectra analysis of titanium sam-
ples interpreted the chemical composition.
The MN1 and MN2 groups demonstrated
distinct hydroxyl (O–H) stretching peaks within
the 3700–3590 cm−1 range [25]. Specically,
MN1 exhibited a peak at 3629.1 cm−1, whereas
MN2 displayed a peak at 3638.1 cm−1 (Figure 3A,
lower panel). X-ray diffraction (XRD) patterns,
which serve as an indication of the chemical
composition of the samples, conrmed that the
MA group did not include titanium dioxide (TiO2)
and sodium titanate. Similarly, the MR group
displayed no detectable peaks associated with the
6
Braz Dent Sci 2025 Jan/Mar;28 (1): e4663
Kartikasari N et al.
In vitro investigation of flower-like micro-nano topography modifications to improve titanium implant surface properties
Kartikasari N et al. In vitro investigation of flower-like micro-nano topography
modifications to improve titanium implant surface properties
Figure 2 - Surface topography of the titanium surfaces. (A) Representative of the macroscopic features of the machine (MA), micro-roughed
(MR), micro-nano roughed 1 (MN1) and micro-nano roughed 2 (MN2) surfaces. (B) Representative scanning electron microscope (SEM) images in
each titanium surfaces at magnification 10.000x (a,b,c,d), 20.000x (a’,b’,c’,d’), and 50.000x (a”, b”,c”,d,”) (C) Representative of Energy Dispersive
X-Ray in each titanium surfaces (N=3).
7
Braz Dent Sci 2025 Jan/Mar;28 (1): e4663
Kartikasari N et al.
In vitro investigation of flower-like micro-nano topography modifications to improve titanium implant surface properties
Kartikasari N et al. In vitro investigation of flower-like micro-nano topography
modifications to improve titanium implant surface properties
phases of sodium titanate. This lack of detectable
peaks indicates that these compounds were absent
from the surfaces of the MA and MR groups. The
findings suggest that the surface treatments
applied to the MN samples effectively facilitated
the formation of rutile crystallite TiO2 structure
and sodium titanate, a process that did not occur
in the MA and MR groups (Figure 3B). Titanium
dioxide (TiO2) has been recognized for its
potential antibacterial properties [26,27], while
sodium titanate, an inorganic ion exchanger, has
also been reported to exhibit both antibacterial
and antifungal activities [28].
Following the modification of titanium
surfaces, all experimental groups exhibited an
increase in surface roughness. The parameters
Sa and Sq indicated that the MN1 group
demonstrated the highest roughness values, with
Sa and Sq measurements being 2.20 and 2.00
times greater, respectively, than those of the MA
group. Nevertheless, the roughness values for the
MN1 group were comparable to those observed
in the MR group. MN2 showed a similar surface
roughness with MR. No signicant differences
were detected in the Sz and St values across the
groups (Figure 4A). Water contact angles were
measured to assess hydrophilicity, serving as an
indicator of surface wettability. Contact angles
exceeding 90° are classied as hydrophobic, those
below 90° as hydrophilic, and measurements
falling beneath 10° as superhydrophilic [13]. In
this investigation, all groups exhibited hydrophilic
characteristics, with the MN1 and MN2 groups
displaying signicantly lower contact angles in
comparison to the other groups (Figure 4B).
Antibacterial activity against
Porphyromonas
gingivalis
was assessed by monitoring the
development of colonies characterized by a
Figure 3 - Chemical composition of the titanium surfaces. (A) Fourier transform infrared (FTIR) spectra of of the machine (MA), micro-roughed
(MR), micro-nano roughed 1 (MN1) and micro-nano roughed 2 (MN2) surfaces. (B) X-ray diffraction (XRD) features in each titanium surfaces. (N=3).
8
Braz Dent Sci 2025 Jan/Mar;28 (1): e4663
Kartikasari N et al.
In vitro investigation of flower-like micro-nano topography modifications to improve titanium implant surface properties
Kartikasari N et al. In vitro investigation of flower-like micro-nano topography
modifications to improve titanium implant surface properties
yellowish-white coloration (Figure 5A). The
MN group exhibited a statistically significant
difference compared to the MA and MR1 groups,
however, no signicant difference was observed
between the MN1 and MN2 groups (Figure 5B).
The highest bacterial ratio, utilized to evaluate
the percentage of bacterial death, was recorded
in the MN2 group. Furthermore, no signicant
disparities were found between the MR and MN1
groups (Figure 5B).
Figure 4 - Surface roughness and wettability of titanium surfaces. (A) Surface roughness of of the machine (MA), micro-roughed (MR), micro-
nano roughed 1 (MN1) and micro-nano roughed 2 (MN2) surfaces from Atomic Force Microscopy (AFM) analysis (first and second raw). Vertical
roughness parameter Sa, Sq, St, and Sz (third raw). (B) Water contact angle of the titanium surfaces. Data presented as means ± standard
deviation (SD) (N = 3). Different letters or asterisks indicate statistically significant differences between them (P < 0.05; Tukey’s honestly
significant difference [HSD] test. arithmetical mean height (Sa), root mean squared height (Sq), maximum height (Sz), and maximum surface
amplitude (St).
9
Braz Dent Sci 2025 Jan/Mar;28 (1): e4663
Kartikasari N et al.
In vitro investigation of flower-like micro-nano topography modifications to improve titanium implant surface properties
Kartikasari N et al. In vitro investigation of flower-like micro-nano topography
modifications to improve titanium implant surface properties
DISCUSSION
Surface modication is critical for enhancing
the topography and composition of implant
surfaces. The primary objectives of these
modications are to decrease wettability, and
increase surface roughness and antibacterial
properties, thereby facilitate osteoblast attachment
and ensure effective osseointegration [29]. The
most widely method for combination of surface
modication is acid etching. Additionally, alkali-
heat treatment may be implemented to further
refine surface characteristics. In this study, a
combination of micro-scale surface modication
through acid etching and alkali-heat treatment
was employed to generate a nano-structured
surface, resulting in a surface that integrates both
micro and nano-scale features. In comparison with
single micro or nano topography, hierarchical
micro-nano topography has a great deal of
potential to promote osteogenesis because it has a
combination of benets. The nano-scale structure
could increase protein adsorption, cell adhesion,
and ultimately osseointegration, while the micro-
scale structure may strengthen the interlocking
of the bone with the implant [22].
The acid-etched treatment produced
uniform, distinct ridges, and pits, resulting in
a honeycomb-like surface texture characterized
by grooves of varying sizes similar to prior
studies [13,15,20,30]. The previous research
indicated that alkaline-heat treatment generated
dense nanosized spikes. Notably, alkaline heat
with 5M NaOH displayed a relatively isotropic
distribution of nanospikes, whereas alkaline
heat with 10M NaOH exhibited an anisotropic
distribution [13-16]. The integration of acid
etching and alkaline-heat treatment yielded a
ower-like structure with distinct morphological
features. Combination of acid etched and alkaline
heat with 5M NaOH (MN1) presented lopodia-
like petal extensions radiating outward, while
combination of acid etched and alkaline heat
with 10M NaOh (MN2) showed a more compact
conguration with thicker, densely clustered petal-
like formations, accompanied by overlapping
Figure 5 - (A) Antibacterial activity against
Porphyromonas gingivalis
. Representative of the macroscopic features of bacterial growth in
the machine (MA), micro-roughed (MR), micro-nano roughed 1 (MN1) and micro-nano roughed 2 (MN2) surfaces. (B) The quantification of
the bacterial growth and bacterial ratio. Data presented as means ± standard deviation (SD) (N = 3). Different letters or asterisks indicate
statistically significant differences between them (P < 0.05; Tukey’s honestly significant difference [HSD] test.
10
Braz Dent Sci 2025 Jan/Mar;28 (1): e4663
Kartikasari N et al.
In vitro investigation of flower-like micro-nano topography modifications to improve titanium implant surface properties
Kartikasari N et al. In vitro investigation of flower-like micro-nano topography
modifications to improve titanium implant surface properties
layers. This ower-like topography, representing
a micro-nano structure, demonstrated signicant
antibacterial properties by imposing physical
stress on bacterial surfaces. The unique
architectural design produced shearing forces
during bacterial adhesion, resulting in substantial
membrane stress that mechanically disrupted
bacterial membranes, thereby enhancing physical
sterilization efcacy [1].
Hydroxyl structures have been identified
in the MN groups, a characteristic typically
associated with alkaline heat treatment. Hydroxyl
groups are recognized for their robust oxidative
properties, which contribute to the generation
of reactive oxygen species (ROS). These ROS
are significant contributors to bacterial cell
toxicity due to the induction of oxidative
stress [24]. Chemical analysis of the MN group
has revealed the presence of TiO2 and sodium
titanate. TiO2 is a material known for its diverse
physical and chemical properties, and it is widely
acknowledged for its strong antibacterial and
antifungal activities against both Gram-positive
and Gram-negative bacteria. This study identied
the rutile crystallite phase of TiO2 within the
MN group, a phase known for its stability and
exceptional antibacterial efficacy [25-27].
Furthermore, sodium titanate was also detected
in the MN group. Previous reports indicate that
sodium titanate enhances surface hydrophilicity
by reducing wettability. Surfaces that exhibit
high hydrophilicity particularly have a high
antibacterial activity [28,31]. The analysis of
the chemical composition was in line with the
ndings of the elemental study, which identied
Ti, O, and Na only within the MN2 group,
therefore, the presence of TiO2 and sodium
titanate was conrmed to be specic to this group.
Surface roughness and wettability are
essential factors that signicantly affect bacterial
adhesion. Extensive research indicates that
bacteria prefer adhering to rough surfaces rather
than smooth ones, with a marked inclination
toward hydrophilic surfaces over hydrophobic
ones [2,7,32]. Although an increase in surface
roughness typically correlates with an enlarged
surface area and a higher potential for bacterial
adherence, under specific conditions it may
conversely contribute to a reduction in bacterial
colonization. Previous report also shown
that micro-nano topographic surfaces have
demonstrated signicant mechano-bactericidal
activity, thereby challenging the conventional
paradigm [33,34]. In this study, the application
of surface treatments resulted in a substantial
increase in roughness compared to the MA
group. The MN surfaces demonstrated a higher
level of roughness than the MR surfaces.
Within the MN group, MN1 exhibited the
greatest roughness, followed by MN2. Acid-
etched surfaces typically displayed hydrophobic
properties, whereas superhydrophilic properties
characterized surfaces subjected to alkaline-heat
treatment [13-16]. Notably, the combination
of acid-etching and alkaline-heat treatment
produced surfaces that were more hydrophilic
than those that were solely acid-etched,
although they did not attain superhydrophilic
characteristics. This is a similar result with
previos study also shown the micro-nano
topography increase the hydrophilicity [23].
Porphyromonas gingivalis
is a Gram-
negative, and anaerobic bacteria which is
the most common pathogen associated peri
implant mucositis and peri implantitis after the
implant placement. The MN group indicated the
antibacterial higher antibacterial activity compare
to the MR and MA. Micro-nano topography is
considered an effective strategy for preventing
bacterial adhesion. Microscale topographical
features, which are comparable in size to
Porphyromonas gingivalis
, typically measuring
approximately 1.51 μm in length and 1 μm
in diameter, facilitate bacterial positioning to
maximize contact with the surface. Conversely,
surfaces with micro-nano or nanoscale features,
which are substantially smaller than bacterial
cells, signicantly reduce adhesion by limiting the
contact area between the bacterial cells and the
surface [7]. Moreover, ower-like topographical
structures have been reported to exhibit enhanced
antibacterial activity [1].
In addition to surface topography, the
chemical composition, surface roughness, and
wettability are critical factors inuencing the
antibacterial activity observed within the MN
group. Surface roughness affects the contact
area available for bacterial adhesion and
increased roughness generally provides more
opportunities for bacterial attachment. However,
the MN surface roughness may counteract
this by reducing the effective contact area,
thereby limiting bacterial colonization [7].
Furthermore, wettability signicantly inuences
the interaction between the bacterial membrane
and the surface.
Porphyromonas gingivalis
11
Braz Dent Sci 2025 Jan/Mar;28 (1): e4663
Kartikasari N et al.
In vitro investigation of flower-like micro-nano topography modifications to improve titanium implant surface properties
Kartikasari N et al. In vitro investigation of flower-like micro-nano topography
modifications to improve titanium implant surface properties
possesses a hydrophobic outer membrane, and
a hydrophilic surface can effectively inhibit the
attachment of these hydrophobic bacteria [32].
Overall the combination of the acid etching and
alkaline heat treatment supported our hypothesis
increase the titanium surface properties and
enhance antibacterial effectiveness.
Despite the promising surface properties
and antibacterial activity of the MN material, its
mechanical properties have not been thoroughly
investigated. This study did not confirm the
material’s effectiveness against Gram-positive
pathogens nor fully elucidate the mechanisms
underlying its antibacterial activity. From a
clinical standpoint, the long-term performance
including the wear resistance, biocompatibility,
and osseointegration of the combined acid-
etching and alkali-heat treatment technique
should be thoroughly evaluated in the future.
We acknowledge the limitations of this study and
emphasize the need for additional research to
comprehensively assess the material’s mechanical
performance, antibacterial mechanisms, and
osseointegration potential to gain a more complete
understanding of its capabilities and applications.
CONCLUSION
In conclusion, the combination of acid etching
and alkaline heat treatment has shown the ability
to create a micro-nano surface with outstanding
properties and signicant antibacterial effects.
This surface modification offers a promising
alternative for enhancing the titanium surfaces
of dental implants, potentially improving both
biological performance and resistance to bacterial
adhesion. However, further research is necessary
to fully investigate its mechanical properties and
long-term effectiveness in clinical applications.
Acknowledgements
The authors would like to express their
sincere gratitude to Tahta Amrillah. for their
invaluable assistance in preparing the titanium
surface and conducting the XRD analysis.
Author’s Contributions
NK: Conceptualization, Methodology,
Validation, Formal Analysis, Investigation, Writing
– Original Draft Preparation, Writing – Review &
Editing, Visualization, Supervision, and Funding
Acquisition. RMS: Conceptualization, Methodology,
Validation, Formal Analysis, Investigation, Writing
– Review & Editing, Visualization, Supervision,
and Funding Acquisition. KM: Investigation,
Writing – Review & Editing, Visualization. SEA:
Investigation, Formal Analysis, Writing – Review
& Editing, Visualization. ARPG: Investigation,
Formal analysis, Writing – Review & Editing. HL:
Investigation, Formal analysis, Writing – Review
& Editing.
Conict of Interest
The authors have no conicts of interest to
declare.
Funding
This study was supported by a grant from
Airlangga Research Fund, Universitas Airlangga,
Indonesia, with grant number 267/UN3/2024.
Regulatory Statement
This study was conducted accordance
with ethical guidance and approved by Health
Research Ethical Clearance Commission, Faculty
of Dental Medicine, Universitas Airlangga,
Number 0524/HRECC.FODM/V/2024.
REFERENCES
1. Ding Y, Yuan Z, Hu JW, Xu K, Wang H, Liu P, et al. Surface
modification of titanium implants with micro–nano-topography
and NIR photothermal property for treating bacterial infection
and promoting osseointegration. Rare Met. 2022;41(2):673-88.
http://doi.org/10.1007/s12598-021-01830-0.
2. Hou C, An J, Zhao D, Ma X, Zhang W, Zhao W,etal. Surface
modification techniques to produce micro/nano-scale
topographies on ti-based implant surfaces for improved
osseointegration. Front Bioeng Biotechnol. 2022;10(3):835008.
http://doi.org/10.3389/fbioe.2022.835008. PMid:35402405.
3. Araujo JCR, Espirito Santo LL, Schneider SG. Influence of
titanium nanotubular surfaces, produced by anodization, on the
behavior of osteogenic cells: in vitro evaluation. Braz Dent Sci.
2022;25(1):1-9. http://doi.org/10.4322/bds.2022.e2528.
4. Al-Khafaji AM, Issa MI, Ismael FJ, Hamad TI, Aljudy HJ. Poly ether
keton keton polymer deposition on laser surface structured
commercial pure titanium using magnetron sputtering. Braz Dent
Sci. 2024;27(2):1-12. http://doi.org/10.4322/bds.2024.e4234.
5. Banu Raza F, Vijayaragavalu S, Kandasamy R, Krishnaswami V,
Kumar VA. Microbiome and the inflammatory pathway in peri-
implant health and disease with an updated review on treatment
strategies. J Oral Biol Craniofac Res. 2023;13(2):84-91. http://
doi.org/10.1016/j.jobcr.2022.11.005. PMid:36504486.
6. Manivasagam VK, Perumal G, Arora HS, Popat KC. Enhanced
antibacterial properties on superhydrophobic micro-
nano structured titanium surface. J Biomed Mater Res A.
12
Braz Dent Sci 2025 Jan/Mar;28 (1): e4663
Kartikasari N et al.
In vitro investigation of flower-like micro-nano topography modifications to improve titanium implant surface properties
Kartikasari N et al. In vitro investigation of flower-like micro-nano topography
modifications to improve titanium implant surface properties
2022;110(7):1314-28. http://doi.org/10.1002/jbm.a.37375.
PMid:35188338.
7. Zhai S, Tian Y, Shi X, Liu Y, You J, Yang Z,etal. Overview of
strategies to improve the antibacterial property of dental
implants. Front Bioeng Biotechnol. 2023;11(9):1267128. http://
doi.org/10.3389/fbioe.2023.1267128. PMid:37829564.
8. Zheng TX, Li W, Gu YY, Zhao D, Qi MC. Classification and research
progress of implant surface antimicrobial techniques. J Dent
Sci. 2022;17(1):1-7. http://doi.org/10.1016/j.jds.2021.08.019.
PMid:35028014.
9. Fernandes VVB Jr, da Rosa PAA, Grisante LAD, Embacher FC,
Lopes BB, de Vasconcellos LGO,etal. Argon plasma application
on the surface of titanium implants: osseointegration study.
Braz Dent Sci. 2023;26(4):1-9. http://doi.org/10.4322/bds.2023.
e3843.
10. Costa PM Fo, Marcantonio CC, de Oliveira DP, Lopes MES,
Puetate JCS, Faria LV,etal. Titanium micro-nano textured surface
with strontium incorporation improves osseointegration: an in
vivo and in vitro study. J Appl Oral Sci. 2024;32:e20240144.
http://doi.org/10.1590/1678-7757-2024-0144. PMid:39292113.
11. Tokmedash AM, Kim C, Chavda AP, Li A, Robins J, Min J. Engineering
multifunctional surface topography to regulate multiple
biological responses. Biomaterials. 2025;319(9):123136. http://
doi.org/10.1016/j.biomaterials.2025.123136. PMid:39978049.
12. Guo Z, Liu H, Wang W, Hu Z, Li X, Chen H,etal. Recent advances
in antibacterial strategies based on TiO2 biomimetic micro/
nano-structured surfaces fabricated using the hydrothermal
method. Biomimetics (Basel). 2024;9(11):656. http://doi.
org/10.3390/biomimetics9110656. PMid:39590228.
13. Kartikasari N, Yamada M, Watanabe J, Tiskratok W, He X, Egusa H.
Titania nanospikes activate macrophage phagocytosis by ligand-
independent contact stimulation. Sci Rep. 2022;12(1):12250.
http://doi.org/10.1038/s41598-022-16214-2. PMid:35851278.
14. Yamada M, Kimura T, Nakamura N, Watanabe J, Kartikasari N,
He X,etal. Titanium nanosurface with a biomimetic physical
microenvironment to induce endogenous regeneration of the
periodontium. ACS Appl Mater Interfaces. 2022;14(24):27703-19.
http://doi.org/10.1021/acsami.2c06679. PMid:35695310.
15. Kartikasari N, Yamada M, Watanabe J, Tiskratok W, He X, Kamano
Y,etal. Titanium surface with nanospikes tunes macrophage
polarization to produce inhibitory factors for osteoclastogenesis
through nanotopographic cues. Acta Biomater. 2022;137:316-30.
http://doi.org/10.1016/j.actbio.2021.10.019. PMid:34673230.
16. Kato E, Yamada M, Kokubu E, Egusa H, Ishihara K. Anisotropic
patterns of nanospikes induces anti-biofouling and mechano-
bactericidal effects of titanium nanosurfaces with electrical cue.
Mater Today Bio. 2024;29(8):101352. http://doi.org/10.1016/j.
mtbio.2024.101352. PMid:39669800.
17. Tan L, Fu J, Feng F, Liu X, Cui Z, Li B,etal. Engineered probiotics
biofilm enhances osseointegration via immunoregulation and
anti-infection. Sci Adv. 2020;6(46):1-10. http://doi.org/10.1126/
sciadv.aba5723. PMid:33188012.
18. Martinez Y, Ausina V, Llena C, Montiel JM. Scientific evidence
on the efficacy of effervescent tablets for cleaning removable
prostheses. A systematic review and meta-analysis. J
Prosthet Dent. 2024;131(6):1071-83. http://doi.org/10.1016/j.
prosdent.2023.01.031. PMid:36870893.
19. Su Y, Komasa S, Sekino T, Nishizaki H, Okazaki J. Nanostructured
Ti6Al4V alloy fabricated using modified alkali-heat treatment:
characterization and cell adhesion. Mater Sci Eng C. 2016;59:617-
23. http://doi.org/10.1016/j.msec.2015.10.077. PMid:26652415.
20. Yamada M, Kato E, Yamamoto A, Sakurai K. A titanium surface
with nano-ordered spikes and pores enhances human dermal
fibroblastic extracellular matrix production and integration of
collagen fibers. Biomed Mater. 2016;11(1):015010. http://doi.
org/10.1088/1748-6041/11/1/015010. PMid:26835848.
21. Jaggessar A, Shahali H, Mathew A, Yarlagadda PKDV. Bio-
mimicking nano and micro-structured surface fabrication for
antibacterial properties in medical implants. J Nanobiotechnology.
2017;15(1):64. http://doi.org/10.1186/s12951-017-0306-1.
PMid:28969628.
22. Gao Q, Hou Y, Li Z, Hu J, Huo D, Zheng H,etal. mTORC2 regulates
hierarchical micro/nano topography-induced osteogenic
differentiation via promoting cell adhesion and cytoskeletal
polymerization. J Cell Mol Med. 2021;25(14):6695-708. http://
doi.org/10.1111/jcmm.16672. PMid:34114337.
23. Chen C, Feng P, Feng F, Zheng Z, Wang J. Micro/nano-surface
modification of titanium implant enhancing wear resistance and
biocompatibility. Int J Mech Sci. 2024;276(May):109385. http://
doi.org/10.1016/j.ijmecsci.2024.109385.
24. Nakamura K, Shirato M, Kanno T, Örtengren U, Lingström P,
Niwano Y. Antimicrobial activity of hydroxyl radicals generated
by hydrogen peroxide photolysis against Streptococcus mutans
biofilm. Int J Antimicrob Agents. 2016;48(4):373-80. http://doi.
org/10.1016/j.ijantimicag.2016.06.007. PMid:27449541.
25. Younis AB, Milosavljevic V, Fialova T, Smerkova K, Michalkova
H, Svec P,et al. Synthesis and characterization of TiO2
nanoparticles combined with geraniol and their synergistic
antibacterial activity. BMC Microbiol. 2023;23(1):207. http://
doi.org/10.1186/s12866-023-02955-1. PMid:37528354.
26. Haghi M, Hekmatafshar M, Janipour MB, Gholizadeh SS, Faraz
MK, Sayyadifar F,etal. Antibacterial effect of TiO2 nanoparticles
on pathogenic strain of E. coli. Int J Adv Biotechnol Res.
2012;3(3):621-4.
27. Serov DA, Gritsaeva AV, Yanbaev FM, Simakin AV, Gudkov
SV. Review of antimicrobial properties of titanium dioxide
nanoparticles. Int J Mol Sci. 2024;25(19):10519. http://doi.
org/10.3390/ijms251910519. PMid:39408848.
28. Almalki AH, Hassan WH, Belal A, Farghali A, Saleh RM, Allah
AE,etal. Exploring the antimicrobial activity of sodium titanate
nanotube biomaterials in combating bone infections: an in vitro
and in vivo Study. Antibiotics (Basel). 2023;12(5):799. http://doi.
org/10.3390/antibiotics12050799. PMid:37237702.
29. Tuikampee S, Chaijareenont P, Rungsiyakull P, Yavirach A. Titanium
surface modification techniques to enhance osteoblasts and
bone formation for dental implants: a narrative review on
current advances. Metals (Basel). 2024;14(5):1-28. http://doi.
org/10.3390/met14050515.
30. Kato E, Sakurai K, Yamada M. Periodontal-like gingival connective
tissue attachment on titanium surface with nano-ordered
spikes and pores created by alkali-heat treatment. Dent Mater.
2015;31(5):e116-30. http://doi.org/10.1016/j.dental.2015.01.014.
PMid:25698416.
31. Joshi B, Regmi C, Dhakal D, Gyawali G, Lee SW. Efficient
inactivation of Staphylococcus aureus by silver and copper
loaded photocatalytic titanate nanotubes. Prog Nat Sci.
2018;28(1):15-23. http://doi.org/10.1016/j.pnsc.2018.01.004.
32. Liu J, Liu J, Attarilar S, Wang C, Tamaddon M, Yang C,etal.
Nano-modified titanium implant materials: a way toward
improved antibacterial properties. Front Bioeng Biotechnol.
2020;8(11):576969. http://doi.org/10.3389/fbioe.2020.576969.
PMid:33330415.
33. Ziegelmeyer T, Martins de Sousa K, Liao TY, Lartizien R, Delay A,
Vollaire J,etal. Multifunctional micro/nano-textured titanium
with bactericidal, osteogenic, angiogenic and anti-inflammatory
properties: insights from in vitro and in vivo studies. Mater
Today Bio. 2025;32(12):101710. http://doi.org/10.1016/j.
mtbio.2025.101710. PMid:40230651.
34. Papa S, Maalouf M, Claudel P, Sedao X, Di Maio Y, Hamzeh-
Cognasse H,etal. Key topographic parameters driving surface
adhesion of Porphyromonas gingivalis. Sci Rep. 2023;13(1):15893.
http://doi.org/10.1038/s41598-023-42387-5. PMid:37741851.
13
Braz Dent Sci 2025 Jan/Mar;28 (1): e4663
Kartikasari N et al.
In vitro investigation of flower-like micro-nano topography modifications to improve titanium implant surface properties
Kartikasari N et al. In vitro investigation of flower-like micro-nano topography
modifications to improve titanium implant surface properties
Date submitted: 2025 Jan 23
Accept submission: 2025 Apr 22
Nadia Kartikasari
(Corresponding address)
Universitas Airlangga, Department of Prosthodontic, Faculty of Dental Medicine,
Surabaya, Jawa Timur, Indonesia.
Email: nadiakartikasari@fkg.unair.ac.id