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.2023.e3843

Braz Dent Sci 2023 Oct/Dec;26 (4): e3843

Argon plasma application on the surface of titanium implants:

osseointegration study

Aplicação de plasma de argônio na superfície de implantes de titânio: estudo de osseointegração

Virgílio Vilas Boas FERNANDES JUNIOR

, Paola Aguiar Afonso da ROSA

, Letícia Adrielly Dias GRISANTE

,

Fábio Colhado EMBACHER

, Bruno Bellotti LOPES

, Luis Gustavo Oliveira de VASCONCELLOS

,

Rogério de Lima ROMEIRO

, Luana Marotta Reis de VASCONCELLOS



1 - Faculdade São Leopoldo Mandic, Instituto São Leopoldo Mandic, Área de Implantodontia. São Paulo, SP, Brazil.

2 - Universidade Estadual Paulista (Unesp), Instituto de Ciência e Tecnologia de São José dos Campos, Departamento de Biociências e

Diagnóstico Oral. São José dos Campos, SP, Brazil.

3 - Engenharia e Soluções a Plasma LTDA. Campinas, SP, Brazil.

4 - Pesquisador independente. São José dos Campos, SP, Brazil.

How to cite: Fernandes VVB Jr, Rosa PAA, Grisante LAD, Embacher FC, Lopes BB, Vasconcellos LGO, et al. Argon plasma application on

the surface of titanium implants: osseointegration study. Braz Dent Sci. 2023;26(4):e3843. https://doi.org/10.4322/bds.2023.e3843

ABSTRACT

Introduction: The development of new biomaterials with improved properties is a trend in regenerative medicine.

The successful healing of implants is related to their osseointegration and the topographic geometry of their

surface. Treatment with argon plasma acts on the surface of the implants, bringing several benets to their

osseointegration in the body. Material and Methods: Previously the in vivo study, the topography implants were

observed by scanning electron microscopy (SEM). Following the implants were inserted in 14 male rats, and one

perforation was made in the right and left tibias for implant placement without the surface treatment (control

group), and with the argon plasma surface treatment (experimental group), respectively. The rats were euthanized

at 4 weeks, a time in which tibia fragments were submitted for histological and histomorphometric examination,

and torque removal test for comparison and analysis of osseointegration. Results: The SEM images showed the

argon plasma surface treatment altered the topography. At the end of the study, both greater bone formation

and better osseointegration were veried in the experimental group, and a statistically signicant difference

between the groups was observed. Conclusion: It was concluded that implants with this surface treatment can

bring more practicality in the rehabilitation treatment, and more comfort in the patients’ postoperative time.

KEYWORDS

Argon plasm; Implant; Osseointegration; Reverse torque; Titanium.

RESUMO

Introdução: O desenvolvimento de novos biomateriais com propriedades aprimoradas é uma tendência na

medicina regenerativa. A cicatrização bem-sucedida dos implantes está relacionada à sua osseointegração e à

geometria topográca de sua superfície. O tratamento com plasma de argônio atua na superfície dos implantes,

trazendo diversos benefícios para sua osseointegração no corpo. Materiais e Métodos: Antes do estudo in vivo, a

topograa dos implantes foi observada por microscopia eletrônica de varredura (MEV). Em seguida, os implantes

foram inseridos em 14 ratos machos, e uma perfuração foi feita nas tíbias direita e esquerda para a colocação

do implante sem o tratamento de superfície (grupo controle) e com o tratamento de superfície de plasma de

argônio (grupo experimental), respectivamente. Os ratos foram sacricados após 4 semanas, momento em que

fragmentos das tíbias foram submetidos a exame histológico e histomorfométrico, além do teste de remoção

de torque para comparação e análise da osseointegração. Resultados: As imagens de MEV mostraram que o

Braz Dent Sci 2023 Oct/Dec;26 (4): e3843

Fernandes Junior VVB et al.

Argon plasma application on the surface of titanium implants: osseointegr ation study

Fernandes Junior VVB et al.

Argon plasma application on the surface of titanium implants:

osseointegration study

INTRODUCTION

Problems related to tooth loss have plagued

the population for centuries. Since antiquity,

attempts have been made to replace missing

teeth. Throughout history, various materials,

including ivory, bone, metals, and precious

stones, have been employed in endeavors to

replicate natural teeth [1]. Metals, in particular,

have been used in attempts to replace lost dental

elements [2]. One of the metals extensively

studied for this purpose was titanium. In 1950,

Branemark conducted pioneering research on

bone integration, fundamentally altering the eld

of dentistry by conceiving that titanium could be

successfully integrated into bone tissue [3]. His

groundbreaking technique became widely known

as the Branemark system, and by 1980, his concept

had revolutionized the clinical practice of dental

implantation [3]. Titanium and its alloys have

proven to be highly compatible with living tissues,

boasting numerous desirable qualities such as

biocompatibility, exceptional mechanical strength,

and resistance to corrosion. Consequently,

titanium has become the preferred material for

dental implants [4].

The clinical success of implants in both the

short and long term hinges on their ability to

achieve osseointegration and the topographical

features of their surfaces [5]. Therefore, surface

modications of dental implants are proposed

to optimize the osseointegration process, as

these modified surfaces can interact more

effectively with adjacent tissue, facilitating

direct bone-to-implant contact [6]. Shortly after

implant placement, brin precipitation initiates

on the implant’s surface, followed by platelet

aggregation, which releases growth factors that

stimulate the recruitment and proliferation

of osteoblastic lineage cells. Subsequently,

osteoblast differentiation occurs, leading to the

deposition of a non-mineralized cellular matrix

that later undergoes calcication [7]. Surface

treatments of titanium are performed to enhance

and expedite these cellular events responsible for

the surrounding ossication process [1].

The long-term success rate of implant

rehabilitation is determined by various factors,

including the establishment and maintenance of

osseointegration, as previously mentioned. Over

the years, research has focused on improving

implant surface treatments to achieve more efcient

and stable cell adhesion, which can subsequently

influence cellular responses due to surface

topography. Various implant surface treatments,

such as argon plasma, have been studied in vitro

for surface decontamination and increased cell

adhesion. According to research ndings, argon

plasma can activate surfaces, thus enhancing cell

proliferation and creating the optimal conditions

for superior cell adhesion [8]. Studies have shown

that implants treated with argon plasma achieve

greater bone-implant contact compared to untreated

implants [9]. Argon plasma works by modifying

the surface without compromising its structural

properties; the application of plasma increases the

surface energy of the treated substrate, promoting

favorable molecular chemical interactions [10].

The use of argon plasma surface treatment is

widespread as the nal step in the manufacturing

process of titanium implant accessories. This

technology operates by activating the electronic

structure of materials through argon spraying

under pressure at room temperature. The primary

microscopic outcome of such activation is the

removal of microbiological contaminants and

pollution from metal surfaces. Additionally,

this process has the capability to modify the

physicochemical properties and, consequently,

the biological characteristics of the implant

surfaces, affecting their interaction with the

surrounding environment [11]. In vitro studies

have assessed the impact of argon plasma on

bone cell cultures on titanium samples [7,11,12].

tratamento de superfície com plasma de argônio alterou a topograa. Ao nal do estudo, foi vericada maior

formação óssea e melhor osseointegração no grupo experimental, e foi observada diferença estatisticamente

signicativa entre os grupos. Conclusão: Concluiu-se que os implantes com esse tratamento de superfície podem

trazer mais praticidade no tratamento de reabilitação e maior conforto no pós-operatório dos pacientes.

PALAVRAS-CHAVE

Plasma de argônio; Implante; Osseointegração; Torque reverso; Titânio.

Braz Dent Sci 2023 Oct/Dec;26 (4): e3843

Fernandes Junior VVB et al.

Argon plasma application on the surface of titanium implants: osseointegr ation study

Fernandes Junior VVB et al.

Argon plasma application on the surface of titanium implants:

osseointegration study

The aim of this study was to assess, in vivo,

the influence of argon plasma application on

implant surfaces on bone regeneration and the

implant xation force in the tibias of rats.

MATERIAL AND METHODS

Implants

The implants were custom-made by the

company Emls® specically for this research

project. Both implants were crafted from grade 5

titanium alloy, measuring 2.5mm by 2.0mm. To

morphologically characterize the implant surface,

scanning electron microscopy (Philips XL-30 FEG,

PHILIPS, LEUVEN, België) was utilized before

and after laser treatment.

Surgical procedure

Fourteen male rats (Rattus norvegicus

albinus; Wistar), each 3 months old and weighing

approximately 400g, were utilized in this study.

Surgical procedures were conducted at São Paulo

State University (UNESP, São José dos Campos,

SP, Brazil), and the study received approval

from the Research Ethics Committee (approval

number: 08/2019-CEUA-ICT-CSJC-UNESP).

All rats were housed in groups of three animals

per cage and provided with ad libitum access

to food and water. The animal model design

adhered to controlled and randomized principles,

following the Animal Research: Reporting In

Vivo Experiments (ARRIVE) guidelines for the

execution and submission of animal studies [13].

Prior to the surgical procedure, the

animals were weighed and then anesthetized

intramuscularly using a solution containing

2.3g/100ml of xylazine hydrochloride (Anasedan®

- Vetbrands, Jacareí - Brazil) and 1.16g/10ml of

ketamine hydrochloride (Dopalen® - Vetbrands,

Jacareí - Brazil). After confirming anesthesia,

trichotomy was performed, and antisepsis was

carried out using iodized alcohol solution in

the target region. An incision was made with a

number 15 scalpel blade to provide access to the

bone tissue, and the entire ap was retracted. In

all 14 animals, perforations were made in both

the right and left tibias using a trephine drill.

These perforations were performed under copious

irrigation with a 0.9% sodium chloride solution

to prevent heat generation from friction between

the drill and bone. Subsequently, each animal

received an untreated implant (control) in the

right tibia and an argon plasma-treated implant

in the left tibia, both of which were immediately

inserted into the surgical cavity. The ap was then

repositioned and sutured using #4 silk thread

(Ethicon/Johnson & Johnson). Following surgery,

all animals were administered an intramuscular

injection of 0.1 mg/kg of sodium phenyl-dimethyl-

pyrazolone-methylamino-methanesulfonate

(dipyrone) for three days. After 21 days, the

animals were euthanized, with the tibias of seven

animals from one group preserved in 10% formalin

for subsequent histological analysis, while the

remaining seven animals were stored in Ringer’s

solution at -20°C for the reverse torque test.

Histological and morphological analysis

The bone fragments containing the defects

were immersed in 10% formaldehyde for a

minimum period of 48 hours. Subsequently, the

samples underwent preparation for conventional

histological analysis using the decalcification

method, which was initiated upon obtaining

the specimens. They were first fixed in 10%

formaldehyde for 48 hours and then subjected to

dehydration in an increasing sequence of alcohol

concentrations (60%, 70%, 80%, 90%, and

100%) [14]. Following these steps, the specimens

were placed in xylene (PA) for clarification.

Subsequently, the samples were embedded in a

resinous solution, utilizing a mixture of methyl

methacrylate and dibutyl phthalate in an 85% to

15% ratio, respectively, along with the addition

of 1g of benzoyl peroxide. They were then placed

in an oven at 37°C for 3 days. Following this

incubation period, the resin block containing

the histological specimen was obtained and

subsequently sectioned [14].

Histological analyses were conducted using

optical microscopy after staining the slides with

toluidine blue, a dye suitable for highlighting

bone tissue, osteoid tissue, and cellular nuclei

such as osteoblasts, osteoclasts, and marrow cells.

This staining method allows for the observation of

bone remodeling that occurs during the process

of implant osseointegration and facilitates the

identication of various cell types [15,16].

To calculate the Bone Area Fraction Occupied

(BAFO), we evaluated the total area of the

threads, determining the area either occupied

by bone tissue or devoid of it. The percentage

of the total thread area lled with bone tissue

Braz Dent Sci 2023 Oct/Dec;26 (4): e3843

Fernandes Junior VVB et al.

Argon plasma application on the surface of titanium implants: osseointegr ation study

Fernandes Junior VVB et al.

Argon plasma application on the surface of titanium implants:

osseointegration study

was quantified in square micrometers (μm

To ensure measurement consistency, a single

examiner assessed two specic regions: the mesial

and distal surfaces of the bone-implant interface.

Optical microscopy at 100x magnication was

utilized, employing Axiophot® equipment (Carl

Zeiss, Oberkochen, Germany) coupled with a

digital camera, AxioCam MRc® 5 (Carl Zeiss,

Oberkochen, Germany), and data transmission

to the computer program AxioVision® Release

4.7.2. The images were coded by another

examiner who was unaware of the groups, thus

enabling impartial data analysis. Following image

acquisition, histomorphometric analysis was

conducted using the publicly available program

NIH ImageJ (National Institute of Health,

Bethesda, Maryland, USA).

Torque and removal test

Subsequent to euthanasia, the left tibias

were excised and preserved in Ringer’s solution

at -20°C until the reverse torque test, which was

conducted at room temperature. The reverse

torque test involved the use of a digital torque

wrench (Mark-10 Corporation, New York, NY,

USA). Counterclockwise rotation was applied,

and we recorded the maximum torque values

(N·cm) required for bone fracture at the bone-

implant interface.

Statistical analysis

All statistical analyses were carried out

utilizing GraphPad Prism version 6.0 software

(GraphPad Software, San Diego, USA).

Descriptive data obtained from the tests were

first plotted and subjected to initial analysis

through the Kolmogorov-Smirnov normality test.

Subsequently, the parametric unpaired Student’s

t-test was applied, treating the control group

and the experimental group as the independent

variable. These statistical procedures were

conducted using GraphPad Prism 6.0 software

(GraphPad Software, San Diego, CA, USA), with a

signicance level of 5% being adopted for all tests.

RESULTS

Implant surfaces

Figure 1 shows the surface of the titanium

implant without plasma treatment after

photomicrography in SEM in panoramic view. SEM

analysis demonstrated topographical differences

between the implant surfaces (Figure 2). The

untreated surface has a smooth appearance

(Figure 2A), while the laser-treated surface shows

an irregular topography formed by valleys, with

different depths and sizes (Figure 2C).

Histological and histomorphometric analysis

Macroscopically, the implants exhibited

bone growth around their surfaces in contact

with cortical bone, regardless of the surface type.

Microscopically, the evaluated sections showed

cross-sections of the tibias of the rats, where the

implants were inserted. This long bone consisted

of compact bone tissue, forming the tibia’s walls,

and medullary tissue in the central region. The

compact bone displayed numerous Haversian

systems, composed of concentric bone lamellae

arranged around a central canal. These lamellae

contained osteocyte lacunae, also arranged in

concentric rings, connected through canaliculi.

Irrespective of the implant surface type, the

anks of the threads were nearly entirely lled

with newly formed bone tissue, and occasionally,

the demarcation line between the pre-existing

tissue and the newly formed bone tissue was

discernible (Figure 3). The bone tissue was

closely in contact with the implant, indicating

successful osseointegration (Figure 4).

The results of bone neoformation around the

implants are presented in Figure 5A and expressed

as a percentage. A statistically signicant difference

was observed between the groups (p = 0.0206),

with the treated group exhibiting higher values

compared to the control group.

Figure 1 - Photomicrograph of the implant at 30x magniﬁcation.

Caption: shows a panoramic view of the titanium implant surface.

Braz Dent Sci 2023 Oct/Dec;26 (4): e3843

Fernandes Junior VVB et al.

Argon plasma application on the surface of titanium implants: osseointegr ation study

Fernandes Junior VVB et al.

Argon plasma application on the surface of titanium implants:

osseointegration study

Analysis of the removal torque test

The torque removal values were quantied

in N·cm and are graphically depicted in

Figure 5B, illustrating the statistical analysis.

The data underwent a Student’s t-test, revealing

a signicant difference between the groups (p =

0.029), with the treated group requiring more

force for displacement in comparison to the

control group.

DISCUSSION

Since the advent of osseointegrated implants,

surface treatments, coupled with minimally

Figure 2 - Photomicrographs depicting surface details of the implants. Caption: A) detail of the surface without argon plasma treatment; B)

surface of the untreated implant at a 500x magniﬁcation; C) surface of the implant treated with argon plasma at a 500x.

Figure 3 - Histomorphometric analysis. Caption: A) area selected for analysis, ﬂank of the ﬁrst implant thread; B) image obtained by histological

section, in which the selected part is the bone tissue, while the part with negative image (*) is the place where the implant was performed;

through this it is possible to identify the intimate contact between implant and bone tissue.

Braz Dent Sci 2023 Oct/Dec;26 (4): e3843

Fernandes Junior VVB et al.

Argon plasma application on the surface of titanium implants: osseointegr ation study

Fernandes Junior VVB et al.

Argon plasma application on the surface of titanium implants:

osseointegration study

traumatic surgical installation techniques and

well-planned functional restorations, have yielded

promising long-term outcomes [17]. According to

Branemark, osseointegration in dentistry hinges

on a comprehensive understanding of the healing

and repair capacities of both hard and soft tissues.

An osseointegrated dental implant represents the

culmination of biological and mechanical xation

on the bone, capable of supporting normal

clinical function. This response must be highly

specialized and organized to meet functional

demands [18,19].

Events that transpire following implant

insertion involve interactions between the

biological environment and the implant surface,

initiating the biological reactions crucial for the

osseointegration process; the success of an implant

largely depends on the balance among all phases

of the clinical part, from planning to insertion and

maintenance, and the interactions with the soft and

hard biological tissues with the synthetic structure

of the implants [20]. The rate and quality of

osseointegration in titanium implants are closely

tied to their surface properties, encompassing

aspects like composition, hydrophilicity, and

roughness [21]. The characteristics of an implant

as a biomaterial have a significant impact on

the quality of osseointegration. Specically, its

surface characteristics can inuence the activation

and differentiation process of osteogenic cells.

Therefore, discussions about modications and

Figure 5 - Bar chart (mean ± standard deviation). Caption: A) bone area fraction occupied (BAFO) new bone formation (%); (B) removal torque

test (N.cm). Statistical diﬀerences are indicated by diﬀerent letters (Student’s t-test, p < 0.05).

Figure 4 - Optical microscope photomicrograph of the area between the implant threads, original 10x magniﬁcation. Caption: A) control

implant; B) experimental implant.

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Fernandes Junior VVB et al.

Argon plasma application on the surface of titanium implants: osseointegr ation study

Fernandes Junior VVB et al.

Argon plasma application on the surface of titanium implants:

osseointegration study

treatments on the implant’s surface have taken

place to enhance properties that facilitate the

interaction between bone and the implant [22].

During the production of titanium implants, a

surface layer is generated that is typically oxidized

and contaminated, often stressed and plastically

deformed, and characterized by non-uniformity.

Such surfaces are ill-suited for biomedical

applications, necessitating surface treatments to

rectify these irregularities [1].

Titanium surfaces have a propensity to

adsorb organic impurities, such as atmospheric

hydrocarbons, leading to an increase in

hydrophobicity [23]. From a physicochemical

perspective, non-thermal plasma treatment,

like argon plasma, enhances surface energy by

reducing the contact angle, rendering it more

hydrophilic and facilitating cell spreading [24].

Moreover, plasma serves to clean the surface,

breaking C-H and C-C bonds, thereby decreasing

the presence of hydrocarbons and organic

molecules deposited on the surface upon exposure

to atmospheric air [25].

Previous studies indicate that non-thermal

plasma treatment tends to increase the surface

energy of titanium, resulting in the formation of

polar groups. Notably, positive OH (hydroxyl)

groups play a crucial role in the chemical

interaction between osteoblasts and the Ti

surface, promoting greater cell adhesion and

spreading [26-28]. This hydroxylation of the

titanium surface also contributes to the reduction

of organic impurities, like hydrocarbons, further

facilitating future cell migration [29,30].

These ndings elucidate the superior outcomes

in our experimental group, where histological and

morphological analyses, specically descriptive

statistics of bone neoformation around the

implants (BAFO), revealed statistically higher

values than the control group. This suggests

greater extracellular bone matrix formation,

potentially attributable to an increased number of

osteoblasts or their heightened activity compared

to the control group.

The results of this study suggest that modify-

ing surface topography and/or physicochemical

properties signicantly impacts titanium sur-

face wettability, potentially enhancing protein

adsorption and subsequent cellular behavior.

Additionally, it’s worth noting that plasma treat-

ment induces a corrosive effect on the surface

[29], explaining the topographical changes

observed in the titanium surface of the experi-

mental group, which increases its interface area

with the adjacent bone.

Hence, this technology holds the potential

to transform the surface of titanium implants,

optimizing osseointegration. This trend was

corroborated by the results of the reverse torque

test, where the experimental group demonstrated

statistically superior values compared to the control

group. Positive outcomes regarding increased

implant xation force have also been documented in

previous studies that employed various techniques

to modify implant surfaces [31,32].

CONCLUSION

Clinical studies are essential to validate

the reduction in the osseointegration timeframe

in dental practice, with the aim of enhancing

the responsiveness of this process, ultimately

beneting patients who may potentially undergo

shorter rehabilitation treatments. Based on the

conducted tests, a notable enhancement in the

characteristics of implants featuring treated

surfaces was observed.

Author’s Contributions

LMRV: Conceptualization. LMRV:

Methodology. VVBFJ, LADG, PAAR: Validation.

VVBFJ, LADG, PAAR: Formal Analysis. VVBFJ,

LADG, PAAR: Investigation. FCE, BBL, LGOV,

RLR: Resources. VVBFJ: Writing – Original Draft

Preparation. PAAR: Writing – Review & Editing.

LADG, LMRV: Visualization. LADG, LMRV:

Supervision. LMRV: Project Administration. FCE,

BBL, LGOV, RLR: Funding Acquisition.

Conict of Interest

No conicts of interest declared concerning

the publication of this article.

Funding

This study generated a scientic initiation

grant for a student, funded by FAPESP (São Paulo

Research Foundation).

Regulatory Statement

This project was submitted to the Research

Ethics Committee of the Institute of Science and

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Argon plasma application on the surface of titanium implants: osseointegr ation study

Fernandes Junior VVB et al.

Argon plasma application on the surface of titanium implants:

osseointegration study

Technology at São José dos Campos Campus/

UNESP, and it was conducted following the

Ethical Principles for Animal Experimentation

adopted by the Brazilian College of Animal

Experimentation (CONCEA). The approval code

for this study is: 08/2019.

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Braz Dent Sci 2023 Oct/Dec;26 (4): e3843

Argon plasma application on the surface of titanium implants:

osseointegration study

Fernandes Junior VVB et al.

Argon plasma application on the surface of titanium implants: osseointegr ation study

Fernandes Junior VVB et al.

Argon plasma application on the surface of titanium implants:

osseointegration study

Date submitted: 2023 Mar 28

Accept submission: 2023 Oct 09

Letícia Adrielly Dias Grisante

(Corresponding address)

Universidade Estadual Paulista (Unesp), Instituto de Ciência e Tecnologia de São José dos

Campos, Departamento de Biociências e Diagnóstico Oral, São José dos Campos, SP, Brazil.

Email: l.cruz@unesp.br