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applied
sciences
Article
In Vitro Preliminary Evaluation of Bacterial Attachment
on Grooved and Smooth Healing Abutments
Ofer Moses 1, Carlos E. Nemcovsky 1, Israel Lewinstein 2, Hasan Zoabi 3, Miron Weinreb 3,
Shifra Levartovsky 2, *,†and Shlomo Matalon 2, †
1Department of Periodontology and Dental Implantology, School of Dental Medicine, Tel Aviv University,
Tel Aviv 6997801, Israel; mosesofer@gmail.com (O.M.); carlos@tauex.tau.ac.il (C.E.N.)
2
Department of Oral Rehabilitation, School of Dental Medicine, Tel Aviv University, Tel Aviv 6997801, Israel;
Lewinsdr@gmail.com (I.L.); matalons@tauex.tau.ac.il (S.M.)
3Department of Oral Biology, School of Dental Medicine, Tel Aviv University, Tel Aviv 6997801, Israel;
Hasanzoabi@gmail.com (H.Z.); Weinreb@tauex.tau.ac.il (M.W.)
*Correspondence: shifra@tauex.tau.ac.il
†These authors contributed equally to this article.
Received: 15 May 2020; Accepted: 24 June 2020; Published: 27 June 2020
Abstract:
This
in vitro
preliminary study investigated the attachment of Fusobacterium nucleatum
and Porphyromonas gingivalis on titanium alloy healing abutments, which differed in their surface
macro-morphology: one was groove-marked while the other was completely smooth. Altogether,
twenty implant-healing abutments, ten of each macro-morphology, were evaluated with a single
type of bacterial strain. Accordingly, four groups of five abutments each were created. The sterilized
healing abutments with the cultured bacteria were placed under anaerobic conditions for 48 h
at 37
◦
C. Afterwards, the abutments were examined with a scanning electron microscope, at a 2500x
magnification. Attached bacteria were quantified in the four vertical quarters within the grooved
abutments and in the two most coronal millimeters of the smooth abutments. The results were
analyzed by applying two-way ANOVA, with square root transformation for a normal distribution.
The bacterial attachment of both strains was statistically significantly larger in the grooved abutment
areas than on the smooth surfaces (p
≤
0.0001), twenty times so for Porphyromonas gingivalis and a
hundred times so for Fusobacterium nucleatum.
Keywords: smooth healing abutment; grooved healing abutment; bacterial growth; SEM
1. Introduction
Healing abutments may be placed on dental implants either following implant placement or in
two-stage surgical protocols, at the time of their exposure to the oral environment. Differing from
dental implants, where micro- and macro-grooving has been shown to enhance soft and hard tissue
integration [
1
], the coronal portion of healing abutments is exposed to the oral cavity and, therefore,
subject to bacterial colonization [
2
]. Abutments’ surface characteristics and the implant–abutment
interface significantly affect supragingival and subgingival biofilm accumulation. An increased surface
roughness facilitates biofilm formation [
3
], which, in turn, affects the peri-implant soft tissues [
3
–
5
].
Healing abutments are placed in a surgical wound area. Enhanced bacterial plaque accumulation
leads to peri-implant soft tissue inflammation, which, in turn, affects soft and hard tissue integration to
the dental implant, especially in the crestal area [
6
–
8
]. Bacterial adhesion and proliferation lead to an
organized biofilm [
9
]; hence, rough surfaces accumulate up to twenty-five times more sub-gingival
plaque than smoother ones, also leading to more difficult removal [10–12].
The consequences of enhanced plaque build-up on healing abutments are not clear; however, it is
evident that bacterial accumulation, especially in a surgical wound healing area, around implants
Appl. Sci. 2020,10, 4426; doi:10.3390/app10134426 www.mdpi.com/journal/applsci
Appl. Sci. 2020,10, 4426 2 of 9
enhances inflammatory reactions in the surrounding gingiva and alveolar mucosa as well as around
teeth [13].
Inflammatory mediators are enhanced in inflamed tissues, thus promoting angiogenesis [
14
] and
pro-inflammatory cytokines [
15
]. Therefore, this inflammatory process within the soft tissue around
healing abutments is also likely to affect implant osseous integration.
Fusobacterium nucleatum and Porphyromonas gingivalis are well known perio-pathogenic bacteria.
Fusobacterium nucleatum belongs to the Bacteroidaceae family. It is a Gram-negative anaerobic species of
the phylum Fusobacteria, numerically dominant in dental plaque biofilms, and important in biofilm
ecology and human infectious diseases [
16
]. Porphyromonas gingivalis is a Gram-negative anaerobic
bacterium [
17
]. Infection by Porphyromonas gingivalis could modulate host immune responses and
ultimately destroy the balance between the normal cell cycle and apoptosis, thereby leading to
periodontal tissue destruction [18,19].
Most healing abutments are made of titanium alloys [
20
]. However, the influence that certain
alterations, such as laser markings or the machining of depth grooves on the abutment surface,
may have on bacterial adhesion has not been evaluated.
This preliminary
in vitro
study aimed to evaluate the bacterial attachment of two strains onto
titanium alloy healing abutments, from the same manufacturer, differing only in their surface
macro-morphology; while one was CNC (Computer Numerical Control) machine-grooved, the other
was completely smooth.
2. Methods
2.1. Tested Materials
Two equal groups of ten titanium grade-V, 5 mm-height and 3.85 mm-diameter implant-healing
abutments (AlphaBio Tec Ltd., Petach Tikva, Israel), were evaluated:
Group I: 10 smooth healing abutments with a machined smooth surface (Figure 1); Group II:
10 healing abutments with a marked (grooved) surface roughness value (Ra) of 0.8 µm (Figure 2).
Appl. Sci. 2020, 10, x FOR PEER REVIEW 3 of 9
Bacterial attachment counts were performed on the four vertical quarters of the grooved
abutments, both in smooth and grooved areas, while on the smooth abutments, they were performed
in the two coronal millimeters (surface area of 958 µm
2
for each quarter) using the Image J 1.4 software
(NIH Bethesda, MD, USA).
2.3. Statistical Analysis
Statistical analysis consisted of two-way ANOVA after square root transformation was applied
for a normal distribution. A p value ≤ 0.05 was considered statistically significant.
Figure 1. Machined smooth surface.
Figure 2. Marked (grooved) surface.
3. Results
The results of the bacterial counts are presented in Table 1 and Figure 3a,b. (1) Similar bacterial
counts for both strains were found on the smooth areas between the grooves of the marked healing
abutments and on the smooth healing abutments. (2) Bacterial adherence was different for each strain,
and F.n attachment to all surfaces was considerably larger than that of P.g. (3) The most important
finding was that the total bacterial counts for both strains in the grooved areas were significantly
Figure 1. Machined smooth surface.
Appl. Sci. 2020,10, 4426 3 of 9
Appl. Sci. 2020, 10, x FOR PEER REVIEW 3 of 9
Bacterial attachment counts were performed on the four vertical quarters of the grooved
abutments, both in smooth and grooved areas, while on the smooth abutments, they were performed
in the two coronal millimeters (surface area of 958 µm
2
for each quarter) using the Image J 1.4 software
(NIH Bethesda, MD, USA).
2.3. Statistical Analysis
Statistical analysis consisted of two-way ANOVA after square root transformation was applied
for a normal distribution. A p value ≤ 0.05 was considered statistically significant.
Figure 1. Machined smooth surface.
Figure 2. Marked (grooved) surface.
3. Results
The results of the bacterial counts are presented in Table 1 and Figure 3a,b. (1) Similar bacterial
counts for both strains were found on the smooth areas between the grooves of the marked healing
abutments and on the smooth healing abutments. (2) Bacterial adherence was different for each strain,
and F.n attachment to all surfaces was considerably larger than that of P.g. (3) The most important
finding was that the total bacterial counts for both strains in the grooved areas were significantly
Figure 2. Marked (grooved) surface.
2.2. Tested Bacteria and Culturing Conditions
The cultured bacterial strains were Fusobacterium nucleatum (ATCC 1594) (F.n) from frozen stock
cultures grown anaerobically in Sherdler broth (Becton, Dickinson and Company, Franklin Lakes,
NJ, USA) and Porphyromonas gingivalis (ATCC 33279) (P.g) from frozen stock cultures grown anaerobically
in Wilkins–Chalgren broth (OXOID Ltd., Basingstoke, Hampshire, UK). Each of these strains
was cultured on five smooth-surfaced and five grooved healing abutments, thus yielding four
experimental groups.
Sterilized abutments were screwed to the inner surface of the microtiter plate cover and immersed
in a 96-well microtiter plate (96-well flat-bottom Nunclon, Copenhagen, Denmark), each well containing
250 microliters of growth medium suitable for each bacterial strain (F.n was in Sherdler broth and P.g,
in Wilkins–Chalgren broth). Into each well, ten microliters of bacterial suspension (O.D 0.6 at 600 nm),
representing 1
×
10
6
cells/mL, were added, and the plates were incubated under anaerobic conditions
for 48 h at 37
◦
C and vortexed automatically for five seconds every 30 min (VesaMax, Molecular Device
Corporation, San Jose, CA, USA) in order to guarantee the homogeneity of the bacterial suspension.
After 48 h (early colonization), the abutments were removed from the plate. Close direct contact
between the bacteria and the abutment surface and bacterial suspension fluids was accomplished by
an evaporation process. The abutments were fixated with 2.5% glutaraldehyde in PBS and gold-plated
for examination with a scanning electron microscope (SEM) (JSM 840A, Jeol Ltd., Tokyo, Japan), at a
2500x magnification and with a 47.9 micrometer horizontal field and accelerating voltage of 25 kV.
Bacterial attachment counts were performed on the four vertical quarters of the grooved abutments,
both in smooth and grooved areas, while on the smooth abutments, they were performed in the
two coronal millimeters (surface area of 958
µ
m
2
for each quarter) using the Image J 1.4 software
(NIH, Bethesda, MD, USA).
2.3. Statistical Analysis
Statistical analysis consisted of two-way ANOVA after square root transformation was applied
for a normal distribution. A pvalue ≤0.05 was considered statistically significant.
3. Results
The results of the bacterial counts are presented in Table 1and Figure 3a,b. (1) Similar bacterial
counts for both strains were found on the smooth areas between the grooves of the marked healing
abutments and on the smooth healing abutments. (2) Bacterial adherence was different for each strain,
and F.n attachment to all surfaces was considerably larger than that of P.g. (3) The most important
finding was that the total bacterial counts for both strains in the grooved areas were significantly higher
Appl. Sci. 2020,10, 4426 4 of 9
(p
≤
0.0001) than those in the smooth areas. The mean P.g counts were ~20 times larger and those of F.n
were over 100 times larger in the grooves compared to on the smooth surfaces.
Table 1. Bacterial counts (mean and SD) in the four experimental groups.
Abutment Type F.n Counts (CFU) P.g Counts (CFU)
Bacteria/Field
Mean SD Mean SD
Abutment Type
Smooth 10.1500 3.33791 192.1750 66.70708
Grooved Within grooves 1043.5500 397.52536 4026.3000 347.55053
Between grooves 11.4500 4.92133 175.7000 44.72210
Appl. Sci. 2020, 10, x FOR PEER REVIEW 4 of 9
higher (p ≤ 0.0001) than those in the smooth areas. The mean P.g counts were ~20 times larger and
those of F.n were over 100 times larger in the grooves compared to on the smooth surfaces.
Scanning Electron Microscopy
Pictures of the grooved and smooth abutment surfaces taken under SEM are shown in Figures
4–7, and a difference in shape between the two bacterial strains may be appreciated. The bacterial
adherence of both strains is clearly larger in the grooved areas of the marked abutments as compared
to in the smooth ones.
Table 1. Bacterial counts (mean and SD) in the four experimental groups.
Abutment Type F.n Counts (CFU) P.g Counts (CFU)
Bacteria/Field
Mean SD Mean SD
Abutment Type
Smooth 10.1500 3.33791 192.1750 66.70708
Grooved Within grooves 1043.5500 397.52536 4026.3000 347.55053
Between grooves 11.4500 4.92133 175.7000 44.72210
(a) (b)
Figure 3. (a) Fusobacterium nucleatum count on smooth surface, in the grooves and in between; (b)
Porphyromonas gingivalis count on smooth surface, in the grooves and in between. CFU (colony-
forming unit)
Figure 3.
(
a
)Fusobacterium nucleatum count on smooth surface, in the grooves and in between;
(
b
)Porphyromonas gingivalis count on smooth surface, in the grooves and in between.
CFU (colony-forming unit).
Scanning Electron Microscopy
Pictures of the grooved and smooth abutment surfaces taken under SEM are shown in Figures 4–7,
and a difference in shape between the two bacterial strains may be appreciated. The bacterial adherence
of both strains is clearly larger in the grooved areas of the marked abutments as compared to in the
smooth ones.
Appl. Sci. 2020,10, 4426 5 of 9
Appl. Sci. 2020, 10, x FOR PEER REVIEW 5 of 9
Figure 4. Fusobacterium nucleatum attached on the border-line between the smooth (dark side of the
figure) and the grooved parts (bright side, the left side of the figure). The larger number of bacteria
within the groove as compared to that in the machined area of the healing cap (right side) is evident
(magnification, × 2500).
Figure 5. High number of Fusobacterium nucleatum found within the grooved part of the healing cap
(magnification, × 2500).
Figure 6. Porphyromonas gingivalis growth on the machined surface. Note the low bacterial count in
this area (magnification, × 2500).
Figure 4.
Fusobacterium nucleatum attached on the border-line between the smooth (dark side of
the figure) and the grooved parts (bright side, the left side of the figure). The larger number of bacteria
within the groove as compared to that in the machined area of the healing cap (right side) is evident
(magnification, ×2500).
Appl. Sci. 2020, 10, x FOR PEER REVIEW 5 of 9
Figure 4. Fusobacterium nucleatum attached on the border-line between the smooth (dark side of the
figure) and the grooved parts (bright side, the left side of the figure). The larger number of bacteria
within the groove as compared to that in the machined area of the healing cap (right side) is evident
(magnification, × 2500).
Figure 5. High number of Fusobacterium nucleatum found within the grooved part of the healing cap
(magnification, × 2500).
Figure 6. Porphyromonas gingivalis growth on the machined surface. Note the low bacterial count in
this area (magnification, × 2500).
Figure 5.
High number of Fusobacterium nucleatum found within the grooved part of the healing cap
(magnification, ×2500).
Appl. Sci. 2020, 10, x FOR PEER REVIEW 5 of 9
Figure 4. Fusobacterium nucleatum attached on the border-line between the smooth (dark side of the
figure) and the grooved parts (bright side, the left side of the figure). The larger number of bacteria
within the groove as compared to that in the machined area of the healing cap (right side) is evident
(magnification, × 2500).
Figure 5. High number of Fusobacterium nucleatum found within the grooved part of the healing cap
(magnification, × 2500).
Figure 6. Porphyromonas gingivalis growth on the machined surface. Note the low bacterial count in
this area (magnification, × 2500).
Figure 6.
Porphyromonas gingivalis growth on the machined surface. Note the low bacterial count in this
area (magnification, ×2500).
Appl. Sci. 2020,10, 4426 6 of 9
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Figure 7. Porphyromonas gingivalis growth on the inner aspect of the grooved surface. Note the high
bacterial count in this area (magnification, × 2500).
4. Discussion
The effect of depth grooves on healing abutments on bacterial adhesion has hardly been
evaluated; the present in vitro study reveals, for the first time, their negative effect, by significantly
enhancing bacterial adhesion. Previous reports have shown that surface modifications on dental
implants and abutments play a relevant role in bacterial adhesion [3]. The chemical and physical
surface characteristics of trans-gingival implant components significantly influence bacterial
attachment. The control of bacterial infection in the peri-implant soft and hard tissues is critical for
the long-term stability of implants supporting oral rehabilitation [4,10,21,22]. There are no previous
reports concerning the relationship of the healing abutment macro-morphology (machined or
grooved), used in dental implant procedures, to clinical and microbiological parameters; however, it
is clear that bacterial plaque negatively affects peri-implant
tissue stability [22].
Our results demonstrate the significantly larger bacterial attachment of both strains in the
grooved areas compared to in the smooth areas of the healing abutments, especially of F.n, which
was 100 times larger. Our results are in accordance with those of Bermejo et al. that showed the
significant accumulation of pathogenic bacteria (F. nucleatum and A. actinomycetemcomitans) on
moderate-roughness compared to minimal-roughness implant surfaces [23].
Peri-implantitis presents a heterogeneous microbiota, where perio-pathogenic bacteria such as
Porphyromonas gingivalis and Prevotella intermedius/nigrescens together with asaccharolytic
anaerobic Gram-positive rods and other Gram-negative rods may be found [24]. The treatment of
peri-implantitis is mainly based on different mechanical and physical means to remove biofilms from
the implant surface [25].
Healing abutments are usually placed following a surgical intervention, such as implant
placement or uncovering. In a recent randomized, controlled study, either micro-grooved or
machined healing abutments were placed. After nonsurgical treatment and the reconstitution of oral
hygiene measures, the incidence of peri-implant mucositis was found to be significantly lower at the
non-grooved abutments [26].
Bacterial colonization, as found in this in vitro study, was enhanced in grooved healing caps.
Bacterial accumulation following implant uncovering might induce pro-inflammatory events in the
mucosa, jeopardizing optimal healing. Epithelial cell attachment may prevent the bacterial invasion
of titanium surfaces; however, early bacterial colonization on these surfaces may, in turn,
downregulate this effect. Peri-implant soft tissue clinical maturity is apparently established as early
as four weeks following implant placement in a one-stage surgical protocol, while the newly created
peri-implant crevices are colonized by bacteria within two weeks [27].
Figure 7.
Porphyromonas gingivalis growth on the inner aspect of the grooved surface. Note the high
bacterial count in this area (magnification, ×2500).
4. Discussion
The effect of depth grooves on healing abutments on bacterial adhesion has hardly been evaluated;
the present
in vitro
study reveals, for the first time, their negative effect, by significantly enhancing
bacterial adhesion. Previous reports have shown that surface modifications on dental implants
and abutments play a relevant role in bacterial adhesion [
3
]. The chemical and physical surface
characteristics of trans-gingival implant components significantly influence bacterial attachment.
The control of bacterial infection in the peri-implant soft and hard tissues is critical for the long-term
stability of implants supporting oral rehabilitation [
4
,
10
,
21
,
22
]. There are no previous reports concerning
the relationship of the healing abutment macro-morphology (machined or grooved), used in dental
implant procedures, to clinical and microbiological parameters; however, it is clear that bacterial plaque
negatively affects peri-implant tissue stability [22].
Our results demonstrate the significantly larger bacterial attachment of both strains in the grooved
areas compared to in the smooth areas of the healing abutments, especially of F.n, which was 100
times larger. Our results are in accordance with those of Bermejo et al. that showed the significant
accumulation of pathogenic bacteria (F. nucleatum and A. actinomycetemcomitans) on moderate-roughness
compared to minimal-roughness implant surfaces [23].
Peri-implantitis presents a heterogeneous microbiota, where perio-pathogenic bacteria such as
Porphyromonas gingivalis and Prevotella intermedius/nigrescens together with asaccharolytic anaerobic
Gram-positive rods and other Gram-negative rods may be found [
24
]. The treatment of peri-implantitis
is mainly based on different mechanical and physical means to remove biofilms from the implant
surface [25].
Healing abutments are usually placed following a surgical intervention, such as implant placement
or uncovering. In a recent randomized, controlled study, either micro-grooved or machined healing
abutments were placed. After nonsurgical treatment and the reconstitution of oral hygiene measures,
the incidence of peri-implant mucositis was found to be significantly lower at the non-grooved
abutments [26].
Bacterial colonization, as found in this
in vitro
study, was enhanced in grooved healing caps.
Bacterial accumulation following implant uncovering might induce pro-inflammatory events in
the mucosa, jeopardizing optimal healing. Epithelial cell attachment may prevent the bacterial invasion
of titanium surfaces; however, early bacterial colonization on these surfaces may, in turn, downregulate
this effect. Peri-implant soft tissue clinical maturity is apparently established as early as four weeks
Appl. Sci. 2020,10, 4426 7 of 9
following implant placement in a one-stage surgical protocol, while the newly created peri-implant
crevices are colonized by bacteria within two weeks [27].
Soft tissue healing and maturation around dental implants placed in a one-stage protocol may be
at greater risk, since bacterial plaque can cause inflammation, which negatively affects osseointegration.
In the present study, only two types of bacterial lines with known morphology were tested to
avoid result masking from full plaque presenting wide morphologic variations. Since bacteria are
present within the implant–abutment connection, one-stage dental implants become contaminated even
during the first two postoperative weeks. These microorganisms can penetrate the sulcus around the
fixtures and disrupt healing and osseointegration [
28
–
30
]. Using machined smooth abutments, as was
demonstrated in the current study, may minimize bacterial adherence, which impairs osseointegration,
especially in one-stage dental implants.
The limitations of the present preliminary
in vitro
investigation are the number of samples, the fact
that only two types of pathogenic bacteria were evaluated and the specific methodology used to mimic
the clinical situation of bacterial adhesion and accumulation.
5. Conclusions
In spite of the limitations of this
in vitro
preliminary study, bacterial adherence was significantly
enhanced on grooved healing compared to machined smooth abutments.
The present
in vitro
study did not evaluate the biological consequences of higher bacterial
colonization on these abutments.
Further studies to investigate the different molecular and cellular reactions to different types of
healing abutments as well as their clinical relevance are still needed.
Author Contributions:
Conceptualization, O.M., C.E.N. and S.M.; Formal analysis, O.M., M.W. and S.M.;
Investigation, I.L., H.Z. and S.M.; Methodology, C.E.N., M.W. and S.L.; Writing—original draft, O.M. and C.E.N.;
Writing—review & editing, S.L. and S.M. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Acknowledgments:
The authors thank Alpha Bio Tec company, Petach Tikva, Israel, for providing both types of
healing caps used in this in-vitro study
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
F.n Fusobacterium nucleatum
P.g Porphyromonas gingivalis
SEM Scanning electron microscope
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