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The microbial colonization of new ceramic materials developed for abutment manufacturing was assessed. The materials used in these experiments were disks of 'as-fired' and 'rectified' ceramic material made of tetragonal zirconia polycrystals stabilized with yttrium (Y-TZP) and commercially pure grade 2 titanium (Ti) with corresponding eluates. They were tested in vitro with the following bacteria: Streptococcus mutans, S. sanguis, Actinomyces viscosus, A. naeslundii, and Porphyromonas gingivalis. Proliferation was evaluated on plates by inhibitory halos around pits, previously inoculated with eluates obtained from the materials. Bacterial adhesion on materials was quantified by spectrophotometric evaluation of the slime production by the same bacteria. Moreover, early bacterial adhesion was evaluated in human volunteers and observed with SEM. No inhibition of bacterial proliferation using eluates was observed. In vitro as-fired and rectified Y-TZP showed significantly more adherent S. mutans than did Ti disks, while S. sanguis seemed to adhere easily to Ti specimens. No differences were noted for Actinomyces spp and P. gingivalis. In vivo Y-TZP accumulated fewer bacteria than Ti in terms of the total number of bacteria and presence of potential putative pathogens such as rods. No differences were observed between rectified and as-fired Y-TZP. Overall, Y-TZP accumulates fewer bacteria than Ti. Y-TZP may be considered as a promising material for abutment manufacturing.
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The International Journal of Oral & Maxillofacial Implants 793
Bacterial Colonization of Zirconia Ceramic Surfaces:
An In Vitro and In Vivo Study
Lia Rimondini, DDS
1
/Loredana Cerroni, PhD, DDS
2
/Antonio Carrassi, DDS, MD
3
/Paola Torricelli, PhD
4
Purpose: The microbial colonization of new ceramic materials developed for abutment manufacturing
was assessed. Materials and Methods: The materials used in these experiments were disks of “as-
fired” and “rectified” ceramic material made of tetragonal zirconia polycrystals stabilized with yttrium
(Y-TZP) and commercially pure grade 2 titanium (Ti) with corresponding eluates. They were tested in
vitro with the following bacteria: Streptococcus mutans, S sanguis, Actinomyces viscosus, A naeslundii,
and Porphyromonas gingivalis. Proliferation was evaluated on plates by inhibitory halos around pits,
previously inoculated with eluates obtained from the materials. Bacterial adhesion on materials was
quantified by spectrophotometric evaluation of the slime production by the same bacteria. Moreover,
early bacterial adhesion was evaluated in human volunteers and observed with SEM. Results: No inhi-
bition of bacterial proliferation using eluates was observed. In vitro as-fired and rectified Y-TZP showed
significantly more adherent S mutans than did Ti disks, while S sanguis seemed to adhere easily to Ti
specimens. No differences were noted for Actinomyces spp and P gingivalis. In vivo Y-TZP accumulated
fewer bacteria than Ti in terms of the total number of bacteria and presence of potential putative
pathogens such as rods. No differences were observed between rectified and as-fired Y-TZP. Discus-
sion: Overall, Y-TZP accumulates fewer bacteria than Ti. Conclusion: Y-TZP may be considered as a
promising material for abutment manufacturing.
(INT J ORAL MAXILLOFAC IMPLANTS 2002;17:793–798)
Key words: bacteria, bacterial colonization, dental abutment, dental implant, plaque, zirconia
S
uprabony tissues, prosthetic structures, and the
oral environment play pivotal roles in achieving
and maintaining dental implant osseointegration.
Transgingival implant components seem to be par-
ticularly important because their characteristics are
essential in the prevention of failures and, at the
same time, can be technologically controlled. Actu-
ally, joint strength and stability, anti-rotational
effect, and sealing at the implant-abutment connec-
tion are achieved because of the rheologic and man-
ufacturing properties of implant abutments and
connecting screws,
1,2
while the external morpho-
logic characteristics of implant-abutment surfaces
may promote or delay plaque accumulation
3,4
and
consequently may help promote or prevent peri-
implant disease.
5–7
In addition, the material compo-
sition of transgingival components seems to influ-
ence the formation of epithelial attachment.
8,9
Their shape and profile are able to guide gingival
contouring
10,11
and, together with the color of the
material, strongly influence the final esthetic results
of dental implant restorations.
Ceramic transgingival components have been
introduced by many manufacturers to provide clini-
cians with more esthetic abutments than those fab-
ricated from titanium (Ti). However, since these
products are generally made of very stiff material
such as alumina, they are often affected by unpleas-
ant technologic problems because of their low resis-
tance to bending forces.
12
1
Consulting Professor, Department of Oral Pathology and Medi-
cine, University of Milan, Milan, Italy.
2
Senior Researcher, Department of Experimental Medicine and
Biochemical Science, University of Rome Tor Vergata, Rome,
Italy.
3
Full Professor and Chairman, Department of Oral Pathology and
Medicine, University of Milan, Italy.
4
Senior Researcher, Department of Experimental Surgery, Istituti
Ortopedici Rizzoli, Bologna, Italy.
Reprint requests: Dr Lia Rimondini, Department of Oral Pathol-
ogy and Medicine, University of Milan, Via Beldiletto 1/3, 20142
Milan, Italy. Fax: + 39 02 89125898. E-mail:
pib1431@iperbole.bologna.it
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794 Volume 17, Number 6, 2002
RIMONDINI ET AL
Much attention has been recently focused on
other ceramic materials commonly used in orthope-
dics, such as zirconia ceramic, which combines bio-
compatibility, pleasant esthetics, and impressive
resistance to fractures.
13
However, no information
is available on how the oral plaque colonizes zirco-
nia surfaces, although such interaction may influ-
ence the success or clinical failure of tissue integra-
tion. In fact, plaque accumulation has been
suggested to be one of the major causes of implant
failure. Anaerobic and Gram-negative species have
been observed to often be associated with peri-
implant disease.
14
The aim of the present in vitro and in vivo study
was to monitor and compare oral bacterial coloniza-
tion on the surfaces of disks fabricated from
machined grade 2 Ti and tetragonal zirconia poly-
crystal stabilized with yttrium (Y-TZP), which were
obtained using various technologies.
MATERIALS AND METHODS
Sample Preparation
Sintered samples of Y-TZP were prepared from
3YB Tosoh powder (Tosoh, Tokyo, Japan) in a liq-
uid suspension, initially pressed uniaxially at 77
MPa to obtain a cylinder fired in air at 1,500°C.
The cylinder, 6 mm in diameter, was cut into 1-
mm-thick disks using a diamond rotating wheel.
The “as-fired” disks were ground and polished
using a diamond rotating wheel.
An aliquot was flattened and highly polished
(“rectified”) on a flat surface using diamond rotat-
ing wheels with different grits in N
2
liquid atmos-
phere. Disks of similar dimensions were obtained by
cutting a bar of grade 2 commercially pure Ti. The
Ti disks were cut and polished using diamond
wheels and grinding papers. Specimens were mor-
phologically characterized with a laser profilometer
(RM 600, Rodenstock, Gottingen, Germany) with
the cutoff set at 0.25 mm and evaluation length at
2.0 mm. The arithmetic mean of the departures of
the profile from the mean line (Ra) and the average
of the 5 largest peak-to-valley heights in the evalua-
tion length (Rtm) were recorded. Profilometric data
are reported in Table 1.
All specimens were sterilized using ethylene
oxide.
An aliquot each of both Ti and as-fired and recti-
fied Y-TZP specimens was incubated in phosphate-
buffered saline (PBS) (0.2 g/mL) for 1, 3, and 5 days
at 37°C to obtain extracting solutions according to
ISO 10993-12:1998.
In Vitro Tests
Microorganisms and Growth Conditions. The organ-
isms used were Porphyromonas gingivalis
(ATCC25175), Streptococcus mutans (ATCC25175),
Streptococcus sanguis (ATCC10556), Actinomyces visco-
sus (ATCC15987), and Actinomyces naeslundii
(ATCC12104). All bacterial cultures were incubated
anaerobically at 37°C.
Agar Bacterial Inhibition Assay. The agar bacter-
ial inhibition assay is a modification of the method
described by Tseng and Wolff.
15
The strains were
pre-cultured in Schaedler broth (1% wt/v Tryptone
soy broth, Sigma-Aldrich Gallarate, Milan, Italy)
for 18 hours, and diluted broth cultures, character-
ized by a spectrophotometric absorbance value of
0.2 recorded at 540 nm, were obtained. Then, a
1:50 dilution of the adjusted bacterial broth culture
was obtained using Schaedler broth. Two hundred
microliters of each broth dilution were uniformly
flooded on agar plate containing Schaedler broth,
5% defibrinated sheep blood, 0.0005% hemin
(Sigma-Aldrich Gallarate), and 0.00005% mena-
dione (Sigma-Aldrich Gallarate). Four wells, each
0.65 mm in diameter, were made in each agar plate,
and the pits were filled with 70 µL of the extracting
solution from all specimens obtained at the differ-
ent times. PBS was used as the negative control and
a standard solution of 0.2% chlorhexidine (Sigma-
Aldrich Gallarate) as the positive control. After-
ward, cultures were incubated anaerobically for 4
days at 37°C. The diameter of the bacterial zone of
inhibition was then measured using a gauge. The
test was repeated 3 times (Fig 1).
Microbial Adhesion In Vitro Test. The amount of
adherent bacteria was quantified through evaluation
of the slime using N-lauryl-sarcosynate (Sarcosyl,
Sigma-Aldrich Gallarate) as the extraction agent
16
according to the following procedure. Standard inoc-
ula of 1 mL were obtained from broth cultures of the
different strains after 18 hours. They were character-
ized by a spectrophotometric absorbance value of 0.4
recorded at 540 nm. Each inoculum was put into the
pits of the 24-well plates, each containing the as-fired
and rectified Y-TZP and Ti specimens. Plates were
then incubated in an anaerobic environment at 37°C
for 36 hours. After this period, specimens were
removed, rinsed in PBS, and fixed in Bouin’s solution
Table 1 Profilometric Mean Values
Material Mean Ra (µm) Mean Rtm (µm)
Machined grade 2 Ti 0.22 2.16
As-fired Y-TZP 0.18 2.16
Rectified Y-TZP 0.04 1.14
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The International Journal of Oral & Maxillofacial Implants 795
RIMONDINI ET AL
at 25°C for 2 hours. They were then rinsed again in
PBS, incubated in crystal-violet, dissolved in 0.01%
PBS for 15 minutes, and rinsed twice with PBS;
finally, 200 µL of N-lauryl-sarcosynate 10% were
added to each well.
16,17
After 5 minutes of incubation
at 37°C, the lysates were finally analyzed with a
microspectrophotometer at 540 nm.
Seven specimens of each material were evaluated
for each bacterial strain.
16,17
In Vivo Experiment
The experiment was performed after recruiting 10
volunteers ranging in age between 20 and 23 years
with good oral and general health conditions and
high standards of oral hygiene. Informed consent
was obtained from each participant. None had
taken antibiotics or used an antibacterial solution
during the 3 months prior to the experimentation.
Silicone stents were produced, and 3 specimens,
1 of each type, were mechanically fixed into the sil-
icone during its polymerization. A stent carrying
the specimens was fixed onto the buccal region of
the molar and premolar of each volunteer using
orthodontic wires. After stent placement, the sub-
jects suspended any oral hygiene procedures for 24
hours. The specimens were then removed and
processed for scanning electron microscopy (SEM)
(840A, Jeol, Tokyo, Japan) as follows: fixed in 2.5%
glutaraldehyde water solution for 2 hours; buffered
in sodium cacodylate 1 mol/L; rinsed in water;
dehydrated in alcohol (50%, 70%, 80%, 90%,
100% for 10 minutes each); dehydrated at CO
2
top
critical point in a bomb (Top Critical Point 30, W.
Pabish, Pero, Milan, Italy); and finally coated with
a 20-nm-thick gold-palladium layer in a coating
unit (Coating Unit E5100, Polaron Equipment,
Watford, United Kingdom). The specimens were
evaluated with SEM as previously described using
the secondary electron mode working between 5
and 15 Kv. A global area of 100 125 µm was
examined for each specimen. The area was the sum
of 5 fields of equal dimensions, which were ran-
domly selected on the specimen surface using a
grid. For each field the following variables were
recorded: presence (= 1) or absence (= 0) of cocci,
short rods (< 10 µm), and long rods (> 10 µm). The
presence of occasionally adherent epithelial cells
was similarly recorded. The cumulative values for
each area were used for statistical evaluation. An
index of the amount of bacteria was calculated for
each area by summing the following scores
recorded in each field: number of bacteria 5,
score = 0; number of bacteria 6 to 30, score = 1;
number of bacteria 31 to 100, score = 2; number of
bacteria more than 100, score = 3.
3,18
Statistical Analysis
One-way analyses of variance (ANOVA) and
Scheffé tests were used to compare slime data. Sta-
tistical analysis of in vivo experimentation was per-
formed using data from the cumulative area with
the Kruskal-Wallis ANOVA exact test followed by
the Mann-Whitney U test to compare substrates
with each other. The Monte Carlo method was used
for probability calculation. The significance level
was set at P < .05.
RESULTS
In Vitro Tests
Analysis of the bacteria assay showed no inhibition
of bacteria proliferation. The behavior of all the
eluates obtained from the different materials was
comparable to that recorded with negative controls.
However, differences were observed in the adhesion
test with respect to the slime production (Table 2).
S mutans was the most adherent strain, producing
more slime on as-fired Y-TZP than on rectified and
Ti specimens (P < .01). In contrast, S sanguis
seemed to adhere to grade 2 Ti easily, with the P
value approaching significance (P < .1). No differ-
ences were observed with regard to Actinomyces spp
and P gingivalis.
In Vivo Experiment
Table 3 shows the index of bacteria density recorded
on the specimen surfaces and the presence of the
different morphotypes. Both Y-TZP surfaces accu-
mulated significantly fewer bacteria than Ti, with a
Fig 1 Example of 1 assay using test materials (Titanium 4-T2P),
positive control (0.2% chlorhexidine), and negative control (PBS).
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796 Volume 17, Number 6, 2002
RIMONDINI ET AL
prevalence of cocci and the absence of long rods.
The Ti surfaces appeared more uniformly coated
with a structured biofilm made of pellicle and bacte-
ria (Fig 2), whereas the Y-TZP surfaces appeared
colonized by clusters of bacteria (Fig 3). No differ-
ences were observed between as-fired and rectified
surfaces in terms of the amount of bacteria and
presence of morphotypes. Epithelial cells were
observed on some rectified Y-TZP specimens.
DISCUSSION
The purpose of the present study was to evaluate in
vitro the capability of different strains of oral bacte-
ria to adhere to and grow on Ti and as-fired Y-TZP
specimens whose Ra roughness values, ranging
from 0.18 to 0.22 µm on average, may be compara-
ble to abutments available commercially (observed
to range from 0.10 to 0.30 µm).
19
Adhesion and
Fig 2 SEM micrograph of bacteria colonizing an as-fired Y-TZP
surface. A cluster of few cocci is observable (original magnifica-
tion 6,000; bar = 1 µm).
Fig 3 SEM micrograph of bacteria colonizing a Ti surface. An
homogeneous layer of cocci covers the specimen surface. Rods
are apparent (original magnification 6,000; bar = 1 µm).
Table 3 Density Index and Presence of Cells on the Different Substrates in Vivo
(Mean ± SD)
Material BDI Cocci Short rods Long rods Keratinocytes
As-fired Y-TZP 6.0 ± 4.9* 3.7 ± 0.8 0.7 ± 1.3* 0.1 ± 0.4* 0.1 ± 0.0
Rectified Y-TZP 8.3 ± 3.1* 5.0 ± 0.3 1.3 ± 1.8 0.0 ± 1.2* 1.0 ± 0.6
Machined Ti 11.3 ± 4.3* 4.3 ± 1.2 3.3 ± 1.8* 0.8 ± 0.9* 0.1 ± 0.0
P
< .5 < .5 < .5 < .5 NS
NS = not significant.
*P < .05, comparison between zirconia and titanium substrates (Mann-Whitney U test with Monte Carlo method to com-
pute probability).
Kruskal-Wallis ANOVA exact test with Monte Carlo method to compute probability.
Table 2 Spectrophotometric Evaluation of Slime (Means ± SDs)
As-fired Rectified Machined P
Bacteria Y-TZP (nm) Y-TZP (nm) Ti (nm) (1-way ANOVA)
S mutans 0.48 ± 0.02* 0.27 ± 0.01* 0.33 ± 0.01* .01
S sanguis 0.09 ± 0.01 0.13 ± 0.01 0.18 ± 0.01 NS
A viscosus 0.15 ± 0.01 0.14 ± 0.01 0.16 ± 0.01 NS
A naeslundii 0.21 = 0.01 0.30 ± 0.01 0.20 ± 0.01 NS
P gingivalis 0.08 ± 0.02 0.09 ± 0.00 0.11 ± 0.01 NS
NS = not significant.
*P < .01 (Scheffe test).
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The International Journal of Oral & Maxillofacial Implants 797
RIMONDINI ET AL
growth on highly polished rectified Y-TZP surfaces,
which are 2 times more flat and polished than those
of commercially available abutments, were also
evaluated. Additionally, this study included an in
vivo experiment to investigate early colonization in
the presence of composite plaque, salivary pellicle,
and removal force related to salivary flow, muscles,
and chewing activity.
No antimicrobial activity was detected for either
Ti or zirconia materials using the killing curve test
in vitro on the strains used. Such a finding is not
surprising. The antimicrobial activity of Ti is very
controversial and directly related to the concentra-
tion of Ti ions and test sensitivity.
20
No previous
data are available on zirconia as far as oral strains
are concerned.
The adhesion tests for the indirect evaluation of
the biofilm amount by assessment of slime and
lipopolysaccharide membranes detached by sarcosyl
showed differences between Ti and ceramic sur-
faces. S mutans adhered to ceramic surfaces more
easily than other pioneer bacteria, such as S sanguis,
which seemed to have more affinity to Ti than to Y-
TZP surfaces. Moreover, no differences were noted
between S sanguis adhesion to highly polished recti-
fied and as-fired Y-TZP surfaces. This latter obser-
vation is apparently at odds with common in vivo
findings, according to which rougher surfaces accu-
mulate more bacteria.
21
This phenomenon is in fact
generally enhanced in vivo because of the sheltering
effect of the rough surfaces against the removal
forces that are lacking under in vitro experimental
conditions. Moreover, other factors, such as chemi-
cal and physical properties of the surfaces (surface
wettability of materials combined with bacterial
strains used), may overrule those chemico-physical
aspects such as roughness,
22
which may modify the
surface chemical composition and wettability,
23,24
especially in short-term tests such as the present
experiment.
The use of an in vivo early colonization model
offers the opportunity to evaluate materials in a
simulated clinical condition presenting composite
plaque, salivary pellicle, and removal forces.
According to the present results, the Y-TZP sur-
faces accumulated fewer bacteria than Ti, with a
prevalence of cocci, few short rods, and no long
rods, which is suggestive of an immature plaque.
25
No significant differences were noted between
as-fired and rectified zirconia in plaque coloniza-
tion, although the rectified surfaces were smoother
than the as-fired surfaces. A possible explanation
may be the roughness threshold of clinical relevance
(about Ra = 0.2 µm) observed in both early
3
and
long-term in vivo models.
4
Finally, the many ectopic epithelial cells occa-
sionally observed on Y-TZP surfaces seem to sug-
gest that Y-TZP might be a promising material
capable of enhancing epithelial attachment forma-
tion, although this hypothesis has not been tested.
CONCLUSION
Within the limits of this study, zirconia ceramic
may be a suitable material for manufacturing
implant abutments, with a low bacterial coloniza-
tion potential. High polishing of zirconia ceramic
does not seem to offer any advantages over the as-
fired material in terms of colonization.
ACKNOWLEDGMENTS
The authors would like to thank Benax s.r.l. (Ancona, Italy),
Sudimplant (Tolouse, France), and Progetto Finalizzato MSTA
II of the National Research Council for financial support, and
Roberto Chiesa (Politecnico-Milan), Giuseppe Magnani (Enea),
Stefano Frangini (Enea), and Corrado Piconi (Enea) for speci-
mens manufacturing and characterization.
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... According to Hauslich, et al. [7], increasing surface roughness increases bacterial adhesion to implant surfaces. However, other studies (12)(13)(14)(15)(16)(17)(18)(19)(20) have shown that surface roughness only affects the early stage of biofilm development. Torsten and Zhao [8,9], on the other hand, have shown that roughness is not the only determinant of bacterial adhesion. ...
... This phenomenon may explain why similar protein-binding properties on titanium and zirconium oxide have been reported and why zirconia did not show any reduced bacterial adhesion [8]. A randomized clinical trial, performed in vivo and in vitro, revealed no significant difference in the colonized surface area in the different discs (p=0.0730) as well as a high percentage of coverage by biofilm on all materials tested (90.9% of the total surface area of zirconia and 84.14% on machined titanium) [18]. Nevertheless, the number of studies on bacterial adhesion on zirconia remains very low, and other studies should be carried out on a larger number of patients to confirm the experimental results found [19][20][21][22][23][24]. ...
... On the other hand, zirconia implants seem to induce less biofilm formation compared to titanium, resulting in less risk of peri-implantitis [20,21]. However, some recent studies suggest a similar effect of zirconium compared to titanium implants regarding its biocompatibility, osteoconductivity, physical properties, allergenicity, and biofilm formation [22][23][24][25][26]. ...
... Therefore, the challenge is to find an ideal surface roughness that combines optimal bone fixation with minimal bacterial retention. In this context, the advantage of Y-TZP is its excellent biocompatibility and its ability to limit bacterial proliferation [20,21]. ...
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The osseointegration of implants is defined as the direct anatomical and functional connection between neoformed living bone and the surface of a supporting implant. The biological compatibility of implants depends on various parameters, such as the nature of the material, chemical composition, surface topography, chemistry and loading, surface treatment, and physical and mechanical properties. In this context, the objective of this study is to evaluate the biocompatibility of rough (Ra = 1 µm) and smooth (Ra = 0 µm) surface conditions of yttria–zirconia (Y-TZP) discs compared to pure zirconia (ZrO2) discs by combining a classical toxicological test, morphological observations by SEM, and a transcriptomic analysis on an in vitro model of human Saos-2 bone cells. Similar cell proliferation rates were observed between ZrO2 and Y-TZP discs and control cells, regardless of the surface topography, at up to 96 h of exposure. Dense cell matting was similarly observed on the surfaces of both materials. Interestingly, only 110 transcripts were differentially expressed across the human transcriptome, consistent with the excellent biocompatibility of Y-TZP reported in the literature. These deregulated transcripts are mainly involved in two pathways, the first being related to “mineral uptake” and the second being the “immune response”. These observations suggest that Y-TZP is an interesting candidate for application in implantology.
... Implant-supported overdentures with three to four implants are sufficient in offering desired results. [4] This in vitro study compared ZrO 2 , PEEK, and ZrO 2 -PEEK telescopic attachments in terms of retention of overdenture. ...
... In addition to the material itself, the characteristics of the surface play a major role in cellular attachment. Cells are known to behave differently on different surface morphologies, and a rough surface favors bacterial attachment [21][22][23]. On polished surfaces, human gingival fibroblasts (HGF) spread without any particular orientation [24]. ...
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Applying antibacterial coatings to dental implant materials seems reasonable but can have negative influences on desired cell adhesion and healing. In this study, zirconia abutment specimens interacting with gingival tissue were used. The aim was to compare the influence of machined or coated zirconia surfaces on the adhesion and proliferation of human gingival fibroblasts (HGF-1). Surface modifications were performed using atmospheric plasma coating with hydroxyapatite, zinc, and copper. Zirconia specimens were divided into four groups: hydroxyapatite, hydroxyapatite with zinc oxide (ZnO), hydroxyapatite with copper (Cu), and an untreated machined surface. After the characterization of the surface conditions, the morphology of adhered HGF-1 was determined by fluorescence staining and subjected to statistical evaluation. The visual analysis of cell morphology by SEM showed flat, polygonal, and largely adherent fibroblast cells in the untreated group, while round to partially flat cells were recorded in the groups with hydroxyapatite, hydroxyapatite + ZnO, and hydroxyapatite + Cu. The cell membranes in the hydroxyapatite + ZnO and hydroxyapatite + Cu groups appeared porous. The results show that HGF-1 adhere and proliferate well on machined zirconia, while plasma coating with hydroxyapatite or hydroxyapatite mixtures does not lead to increased adhesion or proliferation.
... Yttria-stabilized polycrystalline tetragonal zirconia (Y-TZP) has been widely used in dental implants due to its esthetic and mechanical results [7,8] and has also been associated with a significant reduction in the formation of oral biofilms [9][10][11][12][13]. Zirconia implants, as stated in recent systematic and critical reviews, are described as a promising alternative to Titanium implants, for showing similar results in the clinical parameter of direct contact surface between bone and implant, as well as for adequate chewing load dissipation [14][15][16][17]. ...
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The aim of this study was to evaluate gingival fibroblasts and human osteoblasts’ response to textured Nd:YAG laser microgrooves, with different dimensions, on zirconia implant surfaces. A total of 60 zirconia disks (8 mm in diameter and 2 mm in thickness) were produced and divided between four study groups (N = 15): three laser-textured (widths between 125.07 ± 5.29 μm and 45.36 ± 2.37 μm and depth values from 50.54 ± 2.48 μm to 23.01 ± 3.79 μm) and a control group without laser treatment. Human osteoblasts and gingival fibroblasts were cultured on these surfaces for 14 days. FEG-SEM (Field Emission Gun–Scanning Electron Microscope) images showed cellular adhesion at 24 h, with comparable morphology in all samples for both cell types. A similar cell spreading within the grooves and in the space between them was observed. Cell viability increased over time in all study groups; however, no differences were found between them. Additionally, proliferation, ALP (Alkaline phosphatase) activity, collagen type I, osteopontin and interleukin levels were not significantly different between any of the study groups for any of the cell types. Analysis of variance to compare parameters effect did not reveal statistically significant differences when comparing all groups in the different tests performed. The results obtained revealed similar cell behavior based on cell viability and differentiation on different microtopographic laser grooves, compared to a microtopography only established by sandblasting and acid-etching protocol, the reference surface treatment on zirconia dental implants.
... Thus, evaluation of the peri-implant soft-tissue parameters was certainly positive, supporting the observations of previous authors regarding effective adaptation at the gingiva-Zr interface [34]. Furthermore, the absent or scarce plaque accumulation and little sulcus bleeding observed across the cases may be explained by the previously demonstrated lesser bacterial attachment related to Zr implants [35]-ultimately resulting in reduced inflammation and a lesser prevalence of mucositis and/or peri-implantitis as compared to traditional Ti implants. However, these results are to be interpreted with caution, due to the limited sample size and considering that all implants were being evaluated in healthy patients with good oral hygiene and suitable bone volume. ...
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The purpose of this clinical series was to evaluate the clinical and radiographic outcomes of 20 zirconia dental implants, with a minimum follow-up of two years. Patients with at least one zirconia dental implant were included, with a mean follow-up of 34.05 months. The patient complaints such as pain and foreign body sensation, as well as clinical parameters including peri-implant infections with suppuration, implant mobility, gingival index (GI), modified plaque index (mPI), modified sulcus bleeding index (mBI), probing depth (PD) and radiological distance between the implant shoulder to the closest mesial and distal bone-to-implant contact (BIC), were assessed. All zirconia implants were successfully integrated without any pain or foreign body sensation. No mobility was detected in any of the 20 implants. Clinical examination revealed a mean PD of 2.56 mm and a radiological mean distance between the implant shoulder and the initial site of visible bone-to-implant contact (BIC) of 1.44 mm. In addition, GI, mPI and mBI indicated minimal to no inflammation. Results obtained from this series suggest that one-piece zirconia dental implants achieve good clinical and radiographic outcomes over a mean follow-up of 34 months and may be deemed a good option in patients with favorable bone conditions.
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Purpose: A novel implant model consisting of meso-scale cactus-inspired spikes and nano-scale bone-inspired trabeculae was recently developed to optimize meso-scale roughness on zirconia. In this model, the meso-spike dimension had a significant impact on osteoblast function. To explore how different nano-textures impact this model, here we examined the effect of different nano-trabecula sizes on osteoblast function while maintaining the same meso-spike conformation. Methods: Zirconia disks with meso-nano hybrid surfaces were created by laser etching. The meso-spikes were fixed to 40 μm high, whereas the nano-texture was etched as large and small trabeculae of average Feret diameter 237.0 and 134.1 nm, respectively. A polished surface was also prepared. Rat bone marrow-derived and human mesenchymal stromal cell-induced osteoblasts were cultured on these disks. Results: Hybrid rough surfaces, regardless of nano-trabecula dimension, robustly promoted the osteoblastic differentiation of both rat and human osteoblasts compared to those on polished surfaces. Hybrid surfaces with small nano-trabeculae further enhanced osteoblastic differentiation compared with large nano-trabeculae. However, the difference in osteoblastic differentiation between small and large nano-trabeculae was much smaller than the difference between the polished and hybrid rough surfaces. The nano-trabecula size did not influence osteoblast attachment and proliferation, or protein adsorption. Both hybrid surfaces were hydro-repellent. The atomic percentage of surface carbon was lower on the hybrid surface with small nano-trabeculae. Conclusions: Small nano-trabeculae promoted osteoblastic differentiation more than large nano-trabeculae when combined with meso-scale spikes. However, the biological impact of different nano-trabeculae was relatively small compared with that of different dimensions of meso-spikes.
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Animal pathology models play an important role in elucidating pathology, evaluating drug efficacy, and studying therapeutic. This paper presents the creation of rats’ renal failure model by embolizing the renal artery using radiopaque hydrogel microfibers. By using a dual coaxial laminar flow microfluidic device, barium alginate gel microfibers containing zirconia particles were fabricated. Since the zirconia particles are radiopaque, the microfiber can be delivered and embolized while confirming the position of the microfiber in real-time under X-ray imaging. The delivery of microfibers through a catheter into the renal artery of rats to create a renal failure model was tested. The radiopaque microfibers were visualized by X-ray imaging when delivered by a catheter. Furthermore, the microfibers remained stable for 2 weeks after delivery. In addition, by adjusting the number of microfibers, two different rat models with severe and mild renal failure conditions were created. The proposed delivery of radiopaque hydrogel microfibers to create an embolization model is expected to be an effective approach to control the degree of symptoms and elucidate the pathology.
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Objectives This study aimed to assess the independent influence of grooves and pores texturized by milling on gold-standard zirconia implant surfaces. Methods Milled groove and pore textured with equivalent width, depth, and spacing on zirconia discs were produced using press and sintering techniques. All samples were sandblasted and acid-etched (SBAE), and untextured discs were used as controls. Osteoblasts and fibroblasts were cultured on discs for 14 days. Field emission gun-scanning electron microscopy (FEG-SEM) was used to observe cellular adhesion and morphology. Cell viability and proliferation assays were performed. Additionally, alkaline phosphatase activity, collagen type I, and osteopontin were evaluated at pre-defined time points. Results are presented as mean and standard deviation (SD), group comparisons were tested using one-way ANOVA (Tukey’s post-hoc), and significance was set at P <0.05. Results FEG-SEM images revealed cellular adhesion at 24 h in all samples with differences in distribution. Although both cell lines showed increased cell viability and differentiation cell markers such as collagen and osteopontin over time, statistically significant differences between groups were found in none of the quantitative study variables (P >0.05). Conclusion The results suggest similar cellular behavior between different patterns with similar dimensions and between them and microtopography by SBAE protocol currently used as the gold-standard for zirconia dental implants. The addition of pore and groove microtextures to the gold-standard zirconia dental implant surfaces treated with SBAE does not seem to be an asset in the cellular behavior of the hard and soft tissue cells.
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Objectives Oral bacterial adhesion on dental implant materials has been extensively studied using in vitro systems but has yielded results restricted to in vitro growth patterns due to limitations in species selection, sustained fastidious anaerobe growth, and mixed culture longevity. The aim of this study was to develop an oral bacterial biofilm model consisting of colonizers representative of the oral microbiome exhibiting temporal shifts characteristic of plaque development and maturation in vivo. Methods Streptococcus oralis, Actinomyces naeslundii, Aggregatibacter actinomycetemcomitans, Veillonella parvula, Fusobacterium nucleatum, and Porphyromonas gingivalis were grown in monoculture prior to combination in mixed culture. Commercially pure titanium (cpTi) and yttria-stabilized zirconia (ZrO2) disks with polished, acid-etched, or sandblasted surfaces were prepared to evaluate oral bacterial adhesion. After 6 h, 1, 3, 7, 14 and 21 days, genomic DNA from planktonic and adherent bacteria was isolated. Quantitative polymerase chain reaction (qPCR) was used to enumerate the amount and proportion of each species. Results Early-colonizing S. oralis and A. actinomycetemcomitans, dominated after 6 h prior to secondary colonization by F. nucleatum and V. parvula in planktonic (1 day) and sessile (3 days) form. A. naeslundii maintained relatively low but stable bacterial counts throughout testing. After 14 days, late-colonizing P. gingivalis became established in mixed culture and persisted, becoming the dominant species after 21 days. The composition of adherent bacteria across all substrates was statistically similar at all timepoints with notable exceptions including lower S. oralis bacterial counts on polished cpTi (3 days). Significance Within the present model’s limitations, multispecies oral bacterial attachment is similar on surface-treated cpTi and ZrO2.
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Bacterial adhesion to intra-oral, hard surfaces is firmly influenced by the surface roughness of these structures. Previous studies showed a remarkable higher subgingival bacterial load on rough surfaces when compared to smooth sites. More recently, the additional effect of a further smoothening of intra-oral hard surfaces on clinical and microbiological parameters was examined in a short-term experiment. The results indicated that a reduction in surface roughness below Ra=0.2 μm, the so-called “threshold Ra”, had no further effect on the quantitative/qualitative microbiological adhesion or colonisation, neither supra- nor subgingivally. This study aims to examine the long-term effects of smoothening immoral hard transgingival surfaces. In 6 patients expecting an overdenture in the lower jaw, supported by endosseus titanium implants, 2 different abutments (transmucosal part of the implant): a standard machined titanium (Ra=0.2 μm) and one highly polished and made of a ceramic material (Ra=0.06 μm) were randomly installed. After 3 months of intra-oral exposure, supra- and subgingival plaque samples from both abutments were compared with each other by means of differential phase-contrast microscopy (DPCM). Clinical periodontal parameters (probing depth, gingival recession, bleeding upon probing and Periotest-value) were recorded around each abutment. After 12 months. the supra- and subgingival samples were additionally cultured in aerobic, CO,-enriched and anaerobic conditions. The same clinical parameters as at the 3-month interval were recorded after 12 months. At 3 months, spirochetes and motile organisms were only detected subgingivally around the titanium abutments. After 12 months, however, both abutment-types harboured equal proportions of spirochetes and motile organisms, both supra- and sub-gingivally. The microbial culturing (month 12) failed to detect large interabutment differences. The differences in number of colony forming units (aerobic and anaerobic) were within one division of a logarithmic scale. The aerobic culture data showed a higher proportion of Gram-negative organisms in the subgingival flora of the rougher abutments. From the group of potentially “pathogenic” bacteria, only Prevotellu inter-media and Fusobacterium nucleatum were detected after anaerobic culturing and again the inter-abutment differences were negligible. Clinically, the smoothest abutment showed a slightly higher increase in probing depth between months 3 and 12, and more bleeding on probing. The present results confirm the findings of our previous short-term study, indicating that a further reduction of the surface roughness, below a certain “threshold Ra”(0.2 μm), has no major impact on the supra- and subgingival microbial composition.
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Escherichia coli of serotype O157:H7 are Vero cytotoxin-producing enteric pathogens that have been associated recently with sporadic cases and outbreaks of hemorrhagic colitis and with the hemolytic-uremic syndrome. Adherence of many enteropathogenic bacteria to mucosal surfaces is a critical step in the pathogenesis of diarrheal disease. We showed previously that adherence of E. coli O157:H7 strain CL-56 to epithelial cells in vitro is inhibited by outer membranes. In this study we examined whether outer membranes from a series of E. coli O157:H7 strains mediated competitive inhibition of bacterial binding to epithelial cells grown in tissue culture. We also determined which constituents of the outer membrane mediated inhibition of CL-56 adherence. Binding of six O157:H7 strains to HEp-2 cells was determined by quantitating the number of adherent bacteria in the presence and absence of outer membranes which were extracted from each strain with N-lauroyl sarcosinate (1.7%, wt/vol). After separation of outer membranes by gel electrophoresis, four bands (94, 40, 36, and 30 kDa) were collected by electroelution. Immune sera were raised in rabbits to each of the four eluted bands. Outer membrane extracts from each of the six O157:H7 strains inhibited binding of homologous organisms to the HEp-2 cells. At dilutions which did not cause bacterial agglutination, antiserum raised against the 94-kDa outer membrane protein showed maximal inhibition of bacterial adherence (17.0 +/- 7.3% adherence of control levels). Growth of bacteria in iron-depleted broth did not affect their binding to HEp-2 cells, suggesting that iron-regulated outer membranes were not involved. Fluid accumulation in ileal ligated loops of rabbits in response to E. coli O157:H7 challenge was diminished following both parenteral immunization with outer membranes extracted from the homologous strain and coincubation of organisms with immune serum which contained antibodies to outer membrane extracts. These data indicate that outer membranes are competitive inhibitors of E. coli O157:H7 adherence. Specific constituents of the outer membrane may function as bacterial attachment factors (i.e., adhesins) for E. coli O157:H7 adherence to epithelial cell surfaces.
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The aim of the present investigation was to assess the effect of de novo plaque formation on the gingiva and masticatory mucosa around teeth and implants. The study was performed in 5 beagle dogs which at the initiation of the experiment were 15 months old. During a preparatory period, the mandibular right premolars were extracted, 3 fixtures installed, abutment connection performed and a 4-month period of plaque control completed. A clinical examination was performed and biopsies of the second mandibular premolar (P2) and the contralateral implant site (2P) were sampled. The dogs were allowed to form plaque during a period of 3 weeks. The clinical examination was repeated and biopsies harvested from the 2 remaining implants and the contralateral tooth sites. The tissue samples were prepared for histometric and morphometric analysis. Both the masticatory mucosa at implants and the gingiva responded to de novo plaque formation with the development of an inflammatory lesion. The size as well as the composition of the lesions in the 2 tissues had many features in common. It was concluded that the mucosa around implants and the gingiva around teeth had a similar potential to respond to early plaque formation.
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The purposes of this investigation were to evaluate and compare the antimicrobial effect of stannous fluoride (SnF2) gel and other commonly used oral antimicrobial agents/products. The antibacterial inhibitory effect of the various agents was evaluated by their effect against oral plaque bacteria including strains of Streptococcus sanguis, Streptococcus sobrinus, Actinomyces viscosus, Actinobacillus actinomycetemcomitans, Bacteroides gingivalis and Bacteroides intermedius. A lawn of the specific bacteria to be tested was placed onto blood agar plates. Wells were then punched into the agar and each well was filled with 75 microliters of one of the antimicrobial products or control solutions. A positive control was a 0.12% chlorhexidine solution and a negative control was physiological saline. Agar plates were incubated in an anaerobic chamber at 37 degrees C for 5-7 days. Zones of inhibition in the lawn of bacteria were measured by a boley gauge. Each experiment was performed in duplicate and mean zones of bacterial inhibition were determined. Only 0.4% SnF2 and 0.12% chlorhexidine were consistently more effective in inhibiting oral bacteria when compared with other commercial mouth rinses/agents which had any one of a variety of antimicrobial agents as ingredients (alpha = 0.05). This in vitro study demonstrates that stannous fluoride gel is as effective as chlorhexidine in inhibiting the growth of bacteria often found in dental plaque.
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In the present animal experiment, analyses and comparisons were made between the structure and composition of clinically healthy supraalveolar soft tissues adjacent to implants and teeth. 5 beagle dogs were used. The right mandibular premolar region was selected in each dog for placement of titanium implants, while the left mandibular premolar region served as control. Extractions of the mandibular premolars were preformed, healing allowed, following which titanium fixtures were installed in the edentolous premolar region. Abutment connection was carried out 3 months later. After another 2 months of healing, plaque control was initiated and maintained for 8 weeks. At the end of the plaque control period, clinical examinations were performed and biopsies harvested from the implant site and the contralateral premolar tooth region. Following fixation and decalcification, all tissue samples were embedded in EPON and examined by histometric and morphometric means. The result from the analyses demonstrated that the periimplant mucosa which formed at titanium implants following abutment connection had many features in common with gingival tissue at teeth. Thus, like the gingiva, the peri-implant mucosa established a cuff-like barrier which adhered to the surface of the titanium abutment. Further, both the gingiva and the peri-implant mucosa had a well-keratinized oral epithelium which was continuous with a junctional epithelium that faced the enamel or the titanium surface. In the periimplant mucosa, the collagen fibers appeared to commence at the marginal bone and were parallel with the abutment surface. All gingival and periimplant units examined were free from infiltrates of inflammatory cells. It was suggested that under the conditions of study, both types of soft tissues, gingiva and periimplant mucosa, have a proper potential to prevent subgingival plaque formation.
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We have described the morphology of developing plaque on cementum in an in vivo human model. Slabs of cementum obtained from sound teeth, rendered anorganic with 5% sodium hypochlorite, were glued to orthodontic brackets and positioned on the upper canines, premolars and molars in 8 volunteers. The brackets were removed after 2, 4, 8, 24 h and processed for scanning electron microscopy (SEM). Within 2 h, a thin pellicle covered the cementum surface, with few micro-organisms detectable. At 4 and 8 h, coccoid plaques were present. Filaments inserted perpendicularly to the plaque surface were seen at 24 h. The results indicate that early bacterial colonization of human cementum is a selective process, mediated by an organic pellicle and mainly involves cocci.
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Dental implant research has been mostly concerned with the biocompatibility of materials for implantation. In this study the effects of titanium dioxide and other metallic salts on seven bacterial species commonly found in dental plaque, two which are uncommon, and a yeast, were determined by agar incorporation and diffusion techniques, and compared with the effects of a titanium implant abutment. Neither the titanium dioxide nor the implant abutment demonstrated any inhibitory activity, although other compounds such as cobalt used in dental alloys had some effects.
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The sensitivity of the outer and cytoplasmic membranes of Escherichia coli to detergent was examined by isopycnic sucrose density gradient centrifugation. Sodium lauryl sarcosinate (Sarkosyl) was found to disrupt the cytoplasmic membrane selectively under conditions in which Triton X-100 and dodecyl sodium sulfate solubilized all membrane protein. These results were verified by gel electrophoresis; membrane proteins solubilized by Sarkosyl were identical to those of the cytoplasmic membrane. The presence of Mg(2+) during treatment with Sarkosyl was found to afford partial protection of the cytoplasmic membrane from dissolution.
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The purpose of this study was to compare the clinical and microbiological (microscopic) parameters during the development of experimental gingivitis and experimental peri-implant mucositis. Twenty partially edentulous patients were treated for moderate to advanced periodontal disease. Following active periodontal therapy consisting of motivation, instruction in oral hygiene practices, scaling and root planing and periodontal surgery where indicated, IMZ oral implants were incorporated in posterior edentulous areas. After 3 months of healing, the prosthetic abutments were connected, and the patients were closely supervised for another 2 months of healing. At this time, baseline data were obtained. Re-examinations were scheduled at 3 and 6 months. Following this, the patients were asked to refrain from oral hygiene practices for 3 weeks. At all examinations including the end of the period of no oral hygiene, the following clinical parameters were obtained: Plaque Index, Gingival Index and Sulcus Bleeding Index, all modified by Mombelli et al. (1987), probing pocket depths and recession in mm. Furthermore, submucosal/subgingival plaque samples were obtained and analyzed using phase contrast microscopy. At the end of the 3-week period of plaque accumulation, optimal oral hygiene was reinstituted. There were no statistically significant differences between the mean values of all parameters at implant compared to tooth sites at any observation periods. The period of no oral hygiene demonstrated a similar cause-effect relationship between the accumulation of bacterial plaque and the development of peri-implant mucositis as established for the gingival units by the experimental gingivitis model.