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Cytokines and Growth Factors Involved in the

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 Volume 78 • Number 4

Cytokines and Growth Factors Involved

in the Osseointegration of Oral Titanium
Implants Positioned Using Piezoelectric
Bone Surgery Versus a Drill Technique:
A Pilot Study in Minipigs
Giulio Preti,* Germana Martinasso,† Bruno Peirone,‡ Roberto Navone,* Carlo Manzella,*
Giuliana Muzio,† Crescenzo Russo,* Rosa A. Canuto,† and Gianmario Schierano*

Background: Most dental implants are positioned using a

drilling surgery technique. However, dentistry recently experi-
enced the implementation of piezoelectric surgery. This tech-
nique was introduced to overcome some of the limitations
involving rotating instruments in bone surgery. This study
used biomolecular and histologic analyses to compare the
osseointegration of porous implants positioned using tradi-

T
o enhance peri-implant osteogen-
tional drills versus the piezoelectric bone surgery technique. esis and reduce the bone healing
Methods: Porous titanium implants were inserted into mini- time after implant site preparation,
pig tibias. Histomorphology and levels of bone morphogenetic the biologic factors that are involved
protein (BMP)-4, transforming growth factor (TGF)-b2, tumor in these processes must be evaluated.
necrosis factor-alpha, and interleukin-1b and -10 were evalu- Literature supports the hypothesis that a
ated in the peri-implant osseous samples. porous implant surface is more effective
Results: Histomorphological analyses demonstrated that in stimulating peri-implant osteogenesis
more inflammatory cells were present in samples from drilled than is a machined one using traditional
sites. Also, neo-osteogenesis was consistently more active in drilling.1-5 When bone formation was
bone samples from the implant sites that were prepared using evaluated by histologic analysis,5 levels
piezoelectric bone surgery. Moreover, bone around the im- of biologic factors, bone morphogenetic
plants treated with the piezoelectric bone surgery technique protein (BMP)-4 and transforming growth
showed an earlier increase in BMP-4 and TGF-b2 proteins as factor (TGF)-b, increased earlier around
well as a reduction in proinflammatory cytokines. porous implants.
Conclusion: Piezoelectric bone surgery appears to be more BMP-4 contains several proteins that
efficient in the first phases of bone healing; it induced an earlier have bone-inducing capacities. BMPs
increase in BMPs, controlled the inflammatory process better, are key regulators of osteoblast and
and stimulated bone remodeling as early as 56 days post- chondrocyte differentiation during skele-
treatment. J Periodontol 2007;78:716-722. tal development and of osteogenic differ-
entiation in healing fractures.6-9 TGF-b
KEY WORDS
stimulated bone formation in vivo,10,11
Bone morphogenetic proteins; cytokines; dental implants. which may have resulted from the stimu-
lated proliferation of osteoblast precur-
sors rather than stimulated osteoblastic
* Department of Biomedical Sciences and Human Oncology, Section of Prosthetic
Dentistry, School of Dentistry, University of Turin, Turin, Italy. differentiation.12 Thus, it may be postu-
† Department of Experimental Medicine and Oncology, University of Turin. lated that TGF-b could stimulate BMP ac-
‡ Department of Animal Pathology, University of Turin.
tivity in the early phases of bone healing,
just before the BMPs exert their effects.
Dental implants are positioned most
commonly using a surgery technique

doi: 10.1902/jop.2007.060285

716
J Periodontol • April 2007
that uses traditional drilling. Recently, piezoelectric
surgery, which takes advantage of ultrasound vibra-
tions, was introduced in an effort to overcome some
of the limitations of rotating instruments in bone sur-
gery while increasing the cutting precision.13-15
As early as 1961, McFall et al.16 experimented with
ultrasound surgery on bone. In 1975, Horton et al.17
demonstrated that the use of ultrasound was less trau-
matic than the use of rotating instruments in an exper-
imental study on dogs. In 1981, Horton et al.18 used
ultrasound instrumentation on 50 patients to perform
extractions, osteoplastic surgery, and periodontal
bone therapy surgery. They concluded that ultra-
sound surgery allowed for the precise removal of bone
tissue and controlled bleeding.
Vercellotti19 developed an instrument for piezoelec-
tric bone surgery§ that uses ultrasound at the ideal fre-
quencies for bone surgery in orthopedics, maxillofacial
surgery, and periodontal and endodontic surgery.
This instrument was used in research on animals to
compare its ‘‘traumatic effect’’ with that of traditional
orthopedic surgery.20 It allowed for a more precise cut
and a clearer view of the surgical field because of its
control over bleeding. Moreover, at histologic exami-
nation, the cut surfaces showed no signs of necrosis or
pigmentation, whereas the presence of vital osteo-
cytes was observed.20 Robiony et al.21 performed
piezoelectric orthodontic surgery on four patients in
2004, and maxillary osteotomies performed by others
using piezoelectric surgery demonstrated that the in-
strument allowed for precise surgery and protected
delicate anatomic structures.
Therefore, this surgical technique may be used in
place of the drill surgery technique to position im-
plants. Although reports on the clinical use of piezo-
electric surgery documented its effects on histology,
to the best of our best knowledge, no studies have
evaluated its application in preparing dental implant
sites or have considered the biologic effects of this
type of instrumentation on tissues. Therefore, piezo-
electric surgery was tested in the preparation of bone
sites for dental implants and its influence on bone-
healing processes, using porous implants, was evalu-
ated. This study compared the osseointegration of
porous implants positioned using traditional drills
and piezoelectric bone surgery, including histomor-
phological and biomolecular analyses.
MATERIALS AND METHODS
Animals
Eight adult minipigs, weighing 66 to 87 kg and bred in
the Department of Animal Pathology of Turin Univer-
sity, were used in this study. The minipigs were fed
standard pelleted cereal food and were given water
ad libitum. This study was approved by the appropri-
ate Italian committees.
Preti, Martinasso, Peirone, et al.
Implant Insertion
Sixteen titanium implantsi were inserted surgically
into the tibias of the eight minipigs as follows. After
preanesthetic sedation with 2% xylazine¶ (2.3 mg/kg)
and tiletamine/zolazepam# (6.3 mg/kg), surgery was
performed under intubation anesthesia with isoflu-
rane/halothane and O2. The right hind leg was pre-
pared in a standard sterile fashion. The tibia was
exposed and the implant was inserted using a 45
Nw torque. One of two surgical techniques was used:
the drilling technique according to the Brånemark
protocol22 or the piezoelectric bone surgery technique
with specific inserts (patent pending) (Fig. 1). Eight
implants were used for each surgical technique. For
piezoelectric bone surgery, the frequency used was
27,000 to 30,000 kHz, and the specific steel inserts
were 2 and 3 mm in diameter. Tibial bone specimens
were collected at this stage to determine baseline
(time 0) values (Fig. 2).
Seven, 14, 28, and 56 days post-implant, animals
were sacrificed by preanesthesia with 2% xylazine**
(2.2 mg/kg) and tiletamine/zolazepam†† (6.6 mg/
kg) and an intracardiac injection of embutramide,
mebezonium iodide, and tetracaine hydrochloride‡‡
(70 mg/kg). The tibias were exposed and dissected
into slices to evaluate the peri-implant osseous heal-
ing (Fig. 3). Because one animal had fractures of
the operated limb, no samples were collected at
28 days.
Histologic Analysis
To evaluate the degree of inflammation, bone healing,
and remodeling, histologic analysis was performed at
7, 14, and 56 days post-implant.
After the implants were removed the specimens
were fixed in 10% formalin for 24 hours, decalcified
for 3 to 4 days in a mixture of 50% formic acid and
10% sodium citrate tribasic, embedded in paraffin,
sectioned along the longitudinal implant axis using
a microtome,§§ and stained with hematoxylin and
eosin for optical microscopy.ii The amount of inflam-
matory infiltrate was evaluated for each case (expressed
as the number of polymorphonucleated and mononu-
cleated inflammatory cells per high-power field [mean
of 10 fields]) and of osteogenesis (expressed as the
number of osteoblasts per high-power field [mean of
10 fields]).
§ Piezosurgery, Mectron Medical Technology, Carasco, Italy.
i TiUnite MKIII 3.75 · 10, Nobel Biocare Italiana, Agrate Brianza, Milan,
Italy.
¶ Rompun 2%, Bayer, Milan, Italy.
# Zoletil 100-Virbac 20%, Laboratoires Virbac, Carros, France.
** Rompun 2%, Bayer.
†† Zoletil 100-Virbac 20%, Laboratoires Virbac.
‡‡ Tanax, Intervent International, Unterschleibheim, Germany.
§§ Electronic Motorized Microtome, Shandon-Lipshaw, Pittsburgh, PA.
ii DMLB Leica, Solms, Germany.
717
Biologic Factors and Piezoelectric Bone Surgery Technique
Figure 1.
Example of drilled (A) and piezoelectric bone surgery (B) sites.
Figure 2.
Bone sample taken at baseline (0 time).
Figure 3.
Bone sample with implant: lateral (A) and frontal (B) views.
Biomolecular Analysis
To protect the RNA, all specimens (7, 14, and 56
days) were placed in solution¶¶ and stored at -80C
until testing. For testing, the specimens were ground
under a liquid nitrogen stream, and total RNA was ex-
tracted from the bone powder (150 to 200 mg) using
the acid guanidinium thiocyanate-phenol-chloroform
extraction method.23
Polymerase chain reaction (PCR) was performed
using single-stranded cDNA prepared from total
RNA (1 mg) with a kit.## The forward (FW) and reverse
718
Volume 78 • Number 4
(RV) primers (Table 1) were designed using soft-
ware.***
Real-Time PCR
Twenty-five microliters of a PCR mixture, containing
cDNA template equivalent to 80 ng total RNA, 5 pmol
each of the forward and reverse primers, and 2·,†††
were amplified using a PCR mix PCR instrument‡‡‡
to test for BMP-4, TGF-b2, tumor necrosis factor-al-
pha (TNF-a), interleukin (IL)-1b and -10, or GAPDH.
The PCR conditions were an initial melt at 95C for 3
minutes, followed by 45 cycles at 95C for 40 sec-
onds, 52C for 40 seconds, and 72C for 40 seconds.
Each sample was tested six times, and the threshold
cycle (Ct) values were averaged from each reaction.
The fold change was defined as the relative expression
compared to that at time 0, calculated as 2-DDCt,
where DCt = Ctsample - CtGAPDH and DDCt = DCtsample
- DCttime 0.
Statistical Analysis
All data are expressed as mean – SD. The significance
of differences between group means was assessed by
variance analysis, followed by the Newman-Keuls
test.
RESULTS
Histologic Analysis
The peri-implant bone tissues from all implant sites
showed the presence of inflammatory infiltration,
which was greatest at 7 and 14 days and diminished
at 56 days.
At 7 days, the inflammatory infiltrate was charac-
terized by neutrophilic granulocytes, followed at 14
and 56 days by mononuclear cells (lymphocytes,
plasma cells, and monocyte-macrophages) in all sam-
ples. The inflammatory cells (polymorphonucleated
and mononucleated) were more numerous in sam-
ples obtained from the drilled sites (Fig. 4). Neo-
osteogenesis was quantified by considering the
absolute number of osteoblasts per high-power field
(mean of 10 fields) in osteogenetic areas; it was con-
sistently more active in the bone samples from the
implant sites prepared using the piezoelectric bone
surgery technique (Fig. 5). At 7 and 14 days, more
newly formed bone and a larger amount of osteoblasts
were seen in the piezoelectric bone surgery sites (Figs.
6B and 6D) than in the traditionally drilled ones (Figs.
6A and 6C). At 56 days, the rate of neo-osteogenesis
leveled out and was similar for both surgical tech-
niques (Figs. 6E and 6F).
¶¶ RNA Later, Qiagen, Milan, Italy.
## High Capacity cDNA Archive Kit, Applied Biosystems, Foster City, CA.
*** Beacon Designer, Bio-Rad, Hercules, CA.
††† iQ SYBR Green SuperMix, Bio-Rad.
‡‡‡ iCycler PCR instrument, Bio-Rad.
J Periodontol • April 2007 Preti, Martinasso, Peirone, et al.

Table 1. diminished in the piezoelectric

Primers Used for PCR
Gene (accession number)
Homo sapiens GAPDH (NM_002046) FW 59-TGAAGGTCGGAGTCAACGGATTTGGT-39
BMP-4 (Yates et al. )28
Homo sapiens TGF-b2 (AY438979)
Sus scrofa TNF-a (X57321)
Sus scrofa IL-1b (NM_001005149)
Sus scrofa IL-10 (NM_214041)
Primer Sequences
RV 59-CATGTGGGCCATGAGGTCCACCAC-39
FW 59-CTCGCTCTATGTGGACTTC-39
RV 59-ATGGTTGGTTGAGTTGAGG-39
FW 59-GAGACTTGATTGTCCTTCCTTC-39
RV 59-CTCCCCGAACCGTTGAGG-39
RW-59-GCAGGTCTATTTTGGGATCATT-39
RV 59-GGGGGTCTGAAGGAGTAA-39
FW 59-GGGGACTTGAAGAGAGAA-39
RV 59-CATCACACAAGACAGGTACAGA-39
FW 59-CTATTTGTCCTGACTGCCT-39
RV 59-GCTCCCTAGTTTCTCTTCCT-39
bone surgery sites at 56 days
and increased in the drilled sites
(Fig. 7B). An increase in the IL-1b
level was observed at the drilled
sites, whereas in the sites pre-
pared using the piezoelectric
bone surgery technique, an in-
crease in the IL-1b level (2.8-fold
higher than drilled sites) was ob-
served only at 56 days (Fig. 8A).
TNF-a expression increased
in the drilled sites only, and it
was 18.75 times greater than
in the piezoelectric bone sur-
gery sites at 14 days (Fig. 8B).
Although IL-10 expression
was three-fold higher in the pie-
zoelectric bone surgery sites
compared to the drilled sites at
7 days, drilled sites showed a
16-fold higher increase at 14
days and a 13-fold higher in-
crease at 56 days (Fig. 8C).

Figure 4.
Number of inflammatory cells present in peri-implant bone tissues from
the implant sites. The inflammatory cells were counted in 10 fields on
the slide and are means – SD. Means with different letters differ
significantly from one another ( P <0.05) as determined by variance
analysis followed by the Newman-Keuls test.
Biomolecular Analysis
Bone morphogenetic proteins. An early increase in
the BMP-4 level was observed in the sites that had
been prepared with the piezoelectric bone surgery
technique (18.5-fold that of drilled sites at 7 days;
15-fold at 14 days), whereas in drilled sites an in-
crease was observed only at 56 days. By contrast,
BMP-4 levels in the piezoelectric bone surgery sites
had reverted to baseline values at 56 days (Fig. 7A).
Cytokines. During the early phases of testing, a
greater increase in the TGF-b2 level was observed
in sites prepared using the piezoelectric bone surgery
technique compared to drilled sites (3.5-fold at 7
days, 19-fold at 14 days). By contrast, TGF-b2 levels
DISCUSSION
Porous implants were used because they were shown
to be more efficient in stimulating osseointegration
than machined implants.1-5 To monitor the osseointe-
gration process, samples were collected at different
post-implant intervals (7, 14, and 56 days) from sites
prepared using the Brånemark surgical drill or piezo-
electric bone surgery technique. Controls at 7 and 14
days were chosen because these time intervals repre-
sent the early phase of bone healing. The control at 56
days was chosen because it was ascertained previ-
ously that mature bone was present, even at this early
phase, around porous implants.5
The piezoelectric bone surgery technique was
selected because animal and human experiments
showed that it was effective in reducing tissue trauma
and promoting early bone repair.20,24,25
Any differences between the two surgical techni-
ques, with regard to healing and remodeling processes,
were determined by histologic and biomolecular anal-
yses. The piezoelectric bone surgery technique was
more effective in stimulating peri-implant osteogene-
sis, and it also promoted more osteoblasts around
the implant sites than did traditional drilling irrespec-
tive of experimental time. Moreover, neo-osteogenesis
constantly was more advanced in the bone prepared
by the piezoelectric bone surgery technique. This was
confirmed empirically: during bone preparation for
biomolecular analysis, there was less fragmentation
in the bones prepared by the piezoelectric bone sur-
gery technique.

719
Biologic Factors and Piezoelectric Bone Surgery Technique Volume 78 • Number 4

Figure 5.
Number of osteoblasts present in peri-implant bone tissues from the
implant sites. The osteoblasts were counted in 10 fields on the slide
and are the means – SD. Means with different letters differ significantly
from one another ( P <0.05) as determined by variance analysis
followed by the Newman-Keuls test.
Figure 7.
Bone morphogenetic proteins BMP-4 (A) and TGF-b2 (B) mRNA
levels in peri-implant bone tissues from the implant sites prepared
with the traditional drilling technique (Drill) or piezoelectric bone
surgery technique (Piezo). Black line = baseline (time 0).

Figure 6.
Postdecalcification histologic sections. Sample obtained at day 7 from
the piezoelectric bone surgery site (B) shows more newly-formed bone
and a higher number of osteoblasts than the sample from the drilled
site (A). A sample obtained at day 14 from a piezoelectric bone
surgery site (D) shows more newly-formed bone and a higher number
of osteoblasts than the sample from the drilled site (C). Samples
obtained at day 56 from drilled (E) and piezoelectric bone surgery
(F) sites show similarly formed bone. (Hematoxylin and eosin, original Figure 8.
magnification ·100.) Cytokines IL-1b (A), TNF-a (B), and IL-10 (C) mRNA levels in
peri-implant bone tissues from the implant sites prepared with the
traditional drilling technique (Drill) or piezoelectric bone surgery
technique (Piezo). Black line = baseline (time 0).
The amount of inflammatory infiltrate (more exten-
sive in samples obtained from the drilled sites) around
the implants decreased during the experiment period.
The biomolecular analysis confirmed that the piezo-
electric bone surgery technique gave better results. TGF-b2 levels was seen around the same implant
BMP-4 levels increased earlier around the implant sites. These results confirmed that BMP-4 can play
sites that were treated using the piezoelectric bone a role in the bone-healing process, in agreement with
surgery technique. Similarly, an earlier increase in a previous study by Schierano et al.5

720
J Periodontol • April 2007
It was proposed that BMP activity may be stimu-
lated by many growth factors, including the fibroblast
growth factor, TGF-b, and platelet-derived growth
factor, which can be found in bone.26-28
Previously, we hypothesized that TGF-b stimulated
BMP activity in early bone healing,5 a finding sup-
ported by the data obtained from the present study.
TGF-b2 levels were higher than BMP-4 levels in
all sites at the early experimental phase. Moreover,
the piezoelectric bone surgery technique promoted
greater increases in BMP-4 and TGF-b2 production
than did drilling in the first phase of the experiment.
The piezoelectric bone surgery technique provided an
earlier increase in proteins, favoring bone production.
Drilling promoted later new bone synthesis, as dem-
onstrated by the increase in proteins only at 56 days.
TNF-a proinflammatory cytokine, IL-1b proin-
flammatory and bone-resorbing cytokine, and IL-10
anti-inflammatory cytokine were detected in bone
specimens taken from the minipigs at various time in-
tervals. In general, the expression of proinflammatory
cytokines was higher in the early experimental phases
in drilled specimens, which also contained more in-
flammatory cells. The piezoelectric bone surgery sites
showed a higher IL-1b expression at 56 days com-
pared to previous experimental control times and
drilled specimens. This finding might be correlated
with the bone remodeling because IL-1b is involved
in osteoclast differentiation.29,30 Indeed, the data ob-
tained in this study indicate that bone remodeling
takes place earlier with the piezoelectric bone surgery
technique. A higher increase in IL-10 expression over
time was observed in drilled sites compared to piezo-
electric bone surgery sites because of the presence of
a major inflammatory process.
Despite the fact that a limited number of samples
were available for study, the results indicated that
the first step toward ossification began earlier with
porous implants positioned in sites prepared using
the piezoelectric bone surgery technique. The bone
around the implants treated with the piezoelectric
bone surgery technique also had earlier increases in
BMP-4 and TGF-b2 bone repair protein levels and a
reduced expression of proinflammatory cytokines.
The better results obtained with the piezoelectric
bone surgery technique may be explained by the fact
that ultrasound is able to facilitate bone synthesis,
which, in turn, induces the proliferation of osteoblasts
and their differentiation. Such an effect was reported
in studies carried out by us (data not shown) and by
other investigators.31,32
CONCLUSIONS
Within the limitations of this initial study, in the early
phases of the osseointegration process (7 and 14
days) the surgical sites prepared with the piezoelectric
Preti, Martinasso, Peirone, et al.
bone surgery technique showed an increase in neo-
osteogenesis and in the quantity of osteoblasts com-
pared to the sites prepared with drilling. Based on
these results, the piezoelectric bone surgery tech-
nique seemed to be more efficient than the drilling
surgery technique; it promoted earlier bone morpho-
genetic proteins, controlled the inflammatory process
better, and, moreover, bone remodeling already was
evident at 56 days. An understanding of the biologic
factors involved in bone healing around titanium im-
plants is fundamental. This is true in the oral cavity
as well as several other anatomical sites.
ACKNOWLEDGMENTS
This study was supported by grants from Compagnia
di San Paolo, Turin, Italy; the Ministry of University
Research of Italy, Regione-Piemonte, Italy; the Uni-
versity of Turin, Italy; and Nobel-Biocare Italiana,
Agrate Brianza, Italy. The authors thank Mrs. Barbara
Wade, contract professor, University of Turin, for her
linguistic contribution.
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Correspondence: Dr. Gianmario Schierano, Oral Rehabili-
tation and Maxillo-Facial Prosthesis and Dental Implants,
Department of Biomedical Sciences and Human Oncology,
Section of Prosthetic Dentistry, School of Dentistry, Univer-
sity of Turin, Corso Dogliotti 14, 10126 Turin, Italy. Fax: 39-
011-6636489; e-mail: gianmario.schierano@unito.it.
Accepted for publication October 16, 2006.