Abstract

Titanium is a highly biocompatible material and very osteogenic in vivo. However, how titanium regulates osteoblast activity to promote bone formation is incompletely characterized. We, therefore, attempted to get more information by using microRNA (miRNA) microarray techniques to investigate translation regulation in osteoblasts grown on titanium disks. The miRNA oligonucleotide microarray provides a novel method to carry out genome-wide miRNA profiling in human samples. By using miRNA microarrays containing 329 probes designed from the human miRNA sequence, several miRNA were identified in osteoblast-like cell line (MG 63) grown on titanium disks. There were 13 up-regulated miRNAs (ie, mir-23a, mir-222, mir-523, mir-22, mir-23b, mir-143, mir-377, mir-24, mir-422b, mir-26a, mir-29a, mir-17–5p, mir-182) and 2 down-regulated miRNAs (ie, mir-187, mir-339). The data reported are, to our knowledge, the first study on translation regulation in osteoblasts exposed to titanium. The data can be relevant to understand better the molecular mechanism of osteoblast activation and as a model for comparing other materials with similar clinical effects.

Introduction

Several features of the implant surface including composition, topography, roughness, and energy play a relevant role in implant integration with bone.1,21 Little is known about the structural and chemical surface properties that may influence biological responses, especially from a genetic point of view.1 Titanium has been widely used in the biomedical field, but the factors and mechanisms underlying the biological response to titanium are not yet well understood,1 and it is necessary to look for correlations between surface characteristics and response of biological tissues at different levels of resolution and sophistication.1 Because the mechanism by which titanium stimulates osteoblast activity to promote bone formation is incompletely characterized, we therefore attempted to add new information by using microRNA microarray techniques.

MicroRNAs (miRNAs) represent a class of small, functional, noncoding RNAs of 19 to 23 nucleotides (nt) cleaved from 60- to 110-nt hairpin precursors.22,23 Hundreds of miRNAs have been identified in plants and animals. The miRNAs are involved in various biological processes, including cell proliferation and cell death during development, stress resistance, and fat metabolism, through the regulation of gene expression in posttranscriptional RNA silencing pathways.24 The RNA interference (RNAi) and the microRNA (miRNA) pathway, regulate gene expression by inducing degradation and/or translational repression of target messenger RNAs (mRNAs). These pathways are generally initiated by various forms of double-stranded RNA (dsRNA), which are processed by Dicer, an RNase III family endonuclease, to 21 to 22 nt long RNA molecules that serve as sequence-specific guides for silencing.25,26 

MicroRNAs are transcribed as long primary transcripts (pri-miRNAs), which are processed by a nuclear RNase III Drosha-containing complex into short hairpin intermediates (pre-miRNAs). Pre-miRNAs are transported to the cytoplasm where they are further processed by a second RNase III-family enzyme called Dicer to generate 22 base pair (bp) RNA duplexes with 2-nt 3′ overhangs.27,30 

MicroRNAs are loaded onto an Argonaute containing effectors ribonucleoprotein (RNP) complex, referred to as miRNP or RISC (RNA-induced silencing complex), which is capable of recognizing cognate mRNAs and inhibiting protein expression.

We used a recently developed methodology for miRNA gene expression profiling based on the hybridization of a microchip, the Ncode Multi-Species miRNA Microarray (Invitrogen, Carlsbad, Calif), a slide printed with approximately 900 unique probe of miRNA sequences for Homo sapiens, Mus musculus, Rattus norvegicus, Drosophila melanogaster, Caenorhabditis elegans, and Danio rerio.

By the analysis of the 329 human miRNA sequences spotted on the array, we compared miRNA expression and consequently gene regulation in human MG63 cells grown on machined titanium disks with untreated MG63 cells.

Materials and Methods

Cell culture

Osteoblast-like cells (MG63) were cultured in sterile Falcon tissue colture wells (Becton Dickinson, Franklin Lakes, NJ) containing Eagle's minimum essential medium supplemented with 10% fetal calf serum (Sigma Chemical Co, St Louis, Mo) and antibiotics (penicillin 100 U/mL and streptomycin 100 μg/mL, Sigma). Cultures were maintained in a 5% CO2 humidified atmosphere at 37°C.

MG63 cells were collected and seeded at a density of 1 × 105 cells/mL into 9 cm2 (3 mL) wells by using 0.1% trypsin, 0.02% EDTA in Ca++, and Mg++—free Eagle's buffer for cell release. One set of wells contained sterile metal disks of machined grade 3 titanium (diameter 3 cm; test). After 24 hours, when cultures were subconfluent, cells were processed for RNA extraction. Cell cultures were repeated twice.

MicroRNA microarray

MicroRNA was extracted from the cells using the PureLink miRNA Isolation Kit (Invitrogen). Four hundred nanograms of miRNA from each sample (treated and control) were used for hybridization of NCode Multi-Species miRNA Microarray, a slide containing 329 human miRNAs sequences in duplicate.

NCode miRNA Labeling System (Invitrogen) was used for labeling and hybridizing miRNA to the microarray, according to the manufacturer's instructions. Briefly, a poly(A) tail was added to each miRNA, using a poly A polymerase and an optimized reaction buffer. Then, a capture sequence was ligated to the miRNA using a bridging oligo(dT). Following a purification step, the tagged miRNAs were hybridized to the microarray and incubated overnight.

After an incubation of 18–20 hours, the array was washed and hybridized with Alexa Fluor 3 capture reagents (for the control) and Alexa Fluor 5 capture reagents (for the treated cells) in the first experiments, and then the dyes were switched. After another wash, the array was scanned using a standard microarray scanner (Axon Instruments, Sunnyvale, Calif).

After scanning, each spot was identified by means of a GAL (GenePixR Array List) file31 that lists the identities and locations of all probes printed on the array.

Images were quantified by GenePix 6.0 software (Axon Instruments). Signal intensities for each spot were calculated by subtracting local background from total intensities. The data were normalized by using the DNMAD and preprocessing software packages.32,34 This generates an average value of the two spot replicates of each miRNA.

To select for differentially expressed miRNA, the data obtained were analyzed using the SAM package (statistical analysis of microarray).35 

For target predictions and validations, miRNA were processed using miRBase Target,36 a web resource developed by the Enright Lab at the Wellcome Trust Sanger Institute. This source uses an algorithm called miRanda to identify potential binding sites for a given miRNA in genomic sequences.

The gene target list was then processed by FatiGO,37 a web interface which carries out simple data mining using Gene Ontology.38 The data mining consists on the assignation of the most characteristic gene ontology term to each cluster of regulated genes.

Results

Hybridization of miRNA (derived from MG63 cultured on disks of machined grade 3 titanium) to the sequences spotted on the slide allowed us to perform a systemic analysis of miRNAs and to provide primary information with regard to the regulation of the translation process induced by titanium. There were 13 up-regulated miRNAs (ie, mir-23a, mir-222, mir-523, mir-22, mir-23b, mir-143, mir-377, mir-24, mir-422b, mir-26a, mir-29a, mir-17–5p, mir-182) and 2 down-regulated miRNAs (ie, mir-187, mir-339) for FDR (false discovery rate) = 0 and score >9. The Figure is the graphical output of SAM and it shows differentially expressed miRNA. Because miRNA potentially regulates thousands of genes, in this study we selected only the genes related to osteogenesis and bone remodeling (Table). Because we detected some mRNAs regulated in an opposite manner by different miRNAs, these mRNAs with not univocal regulation were excluded from the subsequent analysis since the global effect of microRNAs on these messenger RNAs was not determined, yet.

Figure. SAM (significance analysis of microarray) plot of MG63 cultured for 24 hours on titanium disks

Figure. SAM (significance analysis of microarray) plot of MG63 cultured for 24 hours on titanium disks

Table. Down-regulated genes

Table. Down-regulated genes
Table. Down-regulated genes

Discussion

Recent studies in animal models have demonstrated that the titanium surface is of paramount importance in influencing the timing of bone healing, and rougher surfaces have been demonstrated to present a higher quantity of bone-implant contact and a higher removal torque value.6,7,10,11,21 Moreover, the surface roughness has demonstrated to be able to alter the responsiveness of different types of cells.8,13,14 In addition, previous reports on gene expression have given primary information with regard to the genetic effects of titanium on osteoblast-like cells.39,41 

However, because it is poorly understood how the titanium surface alters osteoblast activity to promote bone formation, we therefore attempted to address this question by using a new method, miRNA microarray, to identified genes that are differently translated in osteoblasts grown on machined grade 3 titanium surfaces.

MicroRNAs are a recently discovered class of small, ∼19 to 23-nucleotide noncoding RNA molecules. They are cleaved from 70 to 110-nucleotide hairpin precursors and play an important role in the posttranscriptional regulatory process. MicroRNAs are not translated into proteins: instead, they regulate the expression of other genes by either cleaving or repressing the translation of their mRNA targets.25,26 

Recent advances in spotted oligonucleotide microarray labeling and detection have enabled the use of this high-throughput technology for miRNA screening.

Microarray is a molecular technology that enables the analysis in parallel on a very large number of DNA or RNA fragments, spanning a significant fraction of the human genome. Gene expression is performed by a process of (1) miRNA extraction, (2) labeling (different dyes are used for reference untreated cells and investigated cells, ie, cultured on titanium disks), and (3) hybridization on slides containing miRNA probes. Then, the slides are scanned with a laser system, and two false color images are generated for each hybridization with miRNA from the investigated and reference cells. The overall result is the generation of a so-called genetic portrait. It corresponds to up- or down-regulated miRNA in the investigated cell system. Hybridization of miRNA derived from MG63 cultured on titanium disks to the sequences spotted on the slide allowed us to perform systemic analysis of miRNAs and to provide primary information with regard to the regulation of translation induced by titanium (Table).

Notable is that miRNAs are up-regulated, and thus, there is a down-regulation of several genes, most of them related to osteogenesis. Down-regulation of genes is caused by the silencing process determined by microRNAs (ie, miRNAs) on messenger RNAs (ie, mRNAs). Several BMPs have a negative transcriptional control like BMP1 (or procollagen C proteinase), BMPR1B (a member of the BMP receptor family), BMP7, and GDF10 (another a member of the BMP family). Another group of mRNAs down-regulated are collagen like COL1A1 (the major component of type I collagen) and COL11A1 (one of the two alpha chains of type XI collagen).

Additional down-regulated mRNAs encode for receptors like CASR (ie, calcium-sensing receptor that functions as a sensor for parathyroid and kidney to determine the extracellular calcium concentration and thus helps to maintain a stable calcium concentration), FGFR3 (a member of the fibroblast growth factor receptor family that binds acidic and basic fibroblast growth hormone and plays a role in bone development and maintenance), and GHRHR (a receptor for growth hormone-releasing hormone).

Other repressed mRNAs encode for hormones and morphogenetic proteins. Insulin growth factor 1 (IGF1 or somatomedin) mediates many of the growth-promoting effects of growth hormone whereas MSX1 encodes a member of the muscle segment homeobox gene family. MSX1 functions as a transcriptional repressor during embryogenesis through interactions with components of the core transcription complex and other homeoproteins. It has roles in limb-pattern formation and craniofacial development, particularly odontogenesis.

The fact that several genes related to bone formation have a translational negative control can be related to the time point analyzed (eg, MG63 cultured for 24 hours until they are subconfluent). This phase is characterized by an elevated cellular kinetics and to a low extracellular matrix production and differentiation.

In the present study a comparison between MG63 cultured on titanium disks vs MG63 cultured on plastic wells was performed. It is well known that titanium is biocompatible, and it is osseointegrated when inserted in bone. This is not the case for plastic. Thus, the comparison produced the maximum contrast with regard to osseointegration associated genetic variables. In addition, the early period of titanium-cell contact was analyzed because immediate loading is the actual target in implantology. Immediate loading means placing the final or provisional prosthetic restoration immediately or within 24 hours of the surgical procedure; early loading means that implants are loaded in a period ranging from 24 hours to 7 days after fixtures are inserted.42 Thus, the choice to study titanium-cell interaction at 24 hours derived from the definition of immediate loading. The detection of increased cell proliferation after 24 hours of titanium-cell contact is relevant because osseointegration depends on 2 phases: cell proliferation and then differentiation. The increase of cell kinetics explains why not sterile conditions can interfere with bone healing process. Proliferation is an additional variable to be considered in immediate loading.

We expected that in a subsequent period (ie, later time points) a differentiation process will start and a different panel of miRNAs will be activated. Additional experiments are needed to get information to subsequent titanium-cell contact periods.

The genes discussed are only a limited number among those differentially regulated by miRNA reported in the Table. We briefly analyzed some of those with a better known function and those that are directly related to bone formation, skeletal development, cartilage remodeling, and bone production.

It is worth noting that MG63 is a cell line and not primary osteoblast cell culture. The advantage of using a cell line is related to the fact that the reproducibility of the data is higher because there is not the variability of the patient studied. Primary cell cultures provide a source of normal cells, but they also contain contaminating cells of different types and cells in variable states of differentiation. Moreover, we have chosen to perform the experiment after 24 hours in order to get information on the early stages of stimulation. It is our knowledge, therefore, that more investigations with other osteoblast-like cell lines, primary cultures, and different time points are needed to get a global comprehension of the molecular events related to titanium action. However, a global effect on bone formation is detected, and the reported data can be a model to compare different substances with similar effects.

Acknowledgments

This work was supported by grants from the University of Ferrara, Italy (F.C.), PRIN 2005 prot. 2005067555-002 (F.C.), Fondazione CARIFE (F.C.), and Fondazione CARISBO (F.P.).

References

References
1
Larsson
,
C.
,
P.
Thomsen
, and
B. O.
Aronsson
.
et al
.
Bone response to surface-modified titanium implants: studies on the early tissue response to machined and electropolished implants with different oxide thicknesses.
Biomaterials
1996
.
17
:
605
610
.
2
Puleo
,
D. A.
and
A.
Nanci
.
Understanding and controlling the bone-implant interface.
Biomaterials
1999
.
20
:
2311
2321
.
3
Bowers
,
K. T.
,
J.
Keller
,
B. A.
Randolph
,
D. G.
Wick
, and
C. M.
Michaels
.
Optimization of surface micromorphology for enhanced osteoblast responses in vitro.
Int J Oral Maxillofac Implants
1992
.
7
:
302
310
.
4
Han
,
C. H.
,
C. B.
Johansson
,
A.
Wennenberg
, and
T.
Albrektsson
.
Quantitative and qualitative investigations of surface enlarged titanium and titanium alloys implants.
Clin Oral Implants Res
1998
.
9
:
1
10
.
5
Cooper
,
L. F.
,
T.
Masuda
,
W.
Whitson
,
P.
Yliheikkila
, and
D. A.
Felton
.
Formation of mineralizing osteoblast cultures on machined, titanium oxide grit-blasted, and plasma-sprayed titanium surfaces.
Int J Oral Maxillofac Implants
1999
.
14
:
37
47
.
6
Klokkevold
,
P.
,
R. D.
Nishimura
,
M.
Adachi
, and
A.
Caputo
.
Osseointegration enhanced by chemical etching of the titanium surface. A torque removal study in the rabbit.
Clin Oral Implants Res
1997
.
8
:
442
447
.
7
Gotfredsen
,
K.
,
A.
Wennerberg
,
C.
Johansson
,
L. T.
Skovgaard
, and
E.
Hjorting-Hansen
.
Anchorage of TiO2-blasted, HA-coated and machined implants: an experimental study with rabbits.
J Biomed Mater Res
1995
.
29
:
1223
1231
.
8
Park
,
J. Y.
and
J. E.
Davies
.
Red blood cell and platelet interactions with titanium implant surfaces.
Clin Oral Implants Res
2000
.
11
:
530
539
.
9
Mustafa
,
K.
,
J.
Wroblewski
,
K.
Hultenby
,
B.
Silva Loprez
, and
K.
Arvidson
.
Effects of titanium surfaces blasted with TiO2 particles on the initial attachment of cells derived from mandibular bone. A scanning electron microscopic and histomorphometric analysis.
Clin Oral Implants Res
2000
.
11
:
116
128
.
10
Wennerberg
,
A.
,
T.
Albrektsson
, and
J.
Lausmaa
.
Torque and histomorphometric evaluation of c.p. titanium screws blasted with 25- and 75 μm-sized particles of Al2O3.
J Biomed Mater Res
1996
.
30
:
251
260
.
11
Wennerberg
,
A.
,
T.
Albrektsson
,
C.
Johansson
, and
B.
Andersson
.
Experimental study of turned and grit-blasted screw-shaped implants with special emphasis on effects of blasting material and surface topography.
Biomaterials
1996
.
17
:
15
22
.
12
Mustafa
,
K.
,
B. S.
Lopez
,
K.
Hultenby
,
A.
Wennenberg
, and
K.
Arvidson
.
Attachment and proliferation of human oral fibroblasts to titanium surfaces blasted with TiO2 particles. A scanning electron microscopic and histomorphometric analysis.
Clin Oral Implants Res
1998
.
9
:
195
207
.
13
Schwartz
,
Z.
,
J. Y.
Martin
,
D. D.
Dean
,
J.
Simpson
,
D. L.
Cochran
, and
B. D.
Boyan
.
Effect of titanium surface roughness on chondrocyte proliferation, matrix production, and differentiation depends on the state of cell maturation.
J Biomed Mater Res
1996
.
30
:
145
155
.
14
Martin
,
J. Y.
,
Z.
Schwartz
, and
T. W.
Hummert
.
et al
.
Effect of titanium surface roughness on proliferation, differentiation, and protein synthesis of human osteoblast-like cells (MG 63).
J Biomed Mater Res
1995
.
29
:
389
401
.
15
Den Braber
,
E. T.
,
J. E.
De Ruijter
,
H. T. J.
Smits
,
L. A.
Ginsel
,
A. F.
Von Recum
, and
J. A.
Jansen
.
Effect of parallel surface microgrooves and surface energy on cell growth.
J Biomed Mater Res
1995
.
29
:
511
518
.
16
Wennenberg
,
A.
,
C.
Hallgren
,
C.
Johansson
, and
S.
Danelli
.
A histomorphometric evaluation of screw-shaped implants each prepared with two surface roughnesses.
Clin Oral Implants Res
1998
.
9
:
11
19
.
17
Feighan
,
J. E.
,
V. M.
Goldberg
,
D.
Davy
,
J. A.
Parr
, and
S.
Stevenson
.
The influence of surface-blasting on the incorporation of titanium-alloy implants in a rabbit intramedullary model.
J Bone Joint Surg
1995
.
77-A
:
1380
1395
.
18
Chehroudi
,
B.
,
D.
McDonnel
, and
D. M.
Brunette
.
The effects of micromachined surfaces on formation of bonelike tissue on subcutaneous implants as assessed by radiography and computer image processing.
J Biomed Mater Res
1997
.
34
:
279
290
.
19
Piattelli
,
A.
,
L.
Manzon
,
A.
Scarano
,
M.
Paolantonio
, and
M.
Piattelli
.
Histologic and morphologic analysis of the bone response to machined and sandblasted titanium implants: an experimental study in rabbit.
Int J Oral Maxillofac Implants
1998
.
13
:
805
810
.
20
Piattelli
,
M.
,
A.
Scarano
,
M.
Paolantonio
,
G.
Iezzi
,
G.
Petrone
, and
A.
Piattelli
.
Bone response to RBM sandblasted titanium implants: an experimental study in rabbit.
J Oral Implantol
2002
.
28
:
2
8
.
21
Wennenberg
,
A.
,
T.
Albrektsson
, and
B.
Andersson
.
An animal study of c.p. titanium screws with different surface topographies.
J Mater Sci Mater Med
1995
.
6
:
302
309
.
22
Moss
,
E. G.
MicroRNAs.
In: Barciszewski J, Erdmann V, eds. Noncoding RNAs: Molecular Biology and Molecular Medicine.. Georgetown, Tex: Landes Bioscience
.
2003
.
98
114
.
23
Schmitter
,
D.
,
J.
Filkowski
, and
A.
Sewer
.
et al
.
Effects of Dicer and Argonaute down-regulation on mRNA levels in human HEK293 cells.
Nucleic Acids Res
2006
.
34
:
4801
4815
.
24
Ambros
,
V.
MicroRNA pathways in flies and worms: growth, death, fat, stress, and timing.
Cell
2003
.
113
:
673
676
.
25
Sontheimer
,
E. J.
and
R. W.
Carthew
.
Silence from within: endogenous siRNAs and miRNAs.
Cell
2005
.
122
:
9
12
.
26
Zamore
,
P. D.
and
B.
Haley
.
Ribo-gnome: the big world of small RNAs.
Science
2005
.
309
:
1519
1524
.
27
Grishok
,
A.
,
A. E.
Pasquinelli
, and
D.
Conte
.
et al
.
Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing.
Cell
2001
.
106
:
23
34
.
28
Hutvagner
,
G.
,
J.
McLachlan
, and
A. E.
Pasquinelli
.
et al
.
A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA.
Science
2001
.
293
:
834
838
.
29
Ketting
,
R. F.
,
S. E.
Fischer
,
E.
Bernstein
,
T.
Sijen
,
G. J.
Hannon
, and
R. H.
Plasterk
.
Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans.
Genes Dev
2001
.
15
:
2654
2659
.
30
Zhang
,
H.
,
F. A.
Kolb
,
L.
Jaskiewicz
,
E.
Westhof
, and
W.
Filipowicz
.
Single processing center models for human Dicer and bacterial RNase III.
Cell
2004
.
118
:
57
68
.
31
GenePixR Array List
Available at: http://www.invitrogen.com/ncode. Accessed June 17, 2008
.
32
Vaquerizas
,
J. M.
,
J.
Dopazo
, and
R.
Díaz-Uriarte
.
DNMAD: web-based diagnosis and normalization for microarray data.
Bioinformatics
2004
.
20
:
3656
3658
.
33
Herrero
,
J.
,
R.
Diaz-Uriarte
, and
J.
Dopazo
.
Gene expression data preprocessing.
Bioinformatics
2003
.
19
:
655
656
.
34
DNMAD and preprocessing software packages
Available at: http://gepas.bioinfo.cipt.es/cgi-bin/tools. Accessed June 17, 2008
.
35
Tusher
,
V. G.
,
R.
Tibshirani
, and
G.
Chu
.
Significance analysis of microarrays applied to the ionizing radiation response.
Proc Natl Acad Sci U S A
2001
.
98
:
5116
5121
.
36
miRBase Target
Available at: http://microrna.sanger.ac.uk/targets/v4/. Accessed June 17, 2008
.
37
FatiGO
Available at: http://fatigo.bioinfo.cnio.es. Accessed June 17, 2008
.
38
Gene Ontology
Available at: http://www.geneontology.org. Accessed June 17, 2008
.
39
Carinci
,
F.
,
F.
Pezzetti
, and
S.
Volinia
.
et al
.
Analysis of MG63 osteoblastic-cell response to a new nanoporous implant surface by means of a microarray technology.
Clin Oral Implants Res
2004
.
15
:
180
186
.
40
Carinci
,
F.
,
F.
Pezzetti
, and
S.
Volinia
.
et al
.
Analysis of osteoblast-like MG63 cells' response to a rough implant surface by means of DNA microarray.
J Oral Implantol
2003
.
29
:
215
220
.
41
Carinci
,
F.
,
S.
Volinia
,
F.
Pezzetti
,
F.
Francioso
,
L.
Tosi
, and
A.
Piattelli
.
Titanium-cell interaction: analysis of gene expression profiling.
J Biomed Mater Res B Appl Biomater
2003
.
66
:
341
346
.
42
Degidi
,
M.
,
A.
Piattelli
,
P.
Felice
, and
F.
Carinci
.
Immediate functional loading of edentulous maxilla: a 5-year retrospective study of 388 titanium implants.
J Periodontol
2005
.
76
:
1016
1024
.

Annalisa Palmieri, PhD, Furio Pezzetti, PhD, and Marzia Arlotti, PhD, are at the Institute of Histology, University of Bologna and Center of Molecular Genetics, Cassa di Risparmio di Bologna Foundation, Bologna, Italy.

Anna Avantaggiato, MD, Laura Guerzoni, PhD, and Francesco Carinci, MD, are at the Department of DMCCC, Section of Maxillofacial Surgery, University of Ferrara, Ferrara, Italy. Address correspondence to Dr Carinci, Department of DMCCC, Section of Maxillofacial Surgery, University of Ferrara, Corso Giovecca, 203, 44100 Ferrara, Italy. (crc@unife.it)

Lorenzo Lo Muzio, MD, is at the Dental Clinic, University of Foggia, Foggia, Italy.

Antonio Scarano, DDS, is at the Dental Clinic, University of Chieti, Chieti, Italy.

Corrado Rubini, MD, is at the Department of Neurosciences, Institute of Pathologic Anatomy and Histopathology, Polytechnic University of the Marche, Ancona, Italy.

Dario Ventorre, MD, is in the Section of Maxillofacial Surgery at the University of Verona, Italy.