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Clinical, histological and histomorphometric evaluation of the healing of
mandibular ramus bone block grafts for alveolar ridge augmentation before
implant placement*
Alessandro ACOCELLA, Oral Surgery Specialist1
, Roberto BERTOLAI, Oral and Maxillo-Facial specialist1
,
Maurizio COLAFRANCESCHI, Professor of Human Anatomy and Pathology2
, Roberto SACCO, Oral Surgery
Programme Resident3
1
Department of Maxillo-Facial Surgery, Faculty of Medicine-University of Florence, Italy; 2
Department of Human
Anatomy and Pathology, Faculty of Medicine-University of Florence, Italy; 3
Department of Odontostomatology, Faculty
of Medicine-University of Sassari, Italy
SUMMARY. Background: Localized bone defects in the maxilla are commonly reconstructed with autologous
mono-cortical bone blocks prior to the placement of dental implants. This study presents a clinical, histological
and histomorphometric analysis on the use of mandibular ramus block grafts for ridge augmentation. Materials
and Methods: mono-cortical bone blocks from the mandibular ramus were grafted in 15 patients. The bone
grafts were left to heal for period varying from 3 to 9 months. Afterwards, 30 implants were inserted and
bone samples were removed for subsequent histological analysis. Results: All the bone grafts were successful
and resorption was minimal. There were no implant failures. At graft placement, mean lateral augmentation was
4.6 ^ 0.73 mm, which, later, at the time of implant insertion, reduced to 4 ^ 0.77 mm. Histological evaluation
indicated signs of active remodelling in all specimens. However, the grafted bone contained substantial
amounts of non-vital bone (NVB) and generally weak neo-vascularization regardless of the time of
biopsies. Conclusions: The outcome of the study suggests that the larger part of osteocytes in mono-cortical
bone do not survive grafting and neo-vascularization of non-vital grafted bone is difficult because of the slow
remodelling process into new vital. Ó 2009 European Association for Cranio-Maxillo-Facial Surgery
Keywords: bone histology, bone histomorphometry, mandibular ramus, maxillary defects, mono-cortical bone
block graft
INTRODUCTION
Bone defects in the human maxilla are common. They
are mostly determined by premature loss of teeth due
to periodontal disease or traumas. They usually cause
reduction of alveolar bone volume, which becomes, in
consequence, inadequate for standard treatments with
osseointegrated implants (Nevins and Mellonig, 1994;
Oikarinen et al., 2003).
Pathological resorption of the maxillary bone may be
the main cause of both functional and aesthetic dissatis-
faction to the patient (Collins and Nunn, 1994).
Moreover, insufficient bone height and width of the al-
veolar ridge make the maxillary anatomy particularly un-
favourable for the conventional placement of dental
implants.
In order to create favourable conditions for implant
placement, the bone must undergo augmentation. Nowa-
days, reconstruction and augmentation of severely
resorbed maxillary alveolar ridges, with the use of differ-
ent grafting materials and techniques, represent a predict-
able procedure for endoosseous implant placement.
(Buser et al., 1995; Oikarinen et al., 2003; Chiapasco
et al., 2007).
There have been experimental in vitro and in vivo
studies in all kinds of animal models in order to assess
the quality of bone defects filled with different test mate-
rials. The majority of these studies evaluated the osteoin-
ductive activities in critical size defect models using
radiography or histology (Bosch et al., 1998; Nidoli
et al., 1999).
Recently, there has been considerable interest in bone
regeneration augmented with stem cells, periosteum-de-
rived cells, or osteoinductive proteins to aid the healing
of large skeletal defects (Boo et al., 2002; Yamada
et al., 2003; Lendeckel et al., 2004; Sakata et al., 2006).
It has been widely accepted that autologous bone is the
most appropriate grafting material. (Listrom and Syming-
ton, 1988; Jensen and Sindet-Pedersen, 1991).
The harvesting sites may be intraoral or extraoral: in
the first case, bone can be harvested from retromolar,
*
The work should be attributed: Department of Maxillo-Facial Sur-
gery, Faculty of Medicine e University of Florence, Italy; Director:
Dr. Roberto Bertolai, address: Largo Palagi,1 Firenze, Italy.
222
Journal of Cranio-Maxillo-Facial Surgery (2010) 38, 222e230
Ó 2009 European Association for Cranio-Maxillo-Facial Surgery
doi:10.1016/j.jcms.2009.07.004, available online at http://www.sciencedirect.com
ramus or symphysis areas (Jensen and Sindet-Pedersen,
1991; Misch, 1997; Widmark et al., 1997; Hunt and
Jovanovic, 1999) while in the second case, bone can be
collected from ilium, calvarium or tibia. (Block and
Kent, 1997).
Both mandibular body and ramus area represent an
ideal donor site as they provide adequate, dense bone
with sufficient volume for implant placement and short
healing times. (Garg et al., 1998).
The advantages of the ascending ramus donor site over
the chin include minimal patient concern for altered fa-
cial contour and low levels of post-operative sensory dis-
turbances and discomfort.
Although mandibular ramus mono-cortical block
grafts for alveolar ridge augmentation have been widely
used with great clinical success, it has not been clearly
explained how they incorporate into the surrounding
bone. The general lack of histological evidence on the
healing of mandibular autogenous bone block grafts
leaves a great number of unanswered questions about
the biological process.
The vitality of osteocytes is believed to be important in
the process of bone remodelling as it seems that these
cells are involved in bone turnover.
This study’s objective was to gain insight into the vi-
tality of bone grafts in the human jaw with emphasis on
the survival of osteocytes used as sign of bone vitality.
Our study has been conducted with an integrated mix
of tests and, among them, histomorphometry which has
only been used in a few studies for the evaluation of os-
teocyte survival. (Blomqvist et al., 1998).
MATERIALS AND METHODS
Patients
Fifteen patients (7 men and 8 women) of ages ranging
from 21 to 53 years were considered for the study
(Table 1). The selected patients had severe bone defects
in the maxillary alveolar ridge, originating from peri-
odontal disease or traumas.
All 15 patients were healthy and had no general con-
traindications for bone block graft surgical treatment;
only four of them were smokers.
First of all, a clinical and radiographic investigation
was carried out and the treatment fully explained to the
patients. The treatment programme included a first oper-
ation for bone augmentation followed by a healing period
varying from 3 to 9 months (Table 1); finally, a second
surgical operation was scheduled for biopsy and implant
placement. All patients gave their informed consent to
donate the bone tissue removed during implant surgery
for histological examination.
First surgical step
The bone augmentation was carried out under local an-
aesthesia in 14 patients and under general anaesthesia
with rhino-tracheal intubation in only one case. The lat-
ter, in fact, was affected by a larger pre-maxilla defect,
which was restored with bilateral ascending ramus grafts.
Full-thickness flaps were reflected in order to allow
a satisfactory exposure of the recipient site (Figs. 1a, 2a).
The remaining crest width was measured with a pair of cal-
lipers to the nearest half-millimeter (pre-augmentation
width) (Table 2).
After administering block anaesthesia to the inferior
alveolar nerve, harvesting of bone from ascending ramus
was performed. An obliqueesagittal incision was made
distal to the third molar following the direction of the ra-
mus and a vertical releasing incision was placed distally
in the ramus area. After reflection of bucco-lingual flaps,
osteotomies were executed with a small fissure bur to
outline the dimensions of the bone block (Figs. 1b, 2b).
Careful techniques were performed to make sure penetra-
tion was only of the cortical layer and to avoid any in-
juries to the alveolar nerve. Once the osteotomy was
completed, a straight elevator was placed along the sag-
ittal cut and the lateral block of bone was green-stick
fractured off (Fig. 2c). The recipient site was prepared
by scoring or perforating through and through (with
a small, straight fissure bur) in order to create bleeding
channels. The block autograft was then fixated to recipi-
ent site with 1 or 2 lag-screws (Osteomed, Addison, TX)
(Figs. 1c, 2d). Sharp edges of the bone blocks were
rounded off (with large diamond burs) and the aug-
mented width of the bony crest was measured again
with a pair of callipers (post-augmentation width).
Gaps around the block grafts were filled with bone chips
harvested from the donor site (Fig. 2e). Once the graft
was adapted to the site, an incision through the perios-
teum at the base of the flap allowed the tissue cover
the graft without tension. The recipient and the donor
site areas were then sutured with vertical mattress sutures
(Vicryl 3-0, Ethicon, Johnson & Johnson Roma, Italy)
without covering the bone graft with a barrier membrane
(Fig. 2f).
Post-operative antibiotic (Zimox, Pfizer, Latina, Italy)
and analgesic (Brufen; Abbott SpA, Campoverde (Lt),
Italy) therapy was routinely prescribed for 7 days. Patients
were also given chlorhexidine digluconate 0.2% mouth-
wash (Curasept; Curaden Health Care Srl, Milano, Italy)
for ten days and sutures were removed after 10e12 days.
Second surgical step
After a healing period of from 3 to 9 months, before the
implant insertion, CT examination was performed in
order to plan implant surgery correctly. The flap was
re-opened with an outline similar to that of the first oper-
ation (Fig. 1d). Once the flap was elevated, the healed
crest width was measured again with a pair of callipers
(re-entry width) (Table 2). Bone block fixation screws
were removed; implant site preparation and insertion
were accomplished according to standard surgical proto-
cols. A total of 30 implants were placed (Table 1) and
primary stability was achieved during this step (maxi-
mum insertion torque was 35 Ncm).
Biopsy procedure
Biopsy was performed at the same time of implant place-
ment including the grafted bone/recipient site interface.
Clinical, histological and histomorphometric evaluation of the healing of mandibular ramus bone block grafts 223
Table 1 e Patient data and results of histomorphometry means
Patient Sex Age No. of implants Healing time (months) Non Vital Bone
(histological %)
1 M 28 6 4 60.7
2 M 34 2 5 34.3
3 F 32 1 4 73.2
4 M 53 2 7 52.7
5 F 37 3 9 38.4
6 F 22 2 6 42.3
7 F 52 2 6 68.2
8 M 43 1 4 73.4
9 F 49 1 3 65.2
10 M 21 2 4 62.3
11 M 38 2 5 69.8
12 F 49 1 5 80.5
13 M 32 2 4 56.2
14 F 30 1 6 42.3
15 F 42 2 6 46.8
Fig. 1 e Incisions to the recipient site (a); osteotomies of the mandibular ramus for harvesting a mono-cortical block (b); fixation of the graft to the
maxillary defect (c); re-entry at 4 months that show no clinical resorption of the graft (d); connection of the implant inserted with a zirconium abutment
and finalization of the case with a zirconium crown (eef).
224 Journal of Cranio-Maxillo-Facial Surgery
Fig. 2 e View of a maxillary narrow ridge after flap elevation (a); bone osteotomies on mandibular buccal shelf (b); the mono-cortical is out-fractured
(c); adaptation and fixation of the graft to the maxillary recipient site (d); gaps around the block grafts were filled with bone chips harvested from the
donor site (e); primary flap closure without tension (f).
Table 2 e Analytical description of the amount of bone augmentation obtained and grafts’ resorption
Case Residual Ridge
Width
Lateral
augmentation at
bone grafting
Post augmentation
width
Re-entry
width
Re-entry lateral
augmentation
Resorption mm Resorption %
1 3.5 mm 5 mm 8.5 mm 8 mm 4.5 mm 0.5 mm 10
2 2.5 mm 6 mm 8.5 mm 8 mm 5.5 mm 0.5 mm 8.3
3 3 mm 5 mm 8 mm 7 mm 4 mm 1 mm 20
4 3 mm 4 mm 7 mm 6.5 mm 3.5 mm 0.5 mm 12.5
5 3.5 mm 4 mm 7.5 mm 7.5 mm 4 mm 0 mm 0
6 4 mm 5 mm 9 mm 7.5 mm 3.5 mm 1.5 mm 30
7 3 mm 5 mm 8 mm 8 mm 5 mm 0 mm 0
8 3.5 mm 4 mm 7.5 mm 7 mm 3.5 mm 0.5 mm 12.5
9 3 mm 5 mm 8 mm 7.5 mm 4.5 mm 0.5 mm 10
10 2.5 mm 5 mm 7.5 mm 7 mm 4.5 mm 0.5 mm 10
11 2 mm 4 mm 6 mm 5.5 mm 3.5 mm 0.5 mm 12.5
12 3 mm 4 mm 7 mm 6 mm 3 mm 1 mm 25
13 4 mm 5 mm 9 mm 8 mm 4 mm 1 mm 20
14 3.5 mm 3 mm 6.5 mm 6 mm 2.5 mm 0.5 mm 16.6
15 3 mm 5 mm 8 mm 7.5 mm 4.5 mm 0.5 mm 10
Clinical, histological and histomorphometric evaluation of the healing of mandibular ramus bone block grafts 225
A bone specimen measuring approximately 2.5 mm di-
ameter and 8e10 mm long was removed with a hollow
trephine burr of 3 mm outer diameter, accompanied by
a copious irrigation. The specimen was carefully re-
moved from the trephine burr and the part of the biopsy
corresponding to the buccal graft’s cortical plate was la-
belled by black Indian ink, for better orientation during
histology examination.
Histology and histomorphometry
All specimens were immediately fixed in 4% formalde-
hyde solution in 0.1 M phosphate buffer (pH 7.3) at
4 
C for 24 h. They were then rinsed three times with
0.1 M phosphate buffer and, finally, stored at 70% etha-
nol at 4 
C, until ready to be embedded. All the biopsies
were cold embedded in methylmethacrylate with 20%
N-plastoid (resin solution). Nondecalcified, 5-mm thick
sections were made along the axis of the biopsy using
a Jung K microtome. All specimens were, then, stained
with haematoxylin and eosin. A Leica DM RA micro-
scope connected to a computer using an electronic stage
table and a Leica DC 200 digital camera were used for
histomorphometrical measurements. Leica QWinÓ
soft-
ware (Leica Microsystems Image Solutions, Rijswijk,
the Netherlands) was used to process and measure the
digitised images. This software allows a selection of parts
of an image by setting a threshold to the colour of the
pixels, which can then, be measured. All measurements
were carried out at Â200 magnification in order to allow
clear distinction between empty and full osteocyte lacu-
nae. The sections stained with haematoxylin and eosin
were used for the measurements of total bone volume
and NVB volume. The mineralized bone tissue containing
areas of empty osteocyte lacunae was defined as NVB.
NVB was expressed as a percentage of the total bone
volume and its measurement was made semi-automati-
cally by outlining the area of empty osteocytes lacunae.
Statistical analysis
All data were analysed using SPSS for Windows 12.0. A
simple regression model was applied to the data in order
to examine the correlation between healing time and
NVB (Model: NVBi ¼ b0 + b1 moni + 3). The data were
presented as means and standard deviations. Significance
was accepted at the p0.05 level.
RESULTS
Clinical
All grafts integrated successfully, guaranteeing sufficient
bone volume area for implant installation. No significant
resorption around the head of the fixation screws was evi-
dent. Only one patient exhibited a small graft exposure but
the soft tissue dehiscence resolved spontaneously showing
no further complications graft infections. Another patient,
who underwent a large bilateral graft from the mandibular
body, experienced a transient hypoesthesia of the lower
lip, which recovered completely within three weeks.
Table 2 analyses the analytical data regarding the
grafting together with rise of bone volume at the time
of grafting and implant placement. The mean bone
resorption occurring during healing time was calculated
according to the measurements of lateral augmentation
at bone grafting and after 3e9 months, at implant place-
ment. Considering all the sites together, mean lateral
augmentation, at grafting, was 4.6 ^ 0.73 mm which re-
duced to a mean of 4 ^ 0.77 mm at implant insertion;
this is equivalent to a mean reduction in the lateral aug-
mentation of 13.1 ^ 8.9% (min 10%, max 30%) during
healing (Table 3).
The number of fixtures placed and the timing of inser-
tion for each patient are indicated in Table 1. After six
months all 30 implants placed were loaded and all fix-
tures were successful, according to Albrektsson criteria
(Albrektsson et al., 1986) (Fig. 1eef). In the subsequent
follow-up (average 12-month follow-up), no major com-
plications were recorded at donor or recipient sites. Soft
tissue healing was uneventful, while pain and swelling
were comparable to conventional dento-alveolar proce-
dures. Wound dehiscence with graft infection and defin-
itive neurosensory deficits at the donor site wase not
detected.
HISTOLOGICAL AND HISTOMORPHOMETRIC
FINDINGS
Histologically, all specimens showed signs of active re-
modelling and all tissues were free of inflammatory cells.
Contemporarily, all sections contained both vital and
NVB tissue, compact osteonic and trabecular bone. The
representation of vital and NVB bone varied consider-
ably according to each individual. Bone classed as non-
vital (with fields of empty osteocyte lacunae) was pre-
dominantly lamellar in type (Fig. 3), while the vital
bone was composed of both lamellar and woven bone.
The vital and NVB were in contact with each other
and, sometimes, the latter was completely surrounded
by vital bone the former first. The margin between the vi-
tal and the NVB coincided with an abrupt change in the
orientation of the lamellae (Fig. 3).
As vital bone was in tight contact with the NVB (os-
teoconductor), the first replaced the second thanks to
creeping substitution process (Fig. 4). Osteoclasts were
rarely detected. Vital bone contained osteocytes in the in-
ner core of an osteon, surrounded by NVB, suggesting
that this was recolonized by blood vessels (BV) and os-
teogenic cells via the Haversian canal (Fig. 3).
Neovasculature was poorly represented in all speci-
mens. At the interface between grafted bone and the re-
cipient site, some specimens showed varying amounts
of fibrous tissue (FT) mixed with new bone formation
(Fig. 4). With the histological analysis alone, very little
qualitative differences could be found among biopsies
at different healing times. The volume of vital and
NVB of all patients and the time of healing is presented
in Table 1. The relative NVB volume (NVB as % of total
bone volume) varied from 80.5% (in patient no. 12) to
34.3% (in patient no. 2) with an overall average of
57.75% of total tissue volume.
226 Journal of Cranio-Maxillo-Facial Surgery
Statistical analysis
The relationship between variables ‘‘NVB’’ (response
variable) and ‘‘Months’’ (explanatory variable, length
of time) looked linear (Fig. 5). A straight line was extrap-
olated from the data by the least squares method. The
estimated slope ^b 1 was negative, indicating that the
estimated decrease in NVB per month is around 5.2%.
The associated R-squared was around 31%, and its
p-value (as well, of course, as the p-value of the wald
test on the slope coefficient) was moderately significant,
showing that the linear relation is good enough. While
most of the data points were clustered towards the left
side of the plot, there is just one an observation point,
which lies far away from the main cluster of the data
(Fig. 5). As this point probably has a major impact on
the regression line, another model omitting this observa-
tion was tried. These data suggest that the amount of
NVB decreased slowly as time of healing increased;
grafted bone resulted completely revascularized and
remodelled after 16.2 months (Fig. 5).
DISCUSSION
Preprosthetic reconstructive surgery of the resorbed max-
illa has to create sufficient, good quality bone for implant
placement. Several procedures have been proposed to
achieve alveolar ridge augmentation in partially edentu-
lous patients. (Buser et al., 1995; de Wijs, 1997).
Bone block graft is the preferred method for many types
of augmentation procedures, since it secures both a source
of osteogenic cells and a rigid structure for mechanical
support. In addition, bone block graft conserves its volume
better than particulate grafting (Lew et al., 1994).
Autologus bone block grafts have a greater flexibility, as
they can be used in all clinical situations (also in cases of
complex), while on the other hand, they show a greater
potential for post-operative morbidity (risk of graft infec-
tion and neurologic sequelae related to bone harvesting
in intra-oral site). In fact, harvesting of grafts from the
mandibular region may cause neurological complications,
due to the risk of damage to the inferior alveolar nerve.
Recently, a sensory deficit in lower lip and mental areas
Table 3 e Mean and standard deviation in mm of lateral bone augmentation measured at the time of bone grafting and surgical re-entry for implant
placement. Mean percentage reduction in lateral augmentation after grafts healing
Lateral augmentation at bone grafting Re-entry lateral augmentation Resorption %
4.6 ^ 0.73 4 ^ 0.77 13.1 ^ 8.19
Fig. 3 e Histology of the external cortical layer of the ascending ramus graft after 4 (a), 5(b), 6(c; d) months. The pictures show the typical compact
osteonic structure of the mandibular ramus but with predominantly empty osteocyte lacunae (NVB) at different healing times. Signs of active
remodelling and new growing vessels are not evident; at a higher magnification vital bone containing osteocytes in the inner core of osteons, surrounded
lamellar non-vital bone suggesting that non-vital bone was recolonized by BV and osteogenic cells via the Haversian canals (d). o ¼ osteocyte,
bv ¼ blood vessel, NVB ¼ non-vital bone; original magnification Â100 (a,b) and Â200 (c,d); specimens stained with haematoxylin and eosin.
Clinical, histological and histomorphometric evaluation of the healing of mandibular ramus bone block grafts 227
Fig. 4 e Grafted bone/recipient site interface after 4 (A) and 6 months (B). (a1) After 4 months a substantial amount of FT, little new vital bone; (a2) at
a higher magnification new vital (VB) bone surrounded by NVB with empty osteocyte lacunae, way of resorption (creeping substitution). (b1) After 6
months: active bone remodelling with presence of larger amount of vital bone; FT is still present and NVB has not been completely resorbed. (b2) At
a higher magnification, a large amount of new vital bone containing osteocytes; original magnification Â100 (a1,b1) and Â200 (a2,b2); specimens
stained with haematoxylin and eosin.
Fig. 5 e Non-vital bone as percentage of total bone volume relative to time.
228 Journal of Cranio-Maxillo-Facial Surgery
of 8.3% in mandibular ramus harvesting was reported,
compared with 16% for the chin as the donor site.
Our clinical findings showed that 9 months post-graft-
ing is a sufficient period of time for the grafted bone to
integrate successfully with the original maxillary bone
and reach the proper stability for an ideal implant
placement. Moreover, in accordance with literature, the
resorption rates reported confirmed the excellent volu-
metric stability of the bone block grafts harvested from
ascending ramus (Braun and Sotereanos, 1984; Misch
et al., 1992; Raghoebar et al., 1996; Cordaro et al.,
2002; Schwartz-Arad and Levin, 2005).
Histologically, this work shows that the grafted bone
contained extended parts of NVB (with empty osteocyte
lacunae) at the time of implant placement. In fact, osteo-
cytes require a 0.1-mm proximity to nutrient vessels
(Ham, 1952) to survive and interruption of the blood sup-
ply results in avascular necrosis (Cruess, 1986). In this
sense, our findings correspond with that of Ellegaard
et al., 1975 who state that with disruption of BVs,
many osteocytes are completely entombed by mineral-
ised bone and do not survive the relocation.
Therefore, the presence of deficient neovasculature at
different healing times suggests that the dense structure
of cortical bone represents a physical obstacle the new
vessel growth both from the soft and hard tissues.
From a clinical point of view, it seems important to
wait for NVB to be replaced by vital bone prior to im-
plant placement, since vital bone has better mechanical
characteristics (Goldberg and Stevenson, 1987). In this
respect, the rate by which NVB is resorbed by osteoclast
activity followed by new bone formation is an important
factor for the osseointegration of implants. There is in-
creasing evidence that osteocytes are mechanosensitive
(Klein-Nulend et al., 1995) and, for this, they play a cru-
cial role in adaptive bone remodelling. Osteocytes may
be involved in the recruitment of osteoclasts or in the
modulation of their activity by secreting signalling factors,
such as nitrogen oxide, prostaglandins (Klein-Nulend
et al., 1995), osteoprotegerin, macrophage colony-stimu-
lating factor (M-CSF) and receptor activator of nuclear
factor kappa B ligand (RANKL) (Zhao et al., 2002) or fac-
tors related to the apoptosis pathway (Bronckers et al.,
1996) induced by ischaemia (Kikuyama et al., 2002).
These factors communicate with the bone surface and con-
trol the activity of bone surface cells such as osteoblasts,
osteoclasts and bone lining cells. (Smit and Burger,
2002; Zhao et al., 2002; Zerbo et al., 2003).
As the sample of patients considered by this work is
not relevant, this study does not contribute extensively
to set new guidelines for the clinical management of
the correct timing of implant placement after grafting.
However, the slow revascularization and remodelling
processes of mandibular bone blocks suggest waiting at
least 4 months after grafts healing before implant inser-
tion. (Aalam and Nowzari, 2007; Felice et al., 2009).
CONCLUSIONS
From a clinical point of view, as supported by literature,
this procedure appears to be simple, safe and effective for
the treatment of localized alveolar ridge defects in par-
tially edentulous patients. Nonetheless, more studies are
needed to explain the internal micro architectural
changes occurring during the incorporation and remodel-
ling processes in order to draw more definitive conclu-
sions about the correct timing of implant insertion and
loading.
ACKNOWLEDGMENTS
The authors would like to acknowledge Paolo Nardi MD, DDS,
Carlo Catelani MD, DDS, Pasquale Paglianiti, DDS for their
clinical contribution and overall assistance and Miss Ilaria Pas-
quinelli for English review.
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osteocyte-like cells support osteoclast formation and activation.
J Bone Miner Res 17: 2068e2079, 2002
Dr. Roberto SACCO
Via Alcide De Gasperi
113 Prato (59100)
Italy
Tel: +39 349 5858220
E-mail: saccoroberto@yahoo.it
Paper received 7 January 2009
Accepted 3 July 2009
230 Journal of Cranio-Maxillo-Facial Surgery

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Histomorfometria mandibular si

  • 1. Clinical, histological and histomorphometric evaluation of the healing of mandibular ramus bone block grafts for alveolar ridge augmentation before implant placement* Alessandro ACOCELLA, Oral Surgery Specialist1 , Roberto BERTOLAI, Oral and Maxillo-Facial specialist1 , Maurizio COLAFRANCESCHI, Professor of Human Anatomy and Pathology2 , Roberto SACCO, Oral Surgery Programme Resident3 1 Department of Maxillo-Facial Surgery, Faculty of Medicine-University of Florence, Italy; 2 Department of Human Anatomy and Pathology, Faculty of Medicine-University of Florence, Italy; 3 Department of Odontostomatology, Faculty of Medicine-University of Sassari, Italy SUMMARY. Background: Localized bone defects in the maxilla are commonly reconstructed with autologous mono-cortical bone blocks prior to the placement of dental implants. This study presents a clinical, histological and histomorphometric analysis on the use of mandibular ramus block grafts for ridge augmentation. Materials and Methods: mono-cortical bone blocks from the mandibular ramus were grafted in 15 patients. The bone grafts were left to heal for period varying from 3 to 9 months. Afterwards, 30 implants were inserted and bone samples were removed for subsequent histological analysis. Results: All the bone grafts were successful and resorption was minimal. There were no implant failures. At graft placement, mean lateral augmentation was 4.6 ^ 0.73 mm, which, later, at the time of implant insertion, reduced to 4 ^ 0.77 mm. Histological evaluation indicated signs of active remodelling in all specimens. However, the grafted bone contained substantial amounts of non-vital bone (NVB) and generally weak neo-vascularization regardless of the time of biopsies. Conclusions: The outcome of the study suggests that the larger part of osteocytes in mono-cortical bone do not survive grafting and neo-vascularization of non-vital grafted bone is difficult because of the slow remodelling process into new vital. Ó 2009 European Association for Cranio-Maxillo-Facial Surgery Keywords: bone histology, bone histomorphometry, mandibular ramus, maxillary defects, mono-cortical bone block graft INTRODUCTION Bone defects in the human maxilla are common. They are mostly determined by premature loss of teeth due to periodontal disease or traumas. They usually cause reduction of alveolar bone volume, which becomes, in consequence, inadequate for standard treatments with osseointegrated implants (Nevins and Mellonig, 1994; Oikarinen et al., 2003). Pathological resorption of the maxillary bone may be the main cause of both functional and aesthetic dissatis- faction to the patient (Collins and Nunn, 1994). Moreover, insufficient bone height and width of the al- veolar ridge make the maxillary anatomy particularly un- favourable for the conventional placement of dental implants. In order to create favourable conditions for implant placement, the bone must undergo augmentation. Nowa- days, reconstruction and augmentation of severely resorbed maxillary alveolar ridges, with the use of differ- ent grafting materials and techniques, represent a predict- able procedure for endoosseous implant placement. (Buser et al., 1995; Oikarinen et al., 2003; Chiapasco et al., 2007). There have been experimental in vitro and in vivo studies in all kinds of animal models in order to assess the quality of bone defects filled with different test mate- rials. The majority of these studies evaluated the osteoin- ductive activities in critical size defect models using radiography or histology (Bosch et al., 1998; Nidoli et al., 1999). Recently, there has been considerable interest in bone regeneration augmented with stem cells, periosteum-de- rived cells, or osteoinductive proteins to aid the healing of large skeletal defects (Boo et al., 2002; Yamada et al., 2003; Lendeckel et al., 2004; Sakata et al., 2006). It has been widely accepted that autologous bone is the most appropriate grafting material. (Listrom and Syming- ton, 1988; Jensen and Sindet-Pedersen, 1991). The harvesting sites may be intraoral or extraoral: in the first case, bone can be harvested from retromolar, * The work should be attributed: Department of Maxillo-Facial Sur- gery, Faculty of Medicine e University of Florence, Italy; Director: Dr. Roberto Bertolai, address: Largo Palagi,1 Firenze, Italy. 222 Journal of Cranio-Maxillo-Facial Surgery (2010) 38, 222e230 Ó 2009 European Association for Cranio-Maxillo-Facial Surgery doi:10.1016/j.jcms.2009.07.004, available online at http://www.sciencedirect.com
  • 2. ramus or symphysis areas (Jensen and Sindet-Pedersen, 1991; Misch, 1997; Widmark et al., 1997; Hunt and Jovanovic, 1999) while in the second case, bone can be collected from ilium, calvarium or tibia. (Block and Kent, 1997). Both mandibular body and ramus area represent an ideal donor site as they provide adequate, dense bone with sufficient volume for implant placement and short healing times. (Garg et al., 1998). The advantages of the ascending ramus donor site over the chin include minimal patient concern for altered fa- cial contour and low levels of post-operative sensory dis- turbances and discomfort. Although mandibular ramus mono-cortical block grafts for alveolar ridge augmentation have been widely used with great clinical success, it has not been clearly explained how they incorporate into the surrounding bone. The general lack of histological evidence on the healing of mandibular autogenous bone block grafts leaves a great number of unanswered questions about the biological process. The vitality of osteocytes is believed to be important in the process of bone remodelling as it seems that these cells are involved in bone turnover. This study’s objective was to gain insight into the vi- tality of bone grafts in the human jaw with emphasis on the survival of osteocytes used as sign of bone vitality. Our study has been conducted with an integrated mix of tests and, among them, histomorphometry which has only been used in a few studies for the evaluation of os- teocyte survival. (Blomqvist et al., 1998). MATERIALS AND METHODS Patients Fifteen patients (7 men and 8 women) of ages ranging from 21 to 53 years were considered for the study (Table 1). The selected patients had severe bone defects in the maxillary alveolar ridge, originating from peri- odontal disease or traumas. All 15 patients were healthy and had no general con- traindications for bone block graft surgical treatment; only four of them were smokers. First of all, a clinical and radiographic investigation was carried out and the treatment fully explained to the patients. The treatment programme included a first oper- ation for bone augmentation followed by a healing period varying from 3 to 9 months (Table 1); finally, a second surgical operation was scheduled for biopsy and implant placement. All patients gave their informed consent to donate the bone tissue removed during implant surgery for histological examination. First surgical step The bone augmentation was carried out under local an- aesthesia in 14 patients and under general anaesthesia with rhino-tracheal intubation in only one case. The lat- ter, in fact, was affected by a larger pre-maxilla defect, which was restored with bilateral ascending ramus grafts. Full-thickness flaps were reflected in order to allow a satisfactory exposure of the recipient site (Figs. 1a, 2a). The remaining crest width was measured with a pair of cal- lipers to the nearest half-millimeter (pre-augmentation width) (Table 2). After administering block anaesthesia to the inferior alveolar nerve, harvesting of bone from ascending ramus was performed. An obliqueesagittal incision was made distal to the third molar following the direction of the ra- mus and a vertical releasing incision was placed distally in the ramus area. After reflection of bucco-lingual flaps, osteotomies were executed with a small fissure bur to outline the dimensions of the bone block (Figs. 1b, 2b). Careful techniques were performed to make sure penetra- tion was only of the cortical layer and to avoid any in- juries to the alveolar nerve. Once the osteotomy was completed, a straight elevator was placed along the sag- ittal cut and the lateral block of bone was green-stick fractured off (Fig. 2c). The recipient site was prepared by scoring or perforating through and through (with a small, straight fissure bur) in order to create bleeding channels. The block autograft was then fixated to recipi- ent site with 1 or 2 lag-screws (Osteomed, Addison, TX) (Figs. 1c, 2d). Sharp edges of the bone blocks were rounded off (with large diamond burs) and the aug- mented width of the bony crest was measured again with a pair of callipers (post-augmentation width). Gaps around the block grafts were filled with bone chips harvested from the donor site (Fig. 2e). Once the graft was adapted to the site, an incision through the perios- teum at the base of the flap allowed the tissue cover the graft without tension. The recipient and the donor site areas were then sutured with vertical mattress sutures (Vicryl 3-0, Ethicon, Johnson & Johnson Roma, Italy) without covering the bone graft with a barrier membrane (Fig. 2f). Post-operative antibiotic (Zimox, Pfizer, Latina, Italy) and analgesic (Brufen; Abbott SpA, Campoverde (Lt), Italy) therapy was routinely prescribed for 7 days. Patients were also given chlorhexidine digluconate 0.2% mouth- wash (Curasept; Curaden Health Care Srl, Milano, Italy) for ten days and sutures were removed after 10e12 days. Second surgical step After a healing period of from 3 to 9 months, before the implant insertion, CT examination was performed in order to plan implant surgery correctly. The flap was re-opened with an outline similar to that of the first oper- ation (Fig. 1d). Once the flap was elevated, the healed crest width was measured again with a pair of callipers (re-entry width) (Table 2). Bone block fixation screws were removed; implant site preparation and insertion were accomplished according to standard surgical proto- cols. A total of 30 implants were placed (Table 1) and primary stability was achieved during this step (maxi- mum insertion torque was 35 Ncm). Biopsy procedure Biopsy was performed at the same time of implant place- ment including the grafted bone/recipient site interface. Clinical, histological and histomorphometric evaluation of the healing of mandibular ramus bone block grafts 223
  • 3. Table 1 e Patient data and results of histomorphometry means Patient Sex Age No. of implants Healing time (months) Non Vital Bone (histological %) 1 M 28 6 4 60.7 2 M 34 2 5 34.3 3 F 32 1 4 73.2 4 M 53 2 7 52.7 5 F 37 3 9 38.4 6 F 22 2 6 42.3 7 F 52 2 6 68.2 8 M 43 1 4 73.4 9 F 49 1 3 65.2 10 M 21 2 4 62.3 11 M 38 2 5 69.8 12 F 49 1 5 80.5 13 M 32 2 4 56.2 14 F 30 1 6 42.3 15 F 42 2 6 46.8 Fig. 1 e Incisions to the recipient site (a); osteotomies of the mandibular ramus for harvesting a mono-cortical block (b); fixation of the graft to the maxillary defect (c); re-entry at 4 months that show no clinical resorption of the graft (d); connection of the implant inserted with a zirconium abutment and finalization of the case with a zirconium crown (eef). 224 Journal of Cranio-Maxillo-Facial Surgery
  • 4. Fig. 2 e View of a maxillary narrow ridge after flap elevation (a); bone osteotomies on mandibular buccal shelf (b); the mono-cortical is out-fractured (c); adaptation and fixation of the graft to the maxillary recipient site (d); gaps around the block grafts were filled with bone chips harvested from the donor site (e); primary flap closure without tension (f). Table 2 e Analytical description of the amount of bone augmentation obtained and grafts’ resorption Case Residual Ridge Width Lateral augmentation at bone grafting Post augmentation width Re-entry width Re-entry lateral augmentation Resorption mm Resorption % 1 3.5 mm 5 mm 8.5 mm 8 mm 4.5 mm 0.5 mm 10 2 2.5 mm 6 mm 8.5 mm 8 mm 5.5 mm 0.5 mm 8.3 3 3 mm 5 mm 8 mm 7 mm 4 mm 1 mm 20 4 3 mm 4 mm 7 mm 6.5 mm 3.5 mm 0.5 mm 12.5 5 3.5 mm 4 mm 7.5 mm 7.5 mm 4 mm 0 mm 0 6 4 mm 5 mm 9 mm 7.5 mm 3.5 mm 1.5 mm 30 7 3 mm 5 mm 8 mm 8 mm 5 mm 0 mm 0 8 3.5 mm 4 mm 7.5 mm 7 mm 3.5 mm 0.5 mm 12.5 9 3 mm 5 mm 8 mm 7.5 mm 4.5 mm 0.5 mm 10 10 2.5 mm 5 mm 7.5 mm 7 mm 4.5 mm 0.5 mm 10 11 2 mm 4 mm 6 mm 5.5 mm 3.5 mm 0.5 mm 12.5 12 3 mm 4 mm 7 mm 6 mm 3 mm 1 mm 25 13 4 mm 5 mm 9 mm 8 mm 4 mm 1 mm 20 14 3.5 mm 3 mm 6.5 mm 6 mm 2.5 mm 0.5 mm 16.6 15 3 mm 5 mm 8 mm 7.5 mm 4.5 mm 0.5 mm 10 Clinical, histological and histomorphometric evaluation of the healing of mandibular ramus bone block grafts 225
  • 5. A bone specimen measuring approximately 2.5 mm di- ameter and 8e10 mm long was removed with a hollow trephine burr of 3 mm outer diameter, accompanied by a copious irrigation. The specimen was carefully re- moved from the trephine burr and the part of the biopsy corresponding to the buccal graft’s cortical plate was la- belled by black Indian ink, for better orientation during histology examination. Histology and histomorphometry All specimens were immediately fixed in 4% formalde- hyde solution in 0.1 M phosphate buffer (pH 7.3) at 4 C for 24 h. They were then rinsed three times with 0.1 M phosphate buffer and, finally, stored at 70% etha- nol at 4 C, until ready to be embedded. All the biopsies were cold embedded in methylmethacrylate with 20% N-plastoid (resin solution). Nondecalcified, 5-mm thick sections were made along the axis of the biopsy using a Jung K microtome. All specimens were, then, stained with haematoxylin and eosin. A Leica DM RA micro- scope connected to a computer using an electronic stage table and a Leica DC 200 digital camera were used for histomorphometrical measurements. Leica QWinÓ soft- ware (Leica Microsystems Image Solutions, Rijswijk, the Netherlands) was used to process and measure the digitised images. This software allows a selection of parts of an image by setting a threshold to the colour of the pixels, which can then, be measured. All measurements were carried out at Â200 magnification in order to allow clear distinction between empty and full osteocyte lacu- nae. The sections stained with haematoxylin and eosin were used for the measurements of total bone volume and NVB volume. The mineralized bone tissue containing areas of empty osteocyte lacunae was defined as NVB. NVB was expressed as a percentage of the total bone volume and its measurement was made semi-automati- cally by outlining the area of empty osteocytes lacunae. Statistical analysis All data were analysed using SPSS for Windows 12.0. A simple regression model was applied to the data in order to examine the correlation between healing time and NVB (Model: NVBi ¼ b0 + b1 moni + 3). The data were presented as means and standard deviations. Significance was accepted at the p0.05 level. RESULTS Clinical All grafts integrated successfully, guaranteeing sufficient bone volume area for implant installation. No significant resorption around the head of the fixation screws was evi- dent. Only one patient exhibited a small graft exposure but the soft tissue dehiscence resolved spontaneously showing no further complications graft infections. Another patient, who underwent a large bilateral graft from the mandibular body, experienced a transient hypoesthesia of the lower lip, which recovered completely within three weeks. Table 2 analyses the analytical data regarding the grafting together with rise of bone volume at the time of grafting and implant placement. The mean bone resorption occurring during healing time was calculated according to the measurements of lateral augmentation at bone grafting and after 3e9 months, at implant place- ment. Considering all the sites together, mean lateral augmentation, at grafting, was 4.6 ^ 0.73 mm which re- duced to a mean of 4 ^ 0.77 mm at implant insertion; this is equivalent to a mean reduction in the lateral aug- mentation of 13.1 ^ 8.9% (min 10%, max 30%) during healing (Table 3). The number of fixtures placed and the timing of inser- tion for each patient are indicated in Table 1. After six months all 30 implants placed were loaded and all fix- tures were successful, according to Albrektsson criteria (Albrektsson et al., 1986) (Fig. 1eef). In the subsequent follow-up (average 12-month follow-up), no major com- plications were recorded at donor or recipient sites. Soft tissue healing was uneventful, while pain and swelling were comparable to conventional dento-alveolar proce- dures. Wound dehiscence with graft infection and defin- itive neurosensory deficits at the donor site wase not detected. HISTOLOGICAL AND HISTOMORPHOMETRIC FINDINGS Histologically, all specimens showed signs of active re- modelling and all tissues were free of inflammatory cells. Contemporarily, all sections contained both vital and NVB tissue, compact osteonic and trabecular bone. The representation of vital and NVB bone varied consider- ably according to each individual. Bone classed as non- vital (with fields of empty osteocyte lacunae) was pre- dominantly lamellar in type (Fig. 3), while the vital bone was composed of both lamellar and woven bone. The vital and NVB were in contact with each other and, sometimes, the latter was completely surrounded by vital bone the former first. The margin between the vi- tal and the NVB coincided with an abrupt change in the orientation of the lamellae (Fig. 3). As vital bone was in tight contact with the NVB (os- teoconductor), the first replaced the second thanks to creeping substitution process (Fig. 4). Osteoclasts were rarely detected. Vital bone contained osteocytes in the in- ner core of an osteon, surrounded by NVB, suggesting that this was recolonized by blood vessels (BV) and os- teogenic cells via the Haversian canal (Fig. 3). Neovasculature was poorly represented in all speci- mens. At the interface between grafted bone and the re- cipient site, some specimens showed varying amounts of fibrous tissue (FT) mixed with new bone formation (Fig. 4). With the histological analysis alone, very little qualitative differences could be found among biopsies at different healing times. The volume of vital and NVB of all patients and the time of healing is presented in Table 1. The relative NVB volume (NVB as % of total bone volume) varied from 80.5% (in patient no. 12) to 34.3% (in patient no. 2) with an overall average of 57.75% of total tissue volume. 226 Journal of Cranio-Maxillo-Facial Surgery
  • 6. Statistical analysis The relationship between variables ‘‘NVB’’ (response variable) and ‘‘Months’’ (explanatory variable, length of time) looked linear (Fig. 5). A straight line was extrap- olated from the data by the least squares method. The estimated slope ^b 1 was negative, indicating that the estimated decrease in NVB per month is around 5.2%. The associated R-squared was around 31%, and its p-value (as well, of course, as the p-value of the wald test on the slope coefficient) was moderately significant, showing that the linear relation is good enough. While most of the data points were clustered towards the left side of the plot, there is just one an observation point, which lies far away from the main cluster of the data (Fig. 5). As this point probably has a major impact on the regression line, another model omitting this observa- tion was tried. These data suggest that the amount of NVB decreased slowly as time of healing increased; grafted bone resulted completely revascularized and remodelled after 16.2 months (Fig. 5). DISCUSSION Preprosthetic reconstructive surgery of the resorbed max- illa has to create sufficient, good quality bone for implant placement. Several procedures have been proposed to achieve alveolar ridge augmentation in partially edentu- lous patients. (Buser et al., 1995; de Wijs, 1997). Bone block graft is the preferred method for many types of augmentation procedures, since it secures both a source of osteogenic cells and a rigid structure for mechanical support. In addition, bone block graft conserves its volume better than particulate grafting (Lew et al., 1994). Autologus bone block grafts have a greater flexibility, as they can be used in all clinical situations (also in cases of complex), while on the other hand, they show a greater potential for post-operative morbidity (risk of graft infec- tion and neurologic sequelae related to bone harvesting in intra-oral site). In fact, harvesting of grafts from the mandibular region may cause neurological complications, due to the risk of damage to the inferior alveolar nerve. Recently, a sensory deficit in lower lip and mental areas Table 3 e Mean and standard deviation in mm of lateral bone augmentation measured at the time of bone grafting and surgical re-entry for implant placement. Mean percentage reduction in lateral augmentation after grafts healing Lateral augmentation at bone grafting Re-entry lateral augmentation Resorption % 4.6 ^ 0.73 4 ^ 0.77 13.1 ^ 8.19 Fig. 3 e Histology of the external cortical layer of the ascending ramus graft after 4 (a), 5(b), 6(c; d) months. The pictures show the typical compact osteonic structure of the mandibular ramus but with predominantly empty osteocyte lacunae (NVB) at different healing times. Signs of active remodelling and new growing vessels are not evident; at a higher magnification vital bone containing osteocytes in the inner core of osteons, surrounded lamellar non-vital bone suggesting that non-vital bone was recolonized by BV and osteogenic cells via the Haversian canals (d). o ¼ osteocyte, bv ¼ blood vessel, NVB ¼ non-vital bone; original magnification Â100 (a,b) and Â200 (c,d); specimens stained with haematoxylin and eosin. Clinical, histological and histomorphometric evaluation of the healing of mandibular ramus bone block grafts 227
  • 7. Fig. 4 e Grafted bone/recipient site interface after 4 (A) and 6 months (B). (a1) After 4 months a substantial amount of FT, little new vital bone; (a2) at a higher magnification new vital (VB) bone surrounded by NVB with empty osteocyte lacunae, way of resorption (creeping substitution). (b1) After 6 months: active bone remodelling with presence of larger amount of vital bone; FT is still present and NVB has not been completely resorbed. (b2) At a higher magnification, a large amount of new vital bone containing osteocytes; original magnification Â100 (a1,b1) and Â200 (a2,b2); specimens stained with haematoxylin and eosin. Fig. 5 e Non-vital bone as percentage of total bone volume relative to time. 228 Journal of Cranio-Maxillo-Facial Surgery
  • 8. of 8.3% in mandibular ramus harvesting was reported, compared with 16% for the chin as the donor site. Our clinical findings showed that 9 months post-graft- ing is a sufficient period of time for the grafted bone to integrate successfully with the original maxillary bone and reach the proper stability for an ideal implant placement. Moreover, in accordance with literature, the resorption rates reported confirmed the excellent volu- metric stability of the bone block grafts harvested from ascending ramus (Braun and Sotereanos, 1984; Misch et al., 1992; Raghoebar et al., 1996; Cordaro et al., 2002; Schwartz-Arad and Levin, 2005). Histologically, this work shows that the grafted bone contained extended parts of NVB (with empty osteocyte lacunae) at the time of implant placement. In fact, osteo- cytes require a 0.1-mm proximity to nutrient vessels (Ham, 1952) to survive and interruption of the blood sup- ply results in avascular necrosis (Cruess, 1986). In this sense, our findings correspond with that of Ellegaard et al., 1975 who state that with disruption of BVs, many osteocytes are completely entombed by mineral- ised bone and do not survive the relocation. Therefore, the presence of deficient neovasculature at different healing times suggests that the dense structure of cortical bone represents a physical obstacle the new vessel growth both from the soft and hard tissues. From a clinical point of view, it seems important to wait for NVB to be replaced by vital bone prior to im- plant placement, since vital bone has better mechanical characteristics (Goldberg and Stevenson, 1987). In this respect, the rate by which NVB is resorbed by osteoclast activity followed by new bone formation is an important factor for the osseointegration of implants. There is in- creasing evidence that osteocytes are mechanosensitive (Klein-Nulend et al., 1995) and, for this, they play a cru- cial role in adaptive bone remodelling. Osteocytes may be involved in the recruitment of osteoclasts or in the modulation of their activity by secreting signalling factors, such as nitrogen oxide, prostaglandins (Klein-Nulend et al., 1995), osteoprotegerin, macrophage colony-stimu- lating factor (M-CSF) and receptor activator of nuclear factor kappa B ligand (RANKL) (Zhao et al., 2002) or fac- tors related to the apoptosis pathway (Bronckers et al., 1996) induced by ischaemia (Kikuyama et al., 2002). These factors communicate with the bone surface and con- trol the activity of bone surface cells such as osteoblasts, osteoclasts and bone lining cells. (Smit and Burger, 2002; Zhao et al., 2002; Zerbo et al., 2003). As the sample of patients considered by this work is not relevant, this study does not contribute extensively to set new guidelines for the clinical management of the correct timing of implant placement after grafting. However, the slow revascularization and remodelling processes of mandibular bone blocks suggest waiting at least 4 months after grafts healing before implant inser- tion. (Aalam and Nowzari, 2007; Felice et al., 2009). CONCLUSIONS From a clinical point of view, as supported by literature, this procedure appears to be simple, safe and effective for the treatment of localized alveolar ridge defects in par- tially edentulous patients. Nonetheless, more studies are needed to explain the internal micro architectural changes occurring during the incorporation and remodel- ling processes in order to draw more definitive conclu- sions about the correct timing of implant insertion and loading. ACKNOWLEDGMENTS The authors would like to acknowledge Paolo Nardi MD, DDS, Carlo Catelani MD, DDS, Pasquale Paglianiti, DDS for their clinical contribution and overall assistance and Miss Ilaria Pas- quinelli for English review. 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