Ana Carolina Hawthorne Immunohistochemical, tomographic,

Samuel Porfı́rio Xavier

Roberta Okamoto
and histological study on onlay bone
Sérgio Luiz Salvador graft remodeling. Part III: allografts
Antonio Azoubel Antunes
Luiz Antonio Salata

Authors’ affiliations:
Ana Carolina Hawthorne, Samuel Porfı́rio Xavier,
Antonio Azoubel Antunes, Luiz Antonio Salata,
Department of Oral and Maxillofacial Surgery,
University of Sao Paulo, Ribeirao Preto, Brazil
Roberta Okamoto, Department of Oral and
Maxillofacial Surgery, University of the State of Sao
Paulo, Aracatuba, Brazil
Sérgio Luiz Salvador, Department of Clinical
Analysis, University of São Paulo, Ribeirao Preto,
Brazil
Corresponding author:
Prof. Dr Luiz A. Salata, BDS, PhD
University of São Paulo at Ribeirão Preto
Faculty of Dentistry
Department of Oral and Maxillofacial Surgery and
Periodontics
Avenida do Café s/n-Campus da USP
14040-904-Ribeirao Preto, SP-Brasil
Tel.: +55 16 3602 4018
Fax: +55 16 3602 4788
e-mail:lasalata@forp.usp.br
Key words: allogeneic bone graft, autogeneous bone graft, histologic evaluation, immunohis-
tochemistry, onlay bone graft, tomographic evaluation
Abstract
Objective: In the last decades aroused the interest for bone tissue bank as an alternative to
autogenous grafting, avoiding donor sites morbidity, surgical time, and costs reduction. The
purpose of the study was to compare allografts (ALg) with autografts (AUg) using histology,
immunochemistry, and tomographic analysis.
Material and methods: Fifty-six New Zealand White rabbits were submitted to surgical procedures.
Twenty animals were donors and 36 were actually submitted to onlay grafting with ALg
(experimental group) and AUg (control group) randomly placed bilaterally in the mandible. Six
animals of each group were sacrificed at 3, 5, 7, 10, 20, and 60 postoperative days. Immunolabeling
was accomplished with osteoprotegerin (OPG); receptor activator of nuclear factor-k ligand
(RANKL); alkaline phosphatase (ALP); osteopontin (OPN); vascular endothelial growth factor
(VEGF); tartrate-resistant acid phosphatase (TRAP); collagen type I (COL I); and osteocalcin (OC).
Density and volume of the grafts was evaluated on tomography obtained at the surgery and
sacrifice.
Results: The ALg and AUg exhibited similar patterns of density and volume throughout the
experiments. The intra-group data showed statistical differences at days 7 and 60 in comparison
with other time points (P = 0.001), in both groups. A slight graft expansion from fixation until day
20 (P = 0.532) was observed in the AUg group and then resorbed significantly at the day 60
(P = 0.015). ALg volume remained stable until day 7 and decreased at day 10 (P = 0.045). The light
microscopy analysis showed more efficient incorporation of AUg onto the recipient bed if
compared with the ALg group. The immunohistochemical labeling picked: at days 10 and 20 with
OPG in the AUg group and at day 7 with TRAP in the ALg group (P = 0.001 and P = 0.002,
respectively).
Conclusions: ALg and AUg were not differing in patterns of volume and density during entire
experiment. Histological data exhibit more efficient AUg incorporation into recipient bed
compared with the ALg group. Immunohistochemistry outcomes demonstrated similar pattern for
both ALg and AUg groups, except for an increasing resorption activity in the ALg group mediated
by TRAP and in the AUg group by higher OPG labeling. However, this latter observation does not
seem to influence clinical outcomes.

Date:
Accepted 04 June 2012
To cite this article:
Hawthorne AC, Xavier SP, Okamoto R, Salvador SL, Antunes
AA, Salata LA. Immunohistochemical, tomographic and
histological study on onlay bone graft remodeling. part iii:
allografts.
Clin. Oral Impl. Res. 24, 2013, 1164–1172
doi: 10.1111/j.1600-0501.2012.02528.x
Several studies on autografts (AUg) have
shown the importance of bone microarchitec-
ture, embryonic origin, type of installation
(“inlay” or “onlay”), periosteum presence,
recipient bed preparation, revascularization
rate, and graft fixation methods to assure the
success of grafting technique (Alberius et al.
1996; Stevenson et al. 1996; Alberius &
Gordh 1998; Ozaki & Buchman 1998; Buch-
man & Ozaki 1999; Tong & Buchman 2000;
Khan et al. 2005). The major drawbacks on
the use of AUg, however, are related to mor-
bidity of donor sites; extended surgical time;
increased blood loss; and higher surgical
costs.
In the last few decades a noticeable interest
in allografts (ALg) from musculoskeletal tis-
sue banks as an alternative to AUg for jaws
reconstructions. The fresh frozen ALg were
recently introduced as an option for augment-

1164 © 2012 John Wiley & Sons A/S


ing areas in sites to receive dental implants,
due to easy acquisition in an unlimited
amount, easy handling, reduced surgical time
and anesthesia, reduced bleeding (Tomford &
Mankin 1999; Gamradt & Lieberman 2003),
decreased postoperative morbidity (Barone
et al. 2009), and the possibility of major
reconstruction under local anesthesia. The
pertinent literature shows few studies inves-
tigating volume maintenance and ALg’s
interaction with recipient bed. Shand et al.
(2002) examined the response of ALgs har-
vested from the parietal region of eight donor
rabbits, processed according to the Local
Bone Bank protocol and installed in defects
made in the 15 receptor rabbits parietal
region. Radiographic evaluations, fluores-
cence microscopy, and histology were used
and after 1 year of follow-up, a positive
process of incorporation and osteoconduction
was observed, which reinforces its use in large
bone reconstruction. Clinical studies have
demonstrated the use of ALg from musculo-
skeletal tissue banks, aiming to rehabilitate
injury caused by trauma, tumors resections,
and congenital deformities, especially in
children where the amount of bone in donor
sites is scarce (Ellis & Sinn 1993). Virolainen
et al. (2003) evaluated a long-term use of fresh
frozen ALg and did not observe any allergenic,
rejection, or antibody unexpected reactions
after the transplant in a period of 30 years.
Similar findings were observed by Aho et al.
(1998) after histological analysis and immune
responses, by Spin-Neto et al. (2011a) in quan-
titative hemogram analysis, and by Reikerås
et al. (2010) in a serum antibody analysis. The
authors did not find signs of antigenic
reactions.
Studies on incorporation and repair of ALg
are scarce, especially when it comes to basic
research. The aims of this investigation are:
to provide a better understanding on onlay
ALg incorporation, remodeling, revasculariza-
tion process, volume maintenance, and com-
pare with AUg using histology, bone proteins
immunohistochemistry, and Computerized
tomography (CT) scans.
Material and methods
Fifty-six male adult New Zealand White rab-
bits, weighing between 3.5 and 4.0 kg were
used for this study. Twenty animals were
used as bone tissue donors and 36 as recep-
tors. The experimental protocol was submit-
ted and approved by the Ethics Committee in
the use of Animals (CEUA), of the University
of São Paulo, under number 07.1.1488.53.6.
© 2012 John Wiley & Sons A/S
All animals were kept under appropriate vet-
erinary care and nutritional support in indi-
vidual cages throughout the experimental
period.
Bone block harvesting from donor animals
In aseptic condition and performed by the
same surgeon, 20 animals were subjected to
sedation with Acepromazine® (2.0 mg/ml;
Univet S/A, Sao Paulo, Brazil) at a dose of
1.0 mg/kg administered subcutaneously
15 min before surgery. Then, by deep intra-
muscular route, general anesthesia was per-
formed using Xylazine® (20 mg/ml; Calier
Laboratories, Barcelona, Spain) at a dose of
3.0 mg/kg mixed with 10% ketamine®
(50.0 mg/ml; SA National Union Pharmaceu-
tical Chemistry, Embuguaçú, Sao Paulo,
Brazil) at a dose of 60 mg/kg.
Animals were submitted to local anes-
thetic infiltration with mepivacaine 2% with
epinephrine 1 : 200,000 (DFL; Trade & Indus-
try SA, Rio de Janeiro, Brazil) to make a lin-
ear incision directly on the skin, measuring
3.0 cm long, and placed on the sagittal suture
on the skull. The galea aponeurotica and fas-
cia were incised followed by periosteal inci-
sion to expose skull cortex. Two bicortical
bone blocks were obtained from the animal’s
calvaria, using a customized surgical guide
and electric handpiece, so that to provide a
standardized size of bone blocks. The osteot-
omies were performed under copious irriga-
tion with sterile saline. After bone blocks
harvesting, donor animals were sacrificed
with overdose of Thiopental (Crystal
Pharma® Laboratories, Belo Horizonte, Brazil)
administered intravenously.
The bone blocks were washed with copious
sterile saline irrigation followed by storage in
sterile Petri plaques and sent immediately to
laboratory for microbiological examinations.
Each block was submitted to microbiological
analysis for fungi, aerobic, and anaerobic bac-
teria. After microbiologic cultures, the bone
blocks were packed in three sterile sealed
plastic bags and stored in 80°C freezer.
Specimens tested negative for microorgan-
isms were sent to the University of Marilia
BTME – UNIOSSTM and subjected to sterili-
zation processing according to the institu-
tion’s own protocol, which follows National
Legislation (ANVISA, RDC # 220, of Decem-
ber 27, 2006, Brazil) for bone banks.
Bone grafting procedure
The AUgs were harvested from the parietal
region of 36 animals, following the same sur-
gical steps mentioned above for ALg attain-
ment. At this time, only one block was
1165 |
Hawthorne et al
Allogenous bone grafts remodeling
removed from the skull. Animals were kept
anesthetized for ALg and AUg blocks graft-
ing, respectively, bilaterally in the mandible.
The AUg specimen was kept in gauze
soaked in saline until the recipient bed was
prepared. The same procedure was performed
for the ALg block. On both sides, monocorti-
cal perforations were performed in the recipi-
ent beds using a customized stainless steel
surgical guide with nine holes of 1.0 mm in
diameter, equidistantly distributed. The sur-
gical approach was through the submandibu-
lar region to place an incision of
approximately 2.0 cm at the mandible base.
Each of the mandible sides received either
AUg (Group I – control) or ALg (Group II –
test) blocks in a random fashion. Both blocks
were fixed with a titanium screw (Synthes®,
Solothurn, Switzerland) measuring 1.5 mm in
diameter and 6.0 mm in length, placed in the
center of the graft (Figs 1 and 2). Surgical
wounds were closed with Vicryl 4.0 (Ethicon,
Johnson & Johnson, São José dos Campos,
São Paulo, Brazil) for muscle layers and
Nylon 4.0 (Ethicon; Johnson & Johnson, São
José dos Campos, Sao Paulo, Brazil) for skin.
In the postoperative period, animals
received prophylactic antibiotic therapy with
oxytetracycline (1.0 g/10 ml) at a dose of
0.2 ml/kg and for 3 subsequent days by intra-
peritoneal route. Animals also received
analgesic Tramadol (Biolab Searle, São Paulo,
Brazil) at a dose of 0.02 mg/kg and
anti-inflammatory (Profenid®; Pharmacy
Fig. 1. AUg fixed in the mandible.
Fig. 2. ALg fixed in the mandible.
Clin. Oral Impl. Res. 24, 2013 / 1164–1172
Hawthorne et al
Allogenous bone grafts remodeling
Distribuidora LTDA Brazil, Jandira SP) at a
dose of 3.0 mg/kg, both intra-muscularly.
Six animals from each group were sacri-
ficed at 03, 05, 07, 10, 20, and 60 days after
surgery by overdose of thiopental 1.0 g
(2.0 ml), intravenously.
CT
Immediately after grafting surgery and sacri-
fice, animals were subjected to CT scans of
the mandible with the objective of analyzing
bone density and volume. The scanning was
performed under intravenous sedation, using
a helicoidal tomography device; model Emo-
tionTM (Siemens, Elrlangen, Germany).
Images were acquired in the coronal plan,
with 2.0 mm increments in continuous bed
motion and 1 mm slice thickness. Recon-
struction was 0.5 mm thick, using a high
spatial resolution filter, resulting in a voxel
size of 0.5 9 0.3 9 0.3 mm.
For density evaluation, the software eFilm
Workstation 2.01 (Merge Healthcare Com-
pany, Milwaukee, WI, USA) was used. A cir-
cular region of interest (ROI) was used to
measure bone density on the CT sections.
The location and dimension of ROI were stan-
dardized for every measurement performed,
regardless of the occurrence or not of graft
resorption. The values measured inside the
ROI were expressed in Hounsfield units. For
this analysis, the five most central tomogra-
phy slices were considered, which enabled the
evaluation of the upper and lower portions of
the graft fixation screw. The density measure-
ment procedure was repeated five times on
each of these sites to minimize errors. Similar
to volume, the bone densities were calculated
at the baseline and sacrifice times in a dou-
ble-blind mode by the same examiner.
For volume evaluation, images were gener-
ated in a Dicom pattern and transferred onto
a PC Workstation equipped with Dicom
Works 1.3.5.b software intended to perform
graft volume measurements. In a standard-
ized fashion, the coronal plan slices were
obtained for each graft, making it possible to
measure an area in millimeter square for
each slice. When added and multiplied by the
number of slices, the total volume of the
graft was obtained (mm3). The gauging proce-
dure was used in a double-blind manner by
the same examiner.
Histology and immunohistochemistry
After sacrifice, mandible bone blocks com-
prising the grafted area without surrounding
soft tissue were reduced in a standardized
manner, maintaining 2 mm beyond anterior
and posterior graft limits.
1166 | Clin. Oral Impl. Res. 24, 2013 / 1164–1172
Specimens were fixed in 10% buffered for-
malin at pH = 7.4 (for at least 24 h). After
fixation, they were decalcified with 4%
EDTA changed twice a week. Next decalcifi-
cation, the bone blocks were washed in water
around 8 h, followed by dehydration through
a series of alcohols with increasing concen-
trations (50–100% every 2 h). After dehydra-
tion, diaphanization was performed by
placing the pieces in xylene (three solutions
of two in 2 h) until they are transparent. Fol-
lowing that impregnation was carried out in
paraffin in an oven at 60°C in three baths of
3 h. The next step consisted in the inclusion
of parts ways with paraffin, which plays with
hardened completely soaked. Paraffin blocks
containing the samples were cut in micro-
tome in a standardized way (5 mm thick),
which were divided interchangeably for histo-
logical and immunohistochemical analysis.
The slides for histological analysis were
stained with Mallory’s Trichrome. The Leika
DMLB-2TM microscope (Leica Microsystems
WetzlarGmbH, Bensheim, Germany) was
used for all measurements.
The immunohistochemical labeling was
carried out using the immunoperoxidase
detection method with the following primary
antibodies (Santa Cruz Biotechnology, Santa
Cruz, CA, USA): anti-OC, anti-osteopontin,
anti-collagen Type I, anti-VEGF, anti-ALP,
anti-receptor activator of nuclear-kb ligand,
anti-TRAP, and anti-osteoprotegerin. The
anti-IgG biotinylated antibody (Jackson
Immunoresearch Laboratories, West Balti-
more Pike, PA, USA) at a 1 : 200 concentra-
tion was used as secondary antibody. Avidin
and biotin (Vector Laboratories, Burlingame,
CA, USA) was used to amplify the signal of
the reaction that was revealed using
diaminobenzidine (Sigma-Aldrich, St Louis,
MO, USA) as a chromogene. The intensity of
labeling in each glass slide was assessed
using a semi-quantitative ranking that ranged
from 0 for no labeling to 4 for intense
labeling. This subjective quantification was
carried out at the interface between resident
bone and the graft, using 9 100 magnifica-
tion on a light microscope (Leica DMLB;
Leica; Bensheim, Germany).
Statistical analysis
To compare the measurements of density
and volume of the grafts between the initial
and sacrifice time points, as well as the grafts
incorporation assessment, a paired t-Student
analysis was applied. To compare the density
and volume differences between allogeneic
and autologous groups and between the six
evaluation time points was used the analysis
of variance (two-way ANOVA – mixed
model). For immunohistochemical analysis,
the non-parametric Kruskal–Wallis and
Dunn’s post hoc were used to compare the
6 days evaluation. The Wilcoxon test was
used for comparison between ALg and AUg
groups. All statistical tests adopted signifi-
cance level of P  0.05.
Results
All animals tolerated the surgical procedures
and the postoperative period was uneventful.
No signs of infection were observed.
CT scan
Bone graft density
No differences were identified between ALg
and AUg groups in terms of bone density.
The AUg group showed relatively constant
bone density until the day 10, when a
decrease and a slight increase were observed.
Contrarily, the ALg group showed an unsta-
ble density variation (Figs 3 and 4).
Bone graft volume
There were no volume differences when
groups’ figures were compared. However,
when intra-group data were analyzed for both
ALg and AUg groups, the volume decreased
significantly from day 7 to day 60 in the ALg
group (P = 0.001), whereas the volume dimin-
ished from day 20 to day 60 in the AUg group
(P = 0.015).
The ALg group showed a continuous reduc-
tion in volume from day 3 to day 60, except
at day 5, in which an increase in volume was
observed. In contrast, the AUg group showed
a volume increase until day 20, when started
a process of volume reduction that lasted
until the end of the experimental time points
(Figs 5 and 6).
Histology
ALg
In the initial time point (day 3), the bone
grafts showed slight differences between
them. The ALg showed empty lacuna and no
viable osteocytes cells (Fig. 7). The cell infil-
trate, especially erythrocytes, was reduced at
the day 5, whereas collagen fibers network
could be detected in the vicinity of the graft
and near the periosteum. The bone graft
appeared preserved, with no suggestive signs
of resorption, remodeling, or new bone depo-
sition (Fig. 8). In the day 7, the collagen
fibers appeared organized together with plu-
ripotent cells, located between the graft and
© 2012 John Wiley & Sons A/S

Fig. 3. Graphic showing the density variation in ALg and AUg groups over time (3, 5, 7, 10, 20, and 60 days).
Fig. 4. CT Scan in coronal plane with density measures
in Dicom Works software.
recipient bed. At this stage it was also possi-
ble to find cells of osteoblastic leanage just
below the periosteum. The graft continued
well preserved, with few lacunae and discreet
presence of osteoclastic-like cells (Fig. 9).
After 10 days, the graft still maintained its
original shape. It was possible to capture
Fig. 5. Graphic showing the volume variation in ALg and AUg groups over time (3, 5, 7, 10, 20, and 60 days).
© 2012 John Wiley & Sons A/S
Hawthorne et al
Allogenous bone grafts remodeling
Fig. 7. Histologic photomicrograph showing cellular
infiltrate (CI) in the interface (IT) between bone graft
(BG) and recipient bed (RB) at 3 postoperative days in
the ALg group. Note the presence of empty osteocyte
lacunae (arrow). Staining: Mallory’s trichrome.
Fig. 8. Histologic photomicrograph showing loosely
organized collagen fibers formation (CF) between the
interface (IT), bone graft (BG), and recipient bed (RB)
with scarce cellular infiltrate (CI) at 5 postoperative
Fig. 6. CT Scan in coronal plane with volume measure-
days in the ALg group. Note: P = perforation, Staining:
ments in Dicom Works software.
Mallory’s trichrome.
incipient new bone formation close to perios-
teum. Resorption areas were minimal, but
there was a greater presence of osteoclasts
(Fig. 10). At the day 20, there was scarce
immature and disorganized new bone forma-
tion inside the graft. There was still a clear
delimitation between graft and recipient bone
Fig. 9. Histologic photomicrograph showing cellular
infiltration (CI) significant reduction with the organiza-
tion of collagen fibers (CF) in the interface at 7 postop-
erative days in the ALg group. Note: BG = bone graft,
RB = recipient bed, P = perforation, and Staining: Mal-
lory’s trichrome.
(Fig. 11). After 60 days, the interface between
recipient bone and ALg was bridged by new
bone originated from the former. In the graft
edges, beneath the periosteum, newly formed
bone presented organized (Fig. 12).
1167 | Clin. Oral Impl. Res. 24, 2013 / 1164–1172
Hawthorne et al
Allogenous bone grafts remodeling
Fig. 10. Histologic photomicrograph showing a disorga-
nized immature new bone (NB) sharing space with
loosely organized collagen fibers network (CF) at the
interface (IT) and perforation (P) areas at 10 postopera-
tive days in the ALg group. Note: BG = bone graft,
RB = recipient bed, and Staining: Mallory’s trichrome.
Fig. 11. Histologic photomicrograph showing new bone
(NB) formation attached to bone graft (BG) to the recipi-
ent bed (RB) at 20 postoperative days in the ALg group.
Note the formation of concentric lamellae. Note:
IT = Interface, P = Perforation, and Staining: Mallory’s
trichrome.
Fig. 12. Histologic photomicrograph showing incorpora-
tion of the bone graft (BG) to the recipient bed (RB),
characterized by mature bone (MB) bridges at 60 postop-
erative days in the ALg group. Note intense mature
bone formation due to periosteum reaction (PR) in the
graft inferior lateral margin. Staining: Mallory’s
trichrome.
AUg
The 3 and 5 days were characterized by large
amount of cellular infiltrate with presence of
erythrocytes, low differentiation of osteo-
blasts (day 3), and evidence of increased pro-
1168 | Clin. Oral Impl. Res. 24, 2013 / 1164–1172
liferation at day 5 (Figs. 13 and 14). The reaction. At the day 60 a discrete resorption
periosteum remained intact, with early orga- was observed in the graft edges, characterized
nization of the collagen fibers. The graft pre- by increased number of Howship’s lacunae.
sented remnants of blood vessels and
osteocytes lacunae with viable cells. A major
cell proliferation in the interface was seen in
day 7 (Fig. 15) with an increased number of
osteoblasts, bone marrow, and new bone for-
mation. The graft/recipient bed interface and
graft edges exhibited deposition of woven
bone, uniting the graft to the recipient bed.
The periosteum presented complete regenera-
tion. In the day 10, osteoblasts were lining
the recipient bed from the periosteum. The
graft morphology remained intact, with few
areas of resorption and bone bridging con-
necting the graft to the recipient bed
(Fig. 16). At the day 20, the interface graft/ Fig. 15. Histologic photomicrograph showing new bone
recipient bed was noted with the presence of formation (arrow) in a loosely organized collagen fibers
fat cells, with intense woven bone formation (CF) extracellular matrix and presence of fat tissue (FT)
inside the bone graft (BG) at 7 postoperative days in the
arising from the perforations, which
AUg group. Note: * = artifact separation, Staining:
increased the graft union (Fig. 17). At this Mallory’s trichrome.
period the periosteum presented remarkable
Fig. 16. Histologic photomicrograph showing new bone
(NB) formation for all over the interface (IT) between
the bone graft (BG) and recipient bed (RB) at 10 postop-
Fig. 13. Histologic photomicrograph showing cellular
erative days in the AUg group. Note new bone deposi-
infiltrate (CI) in the interface (IT) between bone graft
tion (arrow) in a collagen fibers extracellular matrix
(BG), recipient bed (RB), and perforation (P) at 3 postop-
near a perforation (P) area. Staining: Mallory’s
erative days in the AUg group. Note: Harver’s canal in
trichrome.
bone graft (arrow), * = artifact separation, and Staining:
Mallory’s trichrome.
Fig. 17. Histologic photomicrograph showing bone graft
Fig. 14. Histologic photomicrograph showing cellular (BG) incorporation to recipient bed (RB), organized
infiltrate reduction with formation of collagen fibers mature bone (MB) bridges with osteonic lamelae attach-
network (CF) and initial cellular proliferation (arrow) at ing both structures (arrow) at 20 postoperative days in
5 postoperative days in the AUg group. Note: BG = bone the AUg group. Presence of collagen fibers (CF) extracel-
graft, RB = recipient bed, IT = interface, * = artifact sep- lular matrix in perforation (P). Staining: Mallory’s
aration, and Staining: Mallory’s trichrome. trichrome.
© 2012 John Wiley & Sons A/S

The union graft-recipient bone became
mature at this point (Fig. 18).
Grafts incorporation
The bone healing at interface at day 20 and
day 60 are shown in the Fig. 19. The bone
formation seemed to fill the gap from the
recipient bone and/or periosteum covering
the grafts. The AUg underwent remodeling,
whereas the ALg appeared intact at 20 and
60 days. On the day 60 the AUg was com-
pletely incorporated onto the recipient bone,
whereas in the group ALg some bone marrow
yet remained between the graft and recipient
bone.
Immunohistochemistry
All samples of immunohistochemical label-
ing for each protein used in this study are
shown in Figs 20 and 21.
Fig. 18. Histologic photomicrograph showing bone graft
(BG) intense incorporation to the recipient bed (RB) at
60 postoperative days in the AUg group. Note the limit
line between them (arrows). Note: CT = Conjunctive
tissue, Staining: Mallory’s trichrome.
(a)
(c)
Fig. 19. Interface assessment showing A: ALg 20 days, B: AUg 20 days, C: ALg 60 days, D: AUg 60 days.
© 2012 John Wiley & Sons A/S
From the proteins OP, OC, Col I, and
ALP involved in osteogenesis, none pre-
sented statistically significant results. The
ALP, in the ALg group, underwent an
important reduction from 5 to 20 days. In
both groups it was not found differences
between the time points. The OP and COL
I showed no differences when comparing
groups and within groups, in the six experi-
mental time points.
In the OPG and RANKL proteins, involved
in regulation of osteoclastogenesis induction,
OPG showed significant increase in the AUg
group when compared with ALg in the days
10 and 20 (P = 0.034 and P = 0.001, respec-
tively). As to the ALg group a significant
increase was detected at days 5–60. However,
in the AUg group we observe the opposite: a
statistically significant reduction from 20 to
60 days (P = 0.021). No difference was found
in RANKL in the comparison between groups
and between time points.
The TRAP, which is a specific enzyme
present in large quantities at the corrugated
osteoclasts edge that expresses bone
resorption, showed a statistically significant
difference in day 7 between both groups
(P = 0.002), more intensely for the ALg
group. For this group, a significant increase
was found from 3 to 7 days, but in 7–
60 days, there was no significant reduction.
The within AUg group time points compari-
sons showed significant increases in the
respective time points: 3–7, 3–20, 7–10, and
7–20 days.
The labeling for VEGF protein expressed
by osteoblasts and closely linked to the pro-
cess of angiogenesis showed no statistical
(b)
(d)
1169 |
Hawthorne et al
Allogenous bone grafts remodeling
difference between all experimental time
points.
Discussion
This study represents a continuation of two
previous studies by the same researchers’
group that used similar methodology to
assess onlay iliac crest grafts – PART I (Faria
et al. 2008), and biological behavior of calvar-
ial autogenous grafts in the rabbit’s mandible
– PART II (Pedrosa et al. 2009). By adding the
Part III (this study), the authors provide the
readers the comparison of onlay grafts in dif-
ferent aspects, such as variation in grafting
technique and the use of alternative bone
structures.
This investigation aimed at understanding
the incorporation process of calvarial AUg
and ALg using histology, immunohistochem-
istry, and CT scan, the latter intended to
assess density and volume in six different
time points.
The tomographic outcomes revealed no
differences between AUg and ALg in terms
of bone density and volume. However,
when intra-group data on volume parameter
were compared along the experiments, a
slight expansion of the graft from fixation
until day 20 (P = 0.085) was observed in the
AUg group and then resorbed significantly
at the day 60 (P = 0.001). This can be
explained by immunohistochemistry test,
which indicated higher concentration of
OPG at the days 10 and 20 as compared
with AUg group (P = 0.034, P = 0.001,
respectively). The OPG acts as inhibitory
receptors for RANKL, blocks the interaction
between RANK/RANKL, inhibits the ending
stage of osteoclast differentiation, and
thereby results in decreased bone resorption
(Takahashi et al. 2002; Hofbauer 2006;
Baud’huin et al. 2007). Hence, a high con-
centration of osteoclasts at days 10 and 20
and beyond triggered an increment of OPG
in AUg sites through a regulation mecha-
nism. On the other hand, due to its biologi-
cal characteristics, the ALg is somewhat
osteoclast resistant which leads to remodel-
ing as suggested by the histological out-
comes. In the ALg group, the grafts drew a
curve of continuous volume loss from the
time of installation until 60 days, with
marked graft contraction between 7 and
10 days. This effect could be explained by
immunohistochemistry labeling, which
showed a higher TRAP labeling at day 7
(P = 0.002), which may explain the marked
reduction in volume that occurred in this
Clin. Oral Impl. Res. 24, 2013 / 1164–1172
Hawthorne et al
Allogenous bone grafts remodeling
Fig. 20. Immunolabeled proteins (arrows) used in the study. OPG, osteoprotegerin; RANKL, receptor activator of
nuclear factor jB; ALP, alkaline phosphatase; OP, osteopontin; VEGF, vascular endothelial growth factor; TRAP,
tartrate-resistant acid phosphatase; COL I, type I collagen; and OC, osteocalcin.
time point in ALg group. Our data corrobo-
rate with those of Winslow et al. (2006),
who stated that TRAP positive cells appear
from the day 5 after grafting.
The density analysis showed no statistical
differences between the ALg and AUg groups.
The AUg group exhibited increased density
until the day 10 and marked reduction until
the latest time point. Apparently, the ALg
group tended to maintain Hounsfield values
along the entire experiments, whereas the
AUg group, in our study, was more prone to
remodeling. Indeed, in a recent clinical study
using tomography the authors concluded that
ALg was associated with slow remodeling
process (Spin-Neto et al. 2011b).
The VEGF, ALP, OC, COL-1, RANKL, and
OP bone proteins did not vary, suggesting
1170 | Clin. Oral Impl. Res. 24, 2013 / 1164–1172
that they did not interfere in the bone remod-
eling process evaluated in this study. With
regard to VEGF, the immunohistochemistry
tests were equal for both AUg and ALg
groups along with the experiments. Interest-
ingly, Faria et al. (2008) and Pedrosa et al.
2009 described the influence of VEGF when
these authors compared autogenous onlay
grafting (iliac crest and calvarial bone, respec-
tively) in perforate and non-perforated bone
beds. In both situations, the perforation of
bone bed elicited increased VEGF labeling in
both studies. In the present experiment, the
recipient bed was always perforated irrespec-
tively the graft type. This may signify that
VEGF did not play a role in angiogenesis, in
this experimental model. The ALP was not
altered in ALg and AUg groups during the
experiments. Although ALP is considered as
an early osteoblast differentiation label
(Bellows et al. 1991; Liu et al. 1996) – it may
have had no influence in the grafts volume at
early time points. In parallel, the OC – bone
matrix protein that is predominantly
synthesized by osteoblasts (Bronckers et al.
1985) – plays an important role in bone
mineralization and new bone formation
(Garnero and Delmas 1999). The OC is
associated with the turnover phenomenon
when bone resorption and formation are
occurring simultaneously (Ducy et al. 1996).
In our study, OC exhibited a peak concentra-
tion at 3 days in the ALg group compared
with AUg group (P = 0.098) and maintained
higher levels until the day 20 (P = 0.598).
In this study, the AUg was well incorpo-
rated onto the recipient bone and appeared
vital in later periods (20 and 60 days), which
could not be confirmed in the ALg group. At
day 20, the AUg group presented vascular
penetration in the entire length of the graft
and with intense woven bone formation aris-
ing from the perforations, which increased
the graft union. This data corroborates with
Pedrosa et al. 2009; who carried out an
experimental study in rabbits using autoge-
nous calvarial bone grafts in perforated bed.
They observed at day 20 a noticeable incor-
poration process of the graft to the recipient
bed. At day 60, well-established incorpora-
tion was observed, with organized bone
bridges rich in concentric lamellae and ost-
eons. Bone formation is conducted by surviv-
ing cells and bone minerals of the graft and
the ability of bone matrix to induce osteopro-
genitor cells differentiation at the recipient
area. Although the graft and recipient bed
have individual contributions to this process,
the sum of these interactions determines the
graft success or failure (Keith et al. 2006).
Unlike the AUg group, the histology related
to ALg group showed predominance of empty
osteocytes lacunae and poor vascularization.
These histological features are defined as
non-vital bone, in spite of mature bone for-
mation surrounding the graft (Sohn et al.
2009). Indeed, in our study the groups AUg
and ALg were similar histologically in all
parameters assessed, but at 60 days a greater
presence of new bone was observed in the
interface bone graft recipient in the AUg
group but not in ALg group. Similar data
were shown in a clinical study conducted by
Spin-Neto et al. (2011b) comparing AUg and
ALg onlay blocks for maxillary reconstruc-
tion. The grafts were biopsied for histology.
Although both graft types showed clinical
compatibility, the histological analysis
© 2012 John Wiley & Sons A/S
 Hawthorne et al
Allogenous bone grafts remodeling

AUg
1.8
1.0

0.8
1.9
1.5
1
VEGF

ALg
1.3
0.8
1.7
1.2
1.6
1.5
AUg
1.5
1.5
1.1
1.8
1.7
2
COL I

ALg

1.3
2.3
1.1
1.9
1.8
2
AUg

1.6

1.1
1.9
2.6
1

2
Table 1. Medians of scores attributed to each protein for ALg and AUg groups in days 3, 5, 7, 10, 20, and 60

ALg
OC

2.3
2.1
2.3
2.3
2.1
2
AUg
0.9

0.8
1.3
2.4
1

1
OPG

ALg
1.3

1.5

1.3

No statistical difference between groups and time points. Kruskal–Wallis and Dunn tests.
1

1
1
AUg

1.8
2.8
1
2
2

2
ALg

1.8

1.5
OP

2
1
2
AUg

0.8
1.3
1.4
1.3
1.1
1
RANKL

ALg

1.5
1.8
1.4
1.8
1
1
AUg

0.5
0.0
1.0
1.1
0.4
0
TRAP

Fig. 21. Mean scores assigned for each protein (ALP, alkaline phosphatase; TRAP, tartrate-resistant acid phospha- ALg

0.2
1.3
0.7
0.8
tase; RANKL, receptor activator of nuclear factor jB; OP, osteopontin; OPG, osteoprotegerin; OC, osteocalcin;
0

0
COL, collagen; VEGF, vascular endothelial growth factor) according to the assessment time points (3, 5, 7, 10, 20,
Immunolabeling of proteins

and 60 days) in ALg and AUg groups. *Statistical difference between groups.
AUg

1.3
1.7
1
1
1

2
ALg
ALP

showed a large amount of necrotic bone sur-

1.5
1.5

1.3
AUg and ALg groups, except for an increasing
2
2

rounded by few spots of new formed bone in resorption activity in the ALg group medi-
ALg group. ated by TRAP and in the AUg group by
(days)
point
Time

It can be concluded that AUg and ALg did higher OPG labeling. However, this latter
10
20
60
3
5
7

not differ in bone density and volume during
entire experiment. The histological data
exhibited more efficient incorporation of
AUg group into recipient bed as compared
with ALg group. Immunohistochemistry out-
comes demonstrated similar pattern for both
observation does not seem to influence clini-
cal outcomes.
Acknowledgements: The authors
thank Mr Sebastiao Bianco and Ms Adriana
Almeida for the competent technical support.
This study was supported by FAPESP (Sao
Paulo Research Foundation) granted (No.
2008/01382-6) to Professor Luiz A. Salata.

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