M.

Araújo Effect of a xenograft on early bone

E. Linder
J. Lindhe
formation in extraction sockets:
an experimental study in dog

Authors’ affiliations:
Mauricio Araújo, Department of Dentistry,
State University of Maringá, Paraná, Brazil
Elena Linder, Jan Lindhe, Institute of Odontology,
The Sahlgrenska Academy at Göteborg University,
Gothenburg, Sweden
Correspondence to:
M. Araújo
Key words: biomaterial, bone formation, extraction socket, grafting
Abstract
Aim: The aim of this study was to study the effect on early bone formation resulting from
the placement of a xenograft in the fresh extraction socket in dogs.
Material and methods: Five beagle dogs were used. The distal roots of the third and fourth
mandibular premolars were removed. In one quadrant, a graft consisting of Bio-Oss
s

Rua Silva Jardim

15/sala 03 Collagen was placed in the fresh extraction wound, while the corresponding premolar sites
87013-010 Maringá in the contra-lateral jaw quadrant were left non-grafted. After 2 weeks of healing, the dogs
Paraná
Brazil were perfused with a fixative, the mandibles removed, the experimental sites dissected,
Tel.: þ 55 44 3224 6444 demineralized, sectioned in the mesio-distal plane and stained in hematoxyline–eosine.
Fax: þ 55 44 3224 6444
Results: The central portion of the non-grafted sockets was occupied by a provisional
e-mail: odomar@hotmail.com
matrix comprised of densely packed connective tissue fibers and mesenchymal cells. Apical
and lateral to the provisional matrix, newly formed woven bone was found to occupy most
of the sockets. In the apical part of the grafted sockets, no particles of the xenograft could
be observed but newly formed bone was present in this portion of the experimental site.
In addition, limited numbers of woven bone trabeculae occurred along the lateral socket
walls. The central and marginal segments of the grafted sockets, however, were occupied
s
by a non-mineralized connective tissue that enclosed Bio-Oss particles that frequently
were coated by multinucleated cells.
s
Conclusions: The placement of Bio-Oss Collagen in the fresh extraction wound obviously
delayed socket healing. Thus, after 2 weeks of tissue repair, only minute amounts of newly
formed bone occurred in the apical and lateral borders of the grafted sockets, while large
amounts of woven bone had formed in most parts of the non-grafted sites.

Date:
Accepted 5 April 2008
To cite this article:
Araújo M, Linder E, Lindhe J. Effect of a xenograft
on early bone formation in extraction sockets:
an experimental study in dog.
Clin. Oral Impl. Res. 20, 2009; 1–6.
doi: 10.1111/j.1600-0501.2008.01606.x
In previous experiments in dogs (Cardaro-
poli et al. 2003; Araújo & Lindhe 2005), it
was observed that healing of an extraction
socket included (i) early formation of wo-
ven bone (modeling) and (ii) the subse-
quent replacement of this immature bone
with lamellar bone and marrow (remodel-
ing). Healing of the edentulous site was
further characterized by (iii) loss of bundle
bone in the walls of the socket and (iv) a
marked reduction in the height of the
buccal bone crest. Similar observations
were reported from retrospective and pro-
spective studies in man (Pietrokovski &
Massler 1967; Schropp et al. 2003; Pietro-
kovski et al. 2007; for review see Chen
et al. 2004).
Different approaches were used to pre-
vent the post-extraction ridge reductions.
Such methods included the utilization of
various graft materials and application
techniques (e.g. Becker et al. 1998; Artzi
et al. 2000; Froum et al. 2002; Carmagnola
et al. 2003; Nevins et al. 2006).

c 2009 The Authors. Journal compilation 

 c 2009 Blackwell Munksgaard 1
Araújo et al . Early bone formation in extraction sockets
In a recent study by Araújo et al. (2008),
the dog model was used to study healing of
an extraction socket that had been grafted
s tlich , Wolhusen, Switzerland) was placed
with Bio-Oss Collagen. It was observed
that the placement of the xenograft in the
fresh extraction socket after a 3-month
period of healing appeared to have inter-
fered with the process of bone modeling
and remodeling. Thus, while the non-
grafted sites underwent marked resorption
during the 3-month interval, the dimen-
sion of the walls of the grafted sockets as
well as the profile of the edentulous ridge
remained unchanged. The newly formed
hard tissue in the marginal portion of the
grafted sites, however, contained a large
s
number of Bio-Oss particles that were
surrounded by immature woven bone. A
more detailed analysis of the newly formed
tissues in the two differently treated sock-
ets further indicated that the process of
tissue remodeling had progressed further
in the non-grafted than in the grafted sites.
Thus, while about 50% of the tissue vo-
lume in the non-grafted sites was occupied
by bone marrow, in the grafted site bone
marrow represented only about 25% of the
tissue volume. This indicates that the graft
may in fact have delayed healing.
The objective of the present experiment
was to evaluate the effect on early wound
healing and bone modeling of the place-
ment of a xenograft in the fresh extraction
socket in dogs.
Material and methods
The ethical committee of the State Uni-
versity of Maringá approved the research
protocol. Five beagle dogs about 1 year old
and weighing between 10 and 12 kg each
were used. During surgical procedures, the
animals were anesthetized with intrave-
nously administered Ketamin (10%,
8 mg/kg; Agener União, São Paulo, Brazil).
Pocket and crestal incisions were made
in the posterior premolar region in both
quadrants of the mandible. Buccal and
lingual full thickness flaps were elevated
to disclose the alveolar crest. The canal of
the mesial roots of the third and fourth
mandibular premolars was reamed and
filled with gutta-percha. The premolars
were subsequently hemi-sected with the
use of fissure burs. The distal roots were
carefully removed with the use of elevators.
2 | Clin. Oral Impl. Res. 20, 2009 / 1–6
Grafted sites
In one quadrant of the mandible, a graft
s
consisting of Bio-Oss Collagen (Geis-
s
in the fresh extraction wounds (Fig. 1) in a
manner previously described (Araújo et al.
2008). The marginal portion (about 2 mm)
of the buccal bone wall of the distal sockets
was also covered with the biomaterial.
Non-grafted sites
The corresponding premolar sites in the
contra-lateral jaw quadrant were exposed
and managed in the manner described
above. No biomaterial was, however,
placed in the fresh extraction sockets.
The flaps were replaced in the grafted
and non-grafted sites and were retained
with interrupted sutures. The sutures
were removed after 1 week. Mechanical
tooth cleaning was performed on days 3, 7
and 10 after surgery.
After 2 weeks of healing, the dogs were
euthanized with an overdose of Ketamin
(Agener União) and perfused, through the
carotid arteries, with a fixative containing
a mixture of 5% glutaraldehyde and 4%
formaldehyde (Karnovsky 1965). The
mandibles were removed and placed in
the fixative. Each experimental site, in-
cluding the distal socket area, was dis-
sected using a diamond saw (Exact
s
Apparatebeau, Norderstedt, Hamburg,
Germany), and subsequently deminera-
lized in a 10% solution of EDTA. The
Fig. 1. Clinical photographs illustrating the graft of
s
Bio-Oss Collagen placed in the fresh extraction
socket. Note that the graft material also covers the
marginal portion of the buccal wall of the socket.
c 2009 The Authors. Journal compilation 
 c 2009 Blackwell Munksgaard
blocks were dehydrated in ethanol and
isopropanol and embedded in paraffin. Se-
rial sections representing the experimental
sites were cut in a mesio-distal plane and
parallel to its long axis. The microtome
was set at 6 mm. From each site, three
sections, representing the central part of
the socket and about 20 mm apart, were
selected for the histological examination.
The sections were stained in hematoxy-
line–eosine
Histological examination
The histological examinations were per-
s
formed in a Leitz DM-RBE microscope
(Leica, Wetzlar, Germany) equipped with
s
an image system (Q-500 MC ; Leica).
The composition of the newly formed
tissue in the extraction socket was deter-
mined using a point counting procedure.
Thus, a lattice comprising 100 light points
(modified from Schroeder & Münzel-
Pedrazzoli 1973) was superimposed over
the tissue in the healing socket, and the
percentage area occupied by (i) mineralized
bone, (ii) bone marrow, (iii) provisional
matrix, (iv) granulation tissue and (v) Bio-
s
Oss particles was determined (magnifica-
tion  200). In addition, the number of
s
Bio-Oss particles in the socket that were
in direct contact with multinucleated cells
was determined and expressed as percen-
s
tage of the total number of Bio-Oss parti-
cles identified.
Mean values and standard deviations of
the different variables were calculated
using the dog as the statistical unit.
Results
Healing was uneventful at all sites. The
clinical examination of the experimental
sites that was performed after 2 weeks (Fig.
2) disclosed that a slightly invaginated and
modestly inflamed soft tissue covered the
socket entrance.
Gross histological observations
The connective tissue of the mucosa cover-
ing the grafted as well as the non-grafted
sockets was rich in mesenchymal cells,
vascular structures and clusters of inflam-
matory cells.
The central portion of the non-grafted
sockets (control site; Fig. 3) was occupied
by a provisional matrix comprised of

Fig. 2. Clinical photographs illustrating a site
s
grafted with Bio-Oss Collagen after 2 weeks of
healing. Note the slightly invaginated crestal surface
and the slightly inflamed soft tissue.
Fig. 3. Microphotograph of a mesio-distal section
representing a non-grafted site. Note that most of
the alveolar socket is occupied by woven bone and
provisional matrix. PM, provisional matrix; GT,
granulation tissue; WB, woven bone. Hematoxyline–
eosine stain; original magnification: 16.
densely packed connective tissue fibers and
mesenchymal cells (Fig. 4). Apical and
lateral to the provisional matrix, newly
formed woven bone was found to occupy
most of the socket (Fig. 3). Several of the
bone projections formed primary osteons
c 2009 The Authors. Journal compilation 
 c 2009 Blackwell Munksgaard
Araújo et al . Early bone formation in extraction sockets
Fig. 4. Microphotograph representing the provi-
sional matrix in the non-grafted site. Note the
densely packed connective tissue fibers and cells;
V, vessel. Hematoxyline–eosine stain; original mag-
nification: 200.
Fig. 5. Microphotograph representing the minerali-
zation front of the woven bone in the non-grafted
site. Note that the woven projections had initiated
the formation of a primary spongiosa (primary
osteons and enclosed vascular structures). PM, pro-
visional matrix; V, vessel; WB, woven bone. Hema-
toxyline–eosine stain; original magnification:
200.
and enclosed vascular structures (Fig. 5).
The trabeculae of immature bone were
lined with osteoblasts and occasionally
isolated osteoclasts, residing in resorption
bays, could be observed on the surface of
the newly formed bone (Fig. 6).
No particles of the xenograft could be
observed in the apical part of the grafted
sockets (test site; Fig. 7), but newly formed
immature bone was found to occupy this
portion of the post-extraction wound. In
addition, limited numbers of trabeculae of
woven bone occurred along the lateral
walls of the sockets. The central and mar-
ginal segments of the wound were occupied
by a connective tissue that enclosed the
s
Bio-Oss particles (Fig. 7). This connective
Fig. 6. Microphotograph representing the woven
bone in the non-grafted site. The trabeculae of
woven bone were lined with osteoblasts and occa-
sional isolated osteoclasts (arrows). BM, primitive
bone marrow; V, vessel; WB, woven bone. Hema-
toxyline–eosine stain; original magnification:
200.
Fig. 7. Microphotograph of a mesio-distal section
representing a grafted site. Note that the central
and marginal segments of the sockets were occupied
s
by a connective tissue that enclosed Bio-Oss parti-
cles (asterisks). PM, provisional matrix; WB, woven
bone. Hematoxyline–eosine stain; original magnifi-
cation: 16.
tissue had the features of a provisional
matrix (rich in mesenchymal cells, fibers
and vessels) but small amounts of mono-
nuclear leukocytes could consistently also
be observed. In addition, the provisional
matrix harbored a multitude of large multi-
nucleated cells that were in direct contact
with and occupied substantial portions of
the periphery of many of the graft particles
(Figs 8 and 9). Segments of the graft that
3 | Clin. Oral Impl. Res. 20, 2009 / 1–6
Araújo et al . Early bone formation in extraction sockets
Fig. 8. Microphotograph representing the provi-
sional matrix in the grafted site. The provisional
matrix harbored large multinucleated cells (arrows)
that were in direct contact with and occupied differ-
ent portions of the periphery of many of the graft
s multinucleated cells (arrows) but also with provi-
particles. Bp, Bio-Oss particles; PM, provisional
matrix. Hematoxyline–eosine stain; original magni-
fication: 200.
Fig. 9. Microphotograph representing a higher mag-
nification of multinucleated cells (arrows) that
s
coated the entire periphery of a Bio-Oss particle
s
in a grafted site. Bp, Bio-Oss particle. Hematoxy-
line–eosine stain; original magnification: 1000.
were not occupied by the multinucleated
cells were found to be in direct contact
with provisional matrix or newly formed
mineralized bone (Fig. 10). In the lateral
portions of the socket, several adjacent Bio-
s
Oss particles were frequently found to be
‘connected’ (bridged) by zones of immature
bone (Fig. 11). There were no obvious signs
of active degradation of the bovine mineral
and only at a few sites could multinu-
cleated cells be observed in shallow invagi-
nations on the surface of the graft (Fig. 12).
Morphometric measurements
The percentage area occupied by the var-
ious tissues in the alveolar socket is pre-
sented in Table 1. The proportion of
4 | Clin. Oral Impl. Res. 20, 2009 / 1–6
Fig. 10. Microphotograph representing the provi-
sional matrix in a grafted site. Note that the Bio-
s
Oss particle was not only in direct contact with
sional matrix or newly formed woven bone (arrow-
s
heads). a, artifact; Bp, Bio-Oss particles; PM,
provisional matrix; WB, woven bone. Hematoxy-
line–eosine stain; original magnification: 400.
Fig. 11. Microphotograph representing the lateral
portions of the socket in a grafted site. Note few
s retarded in extraction sockets that, imme-
multinucleated cells at the surface of the Bio-Oss
s
particles and that several adjacent Bio-Oss particles
were bridged by newly formed woven bone. Bp, Bio-
s
Oss particles; V, vessel; WB, woven bone. Hema-
toxyline–eosine stain; original magnification:
400.
granulation tissue was 3.7  0.8% (SD)
in the grafted and 3.1  0.5% in non-
grafted sites. The proportions of provi-
sional matrix were 62.7  12.6% (grafted
sites) and 49.1  8.2% (non-grafted sites),
and the corresponding proportions of
mineralized bone were 14.7  5.9% and
s
47.8  15.6%. The Bio-Oss particles oc-
cupied 18.9  9.8% of the tissue present
in the grafted sockets. The percentage of
s
Bio-Oss particles that were in direct con-
tact with multinucleated cells was
53  12.2%.
c 2009 The Authors. Journal compilation 
 c 2009 Blackwell Munksgaard
Fig. 12. Microphotograph representing a higher
magnification of a multinucleated cell present in a
shallow invagination on the surface of a Bio-Oss
s
particle in the provisional matrix of a grafted site.
s
Bp, Bio-Oss particle. Hematoxyline–eosine stain;
original magnification:  1000.
Table 1. Mean values (SD) of the propor-
tions (%) of the different tissues in the
grafted and non-grafted alveolar sockets
Grafted Non-grafted
site site
Granulation 3.7 (0.8) 3.1 (0.5)
tissue
Provisional 62.7 (12.6) 49.1 (8.2)
matrix
Mineralized 14.7 (5.9) 47.8 (15.6)
bone
s
Bio-Oss 18.9 (9.8) –
Discussion
The present experiment indicated that the
early phase of hard tissue formation was
diately after tooth removal, had been
s
grafted with Bio-Oss Collagen. It appeared
that this delayed wound healing and bone
modeling may have been influenced by the
multinucleated cells that occurred in tis-
sues harboring the xenograft. Thus, in the
grafted sites, substantial amounts of newly
formed bone could only be detected in the
apical portion of the socket where the graft
material was absent. In the remaining
portions of the grafted sockets, a mildly
inflamed provisional matrix surrounded
s
the majority of the Bio-Oss particles, the
surface of which was frequently, but not
always, coated with multinucleated cells.
In isolated areas of the grafted sites, multi-
nucleated cells were absent and the foreign
material occasionally surrounded by the
woven bone that bridged adjacent granules
of the bovine bone.

In the non-grafted control sites, large
amounts of woven bone had formed in
most compartments of the socket. This
finding is in agreement with observations
from similar experiments that investigated
tissue modeling and remodeling in extrac-
tion sockets as well as in mechanically
produced defects in the alveolar ridge in
dogs (Cardaropoli et al. 2003, 2005; Araújo
& Lindhe 2005).
Similar amounts of new bone had formed
in the apical segments of the test and
control sites, while in the remaining por-
tions of the grafted sockets, only minute
amounts of immature bone could be found.
There are reasons to assume that following
root extraction and graft placement, the
ensuing flow of blood into the cone-shaped
s
socket forced the Bio-Oss Collagen mate-
rial in marginal direction. A void was
hereby established in the apical zone of
the socket that could house a coagulum
that apparently was unaffected by the
translocated foreign material. Hence, in
this apical portion of the test socket as
well as in the entire non-grafted socket,
tissue modeling evidently followed a nor-
mal patter, i.e. the clot was replaced with
granulation tissue, provisional matrix and
primary spongiosa (Cardaropoli et al.
2003).
In the non-mineralized portions of the
newly formed tissue in the grafted sockets,
large multinucleated cells were found to
surround a substantial number of the Bio-
s
Oss granules. Thus, 450% of the gran-
ules were more or less completely coated
by multinucleated cells. In the remaining
s
portions of the graft, the Bio-Oss particles
were surrounded either by a leukocyte-
containing provisional matrix or occasion-
ally newly formed woven bone. In such
locations, multinucleated cells were con-
spicuous by their absence.
The occurrence of multinucleated cells
s
on the surface of Bio-Oss material was
previously described (e.g. Piatelli et al.
1999; Merkx et al. 2000; Tadjoedin et al.
2003; Houshmand et al. 2007; Simion
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s
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c 2009 The Authors. Journal compilation 
 c 2009 Blackwell Munksgaard
Araújo et al . Early bone formation in extraction sockets
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s
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