IOP PUBLISHING
BIOMEDICAL MATERIALS
doi:10.1088/1748-6041/4/1/015012
Biomed. Mater. 4 (2009) 015012 (6pp)
Viability of osteocytes in bone autografts
harvested for dental implantology
Bernard Guillaume1, Christine Gaudin2, Sonia Georgeault2,
Romain Mallet2, Michel F Baslé2 and Daniel Chappard2
1
CFI—Collège Français d’Implantologie, 6 rue de Rome, 75008 Paris, France
INSERM, U 922—LHEA, Faculté de Médecine, 49045 Angers Cédex, France
2
E-mail: [email protected]
Received 2 September 2008
Accepted for publication 17 November 2008
Published 12 December 2008
Online at stacks.iop.org/BMM/4/015012
Abstract
Bone autograft remains a very useful and popular way for filling bone defects. In maxillofacial
surgery or implantology, it is used to increase the volume of the maxilla or mandible before
placing dental implants. Because there is a noticeable delay between harvesting the graft and
its insertion in the receiver site, we evaluated the morphologic changes at the light and
transmission electron microscopy levels. Five patients having an autograft (bone harvested
from the chin) were enrolled in the study. A small fragment of the graft was immediately fixed
after harvesting and a second one was similarly processed at the end of the grafting period
when bone has been stored at room temperature for a 20 min ± 33 s period in saline. A net
increase in the number of osteocyte lacunae filled with cellular debris was observed (+41.5%).
However no cytologic alteration could be observed in the remaining osteocytes. The viability
of these cells is known to contribute to the success of autograft in association with other less
well-identified factors.
(Some figures in this article are in colour only in the electronic version)
with various biomechanical and osteoconductive properties.
None of them have been found sufficient to restore large bone
defects. Injectable calcium phosphate materials have been
proposed but their long-term effects could not be detectable
in a multicentric orthopedic study [4]. In a recent animal
study conducted in this laboratory, an injectable bone paste
based on βTCP-hydroxyapatite induced osteoconduction with
woven bone apposition but the remodeling process removed
it in a second time, making the rationale of such substitutes
questionable [5]. In addition, synthetic ceramics are often too
brittle to be used in weight-bearing bones or when compressive
stresses are too high.
In orthopedics, bone is commonly harvested from the
iliac crest, as it provides easy access to good quality and
quantity cancellous autograft. Other sources are Gerdy’s
tubercle of the tibia and the distal parts of the radius or tibia
for cancellous bone; autologous cortical bone can be obtained
from the fibula [6], ribs and iliac crest. These sites were
also widely used in maxillofacial surgery from as early as
World War II [7]. However other sites have been favored
Introduction
Materials for replacing bone are necessary in a number of
reconstruction surgeries of the skeleton caused by traumatic,
tumor-resection or congenital defects. The highest demand
concerns orthopedic surgery for the treatment of femoral
prosthesis loosening where wear debris induces a marked
osteolysis. It has been estimated that more than 500 000
bone grafts are done each year in the USA [1]. However,
there is also a growing demand in maxillofacial surgery
and in implantology to restore a sufficient volume of bone
before placing implants in the mandible or the maxillar
[2]. Allogenic bone grafts are common in orthopedics but
require access to a bone bank in some countries [3]. The
high cost of a bone bank, the difficulties of conditioning
bone chips of fragments specially adapted to maxillofacial
surgery and the potential risk of transmission of viral or prior
infections have made allografts seldom used in implantology.
Synthetic phosphocalcic materials (β-tricalcium phosphate,
hydroxyapatite) are numerous in the market but are associated
1748-6041/09/015012+06$30.00
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© 2009 IOP Publishing Ltd Printed in the UK
B Guillaume et al
Biomed. Mater. 4 (2009) 015012
for the reconstruction of maxillofacial bone defects such as
parietal [8] or mandibular bone [9]. These observations
were based on experimental works in animals showing that
membranous bone grafts survive better than endochondral
bone after grafting [10, 11]. Parietal or chin bone grafting has
been proposed in implantology because access is simpler in the
surgical theatre than for other sites. Autologous bone provides
superior results to other methods since it is revascularized
easily and rapidly incorporated into the recipient site. It is
also commonly thought that osteoblasts, osteocytes (OCs) and
lining cells of the graft can survive the transplant and favor
the osseointegration of the graft [12]. However, this has
been seldom documented in the literature. This study was
undertaken to survey the ability of bone cells to survive during
the transplant process starting from harvesting to implantation.
(A)
Material and methods
Patients and surgical protocol
Human mandibular bone samples were obtained from five
patients under general anesthesia during the time course of
grafting for pre-implantation. Each patient has given his/her
informed consent to participate in the present study. The
surgical protocol aims at increasing the bone wall thickness
at the maxilla or mandible in deficient recipient sites prior
to the placement of dental implant(s) of standard diameter.
A broader bone volume ensures an easier position for the
implant’s axis. The grafting zone was operated on first to
appreciate and determine the shape, volume and position of
the graft.
For each patient, the graft was harvested at the chin;
a preliminary radiographic assessment has been done to
check the axis and the thickness of the donor site. One or
two rectangular cortico-cancellous bone samples and corticocancellous cylinders were removed. The intra sulcular incision
was made at the level of the dental collars with two side
incisions at the mesial first premolars. This full thickness
reflection flap is preferred to an incision at the gingival margin
because it allows a better coverage flap of the grafting zone
without any tension and exposure of the graft (figure 1). The
limits of the harvesting graft areas were done with a thin bur
for bone to outline the defect margins on the buccal bone plate
to a 6 mm depth. The harvested graft was separated with a
chisel by progressive cleavage. Chips of cortico-cancellous
bone (mostly cortical) were collected. The bone samples
were immediately placed in sterile saline at room temperature
until used for grafting. One small sample (1 mm × 1 mm
from the chin cortex and containing the endosteal surface) was
immediately fixed in a glutaraldehyde-based fluid. The margin
flaps were closed with interrupted sutures with 5/0 vycril. For
each patient, the amount of the graft corresponded roughly to
1.8 cm3 per cortico-cancellous block graft (usual dimensions:
10 mm × 6 mm × 3 mm). The flow chart of the study appears
in figure 2.
When harvesting bone was achieved, the surgeon began to
release the muccoperiosteal flap; the margin recipient bone site
was revived with a round bur to obtain a slightly hemorrhagic
zone. The site was carved to create a mortise favorable so
(B )
Figure 1. Harvesting the bone graft at the chin. (A) Section of bone
with the thin bur. (B) Mobilization of the cortico-cancellous grafts
with a chisel.
Figure 2. Flow chart of the study from harvesting to grafting. The
specimens collected for histological analysis were taken
immediately and at the end of the grafting period.
to create a primary closure and the anchorage of the graft
was then stabilized with stainless steel screws (the cancellous
bone of the cortico-cancellous harvested graft was placed in
direct contact with the patient’s recipient site) (figure 3(A)).
The cancellous bone chips were placed on all sides around
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Biomed. Mater. 4 (2009) 015012
For each block, five sections (1 μm in thickness)
were obtained semi-serially every 20 μm (this separation
ensures that no osteocyte lacuna profile could appear on
two consecutive sections). A Leica Ultracut S (Leica–
Rueil Malmaison, France) was used with a glass knife and
sections were immediately deposited on a glass slide with a
drop of distilled water. The sections were dried at 60 ◦ C;
then they were incubated for 15 min in periodic acid
44 mM and stained with an extemporaneously prepared
solution combining aqueous methylene blue 1% (1 volume)
and azure II 1% (1 volume) for 20 min at room temperature.
The sections were then thoroughly washed in distilled water
and mounted in NeoEntellanTM (Merck) after air drying.
On each set of sections prepared per block, the total
number of osteocyte lacunae was counted by light microscopy
on the five sections. Lacunae were separately classified as
containing a normal OC with nucleus and cytoplasm with
prolongations, empty lacunae and lacunae containing cellular
debris. The results were expressed as a percentage of the total
lacunae.
(A)
Transmission electron microscopy
Ultrathin sections (80 nm in thickness) were obtained with a
diamond knife and transferred onto nickel grids (mesh 100)
coated with a film of collodion. Grids were then air-dried,
contrasted with sodium metaperiodate, uranyl acetate and lead
citrate. Observations were done with a transmission electron
microscope JEOL 2011 (JEOL-France) at 120 kV.
(B )
Figure 3. (A) The cortico-cancellous grafts are immobilized in the
recipient site by screwing. (B) Clinical aspect of a well-integrated
allograft at 6 months.
Statistical analysis
Statistical study was done using Systat 11 (Systat software
Inc.). Data were expressed as mean ± standard error of the
mean (SEM). Significant differences between samples were
assessed with nonparametric Mann and Whitney’s U test.
Differences were considered as significant when P < 0.05.
and on top of the monocortical block graft. The full thickness
flap was then closed to the primary incisions and sutured with
5/0 vicryl. When the last chip was positioned, the surgeon
placed a remaining bone sample into a new vial containing
the glutaraldehyde fixative. In this way, the effects of storing
in physiological saline at room temperature during a mean of
20 min ± 33 s could be explored on paired specimens. Sutures
were removed two weeks post-operatively. A CT-scan was
performed at 3 and 6 months post-surgery to ensure the bone
graft healing. The graft was deemed successful when a dense
bony reaction could be seen at the grafted site.
Results
The patients were re-examined in the days following the graft,
even in the absence of any complication. The absence of
prolonged pain, edema, purulent discharge, elimination of
sequestrated and devitalized bone fragments in the following
months are in favor of a progressive success of graft fixation.
The palpation of the grafted site showed an increased, stable
and painless relief (figure 3(B)). Radiographs (especially the
CT-scans) confirmed adherence of the graft at the receiver site
by the absence of radiolucent space between the two zones.
The gain in thickness was 5 mm on average and 4 mm implants
could be placed with a satisfactory axis.
The methylene-blue-azure staining allowed a clear-cut
identification of the various types of lacunae. OCs were clearly
evidenced by a blue cytoplasm with processes extending into
the matrix lacunae. Empty lacunae were devoid of any cellular
material while cellular debris could be identified inside or on
the margins of the lacunae and corresponded to remnants of
necrotic cells. The matrix itself was unstained, or appeared
light bluish (figure 4).
Bone microscopy
The fixative was prepared in ready-to-use plastic vials that
were stored in the refrigerator until use. The fixative was
composed of glutaraldehyde 4% in cacodylate buffer, pH 7.4.
Samples were fixed for 1 h at 4 ◦ C in the surgical unit then
rinsed, stored in cacodylate buffer and sent to the laboratory.
The samples were then cut with a clean blade into small
pieces suitable for electron microscopy and post-fixed in 1%
osmium tetroxide. They were dehydrated through a graded
ethanol series and finally embedded in Epon 812 according to
standard methods used for TEM. The blocks were stored at
room temperature until ready to use.
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(A)
(B )
Figure 5. OC count on semi-thin sections. (A) The number of
osteocyte lacunae filled with cell debris increased in all patients.
(B) Cumulative histogram of the three different types of lacunae at
the beginning and the end of the grafting surgery.
Discussion
Cell necrosis can occur rapidly when cells are deprived
of oxygen and essential metabolites such as glucose [13].
The process differs from apoptosis, which is a particular
mechanism induced by various factors (excess or deprivation
of hormones, growth factors or cytokines, p53 . . . ). Necrosis,
unlike apoptosis, can induce an inflammatory reaction.
However, osteoinductive factors released from the graft during
the resorptive process, as well as cytokines released locally by
the inflammatory phase, are thought to contribute to healing
of the graft. It is likely that other factors may play a
key role in the excellent success rate of autografts because
devitalized allografts can release similar molecules. The
presence of osteogenic cells is frequently advocated as the
basis requirement for the success of autografts [12]. Cortical
bone has a higher fractional bone volume (i.e., percentage
of the tissue volume occupied by bone) than cancellous
bone and has a lower porosity. Haversian canals are the
main reservoir of osteogenic cells together with the endosteal
surfaces but the number of osteoblast precursors is reduced
(C )
Figure 4. The different types of osteocyte lacunae in light
microscopy. (A) Lacuna filled with a typical osteocyte with a
well-defined nucleus. (B) Lacuna containing cellular debris.
(C) Empty lacuna. Original magnification ×1000.
OC counts appear in figure 5; counts are presented on the
samples studied at the beginning and the end of the grafting
surgery. The number of empty lacunae remained unchanged
after the period spent in ex vivo conditions. On the other hand,
a significant increase in the number of lacunae containing
debris was observed (+41.5%) and conversely, the number of
lacunae containing an intact osteocyte was reduced.
On TEM sections, the OC lacunae were observed and the
analysis of the fine cytological details of these cells did not
reveal any change (figure 6). Occasionally some osteoblasts
were encountered and no gross abnormalities (e.g., recycling
membrane) were evidenced.
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Biomed. Mater. 4 (2009) 015012
no data have been presented on the potential changes in
viability of bone cells induced by the harvesting and storage
procedures used before grafting. Another way to evaluate the
viability of the osteocytes in the bone chips would have been
to perform a LDH (lactate dehydrogenase) histoenzymatic
analysis. However in the present study, the grafts were
harvested far from the laboratory and we chose to evaluate bone
cell preservation by using morphological EM criteria (absence
of mitochondrial or membrane changes).
Furthermore,
the method necessitates cryosectioning, which is not
compatible with EM techniques [17]. In the present study,
the number of osteoblasts that could be seen on the cortical
morcellized chips was very low, and only OCs were evidenced
in all subjects. OCs have a reduced number of organites
when compared to osteoblasts. Because OCs reside distant
from the blood supply, their metabolic needs are satisfied
by a combination of passive diffusion of fluids through the
matrix and canaliculi and enhanced diffusion arising when the
skeleton is loaded during functional activity [18–20]. Their
metabolic demand is probably lower than other high energy
spending cells and this can explain why a large fraction of OCs
survived the surgical conditions (room temperature, saline,
absence of oxygen and metabolites). In normal humans, a
noticeable fraction of lacunae are found empty, the fraction
is increased in osteoporotic patients [21]. OCs are highly
differentiated cells which differ from osteoblasts [22, 23]. In
vitro studies on the MLO-Y4 osteocytic cell line have found
the possibility of these cells to dedifferentiate into osteoblasts
[22]. Furthermore, the importance of OCs in the success
of bone autografting was stressed in a recent animal study:
bone-grafted particles healed when living OCs were present
and failed when OCs have undergone necrosis [24]. Another
important factor that could explain the superiority of autograft
versus allograft is the total absence of protein denaturation in
the former. The 20 min incubation in saline used in this study is
probably harmless to the patient’s proteins. On the other hand,
protein denaturation is known to occur after deep freezing [25]
or autoclaving [26]. Treatment of bone allografts with various
chemical processes used to clean bone was recently found to
alter some important matrix proteins [27].
This study is the first to report the cellular effects of
the limited storage conditions used surgically to preserve
morcellized bone chips of the chin. A noticeable proportion
of OCs remains healthy without modifications observed at the
electron microscopy level; other OCs died by necrosis. The
success of autografts could result in the preservation of living
OCs together with additional factors such as better protein
preservation.
(A )
(B )
(C )
Figure 6. Transmission electron microscopy of living osteocytes in
a graft sample at the beginning (A) and the end (B) of the surgical
procedure. A group of osteoblasts is also shown at the end (C).
Scale bars: 2 μm.
when compared to cancellous bone [14]. Also, the absence
of hematopoietic tissue is recognized to provide fewer stem
cells in cortical bone autografts. In orthopedic surgery,
cortical bone grafts are favored in loaded areas and they have
been found to be more resistant to vascular ingrowth and
remodeling [15]. Vascularized cortical grafts (i.e., parts of
harvested bone with their vascular pedicle, reanastomosed
at the recipient site) have been proposed to provide bone
with viable cells and restore a local vascularization inside the
graft [16]. Although the method has shown clearly superior
results with large bone defects (e.g. >12 cm), it is associated
with increased morbidity. The continued vascular supply
is thought to allow for faster bone incorporation but clear
histopathologic analyses are lacking. To our knowledge,
Acknowledgment
The authors thank Mrs Laurence Lechat for secretarial
assistance. This work was supported by funds from ‘Pays
de la Loire–Bioregos’ and INSERM.
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