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Histological Analysis Of The Newly Formed Bone Secondary To
Distraction Osteogenesis Of The Mandible In Goat
MOUKHTAR MOUSSA* and MAHMUD NASR**
*Dept. Cytology and Histology1 and Faculty of Veterinary Medicine
** Dept. Dental Surgery National Institute for laser enhanced sciences
Cairo University – Giza, EGYPT
[email protected] & [email protected]
Abstract: A unilateral mandibular distraction osteogenesis (DO) was performed in 16 healthy adult male goats.
The mandibular body was lengthened by gradual distraction. After 10 days of distraction periods, using a protocol
of 0-day latency and a 1-mm/day rate for 10 days, the mandible was held in external fixation for 8 weeks
consolidation. The histological features and pattern of the healing process was described after 4 and 8 weeks
consolidation. Contrary to the prevalent concept, the results indicated that bone formation in the mandible of this
model was principally via the intramembranous-type of ossification. No evidence of cartilage tissue was seen in the
gap healing. Five phases could be observed during the healing process: 1) Initial phase 2) Collagenic phase 3)
Osteogenic Phase 4) Lacuna Formation Phase, and 4) Early modeling - remodeling phase of the bony regenerate.
Key Words: distraction osteogenesis, osteodistraction, mandibular body lengthening, consolidation, osteogenic
cells, intramembranous ossification, Bone remodeling.
1 Introduction
DO is the biologic process of new bone formation
between the surfaces of bone segments that are
gradually separated by incremental traction [1]. The
traction generates tension on the skeletal and
surrounding soft tissue structures, which stimulates
new bone formation parallel to the vector of distraction
[2,3]. DO was first described by Codivilla in 1905 to
elongate the femur [1]. In 1951, Ilizarov developed a
technique for repairing complex fractures of long
bones. This technique was divided into five
sequentional periods: osteotomy, latency, distraction,
consolidation and remodeling [4, 5, 6].
When the
desired or possible length is reached, a consolidation
phase follows to allow maturation of the regenerate
after traction forces are discontinued [7].
Cope and
Samchukov [8] found zones of endochondral
ossification and transchondroid bone formation at 4
weeks of consolidation in dog mandible. In rabbit
mandible, new bone was formed in the distraction gap
(DG) by both intramembranous and endochondral
ossification [5] and by intramembranous ossification
only [6, 9]. Sawaki et al. [7] in children reported new
bone formed by intramembranous ossification with
some fibro-cartilage islands. The aim of this study was
to analyze the sequence of histological features and cell
behaviors during DO of the mandibular body.
2 Materials and Methods Sixteen adult male goats, divided into two groups, were
used in this study. They underwent unilateral
mandibular body DO using a protocol of 0-day latency
and a 1-mm/day rate for 10 days.
The mandible (in one group) was held for 4 and (in the
other group) for 8 weeks consolidation periods to allow
distraction gap regeneration. Plain radiographs were
performed before the mandibles of both groups were
harvested. Also an arteriogram had been made to show
the pattern and distribution of the inferior alveolar
artery in the distraction area. Sagittal sections of The
10 mm distraction regenerate was osteomized and fixed
in 10% buffered formalin for 24 hours. Then
decalcified in 10% EDTA for about 3 weeks before
paraffin embedding. Sections, 5 – 7 Um thick, were
cut and stained with hematoxylin and eosin and
Crossmon’s trichrome stain as outline by Bancroft and
Stevens [10].
3 Results When a fracture induced, the soft and bony
vasculatures were disrupted. A hematoma floods the
wound as a result, with many blood borne elements
providing the first population of cells at the fracture
site. As the clot was formed, platelet deposition
occurred and a fibrin scaffold developed. Too few
mesenchymal cells (Fig. 1) migrated from the
adjacent marrow and bony tissue. They
contributed to the earliest formation of the DR. In
addition, osteoclasts in the form of syncytial
consolidation of monocytes of hematological origin
were observed (Fig. 2). This stage lasted from 1 to 5
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days, at which time the clot was replaced by a
granulation tissue. One week after osteotomy, the
reparative tissue was filling the DG. It was soon
reinvaded by an influx of undifferentiated cells. This
stage lasted for approximately 2 weeks.
As the granulation tissue continued to organize,
endothelial cells and smooth muscle cells migrated
along the fibrin scaffold as vascular buds. They
provided the pool needed for the ongoing
replacement of cells during regeneration. As healing
progressed, ossification centers became apparent to
start an intramembranous-type of bone. They were
randomly scattered in the DR. Each center appeared as
an area of mesenchymal condensation within a
homogeneous ground substance (Fig.3).
Pronounced development occurred when fi-
broblasts differentiated from the mesenchymal cells.
They produced collagen fibers and extracellular
ground substance. Later, randomly scattered collagen
fibers were seen among the cells. Progressive
development of the collagen produced identifiable
bony specules. In the meantime, some of the
mesenchymal cells became osteoprogenitor cells and
preosteoblasts (Fig.4). Soon after, these
preosteoblasts underwent maturation and became
active osteoblasts. Each appeared as a large oval cell
with a basal spherical nucleus, basophilic cytoplasm
and a prominent large pale apical Golgi zone (Fig.4). It
seems possible that maturation of these osteoblasts
was enhanced by the presence of pre-existing
osteoclasts in the ossification centers (Fig.5).
Fig.1: Ossification center – 4 weeks consolidation.
H&E stain, 800 X
Fig.2: Syncytial condensation of monocytes to form
osteoclasts. H&E stain, 600 X
Fig.3: Ossification center – 4 weeks consolidation.
H&E stain, 300 X
Fig.4: Cell behavior during osteogenesis – 4 weeks
consolidation. H&E stain, 600 X
Fig.5: Cell behavior during osteogenesis – 4 weeks
consolidation. H&E stain, 600 X
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An osteoclast appeared as a giant multinucleated cell
(Fig.6). Their nuclei varied in number, 6-15.
Their cytoplasm was "foamy" with many vacuoles. As
osteoblasts deposit initial matrix (osteoid), some of
them became entrapped in their secretion as the
future osteocyte (Fig.7). This osteoid became
mineralized after a time lag the so called osteoid
maturation period. As healing advanced, the
reparative area showed many irregular primitive
trabeculae or specules. Later on, progressive
expansion of these developing trabeculae occurred.
Formations of other new trabeculae made them
branched and united with one another to form
woven bone (Fig.8). This primitive bone enclosing
spaces filled with fibro-cellular vascular elements
which gradually transformed into marrow tissue. In
parallel with this, the peripheral zone of the
regenerate grew rapidly and thickened. It became
more organized to form the future periosteum.
Woven bone was a transient structure during the
reparative process. The early formed osteonal
canal could be seen at this stage of growth.
Gradually, the new regenerate became modeled and
remodeled, by both the osteoblasts and the
osteoclasts, to acquire the architecture of
neighboring bone. Although, newly formed bone was
achieved by the 4th weeks and became more remodeled
by the 8th weeks of consolidation, the reparative gap
did not yet attained the neighboring bony architecture.
Analysis and Cell behaviors in the Distraction
regenerate (DR):
After 4th
and 8th weeks consolidation, the new
regenerate revealed only an intra-membranous-type of
ossification which occurred simultaneously in the
following phases:
2.1 Initial phase: Examination of the newly formed DR revealed
multiple ossification centers scattered randomly in
the reparative framework. These foci could be
considered as the osteogenic forerunner in the
DG. Adjacent to the DR, the outer surface of the
edges of the DG was invested by the periosteum.
Its inner osteogenic layer was sharing actively in
invading the DR by an influx of osteoprogenitor
cells. Each ossification center appeared as an
aggregation of mesenchymal and osteoprogenitor
cells intermingled with minute blood spaces and
randomly arranged collagen fibrils. The mesenchymal
cells appeared crowded and hyperplastic, forming
mesenchymal condensations. A mesenchymal cell had
a small cell body with a few long and thin cell
processes. The nucleus was large spherical with clear
appearance. Many mesenchymal cells begun to
differentiate very rapidly into preosteoblasts. The
cells enlarged and gradually became polyhedral or
rectangular. Their processes were retained or
disappeared. Their cytoplasm stained lightly
basophilic on account of its high content of rER.
Gradually, these centers became more vascular as they
Fig.6: Osteoclast in early ossification center. H&E
stain, 800 X
Fig.7: Osteoblast entrapped in the osteoid. H&E
stain, 600 X
Fig.8: Woven bone. Crossmon's trichrome stain, 150 X
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were actively invaded with numerous capillaries
derived from periosteal and endosteal. These new
capillaries were essential for the ongoing reparative
process. As these ossification foci became highly
vascular, their activity became more pronounced.
2.2 Collagenic phase: The intercellular spaces in the ossification center were
occupied by a ground substance containing wavy
collagen fibers. As more fibroblasts differentiated
from the mesenchymal cells, more collagen fibers
became randomly scattered within the ground
substance. They formed interlacing fibers within
which many cells were located. Moreover, many
collagen fibers coalesced together to form bundles
and became embedded in the initial secretion
(osteoid) of the newly differentiated osteoblasts to
form primitive specule or trabecula. The osteoid was in
the form of a hyaline jelly-like material. This secretion
was partially surrounding the osteoblast and was mostly extending towards the collagen bundles.
2.3 Osteogenic Phase: Osteoclast was the first bone cell observed early in the
regenerate. As the growing ossification centers
initially invaded by the osteoprogenitor cells from the
neighboring tissue, many of these cells differentiated
into preosteoblasts which soon became mature
osteoblasts. This maturation was enhanced by the effect
of the pre-existing osteoclasts. An active osteoblast
deposited the initial matrix, the osteoid amorphous
substance. This secretion was in the form of a hyaline
jelly-like material. It was given off unidirectional from
the cell end away from the nucleus. The osteoid
became mineralized after a time lag the so-called
osteoid maturation period. The osteoid was mostly
extending towards the collagen bundles in the form of
thin bars (Fig. 5) which coalesced together. This
secretion masked the collagen fibers and imparted
them a hyaline or lightly basophilic appearance.
Some osteoblasts became entrapped in their
secretion as the future osteocytes. Adjacent collagenic
bundles embedded in the osteoid with some entrapped
osteoblasts formed a specule or a trabecula of early
forming bone. Numerous osteoblasts congregated on
the surface of this newly formed bony trabecula (Fig.
9). As more osteoblasts became incarcerated within
their secretion as osteocytes, they were replaced by
newly differentiated osteoblasts at the surface of the
trabeculae. The presence of some osteoclasts attached
to the surface of the newly formed trabeculae, in
association with osteoblasts (Fig.7).This supported the
theory that these two cells are working together in a
synchronizing manner and were responsible for
normalizing the newly formed regenerate.
2.4 Lacuna Formation Phase:
The osteoblasts eventually formed a row of cells on the
surface of the developing bony specules. They
deposited their osteoid secretion. Some cells, that
failed to join the surface, became entrapped within the
newly deposited matrix at the periphery of the
trabecula (Fig.10). These trapped osteoblasts became
osteocyte when it was completely encased by matrix in
a lacuna. These first osteocytes assisted in osteonal
development and its mineralization. An osteocyte lay in
its own lacuna could contacting its neighboring
osteocytes through fine primitive canaliculi. This
contact would maintain its vitality by passing nutrients
and metabolites from the blood, regulating ion
homeostasis, and transmitting signals. Maturation of
the osteoblasts into osteocytes was marked by cellular
hypertrophy, lacunar enlargement, and the change in
the lacuna shape from ovoid to flat. More osteoblasts
became embedded within their secretion. This activity
resulted in the formation of more lacunae in which the
osteoblasts resided. The lacunae and the cells within
Fig.9: Osteoblasts congregated on the surface of the
newly formed bony trabecula. H&E stain, 800 X
Fig.10: Trapped osteoblast becomes osteocyte when
completely encased by matrix in a lacuna.
H&E stain, 800 X
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them appeared flattened with their long axes parallel to
the surface of the developing trabecula. The matrix was
generally acidophilic near the surface of the trabecula.
However, some trabeculae showed a mottled
appearance due to the difference in staining properties
of both the core and the edges of the trabecula.
2.5 Early Modeling-remodeling phase:
During the consolidation period, there was continual
modeling and remodeling of the newly formed bony
trabeculae and specules. They were subjected to the
synchronizing activities of both osteoblast and
osteoclast. It is well known that the osteoblast and
osteoclast are integral parts of the bone formation.
Moreover, it was believed that the osteoclast is only
lodging in a concavity called Howship's lacuna. This
lacuna, in the present study, was not found as the
osteoclast was present in a very active form at the early
reparative phase.
2.5.1 Role of osteoblast:
The osteoblasts produced layer after layer of bone
inward on the surface of the developing trabecula. As
the trabeculae enlarged in size, its structure was altered
by internal reconstruction and by remodeling.
Remodeling resulted from alteration in certain areas
and deposition of new bone elsewhere.
2.5.2 Role of osteoclast:
a) Bone remodeling:
Osteoclasts were usually found in contact with the
surface of a collagenic bundle (Fig.11). Osteoclasts
refine the affected bony edges. This enhanced the
newly formed regenerate largely to retain the
architectural shape of the preexisting bone.
b) Haversian canal remodeling:
The lytic or erosion-refining effects of the
osteoclasts resulted in the formation of cylindrical
cavities or tunnels, the forerunner of the Haversian
canal (Fig. 12). The latter were lined by osteoblasts
which deposited successive bony lamellae to increase
the diameter of the canal.
c) Phagocytic activity:
Some osteoclasts acted as a giant cell. This was
illustrated by phagocytosing and digesting osteocyte
together with its surrounding bony lacuna.
Radiographic findings: The regenerate filling the distraction gap after 4 weeks
consolidation, showed a variable density when
compared with the adjacent bone segments (Fig. 13).
After 8 weeks this regenerate showed homogenous
bone densities (Fig. 14).
Fig.11: Osteoclast attached to the surface of a
newly formed bony trabecula. H&E stain,800 X
Fig.12: Haversian canal remodeling. H&E stain, 200
X
Fig.13: X-ray to show bone density of the DR – 4
weeks consolidation.
Fig.14: X-ray to show bone density of the DR – 8
weeks consolidation.
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ISBN: 978-1-61804-111-1 84
Angiographic results: After 4 weeks consolidation, the inferior alveolar artery
appeared patent and thin with remarkable decrease in
its diameter (Fig.15). After 8 weeks consolidation, it
showed a relatively thicker diameter. However, it still
had a less diameter than the normal one (Fig.16).
4 Discussion
Intramembranous ossification was the main and
only mechanism of bone formation in the distraction gap.
There was no evidence of cartilaginous tissue in the
newly formed bone. This finding was also described by
Sencimen et al. [9] in rabbits. The finding of Carter et
al. [11] that distraction was associated with cartilage
only in very small discrete regions at the periphery of
the distraction gap adjacent to the osteotomy ends is
denied by the present study. Also in agreement with
Rahn et al. [12] no inflammatory phase, no cartilaginous
intermediate, nor callus formation were found in
primary bone healing. Wornom and Buchman [13] were
of the opinion that in some instances, bone may undergo
primary healing after a fracture without a cartilage
intermediate. In rabbit mandible, Komuro et al. [5]
reported new bone formed by both intramembranous and
endochondral ossification whereas Sawaki et al. [7]
found new bone formed by intramembranous
ossification, with some fibrocartilage islands. At the
initial time of osteotomy, Ashton and Allen [14] and
Brighton [15] found precursor mesenchymal cells and
osteoprogenitor cells migrating into the gap from the
adjacent muscle, marrow and from the periosteutn.
Tonna and Cronkite [16] mentioned that these cells
contribute to the earliest formation of bone at the
fracture site. The present findingsshowed an influx of
mononuclear cells invading the reparative zone. This
was derived from the marrow, blood and deepest layer
of periosteum. These local precursor cells differentiate
into fibroblasts and osteoblasts to produce collagen and
osteoid in the gap. This finding is parallel with that of
Nemelh et al. [17]. Moreover, these pluripotential cells
differentiate into osteoblasts which underwent
cementing to the cutting edges of bone at the fracture
site. Others congregate on the surface of the newly
formed bony trabeculae. They are responsible for
depositing a new matrix. Their activity results in the
formation of small lacunae in which the osteoblast
resides to become osteocyte. In agreement with Lane et
al. [18] type-1 collagen was initially deposited to form
the framework on which mineralization occurs.
According to Robling, Castillo,and Turner [19] bone
matrix consists mainly of type-I collagen fibers
(approximately 90%) and noncollagenous proteins or
osteocalcin, (makes up 1 % of the matrix). The latter
may play a role in calcium binding and stabilization of
hydroxyapatite in the matrix and/or regulation of bone
formation. Within larnellar bone, the collagenic fibers
are forming arches for optimal bone strength. These
lamellae can be parallel to each other or concentrically
arranged. Our findings supported the view of the
interplay of the three bone cells as a team, which is
called basic multicellular unit (BMU). This BMU is a
mediator mechanism bridging individual cellular
activity to whole bone morphology [20,21,22]. Units of
the osteoclasts and the osteoblasts were activated in
sequences of both bone resorption and formation to
convert the contours of the reparative zone to the
original bony architecture [13]. Recent evidence
suggests that the normal mechanism of internal bone
resorption may occur under the influence of the mature
osteocytes. Osteocytes live in fluid-filled hollows
within the bone matrix and are interconnected by
numerous long cell extensions through microscopic
tunnels. They are believed to be the cells that sense
mechanical strain. Osteocytes respond to this strain
by sending signals that either cause new bone
formation or bone remodeling [23]. Glass, Bialek,
Starbuck and Patel [24] described that osteoblast secrets
collagenous and non-collagenous bone matrix proteins,
including type-I collagen. Osteoblasts also produce a
range of growth factors under a variety of stimuli. The
most prominent features of the osteoclast is the ruffled
Fig.15: Arteriogram – 4 weeks consolidation.
Fig.16: Arteriogram – 8 weeks consolidation.
Recent Researches in Medicine and Medical Chemistry
ISBN: 978-1-61804-111-1 85
border facing the bone matrix. Osteoclasts are able to
produce both phosphatase and protease which are
necessary to dissolve bone minerals and to activate
enzymes that break down collagen fibers. Furthermore,
the cell secretes collagenase and gelatinase-� which
are involved in bone matrix digestion [25]. Attachment
of the osteoclast to the bone surface is essential for bone
resorption. This attachment is performed via integrin
receptors, which bind to specific Arginine-Glycine-
Aspartate (RGD) sequences found in matrix proteins
[26]. This process involves transmembrane adhesion
receptors of the integrin. According to Parfitt [27] and
Weinbaum, Cowin and Zeng [28] integrins attach to
specific amino acid sequences at the surface of the
bone matrix.
Conclusion: The intramembranous type of ossification was the
main mechanism of bone reformation in the distraction
gap in mandible. No evidence of pre-existing cartilage
tissue during gap healing. The term soft or hard callus
is a surgical term and is not longer valid in the field of
histology. Apoptotic and not necrotic changes occur
during induction and healing of fracture. The role of
osteoclast is to refine the bony surface and remodeling
the newly formed bone. Both osteoblast and osteoclast
are doing their work in a synchronizing manner under
a genetic encoding which regulates their functions.
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