THREE MODES OF OSSIFICATION DURING DISTRACTION
OSTEOGENESIS IN THE RAT
NATSUO YASUI,
MOTOHIKO SATO,
HIROHISA KAWAHATA,
TAKAHIRO OCHI,
YUKIHIKO KITAMURA,
TOMOATSU KIMURA,
SHINTARO NOMURA
From Osaka University Medical School, Japan
We developed a rat model of limb lengthening to study
the basic mechanism of distraction osteogenesis, using a
small monolateral external fixator. In 11-week-old male
rats we performed a subperiosteal osteotomy in the
midshaft of the femur with distraction at 0.25 mm every
12 hours from seven days after operation.
Radiological and histological examinations showed a
growth zone of constant thickness in the middle of the
lengthened segment, with formation of new bone at its
proximal and distal ends.
Osteogenic cells were arranged longitudinally along
the tension vector showing the origin and the fate of
individual cells in a single section. Typical endochondral
bone formation was prominent in the early stage of
distraction, but intramembraneous bone formation
became the predominant mechanism of ossification at
later stages. We also showed a third mechanism of ossification, ‘transchondroid bone formation’. Chondroid
bone, a tissue intermediate between bone and cartilage,
was formed directly by chondrocyte-like cells, with
transition from fibrous tissue to bone occurring
gradually and consecutively without capillary invasion.
In situ hybridisation using digoxigenin-11-UTPlabelled complementary RNAs showed that the
chondroid bone cells temporarily expressed type-II
collagen mRNA. They did not show the classical
morphological characteristics of chondrocytes, but were
N. Yasui, MD, PhD, Associate Professor
M. Sato, MD, Postgraduate Student
T. Ochi, MD, PhD, Professor and Chairman
Department of Orthopaedic Surgery
H. Kawahata, Postgraduate Student
S. Nomura, PhD, Associate Professor
Y. Kitamura, MD, Professor and Chairman
Department of Pathology
Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565,
Japan.
T. Kimura, MD, Director of Orthopaedic Surgery
Osaka Rosai Hospital, 1179-3 Nagasonecho, Sakai, Osaka 591, Japan.
Correspondence should be sent to Dr N. Yasui.
©1997 British Editorial Society of Bone and Joint Surgery
0301-620X/97/57423 $2.00
824
assumed to be young chondrocytes undergoing further
differentiation into bone-forming cells.
We found at least three different modes of ossification
during bone lengthening by distraction osteogenesis. We
believe that this is the first report of such a rat model,
and have shown the validity of in situ hybridisation
techniques for the study of the cellular and molecular
mechanisms involved in distraction osteogenesis.
J Bone Joint Surg [Br] 1997;79-B:824-30.
Received 18 November 1996; Accepted after revision 21 March 1997
The common principles of current techniques of limb
lengthening are osteotomy and slow progressive distraction
1,2
by an external fixation device. The type of osteotomy, the
timing and rate of distraction, and the apparatus have varied
3-8
considerably. It has been established that slow distraction
does not break the bony callus, but actually stimulates
osteogenesis and the growth of surrounding soft tissues.
4,5
The principle has been termed the ‘law of tension-stress’,
but the exact mechanism is not understood.
We have previously reported the histological findings in
lengthened segments in rabbits and concluded that new
bone was formed predominantly by endochondral (indirect)
9
ossification. Other reports of canine or ovine experiments
have established that intramembranous (direct) bone formation is the main mechanism of ossification during distrac4,5,10,11
tion osteogenesis.
Factors such as the stability of
fixation, the timing and rate of distraction, and speciesrelated differences may determine the relative share of
endochondral and direct bone formation.
12
Kossmann, Giebel and Glombitza recently described a
rat model of tibial lengthening using a special distraction
apparatus, which is useful for studying the cellular and
molecular mechanisms since modern techniques can be
applied to this animal. We have developed a new model of
rat femoral lengthening using a commercially available
monolateral external fixator. We now describe the surgical
technique and report our radiological and histological findings and the results of in situ hybridisation using complementary RNA (cRNA) probes for type-I, type-II and
type-X collagens.
THE JOURNAL OF BONE AND JOINT SURGERY
THREE MODES OF OSSIFICATION DURING DISTRACTION OSTEOGENESIS IN THE RAT
MATERIALS AND METHODS
We used 14 male 11-week-old Sprague-Dawley rats weighing 380 to 400 g. A small monolateral external fixator
(Hoffmann Mini Lengthening System 5094-0-202, Howmedica, Jaquet, Geneva, Switzerland) was fixed to the
anterolateral aspect of the left femur with four half-pins
(Fig. 1). Under aseptic conditions with general anaesthesia
produced by the intraperitoneal injection of ketamine
(90 mg/kg) and xylazine (10 mg/kg), a lateral longitudinal
skin incision was made over the femur. The fascia was cut
longitudinally and the muscles separated between quadriceps femoris and the hamstrings. The periosteum was
incised longitudinally and carefully retracted. The bone
cortex was drilled using a 1.5 mm bit and four self-tapping
screws 2 mm in diameter were placed at right angles to the
axis of the femoral shaft. An osteotomy was made between
the second and the third screws using a manual saw. The
screws were then clamped to the external fixation device,
so that the two bone ends were reduced. The periosteum
was sutured to cover the osteotomy site.
Distraction started seven days after operation at a rate of
825
0.25 mm every 12 hours (0.5 mm/day), and was stopped at
28 days after operation (21 days of distraction). Radiographs were taken each week and the animals were killed at
various stages to allow histological examination. The femoral arteries were perfused with 4% paraformaldehyde in a
phosphate buffer (pH 7.4) and the femur was fixed for 48
hours in the same solution with the external fixator still in
place. The bone was then decalcified in 20% EDTA (pH
7.4) at 4°C for about three weeks, dehydrated through
increasing concentrations of ethanol, and embedded in
paraffin. Sections of 4 m were cut and stained with
haematoxylin and eosin.
In situ hybridisation was achieved on decalcified sections
13,14
The
using digoxigenin-labelled cRNA as a probe.
cRNA probes for in situ hybridisation of mouse 1 type-I
collagen and 1 type-II collagen were given by Metsäranta
15
et al. These were used after homology between mouse
and rat cRNA had been confirmed by northern blotting.
Type-X collagen cDNA was obtained by reverse transcription of mRNA from newborn rat bone tissue, followed by a
polymerase chain reaction and subcloning into EcoRV site
of pBluescript KS- (Stratagene, La Jolla, California).
Sequencing of the obtained cDNA was confirmed by the
16
method of Sanger, Nicklen and Coulson. The base
sequence was identical to that of the rat type-X collagen
17
cDNA described previously.
RESULTS
Fig. 1
A rat with an external fixator on the left femur.
Fig. 2a
Fig. 2b
Fig. 2c
All 14 rats survived the operation and showed good tolerance of the lengthening. The external fixation devices
caused no additional injuries although several animals were
kept in one cage. The femoral lengthening was successful
at the timing and rate described above and our results were
highly reproducible and homogenous.
Radiological changes. These are shown in Figure 2. After
operation and before lengthening, immature callus formed
around the osteotomy site, but was barely visible on the
radiograph (Fig. 2a). As distraction began, bony callus
Fig. 2d
Fig. 2e
Fig. 2f
Radiographs of the lengthened femur seven days after operation, just before distraction was started (a), after ten days of distraction (b), after 21 days
of distraction when lengthening ceased (c), seven days after completion of distraction (d), 21 days after completion of distraction (e), and 21 days after
pin removal (f).
VOL. 79-B, NO. 5, SEPTEMBER 1997
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N. YASUI,
M. SATO,
T. OCHI,
Fig. 3
ET AL
Fig. 4
Figure 3 – Photomicrograph of the lengthened segment after ten days of distraction. New bone is formed between fibrous tissue and host bone by
endochondral ossification (haematoxylin and eosin 10; BC, bone cortex; BM, bone marrow; BV, blood vessels; PC, periosteal callus; EC, endosteal
callus). Figure 4 – Photomicrograph of intramembranous ossification after 20 days of distraction. New bone columns were formed directly by osteoblasts
(haematoxylin and eosin 33).
appeared and separated into proximal and distal segments
with a radiolucent growth zone between them (Fig. 2b)
which maintained a relatively constant thickness during
distraction (Fig. 2c), but after completion of distraction,
became calcified and fused (Fig. 2d). The resulting bony
callus gradually consolidated (Fig. 2e) and had remodelled
by the end of the experiment (Fig. 2f).
Histological findings. Between operation and lengthening,
the osteotomy site became surrounded by cartilaginous
external callus, and the proximal and distal bone cortices
were covered by periosteal bony callus. The inner cortex at
the osteotomy site was bridged by endosteal callus containing immature bony trabeculae. Callus formation after
osteotomy thus followed the normal course of fracture
healing.
When distraction began, the callus became elongated and
eventually separated into proximal and distal segments. The
distraction gap was filled with longitudinally-orientated
fibrous tissue and newly-formed blood vessels (Fig. 3). At
the proximal and distal ends of the fibrous tissue, this
cartilaginous callus became hypertrophic and new bone
was formed by endochondral ossification. The tissue containing hypertrophic chondrocytes was invaded by capillaries and new bone was deposited on the surface of the
eroded cartilage.
Between 10 and 20 days of distraction, cartilaginous
callus was progressively resorbed and replaced by bone.
After this new bone was formed more directly by intramembranous ossification (Fig. 4). New bone trabeculae
formed longitudinally along the tension vector at the proximal and distal ends of the fibrous growth zone. Osteoblasts, preosteoblasts and fibroblast-like cells were
arranged longitudinally in order of their stage of differentiation. Cell proliferation appeared at the tip of new bone
columns; the latter are assumed to grow longitudinally so
that the fibrous growth zone retained a relatively constant
thickness during progressive distraction.
Between endochondral and intramembranous ossification
we saw a third mechanism by which new bone was formed
directly by chondrocyte-like cells. ‘Chondroid bone’, a
18
tissue intermediate between cartilage and bone, was
observed at the boundary between fibrous tissue and new
Fig. 5
Transchondroid bone formation after 20 days of distraction (haematoxylin and eosin55; F, fibrous tissue; CB,
chondroid bone; B, bone).
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THREE MODES OF OSSIFICATION DURING DISTRACTION OSTEOGENESIS IN THE RAT
Fig. 6a
Fig. 6b
Fig. 6c
Fig. 6d
827
Sequential sections showing endochondral ossification. Figure 6a – Haematoxylin and eosin66 (CH, chondrocytes; OB, osteoblasts; OC, osteocytes).
Figures 6b to 6d – In situ hybridisation using cRNA probes of 1 type-I collagen (b), 1 type-II collagen (c) and 1 type-X collagen (d).
bone. The matrix of this chondroid bone is more like bone
than cartilage, but the cells were indistinguishable from
chondrocytes (Fig. 5). Round chondrocyte-like cells and
smaller osteocyte-like cells coexisted forming columnar
arrangements in the chondroid bone. Transition from
fibrous tissue to bone via chondroid bone occurred gradually and consecutively. Three-dimensional observation of
the chondroid bone in the sequential histological sections
showed no capillary invasion during the transition from
cartilage to bone. This ‘transchondroid bone formation’
was clearly different from endochondral ossification in
which the cartilage is invaded by capillaries and new bone
is deposited on the surface of eroded cartilage.
In some regions all three modes of ossification overlapped; during regular histological examinations, transchondroid bone formation was mistaken for either
endochondral bone formation or direct bone formation.
In situ hybridisation. To determine the collagen phenotypes of the cells involved in the three different modes of
ossification, we used in situ hybridisation achieved with
VOL. 79-B, NO. 5, SEPTEMBER 1997
non-isotopic cRNA probes for type-I, type-II and type-X
collagens. Type-I collagen is a main component of connective tissues, including skin, tendon and bone, while
type-II collagen is cartilage-specific. Type-X collagen is
known to be synthesised by the hypertrophic chondrocytes
of growth cartilage and to have an important role in
19
provisional calcification.
During endochondral ossification (Figs 6a to 6d), a
positive signal for 1 type-I collagen mRNA was detected
in osteoblasts surrounding new bone trabeculae (Fig. 6b).
The same signal was also detected in chondrocytes and
elongated fibroblast-like cells, but not in osteocytes (Fig.
6b). The signal of 1 type-II collagen mRNA was detected
exclusively in differentiated chondrocytes (Fig. 6c), while
hypertrophic chondrocytes expressed 1 type-X collagen
mRNA (Fig. 6d).
During intramembranous ossification (Figs 7a to 7d), the
positive signal of 1 type-I collagen mRNA was detected
in osteoblast, preosteoblast and fibroblast-like cells, but not
in osteocytes (Fig. 7b). Neither 1 type-II collagen mRNA
828
N. YASUI,
M. SATO,
T. OCHI,
ET AL
Fig. 7a
Fig. 7b
Fig. 7c
Fig. 7d
Sequential sections showing intramembranous ossification. Figure 7a – Haematoxylin and eosin66 (OB, osteoblasts; OC, osteocytes). Figures 7b to
7d – In situ hybridisation using cRNA probes of 1 type-I collagen (b), 1 type-II collagen (c) and 1 type-X collagen (d).
(Fig. 7c) nor 1 type-X mRNA (Fig. 7d) was detected in
cells involved in intramembranous ossification.
During transchondroid bone formation (Figs 8a to 8d),
we found a positive signal for 1 type-I collagen mRNA in
fibroblast-like cells, chondrocyte-like cells and young
osteocyte-like cells (Fig. 8b). The well-differentiated osteocytes embedded in dense bone matrix did not show this
signal. The signal for 1 type-II mRNA was detected not
only in chondrocyte-like cells but also in some which did
not have the histological appearance of chondrocytes. A
few cells expressed type-I and type-II collagens simultaneously, suggesting that they were switching collagen phenotypes. A positive signal for 1 type-X collagen mRNA was
occasionally detected in some chondrocyte-like cells (Fig.
8d).
DISCUSSION
We have described a surgical technique for lengthening
small bones using a monolateral external fixation device to
study the cellular and molecular mechanism of distraction
osteogenesis. Our radiological and histological examinations consistently showed a growth zone of constant thickness in the middle of the lengthening segment, with new
bone forming at its proximal and distal ends. A single
histological section showed the origin and the fate of
osteogenic cells because the differentiating cells from a
single lineage were arranged longitudinally along the tension vector in order of maturity. Our model is clearly
different from other experimental models of fracture healing in which the cells at various stages of differentiation are
distributed at random.
Ossification is usually classified into two types: intra20
membranous (direct) and endochondral (indirect). Intramembranous ossification is typically seen during
embryonic development of the cranial vault, but at numerous other sites where there is no pre-existent cartilage
model, new bone is directly formed by differentiated osteoblasts. Typical endochondral ossification is seen during
embryonic development of long bones. New bone formaTHE JOURNAL OF BONE AND JOINT SURGERY
THREE MODES OF OSSIFICATION DURING DISTRACTION OSTEOGENESIS IN THE RAT
Fig. 8a
Fig. 8b
Fig. 8c
Fig. 8d
829
Sequential sections showing transchondroid bone formation. Figure 8a– Haematoxylin and eosin66 (CH, chondrocyte-like cells; OC, osteocytes).
Figures 8b to 8d – In situ hybridisation using cRNA probes of 1 type-I collagen (b), 1 type-II collagen (c) and 1 type-X collagen (d).
tion may be regarded as endochondral when cartilage is
formed first and later replaced by new bone. In the physeal
growth plate, for example, a highly ordered structure of
resting, proliferating, hypertrophic, and calcifying cartilage
is first established by differentiating chondrocytes. The
calcified cartilage matrix is then invaded by capillaries and
new bone is laid down by osteoblasts in the space previously occupied by hypertrophic chondrocytes. The fate of
hypertrophic chondrocytes in calcifying cartilage remains
controversial. Some authors consider that all the cells
21
22
degenerate or are programmed to die, but others believe
that they survive to become osteoblasts under the influence
23,24
of vascularisation.
During distraction osteogenesis in our rat model, endochondral ossification was predominant in the early stage of
distraction especially in the circumferential region where
cartilaginous callus had been formed during the seven days
between osteotomy and distraction. This cartilaginous callus appeared to grow to some extent in the initial stage of
distraction, but was eventually totally replaced by bone,
VOL. 79-B, NO. 5, SEPTEMBER 1997
leaving intramembranous ossification as the main mechanism of new bone formation. In addition to various growth
factors and cytokines released from broken bone matrix,
appropriate strain may contribute to the stimulation of bone
formation during the distraction phase.
The third ossification mechanism produces chondroid
bone. This has not attracted much attention, although it
was identified as an intermediate tissue between cartilage
18
and bone some time ago. More recently, it has been
suggested that some hypertrophic chondrocytes undergo
further differentiation into osteoblast-like cells and partici25-28
If this is the case, a
pate in the initial bone formation.
compound tissue of bone and cartilage should be produced
unless chondrocytes turn into osteoblasts very rapidly. We
found that chondrocyte-like cells and osteocyte-like cells
coexisted in chondroid bone with no clearly distinguishable boundary. The columnar arrangement of these cells
suggested that they were derived from a common mesenchymal cell, and careful observation of sequential histological sections confirmed the gradual transition from
830
N. YASUI,
M. SATO,
cartilage to bone via chondroid bone.
In situ hybridisation using cRNA probes for type-I, typeII and type-X collagens showed that the cells of chondroid
bone temporarily expressed type-II collagen mRNA. These
cells did not necessarily look like chondrocytes in standard
histological sections, but were considered to be young
chondrocytes undergoing further differentiation into boneforming cells. Chondrocytes changing their collagen phenotypes are known to express both type-II and type-I
29,30
collagen.
In our model, young osteocytes expressed
type-I collagen mRNA, but small osteocytes embedded in
dense bone matrix did not express any type of collagen
mRNA, which suggests that they were in the terminal
differentiation stage. Although there is no direct evidence
yet, we consider that the cells involved in transchondroid
bone formation temporarily express cartilage phenotypes
and then turn directly into osteocytes which survive in the
chondroid bone until the tissue is resorbed and remodelled
by true bone. Chondroid bone then temporarily provides
mechanical strength for the callus.
Our rat model of femoral lengthening is a promising tool
for studying distraction osteogenesis because the techniques of molecular biology are applicable to this animal.
In situ hybridisation of non-isotopic cRNA in decalcified
mineralised sections has provided high-resolution results
which allow detection of phenotypic expression of individual cells. We are now using probes other than collagen
mRNAs to investigate further the molecular mechanism of
distraction osteogenesis.
We thank Dr Metsäranta for providing the cRNAs of 1 type-I collagen
and 1 type-II collagen, and Mr A. Fukuyama and Mr K. Morihana for
their help with the preparation of the histological sections.
No benefits in any form have been received or will be received from a
commercial party related directly or indirectly to the subject of this
article.
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