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Transmission of an osteogenic message through intact bone after wounding.

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Transmission of a n “Osteogenic Message” through
Intact Bone after Wounding ’
A. H. MELCHER AND G . E ACCURSI
Faculty of Dentistry, Univemity of Toronto, 124 Edward Street,
Toronto 101, Ontario, Canadti
ABSTRACT
Wounds
1 mm wide were made in the middle third of the
500 gm.
spine in both scapulae of 25 middle-aged Wistar-strain rats weighing
Wounding of the body of the scapula was ;.voided. Some of the animals were
injected with tetracycline, and sections from their scapulae were viewed in ultraviolet light. After one or more weeks of healing, bony callus was deposited on
the dorsal surface of the blade of the scapulae in the vicinity of the wounds.
Discrete deposits of bony callus were also deposited on the corresponding costal
surface of the blade of the scapulae. These deposits, which were i n no way connected with those on the dorsal surface, did not exhibit continuity with the
wound in the spine. This observation has ‘3een interpreted as suggesting the
passage of an “osteogenic message,” whose nature can only be guessed, from
the vicinity of the wound on the dorsal surfaze of the scapula to the corresponding costal surface. Lacunae of osteocytes in the scapulae, lying deep to periosteal
and endosteal callus, fluoresced. This suggests that these osteocytes were activated by the trauma, and deposited mineral salts on the walls of their lacunae.
Differentiation to osteoblasts of precursor cells in periosteum covering mature
hone can be initiated by damage to adjacent periosteum (Haldeman, ’32; Melcher, ’69, ’71), to underlying bone
(‘Kistler, ’34), or to both (Melcher and
Accursi, ’71). Although the precise nature
of the stimulus that leads to differentiation
of osteoblasts after wounding is unknown,
it is conceivable that trauma to bone can
result in transmission of an “osteogenic
message” to progenitor cells at a distance
from the wound. The hypothesis that an
“osteogenic message” can be transmitted
through bone was tested in the present
jnvestigation by attempting to elicit an
osteogenic response on the costal surface
of a rat scapula after wounding the spine
on the dorsal surface of the bone.
-
distance of
4 mm, so as to expose the
spine to its junction with the blade of the
scapula. The soft tissues were retracted
slightly; just sufficient to allow a cut to
be made with a number 57 dental bur
through the width of the spine (fig. 1).
Care was taken to restrict to a minimum
damage to the periosteum covering the
blade of the scapula, and not to penetrate
the blade with the bur. In the few instances
wkere the blade of the scapula was damaged, the bone was not included in the
experiment. The bur, which was rotated at
high speed in a turbine-driven dental handpicce and cooled with water, made a cut
1 mm wide. The muscles were then
clc sed with resorbable sutures and the skin
W E S apposed with clips. Five rats were
killed at each of 3, 5, 7, 14 and 21 days.
Ori every alternate day from the third day
MATERIALS AND METHODS
afier operation, two animals from each
Twenty-five middle-aged Wistar rats tine-period received an intramuscular inweighing
500 gm and aged at least two jection of 37.5 mg tetracycline HCI con.years, were used in the experiments. Under taning 2% (w/v) procaine HCl buffered
fitraperitoneal nembutal anaesthesia, bi- with ascorbic acid.
lateral incisions were made in the skin
Iteceived Nov. 1, ‘71. Accepted Feb. 28, ‘72.
over the spines of both scapulae. Muscle
1 This investigation was supported by grant MAwas separated from the cephalic aspect of 38(13 to A.H.M. and by a n Undergraduate Summer
StLdent Scholarship to G.E.A., both awarded by the
the middle one-third of the spine over a Medical Research Council of Canada.
+
-
~
ANAT. REC., 173: 265-276.
265
266
A. H. MELCHER AND G . E. ACCURSI
RIGHT SCAPULA
View from Superior border
ACROMIAL
PROCESS
SPINE
GLENOID CAVITY
WOUND
BLADE
A
REACT 10 N
CORONOID PROCESS
COSTAL SURFACE
Fig. 1 A diagrammatic representation of the experimental procedure.
The animals were killed by perfusion
with phosphate-buffered formalin, pH 7.4,
while anaesthetized with nembutal. The
scapulae, with surrounding muscle intact,
were excised from the animals, and fixed
for a further 48 hours in the same fixative.
Scapulae from animals that had not received tetracycline were demineralized in
an aqueous solution comprising equal parts
of 45% formic acid and 20% sodium citrate. They were then processed for paraffin sections. Serial sections were cut transversely through each scapula in the region
of the wound so that all of the bony callus
that was deposited during repair was included in the series. All the sections were
stained with haematoxylin and eosin.
Scapulae from animals that had received
tetracycline were not demineralized. They
were dehydrated in an ascending series of
ethanol and then cleared in Bioplastic
Resin without catalyst (Ward’s Natural
Science Establishment Inc., Rochester,
N.Y., U . S . A . ) . Following clearing, the specimens were embedded in complete Bioplastic, and sectioned serially at about 80 p
through the area of the wound. The sections, which were cut with a motor-driven
diamond wheel in the same plane as the
paraffin sections, were then mounted on
slides using Canada Balsam, and examined
immediately, using ultra-violet light.
RESULTS
As has been described widely to occur
following trauma to other bones, a deposit
of periosteal bony callus was found to have
been laid down postoperatively in the vi-
cinity of the wound. This callus was deposited on the spine and dorsal surface
of the blade of the scapulae adjacent to
the wounds (figs. 2, 3 ) . Study of the serial
sections revealed that bony callus deposited
on the spine or dorsal surface of the blade
never extended to the periphery of the bone.
A deposit of subperiosteal callus was
found on the costal surface of the scapulae
of all animals that had healed for at least
seven days (figs. 2, 3 ) . This deposit lay opposite the wound, and followed the distribution of the callus on the dorsal surface
of the blade. However, it usually covered a
smaller area and was thinner than the
deposit on the dorsal surface (figs. 2, 3 , 4 ) .
Study of the serial sections showed that
the callus on the costal surface never
reached the periphery of the blade of the
scapula, nor did it ever become co-extensive
with the callus on the dorsal surface of the
blade (fig. 4). The serial sections also
showed that the callus on the costal surface
of the blade was separated from the wound
in the spine by the intact bone of the blade
of the scapula (fig. 2). Deposits of endosteal callus were also found in the medullary cavity that occupies a part of the junction between the spine and blade of the
scapula (fig. 5). Twenty-one days after
operation the bony callus that had been
laid down in the repair process had been
remodelled to form a compact deposit
(figs. 6, 7, 8).
Confirmation of the belief that the callus
on the costal surface was deposited during
the experimental period was obtained from
the scapulae of animals that had received
“OSTEOGENIC MESSAGE” Ih WOUNDED BONE
tetracycline (figs. 9, 10, 1 1 ) . It was evident from observations on some of the five
day specimens that callus deposited in the
medullary cavity antedated that deposited
on the costal surface (figs. 12, 13). Osteocytes in the old bone of the scapula lying
deep to deposits of both periosteal and
endosteal callus in animals that had received tetracycline were found to fluoresce;
by contrast, the osteocyte lacunae in the
newly deposited callus did not (figs. 9,
LO, 11 ). This observation could be made on
lacunae lying deep to endosteal callus deposited on the walls of the medullary cavity
as early as five days after operation. However, no evidence was obtained to suggest
bhat the osteocyte lacunae became fluorescent earlier than the callus. Fluorescent
endosteal callus was also found on the
walls of channels in the scapulae in the
vicinity of both the wound and the deposits
of callus (figs. 9, 10, 11). These channels
were believed to transmit blood vessels.
DISCUSSION
267
was a necessary prerequisite if it is to be
certain that the “message” that stimulated
osleogenesis at the remote site had passed
thi*ough the bone and had not arisen as a
result of damage to adjacent periosteum.
Axiomatically, the wound and the site predicted for the remote periosteal reaction
could not be so far separated that it would
be unrealistic to expect an “osteogenic
mc ssage” to traverse the distance.
[t is believed that the experimental systern used in the present investigation met
these conditions. Firstly, the experimental
animals were “middle-aged,” and the majority of the cells in the periosteum covering: their scapulae were of flattened appearanze when viewed in the light microscope,
suggesting that they were not active
osieoblasts. Secondly, a wound could be
made in the spine of the scapula through
a !;mall incision, ensuring a minimal disruption of both the surrounding soft tissue
and the bone of the blade, and no interference with the costal surface of the bone
wl-ere the remote reaction was being
sought. Finally, the scapula, having a thin
bl:de, allowed the site of wounding on the
dorsal surface and the site of anticipated
reaction on the costal surface to be located
clcse to one another, while still being
separated by intact bone.
Certain stringent conditions had to be
fulfilled in the design of this experiment:
( a ) Tonna (’65) has shown that, following a fracture, undifferentiated cells of
{he periosteum of femurs in mature mice
must divide before differentiating into
osteoblasts; in young mice the precursor
cells can differentiate directly. Thus, Cause o f osteogenesis on the costal surface
In animals that had healed for at least
periosteum covering a bone in a young,
growing animal, provides an on-going 7 days, isolated deposits of bony callus
osteogenic system, whereas the cells in were consistently found on the costal surperiosteum covering a mature bone must face of the scapulae, and there were no
divide and differentiate de novo before be- apparent connections between these decoming osteogenic. This means that mature posits and the wound in the spine, or beanimals must be used in an investigation, tween these deposits and the periosteal
!juch as the one that is here being described, calus on the dorsal surface of the bone.
where the capacity of cells of the perios- A1,hough the bony callus deposited on the
i:eum to differentiate into osteoblasts after coistal surface was always separated from
.wounding is being examined. If young ani- thi: wound and the callus on the dorsal
mals were used, there could be no cer- surface by a layer of intact bone, it is not
tainty that any osteogenic reaction that was clc ar precisely what provoked the osteofound had not occurred during the normal genic response of the periosteal cells on
the costal surface. A number of possibilities
course of growth.
( b ) The site remote from the wound, in may be considered.
It is evident that muscular pull on the
which the periosteal reaction was expected
to occur in response to the distant trauma, wounded scapula could have resulted in
had to be so located that it could be iso- dicitortion of the bone, and this may have
lated from the direct action of the experi- led to early remodelling of its costal surmental trauma inflicted on the bone, its face. There is also the possibility that alperiosteum and surrounding tissues. This though precautions were taken to avoid
268
A. H. MELCHER AND G . E. ACCURSI
generation of heat during cutting of the
wound in the spine, sufficient heat may
have been produced to damage the bone
in the underlying blade. If this did happen,
repair of the thermally-traumatized areas
of the blade may have involved osteogenesis
by cells present on the costal surface.
Finally, wounding of the spine of the
scapula may itself have been sufficient to
stimulate the osteogenic response by the
cells of the cambium layer of the periosteum covering the costal surface of the
bone.
If distortion of the scapula through muscular pull did occur, it could have stressed
the bone cells directly, which conceivably
could have had the effect of inducing cells
on bone surfaces to differentiate into osteoblasts. Alternatively, distortion of the
scapula could have generated piezoelectric
stimuli; these stimuli may have induced
precursor cells to differentiate into osteoblasts (see, for example Bassett, ’68). However, as far as is known, there is no evidence that either of these stimuli are the
direct cause of differentiation of osteoblasts. That some form of “osteogenic
message” could have been transmitted from
the site of the wound in the spine( and
the site of thermal trauma if it occurred),
to the cells of the cambium layer of the
periosteum covering the costal surface of
the bone, is another possibility that may
be considered. The finding in the present
experiment, that osteocyte lacunae in the
wounded scapula in the vicinity of deposits
of bony callus exhibited fluorescence, provides some evidence to support the idea
that there may be communication between
bone cells. The fluorescent lacunae were
found not only adjacent to callus deposited
on the costal surface, but also adjacent to
that deposited on the walls of the medullary
cavity and on the dorsal surface adjacent
to the wound. It seems unlikely that either
distortion of the scapula or thermal trauma
would produce so regular a distribution of
fluorescent osteocyte lacunae and, in particular, reactive osteocyte lacunae in the
vicinity of the callus adjacent to the wound.
Irrespective of how a wound is made, an
osteogenic reaction by cells of undamaged
periosteum in its vicinity occurs almost
invariably. It is consequently attractive to
consider the possibility that the deposition
of fluorescent material on the walls of
osteocyte lacunae in the vicinity of callus
deposited near the wound was the result of
transmission of some form of “osteogenic
message” between the bone cells. Further
support for the belief that some form of
“message” may have passed from the dorsal
to the costal surface of the bone is contained in the finding in some early specimens that, prior to formation of periosteal
callus on the costal surface, endosteal callus had been deposited on the medullary
cavity which was located between the two
surfaces.
Transmission and nature of an
“osteogenic message”
The routes through which a presumptive
“osteogenic message” could pass most
likely involve the osteocytes or their canalicular system. It is conceivable that debris
from dead or dying osteocytes, or products
of osteocytes, could be transported through
the canaliculi from lacuna to lacuna, and
so influence the osteocytes that occupy
them and the periosteal cells that cover
the bone surface. It is germane to this
suggestion that Arnold et al. (’71) have
recently postulated that osteocytes can act
as bone pumps. An alternative possibility
is suggested by the knowledge that some
cells are electrotonically coupled by gapjunctions (see, for exampIe, Brightman and
Reese, ’69; Payton et al., ’69; Goodenough
and Revel, ’ i ’ O ) , and that Holtrop and
Weinger (’72) have shown in calvaria of
seven day-old mice the presence of “tight
junctions” between osteocytes and between
osteocytes and osteoblasts on the bone surfaces, and have proposed that the osteocytes provide a transport system within
bone. Although, as far as is known, there
is as yet no information on whether the
latter situation pertains in mature bone,
these observations do suggest the possibility that an “osteogenic message” could
be relayed from one bone cell to the next.
In relation to the experiment being described here, it is therefore reasonable to
suppose that the osteocytes in the body of
the scapula could have relayed an “osteogenic message” from the wound in the
spine to the cells in the cambium layer of
269
"Ub 1 b U b B N l C 1VlLbbAbL
the periosteum on the costal surface. Electron microscopic studies on this experimental system are currently being undertaken in an endeavour to detect cytological
changes in bone cells that could indicate
their participation in this way, or that may
a1ternatively indicate that the observed reaction was brought about by thermal
trauma or some other mechanism.
A final question that may be posed concerns the nature of an "osteogenic message"
that could bring about differentiation of
periosteal cells into osteoblasts. It has been
mentioned above that Tonna ('65) has
shown that periosteal cells of mature mice
must divide before differentiating into
osteoblasts in repair of a fracture of a
mature bone. Consequently, a significant
event after wounding of a mature bone is
the division of progenitor cells. It is well
kinown that contact between cells will inhibit their division (see Trinkaus, '69),
aind it has recently been found that a confluent strain of fibroblasts maintained in
v,;tro releases a diffusible factor that sustains the state of contact inhibition of
replication (Yeh and Fisher, '69; Hodgson
and Fisher, '71 ). Similarly, Bullough and
Laurence ('64) have described a diffusible
substance that they call a chalone, that is
produced by, and that inhibits division of
epidermal cells. They have proposed that
wounding of epidermis results in reduced
production of the inhibitor, and that this
alllows division of intact cells in the vicinity. Chalones have also been described
to occur in other tissues (for example, see
Leeson and Voaden, '70; Voaden and Leeson, '70; Houck, Irausquin and Leiken, '71;
Editorial, '71). It is conceivable that an
inhibitor of DNA replication and mitosis
is produced by bone cells, and transported
by osteocytes and periosteal cells to other
bone cells in the vicinity. Death of bone
cells could lead to reduced production of
the inhibitor, and this would allow mitotic
activity in local endosteal and periosteal
cells. Alternatively, the possibility that
damaged cells could produce a substance
that is transported by osteocytes or pumped
through their lacunar system, which stimulaites periosteal and endosteal cells to divide, must also be contemplated.
Participation of osteocytes
in repair of bone
7he finding that fluorescence was exhibited by the walls of osteocyte lacunae
locz ted in the old bone of the scapulae deep
to deposits of bony callus supports the
be1i:f that osteocytes can deposit mineral
(set:, for example, Baud, '68; Vitalli, '68;
Jande and Belanger, '71). It also suggests
thai osteocytes can participate in repair
of bone.
ACKNOWLEDGMENTS
Vie are indebted to Mrs. Wilma Hiddlestort and to Mrs. Shirley Reimers for technical assistance, and to Dr. M. E. Holtrop
for permitting us to read her paper in
maiiuscript.
LITERATURE CITED
Arnold, J. S., H. M. Frost and R. 0. Buss 1971
Clin. Orthopaed., 78: 47-55.
Basrett, C. A. L. 1968 Biologic significance of
piezoelectricity. Calcif. Tissue Res., 1: 252-272.
Baui, C. A. 1968 Submicroscopic structure and
ftnctional aspects of the osteocyte. Clin.
0 ethop., 56: 227-236.
Brightman, M. W., and T. S. Reese 1969 Junctions between intimately apposed cell membi anes in the vertebrate brain. J. Cell Biol., 40:
6L 8-677.
Bullough, W. S., and E. B. Laurence 1964 Mitotic control by internal secretion: The role of
the chalone-adrenalin complex. Expert. Cell
R~:s., 33: 176-194.
Editorial 1971 How is the chalone story ageing?
Nature (New Biol.) 231: 162.
Goolienough, D. A., and J. P. Revel 1970 A fine
st cuctural analysis of intercellular junctions in
the mouse liver. J. Cell Biol., 45: 27S290.
Haltleman, K. 0. 1932 The role of periosteum
in the healing of fractures: a n experimental
stJdy. Arch. Surg. (Lond.), 24: 4 4 0 4 5 0 .
Hod:son, J. R., and H. W. Fisher 1971 Formation of polyribosomes during recovery from contact inhibition of replication. J. Cell Biol., 49:
9L 5-947.
Holtrop, M. E., and J. M. Weinger 1972 Ultrastructural evidence for a transport system in
bc ne. In: Calcium, Parathyroid Hormone and
the Calcitonins. R. V. Talmage and P. L. Munson, eds. Excerpta Medica, Amsterdam, pp. 3653; 4.
Hou-k, J. C . , H. Irausquin and S. Leikin 1971
Lrmphocyte DNA synthesis inhibitlon. Science,
1:'3: 1139-1141.
Jantle, S. S., and L. F. Belanger 1971 Electron
microscopy of osteocytes and the pericellular
matrix in r a t trabecular bone. Calcif. Tissue
R:s., 6: 280-289.
Kist er, G. H. 1934 Formation of bone by periorteum after experimental infarction by embolism of the femur in rabbits. Proc. SOC.Exp.
B 01. Med., 31: 1218-1220.
270
Leeson, S.
lone in
fects of
division
A. H. MELCHER AND G . E. ACCURSI
J., and Mary J. Voaden 1970 A chathe mammalian lens: 11. Relative efadrenaline and noradrenaline on cell
in the rabbit lens. Exp. Eye Res., 9:
67-72.
Melcher, A. H. 1969 Role of the periosteum in
repair of wounds of the parietal bone of the
rat. Arch. Oral Biol., 14: 1101-1109.
1971 Wound healing in monkey (Macaca Irus) mandible: Effect of elevating periosteum on formation of subperiosteal callus.
Arch. Oral Biol., 16: 461464.
Melcher, A. H., and G . E. Accursi 1971 Osteogenic capacity of periosteal and osteoperiosteal
flaps elevated from the parietal bone of the rat.
Arch. Oral Biol., 16: 573-580.
Payton, B. W., M. V. L. Bennett and G. D. Pappas
1969 Permeability and structure of junctional
membranes at a n electrotonic synapse. Science,
166: 1641-1643.
Tonna, E. A. 1965 Protein synthesis and cells
of the skeletal system. In: The Use of Radioautography in Investigating Protein Synthesis.
C. P:Leblond and K. B. Warren, eds. Academic
Press, New York, pp. 215-245.
Trinkaus, J. P. 1969 Cells into organs. The
forces that shape the embryo. Prentice-Hall,
Inc., Englewood Cliffs, N. J., p. 209.
Vitalli, P. H. 1968 Osteocyte activity. Clin.
Orthop., 56: 213-226.
Voaden, M. J., and S. J. Leeson 1970 A chalone
i n the mammalian lens. I. Effect of bilateral
adrenalectomy on the mitotic activity of the
adult mouse lens. Exp. Eye Res., 9: 57-66.
Yeh, J., and H. W. Fisher 1969 A diffusible
factor which sustains contact inhibition of replication. J. Cell Biol., 40: 382-388.
PLATE I
EXPLANATION O F FIGURES
2
A transverse section of a scapula cut through the wound one week
postoperatively. S , spine; W, wound; B, blade; M, medullary cavity;
D, periosteal callus on dorsal aspect of blade and on spine; C, periosteal callus on costal surface of blade. Haematoxylin & eosin. x80.
3
Area of the section illustrated in figure 2 and marked by box. S, spine;
D, periosteal callus o n dorsal aspect of blade; C, periosteal callus o n
costal aspect of blade. Haematoxylin & eosin. x 330.
4
A more peripheral part of the blade of the scapula from the same
section as that illustrated in figures 2 and 3. D, periosteal callus on
dorsal aspect of blade. No callus has been deposited on the costal
surface of this part of the blade (+). Haematoxylin & eosin. ~ 3 3 0 .
5
Periosteal callus deposited on the spine ( D ) and on the costal surface
of the blade ( C ) , and endosteal callus ( E ) deposited on the wall of
the medullary cavity of a scapula one week after wounding. Haematoxylin & eosin. x330.
“OSTEOGENIC MESSAGE” I N WOUNDED BONE
A. I€. Melcher and G. E. Accursi
PLATE 1
PLATE 2
EXPLANATION O F FIGURES
6
A transverse section of a scapula cut through the wound three weeks
postoperatively. S , spine; W, wound; B, blade; M, medullary cavity.
Haematoxylin & eosin. x 90.
7
A n area of the section illustrated in figure 6 . D, periosteal callus de-,
posited on spine. Haematoxylin & eosin. ~ 7 5 0 .
8 An area of the section illustrated i n figure 6. C, periosteal callus deposited on costal surface of blade ( B ) . Haematoxylin & eosin. x 1,000.
272
“OSTEOGENIC M E S S A G E IN WOUNDED BONE
A. H. Melcher and 6 . E. Accursi
PLATE 2
273
PLATE 3
Transverse sections of three different scapulae cut through the
wound three weeks aPter operation. The animals had received tetracycline, and the sections were photographed i n ultraviolet light.
Fluorescent areas appear white. S, spine; W, wound; B, blade;
M, medullary cavity; D, periosteal callus on dorsal aspect of blade
and on spine; C, periosteal callus on costal surface of blade; E,
endosteal callus on wall of medullary cavity; (+) fluorescent osteocyte lacunae in old bone; (-/-/+) non-fluorescent osteocyte lacunae i n new bony callus: w, fluorescent walls of channels in the
scapulae. Figure 9, X 150; figure 10, X 330; figure 11, X 330.
Two photomicrographs of the same transverse section cut through
a wound in a scapula five days after operation. The animal had received tetracycline. Figure 12 was photographed in white-light and
figure 13 i n ultraviolet light. W, wound; M, medullary cavity;
(3)
costal surface of scapula; E, endosteal callus on wall of medullary cavity. Osteocyte lacunae i n the bone of the scarula deep to
the endosteal callus were fluorescent, but insufficiently bright to
be photographed. Figure 12, W 225; figure 13, X 750.
9-11
12-13
EXPLANATION O F FIGURES
A. H. 1\.
. and G . E. A~cu;::
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