Calcif Tissue lnt (1985) 37:431-436
Calcified Tissue
Mechanical Stretching Increases the Number of Cultured Bone Cells
Synthesizing DNA and Alters Their Pattern of Protein Synthesis
Shin Hasegawa, 1 S. Sato, ~ S. Saito, 1 Y. Suzuki, 1 and D. M. Brunette 2
IKanagawa Dental College, Yokosuka, Japan; and 2Faculty of Dentistry, University of British Columbia, Vancouver, Canada
Summary. A simple m e t h o d was devised for ap-
plying mechanical stretching to bone cell cultures.
Bone cells cultured on the flexible plastic mem-
brane of a Petriperm dish are placed over a template
with a convex surface. A lead weight is then placed
on top of the dish which causes the membrane and
the tightly attached cells to be stretched. Mechan-
ical stretching, applied either intermittently or con-
tinuously for a 2-hour period resulted in a 64% in-
crease in the number of cells synthesizing DNA.
Stretching the cells also significantly increased in-
corporation of tritiated proline and tritiated leucine.
To assay the ratio of collagenous to noncollagenous
protein, medium and cell layers of cultures labeled
with tritiated leucine were i n c u b a t e d with colla-
genase and the digests chromatographed on PD 10
columns. The amount of collagen synthesized by
stretched and unstretched cultures did not differ;
but an increased synthesis of noncollagenous pro-
teins was observed in the stretched cultures.
Key words: Mechanical stretching - - Bone remod-
eling - - PGE? - - D N A - - Metalloproteinases
Although mechanical stress has long been recog-
nized as an important factor in the regulation of
bone remodeling, the mechanism underlying these
effects has remained obscure. A number of methods
[1-4] have been devised to apply forces to bone, or
cells derived from bone or periosteum, in order to
investigate the biochemical results of mechanical
Send reprint requests to Dr. D. M. Brunette, The University of
British Columbia, Dept. of Oral Biology, Faculty of Dentistry,
2199 Westbrook Mall, Vancouver, B.C., Canada.
International
9 1985 by Springer-Verlag
stimuli. The effects of mechanical stretching on
cells include increased synthesis of PGE 2 [2], me-
talloproteinases [5], and D N A [1]. In addition, tran-
sient increases in cyclic AMP production and C a 2+
accumulation have been reported [2]. In this pa-
per, we r e p o r t a simple m e t h o d for m e c h a n i -
cally stretching cultured bone cells. M e c h a n i c a l
stretching was found to increase the number of cells
synthesizing D N A and to alter the pattern of pro-
teins synthesized.
Materials and Methods
Bone cells were isolated by a modification of the method of
Wong and Cohn [6]. Calvariae were removed aseptically from
3-5-day-old rats and, as far as possible, stripped of their over-
lying periosteum. The calvariae were minced into pieces of ap-
proximately 2 mm per side, placed into a 5 ml vial (Reactivial-
Pierce Chemical Co., Rodsford IL) and incubated at 37~ with
stirring, in a digestion mixture comprised of 180 U/ml clostridial
collagenase (type 1, Sigma, St. Louis, MO) and 0.05% (w/v)
trypsin (Worthington Diagnostic Systems, Freehold, NJ) dis-
solved in phosphate-buffered saline (PBS) (GIBCO Grand Is-
land, NY). The digestion mixture with released cells was with-
drawn every 20 min to yield six sequentially derived cell popu-
lations. After harvesting, the digestion mixture and cells were
mixed with an equal volume of fetal bovine serum and centri-
fuged at 1000 rpm for 5 rain at room temperature and then re-
suspended in culture medium for plating into 75 cm 2 culture
flasks (Falcon, Div. of Becton, Dickinson and Co., Cockeysville,
MD). The culture medium was c~ MEM (GIBCO) supplemented
with 10% fetal bovine serum (Flow, Cockeysville, MD) and an-
tibiotics 100 p~g/ml penicillin and streptomycin (Sigma, St.
Louis, MO). All cultures were incubated at 37~ in a humidified
atmosphere of 95% air, 5% CO 2. To subculture the primary bone
cell cultures, the medium was decanted and the cells washed
twice with phosphate-buffered saline. Then 10 ml of 0.25% (w/
v) trypsin (Difco, Detroit, Michigan) 1:250 was added and the
cells incubated at 37~ until the majority of the cells had rounded
432 S. H a s e g a w a et al.: Effects of Mechanical Stretching on Cultured Bone Cells

up and detached from the flask. Ten milliliters o f culture m e d i u m

was added and the cell s u s p e n s i o n centrifuged at 1000 rpm for 5
rain. The supernatant was discarded and the pellet r e s u s p e n d e d
in 20 ml of culture m e d i u m which gave a concentration of - 1 . 5
105 cells/ml. Five milliliter of this s u s p e n s i o n was plated into
each 50 m m culture dish which had a flexible plastic growth
surface (Petriperm, Tekmar, Cincinnati, OH).

Application of Mechanical Stretching

The principle of the stretching apparatus is similar to thai of

Harrell et al. [2] in that the physical force is applied to deform
the culture dish to which the cells are firmly attached. Unlike
the apparatus of Harrell et al., however, the deformation of the
dish is not irreversible b e c a u s e the bottom of the Petriperm dish
is elastic. T h u s it is possible to alternate periods of mechanical I1[.
stretching with periods w h e n no stress is applied. A second dif-
ference is the way the dish is stretched. Harrell et al. employed
an orthodontic screw which primarily stretched the dish along
only one axis. As s h o w n in Fig. I, which illustrates our method,
the bottom o f the dish is stretched by placing a template with a
c o n v e x surface u n d e r n e a t h the dish and placing a lead weight on
the top. Twenty dishes can be stretched at the same time by
placing a 40 cm long, 26 cm wide, 0.55 cm thick, plexiglass sheet
over the dishes and then putting two 6 kg weights on the plexi-
glass. To stretch a single dish, two "'lead d o n u t s " (I2R, Chel-
Fig. 1. A s c h e m a t i c r e p r e s e n t a t i o n o f the a p p a r a t u s u s e d to
t e n h a m Pa.), e a c h w e i g h i n g 670 g c a n be u s e d . B e c a u s e the
stretch cells. Cells are cultured on a flexible m e m b r a n e of a
convex surface is uniformly curved, stretching occurs uniformly
Petriperm dish which is placed over a template with a c o n v e x
over the whole bottom of the dish. The third difference between
surface 1. A lead weight placed on top of the dish forces the
the m e t h o d s is that the degree of stretching can be varied by
m e m b r a n e and the tightly attached cells to be stretched (11.111).
using templates with different a m o u n t s of curvature. For the
e x p e r i m e n t s reported here a template producing a 4% increase
in surface area was used. The surface curvature is that of an
arc of 36 ~ with a radius of 7.6 cm. The cell population density m e t h o d d e s c r i b e d p r e v i o u s l y [7]. Following d e v e l o p m e n t the
at the time of stretching averaged 4.4 _+ .6 10~ cells/cm 2 in cells were stained with Gill's hematoxylin.
these experiments.
Nine fields were c o u n t e d to determine the labeling index for
each culture. T h e fields were selected by a stratified r a n d o m
sampling procedure in which the fields are selected blindly but
Measurement of the Number of Cells cover a specified spatial distribution. This is achieved by placing
Synthesizing DNA the slide on a mechanical stage and counting the n u m b e r of la-
beled and unlabeled cells in the field. The stage is then m o v e d
a p r e d e t e r m i n e d d i s t a n c e , a n d a n o t h e r field c o u n t e d a n d so
Tritiated thymidine ([3H]dThd) (New England Nuclear, Boston.
forth. Statistical c o m p a r i s o n s were made using the Students t
MA) at a concentration of 0.1 ixCi/ml and a specific activity of
test.
72 Ci/mmol was added to the bone cell cultures for 2 h. After
i n c u b a t i o n with [3H]dThd, t h e cells w e r e r i n s e d twice with
phosphate-buffered saline (PBS) and fixed with 3% glutaralde- Radiolabeling of Cultures
hyde in PBS at 4~ for 1 h. The fixative was then removed and
the culture washed twice with distilled water and extracted 3 3H leucine (56.5 Ci/mmol, N e w England Nuclear, Boston, MA)
times for 10 min with 5% trichloroacetic acid and finally washed or 3H-proline (13.6 Ci/mmol, N e w England Nuclear) was added
twice more with distilled water. to ~ M E M deficient in either leucine or proline to an activity of
A difficulty e n c o u n t e r e d in using the Petriperm dishes con- 2 - 1 0 p~Ci/ml. 6.5 ml of radiolabel-containing m e d i u m supple-
cerned the m o u n t i n g of the plastic m e m b r a n e for preparation of m e n t e d with 2% fetal bovine s e r u m was added to confluent cul-
autoradiographs. The following procedure was used. Glass slides tures o f bone-derived cells which bad previously been rinsed
were attached to the bottom of the Petriperm dishes on the sur- twice with PBS. After various times, as noted in the text, the
face to w h i c h t h e cells w e r e not a t t a c h e d u s i n g e p o n ( L a d d m e d i u m was r e m o v e d and dialized against distilled water over-
R e s e a r c h I n d u s t r i e s , Burlington, UT). T h e d i s h e s were t h e n night at 4~ to remove unincorporated isotope and centrifuged
placed in an oven at 60~ for 2 days until the epon hardened and at 9000 g for 20 min to remove insoluble matter. The cell layers
the slides were firmly attached to dishes. T h e n the part of the were washed three times with cold PBS, sonicated in 3 ml of a
Petriperm dish attached to the glass slide was cut out using a s o l u t i o n c o n t a i n i n g 4 M g u a n a d i n i u m c h l o r i d e a n d 20 m M
heated scalpel. T h e slides were put into slide holders, dipped in Tris.HCl pH 7.4 using a Fisher (Pittsburgh, PA) Model 300 sonic
emulsion (NTB-2 Kodak, Rochester, NY), and processed by a d i s m e m b r a n a t o r . T h e s o n i c a t e w a s t h e n dialized o v e r n i g h t
S. Hasegawa et al.: Effects of Mechanical Stretching on Cultured Bone Cells
against 25 mM Tris buffer pH 7.4. The amount of radioactivity
was determined by dissolving 250 ~1 samples in 3 ml Aquasol II
(New England Nuclear) and counting on a liquid scintillation
counter (Philips Series PW 4700, Philips Electronics Ltd.,
Hoffdgroep PPS, Eindhoven).
Determination of Collagenous Vs
Noncollagenous Protein
Collagen was determined by a modification of the method of
Peterkofsky and Diegelmann [8] in which 250 pJ samples were
digested with 10 ~g/ml of purified collagenase (Sigma type VII
1110 U/mg protein) in 25 mM Tris-HCl pH 7.4 for 17 h at room
temperature. To separate the small molecular-weight collagen
fragments produced by the digestion from the noncollagenous
proteins, the reaction mixture was applied to PD10 columns (1.5
x 5 cm) (Pharmacia, Uppsala, Sweden) and eluted with 25 mM
Tris-HC1 buffer pH 7.4 Fractions were collected and 250 pJ sam-
ples counted as described above.
SDS Polyacrylamide-Gel Electrophoresis
SDS/Polyacry[amide-gel electrophoresis was carried out by
using the discontinuous buffer system [9] on 5-15% gradient
polyacrylamide slabs prepared as described by Butler et al. [10].
The electrophoresis was conducted for about 4 h at room tem-
perature at a constant voltage of 330 V and was terminated when
the Bromphenol Blue tracking dye had reached the bottom of
the gel. The gels were soaked in Enlightening (New England
Nuclear) for 30 rain and then vacuum dried using a slab gel drier
apparatus (Bio-Rad Laboratories (Canada) Ltd., Mississauga,
Ont., Canada). The dried gels were exposed to 20.3 cm 25.4
cm Kodak SB-5 film for 4 weeks. Densitometer profiles were
obtained by scanning radioautogram tracks at 550 nm (Densi-
tometer 377 Instrumentation Laboratory).
In some experiments, 2.5 p~g of molecular weight standards
(Pharmacia) were run on the gels which were stained overnight
in a solution of 0.05% Coomasie blue (Eastman Kodak, Roch-
ester, NY) and 10% acetic acid. Gels were destained by several
washes in a solution which contained 10% (v/v) acetic acid and
10% (v/v) methanol.
Results
Mechanical stretching caused an increase in the
number of bone-derived cells synthesizing DNA
(Fig. 2). As has been observed for epithelial cells,
[12] a significant (P < 0.05) increase could be dem-
onstrated after just 2 h application of stress. As no
labeled cells were observed in cultures exposed to
[3H]dThd at 4~ mechanical stretching did not
cause an increase in background as a result of pres-
sure or stress effects on the emulsion. In order to
determine if the effect of stretching on bone-derived
cells persisted after the force was removed, the la-
beling index of cultures which had incorporated
[3H]dThd while being stretched was compared with
433
20.0
,-.-..
0 :~i:!
"I~ ,:.:.:.:
~ , i:i:i:i:
I I
' ' i:!:!:i:
U :i:!:!~
~ontrol Stretched
~ ntermittently
and Labelled
Then Labelled
And Labelled
Fig. 2. Percentage of cells (_+95% confidence limits) after 2 h
incubation with tritiated thymidine. Control cultures were not
stretched, stretched and labeled cultures were stretched and la-
beled concurrently, s t r e t c h e d then labeled cultures were
stretched for 2 h, then the weight removed so that the dish could
resume its original shape and were labeled for 2 h. Stretched
intermittently and labeled cultures were subjected to 10 rain cy-
cles of stretching and relaxation for a 2 h period in the presence
of trifiated thymidine.
cultures that received [3H]dThd immediately after
stretching. An increase in the number of cells syn-
thesizing DNA occurred under both labeling pro-
tocols. This observation indicates that once the
bone-derived cells have been stimulated to initiate
DNA synthesis, the process continues even after
the force is removed. Ten minute cycles of force
application followed by relaxation also increased
the number of cells synthesizing DNA to about the
same degree as the other stretching protocols (Fig. 2).
Protein Synthesis
Because collagen is a prominent product secreted
by bone-derived cells in culture [11], it was decided
to study the incorporation of 3H-proline by
stretched and unstretched cultures. After labeling
with 3H-proline for 12 h (Fig. 3), there was a 33%
increase in the amount of proline incorporated into
the cell layer of the stretched cultures. The number
of counts found in the medium overlying the cul-
tures was so low under these labeling conditions
that meaningful comparisons could not be made. A
modification of the method of Peterkofsky and
Diegelman [10] was devised to test the hypothesis
that the increase in protein synthesis by stretched
cultures was the result of an increase in collagen
434
1.5
t-- rl-
o A
Im
o
In
It
o I.O
o
t.-
0
t-
.R
o i f ! 84i;]
0.5 . . . . ii
0.
I
"1"
o3 ~ii!ilililili!)il
Control Stretched
Fig. 3. 3H-proline incorporation (c.p.m. 103) by six stretched
and control cultures of bone-derived cells. Coarse. ,fine, and no
dots corresponds to counts found in the media~ cell layer, and
total respectively.
production. In this method, collagen is identified by
its s u s c e p t i b i l i t y to digestion by purified colla-
genase. The results of one assay are shown in Fig.
4. It can be seen that prior to digestion all counts
from the sonicated cell layer were found to elute in
the void volume of the PD 10 column, whereas after
digestion with collagenase, some 25% were found
to elute in the low molecular weight region. The
data obtained when the sonicated cell layers of leu-
c i n e - l a b e l e d s t r e t c h e d and u n s t r e t c h e d c u l t u r e s
were digested with collagenase are shown in Fig. 5.
It is evident that the amount of collagenase-sensi-
tire material in the unstretched and stretched cul-
tures is nearly identical. However, there is an in-
crease in the material eluting in the void volume. It
thus appears likely that the proteins synthesized by
cultured bone cells in r e s p o n s e to stretching are
predominantly noncollagenous.
In view of the rapid response of the cells to me-
chanical stretching, it was decided to examine the
incorporation of radiolabel into proteins for shorter
time p e r i o d s . To i m p r o v e the sensitivity o f the
assay, 3H-leucine was used as the label, and ~ MEM
deficient in leucine was used as the culture medium.
The incorporation of 3H-leucine into stretched and
unstretched cultures is shown in Fig. 6. Although
no difference was observable after 6 h, there was a
significant (P < 0.05, t test) increase in the amount
of label in the cell layers of the cultures stretched
continuously for 12 h.
A densitometric scan of a fluorograph of the pro-
S. Hasegawa et al.: Effects of Mechanical Stretching on Cultured Bone Cells
6.0
%
g3.o
O
I .5 I0 15 20
Fraction Number
Fig. 4. Example of an assay for collagen production in cultures
labeled with leucine. Extracts of the cell layer were eluted off a
PDI0 column (closed circles). Digestion of the extracts with col-
lagenase (open circles) resulted in the production of lower mo-
lecular weight fragments.
teins from the cell layer which contain radiolabeled
leucine is shown in Fig. 7. As expected from the
data in Fig. 5, more radiolabel was incorporated by
the stretched cultures. For both stretched and un-
stretched cultures, the most prominent bands were
found in areas of the gel that corresponded to mo-
lecular weights of 58,000 and 40,000 daitons. In Fig.
7, it can be seen that stretched cultures also ap-
peared to incorporate more 3H-leucine into material
found in the 29,000 and 22,000 dalton region of the
gel but this observation was not found consistently.
Discussion
The method of culturing bone cells in a dish with a
flexible bottom and applying a stretching force by
means of a template has several advantages. Like
the method of Harrell et al. [2], it is simple but it
has the a d d i t i o n a l b e n e f i t that the a m o u n t of
stretching can be varied by simply changing the ra-
dius o f c u r v a t u r e of the underlying template. A
second benefit is that, because the culture surface
is elastic, stretching can be applied repeatedly by
simply lifting and reapplying the weight. In contrast
to the more complex apparatus designed by Leung
S. H a s e g a w a et al.: Effects of Mechanical Stretching on Cultured Bone Cells 435

6.0 58 K
~, 40 K
"1

0
to
c~J
!/ ~\ i,.~i 29 K 22 K
0
% y "-J -,~.,. . . . . . .,-----,. ~,
x
c 3.0
.__
P
O

O
U -- Control
.... Stretched
Fig. 7. Densitometric scan of a fluorograph of polyacrylamide
.J
gel. Gel u s e d to separate 3H-leucine labeled proteins from the
4-
tO cell layer of stretched (dotted line) and control cultures. Equal
"T v o l u m e s of m e d i a from stretched and u n s t r e t c h e d cultures were
I 5 I0 15 20 loaded on the tracks of the gel.
Fraction Number

min) used by Leung et al. to simulate the forces

O-oControl, 0--0 S t r e t c h e d
experienced by the smooth muscle cells of arteries.
Fig. 5. C o l l a g e n p r o d u c t i o n , as a s s a y e d by t h e c o l l a g e n a s e A final advantage is that cells grown on the Petri-
digestion t e c h n i q u e , for s t r e t c h e d (open circles) a n d control perm membrane can be sectioned and processed for
(closed circles) cultures. histology. The latter property enabled us to perform
autoradiographs of bone-derived cells labeled with
[3H]dThd.
IO.O By preparing autoradiographs of cultures labeled
with [3H]dThd we were able to demonstrate that the
num ber of cells synthesizing DNA increased
t- sharply as a result of the application of mechanical
O
stretching. This approach differs from measuring
the total amount of [3H]dThd incorporated by dul-
tures, a measurement that is influenced both by the
0 5.0
number of cells synthesizing DNA and the rate at
which DNA is synthesized. The effect of stretching
on cellular proliferation as measured by the incor-
a)
..J poration of [3H]dThd appears to vary with cell type.
I Epithelial cells derived from the cell rests of Ma-
"1-
lassez exhibited a 92% increase in the labeling index
after stretching [12] but Leung et al. [13] found no
O consistent increase in [3H]dThd incorporation as
0 :5 6 12 the result of cyclic stretching of smooth muscle
cells. Somjen et al. [1] r e p o r t e d that stretching
Incubation time (hrs.) caused a 45% increase in the amount of [3H]dThd
Fig. 6. 3 H - l e u c i n e i n c o r p o r a t i o n w i t h t i m e into c o l l a g e n incorporated in bone cell cultures, but did not mea-
(squares) and noncollagenous proteins (circles). Closed symbols sure the number of labeled cells. Our results reveal
are control cultures; open symbols are stretched cultures.
a roughly similar size of effect: a 64% increase in
the number of cells synthesizing DNA. It should be
noted that the increase in labeling occurred within
et al. [13] to apply cyclic stretching, our method 2 h of the application of mechanical stretching.
does not require an airtight seal on the side of the Thus the time required for a proliferative response
dish or any elaborate mechanical connections, but to mechanical stretching is much less than that
by the same token it must be admitted that our found for growth factors such as serum where lag
method is not suitable for the rapid cycles (52 x / times of 8 h or longer are usual [14].
436 S. Hasegawa eta[.: Effects of Mechanical Stretching on Cultured Bone Cells
Protein synthesis, as measured by either proline
or leucine incorporation, also increased but the first
significant difference did not occur until after 12 h
of stretching. It is possible, however, that more sen-
sitive methods could detect differences at earlier
times. Nevertheless, with the present methods we
were able to demonstrate that the increase in pro-
tein synthesis after 12 h of stretching was the result
of more noncollagenous proteins being produced;
stretched cultures synthesized the same amount of
collagen as unstretched cultures. Analysis of the
proteins synthesized by polyacrylamide slab gel
electrophoresis indicated that the cell layers from
stretched and unstretched cultures produced a sim-
ilar number of bands but differed in the amount of
labeled material present in the bands. Definitive
identification of any of the bands has not been at-
tempted in this study but it is perhaps of interest
that one of the most prominent bands, which was
found to be increased in stretched cultures, was es-
timated to have a molecular weight (40,000) similar
to osteonectin [15, 16]. It is feasible that bone cells
e x p o s e d to mechanical stress act to reduce the
stress not only by increasing cell number but also
by rearranging their contacts to adjacent structures.
Such a rearrangement could require the production
of attachment factors such as osteonectin [15, 16].
However, the validation of such a hypothesis re-
quires a specific assay for the identification and
quantitation of osteonectin in cultures of bone de-
rived cells.
The cell population used in these studies was re-
leased after 60-120 rain of proteolytic digestion of
rat calvariae. Moreover, the calvariae had been
stripped, insofar as this is possible, of their peri-
osteum prior to digestion, and the cells cultured for
this study probably did not occupy a location on the
surface of the bone prior to culture. There is abun-
dant evidence (see for example [17]) that cultures
derived from bone are heterogeneous with respect to
growth, hormone responses, and other properties.
It is therefore also likely that the population used
in these studies was not homogeneous. The bio-
chemical and cytological responses to stretching re-
ported here may not occur for each type of bone-
derived cell in the culture; some populations of cells
may be more or less responsive to mechanical
stimuli. Nevertheless it should be pointed out that
there is a 64% increase in the num be r of cells
synthesizing DNA in response to mechanical
stretching, so stretching is affecting a significant
proportion of cells in the culture and not just a small
subpopulation. Moreover, aside from the presence
of label in the nucleus, those cells synthesizing
DNA in stretched or unstretched cultures did not
look noticeably different from their unlabeled neigh-
bors. Thus at present, we have no evidence for het-
erogeneity in cell response to tension but more de-
tailed investigation, perhaps using cloned popula-
tions, is required to address this question more
definitively.
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