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Calcif Tissue lnt (1985) 37:431-436

Calcified Tissue
International
9 1985 by Springer-Verlag

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- stimuli. The effects of mechanical stretching on
plying mechanical stretching to bone cell cultures. cells include increased synthesis of PGE 2 [2], me-
Bone cells cultured on the flexible plastic mem- talloproteinases [5], and D N A [1]. In addition, tran-
brane of a Petriperm dish are placed over a template sient increases in cyclic AMP production and C a 2+
with a convex surface. A lead weight is then placed accumulation have been reported [2]. In this pa-
on top of the dish which causes the membrane and per, we r e p o r t a simple m e t h o d for m e c h a n i -
the tightly attached cells to be stretched. Mechan- cally stretching cultured bone cells. M e c h a n i c a l
ical stretching, applied either intermittently or con- stretching was found to increase the number of cells
tinuously for a 2-hour period resulted in a 64% in- synthesizing D N A and to alter the pattern of pro-
crease in the number of cells synthesizing DNA. teins synthesized.
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- Materials and Methods
genase and the digests chromatographed on PD 10
Bone cells were isolated by a modification of the method of
columns. The amount of collagen synthesized by
Wong and Cohn [6]. Calvariae were removed aseptically from
stretched and unstretched cultures did not differ; 3-5-day-old rats and, as far as possible, stripped of their over-
but an increased synthesis of noncollagenous pro- lying periosteum. The calvariae were minced into pieces of ap-
teins was observed in the stretched cultures. 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
Key words: Mechanical stretching - - Bone remod- collagenase (type 1, Sigma, St. Louis, MO) and 0.05% (w/v)
eling - - PGE? - - D N A - - Metalloproteinases 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
Although mechanical stress has long been recog-
mixed with an equal volume of fetal bovine serum and centri-
nized as an important factor in the regulation of fuged at 1000 rpm for 5 rain at room temperature and then re-
bone remodeling, the mechanism underlying these suspended in culture medium for plating into 75 cm 2 culture
effects has remained obscure. A number of methods flasks (Falcon, Div. of Becton, Dickinson and Co., Cockeysville,
[1-4] have been devised to apply forces to bone, or MD). The culture medium was c~ MEM (GIBCO) supplemented
cells derived from bone or periosteum, in order to with 10% fetal bovine serum (Flow, Cockeysville, MD) and an-
investigate the biochemical results of mechanical 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
Send reprint requests to Dr. D. M. Brunette, The University of twice with phosphate-buffered saline. Then 10 ml of 0.25% (w/
British Columbia, Dept. of Oral Biology, Faculty of Dentistry, v) trypsin (Difco, Detroit, Michigan) 1:250 was added and the
2199 Westbrook Mall, Vancouver, B.C., Canada. 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 433

against 25 mM Tris buffer pH 7.4. The amount of radioactivity 20.0


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).
,-.-..
0 :~i:!
"I~ ,:.:.:.:

Determination of Collagenous Vs
Noncollagenous Protein

Collagen was determined by a modification of the method of


~ , i:i:i:i:
Peterkofsky and Diegelmann [8] in which 250 pJ samples were
I I
' ' i:!:!:i:
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 U :i:!:!~
temperature. To separate the small molecular-weight collagen ~ontrol Stretched
fragments produced by the digestion from the noncollagenous ~ ntermittently
proteins, the reaction mixture was applied to PD10 columns (1.5 and Labelled
x 5 cm) (Pharmacia, Uppsala, Sweden) and eluted with 25 mM
Then Labelled
Tris-HC1 buffer pH 7.4 Fractions were collected and 250 pJ sam-
ples counted as described above. 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-
SDS Polyacrylamide-Gel Electrophoresis 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
SDS/Polyacry[amide-gel electrophoresis was carried out by resume its original shape and were labeled for 2 h. Stretched
using the discontinuous buffer system [9] on 5-15% gradient intermittently and labeled cultures were subjected to 10 rain cy-
polyacrylamide slabs prepared as described by Butler et al. [10]. cles of stretching and relaxation for a 2 h period in the presence
The electrophoresis was conducted for about 4 h at room tem- of trifiated thymidine.
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 cultures that received [3H]dThd immediately after
Nuclear) for 30 rain and then vacuum dried using a slab gel drier
apparatus (Bio-Rad Laboratories (Canada) Ltd., Mississauga,
stretching. An increase in the number of cells syn-
Ont., Canada). The dried gels were exposed to 20.3 cm 25.4 thesizing DNA occurred under both labeling pro-
cm Kodak SB-5 film for 4 weeks. Densitometer profiles were tocols. This observation indicates that once the
obtained by scanning radioautogram tracks at 550 nm (Densi- bone-derived cells have been stimulated to initiate
tometer 377 Instrumentation Laboratory). DNA synthesis, the process continues even after
In some experiments, 2.5 p~g of molecular weight standards the force is removed. Ten minute cycles of force
(Pharmacia) were run on the gels which were stained overnight
application followed by relaxation also increased
in a solution of 0.05% Coomasie blue (Eastman Kodak, Roch-
ester, NY) and 10% acetic acid. Gels were destained by several the number of cells synthesizing DNA to about the
washes in a solution which contained 10% (v/v) acetic acid and same degree as the other stretching protocols (Fig. 2).
10% (v/v) methanol.

Protein Synthesis
Results
Because collagen is a prominent product secreted
Mechanical stretching caused an increase in the by bone-derived cells in culture [11], it was decided
number of bone-derived cells synthesizing DNA to study the incorporation of 3H-proline by
(Fig. 2). As has been observed for epithelial cells, stretched and unstretched cultures. After labeling
[12] a significant (P < 0.05) increase could be dem- with 3H-proline for 12 h (Fig. 3), there was a 33%
onstrated after just 2 h application of stress. As no increase in the amount of proline incorporated into
labeled cells were observed in cultures exposed to the cell layer of the stretched cultures. The number
[3H]dThd at 4~ mechanical stretching did not of counts found in the medium overlying the cul-
cause an increase in background as a result of pres- tures was so low under these labeling conditions
sure or stress effects on the emulsion. In order to that meaningful comparisons could not be made. A
determine if the effect of stretching on bone-derived modification of the method of Peterkofsky and
cells persisted after the force was removed, the la- Diegelman [10] was devised to test the hypothesis
beling index of cultures which had incorporated that the increase in protein synthesis by stretched
[3H]dThd while being stretched was compared with cultures was the result of an increase in collagen
434 S. Hasegawa et al.: Effects of Mechanical Stretching on Cultured Bone Cells

1.5 6.0
t-- rl-
o A

Im
o
In
It
o I.O
o
t.- %
0
t-
.R g3.o
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 O
and control cultures of bone-derived cells. Coarse. ,fine, and no I .5 I0 15 20
dots corresponds to counts found in the media~ cell layer, and Fraction Number
total respectively. 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-
production. In this method, collagen is identified by lagenase (open circles) resulted in the production of lower mo-
its s u s c e p t i b i l i t y to digestion by purified colla- lecular weight fragments.
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 teins from the cell layer which contain radiolabeled
the void volume of the PD 10 column, whereas after leucine is shown in Fig. 7. As expected from the
digestion with collagenase, some 25% were found data in Fig. 5, more radiolabel was incorporated by
to elute in the low molecular weight region. The the stretched cultures. For both stretched and un-
data obtained when the sonicated cell layers of leu- stretched cultures, the most prominent bands were
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 found in areas of the gel that corresponded to mo-
were digested with collagenase are shown in Fig. 5. lecular weights of 58,000 and 40,000 daitons. In Fig.
It is evident that the amount of collagenase-sensi- 7, it can be seen that stretched cultures also ap-
tire material in the unstretched and stretched cul- peared to incorporate more 3H-leucine into material
tures is nearly identical. However, there is an in- found in the 29,000 and 22,000 dalton region of the
crease in the material eluting in the void volume. It gel but this observation was not found consistently.
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- Discussion
chanical stretching, it was decided to examine the
incorporation of radiolabel into proteins for shorter The method of culturing bone cells in a dish with a
time p e r i o d s . To i m p r o v e the sensitivity o f the flexible bottom and applying a stretching force by
assay, 3H-leucine was used as the label, and ~ MEM means of a template has several advantages. Like
deficient in leucine was used as the culture medium. the method of Harrell et al. [2], it is simple but it
The incorporation of 3H-leucine into stretched and 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
unstretched cultures is shown in Fig. 6. Although stretching can be varied by simply changing the ra-
no difference was observable after 6 h, there was a dius o f c u r v a t u r e of the underlying template. A
significant (P < 0.05, t test) increase in the amount second benefit is that, because the culture surface
of label in the cell layers of the cultures stretched is elastic, stretching can be applied repeatedly by
continuously for 12 h. simply lifting and reapplying the weight. In contrast
A densitometric scan of a fluorograph of the pro- 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 erogeneity in cell response to tension but more de-
or leucine incorporation, also increased but the first tailed investigation, perhaps using cloned popula-
significant difference did not occur until after 12 h tions, is required to address this question more
of stretching. It is possible, however, that more sen- definitively.
sitive methods could detect differences at earlier
times. Nevertheless, with the present methods we
References
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