- Journal of Biomechanics

Journal of Biomechanics 33 (2000) 63}71
Primary human bone cultures from older patients do not respond at
continuum levels of in vivo strain magnitudes
Clark M. Stanford!,*, Frederic Welsch", Norbert Kastner$, Geb Thomas#,
Rebecca Zaharias!, Kevin Holtman!, Richard A. Brand"
!Dows Institute for Dental Research College of Dentistry, The University of Iowa, Iowa City, IA 52242, USA
"Department of Orthopaedic Surgery, College of Medicine, The University of Iowa, Iowa City, IA 52242, USA
#Department of Industrial Engineering, College of Engineering, The University of Iowa, Iowa City, IA 52242, USA
$Department of Orthopaedic Surgery, University of Graz, 8036 Graz, Austria
Abstract
Osteoporosis is characterized by excessive loss of bone mass, while exercise is believed to maintain or enhance bone mass. Since
exercise marginally a!ects osteoporosis, we wondered whether bone cells from osteoporotic patients would fail to respond to strain.
Primary human bone-like cultures were obtained from females over age 60 with hip arthroplasty procedures performed for either
osteoporotic fracture (n"8) or non-osteoporotic osteoarthrosis (n"5). Cultures (96,000 cell/cm2) were strained in rectangular
optically clear silastic wells. Three periods of uniaxial substratum strain (1000 l-strain, 1 Hz, 10,000 cycles, sine wave) were provided
every 24 h using a four-point bending, computer-controlled device. Results at a frequency of 1 Hz were compared to cultures exposed
to 20 Hz with bone cells derived from one osteoarthritic subject. Alterations in protein level expression of bone-related proteins were
determined using a semi-quantitative confocal approach along with enzyme (alkaline phosphatase) activity and enzyme mRNA copy
number using cRNA RT-PCR. Strain did not alter levels of bone-related protein levels, enzyme activity, or steady state copy number
per cell in response to strain in either group. Strained cultures from osteoporotic patients exhibited little variation from unstrained
controls, while individual cultures from osteoarthritic patients exhibited increases in one protein or the other. The results suggest that
bone cells from older individuals may not be responsive to continuum levels of strain anticipated with vigorous activity. ( 1999
Elsevier Science Ltd. All rights reserved.
1. Introduction
The regularity of cortical and trabecular architecture
and their reproducible responses to altered loading (e.g.,
exercise) or geometry (e.g., malunited fractures) strongly
argue for a formal (mathematical) relationship between
the mechanical environment and the biological response.
Wol! (1892) and Roux (1895) were among the early
investigators who recognized this argument as well as the
therapeutic implications of quantitatively understanding
the stimuli for bone adaptation: the treatment of deformities, the prevention and treatment of osteopenias, the
acceleration of fracture healing, and the optimization of
implant designs. Wol! (1892) postulated the existence of
mathematical laws (i.e., stress or strain) `governinga bone
* Corresponding author. Tel.: 001-319-335-7381; fax: 001-319-3358895.
E-mail address: [email protected] (C.M. Stanford)
remodeling, but did not speci"cally formulate them. Current researchers agree some component of stress or strain
relates to bone adaptation.
In vivo cortical bone strains in a wide variety of species
are remarkably consistent, and range from peaks of
100}1000 l-strain in the majority of daily activities (van
B Cochran, 1992; Lanyon et al., 1975; Keller and Spengler, 1982), to 3000}4000 l-strain in the most vigorous
activities (Rubin and Lanyon, 1984; Gross et al., 1992).
Most investigators assume that strain magnitudes in
these ranges are associated with whatever stress or strain
quantities might relate to disuse atrophy, bone maintenance, and bone modeling or remodeling.
Presuming macro-level strains relate to cell responses,
many studies document proliferative and/or expressive
responses of various dynamically strained osteoblast-like
cells in culture (Harell et al., 1977; Hasegawa et al., 1985;
Buckley et al., 1988; Murray and Rushton, 1990; Neidlinger-Wilke et al., 1994) and bone organ cultures (Dallas et
0021-9290/99/$ - see front matter ( 1999 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 2 1 - 9 2 9 0 ( 9 9 ) 0 0 1 7 3 - 6
64
C.M. Stanford et al. / Journal of Biomechanics 33 (2000) 63}71
al., 1993; Rawlinson et al., 1995; Stanford et al., 1995a,b).
However, it is important to emphasize that strain magnitudes in these studies were either not well characterized
and/or at magnitudes considered supra-physiological for
bone as a tissue (i.e., at levels between 5000 and 50,000
l-strain), well beyond the approximately 3000 l-strain
peak level recorded in most species, and at times beyond
the roughly 25,000 l-strain breaking strain of bone. Few
studies report bone cell responses at magnitudes believed
to be physiological. Brighton et al. (1991) suggested neonatal rat calvarial osteoblast-like cells deformed at 400
l-strain increase DNA production, while decreasing expression of collagen, non-collagenous protein, proteoglycan, and alkaline phosphatase. Jones et al. (1991)
demonstrated primary bovine periosteal-derived cells but
not Haversian-derived osteoblasts responded (increased
proliferation) at 3000 l-strain. Thus, the precise strain
history features (re#ected by activity levels in vivo) relating to bone adaptation are unknown.
Decreasing activity levels after age 55}60, are generally
believed to be one factor in osteoporosis. Some advocate
exercise as a means to maintain or increase bone mass,
although such e!orts have been modestly successful at
best. An appropriate temporal expression of bone
matrix-related proteins establishes and maintains a normal tissue. If exercise fails to increase bone mass in
osteoporosis (although it does so experimentally in normal bone under some conditions of increased strain history), it might be related to the lack of responsiveness of
bone cells in these patients. It is that known aging bone
cells have diminished activity (Fedarko et al., 1995). We
therefore wondered whether bone cells from osteoporotic
patients would respond less satisfactorily to strain than
cells from age-matched controls, postulating a regimen
which maintained bone mass in vivo would increase bone
matrix protein production to a greater degree in nonosteoporotic cells. Since many matrix proteins interact,
alterations in the patterns of protein expression (rather
than merely levels of a given protein) may also play a
role in the abnormal phenotypes (e.g., osteoporosis),
and therefore we also questioned whether the patterns
might be distinct in non-osteoporotic versus osteoporotic
cells.
2. Materials and methods
A strain regimen (1000 l-strain, 10,000 cycles, 1 Hz
sine wave) was based upon in vivo experiments maintaining bone mass and re#ecting the regimen of a very active
women over the age of 60. Data published by several
authors suggest 1000 l-strain peak magnitude, while at
the higher magnitude end of non-athletic activity, maintains bone mass with adequate numbers of cycles and
strain rates. Higher strains (i.e., greater than 1400}2000
l-strain on the periosteal surfaces) results in new bone
formation (McLeod and Rubin, 1992; Turner et al., 1994).
(We recognize magnitude alone is not the sole, and perhaps not even dominant feature of the mechanical environment a!ecting bone adaptation; see Brand and
Stanford, 1994.) Ten thousand cycles per day represents
the number of cycles of activity of a very active human,
while 1 Hz re#ects the dominant frequency of cycle activity. The regimen was intended to simulate a steady state
in bone under two conditions: one where bone is more or
less maintained (OA), and another where (relative to the
other condition) bone is lost (osteoporosis). A sine wave
avoids rapid accelerations and decelerations (which
might simulate impact loading). Such a regimen would
likely re#ect bone strains in an aerobic regimen recommended for osteoporosis. All assays were performed at
the completion of the third strain regime. We used a cell
culture straining system providing uniform uniaxial
strain at speci"ed magnitudes (1000 l-strain), using agematched controls, compared the production of Type
I collagen (Coll), alkaline phosphatase (AP), osteopontin
(OP), osteocalcin (OC), and bone sialoprotein (BSP).
Osteoblast-like cells were derived from surgical material obtained from females undergoing hip arthroplasty
for osteoarthrosis or hemiarthroplasty for hip fracture.
Institutional Review Board approval was obtained as
well as informed consent. Inclusion criteria included
non-smoking females over age 60 with stable weight,
normal clinical laboratory pro"les, and not on estrogen
or other bone-altering drugs. Osteoporosis was assessed
by the method of Singh et al. (1970) and considered
present when the Singh index was 43 or absent when
the index was 55. Using these criteria, "ve non-osteoporotic patients with concurrent osteoarthrosis, and
eight with osteoporosis were identi"ed. Collection and
culture procedures (Hesby et al., 1998; Zaharias et al.,
1998) were similar to those reported by Robey and Termine (1985), Neidlinger-Wilke et al. (1994) and Battmann
et al. (1997). Brie#y, cancellous bone chips were minced
and treated by shaking for 2 h in CMRL media
(Gibco/BRL, Gaithersburg, MD) with collagenase (300
U/mL, Type IV, Sigma Chemical Company, St. Louis,
MO). Cultures were initiated into low Ca`` CMRL
supplemented with antibiotics and 10% FCS (Integen,
Purchase, NY). Primary cultures reached con#uence between three and "ve weeks, at which time they were
treated with trypsin/EDTA (0.05%/0.02%) for preparation of single-cell suspension for plating into #asks. Cultures were maintained in CMRL (with Ca``) #10%
FCS and antibiotics until an adequate number of cells
were available. A su$cient number of cells were obtained
by the second or third passage. Circular cultures (96,000
cells/cm2 over a 0.8 cm2 area) were plated on "bronectin
coated (2] with 2 lg/mL bovine plasma "bronectin)
custom silastic dishes (2 cultures/dish). Cultures were
plated using a 1 mm thick silastic template and allowed
to attach for 2 h (373C) prior to the addition of 5 ml of
C.M. Stanford et al. / Journal of Biomechanics 33 (2000) 63}71
Fig. 1. (a) Cross-sectional diagram of one station of a six-station device
to strain cells. Cells are cultured on the #at surface of the thin crosshatched silicone culture dish. (b) View of strain device from above
showing three PlexiglasTM base plates, and a single silicone well (foreground).
CMRL #10% FCS. Cultures were incubated for 22 h
prior to the "rst strain regimen, by which time the cells
were nearly con#uent.
Cell deformation was accomplished by imparting four-point bending to rectangular silicone culture
dishes seated upon PlexiglasTM base plates (Bottlang et
al., 1997, see Fig. 1 and b). The six-station device was
controlled with a user-developed interface within a commercial program (LabVIEW version 3.1, National Instruments, Austin, TX). The base plates and culture
dishes were strained by shielded electromagnetic motors
(BEI Motion Systems Company, San Marcos, CA) in
which the magnetic radiation was determined to be less
than 0.5 mG (i.e., in the range of room background radiation). The device allows 100}3000 uniaxial l-strain of
the substrate from 0.5 to 30 Hz, using any programmable
wave, and speci"ed duty cycle. Evaluation of the system
insured uniform "delity of the input signal and base-plate
deformation, uniform substrate surface strain "elds, and
reproducible strains from dish to dish (Bottlang et al.,
1997).
Substrate strain was initiated for a period of 2.7 h
(1000 l-strain, 10,000 cycles, 1 Hz sine wave) at 24, 48
65
and 72 h after plating. The same conditions were used in
all reported trials. Since higher frequencies have been
associated with enhanced bone formation (Rubin and
McLeod, 1994), we performed additional experiments in
which the cells were exposed to a higher frequency of
loading (20 Hz, 1000 l-strain, 10,000 cycles). Assays were
performed immediately following the last strain regime
and were identical in all experiments.
The distribution and semi-quanti"cation of matrix
components within each culture was ascertained with
immunolabeling (Zaharias et al., 1998). These assays allowed the evaluation of protein level responses from
multiple cells (i.e., a general response) rather than from
just a few cells within the culture. The presence and
distribution of Type I collagen (COL), osteopontin
(OPN), bone speci"c alkaline phosphatase (AP), osteonectin (OCN), bone sialoprotein (BSP) and a negative
control antibody (Drosophila even-skipped protein,
3C10) were ascertained using monoclonal antibodies
from the Developmental Studies Hybridoma Bank (The
University of Iowa, Iowa City). Antigen location was
determined by secondary labeling with Cy3 conjugated
a$nity pure goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). Planar
images (Noran ConFocal Microscope, Madison, WI) of
the prepared cultures were collected. Two cultures were
assessed per antibody per patient. Ten random images
(10]) were collected from each culture and image analysis performed using NIH Image alpha-9 (Scion Corporation, Fredrick, MD). Each image was calibrated and the
background subtracted. Pixel density/image was calculated using a gray-scale value (0}256) and averaged for
semi-quantitative comparisons between images of label
intensity. A minimum of two cultures per label per trial
were evaluated in this manner. Experimental (strained)
cultures were compared to controls (unstrained) obtained
from the same donor and cultured under identical conditions. Kruskal}Wallis analysis was performed on average
percent increases or decreases in signal intensity. We
presumed an a"0.05 as the level of signi"cance.
A power analysis was conducted for Types I and II errors
(Lachin, 1981).
Given interactions of some matrix proteins, we presumed patterns of expression might be important. That
is, at a given time, the relative amount of a given protein
compared to another in one culture may be di!erent
from those in other cultures. To assess these patterns in
a concise manner, a radar plot diagram (Excel 97, Microsoft, Redman, WA) of the percent change relative to
non-strained controls was created for the osteoarthritic
and osteoporotic cultures. Since a disease process may be
re#ected in altered expression of one or more proteins,
one would expect greater variability in responses. Bartlett's Test for homogeneity was used to test whether the
osteoarthritic and osteoporotic patient had di!erent
variance of protein expression.
66
C.M. Stanford et al. / Journal of Biomechanics 33 (2000) 63}71
Alkaline phosphatase (AP) activity, an indirect
measure of the capacity to hydrolyze inorganic phosphate and thus mineralize tissues, was determined as
previously described (Stanford et al., 1994). Product
formation was normalized to the #uorometrically determined concentration of DNA per respective culture. Cell
proliferation was also determined by direct cell counting
using a Coulter Counter as described (Stanford et al.,
1999).
Total protein expression at any given time point depends on the production, accumulation and degradation
of translatable message. Therefore, we assessed the
steady-state pool of available message transcripts for
translation into protein. Message copy number on a per
cell basis was determined using a cRNA PCR-based
analysis. Human-speci"c primer sets were de"ned and
optimized for single-band amplicon formation (Holtman
et al., 1998). AP PCR primers [P1"5@-ACTCCCACTTCATC TGGAACCGC-3@ (exon 8) and P2"5@AGACATTCTCTCGTTCACCGCCC-3@ (exon 11)],
gave a single-band product of 501 bp (Fig. 5a). To fabricate a cRNA standard for message quantitation in both
the RT and PCR steps, these primers were appended to
a 308 bp DNA stu!er core. The 399 bp resulting product
was cloned (Dual Promoter TA Cloning procedure, Invitrogen, Carlsbad, CA), puri"ed, linearized with a Xho
I digest followed by a Proteinase K treatment, phenol
chloroform extraction, and ethanol precipitation. In vitro
transcription was then performed with SP6 RNA Polymerase. The cRNA was quantitated with A
and
260
diluted serially in H O for competitive RT-PCR,
2
which produced a 354 bp (AP primers) standard
amplicon.
For the PCR quantitation, cytoplasmic mRNA was
isolated and calibrated conditions used for analysis. Total cytoplasmic RNA was isolated with a RNeasy Mini
Kit (Qiagen, Valencia, CA.), and stored at !703C. Total
cytoplasmic RNA was combined with the AP primer (P2)
and the cRNA standard (0.167}2.67 fg) in water. PCR
was then performed as described (Holtman et al., 1998).
PCR products were separated in 3% agarose (0.5X TBE)
and were run with 0.125 lg/mL ethidium bromide in the
gel and bu!er. Gels were digitized with a camera system
[CCD camera (Cohu model 4910, San Diego, CA), with
a LG-3 PCI frame grabber card (Scion, Fredrick, MD),
and 70 nm bandpass "lter centered at 550 nm (Corion,
Holliston, MA)] mounted above a UV transilluminator.
The ratio of AP amplicon/standard amplicon was calculated using Scion Image and macro GelPlot2, and
corrected for the di!erence in size between the AP and
standard amplicons (501 bp/354 bp"1.42). The log of
the ratio of the PCR amplicons was plotted on the y-axis
versus the log of input cRNA standard molecules. Following linear regression, the antilog of the x-intercept
equaled the number of AP mRNA per 12.5 ng total
cytoplasmic RNA. All regression lines crossed the xintercept (mean r2"0.95$0.05).
3. Results
Con#uence occurred at similar times in non-osteoporotic (osteoarthritic) and osteoporotic cultures. The
isolated cells expressed relatively high levels of the "ve
bone-related proteins upon detection by immuno#uorescence (Fig. 2), demonstrating a bone-like phenotype
of the culture model by 72 h under these conditions.
Semi-quantitative image analysis of the labeled cells
demonstrated no di!erences between the strained and
unstrained cultures for Type I collagen (p"0.7), BSP
(p"0.88) and AP (p"0.16) (Fig. 3). Cultures derived
from osteoarthritic samples demonstrated modestly elevated but non-signi"cant levels of OPN (p"0.11) and
OCN (p"0.09) with strain. Power analysis demonstrated at a Type I error of 0.05 (and a Type II
Power"80%) a sample population of '3000 would be
Fig. 2. Confocal immuno#uorescence analysis of osteoarthritic cultures (96,000 cells/cm2) derived from trabecular bone samples grown for 3 days
demonstrated positive intra- and extracellular labeling for Type I collagen (A), alkaline phosphatase (B), and bone sialoprotein (C). (20X magni"cation,
Bar"100lm). The majority of cells show some label, suggesting they are bone-like cells.
C.M. Stanford et al. / Journal of Biomechanics 33 (2000) 63}71
67
Fig. 3. Ratios of bone matrix proteins for strained versus non-strained
(control) cultures. Bars re#ect averages of two cultures/patient sample
imaged 10 times/culture (see methods). Five trials were performed with
cultures derived from osteoarthrosis (empty bars), while the shaded
bars re#ect averages from eight osteoporotic patients. Mean$SEM.
None of the bone matrix proteins was signi"cantly increased (p'0.05)
for either group of cultures.
Table 1
Percent change in protein levels in response to strain relative to nonstrained cultures!
Patient
Age
Coll
BSP
AP
OPN
OCN
OA-1
OA-2
OA-3
OA-4
OA-5
68
69
78
69
71
79
86
121
117
49
107
133
74
129
119
101
112
123
174
109
112
108
165
120
107
91
96
191
144
119
OP-1
OP-2
OP-3
OP-4
OP-5
OP-6
OP-7
OP-8
49
55
86
69
73
52
85
63
82
95
113
71
96
162
52
58
82
112
94
88
112
78
144
42
98
111
108
123
90
101
82
109
113
100
107
105
106
63
73
87
83
98
98
93
95
71
98
76
!Values are the ratio of the average gray-scale value for 10 measurements of two cultures (n"20) for both strained and non-strained
cultures. Strain conditions used were: 1000 l-strain, 1 Hz, 10,000 cycles,
sine wave form.
Fig. 4. Radial plots of protein response to strain determined by a semiquantitative confocal assay. The abnormal polygons for the "ve osteoarthritic (Fig. 4a) patients (OA-1 though OA-5) suggest greater
variability of pattern of protein expression compared to the eight
osteoporotic (Fig. 4b) patients (OP-1 though OP-8).
sured in response to mechanical strain (Fig. 4a). There
were signi"cant di!erences in variance for AP (Bartlett
ratio"7.855, p(0.005) and OPN (Bartlett ratio"
5.924, p(0.015).
Cultures derived from the osteoporotic patients did
not exhibit di!erences in AP activity at 1 versus 20 Hz
(p"0.93), nor was there a di!erence in cellular proliferation over a three day period (p"0.07). Confocal imaging demonstrated no di!erences in response between the
two frequencies (Table 2). Steady-state message for AP
(expressed as a ratio of copy number per unit total
cytoplasmic RNA) also demonstrated no changes in response to the two strain frequencies under the conditions
of this study (Fig. 5).
4. Discussion
needed to demonstrate di!erences less than 0.05 under
the conditions of this study.
The osteoporotic cultures demonstrated a uniform
lack of response of all assays to strain while cultures
derived from the osteoarthritic samples demonstrate patient-speci"c responses to the strain regimen with an
occasionally high response of a given assay (Table 1 and
Fig. 4). The osteoarthritic cultures therefore demonstrated less consistency of expression between patient
samples as exhibited by the distorted polygon relationship between the bone-related phenotypic markers mea-
We found con#uent human bone-like cells from older
non-osteoporotic and osteoporotic patients under
uniaxial physiological (continuum level) strains did not
induce alterations in Type I collagen, osteopontin, alkaline phosphatase, bone sialoprotein and osteonectin.
Control cultures maintained a mature osteoblastic/osteocytic phenotype (Zaharias et al., 1998).
All cell culture experiments have inherent limitations.
Perhaps the most critical is whether cells in vitro mimic
those in vivo. We presume the biological responses are
similar since they express the same proteins in vitro.
68
C.M. Stanford et al. / Journal of Biomechanics 33 (2000) 63}71
Table 2
Human osteoarthritic cultures exposed to 1 versus 20 Hz
AP
Non-strained
1 Hz
20 Hz
Proliferation
Confocal
Nmol PNP/min/lg DNA (]1000) cells/culture
BSP!
OPN!
OCN!
6.6$3.9
6.0$1.3
6.8$2.1
P"0.93
91$5%
87$4%
P"0.44
120$6%
111$5%
P"0.25
98$4%
99$5%
P"0.53
332$19
338$1.2
313$1.4
P"0.07
!Values are cited as a percent change relative to non-strained cultures. N"2 trials, 2 cultures per trial, 10 "elds imaged (10])/culture.
Fig. 5. (a) AP mRNA copy number at 20 Hz versus 1 Hz. Each reaction
(lanes 1}4) started with 12.5 ng total cytoplasmic RNA and (1) 19,246,
(2) 9623, (3) 4812, or (4) 2406 copies of cRNA standard. Target amplicon
is 501 bp and standard amplicon is 354 bp. (M) DNA molecular weight
marker. (b) Message copy number per cell (mean$S.D.). There are no
di!erences (p"0.09) in AP mRNA for strained (1 or 20 Hz) compared
to unstrained controls.
However, we have no a priori assurance the responses to
the mechanical environment are the same in part because
attachment to a substrate may be quite distinct from
attachment to matrix. In addition, we have no knowledge
of the actual magnitude of bone cell deformation
(`straina) in vivo (cell attachment to matrix is not rigid,
and we should not presume cell deformation is identical
to that of the surrounding matrix). Second, bone adaptation in vivo obviously re#ects protein production, maintenance, and degradation. In most experiments such as
ours, investigators consider only protein production. Obviously, a reduction in degradation can alter overall
tissue responses. However, in vitro systems a!ord a controlled manner to isolate, control, and subsequently explore individual mechanisms of responses, including
degradative enzymes. Third, at these small levels of deformation, we have no assurance that substrate strain re-
#ects cell deformation: at 1000 l-strain, the deformation
of a 10 lm cell would be below the level of detection (i.e.,
10 nm) for light microscopy. However, Neidlinger-Wilke
et al. (1994) documented cells exhibited approximately
88}90% substrate strain at 30,000 l-strain using identical
dishes. We presume that if cells maintain attachment at
such high levels of strain, they will maintain attachment
at these much lower levels. Fourth, since longer periods
at con#uence have a greater risk of cell necrosis and
detachment of the cultures from the surface of the wells
we performed assays at 72 h; this short time period
may also limit the impact of mechanotransduction responses. Fifth, the precise mechanical history triggering
responses is unknown. However, the strain magnitude,
frequency, and cycle number in this experiment are
physiologic for bone as a tissue (Lanyon et al., 1975,1979;
Lanyon, 1993) and at magnitudes believed to maintain
bone mass in vivo (McLeod and Rubin, 1992; Turner
et al., 1994).
Numerous in vivo and in vitro studies have been
performed with a variety of culture models, culture conditions, strain devices and outcome measures. Often,
these studies report con#icting results. There are many
potential explanations for these variations. First, cultures
from transformed cell lines, neo-natal primary cells or
aging cells di!er substantially. Brighton et al. (1991), used
a common model of osteoblasts derived from newborn
rat calvarium. Since cell expression diminishes as a function of patient and cellular aging (Fedarko et al., 1995), it
is likely that such results are not comparable to those
with older primary human cell cultures. Second, the
calvarium, with no muscle attachments, is not subjected
to signi"cant loads and experiences very low strains.
Rawlinson et al. (1995), demonstrated distinct cell responses from load-bearing and non-load-bearing bones.
Third, various growth mediums, serum concentrations,
etc., alter the complex responses (Owen et al., 1991). In
the current model system, we have observed where Vitamin D and estrogen (10~8M) reduces protein expres3
sion in the osteoporotic cultures, an observation reversed
by application of the strain regime (data not shown).
These observations suggest that a complex interaction
C.M. Stanford et al. / Journal of Biomechanics 33 (2000) 63}71
occurs between various environmental factors and argues against a conventional bias to consider only large
di!erences in in vitro measured outcome measurements
as constituting favorable responses. Fourth, investigators
report a wide variety of mechanical stimuli, including
magnitude, frequency, cycle number, and even varying
amounts of #uid #ow or inertial forces applied to cells
from overlying moving medium (Brown et al., 1998).
Owan et al. (1997) in an experiment intended to distinguish e!ects of #uid #ow and mechanical stretch concluded #ow, not stretch, stimulated cells. In many
experiments the coexistence of #ow and stretch may
make it di$cult to discern which is the actual stimulus. In
our experiments, the small displacements make substantial #uid #ow unlikely. However, many other factors
make direct comparison of results di$cult.
While some authors report no response to strains
below 3000 l-strain, one group reported modest but
statistically signi"cant increases of bone-related protein
(Type I collagen, non-collagenous protein) expression
and AP activity at 400 l-strain for newborn rat calvarial
osteoblast-like cells (Brighton et al., 1991). With a similar
culture model and higher strain magnitudes MikuniTakagaki et al. (1996) observed either no response or an
increase in response to strain. Fermor et al. (1998) observed variable responses in proliferation of bone cells
derived from osteoarthritic patients. These studies, however, demonstrated substantial variability and seemingly
con#icting results in replicate experiments (e.g., Brighton
et al., 1991; Fermor, et al., 1998). High variability of
primary human bone-like cell responses have also been
noted by Neidlinger-Wilke et al. (1994) although their
strain regimens were substantially higher than those reported here. Using sequential digestion of the bone samples to release more or less distinct cell populations
(osteoblastic versus osteocytic) Mikuni-Takagaki et al.
(1996) suggested the more mature osteocytic cells
were not responsive to even high levels of strain. Jones et
al. (1991) reported that Haversian-derived cells showed
no responses at 1000 and 3000 l-strain, while periosteal cells did respond at the higher levels. Similarly,
we observed no signi"cant increases in protein expression at a high physiological strain magnitude in aging
cells. As a part of the cellular aging process, it is possible
that the increasing mechanical thresholds proposed by
Frost (1987) may provide one explanation for the lack of
a response to the strain conditions utilized in this study.
Recently, Nicolella et al. (1998) and Nicolella (1998)
demonstrated localized areas within trabeculae (e.g.,
around lacunae and canaliculi) experience signi"cantly
greater (more than one order of magnitude) strain magnitudes than previously thought. This suggests osteocytic
cells may be exposed to complex #uid and #uid/matrixinduced forces which are considerably higher than previously considered. However, cell deformation in vivo is
not well understood and as noted earlier, we cannot
69
presume cell deformation in vivo re#ects deformation of
the matrix owing to #exibility and biological `plasticitya
of molecular attachments, even if such pericellular matrix
deformation is substantially higher than that at the tissue
level (Brand, 1992; Brand and Stanford, 1994). Nonetheless, if some cells are experiencing much higher deformations than at average (continuum) tissue levels (and
higher than those used in this study), there may be
relatively small populations of `sensinga cells causing
more widespread in vivo responses via some signaling
pathway to `respondinga cells. This argument may explain reports in which responses are observed at higher
strain magnitudes than are conventionally considered
physiologic for bone as a tissue, and the lack of responses most authors have reported at levels below 3000
l-strain.
The appropriate sequential expression of matrix-related proteins is critical to the orderly and optimal assembly of a matrix that has biological and mechanical
properties capable of normal load bearing capacity (Stein
et al., 1990). Disease states may cause altered mechanical
and architectural properties of bone that in turn may
alter the capacity of osteocytic and/or osteoblastic cells
to sense and respond to the mechanical environment.
Alterations in protein expression may not only in#uence
tissue properties through quantitative changes but also
through alterations in the spatial and temporal patterns
of bone-related protein expression. Descriptive statistical
approaches (e.g., means) may obscure biologically important changes in the variations and/or patterns of expression of various matrix proteins. The radar plot diagram
suggests that cultures derived from osteoarthritic patients tended to have a less uniform pattern of expression
relative to those derived from osteoporotic patients. An
evaluation for subject-to-subject variability (Bartlett
Homogeneity test) demonstrated that there was subject
speci"c responses for AP and OPN (Fig. 4a) in this group
of subjects. This suggests that the osteoporotic state may
be essentially unresponsive to strain, while the osteoarthritic is selectively responsive in the complex patterns of
matrix expression.
In summary, we found that primary bone cultures
from older individuals were non-responsive to a continuum magnitude regimen replicating that of an active
individual. It is possible that the sensing cells in bone
experience magnitudes of strain well exceeding the conditions in this experiment and those recorded by strain
gages in whole bone.
Acknowledgements
Supported in part by NIA Grant AG15197, The Roy J.
Carver Charitable Trust and by the Department of Orthopaedic Surgery and Dows Institute for Dental Research, The University of Iowa.
70
C.M. Stanford et al. / Journal of Biomechanics 33 (2000) 63}71
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