Am J Physiol Cell Physiol 287: C1527–C1536, 2004.
First published August 12, 2004; doi:10.1152/ajpcell.00059.2004.
Invited Review
“Culture shock” from the bone cell’s perspective: emulating physiological
conditions for mechanobiological investigations
Adam M. Sorkin,1,3 Kay C. Dee,2 and Melissa L. Knothe Tate1,3
1
Department of Biomedical Engineering and Department of Mechanical & Aerospace Engineering, Case
Western Reserve University, Cleveland, Ohio 44106; 2Department of Applied Biology and Biomedical
Engineering, Rose-Hulman Institute of Technology, Terre Haute, Indiana 47803; and 3Orthopaedic Research
Center, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195
osteoblast; osteocyte; tissue engineering; mechanobiology; mechanochemical
transduction; fluid flow
BONE CELLS RESIDE in a mechanically active, composite tissue
comprising an “organic-mineral” solid phase and a dynamic
aqueous phase that permeates spaces within the porous solid
matrix and acts as a carrier for ions, proteins, and other
nutrients. Bone’s mechanobiological function modulates its
organization and structure across length scales, e.g., from gross
anatomical features at the organ level to the microarchitecture
of trabeculae at the tissue level, to the anisotropic arrangement
of the canaliculi defining the pericellular space, to the arrangement of extracellular matrix molecules within the pericellular
space and extracellular matrix. More than a century of investigation provides a foundation for organ- and tissue-level
understanding of bone mechanobiology and its role in bone
physiology. In comparison, understanding of bone cell mechanobiology is in its nascency.
An understanding of bone cell mechanobiology is critical for
understanding of bone physiology because the cells are the
“micromachines” that actively model, maintain, and remodel
the tissue (71, 72). Mesenchyme-derived bone-lining cells and
osteoblasts lay down osteoid, an organic matrix, which is
Address for reprint requests and other correspondence: M. L. Knothe Tate,
Dept. of Biomedical Engineering and Dept. of Mechanical and Aerospace
Engineering, Case Western Reserve Univ., 10900 Euclid Ave., Olin 219,
Cleveland, OH 44106 (E-mail: [email protected]).
http://www.ajpcell.org
subsequently mineralized with a hydroxyapatite-like calcium
phosphate. Leukocyte-derived osteoclasts dissolve mineral and
digest matrix, providing space and potentially biochemical
cues for osteoblasts to lay down new bone matrix (135).
Osteoblasts that become embedded in new bone differentiate
into a network of interconnected osteocytes that may play a key
role in mechanotransduction (72). Remodeling provides a
means for repair and adaptation of bone tissue in response to
injury as well as changing mechanical and metabolic demands.
However, though cellular activities associated with remodeling
appear to be highly coordinated, the signaling and timing of
interactions among osteocytes, osteoclasts, osteoblasts, and
pluripotent cells are not clear. Although this has provided
impetus for continued investigation, in vivo and in situ investigations of bone cells and their coordination during modeling
and remodeling activity have been extremely limited.
One major barrier to understanding bone physiology at a
cellular level is the lack of methodology to study bone cells in
their native environment. Contributing to this difficulty are the
fundamentally different experimental approaches used to examine bone physiology/biology on multiple scales. Bone can
be approached as an organ within a system, where observations
of bone function can then be extrapolated empirically to create
informed hypotheses about the functions of the constituent
cells. Alternatively, the cells can be investigated independently
0363-6143/04 $5.00 Copyright © 2004 the American Physiological Society
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Sorkin, Adam M., Kay C. Dee, and Melissa L. Knothe Tate. “Culture shock”
from the bone cell’s perspective: emulating physiological conditions for mechanobiological investigations. Am J Physiol Cell Physiol 287: C1527–C1536, 2004.
First published August 12, 2004; doi:10.1152/ajpcell.00059.2004.—Bone physiology can be examined on multiple length scales. Results of cell-level studies,
typically carried out in vitro, are often extrapolated to attempt to understand tissue
and organ physiology. Results of organ- or organism-level studies are often
analyzed to deduce the state(s) of the cells within the larger system(s). Although
phenomena on all of these scales— cell, tissue, organ, system, organism—are
interlinked and contribute to the overall health and function of bone tissue, it is
difficult to relate research among these scales. For example, groups of cells in an
exogenous, in vitro environment that is well defined by the researcher would not be
expected to function similarly to those in a dynamic, endogenous environment,
dictated by systemic as well as organismal physiology. This review of the literature
on bone cell culture describes potential causes and components of cell “culture
shock,” i.e., behavioral variations associated with the transition from in vivo to in
vitro environment, focusing on investigations of mechanotransduction and experimental approaches to mimic aspects of bone tissue on a macroscopic scale. The
state of the art is reviewed, and new paradigms are suggested to begin bridging the
gap between two-dimensional cell cultures in petri dishes and the three-dimensional
environment of living bone tissue.
Invited Review
C1528
MECHANOBIOLOGICAL CONSIDERATIONS FOR BONE CELL CULTURE
AJP-Cell Physiol • VOL
biology at the organ, tissue, and cellular levels will advance the
ultimate goal of developing new strategies to prevent, cure,
and/or replace diseased bone tissue in the future.
BONE CELL LINES AND CULTURE
An inherent strength of cell culture is that the cells are
derived from a common source that can be passaged as many
as 50 times while maintaining the basic phenotypic characteristics of the line. This aids in study design because a “cohort of
study subjects” is readily available, and it allows for comparison of data from a given cell line that is cultured according to
standardized protocols. However, it should be noted that there
can be significant variation among cells isolated from a single
source; in fact, this is exploited to produce subclones of cell
lines with specifically desired characteristics (95, 113, 139).
Furthermore, comparison of data derived from studies using
different cell lines may not be appropriate, as it is analogous to
comparing outcome measures (e.g., bone remodeling dynamics) between different cohorts of patients or different species.
According to current practice, bone cells are either isolated
from primary human or animal tissue or obtained from an
immortalized or otherwise transformed cell line. Immortalized
cell lines take advantage of genetic instabilities in cells that
have been cultured from pathological source tissue, selected
from late-generation primary cultures, or transfected with specific genes. Osteoblast cell lines are commonly derived from
either animal or human osteosarcoma. A recently isolated
osteocyte-like cell line, murine long bone osteocyte Y-4
(MLO-Y4), exhibits high levels of osteocalcin and low levels
of alkaline phosphatase production as well as expression of gap
junctional proteins such as connexin 43 (Cx43), similar to
primary osteocytes and distinct from primary osteoblasts (66);
hence, this cell line is used almost exclusively as an osteocyte
model (Fig. 1, refer to Table 1 for an overview of bone cell
lines reported in the literature).
Alternatively, primary osteogenic cell cultures can be obtained directly from healthy donor tissue and are, as such, the
“real cells,” transplanted from the endogenous to an exogenous
environment. Major drawbacks of this approach are that considerable variation can exist between cells from different tissue
sources, cell populations obtained from a single tissue source
can be heterogeneic depending on isolation and purification
procedures, and these cell cultures can typically be maintained
for only three to four generations before phenotypic drift
becomes evident. In fact, significant changes in behavior (contractile force) have been reported between cells of successive
generations within only three passages (154). A variety of
methods are employed to isolate primary cells. One common
method involves digestion of the minced bone explanted from
sites such as long bones, (fetal) calvaria, mandibles, or the iliac
crest with collagenase or similar enzymes, followed by subculture of the resulting cells (17, 83, 143). A variation on this
technique entails culture of cells that grow out of undigested
bone chips (41). A potential shortcoming of this approach is
that it does not allow selection or exclusion of particular cell
types; however, the use of improved culture substrates with
better bone fixation (137) and the inclusion of differentiationinducing osteogenic factors such as dexamethasone, ascorbate,
and phosphates in the culture media generate cells that are
distinctly osteogenic (63). Osteogenic precursor cells are oth-
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of their native environment, i.e., outside of the physiological
system, organ, tissue, and perhaps even excluding the extracellular matrix. Macroscopic investigations may succeed in
maintaining the native mechanobiological environment but by
definition fall short in allowing for observation of cell-level
responses. In contrast, a rich armamentarium of techniques has
been developed to measure cell activity outcomes precisely in
vitro. An inherent drawback of in vitro studies is that the cell
is cultured in an exogenous setting. When a cell is removed
from its native environment and reintroduced into an extracorporeal setting, it is expected that the cell will have to adapt to
its new environment; the cell must recondition itself to respond
to different cues. We suggest that that this “culture shock”
(116) can be mitigated by better emulating physiological conditions in vitro.
Some of the distinct advantages of the cell culture methodology include controlled definition of culture environment
conditions, including pH, temperature, osmolarity, and solute
concentration. Control of these parameters allows for the
design of experiments that would not be feasible in situ or in
vivo. This is due to the fact that the biochemical and mechanobiological milieu of bone cells is challenging to observe in
situ and in vivo. Nonetheless, specific, basic environmental
parameters, including pH, osmolarity, and solute concentration, have not been measured extensively in vivo. For example,
it is known that bone extracellular fluid is distinctly different
from that of plasma or interstitial fluid, e.g., in other inner
organs, particularly considering its high content of K⫹ (71). In
the same vein, it is assumed, although not yet measured in situ,
that mechanical loads imparted through bone as a natural
consequence of physiological activity influence the local mechanobiological environment of the cell through both direct
deformation of the pericellular environment and indirect effects of load-induced fluid movement, including drag and shear
stresses at the cell surface, as well as transport of nutrients,
chemokines, and other osteotropic factors (71). Furthermore,
although a number of technologies are being developed to
optimize cell culture environments, no single approach has
been developed to bridge the gap in understanding between
single-cell physiology, the physiology of cells cultured in
groups (e.g., to confluency), and the physiology of cellular
networks in tissues and organs. Several multidisciplinary approaches have been applied recently. These include the use of
knockout mice with musculoskeletal modifications (58) as well
as the identification of genes responsible for regulation of
osteogenic cytokines (13) and genes giving rise to various bone
disorders (38, 84). Furthermore, animal models have been
developed and refined to correlate bone morphology and
damage with apoptosis (20, 92) to study fracture repair
(93), microgravity effects (21), and pathological conditions
(62, 91, 119).
Whereas the local environment of a cell may modulate its
activity at any given time, cell-level communication and transport are coordinated across the tissue and at the organ level
(74). Few studies integrate findings from organ-, tissue-, and
cell-level studies, which hinders translation of new fundamental insights to clinical modalities. Hence, the purpose of this
compendium is to examine current approaches to bone cell
culture to reduce “cellular culture shock” through improvement
of methodologies emulating physiological conditions. Identifying commonalities as well as implications of bone mechano-
Invited Review
C1529
MECHANOBIOLOGICAL CONSIDERATIONS FOR BONE CELL CULTURE
Fig. 1. Osteocyte-like cells in culture vs.
osteocytes in situ. A: murine long bone osteocyte Y-4 (MLO-Y4) cells in culture exhibit morphology and biochemical characteristics distinct from osteoblast-like cell
lines and similar to osteocytes. [Reprinted
with kind permission from ecmjournal.org
and Dr. Brendan Noble (90).] B: 3-dimensional (3-D) reconstruction of a confocal
image stack of osteocytes in situ, from the
ulna of skeletally mature rats that was dissected out, fixed, and embedded in polymethyl methacrylate before histological sectioning.
established without a significant shift in the current paradigm.
However, more routine use of multiple cell lines within similar
experimental contexts may better define the differences among
the cell lines and provide a more complete understanding of the
implications of their use in mechanobiological research (for
further review of bone cell and tissue culture methods, see
Ref. 85a).
ROLE OF THE “THIRD DIMENSION” IN EMULATING THE
CELLULAR ENVIRONMENT
Regardless of their source, bone cells removed from their
native setting occupy an exogenous environment. Although the
environment is not alien enough to kill the cell, the effects of
such environmental changes have not been thoroughly investigated. For example, two-dimensional culture models of osteocytes may not adequately mimic the behavior of native
osteocytes that reside naturally in a disparate three-dimensional
network. The complex mechanochemical environment of the
periosteal and endosteal surfaces, cutting cones, lacunae within
bone, or the marrow cavity is often replaced by culture on flat
plastic surfaces, within a nutrient medium bereft of all but the
most essential ions, peptides, and proteins. Response to these
abrupt and substantial changes, or culture shock, may play a
significant role in altering the response of cultured mammalian
cells and in inducing cellular senescence (116, 144).
Table 1. A selection of osteoblast and osteoclast cell lines used in in vitro models
Cell Line
Phenotype
Source
UMR-106
UMR-108
MC3T3-E1 subclones
4 and 14
MC3T3-E1 subclones
24 and 30
MG-63
HOS TE85
U-2 OS
MLO-Y4
MLO-A5
HOB-01-C1
Osteoblast
Osteoblast
Osteoblast/preosteoblast
Rat osteosarcoma
Rat osteosarcoma
Mouse calvarial fibroblast
Osteoblast/preosteoblast
Osteoblast/preosteoblast
Osteoblast
Osteoblast
Osteocyte
Preosteocyte
Preosteocyte
Characteristics
Ref.
ATCC No.
Responsive to PTH and PGE
Similar to UMR-106, less PTH sensitive
Similar to primary calvarial osteoblasts
35
35
139
Mouse calvarial fibroblast
Poor mineralization
139
Human osteosarcoma
Human osteosarcoma
Human osteosarcoma
Transgenic murine long bone
Transgenic murine long bone
Osteoarthritic human femoral head
Hypotriploidy common, interferon producing
Susceptible to transformation
8
86
103
66
65
10
CRL-1661
CRL-1663
CRL-2593,
CRL-2594
CRL-593,
CRL-2595
CRL-1427
CRL-1543
HTB-96
N/A
N/A
N/A
Similar to primary osteocytes
Potentially postosteoblast/preosteocyte
Responsive to calcitropic hormones
PTH, parathyroid hormone; PGE, prostaglandin E; ATCC, American Type Culture Collection.
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erwise routinely isolated from bone marrow and can become
mature osteogenic cells by the inclusion of osteogenic factors
in the culture medium (50, 134). Many current efforts to
improve the homogeneity of marrow-derived cells include
utilization of protocols that take advantage of antibody-based
detection of select membrane receptors that indicate predisposition to specific cell types (107, 125). Primary osteoclast
culture is also possible via isolation from bone tissue (143)
and, more efficiently, from the hematopoietic stem cells in
bone marrow via exposure to intercellular signaling factors,
including osteoclast growth factor (30), specific interleukins
(44), macrophage-stimulating factor (128), colony-stimulating
factors (76, 120, 126, 147), receptor activator of nuclear
factor-␬B ligand (RANKL) (127, 148), tumor necrosis factor
(81), osteoprotegerin (121), and B lymphocytes (57). Coculture
with osteoblasts and osteocytes known to express these factors
also induces osteoclastogenesis, which can be enhanced, in
turn, by a wide variety of stimulating factors (6a, 29, 67).
Although the use of certain specific cell lines, such as
MC3T3-E1 subclones for osteoblast-like cells and MLO-Y4
cells for osteocyte-like cells (2, 110, 132, 149), is becoming
more commonplace, there remains no standard cell line or
preferred isolation method for bone cell study and no standardized maintenance protocols following culture. Because these
methods tend to be selected for ease of use, for known
responses to particular stimuli, and to model specific stages of
the osteocyte lineage, it is unlikely that standards will be
Invited Review
C1530
MECHANOBIOLOGICAL CONSIDERATIONS FOR BONE CELL CULTURE
INFLUENCES OF CULTURE MEDIUM ON CELL FUNCTIONS
MECHANICAL MANIPULATION OF CELLS: SUBSTRATE
DEFORMATION AND FLUID SHEAR
Application of mechanical stress through gross environmental changes is relatively easy to achieve, but other methods
offer better control and provide for more straightforward ob-
Fig. 2. Cell volume change induced by osmotic pressure. In the hypertonic condition (A), water moves up the concentration
gradient, causing crenation. The cell is at equilibrium with an isotonic environment (B). In a hypotonic medium (C), water swells
the cell. It is possible that ion channels may allow the cell to actively regulate its volume and return to its original state.
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Cell lines derived from murine tissue, a common culture
source, are prone to react poorly to inadequate in vitro conditions. One of the principal possible causes of this is excess
oxidative stress found in culture environments; standard culture conditions (95% air-5% CO2, 37°C) lead to formation of
reactive oxidative species that are normally limited in vivo
(45). Interestingly, although cell biologists have developed and
optimized culture media so that cells remain viable and functional, the chemical composition of bone fluids are not yet well
characterized (71). It is not clear how differences between the
well-defined exogenous medium and the poorly defined endogenous medium could affect cell behavior. Nonetheless, definition of standards for in vitro study allows exploitation of a
cell’s sensitivity to changes in medium composition.
One such technique, varying the normal 285–310 mosmol/l
(42, 133, 146) osmolarity of the medium by altering the
concentration of ionic species (including K⫹, Ca2⫹, and Na⫹,
as well as nonionic species such as complex sugars) yields a
number of effects. Decreasing the osmolarity exerts swelling
pressure on cells; increasing the osmolarity causes the cells to
shrink initially (Fig. 2). Swelling induces membrane stretch,
which can be employed to manipulate the cytoskeleton (42).
This phenomenon has also been used extensively to investigate
the active regulation of volume by osteoblasts and other cells
via regulation of ion flux, useful in defining and exploiting
Ca2⫹-, K⫹-, and Na⫹-regulated signaling pathways (42, 133,
146). The electrical activity resulting from ion flows can be
measured using patch-clamp techniques in which microelectrodes are inserted into cells (46). This approach has been used
to characterize ion channel activity in bone cells (104, 153).
servation and quantification of cellular response. Traditional in
vitro methods typically apply unidirectional, biaxial, radial,
and/or bending loads to the culture substrate. In addition, these
methods often presume uniform deformation of cells, and
allow for quantification of changes in cell physiology such as
Ca2⫹ ion flux (136), regulatory effects of parathyroid hormone
(PTH) (22), integrin expression (23), prostaglandin E2 (PGE2)
production (108), collagen production (48), insulin-like growth
factor (151), and others. However, it is becoming apparent that
direct mechanical strain at physiological levels in bone tissue
may not stimulate a response in cells (14, 97, 151) and that
traditional in vitro methods are not as demonstrative as more
physiologically relevant models could be. Even a seemingly
simple experimental manipulation may have complex repercussions and result in confounding stimuli. For example, substrate deformation in aqueous medium exposes the attached
cell monolayer to an unknown degree of fluid shear (16),
limiting the precise control of culture environment and complicating the interpretation of results, leading to ambiguity in
determining whether the substrate deformation or the associated fluid flow is the critical stimulus.
Interstitial fluid flow is increasingly implicated as a direct
mediator of mechanical forces both throughout bone and to the
cells that inhabit bone. Examination of the effects of flow on
bone physiology have likewise become of interest to physiologists and biologists. Because of practical difficulties in studying bone fluid flow in situ during normal physiological activity,
cell perfusion chambers have been developed to simulate
physiological fluid flow (as seen in locations such as vascular
endothelium) to observe cellular responses in vitro. In particular, the pressure-driven parallel-plate perfusion chamber design has been implemented (36, 59, 61) and optimized (15, 27,
112) for application of steady, oscillatory, or pulsatile fluid
shear stresses to cell monolayers, allowing correlation of fluid
shear stresses and nutrient transport with cell activity and
adaptation (Fig. 3).
Invited Review
MECHANOBIOLOGICAL CONSIDERATIONS FOR BONE CELL CULTURE
C1531
complex chemical gradients into parallel-plate flow chambers,
to provide more a more physiological environment.
FLOW-REGULATED CELLULAR MECHANISMS
OF MECHANOTRANSDUCTION
Variations of the parallel-plate flow chamber design have
become commonplace tools in cell biology research and provide a basis for current in vitro modeling of many physiological flow regimes. Though developed to simulate better understood biological flows, this technology has been embraced by
bone cell experimentalists. Flow regimes believed to be relevant to bone have been studied extensively. Flow chamber
parameters are versatile and have allowed investigation of
many distinct phenomena, including PGE2 upregulation (69,
105), NO䡠 synthesis (69), Ca2⫹ flux within monolayers (142),
and others. In addition to these early bone cell-related applications (59), fluid shear is currently used to investigate phenomena including mechanical regulation of integrin expression
(100), actin cytoskeleton reorganization (25), gap junctional
protein expression (132), and the relationship between shortterm PGE2 production and mineralization (89) as well as to
probe the underlying cellular mechanisms of transduction of
fluid shear (19).
This approach has obvious advantages for investigating
effects of fluid shear in many biomedical arenas, but it is not
known how well these in vitro flow chambers perform, e.g., in
achieving a desired stress at the cell level and in emulating
physiological flow regimes (4, 73). The type of flow (steady,
pulsatile, oscillatory, turbulent, or combinations thereof) plays
a significant role in eliciting a cellular response in vitro that
may or may not be predictive of cellular response in vivo, but
this is often ignored (150). Oscillatory flow might be expected
in a physiological setting, but cell monolayers can often be
more responsive to flow with continuous components (61).
Furthermore, local variations in the flow profile due to uneven
topography of the monolayer are generally not accounted for
(16), and few studies to date have reported how flow regimes
induced through common and commercially available flow
chamber designs compare (4). In addition, although these
chambers have been used to examine chemokinetic modulation
of shear response (5), they provide little control over chemokinetic gradients. However, recent advances in microfabrication techniques (80) could potentially allow incorporation of
independently driven microchannels capable of maintaining
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Fig. 3. Fluid shear in a parallel-plate flow chamber. Fluid flow applies shear
stress to a monolayer of cultured cells. Applied stress is often approximated by
the formula for wall shear stress: ␶wall ⫽ 6␮Q/bh2, where ␮ is the fluid
viscosity, Q is the flow rate, b is the width of the parallel plates, and h is the
separation of the parallel plates. Note that on the microscopic scale, the
monolayer topography is irregular. Most analyses of this experimental system
assume a uniform geometry when, in fact, the irregularities are likely to cause
differential stresses along the cells.
Use of in vitro techniques, and perfusion chambers in
particular, has provided much insight into bone physiology
when taken in context of the better understood processes of
primary and secondary osteogenesis. As osteoblasts lay down
new osteoid, many become embedded in the matrix and differentiate into osteocytes that occupy lacunae in bone. As bone
forms, individual osteocytes retain communication with one
another (as well as with osteoblasts and bone lining cells) via
processes occupying canaliculi that extend throughout the
bone. This results in a syncytium of spatially remote cells that
remain in contact with one another. Fluid movement through
the lacunocanalicular pericellular spaces has been postulated as
a key mediator of mechanotransduction via direct fluid shear
effects as well as the ancillary transport of chemokinetic
factors (71), but the active response of the osteocyte has not
been thoroughly investigated until recently.
Although the manner in which osteocytes sense fluid flow
has not yet been elucidated through use of common in vitro
modeling techniques, the osteocyte’s interaction with the contents of the pericellular space has been identified as a putative
factor for transduction of fluid shear. Ca2⫹ concentration,
important to many bone cell functions, appears to influence
fluid movement directly through osteoblast monolayers (55).
The glycocalyx, or “cell coat” believed to be composed of
hyaluronic acid chains with glycosaminoglycan side chains
that anchor the cell to the lacuna wall, plays a significant, but
not exclusive, role. In cells exposed to fluid flow, after removal
of the hyaluronic acid from the cell surface, prostaglandin
expression is reduced whereas Ca2⫹ flux is not (110). Another
mechanism must exist for sensing flow, whether through another unknown component of the glycocalyx or, possibly, via
a more direct contact with the membrane itself. Other kinds of
membrane deformation in bone cells provoke Ca2⫹ flux, possibly via cytoskeletal deformation (42, 133, 146), which has
been shown to occur rapidly and which may redistribute
intracellular forces (53). Fluid shear disruption of integrin/
ECM ligand interactions, which causes a dynamic unbinding/
binding situation that stimulates production of signaling proteins, has been identified as a mechanotransduction mechanism
in the vascular endothelium (62). It may be possible that a
similar effect is at work in bone; integrin expression and matrix
interaction have already been linked to osteoblast differentiation (7) as well as to Ca2⫹ channel kinetics of osteoblasts (96)
and osteoclasts (33). A new paradigm in which some of these
key in vitro experimental concepts are adapted and applied to
an in vivo or ex vivo system may provide a crucial next step to
decipher mechanotransduction mechanisms in situ through
observation of native osteocytes with intact glycocalyx components, other extracellular bindings, and, presumably, different cytoskeletal conformations reacting to shear from flow
through the lacunocanalicular system.
Modulation of bone cell responses to fluid shear is better
understood than are the underlying mechanisms of bone mechanotransduction. Osteocytes communicate with one another as
well as with osteoblasts and bone lining cells via gap junctions
Invited Review
C1532
MECHANOBIOLOGICAL CONSIDERATIONS FOR BONE CELL CULTURE
POTENTIAL OF THREE-DIMENSIONAL MODELS
Intercellular communication can be examined by two-dimensional planar flow schemes that allow formation of intercellular
junctions, but their ability to simulate other phenomena that are
highly dependent on the porous microarchitecture of bone, such as
flow-related nutrient transport, is necessarily limited. Several new
Fig. 4. Spinner flask bioreactor. Stirring of fluid culture medium overcomes
diffusive limitations of scaffold to promote penetration of cells into a 3-D
scaffold. [Adapted from Ref. 138.]
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Fig. 5. Flow perfusion bioreactor. An applied pressure gradient forces fluid to
perfuse the 3-D structure. Lateral constraints prevent flow out of the sides of
the construct. [Adapted from Ref. 6.]
bioreactor technologies are designed to overcome this limitation
by employing three-dimensional convective fluid flow strategies.
Suspension cultures, in which cells are cultured on microcarrier
substrates, can be maintained in a dynamic fluid environment to
apply shear stresses and altered nutrient gradients (12, 105).
Several methods apply fluid flow through transudant structures.
One of the simplest is the spinner flask, in which a scaffold is
seeded with cells (Fig. 4) and submerged in culture medium that
is continuously stirred by a magnetic bar (138). Though effective
for cartilage tissue engineering, this strategy does not appear to
provide internal nutrient gradients conducive to colonization of
scaffold interiors by osteogenic cells (120). Similar approaches in
which the culture vessel wall rotates can yield poor-quality engineered tissues as well (39). One promising technology, the flow
perfusion bioreactor, directly applies fluid pressure to a variety of
Fig. 6. Device for application of 4-point bending. Deformation due to applied
loads induces fluid flow into a porous specimen, improving penetration of cells
and cell viability within the structure. P, applied load; a, distance between
support and load; L, distance between supports. [Adapted from Ref. 118.]
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(31), characterized by the presence of Cx43 hemichannel
subunits (26, 59, 149) capable of transmitting survival signals
such as ERK (101) and NO䡠 (86). Communication via Cx43
channels is modulated by fluid shear stresses, particularly in
the presence of prostaglandins (64), indicating a clearly defined
system by which fluid flow around one osteocyte regulates
communication between distant cells. Interestingly, PGE2 production by osteocytes is dependent on the actin cytoskeleton
(3) and can alter conformation of the actin fibers (145) in
osteocytes, possibly indicating a cytoskeletal basis for a mechanotransduction pathway as has been postulated elsewhere (18,
114, 152). Osteocytes produce transforming growth factor-␤
(TGF-␤), which can be regulated by Cx43 in other cell types
(56) and which inhibits bone resorption by osteoclasts (52).
This suggests at least one pathway by which fluid flow generated by applied loads can be sensed by cells. The cells, in turn,
can regulate processes crucial to bone remodeling in ways that
are consistent with fracture repair, functional adaptation, and
theories indicating a cellular basis for various pathologies (71).
A reduction in local fluid flow stimulus to osteocytes, whether
due to inactivity, microgravity effects, damage, or a change in
sensory network, can subsequently decrease intercellular communication, which would downregulate the inhibition of osteoclasts, increasing net resorption. Similar effects might also be
expected if reduction of fluid flow stemmed nutrient transport
to osteocytes, making the osteocytic syncytium less viable and
therefore less functional.
Invited Review
MECHANOBIOLOGICAL CONSIDERATIONS FOR BONE CELL CULTURE
AJP-Cell Physiol • VOL
SUMMARY
Cells removed from their native environments and maintained
in vitro by current methods experience culture shock that is poorly
understood. Cell types of many different and distinct lineages are
incorporated into in vitro osteogenic models and yield conflicting
results. Directed differentiation of cells in culture to yield osteoblast-like cells is a commonly used and well-understood technique; native differentiation into, for example, the osteocyte phenotype, which accounts for 90% of cells in healthy bone (72), is
less well understood. Accordingly, a majority of in vitro research
has focused on the investigation of fluid flow-related mechanobiology of osteoblast-like cells, even though the osteocytic syncytium has been indicated as the primary mechanosensory element
in bone. However, new insights into mechanotransductive pathways are paving the way to new experimental methods that more
accurately represent the in vivo environment. An evolving understanding of mechanobiological aspects of bone formation and
functional adaptation on multiple scales— cell, tissue, and organ—is providing a basis for future research able to advance
fundamental science while improving clinical translation.
ACKNOWLEDGMENTS
We gratefully acknowledge Beth Halasz, MFA, and the Dept. of Medical
Illustration, The Cleveland Clinic Foundation, for all illustrations, published
with the permission of The Cleveland Clinic Foundation.
GRANTS
This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant R21 AR-049351-01 (to M. L. Knothe Tate).
REFERENCES
1. Adachi T, Sato K, and Tomita Y. Directional dependence of osteoblastic calcium response to mechanical stimuli. Biomech Model Mechanobiol
2: 73– 82, 2003.
2. Ahuja SS, Zhao S, Bellido T, Plotkin LI, Jimenez F, and Bonewald LF.
CD40 ligand blocks apoptosis induced by tumor necrosis factor alpha,
glucocorticoids, and etoposide in osteoblasts and the osteocyte-like cell line
murine long bone osteocyte-Y4. Endocrinology 144: 1761–1769, 2003.
3. Ajubi NE, Klein-Nulend J, Nijweide PJ, Vrijheid-Lammers T, Alblas
MJ, and Burger EH. Pulsating fluid flow increases prostaglandin
production by cultured chicken osteocytes—a cytoskeleton-dependent
process. Biochem Biophys Res Commun 225: 62– 68, 1996.
4. Anderson E, Sorkin A, and Knothe Tate ML. Performance evaluation
of commercial cell flow chambers: how well is stress controlled at a
cellular level? ASME International Mechanical Engineering Congress,
Anaheim, CA, November 13–19, 2004. In press.
5. Bakker AD, Klein-Nulend J, and Burger EH. Mechanotransduction in
bone cells proceeds via activation of COX-2, but not COX-1. Biochem
Biophys Res Commun 305: 677– 683, 2003.
6. Bancroft GN, Sikavitsas VI, and Mikos AG. Design of a flow perfusion bioreactor system for bone tissue-engineering applications. Tissue
Eng 9: 549 –554, 2003.
6a.Bar-Shavit Z, Teitelbaum SL, Reitsma P, Hall A, Pegg LE, Trial J,
and Kahn AJ. Induction of monocytic differentiation and bone resorption by 1,25-dihydroxyvitamin D3. Proc Natl Acad Sci USA 80: 5907–
5911, 1983.
7. Bennett JH, Carter DH, Alavi AL, Beresford JN, and Walsh S.
Patterns of integrin expression in a human mandibular explant model of
osteoblast differentiation. Arch Oral Biol 46: 229 –238, 2001.
8. Billiau A, Edy VG, Heremans H, Van Damme J, Desmyter J,
Georgiades JA, and De Somer P. Human interferon: mass production in
a newly established cell line, MG-63. Antimicrob Agents Chemother 12:
11–15, 1977.
9. Biot MA. Theory of elasticity and consolidation for a porous anisotropic
solid. J Appl Physiol 26: 182–185, 1955.
10. Bodine PV, Vernon SK, and Komm BS. Establishment and hormonal
regulation of a conditionally transformed preosteocytic cell line from
adult human bone. Endocrinology 137: 4592– 4604, 1996.
287 • DECEMBER 2004 •
www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on June 17, 2017
engineered scaffolds. The scaffold construct is constrained within
a tightly fit cassette. Fluid is pumped through the top, moves
through the entire porous structure, and exits through the bottom.
This design effects a more homogenous distribution of nutrients,
and osteogenic cell penetration is similarly distributed (6). Although only steady flow regimes have been used to date, the
design should allow for potentially more pulsatile and oscillatory
effects to be studied as well (Fig. 5).
Another device simulates physiology to a greater degree
than the perfusion bioreactor by relying on mechanical loading
to induce flow. Tissue scaffolds are analogous to stiff, fluidfilled sponges. Compression of fluid-containing spaces induces
flow out of the scaffold, and relaxation draws flow back into
the scaffold. Applications of this poroelasticity theory have
predicted load-induced flow in bone (9, 101), which has recently been demonstrated experimentally (73). The aforementioned device capitalizes on this concept and utilizes/harnesses
load-induced flow as a means for enhanced nutrient transport:
cyclical four-point bending is applied to a three-dimensional
beam in an aqueous culture environment for extended periods
of time. It can accommodate explanted tissue, synthetic resorbable polymer foams, and biopolymer constructs. Intended for
use with rigid, porous structures similar to bone, it can potentially be used to study induced penetration of fluid into structures and the enhancement of nutrient transport into structures
under various mechanical loading regimes. It has already been
shown to alter penetration and long-term viability of osteogenic cells in a scaffold interior, as well as altering their matrix
mineralization behavior (118) (Fig. 6).
A number of the aforementioned three-dimensional cell culture
approaches lend themselves to application with explanted bone to
study ex vivo effects, potentially a powerful strategy for better
understanding of bone physiology and biology on multiple scales.
Taking this one step further, tissue engineering necessitates the
combination of cell culture with complex three-dimensional structures (scaffolds), requiring the further development of new analytical techniques to investigate cellular function in situ. It is
expected that as these techniques are developed, they could be
adapted readily to study bone per se, either ex vivo or in vivo.
Furthermore, new imaging and immunohistochemical tools that
have been developed to probe microfluidic movement and transport in bone (71) can be applied to an engineered tissue. Finally,
as new fabrication processes provide greater control over the
microarchitecture of tissue engineering scaffolds (115, 141), the
potential exists to develop new, more accurate in vitro models that
1) incorporate structural elements common to various bone tissue
pathologies; 2) provide an experimental test bed to evaluate
analytical models that predict structural properties of bonelike
mechanochemical transduction (123), hydraulic conductance (88),
or streaming potential (47); and 3) integrate more complex fluid
sources that imitate flow into and out of vasculature or mimic
material aspects of bone, such as piezoelectric effects (49). With
the increased availability of technology to observe biophysical
phenomena at the cellular level comes an increasing impetus to
challenge and, in some cases, discard past assumptions. This
should, in turn, improve the understanding of bone mechanobiology and physiology from the cellular to the organ level and vice
versa.
C1533
Invited Review
C1534
MECHANOBIOLOGICAL CONSIDERATIONS FOR BONE CELL CULTURE
AJP-Cell Physiol • VOL
36. Frangos JA, McIntire LV, and Eskin SG. Shear stress induced stimulation
of mammalian cell metabolism. Biotechnol Bioeng 32: 1053–1060, 1988.
37. Gao F and Ma XL. Anion channels influence ECC by modulating L-type
Ca2⫹ channel in ventricular myocytes. J Appl Physiol 93: 1660 –1668, 2002.
38. Garnero P and Delmas PD. Biomarkers in osteoarthritis. Curr Opin
Rheumatol 15: 641– 646, 2003.
39. Goldstein AS, Juarez TM, Helmke CD, Gustin MC, and Mikos AG.
Effect of convection on osteoblastic cell growth and function in biodegradable polymer foam scaffolds. Biomaterials 22: 1279 –1288, 2001.
40. Gomes ME, Sikavitsas VI, Behravesh E, Reis RL, and Mikos AG.
Effect of flow perfusion on the osteogenic differentiation of bone marrow
stromal cells cultured on starch-based three-dimensional scaffolds.
J Biomed Mater Res 67A: 87–95, 2003.
41. Guerriero C, De Santis D, Nocini PF, Gotte P, and Armato U. Tissue
culture of adult human osteoblasts isolated from jaw bones. Ital J Anat
Embryol 100, Suppl 1: 83–93, 1995.
42. Guilak F, Erickson GR, and Ting-Beall HP. The effects of osmotic
stress on the viscoelastic and physical properties of articular chondrocytes. Biophys J 82: 720 –727, 2002.
43. Gurevich O, Vexler A, Marx G, Prigozhina T, Levdansky L, Slavin
S, Shimeliovich I, and Gorodetsky R. Fibrin microbeads for isolating
and growing bone marrow-derived progenitor cells capable of forming
bone tissue. Tissue Eng 8: 661– 672, 2002.
44. Hagenaars CE, van der Kraan AA, Kawilarang-de Haas EW, Visser
JW, and Nijweide PJ. Osteoclast formation from cloned pluripotent
hemopoietic stem cells. Bone Miner 6: 179 –189, 1989.
45. Halliwell B. Oxidative stress in cell culture: an under-appreciated problem? FEBS Lett 540: 3– 6, 2003.
46. Hamill OP, Marty A, Neher E, Sakmann B, and Sigworth FJ. Improved
patch-clamp techniques for high-resolution current recording from cells and
cell-free membrane patches. Pflügers Arch 391: 85–100, 1981.
47. Harrigan TP and Hamilton JJ. Bone strain sensation via transmembrane potential changes in surface osteoblasts: loading rate and microstructural implications. J Biomech 26: 183–200, 1993.
48. Harter LV, Hruska KA, and Duncan RL. Human osteoblast-like cells
respond to mechanical strain with increased bone matrix protein production
independent of hormonal regulation. Endocrinology 136: 528 –535, 1995.
49. Hastings GW and Mahmud FA. Electrical effects in bone. J Biomed
Eng 10: 515–521, 1988.
50. Haynesworth SE, Goshima J, Goldberg VM, and Caplan AI. Characterization of cells with osteogenic potential from human marrow. Bone
13: 81– 88, 1992.
51. Heidemann SR, Lamoureux P, Ngo K, Reynolds M, and Buxbaum
RE. Open-dish incubator for live cell imaging with an inverted microscope. Biotechniques 35: 708 –716, 2003.
52. Heino TJ, Hentunen TA, and Vaananen HK. Osteocytes inhibit
osteoclastic bone resorption through transforming growth factor-beta:
enhancement by estrogen. J Cell Biochem 85: 185–197, 2002.
53. Helmke BP, Thakker DB, Goldman RD, and Davies PF. Spatiotemporal analysis of flow-induced intermediate filament displacement in
living endothelial cells. Biophys J 80: 184 –194, 2001.
54. Hildebrand G, Kunze S, and Driver M. Blood cell adhesion on sensor
materials studied by light, scanning electron, and atomic-force microscopy. Ann Biomed Eng 29: 1100 –1105, 2001.
55. Hillsley MV and Frangos JA. Osteoblast hydraulic conductivity is
regulated by calcitonin and parathyroid hormone. J Bone Miner Res 11:
114 –124, 1996.
56. Hirschi KK, Burt JM, Hirschi KD, and Dai C. Gap junction communication mediates transforming growth factor-␤ activation and endothelial-induced mural cell differentiation. Circ Res 93: 429 – 437, 2003.
57. Horowitz MC and Lorenzo JA. The origins of osteoclasts. Curr Opin
Rheumatol 16: 464 – 468, 2004.
58. Horton WA. Skeletal development: insights from targeting the mouse
genome. Lancet 362: 560 –569, 2003.
59. Hung CT, Pollack SR, Reilly TM, and Brighton CT. Real-time
calcium response of cultured bone cells to fluid flow. Clin Orthop 313:
256 –269, 1995.
60. Ilvesaro J, Vaananen K, and Tuukkanen J. Bone-resorbing osteoclasts
contain gap-junctional connexin-43. J Bone Miner Res 15: 919 –926, 2000.
61. Jacobs CR, Yellowley CE, Davis BR, Zhou Z, Cimbala JM, and
Donahue HJ. Differential effect of steady versus oscillating flow on
bone cells. J Biomech 31: 969 –976, 1998.
62. Jalali S, del Pozo MA, Chen K, Miao H, Li Y, Schwartz MA, Shyy
JY, and Chien S. Integrin-mediated mechanotransduction requires its
287 • DECEMBER 2004 •
www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on June 17, 2017
11. Borke JL, Yu JC, Isales CM, Wagle N, Do NN, Chen JR, and Bollag
RJ. Tension-induced reduction in connexin 43 expression in cranial
sutures is linked to transcriptional regulation by TBX2. Ann Plast Surg
51: 499 –504, 2003.
12. Botchwey EA, Pollack SR, El Amin S, Levine EM, Tuan RS, and
Laurencin CT. Human osteoblast-like cells in three-dimensional culture
with fluid flow. Biorheology 40: 299 –306, 2003.
13. Bouletreau PJ, Warren SM, and Longaker MT. The molecular biology of distraction osteogenesis. J Craniomaxillofac Surg 30: 1–11, 2002.
14. Brand RA, Stanford CM, and Nicolella DP. Primary adult human bone
cells do not respond to tissue (continuum) level strains. J Orthop Sci 6:
295–301, 2001.
15. Brown DC and Larson RS. Improvements to parallel plate flow chambers to reduce reagent and cellular requirements. BMC Immunol 2: 9,
2001. First published September 11, 2001; doi:10.1186/1471-2172-2-9.
16. Brown TD. Techniques for mechanical stimulation of cells in vitro: a
review. J Biomech 33: 3–14, 2000.
17. Burger EH, Boonekamp PM, and Nijweide PJ. Osteoblast and osteoclast precursors in primary cultures of calvarial bone cells. Anat Rec
214: 32– 40, 1986.
18. Burger EH and Klein-Nulend J. Microgravity and bone cell mechanosensitivity. Bone 22, Suppl 5: 127S–130S, 1998.
19. Burger EH and Klein-Nulend J. Mechanotransduction in bone—role of
the lacuno-canalicular network. FASEB J 13, Suppl: S101–S112, 1999.
20. Burr DB and Radin EL. Microfractures and microcracks in subchondral
bone: are they relevant to osteoarthrosis? Rheum Dis Clin North Am 29:
675– 685, 2003.
21. Carmeliet G, Vico L, and Bouillon R. Space flight: a challenge for normal
bone homeostasis. Crit Rev Eukaryot Gene Expr 11: 131–144, 2001.
22. Carvalho RS, Scott JE, Suga DM, and Yen EH. Stimulation of signal
transduction pathways in osteoblasts by mechanical strain potentiated by
parathyroid hormone. J Bone Miner Res 9: 999 –1011, 1994.
23. Carvalho RS, Scott JE, and Yen EH. The effects of mechanical
stimulation on the distribution of ␤1 integrin and expression of ␤1integrin mRNA in TE-85 human osteosarcoma cells. Arch Oral Biol 40:
257–264, 1995.
24. Casser-Bette M, Murray AB, Closs EI, Erfle V, and Schmidt J. Bone
formation by osteoblast-like cells in a three-dimensional cell culture.
Calcif Tissue Int 46: 46 –56, 1990.
25. Chen NX, Ryder KD, Pavalko FM, Turner CH, Burr DB, Qiu J, and
Duncan RL. Ca2⫹ regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts. Am J Physiol Cell Physiol 278:
C989 –C997, 2000.
26. Cheng B, Kato Y, Zhao S, Luo J, Sprague E, Bonewald LF, and
Jiang JX. PGE2 is essential for gap junction-mediated intercellular
communication between osteocyte-like MLO-Y4 cells in response to
mechanical strain. Endocrinology 142: 3464 –3473, 2001.
27. Chotard-Ghodsnia R, Drochon A, and Grebe R. A new flow chamber
for the study of shear stress and transmural pressure upon cells adhering
to a porous biomaterial. J Biomech Eng 124: 258 –261, 2002.
28. Chun L, Yoon J, Song Y, Huie P, Regula D, and Goodman S. The
characterization of macrophages and osteoclasts in tissues harvested from
revised total hip prostheses. J Biomed Mater Res 48: 899 –903, 1999.
29. Deyama Y, Kikuiri T, Ohnishi G, Feng YG, Takeyama S, Hatta M,
Yoshimura Y, and Suzuki K. Histamine stimulates production of osteoclast differentiation factor/receptor activator of nuclear factor-␬B ligand by
osteoblasts. Biochem Biophys Res Commun 298: 240 –246, 2002.
30. Dickson IR and Scheven BA. Regulation of new osteoclast formation
by a bone cell-derived macromolecular factor. Biochem Biophys Res
Commun 159: 1383–1390, 1989.
31. Doty SB. Morphological evidence of gap junctions between bone cells.
Calcif Tissue Int 33: 509 –512, 1981.
32. Dragoo JL, Samimi B, Zhu M, Hame SL, Thomas BJ, Lieberman JR,
Hedrick MH, and Benhaim P. Tissue-engineered cartilage and bone
using stem cells from human infrapatellar fat pads. J Bone Joint Surg Br
85: 740 –747, 2003.
33. Duong LT, Lakkakorpi P, Nakamura I, and Rodan GA. Integrins and
signaling in osteoclast function. Matrix Biol 19: 97–105, 2000.
34. Dziak R and Brand JS. Calcium transport in isolated bone cells. I. Bone
cell isolation procedures. J Cell Physiol 84: 75– 83, 1974.
35. Forrest SM, Ng KW, Findlay DM, Michelangeli VP, Livesey SA,
Partridge NC, Zajac JD, and Martin TJ. Characterization of an
osteoblast-like clonal cell line which responds to both parathyroid hormone and calcitonin. Calcif Tissue Int 37: 51–56, 1985.
Invited Review
MECHANOBIOLOGICAL CONSIDERATIONS FOR BONE CELL CULTURE
63.
64.
65.
66.
67.
68.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
83.
84.
85.
AJP-Cell Physiol • VOL
85a.Majeska RJ. Current methodologic issues in cell and tissue culture. In:
Principles of Bone Biology, edited by Bilezikian JP, Raisz LG, and
Rodan GA. New York: Academic, 1996.
86. McAllister RM, Gardner MB, Greene AE, Bradt C, Nichols WW,
and Landing BH. Cultivation in vitro of cells derived from a human
osteosarcoma. Cancer 27: 397– 402, 1971.
87. McDuffee LA and Anderson GI. In vitro comparison of equine cancellous bone graft donor sites and tibial periosteum as sources of viable
osteoprogenitors. Vet Surg 32: 455– 463, 2003.
88. Mishra S and Knothe Tate ML. Effect of lacunocanalicular architecture on hydraulic conductance in bone tissue: implications for bone
health and evolution. Anat Rec 273A: 752–762, 2003.
89. Nauman EA, Satcher RL, Keaveny TM, Halloran BP, and Bikle DD.
Osteoblasts respond to pulsatile fluid flow with short-term increases in PGE2
but no change in mineralization. J Appl Physiol 90: 1849 –1854, 2001.
90. Nelson RL and Bauer GE. Isolation of osteoclasts by velocity sedimentation at unit gravity. Calcif Tissue Res 22: 303–313, 1977.
91. Newman E, Turner AS, and Wark JD. The potential of sheep for the
study of osteopenia: current status and comparison with other animal
models. Bone 16: 277S–284S, 1995.
92. Noble B. Bone microdamage and cell apoptosis. Eur Cell Mater 21:
46 –56, 2003.
93. Nunamaker DM. Experimental models of fracture repair. Clin Orthop
355, Suppl: S56 –S65, 1998.
94. O’Connor JP. Animal models of heterotopic ossification. Clin Orthop
346: 71– 80, 1998.
95. Ohlsson C, Nilsson A, Swolin D, Isaksson OG, and Lindahl A.
Establishment of a growth hormone responsive chondrogenic cell line
from fetal rat tibia. Mol Cell Endocrinol 91: 167–175, 1993.
96. O’Neill CA and Galasko CS. Calcium mobilization is required for
spreading in human osteoblasts. Calcif Tissue Int 67: 53–59, 2000.
97. Owan I, Burr DB, Turner CH, Qiu J, Tu Y, Onyia JE, and Duncan
RL. Mechanotransduction in bone: osteoblasts are more responsive to
fluid forces than mechanical strain. Am J Physiol Cell Physiol 273:
C810 –C815, 1997.
98. Oyajobi BO, Lomri A, Hott M, and Marie PJ. Isolation and characterization of human clonogenic osteoblast progenitors immunoselected
from fetal bone marrow stroma using STRO-1 monoclonal antibody.
J Bone Miner Res 14: 351–361, 1999.
99. Partridge NC, Alcorn D, Michelangeli VP, Ryan G, and Martin TJ.
Morphological and biochemical characterization of four clonal osteogenic sarcoma cell lines of rat origin. Cancer Res 43: 4308 – 4314, 1983.
100. Pavalko FM, Chen NX, Turner CH, Burr DB, Atkinson S, Hsieh YF,
Qiu J, and Duncan RL. Fluid shear-induced mechanical signaling in
MC3T3-E1 osteoblasts requires cytoskeleton-integrin interactions. Am J
Physiol Cell Physiol 275: C1591–C1601, 1998.
101. Piekarski K and Munro M. Transport mechanism operating between
blood supply and osteocytes in long bones. Nature 269: 80 – 82, 1977.
102. Plotkin LI, Manolagas SC, and Bellido T. Transduction of cell survival
signals by connexin-43 hemichannels. J Biol Chem 277: 8648 – 8657,
2002.
103. Ponten J and Saksela E. Two established in vitro cell lines from human
mesenchymal tumours. Int J Cancer 2: 434 – 447, 1967.
104. Publicover SJ, Preston MR, and El Haj AJ. Voltage-dependent potentiation of low-voltage-activated Ca2⫹ channel currents in cultured rat
bone marrow cells. J Physiol 489: 649 – 661, 1995.
105. Qiu Q, Ducheyne P, Gao H, and Ayyaswamy P. Formation and
differentiation of three-dimensional rat marrow stromal cell culture on
microcarriers in a rotating-wall vessel. Tissue Eng 4: 19 –34, 1998.
106. Quinn JM, Elliott J, Gillespie MT, and Martin TJ. A combination of
osteoclast differentiation factor and macrophage-colony stimulating factor is sufficient for both human and mouse osteoclast formation in vitro.
Endocrinology 139: 4424 – 4427, 1998.
107. Quirici N, Soligo D, Bossolasco P, Servida F, Lumini C, and Deliliers
GL. Isolation of bone marrow mesenchymal stem cells by anti-nerve
growth factor receptor antibodies. Exp Hematol 30: 783–791, 2002.
108. Rawlinson SC, Mosley JR, Suswillo RF, Pitsillides AA, and Lanyon
LE. Calvarial and limb bone cells in organ and monolayer culture do not
show the same early responses to dynamic mechanical strain. J Bone
Miner Res 10: 1225–1232, 1995.
109. Reich KM and Frangos JA. Effect of flow on prostaglandin E2 and
inositol trisphosphate levels in osteoblasts. Am J Physiol Cell Physiol
261: C428 –C432, 1991.
287 • DECEMBER 2004 •
www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on June 17, 2017
69.
dynamic interaction with specific extracellular matrix (ECM) ligands.
Proc Natl Acad Sci USA 98: 1042–1046, 2001.
Jerome CP and Peterson PE. Nonhuman primate models in skeletal
research. Bone 29: 1– 6, 2001.
Jiang JX and Cheng B. Mechanical stimulation of gap junctions in bone
osteocytes is mediated by prostaglandin E2. Cell Commun Adhes 8:
283–288, 2001.
Kato Y, Boskey A, Spevak L, Dallas M, Hori M, and Bonewald LF.
Establishment of an osteoid preosteocyte-like cell MLO-A5 that spontaneously mineralizes in culture. J Bone Miner Res 16: 1622–1633, 2001.
Kato Y, Windle JJ, Koop BA, Mundy GR, and Bonewald LF.
Establishment of an osteocyte-like cell line, MLO-Y4. J Bone Miner Res
12: 2014 –2023, 1997.
Kitazawa S, Kajimoto K, Kondo T, and Kitazawa R. Vitamin D3
supports osteoclastogenesis via functional vitamin D response element of
human RANKL gene promoter. J Cell Biochem 89: 771–777, 2003.
Klein-Nulend J, Roelofsen J, Semeins CM, Bronckers AL, and
Burger EH. Mechanical stimulation of osteopontin mRNA expression
and synthesis in bone cell cultures. J Cell Physiol 170: 174 –181, 1997.
Klein-Nulend J, Semeins CM, Ajubi NE, Nijweide PJ, and Burger
EH. Pulsating fluid flow increases nitric oxide (NO) synthesis by osteocytes but not periosteal fibroblasts— correlation with prostaglandin upregulation. Biochem Biophys Res Commun 217: 640 – 648, 1995.
Klein-Nulend J, van der Plas A, Semeins CM, Ajubi NE, Frangos JA,
Nijweide PJ, and Burger EH. Sensitivity of osteocytes to biomechanical stress in vitro. FASEB J 9: 441– 445, 1995.
Knothe Tate ML. Whither flows the fluid in bone? An osteocyte’s
perspective. J Biomech 36: 1409 –1424, 2003.
Knothe Tate ML, Adamson JR, Tami AE, and Bauer TW. The
osteocyte. Int J Biochem Cell Biol 36: 1– 8, 2004.
Knothe Tate ML and Knothe U. An ex vivo model to study transport
processes and fluid flow in loaded bone. J Biomech 33: 247–254, 2000.
Knothe Tate ML, Niederer P, and Knothe U. In vivo tracer transport
through the lacunocanalicular system of rat bone in an environment
devoid of mechanical loading. Bone 22: 107–117, 1998.
Knothe Tate ML, Steck R, Forwood MR, and Niederer P. In vivo
demonstration of load-induced fluid flow in the rat tibia and its potential
implications for processes associated with functional adaptation. J Exp
Biol 203: 2737–2745, 2000.
Kodama H, Nose M, Niida S, and Yamasaki A. Essential role of
macrophage colony-stimulating factor in the osteoclast differentiation
supported by stromal cells. J Exp Med 173: 1291–1294, 1991.
Koerner RJ, Butterworth LA, Mayer IV, Dasbach R, and Busscher
HJ. Bacterial adhesion to titanium-oxy-nitride (TiNOX) coatings with
different resistivities: a novel approach for the development of biomaterials. Biomaterials 23: 2835–2840, 2002.
Kruyt MC, de Bruijn JD, Wilson CE, Oner FC, van Blitterswijk CA,
Verbout AJ, and Dhert WJ. Viable osteogenic cells are obligatory for tissueengineered ectopic bone formation in goats. Tissue Eng 9: 327–336, 2003.
Kumar TR and Krishnan LK. A stable matrix for generation of
tissue-engineered nonthrombogenic vascular grafts. Tissue Eng 8: 763–
770, 2002.
Li JN, Baskaran H, Dertinger SK, Whitesides GM, Van de Water L,
and Toner M. Neutrophil chemotaxis in linear and complex gradients of
interleukin-8 formed in a microfabricated device. Nat Biotechnol 20:
826 – 830, 2002.
Li P, Schwarz EM, O’Keefe RJ, Ma L, Looney RJ, Ritchlin CT,
Boyce BF, and Xing L. Systemic tumor necrosis factor alpha mediates
an increase in peripheral CD11b high osteoclast precursors in tumor
necrosis factor ␣-transgenic mice. Arthritis Rheum 50: 265–276, 2004.
Li S, Kim M, Hu YL, Jalali S, Schlaepfer DD, Hunter T, Chien S, and
Shyy JY. Fluid shear stress activation of focal adhesion kinase. Linking to
mitogen-activated protein kinases. J Biol Chem 272: 30455–30462, 1997.
Lind M, Deleuran B, Thestrup-Pedersen K, Soballe K, Eriksen EF,
and Bunger C. Chemotaxis of human osteoblasts. Effects of osteotropic
growth factors. APMIS 103: 140 –146, 1995.
Liu YZ, Liu YJ, Recker RR, and Deng HW. Molecular studies of
identification of genes for osteoporosis: the 2002 update. J Endocrinol
177: 147–196, 2003.
Lohmann CH, Schwartz Z, Liu Y, Li Z, Simon BJ, Sylvia VL, Dean
DD, Bonewald LF, Donahue HJ, and Boyan BD. Pulsed electromagnetic fields affect phenotype and connexin 43 protein expression in
MLO-Y4 osteocyte-like cells and ROS 17/2.8 osteoblast-like cells. J Orthop Res 21: 326 –334, 2003.
C1535
Invited Review
C1536
MECHANOBIOLOGICAL CONSIDERATIONS FOR BONE CELL CULTURE
AJP-Cell Physiol • VOL
132. Thi MM, Kojima T, Cowin SC, Weinbaum S, and Spray DC. Fluid
shear stress remodels expression and function of junctional proteins in
cultured bone cells. Am J Physiol Cell Physiol 284: C389 –C403, 2003.
133. Tsuzuki T, Okabe K, Kajiya H, and Habu T. Osmotic membrane
stretch increases cytosolic Ca2⫹ and inhibits bone resorption activity in
rat osteoclasts. Jpn J Physiol 50: 67–76, 2000.
134. Umezawa A, Maruyama T, Segawa K, Shadduck RK, Waheed A,
and Hata J. Multipotent marrow stromal cell line is able to induce
hematopoiesis in vivo. J Cell Physiol 151: 197–205, 1992.
135. Vaananen HK, Zhao H, Mulari M, and Halleen JM. The cell biology
of osteoclast function. J Cell Sci 113: 377–381, 2000.
136. Vadiakas GP and Banes AJ. Verapamil decreases cyclic load-induced
calcium incorporation in ROS 17/2.8 osteosarcoma cell cultures. Matrix
12: 439 – 447, 1992.
137. Van Griensven M, Zeichen J, Tschernig T, Seekamp A, and Pape
HC. A modified method to culture human osteoblasts from bone tissue
specimens using fibrin glue. Exp Toxicol Pathol 54: 25–29, 2002.
138. Vunjak-Novakovic G, Obradovic B, Martin I, Bursac PM, Langer R,
and Freed LE. Dynamic cell seeding of polymer scaffolds for cartilage
tissue engineering. Biotechnol Prog 14: 193–202, 1998.
139. Wang D, Christensen K, Chawla K, Xiao G, Krebsbach PH, and
Franceschi RT. Isolation and characterization of MC3T3-E1 preosteoblast subclones with distinct in vitro and in vivo differentiation/mineralization potential. J Bone Miner Res 14: 893–903, 1999.
140. Wergedal JE and Baylink DJ. Characterization of cells isolated and
cultured from human bone. Proc Soc Exp Biol Med 176: 60 – 69, 1984.
141. Wettergreen MA, Timmer MD, Mikos AG, and Liebschner MAK.
Modification of porogen surface to volume ratio can be used to optimize the
permeability of scaffolds used in tissue engineering. Fifth International Bone
Fluid Flow Workshop, Cleveland, OH, September 17–18, 2003, p. 40.
142. Williams JL, Iannotti JP, Ham A, Bleuit J, and Chen JH. Effects of
fluid shear stress on bone cells. Biorheology 31: 163–170, 1994.
143. Wong GL. Bone cell cultures as an experimental model. Arthritis Rheum
23: 1081–1086, 1980.
144. Wright WE and Shay JW. Historical claims and current interpretations
of replicative aging. Nat Biotechnol 20: 682– 688, 2002.
145. Yamakura K, Watanabe H, Abe S, and Yanagisawa T. Ultrastructural
changes in calvarial osteoblast cytoskeleton after prostaglandin E2 administration in rats. Anat Histol Embryol 30: 301–307, 2001.
146. Yamaguchi DT, Green J, Kleeman CR, and Muallem S. Characterization of volume-sensitive, calcium-permeating pathways in the osteosarcoma cell line UMR-106-01. J Biol Chem 264: 4383– 4390, 1989.
147. Yao GQ, Sun BH, Weir EC, and Insogna KL. A role for cell-surface
CSF-1 in osteoblast-mediated osteoclastogenesis. Calcif Tissue Int 70:
339 –346, 2002.
148. Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M,
Mochizuki S, Tomoyasu A, Yano K, Goto M, Murakami A, Tsuda E,
Morinaga T, Higashio K, Udagawa N, Takahashi N, and Suda T.
Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc
Natl Acad Sci USA 95: 3597–3602, 1998.
149. Yellowley CE, Li Z, Zhou Z, Jacobs CR, and Donahue HJ. Functional
gap junctions between osteocytic and osteoblastic cells. J Bone Miner
Res 15: 209 –217, 2000.
150. Yoshigi M, Clark EB, and Yost HJ. Quantification of stretch-induced
cytoskeletal remodeling in vascular endothelial cells by image processing. Cytometry 55A: 109 –118, 2003.
151. You J, Yellowley CE, Donahue HJ, Zhang Y, Chen Q, and Jacobs
CR. Substrate deformation levels associated with routine physical activity are less stimulatory to bone cells relative to loading-induced oscillatory fluid flow. J Biomech Eng 122: 387–393, 2000.
152. You L, Cowin SC, Schaffler MB, and Weinbaum S. A model for strain
amplification in the actin cytoskeleton of osteocytes due to fluid drag on
pericellular matrix. J Biomech 34: 1375–1386, 2001.
153. Ypey DL, Ravesloot JH, Buisman HP, and Nijweide PJ. Voltageactivated ionic channels and conductances in embryonic chick osteoblast
cultures. J Membr Biol 101: 141–150, 1988.
154. Zaleskas JM, Kinner B, Freyman TM, Yannas IV, Gibson LJ, and
Spector M. Contractile forces generated by articular chondrocytes in
collagen-glycosaminoglycan matrices. Biomaterials 25: 1299 –1308,
2004.
287 • DECEMBER 2004 •
www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on June 17, 2017
110. Reilly GC, Haut TR, Yellowley CE, Donahue HJ, and Jacobs CR. Fluid
flow induced PGE2 release by bone cells is reduced by glycocalyx degradation whereas calcium signals are not. Biorheology 40: 591– 603, 2003.
111. Romanello M, Pani B, Bicego M, and D’Andrea P. Mechanically
induced ATP release from human osteoblastic cells. Biochem Biophys
Res Commun 289: 1275–1281, 2001.
112. Salih V, Greenwald SE, Chong CF, Coumbe A, and Berry CL. The
development of an in-vitro perfusion system for studies on cultured cells.
Int J Exp Pathol 73: 625– 632, 1992.
113. Schmidt J, Strauss GP, Schon A, Luz A, Murray AB, Melchiori A,
Aresu O, and Erfle V. Establishment and characterization of osteogenic
cell lines from a spontaneous murine osteosarcoma. Differentiation 39:
151–160, 1988.
114. Shafrir Y and Forgacs G. Mechanotransduction through the cytoskeleton. Am J Physiol Cell Physiol 282: C479 –C486, 2002.
115. Shastri VP, Martin I, and Langer R. Macroporous polymer foams by
hydrocarbon templating. Proc Natl Acad Sci USA 97: 1970 –1975, 2000.
116. Sherr CJ and DePinho RA. Cellular senescence: mitotic clock or
culture shock? Cell 102: 407– 410, 2000.
117. Shiga M, Kapila YL, Zhang Q, Hayami T, and Kapila S. Ascorbic
acid induces collagenase-1 in human periodontal ligament cells but not in
MC3T3-E1 osteoblast-like cells: potential association between collagenase expression and changes in alkaline phosphatase phenotype. J Bone
Miner Res 18: 67–77, 2003.
118. Shimko DA, White KK, Nauman EA, and Dee KC. A device for long
term, in vitro loading of three-dimensional natural and engineered tissues. Ann Biomed Eng 131: 1347–1356, 2003.
119. Siegel MI and Mooney MP. Appropriate animal models for craniofacial
biology. Cleft Palate J 27: 18 –25, 1990.
120. Sikavitsas VI, Bancroft GN, and Mikos AG. Formation of threedimensional cell/polymer constructs for bone tissue engineering in a
spinner flask and a rotating wall vessel bioreactor. J Biomed Mater Res
62: 136 –148, 2002.
121. Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, Luthy
R, Nguyen HQ, Wooden S, Bennett L, Boone T, Shimamoto G,
DeRose M, Elliott R, Colombero A, Tan HL, Trail G, Sullivan J,
Davy E, Bucay N, Renshaw-Gegg L, Hughes TM, Hill D, Pattison W,
Campbell P, Sander S, Van G, Tarpley J, Derby P, Lee R, and Boyle
WJ. Osteoprotegerin: a novel secreted protein involved in the regulation
of bone density. Cell 89: 309 –319, 1997.
122. Smit TH, Huyghe JM, and Cowin SC. Estimation of the poroelastic
parameters of cortical bone. J Biomech 35: 829 – 835, 2002.
123. Steck R, Niederer P, and Knothe Tate ML. A finite element analysis
for the prediction of load-induced fluid flow and mechanochemical
transduction in bone. J Theor Biol 220: 249 –259, 2003.
124. Sudo H, Kodama HA, Amagai Y, Yamamoto S, and Kasai S. In vitro
differentiation and calcification in a new clonal osteogenic cell line
derived from newborn mouse calvaria. J Cell Biol 96: 191–198, 1983.
125. Sun S, Guo Z, Xiao X, Liu B, Liu X, Tang PH, and Mao N. Isolation
of mouse marrow mesenchymal progenitors by a novel and reliable
method. Stem Cells 21: 527–535, 2003.
126. Takahashi N, Udagawa N, Akatsu T, Tanaka H, Shionome M, and
Suda T. Role of colony-stimulating factors in osteoclast development.
J Bone Miner Res 6: 977–985, 1991.
127. Takahashi N, Udagawa N, and Suda T. A new member of tumor
necrosis factor ligand family, ODF/OPGL/TRANCE/RANKL, regulates
osteoclast differentiation and function. Biochem Biophys Res Commun
256: 449 – 455, 1999.
128. Takayanagi H, Oda H, Yamamoto S, Kawaguchi H, Tanaka S,
Nishikawa T, and Koshihara Y. A new mechanism of bone destruction
in rheumatoid arthritis: synovial fibroblasts induce osteoclastogenesis.
Biochem Biophys Res Commun 240: 279 –286, 1997.
129. Takeuchi T, Tsuboi T, Arai M, and Togari A. Adrenergic stimulation
of osteoclastogenesis mediated by expression of osteoclast differentiation
factor in MC3T3-E1 osteoblast-like cells. Biochem Pharmacol 61: 579 –
586, 2001.
130. Tami AE, Nasser P, Verborgt O, Schaffler MB, and Knothe Tate
ML. The role of interstitial fluid flow in the remodeling response to
fatigue loading. J Bone Miner Res 17: 2030 –2037, 2002.
131. Tami AE, Schaffler MB, and Knothe Tate ML. Probing the tissue to
subcellular level structure underlying bone’s molecular sieving function.
Biorheology 40: 577–590, 2003.