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Cell Motility and the Cytoskeleton 40:13–21 (1998)
Effect of Precise Mechanical Loading
on Fibroblast Populated Collagen Lattices:
Morphological Changes
M. Eastwood,1* V.C. Mudera,2 D.A. McGrouther,2 and R.A. Brown2
1Centre
for Tissue Engineering Research, Department of Technology and Design,
University of Westminster, London, United Kingdom
2University College London, Division of Plastic Surgery Tissue Repair Unit,
London, United Kingdom
The contraction of a collagen lattice by resident fibroblasts causes strains to be
developed within that lattice. These strains can be increased or decreased by
altering the aspect ratio (ratio of length/width/thickness) of the fibroblast populated
collagen lattice, as the cross-sectional area resisting the strain is changed and by the
application of an external load. The fibroblasts align themselves with the direction
of the maximum principle strain; in effect, these cells are ‘‘hiding’’ from the
perceived strain. The direction of the maximum principle strain can be predetermined by the use of a computational finite element analysis. Using the
tensioning-Culture Force Monitor to apply pre-determined loading patterns of
known repeatable magnitudes, as calculated by the finite element analysis, we have
succeeded in aligning fibroblasts into a deliberate predicted orientation. This study
has shown that the resident fibroblast population will respond to changes in strain
resulting from the most subtle of mechanical loads. This may be an important
mechanism in development and repair of connective tissue. Cell Motil. Cytoskeleton 40:13–21, 1998. r 1998 Wiley-Liss, Inc.
Key words: Culture Force Monitor; mechanical loading; fibroblast; collagen gel; cell morphology and
alignment
INTRODUCTION
The biochemical effect of mechanical loading on
fibroblast populations in collagen matrix has been studied
previously. These studies have ranged from the stress
effect on cyclic AMP pathway [He and Grinnell, 1994],
the up-regulation of Tenascin C production [ChiquetEhrismann et al., 1994], to changes in free intercellular
calcium ion concentration [Arora et al., 1994]. Margolis
and Popov [1991] reported that the application of a local
force to mouse embryo fibroblasts induced the formation
of cell processes. The effect of mechanical load on
collagen expression was studied by Carver et al. [1991],
who found that the ratio of collagen type III to collagen
type I was increased in mechanically stretched cells.
Lambert et al. [1992] found that fibroblast function could
be effected by the application of mechanical stress in
terms of collagenase activity and collagen synthesis. Butt
r 1998 Wiley-Liss, Inc.
et al. [1995] reported that fibroblasts subjected to external
mechanical stress resulted in increased cell growth and
proliferation. All of these loading systems suffered from
difficulties in defining the actual applied load, and how it
would act on the resident cells.
Methods of the application of mechanical stimulation differ. Sudden release of the loading across an
attached lattice causes an effect known as strain recovery,
or stress relaxation [Mochitate et al., 1991; He and
Contract grant sponsor: Pearl Assurance Ltd; Contract grant sponsor:
Phoenix Appeal; Contract grant sponsor: EEC; Contract grant number:
BMH4-CT95-0396.
*Correspondence to: Dr. Mark Eastwood, University College London,
Division of Plastic Surgery Tissue Repair Unit, Charles Bell House, 67
Riding House St., London, W1P 7LD United Kingdom.
Received 10 October 1997; accepted 26 January 1998
14
Eastwood et al.
Grinnell, 1994]. In these experiments, the collagen lattice
was allowed to remain attached to the culture well for a
number of days. Fibroblasts become stellate in shape,
contracted the gel thickness, and produced an internal
strain. When the edges of the gel were released from the
culture well, a rapid contraction of the gel was seen.
Grinnell [1994] has likened this to the transition from
granulation tissue to dermis. Morphologically, the fibroblasts change during this strain recovery from large
stellate or bipolar cells to rounded cells with the retraction
of pseudopodia and the collapse of actin bundles [Tomasek et al., 1992; Mochitate et al., 1991; Grinnell, 1994].
The present study has used the tensioning-Culture
Force Monitor (t-CFM) model [Eastwood, 1996]. This
experimental model is a development of the Culture
Force Monitor (CFM) [Eastwood, 1996; Eastwood et al.,
1994, 1996] that enables precise uni-axial mechanical
loads to be applied to fibroblast populated collagen
lattices (FPCL) whilst simultaneously recording the total
mechanical load across the FPCL (i.e., including the
cellular contraction). This has allowed us to define
accurately the magnitude and type of mechanical loading
to which the matrix was subjected, and the resulting
alignment of cells. In turn, this level of precision has
made it possible to estimate the pattern of strain within
the FPCL, through finite element analysis, and to manipulate this to test how cells respond to strain cues within that
matrix.
MATERIALS AND METHODS
The tensioning-Culture Force Monitor (t-CFM)
utilises the same force transducer, data collection system,
and culture well as the Culture Force Monitor (CFM), as
described previously [Eastwood et al., 1994]. Tension
was applied to cultures via a microprocessor controlled
microstepping motor (Micromech, Braintree, UK). The
stepping motor drives a precision ground leadscrew with
a pitch of 0.508 mm that is attached to the moveable table
via a recirculating ball nut. The SX6 microprocessor
controller (Micromech) enabled a resolution of up to
50,800 pulses per revolution of the microstepping motor.
This, in conjunction with the precision ground leadscrew
enabled a positional accuracy of 1 x 10-8 m to be
achieved. The microstepping motor and table are mounted
onto a base of dimensions 200 x 180 x 10 mm constructed
of Perspex; a stainless steel post of 180 x 10 x 10 mm
facilitates attachment of the force transducer in the same
way as the CFM. The culture well is attached to the
t-CFM by a cradle constructed from Perspex. A locking
screw ‘‘nips’’ the petri dish and secures it in position.
Initial positioning of the culture well during the experimental setup is through the moveable ‘‘X-Y’’ table mounted
onto the motorised base.
Control of the microstepping motor is achieved by
the programming of the SX6 (Micromech) controller in
‘‘X-ware4’’ via the IBM computer. The code can be
activated or deactivated by the RP240 instructional set
(Micromech), which is run independently of the main
computer. The t-CFM is shown in Figure 1. Calibration of
the force transducer and data capture from the t-CFM was
identical to the CFM and has been described previously
[Eastwood et al., 1994, 1996; Eastwood, 1996].
Human dermal fibroblasts were cultured from explants of normal skin taken directly from the operating
theatre [Eastwood et al., 1994; Burt and McGrouther,
1992] in Dulbecco’s Modified Eagle’s Medium (DMEM)
Streptomycin/Penicillin (Gibco BRL, Paisley, Scotland)
with 10% foetal calf serum (Advanced Protein Products
[APP], West Midlands, UK). Fibroblast-seeded collagen
gels were prepared by mixing 4 ml of 2.28 mg/ml solution
of NATIVE acid soluble type I rat tail collagen (APP)
with 0.5 ml of 10x strength, DMEM, and neutralised with
1M NaOH prior to the addition of fibroblasts (106 cells/ml
of gel solution). The 5-ml collagen/cell suspension formed
a gel within 5 min, at which stage the cell chamber was
topped with a further 15 ml of culture medium, containing 2.5 mg/ml Amphotericin B (ICN, Paisley, Scotland).
Rectangular FPCLs (75 x 25 mm) were restrained
either by their opposing long or short edges (giving low
or high aspect ratio configurations, respectively). Both
configurations used identical culture wells. FPCLs were
loaded with 120 Dyne force in a cyclic ramp after 8 h in
culture. The loading cycle consisted of 15 min of loading
in a linear progression at a rate of 480 dynes/h, followed
by a 15-min resting phase, then unloading for 15 min at
the same rate of loading prior to a further 15-min resting
phase. The total cycle time being 1 h repeated for 16 h.
Direct observation of collagen gels after 24 h in the
t-CFM involved fixation (and subsequent processing, see
below) by replacement of medium in the t-CFM culture
chamber with 2.5% gluteraldehyde at 40C in 0.1M
phosphate buffer, pH 7.5, at 40C, without the release of
tension [Tomasek et al., 1992] for 1 h followed by
washing in phosphate buffer, pH 7.5, at 4oC. The gels
were stained with 1% toludine blue, (destained with
distilled water) for routine light microscopy and stereomicroscopic examination on an Edge High Definition Stereo
Light Microscope (Edge Scientific Instrument Corporation, Los Angeles, CA) [Greenberg and Boyde, 1993].
For scanning electron microscopy, fibroblast populated
collagen gels were fixed as above, then rapidly cooled in
liquid N2. After freezing, the gels were fractured down the
long axis of the collagen gel, dehydrated, critical point
dried, and sputter coated before examination on a JEOL
(Peabody, MS) 35C electron microscope.
Strain analysis of the collagen lattices was performed using the RASNA software suite of programs
Mechanical Stimulation of Fibroblasts
15
Fig. 1. The tensioning-Culture Force Monitor. Indicated are the force transducer (f), the culture well (w),
and the micro-stepping motor system (m).
(RASNA Corporation, Los Angeles, CA). The collagen
lattice was modelled in both the high aspect ratio of 3:1
(load and cell generated tension being measured along the
short aspect) and the low aspect ratio of 0.33:1 (load and
cell generated tension being measured along the long
aspect). A load was applied that was representative of the
force magnitude applied by the t-CFM. The model
consisted of 3-dimensional elements connected through a
P-Shell formulation, restraints were applied by the removal of the ux, uy, uz, fx fy and fz degrees of freedom
along the grounded side of the model. The load was
applied as uniform pressure acting out from the model
edge. The analysis was computed on a Sun Sparkstation
running a Unix operating system.
RESULTS
External mechanical loading by the t-CFM caused
strains to be developed in the tethered collagen lattice, as
did the force generated by the cells themselves. An
assessment of the effect of external loading of the matrix
was performed by Finite Element Analysis (FEA) to
determine the strain gradients that were present in the
FPCL in both the high and low aspect ratio gels. The
strain gradients generated by the application of the
mechanical loading are shown in Figure 2A,B. The FEA
performed on the low aspect ratio gel (Fig. 2A) showed
that there were no high strain gradients, and that the
iso-strains were orientated parallel with the long axis, i.e.,
at 90o to the direction of the applied mechanical load. By
contrast the strain gradients in the high aspect ratio gels
(identical applied mechanical load) (Fig. 2B) were highly
condensed, and aligned parallel with both the long axis
and the direction of the applied load. Figure 2B also
shows that all regions within the gel had high strain
gradients except for the delta zone starting 4 mm behind
the vyon bars. This zone had minimal strain due to the
stiffness imparted into the collagen gel by its close
proximity to the rigid vyon bar.
The effect of applied mechanical load on cells
within a low aspect ratio FPCL is shown in Figure 3A.
After 16 h of cyclic loading, cells had a mixture of stellate
and bipolar morphologies with no clear orientation,
suggesting that there was no significant orientational cue
in this configuration of the model. In contrast, application
of the identical load in the high aspect ratio configuration
of FPCLs produced a dramatic alignment parallel with
the axis of the applied load and with the iso-strain lines
Fig. 2. Principle strains developed within (A) low and (B) high aspect ratio collagen gels in response to the applied force from mechanical
loading. Inserts: Direction of the applied force. Dotted, dashed, and solid lines show the contours of constant strain. Close lines indicate high
strain gradients.
Mechanical Stimulation of Fibroblasts
predicted by the FEA. In addition shape control of the
resident cells (Fig. 3B) was clearly produced in that most
of the cells in these areas were elongate and approximately bipolar. This load produced a strain of only 0.2%
of the total gel length.
To test the idea that cells had aligned themselves
parallel with the axis of the principle iso-strains, cell
morphology was compared in adjacent areas of lowest
and highest strain gradient, i.e., zone 2 (delta area) and
zone 3 (high strain on the edge of the gel) as shown in
Figure 4A. Figure 4B–D shows the corresponding micrographs of cells from zones 1–3 from those areas. Zone 2
(Fig. 4C) contained non-aligned cells with a mixture of
stellate and bipolar morphologies. Aligned cells with
bipolar morphologies, parallel with the axis of the
principal iso-strain lines, were found in zones 1 (Fig. 4B)
and 3 (Fig. 4D). Removal of all loads from the high aspect
ratio FPCL experiment resulted in a rapid loss of cell
alignment and return to a predominantly stellate morphology (Fig. 4E).
Scanning EM of freeze fractured gel (Fig. 5A,B)
confirmed the bipolar nature of the fibroblasts and their
parallel alignment relative to each other and to the applied
load. It is interesting to note that the cells are aligned in a
predominantly ‘‘nose to tail’’ configuration (Fig. 5B),
rather than in a staggered formation. This produced the
appearance of strings of cells running in long lines
through the matrix. Figure 5C shows a cell free collagen
gel that has been loaded on the by the t-CFM for 24 h
prior to being fixed under tension. After this loading cycle
no alignment of the collagen fibrils is evident.
DISCUSSION
This series of experiments was performed to determine the effect on cell shape and alignment (morphology)
in response to defined mechanical stimulation. Others
[Butt et al., 1995] have reported that fibroblasts become
aligned perpendicular to the direction of the applied force
in a radially strained 2-dimensional system (Flexcell International Corp., McKeesport, PA). However, in this system the
membrane is deformed by a vacuum; therefore, the force acts
at a normal to the membrane surface. In addition, it has also
been reported that fibroblasts in a 3-dimensional, ligament
equivalent collagen matrix subjected to mechanical loading
were aligned parallel with the direction of the applied load
[Huang et al., 1993]. Data generated from this model are
consistent with the proposal that fibroblasts become orientated parallel with the principle iso-strains. The response to
mechanical load by other cell types has also been shown,
notably in the observations of Julius Wolff [1896] and in
his work on bone remodelling, commonly known as
Wolff’s law, where he proposed that bone grew in
response to mechanical load in such a way as to resist the
17
prominent loads. This was later quantitated to the stress
present in the system [Kummer, 1986].
It is predicted that orientation of the fibroblasts
along the maximum principle strain will reduce the
perceived strain across the cell. This will be further
minimised by the long thin bipolar morphology. In effect,
these cells appear to be hiding from the 3-dimensional
strain, such that they only perceive the strain in one
direction. Furthermore, the direction of the iso-strains in a
3-dimensional object will be dependent upon the shape of
that object and can be independent of the direction of the
applied load as in the case of the low aspect ratio
configuration. Indeed, this was evident in this model,
when identical loading of collagen matrices of different
aspect ratios caused iso-strains to be aligned both parallel
with, and at 900 to, the direction of the load.
The FEA indicated that small shear and compressive force elements were also present at the sample zones
and the possible contribution of these has been considered. However, the levels of shear strain were negligible,
relative to the principal strains. Indeed, the centre of the
gel (zone 1 in Fig. 4B) is devoid of all shear strain but is
an area of maximum cell alignment. Low levels of shear
strain were predicted in zone 3 (Fig. 4D) but the patterns
did not match that of the observed cell alignment.
Similarly, the levels of compressive strain are low and
gave a poor match to the observed cell alignment.
Cell processes can be induced by the action of a
local force. Margolis and Popov [1991] induced cell
processes in rounded mouse embryo fibroblasts on coverslips by pulling the cells with two tungsten microelectrodes. This force caused cell processes to be formed
within 20 sec of its application and parallel with the
applied force. It was argued that applying a pulling, or
tensile force, to the membrane reduced the load on the
cytoskeletal components, namely the microtubules. The
model of Margolis and Popov [1991] also supports the
hypothesis of cells being aligned along principal isostrains. The application of the tensile force caused strains
to be generated. The cell processes formed were in the
direction of the applied force, which would also be the
direction of the maximum principal strain.
The alignment of cells including fibroblasts has
been attributed by others [Wojciak-Stothard et al., 1996;
Ejim et al., 1993; Guido and Tranquillo, 1993; Dow et al.,
1987] to contact guidance on substrates including collagen fibrils. This raises the possibility that cells here were
orientated by contact guidance on collagen fibrils, which
had been aligned due to the applied mechanical load.
However, the SEM shown in Figure 5C shows that there
is no alignment in the collagen gel after 24 h of
mechanical loading. This tends to discount the idea of
cells becoming aligned due to contact guidance during
this early time course. Furthermore, it demonstrates that
18
Eastwood et al.
Fig. 3. A: Effect of mechanical loading on a low aspect ratio FPCL; note the slight amount of cellular
alignment. B: Effect on fibroblast morphology of mechanically loading a high aspect ratio FPCL. (Note all
micrographs are the same magnification.)
Mechanical Stimulation of Fibroblasts
19
Fig. 4. A: Diagram of a high aspect ratio FPCL. The zones for
morphological studies are indicated as 1,2, or 3. B: Aligned fibroblasts
from zone 1. C: Non-aligned fibroblasts from zone 2. D: Aligned
fibroblasts from zone 3. E: Loss of cellular alignment and bipolarity
from zone 1 with the removal of the mechanical load. The arrows
indicate the direction of the long axis of the collagen gel. (Note all
micrographs are the same magnification.)
fibroblasts are highly responsive to the most subtle
mechanical loads, and the resident environment.
The principle that fibroblasts respond to minimise
perceived strain within the matrix is perhaps not surpris-
ing as such strains will produce cytoskeletal rearrangements consistent with observed shape changes [Eastwood
et al., 1996; Eastwood, 1996]. However, the cell behaviour of hiding, or shielding, from strain does produce an
20
Eastwood et al.
Fig. 5. Scanning electron micrographs of a high aspect ratio FPCL
that has been freeze fractured along the major axis. A: Random
orientation of collagen fibrils in between two aligned bipolar fibroblasts. B: Highly aligned fibroblasts, indicated with a small arrow is a
cell, whilst the larger arrow indicates the general orientation of the
cells and the direction of the major axis. C: Cell-free collagen gel,
loaded in the t-CFM for 24 h and fixed under tension; note the random
orientation of collagen fibrils.
attractive mechanism to explain the intimate interrelationships between mechanical load and collagen fibre organisation in soft tissues. It is known that orientated fibroblasts tend to deposit collagen, which is aligned, in the
same long axis [Trelstad and Birk,1985; Birk and Trelstad, 1985] (Brown et al., unpublished data). This new
matrix will further tend to shield the resident cells from
habitual strain in that axis, further minimising the perceived strain. Consequently, mechanical forces would be
important in regulating the amount and the orientation of
newly deposited collagen. Such a model would predict
that increases in perceived strain would stimulate collagen production rates in fibroblasts, eventually falling again as
the collagen matrix shielded the resident cells from the strain
of applied loads. Disruption of the collagenous matrix in
injury would then be expected to induce responses in fibroblasts consistent with those seen during soft tissue repair.
Pathological failures of this feedback system may be seen
in keloid and hypertrophic scarring where downregulation of matrix production is not elicited by a dense
mechanically strong collagenous matrix.
The dependence on strain and aspect ratio described
here suggest that shape of a tissue will dictate the
mechanical forces and, hence, the cellular activity. For
example, a tendon has a high aspect ratio, and is subjected
to high tensile loads. Consequently, the iso-strains will
run parallel with the long axis, consistent with the
orientation of the resident fibroblasts. Bipolar fibroblasts
residing between collagen bundles of normal healthy
tendon also have cell processes extending perpendicular
to the main cell body [Squier and Magnes 1983]. These
cell processes, in mature tissues, may act as parts of a
strain sensing or ‘‘damage detection system.’’ Any breakage of the local collagen fibres would immediately place
an unfamiliar strain on the fibroblasts attached to the
surrounding intact collagen bundles, which its configuration is not adapted to minimise or shield. This would
re-activate the cellular strain sensing system. From previous work we have determined that a load of 25–30%
above the normal cell generated force will elicit a
mechanical response from such cells [Eastwood et al.,
1996] (Brown et al., unpublished data).
The observation reported here of fibroblasts being
aligned ‘‘nose to tail’’ rather than in less ordered (e.g.,
staggered) formation has analogies with slip planes,
commonplace in engineering materials [Askeland, 1989].
Mechanical Stimulation of Fibroblasts
Engineering materials fail along these slip planes as it
represents the minimum energy requirement for failure to
occur. When fibroblasts become aligned due to applied
mechanical load they themselves will create local minimum potential energy wells in a similar manner to that
seen in slip planes. It may be then that cells will be
energetically drawn towards the same slip planes, further
increasing the alignment in this head-to-tail manner. This
cell arrangement is seen in the densest collagenous
tissues such as tendons and ligaments where strain
gradients are steepest. It is tempting to speculate that such
energy wells and slip planes may have a developmental
role in the formation of fascicle margins and epitendonous surfaces in vivo.
In this study we have shown that fibroblasts are
responsive to mechanical load, and mechanical stimulation can be a ‘‘cue’’ for the alignment of cells, necessary
for many tissue engineering applications. The ability to
control alignment of cells through strain, via mechanical
load, and tissue shape provides potentially important
insights into how soft tissue architecture is controlled
during growth and repair.
ACKNOWLEDGMENTS
This work was supported by Pearl Assurance Ltd,
the Phoenix Appeal, and EEC grant BMH4-CT95-0396,
Bioartificial Organs and Tissues. We are grateful to Ms.
Kirsty Smith for photography and electron microscopy.
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