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1 Department of Developmental Biology and Anatomy and 2 Department of Surgery, University of South Carolina School of Medicine, and 3 Department of Chemical Engineering, University of South Carolina, Columbia, South Carolina 29208
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In vitro
mechanical cell stimulators are used for the study of the effect of
mechanical stimulation on anchorage-dependent cells. We developed a new
mechanical cell stimulator, which uses stepper motor technology and
computer control to achieve a high degree of accuracy and
repeatability. This device also uses high-performance plastic
components that have been shown to be noncytotoxic, dimensionally stable, and resistant to chemical degradation from common culture laboratory chemicals. We show that treatment with glow discharge for
25 s at 20 mA is sufficient to modify the surface of the rubber to
allow proper adhesion for polymerization of aligned collagen. We show
through finite element analysis that the middle area of the membrane,
away from the clamped ends, is predictable, homogeneous, and has
negligible shear strain. To test the efficacy of the mechanical stretch, we examined the effect of mechanical stimulation on the production of 
1-integrin by neonatal rat cardiac
fibroblasts. Mechanical stimulation was tested in the range of
0-12% stretch and 0-10-cycles/min stretch frequency. The
fibroblasts respond with an increase in 1-integrin at
3% stretch and a decrease at 6 and 12% stretch. Stretch frequency was
found to not significantly effect the concentration of
1-integrin. These studies yield a new and improved
mechanical cell stimulator and demonstrate that mechanical stimulation
has an effect on the expression of 1-integrin.
mechanical cell stimulator; fibroblasts; integrins
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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IT IS WELL DOCUMENTED that cells and tissues in vivo are subjected to various forms of mechanical stimulation while performing their natural function (14, 28, 29). To study this phenomenon in a controlled environment, several types of in vitro mechanical cell stimulators have been developed (14, 24, 27). Most of these rely on a flexible, resilient substrate coated with an extracellular matrix (ECM) material to which cells are attached. Attached cells are then processed through a controlled deformation regime. A variety of biological responses such as cell size, regulation, expression (10, 16, 20-22), synthesis (5-7, 12, 30), and degradation (7, 12, 23) of a variety of contractile and regulatory proteins are measured.
There are several uniaxial and equibiaxial cell stretchers available today in the literature and commercially. Biaxial stretchers allow the cells to be stretched along two axes perpedicular to each other without shear stress. The uniaxial stretchers allow cells to be stretched in one direction while experiencing compression in the perpedicular direction. The complication of shear stress in these systems can be minimized by careful design of the system and substrate geometry. There are distinct advantages to uniaxial cell stretch in approaching a number of biological problems. Previous devices have used a variable speed AC motor connected to an eccentric cam. The cam converts the rotational motion of the motor to a sinusoidal linear motion for the stretcher. The membrane containing the cells is submerged in a glass dish that also contains the culture medium. Two nylon clamps hold the membrane. One clamp is fixed, while the other is attached to the driven cam via a yoke and slide assembly. This type of device has yielded significant biological results but has not been particularly well engineered, primarily because the accuracy and reproducibility of the applied stress-strain have not been defined (4, 27).
The focus of this work was to apply engineering design principles and
analysis to the design and construction of a new linear stretcher with
greater accuracy and functionality. We then demonstrate the
functionality of the stretcher by subjecting neonatal rat cardiac
fibroblasts to well-defined stress-strain programs using the stretch
system and measured the response of 1-integrin to various stretch conditions.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Design of the cell stretcher.
Figure 1 is a photograph of the cell
stretcher assembly. The device applies a linear strain by displacing a
3-cm × 6-cm rectangular membrane that is clamped along the short
sides with the long sides left free. Two versions of the device have
been built: a single and dual unit. The dual-stretch unit is the final
design and will be the focus of this paper. Each device uses standard
150-mm culture dishes as the cell culture vessel.
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Stretcher calibration. To determine the displacement of the stretcher yoke, we applied power to the unit and allowed it to find its mechanical home signal. We measured the inside dimension of the PEEK membrane holding rods using digital calipers (Brown & Sharp Manufacturing). We commanded the unit to move a distance of 5, 10, 15, and 20% stretch. After each move, the inside dimension was measured again and recorded. This procedure was repeated a minimum of three times. The distance traveled by each side of the slider while under load from the silicone membrane was measured to assess the deflection of each side of the cantilevered yolk assembly. The deflection was not detectable using digital calipers.
Membrane treatment. A rectangular membrane of 3 cm wide by 6 cm long by 0.025 cm thick was chosen to fit the stretch device used in the laboratory. For this project, we used the partial differential equations toolbox available for use with MatLab (13). We have shown using the plane stress module of the toolbox finite element analysis that the middle area of the membrane, away from the clamped ends, has predictable tension and compression, is homogeneous, and has negligible shear strain.
Although the clear silicone rubber membranes have many advantages, there has been little success getting extracellular matrix proteins such as collagen and especially aligned collagen gels (24) to polymerize and adhere to the rubber surface. We discovered that pretreatment with glow discharge sufficiently modifies the silicone polymer surface to allow aligned collagen gels to adhere and polymerize to the surface. Briefly, the polymer is made the anode for an electrical discharge over a potential of several thousand volts. The monomers (in this case residual monomer) can form a highly adherent, cross-linked film (19). Pretreatment with glow discharge was found to be effective at enhancing the adhesion performance. Samples to be treated were placed in the treatment chamber of a standard SEM sputtercoating glow discharge unit (model E5100, Bio-Rad). The gold target was removed, and the unit was sealed as usual. Vacuum was applied to roughly 3 × 10
2 mbar of pressure. Dry argon was introduced into the
chamber for 30 s at 1 × 101 mbar of pressure.
Vacuum was restored to 3 × 102 mbar, and glow
discharge was initiated. The applied voltage was roughly 2 kV.
Treatment was tested at various amounts of time and at different
current settings to determine the optimum.
Evaluation of adhesion performance.
Adhesion of aligned collagen gels was evaluated by applying the
collagen to the membrane using the methods of Simpson et al. (24). After the collagen was allowed to polymerize, the
samples were inspected using a phase-contrast microscope and rated for adhesion (arbitrary ranking 1-5, with 1 being poor adhesion and alignment 5 being the best). The rating was done blinded to treatment condition. These data were evaluated using Excel (Microsoft, Redmond, WA) to determine the optimum conditions for best adhesion and best
midpoint alignment. Figure 2 is a
photomicrograph comparison of the 1, 3, and 5 rankings of collagen
polymerization and adhesion. Figure 2A is rank 1, in which the collagen adhesion is extremely poor and the alignment is
nonexistent. Figure 2B is rank 3; there are some
gaps present in the coverage, and the fibers are not aligned parallel
with the length of the membrane. Figure 2C is rank 5, where there is collagen polymerization and adhesion.
The collagen coverage is complete and uniform. The collagen fibers are
parallel to each other lengthwise on the membrane. It is important to
note that, without pretreatment with glow discharge, the collagen coverage on the membranes is unsuitable for use as a cellular substrate.
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Atomic force microscopy. The surface of the silicone membranes was analyzed using atomic force microscopy and was performed before and after treatment; the results are shown in Fig. 2, D and E. Figure 2D is the membrane before glow discharge treatment and shows a relatively homogeneous surface with some surface debris and scratches but no pores. Figure 2E is after treatment at 20 mA for 15 s and shows a series of what appears to be nonrandom pores in the surface. Images were taken in tapping mode in air with uncoded Tapping Probe mode ("TESP") tips with a Digital Instrument Nanoscope MultiMode Scanning Probe Microscope (Santa Barbara, CA). The size of the pores was determined using the manufacturers software. Section analysis of the pores shows a radius of 625 nm and a depth of 7.29 nm. The glow discharge created nanopores in the polymer film that may serve as anchorage points for the collagen fibrils.
Cell culture. All animals were housed in a facility approved by the American Association for Accreditation of Laboratory Animal Care, and protocols were approved by the Institutional Animal Care and Use Committee. Neonatal rat cardiac fibroblasts were isolated using the methods of Carver et al. (24). Silicone rubber stretch membrane (0.01 in. thick, gloss finish; Specialty Manufacturing) was prepared using glow discharge. They were subsequently coated with aligned collagen using the methods of Simpson et al. (24). Approximately 75,000 cells were plated on a 3-cm × 6-cm membrane by placing a cell-retaining ring, 2.36 cm ID and 2.7 cm OD, utilizing ~24% of the available area in the middle of the membrane and adding cells inside the ring. On the basis of the finite element model developed for these membranes, 67% of the area could be utilized with a retaining ring of the appropriate geometry. Cells were cultured in Dulbecco's modified Eagle's medium with 5% fetal bovine serum and 10% newborn bovine serum. The cells were allowed to attach for 24 h and then loaded in the stretcher apparatus to begin stretching. Stretch conditions were 3, 6, and 12% stretch at frequencies of 0 (static), 5, and 10 cycles/min. Cells were stretched for 12 h and then harvested at the end of the 12-h period. All stretch frequencies were continuous throughout the 12-h period. The experimental controls were cells plated on the aligned collagen-coated membranes but not stretched.
Integrin analysis.
To determine the relative concentration of 1-integrin,
the membranes were removed from the stretcher and rinsed three times with Moscona's saline, and the back of the membrane was blotted on a
Kimwipe and transferred to a 100-mm culture dish. The cells were extracted with 150 µl of RIPA buffer solution (in M: 0.15 NaCl,
0.015 NP-40, 0.012 deoxycholate, 0.003 SDS, and 0.5 Tris-base) and
transferred to a cold room (4°C). Phenylmethylsulfonyl fluoride (20 µl/ml) and benzamidine (10 µl/ml) were added to the RIPA buffer and
the cells scraped from the membrane. The solution and the cells were
transferred to Eppendorf tubes and centrifuged at 14,000 rpm for 12 min. The supernatant was transferred to a new tube boiled for 10 min
and stored at 
20°C until analyzed. The pellet was discarded.
1-integrin was separated by gel electrophoresis on a
10% polyacrylamide gel. The loading of the gels was normalized on a
per milligram of protein basis using a Pierce assay (Pierce, Rockford,
IL) with a bovine serum assay (BSA) standard. The proteins were
transferred to nitrocellulose for 1 h at 100 V with ice-pack stirring. The nitrocellulose blots were rinsed once in Tris-buffered saline (TBS)-Tween (T) (1 × TBS, 0.1% Tween 20) and blocked for 1.5 h in TBS-T containing 5% nonfat dry milk. The final
concentration (2 µg/ml) of rabbit anti-1-integrin,
prepared in our laboratory by the method reported in Xenophontos et al.
(27), in 3% BSA and phosphate-buffered saline was
added and incubated overnight at 4°C overnight. The blots were rinsed
three times (5 ml each) in TBS-T and then with rabbit horseradish
peroxidase (Amersham, Piscataway, NJ) diluted 1:2,000 in blocking
solution for 1.5 h. The blots were washed three times with TBS-T,
developed using ECL reagents (Amersham, Piscataway, NJ), and exposed to
film (Kodak Biomax, Eastman-Kodak, Rochester, NY). The exposed gel
spots were analyzed with a spot densitometer (AlphaImager, Alpha
Innotech, Oakland CA). Each experimental trial had a control, a static
condition, and a cyclic condition (in duplicate). The data from each
stretch trial were compared with the control sample for that trial and calculated as percent change from control. The data were then compared
trial to trial on a percentage of control basis.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Stretcher calibration. During calibration, some of the overall capabilities of the stretcher were tested. This particular design is capable of a maximum strain of 24% for a 6-cm membrane. It also has a maximum linear speed of 72 cm/min, which translates into 20% strain at 30 cycles/min and 5% strain at 120 cycles/min on a 6-cm membrane. The linear displacement of the yoke was measured and found to be repeatable. The stretcher can be used on any anchorage-dependent cell type that will adhere to the silicone rubber substrate or an adherent coating on the substrate. Neonatal cardiac myocytes and fibroblasts were tested in this study. The finite element model developed for this project shows the strain on the silicone membrane will be proportional to the displacement of the yoke. This result verifies the accuracy of the displacement expected by the cell stretcher.
Cell culture.
Figure 2C is a photomicrograph of aligned collagen attached
to the silicone membrane before plating the cells. The stretch was
parallel to the axis of collagen fibril orientation. We achieved good
adhesion and stability of the collagen on the silicone. Figure 3 is a photomicrograph comparison of the
fibroblasts on the aligned collagen gel before and after stretching.
The cells aligned themselves with the collagen matrix during the
attachment phase. The cells rotated to be more perpendicular to the
direction of stretch during the stretch treatment. These results are
consistent with what has been reported by previous investigators
(27).
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Integrin analysis.
The Western blot analysis of the stretch experiments compared with
controls are reported in Fig. 5 as percentage of control. Figure
4 shows representative Western blots for
1-integrin. In Fig. 4, lanes A and
B are from a gel containing samples of 3% stretch and 5 cycles/min. Figure 4, lane C, is a control from this
experiment. In Fig. 4, lanes D, E, F, and G are
from another gel containing samples of 6% (D and
E) and 12% (F and G) stretched at 10 and 5 cycles/min, respectively. Lanes H and I are
from a control for this experiment. These bands show an increased
intensity at 3% and decreased intensity at 6 and 12%. Figure
5 is the densitometric analysis of the
Western blots for 1-integrin. Figure 5A is a comparison of all of the data points in the experimental set versus the
percentage stretch. The data are expressed as percentage of control. A
linear regression of these data shows that the percent stretch is a
significant factor in determining the concentration of
1-integrin with P < 0.003, n = 60. Figure 5B is a comparison of all of
the data points versus the stretch frequency. A linear regression of
these data shows that stretch frequency is not a significant factor
determining the concentration of 1-integrin with
P < 0.72, n = 60. Figure 5C
is a comparison of the static stretch data versus percentage stretch. A
linear regression of these data shows that static stretch alone is not
a significant factor determining the concentration of
1-integrin with P < 0.26, n = 20. Figure 5D is a comparison of the
cyclic stretch data versus percentage of stretch. A linear regression
of these data shows that cyclic stretch was a significant factor
determining the concentration of 1-integrin with
P < 0.006, n = 40.
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1-integrin increases with stretch and
that at higher deformations, 6 and 12%, the amount of
1-integrin decreases. Cyclically stretching the cells
gave a much stronger response than did static stretch, although
the actual stretch frequency was not significant. The statically
stretched cells showed the same trend as the cyclically stretched
cells, but the magnitude of response was lower and not significant.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Stretcher design. It is well documented that cardiac cells are stretched in vivo while performing their natural functions in the heart (14, 27, 28). The objective of this study was to design and construct a uniaxial cell stretcher and demonstrate its capabilities in a biological application. Although several uniaxial cell stretchers have been used successfully for biological experimentation, it was felt that a well-designed, accurate, and repeatable apparatus would be very useful in further investigations in the field. The design uses a disposable plastic cell culture dish with a reusable plastic baseplate insert. The design is based on the uniaxial cell stretcher developed and used by Simpson et al. (24) and the cyclical stretcher developed and used by Terracio et al. (28). High-performance plastic materials were required to survive and maintain dimensional stability through all of the commonly used processes for sterilization and incubation. Materials not only had to withstand the autoclave and incubator temperatures, they also had to be chemically resistant to the cell culture media and 70% ethanol commonly used as a localized sterilization technique. The materials selected also had to be ultraviolet (UV) stable to survive UV treatment common in some sterilization procedures. All of the materials tested were found to be noncytotoxic. This made the selection process criteria one of cost, availability, chemical resistivity, and structural suitability rather than toxicity. Although in the original prototypes, polysulphone was selected as the base-plate material, it was later found, during production, to be too brittle for the specified press fit of the PEEK components. Several new material candidates were tried, and the Ultem 100 (polyetherimide) was found to be superior and was selected.
Cell culture.
After the 3% stretch treatment, the cells have realigned themselves to
be roughly 70° off the primary stretch axis. This phenomenon is
similar to previous observations where fibroblasts aligned perpendicular to the direction of stretch after 48-72 h of cyclic stretch (27). It has been proposed that the cells are
responding to an off-axis force vector as a result of uniaxially
stretching the rubber substrate (shear stress) (26). This
hypothesis is inconsistent with the plane stress analysis completed by
the author. In that analysis, there is no force or deformation
vector present to cause the cells to rotate. In the stretch membrane
area of interest, there is only compression and tension that are
perpendicular to each other. The shear stress present is negligible in
the area of the membrane of interest. Shear stress appears most
significantly near the clamped edges of the membrane. The rotation of
the cells is possibly caused by the interaction of the cell with the
collagen 
-helix twisting and untwisting in response to the stretch
than by the rubber substrate deformation.
Integrin analysis.
Integrins are transmembrane proteins that serve as an interface between
the cellular cytoskeleton complex and the extracellular matrix
(3). Integrins play a vital role in the transduction of
mechanical information from the ECM to the cell nucleus via the
cytoskeleton complex (3). Integrins have two components, a
variable -chain and an invariant -chain.
1-Integrin, as well as many associated -integrins,
serve as mechanical signal transducers for the cell and are essential
in the morphogenic process associated with heart development
(1) and play an important role in the cellular development
process. Given the critical mechanical communications role of
1-integrin, it was a good candidate to test the efficacy of the new cell stretcher. There was a definite response of
1-integrin to stretch. The increase of
1-integrin at low deformations and the subsequent
decrease at higher deformations was not originally predicted. It was
predicted that the 1-integrin would increase with
increasing percentage of stretch as the cell attempts to hold on to the
substrate. It could be speculated that at the low deformations the
cells are producing more integrin to improve the anchorage stability.
At high deformations, the cells are cleaving off integrins and are
attempting to adapt the new environment.
1-integrin
concentration. It also showed that although the percentage of stretch
was important, there are likely other factors influencing the
concentration of 1-integrin. The
R2 statistic for these experiments never
exceeded 0.5, suggesting that other factors also have a significant
impact on the concentration of 1-integrin.
In vivo, heart muscle is routinely stretched by up to 20% at
frequencies of 150 cycles/min. However, these data would suggest that
there would be a substantial downregulation of
1-integrin at these conditions. One can only speculate
that the in vivo conditions are sufficiently different from the in
vitro conditions used here that no exact correlation can be drawn
between the degree and rate of stretch in the two conditions. However,
in vitro experiments should provide insight into the in vivo situation,
even though the magnitude and frequency are different.
These studies have produced an accurate and repeatable stretcher
device. The device was demonstrated in a biological application to
yield repeatable results. This device should prove very useful for
biological investigations of the effect of mechanical stimulation on
anchorage-dependent cells.
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ACKNOWLEDGEMENTS |
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We acknowledge the support of the Integrated Microscopy Analysis Facility at the University of South Carolina, Heather Muckenfuss, Robert and Margret Salters, and Limin Fu. We also thank Phillip Page of Devro-Teepak for expert technical assistance in the machine design. Digital Instruments and Dr. Irene Revenko are thanked for assistance with the Atomic Force Microscopy on this project.
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FOOTNOTES |
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This work was supported by Devro-Teepak, Dr. Fredric R. Miller, and Dr. Joseph R. Pounder. This work also supported by National Heart, Lung, and Blood Institute Grants HL-58893, HL-42249, and HL-37669.
Address for reprint requests and other correspondence: M. J. Yost, Dept. of Surgery and Developmental Biology and Anatomy, Univ. of South Carolina, School of Medicine, Columbia, SC 29208 (E-mail: Yost{at}med.sc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 9 April 2000; accepted in final form 22 June 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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