J. Biomedical Science and Engineering, 2011, 4, 357-361
doi:10.4236/jbise.2011.45045 Published Online May 2011 (http://www.SciRP.org/journal/jbise/ JBiSE ).
Published Online May 2011 in SciRes. http://www.scirp.org/journal/JBiSE
Mechanical loading of adipose derived stromal cells causes cell
alignment*
David A. Gonzales, Alice S. Ferng, Chris P. Geffre, Jamie L. Borg, Michael Miller, John A. Szivek
Orthopaedic Research Laboratory, University of Arizona, Tucson, USA.
Received 22 February 2011; revised 7 April 2011; accepted 16 April 2011.
ABSTRACT
Osteoarthritis is a debilitating disease that affects
hundreds of millions of people worldwide. Current
research involving growth and characterization of
adipose derived stromal cells (ADSC) in vitro offers a
potential solution for the treatment of cartilage de-
fects that will allow patients to return to the physical
activities they were involved in. Studies have shown
that fibroblast cells grown in vitro respond to cyclic
mechanical stretching by orienting in a direction
perpendicular to the direction of stretch. ADSCs were
isolated from human peripatellar adipose tissue dis-
cards. Cells were cultured until confluent and seeded
at a density of approximately 105 cells in silicone
wells pretreated with ProNectin-F Plus. After stretch-
ing, relative alignment of the cells was ascertained
using imaging software. Stretching cells for 3, 4, 8
and 12 hours re sulted in noticeable cellular alignment
of approximately 60˚ relative to the direction of load-
ing. Cell alignment is crucial for developing tissue-
engineered cartilage that has similar mechanical
properties to native cartilage. Mechanically loading
cells is one method to achieve cell alignment. Since
cell differentiation will be initiated after alignment,
the resulting chondrocytes will be aligned, leading to
organized collagen formation and resulting in a hya-
line-like cartilage structure.
Keywords: Adipose Derived Stromal Cells; Mechanical
Loading; Chondrog enesis; Osteoarthritis
1. INTRODUCTION
Cartilage defects inevitably lead to osteoarthritis which
is a debilitating d isease that affects hundreds of millions
of people worldwide [1]. Current treatments for focal
cartilage defects have had limited success rates and in-
clude focal drilling or defect perforation (marrow stimu-
lation), osteochondral auto- or allo-graft transplantation
(mosaicplasty), and autologous chondrocyte implanta-
tion (ACI) [2-12]. The standard of care for treatment of
advanced osteoarthritis is total joint replacement. Recent
studies have concluded that adipose tissue is a signifi-
cant source of multipotent cells which can be differenti-
ated into cartilage cells [13,14]. Extension of focal de-
fect repair strategies using adult stem cells instead of
chondrocytes shows promise in the treatment of small
and large cartilage defects and the potential for develop-
ing resurfacing strategies for patients with extensive
damage due to early stages of osteoarthritis. Success in a
preliminary study that used adipose derived stromal cells
placed into the joints of an animal model [15,16] sug-
gested that this cell popu lation was ideally suited for use
in a technique that could be developed to regenerate
hyaline articular cartilage in large cartilage defects. A
successful strategy for regeneration of hyaline articular
cartilage requires a technique which facilitates the for-
mation of cartilage tissue with cells and collag en that are
stratified through the section of the cartilage and ori-
ented in the directions observed in native articular carti-
lage. Techniques reported in the literature to align carti-
lage cells causing them to form aligned tissue include
substrate patterning [17,18] and loading to induce
alignment [19-21]. Successful alignment of fibroblasts
documented by Neidlinger-Wilke et al lead to this study.
The goal of this study was to determine whether adult
stromal cells extracted from adipose tissue could be
aligned by cyclically loading the cells to a sp ecific strain
at a specific frequency and a given time duration. A sec-
ondary goal was to determine how long this cell popu la-
tion remained aligned following loading.
2. MATERIALS AND METHODS
2.1. Silicon Dish Preparation
Silicone dishes were formed in custom designed metal
molds (Figure 1(a)) using Sylgard 184 Silicone Elas-
tomer Base and Sylgard 184Silicone Curing Agent in a
*We would like to thank Howard Hughes Medical Institute; Grant #
52003749 for supporting this research.

D. A. Gonzales et al. / J. Biomedical Science and Engineering 4 (2011) 357-361 358
(a) (b) (c)
Figure 1. The metal mold (a) to form the silicone dishes (b). The cell stretching machine (c).
10 : 1 ratio as per the manufacturer’s procedure (Dow
Corning, Midland, MI). The surfaces of the molds were
polished to a 1200 grit finish. The silicone dishes (Fig-
ure 1(b)) had a smooth rectangular surface area of 15
cm2. The mo lds wer e allo wed to se t for a mi nimum o f 8
hours and then cured for 4 hours at 90˚C. Prior to seed-
ing dishes with cells, the silicone dishes were steam ster-
ilized in an autoclave and then treated with a 0.01
mg/mL solution of ProNectin-F Plus for 5 min
(Sigma-Aldrich, St. Louis, MO). They were also rinsed
with dication-free pho sphate buffered saline ( DCF-PBS)
(Sigma-Aldrich) twice before seeding.
2.2. ADSC Isolation and Culture
Adipose derived stromal cells (ADSC) were obtained
from human peripatellar adipose tissue. The tissues were
obtained from patients according to an IRB approved
protocol. Multiple samples were used from female pa-
tients, with ages ranging from 47 to 75 years of age.
The ADSCs were digested using the technique previ-
ously described by Szivek et al. [22]. The tissue samples
were cut into small 1 - 2 cm cubes and digested in a 50%
ratio with digestion media in a 37˚C shaker bath for 1
hour. Digestion media consisted of 4500 U/mL colla-
genase I (EMD Biosciences, San Diego, CA) and 8
mg/mL Bovine Serum Albumen (Sigma-Aldrich) dis-
solved in dication-free phosphate buffered saline
(DCF-PBS) (Sigma-Aldrich). ADSCs were isolated from
the digested fluid by fractional centrifugation for 4 min
at 700 g. The resulting cell pellet was suspended in Dul-
becco’s Modified Eagle Medium (DMEM) (Sigma-Al-
drich) supplemented with 10% fetal bovine serum (FBS)
(Sigma-Aldrich) 1% penicillin-streptomycin (Sigma-
Aldrich), 0.25 ng/mL Transforming Growth Factor-β1
(TGF-β1), 5 ng/mL Epidermal Growth Factor (EGF),
and 1 ng/mL bovine Fibroblast Growth Factor (bFGF)
and seeded into a T-150 cell culture flask. Cells were
maintained in an incubator at 37˚C and 5% CO2. The
cells were allowed to reach conflu ence, and p assages 3- 4
were used in these experiments.
2.3. Dynamic Cell Loading
ADSCs were seeded onto the ProNectin treated silicone
wells at a minimum density of approximately 6 667
cells/cm2 (approximately 105 cells total per well) as well
as 13 333 cells/cm2 (approximately 2*105 cells total per
well) and allowed to attach for a 24 hour period. Cells
were photographed prior to stretching. Three separate
locations were imaged near the central portion of the
dish. Previous studies utilizing silicone dishes with a
similar design for uniaxial loading demonstrated that the
loading region experiences nearly uniform stress [23].
Dishes were cyclically loaded at 1.0 Hz and 10% strain
for 0, 2, 3, 4, 8 and 12 h using a custom designed cyclic
loading machine (Figure 1(c)) housed within the incu-
bator. At each time period, the dishes were removed and
photographed at the same three locations. In add ition, an
unstretched set of cells was also photographed for com-
parative analysis [24]. The cells were fixed in 10%
methanol for 5 min and stained using haematoxylin and
eosin.
2.4. Analysis of Cell Orientation
Images of stretched and control cells were analyzed
(Figure 2) and the relative cell angles were measured
with respect to the horizontal axis using imaging soft-
ware. The horizontal (stretch direction) was defined to
be 0˚. For each image, cells with a measureable orienta-
tion were evaluated. The average of the total measured
angles for each time point was taken, as well as the stan-
dard deviation between the various samples. A paired
t-test was conducted comparing th e initial control values
with the stretched values for each time point. Images of
stretched cells were also analyzed at 1, 2 and 3 hours
post-stretch and the average angles were measured and
analyzed using the same methods described above.
3. RESULTS
The silicone dishes showed no visible signs of damage
after the loading period, andthe cyclic loading machine
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D. A. Gonzales et al. / J. Biomedical Science and Engineering 4 (2011) 357-361 359
(a) (b)
Figure 2. Randomly oriented cells (a) compared with cells beginning to show alignment after eight hours of loading (b).
maintained stretch frequency and stretch length accu-
rately during the entire loading period. For a loading
period of 2 hours there was no significant difference
between stretched and non-stretched cells (p = 0.4).
There was also no significan t difference in orientation
between cells seeded at 6 667 cells/c m2 and cells seeded
at 13 333 cells/cm2 after stretching cells for a period of 3
hours (p = 0.4). ADSC orientation perpendicular to
stretching direction was apparent in stretched cells after
periods of 3 and 4 hours, and a relatively random orien-
tation was noted in non-stretched cells. For a stretching
period of 3 hours, the average angle relative to the axis
of loading was 57.8˚ ± 5.1, compared to an average con-
trol angle of 45.7˚ ± 4.0 (p < 0.001). For cells stretched
for 4 h ours, the avera ge mea sured angle was 60.1˚ ± 1.6,
with an average control angle of 43.3˚ ± 3.3 (p = 0.004).
Cells stretched for an 8 hour period had an average angle
of 62.5˚ ± 3.6, with an average control angle of 41.8˚ ±
2.6 (p = 0.002). Cells stretched for 12 hours had an av-
erage angle of 59.9˚ ± 3.0, with an average control angle
of 45.7˚ ± 2.6 (p = 0.002) (Figure 3).
After a 3-hour stretching period, cells were reliev ed of
tension and imaged every hour for several hours. After
1-hour post stretch, cells maintained their alignment
compared to the values recorded immediately after
stretching (p = 0.04). However, after two hours, random
cell orientation was apparent as seen in Table 1 (p =
0.17).
4. DISCUSSION
This study was conducted to determine whether the
alignment of ADSCs could be changed when subjected
to cyclic mechanical stretching. Our results indicate that
ADSCs do respond to stretching stimuli, and that this
Figure 3. Graphical representation of the average measured
angles between various time points.
Table 1. T-test analysis of cell orientation after a 3 hours
stretching period.
init 3 hr 3 + 1 hr 3 + 2 hr
well #1 45.7 55.9 51.6 47.8
well #2 46.8 57.1 49.7 56.4
well #3 46.8 54.7 56.3 45.7
well #4 42 57.7 55.1 54.9
average 45.2 56.3 53.6 50.8
t-test 0.006 883 0.038 02 0.167 63
strategy could be used to prime cells prior to use in car-
tilage tissue engineering. In these experiments, complete
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D. A. Gonzales et al. / J. Biomedical Science and Engineering 4 (2011) 357-361 360
perpendicular alignment was not achieved. To achieve
nearly perpendicular cellular alignment in relation to
stretching direction, additional strategies will be as-
sessed, including combinations of faster load rates
and/or longer loading times as well as different cellular
densities. Our results agree with studies by Neidlinger-
Wilke [24] on fibroblasts that demonstrated fibroblast
alignment within three hours. Their work with fibro-
blasts did demonstrate continued alignment for up to
several hours immediately after the stretch period, how-
ever based upon our preliminary studies, ADSCs appear
to demonstrate a degenerative alignment after a 2-hour
period. Further studies will attempt to determine the
mechanism behind this change and whether induction of
chondrogenesis by the addition of growth factors will
halt the degenerative alignment.
Cell alignment is crucial for developing tissue-engi-
neered cartilage that has the same structure as hyaline
articular cartilage. This structure is believed to be the
reason native hyaline cartilage is resilient and has ap-
propriate mechanical properties for long term load bear-
ing [25]. Mechanically stretching cells is one approach
which will induce cell alignment. Growing tissue engi-
neered cartilage may provide an alternative to total joint
replacement as a treatment for widespread cartilage
damage noted in OA patients [26,27], but this will only
be possible if resilien t hya line cartilage can be grown for
the joints of patients. Adipose derived cells collected
from the tissues of patients can be readily accessed and
expanded. Differentiation occurs once cells are placed
into a chondrogenic environment and it is expected that
cell alignment will be retained for so me period fo llowing
alignment. This will ensure hyaline cartilage formation
with the appropriate tissue structure.
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