Mechanical loading of adipose derived stromal cells causes cell alignment

doi:10.4236/jbise.2011.45045

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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.

Email: [email protected]

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

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

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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