Tissue Engineering

Tissue Engineering Manuscript Central: http://mc.manuscriptcentral.com/ten

Reversible adipose tissue enlargement induced by external
tissue suspension: possible contribution of basic fibroblast
growth factor for preservation of enlarged tissuer
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Journal:Tissue Engineering

Manuscript ID:

TEA-2009-0551.R2

Manuscript Type:

Complete List of Authors:

20-Jan-2010

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Date Submitted by the
Author:Original Article

erKato, Harunosuke; University of Tokyo, Plastic Surgery
Suga, Hirotaka; University of Tokyo, Plastic Surgery
Eto, Hitomi; University of Tokyo, Plastic Surgery
Araki, Jun; University of Tokyo, Plastic Surgery
Aoi, Noriyuki; University of Tokyo, Plastic Surgery
Doi, Kentaro; University of Tokyo, Plastic Surgery
Higashino, Takuya; University of Tokyo, Plastic Surgery
Iida, Takuya; University of Tokyo, Plastic Surgery
Tabata, Yasuhiko; Institute for Frontier Medical Sciences, Kyoto
University, Biomaterials
Yoshimura, Kotaro; University of Tokyo, Plastic Surgery

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Abstract:

Various kinds of tissue expansion have been performed clinicallywith internal devices, but external expansion has not previously
been investigated. We applied continuous external force on skintissue in a mouse model. Four weeks of external suspension caused
enlargement of the subcutaneous tissue, particularly adipose tissue,
though the enlargement was reversible. We found that the enlarged
tissue volume could be adequately sustained with controlled releaseof basic fibroblast growth factor (bFGF), administered at the timethe device was removed. Ki67+ proliferating cells, perilipin+ small
adipocytes, lectin+ capillaries, and glycerol-3-phosphate
dehydrogenase activity in the tissue increased during the expansion
process, indicating dynamic adipose remodeling with adipogenesis
and angiogenesis. Histological analyses revealed that vessels had
elongated in the direction of the external force. Adipose-resident
progenitor cells (adipose-derived stem/stromal cells) were theprimary proliferating cell population involved in the remodelingprocess, particularly in the superficial layer. Treatment with b-FGF
did not enhance the small adipocyte number, but promoted
angiogenesis; this mechanism may contribute to the preservation ofew

Keyword:

Biomedical Engineering, Animal Models < Enabling Technologies,
Angiogenesis and Vasculogenesis < Fundamentals of Tissue
Engineering, Adult Stem Cells < Fundamentals of Tissue
Engineering, Adipose < Tissue Engineering Applications

Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801

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enlarged tissue. Our results suggested that external tissuesuspension induced adipose tissue enlargement by activating
resident progenitor cells and that this external suspension
approach, combined with controlled release of bFGF, hastherapeutic potential for soft tissue engineering.

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Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801

Tissue Engineering

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Kato H, et al.

Title: Reversible adipose tissue enlargement induced by external tissue suspension:
possible contribution of basic fibroblast growth factor in the preservation of enlarged
tissueHarunosuke Kato, M.D.,a Hirotaka Suga, M.D.,a Hitomi Eto, M.D.,a Jun Araki, M.D.,aNoriyuki Aoi, M.D.,a Kentaro Doi, M.D., a Takuya Iida, M.D.,a Yasuhiko Tabata,Ph.D.,b Kotaro Yoshimura, M.D.a*
Institutions:aDepartment of Plastic Surgery, University of Tokyo, Tokyo, Japanb
Department of Biomaterials, Institute for Frontier Medical Sciences, Kyoto University
53 Kawara-cho Seigoin, Sakyo-ku, Kyoto 606-8507, Japan.

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Short running head: Tissue enlargement by external suspension

ee*Correspondence:
Kotaro Yoshimura, M.D.
Department of Plastic Surgery, University of Tokyo School of Medicine,
7-3-1, Hongo, Bunkyo-Ku, Tokyo 113-8655, Japan.
TEL: +81-3-5800-8948
FAX: +81-3-5800-8947
E-mail: yoshimura-pla@h.u-tokyo.ac.jp

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Grant numbers and sources of support: The authors declare that they have no

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competing financial interests. This work was supported by a grant from the Japanese
Ministry of Education, Culture, Sports, Science, and Technology (MEXT); Contract
grant numbers: B2- 19592070 and B2- 21592283.

Number of figures and tables: No table, 7 figures and 4 supplemental figures

Keywords:•

adipose-derived stromal cells•

adipose-derived stem cells

Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801

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Kato H, et al.

•

gradual tissue expansion•

mechanotransduction•

external force•

basic fibroblast growth factor•

adipogenesis•

angiogenesis

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Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801

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Kato H, et al.

ABSTRACT
Various kinds of tissue expansion have been performed clinically with internal devices,
but external expansion has not previously been investigated. We applied continuous
external force on skin tissue in a mouse model. Four weeks of external suspension
caused enlargement of the subcutaneous tissue, particularly adipose tissue, though theenlargement was reversible. We found that the enlarged tissue volume could be
adequately sustained with controlled release of basic fibroblast growth factor (bFGF),
administered at the time the device was removed. Ki67+ proliferating cells, perilipin+

Fosmall adipocytes, lectin+ capillaries, and glycerol-3-phosphate dehydrogenase activityrP

in the tissue increased during the expansion process, indicating dynamic adipose
remodeling with adipogenesis and angiogenesis. Histological analyses revealed thateevessels had elongated in the direction of the external force. Adipose-resident progenitor
cells (adipose-derived stem/stromal cells) were the primary proliferating cell populationrR

involved in the remodeling process, particularly in the superficial layer. Treatment withb-FGF did not enhance the small adipocyte number, but promoted angiogenesis; thisevmechanism may contribute to the preservation of enlarged tissue. Our results suggested

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that external tissue suspension induced adipose tissue enlargement by activating
resident progenitor cells and that this external suspension approach, combined with
controlled release of bFGF, has therapeutic potential for soft tissue engineering.

Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801

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Kato H, et al.

INTRODUCTION

Gradual tissue expansion is a natural physiological phenomenon that occurs during
growth, pregnancy, or morbid obesity; however, it is also an established therapeutic
method for treating tissue defects. Tissue expansion is applied therapeutically withspecific devices, including an internal tissue expander or an internal/external distraction
osteogenesis system. Distraction osteogenesis can elongate limbs,1,2 the cranial bone, orFo

the maxillofacial bone3 without bone grafting; an internal tissue expander is widelyrP

used in tissue reconstruction to expand healthy skin or soft tissue adjacent to defects
imparted by trauma or surgery, like those due to mastectomy.4-6 Thus, gradual tissueeeexpansion has been widely used as a reliable way to regenerate tissue, and it isconsidered a viable alternative to transplantation. Tissue expansion procedures can

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expand the target tissue to over twice the volume of the original tissue. The expansion
capacity is based on the potential of target tissues that can expand and regenerate, likeev

the skin, bone, or soft tissues. The regenerating potential has been attributed to the

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Tissue Engineering

number (density) and potential of resident stem/progenitor cells specific to each tissue;thus, irradiated tissue has a limited potential for expansion.

Recently, continuous external tissue expansion was attempted with a device called theBrava® (Brava LLC, Miami, FL).7,8 This proved to be a potent procedure for soft tissue
enlargement. Its primary advantage, compared to other conventional tissue expansion
procedures, is the external application, which obviates any surgical interventions.7-9
The device expands the skin and soft tissue by applying external negative pressure fromthe outside. Edematous change was induced in response to the pressure, and the

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Kato H, et al.

expansion effects could be reversed, but significant expansion effects may be achieved
when the distractive force was continuously applied for a sufficient period of time.7,8

An external force is transduced into cells and subcellular structures through various
mechanical disturbances, including shear, stretch, tension, distraction, andcompression. These mechanical disturbances are converted into biological signals by aseries of mechanotransduction pathways.10 There are many different cellular responses
to mechanical forces, and many different second messengers that mediate theseFo

responses, including integrins or cadherins.10 It has been shown that applied mechanicalforces can affect tissue growth, cellular function, and even survival.11-16 Moreover,rPphysical interactions with the extracellular matrix can significantly influence stem cell
behavior.17

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Studies on gradual tissue expansion are scarce, and, to our knowledge, no experimental
research has been reported for soft tissue expansion in response to an external force.

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Therefore, in this study, we created an original animal model to examine cellular events

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during gradual tissue expansion with external tissue suspension. In any organ, tissueresident or circulation-derived stem/progenitor cells are involved in a variety of tissue
growth, repair, regeneration, and remodeling processes. We hypothesized that tissueresident stem/progenitor cells would play an important role in the expansion process.
We sought to evaluate the therapeutic potential of external tissue suspension for soft
tissue enlargement. In a preliminary study, we found that soft tissue, particularly
adipose tissue, was expanded after 4-weeks of external suspension, but the expansion
was not sustained over a long time. Therefore, we performed an additional experimentto evaluate the effect of administering controlled release basic fibroblast growth factor

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Kato H, et al.

(bFGF) on the expanded tissue in an effort to prevent reversal and preserve tissue
enlargement. Adipose tissue is known to contain multipotent mesenchymal cells,18,19
also referred to as adipose-derived stem, stromal, or progenitor cells (ASC).20,21
Compared to bone marrow stem cells, adipose tissue mesenchymal stem cells are easier
to isolate, require less invasive procedures, and provide higher cell yields. Thus, ASCs
hold promise for therapeutic use in tissue engineering and regenerative medicine.20 In
this study, we also analyzed in detail the involvement of ASCs in the tissue
expansion/remodeling process.

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MATERIALS AND METHODS

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Mouse models for external tissue suspensionrR

Eight-week-old Balb/c nude mice were anesthetized with intraperitoneal pentobarbital(50 mg/kg weight), and the back skin (30 mm diameter) was placed under continuousevsuspension, as follows. A suspension device, originally prepared for this purpose

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Tissue Engineering(Hiroki Corp., Kanagawa, Japan), was placed on a designated site in the back (Fig. 1A).
The device comprised a small round plate (8 mm diameter) with a durable thread
attached, that was placed on the skin. A round, adhesive, 30 mm-diameter film wasplaced over the plate, and the thread was pulled through a hole in the middle of thefilm. The film adhered to the plate and the back skin; thus, a pull on the string lifted
both the plate and the surrounding skin. A plastic cap was then placed over the skin,film, and plate, and the thread was pulled through a hole and attached at the top of theplastic cap (Supplementary Fig.1). Sufficient force was applied to cause suspension of
the patch of skin in contact with the adhesive film. The applied suspension force wasMary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801

Tissue Engineering

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Kato H, et al.

measured with a tension gage (ST02, Sanko Seikohjo Co. Ltd., Tokyo, Japan) and was
1.64±0.26 N (mean±SEM) immediately after device application. The adhesive film
was exchanged every 5 days. The suspended skin and underlying soft tissue washarvested at days 0, 3, 7, 14, or 28 (n= 6 in each group). For the 42-day animals (n=12),
one of two types of gelatin microspheres (see below) were injected into thesubcutaneous tissue at the suspension site on day 28; then the suspension device was
not applied between days 28 and day 42. The harvested tissue samples were weighedand subjected to the assays described below.

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FobFGF-incorporated gelatin microspheres
Gelatin microspheres that contained bFGF or vehicle alone were prepared with aeemodification of a previously described method.22 In brief, 2 mg of gelatin microspheres
were mixed with 20 µl phosphate buffered saline (PBS), with or without bFGF (0.1rRµg). To the resulting 20 µl hydrogel, we added 80 µl PBS, to obtain a 100 µl hydrogel
mixture, containing bFGF or vehicle alone. On day 28, the suspension device wasevremoved, and the 100µl hydrogel was carefully injected into the back skin under

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anesthesia (n=6 in each group, with or without 0.1 µg bFGF).

To confirm the location of injected gelatin microspheres, we prepared frozen sectionsof skin samples. The sections were stained with DAPI (Vectashield®; Vector
Laboratories, Burlingame, CA) to visualize nuclei under a fluorescence microscope(BioZero®; Keyence, Tokyo, Japan).

Glycerol-3-phosphate dehydrogenase (GPDH) assay

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Kato H, et al.

A GPDH Assay Kit (Cell Garage, Tokyo, Japan) was used to detect changes associatedwith adipocyte amount. The protocol was supplied by the manufacturer. In brief,
harvested skin samples were submerged in 5 ml of a 0.25 M sucrose solution. The
mixture was homogenized and then centrifuged at 430 ×g for 5 minutes. A 1 ml aliquotof the aqueous layer was obtained, and this was centrifuged at 17,700 ×g for 5 minutes.
Next, the supernatant was diluted 10× with an enzyme-extracting reagent, the mixture
was dispensed into the wells of a 96-well plate, two volumes of a substrate reagent wasadded, and the optical absorption was measured at 340 nm for 10 minutes. GPDH

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activity was calculated based on the following formula: GPDH (U/ml) =∆OD×0.482

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×5 (∆OD is the change in optical density per minute).

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Whole mount staining of living subcutaneous adipose tissue
Visualization of living skin and subcutaneous tissue was performed with the procedurerR

described by Nishimura et al.23 Briefly, harvested skin samples were cut into 3 mmpieces within 2 hours after sacrifice, and the samples were incubated with the following

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reagents for 30 min: BODIPY TM 558/568 (dilution 1:100, Molecular Probes, Eugene,

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Tissue EngineeringOR) to stain adipocytes, Alexa FluorTM 488-conjugated isolectin GS-IB4 (dilution
1:200, Molecular Probes, Eugene, OR) to stain endothelial cells, and Hoechst 33342(dilution 1:200, Dojindo, Kumamoto, Japan) to stain nuclei. The samples were thenwashed and directly observed with a confocal microscope system (Leica TCS SP2,
Leica Microsystems GmbH, Wetzlar, Germany). Fifteen images, acquired at 2 µm
intervals, were used for reconstructing a 30 µm-thick, three-dimensional image.

Immunohistochemistry

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Harvested skin samples were paraffin-embedded. We prepared 6-µm-thick sections andperformed immunostaining with the following primary antibodies: goat anti-mouse
CD34 (dilution 1:100, Santa Cruz Biotechnology, Santa Cruz, CA), guinea pig antimouse perilipin (dilution 1:200, Progen, Heidelberg, Germany), and rabbit anti-human
Ki67 (clone SP6, dilution 1:200, Thermo Fisher Scientific, Fremont, CA). For double
fluorescence stains, the following secondary antibodies were used: Alexa FluorTM
488-conjugated donkey anti-goat IgG (dilution 1:200, Invitrogen, OR), Alexa FluorTM
488-conjugated goat anti-guinea pig IgG (dilution 1:200, Invitrogen), and Alexa

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FluorTM 594-conjugated donkey anti-rabbit IgG (dilution 1:200, Invitrogen). An

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isotypic antibody was used as a negative control for each antibody stain. Nuclei were
stained with Hoechst 33342 (dilution 1:200, Dojindo). Blood vessels were stained withee

Lectin 488 (dilution1:200, Invitrogen) or Isolectin 594 (dilution1:200, Invitrogen). The
number of capillaries (lectin+), proliferative ASCs (CD34+/Ki67+ nuclei), and smallrR

adipocytes (perilipin+ and less than 30 µm in diameter) were counted by analyzing atleast 3 field images for each sample at low magnification.

Histological detection of apoptosis

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To detect apoptosis in subcutaneous adipose tissue, terminal deoxynucleotidyltransferase dUTP nick-end labeling (TUNEL) was performed with an In-Situ Cell
Death Detection Kit (Roche Diagnostics, Mannheim, Germany). The protocol wasprovided by the manufacture.

Statistical analysis
Results are expressed as mean ± S.D. The statistical significance was determined usinga Welch’s t-test for all variables. Values of p < 0.05 were considered significant.

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Kato H, et al.

RESULTS

Macroscopic and weight changes of suspended tissue samples
After 28 days of continuous tissue suspension, the back skin appeared thickened andwrinkled, with a thick subcutaneous layer that appeared reddish, probably due to anincrease in vasculature, while the skin was flat with a thin, pale-colored subcutaneousFofatty layer at baseline (Fig. 1B). The weight of the suspended tissue increased with timerPand reached maximum enlargement on day 28. However, the weight was substantiallylower on day 42, 14 days after removing the suspension device (Fig. 1C).

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On day 28, the suspension device was removed, and gelatin microspheres containingrR

bFGF or vehicle were injected into the expanded subcutaneous tissue. The presence of
microspheres in the subcutaneous adipose tissue was confirmed by visualization with aev

fluorescence microscope (Supplemental online Fig. 2A). On day 42, the weights of

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Tissue Engineering

harvested skin samples that were treated with bFGF microspheres were significantly
higher (0.600 ± 0.045 g, n=5) than the control skin weights (0.371 ± 0.016 g, n=6) (Fig.
1C).

Histological and adipose volume (GPDH activity) changes in suspended tissues
Hematoxylin and eosin stained sections of tissues harvested during continuous
suspension through day 28 showed a thickening of subcutaneous adipose tissue andmuscle layers; in particular, an enlargement of the adipose tissue was noted (Fig. 2A).
On days 3 and 7, small-sized adipocytes appeared throughout the subcutaneous adipose

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Kato H, et al.

tissue, suggesting ongoing adipogenesis. After removing the suspension device on day
28, the thickened subcutaneous tissue shrank substantially, as noted on day 42 (Fig.
2A).

GPDH activity is known to increase when preadipocytes differentiate into adipocytes.
We found that GPDH activity was elevated in the tissue samples from day 3 to day 28,
but then declined on day 42 (Fig. 2B). The decline in GPDH on day 42 was partly
prevented by administration of bFGF microspheres (0.101 ± 0.016 U/g, n=3) comparedFo

to controls (0.080 ± 0.008 U/g, n=3), but the difference was not statistically significant.

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These results suggested that the volume of subcutaneous adipose tissue increased undereecontinuous external suspension and reversed after releasing the suspension. Moreover,
the treatment with bFGF contributed to preservation of the enlarged adipose tissue.

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Cellular events evaluated by whole mount stainingev

Triple-fluorescence staining allowed three-dimensional analysis. Whole mount

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specimens were immediately stained without any fixing or sectioning procedures; thus,
the obtained images reflected the original tissue structures, including adipocytes andcapillaries. The reversible enlargement of subcutaneous adipose tissues by external
tissue suspension was assessed by the whole mount histology (Fig. 3A). At baseline,
capillaries or vessels ran between mature adipocytes of relatively similar cell size (4060 µm diameters). On days 3, 7, and 14, small adipocytes were frequently observed
among the mature adipocytes. On day 14, preadipocytes that contained multiple lipid
droplets in the cytoplasm, were occasionally observed along capillaries (Supplemental
online Fig. 2B). At baseline, the capillaries ran alongside adipocytes and formed a

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well-organized network. On days 7, 14, and 28, capillaries appeared elongated,distended in the direction of the external suspension. In the group treated with bFGF
microspheres, the enlarged fat tissue volume was retained compared to the control
group, and the increased density of vascular structures was also relatively preserved(Fig. 3B).

Increased number of small adipocytes under suspension
Small adipocytes, suggesting ongoing adipogenesis, were counted in microsections

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stained for perilipin (Fig. 4A). Under external suspension, the number of smallrP

adipocytes increased and peaked on day7; thereafter, it declined to the baseline level on
day28, and treatment with bFGF-incorporated microspheres did not influence thenumber (Fig. 4B).

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Increased number of capillaries in subcutaneous adipose tissue
Lectin-positive capillaries increased in number under tissue suspension and peaked onev

day 7 (Fig. 5A). On day 42, the number of capillaries in subcutaneous adipose tissue

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Tissue Engineering

treated with bFGF (38 ± 13) was significantly higher than that of controls (15 ± 3) (Fig.
5B). These results suggested that angiogenesis was associated with adipogenesis under
external tissue suspension.

Proliferating cells in subcutaneous adipose tissue under suspension
Histological analysis of day 14 samples showed that most Ki67+ proliferating cells
were CD34+ and lectin−, which suggested that they were ASCs (Figs. 6A, 6B). CD34+
ASCs increased in number and were most frequently observed in the superficial
adipose layer immediately under the dermis. Lectin+/CD34+ cells were also

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occasionally observed, perhaps due to the transdifferentiation of cells from ASCs tovascular endothelial cells (Fig. 6C). The Lectin+/CD34+ cells were most frequently
observed on day 14 (Supplementary Fig. 3).

Sequential changes in the frequency of CD34+/Ki67+ cells (proliferating ASCs) was
measured during external suspension (Fig. 7A). The frequency started to increase on
day 7; this suggested a regenerating process like adipogenesis and/or angiogenesis due
to external suspension (Fig. 7B). On day 42, the percentage of CD34+/Ki67+ cells was

Fohigher in the adipose tissue treated with bFGF (11.2 ± 3.3 %) microspheres than in therPcontrols (8.6 ± 1.5 %) (p=0.06; n=3).

ee

Apoptosis in subcutaneous adipose tissue after releasing suspension
To examine acute changes after removing the suspension device, some samples wererR

harvested on day 29. The enlarged tissues showed acute shrinking, and TUNELpositive nuclei were frequently observed (Supplemental online Figs. 4A, 4B). Fewer

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TUNEL-positive nuclei were observed on day 42 (data not shown), suggesting that

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apoptosis may be one of the mechanisms driving the reversal of enlargement in the
adipose tissue volume.

DISCUSSION

Our results showed that application of continuous external suspension induced soft
tissue enlargement, particularly in the subcutaneous adipose layer. However, theenlargement was reversible as far as external suspension was applied for up to 4 weeks.

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Kato H, et al.

This reversible enlargement was also observed in a clinical trial with an external
expansion device.9 From days 14 to 28, the skin samples showed increases in weight,
GPDH, and capillary density, which almost reached a plateau; the interstitial space had
decreased and each emerging adipocyte appeared to be mature by 28 days. The tissue
shrinkage after discontinuation of the external force suggested that the augmented
volume may have been partly due to interstitial fluid pooling (edema), though thehistological findings did not clearly indicate edema and GPDH activity was increased
after tissue suspension. Our results suggested that apoptosis of adipocytes may be one

Foof the mechanisms underlying the reversal of enlarged tissue volume. The animalrPmodel used in this study was novel, because previously, no animal models existed for
non-obese adipose tissue enlargement or for experimental external tissue expansion.

eeThis study also showed that the reversal of enlarged soft tissue may be prevented by arRconcomitant use of controlled release growth factors, like bFGF. This suggested that a
combined use of external expansion and growth factors is a potential therapeutic

evstrategy. It has been shown that adipogenesis requires accompanying angiogenesis,24

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Tissue Engineeringand the extent of angiogenesis may determine the partial pressure of tissue oxygen andaffect metabolic functions of the generated adipocytes. Capillaries increased in density
during the angiogenesis process, and also after treatment of bFGF along withdiscontinuation of external suspension.

During the 4-week expansion period, several dynamic cellular events were observedthat were associated with tissue remodeling, including: the proliferation anddifferentiation of cells, like ASCs; angiogenetic changes, including increased capillarydensity and elongation of vessels along the direction of tissue suspension; andMary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801

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increased adipogenesis, suggested by the increased number of small adipocytes and theincrease in GPDH activity. The histological results indicated that cell proliferation was
promoted from day 7, and that the proliferating population primarily comprised ASCs
(lectin−/CD34+). The role of ASCs in the adipogenesis/angiogenesis process waspreviously shown in the repair process after ischemia-reperfusion injury to adipose
tissue.25 Lectin+/CD34+ cells were also observed in the proliferative area; thissuggested that ASCs may differentiate into lectin+/CD34− vascular endothelial cells.
There have been a number of studies that demonstrated the extraordinary proangiogenic

Fopotential of ASCs, and some have shown that ASCs had potential for differentiatingrP

into vascular endothelial cells. For example, implanted ASCs differentiated into
endothelial cells and smooth muscle cells, which incorporated into newly formedeevessels in several animal models, including carcinogenesis, ischemia, and an adipose
graft model.26-30 However, ASC differentiation into endothelial cells may not occur at ahigh frequency.31,32

evrR

A large body of evidence showed that soluble growth factors are released from ASCs

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Page 16 of 38

and, in addition, can affect the behavior of ASCs. Among the growth factors tested,bFGF appeared to have the largest impact on ASC function and behavior. Basic FGF isreleased from injured tissue immediately after wounding33 and has been reported topromote cell proliferation,34 inhibit apoptosis, stimulate release of angiogenic growth
factors, like hepatocyte growth factor (HGF) ,25 and enhance adipogenic
differentiation.35 In vivo experiments showed subcutaneous implantation of Matrigel
with control release of bFGF induced de novo adipogenesis.23 In addition, bFGF
enhanced ASC survival after transplantation, promoted blood perfusion andadipogenesis, and prevented fibrogenesis.25,29 This study showed that controlled release

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bFGF improved the retention rate of enlarged adipose tissue after continued external
tissue suspension. The numerical measurement of small adipocytes suggested that
adipogenesis was not upregulated by bFGF. Therefore, the upregulation ofangiogenesis and the suppression of adipocyte death may be a mechanism underlying
the effect of bFGF on the preservation of adipose tissue volume. Adipocytes are very
sensitive to hypoxia and undergo apoptosis under hypoxic conditions.36 Therefore, theincreased capillaries associated with bFGF treatment may have led to higher tissue
oxygen levels in adipose tissue, and resulted in the prevention of hypoxia-induced
adipocyte death.

rPFo

In our animal model, the application of external suspension resulted ineemicrodeformation of the tissue. This induced cell proliferation and angiogenesis,
consistent with responses observed with vacuum assisted closure.37 It has been shownrR

that both not only soluble factors but also mechanical forces modulate stem cell
behaviors; for example, the differentiation lineages of mesenchymal stem cells wasev

determined by gradients of mechanical stress.38 The interactions between cells and

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Tissue Engineering

mechanical factors are critical to the health and function of various tissues and organsof the body and may play critical roles in controlling stem cell fate and lineage
determination.17 There are a variety of mechanotransduction molecules that regulate
stem cell differentiation, including the actin cytoskeleton, mechano- and osmoticallysensitive ion channels, and chromatin remodeling enzymes.17 This study provides invivo evidence that mechanical force was able to activate resident stem cells and induce
dynamic tissue remodeling, accompanied by adipogenesis and angiogenesis, although
the detailed mechanical transduction pathways remain to be definitively determined.
Several phenomena observed in this study reflected influences of mechanical forces,

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such as a higher density of proliferating cells in superficial layers and elongated vessels
arranged parallel to the direction of the external force.

In conclusion, continuous external suspension for 4 weeks induced reversible tissue
enlargement, particularly in adipose tissue. Adipose-resident progenitor cells (ASCs)
were highly involved in the dynamic expansion/remodeling process; ASCs actively
proliferated, differentiated, and contributed to adipogenesis and angiogenesis. Although
discontinuation of the external force resulted in a reduction in the enlarged tissue

Fovolume, the enlarged tissue could be preserved and capillary density was increased byrPtreatment of controlled release bFGF. These results suggested that an external force can
induce soft tissue engineering with a clinically useful significance and the combinationeeof tissue suspension with an external device and controlled release of bFGF has
potential as a therapy for soft tissue expansion that does not require cell transplantation.

Acknowledgments

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Page 18 of 38

We thank Ayako Kurata for technical assistance. Recombinant human basic fibroblast
growth factor was kindly provided by Kaken Pharmaceutical Co. Ltd. (Tokyo, Japan).
Devices for the external suspension of mouse skin were manufactured and kindly
provided by Hiroki Corp. (Yokohama, Japan).

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12. Millward-Sadler, S.J.M., and Salter, D.M. Integrin-dependent signal cascades inchondrocyte mechanotransduction. Ann Biomed Eng 32, 435, 2004.
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19. Rodeheffer, M.S., Birsoy, K., and Friedman, J.M. Identification of white adipocyte
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20. Gimble, J.M., Katz, A.J., and Bunnell, B.A. Adipose-derived stem cells forregenerative medicine. Circ Res 100, 1249, 2007.
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Sato, K., Inoue, K., Nagase, T., Koshima, I., and Gonda, K. Characterization of freshly
isolated and cultured cells derived from the fatty and fluid portions of liposuction
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22. Tabata, Y., Miyao, M., Inamoto, T., Ishii, T., Hirano, Y., Yamaoki, Y., and Ikada,Y. De novo formation of adipose tissue by controlled release of basic fibroblast growth
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obesity requires close interplay between differentiating adipocytes, stromal cells, andblood vessels. Diabetes 56, 1517, 2007.
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25. Suga, H., Eto, H., Shigeura, T., Inoue, K., Aoi, N., Kato, H., Nishimura, S., Manabe,I., Gonda, K., and Yoshimura, K. IFATS collection: Fibroblast growth factor-2-induced

eehepatocyte growth factor secretion by adipose-derived stromal cells inhibits postinjury
fibrogenesis through a c-Jun N-terminal kinase-dependent mechanism. Stem Cells 27,
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Clergue, M., Manneville, C., Barreau, C.S., Dueiz, M., Tedgui, A., Levy, B., Penicaud,

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physiological and therapeutic perspectives. Circulation 109, 656, 2004.
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A. Improvement of postnatal neovascularization by human adipose tissue-derived stem
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28. Matsumoto, D., Sato, K., Gonda, K., Takaki, Y., Shigeura, T., Sato, T., Aiba, E.,
Iizuka, F., Inoue, K., Suga, H., and Yoshimura, K. Cell-assisted lipotransfer: supportive
use of human adipose-derived cells for soft tissue augmentation with lipoinjection.
Tissue Eng 12, 3375, 2006.

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29. Bhang, S.H., Cho, S.W., Lim, J.M., Kang, J.M., Lee, T.J., Yang, H.S., Song, Y.S.,
Park, M.H., Kim, H.S., Yoo, K.J., Jang, Y., Langer, R., Anderson, D.G., and Kim, B.S.
Locally-delivered growth factor enhances the angiogenic efficacy of adipose-derived
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factors, and enhancing vessel maturation. Tissue Eng Part A, in press (doi:
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33. Aiba-Kojima, E., Tsuno, N.H., Inoue, K., Matsumoto, D., Shigeura, T., Sato, T.,
Suga, H., Kato, H., Nagase, T., Gonda, K., Koshima, I., Takahashi, K., and Yoshimura,
K. Characterization of wound drainage fluids as a source of soluble factors associatedwith wound healing: comparison with platelet-rich plasma and potential use in cell
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34. Suga, H., Shigeura, T., Matsumoto, D., Inoue, K., Kato, H., Aoi, N., Murase, S.,
Sato, K., Gonda, K., Koshima, I., and Yoshimura, K. Rapid expansion of humanadipose-derived stromal cells preserving multipotency. Cytotherapy 9, 738, 2007.
35. Kakudo, N., Shimotsuma, A., and Kusumoto, K. Fibroblast growth factor-2
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in adipose tissue remodeling and potential use for soft tissue augmentation. Regen Med
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37. Orgill, D.P., Manders, E.K., Sumpio, B.E., Lee, R.C., Attinger, C.E., Gurtner, G.C.,
and Ehrlich, H.P. The mechanisms of action of vacuum assisted closure: more to learn.
Surgery 146, 40, 2009.

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38. Alomruiz, S., and Chen, C.S. Emergence of patterned stem cell differentiationrR

within multicellular structures. Stem Cells 26, 2921, 2008.

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FIGURE LEGENDS

Figure 1. Tissue enlargement by continuous suspension(A) Suspension device applied on the back skin. The skin was continuously suspended
with a device specifically prepared for this purpose (see Supplementary Fig. 1 fordetails). (B) Macroscopic views of tissue samples harvested on days 0 and 28. Cross
sectional (left), external (middle), and internal (right) views of representative tissue
samples. Substantial increases in skin thickness and vascular density were noted afterFo

continuous suspension for 28 days. (C) Weight changes of harvested tissue samples.

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The weight of the samples increased during tissue suspension until day 28, but theincreased weight was lost 14 days after discontinuing the suspension. However,eeadministration of bFGF microspheres on day 28 preserved the tissue enlargement andthese samples showed significantly higher tissue weights than the controls on day 42.

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Data are shown as mean + SD. * p < 0.05.

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Figure 2. Histological and GPDH activity changes in suspended tissue

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Page 24 of 38(A) Histological views (hematoxylin and eosin staining) of suspended skin samples ondays 0, 3, 7, 14, 28, and 42 at low (left column) or high (right column) magnification.
The subcutaneous layer, particularly the adipose layer, expanded over time from day 0
to day 28. On the other hand, the expanded subcutaneous adipose tissue shrunk on day
42, 14 days after the discontinuation of tissue suspension. Scale bars = 200 µm (left) or
50 µm (right). (B) GPDH activity of tissue samples. GPDH activity, reflecting adipose
tissue volume, increased with continuous suspension, but returned to baseline by day
42. Administration of bFGF microspheres caused less reduction of GPDH, though thisdid not reach statistical significance.

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Figure 3. Whole mount histology of tissue samples
Harvested tissue was stained with BODIPY (adipocytes; green), lectin (endothelial
cells; red), and Hoechst 33342 (nuclei; blue). (A) Suspended tissue samples at low (leftcolumn) or high (right column) magnification. With continuous suspension, thesubcutaneous adipose layer thickened; numerous new capillaries formed in the adipose
tissue and growth extended in the direction of the applied force. Scale bars = 200 µm(left column) or 25 µm (right column). (B) Tissue samples on day 42. The enlarged

Fosubcutaneous adipose tissue had shrunken by day 42 in controls; however, therPadministration of bFGF-incorporated microspheres was associated with a thicker layerof adipose and more prominent capillary network compared to the control. Scale bars =ee

200 µm (left column) or 25 µm (right column).

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Figure 4. Measurement of small adipocytes in samples immunostained for perilipin.
(A) Histological sections immunostained for perilipin (plasma membrane of viableev

adipocytes; green), or stained with lectin (endothelial cells; red) and Hoechst 33342

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Tissue Engineering(nuclei; blue). Scale bars = 50 µm. (B) Sequential change in the number of small
adipocytes. The numbers of small adipocytes (diameter of less than 30 µm) per opticalfield were counted. The number of small adipocytes, reflecting ongoing adipogenesis,
peaked on day 7.

Figure 5. Histological measurement of vasculature in tissue samples(A) Histology of subcutaneous adipose tissue stained with lectin (endothelial cells; red)and Hoechst 33342 (nuclei; blue). Tissue was harvested on days 0, 3, 7, 14, and 28;
some animals were removed from the suspension device and samples were harvested

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14 days later (day 42). Some animals received bFGF-incorporated microspheres after
removal of the suspension device, and then samples were harvested on day 42. Scalebars = 25 µm. (B) Quantification of vessels in the subcutaneous adipose tissue. The
number of vessels per field was counted in images at a low magnification. The largest
number of vessels was observed on day 7. A larger number of vessels were counted inbFGF-treated tissues compared to the control on day 42 (* p < 0.05).

Figure 6. Immunohistological analysis of proliferating cells in tissues under suspension

Fo(A) Tissue samples harvested on day14 were triply stained with anti-Ki67 (proliferativerP

cells; red), anti-CD34 (ASCs; green), and Hoechst 33342 (nuclei; blue). Most
proliferating cells were CD34+, suggesting that they were ASCs. Scale bars = 100 µm

ee(yellow); boxed sections were enlarged at 10 µm (white). (B) Tissue samples harvested
on day14 were triply stained with lectin (capillaries; red), anti-Ki67 (proliferative cells;rR

green), and Hoechst 33342 (nuclei; blue). Most of proliferating cells were lectin−,which also suggested that they were ASCs. Scale bars = 100 µm (yellow); boxed

ev

section was enlarged at 25 µm (white). (C) Tissue samples harvested on day14 were

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triply stained with lectin (capillaries; red), anti-CD34 (ASCs; green), and Hoechst
33342 (nuclei; blue). CD34+ cells were most frequently observed in the superficial
adipose layer, immediately under the dermis. In that area, there were lectin+/CD34−
endothelial cells (red arrow heads), lectin−/CD34+ ASCs (green arrow heads), and also
lectin+/CD34+ cells (white arrow heads), which may be ASCs that are transdifferentiateing into vascular endothelial cells. Scale bars = 100 µm (yellow); boxed
section was enlarged at 10 µm (white).

Figure 7. Histological measurement of proliferating ASCs in tissues under suspension

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(A) Tissue samples harvested on days 0, 3, 7, 14, 28, and 42 were triply stained with
anti-Ki67 (proliferative cells; red), anti-CD34 (ASCs; green), and Hoechst 33342(nuclei; blue). Scale bars = 50µm (B) Quantification of CD34+/Ki67+ cells. The double
positive cells (ASCs) increased in number from day 7 to day 28. The number ofproliferating ASCs decreased after cessation of suspension, but the decrease appeared
to be partly compensated by administration of bFGF.

Fo

Supplementary Figure 1. Suspension devicerP

A suspension device specifically designed for this experiment (Hiroki Corp.,
Yokohama, Japan). An adhesive film (diameter =30 mm) combined with a plastic plateee

was placed on the back skin of mice. A suspension thread attached to the plate was
threaded through the hole in a plastic cap placed over the plate. The thread was thenrRpulled upward and fixed to the plastic cap to maintain a suspending force on the skin(see also Figure 1A). The suspension device was exchanged every 5 days throughout
the experiment.

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Supplementary Figure 2. The application of gelatin microspheres by subcutaneous
injection. (A) Gelatin microspheres containing bFGF or PBS were subcutaneously
injected on day 28, and tissue suspension was discontinued. A paraffin section of theharvested tissue was stained with DAPI (nuclei; blue) and the injected auto-fluorescent
microspheres (yellow) were visualized under a fluorescence microscope. Scale bars =100 µm. (B) Preadipocytes observed under external suspension (day 14). Whole
mounts were triply-stained with BODIPY (lipid; green), lectin (endothelial cells; red),

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and Hoechst 33342 (nuclei; blue); preadipocytes located alongside the capillaries (red)contained numerous small lipid droplets (green). Scale bars = 5 µm.

Supplementary Figure 3. Tissue samples were triply-stained with lectin (capillaries;red), anti-CD34 (ASCs; green), and Hoechst 33342 (nuclei; blue).
Samples were harvested and stained on day 0, 3, 7, 14, and 28. Lectin+/CD34+ cells(white arrow heads), which may be cells that trans-differentiated from ASCs into
vascular endothelial cells, were most frequently observed on day 14. Scale bars = 100

Foµm (yellow); boxed sections were enlarged at 25 µm (white).

rP

Supplementary Figure 4. Histological section of tissue samples harvested on day 29ee(A) Hematoxylin and eosin staining of a sample harvested on day 29, one day after
removal of the suspension device. A substantial reduction in tissue volume wasrR

observed compared to day 28. Scale bar = 200 µm. (B) TUNEL staining of tissue
samples harvested on day 29. TUNEL staining (left column) and the same image

ev

merged with DAPI nuclear staining (right column) showed apoptotic changes. Scalebars = 50 µm.

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Figure 1. Tissue enlargement by continuous suspension(A) Suspension device applied on the back skin. The skin was continuously suspended with a device
specifically prepared for this purpose (see Supplementary Fig. 1 for details). (B) Macroscopic viewsof tissue samples harvested on days 0 and 28. Cross sectional (left), external (middle), and internal(right) views of representative tissue samples. Substantial increases in skin thickness and vasculardensity were noted after continuous suspension for 28 days. (C) Weight changes of harvested tissue
samples. The weight of the samples increased during tissue suspension until day 28, but theincreased weight was lost 14 days after discontinuing the suspension. However, administration ofbFGF microspheres on day 28 preserved the tissue enlargement and these samples showedsignificantly higher tissue weights than the controls on day 42. Data are shown as mean + SD. * p< 0.05.
148x225mm (300 x 300 DPI)

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Figure 2. Histological and GPDH activity changes in suspended tissue(A) Histological views (hematoxylin and eosin staining) of suspended skin samples on days 0, 3, 7,
14, 28, and 42 at low (left column) or high (right column) magnification. The subcutaneous layer,
particularly the adipose layer, expanded over time from day 0 to day 28. On the other hand, theexpanded subcutaneous adipose tissue shrunk on day 42, 14 days after the discontinuation of tissuesuspension. Scale bars = 200 µm (left) or 50 µm (right). (B) GPDH activity of tissue samples. GPDH
activity, reflecting adipose tissue volume, increased with continuous suspension, but returned tobaseline by day 42. Administration of bFGF microspheres caused less reduction of GPDH, though
this did not reach statistical significance.
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Figure 3. Whole mount histology of tissue samples
Harvested tissue was stained with BODIPY (adipocytes; green), lectin (endothelial cells; red), andHoechst 33342 (nuclei; blue). (A) Suspended tissue samples at low (left column) or high (rightcolumn) magnification. With continuous suspension, the subcutaneous adipose layer thickened;
numerous new capillaries formed in the adipose tissue and growth extended in the direction of theapplied force. Scale bars = 200 µm (left column) or 25 µm (right column). (B) Tissue samples on
day 42. The enlarged subcutaneous adipose tissue had shrunken by day 42 in controls; however,
the administration of bFGF-incorporated microspheres was associated with a thicker layer of adipose
and more prominent capillary network compared to the control. Scale bars = 200 µm (left column)
or 25 µm (right column).
208x253mm (300 x 300 DPI)

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Figure 4. Measurement of small adipocytes in samples immunostained for perilipin.
(A) Histological sections immunostained for perilipin (plasma membrane of viable adipocytes;
green), or stained with lectin (endothelial cells; red) and Hoechst 33342 (nuclei; blue). Scale bars =50 µm. (B) Sequential change in the number of small adipocytes. The numbers of small adipocytes(diameter of less than 30 µm) per optical field were counted. The number of small adipocytes,
reflecting ongoing adipogenesis, peaked on day 7.
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Figure 5. Histological measurement of vasculature in tissue samples(A) Histology of subcutaneous adipose tissue stained with lectin (endothelial cells; red) and Hoechst
33342 (nuclei; blue). Tissue was harvested on days 0, 3, 7, 14, and 28; some animals were
removed from the suspension device and samples were harvested 14 days later (day 42). Someanimals received bFGF-incorporated microspheres after removal of the suspension device, and thensamples were harvested on day 42. Scale bars = 25 µm. (B) Quantification of vessels in the
subcutaneous adipose tissue. The number of vessels per field was counted in images at a low
magnification. The largest number of vessels was observed on day 7. A larger number of vessels
were counted in bFGF-treated tissues compared to the control on day 42 (* p < 0.05).
166x241mm (300 x 300 DPI)

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Figure 6. Immunohistological analysis of proliferating cells in tissues under suspension(A) Tissue samples harvested on day14 were triply stained with anti-Ki67 (proliferative cells; red),anti-CD34 (ASCs; green), and Hoechst 33342 (nuclei; blue). Most proliferating cells were CD34+,
suggesting that they were ASCs. Scale bars = 100 µm (yellow); boxed sections were enlarged at 10µm (white). (B) Tissue samples harvested on day14 were triply stained with lectin (capillaries; red),anti-Ki67 (proliferative cells; green), and Hoechst 33342 (nuclei; blue). Most of proliferating cells
were lectin−, which also suggested that they were ASCs. Scale bars = 100 µm (yellow); boxed
section was enlarged at 25 µm (white). (C) Tissue samples harvested on day14 were triply stainedwith lectin (capillaries; red), anti-CD34 (ASCs; green), and Hoechst 33342 (nuclei; blue). CD34+
cells were most frequently observed in the superficial adipose layer, immediately under the dermis.
In that area, there were lectin+/CD34− endothelial cells (red arrow heads), lectin−/CD34+ ASCs
(green arrow heads), and also lectin+/CD34+ cells (white arrow heads), which may be ASCs thatare trans-differentiateing into vascular endothelial cells. Scale bars = 100 µm (yellow); boxed
section was enlarged at 10 µm (white).

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Figure 7. Histological measurement of proliferating ASCs in tissues under suspension(A) Tissue samples harvested on days 0, 3, 7, 14, 28, and 42 were triply stained with anti-Ki67(proliferative cells; red), anti-CD34 (ASCs; green), and Hoechst 33342 (nuclei; blue). Scale bars =50µm (B) Quantification of CD34+/Ki67+ cells. The double positive cells (ASCs) increased in
number from day 7 to day 28. The number of proliferating ASCs decreased after cessation ofsuspension, but the decrease appeared to be partly compensated by administration of bFGF.

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Supplementary Figure 1. Suspension device
A suspension device specifically designed for this experiment (Hiroki Corp., Yokohama, Japan). An
adhesive film (diameter =30 mm) combined with a plastic plate was placed on the back skin ofmice. A suspension thread attached to the plate was threaded through the hole in a plastic cap
placed over the plate. The thread was then pulled upward and fixed to the plastic cap to maintain asuspending force on the skin (see also Figure 1A). The suspension device was exchanged every 5days throughout the experiment.
126x138mm (300 x 300 DPI)

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Supplementary Figure 2. The application of gelatin microspheres by subcutaneous injection. (A)Gelatin microspheres containing bFGF or PBS were subcutaneously injected on day 28, and tissuesuspension was discontinued. A paraffin section of the harvested tissue was stained with DAPI
(nuclei; blue) and the injected auto-fluorescent microspheres (yellow) were visualized under afluorescence microscope. Scale bars = 100 µm. (B) Preadipocytes observed under externalsuspension (day 14). Whole mounts were triply-stained with BODIPY (lipid; green), lectin(endothelial cells; red), and Hoechst 33342 (nuclei; blue); preadipocytes located alongside thecapillaries (red) contained numerous small lipid droplets (green). Scale bars = 5 µm.
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Supplementary Figure 3. Tissue samples were triply-stained with lectin (capillaries; red), anti-CD34(ASCs; green), and Hoechst 33342 (nuclei; blue).
Samples were harvested and stained on day 0, 3, 7, 14, and 28. Lectin+/CD34+ cells (white arrowheads), which may be cells that trans-differentiated from ASCs into vascular endothelial cells, weremost frequently observed on day 14. Scale bars = 100 µm (yellow); boxed sections were enlarged
at 25 µm (white).
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Supplementary Figure 4. Histological section of tissue samples harvested on day 29(A) Hematoxylin and eosin staining of a sample harvested on day 29, one day after removal of thesuspension device. A substantial reduction in tissue volume was observed compared to day 28.
Scale bar = 200 µm. (B) TUNEL staining of tissue samples harvested on day 29. TUNEL staining (leftcolumn) and the same image merged with DAPI nuclear staining (right column) showed apoptotic
changes. Scale bars = 50 µm.
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