Yuka Kato,1,2 Takanori Iwata,2 Shunichi Morikawa,3 Masayuki Yamato,2

Teruo Okano,2 and Yasuko Uchigata1

Allogeneic Transplantation of anAdipose-Derived Stem Cell SheetCombined With Artificial SkinAccelerates Wound Healing in a RatWound Model of Type 2 Diabetesand ObesityDiabetes 2015;64:2723–2734 | DOI: 10.2337/db14-1133

One of the most common complications of diabetes isdiabetic foot ulcer. Diabetic ulcers do not heal easily dueto diabetic neuropathy and reduced blood flow, andnonhealing ulcers may progress to gangrene, whichnecessitates amputation of the patient’s foot. This studyattempted to develop a new cell-based therapy for non-healing diabetic ulcers using a full-thickness skin defect ina rat model of type 2 diabetes and obesity. Allogeneicadipose-derived stem cells (ASCs) were harvested fromthe inguinal fat of normal rats, and ASC sheets were cre-ated using cell sheet technology and transplanted intofull-thickness skin defects in Zucker diabetic fatty rats.The results indicate that the transplantation of ASC sheetscombined with artificial skin accelerated wound healingand vascularization, with significant differences observed2 weeks after treatment. The ASC sheets secreted largeamounts of several angiogenic growth factors in vitro, andtransplanted ASCs were observed in perivascular regionsand incorporated into the newly constructed vessel struc-tures in vivo. These results suggest that ASC sheets ac-celerate wound healing both directly and indirectly in thisdiabetic wound-healing model. In conclusion, allogeneicASC sheets exhibit potential as a new therapeutic strat-egy for the treatment of diabetic ulcers.

The population of patients with diabetes is growingworldwide and reached ;400 million in 2013 (1). An

estimated 15–25% of patients with diabetes are at risk fordeveloping a lower-extremity diabetic ulcer in their lifetime(2). Among patients with diabetic foot ulcers, 7–20% willsubsequently require an amputation, and 85% of lower-extremity amputations in diabetic patients are caused byfoot ulcers (3). An urgent need exists to develop new ther-apies for the treatment of diabetic wounds to prevent footulcers from leading to amputations.

Artificial skin is one of the commercially availabletreatments for full-thickness skin defects after debride-ment. Artificial skin typically comprises two layers: an outersilicone sheet layer and an inner collagen sponge layer thatact as the epidermis and dermis, respectively (4). However,difficulties often are experienced with the use of artificialskin treatments for diabetic wounds in patients with neu-ropathy, impaired blood flow, or relatively large wounds. Inthese cases, the formation of neodermal tissue is delayed,thus prolonging the treatment period (5).

Recombinant basic fibroblast growth factor has beenwidely used in wound healing to promote angiogenesisand granulation. Although recombinant basic fibroblastgrowth factor successfully accelerates wound healing, itrequires frequent administration because of its shorthalf-life (6,7).

Cell-based therapy has emerged as a new applicationfor the treatment of ulcers (8,9). In particular, mesenchy-mal stem cells (MSCs) exhibit an excellent potential for

1Diabetic Center, Tokyo Women’s Medical University School of Medicine, Tokyo, Japan2Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s MedicalUniversity, Tokyo, Japan3Department of Anatomy and Developmental Biology, Tokyo Women’s Medical Uni-versity School of Medicine, Tokyo, Japan

Corresponding authors: Teruo Okano, tokano@twmu.ac.jp, and Takanori Iwata,iwata.takanori@twmu.ac.jp.

Received 10 August 2014 and accepted 16 March 2015.

© 2015 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

See accompanying article, p. 2717.

Diabetes Volume 64, August 2015 2723

TECHNOLOGIC

ALADVANCES

increasing the rate of wound healing given their self-renewal capacity, immunomodulatory effects, and abilityto differentiate into various cell lineages (10). Adipose-derived stem cells (ASCs), a type of MSC, exhibit variousadvantageous properties, such as paracrine activity andangiogenic potential (11,12). ASCs have been used exper-imentally in wound-healing applications (13,14).

Single-cell suspensions of MSCs have been directlyinjected around wounds in numerous studies, and accel-erated wound healing has been observed (15,16). Despitereports of improved wound healing in diabetic ulcer mod-els following the injection of single-cell suspensions, theresidence time of transplanted cells at the wound site isunclear. Yang et al. (17) reported that the injection ofsingle-cell suspensions result in the formation of island-like aggregates and visible necrosis of the injected cells.

Cell sheet engineering improves the efficacy of celltransplantation. Okano et al. (18) reported that temperature-responsive culture dishes can be prepared by cova-lently grafting the temperature-responsive polymerN-isopropylacrylamide onto a culture dish surface. Thegrafted polymer layer permits temperature-controlledcell adhesion/detachment on the culture dish surface.Specifically, the surface is hydrophobic at 37°C, allowingcells to adhere and proliferate. In contrast, the surfacebecomes hydrophilic at ,32°C, causing cells to sponta-neously detach from the surface. These cells can be sub-sequently harvested as a contiguous cell sheet with intactcell-cell junctions and extracellular matrix (ECM). Thisapproach avoids the use of proteolytic enzymes, such astrypsin, that damage the ECM, and the cell sheets can beimmediately transplanted to the wound site (19).

Artificial skin has been used to achieve great thera-peutic results, even in chronic wounds such as diabeticwounds (20). In addition, cellular artificial skin has beendeveloped, and a significant body of evidence supports itsuse in the treatment of diabetic wounds (21). As previ-ously stated, ASCs exhibit therapeutic potential; there-fore, ASC sheets with artificial skin have the potentialto accelerate chronic wound healing and offer a feasibletherapeutic strategy for diabetic wound treatment. Theefficacy of ASC sheets with artificial skin requires furtherevaluation in diabetic wounds with exposed bone. In thisstudy, the artificial skin used provided a three-dimensionalframework for ASCs, maintaining transplanted ASCsheets and wounds in a moist environment, preventingwounds from spontaneous contraction, and protectingwounds from infection and external forces (22). Theaim of the current study was to evaluate the efficacy ofallogeneic ASC sheet transplantation with artificial skin topromote the healing and vascularization of full-thicknessskin defects in a rat diabetic wound model.

RESEARCH DESIGN AND METHODS

AnimalsAll experimental protocols were approved by the AnimalWelfare Committee of Tokyo Women’s Medical University

School of Medicine. Zucker diabetic fatty (ZDF) rats(ZDF-Leprfa/CrlCrlj) were used as a type 2 diabetic obesitymodel. Adipose tissue was isolated from Lewis rats (LEW/CrlCrlj) and EGFP rats [SD-Tg (CAG-EGFP)] and used toprepare cell sheets.

Isolation of Rat ASCsRat ASCs (rASCs) were isolated from the inguinal adiposetissue of Lewis rats (20–33 weeks old, male), which wasprocessed according to a previously reported method (23).Briefly, the isolated adipose tissue was enzymaticallydigested with 0.1% type A collagenase (Roche Diagnostics,Mannheim, Germany) at 37°C for 1 h. The stromal vas-cular fraction was collected after centrifugation at 700gfor 5 min. Cells in the stromal vascular fraction wereplated on a 60-cm2 Primaria tissue culture dish (BD Bio-sciences, Franklin Lakes, NJ) and cultured in completeculture medium (MEM a with GlutaMAX [Invitrogen,Carlsbad, CA] with 20% FBS [Moregate Biotech, Bulimba,QLD, Australia] and 1% penicillin/streptomycin [Sigma-Aldrich, St. Louis, MO]) at 37°C in a 5% CO2 incubator.After 24 h, debris was removed by washing with PBS (LifeTechnologies, Grand Island, NY), and fresh medium wasadded. The cells were passaged with 0.25% trypsin-EDTA(Life Technologies) on day 3 and transferred to a new dish.Subcultures were plated at a density of 1.7 3 103 cells/cm2

every 3 days until passage 3.

rASC Culture and CharacterizationrASCs were characterized by measuring their colony-forming, adipogenic, and osteogenic abilities using pre-viously reported methods (24). For each assay, 100 rASCsat passage 3 were plated in a 60-cm2 dish and cultured incomplete medium for 7 days. For the colony-forming as-say, the cells were fixed with 4% paraformaldehyde (PFA)(Muto Pure Chemicals, Tokyo, Japan) and strained with0.5% crystal violet in methanol for 5 min.

For adipogenesis, the medium was switched to anadipogenic medium consisting of complete medium sup-plemented with 0.5 mmol/L dexamethasone (Fuji Pharma,Tokyo, Japan), 0.5 mmol/L isobutyl-1-methyl xanthine(Sigma-Aldrich), and 50 mmol/L indomethacin (Wako PureChemical Industries, Osaka, Japan). After 14 days, the cellswere fixed with 4% PFA for at least 1 h and stained for 2 hwith fresh Oil Red O solution (Wako). For osteogenesis, themedium was switched to calcification medium consistingof complete medium supplemented with 100 nmol/L dexa-methasone, 10 mmol/L b-glycerophosphate (Sigma-Aldrich), and 50 mmol/L ascorbic acid (Wako). The cellswere incubated for 21 days and then stained with 1%alizarin red S solution. The number of stained colonieswas counted for each assay (n = 3).

Flow Cytometry AssayOne million rASCs at passage 4 were suspended in 100 mLPBS containing 10 mg/mL fluorescein isothiocyanate–conjugated primary antibodies to characterize their surfacemarker expression (Table 1). After incubation for 30 min

2724 Allogeneic ASC Sheet Therapy for Diabetic Ulcer Diabetes Volume 64, August 2015

at 4°C, the cells were washed with PBS and then sus-pended in 1 mL PBS for analysis. Cell fluorescence wasevaluated using a Gallios flow cytometer (Beckman Coul-ter, Tokyo, Japan), and data were analyzed using Kaluzafor Gallios software (Beckman Coulter).

Creation of rASC SheetsrASCs derived from Lewis rats or EGFP rats at passage 3were seeded on 35-mm diameter temperature-responsiveculture dishes (UpCell; CellSeed, Tokyo, Japan) at a densityof 1.5 3 105 cells/dish and cultured in complete mediumfor 3 days. The cells were then cultured in complete me-dium with 16.4 mg/mL ascorbic acid for an additional 4–5days. After reducing the temperature to room temperature,the cells spontaneously detached as contiguous cell sheetsand were harvested from the dishes with forceps.

Preparation of the Full-Thickness Skin Defect WoundModel and Transplantation of rASC SheetsZDF rats (n = 48; 16 weeks old; male; 520–600 g) wereused as a rat wound-healing model for type 2 diabetes andobesity. rASC sheets from Lewis rats were used for trans-plantation (Fig. 1A–D). The blood glucose levels of theZDF rats were measured using a blood glucose monitor(Glutest Neo Sensor; Sanwa Kagaku Kenkyusho, Nagoya,Japan), and their body weights were monitored both be-fore the operation and at kill.

ZDF rats were anesthetized by inhalation of 4%isoflurane (Pfizer Japan, Tokyo, Japan) and ventilatedwith a rodent mechanical ventilator (Stoelting, WoodDale, IL). Then, 15 3 10-mm full-thickness skin defectswere created on the heads of the ZDF rats by removingthe cutaneous tissue from the epidermis to the perios-teum (25), and the wound healing of the defects wasobserved at predetermined time points. ZDF rats wererandomly divided into two groups—a control group andan rASC sheet transplantation group—to investigate theefficacy of cell sheets for wound healing. In the transplan-tation group, an rASC sheet was placed on the defect. Thedefects with or without an rASC sheet were covered with15 3 10 mm of artificial skin (PELNAC; Smith & Nephew,Tokyo, Japan). Defects were closed with 10 stitches using5-0 silk sutures (Alfresa, Osaka, Japan) (Fig. 1D). To pro-tect the wound, 20 3 15 mm of nonadhesive dressing(Hydrosite Plus [known as ALLEVYN Non-Adhesive in the

U.S.]; Smith & Nephew) was placed on top of the artificialskin with 5-0 silk sutures to maintain a moist woundenvironment and absorb exudate.

Transplantation of the rASC Sheet From EGFP RatsEGFP-expressing rASCs were obtained from EGFP rats totrack the fate of the transplanted cells. EGFP-expressingrASC sheets were created and transplanted into full-thickness defects in ZDF rats (16–18 weeks old; male; n = 2)using the aforementioned procedure.

Gross Wound Measurement and Complete WoundClosure TimeGross wounds were observed and photographs taken at0, 3, 7, 10, and 14 days (n = 12) after the operation andevery 3–7 days thereafter (n = 6) until complete woundclosure was observed (42 days). The wound area was mea-sured by tracing the wound margin on the photographand calculating the pixel data using ImageJ software (Na-tional Institutes of Health, Bethesda, MD). The time tocomplete wound closure was assessed (n = 6).

Tissue Preparation for ImmunohistochemistryThree rats from each group were killed 3, 7, 10, and 14days after the operation. The wound areas were excisedfor histological analysis, and the blood vessel density wasanalyzed immunohistochemically. After fixation with 4%PFA for 24 h at 4°C, the trimmed specimens were decal-cified with Morse’s solution (10% sodium citrate [Wako]and 22.5% formic acid [Wako]) (26) for 3 days at 4°C withgentle agitation. The Morse’s solution was exchangeddaily. After decalcification, the specimens were rinsedwith PBS, embedded in paraffin, cut into 6-mm–thick sag-ittal sections, and stained with hematoxylin-eosin (H-E).The sections of the wound area were then observed forhistological analysis.

To quantify vascularization within the wound, bloodvessel endothelial cells were immunohistochemicallystained with an anti-CD31 antibody (rabbit polyclonalantibody; Thermo Fisher Scientific Anatomical Pathology,Fremont, CA). The specimens were pretreated throughheating followed by blocking with 1% BSA (Sigma-Aldrich)and then incubated with the anti-CD31 antibody at 4°Covernight. After primary antibody staining, the specimenswere washed with PBS, incubated with a secondary anti-body (Alexa Fluor 488 Goat Anti-Rabbit IgG [H+L] Anti-body; Life Technologies), mounted on coverslips withProlong Gold, and stained with DAPI (Invitrogen). Serialsections of the specimens were observed with a fluores-cence microscope (U-RFL-T; Olympus, Tokyo, Japan), andthe images were analyzed with application software (DP2-BSW; Olympus).

The blood vessel densities of six animals in each groupwere determined 14 days after the operation by measur-ing the area of CD31-positive vessels in the wound area ofthe specimen. The center of the wound in each sectionwas selected. The vessel area in the selected field of eachspecimen was then observed with a microscope (EclipseE800; Nikon, Tokyo, Japan), and the area of CD31-positive

Table 1—Fluorescein isothiocyanate–conjugated antibodiesused for flow cytometry

Target Catalog number Isotype control Source

CD11b 554982 Mouse IgA BD Pharmingen*

CD29 561796 Hamster IgM BD Pharmingen

CD31 MCA1334FA Mouse IgG AbD Serotec†

CD45 MCA43FA Mouse IgG AbD Serotec

CD90 MCA47FA Mouse IgG AbD Serotec

*BD Pharmingen, Franklin Lakes, NJ; †AbD Serotec, Raleigh,NC.

diabetes.diabetesjournals.org Kato and Associates 2725

vessels was quantified using ImageJ software. The relativearea of CD31-positive vessels (VA) was calculated usingEq. 1:

VAð%Þ ¼ VAact=Af3 100 (Eq. 1)

where VAact and Af are the actual area of CD31-positivevessels and the total area of the field, respectively.

EGFP-expressing rASC sheets were transplanted totrace the fate of the transplanted rASC sheets. At 3, 5, 7,and 14 days after the transplantation, the rats’ chestswere opened, and their vasculature was perfused fromthe left ventricle with 100 mL of 2% PFA in 0.01 mol/LPBS (pH 7.4) at a pressure of 120 mmHg. The wound

tissue, including the skull bone and surrounding cutane-ous tissue, was then removed and soaked in 4% PFA for24 h at 4°C. The specimens were subsequently washedwith PBS and decalcified with 10% EDTA weight for vol-ume (Wako) in PBS for 5 days at 4°C with gentle agitation.The EDTA solution was changed daily. After decalcifica-tion, the specimens were rinsed .10 times with PBS,cryoprotected in an ascending series of sucrose-PBS (15and 30 weight for volume percent), embedded in optimalcutting temperature compound (Sakura Finetek Japan,Tokyo, Japan), and snap-frozen in liquid nitrogen. Then,14-mm–thick frozen sections were prepared with a cryo-stat (Leica CM1850; Finetek) and processed forimmunohistochemistry. The frozen sections were first

Figure 1—Schematic of the experimental procedure for transplanting an allogeneic rASC sheet with artificial skin into a rat wound-healingmodel of type 2 diabetes and obesity. A: Rat subcutaneous adipose tissue was surgically excised from Lewis rats or EGFP rats. rASCswere isolated and seeded onto a 60-cm2 Primaria tissue culture dish and cultured at 37°C for 7–8 days. B: Passage 3 rASCs were seededonto a temperature-responsive culture dish and cultured at 37°C for 7–8 days. rASCs were harvested as an rASC sheet with intact cell-celljunctions and ECM by reducing the temperature to 20°C. C: rASC sheets from Lewis rats or EGFR rats were transplanted into a 153 10-mmfull-thickness skin defect with exposed bone created on the heads of ZDF rats (n = 48). D: In the transplantation group, an rASC sheetwas placed on the defect, and a 15 3 10-mm sheet of artificial skin (PELNAC) was overlaid on the transplanted rASC sheet (n = 24). In thecontrol group, only the artificial skin was placed on the defect (n = 24). The defect with the artificial skin was closed with 10 stitches using5-0 silk sutures. GFP, green fluorescent protein.

2726 Allogeneic ASC Sheet Therapy for Diabetic Ulcer Diabetes Volume 64, August 2015

incubated with 4% Block-Ace (Dainippon Sumitomo Seiyaku,Osaka, Japan) and then incubated with rat antiendothelialcell antibody-1 (RECA-1) (Abcam, Cambridge, U.K.) as theprimary antibody at a dilution of 1:200 in PBS containing1% BSA (Sigma-Aldrich) at 4°C overnight. After severalPBS washes, the specimens were incubated with a Cy3-conjugated secondary antibody at a 1:200 dilution (JacksonImmunoResearch Laboratories, West Grove, PA) for 3 h atroom temperature. Finally, the specimens were observedand photographed with a Keyence BZ-9000 fluorescencemicroscope (Keyence Corp., Osaka, Japan).

ELISA of the Supernatant From rASC SheetsrASCs at passage 3 were seeded onto temperature-responsiveculture dishes at a density of 1.5 3 105 cells/dish and cul-tured at 37°C for 72 h. The cells achieved subconfluence (80–90%) at 72 h after seeding, and this time point was definedas day 0. The medium was then collected and replaced withcomplete medium containing 16.4 mg/mL ascorbic acid ondays 0, 2, and 4. The rASC sheets were transplanted on day4 in this study. After the collected medium was centrifugedat 300g for 3 min at 4°C, the supernatant was collected,immediately frozen, and maintained at 280°C for fur-ther experiments.

The number of cultured cells per dish was assessed ondays 0, 2, and 4 (n = 4). The levels of vascular endothelialgrowth factor (VEGF), hepatocyte growth factor (HGF),transforming growth factor-b1 (TGF-b1), IGF-I, epidermalgrowth factor (EGF), and keratinocyte growth factor (KGF)were determined using RRV00, MHG00, MB100B, MG100(R&D Systems, Minneapolis, MN), SE560Ra (Cloud-CloneCorp., Houston, TX), and CSB-E12905r (CUSABIO, Wuhan,China) quantitative ELISA kits, respectively. The proteinconcentrations were measured by duplicate ELISA assays,which were performed according to the manufacturer’sinstructions. The amounts of the various growth factorsin the supernatant were calculated to examine the amountsecreted from the rASC sheets at each time point.

Statistical AnalysisData are expressed as the mean 6 SD. Comparisons be-tween the two groups (i.e., transplant, control) were per-formed using Student t test. P , 0.05 was consideredstatistically significant.

RESULTS

Characterization of rASCsTo evaluate the characteristics of the rASCs, crystal violet,Oil Red O, and alizarin red S staining were performed toconfirm rASC self-renewal, adipogenesis, and osteogenesis,respectively. rASCs exhibited colony-forming potential(Fig. 2A) as well as adipogenic and osteogenic differentia-tion potential in vitro (Fig. 2B and C), suggesting that therASCs used in this study possessed MSC-like properties.

Flow cytometry was used to characterize cell surfacemarkers (Fig. 2D–H). rASCs strongly expressed CD29 andCD90. In contrast, low expression levels of CD11b, CD31,and CD45 were observed.

Blood Glucose Level and Body Weight of 16-Week-OldZDF RatsZDF rats in each group exhibited an average blood glucoselevel .250 mg/dL, and these levels were ;2.5-fold higherthan those observed in 16-week-old nondiabetic rats. ZDFrats exhibited an average body weight of 578.2 g in thetransplantation group and 582.6 g in the control group.These body weights were ;1.5-fold higher than thoseobserved in 16-week-old nondiabetic rats.

Gross Wound AreaDigital photographs were obtained 0, 3, 7, 10, and 14 daysafter the operation and every 3–7 days thereafter untilcomplete wound closure (Fig. 3A–L). The average woundarea was significantly smaller in the transplantation groupthan in the control group beginning on the 7th day(Fig. 4A). The wounds in both groups were digitally photo-graphed and observed until complete wound closure (n =6). The average observed wound closure time was signif-icantly reduced in the transplantation group comparedwith the control group (Fig. 4B).

HistologyIn low-magnification microphotographs of sagittal sec-tions of H-E–stained specimens collected 14 days afterthe operation (Fig. 5A–D), a thin dermal layer was ob-served for the control group (Fig. 5A). In contrast, denseconnective tissue was observed in the transplantationgroup (Fig. 5B). Higher-magnification microphotographsindicated the same results (Fig. 5C and D).

Blood Vessel DensityBlood vessel density was quantified 14 days after theoperation (Fig. 5E–G). The blood vessel density in thewound was increased by ;2.5-fold in the transplantationgroup compared with the control group after 14 days, andthe difference was statistically significant (n = 6) (Fig. 5G).

Number of rASCs Per rASC SheetThe average number of cultured cells per dish was 8.76 3105 cells on day 4 at the time of transplantation (Fig. 6A).

Growth Factor Levels in the rASC Culture SupernatantVEGF, HGF, TGF-b1, IGF-I, EGF, and KGF were presentin the conditioned medium from cultured rASCs at pas-sage 3 (Fig. 6B–G). Specifically, the levels of VEGF, HGF,TGF-b1, IGF-I, EGF, and KGF per rASC sheet were18.60, 3.27, 0.76, 2.78, 0.44, and 0.42 ng/sheet/day atday 4 (n = 4), respectively, as determined using ELISAkits for each protein. These results confirmed that therASC sheets were secreting growth factors at the time oftransplantation.

Fate of the Transplanted rASCsEGFP-expressing rASC sheets were transplanted into thewounds (Fig. 7A and B). Fourteen days after the trans-plantation, the transplanted EGFP-expressing rASCs(stained green [Fig. 7C]) and endothelial cells of regener-ated blood vessels (stained red with RECA-1 [Fig. 7D])were visualized. Overlaid microphotographs revealed

diabetes.diabetesjournals.org Kato and Associates 2727

that EGFP-expressing rASCs were located on the periph-ery of the RECA-1–positive blood vessels (Fig. 7E).

DISCUSSION

Although streptozotocin-induced type 1 diabetic ratswithout obesity are most frequently used for diabeticwound-healing studies, this study used a wound-healingmodel with type 2 diabetes, which is known to affect.90% of patients with diabetes. Specifically, ZDF ratswere used as a wound-healing model of type 2 diabetesand obesity in this study. These rats spontaneously de-velop obesity at ;4 weeks of age and then develop type 2diabetes at 8–12 weeks, exhibiting hyperglycemia com-bined with insulin resistance, dyslipidemia, and hypertri-glyceridemia (27). Delayed wound healing, impeded bloodflow in peripheral blood vessels, and diabetic nephropathyare also observed (28–30).

ASC sheets have been used to accelerate wound healingin previous studies. Lin et al. (31) and McLaughlin andMarra (32) reported that transplantation of human ASC

sheets with fibrin-coated membranes accelerates woundhealing in a 12-mm–diameter full-thickness skin defecton the backs of 6-week-old athymic nude mice. Cerqueiraet al. (33) reported that human ASC sheet transplantationpromotes wound regeneration in 10-mm–diameter full-thickness skin defects on the backs of 5-week-old normalmice. In contrast to previous studies, the current studycreated a wider full-thickness skin defect with exposedbone (wound area 15 mm2) on the parietal region of16-week-old ZDF rats. In addition, the periosteum, whichmay support the regeneration of the skin, was removed toincrease the severity of the wound (25). Diabetic woundswith exposed bone are often observed clinically after de-bridement. To assess the clinical applicability of themethod for severe diabetic foot ulcers, this study attemp-ted to produce a wider full-thickness skin defect modelwith exposed bone and removed periosteum in rats withtype 2 diabetes and obesity.

The differences in wound-healing mechanisms notedbetween humans and rodents are attributed to anatomical

Figure 2—Confirmation of the characteristics and bipotency of rASCs obtained from normal Lewis rats. A: One hundred cells were culturedin a 60-cm2 dish for 7 days and stained with crystal violet to assess the number of cell colonies and confirm the self-renewal ability ofrASCs. rASCs were able to differentiate into adipogenic and osteogenic lineages. B: After culture in adipogenic induction medium for2 weeks, rASCs stained positively with Oil Red O. C: After culture in osteogenic induction medium for 4 weeks, the rASCs stained positivelywith alizarin red S, confirming their osteogenic differentiation potential. D–H: The expression of surface antigens on the rASCs was analyzedby flow cytometry. rASCs at passage 3 exhibited a typical MSC phenotype because they were positive for CD29 and CD90 and negative forCD11b, CD31, and CD45.

2728 Allogeneic ASC Sheet Therapy for Diabetic Ulcer Diabetes Volume 64, August 2015

differences of the skin (e.g., panniculus carnosus layer).Normal rats exhibit contraction-based wound healing,whereas humans display re-epithelialization and granula-tion tissue formation–based wound healing. Typically,wound splinting in rodent models helps to minimizewound contracture, which allows for the gradual forma-tion of granulation tissue (34). Slavkovsky et al. (28)reported that the contraction of diabetic wounds in ZDFrats is impaired, whereas nondiabetic wounds arealmost entirely closed by contraction. Epithelialization also

contributes to repair in this diabetic wound model in ZDFrats. Furthermore, the bilayer artificial skin we used inthe current study prevented wound contraction and pro-moted new connective tissue matrix synthesis, resem-bling the true dermis (35). In the current study,artificial skin was placed on a recipient wound and fixedwith nylon threads to reduce wound contraction and pre-vent wound enlargement as a result of the rat’s loose

Figure 3—Macroscopic images of full-thickness skin defects.A macroscopic photograph of a typical full-thickness skin defectimmediately after the creation of a 15 3 10-mm wound (A); thewound was covered with artificial skin using 10 stitches (B). Mac-roscopic photographs of full-thickness skin defects in the control(C, E, G, I, and K) and transplant (D, F, H, J, and L) groups at 3 (Cand D), 7 (E and F ), and 14 days (G and H) after creation of thewound (n = 12) and at 21 days (I and J) and 28 days (K and L) aftercreation of the wound (n = 6). White arrows indicate the area ofscar-like appearance with complete epithelialization.

Figure 4—A: Measurement and analysis of the wound area with orwithout rASC sheet transplantation. Macroscopic photographs ofthe wounds were digitized, and the wound area at 0, 3, 7, 10, and 14days (n = 12) and 18 and 21 days (n = 6) after surgery was quantifiedusing ImageJ software. The average wound areas at 0, 3, 7, 10, 14, 18,and 21 days after the operation were 1.56, 1.42, 1.23, 1.11, 0.87, 0.62,and 0.38 cm2 in the control group and 1.58, 1.30, 1.16, 0.93, 0.65,0.40, and 0.25 cm2 in the transplantation group, respectively. Theaverage wound area in the transplantation group 7 days after cre-ation of the wound was significantly reduced compared with thecontrol group, indicating accelerated wound healing in the trans-plantation group. B: The times required to complete wound closureof full-thickness skin defects are presented on a scatter diagram ofthe data for both the control and the transplantation groups (n = 6).The red bars on the diagram indicate the mean time to completewound closure for both groups. The mean (range) times to completewound closure were 34.2 (28–42) and 25.6 (18–35) days in thecontrol and transplantation groups, respectively. The time to com-plete wound closure was significantly reduced in the transplantationgroup compared with the control group (P = 0.02). *P < 0.05.

diabetes.diabetesjournals.org Kato and Associates 2729

skin. Even if considerable contraction of the wound was ob-served, it appeared similar across all conditions betweenthe control and the transplantation groups except in thepresence or absence of ASC sheet transplantation. Underthese conditions, significant differences in wound area

and vascular density were observed between the groups.This experiment may be useful for evaluating ASC sheets.

Estrogen deficiency is associated with impaired cuta-neous wound healing. Female rats exhibit a faster rate ofwound healing with a higher rate of wound contractionbecause of their thinner skin (36). Accordingly, to reducepotential hormonal influences on wound healing, onlymale rats were used in this study.

ASCs exhibit several advantages compared with MSCsderived from other tissues (12). Adipose tissue is rela-tively abundant in the human body. These tissues areaccessible and can be collected using a minimally invasiveliposuction procedure. ASCs are also abundant in adiposetissue (11). Moreover, many reports have suggested thatASCs play an important role in accelerating wound heal-ing (37,38). Thus, ASCs are considered a good cell sourcefor stem cell therapies.

This study investigated whether allogeneic ASC sheetstogether with artificial skin are effective at acceleratingwound healing. Xing et al. (39) reported that postopera-tive wound healing after laparotomy is impaired in obeserats, suggesting that the risk of laparotomy dehiscencemay be increased by obesity. In clinical practice, patientswith diabetic ulcers often exhibit severe diabetes compli-cations, including uncontrolled high blood glucose anda high BMI. The collection of adipose tissue from patientswith acute diabetic ulcers is difficult. Furthermore, af-ter obtaining autologous ASCs, ASC sheet preparationrequires several weeks. rASCs from diabetic animals ex-hibit altered properties and impaired functions (40). Infact, our group attempted to create rASC sheets usingrASCs from ZDF rats, but cell proliferation was slow(data not shown). Given that MSCs, including ASCs, sup-press immune responses (41), rASC sheets prepared fromnormal rats were transplanted into defects in ZDF rats tostudy allogeneic transplantation. In the current study,accelerated wound healing was observed in the transplan-tation group, and no obvious clinical signs of immunerejection, such as erythema, other local inflammatorysigns, or visible signs of necrosis as described by Falangaet al. (42) and Briscoe et al. (43), were observed in thetransplantation group during the experimental period.

rASC sheets were successfully created from Lewis ratsexhibiting a wide range of ages (8, 10, 16, 20, and 33weeks) (data not shown), and rASC sheets from 20–33-week-old rats were used for the transplantations. Thisstudy showed that rASC sheets from 33-week-old ratssecreted growth factors and accelerated wound healingcompared with the control group, suggesting that adiposetissue from middle-aged rats created functional rASCsheets. ASC sheets from patients over a wide range ofages could potentially be useful for treatment in clinicalsettings. In clinical practice, the age of patients undergo-ing liposuction operations ranges from young to elderly,and most patients are adults. This study intentionallyused cells from middle-aged rats to simulate actual clinicalpractice.

Figure 5—Histological analysis of wounds treated with rASCsheets. H-E–stained sagittal cross-sections of the control (A) andrASC sheet transplant (B) groups evaluated 14 days after surgery.The arrowheads indicate the epidermal margins. The region be-tween the two arrowheads corresponds to the epidermal defect.Scale bars = 500 mm. C and D: Microphotographs display higher-magnification images of the boxed regions of the dermal layers in Aand B, respectively. Scale bars = 100 mm. E and F: Quantitativeanalysis of neovascularization of the wounds. Control and trans-plant specimens were stained with CD31, a blood vessel endothe-lium cell marker (green). G: Blood vessel density in the wound areawas measured at 14 days after wound creation and was calculatedby dividing the area of CD31-positive vessels by the total area. Theaverage CD31-positive area was 1.39% (SD 0.94) in the controlgroup (E) and 3.77% (SD 1.74) in the transplantation group (F )(n = 6). The average blood vessel density in the transplantationgroup was increased by ;2.5-fold compared with the controlgroup, and this difference was statistically significant (P < 0.001).Scale bar = 50 mm. *P < 0.05.

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In this study, rASC sheets contained ;9 3 105 rASCsat the time of transplantation. A previous study based onthe topical administration of MSCs in a collagen scaffoldreported that 1 3 106 MSCs were transplanted into

a 6-mm–diameter wound, which was the highest dose inthat experiment. The highest percentage of wound closureand angiogenesis was achieved in 1 week with this cell dosecompared with lower doses, suggesting that the wound-healing

Figure 6—The number of rASCs per rASC sheet and the levels of growth factors secreted by an rASC sheet as measured by ELISA. rASCsachieved subconfluency (80–90%) in a 35-mmdiameter dish 72 h after seeding, and this time point was considered as day 0 (A). Themeasured timepoints in the remaining panels (B–G) were the same. A: The number of rASCs per cell sheet was assessed on days 0, 2, and 4 (n = 8). Theaverage number of rASCs per rASC sheet was 3.88 3 105, 6.59 3 105, and 8.76 3 105 cells at days 0, 2, and 4, respectively (n = 8). Thelevels of VEGF, HGF, TGF-b1, IGF-I, EGF, and KGF were determined in vitro with quantitative ELISA kits for these growth factors. B: VEGFlevels were 32.58, 13.90, and 18.60 ng/sheet/day at days 0, 2, and 4, respectively (n = 4). C: HGF levels were 1.32, 3.73, and 3.27 ng/sheet/day at days 0, 2, and 4, respectively (n = 4). D: TGF-b1 levels were 0.01, 0.49, and 0.76 ng/sheet/day at days 0, 2, and 4, respectively (n = 4).E: IGF-I levels were 0.04, 0.77, and 2.78 ng/sheet/day at days 0, 2, and 4, respectively (n = 4). F: EGF levels were 0.32, 0.47, and 0.44 ng/sheet/day at days 0, 2, and 4, respectively (n = 4). G: KGF levels were 0, 0.06, and 0.42 ng/sheet/day at days 0, 2, and 4, respectively (n = 4).

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effects of MSCs may be dose dependent (44). In thecurrent study, approximately the same number of rASCswas transplanted into larger defects (15 3 10 mm) as a cellsheet, and wound closure in the transplantation group wassignificantly accelerated 2 weeks after the operation. Theseresults suggest that cell sheet technology might enhancethe cellular activity and efficacy of cell transplantation becausecell sheets are harvested with intact cell-cell junctions andundamaged ECM and can be immediately transplanted intothe defect site (19). Moreover, a cell sheet is thin and exhibitssufficient flexibility to allow it to adhere well to the roughsurface of a wound. In fact, cell sheet technologies havebeen used for the reconstruction of various tissues, withcurative effects (45,46).

In this study, the time to complete wound closure wassignificantly faster in the transplantation group, andincreased blood vessel density was confirmed in thetransplantation group 14 days after transplantationcompared with the control group. The regeneration andintroduction of capillaries inside the wound are importantbecause tissue regeneration commonly requires blood flowto supply oxygen and nutrition and remove waste.Increased blood vessel density at the wound site mayhelp to promote wound healing.

Wound healing requires complex processes, includinga coordinated interplay between cells and growth factors;it also requires multiple growth factors acting synergisti-cally (47). A previous study reported that the rate ofVEGF secretion from surrounding cells, such as macro-phages and fibroblasts, is reduced in diabetic wounds(48). Numerous physiological factors contribute towound-healing deficiency in individuals with diabetes (48).

In this study, various secreted growth factors (VEGF,HGF, TGF-b1, IGF-I, EGF, and KGF) played importantroles in wound repair and were detected in large quanti-ties in the conditioned medium from rASC sheets. ASCssecrete angiogenic growth factors (49), such as VEGF andHGF (50), and these growth factors could contribute toneovascularization (51,52) and accelerate wound healingin both normal and diabetic rats (15). These findings,which are consistent with the current results, suggestthat the paracrine effects of transplanted rASCs mightenhance neovascularization and wound epithelialization.

In this study, EGFP-expressing rASCs were surroundedwith blood vessel endothelial cells 14 days aftertransplantation, suggesting that the rASCs remainedat the wound site for at least 14 days and were able todifferentiate into mural cells at the wound site and

Figure 7—Transplantation of rASC sheets prepared from rASCsisolated from EGFP rats. Bright-field (A) and dark-field (B) photo-graphs depict the wound immediately after transplanting an EGFP-positive rASC sheet and before overlaying the artificial skin.Fluorescent microphotography (B) confirmed that the transplantedEGFP-positive rASC sheet expressed green fluorescence (green).Scale bars = 10 mm. C: The differentiation fate of the transplanted

rASC sheet, which expressed green fluorescence, was examined.D: Frozen sections of the wounds were immunostained with RECA-1 (red) to visualize blood vessel endothelial cells. E: An overlaidphotograph of C depicting EGFP-positive rASCs (green) and Ddepicting RECA-1–positive endothelial cells (red) revealed that theEGFP-expressing rASCs (green) were located on the periphery ofthe RECA-1–stained areas (red).

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support the reconstitution of new blood vessels (53,54).MSCs originate from and are natively associated withblood vessel walls, suggesting that MSCs belong to a sub-set of perivascular cells (55,56). Some studies reportedthat ASCs have the potential to differentiate into endo-thelial cells (57,58). Also previously reported was that themajority of transplanted ASCs are adjacent to microves-sels and differentiate into pericytes (59,60).

The results from this animal study demonstratea promising approach for the application of ASC sheetstogether with artificial skin to accelerate diabetic woundhealing. However, additional studies in larger animals arenecessary to confirm and validate the efficacy of ASCsheets with artificial skin for clinical trials.

In conclusion, this study demonstrated that allogeneictransplantation of ASC sheets in combination with artificialskin accelerates wound healing in a rat model of type 2diabetes and obesity. The rASC sheets displayed effectiveparacrine activity because they secreted angiogenic growthfactors and potentially differentiated into perivascular cellsto support the construction of new blood vessels. Thus,ASC sheets exhibited therapeutic value in this rat modeland may be useful for accelerating the healing of diabeticulcers in patients with type 2 diabetes and obesity.

Acknowledgments. The authors thank Toshiyuki Yoshida and KaoruWashio of the Institute of Advanced Biomedical Engineering and Science, TokyoWomen’s Medical University, for valuable advice and suggestions; Hozue Kurodaof the Institute of Advanced Biomedical Engineering and Science, Tokyo Women’sMedical University, for excellent technical support; Taichi Ezaki of the Departmentof Anatomy and Developmental Biology, Tokyo Women’s Medical UniversitySchool of Medicine, for expert technical advice and support in immunohisto-chemistry; Yukiko Koga of the Department of Plastic and Reconstructive Surgery,Juntendo University School of Medicine, for practical advice; and Norio Ueno, ofthe Institute of Advanced Biomedical Engineering and Science, Tokyo Women’sMedical University, for English editing and valuable advice. They also thank YukaTsumanuma and Supreda Suphanantachat of Tokyo Medical and Dental Univer-sity; Issei Komatsu and Daisuke Fujisawa of the Institute of Advanced BiomedicalEngineering and Science, Tokyo Women’s Medical University; Nana Mori of theDepartment of Odontology, Periodontology Section, Fukuoka Dental College; andAya Matsui of the Department of Anatomy and Developmental Biology, TokyoWomen’s Medical University School of Medicine, for technical support. Finally,the authors thank Kazuki Ikura of the Diabetic Center, Tokyo Women’s MedicalUniversity School of Medicine, for expert advice on diabetic feet.Funding. This study was supported by the Creation of Innovation Centers forAdvanced Interdisciplinary Research Areas Program of the Project for DevelopingInnovation Systems “Cell Sheet Tissue Engineering Center (CSTEC)” from theMinistry of Education, Culture, Sports, Science and Technology (MEXT), Japan.Duality of Interest. T.O. is a founder and director of the board of CellSeed,Inc., and holds technology licensing and patents from Tokyo Women’s MedicalUniversity. T.O. also is a stakeholder of CellSeed, Inc. Tokyo Women’s MedicalUniversity receives research funds from CellSeed, Inc. No other potential conflictsof interest relevant to this article were reported.Author Contributions. Y.K. wrote the manuscript and researched data.T.I. and S.M. researched data, contributed to the discussions, and reviewed andedited the manuscript. M.Y., T.O., and Y.U. participated in discussions andreviewed and edited the manuscript. T.I. and T.O. are the guarantors of thiswork and, as such, had full access to all the data in the study and take re-sponsibility for the integrity of the data and the accuracy of the data analysis.

Prior Presentation. Parts of this study were presented in abstract form atthe 74th Scientific Sessions of the American Diabetes Association, San Francisco,CA, 13–17 June 2014.

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