potential of allogeneic adipose-derived stem cell

1

Potential of Allogeneic Adipose-Derived Stem Cell – Hydrogel Complex for

Treating Diabetic Foot Ulcers

Running Title: Stem Cells for Diabetic Ulcers

Kyung-Chul Moon, MD, PhD1, *, Hyun-Suk Suh, MD, PhD1, †, Ki-Bum Kim, MD*, Seung-

Kyu Han, MD, PhD*, Ki-Won Young, MD, PhD‡, Jin-Woo Lee, MD, PhD§

*Department of Plastic Surgery, Korea University Guro Hospital, Seoul, South Korea

†Department of Plastic Surgery, Asan Medical Center, Seoul, South Korea

‡Department of Foot and Ankle Surgery, Eulji Medical Center, Seoul, South Korea

§Department of Orthopaedic Surgery, Yonsei University College of Medicine, Seoul,

South Korea

1Kyung-Chul Moon and Hyun-Suk Suh contributed equally to this work.

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Diabetes Publish Ahead of Print, published online January 24, 2019

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Corresponding author:

Seung-Kyu Han, M.D., Ph.D.

Department of Plastic Surgery

Korea University Guro Hospital

148 Guro-Dong, Guro-Ku

Seoul, South Korea (152-703)

Tel: + 82 2 2626 3333; Fax: + 82 2 868 6698

E-mail: [email protected]

Manuscript word count: 3915 words

Abstract word count: 194 words

Number of the table: 4

Number of figures: 4

Keywords: cell therapy, chronic foot ulcer, diabetic foot, stem cell, tissue engineering,

wound healing

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mailto:[email protected]

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Abstract

Mesenchymal stem cells (MSCs) may hold great promise for treating diabetic wounds.

However, it is difficult for a clinician to use MSCs because they have not been

commercialized. Meanwhile, a new commercial drug that contains adipose-derived

stem cells (ASCs) has been developed. The purpose of this study was to examine the

potential of allogeneic ASC sheets for treating diabetic foot ulcers. Fifty-nine patients

with diabetic foot ulcers were randomized to either ASC treatment group (n = 30) or

control group treated with polyurethane film (n = 29). Either allogeneic ASC sheet or

polyurethane film was applied on diabetic wounds weekly. These wounds were

evaluated for a maximum of 12 weeks. Complete wound closure was achieved for 73%

in the treatment group and 47% in the control group at week 8. Complete wound

closure was achieved for 82% in the treatment group and 53% in the control group at

week 12. Kaplan-Meier median times to complete closure were 28.5 and 63.0 days for

the treatment group and the control group, respectively. There were no serious adverse

events related to allogeneic ASC treatment. Thus, allogeneic ASCs might be effective

and safe to treat diabetic foot ulcers.

This trial was registered with ClinicalTrials.gov (registration number: NCT02619877).

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The pathophysiologic relationship between diabetes and impaired wound healing is

complicated. Attenuated activities of cells that play a key role in wound healing

contribute to the impairment of tissue restoration in diabetic ulcers. Keratinocytes and

fibroblasts isolated from diabetic foot ulcers show lower proliferative potential and

attenuated growth factor production (1). Therefore, there is considerable interest in the

treatment of diabetic foot ulcers with biological dressings and/or tissue-engineered

products.

Mesenchymal stem cells (MSCs) may hold great promise for treating diabetic wounds

because they have advantages as allogeneic and autologous cells. MSCs demonstrate

low levels of immunity-assisted rejection with ability to divide without apoptosis (2, 3).

Even after 20 or 30 cycles of cell doubling in culture, they still retain their initial stem

cell properties (4).

Bone marrow stroma has been a main source of MSCs. Previous studies performed by

our group have demonstrated that bone marrow-derived MSCs (BM-MSCs) can

synthesize higher amounts of collagen, fibroblast growth factor (FGF), and vascular

endothelial growth factor (VEGF) in vitro than dermal fibroblasts. Furthermore, they

showed greater activity in terms of granulation tissue formation, epithelialization, and

angiogenesis in vivo, indicating their potential use in accelerated wound healing (2, 5,

6). However, there have been no commercial drugs that contain BM-MSCs to treat

diabetic foot ulcers.

Recently, adipose-derived stem cells (ASCs) have been demonstrated to be one of the

main sources of MSCs (7-10). Kato et al have shown that allogeneic transplantation of

an ASC combined with artificial skin can accelerate wound healing in Zucker diabetic

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fatty rats in vivo (11). McLaughlin et al demonstrated that ASC sheets can increase

wound healing compared with untreated controls in vivo (12). Furthermore, ASCs

showed greater activity in terms of epithelialization, angiogenesis, and secretion of

growth factors (11). Moreover, bioengineered dermal substitute comprised of allogeneic

ASCs has been commercialized to help diabetic wound healing. The purpose of this

study was to examine the potential of hydrogel-based allogeneic ASC sheets for treating

diabetic foot ulcers.

Research Design and Methods

This was a randomized, comparator-controlled, single-blind, parallel-group, multi-

center study in which patients with diabetic foot ulcers were recruited consecutively

from four centers in Korea. This trial was registered with ClinicalTrials.gov

(registration number: NCT02619877). This clinical study protocol and informed

consent document were also approved by appropriate Institutional Review Board for

each participating center and the Food and Drug Administration (FDA) of Korea (study

code: ALLO-ASC-DFU-201).

Patients

The major inclusion criteria were: age between 18 and 80 years; patients with diabetic

type I/II; longer than 4 weeks for the history of ulcer at screening; wound size between

1 and 25 cm2; and wound depth of Wagner grade I and II. Additional criteria were:

blood flow around the ulcer was detectable by a Doppler Test, Ankle Brachial Index

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range of > 0.7 to < 1.3, or transcutaneous oxygen pressure higher than 30 mmHg. Key

exclusion criteria included a change in wound size of more than 30% within one week

from screening, wound infection, human immunodeficiency virus positive, HbA1c

higher than 15%, and postprandial blood sugar level higher than 450 mg/dl.

Once patients were enrolled, they were randomized into one of two treatment groups.

Randomization schedules were stratified according to clinical center using a permuted-

block method with a block size of four to six through Statistical Analysis System.

Between November 2015 and October 2016, 24, 11, 12, and 12 patients with diabetic

foot ulcers from the Korea University Guro Hospital, Eulji General Hospital, Asan

Medical Center, and the Yonsei University Severance Hospital, respectively, were

included in this clinical trial. Of these 59 subjects, five subjects in the control group

were excluded before the study due to withdrawal of consents and ineligibility of

criteria such as small wound size (< 1 cm2). Therefore, assessment of modified intent-

to-treat set (mITT) was conducted on data available from the remaining 54 subjects.

Five subjects in the treatment group and five subjects in the control group were further

excluded before completion of the study due to occurrence of adverse events and

protocol violations such as violation of timeline. Of the remaining 44 subjects, 25 were

assigned to the treatment group and 19 were assigned to the control group. Three

subjects in the treatment group and two subjects in the control group were excluded

because of violations of eligibility criteria such as high serum glucose levels (> 450

mg/dl) during the study period. Therefore, assessment of modified per-protocol set

(mPPS) was conducted on data available from the remaining 39 subjects who

completed the study (Figure 1). Demographics and wound characteristics of subjects in

the two groups at baseline are shown in Table 1.

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Allogeneic ASC Sheet Preparation

Allogeneic ASC sheet (ALLO-ASC-Sheet, Anterogen, Seoul, Korea) is a 5 × 5cm

hydrogel sheet containing allogeneic ASCs. Briefly, ASCs were obtained from

subcutaneous fat tissue of healthy donors who provided informed consents via

liposuction. Obtained adipose tissues were rinsed with phosphate buffered saline

(Hyclone, Logan, UT) in Dulbecco’s Modified Eagle Medium/Ham’s F-12 (DMEM,

Hyclone) containing 0.025% type I collagenase (Invitrogen, Carlsbad, CA) for 80

minutes at 37°C according to the manufacturer’s protocol. The top lipid layer was

removed and the remaining liquid portion was centrifuged at 300 × g for 10 minutes at

4°C. The stromal vascular fraction was collected and cultured in DMEM to obtain

required number of ASCs. ASCs seeded onto the hydrogel matrix were cultured until

the number of ASC reached about 1 × 106 cells/sheet. They were stored frozen at -80°C

according to the manufacturer’s undisclosed protocols.

ASCs were characterized by their expression of stromal cell-associated markers such

as CD10, CD13, CD29, CD44, and CD90. These cells were negative for the expression

of hematopoietic stem cell-associated markers (CD34 and CD45). Genomic stability of

ASCs was evaluated by karyotyping analysis. A series of additional efficacy release

tests were performed for the final product, including confirmation of cell count and

assessment of cell viability. ASCs were subjected to a series of quality controls to

ensure their purity, safety, and potency approved by the FDA of Korea.

Techniques

In the treatment group, the ulcer was cleaned to remove dirt and other debris with 3%

hydrogen peroxide followed by saline solution. The allogeneic ASC sheet which was

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stored at -80°C was thawed at room temperature for 1-2 minutes, tailored according to

the wound size and shape, and applied directly to the wound bed as a primary dressing.

It was then covered with a polyurethane foam (Mepilex, Molnlycke Health Care,

Gothenburg, Sweden) as a secondary dressing. The graft and polyurethane foam were

left on the wound for seven days. Dressing changes were scheduled at weekly intervals.

If found necessary, the patient returned to the hospital 2-3 times per week after

application of the graft so that the wound could be examined. Only the secondary

dressing was changed. In the control group, all conditions including management for

diabetic foot ulcers were set up to be identical to those for the treatment group. A

meshed polyurethane film with silicone adhesive (Mepitel, Molnlycke Health Care) was

applied as primary dressing over the wound. It was then covered with the same

secondary dressing used for the treatment group. Visits and dressing changes for the

control group were scheduled as the same for the treatment group.

All patients with ulcers on weight-bearing sites or sites otherwise subjected to pressure

when wearing shoes had pressure off-loaded using foam dressings with a hole on the

ulcer site and footwear with cushioning insoles.

Evaluation

Wounds were evaluated weekly until the 12th week visit. When the ulcer was

completely healed before the 12th week visit, treatment was stopped but visits were

continued as scheduled until week 12 to evaluate long-term safety. Wound evaluation

was performed in a single-blinded fashion. Patients did not know whether or not their

wounds had been treated with ASC sheets. However, wound evaluators were aware of

the method of treatment. Photographs were captured at baseline, follow-up, and last

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visit using standardized photographic equipment.

The mPPS population was used for primary and secondary efficacy analysis in this

study. The mITT population was used for the safety analysis and summaries which

included all subjects who were treated with ASC sheets and visited the clinic at least

once.

Primary efficacy criterion was the percentage of subjects who achieved complete

wound closure within the 8-week study period. Secondary efficacy criteria were the

proportion of subjects who achieved complete wound closure within 12-week study

period, the time required for complete wound closure in patients who achieved

complete healing within 12 weeks, the rate of wound size reduction from baseline, and

the proportion of subjects by Wagner grade at each week. In addition, post-hoc analyses

were performed to evaluate the efficacy of the treatment and prognostic factors such as

wound depth and location. Wound size was determined using a Visitrak Digital

Planimetry Wound Measurement System (Smith & Nephew, Hull, UK).

For safety study, information on adverse event, adverse drug reaction, serious adverse

event, and serious adverse drug reaction was collected at each visit. Safety was also

monitored at indicated timepoints by evaluating adverse event reports, laboratory

assessments, and vital signs. A systematic immunologic study was conducted to detect

anti-human leukocyte antigen (anti-HLA) panel reactive antibodies at baseline, the 1st

week, and the 12th week after ASC treatment.

Ulcer recurrence and adverse events of healed ulcers were monitored for two years

after healing by reviewing medical records and/or through telephone interviews. This

follow-up study was registered with ClinicalTrials.gov (registration number:

NCT03183804) and the FDA of Korea (study code: ALLO-ASC-DFU-202).

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Statistical Analyses

Based on previous studies (13, 14), the number of subjects was calculated in this study.

Weighted complete healing was the sum of number of subjects with complete healing in

each study divided by the sum of the number of subjects used in each study. The

weighted complete healing was 0.81 in the treatment group and 0.45 in the control

group. The following values were used to calculate the number of subjects: Type I error

(α) = 5%, power of test = 80%, complete healing rate in the treatment group = 0.81, and

complete healing rate in the control group = 0.45. When each complete healing rate was

Pt for the treatment group and Pc for the control group, the two-sided null hypothesis

and alternative hypothesis were as follows.

Null hypothesis: Pt = Pc, alternative hypothesis: Pt ≠ Pc.

In this study, Z0.975 was 1.96 and Z0.80 was 0.84. Therefore, the number of subjects in

each group could be calculated as follows (15).

N =

= 24.28

Considering a drop-out rate of broadly 10%, the number of subjects in each group was

26.97 (=24.28/(1-0.10)) and a sample size of 54 (=27 × 2) randomly assigned subjects

was required. Based on our experience from previous clinical trials for treating diabetic

foot ulcers, approximately 10% of subjects experienced unexpected events related to

diabetes-related complications and high-risk comorbidities. Therefore, we added

another 10% of subjects, bringing the total number of subjects enrolled in this study to

59.

Pearson’s Chi-square test was used to evaluate the proportion of subjects who

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achieved complete wound closure and the safety set. Log-rank test and independent t-

test or Wilcoxon rank-sum test were used to evaluate the time required for complete

closure and the rate of wound size reduction, respectively. The median time to complete

closure was also estimated using the Kaplan-Meier method. A P-value < 0.05 was

considered statistically significant.

Results

The proportion of subjects achieving complete wound closure at week 8 in the

treatment group was 73% (16/22). It was 47% (8/17) in the control group (P = 0.102).

For results of secondary efficacy criteria, the proportion of subjects achieving

complete wound closure at week 12 in the treatment group was 82% (18/22) and 53%

(9/17) in the control group (P = 0.053). The mean time required for complete wound

closure was 40.8 ± 5.3 days in the treatment group and 51.2 ± 3.9 days in the control

group. The Kaplan-Meier median time to complete wound healing was 28.5 days for

the treatment group and 63.0 days for the control group (P = 0.033, Figure 2). The rate

of wound size reduction at week 1 was 49.6 ± 25.7% in the treatment group and 23.0 ±

32.2% in the control group (P = 0.007). The rate of wound size reduction was also

statistically significant between the two groups at nine weeks out of 12 study weeks

(Figure 3). Post-hoc analyses were performed to further explore other prognostic factors

such as baseline wound depth and location that might affect the efficacy of the

treatment (Figure 4, Tables 2 and 3).

Incidence rates of adverse events are shown in Table 4. None of these events were

related to study dressings. There were no significant differences between the two

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groups. The incidence of serious adverse event was similar between treatment and

control groups. Cellulitis on untargeted sites, paresthesia, uncontrolled diabetic mellitus,

and cardiac arrest occurred as serious adverse events. However, none of these serious

adverse events was considered to be related to the treatment. No other serious adverse

events were found in either group. No clinically meaningful changes from baseline

clinical and laboratory parameters including serum chemistry, hematology, urinalysis,

and vital signs occurred in any of these patients.

In the systematic immunologic study, 16 subjects (11 in the treatment group and 5 in

the control group) showed anti-HLA antibodies >1,500 fluorescence intensity values at

the 1st week and the 12th week. Among these subjects, three (27%) in the treatment

group and one (20%) in the control group showed slight elevation of antibodies at the

12th week. However, we observed no obvious clinical signs of rejection such as

erythema, local inflammatory signs, or visible signs of necrosis, as described by

previous studies (16, 17), in these subjects.

Ulcer recurrences and adverse events in healed ulcers were successfully monitored for

two years for 16 subjects in the treatment group and 8 in the control group. We

reviewed medical records of subjects who attended routine appointments for 7 subjects

in the treatment group and 6 in the control group. Nine subjects in the treatment group

and 2 in the control group were interviewed over the telephone. Two subjects in the

treatment group and no subjects in the control group showed ulcer recurrence six

months after completion of the trial, but these recurred ulcers eventually healed. We

noted no adverse events related to wound dressings in either group for two years after

completion of the trial.

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Discussion

The development of advanced wound healing technology has triggered the use of cells

to overcome limitations of conventional methods. Various commercially available

allogeneic cell-scaffold complexes have been developed and widely used for treating

diabetic foot ulcers (18-21). Recently, a new bioengineered dermal substitute composed

of cultured autologous fibroblasts seeded on a hyaluronic acid sheet (Hyalograft 3D;

ChaBio & Diostech, Seoul, Korea) was developed. However, in several cases,

autologous primary cells are not accessible or are unavailable in sufficient number

because they may not have enough proliferative capacity for treatment in diabetic

patients due to cellular senescence in the presence of high glucose concentrations (22-

24).

Therefore, stem cells may hold great promise for addressing the need for viable cell

sources and MSCs have attracted much attention in the bioengineering field. The

therapeutic potential of allogeneic ASCs has been widely explored (11, 12, 25-27).

However, most of these studies are in vitro or in vivo studies. Our study might be the

first report demonstrating the efficacy of a commercially available allogeneic ASCs in

scaffold for treating diabetic foot ulcers. Cultured ASCs are derived from an unrelated

allogeneic donor. They can be stockpiled. Cell Bank enables a large-scale production

and ASC sheet can be stored at -80°C. It is stable for 12 months. The allogeneic ASC

sheet can maintain cell viability and potency up to one year and sustain its

specifications during transport and storage. The sheet is available immediately and

convenient to use for physicians and patients. Unlike autologous ASCs, no biopsy is

necessary. Patients do not need to wait for cells to be processed. The ASC sheet is

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nearly ready-made when the patient arrives at the clinic.

Prior to this phase II clinical trial study, phase I trial was carried out to evaluate early

safety of the treatment. For phase I trial, five subjects were enrolled. There was no

adverse event. Complete wound closure was achieved for all subjects within 8 weeks

(unpublished data). This phase II clinical trial involving allogeneic ASC products also

showed promising results. At 8th and 12th weeks after the treatment, more subjects in the

treatment group had complete wound healing compared to those in the control group.

Statistically, there were borderline differences. Other parameters demonstrated

statistically significant differences. In detail, the mean time to complete wound healing

was shorter for the treatment group compared to that for the control group (P = 0.033).

The rate of wound size reduction was also statistically significant between the two

groups at nine out of 12 weeks of study period. Results of post-hoc analyses indicated

that more subjects with Wagner grade 2 in the treatment group achieved complete

wound closure compared to those in the control group.

The results of the two-year follow-up study showed that ulcer recurrence occurred in

two subjects in the treatment group six months after completion of the trial although

their ulcer locations were the toe tip and the plantar foot, areas that are vulnerable to

pressure. Therefore, ulcer recurrence might not have been related to the ASC treatment.

In addition, we observed no adverse events related to the treatment for two years after

completion of the study.

The exact mechanism of action of allogeneic ASCs remains unknown. They might be

able to exert their effects through several mechanisms, including the release of various

growth factors such as VEGF, hepatocyte growth factor, transforming growth factor-β1,

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insulin-like growth factor-1, epithelial growth factor, and keratinocyte growth factor to

promote angiogenesis, collagen synthesis, and epithelialization (11). ASCs may

produce extracellular matrix proteins, induce proliferation of native fibroblasts, protect

cells from a noxious environment to aid in wound healing, and enhance regeneration of

new tissue. In addition, ASCs have anti-inflammatory effect by inhibiting T cell

activation, thereby inhibiting cellular signaling pathway of immune cells and resulting

in decreased inflammatory molecules such as tumor necrosis factor alpha and interferon

gamma. A combination of these processes is likely to be involved in their action

mechanisms (11, 27, 28).

Diabetic foot ulcers present a difficult treatment problem because the

pathophysiology of the condition involves multiple factors such as peripheral

neuropathy, peripheral vascular disease, repetitive trauma or pressure, and

superimposing foot infection. However, many diabetic foot ulcers are delayed or fail to

heal despite standard treatment such as debridement, infection control, pressure off-

loading, and lower extremity revascularization due to attenuated activities of cells

contributing to wound healing. In diabetic patients, delayed wound healing may result

in serious complications such as osteomyelitis and major/minor amputation (29-32); the

longer the ulcer persists, the greater the possibility that the patient will develop serious

complications that can lead to hospitalization, and delayed wound healing may increase

medical, economic, and social burdens. Therefore, efforts to reduce time to complete

wound closure are important for treating diabetic foot ulcers. In the present study,

subjects in the treatment group showed significantly faster complete wound closure

than did those in the control group (P = 0.033). This study suggested that the time to

complete wound closure could be reduced simply by applying allogeneic ASC sheets in

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diabetic patients. This is the key highlight of our study.

In this study, the follow-up duration was 12 weeks because many previous similar

clinical trials for diabetic wound healing also used 12 weeks for the follow-up duration

(13, 14, 18, 32-35). For precise assessment of the efficacy of the allogeneic ASC

treatment, we excluded external confounding factors that could have affected the

treatment such as vascular insufficiency and infection. Therefore, 12 weeks might be

enough to assess the time to complete wound closure in diabetic patients after

standardized diabetic foot ulcer management. It is important to emphasize that

allogeneic ASC treatment must be used along with other standard principles of diabetic

foot ulcer management, including debridement, infection control, pressure off-loading,

and revascularization (21, 36). Without adhering to these important principles, adding

an active adjunctive modality is unlikely to result in improved healing rates. Patients

with diabetic foot ulcer, who do not exhibit significant signs of wound healing despite

good metabolic control, acceptable vascularity (transcutaneous oxygen pressure > 40

mmHg), adequate pressure off-loading, and absence of infection might be good

responders to allogeneic ASC treatment.

The present study has some limitations. First, our study had all the limitations inherent

to a phase II trial. For example, the sample size was relatively small although the

number of subjects was determined by power calculation. Although acknowledging the

implications of multiplicity adjustments was important for helping to interpret the trial

results, we did not consider correcting the additional significance level using adjustment

for alpha inflation because multiplicity adjustments may not be necessary in exploratory

trials (37, 38). Among parameters of this study, differences in proportion of subjects

with complete wound closure were statistically at borderline. Furthermore, the

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percentage of subjects with amputation history, a risk factor for replication, in the

treatment group was more than two times compared to that in the control group. On the

contrary, more older subjects were enrolled in the control group compared to those in

the treatment group. Therefore, a phase III trial study should be followed with large

sample size of subjects to establish the effect of allogeneic ASCs with certainty. Second,

wound evaluation was performed in a single-blind fashion. Wound evaluators knew

whether or not patients were in the ASC-treated group, which might have affected the

results. However, this was unavoidable because the wound evaluators could

discriminate between ASC sheets and polyurethane films. Therefore, it was difficult to

conduct a double-blind design in this study. To increase transparency, our design

included careful checks of the extent to which evaluators were blinded to group

allocation. Third, further studies are necessary to determine the optimal interval for

ASC application. Application of ASCs more frequently than once a week might reduce

the time to healing.

In summary, results of this study showed that allogeneic ASC sheet might be effective

and safe to treat non-ischemic diabetic foot ulcers without infection.

Acknowledgements

The authors are indebted to Mi-Hyeong Kim who supervised the laboratory analyses.

This research was supported by a grant of the Korea Health Technology R&D Project

through the Korea Health Industry Development Institute. We thank all patients and

health-care professionals who contributed to make the trial possible. We also thank

investigators for their commitment, time, and effort.

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Contributions

S.K.H, K.W.Y., H.S.S., and J.W.L. identified, treated, and monitored study participants

and contributed to data recording. S.K.H. coordinated the work. K.C.M. and S.K.H.

interpreted the data and wrote the final version of the manuscript. K.B.K. contributed to

data analyses and data handling. K.C.M. and H.S.S. contributed equally to this work.

All authors had direct access to original data, critically revised the draft, and approved

the final manuscript.

Guarantor

K.C.M is the guarantor of this work and, as such, has full access to all the data in the

study and takes responsibility for the integrity of the data and the accuracy of the data

analysis.

Conflict of interest

None of the authors have any conflict of interest to disclose.

Funding

This study was supported by grants from Anterogen (Seoul, Korea) and the Ministry

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of Health & Welfare, Republic of Korea (grant number: HI16C1037).

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model of skin rejection. Transplantation 1999;67:1590-918. Marston WA, Hanft J, Norwood P, Pollak R, Dermagraft Diabetic Foot Ulcer Study G. The efficacy and safety of Dermagraft in improving the healing of chronic diabetic foot ulcers: results of a prospective randomized trial. Diabetes Care 2003;26:1701-519. Steinberg JS, Edmonds M, Hurley DP, Jr., King WN. Confirmatory data from EU study supports Apligraf for the treatment of neuropathic diabetic foot ulcers. J Am Podiatr Med Assoc 2010;100:73-720. Han SK, Choi KJ, Kim WK. Clinical application of fresh fibroblast allografts for the treatment of diabetic foot ulcers: a pilot study. Plast Reconstr Surg 2004;114:1783-921. Han SK, Kim HS, Kim WK. Efficacy and safety of fresh fibroblast allografts in the treatment of diabetic foot ulcers. Dermatol Surg 2009;35:1342-822. Hehenberger K, Heilborn JD, Brismar K, Hansson A. Inhibited proliferation of fibroblasts derived from chronic diabetic wounds and normal dermal fibroblasts treated with high glucose is associated with increased formation of l-lactate. Wound Repair Regen 1998;6:135-4123. Moon KC, Lee JS, Han SK, Lee HW, Dhong ES. Effects of human umbilical cord blood-derived mesenchymal stromal cells and dermal fibroblasts on diabetic wound healing. Cytotherapy 2017;19:821-824. You HJ, Han SK. Cell therapy for wound healing. J Korean Med Sci 2014;29:311-925. Kaisang L, Siyu W, Lijun F, Daoyan P, Xian CJ, et al. Adipose-derived stem cells seeded in Pluronic F-127 hydrogel promotes diabetic wound healing. J Surg Res 2017;217:63-7426. Madonna R, Delli Pizzi S, Tartaro A, De Caterina R. Transplantation of mesenchymal cells improves peripheral limb ischemia in diabetic rats. Mol Biotechnol 2014;56:438-4827. Cianfarani F, Toietta G, Di Rocco G, Cesareo E, Zambruno G, et al. Diabetes impairs adipose tissue-derived stem cell function and efficiency in promoting wound healing. Wound Repair Regen 2013;21:545-5328. Shin L, Peterson DA. Human mesenchymal stem cell grafts enhance normal and impaired wound healing by recruiting existing endogenous tissue stem/progenitor cells. Stem Cells Transl Med 2013;2:33-4229. Falanga V. Wound healing and its impairment in the diabetic foot. Lancet 2005;366:1736-4330. Boulton AJ, Vileikyte L, Ragnarson-Tennvall G, Apelqvist J. The global burden of diabetic foot disease. Lancet 2005;366:1719-2431. Bolton LL. Quality Randomized Clinical Trials of Topical Diabetic Foot Ulcer Healing Agents. Adv Wound Care (New Rochelle) 2016;5:137-4732. Veves A, Falanga V, Armstrong DG, Sabolinski ML, Apligraf Diabetic Foot Ulcer S. Graftskin, a human skin equivalent, is effective in the management of noninfected neuropathic diabetic foot ulcers: a prospective randomized multicenter clinical trial. Diabetes Care 2001;24:290-533. DiDomenico LA, Orgill DP, Galiano RD, Serena TE, Carter MJ, et al. Use of an aseptically processed, dehydrated human amnion and chorion membrane improves likelihood and rate of healing in chronic diabetic foot ulcers: A prospective, randomised, multi-centre clinical trial in 80 patients. Int Wound J 2018;34. Tettelbach W, Cazzell S, Reyzelman AM, Sigal F, Caporusso JM, et al. A confirmatory study on the efficacy of dehydrated human amnion/chorion membrane

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dHACM allograft in the management of diabetic foot ulcers: A prospective, multicentre, randomised, controlled study of 110 patients from 14 wound clinics. Int Wound J 2018;35. Park KH, Han SH, Hong JP, Han SK, Lee DH, et al. Topical epidermal growth factor spray for the treatment of chronic diabetic foot ulcers: A phase III multicenter, double-blind, randomized, placebo-controlled trial. Diabetes Res Clin Pract 2018;142:335-4436. Han SK, Kim HR, Kim WK. The treatment of diabetic foot ulcers with uncultured, processed lipoaspirate cells: a pilot study. Wound Repair Regen 2010;18:342-837. Bender R, Lange S. Adjusting for multiple testing--when and how? J Clin Epidemiol 2001;54:343-938. Wason JM, Stecher L, Mander AP. Correcting for multiple-testing in multi-arm trials: is it necessary and is it done? Trials 2014;15:364

Tables

Table 1. Demographic and baseline characteristics of subjects

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Parameter

Treatment

group

N=22

Control

group

N=17

Total

N=39

P-value

Male 14 (63.6) 13 (76.5) 27 (69.3)

Gender (N, %)Female 8 (36.4) 4 (23.5)

12

(30.8)

0.389*

Mean ±

SD59.9 ± 13.3 68.4 ± 9.9

63.6 ±

12.5

Median 60.5 70.0 66.0 0.034*Age (years)

Min,

Max26.0, 80.0 43.0, 79.0 26.0, 80.0

Non-

smoker11 (50.0) 9 (52.9) 20 (51.3)

Ex-

smoker 6 (27.3) 5 (29.4) 11 (28.2)

Smoking history

(N, %)

Smoker 5 (22.7) 3 (17.6) 8 (20.5)

1.000‡

Mean ±

SD167.9 ± 11.4

167.3 ±

7.3

167.7 ±

9.7Height (cm)

Median 166.0 167.5 167.0 0.839*

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Parameter

Treatment

group

N=22

Control

group

N=17

Total

N=39

P-value

Min,

Max143.0, 190.9

152.0,

179.9143.0, 190.9

Mean ±

SD72.9 ± 15.7

73.3 ±

14.2

73.1 ±

14.9

Median 68.5 75.0 70.4 0.938*Body weight (kg)

Min,

Max58.0, 124.3

51.6,

101.0

51.6,

124.3

Mean ±

SD25.8 ± 4.1 26.2 ± 5.0 25.9 ± 4.5

Median 25.4 25.7 25.5 0.787*Body mass index

(kg/m2)

Min,

Max20.6, 36.3 19.4, 40.5 19.4, 40.5

Mean ±

SD7.9 ± 1.6 8.1 ± 1.7 8.0 ± 1.6

Median 7.9 8.0 8.0 0.739*

Glycated hemoglobin

A1c at screening visit

(%)Min,

Max5.3, 10.5 5.5, 10.9 5.3, 10.9

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Parameter

Treatment

group

N=22

Control

group

N=17

Total

N=39

P-value

Mean ±

SD193.0 ± 96.5

208.5 ±

94.8

199.7 ±

94.8

Median 144.0 165.0 163.0 0.618*

Postprandial blood

glucose at screening

visit (mg/dl)Min,

Max65.0, 369.0

91.0,

445.0

65.0,

445.0

Mean ±

SD4.1 ± 0.4 4.1 ± 0.3 4.1 ± 0.4

Median 4.1 4.1 4.1 0.988*Serum albumin at

screening visit (g/dl)

Min,

Max3.3, 5.3 3.7, 4.9 3.3, 5.3

Mean ±

SD205.7 ± 128.0

239.7 ±

117.3

220.5 ±

123.0

Median 186.0 234.0 191.0 0.399*

DM duration

(months)Min,

Max1.0, 496.0

72.0,

433.01.0, 496.0

DM type (N, %) Type 1 1 (4.5) 0 (0) 1 (2.6) 1.000‡

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Parameter

Treatment

group

N=22

Control

group

N=17

Total

N=39

P-value

Type 2 21 (95.5) 17 (100) 38 (97.4)

Yes 13 (59.1) 9 (52.9) 22 (56.4)Eye disorders (N, %)

No 9 (40.9) 8 (47.1) 17 (43.6)0.701§

Yes 7 (31.8) 10 (58.8) 17 (43.6)Renal disorder (N, %)

No 15 (68.2) 7 (41.2) 12 (56.4)0.092§

Yes 18 (81.8) 16 (94.1) 34 (87.2)Cardiovascular

disorder (N, %) No 4 (18.2) 1 (5.9) 5 (12.8)0.363‡

Yes 13 (59.1) 11 (64.7) 24 (61.5)Neuropathy history

(N, %) No 9 (40.9) 6 (35.3) 15 (38.5)

0.721†

Mean ±

SD6.7 ± 11.0

10.2 ±

23.38.2 ± 17.3

Median 3.0 3.0 3.0 0.875†Duration of diabetic

foot ulcers (months)

Min,

Max1.0, 52.0 1.0, 99.0 1.0, 99.0

Wagner grade (N, %) Grade 1 14 (63.6) 11 (64.7) 25 (64.1) 0.945§

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Parameter

Treatment

group

N=22

Control

group

N=17

Total

N=39

P-value

Grade 2 8 (36.4) 6 (35.3) 14 (35.9)

Right 13 (59.1) 8 (47.1) 21 (53.8)

Left 9 (40.9) 9 (52.9) 18 (46.2)0.455§

Dorsal 11 (50.0) 12 (70.6) 23 (59.0)

Plantar 11 (50.0) 5 (29.4) 16 (41.0)0.195§

Forefoot 20 (90.9) 11 (64.7) 31 (79.5)

Midfoot 1 (4.5) 1 (5.9) 2 (5.1)

Ulcer location (N, %)

Hindfoot 1 (4.5) 5 (29.4) 6 (15.4)

0.068‡

Mean

±SD2.0 ± 0.9 2.8 ± 2.0 2.3 ± 1.5

Median 1.7 1.8 1.8 0.335†Wound size (cm2)

Min,

Max1.0, 4.5 1.1, 7.6 1.0, 7.6

Trauma 1 (4.5) 0 (0.0) 1 (2.6)

Cause of ulcer (N, %) Pressure

by shoes16 (72.7) 12 (70.6) 28 (71.8)

0.523‡

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Parameter

Treatment

group

N=22

Control

group

N=17

Total

N=39

P-value

Unknow

n 3 (13.6) 1 (5.9) 4 (10.3)

Others 2 (9.1) 4 (23.5) 6 (15.4)

Toe 10 (45.5) 3 (17.6) 13 (33.3) 0.068§Amputation history

(N, %) Total 11 (50.0) 4 (23.5) 15 (38.5) 0.092§

Yes 14 (63.6) 8 (47.1) 22 (56.4)Surgical history of

target site (N, %) No 8 (36.4) 9 (52.9) 17 (43.6)0.301‡

DM; Diabetic Mellitus

*: Independent t-test

†: Wilcoxon rank sum test

‡: Fisher’s exact test

§: Pearson’s chi-square test

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Table 2. Proportion of subjects with complete wound closure at weeks 8 and 12 by

baseline Wagner grade

Baseline Wagner

Grade

Treatment group

N =22 (%)

Control group

N = 17 (%)

Week 8

Grade 1 11/14 (78.6) 7/11 (63.6)

Grade 2 5/8 (62.5) 1/6 (16.7)

Week 12

Grade 1 12/14 (85.7) 8/11 (72.7)

Grade 2 6/8 (75.0) 1/6 (16.7)

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Table 3. Proportion of subjects with complete wound closure by wound location at

week 12

LocationTreatment group

N =22 (%)

Control group

N = 17 (%)

Dorsal 4/4 (100.0) 3/6 (50.0)

Plantar 7/11 (63.6) 0/5 (0.0)

Border 3/3 (100.0) 6/6 (100.0)

Tip 4/4 (100.0) -

Total 18/22 (81.8) 9/17 (52.9)

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Table 4. Adverse events in treatment and control groups

Treatment group

N= 30 (%)

Control group

N= 24 (%)

P-value

Abdominal pain 1 (3.3) 0 (0.0) 1.000*

Back pain 0 (0.0) 1 (4.2) 0.444*

Blood pressure increased 1 (3.3) 0 (0.0) 1.000*

Cardiac arrest 1 (3.3) 0 (0.0) 1.000*

Cellulitis 0 (0.0) 2 (8.3) 0.193*

Constipation 0 (0.0) 1 (4.2) 0.444*

C-reactive protein increased 0 (0.0) 1 (4.2) 0.444*

Diabetes mellitus inadequate control 1 (3.3) 0 (0.0) 1.000*

Diabetic neuropathy 1 (3.3) 0 (0.0) 1.000*

Dizziness 1 (3.3) 0 (0.0) 1.000*

Dyspnea 1 (3.3) 0 (0.0) 1.000*

Hyperglycemia 1 (3.3) 2 (8.3) 0.579*

Hyperkalemia 0 (0.0) 1 (4.2) 0.444*

Nasopharyngitis 0 (0.0) 1 (4.2) 0.444*

Nephropathy 0 (0.0) 1 (4.2) 0.444*

Edema 1 (3.3) 0 (0.0) 1.000*

Osteoporosis 1 (3.3) 0 (0.0) 1.000*

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Pain in extremity 2 (6.7) 1 (4.2) 1.000*

Paresthesia 1 (3.3) 2 (8.3) 0.579*

Pleural effusion 0 (0.0) 1 (4.2) 0.444*

Proteinuria 1 (3.3) 0 (0.0) 1.000*

Pyelonephritis acute 1 (3.3) 0 (0.0) 1.000*

Pyrexia 0 (0.0) 1 (4.2) 0.444*

Trigger finger 1 (3.3) 0 (0.0) 1.000*

*: Fisher’s exact test

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Figure Legends

Figure 1. A CONSORT diagram illustrating the flow of patients included in this study.

Figure 2. Kaplan-Meier diagram showing results on time to wound closure.

Figure 3. Rate of wound size reduction from baseline over time.

Figure 4. Proportion of Wagner grade at every visit week.

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Figure 1. A CONSORT diagram illustrating the flow of patients included in this study.

165x257mm (300 x 300 DPI)

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Figure 2. Kaplan-Meier diagram showing results on time to wound closure.

187x105mm (300 x 300 DPI)

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Figure 3. Rate of wound size reduction from baseline over time.

142x107mm (300 x 300 DPI)

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Figure 4. Proportion of Wagner grade at every visit week.

212x141mm (300 x 300 DPI)

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potential of allogeneic adipose-derived stem cell

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