Supernatant from Activated Omentum Accelerates Wound Healing in Diabetic mice wound model

概要

主論文の要旨

Supernatant from Activated Omentum Accelerates
Wound Healing in Diabetic mice wound model
活性化大網培養上清を用いた糖尿病潰瘍に与える影響

名古屋大学大学院医学系研究科
運動・形態外科学講座
（指導：亀井 讓
李 宇

総合医学専攻

形成外科学分野
教授）

【Introduction】
Diabetic wounds (DWs) are the most common complications in patients with diabetes
mellitus, which is a result of poor glycemic control, narrowed peripheral vessels, underlying
neuropathy, and poor immune response. Thus far, the available therapeutic approa ches have
limitations and none appear adequate to guarantee successful. Hence, there is an urgent need
for a therapeutic alternative to currently available treatments.
The omentum is a highly vascularized fibrous fatty layer of tissue located in the abdom inal
cavity, serving as a layer of coverage and protection. It is known to possess healing potential
for over 100 years, mediated by omentum transportation, owing to its angiogenic, immunogenic,
and lymphatic properties.
Recently, some studies showed that the activated omentum, with rich of growth factors and
characteristics of stem cells, have ability of tissue regeneration. The present work aimed to
investigate the effect of supernatant from activated omentum condition medium on diabetic
mice skin wound model.
【Methods】
C57BL/6 male mice were randomly divided into three groups: (1) the control group (c),
which served as a negative control without any interference; (2) the saline group (s), where 0.5
mL saline was injected intraperitoneally; and (3) the activated group (a), where 0.5 mL
polydextran particle slurry was injected intraperitoneally16. After 2 weeks, the omentum was
harvested, weighted and then cultured at 37°C. The omentum conditioned medium (OCM) of
three groups were collected after 48 hours culturing, meanwhile, protein density was measured.
Thirty-six 12weeks C57BLKS/J Leprdb (db/db) mice female diabetic mice were randomly
divided into four groups, which were injected with medium (M), saline -OCM (sOCM),
inactived-OCM (iOCM), and actived-OCM (aOCM) subcutaneously. Wound closure (%) were
evaluated on days 0, 3, 5, 7, 9, 11, 14, 21, 28 post-treatments. As for histology analyses, we
performed hematoxylin and eosin staining and Masson's trichrome staining on days 9 and 28
post-treatments for gross observation and collagen volume fraction (CVF) assessment
respectively. To investigate newly vessels and nerve fibers regeneration degree on days 9 post treatments, fluorescent immunostaining and quantification were performed.
【Results】
1. Characteristics of activated omentum and aOCM
The intraperitoneal injection of polydextran particles caused the omentum to spread rapidly
and was activated within 2 weeks. Notably, the omentum of group (a) gained significantly
more weight and higher protein density than that of groups (c) and (s) (Fig. 1A, 1B).
2. aOCM accelerated re-epithelialization in a diabetic murine wound model

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aOCM group showed smaller wound closure (%) than that in the other three groups, thus
demonstrating that aOCM significantly accelerated wound closure from day 7 to day 11
compared to that in the M, iOCM, and sOCM (Fig. 2A). Furthermore, the time taken for 50%
wound healing in aOCM group was significantly faster than that in the other three groups
(p < 0.0001) (Fig. 2B)
3. aOCM promoted angiogenesis, nerve regeneration and collagen density in diabetic
mice model in early wound healing stage
Newly formed vessels at the wound sites were examined by CD31 and α-SMA co-staining.
According to vessel quantification, on day 9 post-treatments, the average vessel number in the
aOCM group was significantly higher than other three groups (p < 0.001) (Fig. 3A).
Additionally, newly formed peripheral nerve fibers were stained with NF-L antibody and
quantified in M, iOCM, sOCM, and aOCM groups at day 9 which were 20.67 ± 5.28, 20.17 ±
4.17, 20.83 ± 8.70, and 36.50 ± 11.0, respectively (p < 0.05) (Fig. 3B). As for CVF, aOCM
administration significantly enhanced CVF as demonstrated by increased blue -stained,
especially on day 9 post-operation. (p < 0.0001) (Fig. 3C).
【Discussion】
In clinics, the omentum-free flap has a wide variety of applications in reconstructive surgery
and has been shown to be a reliable donor tissue. Further, it has been reported that activated
omentum become a rich source for growth factors including fibroblast growth factor and
vascular endothelial growth factor. In this study, we demonstrate for the first time that aOCM
can accelerate wound healing in the early wound healing process in diabetic mice. Animal
experiments indicated that aOCM significantly accelerated the re-epithelialization rate from
days 7 to 11 post-operation. In early wound healing process, aOCM tended to promote
angiogenesis and peripheral nerve fibers regeneration at the edges of t he wound. Moreover,
our findings indicate that aOCM can promote collagen deposition in the early stages of wound
healing in diabetic mice. Furthermore, according to our mass spectrometry analysis, the aOCM
group had abundant proteins, which potentially contributed to re-epithelialization, granulation
tissue formation, inflammatory regulation, neovascularization, and peripheral nerve
regeneration (Table. 1). Nevertheless, further experiments are necessary to determine the
molecular mechanisms underlying these observed effects.
Additionally, despite the fact that the omentum is regarded as a reliable donor site in recent
studies, the donor-site morbidity and complications such as intestinal obstruction and
herniation remain after omentum harvesting. Our study used conditioned medium from the
activated omentum, which is abundant in groups of proteins and potentially contributes to
wound healing in a diabetic mouse wound model. Our findings in animals support the potential
use of aOCM for wound treatment in patients with diabetes. Taken together, the cocktail gel

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or mixture minimizes donor-site morbidity in the future and it may provide a new therapeutic
method for treating DWs.
【Conclusions】
The results of our study suggested that administration of supernatant from activated
omentum enhance the wound closure in early stage compare with other groups. Meanwhile,
activated omentum can obviously promote the angiogenesis, nerve regeneration and col lagen
deposition in diabetic mouse wound model, which may provide us a new therapy for diabetic
ulcer.

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Table 1. Protein enrichment in supernatants and their potential contribution to wound healing.
Accession
O70370

Description
Cathepsin S

MW

PMS (Peptide spectrum match)

Contribution to wound

[kDa]

iOCM

sOCM

aOCM

healing

38.45

1

6

53

Angiogenesis;
Wound closure

Q61292

Laminin subunit beta-2

196.45

2

8

62

Angiogenesis;
Re-epithelization

Q91X72

Hemopexin

51.29

15

12

333

Enhanced peripheral
nerve regeneration

P35441

Thrombospondin-1

129.56

6

17

67

Re-epithelization

Q61554

Fibrillin-1

312.08

30

68

259

Formation of

Q8VCM7

Fibrinogen gamma chain

49.36

12

17

98

granulation
Angiogenesis;
Formation of
granulation

Q61001

Laminin subunit alpha-5

403.79

6

13

44

Re-epithelization;

512

Collagen deposition
Re-epithelization,
Collagen deposition;
angiogenesis

P11276

Fibronectin

273.36

102

Figure 1. Characteristics of activated omentum and aOCM.
A. Omentum weight in each group. ****p < 0.0001.
B. Protein concentration in each group. *p < 0.05.

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161

Figure 2. aOCM accelerated re-epithelialization in a diabetic murine wound model.
A. The wound closure percentage (%) was determined on days 0, 3, 5, 7, 9, 11, 14, 21, 28 post-treatments in each
group. *p < 0.05; **p < 0.01; *** p < 0.001.
B. Average time taken for 50% wound healing in M, iOCM, sOCM, and aOCM groups. ****p < 0.0001.

Figure 3. aOCM promoted angiogenesis, nerve regeneration and collagen density in diabetic mice model in early
wound healing stage.
A. Quantitative analysis of angiogenesis assessed by CD31 and α-SMA co-staining. **p < 0.01.
B. Quantitative analysis of peripheral nerve fiber stained by NF-L on day 9. *p < 0.05.
C. Collagen volume fraction (CVF) against stained section of each group was calculated through collagen-stained
area (blue) relative to total stained area on day 9 post-operation. ****p < 0.0001.

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