The Effect of Mesenchymal Stem Cell Conditioned Medium (Adipose-

Derived and Wharton's Jelly-Derived) on the Prevention of

Hypertrophic Scar Formation

Gita Hening Bunga

, Retno Dwi Utami

, Erlina Pricilla Sitorus

, Novan Adi Setyawan

, Indah

Julianto

1,3

, Moerbono Mochtar

Dermatoveneorology Department Faculty of Medicine Sebelas Maret University, Surakarta, Indonesia

Pathology Anatomy Department Faculty of Medicine Sebelas Maret University, Surakarta, Indonesia

Dermama Biotechnology, Surakarta, Indonesia

Keywords: ADSC-CM, hypertrophic scar, scar elevation index, WJSC-CM

Abstract: The process of wound healing can lead to the formation of varied outcomes, from scarless healing to excessive

fibrosis (hypertrophic) or atrophic scar. Mesenchymal stem cell (MSC) had been shown to prevent the growth

of fibrosis tissue. Conditioned media (CM) is the medium where stem cells are cultured. Adipose-Derived

Stem Cell Conditioned Medium (ADSC-CM) and Wharton’s Jelly Stem Cell Conditioned Medium (WJSC-

CM) are MSC-CM reported to contain growth factors, some of which plays a role in the formation of

hypertrophic scar such as TGFβ1 and VEGF, and some in the prevention of scar formation, such as bFGF.

This in vivo study conducted using 24 mice, divided into 4 groups: group I given Dulbecco's Modified Eagle

Medium (DMEM), group II ADSC-CM, group III WJSC-CM, and group IV without any treatment (control).

Before the injection, wound was made on the back of each mice with a 1 cm punch biopsy. On day 28th,

rebiopsy was done on the scar area, the tissue stained with hematoxyllin eosin staining to assess the Scar

Elevation Index (SEI). The SEI score showed that WJSC-CM group showed the lowest percentage of

hypertrophic scar formation (50%) compared to ADSC-CM (66.67%), DMEM (83.33%) or control

group(100%), although statistically the difference was not significant. This study showed that WJSC-CM

injection had a greater potential in preventing the formation of hypertrophic scar than ADSC-CM. It is thought

to be related to higher bFGF levels as well as lower TGFβ1 in WJSC-CM.

1 INTRODUCTION

The wound healing process can cause the formation

of fibrosis tissue, also known as scar. Scar tissue

consist of a collection of cells (especially fibroblasts)

and an irregular extracellular matrix (mainly

composed of collagen) (Gurtner et al., 2008).

Hypertrophic scar is a fibroproliferative dermis

disorder with typical features of excessive collagen

deposition in the dermis and subcutaneous layer. The

occurrence of hypertrophic scar indicates an

excessive wound healing process, including

migration and cell proliferation, inflammation,

increased of synthesis and secretion of cytokines and

extracellular matrix proteins, also remodeling of new

matrices that form to excessive deposition of

extracellular matrix (Meenakshi et al., 2005).

Several experimental and pre-clinical studies have

reported the potential of mesenchymal stem cells

(MSCs) to preventthe growth of fibrosis tissue (Dong

et al., 2015; Li, Zhang and Fu, 2016). Mesenchymal

stem cells may be present in both embryonic and adult

tissue such as adipose tissue, muscle, peripheral

blood, lung, heart, corneal stroma, dental pulp,

placenta, endometrium, amniotic membrane and

Wharton's jelly (Kalaszczynska et al., 2015).

The use of stem cell-conditioned media,

compared to direct stem cells, provides a better

solution in overcoming the limitations of cell-based

therapies (Jayaraman et al., 2013). Potapova et al

proved that the media used to culture stem cells, so-

called conditioned media, was useful for survival,

proliferation, invasion of extracellular matrix, and in

vitro endothelial cell migration (Potapova et al.,

2007). The conditioned media also contains a number

of cytokines and growth factors that are directly

146

Bunga, G., Utami, R., Sitorus, E., Setyawan, N., Julianto, I. and Mochtar, M.

The Effect of Mesenchymal Stem Cell Conditioned Medium (Adipose-Derived and Wharton’s Jelly-Derived) on the Prevention of Hypertrophic Scar Formation.

DOI: 10.5220/0008152701460149

In Proceedings of the 23rd Regional Conference of Dermatology (RCD 2018), pages 146-149

ISBN: 978-989-758-494-7

related to the wound healing process (Jayaraman et

al., 2013).

Zhang et al showed that adipose-derived stem cell

(ADSC) intralesional injection can reduce the

formation of hypertrophic scar in rabbit ears by

decreasing the expression of α-SMA genes and

collagen type I, as well as reducing collagen

deposition (Zhang et al., 2015). Yun et al also

conducted research using ADSC injection on full-

thickness defects made on pig's back and found that

ADSC injection reduce the size, color and soften the

scars that arise. The ADSC injection also decreases

mast cell activity and inhibits the transformational

growth factor β (TGFβ), and stimulates scar

remodeling by increasing the expression of matrix

metalloproteinase (MMP) (Yun et al., 2012).

Kitajima et al conducted studies using umbilical

cord blood (UCB-MSC) mesenchymal stem cells and

wharton's jelly mesenchymal stem cells (WJ -MSC)

injected in full thickness wounds in nude rats.

Although the results showed no significant difference

between injection of UCB-MSC and WJ-MSC on

scar formation, the scar tissue in the WJ-MSC group

showed smaller and thicker areas (Kitajima et al.,

2016).

The various descriptions above underlay the

researcher to examine the effect of injection of

mesenchymal stem cell conditioned-medium

(adipose-derived and wharton's jelly-derived) in

preventing hypertrophic scar formation.

2 RESEARCH METHODS

This experimental research using mice and was

conducted at the Animal House of Pharmacology

Department Faculty Of Medicine Gajah Mada

University Yogyakarta between April-Mei 2016. As

many as 24 mice were used divided into 3 treatment

groups and 1 control group. Group I was given

Dulbecco's Modified Eagle Medium (DMEM)

injection 0.2 ml perilesion, Group II injected with

ADSC-CM 0.2 ml perilesion, Group III given WJSC-

CM 0,2 ml perilesion, and group IV as negative

control not given any injection. Before the injection

was performed, wound were made on the back of

each mice with a 1 cm diameter punch biopsy (day 0).

On day 28th, re-biopsy was done on the scar area, the

tissue taken was stained with Hematoxyllin Eosin in

Pathology Anatomy Laboratory of Gajah Mada

University and analyzed with Image J to assess the

Scar Elevation Index (SEI) of each scar. If the SEI >

1 = hypertrophic, while if SEI < 1 the scar is normal

(eutrophy). The result were statistically analyzed with

one way analysis of variance (ANOVA) with the

significance value of p < 0.05.

DMEM ADSC-CM

WJSC-CM Negative Control

Figure 1. Histopathology Image and Scar Elevation Index (SEI) Measurement.

The Effect of Mesenchymal Stem Cell Conditioned Medium (Adipose-Derived and Wharton’s Jelly-Derived) on the Prevention of

Hypertrophic Scar Formation

147

Figure 2. Scar Elevation Index Percentage.

3 RESULTS

ANOVA analysis from SEI measurements showed

no significant differences between groups (p >

0.05). For the percentage of scar formed the result

showed that in group I (DMEM) hypertrophic scar

formed as much as 83,33%, group II (ADSC-CM)

66,67%, group III (WJSC-CM) 50% and in group

IV (control) all of the scar was hypertrophic. 100%.

The data showed that WJSC-CM had the lowest

percentage of hypertrophic scar formation

compared to ADSC-CM, DMEM or without

treatment, although statistically the difference was

not significant.

4 DISCUSSION

In the SEI measurements there was no significant

difference between the treatment groups

statistically. However, it can be seen that the

percentage of hypertrophic scar most in group IV

(without treatment) equal to 100% and smallest in

group III (given WJSC-CM) as much as 50%.

The Enzym-Linked Immunosorbent Assay

(ELISA) examination of growth factor content

contained in ADSC-CM and WJSC-CM revealed

the content of bFGF, TGFβ1 and VEGF in both of

the conditioned media. The content of bFGF at

WJSC-CM (7,141 pg / ml) is higher than ADSC-

CM (6.376 pg/ml), whereas the TGFβ1 content of

WJSC-CM (7.596 pg/ml) is lower than ADSC-CM

(8,176 pg/ml) . Similarly, the WJSC VEGF content

(3.645 pg / ml) is lower than ADSC-CM (7.287 pg

/ ml).

Several studies have proven the role of bFGF in

the process of wound healing and prevention of

hypertrophic scar formation (Spaccapelo, 2016).

Ono et al injected basic fibroblast growth factor

(bFGF) in postoperative wounds and showed that

bFGF injection may inhibit the formation of

hypertrophic scars and inhibit the widening of

postoperative scar size (Ono et al., 2007). In this

study it appears that the administration of WJSC-

CM, which contains higher bFGF levels, showed a

lower percentage of hypertrophic scar formation

slightly compared to ADSC-CM, DMEM media or

without treatment. This is in accordance with the

theory that bFGF can prevent the formation of

hypertrophic scars.

Transforming growth factor β1 (TGFβ1) is

known to have an important role in the formation of

hypertrophic scars (Lu et al., 2005). WJSC-CM-

conditioned media known to contain lower levels of

TGFβ1 than ADSC-CM. It is estimated that the

lower TGFβ1 levels are also a factor that plays a

smaller percentage of WJSC-CM hypertrophic

scarred than ADSC-CM.

Vascular endothelial growth factor (VEGF) is a

growth factor that played a role in angiogenesis of

wound healing process. In angiogenesis process it is

estimated that dermis endothelial cells can stimulate

expenditure of TGFβ, CTGF or other profibrotic

factors that can stimulate scar tissue formation by

fibroblasts. The process of remodeling the

DMEM ADSC-CM WJSC-CM Negative

Control

83,33%

66,67%

50%

100%

16,67%

33,33%

50%

Scar Elevation Index (SEI) Percentage

Hypertrophic Scar Eutrophic Scar

RCD 2018 - The 23rd Regional Conference of Dermatology 2018

148

extracellular matrix that accompanies angiogenesis

also indirectly can stimulate the formation of scar

tissue. Thus VEGF can act as a link between

angiogenesis and scar formation, by directly

stimulating both endothelial cells and dermal

fibroblasts (Wilgus et al., 2008). In this study

WJSC-CM, which contained lower VEGF, showed

the lowest percentage of hypertrophic scar. It is

estimated that low levels of VEGF also affect these

results, in addition to higher levels of bFGF and

lower TGFβ1.

5 CONCLUSION

WJSC-CM injection has a greater potential in

preventing the formation of post-wound

hypertrophic scarring than ADSC-CM. It is thought

to be related to higher bFGF levels, as well as lower

TGFβ1 in WJSC-CM used in this study.

ACKNOWLEDGEMENT

This research was supported by Dermama

Biotechnology Laboratory Surakarta. We thank our

colleagues Dermama Biotechnology Laboratory

Surakarta who provided insight and expertise that

greatly assisted the research.

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