The Effect of Advanced Adipose-derived Stem Cell Protein Extract to

Repairment of Collagen Deposition in Cultured Senescent Fibroblast

Erlina Pricilla Sitorus

, Retno Dwi Utami

, Gita Hening Bunga

, Indah Julianto

, Harijono

Karionosentono

, Chandrani Khoirinaya

, Brian Wasita

Dermatology and Venereology Departement of Medical Faculty Sebelas Maret University, Surakarta

Dermama Biotechnology Laboratories, Surakarta

Pathology Anatomy Departement of Medical Faculty Sebelas Maret University, Surakarta

Keywords: Advanced adipose-derived stem cell protein extract, aging skin, collagen deposition

Abstract: Aging skin is progressive process in skin that stimulates by environment damage and influences the

appearance. There are two factors inducing aging, intrinsic i.e genetic and age, and extrinsic i.e sun expose,

air pollutant, smoking, alcoholic and malnutrition. Recently, cell based therapy has been widely reported as

the option for aging skin therapy. Advanced adipose-derived stem cell protein extract (AAPE) is a

conditioned medium cultured under a hypoxia of Adipose-derived stem cell (ADSC), which can stimulate

collagen synthesis and migration dermal fibroblast, thus induces wound healing and wrinkles repairment.

This study samples used cultured fibroblast from amnion, with donor from a 30 year old woman by

cesarean section on her first delivery. There were three samples cultured groups, normal fibroblast (control),

normal fibroblast with 30 minutes UVB exposure in basal media (study group 1), and normal fibroblast with

30 minutes UVB exposure in basal media added AAPE (study group 2). All groups were evaluated in 48

hours and checked the collagen density by using immunocytochemistry assay with collagen antibody-1. The

results were interpreted by Image-J software. Study group 2 showed significant increasing of collagen

density, with p value 0.001. Because the AAPE was derived from ADSC thus it has the same growth factors

as those of ADSC. AAPE had been proven in our study that it stimulated collagen deposition.

1 INTRODUCTION

Aging is a progressive degenerative process of all

organs in the body, including the skin. Aging in

human skin is caused by intrinsic factors such as

genetics and age and extrinsic factors like sun

exposure, air pollutant, smoking, alcoholic and

malnutrition (Yaar and Gilchrest, 2007; Ichihashi

and Ando, 2014), which disturb the function and

structures of epidermal cells and dermis, also the

extracellular matrix. Clinically, skin aging induced

by sun exposed or photoaging exhibits as wrinkles,

mottled pigmentation, rough skin, loss of skin tone,

dryness, sallowness, deep furrows, severe atrophy,

teleangiectasis, laxity, leathery appearance, solar

elastosis, actinic purpura, precancerous lesions, skin

cancer and melanoma (Pandel et al., 2013). The

histological features are epidermis and dermis

atrophy, elastosis in dermis, collagen changes and

elastin fibers fragments. Ultraviolet (UV) exposure

induces enhanced reactive oxygen species (ROS)

production so that the production of the components

and the oxidative destruction increase as well. This

destruction is a significant mechanism in aging

process (Stojiljković et al., 2014).

Cell based therapy has become a promising

therapy since it can induce repairment or cell

regeneration post tissue trauma or organ function

failure (Baer et al., 2016). One of cell based

therapies which show significant outcome is ADSC.

It is an adult stem cells which is also as

mesenchymal stem cell from human fat tissue. It has

ability to differentiate to be their derivates and to

secrete the various growth factors, which can repair

as well as replace the surrounding damaged cells

(Kim et al., 2011). The conditioned medium of

ADSC known as AAPE containing secreted growth

factors, which is beneficial for skin problem i.e face

wrinkles as well as wound repairment. AAPE can

induce collagen synthesis and fibroblast cell

migration into dermis, thus it can be used as

rejuvenation therapy and wound healing (Kim et al.,

2009; Zhou et al., 2016). Recently, the skin

regeneration has become cosmetical and

272

Sitorus, E., Utami, R., Bunga, G., Julianto, I., Kariosentono, H., Khoirinaya, C. and Wasita, B.

The Effect of Advanced Adipose-derived Stem Cell Protein Extract to Repairment of Collagen Deposition in Cultured Senescent Fibroblast.

DOI: 10.5220/0008155502720275

In Proceedings of the 23rd Regional Conference of Dermatology (RCD 2018), pages 272-275

ISBN: 978-989-758-494-7

dermatologist concern as anti aging therapy due to

long term exposure to UV, for various non invasive

therapies to treat the skin aging symtomps (Moon et

al., 2012; Lee et al., 2014).

2 MATERIAL AND METHODS

2.1 Isolation and Culture of Fibroblast

The fibroblast cells were obtained from amnion

tissue from a 30 year old woman with history of her

first delivery by section cesarean as donor, with

informed consent and ethical clearance. After the

mechanical procedure, the tissue was cut in 0,5-1

in size then placed to culture flask, immersed

with small amount of growth medium consisting of

high glucose Dulbecco’s Modified Eagle’s Medium

(DMEM) Gibco

. Then the part of these tissues was

incubated in 37°C and 5% CO

for 48 hours, until

the tissues were attached on the bottom of the well

plate. After 48 hours, when the sections had attached

the medium was exchange with new medium until

the entire of tissue section was submerged. The

medium was changed in 2 days until the fibroblast

growth with 80% confluences.

2.2 Induction of Cellular Senescence

The cultured fibroblast in DMEM was added with

10% FBS, 100 IU/ml Penicillin and 100 μg/ml

Streptomycin with 5% CO

in 37°C. After starvation

for 24 hours, the cells were washed with PBS and

exposed with UVB light dose 100 mJ/cm

in 30

minutes, using applied method by Kim et al. (2009).

After exposure, the PBS was aspirated and replaced

to complete growth medium.

2.3 Preparation of the Advanced

Adipose-derived Stem Cell Protein

Extract (AAPE)

ADSCs (4 x 10

cells) were cultured in DMEM

(Gibco

) serum-free medium. Conditioned medium

of ADSCs was collected after 72 h of culture,

centrifuged at 400 x g for 5 min and filtered using a

100 mm syringe filter (Kim et al., 2009)

2.4 Experiments

2.4.1 Treatment

Normal fibroblast (in DMEM) group as control

study (2 x 10

cells/400 μl). Second group were 2 x

cells/400 μl normal fibroblast + 30 minutes UVB

exposed in DMEM (basal media/without AAPE).

Third group were 2 x 10

cells/400 μl normal

fibroblast + 30 minutes UVB exposed in DMEM

added with AAPE 400 μl. Then all groups were

evaluated in 48 hours.

2.4.2 Immunocytochemistry with Collagen

Antibody-1

The coverslip was taken from the bottom of well

plate and pasted to object glass then was gave 1-3

drops of blocking reagen serum for 15 minutes.

Then dripped with blocking reagent 1-3 drops for 15

minutes, rinse with wash buffer then dried. After

that dripped with HSS-HRP in 30 minutes, rinse

with was buffer in every 2 minutes three times, then

dripped with 100-200 μL DAB until covered entire

tissues section for 20-30 minutes. Rinse in 10

minutes with was buffer for three times. Cover

stained tissue with a coverslip of an appropriate size.

Place slides vertically on a filter paper or towel to

drain excess mounting medium and allow them to

dry. Visualize tissue under a light microscope

(IHC/ICC Protocol Guide, 2014).

2.5 Measurement

2.5.1 Collagen Deposition

The slides which had been stained with collagen

antibody were examined under the microscope,

which one of it lenses used Optilab Olympus CX-

connected to computer installed with Image-J

software which enabled it to detect collagen

deposition and to score it with 0= negative; 1= low

positive; 2= positive; 3= high positive (Varghese et

al., 2014).

2.5.2 Results Analysis using Statistical Data

Statistical analysis using Kruskal Wallis, the

continued with Mann-Whitney assay, with

significant p value are < 0.05. The calculation of this

data using SPSS software.

2.6 Ethical Clearance

This study had received permission from Dr.

Moewardi Hospital’s Ethical Comissions after the

patient signed the informed consent.

The Effect of Advanced Adipose-derived Stem Cell Protein Extract to Repairment of Collagen Deposition in Cultured Senescent Fibroblast

273

Table 1: Differentiation assay of collagen deposition in each group : control, AAPE (-) and AAPE (+)

Collagen

deposition

(fibroblast)

Groups

Total

(n=95)

Control

(

n=30

)

AAPE(-)

(

n=35

)

AAPE (+)

(

n=30

)

Negative 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0.001

Low positive 9 (30.0%) 14 (40.0%) 0 (0.0%) 23 (24.2%)

Positive 21 (70.0%) 21 (60.0%) 30 (100.0%) 72 (75.8%)

3 RESULT

In the table 1, negative category of collagen

deposition was seen in each groups (0%). Low

positive score was observed in AAPE (-) or only

basal media groups (40%) and followed by control

group (30%), but not in AAPE (+) groups. The

increase of collagen deposition with the highest

proportion in AAPE (+) groups (100%) and the

lowest one in AAPE(-) groups (60%).

4 DISCUSSIONS

Changes in mechanical properties of the skin are

generally referred to extracellular aspects such as

alterations in polymerization and cross-linking of

collagen and elastin. In vivo study, the skin fibroblast

changes affect the separation of collagen fibers

leading to decreased collagen production (Schulze et

al., 2012).

AAPE are conditioned medium from

ADSC had specific ability to organize the protein and

secreted growth factors into extracellular

environment and had a relevant affect to various

organ and human body systems (Li et al., 2015).

Because it is derived from adipose mesenchymal

stem cell, this secretome has similar ability to its

source, such as promoting the collagen synthesis and

fibroblast migration in tissue repairment (Zhou et al.,

2016). Both ADSC and AAPE contain various

growth factors and they have ability to repaired and

renew the surrounding damaged cells. Our study

revealed there were significant differences in the

increase of collagen density among the control,

AAPE (-) and AAPE (+) groups. The highest of

collagen deposition was obtained in AAPE (+)

groups, as 100% of samples had shown positive

category in increasing of collagen deposition.

Kim et al. who studied the benefit of ADSC and

AAPE as anti aging through the activation of dermal

fibroblast by its secreted factors. Animal study

demonstrated that adding AAPE and ADSC to skin

fibroblast animal study exposed to UVB, resulting in

increased dermal layer thickness and collagen

amount, as well AAPE can reduced the apoptosis

death cell or UVB induced-apoptosis (Kim et al.,

2009).

Kim et al had studied highest concentration

of various cytokine concentration of AAPE proved

its ability to repair human dermal fibroblast, by

stimulating the wound healing in animal study, and

the last can play role in anti aging process.

Fibroblast +

UVB

exposed 30

minutes

AAPE (-)/ only basal media AAPE (+)

24 hours 48 hours 24 hours 48 hours

Figure 1. The differentiation of collagen deposition in treatment group, after UVB exposure in 30 minutes. After 30 minutes

UVB exposure, many damage fibroblasts were seen. After treatment, in AAPE groups showed repairmen of fibroblasts and

had more density of fibroblast than in basal media/ AAPE (-) groups.

RCD 2018 - The 23rd Regional Conference of Dermatology 2018

274

Analysis of cell cycle show that adding

conditioned medium of ADSC can prevent the

apoptotic cell process induced by reactive oxygen

species, by significant decreasing of sub-G1 phase

of dermal fibroblast cell (Kim et al., 2009).

5 CONCLUSIONS

From all samples (95 photos) there were significant

increasing of collagen deposition in control, AAPE

(-) and AAPE (+) groups, which the highes

deposition showed in AAPE (+) groups, with p

value 0.001. Therefore AAPE can repair the

damaged fibroblast by increasing the collagen

deposition.

ACKNOWLEDGEMENT

There is no conflict of interest while did this study

until we had all the results and did not sponsored by

any party.

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