Differentiation of human fat mesenchymal stem cells

using electromagnetic waves

Nariman Gharari

and Mahsa Laleh

School of Biology, University of Tehran, Tehran, Iran

Department of Biomedical Engineering, Azad University of Science and Research Branch, Tehran, Iran

ABSTRACT

In orthopedic sciences, cartilage regeneration and repairing is an underlying issue investigated by many studies in tis-

sue engineering. Bioactive growth factors, cell implantation and biocompatible scaffolding are the new developments

in this  eld. Keeping the proliferation and differentiation ability of cultured cells is essential in producing extracellular

matrix and cartilage. Adult stem cells can be used due to their high ability to be differentiated in several cell types in

tissue engineering. Till recently hematopoietic stem cells and fat tissue stem cells have been used to repair the tissues.

Studies indicates that using fat tissue stems cells is preferred because of easy access to adipose tissue and maintain-

ing the ability of proliferation and differentiation to cartilage in high passages compared to hematopoietic stem cells.

Therefore, this study has been conducted to analyze the differentiation of human fat mesenchymal stem cells using

electromagnetic waves. In this work simultaneous effect of platelet-rich plasma and electromagnetic waves have been

studied on ef ciency of differentiation of fat mesenchymal stem cells to cartilage. Mesenchymal stem cells have been

extracted from human fat tissue and then, the cells have been differentiated to fat, bone and cartilage tissue cells using

different treatments. Cartilage differentiation was done in 2-D form and single-layer cell in bottom of 6-bed and 3-D

cells in cell falcons. Required tests to estimate ef ciency of cartilage differentiation were done in two general examina-

tions for analysis of evaluation of speci c and pathologic markers such as osteogenicity, angiogenesis and in ammation

parameters by measuring the amount of TNF and VEGF with ELISA technique. The results obtained from this study

showed reasonable use of platelet-rich plasma (PRP) and electromagnetic waves in medicine and tissue engineering.

Although PRP enables cartilage differentiation, it cannot reduce pathologic symptoms.

KEY WORDS: DIFFERENTIATION OF HUMAN FAT MESENCHYMAL STEM CELLS, ELECTROMAGNETIC WAVES, PRP

577

Biomedical

Communication

Biosci. Biotech. Res. Comm. 11(4): 577-586 (2018)

ARTICLE INFORMATION:

Corresponding Authors: narimangharari@gmail.com

Received 1

Oct, 2018

Accepted after revision 21

Dec, 2018

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 CODEN: USA BBRCBA

Thomson Reuters ISI ESC / Clarivate Analytics USA

Mono of Clarivate Analytics and Crossref Indexed

Journal Mono of CR

NAAS Journal Score 2018: 4.31 SJIF 2017: 4.196

Online Contents Available at:

http//www.bbrc.in/

DOI: 10.21786/bbrc/11.4/7

Nariman Gharari and Mahsa Laleh

578 DIFFERENTIATION OF HUMAN FAT MESENCHYMAL STEM CELLS USING ELECTROMAGNETIC WAVES BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

INTRODUCTION

The aim of tissue engineering is normalizing tissue per-

formance through transferring living factors in body

of patients, (Kocan et al. 2017, Bondarava et al. 2017).

Continuous efforts of cell biologists, engineers, mate-

rial engineers, mathematicians, geneticists and physi-

cians should be in way of achievement and success in

new tissue production, (Alonso-Goulart et al. 2017, Klar

et al. 2017).

Nowadays, tissue engineering is being used as a tech-

nique for tissue or organ transplant. The lost and injured

tissues and organs can be treated by engineered biologi-

cal transplant (Zhai et al. 2016). Engineered products and

components should have high performance and have

the ability to form expected functional tissue (Zajdel

et al. 2017). Mesenchymal stem cells (Skeletal Stem Cells

or Bone marrow stromal stem cells) are plastic sticky

non-hepatocytes cells placed in bone marrow stro-

mal vessels (Seong et al. 2014 Ross et al. 2015 and Liu

et al. 2017) and have multi-generation differentiation

with self-renewal capacity (Heo et al. 2016 and Mobini et

al. 2017). Deciding which type of cell should be applied

to repair injured tissue is essential which can be adult

brucellosis, mesenchymal stem cells or protozoal cells

from hypothyroidism, pericardium or cells manipulated

genetically, (Tan et al. 2017). Cartilage is non-vascular

 brous connective tissue having resistant extracellular

matrix, which is stiffer than other connective tissues and

includes intra-matrix chondrocytes, (Choi et al. 2018).

The  rst function of cartilage is protection of soft tis-

sues and helping evolution and growth of long bones.

Fat tissue stem cells are similar to bone marrow stromal

cells in terms of physical form and can be appropriate

replacement. These cells have common features com-

pare with mesenchymal stem cells such as self-renewal

ability, powerful cells,  broblast appearance, ability to

attach to plastic dishes for cell cultivation and ability

to be changed into other mesenchymal types, (Dubois

et al. 2008 and Tan et al. 2017).The differentiation of

these cells from fat, bone, cartilage, skeletal muscular

and cardiovascular cells has been reported in recent

years by different researchers. Fat tissue stem cells, same

as bone marrow stromal cells, can be converted to ecto-

dermal types (like neural and glial cells) by the media-

tion of neural inducers, (Ivan et al. 2017).

Several studies have been conducted in fat tissue stem

cells, especially for treatment of neural system lesions

and revealed that direct injection of fat stem cells to

brain ischemic models can convert these cells to quasi-

neural cells. These cells can improve laboratory models

in the place of lesion with (injured place by) secretion

of neural growth factors (Gugjoo et al. 2016).In order to

prepare mesenchymal cell, fat tissue is a considerable

source, since it is available in large amount (liposuc-

tioned) fat tissue or in fat tissue section

and contains

large amount of stem cells called fat-derived stem cells.

The property has made it be considered as a good can-

didate for 1-step treatment of cartilage defects (Bruder

et al. 1997).

In the past decade, PRP or platelet-rich plasma has

gained many attentions, especially in regenerative medi-

cine, (Liu et al. 2017). PRP can be considered as a part of

blood plasma with platelet density higher than base level

(Xu et al. 2017 Qian et al. 2017). Importance of PRP with

appropriate density of platelet is the abundant growth

factors and proteins used in tissue engineering, (Anitua,

et al. 2007). In 2012, a study was done in China for

regenerating cartilage, which evaluated and compared

mesenchymal stem cells, extracted from bone marrow

and fat tissue placed on the PRP-derived scaffolding.

Findings showed that rich plasma is appropriate bioac-

tive scaffolding with ability of secreting growth factor,

bone marrow stem cells and fat mesenchymal stem cells

-placed on PRP were differentiated from cartilage and

ef cient to repair cell-based cartilage, (Lee et al. 2012).

In a study, the effects of platelet-rich plasma on his-

tological, biochemical and biomechanical properties of

cartilage tissue engineering were studied. Cultured cells

in PRP created 20% thicker cartilage tissue compared

with other cultured cells. Hence, platelet-rich plasma in

culture medium can cause creation of cartilage in vitro

cartilage formation with increased content of glycosa-

minoglycan and more mechanical compressive proper-

ties at the same time with maintaining Phenotype Fea-

tures of Hyaline cartilage (Petrera, et al. 2013). In 2014,

a research team from Spain studied effects of PRP on

human mesenchymal stem cells, which can increase or

limit clinical uses of these cells. PRP can preserve dif-

ferentiated immune of mesenchymal stem cells and can

apparently postpone ageing phenomenon. They also

provide data on exact molecular relation and its mecha-

nisms, (Rubio-Azpeitia et al. 2014).

In 2014, an economic method was proposed to pre-

pare PRP. PRP-derived growth factor-BB was measured

under conditions of using Anticoagulant dextrose solu-

tion A (ACD-A), along with or in absence of Prosta-

glandin E1 (PGE1) (as Platelet aggregation inhibitor).

The new method was successful in analysis of PRP with

growth factor and high BB under all growth conditions

and high volume of PRP is obtained using ACD-A and

PGE1(Fukaya et al. 2014).

In 2014, the effect of PRP on cartilage cells differ-

entiated from rabbit fat-derived stem cells was studied

in vitro. Stem nature of rabbit cells were studied with

differentiation of fat, bone and cartilage types in vitro.

Collagen type 2 expression and agrican expression in

PRP-treated cells was increased to 10% compared to

Nariman Gharari and Mahsa Laleh

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS DIFFERENTIATION OF HUMAN FAT MESENCHYMAL STEM CELLS USING ELECTROMAGNETIC WAVES 579

control group. Hence, the  ndings showed that PRP of

rabbit can take cartilage differentiation in mesenchymal

stem cells extracted from rabbit fat (Elder et al. 2014).

Analysis of the simultaneous effect of PRP and elec-

tromagnetic waves on cartilage differentiation ef ciency

in fat mesenchymal cells has been studied. Mesenchymal

stem cells have been extracted from human fat tissue

and then, the cells were differentiated to types of fat

tissues, bone and cartilage using different treatments

Therefore, these experiment have been conducted to

analyze differentiation of mesenchymal human fat stem

cells using electromagnetic waves.

MATERIALS AND METHODS

Extraction of mesenchymal stem cells from fact

tissue:To transfer fat tissue, fat pieces were placed in

DMEM medium containing Penicillin and streptomycin

3X , amphotericin 1x , without FBS and were transferred

to laboratory in ice. After each time of adding buffer to

2-3 times of tissue volume, centrifuge was taken with

speed of 1200rpm for 20min and buffer solution was

disposed. A thin layer of fat was formed on the solution,

which had to be removed, since it could cause problems

such as choking on cells due to lack of oxygen while

cultivation. In this step, suspended cells were transferred

to  asks 75 containing 10ml culture medium with FBS

and were then heated in incubator.

To make cells face electromagnetic waves, after

counting cells using Neubauer lam method, a part of

culture medium containing 200.000 cells was removed

from culture  asks and was transferred to sterile falcons.

Then, 1ml of culture medium without differentiation fac-

tors and containing Penicillin - Streptomycin and FBS

was added and maintained for 1 day. After the heating

in incubator, cells were accumulated in bottom of the

falcon in rounded form and were prepared to continue

the examination and to expose to electromagnetic radi-

ation. To apply electromagnetic waves on cell culture,

wave winding machine was used. The winding machine

was placed inside the incubator and culture falcon was

placed there to be exposed to electromagnetic waves for

6 hours per day. Frequency of the machine was set in

range 171-173Hz and according to power about 30%,

ultimate and real time power about 50Hz was obtained.

Cells were exposed to such conditions in 14-day period

and the culture medium was changed every 3 days. 5

falcons containing culture medium with 5% PRP were

exposed to electromagnetic waves and other 4 falcons

were not. Measurement of VEGF distributed in differ-

entiated cells was done using supernatant of cultures

in days 7, 14 and 21. The phosphorylation of this factor

was measured by kit based on Sandwich ELISA method.

The Sandwich ELISA method was used to measure the

amount of TNF propagated from differentiating cells.

To this end, the supernatant collected from cell culture

was examined in the days 7, 14 and 21. The  nal results

were analyzed statistically at the con dence level of 95%

to test signi cance of the difference of different groups

using Prism software and using one-way ANOVA and

T-test. Moreover, the diagrams were drawn using Excel

and SPSS software. The results obtained from analysis of

cartilage differentiation gene expression were analyzed

using REST software by normalization with reference

beta-actin gene. Each test was replicated 3 times and the

signi cance was considered lower than 0.05.

RESULTS

Analysis of differentiation of stem cells extracted from

fat tissue to cartilage :In this study, differentiation to

cartilage was analyzed using different staining methods

and Immunocytochemistry test during 14 days of cell

differentiation. Expression of some genes relevant to

cartilage was also studied at the end of the differentia-

tion period.

Proof of cartilage differentiation: The Immunocyto-

chemistry method was used to analyze expression and

production of collagens types 2 and 10. Collagen type 2

was expressed during cartilage differentiation and colla-

gen type 10 was one of the bone and hypertrophy mark-

ers expressed by chondrocytes. Figure 1 has illustrated

expression of these collagens in treated cells in the day

14. The blue color in the  gure is DAPI staining to stain

cell core and red color shows presence of studied col-

lagens in differentiating cells.

Analysis of bone markers during differentiation of

stem cells to cartilage: At the time of applying differ-

entiation factors such as using PRP as a bioactive sub-

stance or electromagnetic radiation to stimulate carti-

lage differentiation in mesenchymal stem cells of fat

tissue, non-speci ed differentiation to bone is also pos-

sible. With taking tests such as measurement of Alka-

line Phosphatase Activity and Calcium sedimentation

measurements, the non-speci ed differentiation was

also examined.

Measurement of Alkaline Phosphatase Activity: To

determine non-speci ed differentiation towards bones,

measurement of alkaline phosphatase enzyme was activ-

ity was done. As it is clear in diagram 1, the amount of

alkaline phosphatase in treatment PRP.W is the highest

level measured equal to 2 units of optical absorption per

mg protein. Then, treatment PRP a with value of 1.44

unit absorption per mg protein shows highest alkaline

phosphatase activity. The lowest level of phosphatase

Nariman Gharari and Mahsa Laleh

580 DIFFERENTIATION OF HUMAN FAT MESENCHYMAL STEM CELLS USING ELECTROMAGNETIC WAVES BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

FIGURE 1. Immunocytochemistry staining of collagens type 2

and 10 in treated cells; A) collagen type 2 in treatment Dif.a; B)

treatment Dif.W and c) collagen 10 in treatment PRP.a (the upper

part is staining core by DAPI and the lower part is collagen stain-

ing); blue color shows cell core and red color refers to surface

place of collagens; the line illustrates size of 0.26mm and magni-

 cation of image ×4.

DIAGRAM 1. Phosphatase alkaline activity per unit optical absorption per mg protein in different treat-

ments

alkaline was observed in treatment base.w at 0.32

unit absorption per mg protein. According to obtained

results, treatments PRO.W and PRP.a motivate differ-

entiation towards bones more than others and this can

be inferred based on activity of alkaline phosphatase in

these treatments. Despite to them, treatment base.w has

caused lowest stimulation towards boning.

VEGF factor secretion : According to diagram 2, highest

secretion level of VEGF was observed in treatments with

base environment with PRP. In the base treatment with

PRP.a, higher level of Endothelial Angiogenesis Growth

Factor was observed compared to base environment in

combination with PRP.w. The lowest secretion level was

detected in base.w and it seems that the effect of electro-

magnetic wave treatments and TGF factor varies due to

medium culture used. For example, in environment with

Dif base, secretion in wave treatment is higher than oth-

ers; although the results differ in base environment and

secretion has been in higher level using TGF treatment.

The effects of PRP have been also con icting. This factor

has shown different effects in combination with differ-

ent culture media exposed to wave and TGF treatments.

However, it seems that it has led to increased secretion

of VEGF by itself, since secretion in treatments with PRP

has been higher than others.

Nariman Gharari and Mahsa Laleh

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS DIFFERENTIATION OF HUMAN FAT MESENCHYMAL STEM CELLS USING ELECTROMAGNETIC WAVES 581

DIAGRAM 2. Secretion of VEGF in different treatments

DIAGRAM 3. TNF level in different treatments of induction of differentiation

to cartilage

Analysis of in ammation with TGF measurement:

Another symptom of cartilage-related articular diseases

is in ammation. According to existing reports, PRP

can cause in ammation in some cases and may inten-

sify that. Moreover, it can inhibit and reduce in am-

matory reactions. The con ict is because of wide range

of in ammatory factors in PRP. In ammatory potential

of engineered tissue has been examined by measure-

ment of TNF. The results have been presented in dia-

gram 3. According to these results, treatments Dif.w and

PRPb.a have shown highest level of in ammatory activ-

ity. On the contrary, treatments Base.w and Base.A and

PRP.W have shown low level of TNF even lower than

ELISA control level. Treatment Dif showed highest level

of in ammation; although same factor showed lowest

level of in ammatory activity in combination with Base

medium. PRP.b factor has also shown different behav-

ior in different combinations of treatment with TGF and

electromagnetic radiation, so that it has shown high

in ammatory activity in combination with TGF and

in ammatory activity lower than control level in com-

bination with electromagnetic radiation. The con icting

responses can be attributed to complexity of compounds

in culture media and PRP compounds, since controlling

Nariman Gharari and Mahsa Laleh

582 DIFFERENTIATION OF HUMAN FAT MESENCHYMAL STEM CELLS USING ELECTROMAGNETIC WAVES BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

FIGURE 2. Agarose gel produced by

PCR reaction for ALK5 M 1--bp marker

gene

FIGURE 3. Agarose gel pro-

duced by PCR reaction for

Pleotrophin M gene 100bp

marker

DIAGRAM 4. Sox9 gene expression during cartilage differentiation

these complex compounds and inhibition of interfering

effects of factors in the combinations is dif cult.

Measurement of cartilage and bone gene expression :

Analysis of cartilage differentiation by gene expression

using RT-PCR

Expression of Pleotrophin and ALK5 genes as markers

in cartilage differentiation samples

and collagen status

type 2 gene were analyzed. Collagen type 2 includes 2

isoforms called ColllA and ColllB created as a result of

alternative array of exon 2. Expression of these markers

during Chondrogenic differentiation caused differentia-

tion ef ciency detection. The image of gels produced by

reaction of these genes is presented in  gures 2 and 3.

Emergence of 250bp band showed expression of

ALK5 gene in samples, which can show cartilage differ-

entiation in these samples. 111bp band emergence also

showed expression of Pleotrophin in cells and con rms

cartilage formation processes. However, no band was

observed in proliferation of isoforms in collagen type

2 and the result of this gene proliferation was negative,

which shows lack expression of this gene in this differ-

entiation steps.

Analysis of speci c cartilage genes expression using Real

Time PCR

In order to analyze expression of speci c cartilage genes

such as sox9, colll and colX using b2m reference gene,

Real Time PCR method was used.

Sox9 cartilage marker: Sox9 marker is the transcription

factor required for expression of cartilage matrix genes

Nariman Gharari and Mahsa Laleh

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS DIFFERENTIATION OF HUMAN FAT MESENCHYMAL STEM CELLS USING ELECTROMAGNETIC WAVES 583

DIAGRAM 5. Colll gene expression during differentiation process

DIAGRAM 6. ColX gene expression during differentiation of stem cells to cartilage

such as collagen type 2. Diagram 4 illustrates sox9 gene

expression during cartilage differentiation process. In

the day 14 of treatment, expression of this gene in Dif

medium exposed to electromagnetic waves was signi -

cantly high; although its expression in medium contain-

ing 5% PRP was not tracked. Moreover, expression of

this gene was not observed or was insigni cant in treat-

ments without wave.

Collagen type 2: The products of this gene were

observed in hyaline cartilage in form of  bril. The pro-

teins can’t form  ber. In this study, proalpha-1 chain has

been detected, which has interfered in partial production

of collagen type 2. According to diagram 5, colll gene

expression has been signi cantly increased under effect

of electromagnetic waves. Moreover, the results show

that medium containing 5% PRP has been unable to

induce expression of this gene and has also had inhibi-

tory effects on wave induction capacity on the other

hand.

Collagen type 10: Alpha-1 collagen chain was coded by

COL10A1 gene. This gene can encode collagen type-10

alpha chain, which is expressed by hypertrophic chon-

drocytes during Endocardal bone formation.

Diagram 6 has illustrated adjustment of existing data

of electromagnetic wave and 55 PRP on both inhibitory

effects on expression of this gene. When the two factors

are applied in same treatment on cells, no expression

can be observed; although colX gene expression is in

high level in presence of the two factors.

DISCUSSION

Mature stem cells can be used for tissue engineering

purposes due to high differentiation ability to several

Nariman Gharari and Mahsa Laleh

584 DIFFERENTIATION OF HUMAN FAT MESENCHYMAL STEM CELLS USING ELECTROMAGNETIC WAVES BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

cell types. Recently, fat tissue stem cells have been used

to repair tissues. The study conducted by Zul et al. (2001)

using stem cell extraction methods based on using col-

lagenase could extract these cells from fat tissue of adult

people, (Hunziker et al. 2002). Moreover, Estes Research

Team used this method to extract stem cells to obtain fat

tissue stem cells to create cartilage phenotype, (Jonstone

et al. 1998).

According to  ndings of Pena et al. (2011), spindle

cells with ability of adherence to bottom of culture dish

and the ability of differentiation to mesodermic cell lines

can be considered as mesenchymal stem cells (Yoo et al.

1998). Adherence ability of cells to plastic surfaces has

been used for early separation of stem cell populations.

Similar results were also observed in study of Tappe

et al. (2009) and Aust et al. (2004), in which nature of

fat tissue was analyzed using this type of staining, (Sol-

chaga et al. 2006, Diduch et al. 2000).

In this study, to analyze the differentiation of stem

cells extracted from fat tissue to cartilage, expression

and production of collagens type 2 and 10 has been

applied using Immunocytochemical method. Similar

results were also obtained by Ogawa, (Williams et al.

2003). In this study, expression of collagen type 2 was

observed during cartilage differentiation and type 10 as

bone and hypertrophy markers in chondrocytes. It seems

that Dif medium and electromagnetic wave treatment

have induced cartilage differentiation and bone mark-

ers have been observed in PRP-contained treatment and

it seems that the treatment has the ability of inducing

hypertrophy and boning. Mishra et al. (2009) showed the

effect of PRP on cartilage differentiation. Further, con-

 icting effects of PRP on cartilage and bone differentia-

tion were revealed (Bosnakovski et al. 2006, Wakitani

et al. 2002).

Alkaline phosphatase enzyme activity can be con-

sidered as one of the most underlying factors of hyper-

trophy and bone formation and when the cartilage cells

are differentiated, they gain hypertrophic mode and start

production of alkaline phosphatase enzyme, (Bruder

et al. 1997). According to the results of this study, treat-

ments PRP.W and PRP.a have stimulated differentiation to

bone more than others and lowest alkaline phosphatase

activity has been observed in treatment Base. W on the

other hand. Measurements showed that highest secretion

of VEGF was observed in treatments with base medium

containing PRP. Lowest secretion level was also detected

in Base. W medium. It is expected that PRP can induce

such reaction because of nature rich of growth factors.

However, there are some reports on inhibitory effect

depended on PRP dose in VEGF secretion (Clin Pediatr

Endocrinol 2014). The con icts can be because of differ-

ence in type of treatments, PRP preparation method and

its source and also type of cells under treatment.

According to results obtained from this study, treat-

ments Dif.W and PRP.a have shown highest level of

in ammatory activity. On the contrary, treatments

Base.W, Base.A and PRPb.W have even shown TNF

level even lower than ELISA control level. It seems that

combination of base medium lays key role in reduction

of in ammatory activities regardless of other inductive

factors.

The effects of electromagnetic radiation or using PRP

showed different results. For example, radiation in com-

bination with Dif medium showed highest in ammation

level; although same factor showed lowest in ammation

activity in combination with base medium. In 3 out of 4

treatments containing electromagnetic wave, TNF level

was low and insigni cant and it could be inferred that

electromagnetic wave can probably reduce in amma-

tory activity regardless of culture medium compounds.

Controlling expression of cartilage matrix gene like

collagen type 2 was taken by transcription factor called

sox9. During cartilage differentiation process, the tran-

scription factor played key role and its expression was

increased in chondrocytes and Chondroprogenitor cells.

According to obtained results, expression of this gene

in Dif medium under electromagnetic radiation was

signi cantly increased; although its expression was

not detected in medium containing 5% PRP. Moreover,

the expression of this gene was not observed or was

insigni cant in treatments without wave. As a result of

increased expression of sox9 gene, its downstream gene

(colll) was also increased in terms of expression. Moreo-

ver, the results showed that medium containing 5% PRP

has not the ability to induce expression of this gene and

has had also inhibitory effects on wave induction capac-

ity on the other hand. Colll expression pattern under

treatment has been signi cantly similar to expression

pattern of control gene (sox9).

Adjustment of data of electromagnetic wave and 5%

PRP has shown that both of them have inhibitory effects

on colX gene expression.

CONCLUSION

The results of the present study showed the ef cient and

logical use of PRP and electromagnetic waves in medi-

cine and tissue engineering. However PRP allow carti-

lage differentiation in the right format, this method need

further study and cannot decrease pathologic symptoms.

ACKNOWLEDGMENT

This research was an independent bachelors project sup-

ported by research affairs University of Tehran, Tehran,

Iran with cooperation of Mrs. Mahsa Laleh at Azad Uni-

versity of Science and Research Branch, Tehran, Iran.

Nariman Gharari and Mahsa Laleh

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS DIFFERENTIATION OF HUMAN FAT MESENCHYMAL STEM CELLS USING ELECTROMAGNETIC WAVES 585

REFERENCES

Agnes S. Klar, Jakub Zimoch, and Thomas Biedermann, Skin

Tissue Engineering: Application of Adipose-Derived Stem

Cells, BioMed Research International, vol. 2017, Article ID

9747010, 12 pages, 2017. doi:10.1155/2017/9747010

Alicja Zajdel, Magdalena Kałucka, Edyta Kokoszka-Mikołaj,

Adam Wilczok (2017). Osteogenic differentiation of human

mesenchymal stem cells from adipose tissue and Wharton’s

jelly of the umbilical cord. Acta Biochimica Polonica. 2017 no.

64 Issue 2 pp 365-369

Anitua, E., et al., (2007) The potential impact of the prepara-

tion rich in growth factors (PRGF) in different medical  elds.

Biomaterials, 2007. 28(31): p. 4551-60.

Beata Kocan, Aleksandra Maziarz, Jacek Tabarkiewicz, (2017)

Trophic Activity and Phenotype of Adipose Tissue-Derived

Mesenchymal Stem Cells as a Background of Their Regenera-

tive Potential, Stem Cells International, Article ID 1653254, 13

pages doi:10.1155/2017/1653254

Bosnakovski, D., et al., (2006) Chondrogenic differentiation of

bovine bone marrow mesenchymal stem cells (MSCs) in dif-

ferent hydrogels: in uence of collagen type II extracellular

matrix on MSC chondrogenesis. Biotechnology and Bioengi-

neering, 93(6): p. 1152-1163.

Bruder, S.P., N. Jaiswal, and S.E. Haynesworth (1997), Growth

kinetics, self‐renewal, and the osteogenic potential of puri ed

human mesenchymal stem cells during extensive subcultiva-

tion and following cryopreservation. Journal of cellular bio-

chemistry, 1997. 64(2): p. 278-294.

Choi JR, Yong KW, Choi JY (2018). Effects of mechanical load-

ing on human mesenchymal stem cells for cartilage tissue

engineering. J Cell Physiol. 233(3):1913-1928. doi: 10.1002/

jcp.26018. Epub 2017 Jun 12. DOI: 10.1002/jcp.26018

Diduch, D.R., et al., (2000) Marrow stromal cells embedded in

alginate for repair of osteochondral defects. Arthroscopy: The

Journal of Arthroscopic & Related Surgery, 16(6): p. 571-577.

Elder, S. and J. Thomason (2014) Effect of Platelet-Rich Plasma

on Chondrogenic Differentiation in Three-Dimensional Cul-

ture. The Open Orthopaedics Journal. 8: p. 78-84.

Fukaya, M. and A. Ito, (2014) A New Economic Method for

Preparing Platelet-rich Plasma. Plastic and Reconstructive

Surgery Global Open, 2(6): p. e162.

Gugjoo, M. B., Amarpal, Sharma, G. T., Aithal, H. P., & Kin-

javdekar, P. (2016). Cartilage tissue engineering: Role of mes-

enchymal stem cells along with growth factors & scaffolds. The

Indian Journal of Medical Research, 144 (3), 339–347. http://

doi.org/10.4103/0971-5916.198724

Heo JS, Choi Y, Kim HS, Kim HO (2015). Comparison of molec-

ular pro les of human mesenchymal stem cells derived from

bone marrow, umbilical cord blood, placenta and adipose

tissue. Int J Mol Med. 2016 Jan;37(1):115-25. doi: 10.3892/

ijmm.2015.2413. Epub 2015 Nov 19.

Hunziker, E., Articular cartilage repair: basic science and clin-

ical progress. A review of the current status and prospects.

Osteoarthritis and cartilage, 2002. 10(6): p. 432-463.

Johnstone, B., et al. (1998) In Vitro Chondrogenesis of Bone

Marrow-Derived Mesenchymal Progenitor Cells. Experimental

cell research, 238(1): p. 265-272.

Judit Iván, Evelin Major, Adrienn (2017). The Short-Chain

Fatty Acid Propionate Inhibits Adipogenic Differentiation of

Human Chorion-Derived Mesenchymal Stem Cells Through

the Free Fatty Acid Receptor 2. Stem Cells Dev. December 1;

26(23): 1724–1733.Doi: 10.1089/scd.2017.0035

Lee, H.R., et al., (2012) Platelet-rich plasma loaded hydrogel

scaffold enhances chondrogenic differentiation and matura-

tion with up-regulation of CB1 and CB2. J Control Release,

159(3): p. 332-7.

Lei Tan, Bin Zhao, Fu-Tao Ge, Da-Hui Sun & Tiecheng Yu

(2017). Shockwaves Inhibit Chondrogenic Differentiation of

Human Mesenchymal Stem Cells in Association with Adeno-

sine and A2B Receptors. Scienti c Reports 7, Article number:

14377 (2017). doi:10.1038/s41598-017-14875-y

Liu Z, Yuan X, Fernandes G, Dziak R, Ionita CN, Li C, Wang

C, Yang S (2017). The combination of nano-calcium sulfate/

platelet rich plasma gel scaffold with BMP2 gene-modi ed

mesenchymal stem cells promotes bone regeneration in rat

critical-sized calvarial defects. Stem Cell Res Ther. 2017 May

25;8(1):122. doi: 10.1186/s13287-017-0574-6. DOI: 10.1186/

s13287-017-0574-6

Liu, X., Ren, W., Jiang, Z., Su, Z., Ma, X., Li, Y.,Yang, X. (2017).

Hypothermia inhibits the proliferation of bone marrow-

derived mesenchymal stem cells and increases tolerance to

hypoxia by enhancing SUM Oylation. International Journal of

Molecular Medicine, 40(6), 1631–1638. http://doi.org/10.3892/

ijmm.2017.3167

Maryna Bondarava, Chiara Cattaneo, Bin Ren, Wolfgang E. Tha-

sler, Volkmar Jansson, Peter E. Müller & Oliver B. Betz (2017).

Osseous differentiation of human fat tissue grafts: From tissue

engineering to tissue differentiation. Scienti c Reports volume

7, Article number: 39712 (2017). doi:10.1038/srep39712

Mobini, S., Leppik, L., Thottakkattumana Parameswaran, V., &

Barker, J. H. (2017). In vitro effect of direct current electrical

stimulation on rat mesenchymal stem cells. PeerJ, 5, e2821.

http://doi.org/10.7717/peerj.2821

Petrera, M., et al., (2011) Supplementation With Platelet-Rich

Plasma Improves the InVitro Formation of Tissue-Engineered

Cartilage With Enhanced Mechanical Properties. Arthroscopy.

29(10): p. 1685-1692.

Qian, Y., Han, Q., Chen, W., Song, J., Zhao, X., Ouyang, Y. Fan,

C. (2017). Platelet-Rich Plasma Derived Growth Factors Con-

tribute to Stem Cell Differentiation in Musculoskeletal Regen-

eration. Frontiers in Chemistry, 5, 89. http://doi.org/10.3389/

fchem.2017.00089

Ross, C. L., Siriwardane, M., Almeida-Porada, G., Porada, C. D.,

Brink, P., Christ, G. J., & Harrison, B. S. (2015). The effect of

low-frequency electromagnetic  eld on human bone marrow

stem/progenitor cell differentiation. Stem Cell Research, 15(1),

96–108. http://doi.org/10.1016/j.scr.2015.04.009

Rubio-Azpeitia, E. and I. Andia, (2014) Partnership between

platelet-rich plasma and mesenchymal stem cells: in vitro

Nariman Gharari and Mahsa Laleh

586 DIFFERENTIATION OF HUMAN FAT MESENCHYMAL STEM CELLS USING ELECTROMAGNETIC WAVES BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

experience. Muscles, Ligaments and Tendons Journal 4(1): p.

52-62.

S.G. Dubois et al. (2008) Isolation of Human Adipose-derived

Stem Cells from Biopsies and Liposuction Specimens 20 8

Seong Y, Moon J, Kim J. Life Sci. 2014 Apr 25; 102(1):16-27.

Epub 2014 Mar 3.

Solchaga, L.A., et al., (2006) A rapid seeding technique for

the assembly of large cell/scaffold composite constructs. Tissue

Engineering 12(7): p. 1851-1863.

Tan, A. R., & Hung, C. T. (2017). Mesenchymal Stem Cells for

Functional Cartilage Tissue Engineering: Taking Cues from

Chondrocyte-Based Constructs. Stem Cells Translational Medi-

cine, 6(4), 1295–1303. http://doi.org/10.1002/sctm.16-0271

Toshimi Michigami (2014) Current Understanding on the

Molecular Basis of Chondrogenesis Clin Pediatr Endocrinol.

2014 Jan; 23(1): p,1–8.

Vivian Alonso-Goulart, Lorraine Braga, Ferreira, (2017).

Mesenchymal stem cells from human adipose tissue and

bone repair: a literature review. Biotechnology Research and

Innovation. Available online 14 November 2017. https://doi.

org/10.1016/j.biori.2017.10.005

Wakitani, HJ et al. (2002) Human autologous culture expanded

bone marrow mesenchymal cell transplantation for repair of

cartilage defects in osteoarthritic knees. Osteoarthritis and car-

tilage 2002. 10(3): p. 199-206.

Williams, C.G., et al., (2003) In vitro chondrogenesis of bone

marrow-derived mesenchymal stem cells in a photopolymer-

izing hydrogel. Tissue Engineering, 2003. 9(4): p. 679-688.

Xu Q, Li B, Yuan L, Dong Z, Zhang H, Wang H, Sun J, Ge S,

Jin Y (2017). Combination of platelet-rich plasma within peri-

odontal ligament stem cell sheets enhances cell differentiation

and matrix production. J Tissue Eng Regen Med. 11(3):627-

636. doi: 10.1002/term.1953. Epub 2014 Sep 4. DOI: 10.1002/

term.1953

Yoo, J.U., et al., (1998) The Chondrogenic Potential of Human

Bone-Marrow-Derived Mesenchymal Progenitor Cells The

Journal of Bone & Joint Surgery 80(12): p. 1745-

Zhai L, Ma XL, Jiang C, Zhang B, Liu ST, Xing GY (2016)

Human autologous mesenchymal stem cells with extra-

corporeal shock wave therapy for nonunion of long

bones. Indian J Orthop [serial online] 2016 [cited 2018 Jan

28;50:543-50. Available from:http://www.ijoonline.com/text.

asp?2016/50/5/543/189602