Evaluation

of

the

Occupational

Doses

of

Interventional

Radiologists

The

aim

of

the

present

study

was

to

determine

whether

there

is

a

linear

relation

between

the

doses

measured

above

and

those

measured

under

the

lead

apron

of

the

radiologists

performing

interventional

procedures

To

monitor

radiation

exposure

the

International

Commission

of

Radiological

Protection

-LRB-

ICRP

-RRB-

recommends

the

use

of

a

single

dosimeter

under

the

protective

apron

To

determine

the

exposure

more

accurately

an

additional

dosimeter

is

recommended

above

the

protective

apron

The

exposure

of

eight

radiologists

was

monitored

with

two

personal

dosimeters

during

consecutive

years

To

measure

the

doses

uniformly

the

two

dosimeters

were

worn

in

a

special

holder

attached

to

the

lead

apron

The

two

personal

dosimeters

were

replaced

every

weeks

on

the

same

day

The

doses

above

and

under

the

protective

aprons

of

seven

radiologists

did

not

differ

significantly

A

significant

lower

dose

above

and

under

the

protective

apron

was

measured

for

one

of

the

radiologists

During

a

period

the

average

dose

measured

above

the

lead

apron

was

mSv

-LRB-

median

mSv

-RRB-

while

that

under

the

lead

apron

was

mSv

-LRB-

median

mSv

-RRB-

The

coefficients

of

the

regression

line

result

in

the

equation

Y

X

with

Y

as

the

dose

under

the

lead

apron

and

X

as

the

dose

above

the

lead

apron

The

statistical

analysis

of

the

data

established

a

linear

relation

between

the

doses

above

and

those

under

the

lead

apron

-LRB-

-RRB-

Before

the

special

holder

was

introduced

it

was

not

possible

to

derive

a

relation

between

the

doses

above

and

those

under

the

lead

apron

as

the

doses

were

measured

at

varying

places

above

and

under

the

lead

apron

There

is

no

evidence

that

the

effective

dose

can

be

estimated

more

accurately

when

an

additional

dosimeter

is

used

The

present

study

revealed

a

threshold

before

doses

under

the

lead

apron

were

measured

Due

to

the

threshold

it

can

be

concluded

that

the

doses

under

the

lead

apron

will

not

be

underestimated

easily

when

doses

above

the

lead

apron

are

used

to

calculate

them

This

is

not

the

case

when

the

doses

above

the

lead

apron

are

calculated

for

the

doses

under

the

lead

apron

Introduction

Like

many

other

hospitals

the

Academic

Medical

Centre

-LRB-

AMC

-RRB-

in

Amsterdam

uses

personal

dosimeters

to

monitor

the

occupational

doses

from

x-ray

procedures

to

clinical

staff

The

occupational

doses

of

the

majority

of

the

clinical

staff

do

not

exceed

mSv

in

a

single

year

However

for

clinical

staff

involved

in

fluoroscopically

guided

interventional

procedures

significant

doses

are

measured

As

a

consequence

of

the

radiation

exposure

the

risk

of

fatal

cancer

for

the

radiologists

performing

interventional

procedures

increases

This

stochastic

risk

can

increase

to

a

value

of

about

for

radiologists

performing

these

procedures

during

many

years

-LSB-

-RSB-

In

recent

years

the

number

of

fluoroscopically

guided

interventional

procedures

at

the

AMC

has

increased

as

has

the

number

of

radiologists

performing

the

procedures

Moreover

the

discipline

to

wear

personal

dosimeters

has

improved

As

a

result

the

number

of

radiologists

with

doses

exceeding

mSv

in

a

single

year

has

increased

Whenever

the

dose

exceeds

mSv

in

a

single

year

a

report

has

to

be

sent

to

the

Dutch

labor

inspection

even

when

the

doses

are

measured

above

the

lead

apron

In

the

report

evidence

has

to

be

given

that

the

limit

for

stochastic

effects

is

not

exceeded

In

order

to

prove

that

the

doses

of

mSv

measured

above

lead

aprons

do

not

exceed

the

stochastic

dose

limit

it

was

decided

to

provide

additional

personal

dosimeters

to

the

radiologists

performing

interventional

procedures

at

the

AMC

These

additional

personal

dosimeters

were

meant

to

monitor

the

occupational

doses

under

the

lead

apron

When

in

it

was

decided

to

provide

the

radiologists

with

an

additional

dosimeter

the

AMC

had

already

substantial

data

of

doses

monitored

on

both

sides

of

the

lead

apron

These

data

were

collected

at

the

Department

of

Cardiology

where

additional

dosimeters

were

introduced

in

Until

the

data

collected

at

the

Department

of

Cardiology

established

no

relation

between

the

doses

measured

above

and

those

measured

under

the

lead

apron

This

was

thought

to

be

caused

by

the

fact

that

the

two

dosimeters

were

often

transposed

that

the

dosimeters

were

not

always

returned

at

the

same

time

and

that

unmistakable

instruction

regarding

the

position

of

the

two

personal

dosimeters

was

missing

To

rule

out

the

influence

of

these

factors

on

the

dose

measured

it

was

decided

to

develop

a

special

holder

for

the

two

dosimeters

at

the

time

the

radiologists

were

provided

with

additional

dosimeters

Moreover

it

was

decided

to

replace

all

personal

dosimeters

in

the

study

on

the

same

day

by

one

person

to

preclude

the

influence

of

differences

in

measuring

periods

and

problems

with

transposed

dosimeters

For

this

department

the

doses

measured

under

the

lead

apron

were

considered

to

be

a

substitute

for

the

effective

dose

as

recommended

by

the

ICRP

-LSB-

-RSB-

while

the

doses

measured

above

the

lead

apron

were

used

to

estimate

the

effective

dose

more

accurately

The

aim

of

the

present

study

was

to

determine

whether

a

relation

exists

between

the

doses

measured

above

and

those

measured

under

the

lead

aprons

of

radiologists

performing

interventional

procedures

and

whether

two

personal

dosimeters

provide

additional

information

compared

to

a

single

dosimeter

Materials

and

Methods

From

onward

the

occupational

doses

of

eight

radiologists

involved

in

interventional

fluoroscopically

guided

procedures

were

monitored

by

means

of

two

personal

dosimeters

The

personal

dosimeters

were

used

to

measure

the

doses

above

and

under

the

lead

aprons

of

the

radiologists

To

receive

consistent

measurements

the

two

dosimeters

were

worn

in

a

specially

designed

holder

that

was

fixed

to

the

lead

apron

-LRB-

Figs.

and

-RRB-

The

holder

with

the

two

personal

dosimeters

was

worn

breast-high

The

two

dosimeters

were

replaced

every

weeks

on

the

same

day

while

the

holder

remained

fixed

to

the

lead

apron

during

the

whole

study

Fig.

specially

designed

holder

fixed

to

the

lead

apron

breast-highFig

specially

designed

holder

with

the

two

personal

dosimeters

The

doses

were

measured

during

periods

of

weeks

in

consecutive

years

At

the

start

of

the

study

six

of

the

radiologists

were

provided

with

two

personal

dosimeters

while

throughout

the

study

another

two

radiologists

were

provided

with

two

dosimeters

Three

radiologists

left

the

AMC

before

the

end

of

the

study

The

personal

dosimeters

were

provided

by

the

Nuclear

Research

and

Consultant

Group

-LRB-

NRG

Arnhem

the

Netherlands

-RRB-

The

personal

dosimeters

were

read

by

the

NRG

and

reported

to

the

AMC

The

dosimeters

contain

LiF

Ti

Mg

-LRB-

-RRB-

detectors

behind

mm

of

aluminum

for

the

measurements

at

a

depth

of

mm

In

the

dose

reports

the

doses

were

presented

by

means

of

the

personal

dose

equivalent

at

a

depth

of

mm

the

Hp

-LRB-

-RRB-

The

doses

were

reported

in

multiples

of

mSv

Doses

below

mSv

were

reported

as

mSv

-LSB-

-RSB-

At

the

Department

of

Radiology

the

interventional

procedures

were

performed

with

two

Philips

Integris

Allura

machines

-LRB-

Philips

Medical

Systems

Best

the

Netherlands

-RRB-

with

the

same

potential

range

of

kVp

During

all

interventional

procedures

the

radiologists

used

wrap-around

lead

aprons

-LRB-

skirts

and

vests

Scanflex

Medical

AB

Täby

Sweden

-RRB-

with

an

overall

lead

equivalence

of

mm

at

kV

The

wrap-around

apron

has

an

overlap

due

to

which

the

actual

thickness

of

the

protective

apron

in

the

front

is

mm

lead

equivalent

The

doses

were

monitored

above

and

under

a

layer

of

mm

lead

equivalence

The

doses

measured

above

and

under

the

lead

apron

were

statistically

analyzed

using

SPSS

software

-LRB-

release

for

Windows

-RRB-

Under

the

lead

apron

doses

mSv

were

treated

as

mSv

for

the

statistical

analysis

Above

the

lead

apron

doses

mSv

were

entered

as

missing

values

For

the

statistical

analysis

the

doses

under

the

lead

apron

were

entered

as

dependent

variables

while

the

doses

above

the

lead

apron

and

the

eight

radiologists

were

independent

variables

Statistical

significant

differences

were

set

at

a

level

of

-LRB-

p

-RRB-

Results

During

the

study

dosimeters

were

provided

to

eight

radiologists

All

dosimeters

were

returned

to

the

NRG

The

doses

were

read

and

reported

in

dose

reports

In

total

doses

were

recorded

above

the

lead

apron

ranging

from

to

mSv

Under

the

lead

apron

the

doses

ranged

from

to

mSv

Five

readings

were

excluded

from

the

study

as

the

doses

measured

above

and

under

the

lead

apron

were

reported

to

be

mSv

In

cases

the

doses

measured

under

the

lead

apron

were

mSv

These

doses

were

treated

as

mSv

in

the

statistical

analysis

In

Tables

and

and

in

Figs.

and

the

mean

dose

median

and

quartiles

above

and

under

the

lead

apron

are

presented

for

the

eight

radiologists

A

Kolmogorov-Smirnov

test

showed

that

the

doses

of

the

eight

radiologists

were

normally

distributed

-LRB-

p

-RRB-

The

doses

measured

above

and

under

the

lead

apron

differed

significantly

among

the

eight

radiologists

-LRB-

one-way

ANOVA

p

-RRB-

The

Levenes

test

established

that

the

variance

of

the

doses

measured

above

and

under

the

lead

apron

of

seven

radiologists

was

equal

-LRB-

p

-RRB-

The

doses

of

these

seven

radiologists

did

not

differ

significantly

-LRB-

one-way

ANOVA

p

-RRB-

The

average

dose

of

the

seven

radiologists

was

mSv

in

weeks

above

the

lead

apron

and

mSv

in

weeks

under

the

lead

apron

The

average

dose

of

radiologist

was

mSv

in

weeks

above

the

lead

apron

and

mSv

under

the

lead

apron

The

dose

above

the

lead

apron

of

radiologist

differed

significantly

from

the

doses

of

radiologists

and

-LRB-

Tamhane

post

hoc

analysis

p

-RRB-

Under

the

lead

apron

the

dose

of

radiologist

differed

significantly

from

the

dose

of

radiologist

-LRB-

Tamhane

post

hoc

analysis

p

-RRB-

Table

number

of

measurements

mean

dose

standard

deviation

and

percentiles

-LRB-

-LSB-

median

-RSB-

and

-RRB-

of

the

doses

measured

above

the

lead

aprons

of

radiologists

to

no.NMean

-LRB-

mSv

-RRB-

-LRB-

mSv

-RRB-

-LRB-

mSv

-RRB-

-LRB-

mSv

-RRB-

Minimum

-LRB-

mSv

-RRB-

Maximum

-LRB-

mSv

-RRB-

143.411.222.163.584.481.884.582343.673.211.323.155.260.0316.78392.331.241.382.173.590.284.00493.652.611.763.564.720.359.245

Note

Doses

followed

by

a

superscript

a

differ

significantly

from

doses

followed

by

a

superscript

b

-LRB-

p

-RRB-

Table

number

of

measurements

mean

dose

standard

deviation

and

percentiles

-LRB-

-LSB-

median

-RSB-

and

-RRB-

of

the

doses

measured

under

the

lead

aprons

of

radiologists

to

no.NMean

-LRB-

mSv

-RRB-

-LRB-

mSv

-RRB-

-LRB-

mSv

-RRB-

-LRB-

mSv

-RRB-

Minimum

-LRB-

mSv

-RRB-

Maximum

-LRB-

mSv

-RRB-

140.180.090.100.170.270.090.292340.130.150.030.110.170.000.83390.060.060.010.070.110.000.15490.140.090.060.150.210.000.245

.070.090.000.060.090.000.496390.120.080.060.110.190.000.377

.200.120.120.180.270.020.508370.130.120.050.120.180.000.66

aThese

doses

differ

significantly

-LRB-

p

-RRB-

Fig.

of

the

doses

-LRB-

mSv

-RRB-

measured

above

the

lead

apron

The

black

line

in

the

red

box

marks

the

median

the

box

demarks

the

and

percentiles

and

the

whiskers

mark

the

minimum

and

maximum

observed

values

that

are

not

statistical

outliers

Extreme

values

are

marked

with

an

asterisk

The

extreme

values

are

more

than

three

box

lengths

from

the

upper

edge

of

the

box

The

box

length

is

the

interquartile

rangeFig

of

the

doses

-LRB-

mSv

-RRB-

measured

under

the

lead

apron

The

black

line

in

the

red

box

marks

the

median

the

box

demarks

the

and

percentiles

and

the

whiskers

mark

the

minimum

and

maximum

observed

values

that

are

not

statistical

outliers

Extreme

values

are

marked

by

an

asterisk

The

extreme

values

are

more

than

three

box

lengths

from

the

upper

edge

of

the

box

The

box

length

is

the

interquartile

range

In

Fig.

the

doses

measured

under

the

lead

apron

are

plotted

against

the

doses

above

the

lead

apron

The

statistical

analysis

of

the

data

established

a

linear

relation

between

the

doses

above

and

those

under

the

lead

apron

-LRB-

ANOVA

p

-RRB-

The

coefficients

of

the

regression

line

representing

the

relation

between

the

doses

measured

above

and

those

measured

under

the

leaded

apron

were

derived

resulting

in

the

equation

Y

X

with

Y

as

the

dependent

variable

-LRB-

dose

under

the

lead

apron

-RRB-

and

X

as

the

predictor

value

-LRB-

dose

above

the

lead

apron

-RRB-

The

confidence

interval

for

the

coefficients

ranged

from

to

The

standard

error

of

the

models

tested

was

The

standard

error

of

the

model

was

lower

than

the

standard

deviation

of

the

average

dose

under

the

lead

apron

-LRB-

-RRB-

The

residuals

were

normally

distributed

-LRB-

p

-RRB-

-LRB-

Fig.

-RRB-

Fig.

of

doses

-LRB-

mSv

-RRB-

measured

above

the

lead

apron

-LRB-

X-axis

-RRB-

and

under

the

lead

apron

-LRB-

Y-axis

-RRB-

The

line

represents

the

equation

for

the

regressionFig

of

the

residuals

-LRB-

mean

standard

deviation

-RRB-

The

linear

relation

was

not

influenced

by

the

doses

measured

for

radiologist

The

equation

remained

the

same

With

the

equation

and

the

lowest

detectable

dose

under

the

lead

apron

-LRB-

mSv

-RRB-

the

corresponding

dose

above

the

lead

apron

was

calculated

mSv

Above

this

threshold

the

relation

between

doses

above

and

those

under

the

lead

apron

was

determined

another

time

The

statistical

analysis

once

again

established

a

linear

relation

-LRB-

ANOVA

p

-RRB-

The

coefficients

of

the

regression

line

remained

the

same

Only

the

strength

of

the

model

was

lower

-LRB-

-RRB-

Discussion

The

dose

measurements

above

and

under

the

lead

apron

established

that

the

occupational

exposure

of

the

radiologists

performing

interventional

procedures

in

the

AMC

is

comparable

There

was

only

one

exception

radiologist

As

radiologist

performed

fewer

procedures

than

his

colleagues

his

exposure

was

expected

to

be

lower

It

was

however

unknown

what

reduction

in

doses

was

to

be

expected

as

data

on

the

number

and

the

length

of

procedures

were

not

collected

for

the

study

Despite

this

it

is

likely

that

the

lower

exposure

of

radiologist

was

caused

by

a

lower

workload

as

the

doses

above

and

under

the

lead

apron

decreased

proportionally

compared

to

the

other

radiologists

and

Results

of

doses

measured

above

and

under

the

lead

apron

of

physicians

performing

interventional

procedures

have

been

published

by

several

authors

Moreover

measurements

for

cardiologists

performing

interventional

procedures

were

carried

out

Renaud

et

al.

-LSB-

-RSB-

reported

measurements

above

and

under

the

lead

aprons

of

in-room

personnel

during

cardiac

catheterization

Vano

et

al.

-LSB-

-RSB-

published

measurements

of

cardiologists

during

a

follow-up

However

differences

in

the

way

radiologists

and

cardiologists

perform

interventional

procedures

make

it

difficult

to

compare

the

doses

of

cardiologists

and

radiologists

A

limited

number

of

clinical

studies

on

doses

above

and

under

the

lead

apron

have

been

carried

out

among

radiologists

Williams

et

al.

-LSB-

-RSB-

reported

average

monthly

doses

above

the

lead

aprons

of

radiologists

that

ranged

from

to

mSv

The

highest

average

dose

in

this

study

was

found

for

a

radiologist

who

performed

all

interventional

procedures

in

patients

with

liver

disease

The

average

dose

of

the

other

radiologists

in

the

study

was

mSv

The

findings

of

Williams

et

al.

are

comparable

to

the

average

dose

above

the

lead

apron

in

the

present

study

where

the

doses

varied

between

and

mSv

Niklason

et

al.

-LSB-

-RSB-

published

annual

radiation

doses

above

the

lead

aprons

of

radiologists

The

average

annual

dose

in

their

study

was

mSv

The

average

annual

dose

in

the

study

by

Niklason

et

al.

was

higher

than

the

average

annual

dose

in

the

present

study

mSv

However

the

average

annual

dose

found

by

Niklason

et

al.

was

lower

than

the

average

annual

dose

when

radiologist

was

excluded

The

average

annual

dose

of

the

seven

remaining

radiologists

was

mSv

In

the

studies

by

Williams

et

al.

and

Nicklason

et

al.

the

doses

under

the

lead

apron

were

also

measured

Williams

et

al.

found

that

the

average

monthly

dose

under

the

lead

apron

ranged

from

to

mSv

As

for

the

results

above

the

lead

apron

Williams

et

al.

reported

that

the

highest

average

dose

was

caused

by

the

differences

in

the

nature

of

work

of

one

of

the

radiologists

The

average

monthly

dose

under

the

lead

apron

of

all

other

radiologists

-LRB-

the

radiologist

with

the

highest

dose

was

excluded

-RRB-

was

mSv

In

the

present

study

the

doses

under

the

lead

apron

varied

between

and

mSv

in

a

period

The

average

dose

mSv

was

higher

than

the

average

dose

reported

by

Williams

et

al.

The

differences

between

the

two

studies

are

possibly

caused

by

the

differences

in

thicknesses

of

the

lead

aprons

between

them

Williams

et

al.

reported

that

the

doses

were

measured

under

lead

aprons

of

and

mm

while

in

the

present

study

doses

were

measured

under

a

layer

of

mm

Niklason

et

al.

measured

doses

under

lead

aprons

of

and

mm

As

for

the

doses

measured

above

the

lead

apron

the

doses

under

the

lead

apron

were

annual

doses

The

average

annual

dose

under

the

lead

apron

reported

by

Niklason

et

al.

was

mSv

while

in

the

present

study

the

average

annual

dose

was

mSv

-LRB-

median

mSv

-RRB-

As

for

the

results

reported

by

Williams

et

al.

the

differences

in

doses

were

probably

caused

by

the

variation

in

thickness

of

the

lead

aprons

worn

by

the

radiologists

In

the

study

reported

by

Williams

et

al.

the

radiologists

ensured

that

the

personal

dosimeters

were

worn

in

all

cases

However

the

personal

dosimeters

under

the

lead

aprons

were

worn

not

only

during

interventional

procedures

but

also

during

other

radiology

procedures

The

authors

described

that

the

dedication

of

the

radiologists

to

wearing

additional

dosimeters

under

the

lead

apron

was

probably

less

consistent

than

for

the

other

dosimeters

In

the

present

study

the

dedication

of

the

radiologists

was

not

tested

as

the

dosimeters

were

simultaneously

worn

in

the

special

holder

Moreover

the

dosimeters

in

this

study

were

worn

exclusively

during

interventional

procedures

The

duration

of

the

measurements

in

the

study

by

Niklason

et

al.

was

approximately

months

while

in

the

present

study

the

doses

of

the

majority

of

the

radiologists

were

measured

during

consecutive

years

The

regression

analysis

revealed

a

linear

relationship

between

the

doses

measured

above

and

those

measured

under

the

lead

apron

The

relation

was

described

by

the

formula

Y

X

The

slope

of

the

line

-LRB-

-RRB-

is

a

measure

of

the

transmission

of

the

lead

aprons

In

the

present

study

a

transmission

of

was

found

for

lead

aprons

of

mm

This

transmission

was

derived

breast-high

at

the

height

of

the

lead

collar

of

radiologists

According

to

Kharrati

et

al.

-LSB-

-RSB-

the

energy

of

the

scattered

radiation

at

the

height

of

the

lead

collar

of

radiologists

is

kVp

The

transmission

fraction

in

the

present

study

-LRB-

-RRB-

is

in

accordance

with

the

data

on

transmission

factors

of

different

primary

beam

energies

found

by

other

authors

Murphy

et

al.

-LSB-

-RSB-

found

a

transmission

factor

of

for

lead

aprons

of

mm

in

the

primary

beam

of

kVp

Vano

et

al.

-LSB-

-RSB-

found

a

fraction

transmitted

through

lead

aprons

of

between

at

kVp

and

at

kVp

Christodoulou

et

al.

-LSB-

-RSB-

reported

about

transmission

fraction

through

lead

aprons

of

mm

at

a

tube

potential

of

kVp

The

transmission

fraction

ranged

from

up

to

From

the

formula

Y

X

the

intercept

of

the

regression

line

can

be

calculated

The

intercept

with

the

X-axis

lies

at

mSv

This

value

theoretically

represents

the

threshold

of

the

doses

above

the

lead

apron

before

doses

under

the

lead

apron

This

means

that

for

the

doses

above

the

lead

apron

of

mSv

only

doses

below

the

diction

limit

are

expected

The

doses

will

be

reported

as

mSv

As

doses

up

to

mSv

are

below

the

detection

limit

of

the

dosimeters

and

are

reported

as

doses

mSv

the

theoretical

threshold

is

even

higher

mSv

versus

mSv

for

the

doses

above

the

lead

apron

As

a

matter

of

fact

in

the

present

study

for

all

measurements

above

the

lead

apron

mSv

the

corresponding

doses

under

the

lead

apron

were

reported

to

be

below

the

detection

limit

of

mSv

The

lowest

dose

in

this

study

above

the

lead

apron

for

which

a

dose

under

the

lead

apron

of

mSv

reported

was

mSv

The

minimum

thickness

recommended

by

the

IAEA

-LSB-

-RSB-

for

lead

aprons

of

radiologists

performing

interventional

procedures

is

mm

In

the

present

study

the

doses

were

measured

at

mm

The

doses

were

measured

under

one

layer

of

wrap-around

lead

aprons

Due

to

the

overlap

of

these

aprons

the

thickness

of

the

lead

apron

in

the

front

of

the

radiologists

was

mm

For

practical

reasons

it

was

decided

to

measure

the

doses

under

one

layer

of

mm

despite

the

fact

that

the

doses

measured

were

higher

than

the

occupational

doses

to

the

body

of

the

radiologists

The

occupational

doses

of

the

radiologists

under

the

lead

apron

in

this

study

are

therefore

overestimated

The

present

study

showed

that

additional

monitoring

under

the

lead

apron

is

not

necessary

to

estimate

the

occupational

exposure

more

accurately

This

result

is

in

agreement

with

the

conclusions

of

Schultz

et

al.

-LSB-

-RSB-

based

on

dose

calculations

with

Monte

Carlo

simulations

A

transmission

of

for

lead

aprons

mm

does

not

underestimate

the

exposure

for

radiologists

In

the

present

study

the

doses

under

the

lead

apron

were

determined

almost

at

the

same

place

as

the

doses

above

the

lead

apron

This

was

possible

as

a

special

holder

was

used

Before

the

special

holder

was

introduced

it

was

not

possible

to

derive

a

relation

between

the

doses

above

and

those

under

the

lead

apron

Conclusion

The

aim

of

the

present

study

was

to

determine

whether

a

relation

exists

between

the

doses

measured

above

the

lead

apron

and

those

measured

under

the

lead

apron

of

radiologists

performing

interventional

procedures

and

whether

two

personal

dosimeters

provide

additional

information

compared

to

a

single

dosimeter

It

might

be

concluded

that

a

linear

relation

between

the

measurements

above

and

those

under

the

lead

apron

is

proved

in

this

study

With

this

there

is

no

evidence

for

a

more

accurate

estimation

of

the

effective

dose

when

an

additional

dosimeter

is

used

As

the

results

of

this

study

were

derived

from

measurements

by

radiologists

performing

interventional

procedures

it

is

not

known

whether

a

factor

of

is

applicable

for

other

specialists

performing

interventional

procedures

such

as

interventional

cardiologists

and

electrophysiologists

For

this

reason

more

investigation

is

needed

to

derive

correction

factors

for

other

physicians

performing

interventional

procedures

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B I O O O O O O O O O B I O O O O O O O O O O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O B I O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O B I O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B I O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B I O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B I O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

dosimetry
fluoroscopy
radiation protection


Effect

of

tissue-harvesting

site

on

yield

of

stem

cells

derived

from

adipose

tissue

implications

for

cell-based

therapies

The

stromal

vascular

fraction

-LRB-

SVF

-RRB-

of

adipose

tissue

contains

an

abundant

population

of

multipotent

adipose-tissue-derived

stem

cells

-LRB-

ASCs

-RRB-

that

possess

the

capacity

to

differentiate

into

cells

of

the

mesodermal

lineage

in

vitro

For

cell-based

therapies

an

advantageous

approach

would

be

to

harvest

these

SVF

cells

and

give

them

back

to

the

patient

within

a

single

surgical

procedure

thereby

avoiding

lengthy

and

costly

in

vitro

culturing

steps

However

this

requires

SVF-isolates

to

contain

sufficient

ASCs

capable

of

differentiating

into

the

desired

cell

lineage

We

have

investigated

whether

the

yield

and

function

of

ASCs

are

affected

by

the

anatomical

sites

most

frequently

used

for

harvesting

adipose

tissue

the

abdomen

and

hip/thigh

region

The

frequency

of

ASCs

in

the

SVF

of

adipose

tissue

from

the

abdomen

and

hip/thigh

region

was

determined

in

limiting

dilution

and

colony-forming

unit

-LRB-

CFU

-RRB-

assays

The

capacity

of

these

ASCs

to

differentiate

into

the

chondrogenic

and

osteogenic

pathways

was

investigated

by

quantitative

real-time

polymerase

chain

reaction

and

-LRB-

immuno

-RRB-

histochemistry

A

significant

difference

-LRB-

P

-RRB-

was

seen

in

ASC

frequency

but

not

in

the

absolute

number

of

nucleated

cells

between

adipose

tissue

harvested

from

the

abdomen

-LRB-

mean

SEM

-RRB-

and

hip/thigh

region

-LRB-

-RRB-

However

within

the

CFUs

derived

from

both

tissues

the

frequency

of

CFUs

having

osteogenic

differentiation

potential

was

the

same

When

cultured

homogeneous

cell

populations

were

obtained

with

similar

growth

kinetics

and

phenotype

No

differences

were

detected

in

differentiation

capacity

between

ASCs

from

both

tissue-harvesting

sites

We

conclude

that

the

yield

of

ASCs

but

not

the

total

amount

of

nucleated

cells

per

volume

or

the

ASC

proliferation

and

differentiation

capacities

are

dependent

on

the

tissue-harvesting

site

The

abdomen

seems

to

be

preferable

to

the

hip/thigh

region

for

harvesting

adipose

tissue

in

particular

when

considering

SVF

cells

for

stem-cell-based

therapies

in

one-step

surgical

procedures

for

skeletal

tissue

engineering

Introduction

Tissue

engineering

is

an

emerging

field

in

modern

medicine

Therapies

involve

the

combination

of

cells

and

scaffold

materials

that

can

be

loaded

with

bioactive

factors

ideally

resulting

in

the

regeneration

or

replacement

of

lost

or

damaged

tissues

and

organs

Multiple

cell

sources

have

been

investigated

for

their

possible

applicability

in

tissue

engineering

Embryonal

stem

cells

are

the

most

potent

stem

cells

however

their

use

is

controversial

and

has

mayor

ethical

considerations

-LRB-

Dresser

-RRB-

Mesenchymal

stem

cells

-LRB-

MSCs

-RRB-

can

be

obtained

from

the

adult

and

are

widely

used

because

of

their

differentiation

potential

In

addition

to

bone

marrow

periosteum

-LRB-

Nakahara

et

al.

-RRB-

muscle

-LRB-

Asakura

et

al.

-RRB-

and

adipose

tissue

-LRB-

Zuk

et

al.

-RRB-

also

appear

to

be

sources

of

MSCs

Subcutaneous

adipose

tissue

is

a

particularly

attractive

reservoir

of

progenitor

cells

because

it

is

easily

accessible

abundant

and

self-replenishing

It

is

derived

from

the

mesodermal

germ

layer

and

contains

a

supportive

stromal

vascular

fraction

-LRB-

SVF

-RRB-

that

can

be

readily

isolated

-LRB-

Gronthos

et

al.

Zuk

et

al.

-RRB-

This

SVF

from

adipose

tissue

consists

of

a

heterogeneous

mixture

of

cells

including

endothelial

cells

smooth

muscle

cells

pericytes

leukocytes

mast

cells

and

pre-adipocytes

-LRB-

Oedayrajsingh-Varma

et

al.

Peterson

et

al.

Prunet-Marcassus

et

al.

-RRB-

In

addition

to

these

cells

the

SVF

contains

an

abundant

population

of

multipotent

adipose-tissue-derived

stem

cells

-LRB-

ASCs

-RRB-

that

possess

the

capacity

to

differentiate

into

cells

of

mesodermal

origin

in

vitro

e.g.

adipocytes

chondrocytes

osteoblasts

and

-LRB-

cardio

-RRB-

myocytes

-LRB-

Erickson

et

al.

Guilak

et

al.

Halvorsen

et

al.

Hattori

et

al.

Planat-Benard

et

al.

Rangappa

et

al.

Zuk

et

al.

-RRB-

Because

of

these

favorable

characteristics

interest

has

been

growing

in

the

application

of

ASCs

for

cell-based

therapies

such

as

tissue

engineering

For

clinical

practice

an

advantageous

approach

would

be

to

harvest

ASCs

and

immediately

give

them

back

to

the

patient

within

the

same

operation

the

so

called

one-step

surgical

procedure

-LRB-

Helder

et

al.

-RRB-

This

overcomes

long-lasting

culture

expansion

of

ASCs

on

the

one

hand

but

necessitates

the

use

of

the

SVF

of

adipose

tissue

on

the

other

hand

since

fast

selection

procedures

for

stem

cells

in

the

SVF

are

not

yet

available

Therefore

the

success

of

this

procedure

requires

SVF-isolates

to

contain

sufficient

ASCs

capable

of

differentiating

into

the

desired

cell

lineage

In

view

of

this

we

have

previously

investigated

the

effect

of

three

different

surgical

procedures

for

the

harvesting

of

adipose

tissue

i.e.

resection

tumescent

or

conventional

liposuction

and

ultrasound-assisted

liposuction

on

the

yield

and

function

of

the

stem

cells

We

have

demonstrated

that

the

SVF

isolates

from

adipose

tissue

harvested

by

ultrasound-assisted

liposuction

contain

fewer

stem

cells

and

that

the

stem

cells

have

a

longer

population

doubling

time

leading

us

to

conclude

that

resection

and

tumescent

liposuction

are

preferable

to

ultrasound-assisted

liposuction

for

harvesting

adipose

tissue

if

the

cells

are

to

be

used

for

tissue-engineering

purposes

-LRB-

Oedayrajsingh-Varma

et

al.

-RRB-

In

the

present

study

we

have

investigated

whether

the

yield

and

functional

characteristics

of

ASCs

in

the

SVF

are

affected

by

the

most

frequently

used

adipose-tissue-harvesting

sites

We

have

previously

demonstrated

that

the

yield

of

nucleated

cells

in

the

SVF

of

the

adipose

tissue

from

these

different

tissue-harvesting

sites

is

similar

-LRB-

Oedayrajsingh-Varma

et

al.

-RRB-

In

the

current

study

the

frequency

of

ASCs

in

the

SVF

cell

isolates

has

been

determined

by

using

limiting

dilution

and

colony-forming

unit

-LRB-

CFU

-RRB-

assays

In

addition

we

have

investigated

the

frequency

of

CFUs

showing

an

osteogenic

differentiation

capacity

SVF

cells

have

been

subsequently

cultured

in

order

to

obtain

homogeneous

cell

populations

and

to

acquire

sufficient

cells

to

determine

their

chondrogenic

differentiation

potential

in

micromass

cultures

Homogeneity

has

been

checked

by

determining

the

growth

kinetics

and

phenotypic

characteristics

of

the

ASCs

To

verify

the

maintenance

of

multidifferentiation

potential

osteogenic

and

chondrogenic

induction

has

been

assessed

in

these

homogeneous

ASC

cultures

Materials

and

methods

Donors

Samples

of

human

subcutaneous

adipose

tissue

were

obtained

as

waste

material

after

elective

tumescent

liposuction

or

resection

and

donated

after

informed

consent

from

healthy

donors

operated

on

at

the

Departments

of

Plastic

Surgery

of

two

clinics

in

Amsterdam

The

Netherlands

Adipose

tissue

was

taken

from

the

abdomen

-LRB-

n

-RRB-

and

the

hip/thigh

region

-LRB-

n

-RRB-

during

cosmetic

surgery

female

donors

were

included

in

this

study

The

average

age

-LRB-

mean

age

range

years

-RRB-

and

body

mass

index

-LRB-

BMI

mean

BMI

range

-RRB-

were

similar

for

both

groups

-LRB-

Table

-RRB-

Table

characteristics

-LRB-

BMI

body

mass

index

THS

tissue-harvesting

site

ASC

adipose-derived

stem

cell

Res

resection

T-LS

tumescent

liposuction

ND

not

determined

-RRB-

DonorAge

-LRB-

years

-RRB-

BMI

-LRB-

-RRB-

THSProcedureASC

yield

-LRB-

-RRB-

Hip/thighT-LS

Hip/thighT-LS

Hip/thighT-LS

Hip/thighT-LS

Hip/thighT-LS

Hip/thighT-LS

Hip/thighT-LS

Hip/thighT-LS

Cell

isolation

and

storage

Isolation

of

the

SVF

from

adipose

tissue

was

performed

as

previously

described

-LRB-

Oedayrajsingh-Varma

et

al.

-RRB-

The

isolation

protocol

included

a

Ficoll

density

centrifugation

step

to

remove

contaminating

erythrocytes

After

isolation

SVF

cells

were

resuspended

in

a

mixture

-LRB-

-RRB-

of

Dulbecco

's

modified

Eagle

's

medium

-LRB-

DMEM

-RRB-

and

cryoprotective

medium

-LRB-

Freezing

Medium

BioWhittaker

Cambrex

Verviers

Belgium

-RRB-

frozen

under

controlled

rate

conditions

in

a

Kryosave

-LRB-

HCI

Cryogenics

Hedel

The

Netherlands

-RRB-

and

stored

in

the

vapor

phase

of

liquid

nitrogen

according

to

standard

practice

at

the

Department

of

Pathology

of

the

VU

University

Medical

Center

and

following

the

guidelines

of

current

Good

Manufacturing

Practice

Limiting

dilution

assay

To

assess

the

frequency

of

ASCs

in

the

SVF

of

adipose

tissue

SVF

cells

were

seeded

in

normal

culture

medium

consisting

of

DMEM

supplemented

with

fetal

bovine

serum

-LRB-

FBS

-RRB-

U/ml

penicillin

μg/ml

streptomycin

and

mM

L-glutamine

-LRB-

Invitrogen

Gibco

Calif.

USA

-RRB-

in

plates

at

cells/well

in

the

upper

row

Two-fold

dilution

steps

of

the

cells

were

made

in

subsequent

rows

All

cultures

were

performed

in

duplicate

Medium

was

changed

twice

a

week

After

weeks

each

well

was

individually

scored

for

the

number

of

cells

A

well

containing

a

cluster

of

at

least

adhered

fibroblast-like

cells

was

considered

as

being

positive

The

frequency

of

ASCs

was

calculated

from

the

rows

of

cells

for

which

of

the

wells

were

scored

as

positive

CFU

assays

CFU

assays

were

performed

to

check

the

consistency

of

the

limiting

dilution

assay

method

and

to

determine

the

frequency

of

CFU

capable

of

differentiating

into

the

osteogenic

lineage

from

the

abdomen

-LRB-

n

-RRB-

and

hip/thigh

region

-LRB-

n

-RRB-

SVF

cells

were

resuspended

in

normal

culture

medium

consisting

of

DMEM

supplemented

with

FBS

U/ml

penicillin

μg/ml

streptomycin

and

mM

L-glutamine

-LRB-

Invitrogen

Gibco

-RRB-

Two

plates

were

prepared

in

which

the

SVF

was

diluted

ten-fold

across

both

columns

resulting

in

a

upper

column

containing

and

a

lower

column

containing

nucleated

SVF

cells

For

the

CFU-fibroblast

-LRB-

CFU-F

-RRB-

assay

the

fixation

time

was

days

depending

on

the

amount

and

growth

kinetics

of

the

colonies

-LRB-

merging

of

colonies

was

avoided

-RRB-

At

the

appropriate

time

point

the

medium

was

removed

and

the

cells

were

washed

with

phosphate-buffered

saline

-LRB-

PBS

-RRB-

fixed

with

formaldehyde

for

min

and

subsequently

colored

in

a

toluidine

blue

solution

in

borax

buffer

for

about

min

Excess

stain

was

washed

off

with

distilled

water

and

colonies

were

counted

Cells

of

the

duplicate

plate

were

submitted

to

a

CFU-alkaline

phosphatase

-LRB-

CFU-ALP

-RRB-

assay

Cultures

were

performed

in

normal

medium

for

days

in

order

to

obtain

colonies

and

to

remove

contaminating

cells

after

which

osteogenic

medium

was

added

for

weeks

Following

this

period

cells

in

the

CFU-ALP

plate

were

rinsed

with

PBS

fixed

in

formaldehyde

and

incubated

for

min

in

a

M

TRIS-hydrochloride

-LRB-

pH

-RRB-

M

calcium

chloride

M

magnesium

chloride

solution

whereafter

a

solution

containing

M

TRIS-hydrochloride

-LRB-

pH

-RRB-

M

calcium

chloride

M

magnesium

chloride

and

μl

nitroblue

was

added

for

min

The

percentage

of

the

colonies

staining

positive

for

ALP

was

determined

Culturing

of

SVF

cells

ASCs

from

the

abdomen

and

hip/thigh

region

were

cultured

up

to

passage

for

an

adequate

and

quantitative

comparison

of

stem-cell

proliferation

and

differentiation

capacity

Single-cell

suspensions

of

cryopreserved

SVF

cells

were

seeded

at

nucleated

in

normal

culture

medium

The

cultures

were

maintained

in

a

incubator

at

°C

in

a

humidified

atmosphere

The

medium

was

changed

twice

a

week

When

reaching

confluency

cells

were

detached

with

mM

trypsin

-LRB-

Invitrogen

-RRB-

for

min

at

°C

and

replated

Cell

viability

was

assessed

by

using

the

trypan

blue

exclusion

assay

A

homogeneous

population

of

ASCs

was

thus

obtained

from

abdomen

and

hip/thigh

region

and

was

subsequently

checked

by

determining

growth

kinetics

and

by

analyzing

the

surface-marker

expression

profile

of

the

ASCs

Growth

kinetics

of

ASCs

To

determine

the

growth

kinetics

of

cultured

ASCs

ten

flasks

per

donor

were

seeded

with

cultured

ASCs

-LRB-

passage

or

-RRB-

At

several

time

points

-LRB-

between

days

and

-RRB-

after

seeding

cells

from

two

duplicate

flasks

were

harvested

and

counted

ASC

numbers

were

plotted

against

the

number

of

days

cultured

and

the

exponential

growing

phase

of

the

cells

was

determined

The

population

doubling

time

was

calculated

by

using

the

formula

where

was

the

number

of

cells

at

the

beginning

of

the

exponential

growing

phase

and

was

the

number

of

cells

at

the

end

of

the

exponential

growing

phase

Flow

cytometry

Single-cell

suspensions

of

cultured

ASCs

from

abdomen

and

hip/thigh

region

were

phenotypically

characterized

by

using

fluorescence-activated

cell

sorting

-LRB-

FACS

FACSCalibur

Becton

Dickinson

USA

-RRB-

as

previously

described

-LRB-

Varma

et

al.

-RRB-

All

monoclonal

antibodies

-LRB-

mAbs

-RRB-

were

of

the

immunoglobulin

-LRB-

-RRB-

isotype

Cells

were

stained

with

fluorescently

labeled

antibodies

-LRB-

conjugated

to

fluorescein

isothiocyanate

phycoerythrin

or

allophycocyanin

-RRB-

against

HLA-DR

and

HLA-ABC

-LRB-

BD

Biosciences

San

José

Calif.

-RRB-

-LRB-

RDI

Research

Diagnostics

Flanders

N.J.

-RRB-

-LRB-

Caltag

Laboratories

Burlingame

Calif.

-RRB-

-LRB-

PharMingen

San

Diego

Calif.

-RRB-

and

-LRB-

Chemicon

Temecula

Calif.

-RRB-

Nonspecific

fluorescence

was

determined

by

incubating

the

cells

with

conjugated

mAb

anti-human

-LRB-

DakoCytomation

Glostrup

Denmark

-RRB-

Chondrogenic

and

osteogenic

differentiation

The

chondrogenic

and

osteogenic

differentiation

capacities

of

the

cultured

ASCs

from

the

abdomen

-LRB-

n

-RRB-

and

hip/thigh

region

-LRB-

n

-RRB-

were

studied

Chondrogenic

differentiation

was

induced

in

cultured

ASCs

as

previously

described

with

some

modifications

-LRB-

Oedayrajsingh-Varma

et

al.

-RRB-

In

short

a

drop

of

a

concentrated

ASC

cell

suspension

-LRB-

passage

-RRB-

was

applied

to

a

glass

slide

and

allowed

to

attach

at

°C

for

h.

Then

μl

chondrogenic

medium

consisting

of

DMEM

plus

ITS

Premix

-LRB-

final

concentration

in

medium

when

diluted

was

μg/ml

insulin

μg/ml

transferrin

ng/ml

selenous

acid

mg/ml

bovine

serum

albumin

-LRB-

BSA

-RRB-

μg/ml

linoleic

acid

BD

USA

-RRB-

ng/nl

transforming

growth

factor

β1

-LRB-

Biovision

ITK-diagnostics

-RRB-

FCS

μM

-LRB-

Sigma

St.

Louis

Mo.

-RRB-

U/ml

penicillin

μg/ml

streptomycin

and

mM

L-glutamine

was

overlaid

gently

Cells

were

maintained

in

a

oxygen

custom-designed

hypoxia

workstation

-LRB-

T.C.P.S.

Rotselaar

Belgium

-RRB-

at

°C

in

a

humidified

atmosphere

as

this

was

shown

to

enhance

chondrogenic

differentiation

-LRB-

data

not

shown

-RRB-

Chondrogenic

media

were

changed

every

days

For

osteogenic

differentiation

ASCs

-LRB-

passage

-RRB-

were

seeded

at

and

cultured

in

monolayer

in

osteogenic

medium

consisting

of

normal

culture

medium

supplemented

with

mM

β-glycerol

phosphate

μg/ml

and

ng/ml

bone

morphogenetic

protein

-LRB-

Peprotech

EC

London

UK

-RRB-

Osteogenic

medium

was

changed

twice

a

week

-LRB-

Immuno

-RRB-

histochemistry

-LRB-

Immuno

-RRB-

histochemistry

was

performed

as

described

previously

-LRB-

Oedayrajsingh-Varma

et

al.

-RRB-

Cell

nodules

that

formed

under

chondrogenic

culture

conditions

were

stained

with

Alcian

blue

-LRB-

Sigma-Aldrich

Zwijndrecht

The

Netherlands

-RRB-

at

acidic

pH

for

detection

of

proteoglycans

For

the

detection

of

collagen

Type

II

staining

was

performed

with

mouse

monoclonal

antibody

-LRB-

Developmental

Studies

Hybridoma

Bank

Iowa

USA

-RRB-

against

human

collagen

Type

II

in

PBS

containing

BSA

Osteogenesis

was

visualized

after

days

of

culture

in

osteogenic

medium

by

Von

Kossa

staining

to

establish

the

formation

of

a

calcified

matrix

typical

for

mature

osteoblasts

The

protocol

used

was

as

described

previously

-LRB-

Varma

et

al.

-RRB-

with

only

one

modification

of

counterstaining

the

cytoplasm

of

the

cells

using

fast

green

Calcified

extracellular

matrix

was

visualized

as

black

spots

Spectrophotometric

ALP

activity

Early

differentiation

of

MSCs

into

immature

osteoblasts

is

characterized

by

ALP

enzyme

activity

with

human

MSCs

expressing

ALP

as

early

as

days

after

induction

and

maximum

levels

being

observed

at

around

days

after

induction

-LRB-

Jaiswal

et

al.

-RRB-

Therefore

cellular

ALP

activity

was

measured

after

culturing

the

ASCs

in

osteogenic

medium

for

days

Cells

were

lysed

with

distilled

water

and

the

ALP

activity

and

protein

content

were

determined

To

determine

ALP

activity

p-nitrophenyl

phosphate

-LRB-

Merck

Darmstadt

Germany

-RRB-

at

pH

was

used

as

the

substrate

as

described

by

Lowry

-LRB-

-RRB-

ALP

activity

was

expressed

as

micromole

per

microgram

of

protein

in

the

cell

layer

The

amount

of

protein

was

determined

by

using

a

BCA

Protein

Assay

reagent

Kit

-LRB-

Pierce

Rockford

Ill.

USA

-RRB-

and

the

absorbance

was

read

at

nm

with

a

microplate

reader

-LRB-

Biorad

Laboratories

Hercules

Calif.

USA

-RRB-

Real-time

polymerase

chain

reaction

RNA

isolation

and

reverse

transcription

were

performed

as

previously

described

-LRB-

Oedayrajsingh-Varma

et

al.

-RRB-

Real-time

polymerase

chain

reactions

-LRB-

PCR

-RRB-

were

performed

by

using

the

SYBRGreen

reaction

kit

according

to

the

manufacturer

's

instructions

-LRB-

Roche

Diagnostics

-RRB-

in

a

LightCycler

-LRB-

Roche

Diagnostics

-RRB-

cDNA

-LRB-

approximately

ng

-RRB-

was

used

in

a

volume

of

μl

PCR

mix

-LRB-

LightCycler

DNA

Master

Fast

Startplus

Kit

Roche

Diagnostics

-RRB-

containing

a

final

concentration

of

pmol

primers

Relative

housekeeping

gene

expression

for

S-rRNA

-LRB-

S

-RRB-

and

relative

target

gene

expression

for

aggrecan

-LRB-

AGG

-RRB-

collagen

Type

II

-LRB-

-RRB-

and

collagen

Type

X

-LRB-

-RRB-

regarding

chondrogenic

differentiation

and

for

collagen

Type

I

-LRB-

-RRB-

osteopontin

-LRB-

OPN

-RRB-

and

runt-related

transcription

factor

-LRB-

-RRB-

regarding

osteogenic

differentiation

were

determined

Primers

-LRB-

Invitrogen

-RRB-

used

for

real-time

PCR

are

listed

in

Table

They

were

designed

by

using

Clone

Manager

Suite

software

program

version

-LRB-

Scientific

Educational

Software

Cary

N.C.

USA

-RRB-

The

amplified

PCR

fragment

extended

over

at

least

one

exon

border

based

on

homology

in

conserved

domains

between

human

mouse

rat

dog

and

cow

except

for

the

S

gene

-LRB-

encoded

by

one

exon

only

-RRB-

Amplified

PCR

products

were

electrophoresed

on

a

agarose

gel

and

stained

with

ethidium

bromide

For

real-time

PCR

the

values

of

relative

target

gene

expression

were

normalized

to

relative

S

housekeeping

gene

expression

Table

PCR

primer

sets

used

for

reverse

transcription/PCR

-LRB-

S

S

subunit

AGG

aggrecan

collagen

Type

I

collagen

Type

II

collagen

Type

X

OPN

osteopontin

runt-related

transcription

factor

-RRB-

Gene

Primer

sets

Accession

number

product

length

-LRB-

bp

-RRB-

S

Forward

GTAACCCGTTGAACCCCATT

Human

bp

Reverse

CCATCCAATCGGTAGTAGCG

AGG

Forward

CAACTACCCGGCCATCC

Human

bp

Reverse

GATGGCTCTGTAATGGAACAC

Forward

AAGCCGAATTCCTGGTCT

Human

bp

Reverse

TCCAACGAGATCGAGATCC

Forward

AGGGCCAGGATGTCCGGCA

Human

bp

Reverse

GGGTCCCAGGTTCTCCATCT

Forward

CACTACCCAACACCAAGACA

Human

bp

Reverse

CTGGTTTCCCTACAGCTGAT

OPN

Forward

TTCCAAGTAAGTCCAACGAAAG

Human

bp

Reverse

GTGACCAGTTCATGAGATTCAT

Forward

ATGCTTCATTCGCCTCAC

Human

bp

Reverse

ACTGCTTGCAGCCTTAAAT

Real-time

PCR

data

analysis

With

the

Light

Cycler

software

-LRB-

version

-RRB-

the

crossing

points

were

assessed

and

plotted

versus

the

serial

dilution

of

known

concentrations

of

the

standards

derived

from

each

gene

by

the

Fit

Points

method

PCR

efficiency

was

calculated

by

Light

Cycler

software

and

the

data

were

used

only

if

the

calculated

PCR

efficiency

was

between

Statistics

Kolmogorov-Smirnov

tests

were

used

to

determine

the

normalcy

of

measurements

and

if

appropriate

their

logarithmics

For

the

evaluation

of

yield

and

growth

kinetics

means

between

two

groups

in

one

variable

were

compared

by

using

the

independent

sample

two-tailed

t-test

Partial

correlation

was

expressed

as

the

Pearson

correlation

coefficient

r.

For

evaluation

of

gene

expression

a

repeated

measures

analysis

of

variance

was

used

to

determine

significant

differences

when

increasing

time-points

in

one

donor

within

one

variable

were

compared

If

levels

of

gene

expression

were

below

the

detection

limit

-LRB-

-RRB-

values

were

set

at

-LRB-

or

log

level

at

-RRB-

All

statistical

tests

used

a

significance

level

of

α

Results

Effects

of

tissue-harvesting

site

on

frequency

of

ASCs

In

a

previous

study

we

demonstrated

that

the

yield

of

nucleated

cells

in

the

SVF

of

adipose

tissue

from

different

tissue-harvesting

sites

was

similar

-LRB-

Oedayrajsingh-Varma

et

al.

-RRB-

To

investigate

whether

the

tissue-harvesting

site

affected

the

frequency

of

ASCs

in

the

SVF

in

the

present

study

limiting

dilution

and

CFU-F

assays

were

performed

The

outcomes

of

both

types

of

assays

were

similar

-LRB-

Fig.

b

-RRB-

When

combined

the

SVF

of

adipose

tissue

harvested

from

the

abdomen

contained

ASCs

-LRB-

mean

SEM

-RRB-

whereas

the

percentage

of

ASCs

in

the

SVF

of

adipose

tissue

harvested

from

the

hip/thigh

region

was

much

lower

-LRB-

Fig.

-RRB-

This

difference

in

ASC

frequency

between

adipose

tissue

from

the

abdomen

and

hip/thigh

region

was

significant

-LRB-

P

-RRB-

Fig.

of

adipose-tissue-harvesting

site

on

the

frequency

of

adipose-derived

stem

cells

-LRB-

ASCs

-RRB-

in

the

stromal

vascular

fraction

-LRB-

SVF

-RRB-

After

isolation

of

the

SVF

from

adipose

tissue

of

both

tissue-harvesting

sites

the

frequency

of

ASCs

in

the

SVF

isolates

was

determined

by

using

-LRB-

a

-RRB-

a

limiting

dilution

assay

-LRB-

LD

-RRB-

and

-LRB-

b

-RRB-

a

colony-forming

unit

-LRB-

CFU

-RRB-

assay

-LRB-

CFU-F

-RRB-

with

similar

results

When

combined

-LRB-

c

-RRB-

a

significant

difference

was

detected

in

ASC

frequency

between

adipose

tissue

harvested

from

the

abdomen

and

adipose

tissue

harvested

from

the

hip/thigh

region

-LRB-

P-values

a

limiting

dilution

P

b

colony-forming

unit

P

c

combined

P

-RRB-

Effect

of

tissue-harvesting

site

on

frequency

of

CFUs

having

osteogenic

differentiation

potential

Parallel

to

the

CFU-F

assay

-LRB-

Fig.

-RRB-

CFU-ALP

assays

were

also

performed

to

determine

the

percentage

of

the

CFUs

capable

of

osteogenic

differentiation

For

adipose

tissue

from

the

abdomen

-LRB-

mean

SEM

-RRB-

of

the

CFUs

stained

positive

for

ALP

whereas

the

CFUs

from

adipose

tissue

of

the

hip/thigh

region

displayed

an

ALP

positivity

of

-LRB-

Fig.

-RRB-

Apparently

no

differences

in

osteogenic

potential

existed

between

ASCs

from

the

two

tissue-harvesting

sites

-LRB-

P

-RRB-

Fig.

of

adipose-tissue-harvesting

site

on

the

osteogenic

diffentiation

capacity

of

CFU

from

the

abdomen

or

hip/thigh

regions

No

significant

difference

is

apparent

in

the

CFU-alkaline

phosphatase

-LRB-

CFU-ALP

-RRB-

frequency

from

the

abdomen

and

hip/thigh

region

when

corrected

for

CFU-fibroblast

-LRB-

CFU-F

-RRB-

Phenotypic

characterization

and

growth

kinetics

of

cultured

ASCs

Cultured

ASCs

-LRB-

passages

to

-RRB-

from

abdomen

and

hip/thigh

regions

were

phenotypically

characterized

ASCs

from

both

tissue-harvesting

sites

were

demonstrated

to

be

homogeneous

populations

staining

positive

for

stem-cell-associated

markers

and

HLA-ABC

and

negative

for

hematopoietic/leukocytic/endothelial

markers

such

as

and

HLA-DR

-LRB-

Table

-RRB-

Table

expression

of

human

cultured

ASC

at

passages

Results

are

expressed

as

mean

fluorescence

-LRB-

MF

-RRB-

with

isotype

control

-LRB-

MF

MF

MF

MF

-RRB-

Cell-surface

markerCultured

ASC

-LRB-

n

-RRB-

HLA-ABC

HLA-DR

To

determine

growth

kinetics

the

population

doubling

time

of

ASCs

from

passages

to

was

determined

When

ASCs

numbers

were

monitored

over

time

a

cell

growing

curve

was

obtained

showing

an

exponential

growing

phase

after

which

the

cells

reached

confluency

-LRB-

Fig.

b

-RRB-

The

mean

population

doubling

time

of

the

ASCs

in

the

exponential

growing

phase

was

about

days

when

the

adipose

tissue

was

harvested

from

the

abdomen

and

hip/thigh

regions

-LRB-

abdomen

hip/thigh

mean

SEM

Fig.

-RRB-

Fig.

of

the

adipose-tissue-harvesting

site

on

growth

kinetics

of

ASCs

in

vitro

a

Growth

kinetics

of

ASCs

of

a

representative

donor

when

adipose

tissue

was

harvested

from

the

abdomen

b

Growth

kinetics

of

ASCs

when

adipose

tissue

was

harvested

from

the

hip/thigh

region

c

Population

doubling

time

was

calculated

from

the

exponential

growing

phase

of

the

cells

There

was

no

significant

difference

in

population

doubling

time

of

ASCs

from

the

abdomen

and

hip/thigh

region

-LRB-

P

independent

Student

t-test

-RRB-

Effect

of

tissue-harvesting

site

on

osteogenic

differentiation

potential

of

ASCs

Differentiation

of

cultured

ASCs

into

the

osteogenic

lineage

was

induced

by

culturing

the

cells

in

monolayer

in

osteogenic

medium

containing

Specific

RNA

expression

of

osteogenically

induced

ASCs

from

all

donors

tested

increased

over

time

being

up-regulated

-LRB-

P

-RRB-

and

being

up-regulated

seven-fold

-LRB-

P

-RRB-

after

days

-LRB-

Fig.

b

-RRB-

OPN

gene

expression

was

increased

after

days

however

this

increase

was

not

significant

-LRB-

Fig.

-RRB-

No

significant

differences

were

detected

in

osteogenic

gene

expression

between

ASCs

derived

from

the

abdomen

and

ASCs

derived

from

the

hip/thigh

region

at

all

three

time

points

tested

Fig.

of

adipose-tissue-harvesting

site

on

the

osteogenic

differentiation

of

cultured

ASCs

in

vitro

a

-LRB-

runt-related

transcription

factor

P

-RRB-

-LRB-

collagen

type

Ia

P

-RRB-

and

OPN

-LRB-

osteopontin

P

-RRB-

gene

expression

was

measured

after

and

days

-LRB-

d

-RRB-

of

osteogenic

induction

by

using

quantitative

real-time

polymerase

chain

reaction

-LRB-

qRT-PCR

-RRB-

No

significant

differences

were

detected

in

osteogenic

gene

expression

between

ASCs

derived

from

the

abdomen

and

ASCs

derived

from

the

hip/thigh

region

at

all

three

time

points

tested

d

ALP

activity

was

significantly

increased

after

days

in

the

osteogenically

stimulated

cells

-LRB-

stim

-RRB-

compared

with

that

in

unstimulated

cells

-LRB-

con

P

-RRB-

No

statistically

significant

difference

was

apparent

in

ALP

activity

between

ASCs

derived

from

abdominal

fat

and

ASCs

from

hip/thigh

fat

e

g

Von

Kossa

staining

of

ASCs

from

abdomen

-LRB-

e

-RRB-

and

hip/thigh

region

-LRB-

f

-RRB-

after

days

of

culture

in

osteogenic

medium

and

in

control

medium

-LRB-

g

-RRB-

showing

mineralized

matrix

visible

as

black

spots

ALP

activity

in

the

ASCs

was

measured

after

days

of

osteogenic

stimulation

ASCs

cultured

in

control

medium

served

as

negative

controls

ALP

activity

in

the

osteogenically

stimulated

cells

was

significantly

increased

-LRB-

P

-RRB-

compared

with

that

in

control

cells

-LRB-

Fig.

-RRB-

No

statistically

significant

difference

was

apparent

in

ALP

activity

between

ASCs

derived

from

abdominal

fat

and

ASCs

from

hip/thigh

fat

Calcification

of

the

osteogenic

matrix

was

confirmed

by

using

Von

Kossa

staining

black

spots

could

be

observed

after

weeks

of

osteogenic

stimulation

of

ASCs

from

both

origins

-LRB-

Fig.

abdomen

Fig.

hip/thigh

-RRB-

No

calcification

was

seen

in

ASC

cultures

expanded

in

control

medium

-LRB-

Fig.

-RRB-

Effect

of

tissue-harvesting

site

on

chondrogenic

differentiation

potential

of

ASCs

The

chondrogenic

differentiation

potential

of

ASCs

was

analyzed

after

culturing

the

cells

in

a

micromass

in

chondrogenic

medium

containing

TGF-β

Within

h

of

culture

most

of

the

cells

formed

nodules

-LRB-

n

-RRB-

PCR-amplified

mRNA

expression

was

detectable

but

not

quantifiable

after

days

in

ASCs

from

both

the

abdomen

and

hip/thigh

region

in

most

but

not

all

donors

-LRB-

n

-RRB-

As

shown

in

Fig.

cells

from

both

tissue-harvesting

sites

displayed

mRNA

Under

non-chondrogenic

conditions

no

could

be

detected

AGG

and

mRNA

expression

in

all

donors

tested

increased

over

time

AGG

being

up-regulated

-LRB-

P

-RRB-

at

day

when

compared

with

day

and

being

up-regulated

four-fold

-LRB-

P

-RRB-

after

days

-LRB-

Fig.

c

-RRB-

No

significant

differences

were

detected

in

chondrogenic

gene

expression

between

ASCs

derived

from

the

abdomen

and

ASCs

derived

from

the

hip/thigh

region

at

all

three

time

points

tested

Fig.

of

the

tissue-harvesting

site

on

the

chondrogenic

differentiation

of

cultured

ASCs

in

vitro

a

Both

abdomen

-LRB-

lane

-RRB-

and

hip/thigh

region

-LRB-

lane

-RRB-

display

mRNA

Under

nonchondrogenic

conditions

no

could

be

detected

-LRB-

lane

-RRB-

b

c

Aggrecan

-LRB-

AGG

P

-RRB-

and

collagen

-LRB-

P

-RRB-

gene

expression

respectively

was

up-regulated

after

days

-LRB-

d

-RRB-

as

measured

by

qRT-PCR

No

significant

differences

were

detected

in

chondrogenic

gene

expression

between

ASCs

derived

from

the

abdomen

and

ASCs

derived

from

the

hip/thigh

region

at

all

three

time

points

tested

d

f

Cartilaginous

matrix

expression

was

visualized

in

both

tissue-harvesting

sites

by

staining

proteoglycans

-LRB-

Alcian

blue

-RRB-

and

-LRB-

antibody

-RRB-

respectively

e

At

higher

magnification

the

ASC

nodules

resembled

cartilage-like

tissue

composed

of

spherical

cells

surrounded

by

lacunae

and

lying

in

a

proteoglycan-rich

extracellular

matrix

Alcian

blue

staining

demonstrated

proteoglycan

deposition

in

the

ASC

nodules

from

both

the

hip/thigh

region

and

abdomen

-LRB-

Fig.

-RRB-

At

higher

magnification

-LRB-

Fig.

-RRB-

the

ASC

nodules

resembled

cartilage-like

tissue

composed

of

round

cells

surrounded

by

lacunae

and

lying

in

a

proteoglycan-rich

extracellular

matrix

that

appeared

positive

for

collagen

Type

II

by

immunostaining

-LRB-

Fig.

-RRB-

Discussion

In

this

study

we

have

investigated

whether

the

yield

and

functional

characteristics

of

ASCs

are

affected

by

the

adipose

tissue-harvesting

site

i.e.

abdomen

and

hip/thigh

regions

We

have

found

a

difference

in

the

frequency

of

ASCs

between

adipose

tissue

harvested

from

the

abdomen

and

the

hip/thigh

regions

SVF

isolates

derived

from

abdominal

fat

contain

significantly

higher

frequencies

of

ASCs

When

cultured

the

growth

kinetics

and

surface-marker

expression

of

ASCs

from

both

tissue-harvesting

sites

are

similar

We

have

detected

no

differences

in

osteogenic

or

chondrogenic

differentiation

potential

between

these

cultured

ASCs

from

the

two

tissue-harvesting

sites

Adipose

tissue

is

a

highly

heterogeneous

tissue

not

only

among

individuals

but

also

when

comparing

different

fat

depots

within

one

individual

Donor-dependent

differences

have

been

demonstrated

to

exist

in

-LRB-

stem

-RRB-

cell

yield

proliferation

and

differentiation

capacity

probably

caused

by

differences

in

age

-LRB-

Hauner

and

Entenmann

van

Harmelen

et

al.

-RRB-

BMI

-LRB-

Aust

et

al.

Hauner

et

al.

Jaiswal

et

al.

van

de

Venter

et

al.

-RRB-

and

diseases

such

as

osteoarthritis

and

diabetes

-LRB-

Barry

Murphy

et

al.

Ramsay

et

al.

-RRB-

Donors

used

in

this

study

for

harvesting

adipose

tissue

were

healthy

female

donors

up

to

years

old

Some

patients

who

will

benefit

from

tissue

engineering

of

cartilage

will

be

older

or

might

suffer

from

disease

and

males

will

also

be

affected

Therefore

future

research

should

include

these

donor

types

to

determine

whether

yields

and

functional

characteristics

are

influenced

by

these

variables

In

our

donor

population

no

significant

correlation

has

been

detected

between

the

frequency

of

ASCs

and

the

age

of

the

donor

-LRB-

P

r

-RRB-

or

between

the

frequency

of

ASCs

and

BMI

-LRB-

P

r

data

not

shown

-RRB-

These

data

are

in

agreement

with

other

studies

that

have

demonstrated

no

correlation

between

BMI

or

age

and

numbers

of

ASCs

per

gram

of

adipose

tissue

-LRB-

Hauner

and

Entenmann

van

Harmelen

et

al.

-RRB-

Most

importantly

this

means

that

these

variables

can

not

be

responsible

for

the

difference

that

we

have

found

between

the

frequency

of

ASCs

and

the

tissue-harvesting

site

In

addition

to

donor-dependent

heterogeneity

intra-individual

differences

between

fat

depots

have

been

demonstrated

e.g.

with

regard

to

the

metabolic

response

of

adipose

tissue

to

various

hormonal

and

neurological

stimuli

-LRB-

Guilak

et

al.

Lacasa

et

al.

Masuzaki

et

al.

Monjo

et

al.

Rodriguez-Cuenca

et

al.

-RRB-

and

the

cellular

composition

of

the

adipose

tissue

-LRB-

Peptan

et

al.

Prunet-Marcassus

et

al.

-RRB-

In

this

study

we

have

focused

on

the

intra-individual

differences

in

the

yield

and

function

of

ASCs

from

two

fat

depots

the

abdomen

and

the

hip/thigh

region

We

have

demonstrated

that

adipose

tissue

derived

from

the

abdomen

contains

significant

higher

frequencies

of

stem

cells

compared

with

adipose

from

the

hip/thigh

region

Whereas

SVF

isolates

from

the

abdomen

in

this

study

contain

about

of

ASCs

the

frequency

of

ASCs

in

SVF

from

the

hip/thigh

region

is

only

Moreover

this

is

the

average

of

a

population

in

which

some

donors

hardly

possess

any

adipose-derived

stem

cells

-LRB-

see

spreading

ASC

frequency

hip/thigh

in

Fig.

-RRB-

Despite

this

more

than

four-fold

difference

the

frequency

of

ASCs

in

adipose

tissue

from

the

hip/thigh

region

is

still

much

higher

compared

with

the

frequency

of

MSCs

in

the

bone

marrow

compartment

which

is

as

low

as

-LRB-

Pittenger

et

al.

-RRB-

thereby

making

the

hip/thigh

region

still

a

much

more

attractive

stem-cell

source

for

tissue

engineering

therapies

What

are

the

implications

of

these

stem-cell

frequencies

for

clinical

practice

We

have

shown

that

SVF

cells

can

be

harvested

from

g

adipose

tissue

an

amount

that

can

easily

be

obtained

from

a

patient

-LRB-

Aust

et

al.

De

Ugarte

et

al.

Oedayrajsingh-Varma

et

al.

Zuk

et

al.

-RRB-

With

an

ASC

frequency

of

the

SVF

isolates

contain

between

stem

cells

which

is

an

amount

that

appears

to

be

sufficient

for

cell-based

therapies

as

compared

with

the

amount

of

cells

used

by

others

-LRB-

Erickson

et

al.

Fan

et

al.

Williams

et

al.

Zheng

et

al.

-RRB-

This

implies

that

time-consuming

culturing

and

expanding

steps

of

the

stem

cells

can

be

avoided

In

comparison

a

bone

marrow

transplant

of

ml

contains

approximately

nucleated

cells

-LRB-

Zuk

et

al.

-RRB-

of

which

only

-LRB-

cells

-RRB-

are

stem

cells

-LRB-

Pittenger

et

al.

-RRB-

To

determine

the

frequency

of

CFU

capable

of

differentiating

into

the

osteogenic

lineage

a

CFU-ALP

assay

has

been

performed

on

cells

from

the

abdomen

and

hip/thigh

region

The

frequencies

of

CFU-ALP

-LRB-

-RRB-

are

almost

comparable

with

those

of

the

CFU-F

-LRB-

Figs.

-RRB-

This

is

higher

than

the

value

that

Mitchell

et

al.

-LRB-

-RRB-

have

found

in

their

clonogenic

assay

of

freshly

isolated

stromal

cells

-LRB-

-RRB-

However

they

have

used

a

different

assay

method

and

shorter

incubation

period

and

their

frequency

increases

to

the

same

level

-LRB-

-RRB-

after

progressive

passaging

-LRB-

Mitchell

et

al.

-RRB-

SVF

cells

have

been

cultured

up

to

passage

to

obtain

a

homogeneous

population

of

ASCs

as

starting

material

for

differentiation

studies

toward

the

chondrogenic

lineage

When

comparing

the

growth

kinetics

of

these

cultured

ASCs

from

the

abdomen

and

hip/thigh

region

the

cell

doubling

time

appears

to

be

similar

being

approximately

days

This

is

in

accordance

with

the

findings

of

others

who

have

compared

the

replication

rate

of

adipocyte

precursor

cells

from

various

tissue-harvesting

sites

-LRB-

Hauner

et

al.

Pettersson

et

al.

Roncari

et

al.

Zuk

et

al.

-RRB-

On

the

other

hand

when

comparing

SVF

cells

from

omental

and

subcutaneous

fat

van

Harmelen

et

al.

-LRB-

-RRB-

have

found

a

difference

in

cell

proliferation

rate

however

this

may

be

explained

by

differences

in

methodological

approach

since

they

use

stromal

cells

instead

of

cultured

ASCs

As

we

have

shown

that

different

fat

depots

contain

different

numbers

of

stem

cells

these

differences

in

proliferation

rate

may

be

caused

by

differences

in

initial

stem-cell

numbers

when

using

SVF

cells

This

is

reflected

in

the

finding

that

in

our

study

SVF

cells

derived

from

abdominal

fat

reach

confluency

within

days

whereas

SVF

cells

derived

from

adipose

tissue

of

the

hip/thigh

region

take

more

than

days

to

reach

confluency

when

seeded

in

the

same

density

-LRB-

data

not

shown

-RRB-

In

addition

to

the

determination

of

growth

kinetics

we

have

phenotypically

characterized

the

cultured

ASCs

The

surface-marker

expression

profile

is

in

accordance

with

those

found

by

others

-LRB-

Mitchell

et

al.

Schaffler

and

Buchler

Varma

et

al.

-RRB-

making

both

tissue

sites

fully

comparable

with

each

other

-LRB-

Tables

-RRB-

The

homogeneous

ASC

population

has

been

induced

to

the

osteogenic

and

chondrogenic

lineages

Having

determined

the

osteogenic

differentiation

capacity

of

ASCs

we

have

shown

significant

up-regulation

of

osteogenic

gene

expression

ALP

activity

and

matrix

mineralization

Interestingly

although

no

significant

difference

has

been

detected

in

ALP

activity

between

ASCs

from

the

abdomen

and

hip/thigh

regions

ASCs

from

the

hip/thigh

region

tend

to

show

higher

values

of

ALP

activity

after

induction

This

might

be

related

to

the

underlying

bone

tissue

thereby

implying

that

the

ASCs

of

the

hip/thigh

region

are

less

multipotent

and

more

committed

to

the

osteogenic

lineage

The

chondrogenic

differentiation

capacity

of

the

ASCs

has

been

demonstrated

by

the

up-regulation

of

AGG

and

gene

expression

and

the

production

of

matrix

proteins

No

difference

has

been

detected

in

the

chondrogenic

differentiation

potential

between

ASCs

from

the

abdomen

and

hip/thigh

regions

However

in

the

PCR

studies

we

have

not

succeeded

in

quantifying

and

mRNA

expression

for

any

of

the

donors

tested

Others

have

obviously

faced

the

same

difficulty

when

trying

to

measure

the

up-regulation

of

genes

in

MSCs

-LRB-

Huang

et

al.

Winter

et

al.

Zuk

et

al.

-RRB-

Although

intending

to

measure

collagen

Type

X

mRNA

expression

as

a

hypertrophic

and

therefore

late

marker

of

chondrogenesis

we

have

noticed

that

this

mRNA

is

expressed

earlier

than

collagen

Type

II

mRNA

This

is

surprising

as

we

would

expect

stem

cells

to

have

to

differentiate

into

chondrocytes

before

they

can

become

hypertrophic

However

this

unexpected

hierarchy

of

chondrogenic

gene

expression

has

also

been

found

by

Mwale

et

al.

-LRB-

-RRB-

Moreover

both

Mwale

et

al.

-LRB-

-RRB-

and

we

have

found

AGG

to

be

constitutively

expressed

in

MSCs

Because

of

this

constitutive

expression

of

AGG

and

the

early

up-regulation

of

Mwale

et

al.

-LRB-

-RRB-

warn

against

using

these

molecules

as

markers

for

chondrogenesis

and

chondrocytic

hypertrophy

We

think

that

these

genes

can

nevertheless

be

used

as

markers

for

differentiation

into

the

chondrogenic

lineage

albeit

being

exclusively

shown

as

the

quantitative

up-regulation

of

gene

expression

and

always

in

combination

with

other

chondrogenic

markers

-LRB-

an

awareness

of

the

possible

difference

in

the

function

of

in

chondrogenesis

in

adult

stem

cells

when

compared

with

embryonic

stem

cells

is

also

necessary

-RRB-

Our

lack

of

detection

of

any

significant

differences

in

the

osteogenic

and

chondrogenic

differentiation

potential

when

comparing

ASCs

from

the

two

tissue-harvesting

sites

seems

to

be

in

contrast

with

studies

of

Hauner

and

Entenmann

-RRB-

who

have

found

differences

in

the

adipogenic

differentiation

potential

between

SVF

cells

from

abdominal

and

femoral

adipose

tissue

However

since

Hauner

and

Entenmann

-RRB-

have

used

fresh

SVF

cells

instead

of

culture-passaged

ASCs

the

variation

in

differentiation

potential

might

be

attributable

to

differences

in

numbers

of

ASCs

in

the

SVF

isolates

of

the

two

regions

as

we

have

shown

in

this

study

Other

factors

responsible

for

the

variation

in

differentiation

potential

can

be

ascribed

to

other

specific

histological

characteristics

of

the

adipose

tissue

at

the

anatomical

site

such

as

vascularity

and

amount

of

fibrous

tissue

-LRB-

Lennon

et

al.

Peptan

et

al.

Pittenger

et

al.

-RRB-

and

to

differences

in

the

regulation

of

gene

expression

-LRB-

Djian

et

al.

Peptan

et

al.

Pittenger

et

al.

-RRB-

We

therefore

conclude

that

the

yield

of

ASCs

is

dependent

on

the

tissue-harvesting

site

In

planning

the

optimal

one-stage

procedure

for

the

regeneration

of

cartilage

tissue

factors

that

can

positively

influence

the

outcome

of

the

operation

must

be

taken

into

account

In

view

of

this

the

abdomen

seems

to

be

preferable

to

the

hip/thigh

region

for

harvesting

ASCs

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

chondrogenic differentiation
osteogenic differentiation
human

adipose-tissue-derived stem cells
colony-forming unit assay
stem-cell yield
tissue-harvesting site