Electrical Stimulation Optimization in Bioreactors for Tissue ...

Electrical Stimulation Optimization in Bioreactors for Tissue Engineering Applications

Paula Pascoal-Faria1,2,a*, Pedro Castelo Ferreira1,b, Abhishek Datta3,4,c, Sandra Amado1, Carla Moura1 and Nuno Alves1,5,d

1Centre for Rapid and Sustainable Product Development, Polytechnic Institute of Leiria, Rua de Portugal, Marinha Grande, 2430-028, Portugal

2Department of Mathematics, School of Technology and Management, Polytechnic Institute of Leiria, Rua General Norton de Matos, Apartado 4133, 2411-901 Leiria, Portugal

3Soterix Medical, Inc. 4City College of New York, NY

5Department of Mechanical Engineering, School of Technology and Management, Polytechnic Institute of Leiria, Rua General Norton de Matos, Apartado 4133, 2411-901 Leiria, Portugal

[email protected] (corresponding author)

Keywords: tissue engineering, bioreactor, scaffold, electrical stimulation, electro-mechanical strain stimulation, optimization.

Abstract. We review here the current research status on bioreactors for tissue engineering with cell electrical stimulation. Depending on the cell types, electrical stimulation has distinct objectives: 1) being employed both to mimic and enhance endogenous electricity measured in the natural regeneration of living organisms and 2) to mimic strain working conditions for contractible tissues (for instance muscle and cardiac tissues). Understanding the distinct parameters involved in electrical stimulation is crucial to optimize its application. The results presented in the literature and reviewed here reveal that the application of electrical stimulation can be essential for tissue engineering applications.

Introduction Tissue Engineering is today seen as the ultimate solution in the treatment and replacement of lost or damaged tissues. Understanding the mechanisms underlying biological tissue differentiation, regeneration and growth is a timely goal. In particular, the proper identification of the relevant parameters involved and the respective possible optimization strategies for each tissue type is a common objective in this field of research. It has been established that the following conditions should generally be satisfied:

i) an adequate mechanical laminar shear stress of the culture medium fluid that not only ensures nutrient and oxygen delivery but removal of cell activity byproducts as well [1,2];

ii) an adequate low voltage and low intensity electrical stimulus that both mimics endogenous real organism conditions [3-5] and selectively enhances or inhibits cell regeneration. This type of stimulus evidently plays an essential role in artificial control of cellular differentiation [6], replication [7], growth [7, 8], maturation [9], adhesion and orientation [10].

iii) for the particular case of muscular and cardiac tissues, electro-mechanical strain stimulation mimicking the biological strain working conditions (contraction and elongation). It should be noted that the electric impulses considered in this study should not be confused with the endogenous regenerated electric fields referred in point (i), instead they refer to the functional activation of muscle and cardiac tissues of biological organisms. The scaffolds for these tissue types are commonly denominated constructs (when requiring a cell culture for construction of the scaffold) being itself constituted by biological tissues. Typically the constructs mimic the elasticity of the final biological

Applied Mechanics and Materials Submitted: 2017-11-30ISSN: 1662-7482, Vol. 890, pp 314-323 Revised: 2018-10-12doi:10.4028/www.scientific.net/AMM.890.314 Accepted: 2018-10-15© 2019 The Author(s). Published by Trans Tech Publications Ltd, Switzerland. Online: 2019-04-09

This article is an open access article under the terms and conditions of the Creative Commons Attribution (CC BY) license(https://creativecommons.org/licenses/by/4.0)

https://doi.org/10.4028/www.scientific.net/AMM.890.314

tissue [11-14]. Mechanical strain alone has also been considered for cartilaginous, blood vessel and ligament tissues;

iv) the existence of a geometric adequate substrate that mimics the final desired shape and material properties. This is commonly solved by cultivating the tissue on a biodegradable scaffold which both in vitro and in vivo shapes the tissue to the intended biological shape [15-17]. Also the geometry of these scaffolds allow for medium perfusion ensuring adequate volumetric nutrient and oxygen delivery [18-20].

Regarding materials, numerous research have proposed using both natural and synthetic materials

for the design and construction of tissues, including naturally occurring polymers, synthetic bioresorbable polymers or ceramics and biodegradable materials. Examples using polycaprolactone (PCL) matrix as reference, PCL reinforced with cellulose nanofibers (CNF) and PCL reinforced with CNF and hydroxyapatite nanoparticles have been developed and used by Morouço and collaborators (see [16]). Moreover, advances have been made in exploring the ideal geometries of scaffolds [21], mostly due to 3D printing methods that have been developed over the years such as fused deposition modelling, selective laser melting or selective laser sintering techniques [22, 23]. More recently, a 4D bioprinting technology that fabricates dynamic structures that improves cartilage and bone regeneration has also been proposed [22, 24].

Different types of stimulation have been used to benefit tissue regeneration applications over the years. Mechanical, electrical and magnetic stimulation have all been explored with promising results. However, numerical studies aiming to optimize the different stimulation parameters are critically needed focusing on the vast applications of tissue engineering. Early modelling studies performed by [25] for mechanical stimulation, by [26-28] and by [29] for electrical and magnetic stimulation have proven to be not only crucial for understanding the neurophysiological mechanics involved but have also helped to optimize the application of stimulation in different type of cells and models.

With the objective of optimizing in vitro tissue engineering for eventual in vivo chirurgical implantation in such a way that it is both economically viable and scalable to industrial levels, it is highly desirable to develop bioreactors that incorporate all the necessary conditions for tissue engineering. This review will therefore mainly focus on the research status of such bioreactors which already contemplate aforementioned culture conditions (i), (ii), (iii) and (iv); specifically we will address studies that deal with the application of electrical stimulation on a scaffold placed inside a bioreactor. Furthermore, this review aims to provide insight on the different parameters involved and suggest what future directions to take.

Methods Information sources and literature search. Studies in English quantitatively assessing the

application of electrical stimulation on a scaffold placed inside a bioreactor for tissue regeneration were identified by searching the Science Direct and PubMed electronic databases on November 12, 2017. The following search string was used to retrieve relevant publications: “("bioreactor" OR "dynamic cell culture") AND ("scaffold" OR "construct") AND ("regenerative medicine" OR "tissue engineering" OR "tissue regeneration") AND (stimuli OR stimulation) AND ("electric" OR "electrical")”. Selection and exclusion criteria. The publications retrieved by using the aforementioned search string were assessed for eligibility. Notably, for an article to be included in the qualitative analysis performed in this review, the following criteria had to be met: (1) published in English, (2) original research reports – e.g., reviews were excluded and (3) used electrical stimulation applied on cells placed on scaffolds inside bioreactors corresponding to cell culture conditions (ii) or (iii).

Additionally, the following exclusion criteria were considered: (1) case studies, (2) different types of stimulation that do not include electrical stimulation and (3) when cell culture was not directly studied. Data extraction. Relevant data were extracted systematically from the selected articles and recorded on an extraction sheet (see Table 1). This procedure was pilot-tested independently by all the authors

Applied Mechanics and Materials Vol. 890 315

to ensure that data extraction occurred consistently. The extraction sheet included several topics, notably information regarding cell preparation, characteristics of the scaffolds and bioreactors and the different parameters involved in stimulation. Identification of articles. After all data were collected, all the authors were involved in the preliminary screening of the papers, which consisted of assessing their specific titles and abstracts for eligibility. Each article was assessed by two authors. Whenever disagreement occurred, the paper was not excluded without both authors reaching an agreement on the reason for exclusion. The full-text version of the remaining articles was then assessed. This second stage eligibility screening was performed by the same authors and used the same method except for the disagreements, which were solved by joint discussions of all the authors.

Results Study Selection. The search strategy described above resulted in 66 results from Science Direct

and 14 from PubMed. After discarding duplicate records, reviews and book chapters, 21 articles were assessed for eligibility on the basis of their titles, abstracts and keywords, with 9 being excluded from the review (see Figure 1). Out of the remaining 12, which had their full text analyzed; an additional 2 papers were also excluded due to non-inclusion of either culture conditions (ii) and (iii). The process was repeated by analyzing the full-text of the remaining 10 articles [14, 30-36, [37,38]. The characteristics of these articles most relevant to this review were collected on an extraction sheet as described above (see Table 1).

Figure 1. Flow chart diagram of the study selection process.

Characteristics of the studies. From the selected studies, [30, 37] correspond to tissue cultures applying low electrical current stimulation (ii) while the studies [14, 31-36, 38] correspond to electro-mechanical strain stimulation (iii).

316 Direct Digital Manufacturing and Polymers

Appl

icatio

nGo

alRe

sults

Adva

ntag

es o

f Ele

ctric

St

imul

atio

nCo

nclu

sions

Refe

renc

esTy

pe o

f Cel

lsCu

lture

Cont

rols

Geom

met

ryPr

oduc

tion

Char

acte

ristic

sM

ater

ials

Char

acte

ristic

sGe

omm

etry

Type

Elec

trod

es[3

0]

Bone

tiss

ue g

raft

(in

vitr

o to

in v

ivo)

.Hu

man

mes

ench

ymal

st

rom

al ce

lls (h

MSC

s).

- Bi

phas

ic e

lect

ric c

urre

nt

(BEC

) can

be

sele

cted

the

ampl

itude

from

2uA

to 3

00uA

in

2-u

A st

eps,

the

dura

tion

and

the

frequ

ency

from

16u

s to

51

2us

in 1

6-us

ste

ps a

nd fr

om

32Hz

to 1

kHz

in 3

2-Hz

ste

ps.

Cylin

drica

l. -

hMSC

s see

ded

on th

e co

llage

n sp

onge

th

at se

rves

as s

caffo

ld to

form

a b

one

tissu

e gr

aft.

Colla

gen.

Impl

anta

ble

bior

eact

or ch

ambe

r with

el

ectr

ode,

cham

ber a

nd st

imul

atio

n ch

ip.

Cylin

drica

l Tef

lon

body

co

ntai

ning

a fl

exib

le

poly

imid

e el

ectr

ode

and

impl

anta

ble

stim

ulat

or.

Biph

asic

elec

tric

curr

ent

stim

ulat

ion.

Bioc

ompa

tible

, fle

xible

po

lyim

ide.

Appl

ied

bio-

favo

rabl

e BE

C to

th

e 3D

in-v

ivo b

iore

acto

r to

enha

nce

cell p

rolif

erat

ion.

In v

itro

and

In v

ivo c

ultu

re o

f hM

SCs

show

ed

19%

and

22%

incr

ease

in c

ell p

rolif

erat

ion

at

stim

ulat

ed g

roup

s, c

ompa

red

to u

nstim

ulat

ed

cont

rol.

Supp

ress

es th

e pH

el

evat

ion

and

prod

uctio

n of

fa

radi

c pr

oduc

ts b

y in

hibi

ting

char

ge

accu

mul

atio

n.

Achi

eved

enh

aced

in v

ivo c

ell

prol

ifera

tion.

[31]

En

gine

ered

card

iac

tissu

e (in

vitr

o).

Card

iac c

ells

from

1–

2day

-old

neo

nata

l Sp

ragu

e–Da

wle

y ra

ts.

High

-glu

cose

Dul

becc

o’s

mod

ified

Eag

le’s

med

ium

(D

MEM

) sup

plem

ente

d w

ith 1

0% fe

tal b

ovin

e se

rum

(FBS

).

Elec

tric

stim

ulat

iono

f: 1.

3 ±

0.3,

2.

5 ±

0.5,

2.7

± 0

.4, 2

.9 ±

0.4

an

d 4.

1 ±

0.7

V/cm

.

Disk

s with

por

osity

of 7

6%.

Poro

us p

oly(

glyc

erol

seba

cate

) (PG

S)

scaf

fold

s wer

e fa

brica

ted

by m

eans

of

a sa

lt-le

achi

ng te

chni

que.

Poro

us, c

hann

elle

d el

asto

mer

sc

affo

lds.

Poro

us p

oly(

glyc

erol

se

baca

te) (

PGS)

.Si

mul

tane

ously

supp

ort n

utrie

nt

perfu

sion,

ele

ctric

al st

imul

atio

n an

d un

con-

stra

ined

(i.e

. not

isom

etric

) tis

sue

cont

ract

ion

Cylin

drica

lEl

ectr

ical s

timul

atio

n: p

ulse

s (3

V/cm

, 3Hz

, mon

o-ph

asic

squa

re w

ave)

.

Carb

on ro

d el

ectr

odes

.C

reat

ed a

n im

plan

tabl

e el

ectri

cal b

iore

acto

r sys

tem

co

mbi

ning

in-v

ivo b

iore

acto

r an

d el

ectri

cal s

timul

atio

n sy

stem

an

d ex

ecut

ed p

relim

inar

y st

udy

of a

bout

cel

l pro

lifer

atio

n at

in-

vitro

and

in-v

ivo

Cel

ls we

re m

ost c

lust

ered

and

mos

t abu

ndan

t in

the

stim

ulat

ed g

roup

. Add

ition

ally

, bot

h th

e pe

rfusi

on a

nd e

lect

rical

stim

ulat

ion

indu

ced

cell e

long

atio

n an

d th

e or

gani

zatio

n of

inte

rnal

ce

ll stru

ctur

es, w

here

as in

non

-stim

ulat

ed

grou

ps th

e ce

lls w

ere

mos

tly ro

unde

d an

d ha

d le

ss o

rgan

ized

inte

rnal

stru

ctur

e. A

fter o

nly

8day

s of

cul

ture

, with

sim

ulta

neou

s m

ediu

m

perfu

sion

and

ele

c- tr

ical

stim

ulat

ion,

the

cells

de

velo

ped

a re

mar

kabl

e le

vel o

f fun

ctio

nality

co

mpa

red

to a

ny c

ontro

l gro

up.

Supp

ress

es th

e pH

el

evat

ion

and

prod

uctio

n of

fa

radi

c pr

oduc

ts b

y in

hibi

ting

char

ge

accu

mul

atio

n.

Sim

ulta

neou

s pe

rfusi

on a

nd

stim

ulat

ion

enha

nce

the

deve

lopm

ent o

f eng

inee

red

card

iac

tissu

e, e

spec

ially

in

term

s of

con

tract

ion

ampl

itude

. Th

ere

appe

ars

to b

e a

syne

rgis

tic e

ffect

bet

ween

thes

e tw

o co

nditi

onin

g st

imul

i, le

adin

g to

enh

ance

d or

gani

zatio

n an

d fu

nctio

nality

of e

ngin

eere

d ca

rdia

c tis

sue.

[32]

Impl

anta

ble

cont

ract

ile

card

iac t

issue

(in

vivo

).2-

day

old

neon

atal

Sp

ragu

e D

awle

y ra

t he

arts

(c

ardi

omyo

cyte

s/fib

rob

last

s an

d ca

rdio

myo

cyte

s).

High

-glu

cose

DM

EM 4

.5

g/L

gluc

ose,

4m

M L

-gl

utam

ine,

10%

Fet

al

Bovin

e Se

rum

, 10m

M

HEPE

S Bu

ffer,

and

100

units

/mL

peni

cillin

and

100

μg

/mL

stre

ptom

ycin

.

For t

he st

imul

ated

gro

ups,

elec

trica

l stim

ulat

ion

was

ap

plie

d as

bip

hasic

squa

re

pulse

s at 2

.5V/

cm, 5

V/cm

and

8V

/cm

, 1Hz

and

2Hz

and

1m

s (p

er p

hase

).

Para

llelip

iped

ic 1

cm le

ngth

wi

th m

icro

groo

ves

(25

x 50

x

100

µm).

Az-c

hito

san

was

synt

hesi

zed

by

cham

ical

ly m

odify

ing

chito

san

with

az

idob

enzo

ic a

cid.

4-a

zido

benz

oid

acid

, 1-e

thyl

-3-(

3-di

men

thyl

-am

inop

ropy

) car

bodi

imid

e hy

droc

hlor

ide

and

N,N,

N',N

'-Te

tram

ethy

leth

ylen

e-di

amin

e we

re

mixe

d in

to a

sol

utio

n in

a d

rop-

wise

m

anne

r. Th

en re

mov

ed th

e un

reac

ted

ABA.

With

micr

ogro

oves

of 1

0 µm

, 20

µm

and

100

µm in

wid

th p

erpe

ndicu

lar t

o el

ectr

odes

.

Colla

gen-

chito

san

hydr

ogel

s.Pe

tri d

ish w

ith sc

affo

ld a

rray

in

side.

Elec

trica

l stim

ulat

ion

(squ

are

pulse

s at

1 H

z an

d 2

Hz a

nd

2.5

V/cm

, 5 V

/cm

and

8

V/cm

).

Two

para

llel c

arbo

n el

ectr

odes

(1/4

in d

iam

eter

; La

dd R

ease

rch

Indu

strie

s, W

illist

on, V

T, U

SA).

Stud

y th

e be

atin

g ca

rdia

c tis

sue

on a

bio

degr

adab

el, c

olla

gen-

chito

san

hydr

ogel

with

m

icro

groo

ves,

with

the

appl

icat

ion

of e

lect

rical

fied

l st

imul

atio

n du

ring

cultiv

atio

n

The

succ

ess

rate

of a

chie

ving

beat

ing

cons

truct

s wa

s hi

gher

with

the

appl

icat

ion

of

elec

trica

l stim

ulat

ion

and

the

form

atio

n of

de

nse

cont

ract

ile c

ardi

ac o

rgan

oids

was

ob

serv

ed in

gro

ups

with

bot

h bi

omim

etic

cue

s.

On

hydr

ogel

s e

lect

rical

fiel

d st

imul

atio

n fu

rther

incr

ease

d ce

ll den

sity

and

enh

ance

d tis

sue

mor

phol

ogy.

Bes

t res

ults

obt

aine

d fo

r 1H

z at

2.5

V/cm

.

Forc

es c

ell a

lignm

ent.

The

biod

egra

dabi

lity o

f the

hy

drog

el s

ubst

rate

allo

ws fo

r the

ra

pid

trans

latio

n of

the

engi

neer

ed, o

rient

ed c

ardi

ac

tissu

e to

clin

ical

app

licat

ions

. Be

st re

sults

with

ele

ctric

st

imul

aito

n wi

th 1

Hz a

t 2.5

V/cm

.

[33]

Car

diac

tiss

ue

engi

neer

ing

(in v

itro

to

in v

ivo).

2-da

y ol

d Sp

ragu

e D

awle

y ra

ts c

ardi

ac

cells

.

high

-glu

cose

DM

EM +

10

% F

BS, 1

% H

EPES

, an

d 1%

pen

icilli

n (1

0,00

0 U/

ml)/

stre

ptom

ycin

(1

0,00

0 μg

/ml).

Mon

opha

sic

pulse

s of

2 m

s du

ratio

n, 3

Hz,

3 V

/cm

.Pa

ralle

lipip

edic

spo

nges

(8

mm

× 1

0 m

m ×

1.5

mm

) wi

th m

icro

chan

nels

for

perfu

sion

stim

ulat

ion.

By s

eedi

ng c

olla

gen

spon

ges

with

ce

ll pop

ulat

ions

. The

col

lage

n sh

eet

was

fold

ed a

roun

d an

aut

ocla

ved

bund

le o

f 14

hollo

w fib

ers,

m

anuf

actu

red

from

rege

nera

ted

cellu

lose

, and

sec

ured

usi

ng a

si

ngle

stit

ch s

utur

e.

Car

diac

pat

ch s

pong

e.C

olla

gen

shee

t.Sc

affo

ld b

etwe

en e

lect

rode

s an

d pe

rfusi

on tu

bing

. Allo

ws b

oth

shea

r st

ress

stim

ulat

ion

and

perfu

sion

em

ploy

ing

hollo

w m

icro

dial

ysis

ce

llulo

se tu

bing

that

mim

ics

capi

llarie

s wi

th in

ters

titia

l med

ium

flo

w th

at p

rote

ct c

ells

from

ex

cess

ive s

hear

stre

ss.

Para

llelip

iped

ic.El

ectri

cal s

timul

atio

n, s

hear

st

ress

stim

ulat

ion

and

capi

llar

perfu

sion

.

Car

bon

rod

elec

trode

.D

evel

oped

a p

orta

ble

devic

e th

at

prov

ides

the

perfu

sion

and

el

ectri

cal s

timul

atio

n ne

cess

ary

to e

ngin

eer c

ardi

ac ti

ssue

in

vitro

, and

to tr

ansp

ort i

t to

the

site

whe

re it

will

be im

plan

tate

d.

Appl

icat

ion

of p

erfu

sion

mai

ntai

ned

mor

e un

iform

, com

pact

tiss

ue, a

s co

mpa

red

to

othe

r gro

ups.

Also

inte

rest

ing

to n

ote

is th

at

elec

trica

lly s

timul

ated

gro

ups

mai

ntai

ned

mor

e ce

lls in

the

mos

t sup

erfic

ial p

ortio

n of

the

scaf

fold

, sug

gest

ing

ther

e m

ay b

e a

syne

rgis

tic e

ffect

of t

he a

pplic

atio

n of

el

ectri

cal s

timul

atio

n an

d ce

ll dis

tribu

tion.

Indi

ces

mor

e ce

lls in

the

mos

t sup

erfic

ial p

ortio

n of

th

e sc

affo

ld.

Furth

er s

tudi

es w

ould

be

need

ed

to v

alid

ate

whet

her e

lect

rical

st

imul

atio

n af

fect

ed c

ell

atta

chm

ent.

[34]

Card

iac t

issue

en

gine

erin

g (in

vitr

o).

Car

diac

cel

ls fro

m

Lewi

s ra

t (m

esen

chym

al s

tem

ce

lls).

L-D

MEM

, 10%

FBS

, ca

rdia

c m

yocy

te g

rowt

h su

pple

men

t (Sc

ienC

ell

Rese

arch

Lab

orat

orie

s),

100

U/m

l pen

icilli

n an

d 10

0μg/

ml s

trept

omyc

in.

20%

mec

hani

cal s

train

and

el

ectri

cal s

timul

atio

n of

5 V

, 1

Hz.

Squa

re s

lices

(20

mm

× 2

0 m

m ×

~3

mm

).Fo

rty fr

esh

pig

hear

ts w

ere

harv

este

d fro

m ~

6-m

onth

old

pig

s an

d fro

m th

e m

iddl

e re

gion

of t

he

ante

rior l

eft v

entri

cula

r wal

l wer

e tri

med

squ

are-

shap

ed m

yoca

rdiu

m

sam

ples

(20

× 20

× ～

3mm

). W

ere

mou

nted

in a

fram

e-pi

n su

ppor

t sy

stem

to p

reve

nt c

ontra

ctio

n of

tis

sue

mac

roge

omet

ry a

nd c

olla

pse

of in

tern

al c

ardi

omyo

cyte

s la

cuna

e.

Then

dec

ellu

lariz

ed in

a ro

tatin

g

3D p

orci

ne m

yoca

rdia

l sca

ffold

s pr

eser

ving

mec

hani

cal a

niso

tropy

an

d va

scul

atur

e te

mpl

ates

.

Fres

h pi

g he

arts

and

pl

astic

fram

es.

Allo

ws fo

r bot

h el

ectri

cal a

nd

mec

hani

cal s

train

stim

ulat

ion.

Squa

re b

ox o

f acr

ylic

or

poly

sulfo

ne.

Elec

tro-M

echa

nica

l: m

onop

hasi

c pu

lses

of 2

ms

dura

tion,

3 H

z, 3

V/c

m.

Elec

trode

s we

re m

ade

from

Te

flonc

oate

d si

lver w

ire o

f 75

μm

dia

met

er (A

-M

Syst

ems,

Car

lsbor

g, W

A).

Show

effe

ctive

ness

and

ef

ficie

ncy

of th

e co

ordi

nate

d si

mul

atio

ns in

pro

mot

ing

tiss

ue re

mod

elin

g

Biax

ial m

echa

nica

l tes

ting

dem

onst

rate

d th

at

posi

tive

tissu

e re

mod

elin

g to

ok p

lace

afte

r 2-

day

bior

eact

or c

ondi

tioni

ng (2

0% s

train

+ 5

V,

1 H

z); p

assi

ve m

echa

nica

l pro

perti

es o

f the

2-

day

and

4-da

y tis

sue

cons

truct

s we

re

com

para

ble

to th

e tis

sue

cons

truct

s pr

oduc

ed

by s

tirrin

g re

seed

ing

follo

wed

by 2

-wee

k st

atic

cu

lture

, im

plyi

ng th

e ef

fect

ivene

ss a

nd

effic

ienc

y of

the

coor

dina

ted

sim

ulat

ions

in

prom

otin

g tis

sue

rem

odel

ing.

Enha

nces

tiss

ue

prol

ifera

tion.

In s

hort,

the

syne

rgis

tic

stim

ulat

ions

mig

ht b

e be

nefic

ial

not o

nly

for t

he q

uality

of

card

iac

cons

truct

dev

elop

men

t, bu

t also

for p

atie

nts

by re

duci

ng

the

waiti

ng ti

me

in fu

ture

clin

ical

sc

enar

ios.

[35,

39]

Card

iac n

iche:

m

yoca

rdiu

m,

card

iom

yocy

tes,

fibro

blas

ts, e

ndot

helia

l ce

lls (i

n vi

tro)

.

Hum

an a

tria

l fib

robl

ast c

ells

(orig

inal

exp

erim

ent

on a

dult

rat

vent

ricul

ar m

yocy

tes)

.

Dul

becc

o’s

mod

ified

Eag

le

med

ium

[DM

EM];

Gib

co 2

2320

+ 1

0% fe

tal b

ovin

e se

rum

[FBS

] + 1

%

pen.

/stre

p.

10%

cycli

c str

ain

at 1

Hz.

Elec

tric

Stim

ulat

ion

of 7

V/cm

, 4m

s bip

olar

pul

ses a

t 1 o

r 2Hz

. Fl

uid

perfu

sion

of 0

.6m

L/m

in.

Para

llelip

iped

ic s

lices

.18

In b

rief,

post

partu

m h

uman

um

bilic

al c

ord

vein

s we

re w

ashe

d wi

th th

e M

-199

med

ium

, fille

d wi

th

the

colla

gena

se s

olut

ion

and

incu

bate

d. T

hen

solu

tion

was

cent

rifug

ed, r

esus

pend

ed, c

ultu

red

and

seed

ed o

n PD

MS

subs

trate

s fo

r cy

clic

stre

tch

treat

men

t. To

faci

litate

ce

ll atta

chm

ent,

PDM

S su

bstra

tes

were

coa

ted

with

fibr

onec

tin a

nd

type

I co

llage

n m

ixtur

e be

fore

se

edin

g.

Con

stan

t stif

fnes

s an

d re

sist

ivity

.Hu

man

um

bilic

al v

ein

endo

thel

ial c

ells

(HUV

EC),

Prom

ocel

l med

ium

(P

rom

ocel

l), P

DMS

subs

trat

es, f

ibro

nect

in a

nd

type

I co

llage

n.

Allo

ws fo

r bot

h el

ectri

cal,

mec

hani

cal s

train

and

she

ar s

tress

st

imul

atio

n.

Para

llelip

iped

ic.El

ectro

-Mec

hani

cal:

cycl

ic

mec

hani

cal a

nd e

lect

rical

st

rain

and

she

ar s

tress

.

Carb

on ro

ds (0

.5m

m

diam

eter

, 5 cm

long

; C

arbo

ntea

m).

Reac

tor t

o re

cons

truc

t the

ph

ysica

l env

ironm

ent f

ound

in

the

card

iac n

iche

for r

outin

ece

ll cu

lture

use

Cells

subj

ecte

d to

10%

stre

tch

at 1

Hz f

or 2

4 h

exhi

bite

d la

rger

are

as o

f org

anize

d, in

terw

oven

fib

rous

sign

als o

f α-S

MA

(79.

3%) t

han

in co

ntro

l gr

oup

(19.

5%).

The

myo

fibro

blas

ts p

opul

atio

n tr

iple

d af

ter s

tret

ch tr

eatm

ent.

Tran

swel

l m

igra

tion

assa

y re

veal

ed o

ver 1

0% o

f the

cells

in

cont

rol g

roup

mig

rate

d to

the

tran

s sid

e of

th

e po

rous

mem

bran

e, b

ut ce

ll m

otili

ty is

sig

nific

antly

redu

ced

to le

ss th

an 5

% in

st

retc

hed

cells

.

Redu

ces

unde

sire

d ce

ll m

otilit

y in

side

the

reac

tor,

corr

ectly

alig

ns c

ells

and

incr

ease

s α-

SMA.

-

[36]

C

ardi

ac ti

ssue

bi

orea

ctor

(in

vitro

).C

ardi

ac c

ells

from

2-

to 3

-day

-old

neo

nata

l Sp

ragu

e–D

awle

y ra

ts

(Car

diom

yocy

tes)

.

Dul

becc

o’s

mod

ified

Ea

gle’

s m

ediu

m, 1

0%

hors

e se

rum

, 2%

feta

l bo

vine

seru

m, 1

%

peni

cillin

–stre

ptom

ycin

, 2

mM

e-a

min

ocap

roic

aci

d,

2 m

g/m

L in

sulin

, and

as

corb

ic a

cid,

50 m

g/m

L.

Mec

hani

cal s

timul

atio

n of

5%

st

rain

with

a 5

0% d

uty

cycl

e at

1

Hz. S

timul

i for

ele

ctric

al

stim

ulat

ion

were

bip

hasi

c re

ctan

gula

r pul

ses,

with

a

dura

tion

of 1

ms

with

1Hz

and

a

supr

a-th

resh

old

volta

ge o

f 3

V/cm

. Syn

chro

nize

d or

el

ectri

cal s

timul

atio

n de

laye

d by

0.0

1 s.

Cilin

dric

al.

Hear

ts w

ere

isol

ated

, min

ced

to

smal

l pie

ces,

and

then

dig

este

d wi

th

colla

gena

se a

t. Fr

eshl

y is

olat

ed

card

iac

cells

wer

e m

ixed

into

a

fibrin

form

ing

solu

tion

and

inje

ct

mol

ded

into

tubu

lar m

olds

whi

ch

were

incu

bate

d to

allo

w th

e fib

rin to

po

lym

eriz

e. C

onst

ruct

s we

re

cultu

red

befo

re tr

ansf

er in

to th

e bi

orea

ctor

.

Con

stan

t stif

fnes

s an

d re

sist

ivity

.2-

to 3

-day

-old

neo

nata

l Sp

ragu

e–D

awle

y ra

ts

hear

ts, b

ovin

e fib

rinog

en,

bovin

e th

rom

bin.

Cily

ndric

al w

ith v

ertic

al e

lect

rode

s.C

ylin

dric

al.

Elec

tro-M

echa

nica

l, m

echa

nica

l, an

d el

ectri

cal.

Car

bon

rods

.C

ompa

risio

n be

twee

n m

echa

nica

l, el

ectri

cal a

nd

elec

tro-m

echa

nica

l stim

uli

Elec

tro-m

echa

nica

l stim

ulat

ed c

onst

ruct

s ha

s a

stat

istic

ally

gre

ater

per

cent

age

of c

ells

(72.

5%–

20.5

%) a

s co

mpa

red

with

the

othe

r st

imul

atio

ns (~

45.1

%–

9.56

%).

Toge

ther

with

mec

hani

cal

stra

in is

the

mor

e ad

vant

age

for c

ell

surv

ival/p

rolif

erat

ion.

The

resu

lts in

dica

te th

at

elec

trica

l and

mec

hani

cal

stim

ulat

ion

alon

e im

prov

ed tw

itch

forc

e si

mila

rly o

ver s

tatic

cul

ture

wi

th m

echa

nica

l stim

ulat

ion

havin

g a

stat

istic

ally

gre

ater

fina

l ce

ll cou

nt in

the

tissu

e.

[14]

Lig

amen

ts, t

endo

ns,

and

skel

etal

mus

cles

tissu

e en

gine

erin

g (in

vi

tro)

.

Tibi

alis

ante

rior

mus

cle fr

om a

mou

se.

Perm

ount

(The

rmoF

ishe

r Sc

ient

ific)

med

ium

.10

V in

20

mse

c pu

lses

+ m

ax

mec

hani

cal s

train

10%

. A

com

pute

r con

trolle

d st

imul

ator

ap

plie

d 20

ms,

10

V sq

uare

wa

ve p

ulse

s at

inte

rval

s of

1 s

in

a 5

-pul

se tr

ain.

Dis

ks.

Cell

cultu

re fo

llow

ed b

y el

ectr

ospu

n hy

drog

el fi

ber g

ener

atio

n te

chni

que

follo

wed

by

scaf

fold

cons

truc

t cu

lture

.

Con

stan

t stif

fnes

s an

d re

sist

ivity

.(B

ioCo

nstr

uct)

Adip

ose-

deriv

ed st

em/s

trom

al ce

lls,

poly

mer

hyd

roge

l and

sa

crifi

cial a

lgin

ate.

Allo

ws fo

r bot

h el

ectri

cal,

mec

hani

cal s

train

and

she

ar s

tress

st

imul

atio

n.

Para

llele

pipe

d.El

ectro

-Mec

hani

cal s

timul

atio

n an

d sh

ear s

tress

stim

ulat

ions

.St

ainl

ess

stee

l hoo

k-sh

aped

ele

ctro

des.

Build

mul

tifun

ctio

nal r

eact

or -

Impr

oves

mus

cle

deve

lopm

ent.

-

[38,

41]

Stem

cell

alig

nmen

t and

m

otili

ty (i

n vi

tro)

.Ra

t adi

pose

-tiss

ue-

deriv

ed st

rom

al ce

lls

(ste

m ce

lls).

high

-glu

cose

Dul

becc

o’s

mod

ified

eag

le m

ediu

m

[DM

EM] (

Hycl

one,

UT,

US

A), 1

0% fe

tal b

ovin

e se

rum

(Inv

itrog

en, C

A,

USA)

, and

100

uni

ts/m

L pe

nici

llin/1

00 lg

/mL

stre

ptom

ycin

(Hyc

lone

, UT

, USA

)] su

pple

men

ted

with

25

mm

ol/L

HEP

ES

(Sig

ma,

MO

, USA

).

Elec

tric

Stim

ulat

ion

of 2

, 4, 6

, an

d 8

V/cm

.D

isks

(dep

osite

d in

co

vers

lips)

.D

istin

ct te

chni

cs fo

r col

lage

n hy

drog

el, c

hito

san

hydr

ogel

and

el

ectro

spin

ning

PLL

A/ge

latin

fibe

rs

fabr

icat

ion.

Con

stan

t stif

fnes

s an

d re

sist

ivity

.Pl

astic

cultu

re d

ishes

and

in

3D-c

ultu

re o

n va

rious

sc

affo

ld m

ater

ials,

inclu

ding

co

llage

n hy

drog

els,

chito

san

hydr

ogel

s and

pol

y(L-

lact

ic ac

id)/

gela

tin e

lect

rosp

inni

ng

fiber

s.

Agar

sal

t brid

ges

work

as

effe

ctive

el

ectro

des

avoi

ding

ioni

zatio

n.Pa

ralle

lepi

ped.

Elec

tric

stim

ulat

ion.

Squa

re a

gar b

ridge

s with

ag

ar sa

lt ge

l con

nect

the

Stei

nber

g’s s

olut

ion

pool

an

d cu

lture

med

ium

poo

l (a

ctua

l ele

ctro

des a

re ca

rbon

ro

ds).

Stud

y el

ectr

ic fie

ld e

ffect

on

cell

galv

anot

axis

beha

vior

s.Re

sults

sho

wed

that

EF

faci

litate

d AD

SC

mor

phol

ogic

al c

hang

es, u

nder

6 V

/cm

EF

stre

ngth

, and

that

AD

SCs

in 2

D-c

ultu

re

alig

ned

verti

cally

to E

F ve

ctor

and

kep

t a g

ood

cell s

urviv

al ra

te. I

n 3D

-cul

ture

, cel

l ga

lvano

taxis

resp

onse

s we

re s

ubje

ct to

the

syne

rgis

tic e

ffect

of a

pplie

d EF

and

sca

ffold

m

ater

ials.

Fas

t cel

l mov

emen

t and

intra

cellu

lar

calc

ium

act

ivitie

s we

re o

bser

ved

in th

e ce

lls o

f 3D

-cul

ture

.

Indu

ces

cell a

ligne

men

t.Th

e re

sear

ch w

ill pr

ovid

e so

me

expe

rimen

tal r

efer

ence

s fo

r the

fu

ture

stu

dy in

cel

l gal

vano

taxis

be

havio

rs.

[37,

40]

Ligam

ent r

egen

erat

ion

(in v

itro)

.Un

rest

ricte

d so

mat

ic hu

man

stem

cells

(s

tem

cells

).

Min

imum

ess

entia

l med

ium

ea

gle

(alp

ha-m

odifi

ed

supp

lem

ente

d wi

th 1

0%

FBS)

(Atla

nta

Biol

ogic

als,

La

wren

cevil

le, G

A, U

SA),

2 m

M L

-glu

tam

ine,

100

un

its/m

l pen

icilli

n an

d 10

0 m

g/m

l stre

ptom

ycin

.

200

mA

outp

ut cu

rren

t was

set

in e

very

hou

se w

ith th

e in

duct

ion

time

of 1

0 m

in o

n an

d 20

min

off.

Rect

angu

lar s

hape

(10

mm

60

mm

) for

nan

ofib

rous

sc

affo

ld, c

ylin

dric

al s

hape

(2

0 m

m 3

0 m

m) f

or s

pong

e m

ats

and

squa

re s

lices

for

reac

tor c

ultu

re.

elec

trosp

inni

ng te

chni

que

and

vapo

r ph

ase

poly

mer

izat

ion

com

bina

tion

met

hod

with

free

ze d

ryin

g.

Unifo

rm b

ilaye

r mat

em

bedd

ed

spon

ge w

ith p

oros

ity o

f 91.

26 ±

0.

55%

.

Silk

fibr

oin/

PEDO

T/Ch

itosa

n.Th

e el

ectri

cal i

nduc

tion

was

mad

e by

a p

olys

tyre

ne b

iore

acto

r w

ith 8

hou

ses

that

can

be

used

for

cell c

ultu

re a

nd p

lace

d in

side

incu

bato

r.

Adja

cent

cube

s of 3

0mm

x 30

mm

x 30

mm

with

squa

re

slice

scaf

fold

fixe

d w

ith

pexig

las.

Elec

trod

es p

aral

lel

fixed

dist

ing

10m

m fr

om

each

othe

r.

Elec

trica

l stim

ulat

ion

(DC

el

ectri

c pu

lses)

.St

ainl

ess s

teel

(316

) el

ectr

odes

(dia

met

er: 1

.4

mm

; len

gth:

25

mm

).

Test

cell

diffe

rent

iatio

n in

to

ligam

ent c

ells

star

ting

from

stem

ce

lls fo

r 3 d

istin

ct cu

lture

case

s:

petr

y di

sh, s

caffo

ld, s

caffo

ld a

nd

elec

tric

stim

ulat

ion.

From

the

RT-P

CR

resu

lts c

oncl

ude

that

lig

amen

t spe

cific

mar

kers

suc

h as

col

lage

n I,

colla

gen

III, d

ecor

in, b

igly

can

& ag

grec

an

gene

s on

ly w

ere

expr

esse

d in

gro

up (I

II) a

nd

as p

rese

nted

ele

ctric

al in

duct

ion

had

sign

ifica

nt s

timul

ator

y ef

fect

s on

tran

scrip

tion

of th

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Types of cell tissues. As for the cell types considered in each of the selected references, six studies deal with cardiac tissue engineering [31-36, 38], one with bone tissue engineering [30], one with muscle/ligament/tendon tissue engineering [14] and two with stem cell tissue engineering, [38]. Regarding the objective of generic cell differentiation, Dodel and his collaborators (see [37]) explicitly show that differentiation into ligament tissue requires electrical stimulation. In all these studies, it has been shown experimentally that the electrical stimulation and electro-mechanic stimulation is beneficial to the cell culture with respect to the control cultures without any electrical or mechanical stimulation. The interpretation is therefore common among all these studies, the stimuli mimic the natural organism conditions of tissues. In particular the cardiac tissue is naturally strain stimulated in living organisms as well as muscle, ligament and tendon tissues. We highlight the study [36] where electric strain, mechanical strain and both electro-mechanical stimulations are compared for cardiac tissue with the conclusion that electro-mechanical strain with electric stimulation when delayed with respect to the mechanical stimulation is the more beneficial stimuli for cell health. Not surprisingly this is indeed the stimulation that more closely resembles the working conditions of cardiac tissue in living organisms.

Scaffold Characteristics. The scaffolds employed in the reactors are commonly made out of a

mixture of both non-biological and biological cell tissues (synthesized and/or natural). The biological components range from generic components as collagen [30, 33], chitosan [37, 38], collagen-chitosan hydrogels [32, 38], poly(glycerol sebacate) [31] and poly(l-lactic acid) [38] to specific tissues adequate as substrate for each target cell culture. These tissues range from fresh pig hearts [34] (cardiac), human umbilical vein endothelial cells [35] (cardiac), rat hearts [33, 36] (cardiac) to adipose-derived stem/stromal cells [14] (ligament differentiation). The non-biological components for the scaffolds are commonly polymers as discussed in the introduction.

Synthetic scaffolds were employed in [30-32, 37]. When required to have a cell culture for the construction of the scaffolds, these are typically denominated as constructs [33-36, 14, 38]. The fabrication process employed includes cell culture after mixture/deposition in the base substrate [33-36, 38] and electrospinning [14, 37].

The specific shape of the scaffolds depend on the application: [31-35] considered parallelipipedic / square slices, [36, 37] considered cylindrical, and finally [14, 38, 37] considered disk shaped scaffolds.

Bioreactor. Each bioreactor has distinct objectives while in [31, 34-36, 14, 38] cell culture was

studied strictly in vitro, in [32] cell culture was studied strictly in vivo, in [30, 33] the goal was to study the viability and optimization of the transfer of in vitro cell culture to living organisms. In particular [30] suggest a bioreactor that enhances cell proliferation in vivo and in [33] it is shown that mechanical perfusion and electrical stimulation in vitro prior to transference of cell cultures to living organisms improves cell survival rate in vivo.

The geometry of the reactors considered in the selected studies is typically either cylindrical [30-32, 36] or parallelipipedic [33-35, 14, 38, 37] (see [39] for detailed description of [35]). When considering mechanical strain stimulation, servo actuators are typically employed [34-36, 14]. As for the electrical electrodes, composition involves either -gold [30] (wire of diameter 300um forming a pattern of 12mm by 3mm), carbon [31-33, 35, 36] (rods with diameter ranging from 0.5mm to 5mm), silver [34] (wire of 75μm diameter), stainless steel [38, 37] (rods of diameter 1.4mm, described in [40]) and salt bridges [38] (square agar bridges, the actual electrode is a carbon rod, described in [41]). It is relevant to note that the direct insertion of electrodes in the culture medium may induce ionization depending on the specific value of the voltage and current intensity. As this is undesirable for the cell culture, one possible solution is to consider salt bridges between the actual culture medium and independent recipients where the actual electrodes are immersed (see for instance [5] and references therein), being [38] the only selected bioreactor study that actually employed salt bridges. Typically for electro-mechanical strain stimulation the electrodes are in direct contact with the culture


medium (through the reactor wells when present) [30-33, 35-37] or in direct contact with the cells/scaffold [34, 14].

Stimulation. Studies included in this review show that different stimulation parameters seem to play a key role on cell differentiation, regeneration and growth. Scaffold stiffness (Young’s modulus) and electric resistivity: Laminar shear stress and perfusion mimicking the volumetric biological vascular systems. Also important is understanding the adhesion of cell cultures and its biodegradability. The geometry and topology of the scaffold is also crucial in the optimization of stimuli application [21, 23]. We highlight the two different types of stimulation that are mostly used and the main parameters involved:

i) Mechanical stimulation: amplitude, frequency and pulse shape, periodicity on/off during culture time and synchronization with respect to electrical stimulation were the main parameters involved;

ii) Electrical stimulation: electrodes characteristics like shape, dimensions and spacing between electrodes varied according to the different applications studied [30-33, 35-38]. Additionally, electric voltage and intensity, frequency and pulse shape, periodicity on/off during culture time and synchronization with respect to mechanical stimulation (when present) are parameters that have been shown to influence the results of electrical stimulation [32, 38].

Discussion In this review paper we report in detail the influence of applying electrical stimulation on cells

placed on a scaffold inside a bioreactor. Most of the papers in the literature to-date have used only mechanical stimulation. However, recently, some studies have shown the advantage of using different types of stimulation, such as, electrical or magnetic either in isolation or in combination [29, 42-44]. In order to understand the neurophysiology of the underlying mechanics, an in-depth study of all stimulation parameters involved should be performed. Initially our plan was to consider both numerical and experimental studies. However after searching on Science Direct and PubMed for relevant papers that address an updated string (the original string with addition of the keywords: simulation, numerical and modelling), we did not find any paper addressing the use of computational simulation to optimize the stimulation parameters. Therefore we decided to focus only on the experimental results. Here, we are mainly interested in understanding which type of parameters have been used for the different tissue engineering applications and possible optimizations on designs, protocols and stimulation parameters that may increase cell differentiation, growth, proliferation and survival rate. It was also possible to conclude that different electrode characteristics were considered empirically for the different applications, for instance, either they were emerged in the medium orthogonally to the scaffold or horizontally aligned with the scaffold. Additionally, electric voltage and intensity, frequency and pulse shape, periodicity on/off during culture time and synchronization with respect to mechanical stimulation (when present) are parameters that have been shown to influence the results of electrical stimulation [32, 38].

Once the relevance and optimization of the physical parameters for each stimulus is experimentally measured, numerical simulations can significantly reduce experimental time and costs [44]. In particular the optimal topologies and configurations of bioreactors can be investigated by numerical simulations prior to new experimental setups. For instance, Pereira et al., show numerically the importance of choosing the adequate mechanical stimuli parameters to improve shear stress value and fluid flow on the scaffold. Further, other numerical studies have investigated the influence of mechanical stimulation on the growth and distribution of cells on scaffolds and tissue differentiation [45-48].

Additionally, the developed bioscaffolds are usually tested and studied in vivo, presenting a significant expensive option thereby necessitating the development of numerical approaches. Therefore computational studies are of paramount importance to optimize the direct digital


manufacturing of scaffolds that can be produced using different geometries, geometry gradients, topologies and also different materials.

Finally, we note that the low number of bioreactor studies including (i), (iii) and (ii) or (iv) reflects both the complexity and multidisciplinary nature of the research field as distinct conditions are required for each organic tissue and the multitude of scaffold materials and respective construction process. It is relevant to note that no studies including both regenerative electrical stimulation and electro-mechanic strain stimulation have been found, it is however expected that further enhancement for contractible tissues may be expected by considering both these stimulations.

Conclusions Only few studies were found that focus on the application of electrical stimulation on cells placed

on a scaffold inside a bioreactor. The results are promising when using an electrical stimuli and also when combining it with mechanical stimulation. An increase in cell proliferation and density was observed as well as an enhancement in tissue morphology. Additionally, further studies must be performed where stimuli responsive scaffolds are used enabling a better cell proliferation, differentiation or tissue growth. This can be achieved through the combination of different types of stimuli to optimize both the response of the intelligent scaffold and cells behavior. Numerical studies can play here a key role to optimize all the stimulation parameters involved and predict cell response which can lead to a reduction of experimental time and costs.

Acknowledgments This work was supported by the Portuguese Foundation for Science and Technology (FCT) through

the Projects references UID/Multi/04044/2013, BIOMATE (PTDC/EMS-SIS/7032/2014) and Stimuli2BioScaffold (02/SAICT/2017, POCI-01-0145-FEDER-032554). It was also funded by PAMI (ROTEIRO/0328/2013; Nº 022158), a Research Infrastructure of the National Roadmap of Research Infrastructures of Strategic Relevance for 2014-2020, co-funded by the Portuguese Foundation for Science and Technology (FCT) and European Union through the Centro2020 and MATIS (CENTRO-01-0145-FEDER-000014 - 3362) funded by the European Union through the PT2020 and Centro2020.

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