Heart

Valve

Tissue

Engineering

Concepts

Approaches

Progress

and

Challenges

Potential

applications

of

tissue

engineering

in

regenerative

medicine

range

from

structural

tissues

to

organs

with

complex

function

This

review

focuses

on

the

engineering

of

heart

valve

tissue

a

goal

which

involves

a

unique

combination

of

biological

engineering

and

technological

hurdles

We

emphasize

basic

concepts

approaches

and

methods

progress

made

and

remaining

challenges

To

provide

a

framework

for

understanding

the

enabling

scientific

principles

we

first

examine

the

elements

and

features

of

normal

heart

valve

functional

structure

biomechanics

development

maturation

remodeling

and

response

to

injury

Following

a

discussion

of

the

fundamental

principles

of

tissue

engineering

applicable

to

heart

valves

we

examine

three

approaches

to

achieving

the

goal

of

an

engineered

tissue

heart

valve

-LRB-

-RRB-

cell

seeding

of

biodegradable

synthetic

scaffolds

-LRB-

-RRB-

cell

seeding

of

processed

tissue

scaffolds

and

-LRB-

-RRB-

in-vivo

repopulation

by

circulating

endogenous

cells

of

implanted

substrates

without

prior

in-vitro

cell

seeding

Lastly

we

analyze

challenges

to

the

field

and

suggest

future

directions

for

both

preclinical

and

translational

-LRB-

clinical

-RRB-

studies

that

will

be

needed

to

address

key

regulatory

issues

for

safety

and

efficacy

of

the

application

of

tissue

engineering

and

regenerative

approaches

to

heart

valves

Although

modest

progress

has

been

made

toward

the

goal

of

a

clinically

useful

tissue

engineered

heart

valve

further

success

and

ultimate

human

benefit

will

be

dependent

upon

advances

in

biodegradable

polymers

and

other

scaffolds

cellular

manipulation

strategies

for

rebuilding

the

extracellular

matrix

and

techniques

to

characterize

and

potentially

non-invasively

assess

the

speed

and

quality

of

tissue

healing

and

remodeling

INTRODUCTION

Potential

applications

of

tissue

engineering

in

regenerative

medicine

range

from

structural

tissues

-LRB-

e.g.

skin

cartilage

bone

-RRB-

to

complex

organs

-LRB-

e.g.

heart

and

other

components

of

the

cardiovascular

system

liver

kidney

pancreas

-RRB-

In

each

case

there

are

limitations

to

conventional

surgical

approaches

and

existing

prosthetic

devices

serious

complications

associated

with

transplantation

and

critical

shortages

of

available

donor

tissues

Cardiovascular

tissue

engineering

has

primarily

considered

blood

vessels

myocardium

and

heart

valves

This

review

focuses

on

the

application

of

tissue

engineering

technology

to

heart

valves

Currently

adults

who

undergo

replacement

of

diseased

valves

by

either

mechanical

prosthetic

or

tissue

valves

-LRB-

including

bioprosthetic

valves

-LSB-

porcine

aortic

valve

or

bovine

pericardial

xenograft

-RSB-

cadaveric

allograft

or

pulmonary-to-aortic

autograft

valves

-LSB-

Ross

procedure

-RSB-

-RRB-

generally

have

enhanced

survival

and

quality

of

life

Nevertheless

each

of

these

valve

types

has

its

limitations

in

particular

mechanical

valves

require

anticoagulation

to

control

thromboembolism

while

bioprosthetic

and

allograft

valves

frequently

undergo

calcification

and

structural

deterioration

Advantages

of

an

engineered

tissue

heart

valve

would

likely

include

nonthrombogenicity

infection

resistance

and

cellular

viability

The

design

criteria

and

characteristics

for

conventional

and

tissue

engineered

replacement

heart

valves

are

summarized

and

compared

in

Table

The

most

immediate

need

for

heart

valve

tissue

engineering

and

regeneration

technology

is

in

the

pediatric

and

young

adult

population

in

which

the

results

of

valve

replacement

are

not

as

favorable

as

those

in

older

adults

Most

exciting

is

the

possibility

of

growth

repair

and

remodeling

as

a

child

recipient

matures

thus

eliminating

the

repetitive

surgeries

typically

necessitated

by

the

inability

of

a

valve

substitute

to

enlarge

as

an

individual

grows

Only

autografts

-LRB-

such

as

Ross

valves

transplanted

from

the

pulmonary-to-aortic

position

in

an

individual

-RRB-

presently

are

viable

but

the

Ross

procedure

is

technically

difficult

risky

only

serves

a

small

patient

subset

and

has

controversial

results

including

uncertainty

over

whether

the

grafts

will

grow

commensurate

with

recipient

growth

TABLE

Design

objectives

for

and

characteristics

of

replacement

heart

valves.Feature

to

optimizeConventional

-LRB-

Mechanical

bioprosthetic

-RRB-

Tissue

engineeredClosure

of

leafletsRapid

and

completeRapid

and

completeSize

of

orifice

areaLess

than

that

of

natural

valvesBetterMechanical

propertiesStableStableSurgical

insertionEasy

and

permanentEasy

and

permanentRisk

of

thrombosisYes

especially

mechanical

valves

which

require

anticoagulation

causing

vulnerability

to

hemorrhageNo

endothelial

surface

to

inhibit

thrombogenesisRisk

of

structural

dysfunctionDegradation

of

synthetic

materials

rare

with

mechanical

valvesResistant

to

degradation

and

calcificationTissue

degradation

and

calcification

of

leaflets

with

bioprosthetic

valvesRisk

of

InfectionEver

presentResistant

to

infectionViabilityNoYes

able

to

repair

injury

remodel

and

potentially

grow

with

patientFIGURE

Specialized

ECM

enables

dynamic

aortic

valve

function

-LRB-

a

-RRB-

Photograph

of

the

aortic

valve

in

open

and

closed

position

-LRB-

from

the

aorta

-RRB-

-LRB-

b

-RRB-

Aortic

valve

histology

emphasizing

trilaminar

structure

and

presence

of

valvular

interstitial

and

endothelial

cells

-LRB-

c

-RRB-

Biomechanical

cooperativity

between

elastin

and

collagen

during

valve

motion

-LRB-

d

-RRB-

Schematic

depiction

of

layered

aortic

valve

cuspal

structure

and

configuration

of

collagen

and

elastin

during

systole

and

diastole

-LRB-

a

-RRB-

and

-LRB-

b

-RRB-

reproduced

by

permission

from

Schoen

FJ

Valvular

heart

disease

General

principles

and

stenosis

IN

Cardiovascular

Pathology

Ed

Silver

MD

Gotlieb

AI

Schoen

FJ

-LRB-

eds

-RRB-

WB

Saunders

pp.

-LRB-

c

-RRB-

and

-LRB-

d

-RRB-

reproduced

by

permission

from

Schoen

FJ

Aortic

valve

structure-function

correlations

Role

of

elastic

fibers

no

longer

a

stretch

of

the

imagination

J

Heart

Valve

Dis

The

goal

to

engineer

functional

heart

valve

tissue

presents

a

unique

combination

of

challenges

Normal

heart

valves

are

vital

and

dynamic

tissues

composed

of

specialized

cells

and

extracellular

matrix

-LRB-

ECM

-RRB-

that

respond

and

remodel

in

response

to

changes

in

local

mechanical

forces

Approximately

million

times

a

year

opening

and

closing

of

the

leaflets

induces

repetitive

changes

in

the

shape

dimensions

and

stress

of

the

leaflets

and

supporting

valvular

structures

-LRB-

Fig.

-RRB-

A

successful

tissue

engineered

valve

and

its

components

must

not

only

accommodate

those

deformations

but

also

have

ongoing

strength

flexibility

and

durability

beginning

at

the

instant

of

implantation

and

continuing

indefinitely

thereafter

possibly

despite

an

evolving

tissue

architecture

This

review

provides

a

contemporary

analysis

of

heart

valve

tissue

engineering

and

regeneration

emphasizing

the

evolving

understanding

of

heart

valve

biology

the

promise

and

difficulties

demonstrated

by

in-vivo

studies

done

to

date

and

the

critical

challenges

that

will

be

encountered

in

translating

the

potential

of

this

exciting

therapeutic

modality

from

the

laboratory

to

the

clinical

realm

To

put

the

technology

and

the

evolving

enabling

science

into

context

we

begin

by

examining

the

complex

and

dynamic

structural

components

that

are

needed

to

accomplish

normal

heart

valve

function

biomechanics

physiological

tissue

maintenance

homeostasis

and

ongoing

health

Our

discussion

of

the

basic

principles

of

tissue

engineering

summarizes

current

principles

of

scaffolding

and

cell

sourcing

Subsequently

we

examine

three

potential

approaches

to

the

field

of

tissue-engineered

heart

valves

-LRB-

-RRB-

cell

seeding

of

biodegradable

synthetic

scaffolds

-LRB-

-RRB-

cell

seeding

of

processed

tissue

scaffolds

and

-LRB-

-RRB-

in-vivo

repopulation

by

circulating

endogenous

cells

of

implanted

substrates

without

prior

in-vitro

cell

seeding

Finally

we

analyze

challenges

to

the

field

and

suggest

future

directions

for

both

preclinical

and

translational

-LRB-

clinical

-RRB-

studies

that

will

be

needed

to

address

key

regulatory

issues

for

safety

and

efficacy

of

the

application

of

tissue

engineering

and

regenerative

approaches

to

heart

valves

Our

primary

goal

is

to

stimulate

thinking

in

the

field

by

introducing

concepts

such

as

outcome

criteria

biomarkers

molecular

imaging

product

release

criteria

questions

raised

by

the

integration

of

engineered

heart

valves

with

the

host

tissues

and

uncertainties

engendered

by

patient-to-patient

heterogeneity

in

key

biological

processes

such

as

tissue

remodeling

capability

Our

discussion

in

this

paper

underscores

that

the

development

and

application

of

innovative

approaches

to

repair

and

regenerate

damaged

or

diseased

heart

valves

requires

the

integration

of

numerous

biological

engineering

and

technological

principles

TABLE

Key

structural

elements

of

heart

valves.ElementSub-structureFunctionExtracellular

matrixCollagenProvides

strength

and

stiffness

to

maintain

coaptation

during

diastole

when

cusp

has

maximal

areaElastinExtends

in

diastole

contracts

in

systole

to

minimize

cusp

areaGlycosaminoglycansAccomodates

shear

of

cuspal

layers

cushions

shock

during

valve

cycleCellsInterstitialSynthesize

ECM

express

MMPs

and

TIMPs

that

mediate

matrix

remodelingEndothelialMaintain

nonthrombogenic

blood-tissue

interface

regulate

immune

and

inflammatory

reactionsBlood

vesselsFew

and

focal

valve

cusps

and

leaflets

sufficiently

thin

to

be

nourished

by

diffusion

from

the

heart

's

bloodNervesPresent

with

uncertain

functionOther

principlesCorrugationsAccordian-like

folds

in

cusps

allows

cuspal

shape

and

dimensions

to

vary

with

cardiac

cycleCrimpMicroscopic

collagen

folding

allows

lengthening

at

minimal

stressAnisotropyPermits

differences

in

radial

and

circumferential

extensibilityCordsMacroscopic

collagen

alignment

transfers

forces

from

cusps

to

aortic

wall

HEART

VALVE

FUNCTION

AND

STRUCTURE

Healthy

native

heart

valves

maintain

unidirectional

blood

flow

via

an

extraordinarily

dynamic

functional

structure

with

several

key

characteristics

viability

sufficient

strength

to

withstand

repetitive

and

substantial

mechanical

stress

and

ability

to

adapt

and

repair

injury

by

connective

tissue

remodeling

A

rational

approach

to

heart

valve

tissue

engineering

depends

on

a

thorough

understanding

of

the

complex

normal

functional

elements

and

their

coordinated

interactions

-LRB-

Table

-RRB-

For

the

trileaflet

semilunar

valves

-LRB-

aortic

and

pulmonary

-RRB-

the

important

structures

are

the

cusps

-LRB-

which

avoid

prolapse

by

substantial

coaptation

-RRB-

the

commissures

and

the

supporting

structures

in

the

aortic

and

pulmonary

roots

For

the

atrioventricular

valves

-LRB-

mitral

and

tricuspid

-RRB-

the

key

components

are

the

leaflets

commissures

annulus

chordae

tendineae

papillary

muscles

and

atrial

and

ventricular

myocardium

Valve

leaflets

and

cusps

have

few

and

only

focal

blood

vessels

-LRB-

vessels

may

be

present

in

the

proximal

portion

near

the

myocardium

-RRB-

likely

because

valves

cusps

and

leaflets

are

sufficiently

thin

to

be

nourished

predominantly

by

diffusion

from

the

heart

's

blood

Valve

leaflets

and

cusps

also

have

nerves

but

their

significance

is

uncertain

The

four

cardiac

valves

have

microstructural

similarities

however

the

aortic

valve

best

illustrates

the

essential

features

and

serves

as

a

paradigm

for

microstructural

and

cellular

adaptation

to

functional

requirements

The

aortic

valve

is

the

most

frequently

diseased

and

also

commonly

transplanted

The

aortic

valve

cusps

undergo

substantial

changes

in

shape

and

dimension

during

the

cardiac

cycle

-LRB-

see

Fig.

-RRB-

Microscopically

the

aortic

valve

is

composed

of

three

distinct

layers

-LRB-

-RRB-

the

ventricularis

closest

to

the

inflow

surface

rich

in

elastin

-LRB-

-RRB-

the

fibrosa

closest

to

the

outflow

surface

primarily

composed

of

densely

packed

collagen

and

-LRB-

-RRB-

the

centrally

located

spongiosa

largely

composed

of

glycosoaminoglycans

-LRB-

GAGs

-RRB-

Together

collagen

elastin

and

GAGs

comprise

the

valvular

ECM

-LRB-

see

Fig.

-RRB-

Studies

of

normal

pathological

and

substitute

valves

have

demonstrated

that

the

principal

determinant

of

valve

durability

is

the

valvular

ECM

whose

quantity

and

quality

depend

on

the

viability

and

function

of

valvular

interstitial

cells

-LRB-

VIC

-RRB-

Recent

research

suggests

that

cell-matrix

interactions

in

heart

valves

are

highly

stress-dependent

and

likely

reciprocal

In

vivo

heart

valve

leaflets

experience

a

dynamic

and

complex

mechanical

stress

state

during

every

cardiac

cycle

sheer

stress

due

to

blood

flow

-LRB-

open

valve

-RRB-

flexure

-LRB-

opening

and

closing

-RRB-

and

tension

-LRB-

closed

valve

-RRB-

Forces

acting

on

the

valve

at

the

macroscopic

level

-LRB-

pressure

sheer

stress

and

tension

-RRB-

are

translated

into

specific

biomechanical

responses

at

the

tissue

level

-LRB-

collagen

uncrimping

reorientation

and

fiber

compaction

-RRB-

which

are

transduced

into

a

VIC

response

at

the

cellular

level

-LRB-

with

intracellular

signaling

events

leading

to

changes

such

as

increased

alpha

smooth

muscle

actin

-LRB-

α-SMA

-RRB-

expression

increased

VIC

stiffness

and

increased

ECM

biosynthesis

-RRB-

Indeed

the

higher

absolute

pressure

and

transvalvular

pressure

gradients

on

the

left

side

of

the

heart

impose

larger

local

tissue

stress

on

the

VICs

than

those

on

the

right

side

which

leads

to

higher

VIC

stiffness

-LRB-

through

α-SMA

content

-RRB-

and

collagen

biosynthesis

in

the

left-sided

valves

Complex

microstructural

rearrangements

and

several

specializations

of

collagen

accommodate

the

cyclical

pressure

fluctuations

during

the

cardiac

cycle

Collagen

fibers

in

a

planar

orientation

in

the

fibrosa

comprise

the

strongest

portion

of

the

leaflet

that

is

responsible

for

bearing

diastolic

stress

GAGs

in

the

spongiosa

probably

serve

predominantly

as

a

shock

and

shear

absorber

The

large

cuspal

deformation

during

the

cycle

between

systole

and

diastole

is

facilitated

by

biomechanical

cooperativity

between

collagen

and

elastin

-LRB-

see

Fig.

-RRB-

Collagen

fibrils

are

inelastic

and

incapable

of

supporting

large

strains

they

therefore

have

adaptations

-LRB-

macroscopic

corrugations

and

microscopic

crimp

-RRB-

that

permit

collagen

stretching

at

minimal

stress

by

unfolding

-LRB-

Fig.

-RRB-

During

valve

opening

elastin

stretches

during

extension

of

collagen

crimp

and

corrugations

When

the

valve

is

closed

the

collagen

is

fully

unfolded

and

the

load

is

shifted

from

elastin

to

collagen

elastin

restores

the

contracted

configuration

of

the

cusp

during

systole

Two

types

of

cells

are

present

in

the

aortic

valve

endothelial

cells

covering

the

surface

and

interstitial

cells

with

variable

properties

of

fibroblasts

smooth

muscle

cells

and

myofibroblasts

in

the

interior

-LRB-

see

Fig.

-RRB-

Like

endothelial

cells

elsewhere

in

the

circulation

valvular

endothelial

cells

-LRB-

VEC

-RRB-

maintain

a

nonthrombogenic

blood-tissue

interface

and

regulate

immune

and

inflammatory

reactions

Nevertheless

endothelial

cells

isolated

from

different

vascular

and

valvular

sources

display

differences

in

gene

expression

and

other

properties

-LRB-

endothelial

cell

heterogeneity

-RRB-

Indeed

heart

valve

endothelial

cells

are

different

from

those

in

the

aorta

For

example

in

response

to

mechanical

stress

porcine

aortic

valve

endothelial

cells

align

perpendicular

to

flow

whereas

endothelial

cells

from

the

aorta

align

parallel

to

flow

and

their

gene

expression

is

different

from

that

of

aortic

endothelial

cells

exposed

to

the

same

mechanical

environment

These

differences

suggest

a

unique

phenotype

of

VEC

not

mimicked

by

vascular

cells

and

could

have

implications

for

cardiovascular

cell

biology

and

cell-source

considerations

for

tissue-engineered

heart

valves

Moreover

different

transcriptional

profiles

are

expressed

by

the

endothelium

on

the

aortic

side

versus

ventricular

side

of

normal

adult

pig

aortic

valves

The

reasons

for

these

differences

are

not

yet

known

VIC

the

most

numerous

valvular

cell

type

synthesize

ECM

and

express

matrix

degrading

enzymes

metalloproteinases

-LRB-

MMPs

-RRB-

and

their

inhibitors

-LRB-

TIMPs

-RRB-

that

mediate

matrix

remodeling

Moreover

VIC

comprise

a

dynamic

population

of

resident

cells

of

diverse

and

dynamic

phenotypes

in

a

spectrum

that

ranges

from

fibroblast-like

to

myofibroblast-like

their

phenotype

is

regulated

by

environmental

conditions

We

and

others

have

evaluated

VIC

phenotypes

in

normal

and

diseased

human

valves

In

adult

heart

valves

in

situ

VIC

are

quiescent

and

display

a

fibroblast-like

phenotype

characterized

by

the

presence

of

vimentin

-LRB-

intermediate

filaments

-RRB-

and

very

low

levels

of

α-SMA

-LRB-

proteolytic

enzymes

-RRB-

and

SMemb

-LRB-

non-muscle

myosin

heavy

chain

-RRB-

Indeed

only

of

normal

adult

VIC

express

α-SMA

which

is

a

marker

of

myofibroblast-like

function

Myofibroblasts

are

activated

fibroblasts

that

synthesize

and

remodel

the

specialized

ECM

facilitate

tissue

remodeling

and

wound

healing

and

play

a

pathological

role

in

fibrotic

disease

In

contrast

as

demonstrated

by

previous

in-vitro

studies

using

isolated

cells

cultured

from

heart

valves

of

cells

are

This

suggests

that

removal

of

cells

from

the

environment

of

the

intact

valve

-LRB-

i.e.

in

culture

-RRB-

or

their

manipulation

stimulates

VIC

Moreover

treatment

of

isolated

VICs

with

TGF-β

strongly

activates

interstitial

cells

to

the

myofibroblast

phenotype

Biomechanical

and

biochemical

factors

play

an

important

and

potentially

synergistic

role

in

determining

the

local

homeostatic

environment

of

the

aortic

VIC

As

demonstrated

in

in-vitro

studies

mechanical

stimulation

-LRB-

tension

-RRB-

and

cytokine

stimulation

-LRB-

-RRB-

were

found

to

synergistically

alter

the

contractile

-LRB-

α-SMA

-RRB-

and

biosynthetic

-LRB-

heat

shock

protein

Hsp

-RRB-

proteins

of

aortic

VICs

to

a

greater

extent

than

each

factor

alone

The

need

for

mechanical

stimulation

and/or

cytokines

and

potentially

other

soluble

factors

for

the

maintenance

of

appropriate

cellular

biosynthetic

activity

will

be

an

important

influence

on

tissue

engineering

efforts

HEART

VALVE

DEVELOPMENT

MATURATION

ADAPTATION

AND

REPAIR

A

thorough

understanding

of

developmental

biology

physiology

and

pathophysiology

of

heart

valves

will

likely

inform

tissue

engineering

Indeed

some

of

the

processes

and

regulatory

pathways

active

in

valvular

development

and

maturation

may

be

recapitulated

in

tissue

engineered

valves

During

embryological

development

the

three

germ

layers

ectoderm

endoderm

and

mesoderm

give

rise

to

cells

that

differentiate

to

form

the

body

's

tissues

and

complex

organs

The

heart

develops

primarily

from

the

embryonic

layer

called

mesoderm

The

initial

commitment

of

mesodermal

precursor

cells

to

a

cardiac

lineage

depends

on

complex

signaling

pathways

Cardiac

myocytes

become

organized

into

a

linear

heart

tube

that

subsequently

undergoes

looping

Growth

of

the

looped

heart

tube

and

development

of

septa

leads

to

the

multichambered

heart

During

cardiac

development

the

valve

cusps

and

leaflets

originate

as

outgrowths

-LRB-

known

as

endocardial

cushions

-RRB-

from

mesodermal

derived

connective

tissue

called

mesenchyme

Endothelial

cells

lining

the

inside

surface

of

the

cushion

forming

area

undergo

an

epithelial-to-mesenchymal

transdifferentiation

-LRB-

EMT

-RRB-

and

migrate

from

the

blood-contacting

internal

heart

surface

deep

into

the

connective

tissue

of

the

subendocardium

-LRB-

called

cardiac

jelly

-RRB-

to

become

precursors

of

mature

VICs

Widespread

in

embryological

development

epithelial

mesenchymal

transitions

comprise

a

series

of

cell

cell

and

cell-matrix

interactions

that

release

epithelial

cells

from

a

surface

and

confer

the

ability

to

move

through

three

dimensional

ECM

and

synthesize

ECM

The

newly

formed

mesenchymal

cells

remodel

the

cushions

into

leaflets

and

cusps

Evidence

for

EMT

is

provided

by

mesenchymal

cellular

expression

of

α-SMA

a

marker

that

is

not

typically

expressed

by

endothelial

cells

Numerous

signaling

pathways

growth

and

transcriptional

factors

-LRB-

including

vascular

endothelial

growth

factor

-LSB-

VEGF

-RSB-

nuclear

factor

in

activated

T

cells

-LSB-

-RSB-

and

Notch

-RRB-

regulate

the

process

of

heart

valve

formation

We

studied

the

subsequent

maturation

and

evolution

of

human

semilunar

valves

in

fetuses

neonates

children

and

adults

We

demonstrated

that

fetal

valves

contain

immature

activated

cells

and

are

dynamic

and

adaptable

structures

the

architecture

collagen

content

and

organization

were

immature

compared

with

that

of

adult

valves

During

valve

development

and

maturation

fetal

VIC

have

a

myofibroblast-like

phenotype

characterized

by

expression

of

α-SMA

and

SMemb

and

continuously

remodel

the

ECM

The

cells

become

quiescent

in

the

normal

valve

post-natally

suggesting

progressive

adaptation

to

the

prevailing

hemodynamic

environment

Moreover

the

cell

density

progressively

decreases

-LRB-

by

nearly

-RRB-

throughout

life

The

role

of

myofibroblasts

in

valvular

wound

healing

adaptation

and

remodeling

is

best

illustrated

by

comparisons

of

normal

diseased

autograft

and

tissue

engineered

valves

In

conditions

of

disease

-LRB-

e.g.

myxomatous

mitral

valve

-RRB-

adaptation

-LRB-

early

pulmonary-to-aortic

autograft

-RRB-

or

remodeling

-LRB-

tissue

engineered

valves

in-vitro

and

in-vivo

-RRB-

VICs

have

an

activated

myofibroblast

phenotype

similar

to

that

of

fetal

valves

Normal

and

pathological

cardiac

valves

respond

to

environmental

conditions

such

as

mechanical

loading

by

cell

activation

and

matrix

remodeling

In

contrast

following

return

of

a

stable

equilibrium

of

mechanical

state

in

development

adaptation

or

remodeling

VIC

return

to

their

normal

quiescent

fibroblast

phenotype

as

exemplified

by

late

pulmonary-to-aortic

autografts

-LRB-

years

postoperative

-RRB-

and

tissue

engineered

valves

implanted

in-vivo

Therefore

heart

valves

respond

to

environmental

change

via

reversible

phenotypic

modulation

of

their

resident

VICs

SCAFFOLDS

FOR

TISSUE

ENGINEERING

GENERAL

CONCEPTS

A

widely

accepted

paradigm

of

tissue

engineering

comprises

-LRB-

-RRB-

a

scaffold

that

is

pre-seeded

with

-LRB-

-RRB-

cells

followed

by

-LRB-

-RRB-

an

in-vitro

stage

of

tissue

formation

typically

conducted

in

a

bioreactor

-LRB-

that

recapitulates

a

physiological

metabolic

and

mechanical

environment

-RRB-

and

subsequently

following

implantation

of

the

construct

-LRB-

-RRB-

an

in-vivo

stage

of

tissue

growth

and

remodeling

-LRB-

Fig.

Pathway

A

-RRB-

The

key

pathophysiological

processes

occurring

during

the

in-vitro

and

in-vivo

phases

are

cell

proliferation

and

migration

ECM

production

and

organization

scaffold

degradation

and

tissue

remodeling

The

in-vivo

but

not

the

in-vitro

phase

can

involve

recruitment

of

the

recipient

's

inflammatory

cells

The

resulting

tissue

engineered

construct

will

likely

contain

some

combination

of

seeded

and/or

recipient-derived

new

cells

An

alternative

pathway

-LRB-

Fig.

Pathway

B

-RRB-

utilizes

an

unseeded

scaffold

that

incorporates

biological

information

designed

to

attract

and

direct

the

formation

of

circulating

endogenous

precursor

cells

-LRB-

potentially

both

endothelial

and

mesenchymal

-RRB-

in-vivo

Both

of

these

pathways

are

considered

in

detail

in

subsequent

sections

of

this

paper

Moreover

host

inflammatory

cells

may

play

a

role

in

the

in-vivo

phase

of

either

approach

In

the

following

sections

we

examine

the

characteristics

of

biodegradable

synthetic

scaffolds

and

natural

allograft

or

xenograft

scaffolds

a

composite

biodegradable

polymer

with

collagen

or

other

natural

material

may

also

be

considered

-LRB-

Table

-RRB-

TABLE

Comparative

analysis

of

scaffolds.Synthetic

scaffoldsNatural

scaffoldsAdvantages

Control

of

material

structure

and

properties

-LRB-

e.g.

pore

size

stability

degradation

rate

-RRB-

Maintain

architecture

of

the

native

tissue

-LRB-

potentially

valve

-RRB-

Easily

reproducedMaintain

biological

information

-LRB-

e.g.

reactive

sites

growth

factors

-RRB-

ResorbablePotentially

resorbableDisadvantages

Difficulty

in

controlling

cell

adhesion

and

tissue

reorganizationDecellularization

may

alter

physical

properties

Inflammation

due

to

incomplete

polymer

degradation

or

lack

of

biocompatibilityDifficulty

of

cell

penetration

into

interior

Space

formerly

occupied

by

polymer

and

its

interstices

is

replaced

by

fibrosis/scarMay

induce

immunologic

reaction

Limited

perfusion

to

deep

cellsPotential

for

calcificationFIGURE

Tissue

engineering

paradigms

-LRB-

Pathway

A

-RRB-

The

conventional

paradigm

of

tissue

engineering

comprises

a

scaffold

that

is

seeded

with

cells

an

in-vitro

stage

of

tissue

formation

typically

conducted

in

a

bioreactor

and

an

in-vivo

stage

of

tissue

growth

and

remodeling

The

key

pathophysiological

processes

occurring

during

the

in-vitro

and

in-vivo

phases

are

cell

proliferation

ECM

production

and

organization

scaffold

degradation

and

tissue

remodeling

The

resulting

tissue

engineered

construct

will

contain

some

combination

of

seeded

and/or

new

cells

A

modified

paradigm

-LRB-

Pathway

B

-RRB-

might

utilize

an

unseeded

scaffold

that

is

fabricated

with

biological

information

designed

to

attract

and

provide

a

suitable

substrate

for

differentiation

of

circulating

precursor

cells

in-vivo

Synthetic

Scaffolds

Synthetic

polymeric

scaffolds

generally

have

well

controlled

and

easily

reproduced

properties

Ideally

a

tissue

engineered

scaffold

should

be

biocompatible

resorbable

have

a

highly

porous

macrostructure

-LRB-

necessary

for

cell

growth

nutrient

supply

and

waste

removal

-RRB-

and

a

surface

conducive

to

cellular

attachment

and

potentially

migration

proliferation

differentiation

matrix

formation

and/or

remodeling

A

bioresorbable

scaffold

serves

as

a

temporary

matrix

until

the

seeded

cells

are

capable

of

producing

their

own

matrix

proteins

the

chemical

and

physical

properties

and

the

rate

of

degradation

are

generally

tailored

to

the

application

and

the

rate

of

new

tissue

evolution

Polyglycolic

acid

-LRB-

PGA

-RRB-

and

polylactic

acid

-LRB-

PLA

-RRB-

and

their

copolymers

are

currently

the

synthetic

biodegradable

polymers

most

widely

considered

for

tissue

engineering

applications

PGA

is

a

highly

crystalline

linear

aliphatic

polyester

with

a

high

melting

point

and

low

solubility

in

organic

solvents

PGA

was

used

as

the

first

totally

synthetic

resorbable

suture

commercially

available

under

the

trade

name

Dexon

since

PGA

is

rapidly

resorbable

thus

Dexon

sutures

tend

to

lose

their

mechanical

strength

rapidly

usually

within

to

weeks

after

implantation

Copolymers

of

the

hydrophilic

PGA

with

the

more

hydrophobic

PLA

increase

the

range

of

material

properties

and

hence

possible

applications

for

example

the

addition

of

PLA

limits

the

water

uptake

and

reduces

the

rate

of

hydrolysis

-LRB-

the

predominant

model

of

degradation

-RRB-

as

compared

to

PGA

alone

Considered

safe

nontoxic

and

biocompatible

these

polymers

are

used

successfully

in

a

number

of

approved

medical

implants

The

use

of

a

synthetic

scaffold

introduces

challenges

in

the

regulation

of

cell

adhesion

and

three-dimensional

tissue

reorganization

since

such

materials

are

usually

isotropic

and

ECM

proteins

are

not

usual

constituents

of

synthetic

polymers

For

example

PGA

PLA

and

their

copolymers

are

generally

considered

poor

substrates

for

cell

growth

Therefore

bioactive

synthetic

biomaterials

are

being

developed

for

use

as

three-dimensional

microenvironments

that

mimic

ECM

function

Recent

advances

include

nanofibrillar

networks

formed

by

self-assembly

of

small

molecules

and

artificial

ECM

networks

from

protein

polymers

or

synthetic

polymers

that

present

bioactive

ligands

and

respond

to

cell-secreted

signals

to

enable

remodeling

An

especially

attractive

concept

is

that

of

smart

biomaterial

scaffolds

which

carry

spatial

and

chemical

information

that

affects

cellular

function

and/or

responds

to

changes

in

the

environment

Smart

scaffolds

are

exemplified

by

biodegradable

elastic

shape-memory

polymers

that

predictably

alter

their

shape

with

changes

in

and

polymers

that

transition

between

hydrophobic

and

hydrophilic

states

in

response

to

electric

potential

Moreover

normal

tissue

culture

dishes

grafted

with

a

temperature

sensitive

polymer

PIPAAm

-LRB-

poly

N-isopropylacrylamide

-RRB-

respond

to

temperature

changes

which

alter

the

adhesion

properties

of

cells

to

this

matrix

At

elevated

temperatures

-LRB-

above

°C

-RRB-

the

dish

surfaces

are

relatively

hydrophobic

and

cells

attach

whereas

at

lower

temperatures

-LRB-

°C

-RRB-

the

polymer

surface

becomes

hydrophilic

and

swells

forming

a

hydration

layer

between

the

dish

surface

and

the

cultured

cells

The

cells

along

with

their

framework

ECM

spontaneously

detach

and

may

be

harvested

as

intact

sheets

As

the

technology

develops

smart

scaffolds

may

prove

important

to

applications

of

stem

cells

in

tissue

engineering

for

example

a

new

three

dimensional

material

with

the

ability

to

direct

the

differentiation

of

neural

progenitor

cells

into

a

specific

lineage

without

the

help

of

growth

factors

has

been

reported

Tissue

engineered

biomaterial

constructs

must

also

exhibit

mechanical

and

structural

properties

comparable

to

the

native

tissue

they

replace

including

dynamic

anisotropic

behaviors

optimized

for

tissue

specific

function

from

the

time

of

implantation

of

a

construct

through

the

lifetime

of

the

recipient

This

implies

central

requirements

of

relatively

constant

mechanical

properties

despite

potentially

considerable

tissue

remodeling

and

scaffold

resorption

Moreover

mechanical

anisotropy

-LRB-

certainly

a

characteristic

of

the

natural

valves

-RRB-

may

be

desirable

towards

this

end

electrospun

scaffolds

were

fabricated

which

exhibited

highly

anisotropic

mechanical

properties

that

resembled

native

pulmonary

heart

valve

cusps

Potential

undesirable

features

of

synthetic

scaffolds

include

local

tissue

inflammation

owing

to

the

foreign

body

reaction

and

slow

and/or

incomplete

polymer

degradation

As

the

scaffold

degrades

the

space

formerly

occupied

by

a

polymer

and

its

interstices

is

progressively

filled

by

cells

and

ECM

which

may

eventuate

in

fibrosis

-LRB-

scar

-RRB-

that

poorly

resembles

specialized

native

tissue

and

may

contract

and

distort

during

maturation

In

some

cases

cells

on

the

scaffold

periphery

are

healthy

and

resemble

native

differentiated

parenchymal

-LRB-

i.e.

function-specific

-RRB-

tissue

whereas

cells

at

the

interior

become

necrotic

due

to

restricted

deep

delivery

of

oxygen

and

nutrients

and

removal

of

wastes

Natural

Scaffolds

Alternative

to

synthetic

scaffolds

natural

scaffolds

usually

comprise

pure

ECM

components

-LRB-

such

as

collagen

or

fibrin

-RRB-

or

decellularized

but

otherwise

intact

allograft

or

xenograft

tissue

-LRB-

such

as

heart

valve

or

small

intestinal

submucosa

-RRB-

In

cardiovascular

tissue

engineering

multi-step

static

seeding

methods

have

been

most

commonly

used

alternatively

efficient

single

step

seeding

may

be

achieved

via

cellular

encapsulation

in

collagen

and

fibrin

gels

Collagen

based

constructs

seeded

with

vascular

smooth

muscle

and/or

endothelial

cells

have

been

utilized

to

generate

tubular

blood

vessels

one-dimensional

strings

to

serve

as

a

component

of

composite

aortic

valve

or

chordae

and

molds

for

heart

valve

leaflets

Cells

entrapped

in

collagen

gels

contract

and

compact

the

gels

in

a

process

similar

to

the

contraction

of

a

wound

during

healing

thus

increasing

their

density

and

enhancing

their

properties

Alternatively

cells

can

be

encapsulated

in

an

autologous

fibrin

gel

which

initially

serves

to

obtain

uniform

cell

distribution

and

to

improve

the

seeding

efficiency

With

gel

encapsulated

cells

newly

synthesized

ECM

accumulates

in

the

immediate

extracellular

space

rather

than

diffusing

into

the

surrounding

medium

Reported

limitations

of

the

encapsulated

cell

approach

to

tissue

engineering

include

excessive

shrinkage

of

the

gel/cell

construct

and

the

possibility

that

the

entrapped

cells

become

necrotic

or

apoptotic

In

one

in-vitro

study

cells

encapsulated

in

a

fibrin

gel

were

seeded

onto

a

synthetic

PGA

scaffold

to

optimize

tissue

formation

and

organization

Following

fibrin

degradation

the

underlying

synthetic

scaffold

contributed

to

the

structural

integrity

of

the

developing

tissue

Additionally

hyaluronan

a

viscoelastic

and

broadly

biocompatible

material

that

plays

a

role

during

embryonic

cardiac

development

is

also

being

explored

as

a

potential

ECM

scaffold

material

Some

investigators

favor

enzymatically

decellularized

tissue

as

a

natural

scaffold

This

treatment

is

done

to

decrease

antigenicity

and

the

risk

of

calcification

-LRB-

both

of

which

are

enhanced

by

cells

and

their

debris

-RRB-

The

hope

with

a

decellularized

scaffold

is

that

preseeded

or

endogenous

circulating

cells

can

repopulate

such

a

scaffold

A

decellularized

allograft

or

xenograft

tissue

scaffold

may

best

serve

as

a

template

for

cellular

attachment

and

retain

many

of

the

mechanical

and

structural

properties

of

native

tissue

such

as

tensile

strength

and

unique

ECM

composition

Decellularized

porcine

small

intestinal

submucosa

-LRB-

SIS

-RRB-

has

been

studied

extensively

as

a

natural

resorbable

scaffold

material

that

does

not

require

cell

seeding

In

both

animal

and

human

clinical

studies

SIS

was

rapidly

remodeled

by

the

host

tissue

Useful

in

implants

ranging

from

particulate

material

related

to

the

bladder

to

sheets

that

might

be

used

to

repair

the

infarcted

left

ventricle

SIS

has

exhibited

good

vascularization

and

tissue

growth

without

excessive

inflammation

and

foreign

body

reaction

The

success

of

SIS

has

been

attributed

to

its

intrinsic

ECM

proteins

GAGs

cytokines

and

growth

factors

-LRB-

VEGF

and

TGF-β

-RRB-

CELLS

FOR

TISSUE

ENGINEERING

GENERAL

CONCEPTS

While

the

ultimate

goal

of

tissue

engineered

heart

valves

is

to

recapitulate

the

matrix

and

cells

found

in

native

tissue

there

exists

variability

in

potential

strategies

and

sources

of

cells

The

predominant

paradigm

provides

cell-seeded

scaffolds

-LRB-

biodegradable

or

natural

-RRB-

with

the

ingredients

and

environment

to

form

tissue

and

mature

in-vitro

in

a

bioreactor

in

order

to

generate

a

construct

which

is

then

implanted

in-vivo

in

the

desired

anatomic

location

-LRB-

recall

Fig.

Pathway

A

-RRB-

Potential

cellular

sources

for

seeding

of

scaffolds

to

fabricate

heart

valves

include

differentiated

tissue-specific

cells

-LRB-

such

as

endothelial

and/or

smooth

muscle

-RRB-

and

stem

cells

that

may

be

autologous

or

allogenic

In

a

clinical

study

an

in-vitro

endothelialization

procedure

in

which

femoropopliteal

arterial

grafts

composed

of

expanded

polytetrafluoroethylene

-LRB-

ePTFE

-RRB-

were

confluently

lined

with

cultured

autologous

endothelial

cells

before

implantation

was

assessed

for

its

ability

to

improve

the

long-term

patency

of

these

prosthetic

bypass

grafts

The

results

-LRB-

regrettably

not

compared

with

concurrent

unseeded

control

grafts

-RRB-

suggested

that

autologous

endothelial

cell

lining

improved

the

patency

of

these

small-diameter

vascular

grafts

Another

option

is

to

harness

the

potential

of

endogenous

cells

by

utilizing

decellularized

biological

scaffolds

that

contain

intact

ECM

and

other

chemical

signals

necessary

to

recruit

the

appropriate

cell

populations

-LRB-

recall

Fig.

Pathway

B

and

see

below

-RRB-

unseeded

scaffolds

that

could

attract

endogenous

cells

in-vivo

to

the

site

of

implantation

might

permit

bypassing

the

in-vitro

stage

of

cell

seeding

by

facilitating

repair

by

endogenous

cells

Nevertheless

presently

used

tissue

heart

valve

replacements

do

not

endothelialize

from

circulating

blood

to

a

degree

sufficient

to

provide

functional

benefit

This

holds

true

for

both

glutaraldehyde-pretreated

porcine

valves

in

which

the

barrier

to

endothelialization

may

result

from

toxicity

induced

by

residual

glutaraldehyde

and

cryopreserved

allograft

heart

valves

The

unique

properties

of

stem

cells

such

as

multipotency

and

capacity

for

self-renewal

make

them

attractive

cells

for

tissue

engineering

Stem

cells

are

found

in

the

bone

marrow

of

adults

including

hematopoietic

stem

cells

-LRB-

which

form

the

mature

blood

cells

-RRB-

endothelial

stem

cells

-LRB-

endothelial

progenitor

cells

which

form

components

of

the

cardiovascular

system

-RRB-

and

mesenchymal

stem

cells

-LRB-

which

form

bone

cartilage

muscle

fat

and

fibroblasts

-RRB-

Bone

marrow-derived

adult

somatic

stem

cells

are

an

attractive

cell

source

because

they

are

multipotential

cells

in

principle

capable

of

differentiation

transformation

and

regeneration

We

will

not

discuss

stem

cells

of

embryonic

origin

owing

to

the

complicated

scientific

ethical

and

moral

issues

surrounding

their

usage

Bone

marrow

comprises

an

ideal

cell

source

for

tissue

engineering

because

its

cells

are

easily

accessible

its

primary

isolate

is

a

cell

suspension

that

is

easier

to

process

and

less

prone

to

contamination

than

solid

tissue

and

bone

marrow

will

likely

be

the

primary

source

of

cells

for

endogenous

repopulation

Throughout

post-natal

life

both

bone

marrow-derived

and

organ-resident

adult

stem

cells

continuously

regenerate

some

tissues

-LRB-

such

as

skin

epithelium

intestinal

epithelium

blood

cells

and

liver

when

stimulated

-RRB-

Nevertheless

increasing

evidence

suggests

that

the

heart

and

the

brain

can

regenerate

some

of

their

mass

defying

the

conventional

wisdom

that

these

organs

can

not

replenish

cells

lost

as

a

result

of

maturation

senescence

and

injury

Endothelial

progenitor

cells

-LRB-

EPCs

-RRB-

are

bone

marrow-derived

hematopoietic

stem

cells

capable

of

differentiating

into

the

endothelial

cells

that

line

the

blood

vessels

and

cardiac

valves

EPCs

maintain

vascular

homeostasis

by

promoting

reendothelialization

after

endothelial

injury

and

neovascularization

after

tissue

ischemia

Endothelial

progenitor-derived

cells

obtained

from

peripheral

blood

have

been

expanded

in-vitro

and

seeded

on

a

three

dimensional

biodegradable

PGA-PLLA

scaffold

When

seeded

alone

they

maintained

an

endothelial

phenotype

for

the

entire

six-week

duration

of

implantation

and

when

co-seeded

with

smooth

muscle

cells

the

endothelial

progenitor

derived

endothelial

cells

formed

microvessels

on

the

scaffold

EPCs

obtained

from

peripheral

blood

have

also

been

used

to

line

small

diameter

vascular

grafts

Mesenchymal

stem

cells

-LRB-

MSCs

-RRB-

have

the

potential

for

differentiating

into

osteogenic

chondrogenic

adipogenic

and

myogenic

lineages

These

can

be

isolated

from

adult

bone

marrow

and

represent

another

adult

stem

cell

population

that

can

be

used

as

a

cellular

source

in

tissue

engineering

The

concept

of

continuous

replacement

of

connective

tissue

with

bone

marrow

mesenchymal

stem

cells

parallels

the

known

continuous

replacement

of

blood

by

bone

marrow

hematopoietic

cells

Cultured

MSCs

display

the

spindle

shape

morphology

characteristic

of

myofibroblasts

and

express

cell

markers

characteristic

of

VIC

Upon

implantation

these

cells

retain

profiles

identical

to

those

seen

in-vitro

Following

myocardial

infarction

in

a

mouse

model

injected

MSCs

improve

recovery

of

the

infarcted

tissue

and

were

thought

to

differentiate

into

cardiac

myocytes

endothelial

cells

and

vascular

smooth

muscle

cells

Marrow

progenitor

cells

or

multipotent

adult

progenitor

cells

-LRB-

MAPCs

-RRB-

have

many

attributes

of

MSCs

but

they

are

reported

to

expand

indefinitely

-LRB-

compared

to

million

fold

expansion

of

MSCs

-RRB-

and

may

have

lineage

potential

that

includes

ectodermal

and

endothelial

cell

types

making

them

similar

to

embryonic

stem

cells

BIOREACTORS

FOR

TISSUE

ENGINEERING

Mechanical

stimulation

through

the

use

of

bioreactors

during

in-vitro

tissue

development

is

widely

utilized

in

cardiovascular

tissue

engineering

for

improving

tissue

formation

organization

and

function

The

bioreactor

exposes

the

developing

tissue

to

mechanical

conditioning

primarily

through

cyclical

flow

and

pressure

changes

that

mimic

physiological

conditions

Bioreactors

have

been

developed

that

use

and

as

their

main

mechanical

cues

to

engineer

blood

vessels

and

heart

valves

For

the

engineering

of

heart

valves

a

diastolic

pulse

duplicator

bioreactor

has

been

developed

to

mimic

only

the

diastolic

phase

of

the

cardiac

cycle

resulting

in

dynamic

tissue

straining

Additionally

electrical

stimulation

-LRB-

designed

to

mimic

native

excitation-contraction

coupling

-RRB-

has

been

used

as

a

cue

to

enhance

the

structure

and

function

of

pulsatile

myocardium

The

optimal

conditioning

protocol

depends

on

numerous

parameters

such

as

the

sensitivity

of

the

cell

phenotype

to

mechanical

cues

the

scaffold

used

the

transfer

of

the

mechanical

cues

from

the

scaffold

to

the

cells

and

the

magnitude

and

type

of

mechanical

cues

KEY

IN-VIVO

STUDIES

IN

HEART

VALVE

TISSUE

ENGINEERING

Many

published

studies

have

used

in-vitro

methods

to

investigate

critical

variables

and

demonstrated

proof-of-principle

of

important

concepts

in

tissue

engineering

germane

to

heart

valves

Only

a

few

key

studies

have

been

conducted

using

synthetic

and

natural

scaffolds

in

animal

models

-LRB-

Table

-RRB-

Early

studies

focused

on

the

design

of

individual

valve

whereas

later

studies

emphasize

design

of

complete

valved

conduits

In

this

section

we

focus

on

studies

in

which

scaffolds

seeded

in-vitro

were

implanted

in-vivo

Following

this

section

we

discuss

the

possibility

that

the

recipient

can

provide

all

the

cells

needed

to

populate

and/or

remodel

a

scaffold

to

yield

a

functional

heart

valve

in-vivo

TABLE

Representative

animal

and

clinical

implant

studies

using

seeded

and

non-seeded

matrices.StudyScaffoldCellsSiteIn-vitro

seeding

-LRB-

A

-RRB-

Shinoka

-LRB-

-RRB-

Polyglycolic

acid

-LRB-

PGA

-RRB-

Autologous

ovine

endothelial

cells

and

fibroblastsReplacement

of

one

pulmonary

valve

-LRB-

PV

-RRB-

leaflet

in

sheep

-LRB-

B

-RRB-

Hoerstrup

-LRB-

-RRB-

-LRB-

-RRB-

coated

PGAAutologous

ovine

endothelial

cells

and

myofibroblastsReplacement

of

all

three

PV

leaflets

in

sheep

-LRB-

C

-RRB-

Steinhoff

-LRB-

-RRB-

Decellularized

pulmonary

sheep

valvesAutologous

ovine

endothelial

cells

and

myofibroblastsPV

conduits

implanted

into

sheep

-LRB-

D

-RRB-

Dohmen

-LRB-

-RRB-

Decellularized

cryopreserved

pulmonary

allograftAutologous

human

vascular

endothelial

cellsReconstruction

of

the

right

ventricular

outflow

tract

-LRB-

RVOT

-RRB-

in

a

human

patient

-LRB-

E

-RRB-

Perry

-LRB-

-RRB-

coated

PGAAutologous

ovine

mesenchymal

stem

cellsIn-vitro

only

no

in

vivo

implantation

-LRB-

F

-RRB-

Iwai

-LRB-

-RRB-

Poly

-LRB-

lactic-co-glycolic

acid

-RRB-

-LRB-

PLGA

-RRB-

compounded

with

collagen

microspongeAutologous

endothelial

and

smooth

muscle

cells

w

and

w/o

in-vitro

precellularizationPatch

implant

in

canine

pulmonary

artery

-LRB-

G

-RRB-

Sutherland

-LRB-

-RRB-

PGA

and

poly-L-lactic

acid

-LRB-

PLLA

-RRB-

Autologous

ovine

mesenchymal

stem

cellsReplacement

of

all

three

PV

leaflets

in

sheepIn-vivo

only

-LRB-

No

in-vitro

seeding

-RRB-

-LRB-

A

-RRB-

Matheny

-LRB-

-RRB-

Porcine

small

intestinal

submucosaN/AReplacement

of

one

PV

leaflet

in

a

pig

-LRB-

B

-RRB-

Elkins

-LRB-

-RRB-

Decellularized

-LRB-

using

SynerGraft

treatment

-RRB-

human

-LRB-

CryoValve

SG

-RRB-

and

sheep

pulmonary

valvesN/ASynerGraft-treated

and

cryopreserved

sheep

PVs

implanted

in

RVOT

in

sheep

CryoValve

SG

human

PVs

implanted

in

patients

-LRB-

C

-RRB-

Simon

-LRB-

-RRB-

Decellularized

porcine

Synergraft

valveN/AImplanted

in

RVOT

in

children

Implant

Studies

Using

Synthetic

Scaffolds

Seeded

In-Vitro

In

this

approach

a

bioabsorbable

polymer

provides

a

temporary

scaffold

until

cells

seeded

in-vitro

produce

their

own

matrix

proteins

-LRB-

see

Fig.

Pathway

A

-RRB-

The

biodegradable

polymer

PGA

and

related

compounds

have

been

used

as

biodegradable

synthetic

polymer

scaffolds

because

this

polymer

-LRB-

albeit

with

suboptimal

mechanical

properties

-RRB-

is

well

characterized

and

approved

by

the

FDA

in

sutures

and

other

devices

for

human

implantation

In

one

of

the

earliest

experiments

in

the

field

an

isolated

tissue

engineered

heart

valve

leaflet

was

implanted

in

the

pulmonary

position

of

sheep

using

a

PGA

scaffold

seeded

with

vascular

wall

cells

Histologic

evaluation

of

the

constructs

showed

development

of

an

ECM

endothelialization

of

the

surface

and

scaffold

remodeling

While

this

preliminary

experiment

showed

that

a

tissue

engineered

valve

leaflet

constructed

from

its

cellular

components

can

function

in

the

pulmonary

valve

position

the

resultant

engineered

tissue

heart

valve

cusps

were

thicker

stiffer

and

less

pliable

than

native

valves

To

alleviate

the

problem

of

scaffold

thickness

and

rigidity

Hoerstrup

et

al.

designed

a

scaffold

composite

that

combined

PGA

with

the

strong

flexible

-LRB-

-RRB-

formed

in

the

configuration

of

a

trileaflet

heart

valve

is

added

to

prolong

mechanical

integrity

because

PGA

degrades

faster

than

-LRB-

approximately

weeks

versus

weeks

respectively

-RRB-

This

scaffold

was

seeded

with

differentiated

autologous

vessel-derived

ovine

endothelial

cells

and

smooth

muscle

cells

To

simulate

a

biomimetic

environment

during

tissue

formation

the

constructs

were

grown

for

varying

time

points

in

an

in-vitro

pulse

duplicator

system

under

gradually

increasing

flow

and

pressure

conditions

while

controls

were

grown

in

static

nutrient

medium

After

days

of

in-vitro

culture

the

constructs

-LRB-

valves

-RRB-

grown

in

the

bioreactor

showed

significantly

increased

DNA

content

higher

formation

of

matrix

proteins

a

more

organized

histological

structure

and

more

favorable

mechanical

properties

than

did

constructs

grown

under

static

culture

conditions

Based

on

these

preliminary

in-vitro

studies

seeded

constructs

that

had

been

matured

in-vitro

in

the

bioreactor

for

days

were

implanted

in-vivo

as

a

pulmonary

valve

replacement

in

an

ovine

animal

model

After

weeks

in

vivo

the

polymer

had

been

degraded

and

replaced

by

a

partially

endothelialized

uniform

layered

tissue

with

layer-specific

ECM

predominance

similar

to

that

of

the

native

valve

including

a

layer

containing

elastin

near

the

inflow

surface

gylcosaminoglycans

centrally

and

a

fibrous

layer

with

abundant

collagen

near

the

outflow

surface

Mechanical

properties

were

comparable

to

those

of

native

tissue

at

weeks

Particularly

exciting

was

the

presence

of

a

trilaminar

structure

resembling

the

native

pulmonary

valve

which

indicates

that

dynamic

growth

remodeling

and

asymmetric

structural

differentiation

had

occurred

in-vivo

probably

regulated

by

the

mechanical

environment

Building

on

these

principles

an

autologous

trileaflet

heart

valve

was

created

from

a

biodegradable

synthetic

scaffold

-LSB-

PGA

and

poly-l-lactic

acid

-LRB-

PLLA

-RRB-

-RSB-

seeded

with

autologous

bone

marrow-derived

MSCs

The

cell-scaffold

construct

was

cultured

in-vitro

then

implanted

into

the

pulmonary

artery

position

of

sheep

for

up

to

months

The

results

showed

that

the

construct

remodeled

in-vivo

to

a

tissue

with

features

that

simulated

those

of

native

valves

In

particular

the

tissue-engineered

valves

displayed

a

trilaminar

distribution

of

cells

and

ECM

analogous

to

those

of

native

valves

myofibroblasts

immediately

below

the

endothelium

fibroblasts

expressing

vimentin

distributed

throughout

the

remainder

and

endothelialization

These

findings

support

the

potential

use

of

bone

marrow

derived

MSCs

as

a

cell

source

for

the

fabrication

of

heart

valves

The

editorial

accompanying

this

paper

emphasized

that

although

proof-of-concept

has

been

demonstrated

based

on

remodeling

of

the

construct

to

a

trilaminar

structure

following

implantation

there

remain

numerous

challenges

to

overcome

before

such

technology

can

be

used

in

human

patients

Implant

Studies

Using

Biologic

Scaffolds

Seeded

In-Vitro

An

especially

attractive

concept

is

that

of

a

native

valve

biological

scaffold

either

decellularized

or

allograft

that

has

been

seeded

with

autologous

cells

One

study

suggested

that

allogenic

decellularized

sheep

matrix

conduits

seeded

with

autologous

myofibroblast

and

endothelial

cells

may

yield

viable

valves

Following

almost

complete

removal

of

cells

control

unseeded

allogenic

acellular

valves

implanted

in

sheep

for

up

to

months

showed

partial

degeneration

and

no

interstitial

tissue

reconstitution

whereas

the

counterparts

seeded

with

autologous

vascular

wall

cells

were

reported

to

show

restitution

of

the

endothelial

cell

surface

myofibroblasts

and

matrix

synthesis

Owing

to

the

antigenicity

of

xenograft

ECM

proteins

xenograft

scaffolds

likely

provide

a

more

inflammatory

stimulus

than

allograft

scaffolds

exemplified

by

the

cryopreserved

homografts

presently

used

clinically

An

inflammatory

reaction

could

weaken

or

scar

the

heart

valve

scaffold

making

it

more

susceptible

to

biomechanical

damage

Therefore

human

allograft

heart

valves

either

decellularized

or

not

have

been

considered

as

an

alternative

scaffold

Presently

used

cryopreserved

allograft

valves

are

effectively

decellularized

following

several

months

of

in-vivo

function

yet

they

do

not

grow

remodel

exhibit

active

metabolic

functions

or

recellularize

even

following

long-term

function

Moreover

decellularization

may

alter

the

physical

properties

of

native

valves

and

newly

seeded

cells

may

initially

have

difficulty

growing

into

a

decellularized

matrix.FIGURE

A

representative

hypothesis

for

the

population

of

a

tissue

engineered

heart

valve

by

endogenous

cells

Key

processes

include

proliferation

differentiation

and

mobilization

of

endothelial

progenitor

cells

within

the

bone

marrow

followed

by

recruitment

in

the

blood

and

adhesion

to

the

valve

Subsequently

recruited

cells

might

undergo

an

epithelial

to

mesenchymal

transdifferentiation

within

the

valve

-LRB-

recapitulating

development

-RRB-

followed

by

differentiation

to

interstitial

cells

that

ultimately

synthesize

and

remodel

the

ECM

HARNESSING

THE

REPARATIVE

POTENTIAL

OF

CIRCULATING

ENDOGENOUS

CELLS

UNSEEDED

SCAFFOLDS

As

discussed

in

the

previous

section

the

basic

paradigm

of

tissue

engineering

uses

a

cell

seeded

scaffold

an

in-vitro

stage

of

tissue

formation

and

an

in-vivo

stage

of

tissue

growth

and

remodeling

-LRB-

see

Fig.

Pathway

A

-RRB-

In

this

section

we

examine

an

alternative

pathway

-LRB-

see

Fig.

Pathway

B

-RRB-

in

which

an

unseeded

scaffold

could

have

the

potential

for

attracting

circulating

precursor

cells

-LRB-

endothelial

and

mesenchymal

-RRB-

in-vivo

-LRB-

Fig.

-RRB-

Accumulating

evidence

suggests

that

circulating

endogenous

cells

can

be

recruited

in-vivo

to

adhere

to

intravascular

sites

via

a

pathway

that

likely

mimics

the

adherence

of

inflammatory

cells

to

the

endothelium

during

physiological

inflammation

EPCs

promote

endothelial

regeneration

in

dog

models

by

covering

implanted

Dacron

and

in

human

studies

by

covering

the

blood-contacting

surfaces

of

implanted

ventricular

assist

devices

and

homing

to

stents

that

have

been

coated

with

antibody

-LRB-

a

marker

found

on

EPCs

-RRB-

Recent

studies

have

suggested

therapeutic

potential

of

EPCs

in

humans

Ischemic

heart

disease

patients

with

naturally

higher

levels

of

EPCs

had

a

reduced

risk

of

death

from

cardiovascular

causes

Following

experimental

myocardial

infarction

bone

marrow

derived

cells

are

recruited

by

selective

homing

to

the

area

of

injury

It

has

been

suggested

that

intracoronary

infusion

of

EPCs

in

patients

with

acute

myocardial

infarction

might

potentially

contribute

to

restoring

myocardial

and

endothelial

function

to

the

damaged

area

Endogenous

stimuli

such

as

tissue

ischemia

and

exogenous

cytokines

promote

mobilization

of

EPCs

Patients

with

vascular

trauma

such

as

acute

myocardial

infarction

display

increased

numbers

of

EPCs

which

is

positively

correlated

to

elevated

plasma

VEGF

VEGF

and

other

angiogenic

growth

factors

such

as

fibroblast

growth

factor

and

stromal

cell-derived

growth

promote

EPC

mobilization

and

recruitment

One

potential

strategy

may

be

to

coat

a

scaffold

with

appropriate

cell-signaling

molecules

in

an

effort

to

encourage

EPC

adhesion

and

differentiation

An

experiment

utilizing

decellularized

porcine

aortic

valves

containing

fibronectin

and

hepatocyte

growth

factor

suggested

that

the

growth

factor

enhances

early

endothelial

cell

recruitment

and

coverage

of

the

grafts

Recent

experimental

evidence

suggests

that

human

bone

marrow

may

be

a

source

of

progenitor

cells

contributing

smooth

muscle-like

cells

to

adult

human

heart

valves

Like

endothelial

cells

smooth

muscle

cells

can

also

be

recruited

to

sites

of

vascular

injury

Experimental

evidence

in

mouse

models

suggests

that

bone

marrow

derived

smooth

muscle

cells

may

be

implicated

in

degenerative

aortic

and

atherosclerosis

Evidence

of

smooth

muscle

cell

recruitment

is

exemplified

by

a

prototype

tissue-engineered

vascular

graft

In

an

experiment

in

which

rat

arteries

were

acellularized

recellularized

with

endothelial

cells

and

implanted

as

grafts

in

the

femoral

artery

for

four

weeks

immunohistochemical

staining

of

explanted

grafts

demonstrated

a

complete

layer

of

endothelial

cells

on

the

luminal

surface

and

smooth

muscle

cell

repopulation

Since

smooth

muscle

cells

were

not

originally

seeded

onto

the

graft

the

authors

suggested

that

they

were

recruited

to

the

graft

from

the

bone

marrow

by

a

mechanism

that

involved

endothelial

cells

Unseeded

scaffolds

have

been

examined

experimentally

with

the

goal

of

achieving

in-vivo

recellularization

by

circulating

endogenous

cells

In

one

experiment

each

of

four

pigs

had

one

pulmonary

valve

leaflet

excised

and

replaced

with

a

leaflet

constructed

from

porcine

SIS

Histology

indicated

that

the

implanted

matrix

was

progressively

resorbed

and

replaced

by

fibrous

connective

tissue

that

had

features

of

adult

valve

Alternatively

a

biodegradable

graft

containing

collagen

microsponge

was

fabricated

and

tested

with

and

without

preseeding

In

both

cases

-LRB-

SIS

and

collagen

microsponge

-RRB-

there

was

no

thrombus

formation

the

scaffold

was

absorbed

and

there

was

endothelialization

parallel

smooth

muscle

cell

alignment

elastin

and

collagen

fibers

These

results

suggested

that

the

patch

promoted

in-situ

cellularization

and

regeneration

of

autologous

tissue

However

an

important

limitation

of

these

studies

was

that

the

implanted

leaflets

and

patches

were

small

cellularization

of

a

large

implant

may

be

less

efficient

CLINICAL

STUDIES

USING

ENGINEERED

MATRICES

AS

HEART

VALVES

In

this

section

we

examine

the

limited

number

of

studies

using

engineered

matrices

in

clinical

settings

One

study

used

a

decellularized

pulmonary

allograft

seeded

with

autologous

endothelial

cells

and

conditioned

in

bioreactor

to

reconstruct

the

right

ventricular

outflow

tract

of

adults

undergoing

the

Ross

procedure

According

to

the

investigators

based

on

a

one-year

follow-up

seeded

endothelial

cells

remained

on

the

construct

and

were

fully

functional

and

the

construct

mechanical

strength

was

maintained

There

was

no

calcification

and/or

thrombogenesis

However

whether

the

seeded

cells

contributed

to

valve

function

is

yet

uncertain

Despite

promising

results

in

animal

experiments

using

decellularized

xenograft

scaffolds

translation

to

humans

has

been

difficult

Clinical

applications

of

implanted

decellularized

xenograft

tissue

heart

valves

have

been

largely

unsuccessful

Histological

examination

of

decellularized

porcine

aortic

SynerGraft

valves

-LRB-

Cryolife

Inc.

-RRB-

implanted

for

months

in

sheep

without

in-vitro

preseeding

suggested

some

growth

of

host

cells

on

intact

leaflets

and

showed

a

lack

of

calcification

Simon

et

al.

used

SynerGraft

decellularized

porcine

heart

valves

as

valve

replacements

in

the

right

ventricular

outflow

tract

during

Ross

procedures

in

children

The

decellularized

valves

were

not

seeded

or

conditioned

in

a

bioreactor

before

implant

with

the

hope

that

the

unseeded

scaffold

could

attract

endogenous

cells

These

valves

had

a

high

rate

of

failure

examination

of

failed

valves

revealed

incomplete

initial

decellularization

lack

of

cell

repopularization

lack

of

endothelialization

severe

inflammation

fibrous

sheath

formation

calcification

and

severe

degeneration

of

both

leaflets

and

wall

A

recent

report

showed

a

case

with

infiltration

of

a

Synergraft

valve

by

inflammatory

cells

-LRB-

neutrophils

and

macrophages

-RRB-

at

weeks

post

implantation

CHALLENGES

FOR

FUTURE

TRANSLATION

TO

THE

CLINIC

Heart

valve

tissue

engineering

has

exciting

potential

but

many

unanswered

questions

and

challenges

remain

before

human

implantation

can

be

considered

A

successful

tissue

engineered

valve

must

be

vital

complex

dynamic

composed

of

specialized

cells

and

ECM

that

remodel

in

response

to

changes

in

local

mechanical

forces

and

have

ongoing

strength

flexibility

and

durability

beginning

at

the

instant

of

implantation

and

continuing

indefinitely

thereafter

A

schema

for

the

interrelationships

among

and

challenges

in

tissue

characterization

for

heart

valve

tissue

engineering

is

summarized

in

Fig.

To

provide

an

agenda

for

translating

the

notion

of

TEHVs

from

an

extraordinarily

interesting

research

curiosity

to

a

clinically

useful

surgical

tool

we

discuss

below

both

the

major

research

goals

i.e.

understanding

mechanisms

defining

animal

models

developing

biomarkers

developing

assays/tools

defining

surrogate

and

true

endpoints

and

the

major

clinical

goals

i.e.

characterizing

and

assuring

quality

tissue

constructs

accommodating

patient-to-patient

heterogeneity

in

tissue

remodeling

and

predicting

outcomes

as

early

as

possible

TABLE

Critical

challenges

to

clinical

translation

of

heart

valve

tissue

engineering.ChallengesStrategy

for

translationTEHV

components

and

function

are

complex

heterogeneous

and

dynamicDevelop

guidelines

for

the

pre-implantation

characterization

of

TEHV

structure

function

and

qualityTEHV

function

depends

upon

patient

response

to

implantation

and

integration

with

the

recipient

's

tissues

more

than

conventional

valve

replacementIdentify/validate

biomarkers

predictive

of

implant

success/failure

and

capable

of

non-invasive

in-vivo

monitoringIndividuals

differ

in

the

speed

and

effectiveness

of

their

tissue

remodelingAssess/control

patient

variability

in

tissue

remodeling

capabilityOwing

to

the

key

role

of

patient

response

animal

models

may

not

reliably

predict

human

outcomesValidate

suitable

animal

models

that

will

test

key

biological

processes

and

correlate

with

human

outcomesRemodeling

processes

after

implantation

may

release

or

change

seeded

cells

and

recruit

host

cellsDevelop

tools

to

monitor

the

fate

of

transplanted

and

endogenous

cells

-LRB-

location

function

viability

phenotype

-RRB-

FIGURE

Paradigm

for

translating

research

in

heart

valve

tissue

engineering

from

the

laboratory

to

the

clinic

Biomarkers

for

cell

and

tissue

characterization

in

conjunction

with

structural

chemical

and

molecular

information

obtained

via

in-vitro

and

in-vivo

models

are

necessary

for

understanding

key

biological

processes

in

tissue

engineering

and

regenerative

medicine

These

concepts

and

data

can

be

used

to

predict

and

measure

patient

success

and

failure

Data

from

clinical

experience

further

informs

the

development

of

appropriate

biomarkers

which

may

result

in

reassessment

of

the

appropriate

characterization

parameters

Numerous

steps

must

be

surmounted

in

the

laboratory

before

heart

valve

tissue

engineered

constructs

can

be

made

clinically

useful

Typical

biomaterial-tissue

interactions

in

medical

devices

such

as

thrombosis

infection

and

inflammatory

interactions

will

have

to

be

acceptable

Another

important

consideration

is

whether

calcification

the

major

pathologic

process

in

bioprosthetic

valve

degeneration

will

be

problematic

Evidence

suggests

that

calcification

may

not

be

a

major

problem

as

long

as

polymer

or

other

scaffold

is

resorbed

and/or

not

intrinsically

mineralizable

the

interstitial

cells

are

viable

and

the

ECM

is

capable

of

remodeling

While

studies

using

animal

models

such

as

sheep

are

promising

further

detailed

studies

will

be

needed

in

these

models

other

animal

models

and

in

humans

There

is

considerable

controversy

over

to

what

extent

results

from

available

animal

models

translate

directly

to

humans

and

the

most

suitable

animal

model

for

testing

tissue-engineered

valves

has

not

yet

been

determined

Sheep

for

example

produce

an

exuberant

fibrotic

response

to

cardiovascular

implants

valves

implanted

in

sheep

generally

overgrow

more

rapidly

with

fibrotic

tissue

than

they

do

in

humans

and

likely

overestimate

tissue

remodeling

relative

to

humans

Owing

to

immunologic

considerations

the

choice

of

an

animal

model

for

preclinical

testing

for

allogenic

or

xenogeneic

cell-based

therapies

presents

unique

challenges

A

key

consideration

is

that

currently

available

heart

valve

replacements

have

predictable

behavior

in

many

recipients

whereas

in-vivo

remodeling

of

tissue

engineered

heart

valves

will

likely

display

considerable

variability

among

patients

owing

to

heterogeneity

among

individuals

in

physiological

tissue

remodeling

potential

As

the

field

of

tissue

engineering

evolves

it

may

become

important

to

apply

principles

analogous

to

those

of

pharmacogenetics

a

field

which

seeks

to

understand

the

role

of

genetics

in

inter-individual

variation

in

drug

metabolism

Simply

stated

some

patients

might

not

appropriately

remodel

their

tissue-engineered

valves

and

this

could

lead

to

failure

-LRB-

Fig.

-RRB-

This

could

be

a

result

of

mutations

or

polymorphisms

in

key

proteins

central

to

remodeling

Indeed

as

implants

have

become

more

interactive

and

integrative

with

the

host

tissues

there

has

arisen

a

corresponding

need

to

understand

and

potentially

control

human

variation

in

different

facets

of

biomaterial-tissue

interaction

and

the

healing

process

To

accommodate

TEHVs

the

usual

mechanism

for

demonstrating

pre-clinical

safety

and

efficacy

of

medical

devices

and

biologics

may

need

to

be

altered

due

to

unpredictability

of

the

engineered

tissue

with

the

recipient

's

native

tissue.FIGURE

A

hypothesis

for

inter-individual

variability

in

tissue

remodeling

While

most

individuals

will

remodel

tissue

with

a

usual

speed

and

quality

of

remodeling

some

people

will

display

slow

and

poor

quality

of

remodeling

while

others

will

show

fast

and

better

quality

of

remodeling

Inadequate

remodeling

could

lead

to

implant

failure

and

its

consequences

for

the

patient

The

threshold

of

properties

needed

for

tissue

engineered

heart

valves

and

the

means

of

conducting

post-implantation

surveillance

of

the

patient

and

graft

need

to

consider

this

variability

Success

or

failure

may

be

followed

and

predicted

non-invasively

To

understand

monitor

and

potentially

control

patient-to-patient

differences

in

wound

healing

and

tissue

remodeling

capability

in-vivo

biomarkers

that

predict

implant

outcomes

must

be

identified

Conventional

and

innovative

invasive

and/or

non-invasive

anatomic

and

functional

imaging

modalities

will

certainly

be

important

tools

to

assess

success

and

failure

Specific

molecular

biomarkers

may

be

identified

and

validated

by

assessing

tissue

healing

and

remodeling

during

in-vitro

and

in-vivo

experiments

suitable

biomarkers

will

need

to

be

followed

in-vivo

possibly

via

chemical

assays

in

the

serum

or

urine

or

via

molecular

imaging

Key

targets

for

characterizing

tissue-engineered

constructs

include

tissue

composition

cellular

gene

expression

and

phenotype

ECM

key

effectors

of

tissue

remodeling

and

tissue

quality

For

example

researchers

are

currently

working

to

identify

serum-specific

biomarkers

of

ECM

remodeling

in

disease

such

as

MMPs

in

acute

coronary

syndromes

and

urine-based

biomarkers

for

cancers

of

the

breast

bladder

and

prostate

These

biomarkers

should

correlate

directly

with

success

and

failure

in

order

to

generate

surrogate

endpoints

namely

outcome

measurements

-LRB-

such

as

laboratory

assays

or

imaging

results

-RRB-

that

substitute

for

but

reflect

the

mechanism

of

a

significant

clinical

event

or

characteristic

-LRB-

such

as

regurgitation

stenosis

thromboembolism

calcification

infection

or

death

-RRB-

Validated

surrogate

end

points

could

be

assessed

in

an

individual

patient

in

order

to

predict

outcome

as

early

as

possible

in

the

patient

's

course

and

influence

necessary

changes

in

management

The

potential

for

molecular

imaging

is

particularly

exciting

in

this

regard

it

requires

the

identification

of

a

molecular

target

selection

of

a

ligand

that

binds

the

target

selection

of

an

appropriate

imaging

system

and

synthesis

of

a

molecular

imaging

agent

to

detect

the

desired

molecular

imaging

has

been

used

to

demonstrate

key

enzymatic

and

cellular

events

in

atherosclerosis

and

thrombosis

For

example

imaging

studies

on

inflammatory

markers

such

as

proteases

-LRB-

cathepsins

and

MMPs

-RRB-

activated

macrophages

-LRB-

expressing

iron

oxide

-RRB-

and

activated

endothelium

-LRB-

intercellular

and

vascular

adhesion

molecules

-RRB-

have

been

performed

in

atherosclerotic

mice

Molecular

imaging

can

probe

polymorphisms

of

ECM

gene

expression

in-vivo

in

models

of

cardiovascular

disease

and

can

potentially

be

translated

to

perform

real-time

in-vivo

characterization

of

scaffold

matrices

-LRB-

either

seeded

or

with

the

potential

of

attracting

endogenous

cells

-RRB-

implanted

in

animal

models

Other

imaging

modalities

such

as

optical

coherence

tomography

-LRB-

OCT

-RRB-

and

intravascular

ultrasound

-LRB-

IVUS

-RRB-

have

been

used

to

assess

collagen

content

of

coronary

atherosclerotic

plaque

allowing

real-time

in-vivo

analysis

without

tissue

sampling

Such

imaging

modalities

may

prove

useful

in

assessing

tissue

remodeling

for

heart

valve

tissue

engineering

applications

Another

important

laboratory

consideration

for

seeded

scaffolds

is

the

origin

of

the

cells

seeded

in-vitro

and

whether

the

seeded

cells

remain

viable

and

attached

to

the

scaffold

following

in-vivo

implantation

In

the

absence

of

in-vitro

cell

labeling

it

is

not

possible

to

ascertain

the

fate

of

preseeded

cells

and

the

precise

origin

of

the

cellular

phenotypes

observed

in

the

explanted

tissue

engineered

valves

Molecular

imaging

could

be

utilized

to

track

the

presence

migration

proliferation

and

function

of

bone

marrow

derived

progenitor

cells

used

to

seed

scaffolds

both

in-vitro

and

Moreover

molecular

resonance

imaging

-LRB-

MRI

-RRB-

of

magnetically

labeled

mesenchymal

stem

cells

injected

into

porcine

myocardium

has

been

performed

in-vivo

a

technique

which

can

potentially

be

expanded

to

study

magnetically

labeled

endothelial

and

mesenchymal

progenitor

cells

seeded

on

a

scaffold

and

implanted

into

an

animal

or

human

model

In

future

experiments

endothelial

progenitor

and

mesenchymal

stem

cells

might

be

labeled

during

the

in-vitro

stage

and

then

analyzed

using

molecular

imaging

to

ensure

that

they

differentiate

into

appropriate

cell

lineages

and

that

they

remain

functional

and

attached

to

the

scaffold

over

time

There

is

a

need

to

develop

clinical

guidelines

that

specify

how

to

characterize

the

safety

efficacy

and

quality

of

a

tissue

engineered

product

before

it

can

be

implanted

in

humans

Demonstration

of

long-term

efficacy

-LRB-

implantability

functionality

long-term

performance

-RRB-

and

safety

-LRB-

biocompatibility

durability

modes

of

failure

ease

of

monitoring

-RRB-

of

these

valves

in

humans

will

be

a

particular

challenge

Risk/benefit

relationships

of

engineered

tissue

may

be

less

predictable

than

those

of

accepted

technology

Since

contemporary

heart

valve

replacements

have

considerable

success

in

most

situations

-LRB-

not

withstanding

the

limitations

and

except

in

pediatrics

-RRB-

acceptance

of

tissue

engineering

by

the

surgical

community

may

be

slow

It

has

been

suggested

that

surgeons

will

consider

the

use

of

a

tissue-engineered

valve

in

a

patient

beyond

appropriately

controlled

clinical

research

only

after

the

lifetime

of

conventional

valve

substitutes

can

be

exceeded

with

a

high

degree

of

certainty

Another

key

need

is

the

development

of

science-based

approaches

to

the

characterization

of

fabricated/manufactured

engineered

tissue

products

in

general

and

heart

valves

in

particular

These

will

likely

include

measurement

of

mechanical

properties

of

the

scaffold

and

the

tissue-scaffold

complex

characterization

of

the

dynamic

cell

phenotypes

and

ECM

components

and

the

evolution

of

the

final

manufactured

product

including

shelf-life

stability

and

shipping

considerations

CONCLUSIONS

The

goal

of

heart

valve

tissue

engineering

is

to

regenerate

a

functional

structure

containing

endothelial

and

interstitial

cells

capable

of

continuously

remodeling

the

ECM

that

functions

structurally

and

biomechanically

as

a

valve

leaflet

Despite

an

exciting

potential

for

tissue

engineered

heart

valves

significant

technical

barriers

must

be

overcome

before

widespread

clinical

application

can

be

envisioned

Further

success

toward

a

clinically

useful

tissue

engineered

heart

valve

will

be

dependent

upon

additional

advances

in

biodegradable

polymers

stem

cell

manipulation

strategies

for

recreating

the

ECM

understanding

how

to

harvest

the

potential

of

endogenous

recruitment

of

cells

and

techniques

to

non-invasively

assess

the

speed

and

quality

of

tissue

healing

and

remodeling

This

need

is

likely

to

engender

a

host

of

novel

testing

strategies

and

methods

which

will

include

in-vitro

safety

studies

ex-vivo

performance

characterization

in

functional

testing

devices

akin

to

bioreactors

and

in-vivo

preclinical

studies

NOTE

ADDED

IN

PROOF

Several

studies

relevant

to

and

that

became

available

during

final

production

of

this

manuscript

may

interest

readers

Visconti

R.P.

Y.

Ebihara

A.C.

LaRue

P.A.

Fleming

T.C.

McQuinn

M.

Masuya

H.

Minamiguchi

R.R.

Markwald

M.

Ogawa

and

C.J.

Drake

An

invivo

analysis

of

hematopoietic

stem

cell

potential

hematopoietic

origin

of

cardiac

valve

interstitial

cells

Circ

Res

To

test

the

hypothesis

that

hematopoietic

stem

cells

-LRB-

HSC

-RRB-

may

be

a

source

of

adult

valve

interstitial

cells

single

lineage-negative

-LRB-

Lin

-RRB-

c-kit

-LRB-

-RRB-

Sca-l

-LRB-

-RRB-

cells

from

the

bone

marrow

of

mice

that

ubiquitously

express

enhanced

green

fluorescent

protein

-LRB-

EGFP

-RRB-

were

transplanted

into

a

lethally

irradiated

congenic

non-EGFP

mouse

Histological

analyses

of

valve

tissue

from

clonally

engrafted

recipient

mice

revealed

the

presence

of

numerous

EGFP

cells

within

host

valves

some

of

which

exhibited

synthetic

properties

characteristic

of

fibroblasts

-LRB-

expression

of

mRNA

for

procollagen

alpha

-RRB-

The

cells

were

shown

to

be

the

result

of

HSC-derived

cell

differentiation

and

not

fusion

with

host

somatic

cells

Together

these

findings

demonstrate

a

contribution

by

HSCs

to

the

adult

valve

interstitial

cell

population

in

mice

Cao

F.

S.

Lin

X.

Xie

P.

Ray

M.

Patel

X.

Zhang

M.

Drukker

S.J.

Dylla

A.J.

Connolly

X.

Chen

I.L.

Weissman

S.S.

Gambhir

and

J.C.

Wu

In

vivo

visualization

of

embryonic

stem

cell

survival

proliferation

and

migration

after

cardiac

delivery

Circulation

As

discussed

in

the

body

of

the

present

manuscript

monitoring

the

trafficking

and

function

of

stem

cells

in

vivo

remains

problematic

owing

to

limitations

of

conventional

histological

assays

and

imaging

modalities

A

recent

study

demonstrated

a

method

by

which

embryonic

stem

-LRB-

ES

-RRB-

cells

could

be

stably

transduced

with

a

lentiviral

vector

carrying

a

novel

triple-fusion

-LRB-

TF

-RRB-

reporter

gene

tracked

by

positron

emission

tomography

and

monitored

for

survival

proliferation

and

migration

This

imaging

platform

should

have

broad

applications

for

basic

research

and

clinical

studies

on

stem

cell

therapy

Kiernan

T.J.

Endothelial

progenitor

cells

in

where

are

we

now

Cardiovasc

Pathol

A

recent

brief

review

of

the

current

status

of

endothelial

progenitor

cells

-LRB-

EPCs

-RRB-

including

their

role

as

biomarkers

and

potential

therapeutic

applications

may

be

useful

to

the

reader

of

the

present

manuscript

The

authors

emphasize

critical

questions

relating

to

the

characterization

of

the

biological

phenotype

of

true

EPCs

and

the

mechanisms

of

interaction

of

EPCs

with

resident

cells

of

the

vascular

wall

heart valve remodeling
engineered tissue heart valves
biodegradable scaffold
circulating stem cells