cell sources for cardiac tissue engineering: in-vivo approach

7/25/2019 Cell Sources for Cardiac Tissue Engineering: In-vivo approach

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Cardiac Tissue Engineering: Proposal for an Improved In Vivo Approach

December 12, 2013

Daniel Houck

Michael Warren

Andrew Vienot

Introduction

In the development o 3D ti!!ue en"ineerin" !olution!, creation o cardiac ti!!ue o

!i"niicant volume will be re#uired$ %hi! will re#uire development! !peciicall& in the area! o

an"io"ene!i! and cell !timulation$ Without an"io"ene!i!, cardiac ti!!ue con!truction i! limited to

material! with cell thickne!! at or ewer than three cell!, ater which due to a lack o diu!ion

cell! will become necrotic '1($ )urrentl&, method! o va!culari*ation ma& be broken into two

main cate"orie!+ intrin!ic va!culari*ation development o blood ve!!el! in!ide the bod&-, and

e.trin!ic '3($ /econdl&, due to ti"htl& interconnected nature o cardiac cell! it i! nece!!ar& to

!timulate them in order or them to orm into the proper !hape$ In thi! area, the current method!

are electrical !timulation and mechanical !timulation 'DDD($ %he lon"er the cell i! the arther it

can contract and the ali"nment o the cell! indicate! how or"ani*ed the contraction! will be

'DDD($ ative heart cell! are "enerall& well ali"ned and elon"ated$ While there are multiple

dierin" avenue! bein" pur!ued in both, no !tudie! are bein" perormed at the uncture o the!e

two ield! the creation o cell con!truct! which are va!culari*ed to improve ho!t acceptance and

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are al!o electromechanicall& optimi*ed in order to improve the contractile re!pon!e and

ultimatel& the eection raction$

Necessity

A! o 2010, heart di!ea!e, in mo!t ca!e! m&ocardial inarction, i! con!i!tentl& the top

cau!e o death in the nited /tate!$ Appro.imatel& 245 o reported death! 2$46 million- were

due to di!ea!e! o the heart, and cardiova!cular di!ea!e co!t! the nited /tate! 272$8 billion

dollar! ever& &ear$ Alarmin"l&, proection! have the!e co!t! increa!in" over 2005 to 919 billion

dollar! b& 2030 '4($ %he advent o lon" term and co!t eective !olution! will become ever more

nece!!ar&$

Physiological effects of Cardiac Disease

%he e.tent o dama"e to the cardiova!cular !&!tem varie! with man& actor!, !uch a!

duration o cardiac incident, ti!!ue re!pon!e, and prior ti!!ue condition$ In order to develop

applicable treatment!, the mechani!m o cellular death and the con!e#uence! mu!t be di!cu!!ed

in detail$ %he mo!t widel& known t&pe o )VD i! acute m&ocardial inarction!, al!o known a!

heart attack!, re!ultin" rom prolon"ed i!chemia within the m&ocardial ti!!ue7-$ %he inarction

occur! re#uentl& due to pla#ue buildup within the coronar& arter&, preventin" down!tream blood

low$ %he patholo"ical mechani!m o m&ocardial necro!i! ollow! throu"h the i!chemic ca!cade

activated in re!pon!e to the reduction o blood low to the aected ti!!ue$ %he lack o o.&"en

prompt! multiple !ide+mechani!m! includin" anaerobic "l&col&!i!, h&drol&*ation b& l&!o!omal

activation, and apopto!i!'7-+9-($ nder anaerobic condition!, m&ocardioc&te! be"in to

breakdown hi"h+ener"& pho!phate! !uch a! adeno!ine tripho!phate A%:- into adeno!ine

dipho!phate! AD:-$ 3

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;i"ure 1 :atho"ene!i! o M&ocardial )ell Death

%he breakdown proce!! continue! until the pho!phate chain i! removed and adeno!ine i!

relea!ed$ %he reduction o A%: and AD: eect! intracellular proce!!e! !peciicall& !huttin"

down the !odium and calcium pump!


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intracellular protein! bindin" within m&ocardioc&te! have brou"ht attention to the po!!ibilit& o

blockin" the!e protein! to enhance ti!!ue !urvival 13-$

%he ran"e o cardiac ti!!ue de!truction varie! primaril& on the duration o i!chemic

condition!$ %&picall& minor inliction! to the cardiac ti!!ue occur ater 18+20 minute!= in

contra!t, maor ti!!ue death occur! ater three to our hour! 14-$ In other i!chemic aliction!,

!uch a! cerebral i!chemia, the e.tent o ti!!ue death i! va!tl& lower than that o cardiac i!chemia

14-$ With current technolo"& it i! po!!ible to retain normal unction ater a cardiova!cular event,

however ri!k! or additional inarction! and cardiac ailure ri!e! !i"niicantl&$ %hereore the

e.tent o ti!!ue death i! likel& to determine the e.tent o treatment$ In ca!e o complete cardiac

ailure, tran!plantation i! the onl& known procedure to prevent death$ With donor or"an !horta"e!

pla"uin" ho!pital! worldwide, new approache! and procedure! are needed in order to combat

)VD$

Disease Treatment

)urrentl&, treatment option! are de!i"ned with the "oal o a- re!torin" blood low, and b-

decrea!in" the amount and eect o the !car ti!!ue '6($

%wo main method! which re!tore blood low are thrombol&!i! and an"iopla!t&$ In

thrombol&!i!, u!in" either mechanical or chemical method!, the blocka"e i! broken down or

removed, re!torin" blood low$ In an"iopla!t&, commonl& known a! balloon, a mechanical device

i! u!ed to e.pand a collap!ed blood ve!!el, once a"ain re!torin" blood low '9($ Method! !uch a!

the!e decrea!e the amount o dama"e done to the m&ocardium, con!e#uentl& lowerin" the

amount o ventricular remodelin" and !car ti!!ue that develop! '


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%he current treatment )aptopril i! the onl& dru" aimin" to inhibit the ormation o !car

ti!!ue, and doe! !o b& inhibitin" an"ioten!in II+ which aid in ibrobla!t and colla"en ormation

'10($ However, the eicac& o thi! dru" i! !till conte!ted '10($

%he!e method! which are approved b& the ;DA, althou"h valuable, do not provide

mechani!m! to rever!e the ne"ative eect! o the inarction+ namel& !car ti!!ue development and

ventricular remodelin"$ )on!e#uentl&, the onl& eective treatment option or patient! with late+

!ta"e heart ailure i! heart tran!plantation '11($ %hi! ha! numerou! i!!ue! includin" the need or

immune+!uppre!!ant!, ailure o the "rat, and i! urther limited b& the low number o donor!

'12($ %he !hortcomin"! o the!e therap& method! provide opportunitie! or ti!!ue en"ineer! to

provide meanin"ul product! with which to improve #ualit& o lie and in a !econdar& manner

provide model! with which to te!t urther product! de!tined or cardiac u!e!$

>ptimization in Tissue Engineering

Electrical timulation

!echanical timulation

%he inherent d&namic! o the heart make mechanical !timulation a clear actor in cardiac

cell "rowth$ %he con!tant mechanical !tre!! applied to natural heart ti!!ue ha! been linked to

man& !i"nal! or "ene !timulation and protein !&nthe!i!$ %here i! evidence that mechanical

!timulation cau!e! !everal "rowth actor! to be created and relea!ed in cardiac ti!!ue$ %he!e

"rowth actor! !i"niicantl& aect the !tructure and unctionalit& o the cardiac ti!!ue$ ;or

en"ineered cardiac ti!!ue, two maor t&pe! o mechanical !timulation have been !tudied applied

pre!!ure and !tretch$ ?oth t&pe! o mechanical !timulation have been !hown to improve the

ormation and behavior o en"ineered cardiac ti!!ue4,2$

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Applied Pressure

Applied pre!!ure !timulation can be evenl& applied to en"ineered cardiac ti!!ue b& a

bioreactor containin" a pi!ton device$ /tudie! have been done comparin" the cellular re!pon!e!

to con!tant compre!!ion, intermittent compre!!ion, and no compre!!ion$ ;i"ure 4 !how! ima"e!

comparin" cardiom&oc&te! that have been !timulated with each t&pe o compre!!ion or our

da&!$ )ontinuou! compre!!ion cau!e! the cardiac cell! to become round$ ?ecau!e the elon"ated

!hape o cardiac cell! i! related to the pul!ation o the ti!!ue, the round !hape i! indicative o

poor cardiac ti!!ue behavior$ %he cell! the e.perienced intermittent compre!!ion became

elon"ated and looked !imilar to the control cell! that received no !timulation$ %he!e cell! are

much more likel& to behave like natural cardiac ti!!ue$

Applied tretching

the eect o mechanical !tretch on en"ineered cardiac ti!!ue i! the other widel& !tudied

t&pe o mechanical !timulation$ It i! believed that the natural beatin" o the heart induce!

!tretchin" which tri""er! the "rowth o cardiom&oc&te!2

$ %i!!ue en"ineer! attempt to mimic thi!

!timulation in cultured cell! to create unctionin" cardiac ti!!ue$ ;i"ure 9 !how! the de!i"n o a

bioreactor u!ed to appl& !tretchin" !timulation to cardiac ti!!ue$ %he reactor wa! u!ed in an

e.periment that wa! ocu!ed on determinin" the eect o chronic !tretch on cardiac ti!!ue$ In the

e.periment, cell! were cultured or our da&!, and then e.po!ed to unidirectional !tretch in thi!

device or a total o !i. da&!$ %he amplitude o the !tretch wa! varied rom 15 o the ori"inal

area to 205 o the area to determine the optimum$ %he eect! o the !tretch on the en"ineered

cardiac ti!!ue were monitored and the re!ult! are di!pla&ed in i"ure! < and 10$ ;i"ure < !how!

an ima"e o a Hemato.&lin+eo!in !tained !ection o the both the !tretched and un+!tretched

cardiom&oc&te!$ %he i"ure !how! that the !tretched cardiom&oc&te! have "rown noticeabl&

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lon"er and more or"ani*ed$ %hi! re!ult i! conirmed b& i"ure 10 which "raphicall& !how! the

width dierence between !tretched and un+!tretched cardiac cell!$ %he hi"her percenta"e o un+

!tretched cell! in the low width ran"e indicate that the !tretchin" induced the cell! to elon"ate

like natural cardiac ti!!ue$ %hi! improved

!tructure i! likel& to po!itivel& aect the cardiac

behavior o the cell!$ %he chronic !tretch wa!

al!o reported to have an eect on the @ADA

and protein cell ratio2$ %hi! i! "enerall&

re"arded to improve the cardiac behavior o

cell!$ %he combined re!ult! o thi! e.periment

indicate that the behavior o cardiom&oc&te!

cultured in vitro are more like native ti!!ue when

the& are applied to chronic !tretch$ /tretchin"

mechanical !timulation appear! to po!itivel&

contribute to the contractile unction o en"ineered

cardiac ti!!ue$

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Figure 9:Image comparing un-stretched (left) and stretched (right) cardiac tissue2

Figure 8:Bioreactor used for strech of

cardiac tissue2

Figure 10:Effect of stretch on cell size2


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Angiogenesis

An"io"ene!i! i! the proce!! b& which new microcirculator& ve!!el! "row and !prout rom

pre+e.i!tin" ve!!el! '2($ %hi! i! dier! rom both embr&onic and po!t+natal va!culo"ene!i! in

which an"io"enic ve!!el! are created either de novo in the ca!e o embr&onic, or b& circulatin"

an"iobla!t!$ Much o the re!earch into an"io"ene!i! ha! been perormed b& re!earcher! in cancer

medicine, a! a ke& i!!ue with tumor cell! i! their abilit& to promote an"io"ene!i!, thu! allowin"

them to "row pa!t diu!ion limitation! '22($ Bectivel&, in the proce!! o an"io"ene!i!

endothelial cell! prolierate under the control o "rowth actor!, mechanical, and chemical

environment!, and !prout new ve!!el! rom pre+e.i!tin" ve!!el! '2($ Ater thi!, the va!cular

network i! !tabili*ed b& !mooth mu!cle cell!$ Multiple va!cular endothelial "rowth actor

i!oorm! VBC;- are nece!!ar& durin" the proce!! alon" with dierin" placental, and ibrobla!t

"rowth actor! '2($ Mana"ement o the!e var&in" ph&!iolo"ical actor! "reatl& complicate

va!culari*ation attempt!, with current attempt! at in vitro va!culari*ation producin" limited,

capillar&+like micro ve!!el!E '3($

In vitro va!culari*ation i! the "oal o creatin" a va!culari*ed ti!!ue, in lab, which can then

be implanted or "rated to the patient$ Ater doin" !o the "oal i! that the e.i!tin" va!culature will

ino!culate the con!truct in a timel& manner !o that the con!truct doe! not become h&po.ic$ %he

method o creatin" va!culari*ed !heet! i! accompli!hed b& !upplementin" cardioc&te !heet! or

!caold! with endothelial cell! and peric&te! '3($ While the!e pre+va!culari*ed cell !heet!

perorm better than !imple !heet!, the& !till ace the !ame i!!ue o needin" timel& ino!culation in

order to !urvive$ Ino!culation i! the proce!! b& which the artiiciall& created con!truct i! oined

into the e.i!tin" circulator& !&!tem$ /tudie! have thi! takin" up to !everal da&!, a timerame

which cau!e! i!chemic cell death '3($ Method! which have been ound to decrea!e cell death

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have been to include an"io"enic actor! !uch a! VBC;, platelet+derived "rowth actor, ibrobla!t

"rowth actor, and other! to decrea!e the time it take! to ino!culate con!truct '3($ In combination

with the development o capillarie!, method! have been ound to "uide the ormation o micro

e!!el! in !uch wa&! that the& are uniorm '2($ %hi! i! a nece!!ar& criteria in the ormation o

uniorm cardiac ti!!ue$ %he primar& method! u!ed or "uidin" va!culari*ation in vitro i! the u!e

o porou! or patterned !caold! which can be !eeded with an"io"enic c&tokine! which create a

pro+an"io"enic microenvironment '2($ %hi! methodolo"& make! the creation ti"htl& controlled

con!truct! po!!ible, a! one can !ee the developin" ti!!ue create more robu!t !caold!= however, it

ace! a ke& !hortcomin" in the inabilit& to properl& ino!culate in a timel& a!hion$ Intrin!ic

va!culari*ation method! aim to combat thi!$

A! the name !u""e!t!, intrin!ic va!culari*ation approache! allow u!e the bod&F! own

an"io"ene!i!, a! current method! aim ir!t to develop a va!cular !&!tem and then add the

nece!!ar& cell!$ )urrentl&, a primar& area o re!earch in thi! methodolo"& i! the u!a"e o

arteriovenou! and low throu"h chamber!$ )onceptuall&, the!e unction b& con!tructin" a

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Figure 1 [16]


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va!cular pedicle, and i!olatin" it rom the !urroundin" ti!!ue u!in" a pol&carbonate chamber '3($

In thi! !cenario, new microcirculator& ve!!el! and va!culature !prout rom the i!olated

va!culature '3($ Ideall&, thi! chamber i! !upplied with !tem or pro"enitor cell!, which would then

orm the cardiac ti!!ue '23($ @e!earcher! ound however that i !tem or pro"enitor are placed in

the chamber at the be"innin" o the e.periment, then h&po.ia occur! a! would be e.pected$ %o

combat thi!, %ilkorn et al$ created a !caold which allow them to dela& the in!ertion o cell! until

the& !aw !uicient va!culari*ation o the con!truct+ around da& 7$ At thi! point, pro"enitor cell!

were inected into the !caold which had the nece!!ar& va!culature to !upport them$ %ilkorn et al

demon!trated that implanted m&obla!t !urvival in an in vivo ti!!ue en"ineerin" con!truct i!

po!itivel& correlated to the va!cularit& o the con!tructE$

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:o!!ibl& the mo!t ambitiou! con!truct methodolo"& i! the decellulari*ed heart$ :ioneered

b& >tt et al, the team h&pothe!i*ed that the be!t va!culari*ation wa! provided b& nature '9($

)on!e#uentl&, the team developed a peru!tion decellulari*ation protocol$ @at heart! were

decellulari*ed u!in" a combination o three deter"ent !olution!, :BC in deioni*ed water, 15

%riton G+100 deioni*ed water, and 15 /D/ in deioni*ed water$ Ater thi! proce!!, all cellular

con!tituent! were removed, while )olla"en! I and III, laminin, and ibronectin remained intact$

Al!o, thi! method circumvent! entirel& the i!!ue o orientation and phenot&pe, a! the orientation

o the m&ocardial e.tracellular matri. wa! pre!erved$ Ater preliminar& te!tin", re!hl& i!olated

neonatal cardiac cell! and a mi.ture o other cell t&pe! were !eeded via intramural inection, and

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endot

helial cell! and media were peru!ed throu"h the va!cular conduit! '9($

Figure 2 [8]

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Ater ei"ht da&!, the recon!tructed or"an !howed electric and contractile re!pon!e!+ two

ke& indicator! o properl& unctionin" cardiac ti!!ue '9($ %he con!truct wa! maintained via ei"ht

dierent con!truct! or 29 da&!, and ater da& 9 mechanical te!tin" !howed that the con!truct!

could "enerate pump unction e#uivalent to 25 o an adult or 285 o a 16+week etal heart$

While thi! method i! promi!in", it !till ace! man& o the !hortcomin"! !een b& other method!$

?eneit! are that it appear! to be the mo!t promi!in" method o creatin" the nece!!ar& va!cular

!&!tem and "eometr&= however, the two maor i!!ue! it mu!t !till ace i! the procurement o cell!,

and al!o the development o mechanical unction to a de"ree that would be viable$ In the

e.periment, neonatal cardiac cell! and endothelial cell! were obtained in amount! which would

be impo!!ible to replicate currentl& in human trial! 2.107 endothelial cell! and a mi.ture o

78.106 neonatal cardiom&oc&te!,ibroc&te!, endothelial cell!, and !mooth mu!cle cell!-$ Al!o,

while the development o mechanical unction wa! notable+ ater ei"ht da&! it had the eection

capabilitie! e#uatin" to 25 o a adult heart, thi! i! a in!uicient level or tran!plantation$ ?a!ed

on the data that thi! e#uated to 16 week!, ba!ed on a linear "rowth model thi! heart would

re#uire appro.imatel& 400 da&! to reach the pump unctionalit& o a workin" adult heart$ A third

i!!ue which i! important i! lowerin" the immuno"enicit& o the !caold !o that it ma& be

po!!ible to re!eed a cadaver heart with autolo"ou! cell! to the per!on in need$

Aim ": Correlate electrical and mechanical stimulation to prior results

Blectromechanical !timulation ha! been !hown to be a maor inluence in the behavior o

developin" cardiac ti!!ue$ %here have been !everal previou! !tudie! characteri*in" the eect o

electrical and mechanical !timulation individuall&$ %he ir!t aim or thi! e.periment i! to

conirm that the procedure u!ed to culture the cardiac ti!!ue corre!pond! to the re!ult! predicted

b& the literature$ %hi! !tep i! intended to !how that the method u!ed to culture the ti!!ue i! valid

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and account or di!turbance! includin" cell !ource!, !caold t&pe, and the method b& which the

cell! are !timulated$ iterature predict! that the optimal electrical !timulation will be at an

amplitude o 3 Vcm and a re#uenc& o 3 H* and that the optimal mechanical !tretch will be at

85 !tretch and 1$8 H*$ %he optimum electromechanical !timulation i! e.pected to be near the!e

value!, but ma& var& dependin" on the !peciic e.periment !etup$

%he ir!t !tep or thi! aim i! to develop a bioreactor capable o !imultaneou!l& providin"

electrical and mechanical !timulation to the "rowin" cardiac ti!!ue$ %he bioreactor will be

con!tructed b& modi&in" a method u!ed b& ;ink B%$ A$ %hi! reactor i! capable o !tretchin"

the cardiac ti!!ue up to 205 o it! ori"inal len"th u!in" !tretchin" rod! attached to the ti!!ue1$

)arbon rod electrode! will be placed len"thwi!e alon" the bioreactor to provide a con!tant

electrical ield "radient acro!! the !caold$ )are will be taken to en!ure that the movement o

the cell! i! not re!tricted and the cell! can be ob!erved throu"hout the e.periment$

Figure 3 Left- stretching apparatus. Right- carbon electrodes

>nce a unctionin" apparatu! to provide the nece!!ar& electromechanical !timulation ha!

be obtained, the optimal parameter! or the !timulation can be determined$ %he electrical and

mechanical !timulation will be individuall& inve!ti"ated and the optimal ma"nitude and

re#uenc& will be obtained or each$ ;or each e.periment a cardiom&oc&te monola&er will be

!eeded into the reactor with /kCM+2 medium and held at 37 o)$

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%he electrical !timulation procedure will be de!i"ned to mimic that o %andon B% A$2

%he ti!!ue monola&er will be !ubu"ated to !#uare electrical pul!e! 24 hour! ater !eedin"$ %he

!timulation will continue or three da&! beore re!ult! are collected$ %he amplitude o the

electrical !timulation will be varied orm 1+9 Vcm and the re#uenc& will be varied rom 1+8 H*$

%he mechanical !timulation procedure will be ba!ed o o the method! u!ed b& ;ink B% A$ to

implement mechanical !tretch$ nidirectional !tretch will be applied to the ti!!ue our da&! ater

the cell! were !eeded in the !caold$ %he !tretchin" will continue or !i. da&! and then the

re!ult! will be collected$ %he !tretch amplitude will be varied rom 15 to 205 o the len"th o

the ti!!ue and the re#uenc& varied rom 1+8 H*$

B.citation thre!hold, ma.imum capture rate, amplitude o contraction, and hi!tolo"& will

be u!ed a! metric! to determine the optimum !timulation or the ti!!ue monola&er!$ %he

e.citation thre!hold i! a mea!urement o the ti!!ueF! electrical e.citabilit&, and i! determined b&

the minimum amplitude re#uired to cau!e the ti!!ue to beat at 60 beat! per minute$ A low

e.citation thre!hold value i! preerred or optimal cardiac ti!!ue$ %he ma.imum capture rate i!

the re#uenc& that the ti!!ue will contract when e.po!ed to electrical pul!e! with an amplitude

1$8 time! the e.citation thre!hold$ A hi"h capture rate value indicate! !tron" intercellular

connectivit& and i! de!ired or cardiac ti!!ue$ %he amplitude o contraction i! mea!ured in

percent chan"e in the ti!!ueF! !urace area durin" a contraction$ A lar"e amplitude o contraction

indicate! more unctional cardiac ti!!ue$ %he hi!tolo"& o the cultured ti!!ue will be compared to

that o natural ti!!ue$ %he hi!tolo"& will be u!ed to "ive a #ualitative anal&!i! o the optimum

ti!!ue$

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Aim #: Com$ine electrical and mechanical stimulation

Ater aim 1 ha! !hown the validit& o the !timulation procedure, the eect o combinin"

the electrical and mechanical !timulation will be inve!ti"ated$ %he cell! will be !eeded in the

reactor, ollowin" the !ame procedure a! in aim 1$ Ater the cell! are cultured or 24 hour! the

ti!!ue will be e.po!ed to electrical !timulation or three da&!$ ;our da&! ater the cell! were

!eeded= unidirectional mechanical !tretch !timulation will be applied in addition to the electrical

!timulation$ %he cell! will be let in the culture or a total o 10 da&! beore the& are lited and

mea!urement! are taken$ %he !ame metric! u!ed in aim 1 will be u!ed to determine the optimum

!timulation or cardiac ti!!ue!$ %he re!ult! o aim 2 will provide novel inormation re"ardin" the

eect that multiple t&pe! o !timulation ha! on cardiac ti!!ue$

Aim %: Increasing Construct Thic&ness and Angiogenesis

Havin" created electromechanicall& optimi*ed cell !heet! via aim! one J two, the "oal o

aim three i! to a create a thicker cell con!ruct b& !tackin" !heet! o cardiom&oc&te!+ with

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!par!el& cultured aortic endothelial cell !heet! placed in between to promote an"io"ene!i!$

Without an& an"io"ene!i! promoter!, a! noted earlier the limit o con!truct thickne!! wa!

appro.imatel& 3 cell! thick$ /chimi*u et alreco"ni*ed the need or va!culari*ation in the!e

con!truct! and be"an e.perimentin" with the u!e o endothelial cell la&er!, with which he wa!

able to create a 8 la&er thick con!truct$ ?uildin" o o thi!, we de!ire to re!earch whether a

!i"niicant increa!e in value i! ound b& perormin" thi! e.periment with electromechanicall&

!timulated cell!$ We will con!ider thi! aim a !ucce!! "ranted we !ee hi"her ma.imum capture

rate, contraction amplitude, and e.citation thre!hold than the control model!$ >ur control model!

will be dierntiated b& lackin" electrochemical optimi*ation$

%he novel method de!i"ned b& /chimi*u relied upon two dierent, temperature

dependent material! ibrino"en "el, which ha! a meltin" point at 37K) and a adherin"

temperature o 20K), and :ol& +i!oproplacr&lamide- which ha! a relea!e temp o 20K) and and

adehe!ion temperature o 32+ 37K)$ Due to the nature o the!e material!, it i! po!!ible to create

lit cell !heet! and !tack them in a or"ani*ed manner$ An overview o the method i! !een in i"ure

GGG$

Aim 4 Animal Model

%hrou"h the indin"! and re!ult! o the previou!

aim!, it i! nece!!ar& to demon!trate the clinical potential u!in" non+

human model!$ :reviou! work ha! !hown promi!e in re"ard! to !howin"

!u!tained development o the tran!planted la&er!$ %alk about previou! workE

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+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

ikewi!e or a pre!ent model, demon!tratin" the abilit& o the optimi*ed cell la&er! to re"enerate

heart unction would be the ultimate "oal$

!aterials and !ethods

Animals

%he !tud& will emplo& u!in" a !mall+animal model con!i!tin" o adult male ewi! rat! around

12+14 week! old$ %he ewi! rat !train i! the mo!t common !train ound within the ti!!ue

en"ineerin" ield and i! readil& available or purcha!e$ cite :h&!iolo"icall&, the rat model will

e.hibit health& condition! !uch a! wei"hin" around 380 "ram! and recordin" a heart rate o

around 400 per minute$ L o animal! needed or "roupin":ower anal&!i!

All animal te!tin" will be conducted in compliance with the Animal Welare Act and the Cuide

or the )are and !e o aborator& Animal!$

Artificial Infarction Procedure

In order to produce an inarction event within the animal model, the u!e o an eicient and

!u!tained procedure i! re#uired$ Man& method! o inducin" an inarction contain hi"h mortalit&

rate! and have let !ome !ubect! unable or te!tin"$ >ne !uch method that i! commonl& u!ed

with improved mortalit& rate i! the li"ation o the let coronar& de!cendin" arter&$

%he !ur"er& i! perormed under "eneral ane!the!ia in which the animal! will under"o intubation$

A thoracothom& i! perormed on the let !ide o the animal, b& cuttin" the ith and !i.th rib!$

%he thoracic cavit& will be arran"ed to be able avoid inur& to the heart$ %he !ur"eon will appl& a

!mall blade an open the pericardial membrane allowin" or a cardiac holder to be placed$$ %he

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AD will be locali*ed 1N2 mm below the unction o the pulmonar& conu! and the let atrial

appenda"e ;i"$ 2and ;i"$ 3-$

%he let atrial appenda"e and the pulmonar& conu! can be identiied b& their appearance and

location above the let ventricle$ %he li"ation will occur u!in" a !uture with a curve needle$

Durin" the in!ertion o the needle, the operator !hould be careul to avoid puncture o the let

atrial appenda"e or the pulmonar& conu!, which can cau!e ma!!ive hemorrha"e$ Ater li"ation

the heart will be returned back to the thoracic cavit& and the che!t will be clo!ed$ :o!toperative

care will be continued u!in" anal"e!ia and hemod&namic monitorin" until e.tubation and ull

recover& rom ane!the!ia$

Transplantation and Post 'P Analysis

%ran!plantation o the abricated cell la&er! will occur ater !ubect! are ull& recovered$ %he

procedure o tran!plantation i! !imilar to previou! work! and will e.pect to produce the !ame

re!ult! in term! o mortalit& rate$

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http://www.sciencedirect.com/science/article/pii/S1553838905000928#fig2http://www.sciencedirect.com/science/article/pii/S1553838905000928#fig2http://www.sciencedirect.com/science/article/pii/S1553838905000928#fig3http://www.sciencedirect.com/science/article/pii/S1553838905000928#fig3http://www.sciencedirect.com/science/article/pii/S1553838905000928#fig2


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Ater the po!t+inarctedtran!plant !ubect! recover, the e.tent o m&ocardial dama"e or recover&

will need to be #uantiied$ %hrou"h the non+inva!ive and applicabilit& to !mall animal model, the

u!e o B)C or Blectrocardio"raph& will be implemented in order to determine the dama"e$

B)CF! !peciicall& mea!ure the !tren"th and timin" o electrical !i"nal! in the heart$ ;rom the!e

mea!urement! the eection raction o the heart can be ound$ ;urther anal&!i! mu!t be

(uture )or&

)on!tant D) electric ield

Mechanical pre!!ure !timulation

Amplitude J re#uenc& o !timuli chan"in" over time

/tackin" thicker cell la&er!

Conclusion

21

cell sources for cardiac tissue engineering: in-vivo approach

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