myocardial tissue reconstruction: the cell sheet ... · cell sheet engineering for myocardial...

171Inflammation and Regeneration　Vol.27 No.3 MAY 2007

Mini Review

Myocardial tissue reconstruction: The cell sheetengineering approach

Hidekazu Sekine1), Tatsuya Shimizu1), Joseph Yang1),Masayuki Yamato1), Eiji Kobayashi2), and Teruo Okano＊,1)1)Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, Tokyo, Japan2)Division of Organ Replacement Research, Center for Molecular Medicine, Jichi Medical School, Tochigi, Japan

Regenerative medicine has currently emerged as one of the most promising therapies for patients suffer-

ing from severe heart failure. Direct implantation of isolated skeletal myoblasts and bone-marrow derived

cells has already been clinically performed and research on fabricating three-dimensional (3-D) cardiac

grafts using tissue engineering technologies has also now been initiated. In contrast to scaffold-based meth-

ods, we have proposed cell sheet-based tissue engineering, which involves stacking confluently cultured

cell sheets to construct 3-D cell-dense tissues. Upon layering, individual cardiomyocyte sheets integrate to

form a single, continuous, cell-dense tissue that resembles native cardiac muscle. When transplanted directly

to host hearts, these engineered myocardial tissues are able to form morphological connections to the host

with the presence of functional gap junctions. The transplantation of layered cardiomyocyte sheets has also

been shown to be able to repair damaged cardiac muscle. As a next step, we have attempted to promote

neovascularization within bioengineered myocardial tissues to overcome the longstanding limitations on

engineered tissue thickness. Finally as a possible advanced therapy, we are now trying to fabricate func-

tional myocardial tubes which may have the potential for circulatory support. Cell sheet engineering tech-

nologies therefore shows enormous promise as a novel approach in the field of myocardial tissue engineer-

ing.

Rec.10/20/2006, Acc.12/18/2006, pp171-176

＊Correspondence should be addressed:Teruo Okano, Ph.D., Institute of Advanced Biomedical Engineering and Science Tokyo Women's Medical University, 8-1Kawada-cho, Shinjuku-ku, Tokyo 162-8666 Japan. e-mail: [email protected]

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Introduction　For the severe heart failure generally associated with ischemic

disease, heart transplantation is the ultimate method of treatment

for patients. However, a shortage in donor organs remains a

longstanding and serious problem throughout the world. In ad-

dition, in the development of artificial heart systems such as

炎症・再生　Vol.23 No.1 2003172

Transplantation

Cell sheet

Biodegradable scaffold

A A

D

C

Direct injection

Mold

B B

Polymer solution + cells

Polymer solution + cells

Fig.1　Myocardial tissue engineering approachesA: Isolated cells are poured into prefabricated, highly porous scaffolds (A).The scaffolds undergo biodegradation, and extracellular matrix (ECM) occu-pies the spaces between the cells, leading to 3-D tissues.B: A mixture of isolated cells and biodegradable molecules is poured into anappropriate mold, and then the molecules are polymerized. The construct isregenerated into tissues.C: Mixtures of cells suspended within polymer solutions are injected directlyinto the damaged myocardium.D: Intact cell sheets released from temperature-responsive culture surfacesare layered. Cell sheets adhere to each other via biological ECM, resulting in3-D tissues containing no biodegradable scaffolds.

mechanical temporary assist devices or left ventricular assist

devices (LVADs), there are also problems related to thromboem-

bolism, infection, and finite durability. Regenerative therapies

have therefore been pursued as an alternative approach and have

presented new possibilities for the repair of injured myocardium.

　Recently, the direct injection of either autologous skeletal

myoblasts or bone-marrow derived cells, has already been ex-

amined in clinical trials as an alternative cell source to cardio-

myocytes1,2). While moderate success has been observed with

the direct injection of dissociated cells, it is often difficult to

control the shape, size and position of the grafted cells. In an

attempt to overcome these problems, research on advanced thera-

pies using functional tissue engineered cardiac grafts has now

begun. Over the past few years, several studies have demon-

strated that bioengineered myocardial tissues are able to improve

cardiac function in animal models of myocardial infarction3). In

this mini review, we present the progress of myocardial tissue

reconstruction, with a focus on our original approach using cell

sheet engineering.

Myocardial tissue engineering　Tissue engineering was originally proposed by Langer and

Vacanti in 19934), as an interdisciplinary research field seeking

to re-create three-dimensional (3-D) tissue structures. This novel

concept for tissue reconstruction has been based on using com-

binations of cells, extracellular matrix (ECM), and growth fac-

tors, ultimately leading to the re-creation of organ-like struc-

tures. Acccording to the methods introduced by Langer and

Vacanti, numerous researchers have applied the use of 3-D bio-

degradable scaffolds, such as poly (lactic-co-glycolic acid) as

substitutes for the ECM components, into which cells could be

seeded. Upon in vitro culture and implantation into the body, the

seeded cells reformed their native structures in accordance with

scaffold biodegradation. From this approach, this original con-

text of bioengineering has been applied for nearly every tissue

type.

　In myocardial tissue engineering, several methods, such as

the use of cardiomyocytes seeded into various polymer scaffolds

(Fig.1-A), myocytes set and molded with liquid collagen (Fig.1-B)

Mini Review　Myocardial tissue reconstruction: The cell sheet engineering approach


and even injectable cell-polymer solutions (Fig.1-C) have been

previously attempted. Importantly, these approaches of cardiac

tissue engineering have shown improved cardiac function after

graft transplantation to ischemic hearts in animal models. Li and

colleagues5) demonstrated that the transplantation of cardiac grafts

created with cardiomyocytes seeded into biodegradable gelatin

meshes was able to improve left ventricular contractile pressures,

in comparison to cell-free grafts. Leor et al. also reported that

bioengineered heart grafts using 3-D porous alginate scaffolds,

could attenuate left ventricular dilatation and deterioration of

heart function after myocardial infarction6). Furthermore, the

group of Zimmermann and associates has engineered contrac-

tile 3-D heart tissues by gelling a mixture of cardiomyocytes

and collagen solution. These engineered heart tissues also pre-

vented further dilatation, induced systolic wall thickening of the

left ventricle infarcted area, and improved fractional shortening

of damaged hearts7). Alternatively, Kofidis et al. reported that in-

jectable constructs of cardiomyocytes and liquid matrix compo-

nents were able to provide improved left ventricular contraction8).

Taken together, these various techniques that have been devel-

oped over the past 10 years, have been able to re-create beating

3-D cardiact tissues, wich can be formed in different shapes and

sizes in a directed fashion.

Cell sheet engineering for myocardialtissue regeneration　In contrast to the previously described technologies using bio-

degradable scaffolds or extacellular matrix components, we have

exploited an original method of tissue engineering that layers

cell sheets for the construction of 3-D tissues (Fig.1-D). Using

novel temperature-responsive culture dishes that are created by

the covalent grafting of the temperature-responsive polymer poly

(N-isopropylacrylamide) (PIPAAm) to ordinary tissue culture

dishes9), we have developed this method of “cell sheet engi-

neering.” Under normal culture conditions at 37℃, the dish sur-

faces are relatively hydrophobic and cells attach, spread, and

proliferate similarly to on commercially-available tissue culture

surfaces. However, upon temperature reduction below the

polymer's lower critical solution temperature (LCST) of 32℃,

the polymer surface becomes hydrophilic and swells, forming a

hydration layer between the dish surface and the cultured cells,

allowing for spontaneous cell detachment without the need for

enzymatic treatments such as trypsinization. By avoiding pro-

teolytic treatment, critical cell surface proteins such as ion chan-

nels, growth factor receptors and cell-to-cell junction proteins

remain intact, and cells can be non-invasively harvested as in-

tact sheets along with their ECM. As a consequence, we can

therefore recreate 3-D structures such as cardiac muscle, by the

layering of individual cell sheets.

　In native cardiac tissue, cell density is considerably higher, in

comparison with other tissues, such as heart valves and blood

vessels. Additionally, within the myocardium, cardiomyocytes

are also tightly interconnected with gap junctions, allowing for

synchronized beatings via electrical communication. Therefore,

in some cases, the use of 3-D biodegradable scaffolds can result

in a reduction of cell-to-cell connections. Similarly, scaffold bio-

degradation can lead to fibrous tissue development containing

excess amounts of ECM, which can pose a serious problem. In

contrast, our technology of layering cardiomyocyte sheets to cre-

ate 3-D cardiac tissues has specific advantages in creating cell-

dense tissues over the use of scaffold-based tissue engineering10).

First, harvested cardiomyocyte sheets consist of only confluently

cultured cells and their biological ECM on the basal surface of

the cell sheets, which can act as an adhesive agent to promote

intimate attachment between the layered cardiomyocyte sheets.

Therefore, these fabricated constructs consist of cell-dense myo-

cardial tissues with little ECM. Second, within these layered

constructs, the formation of gap junctions allows for the rapid

establishment of electrical communication between the cell

sheets, leading to synchronously pulsatile 3-D cell-dense myo-

cardial tissues11). Additionally, when these tissues were trans-

planted into the subcutaneous space of athymic rats, synchro-

nous graft beatings could be observed macroscopically12). Im-

portantly, these implanted tissues also showed long-term sur-

vival of nearly 2 years, with resected grafts demonstrating the

presence of elongated sarcomeres, gap junctions and well-orga-

nized vascular networks within the bioengineered cardiac

tissues13).

　

Cell sheet transplantation to ischemichearts　In terms of transplantation of bioengineered myocardial tis-

sue grafts, communication between the grafts and host hearts is

critical for the synchronized beating that is likely required for

improving impaired heart function. We have therefore previously

focused on the process of morphological connections between

our bioengineered myocardial tissue grafts and hearts. When lay-

ered cardiomyocyte sheets are transplanted directly to host inf-

arcted hearts, they are able to form morphological communica-

tions, with the presence of functional gap junctions in the intact

areas (Fig.2,3)14). The transplantation of layered cardiomyocyte

sheets was also able to repair damaged hearts, with improve-

炎症・再生　Vol.23 No.1 2003174

A

H

C

H

G

B

H

G

E

100um

G

H

D

H

G

100um

F

G

H

500nm500nm

A B

C D

50um

Fig.2 Histology of bioengineered myocardial tis-sue grafts transplanted to hearts

A-D, F: Azan staining. E: Immunofluorescent staining withanti-α-sarcomeric actinin. The surface of the native heartwas covered with a thin monolayer of mesothelial cells (arrowheads) (A). The myocardial tissue graft attached over intactarea of the hearts with some dissociation of the mesothelialcell layer observed 1 day after the procedure (B). At daythree, the mesothelial cell layer disappeared and the borderregion became unclear (C). One week after transplantation,some cells bridged between the grafts and the hearts(arrows) (D). Anti-α-sarcomeric actinin antibody stainingshowed cardiomyocytes were the cells responsible for theobserved bridging between graft and the heart (E). Con-versely no bridging cells were observed between the graftsand the infracted areas containing fibrotic tissues (F). G indi-cates myocardial tissue grafts and H indicates hearts.(Reprinted from reference 15, with permission from Interna-tional Society for Heart and Lung Transplantation)

Fig.3 Detection of gap junction communi-cation between the myocardial tissuegrafts and hearts one week aftertransplantation

Azan staining demonstrates cardiomyocyte bridgingoccured between the grafts and the hearts (A). Co-staining of serial cross-sections (red: anti- green fluo-rescent protein (GFP) antibody, green: anti-connexin43 antibody) shows that graft cells migrated into theheart and that connexin 43 was present at the borderof the graft cells responsible for bridging (arrow head)(B). Transmission electron microscopy demonstratesan intercalated disk between bridging ceardiomyocytes(C). Dye transfer analysis revealed that calcein, whichwas loaded into myocardial tissue grafts, was trans-ferred to the tissue via the bridging cells (arrows) (D).(Reprinted from reference 15, with permission fromInternational Society for Heart and Lung Trans-plantation)



ments in host ejection fraction, and inhibition of left ventricular

dilatation observed after cell sheet transplantation15).

　In addition to cardiomyocyte sheet transplantation, we have

also demonstrated that in myocardial infarction models, layered

skeletal myoblast sheets were able to provide improved left ven-

tricular contraction, reduce fibrosis, and prevent remodeling via

recruitment of hematopoetic stem cells through the release of

various growth factors16). The implantation of myoblast grafts

has also been able to induce restoration of left ventricular dilata-

tion and prolonged life expectancy in dilated cardiomyopathic

animals17). Similarly, mesenchymal stem cell sheets have dem-

onstrated improved cardiac function in impaired hearts, with re-

versal of cardiac wall thinning and prolonged survival after myo-

cardial infarction. This recovery after myocardial infarction sug-

gests that the improvement in cardiac function may be primarily

due to growth factor-mediated paracrine effects and/or a decrease

in left ventricle wall stress by the relatively thick mesenchymal

stem cell sheets18). As a next step, we are now proceeding with

large, clinically relevant animal models of cardiac disease.

The next challenges for myocardialtissue engineering　Although the use of cell sheet engineering has demonstrated

improved cardiac function in animal models, obstacles still exist

in the relatively young field of myocardial tissue reconstruction.

Recently, we have started to introduce neovascularization within

the bioengineered myocardial tissues, to overcome the long-stand-

ing size-limitations due to ischemia. We have observed that co-

cultured endothelial cells initiate endothelial network formation

within cell sheets in vitro, and contribute to rapid in vivoneovascularization19). As an alternative solution for overcoming

the thickness limitation, we have developed a multi-step trans-

plantation procedure of “polysurgery”, in which triple-layer

cell sheet grafts were repeatedly transplanted after allowing for

neovascularization to occur. Using this approach, 10-times

polysurgery has re-created functional myocardial tissues that are

approximately 1 mm in thickness20). Finally, as a further advanced

therapy, instead of bioengineering cardiac patches, we are now

trying to fabricate functional myocardial tubes which may have

the potential for independent circulatory support21). Overall, the

novel approach of cell sheet engineering for cardiac tissue engi-

neering applications, provides a promising alternative for effec-

tive therapies in regenerative medicine.

Acknowledgements　The present work was supported by Grants for the 21COE Program and

the High-Tech Research Center Program from the Ministry of Education,

Culture, Sports, Science, and Technology.

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