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63

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Tissue Engineering for
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Cardiac Valve Surgery
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Danielle Gottlieb • John E. Mayer
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Tissue engineering is a developing science, comprising ele- represents a major source of morbidity for pediatric patients
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ments of engineering and biology, whose aim is to build who must undergo multiple reoperations to replace valves
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replacement tissues de novo, from individual cellular and and/or valved conduits during the period of maximum
structural components. The impetus for our work on tissue- somatic growth.
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engineered cardiovascular structures arises from the need to Tissue engineering is an approach based upon the hypoth-
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replace cardiovascular tissues that failed to develop normally
during embryogenesis or have become dysfunctional as a
consequence of disease. In pediatric patients, the cardiovas-
cular structures most often afflicted by congenital anomalies
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esis that when properly designed and fabricated, these “living”
devices will simulate the biology of normal cardiovascular
structures, thereby overcoming the shortcomings of currently
available heart valve replacements. Of particular relevance to

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involve the cardiac valves and great vessels, and the theoreti- pediatric heart surgery is the long-term function of the engi-
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cal advantages of a tissue-engineered structure containing live neered valve over time and the potential capacity to grow,
cells are the ability to grow, remodel, and repair. Although self-repair, and remodel. This chapter summarizes some of the
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growth potential is not a consideration for the adult popula- progress that has been made in tissue engineering research
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tion, durability of valve structures remains an important issue as it relates to cardiac valves and conduit arteries, and then
for bioprosthetic valves, and a tissue engineering approach outlines the areas where additional efforts must be focused in
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offers the potential to improve durability by providing the order to direct cardiovascular tissue engineering toward clini-
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repair and remodeling capability. cal utility.
Diseases of the heart valves and large “conduit” arteries
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account for approximately 60,000 cardiac surgical proce-
NORMAL HEART VALVE BIOLOGY
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dures each year in the United States, and all of the currently
available replacement devices have significant limitations.1,2
Ideally, any valve or artery substitute would function like
Adult Valve Structure and Function
a normal valve or artery. For valves, this function includes Heart valves open and close approximately 40 million times
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allowing pulsatile blood flow without a transvalvar gradient per year; this coordinated function occurs under the demands
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or valvar regurgitation. The theoretical advantage of engi- of hemodynamic forces of blood pressure and shear stress.
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neered tissue replacements is that they could also display other The normal semilunar valve presents minimal resistance to
desirable characteristics, such as (1) durability, (2) growth opening and no pressure gradient during systolic forward
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(for infants and children), (3) compatibility with blood com- fre
flow. In diastole, the same structure is responsible for rapid
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ponents and the absence of thrombosis or destructive inflam- and complete closure in order to prevent valve regurgitation.
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mation, and (4) resistance to infection. None of the currently Semilunar valves must additionally resist pressure differences
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available devices, constructed from either synthetic or bio- between the diastolic arterial pressure and the diastolic ven-
tricular pressure. The atrioventricular valves must function in
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logic materials, meet these criteria. Mechanical heart valves

are very durable, but they require anticoagulation to reduce a comparable way, opening with minimal resistance during
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the risk of thrombosis and thromboembolism.1,2 Therapeutic diastole and closing to prevent regurgitation during ventricu-
anticoagulation carries associated morbidity, and even among lar systole.
patients who receive therapeutic anticoagulation, the inci- Like many other tissues, valve cusps are composed of cells
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dence of thromboembolic complications of mechanical heart residing within, the extracellular matrix (ECM). In addition,
valve replacement is not zero.1,2 Biologic valves, whether of these same cells act reciprocally on the ECM to which they
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allograft or heterograft origin, remain subject to structural are attached and receive signals from the ECM. The effects of
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deterioration after implantation.2-4 Neither mechanical nor these cellular level mechanical signals on cell phenotype and
behavior have been characterized by Ingber5,6 using the term
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biologic valves have any growth potential, and this limitation

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Part X  Transplant and Mechanical Circulatory Support

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“tensegrity” to describe the interactions between cell adhesion cells separate from the luminal surface, invade the ECM,
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to ECM and the nucleus mediated by the cellular cytoskel- and undergo endothelial-to-mesenchymal transformation

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eton. Semilunar valve cusps are thin, flexible structures with (EMT).17 In early fetal valve development, valve interstitial

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impressive microscopic and molecular complexity. Much of cells are highly proliferative, and reside in a glycosaminoglycan-
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the strength and flexibility of normal heart valve cusps is due rich, homogeneous microenvironment. Over the course of
to the specialized proteins and polysaccharide-protein com- fetal development, cell proliferation slows, and production of
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plexes of the ECM, produced by resident valve interstitial ECM results in valve cusp elongation. During the late stages
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cells.7,8 The microscopic and molecular structure of adult
valves reflects the regional mechanical forces experienced by
the valve cusps; valve cusp ECM is not homogeneous, and
the arrangement of the ECM provides a high degree of flex-
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of fetal development (20 to 36 weeks of human gestation)
and in early postnatal life, valve stratification into a trilaminar
structure occurs, and organization and maturation of colla-
gen fibers begins. At the time of birth, through a series of

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ibility during systole, and a high degree of strength to resist largely undefined molecular steps, changes in oxygenation
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pressure loads during diastole.7 On the surfaces, a specialized and blood pressure distinguish the aortic from the pulmonary

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endothelium prevents thrombosis, but also acts as a trans- sides of the circulation.12 Transitional neonatal circulation has
ducer of mechanical force.9,10 Beneath the endothelium, valve been associated with a change in phenotype from activated to
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interstitial cells receive signals sent by the endothelium, and quiescent interstitial cells in pulmonary, but not aortic, valve
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respond to imposed mechanical signals by secreting suitable cusps.18 Valve maturation and remodeling continue during
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matrix.5,6,11 childhood; cellularity of valve interstitial cells continues to

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Semilunar valve ECM is stratified into layers and its dis- decrease into adulthood.12 Once thought to be passive struc-
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tribution is related to valve mechanics.12 Facing the sinus of tures, increasing evidence shows cardiac valves to be dynamic
Valsalva, where eddy currents occur and diastolic pressure organs. Mechanistically, the finding that cellular and extracel-
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loads are imposed, dense collagen is found in the fibrosa layer.
A middle layer of connective tissue, the spongiosa, is partic-
ularly rich in glycosaminoglycans, large complex molecules
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lular components mature is thought to reflect the dramatic
changes in flow and biomechanical loading conditions from
fetal to adult life.13,19 Further elucidation of the genetic regu-

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which associate with water, and this layer is thought to act as latory events defining valve growth and maturation are likely
a “shock absorber,” bearing largely compressive mechanical to provide important information in designing biomimetic
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loads. The ventricularis, an elastin-rich layer facing the lumen replacement devices.
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and the flow orifice of the semilunar valve is specialized to
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stretch as the cusps elongate during ejection in systole. The
resulting composite structure of the native semilunar valve ENGINEERED VALVE INPUTS
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is anisotropic, with less elasticity in the commissure to com- The fundamental concept of creating a tissue-engineered car-
missure direction and greater elasticity in the annulus to free
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diac valve is to develop a tissue composed of living cells which
edge direction. The orientation of the collagen bundles in the will, at some point, form and remodel their own ECM and
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native semilunar valves have also been demonstrated to affect thus provide durability and growth potential. The biological
normal leaflet motion during systole and diastole.13–15 and engineering challenge lies in how to provide structural
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Valve structure and function are therefore closely related,
and strategies to engineer valves are aimed to mimic normal
valve structure and function, potentially at the subcellular,
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organization for these cells until they are capable of forming
their own mature ECM and to provide sufficient structural
support and mechanical integrity until the cells form their

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cellular, tissue, and whole heart valve levels. own ECM. Therefore, the fundamental variables involved in
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the creation of a living “tissue-engineered” valve involve choice
Embryologic and Postnatal or manipulation of cell phenotype, induction of appropriate
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ECM formation by mechanical and/or biochemical signals,
Development
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and provision of structural integrity and cellular organization

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Valves do not begin development as stratified tissues; as by the scaffold until new “native” ECM formation occurs.
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circulatory patterns evolve in fetal and postnatal life, valve Approaches to tissue-engineered heart valves (TEHV) can
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morphology also changes.12 Embryologic valve development, be divided into two paradigms, based upon the type of struc-
therefore, has relevance to tissue engineering, as a model for tural scaffold employed. One approach is to use bioresorb-
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normal in vivo valve remodeling as programmed by gene able scaffolds (nonwoven felts, electrospun scaffolds; knitted
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regulatory pathways and by applied biomechanical force.
By day 15 of human embryo development, specification
of myocardial and endothelial cardiac cells occurs. At day 21,
formation of a linear heart tube occurs. These events are fol-
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meshes, hydrogel-based, and combinations), and the other
approach involves beginning with decellularized tissue-based
scaffolds. The bioresorbable scaffold approach begins with
seeding cells and culturing under appropriate biomechanical

lowed by rightward (dextro) looping of the heart, resulting in conditions (static flow, pulsatile flow) and nutrient medium.
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orientation of the cardiac chambers into their final adult posi- With regard to tissue mechanical properties, the goal of this
tions, and opposition of two specialized segments of ECM, approach is fabrication of an adequately strong tissue with
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known as cardiac jelly.16 The first evidence of valvulogene- approximately constant mechanical properties, requiring that
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sis occurs in this cardiac jelly, when a subset of endothelial the process of scaffold degradation occurs with a reciprocal
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Chapter 63  Tissue Engineering for Cardiac Valve Surgery

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increase in ECM production. Porosity is an important char- The literature of TEHV demonstrates the selection of a
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acteristic of bioresorbable scaffolds as it provides a permeable variety of cell types, scaffold conditions, and precondition-

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framework for cell migration, nutrient supply, and waste ing regimens, and overall, methods are difficult to compare

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removal. Insufficient porosity leads to nutrient deprivation and results have been inconsistent. Systematic screening of
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and cell death. Once a sufficiently stable tissue is formed, in engineered tissue elements and combinatorial approaches to
vivo implantation would ideally follow, placing a newly syn- building tissues have recently emerged.30,31 A complete review
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thesized tissue, devoid of foreign elements, in the required of past investigations is beyond the scope of this chapter;
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site. An alternative approach involves use of an acellular scaf-
fold material which can then be populated by host tissue,
either by direct in-growth or seeding from the blood stream,
or some combination of these two mechanisms.20,21
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however, a summary of the fundamental elements of syn-
thetic, biomaterial-based TEHV will be considered in detail
in the following sections. In vivo outcomes of these methods
will also be considered.

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This de novo approach using cell-seeded biodegradable
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scaffolds is the one that has been predominantly employed

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Cell Origin and Phenotype
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in our laboratory at Children’s Hospital, Boston.23-29 This
approach treats components of tissue as “building blocks,” As in embryologic valvulogenesis, the “ideal” cell type for a
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constructing valves from a variety of cell types and scaffold TEHV would fulfill the function of both the valve intersti-
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materials. Most in vivo studies have been carried out in tial and endothelial cells. Conceptually, these cells could be
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the lower-pressure pulmonary circulation, which is a more derived from fully differentiated cells capable of ECM syn-
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tolerant system than the systemic circulation. This type of thesis, or from less committed, multipotent or pluripotent
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approach considers several basic questions as its foundation: stem cells with the additional potential for differentiation
(1) What cell type or combination of cell types is necessary into multiple cell types. Alternatively, two separate cell types
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to allow the production and maintenance of an appropriate
ECM? (2) To what extent can cellular phenotype be altered,
guided, or “engineered” to replicate cells found in the normal
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could be potentially used to form a valve structure with valve
endothelial cells on the surface and valve interstitial cells in
the inner layers of the valve. The role of host cell ingrowth

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valve? (3) How can these cells be spatially organized during from adjacent tissues and/or seeding from the blood stream
the development of this tissue-engineered structures until represent other alternative sources of cells.
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the cells in the construct produce sufficient and appropri- The first TEHV experiments were performed with differ-
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ate ECM? (4) What biochemical signals are necessary during entiated cells from artery or vein, including vascular smooth
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the development of these structures to ensure proper ECM muscle cells, fibroblasts, and endothelial cells derived from
production? (5) What mechanical signals are necessary for the vasculature of immature animals.20,23-25 These cells were
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optimal tissue development and growth? (6) Should a tissue- chosen as they were readily accessible, are derived from a
engineered valve construct be completely developed and cardiovascular source, and can synthesize ECM proteins. A
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have the histologic and macroscopic appearance of an adult comparison of myofibroblasts from the wall of the ascending
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valve prior to implantation, or can further maturation of an aorta with those from segments of saphenous vein revealed
engineered construct occur in vivo after implantation? More that the latter cells exhibit superior collagen formation
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recently, we have utilized acellular scaffold materials with
chemical composition and fabrication processes that yield
anisotropic mechanics that closely resemble those of native
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and mechanical strength when cultured on biodegradable
polyurethane scaffolds.32 In our laboratory at Boston Chil-
dren’s Hospital TEHV based on these differentiated cells

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leaflet.22 from systemic blood vessels functioned for periods of up to
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Proponents of the decellularized tissue method base their 4 months in vivo.20,23 However, enhanced collagen forma-
approach on the premise that ECM characteristics and sig- tion may be a double-edged sword. The rapid formation of
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nals direct cell behavior, and that the closest structure to the new tissue in the early culture period could give rise to an
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normal valve scaffold is the normal valve ECM scaffold itself.

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overabundance of matrix elements, leading to tissue stiffness
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Human aortic valve homografts are currently implanted and potential tissue contraction. Early studies with dermal
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without tissue-type matching, are found to become mostly fibroblasts demonstrated that valve cusps constructed from
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acellular after several months in vivo, yet retain their mechan- these cells developed tissue contraction, which limited the
ical properties for longer periods of time. As the exact features ability of valve leaflets to coapt with each other, resulting in
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endowing the aortic valve with this improved, but still limited valve regurgitation.33 In addition to production of excessive
durability are unknown, it is hypothesized that these features or unfavorable types of ECM, mature cells may present a
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are retained after ex vivo decellularization. These decellular- problem of senescence in long-term cell cultures in vitro,
ized scaffolds can either seeded with cells prior to implanta- which limits the ability to quickly produce sufficient num-
tion, or implanted without seeded cells, in the expectation bers of cells to seed a TEHV construct. Finally, the prospect
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that appropriate circulating cell populations will populate the of harvesting segments of artery from an otherwise normal
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scaffolds in vivo. Without seeding of cells prior to implanta- peripheral circulation in order to obtain cells for a TEHV
tion, however, some decellularized matrices are insufficiently represents an undesirable clinical situation, and therefore
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endothelialized by circulating cells or ingrowth from adjacent led to a search for alternative cell sources for engineered
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tissues to resist surface thrombus formation in vivo.21 valves.

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Part X  Transplant and Mechanical Circulatory Support

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The emergence of the field of stem cell biology has the
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potential to change the paradigm for candidate cell types for

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heart valve tissue engineering. As stem cells differentiate from

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their embryonic state, they lose pluripotency with each subse-
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quent step. Multiple steps of differentiation form lineages of
cells. In embryonic development, differentiation down a spe-
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cific lineage occurs with biochemical signaling, occurring in a
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specific mechanical microenvironment.34 How lineage speci-
fication occurs in the developing heart is currently an area of
active research, with the goal of understanding the necessary
cues to replicate differentiation down valve cell lineages. In
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normal development, differentiating cells are subject to a rap-
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idly changing three-dimensional extracellular environment,

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and development is thought to occur by signals originating
outside of the cell, from molecules originating in neighboring
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cells, and in the ECM. The importance of these mechani-
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cal interactions between cells and their immediate environ-
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ment during embryonic development has been emphasized

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by Ingber.6 These reciprocal interactions between cells and
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their environment in developing valves, the regulatory mech- FIGURE 63-1  The tissue engineered pulmonary valve viewed from
anisms which induce cells to secrete and respond to compo- below, before implantation.
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nents of appropriate ECM, is thought to be the underlying
mechanism stratifying valve cusps into their known compart-
ments. These mechanisms are thought to be largely driven by In addition, these cells can be obtained less invasively than dif-
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hemodynamics in developing valves. ferentiated cells.35 Our initial experience with progenitor cells
There is also recent evidence that stem cells with prolifera- in the Mayer laboratory was gained with autologous endo-
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tive and regenerative capacities reside in many adult tissues. thelial progenitor cells (EPCs) isolated from circulating blood
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These stem cells are capable of not only acting locally on the in lambs and seeded onto decellularized arterial segments.20
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tissues in which they reside, but they may also be recruited These seeded arterial grafts were then implanted as an inter-
out of the circulation and enlisted in the regeneration of position graft in the carotid artery of the donor lamb. These
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diverse tissues at distant sites. A recent review details emerg- grafts remained patent and functional for up to 130 days.
ing evidence that bone marrow-derived endothelial, hemato- Subsequent animal studies by Sutherland and associates used
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poietic stem and progenitor cells can also contribute to tissue bone marrow mesenchymal stem cells (MSC) to seed a bio-
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vascularization during both embryonic and postnatal life.35 resorbable scaffold formed into a three-leaflet valve within a
Visconti and coworkers have made the intriguing observation conduit (Fig. 63-1). These valved conduits were implanted as
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that cardiac valve interstitial cells in mice appear to originate
in the bone marrow as hematopoietic cells.36 The idea that
bone marrow contains cells capable of repairing damaged tis-
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valved conduits into the main pulmonary artery of neonatal
sheep, and remained in place for up to eight postoperative
months.27 This study was followed by that of Gottlieb et al,

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sue has been applied to regeneration of cardiac muscle after who implanted valves of similar elements into larger numbers
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myocardial infarction, whereby progenitor cells have been of sheep and followed changes in function with growth of the
isolated from sites outside the heart, then injected back into animals using magnetic resonance imaging (MRI) and echo-
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the heart in an attempt to regain contractile function of isch- cardiography. While the valves had trace to mild regurgitation
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emic or infarcted myocytes. Results of these trials have been

surface area was observed, which correlated with increasing fre
at the time of implantation (Fig. 63-2), a loss of valve leaflets
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equivocal, marginal, or negative, suggesting that the process of

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regeneration does not occur by the simple addition of multi- valve regurgitation over time.29 Importantly, the valve leaflets
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potent cells.37 The early experience with the SynerGraft decel- underwent a remodeling process in vivo after implantation,
lularized heterograft valved conduits which were implanted seen also in earlier experiments using myofibroblasts and
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in children as right ventricle-to-pulmonary artery conduits endothelial cells from systemic arteries, and in both sets of
occurred with the expectation that circulating cells from the experiments, a layered histologic appearance developed after
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bloodstream, or ingrowth from adjacent normal tissue would implantation.23,27

repopulate the grafts and grow. In this instance, repopula- Several types of progenitor cells have been used for tis-
tion of the graft by circulating cells occurred, but did not sue engineering applications in congenital heart disease.
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result in adequate function or tissue growth.38,39 Nonetheless, Cebotari and colleagues have recently reported an initial
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the progenitor cell populations represent an attractive source experience seeding EPCs onto homograft valves followed by
of cells for tissue engineering and regeneration, because they implantation into two children.40 Matsumura and associates
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have the plasticity necessary to fulfill critical cell functions, have shown that when seeded onto a copolymer of lactic acid
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and are potentially programmable for lineage specification. and ε-caprolactone, green fluorescent protein-labeled (GFP)
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Chapter 63  Tissue Engineering for Cardiac Valve Surgery

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controlling differentiation in these environments remains
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incompletely defined. There is evidence that EPCs are able to

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transdifferentiate in response to biochemical signals. Dvorin

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and colleagues showed that in the presence of transforming
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growth factor (TGF)-β1, EPCs express α-smooth muscle
actin (α-SMA), after seeding on a bioresorbable copolymer
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scaffold.45 This behavior is not characteristic of endothelium;
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SMA expression suggests a mesenchymal phenotype, and this
observation is a potentially related phenomenon to EMT
seen in embryologic valvulogenesis.45 Human aortic valve
endothelial cells, but not vascular endothelial cells, respond
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to TGF-β1 in a similar fashion, suggesting that EPCs may
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be suitable as a replacement for valve endothelium. Though

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stem cells of many types remain promising candidates for tis-
sue engineering, their full potential will be harnessed with
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an understanding of their normal generative and regenerative
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roles in vivo.
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A
Structural Scaffold
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Although it has been possible to grow individual cell types
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in culture for decades, it is more difficult to induce cells to
assemble or organize into complex three-dimensional struc-
tural arrangements that are found in normal tissues, and
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to induce cells to produce specific ECM components on
demand. Implantation of engineered tissues requires that
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they have structural integrity at the time of implantation, and
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for this reason, most tissue engineers employ a scaffold that
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provides structural integrity, in addition to cellular material.
Given this fundamental requirement, three main strate-
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gies for development of a three-dimensional tissue have been
used: (1) de novo scaffold synthesis and arrangement into
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a three-dimensional structure with cellularization prior to
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implant, (2) decellularization of a whole (usually xenograft)
tissue, or (3) de novo biodegradable scaffold without preim-
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plant cellularization.
Any scaffold for tissue engineering applications must be
B biocompatible and allow cells to adhere and proliferate. For
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congenital cardiac applications, tissue growth is our target,
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FIGURE 63-2  Representative echocardiogram images of functioning and therefore, the scaffold must either degrade or be remod-
tissue engineered pulmonary valve following implantation. eled in vivo. The advantage of the biopolymer approach is that
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the chemistry of these scaffolds allows for in vivo degradation,
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cells contributed to the histogenesis of their explanted tissue- fre

usually by hydrolysis.46 The disadvantage is that heart valves
are structurally complex and anisotropic; designing the struc-
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engineered vascular graft. These grafts remained patent, and tural features of the normal heart valve de novo has presented
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explanted constructs contained GFP-labeled cells expressing a substantial engineering challenge. The obvious advantage of
both endothelial and mesenchymal markers.33 The group the decellularization approach is that the three-dimensional
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at Tokyo Women’s Medical College has also carried out complexity is largely preserved, however, there is a shortage
implants of tissue-engineered vascular grafts in children with of homograft material relative to the clinical demand, and
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congenital heart disease using whole mononuclear cell frac- immunogenicity remains a concern with xenografts. Perhaps
tions, seeded onto bioresorbable scaffolds.41 This work is now most importantly, the ECM of decellularized xenografts is
ongoing at Yale University, due to encouraging early results. dense, and may prevent the penetration of seeded cells into
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Increasing evidence supports the idea that for cells, “geog- the interstices of the matrix. In our laboratory, we have con-
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raphy or destination is destiny.” Many cell types, including structed trileaflet valved conduits from small intestinal sub-
bone marrow-derived MSC exhibit surprising plasticity, mucosa and seeded them with EPCs.26 After implantation in
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and cell phenotype seems to be related to the microenviron- an ovine model, there was satisfactory short-term function,
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ment in which cells reside.42-44 However, many of the factors but there was no penetration of any cells into the depths of
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Part X  Transplant and Mechanical Circulatory Support

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the small intestinal submucosa scaffold. The ECM proteins, Institute of Technology designed an elastic polymer with a
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when exposed to blood components, can induce inflamma- rapid degradation time, based on sebacic acid, a derivative of

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tion, thrombosis, and calcification, so this material has not castor oil. Polyglycerol sebaceate is a strong but elastomeric

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been pursued further in our laboratory. material, and is currently under investigation for use in heart
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Our primary laboratory efforts to develop heart valves valve tissue engineering applications.52
and large arteries have utilized the approach of seeding cells In addition to their stiffness, polymer-based scaffolds are
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onto a variety of bioresorbable polymer scaffolds. Ideally, thick, relative to normal valve leaflets. A thick scaffold leads
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there exists an inverse relationship between scaffold degrada-
tion and ECM formation by seeded cells. Polymer degrada-
tion is related to polymer chemistry, fabrication method, and
mechanism of degradation, and therefore, polymers used in
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to a nutrient gradient in culture, and many tissue engineer-
ing studies based on nonwoven materials have been limited
by the lack of nutrient delivery to the deepest areas of engi-
neered tissues. Two solutions are proposed to overcome this

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tissue engineering differ in their degradation times. The first limitation: addition of a blood supply, and design of thinner
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generation of scaffolds used in heart valve tissue engineering scaffolds.

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were highly porous, nonwoven felts produced from fibers of Hydrogel-based scaffolds, including collagen, alginate,
polyglycolic acid (PGA). PGA and related polymers continue agarose, gelatin, fibrin, chitosan, polyethylene glycol, hyal-
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to be the most widely used for multiple tissue engineering uronic acid, and dehydrated sheets of ECM have formed
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applications.47-50 The advantage of PGA and related aliphatic the basis of many experiments in tissue engineering. When
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polyesters, including poly-L-lactic acid (PLLA), is their safety, gels become solid, cells are trapped in the cell, providing a
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biocompatibility, lack of toxicity, and commercial availabil- homogeneous distribution of cells embedded in a temporary
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ity. PGA has been used as the commercially available Dexon matrix. In addition, hydrogels can be laid into thin layers.
suture material since the 1970s.51 PGA can be extruded as a A bileaflet heart valve was produced in the Tranquillo labo-
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fiber, which allows fabrication of nonwoven sheets with large,
open pores. Open pore structures facilitate cell delivery and
proliferation by allowing a large surface area for cell attach-
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ratory using a collagen-based scaffold seeded with dermal
fibroblasts.53 Recent advances in drug delivery technologies
and microfluidics have resulted in further control of scaffold

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ment, free diffusion of nutrients and dissolved gases, and characteristics, including orchestration of scaffold polym-
removal of waste products of metabolism. Material properties erization with changes in temperature, pH, or exposure to
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of these nonwoven felts are well established, with reproduc- light; engineering of nano- and microscale cell environments
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ible hydrolytic degradation times; PGA alone degrades in 2 to in order to direct cell distribution throughout a scaffold,
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4 weeks, while the majority of fibers of the more hydropho- and microencapsulation of growth factors and adhesion
bic PLLA degrade within 4 to 6 weeks. These scaffolds lose peptides on scaffolds for improved cell attachment and
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strength prior to losing mass, which challenges tissue engi- proliferation.54-58
neers to seed sufficient numbers of matrix-producing cells so Finally, cells and scaffold have been incorporated and
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as to replace the scaffold strength as it is lost. Our labora- directed in nanoscale fabrication techniques such as electro-
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tory has experimented with PGA coated with the thermo- spinning. Still, no perfect material has been identified for
plastic polymer poly-4-hydroxybutyrate (P4HB), assembled heart valve tissue engineering, and in this regard, the search
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into a trileaflet structure by attaching leaflets to a flat scaf- continues.
fold sheet, then wrapping the scaffold around a mandrel and The complex anisotropy and three-dimensional structure
heat-welding a seam of attachment. Despite promising early of heart valves has provided another challenge for a biomimetic
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results using the PGA/P4HB composite, subsequent studies device. Even if the optimal scaffold material is identified, its
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showed loss of structural integrity with longer periods of in fabrication into a three dimensionally accurate valve geom-
vitro culture, followed by difficulties with suture retention etry is nontrivial. Using normal heart valve anatomy derived
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and hemostasis in vivo. For these reasons, Sutherland and col- from computed tomography images, Sodian employed ste-
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leagues in our laboratory developed a scaffold composed of reolithography to print a three-dimensional model for use
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equal parts of PGA and PLLA fibers. As PGA is a stronger but as a mold for the thermoplastic polymer P4HB.59 The mold
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more rapidly degrading polymer, and PLLA is a less strong was used for curing the polymer into three-dimensional valve
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polymer with a longer degradation time, we expected more anatomy. As improvements in imaging technology yield
uniform strength from the tissue when constructed from this higher spatial and temporal resolution, anatomic definition
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material. The composite nonwoven felt was fabricated into a of thin, moving, anisotropic structures in the heart such as
valved conduit with a trileaflet valve, and had substantially valve leaflets will be more feasible and may be able to account
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improved surgical handling characteristics.27 for the nonuniform cellular and ECM distributions that are
Although satisfactorily strong, polymer fiber-based scaf- found in native leaflets. In addition, evolving microfabrica-
folds are significantly stiffer than normal valve leaflets, and tion methods will further our ability to fabricate microscale
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with the addition of cell-secreted ECM, these constructs are features of valve anatomy. With defined anatomic dimensions
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notably stiff.46 Scaffold stiffness has been shown to effect cell and an understanding of outflow tract, great artery, and leaf-
behavior, and tissue engineers have sought less more flex- let motion and growth, tissue engineers will have clear targets
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ible materials.44 Wang and colleagues at the Massachusetts for three-dimensional fabrication of heart valve scaffolds.
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Chapter 63  Tissue Engineering for Cardiac Valve Surgery

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Biochemical Signals tissue islands that promote cell spreading, they efficiently
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differentiate into bone cells; when plated onto small islands,

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The migration and transdifferentiation of endothelial cells
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the same cells in the same culture medium differentiate into

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in the early stages of valvulogenesis (EMT) has been experi- adipocytes.61
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mentally modeled in an ex vivo chick cardiac cushion explant Clinical observations made from patients undergoing the
system.17 Much progress has been made to understand the Ross procedure has provided further evidence for the respon-
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signals required for the proper sequencing and execution siveness of vascular and valve tissues to hemodynamic forces.
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of these early events. Vascular endothelial growth factor is
one such molecule, thought to regulate EMT in an envi-
ronment of adequate tissue glucose and oxygen saturation.
While VEGF is secreted by endothelial cells, other impor-
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In the Ross procedure, a pulmonary valve from the lower-
pressure pulmonary circulation is transplanted into the aortic
position and subjected to systemic pressure, leading to sig-
nificant changes in the phenotype of valve interstitial cells,

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tant signals, such as bone morphogenetic proteins 2 and 4, including an increase in matrix metalloproteinase activity,
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are expressed by myocardium. Hyaluronic acid, a component indicating ECM remodeling.18

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of the ECM, is thought to regulate downstream signaling
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Biophysical signaling is therefore considered fundamental
through its large, hydrated structure, which regulates ligand to engineered tissue organization and conditioning, or train-
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availability for receptor binding. Therefore, valvulogenesis ing, for the in vivo environment. Mechanical forces can be
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is dependent in vivo upon signals from myocardium, local
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applied to growing engineered tissues in a flow-loop con-
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ECM, and endothelium.12,34 Though very early growth of taining tissue culture medium, known as a bioreactor. Pre-
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endocardial cushions and valve primordia are less under- conditioning of tissues is thought to be important for the
stood, and late regulatory events in valve growth are poorly
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biology and monitoring of engineered cardiovascular tissues.
understood, regulators of endothelial and mesenchymal cell Bioreactors are thought to serve two predominant purposes
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proliferation have been identified. These known pathways
have been largely studied in isolation, and as gene regulation
in organogenesis is a highly complex process, synthesis of
critical gene pathway data is necessary for developing a big-
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for engineered heart valves: first, mechanical forces influ-
ence cell phenotype and gene expression, and therefore, tis-
sue development, and potentially, growth, are controlled by

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biomechanical signals. In experiments from our laboratory
picture view of required events. Current microarray technolo- reported by Hoerstrup and colleagues, flow and pressure were
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gies will yield large volumes of data, and will likely provide demonstrated to increase the production of collagen in tissue-
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additional insight into the critical regulatory steps involved in engineered semilunar valves.23 In separate experiments, Lee
valve growth. This type of information will be high yield in
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and associates demonstrated the variation of ECM gene tran-
future generations of engineered valves. scripts with changes in tightly controlled mechanical strains.
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Vascular smooth muscle cells seeded onto biodegradable scaf-
folds, then subjected to cyclic flexure produced more collagen
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Mechanical Signals and were stiffer than controls.63 Similar findings were repro-
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Identification of a suitable cell phenotype is necessary but duced using MSCs, and porcine heart valves.64,65
not sufficient for engineering replacement tissues. Proper cell In addition, since valves must not be regurgitant at the
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orientation and three-dimensional ECM microstructure are
also required for tissue function; tissues demonstrate orga-
nization of cells and matrix across multiple levels of scale.
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time of implantation, observation of engineered valve
mechanics in a bioreactor prior to implantation has allowed
investigators to monitor and predict in vivo valve function.

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In this regard, engineered tissues, like native tissues, require
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coordination. Hemodynamics are fundamental to the devel-
opment and ongoing function of cardiovascular structures, ENGINEERED VALVE
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and biomechanical signals are epigenetic regulators of tissue
EXPERIMENTAL OUTCOMES
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growth and development. Increasing evidence supports a role

for endothelial cells as mechanotransducers, sending signals Progress toward a clinically translatable engineered heartfre
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through the underlying ECM to the more deeply embed- valve is not possible without in vitro study of valve develop-
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ded valve interstitial cells.19 Endothelial cells respond to ment and remodeling. Developmental biology and in vitro
shear stress and cyclic strains.60 In an environment in which experiments are the fundamental elements of this emerging
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concentrations of soluble growth factors are held constant, field. However, in vivo experiments are the necessary comple-
endothelial cell programs can be switched between growth, ment to in vitro investigation, and represent the true test of
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differentiation, and apoptosis by varying the extent to which engineered device function over time. The in vivo environ-
the cell is spread or stretched.61 As cells are embedded in, ment is more complex than can be approximated in vitro, and
and coupled to, their ECM serves as a vehicle through which remodeling is dependent upon the contributions of circulat-
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signals must pass. Biochemical signals can be sequestered or ing progenitor and immune cells only present in whole living
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amplified by ECM, and as cells bind to surrounding ECM organisms.

via integrin receptors, the binding itself can induce pheno- Four major implantation studies of TEHV have been
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typic changes.34 When human MSCs are plated onto large undertaken using the de novo engineered approach.23,24,29,66
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Part X  Transplant and Mechanical Circulatory Support

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Several groups have implanted decellularized and recellular- the subendothelial layer, while surface cells expressed von
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ized valves into the juvenile lamb circulation,67,68 and two Willebrand factor, suggesting in vivo endothelialization of

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major studies evaluate the growth of vascular conduits in the graft. This study demonstrated the feasibility of implanta-

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the pulmonary circulation.69,70 Several representative stud- tion of a pulmonary valve using stem cells.
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ies will be reviewed in terms of valve function, evaluation of Further work in our laboratory has reproduced some of
valve/conduit growth, and observation of engineered tissue these initial findings, and studied valve function over time in
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maturation. larger numbers of animals.29 Using the same scaffold mate-
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rial (PGA-PLLA composite nonwoven felt) and cells (MSCs),
nineteen animals underwent implantation of valved conduits
In Vivo Valve Function, Growth into the main pulmonary artery of neonatal sheep after resec-
and Maturation tion of the native pulmonary leaflets. Valve function, cusp
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Heart valve tissue engineering was conceptually established and conduit dimensions were evaluated at implantation
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(echocardiography), at the experimental midpoint (MRI),

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through proof-of-concept experiments in large animals. The
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first in vivo TEHV experiment involved implantation of a and at explant at 1 day; 6, 12, or 20 weeks postoperatively. At
implantation, valved conduit function was excellent; maxi-
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single leaflet into the pulmonary circulation.20 Cells were
isolated from ovine arteries and sorted into endothelial and mum transvalvar pressure gradient by Doppler echocardiog-
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fibroblast populations. Cells were labeled, then seeded onto raphy was 17 mm Hg; most valved conduits showed trivial
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PGA scaffold and subsequently implanted. Labeled cells were pulmonary regurgitation. At ≥12 weeks, valved conduit cusps
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visualized in explanted specimens after 6 hours and after 1, 6, were increasingly attenuated and regurgitant. Valved conduit
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7, 9, or 11 weeks. Cells produced ECM, and leaflets persisted diameter remained unchanged over 20 weeks. Dimensional
in the circulation at all time points. measurements by MRI correlated with direct measurement at
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A second set of experiments involved implantation of a
pulmonary valve composed of autologous ovine endothelial
cells and myofibroblasts seeded on a PGA-P4HB composite
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explant. These studies demonstrated autologous engineered
valved conduits that functioned well at implantation, with
subsequent monitoring of dimensions and function in real

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scaffold.23 After 14 days of exposure to gradually increasing time by MRI. The valves underwent structural and functional
remodeling without stenosis, but worsening pulmonary
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flow and pressure conditions in a pulse duplicator, valves were
implanted into sheep for 1 day, 4, 6, 8, 16, and 20 weeks. regurgitation was noted after 6 weeks.
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Valves demonstrated acceptable hemodynamics and had no When fibrin-based valves were implanted in sheep for
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evidence of thrombosis, stenosis, or aneurysm formation for 3 months, valved conduits exhibited the gross appearance
up to 20 weeks. Central valvar regurgitation was noted after of intact tissue, however, leaflets demonstrated pulmonary
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16 weeks. Tissue analysis showed a layered structure with cen- regurgitation due to tissue contraction.66
Most recent experiments have involved implantation of
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tral glycosaminoglycans, collagen on the outflow surface, and
elastin on the inflow surface. Elastin was detectable in the single electrospun anisotropic poly-carbonate urethane urea
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leaflets by 6 weeks in vivo. Over the 20-week study period, (PCUU) leaflets into the ovine pulmonary valve root without
scaffold degradation corresponded to decreased leaflet stiff- pre-implant cellularization. These studies have shown good
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ness. Histological, biochemical, and biomechanical param-
eters were similar to those of native pulmonary artery and
leaflet tissue.
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overall valve function and repopulation of these leaflets with
cells (Fig. 63-3). However, leaflet excursion did decrease with
time, after implant.71

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Using bone marrow-derived MSCs on a PGA/PLLA scaf- To date, no group has implanted valve leaflets that dem-
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fold, investigators fabricated and implanted valved conduits onstrated in vivo growth. In many experiments, however, the
in the pulmonary position of juvenile sheep.27 Valves were microscopic structure of the valve leaflets evolved from a rela-
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evaluated by in vivo echocardiography; explanted tissues were tively homogeneous appearance to a layered structure. The
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analyzed by histology and immunostaining. At the time of mechanisms by which this in vivo evolution of structure and
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implantation, echocardiograms demonstrated a maximum cellular activity occurs remain completely unexplored, but
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these observations suggest that a tissue-engineered valve may
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instantaneous gradient of 17.2 ± 1.33 mm Hg, with estimates

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of regurgitation ranging from trivial to mild. Four animals not have to be in a mature, adult form at the time of implan-
survived the immediate postoperative period. After 4 months tation into the circulation.
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in vivo, there were no statistically significant difference is

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maximum instantaneous gradient, mean gradient or effective

orifice area from the time of implantation. Histologic analy-
sis at eight postoperative months showed explanted tissue
to have a layered organization, with elastin fibers identified
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FUTURE DIRECTIONS
After a decade of effort toward a TEHV replacement, the
clinical need for a more durable valve replacement still exists.

on the inflow surface and collagen dominating the outflow Modes of failure of currently available heart valve replace-
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surface. Glycosaminoglycans were distributed throughout ments are well described and provide insight into strengths
the remainder of the valve. At the time of implantation, cells and weaknesses of valve designs. Though the appropriate cell
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uniformly expressed the mesenchymal cell marker α-SMA, source and scaffold material for heart valve tissue engineering
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but at the time of valve explant, these cells were confined to have been areas of substantial experimental effort, the optimal
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Chapter 63  Tissue Engineering for Cardiac Valve Surgery

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leading to the ultimate goal: a clinically translatable heart
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P valve replacement with the capacity to grow and remain

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A durable for a lifetime of cardiac cycles.

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REFERENCES
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cell type, scaffold material, and in vitro culture conditions
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16. Moore K, Persaud T: The Cardiovascular System: The Developing Human.
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gies will allow a more complete understanding of postimplan- 19. Butcher J, Tressel S, Johnson T, et al: Transcriptional profiles of valvular
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reactor preconditioning has the potential to reduce in vitro 20. Shin’oka T, Bruer CK, Tanel RE, et al: Tissue engineering heart valves:
valve leaflet replacement study in a lamb model. Ann Thorac Surg 1995;
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a controllable environment for evaluation of valve function
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approaches, progress and challenges. Ann Biomed Eng 2006; 34(12):1799.
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leaflets retain function and remodel after implant in ovine pulmonary out-
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occurring in parallel, at the cellular, tissue and organ levels. 23. Hoerstrup SP, Sodian R, Daebritz S, et al: Functional living trileaflet
A detailed understanding of the molecular sequence of criti- heart valves grown in vitro. Circulation 2000; 102(Suppl 3):III-44.
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24. Stock UA, Nagshima M, Khalid PN, et al: Tissue engineered valved
cal developmental events may allow engineering of appro-
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