Fisher John P. e.a. (ed.) Tissue Engineering
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by stacking alternating layers of hyaluronan/elastic sheets with layers of collagen bundles/elastic sheaths
that are oriented as they would be in the native aortic valve (predominantly circumferential). This novel
approach is still in the in vitro developmental stages, but it is envisaged that the multiple layers would
compact, and the embedded cells would synthesize additional ECM, during a period of in vitro dynamic
conditioning.
28.2.6 Cell Origin
It is generally agreed that the source of cells for a TEHV should be autologously derived from the intended
valve recipient. This is particularly feasible given that most valve surgeries are elective, with criteria for
the decisions of surgical timing being continuously reevaluated [5,80]. Autologous cells may perform
best in pediatric cases, given that adult cells may have diminished proliferative ability (cell sources are
discussed extensively in Reference 2). The anatomic source of these autologous cells, however, has not
been firmly established. Shinoka et al. [74] determined that arterial myofibroblasts were superior to
dermal fibroblasts in generating a strong, collagen-rich, and organized neotissue; Schnell et al. [42] found
that venous cells were better yet. Given that saphenous veins are more easily harvested than arteries, the
use of venous cells was therefore considered a promising alternative to arterial cells for seeding a TEHV
scaffold. Although many studies continue to explore the use of cells derived from vascular sources, there
were two recent reports that used either the stromal cells [40] or mesenchymal stem cells [81] from bone
marrow. In the first case, human bone marrow stromal cells (obtained from the sternum) were seeded on
a PGA/P4HB valved conduit, then conditioned in a bioreactor. After 14 days, the cells showed phenotypic
characteristics of myofibroblasts (smooth muscle α-actin and vimentin), but did not show markers for
muscle, osteogenic, or endothelial differentiation. In the second case, ovine bone marrow mesenchymal
stem cells were seeded on the same type of valved conduit, then conditioned in bioreactor. Although
the differentiation characteristics such as cell phenotype were not reported, the cells were able to form a
functional neotissue in vitro.
Very few research groups have actually attempted to use VECs and VICs in the design of TEHVs
[30,75,78]. It appears to have been assumed, if not often explicitly postulated, that whatever cell type is
seeded within the TEHV will soon differentiate and begin to express the phenotypic characteristics of
valvular cells. With rare exception [82], this assumption has not been tested in any depth, even though
recent gene expression work has shown that VECs have many transcriptional differences from vascu-
lar endothelial cells [24]. VECs were also four times more proliferative in culture than were vascular
endothelial cells [24], which may explain why vascular endothelial cells have frequently failed to form a
continuous endothelial coating of the TEHV scaffolds in vitro [71] or in vivo [38,44,63]. VICs seeded in
decellularized porcine leaflets differentiated and segregated into regionally specific myofibroblasts, fibro-
blasts, endothelial cells, and smooth muscle cells after 15 days in vitro [30]. After seeding in porous collagen
sponge-like structures, VICs demonstrated phenotypic similarity to cells in native valve leaflets [78] and
produced many of the PGs normally found in valves [75] A potential source for autologous valvular cells
was proposed by Maish et al. [83], who found that valvular cells could be harvested from an ovine tricuspid
valve without compromising the function of the tricuspid valve in the donor animal. This option may be
important if the restoration of the VIC and VEC phenotype proves necessary and cannot be accomplished
using an alternative cell source.
28.2.7 Bioreactors for Conditioning and Proof of Concept
Bioreactors have been widely used to provide the developing TEHV with strength-building mechanical
stimulation prior to implantation within the animal model. Compared with static controls, bioreactor-
conditioned TEHVs have demonstrated greater production of collagen, greater cell densities, improved
mechanical strength, and more compact matrix organization [39,40,55]. It has also been proposed that
bioreactor conditioning maintains the synthesis of ECM and DNA at levels normally found in the valve
leaflets [84]. Bioreactors can also accelerate the turnover of the biodegradable matrix. Incubation in
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Tissue Engineering of Heart Valves 28-11
FIGURE 28.7 This bioreactor design creates pulsatile flow by cyclically pumping air beneath the silicone diaphragm
separating the medium and air chambers. This action draws medium from a reservoir and propels it through the TEHV
fixed to the silicone tubing above. (Reprinted from Hoerstrup, S.P., Sodian R., Sperling, J.S., Vacanti, J.P., Mayer J. E.,
Tissue Eng., 6, 1, Copyright 2000, with permission from Mary Ann Liebert, Inc.)
a dynamic flexure bioreactor also augmented the bulk degradation of PGA/P4HB and PGA/PLA/P4HB
scaffolds, resulting in significantly less flexural stiffness at 3 weeks compared to unflexed controls [41].
The types of bioreactors developed for TEHV studies can be generally classified into one of two categories.
The first category, which has a continuously circulating supply of tissue culture medium, is predomin-
antly used for the conditioning of intact TEHV and valved conduits. These bioreactors generally consist
of a medium reservoir, a chamber to hold the TEHV, a pressure source, ports for gas exchange, optional
pressure and flow transducers, and often have components to mimic vascular compliance and resist-
ance [85,86] (Figure 28.7 and Figure 28.8). A similar setup was used in a study of the synthesis of
collagen, GAGs, and DNA by valvular cells [84]. Because these bioreactor systems are capable of pro-
ducing pulsatile flows ranging from 50 to 2000 ml/min [85] or even 20 l/min [84], and transvalvular
pressures of 10 to 240 mmHg, they can easily be tailored to the conditioning or testing of aortic or
pulmonary TEHVs. Feedback-driven tensioning systems have also been incorporated into these biore-
actors [87]. The second category of bioreactor tends to be smaller and scalable, and is used more for
proof of concept studies that examine how mechanical stimulation can improve the TEHV charac-
teristics. An example of this was developed by Engelmayr et al. [41], in which scaffolds identical to
those used in certain TEHVs were subjected to flexural conditioning, but were not bathed in circulating
medium.
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28-12 Tissue Engineering
P
C
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A
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LV
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Legend:
C
R
O
V
M
A
LV
E
P
Compliance
Resistance
Reservoir
TE valve
Mechanical valve
Aerator
Left ventricle
External circuit
Pressure transducer
FIGURE 28.8 A bioreactor flow loop that includes components for variable compliance, resistance, and pressure
measurements. (Reprinted from Dumont, K., Yperman, J., Verbeken, E., Segers, P., Mauris, B., Vandenberghe, S.,
Flameng, W., Verdonck, P.R., Artif. Organs, 26, 711, Copyright 2002, with permission from Blackwell Publishing.)
28.2.8 In Vivo Testing in Animal Models
Although many potential TEHV designs have been successfully compared using in vitro and bioreactor
analyses, evaluation of the inflammatory, immunologic, and calcific responses requires an in vivo model.
Canines have been used at least twice [44,88], but the majority of TEHVs have been tested in vivo in
the widely accepted ovine models (either lamb or mature sheep) [33]. TEHVs have been implanted
into the atrioventricular position in the past [77], and a heterotopic implantation in the abdominal
aorta was recently reported [88], but most are tested in the pulmonary position, either as an orthotopic
replacement of the pulmonary valve [38,39,45,51,56,72] or heterotopically interposed between the main
and left pulmonary artery [44]. Single [34,69] and double [36] leaflet replacements within the pulmon-
ary valve have been attempted with more success in the single leaflet replacement. The double leaflet
replacement involved two identical PGA/PLA–PGA sandwich scaffolds: one seeded TEHV leaflet and one
unseeded control. Unfortunately, this otherwise ingenious approach did not work because the stiff, thick
scaffolds were unable to coapt normally and cause pulmonary insufficiency [36]. More recent TEHV
designs are often tested using a valved conduit, containing all three leaflets, in the orthotopic pulmonary
position.
These in vivo studies have often demonstrated an early but very mild inflammatory response to the
TEHV [47], although this response was stronger when allogeneic as opposed to autologous cells were
seeded [34]. A late inflammatory response (9 to 12 weeks) was reported with a reseeded decellularized
porcine TEHV [63]. The remnant muscular shelf of decellularized leaflets has also been shown to invoke
a mild inflammatory and calcific response [62]. Otherwise, reports of calcification of the scaffolds have
been with mixed; there was heavy calcification of reseeded decellularized scaffolds after 12 and 24 weeks
in vivo [64], while a Synergraft decellularized porcine scaffold (not reseeded) showed no calcification after
21 weeks [66].
28.2.9 Clinical Experience
The majority of TEHVs have only been tested in vitro or in animal models; no TEHV has been approved
for clinical use in the United States. The porcine and human Synergrafts, which are technically not TEHVs
since they are not reseeded with cells, have been approved in the United States [46]. As of 2003, these
decellularized human valved conduits have been implanted in more than 150 patients in the United States
and more than 1000 patients worldwide, approximately 84% of which have been in the pulmonary
position (16% aortic). The distribution of patient age in the United States has been approximately 1/3
children and 2/3 adults, with a mean follow-up time of 231 days (range 0 to 788 days). In the two cases
where these pulmonary allograft conduits were explanted (due to external fibrosis at 14 months and
unrelated infection at 3 months), the leaflets and conduit had been repopulated with fibroblasts and
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Tissue Engineering of Heart Valves 28-13
myofibroblasts and demonstrated an endothelial lining and normal leaflet architecture. Function of the
pulmonary Synergraft allografts have been comparable to cryopreserved allografts [46,61], whereas the
aortic Synergraft allografts have evoked humoral antibodies in 25% of patients, but have shown normal
aortic valve function with only trivial regurgitation [46]. The porcine pulmonary Synergrafts have been
implanted in 20 patients [46], with 3 deaths and conduit pseudoaneurysm formation in 3 patients, but
14 other patients have had satisfactory valve performance for a maximum of one year. Unfortunately, the
porcine Synergraft has not been as successful in the pediatric population, with a massive inflammatory
response leading to leaflet degeneration and rupture in at least seven children in Austria and Norway [61].
One Synergraft that was examined after being implanted for one year showed no host recellularization
of the implant at all and the formation of a fibrous capsule around the porcine tissue, which was very
different from the results in a sheep model [45], in which the entire leaflet was repopulated with cells after
one year.
In Germany, decellularized human cryopreserved pulmonary allografts that were reseeded with auto-
logous venous endothelial cells were implanted in at least 6 Ross procedure patients with a mean age of
44 years [60]. All patients demonstrated an improvement of ejection fraction at 3 months. One patient
was reported to be excellent condition after 12 months, and had not demonstrated any fever during this
time. This absence of fever was interpreted as an absence of any antigenic inflammatory or immunologic
response.
28.3 Conclusions and Future Challenges
There are many challenges ahead for the future of TEHVs. The first challenge appears to be the recapitula-
tion of the many aspects of valvular biology in a living TEHV, particularly in designs involving nonvalvular
cells. Quite frankly, it remains to be determined if such recapitulation would even be necessary for TEHV
function, even though that finding could help direct future TEHV research. Moreover, the nascent field
of valvular biology lags far behind vascular biology. Regardless, future TEHV research may wish to build
upon the many studies over the last several years that have examined the contractile, synthetic, signaling,
interactive, and diffusion characteristics of heart valves at the tissue, cellular, and molecular level. Normal
aortic valve leaflets and VICs, for example, will contract slowly in response to stimulation with vasoactive
agents such as serotonin, epinephrine, and endothelin-1 [28,89], potentially to maintain a baseline resting
tension in the normal aortic valve [89]. More recently, the proliferative, migratory, synthetic, signal-
ing, and phenotypic transdifferentiation responses of VICs to members of the renin–angiotensin system
(including TGF-β) and vasoactive agents have been assessed [90–92]. Furthermore, the receptors and/or
mRNA for serotonin, angiotensin II, bradykinin, and angiotensin-converting enzyme have been identified
in VICs [92,93]. In fact, many of the transcriptional and proliferative characteristics of valvular cells have
been shown to be different from those expressed by vascular cells [24,93]. Cell–matrix interactions have
been demonstrated at the molecular level by the characterization of several integrins in bovine and baboon
VICs [94]. Finally, oxygen diffusion within young porcine aortic valves was recently measured [95] and
it was observed that blood vessels were present in most regions of the valve thicker than 0.5 mm. This
finding highlights the differences between pediatric heart valves, which are vascularized [96,97] and even
innervated [98], and adult heart valves, which have far less nerves and vessels, but contain viable, synthetic
cells nonetheless. Exactly how well oxygen and nutrients diffuse to these adult VICs, and likewise to the
cells within a TEHV, is a fertile topic for further research.
Another challenge for the future is the development of TEHV replacements for the aortic, atrioventricu-
lar, and venous valves. The highest clinical need is for the aortic position. To meet the higher pressure
and flow requirements of the systemic circulation, the current pulmonary TEHV designs will need to
be adapted to produce a stronger neotissue. In a recent proof of concept study that explored this need,
a PGA/P4HB scaffold seeded with human venous myofibroblasts became more than three times stiffer
when the maximum cyclic strain was raised from 7 to 10% during bioreactor conditioning [99]. Given
that the atrioventricular valves are more commonly repaired than replaced [5], recent TEHV work in
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that regard has focused on replacement components, as opposed to the entire valve. The chordae, in
particular, frequently rupture in prolapsed mitral valves [80]; torn chordae are currently replaced with
Gore-Tex™ sutures. Because these sutures are several times stiffer than normal chordae [100], Vesely
et al. [79] have been developing tissue-engineered chordae (large collagen fiber bundles) by seeding
NRASMCs in a type 1 collagen gel and allowing the cell-seeded gel to contract uniaxially. Although these
tissue-engineered chordae have not yet been tested in vivo, and are an order of magnitude less stiff than
normal native human chordae, they have approximately normal extensibility [79]. As this avenue of TEHV
research is very young, these preliminary results are likely to improve substantially in the years to come.
Tissue engineered venous valves have been developed in a sheep model using allogeneic decellularized
jugular veins seeded with autologous venous myofibroblasts and endothelial cells; these valves were still
patent at 12 weeks [101].
The final challenge, particularly given the clinical reports of failure of the Synergraft valves in pediatric
patients [61], are the ethical considerations involved as these research findings are eventually translated to
clinical practice [102]. Although there are no current governmental regulatory divisions that are focusing
exclusively on engineered tissues, U.S. and European regulatory agencies are planning centers that will
meet this need. Beyond that goal lies the problem of developing standards to which engineered tissues such
as the TEHV would be held. Furthermore, we are reminded by Sutherland and Mayer in their excellent
review of this topic, “compliance with standards alone carries no guarantee that a given device will be
free from risk” [102]. Applying a risk management approach, with identification and severity grading
of potential hazards, is an appropriate first step along this path. Hazards particularly associated with the
current characteristics of the TEHV described in this chapter could be scaffold-related, such as adverse
reactions to the degradation products of the biodegradable scaffolds, or the potential for transmission
of porcine endogenous retrovirus from the decellularized porcine valve scaffold to the TEHV recipient
[51,102]. Other hazards could be related to the cells involved, particularly if they are not autologous, or
to the performance of the TEHV in vivo.
The field of TEHV research is growing exponentially and has demonstrated many recent advances,
particularly in the development and application of bioreactor technologies. New research in valvular
biology and targeted scaffold development, together with frequent reflection and review [2,26,33], are
certain to drive this field ahead. Although there are many hurdles to overcome before the many promises
of TEHVs become fully realized, the potential to create a living, healing, growing valve continues to inspire
significant effort in this exciting field.
Defining Terms
Allogeneic: Taken from a different individual of the same species
Atrioventricular: Referring to the position between the atrium and the ventricle. The mitral and
tricuspid valves are atrioventricular valves.
Autologous: Taken or derived from the same individual.
Coaptation: Referring to contact between different heart valve leaflets, or regions of the leaflets where
this contact occurs when the valve is closed.
Ejection fraction: The percentage of blood pumped out of an individual’s left ventricle.
Heterotopic: Referring to a region of the body where a structure is not normally located.
Incompetence and insufficiency: Inability of the heart valve to close property.
Orthotopic: Referring to a region of the body where a structure is normallt located.
Ross Procedure: Surgical replacement of a patient’s deficient aortic valve with a pulmonary autograft,
followed by implantation of a cryopreserved pulmonary allograft in the pulmonary position.
Semilunar: Referring to the half-moon shape of the leaflets of the pulmonary and aortic valves.
Tetralogy of Fallot: A congenital heart defect with four predominant symptoms (ventricular septal
defect, pulmonary valve stenosis, right ventricular hypertrophy, and malposition of the aorta over
both ventricles.
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Valvular interstitial cells: An all-encompassing term for the nonendothelial cells that are contained
within the heart valve leaflets. This definition includes cells of variable phenotype.
Xenogenic: Taken from a different species.
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