In the United States alone, over 100,000 heart valve replacement procedures are
performed each year, with approximately 45% of patients below age 65. While current
mechanical and bioprosthetic heart valves are viable options, they have several
limitations. The most significant limitation is for pediatric patients, since neither of
these valve types grow and remodel with the patient. Tissue engineering provides a
methodology to create functional heart valves that can grow and remodel similar to
native tissue once implanted. Several tissue engineering approaches have been
proposed using decellularized native scaffolds, synthetic biopolymers, and biological
polymers seeded with cells. Fibrin provides a scaffold to create tissue-engineered heart
valves (TEHV) that are completely biological with an environment permissive for
extracellular matrix (ECM) deposition. Previous research in our lab has demonstrated
the feasibility of creating a fibrin-based TEHV with neonatal human dermal fibroblast
(nHDF) that yields valve leaflets with structural and mechanical anisotropy similar to
native leaflets. However, the TEHV had sub-optimal tensile mechanical properties and
was thus unable to withstand physiological forces. The development of tissue can be
accelerated by both chemical and mechanical stimulus. Previously, fibrin based TEHV
were cultured with chemical stimulus in the form of growth factors supplemented in the
culture medium resulting in improved ECM deposition by the cells; however, no
mechanical stimulation was applied.
Prior research in both our lab and by other researchers has shown cyclic
stretching with constant strain amplitude is a method to stimulate remodeling of
biological scaffolds seeded with cells. Initial experiments were conducted to evaluate
the effect of cyclic stretching on fibrin-based tubular constructs seeded with porcine
valve interstitial cells (PVIC) and nHDF. Cyclic stretching with 10% constant strain
amplitude applied for 3 weeks led to modest improvement in tensile properties of the
tubular constructs. We hypothesized that long-term cyclic stretching, as was used in
this study, could induce cellular adaptation, minimizing the benefits of cyclic stretching.
This hypothesis was tested in subsequent experiments using tubular constructs cultured
with incremental strain amplitude cyclic stretching, with an average strain of 10% for 3
weeks. Both PVIC and nHDF seeded constructs exhibited a 2-fold improvement in
ultimate tensile strength (UTS) and collagen density over samples conditioned with
constant strain amplitude strteching. To verify that this was the result of a cellular
response, phosphorylation of extracellular signal-regulated kinase (ERK) was measured
by western blot. At 5 weeks, the phosphorylated ERK was 255% higher in incremental
cyclic strained samples compared to constant strain samples.
nHDF-seeded tubular constructs were also used to optimize the use of
transforming growth factor beta (TGF-β). Studies showed that under cyclic stretching
conditions, TGF-β has detrimental effects on total collagen deposition and collagen
maturation. Western blot analysis showed a decrease in p-ERK signaling in TGF-β
treated samples. However, TGF-β use demonstrated a benefit by increasing the elastin
content of the tissue constructs. In subsequent experiments, a sequence of cyclic stretching and TGF-β supplementation was used to optimize tensile mechanical
properties and elastin content of the engineered tissue.
Based on the results with tubular constructs, a novel bioreactor was designed to
apply controlled cyclic stretching to the fibrin-based TEHV. Briefly, the valve was
mounted on two plastic end-pieces with elastic latex tube placed around TEHV. Using a
reciprocating syringe pump, culture medium was cyclically pumped into the bioreactor.
The root distension, which was determined by the stiffer latex was used as a control
parameter, and in turn stretched the leaflets. A separate flowloop (connected to the
bioreactor end-pieces) was used to control nutrient transport to the TEHV. Using an
incremental strain amplitude stretching regime, fibrin-based TEHV were conditioned in
the bioreactor for 3 weeks. Cyclically stretched valves (CS valve) had improved tensile
properties and collagen deposition compared to statically-cultured valves. The
mechanical stiffness (modulus) and anisotropy (measured as ratio of leaflet modulus in
circumferential to radial directions) in the leaflets was comparable to native sheep
pulmonary valve leaflets. Collagen organization/ maturation also improved in CS valves
over statically-cultured valves as observed by picrosirius red staining of tissue crosssections.
In addition, the CS valve root could withstand pressures of up to 150 mmHg
and its compliance was comparable to that of the sheep pulmonary artery at
To assess in vivo remodeling TEHV were implanted in the pulmonary artery of
two sheep for 4.5 weeks with the pulmonary valve either left intact or rendered
incompetent by leaflet excision. Echocardiography immediately after implantation
showed functional coapting leaflets, with normal right heart function. It was also
performed just prior to explantation, revealing functional leaflets although with
moderate regurgitation in both cases and a partial detachment of one leaflet from the
root in one case. The explanted leaflets had thickness and tensile properties comparable
the implanted leaflets. There was endothelialization on the lumenal surface of the
TEHV root. These preliminary results are unprecedented for a TEHV developed from a
biological scaffold; however, many issues remain to be surmounted.
In further development of the TEHV with a fibrin scaffold, photo-cross linking
of the fibrin gel was utilized as a method to stiffen the matrix, thereby inhibiting
excessive cell-induced compaction. Preliminary studies with tubular constructs
demonstrated reduced compaction of cross-linked fibrin gel during cyclic stretching
with no effect on nHDF proliferation or deposited collagen. In addition, a preliminary
investigation using blood outgrowth endothelial cells (BOEC) has been conducted to
assess their adhesion to the remodeled TEHV surface. Studies showed BOEC adhesion
and proliferation on remodeled fibrin surface creating a confluent layer after 4 days of
culture. Successful seeding of sheep BOEC on the TEHV surface prior to implantation
would reduce the risk of clotting.
Overall, the studies presented in this dissertation advance the development of a
completely biological tissue-engineered heart valve. These studies improve our
understanding of the role of cyclic stretching in tissue remodeling and have furthered
the science of mechnotransduction and tissue remodeling.
University of Minnesota Ph.D. dissertation. April 2009. Major: Chemical Engineering. Advisor: Robert T. Tranquillo. 1 computer file (PDF); xii, 215 pages, appendices A-C.
Syedain, Zeeshan Hayder.
Development of a completely biological tissue engineered heart valve..
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