|
44.
BOEC Seeded, Lipid Modified Polyurethane Heart Valve Leaflets: In Vitro
And In Vivo Studies
Stanley J. Stachelek1; Ivan . Alferiev1;
Jeanne M. Connolly1; Richard . Bianco2;
Robert P. Hebbel2; * Robert J. Levy1
1Childrens Hospital of Philadelphia, Philadelphia,
Pennsylvania, United States; 2University of Minnesota,
Minneapolis, Minnesota, United States
|
|
OBJECTIVES: Polyurethane (PU) replacement valves are susceptible to ectopic
calcification and thrombus formation. Seeding prosthetic heart valve leaflets
with autologous endothelial cells would hypothetically mitigate these
problems. Blood outgrowth endothelial cells (BOECs) are a recently identified
circulating endothelial cell derived from bone marrow. Using innovative
bromoalkylation chemistry, we configured polyurethane films with cholesterol
moieties (PU-Chol) that possessed superior levels of BOEC retention under
valvular levels of shear stress. We tested the hypothesis that artificial
valve leaflets fashioned from PU-Chol can retain BOECs and prevent surface
thrombosis and calcification.
|
|
METHODS: Autologous BOECs were prepared from peripheral blood of uniquely
identified sheep and seeded upon both sides of the implantable PU leaflet. A
single leaflet of the pulmonary valve was replaced with a leaflet composed of
PU or PU-Chol +/- seeded BOECs. Leaflets were explanted after 30 or 90 days
and assessed for cell retention and identification of adherent cell type.
|
|
RESULTS: Organized thrombus and calcific nodules were noted on unseeded
unmodified leaflets, but absent on BOEC seeded leaflets. After 30 or 90 days
the number of cells on BOEC seeded leaflets per 200x field was 138.5±25.5 and
172.2 ±19.6 respectively. This was not significantly different than
preimplant numbers (271± 75). However, after 30 days there were significantly
(p= 0.005) fewer cells (17 ± 6) on unseeded control leaflets. Positive PECAM
staining on cell-seeded explants confirmed the continued presence of
endothelial cells.
|
|
CONCLUSIONS: In conclusion, BOEC seeding of PU-Chol represents a state of the
art approach for an endothelialized prosthetic valve leaflet.
|
45. Role Of Cell Phenotype And The Addition Of
ECM Protein On The Mechanical Properties Of Tissue-engineered Mitral Valve
Chordae
Ramakrishnan . Iyer; Yaling . Shi; Ivan . Vesely
University of Southern California, Los Angeles, California, United States
|
|
OBJECTIVES: Repair of mitral valve chordae tendineae is limited by the
availability of artificial materials possessing appropriate biomechanical
properties. Tissue Engineering is a technology that promises limitless supply
of living tissues tailored for a particular function. In an effort to “tissue
engineer” artificial chordae, we have been using the principle of directed
collagen gel shrinkage. In this study, we explored factors such as the choice
of cell phenotype and the addition of fibronectin to the collagen matrix.
Since fibronectin is known to associate with integrins that bind to collagen,
the addition of fibronectin is expected to improve the cell-mediated collagen
gel contraction process.
|
|
METHODS: Neonatal rat aorta was enzymatically digested, minced and placed
in two 6-well plates (labeled A to L) and supplemented with culture medium.
SMCs were isolated from these segments of aorta using the outgrowth method,
and 3 subcultures (B, E & F), selected for best confluence, were then
used to observe the cell-dependent effect on the mechanical strength of our
collagen constructs. Solubilized rat plasma fibronectin (5 ug/ml, 10ug/ml,
20ug/ml, 100ug/ml) was added at the start of the tissue culture process and
mature constructs examined mechanically for ultimate failure strength. Also
periodic addition (three times per week) of fibronectin (10ug/ml) was tested
for its effect on mechanical strength. In each case, cultures were fed with
DMEM/F12 medium containing 20% FBS and 2% antibiotics. Mature constructs were
examined histologically for microstructure, biochemically for cell content,
and tested mechanically to measure their failure strength.
|
|
RESULTS: Collagen constructs cultured using SMCs from subculture B had far
superior mechanical strength that the other two SMC subcultures. The addition
of fibronectin showed a proportional increase in mechanical strength at lower
concentrations (5 ug/ml, 10ug/ml, 20ug/ml), with little or no effect at
higher concentrations (100ug/ml). Constructs with optimal fibronectin had a
failure strength of 2 MPa, significantly greater than controls (1.4 MPa).
|
|
CONCLUSIONS: We have thus shown that cell subculture selection, often through
trial and error, can have a great effect on the mechanics of the final
constructs. The addition of relevant matrix factors can also play a
significant role in increasing the strength of tissue-engineered collagen
constructs.
|
46. Cellular Micro-integrated Elastomeric
Electrospun Scaffolds Under Heart Valve Tissue Engineering
* Michael S. Sacks1; William R. Wagner1;
* John E. Mayer2
1University of Pittsburgh, Pittsburgh, PA, United States;
2Childrens Hospital of Boston, Boston, MA, United States
|
|
OBJECTIVES: Tissue engineered pulmonary valves (TEPV) require scaffolds with
anisotropic mechanical properties that undergo large deformations coupled
with controllable biodegradative and cell-adhesive characteristics. As a next
step in fulfilling these design criteria, we recently synthesized a family of
poly(ester-urethane) ureas (PEUUs), including combination with type I
collagen at various ratios to enhance cell attachment and increase
biodegradation rates.
|
|
METHODS: Smooth muscle cells (SMCs) were fed into a sterilized capillary
charged at 10 kV and located 4 cm from the target mandrel. 12 wt% PEUU/HFIP
solution was fed into a capillary charged at 13KV and located 24 cm from the
target mandrel, which was charged at 4 kV and rotating at 250 rpm while
translating 8 cm along its axis at 8 cm/s.
|
|
RESULTS: Cell Microintegrated electrospun PEUU scaffolds were produced with
biaxial mechanical properties that are remarkably similar to the native
pulmonary valve, including the ability to undergo large physiologic strains
and pronounced mechanical anisotropy. Moreover, a novel cell
micro-integration technique has been developed that allows for successful
integration of the cells directly into the scaffolds at the time of
fabrication, eliminating cellular penetration problems.
|
|
CONCLUSIONS: These encouraging results suggest that ES-PEUU cell
micro-integrated scaffolds can serve as successful TEPV scaffolds. Strategic
combinations of individual mechanical factors relevant to heart valves—cyclic
flexure, strain, and flow—can be determined that optimize ECM organization,
and mechanical properties of cell seeded TEPV. Moreover, use of novel
elastomeric scaffolds can add a critical degree-of-freedom for TEPV designs
by allowing for large strains and highly controllable mechanical anisotropy.
|
