Cardiovascular Tissue Engineering and Repair: Implantation of a Tissue-engineered Heart Valve from Human Fibroblasts Exhibiting Short Term Function in the Sheep Pulmonary Artery

1. Cardiovascular Engineering and Technology, Vol. 2, No. 2, June 2011 (2011) pp. 101–112
DOI: 10.1007/s13239-011-0039-5
ZEESHAN H. SYEDAIN1, MATTHEW T. LAHTI2, SANDRA L. JOHNSON4, PAUL S. ROBINSON4, GEORGE R. RUTH2, RICHARD W. BIANCO2 3,
and ROBERT T. TRANQUILLO 1 4
1Department of Chemical Engineering & Materials Science, University of Minnesota, Minneapolis, MN, USA; 2Experimental Surgical
Services, University of Minnesota, Minneapolis, MN, USA; 3Department of Surgery, University of Minnesota, Minneapolis, MN, USA; and
4Department of Biomedical Engineering, University of Minnesota, 7-114, 312 Church St SE, Minneapolis, MN 55455, USA
Raul Soto
BME501 Tissue Engineering

2. Neonatal human fibroblasts culture,
and preparation of Tissue-Engineered
Valves with Dacron sewing rings
Controlled cyclic stretching bioreactor
Echocardiograms prior to valve
implantation
Explant valve from S1, S2
after 4 weeks. Observed reactive
tissue growth in S1 due to PLA
Explant valve from S3 after 8 weeks.
Observed leaflet tissue degradation
Implant valves on sheep S1, S2, S3
Echocardiograms post-implantation
Treat valve with sodium azide,
implant in S4, explant after 4 weeks
Histology, tensile strength testing,
biochemical properties for S1, S2
Histology, tensile strength testing,
biochemical properties for S3
Histology, tensile strength testing,
biochemical properties for S4

3. The heart consists of four chambers
• Two atria (upper chambers)
• Two ventricles (lower chambers).
Valves are flaps that located on each
end of the two ventricles (lower
chambers of the heart).
Valves prevent the backward flow of
blood.
As the heart muscle contracts and
relaxes, the valves open and shut,
letting blood flow into the ventricles
and atria at alternate times.
http://www.edoctoronline.com/medical-atlas.asp?c=4&id=22190

4. http://en.wikipedia.org/wiki/File:Apikal4D.gif

5. tricuspid valve: located between the
right atrium and the right ventricle
pulmonary valve: located between
the right ventricle and the pulmonary
artery
mitral valve: located between the left
atrium and the left ventricle
aortic valve: located between the left
ventricle and the aorta
http://images.med.cornell.edu/body/greystone/em_0019w.jpg

6. Regurgitation:
• valve(s) does not close completely
• Blood flows backward instead of forward
• turbulent flow erodes tissue
Stenosis:
• valve(s) opening becomes narrowed or does
not form properly
• inhibits ability of the heart to pump blood
• increased force required to pump blood
through the stiff (stenotic) valve(s).
• increased fluid pressure damages tissue
Heart valves can have both malfunctions at the
same time
When valves fail to open and close properly,
the implications for the heart can be serious,
possibly hampering the heart's ability to pump
blood adequately through the body.
http://uvahealth.com/services/cardiac-valve-center/valve-
conditions/pulmonary-regurgitation-
1/resolveuid/34a6422329f28ec4002e775a33089ad6/image_preview
http://www.healthinplainenglish.com/health/cardiovascula
r/mitral_valve_stenosis/mitral-valve-stenosis.jpg

7. http://www.pages.drexel.edu/~nag38/Images/types_of_heart_valves.png
caged
tilting disc
single leaflet
bi-leaflet

8. TEHV before implantation
Macroscopic picture
of autologous tissue
engineered heart
valve (TEHV) based
on vascular-derived
cells integrated into
a self-expanding
nitinol stent, (A)
distal view and (B)
proximal view.
http://www.chir.uzh.ch/cardio/cardiotext/tissueengineering.html

9. Over 95,000 valve replacement surgeries are
now performed annually in the US.
In the pediatric population, 15–25% of the
congenital heart defects (>36,000/ year) are
associated with the pulmonary position,
requiring repair or replacement of the
pulmonary valve.
American Heart Association
Heart Disease and Stroke Statistics 2011
Valvular Heart Disease
ICD-9 424; ICD-10 I34 to I38.
Mortality 23 313
Any-mention mortality 44 149
Hospital discharges 98 000
http://images1.wikia.nocookie.net/__cb20100720160018/logope
dia/images/thumb/7/7c/American_Heart_Association_heart.svg/5
00px-American_Heart_Association_heart.svg.png

10. • S1: control, polylactic acid mesh, previously-existing tissue-engineered valve
• S2, S3 Dacron: this is the experimental treatment
• S2, S3, S4 Dacron: to prevent reactive tissue growth observed in S1 outside the
polylactic acid (PLA) mesh
• S4: Sodium azide pre-treatment: to kill fibroblasts
• In addition to these four sheep, non-operated sheep were used as negative
controls

11. • 1a: VE during static culture on custom Teflon mold
• 1b, c: Side view of VE leaflets in open and closed position
• 1d: End—view of VE prior to implant
• 1e,f: End-view of VE leaflets in open and closed position
• VEs were functional at implantation and at least 4 weeks in vivo

12. • 1k: image of VE leaflets
and root after explants at
4 weeks
• 1h, i: side view of VE
after 4 weeks
implantation
• 1g: Doppler flow
profile of VE after
implant
• 1j: Doppler flow profile of VE
after 4 weeks implantation

13. Echocardiography data, post-implantation: compares values for
• Mean flow velocity (cm/s)
• Peak pressure gradient (mmHg)
• Mean pressure gradienf (mmHg)
• Orifice area (cm2)
Controls:
Native pulmonary valve (healthy, non-operated 6-month old sheep)
VEs were functional at implantation

14. Experimental groups used:
IM0: at implantation
IM4: explanted after 4 wk
IM8: explanted after 8 wk
IM8-az: sheep #4 after 8 wk
Native : negative control,
sheep pulmonary valve
leaflets
Comparison of
Tensile and
biochemical
properties of
explanted VE leaflets
against implant VE
leaflets
• Ultimate strength for explanted VEs after 4 and 8
weeks was higher than pre-implant value
• Thickness and stiffness (as measured by Young’s
Modulus) of explanted VE leaflets after 4 and 8 weeks
was comparable to pre-implant value
• Explanted VE leaflets had increased collagen and
elastin concentrations, and increased cellularization

15. http://www.adl.gatech.edu/classes/dci/structur/loads.jpg
http://aftercorbu.com/wordpress/wp-content/uploads/2007/11/stresses1.gif
54321
1. Compression
2. Tension
3. Bending
4. Torsion
5. Shear

16. Stained cross-
sections of leaflets
• The images compare the explanted VE leaflets with implant VE leaflets and with
pulmonary valve leaflets (native)
• Xenotic implant did not elicit inflammatory or immune responses (under
immunosuppression)
• positive staining by human β2-microglobulin in IM8-az (S4) indicates that human
fibroblasts survived the sodium azide treatment

17. Histology of explanted
root tissue (S3)
4 a: trichrome stain
4 b: vWF stain
4 c: αSMA of tissue near VE
luminal surface
4 d: vWF of tissue within the
VE root
4 e: vWF of tissue within the
pulmonary artery
• Immunostaining showed partial endothelialization after 4 weeks, extensive after 8.
• Luminal surface of explanted VE tissue had been “remodeled” and was similar to the
tissue of pulmonary artery
• higher collagen density, lower fibrin concentration
• microvessels present in root tissue, Young’s modulus, circumferential tensile
strength, cellularity => all similar to what is found in pulmonary artery tissue

18. Experimental groups used:
IM0: at implantation
IM4: explanted after 4 wk
IM8: explanted after 8 wk
IM8-az: sheep #4 after 8 wk
Native : negative control,
sheep pulmonary valve roots
Comparison of
circumferential tensile
and biochemical
properties of explanted
VE roots with implant
VE root
• Ultimate strength for explanted VEs after 4 and 8 weeks
was similar to pulmonary valve roots
• Thickness and stiffness (as measured by Young’s
Modulus) of explanted VE leaflets after 4 and 8 weeks
were higher than implant tissue, similar to pulmonary
tissue
• Explanted VE leaflets had increased collagen but
decrease elastin concentrations
• Cellularity (Mcells/mL) was similar to pulmonary tissue

19. Assessment of Host
Cell Invasion in S4:
Human Fibroblasts
Maintained Viability
and Contractile
Phenotype after 8
Weeks Implantation
• Immunostaining of explanted valves 8 weeks after
implant showed that most of the cells found in the
leaflets were human cells.
• A small amount of ovine cells was located around the
edges of the leaflets.
• The majority of the cells found in the root tissue were
also human.
6a: human β2-
microglobulin
6b: αSMA
6c: CD44
6d: CD45

20. • Table 2: Since echocardiography is non-invasive, they should have
also taken epicardial echocardiograms of S1-S4 before implant, and
then compared pre-implant echo data (blood flow velocity, peak and
mean pressure gradients, orifice area) against their post-implant echo
data for each sheep.
• Once they realized that for animal S3 the leaflets of the tissue-
engineered valves were becoming degraded at some point before 8
weeks, for animal S4, after week 4 they should have performed
echocardiograms every few days, instead of just at Weeks 4 and 8.
That would provide some idea of when the severe leaflet tissue
degradation starts, and the rate at which tissue degradation
progresses.

21. To determine if the presence of human cells of a contractile
phenotype are indeed the cause of leaflet degradation between
weeks 4 and 8 of implantation
• Grow the valves using neonatal fibroblasts
• complete decellularization after fibrin remodeling
• test, compare valves with and without decellularization
• determine if host cells are able to populate the decellularized
valve
• determine if leaflet tissue degradation still occurs after 4 weeks

22. Tissue-Engineered Valves Preparation and Culture
use neonatal fibroblasts (nhDF) to seed a fibrin gel.
inject into molds with Dacron sewing rings
Complete Decellularization After Fibrin Remodeling - manufacture valves
with and without human fibroblasts
Decellularized tissue by lysis in Tris buffer and EDTA, followed by 6h of
solubilization in SDS with orbital mixing, and washing in PBS.
DNA removal by incubation in PBS with Dnase and Rnase
DNA Quantification: Residual DNA in the heart valve tissue was
quantified to confirm decellularization.
this should allow recellularization by host cells of non-contractile
phenotype

23. Bioreactor culturing
Sheep Implant

24. Echocardiography before and 1 hr
after implant: measure mean flow
velocity, peak and mean pressure gradients,
orifice area.
Echos for S1-S4, negative control:
before implant, to obtain baseline
values for all animals
Echos for S1-S4: 1-hr after implant
Echos for S1-S4: 4 weeks after
implant, prior to S1 and S3 explant
Echos for S2, S4: 8 weeks after
implant, prior to explant
compare post-implant S1-S4 values against
the same animal’s pre-implant values,
and against the negative control
results

25. Mechanical / Physical Properties : Uniaxial tensile strength test for leaflets and
roots, thickness, Modulus

26. Biochemical Analysis : concentrations of elastin, collagen, cells

27. Histology
examine explanted valves leaflet and root tissue
determine if leaflet length is appropriate to maintain coaptation
determine if root or leaflet tissue has suffered degradation due to
immunological reaction
examine and evaluate recellularization in S3 and S4 by ovine host cells
Immunohistochemistry
anti-mouse αSMA: identify presence of contractile-phenotype cells
anti-human β2-microglobulin: identify presence of surviving human cells
ovine CD44: identify presence of host ovine cells

28. Paper:
Syedain Z, et al. Implantation of a Tissue-Engineered Heart Valve from Human Fibroblasts
Exhibiting Short Term Function in the Sheep Pulmonary Artery. Cardiovascular Engineering and
Technology. 2011 (2); 101-112.
Websites
American Heart Association: Heart Disease and Stroke Statistics – 2011 Update
http://circ.ahajournals.org/content/123/4/e18.full.pdf
Heart valves : Anatomy and Function, New York Presbyterian Hospital
http://nyp.org/health/heart-valves.html
University of Virginia Health System: Mitral Stenosis, Pulmonary Regurgitation
http://uvahealth.com/services/heart/treatment/11670/?searchterm=stenosis
http://uvahealth.com/services/cardiac-valve-center/valve-conditions/pulmonary-regurgitation-1/pulmonary-
regurgitation/?searchterm=regurgitation
University of Zurich, Division of Surgical Research: Cardiovascular Regenerative Medicine
http://www.chir.uzh.ch/cardio/cardiotext/tissueengineering.html