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

Cardiovascular Engineering and Technology, Vol. 2, No. 2, June 2011 (2011) pp. 101–112DOI: 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 SotoBME501 Tissue Engineering

“The Story”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, S3Echocardiograms 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

Heart ValvesThe 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

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Heart Valves

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

Heart Valve DiseasesRegurgitation: • 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.

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http://www.healthinplainenglish.com/health/cardiovascular/mitral_valve_stenosis/mitral-valve-stenosis.jpg

Artificial Heart Valves

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caged

tilting disc

single leaflet

bi-leaflet

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

DemographicsOver 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 DiseaseICD-9 424; ICD-10 I34 to I38.Mortality 23 313Any-mention mortality 44 149Hospital discharges 98 000

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Table 1

• 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

Figure 1

• 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

Figure 1

• 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

Table 2

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

Figure 2

Experimental groups used:IM0: at implantationIM4: explanted after 4 wkIM8: explanted after 8 wkIM8-az: sheep #4 after 8 wkNative : 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

Types of Mechanical Loads

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1. Compression2. Tension3. Bending4. Torsion5. Shear

Figure 3Stained 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

Figure 4Histology of explanted root tissue (S3)

4 a: trichrome stain 4 b: vWF stain4 c: αSMA of tissue near VE luminal surface4 d: vWF of tissue within the VE root4 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

Figure 5

Experimental groups used:IM0: at implantationIM4: explanted after 4 wkIM8: explanted after 8 wkIM8-az: sheep #4 after 8 wkNative : 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

Figure 6Assessment 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: αSMA6c: CD446d: CD45

Critique

• 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.

Recommended Experimentation

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

Recommended ExperimentTissue-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 RnaseDNA Quantification: Residual DNA in the heart valve tissue was quantified to confirm decellularization.this should allow recellularization by host cells of non-contractile phenotype

Recommended ExperimentBioreactor culturing

Sheep Implant

Recommended ExperimentEchocardiography 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 animalsEchos for S1-S4: 1-hr after implantEchos for S1-S4: 4 weeks after implant, prior to S1 and S3 explantEchos 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

Recommended ExperimentMechanical / Physical Properties : Uniaxial tensile strength test for leaflets and roots, thickness, Modulus

Recommended ExperimentBiochemical Analysis : concentrations of elastin, collagen, cells

Recommended ExperimentHistology

examine explanted valves leaflet and root tissuedetermine if leaflet length is appropriate to maintain coaptationdetermine if root or leaflet tissue has suffered degradation due to immunological reaction examine and evaluate recellularization in S3 and S4 by ovine host cells

Immunohistochemistryanti-mouse αSMA: identify presence of contractile-phenotype cellsanti-human β2-microglobulin: identify presence of surviving human cellsovine CD44: identify presence of host ovine cells

ReferencesPaper:

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 Updatehttp://circ.ahajournals.org/content/123/4/e18.full.pdf

Heart valves : Anatomy and Function, New York Presbyterian Hospitalhttp://nyp.org/health/heart-valves.html

University of Virginia Health System: Mitral Stenosis, Pulmonary Regurgitationhttp://uvahealth.com/services/heart/treatment/11670/?searchterm=stenosishttp://uvahealth.com/services/cardiac-valve-center/valve-conditions/pulmonary-regurgitation-1/pulmonary-regurgitation/?searchterm=regurgitation

University of Zurich, Division of Surgical Research: Cardiovascular Regenerative Medicinehttp://www.chir.uzh.ch/cardio/cardiotext/tissueengineering.html

Questions?