Design of a Biodegradable Aortic Heart Valve

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Design of a Biodegradable Aortic Heart Valve

• Jesse S. Little, MS
• Stony Brook University
• Department of Biomedical Engineering
• 2003 Qualifying Exam Defense
• July 16, 2003

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Aortic Valve Structure 3-Leaflets, 3-Layers
Images Courtesy of http//www.heartlab.robarts.ca/
dissect/dissection2.html
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Valve Pathologies Limit Blood Flow

• Aortic Stenosis
• Occlusion of orifice
• Aortic Regurgitation
• Abnormal backflow

• Symptoms
• Angina Pectoris
• Myocardial Infarction
• Arrhythmias

Image Courtesy of http//www.brisbio.ac.uk
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Limited Options for Valve Replacement

• Mechanical Valves
• Readily available
• Require life-long anticoagulants
• 10 year life expectancy
• Porcine Xenographs
• Good initial haemodynamic performance
• Do not require anticoagulants
• 59 complication rate after 12 years
• (Billiar and Sacks, 2000)

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Tissue Engineered Alternatives

• Degradable scaffold

• SMC and Endothelial Cells

Polyesterurethanes
PGA
PGLA
Carotid Artery Femoral Artery
PGA/P4OH
PVA hydrogels
PHOH
P4OH
Image Courtesy of Hoerstrup et al., 2000
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Why Dont They Work?

• Difficult to form functional three leaflet valve
• Bicuspid valves are more likely to become
  stenotic (Stephen et al., 1997)

• Implanted valves failed to grow in vivo
  (Hoerstrup et al., 2000)
• Are cells receiving wrong mechanical cues?

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Tissue/Polymer Mechanical Profile Disparities
Polymer Bulk Material Properties
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Past Attempts Controlled Porosity

• Porogen Leaching

• Advantages
• - Controlled pore size
• - Controlled porosity
• - Disadvantages
• - No control over general architecture
• - Pores tend to be closed
• - Limits cellular ingrowth
• - Limits cell signalling

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Other Modes of Pore Induction

• Extrusion Fiber Braiding
• Uses high temperature
• Gas Foaming
• No control over architecture
• Electrospinning
• Phase Separation
• Uses organic solvents
• Solid Free-Form Fabrication
• 3D scaffolds built layer-by-layer
• Internal/External architecture predefined

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Design Objectives

• General
• Comprehensive tissue engineering approach to
  valve design
• Specific
• Provide good haemodynamics
• Open and close during proper phases of
  systole/diastole
• Minimize regurgitant volume
• Scaffold should degrade within 8-12 weeks
• Provide mechanical profile closely matching that
  of target tissue
• Constraints
• Avoid use of porogen leaching and electrospinning

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Design General Description

• Composite structure to recreate fibrous and
  GAG-rich layers
• 3-leaflet structure
• Fabricated using indirect solid free-form
  fabrication
• Dynamically seeded with autologous cells

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3-Layer Scaffold Mimicking Native Valve

• Fibrosa Ventricularis
• poly(propylene fumarate) crosslinked to
  poly(ethylene glycol)-dimethacrylate
• PEG-DMA/PPF
• Crosslinks in situ
• Spongiosa
• Chitosan crosslinked to chondroitin sulfate-A
  (CSA)
• Chitosan - polysaccharide
• CSA GAG
• Crosslinks ionically

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Indirect Solid Free-Form Fabrication

• Micro-Stereolithography
• Built in 1 µm thick layers
• Resolution of 10 µm
• Most rapidly advancing SFF type
• Negative scaffold manufactured and used as mold
  for actual scaffold
• Avoids restraints on polymer types
• External geometry defined using Image-Based
  Design (IBD)
• Internal architecture can be optimized and
  defined using CAD (.stl format)

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Optimization of Internal Architecture

• Calculation of effective scaffold and regenerate
  tissue stiffness Ceff CM
• 2 distinct time points
• t 0, Cscaffold eff
  CscaffoldM(d1,d2,d3)scaffold
• t final, Ctissue eff
  CtissueM(d1,d2,d3)tissue

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Minimized Stiffness Function

• Optimization of pore architecture using

• Constraints

Porosity 75, 80, 85, 90, 95
Vpore ?
Vtotal d33
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Calculation of Pore Volume
V3_cyl A B C (I I-II I-II-III
I-III) (II I-II I-II-III II-III) (III
I-III II-III I-II-III) I II III
(I-II) (I-II) (I-II-III) (I-III)
(I-III) (I-IIIII) (II-III) (II-III)
(I-IIIII)
Shared Volume V(AB) (I-II) (I-II-III)
V(AC) (I-III) (I-II-III) V(BC) (II-III)
(I-II-III) V(ABC) (I-II-III)
V3_cyl I II III (I-II) (I-III)
(II-III) V(AB) V(AC) V(BC)
Vactual I II III (I-II) (I-III)
(II-III) (I-II-III)
Vactual V3_cyl V(AB) V(AC) V(BC)
V(ABC)
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Optimal Pore Dimensions
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Scaffold Fabricated in 3 Steps

• Photopolymerization of initial mold
• Low molecular weight monomer capable of
  chain-reacting under radiant energy
• Small spot laser or masked lamp
• Negative ceramic mold created
• Hydroxyapatite (HA) and acrylic slurry
• Sintered
• PEG-DMA/PPF and chitosan/CSA injected into mold
  in layers
• Ventricularis 0.17 mm PEG-DMA/PPF
• Spongiosa 0.176 mm Chitosan/CSA
• Fibrosa 0.30 mm PEG-DMA/PPF
• Heat compression molding if necessary
• Verified Using Mercury Intrusion Porosimetry
• Pore size
• Pore Volume
• Surface Area of Scaffolds

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Pore Size Monomer Ratio Effects on Strength

• Determination of Effective Elastic and Shear
  Moduli
• Tensile and Compressive
• Calculation of Poissons Ratio
• Creep-Recovery
• Stress-Relaxation
• Flexural Strength
• Statistical Analysis of All Results
• n5 for all pore/monomer ratio combinations
• Test both individual layers valve construct
• Normalized data Two-Factor ANOVA w/ post-hoc
  Bonferroni
• Non-normalized data Paired Kruskal-Wallis Signed
  Rank test

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Cell Harvesting, Culturing and Multiplication

• Autologous saphenous vein harvested, minced into
  1 mm2 pieces
• Endothelial cells separated from SMC
• Enzymatic digestion for pure endo. culture
• Removal of endo. lining for pure SMC culture
• Verification using immunofluorescence staining
• Endothelial cells () for PECAM 1 and vWF
• SMC () for smooth muscle actin
• Standard media supplemented with antibiotics

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Dynamic Cell Culturing In vitro

• Pulse Duplicator
• Exposure to controllable shear forces
• Proper alignment of SMC and endothelial cells
• Better cell-to-cell communication
• Functional ECM

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In vitro Biological Testing

• Cellular attachment using ESEM
• Inclusion of adhesion molecules
• t-test
• Cellular proliferation using DNA assay
• Monomer ratios/pore size
• Two-factor ANOVA w/ post-hoc Bonferroni
• ECM formation using 4-hydroxyproline assay
• Monomer ratios/pore size
• Two-factor ANOVA w/ post-hoc Bonferroni
• Degradation rate

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Hydrodynamic Performance Assessment

• Satisfactory forward and regurgitant flow
• Steady and pulsatile flow conditions
• Continuous Digital Particle Image Velocimetry
• Reynolds number (Re), volumetric flow rate (Q),
  pressure drop (?p)
• Bernoulli Eqn.
• Statistical comparison between valve and control
  using Two-Factor ANOVA w/ post-hoc Dunnetts test

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In vivo Implantation in Lamb Model

• Biocompatibility tests
• Lamb model (n6)
• FDA approved valve in control group (n2)
• 20 week study duration
• Echocardiography and angiography
• Statistical comparisons using z-statistic
• Histological examination upon sacrifice
• Cellular organization and proliferation
• Thrombus formation
• Major organs examined for pathology

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Costs and Benefits

• Costs
• One time incidentals for STL equipment, IBD CAD
  software
• Daily technical support for cellular expansion
• Additional surgical procedure
• Benefits
• Structural approach to scaffold design
• More accurate mechanical cues
• Fine control over external shape
• Better functionality and closure
• Avoids the need for anticoagulants
• POTENTIAL FOR GROWTH

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Design Limitations

• Necessitates additional surgical procedure for
  autologous cells
• Allogenic cells could be used
• Increased inflammatory response
• Possible reduction by encapsulating
  fluoroquinolones into scaffold
• Not possible for emergent cases

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Future Directions

• Modifications to optimization procedure
• Degradation using hydrolysis and molecular weight
• Gradient pore size
• Encourage directionality of collagen organization
• Nanoscale surface topography
• Surface etching
• Better cellular attachment
• Inclusion of actual GAG found in spongiosa

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Thank You!