A Tissue Engineered Bioactive Vascular Scaffold

1
A Tissue Engineered Bioactive Vascular Scaffold

• Karen Roberts Biomedical Engineering
• Janell Carter Biomedical Engineering
• Dr. Kenneth Barbee Advisor
• Senior Design Final Presentation
• May 24, 2001

2
Objective

• The broad objective is to develop a bioactive
  vascular scaffold
• Specific Scaffold Geometry
• Mechanical Conditioning
• Dynamic Culturing

3
Proposed Tissue Engineered Artery

• A tissue engineered biodegradable PLAGA
  electrospun cylindrical scaffold seeded with
  smooth muscle cells
• The electrospun scaffold will provide a porous
  environment for cell invasion
• The mechanical properties will be enhanced with
  dynamic mechanical conditioning

4
Agenda

• Significance
• Solutions Available
• Our Proposed Idea
• Electrospinning
• Dynamic Mechanical Conditioning

• Phase I
• Phase II
• Results
• Problems Encounted
• Future Investigations Phase III
• References

5
Significance

• Cardio vascular disease is principle killer in US
• About 58 million American (almost one-fourth of
  the nations population) live with some form of
  cardiovascular disease
• High blood pressure - 50,000,000
• Coronary heart disease - 12,200,000
• Small Artery Graft procedures - 600,000/ yr

6
Solutions Available

• Angioplasty
• Balloon catheter
• Stent
• Small mesh like wire tube
• 95 successful
• 20-25 experience restenosis
• Bypass
• Segment of vein, usually from leg, to by pass
  blockage.

7
Implants Solution In Development

• Endothelial Cell Repair
• Endothelial cell / polymer matrix scaffold
• Help fight restenosis
• Collateral angiogenesis
• Burning tiny holes into heart for vessel growth
• Hormone therapy

8
Pseudo Tissue Engineered Arteries

• Plastic tube surgically placed in abdominal
  cavity
• Fibrous tissue growth
• Tube removed, tissue tube used for bypass
• Animal studies have lasted 12 months

9
Background

• Ideally Tissue engineering is to develop a
  material that is biologically functional
• Synthetic material results in heightened immune
  response
• Bioabsorbable scaffold would guide cells to a
  specific geometry and degrade as the cells
  proliferate

10
Anatomy

• Tunica Intima
• Elastic Mixed Muscular
• Tunica Media
• Elastic greater elastin-collagen content
• Mixed Equal SMC and elastin-collagen content
• Muscular greater SMC content
• Tunica Adventia
• Elastic Mixed Muscular

11
Anatomy Tunica Media

• The number of concentric layers is proportional
  to wall thickness
• Aorta Thin Wall relative to internal diameter
• Coronary Thick walled relative to diameter
• Surrounding elastic lamina is less defined in
  comparison with the internal lamina

Type Internal Diameter Wall Thickness
Elastic 25 mm 2 mm
Mixed 4 mm 1 mm
Muscular 30 mm 20 mm
12
Mechanical Properties

• Visco-elastic Stress-Strain Curve
• Two moduli shows both properties
• Coronary arteries are in most demand
• Physiological pressures
• Systolic 120 mmHg
• Diastolic 90 mmHg

13
Dynamic Mechanical Conditioning

• Repetitive mechanical conditioning in the form of
  cyclic stress
• Inflation and deflation of silicone conduits in a
  bioreactor by filling with cell culturing medium
• This is hypothesized to increase cell growth,
  proliferation, and enhance organization As a
  result mechanical properties will be enhanced
• Studies by Seliktar et al. have had moderate
  success

14
Dynamic Mechanical Conditioning
15
PLAGA

• Components Lactic Acid and Glycolic Acid
• Glycolic acid is naturally occurring in fruit
  acid derived from sugar cane
• Lactic acid is a naturally occurring substance
  found in body
• They form a copolymer when polymerized
• Dexon was first FDA approved totally synthetic
  absorbable suture

16
PLAGA

• Copolymer degrades by hydrolysis
• Macrophages easily consume these particles
• Mechanical properties can be altered by changing
  the concentrations and chain lengths
• Homopolymer combinations are more crystalline
• Copolymers are more amorphous

17
Electrospinning

• A nonwoven porous mesh can be fabricated by
  electrospinning
• The electrospinning process employs the use of
  electrostatic fields to form and accelerate
  liquid jets from the tip of a capillary
• Evaporation of the solvent forms fibers that are
  nanometers in diameter
• The resultant nonwoven mesh is of variable fiber
  diameters and pore size distribution

18
Electrospinning
19
Matrix Characterization

• Tensile test
• Youngs modulus
• Elongation
• Toughness
• Ultimate strength

• Porosity
• Average Pore Size
• SEM
• Mat thickness
• Porosity
• Fiber diameter

20
Mechanical Testing

• Used to determine the stress/ strain data under
  tension, compression, and torsion
• Nanofiber matrices - tensile test are conducted
  because the primary force arteries are subjected
  to in vivo are radial tensile forces
• There is an acceptable amount of error associated
  with this data

21
Specific Aims Phase I

• Electrospin a variety of mats in accordance to
  our design matrix
• Fully characterize the mats by performing
  mechanical and porosity tests

Concentration wt Time hrs Time hrs Time hrs
15 1 3 6
20 1 3 6
25 1 3 6
25-20-25-20 1 hr each 1 hr each 1 hr each
22
Specific Aims Phase II

• Electrospin PLAGA scaffold on to a mandrel of
  characteristic artery shape according to results
  from phase I
• Conduct characterization by SEM

23
Specific Aims Phase III

• Sterilization of scaffolds
• Seed smooth muscle cells on to cylindrical
  scaffold
• Dynamically culture cells and mechanically
  condition scaffold.
• PLAGA degredation studies

24
Goals Achieved

• Phase I
• 20 wt PLAGA Planar Mat
• Phase II
• Cylindrical PLAGA Scaffolds
• 15 wt - 20 wt - 25 wt
• 20 wt - 25 wt layered

25
Preliminary Study Procedure

• Electrospun mat from 20 wt PLAGA in 8020
  THF/DMF solution
• Characterization
• Tensile testing 1x6 cm strips
• SEM 1cm2 gold sputtered
• Porosity Mercury fills pores for density
  readings

26
Secondary Study Procedure

• The primary goal of this study was to achieve a
  variety electrospun 5050 PLAGA scaffold in a
  tubular shape
• 15 wt
• 20 wt
• 25 wt
• Layered 20 25 wt
• Characterization of cylindrical scaffold
• SEM 1cm2 gold sputtered

27
Electrospinning Chamber
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Rotation Device
Polymer solution
Grounded aluminum mandrel
Grounded aluminum mandrel
Positive needle
Silicone mandrel
29
Construct
Aluminum Mandrel
Silicone Sleeve
PLAGA Construct

• A silicone sleeve slid over a grounded aluminum
  mandrel
• The construct was attached to a gearbox with a
  motor
• Construct was rotated at a gear ratio of 807.931

30
Results

• Preliminary Study
• 20 wt PLAGA Planar Mat

31
Tensile Test 20 wt PLAGA Mat
32
Tensile Test 20 wt PLAGA Mat
Mechanical Property Average Value
Ultimate strength 7.793 MPa
Breakage elongation 31.3
Youngs modulus 98.659 MPa
Toughness 1.943 MPa
33
Porosity 20 wt Mat
Calculated Pore Diameter (mm) Computed Pore Diameter (mm) Pressure (Psia) Hg Surface Tension (dynes/cm) Contact Angle of Hg
157.27 159.86 79289 485 130
97.76 97.21 127553 485 130
56.87 57.33 219253 485 130
21.27 21.81 586054 485 130
6.01 6.04 2072563 485 130
34
20 wt PLAGA
Planar Mat Fiber Diameter 170 nm 10 mm Pore
Size 1-100 mm

35
Results

• Secondary Study
• Cylindrical PLAGA Scaffolds
• 15 wt
• 20 wt
• 25 wt
• 20 wt - 25 wt layered

36
15 wt PLAGA
Cross Section Thickness 241mm Fiber
Diameter None Pore size None
37
20 wt PLAGA
Cross Section Thickness 15 mm Fiber
Diameter 170 nm Pore Size 1 5 mm
38
25 wt PLAGA
Cross Section Thickness 60 mm Fiber
Diameter 1-10 mm Pore Size 10 50 mm
39
25 wt PLAGA
Cross Section Thickness 60 mm Fiber
Diameter 1-10 mm Pore Size 10 50 mm
40
25 wt PLAGA
Lateral View Thickness 60 mm Fiber
Diameter 1-10 mm Pore Size 10 50 mm
41
20 wt 25 wt PLAGA Layered
Lateral View Thickness Total 108 mm Thickness
Each Layer 34 mm 38 mm 34 mm 36 mm
42
20 wt 25 wt PLAGA Layered
Cross Section Thickness Total 108 mm Thickness
Each Layer 34 mm 38 mm 34 mm 36 mm
43
20 wt 25 wt PLAGA Layered
44
Problems Encountered

• Phase I II Humidity/Rain Properties of
  Electrospun PLAGA was compromised in these
  conditions i.e. melting

• Phase III Sterilization All forms of
  sterilization melted the PLAGA except UV
  radiation ethylene oxide
• UV radiation Did not completely sterilize all
  of the time
• Money dynamic culturing apparatus upwards of
  40K

45
Future Investigations Phase III

• The scaffold that we designed was for use with
  Dynatek Dalta SVP216 - Small Vascular Prosthesis
  Tester
• This would provide the environment for dynamic
  mechanical conditioning of the cell seeded
  scaffold while maintaining an environment that is
  suitable for cell growth proliferation

46
Cell Culturing

• Seeding cells and incubate for 2 days using
  standard cell culturing techniques
• This is to allow for cell adhesion to PLAGA
• Dynamically condition / culturing for 4 8
  additional days

47
SVP216 - Small Vascular Prosthesis Tester

• Produce data acceptable to the FDA
• Positive displacement pumping system ensures
  known geometric expansion of samples
• All samples submersible in 37 degree C bath
• 2mm-16mm inner diameter grafts

48
Latex and Silicone Precision Mock Arteries

• Known mechanical properties leaves no second
  guessing
• Get the exact fit with precision diameters
• Fit all your products with virtually any shape or
  size

49
Considerations for the Future

• Mechanical conditioning must maintaining the
  correct mechanical properties Smooth muscle
  cells will rearrange within the scaffold as
  mechanical conditioning occurs
• Liquid Chromatography / Mass Spectroscopy
  monitoring of degradation of the polymer matrix
  over time
• A variety of PLAGA mixtures such as 8515 or 9010

50
Special Thanks

• Dr. Kenneth Barbee
• Dr. Frank Ko
• Dr. Attawia

• Yusef Khan Porosity
• Asaf Ali Mechanical Testing
• Dave Rohr SEM

51
References

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6. The Centers for Disease Control and Prevention
   web resources at www.cdc.gov
7. The American Heart Association web resources at
   www.americanheart.org

52
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