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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
May 24,2001
Objective
The broad objective is to develop a bioactive vascular scaffold
Specific Scaffold Geometry
Mechanical Conditioning
Dynamic Culturing
May 24,2001
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
May 24,2001
Agenda
Significance
Solutions Available
Our Proposed Idea Electrospinning Dynamic Mechanical
Conditioning
Phase I
Phase II
Results
Problems Encounted
Future Investigations – Phase III
References
May 24,2001
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
May 24,2001
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.
May 24,2001
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
May 24,2001
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
May 24,2001
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
May 24,2001
Anatomy
Tunica IntimaElastic = Mixed = Muscular
Tunica Media Elastic: greater elastin-
collagen content Mixed: Equal SMC and
elastin-collagen content Muscular: greater SMC
content
Tunica AdventiaElastic = Mixed = Muscular
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Anatomy – Tunica Media
The number of concentric layers is proportional to wall thicknessAorta – Thin Wall relative to
internal diameterCoronary – 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 m 20 m
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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
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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
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Dynamic Mechanical Conditioning
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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
May 24,2001
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
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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
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Electrospinning
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Matrix Characterization
Tensile test Young’s modulus % Elongation Toughness Ultimate strength
Porosity Average Pore Size
SEM Mat thickness Porosity Fiber diameter
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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
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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
15 1 3 6
20 1 3 6
25 1 3 6
25-20-25-20 1 hr each
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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
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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
May 24,2001
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
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Preliminary Study Procedure
Electrospun mat from 20 wt % PLAGA in 80:20 THF/DMF solution
Characterization Tensile testing – 1x6 cm strips SEM – 1cm2 gold sputtered Porosity – Mercury fills pores for density
readings
May 24,2001
Secondary Study Procedure
The primary goal of this study was to achieve a variety electrospun 50:50 PLAGA scaffold in a tubular shape 15 wt% 20 wt% 25 wt% Layered 20 & 25 wt%
Characterization of cylindrical scaffold SEM – 1cm2 gold sputtered
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Electrospinning Chamber
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Rotation Device
Silicone mandrel
Positive needle
Polymer solution
Grounded aluminum mandrel
Grounded aluminum mandrel
May 24,2001
Construct
Aluminum Mandrel
Silicone Sleeve
PLAGA ConstructA silicone sleeve slid over a grounded aluminum
mandrelThe construct was attached to a gearbox with a motorConstruct was rotated at a gear ratio of 807.93:1
May 24,2001
Results
Preliminary Study: 20 wt% PLAGA Planar Mat
May 24,2001
Tensile Test 20 wt% PLAGA Mat
Tensile Test Data for PLAGA Sample #1
0
50
100
150
200
250
300
350
400
450
500
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Displacement (cm)
Fo
rce
(g)
0
1
2
3
4
5
6
7
8
9
Strain (l/l0)
Str
ess
(MP
a)
Load Engineering Stress
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Tensile Test 20 wt% PLAGA Mat
Mechanical Property
Average Value
Ultimate strength 7.793 MPa
Breakage elongation
31.3%
Young’s modulus 98.659 MPa
Toughness 1.943 MPa
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Porosity 20 wt% Mat
Calculated Pore Diameter (m)
Computed Pore Diameter (m)
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
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20 wt% PLAGA
Planar Mat
Fiber Diameter:
170 nm –
10 m
Pore Size:
1-100 m
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Results
Secondary Study:Cylindrical PLAGA Scaffolds
15 wt % 20 wt% 25 wt% 20 wt% - 25 wt% layered
May 24,2001
15 wt% PLAGA
Cross Section
Thickness:
241m
Fiber Diameter:
None
Pore size
None
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20 wt % PLAGA
Cross Section
Thickness:
15 m
Fiber Diameter:
170 nm
Pore Size:
1 – 5 m
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25 wt % PLAGA
Cross Section
Thickness:
60 m
Fiber Diameter:
1-10 m
Pore Size:
10 – 50 m
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25 wt% PLAGA
Cross Section
Thickness:
60 m
Fiber Diameter:
1-10 m
Pore Size:
10 – 50 m
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25 wt % PLAGA
Lateral View
Thickness:
60 m
Fiber Diameter:
1-10 m
Pore Size:
10 – 50 m
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20 wt% & 25 wt% PLAGA Layered
Lateral View
Thickness Total:
108 m
Thickness Each Layer: 34 m38 m34 m36 m
May 24,2001
20 wt% & 25 wt% PLAGA Layered
Cross Section
Thickness Total:
108 m
Thickness Each Layer: 34 m38 m34 m36 m
May 24,2001
20 wt% & 25 wt% PLAGA Layered
May 24,2001
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
May 24,2001
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
May 24,2001
Cell Culturing
Seeding cells and incubate for 2 days using standard cell culturing techniquesThis is to allow for cell adhesion to PLAGA
Dynamically condition / culturing for 4 – 8 additional days
May 24,2001
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
May 24,2001
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
May 24,2001
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 85:15 or 90:10
May 24,2001
Special Thanks
Dr. Kenneth Barbee
Dr. Frank Ko
Dr. Attawia
Yusef Khan – Porosity
Asaf Ali – Mechanical Testing
Dave Rohr – SEM
May 24,2001
References
1. Seliktar D, Black RA, Vito RP, Nerem RM. Dynamic conditioning of collagen-gel blood vessel constructs induces remodeling in vitro. Annals of Biomedical Engineering 2000; 28: 351-362.
2. Bhatnagar RS, Qian JJ, Gough CA. The role in cell binding of a -bend within the triple helical region in collagen 1(I) chain: structural and biological evidence for conformational tautomerism on fiber surface. Journal of Biomolecular Structure & Dynamics 1997; 14(5): 547-560.
3. Bhatnagar RS, Qian JJ, Wedrychowska A, et al. Design of biomimetic habitats for tissue engineering with P-15, a synthetic analogue of collage. Tissue Engineering 1991; 5(1): 53-65.
4. Ibim SM, Uhrich KE, Bronson, R, et al. Poly(anhydride-co-imides): in vivo biocompatibility in rat model. Biomaterials 1998; 19(10): 941-951.
5. Ibim SM, Uhrich KE, Attawia M., et al. Preliminary in vivo report on the osteocompatibility of poly(anhydride-co-imides) evaluated in a tibial model. Journal of Biomedical Materials Research 1998; 43(4): 374-379.
6. The Centers for Disease Control and Prevention web resources at www.cdc.gov7. The American Heart Association web resources at www.americanheart.org
May 24,2001
References
1. Hillebrands JL, van den Hurk BMH, Klatter F., et al. Recipient origin of neointimal vascular smooth muscle cells in cardiac allografts with transplant arteriosclerosis. The Journal of Heart and Lung Transplantation 2000; 19(12): 1183-1192.
2. Bard JBL, Connective Tissue Matrix, Pt. 2 DWL Hukins, Ed., CRC Press, Inc., Boca Raton, FL, pp. 11-43 (1990).
3. Lee EYH, Lee WH, Kaetzel CS, et al. Proceedings of the National Academy of Sciences USA, 82, 1419 (1985).
4. Hay ED, Cell Biology of Extracellular Matrix, Hay ED, Ed., 2d ed., Plenum Press, New York, pp. 419-462 (1991).
5. Deitzel JM, Kleinmeyer J, Harris D., Beck Tan NC. The effect of processing variables on the morphology of electrospun nanofibers and textiles. Polymer 2001; 42(1): 261-272.
6. Rootare HM, Prenzlow CF. Surface areas from mercury porosimeter measurements. Journal of Physical Chemistry 1967; 71(8): 2733-2736.
7. Langer R, Vacanti J. Tissue Engineering. Science 1993; 260: 920-926.8. Procedure number: National Inpatient Profile 1991 Data, Hospital Discharge Survey.