Gary L. Bowlin, Ph.D. Associate Professor Louis and Ruth Harris Exceptional Scholar Professorship Department of Biomedical Engineering Virginia Commonwealth

Gary L. Bowlin, Ph.D.

Associate ProfessorLouis and Ruth Harris Exceptional Scholar

Professorship Department of Biomedical EngineeringVirginia Commonwealth University

Richmond, Virginia 23284

Co-Founding Inventor

NanoMatrix, Inc.

Co-Founding Inventor and Consultant

TraumaCure, Inc.

Bethesda, MD

June 3, 2008

Development of an Electrospun, Acellular, Bioresorbable, Small Diameter Vascular Prosthetic

Reprinted from Principles of Anatomy and Physiology, GJ Tortora and NP Anagnostakos, 1993

Vascular Background

Reprinted from Principles of Anatomy and Physiology, GJ Tortora and SR Grabowski, 1993

3-4 mm

3-4 mm diameter maximum

Vascular Background

Purpose of Blood VesselsArteries - are the vessels that carry oxygenated blood

from the heart to tissues. They are categorized as large, medium, and small.

Arterioles - Deliver blood from the small arteries to the capillaries.Regulate blood flow!

Capillaries - Branch into countless microscopic vessels in the tissue. Substance exchange between the tissue and blood stream occurs through the walls of the capillaries.

Venules - before the capillaries leave the tissue they regroup and dump in to the venules.

Veins - convey the blood back to the heart from the tissue. Run in parallel to the arteries.


Purpose of Blood Vessels


Anatomy of Blood Vessels

Background:Arterial Structure

• Individual components play specific roles:– SMCs: maintain vascular tone– Collagens: provide tensile strength and

prevent vessel rupture– Elastin: confer elasticity to the vessel

and provide the ability to recover from pulsatile deformations

• Dominates the low-strain mechanical response of the vessel to blood flow and prevents pulsatile energy from being dissipated as heat



8 microns

Down to 3 microns

Capillaries

Atherosclerosis

A process in which fatty substances (cholesterol and triglycerides)are deposited in the walls of arteries in response to certain stimuli (hypertension, CO2, dietary cholesterol). Following EC damage,monocytes stick to the tunica interna, develop into macrophages,and take up cholesterol and triglycerides). Smooth muscle cellsin the tunica media ingest cholesterol. This results in the formationof an atherosclerotic plague that decreases the size of the artery lumen.



Atherosclerosis

Ischemia - Reduction of blood flow.

Hypoxia - Ischemia leads to hypoxia, reduced oxygen supply. Weakens cells but does not kill them.

Claudication - The act of limping. Numbness of extremities.

Angina Pectoris - Pain in the chest associated with myocardial ischemia.

Myocardial Infarction - Serious myocardial ischemia. Means death of an area of tissue due to lack of blood supply.

Symptoms/Problems with Atherosclerosis


Possible Treatments

Vasculogenesis, Arteriogenesis and Angiogenesis

1. Soluble Factors

2. ECM

3. BiomechanicsAll have a role in cell migration, proliferation,

differentiation, organization, ECM production, and survival.

Figures from: Carmeliet, P. Nature Medicine, 2000.

Caplice, NM and B Doyle, Stem Cells, 2005

Our “Maestro”

Where inflammatory reactions to biomaterials are concerned, macrophages must be considered as multipotential regulators. Macrophages can play a complex role involving chemotactic factors, growth factors, cytokines, prostaglandins, coagulation, and complement factors. The complex interaction of these various bioactive macrophage products leads to wound healing and cellular ingrowth.

Angiogenesis Role of Activated Mono/Macro:

1. Secretion

Migration, Proliferation, Tube Formation

2. ECM Digestion

Pathways/tunnel for Migration

3. Transdifferentiation

Microvascular EC Phenotype

Myofibroblast

Smooth Muscle Cell?

Synthetic Vascular GraftsDacron (1957)

Polyethylene terephthalate

e-PTFE (1969)

Polytetrafluoroethylene

Tissue Engineering Components

1. Scaffolding (a.k.a. extracellular matrix)GelsFibrous ScaffoldsPorous Structures

2. CellsAutologous CellsAllogenic CellsXenogenic CellsStem CellsUniversal Donor Cells

3. Signaling SystemsChemical (i.e. growth factors)Mechanical (Bioreactors)

4. Time/Physiologic Integration

5. Ethical/Moral/Social Issues

Biodegradable Scaffold and Tissue Engineered Vessel

Scaffold9 MonthsStatic Culture

Tissue Engineering Cell Source

Autologous Cells

Autologous – one’s own.

If one was to use autologous cells for the production of a tissue engineered product,the process would require starting with a biopsy of the tissue/organ which contains the cells required to replicate the desired product.

Advantage: Immune acceptable

Downsides: Limited availability not “off-the shelf” Variability between individuals Expansion in many cases limited or not possible.

Alternative Method: Implantation of an appropriate acellular matrix that has the capacity to recruit and develop the proper cell lines.

Types of Cells in BodyBased on the Proliferative Potential

1. Renewing or Liable (e.g. skin)

Continuous turnover, balances losses

2. Expanding or Stable (e.g. liver)

Capable of proliferation if needed

3. Permanent or Static (e.g. heart)

No proliferative capacity

Tissue Engineering Cell SourceAllogenic Cells – Source same species.

This type of cell source is or could be readily available through tissue/cell banks.

Limitations:1. Tissue donations2. Proliferative capacity in vitro3. Potential for genetic engineering to be immune acceptable

Benefits:1. Can grow and preserve large quantity of cells, thus “off-the-shelf”2. Potentially a reproducible source3. Cost effective

Xenogenic Cells – source different species

Tissue Engineering Cell Source

This type of cell source is or could be readily available through tissue/cell banks.

Limitations:1. Rejection2. Cost – harvest and maintain3. Proliferative capacity in vitro

Genetic engineering could eliminate or reduce these limitations.

Benefits:1. Can grow and preserve large quantity of cells2. Potentially a reproducible source

Main concern with this source is the potential for animal virus transmission.

Tissue Engineering Cell SourceStem Cells – Pluripotent cells

Types of Stem Cells:1. Embryonic stem cells

derived from embryos 2. Adult stem cells

undifferentiated cells in tissues and organs Examples of adult stem cells:

Hematopoietic stem cellsMesenchymal stem cellsNeural stem cellsEpithelial stem cellsBrain stem cells

Limitations:1. In vitro culture capacity2. Differentiation

Big question then is there a single stem cell that exists in the bonemarrow, blood stream, tissues, or organs that can be universally used.

Tissue Engineering Cell SourceUniversal Donor Cells – U.S. Patent 5,705,732

The concept here is to genetically engineer a cell to provide it with protection form hyperacute rejection brought about by complementsystem-based lysis.

At the same time, the genetic engineering causes the cells not to present proteins produced by the class I and II major histocompatibility complex genes on the cell surface. This eliminates attack from the T-cells.

What is created are immunologically neutral cells.

Finally, the cells can be genetically altered with a self-destruction mechanism so that they can be removed from the host when and if desired.

Ethical/Moral/Social Issues

Should we be altering, re-engineering Mother Nature?Who defines “Quality of Life”? Research (academia) vs. Industry – Funding of research/development and control

of Intellectual Property.Does academia need to further develop the field?Will Industry/Venture Capitalists Invest?Minimizing conflicts of interest.Should life saving technology be patented and controlled exclusively?

Are animal models justified?How should human research be performed? Informed consent?

Regulatory IssuesWho determines testing protocols?Who determines what is enough testing?Who and What determines safety and efficacy?

Success with Large Diameter Synthetic Vascular Prosthetics

• e-PTFE (expanded Polytetrafluoroethylene) and Dacron have been successful in replacing Large & Medium Diameter (> 6 mm I.D.) arteries, e.g. the thoracic and abdominal aortas, iliac artery, and common femoral arteries.

• Small Diameter (< 6 mm I.D.) replacements using these materials have failed due to Acute Thrombotic Occlusion and Chronic Anastomotic Hyperplasia.

“Ideal” Vascular Prosthetic “Search for the Holy Grail” – M.S. Conte 1998

Ease of Handling Durable

Suture Retention Packaging and Handling

Ease of Suture Placement After Tissue Ingrowth

Flexible with Kink Resistance Appropriate Remodeling Response

Biocompatible Porous

Non-toxic Leak Resistant but Promote Healing/Regeneration

Non-thrombogenic Compliance Matching Native Artery

Resist Aneurysm Formation

Infection Resistant

Characteristics:

Other Concerns:Easily Manufactured Available in a Variety of Sizes

Easily Stored Lengths, Diameters, and Tapers

Economical

Basically, clinicians are demanding off-the-shelf availability (especially for emergency cases) as provided by current polymeric vascular prosthetics without the short and long-term complications. All these requirements create a daunting challenge.

Bioresorbable Vascular Prosthetic History

The origin of the concept for a bioresorbable graft is credited to Claude Guthrie who in 1919 wrote “To restore and maintain mechanical function an implanted segment only temporarily restores mechanical continuity and serves as a scaffolding or bridge for the laying down of an ingrowth of tissue derived from the host.”

The actual application of a bioresorbable material for use as a vascular prosthetic was first reported by Wesolowski et al. in the early 1960’s utilizing fabrics composed of a variety of Dacron yarns, collagen coatings, and collagen fibers.

Bioresorbable Vascular Prosthetic History (1970’s)

Dr. Ingvar Eriksson and associates in the late 1970’s pioneered the use of PLGA (Vicryl (polyglactin 910)) as a suture mesh graft where the mesh had a fiber diameter of 140 microns with a pore size of 400x400 microns. This large pore size required pre-clotting, but after pre-clotting hemostasis was achieved in minutes after implantation.

The PLGA mesh structure upon implantation in a pig aorta model (4 cm diameter graft) allowed early in-growth of smooth muscle cells (SMCs) from the native aorta to form a neo-media which was completely endothelialized within 20 days. The PLGA mesh almost completely degraded within 40 days, however the neo-artery wall retained sufficient strength through the observed time with very little dispersed elastin deposition.


In the early 1980’s, Dr. Howard Greisler and associates utilized a similar PGA mesh tube (250 micron fiber diameter and 400 micron pore size; length = 2.5 cm) in a rabbit aortic model of regeneration. The results after 7.5 months were that a significant number of animals exhibited mild graft/aorta dilation and hyperplasia with the regenerated graft wall composed of myofibroblasts and dense collagen matrix.

Overall, they concluded that the PGA bioresorbable structure permitted some degree of arterial regeneration. However, at 6 months the histological evaluation revealed lipid laden macrophages and histiocytes, suggesting the early development of arteriosclerosis.


In 1987, Howard Greisler and associates published results utilizing PDS absorbable vascular prosthetics made by the same technique utilized for the PGA scaffolds just discussed (Rabbit model).

Implanted grafts (length = 2.5 cm) were evaluated at 2 weeks up to 12 months with one out of all the 28 evaluated having a small aneurysm and no perigraft hematomas. The myofibroblast migration paralleled the macrophage-mediated degradation of the PDS structure, which was delayed relative to the PGA structures.

A confluent endothelial cell (EC) lining was present within two weeks with the mechanical properties of the explants at 1 year resembling artery elasticity (compliance). Finally, the regenerated aorta segments at 1 year withstood 1200 mm Hg of systolic pressure.

All these implant studies elicit more tissue in-growth of smooth muscle cell- like myofibroblasts and more rapid EC lining development when compared to Dacron or e-PTFE in the animal models.

Speculation by the authors is that the enhanced EC lining is due to transinterstitial migration of capillaries and the overall healing is initiated largely by the macrophages being activated by the polymer interaction.

A 1988 study by this group, verified transinterstitial migration of capillaries to form the neo-intima. Additionally, the PDS based grafts (4 mm diameter) had patency rates significantly higher than Dacron and e-PTFE controls.


Schoen, F.J. Interventional and Surgical Cardiovascular Pathology:Clinical Correlations and Basic Principles , 1989.

Neo-intima Formation

One critical aspect of the historical PLGA, PGA, and PDS bioresorbable prosthetics that seems to have been ignored and not reported in any of the studies is the initial mechanical properties of the prosthetic structure and its comparison to native tissue.

Preliminary evaluation of PDS II (Ethicon, Inc.) monofilament suture with diameters of 100 and 530 microns was performed by uniaxial mechanical testing to obtain the basic material properties of the individual fibers.

Bioresorbable Vascular Prosthetic History

Suture Material Elastic Modulus (MPa) Ultimate Stress (MPa) Strain at Failure (%)

PDS II (530 microns) Dry 989 418 65

PDS II (530 microns) Wetted 827 417 64

PDS II (100 microns) Dry 1125 501 84

PDS II (100 microns) Wetted 1106 489 69

RNA single strand structure: 1 nm DNA double strand helix: 2 nmLipid bilayer: 4 - 5 nm

Virus: 40 nmECM fiber diameter 20-500 nm

Axon 0.1 – 2.0 mBacterium: 0.8 - 1.0 mMammalian cell: 5 - 25 mPlant cell: 70 - 100 mHair Shaft 80 - 100 m

Biologically Important ‘Sizes’

Micrographs courtesy of Judy Williamson, VCU

Platelet: 2,000 – 4,000 nm

RBC: 7,000 nm

Fibrin Mesh: > 80 nm

Hair: 80,000 – 100,000 nm Fly Eye: 300,000 nm Flea: 1,000,000 nm

Native Tissues (Nano-composites):Natural nanofibers (ECM)CellsGround Substance

Process Lab/Industrial Application

Ease of Processing

Advantages Limitations

Self-assembly Lab Difficult Achieves fiber diameters on lowest ECM scale (5-8 nm)

Only short fibers can be created (< 1 micron)

Low yield

Limited to a few polymers

Phase Separation Lab Easy Tailorable mechanical properties, pore size, and interconnectivity

Batch-to-batch consistency

Low yield

Limited to a few polymers

Electrospinning Lab/Industrial Easy Cost effective

Continuous nanofibers

Production of aligned nanofibers

Tailorable mechanical properties, sizes, and shapes

Plethora of polymers may be used

Large nanometer to micron diameter fibers

Use of organic solvents

No control over 3-D pore structure

Nanofiber Processing Techniques

Adapted from: K. Jayaraman, et al., Journal of Nanoscience And Nanotechnology, 2004.

Electrospinning

HVDC

High-VoltageDC Supply

Loscertales, I.G. et al. 2002

2 x 3 Array With Needles

Specific Aim 1: To electrospin bioresorbable polymers to form nano- to micro-fibrous, seamless vascular prosthetic constructs for potential use as an acellular vascular prosthetic.

Specific Aim 2: To perform mechanical characterization of the electrospun nano- to micro-fiber structures. The mechanical evaluations will include stress-strain, burst strength, permeability, and suture retention.

Specific Aim 3: To perform in vivo evaluation of the vascular constructs using a rat subcutaneous model to evaluate biocompatibility and regeneration potential.

Specific Aim 4: To perform in situ evaluation of the vascular constructs using a large animal femoral artery model to evaluate long-term in vivo mechanical performance as well as the thromboresistance and regeneration capacities of the prosthetic.

The hypothesis of this study is that an electrospun composite composed of natural and synthetic polymers will be comparable mechanically (e.g., burst strength and suture retention) to current clinically used prosthetics and/or a native small caliber artery. The corollary is that the in vivo performance of the electrospun composites is capable of promoting the full regeneration of a functional arterial segment. The hypothesis and corollary will be tested using in vitro and in vivo experimental validation.

Overall Research Aims

Electrospinning Biopolymers

Tissue Engineering Matrices

The technology discussed herein is protected by pending United States, Foreign, and International patent applications.

Biodegradable Polymers Spun• D,L-PLA • L-PLA • 50:50 PGA/PLA • PGA• Polycaprolactone (PCL)• Polydioxanone (PDO)• Co-polymers and Blends

y = 0.0101x - 0.2875

R2 = 0.969

0

0.5

1

1.5

2

2.5

25 75 125 175

Concentration (mg/ml)

• A colorless, crystalline resorbable polymer in the polyester family

• Violet Monofilament Suture• Elastic with Memory• Lack of Side Effects

Inflammation

Electrospun Polydioxanone (PDO)

Solvent used - 1,1,1,3,3,3 hexafluoro-2-propanol

Acta Biomaterialia, 1, 115-123, 2005.

Function of the Extracellular Matrix

1. Mechanical Support/Structure/Communication

2. Provides Cell Anchor Sites

3. Directing Cell Orientation

4. Control Cell Activity

5. Aiding in cell-cell and cell-ECM communications

6. Maintaining or Dictating Differentiation

7. Establish Microenvironments

Sequester and Present Regulatory Molecules

8. Guiding Embryonic, Fetal and Somatic Tissue

Development

9. Providing Provisional Wound Healing Substratum

10. Tissue Barrier Formation, Selectively Inhibiting

or Promoting Cell Migration or Proliferation

11. Etc….

Lutolf and Hubbell, Nature Biotechnology, 2005

Importance of the Extracellular Matrix Healing/Regeneration

Freeze Injury vs. Burn Injury

Ehrlich and Hembry, Am. J. Pathol., 1984.

Residual Matrix

Trauma Kills Cells

No Contraction

Minimal Scar Tissue

Matrix Destroyed

Trauma Kills Cells

Wound Contraction

Scar Tissue

Natural Polymers (Proteins) Electrospun• Collagen Type I

Calf Skin, Human Placenta, Rat Tail

• Collagen Type II

Chicken Sternal Cartilage

• Collagen Type III

Human Placenta

• Collagen Type IVHuman Placenta

• Elastin

• FibrinogenHuman and Bovine, Fraction 1 from Plasma

• Hemoglobin

• Myoglobin

• Blends {Globular

Electrospun Collagen Type I

110 40 nm

100 40 nm

Biomacromolecules, 3(2): 232-38, 2002

67 nm Banding

100 nm

0

1

2

3

4

5

6

7

8

30 40 50 60 70 80 90 100

Electrospinning Concentration (mg/ml)

Fib

er

Dia

met

er

(um

)

Electrospun Biopolymer Scaffold

Immune Response


• Dose-response

• Time-course effects

Electrospun Biopolymer ScaffoldMonocyte & Macrophage Interactions

Transforming Growth Factor β ELISA

• Macrophages were seeded on 10 mm circular discs of electrospun Polydioxanone, collagen and blends (50:50, 70:30, 90:10) at a density of 400,000 cells/well in a 48 well plate.

• Cell culture Supernatants were tested for the presence of TGF-β on day 7, 14, 21, 28.

Macrophages on Polydioxanone

0100200300400500600700800900

DAY 7 DAY14 DAY 21 DAY 28

TG

F b

eta

(pg

/ml)

Macrophages on collagen

0

500

1000

1500

2000


TG

F b

eta

(pg

/ml)

PDO COL 50:50 BLEND

0200

400600800

1000

12001400


TG

F b

eta

(pg

/ml)

PDO COL 70:30 BLEND

0

200

400

600

800

1000

1200

1400


TG

F b

eta

(pg

/ml)

PDO COL 90:10 BLEND

0

500

1000

1500

2000


TG

F b

eta

(p

g/m

l)

Monocyte & Macrophage InteractionsTransforming Growth Factor β ELISA

Monocyte & Macrophage InteractionsTransforming Growth Factor β ELISA

0

200

400

600

800

1000

1200

1400

1600

1800

2000


PDO

COL

50-50

70-30

90-10TG

F-b

eta

(pg/

ml)

Electrospun Biopolymer ScaffoldIn Vitro Immune Testing


Material

3 Hour Incubation

WashedsRBC’s

48 well culture

End Points

AFC/ Culture

CellsAgarsRBC

Complement

Culture 4 days

37ºC on rocker 10% CO2, 7%O2, 6 psiMagnified

sRBC around AFC are hemolyzed =PLAQUE

Sheep RBC(AFC)Antibody Forming Cell

Used with Permission; Kimber L. White, Ph.D.

Mishell-Dutton AFC Response


0

1000

2000

3000

4000A

FC

/ c

ultu

re

Media ePTFE Nylon PLA PDO PDO-PCL PGA MTX

****

****

**

0

25

50

75

100

% V

iabi

lity

Media ePTFE Nylon PLA PDO PDO-PCL PGA MTX

** ***

0

250

500

750

1000

1250

AF

C /

cul

ture

Media 100:0 80:20 60:40 40:60 20:80 0:100 MTX

PDO:elastin blend ratio

0255075

100

% V

iabi

lity

*

** ****

**

**


*

CP

M /

2 x

10

5 S

ple

no

cyte

s

0

20000

40000

60000

80000

No Material 100:0 80:20 60:40 40:60 20:80 0:100

PDO:Elastin Blend Ratio

**

**

** ** **

**

**

**

** F(ab')2 (5 g/ml) + IL-4 (10 ng/ml)

F(ab')2 (5 g/ml) + IL-4 (100 ng/ml)

F(ab')2 (5 g/ml) only

Unstimulated

B-Cell Proliferation Assays


Electrospun Collagen/Elastin and Polydioxanone:

Vascular Tissue Engineering

Collagen/Elastin for “bioactivity”

PDO for “strength” and macrophage activation

100% Collagen 75% Collagen – 25% PDO

50% Collagen – 50% PDO

25% Collagen – 75% PDO

100% PDO

PDO – Collagen Blends

Electrospun Small Diameter Vascular Prosthetics

0

20

40

60

80

100

120

140

100 PDS 75 PDS-25 Col 50 PDS-50 Col 25 PDS-75 Col 100 Col

Mod

ulus

(MPa

)

0

2

4

6

8

10

12

14

16

18


Stre

ss (M

Pa)

0%

20%

40%

60%

80%

100%

120%

140%

160%

180%


Stra

in (%

)

Graft Material Elastic Modulus (MPa)

Ultimate Stress (MPa)

Strain at Failure (%)

e-PTFE(30 micron internodal distance)

42 - 60 6 - 15 20 - 30

Saphenous Vein 43 3 11

Femoral Artery 9 - 12 1 – 2 63 - 76

PDO – Collagen BlendsDry

Hydrated

Dry

Hydrated

Dry

Hydrated

Results:PDO:Collagen Mechanical Properties

Results: Uniaxial Tensile Testing

Graft MaterialElastic Modulus

(MPa)Ultimate Stress (MPa) Strain at Failure (%)

Femoral Artery* 9 – 12 1 - 2 63 – 76

e-PTFE (30 micron internodal

distance)**42 – 60 6 - 15 20 - 30

PDO (100:0) 20 ± 0.7 6 ± 0.7 206 ± 39

90:10 5 ± 0.4 5 ± 0.3 194 ± 12

80:20 6 ± 0.8 5 ± 0.8 127 ± 15

70:30 5 ± 0.6 4 ± 0.5 114 ± 4

60:40 6 ± 0.9 3 ± 0.4 78 ± 7

50:50 10 ± 0.7 3 ± 0.2 65 ± 4

* Fung Y C 1984 Blood Flow in Arteries Biodynamics: Circulation (New York: Springer-Verlag)* Hiroshi Y 1970 Strength of Biological Materials (New York: Robert E. Krieger Publishing Company)** Catanese III, J., et al. 1999 Mechanical properties of medical grade expanded polytetrafluoroethylene: the effects of internodal distance, density, and

displacement rate Journal of Biomedical Materials Research 48 187-192

PDO – Elastin Blends

Suture Retention

Utilized 6-0 PDS II Suture

0

2

4

6

8

10

12

14

16

18

20

e-PTFE 42 mg/ml PDS 100 mg/ml PDS 167 mg/ml PDS

Graft Material

Su

ture

Ren

ten

tio

n F

orc

e (N

)

e-PTFE: 800 g-f

100 mg/ml PDO: 880 g-f

Saphenous Vein: 380 g-f (5-0 suture)

Surgeon’s Comments: Suturability Excellent

Wall Thickness: ~ 500 microns

0100200300400500600700800900

1000

PDO:Elastin Ratio

Su

ture

Ret

enti

on

Str

eng

th (

g-f

)

Dry

Hydrated

*Donovan, D.L., et al. 1990 Material and structural characterization of human saphenous vein Journal of Vascular Surgery 12 531-537**Weinberg S L, Cipolletti G B and Turner R J 1982 Human Umbilical Vein Grafts: Physical Evaluation Criteria Biological and Synthetic Vascular Prostheses ed

J C Stanley (New York: Grune and Stratton)

Suture Retention

12

3

42

100

167

0

200

400

600

800

1000

1200

1400

1600

Bursting Pressure (mmHg)

Inner Diameter of Vessels (1=3 mm, 2=6 mm, 3=9 mm)

Concentration (mg/mL)

Preliminary Burst Strength (Hydrated PDO)Wall Thickness: ~ 500 microns

Compliance Measurement

Humphrey J D 2002 Cardiovascular Solid Mechanics: Cells, Tissues, and Organs (New York: Springer) *Tai N R, Salacinski H J, Edwards A, Hamilton G and Seifalian A M 2000 Compliance properties of conduits used in vascular reconstruction British Journal of Surgery 87 1516-1524

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

100:0 90:10 80:20 70:30 60:40 50:50 Artery Vein Dacron ePTFE

Vascular Graft Composition

Co

mp

lian

ce (%

/ 100 m

mH

g)

* ***

)(3

1DSD PPPMAP At MAP = 93.33 mmHg (120/80 mmHg)

Compliance Measurement

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

100:0 90:10 80:20 70:30 60:40 50:50

PDO:Elastin Ratio

Co

mp

lian

ce

(%

/ 10

0 m

mH

g)

90/50

120/80

150/110

4

12

112 10/)(

%

PP

RRRCompliance PPP

ANSI 2000 Cardiovascular implants – Vascular graft prostheses in ANSI/AAMI VP20:1994, Association for the Advancement of Medical Instrumentation

R is the internal radius, P1 is the lower internal pressure, and P2 is the higher internal pressure

Electrospun Vascular Prosthetic Reinforcement

J. Biomaterials Applications, 16: 22-33, 2001.

Fiber Winding System

Fiber Reinforcement

Fiber ReinforcementPDO:Elastin

Results:PDO:Elastin Stability

0

20

40

60

80

100

120

50:50 60:40 70:30 80:20 90:10 100:0

PDO:Elastin

Wei

gh

t R

emai

nin

g (

%)

0 min

15 min

1 hr

1 day

R2 = 0.94

R2 = 0.98

R2 = 0.61

R2 = 0.72

0

20

40

60

80

100

120

0 500 1000 1500 2000

Time (min)W

eig

ht

Per

cen

t

50:50

60:40

70:30

80:20

Results:PDO:Elastin Cross-linking and Stability

0

10

20

30

40

50

60

166.5 mM EDC 30mM Genipin

Cross-linking Reagent

% X

-lin

ked

0

20

40

60

80

100

120

Initial Weight 50-50 EDC 50-50 NO EDC

% W

eig

ht

Rem

ain

ing

Graft Material Peak Stress (MPa) Modulus (MPa)Strain at Break

(mm/mm)

Femoral Artery 1.5 +/- 0.5 11 +/- 1.5 0.7 +/- 0.1

Pig Artery 3.4 +/- 1.6 2.7 +/- 1.3 3.0 +/- 0.8

ePTFE 5.8 +/- 0.2 9.5 +/- 0.6 10 +/- 0.6

50:50 2.3 +/- 0.5 6.5 +/- 0.9 0.9 +/- 0.1

50:50 EDC 1.4 +/- 0.2 5.3 +/- 0.6 0.7 +/- 0.1

50:50 Gen 0.9 +/- 0.1 3.1 +/- 0.3 0.8 +/- 0.1

70:30 3.5 +/- 0.4 4.1 +/- 0.7 1.2 +/- 0.1

70:30 EDC 2.4 +/- 0.2 6.3 +/- 0.6 1.1 +/- 0.2

70:30 Gen 3.8 +/- 0.5 6.3 +/- 0.5 1.0 +/- 0.1

PDO 5.6 +/- 0.7 30 +/- 3.8 0.6 +/- 0.1

0

1

2

3

4

5

6

7

8

50:50 EDC 50:50Genipin


Pig Artery

PDO:Elastin

Tan

gen

tial

Mo

du

lus

(MP

a)

0

1

2

3

4

5

6



Pig Artery

PDO:Elastin

Pea

k S

tres

s (M

Pa)

Results:PDO:Elastin Mechanical Properties

Results:PDO:Elastin Hysteresis Calculation

0

100

200

300

400

500

600

700

800

900



100:0 e-PTFE Pig FemoralArtery

En

erg

y L

os

s (

J/m

^3

)

8th Cycle

200th Cycle

400th Cycle

• Pig femoral artery had smallest amount of energy loss

• 50:50 scaffolds had significantly less hyestersis than e-PTFE grafts

• 100:0 PDO scaffold experienced a significant preconditioning period while elastin containing scaffolds behaved similar to pig femoral artery – very little preconditioning time

•Demonstrates that electrospun elastin is playing role in low strain response of scaffold

Preliminary Cell Culture Electrospun PDO:Collagen

9:1 PDO:CIII 8:2 PDO:CIII 7:3 PDO:CIII100:0 PDO

100:0 PDO 9:1 PDO:CIII 8:2 PDO:CIII 7:3 PDO:CIII

Day

7D

ay 1

Preliminary Cell CulturePDO:Elastin

SEM’s = Day 1Histology = Day 7 (20x)

Prostheses Evaluations

• Biomechanical (SEM, TS, surgical handling)

• Cell culture reaction• Thrombogenicity (AV

shunt model)• Body reaction and

degradation (subcutaneous)

• Vascular replacement

Early Surface Thrombogenicity

Homogenicity of Surface & Cell Deposition

3 and 6 weeks

Subcutaneous Biocompatibility

100% PDO

PDS – 3 Weeks Rat Subcutaneous Implantation

PDS + collagen

50:50 PDS/Collagen

3 Weeks Rat Subcutaneous Implantation

PDO/PLA graft

Quality and patency control after surgery with transit time

flowmeter

1 mm

ePTFE graft

1.5 mm diameter grafts were interposed in the infrarenal abdominal aorta under general anaesthesia. The anastomoses were performed with 10/0 interrupted nylon sutures with an operative microscope. Patency assessment was performed intra-operatively with transit time flow measurement in all animals.

Rat Abdominal Aorta Prosthetic Grafting

Rat 24 3 weeks afteroperation

Rat 24 3 weeks afteroperation

At conclusion of the study, digital subtraction angiography was performed and grafts were harvested for morphologic as well as scanning electron microscopic examination.

Angiographic Patency Evaluation

PDO Graft Interposition(After 3 Weeks Implantation)

ePTFE greft

H&E x25

In Situ EvaluationsElectrospun, PDO/PLA Blended Graft

500 micron wall thickness (1.5 mm I.D.)

3 weeks, rat abdominal aortic replacements

CD31 staining x 200

Non-homogeneous Degradation

In Situ EvaluationsElectrospun, PDO/PLA Blended Graft

500 micron wall thickness (1.5 mm I.D.)

12 weeks, rat abdominal aortic replacements

PDO Grafts Wrapped with Non-Degradable Support (Nylon) (3 Weeks Implantation)

CD31 staining x 200

PDO-PCL (12 weeks)

Angiographic AneurysmsPatency 100%

Prostheses 3 weeks 6 Weeks 12 Weeks

e-PTFE(Control)

0/3 0/3 0/3

PDO 3/3 ---- ----

PDO-PLA 1/3 1/3 1/3

PDO-PCL 0/3 0/3 0/3

SEM After 3 Weeks (PDO)

Neoendothelial Coverage

PDO-PLAPDO-PLA

ePTFEePTFE

Results After 12 Weeks Implantation

Prostheses Aneurysm Occurrence

Endothelialisation(%)

Intimal Hyperplasia

(µm2/µm)

ePTFE(Control) 0/3 5320 169

PDO-PLA 1/3 974* 8638

PDO-PCL 0/3 991* 9123

(*p<0.05 vs. ePTFE; Mann Whitney U test)

Histology (3 weeks): PDO-PCL + SUTURE

PCL After 12 Weeks Implantation

Native Aorta

Lumen

Lumen

Lumen

Extracellular Matrix Remodelling

Time/Physiologic IntegrationOnce we have created tissue engineered products, time and physiologic integration will be the components that will have the attention of the field.

Will immature cells, stem cells, mature and differentiate to maintain tissue?

How will the cells implanted react over time to the new environment?

Will they maintain function indefinitely?

If used in infants or children, will they grow along with the host?

Will structural integrity be maintained or developed?

Is the scaffold degradation rate appropriate?

AcknowledgementsCollaborators:Marcus E. Carr, M.D./Ph.D.David G. Simpson, Ph.D.Gary E. Wnek, Ph.D. (Case Western)Mike McManus, M.D.Beat Walpoth, M.D. (Geneva)Kevin Ward, M.D.Steve Montante, M.DDavid Brand, Ph.D.Matthew Beckman, Ph.D.Thomas Hass, Ph.D.Hu Yang, Ph.D.

Graduate Students:Jamil Matthews, M.D. Eugene D. Boland, Ph.D.

Catherine P. Barnes, M.S. Kristin J. Pawlowski, Ph.D.Scott Sell, M.S. Danielle C. Knapp, M.S.Joel D. Stitzel, Ph.D. Matthew J. Smith, M.S.Charles D. Anderson, M.S. Michael McClure, B.S.

Allison Faucette, M.S. Jared Nimtz, B.S.

Branch Coleman, M.S. Lisa I. Ramdhanie, M.S.

Undergraduate Students:Tara George, B.S. Michelle Park, B.S.

John Layman, B.S. Katherine Neser, B.S.

LaVone Smith, B.S. Jon-Erik Houser, B.S

Colleen McLoughlin, B.S. Josh Grant, B.S.

Lindsay Denault

Funding:The Whitaker FoundationU.S. Department of Defense/ArmyNASA –LangleyAlkermes, Inc.NIH R01 EB003087-01 (DGS)NIH R21 EB003407-01-A1 (GLB)American Heart AssociationNanoMatrix, Inc.Ethicon, Inc.Jeffress Memorial TrustNational Science Foundation

High School Students:Annie Wysock Lindsay Denault

Dan Newton Jennifer Mejia

Teresa Tang

Residents:Michael McManus, M.D. Tommy Miller, M.D.

Paul Espy, M.D. Charles Fields, M.D.

List of Pertinent PublicationsWalpoth, B.H. and G.L. Bowlin. “The Daunting Quest for a Small Diameter Vascular Graft.” Expert Review of Medical Devices, 2

(6), 647-51, 2005.

Sell, S.A. and G.L. Bowlin. “The Potential to Create Small Diameter Bioresorbable Vascular Grafts Through Electrospinning.” Journal of Materials Chemistry, In Press.

Smith, M.J.; McClure, M.J.; Sell, S.A.; Barnes, C.P.; Walpoth, B.H.; Simpson, D.G. and G.L. Bowlin. “A Novel Suture-Reinforced Electrospun Polydioxanone-Elastin Small-Diameter Tube for Use in Vascular Tissue Engineering.” Acta Biomaterialia, In Press.

Smith, M.J.; Smith D.C.; White Jr., K.L. and G.L. Bowlin. “Immune Response Testing of Electrospun Polymers: An Important Consideration in the Evaluation of Biomaterials.” J. Engineered Fabrics and Fibers (Invited Editorial), 2(2), 41-47, 2007.

Barnes, C.P.; Pemble, C.W.; Brand, D.D.; Simpson, D.G. and G.L. Bowlin. “Cross-linking Electrospun Type II Collagen Tissue Engineering Scaffolds with Carbodiimide in Ethanol.” Tissue Engineering, 13(7), 1593-1605, 2007.

Barnes, C.P.; Sell, S.A.; Knapp, D.C.; Walpoth, B.H.; Brand, D.D. and G.L. Bowlin. “Preliminary Investigation of Electrospun Collagen and Polydioxanone for Vascular Tissue Engineering Applications.” International Journal of Electrospun Nanofibers and Applications, 1(1), 73-87, 2007.

Sell, S.A.; McClure, M.J.; Barnes, C.P.; Knapp, D.C.; Simpson, D.G.; Walpoth, B.H. and G.L. Bowlin. “Electrospun Polydioxanone – Elastin Blends: Potential for Bioresorbable Vascular Grafts.” Biomedical Materials, 1, 72-80, 2006.

Boland, E.D., Coleman, B.D.; Barnes, C.P.; Simpson, D.G., Wnek, G.E. and G.L. Bowlin. "Electrospinning Polydioxanone for Biomedical Applications.” Acta Biomaterialia, 1 (1), 115-123, 2005.

Boland, E.D., Matthews, J.A.; Pawlowski, K.J., Simpson, D.G., Wnek, G.E. and G.L. Bowlin. "Electrospinning Collagens and Elastin for Vascular Tissue Engineering.” Frontiers in Biosciences, 9, 1422-1432, May 1, 2004.

Matthews, J.A.; Simpson, D.G.; Wnek, G.E.; and G.L. Bowlin. "Electrospinning of Collagen Nanofibers." Biomacromolecules, 3 (2): 232-238, 2002.

Documents

Gary L. Bowlin, Ph.D. Associate Professor Louis and Ruth Harris Exceptional Scholar Professorship Department of Biomedical Engineering Virginia Commonwealth