Gary L. Bowlin, Ph.D. Associate Professor Louis and Ruth Harris Exceptional Scholar Professorship

1. Development of an Electrospun, Acellular, Bioresorbable, Small Diameter Vascular Prosthetic Gary L. Bowlin, Ph.D. Associate Professor Louis and Ruth Harris Exceptional Scholar Professorship Department of Biomedical Engineering Virginia Commonwealth University Richmond, Virginia 23284 Co-Founding Inventor NanoMatrix, Inc. Co-Founding Inventor and Consultant TraumaCure, Inc. Bethesda, MD June 3, 2008

2. Vascular Background Reprinted from Principles of Anatomy and Physiology, GJ Tortora and NP Anagnostakos, 1993

3. Vascular Background 3-4 mm 3-4 mm diameter maximum Reprinted from Principles of Anatomy and Physiology, GJ Tortora and SR Grabowski, 1993

4. Purpose of Blood Vessels Arteries - 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.

5. Purpose of Blood Vessels Reprinted from Principles of Anatomy and Physiology, GJ Tortora and SR Grabowski, 1993

6. Anatomy ofBlood Vessels Reprinted from Principles of Anatomy and Physiology, GJ Tortora and SR Grabowski, 1993

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

8. Capillaries Down to 3 microns 8 microns Reprinted from Principles of Anatomy and Physiology, GJ Tortora and SR Grabowski, 1993

9. 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 cells in the tunica media ingest cholesterol. This results in the formation of an atherosclerotic plague that decreases the size of the artery lumen. Reprinted from Principles of Anatomy and Physiology, GJ Tortora and NP Anagnostakos, 1993

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

11. Symptoms/Problems with 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.

12. Possible Treatments Reprinted from Principles of Anatomy and Physiology, GJ Tortora and SR Grabowski, 1993

13. Vasculogenesis, Arteriogenesis and Angiogenesis • Soluble Factors • ECM • Biomechanics • All 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

14. Our “Maestro” 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? 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.

15. Synthetic Vascular Grafts Dacron (1957) Polyethylene terephthalate e-PTFE (1969) Polytetrafluoroethylene

16. Tissue Engineering Components • Scaffolding (a.k.a. extracellular matrix) • Gels • Fibrous Scaffolds • Porous Structures • 2. Cells • Autologous Cells • Allogenic Cells • Xenogenic Cells • Stem Cells • Universal Donor Cells • 3. Signaling Systems • Chemical (i.e. growth factors) • Mechanical (Bioreactors) • 4. Time/Physiologic Integration • 5. Ethical/Moral/Social Issues

17. Biodegradable Scaffold and Tissue Engineered Vessel 9 Months Static Culture Scaffold

18. 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.

19. Types of Cells in BodyBased on the Proliferative Potential • 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

20. Tissue Engineering Cell Source Allogenic Cells – Source same species. This type of cell source is or could be readily available through tissue/cell banks. Limitations: 1. Tissue donations 2. Proliferative capacity in vitro 3. 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 source 3. Cost effective

21. Tissue Engineering Cell Source Xenogenic Cells – source different species This type of cell source is or could be readily available through tissue/cell banks. Limitations: 1. Rejection 2. Cost – harvest and maintain 3. Proliferative capacity in vitro Genetic engineering could eliminate or reduce these limitations. Benefits: 1. Can grow and preserve large quantity of cells 2. Potentially a reproducible source Main concern with this source is the potential for animal virus transmission.

22. Tissue Engineering Cell Source Stem 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 cells Mesenchymal stem cells Neural stem cells Epithelial stem cells Brain stem cells Limitations: 1. In vitro culture capacity 2. Differentiation Big question then is there a single stem cell that exists in the bone marrow, blood stream, tissues, or organs that can be universally used.

23. Tissue Engineering Cell Source Universal 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 complement system-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.

24. 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 Issues Who determines testing protocols? Who determines what is enough testing? Who and What determines safety and efficacy?

25. 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.

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

27. 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.

28. 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.

29. Bioresorbable Vascular Prosthetic History (1980’s) 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.

30. Bioresorbable Vascular Prosthetic History (1980’s) 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.

31. Bioresorbable Vascular Prosthetic History (1980’s) 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.

32. Neo-intima Formation Schoen, F.J. Interventional and Surgical Cardiovascular Pathology:Clinical Correlations and Basic Principles, 1989.

33. 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 Bioresorbable Vascular Prosthetic History 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.

34. Biologically Important ‘Sizes’ RNA single strand structure: 1 nm DNA double strand helix: 2 nm Lipid bilayer: 4 - 5 nm Virus: 40 nm ECM fiber diameter 20-500 nm Axon 0.1 – 2.0 m Bacterium: 0.8 - 1.0 m Mammalian cell: 5 - 25 m Plant cell: 70 - 100 m Hair Shaft 80 - 100 m Native Tissues (Nano-composites): Natural nanofibers (ECM) Cells Ground Substance 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 Micrographs courtesy of Judy Williamson, VCU

35. Nanofiber Processing Techniques Adapted from: K. Jayaraman, et al., Journal of Nanoscience And Nanotechnology, 2004.

36. Electrospinning HVDC 2 x 3 Array With Needles High - Voltage DC Supply Loscertales, I.G. et al. 2002

37. Overall Research Aims 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.

38. Electrospinning Biopolymers Tissue Engineering Matrices The technology discussed herein is protected by pending United States, Foreign, and International patent applications.

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

40. Electrospun Polydioxanone (PDO) • A colorless, crystalline resorbable polymer in the polyester family • Violet Monofilament Suture • Elastic with Memory • Lack of Side Effects • Inflammation Solvent used - 1,1,1,3,3,3 hexafluoro-2-propanol Acta Biomaterialia, 1, 115-123, 2005.

41. Function of the Extracellular Matrix • Mechanical Support/Structure/Communication • Provides Cell Anchor Sites • Directing Cell Orientation • Control Cell Activity • Aiding in cell-cell and cell-ECM communications 6. Maintaining or Dictating Differentiation 7. Establish Microenvironments Sequester and Present Regulatory Molecules • Guiding Embryonic, Fetal and Somatic Tissue Development 9. Providing Provisional Wound Healing Substratum • Tissue Barrier Formation, Selectively Inhibiting or Promoting Cell Migration or Proliferation 11. Etc…. Lutolf and Hubbell, Nature Biotechnology, 2005

42. Importance of the Extracellular Matrix Healing/Regeneration Freeze Injuryvs.Burn Injury Residual Matrix Trauma Kills Cells No Contraction Minimal Scar Tissue Matrix Destroyed Trauma Kills Cells Wound Contraction Scar Tissue Ehrlich and Hembry, Am. J. Pathol., 1984.

43. 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 IV Human Placenta • Elastin • Fibrinogen Human and Bovine, Fraction 1 from Plasma • Hemoglobin • Myoglobin • Blends { Globular

44. Electrospun Collagen Type I 100  40 nm 110  40 nm 67 nm Banding 100 nm Biomacromolecules, 3(2): 232-38, 2002

45. Electrospun Biopolymer ScaffoldImmune Response • Dose-response • Time-course effects The technology discussed herein is protected by pending United States, Foreign, and International patent applications.

46. 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.

47. Monocyte & Macrophage Interactions Transforming Growth Factor β ELISA

48. Monocyte & Macrophage Interactions Transforming Growth Factor β ELISA TGF-beta (pg/ml)

49. Electrospun Biopolymer ScaffoldIn Vitro Immune Testing The technology discussed herein is protected by pending United States, Foreign, and International patent applications.

50. Mishell-Dutton AFC Response Antibody Forming Cell (AFC) Sheep RBC End Points Washed sRBC’s AFC/ Culture Material 48 well culture Culture 4 days Cells Agar sRBC Complement 3 Hour Incubation Magnified 37ºC on rocker 10% CO2, 7%O2, 6 psi sRBC around AFC are hemolyzed =PLAQUE Used with Permission; Kimber L. White, Ph.D.