Cardiovascular disease is the major cause of morbidity and mortality in the USA. Most cardiovascular disease is attributable to the effects of atherosclerosis, with myocardial infarction and stroke due to atherosclerotic plaque rupture, thrombosis and/or embolism. Notably, the distribution of atherosclerotic lesions in the blood vessels is not uniform. Lesions tend to form at sites of disturbed flow (e.g., bends, branches and bifurcations). At these sites, there is an early disturbance of normal endothelial functions, which represents the earliest pathological process in the development of atherosclerotic arterial disease.
The endothelium is a delicate monolayer of cells lining blood vessels. A healthy endothelium controls vessel diameter by producing vasodilator substances. Some of these substances, such as nitric oxide and prostacyclin, also inhibit the adhesion of platelets and leukocytes. These and other paracrine substances released by the endothelium prevent vascular thrombosis and inflammation. By contrast, at sites of disturbed flow, the endothelium produces fewer homeostatic factors, and instead elaborates adhesion molecules and chemokines that promote the interaction of circulating blood elements with the vessel wall.
The morphology of endothelial cells (ECs) is a well-known indicator of EC phenotype. Elongated ECs with cytoskeletal elements aligned in the direction of blood flow correspond to a healthy, atheroresistant phenotype. This endothelial morphology is typically observed in straight segments of the arterial tree, where atherosclerotic lesions are less likely to develop. By contrast, ECs with cobblestone morphology and randomly oriented EC cytoskeletons are typically found at sites of disturbed flow, and are atherosusceptible. After vascular injury or disease, EC migration is important in the angiogenesis process to form neovessels in the surrounding tissue. EC migration involves protrusion of filopodia and lamellipodia at the leading edge, forward movement of the cell body and release of the lagging edge of the cell. Therefore, the ability to control EC morphology and motility, with the aim to influence EC biology, might be highly beneficial in the prevention or treatment of vascular disease. Surfaces of patterned topography, with features in the micrometer or nanometer-scale range, have been widely used to investigate the behavior of cells. Nanopatterning, in the form of islands, lanes or grooves, has successfully demonstrated the ability to influence both the morphology and migration of ECs. See Anderson D, Hinds M., Endothelial Cell Micropatterning: Methods, Effects, and Applications, Ann. Biomed. Eng. 39, 2329-2345 (2011); Lauffenburger D A, Horwitz A F., Cell Migration: A Physically Integrated Molecular Process. Cell 84, 359-369 (1996); Li S, Bhatia S, Hu Y L et al., Effects of Morphological Patterning on Endothelial Cell Migration, Biorheology 38, 101-108 (2001); Liliensiek S J, Wood J A, Young J, Auerbach R, Nealey P F, Murphy C J, Modulation of Human Vascular Endothelial Cell Behaviors by Nanotopographic Cues, Biomaterials 31(20), 5418-5426 (2010); Junkin M, Wong P K, Probing Cell Migration in Confined Environments by Plasma Lithography, Biomaterials 32(7), 1848-1855 (2011); Uttayarat P, Chen M, Li M, Allen F D, Composto R J, Lelkes P I, Microtopography and Flow Modulate the Direction of Endothelial Cell Migration, Am. J. Physiol. Heart Circ. Physiol., 294(2), H1027-H1035 (2008); Zorlutuna P, Rong Z, Vadgama P, Hasirci V. Influence of Nanopatterns on Endothelial Cell Adhesion: Enhanced Cell Retention Under Shear Stress, Acta Biomater., 5, 2451-2459 (2009); and Slater J H, Frey W. Nanopatterning of Fibronectin and the Influence of Integrin Clustering on Endothelial Cell Spreading and Proliferation, J. Biomed. Mater. Res. A, 87(1), 176-195 (2008). Commonly used fabrication techniques include soft lithography, photochemistry, inkjet printing or electrospinning. These techniques, however, may have limitations in achieving high-resolution features, reproducibility, translation to 3D surfaces or need expensive fabrication requirements such as described in Anderson D, Hinds M. Endothelial Cell Micropatterning: Methods, Effects, and Applications, Ann. Biomed. Eng., 39, 2329-2345 (2011).
The aligned collagen matrices used here can be made according to the patent applications “Biocomposites and Method of Making the Same” U.S. patent application Ser. No. 12/539,563, (2009), and “Oriented Collagen-Based Materials, Films and Methods of Making Same” World Intellectual Property Organization 2008, WO/2008/131293, the disclosures of which are hereby incorporated by reference in their entirety.
Additionally, the references to Lai E., Huang N., Cooke J., Fuller G. Aligned Nanofibrillar Collagen Regulates Endothelial Organization and Migration, Regen. Med. 7(5), 649-661 (2012); J. E. Kirkwood, G. G Fuller. Liquid Crystalline Collagen: A Self-Assembled Morphology for the Orientation of Mammalian Cells, Langmuir, 25, (5), 3200-3206 (2009), and Lai E., Huang N., Cooke J., Fuller G. Aligned Nanofibrillar Collagen Regulates Endothelial Organization and Migration. Regen., Med. 7(5), 649-661 (2012) are cited herein, or by the method described in the FIG. 13, which is a generalization of the coating method and device described in “Liquid Film Applicator Assembly and Rectilinear Shearing System Incorporating the Same”, World Intellectual Property Organization 2008, WO/2008/063631 to a cylindrical geometry, the entire disclosure of which is hereby incorporated by reference. These matrices can form planar membranes, cylindrical tubular membranes, or general 3D membranes. The membranes can have single or multiple layers with arbitrary orientation of collagen fibrils in each layer. Unlike previous patterning methods used to regulate the cytoskeletal organization of ECs, the aligned collagen matrices do not use physical or biochemical confinement to restrict the cell motion, thereby better mimicking native extracellular matrices (ECMs). The aligned collagen matrices also provide a useful platform to investigate EC migration, where previous migration investigations have been limited to conditions using hemodynamic shear, chemotactic gradients or physical channels.
The typical structures/devices to direct endothelial cell alignment and migration use the groove-like topography like shown in the FIG. 2 and oriented fiber/fibril topography which is similar to the groove-like topography, see FIG. 3 and FIG. 4. The main mechanism of the endothelial cell alignment/orientation on such substrates is the constraints induced by the fibers/fibrils and groove's walls (substrate “contact guidance”). This is consistent with the observation that the “surface feature depth” is “shown to induce greater alignment response on feature depths more than 300 nm” as in Abrams, G. A.; Teixeira, A. I.; Nealey, P. F.; Murphy, C. J., Effects of Substratum Topography on Cell Behavior. In Biomimetic Materials and Design: Biointerfacial Strategies, Tissue Engineering and Targeted Drug Delivery; Dillow, A. K., Lowman, A. M., Eds.; CRC Press: New York, N.Y., USA, 2002; pp. 91-137; and Brody, S.; Anilkumar, T.; Liliensiek, S.; Last, J. A.; Murphy, C. J.; Pandit, A., Characterizing Nanoscale Topography of the Aortic Heart Valve Basement Membrane for Tissue Engineering Heart Valve Scaffold Design, Tissue Eng., 2006, 12, 413-421.
The aligned collagen matrices produced according to disclosures found in Biocomposites and Method of Making the Same, U.S. patent application Ser. No. 12/539,563, (2009), and Oriented Collagen-Based Materials, Films and Methods of Making Same, World Intellectual Property Organization 2008, WO/2008/131293 have quite different surface topography, see also L. Muthusubramaniam, L. Peng, T. Zaitseva, M. Paukshto, G. R. Martin, T. A. Desai, Collagen Fibril Diameter and Alignment Promote the Quiescent Keratocyte Phenotype J Biomed Mater Res A, 100A, (3), 613-621 (2012). The typical example is presented in the FIG. 5. This is a dense fibrillar collagen matrix which is formed by crimped fibrils oriented in one direction. The crimped configurations of collagen fibrils are typical for collagen-based fibrous tissue when external load is reduced, and mimic the woven spiral structure of collagen bundles in relaxed blood vessels, see K. P Arkill, J. Moger, C. P. J. Winlove, The Structure and Mechanical Properties of Collecting Lymphatic Vessels: an Investigation Using Multimodal Nonlinear Microscopy, Anat. 216, (5), 547-55 (2010). The grooves in the FIG. 5 are oriented perpendicular to the fibril direction. Fibroblasts, smooth muscle cells, and endothelial cells plated on this matrix are aligned and migrate along the fibril direction which is perpendicular to the grooves and crimp ridges.
Threads/sutures/fibers made from type I collagen solution have been researched extensively as scaffolds for repair and regeneration and recently for cell delivery applications, see D. Enea, F. Henson, S. Kew, J. Wardale, A. Getgood, et al., Extruded Collagen Fibres for Tissue Engineering Applications: Effect of Crosslinking Method on Mechanical and Biological Properties, J. Mater Sci: Mater Med. 22, 1569-1578 (2011); K G Cornwell, P Lei, S T Andreadis, G D Pins, Crosslinking of Discrete Self-Assembled Collagen Threads Effects on Mechanical Strength and Cell-Matrix Interactions., J. Biomed Mater Res A. 80A, 362-71 (2007); and D I Zeugolis, G R Paul, G. Attenburrow, Cross-linking of Extruded Collagen Fibers a Biomimetic Three-dimensional Scaffold for Tissue Engineering Applications, J. Biomed Mater Res A. 89A, 895-908 (2009). One of the first commercial extruded collagen sutures was manufactured by Ethicon, see A. Smith, Extruded Collagen Ophthalmic Sutures. A clinical survey, Brit. J. Ophthal., 54, 522-527 (1970). Organogenesis, see P. D. Kemp, R M Karr, J G Maresh, J. Cavallaro, J. Gross, Collagen threads, U.S. Pat. No. 5,378,469, (1995) further improved the extrusion process. Since this time the principal parts of the procedure remain the same and the extruded thread/suture/fiber has a shape of a long compact cylinder with near circular cross-section. “The success of these scaffolds has been limited by insufficient tissue ingrowth from the wound margin”, see K G Cornwell, P Lei, S T Andreadis, G D Pins, Crosslinking of Discrete Self-assembled Collagen Threads: Effects on Mechanical Strength and Cell-matrix Interactions., J. Biomed Mater Res A. 80A, 362-71 (2007), because of the collagen high density and crosslinking treatment used to increase the mechanical properties and decrease the degradation rate of these scaffolds.
The novel thread-like collagen construct (scaffold) as described in U.S. patent application Ser. No. 12/539,563, (2009), the entire disclosure of which is hereby incorporated by reference, produced from thin (1-2 um) collagen ribbon has a completely different structure, see FIG. 16 and FIG. 7. It consists of highly aligned collagen fibrils and has a large surface area suitable for cell ingrowth, see FIG. 1.
Purified collagen from animal or human sources is widely used in various medical devices, in research, and in cosmetics. However, the materials prepared from soluble purified collagen lack the diversity in macrostructure and organization observed in tissues. For example, the collagen fibers in tendon are highly aligned for maximal tensile strength, but also have a kinked structure to allow for tissue flexibility. In contrast, the collagen in the cornea is arranged as small parallel transparent fibers. The collagen in the skin is arranged in bundles, not parallel, which allows more expansion and flexibility than seen with tendon. Each structure provides obvious advantages to the tissue it comprises.
Collagen prepared from both human and animal sources has been shown to be safe and of minimal immunogenicity when implanted into humans. Collagen has the advantages that it is biocompatible, can form structures with high tensile strength, that the tensile strength of the constructs can be increased by covalent cross-linking and that the construct is replaced by normal tissue by repair and regeneration.
Methods to deposit collagen molecules in defined structures including aligned, woven and transparent materials for diverse indications are described in U.S. patent application Ser. Nos. 11/951,324, 11/986,263, 12/106,214, and 12/539,563 and paper Lai E., Huang N., Cooke J., Fuller G. Aligned Nanofibrillar Collagen Regulates Endothelial Organization and Migration, Regen., Med. 7(5), 649-661, (2012), all of which are incorporated by reference herein in their entirety. One advantage of these collagen materials is that they closely approximate the natural structures of tissues, are biocompatible and induce the guided growth of cells attached to them. The collagen materials appear to be an excellent substrate for applying endothelial cells to precise tissue sites. While these advances have been made, there is significant need for continued advancement and development of devices, constructs, implants and methods that promote and/or enhance tissue repair and regeneration, particularly constructs for vascular and lymphatic engineering.
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