WO2013101720A1

WO2013101720A1 - Implantable vascular devices and methods of use thereof

Info

Publication number: WO2013101720A1
Application number: PCT/US2012/071188
Authority: WO
Inventors: Song Li; Randall Raphael R. JANAIRO; Jeffrey Henry
Original assignee: The Regents Of The University Of California
Priority date: 2011-12-28
Filing date: 2012-12-21
Publication date: 2013-07-04

Abstract

The present disclosure generally provides an implantable vascular device (IVD) that has one or more of a thrombus-reducing moiety, an endothelialization moiety, and a moiety that promotes recruitment of progenitor cells and/or stem cells attached to at least one surface of the implantable vascular device. The present disclosure provides methods of use of such implantable vascular devices.

Description

IMPLANTABLE VASCULAR DEVICES AND METHODS OF USE THEREOF

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No.

61/580,974, filed December 28, 2011, which application is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Grant No. HL083900 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

[0003] Small diameter synthetic vascular grafts have a high failure rate due to thrombogenic responses. Although tissue-engineered vessels have been created in vitro using cells with or without scaffolds, long construction times generally limit their applications. There is a need in the art for improved synthetic vascular grafts.

Literature

[0004] U.S. Patent Publication No. 2006/0228389; U.S. Patent Publication No. 2007/0269481;

U.S. Patent Publication No. 2008/0220042; U.S. Patent Publication No. 2007/0264306.

SUMMARY

[0005] The present disclosure generally provides an implantable vascular device (IVD) that has one or more of a thrombus-reducing moiety, an endothelialization moiety, and a moiety that promotes recruitment of progenitor cells and/or stem cells attached to at least one surface of the implantable vascular device. The present disclosure provides methods of use of such implantable vascular devices.

[0006] The present disclosure provides an implantable vascular device comprising: a) a

polymeric scaffold; and b) one or more of: i) a thrombus-reducing moiety; ii) an endothelialization and vascular wall remodeling moiety; iii) a moiety that promotes recruitment of progenitor cells and/or stem cells, wherein the moiety is attached to the polymeric scaffold. [0007] In some embodiments, the polymeric scaffold comprises a hollow, tubular structure.

[0008] In some embodiments, polymeric scaffold comprises a single layer, or comprises two or more layers, each layer comprising a different polymer or polymer blend.

[0009] In some embodiments, the thrombus-reducing moiety is mucin. In some embodiments, the thrombus-reducing moiety is heparin, heparan sulfate, heparan sulfate proteoglycan, or combinations thereof. In some embodiments, the thrombus-reducing moiety is hirudin, a hirudin analog, bivalirudin, lepirudin, desirudin, or combinations thereof. In some embodiments, the thrombus-reducing moiety is an aptamer. In some embodiments, the aptamer comprises the nucleotide sequence GGTTGGTGTGGTTGG (SEQ ID NO:9).

[0010] In some embodiments, the endothelialization and vascular wall remodeling moiety is

SDF-1, or an isoform thereof. In some embodiments, the endothelialization and vascular wall remodeling moiety is VEGF, or an isoform thereof.

[0011] In some embodiments, the moiety that promotes recruitment of progenitor and/or stem cells is SDF-1, or an isoform thereof.

[0012] In some embodiments, the polymer scaffold comprises PLLA. In some embodiments, the polymer scaffold comprises PLCG. In some embodiments, the polymer scaffold comprises PCL. In some embodiments, the polymer scaffold comprises a polymer blend comprising PLCG and PCL. In some embodiments, the polymer scaffold comprises PLLA and PCL. In some embodiments, the polymer scaffold comprises PLLA and a polymer blend comprising PLCG and PCL. In some embodiments, the polymer scaffold comprises polyurethane.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 shows details the structure and mechanical properties of bilayered electrospun vascular grafts. (Panel A) SEM image of a bilayered electrospun vascular graft. Scale bar = 500 μιη. (Panels B-C) SEM images of (Panel B) PLLA microfibers on the inner surface and (Panel C) PLCG+PCL nanofibers on the outer surface of the graft. Scale bars = 50 μιη. (Panel D) Representative stress-strain curves of a bilayered vascular graft and a native common carotid artery (CCA). (Panel E) Average elastic modulus of rat CCA and bilayered vascular grafts (n=6). [0014] FIG. 2 shows mucin immobilization and stability. (Panel A) Schematic of mucin conjugation to a fiber via PEG linker. (Panel B) Mucin was conjugated (or adsorbed; not shown) to fibrous scaffolds and incubated with alcian blue solution, exhibiting blue color. (Panel C) PLLA membranes with conjugated or adsorbed mucin were incubated for 48 h at 37°C under static or flow conditions, and the amount of mucin remained on the membranes was quantified. * p<0.05 based on Holm's t-Test, n=3.

[0015] FIG. 3 shows the effects of mucin on coagulation and platelet adhesion. (Panel A)

Anticoagulant activity of mucin, heparin (positive control) and PEG (negative control) in PBS solution. Coagulation time of whole blood at each concentration of mucin, heparin and PEG was measured (n=3). (Panels B - F) Representative SEM images of platelets adhered to (Panel B) untreated, (Panel C) PEGylated (PEG), (Panel D) mucin- conjugated (PEG-MUC), and (Panel E) mucin-adsorbed (MUC) PLLA scaffolds. Scale bar = 10 μιη. (Panel F) Statistical analysis of surface-modification effects on platelet adhesion. * p<0.05 based on Holm's t-Test, n=3.

[0016] FIG. 4 shows cross sections of the explanted grafts at 1 month post-implantation.

(Panels A-E) H&E staining. (Panels F-J) DAPI staining. Neo-tissue / PLCG+PCL border and PLCG+PCL / PLLA border are indicated with dotted lines. In all panels, right side = luminal side. Both scale bars = 100 μιη.

[0017] FIG. 5 shows EC and SMC organization in the explanted grafts at 1 month post- implantation. (Panels A-E) CD31 staining for ECs. Arrows indicate EC presence on the luminal side. (Panels F-J) SM-MHC staining for SMCs. Arrows exemplified SMC staining. Dotted lines indicate neo-tissue / PLCG+PCL border and PLCG+PCL / PLLA border. In all panels, right side = luminal side. Scale bar = 100 μιη.

[0018] FIG. 6 shows the structure and chemical modification of microfibrous vascular grafts.

(Panel A) Structure of microfibers in the vascular grafts. Scale bar=100 μιη. (Panel B) Representative stress-strain curves of a vascular grafts and a rat carotid artery. (Panel C) Schematic illustration of chemical modification of microfibers with SDF-Ι . (Panel D) Immunostaining for SDF-Ι immobilized on the microfibers of a graft. Scale bar=10 μιη. (E) The time course of SDF-Ι release from the grafts. The amount of SDF-l remaining on the grafts is shown.

[0019] FIG. 7 shows morphological characterization of the grafts. (Panel A) A graft before implantation. (Panel B) A graft right after implantation. (Panel C) A heparin-SDF-l - treated graft at 4 weeks after implantation. Arrow indicates microvessels in the wall of the graft. (Panels D-F) H&E staining of an untreated graft (Panel D), a heparin-treated graft (Panel E) and a heparin-SDF- la-treated graft (Panel F) at 4 weeks after

implantation. Arrows in Panel D indicate thrombus formation. Scale bar = 2 mm in Panels A-C; scale bar = 0.5 mm in Panels D-F.

[0020] FIG. 8 shows immunostaining for CD31 and CD34 of the explanted grafts after 1-week implantation. (Panels A-C) En face DAPI staining of nuclei along the entire length of the grafts. (Panels D-L) En face immunostaining for CD31 and CD34 of proximal, middle and distal portions of untreated grafts (Panels D-F), heparin-treated grafts (Panels G-I) and heparin-SDF- la-treated grafts (Panels J-L). Nuclei were stained by DAPI. Scale bars = 1 mm in Panels A-C; Scale bars = 40 μιη in Panels D-L.

[0021] FIG. 9 shows en face immunostaining for CD31 and CD34 of the explanted grafts at 2 weeks (Panels A-I) and 4 weeks (Panels J-R) after implantation. Nuclei were stained by DAPI. Scale bars = 40 μτη.

[0022] FIG. 10 shows immunostaining for SMC markers in cross sections of the explanted grafts. (Panels A-C) Immunostaining for SMA at 1 week. (Panels D-L) Immunostaining for CNN1 and SM-MHC of the cross sections at 1 week (Panels D-F), 2 week (Panels G- I) and 4 weeks (Panels J-L) after implantation. Nuclei were stained by DAPI. Scale bar = 40 um. Dashed lines indicate the border between the graft (top part) and the outer layer.

[0023] FIG. 11 shows SMPC differentiation, collagen I synthesis and the mechanical properties of the explanted grafts. (Panels A-C) SMPCs isolated from explanted grafts were stained for SMA, CNN1 and SM-MHC. Scale bar = 100 μπι. (Panels D-F) After 4-weeks of spontaneous differentiation, SMPCs were stained for SMA, CNN1 and SM-MHC. Scale bar = 100 μιη. (Panels G-I) Immunostaining for collagen I and SM-MHC of the cross sections of untreated grafts (control), heparin-treated grafts and heparin-SDF- la-treated grafts, respectively, at 4 weeks after implantation. Dashed lines indicate the border between the graft (top part) and the outer layer. Scale bar = 40 μιη. Nuclei were stained by DAPI in Panels A-I. (Panel J) Elastic modulus of the grafts at 1, 2 and 4 weeks after implantation. The data were shown as mean ± standard deviation (SD) (n=4). * indicates significant difference compared to untreated group (control) at the same time point.

[0024] Figure 12. Immunostaining of cross sections of the explanted grafts for endothelial cell (EC) marker CD31 and endothelial progenitor cell (EPC) marker CD34. Untreated grafts (control), he arin-treated grafts and heparin-SDF- la-treated grafts were implanted into rat carotid arteries by anastomosis, and explanted at 1 week (A-C), 2 week (D-F) and 4 weeks (G-I) after surgery, followed by immunostaining and confocal microscopy.

Arrows in C indicate CD34⁺ cells (in green) on the luminal surface. Scale bar = 40 μιη.

[0025] Figure 13. Immunostaining of cross sections of the explanted grafts for EC marker CD31 and EPC marker CD 133. Untreated grafts (control), heparin-treated grafts and heparin-SDF- la-treated grafts were implanted into rat carotid arteries by anastomosis, and explanted at 1 week (A-C), 2 week (D-F) and 4 weeks (G-I) after implantation, followed by immunostaining and confocal microscopy. Arrows in C indicate CD133⁺ cells (in red) on the luminal surface. Scale bar = 40 μιη.

[0026] Figure 14. En face DAPI staining of the explanted grafts at 2 weeks after implantation.

The full length of grafts were stained for nuclei with DAPI, followed by confocal microscopy. (A) Untreated grafts (control). (B) Heparin-treated grafts. (C) Heparin- SDF- la-treated grafts.

[0027] Figure 15. En face DAPI staining of the explanted grafts at 4 weeks after implantation.

DEFINITIONS

[0028] "Peptide" refers to a polymer in which the monomers are amino acids and are joined together through amide bonds, alternatively referred to as a polypeptide. Additionally, unnatural amino acids, for example, .beta.-alanine, phenylglycine and homoarginine are also included. Amino acids that are not gene-encoded may also be used in the present invention. Furthermore, amino acids that have been modified to include reactive groups, glycosylation sites, polymers, therapeutic moieties, biomolecules and the like may also be used in the invention. All of the amino acids used in the present invention may be either the D- or L-isomer. In addition, other peptidomimetics are also useful in the present invention. As used herein, "peptide" refers to both glycosylated and

unglycosylated peptides. Also included are peptides that are incompletely glycosylated by a system that expresses the peptide. For a general review, see, Spatola, A. F., in CHEMISTRY AND BIOCHEMISTRY OF AMINO ACIDS, PEPTIDES AND

PROTEINS, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983).

[0029] As used herein, the term "copolymer" describes a polymer which contains more than one type of subunit. The term encompasses polymer which include two, three, four, five, or six types of subunits.

[0030] The term "attached" (used interchangeably herein with "linked"), as used herein

encompasses interaction including, but not limited to, covalent bonding, ionic bonding, chemisorption, physisorption and combinations thereof.

[0031] The term "stem cells", as used herein, refers to cells capable of differentiation into other cell types, including those having a particular, specialized function (i.e., terminally differentiated cells, such as erythrocytes, macrophages, etc.). Stem cells can be defined according to their source (adult/somatic stem cells, embryonic stem cells), or according to their potency (totipotent, pluripotent, multipotent, or unipotent).

[0032] The term "stent", as used herein, is a tube which can be made of, among other things, metal and organic polymers. When the stent is made of an organic polymer, the polymer is not a nanofibrous or microfibrous polymer scaffold as described herein. In some instances, the entire stent is capable of expanding from a first diameter to a second diameter, wherein the second diameter is greater than the first diameter.

[0033] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0034] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. [0035] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[0036] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an implantable vascular device" includes a plurality of such devices and reference to "the thrombus reducing moiety" includes reference to one or more thrombus reducing moieties and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0037] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0038] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. DETAILED DESCRIPTION

[0039] The present disclosure generally provides an implantable vascular device (IVD) that has one or more of a thrombus-reducing moiety, an endothelialization moiety, and a moiety that promotes recruitment of progenitor cells and/or stem cells attached to at least one surface of the implantable vascular device. The present disclosure provides methods of use of such implantable vascular devices.

IMPLANTABLE VASCULAR DEVICE

[0040] The present disclosure generally provides an implantable vascular device comprising: a) a polymeric scaffold; and b) one or more of i) a thrombus-reducing moiety, ii) an endothelialization moiety, iii) a moiety that promotes recruitment of stem cells and/or progenitor cells, wherein the moiety is attached to the polymeric scaffold. A thrombus- reducing moiety and/or an endothelialization moiety may be attached to the polymeric scaffold directly or via a linker.

[0041] The one or more of i) a thrombus-reducing moiety, ii) an endothelialization moiety, iii) a moiety that promotes recruitment of stem cells and/or progenitor cells can be attached directly to the polymeric scaffold, or can be attached via a linker molecule. Suitable linkers include, e.g., peptides; heparin; and the like.

[0042] The one or more of i) a thrombus-reducing moiety, ii) an endothelialization moiety, iii) a moiety that promotes recruitment of stem cells and/or progenitor cells can be attached, directly or indirectly, to the polymeric scaffold via a covalent linkage.

Properties of IVDs

[0043] The IVDs of the present disclosure generally possess anti-thrombogenic characteristics, maintaining patency for a longer duration as compared to other synthetic vascular grafts. In addition, the IVDs of the present disclosure generally provide favorable

biocompatibility and mechanical properties, as well as the ability to recruit endothelial cells, smooth muscle cells, endothelial progenitor cells, smooth muscle progenitor cells, and/or stem cells into the device, any of which facilitate their use in implantable vascular applications, e.g., their use as implantable vascular grafts to replace diseased or damaged tissue in a subject.

[0044] The IVDs of the present disclosure generally provide reduced incidence of thrombus formation, exemplified by, e.g., reduced adsorption of non-specific plasma proteins, reduced non-specific cell adhesion, reduced blood coagulation, reduced platelet adhesion, and/or reduced intimal hyperplasia.

[0045] For example, when evaluated for their ability to cause coagulation of a blood sample in a glass capillary tube, the thrombus-reducing moieties described herein effected an increase in the amount of time required to coagulate the sample compared to controls. Similarly, when evaluated for their ability to cause platelet adhesion to microfibrous polymer scaffolds, the thrombus-reducing moieties described herein effected a decrease in platelet adhesion compared to controls.

[0046] When evaluated in in vivo animal models, the IVDs of the present disclosure

demonstrated improved patency rates as compared to other synthetic vascular grafts. For example, after one month of implantation in the carotid artery of athymic rats, IVDs of the present disclosure displayed increased unobstructed blood flow compared to controls, demonstrating improved patency of the IVDs.

[0047] The IVDs of the present disclosure generally possess mechanical properties that are similar to those of native vascular tissue, which facilitates integration of the IVDs with the surrounding cells and tissues following implantation. For example, IVDs of the present disclosure can be constructed from polymeric materials having similar mechanical properties to the tissue being replaced, e.g., a similar elastic modulus.

[0048] IVDs of the present disclosure also display increased endothelialization on their luminal surface as compared to other synthetic vascular grafts, which facilitates their use in vascular applications. Histological examination of the IVDs as a function of time following in vivo implantation demonstrates that after one month of implantation, the luminal surfaces of the IVDs were almost completely covered with cells, including endothelial cells and smooth muscle cells. These results demonstrate the ability of the IVDs of the present disclosure to recruit endothelial cells, endothelial precursor cells, smooth muscle cells, and smooth muscle precursor cells, as well as stem cells, into the IVDs to facilitate endothelialization and/or tissue remodeling.

[0049] Accordingly, the methods and compositions of the present disclosure find use in a

variety of blood-contacting vascular devices, including, e.g., vascular grafts, stents, shunts, valves, catheters, dialysis tubing, and the like. The methods and compositions provided herein can be used to create IVDs having any of a variety of characteristics suitable for various uses and applications, depending on the problem to be solved. Materials

[0050] IVDs of the present disclosure generally comprise a scaffold made from a suitable

polymeric material. Polymer scaffolds can be fibrous polymer scaffolds, such as microfiber polymer scaffolds or nanofiber polymer scaffolds. These polymer scaffolds can also be micropatterned polymer scaffolds. Polymer scaffolds can optionally be unaligned or they can be aligned, such as longitudinally or circumferentially. Polymer scaffolds can optionally be formed into a shape, such as a sheet, crisscross sheet, conduit, rod, or filled conduit. Polymer scaffolds can have a seam or they can be seamless. Polymer scaffolds can also be modified to include materials such as a biomolecule, or a pharmaceutically acceptable excipient. These alignments, shapes, and additional components can aid in the improvement or regeneration or replacement of biological function, and can be used in tissue engineering to improve, regenerate or replace biological functions.

[0051] In some embodiments, the present disclosure provides fibrous polymer scaffolds. A fibrous polymer scaffold includes a fiber or fibers which can have a range of diameters. In an exemplary embodiment, the average diameter of the fibers in a fibrous polymer scaffold is from about 0.1 nanometers to about 50,000 nanometers. In another exemplary embodiment, the average diameter of the fibers in the fibrous polymer scaffold is from about 25 nanometers to about 25,000 nanometers. In an exemplary embodiment, the average diameter of the fibers in the fibrous polymer scaffold is from about 50 nanometers to about 20,000 nanometers. In an exemplary embodiment, the average diameter of the fibers in the fibrous polymer scaffold is from about 100 nanometers to about 5,000 nanometers. In an exemplary embodiment, the average diameter of the fibers in the fibrous polymer scaffold is from about 500 nanometers to about 2,000 nanometers. In an exemplary embodiment, the average diameter of the fibers in the fibrous polymer scaffold is from about 800 nanometers to about 1,500 nanometers.

[0052] In an exemplary embodiment, a fibrous polymer scaffold is a member selected from a nanofiber polymer scaffold and a micro fiber polymer scaffold. Micro fiber polymer scaffolds have micron-scale features (an average fiber diameter between about 1,000 nanometers and about 50,000 nanometers, and especially between about 1,000 nanometers and about 20,000 nanometers), while nanofiber polymer scaffolds have submicron-scale features (an average fiber diameter between about 10 nanometers and about 1,000 nanometers, and especially between about 50 nanometers and about 1,000 nanometers).

A variety of polymers from synthetic and/or natural sources can be used to compose the polymer scaffolds of the present disclosure. A fiber can be made from a homopolymer, a copolymer, or blended polymers; the homopolymer, copolymer, or blended polymer can be degradable or non-degradable. For example, lactic or polylactic acid or glycolic or polyglycolic acid can be utilized to form poly(lactide) (PLA) or poly(L-lactide) (PLLA) nanofibers or poly(glycolide) (PGA) nanofibers. Fibers can also be made from more than one monomer or subunit thus forming a co-polymer, terpolymer, etc. For example, lactic or polylactic acid and be combined with glycolic acid or polyglycolic acid to form the copolymer poly(lactide-co-glycolide) (PLGA). Other copolymers may include poly(ethyleneco-vinyl) alcohol). In an exemplary embodiment, a fiber comprises a polymer or subunit which is a member selected from an aliphatic polyester, a

polyalkylene oxide, polydimethylsiloxane, polyurethane, expanded

polytetrafluoroethylene (ePTFE), polyvinylalcohol, polylysine, collagen, laminin, fibronectin, elastin, alginate, fibrin, hyaluronic acid, proteoglycans, polypeptides and combinations thereof. In another exemplary embodiment, a fiber comprises two different polymers or subunits which are members selected from an aliphatic polyester, a polyalkylene oxide, polydimethylsiloxane, polyurethane, ePTFE, polyvinylalcohol, polylysine, collagen, laminin, fibronectin, elastin, alginate, fibrin, hyaluronic acid, proteoglycans, polypeptides and combinations thereof. In another exemplary

embodiment, a fiber comprises three different polymers or subunits which are members selected from an aliphatic polyester, a polyalkylene oxide, polydimethylsiloxane, polyurethane, ePTFE, polyvinylalcohol, polylysine, collagen, laminin, fibronectin, elastin, alginate, fibrin, hyaluronic acid, proteoglycans, polypeptides and combinations thereof. In an exemplary embodiment, the aliphatic polyester is linear or branched. In another exemplary embodiment, the linear aliphatic polyester is a member selected from lactic acid (D- or L-), lactide, poly(lactic acid), poly(lactide) glycolic acid, poly(glycolic acid), poly(glycolide), glycolide, poly(lactideco-glycolide), poly(lactic acid-co-glycolic acid), polycaprolactone and combinations thereof. In another exemplary embodiment, the aliphatic polyester may be branched and may comprise at least one member selected from lactic acid (D- or L-), lactide, poly(lactic acid), poly(lactide) glycolic acid, poly(glycolic acid), poly(glycolide), glycolide, poly(lactideco-glycolide), poly(lactic acid-co-glycolic acid), polycaprolactone and combinations thereof which is conjugated to a linker or a biomolecule. In an exemplary embodiment, a polymer may be a polyalkylene oxide selected from polyethylene oxide, polyethylene glycol,

polypropylene oxide, polypropylene glycol and combinations thereof.

[0054] A polymer scaffold can comprise a fiber of at least one composition. In an exemplary embodiment, the fibrous polymer scaffold comprises a number of different types of fibers, and this number is a member selected from one, two, three, four, five, six, seven, eight, nine and ten.

[0055] In some embodiments, the fiber or fibers of a polymer scaffold may be biodegradable. In another exemplary embodiment, the fibers of a polymer scaffold comprise biodegradable polymers. In another exemplary embodiment, the biodegradable polymers comprise a monomer which is a member selected from lactic acid and glycolic acid. In another exemplary embodiment, the biodegradable polymers are poly(lactic acid), poly(glycolic acid) or a copolymer thereof. In another exemplary embodiment, biodegradable polymer scaffolds of the present disclosure can be used to guide the morphogenesis of engineered tissue and gradually degrade after the assembly of the tissue. The degradation rate of the polymers can be tailored by one of skill in the art to match the tissue generation rate. For example, if a polymer that biodegrades quickly is desired, an approximately 50:50 PLGA combination can be selected. Additional ways to increase polymer scaffold biodegradability can involve selecting a more hydrophilic copolymer (for example, polyethylene glycol), decreasing the molecular weight of the polymer, as higher molecular weight often means a slower degradation rate, and changing the porosity or fiber density, as higher porosity and lower fiber density often lead to more water absorption and faster degradation.

[0056] A polymeric scaffold of the present disclosure may comprise any of a variety of

materials, as described above. Exemplary materials include poly(L-lactide) (PLLA), poly(L-lactide-co-caprolactone-co-glycolide) (PLCG), and poly(8-caprolactone) (PCL), and polyurethane. Polymer scaffolds may also comprise polymer blends comprising two or more polymers. In some embodiments, polymer scaffolds comprise a blend of PLCG and PCL. In some embodiments, polymer scaffolds may comprise two or more different polymers, two or more different polymer blends, or one or more polymers and one or more polymer blends. In some embodiments, a polymer scaffold comprises PLLA and PCL. In some embodiments, a polymer scaffold comprises PLLA and a blend of PLCG and PCL.

[0057] Solutions of polymers or polymer blends used to create polymer scaffolds of a subject

IVD generally comprise a suitable amount of one or more selected polymers dissolved in a suitable solvent. For example, in some embodiments, a desired amount of PLLA polymer is dissolved in l ,l,l,3,3-hexafluoro-2-propanol (HFIP) to create a solution having a desired concentration, e.g., a solution consisting of 19% (w/v) of PLLA. In some embodiments, a desired amount of PLCG and PCL is dissolved in HFIP to create a polymer blend solution having a desired concentration of each polymer, e.g., a solution consisting of 10% (w/v) PLCG and 5% (w/v) PCL.

[0058] In addition to the polymers described above, many other suitable polymers may be used to construct IVDs of the present disclosure. For example, other suitable polymers that may be used in the subject IVDs may include ethylene vinyl alcohol copolymer

(commonly known by the generic name EVOH or by the trade name EVAL);

polybutylmethacrylate; poly(hydroxyvalerate); poly(L-lactic acid); polycaprolactone; poly(lactide-co-glycolide); poly(hydroxybutyrate); poly(hydroxybutyrate-co-valerate); polydioxanone; polyorthoester; polyanhydride; poly(glycolic acid); poly(D,L-lactic acid); poly(glycolic acid-co-trimethylene carbonate); polyphosphoester; polyurethane; ePTFE; polyphosphoester urethane; poly(amino acids); cyanoacrylates;

poly(trimethylene carbonate); poly(iminocarbonate); copoly(ether-esters) (e.g.,

PEO/PLA); polyalkylene oxalates; polyphosphazenes; biomolecules, such as fibrin, fibrinogen, cellulose, starch, collagen and hyaluronic acid; polyurethanes; silicones; polyesters; polyolefms; polyisobutylene and ethylene-alphaolefm copolymers; acrylic polymers and copolymers; vinyl halide polymers and copolymers, such as polyvinyl chloride; polyvinyl ethers, such as polyvinyl methyl ether; polyvinylidene halides, such as polyvinylidene fluoride and polyvinylidene chloride; polyacrylonitrile; polyvinyl ketones; polyvinyl aromatics, such as polystyrene; polyvinyl esters, such as polyvinyl acetate; copolymers of vinyl monomers with each other and olefins, such as ethylene- methyl methacrylate copolymers, acrylonitrile-styrene copolymers, ABS resins, and ethylene-vinyl acetate copolymers; polyamides, such as Nylon 66 and polycaprolactam; alkyd resins; polycarbonates; polyoxymethylenes; polyimides; polyethers; epoxy resins; polyurethanes; rayon; rayon-triacetate; cellulose; cellulose acetate; cellulose butyrate; cellulose acetate butyrate; cellophane; cellulose nitrate; cellulose propionate; cellulose ethers; amorphous Teflon; and carboxymethyl cellulose.

[0059] The polymers provided above are merely exemplary, and in no way limit the materials from which the subject IVDs may be constructed.

Thrombus-reducing Moieties

[0060] A subject IVD may include at least one thrombus-reducing moiety attached to at least one surface of the IVD. The thrombus-reducing moieties of the present disclosure generally possess a variety of desired properties, including but not limited to increasing the hydrophilicity of surfaces, decreasing adsorption of non-specific plasma proteins, and decreasing non-specific cell adhesion. Thrombus-reducing moieties may generally be glycoproteins, proteoglycans, nucleic-acid aptamers, or peptides. Non-limiting examples of thrombus-reducing moieties are provided below.

Heparin

[0061] IVDs of the present disclosure may include heparin as a thrombus-reducing moiety.

Heparin is a well-characterized biological polymer that is commercially available from a variety of sources. Heparin generally ranges in molecular weight from about 3 kDa up to about 30 kDa, with most commercially-available preparations having a molecular weight in the range of about 12 kDa to about 15 kDa. Heparin is a member of the

glycosaminoglycan family of carbohydrates (which includes the closely-related molecule heparan sulfate) and consists of a variably-sulfated repeating disaccharide unit. The most common disaccharide unit is composed of a 2-O-sulfated iduronic acid and 6- O-sulfated, N-sulfated glucosamine, IdoA(2S)-GlcNS(6S). For example, this makes up 85% of heparins from beef lung and about 75% of those from porcine intestinal mucosa. Disaccharides containing a 3-O-sulfated glucosamine (GlcNS(3S,6S)) or a free amine group (GlcNH₃+) may also be present in various forms of heparin. [0062] Heparin generally has a high negative charge density and can therefore be used to, e.g., increase the hydrophilicity of surfaces to which it is attached, decrease nonspecific adsorption of plasma proteins, and decrease non-specific cell adhesion. IVDs of the present disclosure may include heparin, heparan sulfate, heparan sulfate

proteoglycan, and combinations thereof. Exemplary heparin molecules include, e.g., heparin, heparan sulfate, heparin-like moieties such as fucoidans, sulfated fucans or heparinoids, i.e., highly acidic, e.g., highly sulfated polysaccharides, or mimetics thereof. In some embodiments, the heparin, heparan sulfate or heparinoid may be composed of from about 3 to about 20 monosaccharide units, e.g., from about 5 to about 15 monosaccharide units, such as pentasaccharides.

Mucin

[0063] IVDs of the present disclosure may include mucin as a thrombus-reducing moiety.

Mucins are heavily glycosylated high molecular weight glycoproteins with

predominantly O-linked oligosaccharide side chains. Mucin molecules generally range in molecular weight from about 0.5 up to about 20 MDa, with carbohydrate composing 50% or more of their dry weight. Carbohydrates found in mucin molecules include, e.g., N-acetylgalactosamine, N-acetylglucosamine, fucose, galactose, sialic acid (N- acetylneutraminic acid), and mannose. The oligosaccharide chains generally comprise 5- 15 monomers, and typically exhibit moderate branching, attaching to the protein core by O-glycosidic bonds to the hydroxyl side chains of serine and threonine residues.

[0064] Mature mucins are generally composed of two distinct regions: the terminal region

(comprising the amino- and carboxyl-termini) and the central, or core, region. The amino- and carboxyl-terminal regions are generally very lightly glycosylated, but rich in cysteine residues. The cysteine residues participate in establishing disulfide linkages within and among mucin monomers. The central region is formed of multiple tandem repeats of 10 to 80 residue sequences in which up to half of the amino acids are serine or threonine. This area becomes saturated with hundreds of O-linked oligosaccharides. N- linked oligosaccharides are also found on mucins, but in less abundance than O-linked sugars.

[0065] Mucin molecules are generally negatively charged and heavily glycosylated, typically forming branched 3-D structures that resemble a bottle brush. Due to this structure, mucins generally increase the hydrophilicity of surfaces to which they are attached, and generally decrease non-specific adsorption of plasma proteins and nonspecific cell adhesion. Mucins are commercially available from a variety of sources. Exemplary mucins include, e.g., GenBank Accession Number CAA03985.1 (SEQ ID. NO: l), AAA60019.1 (SEQ ID:NO. 2), and AAA63229.2 (SEQ ID NO:3). Mucins that may be used in the IVDs of the present disclosure generally include those having at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%, amino acid sequence identity to one of SEQ ID NOS: l , 2, and 3, provided below.

[0066] SEQ ID NO: 1:

MKGARWRRVPWVSLSCLCLCLLPHVVPGTTEDTLITGSKTPAPVTSTGSTTATL

EGQSTAASSRTSNQDISASSQNHQTKSTETTSKAQTDTLTQMMTSTLFSSPSVHN

VMETVTQETAPPDEMTTSFPSSVTNTLMMTSKTITMTTSTDSTLGNTEETSTAGT

ESSTPVTSAVSITAGQEGQSRTTSWRTSIQDTSASSQNHWTRSTQTTRESQTSTLT

HRTTSTPSFSPSVHNVTGTVSQKTSPSGETATSSLCSVTNTSMMTSEKITVTTSTG

STLGNPGETSSVPVTGSLMPVTSAALVTVDPEGQSPATFSRTSTQDTTAFSKNHQ

TQSVETTRVSQINTLNTLTPVTTSTVLSSPSGFNPSGTVSQETFPSGETTISSPSSVS

NTFLVTSKVFRMPISRDSTLGNTEETSLSVSGTISAITSKVSTIWWSDTLSTALSPS

SLPPKISTAFHTQQSEGAETTGRPHERSSFSPGVSQEIFTLHETTTWPSSFSSKGHT

TWSQTELPSTSTGAATRLVTGNPSTGAAGTIPRVPSKVSAIGEPGEPTTYSSHSTT

LPKTTGAGAQTQWTQETGTTGEALLSSPSYSVTQMIKTATSPSSSPMLDRHTSQ

QITTAPSTNHSTIHSTSTSPQESPAVSQRGHTQAPQTTQESQTTRSVSPMTDTKTV

TTPGSSFTASGHSPSEIVPQDAPTISAATTFAPAPTGDGHTTQAPTTALQATPSSH

DATLGPSGGTSLSKTGALTLANSVVSTPGGPEGQWTSASASTSPDTAAAMTHTH

QAESTEASGQTQTSEPASSGSRTTSAGTATPSSSGASGTTPSGSEGISTSGETTRFS

SNPSRDSHTTQSTTELLSASASHGAIPVSTGMASSIVPGTFHPTLSEASTAGRPTG

QSSPTSPSASPQETAAISRMAQTQRTRTSRGSDTISLASQATDTFSTVPPTPPSITSS

GLTSPQTQTHTLSPSGSGKTFTTALISNATPLPVTYASSASTGHTTPLHVTDASSV

STGHATPLP VTSPS S VSTGHTTPLP VTD AS S VSTGHPTP .

[0067] SEQ ID NO:2:

MTPGTQSPFFLLLLLTVLTVVTGSGHASSTPGGEKETSATQRSSVPSSTEKNAVS MTSSVLSSHSPGSGSSTTQGQDVTLAPATEPASGSAATWGQDVTSVPVTRPALG STTPPAHDVTSAPDNKPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDT RPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVT SAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPP AHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPG STAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDT RPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVT SAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPP AHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPG STAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDT RPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVT SAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPP AHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPG STAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDT RPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVT SAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPP AHGVTSAPDNRPALGSTAPPVHNVTSASGSASGSASTLVHNGTSARATTTPASK STPFSIPSHHSDTPTTLASHSTKTDASSTHHSSVPPLTSSNHSTSPQLSTGVSFFFLS FHISNLQFNSSLEDPSTDYYQELQRDISEMFLQIYKQGGFLGLSNIKFRPGSVVVQ LTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAGVP GWGIALLVLVCVLVALAIVYLIALAVCQCRRK YGQLDIFPARDTYHPMSEYPT YHTHGRYVPPSSTDRSPYEKVSAGNGGSSLSYTNPAVAAASANL.

[0068] SEQ ID O:3:

TPTPTPTGTQTPTPTPITTTTTMVTPTPTITSTQTPTPTPITTTTVTPTPTPTSTQRTT

PTSITTTTTVTPTPTPITTTTTVTPTPTPTGTQTPTTTPISTTTTVTPTPTPTGTQTLT

PTPITTTTTVTPTPTPTGTQTPTSTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPT

PTPTGTRYPTPTPITTTTTVTPTPTPTSTQSTTPTPITTTNTVTPTPTPTGTQTPTPTP

ITTTTTMVTPTPTITSTQTPTPTPITTTTVTPTPTPTSTQRTTPTSITTTTTVTPTPT.

Hirudin

[0069] IVDs of the present disclosure may comprise hirudin as a thrombus-reducing moiety.

Hirudin is a well-characterized peptide (approximately 65 amino acids in length) that is commercially available from a variety of sources. Hirudin generally inhibits the pro- coagulant activity of the enzyme thrombin, and therefore generally provides thrombus- reducing activity when used in the IVDs of the present disclosure. IVDs of the present disclosure may include, e.g., hirudin, hirudin analogs, bivalirudin, lepirudin, desirudin, or combinations thereof.

[0070] Wild-type hirudin is organized into a compact N-terminal domain containing three

disulfide bonds and a C -terminal domain which is disordered when the protein is un- complexed in solution. Wild-type hirudin from Hirudo medicinalis contains a mixture of various isoforms (HV1, HV2 and HV3) of the protein. HV1 and HV2 consist of a single polypeptide chain of 65 amino acids in which the amino-terminal apolar core and the strongly acidic carboxyl-terminal tail bind to the apolar binding site and to the anion binding exosite of thrombin, thus preventing it from interacting with fibrinogen. HV3 is identical to HV2 from positions 1 to 32 and then differs from HV1 in the following respects: Gin at position 33 instead of Asp, Lys at position 35 instead of Glu, Asp at position 36 instead of Lys, Gin at position 53 instead of Asp, Pro at position 58 instead of Glu, Asp at position 62 instead of Glu, Ala at position 63 instead of Tyr, Asp at position 64 instead of Leu and Glu at position 65 instead of Gin. Hirudin is also available commercially in a number of hirudin-based anticoagulant pharmaceutical products such as Lepirudin (Refludan™), hirudin derived from Hansenula (Extrauma™) and Desirudin (Revasc/Iprivask™).

[0071] Hirudin can be obtained from Hirudinaria manillensis (GenBank Accession Number CAA51293), Hirudo medicinalis (GenBank Accession Number CAA01205),

Poecilobdella viridis (GenBank Accession Number P84590) or from recombinant technology (e.g., GenBank Accession Number CAA02181 (SEQ ID NO:4)). Hirudin compositions that may be used in the IVDs of the present disclosure generally include those having at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%, sequence identity to SEQ ID NO:4, provided below.

[0072] SEQ ID NO:4:

MTYTDCTESGQNLCLCEGSNVCGQGNKCILGSDGEKNQCVTGEGTPKPQSHND GDFEEIPEE YLQ .

[0073] Hirudin is described in, e.g., Folkers et al, Biochemistry, 28(6): 2601-2617 (1989).

Analogs of hirudin include peptides with one or more mutations, fewer amino acids, more amino acids, chemical modifications to one or more amino acid residues, and combinations thereof. Examples of hirudin include wild-type hirudin, bivalirudin, lepirudin, desirudin, non-sulfated Tyr-63 hirudin, hirudin with the N-terminus modified (ie acetylated), hirudin with the C-terminus modified (ie acetylated), a hirudin fragment with the N-terminal domain deleted (approximately residues 1-53), a hirudin fragment with the C-terminal domain deleted (approximately residues 54-65), [Tyr(SO.sub.3H)- 63]-hirudin fragment 54-65, [Tyr(S0₃H)-63]-hirudin fragment 55-65, acetyl

[Tyr(S0₃H)-63]-hirudin fragment 54-65, acetyl [Tyr(S0₃H)-63]-hirudin fragment 55-65. Hirudin for use herein can be produced from a variety of sources. In some instances, hirudin is isolated from leeches. In others, hirudin is recombinantly produced from bacteria, yeast or fungi. In still others, hirudin is chemically synthesized. Recombinant and chemical syntheses tend to produce homogenous products, while hirudin isolated from leeches can include more than one hirudin analog. Hirudin is commercially available from companies such as Sigma-Aldrich (St. Louis, Mo.).

Aptamers

[0074] IVDs of the present disclosure may include any of a variety of aptamers as thrombus- reducing moieties. Aptamers are generally short nucleic acid sequences (generally ranging in length from 15 to 100 base pairs) that are capable of 3 -dimensional recognition, binding, and inhibition of target proteins. In some embodiments, aptamers that bind to and inhibit thrombogenic factors are used as thrombus-reducing moieties.

[0075] In some embodiments, an aptamer having the sequence GGTTGGTGTGGTTGG (SEQ ID NO: 9) is used as a thrombus-reducing moiety. An aptamer having an internal polarity inversion and the sequence 3 ' -GGT-5 ' -5 ' -TGGTGTGGTTGG-3 ' (SEQ ID NO: 10) can also be used.

[0076] Techniques for selecting aptamers that bind to and interact with target proteins are well known in the art, and include the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) approach for aptamer selection {Science 1990, 249, 505; J. Mol. Biol. 1991, 222, 739). The SELEX approach to aptamer selection involves the synthesis of a DNA oligonucleotide library wherein the oligonucleotides have invariant sequences at each end. The sequence of the middle portion of each oligonucleotide is randomized to create the combinatorial library. The theoretical number of 40-mer combinations is about 1.2x 10 24 , although libraries comprising fewer unique sequences may be used.

[0077] The library, after PCR amplification, can be exposed to target polypeptides or proteins immobilized on beads. Those oligonucleotides that have affinity for the target ligand will remain bound to the bead and get enriched. Non-binding oligonucleotides are partitioned and washed away with buffer. Release of the bound DNAs using strong eluting conditions will separate the beads from the aptamers, which can be PCR amplified. This process constitutes one round of selection. Repetition of this procedure allows for the enrichment of aptamers that have high affinities for a target polypeptide or protein. When necessary, counter selection can be used to eliminate unwanted cross-reactivity. In a counter selection step, the counter selection ligand can be immobilized to the beads so as to remove those aptamers with cross-reactivity with the counter selection ligand. The result of the selection procedures are aptamers having an affinity for a specific target polypeptide or protein. Using this approach, Ka values for aptamer-target binding (in some cases it is the IC₅₀) in the concentration range of low nM to pM are achievable.

[0078] The SELEX approach described above, as well as other methods known in the art, may be used to identify suitable aptamers for use in the IVDs of the present disclosure that bind to specific target peptides or proteins, e.g., aptamers that bind to and modulate the activity of peptides or proteins involved in the blood coagulation process.

Endothelialization and Vascular Wall Remodeling Moieties

[0079] A subject IVD may include at least one endothelialization moiety attached to at least one surface of the IVD. Endothelialization moieties generally facilitate recruitment of endothelial cells (ECs) and endothelial progenitor cells (EPCs). Vascular wall remodeling moieties generally facilitate the recruitment of smooth muscle cells (SMCs), and/or smooth muscle progenitor cells (SMPCs) into the IVD and facilitate extracellular matrix deposition and/or tissue remodeling. Finally the IVD becomes colonized by differentiated endothelial cells, differentiated smooth muscle cells, and/or extracellular matrix.

[0080] Various cellular markers are known to be expressed by endothelial progenitor cells and differentiated endothelial cells. For example, CD34 and CD 133 are expressed on the surface of endothelial progenitor cells, while CD31 is expressed on differentiated endothelial cells. Similarly, smooth muscle progenitor cells can generally be identified by the presence of non-specific markers such as Seal, c-kit, CD34, and CD146, while differentiated smooth muscle cells can generally be identified by the presence of markers such as smooth muscle a-actin (SMA), calponin-1 (C N1), and smooth muscle myosin heavy chain (MHC). Extracellular matrix deposition and tissue remodeling can generally be identified by the presence of type I collagen (collagen-I) and elastin. [0081] Endothelialization moieties generally facilitate recruitment of endothelial cells or their progenitors expressing the above-described markers into the IVDs of the present disclosure, leading to colonization of the IVD by endothelial and smooth muscle cells, as well as deposition of extracellular matrix molecules such as collagen-I. Non-limiting examples of endothelialization moieties are provided below.

Stromal Cell Derived Factor 1

[0082] IVDs of the present disclosure may comprise stromal cell derived factor 1 (SDF-1), or an isoform thereof, as an endothelialization moiety. SDF-1 is a highly-conserved peptide that generally facilitates chemotaxis of ECs, SMCs, EPCs, and SMPCs, as well as neovascularization. SDF-1 and iso forms thereof also generally facilitate recruitment of stem cells. SDF-1 and isoforms thereof are commercially available from a variety of sources. Isoforms of SDF-1 include SDF-1 -alpha and SDF-1 -beta. The amino acid sequences of isoforms SDF-1 -alpha and SDF-1 -beta are provided in GenBank Accession No. NP 954637.1 and GenBank Accession No. NP 000600.1, respectively. The amino acid sequence of SDF-1 -alpha is provided below in SEQ ID NO:5. The amino acid sequence of SDF-l-beta is provided below in SEQ ID NO:6.

[0083] SDF-1 can be attached directly to a surface of an IVD; or can be attached via a heparin moiety.

[0084] SDF-1 molecules that may be used in the IVDs of the present disclosure generally

include those having at least about 85%, at least about 90%, at least about 95%>, at least about 99%, or 100%, amino acid sequence identity to SEQ ID NO:5 or SEQ ID NO:6.

[0085] SEQ ID NO:5:

MNAKVVVVLVLVLTALCLSDGKPVSLSYRCPCRFFESHVARANVKHLKILNTP NCALQIVARLKNNNPvQVCIDPKLKWIQEYLEKALNK.

[0086] SEQ ID NO:6:

MNAKVVVVLVLVLTALCLSDGKPVSLSYRCPCRFFESHVARANVKHLKILNTP

NCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNKRFKM.

VEGF

[0087] IVDs of the present disclosure may comprise VEGF, or an isoform thereof, as an

endothelialization moiety. VEGF is a well-characterized biomolecule that functions as a potent and specific stimulator of endothelial cell migration and proliferation. VEGF and its isoforms are commercially available from a variety of sources. [0088] The broad term "VEGF" refers a number of proteins that result from alternate splicing of mRNA from a single, 8-exon, VEGF gene. Alternate exon splicing impacts the functional and structural properties of the various isoforms of VEGF, e.g., VEGF121, VEGF121b, VEGF 145, VEGF 165, VEGF165b, VEGF 189, VEGF206. In addition, inclusion or exclusion of exons 6 and 7 mediates interactions with heparan sulfate proteoglycans (HSPGs) and neuropilin co-receptors on the cell surface, enhancing their ability to bind and activate the VEGF receptors (VEGFRs).

[0089] Exemplary VEGF sequences can be found in, e.g., GenBank Accession No.

AAA35789.1 and GenBank Accession No. CAC19513.2. The amino acid sequences of two exemplary VEGF molecules are provided below in SEQ ID NO:7 and SEQ ID NO:8.

[0090] VEGF molecules that may be used in the IVDs of the present disclosure generally

include those having at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%, amino acid sequence identity to SEQ ID NO:7 or SEQ ID NO:8.

[0091] SEQ ID NO:7:

MNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRS YCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQI MRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQENPCGPCSERRKHLFVQDPQ TCKCSCKNTDSRCKARQLELNERTCRCDKPRR.

[0092] SEQ ID NO:8:

MTDRQTDTAPSPSYHLLPGRRRTVDAAASRGQGPEPAPGGGVEGVGARGVALK

LFVQLLGCSRFGGAVVRAGEAEPSGAARSASSGREEPQPEEGEEEEEKEEERGPQ

WRLGARKPGSWTGEAAVCADSAPAARAPQALARASGRGGRVARRGAEESGPP

HSPSRRGSASRAGPGRASETMNFLLSWVHWSLALLLYLHHAKWSQAAPMAEG

GGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGC

CNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQE

K SVRGKGKGQKRKRK SRYKSWSVPCGPCSERRKHLFVQDPQTCKCSCKNT

DSRCKARQLELNERTCRCDKPRR.

Moieties that Promote Recruitment of Progenitor Cells and/or Stem Cells

[0093] A subject IVD may include a moiety that promotes recruitment of progenitor cells and/or stem cells attached to at least one surface of the IVD. Such moieties generally facilitate recruitment of endothelial progenitor cells, smooth muscle progenitor cells, and/or stem cells into the IVD and facilitate colonization of the IVD with endothelial and smooth muscle cells, as well as deposition of extracellular matrix.

[0094] Endothelial progenitor cells and smooth muscle progenitor cells can generally be

identified by expression of surface markers. For example, CD34 and CD 133 are generally expressed on the surface of endothelial progenitor cells, while smooth muscle progenitor cells can generally be identified by the presence of markers such as Seal, c- kit, CD34, and CD146.

[0095] Smooth muscle progenitors may also express transcriptional markers such as Sox 10, Sox 17 and Snail and cytoskeletal markers such as nestin and neurofilaments.

[0096] Non-limiting examples of moieties that promote recruitment of progenitor cells and/or stem cells are provided below.

Stromal Cell Derived Factor 1

[0097] IVDs of the present disclosure may comprise stromal cell derived factor 1 (SDF-1), or an isoform thereof, as moiety that promotes recruitment of progenitor cells and/or stem cells. SDF-1 is a highly-conserved peptide that generally facilitates chemotaxis of ECs, SMCs, EPCs, and SMPCs, as well as neovascularization. SDF-1 and iso forms thereof also generally facilitate recruitment of stem cells. SDF-1 and isoforms thereof are commercially available from a variety of sources. Isoforms of SDF-1 include SDF-1 - alpha and SDF-1 -beta. The amino acid sequences of isoforms SDF-1 -alpha and SDF-1 - beta are provided in GenBank Accession No. NP 954637.1 and GenBank Accession No. NP 000600.1, respectively. The amino acid sequence of SDF-1 -alpha is provided in SEQ ID NO:5. The amino acid sequence of SDF-1 -beta is provided in SEQ ID NO:6.

[0098] SDF-1 molecules that may be used in the IVDs of the present disclosure generally

include those having at least about 85%, up to about 90%, up to about 95%>, up to about 99%, or up to about 100% sequence identity to SEQ ID NO:5 or SEQ ID NO:6.

[0099] SDF-1 can be attached directly to a surface of an IVD; or can be attached via a heparin moiety.

METHODS OF MAKING IMPLANTABLE VASCULAR DEVICES

Methods of Making Polymer Scaffolds

[00100] The present disclosure generally provides methods for making polymer scaffolds that may be used to make the subject IVDs. The methods described herein are generally used to create polymer scaffolds that simulate the structure of native vascular tissues, e.g., arteries, veins, and the like, to facilitate integration of the IVDs with surrounding cells and tissues following implantation. The methods provided below are merely exemplary, and are in no way limiting.

[00101] The polymer scaffolds of the present disclosure can be produced in a variety of ways. In an exemplary embodiment, the polymer scaffold can be produced by electrospinning. Electrospinning is an atomization process of a conducting fluid which exploits the interactions between an electrostatic field and the conducting fluid. When an external electrostatic field is applied to a conducting fluid (e.g., a semi-dilute polymer solution or a polymer melt), a suspended conical droplet is formed, whereby the surface tension of the droplet is in equilibrium with the electric field. Electrostatic atomization occurs when the electrostatic field is strong enough to overcome the surface tension of the liquid. The liquid droplet then becomes unstable and a tiny jet is ejected from the surface of the droplet. As it reaches a grounded target, the material can be collected as an interconnected web containing relatively fine, i.e., small diameter, fibers. The resulting films (or membranes) from these small diameter fibers have very large surface area to volume ratios and small pore sizes. A detailed description of electrospinning apparatus is provided in Zong, et al, Polymer, 43(16):4403-4412 (2002); Rosen et al, Ann Plast Surg., 25:375-87 (1990) Kim, K., Biomaterials 2003, 24, (27), 4977-85; Zong, X., Biomaterials 2005, 26, (26), 5330-8. After electrospinninng, extrusion and molding can be utilized to further fashion the polymer scaffolds. To modulate fiber organization into aligned fibrous polymer scaffolds, the use of patterned electrodes, wire drum collectors, or post-processing methods such as uniaxial stretching may be used. Zong, X.,

Biomaterials 2005, 26, (26), 5330-8; Katta, P., Nano Lett 2004, 4, (11), 2215-2218; Li, D, Nano Lett 2005, 5, (5), 913-6.

[00102] The polymer solution can be produced by dissolving the polymer in appropriate solvents. The polymer solution can be subsequently loaded into a syringe assembly.

[00103] The polymer used to form the polymer scaffold is first dissolved in a solvent. The solvent can be any solvent which is capable of dissolving the polymer monomers and/or subunits and providing a polymer solution capable of conducting and being electrospun. Typical solvents include a solvent selected from Ν,Ν-Dimethyl formamide (DMF), tetrahydrofuran (THF), methylene chloride, dioxane, ethanol, hexafluoroisopropanol (HFIP), chloroform, water and combinations thereof. [00104] The polymer solution can optionally contain a salt which creates an excess charge effect to facilitate the electrospinning process. Examples of suitable salts include NaCl, KH₂P0₄, K₂HP0₄, KI0₃, KC1, MgS0₄, MgCl₂, NaHC0₃, CaCl₂ or mixtures of these salts.

[00105] The polymer solution forming the conducting fluid may have a polymer

concentration in the range of about 1 to about 50 weight % (wt %), e.g., from about 1 wt % to about 5 wt %, from about 5 wt % to about 10 wt %, from about 10 wt % to about 15 wt %, from about 15 wt % to about 20 wt %, from about 20 wt % to about 30 wt %, from about 30 wt % to about 40 wt %, or from about 40 wt % to about 50 wt %.

[00106] The electric field created in the electrospinning process can be in the range of about 5 to about 100 kilovolts (kV), e.g., from about 10 kV to about 50 kV. The feed rate of the conducting fluid to the spinneret (or electrode) can be in the range of about 0.1 to about 1,000 microliters/min, e.g., from about 1 to about 250 microliters/min.

[00107] The single or multiple spinnerets may sit on a platform which is capable of being adjusted, varying the distance between the platform and the grounded collector substrate. The distance can be any distance which allows the solvent to essentially completely evaporate prior to the contact of the polymer with the grounded collector substrate. In an exemplary embodiment, this distance can vary from 1 cm to 25 cm. Increasing the distance between the grounded collector substrate and the platform generally produces thinner fibers.

[00108] In electrospinning cases where a rotating mandrel is required, the mandrel is mechanically attached to a motor, often through a drill chuck. In an exemplary embodiment, the motor rotates the mandrel at a speed of between about 50 revolutions per minute (rpm) up to about 1,000 rpm. In an exemplary embodiment, the motor rotation speed of between about 50 rpm up to about 800 rpm.

[00109] Additional embodiments and/or modifications to the electrospinning process and apparatus are described in, e.g., U.S. Patent Publication No. 2007/0269481, which is hereby incorporated by reference in its entirety, and U.S. Patent Publication No.

2008/0220042, which is hereby incorporated by reference in its entirety.

Micropatterned Polymer Scaffolds

[00110] In another aspect, the present disclosure provides micropatterned polymer

scaffolds. With micropatterning, soft lithography is used to topographically or chemically alter the spatial and geometric organization of the polymer and create micron-scale features on substrate surfaces. Taylor, A. M., Nat Methods 2005, 2, (8), 599-605; Dow, J. A, J Cell Sci Suppl 1987, 8, 55-79; Kane, R. S., Biomaterials 1999, 20, (23-24), 2363-76. The polymer scaffolds created by this technique can be used to control many aspects of the subject IVD, including behavior of recruited cells, including cell size, shape, spatial organization, proliferation and survival. Chen, C. S, Science 1997, 276, (5317), 1428-8; Bhatia, S. N, Faseb J 1999, 13, (14), 1883-900; Deutsch, J, J Biomed Mater Res 2000, 53, (3), 267-76; Folch, A., Annu Rev Biomed Eng 2000, 2, 227- 56; Whitesides, G. Μ., Αηηιι Rev Biomed Eng 2001, 3, 335-73. Poly(dimethylsiloxane) (PDMS) is an elastomer that can be micropatterned with high reproducibility and provides a flexible substrate for cell attachment. Wang, N., Cell Motil Cytoskeleton 2002, 52, (2), 97-106.

Alignment of the Polymer Scaffolds

[00111] The polymer scaffolds of the present disclosure can have an aligned orientation or a random orientation. In an aligned orientation, at least 50% of the fibers comprising the polymer scaffold are oriented along an average axis of alignment.

[00112] In an exemplary embodiment, a polymer scaffold has an alignment which is a member selected from essentially longitudinal, essentially circumferential, and crisscross. A longitudinal alignment is present when the fibers are aligned in the direction of the long axis of the conduit, filled conduit or rod shaped polymer scaffolds. A

circumferential alignment is present when the fibers are aligned along the short axis of the polymer scaffold. A criss-cross alignment is present when the fibers of one polymer scaffold in the composition are aligned in such a manner that the average alignment axis of a first polymer scaffold is at an angle relative to the average alignment axis of a second polymer scaffold which is adjacent to the first polymer scaffold. A longitudinally aligned or circumferentially aligned polymer scaffold can have more than one layer of fibers. A criss-cross aligned polymer scaffold requires more than one layer of fibers.

[00113] In another exemplary embodiment, the polymer fibers can have a standard

deviation from the central axis of the fiber bundle. In an exemplary embodiment, the standard deviation of the fiber is a member selected from between about 0° and about 1°C, between about 0° and about 3°C, between about 0° and about 5°C, between about 0° and about 10°C, between about 0° and about 15°C, between about 0° and about 20°C, and between about 0° and about 30°C.

[00114] The direction in which the aligned polymer scaffold is situated may affect the biological function that the aligned polymer scaffold is replacing or improving. For instance, when an aligned polymer scaffold is situated in a wound, wound healing is more rapid when the aligned polymer scaffold is perpendicular, rather than parallel, to the long axis of the wound. In an exemplary embodiment, the central long axis of the bundle of an aligned polymer scaffold is situated perpendicular to the direction of the material which the aligned polymer scaffold is improving or replacing. In another exemplary embodiment, the central long axis of the bundle of an aligned polymer scaffold is situated parallel to the direction of the material which the aligned polymer scaffold is improving or replacing.

[00115] In an exemplary embodiment, the compositions described herein can comprise more than one polymer scaffold. Each of those polymer scaffolds can have an alignment which is the same or different from the other polymer scaffold or scaffolds in the composition.

[00116] In an exemplary embodiment, the composition comprises two polymer scaffolds.

The first polymer scaffold has the shape of a conduit and is longitudinally aligned. The second polymer scaffold surrounds the exterior of the first polymer scaffold and has an orientation which is a member selected from random, circumferential, criss-cross, and longitudinal.

Additional Shapes of Polymer Scaffolds and Methods of Making

[00117] The polymer scaffolds of the present disclosure can be formed into a variety of shapes, depending on the nature of the problem to be solved.

[00118] For example, in some embodiments, polymer scaffolds of the present disclosure can have a variety of dimensions, thus forming a variety of different shapes, such as a tube or a sheet (e.g., a membrane). In an exemplary embodiment, the polymer scaffold is 0.1 mm to 50 cm long. In another exemplary embodiment, the polymer scaffold is 0.1 mm to 1 mm long. In another exemplary embodiment, the polymer scaffold is 1 mm to 1 cm long. In another exemplary embodiment, the polymer scaffold is 1 cm to 10 cm long. In another exemplary embodiment, the polymer scaffold is 10 cm to 50 cm long. In another exemplary embodiment, the polymer scaffold is 1 cm to 5 cm long. In another exemplary embodiment, the polymer scaffold is 2.5 cm to 15 cm long. In another exemplary embodiment, the polymer scaffold is 5 mm to 6 cm long. In another exemplary embodiment, the polymer scaffold is 8 mm to 3 cm long. In another exemplary embodiment, the polymer scaffold is 10 cm to 25 cm long. In another exemplary embodiment, the polymer scaffold is 0.5 cm to 2 cm long. In another exemplary embodiment, the polymer scaffold is 0.1 cm to 2 cm long.

[00119] In an exemplary embodiment, a polymer scaffold has the shape of a sheet or membrane. Polymer scaffold membranes can be made, e.g., through electrospinning. The individual fibers within the membrane can be aligned either during electrospinning using a rotating drum as a collector or after by mechanical uniaxial stretching.

[00120] In another exemplary embodiment, a polymer scaffold has the shape of a crisscross sheet. To form a criss-cross sheet, layers of aligned polymer sheets or membranes can be arranged in relation to each other, at an angle which is a member selected from greater than 20 degrees but less than 160 degrees, greater than 30 degrees but less than 150 degrees, greater than 40 degrees but less than 140 degrees, greater than 50 degrees but less than 130 degrees, greater than 60 degrees but less than 120 degrees, greater than 70 degrees but less than 110 degrees, and greater than 80 degrees but less than 100 degrees.

[00121] There are a variety of ways to make a criss-cross sheet. In one exemplary

embodiment, a rotating metal drum collector is used that does not contain a nonconducting region. An aligned layer of fibers is created on the drum, which is then peeled off the drum. The aligned layer is rotated 90 degrees and then placed back on the drum. Next an additional layer of electrospun fibers is added while the drum rotates at a high speed. Additional criss-cross layers can be added by repeating these steps. In another exemplary embodiment, a drum is used that has a non-conducting region. Here, the drum is rotated slowly for a first period of time so the fibers deposit and align longitudinally on the non-conducting section. Then the drum is spun fast so the fibers are forced to align circumferentially. Additional crisscross layers can be added by repeating these steps.

[00122] In general, selection of the type of polymer or polymer blend used in each layer of a polymer scaffold, as well as the thickness of each layer, is based on the desired properties of the IVD. For example, in some embodiments, a polymer scaffold comprises a first layer selected to provide desirable mechanical properties, and a second layer selected to provide desirable chemical properties that facilitate attachment of a thrombus-reducing and/or endothelialization moiety to the IVD. When an IVD of the present disclosure is used to replace a specific vascular tissue, e.g., a native artery, a polymer or polymer blend having similar mechanical properties to the native tissue may be selected for use in a first layer. For example, in one embodiment, a PLCG + PCL polymer blend consisting of 10% (w/v) PLCG and 5% (w/v) PCL in HFIP is used to form a first layer approximately 800 nanometers thick. Scaffolds containing this PLCG + PCL polymer blend have an elastic modulus that is similar to that of a native artery. The material used as a second layer of the polymer scaffold may be selected to facilitate attachment of a thrombus-reducing and/or endothealization moiety. In some

embodiments, polymer scaffolds comprise a second layer made from PLLA.

[00123] Electrospinning may be used with different collection devices to form polymer scaffolds having different dimensions and/or geometries. For example, in some embodiments, a rotating stainless steel mandrel having a 1 mm diameter and rotating at 50-800 revolutions per minute is used as a collection device to create a polymer scaffold. In some embodiments, a stainless steel mandrel having a diameter of about 0.5mm, lmm, 1.5mm, 2mm, 2.5mm, 3mm, 3.5mm, 4mm, 4.5mm, 5mm, 5.5mm, 6mm, 6.5mm, 7mm, 7.5mm, 8mm, 8.5mm, 9mm, 9.5mm, up to about 10mm is used to produce polymer scaffolds. In some embodiments, the speed of rotation of a stainless steel mandrel ranges from about 100, 200, 300, 400, 500, 600, 700, 800, 900, up to about 1,000 rpms.

[00124] Other collection devices may be used to create polymer scaffolds having

different diameters, different geometries, or other specified properties.

Attachment of Moieties to Polymer Scaffolds

[00125] Polymer scaffolds of the present disclosure may be modified by attaching one or more moieties to one or more surfaces. Moieties may be attached to a polymer scaffold either directly, e.g., via passive adsorption, or may be attached to a polymer scaffold indirectly, e.g., via a linker molecule.

Attachment via Passive Adsorption

[00126] Moieties may be attached to polymer scaffolds by contacting the polymer

scaffold with a solution containing the moiety to be passively adsorbed for a sufficient period of time to allow the moiety to be adsorbed onto the polymer scaffold. For example, in one embodiment, a polymer scaffold is submerged and gently rotated in a solution of 0.1% (w/v) of bovine submaxillary mucin in 2X phosphate buffered saline (PBS) to facilitate passive adsorption of the mucin onto the polymer scaffold. The amount of mucin adsorbed onto the polymer scaffold can be quantified using, e.g., an alcian blue binding assay. Alcian blue dye bonds by electrostatic forces to negatively charged macromolecules, such as mucin, and therefore facilitates quantification of the amount of mucin adsorbed onto the polymer scaffold.

Attachment via Linker Molecules

[00127] Moieties described herein may be attached to polymer scaffolds by covalently bonding the moiety to the polymer scaffold using a linker molecule. Linker molecules are generally covalently linked to the functional groups present on the microfibers of the polymer scaffold, followed by covalent attachment of a desired moiety to the linker molecule.

[00128] Any of a variety of suitable linker molecules may be used for this purpose, and such linker molecules generally comprise a nucleophilic reactive group that can facilitate attachment of a desired moiety. The term, "nucleophilic reactive group" as used herein refers to a chemical functional group, which comprises a nucleophilic reactive group. A nucleophilic reactive group comprises at least one pair of free electrons that is able to react with an electrophile. Non-limiting examples of nucleophilic moieties include sulfur nucleophiles, such as thiols, thiolate anions, anions of thiolcarboxylate, anions of dithiocarbonates, and anions of dithiocarbamates; oxygen nucleophiles, such as hydroxide anion, alcohols, alkoxide anions, and carboxylate anions; nitrogen

nucleophiles, such as amines, azides, and nitrates; and carbon nucleophiles, such as alkyl metal halides and enols.

[00129] Linker molecules may comprise linear, branched, and/or dendrimer molecules that can be used to attach moieties to the polymer scaffolds in a variety of ways. For example, a suitable linker may include a branched PEG molecule that can be used to attach multiple molecules of the same or different moieties to a polymer scaffold. Such linker molecules may be used to, e.g., increase the density of moieties attached to a polymer scaffold. [00130] In some embodiments, di-amino-PEG is used as a linker molecule, and is covalently attached to the carboxylic groups on microfibers of the polymer scaffold using l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N- hydroxysulfosuccinimide (Sulfo-NHS). The density of reactive functional carboxylic groups on the microfibers may be increased by briefly treating the polymer scaffold with a 0.0 IN solution of NaOH, followed by introduction of a di-amino-PEG linker molecule. Once the di-amino-PEG linker molecule has been attached to the polymer scaffold, a moiety is then covalently attached to the free amines on the di-amino-PEG via carbodiimide chemistry. In some embodiments, the density of carboxylic groups can be increased through the addition of a low molecular-weight polymer prior to microfiber formation.

[00131] In some embodiments, a covalently bound moiety itself may serve as an

additional linker molecule that can be used to facilitate attachment of additional moieties. For example, in one embodiment, heparin is covalently bound to a polymer scaffold via a di-amino-PEG linker, as described above. Subsequently, SDF-Ι is introduced to the polymer scaffold as an additional moiety. Since heparin binds to and stabilizes SDF-Ι , heparin functions as a linker molecule to facilitate attachment of SDF-Ι to the polymer scaffold.

[00132] In some embodiments, heparin is covalently bound to a polymer scaffold via a di-amino-PEG linker, as described above. Subsequently, VEGF is introduced to the polymer scaffold as an additional moiety. Since heparin binds to and stabilizes VEGF, heparin functions as a linker molecule to facilitate attachment of VEGF to the polymer scaffold.

METHODS OF USE

Vascular Applications

[00133] The IVDs of the present disclosure generally find use in replacing, regenerating or improving the biological function of vascular tissues (e.g., blood-contacting tissues) in a subject.

[00134] For example, an IVD of the present disclosure may be used to replace or bypass damaged, severed, or altered blood vessels. In an exemplary embodiment, a tube-shaped IVD is used as a vascular graft in coronary artery bypass surgery. In addition, IVDs of the present disclosure may be used to support and stabilize blood vessel aneurysms, e.g., abdominal aortic aneurysms, by either complete replacement of the vessel with an IVD or by creating a sheath-like encasement around a native vessel. Other reinforcement techniques include, e.g., wrapping one or more IVDs around an aneurysm site. Uses are not limited to lower body vessel replacement, but may include other common sites of aneurysms; for example— the Circle of Willis, involving any of the local arteries, including the internal carotid, posterior communicating, posterior cerebral, etc.

[00135] In an exemplary embodiment, the polymer scaffold which is surrounded by a sleeve is used to replace or regenerate a blood vessel. A sleeve can be made to surround the polymer scaffold to improve its mechanical strength, rigidity, compliance or any other physical or chemical property. This sleeve can be placed around a nanofibrous polymer scaffold conduit, for example, such that the underlying nanofibers will have a particular direction of alignment and the sleeve may have the same or different direction of alignment. Unaligned or randomly aligned micro or nanofibers can also serve as the sleeve material or the underlying nanofiber construct. Multiple sleeves can be used to create a multi-layered construct with different physical or chemical properties.

EXAMPLES

[00136] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pi, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c, subcutaneous(ly); and the like. Example 1: Mucin covalently bonded to microfibers improves the patency of vascular grafts

[00137] Polymer scaffolds were made by dissolving poly(L-lactide) (PLLA) (Lactel

Absorbable Polymers, Pelham, AL, 1.09 dL/g inherent viscosity) in 1,1,1,3,3- hexafluoro-2-propanol (HFIP) via sonication for 30 minutes or until all of the PLLA crystals were completely dissolved, resulting in 19% (w/v) solution. A second polymer blend solution (hereinafter referred to as PLCG+PCL solution) consisting of 10%> (w/v) poly(L-lactide-co-caprolactone-co-glycolide) (PLCG) (Sigma Aldrich, St. Louis, MO) and 5% poly(8-caprolactone) (PCL) (Sigma Aldrich) in HFIP was similarly prepared via sonication. Electrospinning was used to spin the PLLA and PLCG+PCL solutions consecutively onto the same collecting mandrel by applying a voltage of 12 kilo volts (kV) via a high voltage generator (Gamma High Voltage, Ormond Beach, FL) to a spinneret that was aimed at a grounded, rotating stainless steel mandrel (1 mm diameter; 150 revolutions per minute). Electrospinning of the PLLA solution was performed first and continued until the scaffold wall thickness reached approximately 50 μιη.

Electrospinning of the PLCG+PCL solution was started immediately after onto the same collecting mandrel and continued until total scaffold wall thickness reached

approximately 100 μιη. The resulting scaffold was then removed from the mandrel and placed into a vacuum desiccator for 24 hours to remove any residual HFIP. Bulk scaffold and fiber quality and dimensions were inspected using a Hitachi TM-1000 Scanning Electron Microscope (SEM). The bulk scaffold was cut into 7 mm length segments, sterilized in 70% ethanol under germicidal ultra-violet light for 30 minutes, and washed five times with sterile, deionized water.

[00138] Mucin modification of the electrospun scaffolds was accomplished in two ways:

1) passive adsorption by submerging and rotating in 0.1 %> (w/v) solution of bovine submaxillary mucin (BSM, hereinafter referred to as mucin) (Sigma Aldrich) in 2x phosphate buffered saline (PBS); and 2) conjugation using l-ethyl-3-(3

dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N- hydroxysulfosuccinimide (sulfo-NHS) (Pierce Biotechnology, Rockford, IL) with di- amino-poly(ethylene glycol) (PEG) as a linker molecule. In addition to untreated control grafts, a control group of scaffolds was also PEGylated, using EDC and sulfo-NHS to conjugate PEG only to the electrospun fibers. [00139] The presence of mucin on scaffolds was verified via alcian blue binding assay

(Sigma Aldrich). Untreated, PEGylated, mucin-adsorbed (MUC), and mucin-conjugated (PEG-MUC) scaffolds were placed in 0.00015% alcian blue solution in 7% acetic acid for 30 minutes at room temperature. The solution for each sample was then extracted, placed in fresh centrifuge tubes, and centrifuged at 10,000 rpm for 5 minutes. The absorbance of each resulting supernatant was measured at 600 nm using a

spectrophotometer (BioRad, Model 550). The amount of dye bound to each treated scaffold was taken as the difference between the absorbance of their supernatant and that of the untreated scaffold and thus reported as AAbsorbance. A higher AAbsorbance correlated to more mucin on the scaffold.

[00140] PEG-MUC and MUC PLLA microfibrous scaffolds, as well as untreated and

PEGylated controls were immobilized onto one plate of a custom-made, parallel-plate, laminar flow chamber and subjected to 24 dynes/cm shear stress produced by flow of PBS for 48 h. The flow chambers were kept in a humidified incubator at 37°C and supplemented with 5% C0₂ throughout the entire flow duration. After the 48 h flow period, relative mucin amounts were detected using the alcian blue binding assay.

Readings were compared to pre-flow readings, as well as readings taken for static controls at 0 h and 48 h.

[00141] To measure the effect of mucin solution on blood coagulation time,

microhematocrit glass capillaries (BD) were loaded via capillary action with 2.5 μΐ_^ οΐ different concentrations of mucin, di-amino-PEG, and heparin salt (Sigma- Aldrich) solutions in PBS. Immediately prior to sacrifice, blood was collected into each solution- loaded capillary tube from the punctured carotid artery of athymic rats by placing the loaded opening of the tube directly adjacent to the puncture site. Enough blood was drawn into the tube such that the final volume in each capillary was 25 μί, as measured by premade markings on the side of each capillary. The chronometer was started at the time of contact between the blood and capillary tube. Immediately after blood draw, the tubes were rotated slowly to allow the blood to flow within a 45 mm length marked on each tube. Time was stopped and recorded once the blood stopped flowing within the capillary tube.

[00142] Whole rat blood was drawn from the athymic rat heart immediately prior to

sacrifice into collection tubes containing ACD Solution A (BD Biosciences) to prevent coagulation. Untreated, PEGylated, PEG-MUC, and MUC PLLA microfibrous membranes were immersed in equal amounts of blood and kept in 37°C for 2 h. Samples were then analyzed via SEM and immunofluorescence staining using anti-rat CD41 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Positive immunofluorescent antibody staining was tallied for each treatment sample and compared. Platelets on the scaffolds were imaged using a Hitachi TM-1000 Scanning Electron Microscope (SEM).

[00143] Adult female athymic rats (8 weeks old, 200 ± 20 g) were anesthetized with 2.0% isoflurane, placed in the supine position, and implanted with an untreated, PEGylated, PEG-MUC, or MUC vascular graft, sutured end-to-end to the ligated left common carotid artery (CCA). No heparin or other anti-coagulant was used at any time during the animal studies. One month following implantation, the rats were euthanized via C0₂ asphyxiation followed by bilateral thoracotomy, and the vascular grafts were explanted and washed with saline to remove any remaining blood.

[00144] Freshly explanted native rat common carotid arteries, pre-implanted grafts, and freshly explanted vascular grafts were cut so that 2 mm segments could be mounted onto and uniaxially loaded by an Instron mechanical testing machine. The stress-strain curve was obtained for each sample using accompanying Bluehill software. The elastic modulus was calculated from the linear portion of the stress-strain curve acquired for each graft.

[00145] Samples were cryopreserved at -20°C in Optimal Cutting Temperature (OCT) compound (TissueTek, Elkhart, IN) and subsequently cryosectioned at 10 μιη thicknesses in the cross-sectional plane of the grafts. Immunohistochemical staining was performed to analyze the presence of ECs via CD31 antibody (BD Biosciences, San Jose, CA) and smooth muscle cells (SMCs) via smooth muscle-myosin heavy chain (SM-MHC) antibody (Santa Cruz Biotechnology), as well as collagen and elastin (via Verhoeff staining) and proliferation marker, Ki67 (Abeam, Cambridge, MA) in the tissue sections. Hematoxylin and Eosin (H&E) and DAPI nuclear stains were also performed to visualize cell presence within the graft sections. Images of all stains were captured with a Zeiss Axioskop 2 MOT microscope.

[00146] Freshly explanted grafts were cut longitudinally to expose the luminal surface, fixed with 4% paraformaldehyde for lh, and soaked in 30% sucrose in PBS overnight. Samples were then washed with PBS, blocked and permeabilized with 5% normal goat serum containing 0.1% Triton X-100, and stained using anti-rat CD31 antibody (BD Biosciences) and Alexa-Fluor 488 secondary antibody (Invitrogen, Carlsbad, CA).

Images of the stained luminal surface were captured using a Zeiss LSM510 Meta confocal microscope.

[00147] The electrospun polymeric vascular grafts exhibited a structure similar to native matrix, marked by fiber morphology and high porosity (Figure IB and C). The average diameter of the PLLA fibers (Figure IB) and PLCG+PCL fibers (Figure 1C) was approximately 1.5 μιη and 800 nm, respectively. PLLA was chosen for the luminal layer because of its easily customizable and reproducible electrospun products, as well as its well-characterized and highly consistent chemical modification method. PLCG+PCL was utilized because of its mechanical properties. The scaffolds containing PLCG+PCL exhibited greater elastic property than scaffolds made from PLLA alone, which on average exhibited elastic moduli of 3.5 MPa. Since the average elastic modulus of the native CCA was 12.6 ± 2.2 MPa, PLCG+PCL was used as the mechanically-supportive outer layer of the vascular grafts due to its similar mechanical properties. Use of PLCG+PCL resulted in a scaffold that matched the stress-strain behavior of the athymic rat CCA (Figure ID) and average elastic modulus (Figure IE). The average elastic modulus of the bilayered electrospun graft was 12.8 ± 2.5 MPa.

[00148] The conjugation scheme is shown in Figure 2A. Successful mucin modification was verified via alcian blue binding assay, which resulted in a visual color change (Figure 2B), as well as a positive AAbsorbance reading at 600 nm (Figure 2C). Mucin concentrations were increased for both passive adsorption and conjugation schemes until a saturation point was reached based on alcian blue binding assay. Saturation

corresponded to a mucin solution of 0.1% (w/v) for both schemes. According to the alcian blue binding assay, despite using a saturated concentration, more mucin was adsorbed than conjugated to the same type of surface (Figure 2C).

[00149] To determine the stability of immobilized mucin under static and hemodynamic environments, mucin release was examined under both static and flow conditions. After 48 h incubation at 37°C, in the absence or presence of flow, the quantity of mucin adsorbed to the PLLA microfibrous membranes decreased significantly, but some still remained, while mucin conjugated to PLLA remained stably attached (Figure 2C). [00150] To determine whether mucin had anticoagulant activity, the effects of mucin, di- amino-PEG (negative control) and heparin (positive control) on coagulation time were measured. An increasing concentration of mucin in PBS correlated with an increase in rat blood coagulation time, albeit not as strongly as the increase of heparin

concentration. Di-amino-PEG solution showed much lower anticoagulant activity than both mucin and heparin (Figure 3 A). The anticoagulant activity of mucin peaked at 1 mg/mL or 0.1% (w/v), which correlated to the mucin concentration that saturated the surface of scaffolds as previously established via alcian blue binding assay.

[00151] To determine the effect of mucin modification on platelet adhesion, untreated,

PEGylated, PEG-MUC, and MUC membranes were incubated with whole rat blood. Mucin modification significantly decreased the ability of rat platelets to adhere to the membranes (Figures 3B-F). Additionally, PEGylated membranes showed reduced platelet adhesion as expected (Figures 3C, 3F). Statistically, modification with PEG alone, as well as in combination with mucin, showed the best ability to prevent platelet adhesion. Figures 3B through 3E show representative SEM images of platelets on untreated, PEGylated, PEG-MUC, and MUC PLLA microfibrous membranes, respectively. More activated platelets, as seen by their spiky protrusions, were present on and within the untreated membranes.

[00152] The patency for untreated, PEGylated, MUC, and PEG-MUC bilayered grafts was determined at 1 month post-implantation in the carotid artery. This was

accomplished by confirming unobstructed blood flow through the graft into the distally- attached native CCA prior to explantation. After 1 month of implantation in athymic rats, 4 out of 6 (66.7%) untreated grafts were patent, 4 of 6 (66.7%>) PEGylated grafts were patent, 4 of 6 (66.7%) MUC grafts were patent, and 6 out of 6 (100%) PEG-MUC grafts were patent, indicating that covalently immobilized mucin, but not adsorbed mucin, significantly improved the patency of vascular grafts.

[00153] To determine whether the mechanical strength of the modified grafts was

affected in-vivo, mechanical tests were performed on rings of the explanted grafts and the average elastic moduli were compared to each other, as well as to the average elastic modulus of pre -implanted grafts. There was no statistical difference in average elastic modulus between each treatment group 1 month post-implantation. In addition, there was no statistically significant difference between the average elastic modulus of each treatment group before implantation and 1 month post-implantation. The elastic modulus of the grafts before implantation was about 12.8 ± 2.5 MPa. After 1 month, the elastic moduli of implanted grafts were: 11.3 ± 0.7 MPa (untreated), 12.4 ± 0.3 MPa (PEG), 13.1 ± 0.6 MPa (PEG-MUC), and 12.3 ± 0.8 MPa (MUC).

[00154] The patent grafts of the four groups (untreated, PEGylated, PEG-MUC, and

MUC) showed similar histological characteristics. For the patent grafts, the minimal thrombosis and/or intimal hyperplasia were consistent throughout each group after 1 month (Figures 4). The H&E stain indicated that neo-tissues formed around all the vascular grafts (Figure 4A-D). Some matrix deposition occurred within the PLLA layer of PEG-MUC and MUC grafts (Figure 4C and 4D). A clogged graft (untreated) is shown in Figure 4E, indicating that many cells are present in the loose matrix inside the occluded lumen.

[00155] To clearly show cell distribution in the grafts, cell nuclei were stained by DAPI

(Figures 4F-J). Cell infiltration had occurred by 1 month, but to different extents in each experimental group and was limited to the luminal layer. PEG-MUC grafts promoted the most cell infiltration. MUC grafts promoted some cell infiltration, but not as much as PEG-MUC grafts. PEGylated grafts had a small amount of cell infiltration, while untreated grafts showed little to none. The extent of cell infiltration correlated well with the amount of matrix deposition in the luminal layers (Figures 4A-D). Ki67 staining of all tissue sections was negative, demonstrating the cells present within the graft walls at 1 month were not proliferative.

[00156] After one month, continuous endothelialization was observed on the luminal surface of the patent grafts from all four groups (Figures 5A-D). SM-MHC staining showed that SMCs were the major cell type in neo-tissues around the grafts (Figures 5F- I). In contrast, non-patent grafts did not have well-organized ECs and SMCs (Figures 4E and 4J). These grafts lacked continuous endothelialization on the luminal surface (Figure 4E), and SMCs formed a layer on the luminal surface and were the major cell type in the loose matrix inside the occluded lumen (Figure 4J).

[00157] Figure 1. Structure and mechanical property of bilayered electrospun vascular grafts. (A) SEM image of a bilayered electrospun vascular graft. Scale bar = 500 μιη. (B-C) SEM images of (B) PLLA microfibers on the inner surface and (C) PLCG+PCL nanofibers on the outer surface of the graft. Scale bars = 50 μιη. (D) Representative stress-strain curves of a bilayered vascular graft and a native CCA. (E) Average elastic modulus of rat CCA and bilayered vascular grafts (n=6).

[00158] Figure 2. Mucin immobilization and stability. (A) Schematic of mucin

conjugation to a fiber via PEG linker. (B) Mucin was conjugated (or adsorbed; not shown) to fibrous scaffolds and incubated with alcian blue solution, exhibiting blue color. (C) PLLA membranes with conjugated or adsorbed mucin were incubated for 48 h at 37°C under static or flow conditions, and the amount of mucin remained on the membranes was quantified. * p<0.05 based on Holm's t-Test, n=3.

[00159] Figure 3. Effects of mucin on coagulation and platelet adhesion. (A)

Anticoagulant activity of mucin, heparin (positive control) and PEG (negative control) in PBS solution. Coagulation time of whole blood at each concentration of mucin, heparin and PEG was measured (n=3). (B - F) Representative SEM images of platelets adhered to (B) untreated, (C) PEGylated (PEG), (D) mucin-conjugated (PEG-MUC), and (E) mucin-adsorbed (MUC) PLLA scaffolds. Scale bar = 10 μιη. (F) Statistical analysis of surface-modification effects on platelet adhesion. * p<0.05 based on Holm's t-Test, n=3.

[00160] Figure 4. Cross sections of the explanted grafts at 1 month post-implantation.

(A-E) H&E staining. (F-J) DAPI staining. Purple or white dotted lines indicate neo- tissue / PLCG+PCL border. Red dotted lines indicate PLCG+PCL / PLLA border. In all images, right side = luminal side. Both scale bars = 100 μιη.

[00161] Figure 5. EC and SMC organization in the explanted grafts at 1 month post- implantation. (A-E) CD31 staining (dark brown) for ECs. Blue arrows indicate EC presence on the luminal side. (F-J) SM-MHC staining (dark brown) for SMCs. Black arrows exemplified SMC staining. Purple dotted lines indicate neo-tissue / PLCG+PCL border. Red dotted lines indicate PLCG+PCL / PLLA border. In all images, right side = luminal side. Scale bar = 100 μιη.

Example 2: Bioactive vascular grafts engineered to recruit endogenous progenitor cells for in situ regeneration of blood vessels

[00162] Microfiber scaffolds were fabricated by using poly(L-lactic acid) (PLLA)

(MW67,400, Sigma-Aldrich) and polycaprolactone (PCL, MW 2,000, Polysciences). The polymer blends (e.g., 19% PLLA and 5% PCL; w/v) were dissolved in 1,1,1,3,3,3- Hexafluoro-2-propanol (HFIP, Aladdin), and electrospun into grafts as described in Example 1 with minor modifications. The mandrel collector was a rotating stainless steel rod (1.0 mm diameter, rotating speed 300 rpm). The structure of the scaffolds was characterized by using a scanning electron microscope (Hitachi TM-1000).

[00163] Heparin-functionalized microfibers were fabricated by using di-amino-PEG as a linker molecule. The density of reactive carboxylic groups on the microfibers was increased by briefly treating the scaffolds with 0.0 IN NaOH (Sigma- Aldrich). Di- amino-PEG molecules (MW 3400, Sigma) were then covalently attached to the carboxylic groups on the microfibers by using EDC and Sulfo-NHS (Pierce

Biotechnology). Heparin was conjugated to the free amines on the di-amino-PEG molecules via EDC and sulfo-NHS. Following heparin conjugation, SDF-Ι (R&D Systems, Inc.) in PBS (500ng/ml) was incubated with the scaffolds over night at 4°C to allow it to bind to heparin and become immobilized on the scaffolds.

[00164] To visualize the uniformity of SDF 1 a to heparin-functionalized scaffolds,

immuno fluorescent staining was performed by using an SDF- la antibody (R&D

System) and Alexa-Fluor 546 labeled secondary antibody.

[00165] To test the stability and in vitro release of bioactive SDF-Ι from microfibrous scaffolds, the treated grafts were incubated in 0.1 N NaOH for 36 hours to dissolve the SDF-Ια, followed by neutralization with 0.1 HCL. An ELISA kit (R&D System) was used to determine the concentration of SDF- la.

[00166] Male Sprague-Dawley rats (weight, 260 to 280 g) were purchased from the

Charles River animal facility. The rats were anesthetized with 2.0% isoflurane in 70% nitrous oxide and 30%> oxygen. The left common carotid artery was dissected, clamped, and transected, and the graft was sutured end to end with 8 uninterrupted stitches by using a 10-0 needle. No heparin or any other anticoagulant was used at any point before, during, or after the implantation procedure.

[00167] Vascular grafts for in-vitro cell isolation were harvested at 1 week post- surgery, and washed three times with sterile phosphate buffered saline (PBS) supplemented with 1% penicillin/streptomycin (P/S). The grafts were then cut open longitudinally. Sterile cotton tips were used to scrape off any blood or tissue attached to the luminal side. The graft was then cut into mm-size pieces and placed onto the surface coated with 1% CellStart (Invitrogen Corp.) in a 35 mm tissue culture dish. The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen Corp.) with 10% fetal bovine serum (FBS, Thermo Fisher Scientific Inc.). Cell cultures were maintained at 37°C in an incubator with 5% C0₂. The medium was changed every other day. Cells started migrating out of the tissue explants within 2 days. After one week, the tissue explants were dislodged and removed from the culture and the cells were passaged to newly coated dishes and cultured as a monolayer. Some cultures were fixed with 4%

paraformaldehyde for characterization, while others were maintained in 10% FBS medium for one month to study the spontaneous differentiation of these cells.

[00168] Samples for histological examination were snap-frozen in optimal cutting

temperature (OCT) Compound (Tissue Tek,), and sectioned into 10-um thickness using cryostat. Immunohistochemical staining was used to analyze the tissue sections with the following primary antibodies: SM-MHC (Santa Cruz Biotechnology Inc.), CNN1 (Epitomics, Inc), SMA (Epitomics, Inc), CD31 (BD Biosciences), CD34 (Santa Cruz Biotechnology Inc.), CD133 (Abeam Inc.). VerhoefP s Staining was performed by using VerhoefP s Elastic Stain kits (American MasterTech Scientific. Inc).

Immunohistochemistry images were captured with a Zeiss confocal microscope

(LSM710).

[00169] The grafts were explanted and fixed with 4% paraformaldehyde for 30 minutes.

Each graft was cut into 3 slices longitudinally using microscissors. Samples were washed with PBS, blocked with 1% bovine serum albumin, and incubated with mouse anti-rat CD31 antibody and goat anti-rat CD34 (or CD 133) antibody as primary antibodies, and then incubated with Alexa-Fluor 488 and Alexa-Fluor 546 labeled secondary antibodies, followed by confocal microscopy.

[00170] The freshly explanted vascular grafts were cut into 1 -mm- wide ring segments.

The tensile strength in the circumferential direction of these rings was tested by using a custom-built soft tissue tester. Two 0.016-inch-diameter stainless steel rods were inserted into the lumen of the ring segment and fixed on mechanical loading grips. The sample was then placed onto the mechanical tester, and the applied deformation (strain rate was 0.1 mm/sec) and force were recorded. The elastic modulus was calculated based on the applied force, graft deformation, and the dimensions (thickness and width) of the rings. [00171] Scanning electron microscopy (SEM) images showed that the electrospun grafts had a porous structure of microfibers (FIG. 6, Panel A). The average diameter of the fibers was approximately 2 μιη. To achieve similar mechanical properties to native arteries, PLLA/PCL polymer blends with various combinations of polymer ratios were used to produce microfibrous vascular grafts and compared with rat native carotid arteries. The addition of low molecular weight of PCL made the scaffolds more flexible, and increased the conjugation sites (carboxylic groups) on the microfibers. The elastic modulus of the grafts made from 19% PLLA and 5% PCL was approximately 5.2 MPa, the same order of magnitude as the native arteries (9.6 MPa) (FIG. 6, Panel B).

[00172] To suppress thrombogenic events, microfibers were functionalized with heparin by using di-amino-poly(ethylene glycol) (di-NH₂-PEG) as a linker molecule. Di-NH₂- PEG was covalently attached to the carboxylic groups on the microfibers by using 1- ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N- hydroxysulfosuccinimide (Sulfo-NHS) (FIG. 6, Panel C). Heparin was then covalently attached to the free amines on the di-NH₂-PEG molecules via carbodiimide chemistry. Since heparin binds to and stabilizes SDF-Ι , it was used as a molecular linker to sequester recombinant SDF-Ι . Immunostaining for SDF-Ι showed that SDF-Ι was evenly conjugated onto the microfibers (FIG. 6, Panel D). ELISA assay measurements showed that SDF-Ι was stably immobilized on the microfibrous scaffolds and a slow release of SDF-Ι was achieved for more than a week in vitro (FIG. 6, Panel E).

[00173] To evaluate the effects of surface modification on the patency of vascular grafts in vivo, untreated grafts, heparin-treated grafts and heparin-SDF- la-treated grafts with a length of 6 mm and an inner diameter of 1 mm (FIG. 7, Panel A) were implanted into the left common carotid artery of rats by anastomosis (FIG. 7, Panel B) and examined at 1, 2, and 4 weeks, with 9 animals per group at each time point. After surgery, blood flow was observed immediately at both the proximal and distal ends of the grafts. After 4 weeks, visible angiogenesis could be observed in the wall of the graft (FIG. 7, Panel C), suggesting the integration of the vascular grafts with the surrounding tissues. All animals (n=81) survived after the artery replacement procedure.

[00174] Necropsy showed that 67% (6 of 9) of untreated grafts, 89% (8 of 9) of heparin- treated grafts, and 89% of (8 of 9) heparin-SDF- la-treated grafts were patent at 1 week after implantation, suggesting that heparin modification improved short-term patency. Similarly, at 2 weeks post-implantation, 67% (6 of 9) of untreated grafts, 89%> (8 of 9) of heparin-treated grafts, and 100% (9 of 9) heparin- SDF- la-treated grafts were patent. At 4 weeks, the patency decreased slightly for untreated grafts (5 of 9; 56%>) and heparin- treated grafts (7 of 9; 78%), but did not change for heparin-SDF- la-treated grafts (8 of 9; 89%)), suggesting that SDF- la helped maintain the long-term patency of the grafts. For patent grafts, hematoxylin and eosin (H&E) staining of the cross-sections in the middle portion of the grafts showed thrombus formation in untreated grafts but not in the heparin-treated and heparin-SDF- la-treated grafts at 4 weeks (FIG. 7, Panels D-F).

[00175] The relationship between maintenance of long-term patency in heparin-SDF- la- treated grafts and endothelialization on the luminal surface of the grafts was then investigated. At 1 week, cells were mostly observed near the proximal end of the grafts in untreated and heparin-treated grafts (FIG. 8, Panels A-B). In contrast, heparin-SDF- la-treated grafts showed much more cells in the middle portion of the grafts (FIG. 8, Panel C).

[00176] To characterize the cells on the luminal surface of the grafts, en face and cross- section staining was performed to detect EPC and EC markers. Untreated and heparin- treated grafts showed none or few cells positive for CD34 (FIG. 8, Panels D-I; Figs. 12A-B) or CD133 (Fig. 13A-B). Patches of ECs from the adjacent carotid arteries were found at the proximal and distal end of the grafts. There were few cells in the middle portion of the grafts, which were blood cells negative for EPC and EC markers. In contrast, heparin-SDF- la-treated grafts recruited many cells to the luminal surface in the middle portion of the grafts, with most of the cells positive for CD34 (Figs. 8J-L, Fig. 12C) and CD133 (Fig. 13C) at 1 week after implantation. These cells did not express CD31, indicating that they were undifferentiated EPCs. At the proximal and distal ends of the grafts, CD31+ ECs migrated from adjacent carotid arteries, and few EPCs were found in these regions.

[00177] At 2 weeks post-implantation, most areas of luminal surfaces in the grafts were covered by cells, with the best cell coverage in heparin-SDF- la-treated grafts (Fig. 14). Further migration of ECs at the proximal and distal ends of grafts was observed (Figs. 9A-I). In the middle portion of the grafts, patches of ECs without mature cell-cell boundaries were found in untreated and heparin-treated grafts (Figs. 9B, E), while an EC monolayer with well-defined cell-cell boundaries had formed in heparin-SDF- la-treated grafts (Fig. 9H). Few cells were positive for EPC markers in untreated and heparin- treated grafts. In contrast, in heparin-SDF-la treated grafts, more than 70% of cells in the middle portion of the grafts were positive for both CD31 and CD34, suggesting that these ECs were derived from EPCs. The staining of cross sections also showed EC and EPC staining on the luminal surface in the middle portion of the grafts (Figs/ 12D-E; Figs. 13D-E), with better endothelialization in heparin-SDF- la-treated grafts.

[00178] At 4 weeks post-implantation, the luminal surfaces of all grafts were almost completely covered by cells (Fig. 15). Most cells were CD31+, but were negative for CD34 or CD133 (Figs. 9J-R; Figs. 12G-I; Figs. 13G-I), suggesting that EPCs, if any, already differentiated into ECs. In untreated and heparin-treated grafts, cells were not fully confluent; ECs had random, disorganized morphology and the cell-cell boundary was not well defined in many areas, indicating that ECs were still in the process of remodeling and a stable monolayer had not formed. In contrast, ECs on heparin-SDF-l grafts showed continuous EC monolayer with well-organized structure and cell alignment in the direction of blood flow, similar to that in native blood vessels (Figs. 9P- R), indicating the maturation of the endothelium.

[00179] In addition to endothelialization, the remodeling of the vascular wall and thus, the mechanical properties of the grafts are critical for the maturation of blood vessels. Accordingly, the recruitment of SMCs to the grafts and the change of mechanical property of grafts as a function of time were determined. At 1 week, cells positive for smooth muscle a-actin (SMA) were recruited to the outer layer of the grafts (Figs. 10A- C), and heparin-SDF-la treated grafts increased the recruitment of SMA+ cells (Figs. 5A-C) and calponin 1 (C N1)+ cells (Figs. 10D-F). However, these cells were negative for smooth muscle myosin heavy chain (MHC) (Figs. 10D-F), suggesting that these cells were not mature SMCs.

[00180] At 2 weeks, more cells in the outer layer became CNN1+MHC-, with most

C N1+MHC- cells found in heparin-SDF-la treated grafts (Figs. 10G-I). At 4 weeks, CNN1+MHC+ were present in the outer layer (Figs. 10J-L), suggesting the

differentiation of the cells into SMCs. Among all three groups, heparin-SDF-la treated grafts had the most SMCs.

[00181] To directly verify that SMPCs were recruited to the grafts, the grafts after 1-week implantation were used for explant culture. The cells derived from all three groups showed the same characteristics. As exemplified in Figures 11 A-C, the isolated cells were SMA+CNN1-MHC- after 1-week culture. Following 1 -month culture, the cells spontaneously differentiated into SMA+C N 1 +MHC+ cells (Figs. 11D-F), and the differentiated cells had larger spreading area and larger nuclei. However, these SMPCs were different from previously identified SMPC because they were negative for markers such as Sca-1, c-kit, CD34 and CD146.

[00182] To investigate the remodeling and extracellular matrix deposition in the

regenerated blood vessels, immunostaining of collagen-I was performed. Heparin-SDF- l -treated grafts showed more and denser collagen-I deposition surrounding the vascular grafts (Figs. 11G-I). Mechanical tests demonstrated that the elastic modulus of the grafts of all three groups increased significantly over time after implantation. SDF-l significantly increased the elastic modulus at 2 weeks and 4 weeks (Fig. 11 J). At 4 weeks, heparin-SDF- la-treated grafts had higher elastic modulus (8.3 MPa) than that of untreated group (6.9 MPa) and heparin-treated group (7.2 MPa) (Fig. 11 J), indicating that immobilized SDF-la improved the mechanical property of the grafts.

[00183] Figure 12. Immunostaining of cross sections of the explanted grafts for

endothelial cell (EC) marker CD31 and endothelial progenitor cell (EPC) marker CD34. Untreated grafts (control), heparin-treated grafts and heparin-SDF- la-treated grafts were implanted into rat carotid arteries by anastomosis, and explanted at 1 week (A-C), 2 week (D-F) and 4 weeks (G-I) after surgery, followed by immunostaining and confocal microscopy. Arrows in C indicate CD34⁺ cells (in green) on the luminal surface. Scale bar = 40 μιη.

[00184] Figure 13. Immunostaining of cross sections of the explanted grafts for EC

marker CD31 and EPC marker CD 133. Untreated grafts (control), heparin-treated grafts and heparin-SDF- la-treated grafts were implanted into rat carotid arteries by

anastomosis, and explanted at 1 week (A-C), 2 week (D-F) and 4 weeks (G-I) after implantation, followed by immunostaining and confocal microscopy. Arrows in C indicate CD133⁺ cells (in red) on the luminal surface. Scale bar = 40 μιη.

[00185] Figure 14. En face DAPI staining of the explanted grafts at 2 weeks after

implantation. The full lengths of grafts were stained for nuclei with DAPI, followed by confocal microscopy. (A) Untreated grafts (control). (B) Heparin-treated grafts. (C) Heparin-SDF- la-treated grafts. [00186] Figure 15. En face DAPI staining of the explanted grafts at 4 weeks after implantation. The full lengths of grafts were stained for nuclei with DAPI, followed by confocal microscopy. (A) Untreated grafts (control). (B) Heparin-treated grafts. (C) Heparin-SDF- la-treated grafts.

[00187] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

CLAIMS What is claimed is:

1. An implantable vascular device (IVD) comprising:

a) a polymeric scaffold; and

b) one or more of:

i) a thrombus-reducing moiety;

ii) an endothelialization and vascular wall remodeling moiety;

iii) a moiety that promotes recruitment of progenitor cells and/or stem cells, wherein the moiety is attached to the polymeric scaffold.

2. The IVD of claim 1, wherein said polymeric scaffold comprises a hollow, tubular structure.

3. The IVD of claim 1, wherein said polymeric scaffold comprises a single layer, or comprises two or more layers, each layer comprising a different polymer or polymer blend.

4. The IVD of claim 1, wherein said thrombus-reducing moiety is mucin.

5. The IVD of claim 1, wherein said thrombus-reducing moiety is heparin, heparan sulfate, heparan sulfate proteoglycan, or combinations thereof.

6. The IVD of claim 1, wherein said thrombus-reducing moiety is hirudin, a hirudin analog, bivalirudin, lepirudin, desirudin, or combinations thereof.

7. The IVD of claim 1, wherein said thrombus-reducing moiety is an aptamer.

8. The IVD of claim 7, wherein said aptamer comprises the nucleotide sequence GGTTGGTGTGGTTGG (SEQ ID NO:9).

9. The IVD of claim 1, wherein said endothelialization and vascular wall remodeling moiety is SDF-1, or an isoform thereof.

10. The IVD of claim 1, wherein said endothelialization and vascular wall remodeling moiety is VEGF, or an isoform thereof.

11. The IVD of claim 1 , wherein said moiety that promotes recruitment of progenitor and/or stem cells is SDF-1, or an isoform thereof.

12. The IVD of claim 1, wherein said polymer scaffold comprises PLLA.

13. The IVD of claim 1, wherein said polymer scaffold comprises PLCG.

14. The IVD of claim 1, wherein said polymer scaffold comprises PCL.

15. The IVD of claim 1, wherein said polymer scaffold comprises a polymer blend comprising PLCG and PCL.

16. The IVD of claim 1, wherein said polymer scaffold comprises PLLA and PCL.

17. The IVD of claim 1, wherein said polymer scaffold comprises PLLA and a polymer blend comprising PLCG and PCL.

18. The IVD of claim 1, wherein said polymer scaffold comprises polyurethane.