WO2019232177A1

WO2019232177A1 - PROMOTING ENDOTHELIAL CELL AFFINITY AND ANTITHROMBOGENICITY OF POLYTETRAFLUOROETHYLENE (ptfe) BY MUSSEL-INSPIRED MODIFICATION AND RGD/HEPARIN GRAFTING

Info

Publication number: WO2019232177A1
Application number: PCT/US2019/034600
Authority: WO
Inventors: Lih-Sheng Turng; Hao-yang MI
Original assignee: Wisconsin Alumni Research Foundation
Priority date: 2018-05-30
Filing date: 2019-05-30
Publication date: 2019-12-05

Abstract

Disclosed herein are methods for modifying a substrate having a hydrophobic surface. Also disclosed are modified hydrophobic substrates. Additionally disclosed herein are novel wavy, multi-component vascular grafts (WMVGs) with a wavy inner layer of rigid biopolymer fibers and an outer layer of elastic biopolymer fibers and a method for preparing WMVGs via electrospinning using a special assembled collector. The modified hydrophobic substrates and methods disclosed herein advantageously improve cell affinity and antithrombogenicity of hydrophobic surfaces such as the inner surfaces of the WMVGs.

Description

PROMOTING ENDOTHELIAL CELL ALLINITY AND ANTITHROMBOGENICITY OL POLYTETRALLUOROETHYLENE (ptfe) BY MUSSEL-INSPIRED MODIFICATION AND

RGD/HEPARIN GRAFTING

STATEMENT OF GOVERNMENT SUPPORT

[0001] This invention was made with government support under HL 134655 awarded by the National Institutes of Health. The government has certain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62/677,939 filed on May 30, 2018 and U.S. Provisional Application No. 62/677,931 filed on May 30, 2018, each of which is hereby incorporated by reference in its entirety.

INCORPORATION OF SEQUENCE LISTING

[0003] A paper copy of the Sequence Listing and a computer readable form of the Sequence Listing containing the file named“Pl80249WO0l_ST25.txt”, which is 1,008 bytes in size (as measured in MICROSOFT WINDOWS® EXPLORER), are provided herein and are herein incorporated by reference. This Sequence Listing consists of SEQ ID NOs:l-4.

BACKGROUND OF THE DISCLOSURE

[0004] The present disclosure relates generally to tissue engineering. In particular, the present disclosure relates to methods for modifying hydrophobic materials and to modified hydrophobic substrates. The modified hydrophobic substrates and methods disclosed herein advantageously improve cell affinity and antithrombogenicity of hydrophobic surfaces.

[0005] Prosthetic vascular grafts, namely polyethylene terephthalate (PET, Dacron) and expanded polytetrafluoroethylene (ePTFE), have been successfully utilized as large-diameter vessel replacements owing to their high mechanical strength, flexibility, biocompatibility, and commercial availability. The massive blood flow in large-diameter blood vessels aids in the prevention of blood clots. However, the long-term patency of prosthetic vascular grafts is discouraging in small diameter vascular grafts (SDVGs) (< 6 mm) due to the high risk of luminal thrombosis that is caused by a lack of endothelial cells and anastomotic intimal hyperplasia. The primary physiological function of endothelial cells is to facilitate blood flow by providing a suitable hemocompatible and antithrombogenic surface. Thus, mimicking the native physiological structure and properties of blood vessels by vascular tissue engineering strategies have been proposed and have become an important topic in biomedical engineering.

[0006] Various biodegradable synthetic materials, such as poly (lactic acid) (PLA), poly (lactic-co-glycolic acid) (PLGA), poly (e-caprolactone) (PCL), and polyurethane (PU), have been employed to fabricate SDVGs. Although these materials have been found to be biocompatible with endothelial cells, they suffer from a slow endothelialization rate and a high risk of thrombosis. Improving the endothelial cell affinity and antithrombogenicity of synthetic SDVGs present challenges for vascular tissue engineering. The major reason for inferior endothelial cell affinity is the lack of bioactive binding sites on these hydrophobic materials. For all of these reasons, SDVGs and methods for preparing SDVGs remain elusive. Accordingly, there exists the need for developing cost-effective SDVGs that can fully mimic the properties of blood vessels, prevent thrombosis, and do not require long maturation times.

[0007] Alternatively and/or in addition, surface modification may be desirable for promoting the bioactivity of synthetic materials since surface modification has the unique advantage of altering the surface chemistry without interfering with the material’s bulk properties. Hydrophobic surfaces are typically difficult to modify, especially in an aqueous environment, due to the lack of hydrophilic functional groups.

[0008] Plasma treatment is a practical physical modification approach for altering a material’s surface energy. Earlier studies demonstrated the positive effect of plasma treatment on improving PTFE biocompatibility. For example, ammonia-plasma-treated PET and PTFE showed the enhanced adhesion and growth of endothelial cells and the slightly upregulated expression of adhesion molecules. Amide- and amine-plasma-treated PTFE showed an enhanced endothelial cell lining and stimulated the formation of an endothelial cell monolayer. However, the functional groups introduced via plasma treatment are limited and the introduced hydrophilic groups are not stable long-term.

[0009] Improving the antithrombogenicity is highly desirable. Introducing an endothelial cell layer provides a solution for the prevention of thrombosis in vascular tissue engineering, but the risk is still present if the surface is not fully covered by an endothelial cell layer. Thus, rapid endothelialization is desirable. The incorporation of heparin is another effective way to improve antithrombogenicity due to its anticoagulation properties. Various heparin-modified materials, such as chitosan/graphene oxide hydrogels, collagen-coated PTFEs, porous PLA membranes, and decellularized matrices, show reduced platelet adhesion. Heparin molecules may gradually release into the blood flow and cause low sustainability in long-term implantation applications. For this reason, fast endothelialization may remedy the gradually decreasing heparin level.

[0010] Arginine-glycine-aspartic acid (RGD), a tri-amino acid sequence, is the most common peptide motif responsible for cell adhesion to the extracellular matrix (ECM) and has been used extensively to enhance cell attachment on biomaterials. Since RGD is readily dissolved in water, it has to be chemically grafted onto a substrate. But, grafting of RGD onto hydrophobic surfaces is fairly difficult. A practical solution is to combine a hydrophobic polymer with a hydrophilic material like alginate or collagen prior to RGD grafting. However, this approach deteriorates the mechanical property advantages of synthetic polymers and increases fabrication cost.

[0011] In view of the foregoing, a need exists for developing cost-effective synthetic biomaterials, and in particular, SDVGs, that can fully mimic the properties of blood vessels, prevent thrombosis, and do not require long maturation times. Additionally and/or alternatively, methods for enhancing endothelial cell affinity and antithrombogenicity of synthetic biomaterials that contain hydrophobic surfaces used in vascular grafts are needed.

BRIEF DESCRIPTION OF THE DISCLOSURE

[0012] Disclosed herein are methods for modifying hydrophobic surfaces of synthetic materials. Also disclosed are modified hydrophobic substrates. The method allows for the attachment of biomolecules on hydrophobic surfaces, which can promote cell affinity and reduce thrombogenicity of synthetic biomaterials used in vascular grafts.

[0013] In particular embodiments, the methods can be used to enhance the biocompatibility of vascular grafts. For example, the present disclosure is further directed to new, wavy, multi-component vascular grafts (WMVGs) composed of hybrid biomaterials with different mechanical properties that resemble the structure and properties of native blood vessels are disclosed. Particularly, the methods of modifying hydrophobic surfaces can be used for surface modification of the WMVGs to enhance the biocompatibility of the inner surface of the wavy- structured rigid biopolymer fibers. Biomolecules can be coated on the wavy-structured rigid biopolymer fibers in an aqueous solution through simple grafting methods based on mussel- inspired chemistry. The modified wavy- structured rigid biopolymer fibers advantageously have a significantly increased cell proliferation and migration rate, as well as increased cell- substrate interactions. The addition of biomolecules also contributes to the dramatically enhanced antithrombogenicity. Along with the enhanced endothelialization on their inner surfaces, the biomimetic WMVGs fabricated provide candidates for CVD treatment

[0014] The WMVG are fabricated by an electrospinning method disclosed in the present disclosure using a custom-designed rotating collector. The resulting WMVG has an inner layer including wavy- structured rigid biopolymer fibers that resemble the properties of collagen in blood vessels and an outer layer including elastic biopolymer fibers that mimic the elastin in blood vessels. During preparation of the WMVG, a first fiber layer of a water soluble polymer is employed as a sacrificial fiber layer that is leached out for easy removal of the resultant electrospun tubular grafts having the rigid biopolymer fiber inner layer and the elastic biopolymer fiber outer layer from the rotating collector. Advantageously, because of the wavy fibrous structure of the inner layer and the material combination, the WMVGs of the present disclosure exhibit the unique non-linear tensile stress-strain behavior of human arteries. The WMVGs of the present disclosure also advantageously prevent thrombosis and can be covered and/or replaced by regenerated tissue based on the biodegradability of the materials used.

[0015] In one aspect, the present disclosure is directed to a method for modifying a hydrophobic surface, the method comprising: treating the hydrophobic surface with oxygen plasma to form an oxygen plasma-treated surface; coating the oxygen plasma-treated surface with a solution comprising dopamine to form a dopamine-coated surface; coating the dopamine- coated surface with a solution comprising polymer comprising a terminal amine to form a polymer coating on the dopamine-coated surface; and immobilizing a bioactive molecule on the polymer coating by contacting the bioactive molecule with the polymer coating.

[0016] In one aspect, the present disclosure is directed to a method for modifying a substrate comprising a hydrophobic surface, the method comprising: treating the hydrophobic surface with oxygen plasma to form an oxygen plasma-treated surface; coating the oxygen plasma-treated surface with a solution comprising dopamine to form a dopamine-coated surface; coating the dopamine-coated surface with a solution comprising a polymer comprising a terminal amine to form a polymer coating on the dopamine-coated surface; and immobilizing a bioactive molecule on the polymer coating by contacting the bioactive molecule with the polymer coating. [0017] In one aspect, the present disclosure is directed to a modified hydrophobic substrate comprising a substrate comprising a hydrophobic surface, a first layer comprising dopamine disposed on the substrate, and a second layer comprising a free amine disposed on the first layer.

[0018] In one further aspect, the present disclosure is directed to a wavy multi- component vascular graft comprising an inner layer comprising rigid biopolymer fibers and an outer layer comprising elastic biopolymer fibers (e.g., thermoplastic polyurethane (TPU) fibers).

[0019] In one aspect, the present disclosure is directed to a method for preparing a wavy multi-component vascular graft. The method includes: electro spinning a first solution comprising a water soluble polymer material to form a first water soluble fiber; collecting the first water soluble fiber on an assembled mandrel that comprises a central tube and a plurality of satellite cylinders surrounding the central tube to form a first water soluble fiber layer; electrospinning a second solution comprising a rigid biopolymer material to form a second fiber; collecting the second fiber on the First water soluble fiber layer to form a rigid biopolymer fiber layer; electrospinning a third solution comprising an elastic polymer material to form a third fiber; collecting the third fibers on the rigid biopolymer fiber layer on the assembled mandrel to form an outer elastic polymer fiber layer; and removing the assembled mandrel to form a wavy multi-component vascular graft.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The disclosure will be better understood, and features, aspects and advantages other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such detailed description makes reference to the following drawings, wherein:

[0021] FIG. 1 is a schematic depicting the surface modification procedure using PTFE. PTFE was first treated with 0₂ plasma to obtain P-PTFE, dopamine (DA) was polymerized on P-PTFE to obtain DA-PTFE, then PEI was immobilized on DA-PTFE, followed by the grafting of RGD or RGD/heparin to obtain RGD-PTFE or R/H-PTFE, respectively.

[0022] FIG. 2 depicts a digital photo of a dopamine-coated PTFE (DA-PTFE) sheet. The left part was protected with tape during the O2 plasma treatment. [0023] FIG. 3 depicts FTIR spectra of PTFE, P-PTFE, DA-PTFE, RGD-PTFE, and R/H-PTFE. Two regions are enlarged for better comparison.

[0024] FIG. 4 depicts XPS survey scans of PTFE, P-PTFE, DA-PTFE, RGD-PTFE, and R/H-PTFE.

[0025] FIG. 5 depicts Gauss-fitted Cls high-resolution scans of PTFE, P-PTFE, DA- PTFE, RGD-PTFE, and R/H-PTFE showing the composition of different carbon bonds. The insets show the chemical structure of polydopamine, RGD, and heparin.

[0026] FIG. 6 depicts SEM images of the surface morphologies of PTFE, P-PTFE, DA-PTFE, RGD-PTFE, and R/H-PTFE samples.

[0027] FIG. 7 depicts three-dimensional AFM images of PTFE, P-PTFE, DA-PTFE, RGD-PTFE, and R/H-PTFE across a 5 pm x 5 pm area. The height differences are marked on the images.

[0028] FIG. 8 depicts cross-sectional AFM images corresponding height profiles from the lines drawn on each image from FIG. 7.

[0029] FIGS. 9A-9C depict SEM images of platelets attached to PTFE, P-PTFE, DA- PTFE, RGD-PTFE, and R/H-PTFE (FIG. 9A), statistical results of the platelet adhesion test (FIG. 9B), and water contact angle results of different PTFE samples (FIG. 9C).

[0030] FIG. 10 depicts HUVEC attachment results after cell seeding for 4 hours. Cell nuclei were stained with DAPI. The lower right diagram shows the statistical results of the number of cells attached to the different substrates.

[0031] FIGS. 11A-11C depict fluorescent images of HUVECs cultured on different PTFE substrates for 7 days (FIG. 11 A), statistical results of cell viability from live/dead assay (FIG. 11B), and statistical results of cell proliferation from MTS assay at day 7 and day 14 time points (FIG. 11C).

[0032] FIG. 12 depicts fluorescent images of HUVECs cultured on different PTFE substrates for 14 days. Bar = 200 pm.

[0033] FIGS. 13A-13C depict fluorescent images showing the cytoskeleton of HUVECs cultured on different PTFE substrates for 7 days (FIG. 13A), measurement results of the average projected area per cell (FIG. 13B), and the average aspect ratio per cell (FIG. 13C).

[0034] FIG. 14 depicts fluorescence images showing the cytoskeleton of HUVECs cultured on different PTFE substrates for 14 days.

[0035] FIG. 15 depicts SEM images of HUVECs cultured on different PTFE substrates for 7 days showing the interaction between cells and substrate.

[0036] FIG. 16 is a schematic depicting the fabrication procedure for wavy multi- component vascular grafts (WMVGs) using a custom-designed rotating collector.

[0037] FIGS. 17A-17J are electron micrographs depicting the microstructure of WMVGs. FIG. 17A depicts a low magnification cross-sectional image of a WMVG removed from the mandrel before poly(ethylene oxide) (PEO) leaching. FIG. 17B depicts a high magnification cross-sectional image of a WMVG removed from the mandrel before PEO leaching. FIG. 17C depicts a low magnification cross-sectional image of a WMVG removed from the mandrel after PEO leaching. FIG. 17D depicts a high magnification cross-sectional image of a WMVG removed from the mandrel after PEO leaching. FIGS. 17E and 17F depict cross-sectional images of the silk/poly(lactic acid) (PLA) inner fiber layer (FIG. 17E) and the TPU outer fiber layer (FIG. 17F). FIGS. 17G and 17H depict the structure of the outer surface (FIG. 17G) and the inner surface (FIG. 17H) of a WMVG. FIGS. 171 and 171 depict enlarged images of the flat region (FIG. 171) and wavy region (FIG. 171) of the inner surface of a WMVG.

[0038] FIGS. 18A-18G depict the mechanical properties of WMVGs. FIG. 18A depicts representative tensile test curves for S/P:T = 1 :2 (S/P: silk/PLA, T: TPU, 1:2 solution blend ratio), S/P:T = 1:1, and S/P:T=2:1 vascular grafts. FIG. 18B depicts suture retention results for S/P:T = 1:2, S/P:T = 1 :1, and S/P:T=2:1 vascular grafts. FIG. 18C depicts burst pressure results for S/P:T = 1:2, S/P:T = 1:1, and S/P:T=2:1 vascular grafts. FIG. 18D depicts cyclic tensile test results of S/P:T = 1:2. FIG. 18E depicts cyclic tensile test results of S/P:T = 1 :1. FIG. 18F depicts cyclic tensile test results of S/P:T=2:1 vascular grafts. FIG. 18G is a schematic illustration depicting the WMVG’s cyclic circumferential expansion behavior mimicking native blood vessels. [0039] FIGS. 19A-19I depict scanning electron micrograph (SEM) images and X-ray photoelectron spectrometer (XPS) survey scans of silk/PLA fiber mat (S/P) (FIGS. 19A-19C), silk/PLA-dopamine (S/P-DA) fiber mat (FIGS. 19D-19F) and silk/PLA-dopamine & heparin (S/P-D&H) fiber mat (FIGS. 19G-19I). FIG. 19A depicts a low resolution SEM image of S/P fiber mat; FIG. 19B depicts a high resolution SEM image of S/P fiber mat; FIG. 19C depicts XPS survey scans of S/P fiber mat; FIG. 19D depicts a low resolution SEM of S/P-DA fiber mat; FIG. 19E depicts a high resolution SEM of S/P-DA fiber mat; FIG. 19F depicts XPS survey scans of S/P-DA fiber mat; FIG. 19G depicts a low resolution SEM of S/P-D&H fiber mat; FIG. 19H depicts a high resolution SEM of S/P-D&H fiber mat; FIG. 41 depicts XPS survey scans of S/P-D&H fiber mat.

[0040] FIGS. 20A-20E depict wettability and platelet adhesion of fiber surfaces. FIG. 20A depicts water contact angle results of different S/P fiber mats. FIG. 20B depicts platelet attachment test results. FIG. 20C depicts representative SEM image of S/P fiber mats; FIG. 20D depicts representative SEM image of S/P-DA fiber mats, and FIG. 20E depicts representative SEM image of S/P-D&H fiber mats.

[0041] FIGS. 21A-21C depict culture of cells on S/P fiber mats. FIG. 21A depicts fluorescence images from the live/dead assay of human umbilical vein endothelial cell (HUVECs) cultured on differently modified S/P substrates. FIG. 21B depicts cell proliferation statistical results from the MTS assay. FIG. 21C depicts cell viability statistical results.

[0042] FIGS. 22A-22B depict cytoskeletal morphology (FIG. 22A) and cellular morphology (FIG. 22B) of HUVECs cultured on differently modified silk/PLA.

[0043] FIG. 23 depicts confocal fluorescence images of HUVECs cultured on dopamine- and heparin-modified WMVGs for 7 and 14 days. Cells were stained with calcein- AM and EthD- 1.

[0044] FIGS. 24 A and 24B depict fiber diameter distribution from the silk/PLA inner fiber layer (FIG. 24 A) and the TPU outer fiber layer (FIG. 24B).

[0045] FIGS. 25A and 25B depict measurement results of the lumen diameter (FIG. 25 A) and the wall thickness of fabricated WDVGs compared to a medium-size human artery and human vein. [0046] FIGS. 26A and 26B depict statistical data of cytoskeleton morphology images. FIG. 26A shows the average projected cell area. FIG. 26B shows the average cell aspect ratio.

DETAILED DESCRIPTION

[0047] 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 the disclosure belongs. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described below.

Methods of promoting endothelial cell affinity and antithrombogenicity

[0048] The present disclosure is directed generally to methods for modifying a hydrophobic surface to improve cell affinity and antithrombogenicity of the surfaces. The method includes: treating a hydrophobic surface with oxygen plasma to form an oxygen plasma- treated surface; coating the oxygen plasma-treated surface with a solution comprising dopamine to form a dopamine-coated surface; coating the dopamine-coated surface with a solution comprising polymer comprising a terminal amine to form a polymer coating on the dopamine- coated surface; and immobilizing a bioactive molecule on the polymer coating by contacting the bioactive molecule with the polymer coating.

[0049] Any suitable method for treating the hydrophobic surface with oxygen plasma can be used. Commercially available plasma etchers (e.g., PlasmaEtch PE-200) can be used to oxygen plasma treat the hydrophobic surfaces.

[0050] The hydrophobic surfaces include polytetrafluoroethylene (PTFE), poly (lactic acid) (PLA), poly (lactic-co-glycolic acid) (PLGA), poly (e-caprolactone) (PCL), polyurethane (PU), polypropylene carbonate (PPC), polyhydroxybutyrate (PHB), and the like, and combinations thereof.

[0051] The dopamine coating can be prepared by contacting the oxygen plasma- treated surface with a solution comprising dopamine to form the dopamine-coated surface. For example, the oxygen plasma-treated surface can be immersed into a dopamine solution for a sufficient period of time to form the dopamine coating. The concentration of dopamine in the dopamine solution can range from about 0.5 mg/mL to about 5 mg/mL. [0052] The method then includes coating the dopamine-coated surface with a solution comprising a polymer having a terminal amine to form a polymer coating on the dopamine-coated surface. Suitable polymers having a terminal amine include, for example, polyethylenimine (PEI), polyethyleneglycol (PEG)-amine, polyalkylene oxide (PAO)-amine, polyallylamine, polyvinylamine, poly(vinylamine-co-vinylformamide), chitosan-amine, and poly(amidoamine). The polyethyleneimine suitably can be linear, dendritic, comb, or branched. To coat the dopamine-coated surface with the polymer, the dopamine-coated surface can be immersed in a solution containing the polymer for a suitable period of time such that a polymer film forms. The polymer is present in the solution in a range of from about 0.1 mg/mL to about 1 mg/mL. The polymer coating introduces amino groups onto the dopamine-coated surface. The thickness of the polymer coating can range from molecular scale to tens of nanometers.

[0053] The method then includes immobilizing a bioactive molecule on the polymer coating. The bioactive molecule is immobilized by contacting the bioactive molecule with the polymer coating. A particularly suitable method is by (l-ethyl-3-(3-dimethylaminopropyl)- carbodiimide) (EDC)/N-hydroxysuccinimide (NHS) coupling chemistry.

[0054] Any bioactive molecule can be immobilized. Suitable bioactive molecules include any biomolecule having carboxyl groups and being water soluble. A suitable bioactive molecule includes a cell adhesion molecule. Suitable cell adhesion molecules include fibronectin, arginine- glycine- aspartic acid (RGD) peptide, arginine-glycine-aspartic acid-serine (RGDS) peptide (SEQ ID NO:l), leucine-aspartic acid-valine (LDV) peptide, fibronectin CS1 region, laminin, tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide (SEQ ID NO:2), proline- aspartic acid-serine-glycine-arginine (PDSGR) peptide (SEQ ID NOG), lysine-arginine- glutamic acid (LRE) peptide, vitronectin, arginine-glycine-aspartic acid-valine (RGDV) peptide (SEQ ID NO:4), and combinations thereof. Other suitable bioactive molecules include anticoagulants. Suitable anticoagulants include heparin, low molecular weight heparin, a coumarin, a directly acting oral anticoagulants (DOACs), fondaparinux, idraparinux, a factor Xa inhibitor, a thrombin inhibitor, hementin, and combinations thereof. Suitable coumarins include warfarin, acenocoumarol, phenprocoumon, atromentin, and phenindione. Suitable directly acting oral anticoagulants (DOACs) include dabigatran, rivaroxaban, apixaban, edoxaban and betrixaban. Suitable factor Xa inhibitors include rivaroxaban, apixaban, edoxaban, betrixaban, darexaban, letaxaban, and eribaxaban. Suitable thrombin inhibitors include hirudin, lepirudin, bivalirudin, argatroban, dabigatran, ximelagatran and combinations thereof. [0055] In one embodiment, at least two bioactive molecules can be immobilized on the polymer coating. Suitably, one of the at least two bioactive molecules is a cell adhesion molecule and the other of the at least two bioactive molecules is an anticoagulant. Suitably, one of the at least two bioactive molecules is a RGD peptide and the other of the at least two bioactive molecules is heparin.

[0056] The method can further include seeding a cell on the modified substrate. Suitable cells include endothelial cells, smooth muscle cells, mesenchymal stem cells, umbilical vein endothelial cells, fibroblast cells, and combinations thereof. The seeded cells can then be cultured for a sufficient period of time for cells to migrate, proliferate and differentiate.

[0057] In one embodiment, the hydrophobic surface is a hydrophobic surface of a vascular graft. Suitable vascular grafts include large diameter vascular grafts, small diameter vascular grafts, and combinations thereof. As used herein,“small-diameter vascular graft” refers to an artificial vascular graft that is made of biocompatible materials and having a lumen diameter less than 6 mm. As used herein,“large- diameter vascular graft” refers to an artificial vascular graft that is made of biocompatible materials and having a lumen diameter greater than 6 mm. In one particular embodiment, the hydrophobic surface is the inner surface of wavy multi- component vascular graft (WMVG) as described below.

[0058] In another aspect, the present disclosure is directed to a method for modifying a substrate comprising a hydrophobic surface, the method comprising: treating the hydrophobic surface with oxygen plasma to form an oxygen plasma-treated surface; coating the oxygen plasma-treated surface with a solution comprising dopamine to form a dopamine-coated surface; coating the dopamine-coated surface with a solution comprising a polymer comprising a terminal amine to form a polymer coating on the dopamine-coated surface; and immobilizing a bioactive molecule on the polymer coating by contacting the bioactive molecule with the polymer coating.

[0059] Suitable substrates include glasses, metals, woods, cotton, plastics, ceramics, and combinations thereof.

[0060] Any suitable method for treating the hydrophobic surface with oxygen plasma can be used. Commercially available plasma etchers (e.g., PlasmaEtch PE-200) can be used to oxygen plasma treat the hydrophobic surfaces. [0061] The hydrophobic surfaces include polytetrafluoroethylene (PTFE), poly (lactic acid) (PLA), poly (lactic-co-glycolic acid) (PLGA), poly (e-caprolactone) (PCL), polyurethane (PU), polypropylene carbonate (PPC), polyhydroxybutyrate (PHB) and combinations thereof.

[0062] The dopamine coating can be prepared by contacting the oxygen plasma- treated surface with a solution comprising dopamine to form the dopamine-coated surface. For example, the oxygen plasma-treated surface can be immersed into a dopamine solution for a sufficient period of time to form the dopamine coating. The concentration of dopamine in the dopamine solution can range from about 0.5 mg/mL to about 5 mg/mL.

[0063] The method then includes coating the dopamine-coated surface with a solution comprising a polymer having a terminal amine to form a polymer coating on the dopamine-coated surface. Suitable polymers having a terminal amine include, for example, polyethylenimine (PEI), polyethyleneglycol (PEG)-amine, polyalkylene oxide (PAO)-amine, polyallylamine, polyvinylamine, poly(vinylamine-co-vinylformamide), chitosan-amine, and poly(amidoamine). The polyethyleneimine suitably can be linear, dendritic, comb, or branched. To coat the dopamine-coated surface with the polymer, the dopamine-coated surface can be immersed in a solution containing the polymer for a suitable period of time such that a the polymer film forms. The polymer in the solution can range from about 0.1 mg/mL to about 1 mg/mL. The polymer coating introduces amino groups onto the dopamine-coated surface. The thickness of the polymer coating can range from molecular scale to tens of nanometers.

[0064] The method then includes immobilizing a bioactive molecule on the polymer coating. The bioactive molecule is immobilized by contacting the bioactive molecule with the polymer coating. A particularly suitable method is by (l-ethyl-3-(3-dimethylaminopropyl)- carbodiimide) (EDC)/N-hydroxysuccinimide (NHS) coupling chemistry.

[0065] Any bioactive molecule can be immobilized. Suitable bioactive molecules include any biomolecule having carboxyl groups and being water soluble. A suitable bioactive molecule includes a cell adhesion molecule. Suitable cell adhesion molecules include fibronectin, arginine- glycine- aspartic acid (RGD) peptide, arginine-glycine-aspartic acid-serine (RGDS) peptide (SEQ ID NO:l), leucine-aspartic acid-valine (LDV) peptide, fibronectin CS1 region, laminin, tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide (SEQ ID NO:2), proline- aspartic acid-serine-glycine-arginine (PDSGR) peptide (SEQ ID NOG), lysine-arginine- glutamic acid (LRE) peptide, vitronectin, arginine-glycine-aspartic acid-valine (RGDV) peptide (SEQ ID NO:4), and combinations thereof. Other suitable bioactive molecules include anticoagulants. Suitable anticoagulants include heparin, low molecular weight heparin, a coumarin, a directly acting oral anticoagulants (DOACs), fondaparinux, idraparinux, a factor Xa inhibitor, a thrombin inhibitor, hementin, and combinations thereof. Suitable coumarins include warfarin, acenocoumarol, phenprocoumon, atromentin, and phenindione. Suitable directly acting oral anticoagulants (DOACs) include dabigatran, rivaroxaban, apixaban, edoxaban and betrixaban. Suitable factor Xa inhibitors include rivaroxaban, apixaban, edoxaban, betrixaban, darexaban, letaxaban, and eribaxaban. Suitable thrombin inhibitors include hirudin, lepirudin, bivalirudin, argatroban, dabigatran, ximelagatran and combinations thereof.

[0066] In one embodiment at least two bioactive molecules can be immobilized on the PEI coating. Suitably, one of the at least two bioactive molecules is a cell adhesion molecule and the other of the at least two bioactive molecules is an anticoagulated. Suitably, one of the at least two bioactive molecules is a RGD peptide and the other of the at least two bioactive molecules is heparin.

[0067] The method can further include seeding a cell on the modified substrate. Suitable cells include endothelial cells, smooth muscle cells, mesenchymal stem cells, umbilical vein endothelial cells, fibroblast cells, and combinations thereof. The seeded cells can then be cultured for a sufficient period of time for cells to migrate, proliferate and differentiate.

[0068] In one further aspect, the present disclosure is directed to a modified hydrophobic substrate comprising a substrate comprising a hydrophobic surface, a first layer comprising dopamine disposed on the substrate, and a second layer comprising a polymer comprising a terminal amine disposed on the first layer.

[0069] In one embodiment, the hydrophobic surface is an oxygen plasma treated surface.

[0070] In one embodiment, the hydrophobic surface is a surface of a substrate. Suitable substrates include glasses, metals, woods, cotton, plastics, ceramics, and combinations thereof.

[0071] The second layer includes a polymer having a terminal amine. Suitable polymers having a terminal amine include, for example, polyethylenimine (PEI), polyethyleneglycol (PEG)-amine, polyalkylene oxide (PAO)-amine, polyallylamine, polyvinylamine, poly(vinylamine-co-vinylformamide), chitosan-amine, and poly(amidoamine). The polyethyleneimine suitably can be linear, dendritic, comb, or branched.

[0072] The polymer having a terminal amine is covalently bonded to the first layer comprising dopamine.

[0073] The modified hydrophobic substrate can further include a third layer having at least one biomolecule. Suitable bioactive molecules include any biomolecule having carboxyl groups and are water soluble. A suitable bioactive molecule includes a cell adhesion molecule. Suitable cell adhesion molecules include fibronectin, arginine-glycine-aspartic acid (RGD) peptide, arginine-glycine-aspartic acid-serine (RGDS) peptide (SEQ ID NO:l), leucine-aspartic acid- valine (LDV) peptide, fibronectin CS1 region, laminin, tyrosine-isoleucine-glycine-serine- arginine (YIGSR) peptide (SEQ ID NO:2), proline- aspartic acid-serine-glycine-arginine (PDSGR) peptide (SEQ ID NOG), lysine-arginine-glutamic acid (LRE) peptide, vitronectin, arginine-glycine-aspartic acid-valine (RGDV) peptide (SEQ ID NO:4), and combinations thereof. Other suitable bioactive molecules include anticoagulants. Suitable anticoagulants include heparin, low molecular weight heparin, a coumarin, a directly acting oral anticoagulants (DOACs), fondaparinux, idraparinux, a factor Xa inhibitor, a thrombin inhibitor, hementin, and combinations thereof. Suitable coumarins include warfarin, acenocoumarol, phenprocoumon, atromentin, and phenindione. Suitable directly acting oral anticoagulants (DOACs) include dabigatran, rivaroxaban, apixaban, edoxaban and betrixaban. Suitable factor Xa inhibitors include rivaroxaban, apixaban, edoxaban, betrixaban, darexaban, letaxaban, and eribaxaban. Suitable thrombin inhibitors include hirudin, lepirudin, bivalirudin, argatroban, dabigatran, ximelagatran and combinations thereof.

Wavy multi-component vascular grafts

[0074] In another aspect, the present disclosure is directed to a wavy multi- component vascular graft comprising an inner fiber layer comprising rigid biopolymer fibers and an outer fiber layer comprising elastic biopolymer fibers. The methods described above for modifying a hydrophobic surface can be used with the surface of the inner layer of the WMVGs to improve cell affinity and antithrombogenicity of the surfaces. While the modification methods described above can be used with the inner layer of the WMVGs of the present disclosure, it should be understood that new WMVGs provided using the methods of the present disclosure can be used without the modification.

[0075] As used herein,“wavy multi-component vascular graft” refers to a vascular graft which, when viewed in cross-section, presents a wavy inner fiber layer morphology and includes at least two biocompatible polymers having different stiffness. As used herein, "wavy" refers to a surface having a series of undulating and wavelike curves. Thus, the inner layer of the wavy vascular grafts of the present disclosure has a series of undulating and wavelike curves when the vascular grafts are viewed in cross-section (see, for example, FIG. 2C).

[0076] As used herein,“small-diameter vascular graft” refers to a vascular graft having a lumen diameter less than 6 mm. Vascular graft is a engineered tubular graft that is intended to replace or bypass a damaged or occluded blood vessel.

[0077] Suitable polymer materials for preparing the inner rigid biopolymer fiber layer include silk, poly(lactic acid) (PLA), poly(L- lactic acid) (PLLA), polycapro lactone (PCL), polylactic-co-glycolic acid (PLGA), poly(glycolic acid) (PGA), PLLA/PLGA copolymer, collagen, chitosan, alginate, and combinations thereof. Particularly suitable rigid biopolymers for the rigid biopolymer fibers is silk/poly(lactic acid) fibers. The rigid fibers include submicron diameter fibers. The rigid fibers comprise an average fiber diameter ranging from about 100 nm to about 1000 nm.

[0078] Suitable polymer materials for preparing the outer elastic biopolymer fiber layer include thermoplastic polyurethane (TPU), polyglycerol sebacate (PGS), poly(ester urethane) urea (PEUU), and combinations thereof. The elastic fibers include nanoscale diameter fibers and submicron diameter fibers. Suitably, elastic biopolymers fibers include an average fiber diameter average fiber diameter ranging from about 50 nm to about 300 nm.

[0079] The wavy multi-component vascular graft can further include a biomolecule. Suitable biomolecules include dopamine, heparin, cell adhesion molecules, growth factors, chemokines, anticoagulants, and combinations thereof. Suitable cell adhesion molecules include fibronectin, arginine- glycine- aspartic acid (RGD) peptide, arginine-glycine-aspartic acid-serine (RGDS) peptide (SEQ ID NO:l), leucine-aspartic acid-valine (LDV) peptide, fibronectin CS1 region, laminin, tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide (SEQ ID NO:2), proline- aspartic acid-serine-glycine-arginine (PDSGR) peptide (SEQ ID NOG), lysine-arginine- glutamic acid (LRE) peptide, vitronectin, arginine-glycine-aspartic acid-valine (RGDV) peptide (SEQ ID NO:4), and combinations thereof. Suitable anticoagulants include heparin, low molecular weight heparin, a coumarin, a directly acting oral anticoagulants (DOACs), fondaparinux, idraparinux, a factor Xa inhibitor, a thrombin inhibitor, hementin, and combinations thereof. Suitable coumarins include warfarin, acenocoumarol, phenprocoumon, atromentin, and phenindione. Suitable directly acting oral anticoagulants (DOACs) include dabigatran, rivaroxaban, apixaban, edoxaban and betrixaban. Suitable factor Xa inhibitors include rivaroxaban, apixaban, edoxaban, betrixaban, darexaban, letaxaban, and eribaxaban. Suitable thrombin inhibitors include hirudin, lepirudin, bivalirudin, argatroban, dabigatran, ximelagatran and combinations thereof. Suitable growth factors include, for example, fibroblast growth factor, vascular endothelial growth factor, transforming growth factor beta, and combinations thereof. Suitable chemokines include, for example, SDF-la, CD47, and combinations thereof.

[0080] The wavy multi-component vascular graft can further include a cell. Suitable cells include endothelial cells, smooth muscle cells, mesenchymal stem cells, umbilical vein endothelial cells, fibroblast cells, and combinations thereof. The seeded cells can then be cultured for a sufficient period of time for cells to migrate, proliferate and differentiate.

[0081] The wavy multi-component vascular graft can include a lumen diameter ranging from about 1.9 mm to about 2.3 mm. The lumen diameter can be made larger by using a larger diameter mandrel. The lumen diameter can be made smaller by using a smaller diameter mandrel.

[0082] The wall thickness of the wavy multi-component vascular graft can be any desirable thickness. Suitable wall thickness can range from about 200 pm to about 500 pm. The wall thickness can be determined by measuring from the inner layer surface facing the lumen to the outermost surface of the wavy multi-component vascular graft of cross-sectional scanning electron micrograph images. The wall thickness of the wavy multi-component vascular graft can be made thicker by depositing more of the rigid biopolymer fibers (to result in a thicker inner fiber layer), by depositing more of the elastic biopolymer fibers (to result in a thicker outer fiber layer), and combinations thereof (to result in a thicker inner fiber layer and a thicker outer fiber layer). The wall thickness of the wavy multi-component vascular graft can be made thinner by depositing less of the rigid biopolymer fibers (to result in a thinner inner fiber layer), by depositing less of the elastic biopolymer fibers (to result in a thinner outer fiber layer), and combinations thereof (to result in a thinner inner fiber layer and a thinner outer fiber layer).

[0083] The wavy multi-component vascular graft can include a suture retention strength ranging from about 1 Newton (N) to about 4 N. Suture retention strength can be by varying the contents of rigid fiber layer and the elastic fiber layer.

[0084] The wavy multi-component vascular graft can include any desired burst pressure. The burst pressure can be increased by increasing the TPU fiber content. Suitable burst pressures range from about from 800 mmHg to about 1800 mmHg.

[0085] The tensile strength and modulus of WMVGs can be increased by increasing the silk/PLA fiber content. The flexibility of WMVGs can be increased by increasing the TPU fiber content. The elongation-at-break of WMVGs can be increased by increasing the TPU fiber content.

[0086] Because of the porous structure, and if biodegradable material components are used, the WMVGs can gradually degrade be replaced by regenerated native blood vessel tissue. The degradation rate of WMVGs can be controlled by selecting biopolymers with different degradation rates. For the silk/PLA and TPU based WMVG, silk and PLA fibers should degrade within 6 months, while the TPU fibers could be maintained for about two years. Generally, the inner fiber layer desirably degrades within about 6 months, while the outer fiber layer is maintained for about 2 years.

Method for preparing a wavy multi-component vascular graft

[0087] In one aspect, the present disclosure is directed to a method for preparing a wavy multi-component vascular graft. The method includes: electro spinning a first solution comprising a water soluble polymer material to form a first water soluble fiber; collecting the first water soluble fiber on an assembled mandrel that comprises a central tube and a plurality of satellite cylinders surrounding the tube to form a first water soluble fiber layer; electro spinning a second solution comprising a rigid biopolymer material to form a second fiber; collecting the second fibers on the first water soluble fiber layer to form an inner rigid biopolymer fiber layer; electrospinning a third solution comprising an elastic biopolymer material to form a third fiber; collecting the third fibers on the rigid biopolymer fiber layer on the assembled mandrel to form an outer elastic biopolymer fiber layer; and removing the assembled mandrel to form a wavy multi-component vascular graft.

[0088] As used herein, “electrospinning” or“electrospun,” refers to any method where materials are streamed, sprayed, sputtered, dripped, or otherwise transported in the presence of an electric field. As known to those skilled in the art, electrospinning generally involves the creation of an electrical field at the surface of a liquid. The resulting electrical forces create a jet of liquid which carries electrical charge. The electrically charged solution is then streamed through an opening or orifice towards a grounded target. As the jet of liquid elongates and travels, it will harden and dry to produce fibers. According to the method of the present disclosure, electrospun material is deposited from the direction of a charged container towards a grounded target (to the assembled mandrel), or from a grounded container in the direction of a charged target (to the assembled mandrel).

[0089] The water soluble polymer material of the first solution, the rigid biopolymer material of the second solution, and the elastic polymer material of the third solution can be dissolved or suspended in a solution or suspension of water, urea, methanol, chloroform, monochloro acetic acid, isopropanol, 2,2,2-trifluoroethanol, l,l,l,3,3,3-hexafluoro-2-propanol (also known as hexafluoroisopropanol or HFP), acetamide, N-methylformamide, N,N- dimethylformamide (DMF), dimethylsulfoxide (DMSO), dimethylacetamide, N-methyl pyrrolidone (NMP), acetic acid, trifluoroacetic acid, ethyl acetate, acetonitrile, trifluoroacetic anhydride, l,l,l-trifluoroacetone, maleic acid, hexafluoroacetone, and combinations thereof.

[0090] Any water soluble polymer material that can be electrospun can be used to form the first water soluble fiber. Suitable water soluble polymer materials include poly(ethylene oxide) (PEO) and poly vinyl alcohol (PVOH). The method can further include removing the first water soluble fiber layer. Suitably, the first water soluble fiber layer can be removed by soaking the grafts in water, saline, and other solutions. The first water soluble fiber layer can also be removed by flowing water, saline, and other solutions through the lumen of the grafts.

[0091] Suitable polymer materials for preparing the inner rigid biopolymer fiber layer include silk, poly(lactic acid) (PLA), poly(L- lactic acid) (PLLA), polycapro lactone (PCL), polylactic-co-glycolic acid (PLGA), poly(glycolic acid) (PGA), PLLA/PLGA copolymer, collagen, chitosan, alginate, and combinations thereof. Particularly suitable rigid biopolymers for the rigid biopolymer fibers is silk/poly(lactic acid) fibers. The rigid fibers include submicron diameter fibers. The rigid fibers comprise an average fiber diameter ranging from about 100 nm to about 1000 nm.

[0092] Suitable polymer materials for preparing the outer elastic biopolymer fiber layer include thermoplastic polyurethane (TPU), polyglycerol sebacate (PGS), poly(ester urethane) urea (PEUU), and combinations thereof. The elastic fibers include nanoscale diameter fibers and submicron diameter fibers. Suitably, elastic biopolymers fibers include an average fiber diameter ranging from about 50 nm to about 300 nm.

[0093] Suitably, a volume ratio of the rigid biopolymer solution to the elastic biopolymer solution can range from about 1:2 to about 2:1.

[0094] Suitable polymer materials for preparing the outer elastic biopolymer fiber layer include thermoplastic polyurethane (TPU), polyglycerol sebacate (PGS), poly(ester urethane) urea (PEUU), and combinations thereof. The elastic fibers include nanoscale diameter fibers and submicron diameter fibers. Suitably, elastic biopolymers fibers include an average fiber diameter average fiber diameter ranging from about 50 nm to about 300 nm.

[0095] The resultant wavy multi-component vascular graft prepared according to the method includes a wavy inner layer of rigid biopolymer fibers and a smooth outer layer of elastic biopolymer fibers.

[0096] The method can further include modifying the inner layer of rigid biopolymer fibers. The inner layer of rigid biopolymer fiber layer can be modified with a biomolecule. Suitable biomolecules include dopamine, heparin, cell adhesion molecules, growth factors, chemokines, anticoagulants, and combinations thereof. Suitable biomolecules include dopamine, heparin, cell adhesion molecules, growth factors, chemokines, anticoagulants, and combinations thereof. Suitable cell adhesion molecules include fibronectin, arginine-glycine-aspartic acid (RGD) peptide, arginine- glycine- aspartic acid-serine (RGDS) peptide (SEQ ID NO:l), leucine- aspartic acid- valine (LDV) peptide, fibronectin CS1 region, laminin, tyrosine-iso leucine- glycine- serine- arginine (YIGSR) peptide (SEQ ID NO:2), proline- aspartic acid-serine-glycine-arginine (PDSGR) peptide (SEQ ID NOG), lysine-arginine-glutamic acid (LRE) peptide, vitronectin, arginine-glycine-aspartic acid-valine (RGDV) peptide (SEQ ID NO:4), and combinations thereof. Suitable anticoagulants include heparin, low molecular weight heparin, a coumarin, a directly acting oral anticoagulants (DOACs), fondaparinux, idraparinux, a factor Xa inhibitor, a thrombin inhibitor, hementin, and combinations thereof. Suitable coumarins include warfarin, acenocoumarol, phenprocoumon, atromentin, and phenindione. Suitable directly acting oral anticoagulants (DOACs) include dabigatran, rivaroxaban, apixaban, edoxaban and betrixaban. Suitable factor Xa inhibitors include rivaroxaban, apixaban, edoxaban, betrixaban, darexaban, letaxaban, and eribaxaban. Suitable thrombin inhibitors include hirudin, lepirudin, bivalirudin, argatroban, dabigatran, ximelagatran and combinations thereof. Suitable growth factors include, for example, fibroblast growth factor, vascular endothelial growth factor, transforming growth factor beta, and combinations thereof. Suitable chemokines include, for example, SDF-la, CD47, and combinations thereof. The inner layer of rigid biopolymer fibers can be modified by immersing the WMVG in a solution containing the biomolecule.

[0097] The method can further include seeding a cell on the wavy multi-component vascular graft. Suitable cells include endothelial cells, smooth muscle cells, mesenchymal stem cells, fibroblasts, and combinations thereof. Cells are suitably seeded by injecting a cell suspension into the lumen of the WMVG followed by adding cell culture media. The seeded WMVG can then be cultured for a sufficient period of time for cells to migrate, proliferate, and differentiate.

[0098] Various functions and advantages of these and other embodiments of the present disclosure will be more fully understood from the examples shown below. The examples are intended to illustrate the benefits of the present disclosure, but do not exemplify the full scope of the disclosure.

EXAMPLE 1

MATERIALS AND METHODS

[0099] Medical-grade PTFE sheets with a thickness of 1 mm were purchased from Scientific Commodities Inc. All other chemicals were purchased from Sigma- Aldrich and used as received. DI water was used throughout the experiment.

PTFE Modification

[0100] PTFE sheets were first cleaned by ultrasonication in a 20% ethanol solution for 30 minutes. The PTFE sheets were then treated with oxygen plasma to enhance their surface hydrophobicity via a plasma etcher (PlasmaEtch PE-200) at an RF power of 200 W for 30 minutes at an oxygen flow rate of 20 cm³/min. The plasma-treated PTFE sheet was named P- PTFE. P-PTFE was further coated with dopamine (DA) by immersing it into a 2 mg/mL dopamine solution with a pH of 8.5 adjusted by 10 mM tris(hydroxymethyl)aminomethane for 16 hours at room temperature. After coating, samples were rinsed with DI water 5 times and dried with nitrogen. Dopamine-coated P-PTFE sheets were named DA-PTFE. To further enhance the surface biocompatibility and anti-thrombogenic properties, RGD and heparin were chemically grafted onto DA-PTFE via a thin layer of PEI molecules. Briefly, PEI was dissolved in a citric acid/sodium phosphate dibasic buffer solution with a pH of 5.5 at a concentration of 0.5 mg/mL. DA-PTFE was immersed in the PEI solution for 1 hour at room temperature, then rinsed with DI water and dried using nitrogen. Another buffer solution containing 20 mM of EDC, 50 mM of NHS, and 0.1 M MES was prepared. An RGD solution (100 pg/mL) and an RGD/heparin solution (100 pg/mL for RGD and 1 mg/mL for heparin) were prepared using the above buffer. PEI-modified samples were soaked separately in these solutions overnight, followed by sufficient washing and drying, to prepare RGD-grafted PTFE, which was named RGD-PTFE, and RGD/heparin- grafted PTFE, which was named R/H-PTFE.

Characterization of Prepared PTFE Sheets

[0101] Fourier transform infrared (FTIR) spectra were recorded in transmittance mode to verify the modifications using a Bruker Tensor 27 spectrometer in the range of 4000- 600 cm ¹, with a resolution of 4 cm ¹. X-ray photoelectron spectroscopy (XPS) measurements of different modified PTFE samples were performed on an X-ray photoelectron spectrometer with a focused, monochromatic K-alpha X-ray source and a monoatomic/cluster ion gun (Thermo Scientific). The Cls core-level signal spectra were Gaussian fitted and the proportion of each bond was determined from the peak area ratios. Scanning electron microscopy (SEM) was used to characterize the morphological properties. Samples were first coated with a thin layer of gold and then imaged using a fully digital LEO GEMINI 1530 SEM (Zeiss, Germany) at a voltage of 3 kV. The surface topography of different modified PTFE samples was analyzed using a Bruker BioScope Catalyst atomic force microscope (AFM) in tapping mode. The wettability of the modified PTFE samples was measured by a video contact angle instrument (Dataphysics, OCA 15) using 7 pL of DI water droplets with the sessile drop method. Platelet Adhesion Test

[0102] Platelet adhesion tests were performed to investigate the antithrombogenicity of the modified PTFE sheets. Platelet-rich -plasma (PRP) was extracted from fresh human blood stabilized with 3.8% sodium citrate as an anti coagulant (Innovative Research). The blood was centrifuged at 1500 rpm for 15 minutes to obtain PRP. For the platelet adhesion test, samples were first incubated in phosphate- buffered saline (PBS) at 37 °C for 1 hour. Then, PBS was aspirated and 500 pL of PRP were added, followed by incubation at 37 °C for 2 hours. After incubation, samples were rinsed three times with PBS and treated with 2.5 wt% glutaraldehyde in PBS at 4 °C for 1 day. After that, samples were subjected to a series of ethanol solution washes (50%, 70%, 80%, 90%, and 100%) and dried in a desiccator overnight, followed by gold coating and imaging using SEM.

Human Umbilical Vein Endothelial Cell (HUVEC) Culture

[0103] Human umbilical vein endothelial cells (HUVECs; Lonza) were maintained on T75 tissue culture-treated polystyrene flasks. Cells were fed every other day with an endothelial cell growth medium EGM-2-MV bullet kit (Lonza). Prepared PTFE sheets were cut to the same size, put in 24-well tissue culture plates (TCPs), and washed in a 20% ethanol solution 5 times, followed by washing 3 times with PBS. They were then sterilized with ultraviolet (UV) light for 30 minutes. HUVECs were detached enzymatically with a trypsin- EDTA solution and seeded on the samples at a density of lxlO⁴ cells/cm² for the live/dead assay and MTS assay. They were seeded at a density of lxlO³ cells/cm² for the cytoskeleton assay. Spent medium was aspirated and replaced with 1 mL of fresh medium daily for screening samples. HUVECs were also cultured on TCPs as a control.

Biological Characterization

[0104] Initial cell attachment was evaluated at 4 hours after cell seeding. The cells were fixed in 4% paraformaldehyde for 15 minutes, followed by a PBS rinse, and then treated with 0.1% Triton-X in PBS for 5 minutes at room temperature. They were rinsed again with PBS and stained with 3 mM 4', 6-diamidino-2-phenylindole (DAPI) for 1 hour at room temperature. Samples were then rinsed with PBS and imaged using a Nikon Eclipse Ti-E inverted fluorescence microscope. [0105] Cell viability was determined after culturing for 7 days and 14 days. Viability was assessed via a live/dead viability/cytotoxicity kit (Life Technologies). Green fluorescent calcein-AM was used to target the esterase activity within the cytoplasm of living cells, while the red fluorescence ethidium homodimer- l(EthD-l) was used to indicate cell death. Stained cells were imaged with a Nikon AlRSi inverted confocal microscope system. The number of collected cells that fluoresced red and green were counted with an Accuri C6 (BD Biosciences) flow cytometer to obtain viability data. Briefly, the stained cells of the live/dead assay were detached from the scaffolds by incubation in 250 pL of trypsin (Life Technologies) per well at 37 °C for 5 minutes. Then the cells were collected and centrifuged at 1000 rpm for 5 minutes. Next, the supernatant was aspirated and the cells were resuspended in 600 pL of PBS and filtered prior to analysis.

[0106] Cell proliferation was assessed at day 7 and day 14 by MTS assay using the CellTiter 96 Aqueous One Solution kit (Promega Life Sciences). Cells were first treated with media containing a 20% MTS solution and allowed to incubate for 1 hour. After incubation, 100 pL of spent media were transferred into a clear 96-well plate. The absorbance of the plates at the 450 nm wavelength was read with a Glomax-Multi+ Multiplate Reader (Promega). The subsequent number of cells was determined relative to the negative control.

[0107] The shape and cytoskeleton organization of the cells were determined by phalloidin-tetramethylrhodamine B isothiocyanate (phalloidin-TMRho, Sigma) staining. For this assay, cells were first fixed following the same procedure in the cell attachment assay. They were then treated with 0.3 pM of phalloidin-TMRho with DAPI for 1 hour at room temperature. Next, samples were washed with PBS and imaged using the same confocal microscope.

[0108] The interaction between cells and substrate was observed using SEM. Briefly, the samples stained with phalloidin/DAPI were dehydrated through a series of ethanol solution (50%, 70%, 80%, 90%, and 100%) washes and sufficiently dried in a desiccator. They were then coated with gold and imaged using SEM.

Statistical Analysis

[0109] All biological results are presented as mean ± standard deviation. All of the values were averaged at least in triplicate. The data were analyzed using the one-way analysis of variance method (ANOVA). The Tukey’s test was then used to evaluate the specific differences of the data, and these differences were considered statistically significant at p < 0.05.

Results and Discussion

[0110] A series of surface modifications were carried out on flat PTFE sheets as shown in FIG. 1. The effect of each modification on the cellular substrate interaction was determined to enhance the bio activity of hydrophobic surfaces. Oxygen plasma was first used to introduce hydrophilic groups on the PTFE surface prior to dopamine (DA) coating. Although it has been reported that dopamine is able to coat any surface, regardless of hydrophobicity, its coating efficiency was greatly improved when the PTFE was first treated with 0₂ plasma. As evidenced in FIG. 2, the plasma-treated PTFE showed a distinctly darker color than the PTFE without plasma treatment, thus indicating that more dopamine was coated on the P-PTFE. To further graft bioactive molecules onto the substrate surface, a very thin layer of polyethylenimine (PEI) was immobilized on the polydopamine to introduce reactive amino groups on the substrate surface. The PEI concentration was controlled at a low level (0.5 mg/mL) to improve cell adhesion and avoid cell death caused by an excess amount of PEI. Chemical grafting of RGD or RGD/heparin was performed using EDC/NHS grafting chemistry. In this grafting process, carboxyl groups on RGD and heparin were reacted while the bioactive component of RGD and the antithrombotic sulfo group of heparin were preserved and exposed on the substrate surface.

[0111] The chemical composition of the modified PTFE sheets was first characterized using FTIR. As shown in FIG. 3, all materials showed very similar peak patterns due to the strong signal from the PTFE substrate. However, the difference among samples can be seen when specific regions are enlarged. The plasma-treated PTFE (P-PTFE) showed the same peak pattern as PTFE, except for a small wide peak at 3300 cm ¹ indicating the introduction of a small amount of hydroxyl groups. The intensity of this peak significantly increased after dopamine coating due to the O-H and N-H bonds of polydopamine. Moreover, another peak attributed to C=0 presented at 1615 cm ¹, and the peak for C=N and C=C was also observed at 1512 cm ¹. These results indicated the successful coating of dopamine on the substrate surface. The RGD-grafted PTFE showed more intense peaks in the enlarged regions. The increase of the peak at 3300 cm ¹ was attributed to the N-H from PEI and RGD. The peaks at 1702 cm ¹ and 1464 cm ¹ corresponded to the amide I and III of RGD. Interestingly, the wavenumbers of these peaks shifted higher compared to the freeze-dried RGD from the references. This might have been due to the formation of hydrogen bonds with dopamine, which would further immobilize the RGD on the substrate surface. When heparin was further grafted onto the PTFE surface, a shoulder peak assigned to S=0 asymmetry vibration presented on the FTIR spectrum at 1018 cm ¹. Additionally, the intensity of the C=0 peak at 1615 cm ¹, and the peak at 3300 cm ¹, increased, thus indicating the successful grafting of heparin.

[0112] Since the strong signal from PTFE in the FTIR measurements may hide some details in the surface chemistry, XPS was used to further characterize the surface layer of the modified samples. From the survey scans (FIG. 4) and atom percentage statistical data (Table 1), it was found that only C and F were detected on PTFE, while 4.1% O was detected on P-PTFE, thus indicating the introduction of hydrophilic groups. Nitrogen was detected on the surface of DA-PTFE and RGD-PTFE at a rate of 5.5% and 8.2%, respectively, suggesting the introduction of bioactive components. On the R/H-PTFE, 2.1% of S was detected, which confirmed the grafting of heparin. The Cls core level scans show the detailed information of carbon-containing bonds on the material surface (FIG. 5). According to statistical data (Table 2), the proportion of CF₂ and CF₃ was reduced by more than half after plasma treatment, and it was less than 5% after dopamine coating, thus suggesting an increase in surface energy. As expected, C-O, C=0, and C-N bonds were present on DA-PTFE, and the proportion of C-N bonds greatly increased after RGD grafting, which was attributed to the massive amide bonds on RGD. Similarly, C-0 bonds dominated when heparin was grafted, which corresponded to the increase in C-O-C linkages from heparin. Therefore, the XPS results further confirmed the success of each modification step. The surface chemistry of PTFE was tuned by dopamine coating and RGD or RGD/heparin grafting.

Table 1. Atom percentage results of PTFE, P-PTFE, DA-PTFE, RGD-PTFE, and R/H-PTFE from XPS survey scans.

RGD-

Atom PTFE P-PTFE DA-PTFE R/H-PTFE

PTFE

C 33.7 36.7 69.9 65.1 60.9

O 0.1 4.1 15.3 17.3 24.2

F 66.2 59.2 9.3 9.4 7.3

N / / 5.5 8.2 5.5

S / / / / 2.1 Table 2. XPS Cls core-level scans of PTFE, P-PTFE, DA-PTFE, RGD-PTFE, and R/H-PTFE showing the binding energy (BE) and percentage of different carbon containing bonds.

PTFE P-PTFE DA-PTFE RGD-PTFE R/H-PTFE

Element

BE % BE % BE % BE % BE %

286.3 13.4 286.3 42.7 284.7 65.0 284.8 55 285.6 30.4

C-N / / / / 285.7 8.0 285.6 20.6 286.4 2.8

C-O / / 286.3 17.3 286.1 17.4 286.4 16.3 287.1 35.7 c=o / / 287.8 0.8 287.9 6.3 287.8 4.0 288.5 29.3

CF 289.9 1.0 289.2 0.1 / / / / / /

CF₂ 293.1 52.9 292.6 25.2 292.0 2.7 292.1 3.0 292.8 1.2

CF₃ 294.4 32.7 293.7 13.9 292.5 0.7 292.4 1.1 294.2 0.6

[0113] The surface morphology of modified PTFE sheets was imaged using SEM. As can be seen from FIG. 6, neat PTFE showed a relatively smooth surface. After 30 minutes of O2 plasma treatment, some cracks showed on the P-PTFE surface, indicating the cleavage of carbon bonds. Dopamine-coated PTFE (DA-PTFE) showed a coating layer with increased surface roughness. It has been reported that self-polymerized polydopamine forms nanoparticles in an aqueous solution. When coated on PTFE, these particles merged to form a film, but some particulate morphology was still observable on the material surface. When RGD was grafted on DA-PTFE with the PEI layer, the surface morphology of RGD-PTFE remained about the same. Remarkably, when heparin was grafted on the surface, R/H-PTFE showed a much rougher surface with many nanoparticles (~50 nm) anchored to the substrate surface. This was because heparin molecules tend to aggregate and form nanoparticles when dried from an aqueous solution. A similar rough surface was also found when heparin was directly coated onto a poly(lactic acid) (PLA) membrane.

[0114] The surface topography of modified PTFE sheets was further characterized using AFM to quantify the change of surface roughness in each modification step. As shown in the 3D AFM images (FIG. 7) and cross-sectional images (FIG. 8), PTFE showed a height difference of 28 nm. The micro grooves observed in the images were caused by the finishing of the pristine PTFE sheets during production. After O2 plasma treatment, the height difference increased to 78 nm, thus indicating that some PTFE had been etched away. A greater increase in height difference (from 18 nm to 135 nm) is observed when PTFE sheets are treated with N₂ plasma for 3 hours. The height difference further increased after dopamine coating and RGD grafting as shown in FIG. 7. Notably, after grafting with RGD and heparin, the height difference increased to 209 nm, which was significantly higher than other samples. This was because of the nanoparticles formed by heparin and was in agreement with SEM observations. The increased surface roughness should be favorable for cell attachment since rough surfaces have been found to facilitate serum protein adsorption and enhance osteoblast cell adhesion.

[0115] Platelet adhesion was evaluated to understand the effect of surface modification on the risk of thrombosis. As shown in FIG. 9A, the platelets adhered on the substrate had round shapes indicating that they generally had a low attachment force to the substrate. The number of attached platelets on P-PTFE, DA-PTFE, and RGD-PTFE was significantly higher than on PTFE and R/H-PTFE as shown in the statistical results (FIG. 9B). Remarkably, almost no platelets adhered to R/H-PTFE. Without being bound by theory, it is believed that the lower platelet adhesion for PTFE was associated with its low surface roughness and high hydrophobicity. As shown in the water contact angle (WCA) measurements (FIG. 9A), the WCA decreased after each modification step because more hydrophilic components were introduced in each step. The surface roughness, on the contrary, gradually increased as demonstrated above. The combination of these two factors caused increased platelet adhesion after modification. However, R/H- PTFE showed very low platelet adhesion although it possessed the lowest WCA and highest surface roughness.

[0116] To investigate the effect of different modifications on cellular- substrate interactions, HUVECs were cultured on pristine PTFE and different PTFE samples. Initial cell attachment was evaluated 4 hours after cell seeding. It was found that the cell attachment on PTFE and P-PTFE samples was significantly lower than on other samples. The dopamine-coated substrate showed improved cell adhesion. The samples grafted with RGD and RGD/heparin had significantly higher cell seeding than the one coated only with dopamine (FIG. 10).

[0117] The viability of HUVECs on different modified PTFE substrates was investigated using a live/dead assay. The assay uses calcein-AM to stain live cells with green fluorescence and EthD-1 to target dead cells with red fluorescence. The fluorescence images showed that HUVECs were able to grow on all substrates (FIGS. 11 A and 12), while the percentage of live cells (FIG. 11B) and the number of cells (FIG. 11C) differed among them. The results indicated that neat PTFE had a very low cell viability (FIG. 11B) and cell population (FIG. 11C) at both day 7 and day 14 time points, indicating that cells were not able to proliferate or proliferated slowly on PTFE. After 0₂ plasma treatment, P-PTFE showed improved cell proliferation and viability at both time points. A significant improvement was achieved when PTFE was coated with dopamine. Compared to PTFE, DA-PTFE showed 4.5 times the cell population of PTFE at day 7, and it further increased to 8.4 times as many on day 14. When RGD or RGD/heparin was grafted, the cell proliferation improved further. Remarkably, at day 14, the number of cells on RGD-PTFE was 11 times that of PTFE, and it was also significantly higher than DA-PTFE and TCP. However, the difference between RGD-PTFE and R/H-PTFE was not significant, suggesting that RGD was the main cause of the increased cell affinity. This trend was maintained at both day 7 and day 14 time points and was consistent with the cell attachment results, which indicated that dopamine and RGD were able to not only facilitate initial cell attachment, but also stimulate cell growth over time. In addition, it was observed that HUVECs cultured on RGD-PTFE and R/H-PTFE showed smaller sizes than cells cultured on other samples (FIG. 11 A). This was because of the significant increase in the cell population over the limited sample area. At day 14, the HUVECs cultured on DA-PTFE and TCP also became smaller compared to day 7 (FIG. 12).

[0118] To investigate cell phenotype, HUVECs were seeded on different PTFE substrates at a low density (5000 cells/well) and cultured for 14 days. The cytoskeletons of the cells were stained red with phalloidin-TMRho and cell nuclei were stained blue with DAPI. FIG. 13 A shows the cytoskeleton of HUVECs cultured on different PTFE substrates for 7 days. As can be seen, both cell size and nuclei size of HUVECs cultured on PTFE were smaller than cells on other samples. The cells were not spread out and the fluorescence intensity was weak, thus indicating that cells did not show a healthy growing state. After a series of modifications, the cellular-substrate interaction greatly improved. For statistical comparison, the average projected cell area and aspect ratio of the cells were measured as shown in FIGS. 13B and 13C. It was found that samples with DA, RGD, and RGD/heparin modification showed significantly larger cell sizes than cells on PTFE and P-PTFE, and the cells were more spread out and stretched on those samples. Notably, the cells on RGD-PTFE and R/H-PTFE showed extremely flourishing growing states with a typical spindle-like cell morphology and filopodia. The average aspect ratio of the cells on RGD-PTFE and R/H-PTFE was over 3, which was significantly larger than those grown on PTFE and TCP (FIG. 13C). Moreover, the filaments in the cells can be clearly seen on cells grown on RGD-PTFE and R/H-PTE, thus indicating highly extended cell morphology. After 14 days of cell culture, the number of cells greatly increased on the DA- PTFE, RGD-PTFE, and R/H-PTFE samples (FIG. 14). In some regions of the RGD-PTFE and R/H-PTFE samples, cells started to grow on top of each other to form dense cell aggregates. Similar to the live/dead assay, the cell size decreased at day 14 due to the great increase in the number of cells, thus the projected cell area was not measured at day 14. All of these results strongly suggest that dopamine coating and RGD and heparin grafting greatly improved the biocompatibility and endothelial cell affinity of PTFE.

[0119] Samples at day 7 were imaged using SEM to observe the morphology of the cells on different substrates. As shown in FIG. 15, cells were rarely present on PTFE samples. Although more cells were observed on P-PTFE compared with PTFE, many of the cells were round and a gap was observed between the cells and the substrate, indicating poor cell adhesion. On the DA-PTFE sample, cells were flattened and showed tight adhesion to the substrate. Many cells were connected with each other by extended filopodia. Meanwhile, the cell boundaries were clearly seen and some cells observed were not fully spread. Remarkably, individual cells and cell boundaries were not observed at low magnification on the RGD-PTFE and R/H-PTFE samples. When observed under high magnification, cell membranes were greatly flattened and extended. The lamellipodia and filopodia were tightly attached to the substrate, and the boundary of the cell membrane was difficult to see, thus indicating strong cellular- substrate interaction.

[0120] The results demonstrate a facile modification method for the functionalization of PTFE with bioactive molecules such as dopamine (DA), RGD, and heparin towards their application as vascular grafts. Oxygen plasma treatment activated hydrophilic groups on PTFE’s surface and facilitated dopamine coating. RGD and heparin were immobilized on DA-PTFE through a thin PEI layer. Successful modification in each step was verified via FTIR and XPS. The surface roughness increased as more components were grafted onto the PTFE surface, and the hydrophilicity increased due to the increased number of hydrophilic groups. Platelet adhesion increased after dopamine and RGD modification, but was decreased dramatically by grafting heparin onto the surface, thereby demonstrating excellent antithrombogenicity. In vitro, HUVEC cultures revealed that all of the modifications had a positive effect on the biocompatibility of PTFE. The initial cell attachment, cell viability, and cell proliferation all improved significantly when dopamine and RGD were grafted onto the PTFE surface, and the incorporation of RGD outperformed dopamine coating alone. Endothelial cells cultured on RGD- and RGD/heparin- grafted PTFE substrates exhibited favorable cell morphologies and strong cell- substrate interactions owing to the significantly enhanced cell affinity. Therefore, the methods described in the present disclosure provide simultaneous improvement of endothelial cell affinity and antithrombogenicity of hydrophobic surfaces. The method advantageously is highly suitable for the modification of SDVGs to stimulate fast endothelialization and effective antithrombosis.

EXAMPLE 2

MATERIALS AND METHODS

[0121] Poly(lactic acid) (PLA, M_w: -60,000) and poly(ethylene oxide) (PEO, M_v: -900,00) were purchased from Sigma-Aldrich. Hexafluoroisopropanol (HFIP) was purchased from CovaChem. Biodegradable elastic thermoplastic polyurethane (TPU) with a molecular weight of 80,000 was synthesized. Silk fibroin was extracted from the cocoons of Bombyx mori silkworms. All other chemicals and solvents were purchased from Sigma-Aldrich. Milli-Q DI water was used throughout the experiment.

[0122] The PEO solution was prepared by dissolving 1 g of PEO powder in deionized (DI) water at 50 °C. The silk/PLA solution was prepared by dissolving 200 mg PLA and 240 mg silk fibroin in 5 mL HFIP at room temperature. The TPU solution was prepared by dissolving 1 g TPU pellets in 10 mL DMF at 60 °C. All solutions were magnetically stirred overnight and used as prepared.

Fabrication of Wavy Multi-Component Vascular Grafts (WMVGs)

[0123] WMVGs were fabricated via electro spinning using a special rotating fiber collection device. The device used an assembled mandrel having a central tube and eight satellite cylinders surrounding the tube. In the fabrication process, as shown in FIG. 16, the satellite cylinders expand (jump ^roP^e effect) when the mandrel starts rotating and electrospun fibers are thus loosely collected on the mandrel. A thin PEO layer was first electrospun on the mandrel, followed by electro spinning of the silk/PLA layer and the TPU layer. As the collected graft thickened, the expansion of the satellite cylinders decreased due to increasing wrapping force from the electrospun fibers. The electro spinning of elastic TPU fibers eventually tighten the graft and mandrel. The electrospun assembly was first treated with methanol vapor overnight to make silk fibroin water insoluble. Afterwards, the inner PEO layer was removed by leaching in a water bath for 2 hours so that the vascular graft sample could be removed. At the end, a vascular graft with a wavy inner layer of silk/PLA fibers and a smooth TPU nanofiber outer layer was prepared. The electrospinning volumes of the silk/PLA solution and the TPU solution were kept at 0.5 mL and 1 mL, 0.75 mL and 0.75 mL, and 1 mL and 0.5 mL, respectively, to alter the blend ratio and thus the mechanical properties of the vascular grafts. The samples were denoted as S/P:T=l:2, S/P:T=l:l, and S/P:T=2:l.

Characterization of WMVGs

[0124] The morphology of WMVGs was imaged using a digital LEO GEMINI 1530 scanning electron microscope (SEM, Zeiss). Samples were quenched in liquid nitrogen, fractured, and sputtered with a thin film of gold for 40 seconds prior to imaging with an accelerating voltage of 3 kV. The fiber diameters were measured from SEM images using Image Pro-plus software. At least 50 fibers were measured for each sample.

[0125] The mechanical properties of the WMVGs were characterized via a universal mechanical property testing instrument (Instron). The tensile properties of the WMVGs were measured in the circumferential direction via two“U”-shaped clamps. Samples were stretched circumferentially at a crosshead speed of 1 mm/min until fracture. Cyclic tensile tests were also performed using the same instrument. All samples were stretched to 30% strain and released for 50 cycles at a crosshead speed of 5 mm/min.

[0126] The suture retention strengths of the WMVGs were measured on samples with a length of 20 mm. One end of the sample was clamped by a fixed clamp, while the other end was pierced through at 2 mm from the edge using a tapered non-cutting needle connected to a movable clamp by a commercial suture (5-0 prolene suture, Ethicon, Inc.). Samples were stretched at 5 mm/min until fractured. The maximum load was recorded as the suture retention strength.

[0127] The burst pressure of WMVGs was measured using a custom-made apparatus consisting of a digital pump, a pressure gage, and a sample holder. A syringe loaded with a phosphate buffered saline (PBS) was connected to a pressure gage which is then connected to one end of the sample. The PBS was continuously injected into the sample and the other end of the sample was sealed by a clamp once PBS started to flow out. The PBS continued to be injected into the sample until it leaked, and the maximum pressure was recorded as the burst pressure. Modification on the Inner Surface of WMVGs

[0128] Dopamine (DA), a mussel adhesive protein-inspired molecule and an important organic chemical that is found in the human brain and body, was first coated on the electrospun silk/PLA fibers to enhance their biocompatibility and endothelial cell affinity while promoting immobilization of heparin. Briefly, silk/PLA fiber mats were cleaned in 20% ethanol followed by several DI water rinses and drying. Samples were then immersed in a 2 mg/mL dopamine solution with a pH of 8.5 adjusted by 10 mM tris(hydroxymethyl)amino methane for 16 hours at room temperature. After coating, samples were rinsed with DI water 5 times and dried with nitrogen. The obtained samples were named S/P-DA.

[0129] Heparin was coated on the S/P-DA to enhance the antithrombogenicity of WMVGs. Briefly, heparin was dissolved in a citric acid/sodium phosphate dibasic buffer solution with a pH of 5.0 at a concentration of 1 mg/mL. S/P-DA was immersed in the heparin solution for 12 hours at room temperature, followed by several rinsing and drying cycles. The obtained samples were named S/P-D&H.

Characterization of Modified Silk/PLA Fiber Mats

[0130] The morphology of the modified silk/PLA fibers was imaged using the same SEM. The surface chemistry was evaluated using an X-ray photoelectron spectrometer (XPS) with a focused, monochromatic K-alpha X-ray source and a monoatomic/cluster ion gun (Thermo Scientific). Water contact angle (WCA) measurements were performed at room temperature with a Dataphysics OCA 15 optical contact angle measuring system using the sessile drop method.

Platelet Adhesion

[0131] Platelet adhesion tests were performed to investigate the antithrombogenicity of the as-spun silk/PLA fibers and modified S/P-DA and S/P-D&H samples. Platelet-rich plasma (PRP) was extracted from fresh human blood stabilized with 3.8% sodium citrate as an anti coagulant (Innovative Research). The blood was centrifuged at 1500 rpm for 15 minutes to obtain PRP. For the platelet adhesion test, samples were first incubated in PBS at 37 °C for 1 hour. Then, PBS was aspirated and 500 pL of PRP were added, followed by incubation at 37 °C for 2 hours. After incubation, samples were rinsed three times with PBS and treated with 2.5 wt% glutaraldehyde in PBS at 4 °C for 1 day. After that, samples were subjected to a series of ethanol solution washes (50%, 70%, 80%, 90%, and 100%) and dried in a desiccator overnight, followed by gold coating and imaging using SEM.

Human Umbilical Vein Endothelial Cell (HUVEC) Culture

[0132] HUVECs (Lonza) were maintained on T75 tissue culture-treated polystyrene flasks. The cells were fed every other day with an endothelial cell growth medium EGM-2-MV bullet kit (Lonza). As-spun silk/PLA and modified silk/PLA samples were punched to the same size, placed in 24-well tissue culture plates, and washed in a 20% ethanol solution five times, followed by washing with PBS three times. They were then sterilized with ultraviolet (UV) light for 30 minutes. HUVECs were detached enzymatically with a trypsin-EDTA solution and seeded on the samples at a density of lxlO⁴ cells/cm² for the live/dead assay and the MTS assay. They were also seeded at a density of lxlO³ cells/cm² for the cytoskeleton assay. Spent medium was aspirated and replaced with 1 mL of fresh medium daily for screening samples. HUVECs were also cultured on glass slides as a control group for the MTS assay.

[0133] HUVECs were also seeded on dopamine- and heparin-modified WMVGs to investigate cell proliferation. For the seeding process, WMVG with a length of 5 mm were sterilized and fixed in a 96-well plate with sterilized polyester double-sided adhesive tape (ARCARE®90l06, Adhesive Research Inc.). HUVEC suspensions were concentrated to 2xl0⁵ cells/mL and 50 pL cell suspensions were injected into the lumen of the WMVG, followed by adding 250 pL of cell culture media. Samples were investigated via live/dead assay on day 7 and day 14.

Live/Dead Assay

[0134] Cell viability was assessed via a Live/Dead viability/cytotoxicity kit (Life Technologies). The staining protocol followed the manufacturer’s instructions. The green fluorescent calcein-AM was used to target the living cells, while the red fluorescence ethidium homodimer- 1 (EthD-l) was used to indicate cell death. Stained cells were imaged with a Nikon Ti-E confocal microscope. Nis-D Elements Advanced Research v.3.22 software was used for image analysis. MTS Assay

[0135] Cell proliferation was evaluated using an MTS assay after culturing for 3, 7, and 14 days using a CellTiter 96 Aqueous One Solution kit following the manufacturer’s instructions (Promega Life Sciences). Upon testing, cells were treated with media containing a 20% MTS solution and incubated for 1 hour. Then, 100 pL of spent media were transferred into a clear 96-well plate. The absorbance of the plates at a wavelength of 450 nm was read with a Glomax-Multi+Multiplate Reader (Promega). The subsequent number of cells was determined relative to the negative control.

Cell Cytoskeleton and Morphology

[0136] The shape and cytoskeleton organization of the cells were determined by phalloidin-tetramethylrhodamine B isothiocyanate (phalloidin-TMRho, Sigma) staining. For this assay, cells were first fixed in 4% paraformaldehyde and then diluted in PBS for 15 minutes at room temperature. Next, they were washed with PBS and permeabilized with 0.1% Triton-X in PBS for 5 minutes. The cells were then washed once again and treated with 0.3 mM of phalloidin-TMRho with 4', 6-diamidino-2-phenylindole (DAPI) for 1 hour at room temperature. Samples were then washed with PBS and imaged using a Nikon AlRSi inverted confocal microscope. After imaging, the cells were dehydrated using a series of ethanol solution washes (50%, 70%, 80%, 90%, and 100%) and dried in a desiccator overnight followed by gold coating and imaging using SEM.

Statistical Analysis

[0137] All biological results are presented as the mean ± the standard deviation. All of the values were averaged at least in triplicate and expressed as the mean ± the standard deviation. The data were analyzed using the one-way analysis of variance method (ANOVA). Tukey’s test was then used to evaluate the specific differences of the data, and these differences were considered statistically significant at p < 0.05.

[0138] FIG. 17A shows the cross-section morphology of the WMVG sample (S/P:T=l:l) removed from the mandrel without leaching of PEO. The inner layer was deformed when pulled out of the satellite cylinders. The PEO layer, which was still attached to the silk/PLA fibers, was clearly observed in the enlarged image (FIG. 17B). The wavy configuration of the inner silk/PLA layer was maintained with the leaching process (FIG. 17C). The enlarged images (FIGS. 17D-17F) show the cross section of the silk/PLA layer and the TPU layer.

[0139] The fabricated WMVGs featured a wavy inner layer made of silk/PLA microfibers and a smooth TPU nanofiber outer layer. The statistical results indicated that the silk/PLA layer was mainly composed of submicron fibers, with an average fiber diameter of 416 nm (see, FIG. 24A). The TPU layer was composed of nano- and submicron fibers, with an average fiber diameter of 163 nm (FIG. 24B). It was also found that the TPU fibers were densely stacked (FIG. 17F), while the silk/PLA fibers were loosely packed due to the electrostatic charge of silk (FIG. 17E). WMVGs with different volumetric blend ratios (i.e., S/P:T=l :2, S/P:T=l:l, and S/P:T=2:l) showed slightly different overall dimensions even though the same total electrospinning volume was used (FIGS. 25A and 25B). The WMVGs possessed smooth outer surfaces (FIG. 17G) and wavy- structured inner surfaces (FIG. 17H). The flat regions of the inner surface (FIG. 171) were composed of fibers that were in contact with the satellite cylinders of the mandrel and the wavy regions of the inner surface (FIG. 17J) were the fibers located between the satellite cylinders. As prepared for these Examples, WMVGs had a lumen diameter ranging from 1.9 mm to 2.3 mm and a wall thickness ranging from 286 pm to 453 pm. The lumen diameter (FIG. 25 A) and wall thickness (FIG. 25B) of fabricated WMVGs were comparable to a medium- size human artery and a human vein.

[0140] FIG. 18A shows the tensile stress-strain curves of three WMVGs. The tensile strength and modulus were higher for the WMVGs with more silk/PLA microfibers since both silk and PLA are relatively more rigid and stronger. As the TPU content was increased, the WMVGs became more flexible as indicated by their reduced strength and modulus as well as increased elongation-at-break (Table 3). Advantageously, unlike many synthetic materials, all WMVGs showed an initial slow increase of stress at low strain and a steep increase of stress at high strain, which resembled the“toe region” of native blood vessels. The statistical results also showed that the tensile strength of WMVGs surpassed native human arteries, and that the toe regions were within the range (9-38%) of human coronary arteries (cf. Table 3). Table 3. Statistical results of the mechanical properties of fabricated WDVGs compared to referenced natural arteries.

[0141] The suture retention strength was measured to verify the applicability of WMVGs for transplant surgery. As shown in FIG. 18B, the suture retention strength increased as the silk/PLA fiber content increased in WMVGs due to the rigidity of the silk and PLA. Furthermore, all of the WMVGs showed superior suture retention strength as compared to mammary arteries and saphenous veins.

[0142] The burst pressure of WMVGs was evaluated to determine if the porous WMVGs would leak in a physiological environment. During the test, PBS was continuously injected into the samples and the Laplace pressure prevented the grafts from leaking. As shown in FIG. 18C, the WMVGs with a higher TPU content showed a higher burst pressure. Without being bound by theory, it is believed this was because the densely packed TPU nanofibers yielded a higher Laplace pressure. However, all WMVGs showed a lower burst pressure than mammary arteries and saphenous veins due to their porous structures. The burst pressure was expected to increase when the grafts were filled with cells. Nevertheless, the burst pressure of WMVGs still exceeded typical human physiological blood pressure (ranging from 800 mmHg to 1400 mmHg). [0143] WMVGs were stretched and released for 50 cycles to determine their cyclic properties and were compared with human internal mammary artery (IMA) data from the literature as shown in FIGS. 18D-18F. WMVGs showed larger hysteresis loops in the first cycle than the latter cycles because the fibers loosened and reoriented in response to the external force. The cyclical stress-strain curves were all located within the upper and lower bounds of the IMA data, indicating that the WMVGs exhibited the non-linear tensile stress-strain behavior of native human arteries. The biomimetic mechanical behavior of WMVGs was attributed to the synergistic effects of biomaterial combinations and the special wavy- structure of WMVGs.

[0144] Modification using dopamine and heparin were employed to enhance the endothelial cell affinity and antithrombogenicity of the inner silk/PLA layer. FIGS. 19A-19I shows the morphology and XPS spectra of modified silk/PLA fibers. The as-spun silk/PLA fibers exhibited submicron-sized smooth fibers (FIGS. 19A & 19B). After dopamine modification, dopamine molecules were self-polymerized into submicron- sized particles and bonded onto silk/PLA fibers as shown in FIGS. 19D & 419E. XPS results (FIGS. 19C & 19F) indicated that the atom percentage of C increased after dopamine modification (FIG. 19F), which was mainly attributed to the aromatic ring of dopamine. After heparin coating, submicron heparin particles were uniformly attached to the silk/PLA fibers, and thin films also formed in some regions (FIGS. 19G & 19H). A percentage of 1.5% S element was detected on the XPS spectrum (FIG. 191), thus indicating the successful coating of heparin on the silk/PLA fibers.

[0145] The water contact angle results (FIG. 20A) showed that the hydrophilicity was dramatically enhanced after dopamine coating, while the contact angle of S/P-D&H was higher than S/P-DA. Although both dopamine and heparin are highly hydrophilic molecules, the significantly increased roughness of the S/P-D&H fiber surface prevented water penetration. Nevertheless, both modified silk/PLA fiber mats were hydrophilic while the as-spun, un modified silk/PLA (S/P) fiber mat was highly hydrophobic. The platelet adhesion test results (FIG. 20B) showed that the number of attached platelets decreased greatly when heparin was coated onto the fiber surface. From the SEM images, platelets mostly appeared round in shape and adhered to the fibers, especially at the intersections of multiple fibers. Some platelets on the unmodified silk/PLA fiber mat displayed a flat shape, thus implying a high thrombosis risk (FIG. 20C). The number of platelets decreased on the S/P-DA fiber mat and flat platelets were absent (FIG. 20D). Remarkably, platelets were barely present on the S/P-D&H fiber mat, indicating high antithrombogenicity (FIG. 20E). [0146] HUVECs were cultured on modified silk/PLA fiber mats for up to 14 days. Immunofluorescence images from the live/dead assay (FIG. 21 A) showed that HUVECs were able to attach and proliferate on all fiber mats, indicating that the fiber mats were all biocompatible with HUVECs. The MTS data (FIG. 21B) showed that S/P-DA and S/P-D&H samples had significantly higher cell populations than silk/PLA samples and control groups, while the difference between S/P-DA and S/P-D&H was not statistically significant. This indicated that the introduction of dopamine enhanced endothelial cell affinity, while the addition of heparin did not further improve endothelial cell affinity. In addition, the cell population increased more than two times from day 3 to day 7 for all samples, while the improvement from day 7 to day 14 was smaller for S/P-DA and S/P-D&H compared to silk/PLA. Presumably because cells already covered almost all of the available area on day 7 on S/P-DA and S/P-D&H, as indicated in FIG. 21 A. Moreover, all three fiber mats showed high cell viability (over 95%), as shown in FIG. 21C.

[0147] The cell phenotype was investigated using phalloidin/DAPI immunofluorescence staining and SEM. The cytoskeletons of HUVECs cultured on modified fiber mats are shown in the fluorescence images (FIG. 22A). Cells on all materials showed a spread morphology with clear fibrils indicating a healthy growing state. The cell proliferation showed the same trend as the MTS and live/dead assays. FIGS. 26 A and 26B depict measurements of the projected cell area (FIG. 26 A) and the cell aspect ratio (FIG. 26B). The results indicated that the cells on S/P-DA and S/P-D&H were larger and more spread out than the cells on silk/PLA at day 7. At day 14, the area and aspect ratio of the cells on S/P-DA and S/P-D&H decreased due to the significantly increased cell population; however, the cells on silk/PLA were more spread out compared to day 7.

[0148] Cell- substrate interactions were analyzed using SEM. As shown in FIG. 22B, HUVECs showed a flat shape on all fiber mats, while the cell size and area for the cells on silk/PLA were smaller than the cells on S/P-DA and S/P-D&H. The cells were tightly bonded to the fibers with pseudopodia extending out, indicating that the cells were able to freely migrate on these materials. The HUVECs on S/P-DA and S/P-D&H covered almost the whole substrate after 14 days of culture. This indicated that endothelial cells quickly formed a cell layer on the modified silk/PLA fibers, which would be beneficial for further improving the material’s mechanical properties (e.g. improving burst pressure) and preventing thromboses. [0149] To investigate whether endothelial cells were able to migrate on tubular grafts, the inner surface of WMVGs were modified with dopamine and heparin and then seeded with HUVECs for up to 14 days. As shown in FIG. 23, HUVECs were able to migrate upward in the lumen of the WMVG, although the cells were cultured in a stationary state. Cells were mostly present at the bottom of the WMVG at day 7 and the depth of cell coverage was 1075 pm. By day 14, cells had migrated upwards and were more uniformly distributed across the tube compared to day 7. The depth of cell coverage increased to 1620 pm and the cell population also greatly increased. Moreover, the cells maintained high viability. These results indicated that the modified WMVGs had excellent endothelial cell affinity and could stimulate fast endothelialization on the lumen side of the WMVGs.

[0150] Effectively preventing thromboses and stimulating fast endothelialization of small diameter vascular grafts remain as critical challenges for artificial SDVGs. Due to the complexity of the structure and composition of blood vessels, it is difficult to mimic their special non-linear tensile stress-strain relationship using only a single biomaterial. Previously, different biomaterials and fabrication methods were combined to prepare multiple-layered SDVGs with different materials and structures in each layer. Although the triple- layered structure and the non linear tensile stress-strain relationship of native blood vessels has been mimicked, the characteristics of the“toe region” has been difficult to achieve. The present disclosure provides a new method and material combination that closely resembles the microstructure and physiological properties of native blood vessels. Silk and PLA are both highly biocompatible, and biodegradable materials with high moduli can resemble the stiffer collagen component of blood vessels. Highly elastic TPU as a biocompatible elastomer is capable of resembling the elastin component of blood vessels. However, simply blending them together would result in a composite whose mechanical properties would lie somewhere in-between. It is known that, while less-wavy elastin plays a major role at low dilation pressures in native blood vessels, collagen fibrils eventually provide the needed strength at high dilation pressures. To achieve this property, a special design for the wavy inner layer was used. The wavy structure mimics the biological configuration of native blood vessels and provides a“toe region” in single- layered WMVGs. The performance of WMVGs of the present disclosure was further enhanced by using multiple materials with properties similar to the components in native blood vessels. As illustrated in FIG. 18G, at low pressure, the wavy silk/PLA fibers oriented and aligned in the same fashion as collagen in blood vessels. The elasticity and recoverability were provided by the elastic TPU layer, which corresponded to the initial“toe region” of native blood vessels. The rigid silk/PLA layer starts to play a major role when the pressure increases further. Through this unique structure, design, and material combination, the special non-linear tensile stress-strain relationship of native blood vessels was successfully mimicked.

[0151] Surprisingly, the silk/PLA solution of the present disclosure resulted in loosely packed fibers due to silk’s electrostatic charge. This special fibrous structure should facilitate cell penetration and tissue regeneration. One common problem faced by electro spinning SDVGs is the removal of samples from the mandrel without interfering with their delicate microstructure. Various methods such as using grease and winding the mandrel with copper wire have been used to assist with graft removal. With the assembled mandrel used in the method of the present disclosure, WMVGs were easily removed by pulling out the central tube first. However, the silk/PLA fibers tended to stick to the satellite cylinders due to electrostatic adhesion. Therefore, a thin PEO layer was electrospun first to assist with removing and harvesting the grafts.

[0152] After achieving the biomimetic mechanical properties of the WMVGs, the antithrombogenicity and endothelial cell affinity was determined. Although silk fibroin has been recognized as the most suitable biodegradable material for vascular grafts, its biocompatibility was further enhanced by incorporating biomolecules as described in the present disclosure. The results of modifying WMVGs by introducing dopamine showed that the HUVEC proliferation rate almost doubled after dopamine coating, and the cells also showed better cell-substrate interactions. The whole substrate was covered by an endothelial cell membrane within 14 days of culture. However, if WMVGs were directly implanted without pre-seeding with endothelial cells, thrombosis may occur since both silk/PLA and S/P-DA showed high platelet adhesion (FIGS. 20B and 20C). Further modification with heparin after dopamine coating dramatically reduced the number of attached platelets and improved antithrombogenicity. Moreover, the fibers modified with dopamine and heparin showed excellent endothelial cell affinity. Therefore, the modified WMVGs can be directly implanted without the need for pre-seeding endothelial cells. Although the heparin may be gradually released in vivo, the WMVG lumen surface should be rapidly covered by an endothelial cell membrane due to the greatly enhanced endothelial cell affinity.

[0153] The enhanced endothelial cell affinity was also demonstrated by the rapid migration of endothelial cells on stationary cultured WMVGs. A specially designed bioreactor is generally required for cell culture on vascular grafts since endothelial cells may find it difficult to migrate on tubular grafts when cultured in a stationary state and under the influence of gravity. Endothelial cells were able to migrate upward on the modified WMVG of the present disclosure in a stationary state due to the high cell affinity of the substrate without the need for any specially designed bioreactor. Therefore, small diameter vascular grafts (SDVG) of the present disclosure closely resemble the non-linear tensile stress-strain relationship of native blood vessels and possesses excellent endothelial cell affinity and antithrombogenicity.

[0154] The present disclosure provides a novel wavy, multi-component vascular graft (WMVG) with a wavy silk/PLA inner layer and an elastic TPU outer layer via electro spinning using a special assembled collector. The fabricated WMVG closely mimicked the non-linear tensile stress-strain relationship of native blood vessels and showed sufficient mechanical strength needed for implantation. Modification of the silk/PLA fibers with dopamine and heparin not only greatly enhanced endothelial cell migration and proliferation, but also gave the grafts antithrombogenicity. The WMVGs, which have biomimetic mechanical properties and endothelial cell affinity, can be mass produced, thus greatly reducing the treatment cost of CVD while increasing treatment efficacy.

Claims

CLAIMS What is claimed is:

1. A method for modifying a hydrophobic surface, the method comprising: treating the hydrophobic surface with oxygen plasma to form an oxygen plasma-treated surface; coating the oxygen plasma-treated surface with a solution comprising dopamine to form a dopamine- coated surface; coating the dopamine-coated surface with a solution comprising polymer comprising a terminal amine to form a polymer coating on the dopamine-coated surface; and immobilizing a bioactive molecule on the polymer coating by contacting the bioactive molecule with the polymer coating.

2. The method of claim 1 wherein the hydrophobic surface comprises polytetrafluoroethylene (PTFE), poly (lactic acid) (PLA), poly (lactic-co-glycolic acid) (PLGA), poly (e-caprolactone) (PCL), polyurethane (PU), polypropylene carbonate (PPC), polyhydroxybutyrate (PHB) and combinations thereof.

3. The method of claim 1, wherein the polymer comprising a terminal amine is present in the solution in an amount ranging from about 0.1 mg/mL to about 1 mg/mL.

4. The method of claim 1, wherein the immobilizing a bioactive molecule comprises immobilizing at least two bioactive molecules.

5. The method of claim 4, wherein the at least two bioactive molecules comprises a cell adhesion molecule and an anticoagulant.

6. The method of claim 1, wherein the bioactive molecule is a cell adhesion molecule selected from the group consisting of fibronectin, arginine-glycine-aspartic acid (RGD) peptide, arginine-glycine-aspartic acid-serine (RGDS) peptide, leucine-aspartic acid-valine (LDV) peptide, fibronectin CS1 region, laminin, tyrosine-iso leucine-glycine-serine-arginine (YIGSR) peptide, proline- aspartic acid-serine-glycine-arginine (PDSGR) peptide, lysine- arginine-glutamic acid (LRE) peptide, vitronectin, arginine-glycine-aspartic acid-valine (RGDV) peptide, and combinations thereof.

7. The method of claim 1, wherein the bioactive molecule is an anticoagulant selected from the group consisting of heparin, low molecular weight heparin, a coumarin, a directly acting oral anticoagulants, fondaparinux, idraparinux, a factor Xa inhibitor, a thrombin inhibitor, hementin, and combinations thereof.

8. The method of claim 1, wherein the hydrophobic surface comprises a vascular graft.

9. The method of claim 8, wherein the vascular graft is selected from a large diameter vascular graft, a small diameter vascular graft and combinations thereof.

10. The method of claim 8, wherein the vascular graft is a wavy multi-component vascular graft.

11. The method of claim 1 further comprising seeding a cell on the modified hydrophobic surface.

12. The method of claim 11, wherein the cell is selected from the group consisting of an endothelial cell, a smooth muscle cell, a mesenchymal stem cell, an umbilical vein endothelial cell, a fibroblast cell, and combinations thereof.

13. A method for modifying a substrate comprising a hydrophobic surface, the method comprising: treating the hydrophobic surface with oxygen plasma to form an oxygen plasma-treated surface; coating the oxygen plasma-treated surface with a solution comprising dopamine to form a dopamine-coated surface; coating the dopamine-coated surface with a solution comprising a polymer comprising a terminal amine to form a polymer coating on the dopamine-coated surface; and immobilizing a bioactive molecule on the polymer coating by contacting the bioactive molecule with the polymer coating.

14. The method of claim 13, wherein the substrate is selected from the group consisting of glass, metal, wood, cotton, plastic, ceramic, and combinations thereof.

15. The method of claim 13, wherein the hydrophobic surface comprises polytetrafluoroethylene (PTFE), poly (lactic acid) (PLA), poly (lactic-co-glycolic acid) (PLGA), poly (e-capro lactone) (PCL), polyurethane (PU), polypropylene carbonate (PPC), polyhydroxybutyrate (PHB) and combinations thereof.

16. The method of claim 13, wherein the polymer comprising a terminal amine is present in the solution in an amount ranging from about 0.1 mg/mL to about 1 mg/mL.

17. The method of claim 13, wherein the immobilizing a bioactive molecule comprises immobilizing at least two bioactive molecules.

18. The method of claim 17, wherein the at least two bioactive molecules comprises a cell adhesion molecule and an anticoagulant.

19. The method of claim 13, wherein the bioactive molecule is a cell adhesion molecule selected from the group consisting of fibronectin, arginine-glycine-aspartic acid (RGD) peptide, arginine-glycine-aspartic acid-serine (RGDS) peptide, leucine-aspartic acid-valine (LDV) peptide, fibronectin CS1 region, laminin, tyrosine-iso leucine-glycine-serine-arginine (YIGSR) peptide, proline- aspartic acid-serine-glycine-arginine (PDSGR) peptide, lysine- arginine-glutamic acid (LRE) peptide, vitronectin, arginine-glycine-aspartic acid-valine (RGDV) peptide, and combinations thereof.

20. The method of claim 13, wherein the bioactive molecule is an anticoagulant selected from the group consisting of heparin, low molecular weight heparin, a coumarin, a directly acting oral anticoagulants, fondaparinux, idraparinux, a factor Xa inhibitor, a thrombin inhibitor, hementin, and combinations thereof.

21. The method of claim 13 further comprising seeding a cell on the modified hydrophobic surface.

22. A modified hydrophobic substrate comprising a substrate comprising a hydrophobic surface, a first layer comprising dopamine disposed on the substrate, and a second layer comprising a terminal amine disposed on the first layer.

23. The modified hydrophobic substrate of claim 22, wherein the second layer comprises a polymer comprising the terminal amine.

24. The modified hydrophobic substrate of claim 23, wherein the polymer comprising a terminal amine is selected from the group consisting of polyethylenimine (PEI), polyethyleneglycol (PEG)-amine, polyalkylene oxide (PAO)-amine, polyallylamine, polyvinylamine, poly(vinylamine-co-vinylformamide), chitosan- amine, and poly(amidoamine).

25. The modified hydrophobic substrate of claim 23, wherein the polymer comprising a terminal amine is covalently bonded to the first layer comprising dopamine.

26. The modified hydrophobic substrate of claim 22 further comprising a third layer comprising at least one biomolecule.

27. The modified hydrophobic substrate of claim 26 wherein the at least one biomolecule is covalently bonded to the terminal amine of the second layer.

28. The modified hydrophobic substrate of claim 22, wherein the hydrophobic surface is an oxygen plasma treated surface.

29. A wavy multi-component vascular graft comprising an inner layer comprising rigid biopolymer fibers and an outer layer comprising elastic biopolymer fibers.

30. The wavy multi-component vascular graft of claim 29 wherein the rigid biopolymer fibers are selected from the group consisting of silk, poly(lactic acid) (PLA), polycaprolactone (PCL), poly(L-lactic acid) (PLLA), polylactic-co-glycolic acid (PLGA), poly(glycolic acid) (PGA), PLLA/PLGA copolymer, collagen, chitosan, alginate, and combinations thereof.

31. The wavy multi-component vascular graft of claim 29 wherein the elastic biopolymer fibers are selected from the group consisting of thermoplastic polyurethane (TPU), polyglycerol sebacate (PGS), poly(ester urethane) urea (PEUU), and combinations thereof.

32. The wavy multi-component vascular graft of claim 29 wherein the rigid biopolymer fibers comprise submicron diameter fibers.

33. The wavy multi-component vascular graft of claim 29 wherein the rigid biopolymer fibers comprise an average fiber diameter ranging from about 100 nm to about 1000 nm.

34. The wavy multi-component vascular graft of claim 29 wherein the elastic biopolymer fibers comprise nanoscale diameter fibers and submicron diameter fibers.

35. The wavy multi-component vascular graft of claim 29 wherein the elastic biopolymer fibers comprises an average fiber diameter ranging from about 50 nm to about 300 nm.

36. The wavy multi-component vascular graft of claim 29 further comprising a biomolecule.

37. The wavy multi-component vascular graft of claim 29 further comprising a cell.

38. The wavy multi-component vascular graft of claim 29 comprising a lumen diameter less than 6 mm.

39. The wavy multi-component vascular graft of claim 29 comprising a wall thickness ranging from about 200 pm to about 500 pm.

40. The wavy multi-component vascular graft of claim 29 comprising a suture retention strength ranging from about 1 N to about 4 N.

41. A method for preparing a wavy multi-component vascular graft, the method comprising: electrospinning a first solution comprising a water soluble polymer material to form a first water soluble fiber; collecting the first water soluble fiber on an assembled mandrel that comprises a central tube and a plurality of satellite cylinders surrounding the tube to form a first water soluble fiber layer; electrospinning a second solution comprising a rigid biopolymer material to form a second fiber; collecting the second fibers on the First water soluble fiber layer to form an inner rigid biopolymer fiber layer; electrospinning a third solution comprising an elastic biopolymer material to form a third fiber; collecting the third fibers on the rigid biopolymer fiber layer on the assembled mandrel to form an outer elastic biopolymer fiber layer; and removing the assembled mandrel to form a wavy multi-component vascular graft.

42. The method of claim 41 further comprising removing the water soluble fiber layer.

43. The method of claim 41 wherein the second solution and the third solution has a ratio ranging from about 1 :2 to about 2:1.

44. The method of claim 41 further comprising modifying the inner rigid biopolymer fiber layer.

45. The method of claim 44, wherein the inner rigid biopolymer fiber layer is modified with a biomolecule.

46. The method of claim 45, wherein the biomolecule is selected from the group consisting of dopamine, heparin, cell adhesion molecules, growth factors, chemokines, anticoagulants, and combinations thereof.

47. The method of claim 41 further comprising seeding a cell on the wavy multi- component vascular graft.

48. The method of claim 47, wherein the cell is selected from the group consisting of an endothelial cell, a smooth muscle cell, a mesenchymal stem cell, a fibroblast cell, and combinations thereof.