WO2019232177A1 - Stimulation de l'affinité des cellules endothéliales et de l'antithrombogénicité du polytétrafluoroéthylène (ptfe) par modification inspirée des moules et greffage rgd/héparine - Google Patents

Stimulation de l'affinité des cellules endothéliales et de l'antithrombogénicité du polytétrafluoroéthylène (ptfe) par modification inspirée des moules et greffage rgd/héparine Download PDF

Info

Publication number
WO2019232177A1
WO2019232177A1 PCT/US2019/034600 US2019034600W WO2019232177A1 WO 2019232177 A1 WO2019232177 A1 WO 2019232177A1 US 2019034600 W US2019034600 W US 2019034600W WO 2019232177 A1 WO2019232177 A1 WO 2019232177A1
Authority
WO
WIPO (PCT)
Prior art keywords
ptfe
cell
dopamine
vascular graft
peptide
Prior art date
Application number
PCT/US2019/034600
Other languages
English (en)
Inventor
Lih-Sheng Turng
Hao-yang MI
Original Assignee
Wisconsin Alumni Research Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wisconsin Alumni Research Foundation filed Critical Wisconsin Alumni Research Foundation
Publication of WO2019232177A1 publication Critical patent/WO2019232177A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/16—Macromolecular materials obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/28—Materials for coating prostheses
    • A61L27/34—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/507—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials for artificial blood vessels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/42—Anti-thrombotic agents, anticoagulants, anti-platelet agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/18—Modification of implant surfaces in order to improve biocompatibility, cell growth, fixation of biomolecules, e.g. plasma treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2420/00—Materials or methods for coatings medical devices
    • A61L2420/08—Coatings comprising two or more layers

Definitions

  • the present disclosure relates generally to tissue engineering.
  • 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.
  • 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.
  • PET polyethylene terephthalate
  • ePTFE expanded polytetrafluoroethylene
  • the massive blood flow in large-diameter blood vessels aids in the prevention of blood clots.
  • 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.
  • SDVGs small diameter vascular grafts
  • endothelial cells The primary physiological function of endothelial cells is to facilitate blood flow by providing a suitable hemocompatible and antithrombogenic surface.
  • vascular tissue engineering strategies 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.
  • 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.
  • 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.
  • 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.
  • the functional groups introduced via plasma treatment are limited and the introduced hydrophilic groups are not stable long-term.
  • 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.
  • 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.
  • modified hydrophobic substrates 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.
  • the methods can be used to enhance the biocompatibility of vascular grafts.
  • 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.
  • WMVGs multi-component vascular grafts
  • 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.
  • the biomimetic WMVGs fabricated provide candidates for CVD treatment
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • TPU thermoplastic polyurethane
  • 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.
  • FIG. 1 is a schematic depicting the surface modification procedure using PTFE.
  • PTFE was first treated with 0 2 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.
  • DA dopamine
  • PEI was immobilized on DA-PTFE
  • 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.
  • FIG. 3 depicts FTIR spectra of PTFE, P-PTFE, DA-PTFE, RGD-PTFE, and R/H-PTFE. Two regions are enlarged for better comparison.
  • FIG. 4 depicts XPS survey scans of PTFE, P-PTFE, DA-PTFE, RGD-PTFE, and R/H-PTFE.
  • 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.
  • FIG. 6 depicts SEM images of the surface morphologies of PTFE, P-PTFE, DA-PTFE, RGD-PTFE, and R/H-PTFE samples.
  • 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.
  • FIG. 8 depicts cross-sectional AFM images corresponding height profiles from the lines drawn on each image from FIG. 7.
  • 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).
  • 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.
  • 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).
  • 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).
  • FIG. 14 depicts fluorescence images showing the cytoskeleton of HUVECs cultured on different PTFE substrates for 14 days.
  • FIG. 15 depicts SEM images of HUVECs cultured on different PTFE substrates for 7 days showing the interaction between cells and substrate.
  • FIG. 16 is a schematic depicting the fabrication procedure for wavy multi- component vascular grafts (WMVGs) using a custom-designed rotating collector.
  • WMVGs wavy multi- component vascular grafts
  • 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.
  • FIGS. 18A-18G depict the mechanical properties of WMVGs.
  • FIG. 18G is a schematic illustration depicting the WMVG’s cyclic circumferential expansion behavior mimicking native blood vessels.
  • 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.
  • SEM scanning electron micrograph
  • XPS X-ray photoelectron spectrometer
  • 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.
  • 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.
  • 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.
  • FIGS. 22A-22B depict cytoskeletal morphology (FIG. 22A) and cellular morphology (FIG. 22B) of HUVECs cultured on differently modified silk/PLA.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • Any suitable method for treating the hydrophobic surface with oxygen plasma can be used.
  • Commercially available plasma etchers e.g., PlasmaEtch PE-200
  • PlasmaEtch PE-200 can be used to oxygen plasma treat the hydrophobic surfaces.
  • 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.
  • PTFE polytetrafluoroethylene
  • PLA poly (lactic acid)
  • PLA poly (lactic-co-glycolic acid)
  • PCL poly (e-caprolactone)
  • PU polyurethane
  • PPC polypropylene carbonate
  • PPC polyhydroxybutyrate
  • the dopamine coating can be prepared by contacting the oxygen plasma- treated surface with a solution comprising dopamine to form the dopamine-coated surface.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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-
  • 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.
  • at least two bioactive molecules can be immobilized on the polymer coating.
  • 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.
  • one of the at least two bioactive molecules is a RGD peptide and the other of the at least two bioactive molecules is heparin.
  • 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.
  • 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.
  • “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.
  • “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.
  • the hydrophobic surface is the inner surface of wavy multi- component vascular graft (WMVG) as described below.
  • WMVG wavy multi- component vascular graft
  • 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.
  • Suitable substrates include glasses, metals, woods, cotton, plastics, ceramics, and combinations thereof.
  • any suitable method for treating the hydrophobic surface with oxygen plasma can be used.
  • Commercially available plasma etchers e.g., PlasmaEtch PE-200
  • 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.
  • the dopamine coating can be prepared by contacting the oxygen plasma- treated surface with a solution comprising dopamine to form the dopamine-coated surface.
  • 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.
  • 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).
  • PEI polyethylenimine
  • PEG polyethyleneglycol
  • PAO polyalkylene oxide
  • polyallylamine polyvinylamine
  • poly(vinylamine-co-vinylformamide) poly(amidoamine)
  • the polyethyleneimine suitably can be linear, dendritic, comb, or branched.
  • 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.
  • 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.
  • 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-
  • 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.
  • At least two bioactive molecules can be immobilized on the PEI coating.
  • 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.
  • one of the at least two bioactive molecules is a RGD peptide and the other of the at least two bioactive molecules is heparin.
  • 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.
  • 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.
  • the hydrophobic surface is an oxygen plasma treated surface.
  • the hydrophobic surface is a surface of a substrate.
  • Suitable substrates include glasses, metals, woods, cotton, plastics, ceramics, and combinations thereof.
  • 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.
  • the polymer having a terminal amine is covalently bonded to the first layer comprising dopamine.
  • 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)
  • 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.
  • 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.
  • “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.
  • “wavy” refers to a surface having a series of undulating and wavelike curves.
  • 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).
  • 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.
  • 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.
  • 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.
  • elastic biopolymers fibers include an average fiber diameter average fiber diameter ranging from about 50 nm to about 300 nm.
  • 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) peptid
  • 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 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • silk and PLA fibers should degrade within 6 months, while the TPU fibers could be maintained for about two years.
  • the inner fiber layer desirably degrades within about 6 months, while the outer fiber layer is maintained for about 2 years.
  • 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.
  • electrospinning refers to any method where materials are streamed, sprayed, sputtered, dripped, or otherwise transported in the presence of an electric field.
  • 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.
  • 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).
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • elastic biopolymers fibers include an average fiber diameter ranging from about 50 nm to about 300 nm.
  • a volume ratio of the rigid biopolymer solution to the elastic biopolymer solution can range from about 1:2 to about 2:1.
  • 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.
  • elastic biopolymers fibers include an average fiber diameter average fiber diameter ranging from about 50 nm to about 300 nm.
  • 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.
  • 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.
  • RGD arginine-glycine-aspartic acid
  • 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.
  • 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.
  • 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 cm 3 /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.
  • DA dopamine
  • Dopamine-coated P-PTFE sheets were named DA-PTFE.
  • 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.
  • RGD solution 100 pg/mL
  • RGD/heparin solution 100 pg/mL for RGD and 1 mg/mL for heparin
  • RGD-PTFE RGD-grafted PTFE
  • R/H-PTFE RGD/heparin- grafted PTFE
  • FTIR Fourier transform infrared
  • 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.
  • 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.
  • PBS phosphate- buffered saline
  • 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.
  • HUVECs were detached enzymatically with a trypsin- EDTA solution and seeded on the samples at a density of lxlO 4 cells/cm 2 for the live/dead assay and MTS assay. They were seeded at a density of lxlO 3 cells/cm 2 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.
  • 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.
  • 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.
  • phalloidin-TMRho phalloidin-tetramethylrhodamine B isothiocyanate
  • 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.
  • PEI polyethylenimine
  • 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.
  • 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.
  • the Cls core level scans show the detailed information of carbon-containing bonds on the material surface (FIG. 5).
  • Table 2 The proportion of CF 2 and CF 3 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.
  • 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.
  • modified PTFE sheets were 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.
  • DA-PTFE Dopamine-coated PTFE
  • RGD-PTFE 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.
  • PLA poly(lactic acid)
  • 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.
  • 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.
  • WCA water contact angle
  • the WCA decreased after each modification step because more hydrophilic components were introduced in each step.
  • the surface roughness gradually increased as demonstrated above.
  • the combination of these two factors caused increased platelet adhesion after modification.
  • R/H- PTFE showed very low platelet adhesion although it possessed the lowest WCA and highest surface roughness.
  • 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).
  • 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.
  • P-PTFE After 0 2 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.
  • FIG. 13 A shows the cytoskeleton of HUVECs cultured on different PTFE substrates for 7 days.
  • 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.
  • 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).
  • 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.
  • the number of cells greatly increased on the DA- PTFE, RGD-PTFE, and R/H-PTFE samples (FIG. 14).
  • 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.
  • 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.
  • 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.
  • 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.
  • the satellite cylinders expand (jump ro P 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.
  • 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.
  • WMVGs 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.
  • 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.
  • 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.
  • 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
  • DA Dopamine
  • 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.
  • 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).
  • XPS X-ray photoelectron spectrometer
  • WCA Water contact angle
  • 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.
  • 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.
  • 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.
  • HUVECs 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.
  • UV ultraviolet
  • HUVECs were detached enzymatically with a trypsin-EDTA solution and seeded on the samples at a density of lxlO 4 cells/cm 2 for the live/dead assay and the MTS assay. They were also seeded at a density of lxlO 3 cells/cm 2 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.
  • HUVECs were also seeded on dopamine- and heparin-modified WMVGs to investigate cell proliferation.
  • WMVG dopamine- and heparin-modified WMVGs
  • 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 2xl0 5 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.
  • 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
  • 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.
  • phalloidin-TMRho phalloidin-tetramethylrhodamine B isothiocyanate
  • phalloidin-TMRho phalloidin-tetramethylrhodamine B isothiocyanate
  • 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.
  • DAPI 6-diamidino-2-phenylindole
  • 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.
  • 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.
  • the fabricated WMVGs featured a wavy inner layer made of silk/PLA microfibers and a smooth TPU nanofiber outer layer.
  • 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 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.
  • 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.
  • 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.
  • the WMVGs became more flexible as indicated by their reduced strength and modulus as well as increased elongation-at-break (Table 3).
  • 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 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.
  • the burst pressure of WMVGs was evaluated to determine if the porous WMVGs would leak in a physiological environment.
  • PBS was continuously injected into the samples and the Laplace pressure prevented the grafts from leaking.
  • 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.
  • 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.
  • 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.
  • IMA human internal mammary artery
  • 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.
  • 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).
  • dopamine molecules were self-polymerized into submicron- sized particles and bonded onto silk/PLA fibers as shown in FIGS. 19D & 419E.
  • XPS results FIGGS. 19C & 19F indicated that the atom percentage of C increased after dopamine modification (FIG.
  • 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.
  • 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.
  • 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.
  • WMVGs were modified with dopamine and heparin and then seeded with HUVECs for up to 14 days.
  • 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.
  • 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.
  • collagen fibrils eventually provide the needed strength at high dilation pressures.
  • 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.
  • WMVGs of the present disclosure were further enhanced by using multiple materials with properties similar to the components in native blood vessels.
  • 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.
  • 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.
  • WMVGs were easily removed by pulling out the central tube first.
  • 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.
  • the antithrombogenicity and endothelial cell affinity was determined.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Dermatology (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Epidemiology (AREA)
  • Public Health (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Vascular Medicine (AREA)
  • Materials For Medical Uses (AREA)

Abstract

L'invention concerne des procédés de modification d'un substrat présentant une surface hydrophobe. L'invention concerne également des substrats hydrophobes modifiés. L'invention concerne en outre de nouvelles greffes vasculaires ondulées à composants multiples (WMVG) présentant une couche interne ondulée de fibres biopolymères rigides et une couche externe de fibres biopolymères élastiques et un procédé de préparation de WMVG par électrofilage à l'aide d'un collecteur assemblé particulier. Les substrats hydrophobes modifiés et les procédés divulgués dans la description améliorent avantageusement l'affinité des cellules et l'antithrombogénicité de surfaces hydrophobes telles que les surfaces internes des WMVG.
PCT/US2019/034600 2018-05-30 2019-05-30 Stimulation de l'affinité des cellules endothéliales et de l'antithrombogénicité du polytétrafluoroéthylène (ptfe) par modification inspirée des moules et greffage rgd/héparine WO2019232177A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201862677939P 2018-05-30 2018-05-30
US201862677931P 2018-05-30 2018-05-30
US62/677,931 2018-05-30
US62/677,939 2018-05-30

Publications (1)

Publication Number Publication Date
WO2019232177A1 true WO2019232177A1 (fr) 2019-12-05

Family

ID=68698987

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2019/034600 WO2019232177A1 (fr) 2018-05-30 2019-05-30 Stimulation de l'affinité des cellules endothéliales et de l'antithrombogénicité du polytétrafluoroéthylène (ptfe) par modification inspirée des moules et greffage rgd/héparine

Country Status (1)

Country Link
WO (1) WO2019232177A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114437402A (zh) * 2020-10-19 2022-05-06 中国石油化工股份有限公司 一种超疏水聚氨酯海绵及其制备方法和应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160302911A1 (en) * 2013-12-27 2016-10-20 Neograft Technologies, Inc. Artificial graft devices and related systems and methods
WO2016187698A1 (fr) * 2015-05-26 2016-12-01 The University Of British Columbia Compositions de revêtement polymères antisalissures
US20170173226A1 (en) * 2013-03-15 2017-06-22 Baxter International Inc. Immobilization of an Active Agent on a Substrate
WO2018053265A1 (fr) * 2016-09-16 2018-03-22 University Of Kansas Vaisseaux sanguins obtenus par ingénierie

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170173226A1 (en) * 2013-03-15 2017-06-22 Baxter International Inc. Immobilization of an Active Agent on a Substrate
US20160302911A1 (en) * 2013-12-27 2016-10-20 Neograft Technologies, Inc. Artificial graft devices and related systems and methods
WO2016187698A1 (fr) * 2015-05-26 2016-12-01 The University Of British Columbia Compositions de revêtement polymères antisalissures
WO2018053265A1 (fr) * 2016-09-16 2018-03-22 University Of Kansas Vaisseaux sanguins obtenus par ingénierie

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MI ET AL.: "Promoting Endothelial Cell Affinity and Antithrombogenicity of Polytetrafluoroethylene (PTFE) by Mussel-Inspired Modification and RGD/Heparin Grafting", JOURNAL OF MATERIALS CHEMISTRY B, vol. 6, no. 21, 9 May 2018 (2018-05-09), pages 3475 - 3485, XP055659451 *
YU ET AL.: "Development of Biomimetic Thermoplastic Polyurethane/Fibroin Small-Diameter Vascular Grafts via a Novel Electrospinning Approach", JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A, vol. 106, no. 4, 5 December 2017 (2017-12-05), pages 985 - 996, XP055628079, DOI: 10.1002/jbm.a.36297 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114437402A (zh) * 2020-10-19 2022-05-06 中国石油化工股份有限公司 一种超疏水聚氨酯海绵及其制备方法和应用
CN114437402B (zh) * 2020-10-19 2023-12-08 中国石油化工股份有限公司 一种超疏水聚氨酯海绵及其制备方法和应用

Similar Documents

Publication Publication Date Title
US11786639B2 (en) Promoting endothelial cell affinity and antithrombogenicity of polytetrafluoroethylene (PTFE) by mussel-inspired modification and RGD/heparin grafting
Zhu et al. Synthesis of RGD-peptide modified poly (ester-urethane) urea electrospun nanofibers as a potential application for vascular tissue engineering
Nazeer et al. Electrospun polycaprolactone/silk fibroin nanofibrous bioactive scaffolds for tissue engineering applications
Avsar et al. Levan based fibrous scaffolds electrospun via co-axial and single-needle techniques for tissue engineering applications
Teo et al. Electrospun scaffold tailored for tissue‐specific extracellular matrix
Yuan et al. Highly aligned core–shell structured nanofibers for promoting phenotypic expression of vSMCs for vascular regeneration
Yuan et al. Hemocompatible surface of electrospun nanofibrous scaffolds by ATRP modification
Cipitria et al. Design, fabrication and characterization of PCL electrospun scaffolds—a review
Mi et al. Promoting endothelial cell affinity and antithrombogenicity of polytetrafluoroethylene (PTFE) by mussel-inspired modification and RGD/heparin grafting
Jacobs et al. Plasma surface modification of biomedical polymers: influence on cell-material interaction
Feng et al. The influence of type-I collagen-coated PLLA aligned nanofibers on growth of blood outgrowth endothelial cells
Amirian et al. Designing of combined nano and microfiber network by immobilization of oxidized cellulose nanofiber on polycaprolactone fibrous scaffold
Zhou et al. Peptide-modified PELCL electrospun membranes for regulation of vascular endothelial cells
US20190365953A1 (en) Wavy multi-component vascular grafts with biomimetic mechanical properties, antithrombogenicity, and endothelial cell affinity
Thomas et al. Electrospinning of Biosyn®-based tubular conduits: structural, morphological, and mechanical characterizations
Ghobeira et al. Plasma surface functionalization of biodegradable electrospun scaffolds for tissue engineering applications
Jing et al. In vitro evaluations of electrospun nanofiber scaffolds composed of poly (ɛ-caprolactone) and polyethylenimine
Junkar Interaction of cells and platelets with biomaterial surfaces treated with gaseous plasma
Karahaliloğlu Electrospun PU-PEG and PU-PC hybrid scaffolds for vascular tissue engineering
Shahriari-Khalaji et al. Advancements in the fabrication technologies and biomaterials for small diameter vascular grafts: A fine-tuning of physicochemical and biological properties
Xu et al. Electrostatic self-assemble modified electrospun poly-L-lactic acid/poly-vinylpyrrolidone composite polymer and its potential applications in small-diameter artificial blood vessels
Kurokawa et al. Electrospinning and surface modification methods for functionalized cell scaffolds
WO2019232177A1 (fr) Stimulation de l'affinité des cellules endothéliales et de l'antithrombogénicité du polytétrafluoroéthylène (ptfe) par modification inspirée des moules et greffage rgd/héparine
Jang et al. Fibroblast culture on poly (L-lactide-co-ɛ-caprolactone) an electrospun nanofiber sheet
Sahebalzamani et al. Enhancement of fibroblasts outgrowth onto polycaprolactone nanofibrous grafted by laminin protein using carbon dioxide plasma treatment

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19811462

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19811462

Country of ref document: EP

Kind code of ref document: A1