Sulfide-based, pro-regenerative, Anti-inflammatory implants

Abstract

The present invention relates to a vascular graft, and more particularly, to a vascular implant having a sulfide-based structure for minimizing inflammation and for encouraging cell tissue regeneration. It describes a design of coaxial nanofibrous systems with slow release H2S to reduce inflammation and encourage cell tissue regeneration.

Description

[0001] Patent Attorney Docket No. UTCB-20718

[0002] SULFIDE-BASED, PRO-REGENERATIVE, ANTI-INFLAMMATORY IMPLANTS

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] The present application claims the benefit of U.S. Provisional Patent Application No. 63 / 696,342, filed on September 18, 2024, which is incorporated herein by reference.

[0005] STATEMENT OF GOVERNMENTAL SUPPORT

[0006] This invention was made with government support under grant number HK119371 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] FIELD OF THE INVENTION

[0008] The present invention relates to H2S-releasing coatings, grafts, or implants, and more particularly, to a vascular implant exhibiting a slow release of H2S within a physiological-relevant range for minimizing inflammation and for encouraging cell tissue regeneration.

[0009] BACKGROUND OF THE INVENTION

[0010] Vascular grafts play a critical role in the contemporary management of a wide range of clinical conditions, including atherosclerotic disease, vascular aneurysm, congenital malformation, vasculitis, arterial bypass, and traumatic injury. While large-sized grafts possess great success rate, small-diameter (<6mm) grafts do not. In most cases, autologous tissue, such as internal thoracic artery or saphenous vein, is the first choice for these grafts. However, prior surgeries and comorbid Patent

[0011] Attorney Docket No. UTCB-20718 medical conditions may limit the availability of a patient’s own vessels, and their harvest can be associated with significant morbidity. The use of synthetic materials for small-diameter grafts, however, has been associated with high rates of thrombus formation and intimal hyperplasia. To facilitate long-term patency, there remains a substantial unfulfilled need for readily-available small-caliber synthetic vascular grafts with reduced rates of thrombosis as well as low intimal hyperplasia and capsule thickness, both of which are associated with inflammation.

[0012] SUMMARY OF THE INVENTION

[0013] This invention is described in preferred embodiments in the following description with reference to the Figures, in which like numbers represent the same or similar elements. Reference throughout this specification to “one embodiment,” “an embodiment,” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

[0014] The described features, structures, or characteristics of the invention may be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details are recited to provide a thorough understanding of embodiments of the invention. One skilled in the relevant art will recognize, however, that the invention may be practiced without one or more of the specific details, or with other methods, components, materials, and so forth. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the invention. Patent

[0015] Attorney Docket No. UTCB-20718

[0016] The present invention relates to H2S-releasing coatings, grafts, or implants, and more particularly, to a vascular implant exhibiting a slow release of H2S within a physiological-relevant range for minimizing inflammation and for encouraging cell tissue regeneration.

[0017] In one embodiment, the invention contemplates a medical implant exhibiting a slow release of H2S. In one embodiment, said slow release comprises within a physiological -relevant range (from low nanomolar to high micromolar concentration) over a period of several days or weeks. In one embodiment, said slow release comprises the low nanomolar range (10'8- 10'9M) to high micromolar concentrations, up to 300 micromolar (10'4M), preferably in the low micromolar range (~ 10’5- 10'6M). In one embodiment, said release of H2S is sustained over a period of time, such as several days or weeks. In one embodiment, said implant comprises a coaxial microfibrous system impregnated with an H2S donor. In one embodiment, said implant comprises a coaxial nanofibrous system impregnated with an H2S donor. In one embodiment, said H2S donor is a polysulfide. In one embodiment, said polysulfide is a natural polysulfide. In one embodiment, said polysulfide is a garlic-derived polysulfide. In one embodiment, said H2S donor comprises diallyl trisulfide (DATS). In one embodiment, said H2S donor comprises diallyl disulfide (DADS). In one embodiment, said H2S donor comprises diallyl tetrasulfide (DATTS). In one embodiment, said H2S donor comprises an organic sulfide such as allyl polysulfides, benzyl polysulfides, and organic disulfides and thiosulfonates. In one embodiment, said implant comprises a coaxial nanofibrous coating. In one embodiment, said implant comprises at least one polyethylene-glycol derivative. In one embodiment, said medical implant comprises a core with a coaxial sheath. In one embodiment, said wherein said medical implant encourages cell tissue regeneration. In one embodiment, said wherein said medical implant reduces inflammation. In one embodiment, said wherein said medical implant reduces cardiometabolic dysfunction. In one embodiment, said Patent

[0018] Attorney Docket No. UTCB-20718 wherein said medical implant treats cancer. In one embodiment, said wherein said treating cancer is associated with selective protein S-thiolation, anti-oxidative anti-inflammatory activity. In one embodiment, said core is a hydrophobic and degradable core. In one embodiment, said medical implant comprises a vascular stent. In one embodiment, said medical implant comprises a vascular graft. In one embodiment, said the concentration of diallyl trisulfide in said implant is between 10 pM and 250 pM. In one embodiment, said the concentration of diallyl trisulfide in said implant is between 25 pM and 150 pM. In one embodiment, said medical implant is selected from the group consisting of vascular graft, heart valve, cardiovascular implants, and tissue-regenerative implant. In one embodiment, said at least one polyethylene-glycol derivative comprises at least one polymer selected from the group consisting of polyethylene-glycol dimethacrylate, polyethylene-glycol modified with thiol-ene, and polyethylene-glycol modified with hydroxy acid groups. In one embodiment, said hydrophobic, degradable core comprises a at least one polymer from the group consisting of poly L-lactide acid, polycaprolactone, polyglycolic acid, poly(lactic-co-glycolic acid), and polyurethane. In one embodiment, said hydrophobic, degradable core comprises a poly L-lactide acid core. In one embodiment, said hydrophobic, degradable core comprises at least one polymer selected from the group consisting of poly L-lactide acid, polycaprolactone, polyglycolic acid, poly(lactic-co-glycolic acid), and polyurethane. In one embodiment, said coating further comprises a controlled release agent. In one embodiment, said coating further comprises a therapeutic agent. In one embodiment, said therapeutic agent comprises an anti-proliferation agent. In one embodiment, said hydrophobic, degradable core provided controlled drug release. In one embodiment, said sheath integrates a surface signaling mechanism. In one embodiment, said surface signaling mechanism that simultaneously aids in regeneration. In one embodiment, said implant further comprises an interactive surface. In one embodiment, said implant further Patent Attorney Docket No. UTCB-20718 comprises sub-millimeter scale fibers. In one embodiment, said sub-millimeter scale fibers comprises nanoscale fibers. In one embodiment, said sub-millimeter scale fibers provide a uniform surface. In one embodiment, said sub-millimeter scale fibers comprise a cell recognition platform. In one embodiment, said implant has no delaminiation. In one embodiment, said the elasticity of said polyethylene-glycol sheath is tunable by varying the photopolymerization time. In one embodiment, said implant comprises crosslinked coaxial nanofiber system. In one embodiment, said nanofiber system comprises poly-£-caprolactone core with a coaxial sheath comprising poly(ethylene glycol) norbornene-RGD. In one embodiment, said nanofiber system comprises coaxial PCL-gelatin (PCL+GEL) nanofiber system, crosslinked with genipin. In one embodiment, said nanofiber system comprises coaxial ng PCL / PEGNB-RGD coaxial fiber system, crosslinked with PEG dithiol. In one embodiment, said nanofiber system is impregnated with diallyl trisulfide. In one embodiment, said implant comprises a coating on an existing implant platform, such as a stent. In one embodiment, said coating comprises an adhesive layer. The present invention is not to be limited by the shape of the implant. In one embodiment, said implant comprises a flat sheet. In one embodiment, said implant is cylindrical. In one embodiment, said implant comprises a 3-D printed shape.

[0019] In one embodiment, the invention contemplates a medical implant with a coating comprising a coaxial micro or nanofibrous system impregnated with H2S donor comprising at least one polyethylene-glycol derivative. In one embodiment, said medical implant comprises a core with a coaxial sheath. In one embodiment, said wherein said medical implant encourages cell tissue regeneration. In one embodiment, said wherein said medical implant reduces inflammation. In one embodiment, said core is a hydrophobic and degradable core. In one embodiment, said medical implant comprises a vascular stent. In one embodiment, said medical implant comprises a vascular Patent

[0020] Attorney Docket No. UTCB-20718 graft. In one embodiment, said medical implant provides a slow release of H2S. In one embodiment, said slow release comprises within a physiological -relevant range (from low nanomolar to high micromolar concentration) over a period of several days or weeks. In one embodiment, said release of H2S is sustained over a period of time, such as several days or weeks In one embodiment, said slow release comprises the low nanomolar range (10‘8- ICT9M) to high micromolar concentrations, up to 300 micromolar (10‘4M), preferably in the low micromolar range (~ 10'5- 10’6M). In one embodiment, said implant comprises a coaxial microfibrous system impregnated with an H2S donor. In one embodiment, said implant comprises a coaxial nanofibrous system impregnated with an H2S donor. In one embodiment, said H2S donor is a polysulfide. In one embodiment, said polysulfide is a natural polysulfide. In one embodiment, said polysulfide is a garlic-derived polysulfide. In one embodiment, said H2S donor comprises diallyl trisulfide (DATS). In one embodiment, said H2S donor comprises diallyl disulfide (DADS). In one embodiment, said H2S donor comprises diallyl tetrasulfide (DATTS). In one embodiment, said H2S donor comprises an organic sulfide such as allyl polysulfides, benzyl polysulfides, and organic disulfides and thiosulfonates. In one embodiment, said implant comprises a coaxial nanofibrous coating. In one embodiment, said the concentration of diallyl trisulfide in said implant is between 10 pM and 250 pM. In one embodiment, said the concentration of diallyl trisulfide in said implant is between 25 pM and 150 pM. In one embodiment, said medical implant is selected from the group consisting of vascular graft, heart valve, cardiovascular implants, and tissue-regenerative implant. In one embodiment, said at least one polyethylene-glycol derivative comprises at least one polymer selected from the group consisting of polyethylene-glycol dimethacrylate, polyethylene-glycol modified with thiol-ene, and polyethylene-glycol modified with hydroxy acid groups. In one embodiment, said hydrophobic, degradable core comprises a at least one polymer from the group Patent Attorney Docket No. UTCB-20718 consisting of poly L-lactide acid, polycaprolactone, polyglycolic acid, poly(lactic-co-glycolic acid), and polyurethane. In one embodiment, said hydrophobic, degradable core comprises a poly L-lactide acid core. In one embodiment, said hydrophobic, degradable core comprises at least one polymer selected from the group consisting of poly L-lactide acid, polycaprolactone, polyglycolic acid, poly(lactic-co-glycolic acid), and polyurethane. In one embodiment, said coating further comprises a controlled release agent. In one embodiment, said coating further comprises a therapeutic agent. In one embodiment, said therapeutic agent comprises an anti-proliferation agent. In one embodiment, said hydrophobic, degradable core provided controlled drug release. In one embodiment, sheath integrates a surface signaling mechanism. In one embodiment, said surface signaling mechanism that simultaneously aids in regeneration. In one embodiment, said implant further comprises an interactive surface. In one embodiment, said implant further comprises submillimeter scale fibers. In one embodiment, said sub-millimeter scale fibers comprises nanoscale fibers. In one embodiment, said sub-millimeter scale fibers provide a uniform surface. In one embodiment, said sub-millimeter scale fibers comprise a cell recognition platform. In one embodiment, said implant has no delaminiation. In one embodiment, said the elasticity of said polyethylene-glycol sheath is tunable by varying the photopolymerization time. In one embodiment, said implant comprises a coating on an existing implant platform, such as a stent. In one embodiment, said coating comprises an adhesive layer. The present invention is not to be limited by the shape of the implant. In one embodiment, said implant comprises a flat sheet. In one embodiment, said implant is cylindrical. In one embodiment, said implant comprises a 3-D printed shape.

[0021] In one embodiment, the invention contemplates a method of producing a medical implant comprising a core with a coaxial sheath comprising: a) providing; i) a core polymer solution Patent Attorney Docket No. UTCB-20718 comprising at least one polymer and at least one said H2S donor in a solvent; ii) a polymer sheath solution in a solvent; and iii) a collecting target; b) extruding said combining said core solution and said sheath solution under conditions to create a hybrid fiber comprising a polymer core with a coaxial sheath polymer; and c) collecting said extruded hybrid fiber on said collecting target to create a medical implant. In one embodiment, said polymer sheath solution further comprises a photoinitiator. In one embodiment, the method further comprises step d) photo-polymerizing said hybrid fiber. In one embodiment, said H2S donor is selected from the group consisting of diallyl trisulfide, diallyl disulfide and diallyl tetrasulfide. In one embodiment, said core polymer solution comprises poly-a-caprolactone. In one embodiment, said sheath polymer solution comprises at least one polyethylene glycol derivative. . In one embodiment, said collecting comprises electrospinning said hybrid fiber upon said collecting target. In one embodiment, said medical implant comprises a vascular stent. In one embodiment, said medical implant is selected from the group consisting of vascular graft, heart valve, cardiovascular implants, and tissue-regenerative implant. In one embodiment, said polyethylene glycol norbornene is RGD-functionalized. In one embodiment, said implant comprises a coating on an existing implant platform, such as a stent. In one embodiment, said coating comprises an adhesive layer. The present invention is not to be limited by the shape of the implant. In one embodiment, said implant comprises a flat sheet. In one embodiment, said implant is cylindrical. In one embodiment, said implant comprises a 3-D printed shape.

[0022] In one embodiment, the invention contemplates a method of producing a medical implant comprising a poly-£-caprol acton e core with a coaxial sheath comprising poly(ethylene glycol) norbomene-RGD comprising: a. providing; i. a core solution comprising diallyl trisulfide (DATS) and poly-s-caprolactone (PCL) in a solvent; ii. a sheath solution comprising polyethylene glycol Patent Attorney Docket No. UTCB-20718

[0023] (PEG), 4-arm poly(ethylene glycol) norbomene (PEG-NB), poly(ethylene glycol) dithiol (PEG- Dithiol), arginylglycylaspartic acid (RGD peptide), and a photoinitiator in a solvent; and iii. a collecting target; b. extruding said combining said core solution and said sheath solution under conditions to create a hybrid fiber comprising a poly-s-caprolactone with diallyl trisulfide core with a coaxial sheath comprising poly(ethylene glycol) norbornene-RGD; c. collecting said extruded hybrid fiber on said collecting target to create a medical implant; and d. photopolymerizing said hybrid fiber. In one embodiment, said method further comprises step e) exposing said implant to anaerobic conditions. In one embodiment, said photo-polymerizing comprises UV exposure. In one embodiment, said collecting comprises electrospinning said hybrid fiber upon said collecting target. In one embodiment, said medical implant comprises a vascular stent. In one embodiment, said medical implant is selected from the group consisting of vascular graft, heart valve, cardiovascular implants, and tissue-regenerative implant. In one embodiment, said polyethylene glycol norbornene is RGD-functionalized. In one embodiment, said implant comprises a coating on an existing implant platform, such as a stent. In one embodiment, said coating comprises an adhesive layer. The present invention is not to be limited by the shape of the implant. In one embodiment, said implant comprises a flat sheet. In one embodiment, said implant is cylindrical. In one embodiment, said implant comprises a 3-D printed shape.

[0024] In one embodiment, the invention contemplates a method of producing a medical implant comprising a poly-s-caprolactone core with a coaxial sheath comprising poly(ethylene glycol) norbornene-RGD comprising: a) providing; i) a core solution comprising diallyl trisulfide (DATS) and poly-s-caprolactone (PCL) in a solvent; ii) a sheath solution comprising gelatin in a solvent; and iii) a collecting target; b) extruding said combining said core solution and said sheath solution under conditions to create a hybrid fiber comprising a poly-s-caprolactone with diallyl trisulfide Patent Attorney Docket No. UTCB-20718 core with a coaxial sheath comprising gelatin; c) collecting said extruded hybrid fiber on said collecting target to create a medical implant; and d) treating said medical implant with a crosslinking agent. In one embodiment, the method further comprises step e) exposing said implant to anaerobic conditions. In one embodiment, said treating comprises exposure to genipin. In one embodiment, said treating comprises immersion exposure to genipin. In one embodiment, said collecting comprises electrospinning said hybrid fiber upon said collecting target. In one embodiment, said medical implant comprises a vascular stent. In one embodiment, said medical implant is selected from the group consisting of vascular graft, heart valve, cardiovascular implants, and tissue-regenerative implant. In one embodiment, said implant comprises a coating on an existing implant platform, such as a stent. In one embodiment, said coating comprises an adhesive layer. The present invention is not to be limited by the shape of the implant. In one embodiment, said implant comprises a flat sheet. In one embodiment, said implant is cylindrical. In one embodiment, said implant comprises a 3-D printed shape.

[0025] In one embodiment, the invention contemplates a method of minimizing inflammation and for encouraging cell tissue regeneration in a subject with vascular condition requiring an intervention comprising: a) a medical implant exhibiting a slow release of H2S; b) implanting said implant. In one embodiment, said implant is within a living subject. In one embodiment, said release of H2S is sustained over a period of time. In one embodiment, said slow release comprises within a physiological-relevant range (from low nanomolar to high micromolar concentration) over a period of several days or weeks. In one embodiment, said slow release comprises the low nanomolar range (10‘8- 10’9M) to high micromolar concentrations, up to 300 micromolar (10’4M), preferably in the low micromolar range (~ 10‘5- 10'6M). In one embodiment, said release of H2S is sustained over a period of time, such as several days or weeks. In one embodiment, said Patent

[0026] Attorney Docket No. UTCB-20718 implant comprises a coaxial microfibrous system impregnated with an H2S donor. In one embodiment, said implant comprises a coaxial nanofibrous system impregnated with an H2S donor. In one embodiment, said H2S donor is a polysulfide. In one embodiment, said polysulfide is a garlic-derived polysulfide. In one embodiment, said polysulfide is a natural polysulfide. In one embodiment, said H2S donor comprises diallyl disulfide (DADS). In one embodiment, said H2S donor comprises diallyl tetrasulfide (DATTS). In one embodiment, said H2S donor comprises an organic sulfide such as allyl polysulfides, benzyl polysulfides, and organic disulfides and thiosulfonates. In one embodiment, said implant comprises a coaxial nanofibrous coating. In one embodiment, method comprises at least one polyethylene-glycol derivative. In one embodiment, said medical implant comprises a core with a coaxial sheath. In one embodiment, said wherein said medical implant encourages cell tissue regeneration. In one embodiment, said wherein said medical implant reduces inflammation. In one embodiment, said core is a hydrophobic and degradable core. In one embodiment, said medical implant comprises a vascular stent. In one embodiment, said medical implant comprises a vascular graft. In one embodiment, said the concentration of diallyl trisulfide in said implant is between 10 pM and 250 pM. In one embodiment, said the concentration of diallyl trisulfide in said implant is between 25 pM and 150 pM. In one embodiment, said medical implant is selected from the group consisting of vascular graft, heart valve, cardiovascular implants, and tissue-regenerative implant. In one embodiment, said at least one polyethylene-glycol derivative comprises at least one polymer selected from the group consisting of polyethylene-glycol dimethacrylate, polyethylene-glycol modified with thiolene, and polyethylene-glycol modified with hydroxy acid groups. In one embodiment, said hydrophobic, degradable core comprises a at least one polymer from the group consisting of poly L-lactide acid, poly caprolactone, polyglycolic acid, poly(lactic-co-glycolic acid), and Patent

[0027] Attorney Docket No. UTCB-20718 polyurethane. In one embodiment, said hydrophobic, degradable core comprises a poly L-lactide acid core. In one embodiment, said hydrophobic, degradable core comprises at least one polymer selected from the group consisting of poly L-lactide acid, polycaprolactone, polyglycolic acid, poly(lactic-co-glycolic acid), and polyurethane. In one embodiment, said hydrophobic, degradable core comprises poly-s-caprolactone. In one embodiment, said coating further comprises a controlled release agent. In one embodiment, said coating further comprises a therapeutic agent. In one embodiment, said therapeutic agent comprises an anti-proliferation agent. In one embodiment, said hydrophobic, degradable core provided controlled drug release. In one embodiment, said sheath integrates a surface signaling mechanism. In one embodiment, said surface signaling mechanism that simultaneously aids in regeneration. In one embodiment, said implant further comprises an interactive surface. In one embodiment, said implant further comprises sub-millimeter scale fibers. In one embodiment, said sub-millimeter scale fibers comprises nanoscale fibers. In one embodiment, said sub-millimeter scale fibers provide a uniform surface. In one embodiment, said sub-millimeter scale fibers comprise a cell recognition platform. In one embodiment, said implant has no delaminiation. In one embodiment, said the elasticity of said polyethylene-glycol sheath is tunable by varying the photopolymerization time. In one embodiment, said implant comprises crosslinked coaxial nanofiber system. In one embodiment, said nanofiber system comprises poly-e-caprolactone core with a coaxial sheath comprising poly(ethylene glycol) norbornene-RGD. In one embodiment, said nanofiber system comprises coaxial PCL-gelatin (PCL+GEL) nanofiber system, crosslinked with genipin. In one embodiment, said nanofiber system comprises coaxial ng PCL / PEGNB-RGD coaxial fiber system, crosslinked with PEG dithiol. In one embodiment, said nanofiber system is impregnated with diallyl trisulfide. In one embodiment, said implant comprises a coating on an existing implant platform, such as a Patent Attorney Docket No. UTCB-20718 stent. In one embodiment, said coating comprises an adhesive layer. The present invention is not to be limited by the shape of the implant. In one embodiment, said implant comprises a flat sheet. In one embodiment, said implant is cylindrical. In one embodiment, said implant comprises a 3-D printed shape.

[0028] In one embodiment, the invention contemplates a method of minimizing inflammation and for encouraging cell tissue regeneration in a vascular condition requiring an intervention with a medical implant comprising: a) a medical implant with a coating comprising a coaxial nanofibrous system impregnated with H2S donor comprising at least one polyethylene-glycol derivative; b) implanting said implant.

[0029] Other objects, advantages, and novel features, and further scope of applicability of the present invention will be set forth in part in the detailed description to follow, taken in conjunction with the accompanying drawings, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.

[0030] DEFINITIONS

[0031] To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims. Patent Attorney Docket No. UTCB-20718

[0032] As used herein, “patency” refers to a condition of being open, expanded, or unobstructed. As used herein, “intima” refers to a layer of a blood vessel wall including arteries and veins. As used herein, “neointima” or neointimal” refers to new or a thickened layer of intima.

[0033] As used herein, “intimal hyperplasia” refers to an abnormal accumulation of cells in the vascular tunica intima. While not intending to be limited to any particular mechanism, it is believed that the cell number is increased because of proliferation and / or migration of vascular wall cells, predominantly smooth muscle cells, often in response to a traumatic stimulus.

[0034] As used herein, "device," "scaffold," "stent", "carrier" and "implant" may be used synonymously. A “biofunctionalized” graft refers to anti-inflammatory, pro- regenerative, and / or anti -thrombotic grafts.

[0035] As used herein, “slow” release refers to releasing H2S within effective, physiological range from low nanomolar range to 300 micromolar concentration, preferably over the course of several days or weeks.

[0036] As used herein, “sustained” release refers to over days and weeks.

[0037] As used herein, an anastomosis is a surgical connection between two structures, and in particular a connection that is created between tubular structures, such as blood vessels. Such treatment may be performed on an artery or vein, and typically involves removal of a small portion or section of the vessel. In a preferred embodiment, after removal, the graft is used to reconnect the vessels.

[0038] DESCRIPTION OF THE FIGURES

[0039] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office Patent Attorney Docket No. UTCB-20718 upon request and payment of the necessary fee.

[0040] Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be consider illustrative rather than restrictive.

[0041] The accompanying figures, which are incorporated into and form a part of the specification, illustrate several embodiments of the present invention and, together with the description, serve to explain the principles of the invention. The figures are only for the purpose of illustrating a preferred embodiment of the invention and are not to be construed as limiting the invention.

[0042] FIG. 1 shows a general example of the custom coaxial electrospinning set-up used to produce fibers used in the present invention.

[0043] FIG. 2A-D shows some characterization of the graphs comprising PCL / PEGNB-RGD coaxial fiber system, crosslinked with PEG dithiol, without (no DATS) or with DATS (+DATS). FIG. 2A-B show the nano- and micro structure from TEM (FIG. 2A) and SEM (FIG. 2B). FIG. 2C shows sulfide release profile of the types of graphs: orange curves and dots showing +DATS graph while blue ones showing no DATS graph. FIG. 2D shows a graph with DATS vs. graph without DATS: hydrogen sulfide (EES) release profile from Day 1 to Day 7. FIG. 3A-C shows structure and H2S release characterization of coaxial PCL-gelatin (PCL+GEL) nanofiber systems.

[0044] FIG. 3 A shows a representative TEM images of the fibers without DATS, or with DATS at different impregnation concentrations of 25 pM (DATS25), 50pM (DATS50), and lOOpM (DATS100); FIG. 3B shows FTIR characterization curve; FIG. 3C shows daily accumulated release curve of sulfide.

[0045] FIG. 4 shows sulfide release from PCL-PEGNB-RGD coaxial system stimulated endothelial growth.

[0046] FIG. 5 shows CCK-8 absorbance analysis: cell proliferation over time under DATS, no Patent Attorney Docket No. UTCB-20718

[0047] DATS, and glass treatments.

[0048] FIG. 6A&B shows sulfide release from PCL-Gelation coaxial fiber system stimulated endothelial growth and migration. FIG. 6A shows endothelial growth on samples with 3 -day culture, which was measured with cck-8 kit. FIG. 6B shows quantitative measure of endothelial migration on the fibrous samples after one-day (DAPI: blue; F-actin: green).

[0049] FIG. 7 shows the effects of sustained H2S release on the cryoprotection of endothelial cells, which is demonstrated by the PCJ / PEGNB-RGD coaxial fiber system. Representative fluorescent images show the susceptibility of endothelial inflammation via measuring the cytoplasm-to- nucleus translocation ratio of NF-KB after the cells’ exposure to pro-inflammatory cytokine TNF- a. Herein, NF-KB was shown in green, and nuclei were in blue.

[0050] FIG. 8 shows anti-inflammatory effects of DATS on NF-KB nuclear translocation in endothelial cells: the figure presents NF-KB nuclear translocation ratios in endothelial cells exposed to TNF-a under three conditions: 100 pM DATS graph, No DATS graph, and MPTS- treated coverslip. The box plots show significantly reduced NF-KB translocation in the 100 pM DATS group, indicating effective anti-inflammatory action. Statistical significance is marked with asterisks (**** p < 0.0001, * p < 0.05).

[0051] FIG. 9A&B shows sustained H2S release from the PCL-gelation coaxial fiber system show its cytoprotection for endothelial cells. The endothelial susceptibility to inflammation depends on the DATS concentration or the available H2S amount. FIG. 9A shows the cytoplasm-to-nucleus translocation ratio of NF-KB was determined and representative images were shown. Immunofluorescent staining was performed after cells were exposed to TNF-a and cultured on the samples for 3 days after (DAPI: blue; NF-KB: green). FIG. 9B shows the ICAM-1 expression level was determined to assess the susceptibility of human endothelial cells to the exposure of TNF-a Patent

[0052] Attorney Docket No. UTCB-20718

[0053] (DAPI: blue; ICAM-1: green).

[0054] FIG. 10 shows immunofluorescent and histological analysis of 1-week explants of PCL / PEGNB-RGD coaxial fiber graphs, showing fast endothelialization (vWF+) and pro-healing M2 type (CD206+) macrophages (CD68+) in DATS grafts. Both grafts show high cell infiltration in the grafts.

[0055] FIG. 11A-D shows one-week implant evaluation on the fascular grafts made of PCL- gelation coaxial fibers with or without DATS impregnation. FIG. 11A shows cells migrated into the different locations of graft after 1-week implantation. Cell number was quantified. Representative images were shown on the right. FIG. 11B shows en face imaging of cell nuclei at the artery-graft anastomosis. FIG. 11C shows representative images of cells in the different anastomotic regions of a DATS graft; cells were stained with PECAM(CD31) FIG. 11D shows representative images of cells located in the upstream region of the two graft types; cells were stained with CD68 macrophage marker.

[0056] FIG. 12 shows representative images from ultrasound doppler imaging showing the comparisons of flow conditions in one-week or 12-week implants of interpositional vascular grafts with or without DATS impregnation.

[0057] FIG. 13 shows a representative image (top panel) and videos (bottom panel) from microCT imaging, showing the evolution of flow conditions over time.

[0058] FIG. 14A&B shows representative images from microCT imaging, for the comparison of the 3D structures of artery-graft from 12-week explants between grafts with (FIG. 14B) or without (FIG. 14A) DATS. In both sets of images, the top panel exhibits two longitudinal views of microCT images and reconstituted 3D views from the arterial end, across the longitudinal axis of the lumen, and from the graft end. Herein, “A” labels the arterial end, and “G” labels the graft end; “I”, “II”, Patent Attorney Docket No. UTCB-20718

[0059] “III” and “IV” label the cross-sectional planes, where the cross-sectional microCT images in the middle plan were taken - in the arterial region (I), in the anastomosis (II), in the graft close to the anastomosis (III) or away from it (IV). The bottom panel shows the intensity profile of microCT signal across the red line in the cross-sectional images. The dotted green and orange lines respectively label approximate intensity values for graft and artery.

[0060] FIG. 16A-C shows proregenerative characteristics of DATS-impregnated grafts, as shown by the histological analyses: FIG. 16A shows multimodality two-photon microscopy (2PM) imaging and FIG. 16B shows immunofluorescence analysis of 12-week graft explants. FIG. 16A shows representative images from histological analyses including hematoxylin and eosin (H&E), Verhoeff-Van Gieson (VVG) and Masson’s tri chrome stains. FIG. 16B shows 2PM images with SHG-MPM dual modality show the matrix production (green for elastin from fluorescence excitation, red for collagen from SHG), and are consistent with corresponding trichrome and VVG stains. FIG. 16C shows immunofluorescent images with vWF / aSMA / DAPI tri-colored staining (green for vWF, red for aSMA, and blue for DAPI).

[0061] FIG. 16 shows comparisons of the cell density in the grafts among 1-week and 12-week PCL / PEGNB-RGD arterial grafts with no DATS and with DATS explanted from rats. Quantitative evaluations of the cell density (the number of cell nuclei per mm2) include the total cell density across the entire graft thickness as well as the cell densities in the luminal half or in the abluminal half.

[0062] FIG. 17 shows H2S release reduced platelet aggression.

[0063] FIG. 18 shows one embodiment of the current invention, a biofunctionalized arteriovenous (AV) graft. The graft has folded ends and has a molecular design comprising coaxially-structured micro / nanofibers with DATS-incorporation in the core and the addition of RGD and VEGF Patent

[0064] Attorney Docket No. UTCB-20718 peptides to the sheath.

[0065] FIG. 19 shows an AV graft that was implanted into a pig subj ect that was sutured connecting an artery and vein.

[0066] FIG. 20 shows various stained microscopic images of the biofunctionalized AV graft, which showed good patency, no inflammation, high cell density, and regenerated functional tissues.

[0067] FIG. 21 shows H&E (hematoxylin and eosin) stain comparing at 10X and 40X comparing native artery, the biofunctionalized AV graft, and a PTFE graft (commercial Gore-Tex graft). The biofunctionalized AV graft showed native cell population along with collagen, with high cell density and no encapsulation. By contrast, the PTFE graft shows far fewer cells and thick encapsulation (a measure related to inflammation).

[0068] FIG. 22 shows photon imaging of extracellular matrix deposition comparing native artery and the biofunctionalized arteriovenous graft. Hierarchical elastin and collagen deposition comparable to native artery was formed with the biofunctionalized graft.

[0069] DETAILED DESCRIPTON OF THE INVENTION

[0070] The present invention relates to a vascular graft, and more particularly, to a vascular implant having a sulfide-based structure for minimizing inflammation and for encouraging cell tissue regeneration.

[0071] 1. Introduction

[0072] Accumulating recent evidence has shown huge therapeutic potential of hydrogen sulfide (H2S) in numerous clinical conditions. These include preservation of donor transplants such as Patent

[0073] Attorney Docket No. UTCB-20718 heart and kidney [1-3], periprocedural treatment for grafting [4], organ protection after ischemia / reperfusion injury [5-7], therapeutics for cardiovascular diseases [8-13], renal diseases

[0014] such as diabetic nephrophathy, and cancer

[0015] , as well as promoting neuroprotection

[0016] , gut health

[0017] , aging and longevity

[0018] , Along with increased clinical applications, cellular and molecular mechanisms underlying the therapeutical roles for H2S are being unraveled. H2S plays critical roles in fighting oxidative stress through redox signaling [19, 20], modulating protein sulfhydration

[0021] , causing genetic and epigenetic alterations

[0022] , or targeting mitochondria

[0023] , Through such molecular mechanisms, H2S regulates cell behaviors such as cell inflammation, apoptosis, differentiation, and proliferation

[0024] , To that end, ThS-based drugs or treatments are under continuous development and translational scrutiny, which encompass H2S pretreatment [3, 4, 25], various types of H2S donors including those from new synthesis routes [26-28], H2S mimetics

[0029] , nanoparticle carriers [30, 31], and conjugation methods to fiber or hydrogel materials [32, 33], Given the short half-life of H2S, one outstanding challenge in translating H2S- based solutions for clinical uses is to provide slow and sustained H2S release over a long period of time

[0010] , Associated to that is the difficulty in measuring low H2S concentrations in a physiological condition. Unfortunately, in most studies, H2S release remain uncharacterized, while “prolonged” H2S releases were shown in experiments for minutes or hours, instead of days [28, 33], Because H2S, even in small amount, was considered as an environmental hazard for decades causing toxicity to organs [34-36], a narrow concentration range exist for its beneficial effects. Thus, the H2S levels require tight control and precise measurement, mimicking endogenous generation of H2S in human tissues. The present invention describes a design of coaxial nanofibrous systems with slow release H2S for vascular graft applications. Novel measurements were utilized to determine both continuous subtle variation and phasic total release of H2S from Patent Attorney Docket No. UTCB-20718

[0074] J S-releasing grafts into physiological environments in vitro or in vivo.

[0075] Vascular grafts play a critical role in the contemporary management of a wide range of clinical conditions, including atherosclerotic disease, vascular aneurysm, congenital malformation, vasculitis, arterial bypass, and traumatic injury

[0037] , While large-sized grafts possess great success rate, small-diameter (<6mm) grafts do not. In most cases, autologous tissue, such as internal thoracic artery or saphenous vein, is the first choice for these grafts. However, prior surgeries and comorbid medical conditions may limit the availability of a patient’s own vessels, and their harvest can be associated with significant morbidity. The use of synthetic materials for small-diameter grafts, however, has been associated with high rates of thrombus formation and intimal hyperplasia. To facilitate long-term patency, there remains a substantial unfulfilled need for readily-available small-caliber synthetic vascular grafts with reduced rates of thrombosis as well as low intimal hyperplasia and capsule thickness, both of which are associated with inflammation. Based on our established coaxial fiber systems, which offer strong mechanical properties and cell compatibility [38-40], this study has further developed the two fibrous systems by impregnating H2S donor for slow, sustained sulfide release in vivo.

[0076] The occurrence of intimal hyperplasia around a vascular graft is often attributed to the endothelial damage and inflammation, leading to late re-endothelization and inflammatory cell infiltration. To address these problems, the present study used coaxially-structured micro / nanofiber grafts that were previously developed [38-40] to impregnate polysulfides for sustained release of H2S. The in vitro and in vivo assessment of grafts impregnated with diallyl trisulfide (DATS), a garlic-derived polysulfide acting as a stable H2S donor, has been performed. In particular, the release profile, cytoprotection, and stimulation of endothelial regeneration through H2S have been demonstrated. The biological production of H2S from DATS occurs when they react with Patent

[0077] Attorney Docket No. UTCB-20718 biological thiols, such as L-glutathione reduced or y-Glu-Cys-Gly (GSH). The study results have shown the great potential of sustained sulfide signal in vascular graft to achieve attenuated oxidative stress, reduced inflammation, and fast endothelization.

[0078] 2. Materials and Methods

[0079] 2.1. Materials

[0080] The DATS graft is fabricated through a coaxial electrospinning technique that offers meticulous control over the biopolymeric matrix's attributes. This process employed a core-sheath fiber architecture comprising a DATS-loaded core and a functional sheath.

[0081] The core solution was formulated by dissolving Poly-s-caprolactone (PCL), molecular weight 70,000-90,000 Da (CAS 24980-41-4, Sigma-Aldrich), in Hexafluoro-isopropanol (HFIP, CovaChem, CAS 920-66-1). Diallyl trisulfide (CAS 2050-87-5, Sigma-Aldrich) was subsequently added to achieve a final concentration of. DATS was mixed with HFIP and vortexed to optimize its solubility, thus forming a DATS stock solution. Subsequently, this stock solution is amalgamated into a 5%wt PCL matrix that has been pre-dissolved in HFIP, resulting in a core solution with a DATS concentration of 100 pM.

[0082] The sheath solution was formulated by first dissolving 24 mg of Poly (ethylene oxide) (PEG, CAS no. 25322-68-3), 158 mg of 4-Arm PEG-norbornene (PEG-NB, from BioChem), and 52.7 mg of PEG-Dithiol (SH-PEG-SH, from Sigma- Aldrich) in 3 ml of hexafluoroisopropanol (HFIP). These components were to be crosslinked to create a stable polymer matrix. To this matrix, 3.5 mg of RGD peptide, a cell adhesion molecule provided by Genscript, was added to enhance cellular interactions. Additionally, 12 mg of Irgacure 2959 (1-2959, Sigma-Aldrich), a photo-initiator essential for UV crosslinking, was dissolved in 2 ml HFIP and incorporated into the sheath solution. Patent Attorney Docket No. UTCB-20718

[0083] This careful, sequential mixing ensured a uniform, homogeneous solution, optimizing the properties necessary for effective electrospinning. To maintain the stoichiometric accuracy of the solution, components are rigorously weighed, ensuring deviations do not surpass a ±5% range from the intended target.

[0084] 2.2 Graft fabrication and Crosslinking

[0085] Electrospinning Setup and Conditions:

[0086] The custom coaxial electrospinning setup, developed in-house, involves a dual syringe system (Pump 11 Plus, Harvard Apparatus, Boston, MA) to simultaneously extrude core and sheath solutions. The core solution, containing diallyl trisulfide (DATS) and Poly-£-caprol acton e (PCL) in hexafluoroisopropanol (HFIP), was extruded at a rate of 0.8 mL / h. The sheath solution, consisting solely of PCL, was extruded at a faster rate of 1.0 mL / h to ensure consistent sheath coverage. Both solutions were administered through a 22 G coaxial needle with a high voltage of 12V (ES30P 10 W Gamma High Voltage Research, Ormond Beach, FL) applied to facilitate fiber formation and precise deposition onto a collecting target.

[0087] Fiber Collection and Mandrel Specifications:

[0088] For in vivo and release applications, the graft was deposited on a mandrel, which rotates consistently at 350 rpm. This systematic rotation, combined with an electrostatic potential of 12V, guaranteed a uniform fiber deposition. Additionally, intermittent compression of the graft using a plastic spatula was employed to achieve desired fiber compaction. Adjustments to collection time and mandrel size were made based on the specific requirements of each experimental application to optimize fiber density and alignment, which significantly impacts the mechanical properties and Patent Attorney Docket No. UTCB-20718 cellular interactions of the final graft.

[0089] For in vivo graft implantation experiments, fibers were electrospun onto a 1.25 mm diameter cylindrical aluminum rod, which is rotated at 350 rpm and situated 11 cm from the needle. This configuration is designed to form a uniformly tubular graft within a 30-minute timeframe, ideal for in vivo applications. For release experiments, a separate set of fibers for release experiments was collected on a 3.175 mm diameter rod under identical conditions, extending the collection to 40 minutes. This adjustment was to construct grafts with tailored release profiles. For in vitro cell studies, the glass coverslips were placed on a thin aluminum foil which is 11 cm away from the needle as the collector to collect the electrospinning coaxial fibers for 30min. This process ensured that the resulting grafts had the precise characteristics necessary for controlled in vitro experiments.

[0090] Final Step with Functionalization:

[0091] After the deposition process, the graft undergoes stabilization processes relevant for both in vitro and in vivo studies. This entails transitioning the graft into an anaerobic state via nitrogen replacement, succeeded by a 40-minute UV exposure for polymer cross-linking. During this process, the tubular grafts constantly rotated to assure uniform cross-linking throughout the graft. Right after the crosslinking process, the vascular grafts were rinsed with phosphate-buffered saline (PBS) and removed from the mandrel. Graft on coverslips for in vitro studies were exposed to UV for 30 minutes with no rotation.

[0092] 2.3. Electron Microscopy Imaging of Fiber Structures

[0093] Transmission electron spectroscopy (TEM) and scanning electron spectroscopy (SEM) Patent

[0094] Attorney Docket No. UTCB-20718 were used to characterize the nanostructure of the electrospun grafts, in particular the coaxial fibers, as described previously

[0039] , The core-sheath morphology was observed using H7650 transmission electron microscope (TEM; Hitachi Ltd, Tokyo, Japan) operated at 80 kV. The electrospun nanofiber samples for TEM observation were prepared by directly depositing an ultrathin layer of as-spun fibers on copper-coated TEM grids.

[0095] SEM sample preparation involved mounting electrospun fibers on aluminum substrates and brass stubs, followed by Pt / Pd sputter-coating using a Cressington Sputter Coater 108auto with a Rotary Vane Vacuum Pump VRL 100-3.5 (Cressington Scientific Instruments, Watford, UK). Imaging was conducted at 5 kV using a field-emission SEM (Hitachi SU3500, Hitachi High Technologies America, Schaumburg, IL) with NPGS e-beam lithography capability.

[0096] 2.4. Hydrogen Sulfide Release Quantification by Ion-Selective Electrode Method

[0097] To delineate the release profile of hydrogen sulfide (H2S) from DATS-impregnated coaxial fiber grafts compared to control grafts, the H>S release behaviors of electrospun fiber mats were examined. DATS-loaded PCL-gel coaxial fibers, collected on a 3.175 mm diameter rod, were cut into 50 mm pieces. Approximately 100 mg of these grafts were soaked in 5 mL of a 2mM Glutathione (GSH) PBS solution (pH = 7.4) at room temperature (20 °C) on a magnetic stirrer. Samples were taken daily from day 1 to day 7.

[0098] GSH was added at 2mM to mimic physiological conditions, as typical cellular concentrations of GSH range from l-2mM. GSH reacts with Diallyl Trisulfide (DATS) to release H2S through thiol-disulfide exchange followed by reduction. Daily replacement of the GSH solution maintains a consistent environment and replenishes the GSH consumed during the reaction. Patent

[0099] Attorney Docket No. UTCB-20718

[0100] The release buffer was mixed with 4M NaOH to create a highly alkaline environment. The concentration of S2ions in the release buffer was then determined using a sulfide ion-selective electrode (UX-27504-28, Cole Palmer Inc.). Continuous readings with the ISE were taken over 7 days. Every 24 hours, the GSH solution was replaced with a fresh one to maintain consistent experimental conditions. The old solution was immediately analyzed to measure total EES content by adding 4M NaOH, which converts all ILS forms to S2ions. The description of electrode calibration and hydrogen sulfide solubility and forms for the detection are available in the supplementary information:

[0101] 2.5. Cell culture, proliferation and growth assays

[0102] Human umbilical vein endothelial cells (HUVECs) with passages 6-10 were used for all experiments. Cells were seeded on circular glass cover slips (12 mm in diameter) with coaxial fiber mats. For the cell proliferation assays, the cell seeding density was 20,000 cells / mL, and for the cell inflammation assays, the seeding density was 10,000 cells / mL. All cultures were maintained in Endothelial Cell Growth Medium-2 BulletKit™ (ECGM) (Lonza), which includes 500 mb of EBMTM-2 Basal Medium (CC-3156) supplemented with 10.00 mL FBS, 0.20 mb hydrocortisone, 2.00 mL hFGF-B, 0.50 mLVEGF, 0.50 mLR3-IGF-l, 0.50 mL ascorbic acid, 0.50 mL hEGF, 0.50 mL GA- 1000, and 0.50 mL heparin from the EGMTM-2 SingleQuots™ Supplement Pack (CC-4176). The cell cultures were incubated at 37 °C with 5% CO2 during all experiments. Prior to cell seeding, the coaxial fiber mats were sterilized by immersing them in 75% ethanol for 30 minutes and then rinsed three times with PBS.

[0103] Cell proliferation on coaxial fiber mats was measured by the Cell Counting Kit 8 (WST-8 / CCK8) (ab228554). After 3 or 5 days post seeding, samples were incubated with 50 pL / well Cell Patent Attorney Docket No. UTCB-20718 counting kit 8 (WST-8) for 5 hours at 37°C. Then 100 pL supernatant was transferred to a 96-well plate, read at an absorbance of 450 nm by a microplate reader (Multi skanMK3, Thermo, USA).

[0104] After the evaluation of cell growth number, samples were washed by warm PBS once and fixed with 4% paraformaldehyde (PFA; Sigma- Aldrich) in PBS for 20 min at room temperature. After rinsed with PBS twice for 5 min, the cells were then permeabilized with 0.5% Triton X-100 in PBS for 15 min. Subsequently, F-actin with FITC-phalloidin (Abl76753; abCam) and 4’, 6- diamidino-2-phenylindole (DAPI, 1 pg / mL; Sigma-Aldrich) diluted in 1.5% BSA in PBS were used to stain the cells for 30 min at room temperature. The samples were rinsed gently with PBS and observed by fluorescence microscope (Olympus 1X51).

[0105] 2.6. Cell inflammation assay

[0106] After culturing the HUVECs on the ECM arrays for 3 days, the cells were treated with 20 ng / mL recombinant bovine TNF-a protein (R&D Systems, Minneapolis, MN) in serum-free media for 6 hours to induce inflammation. After treatment, the cells were washed with warm PBS once and fixed with 4% paraformaldehyde (PFA; Sigma- Aldrich) in PBS for 20 minutes at room temperature. Following fixation, the cells were permeabilized with 0.20% Triton X-100 in PBS for 15 minutes.

[0107] Subsequently, the cells were blocked with 3% BSA in PBS for 30 minutes at room temperature. After blocking, the cells were incubated with a 1 : 50 dilution of polyclonal rabbit anti- bovine NF-KB p65 primary antibody (sc- 109; Santa Cruz Biotechnology Inc., Santa Cruz, CA) in PBS for 1 hour at room temperature. The cells were then washed three times with PBS and incubated with a 1 :50 dilution of FITC-conjugated goat anti-rabbit IgG secondary antibody (m- IgGK BP-FITC) in PBS for 1 hour at room temperature. Finally, the cells were counterstained with Patent

[0108] Attorney Docket No. UTCB-20718

[0109] DAPI at a concentration of 2 drops / mL in PBS for 10 minutes at room temperature. After a final wash with PBS, the samples were mounted on slides using an antifade mounting medium and observed using a fluorescence microscope (Olympus 1X51).

[0110] 2.7. Vascular graft implantation

[0111] Sprague Dawley rats with about 500 grams were purchased from ENVIGO (Indianapolis, IN and anesthetized with 5% isoflurane gas (Vet One) and maintained with 2% isoflurane gas. A midline incision was made to access abdominal aorta. The abdominal aorta was isolated and camped off upstream and downstream of implantation site. The graft with a 1.25 mm diameter was anastomosed and replaced a small section (~lcm) of aorta by using twelve continuous 10G nylon repeating surgical stitches . The wound was then closed in a routine medical protocol with 5G Vicryl sutures. 50 / 50 mix of lidocaine 1-2% with 0.5% bupivacaine was applied onto the on incision line and 4 mg / kg body weight dose of meloxicam (0.3mg / kg) was injected into the animals after the surgery

[0112] Animal experiments were performed according to the IACUC requirements (approval number: 1407.02 at the University of Colorado) and complied with the NIH’s Guidelines for the Care and Use of Laboratory Animals. Two groups of coaxial fiber grafts (n = 4-5 for each group, all successfully explanted) were evaluated. Prior to implantation, grafts were sterilized the same way as done for the in vitro studies. Briefly, after peptide binding, the grafts were processed in the following sequence: rinse with PBS, sterilization with 70% ethanol for 15 min, and rinse again three times with sterile PBS. The grafts were then stored in 2* penicillin-streptomycin and 1% heparin in PBS until their implantation.

[0113] The vascular graft evaluation was performed on Sprague Dawley rats (ENVIGO, Patent

[0114] Attorney Docket No. UTCB-20718

[0115] Indianapolis, IN) at 8-9 months old at the time of the implantation, weighing ~400 g. Anesthesia in rats was induced with 5% isoflurane gas per liter 02 (Vet One) until fully anesthetized and maintained with 2% isoflurane gas. The surgical site was cleaned and disinfected with povidone- iodine (Medline Industries Inc, Northfield, IL). Abdominal incision was made down the midline into the abdominal cavity to access abdominal aorta. The abdominal aorta was dissected away from the inferior vena cava and clamped off upstream and downstream of the implantation site. A small section of aorta was removed to where the graft was anastomosed in an end-to-end manner using twelve continuous surgical stitches at each anastomosis site of the graft. After completion of the anastomosis, the clamps were removed to restore arterial perfusion and the anastomosis examined for leakage. The incision was then closed in a standard manner using running stitches. The animals received 50 / 50 mix of lidocaine 1-2% with 0.5% bupivacaine on incision line as well as 4 mg / kg body weight dose of meloxicam (0.3 mg / kg) after the surgery. Their movements and signs of distress were closely monitored for the first 24 h and twice a day in the following two days. During these examinations, no rats that had been implanted with coaxial fiber vascular grafts were found to experience any obvious discomfort, edema, swelling, weakened thrill, or other neurologic disorders. No complications such as hemorrhage were revealed upon explantation. The grafts were retrieved after 7 days of the implantation. Only two rats that were implanted with grafts made of PCL fibers (control group) had complications and died during the first 48 h.

[0116] For the two study groups (no DATS, +DATS), each group had 4-5 rats that were successfully explanted. At the time of explantation, no aneurysmal changes were observed in any graft implants. The patency rate was 100% for each study group. For the two rats that died during the course of implant evaluation, no blood in abdominal cavity and grafts intact with no blood clotting or narrowing in the graft were noted. Anesthesia in rats was induced with 5% isoflurane Patent Attorney Docket No. UTCB-20718 gas (Vet One) and maintained with 2% isoflurane gas. Surgical site was cleaned and disinfected with povidone-iodine (Medline Industries Inc, Northfield, IL). Abdominal incision was made down the midline into the abdominal cavity to access abdominal aorta. The abdominal aorta was dissected away from the inferior vena cava and camped off upstream and downstream of the implantation site. A small section (~I cm) of aorta was removed to where the graft with a 1.5 mm diameter was anastomosed in an end-to-end fashion using twelve continuous surgical stitches at each anastomosis sit of the graft. After completion of the anastomosis, the clamps were removed to restore arterial perfusion and the anastomosis inspected for leakage. The incision was then closed in a routine fashion using running stitches with 5 G vicryl sutures for the abdominal muscle layer and 9 m (Kent Scientific) wound clips for outer hide closer. The animals received 50 / 50 mix of lidocaine 1-2% with 0.5% bupivacaine on incision line using a 1 ml syringe as well as 4 mg / kg body weight dose of meloxicam (0.3 mg / kg) after the surgery. Follow-up examinations were performed twice a day in the first three days and then weekly after that. During these examinations, no rats were found to experience any obvious discomfort, edema, swelling, weakened thrill or other neurologic disorders. No complications such as hemorrhage were revealed upon explantation.

[0117] 2.9. Ultrasonograph, doppler and graft explantation

[0118] Prior to graft explantation, the vascular graft morphology as well as blood flow pattern and flow velocity through the grafts were obtained with ultrasonographs and doppler colorimetric measurements. Briefly, rats were anesthetized with isoflurane gas after four and a half months of implantation and shaved around the area of implant with ultrasound gel applied thereafter. Ultrasound imaging and measurements were taken with ACUSON SEQUOIATM 512 ultrasound system (Siemens Medical Solutions, Mountain View, CA) after graft sections were located using Patent

[0119] Attorney Docket No. UTCB-20718 color doppler methods. The linear array ultrasound transducer was used to probe graft morphology and flow in both transverse section and cross section from the vertical aspect. After imaging, rats were immediately euthanized via CO2 asphyxiation and samples including the upstream artery, downstream artery, graft and graft-artery anastomosis, were taken. Portions of all samples were saved in All-protect (Qiagen, Hilden, Germany) and 10% Formalin for PCR and histological analyses, respectively.

[0120] Prior to graft explantation, the vascular graft morphology and blood flow through the grafts were obtained with ultrasonographs and Doppler (FIG. 6A&B), which were taken with the ACUSON SEQUOIA 512 ultrasound system (Siemens Medical Solutions, Mountain View, CA). Rats were anesthetized and shaved around the area of implant with ultrasound gel applied. After imaging, rats were immediately euthanized via CO2 asphyxiation, and samples including the graft and neighboring arteries were taken.

[0121] 2.10. Histological Assessment of Graft Explants

[0122] The explanted grafts were embedded with OCT and cryosectioned into 8-20 pm thick sections. The sections were stained with Masson’s trichrome, which was performed by the Histology Core at University of Colorado. Both the middle of the graft and the anastomosis sections were cryosectioned and stained. Imaging was done using a light microscope (Nikon, Melville, NY).

[0123] Samples of the graft, upstream vessel, downstream vessel and neighboring arteries were fixed and stored in 10% formalin (or 3.7% formaldehyde). Samples for immunofluorescence were embedded in cryostat embedding medium (Tissue Tek, Torrence, CA) and sectioned with a cryostat at -20 °C. Samples for histology or histochemistry were embedded in paraffin blocks. Histological Patent Attorney Docket No. UTCB-20718 sections were stained with Hematoxylin and Eosin, Masson trichrome and Verhoeff Van-Gieson stains, and observed under a light microscope at varied magnifications. Sections with 5-10 pm thickness were cut through the transverse plane and collected onto glass slides. Samples slides were stained using the protocol outlined from the Masson trichrome staining kit, Newcomer Supply), and imaged using a Nikon inverted microscope eclipse Ti-E series . H & E staining provided a rough morphological evaluation, including the cell density, while the trichrome and VVG stains revealed the extracellular matrix elastin content with fibrillar collagens evaluated by trichrome and elastin by VVG to better understand matrix remodeling around vascular grafts. Samples for immunofluorescence were embedded in cryostat embedding medium (OCT, Tissue Tek, Torrence, CA) and sectioned with a cryostat at -20 °C into 8-20 pm thick sections. Histological analysis of fibrillar collagens was evaluated with Masson trichrome stain and imaged under a polarized, brightfield microscope performed to better understand matrix remodeling around vascular grafts.

[0124] 2.11. Immunofluorescence

[0125] Slides were rehydrated and antigen retrieval processed. Slides were blocked in appropriate concentration goat serum and probed overnight at 4 °C at a dilution of 1 : 100. To determine the presence of endothelial cells and macrophages in the grafts, immunofluorescent staining was done, respectively, with anti-von Willebrand factor (vWF, ab8822, Abeam, Cambridge, MA) conjugated to FITC (Abeam, Cambridge, MA) and anti-CD68 (6A324, Santa Cruz Biotechnology, Dallas, TX) together with secondary antibody mlgG (Santa Cruz Biotechnology, Dallas, TX). To determine the presence of vascular smooth muscle cells in the grafts, anti-a-SMA stain (ab7817, Abeam, Cambridge, MA). All antibodies were used with 1 : 100 dilution in TBS. Finally, coverslips Patent Attorney Docket No. UTCB-20718 were applied with vectashield mounting medium containing DAPI for counterstain (Vector Laboratories, Burlingame, CA) and imaged with a fluorescent microscope (Nikon, Melville, NY). Coverslips applied with vectasheild mounting medium with DAPI for counter-stain (Vector Laboratories, Burlingame, CA) and imaged with a florescent microscope (Nikon, Instruments Inc., Melville, NY).

[0126] 2.12. Histochemistry, immunofluorescence and immunohistochemistry

[0127] To determine the presence of endothelial cells and macrophages in the grafts, immunofluorescent staining was done, respectively, with anti-von Willebrand factor (vWF, ab8822, Abeam, Cambridge, MA) conjugated to FITC (Abeam, Cambridge, MA) and anti-CD68 (6A324, Santa Cruz Biotechnology, Dallas, TX) together with secondary antibody mlgG (Santa Cruz Biotechnology, Dallas, TX). All antibodies were used with 1 :100 dilution in TBS. Finally, coverslips were applied with vectashield mounting medium containing DAPI for counterstain (Vector Laboratories, Burlingame, CA) and imaged with a fluorescent microscope (Nikon, Melville, NY). To determine the presence of smooth muscle cells in the grafts, immunohistochemistry was performed with anti-a-SMA stain (ab7817, Abeam, Cambridge, MA) and ImmunoCruz® ABC kit (Santa Cruz Biotechnology, Dallas, TX) by following the manufacturer’s protocol. Slides were finally dehydrated with increasing concentrations of ethanol solutions and cleared with Histo-Clear® prior to mounting with coverslips and imaging using a light microscope (Nikon, Melville, NY).

[0128] Immunofluorescent staining of von Willebrand factor (vWF), anti-smooth muscles cells (, and CD68 was obtained using anti-Von Willebrand Factor antibody conjugated to FITC (Abeam, Cambridge, MA) and anti-CD68 (Santa Cruz Biotechnology), respectively. Slides were rehydrated Patent

[0129] Attorney Docket No. UTCB-20718 and antigen retrieval processed following the same method as with immunohistochemistry staining above. Slides were blocked in appropriate concentration goat serum and probed overnight at 4 °C at a dilution of 1 :100. Coverslips applied with vectasheild mounting medium with DAPI for counter-stain (Vector Laboratories, Burlingame, CA) and imaged with a florescent microscope (Nikon, Instruments Inc., Melville, NY).

[0130] 2.13. Dual-modality multiphonton imaging with secondary harmonic generation

[0131] To examine the ECM production in explanted vascular graft implants, dual multiphoton imaging modality, second harmonic generation and two-photon excitation fluorescence (SHG- TPEF) imaging, a dual multiphoton imaging modality, was employed to respectively determine the fibrillar collagen and elastin contents as previously described

[0012] , The dual-modality multiphoton laser scanning microscope system (Radiance 2000 MP, Bio-Rad Laboratories Inc, Hercules, CA) included a femtosecond pulsed laser system (Spectra-Physics, MaiTai wideband, mode-locked Ti: Sapphire laser system) and a dichroic mirror (AT455 DC, Chroma Technology Corp, Bellows Falls, VT). The paraffin-embedded slides were deparaffinized with histoclear, hydrated, and submerged in PBS. A femtosecond pulsed laser system tuned to 860 nm wavelength was used for excitation (Spectra-Physics, MaiTai wideband, modelocked Ti: Sapphire laser system). The response signal was split at 455 nm using a dichroic mirror (AT455 DC, Chroma Technology Corp, Bellows Falls, VT). Collagen SHG signal (400-455 nm) and elastin TPEF (460-610 nm) signals were captured simultaneously using the direct detector system (Bio-Rad). A 535 / 150 nm BrightLine® bandpass filter was used for elastin TPEF capture. Images 1024 x 1024 pixels (305 x 305 pm) were collected at 50 Ips. Each image was generated using Kalman averaging over 3 frames. Patent Attorney Docket No. UTCB-20718

[0132] 2.14. Statistical analysis Data Analysis

[0133] Statistical analysis were performed with analysis of variance (ANOVA) followed by multiple comparison of the means with the Tukey-Kramer Multiple Comparisons Test. Results were given by Mean ± SD. A value of P < 0.05 was set and considered as significance.

[0134] One-way analysis of variance test (ANOVA) was used to analyze the statistical differences among several groups, with each group having a sample size of at least 3 (n > 3). To compare the means between two groups, Student’s / -test was used. The significance levels were set at / ? < 0.05 (*, or $), p < 0.01 (**, or $$), and / ? < 0.001 (***, ###, or $$$). Results were presented as mean ± standard deviation.

[0135] 3. RESULTS:

[0136] Two types of grafts have been developed and evaluated: (1) grafts comprising PCL / PEGNB-RGD coaxial fiber system, crosslinked with PEG dithiol, without (no DATS) or with lOOpM of DATS (+DATS) in the core, and (2) grafts with coaxial PCL-gelatin (PCL+GEL) nanofiber system, crosslinked with genipin, impregnated with DATS at different concentrations (e.g., DATS25 for 25pM of DATS, DATS for 50pM of DATS, and DATS100 for lOOpM of DATS added into the PCL / HFIP solution for DATS / PCL / HFIP solution). The results using the first system were shown first in all figure (e.g., FIG. 2A-D), while those using the second graft type were shown in the FIG. 3A-C. Previous descriptions of similar, non-DATS containing, fibers are found in Kim et al, 2010

[0041] ,

[0137] In this study, two coaxial fiber systems loaded with DATS, which release EES and provide cell-protective and regenerative niches in the grafts have designed, fabricated and optimized. The Patent

[0138] Attorney Docket No. UTCB-20718 core-shell structure of the fiber system allows one to load and release H2S for stable sulfide signaling. At the early stage of graft implantation, stable H2S release was shown to reduce inflammation, promote healing, fast re-endothelization, and inhibit platelet adhesion. In the later stage, grafts were shown to promote vascular graft remodeling and vascular tissue regeneration and reduce capsule thickness around the graft.

[0139] These two types of grafts have unique compositions and structures, and are both soft, bioactive and strong for interpositional grafting in vivo. In both types of grafts, the H2S donors (DATS) were loaded into the PCL core structure of the hybrid fiber which can significantly prolong its release time to mimic the biological generation process of H2S. Based on our knowledge, this is the first study to using DATS in vascular grafts to help vascular graft performances and improve vascular remodeling.

[0140] 3.1. Structures and release profiles of DATS-impregnated hybrid coaxially-spun fibers

[0141] The nano- and micro-structures of the two coaxial fiber systems were characterized. The core-sheath morphology was not significantly affected by the DATS loading. The morphology of coaxial fibers in the fabricated tubular grafts is shown in FIG. 2A-D and FIG. 3A-C. In order to form continuous fibers with the impregnated DATS, the flow rates of sheath and core fluids in accordance with optimal mandrel rotation were identified by performing parametric investigations of the coaxial electrospinning procedure. The development of optimal coaxial PCL-DATS / PEG- NB fibers was demonstrated in both cases. FIG. 2A shows the TEM images of coaxial fibers displaying a sheath of PEG-NB and a core made of PCL impregnated with DATS. There is no significant differences in the diameters of fibers, and their core, the sheath thickness, and the sheath-to-fiber diameter ratios, all of which were obtained from TEM images. It was also observed that the sheath was not entirely uniform through the coaxial fibers, which could potentially be due Patent

[0142] Attorney Docket No. UTCB-20718 to unequal interfacial energies between the sheath and the core constituents, having an impact on their interactions.

[0143] The release profile of hydrogen sulfide (H2S) from DATS-impregnated coaxial fiber grafts was compared to control grafts without DATS over a 7-day period (FIG. 2D). The graph clearly shows that DATS-impregnated grafts exhibited a significantly higher H2S release at all time points compared to the control group, with the release gradually decreasing over time but remaining substantially higher than the control.

[0144] Statistical analysis using the Mann-Whitney U test confirmed the significance of this difference. The P-value of 0.0023 indicates that the difference in H2S release between DATS and No DATS grafts is statistically significant (P < 0.05). The median H2S release for the DATS group was 1.457e-006 mol, substantially higher than the 1.751e-008 mol observed for the control group, with an actual difference of -1.440e-006 mol.

[0145] FIG. 3B shows the FTIR-ATR spectra of the coaxial nanofibers. The spectrum of PCL / Gel presented all the characteristic bonds of the stretching vibration of carbonyl, ether group, hydroxyl, respectively at 1725 cm'1, 1190 cm1, 3410 cm1, and the bending vibration of hydroxyl at 1240 cm'1. For PCL / GEL / DATS, a new allyl group at 987 cm'1emerged, and the peak area grew from 3888 to 4324 to 4714 with the increase of DATS addition from 25 to 50 to 100 pM. It can be concluded that DATS were successfully added into coaxial nanofibers.

[0146] 3.2. Incorporation of sulfide signals into the graft promotes endothelial proliferation and migration in vitro

[0147] Endothelial cells are essential for maintaining graft patency post-implantation. FIG. 4 shows fluorescence microscopy images of cell proliferation and migration at Days 3 and 5 for three Patent Attorney Docket No. UTCB-20718 groups: 100 pM DATS graft, no DATS graft, and MPTS-treated coverslip. On Day 5, the 100 pM DATS group demonstrated a notably higher cell density compared to the no DATS and coverslip groups. Quantitative analysis confirmed this observation.

[0148] FIG. 5 presents the results of a CCK-8 absorbance assay, where absorbance directly correlates with cell density. On Day 3, the absorbance values for the DATS, no DATS, and coverslip groups were 0.2798 ± 0.0178, 0.2620 ± 0.0088, and 0.3283 ± 0.0095, respectively. By Day 5, the DATS group showed an absorbance increase to 0.4181 ± 0.0254, significantly higher than the no DATS group at 0.3106 ± 0.0266 and comparable to the coverslip group at 0.3835 ± 0.0281.

[0149] Statistical analysis using Tukey's multiple comparisons test revealed that the DATS group had a significant mean difference in cell proliferation compared to the no DATS group on both Days 3 and 5, with adjusted p-values of 0.0002 and 0.0013, respectively. The mean difference in absorbance between the DATS and no DATS groups on Day 5 was 0.1076, with a 95% confidence interval ranging from 0.04297 to 0.1722.

[0150] These results indicate that the incorporation of sulfide signals into the graft significantly enhances endothelial proliferation and migration, as evidenced by the higher cell densities and absorbance values in the DATS group compared to controls. The observed effects are likely due to the antioxidant and anti-apoptotic properties of sustained TLS release from the DATS-loaded grafts.

[0151] FIG. 6A&B provides additional data supporting these findings by comparing the effects of different DATS concentrations (25 pM, 50 pM, and 100 pM) on endothelial cell proliferation and migration. FIG. 6A shows that optical density, correlating with cell density, increased significantly with higher DATS concentrations. The optical density for DATS 100 was 0.190 ± 0.010, which was significantly higher than the PCL-GEL control (0.150 ± 0.015) and the lower Patent

[0152] Attorney Docket No. UTCB-20718

[0153] DATS concentrations (DATS25 at 0.165 ± 0.012 and DATS50 at 0.175 ± 0.011).

[0154] FIG. 6B further quantifies cell proliferation by measuring cell numbers, showing that DATS100 resulted in the highest cell count of 230 ± 10 cells / mm2, compared to the PCL-GEL control at 150 ± 12 cells / mm2. This trend supports the observation that increased DATS concentration leads to enhanced endothelial proliferation.

[0155] 3.3. Prolonged sulfide signal plays protective roles in the endothelial inflammation

[0156] The long-term function of vascular grafts relies on the regeneration of endothelial cells with proper functionality to maintain a balanced microenvironment and protect against adverse inflammatory responses. FIG. 7, FIG. 8, and FIG. 9A&B demonstrate the efficacy of the DATS-impregnated grafts in protecting endothelial cells (ECs) from inflammation.

[0157] FIG. 7 presents representative fluorescent images showing NF-KB staining in endothelial cells. NF-KB translocation from the cytoplasm to the nucleus is a marker of inflammatory activation. The images show significantly lower NF-KB nuclear translocation in the 100 pM DATS graft group compared to the no DATS graft and MPTS-treated coverslip groups. The DATS-treated group displayed a more diffuse cytoplasmic NF-KB distribution, indicating reduced inflammation.

[0158] FIG. 8 quantifies these observations with box plots showing NF-K nuclear translocation ratios under the three conditions: DATS, no DATS, and coverslip. The DATS group exhibited the lowest translocation ratio, with a median value of approximately 0.3457, compared to 0.4976 for the no DATS group and 0.5893 for the coverslip group. ANOVA analysis confirmed significant differences among the groups (F = 10.39, p < 0.0001), with Tukey’s multiple comparisons test indicating that the differences between DATS and no DATS (p = 0.0171), as well as between Patent

[0159] Attorney Docket No. UTCB-20718

[0160] DATS and coverslip (p < 0.0001), were statistically significant.

[0161] Further supporting these findings, FIG. 9A&B highlights the protective effects of varying DATS concentrations on NF-KB translocation and fluorescence intensity. The DATS 100 group consistently showed the lowest NF-KB translocation and the highest fluorescence intensity, suggesting reduced activation of inflammatory pathways.

[0162] The data collectively indicate that prolonged sulfide signaling via DATS -impregnated grafts plays a critical role in mitigating endothelial inflammation by reducing NF-KB activation. This anti-inflammatory effect is likely due to the sustained release of FLS, which exerts protective actions on the endothelial cells, thus enhancing the overall stability and function of the vascular grafts.

[0163] 3.4. In vivo evaluation of DATS-impregnated coaxial fibers after one week, showing pro- reendothelialization and anti-inflammation characteristics

[0164] The in vivo implantation of vascular graft in abdominal aorta of rats, using end-to-end anastomosis for interpositional grafting, was performed to evaluate the biological effects of sulfide signaling. FIG. 10 and FIG. 11 A-D respectively show the capacity of DATS group in the two graft systems, after one-week implantation, in reducing inflammation in vivo and speeding up the endothelialization process to form continuous coverage of ECs. In both figures, vWF or PEC AM antibodies were used to identify endothelial cells. A continuous endothelial layer was quickly formed on the inner surface of grafts with DATS, whereas noncontinuous endothelial layer was found on those with no DATS (FIG. 10) Also, the improvements of endothelialization were significant when compared DATS groups with no DATS group (FIG. 11A-D).

[0165] Macrophages were identified with both CD68 and CD206 antibodies. No statistically Patent Attorney Docket No. UTCB-20718 significant differences were found in CD68+cell densities in both graft types; both displayed very few CD68+cells (<1%, in average) showing minimal inflammation. However, for the grafts with DATS, the majority of CD68+cells were also CD206+, M2-like macrophages, which indicated their anti-inflammatory, prohealing nature of the macrophages that are involved in immune homeostasis during inflammation.

[0166] Both types of grafts showed a significant number of cell penetration throughout the graft, an important indicating for tissue regeneration. The H&E stain shows cell nuclei in purplish blue and cytoplasm in pinky red. Masson’s trichrome shows nuclei in black, cytoplasm and muscle fibers in red, and collagen in blue.

[0167] 3.5. Pro-regeneration characteristics of DATS-iinpregnated coaxial fibers — interp ositional evaluation of 12-week graft explants.

[0168] Long-term implantation (12-week) of vascular grafts in adult rats (around 10 months old) has been performed on the grafts comprising PCL / PEGNB-RGD coaxial fiber system. FIG. 12 show that both types of grafts (without or with DATS) had great patency in the short-term and long-term evaluation. Compared to the RGD-only group with no DATS, DATS group in general showed higher in-graft flow velocity (peak and average) at the 12-week time point. For the majority of DATS grafts, the velocity peak reaches the point ~0.9 m / s, while for the majority of RGD-only grafts, the velocity peak reaches the point -0.6 m / s. Only one exception was found. Also, compared to one-week group, 12-week grafts often showed higher in-graft flow velocity. As further illustrated in FIG. 13, the ultrasound doppler images of one- or two- week time points tended to show more disturbance and lower flow velocity, when compared to later time points (e.g., 4-, 6- or 12- weeks). The average peak values of the flow velocity in those earlier times could be Patent

[0169] Attorney Docket No. UTCB-20718 as low as half of those in later times. However, the comparisons should be made on the same animals over the time (longitudinal comparisons) as the values vary with individual animals due to surgical or anatomical differences.

[0170] Results from microCT imaging (FIG. 14) and from the analyses of histology, SHG-MPM and immunofluorescence histology images (FIG. 16A-C) showed that elevated sulfide signaling helped to increase vascular cell and tissue regeneration along the lumen while reducing the capsule thickness in the abluminal space (FIG. 16). The capsule thickness of grafts is another indication of inflammation level in early stage of grafting. In addition, the productions of natural extracellular matrix (ECM), including collagen and elastin were demonstrated in the grafts, with different configurations for those without or with DATS. Overall, the grafts with DATS show mimetic ECM production where dense elastin fibils and collagen fibers were often found in the lumen (FIG. 16A- B), whereas those with no DATS showed higher . Additionally, the cell distribution in the grafts with DATS were more uniform compared to that in those without DATS where cells were found more concentration in the abluminal side of the grafts (FIG. 16C-FIG. 16) together with collagen matrix production in the abluminal side (FIG. 16B). This scenario simulated thin encapsulation of grafts. To further demonstrate the regeneration of vascular cells and Comparison between grafts with (+) or without (-) DATS The VVG stain shows elastic fibers in black, nuclei in blue, collagen in red, and other elements in yellow. Also, results from 1-week grafts showed that the number of cells in the luminal side of the DATS-containing grafts were significantly higher than those in grafts without DATS, which further confirmed the regeneration of vascular endothelium and vascular tissues in the lumen. Patent Attorney Docket No. UTCB-20718

[0171] Materials and Methods

[0172] For FTIR characterization, the chemical compounds of the coaxial fiber was identified by Fourier transform infrared (FTIR Nicolet Instrument Corporation, USA). Spectra were obtained over the range 500-4000 cm1at a resolution of 2 cm

[0173] For platelet adhesion, the study was carried out to assess the blood compatibility of coaxial fiber mats. Fresh bovine venous blood was centrifuged at 1500 rpm for 15 min to obtain bovine platelet-rich plasma (PRP). 200 pl of PRP was acquired to add into each well of a 48-well tissue culture plate with samples on the bottom The adhesion of platelet onto samples was studied by adding 200 pl of bovine platelet-rich plasma (Innovative Research, Novi, MI) to a 48-well tissue culture plate with coaxial fiber mats on the bottom and incubated at 37°C for 2 hr in humid air. After thoroughly washing in PBS to remove unattached platelets, 2.5% glutaraldehyde was added onto each sample to fix the adherent platelets at 4°C for 2hr, and then were rinsed three times in PBS. Samples were then dehydrated and dealcoholized. After it, they were dried overnight in a vacuum chamber and sputter-coated with gold SEM (JEOL JSM6480 LV) was used to observe the morphology and density of platelets adhering to the surface.

[0174] For the H2S detection, the procedures of electrode calibration and hydrogen sulfide solubility and forms are described below:

[0175] The electrode was calibrated using a standard sulfide solution according to the manufacturer's instructions before each series of measurements. Electrochemical methods using ion-selective electrodes (ISE) quantify sulfide in aqueous solutions. However, these measurements are specific to S2ions, which are present in small quantities under physiological conditions. A highly alkaline environment (pH > 12.0) is required to convert all forms of dissolved FLS into S2ions, making them detectable by the ISE. Patent

[0176] Attorney Docket No. UTCB-20718

[0177] Hydrogen sulfide is soluble in water and aqueous solutions such as PBS or plasma. At physiological pH (7.4), H2S exists as H2S (-20%), HS (-80%), and a very small amount of S2ions. The chemical reactions involved in converting PLS to detectable S2ions are:

[0178] H2S + NaOH => NaSH + H2O

[0179] NaSH + NaOH = Na2S + H2O

[0180] These conversions enable the electrode to measure the total amount of H2S released by the graft. Continuous measurements indicated that the detected sulfide amount was proportional to the total H2S. This method provided a detailed and accurate profile of H2S release from the grafts over the study period.

[0181] RESULTS:

[0182] Blood compatibility and Platelet adhesion attenuated by DATS-impregnated coaxial fibers

[0183] At the early stage of implantation, the anticoagulant property of graft is especially important to keep patency, and the influence of nanofibers on platelets adhesion and activation was investigated then. In FIG. 17, a lot of platelets adhered on PCL / GEL surface with most of them accumulated together. In comparison, platelets adhered on DATS surface was less, wherein the DATS 100 had least amounts. Regarding to morphology, platelets in PCL / GEL group were activated in spreading shape, but in DATS25, DATS50 and DATS 100 group, platelets maintained the spherical shape and showed no sign of activation, indicating that H2S released from DATS can prohibit platelets adhesion and activation. Patent Attorney Docket No. UTCB-20718

[0184] Example I

[0185] Implantation of graft into pig subject

[0186] FIG. 18 shows one embodiment of the current invention, a biofunctionalized AV graft. The graft has folded ends and has a molecular design comprising coaxially-structured micro / nanofibers with DATS-incorporation in the core and the addition of RGD and VEGF peptides to the sheath.

[0187] FIG. 19 shows an AV graft that was implanted into a pig subj ect that was sutured connecting an artery and vein.

[0188] FIG. 20 shows various stained microscopic images of the biofunctionalized AV graft, which showed good patency, no inflammation, high cell density, and regenerated functional tissues.

[0189] FIG. 21 shows H&E (hematoxylin and eosin) stain comparing at 10X and 40X comparing native artery, the biofunctionalized AV graft, and a PTFE graft (commercial Gore-Tex graft). The biofunctionalized AV graft showed native cell population along with collagen, with high cell density and no encapsulation. By contrast, the PTFE graft shows far fewer cells and thick encapsulation (a measure related to inflammation).

[0190] FIG. 22 shows photon imaging of extracellular matrix deposition comparing native artery and the biofunctionalized arteriovenous graft. Hierarchical elastin and collagen deposition comparable to native artery was formed with the biofunctionalized graft.

[0191] Example II

[0192] Potential extend release of the H2S

[0193] Usually, the H2S release is transient, which can make its availability and functioning within minutes. Owing to its poor water solubility, the biological activity of DATS as an H2S donor, like many other H2S donors, in the physiological environment is limited. The present invention utilizes Patent

[0194] Attorney Docket No. UTCB-20718 the hierarchical structural design for the delivery of controlled sulfide signals and precise biomolecular motifs. The graft technology consists of micro / nano-fibers coaxially-structured with a biodegradable polymer core, which is encased in a specialized hydrogel sheath. The nanoparticles of sulfide signals are emulsified in the core (FIG. 2A-D and FIG. 3A-C). The sheath allows for precise customization of properties like matrix softness and ligand positioning. In addition, the sheath materials allow one to not only precisely define the ligand and biophysical environments, but also finely tune to H2S donation characteristics, in order to address the antiinflammatory and / or regenerative requirements for specific applications.

[0195] For example, increasing the thickness and stiffness or decreasing the porosity of sheath can theoretically extend the release longer. Alternatively, adding additional layer designs in the microfibers may also efficiently tune the release profiles, for the slow release and long-term effectiveness of H2S.

[0196] Example III

[0197] Potential effective concentration ranges

[0198] At high concentrations, H2S could be toxic. The rate of H2S release from most H2S donors like DATS is too high and uncontrollable, and does not last long. So mimicking endogenous (biological generation) of H2S is difficult. Only making sure that the H2S concentration under physiologically relevant conditions can make H2S efficient.

[0199] According to the recent review

[0042] which cited more than 2000 papers on the topic, animals and humans have dynamic regulated H2S concentrations, but very high (millimolar, or 10’3M) concentrations of H2S impaired many physiological functions and have pathological responses, while the physiological steady-state H2S concentrations, from the low nanomolar range Patent

[0200] Attorney Docket No. UTCB-20718

[0201] (10‘8- 10'9M) to high micromolar concentrations, up to 300 micromolar (10’4M), a difference of several orders of magnitude, are found to be therapeutically efficient and beneficial for many biological functions.

[0202] As described previously, it was found that the concentrations of H2S from the low nanomolar range to high micromolar range (~ 10'4- 10'8M) are effective in that the body functions work well within this range; however, specific tissue concentrations can vary. It was also suggested by the above review that future studies aimed at determining a physiological role of H2S should be performed with H2S in a low micromolar range (~ 10’5- 10'6M). Therefore, the range provided by our micro / nanofiber system can fall right into highly effective physiological range.

[0203] Example IV

[0204] Implant shape and medical applications

[0205] Inflammation and impaired tissue repair occur ubiquitously in tissue grafts, ranging from skin grafts, bone grafts, to vascular grafts. Reducing destructive inflammation and promoting regenerative inflammation are often the common goal for the design of various tissue grafts. As previously discussed, a key factor that leads to vascular graft failure is chronic inflammation derived from improper healing, which result in graft stenosis.

[0206] To make this concept more broadly applicable, such fiber technology does not have to be used as a replacement graft, but also apply as coatings onto existing implant platforms such as stent. Besides implants, these can be used as adhesive layers on other medical devices. For example, H2S has broad effectiveness against cardiometabolic dysfunction (includes a cluster of conditions including abdominal obesity, insulin-resistant glucose metabolism, dyslipidemia, and increased blood pressure). Cardiometabolic dysfunction has also been described as Cardiometabolic Patent Attorney Docket No. UTCB-20718

[0207] Syndrome

[0043] ,

[0208] In addition, H2S are increasingly recognized for its anti-cancer mechanisms associated with its mode of actions including selective protein S-thiolation, anti-oxidative anti-inflammatory activity. Anticancer effect is probably another application area.

[0209] Also, to make the technology more broadly applicable, one embodiment of the present invention, may include other H2S donors, besides DATS. Usually, the development of a delivery system for H2S donors has been hampered by their low aqueous solubility, chemical instability at room temperature, and their pungent smell. These H2S donors include JUS-releasing natural sulfides, like diallyl trisulfide (DATS), diallyl disulfide (DADS), and diallyl tetrasulfide (DATTS), and other organic sulfide such as allyl polysulfides, benzyl polysulfides, and organic disulfides and thiosulfonates. The present invention fiber system overcomes these barriers for slow, sustained release of H2S within a physiological range.

[0210] The fiber system can be used to make implants of many shapes and sizes. In one embodiment, flat implants could be used for subcutaneous implants or skin grafts as well as cylindrical or custom-shaped grafts for bone grafts. It is believed that at least two additional areas of graft applications are skin grafts and bone grafts. In addition, potential 3-D printing or advanced electrospray applications are possible to create any number of sophisticated shapes of implants.

[0211] Thus, specific compositions and methods of sulfide-based, pro-regenerative, antiinflammatory have been disclosed. It should be apparent, however, to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. Moreover, in interpreting the disclosure, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprises" Patent Attorney Docket No. UTCB-20718 and "comprising" should be interpreted as referring to elements, components, or steps in a nonexclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced.

[0212] Although the invention has been described with reference to these preferred embodiments, other embodiments can achieve the same results. Variations and modifications of the present invention will be obvious to those skilled in the art and it is intended to cover in the appended claims all such modifications and equivalents. The entire disclosures of all applications, patents, and publications cited above, and of the corresponding application are hereby incorporated by reference.

[0213] REFERENCES:

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[0215] 2. Ertugrul, I. A. et al. (2021) "Donor Heart Preservation with Hydrogen Sulfide: A Systematic Review and Meta-Analysis," Int. J. Mol. Set. 22(11), 5737.

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[0231] 18. Sokolov, A. S. et al. (2021) "Hydrogen Sulfide in Longevity and Pathologies: Inconsistency Is Malodorous," Ageing Res. Rev. 67, 101262.

[0232] 19. Scammahorn, J. J. et al. (2021) "Fighting Oxidative Stress with Sulfur: Hydrogen Sulfide in the Renal and Cardiovascular Systems," Antioxidants 10(3), 373. 0. Kabil, O. et al. (2014) "H2S and Its Role in Redox Signaling," Biochimica et Biophy sica Acta (BBA) - Proteins and Proteomics 1844( ), 1355-1366. 1. Sen, N. (2017) "Functional and Molecular Insights of Hydrogen Sulfide Signaling and Protein Sulfhydration," J. Mol. Biol. 429(4), 543-561. 2. Liu, J. et al. (2022) "Recent Development of the Molecular and Cellular Mechanisms of Hydrogen Sulfide Gasotransmitter," Antioxidants 11(9), 1788. 3. Vintila, A. R. et al. (2023) "Mitochondrial Sulfide Promotes Life Span and Health Span through Distinct Mechanisms in Developing Versus Adult Treated Caenorhabditis Elegans," P.N.A.S. 120(32), e2216141120. 4. Wang, Y. et al. (2020) "Hydrogen Sulfide Signaling in Regulation of Cell Behaviors," Nitric Oxide 103, 9-19. 5. Smink, A. M. et al. (2020) "The Effect of a Fast-Releasing Hydrogen Sulfide Donor on Patent Attorney Docket No. UTCB-20718

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[0236] 28. Grace, J. P. and Bowden, N. B. (2021) "Synthesis and Hydrogen Sulfide Releasing Properties of Diaminodisulfides and Dialkoxydisulfides," ACS Omega 6(27), 17741- 17747.

[0237] 29. Macabrey, D. et al. (2022) "Sodium Thiosulfate Acts as a Hydrogen Sulfide Mimetic to Prevent Intimal Hyperplasia Via Inhibition of Tubulin Polymerisation," eBioMedicine 78, 103954.

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Claims

1. PatentAttorney Docket No. UTCB-20718CLAIMS:We claim:

1. A medical implant exhibiting a slow release of H2S.

2. The medical implant of claim 1, wherein said implant is within a living subject.

3. The medical implant of claim 1, wherein said release of H2S is sustained over a period of time.

4. The medical implant of claim 3, wherein said period of time comprises several days or weeks.

5. The medical implant of claim 3, wherein said implant comprises a coaxial nanofibrous system impregnated with an H2S donor.

6. The medical implant of claim 5, wherein said H2S donor is a polysulfide.

7. The method implant of claim 6, wherein said polysulfide is a garlic-derived polysulfide.

8. The medical implant of claim 5, wherein said H2S donor comprises diallyl trisulfide.

9. The medical implant of claim 5, wherein said H2S donor is selected from the group comprising: diallyl disulfide and diallyl tetrasulfide.Patent Attorney Docket No. UTCB-2071810. The medical implant of claim 1, wherein said slow release comprises a release of H2S in the range low nanomolar up to 300 micromolar over a period of weeks.

11. The medical implant of claim 1 comprising at least one polyethylene-glycol derivative.

12. The medical implant of claim 5, wherein said medical implant comprises a core with a coaxial sheath.

13. The medical implant of claim 12, wherein said wherein said medical implant encourages cell tissue regeneration.

14. The medical implant of claim 12, wherein said wherein said medical implant reduces inflammation.

15. The medical implant of claim 12, wherein said core is a hydrophobic and degradable core.

16. The medical implant of claim 5, wherein said medical implant comprises a vascular stent.

17. The medical implant of claim 5, wherein said medical implant comprises a vascular graft.

18. The medical implant of claim 8, wherein said the concentration of diallyl trisulfide in said implant is between 10 pM and 250 pM.Patent Attorney Docket No. UTCB-2071819. The medical implant of claim 8, wherein said the concentration of diallyl trisulfide in said implant is between 25 pM and 150 pM.

20. The medical implant of claim 5, wherein said medical implant is selected from the group consisting of vascular graft, heart valve, cardiovascular implants, and tissue-regenerative implant.

21. The medical implant of claim 11, wherein said at least one polyethylene-glycol derivative comprises at least one polymer selected from the group consisting of polyethylene-glycol dimethacrylate, polyethylene-glycol modified with thiol-ene, and polyethylene-glycol modified with hydroxy acid groups.

22. The medical implant of claim 15, wherein said hydrophobic, degradable core comprises a at least one polymer from the group consisting of poly L-lactide acid, polycaprolactone, polyglycolic acid, poly(lactic-co-glycolic acid), and polyurethane.

23. The medical implant of claim 15, wherein said hydrophobic, degradable core comprises a poly L-lactide acid core.

24. The medical implant of claim 15, wherein, said hydrophobic, degradable core comprises at least one polymer selected from the group consisting of poly L-lactide acid, polycaprolactone, polyglycolic acid, poly(lactic-co-glycolic acid), and polyurethane.Patent Attorney Docket No. UTCB-2071825. The medical implant of claim 12, wherein said coating further comprises a controlled release agent.

26. The medical implant of claim 12, wherein said coating further comprises a therapeutic agent.

27. The medical implant of claim 26, wherein said therapeutic agent comprises an antiproliferation agent.

28. The medical implant of claim 15, wherein said hydrophobic, degradable core provided controlled drug release.

29. The medical implant of claim 12, wherein said sheath integrates a surface signaling mechanism.

30. The medical implant of claim 29, wherein said surface signaling mechanism that simultaneously aids in regeneration.

31. The medical implant of claim 12, wherein said implant further comprises an interactive surface.

32. The medical implant of claim 12, wherein said implant further comprises sub-millimeter scale fibers.

33. The medical implant of claim 32, wherein said sub-millimeter scale fibers comprises nanoscale fibers.Patent Attorney Docket No. UTCB-2071834. The medical implant of claim 32, wherein said sub-millimeter scale fibers provide a uniform surface.

35. The medical implant of claim 32, wherein said sub-millimeter scale fibers comprise a cell recognition platform.

36. The medical implant of claim 12, wherein said implant has no delaminiation.

37. The medical implant of claim 12, wherein said the elasticity of said polyethylene-glycol sheath is tunable by varying the photopolymerization time.

38. The medical implant of claim 1, wherein said implant comprises crosslinked coaxial nanofiber system.

39. The medical implant of claim 38, wherein said nanofiber system comprises poly-s- caprolactone core with a coaxial sheath comprising poly(ethylene glycol) norbornene-RGD.

40. The medical implant of claim 38, wherein said nanofiber system comprises coaxial PCL- gelatin (PCL+GEL) nanofiber system, crosslinked with genipin.

41. The medical implant of claim 38, wherein said nanofiber system comprises coaxial ngPCL / PEGNB-RGD coaxial fiber system, crosslinked with PEG dithiol.PatentAttorney Docket No. UTCB-2071842. The medical implant of claim 38, wherein said nanofiber system is impregnated with diallyl tri sulfide.

43. A method of producing a medical implant comprising a core with a coaxial sheath comprising: a) providing; i) a core polymer solution comprising at least one polymer and at least one said H2S donor in a solvent; ii) a polymer sheath solution in a solvent; and iii) a collecting target; b) extruding said combining said core solution and said sheath solution under conditions to create a hybrid fiber comprising a polymer core with a coaxial sheath polymer; and c) collecting said extruded hybrid fiber on said collecting target to create a medical implant.

44. The method of claim 43, wherein said polymer sheath solution further comprises a photoinitiator.

45. The method of claim 44, further comprising step d) photo-polymerizing said hybrid fiber.

46. The method of claim 43, wherein said H2S donor is selected from the group consisting of diallyl trisulfide, diallyl disulfide and diallyl tetrasulfide.Patent Attorney Docket No. UTCB-2071847. The method of claim 43, wherein said core polymer solution comprises poly-s-caprolactone.

48. The method of claim 43, wherein said sheath polymer solution comprises at least one polyethylene glycol derivative.

49. A method of producing a medical implant comprising a poly-s-caprolactone core with a coaxial sheath comprising poly(ethylene glycol) norbomene-RGD comprising: a) providing; i) a core solution comprising diallyl trisulfide (DATS) and poly-s-caprolactone (PCL) in a solvent; ii) a sheath solution comprising polyethylene glycol (PEG), 4-arm poly(ethylene glycol) norbomene (PEG-NB), poly(ethylene glycol) dithiol (PEG-Dithiol), arginylglycylaspartic acid (RGD peptide), and a photoinitiator in a solvent; and iii) a collecting target; b) extruding said combining said core solution and said sheath solution under conditions to create a hybrid fiber comprising a poly-s-caprolactone with diallyl trisulfide core with a coaxial sheath comprising poly(ethylene glycol) norbornene-RGD; c) collecting said extruded hybrid fiber on said collecting target to create a medical implant; and d) photo-polymerizing said hybrid fiber.

50. The method of claim 49, further comprising step e) exposing said implant to anaerobic conditions.Patent Attorney Docket No. UTCB-2071851. The method of claim 49, wherein said photo-polymerizing comprises UV exposure.

52. The method of claim 49, wherein said extruding comprises extruding said core solution surrounded by said sheath solution.

53. The method of claim 49, wherein said collecting comprises electrospinning said hybrid fiber upon said collecting target.

54. The method of claim 49, wherein said medical implant comprises a vascular stent.

55. The method of claim 49, wherein said medical implant is selected from the group consisting of vascular graft, heart valve, cardiovascular implants, and tissue-regenerative implant.

56. The method of claim 49, wherein said polyethylene glycol norbornene is RGD-functionalized.

57. A method of producing a medical implant comprising a poly-e-caprolactone core with a coaxial sheath comprising poly(ethylene glycol) norbomene-RGD comprising: a) providing; i) a core solution comprising diallyl trisulfide (DATS) and poly-s-caprolactone(PCL) in a solvent; ii) a sheath solution comprising gelatin in a solvent; and iii) a collecting target;Patent Attorney Docket No. UTCB-20718 b) extruding said combining said core solution and said sheath solution under conditions to create a hybrid fiber comprising a poly-s-caprolactone with diallyl trisulfide core with a coaxial sheath comprising gelatin; c) collecting said extruded hybrid fiber on said collecting target to create a medical implant; and d) treating said medical implant with a crosslinking agent.

58. The method of claim 57, further comprising step e) exposing said implant to anaerobic conditions.

59. The method of claim 57, wherein said treating comprises exposure to genipin.

60. The method of claim 57, wherein said treating comprises immersion exposure to genipin.

61. The method of claim 57, wherein said collecting comprises electrospinning said hybrid fiber upon said collecting target.

62. The method of claim 57, wherein said medical implant comprises a vascular stent.

63. The method of claim 57, wherein said medical implant is selected from the group consisting of vascular graft, heart valve, cardiovascular implants, and tissue-regenerative implant.

64. A method of minimizing inflammation and for encouraging cell tissue regeneration in a subjectPatent Attorney Docket No. UTCB-20718 with vascular condition requiring an intervention comprising: a) a medical implant exhibiting a slow release of H2S; b) implanting said implant.

65. The method of claim 64, wherein said implant is within a living subject.

66. The method of claim 64, wherein said release of H2S is sustained over a period of time.

67. The method of claim 66, wherein said period of time comprises several days or weeks.

68. The method of claim 64, wherein said slow release comprises a release of H2S in the range low nanomolar up to 300 micromolar over a period of weeks.

69. The method of claim 66, wherein said implant comprises a coaxial nanofibrous system impregnated with an H2S donor.

70. The method of claim 69, wherein said H2S donor is a poly sulfide.

71. The method of claim 70, wherein said polysulfide is a garlic-derived polysulfide.

72. The method of claim 69, wherein said H2S donor comprises diallyl trisulfide.

73. The method of claim 69, wherein said H2S donor is selected from the group comprising: diallylPatentAttorney Docket No. UTCB-20718 disulfide and diallyl tetrasulfide.

74. The method of claim 64, comprising at least one polyethylene-glycol derivative.

75. The method of claim 66, wherein said medical implant comprises a core with a coaxial sheath.

76. The method of claim 75, wherein said wherein said medical implant encourages cell tissue regeneration.

77. The method of claim 75, wherein said wherein said medical implant reduces inflammation.

78. The method of claim 75, wherein said core is a hydrophobic and degradable core.

79. The method of claim 69, wherein said medical implant comprises a vascular stent.

80. The method of claim 69, wherein said medical implant comprises a vascular graft.

81. The method of claim 72, wherein said the concentration of diallyl trisulfide in said implant is between 10 pM and 250 pM.

82. The method of claim 72, wherein said the concentration of diallyl trisulfide in said implant is between 25 pM and 150 pM.Patent Attorney Docket No. UTCB-2071883. The method of claim 69, wherein said medical implant is selected from the group consisting of vascular graft, heart valve, cardiovascular implants, and tissue-regenerative implant.

84. The method of claim 74, wherein said at least one poly ethyl ene-gly col derivative comprises at least one polymer selected from the group consisting of polyethylene-glycol dimethacrylate, polyethylene-glycol modified with thiol-ene, and polyethylene-glycol modified with hydroxy acid groups.

85. The method of claim 78, wherein said hydrophobic, degradable core comprises a at least one polymer from the group consisting of poly L-lactide acid, polycaprolactone, polyglycolic acid, poly(lactic-co-glycolic acid), and polyurethane.

86. The method of claim 78, wherein said hydrophobic, degradable core comprises a poly L-lactide acid core.

87. The method of claim 78, wherein, said hydrophobic, degradable core comprises at least one polymer selected from the group consisting of poly L-lactide acid, polycaprolactone, polyglycolic acid, poly(lactic-co-glycolic acid), and polyurethane.

88. The method of claim 78, wherein, said hydrophobic, degradable core comprises poly-s- caprolactone.

89. The method of claim 75, wherein said coating further comprises a controlled release agent.Patent Attorney Docket No. UTCB-2071890. The method of claim 75, wherein said coating further comprises a therapeutic agent.

91. The method of claim 90, wherein said therapeutic agent comprises an anti -proliferation agent.

92. The method of claim 78, wherein said hydrophobic, degradable core provided controlled drug release.

93. The method of claim 75, wherein said sheath integrates a surface signaling mechanism.

94. The method of claim 93, wherein said surface signaling mechanism that simultaneously aids in regeneration.

95. The method of claim 75, wherein said implant further comprises an interactive surface.

96. The method of claim 75, wherein said implant further comprises sub-millimeter scale fibers.

97. The method of claim 96, wherein said sub-millimeter scale fibers comprises nanoscale fibers.

98. The method of claim 96, wherein said sub-millimeter scale fibers provide a uniform surface.

99. The method of claim 96, wherein said sub-millimeter scale fibers comprise a cell recognition platform.Patent Attorney Docket No. UTCB-20718100. The method of claim 75, wherein said implant has no delaminiation.

101. The method of claim 75, wherein said the elasticity of said polyethylene-glycol sheath is tunable by varying the photopolymerization time.

102. The method of claim 64, wherein said implant comprises crosslinked coaxial nanofiber system.

103. The method of claim 102, wherein said nanofiber system comprises poly-s-caprolactone core with a coaxial sheath comprising poly(ethylene glycol) norbornene-RGD.

104. The method of claim 102, wherein said nanofiber system comprises coaxial PCL-gelatin (PCL+GEL) nanofiber system, crosslinked with genipin.

105. The method of claim 102, wherein said nanofiber system comprises coaxial ng PCL / PEGNB-RGD coaxial fiber system, crosslinked with PEG dithiol.

106. The method of claim 102, wherein said nanofiber system is impregnated with diallyl tri sulfide.

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