Laser assisted rolling fabrication technique to create a mesoporous, compliance-matched and drug eluting vascular graft

Abstract

The disclosed concept includes the use of additive and / or subtractive manufacturing of mesoporous structures on two-dimensional sheets that are mechanically tunable, to provide a vascular graft that controls biodegradable graft host interactions, to improve graft patency and functionality long-term. The vascular graft includes the two-dimensional sheets in a rolled or tubular configuration to form a lumen wherein a surface and / or bulk modification is applied to the flat sheet. In addition, a plurality of mesoporous structures (e.g., gaps) are formed in the flat sheet using a subtractive manufacturing technique.

Description

AN ADDITIVE AND SUBTRACTIVE LASER ASSISTED ROLLING FABRICATION TECHNIQUE TO CREATE A MESOPOROUS COMPLIANCE MATCHED AND DRUG ELUTING VASCULAR GRAFTCROSS-REFERENCE TO RELATED PATENT APPLICATIONSThis patent application claims the benefit of priority to provisional patent application U.S. Patent Application Serial No. 63 / 702,823, entitled “AN ADDITIVE AND SUBTRACTIVE LASER ASSISTED ROLLING FABRICATION TECHNIQUE TO CREATE A MESOPOROUS COMPLIANCE MATCHED AND DRUG ELUTING VASCULAR GRAFT”, filed on October 3, 2024, the contents of which are incorporated herein by reference.GOVERNMENT SUPPORT

[0001] This invention was made with government support under grant # HL157017 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD OF THE INVENTION

[0002] The disclosed concept includes the use of additive and / or subtractive manufacturing of mesoporous structures on two-dimensional sheets that are mechanically tunable, to provide a novel approach to implement a tissue engineered vascular graft and control biodegradable graft host interactions, to improve graft patency and functionality long-term.BACKGROUND

[0003] Cardiovascular diseases (CVDs), including coronary artery disease (CAD), stand as the primary contributor to global mortality and play a significant role in diminishing the quality of life. Coronary artery bypass grafting (CABG) is a common treatment for CAD. The growing incidence of cardiovascular diseases has led to an escalating demand for cardiac bypass grafting (CABG), making CABG surgery the most commonly conducted cardiac surgical procedure. While less invasive procedures, such as percutaneous coronary intervention (PCI), offer an alternative for revascularization, patients undergoing CABG experience a lower risk of postoperative complications such as heart attacks and strokes. However, the failure rate in CABG surgery remains as high as 42.8%, with only 50% to 60% maintaining patency after a decade.

[0004] Further, patients with multi- vessel disease face a scarcity of viable healthy vessels available for CABG procedures; graft failure in CABG procedures occurs when the graft cannotadequately supply blood flow to downstream tissues. It is crucial for the graft to remain open and functional over time. Vascular graft failures manifest in both early and late stages. Thrombosis is typically involved in early-stage failures, while late-stage failures involve intimal hyperplasia (IH) and lack of host integration of the graft (lack of vascular smooth muscle cell (VSMC) infiltration). The scarcity of healthy host tissue (e.g., saphenous vein, mammary artery) and current failure rates when using host tissues as bypass grafts establish a dire need for the development of an off-the-shelf functional vascular graft.

[0005] A functional tissue engineered vascular graft (TEVG) can be antithrombogenic, compliance matched, suturable / deliverable, and remain patent for the lifetime of the device. If biodegradable, there is an additional requirement that the vascular graft be infiltrated with host cells that eventually become the cells required in a functional vessel (e.g., endothelial cells for antithrombogenicity and VSMCs for contractility and ECM maintenance).

[0006] In the context of vascular grafts, compliance refers to the ability of the graft to expand and contract in response to changes in blood pressure (hoop compliance). Compliance matching is essential between grafts and host native vessels to promote appropriate hemodynamics and prevent IH. Mismatched compliance results in alterations in hemodynamics which include oscillating shear stress and shear stress gradients. Additionally, compliance mismatch at the anastomotic sites causes differential non-homeostatic vascular wall stresses. Disruptions in these stresses, especially at the anastomosis sites, triggers a cellular response leading to IH. Given that 60% of grafts fail due to IH within a 10-year period, finding methods to mitigate IH could uphold graft functionality over time.

[0007] The infiltration of VSMCs into fully biodegradable vascular grafts is crucial for prolonging their function. Without the infiltration of VSMCs and other immune cells, grafts are structurally weaker and more prone to mechanical failure or rupture. Additionally, without cell infiltration, the graft may lack the ability to undergo necessary structural changes over time, compromising its long-term patency and functionality. Enhancing cell infiltration within fully biodegradable vascular- grafts is essential for optimal graft integration, remodeling, and longterm function.

[0008] Further, thrombosis accounts for approximately 35% of CABG procedure failures within the initial two years. Platelet adhesion is then followed by a cascade of events, termed thecoagulation cascade, leading to thrombus formation. Graft thrombosis is initiated by the activation and adhesion of platelets to the luminal surface of the graft. Reducing platelet deposition and thrombotic potential of a vascular graft, especially as it degrades, is critical to ensure long term graft patency.

[0009] TEVGs fabricated from biomaterials may mitigate the risk of graft failure and improve long-term patency rates. An important limitation of current fully biodegradable grafts is the lack of controllable porosity within the graft, both radially and axially along the graft. Current approaches for including porosity in TEVGs include electrospinning, thermally induced phase separation (TIPS), porogen leaching, and spray phase separation. All these methods require the use of a random uncontrolled process to generate graft porosity. Modulation of porosity using electrospinning typically either results in dense fiber mats with very small pores but excellent mechanical integrity (to aid in deliverability), or very large pores in constructs that are not deliverable due to a lack of mechanical integrity. Designed radial distributions of pore sizes can be implemented using electrospinning; however, modulation of axial distributions of porosity is not possible. TIPS, porogen leaching, and spray phase separation inherently rely on homogenous distributions of pore size in a randomized manner. All these approaches lack the ability to control pore size independently in the radial and axial direction. These limitations therefore do not allow novel designs spatially controlling cell infiltration, topographical cues, drug elution, or mechanical support.

[0010] VSMCs play an essential role in producing extracellular matrix (ECM) proteins that stabilize the graft, but their migration typically lags behind graft degradation, resulting in graft instability. Porosity in the graft material is crucial for promoting VSMC infiltration, yet current methods for increasing graft porosity — such as salt leaching and thermally induced phase separation (TIPS) — do not provide precise control over pore size and location. Recent advancements in two-photon subtractive manufacturing provide the ability to precisely control pore size and location within the graft. This level of control is essential for optimizing VSMC migration, ECM deposition, and overall graft integration.

[0011] Accordingly, there is a need in the art to design, develop, fabricate and implement biodegradable vascular grafts (e.g., TEVGs) that exhibit the ability to control pore size, independently in the radial and axial directions, within the graft. The novel vascular graft designsof the disclosed concept allow for spatially controlling cell infiltration, topographical cues, drug elution, or mechanical support within the grafts, which can improve graft patency and long-term graft functionality.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1A is a schematic that shows porous fabricated sheets with cut shapes from a binary image, FIG. IB is a schematic that shows pores of the fabricated sheet loaded with drug / chemokine, and FIG. 1C is a schematic that shows the process of rolling the cut sheet onto a mandrel, adhering adjacent layers of cut sheets, and forming a drug loaded TEVG, in accordance with certain embodiments of the inventive concept.

[0013] FIG. 2 is an image that shows binary masks of varying complexity to assess cutting precision and repeatability as follows: Gradient I, Gradient II, Stent Mesh, and Dodecagon Mesh, according to certain embodiments of the inventive concept.

[0014] FIG. 3 is an image that shows cut images of the mesoscopic porous sheets that correspond to the laser cut binary masks shown in FIG. 2, i.e., Gradient 1, Gradient 11, Stent Mesh and Dodecagon Mesh, respectively, to produce mesoscopic porous vascular grafts / TEVGs, in accordance with certain embodiments of the invention.

[0015] FIG. 4 is a plot that shows significant differences in Dice similarity coefficients (p<0.05) for the two-photon cut images shown in FIG. 3, i.e., Gradient I, Gradient II, Stent Mesh and Dodecagon Mesh, in accordance with certain embodiments of the invention.

[0016] FIG. 5 is a plot wherein the coefficient of variation shows repeatability for the two- photon cut patterns shown in FIG. 3, i.e., Gradient I, Gradient II, Stent Mesh and Dodecagon Mesh, in accordance with certain embodiments of the invention.

[0017] FIGS . 6A and 6B show binary images representing decreasing pore sizes for two complexities, i.e., Gradient I (280-100 pm) and Gradient II (90-10 pm), respectively, in accordance with certain embodiments of the invention.

[0018] FIG. 7A shows the Gradient I binary input image (original), FIG. 7B shows the Gradient I post cutting image with artifacts, FIG. 7C shows the Gradient I post cutting image with artifactsremoved, and FTG. 7D shows the Gradient T binary input image overlayed on post cutting image with artifacts removed, according to certain embodiments of the invention.

[0019] FIG. 8A shows the Gradient II binary input image (original), FIG. 8B shows the Gradient II post cutting image with artifacts, FIG. 8C shows the Gradient II post cutting image with artifacts removed, FIG. 8D shows the Gradient II binary input image overlayed on post cutting image with artifacts removed, according to certain embodiments of the invention.SUMMARY OF THE INVENTION

[0020] In one aspect, the disclosed concept provides a tissue engineered vascular graft that includes at least one two-dimensional flat sheet in a rolled or tubular- configuration to form a lumen, including a material selected from the group consisting of a single material, a blend of materials, a weave, a laminate, or a composite of two or more materials; a luminal surface; an inner diameter; one or more of a surface modification and bulk modification applied to the flat sheet; and a plurality of mesoporous gaps formed in the flat sheet.

[0021] In certain embodiments, the surface modification includes a coating that includes a biocompatible and / or biodegradable material applied to the luminal surface. The coating improves cell attachment and / or infiltration or reduces platelet adhesion. The coating may fully or partially cover the luminal surface.

[0022] In certain embodiments, the bulk modification includes inclusion of one or more agents within a bulk of the flat sheet. The one or more agents may be selected from sulfubutane groups, and included throughout a thickness, or entire thickness, of the luminal surface to reduce platelet adhesion.

[0023] In certain embodiments, the subtractive manufacturing technique is selected from singlephoton or two-photon laser ablation using a binary mask computationally optimized for vascular graft functionality. The two-photon microscopy can be selected to create mesopores within the flat sheet that are fully bound by the material above and below. The plurality of mesoporous gaps may include a drug or chemokine or growth factor therein.

[0024] In certain embodiments, the material includes synthetic and / or native extracellular matrix proteins. The material may be selected from the group consisting of polycaprolactone (PCL),poly(glycolic acid) (PGA), poly(L-lactide-co-e-caprolactone) (PLCL), polyvinyl alcohol (PVA), polylactic acid (PLA), poly(lactic-co-glycolic) acid (PLGA), poly-L-lactic acid (PLLA), polyethylene oxide (PEG), polyurethanes, silk, silk elastin, gelatin, collagen (all types), elastin (all types), cellulose, fibronectin, laminin, fibrinogen, vitronectin, tropoelastin, fibrin (including fibrin gels), polyethylene glycol (PEG), hyaluronic acid (HA), nidogens, heparan, heparan sulfate proteoglycans, glycosaminoglycans (GAGs), PEG hydrogels, and methacrylated (and therefore, single or two photon light-crosslinkable) synthetic or native extracellular matrix of any type.

[0025] In certain embodiments, the graft can further comprise one or more of a crosslinker and photoactivator.

[0026] In certain embodiments, the synthetic and / or native extracellular matrix proteins include small intestine submucosa and / or urinary bladder matrix.

[0027] In certain embodiments, the inner diameter matches a host vessel replaced with the tissued engineered vascular graft. The inner diameter can be from about 1 mm to about 5 mm.

[0028] In certain embodiments, the wall thickness can match a host vessel replaced with the tissued engineered vascular graft The wall thickness may be from about 100 um to about 500 m.

[0029] In certain embodiments, the material includes one or more of (i) a low temperature melting polymer that can be thermoformed, (ii) a chemical moiety that can be adhered to itself using a biological glue or adhesive, and (iii) a chemical moiety that can be adhered to itself using a two-photon polymerization process.

[0030] In another aspect, the disclosed concept provides a method of preparing a tissue engineered vascular graft. The method includes fabricating the graft, including rolling at least one two-dimensional flat sheet into a tubular configuration to form a lumen, including a material selected from the group consisting of a single material, a blend of materials, a weave, a laminate, or a composite of two or more materials; a luminal surface; and an inner diameter; applying a surface and / or bulk modification to the flat sheet to form a fabricated graft; and forming a plurality of mesoporous gaps in the flat sheet using a subtractive manufacturing technique.

[0031] The rolling step can include applying at least two flat sheets onto a mandrel having a shape corresponding to a patient vascular bypass.

[0032] In certain embodiments, the fabricated graft includes multiple flat sheets layered in a stacked configuration. The multiple flat sheets can be rolled, and adjacent sheets subsequently joined together by thermoforming or chemical bonding.DETAILED DESCRIPTION

[0033] The inventive concept includes the use of additive and / or subtractive manufacturing of mesoporous structures on two-dimensional (2D) sheet(s) to provide a novel approach to control graft host interactions for vascular grafts, such as but not limited to, tissue engineered vascular grafts (TEVGs). The inventive concept provides vascular grafts with improved graft patency and long-term functionality.

[0034] The vascular grafts according to the inventive concept are structured to provide controllable porosity within the graft, both radially and axially along the graft. In addition, the vascular grafts according to the inventive concept provide novel designs that spatially control cell infiltration, topographical cues, drug elution, and / or mechanical support.

[0035] The inventive concept includes electrospinning a solution, e.g., polycaprolactone (PCL), onto a mandrel to fabricate fibrous 2D sheet(s); cutting the 2D sheet(s), e.g., using two-photon cutting, to form mesoscopic porosity within the 2D sheet(s); subsequently rolling the porous 2D sheet(s), e.g., onto a mandrel; self-adhering the rolled porous 2D sheet(s), e.g., using heat or light, with a balloon catheter to form the vascular graft structure, e.g.,, TEVG.

[0036] The inventive concept provides a method for preparing a vascular graft that includes an improved level of control for forming mesoporous structures not previously known for synthetic or natural biopolymers, or combinations thereof. The 2D sheet(s) is / are also mechanically tunable, allowing modulation of inherent material stiffness in addition to the structural stiffness from the 2D cut (and optimized) design prior to rolling.

[0037] Unless otherwise noted, technical terms are used according to conventional usage. Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Thesingular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. The term “comprises” means “includes.” The abbreviation, “e.g.” is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.” Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0038] As used herein, the term “vascular graft” means a tubular- member which acts as an artificial vessel. A vascular graft can include a single material, a blend of materials, a weave, a laminate or a composite of two or more materials.

[0039] As used herein, the term “tissue-engineered vascular graft” (“TEVG”) means a vascular graft that is constructed of, or at least includes, a tissue-engineered material.

[0040] As used herein, the term “lumen” means the central cavity or inside open space of the tubular member.

[0041] As used herein, the term “luminal” means the inner surface of the lumen.

[0042] As used herein, the term “abluminal” means the outside of, or outer surface of, the lumen.

[0043] In further examples, the disclosed methods of fabrication of a vascular graft I TEVG include coating a surface of a biodegradable scaffold, such as a surface of a biodegradable polyester tubular core with a biocompatible and / or biodegradable material. Suitable biocompatible and / or biodegradable materials for use in the disclosed concept include those that are known in the ail for such applications and use. In certain embodiments, the inner luminal surface of the biodegradable scaffold is coated with a biocompatible and / or biodegradable material. It is contemplated that such coating may be completely or partially cover the surface of the biodegradable scaffold.

[0044] The various dimensions of a disclosed vascular graft / TEVG may vary according to the desired use. In certain embodiments, the method of fabrication is performed to generate a vascular graft / TEVG with an inner diameter which matches that of the host vessel to be replaced. In some examples, the inner diameter is from about 1 mm to about 5 mm. In someexamples, the method of fabrication is performed to generate a vascular graft I TEVG with a wall thickness which matches that of the host vessel to be replaced. However, it is contemplated that the graft wall is fabricated with a thicker or thinner wall than that which is being replaced, if desired. In some examples, a disclosed vascular graft is fabricated to have a wall thickness from about 100 pm to about 500 pm.

[0045] The disclosed scaffolds / vascular grafts / TEVGs are useful to guide host tissue remodeling in many different tissues, including any tissue that has progenitor cells. The disclosed biodegradable scaffolds / vascular grafts / TEVGs can be used to facilitate tissue regeneration in vivo by providing a structural frame for which tissue regeneration will occur. In some examples, the scaffolds / vascular grafts / TEVGs are constructed to allow and facilitate the infiltration of host cells including progenitor cells. In some examples, the scaffolds I vascular grafts / TEVGs allow and facilitate host remodeling of the biodegradable structure, so that eventually the polymeric structure is replaced by the desirable host tissue.

[0046] In certain embodiments, the overall process for fabricating and implementing biodegradable grafts (e.g., TEVGs) that exhibit the ability to control pore size independently in the radial and axial directions, includes the following steps and features. A fully biodegradable flat sheet, of any mixture / combination of any number of synthetic and / or native extracellular matrix (ECM) proteins, is generated. Non-limiting examples of the synthetic and / or native extracellular matrix proteins include but are not limited to polycaprolactone (PCL), poly(glycolic acid) (PGA), poly(L-lactide-co-s-caprolactone) (PLCL), polyvinyl alcohol (PVA), polylactic acid (PLA), poly(lactic-co-glycolic) acid (PLGA), poly-L-lactic acid (PLLA), polyethylene oxide (PEG), polyurethanes, silk, silk elastin, gelatin, collagen (all types), elastin (all types), cellulose, fibronectin, laminin, fibrinogen, vitronectin, tropoelastin, fibrin (including fibrin gels), polyethylene glycol (PEG), hyaluronic acid (HA), nidogens, heparan, heparan sulfate proteoglycans, glycosaminoglycans (GAGs), PEG hydrogels, and methacrylated (and therefore, single or two photon light-crosslinkable) synthetic or native extracellular matrix of any type.

[0047] The flat sheet is fabricated using any number of biofabrication techniques, including but not limited to, one or more of electrospinning, TIPS, freeze drying, porogen leaching, solvent casting, spin coating, or 3D printing using either single or two photon light activated polymerization. If light polymerization is utilized, then inclusion of any number or variety ofcrosslinkers and photoactivators will also be contained within the flat sheet. Additionally, one or more commercially available sheets of dcccllularizcd ECM, including but not limited to, small intestine submucosa (SIS) and urinary bladder matrix (UBM) are suitable for use as the sheets / layers to be cut utilizing this disclosed concept.

[0048] In certain embodiments, the flat sheet is fabricated using a technique that includes one or more of electrospinning and 3D printing with two photon light activated polymerization.

[0049] The flat sheet contains (i) a low temperature melting polymer, selected from such polymers that are known in the art, that can be thermoformed (adhered to itself using moderate temperatures) and / or (ii) the presence of chemical moieties that can be adhered to itself using, for example, a biological glue or adhesive (example being native extracellular matrix), and / or (iii) the presence of chemical moieties that can be adhered to itself using a two photon polymerization process (e.g., methacrylated native or synthetic polymer).

[0050] A surface and / or bulk modification (adsorption of chemical covalent modification, bulk inclusion) of the flat sheet may be performed. Examples of surface modifications include, but are not limited to, surface coatings for improving cell attachment and / or infiltration or, for example, reducing platelet adhesion. Since the treatment is performed on a flat sheet, precise treatments can be easily and efficiently distributed to any particular location on the flat sheet. This spatial control is a novel feature of the disclosed concept. For example, platelet repulsing treatments can be applied to only the portion of the flat sheet that will become the lumen of the scaffolds / vascular grafts / TEVGs , whereas immune cell modulatory and / or VSMC chemokines and agents can be applied to the remainder of the graft (that would become the medial I adventitial layers of the scaffolds / vascular grafts / TEVGs).

[0051] In certain embodiments, in addition to surface modification, the inclusion of agents within the bulk of the fabricated sheet may also be included. An example of an inclusion includes, but is not limited to, sulfubutane groups throughout the entire thickness of the luminal portion of the sheet to reduce platelet adhesion. The use of bulk inclusion will allow these agents to be present throughout the entire degradation lifetime of the fully biodegradable scaffolds / vascular grafts / TEVGs.

[0052] After the flat sheet has been fabricated, it is cut using a known process such as a subtractive manufacturing technique, including but not limited to, single-photon or two-photonlaser ablation that uses a binary mask that is computationally optimized for vascular graft functionality. This technique creates mcsoporous gaps in the flat sheet that arc designed for a particular purpose. The use of a laser-assisted subtractive manufacturing approach allows “any” shape - as dictated by the magnification of the optical objective - to be cut into the flat sheet. Further, two-photon microscopy has the capability to create pores within the sheet that are fully bound by material above and below (as two-photon subtractive manufacturing uses a light focal spot isolate in 3D).

[0053] Subsequently, the flat sheet is loaded with a drug or chemokine or growth factor that is of particular interest to improve scaffolds / vascular grafts / TEVGs functionality and placed within mesopores that were created in the flat sheet, using subtractive manufacturing as described above. In certain embodiments, the flat sheet is loaded with synthetic or native ECM hydrogel including, but not limited to, PEG hydrogel, fibrin gel, collagen gel, tropoelastin gel, gelatin and the like. The flat sheet provides full control over what drugs / factors are spatially distributed in which locations - for designed intent. For example, as mentioned above for bulk or surface modification, there can be anti-platelet or pro-endothelial agents in the luminal locations and pro- regenerative chemokines / growth factors (e.g., immunomodulatory and / or VSMC chemoattractants or promoters) in the medial / abluminal locations. This approach for drug / chemokine / growth factor inclusion allows improved control of the concentration of agent (e.g., the ability to deliver significantly more), as well as improved distribution of agent than any currently available TEVG fabrication technique (e.g., porogen leaching, TIPS, spray phase separation, and electrospinning).

[0054] In certain embodiments, multiple flat sheets as described above are layered, e.g., stacked, if desired - one on top of the other. This is useful to improve and / or make more efficient the thermoforming and / or chemical crosslinking that will be performed following rolling of the sheets, as well as provide the scaffolds / vascular grafts / TEVGs with a biomimetic layered micro structure that is similar to native arterial tissues (e.g., layers of elastin or tropoelastin).

[0055] In certain embodiments, the inventive concept includes an electrospun sheet that is cut using two-photon subtractive manufacturing, rolled, and thermoformed to create a smalldiameter scaffold I vascular graft / TEVG. This process allows for a highly controlled, mesoscopic porosity, e.g., ranging from 10 to 280 um, enhancing the graft’s performancecompared to known fabrication methods. In certain embodiments, the two-photon subtractive manufacturing method creates pores for improving functionality of the scaffold I vascular graft / TEVG.

[0056] The TEVG fabrication process, in accordance with certain embodiments of the inventive concept, is shown in FIGS. 1A, IB and 1C. FIG. 1A shows binary images (left side) with shaped-cuts formed therein to form fabricated sheets having luminal and abluminal portions with the shaped-cuts (right side) from the binary images made in the abluminal portion to form mesopores. FIG. IB shows the porous fabricated sheet with drug / chemokine (e.g., in the form of a gel) loaded into the mesopores (shaped-cuts) to form a loaded fabricated sheet. FIG. 1C shows a process that includes (from left to right) a mandrel, the loaded fabricated sheet (as shown in FIG. IB), applying the loaded fabricated sheet onto the mandrel to form a tubular sheet, thermoforming the tubular sheet, or adhering adjacent layers of tubular sheets, with a balloon catheter to form a drug loaded TEVG construct. In certain embodiments, the fabricated sheet is prepared using an electrospinning apparatus to produce electropun fibers that are collected on a mandrel to form a fibrous sheet; the fibrous sheet is cut using a two-photon cutting mechanism to form a mesoscopic porous sheet; the mesoscopic porous sheet is then rolled onto a mandrel to form a tubular' mesoscopic porous sheet. The mandrel is selected according to the desired diameter of the tubular mesoscopic porous sheet. The tubular mesoscopic porous sheet is then thermoformed, e.g., with a balloon catheter, to form a TEVG construct.

[0057] In certain embodiments, the fabricated sheet has luminal and abluminal portions wherein spherical-shaped cuts are made in the abluminal portion to form mesopores. In certain embodiments, a drug and / or chemokine is / are loaded into the mesopores or a plurality of mesoporous gaps; the sheet is then rolled onto a mandrel; adjacent layers of the rolled sheet(s) is / are adhered together by thermoforming, and a drug-loaded vascular graft / TEVG is formed.

[0058] In certain embodiments, the flat, e.g., fibrous, sheet(s) are rolled or similarly layered onto a mandrel whose shape is specifically chosen for a particular vascular bypass need. For example, if the clinical need is a graft of similar inner diameter both proximal and distal to the stenosis to be bypassed, then a right circular cylinder will be used to perform the rolling. If, however, there is a need for a desired taper in the diameter of the graft proximal to distal, then a mandrel of decreasing diameter can be used. In certain embodiments, the sheets’ shape is changed to benon-rectangular such that rolling around a tapered rod creates no gaps or wrinkles (e.g., analogous to a map of the world globe being distorted for display of a spherical surface on a flat sheet), the one or more sheet(s) are cut in a distorted manner to allow smooth rolling of a non- prismatic rod.

[0059] In certain embodiments, once the sheet(s) have been rolled, adjacent layers in direct contact with each other are thermally or chemically bonded to themselves, such as, employing homogenous gentle heating for short times and low temperatures that have been computationally designed to not denature any bioactive included agent. Alternatively, thermal bonding and / or crosslinking using light is used to crosslink or thermoform adjacent layers of the rolled sheet(s) using either single or two photon laser ablation. In certain embodiments, wherein two photon laser excitation is used, light is focused in a very small Z focal spot that makes light ablation / thermoforming or crosslinking occur only in specific radial-locations within the scaffolds / vascular grafts / TEVGs (thus, avoiding thermal damage to bioactive agents).

[0060] Following graft fabrication, graft design parameters including the concentration of various polymers, concentration or time of crosslinking, laser light ablation energy, or number and thickness of sheet layers within the rolled scaffolds / vascular grafts / TEVGs are computationally tuned to produce sheets whose stiffness can be controlled across a very wide range of values. Tubular biaxial testing in combination with computational simulation is used to determine the stiffness values. In certain embodiments, computational optimization procedures are used to generate TEVGs whose compliance is tunable to match the host artery location.

[0061] In certain embodiments, the end-result of the inventive concept is a compliance tunable vascular graft / TEVG that contains controlled mesoporous microstructures that can be used to (i) encourage host immune / endothelial / VSMC / fibroblast infiltration; and (ii) load heretofore unattainable amounts of drug / growth factor / chemokine efficiently in particular radial and axial locations within the vascular graft I TEVG.

[0062] In accordance with certain embodiments of the invention, a two-photon workflow or method is used. A laser machine that performs two-photon subtractive cutting / manufacturing is selected. A binary mask is positioned within the laser machine for cutting. As a result, there is produced a binary mask with holes cut by the laser machine. The binary mask with the laser cut holes is used to create the mesoscopic porous sheet.

[0063] In accordance with certain embodiments of the invention, FIG. 2 includes images that show binary masks of varying complexity to assess cutting precision and repeatability. The complexity is described as follows (from left to right): Gradient I, Gradient II, Stent Mesh, and Dodecagon Mesh.

[0064] In accordance with certain embodiments of the invention, FIG. 3 includes images that show the cut images of the mesoscopic porous sheets that correspond to the laser cut binary masks shown in FIG. 2, i.e., Gradient 1, Gradient 11, Stent Mesh, and Dodecagon Mesh, respectively, to produce mesoscopic porous vascular grafts / TEVGs in accordance with certain embodiments of the invention.

[0065] FIG. 4 is a plot wherein the Welch’s ANOVA (n=4 per group) shows significant differences in Dice similarity coefficients (p<0.05) for the two-photon cut images shown in FIG.3, i.e., Gradient I, Gradient II, Stent Mesh, and Dodecagon Mesh, respectively, in accordance with certain embodiments of the invention.

[0066] FIG. 5 is a plot wherein the CV shows the repeatability for the two-photon cut patterns shown in FIG. 3, i.e., Gradient I, Gradient II, Stent Mesh, and Dodecagon Mesh, respectively, in accordance with certain embodiments of the invention. According to FIG. 5, Gradient II is most variable.

[0067] Non-limiting applications and advantages of the disclosed concept include the following.

[0068] 1) It is difficult to control the radial and axial distribution of porosity of scaffolds / vascular grafts / TEVGs using existing technology. Known techniques require a process that is inherently random. Radial and axial gradients in the highly controlled mesoporous structures of the inventive concept allow precise control of cell infiltration as well as scaffolds I vascular grafts / TEVGs biodegradation. Combinations of axial and radial location and shape of mesopores are easily implemented using the inventive concept, for optimum performance of the scaffolds / vascular grafts I TEVGs.

[0069] 2) In an embodiment, controlled mesoporosity (especially through the radial thickness and along the axial dimension of the vascular graft) is used to generate a vascular graft that is designed specifically to allow host peripheral blood infiltration. This is achieved using computational fluid dynamics (CFD) models incorporating predictions of the blood clottingprocess. In such a way, the graft is intentionally infiltrated with host circulating cells in clinically reasonable time frame. Controlled elution of agents is released from subsets of mcsoporcs (isolated from interconnected pores designed for blood infiltration) that arc specifically designed to modulate host cell differentiation. One example leverages established in-vitro cell culture protocols that create and subsequently differentiate human pluripotent stem cells (hiPSCs) from peripheral blood mononuclear cells without genetic manipulation. The growth factors and agents used in these protocols are included in the vascular graft, e.g., TEVG, (within isolated noninterconnected pores) and released at different times using existing controlled release technology. This allows peripheral blood host infiltration immediately upon graft implantation and subsequently differentiates derived progenitors into a desired vascular cell type or phenotype (e.g., endothelial cells, contractile VSMCs) using these eluted bioactive agents.

[0070] 3) In another embodiment, mesoporous structures are used to alter the cell alignment of infiltrating host cells. Subtractive manufacturing provides cues (holes created with directionality) that structurally encourage cell alignment and subsequent function. For example, ECs and VSMC layers are independently formed to have different alignments through the thickness of the vascular graft - as is the case in native arteries.

[0071] 4) Radial control of mesoporous structures is also leveraged to promote vascular graft I TEVG endothelialization. The known vascular graft I TEVG approach requires endothelial cells to migrate axially from the proximal / distal anastomoses. The limited replicability and proliferation of these cells makes endothelialization of long bypass grafts / TEVGs (as are typically used in CABG procedures) difficult. The inventive concept creates mesopores that are specifically designed to recruit and promote endothelialization of the vascular graft I TEVG transradially from adjacent fatty tissues. This allows mapping of specific avenues for endothelial cell migration, as well as the capability to easily include factors known to promote endothelial cell migration (e.g., vascular endothelial growth factor (VEGF)). In addition, one or more of host cell infiltration, differentiation, and future viability is / are improved by using the inventive concept, as microvasculature is useful to perfuse and thus promote graft healing throughout the axial length of the vascular graft I TEVG.

[0072] 5) In certain embodiments, a biomimetic layered vascular graft / TEVG is generated using the inventive concept; the appropriate number and types of layers of a vessel are created(e.g., endothelium, media, adventitia), and the control of structures that promote intralayer communication is achieved. In certain embodiments of the inventive concept, fenestrated clastic laminae (holes within the elastic layers of the artery) are created that are known to influence both mechanical and mass transport properties within native arteries.EXAMPLES

[0073] Electrospun sheets were fabricated using a 10% (w / v) solution of 80,000 g / mol polycaprolactone (PCL) in Hexafluoropropylene (HFP) as the solvent. The PCL solution was prepared by dissolving PCL in HFP and stirred at room temperature for 24 hours to ensure complete dissolution. The solution was then loaded into a 5 mL syringe and electrospun using an electrospinning setup (IME Technologies, Inc.), equipped with a high-voltage power supply (15 kV), a syringe pump with a flow rate of 10 pL / min, and a rotating collector. The electrospinning process was carried out under 30% humidity and 22° C temperature conditions. The PCL solution was dispensed onto a 9 cm diameter rotating mandrel collector, which was set to rotate at 300 rpm, allowing the formation of a thin fibrous sheet with an approximate thickness of 180 pm over a period of 45 minutes. For pore size gradient generation, binary images representing decreasing pore sizes were created using the circle packing function in MATLAB. The binary images were designed to simulate a gradient of pore sizes suitable for promoting cellular infiltration. FIGS. 6A and 6B show binary images representing decreasing pore sizes for two complexities, i.e., Gradient I (280-100 pm) and Gradient II (90-10 pm), respectively. The gradients were used as templates for two-photon laser cutting.

[0074] Two-photon subtractive manufacturing (Miltenyi Biotec B.V.) was performed on the electrospun sheet using an Olympus XL Fluor 4x objective. The cutting parameters were determined using a custom Python script, which dictated top-to-bottom cutting with an advancing step size of 10 pm with a Z-range of 200 pm. The cuts ranged from 10-280 pm to create the pore size gradient within the electrospun sheet. The image analysis method (MATLAB) involved processing an input image by converting it to grayscale, applying adaptive thresholding to binarize it, and then complementing the binary image to emphasize pores as white and the background as black. Artifacts were filtered out based on size and shape criteria,preserving only circular pores that match the intended laser-cut size range. Pixel counts of white areas arc then calculated before and after filtering to evaluate the differences. Images taken using TrimScope II (Miltenyi Biotec B.V.)RESULTS

[0075] For Gradient I complexity, the original count (input) of white pixels was 1,623,138 which represents the area designated for laser cutting in the binary input image. After the two-photon laser cutting, the white pixel count decreased to 1,561,263. FIG. 7A shows the Gradient I binary input image (original); FIG. 7B shows the Gradient I post cutting image with artifacts; FIG. 7C shows the Gradient I post cutting image with artifacts removed; FIG. 7D shows the Gradient I binary input image overlayed on post cutting image with artifacts removed - the percent error was approximately 3.81%, scale bar = 200 pm.

[0076] For Gradient II complexity, the original count of white pixels was 179,119 which represents the area designated for laser cutting in the binary input image. After the two-photon laser cutting, the white pixel count increased to 195,657. FIG. 8A shows the Gradient 11 binary input image (original); FIG. 8B shows the Gradient II post cutting image with artifacts; FIG. 8C shows the Gradient II post cutting image with artifacts removed; FIG. 8D shows the Gradient II binary input image overlayed on post cutting image with artifacts removed - the percent error was approximately 9.24%, scale bar = 100 pm.DISCUSSION

[0077] The results of the two-photon subtractive manufacturing process reveal variation in the outcomes for both Gradient 1 and Gradient 11 complexities. In Gradient 1, the white pixel count decreased from 1,623,138 to 1,561,263, with a percent error of 3.81%. This reduction suggests successful material removal but also reflects potential imperfections, such as incomplete cuts or overexposure in some areas. The percent error is relatively low, indicating an overall efficient process, however, optimization in laser intensity and exposure time could minimize such discrepancies.

[0078] In contrast, Gradient II showed an increase in white pixels, from 179, 119 to 195,657, indicating material may have been added or altered. This may result from excess laser energy causing surface roughening or melting. More precise control over laser parameters, such as power and focal length, would ensure accurate material removal.

Claims

We claim:

1. A tissue engineered vascular graft, comprising: at least one two-dimensional flat sheet in a rolled or tubular configuration to form a lumen, comprising: a material selected from the group consisting of a single material, a blend of materials, a weave, a laminate, or a composite of two or more materials; a luminal surface; an inner diameter; one or more of a surface modification and bulk modification applied to the flat sheet; and a plurality of mesoporous gaps formed in the flat sheet.

2. The graft of claim 1, wherein the surface modification comprises a coating comprising a biocompatible and / or biodegradable material applied to the luminal surface.

3. The graft of claim 2, wherein the coating improves cell attachment and / or infiltration, or reduces platelet adhesion.

4. The graft of claim 1, wherein the bulk modification comprises inclusion of one or more agents within a bulk of the flat sheet.

5. The graft of claim 4, wherein the one or more agents are selected from sulfubutane groups and are included throughout a thickness, or entire thickness, of the luminal surface to reduce platelet adhesion.

6. The graft of claim 5, wherein the sulfubutane groups are present throughout an entire degradation lifetime of the graft.

7. The graft of claim 1, wherein the mesoporous gaps within the flat sheet are fully bound by the material above and below.

8. The graft of claim 1, wherein the plurality of mesoporous gaps comprises a drug or chemokine or growth factor therein.

9. The graft of claim 1, wherein the material comprises synthetic and / or native extracellular matrix proteins.

10. The graft of claim 9, wherein the material is selected from the group consisting of polycaprolactone, poly(glycolic acid), poly(L-lactide-co-8-caprolactone), polyvinyl alcohol, polylactic acid, poly(lactic-co-glycolic) acid, poly-L-lactic acid, polyethylene oxide, polyurethanes, silk, silk elastin, gelatin, collagen, elastin, cellulose, fibronectin, laminin, fibrinogen, vitronectin, tropoelastin, fibrin, polyethylene glycol, hyaluronic acid, nidogens, heparan, heparan sulfate proteoglycans, glycosaminoglycans, PEG hydrogels, and methacrylated synthetic or native extracellular matrix.

11. The graft of claim 1, further comprising one or more of a crosslinker and photoactivator.

12. The graft of claim 11, wherein the synthetic and / or native extracellular matrix proteins comprise small intestine submucosa and / or urinary bladder matrix.

13. The graft of claim 1, wherein the inner diameter matches a host vessel replaced with the tissued engineered vascular graft.

14. The graft of claim 13, wherein the inner diameter is from about 1 mm to about 5 mm.

15. The graft of claim 1, wherein the wall thickness matches a host vessel replaced with the tissued engineered vascular graft.

16. The graft of claim 15, wherein the wall thickness is from about 100 pm to about 500 pm.

17. A method of preparing a tissue engineered vascular graft, comprising: fabricating the graft, comprising: rolling at least one two-dimensional flat sheet into a tubular configuration to form a lumen, comprising:a material selected from the group consisting of a single material, a blend of materials, a weave, a laminate, or a composite of two or more materials; a luminal surface; and an inner diameter; applying a surface and / or bulk modification to the flat sheet to form a fabricated graft; and forming a plurality of mesoporous gaps in the flat sheet using a subtractive manufacturing technique.

18. The method of claim 17, wherein the rolling step comprises applying the at least two flat sheets onto a mandrel having a shape corresponding to a patient vascular bypass.

19. The method of claim 17, wherein the fabricated graft comprises multiple flat sheets layered in a stacked configuration.

20. The method of claim 19, wherein the multiple flat sheets are rolled, and adjacent sheets are subsequently joined together by thermoforming or chemical bonding.

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