Patent Application
20240042103
Coronary artery disease (CAD) is a leading cause of death or impaired quality of life for millions of individuals, resulting in more than half a million coronary artery bypass surgeries per year, Gui et al., 2009; Sundaram et al., 2014; Thompson et al., 2002; Mozaffarian et al., 2015, with treatment costs of over $100,000 per procedure. Gokhale, 2013. The standard treatment for CAD, which afflicts small-diameter arteries, is the use of autologous tissue as a bypass graft. Gui et al., 2009.
Autografts, however, have several disadvantages, including the requirement of a secondary surgical site to harvest the donor graft, as well as insufficient availability in patients with widespread atherosclerotic vascular disease or previously harvested vessels. While artificial grafts made of Gore-Tex®, Dacron®, and polyurethanes are the most common for vascular bypass surgeries that require grafts greater than 6 mm in diameter, synthetic polymer small diameter arterial grafts (sdVG, less than 6 mm in diameter) have yet to show clinical effectiveness. Lee et al., 2014. Despite the need for, and extensive literature on, sdVGs, Buttafoco et al., 2006; Zhang et al., 2009; Williams and Wick, 2004; Neumann et al., 2003; Hahn et al., 2007, a functional graft has remained elusive due to post-implantation challenges, including thrombogenicity, poor mechanical properties, aneurysmal failure, calcification, and intimal hyperplasia. Buttafoco et a 1., 2006; Hahn et al., 2007; Niklason et al, 1999.
Graft failure due to thrombosis is a key impediment and common challenge for clinical translation of engineered grafts, likely due to the lack of endothelial barrier function. Bilodeau et al., 2005; Sivarapatna et al., 2015. Systemic combination antithrombotic drug therapy treatments are not useful in clinical applications due to increased bleeding complications. Hess et al., 2017. Meanwhile, it has previously been shown that heparin-coated vascular stents minimally improve outcomes for CAD patients and that these coatings can be unreliable. Haude et al., 2003. A more applicable, local delivery approach is needed to minimize thrombosis in vascular grafts.
In some aspects, the presently disclosed subject matter provides a method for preparing a vascular graft, the method comprising: (a) conjugating one or more therapeutic agents to a protein to form a therapeutic agent-protein conjugate; (b) electrospinning a mixture of the therapeutic agent-protein conjugate and the protein to form a plurality of microfibers having the one or more therapeutic agents embedded therein; (c) forming one or more sheets of the plurality of microfibers having the one or more therapeutic agents embedded therein; and (d) forming a hollow tube comprising the one or more sheets of the plurality of microfibers having the one or more therapeutic agents embedded therein.
In some aspects, the one or more therapeutic agents comprises a compound having at least one carboxyl group. In some aspects, the one or more therapeutic agents is selected from the group consisting of an anticoagulant, an antiplatelet, an antihistamine, an antihypertensive, a nonsteroidal anti-inflammatory drug (NSAID), a statin, an antibiotic, a growth factor, factor Xa inhibitors, direct thrombin inhibitors, an anti-proliferative drug, and combinations thereof. In certain aspects, the anticoagulant comprises heparin. In particular aspects, the heparin comprises a low molecular weight heparin (LMWH). In more particular aspects, the LMWH is selected from the group consisting of bemiparin, nadroparin, reviparin, enoxaparin, parnaparin, certoparin, dalteparin, tinzaparin, ardeparin, and pharmaceutically acceptable salts and combinations thereof.
In some aspects, the protein is selected from the group consisting of fibrinogen, collagen, elastin, gelatin, hyaluronic acid, and combinations thereof.
In some aspects, the mixture of the therapeutic agent-protein conjugate is electrospun into a rotating bath. In some aspects, the one or more therapeutic agents comprises a LMWH, the protein comprises fibrinogen, and the rotating bath comprises thrombin, thereby forming a heparinized fibrin microfiber. In certain aspects, the method further comprises rastering a spinneret back and forth, for example along a linear platform, to form the sheet of microfibers having the one or more therapeutic agents embedded therein.
In some aspects, the method further comprises rolling the one or more sheets of microfibers having the one or more therapeutic agents embedded therein to form the hollow tube. In certain aspects, the method further comprises combining or alternating one or more sheets of microfibers having the one or more therapeutic agents embedded therein with one or more sheets comprising the protein alone, or sheets comprising one or more additional therapeutic agents.
In some aspects, the one or more therapeutic agents comprises a low molecular weight heparin (LMWH) and the protein comprises fibrinogen, and the method further comprises activating the LMWH and then conjugating the activated LMWH with the fibrinogen to form a LMWH-fibrinogen conjugate. In certain aspects, the LMWH is activated with 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS).
In some aspects, the LMWH-fibrinogen conjugate is purified by centrifugal filtration and dialysis to remove non-conjugated LMWH. In certain aspects, the dialysis comprises a first solution comprising saline and a second solution against which the dialysis occurs comprising reverse osmosis (RO) H2O. In more certain aspects, the dialysis comprises a first solution comprising sucrose, polyethylene oxide (PEO), or a combination of sucrose and PEO in saline and a second solution against which the dialysis occurs comprising sucrose, PEO, or a combination of sucrose and PEO in RO H2O.
In some aspects, the method further comprises freezing and lyophilizing the purified LMWH-fibrinogen conjugate to form a powdered LMWH-fibrinogen conjugate.
In other aspects, the presently disclosed subject matter provides a vascular graft, microfibers, sheet, hollow tube, or mesh prepared by any of the presently disclosed methods.
In some aspects, the presently disclosed subject matter provides a vascular graft comprising one or more sheets or hollow tubes comprising a plurality of microfibers having one or more therapeutic agents embedded therein.
In some aspects, the one or more therapeutic agents comprises a compound having at least one carboxyl group. In certain aspects, the one or more therapeutic agents is selected from the group consisting of an anticoagulant, an antiplatelet, an antihistamine, an antihypertensive, a nonsteroidal anti-inflammatory drug (NSAID), a statin, an antibiotic, a growth factor, factor Xa inhibitors, direct thrombin inhibitors, an anti-proliferative drug, and combinations thereof.
In particular aspects, the anticoagulant comprises heparin. In certain aspects, the heparin comprises a low molecular weight heparin (LMWH). In more certain aspects, the LMWH is selected from the group consisting of bemiparin, nadroparin, reviparin, enoxaparin, parnaparin, certoparin, dalteparin, tinzaparin, ardeparin, and pharmaceutically acceptable salts and combinations thereof.
In some aspects, the plurality of microfibers further comprise a protein selected from the group consisting of fibrinogen, collagen, elastin, gelatin, hyaluronic acid, and combinations thereof.
In some aspects, the vascular graft comprises a tubular scaffold comprising a hollow core surrounded by one or more sheets comprising a plurality of microfibers having one or more therapeutic agents embedded therein. In certain aspects, the hollow core has an inner diameter having a range from about 0.1 mm to about 6 mm. In certain aspects, the one or more sheets have a combined thickness having a range from about 5 nm to about 10,000 μm.
In yet other aspects, the presently disclosed subject matter provides a method for treating vascular damage, the method comprising administering a vascular graft disclosed herein or prepared by any of the methods disclosed herein, to a subject having vascular damage.
In some aspects, the vascular graft is administered by vascular bypass surgery. In some aspects, the vascular damage is to an artery or vein. In some aspects, the vascular damage is caused by a disease or trauma. In certain aspects, the disease is selected from the group consisting of congenital cardiovascular defect (CCD), coronary artery disease (CAD), or peripheral artery disease (PAD).
In some aspects, the presently disclosed subject matter provides a kit comprising a powdered LMWH-fibrinogen conjugate, or reagents for preparing the powdered LMWH-fibrinogen conjugate, and solvents for reconstituting the powdered LMWH-fibrinogen conjugate for use in electrospinning.
In some aspects, the presently disclosed subject matter provides a kit comprising a vascular graft or scaffold prepared by the presently disclosed methods, wherein the vascular graft or scaffold is in a dehydrated or hydrated state, and optionally solutions for rehydrating the vascular grafts or scaffolds before use.
Certain aspects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying Examples and Figures as best described herein below.
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 upon request and payment of the necessary fee.
Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:
The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Figures, in which some, but not all embodiments of the inventions are shown. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated Figures. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.
To overcome limitations of vascular grafts known in the art, including thrombus formation, the presently disclosed subject matter provides a technique to conjugate drugs to the proteins used in the electrospinning process and fabricate grafts wherein the drugs, such as low molecular weight heparin (LMWH), are conjugated within the microfiber scaffold. LMWHs are anticoagulant drugs used in combination with dual-antiplatelet therapy (DAPT) clinically to treat acute coronary syndrome. Ostadal et al., 2008; Heart.org (2017). LMWHs are safer and more effective than unfractionated heparin. Ostadal et al., 2008; Tasatargil et al., 2005.
The presently disclosed method of sustained delivery of anti-coagulant drugs via controlled locations and dosages within the sdVG will provide a more effective and safer approach to alleviate acute clot formation. This approach will overcome the significant drawbacks of global heparin therapy and heparin coating of vascular grafts.
Referring now to
Fabrication of LMWH-embedded sdVGs first requires synthesis of LMWH-fibrinogen (LMWH-F), which involves conjugation of fibrinogen with LMWH using carbodiimide chemistry and purification of the LMWH-F to prevent bulk release of the anticoagulant into systemic circulation (
The conjugation of LMWH to fibrinogen was enhanced by first using an elemental analysis to ensure the ratio of carboxyl groups to EDC/NHS was ideal, which resulted in increasing the concentrations of EDC and NHS for carbodiimide crosslinking. Additionally, the LMWH was set to be in large molar excess to fibrinogen (46×). Due to LMWH (mean molecular weight (MW) 4.5 kDa) being a highly negatively charged molecule, Ostadal et al., 2008; Zhang et al., 2010; Ouyang et al., 2019; Barradell and Buckley, 1992, centrifugal filtration through a 30 kDa filter was used to remove non-conjugated LMWH, while dialysis through 25 kDa MWCO tubing was primarily used to remove saline from the LMWH-F solution. Lyohpilization of the synthesized compound enabled storage for later use. The percent yield of LMWH-F using this synthesis protocol was 63.89±12.46% (n=11). The modified synthesis protocol significantly improved the LMWH concentration from the previously published 42.73 mg/g to 551.72±438.83 mg/g (n=7). Yang et al., 2010.
With the presently disclosed electrospinning process, the location of the drug within the graft can be controlled by modulating which of the longitudinally or circumferentially oriented electrospun fibrin sheets wrapped around the mandrel contain LMWH (
HNMR and SDS PAGE were used to assess the LMWH-F conjugation. Zhang et al., 2010. HNMR indicates that the LMWH has unique peaks at 4.40-3.50 and 3.25-3.10 ppm relative to fibrinogen. These peaks were 22 times higher in the LMWH-F compared to the fibrinogen control (
Dynamic incubation of porcine PRP with 0.5-U/mL thrombin for 1 hr at 37° C. on electrospun sheets made with pure fibrinogen or 40% LMWH-F was used as an in vitro thrombogenesis assay. Stevens, 2004; Badimon et al., 2012. The number of activated platelets adhered to the pure fibrin sheets was 1.5 times higher than the 40% heparin-fibrin sheets (
The commonly used porcine model is excellent to assess graft function and clinical-applicability due to the pig's similarity with the human cardiovascular anatomy, physiology, and thrombosis mechanisms. Pashneh-Tala et al., 2015; Stacy et al., 2014; Hoerstrup et al., 2006. The porcine model will enable a more strict assessment of plaque formation and thrombogenicity than previously used mouse models, which have different clotting mechanisms than humans. Pashneh-Tala et al., 2015.
Heparinized and fibrin grafts were implanted in an interpositional porcine carotid artery model for 4 weeks (
To further improve the patency of the heparinized sdVGs, the LMWH-fibrinogen synthesis process was further modified to improve the solubility of the glycoprotein (
Additionally, the PEO was added as this has been able to dissolve fibrinogen completely for electrospinning in the past, even in the absence of saline. Elliott et al., 2019. To ensure sucrose stayed in the dialysis tubing and to prevent excess osmosis, the solution against which the dialysis occurs was also altered to be 100-mM sucrose in 0.2% PEO in RO H2O instead of just RO H2O (
To assess if the improved solubility of LMWH-F reduced thrombogenicity, a thrombin generation assay was performed on porcine PRP with 0.1 U/mL thrombin that had been incubated on 2D sheets made of fibrin, heparinized fibrin made from LMWH-fibrinogen dialyzed against PEO and sucrose (HF), or heparinized fibrin made from LMWH-fibrinogen with reduced solubility dialyzed against RO H2O only (HF RS). Collagen I coated glass coverslips (Col I) were used as a positive control. The more thrombogenic Col I samples had significantly increased peak thrombin generation, reduced time to peak thrombin generation, and faster rate of thrombin generation relative to all the samples. The HF scaffolds appear to reduce the rate of thrombin generation relative to fibrin and HF RS scaffolds (
Accordingly, in some embodiments, the presently disclosed subject matter provides a method for preparing a vascular graft, the method comprising:
In some embodiments, the vascular graft comprises a small diameter vascular graft (sdVG). As used herein, the term “small diameter vascular graft (sdVG)” is small-diameter vascular graft having an inner diameter less than about 6 mm. The vascular graft may taper or vary in size, including variations in length, diameter, and wall thickness, to match the existing vasculature and subject needs.
By “microfiber” is meant a solid tubular structure made up of a bundle of nanofibers.
A “tubular scaffold” generally means a structure comprising a sheet of nanofibers or microfibers forming a circumference around a hollow core.
In some embodiments, the one or more therapeutic agents comprises a compound having at least one carboxyl group. As used herein, the term “carboxyl group” is a functional group consisting of a carbon atom double-bonded to an oxygen atom and singly bonded to a hydroxyl group and comprises the R—C(═O)—OH group. Representative therapeutic agents having a carboxyl group include, but are not limited to, LMWH heparins, such a nadroparin calcium and enoxaparin sodium as disclosed herein; factor Xa inhibitors, such as fondaparinux, rivaroxaban, rapixaban and edoxaban; direct thrombin inhibitors, such as argatroban, inogatran, melagatran (and its prodrug ximelagatran), and dabigatran; antiplatelet drugs, such as clopidogrel and prasugrel, and antihypertension drugs, such as azilsartan, candesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan, and valsartan. The role of the carboxyl group in pharmaceutical compounds and representative pharmaceutical compounds having a carboxyl group are disclosed in Lamberth and Dinges, 2016, which is incorporated by reference in its entirety.
In some embodiments, the one or more therapeutic agents is selected from the group consisting of an anticoagulant, an antiplatelet, an antihistamine, an antihypertensive, a nonsteroidal anti-inflammatory drug (NSAID), a statin, an antibiotic, a growth factor, factor Xa inhibitors, direct thrombin inhibitors, an anti-proliferative drug like rapamycin, and combinations thereof. In certain embodiments, the anticoagulant comprises heparin. In particular embodiments, the heparin comprises a low molecular weight heparin (LMWH).
Heparin is a naturally occurring polysaccharide that inhibits coagulation. Natural heparin consists of molecular chains of varying molecular weights from about 5 kDa to over 40 kDa. In contrast, LMWHs consist of only short chains of polysaccharide and are defined as heparin salts having an average molecular weight of less than 8 kDa and for which at least 60% of all chains have a molecular weight less than 8 kDa. Representative embodiments of LMWH along with their average molecular weights are provided in Table 1.
Accordingly, in yet more particular embodiments, the LMWH is selected from the group consisting of bemiparin, nadroparin, reviparin, enoxaparin, parnaparin, certoparin, dalteparin, tinzaparin, ardeparin, and pharmaceutically acceptable salts and combinations thereof, including, for example sodium, potassium, calcium, ammonium, lithium, tosylates, and the like.
In some embodiments, the protein is selected from the group consisting of fibrinogen, collagen, elastin, gelatin, hyaluronic acid, and combinations thereof.
In some embodiments, the mixture of the therapeutic agent-protein conjugate is electrospun into a rotating bath.
In some embodiments, the one or more therapeutic agents comprises a LMWH, the protein comprises fibrinogen, and the rotating bath comprises thrombin, thereby forming a heparinized fibrin microfiber. In certain embodiments, the method further comprises rastering a spinneret, e.g., an electrospinning needle and the like, back and forth, for example along a linear platform, to form the sheet of microfibers having the one or more therapeutic agents embedded therein.
In some embodiments, the method further comprises rolling the one or more sheets of microfibers having the one or more therapeutic agents embedded therein to form the hollow tube. In certain embodiments, the method further comprises combining or alternating one or more sheets of microfibers having the one or more therapeutic agents embedded therein with one or more sheets comprising the protein alone, or sheets comprising one or more additional therapeutic agents.
In some embodiments, the one or more therapeutic agents comprises a low molecular weight heparin (LMWH) and the protein comprises fibrinogen, and the method further comprises activating the LMWH and then conjugating the activated LMWH with the fibrinogen to form a LMWH-fibrinogen conjugate. In certain embodiments, the LMWH is activated with 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS).
In some embodiments, the LMWH-fibrinogen conjugate is purified by centrifugal filtration and dialysis to remove non-conjugated LMWH. In certain embodiments, the dialysis comprises a first solution comprising sucrose, polyethylene oxide (PEO), or a combination of sucrose and PEO in saline and a second solution against which the dialysis occurs comprising sucrose, PEO, or a combination of sucrose and PEO in RO H2O.
In some embodiments, the method further comprises freezing and lyophilizing the purified LMWH-fibrinogen conjugate to form a powdered LMWH-fibrinogen conjugate.
In other embodiments, the presently disclosed subject matter provides a vascular graft, microfibers, including a solid bundle, sheet, hollow tube, or mesh prepared by any of the presently disclosed methods.
In some embodiments, the presently disclosed subject matter provides a vascular graft comprising one or more sheets or hollow tubes comprising a plurality of microfibers having one or more therapeutic agents embedded therein.
In some embodiments, the one or more therapeutic agents comprises a compound having at least one carboxyl group. In certain embodiments, the one or more therapeutic agents is selected from the group consisting of an anticoagulant, an antiplatelet, an antihistamine, an antihypertensive, a nonsteroidal anti-inflammatory drug (NSAID), a statin, an antibiotic, a growth factor, factor Xa inhibitors, direct thrombin inhibitors, an anti-proliferative drug, and combinations thereof.
In particular embodiments, the anticoagulant comprises heparin. In certain embodiments, the heparin comprises a low molecular weight heparin (LMWH). In more certain embodiments, the LMWH is selected from the group consisting of bemiparin, nadroparin, reviparin, enoxaparin, parnaparin, certoparin, dalteparin, tinzaparin, ardeparin, and pharmaceutically acceptable salts and combinations thereof.
In some embodiments, the plurality of microfibers further comprise a protein selected from the group consisting of fibrinogen, collagen, elastin, gelatin, hyaluronic acid, and combinations thereof.
In some embodiments, the vascular graft comprises a tubular scaffold comprising a hollow core surrounded by one or more sheets comprising a plurality of microfibers having one or more therapeutic agents embedded therein. In some embodiments, the hollow core has an inner diameter having a range from about 0.1 mm to about 6 mm, including 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, and 6 mm.
In certain embodiments, the one or more sheets have a combined thickness having a range from about 5 nm to about 10,000 μm, including 5 nm, 10 nm, 50 nm, 100 nm, 500 nm, 1 μm, 10 μm, 100 μm, 500 μm, 1000 μm, 2000 μm, 3000 μm, 4000 μm, 5000 μm, 6000 μm, 7000 μm, 8000 μm, 9000 μm, and 10,000 μm.
In yet other embodiments, the presently disclosed subject matter provides a method for treating vascular damage, the method comprising administering a vascular graft disclosed herein or prepared by any of the methods disclosed herein, to a subject having vascular damage.
As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
The “subject” treated by the presently disclosed methods in their many embodiments is desirably a human subject, although it is to be understood that the methods described herein are effective with respect to all vertebrate species, which are intended to be included in the term “subject.” Accordingly, a “subject” can include a human subject for medical purposes, such as for the treatment of an existing condition or disease or the prophylactic treatment for preventing the onset of a condition or disease, or an animal subject for medical, veterinary purposes, or developmental purposes. Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, and the like. An animal may be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Further, a “subject” can include a patient afflicted with or suspected of being afflicted with a condition or disease. Thus, the terms “subject” and “patient” are used interchangeably herein. The term “subject” also refers to an organism, tissue, cell, or collection of cells from a subject.
In some embodiments, the vascular graft is administered by vascular bypass surgery.
In some embodiments, the vascular damage is to an artery or vein.
In some embodiments, the vascular damage is caused by a disease or trauma. In certain embodiments, the disease is selected from the group consisting of congenital cardiovascular defect (CCD), coronary artery disease (CAD), or peripheral artery disease (PAD).
In some embodiments, the presently disclosed subject matter provides a kit comprising a powdered LMWH-fibrinogen conjugate, or reagents for preparing the powdered LMWH-fibrinogen conjugate, and solutions for reconstituting the powdered LMWH-fibrinogen conjugate for use in electrospinning. The kits also can include vascular grafts or scaffolds prepared by the presently disclosed methods, which can be either dehydrated or hydrated. In such embodiments, the kits also can include solutions for rehydrating the vascular grafts or scaffolds before use. The component(s) of the kits may be packaged either in aqueous media or in lyophilized form or frozen form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. Various combinations of components may be comprised in a single vial. The kits of the present invention also will typically include a means for containing the components of the kits and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
When the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. The components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means. The kit also can include instructions for use.
In sum, the presently disclosed methods can used to electrospin drug-conjugated proteins to make fibrin microfiber scaffolds, including individual microfibers, flat sheets, and hollow tubes. In general, any drug with a carboxyl group can be incorporated into the scaffold due to the use of carbodiimide chemistry. The graft prepared by the presently disclosed methods provide sustained, local drug (e.g., anticoagulant) release while the graft degrades. Varying concentrations of drug can be electrospun into the fibrin microfibers. The location of the drug and drug concentration within the scaffold can be controlled by modulating which sheets are used to build the scaffold. The embedded heparin remains functional after incorporation into the scaffold and will provide more reliable local administration of drugs, especially in a vascular setting.
Following long-standing patent law convention, the terms “a,” “an,” and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a subject” includes a plurality of subjects, unless the context clearly is to the contrary (e.g., a plurality of subjects), and so forth.
Throughout this specification and the claims, the terms “comprise,” “comprises,” and “comprising” are used in a non-exclusive sense, except where the context requires otherwise. Likewise, the term “include” and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.
For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing amounts, sizes, dimensions, proportions, shapes, formulations, parameters, percentages, quantities, characteristics, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about” even though the term “about” may not expressly appear with the value, amount, or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are not and need not be exact, but may be approximate and/or larger or smaller as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art depending on the desired properties sought to be obtained by the presently disclosed subject matter. For example, the term “about,” when referring to a value can be meant to encompass variations of, in some embodiments, ±100% in some embodiments±50%, in some embodiments±20%, in some embodiments±10%, in some embodiments±5%, in some embodiments±1%, in some embodiments±0.5%, and in some embodiments±0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.
Further, the term “about” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries above and below the numerical values set forth. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.
The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The synthetic descriptions and specific examples that follow are only intended for the purposes of illustration, and are not to be construed as limiting in any manner to make compounds of the disclosure by other methods.
Fibrin hydrogel microfiber sheets were prepared as previously described by electrospinning 2.0 wt % fibrinogen solution co-dissolved in 0.2 wt % PEO in water under the effects of an applied electric field (4.5 kV) to propel the resultant fiber jet across an air gap of 2 cm and onto a rotating collection bath (45 rpm) containing 50-mM calcium chloride and 20-U/mL thrombin. Elliott et al., 2019. The landing position of the spinning jet was rastered back and forth via use of a linear stage during the spinning step to yield a uniform aligned fibrin sheet.
Hollow fibrin tubes with multidirectional alignment were formed by rolling sheets arranged first parallel, then perpendicular, and again parallel to the fiber orientation onto polytetrafluoroethylene (PTFE)-coated stainless-steel mandrels to generate tubes. This process created alternating layers of longitudinally, circumferentially, and longitudinally aligned fibrin microfibers. Tube wall thickness was controlled by altering the number of wraps around the mandrel. To alter the inner diameter of the graft, the diameter of the mandrel used to collect the fibrin sheets was changed. To increase the length of the graft, the width of the fibrin sheet was increased by increasing the path length of the rastering needle. Following wrapping, fibrin tubes were crosslinked for 15 hours in 40-mM EDC/100 mM NHS dissolved in PBS and dehydrated in a series of 25, 50, 60, 70, 80, 90, 95, 100, 100, and 100% EtOH solutions for a minimum of 15 minutes per step and then allowed to air dry. Dried fibrin tubes were removed from the PTFE mandrels following dehydration.
LMWH was pre-activated in 0.05-M 2-morpholinoethanesulfonic acid (MES) in MilliQ H2O (pH 6.0). The LMWH at 1 mg/mL was combined with 1.07-mM EDC and 1.17-mM NHS to activate overnight while stirring. Yang et al., 2010. To conjugate LMWH and fibrinogen (
Alternatively, dialysis against 100-mM sucrose in 0.2% PEO in RO H2O was performed to enhance the solubility of LMWH-Fibrinogen (LMWH-F) (
Fabrication of LMWH-embedded scaffolds involves synthesis of LMWH-F and co-dissolving LMWH-F with fibrinogen for electrospinning (
Following the electrospinning procedure as previously described, Zhang et al., 2009, two-dimensional (2D) sheets for heparin concentration testing were fabricated. Square frames with a side length of 1 cm were flipped through the electrospun sheet to create 15-30 layers (
For all platelet assays, the 3.8% sodium citrate, human or porcine platelet rich plasma (PRP) (BioIVT) was thawed from −80° C. to 4° C. prior to use. The PRP was then incubated with the hydrogel scaffold for 1 hour at 37° C. Stacy et al., 2014.
For the 2D sheets, 500-μL porcine PRP was incubated with 0.1- or 0.5-U/mL thrombin (BioPharm Labs, 91-030) with the sheet in a 24-well, non-tissue culture treated, PDMS coated plate, which was slowly rotated. Samples were rinsed in PBS and fixed for confocal microscopy to assess platelet adhesion. PRP supernatant was collected for the Technothrombin® thrombin generation assay (TGA, Technoclone) to assess platelet activation following the manufacturer's instructions for the RC High reagent. Yao et al., 2020. Bovine collagen I (0.1 mg/mL, Advanced Biomatrix) coated coverslips in a 24-well, tissue culture treated plate served as a positive control for the TGA.
For the 0.6-mm and 5-mm inner diameter grafts, the human PRP was injected into the lumen of the graft in a LumenGen bioreactor (Bangalore Integrated System Solutions Ltd., Bangalore, India) and the bioreactor was slowly rotated inside the incubator to coat all surfaces of the lumen. The hydrogel scaffolds were washed with PBS 3 times to remove unattached platelets.
The platelets were fixed with 3.7% PFA (Thermo Fisher Scientific, F79-1) for 30 minutes, permeabilized with 0.1% Triton X-100 (Thermo Fisher Scientific, 85111) for 20 minutes, washed with PBS for 3 minutes, and blocked in 1% BSA (Sigma-Aldrich, A3059-50 g) overnight. Elliott et al., 2019. The samples were washed in PBS, incubated in rabbit anti-CD41 primary antibody overnight at 4° C., washed with PBS, incubated in phalloidin and anti-rabbit secondary antibody for 2 hours at room temperature, washed with PBS, incubated in DAPI for 15 minutes, and washed with PBS. The sheets were then stored in Milli-Q H2O at 4° C. Finally, the sheets or grafts were imaged using confocal microscopy (Carl Zeiss AG, LSM 780).
Platelet quantification was conducted using the spot package in Imaris software (Bitplane). Platelets were identified using a threshold of 5-μm diameter spheres on the fluorescence from phalloidin (
1.7 Implantation of sdVGs
Fibrin and heparinized sdVGs measuring 5-mm inner diameter and 2-cm length with a 500-μm thick heat-treated poly(F-caprolactone) (PCL) surgical sheath, Elliott et al., 2019, were implanted as carotid artery interposition grafts in Yorkshire pigs (46±6 kg) (
Statistical analysis was performed with Prism 9.0.0 (GraphPad Software). Unpaired t tests, one-way ANOVA with Tukey's posttest, or two-way ANOVA with Tukey's or Sidak's posttest were used where appropriate. Unless otherwise indicated, graphical data were reported as mean±SD for sample size larger than one. Significance levels were represented by *p<0.05, **p<0.01, ****p<0.0001.
Off-the-Shelf, Heparinized sdVG Supports Patency and Remodeling in a Porcine Model
Vascular bypass prostheses development research has been ongoing for over 50 years, but thrombogenicity continues to pose a serious challenge to the clinical translation of engineered grafts. Previously we established natural polymer-based small-diameter vascular grafts (sdVG) composed of fibrin hydrogel microfiber tubes (FMT) with an external poly(8-caprolactone) (PCL) sheath, capable of supporting patency in mice. Towards their clinical translation, we report the FMT's shelf stability, scale-up to a size suitable for human application, and successful conjugation of an antithrombotic to the fibrin scaffold to improve patency in a porcine model. The FMT was stable when stored for up to one year at −20° C., 4° C., and 23° C. with minimal changes in hydrogel mechanical properties and swelling ratio, indicating off-the-shelf availability of the FMT. An external PCL sheath provides mechanical strength for implantation of the FMT in a carotid artery interposition porcine model without rupture. However, one in six Fibrin-PCL grafts and the GORE-TEX® expanded polytetrafluoroethylene control graft had complete lumen occlusion due to clot formation at 2 weeks post-implantation. To reduce thrombogenicity, we conjugated low molecular weight heparin to the protein backbone of the fibrin scaffold, enabling local and sustained anticoagulant delivery. We demonstrate that the low molecular weight heparin embedded in the fibrin scaffold remains active in vitro through platelet adhesion and activation reduction. Heparin conjugation also improved performance in vivo by reducing thrombogenicity and reliably extending the timeframe of patency beyond 2 weeks post-implantation. Patent sdVGs underwent neotissue formation, supporting extensive cell infiltration as the fibrin layer degraded. By 4-5 weeks post-implantation, all four of the heparinized Fibrin-PCL grafts had stenosis due to neointimal hyperplasia Fibrin-PCL comparable to the currently clinically used non-biodegradable GORE® PROPATEN® vascular grafts. This hyperplasia has no relation to the heparin coatings. The presence of endothelial cells on the luminal surface of our sdVGs at 4-5 weeks post-implantation is promising, and incorporation of an anti-proliferative drug may prolong patency and enable the formation of a complete tunica intima. This study establishes a heparinized Fibrin-PCL sdVG with off-the-shelf availability and reduced thrombogenicity, providing a pro-regenerative alternative to autologous bypass vessels with limited availability and thrombotic synthetic polymer scaffolds.
Cardiovascular disease accounts for one-third of deaths worldwide and is the leading cause of death in the United States, resulting in a death every 37 seconds. Satterhwaite et al., 2005; Westein et al., 2013; Atheroscloerosis, 2014; Heart Disease Facts, 2020. Atherosclerosis, or plaque buildup within the vessel wall that restricts or occludes blood flow, is a significant underlying cause of cardiovascular disease. Common presentations include coronary artery disease (CAD), cerebrovascular disease, and peripheral artery disease (PAD). Gallino et al., 2014; Ross, 1999. Standard initial treatments for this disease include lifestyle changes and drug therapies. Westein et al., 2013; Atheroschlerosis, 2014. However, of the 10 million people in the United States who have PAD, 26% of these patients have adverse limb outcomes from continued plaque buildup. Kullo and Rooke, 2016; Varu et al., 2010. The “end-stage” of PAD is critical limb ischemia (CLI), which can lead to surgery for limb salvage, amputation, or death. Kullo and Rooke, 2016; Varu et al., 2010; Norgren et al., 2007. Surgical procedures to restore blood flow include endovascular procedures such as angioplasty, stent insertion, or atherectomy. In patients with severe vascular stenosis (narrowing), arterial bypass surgery re-establishes blood flow in the coronary and peripheral arteries. Bypass surgery is the optimal choice for patients requiring a long-term revascularization solution. Elliot et al., 2019; Houstan et al., 2001. Autografts, like the patient's saphenous vein (SV) or internal thoracic artery (ITA), are the clinical gold standard for bypass grafts. Unfortunately, autografts require a secondary surgical site and are unavailable in patients with widespread atherosclerosis or previously harvested vessels. For CLI patients, secondary vein graft failure occurs in 20% of patients by one year, and inpatient hospital treatment for the first year after bypass costs over $29,000. Varu et al., 2010. Thus, there is an urgent clinical need to develop engineered grafts that provide long-term patency.
To this end, we previously developed natural polymer-based small-diameter vascular grafts (sdVGs, <6 mm in diameter) composed of fibrin hydrogel microfiber tubes (FMT) that mimicked the ECM and supported the formation of a confluent, stable endothelium both in vitro and in vivo. Elliott et al., 2019; Barreto-Ortiz et al., 2013; Zhang et al., 2014; Barreto-Ortiz et al., 2015. With an external, ultrathin poly(8-caprolactone) (PCL) surgical sheath, the FMTs were able to support blood flow and maintain patency for at least 24 weeks as interposition grafts in the abdominal aorta of a mouse. Elliott et al., 2019. The host tissue also remodeled the fibrin scaffold to resemble the native abdominal aorta structural and mechanical features. Elliott et al., 2019. Here, we will assess the FMT shelf-life and Fibrin-PCL sdVG functionality in a large animal model.
Off-the-shelf availability of sdVGs is critical to patients needing emergency arterial bypass. Advantages of off-the-shelf, engineered, acellular sdVGs include increased availability, decreased fabrication costs, decreased potential complications relative to cellularized sdVGs, and no secondary surgical sites. Other important factors for hospitals focused on cost reduction are the storage conditions and product expiration date. Robinson, 2008. Medical device choices, including items for cardiovascular surgery, highly affect hospitals' supply-chain efficiency and revenue. Robinson, 2008. To best serve the patient, surgeon, and hospital, it is crucial to understand the effects of long-term storage on our natural polymer-based scaffolds.
Another impediment and common challenge to clinical translation of engineered grafts is failure due to thrombosis, or clot formation, likely caused by the lack of endothelial barrier function. Bilodeau et al., 2005; Sivarapatna et al., 2015. Prevention of clotting with systemic combination antithrombotic drug therapy treatments is not useful in clinical applications due to increased bleeding complications. Hess et al., 2017. Extensive research focuses on coating the luminal surface of sdVGs with heparin, an anticoagulant drug, to address the thrombosis issue. Dimitrievska et al., 2015; Hoshi et al., 2013; Qiiu et al., 2017. However, heparin-coated vascular stents and grafts only minimally improve outcomes for CAD patients relative to non-coated devices. Haude et al., 2003; Lindholt et al., 2011. Further, the widely, clinically used GORE® PROPATEN® heparin-coated ePTFE graft has a 17% reduced primary patency at 48 months relative to the autologous SV. Dorigo et al., 2011. A more practical, local drug delivery approach combined with a pro-regenerative scaffold is needed to minimize thrombosis in vascular grafts.
We propose to chemically conjugate low molecular weight heparin (LMWH) to the protein backbone of our FMT. This approach permits embedding the LMWH throughout the entire graft, which will yield a more reliable, sustained presence of the anticoagulant drug and thereby reduce graft thrombosis. LMWHs are safer and more effective anticoagulant drugs than unfractionated heparin, both of which are glycosaminoglycans (GAGs). Zhang et al., 2010; Ostadal et al., 2008; Tasatargil et al., 2005. In its active state, LMWH binds to antithrombin III (ATIII) to enhance the ability of ATIII to inactivate coagulation enzymes like thrombin (factor IIa) and the platelet surface factor Xa, thereby preventing platelet activation within the coagulation cascade. Hirsh and Levine, 1992. We compared the patency of Fibrin-PCL and heparinized Fibrin-PCL grafts in a porcine carotid artery interposition model. The commonly used porcine model is excellent for assessing graft function and clinical applicability due to the pig's similarity with the human cardiovascular anatomy, physiology, and thrombosis mechanisms. Pashneh-Tala et al., 2015; Stacy et al., 2014; Hoerstrup et al., 2006. The first 4 weeks were critically important given that grafts undergo maximum thrombus formation during this period. Fleser et al., 2004. Ultimately, we established an off-the-shelf, pro-regenerative sdVG with improved acute patency for arterial bypass applications.
Multidirectional Fibrin Grafts were fabricated as previously and dehydrated using increasing, serial ethanol (EtOH) dilutions. Elliott et al., 2019. Dehydrated FMTs were stored in a sealed, light-protected container in either a refrigerator (4° C.), freezer (−20° C.), or room temperature (23° C.) for 1, 3, 6, or 12 months. The temperature and humidity were recorded randomly 1-3 times each week. Control FMTs were tested within 5 days of dehydration and were kept at room temperature. Abdominal aortas from female Fox Chase severe combined immunodeficient Beige mice (CB17.Cg-PrkdcscidLystbg-J/Crl) were used as native control tissue. The Institutional Animal Care and Use Committee of Johns Hopkins University reviewed and approved the protocol for the murine study (MO19E454).
Dehydrated FMTs were also stored in humidity-controlled incubators for accelerated aging. Elevated temperatures of 37° C. were used to simulate longer-term storage at −20° C. and 4° C., while 47° C. was used to simulate storage at 23° C. The accelerated aging time was calculated using the ASTM International F1980-16 standards and a conservative aging factor of 2 33,34. It was assumed that 1 month was 30 days in length. After storage, FMTs were rehydrated and immediately underwent circumferential tensile testing using an electromechanical puller, as previously 10. MatLab (MathWorks) code was used to calculate circumferential ultimate tensile stress (UTS), strain to failure (STF), Young's modulus, modulus of toughness, and modulus of resilience, using:
stress=force/(2*length*wall thickness)
strain=displacement/(inner diameter)
Diameter and wall thickness were calculated from area measurements of cross-sectional images of FMT rings using Image J (NIH) and the assumption that the FMT was circular. Toughness was the area under the stress-strain curve. Young's modulus was the slope of the linearly elastic region before the yield point (R2≥0.95), and the modulus of resilience was the area under this linearly elastic region of the curve. The mass-swelling ratio of the FMT was calculated as the ratio of the wet to dry weight. Caliari and Burdick, 2016.
LMWH was pre-activated by stirring 1 mg/mL LMWH in 0.05M 2-morpholinoethanesulfonic acid (MES, pH=6) in MilliQ H2O with 1.07 mM N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and 1.17 mM N-Hydroxysuccinimide (NHS) overnight. Yang et al., 2010. Fibrinogen was solubilized in 10×PBS (pH=˜7.4, 0.83 mg/mL) and mixed with the activated-LMWH solution in a 2:1 ratio for 2 days. Based on elemental analysis, we increased the concentrations of EDC and NHS for carbodiimide crosslinking and ensured LMWH was in large molar excess to fibrinogen (0.0741 mM vs. 0.0016 mM, respectively). Due to LMWH (mean molecular weight 4.5 kDa) being a highly negatively charged molecule, Zhang et al., 2010; Ostadal et al., 2008; Ouyang et al., 1992; Barradell and Buckley, 1992, we altered purification to include centrifugal filtration through a 30 kDa filter at 3500 g until approximately 10% of the original volume remained, to which sucrose was added for a final concentration of 100 mM. Subsequently, we dialyzed this solution through 25 kDa molecular weight cut-off tubing against 100 mM sucrose in reverse osmosis (RO) H2O for 4 days. Dialysis was performed with sucrose in the tubing and bath to protect the protein during desalinization, drying, and storage. Lee and Timasheff, 1981; Mensink et al., 2017. All synthesis steps were performed at 4° C. Lastly, the LMW,H-fibrinogen (LMWH-F) solution was frozen at −80° C., then lyophilized until dry for storage at 4° C. and future electrospinning.
For electrospinning heparinized fibrin, the concentration of LMWH was controlled by altering the LMWH-F:fibrinogen co-dissolved ratio in 0.2 wt % PEO. Fabrication of 0.6 mm inner diameter FMTs was otherwise performed as previously. Elliott et al., 2019. The location of the drug within the FMT can be altered by modulating which of the longitudinally or circumferentially oriented electrospun fibrin sheets wrapped around the mandrel contain LMWH. The concentration of LMWH in the FMT can be controlled by not only altering the ratio of LMWH-F:fibrinogen used in electrospinning but also by changing the number of fibrin sheets that contain LMWH-F. Here, a 2:3 ratio of LMWH-F:fibrinogen was used to make heparinized scaffolds with LMWH-F incorporated in every layer.
Two-dimensional (2D) heparinized fibrin or fibrin scaffolds were fabricated for in vitro thrombogenicity assays by flipping 1 cm square, 3D-printed frames through the electrospun sheet for a total of 25 layers. After collecting the heparinized fibrin or fibrin sheets, the scaffolds were crosslinked in EDC/NHS overnight; dehydrated using increasing, serial EtOH solutions; and immediately rehydrated without air drying to prevent cracking the sheets. Elliott et al., 2019.
For the 5 mm inner diameter grafts, the path length of the rastering needle was increased to create a 4 cm wide sheet. The fibrin or heparinized fibrin sheets were rolled onto a 5 mm diameter polytetrafluoroethylene (PTFE) mandrel for eight longitudinally oriented layers; one 79 cm long circumferentially oriented layer; and eleven longitudinally oriented layers. The 5 mm inner diameter FMTs were crosslinked with EDC/NHS; dehydrated in increasing, serial EtOH solutions for 30 mins each; and stored at 4° C., as previously described. Elliott et al., 2019. PCL sheaths with 500 μm thick walls were prepared as previously by electrospinning a 16% w/v PCL solution in 10% w/v dimethylformamide (DMF) and 90% w/v dichloromethane (DCM) onto a rotating 8 or 9 mm diameter aluminum mandrel (100 rotations/min). Elliott et al., 2019. The electric field (17 kV) was applied to a 27-gauge blunt-tipped needle with a 6-12 cm air gap between the needle and mandrel. The sheaths were fitted to the FMTs by heat treatment, as previously, Elliott et al., 2019, to ensure no diameter mismatch.
The concentration of LMWH in the heparinized FMTs (0.6 mm inner diameter) was determined using the dimethyl methylene blue (DMMB) colorimetric assay for sulfated GAGs described by Dunham et al., 2021. After measuring the wet and dry weight, the heparinized FMTs were digested in 1 mL of papain solution for 18 hours at 65° C. The digested samples (105 μL/well) and DMMB solution (438 μL/well) were plated on a 96-well plate. The sample absorbance (525 nm) was measured immediately in triplicate using a plate reader. A standard linear curve (adjusted R2≥0.95) made from chondroitin sulfate (0-30 μg/mL in papain, 5 μg/mL increments) was used to calculate the concentration of sulfated GAGs. FMTs were used as a negative control for all drug concentration and release assessments.
A modified DMMB assay was used to quantify the cumulative sulfated GAG release over time via hydrolytic and enzymatic degradation. Saito and Tabata, 2012. To assess the LMWH and total protein released by hydrolytic degradation, heparinized FMTs were incubated in 1 mL of PBS at 37° C. while agitating (100 rpm). Lim et al., 2007; Zhu et al., 2021. The supernatant was exchanged entirely at 1, 2, 4, 8, 24, 48, 96, and 168 hours, then weekly until the sample fully degraded. Accelerated in vitro release was accomplished by incubating samples in 1 mL of 0.5 CU/mL plasmin in PBS at 37° C. while agitating (100 rpm). Barreto-Ortiz et al., 2013; Kamberi et al., 2009; Shen and Burgess, 2012; Matsuzaki et al., 2021. The supernatant was exchanged entirely at 0.5, 1, 2, 4, 8, 12, 24, 36, 48, and 72 hours, then every other day until the sample fully degraded. A standard linear curve (adjusted R2≥0.95) made from LMWH in PBS or plasmin solutions (0-30 μg/mL), as appropriate, was used to calculate the concentration of released sulfated GAGs. For both release assays, the total protein released at each time point was quantified using the Pierce™ BCA Protein Assay Kit following the manufacturer's instructions. The sample absorbance (562 nm) was measured in triplicate using a plate reader, and a standard quadratic curve (adjusted R2≥0.99) made from fibrinogen (0-2000 μg/mL) in PBS or plasmin solutions, as appropriate, was used to calculate the concentration of released protein.
To determine if the LMWH remained active in the heparinized scaffolds, 2D scaffolds were incubated in 500 μL of Yorkshire porcine (72,000/μL) or human (24,000/μL) platelet-rich plasma (PRP) with high-purity bovine thrombin (0.1 U/mL) for 1 hour at 37° C. on a gently moving rocker. Fibrin 2D scaffolds were used as a control. All scaffolds were placed in a polydimethylsiloxane (PDMS, 1:7 ratio) coated non-tissue culture treated 24-well plate for incubation. Scaffolds were rinsed three times in PBS to remove non-adhered platelets.
The lactate dehydrogenase (LDH) assay assessed platelet adhesion to the 2D scaffold. Matsuzaki et al., 2021; Yao et al., 2020. Platelets adhered to the scaffolds were lysed by incubating the scaffold in 1 mL of 1% Triton X-100 in PBS for 1 hour at 37° C. Subsequently, 100 μL of the lysis supernatant was combined with 100 μL of the freshly prepared reaction mixture in each well of a flat, clear-bottom 96-well plate. After incubation for 20 minutes at room temperature under light-protected conditions, the sample absorbance (490 nm) was read in triplicate using a plate reader, as directed by the kit manufacturer.
For the Technohrombin® thrombin generation assay (TGA), Yao et al., 2020, 40 μL of PRP supernatant was combined with 10 μL of TGA RC High and 50 μL of TGA Substrate in each well of a MaxiSorp, black 96-well plate. The sample fluorescence (360 nm/460 nm) was read in duplicate for 1 hour at 1-minute intervals at 37° C. using a plate reader, as directed by the kit manufacturer. Bovine collagen type I (0.1 mg/mL) coated glass coverslips in tissue culture plastic 24-well plate were used as a positive control. 2.3.6 Mechanical Testing and Porcine Implantation of sdVGs Fibrin-PCL sdVGs (5 mm inner diameter) underwent circumferential tensile testing using an electromechanical puller following the International Organization for Standardization (ISO) 7198:2016(E) Section A.5.2.4.4 (performed by Nanofiber Solutions Inc.). The radial force was applied at a 50 mm/min rate until failure. In addition to circumferential UTS and STF, maximum circumferential tensile strength (CTS) was calculated as maximum force per unit length divided by 2. Suture retention strength (SRS), or the maximum force required to achieve suture pull-out, was measured following ISO 7198:2016(E) Section A.5.7.4.1. A 6-0 polypropylene monofilament suture (Surgipro™ II, Covidien) was placed through one wall at a distance of 2 mm from the graft end and axially pulled at a rate of 13 mm/min. Heat-treated PCL sheaths, a GORE-TEX® expanded PTFE (ePTFE) graft, GORE® PROPATEN®, porcine native carotid arteries, and porcine native jugular veins were tested as controls. For scanning electron microscopy (SEM), critical point dried FMTs were sputter-coated with platinum for 12 seconds and imaged using an electron microscope.
The Institutional Animal Care and Use Committee of The University of Chicago reviewed and approved the protocol for the porcine study (72605). Bilateral implantations were performed where possible to reduce animal numbers. A GORE-TEX® ePTFE graft and two GORE® PROPATEN® grafts were implanted as clinical controls. Briefly, the pigs were anesthetized by continuous gas anesthesia with isoflurane. The Fibrin-PCL and heparinized Fibrin-PCL sdVGs were implanted in the carotid artery of White Yorkshire x Landrace pigs (45.9±5.2 kg). A portion of the carotid artery was exposed, cross-clamped, and truncated. A 2 cm graft length was inserted as an interposition graft using 6-0 monofilament suture for the end-to-end proximal and distal anastomoses. Finally, the muscle, subcutaneous tissue, and skin were closed with absorbable monofilament sutures. The pigs received heparin (100 U/kg IV) just before clamping the carotid artery to implant sdVGs and dual antiplatelet therapy (DAPT) of aspirin (325 mg/day) and Plavix (75 mg/day) until harvest. Hess et al., 2017.
The endpoints for evaluation were 4 weeks following implantation, with non-invasive color Doppler sonography performed 2 weeks postoperatively compared to the GORE-TEX® ePTFE graft, GORE® PROPATEN® grafts, and native carotid artery controls to assess patency. Circumferential tensile testing was performed within 24 hours on harvested sdVG segments, stored in endothelial cell media at 4° C. until testing. Histology and immunohistochemistry (IHC) were used to assess graft integration and remodeling, as previously. Elliott et al., 2019. Briefly, harvested tissue rings were rinsed and flushed with saline before being fixed with formalin; dehydrated in serial EtOH (70%-100%); embedded in paraffin; serially cross-sectioned at 5 μm along the length; and stained. Hematoxylin and eosin (H&E), Masson's trichrome (MT), Verhoeff van Gieson (VVG), and von Kossa staining were performed by the Johns Hopkins University Oncology Tissue Services and Reference Histology Cores. As previously for IHC staining, Shen et al., 2016, paraffin-embedded tissue sections were primary stained with Rabbit anti-mouse/human CD31 (1:1500) or Rabbit anti-mouse αSMA (1:2000); counterstained with ImmPRESS HRP anti-rabbit IgG, ImmPACT DAB Peroxidase substrate (Vector Laboratories); and hematoxylin stained. Images were taken with an upright light microscope and camera.
All porcine implantations of sdVGs were performed with at least 3 biological replicates. The sample size is detailed for each experiment throughout the figure legends. Statistical analysis was performed using GraphPad Prism 9.2.0. Unpaired t-tests, One-Way ANOVA with Tukey's posttest, the mixed-effects model with Tukey's or S̆idák's posttest, or Two-Way ANOVA with Tukey's or S̆idák's posttest were used where appropriate, in which significance levels were set at *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. All graphical data were reported as mean±standard deviation unless otherwise indicated.
To determine the shelf-life of natural polymer-based sdVGs, we fabricated FMTs as previously, Elliott et al., 2019, and dehydrated them with serial EtOH solutions for long-term storage in the freezer, refrigerator, or room temperature (
Next, we further assessed the biological relevance of the mechanical property changes in the stored biopolymer FMTs. We found that storage in the freezer resulted in the most stable circumferential UTS, circumferential STF, and modulus of toughness over time (
We used an accelerated aging model to determine if the changes in FMTs mechanical properties resulting from storage could be reproduced in a shorter timescale. In this model, devices are stored at elevated temperatures for short periods to simulate storage at ambient temperatures for more extended periods (
In our previous work, we compared acellular Fibrin-PCL sdVGs to sdVGs seeded with a luminal monolayer of endothelial colony forming cells in an abdominal aorta interposition mouse model and found that endothelialized sdVGs had a more controlled remodeling process with enhanced neotissue formation. Elliott et al., 2019. Other groups have shown that endothelial cells (ECs) are antithrombotic and prevent intimal hyperplasia, Fleser et al., 2004; van Hinsbergh, 2012; Brisbois et al, 2015; Elliott and Gerecht, 2016, critical to sdVG applications. To provide these same benefits while maintaining the off-the-shelf availability of our sdVGs, Ostadal et al., 2008; Tasatargil et al., 2005; Beamish et al., 2009; Saitow et al., 2011, we developed LMWH-embedded sdVGs. We hypothesized that direct conjugation of LMWH to the protein backbone within the fibrin scaffold would allow sustained and local release of the anticoagulant while the scaffold degrades. Fabrication of LMWH-embedded sdVGs first requires synthesis of LMWH-fibrinogen (LMWH-F), which we achieved by conjugation of fibrinogen with LMWH using carbodiimide chemistry (
We next assessed the cumulative LMWH and fibrinogen released due to hydrolytic and enzymatic degradation. A modified DMMB assay showed no significant difference in the cumulative release of sulfated GAGs in PBS between the HF and Fibrin tubes until one week (
During enzymatic degradation of the tubes with plasmin in PBS, there was a significant increase in the cumulative sulfated GAGs released by the HF tubes by 2 days, and the release plateaued at 13 days (
We used the mass-swelling ratio and mechanical properties to assess how the incorporation of LMWH in scaffolds altered graft structural integrity. There was no significant difference in the mass-swelling ratio between the HF and Fibrin tubes (
We utilized a dynamic incubation of platelets activated with 0.1 U/mL thrombin on electrospun 2D scaffold sheets as an in vitro thrombogenesis assay (
2.4.4 Scale-Up of sdVGs
To increase clinical relevancy of the off-the-shelf sdVGs, we scaled-up fabrication of FMTs from 0.6 mm inner diameter and 1 cm length to 5 mm inner diameter and 4 cm length (
We next optimized the ultra-thin, external PCL surgical sheath. The PCL solution was electrospun at different relative humidities and air gap distances (AGDs) (
We matched the inner diameter of the Fibrin-PCL sdVGs to the pressurized diameter of the native porcine carotid artery, which contracted significantly during surgery and after harvest to 1.86±1.43 mm (
2.4.5 Implantation and Patency of sdVGs in Porcine Model
A porcine model enables a more strict thrombogenicity assessment than a murine model, which has different clotting mechanisms than humans. Pashneh-Tala et al., 2015. We implanted a 2 cm length of the sdVGs with a size suitable for human application in the porcine carotid artery as an interposition graft (
We assessed patent sdVGs harvested 4-5 weeks after implantation for neotissue formation (
This study established the off-the-shelf availability of FMTs and anticoagulant embedded sdVGs with a size and mechanical properties suitable for human applications. We show that the FMT, a natural polymer-based scaffold, has a shelf-life of 12 months when stored in the refrigerator, freezer, or at room temperature. This flexibility will ensure the grafts can be easily shipped to and stored by urban and rural hospitals before use in emergency clinical cases. These grafts with off-the-shelf availability could also be used for limb salvage in combat casualties, either as temporary vascular shunts in austere conditions or for definitive vascular repair after evacuation. Rasmussen et al., 2018. While changes in FMT structure caused by long-term storage should be investigated, we found that the scaffold remains functional with a mechanically stable structure that enables immediate implantation after long-term storage. The fresh and stored FMT's reduced strength relative to the native abdominal aorta confirms the need for the synthetic polymer surgical sheath to provide mechanical strength. Interestingly, the accelerated aging of hydrogel scaffolds in controlled temperature, and humidity environments led to the increased strength of the FMT. In the future, we will investigate how this accelerated aging process may be harnessed to alter mechanical properties and eventually remove the need for the synthetic polymer surgical sheath. This would aid the neotissue formation process as our study and others have shown the potential for PCL calcification. De Valence et al., 2012. Computational modeling has previously been used to improve scaffold design and accurately predict clinical outcomes. Szafron et al., 2018; Drews et al., 2020; Lee et al., 2007. It thus should be considered for the optimization of graft design, including geometry and material composition, to balance maintenance of structural integrity with host cell remodeling.
To provide antithrombotic benefits while maintaining off-the-shelf availability of our engineered bypass graft, we developed a heparinized FMT by chemically conjugating LMWH to the scaffold's protein backbone. This novel method for local anticoagulant drug delivery embeds the LMWH throughout the scaffold and enables a more sustained delivery than physical encapsulation techniques, with over 95% of the drug-releasing in 24 hours. Matsuzaki et al., 2021. The drug release profile for the HF tubes indicates no burst release of LMWH occurs during hydrolytic degradation. We anticipated the LMWH would be available as long as the fibrin scaffold was present; indeed, the enzymatic drug release profile indicates that the LMWH is released during the entire 2 weeks of HF tube degradation. Notably, the conjugated LMWH remained active, as shown by the decreased adhesion of porcine platelets to the HF scaffold surface and the reduced porcine and human platelet activation. In vitro thrombogenicity assays with porcine PRP indicated a reduction in the thrombin generation profile that was more substantial than the anticoagulant effect seen in fucoidan coated grafts, which maintained patency in a rabbit model for 4 weeks. Yao et al., 2020. The HF and Fibrin tubes also had similar hydrogel swelling and mechanical properties.
Scale-up of sdVGs to structures sized for human applications required optimizing the PCL sheath for surgical utility, enabling suturability and improved mechanical properties. Electrospinning air gap distance and heat treatment were critical parameters to improve suture retention strength and Fibrin-PCL layer diameter matching. The pre-implant mechanical properties of the larger diameter HF- and Fibrin-PCL sdVGs were similar to the native porcine carotid artery. The need for the PCL sheath to prevent rupture in the large animal model validated the in vitro shelf-life assessments of FMTs. The LMWH in the HF-PCL sdVGs reliably extended patency in the porcine model beyond 2 weeks. By that time, the clinically available GORE-TEX® ePTFE graft and one of the Fibrin-PCL sdVGs were occluded due to thrombus formation. By 4-5 weeks post-implantation, HF-PCL sdVGs were stenosed due to neointimal hyperplasia, similar to the clinically available GORE® PROPATEN® grafts. Based on the in vitro enzymatic degradation cumulative drug release model, we theorize that LMWH was still available in the fibrin layer at 4-5 weeks in vivo, which is desired until a stable tunica intima is formed. Tissue overgrowth on the luminal surface of the Fibrin-PCL sdVGs indicates the hyperplasia leading to severe stenosis has no relation to the embedded LMWH or PROPATEN® coating. Incorporating an anti-proliferative drug like rapamycin may enhance control of the remodeling process by preventing hyperplasia, Yang et al., 2020, reducing stenosis, and prolonging patency beyond 4-5 weeks until a stable tunica intima is formed.
Fibrin mediated neotissue formation, as previously, by supporting extensive host cell infiltration during scaffold degradation. The GORE® PROPATEN® vascular grafts also helped host cell infiltration, but the scaffold will not degrade over time. Patent sdVGs showed that fibrin supported endothelialization by 4-5 weeks post-implantation. The presence of ECs is auspicious for long-term patency after the LMWH is gone. The irregular medial layer and SMC hyperplasia would also benefit from incorporating an anti-proliferative drug. Future efforts should assess the host immune cells, including macrophages, that are involved in acutely remodeling the fibrin and PCL sheath. Ultimately, the HF tube provided an antithrombotic, pro-regenerative scaffold for neotissue formation, while the synthetic polymer layer provided mechanical stability. The HF-PCL sdVG has exciting potential to remodel towards a healthy native vessel structure and thereby overcome limitations of using autologous vascular tissue harvested from the patient and synthetic polymer grafts. Elliott et al., 2019; Matsuzaki et al., 2021. We have anticipated the human condition and developed anticoagulant embedded, biodegradable sdVGs with off-the-shelf availability to mitigate the effects of prothrombotic environments and progress the clinical and commercial utility of our sdVGs.
All publications, patent applications, patents, and other references mentioned in the specification are indicative of the level of those skilled in the art to which the presently disclosed subject matter pertains. All publications, patent applications, patents, and other references are herein incorporated by reference to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. It will be understood that, although a number of patent applications, patents, and other references are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art. In case of a conflict between the specification and any of the incorporated references, the specification (including any amendments thereof, which may be based on an incorporated reference), shall control. Standard art-accepted meanings of terms are used herein unless indicated otherwise. Standard abbreviations for various terms are used herein.
Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims.
This invention was made with government support under grant nos. CBET1054415 and DMR1410240 awarded by the National Science Foundation. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US21/63128 | 12/13/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63124202 | Dec 2020 | US |
