WO2017083381A1

WO2017083381A1 - Engineered polymeric valves and systems, methods for generating the same, and uses of the same

Info

Publication number: WO2017083381A1
Application number: PCT/US2016/061129
Authority: WO
Inventors: Andrew K. CAPULLI; Kevin Kit Parker
Original assignee: President And Fellows Of Harvard College
Priority date: 2015-11-09
Filing date: 2016-11-09
Publication date: 2017-05-18
Also published as: WO2017083381A8

Abstract

The present invention provides engineered valves comprising oriented polymeric fibers, e.g., nano fibers, methods of fabricating such structures, and systems and devices for fabricating such structures. Such valve scaffolds are fabricated using a multi-part mandrel assembly upon which micron, submicron, and nanometer dimension polymer fibers are collected. The valve scaffolds include one or more sinuses corresponding to at least one leaflet within the valve scaffold.

Description

ENGINEERED POLYMERIC VALVES AND SYSTEMS, METHODS FOR GENERATING THE SAME, AND USES OF THE SAME

RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62/252,782, filed on November 9, 2015, and to U.S. Provisional Patent Application No. 62/307,795, filed on March 14, 2016. The entire contents of each of the foregoing applications are hereby incorporated herein by reference.

GOVERNMENT SUPPORT

[0002] This invention was made with government support as a subcontract issued under Prime Contract Number DE-AC52-06NA25396 between Los Alamos National Laboratory and the United States Department of Energy (DOE) and the National Nuclear Science Administration (NNSA). The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Over 150,000 surgical procedures are performed worldwide each year to replace damaged or diseased cardiac valves. For example, mechanical heart valve prostheses can be used to replace any naturally occurring cardiac valves. Operating much like a rigid mechanical check valve, mechanical heart valves are robust and long-lived. However, mechanical valves suffer from the disadvantage that they are thrombogenic and, thus, the patient requires lifetime anticoagulant therapy which is expensive and potentially dangerous in that it may cause abnormal bleeding which, in itself, can cause a stroke if the bleeding occurs within the brain. In addition, mechanical valves also generate a clicking noise when the mechanical closure seats against the associated valve structure at each beat of the heart.

[0004] One alternative to mechanical valves is tissue-type or "bioprosthetic" valves.

Bioprosthetic valves are generally made from naturally-derived xenogeneic tissues fixed with glutaraldehyde-based processes. Bioprosthetic valves are constructed either by sewing pig aortic valves to a stent to hold the leaflets in proper position, or by constructing valve leaflets using pericardial sac, such as bovine-derived pericardium, and sewing the leaflets to a stent. The stents can be rigid or slightly flexible and are covered with cloth, usually a synthetic material. The major disadvantage of bioprosthetic valves is that they lack the long-term durability of mechanical valves. In addition, naturally occurring processes within the human body can attack and stiffen or "calcify" the tissue leaflets of the bioprosthetic valve over time, particularly at high-stress areas of the valve, such as at the commissure junctions between the valve leaflets and at the peripheral leaflet attachment points or "cusps" at the outer edge of each leaflet. Further, bioprosthetic valves are subject to stresses from constant mechanical operation within the body.

[0005] Another alternative is tissue-engineered heart valves that have been proposed by physicians and scientists alike to be the ultimate solution for treating valvular heart disease. Rather than replacing a diseased or defective native valve with a mechanical or animal tissue-derived artificial valve, a tissue-engineered valve is a living organ, able to respond to growth and physiological forces in the same way that the native valve does. In particular, a whole porcine aortic valve that has been previously cleaned of all pig cells is implanted in a subject leaving an intact, mechanically sound connective tissue matrix. The cells of the patients are expected to repopulate and revitalize the acellular matrix, creating living tissue that already has the complex micro structure necessary for proper function and durability. Although these tissue-engineered valves can overcome the need for anticoagulants as well as the need for open-heart surgery, their very composition sacrifices durability. On average, these heart valves are only expected to last 15 years in the patient before the over 600 million open-close cycles within this time frame cause significant fatigue that warrants replacement. Although the lifecycle of these tissue-engineered valves may be less relevant for the aging patient population that requires replacement semilunar valves as a result of atherosclerosis or calcification, it is a significant problem for children born with congenital valve defects. One out of every 110 babies born in the US suffers from congenital heart disease, with valve malformation among the most common abnormalities. Because these current animal sourced valves are non-regenerative and eventually fatigue, children needing valve repair or replacement will have to undergo numerous surgeries throughout their lifetime. In addition, current tissue-engineered valves consist of foreign body material.

[0006] In order to overcome the foregoing limitations of tissue-engineered valves, there has been a focus on replacing the fixed-tissue basis of the valve with a degradable scaffold material that is seeded with stem cells to promote endogenous repair and remodeling once implanted. Although many of these studies have shown promising results of tissue remodeling and integration into the body, there still remains a significant gap between research usage and commercial adoption of this type of tissue engineered valve. Typical scaffolding materials, however, are still far from ideal, and are often expensive, potentially immunogenic, and show toxic degradation and inflammatory reactions. In addition, they are of poor resorbability. As a consequence, there is a lack of growth and risk of thromboembolic complications, degeneration and infections. Furthermore, tissue engineering of highly customized valve scaffolds doped with growth factors or seeded with stem cells and cultured in the lab prior to implantation results in a very costly and specialized valve implant.

Prosthetic engineered tissue valves have recently been fabricated using polymeric nanofibers (see, e.g, U.S. Patent Publication No. 2015/0182679, the entire contents of which is incorporated herein by reference). These valves, although mechanically sound and biocompatible, require several fabrication steps. The separate fabrication steps of the valve leaflets and conduit require the valve leaflets and conduit to be connected via suture, adhesive, heat-weld, or some other fixation technique, such as suturing, at connection points. The connection points are high load bearing points of the device and are also points where the device has been weakened by forming the connection. As such, there may be an increased risk of leakage, valve failure at the connection points, and/or lack of tissue remodeling as native cells fail to repopulate properly at the connection points. Furthermore, to deliver these valves via a minimally invasive procedure, pieces of the valve (e.g., valve leaflets) may need to be cut and sutured onto a stent by a skilled surgeon, which increases the complexity and time required for the procedure.

[0007] Accordingly, there is a need in the art for improved valves that are durable and can mimic the structure and function of the human cardiac valves in vivo and overcome the issues associated with current devices.

SUMMARY

[0008] In accordance with embodiments of the present disclosure, an engineered valve configured for flow from an upstream first end to a downstream second end is disclosed. The engineered valve includes a tubular wall including micron, submicron, or nanometer dimension polymer fibers defining a shape of the tubular wall, the tubular wall having an inner surface, a first end, and a second end. The engineered valve also includes a plurality of leaflets integral with the inner surface of the tubular wall, each extending from the inner surface of the tubular wall radially inward and toward the second end of the tubular wall, the plurality of leaflets including micron, submicron, or nanometer dimension polymer fibers defining the shape of the plurality of leaflets, the plurality of leaflets configured for flow through the tubular wall from the first end of the tubular wall downstream to the second end of the tubular wall. The tubular wall of the engineered valve includes a plurality of outward bulging portions, each outward bulging portion forming a sinus for a corresponding leaflet of the plurality of leaflets.

[0009] In some embodiments, the each outward bulging portion extends downstream of a portion of the tubular wall that connects with the corresponding leaflet.

[0010] In some embodiments, the valve includes two leaflets and two corresponding bulging portions forming sinuses. In other embodiments, the valve includes three leaflets and three corresponding bulging portions forming sinuses.

[0011] In some embodiments, the valve may include bulging portions forming sinuses corresponding to some, but not all, of the leaflets. For example, in some embodiments, the valve includes three leaflets and bulging portions forming sinuses corresponding to two of the three leaflets.

[0012] In some embodiments, each of the plurality of leaflets are substantially the same size. In other embodiments, each of the plurality of leaflets is a different size. In some

embodiments, for example, when the engineered valve comprises three leaflets, two of the leaflets may be substantially the same size and the third leaflet may be smaller than the other two leaflets, e.g., to mimic the aortic valve.

[0013] In some embodiments, at least some of the micron, submicron or nanometer dimension polymer fibers of the tubular wall interpenetrate with at least some of the micron, submicron or nanometer dimension polymer fibers of the plurality of leaflets.

[0014] In some embodiments, the micron, submicron, or nanometer dimension polymer fibers include at least one biogenic polymer, such as, for example, poly-4-hydroxybuyrate, collagen, and gelatin. In other embodiments, the micron, submicron, or nanometer dimension polymer fibers include a combination of biogenic polymers, e.g., poly-4-hydroxybuyrate and gelatin.

[0015] In some embodiments, the micron, submicron, or nanometer dimension polymer fibers have a diameter between about 10 nanometers and about 10 microns. In some embodiments, the micron, submicron, or nanometer dimension polymer fibers have a diameter between about 500 nanometers and about 1.5 microns, for example, a diameter of between about 550 nanometers and about 1.5 microns, a diameter of between about 600 nanometers and about 1.5 microns, a diameter of between about 650 nanometers and about 1.5 microns, a diameter of between about 700 nanometers and about 1.5 microns, a diameter of between about 750 nanometers and about 1.5 microns, a diameter of between about 800 nanometers and about 1.5 microns, a diameter of between about 850 nanometers and about 1.5 microns, a diameter of between about 900 nanometers and about 1.5 microns, a diameter of between about 950 nanometers and about 1.5 microns, a diameter of between about 1.0 microns and about 1.5 microns, a diameter of between about 500 nanometers and about 1.45 microns, a diameter of between about 500 nanometers and about 1.40 microns, a diameter of between about 500 nanometers and about 1.35 microns, a diameter of between about 500 nanometers and about 1.3 microns, a diameter of between about 500 nanometers and about 1.25 microns, a diameter of between about 500 nanometers and about 1.20 microns, a diameter of between about 500 nanometers and about 1.15 microns, a diameter of between about 500 nanometers and about 1.10 microns, a diameter of between about 600 nanometers and about 1.4 microns, a diameter of between about 700 nanometers and about 1.3 microns, a diameter of between about 800 nanometers and about 1.2 microns, a diameter of between about 900 nanometers and about 1.0 microns. Ranges and values intermediate to the above recited ranges and values are also contemplated to be part of the invention, for example, a diameter of between about 950 nanometers and about 1.2 microns, or a diameter of between about 850 nanometers and 1.1 microns.

[0016] In some embodiment, the tubular wall of the engineered valves of the invention is substantially the same thickness over the entire length of the valve. In other embodiments, the thickness of the tubular wall is greater at the portion of the tubular wall that includes the integral leaflets and bulging portions forming sinuses than the portion of the tubular wall that does not include the integral leaflets and bulging portions forming sinuses. For example, in some embodiments, the tubular wall of the engineered valves of the invention has a thickness of between about 10 microns and about 500 microns or between about 50 microns and about 300 microns, e.g., between about 50 microns and about 275 microns, between about 50 microns and about 250 microns, between about 50 microns and about 225 microns, between about 50 microns and about 200 microns, between about 50 microns and about 175 microns, between about 50 microns and about 150 microns, between about 50 microns and about 125 microns, between about 50 microns and about 100 microns, between about 50 microns and about 75 microns. In other embodiments, the thickness of the tubular wall which includes the leaflets and bulging portions forming sinuses has a thickness of about 200 microns to about 400 microns, e.g., about 200, 225, 250, 275, 300, 325, 350, 375, or about 400 microns. Ranges and values intermediate to the above recited ranges and values are also contemplated to be part of the invention.

[0017] In some embodiments, a diameter of the first end of the tubular structure corresponds to a diameter of a pulmonary valve.

[0018] In some embodiments, the tubular wall further comprises a stent. In one embodiment, the stent is embedded in the micron, submicron or nanometer dimension polymer fibers forming the tubular wall. In other embodiments, the valve is disposed in a conduit of a stent.

[0019] In some embodiments, the micron, submicron or nanometer dimension polymer fibers of the tubular wall and the micron, submicron or nanometer dimension polymer fibers of the plurality of leaflets are configured to form a polymeric fiber scaffold for cellular ingrowth.

[0020] In accordance with other embodiments of the present disclosure, a mandrel assembly for making a valve including a tubular wall and at least three leaflets is disclosed. The mandrel assembly has an axis of rotation and includes a first mandrel having a first end portion, a second end portion, and an outer surface, the outer surface having a tubular wall- forming region at the first end portion and at least three concave leaflet-forming regions at the second end portion, each concave leaflet-forming region configured to define a shape of an upstream surface of a leaflet in the resulting valve. The mandrel assembly also includes a second mandrel structure. The second mandrel structure includes a member including a base portion and at least three spacing portions extending parallel to the axis of rotation from the base portion. The second mandrel structure also includes at least three sinus-forming bodies, each sinus-forming body having a first end portion and a second end portion and configured to be fastened to the member with the first end portion of the sinus-forming body adjacent the base portion of the member and with the each sinus-forming body separated from an adjacent sinus-forming body by one of the at least three spacing portions. The member and the sinus- forming bodies are configured such that, when fastened together, an outward facing surface of the member and outward facing surfaces of the first end portions of each of the sinus- forming bodies collectively form a tubular wall- forming region of an outer surface of the second mandrel structure, an outward facing surface of the second end portion of each sinus- forming body bulges outward to define a shape of a sinus of the resulting valve, and an inward facing surface of the second end portion of each sinus-forming body bulges inward to define a shape of a downstream surface of a leaflet in the resulting valve. [0021] In some embodiments, the first mandrel includes three concave leaflet-forming regions and the second mandrel structure includes three sinus-forming bodies configured to form a resulting valve having three leaflets and three sinuses.

[0022] In accordance with other embodiments of the present disclosure, a mandrel assembly for making a valve including a tubular wall and at least three leaflets is disclosed. The mandrel assembly has an axis of rotation and includes a first mandrel having a first end portion, a second end portion, and an outer surface, the outer surface having a tubular wall- forming region at the first end portion and at least three concave leaflet-forming regions at the second end portion, each concave leaflet-forming region configured to define a shape of an upstream surface of a leaflet in the resulting valve. The mandrel assembly also includes a second mandrel structure. The second mandrel structure includes a member including a base portion and at least two spacing portions extending parallel to the axis of rotation from the base portion. The second mandrel structure also includes at least two sinus-forming bodies and a tubular wall-forming body. Each sinus-forming body has a first end portion and a second end portion and is configured to be fastened to the member with the first end portion of the sinus-forming body adjacent the base portion of the member and with the each sinus- forming body separated from an adjacent sinus-forming body or from the tubular-wall forming body by one of the at least two spacing portions. The a first end portion of the tubular-wall forming body is configured to be fastened to the member. The member the sinus-forming bodies, and the tubular-wall forming body are configured such that, when fastened together, an outward facing surface of the member, outward facing surfaces of the first end portions of each of the sinus-forming bodies, and an outward facing surface of the first end portion of the tubular wall- forming body collectively form a tubular wall- forming region of an outer surface of the second mandrel structure, an outward facing surface of the second end portion of each sinus-forming body bulges outward to define a shape of a sinus of the resulting valve, and an inward facing surface of the second end portion of each sinus- forming body and an inward facing surface of a second end portion of the tubular-wall forming body bulges inward to define a shape of a downstream surface of a leaflet in the resulting valve.

[0023] In some embodiments, the first mandrel includes three concave leaflet-forming regions and the second mandrel structure includes two sinus-forming bodies configured to form a resulting valve having three leaflets and two sinuses. [0024] In some embodiments, each concave leaflet-forming region of the outer surface of the first mandrel is configured to at least partially receive a second-end portion of a

corresponding sinus-forming body when the sinus-forming body is fastened to the member. In some embodiments, at least some of the concave leaflet- forming regions of the outer surface of the first mandrel are configured to at least partially receive a second-end portion of a corresponding sinus-forming body when the sinus-forming body is fastened to the member.

[0025] In some embodiments, a diameter of a first end portion of the first mandrel is substantially equal to a diameter of tubular wall- forming region of an outer surface of the second mandrel structure.

[0026] In some embodiments, the first mandrel is configured to be rotated around a rotation axis to receive a coating of micron, submicron, or nanometer dimension polymer fibers thereby forming an upstream surface of each leaflet in the resulting valve and an inner surface of an upstream tubular wall of the resulting valve.

[0027] In some embodiments, the first mandrel and the second mandrel structure are configured to be fastened together to form a combined mandrel, the combined mandrel configured to be rotated around the axis of rotation to receive a coating of micron, submicron, or nanometer dimension polymer fibers thereby forming sinuses and a downstream tubular wall of the resulting valve.

[0028] In some embodiments, the first mandrel is configured to be withdrawn e.g., in pieces, from an upstream end of the resulting valve without damaging a tubular wall formed on the tubular wall-forming region at the first end portion of the first mandrel after collection of the coating of micron, submicron, or nanometer dimension polymer fibers on the combined mandrel.

[0029] In some embodiments, the second mandrel structure is configured to be disassembled and then withdrawn from a downstream end of the resulting valve in pieces without damaging a tubular wall formed on the tubular wall-forming region of the outer surface of the second mandrel structure after collection of the coating of micron, submicron, or nanometer dimension polymer fibers on the combined mandrel.

[0030] In accordance with other embodiments of the present disclosure, a method of forming an engineered valve including a tubular structure, at least three sinuses, and at least three leaflets is disclosed. The method includes forming micron, submicron, or nanometer dimension polymer fibers by ejecting or flinging a polymer from a reservoir. The method also includes collecting a first portion of micron, submicron, or nanometer dimension polymer fibers on a rotating first mandrel having a first end portion, a second end portion, and an outer surface with a tubular wall-forming region at the first end portion and at least three concave leaflet- forming regions at the second end portion, the tubular-wall forming region of the outer surface having a shape corresponding to an inner surface of a first portion of the tubular resulting engineered valve, each of the at least three concave leaflet-forming regions having a shape corresponding to an upstream surface of a corresponding leaflet in the resulting valve. The method also includes coupling a second mandrel structure to the first mandrel to form a combined mandrel, the second mandrel structure comprising a member including a base portion and as many spacing portions as leaflets, each spacing portion extending parallel to the axis of rotation from the base portion and at least three sinus- forming bodies each having a first end portion and a second end portion, each sinus-forming body fastened to the member and separated from an adjacent sinus-forming body by one of the at least three spacing portions. An outward facing surface of the member and outward facing surfaces of the second end portions of each of the sinus-forming bodies collectively form a tubular wall- forming region of an outer surface of the second mandrel structure. An outward facing surface of the first end portion of each sinus-forming body bulges outward to define a shape of a sinus of the resulting valve, and an inward facing surface of the first end portion of each sinus-forming body bulges inward to define a shape of a downstream surface of a leaflet in the resulting valve scaffold. The method also includes collecting a second portion of micron, submicron, or nanometer dimension polymer fibers on the combined mandrel by rotating the combined mandrel about a rotation axis in the path of the ejected micron, submicron, or nanometer dimension polymer fibers to form a remainder of the resulting valve scaffold having a first end and a second end, at least three leaflets and at least three sinuses.

[0031] In accordance with other embodiments of the present disclosure, a method of forming an engineered valve including a tubular structure, at least two sinuses, and at least three leaflets is disclosed. The method includes forming micron, submicron, or nanometer dimension polymer fibers by ejecting or flinging a polymer from a reservoir. The method also includes collecting a first portion of micron, submicron, or nanometer dimension polymer fibers on a rotating first mandrel having a first end portion, a second end portion, and an outer surface with a tubular wall-forming region at the first end portion and at least three concave leaflet- forming regions at the second end portion, the tubular-wall forming region of the outer surface having a shape corresponding to an inner surface of a first portion of the tubular resulting engineered valve, each of the at least three concave leaflet-forming regions having a shape corresponding to an upstream surface of a corresponding leaflet in the resulting valve. The method also includes coupling a second mandrel structure to the first mandrel to form a combined mandrel, the second mandrel structure comprising a member including a base portion and as many spacing portions as leaflets, each spacing portion extending parallel to the axis of rotation from the base portion and at least two sinus-forming bodies each having a first end portion and a second end portion. The second mandrel structure also including at least two sinus-forming bodies and a tubular wall-forming body, each sinus-forming body and tubular wall-forming body fastened to the member and separated from an adjacent sinus-forming body or tubular wall- forming body by one of the at least two three portions. An outward facing surface of the member, outward facing surfaces of the second end portions of each of the sinus-forming bodies, and an outward facing surface of the tubular-wall forming body collectively form a tubular wall- forming region of an outer surface of the second mandrel structure. An outward facing surface of the first end portion of each sinus-forming body bulges outward to define a shape of a sinus of the resulting valve. An inward facing surface of the first end portion of each sinus-forming body and an inward facing surface of the tubular wall-forming body bulges inward to define a shape of a downstream surface of a leaflet in the resulting valve scaffold. The method also includes collecting a second portion of micron, submicron, or nanometer dimension polymer fibers on the combined mandrel by rotating the combined mandrel about a rotation axis in the path of the ejected micron, submicron, or nanometer dimension polymer fibers to form a remainder of the resulting valve scaffold having a first end and a second end, at least three leaflets and at two sinuses.

[0032] In some embodiments, the method also includes uncoupling the first mandrel and the second mandrel structure and withdrawing the first mandrel from the first end of the resulting valve scaffold.

[0033] In some embodiments, the method also includes unfastening the sinus-forming bodies from the member and withdrawing the member from the second end of the resulting valve scaffold.

[0034] In some embodiments, the method also includes withdrawing the sinus-forming bodies from the second end of the resulting valve scaffold after withdrawing the member from the second end of the resulting valve scaffold. [0035] In some embodiments, the method also includes removing excess submicron, or nanometer dimension polymer fibers from the first mandrel before coupling the second mandrel structure to the first mandrel, e.g., to separate two or more leaflets before coupling the second mandrel to the first mandrel.

[0036] In some embodiments, the leaflets composed of the micron, submicron, or nanometer dimension polymer fibers that are formed during collection of the first portion of micron, submicron, or nanometer dimension polymer fibers are covered by a portion of the second mandrel structure after coupling the first mandrel and the second mandrel structure to form the combined mandrel.

[0037] In some embodiments, the micron, submicron, or nanometer dimension polymer fibers have a diameter of between about 10 nanometers and about 1.5 microns. In some embodiments, the micron, submicron, or nanometer dimension polymer fibers have a diameter of between about 500 nanometers and about 1.5 microns, for example a diameter of between about 550 nanometers and about 1.5 microns, a diameter of between about 600 nanometers and about 1.5 microns, a diameter of between about 650 nanometers and about 1.5 microns, a diameter of between about 700 nanometers and about 1.5 microns, a diameter of between about 750 nanometers and about 1.5 microns, a diameter of between about 800 nanometers and about 1.5 microns, a diameter of between about 850 nanometers and about 1.5 microns, a diameter of between about 900 nanometers and about 1.5 microns, a diameter of between about 950 nanometers and about 1.5 microns, a diameter of between about 1.0 microns and about 1.5 microns, a diameter of between about 500 nanometers and about 1.45 microns, a diameter of between about 500 nanometers and about 1.40 microns, a diameter of between about 500 nanometers and about 1.35 microns, a diameter of between about 500 nanometers and about 1.3 microns, a diameter of between about 500 nanometers and about 1.25 microns, a diameter of between about 500 nanometers and about 1.20 microns, a diameter of between about 500 nanometers and about 1.15 microns, a diameter of between about 500 nanometers and about 1.10 microns, a diameter of between about 600 nanometers and about 1.4 microns, a diameter of between about 700 nanometers and about 1.3 microns, a diameter of between about 800 nanometers and about 1.2 microns, a diameter of between about 900 nanometers and about 1.0 microns. Ranges and values intermediate to the above recited ranges and values are also contemplated to be part of the invention, for example a diameter of between about 950 nanometers and about 1.2 microns, or a diameter of between about 850 nanometers and 1.1 microns. [0038] In some embodiments, the micron, submicron, or nanometer dimension polymer fibers include a biogenic polymer.

[0039] In some embodiments, a spacing between each of the concave leaflet-forming regions of the outer surface of the first mandrel and the corresponding inward facing surface the second end portion of each sinus-forming body in the second mandrel structure of the combined mandrel is set to form a leaflet in the resulting valve scaffold having a thickness between about 50 microns and about 400 microns, e.g., between about 50 microns and about 375 microns, between about 50 microns and about 350 microns, between about 50 microns and about 325 microns, between about 50 microns and about 300 microns, between about 50 microns and about 275 microns, between about 50 microns and about 250 microns, between about 50 microns and about 225 microns, between about 50 microns and about 200 microns, between about 50 microns and about 175 microns, between about 50 microns and about 150 microns, between about 50 microns and about 125 microns, between about 50 microns and about 100 microns, or between about 50 microns and 75 microns. Ranges and values intermediate to the above recited ranges and values are also contemplated to be part of the invention.

[0040] In some embodiments, the micron, submicron, or nanometer dimension polymer fibers are ejected through an orifice of a rotating reservoir.

[0041] In some embodiments, the first mandrel and the combined mandrel are rotated about an axis that is inclined at an angle with respect to an axis of rotation of the rotating reservoir during collection of the first portion and the second portion of micron, submicron, or nanometer dimension polymer fibers.

[0042] In some embodiments, the first mandrel and the combined mandrel are rotated about an axis that is inclined between about 0 degrees and about 45 degrees with respect to the axis of rotation of the rotating reservoir, such as about 0 degrees and about 40 degrees, about 0 degrees and about 35 degrees, about 0 degrees and about 30 degrees, about 0 degrees and about 25 degrees, about 0 degrees and about 20 degrees, about 0 degrees and about 15 degrees, about 0 degrees and about 10 degrees, or about 0 degrees and about 5 degrees. Ranges and values intermediate to the above recited ranges and values are also contemplated to be part of the invention.

[0043] In some embodiments, the first portion of micron, submicron, or nanometer dimension polymer fibers is collected with an axis of rotation of the first mandrel oriented at a first angle with respect to an axis of rotation of the rotating reservoir, and then, in the combined mandrel, the orientation of the first mandrel is flipped by 180 degrees prior to collection of the second portion of micron, submicron, or nanometer dimension polymer fibers on the combined mandrel.

[0044] In some embodiments, the polymer is ejected from the reservoir by rotating the reservoir at a speed of between about 1,000 and about 10,000 rpm. In some embodiments, the polymer is ejected from the reservoir by rotating the reservoir at a speed of between about 1,000 and about 5,000 rpm, e.g., about 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,250, 3,500, 3,750, 4,000, 4,250, 4,500, 4,750, or about 5,000. In some

embodiments, the polymer is ejected from the reservoir by rotating the reservoir at a speed of between about 5,000 and about 50,000 rpm, e.g., between about 5,000 and about 45,000 rpm, between about 5,000 and about 40,000 rpm, between about 5,000 and about 35,000 rpm, between about 5,000 and about 30,000 rpm, between about 10,000 and about 50,000 rpm, between about 15,000 and about 50,000 rpm, between about 20,000 and about 50,000 rpm, between about 25,000 and about 50,000 rpm, or between about 30,000 and about 50,000 rpm. In some embodiments, the polymer is ejected from the reservoir by rotating the reservoir at a speed of between about 20,000 and about 100,000 rpm. Ranges and values intermediate to the above recited ranges and values are also contemplated to be part of the invention.

[0045] In some embodiments, collecting micron, submicron, or nanometer dimension polymer fibers on the rotating first mandrel includes rotating the first mandrel about a rotation axis in a path of the ejected polymer fibers.

[0046] In some embodiments, collecting micron, submicron, or nanometer dimension polymer fibers on the rotating first mandrel includes translating the first mandrel along a path substantially parallel to the axis of rotation of the rotating reservoir.

[0047] In some embodiments, collecting micron, submicron, or nanometer dimension polymer fibers on the combined mandrel includes translating the combined mandrel along a path parallel to the axis of rotation of the rotating reservoir.

[0048] In some embodiments, collecting the second portion of micron, submicron, or nanometer dimension polymer fibers on the combined mandrel by rotating the combined mandrel about a rotation axis in the path of the ejected micron, submicron, or nanometer dimension polymer fibers to form a remainder of the resulting valve scaffold includes collecting part of the second portion of micron, submicron, or nanometer dimension polymer fibers on the combined mandrel by rotating the combined mandrel about a rotation axis in the path of the ejected micron, submicron, or nanometer dimension polymer fibers; positioning a stent over the part of the second portion of micron, submicron, or nanometer dimension polymer fibers collected on the combined mandrel; and collecting a remainder of the second portion of micron, submicron, or nanometer dimension polymer fibers on the stent and the combined mandrel by rotating the combined mandrel about a rotation axis in the path of the ejected micron, submicron, or nanometer dimension polymer fibers to form a remainder of the resulting valve scaffold.

[0049] In accordance with other embodiments, the present invention also provides engineered valves prepared according to the methods of the invention and/or using the mandrel assemblies of the present invention.

[0050] In some embodiments, the valves are lyophilized, e.g., for storage, prior to use.

[0051] In accordance with other embodiments of the present disclosure, a method for treating a subject having a defective or weakened cardiac valve is disclosed. The method includes providing an engineered valve including a tubular wall including micron, submicron or nanometer dimension polymer fibers defining a shape of the tubular wall, the tubular wall having an inner surface, a first end and a second end. The engineered valve also includes a plurality of leaflets integral with the inner surface of the tubular wall, each extending from the inner surface of the tubular wall radially inward and toward the second end of the tubular wall. Each of the leaflets includes micron, submicron, or nanometer dimension polymer fibers defining the shape of the leaflet, the leaflets configured for flow through the tubular wall from the first end of the tubular wall downstream to the second end of the tubular wall. The tubular wall of the engineered valve also includes a plurality of outward bulging portions, each outward bulging portion forming a sinus for a corresponding leaflet of the plurality of leaflets. The method also includes replacing the weakened or defective valve in the subject with the engineered valve, thereby treating the subject.

[0052] Additional features and advantages are realized through the techniques of the present disclosure. Other embodiments and aspects of the disclosure are described in detail herein and are considered part of the invention. The recitation herein of desirable objects, which are met by various embodiments of the present disclosure, is not meant to imply or suggest that any or all of these objects are present as essential features, either individually or collectively, in the most general embodiment of the present disclosure, or in any of its more specific embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The features and advantages of the present disclosure will be more fully understood from the following description of exemplary embodiments when read together with the accompanying drawings, in which:

[0054] FIG. 1 is an exploded perspective view of an example mandrel assembly including a first mandrel and a second mandrel structure, according to embodiments of the present disclosure.

[0055] FIG. 2 is a perspective view of the example mandrel assembly of FIG. 1 in a combined mandrel configuration, according to embodiments of the present disclosure.

[0056] FIG. 3A is a perspective view of an example first mandrel, according to embodiments of the present disclosure.

[0057] FIG. 3B is a top view of the first mandrel of FIG. 3 A, according to embodiments of the present disclosure.

[0058] FIG. 4A is a perspective view of an example second mandrel structure, according to embodiments of the present disclosure.

[0059] FIG. 4B is a bottom view of the second mandrel structure of FIG. 4A, according to embodiments of the present disclosure.

[0060] FIG. 5 is a side view of the second mandrel structure of FIG. 4A, according to embodiments of the present disclosure.

[0061] FIG. 6 is a side view of the first mandrel of FIG. 3 A, according to embodiments of the present disclosure.

[0062] FIG. 7A is an exploded perspective view of an example mandrel assembly including two sinus forming bodies, according to embodiments of the present disclosure.

[0063] FIG. 7B is a perspective view of the mandrel assembly of FIG. 7A in a combined mandrel configuration, according to embodiments of the present disclosure.

[0064] FIG. 8A is a top view of the combined mandrel of FIG. 7B showing two protrusions corresponding to the two sinus forming bodies, according to embodiments of the present disclosure. [0065] FIG. 8B is a bottom view the combined mandrel of FIG. 7B showing the two protrusions, according to embodiments of the present disclosure.

[0066] FIG. 8C is a side view of the example combined mandrel of FIG. 7B, according to embodiments of the present disclosure.

[0067] FIG. 8D is another side view of the example combined mandrel of FIG. 8C, according to embodiments of the present disclosure.

[0068] FIG. 8E is another side view of the example combined mandrel of FIG. 8C, according to embodiments of the present disclosure.

[0069] FIG. 8F is another side view of the example combined mandrel of FIG. 8C, according to embodiments of the present disclosure.

[0070] FIG. 9 is a perspective view of an engineered valve, according to embodiments of the present disclosure.

[0071] FIG. 10 is a top view of the engineered valve of FIG. 9, according to embodiments of the present disclosure.

[0072] FIG. 11 schematically depicts an example fiber spinning system for forming an engineered valve, according to embodiments of the present disclosure.

[0073] FIG. 12A is a scanning electron micrograph of the structural fibrosa layer of a native valve 1201, showing that the structural fibrosa layer is primarily composed of

circumferentially aligned collagen bundles to withstand closing diastolic pressures. This example figure is a hematoxylin and eosin stain of an ovine pulmonary leaflet.

[0074] FIG. 12B is an enlarged view of an area of the structural fibrosa layer of the native valve of FIG. 12A with a decellularized leaflet. The magnified inset 1203 has a scale bar of 10 microns.

[0075] FIG. 13 A is an image of a top view of an example engineered valve produced according to embodiments of the present disclosure showing that the engineered valves recapitulate the macroscopic properties of the native valve.

[0076] FIG. 13B is an enlarged view of an area of the example engineered valve of FIG. 13A showing that the engineered valves recapitulate the microscopic fibrous properties of the native valve. [0077] FIG. 14A is an image of mandrel assembly components of varying sizes and engineered valves prepared using the variously sized components, according to embodiments of the present disclosure.

[0078] FIG. 14B is an enlarged view of one of the valve scaffolds in the image of FIG. 14A, according to embodiments of the present disclosure.

[0079] FIG. 15A is a bar graph showing fiber diameter as a function of protein content, according to embodiments of the present disclosure.

[0080] FIG. 15B is a bar graph showing percent porosity as a function of protein content, according to embodiments of the present disclosure.

[0081] FIG. 16A includes scanning electron micrographs of native valve leaflets and engineered valve leaflets prepared as described herein.

[0082] FIG. 16B is a bar graph showing a comparison of polymeric fiber alignment between a native leaflet fibrous structure and an example polymeric valve scaffold formed according to embodiments of the present disclosure.

[0083] FIG. 17A is a bar graph of low strain (0-10%) parallel stiffness of scaffolds with different levels of protein content illustrating how increased low strain parallel stiffness of scaffolds corresponds to increased protein content, according to embodiments of the present disclosure.

[0084] FIG. 17B is a bar graph of the high strain (10-20%) parallel stiffness of scaffolds with different levels of protein content illustrating how increased high strain parallel stiffness of scaffolds corresponds to increased protein content, according to embodiments of the present disclosure.

[0085] FIG. 17C is a bar graph of the low strain (0-10%) perpendicular stiffness of scaffolds with different levels of protein content illustrating how increased low strain perpendicular stiffness of scaffolds corresponds to increased protein content, according to embodiments of the present disclosure.

[0086] FIG. 17D is a bar graph of the high strain (10-20%) perpendicular stiffness of scaffolds with different levels of protein content illustrating how increased high strain perpendicular stiffness of scaffolds corresponds to increased protein content, according to embodiments of the present disclosure. [0087] FIG. 18 is a graph of stress versus strain for mechanical testing of valve scaffolds and tissue done by equibiaxial loading of samples in the primary axis of fiber alignment, according to embodiments of the present disclosure.

[0088] FIG. 19A is a graph of in vitro valve scaffold performance as measured by arterial pressure, according to embodiments of the present disclosure.

[0089] FIG. 19B is a graph of in vitro valve scaffold performance as measured by ventricular pressure, according to embodiments of the present disclosure.

[0090] FIG. 19C is a graph of in vitro valve scaffold performance as measured by flow, according to embodiments of the present disclosure.

[0091] FIG. 20A is an example graph showing parameter distribution for process capability of seven batches of valve scaffolds formed according to embodiments of the present disclosure.

[0092] FIG. 20B is another example graph showing parameter distribution for nine different parameters in seven batches of valve scaffolds formed according to embodiments of the present disclosure.

[0093] FIG. 21A includes images showing the effect of lyophilization storage on a valve scaffold produced according to embodiments of the present disclosure.

[0094] FIG. 21B includes images showing the effect of dry refrigeration storage on a valve scaffold produced according to embodiments of the present disclosure.

[0095] FIG. 22A is an image of an example valve scaffold crimped to approximately 24 mm in diameter, according to embodiments of the present disclosure.

[0096] FIG. 22B is an image of an example valve scaffold crimped to approximately 9 mm in diameter, according to embodiments of the present disclosure.

[0097] FIG. 23 is a graph of percent fiber surface composition as a function of time in days, according to embodiments of the present disclosure.

[0098] FIG. 24 shows an Echo/Doppler image assessing the in vivo valvular competency of a scaffold according to embodiments of the present disclosure.

[0099] FIG. 25A shows an arterial view of an example valve scaffold upon explantation, according to embodiments of the present disclosure. [00100] FIG. 25B shows a cross-sectional view of an example valve scaffold upon explantation, according to embodiments of the present disclosure.

[00101] FIG. 25C shows an enlarged view of a portion of the example valve scaffold of FIG. 25B, according to embodiments of the present disclosure.

[00102] FIG. 26A shows the results of a VG-Elastin staining test performed on an example explanted valve scaffold, according to embodiments of the present disclosure.

[00103] FIG. 26B shows the results of a Vimentin staining test performed on an example explanted valve scaffold, according to embodiments of the present disclosure.

[00104] FIG. 26C shows the results of a CD31 staining test performed on an example explanted valve scaffold, according to embodiments of the present disclosure.

[00105] FIG. 26D shows the results of an alpha smooth muscle actin staining test performed on an example explanted valve scaffold, according to embodiments of the present disclosure.

[00106] FIG. 27 is a flow chart schematically depicting an example method for fabricating a valve structure from micron, submicron, or nanometer dimension polymeric fibers, according to an embodiment of the present disclosure.

DETAILED DESCRIPTION

[00107] The present disclosure is based, at least in part, on the discovery of improved engineered cardiac valves comprising oriented polymeric fibers, also referred to herein as valve scaffolds. More specifically, to better recapitulate in vivo valve leaflet closing dynamics in an engineered valve, it has been discovered that by including sinuses or exterior bulges that correspond to each of the valve leaflets in the engineered valve, when the leaflets close during diastole the back-flow of blood ensures tight and leak-resistant closure of the valve leaflets by swirling in the sinuses which causes the leaflets to be pushed tightly closed. This swirling not only ensures a tight leaflet coaptation but also distributes the load (pressure and shear) of the blood across the leaflets and the sinuses and, thus, prevents valve failure at the points where the leaflets integrate into the wall of the valve.

[00108] In order to prepare such engineered valves including sinuses that protrude outward from the tubular wall of the valve, the second mandrel structure used in forming the bulging sinus portions of the polymeric fiber valve scaffolds described herein includes a number of separable components that can be removed piecewise from the fibrous valve structure or scaffold. Otherwise, removal of all second mandrel structure components together at one time poses challenges and would damage the tubular wall structure of the scaffold downstream of the sinuses because the bulges of the mandrel used to form the scaffold sinuses would not fit through the narrower downstream portion of the tubular wall of the valve scaffold.

[00109] Micron, submicron, or nanometer dimension polymeric fibers are an ideal scaffold material for tissue engineering because of their small diameters and pore sizes, large surface area to volume ratio, and tunable properties. When spun from different polymers, micron, submicron, or nanometer dimension polymeric fibers exhibit a range of elasticities and scaffold mechanics, and permit cell ingrowth. Accordingly, the present invention provides engineered valves that include a tubular wall comprising micron, submicron, or nanometer dimension polymer fibers, a plurality of leaflets integral with an inner surface of the tubular wall, and outward bulging portions of the tubular wall that form sinuses for corresponding leaflets, mandrel assemblies, methods for forming such valves, and methods of use of such valves to treat subject in need thereof, e.g., a subject having a weakened or defective cardiac valve.

I. Engineered Valves of the Invention

[00110] Example valves with a plurality of integral leaflets and bulging portions forming sinuses were prepared using the methods described herein. A resulting engineered valve scaffold is depicted in FIGS. 9-10. FIG. 9 is a perspective view of an engineered valve, according to embodiments of the present disclosure, prepared using the mandrel assemblies and methods described herein. FIG. 10 shows a top view of the engineered valve of FIG. 9 with three leaflets visible within the engineered valve. The engineered valve 900 includes a tubular wall 905 that includes micron, submicron, or nanometer dimension polymer fibers defining a shape of the tubular wall 905. The tubular wall 905 has an inner surface 907, a first end 901, and a second end 903. According to exemplary embodiments, the engineered valve 900 is configured for flow through the tubular wall 905 from the upstream first end 901 to the downstream second end 903. As can be seen in this example, the engineered valve 900 includes a plurality of leaflets 909 integral with the inner surface 907 of the tubular wall, each leaflet 909 extending from the inner surface 907 of the tubular wall 905 radially inward and toward the second end 903 of the tubular wall 905. The plurality of leaflets 909 include micron, submicron, or nanometer dimension polymer fibers defining the shape of the leaflets 909. In this particular embodiment, the engineered valve 900 includes three leaflets 909. The leaflets 909 are configured to allow fluid flow through the tubular wall 905 from the upstream first end 901 to the downstream second end 903. The tubular wall 905 also includes a plurality of outward bulging portions 911, each outward bulging portion 911 forming a sinus 913 for a corresponding leaflet 909. In some embodiments, each outward bulging portion 911 extends downstream of a portion of the tubular wall 905 that connects with the corresponding leaflet 909.

[00111] In exemplary embodiments, the micron, submicron, or nanometer dimension polymer fibers include a biogenic polymer. The micron, submicron, or nanometer dimension polymer fibers can have a diameter between about 500 nanometers and about 1.5 microns, and can have a thickness between about 50 and about 400 microns, in various embodiments. At least some of the micron, submicron, or nanometer dimension polymer fibers of the tubular wall 905 interpenetrate with at least some of the micron, submicron or nanometer dimension polymer fibers of the one or more leaflets 909. In some embodiments, the diameter of the first end 901 of the tubular wall 905 can correspond to a diameter of a pulmonary valve. The micron, submicron or nanometer dimension polymer fibers of the tubular wall 905 and the micron, submicron or nanometer dimension polymer fibers of the plurality of leaflets 909 can be configured to form a polymeric fiber scaffold for cellular ingrowth, in some embodiments.

[00112] FIG. 13A shows a top view of an example engineered valve 1301 of the invention formed using the mandrel assemblies and methods described herein. FIG. 13B is an enlarged view of an area of the example engineered valve of FIG. 13 A viewed with a scanning electron microscope (SEM). For comparison, FIG 12A shows a scanning electron micrograph of a hematoxylin and eosin stained ovine pulmonary valve leaflet showing the structural fibrosa layer of the native valve 1201, which is primarily composed of

circumferentially aligned collagen bundles that withstand closing diastolic pressures, and FIG. 12B is an enlarged view of an area of an ovine pulmonary valve leaflet which has been decellularized. As can be seen in the image of the engineered valve 1301 in FIG. 13 A and the SEM inset 1303, which has a scale bar of 25 microns, in FIG. 13B as compared with the images in FIG 12A and 12B, the engineered valves of the present disclosure recapitulate both the macroscopic and microscopic structural properties of a native valve. Methods for forming suitable polymeric fibers and valve scaffolds are discussed in detail below.

[00113] FIG. 14A shows mandrel components 1401 for forming engineered valve scaffolds and engineered valve scaffolds 1403 of varying sizes formed using the mandrel components, according to embodiments of the present disclosure. In exemplary embodiments, a valve scaffold size can be determined based on the size of the mandrel components used to collect the fibers. In the examples shown in FIG. 14A, the mandrel assemblies produced semilunar engineered valve scaffolds having diameters of 30 mm, 10 mm, 5 mm, and 3 mm. FIG. 14B shows an enlarged view of one of the engineered valve scaffolds of FIG. 14A, according to embodiments of the present disclosure. Inset 1405 shows an example engineered valve scaffold having a diameter of about 3 mm. As will be appreciated, the mandrel components and engineered valve scaffolds can be scaled to various sizes, depending on the patient or the desired use of the engineered valve scaffold, and the measurements provided here, as well as the measurements provided with respect to FIGS. 3B-6, are for illustrative purposes only.

[00114] The engineered polymeric fiber valves may include one or more embedded elements. The one or more embedded elements include, but are not limited to, metal mesh structures, support meshes, pressure transducers, strain transducers, time-released therapeutic agents (e.g., in capsules or pouches), flow sensors, actuators, optical tracers (e.g. in capsules or pouches, which could be activated or released for imaging). The one or more elements may be embedded in the resulting engineered polymeric fiber valves by collecting a first portion of polymeric fibers on the collection device, positioning at least one or some of the elements on the collection device over the first portion of the polymeric fibers, and then depositing a second portion of the polymeric fibers over the elements.

[00115] In some embodiments, the tubular wall 905 further comprises a stent. In one embodiment, the stent is embedded in the micron, submicron or nanometer dimension polymer fibers forming the tubular wall, e.g., including the bulging portions. In other embodiments, the valve is disposed in a conduit of a stent, e.g., including the bulging portions.

II. Mandrel Assemblies, Systems, and Methods for Generating Engineered Valve Structures

[00116] Methods for generating engineered valves of the invention may include configuring micron, submicron, or nanometer dimension polymeric fibers in a desired shape using a mandrel or mandrel assembly. Systems and methods for forming suitable micron, submicron or nanometer dimension polymeric fibers are described below.

[00117] FIG. 1 is an exploded view of a mandrel assembly 100, according to embodiments of the present disclosure. In this particular example, the mandrel assembly 100 includes first mandrel 101, also known as a lower leaflet mandrel, and components of a second mandrel structure 119. The second mandrel structure 119 includes three sinus- forming bodies 103, and a member 107. The member 107 includes a base portion 108 and three spacing portions 109, each extending parallel to an axis of rotation 102 from the base portion 108.

[00118] Each sinus-forming body 103 includes a first end portion 104a and a second end portion 104b. The first end portion 104a of each sinus-forming body is configured to be fastened to the member 107 at the base portion 108. The sinus-forming bodies 103 each include a protrusion 105 at the second end portion 104b of the sinus-forming body that bulges radially outward at least partially beyond the radius of the member 107. In this particular embodiment, each protrusion 105 extends an equal distance beyond the radius of the member 107. However, in alternative embodiments one or more of the protrusions 105 can extend radially outward beyond one or more of the other protrusions 105.

[00119] Each sinus-forming body 103 can be fastened or secured to the member 107 using respective fasteners 115, such as screws. The first mandrel 101 includes a hole, channel, or bore 117 extending at least partially through its center axis and configured to align with a perforation 113 in the member 107. The member 107 also includes holes 111 passing through a base portion 108 and configured to align with holes 113 in a first end portion of the sinus-forming bodies 103. Each fastener 115 is configured to extend through one of the holes 111 in the member 107 and at least partially extend within a corresponding hole 113 in one of the sinus-forming bodies 103. One of ordinary skill in the art will appreciate other mechanisms and other elements can be employed to fasten the sinus-forming bodies 103 and the member 107 together.

[00120] As shown in FIG. 2, when the sinus-forming bodies 103 are attached to the member 107, the three spacing portions 109 extending from the base portion 108 parallel to the axis of rotation 102 separate each sinus-forming body 103 from its adjacent sinus-forming body 103. Specifically, each sinus forming body 103 is configured to be fastened to the member 107 with the first end portion 104a of the sinus-forming body adjacent the base portion 108 of the member and with each sinus-forming body 103 separated from an adjacent sinus-forming body 103 by a spacing portion 109. Spacing portions 109 provide suitable spacing between each sinus-forming body 103 such that, once the valve scaffold is formed around the mandrel and dried, the sinus-forming bodies 103 can be detached from the member 107, which then can be withdrawn from the downstream end of tubular wall and removed from the valve scaffold to allow space within the valve scaffold for the sinus- forming bodies 103 to be removed without damaging the valve scaffold. In exemplary embodiments, the various mandrel components are machined from Teflon to allow for easy removal of components from either end of the scaffold or valve structure.

[00121] As shown in FIGs. 1 and 2 the first mandrel includes three leaflet fins 121 that are designed to align with the spacing portions 109 between the protrusions 105 of the sinus- forming bodies 103. Once the sinus-forming bodies 103 and upper member 107 are fastened together, they form a second mandrel structure 119 that may be positioned over and fastened to the first mandrel 101 using a fastener (not shown) that can extend through the hole 113 in the upper member 107 and at least partially extend within the hole 117 in the first mandrel 101.

[00122] When the member 107 and the sinus-forming bodies 103 are fastened together to form the second mandrel structure 119 as shown in FIG. 2, an outward facing surface of the member 107 (e.g. , outward facing surfaces of the base 108 and the spacing portions 109) and outward facing surfaces of the first end portions 104a of each of the sinus-forming bodies collectively form a tubular wall- forming region of an outer surface of the second mandrel structure 119. An outward facing surface of the second end portion 104b of each sinus- forming body bulges outward at protrusions 105 to define a shape of a sinus of the engineered valve. An inward facing surface of the second end portion 104b of each sinus-forming body bulges inward to define a shape of a downstream surface of a leaflet in the resulting engineered valve (see FIGS. 4A and 4B). As shown in FIG. 2, the second mandrel structure 119 is positioned over the first mandrel 101 such that each leaflet fin 121 aligns with a spacing portion 109 between two sinus-forming bodies 103.

[00123] FIG. 3A shows a perspective view and FIG. 3B shows a top view of an example first mandrel 101, according to embodiments of the present disclosure. As can be seen in this example, the first mandrel 101 has a first end portion 106a, a second end portion 106b, and an outer surface including concave leaflet-forming regions 122 at the second end portion 106b, each concave leaflet-forming region 122 located between leaflet fins 121. These concave leaflet-forming regions 122 are configured to align with and at least partially receive the second end portions 104b of the sinus-forming bodies when the sinus-forming bodies 103 are fastened to the member 107 and the first mandrel 101 and second mandrel structure 119 are combined to form the combined mandrel 120. In exemplary embodiments, the concave leaflet-forming regions 122 are configured to at least partially receive the second end portions of the sinus-forming bodies 103 when fibers have been spun on the first mandrel 101 and the second mandrel structure 119 has been positioned over the first mandrel 101. The concave leaflet- forming regions 122 are configured to define a shape of an upstream surface of a leaflet in the resulting engineered valve.

[00124] The first mandrel also includes an end surface 124. The end surface 124 can also form a surface which the spacing portions 109 of the second mandrel structure, disclosed above, may contact or abut.

[00125] In this particular example, each leaflet fin 121 has a thickness 127 of 1.0 mm and the hole 117 has a diameter of 5.0 mm. As will be appreciated, the measurements disclosed herein are for explanation purposes only and these measurements can be scaled and varied, as desired.

[00126] FIG. 4A is a perspective view and FIG. 4B is a bottom view of the example second mandrel structure 119, according to embodiments of the present disclosure. As can be seen in this example, the second mandrel structure 119 includes three protrusions 105 corresponding to the second end portions of each sinus-forming body 103 and extending beyond the radius of the upper portion of the second mandrel structure. The inward facing surfaces 126 of the end portions 104b of each of the sinus-forming bodies 103 of the second mandrel structure 119 are designed to align with the concave leaflet-forming regions 122 of the outer surface of the first mandrel 101, allowing sufficient space such that the inward facing surfaces 126 do not crush or apply unwanted pressure to the fibers that are spun onto the leaflet forming regions 122 of the first mandrel 101. An outward facing surface 125 of each of the second end portions 104b of each sinus-forming body 103 bulges outward to define a shape of a sinus of the resulting engineered valve.

[00127] As can be seen in this example, the protrusions 105 correspond to the second end portions of each sinus-forming body 103 and extend partially radially outward beyond the radius of the upper member, and each protrusion 105 is separated from its adjacent sinus by a separation length 123. An inward facing surface 126 of the second end portions 104b of each sinus-forming body bulges inward to define a shape of a downstream surface of a leaflet in the resulting engineered valve. In this particular embodiment, the separation length 123 is about 4.5 mm. However, in alternative embodiments, the separation length 123 may be greater or smaller, and may vary between each protrusion 105. [00128] FIG. 5 is a side view of the second mandrel structure 119, according to embodiments of the present disclosure. In this particular embodiment, the second mandrel structure height 217 is 34.5 mm, and the second mandrel structure diameter 215 is 30 mm. The second mandrel structure 119 also includes an upper lip 225 to assist in removal of the upper member 107 from the valve scaffold once dried. In this example embodiment, the upper lip 225 has a height of 2.5 mm. The second mandrel structure 119 can also include a sample cut line 223 that provides a guide for cutting the collected fibers to form the downstream end of the resulting valve. In this particular embodiment, the sample cut line 223 has a depth of 1.0 mm. The protrusions 105 of the second mandrel structure 119 shown in FIG. 5 extend or protrude beyond the radius of the upper portion of the second mandrel structure 119 a protrusion distance 221 of 4 mm. In other embodiments, the sinuses can protrude any distance between about 1 mm to about 1 cm beyond the radius of the upper portion of the second mandrel structure 119. As discussed above, in some embodiments each sinus of the second mandrel structure 119 can protrude the same distance, and in other embodiments one or more sinuses can be designed to protrude a different distance from the other sinuses. The second mandrel structure also includes a cylinder height 219 of 19.5 mm, in this particular example. The cylinder height 219 is the distance from the outward facing surface of the base 108 to the end surface of the spacing portions 109, discussed above in reference to FIG. 1.

[00129] FIG. 6 is a side view of a first mandrel 101, according to embodiments of the present disclosure. In this particular embodiment, the first mandrel 101 has a first mandrel diameter 201 of 30 mm, which forms a tubular wall- forming region 130 of the first mandrel 101. In some embodiments, the first mandrel diameter is to the same as the second mandrel diameter 215, which forms a tubular wall-forming region of the second mandrel structure. The first mandrel 101 has a first mandrel height 203 of 34.5 mm in some embodiments. In this example, because the end surface of the spacing portions 109 of the second mandrel structure 119 are configured to rest on the end surface 124 of the first mandrel 101, the total height of the combined mandrel is equal to the first mandrel height 203 plus the cylinder height 219. The first mandrel 101 has a leaflet height 205 of 14 mm, a coaptation height 207 of 4.48 mm, and a radius of a curvature filet 209 of 9.5 mm. The curvature filet 209 radius defines the geometry of the concave leaflet- forming regions 122. In exemplary

embodiments, the separation length 123 between each sinus, the shape of the sinuses, cylinder height 219, leaflet height 205, curvature filet radius 209, leaflet fin thickness, etc. are chosen such that when the second mandrel structure 119 is positioned over and fastened to a first mandrel 101 the inward facing surfaces 126 of the sinus-forming bodies do not crush or apply unwanted pressure to the fibers that are spun onto the leaflet forming regions 122 of the first mandrel 101. The first mandrel 101 also includes a lower lip 213 to assist in removal of the first mandrel from the valve scaffold once dried. In this example embodiment, the lower lip 213 has a height of 2.5 mm. The first mandrel 101 can also include a sample cut line 211 as a guide for cutting collected fibers to form an upstream end of the resulting valve. In this particular embodiment the sample cut line 211 has a depth of 1.0 mm.

[00130] FIG. 7A is an exploded view of a mandrel assembly 700 for forming an engineered valve having three leaflets and two sinuses, according to some embodiments of the present disclosure. In this particular example, the mandrel assembly 700 includes first mandrel 701, also known as a leaflet mandrel, and a second mandrel structure 719 including two sinus-forming bodies 703, a tubular wall- forming body 704, and a member 707. The first mandrel 701 and the member 707 can be substantially similar to the first mandrel 101 and member 107 described above in reference to FIG. 1. Similarly, the sinus-forming bodies 703 can be substantially similar to the sinus-forming bodies 103 described above in reference to FIG. 1. In this example embodiment, the sinus-forming bodies 703 can be configured to fasten to the member 707 and can each include a protrusion 705 that bulges radially outward at least partially beyond the radius of the member 707. In this particular embodiment, each protrusion 705 extends an equal distance beyond the radius of the member 707. In contrast, the tubular wall-forming body 704, which is also configured to fasten to the member 707, does not bulge or protrude radially outward beyond the radius of the member 707. The sinus- forming bodies 703 and the tubular wall-forming body 704 can be fastened or secured to the upper member 707 using respective fasteners, such as screws, as described above in reference to FIG. 1. The tubular wall- forming body 704 has a first end portion configured to be secured to the member 707 and a second end portion and includes an outward facing surface that forms part of a tubular wall-forming region of the second mandrel structure. The second end portion of the tubular wall-forming body 704 has an inward facing surface that bulges inward to define a shape of a downstream surface of a leaflet in the resulting valve. In exemplary embodiments, the various mandrel components are machined from Teflon to allow for easy removal of components from either end of the scaffold or valve structure. Once the sinus-forming bodies 703, the tubular wall-forming body 704, and the upper member 707 are fastened together, they form a second mandrel structure 719 that may be positioned over and fastened to the first mandrel 701.

[00131] FIG. 7B shows a perspective view of an example combined mandrel 720 including the first mandrel 701 and the second mandrel structure 719, according to embodiments of the present disclosure. The second mandrel structure 719 includes the member 707, the sinus-forming bodies 703, and the tubular wall-forming body 704, fastened together as described in FIG. 7A. When fastened together, an outward facing surface of the upper member 707 and outward facing surfaces of the first end portions of each of the sinus- forming bodies 703 and the tubular wall- forming body 704 collectively form a tubular wall- forming region of an outer surface of the second mandrel structure 719. An outward facing surface of the second end portion of each sinus-forming body 703 bulges outward to define a shape of a sinus of the engineered valve. However, an outward facing surface of the second end of the tubular wall-forming body 704 does not bulge out significantly beyond the radius of the upper member 707. An inward facing surface of the second end portion of each sinus- forming body 703 and the tubular wall-forming body 704 bulges inward to define a shape of a downstream surface of a leaflet in the resulting engineered valve.

[00132] FIGS. 8A-8F collectively show various views of the combined mandrel 720 including a first mandrel 701 and a second mandrel structure 719, according to embodiments of the present disclosure. As described above, the second mandrel structure 719 includes the member 707, the sinus-forming bodies 703, and the tubular wall-forming body 704, fastened together as described in FIGS. 7A-7B. The second mandrel structure 719 is placed over the first mandrel 701 as also described above in reference to FIGS. 7A-7B.

[00133] FIG. 8A shows a top view of the example combined mandrel 720 showing two protrusions 705 bulging radially outward beyond the radius of the upper member, according to embodiments of the present disclosure. FIG. 8B shows a bottom view of the example combined mandrel 720 showing two protrusions 705 bulging radially outward beyond the radius of the first mandrel 701, according to embodiments of the present disclosure. FIGS. 8C-8F show various side views of the example combined mandrel showing two protrusions, according to embodiments of the present disclosure. As can be seen in these embodiments, the two protrusions 705 of the sinus-forming bodies bulge radially outward beyond the radius of the first mandrel 701. However, the tubular wall- forming body 704 does not bulge radially outward beyond the radius of the first mandrel 701. [00134] FIG. 11 schematically depicts a system 1100 for forming a valve scaffold including micron, submicron or nanometer dimension polymeric fibers, in accordance with some embodiments. System 1100 includes a rotating reservoir 1101 that rotates about an axis 1102, which is referred to as the deposition rotation axis. In this specific example, the reservoir is rotated using a rotary jet spinning (RJS) motor or extrusion motor 1103. In FIG. 11, the axis of rotation 1102 of the rotating reservoir is parallel to the vertical z-axis. One of ordinary skill in the art, in view of the present disclosure, would recognize that another convention may be chosen for describing an orientation of the deposition rotation axis, and that the deposition rotation axis need not be aligned with a vertical axis. In exemplary embodiments, a polymer solution 1105 is introduced into the reservoir 1101, and the reservoir 1101 has orifices through which a polymer is ejected, forming polymeric fibers 1106 that are extruded along a fiber extrusion plane 1107. In this example, a collection device 1109 (e.g. , the mandrels discussed above in reference to FIGS. 1-8F) can be inserted into the fiber extrusion plane 1107. In some embodiments, the collection device is rotated about an axis 1104, which is referred to as the collection rotation axis. In this particular embodiment, the collection device is rotated via a mandrel motor 1111. When the collection mandrel 1109 is in the path of the polymeric fibers 1106 ejected from the rotating reservoir 1101, the polymeric fibers 1106 are wrapped around the collection mandrel 1109 via rotation of the mandrel about the collection rotation axis. In one embodiment, the collection device can be translated vertically so that the entire outer surface of the collection mandrel 1109 passes through the extrusion plane 1107. In this particular example, a linear actuator or motor 1113 allows the collection mandrel 1109 to translate vertically, or parallel to, the deposition rotation axis. In exemplary embodiments, the first mandrel 101 is configured to be rotated around the collection rotation axis to receive a coating of micron, submicron, or nanometer dimension polymer fibers 1106, thereby forming an upstream surface of each leaflet in the resulting engineered valve and an inner surface of an upstream tubular wall of the resulting engineered valve. The first mandrel 101 and the second mandrel structure 119 can be fastened together to form a combined mandrel 120, and the combined mandrel 120 can be rotated about the collection rotation axis 1104 to receive a coating of micron, submicron, or nanometer dimension polymer fibers, thereby forming sinuses and a downstream tubular wall of the resulting engineered valve as discussed in further detail below in connection with exemplary methods. [00135] Control over the rate of translation of the collection device along the collection rotation axis 1104 (or parallel to the deposition rotation axis 1102) and orientation of the collection rotation axis 1104 relative to the deposition rotation axis 1102 provides control over an orientation of fibers deposited on the collection device (e.g., the collection mandrel 1109, which may be the first mandrel or the combined mandrel), as depicted in FIG. 11. The rotation of the collection mandrel 1109 may be opposite the rotation of the reservoir, or the rotation of the collection mandrel may be the same direction as the rotation of the reservoir 1101, according to various embodiments. FIG. 11 schematically illustrates collection of fibers with the axis of rotation 1104 of the collection mandrel 1109 oriented at an angle with respect to the rotation axis 1102 of the reservoir 1101. In some exemplary embodiments, the collection mandrel is oriented at an angle of 45 degrees with respect to the axis of rotation of the reservoir 1102. The mandrel may be moved manually or automatically in various embodiments. In alternative embodiments, increasing the speed of translation and/or rotating the mandrel at a nonzero angle with respect to the deposition rotation axis 1102 can produce cross or "x" shaped weaves in collected polymeric fibers.

[00136] In some exemplary embodiments, fibers are spun onto a first mandrel to form the three leaflets of a semilunar valve. Once fibers are spun on the first mandrel, excess fibers may be removed from the first mandrel and a second mandrel structure is assembled, which includes three sinus-forming bodies, each including a sinus that extends radially outward beyond the radius of the first mandrel. These sinuses form the mold over which additional fibers will be spun to form the sinuses of a valve scaffold. In one embodiment, the second mandrel structure is assembled by fastening the three sinus-forming bodies to an upper cylinder component (e.g., the member 107). In one such example, the upper cylinder component (e.g., the member 107) has a radius equal to the radius of the first mandrel. Once the second mandrel structure is assembled, it can be placed over the fiber- wrapped first mandrel. The second mandrel structure and first mandrel are then wrapped in additional fibers to create the exterior conduit in which the leaflets are housed. Once dried, the first mandrel may be removed by sliding it out of the bottom of the conduit or valve scaffold. The sinus-forming bodies may be detached from the upper cylinder component (e.g., the member 107) so that the upper cylinder component (e.g., the member 107) can be removed from the top of the conduit. Once the upper cylinder is removed from the conduit or valve scaffold, sufficient space is available within the conduit to allow for easy removal of each sinus- forming body. [00137] FIG. 27 illustrates an example method for fabricating a valve structure from micron, submicron, or nanometer dimension polymeric fibers, according to an embodiment of the present disclosure. Solely for illustrative purposes, the example method is described below with respect to reference numbers used for mandrel assembly 100 shown in FIGs. 1-6 and with respect to valve 900 shown in FIGs. 9-10.

[00138] In step 2701, micron, submicron or nanometer dimension polymer fibers are formed by ejecting a polymer from a rotating reservoir or by flinging a polymer from a deposit. In exemplary embodiments, rotary jet spinning (RJS) may be used to create the micron, submicron, or nanometer dimension polymeric fibers collected on the first mandrel or on the combined mandrel. Suitable RJS devices and uses of the devices for fabricating the micron, submicron, or nanometer dimension polymeric fibers are described in U.S. Patent Publication No. 2012/0135448, U.S. Patent Publication No. 2013/0312638, U.S. Patent Publication No. 2014/0322515, the entire contents of each of which are incorporated in their entirety by reference. In some exemplary embodiments, the polymeric fibers may be flung using a pull spinning technique.

[00139] In step 2702, a first portion of micron, submicron, or nanometer dimension polymer fibers is collected on a rotating first mandrel 101. The first mandrel 101 has a first end portion 106a, a second end portion 106b, and an outer surface with a tubular wall- forming region at the first end portion 106a and at least three concave lealet-forming regions 122 at the second end portion 106b. The tubular-wall forming region 130 of the outer surface has a shape corresponding to an inner surface 907 of the first portion of the tubular structure 905 of the resulting engineered valve 900. Each of the at least three concave leaflet-forming regions 122 has a shape corresponding to an upstream surface of a corresponding leaflet 909 in the resulting valve 900.

[00140] In some embodiments, the first portion of fibers are collected on the first mandrel 101 while the leaflet fins 121 and leaflet-forming regions 122 are oriented downward, such that a larger proportion of fibers are collected on the leaflet-forming regions 122 than on a base portion of the first mandrel 101. This technique can provide increased uniformity in fiber thickness of the valve scaffold because additional fibers will be spun over the base portion of the first mandrel 101 once the second mandrel structure 119 is positioned over the first portion of collected polymeric fibers on first mandrel. The first mandrel 121 can be translated vertically or parallel to the deposition rotation axis 1102 during collection of the first portion of the fibers in order to cover the mandrel with fibers. The first portion of polymer fibers may be collected on the first mandrel for about six minutes, in some examples.

[00141] In some embodiments, the method includes step 2703, in which excess fibers are removed from the first mandrel 101. During collection of the first portion of the polymer fibers, fibers may be collected over the end surface 124 of the first mandrel as well as over the over the leaflet fins 121 and the concave leaflet-forming regions 122 of the first mandrel. Excess fibers extending over the end surface 124 of the first mandrel 101 should be cut away, or otherwise removed, in order to ensure proper mobility of the leaflets in the resulting valve scaffold. Fibers collected on the tubular wall- forming region 130 of the outer surface of the first mandrel 101 form an inner surface of a first portion of the resulting tubular engineered valve. Fibers collected on each concave leaflet-forming region 122 form the upstream surface of a corresponding leaflet 909 in the resulting valve scaffold 900.

[00142] In some embodiments, a second mandrel structure 119 may be pre-assembled,

In other embodiments, in step 2705, the second mandrel structure 119 is assembled, as described above with respect to FIGs. 1 and 2, by fastening the sinus-forming bodies 103 to the member 107.

[00143] In step 2707, the assembled second mandrel structure 119 is coupled to the first mandrel 101 on which the first portion of polymer fibers has been collected to form a combined mandrel 120 including both the second mandrel structure 119 and the first mandrel 101. A portion of the leaflets is covered by a portion of the second mandrel structure 119 after coupling the first mandrel 101 and the second mandrel structure 119 to form the combined mandrel 120.

[00144] The second mandrel structure 119 has a member 107 including a base portion

108 and at least three spacing portions 109 extending parallel to the axis of rotation 102 from the base portion 108 and at least three sinus-forming bodies 103. Each sinus-forming body 103 has a first end portion 104a and a second end portion 104b and is fastened to the member 107 and separated from an adjacent sinus-forming body 103 by one of the at least three spacing portions 109. An outward facing surface of the member 108 and outward facing surfaces of the first end portions 104a of each of the sinus-forming bodies collectively form a tubular wall- forming region of an outer surface of the second mandrel structure 119. An outward facing surface 125 of the second end portion 104b of each sinus-forming body bulges outward to define a shape of a sinus 913 of the resulting valve scaffold 900, and an inward facing surface 125 of the first end portion 104a of each sinus-forming body bulges inward to define a shape of a downstream surface of a leaflet 909 in the resulting valve scaffold.

[00145] In exemplary embodiments, the spacing between each of the concave leaflet- forming regions 122 of the outer surface of the first mandrel 101 and the corresponding inward facing surfaces 126 of the second end portion 104b of each sinus-forming body 103 in the second mandrel structure 119 of the combined mandrel 120 is set to form a leaflet 909 in the resulting valve scaffold having a thickness between about 50-300 microns.

[00146] In step 2709, a second portion of micron, submicron, or nanometer dimension polymer fibers is collected on the combined mandrel 120 by rotating the combined mandrel

120 about a rotation axis 1104 in the path of the ejected micron, submicron, or nanometer dimension polymer fibers to form a reminder of the resulting valve scaffold 900 having a first end 901, a second end 903, at least three leaflets 909 and at least three sinuses 913. In some exemplary embodiments, once the second mandrel structure 119 is coupled to the fiber-spun first mandrel 101, forming a combined mandrel 120, the combined mandrel 120 can be oriented such that the sinus forming bodies 103 are extending downward and the leaflet fins

121 are extending upward for further spinning and fiber collection. In one embodiment, the combined mandrel is angled at 45 degrees and oriented such that the sinus-forming bodies 103 extend downward during collection of the second portion of the polymer fibers. As with the first mandrel, the combined mandrel can be translated vertically, or along the axis of rotation of the reservoir, in order to cover the entire combined mandrel with fibers. As noted above, in some embodiments, increasing the speed of translation and/or rotating the mandrel at a nonzero angle with respect to the deposition rotation axis 1102 can be used to produce cross or "x" shaped weaves in collected polymeric fibers. According to exemplary embodiments, proper alignment of the particles resulting from the fiber spinning techniques described herein can provide the biaxial properties of a native valve.

[00147] Once the first and second portions of polymer fibers have been spun on the combined mandrel and the engineered valve scaffold is dried, the second mandrel structure 119 is uncoupled or unfastened from from the first mandrel 101 in step 2711 in order to remove the mandrel components. In step 2713, the first mandrel 101 is withdrawn from a first end portion of the resulting valve scaffold. In some embodiments, a lower lip protruding radially outward from the first mandrel assists in removing the first mandrel from the valve scaffold. In some embodiments, the polymer fibers are cut along the sample cut line 211 prior to withdrawing the first mandrel 101.

[00148] In step 2715, the sinus-forming bodies 103 are unfastened from the member

107 so that the member 107 can be withdrawn from a second end portion of the valve scaffold separately from the sinus-forming bodies 103. In some embodiments, the polymer fibers are cut along with sample cut line 223 prior to unfastening the sinus-forming bodies 103 from the member 107.

[00149] In step 2717, the member 107 is withdrawn from the second end portion of valve scaffold, leaving sufficient space within the valve scaffold such that the sinus-forming bodies 103 may be removed one at a time without damaging the valve scaffold. In some embodiments, the member 107 includes an upper lip protruding radially outward to assist in removal of the member 107. In step 2719, the sinus-forming bodies 103 are withdrawn from the dried valve scaffold.

[00150] Exemplary polymers for use in the present invention may be biocompatible or non-biocompatible, synthetic or natural, or combinations thereof.

[00151] Exemplary synthetic polymers include, for example, poly(urethanes), poly(siloxanes) or silicones, poly( ethylene), poly( vinyl pyrrolidone), poly(2-hydroxy ethyl methacrylate), poly(N- vinyl pyrrolidone), poly(methyl methacrylate), poly( vinyl alcohol), poly(acrylic acid), polyacrylamide, poly(ethylene-co-vinyl acetate), poly(ethylene glycol), poly(methacrylic acid), polylactides (PLA), polyglycolides (PGA), poly(lactide-co- glycolides) (PLGA), polyanhydrides, polyphosphazenes, polygermanes, polyorthoesters, polyesters, polyamides, polyolefins, polycarbonates, polyaramides, polyimides,

polycaprolactone (PCL), and copolymers and derivatives thereof, and combinations thereof.

[00152] Exemplary polymers for use in the methods of the invention may also be naturally occurring polymers e.g., biogenic polymers (or bio-derived polymers), e.g., proteins, polysaccharides, lipids, nucleic acids or combinations thereof. Exemplary biogenic polymers, e.g. , polymers made in a living organism, e.g. , fibrous proteins, for use in the devices and methods of exemplary embodiments include, but are not limited to, silk (e.g. , fibroin, sericin, etc.), keratins (e.g. , alpha-keratin which is the main protein component of hair, horns and nails, beta-keratin which is the main protein component of scales and claws, etc.), elastins (e.g. , tropoelastin, etc.), fibrillin (e.g. , fibrillin- 1 which is the main component of microfibrils, fibrillin-2 which is a component in elastogenesis, fibrillin-3 which is found in the brain, fibrillin-4 which is a component in elastogenesis, etc.), fibrinogen/fibrins/thrombin (e.g., fibrinogen which is converted to fibrin by thrombin during wound healing), fibronectin, laminin, collagens (e.g., collagen I which is found in skin, tendons and bones, collagen II which is found in cartilage, collagen III which is found in connective tissue, collagen IV which is found in extracellular matrix protein, collagen V which is found in hair, etc.), vimentin, neurofilaments (e.g., light chain neurofilaments NF-L, medium chain

neurofilaments NF-M, heavy chain neurofilaments NF-H, etc.), amyloids (e.g. , alpha- amyloid, beta-amyloid, etc.), actin, myosins (e.g. , myosin I-XVII, etc.), titin which is the largest known protein (also known as connectin), gelatin, chitin which is a major component of arthropod exoskeletons, hyaluronic acid which is found in extracellular space and cartilage (e.g., D-glucuronic acid which is a component of hyaluronic acid, D-N-acetylglucosamine which is a component of hyaluronic acid, etc.), etc, and combinations thereof.

[00153] Exemplary biogenic polymers, e.g., glycosaminoglycans (GAGs)

(carbohydrate polymers found in the body), for use in the present invention include, but are not limited to, heparan sulfate founding extracelluar matrix, chondroitin sulfate which contributes to tendon and ligament strength, keratin sulfate which is found in extracellular matrix, etc.

[00154] In one embodiment, the polymers may be mixtures of two or more polymers and/or two or more copolymers. In one embodiment, the polymers may be a mixture of one or more polymers and/or copolymers. In another embodiment, the polymers for use in the devices and methods of the invention may be a mixture of one or more synthetic polymers and one or more naturally occurring polymers. In one embodiment, the polymers for use on the present invention may be a mixture of a biogenic polyester and a protein. For example, in one embodiment, the polymers are a mixture of collagen and polycarpolactone. In another embodiment, the polymers are a mixture of poly-4-hydroxybutyrate (P4HB), polyglycolic acid (PGA), and gelatin/collagen. In yet another embodiment, the polymers are a mixture of polycapro lactone and gelatin, e.g. , uncrosslinked gelatin. In still other embodiments, the polymers are a mixture of poly-4-hydroxybutyrate (P4HB) and gelatin. In one embodiment, the polymers for use on the present invention may be a 60:40 mixture of a biogenic polyester and a protein. In alternative embodiments, the ratio of biogenic polyester and protein can be 100:0, 90: 10, 80:20, 70:30, 40:60, 20:80, or 0: 100.

[00155] Exemplary materials may also include other suitable materials, e.g. , metallic or ceramic materials. [00156] In some embodiments, suitable devices for fabricating the micron, submicron or nanometer dimension polymeric fibers configured in a desired shape as described herein generally include a reservoir for holding a polymer, the reservoir including one or more orifices for ejecting the polymer during fiber formation, thereby forming a micron, submicron or nanometer dimension polymeric fiber and a collection device (e.g., a mandrel or mandrel assembly) for accepting the formed micron, submicron or nanometer dimension polymeric fiber, wherein at least one of the reservoir and the collection device employs rotational motion during fiber formation, and the device is free of an electrical field, e.g. , a high voltage electrical field.

[00157] The device may include a rotary motion generator for imparting a rotational motion to the reservoir and, in some exemplary embodiments, to the collection device, e.g. , mandrel, e.g. , mandrel assembly. In some embodiments, a flexible air foil is attached to a shaft of the motor above the reservoir to facilitate fiber collection and solvent evaporation.

[00158] Rotational speeds of the reservoir and/or the mandrel in exemplary

embodiments may range from about 1,000 rpm to about 50,000 rpm, about 1,000 rpm to about 40,000 rpm, about 1,000 rpm to about 20,000 rpm, about 1,000 rpm to about 15,000 rpm, about 1,000 rpm to about 12,500 rpm, about 1,000 rpm to about 10,000 rpm, about 1,000 rpm to about 7,500 rpm, about 1,000 rpm to about 5,000 rpm, about 1,000 rpm to about 2,500 rpm, about 5,000 rpm- 20,000 rpm, about 5,000 rpm to about 15,000 rpm, e.g. , about 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 20,500, 21,000, 21,500, 22,000, 22,500, 23,000, 23,500, 24,000, 25,500, 26,000, 26,500, 27,000, 27,500, 28,000, 28,500, 29,000, 29,500, 30,000, 30,500, 31,000, 31,500, 32,000, 32,500, 33,000, 33,500, 34,000, 35,500, 36,000, 36,500, 37,000, 37,500, 38,000, 38,500, 39,000, 39,500, 40,000, 40,500, 41,000, 41,500, 42,000, 42,500, 43,000, 43,500, 44,000, 45,500, 46,000, 46,500, 47,000, 47,500, 48,000, 48,500, 49,000, 49,500, or about 50,000 rpm. Ranges and values intermediate to the above recited ranges and values are also contemplated to be part of the invention.

[00159] In certain embodiments, for example, when sufficient shear forces are required to induce protein unfolding and facilitate, e.g., in vitro fibrillogenesis to fabricate insoluble polymer fibers comprising a biogenic polymer (e.g., fibronectin, collagen, etc.), rotational speeds of the rotating reservoir may be about 50,000 rpm-400,000 rpm are intended to be encompassed by the invention. In one embodiment, devices employing rotational motion may be rotated at a speed greater than about 50, 000 rpm, greater than about 55,000 rpm, greater than about 60,000 rpm, greater than about 65,000 rpm, greater than about 70,000 rpm, greater than about 75,000 rpm, greater than about 80,000 rpm, greater than about 85,000 rpm, greater than about 90,000 rpm, greater than about 95,000 rpm, greater than about 100,000 rpm, greater than about 105,000 rpm, greater than about 110,000 rpm, greater than about 115,000 rpm, greater than about 120,000 rpm, greater than about 125,000 rpm, greater than about 130,000 rpm, greater than about 135,000 rpm, greater than about 140,000 rpm, greater than about 145,000 rpm, greater than about 150,000 rpm, greater than about 160,000 rpm, greater than about 165,000 rpm, greater than about 170,000 rpm, greater than about 175,000 rpm, greater than about 180,000 rpm, greater than about 185,000 rpm, greater than about 190,000 rpm, greater than about 195,000 rpm, greater than about 200,000 rpm, greater than about 250,000 rpm, greater than about 300,000 rpm, greater than about 350,000 rpm, or greater than about 400,000 rpm. Ranges and values intermediate to the above recited ranges and values are also contemplated to be part of the invention.

[00160] Exemplary devices employing rotational motion may be rotated for a time sufficient to form a desired polymeric fiber, such as, for example, about 1 minute to about 100 minutes, about 1 minute to about 60 minutes, about 10 minutes to about 60 minutes, about 30 minutes to about 60 minutes, about 1 minute to about 30 minutes, about 20 minutes to about 50 minutes, about 5 minutes to about 20 minutes, about 5 minutes to about 30 minutes, or about 15 minutes to about 30 minutes, about 5-100 minutes, about 10-100 minutes, about 20-100 minutes, about 30-100 minutes, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 minutes, or more. Times and ranges intermediate to the above-recited values are also intended to be part of this invention.

[00161] An exemplary reservoir may have a volume ranging from about one nanoliter to about 1 milliliter, about one nanoliter to about 5 milliliters, about 1 nanoliter to about 100 milliliters, or about one microliter to about 100 milliliters, for holding the liquid material. Some exemplary volumes include, but are not limited to, about one nanoliter o about 1 milliliter, about one nanoliter to about 5 milliliters, about 1 nanoliter to about 100 milliliters, one microliter to about 100 microliters, about 1 milliliter to about 20 milliliters, about 20 milliliters to about 40 milliliters, about 40 milliliters to about 60 milliliters, about 60 milliliters to about 80 milliliters, about 80 milliliters to about 100 milliliters, but are not limited to these exemplary ranges. Exemplary volumes intermediate to the recited volumes are also part of the invention. In certain embodiment, the volume of the reservoir is less than about 5, less than about 4, less than about 3, less than about 2, or less than about 1 milliliter. In other embodiments, the physical size of an unfolded polymer and the desired number of polymers that will form a fiber dictate the smallest volume of the reservoir.

[00162] In one embodiment, a polymer is fed into a reservoir as a polymer solution, i.e., a polymer dissolved in an appropriate solution. In this embodiment, the methods may further comprise dissolving the polymer in a solvent prior to feeding the polymer into the reservoir. In other embodiments, a polymer is fed into the reservoir as a polymer melt. In such embodiment, the reservoir is heated at a temperature suitable for melting the polymer, e.g., is heated at a temperature of about 100°C to about 300°C, 100-200°C, about 150-300°C, about 150-250°C, or about 150-200°C, or about 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, or about 300°C.

[00163] The reservoir may include one or more orifices through which one or more jets of the material solution (e.g., polymer solution) are forced to exit the reservoir by the motion of the reservoir during fiber formation. One or more exemplary orifices may be provided on any suitable side or surface of the reservoir including, but not limited to, a bottom surface of the reservoir that faces the collection device, a side surface of the reservoir, a top surface of the reservoir that faces in the opposite direction to the collection device, etc. Exemplary orifices may have any suitable cross-sectional geometry including, but not limited to, circular, oval, square, rectangular, etc. In an exemplary embodiment, one or more nozzles may be provided associated with an exemplary orifice to provide control over one or more characteristics of the material solution exiting the reservoir through the orifice including, but not limited to, the flow rate, speed, direction, mass, shape and/or pressure of the material solution. The locations, cross-sectional geometries and arrangements of the orifices on the reservoir, and/or the locations, cross-sectional geometries and arrangements of the nozzles on the orifices, may be configured based on the desired characteristics of the resulting fibers and/or based on one or more other factors including, but not limited to, viscosity of the material solution, the rate of solvent evaporation during fiber formation, etc. [00164] Exemplary orifice lengths that may be used in some exemplary embodiments range between about 0.001 m and about 0.1 m, e.g., 0.0015, 0.002, 0.0025, 0.003, 0.0035, 0.004, 0.0045, 0.005, 0.0055, 0.006, 0.0065, 0.007, 0.0075, 0.008, 0.0085, 0.009, 0.0095, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, or 0.1 m. Ranges and values intermediate to the above recited ranges and values are also contemplated to be part of the invention.

[00165] Exemplary orifice diameters that may be used in some exemplary

embodiments range between about 0.1 μιη and about 10 μιη, about 50 μιη to about 500 μιη, about 200 μιη to about 600 μιη, about 200 μιη to about 1,000 μιη, about 500 μιη to about 1,000 μιη, about 200 μιη to about 1,500 μιη, about 200 μιη to about 2,000 μιη, about 500 μιη to about 1,500 um, or about 500 μιη to about 2,000 um, e.g. , about 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,050, 1, 100, 1, 150, 1,200, 1,250, 1,300, 1,350, 1,400, 1,450, 1,500, 1,550, 1,600, 1,650, 1,700, 1,750, 1,800, 1,850, 1,900, 1,950, or about 2,000 um. Ranges and values intermediate to the above recited ranges and values are also contemplated to be part of the invention.

[00166] In other embodiments, a suitable device for the formation of a micron, submicron or nanometer dimension polymeric fibers includes a reservoir for holding a polymer, the reservoir including one or more orifices for ejecting the polymer during fiber formation, thereby forming micron, submicron or nanometer dimension polymeric fibers, a collection device, and an air vessel for circulating a vortex of air around the formed fibers to wind the fibers into one or more threads. In one embodiment, a suitable device further comprises a component suitable for continuously feeding the polymer into the rotating reservoir, such as a spout or syringe pump.

[00167] An exemplary method to fabricate the micron, submicron or nanometer dimension polymeric fibers may include imparting rotational motion to a reservoir holding a polymer, the rotational motion causing the polymer to be ejected from one or more orifices in the reservoir to form a micron, submicron or nanometer dimension polymeric fiber, and collecting the formed fibers on a mandrel assembly, as described herein, to form the micron, submicron or nanometer dimension polymeric fibers in the desired shape.

[00168] In one embodiment, a mandrel assembly (or portion of a mandrel assembly) is positioned in the path of the fibers ejected from the reservoir from the rotating structure and rotated, angled and/or vertically maneuvered such that the fibers are accepted on the mandrel at a desired thickness and pattern. In one embodiment, the maneuvering of the mandrel is automated.

[00169] In other example embodiments, devices and uses of the devices for generating micron, submicron, or nanometer dimension polymeric fibers and engineered valve scaffolds disclosed herein do not employ or require a nozzle for ejecting the liquid material, a spinneret or rotating reservoir containing and ejecting the liquid material, or an electrostatic voltage potential for forming the fiber. Such exemplary devices and uses are described in, for example, U.S. Provisional Patent Application No. 61/561,185, filed on November 17, 2011, U.S. Patent Publication No. 2014/0322515, and PCT Publication No. WO 2013/115896, the entire contents of each of which are hereby incorporated herein by reference. In one such example, suitable devices for fabricating the micron, submicron or nanometer dimension polymeric fibers include a platform for supporting a stationary deposit of a polymer, a rotating structure disposed vertically above the platform and spaced from the platform along a vertical axis, and a combined mandrel assembly (or portion thereof), as disclosed herein. The rotating structure includes a central core rotatable about a rotational axis, and one or more blades affixed to the rotating core. The rotating structure is configured and operable so that, upon rotation, the one or more blades or bristles contact a surface of the polymer to impart sufficient force in order to decouple a portion of the polymer from contact with the one or more blades of the rotating structure and to fling the portion of the polymer away from the contact with the one or more blades and from the deposit of the polymer, thereby forming a micron, submicron and/or nanometer dimension polymeric fiber. Unlike in the RJS system, the polymer reservoir in pull spinning systems is static, and the extruded polymer jet is predominately moving unidirectionally and not circumferentially towards the collector. We reason that in spite of these differences, jet extension during pull spinning is governed by a balance of viscous and centrifugal forces, similar to the RJS. In one example embodiment, a pull spinning bristle can be 0.531 cm long, 0.1 cm in diameter, and can be fixed

perpendicularly onto a bristle holder or rotating disk that is 1.754 cm in diameter. Such exemplary devices may be compact (14.0cm x 2.0cm x 2.0cm), and generally include a highspeed rotating bristle that dips into a polymer reservoir and pulls a droplet from solution into a network of insoluble fibers. Unlike other nanofiber fabrication systems, such as

electro spinning or melt spinning, pull spinning can involve a manufacturing technique that operates under ambient conditions and functions independently of system parameters such as speed, collector distance, electric potential, or temperature. In some examples, pull spinning can produce uniform, substantially defect-free fibers with diameters of between 250 to 2,500 nm, depending on solution concentration. Collected nanofibers may be collected on a rotating collection mandrel or cylindrical structure.

III. Methods for Generating Engineered Tissues

[00170] In certain embodiments, the engineered valves may comprise an engineered tissue which is fabricated by seeding cells onto the polymeric fibers of a valve , and culturing the cells to form a functional tissue. For example, in some embodiments, an engineered valve fabricated as described herein is seeded with cells which are cultured under suitable conditions to form a functional tissue prior to implantation into a subject as a replacement valve.

[00171] In other embodiments, the engineered valves fabricated as described herein are not seeded with cells that are cultured to form a functional tissue prior to implantation into a subject as a replacement valve, thereby significantly simplifying the fabrication process of a replacement valve and significantly reducing the costs for fabrication of a replacement valve. In such embodiments, native cells migrate into the engineered valve when implanted in a subject, grow, and form a functional tissue.

[00172] Accordingly, in some embodiments of the methods of the invention, a valve, as described above, is seeded with cells and cultured in an incubator under physiologic conditions {e.g., at 37°C) until the cells form an engineered tissue.

[00173] Any appropriate cell culture method may be used. The seeding density of the cells will vary depending on the cell size and cell type, but can easily be determined by methods known in the art. In one embodiment, cells are seeded at a density of between about 1 x 10⁵ to about 6 x 10⁵ cells/cm², or at a density of about 1 X 10⁴, about 2 X 10⁴, about 3 X 10⁴, about 4 X 10⁴, about 5 X 10⁴, about 6 X 10⁴, about 7 X 10⁴, about 8 X 10⁴, about 9 X 10⁴, about 1 X 10⁵, about 1.5 X 10⁵, about 2 X 10⁵, about 2.5 X 10⁵, about 3 X 10⁵, about 3.5 X 10⁵, about 4 X 10⁵, about 4.5 X 10⁵, about 5 X 10⁵, about 5.5 X 10⁵, about 6 X 10⁵, about 6.5 X 10⁵, about 7 X 10⁵, about 7.5 X 10⁵, about 8 X 10⁵, about 8.5 X 10⁵, about 9 X

10⁵, about 9.5 X 10⁵, about 1 X 10⁶, about 1.5 X 10⁶, about 2 X 10⁶, about 2.5 X 10⁶, about 3 X 10⁶, about 3.5 X 10⁶, about 4 X 10⁶, about 4.5 X 10⁶, about 5 X 10⁶, about 5.5 X 10⁶, about 6 X 10⁶, about 6.5 X 10⁶, about 7 X 10⁶, about 7.5 X 10⁶, about 8 X 10⁶, about 8.5 X

10⁶, about 9 X 10⁶, or about 9.5 X 10⁶. Values and ranges intermediate to the above-recited values and ranges are also contemplated by the present invention. [00174] In some embodiments, a valve is contacted with living cells during the fabrication process such that a structure populated with cells or fibers surrounded (partially or totally) with cells are produced. The valve may also be contacted with additional agents, such as proteins, nucleotides, lipids, drugs, pharmaceutically active agents, biocidal and antimicrobial agents during the fabrication process such that functional micron, submicron or nanometer dimension polymeric fibers are produced which contain these agents. For example, fibers comprising living cells may be fabricated by providing a polymer and living cells in a solution of cell media at a concentration that maintains cell viability.

[00175] Suitable cells for use in the invention may be normal cells, abnormal cells

(e.g., those derived from a diseased tissue, or those that are physically or genetically altered to achieve an abnormal or pathological phenotype or function), normal or diseased muscle cells, stem cells (e.g., embryonic stem cells), or induced pluripotent stem cells. Suitable cells include umbilical endothelial cells, vascular endothelial cells, mesenchymal stem cells, primary valve harvest endothelial/interstitial cells, and cardiomycocytes. Such cells may be seeded on the scaffold including leaflets and cultured to form a functional tissue, such as a functional valve tissue.

[00176] Cells for seeding can be cultured in vitro, derived from a natural source, genetically engineered, or produced by any other means. Any natural source of prokaryotic or eukaryotic cells may be used. Embodiments in which a valve is implanted in an organism can use cells from the recipient, cells from a conspecific donor or a donor from a different species, or bacteria or microbial cells.

[00177] The term "progenitor cell" is used herein to refer to cells that have a cellular phenotype that is more primitive (e.g., is at an earlier step along a developmental pathway or progression than is a fully differentiated cell) relative to a cell which it can give rise to by differentiation. Often, progenitor cells also have significant or very high proliferative potential. Progenitor cells can give rise to multiple distinct differentiated cell types or to a single differentiated cell type, depending on the developmental pathway and on the environment in which the cells develop and differentiate.

[00178] The term "progenitor cell" is used herein synonymously with "stem cell."

[00179] The term "stem cell" as used herein, refers to an undifferentiated cell which is capable of proliferation and giving rise to more progenitor cells having the ability to generate a large number of mother cells that can in turn give rise to differentiated, or differentiable daughter cells. The daughter cells themselves can be induced to proliferate and produce progeny that subsequently differentiate into one or more mature cell types, while also retaining one or more cells with parental developmental potential. The term "stem cell" refers to a subset of progenitors that have the capacity or potential, under particular circumstances, to differentiate to a more specialized or differentiated phenotype, and which retains the capacity, under certain circumstances, to proliferate without substantially differentiating. In one embodiment, the term stem cell refers generally to a naturally occurring mother cell whose descendants (progeny) specialize, often in different directions, by differentiation, e.g. , by acquiring completely individual characters, as occurs in progressive diversification of embryonic cells and tissues. Cellular differentiation is a complex process typically occurring through many cell divisions. A differentiated cell may derive from a multipotent cell which itself is derived from a multipotent cell, and so on. While each of these multipotent cells may be considered stem cells, the range of cell types each can give rise to may vary considerably. Some differentiated cells also have the capacity to give rise to cells of greater developmental potential. Such capacity may be natural or may be induced artificially upon treatment with various factors. In many biological instances, stem cells are also "multipotent" because they can produce progeny of more than one distinct cell type, but this is not required for "sternness." Self-renewal is the other classical part of the stem cell definition. In theory, self- renewal can occur by either of two major mechanisms. Stem cells may divide

asymmetrically, with one daughter retaining the stem state and the other daughter expressing some distinct other specific function and phenotype. Alternatively, some of the stem cells in a population can divide symmetrically into two stems, thus maintaining some stem cells in the population as a whole, while other cells in the population give rise to differentiated progeny only. Formally, it is possible that cells that begin as stem cells might proceed toward a differentiated phenotype, but then "reverse" and re-express the stem cell phenotype, a term often referred to as "dedifferentiation" or "reprogramming" or "retrodifferentiation".

[00180] The term "embryonic stem cell" is used to refer to the pluripotent stem cells of the inner cell mass of the embryonic blastocyst (see US Patent Nos. 5,843,780, 6,200,806, the contents of which are incorporated herein by reference). Such cells can similarly be obtained from the inner cell mass of blastocysts derived from somatic cell nuclear transfer (see, for example, US Patent Nos. 5,945,577, 5,994,619, 6,235,970, which are incorporated herein by reference). The distinguishing characteristics of an embryonic stem cell define an embryonic stem cell phenotype. Accordingly, a cell has the phenotype of an embryonic stem cell if it possesses one or more of the unique characteristics of an embryonic stem cell such that that cell can be distinguished from other cells. Exemplary distinguishing embryonic stem cell characteristics include, without limitation, gene expression profile, proliferative capacity, differentiation capacity, karyotype, responsiveness to particular culture conditions, and the like.

[00181] The term "adult stem cell" or "ASC" is used to refer to any multipotent stem cell derived from non- embryonic tissue, including fetal, juvenile, and adult tissue. Stem cells have been isolated from a wide variety of adult tissues including blood, bone marrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle, and cardiac muscle. Each of these stem cells can be characterized based on gene expression, factor responsiveness, and morphology in culture. Exemplary adult stem cells include neural stem cells, neural crest stem cells, mesenchymal stem cells, hematopoietic stem cells, and pancreatic stem cells.

[00182] In one embodiment, progenitor cells suitable for use in the claimed devices and methods are Committed Ventricular Progenitor (CVP) cells as described in PCT

Application No. WO 2010/042856, entitled "Tissue Engineered Mycocardium and Methods of Productions and Uses Thereof, filed October 9, 2009, the entire contents of which are incorporated herein by reference.

IV. Use of Engineered Valves of the Invention

[00183] The engineered valves may be used in vitro or in vivo. The engineered valves described herein have various applications. For example, they may be used as engineered tissues and/or for implantation, repair and replacement of biological tissues and organs.

[00184] For example, the engineered valves of the invention may be used as replacement valves in any subject having a congenital or acquired valvular heart disease, such as a subject having a defective or weakened tricuspid valve, mitral valve, semilunar valve and/or venous valve, such as a subject having valve insufficiency or stenosis. In such examples, a subject may be treated by replacing a weakened or defective valve with an engineered valve.

[00185] For in vivo applications, the exact size and shape of the valves are species and patient specific. For example, pediatric patients may require smaller valves than adult patients. Pediatric aortic diameters can range from about 10 mm- 20 mm {i.e., the diameter of the aortic annuls). Adults can have an aortic annulus ranging from 20-35 mm in diameter. [00186] One benefit of the valves of the present invention is that they can be fabricated and custom-sized to fit the subject (see, e.g., FIG. 14A).

[00187] Additionally, the valves of the present invention do not require that cells be seeded and cultured prior to implantation into a subject, as the valves are configured to permit native cells to populate the valves.

[00188] In addition, in embodiments in which cells are seeded and cultured prior to implantation of the valve into a subject, the use of autologous cells to seed the valves permits the subject to forego immunosuppressive therapy. Moreover, the engineered tissues will be integrated into the natural tissue as cells from the subject will integrate into the polymeric scaffold of the valves and remodel the scaffold. Any suitable means for accessing the subject's heart and attaching the devices may be used, such as thoracic surgery or

transmyocardial catheter delivery.

V. Example Engineered Valves

[00189] Exemplary engineered valve scaffolds with integral leaflets were made using a method similar to that described above with respect to FIG. 27. FIGS. 15A-17D show that the polymer composition of the fibers used to make the valves is tuned to mimic the structure and mechanics of a native valve. For example, FIG. 15A is a bar graph showing polymer fiber diameter as a function of protein content, according to embodiments of the present disclosure. FIG. 15B is a bar graph showing percent porosity as a function of protein content, according to embodiments of the present disclosure. In this particular example, 1501 and 1511 show results from an example valve formulated from a solution of 100% P4HB, 1503 and 1513 show results from an example valve formulated from a solution of 80% P4HB and 20% gelatin, 1505 and 1515 show results from an example valve formulated from a solution of 60% P4HB and 40% gelatin, 1507 and 1517 show results from an example valve formulated from a solution of 40% P4HB and 60% gelatin, and 1509 and 1519 show results from an example valve formulated from a solution of 20% P4HB and 80% gelatin, each spun at 30,000 RPM fiber extrusion rate, 4% w/v. As can be seen in this graph, the examples had decreased fiber diameter and increased percent scaffold porosity as a function of increased protein content (*p<0.5).

[00190] FIG. 16A includes images illustrating a comparison of a native leaflet and an example valve scaffold spun at 45 degrees with a mandrel collection rate of 3,000 RPM, in which the images have a scale bar of 50 microns. In this particular example, the fiber anisotropy of the scaffold recapitulated the collagen alignment of native leaflets, as measured by Orientational Order Parameter (OOP), which is measured between 0 and 1 with 0 being complete disorder (no alignment) and 1 being perfect order (i.e., perfect alignment). FIG. 16B is a bar graph showing a comparison of alignment between a native leaflet structure (1603) and an example valve scaffold (1601) formed according to embodiments of the present disclosure. As can be seen in this example embodiment, there is no significant difference in alignment between the valve scaffold (1601) and the native leaflet (1603).

[00191] FIG. 17A is a bar graph illustrating the parallel stiffness of scaffolds as a function of increased protein content under low strain (0-10%) conditions, according to embodiments of the present disclosure. FIG. 17B is a bar graph illustrating the parallel stiffness of scaffolds as a function of increased protein content under high strain (10-20%) conditions, according to embodiments of the present disclosure. FIG. 17C is a bar graph illustrating the perpendicular stiffness of scaffolds as a function of increased protein content under low strain (0-10%) conditions, according to embodiments of the present disclosure. FIG. 17D is a bar graph illustrating the perpendicular stiffness of scaffolds as a function of increased protein content under high strain (10-20%) conditions, according to embodiments of the present disclosure. Increasing protein content of spun scaffolds, in these examples, decreased the biaxial global stiffness of the scaffolds under both low strain and high strain conditions. Examples having 60:40 and 40:60 P4HB:gelatin ratios matched native leaflet stiffness within the biaxial strain regions tested (*p<0.5).

[00192] Engineered valves, i.e., semilunar valves with integral leaflets fabricated as described herein, were evaluated in vitro under conditions mimicking those found in the adult human heart using a commercially available flow loop system. The valves were formed with dimensions corresponding to human semilunar valves according to the method described above with respect to FIG. 27.

[00193] FIG. 18 is a graph showing mechanical testing of an exemplary engineered valve scaffold and tissue done by equibiaxial loading of samples in the primary axis of fiber alignment, with strain shown on the x-axis and shear stress shown in the y-axis in kPa. The testing was done mimicking the circumferential alignment of native leaflets 1801, and the perpendicular axis of alignment 1803 mimicking the radial leaflet axis. Valve scaffolds tested in this example had a 60:40 P4HB:gelatin blend ratio. According to example embodiments, proper alignment of the fibers provides the biaxial properties of a native valve as a result of the fiber spinning processes described herein. [00194] FIG. 19A is a graph of in vitro valve scaffold performance as measured by arterial (distal) pressure as a function of time, according to embodiments of the present disclosure. FIG. 19B is a graph of in vitro valve scaffold performance as measured by ventricular (proximal) pressure as a function of time, according to embodiments of the present disclosure. FIG. 19C is a graph of in vitro valve scaffold performance as measured by flow as a function of time, according to embodiments of the present disclosure. As can be seen in FIGS. 19A-19C, the valve scaffold functional performance under pulmonary conditions was evaluated prior to implantation studies using a pulse duplicator system.

Pressure and flow recordings were made at 48 hours in systole (opened) and diastole (closed) conditions. In the examples tested, arterial (distal) pressure varied between -10 and -80 mmHg, while ventricular (proximal) pressure varied from—5 and -60 mmHg, resulting in approximately 20 mmHg transvalvular pressure during diastole (FDA waveform). Flow through the valve scaffold reached -200 ml/s during peak systole with complete valve closure during diastole, resulting in no flow.

[00195] Process capability, Cp was calculated for each batch of valve scaffolds produced using measurements of test sample strips incorporated into the scaffold

manufacturing process. Acceptable manufacturing Upper Control Limits (UCL) and Lower Control Limits (LCL) were set for each capability criterion. In the examples tested in FIGS. 19-20, Cp was defined as the minimal ratio of the distance from the mean (μ) to each control limit, and the allowable variance (σ), commonly taken as three standard deviations away from the mean in industrial manufacturing. A process capability Cp of one or greater is taken as acceptable, while a process capability Cp less than one in any test criteria results in batch rejection, due to high variance and valve inconsistency.

[00196] FIG. 20A is an example graph showing parameter distribution of seven batches of valve scaffolds formed according to embodiments of the present disclosure. FIG. 20B is another example graph showing parameter distribution of seven batches of valve scaffolds formed according to embodiments of the present disclosure. FIGS. 20A-20B constitute an example Cp report of seven batches of valve scaffolds, demonstrating the clarity of quality, go/no-go decision making process after calculating Cp, and the various structural, mechanical, and compositional parameters of the scaffolds. In the particular report shown in FIGS. 20A-20B, batches 1 and 5 were rejected. Batch 1 was rejected for having unacceptable thickness and alignment, while batch 5 was rejected for having unacceptable fiber diameter and thickness. [00197] FIG. 21A shows the effect of one month of lyophilization storage on a valve scaffold, according to embodiments of the present disclosure. FIG. 21B shows the effect of one month of dry refrigeration storage on a valve scaffold, according to embodiments of the present disclosure. Due to significant protein content of the biohybrid valve scaffold, and pre-stresses of wrapped fibers within scaffolds, storage conditions had varied effects on the micro structure of the semilunar valve shape. Hydration/dehydration cycling can cause scaffold stiffening and shrinkage, as uncrosslinked protein is lost and pre-stressed fibers compact once removed from their mandrels. As can be seen in FIG. 21A, lyophilization of scaffolds allowed for non-destructive storage conditions up to one month, maintaining scaffold size and pliability. In contrast, as can be seen in FIG. 21B, dry refrigeration storage resulted in significant compaction and stiffening of the valve scaffold, as evidenced by the size mismatch of the scaffold and appropriate stent.

[00198] FIG. 22A shows an example valve scaffold crimped approximately 24 mm in diameter, according to embodiments of the present disclosure. FIG. 22B shows an example valve scaffold crimped approximately 9 mm in diameter, according to embodiments of the present disclosure. When prepared for implantation, in some embodiments, some scaffolds were hydrated and anchored into a conduit of stent via suturing and stored in sterile saline until the time of surgery. Example scaffolds had to withstand crimping down to 9 mm from about 30 mm. According to exemplary embodiments, various types of stents can be used, including stents having bulges or gaps, which would allow the sinuses of the valve to extend beyond the diameter of the valve inlet or outlet without being obstructed by the stent.

[00199] FIG. 23 shows a graph of percent fiber surface composition as a function of storage time in days. Although not cross-linked, over the course of one week, example scaffolds maintained at least their original surface gelatin content 2303 from dry 2307 and wetted 2309 conditions, and lost detectable amounts of potentially harmful solvent 2305 once wetted. Surface gelatin content 2303 initially spiked once wetted, and surface P4HB content 2301 initially dropped, as gelatin molecules physically diffused from the fiber core to the surface. Surface gelatin content 2303 subsequently fell as gelatin diffused into the solution.

[00200] FIG. 24 shows an Echo/Doppler image assessing the valvular competency of an in vivo scaffold, according to embodiments of the present disclosure. In this particular example, semilunar valve scaffolds were transapically implanted into the pulmonary valve position in an ovine model, and Echo/Doppler was used to assess valvular competency at the time of implantation and explantation at 15 hours. Throughout implantation, the valve scaffold remained functional, fully opening during systole 2401 to allow forward flow from the right ventricle to the pulmonary artery. A side view of forward Doppler flow is shown at 2401, with a 3D echocardiography arterial view of fully opened leaflets shown at inset 2403. During diastole 2405, full coaptation of the leaflets was observed with only a small closing volume at the beginning of diastole. A side view of leaflet coaptation is shown at 2405, with a 3D echocardiography arterial view of fully closed leaflets shown at inset 2407.

[00201] FIG. 25A shows an arterial view of an example valve scaffold upon explantation, according to embodiments of the present disclosure. An arterial view shown in FIG. 25A shows no substantial clotting in the valve scaffold. FIG. 25B shows a cross- sectional view of a portion of an example valve scaffold upon explantation, according to embodiments of the present disclosure. FIG. 25C shows an enlarged view of a portion of the example valve scaffold of FIG. 25B, according to embodiments of the present disclosure. FIG. 25C shows a hematoxylin and eosin stain (H&E stain) analysis of the example valve scaffold, revealing full cellular (neutrophil) infiltration after 15 hours of implantation. Upon explantation of the scaffolds, full hydration of the scaffolds was observed with no major clotting.

[00202] FIG. 26A shows the results of a VG-Elastin staining test performed on an example explanted valve scaffold, according to embodiments of the present disclosure. The VG-Elastin staining test revealed elastin expression on the ventricular side of leaflets. FIG. 26B shows the results of a Vitemin staining test performed on an example explanted valve scaffold, according to embodiments of the present disclosure. Positive Vimentin staining shown in this figure is suggestive of mesenchymal cell infiltration on both leaflet sides. FIG. 26C shows the results of a CD31 staining test performed on an example explanted valve scaffold, according to embodiments of the present disclosure. Positive CD31 stains at the leaflet surfaces is indicative of early endothelial cell recruitment. FIG. 26D shows the results of an alpha smooth muscle actin staining test performed on an example explanted valve scaffold, according to embodiments of the present disclosure. The alpha smooth muscle actin staining was negative in this particular example.

[00203] In describing exemplary embodiments, specific terminology is used for the sake of clarity. For purposes of description, each specific term is intended to at least include all technical and functional equivalents that operate in a similar manner to accomplish a similar purpose. Additionally, in some instances where a particular exemplary embodiment includes a plurality of system elements or method steps, those elements or steps may be replaced with a single element or step. Likewise, a single element or step may be replaced with a plurality of elements or steps that serve the same purpose. Further, where parameters for various properties are specified herein for exemplary embodiments, those parameters may be adjusted up or down by l/20th, 1/lOth, l/5th, l/3rd, ½, etc., or by rounded-off

approximations thereof, unless otherwise specified. Moreover, while exemplary

embodiments have been shown and described with references to particular embodiments thereof, those of ordinary skill in the art will understand that various substitutions and alterations in form and details may be made therein without departing from the scope of the invention. Further still, other aspects, functions and advantages are also within the scope of the invention.

[00204] The contents of all references, including patents and patent applications, cited throughout this application are hereby incorporated herein by reference in their entirety. The appropriate components and methods of those references may be selected for the invention and embodiments thereof. Still further, the components and methods identified in the Background section are integral to this disclosure and can be used in conjunction with or substituted for components and methods described elsewhere in the disclosure within the scope of the invention.

Claims

We claim:

1. An engineered valve configured for flow from an upstream first end of the valve to a downstream second end of the valve, the engineered valve comprising: a tubular wall comprising micron, submicron, or nanometer dimension polymer fibers defining a shape of the tubular wall, the tubular wall having an inner surface, a first end, and a second end; and a plurality of leaflets integral with the inner surface of the tubular wall, each extending from the inner surface of the tubular wall radially inward and toward the second end of the tubular wall, the plurality of leaflets comprising micron, submicron, or nanometer dimension polymer fibers defining the shape of the plurality of leaflets, the plurality of leaflets configured for flow through the tubular wall from the first end of the tubular wall downstream to the second end of the tubular wall; the tubular wall having a plurality of outward bulging portions, each outward bulging portion forming a sinus for a corresponding leaflet of the plurality of leaflets.

2. The engineered valve of claim 1, wherein the each outward bulging portion extends downstream of a portion of the tubular wall that connects with the corresponding leaflet.

3. The engineered valve of claim 1, comprising two leaflets and two outward bulging portions forming two sinuses.

4. The engineered valve of claim 1, comprising three leaflets and three outward bulging portions forming three sinuses.

5. The engineered valve of claim 1, wherein at least some of the micron, submicron or nanometer dimension polymer fibers of the tubular wall interpenetrate with at least some of the micron, submicron or nanometer dimension polymer fibers of the plurality of leaflets.

6. The engineered valve of claim 1, wherein the micron, submicron, or nanometer dimension polymer fibers comprises a biogenic polymer.

7. The engineered valve of claim 1, wherein the micron, submicron, or nanometer dimension polymer fibers have a diameter between about 10 nanometers and about 10 microns.

8. The engineered valve of claim 1, wherein each of the plurality of leaflets

independently has a thickness between about 10 and about 300 microns.

9. The engineered valve of claim 1, wherein a diameter of the first end of the tubular wall corresponds to a diameter of a pulmonary valve.

10. The engineered valve of claim 1, wherein the tubular wall further comprises a stent.

11. The engineered valve of claim 1, wherein the micron, submicron or nanometer dimension polymer fibers of the tubular wall and the micron, submicron or nanometer dimension polymer fibers of the plurality of leaflets are configured to form a polymeric fiber scaffold for cellular ingrowth.

12. A mandrel assembly for making a valve including a tubular wall and at least three leaflets, the mandrel assembly having an axis of rotation and comprising: a first mandrel having a first end portion, a second end portion, and an outer surface, the outer surface having a tubular wall-forming region at the first end portion and at least three concave leaflet- forming regions at the second end portion, each concave leaflet-forming region configured to define a shape of an upstream surface of a leaflet in the resulting valve; and a second mandrel structure comprising: a member including a base portion and at least three spacing portions extending parallel to the axis of rotation from the base portion; and at least three sinus-forming bodies, each sinus-forming body having a first end portion and a second end portion and configured to be fastened to the member with the first end portion of the sinus-forming body adjacent the base portion of the member and with each sinus-forming body separated from an adjacent sinus-forming body by one of the at least three spacing portions, the member and the sinus-forming bodies configured such that, when fastened together, an outward facing surface of the member and outward facing surfaces of the first end portions of each of the sinus- forming bodies collectively form a tubular wall- forming region of an outer surface of the second mandrel structure, an outward facing surface of the second end portion of each sinus-forming body bulges outward to define a shape of a sinus of the resulting valve, and an inward facing surface of the second end portion of each sinus-forming body bulges inward to define a shape of a downstream surface of a leaflet in the resulting valve.

13. The mandrel assembly of claim 12, wherein each concave leaflet-forming region of the outer surface of the first mandrel is configured to at least partially receive a second end portion of a corresponding sinus-forming body when the sinus-forming body is fastened to the member.

14. The mandrel assembly of claim 12, wherein the first mandrel includes three concave leaflet-forming regions and the second mandrel structure includes three sinus-forming bodies configured to form a resulting valve having three leaflets and three sinuses.

15. The mandrel assembly of claim 12, wherein a diameter of a first end portion of the first mandrel is substantially equal to a diameter of tubular wall- forming region of an outer surface of the second mandrel structure.

16. The mandrel assembly of claim 12, wherein the first mandrel is configured to be rotated around a rotation axis to receive a coating of micron, submicron, or nanometer dimension polymer fibers thereby forming an upstream surface of each leaflet in the resulting valve and an inner surface of an upstream tubular wall of the resulting valve.

17. The mandrel assembly of claim 12, wherein the first mandrel and the second mandrel structure are configured to be fastened together to form a combined mandrel, the combined mandrel configured to be rotated around the axis of rotation to receive a coating of micron, submicron, or nanometer dimension polymer fibers thereby forming sinuses and a

downstream tubular wall of the resulting valve.

18. The mandrel assembly of claim 17, wherein the first mandrel is configured to be withdrawn from an upstream end of the resulting valve without damaging a tubular wall formed on the tubular wall-forming region at the first end portion of the first mandrel after collection of the coating of micron, submicron, or nanometer dimension polymer fibers on the combined mandrel.

19. The mandrel assembly of claim 12, wherein the second mandrel structure is configured to be disassembled and then withdrawn from a downstream end of the resulting valve in pieces without damaging a tubular wall formed on the tubular wall- forming region of the outer surface of the second mandrel structure after collection of the coating of micron, submicron, or nanometer dimension polymer fibers on the combined mandrel.

20. A method of forming an engineered valve including a tubular structure, at least three sinuses, and at least three leaflets, the method comprising: forming micron, submicron, or nanometer dimension polymer fibers by ejecting or flinging a polymer from a reservoir; collecting a first portion of micron, submicron, or nanometer dimension polymer fibers on a rotating first mandrel having a first end portion, a second end portion, and an outer surface with a tubular wall-forming region at the first end portion and at least three concave leaflet-forming regions at the second end portion, the tubular-wall forming region of the outer surface having a shape corresponding to an inner surface of a first portion of the tubular structure of the resulting valve scaffold, each of the at least three concave leaflet-forming regions having a shape corresponding to an upstream surface of a corresponding leaflet in the resulting valve; coupling a second mandrel structure to the first mandrel to form a combined mandrel, the second mandrel structure comprising a member including a base portion and at least three spacing portions extending parallel to the axis of rotation from the base portion and at least three sinus-forming bodies each having a first end portion and a second end portion, each sinus-forming body fastened to the member and separated from an adjacent sinus-forming body by one of the at least three spacing portions, an outward facing surface of the member and outward facing surfaces of the first end portions of each of the sinus-forming bodies collectively form a tubular wall- forming region of an outer surface of the second mandrel structure, an outward facing surface of the second end portion of each sinus-forming body bulges outward to define a shape of a sinus of the resulting valve, and an inward facing surface of the second end portion of each sinus-forming body bulges inward to define a shape of a downstream surface of a leaflet in the resulting valve scaffold; and collecting a second portion of micron, submicron, or nanometer dimension polymer fibers on the combined mandrel by rotating the combined mandrel about a rotation axis in the path of the ejected micron, submicron, or nanometer dimension polymer fibers to form a remainder of the resulting valve scaffold having a first end and a second end, at least three leaflets and at least three sinuses.

21. The method of claim 20, further comprising uncoupling the first mandrel and the second mandrel structure and withdrawing the first mandrel from the first end of the resulting valve scaffold.

22. The method of claim 21, further comprising unfastening the at least three sinus- forming bodies from the member and withdrawing the member from the second end of the resulting valve scaffold.

23. The method of claim 22, further comprising withdrawing the at least three sinus- forming bodies from the second end of the resulting valve scaffold after withdrawing the member from the second end of the resulting valve scaffold.

24. The method of claim 20, further comprising removing excess submicron, or nanometer dimension polymer fibers from the first mandrel before coupling the second mandrel structure to the first mandrel.

25. The method of claim 20, wherein the at least three leaflets composed of the micron, submicron, or nanometer dimension polymer fibers that are formed during collection of the first portion of micron, submicron, or nanometer dimension polymer fibers are covered by a portion of the second mandrel structure after coupling the first mandrel and the second mandrel structure to form the combined mandrel.

26. The method of claim 20, wherein the micron, submicron, or nanometer dimension polymer fibers have a diameter between about 10 nanometers and about 10 microns.

27. The method of claim 20, wherein the micron, submicron, or nanometer dimension polymer fibers comprise a biogenic polymer.

28. The method of claim 20, wherein a spacing between each of the at least three concave leaflet-forming regions of the outer surface of the first mandrel and the corresponding inward facing surface the second end portion of each sinus-forming body in the second mandrel structure of the combined mandrel is set to form a leaflet in the resulting valve scaffold having a thickness between about 50 and about 300 microns.

29. The method of claim 20, wherein the micron, submicron, or nanometer dimension polymer fibers are ejected through an orifice of a rotating reservoir.

30. The method of claim 29, wherein the first mandrel and the combined mandrel are rotated about an axis that is inclined at an angle with respect to an axis of rotation of the rotating reservoir during collection of the first portion and the second portion of micron, submicron, or nanometer dimension polymer fibers.

31. The method of claim 30, wherein the first mandrel and the combined mandrel are rotated about an axis that is inclined between about 0 degrees and about 45 degrees with respect to the axis of rotation of the rotating reservoir.

32. The method of claim 20, wherein the polymer is ejected from the reservoir by rotating the reservoir at a speed of between about 20,000 and about 60,000 rpm.

33. The method of claim 20, wherein collecting micron, submicron, or nanometer dimension polymer fibers on the rotating first mandrel comprises rotating the first mandrel about a rotation axis in a path of the ejected polymer fibers.

34. The method of claim 20, wherein collecting micron, submicron, or nanometer dimension polymer fibers on the rotating first mandrel comprises translating the first mandrel along a path substantially parallel to the axis of rotation of the rotating reservoir.

35. The method of claim 20, wherein collecting micron, submicron, or nanometer dimension polymer fibers on the combined mandrel comprises translating the combined mandrel along a path parallel to the axis of rotation of the rotating reservoir.

36. The method of claim 20, wherein collecting the second portion of micron, submicron, or nanometer dimension polymer fibers on the combined mandrel by rotating the combined mandrel about a rotation axis in the path of the ejected micron, submicron, or nanometer dimension polymer fibers to form a remainder of the resulting valve scaffold comprises: collecting part of the second portion of micron, submicron, or nanometer dimension polymer fibers on the combined mandrel by rotating the combined mandrel about a rotation axis in the path of the ejected micron, submicron, or nanometer dimension polymer fibers; positioning a stent over the part of the second portion of micron, submicron, or nanometer dimension polymer fibers collected on the combined mandrel; and collecting a remainder of the second portion of micron, submicron, or nanometer dimension polymer fibers on the stent and the combined mandrel by rotating the combined mandrel about a rotation axis in the path of the ejected micron, submicron, or nanometer dimension polymer fibers to form a remainder of the resulting valve scaffold.

37. An engineered valve configured for flow from an upstream first end to a downstream second end, the engineered valve formed according to the method of claim 20.

38. A method for treating a subject having a defective or weakened cardiac valve, comprising providing an engineered valve comprising: a tubular wall comprising micron, submicron or nanometer dimension polymer fibers defining a shape of the tubular wall, the tubular wall having an inner surface, a first end and a second end; a plurality of leaflets integral with the inner surface of the tubular wall, each extending from the inner surface of the tubular wall radially inward and toward the second end of the tubular wall, each leaflet comprising micron, submicron, or nanometer dimension polymer fibers defining the shape of the leaflet, the leaflets configured for flow through the tubular wall from the first end of the tubular wall downstream to the second end of the tubular wall; and the tubular wall having a plurality of outward bulging portions, each outward bulging portion forming a sinus for a corresponding leaflet of the plurality of leaflets; and replacing the weakened or defective valve in the subject with the engineered valve, thereby treating the subject.