External support for a tissue implant

Abstract

The present invention is an external support or frame for a constructed tissue that together can be implanted.

Description

External Support for a Tissue ImplantField of the Invention

[0001] The invention relates to products formed from proprietary regenerative tissue, products made from the tissue, an implant comprising this tissue, and methods of treating conditions and / or disorders using such tissue.Background of the Invention

[0002] A relatively new field of medicine -- since the early 1990s - is the field of Regenerative Medicine. Regenerative Medicine is the process of creating functional tissues to repair, replace, or restore tissue or organ structure and function lost due to age, disease, damage, or congenital defects. This field of medicine uses new methods and products including tissue engineering or constructed tissue (CT).

[0003] The use of prepared heterogenous graft material for human surgical implantation is well known. More specifically, the use of treated animal tissue as human tissue grafts, replacement valves, and similar implantation surgical procedures is well known. However, problems of immunogenicity, thrombogenicity, calcification, material strength, and size have not been adequately addressed in the art.

[0004] Since the 1930's, medical researchers have attempted to develop suitable natural and synthetic alternatives for obtaining small diameter grafts useful in vascular surgery. Historically, attempts to fabricate such tubular grafts from manmade materials have been somewhat unsuccessful. Homologous tissues are not always readily available and are not always readily available in the size the surgeon needs. Furthermore, some of these tissues may be immunogenic and therefore may require processing or certain treatments to reduce their immunogenicity.

[0005] Coronary artery bypass grafts (CABG) remain the mainstay of revascularization for multivessel coronary artery disease (CAD) (2). The most widely used conduits are autologous internal thoracic arteries, radial arteries and saphenous veins, which provide excellent mechanical stability and natural anti- thrombogenicity (2). Patients currently undergoing coronary revascularization surgery present with an increasing age and risk profile (2). In patients with comorbidity of other vascular diseases, the quality of saphenous vein grafts may beimpaired if they are available at all (2, 3). Therefore, there continues to be a need for alternative bypass conduits for the coronary revascularization surgery (4-6). Further, even in patients with suitable veins, the procedure to harvest, ensuring all branches are tied and lumen diameter matches to native coronary, is associated with additional complications.

[0006] Several clinical studies have evaluated non-autologous grafts for coronary artery bypass graft (CABG) surgery with limited success (4-6). All of these non- autologous materials were either animal-derived or cadaver-based, both of which have poor regenerative capacity and ability to endothelialize. The new approach of tissue engineering a blood vessel from cells in a laboratory, decellularizing and using the cell produced extracellular matrix as a conduit has been shown to regenerate and remodel both in preclinical non-human primate studies (7, 8) and clinical studies (9-13).

[0007] Humacyte Inc has pioneered one of these approaches of tissue engineering a vascular graft by seeding vascular cells on a synthetic biodegradable polymer, subsequently culturing it in a bioreactor to produce final acellular ECM tube (8). Humacyte has implanted their vascular conduits in patients needing vascular grafts for dialysis, for femoral artery and for trauma injuries (9-11 , 13, 14). In the above knee femoral-to-popliteal artery implant in 20 patients, 6 mm internal diameter conduits of 35-42 cm length were followed up to 24 months. Primary and secondary patency was 58% and 74% respectively. Further, immunological panel testing showed no change in panel reactive antibodies after implant. The most common cause for loss of primary patency was thrombosis and anastomosis stenosis (10). Humacyte recently reported the outcomes using the conduits for coronary artery bypass in a non-human primate model (AHA Conference 2022).

[0008] A variety of materials are available, but all have shortcomings, and none are regenerative. There remains a need for a solution that combines the benefits of native collagenous tissue having regenerative properties with those of manufacturing at scale and in the proper form (i.e., conduit).

[0009] Vascular graft failures are commonly associated with thrombosis, intimal hyperplasia, atherosclerosis, and / or infection. Implanted grafts of the present invention show no signs of any of these failure signs.

[0010] Notwithstanding the usefulness of the above-described methods, a need still exists for increasing patency; making an implant less thrombotic in structure and / or function; minimizing calcification; and increasing the useful life of the implant.Summary of the Invention

[0011] One embodiment of the invention is the preparation and use of constructed tissue (CT) to make implants, their use as implants, and their use in mediating treatment or therapy. In preferred embodiments of the invention, the implant is a coronary artery bypass graft (CABG), a peripheral vascular graft, or a saphenous vein conduit.

[0012] In the most preferred embodiments of the invention, the implant includes an external support structure (ESS).

[0013] The present invention utilizes a novel tissue engineering approach that uses human fibroblasts seeded in a biological polymer followed by bioreactor culture leading to a final acellular ECM vascular conduit that is completely biological with mechanical properties comparable to the internal mammary artery (7, 15). The tissue may be characterized in part by beneficial collagen alignment; anisotropy; anisotropy in vivo; acellular; compatible with terminal sterilization; non-chemically crosslinked; and typical moisture content about 85%. In preferred embodiments of the invention, the tissue includes non-uniform collagen layers, typically, a high-density collagen layer, then a porous layer, then a high-density layer, then another porous layer. In some embodiments, the non-uniform collagen layer comprises a high-density layer sandwiched between two or more porous layers. The inventors believe that these non-uniform collagen layers provide many advantages, including but not limited to providing more strength to the finished tissue, e.g., greater burst strength. The vascular conduit (6 mm diameter conduit) has been implanted in 16 patients in two trials to date for use in hemodialysis access (12). In the first-in-human, 6-month study showed favorable patency and low complication rate establishing the initial safety and feasibility of the tissue’s use for dialysis access in patients with end-stage kidney disease. The mechanical durability and lack of immune response established the tissue as a potential regenerative material for clinical use in dialysis and other vascular applications.

[0014] An advantage of the invention is the starting material itself.

[0015] The tissue of the present invention is a completely biological, acellular collagenous matrix produced by allogeneic, neonatal, human dermal fibroblasts (nHDF). The nHDF are mixed with thrombin and fibrinogen and molded into the desired shape, typically a tube or sheet. The molded structure is then cultured in a bioreactor where the nHDF degrades the fibrin gel and secretes collagen and other extracellular matrix (ECM). The resulting tissue is then decellularized, yielding an acellular human collagenous matrix.

[0016] The products, uses, and processes of the present invention are suitable for treating diseases and conditions that would benefit from regenerative engineered tissues, especially those involving tubular tissue constructs. One such use is for coronary artery bypass grafts (CABG).

[0017] Some embodiments of the invention include the manufacture of a graft that is durable and has been demonstrated in both animal and human trials to withstand the rigorous mechanical requirements of the vascular system (8).

[0018] The biological materials according to the present invention, are processed to modify (e.g., reduce or eliminate) size and shape, thinness, collagen content, and other characteristics and properties that will become clear from the description of the invention. The methods, uses, and products of the present invention are intended for implant in a mammal, preferably a human. All of the biological materials, processed according to the present invention, are appropriate for use in an in vivo environment, and include one or more of the following desirable properties for graft material suitable for implantation: a) size compatibility with surrounding vessels to which it will be anastomosed; b) sutureability, kink resistance, softness, radial and longitudinal compliance, and flexibility (a softer hand); c) non-thrombogenicity or low levels of thrombogenicity, particularly after regeneration or recellularization; d) durability; e) ease of sterilization; f) readily available, and available in diameters and lengths appropriate for surgical procedures; g) shelf life appropriate for market conditions (typically greater than three years); h) resistant to infection; i) sufficient strength to resist aneurysm formation; j) non-immunogenic; k) resistant to degradation; I) resistant to formation of neointimal hyperplasia; m) tactile, as expressed by surgeons using the tissue and / or grafts of the present invention, particularly suitable for microsurgery; n) is anti-bacterial; o) is non-infectious; p) is anti-microbial; and q) under circumstances when the implant becomes infected, the implant of the present invention may not become infected or resists infection (e.g., the host / recipient cellsbecome infected exclusive of the implant); r) reduced or eliminated calcification characteristics; s) improved or biologically appropriate or desirable resorbability; t) becomes or transitions into living tissue; and u) in some embodiments, capable of endothelialization. As used herein, living tissue refers to a tissue that exhibits the presence of active cells (originating from the implant recipient), e.g., including but not limited to producing elastin, producing ECM; and / or an un-paralleled lack of immunogenicity.

[0019] Surgeons have also recognized the superior quality of the tissue of the present invention in terms of a tactile response, specifically in comparison to typical vasculature (usually autologous, e.g., venous grafts during coronary artery bypass procedures).

[0020] An implant of the present invention provides several other advantages: the graft allows revascularization of the diseased heart using a tissue that exhibits little or no discernable immune response; isodiametrically uniform, which contributes to excellent laminar flow within the graft, which in turn reduces turbulence and turbulence-related thrombus formation; a graft that grows with the body, making this graft useful and beneficial for pediatric patients; the graft eliminates the need for vein and artery harvesting procedures, thereby eliminating the need for a second surgery; provides a small diameter, consistently uniform sized graft; and provides an excellent graft with high patency rates and mechanical characteristics.

[0021] Although specific advantages have been enumerated above, various embodiments may include some, none, or all of the enumerated advantages.

[0022] Other technical advantages may become readily apparent to one of ordinary skill in the art after review of the following figures and description.

[0023] It should be understood at the outset that, although exemplary embodiments are illustrated in the figures and described below, the principles of the present disclosure may be implemented using any number of techniques, whether currently known or not. The present disclosure should in no way be limited to the exemplary implementations and techniques illustrated in the drawings and described below.

[0024] With the following enabling description of the drawings, the apparatus should become evident to a person of ordinary skill in the art.Brief Description of the Figures

[0025] Figure 1 shows an implant of the present invention comprising a tissue tube supported by an external support structure.

[0026] Figure 2 shows a close-up view of a portion of an external support structure encasing a tissue biomaterial.

[0027] Figure 3 shows a flat non-expanded representation of a support structure of the present invention.

[0028] Figure 4 shows an expanded and finished cell within the support structure shown in Figure 3.

[0029] Figure 5 shows an end portion of an external support structure.

[0030] Figure 6 shows a close-up view of a preferred ESS (A) as compared to a close-up view of a less preferred and less flexible ESS (B).

[0031] Figure 7 shows the exemplary placement of a CABG implant in relation to the structures of an anatomical heart model.

[0032] Figure 8 is a chart showing the primary and secondary patency of a tissue of the present invention.Detailed Description of the Invention

[0033] The present invention is a coronary artery bypass graft (CABG) formed from constructed tissue (CT) produced according to the present invention. In preferred embodiments of the invention, the graft includes an external support to prevent the graft from kinking. In the most preferred embodiments, the external support prevents / reduces kinking, prevents / reduces abrasion or wear on the outside portion of the graft, prevents or controls any tissue expansion or dilation, and maintains lumen uniformity.

[0034] The graft of the present invention may be used as a vascular conduit anywhere it might be beneficial in vivo in a mammal, including a human. Exemplary uses include but are not limited to CABG surgery, preferably for bypassing a blocked or damaged portion of any coronary artery, for restoring blood flow to the heart; a peripheral vascular graft; or a saphenous (superficial or deep) vein conduit, including but limited to above the knee, around the knee, or below the knee.

[0035] In preferred embodiments of the invention, the tissue is used as a bypass to a second or third target (non-LAD coronary artery) in patients needing coronary artery bypass grafting (CABG). The implant is a tubular bypass conduit that createsa new path for blood to flow around a blocked or partially blocked artery in the heart, improving blood flow to the heart muscle.

[0036] In some embodiments of the invention, the graft or conduit also includes an external support. Examples of suitable stents, that in accordance with the present invention, have been used as external supports, include but are not limited to a Gore Tigris, Terumo Misago, Gore ViaBahn, Boston Scientific Innova™, Abbott Supera, Biotronik Pulsar-18; Medtronic Everflex, and Cook Zilver.

[0037] In the most preferred embodiments of the invention, the external support is a support as shown in Figures 1-7.

[0038] A support of the present invention may be used with any tissue or conduit intended to establish a fluid flow path between one part of the body and another part, e.g., from a coronary artery to a lung artery, between one coronary artery and another, etc.

[0039] In accordance with some embodiments of the invention, a tissue, conduit, or tube of the present invention may be implanted in a first step, and in a second step, the support may be added around the implant.

[0040] In the most preferred embodiments of the invention, the external support has one or more of the following characteristics: is laser cut; radially flexible, biocompatible, sterile, radially strong, radially stiff, radially stiff to promote or support crush resistance; fatigue resistance, non-corrosive, precise diameter, non-toxic, precise thickness, electropolished, axially flexible, medical grade material; diameter sufficient to prevent conduit kinking; provides sufficient support to prevent conduit kinking under anatomical (in vivo) conditions (e.g., cyclic axial motion); sufficiently rigid to prevent kinking in the typically curved conditions (curvature of the heart or other anatomy, and under pulsatile flexion of an artery); sufficiently flexible to curve in the typically curved conditions of the target anatomy (e.g., curvature of the heart or other anatomy, and under pulsatile flexion of an artery); supports vascular compliance; diameter sufficiently larger than conduit to allow conduit dilation (e.g., typical of pulsatile blood flow in coronary arteries); to prevent, reduce, or control deleterious dilation, e.g., an aneurysm; of a size and diameter that does not snuggly fit the conduit; of a size and diameter that permits sliding the ESS over a conduit; can slide manually over the conduit; sufficiently axially compliant to not push the bend toward an end of the implant (e.g., ESS / CAB junction); flexible enough to expand with pulsatile expansion and contraction; maintain integrity through beatingheart motion; axially compressible sufficient to allow exposure of either or both ends of the conduit, permitting ease of creating an anastomosis; atraumatic edges and structure sufficient to prevent conduit puncture or abrasion; atraumatic edges and structure sufficient to prevent anatomy (e.g., heart) puncture or abrasion; sterilizable; and / or durable; MRI compatible; a support structure, design, and configuration that does not inhibit the integrity of the supported tissue or tube; a support structure, design, and configuration that does not damage or abrade the outer surface of the supported tissue or tube; a support structure, design, and configuration that does not damage or cause surface abrasions of any anatomy or organ contacted by the implant; a support that is fitted to the tissue or tissue tube; a support that is fitted to the tissue or tissue tube tight enough so that the frame or support does not substantially axially move under the cyclic beating heart movement; crush resistant; durable; maintain uniform lumen diameter; and prevent excessive dilation.Tubular Frame Structure

[0041] The tubular frame can be a ring-like elliptical, or cylindrical configuration or shape made from a durable, biocompatible structural material such as Nitinol or similar alloy, wherein the tubular frame is formed by manufacturing the structural material as a braided wire frame, a laser-cut frame, or a wire loop. The frame or support is sized in length and diameter to enclose a tissue material. In length, the support may be any length, again sized to the tissue material. In some embodiments, the support is the length of the tissue material. In other embodiments, one or both ends of the support is shorter than the tissue material. In the most preferred embodiments, the support is compressible so that one or both ends of the tissue material can be exposed.

[0042] In some embodiments of the invention, the support may be up to about 300 mm in length, preferably between about 80 mm and about 120 mm.

[0043] In some exemplary embodiments, a typical inner diameter is up to about 24 mm in diameter; preferably greater than about 4.0 mm, and in a typical range from about 4 mm to about 30 mm. In some exemplary embodiments, a typical outer diameter is up to about 30 mm in diameter; preferably greater than about 4.0 mm, and in a typical range from about 4 mm to about 30 mm. As stated, the tubular frame can have a side-profile of a ring shape, cylinder shape, but may also have a sideprofile of a convex cylinder (walls bulging out), a ring or cylinder having a flared top, flared bottom, or both.

[0044] In one preferred embodiment, the tubular frame used in a CABG implant may have a complex shape determined by the anatomical structures where the implant is being inserted. For example, in the CABG implant, the circumference of the ESS may be substantially circular, slightly larger in diameter than the underlying tissue tube, incorporating sufficient flexibility to enlarge and shrink according to the variability in all normal and diseased vasculature. Accordingly, a CABG implant with ESS may start in a roughly tubular configuration, and be heat-shaped to diameter and length to provide adequate support for the conduit, with the annular tubular section having a uniform cross-section for most or all of its length; and having atraumatic edges along the posterior and anterior annular transition segments where it is intended that the tissue is not supported (e.g., bare conduit transition at one or both of the anterior or posterior ends of the tissue).

[0045] In an alternative embodiment, the ESS may be configured in a clamshell configuration. In this configuration, instead of sliding the ESS onto the vessel (as above), in this embodiment, a tissue is placed within a longitudinally open support, which can then be wrapped, clamped, or closed over the tissue.

[0046] A scaffold, support, or frame of the present invention is flexible and capable of manipulation so that the frame or support can be bent or wrapped around a tissue tube or conduit, without breaking or damaging the tissue. A wrap of the present invention may also be flexible enough to slide over, enclose, or cover a tissue material.

[0047] As noted above, a wrap or tissue product of the present invention is a tube or conduit, or tubular is shape. A preferred embodiment is a continuous tubular shape. Other preferred embodiments are shown in the Figures. Typically, these shapes include or enclose a lumen through the longitudinal length of the construct. The lumen can be open or closed at one or both ends.Tubular Frame Purpose

[0048] The tubular frame has a central axial lumen where a tissue material, such as a tube, may be placed within the inside diameter of the frame.Frame Structure

[0049] Preferably, the frame is made from superelastic metal wire, such as Nitinol (TM) wire or other similarly functioning material or alloy. The material may be used for the frame or support. It is contemplated as within the scope of the invention to use other shape memory alloys such as Cu - Zn - Al — Ni alloys, Cu - A1 Ni alloys, as well as polymer composites including composites containing carbon nanotubes, carbon fibers, metal fibers, glass fibers, and polymer fibers. It is contemplated that the frame may be constructed as a braided wire frame or as a laser cut wire frame. Such materials are available from any number of commercial manufacturers, such as Pulse Systems. Laser cut wire frames are preferably made from Nickel - Titanium (Nitinol (TM)), but also without limitation made from stainless steel, cobalt chromium, titanium, and other functionally equivalent metals and alloys, or Pulse Systems braided frame that is shape - set by heat treating on a fixture or mandrel.

[0050] One key aspect of the frame design is that it is compressible and when released can return to its original (uncompressed) shape. This requirement may limit the potential material selections to metals and plastics that have shape memory properties. With regards to metals, Nitinol has been found to be especially useful since it can be processed to be austenitic, martensitic, or super elastic. Martensitic and super elastic alloys can be processed to demonstrate the required compression features. The inventors have found that supports that are axially compressible from about 0% to about 40% of the length are suitable in the practice of this invention. In preferred embodiments of the invention, any amount of axial compression may be selected as long as a relatively uniform tissue lumen is maintained. More preferred are supports that are compressible between about 10% and about 30% of the length.

[0051] In accordance with an embodiment of the invention, supports that are less compressible or not compressible, e.g., compressible from about 0% to about 5%, may be further configured in length so that the tissue material extends beyond the end(s) of the support.

[0052] In the most preferred embodiments, the frame is compressible to the extent that one strut can nest into an adjacent strut without contacting or binding the adjacent strut. Compare, for example, Figure 6A and 6B.

[0053] In accordance with various embodiments of the invention, compressibility and nesting may be accomplished by using a wide variety of strut angles andshapes, all of which is within the expertise of one skilled in the art. In the exemplary designs shown in the figures, the supports have strut angles from about 15° to about 25°. The exemplary configuration shown in Figures 2-4 have a strut angle of about 20°

[0054] Alternatively, the support may be synthetic, made from a plastic and / or fabric material, or the tissue could be printed. In these embodiments, the support should still be compressible and flexible, in accordance with the teachings of this invention.Laser Cut

[0055] One possible construction of the wire frame envisions the laser cutting of a thin, isodiametric Nitinol tube. The laser cuts form regular cutouts in the thin Nitinol tube.

[0056] Secondarily the tube is placed on a mold of the desired shape, heated to the Martensitic temperature and quenched. The treatment of the wire frame in this manner will form a device that has shape memory properties and will readily revert to the memory shape at the calibrated temperature.

[0057] The frames are support structures that comprise a number of struts or wire portions arranged relative to each other to provide a desired compressibility and strength. In general terms, the stents or stent frames of the present disclosure are generally tubular support structures having an internal area in which tubular tissue material may be positioned.

[0058] Some embodiments of the stent frames can be a series of wires or wire segments arranged such that they are capable of self-transitioning from the compressed or collapsed arrangement to the normal, radially expanded arrangement.

[0059] In some constructions, a number of individual wires comprising the stent frame support structure can be formed of a metal or other material. These wires are arranged in such a way that the stent frame support structure allows for folding or compressing or crimping to the compressed arrangement in which the internal diameter is smaller than the internal diameter when in the normal, expanded arrangement. In the compressed arrangement, such a stent frame support structure enclosing a tissue tube, can be mounted onto a delivery device. The stent frame support structures are configured so that they can be changed to their normal,expanded arrangement when desired, such as by the relative movement of one or more outer sheaths relative to the length of the stent frame.

[0060] The wires of these stent frame support structures in embodiments of the present disclosure can be formed from a shape memory material such as a nickel titanium alloy (e.g., Nitinol™). With this material, the support structure is selfexpandable from the compressed arrangement to the normal, expanded arrangement, such as by the application of heat, energy, and the like, or by the removal of external forces (e.g., compressive forces). This stent frame support structure can also be compressed and re-expanded multiple times without damaging the structure of the stent frame. In addition, the stent frame support structure of such an embodiment may be laser-cut from a single piece of material or may be assembled from a number of different components. For these types of stent frame structures, one example of a delivery device that can be used includes a catheter with a retractable sheath that covers the stent frame until it is to be deployed, at which point the sheath can be refracted to allow the stent frame to self-expand.

[0061] The present invention is a product and process for use in any in vivo treatment in which endothelization and / or recellularization provides a beneficial result for the patient / recipient.

[0062] As used herein, recellularization refers to the repopulation or growth of cells and structures near the implant site aiming to reconstitute and recreate the natural tissue-specific function. In some embodiments of the invention recellularization includes the tissue growing, including somatic growth, with the patient.

[0063] As used herein, healing refers to the ability of living organisms to replace damaged or lost tissue with new cells, heal damaged tissue, restoring their structure and function, that is, becoming living tissue. Healing includes repopulation leading to restoration of tissue and / or body functions, including but not limited to restoration of cells, other biological molecules, and biological structures, including acellular ECM scaffolds. Healing is the process of renewal, regrowth, or restoration of a tissue, organ, or organism after damage, injury, or disease.

[0064] As used herein, remodeling refers to the process by which the body adapts to and integrates an implanted medical device. This process involves the interaction between the implant and the surrounding tissues, which can lead to changes in the structure and composition of the tissue, and in healing.

[0065] As used herein, recellularization and remodeling are considered to be within the definition of regeneration and healing.

[0066] In the most preferred embodiments, the tissue of the present invention, when implanted, does not mechanically weaken during the healing process. One of the benefits of a tissue of the present invention is that it does not need a scaffold or the like. This feature is in contrast to products that have a synthetic or biological portion that is specifically degradable, e.g., a scaffold. This feature is also distinct from the tissue as it is being manufactured. During formation, the ECM-producing cells degrade fibrin until a collagenous tissue is formed. The cell-containing tissue contains no, very little, or not detectable amounts of fibrin.

[0067] An embodiment of the invention includes but is not limited to a tissue of the present invention that recellularizes without first degrading.

[0068] An embodiment of the invention includes a product or implant formed from the tissue of the present invention, wherein the tissue includes an external support structure.

[0069] Embodiments of the invention include but are not limited to the form of tissue delivery. Examples include endovascular delivery and surgical implant.

[0070] A tube or tubular material of the present invention may be positioned to replace, surround, contain, or enclose a body structure. Examples include but are not limited to one or more coronary arteries.

[0071] The graft or implant of the present invention may be used in a method of treating a patient having a wide variety of conditions, diseases, or injuries, the common theme of which is that the treatment involves a tissue implant. Exemplary diseases and conditions include but are not limited to arrythmia, wound infections, kidney / renal failure, thrombosis / embolization, aneurysm, patient infection, and patient immune response.

[0072] In another embodiment of the invention, the tissue may include structures or agents (e.g., active agents) within the surface of the CT tissue.

[0073] The tissue of the present invention may be any size or shape. In a preferred embodiment, the tissue is the form of a wrap, conduit, sheet, cover, envelope, or tube.

[0074] In accordance with the present invention, the tissue or tube may be enclosed or covered by an external support structure (ESS). In the most preferred embodiments, the ESS substantially covers the tissue from one end to the other. Insome embodiments, the ESS is longitudinally flexible or compliant enough to expose one or both ends of the tube, thereby allowing access to one or both tube ends for surgical procedures.

[0075] In another embodiment, the ESS is of a size and dimension wherein one or both tube ends are exposed or not covered by the ESS.

[0076] The present invention is a graft, prosthesis, or covering formed from constructed (CT) regenerative, and / or engineered tissue. As used herein, constructed or engineered refers to the fact that the inventors and others may produce or construct the tissue, e.g., the tissue is not a product of nature. In preferred embodiments of the invention, the tissue mediates regeneration without causing degradation of tissue and other biological material in the area of the implant site. The invention includes methods of making the tissue and methods of making the graft or prosthesis.

[0077] The tissue may be formed by combining ECM-producing cells in the presence of fibrinogen and thrombin under conditions that permit the formation of regenerative tissue. Typically, the process involves forming a cell-seeded suspension comprising ECM producing cells, fibrinogen, and thrombin. The suspension is then cast over a form and allowed to incubate. During incubation, an ECM / fibrin / collagen tissue begins to form. As part of that process, compaction and fiber alignment may occur, leading to remodeling of the ECM / collagen / fibrin tissue as it forms. The tissue is then cultured until it matures, e.g., is substantial enough to be used for its intended purpose. The resulting cell-containing tissue is the decellularized. The tissue of the present invention is cultured from completely biological raw materials and allogeneic dermal cells. For example, see the patents and patent applications listed below.

[0078] In preferred embodiments of the invention, the hydrogel - the starting ingredients in a suspension - allows the tissue to grow in a volumetric 3-D process also known as casting. In many ways, the process from initial ingredients to a complete tissue represents a remodeling continuum. In contrast, most if not all typical tissue engineering methods use a synthetic and / or immunogenic scaffold or the like to grow the tissue in a 2-D manner (cell suspension seeded on the surface). The growth eventually produces a 3-D construct, but the growth in scaffold-based constructs is different that the volumetric 3-D casting growth in tissues of the present invention.

[0079] A preferred embodiment of the invention is any structure or shape formed from the tissue of the present invention, including but not limited to a tubular graft.

[0080] In accordance with embodiments of the present invention, any prosthesis may be formed in whole or in part using regenerative tissue (RT) or engineered tissue. RT, as used herein, refers to tissue formed or processed as disclosed in the following: 2007 / 061800; WO 2007 / 092902; 2016 / 0203262; WO / 2004 / 018008; WO 2004 / 101012; PCT / US21 / 62709 (filed 09 December 2021 ); PCT / US2017 / 026204 (filed 5 April 2017); U.S. Patent 10,111 ,740; U.S. Patent 10,105,208; U.S. Patent 10,893,928; U.S. Patent 8,192,981 ; U.S. Patent 8,399,243; U.S. Patent 8,617,237; U.S. Patent 8,636,793; U.S. Patent 9,034,333; U.S. Patent 9,126,199; U.S. Serial No. 17 / 139,575 filed 12 / 31 / 2020 (issue fee paid); U.S. Serial No. 16 / 500,147 filed 10 / 02 / 2019 (issue fee paid); U.S. Serial No. 10 / 523,618; U.S. Serial No. 10 / 556,959; U.S. Serial No. 13 / 771 ,676; 2015 / 0012083; 2009 / 0319003; 2011 / 0020271 ;2012 / 0230950; 2013 / 0013083; 2014 / 0330377; 2014 / 035805; 2017 / 0135805; 2017 / 0296323; 2017 / 0306292; USP 8198245; USP 9127242; USP 9556414; USP 9657265; and USP 9650603; all of which are hereby incorporated in the entirety be reference.

[0081] In one embodiment of the invention, the bioengineered tissue may be made according to U.S. Patent 10,111 ,740; U.S. Patent 10,105,208; U.S. Patent 10,893,928; and U.S. Patent 11 ,589,982, all Tranquillo, et al., each incorporated in its entirety be reference. Any process or method for producing engineered tissue involving ECM-producing cells in a hydrogel is included within the scope of the present invention.

[0082] The CT of the present invention, may be characterized by lack of evidence of patient infection (in vivo)', lack of evidence of patient immune response (in vivo)', lack of evidence of toxicity; lack of evidence of implanted tissue degradation; lack of evidence of residual cellular debris (e.g., particle shedding from the tissue, in contrast to polymer degradation and erosion); modified (e.g., reduce or eliminate) inflammation, calcification characteristics, resorbability, resorption, absorption, suture retention, size and shape, thinness (e.g. dilatation or aneurysm formation), collagen content, and other characteristics and properties that will become clear from the description of the invention.

[0083] The CT of the present invention is distinct from certain other kinds of constructed tissue in the use of completely biological raw materials and allogeneicdermal cells; and in the use of crosslinked fibrinogen that is later degraded during the culturing process. Also, the CT of the present invention can be contracted or allowed to contract, for example, in the longitudinal direction and / or in the radial direction, among others. In accordance with some embodiments of the invention, the fibers in the tissue may align or become aligned, believed to be partially due to fibrin having no or little resistance to contraction that occurs naturally as part of the collagen / ECM formation process. The inventors also believe that radial and / or longitudinal contraction occurs in part naturally as an inherent function of tissue forming as described herein. In another embodiment of the invention, the contraction may be scalable or intentionally controlled to enhance, promote, or achieve one or more tissue characteristics, e.g., fiber alignment, or tensile strength, or suturability. Furthermore, the CT of the present invention does not include any synthetic materials, as is typical in other processes that use PLA, PGA, or the like.

[0084] The CT of the present invention may be characterized by one or more of the following: non-oriented fibers; oriented fibers; thickness up to about 2 mm, preferably between about 100 pm and about 800 pm; diameters greater than about 1 mm; diameters from about 1 mm to about 40 mm, preferably from about 2 mm to about 25 mm, most preferably from about 3 mm to about 16 mm; lengths greater than about 1 cm; lengths from about 1 cm to about 100 cm, preferably 10 cm to 30 cm, and most preferably about 12 cm to about 22 cm; non-immunogenic or minimally immunogenic; a tissue, sheet or shape that is anisotropic; a tissue, sheet, or shaped structure produced by a process that includes scaled contraction (as described above); a sheet or shape that is suitable for cutting into shapes, e.g., by scalpel, die, or laser; suppleness; suturability; no or little calcification during life of implant; crosslink density, or variations of crosslink density through the material thickness; collagen concentration; collagen density, or variation of crosslink density through the material thickness; remodeling proclivity; absorption; resorption; degradability, regions of greater stiffness; regions of greater flexibility,.

[0085] In some embodiments of the invention, the diameter and diameter range may be determined by the type of graft or implant being used. The diameter may be any diameter, and typically less than about 6 mm (but it can also be greater). For example, a CABG graft preferably has a diameter from about 3 mm to about 6 mm.

[0086] In some embodiments of the invention, the length and length range may be determined by the type of graft or implant being used. The length may be anylength. For example, a CABG graft preferably has a length from about 15 cm to about 25 cm, which is then sized as needed during the implant procedure, typically from about 8 cm to about 15 cm.

[0087] In accordance with some embodiments of the invention, either or both of the diameter and the length can be tapered.

[0088] In some embodiments of the invention, the thickness and thickness range may be determined by the type of graft or implant being used. For example, a CABG graft preferably has a thickness from about 0.3 mm to about 0.8 mm; an AV access graft may have a thickness from about 0.3 mm to about 1 .0 mm.

[0089] In accordance with the present invention, the external support structure is sized to fit around an external surface of the graft.

[0090] The tissue or a product of the present invention may be used to treat or repair other types of tissues including but not limited to tubular non-vascular applications, muscle, tendon, organs (e.g., kidneys and liver) skin, trachea, ureter, vasculature, bladder, fascia, and uterus.

[0091] Now referring to the Figures, Figure 1 shows an implant 10 comprising a tissue material 11 covered by an external support structure 12. This figure shows the implant configured (e.g., curved) as might be beneficial when placed near human anatomical structures in vivo (not shown). This view illustrates exposing the anterior end (13) and the posterior end (14) of the tissue. The uncovered or exposed ends may be used as anastomosis sites to attach to a portion of the tissue or implant to the vascular system.

[0092] Figure 2 shows a close-up view of a portion of the ESS.

[0093] Figure 3 shows a construction drawing (flat pattern view) of an unexpanded ESS of the present invention. Figure 4 shows one expanded cell of the ESS shown in Figure 3.

[0094] Figure 5 shows an end 50 of an exemplary ESS 51 . In this embodiment, in comparing cell 52 to cell 53, cell 52’s struts are more widely spaced, allowing variation in flexibility. The inventors have found that longer struts tended to make the frame less flexible (e.g., end segment 52), that repeating segment 53 tended to make the frame more flexible. The flexibility or bending ability in the frame occurs at the connection points between the struts and the connectors, so the more connection points per length of frame, the more places for the frame to bend, all of which leads to greater flexibility.

[0095] Figure 6 compares two different configurations of an ESS of the present invention. Figure 6A shows a preferred embodiment wherein one lobe 60 nests within another lobe 61 without individual struts contacting. In Figure 6B the struts of a first lobe contact 62 the struts of a second lobe. Configurations such as Figure 6A are more flexible than configurations such as Figure 6B.

[0096] Figure 7 shows the exemplary placement of an implant 70 in relation to the various anatomical structures of a model heart. Implant 70 has a tubular tissue tube 71 partially covered by an ESS 72 of the present invention. Implant 70 also shows two ends 73 and 74 of the tissue uncovered or exposed - these are both anastomosis sites.

[0097] Figure 8 is a Kaplan Meier plot of primary and secondary patency of a graft of the present invention.VARIOUS METHOD STEPS:

[0098] By mimicking the extra cellular matrix of the natural environment, tissues of the present invention can be and are grown having desirable or beneficial structural properties, which eventually develop toward a native-like architecture (i.e. , the tissues of the present invention are a biomimetic material). In preferred embodiments of the invention, the tissue of the present invention may be handled in a similar way like a native vein or artery when surgically implanted.

[0099] Some embodiments of the invention may further include storing and / or sterilizing a medical device or tissue of the present invention. These embodiments may include preselected storage solution; preselected sterilization solution or technique; storage packaging; and / or sterilization packaging. In one embodiment, the tissue may be stored in PBS and refrigerated until use. In another embodiment, the tissue may be partially or fully dehydrated. In one embodiment, the storage is in a sterile dry container. Other storage / sterilization processes may include one or more additives known to those with skill in the art. In another embodiment, the tissue may be E-beam sterilized in PBS alone.

[0100] One skilled in the art will recognize that other storage and sterilization protocols may be used with the tissue of the present invention.

[0101] In some embodiments of the invention, the tissue when implanted in humans did not degrade prior to recellularization or body cell infiltration.

[0102] In some embodiments, a CABG tissue conduit is intended to be used with an outer metal support providing outer support, preventing or reducing conduit kinking; prevents or reduces tissue abrasion or wear; maintains uniform lumen diameter; and prevents or controls tissue dilation. The external support may also reduce or prevent abrasion to other tissues or parts of the anatomy that come into contact with the implant. The outer or external stent may be a standalone, commercially available product.OTHER EMBODIMENTS:

[0103] A tissue of the present invention may also define or enclose a total volume from about 3 mm3to about 65,000 mm3.

[0104] The tissue of the present invention may include one or more collagens, including but not limited to collagen types I, III, and VI; tenascin, and fibronectin.

[0105] A tissue product of the present invention may be unsupported or may further include a support member, such as a nitinol stent or scaffold. The support member many be internal, external, or embedded in the tissue. In preferred embodiments of the invention, the implant is a tissue including an external stent that provides the benefits described above.

[0106] As noted above, a tissue product of the present invention may be a tube or conduit, or tubular in shape. A preferred embodiment is a continuous tubular shape. Typically, these shapes include or enclose a lumen through the longitudinal length of the construct. The lumen can be open or closed at one or both ends and may be shaped at one or both ends.

[0107] In some embodiments of the invention, the implanted tissue, graft, or implant is capable of endothelialization, even substantial endothelialization, a feature that provides evidence of the long-term biocompatibility of the tissue and mediation in vascular repair.

[0108] In various embodiments of the invention, including but not limited to CABG, pre-clinical studies showed exemplary hemodynamics and biomimetic function.

[0109] In preferred embodiments of the invention, CT tissue grafts have been shown to provide anatomical and functional characteristics that mimic native structures, e.g., native vessels around the heart and saphenous veins.

[0110] In preferred embodiments of the invention the CT tissue was evaluated for its mechanical properties and hemodynamics. All of these evaluations showed that the CT tissue of the present invention has functional compliance and “hand” to native structures.

[0111] In some embodiments of the invention, the CABG is a sterile, ~4-6 mm inner diameter, ~15 cm long tubular conduit composed of a naturally produced acellular human collagen matrix. The collagen matrix is produced by culturing human dermal fibroblast (HDF) cells in a controlled process. The product possesses structural integrity with burst strength and suture retention strengths similar to native arteries and veins (e.g., 1000-5000 mmHg burst strength).

[0112] The present invention also is a surgical kit comprising one or more of the following: a regenerative tissue implant or graft processed or produced according to the present invention; one or more instruments for implanting the graft; a rinse tray; a rinse solution, e.g., heparin; and suture material.

[0113] One of the embodiments of the present invention is a sterile closed package containing a biological material of the present invention and an external support structure configured to slide over the biological material. Typically, a separate container may hold individual or multiple samples having known size or dimensions.

[0114] The present invention also includes tubular grafts in a variety of diameters and lengths as needed to match the anatomy.

[0115] An implant of present invention maybe delivered in any medically acceptable manner. In a preferred embodiment, the tissue is surgically delivered. In another embodiment, the tissue is delivered via catheter or tube.

[0116] One skilled in the art will recognize that the processes steps described herein may be variously modified and a wide variety of ways in order to achieve a tissue with certain properties, according to present invention.Definitions

[0117] The following definitions are used in reference to the invention:

[0118] As indicated herein, a decellularized vessel consists essentially of the extracellular matrix (ECM) components of the vascular tree. ECM components can include any or all of the following: fibronectin, fibrillin, laminin, elastin, members of the collagen family (e.g., collagen I, III, and IV), glycosaminoglycans, groundsubstance, reticular fibers and thrombospondin, which can remain organized as defined structures such as the basal lamina. Successful decellularization is defined as the absence of detectable myofilaments, endothelial cells, smooth muscle cells, and nuclei in histologic sections using standard histological staining procedures.

[0119] As used herein, biomimetics or biomimicry refer to imitating the models, systems, and elements of nature for the purpose of solving complex human or animal problems. In the present invention, biomimetics is used for therapeutic purposes.References:

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[0160] 41 . Connolly et al., “Rivaroxaban with or without aspirin in patients with stable coronary artery disease: an international, randomised, double-blind, placebo- controlled trial,” Lancet, 391 :205-18 (2018).

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[0162] 43. Avezum et al., “Apixaban in Comparison With Warfarin in Patients With Atrial Fibrillation and Valvular Heart Disease: Findings From the Apixaban for Reduction in Stroke and Other Thromboembolic Events in Atrial Fibrillation (ARISTOTLE) Trial,” Circulation, 132:624-32 (2015).

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[0164] 45. McAllister et al., “Effectiveness of haemodialysis access with an autologous tissue-engineered vascular graft: a multicentre cohort study,” Lancet, 373:1440-46 (2009).EXAMPLESExample 1

[0165] A tissue of the present invention was implanted as a CABG graft into an ovine model, and after one year showed long-term performance and regeneration into a living blood vessel.

[0166] A tissue of the present invention was implanted as a pediatric vascular conduit into a lamb model, and after one year showed somatic growth and regeneration.Example 2

[0167] The flexibility or bend compliance of an exemplary support of the present inventions was compared to different designs and various commercially available stents. The preferred design of the present invention was found to be more flexible and bend compliant than the Innova™ commercially available stent which has been previously evaluated with the tissue of the present invention. The flexibility of the present invention was found to have bend compliance that falls between Innova™ and Pulsar-18, both commercially available stents that have been proven safe and effective as permanent implant in patients. This gives the present invention the flexibility to curve with the anatomy seen of the CABG graft while remaining durable enough to be safe for permanent implant in patients.Example 3

[0168] Table 1 . Summary of Human Vessel and Comparable BiologicallyEngineered Vascular Grafts* Only ranges are reported** probe burst test in place of pressurized burst to decrease sample volume requirement.Example 4

[0169] Tissue recovered from the animals were fixed in formalin and processed for staining.• H&E-stained sections show small aggregates of inflammatory / immune cells including eosinophils, PMNs, lymphocytes, and macrophages within the abluminal area of the conduits, but rated as absent to minimal / diffuse in all 6- month explants. The 3-month explant (BAVG 7), where they were rated as marked / focal in the abluminal areas of the graft in some sections.• Most trichrome-stained sections showed elongated cells with circumferential orientation in both the 3-month and 6-month explants, indicative of positive remodeling. The conduits all possessed abundant collagen as seen in the trichrome- stained sections.• Fibrin immunohistochemical staining confirmed a thin thrombus layer on the luminal surface of the 3-month explant, also evident in the trichrome staining. Minimal fibrin staining was evident in the 6-month explants.• Verhoeff-van Gieson stained sections revealed mature elastic fibers within the implant near the proximal anastomosis in two of the 6-month explanted conduits, but not seen in other sections.• All conduits at 3 and 6-month stained negative for calcification with Von Kossa stain.• Many cells populating the interstitium of the conduits stained positive for vimentin in all sections for both 3 and 6 months.• The majority of elongated cells in a distinct region adjacent to the luminal surface of many sections stained positive for alpha-smooth muscle actin (aSMA) at both time points. The thickness of this region, when clearly identifiable, was highly variable but commonly between 100-300 pm.• There was sporadic smoothelin staining of the elongated cells along the base of the aforementioned region.• At 3 months, CD31 staining showed limited endothelium on the distal conduit region with none noted mid-conduit. Whereas the 6-month sections showed complete endothelium in the proximal and distal regions in 2 of 3 patent conduits, and one mid-conduit region, while the remaining sections showed some level of incomplete endothelium coating.• Consistent with the ultrasonography, no stenosis was evident at the distal anastomosis based on histological assessment, with minimal to mild (generally < 250 urn in thickness) intimal thickening and fibrosis in the vein at the junction with the implant.

[0170] Overall, the immunological analysis shows no adverse acute or chronic immune reaction or sensitization to implanted engineered tissue.Example 5

[0171] In Gen 1 study, 4 animals survived past 6 months with 3 planned explants accessed for angiography, gross pathology, and histopathology to evaluate remodeling of a CABG graft and any adverse outcomes in myocardial tissue.Animals 22S0026, 22S0032 and 22S0034 all had a patent conduit, no abnormal findings in gross pathology and no myocardial tissue damage or evidence of emboli.

[0172] The angiogram at 180 days showed uniform diameter transition from a CABG into the native coronary. Further, gross examination showed distal anastomosis without evidence of hyperplastic response. The anastomotic sutures were visible in the 180-day explants. A cross-section of the graft showed remodeled tissue with areas of glossy lumen surface and areas with thin layer of thrombus. Explanted tissue for n=2 conduit was mechanically tested with an average lumen burst strength of 3500±1405 mm Hg, in comparison to implant lumen burst strength of 3109±624 mm Hg, confirming remodeling of the graft without any weakening of the tissue mechanical strength.

[0173] Histology of explants at early timepoint confirmed a lack of overt immune response with only few neutrophils migrating at day 8. After 180 days of implant, the implanted graft was fully remodeled with interstitial cells and evidence of endothelial cells in the proximal third of the conduit. While lumen endothelium lacked in the entire length of implant, the interstitial cell remodeling looked similar along the length of implant. Further, remodeling was seen with the graft in coronary implant is comparable to previous reported remodeling with the Baboons after 6 months implant as arteriovenous conduit and in sheep as femoral artery implant.Example 6

[0174] For 22S0051 , a 39-day explant permitted gross examination of the external support and any gross differences from the graft Gen 1 implants (non-stentsupported tissue conduit). Grossly, the stent was well fused within the scar tissue around the heart. There was no evidence of any inflammatory or fibrotic response. Once exposed and removed from the heart, there was only a thin layer of remodeled tissue around the external stent. The dissected lumen surface showed predominantly fresh clots with one area of thrombus 2-3 cm from distal anastomosis. There was no evidence of stenosis or hyperplastic response at either anastomosis. The explanted heart sections also showed no gross infraction.

[0175] Histological examination of the mid conduit from 22S0051 showed remodeling of the implanted graft comparable to Gen 1 explants within the same time duration. The lumen surface shows spare inflammatory cells, mostly neutrophils. The tissue around the stent external to the conduit shows remodeled collagenous structure with no overt immune response.

[0176] A graft (Gen 2) with external stent support provides the benefit of no kink or sharp bend seen up to 30 days post-implant. Further, there was no complication in implant procedure. Similar stent support has been used in at least two major clinical trials to support autologous saphenous vein (30, 31 ). The remodeling both grossly and histologically shows no immediate adverse effects of the external support. Overall, the initial data supports the use of an external stent with a CABG to prevent adverse events.Example 7

[0177] In the studies of a tissue conduit in the baboon model and the ovine model, all explanted grafts were histologically evaluated for remodeling of the graft tissue. In the ovine model, in explanted grafts at 4 and 8 weeks, an acellular band of original graft was surrounded by remodeled matrix tissue on both luminal and abluminal surfaces. By 6 months, this acellular region had much reduced width suggesting remodeling of the original tissue matrix by invading host cells. The explanted tissue at 180 days was evaluated for mechanical properties and exceeded the properties of implant material, suggesting host tissue formation leading to a stronger graft. As the original matrix and remodeled matrix are both collagenous in nature, no immunostaining is available to differentiate and quantitate the new cell- produced collagen from the implanted collagen.

[0178] In the baboon model implanted with tissue grafts, the graft was fully cellularized by 3 months, suggesting complete remodeling of the implanted graft byhost cells. This was confirmed by an increase in mechanical burst pressure strength of the explanted graft, which was 136% of implanted grafts. At 6 months, the explanted grafts were also stronger by 156% compared to implanted grafts, including areas with repeated puncture using a 16G dialysis needle. The ECM remodeling is a natural phenomenon and does not include any chemical processes and products that would not occur naturally.Example 8

[0179] Tissue conduit with external support spanning 80% of the conduit length was implanted for a duration of 12 months. The angiogram assessment during the 12 months showed uniform conduit diameter within the externally supported segment with no kink, no dilation and maintained uniform diameter. The section not covered by the external support showed evidence of dilation and nonuniform diameter.

[0180] Modifications, additions, or omissions may be made to the systems, apparatuses, and methods described herein without departing from the scope of the disclosure. For example, the components of the systems and apparatuses may be integrated or separated. Moreover, the operations of the systems and apparatuses disclosed herein may be performed by more, fewer, or other components and the methods described may include more, fewer, or other steps. Additionally, steps may be performed in any suitable order. As used in this document, “each” refers to each member of a set or each member of a subset of a set.

[0181] While the invention has been described in some detail by way of illustration and example, it should be understood that the invention is susceptible to various modifications and alternative forms and is not restricted to the specific embodiments set forth in the Examples. It should be understood that these specific embodiments are not intended to limit the invention but, on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.

Claims

CLAIMS:1 . An implant suitable for use in humans and animals comprising a conduit engineered or constructed from tissue formed using a composition comprising an ECM producing cell, thrombin, and fibrinogen; and an external support structure.

2. The implant of claim 1 wherein said external support comprises one or more of the following: formed from superelastic metal; laser cut; compressible longitudinally; compressible sufficient to expose one or both ends of a tissue enclosed by the support; MRI compatible; sufficiently rigid to prevent kinking; kink resistance as the tissue or implant curves; sufficiently flexible axially and longitudinally to curve around anatomical structures; abrasion resistant; atraumatic edges; slidable over a tissue, including a tissue tube or conduit; up to about 300 mm in length.

3. The implant of claim 1-2 wherein the external support includes struts that are configured to nest within an adjoining strut.

4. The implant of claim 1 wherein the implant is a coronary bypass graft; a peripheral bypass graft; a vascular bypass graft; or a pediatric bypass graft.

5. A coronary artery bypass implant comprising a constructed tissue formed from a composition comprising ECM-producing cells, fibrinogen, and thrombin; and an external support.

6. The implant of claim 1 wherein the tissue is acellular.

7. The implant of claim 1 wherein the tissue induces recipient cellular regeneration or remodeling at or near the site of implant.

8. The implant of claim 1 wherein the tissue or the implant is biomimetic.

9. The implant of claim 1 wherein the implant is a pediatric bypass.

10. The implant of claim 1 wherein the tissue comprises non-uniform layers of collagen.

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