Biomatrix-impregnated porous conduits for tissue engineering.

Abstract

Disclosed herein are conduits and methods of making conduits, including tubular structures that include a porous biocompatible polymer embedded in a biocompatible extracellular matrix material and an internal luminal space. The conduits are for use in devices such as artificial tissues and organs. The method of making the conduits may include providing a tubular structure having an internal luminal space and including a porous biocompatible polymer, perfusing the pores and luminal space of the tubular structure with a first solution, replacing the first solution by perfusing the pores and luminal space of the tubular structure with an aqueous solution that includes a liquefied extracellular matrix material, and polymerizing the liquefied extracellular matrix material to embed the tubular structure in the extracellular matrix material to form the conduit.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 152,749 (filed 02 / 23 / 2021, the contents of which are incorporated by reference herein in their entirety). [Background technology]

[0002] Vascular perfusion supplies organs with blood, enabling functions such as gas and solute exchange. Similar luminal structures (i.e., "tubes") enable similar functions such as waste removal. In natural tissues, these tubes rely on the highly organized structure of their collagen fibers to provide mechanical strength and durability. These highly sophisticated mechanisms allow for thin vessel walls that can withstand pressures of hundreds, sometimes thousands of mmHg, and can be sutured without tearing, yet remain compliant, soft, and permissive for cell growth.

[0003] To develop engineered tissues, specifically vasculatures that can recapitulate and replace native function, a similarly robust conduit structure for engineered scaffolds is needed. This has proven extremely challenging, and progress to date has been limited. However, sophisticated approaches toward standalone engineered vascular grafts and blood vessels are beginning to show promise (see Dahl, et al (2011) Science Translational Medicine, 3(68), 68ra9-68ra9). These grafts are fabricated with a collagen layer similar to native blood vessels, which gives them substantial strength. However, the methods by which they are made prohibit integration and / or co-fabrication with engineered organ tissues or cells. Despite their sophisticated nature, these grafts are not integrated with a scaffold structure that could provide function, and in fact have been purposefully developed only as standalone blood vessels for anastomosis with native vasculature. To create organ scaffolds with fully integrated vasculature that can meet the demands of native physiology, it is necessary to develop blood vessels of similar composition that can be fabricated in parallel or ideally in situ within engineered organ scaffolds. This poses some significant challenges, as the materials most suitable for tissue engineering (hydrogels and natural extracellular matrix derivatives) are unsuitable for constructing mechanically robust conduits. Enhancement attempts have focused on synthetic meshes and other materials that provide support structures, but the macroscale nature of the material structures and interfaces with hydrogels has hindered the development of integrated tubular structures, and these implants suffer from mechanical instability. These limited attempts to develop tubular structures that interface with engineered scaffolds have left the most sophisticated engineered tissue scaffolds dependent on surrounding chambers or other artificial, foreign constructs for sustained perfusion (see Homan, et al. (2016) Sci Rep 6, 34845). Attempts to anchor synthetic or bioartificial tubes to engineered scaffolds fail due to poor bonding and mechanical mismatch between the tubes and bulk materials. To advance the development of engineered, fully biological vasculatures and organ scaffolds, it is important to develop approaches for vascular construction that are flexible and easily integrated into a variety of fabrication approaches. Beyond the recapitulation of vascular and luminal structures in engineered tissues, there are many potential in vitro applications for mechanically stable hydrogel-based engineered tubular structures, e.g., recapitulating biological functions, physiology, or developmental processes. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Dahl,et al(2011)Science Translational Medicine,3(68),68ra9-68ra9 [Non-Patent Document 2] Homan,et al.(2016)Sci Rep 6,34845 Summary of the Invention [Means for solving the problem]

[0005] Herein, we present a novel solution whereby mechanically robust tubular structures are fabricated using highly microporous (up to 80% and above porosity, 0.1-0.5 μm pore size) support structures of expanded synthetic polymers such as polytetrafluoroethylene tubing (ePTFE). Low porosity, thick-walled configurations of ePTFE have been used and implanted as acellular vascular grafts for decades, providing a deep pool of data on biocompatibility, making ePTFE an ideal material for tissue engineering. In addition to the standalone construction of tubular structures, this novel process allows for the integration of support structures and co-fabrication of tubular vessels in a holistic manner within the fabrication process of engineered tissue scaffolds or constructs. This results in an integrated end product with mechanically stable vasculature and tubular structures that can meet performance requirements for implantation, but that are also mechanically and materially cohesive structures. This novel solution overcomes current challenges for perfusion of engineered vessels and engineered tissues.

[0006] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0007] [Figure 1] 1 shows a schematic diagram for manufacturing a conduit according to one embodiment of the present invention, the method includes step A: providing a tubular structure having an internal luminal space and comprising a porous biocompatible polymer, step B: perfusing the pores and luminal space of the tubular structure with a first solution, step C: replacing the first solution by perfusing the pores and luminal space of the tubular structure with an aqueous solution comprising a liquefied extracellular matrix material, and step D: embedding the tubular structure in the extracellular matrix material by polymerizing the liquefied extracellular matrix material to form a conduit. [Figure 2A]Figure 2 shows the porosity of an ePTFE tube. Figure 2A provides SEM and optical microscope images of the ePTFE tube. Figure 2B shows the porosity of the ePTFE tube as a function of density and pore size. [Figure 2B] Figure 2 shows the porosity of an ePTFE tube. Figure 2A provides SEM and optical microscope images of the ePTFE tube. Figure 2B shows the porosity of the ePTFE tube as a function of density and pore size. [Diagram 3] Histology (cross section) of fabricated tubular constructs in which GFP+ HUVEC cells were seeded and cultured to provide complete endothelial coverage. [Figure 4] 1 shows images of an embodiment of an engineered construct of the present invention fabricated using ePTFE tubular conduits. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] Some aspects of the present disclosure relate to conduits having a tubular structure comprising a porous biocompatible polymer embedded in a biocompatible extracellular matrix material and an interior luminal space.

[0009] The porous biocompatible polymer is not limited and can be any suitable porous biocompatible polymer. In some embodiments, the porous biocompatible polymer comprises or consists of polytetrafluoroethylene, polydimethylsiloxane, polycarbonate, or silicone. In some embodiments, the porous biocompatible polymer is expanded polytetrafluoroethylene.

[0010] The pore size of the pores in the biocompatible polymer is not limited and can be any suitable pore size. In some embodiments, the average or median pore size (i.e., pore diameter) is about 0.1 μm, 0.15 μm, 0.2 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, or about 0.5 μm. In some embodiments, the average or median pore size is about 0.1-0.5 μm. The porosity (i.e., the ratio of void volume to total volume) of the porous biocompatible polymer is also not limited and can be any suitable porosity. In some embodiments, the porosity is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more. In some embodiments, the porosity is greater than 50%. In some embodiments, the porosity is between 50% and 60%, between 50% and 70%, or between 50% and 80%.

[0011] The biocompatible extracellular matrix material can be any suitable biocompatible extracellular matrix material known in the art, without limitation. The biocompatible extracellular matrix material can be a biological, synthetic, or composite material, such as collagen, decellularized natural extracellular matrix, gelatin, gelatin methacryloyl, other hydrogels, cellulose, or other materials. In some embodiments, the biocompatible extracellular matrix material is polymerized. In some embodiments, the biocompatible extracellular matrix material comprises a polymerized material selected from gelatin, gelatin methacryloyl, collagen, or decellularized natural extracellular matrix.

[0012] The interior luminal space of the conduit may be of any suitable shape for connection to a biological fluid supply (e.g., blood vessels, lymphatic vessels, renal collecting ducts, bronchioles, endocrine or hormonal ducts, etc.). In some embodiments, the interior luminal space has a long axis. In some embodiments, the interior luminal space is cylindrical or approximately cylindrical. In some embodiments, the interior luminal space has a diameter (e.g., on the long axis) of 0.1 to 10 cm. In some embodiments, the interior luminal space has a diameter (e.g., on the long axis) of 0.1 to 1 cm. In some embodiments, the interior luminal space has a diameter (e.g., on the long axis) of 0.1 to 0.5 cm. In some embodiments, the interior luminal space has a diameter (e.g., on the long axis) of at least 0.1 cm, 0.2 cm, 0.3 cm, 0.5 cm, or 1 cm.

[0013] In some embodiments, the conduit further comprises mammalian cells. The mammalian cells are not limited and can be any suitable mammalian cells. In some embodiments, the mammalian cells are epithelial or endothelial cells. In some embodiments, the mammalian cells are selected from smooth muscle cells, fibroblasts, and / or endothelial cells. In some embodiments, the mammalian cells comprise at least two, at least three, at least four, or more cell types. In some embodiments, the mammalian cells comprise or consist of one or more of human vascular endothelial cells, human renal epithelial cells, human intestinal epithelial cells, human endocrine cells, or human epithelial cells.

[0014] In some embodiments, the conduit is not attached to another structure (e.g., the conduit prior to use). In some embodiments, one end of the conduit is attached (e.g., surgically anastomosed) to an artificial tissue scaffold. In some embodiments, the conduit is attached at one end to native tissue (e.g., blood vessels, lymphatic vessels, renal collecting ducts, bronchioles, endocrine or hormonal ducts, etc.). In some embodiments, the conduit is attached at both ends to native tissue (e.g., blood vessels, lymphatic vessels, renal collecting ducts, bronchioles, endocrine or hormonal ducts, etc.). In some embodiments, one or both ends of the conduit are surgically anastomosed to native vasculature in vivo.

[0015] Some aspects of the present disclosure relate to a device comprising a conduit as described herein. In some embodiments, the device comprises a conduit as described herein having a first end embedded (e.g., embedded and integrated) in a tissue scaffold. In some embodiments, the tissue scaffold comprises the same biocompatible extracellular matrix material as the conduit and a vascular channel system. In some embodiments, the luminal space of the conduit is configured to be in fluid communication with the vascular channel system. The tissue scaffold is not limited. In some embodiments, the tissue scaffold is a tissue scaffold described in PCT Publication No. WO2018 / 112480 (published June 21, 2018, which is incorporated herein by reference in its entirety). In some embodiments, the tissue scaffold comprises one or more mammalian cell types. The mammalian cell types are not limited and may be mammalian cell types described herein.

[0016] Some aspects of the present disclosure relate to a device including a tissue scaffold and a conduit, where the tissue scaffold includes a biocompatible extracellular matrix material and a vascular channel system, and the conduit includes a tubular structure including a porous biocompatible polymer embedded in the biocompatible extracellular matrix material and an internal luminal space, where the tubular structure has a first end embedded and integrated in the biocompatible extracellular matrix material of the tissue scaffold and a second end configured to connect to a fluid supply, where the luminal space is configured to be in fluid communication with the fluid supply and the vascular channel system. The conduit is not limited to any suitable conduit as described herein. The tissue scaffold is also not limited to any suitable tissue scaffold as described herein. The biocompatible extracellular matrix material is also not limited to any suitable biocompatible extracellular matrix material as described herein. In some embodiments, the tissue scaffold includes one or more mammalian cell types. The mammalian cell types are not limited to any mammalian cell types as described herein.

[0017] The porous biocompatible polymer can be any porous biocompatible polymer described herein, without limitation. In some embodiments, the porous biocompatible polymer is expanded polytetrafluoroethylene.

[0018] The pore size of the porous biocompatible polymer is not limited and can be any pore size described herein. In some embodiments, the porous biocompatible polymer comprises pores having a pore size of 0.1 to 10 μm in diameter and a porosity of more than 50%.

[0019] In some embodiments, the biocompatible extracellular matrix material is polymerized and comprises gelatin, gelatin methacryloyl, collagen, or decellularized natural extracellular matrix.

[0020] The interior luminal space of the conduit may be of any suitable shape for connection to a biological fluid supply (e.g., blood vessels, lymphatic vessels, renal collecting ducts, bronchioles, endocrine or hormonal ducts, etc.). In some embodiments, the interior luminal space has a long axis. In some embodiments, the interior luminal space is cylindrical or approximately cylindrical. In some embodiments, the interior luminal space has a diameter (e.g., on the long axis) of 0.1 to 10 cm. In some embodiments, the interior luminal space has a diameter (e.g., on the long axis) of 0.1 to 1 cm. In some embodiments, the interior luminal space has a diameter (e.g., on the long axis) of 0.1 to 0.5 cm. In some embodiments, the interior luminal space has a diameter (e.g., on the long axis) of 0.3 to 0.7 cm. In some embodiments, the interior luminal space has a diameter (eg, on the long axis) of at least 0.1 cm, 0.2 cm, 0.3 cm, 0.5 cm, 0.6 cm, 0.8 cm, 0.9 cm, or 1 cm.

[0021] In some embodiments, the conduit further comprises mammalian cells, optionally selected from smooth muscle cells, fibroblasts, and / or endothelial cells.

[0022] The volume of the vascular channel system is not limited and can be any suitable volume for use when implanted in vivo or for use in ex vivo or in vitro applications. In some embodiments, the vascular channel system has a volume of about 0.01 mL to about 10 L. In some embodiments, the vascular channel system has a volume of about 0.01 mL to about 100 mL, about 0.1 mL to about 10 mL, or 1 mL to 100 mL, or any range therebetween. In some embodiments, the vascular channel system is described in WO2018 / 112480 (published June 21, 2018) or WO2018 / 227026 (published December 13, 2018).

[0023] The disclosed devices can have a single conduit (e.g., where a biological fluid diffuses into and out of the cell scaffold through the conduit, e.g., recapitulating an endocrine gland function) or more than one conduit. In some embodiments, the device includes a second conduit having a second luminal space, a first end embedded (e.g., embedded and integrated) in a biocompatible extracellular matrix material of the tissue scaffold, and a second end configured to connect to a fluid outlet, where the luminal space, vascular channel system, and second luminal space are configured to be in fluid communication with each other, a fluid supply, and a fluid outlet. Such devices can be used, for example, to shunt a biological fluid, such as blood, through the vascular channel system to add a desired substance, such as a hormone, cytokine, or endocrine substance produced by cells in the vascular channel system.

[0024] In some embodiments, the device includes a membrane separating the vascular channel system from the second vascular channel system. The membrane is not limited and can be any suitable membrane. In some embodiments, the membrane is described in WO2018 / 112480 (published June 21, 2018), or WO2018 / 227026 (published December 13, 2018), which are incorporated herein by reference in their entirety. In some embodiments, the second vascular channel system is fluidly connected to a conduit as described herein. In some embodiments, the second vascular system is fluidly connected to two conduits as described herein. In some specific embodiments, the device further includes a membrane, a second vascular channel system, a third conduit having a third luminal space, and a fourth conduit having a fourth luminal space, where the vascular channel system and the second vascular channel system are configured in fluid communication across the membrane, the third conduit has a first end embedded (e.g., embedded and integrated) in the biocompatible extracellular matrix of the tissue scaffold and a second end configured to connect to a second fluid supply, and the fourth conduit has a first end embedded (e.g., embedded and integrated) in the biocompatible extracellular matrix of the tissue scaffold and a second end configured to connect to a second fluid outlet, and the third conduit space, the second vascular channel system, and the fourth luminal space are configured to be in fluid communication with each other, the second fluid supply, and the second fluid outlet. In some embodiments, the device further comprises mammalian cells as described herein in one or more of the first through fourth conduits, the first vascular channel system, and the second vascular channel system, In some embodiments, the device is surgically anastomosed into a vascular or other channel system of a subject, for example, to replace or augment renal function, pulmonary function, endocrine function, gastrointestinal function, lymphatic function, etc.

[0025] Some aspects of the present disclosure relate to a method of manufacturing a conduit or device as described herein. In some embodiments, the method of manufacturing a conduit includes providing a tubular structure having an internal luminal space and comprising a porous biocompatible polymer, perfusing the pores and luminal space of the tubular structure with a first solution, replacing the first solution by perfusing the pores and luminal space of the tubular structure with an aqueous solution comprising a liquefied extracellular matrix material, and embedding the tubular structure in the extracellular matrix material by polymerizing the liquefied extracellular matrix material to form a conduit. In some embodiments, the method further includes providing a tissue scaffold (e.g., a tissue scaffold as described herein) comprising the extracellular matrix material, and embedding (e.g., embedding and integrating) a first end of the conduit in the extracellular matrix material of the tissue scaffold. In some embodiments, the tissue scaffold further includes a vascular channel system, and the first end is embedded in the extracellular matrix material such that the luminal space is configured to be in fluid communication with the vascular channel system.

[0026] In some alternative embodiments, the steps of perfusing the pores and luminal space of the tubular structure with a first solution and replacing the first solution by perfusing the pores and luminal space of the tubular structure with an aqueous solution comprising liquefied extracellular matrix material are replaced with a single step, where the first solution perfusates the pores and luminal space of the tubular structure and contains the liquefied extracellular matrix material (e.g., the first solution is an aqueous solution comprising liquefied extracellular matrix material and a surfactant in a concentration sufficient to allow perfusion into the pores of the tubular structure).

[0027] The first solution is not limited to, and may be any suitable solution that is compatible with the tubular structure (e.g., does not damage the tubular structure) and can perfuse into the pores and lumen space (e.g., a solution with sufficiently low surface tension, viscosity, etc.). In some embodiments, the first solution is an aqueous solution and includes a surfactant at a sufficient concentration to allow perfusion into the pores. The surfactant is not limited to, and may be any suitable surfactant. In some embodiments, the surfactant is sodium lauryl sulfate, dioctyl sodium sulfosuccinate, a polysorbate-type nonionic surfactant (e.g., TWEEN®-20, TWEEN®-80), Triton X-100, or a combination thereof. In some embodiments, the surfactant is a medically acceptable surfactant. In some embodiments, the first solution is non-polar and miscible in the aqueous solution containing the liquefied extracellular matrix material.

[0028] The porous biocompatible polymer can be any suitable porous biocompatible polymer described herein, without limitation. In some embodiments, the porous biocompatible polymer is expanded polytetrafluoroethylene. In some embodiments, the porous biocompatible polymer comprises pores having a pore size of 0.1-10 μm in diameter and a porosity of greater than 50%. In some embodiments, the biocompatible extracellular matrix material is polymerized and comprises gelatin, gelatin methacryloyl, collagen, or decellularized natural extracellular matrix.

[0029] In some embodiments, the interior luminal space of the conduit has a diameter of between 0.1 and 10 cm.

[0030] In some embodiments, the method further comprises adding (e.g., seeding) mammalian cells to the vascular channel system and / or conduit. The mammalian cells can be, without limitation, any mammalian cell or mixture of mammalian cells described herein. In some embodiments, the mammalian cells are selected from smooth muscle cells, fibroblasts, and / or endothelial cells.

[0031] Some embodiments of the methods disclosed herein include the following steps.

[0032] In step 1, a mechanically robust tubular porous ePTFE structure is procured, fabricated, or manufactured using standard techniques. This structure may be purely tubular in nature or may have additional features such as ribbing, flanges, fastening points, or other such structural additions designed to enhance function or integration with other structures. The amount of void space in this structure, which is equivalent to the porosity of a tube, is adjustable and may be uniform or locally tailored to vary the amount and location of porosity and stiffness in the final composition.

[0033] In step 2, the porous structure may be treated with other materials or processes to modify chemical, biological, or physical properties, such as improving hydrophilicity or material surface energy, bioactivity, or other desired properties to aid fabrication or enhance the final product.

[0034] In step 3, the entire void space in the porous tubular structure is impregnated with an initial non-polar solution that is miscible with the aqueous solution. This can be done by immersion or by flowing the solution through the porous structure with the aid of vacuum, pressure, sonication, or other techniques.

[0035] In step 4, the original non-polar solution is replaced with the primary aqueous solution. This can be done by similar techniques as described in step 3.

[0036] In step 5, a secondary material solution is used to replace the aqueous solution. This secondary material may or may not contain cells or other biological materials as determined by the final application. This secondary material may be biological, polymeric, composite, or other composition, but must be in liquid form. This may be done through a process similar to that described in steps 3 and 4, and may occur in isolation, or in conjunction with the fabrication of other structures or engineered scaffolds to integrate the tubular structures into a perfusable construct.

[0037] In step 6, the material is polymerized, gelled, crosslinked, bonded or otherwise solidified within the interconnected porous structure of the ePFTE tube. If desired, the lumen of the tube may be cleared before or after solidification of the secondary material.

[0038] In step 7, the tubular structure of this patent can be used as is, or can be further processed or incorporated into subsequent structures or engineered tissues. This can be done by molding, casting, or other such techniques. During this process, a secondary material can be impregnated into the porous structure, contacting similar materials in the engineered construct, and then crosslinking, bonding, curing, or otherwise solidifying so that it forms a coherent, uniform single piece of material that permeates both the porous structure and some or all of the engineered scaffold. Parts or components of this scaffold can be fabricated prior to the integration of the tubular structure, or the tubular structure can act as a basis or component for the fabrication of the scaffold.

[0039] At step 8, the fabricated scaffold is complete and the lumen of the tubular structure may be accessed via barbed connections, cannulation, surgical anastomosis, or other such means. The porous tubular structure provides a mechanically stable structure for robust connections that allow for the flow of fluids or gases into or out of the lumen and the anastomosed or attached tissue or blood vessels. ***

[0040] All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that may be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or prior publication, or for any other reason. All statements as to the date or indication as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the accuracy of the dates or contents of these documents.

[0041] Those skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the aims and advantages mentioned, as well as those inherent therein. The details of the description and examples herein are representative of specific embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention. It will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0042] The articles "a" and "an" as used herein in the specification and claims should be understood to include plural referents unless clearly indicated to the contrary. A claim or description including "or" between one or more members of a group is deemed to be satisfied when one, more than one, or all of the group members are present in, used in, or otherwise relevant to a given product or process, unless indicated to the contrary or otherwise clear from the context. The invention includes embodiments in which exactly one member of a group is present in, used in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, used in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the present invention provides all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc. from one or more of the enumerated claims are introduced into another claim (or any other claim, if relevant) that is dependent on the same base claim, unless otherwise indicated or unless it is obvious to one of ordinary skill in the art that a contradiction or inconsistency would result. It is contemplated that all embodiments described herein are applicable to all different aspects of the present invention, where appropriate. It is also contemplated that any embodiment or aspect may be freely combined with one or more other such embodiments or aspects, as appropriate. It is to be understood that when elements are presented as lists, e.g., in a Markush group or similar format, each subgroup of elements is also disclosed, and any element may be removed from the group. In general, when the invention, or aspects of the invention, are referred to as including certain elements, features, etc., it should be understood that the particular embodiment or aspect of the invention consists of or consists essentially of such elements, features, etc. For purposes of brevity, those embodiments will not in all cases be specifically described in so many words herein.It should also be understood that any embodiment or aspect of the invention may be explicitly excluded from the claims, regardless of whether a specific exclusion is set forth herein, For example, any one or more active agents, additives, ingredients, optional agents, organism types, disorders, subjects, or combinations thereof may be excluded.

[0043] Where a claim or description is directed to a composition of matter, it is understood that methods of making or using the composition of matter by any method disclosed herein, and methods of using the composition of matter for any purpose disclosed herein, are aspects of the invention, unless otherwise indicated or it is apparent to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where a claim or description is directed to a method, it is understood that, for example, methods of making compositions useful for carrying out the method, and products produced according to the method, are aspects of the invention, unless otherwise indicated or it is apparent to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[0044] When ranges are given herein, the invention includes embodiments in which the endpoints are included, in which both endpoints are excluded, and in which one endpoint is included and the other is excluded. Unless otherwise indicated, it should be presumed that both endpoints are included. Furthermore, unless otherwise indicated or clear from the context and the understanding of one of ordinary skill in the art, it should be understood that values ​​expressed as ranges can take any particular value or subrange within the stated range in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly indicates otherwise. It should also be understood that when a series of numerical values ​​is described herein, the invention includes embodiments that similarly relate to any intermediate value or range defined by any two of the values ​​in the series, with the smallest value being the minimum and the largest value being the maximum. Numeric values ​​used herein include values ​​expressed as a percentage. For any embodiment of the invention in which a numerical value is prefaced by "about" or "approximately," the invention includes embodiments in which the exact value is described. For any embodiment of the invention where a numerical value is not prefaced by "about" or "approximately," the invention includes embodiments where the value is prefaced by "about" or "approximately."

[0045] As used herein, "A and / or B" generally means at least one of A, B, or both A and B, where A and B are different claim terms. For example, a sequence that is complementary to and / or hybridizes to another sequence includes (i) a sequence that is complementary to the other sequence, even though the one sequence may not necessarily hybridize to the other sequence under all conditions, (ii) a sequence that hybridizes to the other sequence, even though the one sequence is not fully complementary to the other sequence, and (iii) a sequence that is both complementary to and hybridizes to the other sequence.

[0046] "Approximately" or "about" generally includes numbers in either direction (greater or lesser than the number) within 1%, or in some embodiments, within 5% of the number, or in some embodiments, within 10% of the number, unless otherwise stated or clear from the context (unless such number unacceptably exceeds 100% of possible values). Unless expressly indicated to the contrary, in any method claimed herein that includes more than one act, the order of the method acts is not necessarily limited to the order in which the method acts are described, but it should be understood that the invention includes embodiments in which the order is so limited. It should also be understood that any product or composition described herein can be considered to be "isolated" unless otherwise stated or clear from the context.

[0047] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, and their respective components that are essential to the present invention, and still permit the inclusion of unspecified elements, whether essential or not.

[0048] As used herein, the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristics of that embodiment of the invention.

[0049] The term "consisting of" refers to compositions, methods, and their respective components described herein, which exclude any element not recited in that description of an embodiment. EXAMPLES

[0050] Example 1 In one example, the porous tubular structure is a length of expanded polytetrafluoroethylene (ePTFE), a commercially available, highly tunable, biologically inert material originally developed by Gore. By varying the rate and acceleration of expansion during processing (among other parameters), it is possible to achieve tubes with porosities of up to and beyond 80%, which provides a substantial amount of void space for the integration of secondary materials. The ePTFE tube has an inner diameter of 3 mm and an outer diameter of 6 mm, and is 50 mm long. One end of the ePTFE tube is placed onto a barbed fitting, and the other end is capped with a barbed fitting and closure. The open end of the tube is connected to a syringe, and the tube is manually filled with a 70% isopropanol solution warmed to 37° C. Pressurizing the lumen of the tube forces the solution through the tube wall, impregnating the void space and completely excluding any air from the tube pores. The tube is then attached to a syringe or pump system and pressurized with a solution containing 10% by weight gelatin at 37° C. Pressurizing the tube forces the solution through the walls of the tube and into the void space occupied by the isopropanol solution, mixing and ultimately totally replacing the solution in the void space with the gelatin solution. A sufficient volume is passed through the tube to ensure complete displacement of the isopropanol.

[0051] The cap of the tube is then removed and the tube is removed from the pump, allowing the gelatin solution to drain from the tube. The syringe is then reattached, the other end of the tube is left open, and a bolus of 37°C saline is passed through the tube to remove any separate layers of gelatin that remain inside the tube and may later delaminate. The tube is then removed from the barbed fitting and placed in a 4°C bath of saline to cause thermal crosslinking and gelation of the gelatin solution remaining in the void spaces of the porous tube. At this point, the tube can be stored or trimmed and further manipulated for inclusion in downstream fabrication.

[0052] Gelatin-impregnated tubes are incorporated into tissue scaffolds. Specifically, the lumens of the tubes are brought together with a sacrificial material that forms channel structures in the scaffold, and the entire device is molded in a single or series of steps that generate a scaffold with tubular structures embedded in a similar gelatin material. During the molding process, the gelatin in the scaffold becomes integrated with the gelatin in the tubular structures, becomes sticky, and can then be crosslinked using enzymatic, chemical, or other means. The end result is a gelatin-based scaffold with a single integrated hydrogel material that permeates the highly porous tubular support structure, resulting in a mechanically robust tubular structure that is fabricated in situ during scaffold construction.

[0053] In a further example of the present invention, the gelatin solution contains cells, such as smooth muscle cells, fibroblasts, and endothelial cells.

[0054] In additional examples of the invention, the tube is impregnated with GelMA, collagen, decellularized native ECM, or other such biomaterials in addition to or instead of the gelatin solution.

[0055] In an additional embodiment of the invention, the tube is impregnated with different solutions in different regions to achieve variable function or transition for each tissue type or configuration.

[0056] In an additional example of the present invention, the ePTFE tube has a flange and / or barbed feature on one end to improve the interfacial surface area and overall integration with the larger construct.

[0057] In an additional example of the invention, the ePTFE tube has variable properties along its length, such as variable porosity or stiffness, to improve function.

[0058] In an additional embodiment of the invention, the ePTFE tube has branching or tapering along its length to regulate fluid perfusion and distribution.

Claims

1. 1. A device comprising a tissue scaffold and a conduit, a. the tissue scaffold comprises a biocompatible extracellular matrix material and a vascular channel system; b. the conduit comprises a tubular structure comprising a porous biocompatible polymer embedded in the biocompatible extracellular matrix material and an interior luminal space, wherein the porous biocompatible polymer comprises a porosity of greater than 50%; A device wherein the tubular structure has a first end embedded and integrated in the biocompatible extracellular matrix material of the tissue scaffold and a second end configured to connect to a fluid supply, and the luminal space is configured to be in fluid communication with the fluid supply and the vascular channel system.

2. The device of claim 1 , wherein the porous biocompatible polymer is expanded polytetrafluoroethylene.

3. 3. The device of any one of claims 1 to 2, wherein the porous biocompatible polymer comprises pores having a pore size of 0.1 to 10 μm in diameter, and optionally the internal luminal space has a diameter of 0.1 to 10 cm.

4. The device of any one of claims 1 to 3, wherein the biocompatible extracellular matrix material is polymerized and comprises gelatin, gelatin methacryloyl, collagen, or decellularized natural extracellular matrix.

5. 5. The device of any one of claims 1 to 4, wherein the conduit and / or the vascular channel system further comprises mammalian cells, optionally selected from smooth muscle cells, fibroblasts, and / or endothelial cells.

6. The device according to any one of claims 1 to 5, wherein the vascular channel system has a volume of about 0.01 mL to about 10 L.

7. 7. The device of any one of claims 1 to 6, further comprising a second conduit having a second luminal space, a first end embedded in the biocompatible extracellular matrix material of the tissue scaffold and a second end configured to connect to a fluid outlet, wherein the luminal space, vascular channel system and second luminal space are configured to be in fluid communication with each other, the fluid supply, and the fluid outlet.

8. a membrane, a second vascular channel system, a third conduit having a third luminal space, and a fourth conduit having a fourth luminal space; a. the vascular channel system and the second vascular channel system are configured to be in fluid communication across the membrane; b. the third conduit has a first end embedded in the biocompatible extracellular matrix of the tissue scaffold and a second end configured to connect to a second fluid supply; c. the fourth conduit having a first end embedded in the biocompatible extracellular matrix of the tissue scaffold and a second end configured to connect to a second fluid outlet; d. The device of any one of claims 1-7, wherein the third luminal space, the second vascular channel system, and the fourth luminal space are configured in fluid communication with each other, with a second fluid supply, and with a second fluid outlet.

9. A conduit comprising a tubular structure including a porous biocompatible polymer embedded in a biocompatible extracellular matrix material and an internal luminal space, wherein the porous biocompatible polymer has a porosity of greater than 50%.

10. The conduit of claim 9, wherein the porous biocompatible polymer is expanded polytetrafluoroethylene.

11. A conduit described in any one of claims 9 to 10, wherein the porous biocompatible polymer contains pores having a pore size of 0.1 to 0.5 μm in diameter, and optionally the internal luminal space has a diameter of 0.1 to 10 cm.

12. A conduit described in any one of claims 9 to 11, wherein the biocompatible extracellular matrix material is polymerized and comprises gelatin, gelatin methacryloyl, collagen, or decellularized natural extracellular matrix.

13. A conduit described in any one of claims 9 to 12, further comprising mammalian cells, optionally selected from smooth muscle cells, fibroblasts, and / or endothelial cells.

14. A conduit as described in any one of claims 9 to 13, wherein one or both ends of the conduit are surgically anastomosed to the natural vasculature in vivo.

15. A device comprising a conduit described in any one of claims 9 to 14 having a first end embedded in a tissue scaffold comprising a biocompatible extracellular matrix material and a vascular channel system, wherein the luminal space of the conduit is configured to be in fluid communication with the vascular channel system.

16. 1. A method of manufacturing a conduit, comprising the steps of: a. providing a tubular structure having an interior luminal space and comprising a porous biocompatible polymer comprising greater than 50% porosity; b. perfusing the pores and luminal space of the tubular structure with a first solution; c. replacing the first solution by perfusing the pores and luminal space of the tubular structure with an aqueous solution comprising liquefied extracellular matrix material; and d) embedding said tubular structures in extracellular matrix material by polymerizing said liquefied extracellular matrix material to form said conduit.

17. The method of claim 16, further comprising providing a tissue scaffold comprising the extracellular matrix material; and embedding a first end of the conduit in the extracellular matrix material of the tissue scaffold, optionally wherein the tissue scaffold further comprises a vascular channel system, the first end being embedded in the extracellular matrix material such that the luminal space is configured to be fluidly connected with the vascular channel system.

18. 18. The method of any one of claims 16 to 17, wherein the first solution comprises a surfactant that allows perfusion into the pores and / or the first solution is non-polar and miscible in aqueous solution.

19. The method according to any one of claims 16 to 18, wherein the porous biocompatible polymer is expanded polytetrafluoroethylene.

20. 20. The method of any one of claims 16 to 19, wherein the porous biocompatible polymer comprises pores having a pore size of 0.1 to 10 μm in diameter, and optionally the internal luminal space has a diameter of 0.1 to 10 cm.

21. The method of any one of claims 16 to 20, wherein the biocompatible extracellular matrix material is polymerized and comprises gelatin, gelatin methacryloyl, collagen, or decellularized natural extracellular matrix.

22. The method of any one of claims 16 to 21, further comprising adding mammalian cells to the vascular channel system and / or the conduit.

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