Artificial implants that promote neovascularization

Abstract

Systems and methods for providing artificial implants that promote neovascularization using an implant body enclosing biomaterial and a length of an arteriovenous (AV) bundle. The AV bundle can be configured to have closed ends that reside in or outside the internal chamber of the implant body so as to not form a blood flow path therebetween. The biomaterial can include decellularized human pancreatic extracellular material in the internal chamber. The biomaterial can be pre-formed or pre-assembled or injected into the internal chamber of the implant body or the biomaterial or inserted onsite during or prior to a surgical procedure associated with implantation of the device.

Description

ARTIFICIAL IMPLANTS THAT PROMOTE NEOVASCULARIZATIONRELATED APPLICATIONS

[0001] This patent application claims the benefit of and priority to U.S. Provisional Application Serial Number 63 / 637,003 filed April 22, 2024, the contents of which are hereby incorporated by reference as if recited in full herein.FIELD OF THE INVENTION

[0002] The present invention relates to artificial implants and may be particularly suitable for an artificial pancreas that can provide insulin-secreting cells.BACKGROUND

[0003] Type 1 diabetes is caused by an auto-immune destruction of beta cells leading to insulinopenia. The management of Type 1 diabetes relies on injections of insulin.

[0004] Islet transplantation or cell therapy of Type 1 diabetes represents a hope for millions ot Type 1 diabetes patients. However, to date, survival rates of transplanted islets (islets apoptosis after transplantation) have been poor which is believed to be due to host immune rejection and / or lack of suitable vascularization.

[0005] There is a long-felt need for islet transplantation devices with suitable vascularization to promote survivability and functionality of islets.SUMMARY

[0006] Embodiments of the present invention provide artificial implants that have an implant body and an internal chamber or core with biomaterial that includes target (transplant) cells and which is configured to encase part of an arteriovenous bundle.

[0007] Embodiments of the present invention provide an artificial pancreas implant having an implant body with an internal chamber; an arteriovenous bundle extending at least partially into the internal chamber; and biomaterial in the implant body that supports and / or includes human pancreatic islets.

[0008] The artificial implants can be configured to have branches of vascularization extending from the AV bundle toward an inner surface of the implant body through the biomaterial at one week post implantation.

[0009] Embodiments of the present invention are directed to an artificial pancreas implant that includes: an implant body with an internal chamber; an arteriovenous (AV) bundleextending at least partially into the internal chamber; and a biomaterial in the implant body that includes pancreatic islets.

[0010] The implant body can be gas permeable and be formed with a synthetic, non- resorbable material.

[0011] The implant body can be formed of (medical grade) silicone.

[0012] The internal chamber can have a volume in a range of about 5 mL to about 10 mL, typically about 5 mL.

[0013] The implant body can have an axially extending length with a first port on a first end sized and configured to slidably receive the AV bundle.

[0014] The implant body can have a second port on a second end that is axially opposed to the first end.

[0015] Branches of (neo)vascularization can extend from the AV bundle toward an inner surface of the implant body through the biomaterial one week post implantation.

[0016] The biomaterial with pancreatic islets can include a decellularized extracellular matrix (dECM)-fibrin hydrogel with human pancreatic islets in a therapeutically effective amount.

[0017] The AV bundle can have an artery segment with a first end and a second end and a vein segment with a first end and a second end. The first end of the artery segment can be attached to an artery of a patient outside the implant body and the first end of the vein segment can be attached to a vein of the patient outside the implant body. The artery segment and the vein segment can both extend at least partially into the internal chamber.

[0018] The second end of the artery segment and the second end of the vein segment can both have closed ends that are both held inside the internal chamber or that are both held outside the internal chamber. The second ends can both be held adjacent an end wall of the implant body at a location spaced apart from an entry port of an axially spaced apart end wall.

[0019] Optionally, the second end of the artery segment and the second end of the vein segment are coupled together (attached) but do not provide a blood flow path therebetween.

[0020] The AV bundle can have a length that is greater than a length of the implant body.

[0021] The AV bundle can have a length that is 2 cm-4 cm longer than the length of the implant body.

[0022] The AV bundle can be provided as first and second AV bundles and both can be positioned to extend substantially an entire length of the implant body.

[0023] The AV bundles can both have a length that extends outside both distal and proximal ends of the implant body.

[0024] The implant body can have an axial length and axially spaced apart first and second ports. The first port can have a width or diameter that is greater than a width or diameter of the second port.

[0025] The implant body can have first and second axially spaced apart end walls that provide the respective first and second ports. The first end wall can have a thickness that is less than a thickness of the second end wall.

[0026] The first and second ports can optionally include a sealant or cover configured to close the first and second ports to prevent the biomaterial in the internal chamber from migrating or moving out of the internal chamber through the first and second ports (at least during initial fill).

[0027] The AV bundle can have an artery segment with a first end and a second end and a vein segment with a first end and a second end. The first end of the artery segment can be attached to an artery of a patient outside the implant body and the first end of the vein segment can be attached to a vein of the patient outside the implant body. The second end of the artery segment and the second end of the vein segment can both be closed ends held in or adjacent the second port and do not provide a blood flow path therebetween.

[0028] The AV bundle can be arranged so that at least a major portion (50% or greater) of a vein segment and at least a major portion of an artery segment thereof are parallel and inside the internal chamber of the implant body.

[0029] The artificial pancreas implant can further include tissue attachment features for anchoring the implant body in a desired position in a patient to prevent undue movement or migration post-implantation.

[0030] The artificial pancreas implant can further include a radiopaque marker or material defining an access location thereby providing an intrabody visualization feature postimplantation.

[0031] Still other embodiments are directed to medical kits that include an artificial pancreas implant body comprising an internal chamber and a biomaterial comprising pancreatic islets and a carrier.

[0032] The medical kit can further include a catheter sized and configured to pull an arteriovenous bundle at least partially into the artificial pancreas implant body.

[0033] The artificial pancreas implant body can have a length and first and second ports spaced apart in the length dimension.

[0034] The first port can have a width or diameter that is greater than a width or diameter of the second port.

[0035] The internal chamber can have a volume in a range of about 5 cubic centimeters to about 10 cubic centimeters, optionally about 5 cubic centimeters.

[0036] The artificial pancreas implant body can have first and second axially spaced apart end walls that provide the respective first and second ports and the first end wall can have a thickness that is less than a thickness of the second end wall.

[0037] The carrier of the biomaterial can include decellularized pancreatic extracellular matrix dECM material.

[0038] Yet additional embodiments are directed to an implant for providing cell therapy to a subject. The implant includes: an implant body; an arteriovenous (AV) bundle in the implant body; and target cells in a therapeutic amount in a carrier in the implant body.

[0039] The AV bundle can be provided as a plurality of AV bundles with a portion of each held in the implant body and with one end portion extending out of the implant body, coupled to respective artery and vein segments of the subject.

[0040] Other aspects of the present inventive concept are directed to methods of providing human islets, that includes: providing an implant body comprising an internal chamber; providing a biomaterial comprising human islets; forming an arteriovenous (AV) bundle from harvest site of a subject; positioning a portion of the AV bundle inside the internal chamber of the implant body; introducing the biomaterial with the human islets in the internal chamber of the implant body before, during or after positioning the AV bundle in the implant body; and implanting the implant body before or after introducing the biomaterial into the implant body thereby providing human islets to the subject.

[0041] The methods can include vascularizing the AV bundle inside the internal chamber whereby branches of vascularization grow and extend from the AV bundle toward an inner surface of a wall bounding the internal chamber within one-week post-implantation thereby promoting survivability of transplanted human islets for long term viability.

[0042] The methods can further include transferring at least some of the biomaterial to the internal chamber of the implant body at a surgical site of the implantation before the implanting step.

[0043] The methods can further include anchoring the implant body at an iliac location adjacent a groin area as a target implant location.

[0044] The biomaterial can include decellularized pancreatic extracellular matrix dECM material.

[0045] The positioning can be carried out by placing an artery segment and a vein segment of the AV bundle inside the internal chamber in a substantially parallel arrangement over at least 50% of a length of the internal chamber.

[0046] The forming the AV bundle can include closing off end portions of each of an artery segment and a vein segment so that no blood flow path is formed therebetween.

[0047] The closed end portions can be attached to an end portion of the implant body.

[0048] The closed end portions can be coupled together.

[0049] The closed end portions can extend outside an end wall of the internal chamber and optionally terminate adjacent thereto.

[0050] The AV bundle can have a length that is 2 cm to 4 cm longer than a length of the implant body.

[0051] The AV bundle can have a sub-length that extends out of the implant body with end portions thereof attached to a respective vein and artery of the subject.

[0052] Further features, advantages and details of the present invention will be appreciated by those of ordinary skill in the art from a reading of the figures and the detailed description of the preferred embodiments that follow, such description being merely illustrative of the present invention.

[0053] It is noted that aspects of the invention described with respect to one embodiment, may be incorporated in a different embodiment although not specifically described relative thereto. That is, all embodiments and / or features of any embodiment can be combined in any way and / or combination. Applicant reserves the right to change any originally filed claim or file any new claim accordingly, including the right to be able to amend any originally filed claim to depend from and / or incorporate any feature of any other claim although not originally claimed in that manner. These and other objects and / or aspects of the present invention are explained in detail in the specification set forth below.BRIEF DESCRIPTION OF DRAWINGS

[0054] FIG. 1A is an enlarged, schematic section view of an example implant with an arteriovenous bundle extending into and at least partially through the interior chamber of the implant with a biomaterial at time of placement according to embodiments of the present invention.

[0055] FIG. IB shows the implant of FIG. 1A at a subsequent time, schematically prophetically illustrating rapid vascularization branching from the AV bundle of the implanttoward the inner surface of the implant body according to embodiments of the present invention.

[0056] FIG. 1C and ID illustrate alternative arrangements of the AV bundle in the implant body according to embodiments of the present invention.

[0057] FIG. IE is a schematic partial section view of an example implant using a plurality of AV bundles according to embodiments of the present invention.

[0058] FIG. 2A is a schematic illustration of a kit for providing the example implant shown in FIG. 1A according to embodiments of the present invention.

[0059] FIG. 2B is a schematic illustration of the cannula in the kit shown in FIG. 2A forming a channel in the implant body for receiving an AV bundle according to embodiments of the present invention.

[0060] FIG. 3 is a schematic illustration of another embodiment of a kit for providing an implant according to embodiments of the present invention.

[0061] FIG. 4A is a side perspective view of an example implant comprising an outer cover or membrane on the implant according to embodiments of the present invention.

[0062] FIG. 4B is a section view of the device shown in FIG. 4A.

[0063] FIG. 5 is a schematic illustration of an example placement of an implant and AV bundle connections to a target subject according to embodiments of the present invention.

[0064] FIG. 6A is a schematic illustration of a kit that can provide the implant according to embodiments of the present invention.

[0065] FIG. 6B is a schematic section view of components of the kit shown in FIG. 6A assembled together to form the implant according to embodiments of the present invention.

[0066] FIG. 6C is a schematic sectional view of the implant shown in FIG. 6B, 1 (one) week post implantation illustrating prophetic vascularization in the implant body.

[0067] FIG. 7A is an enlarged schematic section view of another embodiment of an implant body comprising a three-dimensional scaffold according to embodiments of the present invention.

[0068] FIG. 7B is an enlarged schematic section view of the implant body shown in FIG. 7A with biomaterial and an AV bundle held therein according to embodiments of the present invention.

[0069] FIGS. 8A-8D are schematic illustrations of additional example shapes of the implant body according to embodiments of the present invention.

[0070] FIG. 9 is a schematic illustration of an implant body comprising suture attachment features for providing attachment to local tissue for implant stabilization according to embodiments of the present invention.

[0071] FIG. 10 is a schematic illustration of another example kit of components for providing the implant according to embodiments of the present invention.

[0072] FIG. 11 is a schematic illustration of another example of an implant body according to embodiments of the present invention.

[0073] FIG. 12 is a flow chart of example actions that can be used to provide an implant with biomaterial with an AV bundle (optionally an artificial pancreas) according to embodiments of the present invention.

[0074] FIGS. 13A-13C illustrate vascularization of an osteogenic implant after 1-week transplantation in rats. FIGS. 13A / B show E staining, along with black stained vessels (Indian Ink) throughout the entire construct. Microvessel expansion from the AV bundle (blue dotted circle) to the device rim (red dotted circle; ~4 mm distance from AV bundle) can be visualized. FIG. 13C shows a Micro-tomographical 3D reconstruction showing a vessel tree originating from the AV bundle at 8 weeks post-implantation.DETAILED DESCRIPTION

[0075] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Like numbers refer to like elements throughout. The abbreviation “FIG.” may be used interchangeably with “Fig.” and the word “Figure” in the specification and figures. It will be appreciated that although discussed with respect to a certain embodiment, features or operation of one embodiment can apply to others.

[0076] In the drawings, the thickness of lines, layers, features, components and / or regions may be exaggerated for clarity and broken lines (such as those shown in flow diagrams) illustrate optional features or operations, unless specified otherwise. In addition, the sequence of operations (or steps) is not limited to the order presented in the claims unless specifically indicated otherwise.

[0077] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms"a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0078] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. Well- known functions or constructions may not be described in detail for brevity and / or clarity.

[0079] It will be understood that when a feature, such as a layer, region or substrate, is referred to as being "on" another feature or element, it can be directly on the other element or intervening elements may also be present. In contrast, when an element is referred to as being "directly on" another feature or element, there are no intervening elements present. It will also be understood that, when a feature or element is referred to as being "connected" or "coupled" to another feature or element, it can be directly connected to the other element or intervening elements may be present. In contrast, when a feature or element is referred to as being "directly connected" or "directly coupled" to another element, there are no intervening elements present. Although described or shown with respect to one embodiment, the features so described or shown can apply to other embodiments. The term “about” with respect to a number means that the noted number can vary by + / - 20%. The term “subject” refers to animal and human subjects.

[0080] Referring to FIGS. 1A and IB, an example implant 10 is shown in section view. The implant 10 has an implant body 15. The implant body 15 has an internal chamber 20. The internal chamber 20 holds a volume of a biomaterial 25 and an arteriovenous (AV) bundle 30. The term “biomaterial” refers to a biological material comprising target cells such as islets in a therapeutic amount. The target cells provide a target therapy such as insulin (for islets) or other therapies such as enzymes, proteins, and / or hormones for other cell therapies. The biomaterial 25 can also comprise a carrier that can support the viability of the target cells in vivo and can promote or at least not preclude vascularization. The biomaterial 25 can providea therapeutic amount of target cells as the active component for therapy that can be provided in a carrier that supports the target cells and provides a medium for vascularization.

[0081] Once the implant body 15 has the AV bundle 30 and is in position in a subject, it forms the implant 10.

[0082] FIG. 1A illustrates the implant 10 at an initial day of implantation and FIG. IB schematically illustrates a prophetic view of the implant 10 at a subsequent relatively short time period, such as at day 7 or about 1 week post implantation, whereby the implant 10 includes neovascularization V with branches Vb of vascularization extending from the AV bundle 30 in the internal chamber 20 outward through the biomaterial 25 toward the inner surface 15i of the wall 15w of the implant body 15. The wall 15w bounds the internal chamber 20. Free ends Vf of the neovascularization V, post implantation, such as at day 7-14 post implantation, can reside adjacent the inner surface 15i of the wall 15w, such as within about 0-4 mm of the inner surface 15i of the wall 15w. The AV bundle 30 comprises an artery segment 31 and a vein segment 32. As shown, a length of the artery segment 31 and a length of the vein segment 32 extend at least partially into the internal chamber 20 for at least a major portion (at least 50%) of a length L of the internal chamber 20.

[0083] The implant body 15 can have a length that corresponds to the length L of the internal chamber 20 plus the thickness of the end walls 16, 17. The implant body 15 can have a width to length ratio, W:L, that is less than one (1) whereby the lateral width or diameter is less than the length, typically by at least 30%, with the AV bundle 30 extending in the length direction for at least 50% of the length of the internal chamber 20 which may promote neovascularization and / or oxygen exchange between local tissue, the biomaterial 25 and / or the AV bundle 30.

[0084] There may be a critical distance to which the neovascular network can extend from the AV bundle 30 in the chamber 20 while providing sufficient volume in the chamber 20 for biomaterial 25 and / or vascularization to promote target cell viability. This critical distance can limit the width or diameter of the implant body 15. Axial or length dimension upscaling may be desirable over diameter or width.

[0085] In some embodiments, the artery segment 31and the vein segment 32 inside the internal chamber 20 are parallel to each other for at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or even 100% of the length L of the internal chamber 20. Thus, the artery segment 31 and the vein segment 32 can be parallel to each other inside the internal chamber 20 over at least a majority (50% or more) of their length in the internal chamber 20. The artery segment 31 and the vein segment 32 inside the internal chamber 20 can bearranged to extend along, aligned with and parallel to, a longitudinally extending centerline A-A of the implant body 15. In some embodiments, the artery segment 31 and the vein segment 32 can be in a straight linear arrangement in the internal chamber 20 for at least 50% of the length L of the internal chamber 20. As shown in FIGS. 1A and IB, the artery segment and the vein segment, 31, 32, respectively, inside the internal chamber 20 may be coaxial. This arrangement can be particularly suitable for some embodiments such as for implant bodies 15 having a cylindrical, frustoconical, oval or circular / spherical shape.

[0086] FIGS. 1C and ID illustrate that the AV bundle 30 can be arranged to have a curvilinear shape 30c inside the chamber 20, that merges into respective straight segments at opposing ends of the chamber 20. FIG. ID shows that the curvilinear shape can extend on both sides of the axial center line of the chamber at segments along its length.

[0087] FIG. IE illustrates that a plurality of AV bundles 30i, 30i, can be arranged in a single implant body 15 according to embodiments of the present invention. The AV bundles 30i, 30i can be attached together or remain separate and attached separately to an end portion of the implant body 15. Use of multiple AV bundles can provide redundancy if one fails or improve function / vascularization, potentially increased insulin or other therapy delivery.

[0088] FIGS. 1C and ID also illustrate that one or more radiopaque markers 50 may be provided on or in the implant body 15 so that visualization using X-ray or other imaging modality to readily identify location of the implant 10, 10’ and / or port 21, 24 thereof.

[0089] The AV bundle 30 can be formed in situ during the surgical procedure for subcutaneously implanting the implant body 15. The AV bundle 30 can be formed using natural artery and vein segments 31, 32 harvested from a subject, e.g., a subject intended to receive the implant body 15 (namely, the patient in need for the islet transplantation).

[0090] Referring to FIGS. 5, 6A, in some embodiments, the implant body 15 can be placed in a groin area and a superficial inferior epigastric artery and vein can be dissected from this area (with the help of a microsurgical microscope). However, there are numerous sites (subcutaneous, muscular flap areas) which may be used for the implantation. More than one implant 10 in more than one implant location for a respective subject can be used at any one time. The selected implant site of the implant 10 and / or of the graft may be subject to other parameters such as protection of the graft, mobility and the like.

[0091] The AV bundle 30 can be harvested / derived from many locations. The use of the AV bundle 30 with the implant body 15 can allow a site(s) of implantation that is not limited to the local environment / innate degree of vascularization allowing the implant 10 to have a“ubiquitous” presence, even locations that are poorly vascularized which may be a game changer for many subjects.

[0092] The AV bundle 30 can be ligated distally, transected and pulled or otherwise placed inside (and optionally through) the implant body 15, using a surgical instrument such as a catheter configured to position the AV bundle 30 in the internal chamber 20.

[0093] A subcutaneous pocket or pouch in the subject can be created (e.g., by blunt dissection) and the implant body 15 positioned therein with the AV bundle 30 assembled in the implant body 15 and attached to an artery 131 and a vein 132 of the subject. The implant body 15 can be anchored in position using a suture or other anchoring device.

[0094] The biomaterial 25 with the target cells can be deposited in the internal chamber 20 of the implant body 15 at a clinical site (e.g., surgical site such as a hospital), prior to implantation and / or during the surgical implant procedure. The term “deposit” and derivatives thereof, is used broadly and encompasses any manner of providing at least some biomaterial 25 in the internal chamber 20.

[0095] In some particular embodiments, as shown in FIG. 6A, the biomaterial 25 comprises a therapeutic amount of pancreatic islets as the target cells 325 and dECM-fibrin hydrogel 425 as the carrier, both of which can be provided as a mixture in a syringe 125 for introducing into the chamber 20 of the implant body 15 to provide the implant 10 with the AV bundle 30 as shown in FIG. 6B. FIG. 6C shows the prophetic vascularization V, about 1 (one) week after implantation.

[0096] The sequence of assembling the implant 10 can be carried out in different ways. For example, the biomaterial 25 comprising the target cells, such as an islet-hydrogel mixture, can be loaded in the chamber 20 of the implant body 15, then the AV bundle 30 can be pushed or pulled through and / or into the chamber 20. Alternatively, the AV bundle 30 can be placed in the chamber 20 first, then the biomaterial 25 comprising the target cells, e.g., the islet-hydrogel mixture, can be introduced into the chamber 20. The latter may be simpler and the interaction of the AV bundle 30 with the biomaterial 25 may be in closer proximity, e.g., tighter, at implantation. If the former, referring to FIG. 2B, the biomaterial 25 comprising the target cells, e.g., the islet-hydrogel, can be introduced after placing a canula 225 axially in a center of the chamber 20, then the cannula 225 is removable to create an elongate channel 15c in the implant body 15 for holding the AV bundle 30.

[0097] The biomaterial 25 comprising the target cells, e.g., islet-hydrogel mixture, can be loaded into the chamber 20 of the implant body 15 outside a body of the subject to be treated, either during the surgical procedure in the OR (operating room) or it can be pre-loaded in thechamber 20 for use during the surgery with the cannula 125 in position to provide the location for the AV bundle 30.

[0098] The AV bundle 30 can have a length that is in a range of about 2 cm to about 4 cm longer than a length L of the internal chamber 20 and / or a length of the implant body 15 for humans and certain large animal uses. The AV bundle 30 can have a length of about 1 cm for rats. The AV bundle 30 may have a maximum length of about 10 cm for humans and certain larger animal uses.

[0099] The implant body 15 can be flexible but sufficiently rigid to be able to maintain a 3-D shape ex vivo and during and after placement and may be malleable and / or sufficiently flexible to be suitable for subcutaneous placement. The implant body 15 can be compressible, optionally elastically compressible, under normal loading forces applied during normal activity over time when in position in the body of a subject for facilitating patient comfort.

[0100] The implant body 15 can be oxygen permeable to allow the transport of oxygen in and / or out of the implant body 15.

[0101] The implant body 15 can be non-resorbable and synthetic such as a polymer or non- resorbable and formed of a biological synthetic material(s).

[0102] The implant body 15 can be absorbable or resorbable in situ, for example, an osteogenic material or PLLA, PCI material may be used.

[0103] The implant body 15 can be highly oxygen permeable. A high oxygen permeability aids and / or facilitates the biomaterial 25 to be able to receive sufficient oxygen to promote long term viability (months or years) of target cells such as islets.

[0104] Referring to FIGS. 1A, IB and 5, the artery segment 31 and the vein segment 32 can extend entirely through the internal chamber 20 positioning the respective second end portions 31ei, 32ei outside the internal chamber 20 adjacent an end wall 16 of the implant body 15. The artery segment 31 can have a first end portion 31ei that resides outside the internal chamber 20 and couples to an artery 131 of the subject (FIG. 5). The vein segment 32 can have a first end portion 32ei that resides outside the chamber 20 and couples to a vein 132 of the subject (FIG. 5).

[0105] The AV bundle 30 can be configured so that one end portion thereof 33 couples the artery segment 31 and the vein segment 32 together.

[0106] The second end portions 31ei and 32ei can both be closed off such that no blood flow path is provided between these second end portions 31ei, 32ei. These second endportions 31ei, 32ei can be sutured or tied off, optionally coupled for ease of positioning and attachment to the implant body 15.

[0107] Still referring to FIGS. 1A and IB, the implant body 15 can have first and second end walls 16, 17 that are spaced apart in a length dimension L. The first end wall 16 can have a first port 21 and the second end wall 17 can have a second port 24. The first and second ports 21, 24 can be aligned.

[0108] The first port 21 can be larger than the second port 24. The first port 21 can be used to pull the AV bundle 30 toward the opposing second port 24 so a larger port can facilitate this action. Graspers can be used to extend through the first port 21 to pull the AV bundle into the second port and toward the second port 24. The ports 21, 24 can have different lateral widths / sizes because where the legs 31el, 31e2 of the AV bundle 30 penetrate from the connected artery / vein segments, more freedom may be desired to accommodate if the implant body 15 moves, so that the AV bundle 30 doesn’t break or thrombose. However, in other embodiments, the ports 21, 24 can be she same size and are not required to be axially aligned.

[0109] The first end wall 21 can have a smaller thickness “dl” than the thickness d2 of the second end wall 17.

[0110] A cover, sealant and / or adhesive (plug) 40 can be placed over the first port to inhibit migration or discharge of the biomaterial 25 out through the first port 21. A cover, sealant and / or adhesive (plug) 42 can be placed over the second port 24 to anchor the end portion 33 of the AV bundle 30 to the implant body 15 and / or to inhibit migration or discharge of the biomaterial out the second port 24.

[0111] For certain embodiments, the implant body 15 can be configured so that the internal chamber 20 has a volume in a range of about 5 cubic centimeters to about 15 cubic centimeters, at least for human uses. However, it is contemplated that the volume of the chamber 20 is indirectly dependent on the length of the AV bundle 30 and kinetics of vascularization which is preferably sufficient to deliver oxygen / nutrients to target cells from the biomaterial in the implant body 15. Thus, the chamber volume can vary, and the present invention is not limited to the example volume range.

[0112] For animal studies, the implant body 15 can have an internal volume such as in a range of about 500 U1 to about 1 cubic centimeter, such as about 785 pL for some uses such as mouse / rat uses. The biomaterial 25 can be provided in a volume of about 5 cubic centimeters for a therapeutic dose of transplanted islets.

[0113] The therapeutic dose of islets in the biomaterial 25 can be in a range of about 5,000 to about 20,000 lEQ / Kg for human uses, such as about 10,000 lEQ / kg.

[0114] As will be discussed further below, the biomaterial 25 can comprise islets as the target calls and fibrin / dECM as a carrier. However, it is contemplated that the implant body 15 can be configured to be used with the AV bundle 30 and other target cells for a cell therapy and / or other carriers such as other hydrogels.

[0115] As discussed above, in some embodiments, the biomaterial 25 can be pre-loaded in the implant body 15, and / or provided in a medical kit 100 in a ready to use configuration for use during a surgical implantation procedure.

[0116] FIG. 2A shows an example medical kit 100 comprising the implant body 15 and also with a biomaterial 25 in a syringe 125 for depositing in the internal chamber 20. The syringe 125 can be configured to fluidly inject the biomaterial 25 into the internal chamber 20 via a port 21 in an end wall 17 of the implant body 15. The kit 100 may also optionally include a cannula 225 with a length that can be greater than the length of the implant body 15 and used to create a channel 15c (FIG. 2B) for the AV bundle 30 as discussed above. The cannula 225 can be hollow or solid with closed or open ends.

[0117] FIG. 3 shows the kit 100 can include an outer membrane 35 that can be applied in situ to cover the outer wall surface 15s of the implant body 15 and optionally one or more of the end walls, 16, 17. FIGS. 4A, 4B show the implant body 15 with the membrane 35 attached after the biomaterial 25 is placed / deposited inside the internal chamber 20.

[0118] FIG. IB schematically illustrates that the inner surface 15i of the wall 15w of the implant body 15 can optionally comprise a scaffold 150 that is compatible with target cells such as islets and provides a structure or surface to facilitate at least one of a biomaterial retention in the internal chamber, function and / or viability of the target cells. The scaffold 150 is optional and may not be used for islet cells. The scaffold 150, where used, may be resorbable or non-resorbable and may also or alternatively facilitate the retention of the biomaterial 25 comprising islets inside the internal chamber 20.

[0119] FIGS. 7A and 7B show a scaffold 150 that is three-dimensional and that can extend across and along the internal chamber 20 prior to placement of the biomaterial 25 (FIG. 7B). The scaffold 150 can comprise a “net-like” structure such as an open cell foamed substrate with open cell features 150c. FIG. 7A shows the scaffold and biomaterial in the implant body 15.

[0120] As shown in FIGS. 4A and 4B, an optional outer membrane and / or coating 35 can be applied to an outer surface 15s of the wall 15w of the implant body 15. This outermembrane and / or coating 35 can be configured to promote local stabilization via adhesion to adjacent tissue and / or prevent ingrowth of host cells from adjacent surrounding tissue in vivo. One example outer membrane material is a semipermeable inorganic-based silicone membrane (BIOBRANE®). However, it is also contemplated that other synthetic or biological synthetic materials can be used or the implant body 15 can be formed such that no coating or membrane is used at all.

[0121] The implant body 15 can comprise any biocompatible material. The implant body 15 can comprise synthetic materials such as (bi-component) silicone, other polymers and / or copolymers. The implant body 15 can comprise biological synthetic materials comprising or formed from biological materials such as decelluarized tissue, hydrogel, collagen, devitalized bone matrix, electrospun tissue or collagen or even plant-based materials. The implant body 15 can be made by casting, 3-D printed, molded or otherwise fabricated. Combinations of the different materials may also be used to form the implant body 15.

[0122] The implant body 15 can be autoclaved or ethylene oxide (ETO) sterilized for medical grade sterility and provided in a medical kit 100, 100’ (FIGS. 2A, 3, 6A, 10). The inner 15i and / or outer surface 15o of the implant body 15 can be smooth, roughened or have a texture and / or patterned surface and can be configured to facilitate surface adhesion.

[0123] It is contemplated that biomaterial 25 post-implantation may be “reloadable” using a needle and reloading access port in a minimally invasive surgical procedure. The reloading port can be provided by the first port 21, the second port 24 or a side port 151 (FIG. 6C) formed in the wall 15w of the implant body 15. However, if islets of the biomaterial 25 get exhausted so as not to generate sufficient insulin, the reloading must be done without damaging the vascular tree in the chamber 20. Thus, it may be that a new implant is preferable over reloading the existing implant.

[0124] In some embodiments, the implant 10 can be implanted at a first point in time and used until the target cells of the biomaterial 25 are no longer sufficiently viable. For example, the target cells islets are the (typically only) active therapeutic ingredient / component of the biomaterial 25 and if they stop producing insulin (or for other cell therapies, protein / enzyme / hormone) below a critical level, then they need replacement. The carrier (fibrin / ECM) is a supportive material that promotes the active cells / islets and vascularization at appropriate levels. A second implant 10 can replace the first implant 10 and can be positioned in the same or a different location as the first implant 10. For example, like a kidney transplant a first implant 10 can be placed on a right side at a first point in timeand a second implant 10 can be subsequently placed on a left side without removing the first implant 10.

[0125] FIGS. 8A-8D show example alternate shapes of the implant body 15. FIG. 8A shows a frustoconical shape. FIG. 8B shows a rectangular shape. FIG. 8C shows an oval shape. FIG. 8D shows a curvilinear shape with a bulbous end portion merging into a neck portion which may correspond to a portion of a natural (human) pancreas.

[0126] FIG. 9 illustrates that the implant body 15 can have anchors 200 that may also provide suture attachment features.

[0127] FIG. 10 illustrates another example of a medical kit 100’ provided in medical grade sterile packaging. This kit 100’ includes a catheter 115 configured with a distal end portion that provides a grasper 116 coupled to a handle control 120 for placing, optionally pulling the AV bundle 30, in the internal chamber 20 of the implant body 15. The grasper 116 can reside at the end of a narrow segment 117 with a length L2 that is greater than the length L of the internal chamber 20 of the implant body 15. The biomaterial 25 can be provided in the kit 100 in a container 25c along with a syringe 125 and the implant body 15. The biomaterial 25 may be provided in the same kit or a separate kit and be configured to be inserted into the internal chamber 20 at a clinical or research site.

[0128] FIG. 11 illustrates that the implant body 15 can have an internal attachment structure29 under a closed end wall 16 for capturing the (attached) ends 31ei, 32ei of the AV bundle 30. The internal attachment structure 29 can comprise a frictional engagement or adhesively or sticky engagement structure to be able to grasp / hold the end portion 33 of the AV bundle30 inside the internal chamber 20.

[0129] The biomaterial 25 can comprise a carrier comprising an extracellular matrix (ECM)-based hydrogel such as an ECM-based hydrogel derived and / or obtained from the Revivicor genetically modified porcine pancreas or from a donor human pancreas. For the ECM-based hydrogel, decellularization of the pancreas generates an acellular ECM (dECM) powder that, when added to alginate for microencapsulation of human islets, has been shown to significantly improve stimulation index and total insulin production and the ability to maintain islets in culture for up to 58 days. See, e.g., Asthana et al., Decellularized human pancreatic extracellular matrix-based physiomimetic microenvironment for human islet culture, Acta Biomaterialialial71 (2023); 261-272, the contents of which are hereby incorporated by reference as if recited in full herein. See also, US PGPUB 2022 / 0168232, the contents of which are hereby incorporated by reference as if recited in full herein.

[0130] Moreover, the dECM powder in alginate biomaterial contains angiogenic factors that induce remarkable neoangiogenesis in the corioallantoidea membrane model. Embodiments of the present invention can apply the method of manufacturing dECM from human pancreases to those obtained from the genetically modified Revivicor pig, a breed that is currently being tested in kidney. See, Loupy A et al., Immune response after pig-to-human kidney xenotransplantation: a multimodal phenotyping study. Lancet. 2023 Sep 30;402(10408): 1158-1169. doi: 10.1016 / 80140-6736(23)01349-1. Epub 2023 Aug 17; (PMID: 37598688) and heart xenotransplantation; see. Loupy A et al.„ Immune response after pig-to-human kidney xenotransplantation: a multimodal phenotyping study. Lancet. 2023 Sep 30;402(10408): 1158-1169. doi: 10.1016 / S0140-6736(23)01349-L Epub 2023 Aug 17. (PMID37393920), the contents of these references are hereby incorporated by reference as if recited in full herein.

[0131] It is believed that in the past, the hydrogels / materials used for encapsulating, embedding or delivering islets in grafts generally lack a human pancreas-specific microenvironment that is essential for supporting optimal islet health and function. Embodiments of the present invention contemplate that incorporation of dECM can provide biochemical cues beneficial for maintaining long-term islet viability and function within the graft. Current decellularization protocols include harsh detergents that remove many critical components of the ECM matrisome (e.g. glycoaminoglycans, GAGs). This exacerbates the loss of important cell signaling mediators like growth factors and cytokines. Using a detergent-free, DI water based decellularization process that is adept to removing most of the cellular components while avoiding the extraction of important GAGs can be used to generate the carrier for the biomaterial 25. Seem Minimal Processing of Method for Decellularization of Tissues”, PCT / US2020 / 031995, the contents of which are hereby incorporated by reference as if recited in full herein. This decellularized ECM can be solubilized with pepsin-HCl, followed by neutralization and centrifugation, with the supernatant undergoing subsequent lyophilization and cryomilling to produce a fine solubilized ECM powder (fdECM”). See, Asthana et al., Comprehensive characterization of the human pancreatic proteome for bioengineering applications, Biomaterials 270 (2021) 120613; and Tamburrini et al., Detergent-Free Decellularization of the Human Pancreas for Soluble Extracellular Matrix (ECM) Production, J Vis Exp (163) (2020). The contents of which are hereby incorporated by reference as if recited in full herein.

[0132] The dECM was enriched for protein families that are believed to be critical in the regulation of pancreatic beta cell differentiation / proliferation and pancreatic developmental processes. Also, a recent study demonstrated that incorporation of this (pancreatic) dECM into alginate (1.5% Ultra-Pure Low Viscosity Mannuronate; UP-LVM) capsules at an optimized concentration of 0.1 mg / ml can significantly improve long-term human islet (HI) viability and function. See, Asthana et al., Decellularized human pancreatic extracellular matrix-based physiomimetic microenvironment for human islet culture, Acta Biomaterialia 171 (2023) 261-272, the contents of which are hereby incorporated by reference as if recited in full herein.

[0133] His encapsulated in dECM-alginate showed a significant increase in both GSI and total insulin secreted, compared to free His and His encapsulated in only alginate. dECM supplementation also resulted in long-term (58 days) maintenance of GSI levels comparable to free islets at the first time point (day 5). It is believed that the addition of human pancreatic dECM within the capsules provides essential biochemical cues to preserve islet functionality during long-term in vitro culture. Notably, higher total insulin secretion (compared to both free islets and only alginate) may lower the islet dosage (therapeutic amount) needed to restore euglycemia, reducing the construct size or the number of constructs (for multiple implants) required to deliver islets.

[0134] Embodiments of the present invention contemplate that the best way to develop vascularized tissues is through self-vascularization within bioengineered tissue constructs. This is a slow process and the time required for the assembly and maturation of a perfusable vascular network throughout a graft is often longer than its survival time. Often, graft tissue necrosis occurs early during the engraftment period due to insufficient oxygen supply. Conventionally, different growth factors and cytokines that provide cues for angiogenesis have been added to islet constructs. Advantageously, the dECM may provide an advantage over singular factors as it contains multiple bioactive molecules that can act synergistically to create a more physiologically relevant microenvironment, not only for islet health but also for attracting the host vasculature. The biodegradable fibrin hydrogel already provides a fertile substrate for EC attachment, migration and remodeling. The addition of dECM to fibrin can supplement it with biochemical cues from the native ECM similar to the in vivo pancreatic niche, resulting in enhanced pro-angiogenic properties. Proteomic characterization of the dECM demonstrated enrichment of protein families responsible for the regulation of angiogenesis (VEGFA, SPP1, COL3A1, COL5A2, FSTL1, SERPINA5), cell adhesion, and function. Other studies have shown thatdecellularized ECM-based scaffolds and hydrogels contain chemoattractants that promote migration of host ECs inducing neovascularization upon implantation. The highly vascularized extraembryonic chorioallantoic membrane (CAM) of fertilized chicken eggs offers a simple, easily accessible, and cheap angiogenic screening tool.

[0135] It is contemplated that solubilized dECM is uniquely suitable for incorporation in beta-cell replacement constructs to both provide a pancreas-specific biochemical niche and support graft revascularization. The dECM has other clinically relevant advantages. First, it exists in a powder form (instead of a hydrogel) and can be prepared in a variety of clinically relevant buffers. Addition of the powder has no effect on base hydrogel crosslinking and structural properties as previously demonstrated

[0046] , Second, the dECM exhibiting desirable bioactivity at a relatively low concentration (0.1 mg / ml) is a testament to the efficiency of the decellularization method. This is a significant result related to scale production, as one human pancreas produces around 2g of dECM. One challenge is that the dECM can have compositional differences due to donor variability. This is reduced by a combination of 1) stringent selection criterion (disease-free donors including diabetes mellitus and a BMI<30); 2) pooling the dECM in a batch of multiple (e.g., five) donor pancreata.

[0136] The internal chamber 20 of the implant body 15 holding the AV bundle 30 can comprise osteogenic materials and / or a fibrin hydrogel, depending on a specific clinical application. It is believed that this implant 10 can induce potent neovascularization as early as one week post implantation.

[0137] In some embodiments, the implant 10 may be configured to be able to reverse diabetes and maintain long-term eugly cemia, which can be shown in a preclinical rodent model. Once successful, the implant 10 can be used to generate an artificial pancreas of clinically-relevant size, such as in a range of 5-10 ml or larger such as 10-20 ml.

[0138] FIG. 12 is a flow chart of example actions that can be used to provide an artificial implant such as an artificial pancreas implant. An implant body comprising an internal chamber with biomaterial comprising target cells in a therapeutic amount is provided (optionally to provide islets for transplantation) (block 300). The implant body with the biomaterial is subcutaneously implanted at a target location inside a subject (block 310). A portion of the AV bundle is positioned inside the implant body with the biomaterial before, during or after the implanting (block 320). The AV bundle inside the internal chamber is vascularized whereby branches of vascularization grow to extend from the AV bundle toward an inner surface of a wall bounding the internal chamber within one-week post-implantationthereby promoting survivability of transplanted human islets for long term viability (block 330).

[0139] At least some of the biomaterial is transferred from a container to the internal chamber at a surgical site to provide human islets for transplantation (block 302).

[0140] The biomaterial can be placed in the internal chamber at a manufacturing or assembly site and shipped in a ready to implant condition in a kit for use onsite (block 304).

[0141] The closed end portions of the AV bundle can be attached to the implant body (block 333).

[0142] The closed end portions of the AV bundle can be coupled together and extend outside an end wall of the internal chamber (block 335).

[0143] The AV bundle can have a length that is in a range of about 2 cm to 4 cm longer than a length of the implant body (block 338).

[0144] The implant body can be anchored to internal local tissue, optionally at an iliac location adjacent a groin area as the target location (block 312).

[0145] The biomaterial can comprise decellularized human or porcine pancreatic extracellular matrix dECM material (block 306).

[0146] The positioning can include forming the AV bundle in situ and placing an artery segment and a vein segment of the AV bundle inside the chamber in a substantially parallel arrangement over at least 50% of a length of the internal chamber. The forming the AV bundle can include closing off end portions of each of an artery segment and a vein segment so that no blood flow path is formed therebetween (block 322).

[0147] The biomaterial 25 and / or the implant body 15 can include a secondary therapeutic agent. The term “therapeutic agent” refers to any substance used to treat a desired condition and / or subject. The term “therapeutic agent” can be used interchangeably with “drug”.

[0148] " Treat," "treating" or "treatment of' (and grammatical variations thereof) as used herein refer to any type of treatment that imparts a benefit to a subject and may mean that the severity of the subject’s condition is reduced, at least partially improved or ameliorated and / or that some alleviation, mitigation or decrease in at least one clinical symptom associated with delivery and / or administration of the therapeutic agent is achieved and / or there is a delay in the progression of the symptom. The therapeutic agent can be provided target cells alone, or target cells in combination with one drug, multiple drugs, one drug with a drug carrier (not necessarily a drug), or multiple drugs with multiple carriers. The target cells and any additional therapeutic agent may be formulated for administration in a pharmaceutical carrier in accordance with known techniques See the above referencesregarding the fibrin / hydrogel carriers for target cells such as islets. See, also., Remington, The Science And Practice of Pharmacy (9th Ed. 1995). In the manufacture of a pharmaceutical formulation according to the invention, a therapeutic agent (including a physiologically acceptable salt thereof) is typically admixed with, inter alia, an acceptable carrier. The carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the subject. The carrier may be a solid or a liquid, or both, and may be formulated with a therapeutic agent as a unitdose formulation, for example, a solution (e.g., that may dry to form a coating) and / or powder, which may contain from 0.01 or 0.5% to 95% or 99% by weight of the therapeutic agent. In some embodiments, the carrier may be a biomaterial. One or more therapeutic agents may be incorporated in the formulations of the biomaterial or applied directly to a surface of the implant body 15, which may be prepared by any of the well-known techniques of pharmacy comprising admixing the components, optionally including one or more excipients.

[0149] The therapeutic agent comprises target cells alone or with any anti-proliferative drug, anti-inflammatory drug, anti -migratory drug, any excipient, vitamin, protein or other substance, including combinations thereof.

[0150] The therapeutic agent can be configured or selected to target one or more of proliferation, migration and inflammation and the therapeutic agent can be coated, optionally in a therapeutically effective amount, onto the inner surface and / or the outer surface of the implant body 15.

[0151] As used herein, the term "therapeutically effective amount" or “therapeutic dose” refers to an amount of a therapeutic agent that elicits a therapeutically useful response in a subj ect. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.

[0152] Embodiments of the present inventive concept can provide rapid vascularization in grafts of clinically-relevant sizes for animal and human uses. Once implanted, the fibrotic encapsulation on the external device surface should not hamper the viability and function of the islets embedded within, as is the case with conventional macro devices. On the contrary, the fibrosis could help with adhering / integrating the implant 10 with the host tissues, reducing its movement and constraining the AV bundle 30 for increased stability and limiting movement in the body.

[0153] Advantages of the implants 10 contemplated by the present invention include that, as the AV bundle 30 passes through the core of the chamber 20 along its axis A-A, the setup canbe considered similar to an intravascular device, without the associated issues potential risks, such as clotting, thrombosis and hemorrhage. Moreover, intravascular devices generally contain a central artificial vascular channel / conduit that can only serve the cells / islets in its vicinity. This is not the case in the implants 10 contemplated by embodiments of the present invention that provide microvascular network formation from the central AV bundle 30, which can nutritionally support embedded islets as the target cells of the biomaterial 25 even in the peripheral region of the implant body 15 (easily up to about 1 cm diameter or even larger). It is contemplated that the implant 10 can scale up along its axis in a manner that is easily achievable, as it depends on the length of the AV bundle 30. In rats, the bundles are typically 1 cm in length. In humans, serratus AV bundles have been generated that were ~6 cm long, with a maximum possible length believed to be about ~10 cm. An implant body 15that is 6 cm in length, with an inner diameter of about 1 cm provide a core / chamber volume of ~5 ml, which is contemplated can easily accommodate a clinically-applicable therapeutic amount / dose of islets in a single implant 10. As the AV bundle 30 has a ubiquitous presence, numerous alternative sites including subcutaneous, and muscular flap can be easily explored for islet transplantation, irrespective of the degree of their innate vascularization. This can provide less invasive implantation site options that can also allow easier retrieval, if necessary.

[0154] Embodiments of the invention will be described further below by way of the following non-limiting Examples.EXAMPLES

[0155] Studies will be carried out to validate the clinical efficacy of the implants described hereinabove. One study will generate the implant using three components: 1) An outer chamber which may be generated by 3D printing or cast-molding a medical grade material, contemplated to comprise a bi-component silicone, which is highly gas-permeable and allows optimal diffusion of oxygen. 2) An inner core where islets embedded in a dECM-fibrin hydrogel will be housed; the volume of the core will be about 785 pl, which of note will be suitable for transplantation of curative islet doses in a rat model. 3) An AV bundle, axially implemented to provide a strong and reliable vascularization of the graft, that will be for neovascularization. The study will use human islets.

[0156] Porcine pancreases for decellularization will be purchased from Revivicor, Inc. and processed to make ECM-based biomaterial which will be added to fibrin to create an islet- supportive as well as vascularizing microenvironment. Human islets will be mixed in this dECM-fibrin hydrogel and loaded into in the inner core of the chamber using a syringe. Aftergel polymerization, an AV bundle will be created during the implantation and inserted through the chamber.

[0157] It is believed that the islets housed in the vascularization device (implant body 15) with an AV bundle in dECM-fibrin hydrogel will rapidly vascularize and maintain high viability and function, after transplantation in nude rats.

[0158] Proposed experiments : he combination of AV bundle and the biochemical factors present in the dECM will be evaluated to see if they can drive rapid microvascular network formation within the device, thus protecting the His from hypoxia during the revascularization phase, and maintaining islet viability and function. The device will be implanted in the iliac fossa in non-diabetic nude rats (RNU Nude Rat Crl:NIH-Foxnl™") with a HI dose of 4000 lEQ / rat, with 6 recipients per group per time point (based on previous studies). In the experimental condition, the vascularization chamber will contain both the dECM-fibrin hydrogel and the AV bundle, while the control will be without the AV bundle. The grafts will be analyzed at two time points after transplantation: 1) post-operative day (POD) 7 - to assess and compare islet survival, early graft revascularization, and hypoxia and 2) POD 30 - to assess long-term in vivo functionality. Longitudinal monitoring of human islet functionality will be performed by measuring human C-peptide in peripheral blood by ELISA.

[0159] Methods'.

[0160] Pancreas decellularization, solubilization and dECM-fibrin hydrogel preparation: Briefly, human pancreas from disease-free organ donors will be obtained from the local OPO. After the removal of the peripancreatic tissue and all visible vascular structures, it will be chopped into 1 cm3pieces. Decellularization will be accomplished using our non-detergent- based protocol published recently. Thereafter, the cubes will be lyophilized, cryomilled, and then solubilized with pepsin-HCl for 48 hours at room temperature and neutralized with 0.1N NaOH and 10X PBS to obtain a pH of 7.4 at 4°C. The resulting solution will be further centrifuged, and the supernatant will undergo another series of lyophilization and cryomilling to produce the solubilized growth factor-rich dECM powder. This solubilized dECM will be mixed with fibrin at the previously optimized concentration of 0.1 mg / ml to form the composite dECM-fibrin hydrogel.

[0161] Islet culture: HI will be procured from Prodo Labs and cultured overnight. A day after their arrival, His will be counted. Some free His will be used for GSIS to validate functionality. Remaining His will be mixed in the dECM-fibrin hydrogel and loaded into the inner core of the chamber (4000 IEQS) using a syringe.

[0162] Device implantation: Animal care, housing, and procedures will be performed in accordance with the protocol approved by the Animal Care and Use Committee of WFIRM. 6-7 weeks old nude rats (RNU Nude Rat Crl:NIH-Foxnl™") weighing -200 - 230 g will be obtained from the Charles River Laboratories. An approximately 15 mm incision will be made along the groin and the superficial inferior epigastric artery and vein will be dissected free with the help of a microsurgical microscope. This AV bundle will be ligated distally, transected and pulled inside and through the core of the device with help of the previously placed venous catheter. A subcutaneous pouch will be then created by blunt dissection and the construct carefully inserted. Eventually, the chamber will be secured with a silk stitch to the fascia. Rats will be monitored every other day for their blood glucose levels, weight, and general health assessment.

[0163] Quantification of islet survival, graft revascularization and hypoxia'. Animals will be euthanized at POD 7 and 30 post-implantation and grafts will be retrieved. Harvested grafts will be formalin-fixed grafts, embedded in paraffin, thin sectioned (5 pm in thickness), and processed for immunostaining.

[0164] TUNEL assay will be conducted to determine the percentage of viable cells. Immunofluorescence staining of the grafts will be performed for insulin, glucagon, CD31 and aSMA, followed by counterstaining with DAPI and confocal microscopy. For quantitative analysis, all nuclei will be classified as INS GLU p cells, or INS GLU a cells, or CD31+endothelial cells with the absence of aSMA+cells (immature vessels), or CD31+endothelial cells with the presence of aSMA+cells (mature vessels). In some animals, we will inject fluorescein Griffonia simplicifolia Lectin I (GSL I), which binds to rodent endothelial cells, intravenously prior to graft explant. The whole mount will then be imaged with multiphoton microscopy. AngioTool will be used to analyze microscopy images for vessel morphometric and spatial parameters, including vessel length, density, and branching index. For quantification of graft hypoxia, immunofluorescence staining will be performed using antibodies against HIF-1 alpha.

[0165] C-peptide measurement - Blood will be drawn from rat tail on PODs 0, 2, 4, 7, 14, 21 and 28. Human C-peptide will be measured in peripheral blood by ELISA (Mercodia).

[0166] Ex vivo functionality of human islets'. Harvested grafts (not fixed) will be assessed through a standard ex vivo glucose-stimulated insulin secretion (GSIS) with sequential stimulation with 2.2mM, 16.7mM, 2.2mM glucose and quantified for human insulin content by ELISA (Mercodia).

[0167] Expected Outcomes: It is expected that the islets in the implant (vascular device), VD, with the AV bundle to be functional for a longer time as detected by the presence / higher concentration of human C-peptide in peripheral blood, compared to those in the device without the AV bundle. The device with the AV bundle is also expected to display a higher density of mature blood vessels compared to controls. While the device without the AV bundle will likely display necrotic cores as well as positive staining of HIF-lalpha, constructs including the AV bundle are expected to display less severe hypoxia-related central necrosis, demonstrating the effect of the AV bundle in enabling rapid vascularization to counteract hypoxia-mediated cell death. At the completion of this aim, we will identify a supportive niche for islets as well as vascularization, which will be used for Aim 2 experiments where the grafts will be tested for their capability to promote long-term diabetes reversal after transplantation.

[0168] If expected results are not obtained, a higher concentration of dECM can be tested for its effect on islet function as well as vascularization. Fibrin is being used here because it has been successfully used it in a prior VD. However, other hydrogels such as Collagen can also be explored.

[0169] Rat tail collagen can be used for proof of concept studies, which can be easily be replaced in future with marine (jellyfish) or recombinant human collagen for translation. Other pro-angiogenic factors can be used / included (VEGF and / or FGF-2) in the hydrogel to enhance vascularization. The release of FGF-1 incorporated in alginate microcapsules can result in enhancement of graft neovascularization. Moreover, if needed, VEGF or its artificially synthesized mimetic peptide (QK) can be incorporated in the hydrogel to provide additional signals for enhancing vascularization. The QK peptide has been used in many studies to improve angiogenic properties of different implant materials, resulting in significantly improved migration and proliferation of ECs and promoting neovascularization. Autologous vascular adipose-derived cells can also be included, which have been shown in previous studies to further speed up as well as increase the density of the newly-formed vascular network.

[0170] Aim 2: Test the vascularization device with human islets in diabetic nude rats

[0171] Hypothesis: It is hypothesized that His suspended in a dECM-fibrin hydrogel, housed in the vascularization chamber along with an AV bundle will rapidly vascularize and integrate with the host, resulting in long-term euglycemia after implantation in nude diabetic rats.

[0172] Proposed experiments: The combination of the vascularization chamber, AV bundle and the dECM hydrogel will be assessed to see if they can protect His from hypoxia during the revascularization phase, form a microvascular network within the device and promote islet engraftment, resulting in faster diabetes reversal after transplantation, and longer maintenanceof euglycemia, compared to controls. The experimental and control groups will be same as Aiml, except that the nude rats will be rendered diabetic by streptozotocin (STZ) injection, prior to transplantation. An intraperitoneal glucose tolerance test (IPGTT) will be conducted on POD 30, 60, and 100. The median diabetes reversal time (MDRT) and the duration of euglycemia (blood glucose <200 mg / dL and glucose tolerance compared to non-diabetic rats) will be evaluated for up to 100 days after transplantation. Quantification of islet survival, and graft revascularization will be performed (as in Aiml) at POD 100.

[0173] Methods'.

[0174] Diabetes induction, islet transplantation, and diabetes monitorins:

[0175] 7-8 weeks old nude rats (RNU Nude Rat Crl:NIH-Foxnl™“) weighing -280 - 300 g will be obtained from the Charles River Laboratories. Diabetes will be induced in rats via a single dose of STZ in citrate buffer at a pH of 4.5 and injected intraperitoneally at a dose of 60 mg / kg. Rats will be considered diabetic after three consecutive fasting blood glucose readings greater than 250 mg / dL. Blood glucose will be measured via tail vein sampling using a portable glucometer. Transplantation will be conducted on diabetic animals with the same methods as described in aim 1. Rats will be monitored every other day for their blood glucose levels, weight, and general health assessment. An intraperitoneal glucose tolerance test (IPGTT) will be conducted on post-operative days (POD) 30, 60, and 100. Briefly, rats will be fasted overnight prior to IPGTT, then a 30-pl blood sample will be collected via tail vein prick just before IPGTT. Rats will then be injected intraperitoneally with 2 g / kg of glucose. Blood glucose measurements will be taken at time 0, 10, 30, 60, and 120 minutes after administration. A 30-pl blood sample will be collected before glucose injection and at 30 minutes after glucose administration via tail vein prick and assayed for serum human C peptide by ELISA.

[0176] Quanti fication of islet survival, raft revascularization and hypoxia'. Recipient rats will be euthanized at POD 100 and grafts retrieved. Quantification of islet survival, graft revascularization and hypoxia will be performed as described in aim 1.

[0177] Expected outcomes: It is expected that the combination of the AV bundle and dECM to promote engraftment of His in rats, exhibiting decreased MDRT and decreased islet hypoxia in grafts analyzed at POD 100, compared to the control group (without AV bundle). The proposed combination approach will hopefully result in maintenance of long-term euglycemia and improved glucose tolerance as demonstrated by decreased AUC and higher levels of stimulated c-peptide during the IPGTT, compared to control.

[0178] Potential pitfalls / alternative approaches: If the combination of AV bundle and incorporation of dECM in fibrin does not mitigate islet death or result in diabetes reversal, thenthere are three primary parameters that can be altered. First, islets can be encapsulated in alginate supplemented with dECM and cultured in vitro, prior to adding encapsulated islets in the implant body 15 / VD for implantation. Encapsulation in alginate-dECM can provide human islets with essential biochemical cues to preserve their long-term islet viability and function. Second, islets can be preconditioned by co-culturing them with human mesenchymal stem cells to improve their viability and function, prior to transplantation. Third, multiple AV bundles can be introduced within one device to further improve the density of the microvascular network and provide nutrient / oxygen and insulin exchange to serve islets residing even in the peripheral parts of the VD, eliminating any dead zones.

[0179] A. Advantages over alternative approaches that would address goal.

[0180] Example advantages and innovative aspects of the novel strategy over traditional approaches are summarized below.

[0181] The approach uses a device that offers ease of surgical implantation and retrieval in case of adverse events, overcoming a potential regulatory hurdle associated with cell therapy.

[0182] The ubiquitous presence of the AV bundle makes the strategy independent of implantation sites, which can often be limited based on their innate degree of vascularization. Implantation sites including, subcutaneous and muscular flap, that are generally considered as poorly vascularized and therefore not considered ideal for conventional implants can be successfully used with our approach, offering less invasive alternatives to traditional sites.

[0183] Longer AV bundles can be harnessed for device scale up in the axial direction (rather than radial), to easily accommodate a therapeutic islet dose, while ensuring that the proximity distribution of islets from the AV bundle remains similar along the entire axis of the device.

[0184] With the implant device configuration, fibrotic encapsulation on the external surface of the device should not have any direct impact on the viability or function of islets within, which could be prevalent and ultimately lead to failure in currently used macro devices. Conversely, fibrosis on the external surface can be turned into an advantage, as it can adhere / integrate the device to the host tissue, which in turn could constrain the AV bundle, thus improving its stability.

[0185] Incorporation of human pancreatic dECM that can support long-term islet viability and function and also provide biochemical factors to support rapid vascularization.

[0186] Use of the patented detergent-free DI water-based decellularization methods can provide an enriched dECM demonstrating functional bioactivity at a relatively lowconcentration. As a powder, the dECM can be seamlessly incorporated into new and established hydrogels without altering their chemical, physical and mechanical properties.

[0187] The proposed strategy is built around easily approved soluble factors and the body’s innate neo-vascularization capability and mechanisms for graft revascularization rather than the regulatory challenge of additional exogenous cell sources (endothelial, mesenchymal) in the hydrogel / device. A single cell source will simplify regulatory approval and clinical translation.

[0188] Currently naked islets are being proposed for implantation, however, after successful proof of concept, these can be seamlessly replaced with conformal / nano-coated or microencapsulated islets to provide immune protection for allogenic grafts.

[0189] B. Future plans if research is successful.

[0190] This project will provide the proof of concept that the proposed device is able to reverse diabetes and maintain long-term eugly cemia in a preclinical rodent model. Once successful, the next step can be to upscale the approach to generate an artificial pancreas of clinically-relevant size (5-10 ml) to be tested in non-human primates (NHP), prior to final initiation of a first-in-human trial. In terms of implantation site and vascular bundle, the right iliac fossa (as in renal transplantation) and the superficial epigastric vessels respectively, are currently contemplated as preferred sites because they are easy to access and manipulate. Longterm goals also include testing the device with stem cell-derived islet-like clusters (ILCs). The decellularization process has already been scaled up in the GLP / cGMP facility in Wake Forest Regenerative Medicine Clinical Centre (RMCC), where the development has been refined and approved with appropriate SOPs.

[0191] Neovascularization is believed to occur immediately, ensuring the survival and functionality of the islets. Moreover, the envisioned size for an islet graft is in the range of 5 ml for a therapeutic dose of transplanted islets (10,000 lEQ / kg), which is below what we have successfully demonstrated for engineered bone (several tens of cm3). It is believed that human islets suspended in a dECM-fibrin hydrogel, housed in the vascularization chamber along with an arteriovenous bundle will rapidly revascularize and integrate with the host, maintaining high islet viability and function, and resulting in long-term euglycemia after transplantation in nude diabetic rats.

[0192] FIGS. 13A-13C illustrate vascularization of the osteogenic VD after 1-week transplantation in rats. FIGS. 13A / B show E staining, along with black stained vessels (Indian Ink) throughout the entire construct. Microvessel expansion from the AV bundle (blue dotted circle) to the device rim (red dotted circle; ~4 mm distance from AV bundle) can be visualized.FIG. 13C shows a Micro-tomographical 3D reconstruction showing a vessel tree originating from the AV bundle at 8 weeks post-implantation.

[0193] While not wishing to be bound to any particular theory, maintenance of long-term euglycemia and improved glucose tolerance may be demonstrated by decreased AUC and higher levels of stimulated c-peptide during the IPGTT, compared to controls, postimplantation of the implant(s) contemplated by the present invention.

[0194] The drawings and specification, there have been disclosed embodiments of the invention and, although specific terms are employed, they are used in a generic and descriptive sense only and not for purposes of limitation, the scope of the invention being set forth in the following claims.

[0195] Thus, the foregoing is illustrative of the present invention and is not to be construed as limiting thereof. More particularly, the workflow steps may be carried out in a different manner, in a different order and / or with other workflow steps or may omit some or replace some workflow steps with other steps. Although a few exemplary embodiments of this invention have been described, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention.

[0196] Accordingly, all such modifications are intended to be included within the scope of this invention as defined in the claims. In the claims, means-plus-function clauses, where used, are intended to cover the structures described herein as performing the recited function and not only structural equivalents but also equivalent structures. Therefore, it is to be understood that the foregoing is illustrative of the present invention and is not to be construed as limited to the specific embodiments disclosed, and that modifications to the disclosed embodiments, as well as other embodiments, are intended to be included within the scope of the appended claims. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

THAT WHICH IS CLAIMED:

1. An artificial pancreas implant, comprising: an implant body comprising an internal chamber; an arteriovenous (AV) bundle extending at least partially into the internal chamber; and a biomaterial in the implant body comprising pancreatic islets.

2. The artificial pancreas implant of Claim 1, wherein the implant body is gas permeable and comprises a synthetic, non-resorbable material.

3. The artificial pancreas implant of Claim 1, wherein the implant body comprises silicone.

4. The artificial pancreas implant of Claim 1, wherein the internal chamber has a volume in a range of about 5 mL to about 10 mL, optionally about 5 mL.

5. The artificial pancreas implant of Claim 1, wherein the implant body has an axially extending length with a first port on a first end sized and configured to slidably receive the AV bundle.

6. The artificial pancreas implant of Claim 1, wherein the implant body comprises a second port on a second end that is axially opposed to the first end.

7. The artificial pancreas implant of Claim 1, wherein branches of (neo)vascularization extend from the AV bundle toward an inner surface of the implant body through the biomaterial one week post implantation.

8. The artificial pancreas implant of Claim 1, wherein the biomaterial comprising pancreatic islets comprises a decellularized extracellular matrix (dECM)-fibrin hydrogel with human pancreatic islets in a therapeutically effective amount.

9. The artificial pancreas implant of Claim 1, wherein the AV bundle comprises an artery segment with a first end and a second end and a vein segment with a first end and a second end, wherein the first end of the artery segment is attached to an artery of a patient outside theimplant body and the first end of the vein segment is attached to a vein of the patient outside the implant body, and wherein the artery segment and the vein segment extend at least partially into the internal chamber.

10. The artificial pancreas implant of Claim 9, wherein the second end of the artery segment and the second end of the vein segment are both closed ends that are both held inside the internal chamber or that are both held outside the internal chamber, optionally adjacent an end wall of the implant body at a location spaced apart from an entry port of an axially spaced apart end wall, and further optionally wherein the second end of the artery segment and the second end of the vein segment are coupled together but do not provide a blood flow path therebetween.

11. The artificial pancreas implant of Claim 1, wherein the AV bundle has a length that is greater than a length of the implant body, optionally 2 cm-4 cm longer than the length of the implant body.

12. The artificial pancreas implant of Claim 1, wherein the AV bundle is provided as first and second AV bundles, optionally each positioned to extend at least an entire length of the implant body.

13. The artificial pancreas implant of Claim 1, wherein the implant body has an axial length and axially spaced apart first and second ports, and wherein the first port has a width or diameter that is greater than a width or diameter of the second port.

14. The artificial pancreas implant of Claim 13, wherein the implant body has first and second axially spaced apart end walls that provide the respective first and second ports, and wherein the first end wall has a thickness that is less than a thickness of the second end wall.

15. The artificial pancreas implant of Claim 14, wherein the first and second ports comprise a sealant or cover configured to close the first and second ports to prevent the biomaterial in the internal chamber from migrating or moving out of the internal chamber through the first and second ports.

16. The artificial pancreas implant of Claim 1, wherein the AV bundle comprises an artery segment with a first end and a second end and a vein segment with a first end and a second end, wherein the first end of the artery segment is attached to an artery of a patient outside the implant body and the first end of the vein segment is attached to a vein of the patient outside the implant body, wherein the second end of the artery segment and the second end of the vein segment are both closed ends held in or adjacent each other, optionally adjacent a port of the implant body, and wherein the closed ends do not provide a blood flow path therebetween.

17. The artificial pancreas implant of Claim 1, wherein the AV bundle is arranged so that at least a major portion (50% or greater) of a vein segment and at least a major portion of an artery segment thereof are parallel and inside the internal chamber of the implant body.

18. The artificial pancreas implant of Claim 1, further comprising tissue attachment features for anchoring the implant body in a desired position in a patient to prevent undue movement or migration post-implantation.

19. The artificial pancreas implant of Claim 1, further comprising a radiopaque marker or material defining an access location thereby providing an intrabody visualization feature post-implantation.

20. A medical kit comprising: an artificial pancreas implant body comprising an internal chamber; and a biomaterial comprising pancreatic islets and a carrier.

21. The medical kit of Claim 20, further comprising a catheter sized and configured to pull an arteriovenous bundle at least partially into the artificial pancreas implant body.

22. The medical kit of Claim 20, wherein the artificial pancreas implant body has a length and first and second ports spaced apart in the length dimension.

23. The medical kit of Claim 22, wherein the first port has a width or diameter that is greater than a width or diameter of the second port.

24. The medical kit of Claim 21, wherein the internal chamber has a volume in a range of about 5 cubic centimeters to about 10 cubic centimeters.

25. The medical kit of Claim 23, wherein the artificial pancreas implant body has first and second axially spaced apart end walls that are configured with the respective first and second ports, and wherein the first end wall has a thickness that is less than a thickness of the second end wall.

26. The medical kit of Claim 21, wherein the carrier of the biomaterial comprises decellularized pancreatic extracellular matrix dECM material.

27. An implant for providing cell therapy to a subject, comprising: an implant body; an arteriovenous (AV) bundle in the implant body; and target cells in a therapeutic amount in a carrier in the implant body.

28. The implant of Claim 27, wherein the AV bundle is provided as a plurality of AV bundles with a portion of each held in the implant body and with one end portion extending out of the implant body, coupled to respective artery and vein segments of the subject.

29. A method of providing human islets, comprising: providing an implant body comprising an internal chamber; providing a biomaterial comprising human islets; forming an arteriovenous (AV) bundle from harvest site of a subject; positioning a portion of the AV bundle inside the internal chamber of the implant body; introducing the biomaterial comprising the human islets in the internal chamber of the implant body before, during or after positioning the AV bundle in the implant body; and implanting the implant body before or after introducing the biomaterial into the implant body thereby providing human islets to the subject.

30. The method of Claim 29, further comprising vascularizing the AV bundle inside the internal chamber whereby branches of vascularization grow and extend from the AV bundle toward an inner surface of a wall bounding the internal chamber within one-week post-implantation thereby promoting survivability of transplanted human islets for long term viability.

31. The method of Claim 29, wherein the method further comprises transferring at least some of the biomaterial to the internal chamber of the implant body at a surgical site of the implantation during or before the implanting step.

32. The method of Claim 29, further comprising anchoring the implant body at an iliac location adjacent a groin area as a target implant location.

33. The method of Claim 29, wherein the biomaterial comprises decellularized pancreatic extracellular matrix dECM material.

34. The method of Claim 29, wherein the positioning comprises placing an artery segment and a vein segment of the AV bundle inside the internal chamber in a substantially parallel arrangement over at least 50% of a length of the internal chamber.

35. The method of Claim 29, wherein the forming the AV bundle includes closing off end portions of each of an artery segment and a vein segment so that no blood flow path is formed therebetween.

36. The method of Claim 35, wherein the closed end portions are attached to an end portion of the implant body.

37. The method of Claim 35, wherein the closed end portions are coupled together and extend outside an end wall of the internal chamber, and optionally terminate adjacent thereto.

38. The method of Claim 29, wherein the AV bundle has a length that is 2 cm to 4 cm longer than a length of the implant body.

39. The method of Claim 38, wherein the AV bundle has portion formed as a sub-length of the AV bundle that extends out of the implant body with end portions thereof attached to a respective vein and artery of the subject.

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