Tissue regenerative multi-drug cocktail and apparatus for delivery thereof

Abstract

To promote regeneration of a tissue on a subject such as a wounded, damaged, or injured appendage or a tissue within a subject such as a wounded, damaged, or injured organ.SOLUTION: Disclosed are apparatus, compositions, and methods for promoting regeneration of a tissue on a subject such as a wounded, damaged, or injured appendage, or a tissue within a subject such as a wounded, damaged, or injured organ. The disclosed apparatus, composition, and methods include or utilize wearable sleeves and regenerative compositions.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 944,707, filed on December 6, 2019, the entire content of which is incorporated herein by reference.

[0002] Description of Research and Development Sponsored by the Federal Government This invention was made with government support under grant number AR055993 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Technical Field The field of the present invention relates to regenerative medicine. More particularly, the present disclosure relates to devices, compositions, and methods for promoting tissue regeneration in or within a subject in need thereof. The disclosed devices include wearable sleeves, and the disclosed compositions include regenerative compositions.

Background Art

[0004] The prevalence of limb loss in the United States alone is expected to increase significantly over the next 30 years, with 3.6 million individuals affected per year by 2050 (Ziegler - Graham et al., 2008), and individuals suffering from diabetes, war veterans, survivors of traumatic brain injury, and those with peripheral arterial disease who have limited options in amputation events will be left as they are.

[0005] Previous efforts have included the use of electrical stimulation (Borgens (1982) Science 217,747 - 750; Leppik et al. (2015) Sci.Rep. ,5,18353; Smith, (1981) Bioelectrochem.and Bioenergetics ,8(6),661 - 670), tissue - guiding biomaterials (Suckow et al. (1999) J.of Invest.Surg., 12(5), 277 - 287), progenitor cell transplantation (Lin et al., (2013) Dev.Cell , 24(1), 41 - 51), and the regulation of key molecular pathways (Kawakami et al., (2006) Genes & Dev. , 20(23), 3232 - 3237; Yokoyama et al., (2001) Dev.Biology , 233(1), 72 - 79) are intended to induce limb regeneration. However, they have not achieved great results in restoring significant growth and pattern formation of new limbs.

[0006] Tissue regeneration involves a series of biological events that are overall associated with restoring excised organs or appendages lost during mental trauma or amputation. There are distinct differences between typical wound healing responses and regeneration responses. Although these two processes are similar in many embodiments, they result in completely different end products. In the process of normal wound healing, many complex biological structures, such as sweat glands, ducts, and hair follicles, cannot be restored because the biological machinery for doing so is not available. In typical adult mammalian skin wounds, these structures are not regenerated because the development of these tissues and organs involves highly specific biological processes. Also, normal wound closure and scar formation do not provide an environment suitable for these structures to regenerate. Regenerative addition, on the other hand, is a process in which all prototype structures are replaced by replication of the prototype.

[0007] Some complex animals, such as axolotls, can naturally regenerate their limbs, eyes, and all other organs throughout their bodies, while mammals generally exhibit limited regeneration and generally do not show such flexibility and transdifferentiation ability as possessed by urodeles. Adult anurans can regenerate their amputated or injured limbs when exposed to regeneration-inducing factors delivered through sustained-release beads implanted in the transected tissue. Tissue progenitor cells for inducing the regeneration process may also have uses; it has been shown that larval limb progenitor cells activate the same Wnt and Shh signaling to promote patterning.

[0008] However, how Xenopus can function well as a useful animal model for regeneration after complete non-regenerative metamorphosis (adult) is not known because they cannot regenerate their hindlimbs during amputation but instead produce cartilage spikes characteristic of adult frogs (Suzuki et al., (2006) Sci.World J. ,6:26-37).

[0009] Regeneration sleeves have been used for tissue regeneration of wounded tissues. Conventional sleeves include a diaphragm that allows a needle to repeatedly exchange the medium within the wound space, a liquid reservoir that keeps the wound moist, and a liquid-tight seal that prevents the liquid from exiting the sleeve. However, such conventional sleeves are subject to several limitations. In some cases, the liquid-tight seal may be too strong and lead to tissue necrosis. Replacement of the medium through the needle may directly affect the wound bed.

[0010] Furthermore, conventional tissue regeneration devices are typically attached to a subject using a glue that is not preferred for long-term treatment. Attaching a regeneration device using glue has two main disadvantages. The glue lacks the flexibility adjustment for the device after attachment. Also, there are handling complications during surgery and with the device.

[0011] Therefore, improved devices and regeneration compositions are desired and needed.

Summary of the Invention

[0012] Disclosed are devices, compositions, and methods for promoting the regeneration of tissues, such as wounded, damaged, or injured sites in the tissues or organs of the attached organs of a survival target, or in the tissues within the organs. The disclosed devices, compositions, and methods include or utilize a wearable sleeve and a regeneration composition.

[0013] In one aspect, the present disclosure provides a therapeutic composition for promoting tissues. In some embodiments, the regeneration composition may include a plurality of components. In certain embodiments, the disclosed regeneration composition is utilized in the disclosed device as a therapeutic composition present in the material of the inner sleeve that contacts the wounded, damaged, or injured tissue.

[0014] In one aspect, the therapeutic composition includes a growth factor, an inhibitor of the prolyl hydroxylase domain (PHD) enzyme, vitamin A or its derivatives, and a lipid mediator.

[0015] In certain embodiments, the therapeutic composition includes a growth factor selected from brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), and leukemia inhibitory factor (LIF), and combinations thereof. In certain embodiments, the therapeutic composition includes nerve growth factor. In certain embodiments, the therapeutic composition includes brain-derived neurotrophic factor (BDNF).

[0016] In some embodiments, the therapeutic composition comprises an inhibitor of a PHD enzyme selected from the group consisting of 4,4α-dihydro-4-oxo-1,10-phenanthroline-3-carboxylic acid (1,4-DPCA), N-[(1,3-dicyclohexylhexahydro-2,4,6-trioxo-5-pyrimidinyl)carbonyl]-glycine, 6-amino-1,3-dimethyl-5-[(2-pyridinylthio)acetyl]-2,4(1H,3H)-pyrimidinedione, 6-amino-1,3-dimethyl-5-[[2-(2-pyridinyl)-4-quinolinyl]carbonyl]-2,4(1H,3H)-pyrimidinedione, N-[(4-hydroxy-1-methyl-7-phenoxy-3-isoquinolinyl)carbonyl]-glycine, an iron chelating agent, and combinations thereof. In certain embodiments, the PHD inhibitor is 4,4α-dihydro-4-oxo-1,10-phenanthroline-3-carboxylic acid (1,4-DPCA). In certain embodiments, the therapeutic composition comprises 4,4α-dihydro-4-oxo-1,10-phenanthroline-3-carboxylic acid (1,4-DPCA).

[0017] In some embodiments, the therapeutic composition comprises a derivative of vitamin A selected from the group consisting of retinoic acid, retinol, retinyl carboxylic acid, tretinoin, tazarotene, and combinations thereof.

[0018] In some embodiments, the therapeutic composition comprises a lipid mediator selected from the group consisting of resolvin, metabolites of omega-3 fatty acids, derivatives of eicosapentaenoic acid, derivatives of docosahexaenoic acid, and combinations thereof. In certain embodiments, the lipid mediator is resolvin. In certain embodiments, the therapeutic composition comprises resolvin 5, interleukin 6 (IL-6), interleukin 4 (IL-4), tumor necrosis factor-alpha (TNF-alpha), nuclear factor kappa-light-chain enhancer of activated B cells (NF-kB), and resolvin selected from the group consisting of combinations thereof.

[0019] In some embodiments, the therapeutic composition comprises an agent that functions in proximal-distal positional information, and the agent is selected from the group consisting of bone morphogenetic protein 9 (BMP9), nodal growth differentiation factor, activin, transforming growth factor-beta (TGF-β), fibroblast growth factor 8 (FGF8), and combinations thereof.

[0020] In some embodiments, the therapeutic composition comprises a peptide or protein hormone. In some embodiments, the therapeutic composition comprises a peptide hormone selected from the group consisting of growth hormone (GH), insulin-like growth factor-1 (IGF-1), transforming growth factor-beta-1 (TGFβ-1), epidermal growth factor (EGF), granulocyte-colony stimulating factor (G-CSF), fibroblast growth factor FGF, and combinations thereof.

[0021] In some embodiments, the growth factor is present in the therapeutic composition at a dosage of 0.1 μg / ml to 1 μg / ml. In some embodiments, the PHD inhibitor is present in the therapeutic composition at a dosage of 0.004 μg / ml to 0.024 μg / ml. In some embodiments, vitamin A or its derivative is present in the therapeutic composition at a dosage of 0.03 μg / ml to 0.27 μg / ml. In some embodiments, the lipid mediator is present in the therapeutic composition at a dosage of 0.006 μg / ml to 0.054 μg / ml. In some embodiments, the peptide or protein hormone is present in the therapeutic composition at a dosage of 0.1 μg / ml to 1.0 μg / ml.

[0022] In another aspect, the present disclosure provides a method for promoting tissue regeneration in a mammal, the method comprising administering to the mammal a therapeutic composition provided in an amount sufficient to promote tissue regeneration in the mammal.

[0023] In yet another aspect, the present disclosure provides the use of a therapeutic composition for stimulating the regeneration of tissue in a mammal in need thereof.

[0024] In another aspect, the disclosed device includes an outer sleeve having a tissue receiving and / or insertion end, a pressing member receiving end opposite the tissue receiving end, and an internal chamber configured to receive and / or contain tissue. The device further includes an inner sleeve disposed within the outer sleeve, the inner sleeve having an end for receiving a wounded, damaged, or injured tissue or appendage, a fitting receiving end for fitting a pressing member, and an internal chamber configured to receive and / or contain a wounded, damaged, or injured tissue. The pressing member is configured to extend through the pressing member receiving end into the internal chamber of the outer sleeve and bias the fitting receiving end of the inner sleeve toward the tissue such that at least a portion of the subject's wounded, damaged, or injured tissue is placed in contact with a portion of the internal chamber of the inner sleeve. The inner sleeve may include and / or be formed from a polymeric material including, but not limited to, a silk hydrogel material. The device further includes a first end cap having an opening configured to receive and maintain tissue within the device and being matingly engageable with the tissue receiving end of the outer sleeve, and a second end cap matingly engageable with the pressing member receiving end of the outer sleeve.

[0025] In some embodiments, the tissue is an appendage or a portion of an organ.

[0026] In some embodiments, the device includes a first threaded adapter that can be installed within the tissue receiving end of the outer sleeve for selectively coupling the first end cap matingly engageable with the tissue receiving end of the outer sleeve. In some embodiments, the first end cap includes a groove configured to receive the threads of the first threaded adapter.

[0027] In some embodiments, the device includes a second screw adapter that is installable within a pressure member receiving end of an outer sleeve for selectively coupling an outer sleeve to a second end cap that is engageable with the pressure member. In some embodiments, the second end cap includes a groove configured to receive the threads of the second screw adapter.

[0028] In certain embodiments, the pressure member includes a seat portion configured to receive the mating receiving end of the inner sleeve, or includes a mating end portion configured to be at the seat portion of the second end cap.

[0029] In some embodiments, the mating receiving end of the inner sleeve includes a porous filter medium that closes the internal chamber by sealing at the mating receiving end. In some embodiments, the porous filter medium is a synthetic or polymeric membrane. In some embodiments, the device includes a compressible member positioned between the porous filter medium and the pressure member. In some embodiments, the compressible member includes cotton or encapsulated gel.

[0030] In some embodiments, the inner sleeve includes a protein or polymeric matrix that at least partially fills the internal chamber of the inner sleeve. In some embodiments, the protein or polymeric matrix includes a three-dimensional porous scaffold. In some embodiments, the porous scaffold may include pores that form a directional pattern. In some embodiments, the porous scaffold includes aligned pores that form substantially aligned channels. The aligned channels in the protein or polymeric matrix may be arranged parallel to the longitudinal axis of the inner sleeve. In some embodiments, the protein or polymeric matrix is selected from silk fibroin, collagen, or combinations thereof.

[0031] In some embodiments, the device includes an electrical stimulation device including an anode and a cathode, the anode and the cathode being configured to be electrically connected to corresponding terminals of a power source, and a portion of the cathode being disposed within an inner sleeve.

[0032] In another aspect, the disclosed device includes a pressing member that is axially movable, for example, toward and / or away from a wounded, damaged, or injured tissue site in response to growth at the site of the wounded, damaged, or injured tissue. In some embodiments, the pressing member biases the fitting receiving end of the inner sleeve toward the tissue such that at least a portion of the targeted wounded, damaged, or injured tissue is placed in contact with a portion of the inner chamber of the inner sleeve, and when the tissue regenerates and / or the wounded, damaged, or injured tissue heals, the regenerated and / or healed tissue moves the pressing member axially away from the wound to contact the fitting receiving end directly or indirectly.

[0033] In some embodiments, the pressing member is elastic or includes an elastic member that compresses in response to growing tissue. The elastic member may be a spring.

[0034] In some embodiments, the device includes a threaded adapter that can be installed within a pressing member receiving end of an outer sleeve for selectively connecting a second end cap that can be mated with the pressing member to the outer sleeve. The second end cap may include a groove configured to receive the threads of the threaded adapter. The elastic member may extend between a seat portion of the threaded adapter and a seat portion of the pressing member.

[0035] In some embodiments of the disclosed device, the inner sleeve of the disclosed device contacts the traumatized, damaged, or injured tissue when the traumatized, damaged, or injured tissue is inserted into the device, and contains a material within a reservoir in the inner sleeve. The material of the inner sleeve may include a hydrogel that moistens the traumatized, damaged, or injured tissue. The material may further include a therapeutic composition that promotes tissue regeneration and / or healing, and / or an antibacterial agent.

[0036] In yet another aspect, the disclosed device includes an inner sleeve that includes an end portion having a first opening sized to accommodate traumatized, damaged, or injured tissue, and a mating receiving end portion having a second opening, the inner sleeve defining an internal chamber extending between the first opening and the second opening, the internal chamber being sized to accommodate the traumatized, damaged, or injured tissue of the subject. The device further includes a matrix disposed within the internal chamber of the inner sleeve, the matrix including a porous scaffold having pores that form substantially aligned channels. The matrix may include, but is not limited to, any suitable form of collagen and silk fibroin, may include materials including, but not limited to, polymeric materials such as proteins, and / or may be formed therefrom. The aligned channels of the porous scaffold may be disposed parallel to the longitudinal axis of the inner sleeve. The matrix may include a therapeutic composition disclosed herein, such as a regenerative composition or a regenerative cocktail.

[0037] In some embodiments, the substantially aligned channels are substantially parallel or parallel to the longitudinal axis of the inner sleeve that is orthogonal to the first and second openings. In some embodiments, the internal chamber includes a reservoir of an aqueous solution or a dispersion medium between a filter medium and a protein matrix.

[0038] In some embodiments, the device further includes a filter medium sized to enclose a second opening at the fitting receiving end of the inner sleeve. In some embodiments, the filter medium has a pore size large enough to allow air to pass through the device but small enough to prevent microorganisms from entering the device.

[0039] In some embodiments, the inner chamber of the inner sleeve of the provided device contains the provided therapeutic composition.

[0040] In another aspect, the present disclosure provides a method for promoting tissue regeneration in a mammal. The method includes attaching the provided device to a wounded accessory organ or tissue of a mammal.

[0041] In another aspect, the present disclosure provides the use of the above-described device for stimulating the regeneration of tissue in a mammal in need thereof.

[0042] The above and other embodiments and advantages of the present disclosure will be apparent from the following detailed description. In the detailed description, reference is made to the accompanying drawings, which form a part hereof, and which illustrate, by way of non-limiting example, embodiments. This embodiment does not necessarily present the full scope of the present disclosure, but the entire disclosure herein is hereby incorporated by reference for the purpose of interpreting the scope of the present disclosure.

[0043] The present disclosure is described below with reference to the accompanying drawings, in which like reference numerals indicate like elements.

Brief Description of the Drawings

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[0066] The disclosures of these patents, patent applications, and published publications are hereby incorporated by reference in their entirety into this application to more fully describe the state of the art as known to those skilled in the art as of the date of the invention described and claimed herein. In the event of any conflict between such patents, patent applications, and published publications and the present disclosure, the present disclosure shall control.

[0067] Before explaining any embodiment of the present disclosure in detail, it is to be understood that the present disclosure is not limited in its application to the details of the construction and arrangement of components set forth in the following detailed description or shown in the following figures. The present disclosure is capable of other embodiments and of being practiced or carried out in various ways.

[0068] It should be understood that the expressions and terms used in this specification are for illustrative purposes and should not be regarded as limiting. The use of "including", "comprising", or "having" and their variants in this specification is intended to include the items listed hereinafter and their equivalents, as well as additional items. Unless otherwise specified or limited, the terms "mounted", "connected", "supported", and "coupled", and their variants are used broadly and include both direct and indirect mounting, connection, support, and coupling. Further, "connected" and "coupled" are not limited to physical or mechanical connection or coupling.

[0069] As used herein, the term "tissue" is defined as an aggregate of similar cells from the same origin and their extracellular matrix that together perform a specific function. As used herein, "tissue" can be present in, but is not limited to, phalanges (e.g., fingers and toes), arms, legs, and accessory organs including those of the same kind. As used herein, "tissue" can be present in organs (e.g., liver, lung, pancreas, and the like).

[0070] The terms "therapeutic composition", "regenerative composition", "regenerative cocktail", and "multi-drug therapeutic compound" or "MDT" include formulations that contain combinations of therapeutic agents that stimulate or initiate tissue regeneration and, in some embodiments, have a synergistic effect when administered to a subject.

[0071] The following considerations are presented to enable one skilled in the art to make and use embodiments of the present disclosure. Various modifications to the embodiments shown will be readily apparent to one skilled in the art, and the general principles herein may be applied to other embodiments and applications without departing from the scope of the present disclosure. Accordingly, the embodiments of the present disclosure are not intended to be limited to the embodiments shown, but the broadest scope consistent with the principles and features disclosed herein should be given. The following detailed description should be read with reference to the drawings, and like elements in different drawings have like reference numerals. The drawings are not necessarily to scale and show selected embodiments, and are not intended to limit the scope of the embodiments of the present disclosure. One skilled in the art will recognize that the examples provided herein have many useful alternatives and are within the scope of the embodiments of the present disclosure.

[0072] Device for tissue regeneration The present disclosure provides a device for assisting tissue regeneration. Referring to FIGS. 1-3, a device 200 for stimulating tissue regeneration of a subject 1 is shown. In some embodiments, the device 200 is used to enclose a wounded or injured tissue 9 of the subject 1. The wounded or injured tissue 9 may be located at an outer or inner location of the subject 1. In the embodiment shown, the device 200 is described with respect to stimulating tissue regeneration of a wounded tissue 9 located in an accessory organ 3 (e.g., a mouse finger), and the tip 2 has been cut along a line 5 through at least a portion of the distal phalanx 4, whereby the regenerated tissue may include, among other tissues, bone tissue 6, muscle tissue 7, and skin tissue 8. For illustrative purposes in describing FIGS. 1-7, the "accessory organ" may be referred to as including an exemplary "tissue".

[0073] In some embodiments, the wounded or injured tissue 9 for stimulation of tissue regeneration using the device 200 includes epithelial tissue, connective tissue, muscle tissue, or nerve tissue. Exemplary wounded or injured tissues 9 for regeneration include, but are not limited to, squamous epithelium, cuboidal epithelium, transitional epithelium, stratified columnar epithelium, columnar epithelium, glandular epithelium, bone, tendon, ligament, fat, loose connective tissue, blood tissue, visceral muscle, smooth muscle, skeletal muscle, cardiac muscle, and nerve tissue.

[0074] Referring to FIGS. 1-2, the device 100 includes an outer sleeve 202 that extends between an accessory organ receiving end 204 and a pressing member receiving end 206. In some embodiments, the outer sleeve 202 is a hollow cylinder that defines an internal chamber 208 sized to receive the tissue 3 (e.g., accessory organ) to be treated and the wounded or injured tissue 9. The internal chamber 208 forms a passageway that extends between a first opening at the accessory organ receiving end 204 and a second opening at the pressing member receiving end 206. In some embodiments, the first opening at the accessory organ receiving end 204 is sized to receive the accessory organ 3, and the second opening is sized to receive the pressing member 214.

[0075] The outer sleeve 202 can be formed from a transparent material that allows observation of the wound 9 while the device 200 is in use. In some embodiments, the outer sleeve 202 is rigid enough to prevent any flexure or indentation of the body wall during use to reliably maintain the desired wound space volume and to protect the wounded or injured tissue 9. Exemplary materials for the structure of the outer sleeve 202 include, but are not limited to, transparent nylon tubing. The outer sleeve 202 may include one or more openings (not shown) that facilitate replacement of the fluid within the internal chamber 208 of the outer sleeve 202. For example, the one or more openings may include a diaphragm that allows a needle to introduce and replace fluid within the internal chamber 208.

[0076] Device 200 includes an inner sleeve 216 that extends between a wound receiving end 218 and a mating receiving end 220 that is on the opposite side of the wound receiving end 218. In some embodiments, the inner sleeve 216 is a hollow cylinder that defines an internal chamber 222 sized to receive wound 9. The internal chamber 222 forms a passageway that extends between a first opening at the wound receiving end 218 and a second opening at the mating receiving end 220. When the device 200 is assembled, the inner sleeve 216 is configured to enclose the wound 9, and the pressing member 214 is configured to bias the mating receiving end 220 toward the accessory organ 3 such that the wound 9 is placed in contact with at least a portion of the internal chamber 222. In some embodiments, the pressing member 214 places the wound 9 in contact with a protein matrix 228 that is disposed within the internal chamber 222 of the inner sleeve 216. The protein matrix 228 can direct tissue growth and / or deliver a therapeutic agent to the wound 9 to stimulate tissue regeneration.

[0077] In some embodiments, the mating receiving end 220 includes a porous filter medium 230 that seals the second opening of the inner sleeve 216. Incorporating the porous filter medium 230 into the device 200 prevents contamination and allows for air and media exchange with the surrounding environment. The porous filter medium 230 helps to moisten the wound 9 and maintain high cell viability while reducing necrosis. A compressible member or media exchange member 232 can be positioned between the filter medium 230 and the pressing member 214 to provide a reservoir for an aqueous solution or dispersion medium that is in fluid communication with the internal chamber 222. In some embodiments, the outer sleeve 202 can contain an aqueous solution or dispersion medium and be placed in fluid communication with the internal chamber 222 of the inner sleeve 216 via the filter medium 230. In some embodiments, the compressible member or media exchange member 232 includes a gel that contains an aqueous solution or dispersion medium, or cotton that is optionally moistened with an aqueous solution or dispersion medium. In some embodiments, the protein matrix 228 is displaced from the filter medium 230 by a reservoir of an aqueous solution or dispersion medium.

[0078] Suitable aqueous solutions or dispersion media include, but are not limited to, water, cell culture media, buffers (e.g., phosphate buffered saline), polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. In some embodiments, the dispersion medium comprises a therapeutic agent.

[0079] The device 200 includes a first end cap 234 that is engageable with the accessory organ receiving end 204 of the outer sleeve 202. The first end cap 234 includes an opening 236 sized to receive the accessory organ 3 of the subject 1. In some embodiments, a gasket or diaphragm 238 may be positioned between the first end cap 234 and the accessory organ receiving end 204 of the outer sleeve 202. The gasket or diaphragm 238 includes a through hole 240 sized to receive the accessory organ 3 and provides a seal that prevents liquid from avoiding the inner chamber 208 of the outer sleeve 202. In some embodiments, the diaphragm 238 includes a flexible side portion 242 composed of silicon and a rigid side portion 244 including polytetrafluoroethylene (PTFE).

[0080] The device 200 may include a first adjustable adapter 246 that is installable within the accessory organ receiving end 204 for selectively coupling the first end cap 234 engageable with the accessory organ receiving end 204 to the outer sleeve 202. In some embodiments, the adjustable adapter 246 is a threaded adapter and the first end cap 234 includes a groove 248 sized to receive the threads 250 of the adjustable adapter 246. The first end cap 234 may be tightened to fix the accessory organ 3 within the outer sleeve 202 by placing the gasket or diaphragm 238 in contact with the tip 252 of the adjustable adapter 246 when assembled.

[0081] The device 200 includes a second end cap 254 that is engageable with the pressure member receiving end 206 of the outer sleeve 202. The second end cap 254 is coupled to the pressure member 214 to bias the pressure member 214 toward the mating receiving end 220 of the inner sleeve 216. Referring to FIG. 1, the pressure member 214 may be directly attached to or form a part of the second end cap 254. Referring to FIG. 2, the pressure member 214 may be separated from the second end cap 254. In some embodiments, the pressure member 214 includes a mating end 256 configured to be in the sheet portion 258 of the second end cap 254. In some embodiments, the pressure member 214 includes a sheet portion 260 on the opposite side of the mating end 256 configured to receive the mating end 220 of the inner sleeve 216. The pressure member 214 may be formed of a rigid or elastic material that deforms or compresses in response to tissue growth at the wound 9.

[0082] The device 200 may include a second adjustable adapter 262 that is installable within the pressure member receiving end 206 for selectively coupling the second end cap 254 engageable with the pressure member receiving end 206 to the outer sleeve 202. In some embodiments, the second adjustable adapter 262 is a threaded adapter and the second end cap 254 includes a groove 264 configured to receive the threads 266 of the second adjustable adapter 262. The second end cap 254 may be adjusted to control the pressure at the wound 9. One disadvantage of conventional devices is the lack of control of the pressure at the wound 9 interface, which leads to variations in the outcome of tissue regeneration when any type of gap (fluid collection, air, etc.) is present. The device 200 advantageously applies an adjustable pressure sufficient to hold the protein matrix 228 in contact with the wound 9. Also, unlike conventional devices, the device 200 is adjustable to facilitate long-term attachment (weeks, months, years, or more) and the growth of the tissue during regeneration.

[0083] Referring to FIGS. 4 - 5, in some embodiments, device 200 includes a movable or adjustable pressing member 214. For example, the pressing member 214 may be able to move or extend in response to tissue growth (shown as Δx in FIGS. 4 and 5). In some embodiments, the elastic member 268 is configured to extend between the sheet portion 270 in the pressing member 214 and the sheet portion 272 in the second adjustable adapter 262. In some embodiments, the elastic member 268 (e.g., a spring, compressible material, deformable material) creates a resistance force that produces a linear movement rearward (e.g., when the screw of the second end cap 254 is removed). In some embodiments, the adjustable pressing member 214 includes an interlocking spacer that can extend over time in response to tissue growth, or a screw extension system.

[0084] Referring to FIGS. 6 - 7, in some embodiments, device 200 also includes an electrical stimulation device 300 that is configured to establish a longitudinal electric field through wound site 9, which is said to impart an inner wound edge flow and provide an electrical guidance cue for nerve innervation and migration of cell types near the wound site. The electrical stimulation device 300 includes an anode 302 and a cathode 304 that are electrically connected to corresponding terminals 306 of a power source through leads 308, 310.

[0085] In some embodiments, the cathode 304 is in the form of a stainless - steel wire placed adjacent to the wound 9. A portion of the cathode 304 exists outside the device 200 and is connectable to the lead 308, and a portion of the cathode 304 exists within the inner chamber 222 of the inner sleeve. The anode 302 is a conductive wire that can be inserted into the subject 1 at a location far from the wound site 9. In the illustrated embodiment where the device 200 is placed on the accessory organ 3, the anode 302 is placed on the upper part of the limb (forelimb) where the accessory organ 3 extends. The anode 302 may include a platinum / indium alloy wire connected to the power source 306 via the lead 310. The anode 302 may be permanently implanted or temporarily inserted as needed.

[0086] Power source 306 includes battery pack 312 and electrical circuit 314, both of which are enclosed within housing 316 and are configured to impart a constant low level of current to electrodes 302, 304 when connected. In the embodiment shown in FIG. 6, power source 306 is located outside of object 1 and electrodes 302, 304 are configured to be removably connectable to power source 306. In this arrangement, when electrical stimulation is used, cathode 304 and anode 302 are electrically connected to power source 306 during the duration of the electrical stimulation treatment and are then disconnected between electrical stimulation treatments. Since power source 306 and leads 308, 310 can each be separated from electrodes 302, 304, this arrangement advantageously reduces the overall profile of combined device 200 and electrical stimulation device 300 during treatment paradigms in which electrical stimulation is used only intermittently.

[0087] In some embodiments, protein matrix 228 includes a biocompatible polymer. Biocompatible polymers suitable for use with device 100 include, but are not limited to, polyethylene oxide (PEO), polyethylene glycol (PEG), collagen, fibronectin, keratin, polyaspartic acid, polylysine, alginate, chitosan, chitin, hyaluronic acid, pectin, polycaprolactone, polylactic acid, polyglycolic acid, polyhydroxyalkanoate, dextran, polyanhydrides, polymers, PLA - PGA, polyanhydrides, polyorthoesters, polycaprolactone, polyfumarate, collagen, silk fibroin, chitosan, alginate, hyaluronic acid, and other biocompatible and / or biodegradable polymers. In some embodiments, protein matrix 228 is silk fibroin and / or collagen.

[0088] In some embodiments, the protein matrix 228 is processed from a silk solution (e.g., an aqueous solution) having a silk solution concentration of about 1% silk to about 50% silk. In some embodiments, the silk fibroin-based material is processed from the silk solution to form various material formats, such as fibers, foams, particles, films, and / or hydrogels.

[0089] In some embodiments, the protein matrix 228 is porous or has porosity. As used herein, the term "porosity" can refer to the measure of voids in a material and is the volume or fraction of pores relative to the total volume as a percentage from 0 to 100%. The determination of porosity is known to those skilled in the art using standard techniques such as mercury porosimetry and gas adsorption (e.g., nitrogen adsorption).

[0090] In some embodiments, the protein matrix 228 has pores that are sized to fit the surface area (size) of the tissue secured in the device 200 to optimize the regrowth of wounded or injured tissue. In some embodiments, the protein matrix 228 has a pore size of from about 1 μm to about 1500 μm, or about 350 μm, about 400 μm, about 450 μm, about 500 μm, about 550 μm, about 600 μm, about 650 μm, about 700 μm, about 750 μm, about 800 μm, about 850 μm, about 900 μm, about 950 μm, about 1000 μm, about 1050 μm, about 1100 μm, about 1150 μm, about 1200 μm, about 1300 μm, about 13350 μm, about 1400 μm, about 1450 μm, or about 1500. In terms of pore size, generally, from about 100 μm to about 300 μm or about 100 μm, about 150 μm, about 200 μm, about 250 μm, or about 300 μm are suitable for providing gaps suitable for cell and tissue growth while supporting adequate oxygen, nutrient, and waste transport. In other embodiments, smaller pore sizes, for example, from about 50 μm to about 100 μm, or about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, or about 100 μm are suitable for smaller tissues, such as nerves or blood cells. Higher porosity can facilitate improved tissue outcomes due to improved nutrient movement and waste removal.

[0091] The protein matrix 228 may have pores that form a directional pattern used to guide tissue growth in the wound 9. In some embodiments, the directional pattern includes aligned pores that form substantially aligned channels. The aligned pores may be arranged to be parallel to the longitudinal axis of the inner sleeve 216. In some embodiments, the aligned pores are formed by freezing a protein solution (e.g., silk or collagen solution) in a conductive substrate (e.g., an aluminum plate) using a steep temperature gradient induced by integrating the conductive substrate with a cold heat source (e.g., liquid nitrogen). It is expected that finger-like columns of ice crystals growing from the cold face create channel-like structures inside the frozen protein. The frozen protein is then lyophilized over a duration (e.g., 24 hours) to remove water. The resulting product is the protein matrix 228 having substantially aligned pores.

[0092] In some embodiments, the protein matrix comprises silk fibroin. As used herein, "silk fibroin" or "SF" can refer to a biopolymer produced from silkworm fibroin and the proteins of insect or spider silk. For example, silk fibroin useful in the present disclosure includes, without limitation: Antheraea mylitta; Antheraea pernyi; Antheraea yamamai; Galleria mellonella; Bombyx mori; Bombyx mandarina; Galleria mellonella; Nephila clavipes; Nephila senegalensis; Gasteracantha mammosa; Argiope aurantia; Araneus diadematus; Latrodectus geometricus; Araneus bicentenarius; Tetragnatha versicolor; Araneus ventricosus; Dolomedes tenebrosus; Euagrus chisoseus; Plectreurys tristis; Argiope trifasciata; and may be produced by a number of species including Nephila madagascariensis. Alternatively, the silk utilized in the present disclosure can be prepared through genetic manipulation of an artificial process, such as cells or organisms (e.g., non-human organisms including genetically engineered bacteria, yeast, mammalian cells, animals, or transgenic plants).

[0093] SF has unique characteristics and is a structural protein like collagen: it is generated from the ejection of amino acid solutions by complex organisms surviving in the external environment, while collagen is produced in vivo in the extracellular space by the self-assembly of cell-produced monomers and is not secreted into the external environment. The properties of SF result from its structure, which consists of hydrophobic blocks arranged alternately by hydrophilic acidic spacers. In its natural state, SF is structured into a semi-crystalline material by β-sheet crystals alternating with amorphous regions that impart strength and elasticity to the proteinaceous material formed from the protein. The diversity of forms in which regenerated SF can be processed at low to high protein concentrations and low to high molecular weights makes it attractive for several high-tech applications.

[0094] The processing of SF generally involves partial or total dehydration of a fibroin solution (protein content of about 1 wt% to about 15 wt%) to form, for example, films, sponges, gels, spheres (micron to nano-sized), and foams, by a number of techniques (e.g., solvent casting, freeze-drying, salt leaching, sonication). These fabrication processes provide robust materials that combine mechanical strength with biochemical properties.

[0095] The silk fibroin solutions used in the methods and compositions provided herein may be obtained, for example, from solutions in which silk from Bombyx mori, such as silkworms, has been dissolved. Alternatively, the silk fibroin solution may be obtained, for example, from a solution in which spider silk from Nephila clavipes, etc., has been dissolved. The silk fibroin solution may also be obtained, for example, from a solution containing recombinant silk from bacteria, yeast, mammalian cells, transgenic animals or transgenic plants. See, for example, WO 97 / 08315 pamphlet and US Patent No. 5,245,012. Recombinant silk may also include, for example, fusion proteins with therapeutic agents such as cytokines, enzymes, or any number of hormones or peptide drugs, antibacterial agents and related substrates.

[0096] The silk fibroin solution may be prepared by any conventional method known to those skilled in the art. In some embodiments, the silk solution is an aqueous silk solution. In other embodiments, the silk solution may contain a second polymer that facilitates transfer to a solid state (e.g., polyethylene glycol, collagen, hyaluronic acid, and the like).

[0097] Silk from silkworm cocoons contains two structural proteins, fibroin heavy chain (about 350 kDa (5.81×10 -19 g); and a fibroin light chain (about 25 kDa (4.15×10 -20 g) related to a family of non-structural proteins called sericin that adhere the fibroin chains together when forming the cocoon. The heavy and light fibroin chains are linked by disulfide bonds at the C-terminus of two subunits (see Takei, et al., J.Cell Biol. , 105:175, 1987; Tanaka, et al., J. Biochem. 114:1, 1993; Tanaka, et al., Biochim.Biophys.Acta ., 1432:92, 1999; Kikuchi, et al., Gene, see also 110:151, 1992). Sericin is a high molecular weight soluble glycoprotein component of silk that imparts adhesiveness to materials. These glycoproteins are hydrophilic and can be easily removed from the cocoon by boiling in water “degumming”).

[0098] In some embodiments, the silk polypeptide composition utilized by the present composition is substantially free of sericin (e.g., contains no detectable sericin or contains sericin at levels that one of ordinary skill in the art would consider negligible for a particular use).

[0099] In one exemplary method of obtaining a silk polypeptide composition, Bombyx mori (B. mori) is boiled in an aqueous solution such as, but not limited to, about 0.02 M Na2CO3 for about 30 minutes. The boiling (degumming) time is in the range of about 5 minutes to about 120 minutes, and the boiling (degumming) temperature is in the range of about 30 °C (303.15 K) to about 120 °C (393.15 K). The cocoon may be washed, for example, with water to extract the sericin protein, and the extracted silk is dissolved in an aqueous salt solution. Exemplary non-limiting salts useful for this purpose include lithium bromide, lithium thiocyanate, calcium nitrate, and other chemicals capable of solubilizing silk. For example, the extracted silk is dissolved in a solution of about 9 M to about 12 M LiBr. The salt is then removed, for example, by dialysis.

[0100] Optionally, the solution may then be concentrated using any method known in the art. For example, dialysis can be performed against a hygroscopic polymer such as PEG, polyethylene oxide, amylose, or sericin. PEG has a molecular weight of about 8,000 g / mol to about 10,000 g / mol and a concentration of about 25% to about 50%. Any dialysis system, such as a Slide-a-lyzer dialysis cassette (Pierce, MW CO3500), can be used. The solution is dialyzed for a period sufficient to result in a final concentration of the silk solution of about 1% to about 30%. In some cases, dialysis for about 2 hours to about 12 hours is sufficient.

[0101] In some embodiments, the present disclosure provides a method of attaching the device 200 to an accessory organ or tissue of a subject in need of tissue regeneration. The method includes contacting the damaged accessory organ or tissue 9 of the subject 1 with the wound receiving end 218 of the inner sleeve 216. The damaged accessory organ or tissue 9 may be placed in contact with or adjacent to a protein matrix 228 that contains a therapeutic composition to be provided. In some embodiments, before contacting the damaged accessory organ or tissue 9 with the protein matrix 228, the damaged accessory organ or tissue 9 is slid through an opening 236 of a gasket or diaphragm 238 and a first end cap 234.

[0102] The method further includes placing the accessory organ 3 of the subject through the accessory organ receiving end 204 of the outer sleeve 202 such that the inner sleeve 216 is positioned within the inner chamber 208 of the outer sleeve 202. In some embodiments, the method includes selectively fitting a first end cap 234 and a second end cap 254 to the outer sleeve 202 such that a pressing member 214 biases a fitting receiving end 220 toward the accessory organ 3. In some embodiments, the method includes biasing the pressing member 214 toward the accessory organ 3 such that the wound 9 is placed in contact with at least a portion of the inner chamber 222 of the inner sleeve. The contact pressure between the damaged accessory organ or tissue 9 and the protein matrix 228 can be adjusted by selectively fitting or releasing the second end cap 254 (e.g., tightening or loosening the second end cap 254 via a groove 264 and a thread 266).

[0103] In some embodiments, the traumatized accessory organ or tissue 9 is maintained within the device 200 for a duration that promotes tissue regeneration. In some embodiments, the duration is about 1 minute, or about 10 minutes, or about 30 minutes, or about 1 hour, or about 2 hours, or about 3 hours, or about 4 hours, or about 5 hours, or about 6 hours, or about 12 hours, or about 24 hours, or 2 days, or about 3 days, or about 4 days, or about 5 days, or about 1 week, or about 2 weeks, or about 3 weeks, or about 1 month, or about 6 months, or about 1 year, or within a range of durations bounded by any of these values. During the course of the duration, the protein matrix 228 can be moistened and maintained by adding or replacing the buffer solution within the inner sleeve 216.

[0104] Therapeutic compositions for tissue regeneration Also disclosed herein are therapeutic compositions for tissue regeneration and "multi-drug treatment" compositions (MDT). The therapeutic compositions may be used alone or in combination with the disclosed devices.

[0105] The therapeutic compositions used with the device according to the present invention can be any composition that stimulates, initiates, or directly or indirectly aids tissue regeneration. Alternatively, the therapeutic compositions according to the present invention can be a combination of components that act synergistically to stimulate, initiate, or directly or indirectly aid tissue regeneration. For example, the components provided in the therapeutic composition (e.g., two or more of components such as growth factors, inhibitors of prolyl hydroxylase domain (PHD) enzymes, vitamin A or its derivatives, lipid mediators, or peptide / protein hormones) act synergistically to increase the rate of regeneration at the traumatized accessory organ or tissue site compared to an untreated control experiment (e.g., increased soft tissue length, bone length, bone mass, increased contact response, number of ATT+ nerve bundles, diameter of ATT+ nerve bundles, complexity of regenerative particles by fibronectin expression, number of laminin / SMA+ bundles, reduction in wound diameter at the start of treatment, increased tissue regeneration as measured by the number of SOX2+ cells).

[0106] In some embodiments, at least two of the components in the provided therapeutic composition act synergistically to increase the rate of regeneration, or at least three of such components, or at least four of such components, or at least five of such components, or all of such components act synergistically to increase the rate of regeneration.

[0107] In one example, the disclosed device may contain a therapeutic composition within an inner sleeve (e.g., within a reservoir in the inner sleeve). The therapeutic composition may be present in a material or matrix that contacts the wound site, and the therapeutic composition may be delivered to the wound site when the device is attached to an accessory organ of the subject. Wearable devices that contain and deliver therapeutic compositions are known in the art. (See, e.g., Herrera-Rincon et al., Cell Reports 25,1593-1609 (2018).

[0108] The disclosed therapeutic composition may be present in a polymeric material such as, but not limited to, a silk hydrogel material. Methods of loading therapeutic compositions and drugs into hydrogel materials are known in the art. For example, a silk hydrogel material loaded with a therapeutic composition can be prepared as follows. The therapeutic composition can be added to a silk solution (e.g., a 3% w / v silk solution), and then a reagent, such as horseradish peroxidase (e.g., in a silk solution at a concentration of about 20 U / ml), is induced to gel via addition with hydrogen peroxide (e.g., at a concentration of up to 0.01% w / v). Silk can also be gelled by this enzymatic reaction via a drop in pH, addition of energy, for example, among many options, via sonication or vortexing, application of an electric field, or addition of methanol.

[0109] The disclosed therapeutic composition may include another agent that increases axonal / dendritic growth and / or normal cell proliferation. Preferably, the disclosed therapeutic composition does not promote pluripotency in cells and / or does not lead to teratoma formation.

[0110] The disclosed therapeutic composition may include one or more agents that promote tissue regeneration and / or healing. In some embodiments, the therapeutic composition includes one or more of a growth factor, an agent that inhibits an inhibitor of hypoxia-inducible factor 1-alpha (HIF1-alpha), vitamin A or a derivative thereof, a lipid mediator, such as a metabolite of an omega-3 fatty acid, which may be eicosapentaenoic acid or docosahexaenoic acid, a growth hormone, a steroid, and a depolarizing agent.

[0111] In some embodiments, the disclosed therapeutic composition may include a growth factor, such as a neurotrophic factor. A neurotrophic factor is a protein that promotes the growth and survival of nerve cells during development and promotes the maintenance of adult nerve cells. (See, for example, Terenghi, J.Anat . 1999;194(Pt1):1-14. Exemplary neurotrophic factors include, but are not limited to, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), and combinations thereof. The growth factor may be present in the therapeutic composition at an in-use dose within a dosage range bounded by at least about 0.1 μg / ml, about 0.2 μg / ml, about 0.3 μg / ml, about 0.4 μg / ml, about 0.5 μg / ml, about 0.6 μg / ml, about 0.7 μg / ml, about 0.8 μg / ml, about 0.9 μg / ml, or about 1.0 μg / ml or any of these values. When the growth factor is present in a component of the disclosed device (e.g., when the growth factor is loaded into the inner sleeve or a component of the inner sleeve), the device may include the growth factor at a concentration within a concentration range bounded by at least about 0.1 μg / device, about 0.2 μg / device, about 0.3 μg / device, about 0.4 μg / device, about 0.5 μg / device, about 0.6 μg / device, about 0.7 μg / device, about 0.8 μg / device, about 0.9 μg / device, or about 1.00 μg / device or any of these values. The growth factor promotes the growth of one or more tissue types.

[0112] The disclosed therapeutic composition may, for example, contain a prolyl hydroxylase domain (PHD) enzyme inhibitor (i.e., a PHD inhibitor) to stabilize the constitutive expression of the HIF-1α protein. (For example, Ariazi et al., J.Pharmacol.Expt.Therap .(2017), 363(3) 336-347; and Nangaku et al., Arterioscler., Thromb. Vas.Biol. (See 2007;27:2548-2554). Suitable PHD inhibitors include, but are not limited to, 4,4α-dihydro-4-oxo-1,10-phenanthroline-3-carboxylic acid (1,4-DPCA), N-[(1,3-dicyclohexylhexahydro-2,4,6-trioxo-5-pyrimidinyl)carbonyl]-glycine (i.e., GSK1278863 or Daprodustat), 6-amino-1,3-dimethyl-5-[(2-pyridinylthio)acetyl]-2,4(1H,3H)-pyrimidinedione (i.e., TM6089), 6-amino-1,3-dimethyl-5-[[2-(2-pyridinyl)-4-quinolinyl]carbonyl]-2,4(1H,3H)-pyrimidinedione (i.e., TM60008), N-[(4-hydroxy-1-methyl-7-phenoxy-3-isoquinolinyl)carbonyl]-glycine (i.e., FG4592 or Roxadustat), iron chelating agents, and combinations thereof. Optionally, the PHD inhibitor may be present in the therapeutic composition at an internal dose within a dosage range bounded by at least about 0.004 μg / ml, about 0.006 μg / ml, about 0.008 μg / ml, about 0.010 μg / ml, about 0.012 μg / ml, about 0.014 μg / ml, about 0.016 μg / ml, 0.018 μg / ml, about 0.020 μg / ml, about 0.022 μg / ml, or 0.024 μg / ml or any of these values. When the PHD inhibitor is present in a component of the disclosed device (e.g., when the PHD inhibitor is introduced into the inner sleeve or a component of the inner sleeve), the device may contain the PHD inhibitor at a concentration within a concentration range bounded by at least about 0.087 μg / device, about 0.092 μg / device, about 0.097 μg / device, about 0.102, about 0.107 μg / device, about 0.112 μg / device, about 0.117 μg / device, about 0.122 μg / device, about 0.127 μg / device, or about 0.132 μg / device or any of these values. The PHD inhibitor controls excessive collagen deposition at the wound site.

[0113] The disclosed composition may contain vitamin A or its metabolites or derivatives or any agent that functions in proximal-distal positional information. Exemplary derivatives of vitamin A include, but are not limited to, retinoic acid, retinol, retinyl carboxylate (e.g., retinyl acetate, retinyl propionate, and retinyl palmitate), tretinoin, as well as tazarotene, and combinations thereof. Agents that can function in proximal-distal position include, but are not limited to, bone morphogenetic protein 9 (BMP9), nodal growth differentiation factor (i.e., HTX5 or NODAL), activin, transforming growth factor-beta (TGF-β), and fibroblast growth factor 8 (FGF8). Vitamin A or its metabolites or derivatives or any agent that functions in proximal-distal positional information may be present in a therapeutically effective amount within a dosage range bounded by at least about 0.03 μg / ml, about 0.06 μg / ml, about 0.09 μg / ml, about 0.12 μg / ml, about 0.15 μg / ml, about 0.18 μg / ml, about 0.21 μg / ml, about 0.24 μg / ml, or about 0.27 μg / ml or any value therebetween. When vitamin A or its derivative (or an agent that functions in proximal-distal positional information) is present in a component of the disclosed device (e.g., when vitamin A or its derivative or an agent that functions in proximal-distal positional information is introduced into the inner sleeve or a component of the inner sleeve), the device may contain vitamin A or its derivative or an agent that functions in proximal-distal positional information at a concentration within a concentration range bounded by at least about 0.03 μg / device, about 0.06 μg / device, about 0.09 μg / device, about 0.12 μg / device, about 0.15 μg / device, about 0.18 μg / device, about 0.21 μg / device, about 0.24 μg / device, or about 0.27 μg / device or any value therebetween.

[0114] The disclosed therapeutic composition may include lipid mediators and / or metabolites (i.e., anti-inflammatory agents) by products of omega-3 fatty acids that promote the resolution of the inflammatory response. Suitable lipid mediators may include derivatives of omega-3 aliphatics (e.g., metabolites by products), and / or derivatives of eicosapentaenoic acid or docosahexaenoic acid that promote the resolution of the inflammatory response (i.e., anti-inflammatory). Exemplary lipid mediators include, but are not limited to, resolvins, such as resolvin 5, interleukin 6 (IL-6), interleukin 4 (IL-4), tumor necrosis factor-alpha (TNF-alpha), nuclear factor kappa-light-chain enhancer of activated B cells (NF-kB), and combinations thereof. Optionally, the lipid mediator may be present in the therapeutic composition at an internal dose within a dosage range bounded by at least about 0.006 μg / ml, about 0.012 μg / ml, about 0.018 μg / ml, about 0.024 μg / ml, about 0.030 μg / ml, about 0.036 μg / ml, about 0.042 μg / ml, about 0.048 μg / ml, or about 0.054 μg / ml or any of these values. When the lipid mediator is present in a component of the disclosed device (e.g., when the lipid mediator is introduced into the inner sleeve or a component of the inner sleeve), the device may include the lipid mediator at a concentration within a concentration range bounded by at least about 0.005 μg / device, about 0.011 μg / device, about 0.017 μg / device, about 0.023 μg / device, about 0.029 μg / device, about 0.035 μg / device, about 0.041 μg / device, about 0.047 μg / device, or about 0.053 μg / device or any of these values.

[0115] The disclosed therapeutic composition may include peptide hormones, such as peptide hormones that stimulate growth, cell proliferation, and cell regeneration. (e.g., Schmidmaier et al., Bone (2002) 31(1):165 - 72; and Schneider et al., J.Clin.Invest. Refer to 115(8):2083 - 98. Exemplary hormone peptides or proteins include, but are not limited to, growth hormone (GH), insulin - like growth factor - 1 (IGF - 1), transforming growth factor - beta - 1 (TGFβ - 1), epidermal growth factor (EGF), granulocyte - colony stimulating factor (G - CSF), and fibroblast growth factor FGF. The growth hormone or steroid may be present in the therapeutic composition at an internal dosage within a dosage range bounded by at least about 0.1 μg / ml, about 0.2 μg / ml, about 0.3 μg / ml, about 0.4 μg / ml, about 0.5 μg / ml, about 0.6 μg / ml, about 0.7 μg / ml, about 0.8 μg / ml, about 0.9 μg / ml, or about 1.0 μg / ml or any of these values. When the growth hormone or steroid is present in a component of the disclosed device (e.g., when the growth hormone or steroid is introduced into the inner sleeve or a component of the inner sleeve), the device may contain the growth hormone or steroid at a concentration within a concentration range bounded by at least about 0.1 μg / device, about 0.2 μg / device, about 0.3 μg / device, about 0.4 μg / device, about 0.5 μg / device, about 0.6 μg / device, about 0.7 μg / device, about 0.8 μg / device, about 0.9 μg / device, or about 1.0 μg / device or any of these values.

[0116] The disclosed therapeutic composition may contain a depolarizing agent. Suitable depolarizing agents include, but are not limited to, isophorone (e.g., an ion channel opener or blocker). Suitable depolarizing agents include, but are not limited to, monensin, potassium gluconate, sodium gluconate, and the like.

[0117] The disclosed therapeutic composition can be used to treat a subject in need of treatment. As used herein, "subject" means a human or an animal. Usually, the animal is a vertebrate, for example, a primate, a rodent, a domestic animal or a game animal. Examples of primates include chimpanzees, cynomolgus monkeys, rhesus monkeys, and macaques, such as the rhesus macaque. Examples of rodents include mice, rats, ground squirrels, ferrets, rabbits and hamsters. Examples of domestic and game animals include cows, horses, pigs, deer, bison, water buffalo, felines, such as domestic cats, canines, such as dogs, foxes, wolves, birds, such as chickens, emus, ostriches, and fish, such as trout, catfish and salmon. In certain embodiments of the aspects described herein, the subject is a mammal, for example, a primate, for example, a human. The subject can be female or male. Preferably, the subject is a mammal. The mammal can be a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow, but is not limited to these examples. Non-human mammals can be used as subjects representing animal models for tissue repair, regeneration and / or reconstitution. Also, the methods and compositions described herein can be used to treat domestic animals and / or pets.

[0118] In some embodiments, the disclosed therapeutic composition can be used to treat a wounded or injured accessory organ or tissue of a subject in need of stimulation of tissue regeneration. The tissue or accessory organ can be inside or outside the subject. Exemplary wounded or injured tissues in a subject in need of regeneration include, but are not limited to, squamous epithelium, cuboidal epithelium, transitional epithelium, stratified columnar epithelium, columnar epithelium, glandular epithelium, bone, tendon, ligament, adipose, loose connective tissue, blood tissue, visceral muscle, smooth muscle, skeletal muscle, cardiac muscle, and nerve tissue.

[0119] In some embodiments, the present disclosure provides a method of administering to a subject a disclosed therapeutic compound comprising a compound disclosed in an effective amount to regenerate at least a portion of a traumatized or injured accessory organ or tissue. In some embodiments, the method comprises contacting the traumatized or injured accessory organ or tissue with a therapeutic compound that may or may not be present in a provided hydrogel. In some embodiments, the provided therapeutic compound or the provided hydrogel is contacted with the traumatized or injured accessory organ or tissue within the device provided herein. The disclosed devices and / or therapeutic compositions promote tissue regeneration. Tissue regeneration can be measured by any method known in the art, e.g., but not limited to, measuring the expression of the Yamanaka factors, Sox2, Oct3 / 4, Klf4, and / or c-Myc in tissue treated with the devices and / or therapeutic compositions disclosed herein versus tissue not treated with the devices and / or therapeutic compositions disclosed herein.

[0120] Animal Model Studies Juvenile Xenopus that have matured towards adulthood can regenerate their amputated or injured limbs when exposed to a regeneration-inducing factor delivered through a sustained-release bead implanted into the transected tissue. However, fully non-regenerative, metamorphosed (adult) Xenopus cannot regenerate their hindlimbs during amputation, instead producing a featureless cartilage spike (Suzuki et al., (2006) TheScientificWorldJOURNAL,6 .). This model was used to test whether a regeneration-inducing factor could stimulate regeneration.

[0121] As described in the following examples, complex interventions in the hindlimb amputation of adult Xenopus were tested to address several aspects of limb regeneration. A wearable bioreactor ("biodome") was used to gain control over the local microenvironment of the wound in vivo. When exploring the mechanism, a short exposure period to the regeneration cocktail was found to initiate a long-term endogenous morphogenetic cascade without continuous micromanagement. Various stimuli that induce pre-regenerative activity were selected, such as agents that reduce inflammation, promote nerve preservation, and induce overall growth.

[0122] A short exposure period (e.g., 24 hours) to a silk-containing wearable bioreactor injected with several small molecule compounds was found to induce dramatic elongation, pattern formation, and sensorimotor function after amputation in Xenopus. The treated animals showed a marked delay in wound closure, followed by long-term (about 16 months) growth outcomes including increased bone length, soft tissue pattern formation, and neuromuscular repair. Histologically, the new limbs contained smooth muscle indicating nerves and blood vessels, and the restructuring of extracellular matrix proteins included limb reforming. Transcriptomic analysis identified transcriptional targets of the intervention in intermediate and short-term pathways as well as in blastemas. RNA-seq testing also revealed a rapid response (compared to sham controls) in the brain to the fully treated device. The regenerated bone showed anatomical features characteristic of the wild-type form, and the soft tissue of the distal limb showed finger-like protrusions. Also, the animals walked around using the newly formed limbs, similar to wild-type frogs. Additionally, it was shown that the sensorimotor pathway was restored in animals exposed to the fully treated condition, and tissue-repattern formation included re-extension or regrowth of the afferent nerves as well as the neuromuscular tissue interface.

[0123] These data demonstrate that in adult Xenopus, a very significant long-term regenerative response can be induced by a simple trigger without the need for gene therapy or stem cell transplantation, and also reveal the molecular, cellular, and tissue-level components of this process occurring in the wound and distal brain.

[0124] The following examples describe in detail the methods by which the present disclosure can be used or implemented, which will enable those skilled in the art to more easily understand the principles of the present disclosure. The following examples are presented as illustrations and are not intended to be limiting in any way.

Example

[0125] Animal Adult male African clawed frogs (n = 115) (Nasco, Fort Atkinson, WI) measured 5 cm to 6.25 cm (from nose to tail) were acclimated to storage tanks for 2 weeks prior to the experiment. The animals were maintained at 18 °C (291.15 K) in 10 L plastic tanks containing regular frog water (Leaf Solution, Seachem Laboratories, conductivity of approximately 1.65 kΩ, pH of 7.8 - 8.0) and exposed to a 12-hour light-dark cycle. Prior to the experiment, the animals were immersed in broad-spectrum gentamicin antibiotic for 2 hours (Gibco, Fisher Scientific, USA) to minimize bacterial contamination of the limb stumps after amputation.

[0126] Limb amputation The surgery for hindlimb amputation was performed according to the previously established protocol published by Golding et al. (2016), PLoS One 11 , e0155618, and Herrera et al. (2018), Cell Rep 25 , 1593-+. Briefly, the animals were first anesthetized by whole-body immersion in buffered frog water containing 0.05% benzocaine. When the reflex action was lost when the fingertip was pinched, 75 mg / kg buprenorphine was injected subcutaneously directly under the lateral line on the opposite side of the leg to be amputated. The right hindlimb was cut in the middle of the tibia and fibula with a sterile microsurgical blade using a straight cut. Bone resection was not performed, and the tissue flap over the wound site was not sutured either. After hemostasis, the animals were allowed to regain consciousness and were recovered in sterile frog water for at least 60 minutes.

[0127] Device attachment Animals were randomly assigned to one of three treatment conditions: no device, biodome only, or biodome with a cocktail treatment (described below in “Biodome Fabrication and Cocktail Composition”). A second anesthesia (0.05% benzocaine soak, 75 mg / kg buprenorphine) was administered prior to the device attachment procedure. The unconscious animals were then fitted with a device secured to the stump of the amputation site using monofilament surgical sutures (7-0 Monosof, 18” P-16 cutting, Covidien, USA). Two sutures were placed through the cortex on either side of the leg. These stitches were sufficient to hold the device in place and did not damage the underlying deep fascia layer. After attachment, the animals were returned to the home tank where they regained consciousness and were allowed to swim freely. Control animals were treated similarly to those that received the device, but the device was not attached to the wound stump. The results presented herein were generated using a sutured biodome.

[0128] Alternatively, an adjustable biodome may be used. The attachment procedure for the adjustable biodome involves pushing the amputated limb through the cap and donut-shaped diaphragm into a suitable position and securing the limb using a bioadhesive (Skin-Tite Bioadhesive, Smooth-On, PA). A scaffold-containing biodome insert was attached to the amputated digit and secured with a small amount of adhesive. An acrylic protective cap was threaded into the top cap, followed by using a custom washer to push the insert closer to the wound bed and closing it with a second cap. Approximately 4 μL of sterile phosphate-buffered saline (PBS) 1X was slowly injected into the insert using a 31G needle to keep the tissue moist and maintained over the attachment period. The PBS was changed every two days to remove cellular debris and keep the tissue fresh. The device was attached and maintained until the animals were sacrificed for later analysis.

[0129] Biodome Fabrication The suture biodome was composed of a soft silicone insert containing silk hydrogel as a controlled release substrate and drug carrier. The device was fabricated as reported elsewhere (Golding et al., (2016) PLoS One 11 ,e0155618). Briefly, the outer cylindrical silicone sleeve (20 mm height × 18 mm diameter) was designed using CAD software (Solidworks, Waltham, MA, USA) and fabricated by casting a silicone elastomer (Dragon skin 10, Smooth-on, Macungie, PA) against a 3D printed mold printed using a Formlab 3D printer (Somerville, MA, USA).

[0130] The adjustable biodome was constructed from acrylic tubes (#8532K13, Mcmaster-Carr, Elmhurst, IL) cut into 1-cm lengths and used as the main body of device 100. A screw-type adapter was designed using 3D CAD software (Inventor Professional, Autodesk, San Rafael, CA) and printed using a 3-D printer by stereolithography (Form2, Formlab, Somerville, MA). The adapter was adhered to the acrylic tube using a medical-grade superglue. A 2-ml HPLC vial cap (Agilent, Santa Clara, CA) had its PTFE / silicone septum perforated by a 3-mm biopsy punch to create an access port for the animal's extremities. The septum was cut along the central hole in four places, in four semi-circles spaced apart from each other, to provide additional room for the animal's leg to pass through the port. A custom washer was made from PDMS using soft lithography or 3D printed using a 3D printer. The cylindrical wall portion of the insert of device 100 was made from a transparent polyester membrane filter (0.45-μm pore size, 12-μm thickness, #1300016, Sterlitech, Kent, WA). The filter was cut into a rectangle (7 mm × 5 mm) and wound around a 1.5-mm diameter metal rod (#8907K62, McMaster). The wall portion was then adhered to the silicone bottom using a silicone adhesive (Dragon Skin 10 FAST, Smoothon, Macungie, PA) to complete the insert.

[0131] The protective cage was assembled from a screw-on cap, a transparent acrylic body, and an adapter. A donut-shaped diaphragm that only allows bending in one direction was provided to prevent separation of the device due to animal movement and interference. The custom washer, together with the bottom cap, applies an adjustable pressure sufficient to firmly hold the scaffold insert against the wound bed and maintain this position stably over long-term experiments. This is also equipped with an inspection port for media exchange. The device insert includes a membrane-forming sidewall and a silicone bottom. The sidewall can hold the liquid necessary to keep the tissue moist and facilitate gas exchange, and the silicone bottom functions as a diaphragm for the insertion of a needle for media exchange.

[0132] Device Removal and Maintenance After 24 hours, the animals were anesthetized and treated with an analgesic as described previously. The device was then removed by cutting a single suture on either side of the leg, and the frogs were placed back into a tank containing a fungicide (Cordon methylene blue at a concentration of 1 mL / 10 L frog water). After an additional 24 hours, the water was replaced with fresh 100% frog water. Once these devices were removed, the animals were maintained in frog water for 18 months and this water was changed daily. Terminal euthanasia was performed by immersing the whole body in frog water with 0.2% benzocaine. The regenerates, contralateral limbs, and brain tissue were collected and processed for histological analysis.

[0133] Silk Treatment The silk fibroin solution was prepared by cutting and degumming 5 g of Bombyx mori cocoons (Tajima Shoji, Yokohama, Japan) in a 0.02 M sodium carbonate (Na2CO3) solution for 45 minutes to remove non-essential protein substrates (i.e., sericin). The fibers were washed several times with deionized (DI) water to remove Na2CO3 and then dried overnight in a draft at 22 °C (295.15 K). The dried silk fiber was then dissolved in a 20% (w / v) solution of 9.3 M lithium bromide (Sigma-Aldrich, St. Louis, MO) and placed in an oven set at 60 °C (333.15 K) for 4 hours. The solution was then dialyzed against DI water with gentle stirring using a dialysis cassette (molecular weight cut-off of 3.5 kDa (5.81×10 -21 g), Thermo Fisher Scientific, Waltham, MA). The water was changed 6 times over a period of 48 hours. The dialyzed solution was centrifuged 3 times at 13,000 g for 20 minutes at 4 °C (277.15 K) and then filtered through a cell filter (40 μm pore size, Thermo Fisher) to remove impurities. To determine the concentration of the filtered solution, a 0.5 ml sample was dried completely in an oven overnight. Once the water had evaporated, the dried silk was weighed and the % concentration (wt / v) was calculated as the ratio of the weight of the dried silk to the initial volume of 0.5 ml.

[0134] Silk hydrogels were formed by crosslinking liquid silk fibroin. 45 μl of silk (3% w / v) and horseradish peroxidase (HRP) solution (20 U / ml) were cast in a 24-well plate and incubated at 37 °C (310.15 K) for 45 minutes to complete gelation. The gel compression strength and modulus of the gels were tested according to the method of Golding et al. (2016), PLoS One 11 ,e0155618.

[0135] Scaffold with aligned pores A silk scaffold with aligned pores was fabricated using 5 μl of 4% (wt / v) silk solution. The solution was placed on top of an aluminum plate, and a steep temperature gradient was induced by fusing the plate in liquid nitrogen (LN2). Finger-like columnar ice crystals growing from the cold face remained solidified and created a channel-like structure inside the silk solution. After 10 minutes of cooling, the frozen solution was lyophilized for 24 hours to remove water. The sponge was trimmed to fit the biodome as an insert, sterilized in ethylene oxide, and stored at room temperature in a sterile state until use.

[0136] A collagen scaffold with an aligned channel-like porous structure was fabricated by controlled directional freezing and lyophilization of a 1.5% (wt / wt) collagen solution (in a manner similar to the silk scaffold). The scaffold was cut into a cylindrical shape (4 mm in length and 1.5 mm in diameter) and placed inside the biodome insert using scissors.

[0137] Characterization of Materials The morphology of the scaffolds was characterized using a scanning electron microscope (SEM) and a fluorescence microscope. For SEM imaging, the scaffolds were cut in half using a razor to expose the internal geometry of the pores. Before imaging, the scaffolds were sputter-coated with gold to increase conductivity. SEM imaging was performed on a microscope (Zeiss EVO MA10) set at 5 kV. For fluorescence imaging, the scaffolds were stained with 2 μg / ml fluorescein isothiocyanate (FITC) in PBS and imaged using a Keyence microscope (BZ-X800, Keyence, Japan). The compressive stiffness and elastic modulus of the scaffolds were determined using an Instron test system. Figure 8A is a silk scaffold with channel-like pores aligned along the long axis. Figure 8B is a silk scaffold with pores aligned perpendicular to the long axis. Figure 8C is an SEM image of a collagen scaffold with pores aligned along the long axis. Figure 8D is a collagen scaffold with pores aligned perpendicular to the long axis.

[0138] Therapeutic Compositions The hydrogel was prepared with a final concentration of 3% (w / v) silk solution, 20 U / ml horseradish peroxidase (HRP), and 0.01% wt / v hydrogen peroxide (H2O2). The liquid solution was poured into a silicone sleeve and gelled for 30 minutes before being attached to the stump of the animal's limb. In the cocktail delivery device, 0.014 μg / ml of 1,4(dihydroxyphenone serine-4-one-3-carboxylic acid) DPCA (Catalog number 71220, Caymen Chemicals, MI, USA), 0.5 μg / ml of brain-derived neurotrophic factor (BDNF) (Catalog number 450-02, Peprotech, MA, USA), 0.5 μg / ml of growth hormone (GH) (Catalog number 100-40, Peprotech, MA, USA), 0.036 μg / ml of resorcin D5 (Catalog number 10007280, Caymen Chemicals, MI, USA), and 0.015 μg / ml of retinoic acid (Catalog number 11017, Caymen Chemicals, MI, USA) were introduced into the liquid silk solution and then inserted into the silicone sleeve for gelling.

[0139] In vitro release study To determine the drug release profile of the drug used in this example, 50 μL of each hydrogel solution containing a specific amount of a specific drug was added to 1.5 mL microcentrifuge tubes and incubated at 37 °C (310.15 K) for 45 minutes to complete gelation. Then, 1 mL of Dulbecco's phosphate-buffered saline (DPBS 1X, Gibco) was added to each vial, followed by incubation at 37 °C (310.15 K). At the determined time points, 300 μL of the supernatant was collected for analysis. A drug-free silk hydrogel release solution was used as a control. A calibration curve was obtained by measuring the optical density of solutions with known concentrations. All release experiments were performed in triplicate to ensure accuracy. For RA and 1,4-DPCA, the optical density of the release solution was determined in a UV-transparent 96-well plate (Corning, Corning, NY) using a microplate reader SpectraMax M2 (Molecular Devices, San Jose, CA) operated by SoftMax Pro6 software. Detection was performed at wavelengths of 280 nm and 350 nm for 1,4-DPCA and RA solutions, respectively.

[0140] The concentrations of BDNF and GH in the release samples were determined using enzyme-linked immunosorbent assay (ELISA) kits (#BGK23560 and #BGK01241, Peprotech, Rocky Hill, NJ, USA) containing monoclonal antibodies designed for BDNF and GH. Sample preparation and measurement were performed according to the manufacturer's protocol. The optical density of the prepared samples was read at 450 nm using a SpectraMax M2 plate reader. The release samples of resorcin D5 were filtered through a protein filtration column (MWCO = 3 kDa (4.98×10 -21 g), #UFC500324, Fisher) to remove high-molecular-weight fibroin components. Then, the optical density of the samples was determined at a wavelength of 244 nm using a SpectraMax M2 plate reader.

[0141] Soft tissue imaging At regular intervals over an 18-month maintenance period, the animals were evaluated for soft tissue repatterning and bone regrowth. As previously described, the animals were anesthetized and high-resolution images of these wound sites and regenerate dimensions were acquired using a DSLR camera (Canon EOS Rebel T7i). To ensure reproducibility, the amputation plane functioned as a standard reference point for all measurements. The site of amputation was easily identified by the sure tapering of the limb at the incision point. Each measurement consisted of a linear assessment of the length between the amputation site and the most distal end of the regenerate.

[0142] In vivo X-ray and micro-CT bone imaging In addition to soft tissue measurements, bone length was measured according to the imaging protocols of Golding et al. (2016) PLoS One 11 , e0155618, and Herrera-Rincon, et al., (2018) Cell Rep 25, 1593-+, using a handheld X-ray device (Nomad Pro 2TM) with standard imaging settings of 60 kV and 2.5 mA at an exposure time of 0.20 seconds. Each animal was subjected to the same dose (0.12 mSv) at defined time points. Computed tomography (CT) was performed on a viva CT40 scanner (Scanco Medical, Switzerland) to visualize the detailed microstructure of the bone at the end of the 18-month regeneration period after euthanasia with an overdose of benzocaine (0.2% total body immersion). Distal trabecular and midshaft cortical bone slices (615 slices / animal, 76 μm / slice, 300 ms integration time) were visualized and further quantified as 3D images. The radiation dose was according to the manufacturer's guidelines established using the local CT dose index (CTDI) within the range of 453 mGy to 1255 mGy.

[0143] Histology and immunohistochemistry To characterize the effect of treatments on limb regrowth and repatterning after amputation, histological analysis was performed at defined intervals over time in the regenerates and contralateral limbs. Tissues were harvested at 18 mPa. Long-term regenerate tissues were fixed overnight in 4% paraformaldehyde (PFA) in PBS and decalcified for 2 weeks by exposure to increasing concentrations (10% - 15%) of ethylenediaminetetraacetic acid (EDTA) (pH 7.4). Once decalcified (confirmed using x-rays), tissues were gradually equilibrated in 30% sucrose and then embedded in OCT (Sakura FInetek, USA). Samples were frozen in liquid nitrogen. Limb tissues were sequentially sectioned at 14 μm using a cryostat (Leica CM1850) and placed on glass slides. Cross-sections were taken at 14 μm intervals across the limb from the tibiofibular region above the original amputation site to the patterned region at the end of the limb. To visualize patterning, the ends of the limbs were sectioned horizontally at 14 μm intervals. Sections were dried for at least 1 hour and then stored at -80 °C (193.15 K).

[0144] For immunohistochemistry, slides were equilibrated at room temperature for at least 2 hours and then stained. Slides were post-fixed in 4% PFA for 5 minutes and then blocked in blocking buffer (PBS containing 0.1% Triton X-100 and 10% normal goat serum) for 1 hour. Primary antibodies were used against acetylated α-tubulin (1:100), TGF-β (1:250), smooth muscle actin (1:100), laminin (1:100), fibronectin (1:500), and phospho-histone H3 (1:250). Slides were stained individually with each antibody except anti-smooth muscle actin and anti-laminin, and these were stained together. Primary antibodies were incubated overnight on the slides. After washing in PBS, alexa-fluor secondary antibody (1:500, ThermoFisher Scientific) was applied in blocking buffer for 2 hours. Slides were washed again in PBS and stained with DAPI 1:200 in PBS for 20 minutes. Slides were mounted in Fluoromount-G (ThermoFisher Scientific) and allowed to cure for at least 24 hours before imaging.

[0145] The sections were imaged using an EVOS FL automated imaging system (ThermoFisher Scientific). Whole sections were collected and stitched together for analysis.

[0146] Analysis of immunostained sections All statistical analyses were performed in IBM SPSS version 20 software. The assumption of normality was tested before using parametric tests, including ANOVA, t-tests, and related analyses (Pearson's r). Non-parametric analyses, the Mann-Whitney Wilcoxon test or the Kruskal-Wallis test, were performed for comparisons where the data were not normally distributed. A significant difference was assumed when the p-value was less than the threshold of 0.05 (two-sided hypothesis testing).

[0147] Assessment of sensorimotor thresholds To evaluate the sensorimotor ability of the regenerates, the animals were evaluated 18 months after amputation. Each animal was placed in a glass tank filled with 2 L of frog water and allowed to acclimate for 5 minutes until movement completely stopped. A video camera (iPod (registered trademark) Touch 5th generation, Apple, CA, USA) was placed on the housing to acquire a record of the test procedure. The sensory threshold of the regenerate was evaluated using standardized von Frey filaments (Touch Test, Stoelting, IL, USA). Filaments ranging in force from 0.008 g to 300 g were applied to the distal part of the regenerate, starting from the lowest force to the highest force. The first filament that induced an obvious response (movement from the resting position) was recorded. The animals were tested twice over a 2-day period, and the mean threshold was reported.

[0148] Statistical analysis All statistical analyses were performed in IBM SPSS v20. First, the data were tested for homogeneity of variance by Levene's test. Single gene analysis was performed on normally distributed data by unpaired, two-sided Student's t-test (for two independent groups), or one-way ANOVA test (for multiple independent groups), followed by post hoc Scheffe's test (when P < 0.05). When considering the variable "time", two-factor analysis was performed by two-way ANOVA. Statistical significance between treatment groups (no device, biodome only, and cocktail treatment) at each specific time point was determined by using Student's t-test. In non-normally distributed data, the Kruskal-Wallis test, followed by post hoc Dunn's test (when P < 0.05), was performed. The significance level was set at 0.05 in all cases. Statistical values were reported as mean ± standard deviation or mean ± standard error of the mean when indicated. Where appropriate, dot or scatter plots were used to emphasize the individual variability within the experimental groups respectively.

[0149] RNA extraction After transection, device attachment and removal, and 24 hours of treatment, the regenerated tissue was harvested at 11-, 24-, and 72 hours post-transection for next-generation sequencing (NGS). Samples consisted of 1 cm thick tissue blocks from the distal wound site. Brains were also collected and flash frozen. Tissues were extracted using TRIzol (ThermoFisher Scientific) according to the manufacturer's protocol, and the quality and quantity of total RNA were evaluated using a Nanodrop spectrophotometer (ThermoFisher Scientific).

[0150] Next-generation sequencing (NGS) 1.1 μg of total RNA was sent to the Tufts Genomic Core. The quality of the RNA was evaluated via a Bioanalyzer, and high-quality RNA was used for library preparation with the TruSeq Stranded Total RNA Library Prep Kit with RiboZero Gold (Invitrogen). The libraries were then multiplexed and single-end, 50 nt sequencing was performed on an Illumina HiSeq2500. The raw read files were sent to the Bioinformatics & Biostatistics Core at the Joslin Diabetes Center.

[0151] NGS analysis The Xenopus tropicalis reference genome was downloaded from the NCBI Genome database, assembly GCA_001663975.1. Reads were aligned using STAR aligner (Dobin et al., Bioinform .(2013);29(1):15 - 21.doi:10.1093 / bioinformatics / bts635.Epub Oct 25.PubMed PMID:23104886;PubMed Central PMCID:PMC3530905.), and the aligned reads were counted using featureCounts (Liao et al., (2014) Bioinform .30(7):923 - 30). Genes with expression counts of more than 1 count per million (cpm) in at least 3 samples were included in the analysis and normalized by the trimmed mean of M - values (TMM, Robinson et al., (2010) Genome Biol .11,R25.). A voom transformation was performed (Law et al., Genome Biol .15,R29.) to convert the counts to logCPM: CPM = 1e+6 * gene counts / (total sample counts * sample normalization factor). The voom transformation also estimates the mean - variance relationship and uses this to calculate observation - level weights, giving more weight for greater read depth. To further weight outliers, sample - specific quality weights (Ritchie et al., (2006) BMCBioinform .7,261.) was collected and combined with the observed level weights.

[0152] Differentially expressed genes were identified using limma (Ritchie et al., Nucl. Acids Res. 43, e47.). A moderated t-test was performed to detect genes differentially expressed between the two groups. Genes with FDR < 0.25 were considered significantly changed.

[0153] Gene sets for pathway analysis were obtained from the MSigDB collection, and gene sets belonging to canonical pathways (CP) or gene ontology (GO) were selected. The analysis was performed by the Fry function in the rotation gene set test (Roast) in the limma R package (Wu et al., (2010) Bioinform .26, 2176 - 2182.). All coordinated up, coordinated down, and mixed gene sets were considered significant when P < 0.05 and FDR < 0.05.

[0154] Network analysis Gene modules were identified by co-expression analysis in CeMiTool (Russo et al., 2018) based on logCPM values for the control and cocktail-treated groups at 11 hours, 24 hours, and 7 days after limb amputation. In CeMiTool, variance-stabilizing transformation was applied to remove the difference between the mean and variance parameters, and genes were filtered based on expression levels with a threshold of p < 0.1. Modules of co-expressed genes were identified within the dataset based on the automatically generated scaling value beta (β = 10) of 30 genes and the minimum module size. To evaluate how the enrichment of these modules changed over time and between groups, module enrichment was calculated based on sample annotation. To determine which biological functions were associated with each module, overrepresentation analysis was performed using the pathway database from Reactome, and pathways were considered significant at p < 0.05. Annotated modules were used to graph combined gene-gene interaction data from the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Chemical and Genetic Perturbations (CGP), mapping the interacting genes included in each module.

[0155] qPCR method The same total RNA submitted for RNAseq analysis was DNase treated using an RQ1 RNase-Free DNase Kit (Promega Corp., Madison, WI, USA). The resulting RNA (0.5 μg) was subjected to a second DNase treatment followed by cDNA synthesis using a Verso cDNA Synthesis Kit (ThermoFisher Scientific). Quantitative analysis of the amount of gene product was performed using a Step One Plus Real Time System (Applied Biosystems, USA). Duplicate 10 μl reactions were set up which contained: 5 μl of 2x PowerUp SYBR Green Master Mix (Applied Biosystems, USA), 0.5 μl each of 10 μM forward and reverse gene specific primers, and 1.33 μl of diluted DNA template. Relative expression was analyzed using the delta-delta Ct method using the mean of all ND expression over time as a calibrator for all samples.

[0156] Results 1. Induction of leg regeneration by multi-drug reconditioning Adult Xenopus hindlimbs were amputated at the midpoint along the tibia and fibula and a biodome device containing either a silk-based hydrogel or hydrogel alone with a 5-drug multi-drug treatment (MDT) was attached. Control animals were amputated without treatment. After 24 h exposure in the biodome, the device was removed and the animals were maintained for up to 18 months with regular assessment of hindlimb regenerate regeneration and repatterning. The extended observation window was selected based on the calculation of Alibardi (2018) which predicts that it takes approximately 1.5 years to regenerate anuran limbs based on projection modeling using the stump diameter of the newt.

[0157] X-ray images were obtained and limb lengths were measured as a function of the number of months post-amputation. The X-rays showed that the regenerates associated with both the biodome only and the multi-drug treatment (MDT) conditions were longer than the control (device-free) regenerates at 4 months, with the effect appearing at 4 months and then again at 8 months and persisting over time (two-way ANOVA, between-subjects factor treatment exposure, within-subjects factor regeneration time F(2,19)=61.9, p<.05). Limb lengths compared to the amputation site were greater for the MDT group compared to other groups that were as early as 0.5 months post-amputation (mpa) and persisted for 4 mpa (p<0.05), suggesting that MDT increased the growth rate early. At 6 - 8 months, growth was significantly delayed and the hindlimb regenerates associated with the biodome only group achieved comparable lengths compared to MDT-exposed animals (p>0.05). The secondary increase in late growth after 9 mpa showed that the MDT group again displayed longer limbs compared to other groups, and the effect persisted until the final measurement at 18 mpa (p<0.01). Thus, the MDT group not only shows a final increase in leg length compared to other treatment groups, but also shows a secondary growth period that does not exist in other treatment groups, particularly the device-free group. These data suggest that MDT and short exposure to the biodome facilitate the regrowth of longer legs.

[0158] Not only did the leg lengths increase, but 76% of the animals exposed to MDT also displayed a thicker, more complex regenerative morphology compared to the severely pigmented spikes characteristic of the device-free group. Specifically, the distal segments of the hindlimb regenerates associated with the MDT group protruded buds characteristic of fingers and presented a flat paddle-like structure. In contrast, the device-free and biodome only conditions were exclusively associated with featureless regenerative spikes, and the MDT condition provided resulted in a reliably formed paddle-like structure with distal buds. The biodome group had an intermediate phenotype, with 20% of the biodome animals displaying thicker cartilage spikes and hook-like distal protrusions, but pattern formation was limited. In the device-free group, none of the animals displayed a distinct phenotype.

[0159] 2. The induced leg exhibits sensory function Next, to evaluate whether the 18 mpa regenerate regained sensory function, a sensorimotor assessment was performed. Using a standardized Von Frey (VF) filament (minimum force: 0.008 g; maximum force: 300 g), the regenerated right hindlimb was examined at the most distal end by increasing the filament strength until the maximum force was applied. The first filament that induced an obvious response (movement from the rest position) was recorded, and the behavioral assay was averaged over 2 days. The MDT-treated hindlimb regenerates displayed a stimulus-response pattern comparable to that of the non-transected group (p > 0.01; Figure 10), indicating significant reinnervation and neuromuscular reconstruction. In the biodome group, the responses varied significantly among individual animals in the group that performed at nearly normal levels and the group that could not detect the stimulus (56.6 ± 98.8 g), while the untreated animals were definitely unable to display any response to the application of force at any value up to 300 g (inclusive). Therefore, the inventors concluded that the MDT treatment facilitated sensorimotor integration similar to that of the non-transected limbs. Interestingly, the biodome-alone group may also have been able to facilitate the regrowth of limbs with an intact sensory system. This indicates that hydration and structural support may be important for maintaining innervation and facilitating nerve regrowth.

[0160] 3. MDT exposure increased hindlimb bone length and complexity The internal structure of the regenerates was characterized using micro-CT and X-ray images. Since the overall hindlimb length was significantly improved by the treatment (Figure 9), the study of the bone components of the regenerates was conducted later. Micro-CT and X-ray images of the regenerates revealed an increase in bone mass and length in relation to the MDT composition provided, compared to biodome only and the control.

[0161] Using X-ray images, the presence of complex morphology and increased bone length were confirmed in the MDT regenerates, compared to the biosphere only and device-free control experiments. Starting from the measurements at 2.5 mpa, a dense tissue protruding outward from the osteotomy site was observed in the MDT and biosphere groups, but not in the device-free control. The MDT group also showed dense, segmented bone fragments at the distal end of the regenerate. As predicted, the MDT condition showed increased bone length compared to other conditions starting at 4 mpa with an inflection at approximately 8 mpa (similar to the soft tissue measurements) (p<0.05). As shown in Figure 11, the difference in bone length between the MDT and biosphere groups was periodically observed, and the difference in length between these groups and the device-free control was maintained overall (p<0.05).

[0162] To obtain a better understanding of the microstructure of the regenerated bone, MicroCT was performed at 18 mpa. MicroCT imaging enabled visualization of the three-dimensional rendering of the underlying bone and measurement of bone mass without inhibiting the outer soft tissue. MicroCT data confirmed the presence of bone fragmentation in the distal region of the MDT hindlimbs. In particular, fragmentation did not occur at the amputation site but instead spontaneously appeared after the growth period at points along the bone equivalent to the contralateral joint. These re-patterned segmentations were common within the MDT group. As shown in Figure 12, the bone mass measured through microCT data was greater in the MDT group hindlimb regenerates compared to the other groups. Quantification by volume measurement confirmed an increase in growth in the provided MDT regenerates (one-way ANOVA (F(2,15)=11.15, p<0.001)). Micro-CT scans also revealed a marked similarity between the non-amputated and MDT-treated bone anatomies. Repatterning was prominent in the provided MDT regenerates, including the re-expression of bone features normally associated with muscle tissue attachment. Specifically, at 17 mpa, the MDT group displayed bone features characteristic of muscle attachment and the presence of joint segmentation. These data indicate that the MDT condition is associated with a significant reformation of bone anatomy, consistent with an active process that ultimately achieves the morphological complexity of features approximating the reshaped and pre-amputated limb.

[0163] 4. Complexity of the regenerated limbs The MDT-treated animals showed increased bone growth, and molecular changes at the cellular level in the regenerated limbs were also evaluated. Immunohistochemistry was performed to evaluate tissue structures related to regeneration and repatterning 18 mpa later. As shown in Figure 13, reinnervation measured by acetylated α-tubulin (AAT) staining revealed a significant increase in the number of AAT+ nerve bundles associated with the 24-hour MDT condition compared to the no-device group (U = 13, P = 0.0014). The number of AAT bundles was comparable when compared to the no-device and biodome-only groups, indicating that the MDT treatment affected nerve bundle regrowth and innervation in the regenerates. As shown in Figure 14, not only were additional bundles present, but the 24-hour MDT group also showed a significantly larger AAT bundle diameter compared to the no-device group. There was no significant increase in bundle size between the biodome-only and no-device groups.

[0164] The Kruskal-Wallis test showed that the number of AAT-positive bundles at 18 mpa differed significantly across conditions (H(15.12) = p < 0.005). Mann-Whitney U post hoc analysis revealed that the main source of variance was the increase in AAT bundles associated with the 24-hour MDT condition compared to uninjured animals (U = 13, P = 0.0014). The number of AAT bundles was comparable when compared to uninjured animals and the biodome-only group (p = 0.1812). The inventors then examined the ATT bundle dimensions and revealed significant differences across conditions (H(11.74) = p = 0.0028). The 24-hour MDT group showed a larger ATT bundle diameter compared to uninjured animals. Also, no significance was identified when compared to the biodome-only and uninjured animal groups.

[0165] To evaluate changes to the connective tissue structure, the inventors evaluated the fibronectin expression pattern after 18 mpa. As shown in Figure 15, the increased complexity of the particles in the MDT-treated regenerates reflected the degree of structural complexity observed under the MDT conditions. Specifically, the fluorescence images revealed increased nerve regeneration and extracellular matrix repatterning in the regenerates at 18 months after the initial surgery and exposure to the multi-drug treatment. Consistent with other results, the 24-hour MDT group showed an increase in the complexity of the particles compared to both the biodome-only and device-free groups. This indicates that MDT itself tends to facilitate the assimilation of cartilage and connective tissue in the regenerates.

[0166] To evaluate angiogenesis in the regenerates, laminin and smooth muscle actin (SMA) expression at 18 mpa was compared across conditions as shown in Figure 16. Fluorescence images of cross-sections of the regenerates obtained 18 months after surgery were double-stained for two markers of angiogenesis: one related to smooth muscle (SMA-red) and the other related to the basement membrane (green). Comparison of the sections revealed a significant increase in SMA / laminin-positive bundles in the multi-drug treatment group compared to the device-only or untreated groups (H(19.84)=p<0.001). The number of blood vessels identified in each section was significantly higher in the MDT-treated group compared to both the biodome-only and device-free groups (H(19.84)=p<0.001). This indicates that MDT facilitates angiogenesis in the regenerates rather than doubling the number of blood vessels in the MDT-treated regenerates compared to the device-free conditions.

[0167] 5. Wound closure, increased Sox2 expression, and re-epithelialization Considering the significant long-term outcomes, the effect of MDT at an early time point after amputation was evaluated during the initial period of this process. The inventors first noticed that wound closure at 0.5 mPa was significantly reduced in the MDT group compared to animals without the device (p < 0.05; Figure 17). MDT-exposed animals exhibited an average wound diameter of 2.02 cm ± 0.40 cm, which was significantly larger than that of the biodome group (1.25 cm ± 0.41 cm) or the group without the device (0.56 cm ± 0.17 cm). Delayed wound closure predicted the success of repatterning at 18 mPa, but animals with the largest wound sites at 0.5 mPa showed the greatest growth and repatterning at 18 mPa.

[0168] Without being bound by a particular theory, it is expected that the larger the wound site, the larger the blastema can be provided and the more material can contribute to limb regeneration. As shown in Figure 18, blastema proliferation was evaluated via immunohistochemistry for the proliferative cell marker SOX2. There were significantly more SOX2-expressing cells in the MDT group compared to the other groups (p < 0.05). This indicates the presence of more proliferative tissue that gives rise to new limbs. And although the soft tissue at 2.5 months was similarly predicted, the tissue was thicker and the bone length increased in the MDT hindlimbs compared to the control. Fluorescent images obtained at 2.5 mPa revealed that 24-hour exposure to the MDT composition increased stemness and that inhibition of wound formation resulted in longer regenerates in the early regeneration process. Increased soft tissue growth and re-epithelialization were also observed at 2.5 months post-amputation in the MDT-exposed group, reflecting an increase in the length of bone tissue confirmed by x-ray imaging.

[0169] 6. Transcriptomic Analysis of Regeneration Induction To gain a more careful understanding of gene expression changes in response to acute exposure to MDT treatment, the transcriptional machinery downstream of the intervention was characterized. RNA-sequencing (RNAseq) was performed by comparing the transcriptomes of buds obtained from MDT-treated versus untreated animals at 11 hours, 24 hours, and 7 days after amputation. A heatmap comparing gene expression levels in MDT animals compared to no-device treatment shows significant differential gene expression at 11 HPA that persists until 24 HPA. At 7 DPA, however, the levels of dynamic gene expression return to normal, indicating a period of dynamic gene expression within 24 hours of amputation.

[0170] The number of differentially expressed genes was determined after correcting the p-value by multiple tests. The Q-value was set to a false discovery rate (FDR) of 0.05, and differentially expressed genes were considered those transcripts that passed this FDR and showed a log2 fold change of 2. When comparing the buds of MDT animals to untreated animals, there were large dynamic changes in the expression profile over 7 days after amputation. The same genes that were overexpressed or underexpressed were allowed to pass 24 hours after the switch in activity 7 days later. To narrow down the dramatically altered expression of these genes, the FDR was set to a log3 or fold change of 3, and the top 15 differentially expressed genes were compared between groups. When comparing MDT-treatment to wild-type bud tissue, the top 15 highly upregulated genes were found to be related to the brain, with the highest expression at 11 hours after amputation (e.g., brain-specific kinase (BRSK), neuropeptide FF, D1C dopamine receptor, neuroligin). This expression level decreased at 24 hours and 7 days after amputation (dpa). The gene Wnt7a, which is involved in the development of the anterior-posterior axis, was also upregulated at 11 hours after amputation (hpa) and then increased at 7 days after amputation. Conversely, the major downregulated genes were mainly related to muscle structure (myosin-4, microfibril-associated glycoprotein) and metabolism (e.g., sarcolipin). The pattern of genes differentially expressed between MDT-treated and control buds was the opposite of that of upregulated genes in that the downregulated genes increased from 11 hpa to 7 dpa.

[0171] Highly regulated genes in MDT animals were compared to genes in animals without devices. Upregulated genes included nervous system-specific transcripts suggesting an important role for neuroprotective proteins immediately after transection. Downregulated genes included metabolic and muscle-related transcripts, suggesting that resources are directed away from muscle maintenance towards tissue stabilization. GO analysis of metabolic and biosynthetic pathways reveals early downregulation (at 11HPA and 24HPA) with increased rate at 7dpa. Table 1 shows gene expression levels in MDT animals compared to device-free treatment at 11hpa, 24hpa, and 7dpa.

Table 1

[0172] Enrichment analysis identifying gene classes showed significant differences in the profiles of biological processes between groups. The web-based version of CEMiTool (co-expression module identification tool) was used to identify co-varying gene sets in MDT (CT) and device-free (ND) animals. Co-varying gene sets with high fold changes were classified into modules (M1-M4).

[0173] There were additional genes involved in different metabolic regulations between MDT and controls early after transection (11hpa). Also, there was additional dynamic gene expression at this time point. This resolves the late (7dpa) and significant changes between MDT and tissue restructuring or resetting of the cellular functional landscape.

[0174] To identify the types of processes regulated by MDT-exposure, enriched pathways were grouped considering "large-scale functions". Co-expression analysis identified four gene modules across control and cocktail-treated groups at 11 hours, 24 hours, and 7 days post-amputation. These modules represent categorizations of genes based on shared expression levels and statistical significance. Module 1 included extracellular matrix organization, collagen formation, collagen biosynthesis and modification enzymes, which may represent tissue destruction after injury, and 607 genes significantly represented (p<0.00509) within the hemostasis pathway. Module 2 included 142 genes significantly present (p<0.00009) within pathways related to cell adhesion organization, laminin interaction, cell communication, apoptotic cleavage of cell adhesion proteins, and non-integrin membrane ECM interaction, which may represent cell-cell communication and adhesion. The 105 genes in Module 3 were significantly present (p<0.01096) in muscle contraction, acetylcholine activity, and myogenesis pathways. Module 4 included 54 genes significantly over-represented (p<0.00142) in glucose metabolism, muscle contraction, gluconeogenesis, and glycolysis pathways. When Modules 2, 3, and 4 were significantly enriched (p<0.00024) and upregulated in MDT-treated blastemas over all time points, it suggested that frogs receiving the pre-regeneration cocktail had persistent gene upregulation related to cell communication, myogenesis, and glucose metabolism compared to controls with biosphere only and no device. In contrast, these modules were enriched (p<0.012) but downregulated in groups not housing the treatment device at all time points evaluated. However, at the 7-day time point, Module 2 was significantly upregulated (p=0.00074) in the same non-treated group, suggesting little difference in enrichment of the cell communication pathway between treated and non-treated groups at 1 week post-amputation. The ECM and collagen-enrichment Module 1, upregulated (p<0.011) in the non-treated group, was downregulated (p<0.00027) in non-treated samples over all time points.

[0175] Generally, M2, M3, and M4 were upregulated over time in the MDT condition versus the ND condition and peaked at 24 HPA. This excludes M1, which was upregulated to the highest degree at 11 HPA and then downregulated in the MDT condition, decreasing until 7 DPA.

[0176] 7. Cumulative drug release from the provided MDT composition To evaluate the cumulative release regarding multi-drug treatment (MDT) in the device hydrogel, hydrogels loaded with each of the MDT drugs were suspended in 1xDPBS (ThermoFisher) and incubated at 37 °C (310.15 K) for 25 minutes. The supernatant was collected every 5 minutes, and the concentration of each drug was determined via a microplate reader. Each drug / hydrogel mixture showed similar release kinetics, releasing approximately 70% of the total drug concentration within 10 minutes and not exceeding 80% of the total amount loaded (except for retinoic acid, which released all of the drug within 25 minutes).

[0177] Other features, objects, and advantages of the present disclosure will be apparent from the following detailed description. However, it should be understood that the detailed description shows embodiments of the present disclosure but is provided only by way of example and without limitation. Various changes and modifications within the scope of the present disclosure will become apparent to those skilled in the art from the detailed description.

Claims

1. An apparatus for stimulating tissue regeneration at a site in a target tissue, comprising: An outer sleeve having a tissue receiving end, a pressing member receiving end opposite to the tissue receiving end, and an inner chamber configured to receive the tissue; A pressing member; An inner sleeve disposed within the outer sleeve, having an end for receiving the site of the tissue for regeneration, a fitting receiving end for fitting the pressing member opposite to the end for receiving the site of the tissue for regeneration, and an inner chamber configured to receive the site of the tissue for regeneration; The pressing member is configured to extend through the pressing member receiving end into the inner chamber of the outer sleeve and bias the fitting receiving end of the inner sleeve towards the tissue; The pressing member places at least a portion of the site of the tissue for regeneration in contact with a portion of the inner chamber of the inner sleeve; A first end cap that is engageable with the tissue receiving end of the outer sleeve and is configured to receive the tissue; and A second end cap that is engageable with the pressing member receiving end of the outer sleeve, the second end cap being connected to the pressing member.

2. The apparatus according to claim 1, wherein the tissue is an accessory organ or a part of an organ.

3. The apparatus according to claim 1, further comprising a first threaded adapter that can be installed within the tissue receiving end of the outer sleeve for selectively connecting the first end cap engageable with the tissue receiving end to the outer sleeve, the first end cap having a groove configured to receive the thread of the first threaded adapter.

4. The apparatus according to claim 1, further comprising a second threaded adapter that can be installed within the pressing member receiving end of the outer sleeve for selectively connecting the second end cap engageable with the pressing member to the outer sleeve, the second end cap having a groove configured to receive the thread of the second threaded adapter.

5. The apparatus according to claim 1, wherein the pressing member includes a sheet portion configured to accommodate the fitting and receiving end portion of the inner sleeve, or includes a fitting end portion configured to be in the sheet portion of the second end cap.

6. The apparatus according to claim 1, wherein the fitting and receiving end portion of the inner sleeve includes a porous filter medium that closes the internal chamber by sealing at the fitting and receiving end portion.

7. The apparatus according to claim 6, wherein the porous filter medium is a synthetic or polymeric membrane.

8. The apparatus according to claim 6, further comprising a compressible member positioned between the porous filter medium and the pressing member.

9. The apparatus according to claim 8, wherein the compressible member includes cotton or a encapsulated gel.

10. The apparatus according to claim 1, wherein the inner sleeve includes a protein or polymeric matrix that at least partially fills the internal chamber of the inner sleeve.

11. The apparatus according to claim 10, wherein the protein or polymeric matrix includes a three-dimensional porous scaffold, and the porous scaffold includes pores that form a directional pattern.

13. The apparatus according to claim 11, wherein the porous scaffold includes aligned pores that form substantially aligned channels, and the aligned channels in the protein or polymeric matrix are arranged parallel to the longitudinal axis of the inner sleeve.

15. The apparatus according to claim 12, wherein the protein or polymeric matrix is selected from the group consisting of silk fibroin and collagen, or a polymer that forms aligned pores in a 3D matrix.

17. The apparatus according to claim 1, wherein the pressing member is movable in response to growth at the site of the tissue for regeneration.

19. The apparatus according to claim 14, wherein the pressing member includes an elastic member that compresses in response to growing tissue.

21. The apparatus according to claim 15, wherein the elastic member includes a spring.

23. In the screw adapter that can be installed in the pressing member accommodating end of the outer sleeve for selectively connecting the second end cap that can be fitted with the pressing member to the outer sleeve, the screw adapter having a groove configured to accommodate the thread of the screw adapter; and An elastic member extending between the seat portion of the screw adapter and the seat portion of the pressing member The device according to claim 14, further comprising.

18. The device according to claim 10, wherein the protein or polymeric matrix contains a therapeutic agent.

19. The device according to claim 1, further comprising an electrical stimulation device including an anode and a cathode, wherein the anode and the cathode are configured to be electrically connected to corresponding terminals of a power source, and a portion of the cathode is installed in the inner sleeve.

20. The device according to any one of claims 1 to 19, wherein the internal chamber of the inner sleeve further contains a therapeutic composition.

21. The therapeutic composition is: Growth factor; An inhibitor of prolyl hydroxylase domain (PHD) enzyme; Vitamin A or a derivative thereof; and Lipid mediator The device according to claim 20, comprising.

22. The device according to claim 21, wherein the growth factor is selected from the group consisting of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), glial-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), and leukemia inhibitory factor (LIF), and combinations thereof.

23. The device according to claim 22, wherein the growth factor is BDNF.

24. The inhibitor of the PHD enzyme is selected from the group consisting of 4,4α-dihydro-4-oxo-1,10-phenanthroline-3-carboxylic acid (1,4-DPCA), N-[(1,3-dicyclohexylhexahydro-2,4,6-trioxo-5-pyrimidinyl)carbonyl]-glycine, 6-amino-1,3-dimethyl-5-[(2-pyridinylthio)acetyl]-2,4(1H,3H)-pyrimidinedione, 6-amino-1,3-dimethyl-5-[[2-(2-pyridinyl)-4-quinolinyl]carbonyl]-2,4(1H,3H)-pyrimidinedione, N-[(4-hydroxy-1-methyl-7-phenoxy-3-isoquinolinyl)carbonyl]-glycine, an iron chelating agent, and combinations thereof, the apparatus according to claim 21.

25. The inhibitor of the PHD enzyme is 4,4α-dihydro-4-oxo-1,10-phenanthroline-3-carboxylic acid (1,4-DPCA), the apparatus according to claim 21.

26. The derivative of vitamin A is selected from the group consisting of retinoic acid, retinol, retinyl carboxylic acid, tretinoin, tazarotene, and combinations thereof, the apparatus according to claim 25.

27. The lipid mediator is selected from the group consisting of resolvin, a metabolite of omega-3 fatty acid, a derivative of eicosapentaenoic acid, a derivative of docosahexaenoic acid, and combinations thereof, the apparatus according to claim 21.

28. The therapeutic composition contains resolvin, and the resolvin is selected from the group consisting of resolvin 5, interleukin 6 (IL-6), interleukin 4 (IL-4), tumor necrosis factor-alpha (TNF-alpha), nuclear factor kappa-light chain enhancer of activated B cells (NF-kB), and combinations thereof, the apparatus according to claim 21.

29. The therapeutic composition contains an agent that functions in proximal-distal positional information, and the agent is selected from the group consisting of bone morphogenetic protein 9 (BMP9), nodal growth differentiation factor, activin, transforming growth factor-beta (TGF-β), fibroblast growth factor 8 (FGF8), and combinations thereof, the apparatus according to claim 21.

30. The device according to claim 21, wherein the therapeutic composition contains a peptide or a protein hormone.

31. The device according to claim 30, wherein the peptide hormone is selected from the group consisting of growth hormone (GH), insulin-like growth factor-1 (IGF-1), transforming growth factor-beta-1 (TGFβ-1), epidermal growth factor (EGF), granulocyte-colony stimulating factor (G-CSF), fibroblast growth factor FGF, and combinations thereof.

32. The device according to claim 21, wherein the growth factor is present in a therapeutic composition internal dose of 0.1 μg / ml to 1 μg / ml.

33. The device according to claim 21, wherein the inhibitor of the PHD enzyme is present in a therapeutic composition internal dose of 0.004 μg / ml to 0.024 μg / ml.

34. The device according to claim 21, wherein the vitamin A or its derivative is present in a therapeutic composition internal dose of 0.03 μg / ml to 0.27 μg / ml.

35. The device according to claim 21, wherein the lipid mediator is present in a therapeutic composition internal dose of 0.006 μg / ml to 0.054 μg / ml.

36. The device according to claim 30, wherein the peptide or protein hormone is present in a therapeutic composition internal dose of 0.1 μg / ml to 1.0 μg / ml.

37. The device according to any one of claims 1 to 36, for use in a method of stimulating tissue regeneration in a mammal in need thereof.

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