Tissue regenerative multi-drug cocktails and devices for their delivery

Abstract

Disclosed are devices, compositions, and methods for promoting regeneration of tissue in a subject, such as a wounded, injured, or injured appendage, or tissue within a subject, such as a wounded, injured, or injured organ. The disclosed devices, compositions, and methods include or utilize a wearable sleeve and a regenerative composition.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 944,707, filed December 6, 2019, the entire contents of which are incorporated herein by reference.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grant No. 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 a wearable sleeve, and the disclosed compositions include a regenerative composition. [Background technology]

[0004] The prevalence of human limb loss in the United States alone is expected to increase significantly over the next 30 years, affecting 3.6 million individuals per year by 2050 (Ziegler-Graham et al., 2008), leaving diabetics, war veterans, and survivors of trauma (and individuals with peripheral arterial disease) with limited options in the event of amputation.

[0005] Previous efforts have focused on 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 Many studies have attempted to induce limb regeneration, including several other methods (e.g., 233(1), 72-79), but these have been limited in their ability to restore significant growth and patterning of the new limb.

[0006] Tissue regeneration involves a series of biological events that all lead to the restoration of an excised organ or appendage lost during trauma or amputation. There is a clear difference between a typical wound healing response and a regenerative response. These two processes are similar in many embodiments, but result in completely different end products. In the normal wound healing process, many complex biological structures, such as sweat glands, ducts, and hair follicles, cannot be restored because the biological machinery to do so is not available. In a typical adult mammalian skin wound, 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 a suitable environment for these structures to regenerate. Additive regeneration, on the other hand, is a process in which all prototype structures are replaced by copies of the prototype.

[0007] While some complex animals, such as the axolotl, naturally regenerate limbs, eyes, and all other organs throughout their life, mammals generally exhibit limited regeneration and do not generally exhibit such plasticity and transdifferentiation capacity as urodeles have. Anurans that mature toward adolescence can regenerate their amputated or injured limbs when exposed to regeneration-inducing factors delivered through slow-release beads embedded in the amputated tissue. Tissue progenitor cells for induction of the regeneration process may also have uses; larval limb progenitor cells have been shown to activate the same Wnt, Shh signaling to promote pattern formation.

[0008] However, how well the completely non-regenerative post-metamorphic (adult) Xenopus might serve as a useful animal model for regeneration remains unclear, as adult frogs are unable to regenerate their hindlimbs upon amputation, instead producing featureless cartilage spikes (Suzuki et al., (2006) Sci. World J. , 6:26-37) and therefore is unknown.

[0009] Regenerative sleeves have been used for tissue regeneration of wounded tissue. Conventional sleeves include a septum that allows the needle to repeatedly exchange media within the wound space, a liquid reservoir that keeps the wound moist, and a liquid-tight seal that prevents liquid from exiting the sleeve. However, such conventional sleeves suffer from several limitations. In some cases, the liquid-tight seal may be too strong, leading to tissue necrosis. The replacement of media through the needle may directly affect the wound bed.

[0010] Furthermore, conventional tissue regeneration devices are typically attached to subjects using glue, which is not suitable for long-term treatment. Attaching regeneration devices using glue has two main disadvantages: glue lacks the flexibility adjustment it provides to the device after attachment. Also, there are complications due to handling during surgery and the device.

[0011] Therefore, improved devices and regenerative compositions are desirable and needed. Summary of the Invention

[0012] Disclosed are devices, compositions, and methods for promoting regeneration of tissue within a subject, such as a wounded, damaged, or injured site in an appendage of a living subject, or a wounded, damaged, or injured site in or within an organ. The disclosed devices, compositions, and methods include or utilize a wearable sleeve and a regenerative composition.

[0013] In one aspect, the present disclosure provides a therapeutic composition for promoting tissue. In some embodiments, the regenerative composition may include multiple components. In certain embodiments, the disclosed regenerative compositions are utilized in the disclosed devices, for example, as a therapeutic composition present in the material of the inner sleeve that contacts the wounded, damaged, or injured tissue.

[0014] In one embodiment, the therapeutic composition comprises a growth factor, an inhibitor of a prolyl hydroxylase domain (PHD) enzyme, vitamin A or a derivative thereof, and a lipid mediator.

[0015] In certain embodiments, the therapeutic composition comprises a growth factor selected from 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. In certain embodiments, the therapeutic composition comprises nerve growth factor. In certain embodiments, the therapeutic composition comprises brain-derived neurotrophic factor (BDNF).

[0016] In some embodiments, a 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 chelator, 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 carboxylate, tretinoin, tazarotene, and combinations thereof.

[0018] In some embodiments, the therapeutic composition comprises a lipid mediator selected from the group consisting of resolvins, metabolites of omega-3 fatty acids, derivatives of eicosapentaenoic acid, derivatives of docosahexaenoic acid, and combinations thereof. In certain embodiments, the lipid mediator is a resolvin. In certain embodiments, the therapeutic composition comprises a resolvin 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.

[0019] In some embodiments, the therapeutic composition comprises an agent that functions in proximal-distal positioning, the agent being 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, hi 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 dose of 0.1 μg / ml to 1 μg / ml. In some embodiments, the PHD inhibitor is present in the therapeutic composition at a dose of 0.004 μg / ml to 0.024 μg / ml. In some embodiments, the vitamin A or derivatives thereof is present in the therapeutic composition at a dose of 0.03 μg / ml to 0.27 μg / ml. In some embodiments, the lipid mediator is present in the therapeutic composition at a dose 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 dose of 0.1 μg / ml to 1.0 μg / ml.

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

[0023] In yet another aspect, the disclosure provides a use of a therapeutic composition for the stimulation of 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 pressure 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 wounded, damaged, or injured tissue or appendage, a mating receiving end for mating with the pressure member, and an internal chamber configured to receive and / or contain wounded, damaged, or injured tissue. The pressure member is configured to extend through the pressure member receiving end into the internal chamber of the outer sleeve and to bias the mating receiving end of the inner sleeve toward the tissue such that at least a portion of the wounded, damaged, or injured tissue of the subject 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 matable with the tissue receiving end of the outer sleeve and having an opening configured to receive tissue and maintain the tissue in the device, and a second end cap matable with the push member receiving end of the outer sleeve.

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

[0026] In some embodiments, the device includes a first threaded adapter installable within the tissue receiving end of the outer sleeve to selectively couple a first end cap to the outer sleeve, the first end cap being matable with the tissue receiving end, hi some embodiments, the first end cap includes a groove configured to receive the threads of the first threaded adapter.

[0027] In some embodiments, the apparatus includes a second threaded adapter mountable within the push member receiving end of the outer sleeve for selectively connecting a push member matable second end cap to the outer sleeve, hi some embodiments, the second end cap includes a groove configured to receive the threads of the second threaded adapter.

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

[0029] In some embodiments, the mating receiving end of the inner sleeve includes a porous filter medium sealingly closing the interior chamber 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 pressing member. In some embodiments, the compressible member includes cotton or an encapsulated gel.

[0030] In some embodiments, the inner sleeve comprises a protein or polymeric matrix at least partially filling the inner sleeve's interior chamber. In some embodiments, the protein or polymeric matrix comprises a three-dimensional porous scaffold. In some embodiments, the porous scaffold may comprise pores that form a directional pattern. In some embodiments, the porous scaffold comprises aligned pores that form substantially aligned channels. The aligned channels in the protein or polymeric matrix may be disposed parallel to the longitudinal axis of the inner sleeve. In some embodiments, the protein or polymeric matrix is ​​selected from silk fibroin, collagen, or a combination thereof.

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

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

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

[0034] In some embodiments, the device includes a threaded adapter mountable within the push member receiving end of the outer sleeve for selectively connecting a push member matable second end cap to the outer sleeve. The second end cap can include a groove configured to receive the threads of the threaded adapter. The resilient member can extend between a seat portion of the threaded adapter and a seat portion of the push member.

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

[0036] In yet another embodiment, the disclosed device includes an inner sleeve including an end with a first opening sized to accommodate wounded, damaged, or injured tissue, and a mating receiving end with a second opening, the inner sleeve defining an interior chamber extending between the first and second openings, the interior chamber sized to accommodate wounded, damaged, or injured tissue of a subject. The device further includes a matrix disposed in the interior chamber of the inner sleeve, the matrix including a porous scaffold having pores forming substantially aligned channels. The matrix may include and / or be formed from materials including, but not limited to, polymeric materials such as proteins, which may include, but are not limited to, collagen and silk fibroin in any suitable form. 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 cocktail.

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

[0038] In some embodiments, the device further includes a filter media sized to enclose the second opening at the mating receiving end of the inner sleeve, hi some embodiments, the filter media 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 interior chamber of the inner sleeve of the provided device contains a provided therapeutic composition.

[0040] In another aspect, the present disclosure provides a method of promoting tissue regeneration in a mammal, the method comprising attaching a provided device to a wounded appendage or tissue of the mammal.

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

[0042] These and other embodiments and advantages of the present disclosure will be apparent from the following detailed description, in which reference is made to the accompanying drawings, which form a part hereof, and in which non-limiting example embodiments are shown by way of illustration, which do not necessarily represent the full scope of the disclosure, but reference is therefore made to the full disclosure herein for interpreting the scope of the disclosure.

[0043] The present disclosure will now be described with reference to the accompanying drawings, in which like reference numbers indicate like elements. [Brief description of the drawings]

[0044] [Figure 1] FIG. 1 is a schematic representation of a side cross-sectional, partially enlarged view of an apparatus for stimulation of tissue regeneration at a wound site of a subject, according to some embodiments of the present disclosure.

[0045] [Diagram 2]FIG. 2 is a schematic representation of a side cross-sectional, partially enlarged view of an apparatus for stimulation of tissue regeneration at a wound site of a subject, according to some embodiments of the present disclosure.

[0046] [Diagram 3] FIG. 3 is a diagrammatic representation of a mouse finger showing the amputation line.

[0047] [Figure 4] FIG. 4 is a schematic representation of a side cross-sectional view of an apparatus for stimulation of tissue regeneration at a wound site of a subject having a movable pushing member, according to some embodiments of the present disclosure.

[0048] [Diagram 5] FIG. 5 is a schematic representation of a cross-sectional side view of the device of FIG. 4 in a compressed state, according to some embodiments of the present disclosure.

[0049] [Figure 6] FIG. 6 is a schematic representation of the apparatus of FIG. 1 but including an electrical stimulation device, according to some embodiments of the present disclosure.

[0050] [Figure 7] FIG. 7 is a diagrammatic representation of the device of FIG. 6 mounted on a subject, according to some embodiments of the present disclosure.

[0051] [Figure 8A] FIG. 8A is an SEM representation showing a silk fibroin scaffold with channel-like pores aligned along its long axis, according to some embodiments of the present disclosure.

[0052] [Figure 8B] FIG. 8B is an SEM representation showing a silk scaffold with pores aligned perpendicular to the long axis, according to some embodiments of the present disclosure.

[0053] [Figure 8C] FIG. 8C shows a collagen scaffold with pores aligned along its long axis, according to some embodiments of the present disclosure.

[0054] [Figure 8D] FIG. 8D shows a collagen scaffold with pores aligned perpendicular to the long axis, according to some embodiments of the present disclosure.

[0055] [Figure 9] FIG. 9 is a graphical representation of right hind limb length as a function of months post amputation (mpa) following exposure to one of the following treatment conditions: (i) after the first 24 hours of exposure to a provided multi-drug treatment (MDT) composition, (ii) after 24 hours of exposure to the Bio-Dome device, and (iii) no treatment.

[0056] [Figure 10] 10 is a graphical representation of contact response after 17 mpa as assessed by Von Frey filaments at the distal tip of regenerants exposed to one of the following treatment conditions: (i) after the first 24 hours of exposure to a provided multi-drug treatment (MDT) composition, (ii) after 24 hours of exposure to the Bio-Dome device, and (iii) no treatment. Means and SDs are presented.

[0057] [Figure 11] FIG. 11 is a graphical representation of right hind limb bone length as assessed by micro-CT and X-ray images as a function of months post-amputation (mpa) following exposure to one of the following treatment conditions: (i) after the first 24 hours of exposure to the provided multi-drug treatment (MDT) composition, (ii) after 24 hours of exposure to the Bio-Dome device, and (iii) no treatment.

[0058] [Figure 12] FIG. 12 is a graphical representation of bone mass in the right hind limb as assessed by micro-CT and X-ray images as a function of months after amputation following exposure to one of the following treatment conditions: (i) after an initial 24-hour exposure to a provided multi-drug treatment (MDT) composition, (ii) after 24 hours of exposure to the Bio-Dome device, and (iii) no treatment.

[0059] [Figure 13] FIG. 13 is a graphical representation of the number of ATT+ nerve bundles as measured by acetylated α-tubulin (AAT) staining in the right hind paw 18 mpa after amputation and after exposure to one of the following initial treatment conditions: (i) the first 24 hours of exposure to a provided multi-drug treatment (MDT) composition, (ii) after 24 hours of exposure to the Bio-Dome device, and (iii) no treatment.

[0060] [Figure 14] FIG. 14 is a graphical representation of nerve bundle diameter (mm) as measured by acetylated α-tubulin (AAT) staining in the right hind paw after 18 mpa and after exposure to one of the following initial treatment conditions: (i) the first 24 hour exposure to the provided multi-drug treatment (MDT) composition, (ii) after 24 hours exposure to the Bio-Dome device, and (iii) no treatment.

[0061] [Figure 15] FIG. 15 is a graphical representation of particle complexity (pixel 2) as assessed by fibronectin expression after 18 mpa and after exposure to one of the following initial treatment conditions: (i) the first 24 hours of exposure to the provided multi-drug treatment (MDT) composition, (ii) after 24 hours of exposure to the Bio-Dome device, and (iii) no treatment.

[0062] [Figure 16] FIG. 16 is a graphical representation of the number of laminin / smooth muscle actin (SMA+) bundles, as measured by laminin and SMA+ expression in regenerates, obtained after exposure to 18 mpa and one of the following initial treatment conditions: (i) the first 24 hours of exposure to a provided multi-drug treatment (MDT) composition, (ii) after 24 hours of exposure to the Bio-Dome device, and (iii) no treatment.

[0063] [Figure 17]FIG. 17 is a graphical representation of wound closure diameter (cm) obtained after exposure to 0.5 mpa and one of the following initial treatment conditions: (i) the first 24 hours of exposure to a provided multi-drug treatment (MDT) composition, (ii) after 24 hours of exposure to the Bio-Dome device, and (iii) no treatment.

[0064] [Figure 18] FIG. 18 is a graphical representation of the number of SOX2+ cells at the wound site obtained after exposure to 0.5 mpa and one of the following initial treatment conditions: (i) the first 24 hours of exposure to a provided multi-drug treatment (MDT) composition, (ii) after 24 hours of exposure to the Bio-Dome device, and (iii) no treatment.

[0065] [Figure 19] FIG. 19 is a graphical representation of the cumulative release of drug from provided MDT compositions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0066] The disclosures of these patents, patent applications, and publications are hereby incorporated by reference in their entireties in order to more fully describe the state of the art as believed to be known to those of skill 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 publications and this disclosure, the present disclosure controls.

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

[0068] It is to be understood that the phraseology and terminology used herein are for descriptive purposes and should not be regarded as limiting. The use of "including," "comprising," or "having" and variations thereof herein is intended to encompass the items listed thereafter and equivalents thereof, as well as additional items. Unless otherwise specified or limited, the terms "mounted," "connected," "supported," and "coupled," and variations thereof, are used broadly and encompass both direct and indirect mounting, connecting, supporting, and coupling. Furthermore, "connected" and "coupled" are not restricted to physical or mechanical connections or couplings.

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

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

[0071] The following discussion is presented to enable those skilled in the art to make and use the embodiments of the present disclosure. Various modifications to the illustrated embodiments will be readily apparent to those skilled in the art, and the general principles herein may be applied to other embodiments and applications without departing from the embodiments of the present disclosure. Thus, the embodiments of the present disclosure are not intended to be limited to the illustrated embodiments, but should be accorded the widest scope consistent with the principles and features disclosed herein. The following detailed description should be read with reference to the figures, in which like elements in different figures have like reference numerals. The figures, which are not necessarily to scale, depict selected embodiments and are not intended to limit the scope of the embodiments of the present disclosure. Those 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] Devices for tissue regeneration The present disclosure provides an apparatus for assisting in tissue regeneration. With reference to FIGS. 1-3, an apparatus 200 for stimulation of tissue regeneration in a subject 1 is shown. In some embodiments, the apparatus 200 is used to encapsulate wounded or injured tissue 9 in the subject 1. The wounded or injured tissue 9 may be located at an external or internal location on the subject 1. In the illustrated embodiment, the apparatus 200 is described with respect to stimulating tissue regeneration of wounded tissue 9 located in an appendage 3 (e.g., a mouse finger) where the tip 2 has been cut along a line 5 through at least a portion of the distal phalange 4, such that the regenerated tissue may include, among other tissues, bone tissue 6, muscle tissue 7, and skin tissue 8. For purposes of explanation in describing FIGS. 1-7, reference may be made to an "appendage" as including exemplary "tissues."

[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, pseudostratified 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] 1-2, device 100 includes an outer sleeve 202 extending between an appendage housing end 204 and a push member housing end 206. In some embodiments, outer sleeve 202 is a hollow cylinder defining an internal chamber 208 that is sized to accommodate the tissue 3 to be treated (e.g., an appendage) and the wounded or injured tissue 9. Internal chamber 208 forms a passageway extending between a first opening at appendage housing end 204 and a second opening at push member housing end 206. In some embodiments, the first opening at appendage housing end 204 is sized to accommodate the appendage 3 and the second opening is sized to accommodate the push member 214.

[0075] The outer sleeve 202 may be formed from a transparent material that allows for observation of the wound 9 while the device 200 is in use. In some embodiments, the outer sleeve 202 is sufficiently rigid to prevent any flexing or indentation of the body wall during use to ensure a desired wound space volume is maintained and to protect the wounded or injured tissue 9. Exemplary materials of construction for 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 the replacement of fluid within the inner chamber 208 of the outer sleeve 202. For example, the one or more openings may include a septum that allows a needle to enter and replace fluid within the inner chamber 208.

[0076] The device 200 includes an inner sleeve 216 extending between a wound accommodation end 218 and a mating accommodation end 220 opposite the wound accommodation end 218. In some embodiments, the inner sleeve 216 is a hollow cylinder defining an internal chamber 222 sized to accommodate the wound 9. The internal chamber 222 forms a passageway extending between a first opening at the wound accommodation end 218 and a second opening at the mating accommodation 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 accommodation end 220 towards the appendage 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 disposed within the internal chamber 222 of the inner sleeve 216. The protein matrix 228 may guide tissue growth in a direction and / or deliver therapeutic agents to the wound 9 to stimulate tissue regeneration.

[0077] In some embodiments, the mating receiving end 220 includes a porous filtration medium 230 that seals the second opening of the inner sleeve 216. Incorporating the porous filtration medium 230 into the device 200 prevents contamination and allows air and media exchange with the surrounding environment. The porous filtration medium 230 helps keep the wound 9 moist and cell viability high while reducing necrosis. The compressible member or media exchange member 232 may be positioned between the filter medium 230 and the pressing member 214 to provide a reservoir of aqueous solution or dispersion medium in fluid communication with the interior chamber 222. In some embodiments, the outer sleeve 202 may contain an aqueous solution or dispersion medium and be placed in fluid communication with the interior chamber 222 of the inner sleeve 216 via the filtration medium 230. In some embodiments, the compressible member or media exchange member 232 includes a gel including an aqueous solution or dispersion medium, or cotton optionally moistened with an aqueous solution or dispersion medium. In some embodiments, the protein matrix 228 is offset from the filtration 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 matable with the appendage-receiving end 204 of the outer sleeve 202. The first end cap 234 includes an opening 236 sized to receive the appendage 3 of the subject 1. In some embodiments, a gasket or diaphragm 238 may be positioned between the first end cap 234 and the appendage-receiving end 204 of the outer sleeve 202. The gasket or diaphragm 238 includes a through hole 240 sized to receive the appendage 3 and provides a seal to prevent liquid from escaping the interior chamber 208 of the outer sleeve 202. In some embodiments, the diaphragm 238 includes a flexible side 242 constructed from silicone and a rigid side 244 comprising polytetrafluoroethylene (PTFE).

[0080] The device 200 may include a first adjustable adaptor 246 that is installable within the appendage receiving end 204 for selectively connecting a first end cap 234 that is matable with the appendage receiving end 204 to the outer sleeve 202. In some embodiments, the adjustable adaptor 246 is a threaded adaptor and the first end cap 234 includes a groove 248 that receives threads 250 of the adjustable adaptor 246. When assembled, the first end cap 234 may be tightened such that a gasket or septum 238 is placed in contact with a tip 252 of the adjustable adaptor 246 to secure the appendage 3 within the outer sleeve 202.

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

[0082] The device 200 may include a second adjustable adaptor 262 that is mountable within the push member receiving end 206 for selectively connecting the push member receiving end 206 matable second end cap 254 to the outer sleeve 202. In some embodiments, the second adjustable adaptor 262 is a threaded adaptor and the second end cap 254 includes a groove 264 configured to receive the threads 266 of the second adjustable adaptor 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 pressure at the wound 9 interface, which leads to variable tissue regeneration outcomes 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, apparatus 200 is adjustable to facilitate long-term attachment (weeks, months, years or longer) and to facilitate regenerative tissue growth.

[0083] 4-5, in some embodiments, the device 200 includes a movable or adjustable pressing member 214. For example, the pressing member 214 may be movable or extendable in response to tissue growth (denoted by Δx in FIGS. 4 and 5). In some embodiments, the elastic member 268 is configured to extend between a seat 270 on the pressing member 214 and a seat 272 on the second adjustable adapter 262. In some embodiments, the elastic member 268 (e.g., a spring, a compressible material, a deformable material) creates a resistive force that creates a linear movement backwards (e.g., when the second end cap 254 is unscrewed). In some embodiments, the adjustable pressing member 214 includes an interlocking spacer or screw extension system that can extend over time in response to tissue growth.

[0084] 6-7, in some embodiments, the apparatus 200 also includes an electrical stimulation device 300 that establishes a longitudinal electric field through the wound site 9 to purportedly provide medial wound stump flow and electrical guidance cues for 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 that is placed adjacent to the wound 9. A portion of the cathode 304 is on the outside of the device 200 and can be connected to a lead 308, and a portion of the cathode 304 is within the inner sleeve's internal chamber 222. The anode 302 is a conductive wire that can be inserted into the subject 1 at a point remote from the wound site 9. In the embodiment shown where the device 200 is placed on the appendage 3, the anode 302 is placed on the top of the limb from which the appendage 3 extends (the front leg). The anode 302 may comprise a platinum / indium alloy wire that is connected to a power source 306 via a lead 310. The anode 302 may be permanently implanted or temporarily inserted as needed.

[0086] The power source 306 includes a battery pack 312 and electrical circuitry 314, both of which are enclosed in a housing 316 and are configured to provide a constant, low level of electrical current to the electrodes 302, 304 when connected. In the embodiment shown in FIG. 6, the power source 306 resides outside the subject 1, and the electrodes 302, 304 are configured to be removably connectable to the power source 306. In this arrangement, when electrical stimulation is used, the cathode 304 and anode 302 are electrically connected to the power source 306 for the duration of the electrical stimulation treatment, and then disconnected between electrical stimulation treatments. Because the power source 306 and leads 308, 310 can be detached from the electrodes 302, 304, respectively, this arrangement advantageously reduces the overall bulk of the combined apparatus 200 and electrical stimulation device 300 during treatment paradigms in which electrical stimulation is only intermittently used.

[0087] In some embodiments, the protein matrix 228 comprises a biocompatible polymer. Biocompatible polymers suitable for use with the 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, the 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 a variety of material formats, such as fibers, foams, particles, films, and / or hydrogels.

[0089] In some embodiments, protein matrix 228 is porous or has porosity. As used herein, the term "porosity" may refer to a measure of voids in a material, the fraction of volume or pores relative to the total volume, as a percentage from 0 to 100%. Determination of porosity is known to those of skill 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 match the surface area (size) of the tissue immobilized in the device 200 to optimize regrowth of the wounded or injured tissue. In some embodiments, the protein matrix 228 has a pore size of 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 μm. In terms of pore size, generally, 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 to support sufficient oxygen, nutrient and waste transport while providing suitable clearance for cell and tissue growth. In other embodiments, smaller pore sizes, for example, 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 nerve or blood cells. Higher porosity may facilitate improved tissue outcome 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 forming 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) on a conductive substrate (e.g., an aluminum plate) using a steep temperature gradient induced by integrating the conductive substrate with a cold source (e.g., liquid nitrogen). Finger-like columns of ice crystals growing from the cold surface are expected to create channel-like structures internally within the frozen protein. The frozen protein is then freeze-dried for a sustained period (e.g., 24 hours) to remove water. The resulting product is a protein matrix 228 with substantially aligned pores.

[0092] In some embodiments, the protein matrix comprises silk fibroin. As used herein, "silk fibroin" or "SF" may refer to a biopolymer produced from silkworm fibroin and insect or spider silk proteins. For example, silk fibroin useful in the present disclosure may be any of the following silk fibroins, including, but not limited to: 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. The silk may be produced by a number of species, including Araneus geometricus; Araneus bicentenarius; Tetragnatha versicolor; Araneus ventricosus; Dolomedes tenebrosus; Euagrus chisoseus; Plectreurys tristis; Argiope trifasciata; and Nephila madagascariensis. Alternatively, the silk utilized in the present disclosure may be prepared through artificial processes, such as the genetic engineering of cells or organisms (e.g., genetically modified bacteria, yeast, mammalian cells, non-human organisms including animals, or transgenic plants).

[0093] SF is a collagen-like structural protein, although with unique characteristics: it is produced from the exhalation of amino acidic solutions by complex organisms living in an external environment, whereas 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 come from its structure, consisting of hydrophobic blocks interleaved by hydrophilic acidic spacers. In its native state, SF is structured into a semi-crystalline material, with β-sheet crystals alternating with amorphous regions, which give strength and elasticity to the proteinaceous material formed from it. The diversity of forms into 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] Processing of SF generally involves partial or total dehydration of fibroin solutions (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 solution used in the methods and compositions provided herein may be obtained from a solution in which silkworm silk, such as from the silkworm Bombyx mori, is dissolved. Alternatively, the silk fibroin solution may be obtained from a solution in which spider silk, such as from Nephila clavipes, is dissolved. The silk fibroin solution may also be obtained from a solution containing recombinant silk, such as from bacteria, yeast, mammalian cells, transgenic animals, or transgenic plants. See, for example, WO 97 / 08315 and U.S. Pat. No. 5,245,012. The recombinant silk may also include, for example, a therapeutic agent, such as a cytokine, an enzyme, or a fusion protein with any number of hormones or peptide-based drugs, antimicrobial 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 the transition to a solid state (e.g., polyethylene glycol, collagen, hyaluronic acid, and the like).

[0097] Silk from silkworm cocoons is made up of two structural proteins: fibroin heavy chains (approximately 350 kDa (5.81 × 10 -19 g); and fibroin light chains (approximately 25 kDa (4.15 × 10 -20 g). The heavy and light fibroin chains are linked by disulfide bonds at the C-terminus of the two subunits (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 J. Appl. Physiol. 1995, 110:151, 1992). Sericin is a high molecular weight soluble glycoprotein component of silk that gives the material its sticky properties. These glycoproteins are hydrophilic and can be easily removed from the cocoons by boiling in water "degumming").

[0098] In some embodiments, the silk polypeptide compositions utilized by the present compositions are substantially free of sericin (e.g., contain no detectable sericin or contain sericin at a level that one of 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 for about 30 minutes in an aqueous solution, such as, but not limited to, about 0.02 M Na2CO3. 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 cocoons may be rinsed, for example, with water to extract the sericin proteins, 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 LiBr solution of about 9 M to about 12 M. The salts are then removed, for example, by dialysis.

[0100] If desired, the solution may then be concentrated using any method known in the art. For example, dialysis against a hygroscopic polymer, such as PEG, polyethylene oxide, amylose, or sericin, may be performed. The 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 may be used, such as a Slide-a-lyzer dialysis cassette (Pierce, MW CO3500). The solution is dialyzed for a period of time sufficient to result in a final concentration of 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 appendage or tissue of a subject in need of tissue regeneration. The method includes contacting a wounded appendage or tissue 9 of a subject 1 with the wound receiving end 218 of the inner sleeve 216. The wounded appendage or tissue 9 may be placed in contact with or adjacent to a protein matrix 228 that includes a provided therapeutic composition. In some embodiments, prior to contacting the wounded appendage or tissue 9 with the protein matrix 228, the wounded appendage or tissue 9 is slid through a gasket or septum 238 and an opening 236 in a first end cap 234.

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

[0103] In some embodiments, the wounded appendage 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 about 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 duration range bounded by any of these values. During the course of the duration, the protein matrix 228 may be kept moist by adding or replacing a buffer solution within the inner sleeve 216.

[0104] Therapeutic Compositions for Tissue Regeneration Therapeutic compositions for tissue regeneration and "multiple drug treatment" compositions (MDT) are also disclosed herein. Therapeutic compositions may be utilized alone or in conjunction with the disclosed devices.

[0105] The therapeutic composition used with the device according to the present invention may be any composition that stimulates, initiates, or directly or indirectly supports tissue regeneration. Alternatively, the therapeutic composition according to the present invention may be a combination of components that act synergistically to stimulate, initiate, or directly or indirectly support tissue regeneration. For example, the components provided in the therapeutic composition (e.g., two or more of the components, e.g., 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 wounded appendage or tissue site (e.g., increase tissue regeneration as measured by soft tissue length, bone length, bone mass, increased contact response, number of ATT+ nerve bundles, diameter of ATT+ nerve bundles, complexity of regeneration particles by fibronectin expression, number of laminin / SMA+ bundles, reduction in wound diameter at the start of treatment, number of SOX2+ cells) compared to a control experiment with no treatment.

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

[0107] In one example, the disclosed device may contain a therapeutic composition within the 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 worn on the subject's appendage. 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 compositions may be present in a polymeric material, such as, but not limited to, a silk hydrogel material. Methods for loading therapeutic compositions and drugs into hydrogel materials are known in the art. For example, a silk hydrogel material loaded with a therapeutic composition may be prepared as follows: The therapeutic composition may be added to a silk solution (e.g., a 3% w / v silk solution), which is then induced to gel via the addition of a reagent, such as horseradish peroxidase (e.g., a silk solution at a concentration of about 20 U / ml) along with hydrogen peroxide (e.g., a concentration of up to 0.01% w / v). Silk can also be gelled by this enzymatic reaction, via lowering the pH, the addition of energy, such as sonication or vortexing, application of an electric field, or the addition of methanol, as some of many options.

[0109] The disclosed therapeutic compositions may include another agent that increases axonal / neurite outgrowth and / or general cell proliferation. Preferably, the disclosed therapeutic compositions do not promote pluripotency in cells and / or lead to teratoma formation.

[0110] The disclosed therapeutic compositions may include one or more agents that promote tissue regeneration and / or healing. In some embodiments, the therapeutic compositions include one or more of growth factors, agents that inhibit inhibitors of hypoxia inducible factor 1-alpha (HIF1-alpha), vitamin A or a derivative thereof, lipid mediators, such as metabolic products of omega-3 fatty acids, and may be derived from eicosapentaenoic acid or docosahexaenoic acid, growth hormones, steroids, and depolarizing agents.

[0111] In some embodiments, the disclosed therapeutic compositions may include growth factors, such as neurotrophic factors. Neurotrophic factors are proteins that promote the growth and survival of neural cells during development and the maintenance of adult neural cells. (See, e.g., Terenghi, J.Anat See, 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-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), and combinations thereof. The growth factor may be present in a dosage within the therapeutic composition that is 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 within a dosage range bounded by any of these values. When growth factors are present in a component of the disclosed devices (e.g., when growth factors are loaded into the inner sleeve or into a component of the inner sleeve), the device may comprise a concentration of growth factor of 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 within a concentration range bounded by any of these values. Growth factors promote the growth of one or more tissue types.

[0112] The disclosed therapeutic compositions may include prolyl hydroxylase domain (PHD) enzyme inhibitors (i.e., PHD inhibitors) to stabilize constitutive expression of the HIF-1α protein, for example. (See, e.g., Ariazi et al., J Pharmacol Expt Therap (2017), 363(3) 336-347; and Nangaku et al., Arterioscler., Thromb. Vas.Biol2007;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)-pyridinylthio]-2,4(1H,3H)-pyridinylthio]-2,5(1H,3H)-pyridinylthio]-2,6(1H,3H)-pyridinylthio]-2,7(1H,3H)-pyridinylthio]-2,8(1H,3H)-pyridinylthio]-2,9(1H,3H)-pyridinylthio]-2,8(1H,3H)-pyridinylthio]-2,9(1H,3H)-pyridinylthio]-2,5 ... Rimidinedione (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 chelators, and combinations thereof. Optionally, the PHD inhibitor may be present in a dosage within the therapeutic composition that is 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 within a dosage range bracketed by any of these values. When a PHD inhibitor is present in a component of the disclosed device (e.g., when a PHD inhibitor is loaded into the inner sleeve or into a component of the inner sleeve), the device may comprise a concentration of PHD inhibitor of 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 within a concentration range bounded by any of these values. The PHD inhibitor controls excess collagen deposition at the wound site.

[0113] The disclosed compositions may include vitamin A or its metabolites or derivatives or any agent that functions at the proximal-distal location. 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, and tazarotene, and combinations thereof. Agents that may function at the proximal-distal location 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 a metabolite or derivative thereof or any agent that functions in a proximal-distal coordinate system may be present in the therapeutic composition at a dosage of 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 within a dosage range bounded by any of these values. When vitamin A or a derivative thereof (or an agent that functions at proximal-distal location information) is present in a component of the disclosed device (e.g., when vitamin A or a derivative thereof or an agent that functions at proximal-distal location information is loaded into the inner sleeve or into a component of the inner sleeve), the device may contain a concentration of vitamin A or a derivative thereof or an agent that functions at proximal-distal location information of 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 within a concentration range bounded by any of these values.

[0114] The disclosed therapeutic compositions may include lipid mediators and / or metabolites of omega-3 fatty acid products that promote resolution of inflammatory responses (i.e., anti-inflammatory agents). Suitable lipid mediators may include derivatives of omega-3 fatty acids (e.g., metabolites of products) and / or derivatives of eicosapentaenoic acid or docosahexaenoic acid that promote resolution of inflammatory responses (i.e., anti-inflammatory). Exemplary lipid mediators may 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 a dosage of 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 within a dosage range bounded by any of these values. When a lipid mediator is present in a component of the disclosed devices (e.g., when the lipid mediator is loaded into the inner sleeve or into a component of the inner sleeve), the device may comprise a concentration of lipid mediator of 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 within a concentration range bounded by any of these values.

[0115] The disclosed therapeutic compositions can include peptide hormones, such as peptide hormones that stimulate growth, cell proliferation, and cell regeneration (see, e.g., Schmidmaier et al., Bone (2002) 31(1):165-72; and Schneider et al., J.Clin.Invest115(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 a dosage within the therapeutic composition that is 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 within a dosage range bounded by any of these values. When a growth hormone or steroid is present in a component of the disclosed devices (e.g., when the growth hormone or steroid is loaded into the inner sleeve or into a component of the inner sleeve), the device may contain a concentration of growth hormone or steroid of 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 a concentration range bounded by any of these values.

[0116] The disclosed therapeutic compositions may include 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 compositions can be used to treat subjects in need of treatment. As used herein, "subject" means a human or an animal. Typically, an animal is a vertebrate, such as a primate, a rodent, a livestock or a game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Livestock and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species, such as domestic cats, canine species, 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, such as a primate, such as a human. The subject can be female or male. Preferably, the subject is a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. Mammals other than humans may be used as subjects that represent animal models of tissue repair, regeneration, and / or reconstruction. The methods and compositions described herein may also be used to treat livestock animals and / or pets.

[0118] In some embodiments, the disclosed therapeutic compositions can be used to treat wounded or injured appendages or tissues of a subject in need of stimulated tissue regeneration. The tissue or appendage can be internal or external to 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, pseudostratified 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.

[0119] In some embodiments, the present disclosure provides a method of regenerating at least a portion of a wounded or injured appendage or tissue by administering to a subject an effective amount of a disclosed therapeutic compound, including a disclosed compound. In some embodiments, the method includes contacting the wounded or injured appendage or tissue with a therapeutic compound, which may or may not be present in a provided hydrogel. In some embodiments, a provided therapeutic compound or a provided hydrogel is contacted with the wounded or injured appendage or tissue in a device provided herein. The disclosed devices and / or therapeutic compositions promote tissue regeneration, which may be measured by any method known in the art, including, but not limited to, by measuring the expression of Yamanaka factors, Sox2, Oct3 / 4, Klf4, and / or c-Myc in tissue treated with a device and / or therapeutic composition according to the present disclosure versus tissue not treated with the disclosed device and / or therapeutic composition.

[0120] Animal model testing Anurans maturing towards adolescence are able to regenerate their amputated or injured limbs when exposed to regeneration-inducing factors delivered through slow-release beads embedded in the amputated tissue. However, completely non-regenerative postmetamorphic (adult) Xenopus are unable to regenerate their hind limbs upon amputation, instead producing featureless cartilage spikes (Suzuki et al., (2006) The Scientific World Journal, 6 .) This model was used to test whether regeneration-inducing factors could stimulate regeneration.

[0121] As described in the following examples, complex interventions in adult Xenopus hind limb amputations were tested to address several aspects of limb regeneration. A wearable bioreactor ("Bio-Dome") was used to gain control over the local microenvironment of the wound in vivo. Mechanisms were explored, showing that a short period of exposure to a regenerative cocktail initiates a long-term endogenous morphogenetic cascade without continuous micromanagement. Various stimuli were selected to induce pro-regenerative activity, including agents that reduce inflammation, promote neural sparing, and induce overall growth.

[0122] A short exposure period (e.g., 24 hours) to silk-containing wearable bioreactors infused with several small molecule compounds was found to induce dramatic elongation, patterning, and sensorimotor function after amputation in Xenopus. Treated animals showed a significant delay in wound closure followed by long-term (approximately 16 months) growth outcomes including increased bone length, soft tissue patterning, and neuromuscular repair. Histologically, the new limb contained smooth muscle indicative of nerves, blood vessels, and restructuring of extracellular matrix proteins involved limb remodeling. Transcriptomics analysis identified intermediate and short-term pathways as well as transcriptional targets of intervention in the blastema. RNA-seq studies also revealed a rapid response to the full treatment device in the brain (compared to sham controls). Regenerated bones displayed anatomical features characteristic of wild-type morphology, and soft tissues in the distal limbs displayed finger-like protrusions. Animals also used the newly formed limbs to ambulate similarly to wild-type frogs. Additionally, sensorimotor pathways reverted in animals exposed to full treatment conditions, indicating that tissue-repatterning involved re-extension or regrowth of afferent nerves as well as the neuromuscular tissue interface.

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

[0124] The following examples are provided to describe in detail how the present disclosure can be used or practiced, and will enable those skilled in the art to more readily understand the principles of the present disclosure. The following examples are presented by way of illustration and are not intended to be limiting in any way. EXAMPLES

[0125] animal Adult male Xenopus laevis (n=115) (Nasco, Fort Atkinson, WI), measuring 5 cm-6.25 cm (nose to tail), were acclimated to holding tanks for 2 weeks prior to experimentation. Animals were maintained at 18°C ​​(291.15 K) in 10 L plastic tanks containing defined frog water (reef salts, Seachem Laboratories, conductivity approx. 1.65 kΩ, pH 7.8-8.0) and exposed to a 12-h light-dark cycle. Prior to experimentation, animals were immersed in broad-spectrum gentamicin antibiotic for 2 h to minimize bacterial contamination of limb stumps following amputation (Gibco, Fisher Scientific, USA).

[0126] limb amputation Hind limb amputation surgery was performed according to Golding et al. (2016), PLoS One 11 ,e0155618, and Herrera et al. (2018), Cell Rep 25 The procedure was performed according to a previously established protocol published by the National Institute of Animal Health, 1593-+. Briefly, animals were first anesthetized by total body immersion in buffered frog water containing 0.05% benzocaine. Upon loss of fingertip pinch reflex, 75 mg / kg buprenorphine was injected subcutaneously just below the lateral line contralateral to the leg to be amputated. The right hind limb was amputated midway through the tibiofibular bone with a sterile microsurgical blade using a straight cut. No bone resection was performed and no tissue flap over the wound site was sutured. After hemostasis, animals were allowed to regain consciousness and recover in sterile frog water for a minimum of 60 min.

[0127] Device installation Animals were randomly assigned to one of three treatment conditions: no device, biodome only, or biodome with cocktail treatment (described below in "Biodome Fabrication and Cocktail Composition"). The device attachment procedure was preceded by a second anesthesia (0.05% benzocaine soak, 75 mg / kg buprenorphine). The unconscious animals were then fitted with a device secured to the amputation site stump using monofilament surgical sutures (7-0 Monosof, 18" P-16 cutting, Covidien, USA). Two sutures were made through the dermal layer on either side of the leg. These stitches were sufficient to hold the device in place and did not damage the underlying deep fascial layer. After attachment, animals were returned to their home tank, at which time they were allowed to regain consciousness and swim freely. Control animals were handled similarly to those housed with the device, but did not have a device attached to the wound stump. The results presented herein were generated using a sutured biodome.

[0128] Alternatively, an adjustable biodome may be used. The adjustable biodome attachment procedure involves pushing the amputated limb in place through the cap and donut-shaped septum, and securing the limb using a bioadhesive (Skin-Tite Bioadhesive, Smooth-On, PA). The scaffold-containing biodome insert was attached to the amputated finger and secured with a small amount of adhesive. An acrylic protective cap was screwed into the top cap, followed by pushing the insert closer to the wound bed with a custom washer and closing with a second cap. To keep the tissue moist throughout the attachment period, approximately 4 μL of sterile phosphate buffered saline (PBS) 1X was slowly injected into the insert using a 31G needle. The PBS was changed every 2 days to remove cellular waste and keep the tissue fresh. The device was maintained attached until the animals were sacrificed for later analysis.

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

[0130] The adjustable biodome was constructed from acrylic tubing (#8532K13, Mcmaster-Carr, Elmhurst, IL) cut into 1 cm lengths of cylindrical tubing and used as the body of the device 100. A threaded adapter was designed using 3D CAD software (Inventor Professional, Autodesk, San Rafael, CA) and printed using a stereolithography 3-D printer (Form2, Formlab, Somerville, MA). The adapter was glued to the acrylic tube using medical grade superglue. 2 ml HPLC vial caps (Agilent, Santa Clara, CA). The PTFE / silicone septum was punched with a 3 mm biopsy punch to create an access port for the animal's limbs. The septum was cut along the center hole in 4 locations, in separate quadrants, to provide additional room for the animal's leg to pass through the port. Custom washers were fabricated from PDMS using soft lithography or 3D printed using a 3D printer. The cylindrical walls of the insert of the device 100 were fabricated from a clear polyester membrane filter (0.45 μm pore size, 12 μm thick, #1300016, Sterlitech, Kent, WA). The filter was cut into a rectangle (7 mm x 5 mm) and wrapped around a 1.5 mm diameter metal rod (#8907K62, McMaster). The walls were then glued with silicone adhesive (Dragon Skin 10 The insert was then completed by gluing it to a silicone bottom using a 350-mm thick 100% polyester tipped adhesive (FAST, Smoothon, Macungie, PA).

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

[0132] Device Removal and Maintenance After 24 hours, animals were anesthetized and treated with analgesics as previously described. The devices were then removed by cutting a single suture on either side of the leg, and the frogs were placed back into a tank containing a disinfectant (cordon methylene blue at a concentration of 1 mL / 10 L frog water. After a further 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 whole-body immersion in frog water with 0.2% benzocaine. Regenerants, contralateral limbs, and brain tissue were collected and processed for histological analysis.

[0133] Silk Treatment Silk fibroin solution was prepared by cutting and degumming 5 g of Bombyx mori cocoons (Tajima Shoji, Yokohama, Japan) in 0.02 M sodium carbonate (Na2CO3) solution for 45 min 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 fume hood at 22 °C (295.15 K). The dried silk fibers were 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 h. The solution was then transferred to a dialysis cassette (3.5 kDa (5.81 × 10 -21 The silk was dialyzed in DI water with gentle stirring using a 1000 g molecular weight cut-off, Thermo Fisher Scientific, Waltham, MA). The water was changed six times over a period of 48 h. The dialyzed solution was centrifuged three times at 13,000 g at 4° C. (277.15 K) for 20 min each, followed by filtration through a cell strainer (40 μm pore size, Thermo Fisher) to remove impurities. To determine the concentration of the filtered solution, a 0.5 ml sample was completely dried overnight in an oven. Once the water was 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 cross-linking liquid silk fibroin. 45 mb silk (3% w / v) and horseradish peroxidase (HRP) solution (20 U / ml) were cast into a 24-well plate and incubated at 37 °C (310.15 K) for 45 min to complete gelation. The gel compressive strength and modulus of the gels were measured according to Golding et al. (2016), PLoS One 11 ,The test was performed according to the method of e0155618.

[0135] Scaffolds with Aligned Pores Silk scaffolds with aligned pores were 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 columns of ice crystals growing from the cold surface created channel-like structures inside the silk solution as they remained solidified. After 10 min of cooling, the frozen solution was lyophilized for 24 h to remove water. The sponges were trimmed to fit the biodome as inserts, sterilized in ethylene oxide, and stored at room temperature in sterile conditions until use.

[0136] Collagen scaffolds with aligned channel-like porous structures were fabricated by controlled directional freezing and freeze-drying of a 1.5% (wt / wt) collagen solution (in a similar manner to the silk scaffolds). The scaffolds were cut into cylindrical shapes (4 mm length and 1.5 mm diameter) and placed into the biodome inserts using scissors.

[0137] Material characterization The morphology of the scaffolds was characterized using scanning electron microscopy (SEM) and fluorescence microscopy. For SEM imaging, the scaffolds were cut in half using a razor to expose the internal geometry of the pores. Prior to 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 testing 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. FIG. 8D is a collagen scaffold with pores aligned perpendicular to the long axis.

[0138] therapeutic composition Hydrogels were prepared with a final concentration of 3% (w / v) silk solution, 20 U / ml horseradish peroxidase (HRP), and 0.01% wt / v hydrogen peroxide (HO). The liquid solution was poured into silicone sleeves and allowed to gel for 30 min before being attached to the animal's limb stump. For the cocktail-dosing device, 0.014 μg / ml of 1,4(dihydrophenonethrol-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 resolvin D5 (catalog number 10007280, Caymen Chemicals, MI, USA), and 0.015 μg / ml of retinoic acid (catalog number 11017, Caymen Chemicals, MI, USA) were dosed into the liquid silk solution and then inserted into a silicone sleeve to gel.

[0139] In vitro release studies To determine the release profile of the drugs used in this example, 50 μL of each hydrogel solution loaded with a specific amount of a specific drug was added to a 1.5 ml microcentrifuge tube 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 designated time points, 300 μl of the supernatant was collected for analysis. Drug-free silk hydrogel release solution was used as a control. Calibration curves were determined 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 carried out 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 measurements were performed according to the manufacturer's protocols. The optical density of the prepared samples was read at 450 nm using a SpectraMax M2 plate reader. The release samples of Resolvin D5 were filtered through a protein filtration column (MWCO = 3 kDa (4.98 × 10 -21 The high molecular weight fibroin fraction was removed by filtration through a 500 mL filtration filter (UFC500324, Fisher) and the optical density of the sample was then determined at a wavelength of 244 nm using a SpectraMax M2 plate reader.

[0141] soft tissue imaging At regular intervals over the 18-month maintenance period, animals were evaluated for soft tissue repatterning and bone regrowth. Animals were anesthetized as previously described, 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 served as a standard reference point for all measurements. The site of amputation was easily identified by the consistent 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 Golding et al. (2016) PLoS One 11 ,e0155618, and Herrera-Rincon,et al.,(2018) Cell Rep The bones were evaluated using a handheld X-ray device (Nomad Pro 2TM) using standard imaging settings of 60 kV, 2.5 mA with an exposure time of 0.20 seconds, according to the 25,1593-+ imaging protocol. All animals were subjected to the same dose (0.12 mSv) at the defined time points. Computed tomography (CT) on a viva CT40 scanner (Scanco Medical, Switzerland) was performed to visualize the detailed microarchitecture of the bone at the end of the 18-month regeneration period after euthanasia with benzocaine overdose (0.2% total body immersion). Distal trabecular and mid-axial cortical bone sections (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 regional CT dose index (CTDI), ranging from 453 mGy to 1255 mGy.

[0143] Histology and immunohistochemistry To characterize the effect of treatment on limb regrowth and re-patterning after amputation, histological analysis was performed on regenerates and contralateral limbs at defined intervals over time. Tissues were collected 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-ray), tissues were gradually equilibrated to 30% sucrose before embedding in OCT (Sakura FInetek, USA). Samples were frozen in liquid nitrogen. Limb tissues were serially sectioned at 14 μm using a cryostat (Leica CM1850) and placed on glass slides. Cross sections were taken at 14 um 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 pattern formation, the limb ends were sectioned horizontally at 14 um intervals. Sections were dried for at least 1 hour and then stored at -80°C (193.15K).

[0144] For immunohistochemistry, slides were equilibrated at room temperature for at least 2 hours before staining. Slides were post-fixed in 4% PFA for 5 minutes and then blocked in blocking buffer (PBS with 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 phosphohistone H3 (1:250). Slides were stained with each antibody individually, except for anti-smooth muscle actin and anti-laminin, which were stained together. Primary antibodies were incubated on the slides overnight. 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 min. Slides were mounted in Fluoromount-G (ThermoFisher Scientific) and allowed to cure for at least 24 h before imaging.

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

[0146] Analysis of immunostained sections All statistical analyses were performed in IBM SPSS version 20 software. Normality assumptions were tested prior to the use of parametric tests, including ANOVA, t-tests, and related analyses (Pearson's r). Non-parametric analyses, Mann-Whitney-Wilcoxon tests or Kruskal-Wallis tests, were performed for comparisons where data were not normally distributed. Significant differences were assumed when p-values ​​were below a threshold of 0.05 (two-sided hypothesis testing).

[0147] Sensorimotor Threshold Assessment To assess the sensorimotor abilities of the regenerates, 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 min until movement completely ceased. A video camera (iPod® Touch 5th generation, Apple, CA, USA) was placed on the enclosure to capture a recording of the test procedure. Standardized Von Fry filaments (Touch Test, Stoelting, IL, USA) were used to assess the sensory threshold of the regenerates. Filaments ranging in force from 0.008 g to 300 g were applied to the distal part of the regenerate, from the lowest to the highest force. The first filament that elicited a clear response (movement from a resting position) was recorded. Animals were tested twice over a 2-day period and the average threshold was reported.

[0148] statistical analysis All statistical analyses were performed in IMB SPSS v20. Data were first tested for homogeneity of variance by Levene's test. Single-gene analyses were performed on normally distributed data by unpaired, two-tailed Student's t-test (two independent groups) or one-way ANOVA test (multiple independent groups) followed by post-hoc Scheffe's test (when P<0.05). When the variable "time" was considered, two-factorial analyses were 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. For non-normally distributed data, Kruskal-Wallis test was performed followed by post-hoc Dunn's test (when P<0.05), respectively. The significance level was set at 0.05 in all cases. Statistics are reported as mean ± standard deviation or mean ± standard error of the mean, where indicated. When appropriate, dot or scatter plots are used to highlight individual variations within experimental groups, respectively.

[0149] RNA extraction Following amputation, device attachment and removal, and 24 hours of treatment, regenerative tissues were harvested at 11-, 24-, and 72-hours post-amputation 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) per manufacturer's protocol, and total RNA quality and quantity were assessed using a Nanodrop spectrophotometer (ThermoFisher Scientific).

[0150] Next Generation Sequencing (NGS) 1.1 μg of total RNA was sent to Tufts Genomic Core. RNA quality was assessed via bioanalyzer, and high quality RNA was used for library preparation with TruSeq Standard RNA Library Prep Kit with RiboZero Gold (Invitrogen). Libraries were then multiplexed and single-ended, 50nt sequencing was performed on an Illumina HiSeq2500. Raw read files were sent to the Bioinformatics & Biostatistics Core at Joslin Diabetes Center.

[0151] NGS analysis The Xenopus laevis reference genome was downloaded from the NCBI genome database, assembly GCA_001663975.1. Reads were aligned using the 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.) The aligned reads were counted using featureCounts (Liao et al., (2014) Bioinform 30(7):923-30). Genes with expression counts >1 count per million (cpm) in at least three samples were included in the analysis and normalized by the weighted trimmed mean of the M values ​​(TMM, Robinson et al., (2010) Genome Biol .11, R25.) Using the Voom transformation (Law et al., Genome Biol .15,R29.) Counts converted to logCPM: CPM=1e+6 * Gene count / (sample total count * The Voom transformation also estimates the mean-variance relationship and uses this to calculate the observation-level weights, so additional read depth gives additional weight. To further weight outliers, we use sample-specific good quality weights (Ritchie et al., (2006) BMC Bioinform .7,261.) were collected and combined with the observed level weight.

[0152] Differentially expressed genes were identified using Limma (Ritchie et al., Nucl. Acids Res. 43, e47.). Moderated t-tests were performed to detect genes that were 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 (CPs) or gene ontology (GO) were selected. The analysis was performed using the rimma R package (Wu et al., (2010) Bioinform A rotated gene set test (Roast) was performed using the Fry function in the genomics section (Schmidt et al., 2013). Cooperatively up, cooperatively down, and mixed gene sets were all considered significant when P<0.05 and FDR<0.05.

[0154] Network Analysis Gene modules were identified by co-expression analysis in CeMiTool in R (Russo et al., 2018) based on logCPM values ​​for control and cocktail-treated groups at 11 h, 24 h, and 7 days after limb amputation. In CeMiTool, a variance-stabilizing transformation was applied to remove differences between 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 an automatically generated scaling value beta (β=10) of 30 genes and a minimum module size. To assess how enrichment of these modules changed over time and groups, module enrichment was calculated based on sample annotation. To determine which biological functions were associated with each module, an over-representation analysis was performed using the pathway database from Reactome, and pathways were considered significant at p<0.05. The annotated modules are used to graph combined gene-gene interaction data from the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Chemical and Genetic Perturbations (CGP) and map interacting genes contained in each module.

[0155] qPCR method The same total RNA submitted for RNAseq analysis was DNase treated using the RQ1 RNase-free DNase kit (Promega Corp., Madison, WI, USA). The resulting RNA (0.5 μg) underwent a second DNase treatment followed by cDNA synthesis using the Verso cDNA synthesis kit (ThermoFisher Scientific). Quantitative analysis of gene product amounts using the Step One Plus Real Time System (Applied Biosystems, USA). Reactions of 10 μl each were performed in duplicate, containing: 5 μl of 2x PowerUp SYBR Green Master Mix (Applied Biosystems, USA), 0.5 μl 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, with the average of all ND expression across time points used as a calibrator for all samples.

[0156] result 1. Induction of limb regeneration by multidrug regeneration treatment Adult Xenopus hind limbs were amputated at the midpoint along the tibia and fibula and fitted with a biodome device containing either a silk-based hydrogel with a 5-drug multidrug treatment (MDT) or hydrogel alone. Control animals were amputated without treatment. After 24 h exposure to the biodome, the device was removed and animals were maintained for up to 18 months with regular assessment of regeneration and repatterning of the hindlimb regenerate. The prolonged observation window was chosen based on Alibardi's (2018) calculations, which predicted that it would take approximately 1.5 years to regenerate an anuran limb based on projection modeling using newt stump diameters.

[0157] X-ray images were obtained and limb length was measured as a function of months post-amputation. X-rays showed that regrowths associated with both the biodome only and multidrug treatment (MDT) conditions were longer than control (no device) regrowths after 4 months, an effect that persisted over time, emerging after 4 months and then again at 8 months (two-way ANOVA, between-subjects factor treatment exposure, within-subjects factor regeneration time F(2,19)=61.9, p<.05). Limb length relative to the amputation site was greater for the MDT group compared to other groups as early as 0.5 months post-amputation (mpa) and persisting at 4mpa (p<0.05), suggesting that MDT increased growth rate early. At 6-8 months, growth was significantly delayed, with hind limb regrowths associated with the biodome only group achieving comparable length compared to MDT-exposed animals (p>0.05). A secondary increase in late growth after 9 mpa indicates that the MDT group redisplayed longer limbs compared to the other groups, an effect that persisted until the final measurement at 18 mpa (p<0.01). Thus, the MDT group not only demonstrates a final increase in leg length compared to the other treatment groups, but also a secondary period of growth that is not present in the other treatment groups, particularly the no-device group. These data suggest that brief exposure to MDT and the Biodome facilitates regrowth of longer limbs.

[0158] Not only was leg length increased, but as many as 76% of animals exposed to MDT displayed thicker, more complex regenerate morphology compared to the featureless heavily pigmented spikes of the no-device group. Specifically, the distal segments of hindlimb regenerates associated with the MDT group presented flattened paddle-like structures with protruding blastemas characteristic of digits. In contrast, the no-device and biodome-only conditions were exclusively associated with featureless regenerate spikes, while the provided MDT condition reliably produced patterned paddle-like structures with distal blastemas present. The biodome group had an intermediate phenotype, with 20% of the biodome animals displaying thicker cartilage spikes and hook-like distal protrusions, but the patterning was not limited. None of the animals in the no-device group displayed the distinctive phenotype.

[0159] 2. The induced limb shows sensory function Sensorimotor assessments were then performed to assess whether the 18 mpa regenerates had regained sensory function. Using standardized Von Frey (VF) filaments (minimum force: 0.008 g; maximum force: 300 g), the regenerated right hindlimb was probed at its most distal end with increasing filament strength until maximum force was applied. The first filament eliciting a clear response (movement from a resting position) was recorded and behavioral assays were averaged over two days. MDT-treated hindlimb regenerates displayed a comparable stimulus-response pattern to the non-amputation group (p>0.01; FIG. 10), indicating significant reinnervation and neuromuscular reconstruction. In the Biodome group, responses varied significantly between individual animals with groups performing at near-normal levels and groups with no detectable stimulation (56.6±98.8 g), whereas non-treated animals reliably failed to display any response to force application at any value up to 300 g (inclusive). Therefore, we conclude that MDT treatment facilitated sensorimotor integration similar to that of the unamputated limb. Interestingly, the Biodome alone group was also able to facilitate regrowth of the limb with an intact sensory system in some cases. This indicates that hydration and structural support may be important to maintain innervation and facilitate 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 imaging. As overall hind limb length was significantly improved by treatment (FIG. 9), a study of the bone components of the regenerates was subsequently investigated. Micro-CT and X-ray images of the regenerates reveal increased bone mass and length associated with the provided MDT composition compared to the biodome alone and the control.

[0161] X-ray images confirmed the presence of complex morphology and increased bone length in the MDT regenerates compared to the Biodome only and No Device control experiments. Starting with measurements at 2.5 mpa, dense tissue protruding outward from the amputation site was observed in the MDT and Biodome groups, but not in the No Device control. The MDT group also displayed dense, segmented bone fragments at the distal end of the regenerate. As expected, the MDT condition displayed increased bone length compared to the 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 FIG. 11, the difference in bone length between the MDT and Biodome groups was monitored periodically, and the difference in length between these groups and the No Device control was maintained throughout (p<0.05).

[0162] To obtain a better understanding of the microarchitecture of the regenerated bone, MicroCT was performed at 18 mpa. MicroCT imaging allowed visualization of a three-dimensional rendering of the underlying bone and measurements of bone mass without disturbing the outer soft tissue. MicroCT data confirmed the presence of bone fragmentation in the distal region of the MDT hindlimb. Notably, fragmentation does not occur at the amputation site, but instead spontaneously appears after a period of growth at a point along the bone equivalent to the contralateral joint. These re-patterned segmentations were common within the MDT group. As shown in Figure 12, bone mass measured through microCT data was greater in the hindlimb regenerates of the MDT group compared to the other groups. Volumetric quantification confirmed the increased growth in the provided MDT regenerates (One-way ANOVA (F(2,15)=11.15, p<0.001)). X-ray tomography also revealed striking similarities between the non-amputation and MDT-treated bone anatomy. Re-patterning was evident in the provided MDT regenerates, including 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, as well as the presence of joint segmentation. These data indicate that the MDT condition is associated with significant remodeling of bone anatomy, consistent with an active process of remodeling bone and ultimately achieving a morphological complexity approximating the characteristics of the previously amputated limb.

[0163] 4. Complexity of regenerated limbs While MDT-treated animals showed increased bone growth, molecular changes at the cellular level in the regenerated limbs were also evaluated. Immunohistochemistry was performed to evaluate tissue structures associated with regeneration and re-patterning after 18 mpa. As shown in FIG. 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 MDT treatment affected nerve bundle regrowth and innervation of the regenerate. As shown in FIG. 14, not only were there more bundles, but the 24-hour MDT group also showed 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] Kruskal-Wallis test showed that the number of AAT positive bundles at 18 mpa was significantly different 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-h MDT condition compared to uninjured animals (U=13, P=0.0014). The number of AAT bundles was comparable compared to uninjured animals and the biodome only group (p=0.1812). We then examined ATT bundle dimensions, which revealed significant differences across conditions (H(11.74)=p=0.0028). The 24-h 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 connective tissue architecture, we evaluated fibronectin expression patterns after 18 mpa. As shown in Figure 15, the increased particle complexity in MDT-treated regenerates reflected the degree of structural complexity observed in MDT conditions. Specifically, fluorescent images revealed increased nerve regeneration and extracellular matrix repatterning in regenerates 18 months after initial amputation and exposure to multi-drug treatment. Consistent with other results, the 24-hour MDT group displayed increased particle complexity compared to both the Biodome only and no-device groups. This indicates that MDT itself tends to facilitate the assimilation of cartilage and connective tissue in regenerates.

[0166] To assess vascularization of regenerates, laminin and smooth muscle actin (SMA) expression at 18 mpa was compared across conditions, as shown in FIG. 16. Fluorescent images of regenerate cross sections obtained 18 months after amputation were double stained for two markers of angiogenesis: one associated with smooth muscle (SMA-red) and the other with basement membrane (green). Comparison of sections revealed a significant increase in SMA / laminin positive bundles in the multi-drug treatment group compared to the device only or no treatment groups (H(19.84)=p<0.001). The number of blood vessels identified in each section was significantly greater in the MDT-treated group compared to both the biodome only and no device groups (H(19.84)=p<0.001). This indicates that MDT facilitates vascularization of regenerates, more than doubling the number of blood vessels in MDT-treated regenerates versus the no device condition.

[0167] 5. Wound closure, increased Sox2 expression, and re-epithelialization Given the significant long-term outcomes, the effect of MDT at early time points after amputation was evaluated early during this process. We first noted that wound closure at 0.5 mpa was significantly reduced in the MDT group compared to animals without the device (p<0.05; FIG. 17). MDT-exposed animals displayed a mean wound diameter of 2.02 cm±0.40 cm, significantly larger than the Biodome group (1.25 cm±0.41 cm) or the No Device group (0.56 cm±0.17 cm). Delayed wound closure was predictive of successful re-patterning at 18 mpa, while animals with the largest wound area at 0.5 mpa showed the greatest growth and re-patterning at 18 mpa.

[0168] Without being bound to a particular theory, it is expected that a larger wound site may provide a larger blastema and contribute more material to the limb regrowth. As shown in FIG. 18, blastema proliferation was assessed 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 that there is more proliferative tissue giving rise to the new limb. And, the soft tissue at 2.5 months was similarly predicted, but the tissue was thicker and bone length increased in the MDT hind limbs compared to the control. Fluorescence images obtained at 2.5 mpa revealed that 24-hour exposure to the MDT composition increased stem cell-likeness and inhibition of wound formation resulted in longer regenerants in the early regeneration process. Increased soft tissue growth and re-epithelialization were also observed in the MDT-exposed group at 2.5 months after amputation, mirroring the increased bone tissue length confirmed by x-ray imaging.

[0169] 6. Transcriptomic Analysis of Induced Regeneration To gain a more careful understanding of gene expression changes in response to acute exposure to MDT treatment, the transcriptional mechanisms downstream of the intervention were characterized. RNA-sequencing (RNASeq) was performed comparing transcriptomes from blastemas obtained from MDT versus non-treated animals at 11 hours, 24 hours, and 7 days after amputation. Heatmaps comparing gene expression levels in MDT animals compared to no device treatment show significant differential gene expression at 11 HPA that persists through 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 p-values ​​by multiple testing. The Q value was set at a false discovery rate (FDR) of 0.05 and differentially expressed genes were considered as those transcripts that passed this FDR and showed a log2 fold change of 2. When comparing the blastema of MDT animals with untreated animals, there was a large dynamic change in expression profile over 7 days after amputation. The same genes overexpressed or underexpressed were allowed to age for 24 hours after switching their activity after 7 days. To narrow in on the dramatically altered expression of these genes, the FDR was set at a log3 or fold change of 3 and the apical 15 differentially expressed genes were compared between groups. When comparing MDT-treated with wild-type blastema tissue, it was found that the apical 15 highly upregulated genes were related to neuromodulation (e.g., brain-specific kinase (BRSK), neuropeptide FF, D1C dopamine receptor, neuroligin) brain, which were most highly expressed at 11 hours after amputation. This expression level decreased at 24 hours and 7 days post-amputation (dpa). Wnt7a, a gene involved in the development of the anterior-posterior axis, was also upregulated at 11 hours post-amputation (hpa) and then increased at 7 days post-amputation. Conversely, the major downregulated genes were primarily related to muscle structure (myosin-4, microfibril-associated glycoprotein) and metabolism (e.g., sarcolipin). The pattern of differentially expressed genes between MDT-treated and control blastemas was the opposite of that of the upregulated genes, in that the downregulated genes increased from 11 hpa to 7 dpa.

[0171] Highly regulated genes in MDT animals were compared to those in animals without the device. Upregulated genes include nervous system specific transcripts suggesting an important role for neuroprotective proteins immediately after amputation. Downregulated genes include metabolic and muscle related transcripts suggesting resources are diverted away from muscle maintenance towards tissue stabilization. GO analysis of metabolic and biosynthetic pathways reveals early downregulation (at 11HPA and 24HPA) with increasing rates at 7dpa. Table 1 shows gene expression levels in MDT animals compared to no device treatment at 11hpa, 24hpa, and 7dpa. [Table 1]

[0172] Enrichment analysis to identify classes of genes showed significant differences in the profiles of biological processes between groups. A web-based version of CEMiTool (Co-expression Module Identification Tool) was used to identify covariant gene sets in MDT (CT) and non-device (ND) animals. Covariant gene sets with high fold changes were classified into modules (M1-M4).

[0173] Early after amputation (11 hpa), there were additional genes involved in metabolic regulation that differed between MDT and controls, and at this time point there is additional dynamic gene expression that resolves the late (7 dpa) and drastic changes between MDT and tissue restructuring or reconfiguration of the cell function landscape.

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

[0175] In general, M2, M3, and M4 were upregulated in MDT vs. ND conditions across all time points, peaking at 24 HPA, except for M1, which was downregulated in MDT conditions, most highly at 11 HPA and decreasing by 7 DPA.

[0176] 7. Cumulative release of drug from provided MDT compositions To assess the cumulative release of multiple drug treatments (MDT) in device hydrogels, hydrogels loaded with each of the MDT drugs were suspended in 1xDPBS (ThermoFisher) and incubated at 37°C (310.15K) for 25 min. Supernatants were collected every 5 min 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 min and not more than 80% of the total amount loaded (except for retinoic acid, which released all of the drug by 25 min).

[0177] Other features, objects, and advantages of the present disclosure will become apparent in the detailed description which follows. It should be understood, however, that the detailed description, while indicating embodiments of the present disclosure, is given by way of illustration only and not by way of 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. 1. A device for stimulation of tissue regeneration at a site in a tissue of a subject, comprising: an outer sleeve having a tissue receiving end, a pusher member receiving end opposite the tissue receiving end, and an interior chamber configured to receive the tissue; A pressing member; an inner sleeve disposed within the outer sleeve, the inner sleeve having an end for receiving the portion of the tissue for regeneration, a mating receiving end opposite the end for receiving the portion of the tissue for regeneration, the mating receiving end for receiving the pressing member, and an internal chamber configured to receive the portion of the tissue for regeneration; the biasing member is configured to extend through the biasing member receiving end into the interior chamber of the outer sleeve and to bias the mating receiving end of the inner sleeve toward the tissue; the inner sleeve, the pressing member placing at least a portion of the tissue site for regeneration in contact with a portion of the interior chamber of the inner sleeve; a first end cap matable with the tissue receiving end of the outer sleeve and including an opening configured to receive the tissue; and an end cap matable with the push member receiving end of the outer sleeve, the end cap being coupled to the push member;

2. The device of claim 1 , wherein the tissue is an appendage or part of an organ.

3. 2. The device of claim 1, further comprising a first threaded adapter installable within the tissue receiving end of the outer sleeve for selectively connecting the first end cap matable with the tissue receiving end to the outer sleeve, the first end cap having a groove configured to receive the threads of the first threaded adapter.

4. 2. The apparatus of claim 1, further comprising a second threaded adapter installable within the push member receiving end of the outer sleeve for selectively connecting the second end cap matable with the push member to the outer sleeve, the second end cap having a groove configured to receive the threads of the second threaded adapter.

5. 2. The apparatus of claim 1, wherein the pressing member includes a seat portion configured to receive the mating receiving end of the inner sleeve or includes a mating end configured to rest in a seat portion of the second end cap.

6. The apparatus of claim 1 , wherein the mating receiving end of the inner sleeve includes a porous filter media sealingly closing the interior chamber at the mating receiving end.

7. The device of claim 6 , wherein the porous filter medium is a synthetic or polymeric membrane.

8. The apparatus of claim 6 , further comprising a compressible member positioned between the porous filter media and the compression member.

9. The device of claim 8 , wherein the compressible member comprises cotton or an encapsulated gel.

10. The device of claim 1 , wherein the inner sleeve comprises a protein or polymeric matrix that at least partially fills the interior chamber of the inner sleeve.

11. The device of claim 10 , wherein the proteinaceous or polymeric matrix comprises a three-dimensional porous scaffold, the porous scaffold comprising pores that form a directional pattern.

12. The device of claim 11 , wherein the porous scaffold comprises aligned pores forming substantially aligned channels, the aligned channels in the protein or polymeric matrix being arranged parallel to the longitudinal axis of the inner sleeve.

13. 13. The device of claim 12, wherein the protein or polymeric matrix is ​​selected from the group consisting of silk fibroin and collagen, or polymers that form aligned pores in a 3D matrix.

14. The device of claim 1 , wherein the pushing member is movable in response to growth of the tissue at the site for regeneration.

15. The device of claim 14 , wherein the biasing member comprises a resilient member that compresses in response to growing tissue.

16. The apparatus of claim 15 , wherein the resilient member comprises a spring.

17. a threaded adapter installable within the push member receiving end of the outer sleeve for selectively connecting the push member matable second end cap to the outer sleeve, the second end cap having a groove configured to receive the threads of the threaded adapter; and An elastic member extending between the seat portion of the screw-type adapter and the seat portion of the pressing member The apparatus of claim 14 further comprising:

18. The device of claim 10 , wherein the proteinaceous or polymeric matrix comprises a therapeutic agent.

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

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

21. The therapeutic composition comprises: Growth factors; Inhibitors of prolyl hydroxylase domain (PHD) enzymes; Vitamin A or its derivatives; and Lipid Mediators 21. The apparatus of claim 20, comprising:

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

24. 22. The device of claim 21, wherein the inhibitor of a 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 chelator, and combinations thereof.

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

26. 26. The device of claim 25, wherein the derivative of vitamin A is selected from the group consisting of retinoic acid, retinol, retinyl carboxylate, tretinoin, tazarotene, and combinations thereof.

27. 22. The device of claim 21, wherein the lipid mediator is selected from the group consisting of resolvins, metabolites of omega-3 fatty acids, derivatives of eicosapentaenoic acid, derivatives of docosahexaenoic acid, and combinations thereof.

28. 22. The device of claim 21, wherein the therapeutic composition comprises a resolvin, the resolvin being 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.

29. 22. The device of claim 21, wherein the therapeutic composition comprises an agent that functions in proximal-distal positioning, the agent being 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.

30. 22. The device of claim 21, wherein the therapeutic composition comprises a peptide or protein hormone.

31. 31. The device of 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. 22. The device of claim 21, wherein the growth factor is present in a dosage within the therapeutic composition of from 0.1 μg / ml to 1 μg / ml.

33. 22. The device of claim 21, wherein the inhibitor of a PHD enzyme is present in a dosage within the therapeutic composition of from 0.004 μg / ml to 0.024 μg / ml.

34. 22. The device of claim 21, wherein the Vitamin A or a derivative thereof is present in a dosage within the therapeutic composition of from 0.03 μg / ml to 0.27 μg / ml.

35. 22. The device of claim 21, wherein the lipid mediator is present in a dosage within the therapeutic composition of from 0.006 μg / ml to 0.054 μg / ml.

36. 31. The device of claim 30, wherein the peptide or protein hormone is present in a dosage within the therapeutic composition of from 0.1 μg / ml to 1.0 μg / ml.

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

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