Tissue regenerative multi-drug

Abstract

Devices, compositions, and methods for promoting regeneration of tissue on a subject, such as a wounded, damaged, or injured appendage, or tissue within a subject, such as a wounded, damaged, or injured organ, are disclosed. The disclosed devices, compositions, and methods include or utilize wearable cannulas and regenerative compositions.

Description

[0001] This application is a divisional application of a Chinese patent application with an application date of December 7, 2020, an application number of 202080093526.2, and an invention title of "Tissue Regeneration Multi-Drug Mixtures and Delivery Devices Thereof".

[0002] Cross - reference to related applications

[0003] This application claims the priority benefit of U.S. Provisional Application Serial No. 62 / 944,707, filed on December 6, 2019, the entire content of which is incorporated herein by reference.

[0004] Statement regarding federally - funded research or development

[0005] This invention was made with government support under grant AR055993 awarded by the National Institutes of Health. The government has certain rights in this invention. Technical Field

[0006] The field of the present invention relates to regenerative medicine. More specifically, the present disclosure relates to devices, compositions, and methods for promoting tissue regeneration on the surface of a subject or within a subject in need thereof. The disclosed devices include wearable sheaths, and the disclosed compositions include regenerative compositions. Background Art

[0007] In the United States alone, the incidence of limb loss in humans is expected to increase significantly over the next 30 years, affecting 3.6 million people per year by 2050 (Ziegler-Graham et al., 2008), leaving diabetics, war veterans, trauma survivors, and those with peripheral artery disease (with limited options in the case of amputation).

[0008] Previous efforts have attempted to induce limb regeneration, including the use of electrical stimulation (Borgens (1982) Science 217,747-750; Leppik et al. (2015) Sci.Rep. ,5,18353; Smith, (1981) Bioelectrochem.andBioenergetics ,8(6),661-670), tissue-guided biomaterials (Suckow et al. (1999) J.ofInvest.Surg. ,12(5),277-287), progenitor cell transplantation (Lin et al., (2013) Dev.Cell ,24(1),41-51) and modulating key molecular pathways (Kawakami et al., (2006) Genes&Dev. ,20(23),3232-3237; Yokoyama et al., (2001)Dev.Biology , 233(1), 72 - 79). However, their success in restoring significant growth and patterning of new limbs has been limited.

[0009] Tissue regeneration involves a series of biological events that are fully combined to reconstruct excised parts or appendages lost during trauma or amputation. There are distinct differences between a typical wound healing response and a regeneration response. Although these two processes are similar in many embodiments, they produce completely different end products. During normal wound healing, many complex biological structures such as sweat glands, ducts, and hair follicles cannot be reconstructed because the biological mechanisms to do so are not available. In a typical adult mammalian skin wound, these structures do not regenerate because the development of these tissues and organs involves highly specific physiological processes. Additionally, normal wound closure and scar formation do not provide an appropriate environment for the regeneration of these structures. On the other hand, epimorphic regeneration is a process in which all original structures are replaced by replicas of the original structures.

[0010] Although some complex animals such as axolotls can naturally regenerate limbs, eyes, and other entire organs throughout their lives, mammals generally exhibit limited regeneration and typically do not exhibit the plasticity and transdifferentiation capabilities of urodeles. When exposed to regeneration inducers delivered by slow - release beads implanted into amputated tissue, post - pubescent anurans can regenerate their truncated or injured limbs. Tissue progenitor cells used to induce the regeneration process may also be useful; larval limb progenitor cells have been shown to activate the same Wnt, Shh signaling to promote patterning.

[0011] However, it is unclear how the fully non - regenerative, highly metamorphosed (adult) African clawed frog (Xenopus) can serve as a useful regeneration animal model because adult frogs cannot regenerate their hindlimbs after amputation but instead produce featureless cartilaginous spikes (Suzuki et al., (2006) Sci.WorldJ. , 6:26 - 37).

[0012] Regeneration sleeves have been used for tissue regeneration of traumatized tissues. Conventional sleeves include septa that allow repeated exchange of media within the wound space, a liquid reservoir to keep the wound moist, and a liquid seal to prevent liquid from leaving the sleeve. However, conventional sleeves are subject to several limitations. In some cases, the liquid seal is too tight and may cause tissue necrosis. Changing the media through a needle may directly affect the wound bed.

[0013] In addition, conventional tissue regeneration devices typically adhere to the subject using glue, which is not conducive to long - term treatment. Using glue to attach the regeneration device has two main drawbacks. Glue lacks the flexibility to adjust the device after attachment. Additionally, there are operational complexities during surgery and device procedures.

[0014] Accordingly, improved devices and regenerative compositions are desirable and needed. SUMMARY OF THE INVENTION

[0015] Devices, compositions, and methods for promoting the regeneration of tissue are disclosed, where the tissue is, for example, a traumatized, damaged, or injured site present on an appendage of a living subject or within the subject, such as a traumatized, damaged, or injured site present on or within an organ. The disclosed devices, compositions, and methods include or utilize a wearable cannula and a regenerative composition.

[0016] In one aspect, the present disclosure provides a therapeutic composition for promoting tissue. In certain embodiments, the regenerative composition can include a plurality of components. In certain embodiments, the disclosed regenerative composition is used as a therapeutic composition in the disclosed device, for example, it is present in the material of the inner cannula that contacts the traumatized, damaged, or injured tissue.

[0017] In one aspect, 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.

[0018] 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 cell line-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).

[0019] In certain embodiments, the therapeutic composition comprises an inhibitor of a PHD enzyme, the inhibitor being selected from 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-pyridylthio)acetyl]-2,4(1H,3H)-pyrimidinedione, 6-amino-1,3-dimethyl-5-[[2-(2-pyridyl)-4-quinolyl]carbonyl]-2,4(1H,3H)-pyrimidinedione, N-[(4-hydroxy-1-methyl-7-phenoxy-3-isoquinolyl)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).

[0020] In certain embodiments, the therapeutic composition comprises a derivative of vitamin A, the derivative being selected from retinoic acid, retinol, retinyl carboxylate, tretinoin, tazarotene, and combinations thereof.

[0021] In certain embodiments, the therapeutic composition comprises a lipid mediator, the lipid mediator being selected from resolvin, metabolites of ω-3 fatty acids, derivatives of eicosapentaenoic acid, derivatives of docosahexaenoic acid, and combinations thereof. In certain embodiments, the lipid mediator is resolvin. In certain embodiments, the therapeutic composition comprises a resolvin selected from resolvin 5, interleukin 6 (IL-6), interleukin 4 (IL-4), tumor necrosis factor-α (TNF-α), nuclear factor kappa-light-chain enhancer of activated B cells (NF-kB), and combinations thereof.

[0022] In certain embodiments, the therapeutic composition comprises an agent that functions in proximal-distal positional information, wherein the agent is selected from bone morphogenetic protein 9 (BMP9), nodal growth differentiation factor, activin, transforming growth factor-β (TGF-β), fibroblast growth factor 8 (FGF8), and combinations thereof.

[0023] In certain embodiments, the therapeutic composition comprises a peptide or protein hormone. In certain embodiments, the therapeutic composition comprises a peptide hormone selected from growth hormone (GH), insulin-like growth factor-1 (IGF-1), transforming growth factor-β-1 (TGFβ-1), epidermal growth factor (EGF), granulocyte-colony stimulating factor (G-CSF), fibroblast growth factor FGF, and combinations thereof.

[0024] In certain embodiments, the growth factor is present in the therapeutic composition at a dose from 0.1 μg / ml to 1 μg / ml. In certain embodiments, the PHD inhibitor is present in the therapeutic composition at a dose from 0.004 μg / ml to 0.024 μg / ml. In certain embodiments, vitamin A or its derivative is present in the therapeutic composition at a dose from 0.03 μg / ml to 0.27 μg / ml. In certain embodiments, the lipid mediator is present in the therapeutic composition at a dose from 0.006 μg / ml to 0.054 μg / ml. In certain embodiments, the peptide or protein hormone is present in the therapeutic composition at a dose from 0.1 μg / ml to 1.0 μg / ml.

[0025] In another aspect, the present disclosure provides a method for promoting tissue regeneration in a mammal, wherein the method comprises administering the provided therapeutic composition to the mammal in an amount sufficient to promote tissue regeneration in the mammal.

[0026] In another aspect, the present disclosure provides the use of the therapeutic composition for stimulating tissue regeneration in a mammal in need thereof.

[0027] In another aspect, the disclosed instrument includes an outer cannula having a tissue receiving and / or insertion tip, a push member receiving tip opposite the tissue receiving tip, and an internal chamber configured to receive and / or contain the tissue. The instrument further includes an inner cannula disposed within the outer cannula, the inner cannula having a tip for receiving traumatized, damaged, or injured tissue or appendage, a engagement receiving tip for engaging a push member, and an internal chamber configured to receive and / or contain traumatized, damaged, or injured tissue. The push member is configured to extend through the push member receiving tip into the internal chamber of the outer cannula and bias the engagement receiving tip of the inner cannula toward the tissue such that at least a portion of the traumatized, damaged, or injured tissue of the subject is placed in contact with a portion of the internal chamber of the inner cannula. The inner cannula may comprise and / or may be formed of a polymeric material, the polymeric material including, but not limited to, a silk hydrogel material. The instrument further includes a first end cap and a second end cap, the first end cap being engagable with the tissue receiving tip of the outer cannula and including an opening configured to receive tissue and maintain the tissue within the device, the second end cap being engagable with the push member receiving tip of the outer cannula.

[0028] In certain embodiments, the tissue is a part of an appendage or an organ.

[0029] In certain embodiments, the instrument includes a first threaded adapter that may be disposed within the tissue receiving tip of the outer cannula for selectively coupling a first end cap engagable with the tissue receiving tip to the outer cannula. In certain embodiments, the first end cap includes a groove configured to receive the threads of the first threaded adapter.

[0030] In certain embodiments, the instrument includes a second threaded adapter that may be disposed within the push member receiving tip of the outer cannula for selectively coupling a second end cap engagable with the push member to the outer cannula. In certain embodiments, the second end cap includes a groove configured to receive the threads of the second threaded adapter.

[0031] In certain embodiments, the push member includes a seat portion configured to receive the engagement receiving tip of the inner cannula or includes an engagement tip configured to be seated on a seat portion of the second end cap.

[0032] In certain embodiments, the engagement receiving tip of the inner cannula includes a porous filter medium that sealingly encapsulates the internal chamber at the engagement receiving tip. In certain embodiments, the porous filter medium is a synthetic or polymeric membrane. In certain embodiments, the instrument includes a compressible member positioned between the porous filter medium and the push member. In certain embodiments, the compressible member comprises cotton or encapsulated gel.

[0033] In some embodiments, the inner cannula comprises a protein or polymer matrix that at least partially fills the internal chamber of the inner cannula. In some embodiments, the protein or polymer matrix comprises a three-dimensional porous scaffold. In some embodiments, the porous scaffold may include pores that form an oriented pattern. In some embodiments, the porous scaffold includes aligned pores that form substantially aligned channels. The aligned channels in the protein or polymer matrix may be arranged parallel to the longitudinal axis of the inner cannula. In some embodiments, the protein or polymer matrix is selected from silk fibroin, collagen, or a combination thereof.

[0034] In some embodiments, the device includes an electrical stimulation device comprising an anode and a cathode, the anode and the cathode being configured to be electrically connected to corresponding terminals of a power source, and a portion of the cathode being disposed within the inner cannula.

[0035] In another aspect, the disclosed device includes a pressing member that is movable in response to growth at a site of traumatized, damaged, or injured tissue, e.g., in an axial direction toward and / or away from the traumatized, damaged, or injured tissue. In some embodiments, the pressing member biases the engagement receiving end of the inner cannula toward the tissue such that at least a portion of the subject's traumatized, damaged, or injured tissue is placed in contact with a portion of the internal chamber of the inner cannula, and as the tissue regenerates and / or the traumatized, damaged, or injured tissue heals, the regenerated tissue and / or the healed tissue axially moves the pressing member away from the trauma to contact the engagement receiving end directly or indirectly.

[0036] In some embodiments, the pressing member is elastic or includes an elastic member that compresses in response to growing tissue. The elastic member may be a spring.

[0037] In some embodiments, the device includes a threaded adapter that may be disposed within the pressing member receiving end of the outer cannula for selectively coupling a second end cap that may engage the pressing member to the outer cannula. The second end cap may include a groove that is configured to receive the threads of the threaded adapter. The elastic member may extend between a seat portion of the threaded adapter and a seat portion of the pressing member.

[0038] In certain embodiments of the disclosed device, the inner cannula of the disclosed device includes a material within a reservoir in the inner cannula that contacts traumatized, damaged, or injured tissue when the traumatized, damaged, or injured tissue is inserted into the device. The material of the inner cannula can include a hydrogel that wets the traumatized, damaged, or injured tissue. The material can additionally include a therapeutic composition and / or an antimicrobial agent that promotes tissue regeneration and / or healing.

[0039] In another aspect, the disclosed device includes an inner cannula that includes a distal end having a first opening and a mating receiving end having a second opening, the first opening sized to receive traumatized, damaged, or injured tissue, the inner cannula defining an internal chamber extending between the first opening and the second opening, the internal chamber sized to receive the traumatized, damaged, or injured tissue of the subject. The device further includes a matrix disposed within the internal chamber of the inner cannula, wherein the matrix includes a porous scaffold having pores that form substantially aligned channels. The matrix can include and / or can be formed from materials including, but not limited to, polymeric materials such as proteins, which can include, but are not limited to, any suitable form of collagen and fibrin. The aligned channels of the porous scaffold can be arranged parallel to the longitudinal axis of the inner cannula. The matrix can include a therapeutic composition such as a regenerative composition or regenerative cocktail disclosed herein.

[0040] In certain embodiments, the substantially aligned channels are substantially parallel or parallel to the longitudinal axis in the inner cannula and orthogonal to the first and second openings. In certain embodiments, the internal chamber includes a reservoir of an aqueous or dispersion medium between the filtration medium and the protein matrix.

[0041] In certain embodiments, the device further includes a filtration medium sized to encapsulate the second opening at the mating receiving end of the inner cannula. In certain embodiments, the pore size of the filtration medium is large enough to allow air to enter the device but small enough to prevent microorganisms from entering the device.

[0042] In certain embodiments, the internal chamber of the inner cannula of the provided device includes the provided therapeutic composition.

[0043] In another aspect, the present disclosure provides a method of promoting tissue regeneration in a mammal. The method includes attaching the provided device to a traumatized appendage or tissue of a mammal.

[0044] In another aspect, the present disclosure provides the use of the device for stimulating tissue regeneration in a mammal in need thereof.

[0045] The foregoing and other embodiments and advantages of the present disclosure will become apparent from the following description. In the description, reference is made to the accompanying drawings, which form a part hereof, and in which is shown, by way of illustration, a non-limiting exemplary embodiment. However, the embodiment does not necessarily represent the full scope of the present disclosure, and thus the scope of the present disclosure is to be construed in light of the entire disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The present disclosure will be described below with reference to the accompanying drawings, in which like reference numerals represent like elements.

[0047] Figure 1 is a schematic side sectional, partially exploded view of an apparatus for stimulating tissue regeneration at a wound site of a subject, according to some embodiments of the present disclosure.

[0048] Figure 2 is a schematic side sectional, partially exploded view of an apparatus for stimulating tissue regeneration at a wound site of a subject, according to some embodiments of the present disclosure.

[0049] Figure 3 is a graphical representation of a mouse toe showing an amputation line.

[0050] Figure 4 is a schematic side sectional view of an apparatus for stimulating tissue regeneration at a wound site of a subject, according to some embodiments of the present disclosure, the apparatus having a movable pressing member.

[0051] Figure 5 is, according to some embodiments of the present disclosure, in a compressed state Figure 4 of a schematic side sectional view of the apparatus.

[0052] Figure 6 is Figure 1 a schematic view of the apparatus, but including an electrical stimulation device according to some embodiments of the present disclosure.

[0053] Figure 7 is a graphical representation of an apparatus installed on a subject, according to some embodiments of the present disclosure Figure 6 of.

[0054] Figure 8A is a SEM image showing a fibrin scaffold having channel-shaped pores aligned along the long axis, according to some embodiments of the present disclosure.

[0055] Figure 8B is a SEM image showing a silk scaffold having pores aligned perpendicular to the long axis, according to some embodiments of the present disclosure.

[0056] Figure 8C It is explained that some embodiments according to the present disclosure have a collagen scaffold with pores aligned along the long axis.

[0057] Figure 8D It is explained that some embodiments according to the present disclosure have a collagen scaffold with pores aligned perpendicular to the long axis.

[0058] Figure 9 Illustration of the right hindlimb length as a function of months post-amputation (mpa) after exposure to one of the following treatment conditions: (i) after an initial 24-hour exposure to the provided multi-drug therapy (MDT) composition, (ii) after a 24-hour exposure to the biodome device, and (iii) no treatment.

[0059] Figure 10 Illustration of the tactile response at 17 mpa, as evaluated by Von Frey Filaments, at the distal tip of the regenerate that has been exposed to one of the following treatment conditions: (i) after an initial 24-hour exposure to the provided multi-drug therapy (MDT) composition, (ii) after a 24-hour exposure to the biodome device, and (iii) no treatment. Mean and SD are presented.

[0060] Figure 11 Illustration of the right hindlimb bone length, as evaluated by micro-CT and X-ray images, as a function of months post-amputation (mpa) after exposure to one of the following treatment conditions: (i) after an initial 24-hour exposure to the provided multi-drug therapy (MDT) composition, (ii) after a 24-hour exposure to the biodome device, and (iii) no treatment.

[0061] Figure 12 Illustration of the right hindlimb bone volume, as evaluated by micro-CT and X-ray images, as a function of months post-amputation after exposure to one of the following treatment conditions: (i) after an initial 24-hour exposure to the provided multi-drug therapy (MDT) composition, (ii) after a 24-hour exposure to the biodome device, and (iii) no treatment.

[0062] Figure 13 Illustration of the number of ATT+ nerve fascicles measured by acetylated α-tubulin (AAT) staining in the right hindlimb after amputation at 18 mpa and after exposure to one of the following initial treatment conditions: (i) an initial 24-hour exposure to the provided multi-drug therapy (MDT) composition, (ii) after a 24-hour exposure to the biodome device, and (iii) no treatment.

[0063] Figure 14Graph of the nerve bundle diameter (mm) measured by acetylated α-tubulin (AAT) staining in the right hindlimb after 18 mPa and after exposure to one of the following initial treatment conditions: (i) initial 24-hour exposure to the provided multi-drug therapy (MDT) composition, (ii) after 24-hour exposure to the biodome device, and (iii) no treatment.

[0064] Figure 15 Graph of the granule complexity (pixels 2 ) evaluated by fibronectin expression after 18 mPa and after exposure to one of the following initial treatment conditions: (i) initial 24-hour exposure to the provided multi-drug therapy (MDT) composition, (ii) after 24-hour exposure to the biodome device, and (iii) no treatment.

[0065] Figure 16 Graph of the number of laminin / smooth muscle actin (SMA+) bundles measured by laminin and SMA+ expression in the regenerates obtained at 18 mPa and after exposure to one of the following initial treatment conditions: (i) initial 24-hour exposure to the provided multi-drug therapy (MDT) composition, (ii) after 24-hour exposure to the biodome device, and (iii) no treatment.

[0066] Figure 17 Graph of the wound closure diameter (cm) obtained at 0.5 mPa and after exposure to one of the following initial treatment conditions: (i) initial 24-hour exposure to the provided multi-drug therapy (MDT) composition, (ii) after 24-hour exposure to the biodome device, and (iii) no treatment.

[0067] Figure 18 Graph of the number of SOX2+ cells in the wound site obtained at 0.5 mPa and after exposure to one of the following initial treatment conditions: (i) initial 24-hour exposure to the provided multi-drug therapy (MDT) composition, (ii) after 24-hour exposure to the biodome device, and (iii) no treatment.

[0068] Figure 19 Graph of the cumulative release of the drug from the provided MDT composition. Detailed Description

[0069] The disclosures of these patents, patent applications, and publications are hereby incorporated by reference in their entirety into this application to more fully describe the state of the prior art known to those skilled in the art as of the date of the invention described and claimed herein. In the event of any inconsistency between the patents, patent applications, and publications and this disclosure, this disclosure shall control.

[0070] Before explaining any embodiments of the present disclosure in detail, it should be understood that the application of the present disclosure is not limited to the details of the construction and arrangement of components set forth in the following description or illustrated in the following drawings. The present disclosure is capable of other embodiments and of being practiced or carried out in various ways.

[0071] It should be understood that the phrases and terms used herein are for the purpose of description and should not be regarded as restrictive. The use of "comprising", "including" or "having" and their variants herein is intended to include the items listed hereinafter and their equivalents as well as additional items. Unless otherwise stated or limited, the terms "mounted", "connected", "supported" and "coupled" and their variants are used broadly and include direct and indirect mounting, connection, support and coupling. Further, "connected" and "coupled" are not limited to physical or mechanical connection or coupling.

[0072] As used herein, the term "tissue" is defined as a collection of similar cells and their extracellular matrix from the same source that together perform a specific function. The "tissue" as used herein can be present on appendages, which include, but are not limited to, phalanges (such as fingers and toes), arms, legs, etc. The "tissue" as used herein can be present on organs (such as the liver, lungs, pancreas, etc.).

[0073] The terms "therapeutic composition", "regenerative composition", "regenerative admixture" and "multi-drug therapeutic compound" or "MDT" include such formulations that contain a combination of therapeutic agents that stimulate or initiate tissue regeneration and, in certain embodiments, have a synergistic effect when administered to a subject.

[0074] The following discussion is presented to enable a person skilled in the art to make and use 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 can 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 embodiments shown, but rather to conform to the broadest scope consistent with the principles and features disclosed herein. The following detailed description will be read with reference to the accompanying drawings, in which like elements in different drawings have the same reference numerals. The drawings, which are not necessarily to scale, depict selected embodiments and are not intended to limit the scope of the embodiments of the present disclosure. Skilled artisans will recognize that the examples provided herein have many useful alternative embodiments and fall within the scope of the embodiments of the present disclosure.

[0075] Devices for tissue regeneration

[0076] The present disclosure provides an instrument for assisting tissue regeneration. Refer toFigures 1 - 3 , shows an instrument 200 for stimulating tissue regeneration in a subject 1. In certain embodiments, the instrument 200 is used to encapsulate traumatized or injured tissue 9 of the subject 1. The traumatized or injured tissue 9 can be located at an external or internal location of the subject 1. In the illustrated embodiment, the instrument 200 is described with respect to stimulating tissue regeneration of traumatized tissue 9 located on an appendage 3 (e.g., a mouse toe), where the tip 2 has been truncated along a line 5 passing through at least a portion of the distal phalanx 4, whereby the regenerated tissue can include bone tissue 6, muscle tissue 7, skin tissue 8, and other tissues. In the description Figures 1 - 7 , for exemplary purposes, "appendage" can be referred to as including exemplary "tissue".

[0077] In certain embodiments, the traumatized or injured tissue 9 for using the instrument 200 to stimulate tissue regeneration includes epithelial tissue, connective tissue, muscle tissue, or nerve tissue. Exemplary traumatized or injured tissue 9 for regeneration includes, but is not limited to, squamous epithelium, cuboidal epithelium, transitional epithelium, pseudostratified columnar epithelium, columnar epithelium, glandular epithelium, bone, tendon, ligament, fat, areolar tissue, blood tissue, visceral muscle, smooth muscle, skeletal muscle, cardiac muscle, and nerve tissue.

[0078] Referring to Figures 1 - 2 , the instrument 100 includes an outer sheath 202 extending between an appendage receiving end 204 and a pressing member receiving end 206. In certain embodiments, the outer sheath 202 is a hollow cylinder defining an internal chamber 208, the size of the internal chamber 208 being designed to receive the tissue 3 (e.g., an appendage) to be treated and the traumatized or injured tissue 9. The internal chamber 208 forms a channel extending between a first opening on the appendage receiving end 204 and a second opening on the pressing member receiving end 206. In certain embodiments, the size of the first opening on the appendage receiving end 204 is designed to receive the appendage 3, and the size of the second opening is designed to receive the pressing member 214.

[0079] The outer sheath 202 can be formed of a transparent material to allow observation of the trauma 9 during use of the instrument 200. In certain embodiments, the outer sheath 202 has sufficient rigidity to prevent any deflection or indentation of the body wall during use to ensure maintenance of the desired trauma space volume and to protect the traumatized or injured tissue 9. Exemplary materials for the construction of the outer sheath 202 include, but are not limited to, transparent nylon tubing. The outer sheath 202 can include one or more openings (not shown) to facilitate replacement of the fluid within the internal chamber 208 of the outer sheath 202. For example, one or more openings can include septa that allow a needle to enter and replace the fluid within the internal chamber 208.

[0080] The instrument 200 includes an inner sleeve 216 that extends between a wound receiving end 218 and an engagement receiving end 220 that is opposite the wound receiving end 218. In certain embodiments, the inner sleeve 216 is a hollow cylinder that defines an internal chamber 222, and the internal chamber 222 is sized to receive the wound 9. The internal chamber 222 forms a passage that extends between a first opening at the wound receiving end 218 and a second opening at the engagement receiving end 220. When the instrument 200 is assembled, the inner sleeve 216 is configured to encapsulate the wound 9, and the pressing member 214 is configured to bias the engagement receiving end 220 toward the appendage 3 such that the wound 9 is placed in contact with at least a portion of the internal chamber 222. In certain embodiments, the pressing member 214 causes the wound 9 to contact a protein matrix 228 disposed within the internal chamber 222 of the inner sleeve 216. The protein matrix 228 can directionally guide tissue growth and / or provide a therapeutic agent to the wound 9 to stimulate tissue regeneration.

[0081] In certain embodiments, the engagement receiving end 220 includes a porous filter medium 230 that seals the second opening of the inner sleeve 216. Incorporating the porous filter medium 230 in the instrument 200 prevents contamination and allows air and media exchange with the surrounding environment. The porous filter medium 230 helps to keep the wound 9 moist and with high cell viability while reducing necrosis. A compressible member or media exchange member 232 can be positioned between the filter medium 230 and the pressing member 214 to provide a reservoir of an aqueous solution or dispersion medium that is in fluid communication with the internal chamber 222. In certain embodiments, the outer sleeve 202 can contain an aqueous solution or dispersion medium that can be placed in fluid communication with the internal chamber 222 of the inner sleeve 216 through the filter medium 230. In certain embodiments, the compressible member or media exchange member 232 comprises a gel or cotton optionally wetted with an aqueous solution or dispersion medium. In certain embodiments, the protein matrix 228 is replaced from the filter medium 230 by the reservoir of the aqueous solution or dispersion medium.

[0082] 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, etc.), and suitable mixtures thereof. In certain embodiments, the dispersion medium includes a therapeutic agent.

[0083] The instrument 200 includes a first end cap 234 that can engage with the appendage receiving end 204 of the outer cannula 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 spacer 238 can be positioned between the first end cap 234 and the appendage receiving end 204 of the outer cannula 202. The gasket or spacer 238 includes a through hole 240 sized to receive the appendage 3 and provide a seal to prevent liquid from escaping from the inner chamber 208 of the outer cannula 202. In some embodiments, the spacer 238 includes a flexible side 242 composed of silicon and a rigid side 244 containing polytetrafluoroethylene (PTFE).

[0084] The instrument 200 can include a first adjustable adapter 246 that can be disposed within the appendage receiving end 204 for selectively coupling the first end cap 234, which can engage with the appendage receiving end 204, to the outer cannula 202. In some embodiments, the adjustable adapter 246 is a threaded adapter and the first end cap 234 includes a groove 248 to receive the threads 250 of the adjustable adapter 246. When assembled, the first end cap 234 can be tightened such that the gasket or spacer 238 is placed in contact with the leading end 252 of the adjustable adapter 246 to secure the appendage 3 within the outer cannula 202.

[0085] The instrument 200 includes a second end cap 254 that can engage with the push member receiving end 206 of the outer cannula 202. The second end cap 254 is coupled to the push member 214 to bias the push member 214 toward the engagement receiving end 220 of the inner cannula 216. Refer to Figure 1 , the push member 214 can be directly attached to the second end cap 254 or form a part of the second end cap 254. Refer to Figure 2 , the push member 214 can be separated from the second end cap 254. In some embodiments, the push member 214 includes an engagement end 256 configured to be seated on the seat portion 258 of the second end cap 254. In some embodiments, the push member 214 includes a seat portion 260 opposite the engagement end 256, which is configured to receive the engagement end 220 of the inner cannula 216. The push member 214 can be formed of a rigid material or an elastic material that deforms or compresses in response to tissue growth at the wound 9.

[0086] The instrument 200 may include a second adjustable adapter 262 that may be disposed within the press member receiving end 206 for selectively coupling a second end cap 254 that may engage the press member receiving end 206 to the outer cannula 202. In certain embodiments, the second adjustable adapter 262 is a threaded adapter and the second end cap 254 includes a groove 264 configured to receive the threads 266 of the second adjustable adapter 262. The second end cap 254 may be adjusted to control the pressure at the wound 9. A disadvantage of conventional devices is the lack of control over the pressure at the wound 9 interface, which can lead to variability in tissue regeneration outcomes if there are any type of gaps (fluid collection, air, etc.). The instrument 200 advantageously provides adjustable pressure sufficient to keep the protein matrix 228 in contact with the wound 9. Additionally, unlike conventional devices, the instrument 200 facilitates long-term attachment (weeks, months, years, or longer) and may be adjusted to promote the growth of regenerated tissue.

[0087] Reference Figures 4 - 5 , in certain embodiments, the instrument 200 includes a movable or adjustable press member 214. For example, the press member 214 may move or extend in response to tissue growth (represented by Δx in Figure 4 and 5 ). In certain embodiments, an elastic member 268 is configured to extend between a seat portion 270 on the press member 214 and a seat portion 272 on the second adjustable adapter 262. In certain embodiments, the elastic member 268 (e.g., a spring, compressible material, deformable material) creates a resistance that causes a backward linear translation (e.g., when unscrewing the second end cap 254). In certain embodiments, the adjustable press member 214 includes an interlocking spacer or screw extension system that may extend over time in response to tissue growth.

[0088] Reference Figures 6 - 7 , in certain embodiments, the instrument 200 also includes an electrical stimulation device 300 to establish a longitudinal electric field across the wound site 9, which is believed to provide an internal wound stump current and provide an electrical guidance signal to innervate and migrate 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 of a power source 306 by leads 308, 310.

[0089] In certain embodiments, the cathode 304 is in the form of a stainless steel wire disposed near the wound 9. A portion of the cathode 304 is disposed outside the instrument 200 and may be connected to the lead 308, and a portion of the cathode 304 is disposed within the inner chamber 222 of the inner cannula. The anode 302 is a wire that can be inserted into the subject 1 at a location remote from the wound site 9. In the illustrated embodiment in which the instrument 200 is disposed on the appendage 3, the anode 302 is disposed at the upper portion of the limb (hind leg) from which the appendage 3 extends. The anode 302 may comprise a platinum / iridium alloy wire, which is connected to the power source 306 via the lead 310. The anode 302 may be permanently implanted or temporarily inserted as needed.

[0090] The power source 306 includes a battery pack 312 and a circuit 314, both of which are encapsulated in a housing 316 and are configured to provide a constant low-level current to the electrodes 302, 304 when connected to the electrodes 302, 304. In Figure 6 the illustrated embodiment, the power source 306 is disposed outside the subject 1, and the electrodes 302, 304 are configured to be detachably connected to the power source 306. In this arrangement, when electrical stimulation is used, the cathode 304 and the anode 302 are electrically connected to the power source 306 during the electrical stimulation treatment and then disconnected between the electrical stimulation treatments. Since the power source 306 and the leads 308, 310 are detachable from the respective electrodes 302, 304, this arrangement conveniently reduces the overall volume of the combined instrument 200 and the electrical stimulation device 300 in a treatment paradigm where electrical stimulation is used only intermittently.

[0091] In certain embodiments, the protein matrix 228 comprises a biocompatible polymer. Biocompatible polymers suitable for use with the instrument 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, polyhydroxyalkanoates, dextran, polyanhydrides, polymers, PLA-PGA, polyanhydrides, polyorthoesters, polycaprolactone, polyfumarates, collagen, silk fibroin, chitosan, alginate, hyaluronic acid, and other biocompatible and / or biodegradable polymers. In certain embodiments, the protein matrix 228 is silk fibroin and / or collagen.

[0092] In certain embodiments, the protein matrix 228 is processed from a silk solution (e.g., an aqueous solution) having a silk solution concentration between about 1% silk and about 50% silk. In certain embodiments, silk fibroin-based materials are processed from silk solutions to form different material forms, such as fibers, foams, particles, films, and / or hydrogels.

[0093] In certain embodiments, the protein matrix 228 is porous or has porosity. The term "porosity" as used herein can represent a measure of void space in a material and is the fraction of the volume of voids to the total volume, expressed as a percentage between 0 and 100%. It is known to those skilled in the art to determine porosity using standard techniques such as mercury porosimetry and gas adsorption (e.g., nitrogen adsorption).

[0094] In certain embodiments, the protein matrix 228 includes pores that match the tissue surface area (size) of the tissue attached thereto in the device 200 to optimize the regrowth of traumatized or injured tissue. In certain embodiments, the protein matrix 228 has a pore diameter in the range 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 diameter, 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 is suitable for supporting sufficient oxygen, nutrient, and waste transport while providing a suitable niche for cell and tissue growth. In other embodiments, smaller pore diameters such as about 50 μm to about 100 μm or about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, or about 100 μm are suitable for smaller tissues such as nerves or blood cells. Higher porosity can promote improved tissue outcomes due to improved nutrient transfer and waste removal.

[0095] The protein matrix 228 can have pores that form an oriented pattern for guiding tissue growth at the wound 9. In certain embodiments, the oriented pattern includes aligned pores that form substantially aligned channels. The aligned pores can be arranged parallel to the longitudinal axis of the inner cannula 216. In certain 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) with a steep temperature gradient, which is induced by combining the conductive substrate with a cold source (e.g., liquid nitrogen). It is considered that finger-like ice crystal columns growing from the cold surface create channel-like structures within the frozen protein. The frozen protein is then lyophilized under low pressure for a period of time (e.g., 24 hours) to remove water. The resulting product is the protein matrix 228 with substantially aligned pores.

[0096] In some aspects, the protein matrix comprises fibroin. As used herein, "fibroin" or "SF" can refer to biopolymers produced from silk fibroin and insect or spider silk proteins. For example, fibroin that can be used in the present disclosure can be fibroin produced by many species, 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; 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 can be prepared by artificial methods, e.g., involving genetic engineering of cells or organisms (e.g., genetically engineered bacteria, yeast, mammalian cells, non-human organisms, including animals or transgenic plants).

[0097] Like collagen, SF is a structural protein but has unique properties: it is produced by a living complex organism extruding an amino acid solution into the external environment, while collagen is produced in vivo by the self-assembly of monomers produced by cells in the extracellular space and is not secreted into the external environment. The properties of SF stem from its structure, which consists of hydrophobic blocks interleaved with hydrophilic acidic spacers. In its natural state, SF is organized into a semi-crystalline material where β-sheet crystals alternate with amorphous regions, providing strength and elasticity to the proteinaceous material formed by the protein. The multiple forms of regenerated SF that can be processed at low to high protein concentrations and low to high molecular weights make it attractive for several high-tech applications.

[0098] The processing of SF typically involves partial or complete dehydration of a fibroin solution (protein content of about 1 wt% to about 15 wt%) using numerous techniques such as solvent casting, freeze drying, salt leaching, sonication to form, for example, films, sponges, gels, spheres (micron to nano scale), and foams. These fabrication processes provide robust materials that combine mechanical strength with biochemical properties.

[0099] The fibroin solutions used in the methods and compositions provided herein can be obtained from solutions containing dissolved silk (e.g., from Bombyx mori). Alternatively, the fibroin solution can be obtained from a solution containing dissolved spider silk (e.g., from Nephila clavipes). The fibroin solution can also be obtained from a solution containing genetically engineered silk (e.g., from bacteria, yeast, mammalian cells, transgenic animals or transgenic plants). See, e.g., WO 97 / 08315 and U.S. Patent 5,245,012. Genetically engineered silk can also contain, for example, therapeutic agents such as fusion proteins with cytokines, enzymes or any number of hormone or peptide-based drugs, antimicrobials and related substrates.

[0100] Fibroin solutions can be prepared by any conventional method known to those skilled in the art. In certain embodiments, the silk solution is an aqueous silk solution. In other embodiments, the silk solution can contain a second polymer to facilitate the transition to the solid state (e.g., polyethylene glycol, collagen, hyaluronic acid, etc.).

[0101] Cocoon silk contains two structural proteins: fibroin heavy chain (about 350 kDa); and fibroin light chain (about 25 kDa), which are associated with a family of non-structural proteins called sericin, which glue the fibroin chains together to form the cocoon. The heavy and light fibroin chains are linked by disulfide bonds at the C-terminus of the two subunits (see Takei, et al., J.CellBiol ., 105:175, 1987; also see Tanaka, et al.,. J. Biochem. 114:1, 1993; Tanaka, et al., Biochim.Biophys.Acta. , 1432:92, 1999; Kikuchi, et al., Gene, 110:151, 1992). Sericin is the high molecular weight soluble glycoprotein component of silk, which imparts stickiness to the material. These glycoproteins are hydrophilic and can be easily removed from the cocoon by "degumming" in boiling water).

[0102] In certain embodiments, the silk polypeptide composition utilized in the compositions of the present invention is substantially free of sericin (e.g., contains no detectable sericin, or contains a level of sericin that is considered negligible by one of ordinary skill in the relevant art for a particular use).

[0103] In an exemplary method of obtaining a silk polypeptide composition, Bombyx mori cocoons are boiled in an aqueous solution (such as, but not limited to, about 0.02M Na2CO3) for about 30 minutes. The boiling (degumming) time is in the range of about 5 minutes to about 120 minutes, and the boiling (degumming) temperature is in the range of about 30°C to about 120°C. The cocoons can be rinsed, for example, with water to extract sericin, and the extracted silk is dissolved in a saline solution. Exemplary non-limiting salts for this purpose include lithium bromide, lithium thiocyanate, calcium nitrate, and other chemicals capable of dissolving silk. For example, the extracted silk is dissolved in a solution of about 9M to about 12M LiBr. The salt is then removed, for example, by dialysis.

[0104] If desired, the solution can then be concentrated using any method known in the art. For example, dialysis can be performed against a hygroscopic polymer (such as, PEG, polyethylene oxide, amylose, or sericin). The PEG has a molecular weight of about 8,000 g / mol to about 10,000 g / mol and has a concentration of about 25% to about 50%. Any dialysis system can be used, for example, a slide-a-lyzer dialysis cassette (Pierce, MW CO 3500). The solution is dialyzed for a sufficient period of time to produce a final concentration of an aqueous silk solution between about 1% and about 30%. In some cases, dialysis for about 2 hours to about 12 hours is sufficient.

[0105] In certain embodiments, the present disclosure provides a method of attaching an apparatus 200 to an appendage or tissue of a subject in need of tissue regeneration. The method includes contacting a traumatized appendage or tissue 9 of the subject 1 with a trauma-receiving end 218 of an inner cannula 216. The traumatized appendage or tissue 9 can be placed in contact with or adjacent to a protein matrix 228 containing the provided therapeutic composition. In certain embodiments, before contacting the traumatized appendage or tissue 9 with the protein matrix 228, the traumatized appendage or tissue 9 is slid through an opening 236 of a washer or spacer 238 and a first end cap 234.

[0106] The method further includes placing the appendage 3 of the subject through the appendage receiving end 204 of the outer cannula 202 such that the inner cannula 216 is positioned within the inner chamber 208 of the outer cannula 202. In certain embodiments, the method includes selectively engaging the first end cap 234 and the second end cap 254 to the outer cannula 202 such that the pressing member 214 biases the engagement receiving end 220 towards the appendage 3. In certain embodiments, the method includes biasing the pressing member 214 towards the appendage 3 such that the wound 9 is placed in contact with at least a portion of the inner chamber 222 of the inner cannula. The contact pressure between the traumatized appendage or tissue 9 and the protein matrix 228 can be adjusted by selectively engaging or disengaging the second end cap 254 (e.g., tightening or loosening the second end cap 254 via the groove 264 and the threads 266).

[0107] In certain embodiments, the traumatized appendage or tissue 9 is maintained within the instrument 200 for a period of time to promote tissue regeneration. In certain embodiments, the duration is about 1 minute, or about 10 minutes, or about 30 minutes, or about 1 hour, or about 2 hours, or about 3 hours, or about 4 hours, or about 5 hours, or about 6 hours, or about 12 hours, or about 24 hours, or 2 days, or about 3 days, or about 4 days, or about 5 days, or about 1 week, or about 2 weeks, or about 3 weeks, or about 1 month, or about 6 months, or about 1 year, or within a duration range defined by any one of these values. During the duration, the protein matrix 228 can be kept moist by adding or replacing the buffer solution within the inner cannula 216.

[0108] Therapeutic compositions for tissue regeneration

[0109] Also disclosed herein are therapeutic compositions and "multi-drug therapy" compositions (MDT) for tissue regeneration. The therapeutic compositions can be used alone or in combination with the disclosed instruments.

[0110] The therapeutic composition used with the device according to the present invention can be any composition that stimulates, initiates, or directly or indirectly aids in tissue regeneration. Alternatively, the therapeutic composition according to the present invention can be a combination of components that act synergistically to stimulate or initiate or directly or indirectly aid in tissue regeneration. For example, the provided components in the therapeutic composition (e.g., two or more components such as growth factors, inhibitors of prolyl hydroxylase domain (PHD) enzymes, vitamin A or its derivatives, lipid mediators, or peptide / protein hormones) act synergistically to increase the rate of regeneration at the traumatized appendage or tissue site relative to untreated control experiments (e.g., increased tissue regeneration as measured by soft tissue length, bone length, bone volume, increased tactile response, number of ATT+ nerve bundles, diameter of ATT+ nerve bundles, complexity of regenerative granulation as assessed by fibronectin expression, number of laminin / SMA+ bundles, decreased wound diameter at the start of treatment, number of SOX2+ cells).

[0111] In certain embodiments, at least two components in the provided therapeutic composition act synergistically to increase the rate of regeneration, or at least three components, or at least four components, or at least five components, or all components act synergistically to increase the rate of regeneration.

[0112] In one embodiment, the disclosed device can contain a therapeutic composition within an inner cannula (e.g., within a reservoir in the inner cannula). The therapeutic composition can be present in a material or matrix that contacts the wound site, and when the device is worn on a subject's appendage, the therapeutic composition can be delivered to the wound site. Wearable devices that contain and deliver therapeutic compositions are known in the art. (See, e.g., Herrera-Rincon et al., CellReports 25, 1593 - 1609 (2018).

[0113] The disclosed therapeutic composition can 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 can be prepared as follows. The therapeutic composition can be added to a silk solution (e.g., a 3% w / v silk solution), and then its gelation can be induced by adding reagents such as horseradish peroxidase (e.g., to a concentration of about 20 U / ml of the silk solution) and hydrogen peroxide (e.g., to a concentration of 0.01% w / v). As some of many options, silk can also be gelled by this enzymatic reaction, by lowering the pH, adding energy (such as by sonication or vortexing), applying an electric field, or adding methanol.

[0114] The disclosed therapeutic compositions can comprise one or more agents that increase axonal / neurite growth and / or general cell proliferation. Preferably, the disclosed therapeutic compositions do not promote pluripotency in cells and / or result in teratoma formation.

[0115] The disclosed therapeutic compositions can comprise one or more agents that promote tissue regeneration and / or healing. In certain embodiments, the therapeutic composition comprises one or more of the following: growth factors, agents that inhibit inhibitors of hypoxia-inducible factor 1-alpha (HIF1-α), vitamin A or its derivatives, lipid mediators such as metabolites of omega-3 fatty acids, and can be derived from eicosapentaenoic acid or docosahexaenoic acid, growth hormone, steroids, and depolarizing agents.

[0116] In certain embodiments, the disclosed therapeutic compositions can include growth factors such as neurotrophic factors. Neurotrophic factors are proteins that promote the growth and survival of nerve cells during development and promote the maintenance of adult nerve cells (see, e.g., Terenghi, J.Anat . 1999;194(Pt 1):1-14. Exemplary neurotrophic factors include, but are not limited to, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), and combinations thereof. The growth factor can be present in the therapeutic composition at a dose of 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 in a dose range defined by any one of these values. When the growth factor is present in a component of the disclosed device (e.g., when the growth factor is loaded in the inner cannula or a component of the inner cannula), the device can comprise 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 a growth factor concentration in a concentration range defined by any one of these values. The growth factor promotes the growth of one or more tissue types.

[0117] The disclosed therapeutic compositions can include prolyl hydroxylase domain (PHD) enzyme inhibitors (i.e., PHD inhibitors), e.g., to stabilize the constitutive expression of HIF-1α protein (see, e.g., Ariazi et al., J.Pharmacol.Expt.Therap.(2017), 363(3) 336 - 347; and Nangaku et al., Arterioscler., Thromb. Vas.Biol . 2007; 27: 2548 - 2554). Suitable PHD inhibitors can 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 - pyridylthio)acetyl] - 2,4(1H,3H) - pyrimidinedione (i.e., TM6089), 6 - amino - 1,3 - dimethyl - 5 - [[2 - (2 - pyridyl) - 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 can be present in the therapeutic composition at a dose of 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 dose range defined by any one of these values. When the PHD inhibitor is present in a component of the disclosed device (e.g., when the PHD inhibitor is loaded in the inner cannula or a component of the inner cannula), the device can contain 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 a PHD inhibitor concentration within a concentration range defined by any one of these values. The PHD inhibitor controls excessive collagen deposition at the wound site.

[0118] The disclosed compositions can include vitamin A or its metabolites or derivatives or any agent that functions in proximal-distal positional information. Exemplary derivatives of vitamin A include, but are not limited to, retinoic acid, retinol, retinyl carboxylates (e.g., retinyl acetate, retinyl propionate, and retinyl palmitate), tazarotene, and combinations thereof. Agents that can function in proximal-distal positional information include, but are not limited to, bone morphogenetic protein 9 (BMP9), nodal growth differentiation factor (i.e., HTX5 or NODAL), activin, transforming growth factor-β (TGF-β), and fibroblast growth factor 8 (FGF8). Vitamin A or its metabolites or derivatives or any agent that functions in proximal-distal positional information can be present in the therapeutic composition at a dose 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 defined by any of these values. When vitamin A or its derivatives (or an agent that functions in proximal-distal positional information) is present in a component of the disclosed device (e.g., when vitamin A or its derivatives or an agent that functions in proximal-distal positional information is loaded in the inner cannula or a component of the inner cannula), the device can contain 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 a concentration of vitamin A or its derivatives or an agent that functions in proximal-distal positional information within a concentration range defined by any of these values.

[0119] The disclosed therapeutic compositions can include lipid mediators and / or omega-3 fatty acid metabolites (i.e., anti-inflammatory agents) that promote the resolution of the inflammatory response. Suitable lipid mediators can include derivatives (e.g., metabolites) of omega-3 fatty acids that promote the resolution of the inflammatory response (i.e., are anti-inflammatory), and / or derivatives of eicosapentaenoic acid or docosahexaenoic acid. Exemplary lipid mediators can include, but are not limited to, resolvins such as resolvin D5, interleukin 6 (IL-6), interleukin 4 (IL-4), tumor necrosis factor-alpha (TNF-α), nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB), and combinations thereof. Optionally, the lipid mediator can be present in the therapeutic composition at a dose 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 dose range defined by any one of these values. When the lipid mediator is present in a component of the disclosed device (e.g., when the lipid mediator is loaded in the inner cannula or a component of the inner cannula), the device can contain 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 a lipid mediator concentration within a concentration range defined by any one of these values.

[0120] The disclosed therapeutic compositions can include peptide hormones, e.g., 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.Invest.115(8):2083-98. Exemplary hormone peptides or proteins include, but are not limited to, growth hormone (GH), insulin-like growth factor-1 (IGF-1), transforming growth factor-β-1 (TGFβ-1), epidermal growth factor (EGF), granulocyte-colony stimulating factor (G-CSF), and fibroblast growth factor FGF. The growth hormone or steroid can be present in the therapeutic composition at a dose of 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 defined by any one of these values. When the growth hormone or steroid is present in a component of the disclosed device (e.g., when the growth hormone or steroid is loaded in the inner cannula or a component of the inner cannula), the device can contain 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 growth hormone or steroid concentration within a concentration range defined by any one of these values.

[0121] The disclosed therapeutic composition can include a depolarizing agent. Suitable depolarizing agents can include, but are not limited to, ionophores (e.g., ion channel openers or blockers). Suitable depolarizing agents can include, but are not limited to, monensin, potassium gluconate, sodium gluconate, etc.

[0122] The disclosed therapeutic composition can be used to treat a subject in need of treatment. As used herein, "subject" refers to a human or an animal. Generally, the animal is a vertebrate such as a primate, rodent, domestic animal, or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., rhesus monkeys. Rodents include mice, rats, groundhogs, ferrets, rabbits, and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species (e.g., domestic cats), canine species (e.g., dogs, foxes, wolves), avian species (e.g., chickens, emus, ostriches), and fish (e.g., trout, catfish, and salmon). In certain embodiments of the aspects described herein, the subject is a mammal, e.g., a primate, e.g., a human. The subject can be male or female. Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be used as subjects in animal models representing tissue repair, regeneration, and / or reconstruction. In addition, the methods and compositions described herein can be used to treat domesticated animals and / or pets.

[0123] In certain embodiments, the disclosed therapeutic compositions can be used to treat a traumatized or injured appendage or tissue of a subject in need of stimulating tissue regeneration. The tissue or appendage can be internal or external to the subject. Exemplary traumatized or injured tissues in need of regeneration in a subject include, but are not limited to, squamous epithelium, cuboidal epithelium, transitional epithelium, pseudostratified columnar epithelium, columnar epithelium, glandular epithelium, bone, tendon, ligament, fat, areolar tissue, blood tissue, visceral muscle, smooth muscle, skeletal muscle, cardiac muscle, and nerve tissue.

[0124] In certain embodiments, the present disclosure provides a method of administering to a subject an effective amount of the disclosed therapeutic compound (comprising the disclosed compound) to regenerate at least a portion of a traumatized or injured appendage or tissue. In certain embodiments, the method comprises contacting the traumatized or injured appendage or tissue with a therapeutic compound, which may or may not be present in a provided hydrogel. In certain embodiments, the provided therapeutic compound or the provided hydrogel contacts the traumatized or injured appendage or tissue within an instrument provided herein.

[0125] The disclosed instrument and / or therapeutic composition promotes tissue regeneration. Tissue regeneration can be measured by any method known in the art, such as, but not limited to, measuring the expression of Yamanaka factors, Sox2, Oct3 / 4, Klf4, and / or c-Myc in a tissue treated with an instrument and / or therapeutic composition according to the present disclosure relative to a tissue not treated with the disclosed instrument and / or therapeutic composition.

[0126] Animal model tests

[0127] When exposed to a regeneration inducer delivered by slow-release beads implanted into amputated tissue, an anuran that has matured to puberty can regenerate its truncated or injured limb. However, a completely non-regenerative, highly metamorphosed (adult) African clawed frog cannot regenerate its hindlimb after amputation, but instead produces featureless cartilaginous spikes (Suzuki et al., (2006) TheScientificWorldJOURNAL,6 .). This model is used to test whether a regeneration inducer can stimulate regeneration.

[0128] As described in the following examples, complex interventions for hindlimb amputation in adult African clawed frogs were tested to address several aspects of limb regeneration. A wearable bioreactor ("BioDome") was used to achieve control of the local microenvironment of the in vivo wound. A mechanism was sought whereby a brief exposure phase to a regenerative cocktail would initiate a long endogenous morphogenetic cascade without continuous microscopic management. A variety of stimuli were selected that induced pro-regenerative activity, such as agents that reduce inflammation, promote neural sparing, and induce overall growth.

[0129] It was found that a brief exposure phase (e.g., 24 hours) to the wearable bioreactor induced significant growth halos, patterning, and sensorimotor function after amputation in African clawed frogs, the bioreactor containing filaments infused with several small molecule compounds. Treated animals showed a marked delay in wound closure, followed by long-term (about 16 months) growth outcomes, including increased bone length, soft tissue patterning, and neuromuscular repair. Histologically, the new limb contained nerves, smooth muscle indicative of blood vessels, and reorganization of extracellular matrix proteins involved in limb remodeling. Transcriptomic analysis identified immediate and short-term pathways and transcriptional targets of blastema intervention. RNA-seq assays also revealed a rapid response in the brain to the intact treatment device (compared to sham controls). The regenerated bone exhibited anatomical features characteristic of wild-type morphology, and the distal limb soft tissue showed digit-like protrusions. In addition, the animals used the newly formed limb to walk, similar to wild-type frogs. Furthermore, in animals exposed to the fully treated condition, sensorimotor pathways were restored, indicating that tissue remodeling included re-extension or regrowth of sensory afferent nerves and the neuromuscular tissue interface.

[0130] These data confirm that adult African clawed frogs can be induced to produce a very significant, long-lasting regenerative response by brief triggering, without gene therapy or stem cell implantation, and reveal the molecular, cellular, and tissue-level components of this process occurring at the wound site and distally in the brain.

[0131] The following examples elaborate on ways in which the present disclosure can be used or implemented and will make it easier for those of ordinary skill in the art to understand its principles. The following examples are presented by way of illustration and are not meant to be limiting in any way.

[0132] Examples

[0133] Animals

[0134] Adult female African clawed frogs (Xenopus laevis) (n = 115) measuring 5 cm to 6.25 cm from nose to tail (Nasco, Fort Atkinson, WI) were allowed to acclimate to the holding tanks for 2 weeks prior to the experiment. The animals were maintained at 18 °C in 10 L plastic tanks containing a defined frog water (Reef Salt, Seachem Laboratories, conductivity of approximately 1.65 kΩ, pH 7.8 - 8.0) and exposed to a 12-hour light-dark cycle. Prior to the experiment, the animals were immersed in a broad-spectrum gentamicin antibiotic for 2 hours (Gibco, Fisher Scientific, USA) to minimize bacterial contamination of the stump after amputation.

[0135] Limb amputation

[0136] According to Golding et al. (2016), PLoSOne11 , e0155618 and Herrera et al. (2018), CellRep25 , 1593-+ a previously established protocol was used for hindlimb amputation surgery. Briefly, the animals were first anesthetized by immersing them entirely in buffered frog water containing 0.05% benzocaine. After the toe pinch reflex disappeared, 75 mg / kg of buprenorphine was injected subcutaneously just below the lateral line on the contralateral side of the leg to be amputated. The right hindlimb was amputated at the midpoint of the tibiofibula using a sterile microsurgical blade with a straight incision. No bone resection was performed, and no tissue flap was sutured at the wound site. After hemostasis, the animals regained consciousness and were allowed to recover in sterile frog water for at least 60 minutes.

[0137] Device attachment

[0138] The animals were randomly assigned to one of three treatment conditions: no device, BioDome only, or BioDome with a mixture treatment (described in "BioDome Fabrication and Mixture Composition" below). Prior to the device attachment procedure was a second anesthesia (0.05% benzocaine soak, 75 mg / kg buprenorphine). Then the unconscious animals were equipped with the device, which was attached to the stump of the amputation site using monofilament surgical sutures (7-0 Monosof, 18” P-16 cutting, Covidien, USA). Two sutures were passed through the dermis layer on either side of the leg. These sutures were sufficient to keep the device in place and did not damage the underlying deep fascia layer. After attachment, the animals were returned to their holding tanks, where they regained consciousness and were allowed to swim freely. Control animals were treated similarly to animals receiving the device, but no device was attached to the traumatic stump. The results presented herein were generated using sutured biodomes.

[0139] Alternatively, an adjustable biodome can be used. The attachment procedure for the adjustable BioDome involves pushing the transected limb through the cap and the annular septum into place and securing the limb using the bioadhesive Skin-Tite Bioadhesive, Smooth-On, PA). The biodome insert containing the scaffold is attached to the transected toe and secured with a small amount of adhesive. The acrylic protective cap is screwed onto the top cap, and then the insert is pushed close to the wound bed using a custom washer and sealed with a second cap. To keep the tissue moist during attachment, approximately 4 μL of sterile phosphate-buffered saline (PBS) 1X is slowly injected into the insert using a 31G needle. The PBS is changed every 2 days to remove cellular waste and keep the tissue fresh. The device is kept attached until the animal is sacrificed for subsequent analysis.

[0140] BioDome manufacturing

[0141] The sutured BioDome contains a soft silicone insert that in turn contains silk hydrogel as a controlled-release matrix and drug carrier. The fabrication of the device has been reported elsewhere (Golding et al., (2016) PLoSOne11 , e0155618). Briefly, the outer cylindrical silicone sleeve (20-mm H x 18-mm D) is fabricated by casting silicone elastomer (Dragon skin 10, Smooth-on, Macungie, PA) onto a 3D printed mold designed using CAD software (Solidworks, Waltham, MA, USA) and printed using a Formlab 3D printer (Somerville, MA, USA).

[0142] The adjustable biodome consists of acrylic tubes (#8532K13, Mcmaster-Carr, Elmhurst, IL) cut into 1-cm long cylindrical tubes, which are used as the body of the device 100. The threaded adapter was designed using 3-D 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 super glue. 2-ml HPLC vial caps (Agilent, (Santa Clara, CA). The PTFE / silicone septum was punched with a 3-mm biopsy punch to create an entry hole for the animal limb. To provide more space for the animal's leg to pass through the hole, the septum was cut at four positions along the central hole, each quarter circle apart. Custom washers were either made from PDMS using soft lithography or 3D printed using a 3D printer. The cylindrical wall of the device 100 insert was made from a transparent 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 rolled around a 1.5-mm diameter metal rod (#8907K62, McMaster). The wall was then glued to the silicone bottom using a silicone adhesive (Dragon Skin 10 FAST, Smoothon, Macungie, PA) to complete the insert.

[0143] The protection cage is assembled from a threaded cap, a transparent acrylic body, and an adapter. An annular septum that allows only one-way bending is provided to prevent the device from detaching due to animal movement and tampering. The custom washer, together with the bottom cap, provides adjustable pressure sufficient to hold the stent insert tightly against the wound bed and maintain its position stable during long-term experiments. It also has an entry hole for media exchange. The device insert contains a membranous sidewall and a silicone bottom. The sidewall can hold the liquid required to keep the tissue moist and promote gas exchange, and the silicone bottom acts as a septum for inserting a needle for media exchange.

[0144] Device Disassembly and Maintenance

[0145] After 24 h, the animals were anesthetized and treated with analgesics as described previously. The device was then removed by cutting a single suture on either side of the leg, and the frogs were returned to a tank containing fungicide (Kordon methylene blue, at a concentration of 1 mL / 10 L frog water). After an additional 24 h, the water was replaced with fresh 100% frog water. Once their devices were removed, the animals were maintained in frog water that was changed daily for 18 months. Terminal euthanasia was performed by whole-body immersion in frog water containing 0.2% benzocaine. The regenerates, contralateral limbs, and brain tissues were collected and processed for histological analysis.

[0146] Silk processing

[0147] A fibrinogen solution was prepared as follows: 5 g of silkworm (Bombyx mori) cocoons (Tajima Shoji, Yokohama, Japan) were cut and degummed in a 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 in deionized (DI) water to remove the Na2CO3 and then dried overnight at 22 °C in a fume hood. The dry 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 to 60 °C for 4 h. The solution was then dialyzed in deionized water using a dialysis cassette (molecular weight cut-off of 3.5 kDa, Thermo Fisher Scientific, Waltham, MA) with gentle stirring. The water was changed six times during the 48-h period. The dialyzed solution was centrifuged three times at 13,000 g and 4 °C for 20 min each time and then filtered through a cell strainer (40-μm pore size, Thermo Fisher) to remove impurities. To determine the concentration of the filtered solution, 0.5 ml of the sample was completely dried in an oven overnight. After the water had evaporated, the dried silk was weighed, and the concentration in % (wt / v) was calculated as the ratio of the weight of the dried silk to its initial volume of 0.5 ml.

[0148] A silk hydrogel was formed by crosslinking liquid fibrinogen. A 45-mb silk (3% w / v) and horseradish peroxidase (HRP) solution (20 U / ml) were poured into a 24-well plate and incubated at 37 °C for 45 min to complete gelation. According to Golding et al. (2016), PLoSOne11 , the gel compression strength and modulus of the gel were tested by the method of e0155618.

[0149] Scaffolds with alignment holes

[0150] A silk scaffold with aligned pores was fabricated using 5 μl of a 4% (wt / v) silk solution. The solution was placed on top of an aluminum plate, and a steep temperature gradient was induced by plunging the plate into liquid nitrogen (LN2). Finger-like ice crystal columns growing from the cold surface established a channel-like structure within the silk solution as it solidified. After cooling for 10 minutes, the frozen solution was lyophilized under low pressure for more than 24 hours to remove water. The sponge was trimmed to fit as an insert into the Biodome, sterilized in ethylene oxide, and stored at room temperature under aseptic conditions until use.

[0151] A collagen scaffold with an aligned channel-like porous structure was fabricated by controlled directional freezing and freeze-drying of a 1.5% (wt / wt) collagen solution (in a manner similar to the silk scaffold). The scaffold was cut into a cylindrical shape (4-mm long and 1.5-mm diameter) and placed inside the biodome insert using forceps.

[0152] Material characterization

[0153] The morphology of the scaffolds was characterized using scanning electron microscopy (SEM) and fluorescence optical microscopy. For SEM imaging, the scaffolds were cut in half using a razor blade 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. Figure 8D is a collagen scaffold with pores aligned perpendicular to the long axis.

[0154] Therapeutic composition

[0155] Hydrogels were prepared using a final concentration of 3% (w / v) silk solution, 20 U / ml horseradish peroxidase (HRP), and 0.01% wt / v hydrogen peroxide (H2O2). The liquid solution was poured into a silicone sleeve and allowed to gel for 30 minutes, then attached to the animal's limb stump. For the device loaded with the mixture, 0.014 μg / ml of 1,4(dihydrophenanthroline-4-one-3-carboxylic acid) DPCA (Catalog No. 71220, Caymen Chemicals, MI, USA), 0.5 μg / ml of brain-derived neurotrophic factor (BDNF) (Catalog No. 450-02, Peprotech, MA, USA), 0.5 μg / ml of growth hormone (GH) (Catalog No. 100-40, Peprotech, MA, USA), 0.036 μg / ml of resolvin D5 (Catalog No. 10007280, Caymen Chemicals, MI, USA), 0.015 μg / ml of retinoic acid (Catalog No. 11017, Caymen Chemicals, MI, USA) were loaded into the liquid silk solution, then inserted into the silicone sleeve and gelled.

[0156] In vitro release studies.

[0157] To determine the release profiles 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 for 45 min to complete gelling. Then, 1 ml of Dulbecco's phosphate-buffered saline (DPBS 1X, Gibco) was added to each vial and then incubated at 37 °C. At fixed time points, 300 μl of supernatant was collected for analysis. The release solution of the drug-free silk hydrogel was used as a control. The standard curve was 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 measured on a UV-transparent 96-well plate (Corning, Corning, NY) using a microplate reader SpectraMax M2 (Molecular Devices, San Jose, CA) run by SoftMax Pro 6 software. The 1,4-DPCA and RA solutions were detected at wavelengths of 280 nm and 350 nm, respectively.

[0158] The concentrations of BDNF and GH in the released samples were determined using an enzyme-linked immunosorbent assay (ELISA) kit containing monoclonal antibodies designed for BDNF and GH (#BGK23560 and #BGK01241, Peprotech, Rocky Hill, NJ, USA). Sample preparation and measurement were performed according to the manufacturer's protocol. The optical density of the prepared samples was read at 450 nm using a SpectraMax M2 plate reader. The released samples of resolvin D5 were filtered through a protein filtration column (MWCO = 3 kDa, #UFC500324, Fisher) to remove high-molecular-weight fibroin contents. Then, the optical density of the samples was measured at a wavelength of 244 nm using a SpectraMax M2 plate reader.

[0159] Soft tissue imaging

[0160] The soft tissue remodeling and bone regrowth of the animals were regularly evaluated during the 18-month maintenance period. The animals were anesthetized as described previously, and high-resolution images of their wound sites and the dimensions of the regenerates were captured using a DSLR camera (Canon EOS Rebel T7i). To ensure reproducibility, the amputation plane was used as the standard reference point for all measurements. The amputation site was easily identifiable as the limb tapered 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.

[0161] In vivo, X - ray and micro - CT bone imaging

[0162] In addition to soft tissue measurements, according to Golding et al. (2016) PLoSOne11 , e0155618 and Herrera-Rincon, et al., (2018) CellRep 25, 1593-+, the bone length was evaluated using a handheld x-ray device (Nomad Pro2TM) with standard imaging settings of 60 kV, 2.5 mA, and an exposure time of 0.20 s. Each animal received the same dose (0.12 mSv) at fixed time points. Computed tomography (CT) was performed in a viva CT 40 scanner (Scanco Medical, Switzerland) to visualize the detailed microstructure of the bone at the end of the 18-month regeneration phase after euthanasia with an overdose of benzocaine (0.2% systemic immersion). Distal trabecular bone and midshaft cortical bone slices (615 slices / animal, 76 μm / slice, integration time of 300 ms) were visualized and presented as 3-D images for further quantification. The radiation dose was in accordance with the established manufacturer's guidelines using the local CT dose index (CTDI), ranging from 453 mGy to 1255 mGy.

[0163] Histology and immunohistochemistry

[0164] To characterize the effects of treatment on limb regeneration and remodeling after amputation, histological analyses of the regenerates and contralateral limbs were performed at fixed intervals over time. Tissues were harvested at 18 mpa. Long-term regenerated tissues were fixed overnight in 4% paraformaldehyde (PFA) in PBS and decalcified for 2 weeks by exposure to increasing concentrations (10%-15%) of ethylenediaminetetraacetic acid (EDTA) (pH 7.4). Once decalcified (confirmed using x-rays), tissues were gradually equilibrated to 30% sucrose and then embedded in OCT (Sakura FInetek, USA). Samples were frozen in liquid nitrogen. Limb tissues were sectioned serially at 14 μm using a cryostat (Leica CM1850) and placed on slides. Transverse sections were taken across the limb at 14-μm intervals from the tibiofibular region above the original amputation site to the remodeling region at the limb tip. To visualize remodeling, horizontal sections were taken at 14-μm intervals of the distal portion of the limb. Sections were dried for at least 1 hour before storage at -80 °C.

[0165] For immunohistochemistry, slides were equilibrated to 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 containing 0.1% Triton X-100 and 10% normal goat serum) for 1 hour. Primary antibodies against acetylated α-tubulin (1:100), TGF-β (1:250), smooth muscle actin (1:100), laminin (1:100), fibronectin (1:500), and phosphorylated histone H3 (1:250) were used. Except for anti-smooth muscle actin and anti-laminin (which were stained together), slides were stained individually with each antibody. 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 minutes. Slides were mounted in Fluoromount-G (ThermoFisher Scientific) and cured for at least 24 hours before imaging.

[0166] Sections were imaged using an EVOS FL automated imaging system (ThermoFisher Scientific). All sections were collected and stitched together for analysis.

[0167] Analysis of immunostained sections

[0168] All statistical analyses were performed in IBM SPSS version 20. The normality assumption was tested before using parametric tests (including ANOVA, t-tests, and correlation analysis (Pearson’s r)). Nonparametric analyses, Mann Whitney Wilcoxon tests, or Kruskal-Wallis tests were performed to compare data with non-normal distributions. A significant difference was assumed if the p-value was below the threshold of 0.05 (two-tailed hypothesis testing).

[0169] Assessment of sensorimotor thresholds

[0170] To evaluate the sensorimotor ability of the regenerates, the animals were evaluated 18 months after amputation. Each animal was placed in a glass jar containing 2 L of frog water and allowed to acclimate for 5 minutes until movement had completely ceased. A camera (iPod Touch 5th generation, Apple, CA, USA) was placed above the chamber to capture a record of the test procedure. Standardized von Frey filaments (Touch Test, Stoelting, IL, USA) were used to evaluate the sensory threshold of the regenerates. Filaments in the force range of 0.008 g to 300 g were applied to the distal portion of the regenerate, with the force starting from the lowest to the highest. The first filament that elicited a clear response (movement from a stationary position) was recorded. The animals were tested twice within a 2-day period, and the mean threshold was reported.

[0171] Statistical analysis

[0172] All statistical analyses were performed using IMB SPSS v20. First, the homogeneity of variance of the data was tested by Levene's test. Univariate analysis of normally distributed data was performed by unpaired two-tailed student's t-tests (for two independent groups) or one-way ANOVA tests (for multiple independent groups) and subsequent post hoc Scheffe's tests (when P < 0.05). When considering the variable "time", two-way analysis was performed by two-way ANOVA. Student's t-tests were used to determine the statistical significance between treatment groups (no device, Biodome only, and mixture treatment) at each specific time point. In non-normally distributed data, Kruskal-Wallis tests and subsequent post hoc Dunn tests (when P < 0.05) were performed separately. In all cases, the significance level was set at 0.05. Statistical values were reported as mean ± standard deviation or mean ± standard error of the mean, where indicated. Dot plots or scatter plots were used, where appropriate, to highlight the individual variability within each experimental group.

[0173] RNA extraction

[0174] After amputation, device attachment and removal, and 24-hour treatment, regenerated tissue was 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 snap-frozen. Tissue was extracted using TRIzol (ThermoFisher Scientific) according to the manufacturer's protocol, and total RNA quality and quantity were evaluated using a Nanodrop spectrophotometer (ThermoFisher Scientific).

[0175] Next - generation sequencing (NGS)

[0176] 1.1 μg of total RNA was sent to the Tufts Genomic Core. RNA quality was evaluated by a Bioanalyzer, and high-quality RNA was used for library preparation using the TruSeq Stranded RNA Library Preparation Kit with RiboZero Gold (Invitrogen). The libraries were then multiplexed and single-end 50-nt sequencing was performed on an Illumina HiSeq 2500. The raw read files were sent to the Bioinformatics and Biostatistics core at Joslin Diabetes Center.

[0177] NGS analysis

[0178] The reference genome of Xenopus laevis was downloaded from the assembly GCA_001663975.1 of the NCBI Genomic Database. 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.) and the aligned reads were counted using featureCounts (Liao et al., (2014) Bioinform. 30(7):923-30). Genes with expression counts of more than one count per million (cpm) in at least 3 samples were included in the analysis, and the counts were normalized by the trimmed mean of M-values (TMM, Robinson et al., (2010) GenomeBiol .11, R25.). A Voom transformation was performed (Law et al., GenomeBiol.15, R29), to transform the counts into logCPM, where CPM = 1e+6 * gene count / (total count of the sample * normalization factor of the sample). The voom transformation also estimates the mean-variance relationship and uses it to calculate appropriate observation-level weights, such that greater read depth gives greater weights. To further downweight outliers, sample-specific quality weights are collected (Ritchie et al., (2006) BMC Bioinform .7, 261.) and combined with the observation-level weights.

[0179] Differentially expressed genes are identified using limma (Ritchie et al., Nucl.AcidsRes .43, e47.). Moderated t-tests are performed to detect differentially expressed genes between two groups. Genes with FDR < 0.25 are considered to have significant changes.

[0180] Gene sets for pathway analysis are obtained from MSigDB Collections, and gene sets belonging to canonical pathways (CP) or gene ontology (GO) are selected. Analyses are performed using the Fry function in the Rotation Gene Set Test (Roast) in the limma R package (Wu et al., (2010) Bioinform .26, 2176 - 2182.). Gene sets that are coordinately up, coordinately down, and mixed are considered significant if P < 0.05 and FDR < 0.05.

[0181] Network analysis

[0182] Gene modules were identified by co-expression analysis in CeMiTool in R (Russo et al., 2018) based on the logCPM values of the control and mixture treatment groups at 11 hours, 24 hours, and 7 days post-amputation. In CeMiTool, variance stabilizing transformation was applied to remove the dependence between the mean and variance parameters, and genes were filtered based on the expression level at a threshold of p < 0.1. Modules of co-expressed genes were identified in the dataset based on the automatically generated scaling value β (β = 10) and a minimum module size of 30 genes. To evaluate how the richness of these modules varied over time and groups, module richness was calculated according to the sample annotation. To determine which biological functions were associated with each module, overrepresentation analysis was performed using the pathway database from Reactome, and pathways were considered significant at p < 0.05. Annotated module graphs combined gene-gene interaction data from the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Chemical and Genetic Perturbations (CGP) to plot the interacting genes included in each module.

[0183] qPCR method

[0184] The same total RNA submitted for RNAseq analysis was treated with DNase using the RQ1 RNase-Free DNase Kit (Promega Corp., Madison, WI, USA). The resulting RNA (0.5 μg) was treated with DNase a second time and then used for cDNA synthesis using the Verso cDNA Synthesis Kit (ThermoFisher Scientific). Quantitative analysis of the amount of gene product was performed using the Step OnePlus Real Time System (Applied Biosystems, USA). Each 10 μl reaction was run in duplicate and contained: 5 μl of 2xPowerUp 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 cDNA template. Relative expression was analyzed using the δ-δCt method, where the mean of all ND expression across time points was used as the calibrator for all samples.

[0185] Results

[0186] 1. Inducing leg regeneration by multi-drug regeneration treatment

[0187] Adult African toads were amputated at the midpoint of the tibia and fibula of the hindlimbs and equipped with a biodome device that contained a silk-based hydrogel with 5-drug multi-drug therapy (MDT) or hydrogel only. Control animals were amputated and untreated. After 24 h of exposure to the BioDome, the device was removed, and the animals were maintained for up to 18 months, and the regeneration and remodelling of the hindlimb regenerates were evaluated regularly. The extended observation window was chosen based on the calculations of Alibardi (2018), which were based on a projection model using the diameter of the salamander stump to predict that it would take approximately 1.5 years to regenerate an anuran limb.

[0188] X-ray images were taken to measure limb length as a function of months post-amputation. The X-rays showed that at 4 months, the regenerates associated with the biodome and multi-drug therapy (MDT) conditions only were longer than the control (no device) regenerates, an effect that disappeared after 4 months and then reappeared at 8 months and persisted over time (two-way ANOVA, between-subjects factor treatment exposure, within-subjects factor regeneration time F(2,19) = 61.9, p < 0.05). As early as 0.5 months post-amputation (mpa) and continuing until 4 mpa, the limb lengths (relative to the amputation site) of the MDT group were greater than those of the other groups (p < 0.05), suggesting that MDT increased the early growth rate. Between 6 - 8 months, growth slowed significantly, and the hindlimb regenerates associated with the biodome-only group reached lengths comparable to those of animals exposed to MDT (p > 0.05). The secondary increase in late growth after 9 mpa showed that the MDT group again had longer limbs relative to the other groups, an effect that persisted until the final measurement at 18 mpa (p < 0.01). Thus, compared to the other treatment groups, the MDT group not only showed a final increase in leg length but also a secondary growth phase that was absent in the other treatment groups, especially the no-device group. These data suggest that brief exposure to MDT and the biodome contributed to longer leg regrowth.

[0189] Not only was there an increase in leg length, but 76% of the animals exposed to MDT also showed a thicker and more complex regenerate morphology compared to the featureless, heavily pigmented spikes of the no-device group. Specifically, the distal segments of the hindlimb regenerates associated with the MDT group presented a flattened paddle-like structure with toe-specific protruding buds. In contrast, the no-device and biodome-only conditions were only associated with featureless regenerate spikes, and the MDT condition provided reliably produced a formed paddle-like morphology with distal buds. The biodome group had an intermediate phenotype, where 20% of the biodome animals showed thicker cartilaginous spikes and hooked distal protrusions but limited formation. None of the animals in the no-device group showed a distinct phenotype.

[0190] 2. The induced legs exhibited sensory function

[0191] Next, to evaluate whether the 18 mpa regenerates had regained sensory function, a sensorimotor assessment was performed. Using standardized von Frey (VF) filaments (minimum force: 0.008 g; maximum force: 300 g), the regenerated right hindlimb was probed with filaments of increasing strength at the most distal end until the maximum force was applied. The first filament that elicited a clear response (movement from the rest position) was recorded, and the behavioral determination was averaged over 2 days. The MDT-treated hindlimb regenerates showed a stimulus-response pattern comparable to that of the non-amputated group (p > 0.01; Figure 10 ), indicating significant nerve reinnervation and neuromuscular reintegration. In the biodome group, the responses were significantly different among the individual animals, with one group showing near-normal levels and the other group showing no detectable response to the application of forces of any value up to and including 300 g, while the untreated animals reliably failed to show any response to the application of forces of any value up to and including 300 g. Therefore, we conclude that MDT treatment promoted sensorimotor integration similar to that of unamputated limbs. Intriguingly, the biodome group alone was sometimes also able to promote the regrowth of limbs with an intact sensory system, suggesting that hydration and structural support may be important for maintaining innervation and promoting nerve regrowth.

[0192] 3. MDT exposure increases hindlimb bone length and complexity

[0193] Micro-CT and X-ray images were used to characterize the internal structure of the regenerates. Since the treatment significantly improved the total hindlimb length ( Figure 9 ), the bone components of the regenerates were subsequently studied. Micro-CT and X-ray images of the regenerates showed an increase in bone volume and length associated with the MDT composition provided, compared to biodome alone and control.

[0194] X-ray images confirmed the presence of complex morphology and increased bone length in the MDT regenerates relative to biodome alone and device-free control experiments. Dense tissue protruding outward from the amputation site was observed in the MDT and biodome groups starting from the measurements at 2.5 mpa, but not in the device-free control group. The MDT group also showed dense segmented bone fragments at the distal end of the regenerates. As predicted, the MDT condition showed increased bone length relative to the other conditions, starting from 4 mpa (p < 0.05), with an inflection point at approximately 8 mpa (similar to the soft tissue measurements). As shown in Figure 11 , the differences in bone length between the MDT and biodome groups were observed regularly, but the length differences between those groups and the device-free control were consistently maintained (p < 0.05).

[0195] To better understand the microstructure of regenerated bone, micro-CT was performed at 18 mpa. Micro-CT imaging allows visualization of 3D rendering of the underlying bone and measurement of bone volume without disturbing external soft tissues. Micro-CT data confirmed the presence of significant bone fragments in the distal region of the MDT hindlimb. Notably, the fragments do not occur at the amputation site but spontaneously appear at skeletal points equivalent to the contralateral joint after a period of growth. These reformed segments are common in the MDT group. As shown in Figure 12 With respect to the hindlimb regenerates of the MDT group, the bone volume measured by micro-CT data was greater relative to other groups. Volume quantification confirmed increased growth in the provided MDT regenerates (one-way ANOVA (F(2,15) = 11.15, p < 0.001)). Micro-CT images also showed significant similarities between the non-amputated and MDT-treated skeletal anatomies. Remodeling was evident in the provided MDT regenerates, including re-expression of bone features typically associated with muscle attachment. Specifically, at 17 mpa, the MDT group showed bone features characteristic of muscle attachment and the presence of joint segments. These data suggest that the MDT condition is associated with significant remodeling of the skeletal anatomy, consistent with an active process of remodeling the bone and ultimately achieving morphological complexity approaching that of the pre-amputation limb.

[0196] 4. Complexity of the regenerated limb

[0197] Although the MDT-treated animals did show increased bone growth, molecular changes at the cellular level in the regenerated limb were also evaluated. Immunohistochemistry was performed to assess tissue structures associated with regeneration and remodeling at 18 mpa. As shown in Figure 13 With respect to nerve reinnervation measured by acetylated α-tubulin (AAT) staining, the number of AAT+ nerve bundles associated with the 24-hour MDT condition was significantly increased compared to the no-device group (U = 13, P = 0.0014). When comparing the no-device and biodome-only groups, the number of AAT bundles was comparable, indicating that MDT treatment affects nerve bundle regrowth and innervation in the regenerates. Not only were there more bundles, but the 24-hour MDT group also showed a significantly greater AAT bundle diameter relative to the no-device group, as shown in Figure 14 There was no significant increase in bundle size between the biodome-only and no-device groups.

[0198] The Kruskal-Wallis test indicated that the number of AAT-positive bundles at 18 mPa was significantly different under different conditions, H(15.12) = p < 0.005. Mann-Whitney U post hoc analysis revealed that the main source of variance was the increase in AAT bundles associated with the 24-hour MDT condition compared to non-injured animals, U = 13, P = 0.0014. When comparing non-injured animals and the biodome-only group, the number of AAT bundles was comparable (p = 0.1812). Next, we examined the ATT bundle size and revealed significant differences under different conditions, H(11.74) = p = 0.0028. The 24-hour MDT group showed a larger ATT bundle diameter relative to non-injured animals. No significant differences were identified when comparing the biodome-only and non-injured animal groups.

[0199] To evaluate the changes in connective tissue structure, we evaluated the fibronectin expression pattern after 18 mPa. As shown in Figure 15 the increased granule complexity in the MDT-treated regenerates reflected the degree of structural complexity observed under the MDT condition. Specifically, fluorescence images revealed increased nerve regeneration and extracellular matrix remodeling in the regenerates 18 months after initial amputation and exposure to multi-drug treatment. Consistent with other results, the 24-hour MDT group showed increased granule complexity relative to the biodome-only and device-free groups, suggesting that MDT itself may contribute to the refinement of cartilage and connective tissue in the regenerates.

[0200] To evaluate angiogenesis in the regenerates, laminin and smooth muscle actin (SMA) expression at 18 mPa was compared between different conditions, as shown in Figure 16 The cross-sections of the regenerates obtained 18 months after amputation were double-stained for two markers of angiogenesis, one marker related to smooth muscle (SMA - red) and the other marker related to the basement membrane (green). Comparison of the sections revealed a significant increase in SMA / laminin-positive bundles in the multi-drug treatment group compared to the device-only or untreated groups (H(19.84) = p < 0.001). The number of blood vessels identified in each section was significantly greater in the MDT-treated group compared to the biodome-only and device-free groups (H(19.84) = p < 0.001), indicating that MDT promoted angiogenesis in the regenerates, with more than twice the number of blood vessels in the MDT-treated regenerates compared to the device-free condition.

[0201] 5. Wound Closure, Increased Sox2 Expression, and Re-epithelialization

[0202] Considering the significant long-term outcomes, the impact of MDT at early time points post-amputation was evaluated during the early stages of the process. We first noted that compared to device-free animals, wound closure was significantly reduced at 0.5 mPa in the MDT group (p<0.05; Figure 17 ). Animals exposed to MDT showed an average wound diameter of 2.02 cm ± 0.40 cm, significantly larger than the biodome group (1.25 cm ± 0.41 cm) or the device-free group (0.56 cm ± 0.17 cm). Delayed wound closure predicted successful remodeling at 18 mPa, with animals having the largest wound sites at 0.5 mPa showing the most growth and remodeling at 18 mPa.

[0203] Without being limited to a particular theory, it is considered that a larger wound site will provide a larger blastema, which may contribute more material for limb regrowth. As shown in Figure 18 , blastema proliferation was evaluated by immunohistochemistry of SOX2, a proliferative cell marker. Significantly more SOX2-expressing cells were present in the MDT group compared to the other groups (p<0.05), indicating that more proliferative tissue gives rise to a new limb. Finally, the soft tissues at 2.5 months were similarly predictive compared to the control group, with the tissues of the MDT hindlimbs being thicker and the bone length increased. Fluorescent images obtained at 2.5 mPa revealed that a 24-hour exposure to the MDT composition increased the dryness, and the inhibition of wound formation led to longer regenerates during the early regeneration process. Increased soft tissue growth and re-epithelialization were also observed at 2.5 months post-amputation in the MDT-exposed group, which reflected an increase in bone tissue length confirmed by x-ray imaging.

[0204] 6. Transcriptome analysis of regeneration induction

[0205] To more closely understand the 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, which compared the transcriptomes of blastemas obtained from MDT at 11 hours, 24 hours, and 7 days post-amputation relative to untreated animals. A heatmap comparing the gene expression levels of MDT animals with device-free treatment showed significant differences in gene expression at 11 hpa, which persisted until 24 hpa. However, the dynamic gene expression levels returned to normal by 7 dpa, indicating a phase of dynamic gene expression within 24 hours of amputation.

[0206] After multiple testing corrections of the p-value, the number of differentially expressed genes was determined. The Q-value was set at a false discovery rate (FDR) of 0.05, and differentially expressed genes were considered those transcripts passing this FDR and those transcripts showing a log2 fold change of 2. When comparing the blastemas of MDT animals with untreated animals, there were large dynamic changes in the expression profiles within 7 days after amputation. After the activity switch at 7 days, the same genes that were overexpressed or underexpressed underwent 24 hours. To narrow down the significantly altered expression of these genes, the FDR was set at a log3 or 3-fold change, and the top 15 differentially expressed genes were compared between groups. When comparing the MDT-treated blastema tissue with wild-type blastema tissue, the top 15 highly upregulated genes were related to neuromodulation in the brain (e.g., brain-specific kinase (BRSK), neuropeptide FF, D1C dopamine receptor, neuroligin), which had the highest expression 11 hours after amputation. This expression level decreased at 24 hours and 7 days (dpa) after amputation. Wnt7a, a gene involved in anteroposterior axis development, was also upregulated 11 hours (hpa) after amputation and then increased at 7 days after amputation. In contrast, the major downregulated genes were mainly related to muscle structure (myosin-4, microfibril-associated glycoprotein) and metabolism (e.g., myolipin). The pattern of these differentially expressed genes between the MDT-treated blastema and the control blastema was opposite to the pattern of upregulated genes, as the expression of downregulated genes increased from 11 hpa to 7 dpa.

[0207] Highly regulated genes in MDT animals were compared with genes in sham animals. Upregulated genes included nervous system-specific transcripts, which suggested an important role of neuroprotective proteins immediately after amputation. Downregulated genes included metabolism- and muscle-related transcripts, thus suggesting that resources were shifting from muscle maintenance to tissue stabilization. GO analysis of metabolic and biosynthetic pathways revealed early downregulation (at 11 HPA and 24 HPA), the rate of which increased at 7 dpa. Table 1 explains the gene expression levels of MDT animals compared with sham treatment at 11 hpa, 24 hpa, and 7 dpa.

[0208] Table 1

[0209]

[0210]

[0211] Enrichment analysis of identified gene classes showed significant differences in the profiles of biological processes between groups. The network-based CEMiTool (co-expression module identification tool) version was used to identify co-varying gene sets in MDT (CT) and sham (ND) animals. Co-varying gene sets with high fold changes were classified into modules (M1 - M4).

[0212] In the early stage after amputation (11 hpa), more genes were involved in metabolic regulation, which was different between MDT and the control. Additionally, there was more dynamic gene expression at this time point. This would subside later (7 dpa), along with significant changes between MDT and the regenerated tissue or the reset cell function landscape.

[0213] To reveal the types of processes regulated by MDT exposure, "large-scale functions" were considered to group the enriched pathways. Co-expression analysis identified 4 gene modules across the control group and the mixture treatment group at 11 hours, 24 hours, and 7 days after amputation. These modules represent gene classifications based on their shared expression levels and statistical significance. Module 1 contains 607 genes that were significantly present (p < 0.00509) in extracellular matrix organization, collagen formation, collagen biosynthesis and modification enzymes, and the hemostasis pathway, and they may be representative of tissue destruction after injury. Module 2 contains 142 genes that were significantly present (p < 0.00009) within pathways related to cell junction organization, laminin interaction, cell-cell communication, apoptotic cleavage of cell adhesion proteins, and integrin-free membrane ECM interaction, and they may be representative of cell-cell communication and adhesion. The 105 genes in Module 3 were significantly present (p < 0.01096) in muscle contraction, acetylcholine activity, and myogenesis pathways. Module 4 contains 54 genes that were significantly (p < 0.00142) overrepresented in glucose metabolism, muscle contraction, gluconeogenesis, and glycolysis pathways. When Modules 2, 3, and 4 were significantly enriched (p < 0.00024) and upregulated in the MDT-treated blastemas at all time points, it indicated that frogs receiving the pro-regenerative mixture had a sustained upregulation of genes related to cell communication, myogenesis, and glucose metabolism compared to the biodome-only and device-free controls. In contrast, in the group without the treatment device at all time points evaluated, these modules were enriched (p < 0.012) but downregulated. However, at the 7-day time point, Module 2 was significantly upregulated (p = 0.00074) in the same untreated group, indicating that there was little difference in the richness of the cell-cell communication pathway between the treated and untreated groups 1 week after amputation. At all time points, the ECM- and collagen-enriched Module 1 was upregulated (p < 0.011) in the untreated group and downregulated (p < 0.00027) in the untreated samples.

[0214] Generally, compared to the ND condition at all time points, M2, M3, and M4 were upregulated under the MDT condition and peaked at 24 HPA. The exception was M1, which was downregulated under the MDT condition, reached the highest at 11 HPA, and decreased until 7 DPA.

[0215] 7. Cumulative Release of Drugs in the Provided MDT Compositions

[0216] To evaluate the cumulative release of multi-drug therapy (MDT) in the device hydrogels, the hydrogels loaded with each MDT drug were suspended in 1x DPBS (ThermoFisher) and incubated at 37 °C for 25 minutes. Supernatants were collected every 5 minutes, and the concentration of each drug was determined by a microplate reader. Each drug / hydrogel mixture exhibited similar release kinetics, releasing approximately 70% of the total drug concentration within 10 minutes and no more than 80% of the total load (except for retinoic acid, which released all of its drug before 25 minutes).

[0217] Other features, objects, and advantages of the present disclosure will be apparent from the following detailed description. However, it should be understood that the detailed description, while indicating embodiments of the present disclosure, is given by way of illustration only and not 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.

[0218] Embodiments of the present invention also include:

[0219] Embodiment 1. An instrument for stimulating tissue regeneration at a tissue site of a subject, the instrument comprising:

[0220] An outer cannula having a tissue receiving end, a pressing member receiving end opposite the tissue receiving end, and an internal chamber configured to receive tissue;

[0221] A pressing member;

[0222] An inner cannula disposed within the outer cannula, the inner cannula having: an end for receiving a regenerating tissue site, a mating receiving end opposite the end for receiving the tissue site for regeneration and configured to engage the pressing member, and an internal chamber configured to receive the tissue site for regeneration,

[0223] wherein the pressing member is configured to extend through the pressing member receiving end into the internal chamber of the outer cannula and bias the mating receiving end of the inner cannula towards the tissue such that at least a portion of the tissue site for regeneration is placed in contact with a portion of the internal chamber of the inner cannula;

[0224] A first end cap that can engage the tissue receiving end of the outer cannula and includes an opening configured to receive the tissue; and

[0225] A second end cap that can engage the pressing member receiving end of the outer cannula.

[0226] Embodiment 2. The device according to Embodiment 1, wherein the tissue is part of an appendage or an organ.

[0227] Embodiment 3. The device according to Embodiment 1, the device further comprising a first threaded adapter that can be disposed within the tissue receiving end of the outer cannula for selectively coupling a first end cap that can engage the tissue receiving end to the outer cannula, the first end cap having a groove configured to receive the threads of the first threaded adapter.

[0228] Embodiment 4. The device according to Embodiment 1, the device further comprising a second threaded adapter that can be disposed within the pressing member receiving end of the outer cannula for selectively coupling a second end cap that can engage the pressing member to the outer cannula, the second end cap having a groove configured to receive the threads of the second threaded adapter.

[0229] Embodiment 5. The device according to Embodiment 1, wherein the pressing member includes a seat portion configured to receive the engaging receiving end of the inner cannula, or includes an engaging end configured to be disposed on the seat portion of the second end cap.

[0230] Embodiment 6. The device according to Embodiment 1, wherein the engaging receiving end of the inner cannula includes a porous filter medium that hermetically encapsulates an internal chamber on the engaging receiving end.

[0231] Embodiment 7. The device according to Embodiment 1, wherein the porous filter medium is a synthetic or polymeric membrane.

[0232] Embodiment 8. The device according to Embodiment 1, the device further comprising a compressible member positioned between the porous filter medium and the pressing member.

[0233] Embodiment 9. The device according to Embodiment 1, wherein the compressible member comprises cotton or encapsulated gel.

[0234] Embodiment 10. The device according to Embodiment 1, wherein the inner cannula comprises a protein or polymer matrix that at least partially fills the internal chamber of the inner cannula.

[0235] Embodiment 11. The device according to Embodiment 10, wherein the protein or polymer matrix comprises a three-dimensional porous scaffold, wherein the porous scaffold includes pores forming an oriented pattern.

[0236] Embodiment 12. The device according to Embodiment 11, wherein the porous scaffold comprises aligned pores that form substantially aligned channels, and wherein the aligned channels in the protein or polymer matrix are arranged parallel to the longitudinal axis of the inner cannula.

[0237] Embodiment 13. The device according to Embodiment 12, wherein the protein or polymer matrix is selected from fibrin and collagen, or a polymer that forms aligned pores in a 3-D matrix.

[0238] Embodiment 14. The device according to Embodiment 12, wherein the protein or polymer matrix contains a therapeutic agent.

[0239] Embodiment 15. The device according to Embodiment 1, the device further comprising an electrical stimulation device comprising an anode and a cathode, the anode and the cathode being configured to be electrically connected to corresponding terminals of a power source, and a portion of the cathode being disposed within the inner cannula.

[0240] Embodiment 16. The device according to Embodiment 1, wherein the inner chamber of the inner cannula further contains a therapeutic composition.

[0241] Embodiment 17. The device according to Embodiment 16, wherein the therapeutic composition comprises:

[0242] Growth factors;

[0243] Inhibitors of prolyl hydroxylase domain (PHD) enzymes;

[0244] Vitamin A or its derivatives; and

[0245] Lipid mediators.

[0246] Embodiment 18. The device according to Embodiment 17, wherein the therapeutic composition contains a growth factor selected from brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), and leukemia inhibitory factor (LIF), and combinations thereof.

[0247] Embodiment 19. The device according to Embodiment 18, wherein the therapeutic composition contains BDNF.

[0248] Embodiment 20. The device according to Embodiment 17, wherein the therapeutic composition comprises an inhibitor of PHD enzyme, and the inhibitor is selected from 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-pyridylthio)acetyl]-2,4(1H,3H)-pyrimidinedione, 6-amino-1,3-dimethyl-5-[[2-(2-pyridyl)-4-quinolyl]carbonyl]-2,4(1H,3H)-pyrimidinedione, N-[(4-hydroxy-1-methyl-7-phenoxy-3-isoquinolyl)carbonyl]-glycine, an iron chelator, and combinations thereof.

[0249] Embodiment 21. The device according to Embodiment 20, wherein the therapeutic composition comprises 4,4α-dihydro-4-oxo-1,10-phenanthroline-3-carboxylic acid (1,4-DPCA).

[0250] Embodiment 22. The device according to Embodiment 17, wherein the therapeutic composition comprises a derivative of vitamin A, and the derivative is selected from retinoic acid, retinol, retinyl carboxylate, tretinoin, tazarotene, and combinations thereof.

[0251] Embodiment 23. The device according to Embodiment 22, wherein the therapeutic composition comprises a lipid mediator, and the lipid mediator is selected from resolvins, metabolites of ω-3 fatty acids, derivatives of eicosapentaenoic acid, derivatives of docosahexaenoic acid, and combinations thereof.

[0252] Embodiment 24. The device according to Embodiment 23, wherein the therapeutic composition comprises a resolvin, and the resolvin is selected from resolvin 5, interleukin 6 (IL-6), interleukin 4 (IL-4), tumor necrosis factor-α (TNF-α), nuclear factor κ-light chain enhancer of activated B cells (NF-kB), and combinations thereof.

[0253] Embodiment 25. The device according to Embodiment 17, wherein the therapeutic composition comprises an agent that functions in proximal-distal positional information, and the agent is selected from bone morphogenetic protein 9 (BMP9), nodal growth differentiation factor, activin, transforming growth factor-β (TGF-β), fibroblast growth factor 8 (FGF8), and combinations thereof.

[0254] Embodiment 26. The device according to Embodiment 17, wherein the therapeutic composition comprises a peptide or protein hormone.

[0255] Embodiment 27. The device according to embodiment 26, wherein the therapeutic composition comprises a peptide hormone selected from growth hormone (GH), insulin-like growth factor-1 (IGF-1), transforming growth factor-β-1 (TGFβ-1), epidermal growth factor (EGF), granulocyte-colony stimulating factor (G-CSF), fibroblast growth factor FGF, and combinations thereof.

[0256] Embodiment 28. The device according to embodiment 17, wherein the growth factor is present in the therapeutic composition at a dose from 0.1 μg / ml to 1 μg / ml.

[0257] Embodiment 29. The device according to embodiment 18, wherein the inhibitor of the PHD enzyme is present in the therapeutic composition at a dose from 0.004 μg / ml to 0.024 μg / ml.

[0258] Embodiment 30. The device according to embodiment 18, wherein the vitamin A or its derivative is present in the therapeutic composition at a dose from 0.03 μg / ml to 0.27 μg / ml.

[0259] Embodiment 31. The device according to embodiment 18, wherein the lipid mediator is present in the therapeutic composition at a dose from 0.006 μg / ml to 0.054 μg / ml.

[0260] Embodiment 32. The device according to embodiment 18, wherein the peptide or protein hormone is present in the therapeutic composition at a dose from 0.1 μg / ml to 1.0 μg / ml.

[0261] Embodiment 33. The device according to embodiment 1, wherein the pressing member is movable in response to growth at the tissue site for regeneration.

[0262] Embodiment 34. The device according to embodiment 33, wherein the tissue is part of an appendage or an organ.

[0263] Embodiment 35. The device according to embodiment 33, wherein the pressing member comprises an elastic member that compresses in response to the growing tissue.

[0264] Embodiment 36. The device according to embodiment 33, wherein the elastic member comprises a spring.

[0265] Embodiment 37. The device according to embodiment 33, the device further comprising:

[0266] A threaded adapter that can be disposed within the pressing member receiving end of the outer sleeve for selectively coupling a second end cap that can engage the pressing member to the outer sleeve, the second end cap having a groove configured to receive the threads of the threaded adapter; and

[0267] An elastic member extending between the seat portion of the threaded adapter and the seat portion of the pressing member.

[0268] Embodiment 38. The instrument according to Embodiment 33, wherein the pressing member is elastic.

[0269] Embodiment 39. The instrument according to Embodiment 33, wherein the inner chamber of the inner sleeve further contains a therapeutic composition.

[0270] Embodiment 40. The instrument according to Embodiment 39, wherein the therapeutic composition comprises:

[0271] Growth factors;

[0272] Inhibitors of prolyl hydroxylase domain (PHD) enzymes;

[0273] Vitamin A or its derivatives; and

[0274] Lipid mediators.

[0275] Embodiment 41. The instrument according to Embodiment 40, wherein the therapeutic composition contains growth factors selected from brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), and leukemia inhibitory factor (LIF), and combinations thereof.

[0276] Embodiment 42. The instrument according to Embodiment 41, wherein the therapeutic composition contains nerve growth factor.

[0277] Embodiment 43. The instrument according to Embodiment 41, wherein the therapeutic composition contains BDNF.

[0278] Embodiment 44. The device according to Embodiment 40, wherein the therapeutic composition comprises an inhibitor of PHD enzyme, and the inhibitor is selected from 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-pyridylthio)acetyl]-2,4(1H,3H)-pyrimidinedione, 6-amino-1,3-dimethyl-5-[[2-(2-pyridyl)-4-quinolyl]carbonyl]-2,4(1H,3H)-pyrimidinedione, N-[(4-hydroxy-1-methyl-7-phenoxy-3-isoquinolyl)carbonyl]-glycine, iron chelators, and combinations thereof.

[0279] Embodiment 45. The device according to Embodiment 44, wherein the therapeutic composition comprises 4,4α-dihydro-4-oxo-1,10-phenanthroline-3-carboxylic acid (1,4-DPCA).

[0280] Embodiment 46. The device according to Embodiment 40, wherein the therapeutic composition comprises a derivative of vitamin A, and the derivative is selected from retinoic acid, retinol, retinyl carboxylate, tretinoin, tazarotene, and combinations thereof.

[0281] Embodiment 47. The device according to Embodiment 46, wherein the therapeutic composition comprises retinoic acid.

[0282] Embodiment 48. The device according to Embodiment 40, wherein the therapeutic composition comprises a lipid mediator, and the lipid mediator is selected from resolvins, metabolites of ω-3 fatty acids, derivatives of eicosapentaenoic acid, derivatives of docosahexaenoic acid, and combinations thereof.

[0283] Embodiment 49. The device according to Embodiment 40, wherein the therapeutic composition comprises a resolvin.

[0284] Embodiment 50. The device according to Embodiment 40, wherein the therapeutic composition comprises a resolvin, and the resolvin is selected from resolvin 5, interleukin 6 (IL-6), interleukin 4 (IL-4), tumor necrosis factor-α (TNF-α), nuclear factor κ-light chain enhancer of activated B cells (NF-kB), and combinations thereof.

[0285] Embodiment 51. The device according to Embodiment 40, wherein the therapeutic composition comprises an agent that acts on proximal-distal positional information, and the agent is selected from bone morphogenetic protein 9 (BMP9), nodal growth differentiation factor, activin, transforming growth factor-β (TGF-β), fibroblast growth factor 8 (FGF8), and combinations thereof.

[0286] Embodiment 52. The device according to embodiment 40, wherein the therapeutic composition comprises a peptide or protein hormone.

[0287] Embodiment 53. The device according to embodiment 52, wherein the therapeutic composition comprises a peptide hormone selected from growth hormone (GH), insulin-like growth factor-1 (IGF-1), transforming growth factor-β-1 (TGFβ-1), epidermal growth factor (EGF), granulocyte-colony stimulating factor (G-CSF), fibroblast growth factor FGF, and combinations thereof.

[0288] Embodiment 54. The device according to embodiment 40, wherein the growth factor is present in the therapeutic composition at a dose from 0.1 μg / ml to 1 μg / ml.

[0289] Embodiment 55. The device according to embodiment 40, wherein the inhibitor of the PHD enzyme is present in the therapeutic composition at a dose from 0.004 μg / ml to 0.024 μg / ml.

[0290] Embodiment 56. The device according to embodiment 40, wherein the vitamin A or its derivative is present in the therapeutic composition at a dose from 0.03 μg / ml to 0.27 μg / ml.

[0291] Embodiment 57. The device according to embodiment 40, wherein the lipid mediator is present in the therapeutic composition at a dose from 0.006 μg / ml to 0.054 μg / ml.

[0292] Embodiment 58. The device according to embodiment 40, wherein the peptide or protein hormone is present in the therapeutic composition at a dose from 0.1 μg / ml to 1.0 μg / ml.

[0293] Embodiment 59. A method for promoting tissue regeneration in a mammal, the method comprising attaching a device according to any one of embodiments 1-58 to a mammal.

[0294] Embodiment 60. Use of a device according to any one of embodiments 1-58 for stimulating tissue regeneration in a mammal in need thereof.

[0295] Embodiment 61. A device for stimulating tissue regeneration at a tissue site of a subject, the device comprising:

[0296] An inner cannula, which includes a distal end having a first opening and an opposite engaging receiving end having a second opening, the first opening being sized to receive a tissue site for regeneration, the inner cannula defining an internal chamber extending between the first opening and the second opening, the internal chamber being sized to receive a tissue site for regeneration;

[0297] A protein matrix disposed within the internal chamber of the inner cannula, the protein matrix comprising a porous scaffold having pores forming substantially aligned channels; and

[0298] A filter medium that encapsulates the second opening on the engaging receiving end of the inner cannula.

[0299] Embodiment 62. The device according to Embodiment 61, wherein the substantially aligned channels are substantially parallel to the longitudinal axis in the inner cannula and orthogonal to the first opening and the second opening.

[0300] Embodiment 63. The device according to Embodiment 61, wherein the internal chamber comprises a reservoir of an aqueous solution or a dispersion medium between the filter medium and the protein matrix.

[0301] Embodiment 64. The device according to Embodiment 61, wherein the inner cannula internal chamber further comprises a therapeutic composition that can be delivered to the wound and promote tissue regeneration.

[0302] Embodiment 65. The device according to Embodiment 61, wherein the inner cannula internal chamber further comprises a therapeutic composition.

[0303] Embodiment 66. The device according to Embodiment 65, wherein the therapeutic composition comprises:

[0304] Growth factors;

[0305] An inhibitor of prolyl hydroxylase domain (PHD) enzymes;

[0306] Vitamin A or its derivatives; and

[0307] Lipid mediators.

[0308] Embodiment 67. The device according to Embodiment 66, wherein the therapeutic composition comprises growth factors selected from brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), and leukemia inhibitory factor (LIF), and combinations thereof.

[0309] Embodiment 68. The device according to Embodiment 67, wherein the therapeutic composition comprises BDNF.

[0310] Embodiment 69. The device according to Embodiment 66, wherein the therapeutic composition comprises an inhibitor of a PHD enzyme, and the inhibitor is selected from 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-pyridylthio)acetyl]-2,4(1H,3H)-pyrimidinedione, 6-amino-1,3-dimethyl-5-[[2-(2-pyridyl)-4-quinolyl]carbonyl]-2,4(1H,3H)-pyrimidinedione, N-[(4-hydroxy-1-methyl-7-phenoxy-3-isoquinolinyl)carbonyl]-glycine, an iron chelator, and combinations thereof.

[0311] Embodiment 70. The device according to Embodiment 69, wherein the therapeutic composition comprises 4,4α-dihydro-4-oxo-1,10-phenanthroline-3-carboxylic acid (1,4-DPCA).

[0312] Embodiment 71. The device according to Embodiment 66, wherein the therapeutic composition comprises a derivative of vitamin A, and the derivative is selected from retinoic acid, retinol, retinyl carboxylate, tretinoin, tazarotene, and combinations thereof.

[0313] Embodiment 72. The device according to Embodiment 66, wherein the therapeutic composition comprises a resolvin, and the resolvin is selected from resolvin D5, interleukin 6 (IL-6), interleukin 4 (IL-4), tumor necrosis factor-α (TNF-α), nuclear factor kappa-light-chain enhancer of activated B cells (NF-kB), and combinations thereof.

[0314] Embodiment 73. The device according to Embodiment 66, wherein the therapeutic composition comprises an agent that functions in proximal-distal positional information, and the agent is selected from bone morphogenetic protein 9 (BMP9), nodal growth differentiation factor, activin, transforming growth factor-β (TGF-β), fibroblast growth factor 8 (FGF8), and combinations thereof.

[0315] Embodiment 74. The device according to Embodiment 66, wherein the therapeutic composition comprises a peptide or a protein hormone.

[0316] Embodiment 75. The device according to Embodiment 74, wherein the therapeutic composition comprises a peptide hormone selected from growth hormone (GH), insulin-like growth factor-1 (IGF-1), transforming growth factor-β-1 (TGFβ-1), epidermal growth factor (EGF), granulocyte-colony stimulating factor (G-CSF), fibroblast growth factor FGF, and combinations thereof.

[0317] Embodiment 76. The device according to Embodiment 66, wherein the growth factor is present in the therapeutic composition at a dose from 0.1 μg / ml to 1 μg / ml.

[0318] Embodiment 77. The device according to Embodiment 66, wherein the inhibitor of the PHD enzyme is present in the therapeutic composition at a dose from 0.004 μg / ml to 0.024 μg / ml.

[0319] Embodiment 78. The device according to Embodiment 66, wherein vitamin A or a derivative thereof is present in the therapeutic composition at a dose from 0.03 μg / ml to 0.27 μg / ml.

[0320] Embodiment 79. The device according to Embodiment 66, wherein the lipid mediator is present in the therapeutic composition at a dose from 0.006 μg / ml to 0.054 μg / ml.

[0321] Embodiment 80. The device according to Embodiment 66, wherein the peptide or protein hormone is present in the therapeutic composition at a dose from 0.1 μg / ml to 1.0 μg / ml.

[0322] Embodiment 81. A method for promoting tissue regeneration in a mammal, the method comprising attaching a device according to any one of Embodiments 61-80 to a mammal.

[0323] Embodiment 82. Use of a device according to any one of Embodiments 61-80 for stimulating tissue regeneration in a mammal in need thereof.

Claims

1. An apparatus for stimulating tissue regeneration at a tissue site of a subject, the apparatus comprising: An inner cannula including a distal end having a first opening and an opposite engaging receiving end having a second opening, the first opening sized to receive a tissue site for regeneration, the inner cannula defining an internal chamber extending between the first opening and the second opening, the internal chamber sized to receive a tissue site for regeneration; A protein matrix disposed within the internal chamber of the inner sleeve, the protein matrix comprising a porous scaffold having pores forming substantially aligned channels; and A filter medium encapsulating a second opening on the mating receiving end of the inner sleeve.

2. The apparatus according to claim 1, wherein the substantially aligned channels are substantially parallel to a longitudinal axis in the inner cannula and orthogonal to the first and second openings.

3. The apparatus according to claim 1, wherein the internal chamber contains a reservoir of an aqueous solution or dispersion medium between the filter medium and the protein matrix.

4. The apparatus according to claim 1, wherein the inner cannula internal chamber further contains a therapeutic composition that can be delivered to the wound and promote tissue regeneration.

5. The apparatus according to claim 1, wherein the inner cannula internal chamber further contains a therapeutic composition.

6. The apparatus according to claim 5, wherein the therapeutic composition comprises: Growth factors; Inhibitors of prolyl hydroxylase domain (PHD) enzymes; Vitamin A or its derivatives; and Lipid mediators.

7. The apparatus according to claim 6, wherein the therapeutic composition contains growth factors selected from brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), and leukemia inhibitory factor (LIF), and combinations thereof.

8. The apparatus according to claim 7, wherein the therapeutic composition contains BDNF.

9. The device according to claim 6, wherein the therapeutic composition comprises an inhibitor of PHD enzyme, and the inhibitor is selected from 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-pyridylthio)acetyl]-2,4(1H,3H)-pyrimidinedione, 6-amino-1,3-dimethyl-5-[[2-(2-pyridyl)-4-quinolyl]carbonyl]-2,4(1H,3H)-pyrimidinedione, N-[(4-hydroxy-1-methyl-7-phenoxy-3-isoquinolinyl)carbonyl]-glycine, iron chelators, and combinations thereof.

10. The device according to claim 9, wherein the therapeutic composition comprises 4,4α-dihydro-4-oxo-1,10-phenanthroline-3-carboxylic acid (1,4-DPCA).

11. The device according to claim 6, wherein the therapeutic composition comprises a derivative of vitamin A, and the derivative is selected from retinoic acid, retinol, retinyl carboxylate, tretinoin, tazarotene, and combinations thereof.

12. The device according to claim 6, wherein the therapeutic composition comprises resolvins, and the resolvins are selected from resolvin D5, interleukin-6 (IL-6), interleukin-4 (IL-4), tumor necrosis factor-α (TNF-α), nuclear factor κ-light chain enhancer of activated B cells (NF-kB), and combinations thereof.

13. The device according to claim 6, wherein the therapeutic composition comprises an agent that acts on proximal-distal positional information, and the agent is selected from bone morphogenetic protein 9 (BMP9), nodal growth differentiation factor, activin, transforming growth factor-β (TGF-β), fibroblast growth factor 8 (FGF8), and combinations thereof.

14. The device according to claim 6, wherein the therapeutic composition comprises a peptide or a protein hormone.

15. The device according to claim 14, wherein the therapeutic composition comprises a peptide hormone, and the peptide hormone is selected from growth hormone (GH), insulin-like growth factor-1 (IGF-1), transforming growth factor-β-1 (TGFβ-1), epidermal growth factor (EGF), granulocyte-colony stimulating factor (G-CSF), fibroblast growth factor FGF, and combinations thereof.

16. The device according to claim 6, wherein the growth factor is present in the therapeutic composition at a dose of from 0.1 μg / ml to 1 μg / ml.

17. The device according to claim 6, wherein the inhibitor of the PHD enzyme is present in the therapeutic composition at a dose from 0.004 μg / ml to 0.024 μg / ml.

18. The device according to claim 6, wherein the vitamin A or its derivative is present in the therapeutic composition at a dose from 0.03 μg / ml to 0.27 μg / ml.

19. The device according to claim 6, wherein the lipid mediator is present in the therapeutic composition at a dose from 0.006 μg / ml to 0.054 μg / ml.

20. The device according to claim 6, wherein the peptide or protein hormone is present in the therapeutic composition at a dose from 0.1 μg / ml to 1.0 μg / ml.

21. Use of the device according to any one of claims 1-20 for stimulating tissue regeneration in a mammal in need thereof.

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