Tissue regeneration multi-drug mixture and delivery device thereof

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application Serial No. 62 / 944,707, filed on December 6, 2019, the entire contents of which are incorporated herein by reference.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] 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. Field of the Invention

[0005] The field of the 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 sleeves, and the disclosed compositions include regenerative compositions.

[0006] background

[0007] In the United States alone, the incidence of human limb loss is projected to increase dramatically over the next 30 years, affecting 3.6 million people annually by 2050 (Ziegler-Graham et al., 2008), leaving behind diabetics, war veterans, trauma survivors, and those with peripheral arterial 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.and Bioenergetics , 8(6), 661-670), tissue-guided biomaterials (Suckow et al. (1999) J.of Invest.Surg. , 12(5), 277-287), progenitor cell transplantation (Lin et al., (2013) Dev.Cell , 24(1), 41-51) and regulate key molecular pathways (Kawakami et al., (2006) Genes&Dev. ,20(23),3232-3237; Yokoyama et al.,(2001) Dev.Biology, 233(1), 72-79). However, they have had limited success in restoring significant growth and patterning of new limbs.

[0009] Tissue regeneration involves a series of biological events, which are fully combined to rebuild the excision or appendage lost in the process of wound or amputation. There are obvious differences between typical wound healing response and regeneration response. Although these two processes are similar in many embodiments, they produce completely different end products. In normal wound healing process, many complex biological structures such as sweat glands, ducts and hair follicles cannot be rebuilt because the biological mechanism of doing so is unavailable. In typical adult mammal skin wounds, these structures will not regenerate because the development of these tissues and organs involves highly specific physiological processes. In addition, normal wound closure and scar formation will not provide appropriate environment for the regeneration of these structures. On the other hand, incision regeneration is the process that all original structures are replaced by replicas of the original structure.

[0010] Although some complex animals such as axolotls naturally regenerate limbs, eyes, and other entire organs throughout their lives, mammals generally show limited regeneration and generally do not show the plasticity and transdifferentiation capabilities of anurans. Anurans that have matured toward puberty can regenerate their amputated or injured limbs when exposed to regeneration inducers delivered by slow-release beads implanted in amputated tissue. 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 molding.

[0011] However, it is unclear how the completely non-regenerative, strongly metamorphosed (adult) African toad (Xenopus) can serve as a useful animal model of regeneration, as adult frogs are unable to regenerate their hind limbs after amputation, but instead produce featureless cartilaginous spikes (Suzuki et al., (2006) Sci.World J. ,6:26-37).

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

[0013] In addition, conventional tissue regeneration devices are usually attached to the subject using glue, which is not conducive to long-term treatment. Using glue to attach the regeneration device has two major disadvantages. The glue lacks the flexibility to adjust the device after attachment. In addition, there are operational complexities during the surgical procedure and the device.

[0014] Therefore, improved devices and regeneration compositions are desirable and needed. Summary of the invention

[0015] Disclosed are devices, compositions and methods for promoting regeneration of tissue, such as a traumatized, damaged or injured site present on an appendage of a living subject or within a 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 sleeve and a regenerative composition.

[0016] In one aspect, the present disclosure provides therapeutic compositions for promoting tissue. In certain embodiments, the regenerative compositions may include a variety of components. In certain embodiments, the disclosed regenerative compositions are used as therapeutic compositions in disclosed devices, for example, in the material of an inner sleeve that contacts a 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 the group consisting of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), glial cell-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), and leukemia inhibitory factor (LIF), and combinations thereof. In certain embodiments, the therapeutic composition comprises a 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 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 selected from the group consisting of retinoic acid, retinol, retinyl carboxylate, tretinoin, tazarotene, and combinations thereof.

[0021] In certain embodiments, the therapeutic composition comprises a lipid mediator selected from the group consisting of 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 a resolvin. In specific embodiments, the therapeutic composition comprises a resolvin selected from the group consisting of resolvin 5, interleukin 6 (IL-6), interleukin 4 (IL-4), tumor necrosis factor-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 acts in proximo-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 of from 0.1 μg / ml to 1 μg / ml. In certain embodiments, the PHD inhibitor is present in the therapeutic composition at a dose of from 0.004 μg / ml to 0.024 μg / ml. In certain embodiments, the vitamin A or its derivative is present in the therapeutic composition at a dose of from 0.03 μg / ml to 0.27 μg / ml. In certain embodiments, the lipid medium is present in the therapeutic composition at a dose of 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 of from 0.1 μg / ml to 1.0 μg / ml.

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

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

[0027] In another aspect, the disclosed apparatus comprises an outer sleeve with tissue receiving and / or insertion end, a pressing member receiving end relative to the tissue receiving end, and an internal chamber configured to receive and / or accommodate the tissue. The apparatus further comprises an inner sleeve disposed in the outer sleeve, the inner sleeve having an end for receiving traumatized, damaged or injured tissue or appendage, a joint receiving end for engaging a pressing member, and an internal chamber configured to receive and / or accommodate traumatized, damaged or injured tissue. The pressing member is configured to extend into the internal chamber of the outer sleeve through the pressing member receiving end, and the joint receiving end of the inner sleeve is biased toward the tissue so 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 sleeve. The inner sleeve may contain a polymeric material and / or may be formed by a polymeric material, and the polymeric material includes, but is not limited to, a silk hydrogel material. The instrument further includes a first end cap engageable with the tissue receiving end of the outer cannula and including an opening configured to receive tissue and maintain the tissue in the device, and a second end cap engageable with the compression member receiving end of the outer cannula.

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

[0029] In some embodiments, the instrument includes a first threaded adapter that can be disposed within the tissue receiving end of the outer sleeve, which is used to selectively couple a first end cap that can engage with the tissue receiving end to the outer sleeve. In some embodiments, the first end cap includes a groove that is configured to receive threads of the first threaded adapter.

[0030] In some embodiments, the instrument includes a second threaded adapter that can be disposed within the pressing member receiving end of the outer sleeve, which is used to selectively couple a second end cap that can engage with the pressing member to the outer sleeve. In some embodiments, the second end cap includes a groove that is configured to receive the threads of the second threaded adapter.

[0031] In certain embodiments, the pressing member includes a seat portion configured to receive an engagement receiving end of the inner sleeve, or includes an engagement end configured to seat on a seat portion of the second end cap.

[0032] In some embodiments, the engagement receiving end of the inner sleeve includes a porous filter medium that seals the internal chamber on the engagement receiving end. In some embodiments, the porous filter medium is a synthetic or polymeric membrane. In some embodiments, the apparatus includes a compressible member positioned between the porous filter medium and the pressing member. In some embodiments, the compressible member comprises cotton or an encapsulated gel.

[0033] In certain embodiments, the inner sleeve comprises a protein or polymer matrix that at least partially fills the inner chamber of the inner sleeve. In certain embodiments, the protein or polymer matrix comprises a three-dimensional porous support. In certain embodiments, the porous support may include a hole that forms an oriented pattern. In certain embodiments, the porous support includes aligned holes that form substantially aligned passages. The aligned passages in the protein or polymer matrix may be arranged parallel to the longitudinal axis of the inner sleeve. In certain embodiments, the protein or polymer matrix is ​​selected from silk fibroin (silk fibroin), collagen or a combination thereof.

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

[0035] In another aspect, the disclosed apparatus includes a pressing member that can move in response to growth at the site of the traumatized, damaged or injured tissue, for example, in an axial direction toward and / or away from the traumatized, damaged or injured tissue. In certain embodiments, the pressing member biases the engagement receiving end of the inner sleeve toward the tissue so that at least a portion of the subject's traumatized, damaged or injured tissue is placed in contact with a portion of the inner chamber of the inner sleeve, and as the tissue regenerates and / or the traumatized, damaged or injured tissue heals, the regenerated tissue and / or the healing tissue causes the pressing member to move axially away from the trauma to directly or indirectly contact the engagement receiving end.

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

[0037] In some embodiments, the instrument includes a threaded adapter that can be disposed within the pressing member receiving end of the outer sleeve, which is used to selectively couple a second end cap that can engage with the pressing member to the outer sleeve. The second end cap may include a groove that is configured to receive the threads of the threaded adapter. The resilient 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 apparatus, the inner cannula of the disclosed apparatus includes a material within a reservoir in the inner cannula that contacts the traumatized, damaged, or injured tissue when the traumatized, damaged, or injured tissue is inserted into the apparatus. The material of the inner cannula may include a hydrogel that wets the traumatized, damaged, or injured tissue. The material may additionally include a therapeutic composition and / or an antimicrobial agent that promotes tissue regeneration and / or healing.

[0039] In another aspect, the disclosed apparatus comprises an inner sleeve, the inner sleeve comprises an end with a first opening and a joint receiving end with a second opening, the size of the first opening is designed to receive traumatized, damaged or injured tissue, the inner sleeve is limited to an inner chamber extending between the first opening and the second opening, and the size of the inner chamber is designed to receive traumatized, damaged or injured tissue of the experimenter. The apparatus further comprises a matrix arranged in the inner chamber of the inner sleeve, wherein the matrix comprises a porous support, and the porous support has a hole forming a substantially aligned passage. The matrix may comprise the following materials and / or may be formed by the following materials, including, but not limited to polymeric materials such as albumen, which may include, but not limited to collagen and silk fibroin of any suitable form. The aligned passages of the porous support may be arranged parallel to the longitudinal axis of the inner sleeve. The matrix may comprise therapeutic compositions disclosed herein such as regenerative compositions or regenerative cocktails (cocktail).

[0040] In certain embodiments, the substantially aligned channels are substantially parallel or parallel to the longitudinal axis in the inner sleeve and orthogonal to the first and second openings. In certain embodiments, the interior chamber contains a reservoir of aqueous solution or dispersion medium between the filter medium and the protein matrix.

[0041] In some embodiments, the device further comprises a filter medium sized to enclose the second opening on the engagement receiving end of the inner sleeve. In some embodiments, the pore size of the filter 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 interior lumen of the inner cannula of a provided device includes a provided therapeutic composition.

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

[0044] In another aspect, the present disclosure provides 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 emerge from the following description. In the description, reference is made to the accompanying drawings which form a part thereof and in which a non-limiting example embodiment is shown by way of illustration. However, this embodiment does not necessarily represent the full scope of the present disclosure, and therefore the scope of the present disclosure is interpreted with reference to the entire disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The present disclosure will hereinafter be described with reference to the drawings, wherein like reference numerals refer to like elements.

[0048] Figure 1 is a schematic diagram of a side cross-sectional, partially exploded view of an apparatus for stimulating tissue regeneration at a wound site in a subject, according to some embodiments of the present disclosure.

[0049] Figure 2 is a schematic diagram of a side cross-sectional, partially exploded view of an apparatus for stimulating tissue regeneration at a wound site in a subject, according to some embodiments of the present disclosure.

[0050] Figure 3 is a diagrammatic representation of a mouse digit showing the amputation line.

[0051] Figure 4 is a schematic diagram of a side cross-sectional view of an apparatus for stimulating tissue regeneration at a wound site of a subject, the apparatus having a movable compression member, according to some embodiments of the present disclosure.

[0052] Figure 5 is in a compressed state according to some embodiments of the present disclosure Figure 4 Schematic diagram of a side cross-sectional view of the device.

[0053] Figure 6 yes Figure 1 Schematic diagram of an apparatus of the present disclosure, but including an electrical stimulation device according to some embodiments of the present disclosure.

[0054] Figure 7 is installed in a subject according to some embodiments of the present disclosure Figure 6 A graphical representation of the device.

[0055] Fig. 8A is a SEM image showing a silk fibroin scaffold having channel-like pores aligned along the long axis according to some embodiments of the present disclosure.

[0056] 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.

[0057] Figure 8C Collagen scaffolds with pores aligned along the long axis are illustrated according to some embodiments of the present disclosure.

[0058] Fig.8D Collagen scaffolds with pores aligned perpendicular to the long axis are illustrated according to some embodiments of the present disclosure.

[0059] Fig. 9 is a graphical representation of right hindlimb length as a function of months post-amputation (mpa) after exposure to one of the following treatment conditions: (i) after the 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.

[0060] Fig.10 It is a graphic representation of the tactile response after 17 MPa assessed by Von Frey Filaments at the distal tip of regenerants that have been exposed to one of the following treatment conditions: (i) after the 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. Mean values ​​and SD are presented.

[0061] Fig.11 is a graphical representation of right hindlimb bone length assessed 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 the 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.

[0062] Fig.12 Figure 2 is a graphical representation of right hindlimb bone volume assessed 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 the 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.

[0063] Fig.13 is a graphical representation of the number of ATT+ nerve bundles measured by acetylated α-tubulin (AAT) staining in the right hindlimb following 18 MPa amputation 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] Fig.14is a graphical representation of 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.

[0065] Fig.15 is the particle complexity (pixels) assessed by fibronectin expression after 18 MPa and after exposure to one of the following initial treatment conditions: 2 ) are graphical representations of: (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] Fig.16 is a graphical representation of the number of laminin / smooth muscle actin (SMA+) bundles measured by laminin and SMA+ expression in regenerants 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.

[0067] Fig.17 is a graphical representation of wound closure diameters (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.

[0068] Fig.18 is a graphical representation 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.

[0069] Fig.19 is a graphical representation of the cumulative release of drug from the provided MDT compositions.

[0070] Detailed Description

[0071] The disclosures of these patents, patent applications, and publications are hereby incorporated by reference into this application in their entireties in order to more fully describe the state of the 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 patents, patent applications, and publications and the present disclosure, the present disclosure shall prevail.

[0072] 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 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 can be practiced or carried out in various ways.

[0073] It should be understood that the phrases and terms used herein are for descriptive purposes and should not be construed as limiting. The use of "including," "comprising," or "having," and variations thereof, herein is intended to include the items listed thereafter and their equivalents as well as additional items. Unless otherwise specified or limited, the terms "mounted," "connected," "supported," and "coupled," and variations thereof, are used broadly and include direct and indirect mountings, connections, supports, and couplings. Further, "connected" and "coupled" are not limited to physical or mechanical connections or couplings.

[0074] The term "tissue" as used herein is defined as a collection of similar cells and their extracellular matrix from the same source that together perform a specific function. "Tissue" as used herein may be present on an appendage including, but not limited to, phalanges (such as fingers and toes), arms, legs, etc. "Tissue" as used herein may be present on an organ (such as the liver, lungs, pancreas, etc.).

[0075] The terms "therapeutic composition," "regenerative composition," "regenerative cocktail," and "multi-drug therapeutic compound" or "MDT" include formulations comprising a combination of therapeutic agents that stimulate or initiate tissue regeneration and, in certain embodiments, have a synergistic effect when administered to a subject.

[0076] The following discussion is presented to enable those skilled in the art to make and use the embodiments of the present disclosure. Various modifications to the illustrated embodiments will be readily apparent to those skilled in the art, and the general principles herein may be applied to other embodiments and applications without departing from the embodiments of the present disclosure. Therefore, the embodiments of the present disclosure are not intended to be limited to the illustrated embodiments, but to conform to the widest range consistent with the principles and features disclosed herein. The following detailed description will be read with reference to the accompanying drawings, wherein the same elements in the different drawings have the same reference numerals. The accompanying drawings, which are not necessarily drawn to scale, depict selected embodiments, and are not intended to limit the scope of the embodiments of the present disclosure. Those skilled in the art will recognize that the embodiments provided herein have many useful alternative embodiments and fall within the scope of the embodiments of the present disclosure.

[0077] Devices for tissue regeneration

[0078] The present disclosure provides devices for assisting tissue regeneration. Figure 1-3 , shows a device 200 for stimulating tissue regeneration in a subject 1. In certain embodiments, the device 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 device 200 is described with respect to stimulating tissue regeneration of traumatized tissue 9 located on an appendage 3 (e.g., a mouse toe), wherein the tip 2 has been severed along a line 5 passing through at least a portion of a distal phalanx 4, whereby the regenerated tissue may include bone tissue 6, muscle tissue 7, and skin tissue 8, as well as other tissues. In the description Figure 1-7 In the present invention, for exemplary purposes, reference may be made to an "appendage" as comprising an exemplary "tissue."

[0079] In certain embodiments, the traumatized or injured tissue 9 for stimulating tissue regeneration using the device 200 includes epithelial tissue, connective tissue, muscle tissue, or neural tissue. Exemplary traumatized or injured tissue 9 for regeneration include, but are not limited to, squamous epithelium, cuboidal epithelium, transitional epithelium, pseudostratified columnar epithelium, columnar epithelium, glandular epithelium, bone, tendon, ligament, fat, cellulite, blood tissue, visceral muscle, smooth muscle, skeletal muscle, cardiac muscle, and neural tissue.

[0080] refer to Figure 1-2, the device 100 includes an outer sleeve 202 extending between an appendage receiving end 204 and a pressing member receiving end 206. In some embodiments, the outer sleeve 202 is a hollow cylinder defining an internal chamber 208 that is sized to receive the tissue 3 to be treated (e.g., an appendage) and the traumatized or injured tissue 9. The internal chamber 208 forms a passage extending between a first opening on the appendage receiving end 204 and a second opening on the pressing member receiving end 206. In some embodiments, the first opening on the appendage receiving end 204 is sized to receive the appendage 3, and the second opening is sized to receive the pressing member 214.

[0081] The outer sleeve 202 can be formed of a transparent material to allow observation of the wound 9 when the instrument 200 is used. In certain embodiments, the outer sleeve 202 has sufficient rigidity to prevent any deflection or depression of the body wall during use to ensure that the desired wound space volume is maintained and the wounded or injured tissue 9 is protected. Exemplary materials for the construction of the outer sleeve 202 include, but are not limited to, transparent nylon tubing. The outer sleeve 202 may include one or more openings (not shown) to facilitate replacement of fluids within the internal chamber 208 of the outer sleeve 202. For example, one or more openings may include a septum that allows a needle to enter and replace the fluid within the internal chamber 208.

[0082] The device 200 includes an inner sleeve 216 extending between a wound receiving end 218 and an engagement receiving end 220 opposite the wound receiving end 218. In some embodiments, the inner sleeve 216 is a hollow cylinder defining an internal chamber 222, the internal chamber 222 being sized to receive the wound 9. The internal chamber 222 forms a passage extending between a first opening on the wound receiving end 218 and a second opening on the engagement receiving end 220. When the device 200 is assembled, the inner sleeve 216 is configured to enclose the wound 9, and the pressing member 214 is configured to bias the engagement receiving end 220 toward the appendage 3 so that the wound 9 is placed in contact with at least a portion of the internal chamber 222. In some embodiments, the pressing member 214 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.

[0083] In some embodiments, the joint 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 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 fluidly connected to the internal chamber 222. In some 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 some embodiments, the compressible member or media exchange member 232 contains a gel or contains cotton that is optionally wetted with an aqueous solution or dispersion medium. In some embodiments, the protein matrix 228 is replaced from the filter medium 230 by the reservoir of the aqueous solution or dispersion medium.

[0084] 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.

[0085] The apparatus 200 includes a first end cap 234 that can be engaged with the appendage receiving end 204 of the outer cannula 202. The first end cap 234 includes an opening 236 that is sized to receive the appendage 3 of the subject 1. In some embodiments, a gasket or septum 238 can be positioned between the first end cap 234 and the appendage receiving end 204 of the outer cannula 202. The gasket or septum 238 includes a through hole 240 that is sized to receive the appendage 3 and provides a seal that prevents liquid from escaping the interior chamber 208 of the outer cannula 202. In some embodiments, the septum 238 includes a flexible side 242 composed of silicon and a rigid side 244 that includes polytetrafluoroethylene (PTFE).

[0086] The instrument 200 may include a first adjustable adapter 246 that may be disposed within the appendage receiving end 204 for selectively coupling a first end cap 234 that may engage with the appendage receiving end 204 to the outer sleeve 202. In certain 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 may be tightened so that the washer 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 sleeve 202.

[0087] The instrument 200 includes a second end cap 254 that is engageable with the pressing member receiving end 206 of the outer sleeve 202. The second end cap 254 is coupled to the pressing member 214 to bias the pressing member 214 toward the engagement receiving end 220 of the inner sleeve 216. Figure 1 , the pressing member 214 may be directly attached to the second end cap 254 or form a part of the second end cap 254. Figure 2 , the pressing member 214 can be separated from the second end cap 254. In some embodiments, the pressing member 214 includes an engagement end 256 configured to be seated on a seat portion 258 of the second end cap 254. In some embodiments, the pressing member 214 includes a seat portion 260 opposite the engagement end 256, which is configured to receive the engagement end 220 of the inner sleeve 216. The pressing member 214 can be formed of a rigid material or an elastic material that deforms or compresses in response to the growth of tissue at the wound 9.

[0088] The instrument 200 may include a second adjustable adapter 262 that may be disposed within the pressing member receiving end 206, which is used to selectively couple a second end cap 254 that may be engaged with the pressing member receiving end 206 to the outer sleeve 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 a thread 266 of the second adjustable adapter 262. The second end cap 254 may be adjusted to control the pressure at the wound 9. One disadvantage of conventional devices is the lack of control over the pressure at the wound 9 interface, which may result in variability in tissue regeneration results if there is any type of gap (fluid collection, air, etc.). The instrument 200 advantageously provides an adjustable pressure sufficient to keep the protein matrix 228 in contact with the wound 9. In addition, unlike conventional devices, the instrument 200 facilitates long-term attachment (weeks, months, years or longer) and can be adjusted to promote the growth of regenerated tissue.

[0089] refer to Figure 4-5 In some embodiments, the device 200 includes a movable or adjustable compression member 214. For example, the compression member 214 can move or extend in response to tissue growth (in Figure 4 and 5 denoted by Δx in the figure). In some embodiments, the resilient member 268 is configured to extend between the seat portion 270 on the pressing member 214 and the seat portion 272 on the second adjustable adapter 262. In some embodiments, the resilient member 268 (e.g., a spring, a compressible material, a deformable material) creates a resistance to rearward linear translation (e.g., when the second end cap 254 is screwed off). In some embodiments, the adjustable pressing member 214 includes an interlocking spacer or screw extension system that can extend over time in response to tissue growth.

[0090] refer to Figure 6-7 In some embodiments, the apparatus 200 also includes an electrical stimulation device 300 to establish a longitudinal electric field through the wound site 9, which is believed to provide internal wound stump current and provide electrical guidance signals to innervate and migrate cell types near the wound site. The electrical stimulation device 300 includes an anode 302 and a cathode 304, which are electrically connected to corresponding terminals of a power source 306 through leads 308, 310.

[0091] In certain embodiments, cathode 304 is in the form of a stainless steel wire disposed proximate to wound 9. A portion of cathode 304 is disposed externally to instrument 200 and may be connected to lead 308, and a portion of cathode 304 is disposed within inner chamber 222 of inner cannula. Anode 302 is a lead wire that may be inserted into subject 1 at a location distal to wound site 9. In the illustrated embodiment in which instrument 200 is disposed on appendage 3, anode 302 is disposed on the upper portion of the limb (hind leg) from which appendage 3 extends. Anode 302 may comprise a platinum / iridium alloy wire that is connected to power source 306 via lead 310. Anode 302 may be permanently implanted, or temporarily inserted as needed.

[0092] The power source 306 includes a battery pack 312 and a circuit 314, both of which are enclosed 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. Figure 6 In the illustrated embodiment, power source 306 is disposed externally to subject 1, and electrodes 302, 304 are configured to be removably connected to power source 306. In this arrangement, when electrical stimulation is used, cathode 304 and anode 302 are electrically connected to power source 306 during electrical stimulation treatment, and then disconnected between electrical stimulation treatments. Because power source 306 and leads 308, 310 are removable from corresponding electrodes 302, 304, this arrangement conveniently reduces the overall volume of combined apparatus 200 and electrical stimulation device 300 in treatment paradigms where electrical stimulation is only used intermittently.

[0093] In certain embodiments, protein matrix 228 comprises biocompatible polymer. Suitable biocompatible polymers for use with apparatus 100 include, but are not limited to polyethylene oxide (PEO), polyethylene glycol (PEG), collagen, fibronectin, keratin, polyaspartic acid, polylysine, alginate, chitosan, chitin, hyaluronic acid, pectin, polycaprolactone, polylactic acid, polyglycolic acid, polyhydroxyalkanoate, dextran, polyanhydride, polymer, PLA-PGA, polyanhydride, polyorthoester, polycaprolactone, polyfumarate, collagen, silk fibroin, chitosan, alginate, hyaluronic acid and other biocompatible and / or biodegradable polymers. In certain embodiments, protein matrix 228 is silk fibroin and / or collagen.

[0094] In certain embodiments, protein matrix 228 is processed by silk solution (for example, aqueous solution), and described silk solution has the silk solution concentration between about 1% silk to about 50% silk.In certain embodiments, the material based on silk-fibroin(s) is processed by silk solution to form different material forms, such as fiber, foam, particle, film and / or hydrogel.

[0095] In certain embodiments, protein matrix 228 is porous or has porosity. As used herein, the term "porosity" can refer to a measure of void space in a material and is the fraction of the volume of void space to the total volume, expressed as a percentage between 0 and 100%. Determining porosity using standard techniques, such as mercury porosimetry and gas adsorption (e.g., nitrogen adsorption) is known to the skilled artisan.

[0096] In some 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 re-growth of traumatized or injured tissue. In some embodiments, the protein matrix 228 has a pore size between about 1 μm and 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. With regard to pore size, generally, about 100 μm to about 300 μm or about 100 μm, about 150 μm, about 200 μm, about 250 μm or about 300 μm are suitable for supporting enough oxygen, nutrients 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 results due to improved nutrient transfer and waste removal.

[0097] Protein matrix 228 can have holes forming directional patterns, which are used to guide tissue growth at wound 9. In certain embodiments, the directional pattern includes aligned holes, which form substantially aligned channels. The aligned holes can be arranged to be parallel to the longitudinal axis of inner sleeve 216. In certain embodiments, aligned holes are formed by freezing protein solutions (e.g., silk or collagen solutions) with steep temperature gradients on conductive substrates (e.g., aluminum plates), which are induced by merging conductive substrates with cold sources (e.g., liquid nitrogen). It is considered that the finger-shaped ice crystal columns grown from cold surfaces produce channel-shaped structures inside frozen protein. The frozen protein is then lyophilized for a period of time (e.g., 24 hours) to remove water. The resulting product is a protein matrix 228 with substantially aligned holes.

[0098] In certain aspects, the protein matrix comprises silk fibroin. "Silk fibroin" or "SF" as used herein may refer to a biopolymer produced from silkworm fibroin and insect or spider silk proteins. For example, the silk fibroin that can be used in the present disclosure can be the silk 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 Nephilamada gascariensis. Alternatively, the silk utilized in the present disclosure can be prepared by artificial methods, for example, involving genetic engineering of cells or organisms (e.g., genetically engineered bacteria, yeast, mammalian cells, non-human organisms, including animals or transgenic plants).

[0099] Like collagen, SF is a structural protein but has unique properties: it is produced by a living complex organism by extrusion of an amino acid solution into the external environment, whereas collagen is produced in vivo in the extracellular space by self-assembly of monomers produced by cells and is not secreted into the external environment. SF properties derive from its structure, which consists of hydrophobic blocks interlaced with hydrophilic acidic spacers. In its natural state, SF is organized into a semi-crystalline material in which β-sheet crystals alternate with amorphous regions, which provide strength and elasticity to the protein 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.

[0100] Processing of SF generally involves partial or complete dehydration of a silk fibroin solution (about 1 wt % to about 15 wt % protein content) using a variety of techniques (e.g., solvent casting, freeze drying, salt leaching, sonication) to form, for example, films, sponges, gels, spheres (micrometer to nanometer size), and foams. These manufacturing processes provide a strong material that combines mechanical strength with biochemical properties.

[0101] The silk-fibroin solution used in the method and composition provided herein can be obtained from a solution containing dissolved silk (for example, from silkworm). Alternatively, the silk-fibroin solution can be obtained from a solution containing dissolved spider silk (for example, from Nephila cinerea). The silk-fibroin solution can also be obtained from a solution containing genetically engineered silk (for example, from bacteria, yeast, mammalian cells, transgenic animals or transgenic plants). See, for example, WO 97 / 08315 and United States Patent (USP) 5,245,012. The silk of genetically engineered can also include, for example, a therapeutic agent, for example, a fusion protein with a cytokine, an enzyme or any number of hormone- or peptide-based medicines, antimicrobials and related substrates.

[0102] Silk-fibroin(s) solution can be prepared by any conventional method well known to persons skilled in the art. In certain embodiments, silk solution is a silk aqueous solution. In other embodiments, silk solution can contain a second polymer to promote solid-state transformation (for example, polyethylene glycol, collagen, hyaluronic acid etc.).

[0103] Silkworm cocoon silk contains two structural proteins: heavy fibroin chains (about 350 kDa); and light fibroin chains (about 25 kDa), which are associated with a family of nonstructural proteins called sericins that glue the fibroin chains together to form the cocoon. The heavy and light fibroin chains are linked by disulfide bonds at the C-termini of the two subunits (see Takei, et al., J.Cell Biol ., 105:175, 1987; see also 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 that imparts stickiness to the material. These glycoproteins are hydrophilic and can be easily removed from the cocoons by "degumming" by boiling in water).

[0104] In certain embodiments, silk polypeptide compositions utilized in accordance with the present compositions contain substantially no sericin (eg, contain no detectable sericin, or contain levels of sericin that one of ordinary skill in the relevant art would consider negligible for a particular use).

[0105] In an exemplary method of obtaining a silk polypeptide composition, B. 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.

[0106] If necessary, the solution can then be concentrated using any method known in the art. For example, a hygroscopic polymer (e.g., PEG, polyethylene oxide, amylose or sericin) can be dialyzed. 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 sufficiently long period of time to produce a final concentration of a silk aqueous solution between about 1% to about 30%. In some cases, dialysis for about 2 hours to about 12 hours is sufficient.

[0107] In certain embodiments, the present disclosure provides a method of attaching a device 200 to an appendage or tissue of a subject in need of tissue regeneration. The method includes contacting a wounded appendage or tissue 9 of a subject 1 to a wound receiving end 218 of an inner cannula 216. The wounded appendage or tissue 9 can be placed in contact with or adjacent to a protein matrix 228 containing a provided therapeutic composition. In certain embodiments, before contacting the wounded appendage or tissue 9 to the protein matrix 228, the wounded appendage or tissue 9 is slid through a gasket or spacer 238 and an opening 236 of a first end cap 234.

[0108] The method further includes placing the appendage 3 of the subject through the appendage receiving end 204 of the outer sleeve 202 such that the inner sleeve 216 is positioned within the interior chamber 208 of the outer sleeve 202. In some embodiments, the method includes selectively engaging the first end cap 234 and the second end cap 254 to the outer sleeve 202 such that the pressing member 214 biases the engagement receiving end 220 toward the appendage 3. In some embodiments, the method includes biasing the pressing member 214 toward the appendage 3 such that the wound 9 is placed in contact with at least a portion of the interior chamber 222 of the inner sleeve. 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), the contact pressure between the wounded appendage or tissue 9 and the protein matrix 228 can be adjusted.

[0109] In some embodiments, the wounded appendage or tissue 9 is maintained in the apparatus 200 for a period of time to promote tissue regeneration. In some embodiments, the duration is about 1 minute, or about 10 minutes, or about 30 minutes, or about 1 hour, or about 2 hours, or about 3 hours, or about 4 hours, or about 5 hours, or about 6 hours, or about 12 hours, or about 24 hours, or 2 days, or about 3 days, or about 4 days, or about 5 days, or about 1 week, or about 2 weeks, or about 3 weeks, or about 1 month, or about 6 months, or about 1 year, or within the 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 in the inner sleeve 216.

[0110] Therapeutic compositions for tissue regeneration

[0111] 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 devices.

[0112] The therapeutic composition used with the apparatus according to the present invention can be any composition that stimulates, initiates, or directly or indirectly helps 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 help tissue regeneration. For example, the components provided 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 regeneration rate at the site of the wounded appendage or tissue relative to an untreated control experiment (e.g., increase tissue regeneration, such as measured by soft tissue length, bone length, bone volume, increased tactile response, ATT+ nerve bundle number, ATT+ nerve bundle diameter, regeneration particle complexity assessed by fibronectin expression, the number of laminin / SMA+ bundles, the wound diameter at the start of treatment, the number of SOX2+ cells).

[0113] In certain embodiments, at least two components of provided therapeutic compositions act synergistically to increase the regeneration rate, or at least three components, or at least four components, or at least five components, or all components act synergistically to increase the regeneration rate.

[0114] In one embodiment, the disclosed device may contain a therapeutic composition within an inner cannula (e.g., within a reservoir within the inner cannula). The therapeutic composition may be present in a material or matrix that contacts the wound site, and when the device is worn on an appendage of a subject, the therapeutic composition may 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., Cell Reports 25,1593-1609(2018).

[0115] 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 induced to gel by adding reagents such as horseradish peroxidase (e.g., to a concentration of about 20 U / ml 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 with this enzymatic reaction, by lowering pH, adding energy (such as by sonication or vortexing), applying an electric field, or adding methanol.

[0116] The disclosed therapeutic compositions may include one or more agents that increase axon / neurite growth and / or general cell proliferation. Preferably, the disclosed therapeutic compositions do not promote multipotency in cells and / or cause teratoma formation.

[0117] The disclosed therapeutic compositions may include one or more agents that promote tissue regeneration and / or healing. In certain embodiments, the therapeutic composition includes one or more of the following: growth factors, agents that inhibit inhibitors of hypoxia-inducible factor 1-α (HIF1-α), vitamin A or its derivatives, lipid mediators such as metabolites of ω-3 fatty acids, and may be derived from eicosapentaenoic acid or docosahexaenoic acid, growth hormone, steroids, and depolarizing agents.

[0118] In certain embodiments, the disclosed therapeutic compositions may include growth factors, such as neurotrophic factors. Neurotrophic factors are proteins that promote the growth and survival of neural cells during development and promote the maintenance of adult neural 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-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), and combinations thereof. The growth factor may be present in the therapeutic composition at 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 dose range defined by any of these values. When the growth factor is present in a component of the disclosed devices (e.g., when the growth factor is loaded in the inner cannula or in a component of the inner cannula), the device can contain a growth factor concentration of at least about 0.1 μg / device, about 0.2 μg / device, about 0.3 μg / device, about 0.4 μg / device, about 0.5 μg / device, about 0.6 μg / device, about 0.7 μg / device, about 0.8 μg / device, about 0.9 μg / device, or about 1.00 μg / device, or within a concentration range defined by any of these values. The growth factor promotes the growth of one or more tissue types.

[0119] The disclosed therapeutic compositions may include prolyl hydroxylase domain (PHD) enzyme inhibitors (i.e., PHD inhibitors), for example, to stabilize 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 may 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-quinolyl]carbonyl]-2,4(1H,3H)-pyrimidinedione (i.e., TM60008), N-[(4-hydroxy-1-methyl-7-phenoxy-3-isoquinolyl)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 dosage 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 dosage range defined by any 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 sleeve or in a component of the inner sleeve), the device can contain a concentration of at least about 0.087 μg / device, about 0.092 μg / device, about 0.097 μg / device, about 0.102, about 0.107 μg / device, about 0.112 μg / device, about 0.117 μg / device, about 0.122 μg / device, about 0.127 μg / device, or about 0.132 μg / device, or within a concentration range defined by any of these values. The PHD inhibitor controls excess collagen deposition at the wound site.

[0120] The disclosed composition may include vitamin A or its metabolites or derivatives or any agent that plays a role in proximal-distal position 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), tretinoin and tazarotene, and combinations thereof. Agents that may play a role in proximal-distal position information include, but are not limited to, bone morphogenetic protein 9 (BMP9), nodular 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 metabolite or derivative, or any agent that plays a role in proximal-distal location information, may be present in the therapeutic composition at a dosage of at least about 0.03 μg / ml, about 0.06 μg / ml, about 0.09 μg / ml, about 0.12 μg / ml, about 0.15 μg / ml, about 0.18 μg / ml, about 0.21 μg / ml, about 0.24 μg / ml, or about 0.27 μg / ml, or within a dosage range defined by any of these values. When vitamin A or its derivatives (or an agent that plays a role in proximal-distal position information) is present in a component of the disclosed device (e.g., when vitamin A or its derivatives or an agent that plays a role in proximal-distal position information is loaded in the inner sleeve or in a component of the inner sleeve), the device can contain a concentration of vitamin A or its derivatives or an agent that plays a role in proximal-distal position information of at least about 0.03 μg / device, about 0.06 μg / device, about 0.09 μg / device, about 0.12 μg / device, about 0.15 μg / device, about 0.18 μg / device, about 0.21 μg / device, about 0.24 μg / device, or about 0.27 μg / device or within a concentration range defined by any of these values.

[0121] The disclosed therapeutic compositions may include lipid mediators and / or ω-3 fatty acid metabolic byproducts (i.e., anti-inflammatory agents) that promote resolution of inflammatory responses. Suitable lipid mediators may include derivatives (e.g., metabolic byproducts) of ω-3 fatty acids that promote resolution of inflammatory responses (i.e., anti-inflammatory), and / or derivatives of eicosapentaenoic acid or docosahexaenoic acid. Exemplary lipid mediators may include, but are not limited to, resolvins such as resolvin 5, interleukin 6 (IL-6), interleukin 4 (IL-4), tumor necrosis factor-α (TNF-α), nuclear factor kappa-light chain enhancer of activated B cells (NF-kB), and combinations thereof. Optionally, the lipid medium can be present in the therapeutic composition at a dosage of at least about 0.006 μg / ml, about 0.012 μg / ml, about 0.018 μg / ml, about 0.024 μg / ml, about 0.030 μg / ml, about 0.036 μg / ml, about 0.042 μg / ml, about 0.048 μg / ml, or about 0.054 μg / ml, or within a dosage range defined by any of these values. When the lipid medium is present in a component of the disclosed device (e.g., when the lipid medium is loaded in the inner sleeve or in a component of the inner sleeve), the device can contain a lipid medium concentration of at least about 0.005 μg / device, about 0.011 μg / device, about 0.017 μg / device, about 0.023 μg / device, about 0.029 μg / device, about 0.035 μg / device, about 0.041 μg / device, about 0.047 μg / device, or about 0.053 μg / device, or within a concentration range defined by any of these values.

[0122] The disclosed therapeutic compositions may 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 may 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 dose range defined by any of these values. When the growth hormone or steroid is present in a component of the disclosed devices (e.g., when the growth hormone or steroid is loaded in the inner sleeve or in a component of the inner sleeve), the device can contain a growth hormone or steroid concentration of at least about 0.1 μg / device, about 0.2 μg / device, about 0.3 μg / device, about 0.4 μg / device, about 0.5 μg / device, about 0.6 μg / device, about 0.7 μg / device, about 0.8 μg / device, about 0.9 μg / device, or about 1.0 μg / device, or within a concentration range bounded by any of these values.

[0123] The disclosed therapeutic compositions may include a depolarizing agent. Suitable depolarizing agents may include, but are not limited to, ionophores (e.g., ion channel openers or blockers). Suitable depolarizing agents may include, but are not limited to, monensin, potassium gluconate, sodium gluconate, and the like.

[0124] The disclosed therapeutic compositions can be used to treat a subject in need of treatment. "Subject" as used herein refers to a human or an animal. Typically, the animal is a vertebrate such as a primate, a rodent, a domestic animal, or a game animal. Primates include chimpanzees, crab-eating monkeys, spider monkeys, and macaques, for example, rhesus monkeys. Rodents include mice, rats, marmots, ferrets, rabbits, and hamsters. Domestic and game animals include cattle, 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 aspects described herein, the subject is a mammal, for example, a primate, for example, a human. The subject can be male or female. Preferably, the subject is a mammal. The mammal can be a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow, but is not limited to these examples. Mammals other than humans can be used as subjects representing animal models of tissue repair, regeneration and / or reconstruction. In addition, the methods and compositions described herein can be used to treat domesticated animals and / or pets.

[0125] 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, cellulite, blood tissue, visceral muscle, smooth muscle, skeletal muscle, cardiac muscle, and neural tissue.

[0126] In certain embodiments, the disclosure provides methods of administering an effective amount of a disclosed therapeutic compound (including a disclosed compound) to a subject 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, a provided therapeutic compound or a provided hydrogel is contacted with a traumatized or injured appendage or tissue within a device provided herein.

[0127] The disclosed devices and / or therapeutic compositions promote 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 tissue treated with a device and / or therapeutic composition according to the present disclosure relative to tissue not treated with the disclosed devices and / or therapeutic compositions.

[0128] Animal model experiments

[0129] Anurans that have matured to puberty can regenerate their amputated or injured limbs when exposed to regeneration-inducing agents delivered via slow-release beads implanted in the amputated tissue. However, the completely non-regenerative, strongly post-metamorphosed (adult) African toads are unable to regenerate their hind limbs after amputation and instead produce featureless cartilaginous spikes (Suzuki et al., (2006) TheScientificWorldJOURNAL,6 .). This model is used to test whether regeneration inducers can stimulate regeneration.

[0130] As described in the following examples, complex interventions were tested on adult African toad hind leg amputations 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 lengthy endogenous morphogenetic cascade without ongoing micromanagement. Multiple stimuli were selected that induced pro-regenerative activity, such as agents that reduced inflammation, promoted neural sparing, and induced overall growth.

[0131] It was found that a short exposure period (e.g., 24 hours) to a wearable bioreactor containing silk injected with several small molecule compounds induced significant growth halos, shaping, and sensorimotor function after amputation in African frogs. Treated animals showed a significant delay in wound closure, followed by long-term (about 16 months) growth results, including increased bone length, soft tissue shaping, and neuromuscular repair. Histologically, the new limbs contained nerves, smooth muscle indicating 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 experiments also revealed a rapid response to the complete treatment device (compared to sham controls) in the brain. Regenerated bones showed anatomical features unique to wild-type morphology, and distal limb soft tissues showed toe-like protrusions. In addition, the animals used the newly formed limbs to walk, similar to wild-type frogs. In addition, in animals exposed to full treatment conditions, sensorimotor pathways were restored, indicating that tissue remodeling includes the re-extension or regrowth of sensory afferent nerves and neuromuscular tissue interfaces.

[0132] These data demonstrate that a highly dramatic, prolonged regenerative response can be induced in adult Xenopus by brief triggering, without the need for gene therapy or stem cell implantation, and reveal the molecular, cellular, and tissue-level components of this process both at the site of injury and in the distal brain.

[0133] The following examples illustrate in detail the manner in which the present disclosure may be used or implemented and will enable one of ordinary skill in the art to more readily understand its principles. The following examples are presented by way of illustration and are not meant to be limiting in any way. Example

[0134] animal

[0135] Adult female African clawed frogs (Xenopus laevis) (n=115) measuring 5 cm to 6.25 cm (nose to tail) (Nasco, Fort Atkinson, WI) were allowed to acclimate to the tanks for 2 weeks prior to the experiment. The animals were maintained in 10 L plastic tanks containing prescribed frog water (Reef Salt, Seachem Laboratories, approximately 1.65 kΩ conductivity, 7.8-8.0 pH) at 18°C ​​and exposed to a 12-hour light-dark cycle. Prior to the experiment, the animals were soaked in a broad-spectrum gentamicin antibiotic for 2 hours (Gibco, Fisher Scientific, USA) to minimize bacterial contamination of the amputated stump.

[0136] Limb Amputation

[0137] According to Golding et al. (2016), PLoS One 11 ,e0155618 and Herrera et al. (2018), Cell Rep 25 Hindlimb amputation was performed according to previously established protocols published by 1593-+. Briefly, animals were first anesthetized by systemic immersion in buffered frog water containing 0.05% benzocaine. After loss of the toe pinch reflex, 75 mg / kg 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 tibia and fibula using a straight incision with a sterile microsurgical blade. No bone resection was performed, nor was a tissue flap sutured at the site of the trauma. After hemostasis, the animal regained consciousness and was allowed to recover in sterile frog water for at least 60 minutes.

[0138] Device Attachment

[0139] Animals were randomly assigned to one of three treatment conditions: no device, BioDome only, or BioDome with a cocktail treatment (described below in "BioDome Manufacturing and Cocktail Composition"). The device attachment procedure was preceded by a second anesthesia (0.05% benzocaine soak, 75 mg / kg buprenorphine). The unconscious animals were then fitted with the device, which was attached to the amputation site stump using monofilament surgical sutures (7-0 Monosof, 18" P-16 cutting, Covidien, USA). Two sutures passed through the dermis on either side of the leg. These sutures were sufficient to keep the device in place without damaging the underlying deep fascia layer. After attachment, the animals were returned to their housing tanks, where they regained consciousness and were allowed to swim freely. Control animals were treated similarly to animals receiving the device, but the wound stump did not have the device attached. The results presented herein were produced using a sutured biodome.

[0140] Alternatively, an adjustable biodome can be used. The attachment procedure of the adjustable BioDome includes pushing the amputated limb through the cap and the annular spacer into the appropriate position and fixing the limb using a bioadhesive Skin-Tite Bioadhesive, Smooth-On, PA). The biodome insert containing the bracket is attached to the amputated toe and fixed with a small amount of adhesive. The acrylic protective cap is screwed into the top cap, and then the insert is pushed close to the wound bed with a custom gasket and closed with a second cap. In order to keep the tissue moist during attachment, about 4 μL of sterile phosphate buffered saline (PBS) 1X is slowly injected into the insert using a 31G needle. PBS is replaced every 2 days to remove cell waste and keep the tissue fresh. The device is kept attached until the animal is killed for subsequent analysis.

[0141] BioDome Manufacturing

[0142] The sutured BioDome consists of a soft silicone insert that contains silk hydrogel as a controlled release matrix and drug carrier. The fabrication of the device has been reported elsewhere (Golding et al., (2016) PLoS One 11 Briefly, an outer cylindrical silicon sleeve (20-mm H x 18-mm D) was fabricated by casting a silicon elastomer (Dragon skin 10, Smooth-on, Macungie, PA) on a 3D printed mold designed using CAD software (Solidworks, Waltham, MA, USA) and printed using a Formlab 3D printer (Somerville, MA, USA).

[0143] The adjustable biodome consisted of acrylic tubing (#8532K13, Mcmaster-Carr, Elmhurst, IL) cut into 1-cm long cylindrical tubes, which was 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 septa were punched with a 3-mm biopsy punch to create entry holes for the animal's limbs. To provide more room for the animal's legs to pass through the holes, the septa were cut along the center hole at four locations, one-quarter circle apart from each other. Custom gaskets 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 clear polyester membrane filter (0.45 μm pore size, 12-μm thickness, #1300016, Sterlitech, Kent, WA). The filter was cut into rectangles (7 mm x 5 mm) and rolled around a 1.5-mm diameter metal rod (#8907K62, McMaster). The wall was then bonded to the silicone bottom portion using a silicone adhesive (Dragon Skin 10FAST, Smoothon, Macungie, PA) to complete the insert.

[0144] The protective cage is assembled from a threaded cap, a clear acrylic body, and an adapter. An annular septum that allows only one-way bending is provided to prevent the device from being dislodged due to animal movement and tampering. A custom gasket together with the bottom cap provides adjustable pressure that is sufficient to hold the stent insert against the wound bed and maintain its position stable during long-term experiments. It also has an access hole for media exchange. The device insert contains a membranous side wall and a silicone bottom. The side wall can contain the liquid required to keep the tissue moist and promote gas exchange, and the silicone bottom serves as a septum for inserting a needle for media exchange.

[0145] Device Disassembly and maintenance

[0146] After 24h, the animals were anesthetized and treated with analgesics as described above. The device was then removed by cutting the single suture on either side of the leg, and the frog was returned to a jar containing a fungicide (Kordon methylene blue, a concentration of 1mL / 10L frog water). After another 24 hours, the water was replaced with fresh 100% frog water. Once their device was removed, the animals were maintained in frog water that was changed every day for 18 months. Terminal euthanasia was performed by full body immersion in frog water containing 0.2% benzocaine. Regeneration, contralateral limbs and brain tissue were collected and processed for histological analysis.

[0147] Wire processing

[0148] Prepare silk fibroin solution as follows: 5g silkworm (Bombyx mori) cocoon (Tajima Shoji, Yokohama, Japan) is cut and degummed in 0.02M sodium carbonate (Na2CO3) solution for 45min to remove non-essential protein substrate (i.e., sericin). Fiber is washed several times in deionized (DI) water to remove Na2CO3, and then dried overnight in a fume hood at 22°C. Dry silk fiber is then dissolved in 20% (w / v) solution of 9.3M lithium bromide (Sigma-Aldrich, St.Louis, MO), and placed in an oven set to 60°C for 4 hours. Then the solution is dialyzed in deionized water using a dialysis cassette (molecular weight cutoff of 3.5kDa, Thermo Fisher Scientific, Waltham, MA), while gently stirring. Change water six times in the 48 hour stage. The dialyzed solution was centrifuged 3 times at 13,000 g, 4° C. for 20 minutes each time, and then filtered through a cell strainer (40-μm pore size, ThermoFisher) to remove impurities. To determine the concentration of the filtered solution, a 0.5 ml sample was completely dried overnight in an oven. 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 0.5 ml initial volume.

[0149] Silk hydrogels were formed by cross-linking liquid silk fibroin. 45-mb silk (3% w / v) and horseradish peroxidase (HRP) solution (20 U / ml) were poured into a 24-well plate and incubated at 37°C for 45 minutes to complete gelation. According to Golding et al. (2016), PLoS One 11 , method e0155618 tests the gel compression strength and modulus of the gel.

[0150] Bracket with alignment holes

[0151] Silk scaffolds with aligned holes were made 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 merging the plates in liquid nitrogen (LN2). Finger-like ice crystal columns grown from the cold surface established channel-like structures inside the silk solution as it solidified. After cooling for 10 minutes, the frozen solution was lyophilized 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 sterile conditions until use.

[0152] Collagen scaffolds with aligned channel-like porous structures were fabricated by controlled directional freezing and freeze drying of 1.5% (wt / wt) collagen solution (in a similar manner to silk scaffolds). Scaffolds were cut into cylindrical shapes (4-mm length and 1.5-mm diameter) and placed in a biodome insert using forceps.

[0153] Material characterization

[0154] The morphology of the support was characterized using scanning electron microscopy (SEM) and fluorescent optical microscopy. For SEM imaging, the support was cut in half using a razor blade to expose the internal geometry of the hole. Before imaging, the support was sputtered gold-plated to increase conductivity. SEM imaging was performed on a microscope (Zeiss EVO MA10) set to 5kV. For fluorescent imaging, the support was stained with 2 μg / ml fluorescein isothiocyanate (FITC) in PBS and imaged using a Keyence microscope (BZ-X800, Keyence, Japan). The compression stiffness and elastic modulus of the support were determined using Instron Testing System. Fig. 8A It is a silk scaffold with channel-like pores aligned along the long axis. Figure 8B It is a silk scaffold with holes aligned perpendicular to the long axis. Figure 8C is a SEM image of a collagen scaffold with pores aligned along the long axis. Fig.8D It is a collagen scaffold with pores aligned perpendicular to the long axis.

[0155] Therapeutic Compositions

[0156] The hydrogel was 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 silicon cannula and allowed to gel for 30 minutes before being 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), and 0.015 μg / ml of retinoic acid (Catalog No. 11017, Caymen Chemicals, MI, USA) were loaded into the liquid silk solution, and then the silicon cannula was inserted and gelled.

[0157] In vitro release studies.

[0158] In order to determine the release curve of the drug used in this embodiment, 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 gelation. Then, 1 ml of Dulbecco's phosphate buffered saline (DPBS 1X, Gibco) was added to each bottle and then incubated at 37°C. At a fixed time point, 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 a solution with a known concentration. All release experiments were performed in triplicate to ensure accuracy. For RA and 1,4-DPCA, the optical density of the release solution was determined on a UV-transparent 96-well plate (Corning, Corning, NY) using a microplate reader SpectraMaxM2 (Molecular Devices, San Jose, CA) run by SoftMax Pro 6 software. 1,4-DPCA and RA solutions were detected at 280nm and 350nm wavelengths, respectively.

[0159] The concentration of BDNF and GH in the released samples was 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 using a protein filter column (MWCO = 3 kDa, #UFC500324, Fisher) to remove high molecular weight silk fibroin content. The optical density of the samples was then measured at a wavelength of 244 nm using a SpectraMax M2 plate reader.

[0160] Soft tissue imaging

[0161] The soft tissue remodeling and bone regrowth of animals were regularly evaluated during the maintenance period of 18 months. Animals were anesthetized as previously described, and a DSLR camera (Canon EOS Rebel T7i) was used to capture high-resolution images and regenerated body sizes of their wound sites. In order to ensure reproducibility, the amputation plane was used as the standard reference point for all measurements. The amputation site was easily identified because the limbs gradually became thinner at the incision point. Each measurement consisted of a linear assessment of the length between the farthest end of the amputation site and the regenerated body.

[0162] in vivo , X-ray and micro-CT bone imaging

[0163] In addition to soft tissue measurements, according to Golding et al. (2016) PLoS One 11 ,e0155618 and Herrera-Rincon, et al., (2018) Cell Rep 25,1593-+ imaging scheme, using handheld x-ray device (Nomad Pro2TM) using 60kV, 2.5mA standard imaging settings and 0.20s exposure time assessment bone length.Each animal receives the same dose (0.12mSv) at a fixed time point.Computerized tomography (CT) is carried out in viva CT 40 scanners (Scanco Medical, Switzerland) to visualize the detailed microstructure of bone at the end of the 18-month regeneration stage after benzocaine overdose (0.2% whole body immersion) euthanasia.Distal trabecular bone and axial bone cortical slices (615 slices / animal, 76 μm / slice, 300ms integration time) are visualized and presented as 3-D images to further quantify.Radiation dose uses local CT dose index (CTDI) according to established manufacturer's guidelines, ranging from 453mGy to 1255mGy.

[0164] Histology and immunohistochemistry

[0165] In order to characterize the effect of treatment on limb regeneration and remodeling after amputation, histological analysis was performed on the regenerated body and the contralateral limb at fixed intervals over time. Tissues were collected at 18mpa. 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) (pH7.4). Once decalcified (confirmed using x-rays), the tissue was gradually balanced to 30% sucrose and then embedded in OCT (Sakura FInetek, the United States). The samples were frozen in liquid nitrogen. Limb tissue was sliced ​​continuously with 14 μm using a cryostat (Leica CM1850) and placed on a slide. From the tibia and fibula region above the original amputation site to the molding region at the end of the limb, a cross-section was taken across the limb at intervals of 14 μm. In order to visualize the molding, horizontal slices were made to the end portion of the limb at intervals of 14 μm. The slices were dried for at least 1 hour before being stored at -80°C.

[0166] 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 for acetylated α-tubulin (1:100), TGF-β (1:250), smooth muscle actin (1:100), laminin (1:100), fibronectin (1:500) and phospho-histone H3 (1:250) were used. Except for anti-smooth muscle actin and anti-laminin (which were stained together), slides were stained separately with each antibody. The primary antibodies were incubated on the slides overnight. After washing in PBS, alexa-fluor secondary antibodies (1:500, ThermoFisherScientific) were applied in blocking buffer for 2 hours. Slides were washed again in PBS and stained with DAPI 1:200 in PBS for 20 min. Slides were mounted in Fluoromount-G (ThermoFisher Scientific) and cured for at least 24 h before imaging.

[0167] Sections were imaged using the EVOS FL automated imaging system (ThermoFisher Scientific). All sections were collected and stitched together for analysis.

[0168] Analysis of immunostained sections

[0169] All statistical analyses were performed in IBM SPSS version 20 software. Normality assumptions were tested prior to using parametric tests, including ANOVA, t-tests, and correlation analyses (Pearson's r). Nonparametric analyses, Mann Whitney Wilcoxon tests, or Kruskal-Wallis tests were performed to compare data that were not normally distributed. Significant differences were assumed if the p value was below a threshold of 0.05 (two-tailed hypothesis testing).

[0170] Assessment of sensorimotor thresholds

[0171] In order to evaluate the sensory motor ability of the regenerator, the animals were evaluated 18 months after amputation. Each animal was placed in a glass jar equipped with 2L frog water and adapted for 5 minutes until the movement stopped completely. A video camera (iPod Touch the 5th generation, Apple, CA, the U.S.) was placed above the shell to capture the record of the test procedure. Standardized Von Fry filaments (Touch Test, Stoelting, IL, the U.S.) were used to evaluate the sensory threshold of the regenerator. The filaments within the range of 0.008g to 300g force were applied to the distal portion of the regenerator, and the force was from the lowest to the highest. The first filament causing a clear response (moving from a static position) was recorded. Animals were tested twice in the 2-day stage, and average thresholds were reported.

[0172] Statistical analysis

[0173] All statistical analyses were performed using IMB SPSS v20. First, data were tested for homoscedasticity by Levene's test. Single factor analysis was performed on normally distributed data by unpaired two-tailed student's t-test (two independent groups) or one-way ANOVA test (multiple independent groups) and subsequent post hoc Scheffe's test (when P<0.05). When the variable "time" was considered, a two-way ANOVA was performed. The statistically significant differences between the treatment groups (no device, Biodome and mixture treatment only) at each specific time point were determined by using student's t-test. In non-normally distributed data, Kruskal-Wallis test and subsequent post hoc Dunn test (when P<0.05) were performed respectively. In all cases, the significance level was set to 0.05. Statistical values ​​were reported as mean ± standard deviation or mean ± standard error of the mean, where indicated. Where appropriate, dot plots or scatter plots were used to highlight the individual variability within each experimental group.

[0174] RNA extraction

[0175] After amputation, device attachment and removal, and 24 hours of treatment, regenerated tissue was harvested for next generation sequencing (NGS) at 11, 24, and 72 hours after amputation. Samples consisted of 1 cm thick tissue blocks from distal trauma sites. Brains were also collected and flash frozen. Tissues were extracted using TRIzol (ThermoFisher Scientific) according to the manufacturer's protocol, and total RNA quality and quantity were assessed using a Nanodrop spectrophotometer (ThermoFisher Scientific).

[0176] Next Generation Sequencing (NGS)

[0177] 1.1 μg of total RNA was sent to Tufts Genomic Core. RNA quality was assessed by 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 50nt sequencing was performed on an Illumina HiSeq 2500. The raw read files were sent to the Bioinformatics and Biostatistics core at Joslin Diabetes Center.

[0178] NGS analysis

[0179] The reference genome of Xenopus laevis was downloaded from the NCBI genome database collection GCA_001663975.1. The genome was aligned using the STAR aligner (Dobin et al., Bioinform .(2013);29(1):15-21.doi:10.1093 / bioinformatics / bts635.Electronic publication on October 25.PubMed PMID:23104886;PubMed Central PMCID:PMC3530905.) calibrated the reads and compared the calibrated reads with featureCounts (Liao et al., (2014) Bioinform. 30(7):923-30). Genes with expression counts exceeding 1 count per million (cpm) in at least 3 samples were included in the analysis, and counts were weighted by trimmed mean of M-values ​​(TMM, Robinson et al., (2010) Genome Biol .11, R25.) normalized. Voom transformation (Law et al., Genome Biol.15, R29) to convert counts to logCPM, where CPM = 1e+6*gene counts / (total counts of sample*normalization factor of sample). Voom transformation also estimates the mean-variance relationship and uses it to calculate appropriate observation-level weights, so that greater read depth gives greater weight. To further reduce the weight of outliers, sample-specific quality weights (Ritchie et al., (2006) BMC Bioinform .7,261.) and combined with observation-level weights.

[0180] Differentially expressed genes were identified using limma (Ritchie et al., Nucl.Acids Res .43,e47.). A moderated t-test was performed to detect differentially expressed genes between the two groups. Genes with a FDR < 0.25 were considered to have significantly changed.

[0181] Gene sets for pathway analysis were obtained from MSigDB Collections, and gene sets belonging to canonical pathways (CP) or gene ontology (GO) were selected. Analysis was 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.). If P < 0.05 and FDR < 0.05, the coordinated up, coordinated down, and mixed gene sets were considered significant.

[0182] Network analysis

[0183] Gene modules were identified by co-expression analysis in CeMiTool in R (Russo et al., 2018) based on logCPM values ​​of control and cocktail-treated groups at 11 h, 24 h, and 7 days after amputation. In CeMiTool, a variance stabilizing transformation was applied to eliminate dependencies between mean and variance parameters, and genes were filtered based on expression levels at a threshold of p < 0.1. Modules of co-expressed genes were identified in the dataset based on an automatically generated scaling value β (β = 10) and a minimum module size of 30 genes. To assess how the richness of these modules varied over time and across groups, module richness was calculated based on sample annotations. To determine which biological functions were associated with each module, over-representation analysis was performed using the pathway database from Reactome, and pathways were considered significant at p < 0.05. The annotated module map combined gene-gene interaction data from the Kyoto Gene and Genome Database (KEGG) and Chemical and Genetic Perturbations (CGP) to plot the interacting genes contained in each module.

[0184] qPCR method

[0185] The same total RNA submitted for RNAseq analysis was treated with DNA enzyme using RQ1 RNAse-free DNA enzyme kit (PromegaCorp., Madison, WI, USA). The obtained RNA (0.5 μg) was treated with DNA enzyme for the second time, and then cDNA synthesis was performed using Verso cDNA synthesis kit (ThermoFisher Scientific). The amount of gene product was quantitatively analyzed using 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 value of all ND expressions across time points was used as a calibrator for all samples.

[0186] result

[0187] 1. Inducing leg regeneration through multi-drug regeneration treatment

[0188] Adult Xenopus hindlimbs were amputated at the midpoint of the tibia and fibula and equipped with a biodome device containing either a silk-based hydrogel with a 5-drug multidrug therapy (MDT) or a hydrogel alone. Control animals were left untreated following amputation. After 24 h of exposure to the BioDome, the device was removed and the animals were maintained for up to 18 months with regular assessment of regeneration and remodeling of the hindlimb regenerators. The extended observation window was chosen based on calculations by Alibardi (2018) that predicted that approximately 1.5 years would be required to regenerate anuran limbs based on a projection model using the diameter of the salamander stump.

[0189] X-ray images were obtained to measure limb length as a function of months post-amputation. X-rays showed that regenerators associated with the biodome-only and multidrug treatment (MDT) conditions were longer than control (no device) regenerators after 4 months, an effect that disappeared after 4 months and then reappeared at 8 months and persisted over time (two-way ANOVA, between-subject factor treatment exposure, within-subject factor regeneration time F(2,19)=61.9, p<0.05). As early as 0.5 months post-amputation (mpa) and persisting to 4 mpa, the MDT group had greater limb length (relative to the amputation site) relative to the other groups (p<0.05), suggesting that MDT increased early growth rate. Growth slowed significantly at 6-8 months, and hindlimb regenerators associated with the Biodome-only group reached lengths comparable to animals exposed to MDT (p>0.05). The secondary increase in late growth after 9 MPa indicated that the MDT group again showed longer limbs relative to the other groups, an effect that persisted until the final measurement at 18 MPa (p<0.01). Thus, the MDT group showed not only a final increase in leg length compared to the other treatment groups, but also a secondary growth phase that was not present in the other treatment groups, particularly the no device group. These data suggest that brief exposure to MDT and the BioDome facilitates longer leg regrowth.

[0190] Not only was leg length increased, but 76% of animals exposed to MDT also exhibited thicker, more complex regenerator morphology compared to the featureless, heavily pigmented spikes of the No Device group. Specifically, the distal segments of the hindlimb regenerators associated with the MDT group exhibited a flattened, paddle-like structure with protruding buds that are characteristic of the digits. In contrast, the No Device and Biodome-only conditions were only associated with featureless regenerator spikes, provided that the MDT condition reliably produced a well-formed, paddle-like morphology with the presence of distal buds. The BioDome group had an intermediate phenotype, with 20% of BioDome animals exhibiting thicker cartilaginous spikes and hook-like distal protrusions with limited shaping. None of the animals in the No Device group displayed a unique phenotype.

[0191] 2. Induced legs show sensory function

[0192] Next, to assess whether the 18 MPa regenerators had recovered 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 maximum force was applied. The first filament that elicited a clear response (movement from a static position) was recorded, and the behavioral measurements were averaged over 2 days. MDT-treated hindlimb regenerators showed a stimulus-response pattern comparable to that of the non-amputee group (p>0.01; Fig.10 ), indicating significant reinnervation and neuromuscular reintegration. In the biodome group, the response was significantly different between the animals, with one group showing near-normal levels, another group unable to detect stimulation (56.6 ± 98.8g), and untreated animals reliably failing to show any response to the application of any value of force up to and including 300g. Therefore, we conclude that MDT treatment promotes sensory motor integration similar to that of unamputed limbs. Interestingly, the biodome group alone is sometimes also able to promote the re-growth of limbs with intact sensory systems, suggesting that hydration and structural support may be important for maintaining innervation and promoting nerve re-growth.

[0193] 3. MDT exposure increases hindlimb bone length and complexity

[0194] Micro-CT and X-ray images were used to characterize the internal structure of the regenerated bodies. Fig. 9 ), and the bone component of the regenerants was subsequently investigated. Micro-CT and X-ray images of the regenerants showed an increase in bone volume and length associated with the provided MDT composition compared to the biodome alone and the control.

[0195] X-ray images confirmed the presence of complex morphology and increased bone length in the MDT regenerates relative to the biodome-only and no-device control experiments. Starting with measurements at 2.5 MPa, dense tissue protruding outward from the amputation site was observed in the MDT and biodome groups but not in the no-device control 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 at 4 MPa (p<0.05) with an inflection point at approximately 8 MPa (similar to soft tissue measurements). As shown in Fig.11 As shown in , differences in bone length between the MDT and Biodome groups were regularly observed, but the length differences between those groups and the device-free controls were consistently maintained (p<0.05).

[0196] To better understand the microarchitecture of the regenerated bone, microCT was performed at 18 MPa. MicroCT imaging allows for visualization of the underlying bone in 3 dimensions and measurement of bone volume without disturbing the external soft tissue. The microCT data confirmed the presence of significant bone fragments in the distal region of the limb following MDT. Notably, the fragments did not occur at the amputation site but rather spontaneously arose after a segment of growth at a bone point equivalent to the contralateral joint. These remodeled segments were common in the MDT group. As shown in Fig.12 As shown in , the bone volume measured by microCT data was larger for the hindlimb regenerations of the MDT group relative to the other groups. Volume quantification confirmed the increased growth in the provided MDT regenerations (one-way ANOVA (F(2,15)=11.15, p<0.001)). X-ray tomograms also showed significant similarities between the skeletal anatomy of the unamputed and MDT-treated. Remodeling was evident in the provided MDT regenerations, including the re-expression of bone features typically associated with muscle attachment. Specifically, at 17 MPa, the MDT group showed bone features unique to muscle attachments and the presence of joint segmentation. These data indicate that the MDT condition is associated with significant remodeling of the skeletal anatomy, consistent with the active process of remodeling the bone, and ultimately achieves a morphological complexity close to the features of the amputated forelimb.

[0197] 4. Complexity of the regenerated limb

[0198] While MDT-treated animals did show increased bone growth, molecular changes at the cellular level in the regenerating limb were also assessed. Immunohistochemistry was performed to assess tissue architecture associated with regeneration and remodeling after 18 MPa. Fig.13 As shown in , reinnervation measured by acetylated α-tubulin (AAT) staining showed a significant increase in the number of AAT+ nerve bundles associated with the 24-hour MDT condition compared to the no device group (U=13, P=0.0014). The number of AAT bundles was comparable when comparing the no device and biodome-only groups, suggesting that MDT treatment affects nerve bundle regrowth and innervation of the regenerators. Not only were there more bundles, but the 24-hour MDT group also exhibited significantly larger AAT bundle diameters relative to the no device group, as shown in Fig.14 There was no significant increase in bundle size between the biodome-only and no device groups.

[0199] Kruskal-Wallis test indicated that the number of AAT positive fascicles at 18 MPa was significantly different between 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 fascicles associated with the 24-hour MDT condition compared to uninjured animals, U=13, P=0.0014. When comparing uninjured animals and the biodome-only group, the number of AAT fascicles was comparable (p=0.1812). Next, we examined ATT fascicle size, which revealed significant differences between conditions, H(11.74)=p=0.0028. The 24-hour MDT group exhibited larger ATT fascicle diameters relative to uninjured animals. Again, no significant differences were identified when comparing the biodome-only and uninjured animal groups.

[0200] To assess changes in connective tissue architecture, we evaluated the expression pattern of fibronectin after 18 MPa. Fig.15 As shown in Figure 2, increased particle complexity in MDT-treated regenerants mirrored the degree of structural complexity observed under MDT conditions. Specifically, fluorescent images revealed increased neural regeneration and extracellular matrix remodeling in regenerants 18 months after initial amputation and exposure to multi-drug treatment. Consistent with other results, the 24-hour MDT group showed increased particle complexity relative to the biodome-only and no-device groups, suggesting that MDT itself may contribute to the elaboration of cartilage and connective tissue in the regenerants.

[0201] To assess vascularization of the regenerates, laminin and smooth muscle actin (SMA) expression at 18 MPa was compared between the different conditions, as in Fig.16 As shown in . Fluorescent images of cross-sections of regenerants obtained 18 months after amputation were double stained for two markers of vascularization, one associated with smooth muscle (SMA-red) and the other associated with 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 no-device groups (H(19.84)=p<0.001), indicating that MDT promoted vascularization of the regenerants, with the number of blood vessels in the MDT-treated regenerants being more than twice that in the no-device condition.

[0202] 5. Wound closure, increased Sox2 expression, and re-epithelialization

[0203] Given the significant long-term outcomes, the effects of MDT at early time points after amputation were evaluated early in the procedure. We first noted that wound closure at 0.5 MPa was significantly reduced in the MDT group compared to non-device animals (p < 0.05; Fig.17 Animals exposed to MDT showed a mean wound diameter of 2.02 cm ± 0.40 cm, significantly larger than the biodome group (1.25 cm ± 0.41 cm) or the no device group (0.56 cm ± 0.17 cm). Delayed wound closure predicted successful remodeling at 18 MPa, with animals with the largest wound sites at 0.5 MPa showing the most growth and remodeling at 18 MPa.

[0204] Without being bound by a particular theory, it is thought that a larger wound site will provide a larger bud base, which may contribute more material to limb regeneration. Fig.18 As shown in , blastema proliferation was assessed by immunohistochemistry of SOX2 (a proliferative cell marker). Compared with other groups, there were significantly more cells expressing SOX2 in the MDT group (p < 0.05), indicating that more proliferative tissues produced new limbs. Finally, compared with the control group, the soft tissue at 2.5 months had similar predictability, with thicker tissue and increased bone length in the MDT hind limbs. The fluorescent images obtained at 2.5 MPa revealed that 24-hour exposure to the MDT composition increased dryness, and the inhibition of wound formation led to longer regenerated bodies in the early regeneration process. Increased soft tissue growth and re-epithelialization were also observed in the MDT exposure group 2.5 months after amputation, which reflected the increase in bone tissue length confirmed by x-ray imaging.

[0205] 6. Transcriptome Analysis of Regeneration Induction

[0206] 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 transcriptome of blastema obtained from MDT relative to untreated animals at 11 hours, 24 hours, and 7 days after amputation. Heatmaps comparing gene expression levels in MDT animals to no device treatment showed significant differences in gene expression at 11HPA that persisted until 24HPA. However, by 7DPA, dynamic gene expression levels returned to normal, indicating that there is a phase of dynamic gene expression within 24 hours of amputation.

[0207] After multiple trials corrected 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 to be those transcripts that passed the FDR and those transcripts that showed a log2 fold change of 2. When the blastema of MDT animals was compared with untreated animals, the expression profile had large dynamic changes within 7 days after amputation. After the activity conversion after 7 days, the same genes that were over-expressed or under-expressed experienced 24 hours. In order to narrow the expression of the significant changes of these genes, the FDR was set to a fold change of log3 or 3, and the first 15 differentially expressed genes were compared between groups. When the blastema tissue processed by MDT was compared with the wild-type blastema tissue, it was found that the first 15 highly upregulated genes were related to neuroregulation in the brain (e.g., brain-specific kinase (BRSK), neuropeptide FF, D1C dopamine receptor, neuroligin), which had the highest expression 11 hours after amputation. The expression level decreased at 24 hours and 7 days (dpa) after amputation. Wnt7a, a gene involved in the development of the anterior-posterior axis, was also upregulated at 11 h post amputation (hpa) and subsequently increased at 7 dpa. In contrast, the major genes downregulated were mainly related to muscle structure (myosin-4, microfibril-associated glycoprotein) and metabolism (e.g., myofasciin). The pattern of these differentially expressed genes between MDT-treated and control blastemas was opposite to that of the upregulated genes, as the expression of the downregulated genes increased from 11 hpa to 7 dpa.

[0208] Highly regulated genes in MDT animals were compared to genes in non-device animals. Upregulated genes included nervous system-specific transcripts, suggesting an important role for neuroprotective proteins just after amputation. Downregulated genes included metabolism- and muscle-related transcripts, suggesting that resources are being diverted from muscle maintenance to tissue homeostasis. GO analysis of metabolic and biosynthetic pathways revealed early downregulation (at 11 and 24 hpa), with rates increasing at 7 dpa. Table 1 explains gene expression levels in MDT animals compared to non-device treatments at 11 hpa, 24 hpa, and 7 dpa.

[0209] Table 1

[0210]

[0211]

[0212] Richness analysis of identified gene classes showed significant differences in the profiles of biological processes between groups. A web-based version of CEMiTool (Co-Expression Module Identification Tool) was used to identify co-varying gene sets in MDT (CT) and device-free (ND) animals. Co-varying gene sets with high fold changes were classified into modules (M1-M4).

[0213] Early after amputation (11hpa), there are more genes involved in metabolic regulation that differ between MDT and controls. In addition, there is more dynamic gene expression at this time point. This subsides later (7dpa), as well as significant changes between MDT and reorganized tissue or reset cell function landscape.

[0214] To reveal the types of processes modulated by MDT exposure, enriched pathways were grouped considering “large-scale functions”. Co-expression analysis identified four gene modules across control and cocktail-treated groups at 11 hours, 24 hours, and 7 days after amputation. These modules represent classifications of genes based on their shared expression levels and statistical significance. Module 1 contained 607 genes that were significantly represented in extracellular matrix organization, collagen formation, collagen biosynthesis and modification enzymes, and hemostasis pathways (p<0.00509), which may represent tissue destruction after injury. Module 2 contained 142 genes that were significantly represented within pathways related to cell junction organization, laminin interactions, cell-cell communication, apoptotic cleavage of cell adhesion proteins, and non-integrin membrane ECM interactions (p<0.00009), which may represent cell-cell communication and adhesion. 105 genes in module 3 were significantly represented in muscle contraction, acetylcholine activity, and myogenesis pathways (p<0.01096). Module 4 contained 54 genes that were significantly (p<0.00142) over-represented in glucose metabolism, muscle contraction, gluconeogenesis, and glycolysis pathways. When modules 2, 3, and 4 were significantly enriched (p<0.00024) and up-regulated in MDT-treated blastema at all time points, it was shown that frogs receiving the pro-regeneration cocktail had a consistent up-regulation of genes related to cell communication, myogenesis, and glucose metabolism relative to the biodome alone and no device controls. In contrast, these modules were enriched (p<0.012) but down-regulated in the group that did not receive the treatment device at all time points evaluated. However, at the 7-day time point, module 2 was significantly up-regulated in the same untreated group (p=0.00074), indicating that there was little difference in the richness of cell-cell communication pathways between the treated and untreated groups 1 week after amputation. At all time points, ECM- and collagen-enriched module 1 was upregulated in the untreated group (p<0.011) and downregulated in untreated samples (p<0.00027).

[0215] In general, M2, M3, and M4 were upregulated under MDT conditions compared with ND conditions at all time points, reaching a peak at 24 hpa. The exception was M1, which was downregulated under MDT conditions, reaching a maximum at 11 hpa and decreasing until 7 dpa.

[0216] 7. Cumulative release of drugs in the provided MDT compositions

[0217] To evaluate the cumulative release of multi-drug therapy (MDT) in the device hydrogel, the hydrogel loaded with each MDT drug was suspended in 1x DPBS (ThermoFisher) and incubated at 37°C for 25 minutes. The supernatant was collected every 5 minutes, and the concentration of each drug was determined by a microplate reader. Each drug / hydrogel mixture showed similar release kinetics, releasing about 70% of the total drug concentration within 10 minutes, and the total amount of the release load did not exceed 80% (except retinoic acid, which released all drugs before 25 minutes).

[0218] Other features, purposes and advantages of the present disclosure are apparent in the following detailed description. However, it should be understood that the detailed description, although indicating the embodiments of the present disclosure, is provided only in an illustrative and non-limiting manner. According to the detailed description, various changes and modifications within the scope of the present disclosure will become apparent to those skilled in the art.

Claims

1. A device for stimulating tissue regeneration at a tissue site of a subject, the device comprising an outer cannula having a tissue receiving end, a compression member receiving end opposite the tissue receiving end, and an interior chamber configured to receive tissue; Pressing member; an inner sleeve disposed within the outer sleeve, the inner sleeve having: an end for receiving a tissue part for regeneration, an engagement receiving end opposite to the end for receiving the tissue part for regeneration and for engaging a pressing member, and an inner chamber configured to receive the tissue part for regeneration, wherein the pressing member is configured to extend through the pressing member receiving end into the interior chamber of the outer cannula and bias the engagement receiving end of the inner cannula toward the tissue so that at least a portion of the tissue site for regeneration is placed in contact with a portion of the interior chamber of the inner cannula; a first end cap engageable with the tissue receiving end of the outer cannula and including an opening configured to receive the tissue; and A second end cap is engageable with the pressing member receiving end of the outer sleeve.

2. The apparatus of claim 1, wherein the tissue is a portion of an appendage or an organ.

3. The instrument according to claim 1 further comprises a first threaded adapter which can be set in the tissue receiving end of the outer sleeve, and is used to selectively connect a first end cap which can be engaged with the tissue receiving end to the outer sleeve, and the first end cap has a groove, and the groove is configured to receive the thread of the first threaded adapter.

4. The instrument according to claim 1 further comprises a second threaded adapter which can be set in the pressing member receiving end of the outer sleeve, which is used to selectively connect a second end cap which can engage with the pressing member to the outer sleeve, and the second end cap has a groove which is configured to receive the thread of the second threaded adapter.

5. The instrument of claim 1, wherein the pressing member includes a seat portion configured to receive an engagement receiving end of the inner sleeve, or includes an engagement end configured to be seated on a seat portion of the second end cap.

6. The apparatus of claim 1, wherein the engagement receiving end of the inner cannula includes a porous filter media sealingly enclosing an interior chamber on the engagement receiving end.

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

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

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

10. The device of claim 1, wherein the inner cannula comprises a protein or polymer matrix that at least partially fills an interior lumen of the inner cannula.

11. The device of claim 10, wherein the protein or polymer matrix comprises a three-dimensional porous scaffold, wherein the porous scaffold comprises pores forming a directional pattern.

12. The device of claim 11, wherein the porous scaffold comprises aligned pores that form aligned channels, and wherein the aligned channels in the protein or polymer matrix are arranged parallel to the longitudinal axis of the inner sleeve.

13. The device of claim 12, wherein the protein or polymer matrix is ​​selected from silk fibroin and collagen, or a polymer that forms aligned pores in a 3-D matrix.

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

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

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

17. The apparatus of claim 14, further comprising: a threaded adapter disposeable within a pressing member receiving end of the outer sleeve for selectively coupling a second end cap engageable with the pressing member to the outer sleeve, the second end cap having a recess configured to receive threads of the threaded adapter; and An elastic member extends between the seat portion of the threaded adapter and the seat portion of the pressing member.

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

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

20. The device of claim 1, wherein the interior chamber of the inner cannula further comprises a therapeutic composition.

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

22. The device according to claim 21, wherein the growth factor is 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.

23. The apparatus of claim 22, wherein the growth factor is brain-derived neurotrophic factor.

24. The device of claim 21, wherein the inhibitor of the prolyl hydroxylase domain enzyme is selected from the group consisting of 4,4α-dihydro-4-oxo-1,10-phenanthroline-3-carboxylic acid (1,4-DPCA), N-[(1,3-dicyclohexylhexahydro-2,4,6-trioxo-5-pyrimidinyl)carbonyl]-glycine, 6-amino-1,3-dimethyl-5-[(2-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.

25. The device of claim 24, wherein the inhibitor of the prolyl hydroxylase domain enzyme is 4,4a-dihydro-4-oxo-1,10-phenanthroline-3-carboxylic acid (1,4-DPCA).

26. The device of claim 21, wherein the derivative of vitamin A is selected from the group consisting of retinoic acid, retinol, retinyl carboxylate, tretinoin, tazarotene, and combinations thereof.

27. The device of claim 26, wherein the lipid mediator is selected from the group consisting of resolvins, metabolites of omega-3 fatty acids, derivatives of eicosapentaenoic acid, derivatives of docosahexaenoic acid, and combinations thereof.

28. The apparatus of claim 20, wherein the therapeutic composition comprises an agent that acts in proximal-distal position 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.

29. The device of claim 28, wherein the therapeutic composition comprises a peptide or protein hormone.

30. The device according to claim 29, wherein the peptide or protein 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.

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

32. The device of claim 21, wherein the inhibitor of a prolyl hydroxylase domain enzyme is present in the therapeutic composition at a dosage of from 0.004 μg / ml to 0.024 μg / ml.

33. The device of claim 21, wherein the vitamin A or derivative thereof is present in the therapeutic composition at a dosage of from 0.03 μg / ml to 0.27 μg / ml.

34. The device of claim 21, wherein the lipid medium is present in the therapeutic composition at a dosage of from 0.006 μg / ml to 0.054 μg / ml.

35. The device of claim 29, wherein the peptide or protein hormone is present in the therapeutic composition at a dosage of from 0.1 μg / ml to 1.0 μg / ml.

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