Human umbilical cord derived compositions and their use for treating neurological disorders

Abstract

The present disclosure provides improved biomaterials extracted from human umbilical cord (hUC) material, which are mechanically milled to produce micronized particles, further treated with proteases, and optionally mixed with gel-forming agents. These materials may have improved inflammatory / anti-inflammatory profiles and may provide particular utility in the treatment of peripheral neuropathy by topical administration of hUC extracts.

Description

[Technical field]

[0001] The present disclosure relates generally to the fields of neurobiology, medicine, and medical treatment, and more specifically to improved biomaterials extracted from human umbilical cord (hUC) material having improved inflammatory / anti-inflammatory profiles, and their use in the treatment of neurological disorders. [Background technology]

[0002] Painful neuropathy is primarily treated non-surgically, often with systemic administration of analgesics or NSAIDs to treat pain. Depending on the type of injury, analgesics and NSAIDs may resolve neuropathic pain as a first line of defense. However, when painkillers and NSAID treatments do not resolve the pain, second-line treatment options are more invasive or risky, such as treatment with anti-inflammatory steroids (e.g., corticosteroids), anticonvulsants (e.g., pregabalin), or surgery.

[0003] Unfortunately, the limitations of current first-line treatments such as painkillers and NSAIDs are that they are not effective in resolving painful neuropathy in many patients. Many patients end up undergoing second-line treatment options such as surgery or medications (i.e., steroids or anticonvulsants) that carry a higher risk of side effects. Common side effects experienced by most patients treated with these more dangerous drugs include fluid retention, weight gain, headaches, stomach pain, and swelling. Thus, there is a need for improved compositions and methods for treating neuropathy. Summary of the Invention

[0004] In at least one embodiment, the present disclosure provides a physiologically buffered human umbilical cord (hUC) extract composition comprising micronized particles of hUC tissue treated with an ECM-degrading protease. The hUC tissue may comprise, consist of, or consist essentially of hUC membrane, hUC stroma, or a combination of hUC membrane and hUC stroma. The composition may further comprise one or more gel forming agents, crosslinking agents, biomolecules, enzymes, and / or buffers. For example, the composition may include an in situ polymerizing gel former. The in situ polymerizing gel former may be present at about 0.1-8 mg / ml.

[0005] The composition may further comprise a cross-linking agent, such as genipin or transglutaminase, among other suitable cross-linking agents. The gel-forming agent, e.g., the polymerizing gel-forming agent, e.g., the thermally polymerizing gel-forming agent, may be or may include one or more of fibrin, collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VIII, collagen X, collagen XI, collagen XXIV, or collagen XXVII. In some examples, the hUC tissue treated with the ECM-degrading protease comprises a hUC membrane and the thermally polymerizing gel-forming agent is not fibrin. The composition may be a saline-based suspension buffered to about pH 7.2-7.4.

[0006] The composition may further comprise one or more biomolecules, such as hyaluronic acid, chondroitin sulfate, chitosan, PEG, collagen VI, collagen VII, collagen IX, collagen XII, collagen XIII, collagen XIV, collagen XV, collagen XVI, collagen XVII, collagen XVIII, collagen XIX, collagen XX, collagen XXI, collagen XXII, collagen XXIII, collagen XXVI and / or collagen XXVIII.In at least one example, the composition does not comprise chondroitin sulfate.The composition may comprise micronized particles.For example, the majority of the micronized particles may have a diameter between about 140 nm and about 160 nm.

[0007] Also provided are methods for producing such compositions. For example, the methods can produce a human umbilical cord (hUC) extract, the methods comprising the steps of: (a) providing hUC membrane and / or stroma, (b) mechanically impacting the hUC membrane and / or stroma to produce micronized particles, and (c) treating one or more of: (i) the composition of step (a) prior to mechanical impaction; (ii) the composition of step (b) during mechanical impaction; and (iii) the micronized particles resulting from step (b) with an ECM-degrading protease.

[0008] The method herein may further comprise inactivating the protease. After inactivating the ECM-degrading protease, an in situ gel former, for example, a polymerizing gel former, may be added. The method may further comprise polymerizing the gel former. The polymerization may be performed in the presence of a cross-linking agent, such as genipin or transglutaminase.

[0009] The gel forming agent may be or may include one or more of fibrin, collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VIII, collagen X, collagen XI, collagen XXIV, or collagen XXVII. The in situ gel forming agent, e.g., polymeric gel forming agent, may be present at about 0.1 mg / ml to 8 mg / ml. About 0.5 to 1.0 cm containing hUC membrane and / or stroma 2 hUC tissue may be provided in step (a). The hUC membrane and / or stroma provided in step (a) may be dispersed in a saline-based suspension buffered to about pH 6.0-8.0.

[0010] The mechanical impingement may be performed for 1 to about 5 cycles. Further, for example, the mechanical impingement may be performed at a speed ranging from about 3400 RPM to about 3700 RPM, for example, with a duration of about 60 seconds per cycle. The method herein may further include centrifuging the micronized particles, for example, at a speed of about 5000×g, prior to ECM-degrading protease treatment.

[0011] The protease, e.g., the ECM-degrading protease, can be a collagenase or a matrix metalloproteinase (MMP), such as collagenase I, collagen III, MMP-2, MMP-3, or MMP-7. One or more of hyaluronic acid, chondroitin sulfate, chitosan, PEG, collagen VI, collagen VII, collagen IX, collagen XII, collagen XIII, collagen XIV, collagen XV, collagen XVI, collagen XVII, collagen XVIII, collagen XIX, collagen XX, collagen XXI, collagen XXII, collagen XXIII, collagen XXVI, and / or collagen XXVIII can be added to the composition, e.g., after inactivation of the ECM-degrading protease. Mechanical impaction can provide a majority of micronized particles having a diameter of about 140 nm to about 160 nm, e.g., a particle size distribution having an average diameter in the range of about 140 nm to about 150 nm.

[0012] The present disclosure also includes a method of treating peripheral neuropathy, comprising administering, e.g., injecting, a composition as described herein at or near the site of peripheral neuropathy in a subject. For example, a method of treating peripheral neuropathy can include injecting a composition made by a method as defined herein at or near the site of peripheral neuropathy in a subject. The subject can be a human, a primate, a non-human mammal, or other vertebrate or animal. The method can further include treating the subject with a second therapy, such as analgesic therapy, NSAID therapy, and / or an anticonvulsant. The method herein can further include administering, e.g., injecting, the composition to the subject at least two, three, four, or five times, or continuously or permanently, chronically.

[0013] As used herein, "a" means one or more. As used herein, when used in conjunction with the word "comprising," the word "a" means one or more.

[0014] Use of the term "or" is used to mean "and / or," unless expressly stated to refer to alternatives only or the alternatives are mutually exclusive, but the present disclosure supports a definition that refers only to alternatives and "and / or." As used herein, "another" means at least a second or more than one.

[0015] Throughout this application, the term "about" is used to indicate that a value includes the variation of error inherent in the device, the variation of error inherent in the method used to determine the value, or the variation that exists among study subjects. Unless otherwise specified, the term "about" should be understood to encompass ±5% of a particular amount or value.

[0016] Other objects, features and advantages of the present disclosure will become clear from the following detailed description. However, it should be understood that the detailed description and specific examples, while showing specific embodiments of the present invention, are provided for illustrative purposes only, since various changes and modifications within the spirit and scope of the present invention will be apparent to those skilled in the art from this detailed description. It should be noted that just because a particular compound belongs to one particular general formula, it does not mean that it cannot belong to another general formula.

[0017] The following drawings form part of this application and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of exemplary embodiments presented herein.

[0018] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication containing color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0019] [Figure 1A]1A-1B show the preparation of hUC membrane extracts as described in Example 1. (FIG. 1A) Pretreated hUC in a tube loaded with approximately 0.7 cm2 of debrided hUC with 700 μL of 1×PBS. [Figure 1B] 1 shows the preparation of hUC membrane extract as described in Example 1. Homogenized hUC when homogenized in a CoolPrep™ sample holder at a speed setting of 6 m / sec for 3 cycles of 60 seconds, followed by centrifugation at 5000×G for 10 minutes. [Diagram 2] 1 shows hUC membrane extract expelled from a syringe with a 26G needle, as described in Example 2. [Diagram 3] 1 shows polymerization of collagen gel and hUC membrane extract as described in Example 2, including incubation at 37° C. for 30 minutes in a circular mold. [Figure 4] 1 shows the polymerization of collagen gel and hUC membrane extract as described in Example 2. The prepolymerized gel extraction mixture was expelled from a syringe with a 26G needle and subsequently incubated at 37° C. for 60 minutes. [Diagram 5] 5-6 show the polymerization profile described in Example 3. Gels loaded with hUC extract polymerized within 10 minutes at 37° C. without the addition of gel crosslinker and within 8 minutes with the addition of crosslinker, indicating rapid in situ gel polymerization. [Figure 6] Same as above. [Figure 7] 1 shows the gel degradation profile described in Example 2. The polymerized gel retained more than 50% of its mass under physiological conditions for 1 day without added crosslinker and for up to 36 days with added gel crosslinker. [Figure 8A] Figures 8A-8B show the particle size distribution described in Example 3. (Figure 8A) The hUC extract contained a monodisperse particle suspension with the majority of particles in the 160-180 nm diameter range. [Figure 8B] The extracts contained varying particle dispersity and size distribution profiles depending on the preparation conditions. [Figure 9]Figure 1 shows the results of the immune modulation assay described in Example 4. hUC extracts were observed to modulate the immune response of human peripheral blood mononuclear cells in vitro during induced inflammation by altering the secretory response of immune biomarkers: IL-1b, IL-10, and MMP-9. [Figure 10A] 10A-10B show immune response modulation in human U-937 cell line derived macrophage-like cells as described in Example 4. hUC extracts modulated immune responses of human U937 cell line derived macrophage-like cells in vitro during inflammatory induction by altering the secretory responses of immune biomarkers: IL-1β and IL-10. [Figure 10B] Same as above. [Figure 11] 1 shows collagenase disruption of collagen polymer formation as described in Example 5. The concentration of disrupted collagen polymer fragments was reduced by treatment with collagenase. [Figure 12] 1 shows an exemplary schematic diagram for preparing a hUC gel composition. The hUC composition can be prepared by mechanically processing hUC tissue to obtain an extract, purifying the extract (e.g., removing cellular debris), treating the purified extract with proteases to reduce inflammatory components, and formulating the processed / purified hUC extract as a hydrogel suitable for injection. [Figure 13] 1 shows the reduction in expression of decorin as described in Example 6. hUC compositions treated with MMP-7 showed reduced expression of decorin (DCN) compared to untreated controls. [Figure 14A] 1 shows immune response regulation in macrophage-like cells derived from the human U-937 cell line as described in Example 7. [Figure 14B] The total protein content of treated and untreated hUC extracts as described in Example 7 is reported. [Figure 15] FIG. 1 shows an exemplary schematic for carrying out the immune modulation assay described in Example 7. [Figure 16A] 16A-16B show the results of an immunomodulatory assay of protease-treated hUC extracts as described in Example 7. [Figure 16B] Same as above. [Figure 17] 1 shows the reduction in expression of decorin in protease-treated hUC extracts as described in Example 7. [Figure 18A] 18A-18B show the results of another immunomodulatory assay of protease-treated hUC extracts as described in Example 7. [Figure 18B] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Detailed Description As mentioned above, the use of amniotic membrane / birth material is a promising means of treating tissue regeneration, including in areas of neurological impairment. The present disclosure provides methods of producing improved biomaterials and methods of use thereof. In general, the present disclosure is directed to a saline-based suspension from human umbilical cord (hUC) material produced by mechanical impaction (e.g., bead-beating homogenization) of hUC membrane and hUC stromal sections. This manufacturing technique is designed to reduce the soluble content of inflammatory components of the hUC membrane and stroma, such as DNA, cytosolic DAMPs (damage-associated molecular patterns), and ECM protein fragments in the saline-based suspension, while releasing abundant soluble bioactive components associated with regulating inflammation and restoring healthy tissue into the same suspension. The suspension can be further processed to reduce inflammatory protein fragments by incubation with ECM-protease enzymes (e.g., collagenase or matrix metalloproteinases (MMPs) such as MMP-7). The suspension can also be supplemented with collagen gels of various concentrations to facilitate sustained delivery of therapeutic agents to local tissues. The hUC membrane and stroma derived saline-based suspension can be delivered to sites of tissue damage (e.g., neuropathy), such as by injection. These and other features of the present disclosure are described in detail below.

[0021] I. Neuropathy Neuropathy generally refers to nerve damage. Peripheral neuropathy refers to damage to nerves outside the central nervous system, i.e., outside the brain and spinal cord. For example, peripheral neuropathy includes damage to the sensory and motor nerves that connect the brain and spinal cord to the rest of the body. (Peripheral nerve damage can impair sensation, movement, and function depending on the extent of the damage and the peripheral nerve affected. Peripheral neuropathy includes reversible or permanent damage, and the effects can be acute with sudden onset, rapidly progressive, or chronic with symptoms that start subtle and progress over time. The cause of peripheral neuropathy can be hereditary or idiopathic (unknown cause) and may be associated with other medical conditions or prescription medications.

[0022] II. Umbilical Cord Material and Method for Extracting It The compositions herein are derived from umbilical cord tissue, including membrane and / or stroma. Umbilical cord extracts may have advantages in treating neurological disorders over materials derived from other types of tissue. Without being bound by theory, it is believed that, for example, umbilical cord tissue lacks human leukocyte antigen (HLA), and thus the compositions herein derived from umbilical cord tissue may result in a reduced immune response. Furthermore, umbilical cord tissue contains high levels of bioactive growth factors, stem cells, floating proteins, and glucosamine, which may be beneficial in promoting neurological repair.

[0023] The compositions described herein can be prepared from human umbilical cord materials as described herein. These materials can be obtained from any suitable source. For example, at least one of the components can be obtained from human tissue. The components can also be obtained from commercial sources. The components can be purified, substantially purified, partially purified, or non-purified.

[0024] Human umbilical cord tissue can be obtained, for example, from commercial sources or from hospitals or surgical / birthing centers. The tissue is usually obtained fresh or frozen. The tissue can be washed to remove excess storage buffer, blood, or contaminants. Excess liquid can be removed, for example, using a brief centrifugation step or by other means. The tissue can be frozen, for example, using liquid nitrogen or other cooling means, to facilitate subsequent homogenization. The source of umbilical cord tissue can be human umbilical cord (hUC). The entire hUC material can be debrided to remove material unrelated to the membrane and / or stroma, for example, through the use of a surgical cutting tool, a manual cutter, or an automated cutter.

[0025] hUC can also be processed to produce extracts using mechanical impaction, such as the "bead beating" technique. Such processing can remove cellular debris. Bead beating is a laboratory-scale mechanical method for processing biological samples using glass, ceramic, or steel beads mixed with the sample suspended in an aqueous medium. The sample-bead mixture is agitated, for example, by stirrer or shaking. The beads collide with the tissue material, mechanically disrupting the tissue and releasing therapeutic bioactive molecules. This has the advantages over other mechanical processing methods in that it produces a micronized extract with a unique distribution of bioactive molecules and small particles, allows many samples to be processed at once without concern for cross-contamination, and does not release potentially harmful aerosols during the process.

[0026] In one example of this method, a volume of beads, e.g., an equal volume compared to the volume of tissue, is added to a tissue suspension in a container, and the sample is vigorously mixed in a laboratory vortex mixer. Processing time is relatively slow, taking 3-10 times longer than specialized shakers, but it effectively processes tissues and is inexpensive. Scale-up procedures with larger volumes and faster processing times are being considered.

[0027] Successful bead beating can depend not only on shaker design features (taking into account shaking oscillations per minute, shaking throw or distance, shaking direction, and vial orientation), but also on selection of the correct bead size, bead composition (glass, ceramic, steel), and bead load in the vial.

[0028] High energy bead-beating devices typically warm the sample by frictional collision of beads during homogenization. Cooling of the sample during or after bead-beating may be necessary to prevent damage to heat-sensitive proteins such as enzymes. Warming of the sample can be controlled by bead-beating in short time intervals and / or cooling on ice / dry ice between each interval, processing the sample in a pre-chilled aluminum vial holder, circulating a gaseous coolant through the device during bead-beating. In some examples herein, the sample in the processing chamber is cooled with dry ice, for example using an apparatus from MP Biomedicals.

[0029] Another bead beater configuration, suitable for larger sample volumes, uses a fluorocarbon rotor rotating in a 15, 50, or 200 ml chamber to agitate the beads. In this configuration, the chamber can be surrounded by a static cooling jacket. Using this same rotor / chamber configuration, larger commercial machines can be used to process many liters of cell suspension. Currently, these machines are limited to processing single-cell organisms such as yeast, algae, and bacteria.

[0030] This initial processing may produce micronized particles of hUC tissue. For example, the hUC extract may contain particles with a unimodal or bimodal particle size distribution, depending on the processing conditions. In some examples herein, the composition (before and / or after treatment with proteases, as described further below) may have an average diameter ranging from about 50 nm to about 500 nm, e.g., from about 70 nm to about 250 nm, from about 80 nm to about 180 nm, from about 120 nm to about 350 nm, from about 150 nm to about 300 nm, from about 165 nm to about 200 nm, from about 140 nm to about 160 nm, from about 200 nm to about 275 nm, from about 175 nm to about 325 nm, or from about 250 nm to about 450 nm. In some examples, particles above a given threshold may be removed (e.g., removing large proteoglycans and / or large tissue fragments released during homogenization, etc.), resulting in a desired unimodal or bimodal particle size distribution. Particle size can be measured, for example, by nanoparticle tracking analysis (NTA) or multi-angle dynamic light scattering (MADLS).

[0031] Optionally, the tissue can be frozen prior to the impingement process. The freezing step can be performed by any suitable cooling process. For example, the tissue can be flash frozen using liquid nitrogen. Alternatively, the material can be placed in an isopropanol / dry ice bath or flash frozen with other coolants. Commercially available flash freezing processes can be used. Additionally, rather than flash freezing the material can be placed in a freezer and allowed to equilibrate to the storage temperature more slowly. The tissue can be stored at any temperature. For example, -20°C, -80°C, or other temperatures can be used for storage. Destruction of the tissue during freezing rather than prior to freezing is one optional method for preparing the tissue.

[0032] The hUC preparation may be in liquid, suspension, or dry (including but not limited to lyophilized) form. Antimicrobial agents, such as antibiotics and antifungals, may be added. The material may be packaged and stored, for example, at room temperature, or, for example, at -20°C or -80°C, prior to use.

[0033] In some embodiments, the hUC material used to prepare the compositions herein, e.g., gel compositions, exists as a dry formulation. Dry formulations can be stored in smaller volumes and may not require the same cryogenic storage requirements to prevent deterioration of the formulation over time. Dry formulations can be stored and reconstituted before use. Dry formulations can be prepared, for example, by preparing frozen morselized hUC as described herein and then removing at least a portion of the water in the composition. Water can be removed from the preparation by any suitable means. An exemplary method of removing water is by the use of lyophilization using commercially available freeze dryers. Suitable equipment can be found, for example, through Virtis, Gardiner, NY, FTS Systems, Stone Ridge, NY, and Speed ​​Vac (Savant Instruments Inc., Farmingdale, NY). In certain embodiments, the moisture content of the dry formulation will be less than about 20% by weight, up to about 10% by weight, up to about 5% by weight, or up to about 1% by weight of the formulation. In some embodiments, substantially all of the moisture is removed. The lyophilized composition can then be stored. Storage temperatures can vary from below about −196° C., below −80° C., below −50° C., or below −20° C. to above about 23° C. If desired, the composition can be characterized (weight, protein content, etc.) prior to storage.

[0034] The lyophilized composition can be reconstituted with an appropriate solution or buffer prior to use. Exemplary solutions include, but are not limited to, phosphate buffered saline (PBS), Dulbecco's modified Eagle's medium (DMEM), and balanced salt solution (BSS). The pH of the solution can be adjusted as needed. The concentration of hUC can vary as needed. In some examples herein, more concentrated hUC solutions are useful, while in other examples, solutions containing lower concentrations of hUC are useful. Additional compounds can be added to the solution. Exemplary compounds that can be added to the reconstituted formulation include, but are not limited to, pH adjusters, buffers, collagen, hyaluronic acid (HA), antibiotics, surfactants, stabilizers, proteins, and the like (discussed further below).

[0035] II. Umbilical Cord Extract Composition According to the present disclosure, hUC compositions as described above are provided.These compositions may be further processed or supplemented with other materials as described herein, including but not limited to one or more gel-forming agents, cross-linking agents, biomolecules, enzymes, and / or buffers.The compositions herein may be formulated for administration to subjects, such as in the form of a solution or gel.

[0036] A. Gel-Forming Agents The compositions herein may include one or more gel-forming agents. Suitable gel-forming agents may be thermally polymerizable at human body temperature, approximately 37°C (98-99°F). Exemplary gel-forming agents include, but are not limited to, collagen I, II, III, IV, V, VIII, X, XI, XXIV, and XXVII; polyethylene glycol (PEG); poly(lactic-glycolic acid) (PLGA); poly(ethylene glycol) diacrylate (PEGDA); gelatin methacryloyl (GelMA); methacrylated hyaluronic acid (MeHA); and fibrin. Fibrin, also known as factor Ia, is a fibrous, non-globular protein involved in blood clotting.

[0037] The amount of gel forming agent can typically range from about 0.1 g / mL to about 8 mg / mL, for example, from about 0.5 g / mL to about 5 mg / mL, from about 1 mg / mL to about 4 mg / mL, or from about 3.5 mg / mL to about 4.5 mg / mL.

[0038] collagen In at least one embodiment, the composition comprising the hUC extract may comprise one or more collagen types. Fibril-forming or network-forming collagens, including types I, II, III, IV, V, VIII, X, XI, XXIV, or XXVII, may be used as in situ polymerizing gel formers (see below). Other collagens may be included in the composition as well.

[0039] In another embodiment, the composition comprising an hUC extract may contain one or more collagen types, none of which are fibril-forming or network-forming. Collagen is the major structural protein found in the extracellular matrix of various connective tissues in the body. As the main component of connective tissue, it is the most abundant protein in mammals, accounting for 25%-35% of the total body protein content. Collagen is made up of amino acids that combine to form elongated fibril triple helices known as collagen helices. It is found primarily in fibrous tissues such as tendons, ligaments, and skin.

[0040] Any one or more of the following may be included in the composition of the hUC extract: Fibrous (types I, II, III, V, XI, XXIV, XXVII) Nonfibrous FACIT (Fibril Associated Collagens with Interrupted Triple Helices) (Types IX, XII, XIV, XVI, XIX, XX, XXI, XXII) Network-forming collagen (types VIII, IV, and X) Multiplexins (multiple interrupted triple-helical domains) (types XV and XVIII) MACIT (Membrane Associated Collagens with Interrupted Triple Helices) (Types XIII and XVII) Transmembrane collagen (XXIII) Others (Types VI, VII, XXVI, XXVIII) The five most common types are type I (skin, tendons, vasculature, organs, bone (main component of the organic part of bone), type II (cartilage, the main collagen component of cartilage), type III (reticular, main component of reticular fibers, commonly found alongside type I), type IV (forming the basal layer, the epithelial secretory layer of the basement membrane), and type V (cell surfaces, hair, placenta).

[0041] Throughout the four stages of wound healing, collagen is understood to perform some or all of the following functions in wound healing: Guidance function Collagen fibers act as guides for fibroblasts, which migrate along the connective tissue matrix.

[0042] Chemotactic properties The large surface area available on collagen fibers can attract fibrogenic cells, which aids in healing. nucleation : Collagen, in the presence of certain neutral salt molecules, can act as a nucleating agent that triggers the formation of fibrillar structures. Collagen wound dressings can act as guides to direct the deposition of new collagen and the growth of capillaries.

[0043] Hemostatic properties Platelets interact with collagen to form a hemostatic plug. B. Crosslinking Agent The compositions herein may additionally or alternatively include a cross-linking agent. Cross-linking agents generally provide bonds that connect one polymer chain to another. These bonds can be in the form of covalent or ionic bonds, and the polymers can be either synthetic or natural polymers (such as proteins). In polymer chemistry, "cross-linking" usually refers to the use of cross-linking to promote changes in the physical properties of a polymer. When "cross-linking" is used in biology, it usually refers to the use of agents that bind proteins together to check protein-protein interactions or to create overall strengthening of biological materials.

[0044] Examples of cross-linking agents useful in the present disclosure include, but are not limited to, genipin, transglutaminase, imidoester cross-linking agent dimethylsuberimidate, N-hydroxysuccinimide ester cross-linking agent BS3, and formaldehyde.Furthermore, for example, the compositions herein may include one or more photocross-linking components, for example, UV light may be used to initiate cross-linking.

[0045] The crosslinkers dimethylsuberimidate, the N-hydroxysuccinimide ester crosslinker BS3, and formaldehyde typically form bonds by inducing nucleophilic attack of the amino group of lysine followed by covalent attachment via the crosslinker. The zero-length carbodiimide crosslinker EDC functions by converting a carboxyl to an amine-reactive isourea intermediate that couples to lysine residues or other available primary amines. SMCC or its water-soluble analog, Sulfo-SMCC, can be used to prepare antibody-hapten conjugates for antibody development.

[0046] Genipin is a compound found in gardenia fruit extracts. It is an aglycone derived from an iridoid glycoside called geniposide, found in the fruit of Gardenia jasminoides. Genipin is a natural cross-linking agent for cross-linking proteins, collagen, gelatin, and chitosan. It has low acute toxicity and an LD50 in mice. 50iv is 382 mg / kg and is therefore much less toxic than glutaraldehyde and many other commonly used synthetic cross-linking reagents. Furthermore, genipin can be used as a modulator of drug delivery and as an intermediate in alkaloid synthesis. In vitro experiments have shown that genipin blocks the action of the enzyme uncoupling protein 2.

[0047] Another class of cross-linking agents is transglutaminases, which naturally cross-link the γ-carboxamide group (-(C=O)NH 2 ) and the ε-amino group of the lysine residue side chain (-NH 3 ) followed by the formation of an isopeptide bond between the 3 ) for this cross-linking (between separate molecules) or intramolecular (within the same molecule) reaction to occur, lysine and glutamine residues must be linked to a peptide or protein. The bonds formed by transglutaminases are highly resistant to proteolysis. These enzymes can also deamidate glutamine residues to glutamic acid residues in the presence of water. For example, transglutaminase isolated from the bacterium Streptomyces mobaraensis is a calcium-independent enzyme. Among transglutaminases, mammalian transglutaminases require Ca as a cofactor. 2+ ions are required.

[0048] Transglutaminases form extensively cross-linked, usually insoluble, protein polymers. These biopolymers are essential for organisms to form barriers and stable structures. Examples are blood clots (clotting factor XIII), and skin and hair. The catalytic reactions are usually considered irreversible and must be carefully monitored through control mechanisms.

[0049] The amount of crosslinker can range from about 0.1 mM to about 5 mM, for example, from about 0.5 mM to about 3 mM, from about 3 mM to about 4 mM, from about 1.5 mM to about 2.5 mM, or from about 1 mM to about 2 mM. C. Other biomolecules In addition to the agents already mentioned, the hUC compositions may further comprise one or more components such as, for example, hyaluronic acid, chondroitin sulfate, chitosan, and / or polyethylene glycol (PEG).

[0050] Hyaluronan, also called hyaluronan, is an anionic, nonsulfated glycosaminoglycan that is widely distributed throughout connective, epithelial, and neural tissues. It is unique among glycosaminoglycans in that it is not sulfated, is formed at the plasma membrane instead of the Golgi apparatus, and can be very large. Human synovial HA averages about 7 million Da, or about 20,000 disaccharide monomers per molecule; other sources cite 3 to 4 million Da. As one of the major components of the extracellular matrix, hyaluronan contributes significantly to cell proliferation and migration and may also be involved in the progression of some malignant tumors.

[0051] Chondroitin sulfate is a sulfated glycosaminoglycan (GAG) that contains chains of alternating sugars (N-acetylgalactosamine and glucuronic acid). It is usually attached to proteins as part of proteoglycans. Chondroitin chains can contain over 100 individual sugars, and the position and amount of sulfated each sugar varies.

[0052] In some examples herein, the composition comprises a reduced amount of one or more proteoglycans (e.g., biglycan, decorin, versican, etc.) and / or sulfated GAGs associated with native hUC tissue. In some examples, the composition does not comprise one or more proteoglycans and / or sulfated GAGs associated with native hUC tissue. For example, the composition herein may have a reduced amount of biglycan, decorin, and / or versican compared to native hUC tissue. In some examples, the composition herein does not comprise (e.g., has a level below detection) one or more proteoglycans present in native hUC tissue, such as biglycan, decorin, and / or versican.

[0053] Chitosan is a linear polysaccharide composed of randomly distributed β-(1→4)-linked D-glucosamine (deacetylated units) and N-acetyl-D-glucosamine (acetylated units) derived from chitin by treating the chitinous shells of shrimp and other crustaceans with alkaline substances such as sodium hydroxide.

[0054] Polyethylene glycol (PEG) is a polyether compound also known as polyethylene oxide (PEO) or polyoxyethylene (POE) depending on its molecular weight. The structure of PEG is generally H-(O-CH 2 -CH 2 ) n It is represented as -OH. The possibility of using PEG for axonal fusion is being investigated by researchers studying peripheral nerve and spinal cord injuries.

[0055] D. Enzyme Treatment After one or more processing steps of hUC tissue (e.g., mechanical bombardment by bead-beating), the resulting hUC extract may contain disrupted protein fragments, such as protein monomers and ECM fragments. Such components may cause side effects or have adverse effects when administered to a patient. For example, certain components of the hUC extract may induce or be associated with an inflammatory and / or immune response when injected at the site of nerve injury. The compositions herein may be prepared by treatment with proteases that selectively remove, reduce, or inactivate certain components while simultaneously retaining bioactive components useful for promoting nerve repair. Components that may be at least partially, substantially, or completely removed may include, for example, natural proteoglycans, such as decorin, biglycan, and / or versican.

[0056] Without being bound by theory, it is believed that protease treatment may cause degradation or reduced expression of proteoglycans, such as decorin, present in the ECM associated with inflammation via the TLR4 pathway. For example, decorin is believed to be recognized as a DAMP by inflammatory cells and influence inflammatory signaling events. By removing or reducing the amount of proteoglycans, such as decorin, the risk of an inflammatory response by a subject when administered a composition herein can be reduced. Embodiments of the present disclosure can effectively induce the immune system to promote healing while reducing or preventing potentially harmful aspects of the immune response.

[0057] For example, production of the hUC compositions herein can include treatment with one or more ECM-degrading proteases. The proteases can be collagenases, such as collagenase I, II, III, IV, V, VI, or VII, or other suitable proteases, including but not limited to MMPs, such as MMP-2, MMP-3, or MMP-7. The enzyme treatment can occur prior to the introduction of a gel-forming agent (described above) and / or prior to the further introduction of one or more other components, including collagens of the types described above and / or other gel-forming agents.

[0058] Collagenases are enzymes that cleave peptide bonds in collagen. They aid in the destruction of extracellular structures in the pathogenesis of bacteria such as Clostridium. They are considered a virulence factor that promotes the spread of gas gangrene. Usually, they target connective tissues in muscle cells and other body organs. Collagen, an important component of the extracellular matrix of animals, is formed by cleavage of procollagen by collagenases after it is secreted from cells. This prevents the formation of larger structures inside the cells themselves. Collagenases are not only produced by some bacteria, but also by the body as part of the normal immune response. This production is induced by cytokines that stimulate cells such as fibroblasts and osteoblasts, which can cause indirect tissue damage.

[0059] The concentration of the protease may be in the range of about 0.1 μg / mL to about 25 μg / mL, for example, about 0.5 μg / mL to about 20 μg / mL, about 1 μg / mL to about 15 μg / mL, about 0.5 μg / mL to about 5 μg / mL, about 1 μg / mL to about 2 μg / mL, about 2.5 μg / mL to about 5 μg / mL, about 3 μg / mL to about 8 μg / mL, about 5 μg / mL to about 15 μg / mL, about 10 μg / mL to about 18 μg / mL, or about 15 μg / mL to about 22 μg / mL. Further, for example, the hUC extract may be treated with a protease for a time ranging from about 5 minutes to about 18 hours, e.g., from about 1 hour to about 16 hours, from about 4 hours to about 12 hours, from about 8 hours to about 16 hours, from about 12 hours to about 16 hours, from about 2 hours to about 8 hours, from about 30 minutes to about 5 hours, or from about 5 minutes to about 2 hours. In a non-limiting example, the hUC extract may be treated with about 0.5 μg / mL to about 5 μg / mL of protease for a time ranging from about 1 hour to about 16 hours.

[0060] According to some examples herein, the method of preparing the composition includes at least partially inactivating the protease. For example, an agent such as ethylenediaminetetraacetic acid (EDTA), Ilomastat (e.g., GM-6001 or Galardin™), or TIMP metallopeptidase inhibitor 1 (TIMP-1) can be added to inactivate the enzyme. Such an agent can be selected to target the enzyme without damaging or minimizing damage to the bioactive components in the hUC extract.

[0061] E. Buffer The compositions herein, such as modified extracts, can be advantageously combined with buffers to maintain a target pH. Buffers (e.g., pH buffers and hydrogen ion buffers) are typically aqueous solutions of a mixture of a weak acid and its conjugate base, or vice versa. The pH of a buffer changes little with the addition of small amounts of a strong acid or strong base. Buffers are used in a variety of chemical applications to keep the pH at a nearly constant value.

[0062] The pH of a solution containing a buffer will usually vary within a limited range, regardless of what else is present in the solution. In biological systems, this allows enzymes to perform their intended functions. If the pH value of a solution increases or decreases too much, the effectiveness of the enzymes decreases, sometimes irreversibly, in a process known as denaturation. Most biological samples used in research are stored in a buffer at around pH 7.4, often phosphate-buffered saline (PBS).

[0063] Some exemplary buffers relevant to physiological pH include citrate and KH 2 PO 4 Contains pK a By combining substances whose pK values ​​differ by less than 2 and adjusting the pH, a wide range of buffer solutions can be obtained. Citric acid has three pK values, spaced less than 2 apart. a Na is a useful component of buffer mixtures because it has a high pH value. The buffer range can be expanded by adding other buffering agents. 2 HPO 4 Various McIlvaine buffers, composed of acetic acid and citric acid, have a buffer range of pH 3 to 8. Other buffers useful in biological systems suitable for the present disclosure include Ringer's lactate (LRS), tris(hydroxymethyl)aminomethane (TRIS), Hank's balanced salt solution (HBSS), Gey's balanced salt solution (GBSS), TAPSO, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES), 3-(N-morpholino)propanesulfonic acid (MOPS), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), cacodylate, and 2-(N-morpholino)ethanesulfonic acid (MES).

[0064] 12 shows an exemplary schematic for preparing a hUC gel composition in accordance with the above discussion and the Examples below. As shown, the composition can be prepared by mechanical processing (e.g., bead beating or other suitable techniques) to obtain an extract of hUC tissue, purification of the extract (which may include, for example, removing cellular debris), treating the purified extract with a protease to reduce inflammatory components, and formulating the processed / purified hUC extract as a hydrogel suitable for injection.

[0065] IV. Treatment method The present disclosure also provides a method for treating tissue damage in a subject, including but not limited to muscle, tendons, ligaments, etc., and in particular a method for treating neuropathy. The method is designed to provide superior treatment of painful sites, such as peripheral neuropathy, and restore local tissue to a healthy state with normal function, compared to pain treatments and currently available amniotic / birth tissue derived flowable products (e.g., OrthoFlo by Mimedx, etc.). This is intended to provide symptom relief for patients who may undergo surgical intervention, as well as those who do not. As an injection therapy, the method is designed to be minimally invasive. The minimal invasiveness and versatility of this treatment as an injection therapy allows access to treat various anatomical regions of painful neuropathy within the body.

[0066] Thus, in at least one embodiment, the method involves injection of hUC-derived material into the site of injury. A medical professional can assess the appropriate site based on symptoms and diagnosis for the foot, hand, and joints (such as shoulder, elbow, wrist, knee, etc.). Similarly, a physician can determine the appropriate surgical procedure for delivery of the agent, such as combining injection with image-guided delivery or surgical removal of the affected area.

[0067] As mentioned above, the compositions herein can be formulated as gels, e.g., hydrogels, for injection at or near the site of injury or the site where treatment is required.Formulating the composition as a gel allows for longer periods of treatment, e.g., the gel may remain at the intended target site longer due to factors such as its viscosity, cohesiveness, etc. Thus, sustained release can be achieved by using gel compositions.Gels can be formulated to provide desired properties such as degradation rate, density, stiffness, and / or cargo loading.

[0068] The methods can include multiple treatments over a period of time, e.g., continuous or permanent, chronic treatments. Any number of treatments can be administered, including, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more treatments. Such treatments can be spaced apart by days, weeks, months, or even years.

[0069] The method may also include combination therapy using administration of the hUC compositions described herein in conjunction with one or more recognized treatments for neuropathy, such as dietary modification and / or administration of NSAIDs, analgesics or anticonvulsants (described in detail above and incorporated herein by reference).

[0070] V. Working Examples The following examples are included to demonstrate exemplary embodiments of the present disclosure, but are not limiting in nature. It is understood that the present disclosure encompasses additional embodiments consistent with the above description and the following examples. Those skilled in the art will understand that the techniques disclosed in the following examples represent techniques that the inventors have discovered to work well in the implementation of the present disclosure, and therefore can be considered to constitute exemplary modes for its implementation. However, those skilled in the art will understand in light of the present disclosure that many changes can be made to the specific embodiments disclosed and still obtain similar results without departing from the spirit and scope of the present disclosure.

[0071] Example 1 Preparation of hUC extracts A composition was prepared from the hUC extract as follows: a sample of hUC membrane tissue was loaded into a tube and 700 μL of 1×PBS was added ( FIG. 1A ). The sample was then homogenized in a CoolPrep™ sample holder at a speed setting of 6 m / s for 3 cycles of 60 seconds, followed by centrifugation at a speed of 5000×g for 10 minutes ( FIG. 1B ).

[0072] Example 2 Preparation of hUC gel A study was conducted to evaluate the formation of gels with hUC extract and gel formers. hUC extract prepared according to Example 1 was loaded into a syringe, extruded through a 26G needle (FIG. 2), and polymerized at 37° C. with collagen as a gel former into a gel (e.g., a formulation suitable for use as a minimally invasive injectable therapy). FIG. 3 shows the polymerization of collagen gel and hUC membrane extract after incubation at 37° C. for 30 minutes in a circular mold. FIG. 4 shows the polymerization of collagen gel and hUC membrane extract, where the prepolymerized gel extraction mixture was expelled from the syringe with a 26G needle and then incubated at 37° C. for 60 minutes.

[0073] Samples were prepared using various amounts of collagen as gel former (1 mg / mL, 2 mg / mL, and 4 mg / mL) with and without genipin (2 mM) as cross-linker over a 30 min incubation period to determine the effect on polymerization time. Absorbance at 410 nm was used as an indicator of the degree of polymerization. Results are shown in Figures 5 and 6. Gels loaded with hUC extract were observed to polymerize within 8 min at 37°C without the addition of gel cross-linker and within 10 min with the addition of cross-linker, indicating immediate in situ gel polymerization. Faster polymerization was observed with increasing amounts of gel former in the absence of cross-linker. Samples prepared with 4 mg / mL collagen and without genipin showed the fastest polymerization time, followed by samples prepared with 2 mg / mL collagen and without genipin. Samples prepared with 4 mg / mL and 2 mg / mL collagen and 2 mM genipin showed similar polymerization times. The slowest polymerization was exhibited by the sample prepared with 1 mg / mL collagen and 2 mM genipin.

[0074] Further studies were performed to examine the degradation of polymerized gels over time (64 days in total) using samples prepared with hUC extracts combined with 4 mg / mL collagen as a gel former and various amounts of genipin (0, 0.5 mM, 1 mM, 2 mM, and 5 mM) as a crosslinker. Samples were incubated in 1 mL of digestive collagenase enzyme solution at 37 °C for 30 min over a period of 6 weeks. The study was performed after polymerization and removal of excess liquid, followed by weekly weighing to determine the starting mass and to assess the ability to resist degradation. Fresh enzyme solution was used after each weighing. Polymerized gels were observed to retain more than 50% of their mass for 1 day in physiological conditions without the addition of crosslinker and for up to 36 days with the addition of gel crosslinker (Figure 7).

[0075] Example 3 Characterization of hUC extract particle size hUC extracts prepared according to Example 1 were analyzed to determine particle size distribution. Mean diameter by multi-angle dynamic light scattering (MADLS) was measured using a Malvern Zetasizer Ultra analyzer. Figure 8A shows the results of samples where hUC membrane tissue (umbilical cord membrane (UCM)) was homogenized for 1, 2, and 3 cycles at 4 m / s, 60 s per cycle, showing that the particle size distribution becomes slightly broader as homogenization time increases. These data support consistency in sample preparation, e.g., consistent mixing of components from homogenized hUC membrane tissue. Samples contained monodisperse particle suspensions with the majority of particles in the 160-180 nm diameter range (Figure 8A). Further studies were performed varying both speed (4 m / s, 5 m / s, or 6 m / s) and number of cycles (1, 3, or 5 cycles, 60 s per cycle) to examine the effect on particle size. The extracts contained particle dispersity and size distribution profiles that varied depending on the preparation conditions (Figure 8B).

[0076] Example 4 Immunomodulation by hUC extracts The ability of hUC extracts to modulate the immune response of human peripheral blood mononuclear cells derived from the human U937 cell line was investigated in vitro during two inflammatory challenges by altering the secretory responses of the following immune biomarkers: IL-1β, IL-10, and MMP-9 (Figures 9 and 10A-10B). In the first study (Figure 9), hUC extracts were prepared by homogenizing hUC membrane tissue at 4000 rpm for 3 min per cycle, for a total of 4-5 cycles. In the second study (Figures 10A-10B), hUC extracts were prepared by homogenizing hUC membrane tissue at 6 m / s for 60 s per cycle, for a total of 3 cycles. In the immune modulation assay, U937 cell cultures were treated with 20 ng / mL phorbol 12-myristate 13-acetate (PMA) for 48 hours to generate macrophage-like cells and then given an M1 differentiation stimulus (50 ng / mL LPS + 10 ng / mL IFN-γ) ("Stim") or no stimulus ("Unstim"). For comparison, M1 cultures were treated with respective hUC extracts ("UC"). The results show that hUC extracts modulate the immune response of human U937 cell line-derived macrophage-like cells during inflammation induction in vitro by altering the secretory response of immune biomarkers IL-1β and IL-10. Figure 9 shows the results at 48 hours. Figures 10A-10B show the results at 24, 48, 72, and 96 hours.

[0077] Example 5 Protease (collagenase) treatment of hUC extracts A study was performed using collagenase treatment (25 μg / mL) for comparison to the control and the results are shown in Figure 11. As shown, the concentration of disrupted collagen polymer fragments was reduced by treatment with collagenase compared to the untreated control. Collagenase-treated and control samples were homogenized at speed settings of 4 m / sec (1 cycle of 60 min) and 6 m / sec (3 cycles of 60 min), and at both speed settings, the collagenase group had reduced collagen polymer fragments compared to the control group.

[0078] Example 6 Protease (MMP-7) treatment of hUC extracts To study treatment with MMP-7 protease, modified / treated hUC extracts were prepared. hUC tissues were mechanically processed by homogenization at 6 m / sec for 1 min per cycle for a total of 3 cycles, and then treated with MMP-7 (0.8 μg / mL) for 16 hours ("treated UC"). This is shown diagrammatically in the process in Figure 12. Another control ("UC") without MMP-7 treatment was prepared. As shown in Figure 13, MMP-7 treated extracts showed a decrease in the expression of decorin (DCN) compared to the untreated control. Decorin was measured using an ELISA kit (ThermoFisher Scientific).

[0079] Additional hUC extracts were prepared and treated with MMP-7 under the same conditions as in Figure 13. Decorin expression levels shown in Figure 14A confirm a reduction of approximately 45% of decorin by protease treatment. Differences in the relative amounts of decorin may be related to variability between tissues obtained, for example, from different donors or within the same tissue source. Figure 14B shows the total protein content (μg / mL) measured for treated and control samples, showing similar levels. Total protein content was measured using a BCA (Bicinchoninic Acid) Protein Assay Kit (ThrmoFisher Scientific), testing 600 different proteins, of which 448 protein biomarkers were identified. This suggests that MMP-7 treatment was successful in removing the decorin content without significant damage to other proteins, including potentially beneficial components.

[0080] Example 7 Immunomodulation by protease-treated hUC extracts The samples in Figures 14A-14B prepared according to Example 6 were further investigated in an immune modulation assay to determine their effect on immune biomarkers IL-1β and IL-10. U937 cell cultures were treated with 20 ng / mL phorbol 12-myristate 13-acetate (PMA) for 48 hours to generate macrophage-like cells and then given an M1 differentiation stimulus (50 ng / mL LPS + 10 ng / mL IFN-γ) ("Stim") or no stimulus ("Unstim"). M1 cultures were also treated with untreated hUC extract ("UC") and MMP-7 treated hUC extract ("Treated UC"). The assay is shown diagrammatically in Figure 15. The results in Figures 16A and 16B show that protease treatment led to a reduced secretory response of IL-1β and IL-10. Without wishing to be bound by theory, it is believed that the lower amount of decorin in treated hUC extract reduces the inflammatory response.

[0081] Additional studies were performed to investigate the effect of residual proteases from treated hUC extracts on IL-1β and IL-10. hUC extracts were prepared and treated with MMP-7 as described in Example 6. Decorin levels in untreated hUC extract control ("UC") and MMP-7 treated hUC extract ("treated UC") were measured as described in Example 6. The results, shown in Figure 17, confirm the reduction of decorin in treated hUC extracts.

[0082] Treated and untreated hUC extract samples were subjected to immune modulation assays as described above. Figures 18A and 18B report the levels of IL-1β and IL-10 for unstimulated cultures without hUC extract ("Unstim"), simulated cultures without hUC extract ("Stim"), simulated cultures with untreated hUC extract ("UC"), simulated cultures with MMP-7 treated hUC extract ("Treated UC"), and stimulated cultures without hUC extract and with MMP-7 (0.8 μg / mL) ("MMP-7"), respectively. These results show that protease treatment of hUC extract resulted in a significant reduction of IL-1β and IL-10, consistent with the results of Figures 16A-16B. MMP-7 alone was not observed to affect IL-1β compared to the "Stim" control (Figure 18A), and caused a small (not statistically significant) decrease in IL-10 compared to the control (Figure 18B), suggesting that the reduced inflammatory response was not due to residual proteases, supporting the conclusion that removal of decorin by protease treatment reduces the immune response.

[0083] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the disclosure and the knowledge of one of ordinary skill in the art. Although the compositions and methods of the present disclosure have been described with reference to exemplary embodiments, it will be apparent to one of ordinary skill in the art that variations can be applied to the methods and the steps or sequence of steps of the described methods without departing from the concept, spirit and scope of the present disclosure. More specifically, it will be apparent that certain agents that are chemically and physiologically related may be substituted for the agents described herein with the same or similar results. All such similar substitutes and modifications apparent to those of ordinary skill in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

Claims

1. A physiologically buffered human umbilical cord (hUC) extract composition comprising micronized particles of hUC that have been treated with an ECM-degrading protease.

2. The composition of claim 1 , wherein the hUC comprises an hUC membrane, an hUC stroma, or a combination of an hUC membrane and an hUC stroma.

3. The composition of claim 1 further comprising a gel former, e.g., an in situ polymerizing gel former.

4. The composition of claim 3, wherein the gel forming agent is present at about 0.1 to 8 mg / ml.

5. 10. The composition of claim 1, further comprising a cross-linking agent, such as genipin or transglutaminase.

6. 4. The composition of claim 3, wherein the gel forming agent comprises one or more of fibrin, collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VIII, collagen X, collagen XI, collagen XXIV, or collagen XXVII, polyethylene glycol, poly(lactic-co-glycolic acid), poly(ethylene glycol) diacrylate, gelatin methacryloyl, and methacrylated hyaluronic acid.

7. The composition of claim 1 , wherein the ECM-degrading protease-treated hUC comprises a hUC membrane and the gel-forming agent is not fibrin.

8. 10. The composition of claim 1, wherein the composition is a saline-based suspension buffered to about pH 7.2-7.4, or the composition is formulated as a gel, such as a hydrogel.

9. 2. The composition of claim 1, further comprising one or more of hyaluronic acid, chondroitin sulfate, chitosan, PEG, collagen VI, collagen VII, collagen IX, collagen XII, collagen XIII, collagen XIV, collagen XV, collagen XVI, collagen XVII, collagen XVIII, collagen XIX, collagen XX, collagen XXI, collagen XXII, collagen XXIII, collagen XXV, collagen XXVI and / or collagen XXVIII.

10. 2. The composition of claim 1, wherein the majority of the micronized particles have a diameter between about 80 nm and about 180 nm, such as between about 140 nm and about 160 nm.

11. 1. A method for producing a human umbilical cord (hUC) extract, comprising: (a) providing a hUC membrane and / or hUC stroma; (b) generating micronized particles by mechanically impacting the hUC membranes and / or hUC stroma; (c) treating one or more of: (i) the composition of step (a) prior to mechanical impaction; (ii) the composition of step (b) during mechanical impaction; and (iii) the micronized particles resulting from step (b) with an ECM-degrading protease. The method includes:

12. The method of claim 11, further comprising inactivating the protease.

13. 13. The method of claim 12, wherein after inactivating the ECM-degrading proteases, a gel-forming agent, for example an in situ polymerizing gel-forming agent, is added.

14. The method of claim 13, further comprising polymerizing the in situ polymerizing gel former.

15. The method of claim 14, wherein the polymerization is carried out in the presence of a crosslinking agent.

16. 16. The method of claim 15, wherein the cross-linking agent is genipin or transglutaminase.

17. 14. The method of claim 13, wherein the gel forming agent comprises one or more of fibrin, collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VIII, collagen X, collagen XI, collagen XXIV, or collagen XXVII, polyethylene glycol, poly(lactic-co-glycolic acid), poly(ethylene glycol) diacrylate, gelatin methacryloyl, and methacrylated hyaluronic acid.

18. The method of claim 13, wherein the gel forming agent is present at about 0.1 to 8 mg / ml.

19. 0.5-1.0 cm ２ The method of claim 11, wherein a hUC membrane and / or hUC stroma of is provided in step (a).

20. 12. The method of claim 11, wherein the hUC membranes and / or hUC stroma provided in step (a) are dispersed in a saline-based suspension buffered to about pH 6.0-8.

0.

21. 12. The method of claim 11, wherein the mechanical impingement is performed at a speed ranging from about 3400 RPM to about 3700 RPM for 1 to about 5 cycles with a duration of about 60 seconds per cycle.

22. 22. The method of claim 21, further comprising centrifuging the micronized particles prior to ECM-degrading protease treatment.

23. The method of claim 11 , wherein the ECM-degrading protease is a collagenase or a matrix metalloproteinase (MMP).

24. 24. The method of claim 23, wherein the protease is one or more of collagenase I, collagenase II, collagenase III, collagenase IV, collagenase V, collagenase VI, collagenase VII, MMP-2, MMP-3, and MMP-7.

25. 24. The method of claim 23, wherein the collagenase is one or more of collagenase I and collagenase III.

26. 12. The method of claim 11, wherein one or more of hyaluronic acid, chondroitin sulfate, chitosan, PEG, collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VI, collagen VII, collagen IX, collagen XII, collagen XIII, collagen XIV, collagen XV, collagen XVI, collagen XVII, collagen XVIII, collagen XIX, collagen XX, collagen XXI, collagen XXII, collagen XXIII, collagen XXVI, and collagen XXVIII are added to the composition after inactivation of the ECM degrading proteases.

27. 12. The method of claim 11, wherein the mechanical collision provides a majority of micronized particles having a diameter between about 80 nm and about 180 nm, such as between about 140 nm and about 160 nm.

28. 1. A composition comprising micronized particles of human umbilical cord (hUC) tissue and a buffer, the composition being formulated for injection into a subject.

29. 30. The composition of claim 28 formulated as a gel.

30. 30. The composition of claim 28, wherein the hUC tissue is treated with a protease.

31. 30. The composition of claim 28, comprising less than 1.0 μg / mL decorin.

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