Method for treating osteoarthritis with combination of mesenchymal stem cell exosomes, synovial mesenchymal stem cells, and scaffolds

Abstract

To provide a method for restoring articular cartilage to a normal physiologic condition.SOLUTION: Provided is a method of treating, inhibiting, reducing, ameliorating and / or preventing a disease, a disorder, injury or symptoms thereof affecting one or more joints in a subject, comprising administering to a subject a therapeutically effective amount of a mesenchymal stem cell (MSC) exosome preparation.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Application No. 62 / 908,853, filed October 1, 2019, and U.S. Provisional Application No. 62 / 802,310, filed February 7, 2019, which are incorporated by reference in their entireties herein. Osteoarthritis (OA) is a disorder of any joint in the body that leads to symptoms such as inflammation, pain, and limited function. It is the most common chronic disease in the United States. Most arthritis occurs in the knee or hip, followed by the ankle and shoulder joints. In addition, arthritis of the interphalangeal joints and the base of the thumb is very common.

[0002] OA, also called wear and tear arthritis, is by far the most common cause of arthritis. Over the last 65 years, approximately one-third of adults in the United States have suffered from arthritis to the point of needing regular medication. Symptoms include joint pain during or after activity, tenderness when compressing the joint, a creaking or grinding sound in the bone, and a feeling of stiffness in the joint after periods of inactivity, such as sleeping or sitting. [Background technology]

[0003] The pathogenesis of knee OA has been linked to biomechanical and biochemical changes in the cartilage of the knee joint, such as the inability to withstand normal mechanical stress, limited supply of nutrients and oxygen, insufficient synthesis of extracellular matrix components, increased synthesis of tissue-destructive proteinases, such as matrix metalloproteinases and aggrecanase, and global apoptosis of chondrocytes. Recently, synovitis has also been recognized as a factor limiting knee cartilage repair. Furthermore, it is associated with clinical signs of knee OA, such as knee swelling and inflammatory pain. Synovitis is thought to be a reaction of synovial macrophages to cartilage debris and degradative mediators entering the synovial cavity.

[0004] Articular cartilage is both aneural and avascular. Cartilage, by itself, cannot directly cause pain, inflammation, stiffness, or any of the symptoms that OA patients typically complain of. Although all of the above is known information, the exact cause of OA pain is not fully understood.

[0005] Therefore, there is a need for a method to restore articular cartilage to its normal physiological state so as to halt synovitis and thereby relieve the pain of OA. Summary of the Invention

[0006] Methods and compositions relating to mesenchymal stem cell (MSC) exosome compositions for use in treating diseases, disorders and injuries affecting joints are disclosed.

[0007] In one aspect, disclosed herein is a method of treating, inhibiting, reducing, ameliorating, and / or preventing a disease, disorder, injury (e.g., osteoarthritis, juvenile arthritis, psoriatic arthritis, infectious arthritis, rheumatoid arthritis, ankylosing spondylitis, gout, bursitis, tendinosis, tendonitis, sprains, labral tears, tendon ruptures, and / or ligament ruptures, etc.) affecting one or more joints (e.g., ankles, knees, hips, writs, elbows, shoulders, knuckles, and / or neck, etc.) in a subject or a symptom thereof (e.g., pain, inflammation, and / or swelling, etc.), comprising administering to the subject a therapeutically effective amount of a mesenchymal stem cell (MSC) exosome preparation.

[0008] Disclosed herein is a method according to any of the above aspects, wherein an MSC exosome preparation is administered to each joint affected by the disorder, disease, or injury.

[0009] In one aspect, the MSC exosome preparation comprises MSC-derived growth factors (e.g., prostaglandin E2 (PGE2), transforming growth factor beta 1 (TGF-β1), hepatocyte growth factor (HGF), stromal cell-derived factor-1 (SDF-1), nitric oxide, indoleamine 2,3-dioxygenase, interleukin-4 (IL-4), IL-6, interleukin-10 (IL-10), IL-1 receptor antagonist and soluble TNF-α receptor, insulin-like growth factor, fibroblast growth factors (FGF) 1-23 (particularly FGF1 and FGF2), bone morphogenetic proteins (BMP) 1-15, epidermal growth factor (EGF), transforming growth factor-α (TGF-α), macrophage stimulating protein (MSP), and / or inflammatory cytokines (IGCs) (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-231, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-39, IL-39, IL-39, IL-39, IL-39, IL-39, IL-39, IL-39, Disclosed herein are methods of any of the above aspects, further comprising a hormone, including a stimulant, such as a stimulant for the treatment of atopic dermatitis (ACAD), ...

[0010] Also disclosed herein is a method according to any of the above aspects, wherein the MSC exosomes are administered via injection, a scaffold carrying the MSC exosomes, a hydrogel, and / or a topical medication or ointment.

[0011] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and, together with the specification, explain the disclosed compositions and methods. [Brief description of the drawings]

[0012] [Figure 1] Lower BPI and ODI indicate improvement, while higher LEFS scores indicate improvement. [Diagram 2] FIG. 1 shows a pre-treatment x-ray of the shoulder demonstrating Kellgren-Lawrence grade 4 glenohumeral osteoarthritis. [Diagram 3]Lower BPI and QuickDash indicate improvement, while higher UFEI scores indicate improvement. [Figure 4] A diagram of MSC exosome treatment for OA is shown. [Diagram 5] An x-ray of the pelvis is shown. [Figure 6] Figure 6A shows an x-ray of a right elbow showing Kellgren-Lawrence grade 3 changes in the radioulnar and humeroulnar joints, and Figure 6B shows a normal x-ray of a left elbow. [Figure 7] Shows the range of motion of the elbow after EVIP administration. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Before the present compounds, compositions, articles, devices, and / or methods are disclosed and described, it is to be understood that these are not limited to particular synthetic methods, or to particular recombinant biotechnology methods, unless otherwise specified, or to particular reagents, which can, of course, vary accordingly. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0014] A.Definition As used in the specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly indicates otherwise. Thus, for example, reference to a "pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

[0015] Ranges may be expressed herein as from one particular value preceded by "about" and / or to another particular value preceded by "about." When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations by use of the antecedent "about," it is understood that the particular value forms another embodiment. It will be further understood that each end of the range is significant relative to the other end, and independent of the other end. It is also understood that there are more than one value disclosed herein, and that each value is also disclosed herein as "about" that particular value in addition to the value itself. For example, if a value of "10" is disclosed, then "about 10" is also disclosed. It is also understood that when a value is disclosed, "less than or equal to," "greater than or equal to," and possible ranges between values ​​are also disclosed, as would be understood by one of skill in the art. For example, if a value of "10" is disclosed, then not only less than "10" but also greater than "10" is disclosed. It is also understood that throughout the application, data is provided in several different formats and that this data represents endpoints and starting points and ranges of any combination of the data points. For example, if a specific data point "10" and a specific data point 15 are disclosed, it is understood that greater than 10 and 15, greater than or equal to 10 and 15, less than 10 and 15, less than or equal to 10 and 15, and equal to 10 and 15 are also contemplated, as well as between 10 and 15. It is also understood that each unit between two specified units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0016] As used herein, the term "subject" is defined to include animals, such as mammals, including, but not limited to, primates (e.g., humans), cows, horses, pigs, sheep, goats, dogs, cats, rabbits, rats, mice, etc. In some embodiments, the subject is a dog.

[0017] "Administering" to a subject includes any route of introducing or delivering a drug to a subject. Administration can be by any suitable route, including oral, topical, intravenous, subcutaneous, transdermal, intramuscular, intraarticular, parenteral, intraarteriolar, intraarticular, intradermal, intracerebroventricular, intracranial, intraperitoneal, intralesional, intranasal, intrarectal, intravaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injection or infusion techniques), and the like. As used herein, "co-administration," "combined administration," "co-administration," or "administered simultaneously" refers to administration of each compound at the same time or substantially immediately after each other. In the latter case, the two compounds are administered at a time sufficiently close that the results achieved are not different from those achieved if both compounds were administered at the same time. "Systemic administration" refers to the introduction or delivery of an agent to a subject via a route that introduces or delivers the agent to a wide area of ​​the subject's body (e.g., greater than 50% of the body), for example, via an entry into the circulatory or lymphatic system. In contrast, "local administration" refers to the introduction or delivery of an agent to a subject via a route that introduces or delivers the agent to the area of ​​the point of administration or the area immediately adjacent to the point of administration, but does not introduce the agent systemically in therapeutically significant amounts. For example, a locally administered agent is readily detectable in the local vicinity of the point of administration, but is undetectable or only detectable in small amounts in distal parts of the subject's body. Administration includes self-administration and administration by another.

[0018] "Biocompatible" generally refers to a material, and any metabolic or decomposition products thereof, that is generally non-toxic to the recipient and does not cause significant adverse effects in the subject.

[0019] "Comprising" is intended to mean that the composition, method, etc. includes the recited elements, but does not exclude others. When used to define compositions and methods, "consisting essentially of" is intended to mean including the recited elements, but excluding any other elements that are of essential importance to the combination. That is, a composition consisting essentially of the elements as defined herein shall not exclude trace contaminants from the isolation and purification methods, as well as pharma- ceutically acceptable carriers, e.g., phosphate buffered saline, preservatives, and the like. "Consisting of" is intended to mean excluding trace elements of other materials and more than substantial method steps for administering the compositions of the invention. Embodiments defined by each of these transitional phrases are within the scope of the invention.

[0020] A "control" is another control or sample used in an experiment for purposes of comparison. There can be "positive" or "negative" controls.

[0021] An "effective amount" of a drug refers to an amount of the drug sufficient to produce a desired effect. The amount of a drug that is "effective" will vary between subjects and will depend on a number of factors, such as the age and general condition of the subject, the specific drug, etc. That is, it is not always possible to specify a quantified "effective amount". However, an appropriate "effective amount" in any subject's case may be determined by one of ordinary skill in the art through routine experimentation. Also, as used herein, and unless otherwise specified, an "effective amount" of a drug may refer to an amount that covers both a therapeutically effective amount and a prophylactically effective amount. The "effective amount" of a drug that is necessary to achieve a therapeutic effect may vary depending on factors such as the age, sex, and weight of the subject. The dosing regimen may be adjusted to provide an optimal therapeutic response. For example, several divided doses may be administered daily, or the dosage may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0022] "Reduce" refers to less gene expression, protein production, or the presence or absence of a condition, disease, composition, It may refer to any change that results in the amount of a condition or activity. A substance is also understood to reduce the genetic output of a gene when the genetic output of a gene product containing the substance is less when compared to the output of the gene product without the substance. For example, a reduction may be a change in symptoms of a disorder such that the symptoms are less than previously observed. A reduction may be any individual, mean, or average decrease in a condition, symptom, activity, or composition that is a statistically significant amount. That is, the reduction may be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% reduction, so long as the reduction is statistically significant.

[0023] "Inhibit," "inhibiting," and "inhibition" refer to a decrease in an activity, response, symptom, disease, or other biological parameter. This can include, but is not limited to, a complete elimination of the activity, response, symptom, or disease. It can also include, for example, a 10% decrease in the activity, response, symptom, or disease compared to native or control levels. That is, the decrease can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% decrease, or any amount between, compared to native or control levels.

[0024] As used herein, "treat", "treating", "treatment" and grammatical variations thereof include administration of a composition intended or intended to partially or completely prevent, delay, ameliorate, cure, alleviate, relieve, alter, improve, ameliorate, enhance, stabilize, quell, and / or reduce the intensity or frequency of one or more diseases or conditions, symptoms of a disease, disorder, injury or condition, or the underlying causes of a disease or condition. Treatment according to the present invention may be applied preventatively, prophylactically, palliatively, or salvageably. Prophylactic treatment is administered to a subject before onset (e.g., before overt signs of cancer), during early onset (e.g., concomitant with early signs and symptoms of cancer), or after the development of cancer has been established. Prophylactic administration can occur days to years prior to the onset of symptoms of infection.

[0025] As used herein, the terms "prevent", "preventing", "prevention" and grammatical variations thereof refer to a method of partially or completely delaying or preventing the onset or recurrence of a disease and / or one or more of its associated symptoms, or a method of partially or completely preventing a subject from acquiring or re-acquiring a disease, or a method of partially or completely reducing a subject's risk of acquiring or re-acquiring a disease or one or more of its associated symptoms.

[0026] A "pharmaceutical acceptable" ingredient may refer to an ingredient that is not biologically or otherwise undesirable, i.e., the ingredient may be incorporated into a pharmaceutical formulation of the invention and administered to a subject as described herein without causing significant undesirable biological effects or adversely interacting with any of the other ingredients in the formulation in which it is contained. When used in reference to administration to humans, the term generally implies that the ingredient has met the necessary standards of toxicity and manufacturing testing or its inclusion in the Inactive Ingredients Guide by the U.S. Food and Drug Administration.

[0027] A "pharmaceutical acceptable carrier" (sometimes referred to as a "carrier") refers to a carrier or excipient that is generally safe and non-toxic and useful in formulating pharmaceutical or therapeutic compositions, including carriers that are acceptable for veterinary and / or human pharmaceutical or therapeutic use. The term "carrier" or "pharmaceutical acceptable carrier" can include, but is not limited to, phosphate buffered saline solutions, water, emulsions (e.g., oil / water or water / oil emulsions, etc.), and / or various types of wetting agents. As used herein, the term "carrier" includes, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material used in pharmaceutical formulations as is well known in the art and further described herein.

[0028] "Pharmacologically active" (or simply "activity"), as in "pharmacologically active" derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, complex, metabolite, isomer, fragment, etc.) that has the same type and approximately the same degree of pharmacological activity as the parent compound.

[0029] "Therapeutic agent" refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., the treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., the prevention of a disorder or other undesirable physiological condition (e.g., non-immunogenic cancer). The term also encompasses pharmaceutically acceptable, pharmacologically active derivatives of the beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. And, when the term "therapeutic agent" is used, or when a particular agent is specifically identified, it is understood that the term includes not only the agent itself, but also pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, complexes, active metabolites, isomers, fragments, analogs, and the like.

[0030] A "therapeutically effective amount" or "therapeutically effective dose" of a composition (e.g., a composition containing an agent) refers to an amount that is effective to achieve a desired therapeutic outcome. In some embodiments, the desired therapeutic outcome is the control of type I diabetes. In some embodiments, the desired therapeutic outcome is the control of obesity. The therapeutically effective amount of a given therapeutic agent will typically vary depending on factors such as, for example, the type and severity of the disorder or disease being treated, as well as the age, sex, and weight of the subject. The term may also refer to the amount of therapeutic agent, or the rate of delivery of the therapeutic agent (e.g., amount over time), that is effective to promote a desired therapeutic effect, such as, for example, the relief of pain (i.e., pain sensation). The exact desired therapeutic effect will vary depending on the condition being treated, the tolerance possessed by the subject, the agent and / or agent formulation being administered (e.g., the potency of the therapeutic agent, the concentration of the agent in the formulation, etc.), as well as a variety of other factors recognized by those skilled in the art. In some instances, the desired biological or medical response is achieved after administering multiple doses of the composition to the subject over a period of days, weeks, or years.

[0031] In this specification and in the claims that follow, reference will be made to certain terms that shall be defined to have the following meanings:

[0032] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs as well as instances where it does not occur.

[0033] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The disclosed references are also individually and specifically incorporated by reference herein for the material contained therein that is discussed in the sentence in which the reference is cited.

[0034] Disclosed are the components used to prepare the disclosed compositions, as well as the compositions themselves used within the methods disclosed herein. These and other substances are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these substances are disclosed, it is understood that each is specifically contemplated and described herein, although specific references to various individual and collective combinations and permutations of these compounds may not be expressly disclosed. For example, certain MSC exosomes (with or without growth factors) referred to herein as extracellular vesicle isolate products (EVIPs) are disclosed and discussed, and several modifications that can be made to several molecules, including EVIPs, are discussed, and in particular, each and every combination and permutation of EVIPs and possible modifications are contemplated, unless specifically indicated to the contrary. That is, if classes of molecules A, B and C are disclosed, and classes of molecules D, E and F are disclosed, and an example of a combination molecule AD is disclosed, each is considered individually or collectively, even if each is not individually described, which means that combinations AE, AF, BD, BE, BF, CD, CE and CF are considered and disclosed. Similarly, any subset or combination of these is also disclosed. That is, for example, subgroups of AE, BF and CE are considered and disclosed. This concept applies to all aspects of the application, including but not limited to steps in the methods of making and using the disclosed compositions. That is, if there are various additional steps that can be performed, it is understood that each of these additional steps can be performed with any particular embodiment or combination of embodiments of the disclosed methods.

[0035] B. Treatment methods for osteoarthritis In contrast to articular cartilage, the synovial and joint capsules are well innervated and therefore appear to be the major source of pain in OA. The meningeal response in OA includes synovial hyperplasia, fibrosis, bursal hypertrophy, activated synovial cells, and in some cases lymphocytic infiltration (B and T cells, as well as plasma cells). The synovium is clearly of relevance as one of the most densely innervated structures of the joint. Causes of synovial pain include stimulation of sensory nerve endings in the synovium from osteophytes, and synovitis due at least in part to release of prostaglandins, leukotrienes, proteinases, neuropeptides, and cytokines. Proinflammatory examples include interleukins 1, 6, and 8, as well as various tumor necrosis factors. Semiquantitative measurements of synovitis from the infrapatellar fat pad are associated with pain severity. Any reduction in synovitis is associated with reduced pain severity in OA.

[0036] Despite the fact that bone marrow is considered to be the widely known source of MSCs, various studies have reported that MSCs can be isolated from various adult mesenchymal tissues, including synovium. Studies have shown that synovium-derived MSCs have high proliferation and multilineage differentiation potential in vitro. Studies have also determined that synovium-derived MSCs have higher proliferation and chondrogenic potential in vitro than MSCs from other tissues, comparing human MSCs derived from bone marrow, synovium, periosteum, adipose tissue, and muscle. This suggests that synovium-derived MSCs are a superior source for cartilage regeneration.

[0037] Studies have shown that synovial MSCs can be dissociated from the synovial membrane using scraping techniques, resulting in a significant increase in the number of synovial MSCs present in the synovial fluid.

[0038] In one aspect, disclosed herein is a method of treating, inhibiting, reducing, ameliorating, and / or preventing a disease, disorder, injury (e.g., osteoarthritis, juvenile arthritis, psoriatic arthritis, infectious arthritis, rheumatoid arthritis, ankylosing spondylitis, gout, bursitis, tendinosis, tendinitis, sprains, labral tears, tendon ruptures, and / or ligament ruptures, etc.) affecting one or more joints (e.g., ankles, knees, hips, writs, elbows, shoulders, knuckles, and / or neck) in a subject or a symptom thereof (e.g., pain, inflammation, and / or swelling, etc.), comprising administering to the subject a therapeutically effective amount of a mesenchymal stem cell (MSC) exosome preparation.

[0039] In one aspect, a method is provided for treating, inhibiting, reducing, ameliorating, and / or preventing a disease, disorder, injury (e.g., osteoarthritis, juvenile arthritis, psoriatic arthritis, infectious arthritis, rheumatoid arthritis, ankylosing spondylitis, gout, bursitis, tendinosis, tendinitis, sprains, labral tears, tendon tears, and / or ligament tears, etc.) or symptoms thereof (e.g., pain, inflammation, and / or swelling, etc.) affecting one or more joints (e.g., ankles, knees, hips, writs, elbows, shoulders, knuckles, and / or neck) in a subject, the method comprising administering to the subject an exosome preparation (also referred to herein as EVIP) that is capable of inhibiting or reducing the production of MSC-derived growth factors (e.g., prostaglandin E2 (PGE2), transforming growth factor β1 (TGF-β1), hepatocyte growth factor (HGF), stromal cell-derived factor-1 (SDF-1), nitric oxide, indoleamine 2,3 Disclosed herein are methods for treating the inflammatory bowel disease, further comprising administering to the patient an effective amount of a therapeutically effective amount of a inflammatory bowel disease inhibitor, such as erythrocyte-derived leukocyte colony-stimulating factor (ECG), insulin, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and / or hormones, including estrogen, thyroid hormone, erythrocyte-derived leukocyte colony-stimulating factor (EGF), fibroblast growth factor (FGF) 1-23 (particularly FGF1 and FGF2), bone morphogenetic protein (BMP) 1-15, epidermal growth factor (EGF), transforming growth factor-α (TGF-α), macrophage stimulating protein (MSP), platelet-derived growth factor (PLGF), vascular endothelial growth factor (VEGF), macrophage colony-stimulating factor (M-CSF), insulin, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and / or hormones, including estrogen, and thyroid hormone.

[0040] It is understood and contemplated herein that the disclosed MSC exosome treatments may not be capable of curing a disease, disorder, injury or condition to a joint, and may reduce or inhibit the injury, disease or disorder. In one aspect, the MSC exosome preparations reduce symptoms of a disease, disorder and / or injury (e.g., pain, inflammation and / or swelling) rather than being capable of curing or repairing a disease, disorder or injury. That is, in one aspect, disclosed herein is a method of treating, inhibiting, reducing, preventing and / or ameliorating pain, inflammation and / or swelling associated with a disease, disorder and / or injury affecting one or more joints in a subject, comprising administering to the subject any of the MSC exosome preparations disclosed herein (including, in some cases, growth factors derived from MSCs).

[0041] It is understood that the administration may be directly to one or more affected joints; As discussed herein, administration of the disclosed MSC-derived exosomes and / or growth factors can be by any method known to one of skill in the art. Accordingly, disclosed herein are methods for treating, inhibiting, reducing, ameliorating, and / or preventing a disease, disorder, injury (e.g., osteoarthritis, juvenile arthritis, psoriatic arthritis, infectious arthritis, rheumatoid arthritis, ankylosing spondylitis, gout, bursitis, tendinosis, tendinitis, sprains, torn labrum, torn tendon, and / or torn ligament, etc.) affecting one or more joints (e.g., ankles, knees, hips, writs, elbows, shoulders, knuckles, and / or neck, etc.) in a subject, or a symptom thereof (e.g., pain, inflammation, and / or swelling, etc.), comprising administering to the subject a therapeutically effective amount of a mesenchymal stem cell (MSC) exosome preparation, wherein the MSC exosomes are administered via injection, MSC exosome-bearing scaffold, hydrogel, and / or topical application or ointment. As the field of tissue engineering advances, the need for novel scaffold structures and reproducible manufacturing techniques has become paramount. The use of biodegradable polymers, such as polylactic acid (PLA), has become widespread, but these polymers can be engineered to allow tailoring of the final properties of the scaffold in an additive-like manner during manufacturing.

[0042] Poly-hydroxyl acids, such as PLA and polylactic-glycolic acid (PLGA), have been widely used in tissue engineering procedures because these materials undergo bulk degradation by hydrolysis, resulting in controllable drug release and degradation profiles suitable for tissue growth. Through careful use of molecular weight, crosslinks, and side chains, materials can be produced with tailored properties that make them ideal for use in tissue engineering matrices. Additionally, polyhydroxy acid materials also have a long history of use in vivo as degradable sutures, drug delivery devices, and biodegradable surgical components.

[0043] Existing scaffold types include high-pressure CO2 foamed scaffolds, injectable scaffolds, and novel custom-made scaffolds. These can be further modified with growth factors, material zonation, and plasma polymerization deposition. Scaffolds enhance the survival of synovial MSCs adjacent to articular cartilage, which can be increased by the addition of cytokines. For example, PLGA with transforming growth factor-β3 (TFG-β3) enhances the differentiation of MSCs into chondrocytes, while implantation of PLGA with stromal cell-derived factor-1α (SDF-1α) results in repair of articular cartilage. Thus, implantation of PLGA in combination with various cytokines enhances more efficient differentiation of synovial MSCs into articular cartilage. This technique delivers concentrated MSC growth factors and RNA to synovial MSCs present in the periarticular scaffold, maximizing their differentiation into chondrocytes.

[0044] By way of example, some embodiments of the present invention include a method of treating osteoarthritis, comprising a one-step arthroscopic procedure to restore arthritic articular cartilage to a normal physiological state. In particular, the method may comprise abrading synovial mesenchymal stem cells (MSCs) from the synovial membrane using an abrasion device, covering the articular cartilage with a scaffold, and placing enriched MSC exosomes in the affected joint, such as the knee joint, to stimulate differentiation of synovial MSCs into articular chondrocytes.

[0045] In embodiments, the abrading device may be an arthroscopic brush specifically designed for use in conjunction with a particular joint.

[0046] When the scaffold is placed in a joint, such as the knee, under arthroscopic guidance to cover the articular cartilage, the MSCs floating in the joint can attach to the scaffold around the joint. By placing MSC exosomes in the joint, they can supply growth factors and various RNAs, stimulating the differentiation of synovial MSCs into chondrocytes. As a result, damaged arthritic articular cartilage can be repaired.

[0047] C. Mesenchymal stem cells As described throughout, the therapeutic compositions disclosed herein may utilize exosomes and / or growth factors derived from mesenchymal stem cells (MSCs). Although existing autologous and allogeneic MSCs contained in bone marrow, bone marrow concentrates, synovium-derived mesenchymal stem cells (MSCs) or adipose-derived stromal vascular fraction (SVF), or various postnatal products from umbilical cord, placenta or amniotic membrane, expanded MSC cultures are currently used to treat injuries, orthopedic and spinal lesions, existing treatments do not contain significant amounts of the MSC secretome (including but not limited to growth factors, cytokines, chemokines, exosomes, extracellular vesicles and / or extracts). Moreover, despite evidence in the art that stem cell-containing treatments (including injection treatments) can help prevent aging and treat scarring and uneven pigmentation, existing skin products, such as creams, lotions, serums, cosmetics, etc., contain materials that may help treat and strengthen the skin, other topical products, but do not penetrate the epidermis and, above all, do not contain human MSCs or MSC-derived growth factors and proteins. In fact, prior to the present disclosure, no active MSC growth factor products have been developed that can be used for these applications. Thus, in one aspect, disclosed herein is an MSC secretome composition (including, but not limited to, MSC growth factors, MSC exosomes, extracellular vesicles containing MSC extracts and / or compositions) for use in treating injury, orthopedic disorder, orthopedic injury, ophthalmic, spinal injury or spinal disorder, the therapeutic composition comprising (i) a powdered additive of growth factors comprising a preparation derived from mesenchymal stem cells (MSCs) and (ii) a pharmaceutically acceptable carrier.

[0048] As described above, MSCs are multipotent cells that have the ability to differentiate into multiple cell types, including myocytes, chondrocytes, adipocytes, and osteoblasts. These cells can typically be found in the placenta, umbilical cord blood, adipose tissue, bone marrow, or amniotic fluid, including perivascular tissue. As used herein, "MSCs" refers to non-terminally differentiated cells, including but not limited to pluripotent stem cells, pluripotent stromal cells, stromal vascular cells, pericytes, perivascular cells, stromal cells, pluripotent cells, multipotent cells, adipose-derived fibroblast-like cells, adipose-derived stromal vascular cells, adipose-derived MSCs, bone marrow-derived fibroblast-like cells, bone marrow-derived stromal vascular cells, bone marrow-derived MSCs, tissue-derived fibroblast-like cells, mature stem cells, mature stromal cells, keratinocytes, and / or melanocytes.

[0049] In addition to their differentiation potential, MSCs have long been recognized to have immune-modulating capabilities resulting in the expression of a number of different cytokines and growth factors. As used herein, "MSC preparation" or "MSC secretome composition" refers to a composition comprising MSC growth factors, MSC exosomes, extracellular vesicles or cell-free extracts of MSCs, or MSC lysates obtained from human MSCs, fibroblast-like cells, and non-human animal MSCs, including but not limited to MSCs from horses, cows, pigs, sheep, non-human primates, dogs, cats, rabbits, rats, and mice. In embodiments, the MSCs may be derived from the patient to whom the composition will be applied (autologous) or from another individual (allogeneic). The MSCs may be cultures expanded to harvest conditioned medium or to increase the amount of cells to be used fresh for lysates or before incorporation into the compositions of the present disclosure.

[0050] The MSC secretome composition (including but not limited to MSC growth factors, MSC exosomes, MSC extracts and / or extracellular vesicles comprising the composition) may comprise about 0.00001 to about 20% by weight of a mesenchymal stem cell (MSC) extract, MSC exosomes, or MSC growth factor preparation, such as about 0.01 to about 10% by weight. The MSC preparation may comprise either MSC conditioned medium or an MSC lysate from MSCs grown in cell culture. In some embodiments, the composition may further comprise about 0.01 to about 10% by weight of cell-free medium conditioned by growth of MSCs or MSC-lineage cells, the cells being cultured under normal hyperoxic culture conditions or under artificial wound healing conditions.

[0051] As disclosed herein, the MSCs used to produce the disclosed MSC additives (including growth factor secretome composition additives, whether frozen or powdered) can be selectively stimulated to produce MSC growth factors, secretomes, cytokines, chemokines, mesenchymal stem cell proteins, peptides, glycosaminoglycans, extracellular matrix (ECM), proteoglycans, secretomes, and exosomes. As used herein, MSC growth factors include prostaglandin E2 (PGE2), transforming growth factor β1 (TGF-β1), hepatocyte growth factor (HGF), stromal cell-derived factor-1 (SDF-1), nitric oxide, indoleamine 2,3-dioxygenase, interleukin-4 (IL-4), IL-6, interleukin-10 (IL-10), IL-1 receptor antagonist and soluble TNF-α receptor, insulin-like growth factor, fibroblast growth factor (FGF) 1-23 (particularly FGF1 and These include, but are not limited to, FGF2), bone morphogenetic proteins (BMP) 1-15, epidermal growth factor (EGF), transforming growth factor-α (TGF-α) macrophage stimulating protein (MSP), platelet-derived growth factor (PLGF), vascular endothelial growth factor (VEGF), macrophage colony-stimulating factor (M-CSF), insulin, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and hormones including estrogen and thyroid hormone.

[0052] In one aspect, the MSC preparation (e.g., MSC secretome composition, etc.) comprises MSC growth factors, MSC exosomes, and / or cellular extracts of MSCs, or MSC lysates obtained from MSCs cultured under standard hyperoxic culture conditions (e.g., 21% oxygen) or under artificial wound healing conditions (e.g., 0.1% to about 5% oxygen in the presence of inflammatory cytokines, angiogenic factors and reduced glucose).

[0053] As disclosed herein, artificial wound healing conditions simulate the growth conditions found in real wounds, where there is a reduced supply of nutrients and reduced removal of waste products, usually resulting from disruption of local blood circulation. This creates a harsh environment for cells until new blood vessels form and blood circulation is restored. Thus, artificial wound healing conditions used to culture MSCs may include one or more of the following growth conditions: reduced glucose availability, reduced oxygen tension, reduced pH, and increased temperature.

[0054] In one aspect, glucose availability can be reduced compared to normal controls. The culture medium modified to reduce glucose but not damage the cells can have between 0-50% reduced glucose, more preferably between about 5%-40% reduced glucose. For example, the MSC artificial wound healing culture conditions may include a glucose reduction of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50%, such as a glucose reduction of about 5% to about 15%, about 10% to about 20%, about 15% to about 25%, about 20% to about 30%, or about 25% to about 35%.

[0055] In one embodiment, the oxygen partial pressure can be reduced to a hypoxic condition. Since normal atmospheric oxygen is about 21%, any reduction is considered hypoxic. That is, in one embodiment, MSCs can be cultured under artificial wound healing conditions at 0.0% to 20.9% oxygen, about 0.1% to about 0.5% oxygen, about 0.1% to about 2.0% oxygen, about 0.1% to about 5.0% oxygen, about 0.5% to 5.0%, about 1.0% to about 10% oxygen, about 5.0% to about 10.0% oxygen, and about 10.0% to about 15.0% oxygen. Preferably, the oxygen tension in the MSC wound healing culture conditions is, for example, 0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.7, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 3.10, 3.11, 3.12, 3.13, 3.14, 3.15, 3.16, 3.17, 3.18, 3.19, 3.20, 3.21, 3.22, 3.23, 3.24, 3.25, 3.26, 3.27, 3.28, 3.29, 3.30, 3.31, 3.32, 3.33, 3.34, 3.35, 3.36, 3.37, 3.38, 3.39, 3.40, 3.41, 3.42, 3.43, 3.44, 3.45, 3.46, 3.47, 3.48, 3.49, 3.50, 3.51, 3.52, 3.53, 3.54, 3.55, 3.56, 3.57, 6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0 ,6.1,6.2,6.3,6.4,6.5,6.6,6.7,6.8,6.9,7.0,7.1,7.2,7.3,7.4,7.5,7.6,7.7,7.8,7.9,8.0,8.1,8.2,8.3,8.4,8.5,8.6,8.7,8.8,8.9,9.0,9.1,9.2,9.3,9.4 , 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, or 20.5% oxygen, such as about 0.5% to 20.5% oxygen.

[0056] The pH may also be decreased under wound healing conditions. Physiological pH is maintained very strictly, so it is usually very close to neutral pH=7.2±0.2 (7.0-7.4). However, in a wound, the acidic environment may be pH=6.2±0.2 (i.e., pH 6.0 to about 6.4). That is, under artificial wound healing culture conditions, the pH may be about 6.0 to about 7.4, such as 6.0 to about 6.4, about 6.2 to about 6.4, about 6.2 to about 6.6, about 6.4 to about 6.6, about 6.4 to about 6.8, or about 6.6 to about 7.0, such as 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, or 7.4.

[0057] Under artificial wound healing culture conditions, the temperature of the culture environment may be elevated to mimic the temperature rise at the wound site. Physiological homeostatic temperature is maintained at 37°C (98.6°F). Small increases or decreases can cause significant changes to cellular metabolism. It is possible to create a "hot" environment by increasing the temperature above 37°C to any temperature up to about 40°C (104°F). That is, in one aspect, artificial wound healing culture conditions for MSCs may include about 35°C to about 39°C, about 35°C to about 36°C, about 36°C to about 37°C, about 37°C to about 38°C, about 38°C to about 39°C, and about 39°C to about 40°C. In one embodiment, the temperature of the artificial wound healing culture is 35.0, 35.1, 35.2, 35.3, 36.4, 35.5, 35.6, 35.7, 35.8, 35.9, 36.0, 36.1, 36.2, 36.3, 36.4, 36.5, 36.6, 36.7, 36.8, 36.9, 37.0, 37.1, 37.2, 37.3, It may be 37.4, 37.5, 37.6, 37.7, 37.8, 37.9, 38.0, 38.1, 38.2, 38.3, 38.4, 38.5, 38.6, 38.7, 38.8, 38.9, 39.0, 39.1, 39.2, 39.3, 39.4, 39.5, 39.6, 39.7, 39.8, 39.9 or 40.0°C.

[0058] In one aspect, the MSC secretome composition (including but not limited to, extracellular vesicles containing MSC growth factors, MSC exosomes, MSC extracts and / or compositions) may further comprise a protective coating (e.g., oligosaccharides of cryoprotectants, and protein solutions, etc.) to reduce degradation of the growth factors. It is understood and contemplated herein that the protective coating can be modified as a polymer. A "polymer" refers to a relatively high molecular weight natural or synthetic organic compound whose structure can be expressed in repeating small unit monomers. Non-limiting examples of polymers include polyethylene, rubber, and cellulose. Synthetic polymers are typically formed by addition or condensation polymerization of monomers. The term "copolymer" refers to a polymer formed from two or more different repeating units (monomer residues). By way of example and not limitation, the copolymer may be an alternating copolymer, a random copolymer, a block copolymer, or a graft copolymer. It is also contemplated that in some aspects, the various block segments of a block copolymer may themselves comprise copolymers. The term "polymer" encompasses all forms of polymers, including, but not limited to, natural polymers, synthetic polymers, homopolymers, heteropolymers, or copolymers, addition polymers, etc. In one embodiment, the gel matrix may comprise copolymers, block copolymers, diblock copolymers, and / or triblock copolymers.

[0059] In one embodiment, the protective coating may include a biocompatible polymer. In one embodiment, the biocompatible polymer may be crosslinked. The polymer may also serve to slow release lipid browning agents and / or fat modifying agents into the tissue. As used herein, biocompatible polymers include polysaccharides; hydrophilic polypeptides; polyamino acids, such as poly-L-glutamic acid (PGS), gamma-polyglutamic acid, poly-L-aspartic acid, poly-L-serine, or poly-L-lysine; polyalkylene glycols and polyalkylene oxides, such as polyethylene glycol (PEG), polypropylene glycol (PPG), and polyethylene oxide (PEO); polyoxyethylated polyols; polyolefin alcohols; polyvinylpyrrolidone; polyhybrids; hydroxyalkyl methacrylamides; polyhydroxyalkyl methacrylates; polysaccharides; polyhydroxy acids; polyvinyl alcohols; polyhydroxy acids such as polylactic acid, polyglycolic acid and lactic acid-glycolic acid copolymers; polyhydroxyalkanoates such as poly 3-hydroxybutyrate or poly 4-hydroxybutyrate; polycaprolactones; polyorthoesters; polyanhydrides; polyphosphazenes; lactide-caprolactone copolymers; polycarbonates such as tyrosine polycarbonate; polyamides. Polyethers and polyoxyethylene copolymers include, but are not limited to, polyamides (including synthetic and natural polyamides), polypeptides, and polyamino acids; polyesteramides; polyesters; polydioxanones; polyalkylene alkylates; hydrophobic polyethers; polyurethanes; polyetheresters; polyacetals; polycyanoacrylates; polyacrylates; polymethyl methacrylates; polysiloxanes; polyoxyethylene / polyoxypropylene copolymers; polyketals; polyphosphates; polyhydroxyvalerates; polyalkylene oxalates; polyalkylene succinates; and polymaleic acids as well as copolymers thereof.Biocompatible polymers also include polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates, polyvinyl alcohols (PVA), methacrylate PVA (m-PVA), polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidones, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocelluloses, polymers of acrylic and methacrylic acid esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, cellulose triacetate, cellulose sulphate sodium salt), polymethyl methacrylate, polyethyl methacrylate, polybutyl methacrylate, polyisobutyl methacrylate, polyhexyl methacrylate, polyisodecyl methacrylate, polylauryl methacrylate, polyphenyl methacrylate, polymethyl acrylate, polyisopropyl acrylate, polyisobutyl acrylate, polyoctadecyl acrylate, polyethylene, polypropylene, polyethylene glycol, polyethylene oxide, polyethylene terephthalate, polyvinyl alcohol, polyvinyl acetate, polyvinyl chloride, polystyrene and polyvinylpyrrolidone, derivatives thereof, linear copolymers, branched copolymers and block copolymers thereof, and mixtures thereof.Exemplary biodegradable polymers include polyesters, polyorthoesters, polyethyleneamines, polycaprolactones, polyhydroxybutyrates, polyhydroxyvalerates, polyanhydrides, polyacrylic acids, polyglycolides, polyurethanes, polycarbonates, polyphosphates, polyphospliazenes, derivatives thereof, linear and branched copolymers and block copolymers thereof, and mixtures thereof.

[0060] In some embodiments, the protective coating comprises a carbohydrate polymer such as, for example, a disaccharide or polysaccharide, including, but not limited to, a monosaccharide carbohydrate structure, as well as a non-reducing polysaccharide or disaccharide, as well as any combination thereof. Examples of carbohydrates that can be used in the protective coating include glucose, aldoses (D-allose, D-altrose, D-mannose, etc.), glucopyranose, pentahydroxyhexanal, α-D-glucopyranosyl-D-glucose, α-D-glucopyranosyl-dihydrate, polymers of β-D-glucopyranosyl units, β-D-fructofuranosyl α-D-glucopyranoside (anhydrous / dihydrate), β-D-galactopyranosyl-D-glucose, α-D-glucopyranosyl-α-D-glucopyranoside (anhydrous / dihydrate), galactose, pentoses (ribose, xylose, lyxose), dextrorotatory glucose, dodecacarbon monodecahydrate, and the like. monodecahydrate), fructose, sucrose, lactose, maltose, trehalose, agarose, D-galactosyl-β-(1-4)-anhydro-L-galactosyl, cellulose, polymers of β-D-glycopyranosyl units, and starch, as well as polyhydric alcohols, polyalcohols, alditol, erythritol, glycitol, glycerol, xylitol, and sorbitol.

[0061] In some embodiments, the protective coating comprises biocompatible and / or biodegradable polyesters or polyanhydrides, such as polylactic acid, polyglycolic acid, and lactic acid-glycolic acid copolymers. The particles can comprise one or more of the following polyesters: homopolymers including glycolic acid units, referred to herein as "PGA", and homopolymers of lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and poly-D,L-lactide 5, collectively referred to herein as "PLA", and homopolymers of caprolactone units, such as poly-e-caprolactone, collectively referred to herein as "PCL", and copolymers containing lactic acid and glycolic acid units, such as various forms of lactic acid-glycolic acid copolymers and lactide-glycolide copolymers characterized by the ratio of lactic acid:glycolic acid, collectively referred to herein as "PLGA", and polyacrylates and derivatives thereof. Exemplary polymers also include copolymers of polyethylene glycol (PEG) with the aforementioned polyesters, such as various forms of PLGA-PEG or PLA-PEG copolymers, collectively referred to herein as "PEGylated polymers." In certain embodiments, the PEG region can be covalently attached to the polymer to obtain a "PEGylated polymer" through a cleavable linker. In one aspect, the polymer comprises at least 60, 65, 70, 75, 80, 85, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent pendant acetal groups.

[0062] The triblock copolymers disclosed herein include core polymers such as polyethylene glycol (PEG), polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone (PVP), polyethylene oxide (PEO), vinylpyrrolidone-vinyl acetate copolymer, polymethacrylic acids, polyoxyethylene alkyl ethers, polyoxyethylene castor oils, polycaprolactam, polylactic acid, polyglycolic acid, lactic acid-glycolic acid copolymer, lactic acid-glycolic acid copolymer (PLGA), and cellulose derivatives such as hydroxymethylcellulose and hydroxypropylcellulose.

[0063] Examples of diblock copolymers that can be used in the protective coatings disclosed herein include polymers such as, for example, polyethylene glycol (PEG), polyvinyl acetate, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyethylene oxide (PEO), vinylpyrrolidone-vinyl acetate copolymers, polymethacrylic acids, polyoxyethylene alkyl ethers, polyoxyethylene castor oils, polycaprolactam, polylactic acid, polyglycolic acid, polylactic-glycolic acid, and polylactic-glycolic acid copolymers (PLGA).

[0064] In one embodiment, the protective coating comprises, i.e., encapsulated, the encapsulated composition may further comprise lecithin or hydrolyzed lecithin as a carrier or as an encapsulating material. As used herein, lecithin and / or hydrolyzed lecithin coatings include coatings containing phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine and phosphatidic acid. The lecithin may be of pnat or animal origin.

[0065] In one aspect, any of the polymers, monosaccharides, disaccharides, or polysaccharides used to form the protective coating formed by the inclusion of the MSC additive in the encapsulation solution can be present at a suitable concentration to form the protective coating. For example, the polymers, monosaccharides, disaccharides, or polysaccharides can be present at any concentration between 0.01 mM and 10.0 M, such as about 0.01 M to about 0.1 M, about 0.1 mM to about 1.0 M, about 1.0 M to about 10.0 M. Exemplary concentrations include 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.4, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 81 Examples include 0, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 450, 500, 600, 700, 800, 900 mM, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, and 10 M.

[0066] As shown in Figures 1 and 2, exosomes and extracellular vesicles were produced in the disclosed MSC secretome composition.

[0067] In one aspect, one method of treating wounds is via administration of an MSC secretome composition (including but not limited to MSC growth factors, MSC exosomes, MSC extracts and / or extracellular vesicles comprising the composition) subcutaneously, intramuscularly, intravenously, topically (e.g., through the use of salvesicles, creams and / or ointments, etc.) impregnated with the MSC secretome composition. It is understood and contemplated herein that the medicated stent, sponge, matrix, scaffold, bandage, covering, suture, graft, surgical drape, surgical adhesive, and / or staple may also be used. That is, in one aspect, a medicated stent, scaffold, sponge, matrix, adhesive bandage, wound dressing, graft, surgical drape, suture, salve, cream, or wound adhesive is disclosed herein, comprising a therapeutically effective amount of the MSC secretome composition. The above-described MSC secretome composition (including but not limited to MSC growth factors, MSC exosomes, extracellular vesicles containing MSC extracts and / or compositions) can be administered topically and applied to the face, neck, hands, or other desired parts of the body. When applied to an adhesive bandage, wound dressing, graft, surgical drape, salve, scaffold, matrix, sponge, or stent, the MSC secretome composition can be applied as a powder.

[0068] In one aspect, the MSC secretome composition disclosed herein (including but not limited to MSC growth factors, MSC exosomes, MSC extracts and / or extracellular vesicles containing the composition) may include any known material typically found in the wound healing field, such as oils, waxes, or other standard fatty substances, or conventional gelling agents and / or thickening agents; emulsifiers; moisturizers; emollients; sunscreens; hydrophilic or lipophilic active agents, such as ceramides; agents for combating free radicals; bactericides; sequestering agents; preservatives; basifying or acidifying agents; fragrances; surfactants; bulking agents; natural products or extracts of natural products, such as aloe or green tea extracts; vitamins; or colorants. Other materials that may be combined with the powder may include antioxidants, which may be selected from a variety of antioxidants. Suitable antioxidants include vitamins such as, for example, Vitamin C (L-ascorbate, Ascorbate-2 Phosphate magnesium salt, Ascorbyl palmitate, Tetrahexyldecyl ascorbate), Vitamin E (tocotrienol), Vitamin A (retinol, retinal, retinoic acid, provitamin A carotenoids such as beta-carotene), N-acetylglucosamine, or other derivatives of glucosamine. Other materials may include at least one essential fatty acid, such as, for example, Omega-3, Omega-6, and Omega-9, polyunsaturated fatty acids such as, for example, linoleic acid (LA), gamma-linoleic acid (GLA), alpha-linoleic acid (ALA), dihomo-y-linolenic acid (DGLA), arachidonic acid (ARA), and others. The fatty acids may be derived from a variety of sources, including evening primrose oil, blackcurrant oil, borage oil, or GLA modified safflower seed. Other materials may include at least one material that supports ECM production and hyaluronic acid production, such as platelet rich fibrin matrix, N-acetylglucosamine or other derivatives of glucosamine, ultra low molecular weight (ULMW) hyaluronic acid, chondroitin sulfate, or keratin sulfate.

[0069] It is understood and contemplated herein that the MSC secretome compositions disclosed herein can provide for the restoration, enhancement of wound healing, and improvement or repair of skin tissue. The compositions may also be used as injectables in the treatment of arthritis and degenerated spinal discs. Furthermore, embodiments of the compositions may not require the inclusion of additional growth factors or hormones, such as, for example, insulin, insulin-like growth factors, thyroid hormones, fibroblast growth factors, estrogen, retinoic acid, etc. In some aspects, the disclosed stem cell growth factor compositions can include additional growth factors, such as, for example, insulin, insulin-like growth factors, thyroid hormones, fibroblast growth factors, estrogen, retinoic acid, etc., as well as additional active materials, including, but not limited to, antibiotics, anti-acne agents, liposomes, antioxidants, platelet-rich fibrin matrix, analgesics, and anti-inflammatory agents. The additional active ingredients can be mixed with the stem cell growth factor and extracellular vesicle compositions disclosed herein as well as MSC conditioned medium, MSC lysates and MSC derived products, and then thawed or dissolved, mixed or suspended in a mixture of emulsifying lanolin alcohol, waxes and oils, or a mixture of petrolatum or mineral oil, a quaternary ammonium compound, a fatty alcohol, and a fatty acid ester emollient, or a lotion substantially similar in composition.

[0070] D. Working Example The following examples are presented to provide one of ordinary skill in the art with a complete disclosure and description of how to make and evaluate the compounds, compositions, articles, devices and / or methods claimed herein, and are intended to be purely illustrative and not intended to limit the present disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, temperature is in ° C. or is at ambient temperature, and pressure is at or near atmospheric pressure.

[0071] 1. Example 1: Intra-articular injection of extracellular vesicle isolate to treat hip labral tears Diarthrodial joints, such as the knee, hip, and shoulder, are composed of articular cartilage, bursae, and fibrocartilage structures that increase the stability of the joint. The knee has both medial and lateral menisci. A patient's shoulder and hip have labrums that increase the glenoid cavity and acetabular cavity, respectively. All of these fibrocartilage structures have innervation and can be very painful when torn. These structures have limited blood supply and minimal natural healing potential when injured. These structures can be torn by acute injury or by chronic degenerative processes. The acetabular labrum is a fibrocartilage seal around the bone-like acetabulum. The labrum increases the depth, surface area, volume, conformity, and stability of the hip joint. The labrum has been shown to increase the articular surface area of ​​the hip joint by an average of 22%. The labrum reduces stress on the articular cartilage by regulating the transport of fluid between the central and peripheral components and by distributing contact pressure over the entire articular surface area. This fluid seal is one of the most important functions of the labrum as it creates a negative intra-articular pressure, significantly enhancing joint stability.

[0072] If the shapes of the femoral head and acetabulum are not perfectly matched, femoroacetabular impingement can occur. It is a genetic condition that damages the hip labrum. Hip labral tears are associated with certain activities such as gymnastics, running, jumping, tennis, soccer, and cycling. The diagnosis is suspected when hip ROM (range of motion), especially with the forced friction of adduction, reproduces the groin pain that the patient normally experiences. The diagnosis is confirmed with an MRI scan.

[0073] Non-operative treatments for hip labral tears are lacking. The most common effective solution involves reducing or avoiding the activity causing the pain. Physical therapy has little effect. Surgical options are typically performed arthroscopically. Surgical options include either repairing the labral tear or, more commonly, removing the torn labrum. Krych et al. reported the results of arthroscopic treatment of the hip for labral tears. They reported 59 patients, with a mean age of 46 years and a mean follow-up of 5 years. They reported a clinical failure rate of 45%, with 20% requiring repeat surgery and 25% experiencing ongoing pain.

[0074] Published reports have shown that the treatment of shoulder labral tears using injections of autologous bone marrow concentrates (BMCs) containing mesenchymal stem cells (MSCs) is safe and effective. MSCs are unique cells that can differentiate into chondroblasts, osteoblasts, or fibroblasts. Differentiation has historically been thought to be the mechanism by which MSC injections treat joint pathologies. There is increasing recognition that the efficacy of MSC injections to treat various conditions is due to their paracrine function of releasing anti-inflammatory growth factors (GFs) and exosomes. These GFs and exosomes can be injected without any live cells. In this case report, we introduce the concept of treating hip labral tears using acellular bone marrow-derived MSC GFs and exosomes, described as extracellular vesicle isolates (EVIPs). This report describes the rationale why acellular may replace all current cellular biological therapies, both autologous and allogeneic, for the treatment of various joint pathologies.

[0075] a) Materials and methods This is a case report of EVIP injection for the treatment of a torn hip labrum. Characterized by locking, clicking, or catching of the hip joint. Pain may be felt in the groin or radiate into the trochanteric area. Patients may report stiffness or limited range of motion of the hip joint. MRI scans are used to diagnose hip labral tears. The patient is a 50-year-old highly active triathlete, personal trainer, and health instructor. After several months of untreated groin pain, she presented on 6 / 11 / 19 with increasing right groin and buttock pain radiating down to the thigh. Her symptoms were exacerbated by activities requiring hip ROM. An MRI scan of the hip on 6 / 4 / 19 revealed a superior labral tear, wear of the superior anterior labrum, a ruptured gluteus minimus tendon, inflammation of the trochanteric capsule, and common hamstring tendonitis. The hip joint was not osteoarthritis. The patient had been treated with NSAIDs, weekly active release, and chiropractic care. She had a total loss of hip ROM of 20% on physical examination. Forced adduction and flexion reproduced and severely exacerbated her pain. Overall, the patient felt that her right hip was less than 50% normal compared to the contralateral hip. The patient was assessed with the Brief Pain Inventory (BPI), Lower Extremity Functional Index (LEFI), and Oswestry Disability Index (ODI) before injections and at follow-up.

[0076] (1) Hip joint injection After counseling and consent, the patient underwent a hip injection on June 11, 2019. The right groin and gluteal area was sterilized with betadine skin preparation. A 20-gauge needle was placed in the hip joint via an anterior approach. Needle placement was confirmed with fluoroscopy. At this time, 2 cc of frozen EVIP (ExoFlo, Direct Biologics, St. Louis, MO) was thawed to room temperature and placed into the joint.

[0077] b) Clinical results The patient was restricted in physical activity for one week after the procedure. Low-resistance passive range of motion was encouraged immediately. The patient returned to full activity on the 10th day. Six weeks after the procedure, the patient was able to run seven miles, kayak for two hours, and swim 600 yards per day. She slept as soundly as a "rock" and was able to wake up without pain. The patient states that she experienced an overall 75% improvement in her hip labral tear symptoms compared to pre-treatment.

[0078] (1) Follow-up MRI scans A follow-up MRI scan with gadolinium arthrogram of the right hip was performed on August 28, 2019. The scan did not reveal any isolated labral tears. There was no evidence of gluteus minimus tendinosis or intratissue tears. There was no inflammation of the trochanteric capsule. Findings were MRI negative for the right hip. The clinical outcomes for this patient from pre-procedure through 12 weeks follow-up (FU) are shown in both Figure 1 and Table 1. The results detail the improvements noted in her Brief Pain Questionnaire (lower scores are better), Oswestry Disability Index (lower scores are better), and her Lower Extremity Functional Score (higher scores are better).

[0079] [Table 1]

[0080] c) Discussion The patient is an active 50-year-old triathlete with a history of increasing pain from a confirmed hip labral tear. She was injected with 2 cc of bone marrow-derived mesenchymal stem cells containing active growth factors and exosomes, EVIP (ExoFlo, Direct Biologics, St. Louis, MO) in a single anterior hip injection. Two weeks later, her hip labral tear symptoms had improved by 75%. This improvement has been maintained over three months. Her pre-injection MRI arthrogram confirmed a superior labral tear, superior anterior labral attrition, gluteus minimus tendon tear, trochanteric capsule inflammation, and common hamstring tendinitis. The hip was not osteoarthritic. Her post-injection MRI arthrogram confirmed no evidence of a labral tear. There was no evidence of gluteus minimus tendinosis or intratissue tears. There was no trochanteric capsule inflammation. The right hip was MRI negative. She will continue to be closely monitored for two years.

[0081] Non-operative treatment of symptomatic labral tears essentially involves avoiding the activity causing the pain. Surgical options include either arthroscopic removal of the labrum or labral repair. Published results show a 45% failure rate with arthroscopic hip labral surgery.

[0082] Based on the understanding of the biology of labral tears, a non-surgical treatment would be the injection of acellular MSC-derived growth factors and especially exosomes. The exosomes are tiny 30-150 nanometer sized (one billionth of a meter) phospholipid bilayer encapsulated structures produced by endosomes. MSCs (12-18 microns) are 1,000 times larger than exosomes. The diameter of a single hair is 80,000 nanometers. Exosomes contain growth factors, signaling lipids, and micro- and messenger RNA. These paracrine factors can be introduced into any joint at concentrations 100,000 times or more higher than those of any cellular MSC treatment. These growth factor proteins and exosomes function in a paracrine manner, either directly or indirectly, to change the inflammatory environment of the painful labral tear back to a normal pain-free physiological environment and stimulate fibrocartilage healing.

[0083] Future acellular treatment of labral tears will involve a two-step process: First, highly concentrated anti-inflammatory bone marrow-derived MSC growth factors and exosomes are injected into the painful joint. Second, these growth factors and exosomes enter the recipient synovial MSCs and stimulate the production of new chondroblasts and anti-inflammatory secretomes, chemokines and cytokines. These actions have been shown to heal the fibrocartilage tears. All this acellular biological therapy can be achieved with a single joint injection. The future of regenerative medicine in orthopedics and spine will likely utilize highly concentrated acellular MSC-derived growth factors and especially exosomes.

[0084] 2. Example 2: Intra-articular injection of extracellular vesicle isolates to treat osteoarthritis of the shoulder in athletes Osteoarthritis (OA) of the shoulder has been documented in cadaveric and radiographic studies to affect up to 33% of patients over the age of 60. Patients with shoulder OA experience pain, frictional friction, reduced motion, and reduced ability to position their hand at a desired point in space. Limited ability to position one's hand in a desired position severely impairs activities of daily living. Non-surgical treatments for shoulder OA include shoulder exercises, analgesics, and the use of nonsteroidal anti-inflammatory drugs in an attempt to maintain range of motion. When these non-surgical treatments fail to alleviate OA dysfunction, total shoulder arthroplasty (TSA) is the default surgical treatment.

[0085] In recent years, researchers and physicians have increasingly come to understand the clinical efficacy of using mesenchymal stem cells (MSCs) to treat osteoarthritis (OA). Furthermore, it is becoming clear that the release is not dependent on the cells differentiating into articular cartilage, but rather on the paracrine release of growth factors (GFs) and exosomes as a whole, which may interfere with the healing process. Active growth factors and exosomes can function without cells when delivered into an arthritic joint. This is a case report of acellular MSC-derived extracellular vesicle isolates (EVIPs) for the treatment of osteoarthritis of the shoulder. OA of the shoulder is defined by pain and stiffness in the shoulder joint, exacerbated by movement, and reduced range of motion of the shoulder. This case report introduces the concept of using EVIPs containing active growth factors and exosomes to treat OA and the principles of why acellular biologic therapy may replace current autologous and allogeneic cellular biologic therapy.

[0086] a) Method The patient was a right-handed, 57-year-old male athlete who presented with a >14-year history of increasing bilateral shoulder pain that was much worse on the right side than on the left. Pre-procedure x-rays and MRI scans were used to identify glenohumeral osteoarthritis from rotator cuff abnormalities. A previous MRI scan of the shoulder obtained on January 5, 2006 (13 years prior to this procedure) revealed a Kellgren-Lawrence score of 4, which is indicative of bone-on-bone osteoarthritis of the glenohumeral joint.

[0087] The patients were further evaluated at each visit by shoulder examination and shoulder function measures QuickDASH (QD), with lower scores being ideal; Upper Extremity Functional Index (UEFI), with higher percentages being better; Brief Pain Questionnaire (BPI), with higher scores indicating more pain; and patient self-assessment of overall shoulder improvement. These physical assessment procedures were performed at three different visits: pre-treatment, and then 6 and 12 weeks after treatment. At the first visit, a mean loss of 75% of right glenohumeral medial, lateral, and abduction was observed. All range of motion assessments were associated with severe pain, friction sounds, and bone-on-bone clunking. Pre-treatment x-rays of the shoulder are shown in Figure 2.

[0088] (1) Shoulder injection After counseling and consent, the patient's right shoulder was prepped with betadine skin solution. A 20-gauge needle was placed in the glenohumeral joint in an anterior approach. The needle was placed within the shoulder capsule against the humoral head. Needle position was confirmed by medial and lateral rotation of the humeral head to translate the needle. A 2.0 cc preparation of frozen EVIP (ExoFlo, Direct Biologics, St. Louis, MO) was thawed to room temperature and placed into the glenohumeral joint.

[0089] Patients had limited physical activity for one week after the procedure. Low-resistance passive range of motion was encouraged immediately after. Patients returned to full activity two weeks after the procedure.

[0090] b) Results Patient self-assessment of overall shoulder improvement was 75% from 2 weeks after injection to the evaluation at week 12. QuickDASH, Upper Extremity Functional Index and Brief Pain Questionnaire all improved within 2 weeks and the improvements were well maintained through week 12, as shown graphically in Figure 3 and with values ​​shown in Table 2.

[0091] [Table 2] Overall shoulder improvement was 75% by self-assessment. Post-treatment glenohumeral range of motion improved 33% based on physical examination.

[0092] c) Discussion The patient is a 57-year-old active male athlete with a 14-year history of increasing right-sided shoulder pain. He was diagnosed with bone-on-bone glenohumeral osteoarthritis with a Kellgren-Lawrence score of 4 without rotator cuff abnormalities. No regeneration of articular cartilage is expected. Follow-up x-rays will remain unchanged. He received a single injection of 2 cc of bone marrow-derived EVIP containing active growth factors and exosomes. The injection was performed without complications. Within 2 weeks, the patient was more than 50% improved based on the BPI, UEFI, and QD measurement scales. He rated his shoulder as 75% improved overall. He was back to full activity by week 2. These results were well maintained through week 12. The patient will continue to be followed for follow-up time points. The observed pain relief is felt to be due to decreased bursal inflammation.

[0093] The shoulder is a diarthrodial joint with a synovial lining and a joint capsule. The bursa contains a large number of synovial MSCs (more than found in bone marrow or fat). These MSCs have more chondrogenic potential than bone or fat MSCs. During the development of OA, proinflammatory and degradative growth factors are produced by these synovial MSCs. This creates a chronic inflammatory pain and degenerative joint environment. Bone marrow concentrates (BMCs) contain only about 2,500 MSCs per cc on average. Despite the very low number of MSCs present in BMCs, there is an extensive literature reporting clinical efficacy of using BMCs for OA treatment. This effect cannot depend on the survival or differentiation of BMC / MSC cells. The effective effect must be due to the release of acellular paracrine factors. The future of biological treatment of OA will utilize acellular MSC-derived growth factors and especially exosomes. The exosomes are tiny 30-150 nanometer sized (one billionth of a meter) phospholipid bilayer encapsulated structures produced inside the cytoplasm of cells. MSCs (12-30 microns) are approximately 1,000 times larger than exosomes. For reference, the diameter of a single hair is 80,000 nanometers or 80 microns. Exosomes contain growth factors, signaling lipids, and microRNA and messenger RNA (miRNA and mRNA). The RNA content in exosomes mediates most of their anti-inflammatory effects. RNA, along with many growth factors, is naturally encapsulated in exosomes by cells. These cell-derived paracrine factors can be introduced into any joint at concentrations 100,000 times or more higher than the concentrations of any cellular MSC treatment and can function, by paracrine action, directly or indirectly, to change the inflammatory environment of any painful arthritic joint back to a normal pain-free physiological environment. Figure 4 illustrates this process.

[0094] Future acellular treatments for OA will involve a two-front attack. First, high concentrations of anti-inflammatory MSC-derived growth factors will be injected into the arthritic joint. These growth factors will enter the nuclei of the recipient synovial MSCs. The EVIP growth factors will stimulate the transcription of mRNAs containing cellular instructions to produce a continuous anti-inflammatory and renewable secretome, chemokines, and cytokines, which will be released from the recipient cells into the synovial fluid. Second, high concentrations of exosomes from EVIP will enter the recipient cells and deliver their mRNAs and miRNAs. The delivered mRNAs will be directly translated in the recipient synovial MSC ribosomes to produce an anti-inflammatory and renewable secretome (growth factors, cytokines, chemokines, and extracellular vesicles). These beneficial effects may last for months or years. All this acellular biological therapy can be accomplished with a single injection into the arthritic joint, avoiding the morbidity and expense of obtaining autologous MSCs. The future of regenerative medicine in orthopedics and spine will likely utilise high concentrations of acellular MSC bone marrow derived growth factors and especially exosomes.

[0095] 3. Example 3: Intra-articular injection of extracellular vesicle isolates to treat osteoarthritis of the hip Osteoarthritis (OA) of the hip has been documented in both cadaveric and radiographic studies to affect up to 55 million patients over the age of 60. Patients with hip OA experience pain, frictional friction, decreased movement, and decreased ability to bear weight or ambulate. Limited ability to ambulate severely impairs activities of daily living. Non-surgical treatments for hip OA according to the American Academy of Orthopedic Surgeons (AAOS) include weight loss, mild exercise, and the use of nonsteroidal anti-inflammatory drugs. Surgical treatments for hip OA include total hip arthroplasty (THA). The AAOS does not recommend the use of hip arthroscopy or hyaluronic acid injections.

[0096] Over the last few years, researchers and clinicians have increasingly come to understand that the clinical efficacy of using mesenchymal stem cells (MSCs) to treat osteoarthritis (OA) does not depend on cell differentiation into articular cartilage, but entirely on the paracrine release of growth factors (GFs) and exosomes from MSCs. Viable MSCs are not required to achieve the release of GFs and exosomes to the arthritic joint. In this case report, we present the concept of using acellular MSC-derived extracellular vesicle isolates (EVIPs) containing active growth factors and exosomes to treat hip OA, and the principles of why acellular could replace all of the current cellular biological therapies, both autologous and allogeneic, currently in use.

[0097] a) Materials and methods This is a case report of EVIP injection for the treatment of osteoarthritis of the hip joint. OA is defined by swelling, pain, and stiffness in the hip joint. Symptoms are typically impaired weight bearing and ambulation. X-rays and MRI scans were used to grade the osteoarthritis of the hip joint on the Kellgen-Lawrence scale of 1 to 4.

[0098] The patient was a 63-year-old retired Chicago firefighter. He presented with increasing pain in his left hip joint and progressive loss of ability to continue his daily fitness routine at the health club. He experienced progressive loss of hip mobility. An MRI scan and x-ray of the left hip joint were consistent with Kellgren-Lawrence grade 3 arthritis in the left hip joint. He had anti-pain staggering on examination and a positive Trendelenburg sign. Passive ROM of the hip joint was associated with reappearance of hip pain, friction crackles, and reduced adduction. The patient had a BMI of 27. NSAIDs did not provide adequate pain relief. The patient was seriously considering total hip replacement. To avoid surgery, we elected to inject EVIP containing active GFs and exosomes into the patient's hip joint.

[0099] An x-ray of the pelvis is shown in Figure 5. This x-ray shows the right hip, which is normal, and the left hip, which has grade 3 OA. The left hip was sterilized with betadine skin preparation. A 20-gauge spinal needle was placed in an anterolateral approach to the hip joint. Needle placement was confirmed with fluoroscopy. At this point, 2 cc of frozen EVIP (ExoFlo, Direct Biologics, St. Louis, MO) was thawed to room temperature and placed intra-articularly. The patient was placed on physical activity restriction for 1 week after the procedure. Low-resistance passive range of motion was encouraged immediately. The patient resumed full activity at 2 weeks.

[0100] clinical results At the 3-month follow-up, the patient had returned to his previous fitness routine without restriction. He no longer had a restricted movement profile and enjoyed functional, pain-free restoration of hip ROM. He returned to full activity without restriction.

[0101] b) Discussion The hip joint is a diarthrodial joint with a synovial lining and a joint capsule. The bursa contains a large number of synovial MSCs (more than those found in bone marrow or adipose tissue). These MSCs have a higher chondrogenic potential than bone or adipose MSCs. During the development of OA, proinflammatory growth factors are produced by these synovial MSCs. This creates a chronic inflammatory painful joint environment. Bone marrow concentrates (BMCs) contain only about 2,500 MSCs per cc on average. Despite the very low number of MSCs present in BMCs, there is an extensive literature reporting the clinical efficacy of using BMCs in animals and humans for the treatment of OA. This effect cannot be dependent on the survival or differentiation of BMC / MSC cells. This efficacy must be due to the release of acellular paracrine factors. The future of biological treatment of OA will utilize acellular MSC-derived growth factors and especially exosomes.

[0102] Exosomes are tiny 30-150 nanometer sized (one billionth of a meter) phospholipid bilayer encapsulated structures produced by the Golgi apparatus or Golgi body. MSCs (12-18 microns) are 1,000 times larger than exosomes. A single hair has a diameter of 80,000 nanometers. Exosomes contain growth factors, signaling lipids, as well as microRNA and messenger RNA. The RNA content within exosomes mediates most of their anti-inflammatory effects. RNA enters exosomes in addition to numerous peptide growth factors. These paracrine factors can be introduced into any joint at concentrations 100,000 times or more higher than the concentrations of any cellular MSC treatment. These growth factor proteins and exosomes will function in a paracrine manner, directly or indirectly, to change the inflammatory environment of any painful arthritic joint back to a normal pain-free physiological environment.

[0103] Future acellular treatments for OA will involve a two-front attack. First, high concentrations of anti-inflammatory MSC-derived growth factors will be injected into arthritic joints. These growth factors will enter the nuclei of the recipient synovial MSCs. EVIP growth factors bind to mRNAs that contain instructions for the production of a sustained anti-inflammatory secretome, chemokines, and cytokines. Secondly, highly concentrated exosomes from EVIP enter the recipient synovial MSCs and deliver their mRNA, which is directly translated in the recipient synovial MSC ribosomes to produce anti-inflammatory secretome, cytokines and chemokines.

[0104] These beneficial effects can last for months or years. All of this acellular biological therapy can be achieved by a single injection into the arthritic joint, without the morbidity and expense of obtaining autologous MSCs. The future of regenerative medicine in orthopedics and spine will likely utilize high concentrations of acellular MSC-derived growth factors and especially exosomes.

[0105] 4. Example 4: Treatment of Elbow Arthritis with Intra-Articular Injection of Extracellular Vesicle Isolate Primary osteoarthritis (OA) of the elbow is a rare condition associated with a genetic predisposition. It occurs primarily within the ulnar-humeral joint of the dominant limb in patients engaged in strenuous sports or work. Post-traumatic elbow OA is by far the most common. Various traumatic injuries can ultimately result in specific forms of post-traumatic OA to the elbow. The most common complaints of patients with elbow arthritis, whether primary or post-traumatic, are pain and / or reduced movement. Reduced extension and pronation can be more easily compensated for than reduced friction and supination.

[0106] For OA of the elbow, non-operative procedures remain the mainstay of initial treatment. These typically include elbow sleeves, nonsteroidal anti-inflammatory drugs, and intra-articular corticosteroid injections. Avoiding active flexion and extension movements of the limb can result in significant pain relief (weightlifting, boxing, etc.). Typically, a course of rehabilitation supervised by a qualified therapist is reserved for patients with acute symptoms versus chronic symptoms with associated effusion and limitation of movement.

[0107] Surgical treatment is indicated for patients with severe pain or significant loss of mobility of the elbow with consequent impairment of upper limb function and limitation of daily activities. Proceeding to elbow arthroscopy has resulted in favorable outcomes and has completely replaced any open surgical debridement. Elbow arthroscopic debridement has reasonable results in terms of improvement in pain and range of motion, especially in a younger patient population. It is important that the procedure presented be performed by a surgeon with considerable experience in safe and meticulous elbow arthroscopic technique. Total elbow replacement is best suited for older patients (over 60 years) with low demands, with inflammatory, traumatic or primary elbow arthritis. In the setting of elbow OA in a younger, typically male population, total elbow replacement is rarely indicated.

[0108] There is a huge gap between non-operative and operative treatment of elbow OA.This is a case report of a young, very active chiropractor with elbow OA.She was treated with a single intra-articular injection of bone marrow-derived mesenchymal stem cell (MSC) extracellular vesicle isolate containing active growth factors and exosomes.

[0109] a) Materials and Methods: The patient is a very active and healthy 60-year-old practicing chiropractor. She initially injured her dominant right elbow in a serious water skiing accident at age 16. She suffered a complete dislocation of the elbow, including the ulna and radius, which are located next to the humerus. There was no neurovascular injury. The elbow was reduced under general anesthesia. After recovery, she had lost 15° of full extension and 10° of full flexion. There was no loss of supination or pronation. She suffered a second injury at age 58, resulting in a complete dislocation of the radial head. Following this injury, she developed traumatic arthritis of the radiocapitellar joint with daily pain and joint swelling that worsened with activity. The patient's ability to perform acupressure therapy was severely limited. She was regularly taking NSAIDs and ice therapy after work. Examination of the patient's elbow revealed a loss of 15° of elbow extension and 10° of flexion. She had a loss of 10° of supination and pronation. There was 1+ swelling in the elbow joint. X-rays of this patient reveal Kellgren-Lawrence grade 3 osteoarthritis of the elbow joint (Figures 6A and 6B).

[0110] (1) Elbow injection: After counseling and consent, the patient underwent an elbow injection on June 11, 2019. The right elbow joint area was sterilized with betadine skin preparation. A 20-gauge needle was placed in the elbow joint with a lateral and medial approach. At this point, 3 cc of frozen EVIP (ExoFlo, Direct Biologics, St. Louis, MO) was thawed to room temperature and placed in the medial and lateral sides of the elbow joint. The patient experienced no adverse events. The entire procedure took 15 minutes.

[0111] b) Results After the injection, the elbow joint was painful and swollen for several days. The patient was unable to work for one week. The patient's symptoms improved over the next four weeks. By six weeks after the injection, she felt that her elbow was 50% better. This improvement continued for three months, and the elbow is now 70% better than it was before the injection. Elbow range of motion did not change after the EVIP injection (Table 3 and Figure 7).

[0112] [Table 3]

[0113] c) Discussion This is a case study of a 60-year-old active practicing chiropractor with an elbow marked by years of history of increasing symptomatic traumatic osteoarthritis. She suffered a severe dislocation of the posterior elbow at age 16. Her symptoms worsened significantly with work to the point that she considered retirement. This was a major decision as she was in private practice with overhead financially. Her surgical option was total elbow arthroplasty. After this surgery, the patient may not have been able to return to practicing chiropractic. The goal of the surgery was to try to improve her activities of daily living, not to return to strenuous work. In an effort to avoid surgery and continue practicing chiropractic, she elected to try an elbow injection of EVIP (ExoFlo, Direct Biologics, St. Louis, MO), which contains bone marrow-derived MSCs, active growth factors, and exosomes. Three months after the injection, the patient's elbow had improved by 70% and she was working full time.

[0114] The elbow is a diarthrodial joint with a synovial lining and a joint capsule. The bursa contains a large number of synovial MSCs (more than found in bone marrow or fat). These MSCs have more chondrogenic potential than bone or fat MSCs. During the development of OA, proinflammatory growth factors are produced by these synovial MSCs. This creates a chronic inflammatory painful joint environment. Bone marrow concentrates (BMCs) contain only about 2,500 MSCs per cc on average. Despite the very low number of MSCs present in BMCs, there is an extensive literature reporting the clinical efficacy of using BMCs in animals and humans for the treatment of OA. This effect may not be dependent on the survival or differentiation of BMC / MSC cells. The effective effect must be due to the release of acellular paracrine factors. The future of biological treatment of OA will be to utilize acellular MSC-derived growth factors and especially exosomes. Exosomes are tiny 30-150 nanometer sized (one billionth of a meter) phospholipid bilayer encapsulated structures produced by the Golgi apparatus or Golgi body. MSCs (12-18 microns) are 1,000 times larger than exosomes. The diameter of a single hair is 80,000 nanometers. Exosomes contain growth factors, signaling lipids, and microRNA and messenger RNA. The RNA content in exosomes mediates most of their anti-inflammatory effects. RNA enters exosomes in addition to numerous peptide growth factors. These paracrine factors can be introduced into any joint at concentrations 100,000 times or more higher than the concentrations of any cellular MSC treatment. These growth factor proteins and exosomes will function in a paracrine manner, directly or indirectly, to change the inflammatory environment of any painful arthritic joint back to a normal pain-free physiological environment.

[0115] Future acellular treatments for OA will involve an attack on two fronts. First, High concentrations of anti-inflammatory MSC-derived growth factors are injected into the arthritic joint. These growth factors will enter the nuclei of the recipient synovial MSCs. The EVIP growth factors will stimulate the transcription of mRNAs containing instructions for the continued production of anti-inflammatory secretomes, chemokines, and cytokines. These will be released from the recipient synovial MSCs into the synovial fluid. Secondly, high concentrations of exosomes from EVIP will enter the recipient synovial MSCs and deliver their mRNAs. These delivered mRNAs will be directly translated in the recipient synovial MSC ribosomes to produce anti-inflammatory secretomes, cytokines, and chemokines. These beneficial health effects can last for months or years. All of this acellular biological therapy can be achieved by a single injection into the arthritic joint, without the morbidity and expense of obtaining autologous MSCs. The future of regenerative medicine in orthopedics and spine will likely utilise highly concentrated acellular MSC bone marrow derived growth factors and especially exosomes.

[0116] 5. Example 5: Treatment of osteoarthritis by administration of EVIP Extracellular vesicle isolates (EVIPs) derived from bone marrow mesenchymal stem cells contain active growth factors (>800) and exosomes (>10 billion per cc). This is the first IRB-sponsored study to report on the safety and efficacy of injecting EVIP to treat OA. Thirty-three former US Navy SEALs injected EVIP into OA of the knee (n=58), shoulder (n=32), elbow (n=16), hip (n=12), ankle (n=8), or wrist (n=6). At the 3-month follow-up, the average knee patient had improved 70% on the Brief Pain Questionnaire (BPI), 67% on the Oswestry Disability Index (ODI), and 62% on the Lower Extremity Functional Scale (LEFS); the average shoulder patient had improved 68% on the BPI, 82% on the ODI, 74% on the QD, and 68% on the UEFS; the average elbow patient had improved 76% on the BPI, 81% on the QD, and 76% on the UEFS; the average hip patient had improved 70% on the BPI, 72% on the ODI, and 64% on the LEFS; the average ankle patient had improved 70% on the BPI, 72% on the ODI, and 64% on the LEFS; and the average wrist patient had improved 68% on the BPI, 64% on the QD, and 74% on the UEFS. All improvements were attributable to p<0.001. There were no complications and no patients worsened with EVIP injection. At 3-month follow-up, EVIP injection for OA appears to be safe and effective and should be considered before joint replacement surgery.

[0117] a) EVIP feature analysis: The product is derived from the bone marrow of a 22-year-old female donor whose MSCs are master preserved and FDA registered. The final product from these cells contains over 800 different active growth factors and over 30 billion exosomes per cc. The product is used in frozen form. Sterilization was achieved by ultrafiltration, not irradiation. Acellular exosomes derived from bone marrow MSCs have been shown to be resistant to advanced particle analysis, proteomic evaluation, and USP sterilization. <71> It provides a uniform product with extensive characterization including sterility assurance. Identification and quantification of cytokines and growth factors are also performed. It considers acellular exosomes as a therapeutic grade quality product that is uniform, standardized, and quality tested for dose and activity.

[0118] (1) Patient demographic factors: The number of patients with mean BMI and mean age treated for the knee, shoulder, elbow, hip, ankle and wrist are shown in Table 4.

[0119] Injection Technique: All joints to be injected were sterilized with betadine skin preparation. Under fluoroscopic guidance, a 20-gauge needle was placed into the arthritic joint. Needle position was confirmed under fluoroscopic control. At this point, 2 cc of frozen EVIP (ExoFlo, Direct Biologics, St. Louis, MO) was thawed to room temperature and placed into the joint. The entire procedure took an average of 30 minutes with four joints injected per patient.

[0120] The patient was not prescribed any pain medication. The patient had limited physical activity for two weeks after the procedure. Immediately, low-resistance passive range of motion was encouraged. After two weeks, the patient was allowed to return to full activity.

[0121] (2) Statistical testing Univariate data comparisons were analyzed by two-tailed Student's t-test with 95% confidence intervals (α = 0.05, Microsoft Excel). Multivariate data were evaluated by analysis of variance (ANOVA) using JMP 9 statistical analysis software (SAS Institute, Cary, NC).

[0122] b) Results Each patient was contacted at 12 hours, 24 hours, 48 ​​hours, and at 2 weeks, 6 weeks, and 3 months to discuss any and all side effects from the EVIP injections. No patients reported any side effects from the EVIP injections. No patients experienced clinical deterioration from the EVIP injections.

[0123] [Table 4]

[0124] Knee=58, shoulder=32, elbow=16, hip=12, ankle=8, wrist=6, total number of joints injected=132 E. References JPEG2025066709000006.jpg202147 JPEG2025066709000007.jpg203146 JPEG2025066709000008.jpg218145 JPEG2025066709000009.jpg139148

Claims

1. 1. A method for treating, inhibiting, reducing, ameliorating, and / or preventing a disease, disorder, injury, or a symptom thereof affecting one or more joints in a subject, comprising administering to the subject a therapeutically effective amount of a mesenchymal stem cell (MSC) exosome preparation.

2. 2. The method of claim 1, wherein the disease or disorder comprises osteoarthritis, juvenile arthritis, psoriatic arthritis, infectious arthritis, rheumatoid arthritis, ankylosing spondylitis, gout, bursitis, tendinosis, tendinitis, sprains, labral tears, tendon tears, and / or ligament tears.

3. The method of claim 1 or 2, wherein the joints include ankles, knees, hips, wrists, elbows, shoulders, knuckles and / or neck.

4. The method of any one of claims 1 to 3, wherein the MSC exosome preparation is administered to each joint affected by the disorder, disease, or injury.

5. The method of claim 1 , wherein the MSC exosome preparation further comprises growth factors obtained from MSCs.

6. The method of any one of claims 1 to 5, wherein the MSC exosomes are administered by injection, a scaffold carrying the MSC exosomes, a hydrogel, and / or a topical medication or ointment.

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