Decellularized placenta extracellular matrix and uses thereof

Abstract

Provided herein is a decellularized extracellular matrix derived from placenta that is substantially free of residual material, having applications in treating a musculoskeletal or orthopedic disease or condition.

Description

DECELLULARIZED PLACENTA EXTRACELLULAR MATRIX AND USES THEREOFPRIORITY CLAIM

[0001] This application claims priority to US Provisional Application 63 / 612,557 filed 20 December 2023, which is hereby incorporated by reference herein in its entirety.FIELD OF INVENTION

[0002] Disclosed herein is heretofore unknown decellularized placenta extracellular matrices (ECM) and pharmaceutical compositions thereof for the treatment of degenerative diseases or conditions Involving degradation of extracellular matrices such as cartilage, bone tissue, etc. Also disclosed herein are methods of treating a musculoskeletal or orthopedic disorder, disease or condition, such as osteoarthritis, degenerative disc disease, cartilage deficits or damage, soft tissue Injury, physical trauma and orthopedic surgery with a decellularized extracellular matrix of the instant disclosure, as well as with a pharmaceutical compositions disclosed herein.BACKGROUND

[0003] Osteoarthritis (OA) Is an example of a musculoskeletal or orthopedic disorder or disease, or condition that affects cartilage, subchondral bone, synovium, synovial fluid, or any combination thereof, and has limited treatment options. It is the most common form of arthritis and affects an estimated 30.5 million adults in the US. Approximately 14 million OA patients experience OA in their knee and is referred to as "symptomatic knee OA". Women are nearly twice as likely to develop OA than are men. It is estimated that over 27 million adults in the US have clinical osteoarthritis (OA) of at least one joint, which translates into an annual impact of over $120 billion in the US alone in terms of health care costs, days of work loss, lost productivity, and other economic costs.

[0004] Focal lesions in cartilage are often caused by acute traumas that occur mostly in the knee and involve damage to articular cartilage of a joint or the meniscal surface in the knee. A majority of focal lesions are caused by either trauma, osteochondritis dissecans (OCD) or osteonecrosis. A high procedure volume associated with small / medium focal lesions (over 882,584), avascular meniscal lesions (1,006,749), small degenerative lesions (1,030,000) and medium / large degenerative lesions (1,400,000), has recently been observed in the population, particularly due to an increased elderly population. Approximately 73% of focal lesions in cartilage result from trauma to a location or cartilage in a joint.

[0005] Osteochondritis Dissecans (OCD) is another disease or condition that affects cartilage for which there are limited treatment options available. In particular, OCD is ajoint condition in which bone underneath the cartilage of a joint dies due to lack of blood flow. This bone and cartilage can then break loose, causing pain and possibly hindering joint motion. It occurs most often In children and adolescents and Is more common in males than females. In the US, it affects approximately 48,000-92,000 patients. Over 75% of those patients experience OCD in the knee, about 4% experience it In the ankle, approximately 6% in the elbow, and 15% in other joints.

[0006] Moreover, simple long term "wear* and "tear* use of various joints can result in degeneration of cartilage and subchondral bone in joints, forming degenerative lesions. These lesions often lead to ligament instability malalignment and are highly correlated with osteoarthritis (OA).

[0007] Post-traumatic Osteoarthritis (PTOA), which is also a disease or condition that affects bone, synovium, synovial fluid, or a combination thereof, is an osteoarthritis that results from a previous joint injury / instability. Approximately 12% of overall prevalence of lower extremity OA Is attributable to previous trauma. PTOA is most prevalent in highly active populations e.g., athletes and military personnel, and approximately greater than 40% of Individuals who sustain significant injuries develop PTOA.

[0008] Treatment decisions and goals with respect to diseases or conditions that affect cartilage, subchondral bone, synovium, synovial fluid, or a combination thereof, and can result In focal and / or degenerative lesions depend on the size of the lesion because the effectiveness of some cartilage repair treatments greatly decreases when used in treating lesions having a size greater than about 2 cm. Age is also a factor that may be considered when making such treatment decisions. Patients greater than 50 years old tend to have more limited treatment options due to increased biological age of the joint. The primary goal of such treatments for patients less than about 50 years old is to restore degenerated cartilage and prevent the early onset of arthritis. For patients greater than 50 years old, the aim for treating a focal lesion Is to restore the damaged cartilage / joint to its original form as dose as possible. For younger patients <50 years old, the primary goal is to restore / and prevent early onset of arthritis.

[0009] One treatment option available for a disease or condition that affects cartilage, such as OA, is to focus on providing temporary pain relief and reducing inflammation during the early stages of the disease or condition without affecting the course of the disease or condition. Example of such treatment options include NSAIDs such as aspirin, acetaminophen, ibuprofen, naproxen, as well as glucosamine & chondroitin sulphate, Diacerin (IL - 1 inhibitor), Hyaluronic Acid (HA) injections, steroid injections, autologous chondrocyte transplantation, and ultimately total joint replacement.

[0010] Other treatment modalities are based on targeting pro-inflammatory pathways of Inflammatory cytokines that stimulate the production of inflammatory mediators such as prostaglandin E, nitric acid synthase, chemokines, and other cytokines in the jointmicroenvironment. Particular examples of such cytokines are Interleukin (IL)-1 and Tumor Necrosis Factor (TNF). They also directly promote the expression of matrix metalloproteinases (MMPs) and other matrix-degrading enzymes Involved in cartilage degeneration. Unfortunately, clinical trials on therapeutic candidates that block these pro-inflammatory pathways have had little or no success despite the fact that these candidates effectively suppress the inflammatory phenotypes in chondrocytes in vitro. Examples include intra-articular injection of anakinra, an IL-1 receptor antagonist that obstructs the receptor binding of both IL- 1α and IL-1β, into 160 individuals with knee OA did not reduce OA-associated pain or cartilage turnover during weeks 4-12 of administration In a random controlled trial (NCT00110916, phase II clinical trial). Likewise, a randomized double-blind controlled trial (NCT00110942, phase II clinical trial) of AMG108, which is a monoclonal antibody against IL-1 receptor type I that blocks the receptor binding of both IL- 1α and IL-1β, did not provide sufficient clinical benefits. ABT- 981 (a dual neutralizing antibody against IL- 1α and IL-1β) was tested In patients with hand or knee OA. Neither phase II clinical trials (NCT02384538 and NCT02087904) showed substantially improved outcomes, indicating that ABT-981 is Ineffective In treating OA. In a clinical trial Involving 43 hand OA patients with random allocation to groups administered adalimumab or placebo for 12 weeks, no significant difference in hand pain was noted between the two groups. Similarly, In a trial of 90 patients with hand OA, etanercept (a decoy receptor that binds to TNF) did not differ from placebo In alleviating pain after 24 weeks of administration.

[0011] US published patent application US20210154240A1 (the US'240 publication) discloses placental tissue particulate comprising amnion membrane particulates, chorion membrane particulates and umbilical cord particulates, and methods of treating a musculoskeletal or orthopedic condition, such as osteoarthritis, degenerative disc disease, cartilage deficits or damage, soft tissue injury, physical trauma and orthopedic surgery with compositions of the extracellular matrix disclosed in the US'240 publication (hereafter "PTP"). The US'240 publication goes on to explain in paragraph

[0076] that FTP compositions described therein contain a combination of beneficial anti-inflammatory, anti-catabolic and pro-anabolic proteins and growth factors, and that the release of such factors from the PTP composition administered to patients can, among other actions, reproducibly inhibit the production of one of the principal collagenases, MMP-13, expressed by human articular cartilage cells (chondrocytes) that have been isolated in culture from donor arthritic cartilage. The US'240 application further explains that a PTP described therein can contain quantifiable amounts of βFGF, ILlRα, IL- 1α, TIMP-1, TIMP-2, TIMP- 3, and fibronectin.

[0012] The citation of any reference herein should not be deemed as an admission that such reference Is available as prior art to the instant disclosure.SUMMARY OF THE DISCLOSURE

[0013] The present disclosure is based upon the discovery that, surprisingly and unexpectedly, a decellularized placenta derived extracellular matrix that: (a) is substantially free of residual material such as (1) cells, (2) cell debris, (3) nucleic acid molecules, (4) nucleotides, (5) growth factors, (6) cytokines, (7) chemoklnes, (8) hormones, or (9) any combination of (l)-(8); and does not include any umbilical cord tissue, exhibits an efficacious result when used to treat a musculoskeletal or orthopedic disease or condition such as osteoarthritis, degenerative disc disease, cartilage deficits or damage, soft tissue Injury, physical trauma, plantar fasciitis, tendonitis, orthopedic surgery, OCD, or PTOA, to name only a few.

[0014] Broadly, the present disclosure extends to a decellularized placenta extracellular matrix (ECM) derived from placenta and substantially free of residual material. In a particular embodiment, residual material is not quantifiable in a decellularized placenta ECM of the Invention or Is present in an amount less than the limit of detection. Examples of such residual material include (1) cells, (2) cell debris, (3) nucleic acid molecules, (4) nucleotides, (5) growth factors, (6) cytokines, (7) chemoklnes, (8) hormones, or (9) any combination of (1)- (8). The present disclosure further extends to a decellularized placenta extracellular matrix In which the residual material comprises chemoklnes, cytokines, or growth factors, or any combination thereof, which are not detectable In the decellularized placenta extracellular matrix or are present in the decellularized placenta extracellular matrix at an amount less than the limit of detection.

[0015] In a particular embodiment of a decellularized placenta extracellular matrix of the instant disclosure, the following chemoklnes, cytokines and growth factors are not detected, or are present in an amount less than limit of detection, and thus, not quantifiable:(a) Platelet Derived Growth Factor- AA (PDGF-AA), less than the limit of detection;(b) platelet Derived Growth Factor-BB (PDGF-BB), not detected;(c) Basic Fibroblast Growth Factor bets (Beta FGF), not detected;(d) Epidermal Growth Factor (EGF), not detected;(e) Transforming Growth Factor beta 1 (TGF-betal), not detected;(f) Hepatocyte Growth Factor (HGF), not detected;(g) Vascular Endothelial Growth Factor (VEGF), not detected;(h) Insulin Like Growth Factor-1 (IGF-1), not detected;(i) Growth and Differentiation Factor-15, less than the limit of detection;0) intercellular Adhesion Molecule-one (ICAM-1), not detected;(k) lnterieukln-10 (IL- 10), not detected;(l) Interleukin-1 Receptor Antagonist (IL1-RA), not detected;(m) Interleukin-6 (IL-6), not detected;(n) Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF), not detected;(o) Interluekin-12p40 subunit (IL-12p40), not detected;(p) Interleukin-1 beta (IL-lbeta), not detected;(q) Interleukin-5 (IL-5), not detected;(r) Interleukin-8 (IL-8), not detected;(s) Interleukin-13 (IL-13), not detected;(t) Interieukin-15 (IL-15), not detected;(u) Monocyte Chemoattractant Protein-1 (MCP-1), not detected;(v) macrophage Inflammatory Protein-1 alpha (MIP-lalpha), not detected;(w) Regulated on Activation, Normal T Expressed and Secreted (RANTES), less than the limit of detection;(x) Tumor Necrosis Factor alpha (TNFalpha), not detected;(y) Human Chorionic Gonadotropin (hCG), not detected;(z) Follicle Stimulating Hormone (FSH), not detected;(aa) Lactogen, not detected;(bb) Estrogen, less than the limit of detection;(cc) Progesterone, not detected; and(dd) Oxytocin, less than the limit of detection.Optionally, a decellularized placenta derived ECM of the instant disclosure can be in the form of particles. In a particular embodiment, such particles have a size of:(i) about 5 microns to about 2000 microns;(ii) about 5 microns to about 300 microns;(iii) about 10 microns to about 250 microns;(iv) about 20 microns to about 200 microns; or(v) any combination of (i) - (iv).

[0016] In a particular embodiment, a decellularized placenta extracellular matrix of the instant disclosure Is sterile.

[0017] Furthermore, the present disclosure extends to a pharmaceutical composition comprising a decellularized placenta extracellular matrix disclosed herein, and a pharmaceutically acceptable carrier.

[0018] Moreover, the instant disclosure extends to a decellularized placenta derived extracellular matrix of the instant disclosure for use in the treatment of a musculoskeletal or orthopedic disease or condition In a subject in need thereof. Examples of a musculoskeletal or orthopedic disease or condition that a decellularized placenta extracellular matrix disclosed herein can be used to treat include, but certainly are not limited to, osteoarthritis, degenerative disc disease, cartilage deficits or damage, softtissue injury, physical trauma, plantar fasciitis, tendonitis, orthopedic surgery, OCD, or PTOA, to name only a few. A pharmaceutical composition of the instant disclosure also has applications In treating the pain and dysfunction associated with a musculoskeletal or orthopedic disease or condition, preventing or reducing scarring or Inflammation, and may delay its progression. Pharmaceutical compositions comprising resuspended decellularized placenta derived extracellular matrix of the instant disclosure also may be administered by injection or localized placement for protection of cartilage and / or for treatment of pain caused by a musculoskeletal or orthopedic disease or condition, such as osteoarthritis, degenerative disc disease, cartilage deficits or damage, soft tissue injury, physical trauma, plantar fasciitis, tendonitis, orthopedic surgery, OCD, or PTOA, to name only a few.

[0019] It is possible the ease and time of resuspension of dehydrated decellularized placenta derived extracellular matrix of the instant disclosure may be affected by a number of factors, since dehydrated particles of a decellularized placenta derived extracellular matrix of the Instant disclosure may clump upon wetting and become difficult to disperse Into a homogenous mixture. Such factors include resuspension technique, resuspension solution, design and composition of the vial, lyophilization method, irradiation level, presence of additives to improve wettability, and means of agitation. Any of these factors or combination of these and / or other factors known In the art may be employed to enhance the ease of resuspension of a pharmaceutical composition of the Instant disclosure. For example, in one embodiment, e-beam irradiation is used to improve resuspension characteristics. In another embodiment, a surfactant or bulking agent is added to a decellularized placenta derived extracellular matrix of the instant disclosure prior to lyophilization, which reduces the likelihood of particle clumping. In another embodiment, beads, such as glass beads or stainless steel beads may be added into the vial prior to lyophilization that can subsequently be used to agitate a decellularized placenta derived extracellular matrix of the instant disclosure upon resuspension and break up any clumps that have formed. The beads are sized to be too large to be drawn up into an appropriately sized syringe for delivery of a pharmaceutical composition of the instant disclosure and are not intended for injection or implantation. A syringe with a luer locking tip, including but not limited to a sterile BD LEUR-LOK syringe, either 10 ml or 3 ml, depending on the volume of the product, packaged in a double foil pouch, sealed, and provided within a carton as a single use kit along with a second empty sterile luer lock syringe, one luer lock connector and one flexible injector (Becton Dickinson and Company, New Canaan, CT) can be used to apply a physical motion of mixing a dry particulate contained within one syringe when connected via a luer lock connector with a second syringe containing equal volume of sterile saline at a surgery site prior to administration. Several back and forth syringemovements will allow mixing of the particulate with saline to form a homogenous pharmaceutical composition of the instant disclosure.

[0020] In addition, the instant disclosure extends to a method of treating a musculoskeletal or orthopedic disease or condition in a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of a decellularized placenta extracellular matrix as disclosed herein. There are numerous ways to administer to a subject the therapeutically effective amount of a decellularized placenta extracellular matrix in a method of the instant disclosure. In a particular embodiment, the step of administering a therapeutically effective amount of a decellularized placenta extracellular matrix of the instant disclosure comprises injecting a particular placental decellularized placenta extracellular matrix of the instant disclosure into a desired site in the subject. Optionally, administration can occur more than once over a period of time so that the multiple administrations are staggered. In a particular embodiment of a method of the instant disclosure, administering a therapeutically effective amount a decellularized placenta extracellular matrix can occur 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more times, and time between administrations can be about 1 day, about 1 month, about 3 months, about 6 months, about 12 months, or more.

[0021] A method of treating a musculoskeletal or orthopedic disease or condition of the Instant disclosure can further comprise the step of administering to the subject a therapeutically effective amount of a therapeutic agent having applications In treating a musculoskeletal or orthopedic disease or condition, or a symptom thereof. Examples of such therapeutic agents include but certainly are not limited to: (a) an NSAID, (b) glucosamine and chondroitin sulphate, (c) DIACE RIN, (d) a hyaluronic acid injection, (e) a steroid , (f) an opioid, or (g) a biologic, (e.g., cells / cell conditioned media, exosomes, plasma, nucleic acid molecules (such as DNA, mRNA, siRNA, etc.), an antibody (fully human, humanized, fragment (fab, f(ab')2, etc.), an ScFv)), (h) an antibiotic, (i) an antifungal agent, or any combination of (a) - (I). Such administration of a therapeutic agent as described above can be topical, systemic, or directly to the affected tissue.

[0022] In a method of treating a musculoskeletal or orthopedic disease or condition as described herein, further comprising the step a therapeutically effective amount therapeutic agent having applications in treating a musculoskeletal or orthopedic disease or condition, the therapeutic agent can be administered simultaneously, concomitantly, or serially with a pharmaceutical composition of the instant disclosure. If a therapeutic agent and pharmaceutical composition of the instant disclosure are administered serially or concomitantly, either one can be administered first.

[0023] In another aspect of the technology, kits containing one or more aliquots of sterilized, dehydrated decellularized placenta derived extracellular matrix of the Instant disclosure In powder form with or without additional components and a separate andappropriate amount of solution for resuspending a decellularized placenta derived extracellular matrix of the instant disclosure, e.g., 0.9% saline are provided. The kits may include an appropriate device for delivery of a preparation or pharmaceutical composition of a decellularized placenta derived extracellular matrix as disclosed herein, e.g., a vial adapter and / or a syringe and appropriately sized needle. The kits may also include instructions for resuspension and administration, as well as a dual syringe as discussed above

[0024] A decellularized, placenta derived extracellular matrix, as well as pharmaceutical compositions thereof of the instant disclosure, can be administered directly to a treatment site (e.g., joints, surgical site, tendon), via an Intra -articular route, direct injection Into a tendon, parenterally (e.g., subcutaneous, via liposomal deliver, diffusion from a device Impregnated with a pharmaceutical composition of the Instant disclosure, via microemulsion-based transdermal delivery, as well as other routes of administration well known to those of ordinary skill in the art.

[0025] Alternatively, a decellularized placenta derived extracellular matrix of the instant disclosure can be applied to a target site, e.g., a joint or tendon, in a dry form by sprinkling a dosage of the extracellular matrix onto the target site. Moreover, following rehydratlon of a decellularized placenta derived extracellular matrix of the instant disclosure to a pastelike consistence, can be applied and spread onto a target site.

[0026] These and other aspects of the present disclosure will be better appreciated by reference to the following drawings and Detailed Description.BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1 are histograms showing examples of the particle size analysis of cryomilled placental ECM of the instant disclosure that was conducted using the Beckman Coulter Multisizer 4e Particle Analyzer. Two lots of cryomilled placental ECM were tested, and the figure above shows number of particulates greater than 300pm to be 0 for both lots with more than 90,000 particles detected by the machine. The Cryomilling program stated above is able to generate a final particulate form of placental ECM with particle size about 5 to 300 microns, preferably about 10 to 250 microns or more preferably 20 to 200 microns in diameter.

[0028] FIG. 2 are histograms showing particle analyses of 2 lots of micronized chorionic plate (FMCP).

[0029] FIG. 3 shows the evaluation of cellular functionalities on ECM (A, B). Remediated ECM samples showed similar cell proliferation and viability as that of tissue culture polystyrene plate (TCP) at all time points. (C, D) non-remediated ECM samples showed higher cell proliferation compared to TCP at days 3 and 7 and (E, F). FMCP samples showed lower cell proliferation compared to TCP on days 3 and 7.

[0030] FIG. 4 shows an evaluation of anti-inflammatory properties of ECM (A remediated and B non-remediated): (A) ECM remediated samples showed significant down regulation of MMP-13 on day 7; (B) ECM remediated samples showed significant down regulation of MMP-13 on day 3; and (B) lyophilized Connective Tissue Matrix (CTM) samples showed significant down regulation of MMP-13 on day 7. CTM is an established and available product sold under the mark INTERFYL.

[0031] FIG. 5 shows (A, B) Monocyte stained with actin-Hoechst on CTM and TCP on day 1 showed increased adhesion of monocytes on CTM compared to TCP. (C) Monocyte viability measured using ALAMARBLUE assay at days 1 and 3 showed higher cell viability on CTM compared to TCP.

[0032] FIG. 6 Gene expression profiles of M1 / M2 markers on CTM or TCP. The monocyte activation on CTM, TCP and GM-CSF was monitored by the expression of Ml markers (TNF-alpha, IL-1β, ILS) and M2 markers (CD206, CCL22, CCL18). CTM showed transient expression of Ml markers and a sustained expression of M2 markers.

[0033] FIG. 7: Study design of Sprague Dawley Rat MIA Model of Osteoarthritis Study#1.

[0034] FIG. 8: Chondrogenesis Induction by CTM in monoiodoacetate induced rat osteo arthritis (MIA OA) Rat Joint.

[0035] FIG. 9: Study design of Sprague Dawley Rat MIA Model of Osteoarthritis Study#2.

[0036] FIG. 10: Chondrogenesis / Repair of Damaged Cartilage Surfaces (% Width).

[0037] FIG. 11: Chondrogenesis results in OA joint of CTM treated MIA OA rats.Micrographs showing increased toluidine blue staining indicating enhanced chondrogenesis in CTM treated MIA OA rats.

[0038] FIG. 12: Monocyte Chemotactic Protein-1 (MCP-1) levels observed in synovial fluid of MIA rats treated with CTM.

[0039] FIG. 13: Dynamic weight bearing results show a trend toward normal weight distribution at study termination on Day 60 for all animals treated with CTM.DETAILED DESCRIPTION

[0040] The present disclosure is based upon the discovery that surprisingly and unexpectedly, a decellularized placenta derived extracellular matrix of the instant disclosure substantially free of residual materials such as DNA, cells, cell debris, growth factors, DNA, cytokines, etc. readily has applications in treating a musculoskeletal or orthopedic disease or condition, such as osteoarthritis, and does not elicit any deleterious immune response. This discovery is contrary to heretofore known placenta extracellular matrix compositions which: (a) do not disclose decellularized extracellular matrix, (b) teach residual materials such as cytokines, proteins, chemokines, etc., are present inquantifiable amounts and indeed provide a therapeutic benefit; and (c) include umbilical cord material.

[0041] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure.

[0042] All numerical designations, e.g. volume, mass, number of particles, etc. are approximations which are varied by (+) or (-) by increments of 1.0 or 0.1, as appropriate. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term "about".

[0043] Numerous terms and phrases are used throughout the instant specification and claims and are defined below.

[0044] "About* and "approximately* are interchangeable and mean plus or minus a percent (e.g., ±5%) of the number, parameter, or characteristic so qualified, which would be understood as appropriate by a skilled artisan to the scientific context in which the term Is utilized.

[0045] As used here, the singular form "a", "an" and "the" Include plural reference unless the context clearly dictates otherwise.

[0046] As used herein, the terms "comprising," "comprises" and "comprise" are Intended to mean that the compositions, preparations and methods disclosed herein Include recited elements, but do not exclude others.

[0047] As used herein, the term "concomitant" refers to a short duration between the administration of a first therapeutic agent and a second therapeutic agent. In the instant disclosure, It is not critical whether a pharmaceutical composition of the instant disclosure or a therapeutic agent is administered first in concomitant administration.

[0048] The terms "delivery" and "administration" are used interchangeably herein and mean and include providing a "pharmaceutical composition" or "biologically active agent" or "active agent formulation" to a treatment site, e.g., damaged tissue, through any method appropriate to deliver the functional agent or formulation or pharmaceutical composition to the treatment site. Non-limiting examples of delivery methods include direct injection, percutaneous delivery and topical application at the treatment site.

[0049] As used herein, the terms "extracellular matrix" and "ECM" are used interchangeably herein and mean and include a collagen-rich substance that is found in between cells in mammalian tissue, and any material processed therefrom, e.g., decellularized ECM. In a particular embodiment, ECM material is derived from human placenta.

[0050] As used herein the phrase "musculoskeletal or orthopedic disease or condition" refers to diseases, disorders or conditions that impact the function of joints In a subject.Examples include, but certainly are not limited to osteoarthritis, degenerative disc disease, cartilage deficits or damage, soft tissue injury, physical trauma, plantar fasciitis, tendonitis, orthopedic surgery, OCD, or PTOA, to name only a few.

[0051] As used herein, the terms "lyophilized" and "dehydrated" are used interchangeably, and refer to the state, but not the method of having water removed as a means of preservation.

[0052] The term "percutaneous", as used herein, means and includes any penetration through the skin of a subject, whether in the form of a small cut, incision, hole, cannula, tubular access sleeve or port or the like. In a particular embodiment, decellularized placenta extracellular matrix is a lyophilized or dehydrated form and is resuspended with an excipient to form a pharmaceutical composition for injection directly to the target site.

[0053] The terms "patient" and "subject" are used Interchangeably herein, include warm blooded mammals, humans and primates; avians; domestic household or farm animals, such as cats, dogs, sheep, goats, cattle, horses and pigs; laboratory animals, such as mice, rats and guinea pigs; fish; reptiles; zoo and wild animals; and the like.

[0054] As used herein, the term "placenta” refers to a disc of tissue that connects a mother's uterus to the umbilical cord and is ultimately responsible for delivering nutrients and oxygen to a fetus. Three layers of membranes make up the placenta : (1) the amnion, which Is a single layer of ectodermal epithelium completely enclosing the embryo; (2) the chorion which surrounds the amniotic sac and Includes the villi and trophoblast; and (3) the decidua of the maternal endometrium. The umbilical cord is the conduit between the placenta and the mother's circulatory system, and consequently, is not a part of the placenta.

[0055] As used herein, a "prophylactically effective amount" as used herein means that amount of a "pharmaceutical composition" and / or "biologically active agent and / or "active agent formulation" administered is a sufficient quantity to prevent or delay the onset of a disease, disorder or condition in a patient susceptible to or at risk of a particular disease, disease or condition. As with a therapeutically effective amount, the prophylactically effective amount will depend on the patient's state of health, age, weight, and the like. It is considered well within the skill of the art for one to determine such prophylactically effective amount by routine experiment (e.g., a dose escalation clinical trial).

[0056] As used herein, the term "resuspended" refers to the addition of a liquid, e.g., a diluent, to a dehydrated material in order suspend the material in a solution and to allow for injection through an adequately sized needle.

[0057] As used herein, the terms "sprinkle" or "sprinkling" refer to scattering or pouring a decellularized placenta derived extracellular matrix or pharmaceutical composition thereof onto a target site.

[0058] As used herein, the term "stagger" or "staggered" with respect to administration of a first and second therapeutic agent means administration of one of the agents occurs at a different time than the administration of the administration of the second agent, such that the duration between each of the administrations is greater than the duration between the concomitant administration of two therapeutic agents.

[0059] As used herein, the term "simultaneous" or "simultaneously" with respect to the administration of a therapeutic agent as described above refers to administration of a therapeutic agent and administration of a decellularized placenta derived extracellular matrix of the instant disclosure, or pharmaceutical composition thereof, occurring at exactly the same time.

[0060] As used herein, the phrase "substantially free of residual material" refers to an extracellular matrix that does not contain a quantifiable amount of residual material, e.g., (1) cells, (2)cell debris, (3) nucleic acid molecules, (4) nucleotides, (5) growth factors, (6) cytokines, (7) chemokines, (8) hormones, or (9) any combination of (l)-(8).

[0061] As used herein, the phrase "therapeutically effective amount" means that the amount of a "pharmaceutical composition" and / or "biologically active agent” and / or "active agent formulation" administered Is of sufficient quantity to ameliorate one or more causes, symptoms, or sequelae of a disease or condition. Such amelioration only requires a reduction or alteration, not necessarily elimination, of the cause, symptom, or sequelae of a disease, disease or condition. A therapeutically effective amount will depend on several factors including severity and course of the disease or condition, previous therapy, the patient's health status, age, weight, and response to the drugs. It is considered well within the skill of the art for one to determine therapeutically effective amounts by routine experimentation (including, but not limited to, a dose escalation clinical trial).

[0062] A pharmaceutical composition of the present disclosure may be resuspended for injection at the point of care or may be mixed with an excipient and stored at a cold temperature, such as from about 2° C to about 8° C. An example of a pharmaceutically acceptable excipient in liquid form that can be used to resuspend a decellularized placenta extracellular matrix of the instant invention, and thus forming a pharmaceutical composition of the instant disclosure, may be a sterile sodium chloride solution, for example about 0.9% sodium chloride (hereinafter referred to as "saline") Other such pharmaceutically acceptable excipients having applications herein are discuss infra. A therapeutically effective amount of a decellularized placenta extracellular matrix contained within a pharmaceutical composition of the instant disclosure can vary, depending upon the damage the subject has suffered, and the size of the joint. Generally, the amount can be about 25 mg, about 100 mg, about 150 mg, about 175 mg, about 200 mg, about 250 mg, about 300 mg, or about 500 mg or more. The volume of liquid used to resuspend a therapeutically effective amount of a pharmaceutical composition of the Instantdisclosure can also vary, from about 1 ml, about 2 ml, about 3 ml, about 4 ml, about 5 ml, about 6 ml, about 7 ml, about 8 ml, about 9 ml, or about 10 ml or more. A particular dosage unit is about 50 mg / ml. Naturally though, the amount of a decellularized placenta derived extracellular matrix of the Instant disclosure contained within a pharmaceutical composition of the instant disclosure, as well as the volume of such a pharmaceutical composition administered may be varied as needed.

[0063] A pharmaceutical composition of the instant disclosure can be administered directly to a target site (e.g., joints, surgical site, tendon). The administration of medical compositions via intra -articular route and direct injection into a tendon, for example, are well-known in the art. Administration can also be parenteral or percutaneous or subcutaneous. Other methods of delivery, e.g., liposomal delivery, diffusion from a device impregnated with the rehydrated composition, and microemulsion-based transdermal delivery in pharmaceutical applications, are known in the art. A decellularized placenta derived extracellular matrix of the instant disclosure may be resuspended forming a pharmaceutical composition as disclosed herein for injection to treat a musculoskeletal or orthopedic disease or condition or a symptom thereof, e.g., pain and dysfunction associated therewith. Administration of a pharmaceutical composition of the instant disclosure may delay its progression and decrease pain associated therewith.

[0064] Alternatively, a pharmaceutical composition of the Instant disclosure can be applied to a target site, e.g., a joint or tendon in a dry form, by 'sprinkling' a dosage of the dry pharmaceutical composition onto the target site. In another embodiment, a pharmaceutical composition of the instant disclosure, following rehydration to have a paste-like consistency, can also be applied directly to a target site, e.g., smeared onto the target site.

[0065] The concentration of a decellularized placenta derived extracellular matrix as disclosed herein in a pharmaceutical composition of the instant disclosure can be varied as needed. In some procedures a more concentrated preparation is useful, whereas in other procedures, a solution with a lower concentration is useful. Moreover, additional compounds or components can be added to a pharmaceutical composition of the instant disclosure. Exemplary compounds that can be added to a resuspended formulation of a decellularized placenta extracellular matrix include, but certainly not limited to an additional therapeutic agent, pH modifiers, buffers, collagen, surfactants, stabilizers, proteins, and the like. Antimicrobial agents such as antibiotics or anti-fungal agents may be added. Other substances can be added to stabilize and / or preserve a pharmaceutical composition as described herein, if needed. Moreover, a pharmaceutical composition as described herein can be packaged and stored, for example, at room temperature, under refrigeration, or for example, at -20° C. or -80° C. prior to use.

[0066] A pharmaceutical composition of the instant disclosure can also be administered for prophylactic and / or therapeutic treatments. For prophylactic treatments, a composition of the instant disclosure may be administered to a patient susceptible to or at risk of a particular disease, disease or condition. As with a therapeutic dose, a " prophy tactically effective amount or dose" will depend on the patient's state of health, age, weight, and the like. It is considered well within the skill of the art for one to determine such prophylactically effective amounts by routine experimentation (e.g., a dose escalation clinical trial).

[0067] In certain embodiments, a pharmaceutical composition as disclosed herein Includes a pharmaceutically acceptable dlluent(s), exclpient(s), or carrier(s). In addition, the resuspended decellularized placenta derived extracellular matrix preparations and pharmaceutical compositions as described herein can be administered as pharmaceutical compositions in which a pharmaceutical composition of the instant disclosure is mixed with other therapeutic agents, as in combination therapy. In some embodiments, the pharmaceutical compositions may Include other medicinal or pharmaceutical agents, carriers, adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure, and / or buffers. In addition, the pharmaceutical compositions can also contain other therapeutically valuable substances.

[0068] Usually, resuspension of a lyophilized decellularized placenta derived extracellular matrix of the Instant disclosure, or a pharmaceutical composition as disclosed herein, does not require more than manual agitation of the vial containing the decellularized placenta derived extracellular matrix and a liquid pharmaceutically acceptable excipient. Generally, the decellularized placenta derived extracellular matrix resuspends within about one minute or less with manual agitation and is suitable for injection or extrusion from a syringe. Resuspended formulations may include suspensions, pastes and putties that are extrudable from a syringe. For intra-articular Injection, for example, the resuspended product is preferably readily deliverable through, for example, a 22 or 26 gauge needle.

[0069] A pharmaceutical composition as described herein may be in unit dosage forms suitable for single administration of dosages. In unit dosage form, the formulation is divided into unit doses containing appropriate quantities of a decellularized placenta derived extracellular matrix of the instant disclosure, e.g., about 100 mg or about 200 mg. The unit dosage may be in the form of a package containing discrete quantities of a decellularized placenta derived extracellular matrix disclosed herein, or a pharmaceutical composition thereof. A non-limiting example Includes a lyophilized decellularized placenta derived extracellular matrix of the Instant disclosure in powder form housed in vials or ampules.

[0070] The dosages for a pharmaceutical composition of the instant disclosure range from about 25 to about 500 mg / jolnt or tendon or other target site, for example, about 25 toabout 50 mg, from about 25 to about 200 mg, from about 50 to 150 mg, from about 100 to about 200 mg, from about 150 to about 300 mg, from about 200 to about 400 mg, or from about 300 to about 500 mg per joint or tendon or other target site, or any range between, conveniently administered in single dose, optionally with follow up doses administered later in time. Moreover, an effective minimum dose of a pharmaceutical composition of the instant disclosure for the treatment of pain, such as, for example, joint pain associated with osteoarthritis, is a dosage amount of greater than about 25 mg, such as about 50 mg, about 100 mg, about 150 mg, about 200 mg, for example. A dosage regimen for the treatment of pain associated with musculoskeletal or orthopedic disease or condition may Include intra -articular Injection of about 50 mg to about 200 mg of a decellularized placenta derived extracellular matrix of the instant disclosure, optionally followed by a repeat dosage via intra-articular injection within one week to six months, as needed. The treatment regimen may include further repeat dosages as needed. Single dose administration of greater than about 25 mg, such as about 50 mg, about 100 mg, about 150 mg, about 200 mg of a decellularized placenta derived extracellular matrix of the Instant disclosure or a pharmaceutical composition thereof results in significant relief of pain associated with, for example, osteoarthritis, while repeat dosage provides a prolonged and significant relief of pain associated with a musculoskeletal or orthopedic disease or condition.

[0071] For the treatment of non-paln related symptoms of a musculoskeletal or orthopedic disease or condition, such as prevention or inhibition of the disorder per se, or for the treatment of surgical sites, a higher dosage amount than used for the treatment of pain is generally used. Typically, a dosage amount of from about 100 mg to about 300 mg of a decellularized placenta derived extracellular matrix of the instant disclosure or a pharmaceutical composition thereof, particularly about 100 to about 250 mg, more particularly about 100 to about 200 mg, is administered via injection, e.g., into the affected tissue, joint, or surgical site. In certain embodiments, the desired dose may be administered as a single dose or as divided doses administered simultaneously (or concomitantly). Alternatively, staggered doses may be administered at appropriate intervals, for example as two or more doses administered about one week to about six months apart, such as about two weeks to about three months apart, about two weeks to about one month apart, etc., and optionally followed by one or more repeat doses as needed. In a particular embodiment, at least two doses, e.g., each of about 100 to about 200 mg, are administered two to four weeks apart. Further doses of a pharmaceutical composition of the instant disclosure are administered as needed.

[0072] The foregoing ranges and timing of doses are merely suggestive, as the number of variables In regard to an individual treatment regime is large. The timing of administration and amount of each dose may vary, depending on several factors Including severity andcourse of the disease or condition, previous therapy, the patient's health status, age, weight, and response to the drugs. It is considered well within the skill of the art for one to determine a therapeutically effective dosing.

[0073] A decellularized placenta derived extracellular matrix as described herein or a pharmaceutical composition thereof may be In unit dosage forms suitable for single administration of dosages. In unit dosage form, the formulation is divided into unit doses containing appropriate quantities of a pharmaceutical composition as described herein, e.g., 100 mg or 200 mg of a decellularized placenta derived extracellular matrix of the instant disclosure. The unit dosage may be in the form of a package containing discrete quantities of the formulation. A non-limiting example includes lyophilized powder of a decellularized extracellular matrix of the instant disclosure in vials or ampules.

[0074] A pharmaceutical composition of the instant disclosure may be formulated in a conventional manner using one or more physiologically acceptable carriers including excipients and auxiliaries which facilitate processing a decellularized placenta derived extracellular matrix of the Instant disclosure into a pharmaceutical composition as described herein that can be used pharmaceutically. Formulation is dependent upon the desired route of administration. Any of the well-known techniques, carriers, and excipients may be used as suitable and as understood in the art. A summary of pharmaceutical compositions described herein may be found, for example, In Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa. Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins; 1999), all of which are hereby incorporated by reference herein in their entireties. In a particular embodiment, a pharmaceutical composition of the instant disclosure, resuspended with a liquid pharmaceutically acceptable excipient, can be mixed with one or more therapeutic agents, as in combination therapeutic. A pharmaceutical composition of the instant disclosure may include other medicinal or pharmaceutical agents, excipients, adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating osmotic pressure, buffers, or any combination thereof. Additionally, a resuspended solution of a decellularized placenta derived extracellular matrix as disclosed herein can be co-delivered with one or more solutions containing additional therapeutic agents, such as, for example, a hyaluronic acid (HA) solution, a solution containing antiinflammatory agent(s), or Stromal Vascular Fraction (SVF), Bone Marrow Aspirate (BMAC), Platelet Rich plasma (PRP) or an antibody, to name only a few, using a dual barrel syringe, such as Becton Dickinsons LEUR-LOK syringe discussed above, for example, to simultaneously deliver the different solutions. Particular examples of excipients havingapplications in a pharmaceutical composition of the Instant disclosure include normal saline, a physiological salt solution (phosphate buffered saline; PBS), Dulbecco's Modified Eagle Solution (DMEM), water, any autologous preparation (such as platelet rich plasma (PRP), bone marrow aspirate concentrate (BMAC), stromal vascular fraction (SVF)), or a balanced salt solution (BSS), to name only a few. A pharmaceutically acceptable excipient having applications in a pharmaceutical composition of the instant disclosure can be a liquid or a solid.

[0075] Optionally, a pharmaceutical composition of the Instant disclosure can be administered separately from a pharmaceutical composition comprising a therapeutic agent. These two pharmaceutical compositions can be given simultaneously, concomitantly, or In staggered or serial form wherein the administration of one follows the administration of another. Moreover, the time that elapses between the administration of each of these pharmaceutical compositions can vary from a few seconds apart up to about hours or days apart. The order in which such pharmaceutical compositions are administered Is not critical. However, If the subject is in pain, it may be advisable to administer initially a therapeutic agent that addresses pain, and then administer a pharmaceutical composition of the Instant disclosure.

[0076] The present disclosure may be better understood by reference to the following nonlimiting examples, which are provided as exemplary of the disclosure. The following examples are presented in order to more fully illustrate particular embodiments of the disclosure. They should in no way be construed, however, as limiting the broad scope of the disclosure.EXAMPLE

[0077] Provided herein is evidence of development of novel decellularized placenta derived extracellular matrix (ECM) and pharmaceutical composition thereof with a novel method to improve the efficacy of the placenta derived ECM for the treatment of inflammatory conditions associated with joints. Decellularizatlon of placental tissue is a critical factor in producing extracellular matrix that Is free of residual materials, such as cells, cell debris, DNA, growth factors and cytokines. The decellularized placental ECM has a characteristic profile and demonstrates that a decellularized placenta derived ECM produced with this decellularizatlon method can mitigate the symptoms of OA using rat monolodoacetate (MIA) induced disease models, where articular cartilage base chondrocytes are chemically rendered non-viable within 3 hours of MIA injection into the knee, resulting in excessive inflammatory response within the joint and synovial fluid causing joint surface degeneration. Also provided are In vitro data revealing the molecular mechanisms of actions (MOA) of a decellularized placenta derived ECM of the instant disclosure to treat osteoarthritis (OA). The MOA is predominantly achieved by promoting chondrocyte cellproliferation, and by anti-inflammatory activities. The presence of a decellularized placenta derived ECM as disclosed herein decreases the levels of proteolytic degradation of the cartilage matrix. The decellularized placenta derived ECM showed unique ability to shift Ml macrophages, which are pro-inflammatory, to M2 phenotype, which are antiinflammatory and pro-regenerative.

[0078] Decellularized placenta derived ECM of the instant disclosure represents a novel therapy for inflammatory joint conditions, manufactured using a process designed to ensure complete decellularization of the decellularized placenta derived ECM, therefore reducing the potential of any adverse inflammatory or immune interactions. Commercially available decellularized products have been shown to exhibit a wide range of DNA content and evidence suggests a correlation of the quantity or localization of remaining DNA in decellularized ECM products with the severity of adverse host Inflammatory response. DNA complexes can elicit innate immunity through activation of Toll Like Receptors (TLRs) or TLR-independent mechanisms. DNA quantification and localization have historically been used as measures of cellularity. Therefore, they, along with DNA fragment-length analysis, can be measures of decellularization by serving as a representative of all nuclear material, even if there Is a non-specific link to the immune response. In measuring DNA, the analysis may exclude single stranded nucleotide chains (for example, ssDNA) and very short fragments of double-stranded nucleotide chains.Materials and MethodsPlacental sourcing:

[0079] The placenta starting material is sourced by established methods designed to reduce the risk of additional contamination and prevent mislabeling and / or tracking errors. These methods are consistent with regulatory requirements outlined in 21 Code of Federal Regulations (CFR) 1271, Subpart C (Donor Eligibility) and Subpart D (Good Tissue Practices). As explained above, for purposes of the instant disclosure, the umbilical cord is not a part of the placental, and thus no material derived from the umbilical cord is contained in a decellularized placenta derived extracellular matrix as disclosed herein. The tissue originates from accredited hospitals, with collections performed by licensed health care professionals. The tissue collection kit utilized includes sufficient containers and barcoded labels for biomaterials along with appropriate documentation and instructions for collection. Furthermore, the placental tissue is screened for the negative presence of transfusion transmissible viruses in accordance with the FDA Guidance for Industry: Eligibility Determination for Donors of Human Cells, Tissues and Cellular and Tissue-Based Products (HCT / Ps) - August 2007.Decellularization of Chorion for Finely Milled Chorionic Plate (FMCP):

[0080] Processing / decellularization of chorionic plate is conducted following ASTM-F3354 standard guide, using aseptic technique in a Class 100 BSC (biological safety cabinet) within an ISO Class 7 cleanroom, using sterile, single-use consumables. The process begins with the procurement of placenta, obtained from a normal, full-term delivery. Within 48 hours of delivery of a baby, the placenta is soaked in hypertonic sodium chloride (NaCI) solution and quarantined until viral safety test results are received.

[0081] Following quarantine release, the placenta is transferred to a Biosafety hood and the chorionic plate is separated from the placenta.

[0082] The chorionic plate is mechanically dissected from the placenta. Following dissection, the plate is mechanically scraped to remove the residual placental tissue. The chorionic plate is then placed in a bottle with 2 molar (M)sodlum chloride (NaCI) solution and incubated In an orbital shaker for greater than 4 hours. The solution is then changed to sterile water and Incubated in an orbital shaker for up to 24 hours. The exposure of the chorionic plate to a high concentration of NaCI, followed by water constitutes an "osmotic shock” to the tissue. The osmotic shock serves to lyse cells, remove blood, blood components, cells, and cell debris from the tissue. To remove embedded cells (e.g., fetal trophoblasts or epithelial cells), the tissue Is rinsed overnight in 2% deoxycholic acid. The deoxycholic acid Is then removed from the tissue with several sterile water washes. At this point of manufacturing, the in-process product Is composed of a densely packed, highly organized native extra-cellular matrix. The wet tissue is then homogenized In a commercial blender and lyophilized by first freezing the blended chorionic plate at -80°C until the tissue is frozen and freeze-dried in a lyophilizer for 16 - 18 hours. The lyophilized Connective Tissue Matrix (CTM) product, which is called CTM wafer, is then cryomilled using a method developed and detailed in the following sections to produce Finely Milled Chorionic Plate (FMCP).Decellularization of Placental ECM for pECM :

[0083] Processing / decellularization of the placenta is conducted following ASTM-F3354 standard guide, using aseptic technique in a Class 100 BSC (biological safety cabinet) in an ISO 7 cleanroom, using sterile, single-use consumables. The process begins with the procurement of placenta, obtained from a normal, full-term delivery. Upon arrival at the point of processing, which occurs within 48 hours of delivery of a baby, the umbilical cord is separated from the placenta and the placenta is quarantined. The quarantine of the placenta is done by freezing the placenta at -80°C until viral safety test and endotoxin results are received.

[0084] The ECM is sourced from whole human placentas. The frozen placenta is released from quarantine for processing and Is thawed at 4 - 8°C for up to 96 hours and transferred to a Biological Safety Cabinet (BSC) . The placenta is removed from the storage containerand placed on a sterile disposable tray at controlled room temperature (about 20 to about 25°C). Each placenta Is processed as a separate lot. The placenta is cleaned to remove excess blood and blood clots and cut into small segments. The cut placental segments are suspended in sterile water and are homogenized using an OMNI mixer homogenizer (with a 2-inch rotor knife probe). The homogenization step serves to generate small tissue particulates, with increased surface area, allowing for more effective separation and removal of cells and cellular debris from the placental tissue. The homogenized tissue from a single placenta is transferred into a sterile processing container of sodium chloride (NaCI) solution. The tissue is washed repeatedly with sterile NaCI by shaking on an orbital shaker; followed by replacement with additional sterile NaCI solution. The placental tissue is Incubated at controlled room temperature (about 20 to about 25°C) for up to about 24 hours while shaking in sterile NaCI solution, followed by sterile water wash. The exposure of the placental tissue to a high concentration of NaCI, followed by water constitutes an "osmotic shock' to the tissue. The osmotic shock serves to remove blood, blood components, cells, and cell debris from the tissue. The placental tissue is subjected to a second osmotic shock before a nucleic acid removal step using BENZONASE endonuclease treatment at an effective pH range. BENZONASE endonuclease degrades all forms of nucleic acids (DNA & RNA) into shorter polynucleotide fragments that are further cleared and removed by subsequent sterile water washes.

[0085] Following removal of the BENZONASE endonuclease with extensive sterile water rinses, the tissue is subjected to detergent washes with sodium deoxycholate (DOC) solution first at a high concentration for up to about 24 hours followed by low concentration for up to an additional about 72 hours. Sodium deoxycholate (DOC) is a water soluble, bile-acid derived, mild ionic detergent used to remove residuals such as cells, cell membranes, and other cell debris.

[0086] Upon completion of the detergent washes, the tissue is washed with sterile water to remove debris and residual DOC. To enrich Collagen content, the placental ECM is treated with a high pH sodium hydroxide (NaOH) solution to denature and remove any soluble non-crosslinked proteins, l.e., cytokines / chemokines, growth factors, and hormones. Thereafter, the ECM is subjected to low pH hydrochloric acid (HCI) solution to neutralize the NaOH and remove the above-mentioned impurities, as well as calcium phosphate deposits, which may be present in the placenta. The incubation of NaOH followed by HCI is referred to as the "remediation step' following the ECM deceliularization process. Iterations of ECM with and without the remediation step have been conducted. The ECM generated is labeled as ECM-remediated and ECM-non remediated, respectively. Where remediated, the ECM is subjected to extensive sterile water washes prior to centrifugation of the suspension to neutralize the ECM, minimize hydrolysis of the Collagen, and to further facilitate the removal of solubilized product residual materialssuch as cells, cellular debris, nucleic acids, growth factors, chemokines / cytokines, hormones, and process reagents, such as salt, DOC, and Benzonase. The decellularlzed placental ECM is then transferred into a sterile disposable tray, and frozen at about -80°C, followed by lyophilization. The lyophilized ECM wafer is stored at room temperature in an enclosed container until further processing by cryomilling to a finely powdered form- placental ECM (pECM). Decellularlzed placenta derived ECM of the instant disclosure Is thus generated and used in studies for the final product attributes in relation to cell proliferation and anti-inflammatory properties.Crvomillino of Ivoohilized ECM and Ivoohilized Chorionic Plate:

[0087] After lyophilizing the decellularlzed ECM or Chorionic plate, to produce micronized decellularlzed placenta and micronized chorionic plate (FMCP), the respective wafers are broken into small pieces about ¼ " In width, while maintaining sterility of the product. The lyophilized matrix Is subjected to cryomilling using cooling and grinding cycle of approximately 2 to about 5 minutes, depending on the particulate size required. The milled particles must be suitable for Injection through a pre-determined syringe gauge. In particular, the milled particulates are extrudable through a 22-gauge needle or higher, a 25-gauge needle or higher, or a 27 gauge needle or higher when in suspension in liquid. Examples of acceptable liquid Include but is not limited to saline, hyaluronic acid, platelet rich plasma. The method used to grind both ECM and CTM wafer Involves up to five alternating cooling followed by grinding cycles at frequencies specific to the make / model of cryomill, with each cycle lasting between about 2 to about 5 mins.Table 1: Cryomilling Program used for micronizing ECM & Chorionic Plate

[0088] After running cryomilling programs, the final finely powdered form of the product Is collected using a sterile spatula and placed In a sterile container. The final weight of the cryomilled product Is recorded and recovery of the cryomilling process Is calculated. The aliquots of lyophilized product may be distributed in individual containers such as glass vials that are stoppered under vacuum and capped to create a hermetic seal, for example. The aliquoted, lyophilized tissue is optionally sterilized by E-Beam irradiation, gamma Irradiation, UV light or exposure to ethylene oxide, supercritical carbon dioxide or other suitable sterilant known to those in the art. E-beam irradiation Is a preferred method ofsterilization. The dehydrated, sterilized decellularized particulate compositions are substantially free of blood residuals and foreign matter.

[0089] The source of decellularized placental tissue could be derived from part of the placenta or full placental tissue as indicated in Table 2.Table 2: Cryomilling Program used for mlcronizlng ECM and Chorionic Plate

[0090] It has been discovered that these particle sizes provide significant therapeutic effects in the treatment of a musculoskeletal or orthopedic disease or condition.Characterization of lyophilized placental ECM

[0091] Decellularization of a placenta derived extracellular matrix that involves subjecting placental tissue to stringent decellularization process steps, including osmotic shock and mild detergent treatment, results In removal of process and product residual materials, such as cell debris, residual cells, host residual growth factors and cytokines, nucleic acids, and residual detergent. At the same time, these process steps are enriching for proteins of the decellularized placenta derived ECMs, such as collagen and elastin, and have beneficial effects on the decrease of inflammatory responses. The biochemical composition of a decellularized placenta derived extracellular matrix is disclosed in Table 3.Table 3: Biochemical Composition of Lyophilized Placental ECM

[0092] The processing of ECM ensures removal of all cells, any cellular debris, residual nucleic acids, and processing reagents.Table 4: Residual Testing of Lyophilized Placental ECM

[0093] In contrast to heretofore known extracellular matrix formulations, such as, for example, as described the US'241 publication, which Includes umbilical cord tissue and contains residual materials such as growth factors and cytokines, for example, each placental component has measurable levels of pro-anabolic (PFGF), or anti-inflammatory (IL-IRu) growth factors and cytokines; and consistently quantifiable levels of inflammatory factors, such as, for example IL-lo, the process described herein is designed to result in removal of numerous residual materials, such as cytokines / chemokines, growth factors and major regulators of wound healing, scarring and fibrosis, as well as anti- and pro- inflammatory factors, and hormones, to name only a few.Table 6: Calculated Values of Cytokines / Chemokines and Growth Factors in pg / mg Tissue in the Unprocessed Placenta as Compared to Processed ECM

[0094] Moreover, the presence of any hormones such as human chorionic gonadotropin (hCG), Follicle-stimulating Hormone (FSH), Lactogen, Estrogen, Progesterone and Oxytocin in the postpartum unprocessed placenta and final ECM were also evaluated. Samples extracted in buffer containing a protease inhibitor cocktail were tested using individual hormone specific ELISA kits from vendors, and analyzed using a Microplate reader (Biotek, Synergy H4 model). The results showed these hormones were detected in the unprocessed placenta however were below the LOD or ND in the processed ECM Table 7.Table 7: Hormone Analysis of Lyophilized Placental ECM

[0095] Figure 1 shows examples of the particle size analysis of cryomilled placenta ECM that was conducted using the Beckman Coulter Multlslzer 4e Particle Analyzer. Two lots of cryomilled placental ECM were tested, and the figure above shows number of particulates greater than 300 pm to be 0 for both lots with more than 90,000 particles detected by the machine. The Cryomilling program stated above is able to generate a final particulate form of placental ECM with particle size about 5 to 300 microns, preferably about 10 to 250 microns or more preferably 20 to 200 microns in diameter.Characterization of Finely Milled Chorionic Plate fFMCPl

[0096] The biochemical composition of the resultant FMCP has been evaluated.Table 8: Biochemical Composition of FMCP & CTM

[0097] The processing of chorionic plate also includes steps to ensure removal of > 95% Intact cells, any cellular debris, and processing reagents such as salt, and DOC. The process also guarantees sterility of the product, and this is concluded through the endotoxin and sterility tests. These tests are conducted on all FMCP lots.Table 9: Residual Testing of FMCP & CTMTable 10: Quality Attributes of FMCP & CTM

[0098] Furthermore, the presence of any hormones such as human chorionic gonadotropin (hCG), Follicle-stimulating Hormone (FSH), Lactogen, Estrogen, Progesterone and Oxytocin in the postpartum unprocessed placenta and final FMCP was also evaluated. The results showed all hormones tested besides FSH were detected in the unprocessed placenta, and after FMCP processing lactogen & oxytocin levels were below limit of detection. HCG, estrogen and progesterone were detected in the unprocessed chorion In very minimal quantity but FMCP processing significantly reduced the amount HCG, estrogen, and progesterone.Table 11: Hormone Characterization of FMCP

[0099] Figure 2 shows the particle analysis of FMCP that was conducted using the Beckman Coulter Multisizer 4e Particle Analyzer. Two lots of FMCP were tested, and the figure above shows number of particulates greater than about 300 μm to be 0 for both lots with more than 55,000 particles detected by the machine. The Cryomilling program stated above Is able to generate a final particulate form of FMCP with particle size ≤ about 300 μm in diameter.Delivery of therapeutic pharmaceutical compositions of decellularized placental ECM

[0100] Prior to therapeutic application, particulate decellularized placenta derived ECM of the Instant disclosure can be resuspended and co-dellvered with one or more solutions containing additional therapeutic agents. For example, the resuspended placental ECM compositions may be co-delivered with an HA solution, a solution containing anti- inflammatory agent(s), or SVF, BMAC, or PRP for example, using a dual barrel syringe for example to simultaneously deliver the different solutions.

[0101] The concentration of particulate decellularized placenta derived ECM of the instant disclosure particulate a composition of the instant disclosure can be varied as needed. In some procedures a more concentrated preparation is useful, whereas in other procedures, a solution with a lower concentration is useful. In various embodiments of the invention, additional compounds or components can be added to the pharmaceutical composition. Exemplary compounds, diluents, excipients, etc. that can be added to a resuspended pharmaceutical composition of the instant disclosure include, but certainly are not limited to pH modifiers, buffers, collagen, HA, anti-inflammatories, surfactants, stabilizers, proteins, exosomes, cells cell conditioned media, and the like. Antimicrobial agents such as antibiotics or anti-fungal agents may be added. Other substances can be added to the pharmaceutical compositions to stabilize and / or preserve the pharmaceutical compositions if needed. A pharmaceutical composition of the instant disclosure can be packaged and stored, for example, at room temperature, under refrigeration, or for example, at about -20°C or about -80° C prior to use.

[0102] Pharmaceutical compositions as disclosed herein may be formulated in a conventional manner using one or more physiologically acceptable carriers including excipients and auxiliaries which facilitate processing of a decellularized placenta derived ECM pharmaceutical composition into a preparation which can be used pharmaceutically. Formulation is dependent upon the desired route of administration. As discussed previously, any of the well-known techniques, carriers, and excipients may be used as suitable and as understood in the art. A summary of pharmaceutical compositions described herein may be found, for example, In Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975;Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins; 1999), all of which are hereby incorporated by reference in their entireties.Evaluation of cellular functionalities (cell viability and proliferation) on ECM f remediated and non-remediated) - Experimental Design

[0103] Human mesenchymal stem cells (hMSCs) purchased from Stem cell technologies were expanded in MSC media following the manufacturer's instructions. For cell proliferation and viability, 48 well plates were coated with FMCP, ECM remediated and non- remediated (0.5 - 5 mg). Before cell seeding, the wells coated with ECM and FMCP were gently rinsed with 0.5 mU / well of phosphate buffered saline (PBS) once. Human MSCs at passage 4 (P4) were seeded at a density of 10,000 cells on FMCP, ECM coated wells, ultra- low attachment plate (ULAP) and tissue culture polystyrene plate (TCP). Cell proliferation was measured at day 1, 3 and 7 using PRESTOBLUE Cell Viability Reagent (Invitrogen) following manufacturer's instructions. Briefly, the media was removed and incubated with PRESTOBLUE reagent for 30 minutes. Fluorescent Intensity was measured using a microplate reader. Calcein staining was used to visualize the distribution of live cells on the FMCP, ECM on day 7. Briefly, the media was removed, and cells were washed with PBS and treated with 4mM calcein stain for 20-30 minutes. Cells were washed twice with PBS and imaged under a fluorescent microscope.Results

[0104] ECM remediated samples: The cell proliferation as measured by PRESTOBLUE showed that cell proliferation increased from day 1 to day 7 for all groups. (Figure 3A). The cell proliferation on ECM and TCP was found to be significantly higher than ULAP at all time points (day 1-7). Compared to TCP, cell proliferation in ECM coated wells was found to be similar to TCP at all time points. (Figure 3A). Calcein staining on day 7 showed MSC attached well to the ECM coated wells and were viable In both TCP and ECM coated wells. Cell viability was noticed to be similar in both TCP and ECM coated wells, indicating that ECM has the potential to maintain cellular viability for 7 days post seeding. (Figure 3B)

[0105] ECM non-remediated samples: The cell proliferation as measured by PRESTOBLUE showed that cell proliferation increased from day 1 to day 7 for all groups. (Figure 3C). The cell proliferation on ECM and TCP was found to be significantly higher than ULAP at all time points (day 1-7). Compared to TCP, cell proliferation in ECM coated wells was found to be similar to TCP on day 1, however, the cell proliferation on ECM coated wells increased in comparison to TCP on days 3 and 7. (Figure 3C). Calcein staining on day 7 showed MSC attached well to the ECM coated wells and were viable in both TCP and ECM coated wells.Cell viability was noticed to be similar in both TCP and ECM coated wells, Indicating that ECM has the potential to maintain cellular viability for 7 days post seeding. (Figure 3D).

[0106] FMCP samples: The cell proliferation as measured by PRESTOBLUE showed that cell proliferation increased from day 1 to day 7 for both TCP and FMCP. However, the proliferation was found to be lower in FMCP compared to TCP at both days 3 and 7. (Figure 3E) Calcein staining showed FMCP samples supported cellular attachment and viability. (Figure 3F)

[0107] Overall, the results indicate that all FMCP, ECM remediated, and non-remedlated samples have the potential to support cellular functionalities such as cell proliferation and viability.

[0108] Figure 4 discloses the evaluation of cellular functionalities on ECM (A, B). Remediated ECM samples showed similar cell proliferation and viability as that of TCP at all time points. (C, D) non-remedlated ECM samples showed higher cell proliferation compared to TCP at days 3 and 7 and (E, F) FMCP samples showed lower cell proliferation compared to TCP on days 3 and 7.Evaluation of antl-Inflammatorv properties of CTM. ECM f remediated and non-remedlated) - Experimental Design

[0109] Anti-Inflammatory properties of ECM were studied using an in vitro model of osteoarthritis. Human chondrocytes were obtained from SclenCell Research Laboratories and maintained in chondrocyte proliferation media following manufacturer's instructions. ECM and CTM were coated (1.25 -5 mg) on 24 well plates, followed by PBS washes. Chondrocytes were plated into ECM and CTM coated wells and TCP at a density of 250,000 cells per well and cultured for 24 h. The culture medium was then refreshed with chondrocyte media containing 1 ng / mL IL-1β to induce chondrocyte inflammatory responses. After day 1, 3 and 7, cells were lysed with RNA Lysis buffer. Total RNA of human chondrocytes treated with ECM, or other conditions, was isolated and used for gene expression analysis. Primers for this analysis were for selected genes and purchased from Thermo-Fisher. GAPDH gene is used as control for normalization of relative abundance of each gene and is expressed as fold of change comparing to control conditions.Results:

[0110] OA is known to be induced by synovitis during which pro-inflammatory cytokines such as IL-1β are produced which cause the expression of metalloproteinase (MMP) including MMP13. These MMPs are responsible for the degradation of cartilage resulting in knee pain. We evaluated whether our ECM of the Instant disclosure has the potential to suppress IL-1β Induced inflammation and induction of MMP-13 in activated human chondrocytes. Human chondrocytes are treated with IL-1β and ECM of the Instantdisclosure (remediated and non-remediated) and the expression of MMP13 was evaluated for their mRNA expression by qRT-PCR. Figure 4A, B and C showed that expression of MMP-13 significantly increased upon IL-1β treatment. It was also noted that CTM and ECM remediated samples showed significant downregulation of MMP-13 on day 7 compared to TCP- IL-1β (Figure 4A, C). It was found that ECM non-remediated showed significant downregulation of MMP-13 at an earlier time point (day 3) compared to TCP- IL-1β (Figure 48).

[0111] These results indicate that both CTM and ECM (remediated and non-remediated) samples have the potential to suppress inflammation in IL-1β activated human chondrocytes.

[0112] Evaluation of anti-inflammatory properties of ECM (A remediated and B non- remediated) are disclosed in Figure 4. Figure 4(A) discloses that ECM remediated samples showed significant down regulation of MMP-13 on day 7, and In Figure 4(B) ECM remediated samples showed significant down regulation of MMP-13 on day 3, while CTM samples showed significant down regulation of MMP-13 on day 7.Effect of CTM on human monocvte viability and macrophage polarization (Ml to M2 transition) Rationale:

[0113] Previous studies have demonstrated the Importance of macrophages In tissue repair and regeneration, particularly in applications involving biologic scaffolds. Macrophages are responsible, in part, for the degradation of ECM scaffolds. Depletion of circulating macrophages severely attenuates scaffold degradation and the associated constructive remodeling response . Non-crosslinked biologic scaffolds promote the expression of an M2 macrophage phenotype at the site of remodeling although a population of macrophages expressing markers of an Ml phenotype persists. Tidball has demonstrated that it is the switch from an Ml to M2 phenotype that stimulates progenitor cell differentiation and constructive tissue remodeling while an early Ml response stimulates progenitor cell recruitment and proliferation. The ability of biologic scaffolds to promote expression of an M2 macrophage phenotype may therefore be critical to their ability to promote constructive tissue remodeling, specifically within the inflammatory joint conditions.Experimental design

[0114] Monocyte viability: Human monocytes were purchased from Stem Cell Technologies and cultured on CTM and tissue culture plate (wells were coated with 0.1 mg / mL collagen 1 in PBS for 3 hours at 37C) for day 1 and 3. For cell viability experiments, 20,000 cells were seeded onto the scaffolds and an ALAMARBLUE assay (ThermoFisher Scientific)performed. For this assay, the cell media was removed and replaced by a solution containing 90% cell media and 10% ALAMARBLUE solution. Background values for each media were placed in a 96 well plate placed into the Incubator with the cells. The cells were incubated in this for two hours, at which time the media was transferred to a 96 well plate. The fluorescence was read on a Tecan Infinite M200 with an excitation wavelength of 560 nm and emission wavelength of 590 nm. For actin / nuclear staining, cells were washed once with PBS and then fixed for 15 minutes in 4% formaldehyde. The cells were then washed twice with PBS and then stained for 30 minutes with rhodamine Phalloidin (ThermoFisher Scientific) dissolved 1:500 and Hoechst Dye 33258 (1:500). It was washed twice more with PBS and then imaged.

[0115] Macrophage polarization: Monocytes were cultured on CTM In 24-well tissue culture plates (Coming) at 0.5x106 cells / mL in complete RPMI media with 10% fetal bovine serum (FBS). For Control surfaces, collagen type I-coated plates (COL) and GM-CSF treated groups were used. Monocytes were differentiated with 100 ng / mL recombinant GM-CSF for 3 days, after which, the medium was removed and replaced with complete RPMI medium containing 10 ng / mL LPS and 50 ng / mL IFN-y. Cell lysates for gene expression were collected after 1, 3 and 6 days. Gene expression for Ml markers (TNF-alpha, IL-1β B, IL8) and M2 markers (CD206, CCL22, CCL18) were analyzed at days 1, 3 and 6Results

[0116] Monocyte viability: Monocyte viability was found to be higher on CTM compared to TCP on both days 1 and 3. (Figure 5 C) In addition actin-DAPI staining showed increased adhesion of monocytes on CTM compared to TCP. (Figure 5 A,B)

[0117] Macrophage polarization: Analysis of day 1 to 6 culture was performed to examine the temporal regulation of monocyte gene expression. Genes associated with Ml macrophages included TNF, IL-1β, and IL8, while genes associated with M2 macrophages included CD206, CCL22, and CCL18. Comparing CTM and Collagen coated wells on day 1, there was an increase in the expression of genes associated with Ml macrophages, specifically IL- 1β, and IL-8 in CTM. (Figure 6) However, CTM transiently induced pro inflammatory (Ml) factors important for wound healing, and their levels (IL-8, IL-1β) were reduced by day 6 of differentiation. (Figure 6) Interestingly, CTM showed more sustained expression of M2 markers with their levels remaining high at day 3 and 6 compared to day 1 of differentiation (CCL-22, CCL-18). (Figure 6)

[0118] The data shows that CTM supports higher monocyte proliferation, a transient increase in expression of genes associated with Ml macrophages (pro-inflammatory) and a more sustained expression of those associated with M2 macrophages (antiinflammatory). These results indicate that the CTM may serve as an effective scaffold to help guide Important Immune cell types through the regenerative process.

[0119] Figure 5 (A, B) discloses Monocyte stained with actin-Hoechst on CTM and TCP on day 1 showed increased adhesion of monocytes on CTM compared to TCP. In Figure 5(C), monocyte viability measured using ALAMARBLUE assay at days 1 and 3 showed higher cell viability on CTM compared to TCP.

[0120] In Figure 6, gene expression profiles of M1 / M2 markers on CTM or TCP are graphically shown. The monocyte activation on CTM, TCP and GM-CSF was monitored by the expression of Ml markers (TNF-alpha, IL-1β, IL8) and M2 markers (CD206, CCL22, CCL18). CTM showed transient expression of Ml markers and a sustained expression of M2 markersRat OA model

[0121] Two in vivo osteoarthritis (OA) studies were run to evaluate CTM in male Sprague Dawley (SD) rats. OA was induced by Intraarticular (IA) administration of a 1 milligram (mg) dose of mono-iodoacetate (MIA) into the right knee joint. In the first study, MIA was injected on Day 0 and the rats were treated with a single IA injection of Vehicle (Saline, N=10), CTM (8.3mg, n=10), CTM (4.15mg, n=10) or triamcinolone (0.06mg, n=10) on Day 7. Five naive controls (no MIA, saline injection) were included as controls. Rats were evaluated for pain by von Frey filaments and dynamic weight bearing on Days 8, 14, 21, and 28. At study termination on Day 28 synovial fluid and the affected knee joint were collected for cytokine profiling and histopathology (including chondrogenesis) respectively.

[0122] In the second study, rats received MIA to induce OA on Day 0 and one day later were randomized into six treatment groups and treated as follows: Vehicle (Saline; n=20), CTM l.lmg (n=20), CTM 0.55mg (n=20), CTM 0.275mg (n=20), triamcinolone 0.06mg (n=20). Five naive rats, without MIA OA induction, treated with saline were included as controls. Test articles were delivered via single IA injections into the affected knee joint on Day 1. Pain was evaluated using von Frey filaments and dynamic weight bearing analyses on days 7, 14, 28, and 60. Five mice per group were euthanized on days 3, 7, 14, and 60. At each euthanasia timepoint, synovial fluid for cytokine profiling and the right knee joint for histopathology was collected.Results1. In vivo study# 1: CTM stimulates chondrogenesis in MIA OA Rats

[0123] CTM was evaluated in rats by the ability to provide benefit in a rat chemically induced model of OA (Figure 7). Histopathology analysis revealed an increase in the percent area of chondrogenesis in the OA joints of the rats treated with the high dose (8.3mg) of CTM. Figure 8 shows that while not significant, an increase in chondrogenic activity was observed compared to the vehicle treated rats (MIA+saline) (p=0.056 by two- tailed T-test) at study termination on Day 28.Table 12: Group and Treatment InformationTable 12: Group and Treatment Information

[0124] Figure 7 discloses the study design of Sprague Dawley Rat MIA Model of Osteoarthritis Study #1. Figure 8 graphically discloses chondrogenesis induction by CTM in MIA OA Rat Joint.2. In vivo study #2: Decellularized Placenta Derived Extracellular Matrix of the instant disclosure (CTM) stimulates chondrooenesis in MIA OA Rats

[0125] To evaluate the potential of CTM to provide benefit In osteoarthritis a chemically induced (MIA) rat model (Figure 9) was utilized, whereby rats received a single intraarticular Injection of different doses of a decellularized placenta derived extracellular matrix (CTM) of the instant invention. Two in vivo MIA induced OA studies were run each with slightly different study designs.Table 13: Group and Treatment InformationTable 13: Group and Treatment InformationFigure 9 provides the study design of Sprague Dawley Rat MIA Model of the Osteoarthritis Study #2

[0126] Chondrogenesis was evaluated as one of the study endpoints of the MIA treated knee via Toluidine Blue (TB) staining. Chondrogenesis was evaluated at each sacrifice timepoint of Day 3, 7, 14, and at study termination on Day 60.

[0127] Figure 10 shows that In rats treated with the high dose of CTM (Group 3, 1.1 mg) a trend of increasing chondrogenesis is seen at Day 14 and a statistically significant increase (p = 0.031) In chondrogenesis is observed when comparing the high dose CTM to the vehicle control (Group 2, MIA induction + saline treatment) by two-tailed t-test at Day 60. Chondrogenesis / repair measures of the area at risk (the 4 surfaces of the knee) and then the lengths where the chondrogenesis occurs. There was a statistically significant (p=0.031 t-test) increase in chondrogenesis in the CTM 1.1 mg group, as compared to MIA control at day 60.

[0128] Figure 11 shows chondrogenesis results in an OA joint of CTM treated MIA OA rats. Micrographs showing increased toluidine blue staining indicating enhanced chondrogenesis in CTM treated MIA OA rats.

[0129] Toluidine blue staining of the OA joint revealed minimal chondrogenesis In vehicle control rats in both the lateral and medial knee section of the OA joint, with 12% and 21% respectively for rat#l and rat#3. In contrast, the rats treated with l.lmg of CTM showed 31% and 40% chondrogenesis in the medial and lateral knee sections for rat#l and rat#5, respectively.

[0130] Synovial fluid was collected and evaluated for the presence of rat cytokines associated with osteoarthritis. Figure 12 shows the results of the synovial fluid cytokine profiling. A significant decrease in rat Monocyte-Chemotactic Protein- 1 (MCP-1) was seen In rats that received both the mid (0.55mg) and low (0.275mg) dose of CTM at Study Day 3, 2 days post CTM treatment. While significance was not reached in the high dose CTM, a clear reduction of MCP-1 was observed. Binding of MCP-1 to receptors on the surface of OA chondrocytes can induce the production of matrix metalloproteinases (MMPs) and damage articular cartilage.

[0131] Dynamic weight bearing was used to evaluate pain and any pain reduction that was observed with CTM treatment. A clear trend of an equal distribution of weight was observed at the study endpoint. Each treatment group started the study with 20 animals. As animals were sacrificed along the course of the study, the total number of animals at study termination was only five. This reduced number of rats at study termination resulted in a strong trend of efficacy with 1.1 mg of CTM, very close to the untreated control animals, however, not being significant due to a reduced number of animals. Figure 13 shows the results of dynamic weight bearing observed in this study.

[0132] Figure 13 graphically discloses dynamic weight bearing results, which show a trend toward normal weight distribution at study termination on Day 60 for all animals treated with CTM, indicative of a strong trend of efficacy.Collagenase-Induced Tendlnltis / Tendlnooathv Model in Rats

[0133] An in vivo rat tendinitis model will be conducted wherein unilateral tendinitis was induced by direct injection of collagenase (0.3mg in PBS; Sigma, St. Louis, MO) into the Achilles tendon on Day O and Day 1 of the study. A separate group of animals served as a sham control (saline injection only) for histopathology analysis. On Day 5 of the study, animals which had received a collagenase Injection were treated with an intra-tendon injection (30 μL) of saline, or decellularlzed placental matrix particulate resuspended in saline (30 μL; 1.5 mg / tendon), or steroid control in saline (0.3mg / tendon). Pain measurements assessing hindlimb weight-bearing (incapacitance testing) or mechanical allodynia (Von Frey analysis), will be taken at 2, 4, 9, 16, 23, and 30 days post-treatment. At the end of the study, tendons will be sectioned for histopathologic assessment to determine extent of tendon damage, inflammation, and character of tendon repair. Animals which received a single 1.5 mg / tendon dose of decellularlzed placental matrix particulate, are expected to demonstrate a significant reduction in weight- bearing pain (hindlimb weight Imbalance) relative to saline treated animals as early as 4 days after dosing. Histopathologic changes will be assessed and scored as follows:• Tendon Damage Score: scored from 0-5, wherein 0 = normal / no damage; 5 = severe focal or multifocal areas of damage (affecting >75% of the tendon area;• Tendon Inflammation Score: scored from 0-5, where 0=normal, 5=severe diffuse infiltration of Inflammatory cells In tendon or tendon sheath, with severe extension into peripheral adipose tissue (affects >75% of adipose tissue, dense infiltration;• Tendon Repair Score: scored from 0-6, with 0 meaning no repair / proliferative tissue due to no damage ever present, l=minimal repair with minimal collage fibril deposition and vascularization, and 6=normal tissue as a result of total or near total repair with well aligned connective tissue bundles.

[0134] Animals that will be treated with a decellularlzed placenta derived extracellular matrix of the instant disclosure relative to saline treatment, are expected to show significantly reduced tendon damage and inflammation with a substantial level of tendon repair.References1. Lawrence RC, Felson DT, Helmick CG, Arnold LM, Choi H, Deyo RA, et al. Estimates of the prevalence of arthritis and other rheumatic conditions in the United States. Part II. Arthritis Rheum 2008; 58(1): 26-35.2. Hunter DJ. Osteoarthritis. Best Pract Res Clin Rheumatol 2011; 25(6): 801-14.3. Timothy J. Keane, Ricardo Londono, Neill J. Turner, and Stephen F. Badylak. "Consequences of ineffective decellularization of biologic scaffolds on the host response," Biomaterials 2012:33:1771-1781.4. Badylak SF, Valentin JE, Ravindra AK, McCabe GP, Stewart-Akers AM. Macrophage phenotype as a determinant of biologic scaffold remodeling. Tissue Engineering Part A 2008; 14(11): 1835e42.5. Seif-Naraghi SB, et al. Safety and Efficacy of an Injectable Extracellular Matrix Hydrogel for Treating Myocardial Infarction. Sci Trans) Med 2013; 5:173.6. Gilbert TW, Freund J, and Badylak SF. Quantification of DNA in Biologic Scaffold Materials. J Surg Res 2009; 152(1): 135-139.7. Yasuda K, Ogawa Y, Yamane I, Nishlkawa M, and Takakura Y. Macrophage activation by a DNA / cationic liposome complex requires endosomal acidification and TLR9-dependent and -independent pathways. Journal of Leukocyte Biology 2006; 77: 71-79.8. ASTM -F3354 -19 Standard Guide for Evaluating Extracellular Matrix Decellularization Processes.9. Hu Q, Ecker M. Overview of MMP-13 as a Promising Target for the Treatment of Osteoarthritis. Int J Mol Sci 202;22(4):1742.10. Nl F, Zhang Y, Peng X. et al. Correlation between osteoarthritis and monocyte chemotactic protein-1 expression: a meta-analysis. J Orthop Surg Res 2020; 15: 51611. Verrico CD et al., A randomized, double-blind, placebo-controlled study of dally cannabidiol for the treatment of canine osteoarthritis pain. Pain. 2020; 161(9): 2191- 2202.12. Hlelm-Bjorkman AK, Rita H, Tulamo RM. Psychometric testing of the Helsinki chronic pain index by completion of a questionnaire in Finnish by owners of dogs with chronic signs of pain caused by osteoarthritis. Am J Vet Res 2009; 70(6): 727-734.13. T. Pham et al., OMERACT-OARSI Initiative: Osteoarthritis Research Society International set of responder criteria for osteoarthritis clinical trials revisited. OsteoArthritis and Cartilage (2004) 12, 389-39914. Cho Y. et al., Disease-modifying therapeutic strategies in osteoarthritis: current status and future directions. Exp Mol Med. 2021 Nov; 53(11): 1689-1696.15. Chevalier X, et al. Intraarticular injection of anakinra in osteoarthritis of the knee: a multicenter, randomized, double-blind, placebo-controlled study. Arthritis Rheumatol. 2009;61:344-352.

[0135] The present disclosure is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the disclosure in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A decellularized placenta derived extracellular matrix substantially free of residual material.

2. The decellularized extracellular matrix of Claim 1, wherein the residual material is not quantifiable as being not detectable or present at an amount less than the limit of detection.

3. The decellularized extracellular matrix of either of Claims 1 or 2, wherein the residual material comprises: (1) cells, (2) cell debris, (3) nucleic acid molecules, (4) nucleotides, (5) growth factors, (6) cytokines, (7) chemokines, (8) hormones, or (9) any combination of (1)- (8).

4. The decellularized extracellular matrix of any one of Claims 1-3, wherein the residual material comprises chemokines and cytokines that are not detectable in the decellularized extracellular matrix or are present In the decellularized extracellular matrix at an amount less than the limit of detection.

5. The decellularized extracellular matrix of any one of Claims 1-3, wherein the following chemokines, cytokines and growth factors are not detectable in the decellularized extracellular matrix or are present at an amount less than the limit of detection:(a) Platelet Derived Growth Factor-AA (PDGF-AA), less than the limit of detection;(b) platelet Derived Growth Factor-BB (PDGF-BB), not detected;(c) Basic Fibroblast Growth Factor bets (Beta FGF), not detected;(d) Epidermal Growth Factor (EGF), not detected;(e) Transforming Growth Factor beta 1 (TGF-betal), not detected;(f) Hepatocyte Growth Factor (HGF), not detected;(g) Vascular Endothelial Growth Factor (VEGF), not detected;(h) Insulin Like Growth Factor-1 (IGF-1), not detected;(I) Growth and Differentiation Factor-15, less than the limit of detection;(j) Intercellular Adhesion Molecule-one (ICAM-1), not detected;(k) interleukin-10 (IL- 10), not detected;(I) Interleukin- 1 Receptor Antagonist (IL1-RA), not detected;(m) Interleukin-6 (IL-6), not detected;(n) Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF), not detected;(o) Interluekin-12p40 subunit (IL-12p40), not detected;(p) Interleukin-1 beta (IL-lbeta), not detected;(q) Interleukin-5 (IL-5), not detected;(r) Interleukin-8 (IL-8), not detected;(s) Interleukin-13 (IL-13), not detected;(t) Interieukin-15 (IL-15), not detected;(u) Monocyte Chemoattractant Protein-1 (MCP-1), not detected;(v) macrophage Inflammatory Protein- 1 alpha (MIP-lalpha), not detected;(w) Regulated on Activation, Normal T Expressed and Secreted (RANTES), less than the limit of detection;(x) Tumor Necrosis Factor alpha (TNFalpha), not detected;(y) Human Chorionic Gonadotropin (hCG), not detected;(z) Follicle Stimulating Hormone (FSH), not detected;(aa) Lactogen, not detected;(bb) Estrogen, less than the limit of detection;(cc) Progesterone, not detected; and(dd) Oxytocin, less than the limit of detection.

6. The decellularized extracellular matrix of any one of Claims 1-4, wherein decellularized extracellular matrix Is in the form of particles.

7. The decellularized extracellular matrix of Claim 6, wherein the particles have a size of:(i) about 5 microns to about 2000 microns;(H) about 5 to about 300 microns;(iii) about 10 to about 250 microns;(iv) about 20 microns to about 200 microns; or(v) any combination of (i) - (Iv).

8. The decellularized extracellular matrix of any one of Claims 1-7, wherein the decellularized extracellular matrix is sterile.

9. A pharmaceutical composition comprising the decellularized placental derived extracellular matrix of any one of Claims 1-8 and a pharmaceutically acceptable carrier.

10. The use of a decellularized placenta derived extracellular matrix of any one of Claims 1-8 In a pharmaceutical composition for the treatment of a musculoskeletal or orthopedic disease or condition In a subject in need thereof.

11. The use of a decellularized placenta derived extracellular matrix Claim 10, wherein the musculoskeletal or orthopedic disease or condition is selected from the group consisting of osteoarthritis, degenerative disc disease, cartilage deficits or damage, soft tissue injury, physical trauma, plantar fasciitis, tendonitis or orthopedic surgery.

12. The use of a decellularized placenta derived extracellular matrix of either of Claims 10 or 11, wherein the musculoskeletal or orthopedic disease or condition is osteoarthritis.

13. The use of the pharmaceutical composition of Claim 9 for the treatment of a musculoskeletal disease or orthopedic condition in a subject in need thereof.

14. The use of the pharmaceutical composition of Claim 13, wherein the musculoskeletal or orthopedic disease or condition Is selected from the group consisting of osteoarthritis, degenerative disc disease, cartilage deficits or damage, soft tissue injury, physical trauma, plantar fasciitis, tendonitis, orthopedic surgery, OCD, or PTOA.

15. The use the pharmaceutical composition of either of Claims 13 or 14, wherein the musculoskeletal or orthopedic disease or condition is osteoarthritis.

16. A method of treating a musculoskeletal or orthopedic disease or condition in a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of a decellularized placenta derived extracellular matrix of any one of Claims 1-8, or a pharmaceutical composition Claim 9.

17. The method of Claim 16, wherein administrating the therapeutically effective amount comprises Injecting the decellularized placenta derived extracellular matrix or pharmaceutical composition into a desired site in the subject.

18. The method of any one of Claims 16-17, wherein administering the therapeutically effective amount of the decellularized placenta derived extracellular matrix or pharmaceutical composition occurs 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more times.

19. The method of Claim 18, wherein the time between administering more than one therapeutically effective amount of the decellularized extracellular matrix or the time between administering more than one pharmaceutical composition Is about 1 day, about 1 month, about 3 months, about 6 months, about 12 months or more.

20. The method of any one of Claims 16-19, further comprising the step of administering to the subject a therapeutically effective amount of a therapeutic agent.

21. The method of Claim 20, wherein the therapeutic agent (a) an NSAID, (b) glucosamine and chondroitin sulphate, (c) DIACERIN, (d) a hyaluronic acid Injection, (e) a steroid , (f) an opioid, or (g) a biologic, (e.g., cells / cell conditioned media, exosomes, plasma, nucleic acid molecules (such as DNA, mRNA, siRNA, etc.), an antibody (fully human, humanized, fragment (fab, f(ab')z, etc.), or an ScFv)), (h) an antibiotic, (I) an anti-fungal agent or any combination of (a) - (I)22. The method of either of Claims 20 or 21, wherein the decellularized extracellular matrix and the therapeutic agent are administered simultaneously, concomitantly, or serially.

23. A kit comprising one or more doses of a pharmaceutical composition a therapeutically effective amount of a decellularized placenta derived extracellular matrix of any of Claims 1-8 a pharmaceutically acceptable excipient.

24. The kit of Claim 23, further comprising an appropriately sized syringe for delivery of the dose(s) of the pharmaceutical composition.

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