Human umbilical cord compositions for use in the treatment of Peyronie's disease

Abstract

A processed human umbilical cord composition for treating Peyronie's disease by intracorporeal injection of an effective amount into a subject in need thereof, the composition comprising an aqueous human umbilical cord filtrate containing endogenous hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycans (sGAGs), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), transthyretin, tissue inhibitor of metalloproteinase 1 (TIMP-1), aggrecan, or a combination thereof, in an amount effective to reduce the size of Peyronie's disease plaques.

Description

[Technical Field]

[0001] The present invention relates generally to the field of umbilical cord-derived compositions, and more particularly to non-immunogenic compositions derived from human umbilical cord used in the treatment of Peyronie's disease. [Background technology]

[0002] The human penis is the external segment of the male reproductive system, consisting of three chambers of spongy tissue surrounded by the tunica albuginea.

[0003] During sexual arousal, the penile tissues fill with blood perfused from elsewhere in the body, resulting in a physiological response that increases penile rigidity. Penile disorders, including erectile dysfunction (ED) and Peyronie's disease (PD), affect and / or interfere with this physiological response. ED is generally defined as the inability to achieve and / or maintain an erection for sexual intercourse. ED is common, affecting approximately half of American men over the age of 40. Causes of ED include vascular disorders, neurological disorders, psychological stress, penile injury, chronic disease, and unhealthy lifestyles.

[0004] In addition to the causes of ED mentioned above, ED can also be due to Peyronie's disease, which is the formation of plaques (i.e., scar tissue) inside the tunica albuginea of ​​the penis. When the plaques are numerous and / or cover a large surface area within the penis, they can cause painful, curved erections, which often lead to ED, making sexual intercourse very painful, difficult, and / or impossible.

[0005] In the United States, approximately 1 in 100 men over the age of 18 have been diagnosed with Peyronie's disease, and the likelihood of developing Peyronie's disease increases with age. A significant number of men with Peyronie's disease develop erectile dysfunction. The exact cause of PD is unknown, but it is thought to be due to injury.

[0006] Scarring (plaque) can develop in the elastic layer surrounding the erectile tissue, reducing the elasticity of the penis in the affected area. Symptoms of PD include scar tissue felt under the skin of the penis, significant bending or curvature of the penis, difficulty achieving or maintaining an erection, penile pain, and penile shortening. Surgical treatments include plaque removal, shortening of the tissue opposite the plaque to even out the curvature, and penile implantation. These procedures carry the risk of loss of erectile function or permanent shortening of the penis.

[0007] Non-surgical treatments involve the injection of various compositions to soften the plaque and correct the curvature. These compositions currently include XIAFLEX®, which is made from bacterial collagenase to dissolve the plaque collagen. XIAFLEX® carries the risk of corporal lacerations, penile hematoma, erectile dysfunction, and hematuria. Verapamil is a calcium channel inhibitor used to halt the progression of plaque formation. Side effects include dizziness, weakness, nausea, and sweating. Verapamil also has a long list of drug interactions. Steroid injections have been used for many years. Many patients find the injections painful. Side effects of corticosteroids include a weakened immune system, infection at the injection site, reduced penile tissue size, thinning of the skin, and complications with future surgical procedures. Radiation therapy is ineffective unless administered early in PD and is used to halt its progression. Studies of this treatment lack robust control groups, and it exposes reproductive tissue to radiation, making many patients hesitant to try it. Summary of the Invention

[0008] In light of the above-described problems with current Peyronie's disease treatments, there is a need for a penile disorder (e.g., Peyronie's disease) treatment that resolves penile dysfunction while minimizing side effects. The compositions and methods disclosed herein achieve this goal by using a processed human umbilical cord composition as an injectable treatment for Peyronie's disease in a subject in need of treatment (e.g., a human subject in need of treatment). Furthermore, it is an object of the present invention to provide human umbilical cord-derived compositions that mimic, contain, and / or maintain a cellular and / or extracellular profile similar to the endogenous profile of human umbilical cord (e.g., in vivo), particularly in comparison to the various umbilical cord isolates described above. These compositions are prepared using fresh human umbilical cord (collected and processed within 48-72 hours of removal from the human subject) and, unlike prior art compositions, advantageously do not undergo biochemical and / or enzymatic digestion, resulting in compositions that contain and / or preserve a significant portion of the cellular and / or extracellular profile (of human umbilical cord in vivo).

[0009] In certain embodiments, a processed human umbilical cord composition is disclosed for treating Peyronie's disease by injecting an effective amount of the composition into a subject in need thereof, the composition comprising an aqueous human umbilical cord filtrate having endogenous hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycans (sGAGs), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), transthyretin, tissue inhibitor of metalloproteinase 1 (TIMP-1), aggrecan, or a combination thereof, in an amount effective to reduce the size of Peyronie's disease plaques. In certain embodiments, the aqueous human umbilical cord filtrate is a solution that does not exhibit settling, separation, and / or precipitation upon storage for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or more. In certain embodiments, exogenous enzymes are not introduced into the composition during its preparation, thereby avoiding exogenous enzymatic degradation / digestion.

[0010] In certain embodiments, the aqueous human umbilical cord filtrate comprises cellular Wharton's jelly, hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycans (sGAGs), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), transthyretin, tissue inhibitor of metalloproteinase 1 (TIMP-1), aggrecan, or a combination thereof. In certain embodiments, the aqueous human umbilical cord filtrate comprises cellular Wharton's jelly, a 3.0 x 10 6 pg / mL ~ 4.0 × 10 8 pg / mL, more preferably 4.5 × 10 6 pg / mL ~ 3.15 × 10 8 pg / mL hyaluronic acid (HA), with a concentration of 2.0 × 10 3 pg / mL ~ 6.0 × 10 6 pg / mL, more preferably 2.25 × 10 3 pg / mL ~ 4.7 × 10 6 pg / mL insulin growth factor binding protein-1 (IGFBP-1), with a concentration of 2.0 × 10 7 pg / mL ~ 3.0 × 10 9 pg / mL, more preferably 2.8 × 10 7 pg / mL ~ 2.3 × 10 9 pg / mL sulfated glycosaminoglycans (sGAGs), with a concentration of 2.0 × 10 2 pg / mL ~ 4.0 × 10 4 pg / mL, more preferably 3.75 × 10 2 pg / mL ~ 3.0 × 10 4 pg / mL interleukin-1 receptor antagonist (IL-1ra), with a concentration of 6.0 × 10 2 pg / mL ~ 6.0 × 10 5 pg / mL, more preferably 7.5 × 10 2 pg / mL ~ 5.25 × 10 5 pg / mL transthyretin, concentration 3.0 × 10 3 pg / mL ~ 8.0 × 10 5 pg / mL, more preferably 4.0 × 10 3 pg / mL ~ 7.75 × 10 5pg / mL tissue inhibitor of metalloproteinase 1 (TIMP-1), with concentrations ranging from 0 pg / mL to 8.0 × 10 4 pg / mL, more preferably 1×10 1 pg / mL ~ 7.5 × 10 4 pg / mL aggrecan, or a combination of these.

[0011] In certain embodiments, the aqueous human umbilical cord filtrate further comprises an isotonic solution, which may be included in the disclosed compositions as a carrier and / or diluent.

[0012] In certain embodiments, the isotonic solution is phosphate buffered saline (1×PBS), lactated Ringer's (pH 6.5, 6 g / L NaCl, 3.1 g / L sodium lactate, 0.3 g / L KCl, and 0.2 g / L CaCl), isotonic saline (0.9 wt% NaCl), Plasma-Lyte® (pH 7.4, 5.26 g / L NaCl, 0.37 g / L KCl, 0.30 g / L magnesium chloride hexahydrate, 3.68 g / L sodium acetate trihydrate, 5.02 g / L sodium gluconate).

[0013] In certain embodiments, the aqueous human umbilical cord filtrate has particles of human umbilical cord tissue therein that are less than 100 μm in diameter, preferably less than 50 μm in diameter, more preferably less than 25 μm in diameter, and even more preferably less than 10 μm in diameter.

[0014] In certain embodiments, the aqueous human umbilical cord filtrate is sterile.

[0015] In certain aspects, the aqueous human umbilical cord filtrate is non-immunogenic.

[0016] Also disclosed are kits containing processed human umbilical cord compositions for treating Peyronie's disease by internal injection into a subject in need of treatment. In certain embodiments, the compositions are pre-packaged in vials (sterile vials), ampoules (sterile ampoules), or pre-filled syringes (sterile pre-filled syringes) in predetermined amounts and contain, for example, endogenous hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycans (sGAGs), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), transthyretin, tissue inhibitor of metalloproteinase 1 (TIMP-1), aggrecan, or a combination thereof, at predetermined concentrations to reduce the size of Peyronie's disease plaques. In certain embodiments, the predetermined volume is 0.25 mL to 20.0 mL, preferably 0.5 mL to 10.0 mL, and most preferably 1.0 to 5.0 mL, and may be used for 1 to 10 treatments (injections) with the composition for treating Peyronie's disease plaque, or as determined by a physician. Also, in certain embodiments, the aqueous human umbilical cord filtrate is a cellular Wharton's jelly, having a concentration of 3.0 x 10 6 pg / mL ~ 4.0 × 10 8 pg / mL, more preferably 4.5 × 10 6 pg / mL ~ 3.15 × 10 8 pg / mL hyaluronan (HA), with a concentration of 2.0 × 10 3 pg / mL ~ 6.0 × 10 6 pg / mL, more preferably 2.25 × 10 3 pg / mL ~ 4.7 × 10 6 pg / mL insulin growth factor binding protein-1 (IGFBP-1), with a concentration of 2.0 × 10 7 pg / mL ~ 3.0 × 10 9 pg / mL, more preferably 2.8 × 10 7 pg / mL ~ 2.3 × 10 9 pg / mL sulfated glycosaminoglycans (sGAGs), with a concentration of 2.0 × 10 2 pg / mL ~ 4.0 × 10 4 pg / mL, more preferably 3.75 × 10 2pg / mL ~ 3.0 × 10 4 pg / mL interleukin-1 receptor antagonist (IL-1ra), with a concentration of 6.0 × 10 2 pg / mL ~ 6.0 × 10 5 pg / mL, more preferably 7.5 × 10 2 pg / mL ~ 5.25 × 10 5 pg / mL transthyretin, concentration 3.0 × 10 3 pg / mL ~ 8.0 × 10 5 pg / mL, more preferably 4.0 × 10 3 pg / mL ~ 7.75 × 10 5 pg / mL tissue inhibitor of metalloproteinase 1 (TIMP-1), with concentrations ranging from 0 pg / mL to 8.0 × 10 4 pg / mL, more preferably 1×10 1 pg / mL ~ 7.5 × 10 4 pg / mL aggrecan, or a combination of these.

[0017] In certain aspects, the aqueous human umbilical cord filtrate is preferably not subjected to exogenous enzymatic digestion.

[0018] In certain embodiments, the aqueous human umbilical cord filtrate of the kit further comprises an isotonic solution.

[0019] In certain embodiments, the isotonic solutions of the kit are phosphate buffered saline (1×PBS), lactated Ringer's (pH 6.5, 6 g / L NaCl, 3.1 g / L sodium lactate, 0.3 g / L KCl, and 0.2 g / L CaCl), isotonic saline (0.9 wt% NaCl), Plasma-Lyte® (pH 7.4, 5.26 g / L NaCl, 0.37 g / L KCl, 0.30 g / L magnesium chloride hexahydrate, 3.68 g / L sodium acetate trihydrate, and 5.02 g / L sodium gluconate).

[0020] In certain embodiments, the aqueous human umbilical cord filtrate of the kit comprises particles of human umbilical cord tissue therein having a diameter of less than 100 μm, preferably less than 50 μm, more preferably less than 25 μm, and even more preferably less than 10 μm. In certain embodiments, the aqueous human umbilical cord filtrate is a solution that does not exhibit settling, separation, and / or precipitation upon storage for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or more.

[0021] In certain embodiments, the aqueous human umbilical cord filtrate of the kit is sterile.

[0022] In certain embodiments, the aqueous human umbilical cord filtrate of the kit is non-immunogenic.

[0023] In certain embodiments, methods for reducing the size of Peyronie's disease plaques in a human subject in need of treatment are also disclosed, the methods comprising contacting the Peyronie's disease plaques with a predetermined amount of the composition. In certain embodiments, the compositions or kits used in accordance with the present invention are used to reduce the size of Peyronie's disease plaques in a human subject in need of treatment, the treatment comprising the step (a) of contacting the Peyronie's disease plaques with a predetermined amount of the composition. In certain embodiments, the contacting step comprises injecting a predetermined amount of the composition containing a predetermined concentration of, for example, endogenous hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycan (sGAG), exosome, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), transthyretin, tissue inhibitor of metalloproteinase 1 (TIMP-1), aggrecan, or a combination thereof, into a subject (human subject) in need of treatment at a predetermined frequency to reduce plaque size. In certain embodiments, the above steps are repeated at a predetermined frequency to further reduce plaque size and / or to continue reducing plaque in the subject.

[0024] In another aspect, a method for producing a processed human umbilical cord composition for treating Peyronie's disease by internal injection into a subject in need thereof is also disclosed, the method comprising: (a) providing a human umbilical cord; (b) washing the human umbilical cord with an isotonic solution; (c) pulverizing the washed human umbilical cord of step (b) to produce pulverized human umbilical cord tissue; (d) separating the pulverized human umbilical cord tissue of step (c) into a solid residue and an aqueous human umbilical cord supernatant, optionally further processing the solid residue of step (d) into a micronized human umbilical cord composition; and (e) filtering and diluting the aqueous human umbilical cord supernatant to produce an aqueous human umbilical cord filtrate, wherein the aqueous human umbilical cord filtrate has particles of pulverized human umbilical cord tissue, if any, less than 100 μm in diameter therein. In this aspect, none of steps (a)-(e) includes the introduction of an exogenous enzyme that results in exogenous enzymatic degradation / digestion.

[0025] In certain embodiments, the human umbilical cord of the present method is obtained from a subject, and steps (a)-(c) are then performed within 48-96 hours, more preferably within 48-72 hours, after birth and / or cesarean section and / or removal of the human umbilical cord.

[0026] In certain embodiments, the method further comprises removing a thrombus present within the human umbilical cord during steps (a)-(c).

[0027] In certain embodiments, step (b) is repeated 1 to 5 times by discarding the used isotonic solution, providing new isotonic solution, and re-washing the human umbilical cord with the new isotonic solution.

[0028] In one embodiment, in step (a), a human umbilical cord weighing 15 to 80 grams is provided, after which steps (b) through (f) are performed.

[0029] In certain embodiments, the isotonic solution is phosphate buffered saline (or one of lactated Ringer's (pH 6.5, 6 g / L NaCl, 3.1 g / L sodium lactate, 0.3 g / L KCl, 0.2 g / L CaCl), isotonic saline (0.9 wt% NaCl), Plasma-Lyte® (pH 7.4, 5.26 g / L NaCl, 0.37 g / L KCl, 0.30 g / L magnesium chloride hexahydrate, 3.68 g / L sodium acetate trihydrate, 5.02 g / L sodium gluconate)) and is provided in a volume of 300 mL to 1000 mL per wash step (b).

[0030] In certain embodiments, step (c) uses a comminution tool configured to crush and / or pulverize the washed human umbilical cord at a speed of 40 to 200 revolutions per minute (RPM) until the cord is completely crushed.

[0031] In one aspect, prior to step (d), the method includes contacting the comminuted umbilical cord tissue produced in step (c) with a filter and then filtering the comminuted umbilical cord tissue to produce a solid residue retained on the filter and an aqueous human umbilical cord supernatant of step (d) that passes through the filter. In this aspect, the filter has pores of 100 μm to 200 μm.

[0032] In certain embodiments, there is a step of further processing the solid residue, which is a crushing process, a freeze-drying process, or a dehydration process to produce the micronized human umbilical cord composition of step (e), wherein the micronized human umbilical cord composition has a particle size of greater than 1 μm to less than 300 μm, more preferably greater than 1 μm to 100 μm, even more preferably greater than 1 μm to 50 μm, and even more preferably greater than 1 μm to 25 μm. Further, the particles of the micronized human umbilical cord are polydisperse.

[0033] In certain aspects, there is a step of further processing the solid residue, which is a freeze-grinding process in which the solid residue of step (d) is placed in a liquid nitrogen-cooled freeze-grinding chamber and ground therein to produce a micronized human umbilical cord composition, wherein the micronized human umbilical cord composition has a particle size of greater than 1 μm to less than 300 μm, more preferably greater than 1 μm to 100 μm, even more preferably greater than 1 μm to 50 μm, and even more preferably greater than 1 μm to 25 μm. Further, the particles of the micronized human umbilical cord are polydisperse.

[0034] In certain embodiments, step (e) comprises multiple filtration steps, including (i) filtering the aqueous human umbilical cord supernatant through a first filter having pores of 30 μm to 40 μm to produce a second human umbilical cord supernatant, (ii) filtering the second human umbilical cord supernatant through a second filter having pores of 12.5 μm to 25 μm to produce a third human umbilical cord supernatant, and (iii) filtering the third human umbilical cord supernatant through a third filter having pores of 4 μm to 10 μm to produce an aqueous human umbilical cord filtrate. Thus, in certain embodiments, the second human umbilical cord supernatant can contain human umbilical cord tissue particles of less than 40 μm, the third human umbilical cord supernatant can contain human umbilical cord tissue particles of less than 25 μm, and the aqueous human umbilical cord filtrate can contain human umbilical cord tissue particles of less than 10 μm. In certain embodiments, the aqueous human umbilical cord filtrate is a solution that does not exhibit settling, separation, and / or precipitation upon storage for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or more.

[0035] In certain embodiments, the micronized human umbilical cord composition of the present method comprises collagen, fibronectin, IGFBP-1, sGAG, hyaluronan, or any combination thereof.

[0036] In certain embodiments, the micronized human umbilical cord of the method compositions is dry micronized human umbilical cord tissue having a particle size of greater than 1 μm to 300 μm and comprising collagen, fibronectin, IGFBP-1, sGAG, hyaluronan, or any combination thereof. In certain embodiments, the micronized human umbilical cord of the method compositions is dry micronized human umbilical cord tissue having a particle size of greater than 1 μm to 300 μm and comprising collagen, fibronectin, IGFBP-1 at a concentration of 1500 pg / mL to 9000 pg / mL, hyaluronan at a concentration of 0.1×10 6 pg / ml ~ 3.0 × 10 7 pg / mL sGAG, concentration 1.5 × 10 9 Particles / mL~4.0×10 9 and exosomes having a particle size of 50 nm to 200 nm, or any combination thereof.

[0037] In certain embodiments, the aqueous human umbilical cord filtrate of the present methods comprises cellular Wharton's jelly, hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycans (sGAGs), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), transthyretin, tissue inhibitor of metalloproteinase 1 (TIMP-1), aggrecan, or a combination thereof. In certain embodiments, the aqueous human umbilical cord filtrate comprises cellular Wharton's jelly, a filtrate having a concentration of 3.0 x 10 6 pg / mL ~ 4.0 × 10 8 pg / mL, more preferably 4.5 × 10 6 pg / mL ~ 3.15 × 10 8 pg / mL hyaluronan (HA), with a concentration of 2.0 × 10 3 pg / mL ~ 6.0 × 10 6 pg / mL, more preferably 2.25 × 10 3 pg / mL ~ 4.7 × 10 6 pg / mL insulin growth factor binding protein-1 (IGFBP-1), with a concentration of 2.0 × 10 7 pg / mL ~ 3.0 × 10 9 pg / mL, more preferably 2.8 × 10 7pg / mL ~ 2.3 × 10 9 pg / mL sulfated glycosaminoglycans (sGAGs), with a concentration of 2.0 × 10 2 pg / mL ~ 4.0 × 10 4 pg / mL, more preferably 3.75 × 10 2 pg / mL ~ 3.0 × 10 4 pg / mL interleukin-1 receptor antagonist (IL-1ra), with a concentration of 6.0 × 10 2 pg / mL ~ 6.0 × 10 5 pg / mL, more preferably 7.5 × 10 2 pg / mL ~ 5.25 × 10 5 pg / mL transthyretin, concentration 3.0 × 10 3 pg / mL ~ 8.0 × 10 5 pg / mL, more preferably 4.0 × 10 3 pg / mL ~ 7.75 × 10 5 pg / mL tissue inhibitor of metalloproteinase 1 (TIMP-1), with concentrations ranging from 0 pg / mL to 8.0 × 10 4 pg / mL, more preferably 1×10 1 pg / mL ~ 7.5 × 10 4 pg / mL aggrecan, or a combination of these.

[0038] In certain embodiments, both the micronized human umbilical cord composition and the aqueous human umbilical cord filtrate of the present methods are sterile.

[0039] In certain embodiments, both the micronized human umbilical cord composition and the aqueous human umbilical cord filtrate of the present methods are non-immunogenic.

[0040] In certain embodiments, the method further comprises (f) sealing the aqueous human umbilical cord filtrate composition in a sterile container for subsequent use. For example, the aqueous human umbilical cord filtrate may be pre-filled in a vial (sterile vial), ampoule (sterile ampoule), or pre-filled syringe (sterile pre-filled syringe) at a predetermined volume, containing, for example, endogenous hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycans (sGAGs), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), transthyretin, tissue inhibitor of metalloproteinase 1 (TIMP-1), aggrecan, or a combination thereof at a predetermined concentration to reduce the size of Peyronie's disease plaques. In certain embodiments, the predetermined volume is between 0.25 mL and 20.0 mL, preferably between 0.5 mL and 10.0 mL, and most preferably between 1.0 and 5.0 mL, and may be used for 1 to 10 treatments (injections) with the composition to treat Peyronie's disease plaques, or as determined by a physician.

[0041] Embodiments of the invention may include one or more, or any combination of, the above-described features and configurations.

[0042] Other features, aspects, and advantages of the present invention are set forth in the following detailed description, and in part will become readily apparent to those skilled in the art upon reading the description, or may be learned by practicing the invention as described herein. It is to be understood that both the foregoing general description and the following detailed description, present various embodiments of the invention, and are intended to provide an overview or framework for understanding the nature and characteristics of the invention as claimed. The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification. [Brief explanation of the drawings]

[0043] These and other features, aspects and advantages of the present invention will be better understood by reading the following detailed description of the invention when taken in conjunction with the accompanying drawings.

[0044] [Figure 1] FIG. 1 is a schematic representation of the steps involved in producing the disclosed compositions. [Figure 2] Figure 2 is a graph showing the concentration profiles of hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycans (sGAGs), interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), transthyretin, tissue inhibitor of metalloproteinases 1 (TIMP-1), and aggrecan in aqueous human umbilical cord filtrate. DETAILED DESCRIPTION OF THE INVENTION

[0045] The present invention will now be described more fully with reference to the accompanying drawings, in which exemplary embodiments of the invention are shown. The present invention may, however, be embodied in many different forms and should not be construed as limited to the representative embodiments set forth herein. The exemplary embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention and enable those skilled in the art to make, use, and practice the invention. Like reference numerals refer to like elements in the various drawings. Furthermore, in this specification and in the claims that follow, reference will be made to a number of terms that will be defined to have the following meanings. It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural unless the context clearly dictates otherwise.

[0046] Concentrations, amounts, and other numerical data may be expressed or presented in range format herein. It should be understood that such range format is used merely for convenience and brevity and, therefore, should be interpreted flexibly to include not only the numerical values ​​explicitly recited as range limits, but also all individual numerical values ​​or subranges subsumed within that range, as if those numerical values ​​and subranges were explicitly recited. As an example, a numerical range of "about 1 to 5" should be interpreted to include not only the numerical value explicitly recited as "about 1 to 5," but also each individual numerical value and subrange within that range. Thus, included within this numerical range are individual values ​​such as 2, 3, and 4, subranges such as 1 to 3, 2 to 4, and 3 to 5, as well as the individual values ​​1, 2, 3, 4, and 5. The same principle applies to ranges reciting only a single numerical value as a minimum or maximum value. Furthermore, such interpretation should be applied regardless of the breadth of the range or the properties described.

[0047] The compositions and methods described herein can comprise, consist of, or consist essentially of the essential elements and limitations described herein, as well as any other or optional ingredients, components, or limitations described herein. Human umbilical cord composition

[0048] Disclosed herein are compositions derived from human umbilical cord that mimic, contain, and / or preserve a cellular and / or extracellular profile resembling the endogenous profile of human umbilical cord in vivo, particularly when compared to various conventional umbilical cord-isolated compositions. The disclosed compositions are prepared using fresh human umbilical cord (harvested and processed within 48-72 hours of removal from the human subject) and, unlike prior art compositions, advantageously do not undergo biochemical and / or enzymatic digestion, resulting in compositions that contain and / or preserve a significant portion of the cellular and / or extracellular profile (compared to the endogenous profile of human umbilical cord in vivo). Furthermore, these compositions may be used to treat Peyronie's disease.

[0049] In certain embodiments, a processed human umbilical cord composition is disclosed for use in treating Peyronie's disease by injecting an effective amount of the composition into a subject in need thereof, the composition comprising an aqueous human umbilical cord filtrate having endogenous hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycans (sGAGs), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), transthyretin, tissue inhibitor of metalloproteinase 1 (TIMP-1), aggrecan, or a combination thereof, in an amount effective to reduce the size of Peyronie's disease plaques. In certain embodiments, the aqueous human umbilical cord filtrate is a solution that does not exhibit settling, separation, and / or precipitation upon storage for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or more. In certain embodiments, no exogenous enzymes are introduced during the preparation of the compositions, thereby avoiding exogenous enzymatic degradation / digestion. That is, the compositions disclosed herein are prepared such that the aqueous human umbilical cord filtrate is not subjected to exogenous enzymatic digestion. The aqueous human umbilical cord filtrate and / or processed human umbilical cord compositions disclosed herein do not contain particulates greater than 100 μm in diameter. The aqueous human umbilical cord filtrate and / or processed human umbilical cord compositions contain cellular Wharton's jelly, a concentration of 3.0×10 6 pg / mL ~ 4.0 × 108 pg / mL, more preferably 4.5 × 10 6 pg / mL ~ 3.15 × 10 8 pg / mL hyaluronan (HA), with a concentration of 2.0 × 10 3 pg / mL ~ 6.0 × 10 6 pg / mL, more preferably 2.25 × 10 3 pg / mL ~ 4.7 × 10 6 pg / mL insulin growth factor binding protein-1 (IGFBP-1), with a concentration of 2.0 × 10 7 pg / mL ~ 3.0 × 10 9 pg / mL, more preferably 2.8 × 10 7 pg / mL ~ 2.3 × 10 9 pg / mL sulfated glycosaminoglycans (sGAGs), with a concentration of 2.0 × 10 2 pg / mL ~ 4.0 × 10 4 pg / mL, more preferably 3.75 × 10 2 pg / mL ~ 3.0 × 10 4 pg / mL interleukin-1 receptor antagonist (IL-1ra), with a concentration of 6.0 × 10 2 pg / mL ~ 6.0 × 10 5 pg / mL, more preferably 7.5 × 10 2 pg / mL ~ 5.25 × 10 5 pg / mL transthyretin, concentration 3.0 × 10 3 pg / mL ~ 8.0 × 10 5 pg / mL, more preferably 4.0 × 10 3 pg / mL ~ 7.75 × 10 5 pg / mL tissue inhibitor of metalloproteinase 1 (TIMP-1), with concentrations ranging from 0 pg / mL to 8.0 × 10 4 pg / mL, more preferably 1.0 × 10 1 pg / mL ~ 7.5 × 10 4The processed human umbilical cord composition comprises a predetermined volume of 0.25 mL to 20.0 mL, preferably 0.5 mL to 10.0 mL, and most preferably 1.0 mL to 5.0 mL, and may be used for 1 to 10 treatments (injections) of the composition to treat Peyronie's disease plaques, or as prescribed by a physician. In certain embodiments, the aqueous human umbilical cord filtrate further comprises an isotonic solution, which is phosphate-buffered saline. In certain embodiments, the aqueous human umbilical cord filtrate comprises particles of human umbilical cord tissue less than 100 μm in diameter. In certain embodiments, the processed human umbilical cord composition is non-immunogenic, sterile, or both. In certain embodiments, the composition or kit used in accordance with the present invention is used to reduce the size of Peyronie's disease plaques in a human subject in need of treatment, wherein the treatment comprises the step (a) of contacting the Peyronie's disease plaques with a predetermined amount of the composition.

[0050] Without being bound by theory, it is believed that the effective concentrations of hyaluronic acid, sulfated glycosaminoglycans, aggrecan, IGFBP-1, IL-1ra, tissue inhibitor of metalloproteinase 1 (TIMP-1), and transthyretin contained in the disclosed processed human umbilical cord compositions treat Peyronie's disease plaques by repairing the ECM, remodeling the matrix, and increasing hydration / water retention, which, combined with the anti-inflammatory properties of one or more of the above ingredients, leads to the reduction and / or elimination of Peyronie's disease plaques. Methods for Producing Human Umbilical Cord Compositions

[0051] Figure 1 shows a schematic diagram of the steps involved in producing the compositions disclosed herein, and as further shown in Figure 1, none of the steps involve the introduction of exogenous enzymes that result in exogenous enzymatic degradation / digestion. A method according to the present invention for producing the disclosed compositions comprises the following steps: (a) Preparing a human umbilical cord (b) Washing the human umbilical cord with an isotonic solution (c) pulverizing the washed human umbilical cord tissue of step (b) to produce pulverized human umbilical cord tissue. (d) separating the pulverized human umbilical cord tissue of step (c) into a solid residue and an aqueous human umbilical cord supernatant, and optionally further processing the solid residue of step (d) into a micronized human umbilical cord composition. (e) filtering the aqueous human umbilical cord supernatant to produce an aqueous human umbilical cord filtrate, the aqueous human umbilical cord filtrate having particles of comminuted human umbilical cord tissue less than 100 μm in diameter. None of steps (a)-(e) involve the introduction of exogenous enzymes that result in exogenous enzymatic degradation / digestion.

[0052] The disclosed method of producing the composition includes steps (a)-(f), which are described immediately below. Prior to step (a), the umbilical cord / cord tissue is tested for communicable diseases to ensure it is healthy / free of disease and to minimize risk during preparation and end use of the two-part coagulation composition. After the cord / cord tissue is determined to be healthy / free of disease, the umbilical cord is maintained at a temperature of 4°C-8°C before processing the cord in steps (a)-(f).

[0053] In one aspect, a method of producing the disclosed compositions according to the present invention comprises obtaining a human umbilical cord from a subject, and then performing steps (a)-(c) within 48-72 hours after birth and / or cesarean section.

[0054] As shown in Figure 1, step (a) involves preparing the human umbilical cord, preferably within 24 to 96 hours, more preferably within 24 to 72 hours, after removal from the human subject to ensure freshness of the human umbilical cord (i.e., the tissue and cells comprising the tissue) and minimize degradation (enzymatic degradation) due to necroptosis and / or apoptosis. In this step, the method described below is preferably performed on 15 to 80 gram portions of the human umbilical cord, more preferably 30 to 60 gram portions, with an average portion being approximately 40 grams, to ensure proper grinding / pulverization (subsequent step (c)).

[0055] Following step (a), step (b) is performed, which involves placing one or more umbilical cord portions (15-80 grams) into a container containing a predetermined volume (e.g., 300 mL-1000 mL, preferably 500 mL) of isotonic solution, preferably phosphate buffered saline (PBS) (i.e., 1x PBS) (or one of lactated Ringer's (pH 6.5, 6 g / L NaCl, 3.1 g / L sodium lactate, 0.3 g / L KCl, 0.2 g / L CaCl), isotonic saline (0.9 wt% NaCl), or Plasma-Lyte® (pH 7.4, 5.26 g / L NaCl, 0.37 g / L KCl, 0.30 g / L magnesium chloride hexahydrate, 3.68 g / L sodium acetate trihydrate, 5.02 g / L sodium gluconate)). Place the container on a stirring plate, insert a stirring rod into the container (containing the PBS and umbilical cord portion), and stir (medium to high speed) the umbilical cord portion in the isotonic solution for 5 to 15 minutes to wash the umbilical cord portion. Next, wash step (b) is repeated 1 to 5 times by transferring the "used" isotonic solution and pouring a predetermined amount (e.g., 300 mL to 1000 mL, preferably 500 mL) of new isotonic solution into the container. Either prior to step (a), during step (a), prior to step (b), or during step (b), the human umbilical cord and / or umbilical cord portion is further checked for the presence of blood clots and / or blood pools, and if present, these blood clots are removed using suction or other mechanical removal means (e.g., scalpel and forceps) to ensure that immunogenic components (e.g., hemoglobin and / or heme-related components from the umbilical cord donor) are minimized in the resulting micronized human umbilical cord composition and aqueous human umbilical cord filtrate. It is essential that a sterile and / or aseptic working environment is maintained during these washing steps to prevent and / or reduce the introduction of contaminants during the production of the disclosed compositions.

[0056] Following step (b), step (c) is performed, in which the washed umbilical cord / umbilical cord portion (encased in a predetermined volume (e.g., 75 mL to 125 mL, preferably 100 mL) of isotonic solution) is transferred to a grinding and / or pulverizing device, such as those disclosed in U.S. Patent No. 716,601 entitled "Tissue Mincing Tool" and / or U.S. Patent No. 8,967,512 entitled "Systems And Methods For Processing Cells," both of which are incorporated herein by reference. The washed umbilical cord / umbilical cord portion is then ground and / or pulverized by a grinding / pulverizing tool, the grinding / pulverizing tool head of which rotates at 40 to 200 revolutions per minute (RPM), until the umbilical cord is completely ground into pulverized human umbilical cord tissue. Maintaining a sterile working environment during this grinding / pulverizing step is essential to prevent and / or reduce the introduction of contaminants during the production of the micronized human umbilical cord composition and aqueous human umbilical cord filtrate. In certain embodiments, the grinding / comminuted tool may be directly connected to the apparatus (i.e., a closed system environment such as that disclosed in U.S. Patent No. 8,967,512) for further performance of steps (d)-(f) described below, and for maintaining sterility and / or minimizing the introduction of contaminants during the production of the micronized human umbilical cord composition and aqueous human umbilical cord filtrate. Alternatively, steps (d)-(f) may be performed in an open system / laboratory environment during the performance of each of the following steps.

[0057] After step (c) is completed, step (d) is performed, in which the pulverized / comminuted human umbilical cord tissue of step (c) is separated into a solid residue and an aqueous human umbilical cord supernatant. In one aspect, prior to step (d), the pulverized umbilical cord tissue produced in step (c) is contacted with a filter, and the pulverized umbilical cord tissue is then filtered to produce a solid residue remaining on the filter and an aqueous human umbilical cord supernatant of step (d) that passes through the filter. This initial separation step may be performed by a filtration process (either positive or negative pressure). For example, the pulverized / ground human umbilical cord tissue (from step (c) in a predetermined volume (e.g., 75 mL to 125 mL, preferably 100 mL) of isotonic solution) may be placed directly on a filter having the desired pore size (e.g., 200 μm, 150 μm, or 100 μm, such as a qualitative or quantitative grade mesh filter or net filter), after which force (either positive or negative pressure) may or may not be applied so that the solid residue (solids greater than 200 μm, 150 μm, or 100 μm in size) remains on the filter, while the aqueous human umbilical cord supernatant (having solids less than 200 μm, 150 μm, or 100 μm in size) passes through the filter. The filtration step generally takes between 15 seconds and 2 minutes. Again, maintaining a sterile and / or aseptic working environment is essential to prevent and / or reduce the introduction of contaminants during step (d).

[0058] After step (d), an optional step is carried out in which the solid residue of step (d) is further processed to form a micronized human umbilical cord composition by subjecting the solid residue to a crushing process, a freeze-drying (freeze-grinding) process, and / or a dehydration (lyophilization) process configured to obtain particles having a size of greater than 1 μm to 300 μm, preferably greater than 1 μm to 100 μm, more preferably greater than 1 μm to 50 μm, and even more preferably greater than 1 μm to 25 μm (polydisperse particles). In certain aspects, this optional step is a freeze-grinding process (e.g., as described in US20160287749, US20170203004, and US10105398, which are incorporated by reference in their entireties) in which the solid residue from step (d) is placed in a liquid nitrogen-cooled freeze-grinding chamber and ground therein to produce a micronized human umbilical cord composition having a particle size of greater than 1 μm to less than 300 μm, preferably greater than 1 μm to 100 μm, more preferably greater than 1 μm to 50 μm, and even more preferably greater than 1 μm to 25 μm. The micronized human umbilical cord composition comprises collagen, fibronectin, hyaluronan, elastin, or any combination thereof. In one embodiment, the micronized human umbilical cord composition has a particle size of more than 1 μm to less than 300 μm, and contains collagen, fibronectin, IGFBP-1 at a concentration of 1500 pg / mL to 9000 pg / mL, and 0.1×10 6 pg / ml ~ 3.0 × 10 7 pg / mL sGAG, concentration 1.5 × 10 9 Particles / mL~4.0×10 9 and a dried, micronized human umbilical cord composition containing exosomes having a particle size of 50 nm to 200 nm, or any combination thereof.

[0059] After step (d) (and any optional steps), the aqueous human umbilical cord supernatant is subjected to step (e). Step (e) preferably includes multiple filtration steps, including (i) filtering the aqueous human umbilical cord supernatant through a first filter having pores of 30 μm to 40 μm to produce a second human umbilical cord supernatant, (ii) filtering the second human umbilical cord supernatant through a second filter having pores of 12.5 μm to 25 μm to produce a third human umbilical cord supernatant, and (iii) filtering the third human umbilical cord supernatant through a third filter having pores of 4 μm to 10 μm to produce an aqueous human umbilical cord filtrate. The filtration steps are completed in approximately 15 seconds to 2 minutes using a vacuum of 1 to 5 psi. In one embodiment, the applied force is negative pressure (vacuum), which is preferred because such negative pressure is less likely to damage the filter and cause subsequent quality control issues with the resulting aqueous human umbilical cord filtrate disclosed herein. The aqueous human umbilical cord filtrate preferably contains acellular endogenous Wharton's jelly, endogenous hyaluronic acid (HA) and / or hyaluronan, endogenous fibronectin, endogenous insulin growth factor binding protein-1 (IGFBP-1), endogenous sulfated glycosaminoglycans (sGAGs), endogenous exosomes, endogenous interleukin-1 receptor antagonist (IL-1ra), endogenous hepatocyte growth factor (HGF), endogenous transthyretin, endogenous tissue inhibitor of metalloproteinase 1 (TIMP-1), endogenous aggrecan, or any combination thereof, in an amount effective to reduce the size of Peyronie's disease plaques in a subject in need of treatment when administered by internal injection. In certain embodiments, the aqueous human umbilical cord filtrate comprises acellular Wharton's jelly, endogenous hyaluronic acid (HA) and / or hyaluronan, endogenous fibronectin, endogenous insulin growth factor binding protein-1 (IGFBP-1), endogenous sulfated glycosaminoglycans (sGAGs), endogenous exosomes, endogenous interleukin-1 receptor antagonist (IL-1ra), endogenous hepatocyte growth factor (HGF), endogenous transthyretin, endogenous tissue inhibitor of metalloproteinase 1 (TIMP-1), endogenous aggrecan, or any combination thereof. In certain embodiments, the aqueous human umbilical cord filtrate comprises acellular Wharton's jelly, endogenous hyaluronic acid (HA), endogenous hyaluronan ... 6 pg / mL ~ 4.0 × 10 8 pg / mL hyaluronan (HA), with a concentration of 2.0 × 10 3 pg / mL ~ 6.0 × 10 6 pg / mL insulin growth factor binding protein-1 (IGFBP-1), with a concentration of 2.0 × 10 7 pg / mL ~ 3.0 × 10 9 pg / mL sulfated glycosaminoglycans (sGAGs), with a concentration of 2.0 × 10 2pg / mL ~ 4.0 × 10 4 pg / mL interleukin-1 receptor antagonist (IL-1ra), with a concentration of 6.0 × 10 2 pg / mL ~ 6.0 × 10 5 pg / mL transthyretin, concentration 3.0 × 10 3 pg / mL ~ 8.0 × 10 5 pg / mL tissue inhibitor of metalloproteinase 1 (TIMP-1), with concentrations ranging from 0 pg / mL to 8.0 × 10 4 pg / mL aggrecan, or a combination of these.

[0060] Each filtration step is completed in approximately 15 seconds to 2 minutes at a vacuum of 1 to 5 psi. Furthermore, the aqueous human umbilical cord filtrate obtained as a result of the above filtration steps is a solution that does not exhibit settling, separation, and / or precipitation even after storage for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or more.

[0061] In certain embodiments, both the micronized human umbilical cord composition and the aqueous human umbilical cord filtrate are sterile, and both the micronized human umbilical cord composition and the aqueous human umbilical cord filtrate are non-immunogenic.

[0062] After completion of step (e), in step (f), the aqueous human umbilical cord filtrate (from step (e)) is placed into and sealed in a sterile container (e.g., a vial, an ampoule, a pre-filled syringe) for subsequent use in internal injection into a subject in need of treatment to treat Peyronie's disease.

[0063] In certain embodiments, the micronized human umbilical cord composition and the aqueous human umbilical cord filtrate are both sterile and configured to form a paste or lotion when mixed together. In this embodiment, the micronized human umbilical cord composition may be pre-filled into a sterile syringe, and the aqueous human umbilical cord filtrate may be pre-filled into a separate sterile container or syringe from the micronized human umbilical cord composition, each of which is sterilized and configured to aseptically mix the two components together as desired to produce the two-part solidification composition for wound packing and / or dental purposes and / or dental treatments disclosed herein. Kits containing processed human umbilical cord compositions for treating Peyronie's disease

[0064] Also disclosed herein is a kit containing a processed human umbilical cord composition for use in treating Peyronie's disease by internal injection into a subject in need of treatment. In certain embodiments, the composition is pre-packaged in a vial (sterile vial), ampoule (sterile ampoule), or pre-filled syringe (sterile pre-filled syringe) in a predetermined amount, and preferably contains, for example, endogenous hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycan (sGAG), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), transthyretin, tissue inhibitor of metalloproteinase 1 (TIMP-1), aggrecan, or a combination thereof, at a predetermined concentration to reduce the size of Peyronie's disease plaques. In certain embodiments, the predetermined volume is 0.25 mL to 20.0 mL, preferably 0.5 mL to 10.0 mL, and most preferably 1.0 to 5.0 mL, and may be used for 1 to 10 treatments (injections) with the composition to treat Peyronie's disease plaque every 2 to 6 weeks, or as determined by a physician. In certain embodiments, the aqueous human umbilical cord filtrate is acellular Wharton's jelly, having a concentration of 3.0 x 10 6 pg / mL ~ 4.0 × 10 8 pg / mL, more preferably 4.5 × 10 6pg / mL ~ 3.15 × 10 8 pg / mL hyaluronan (HA), with a concentration of 2.0 × 10 3 pg / mL ~ 6.0 × 10 6 pg / mL, more preferably 2.25 × 10 3 pg / mL ~ 4.7 × 10 6 pg / mL insulin growth factor binding protein-1 (IGFBP-1), with a concentration of 2.0 × 10 7 pg / mL ~ 3.0 × 10 9 pg / mL, more preferably 2.8 × 10 7 pg / mL ~ 2.3 × 10 9 pg / mL sulfated glycosaminoglycans (sGAGs), with a concentration of 2.0 × 10 2 pg / mL ~ 4.0 × 10 4 pg / mL, more preferably 3.75 × 10 2 pg / mL ~ 3.0 × 10 4 pg / mL interleukin-1 receptor antagonist (IL-1ra), with a concentration of 6.0 × 10 2 pg / mL ~ 6.0 × 10 5 pg / mL, more preferably 7.5 × 10 2 pg / mL ~ 5.25 × 10 5 pg / mL transthyretin, concentration 3.0 × 10 3 pg / mL ~ 8.0 × 10 5 pg / mL, more preferably 4.0 × 10 3 pg / mL ~ 7.75 × 10 5 pg / mL tissue inhibitor of metalloproteinase 1 (TIMP-1), with concentrations ranging from 0 pg / mL to 8.0 × 10 4 pg / mL, more preferably 1×10 1 pg / mL ~ 7.5 × 10 4 pg / mL aggrecan, or a combination of these. How to use

[0065] Without being bound by theory, it is believed that the particular compositions described above in the "Methods for Producing Human Umbilical Cord Compositions" section are particularly useful in wound packing, clotting, and wound healing, and advantageously, do not elicit a significant immunogenic response due to the non-immunogenic properties / nature of the compositions.

[0066] The collagen, fibronectin, and hyaluronan in the composition provide a substrate for coagulation factors to bind and signal for cell adhesion and proliferation. VEGFR1, HGF, interleukin antagonist (IL-1ra), bFGF, and PDGF-BB transmit cell proliferation signals and have anti-inflammatory effects, resulting in various therapeutic effects such as wound healing.

[0067] This is advantageous because the composition is easy to use for a desired purpose (e.g., easy to prepare and easy to mix the micronized human umbilical cord composition with aqueous human umbilical cord filtrate to modify the viscosity of the composition). In certain embodiments, the viscosity may be modified by adding an isotonic solution, such as those disclosed herein.

[0068] For example, the compositions disclosed herein are contemplated for use in dental applications (periodontal and / or endodontic treatment), more specifically, for packing a subject's gums after tooth extraction. In this particular application, the subject's gums are packed with the compositions disclosed herein immediately after tooth extraction. In this embodiment, a micronized human umbilical cord composition and an aqueous human umbilical cord filtrate are mixed (aseptically mixed) to a viscosity effective for clotting blood, and the hole in the subject's gums created by the tooth extraction is filled with the mixed composition (sterile composition), allowing blood to clot within the hole. In a specific embodiment, both the micronized human umbilical cord composition and the aqueous human umbilical cord filtrate are sterile and non-immunogenic. In this method, the micronized human umbilical cord composition and the aqueous human umbilical cord filtrate are mixed in a ratio of 2:1 to 1:2. If a highly viscous mixed composition is desired, more micronized human umbilical cord composition is mixed with less aqueous human umbilical cord filtrate, and conversely, if a less viscous mixed composition is desired, more aqueous human umbilical cord filtrate is mixed with less micronized human umbilical cord composition. The above-described packing process may be repeated as necessary.

[0069] While the above specifically contemplates dental applications, the disclosed compositions are also contemplated for more general medical applications, such as general wound packing (occurring in surgical procedures and / or acute trauma resulting in open external and / or internal wounds) and / or wound healing. In this embodiment, for example, a wound is first assessed to roughly determine the overall viscosity and thickness of the (mixed) two-part composition needed to pack and / or treat the subject's wound. The composition is then aseptically mixed to a viscosity effective to induce blood clotting, and the aseptically mixed composition is then aseptically packed against the subject's wound, resulting in blood clotting within the aseptically packed wound. In certain embodiments, both the micronized human umbilical cord composition and the aqueous human umbilical cord filtrate are sterile and non-immunogenic, and in this procedure, the micronized human umbilical cord composition and the aqueous human umbilical cord filtrate are mixed in a ratio of 2:1 to 1:2. If a highly viscous mixed composition is desired, more micronized human umbilical cord composition is mixed with less aqueous human umbilical cord filtrate, and conversely, if a less viscous mixed composition is desired, more aqueous human umbilical cord filtrate is mixed with less micronized human umbilical cord composition. The above-described packing process may be repeated as necessary.

[0070] In certain embodiments, methods of treating orthopedic and / or podiatric diseases / conditions are also disclosed. For example, in certain embodiments, plantar fasciitis and / or heel conditions may be treated by injecting a mixed two-part composition disclosed herein directly into a subject's foot (subcutaneously between the ball of the foot and the heel) and / or adjacent to the bony structure forming the subject's heel. The method includes (a) aseptically mixing a composition disclosed herein to a viscosity effective for treating a subject suffering from an orthopedic and / or podiatric disease / condition (e.g., plantar fasciitis), and (b) aseptically injecting the sterilely mixed composition of step (a) into and / or adjacent to the site of the orthopedic and / or podiatric disease / condition in the subject to treat the disease / condition. In this embodiment, the micronized human umbilical cord composition and aqueous human umbilical cord filtrate are both sterile and non-immunogenic, and are mixed in a ratio of 2:1 to 1:2 to achieve a viscosity sufficient to treat the disease / disorder. For example, when treating plantar fasciitis with the above method and composition, the mixed composition has a thickness and viscosity sufficient to provide a cushion (subcutaneous cushion) to treat and relieve pain associated with plantar fasciitis. In particular, the high viscosity of the filtrate provides the desired cushioning effect, and the presence of Wharton's jelly (mucopolysaccharides and proteoglycans) in the filtrate further enhances the cushioning and protective properties of the method.

[0071] In certain embodiments, each component of the disclosed two-part solidification composition may be used individually (alone) for a specific purpose. For example, when using the disclosed filtrate individually, the purpose of using the entire filtrate (filtrate alone) would be to provide growth factors and exosomes within the filtrate without introducing matrix or interstitial matrix. For example, the filtrate could be used to provide cushioning components for a modified heel pad, or the filtrate could be mixed with commercially available bone microparticles for use in a dental socket. As another example, when using the disclosed micronized composition individually (alone), the purpose of using all particles would be to pack a moist wound bed or dental socket when the site is too wet to add additional filtrate or when another filtrate, such as platelet-rich plasma (PRP), is desired.

[0072] In certain other embodiments, the processed human umbilical cord compositions disclosed herein, including aqueous human umbilical cord filtrate (but not micronized human umbilical cord composition), can be used to treat Peyronie's disease by internal injection into a subject in need of treatment. In particular, treatment for reducing the size of Peyronie's disease plaques in a human subject in need of treatment includes (a) contacting (by internal injection) Peyronie's disease plaques in the subject in need of treatment (the human subject) with a predetermined amount of processed human umbilical cord composition having a predetermined concentration of, for example, endogenous hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycans (sGAGs), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), transthyretin, tissue inhibitor of metalloproteinase 1 (TIMP-1), aggrecan, or a combination thereof, at a predetermined frequency necessary to reduce plaque size. For example, the processed human umbilical cord compositions disclosed herein may be used for 1 to 10 treatments (injections) or as determined by a physician with the compositions to treat Peyronie's disease plaques in the amounts and concentrations disclosed herein every 2 to 6 weeks or as determined by a physician.

[0073] In certain embodiments, the aqueous human umbilical cord filtrate of the processed human umbilical cord composition comprises acellular Wharton's jelly, at a concentration of 3.0 x 10 6 pg / mL ~ 4.0 × 10 8 pg / mL, more preferably 4.5 × 10 6 pg / mL ~ 3.15 × 10 8 pg / mL hyaluronan (HA), with a concentration of 2.0 × 10 3 pg / mL ~ 6.0 × 10 6 pg / mL, more preferably 2.25 × 10 3 pg / mL ~ 4.7 × 10 6 pg / mL insulin growth factor binding protein-1 (IGFBP-1), with a concentration of 2.0 × 10 7 pg / mL ~ 3.0 × 10 9 pg / mL, more preferably 2.8 × 10 7 pg / mL ~ 2.3 × 10 9 pg / mL sulfated glycosaminoglycans (sGAGs), with a concentration of 2.0 × 10 2 pg / mL ~ 4.0 × 10 4 pg / mL, more preferably 3.75 × 10 2 pg / mL ~ 3.0 × 10 4 pg / mL interleukin-1 receptor antagonist (IL-1ra), with a concentration of 6.0 × 10 2 pg / mL ~ 6.0 × 10 5 pg / mL, more preferably 7.5 × 10 2 pg / mL ~ 5.25 × 10 5 pg / mL transthyretin, concentration 3.0 × 10 3 pg / mL ~ 8.0 × 10 5 pg / mL, more preferably 4.0 × 10 3 pg / mL ~ 7.75 × 10 5 pg / mL tissue inhibitor of metalloproteinase 1 (TIMP-1), with concentrations ranging from 0 pg / mL to 8.0 × 10 4 pg / mL, more preferably 1×10 1 pg / mL ~ 7.5 × 10 4pg / mL aggrecan, or a combination thereof. In certain embodiments, the above is repeated at a predetermined frequency to further reduce plaque size and / or to continue reducing plaque in the subject.

[0074] The foregoing description provides exemplary embodiments of the present invention only. It is envisioned that other embodiments may perform similar functions and / or achieve similar results. Any and all such equivalent embodiments and examples are intended to be within the scope of the present invention and covered by the appended claims.

[0075] (Section 1) 1. A processed human umbilical cord composition for use in treating Peyronie's disease by internal injection of an effective amount into a subject in need of treatment, comprising: (a) A composition comprising an aqueous human umbilical cord filtrate containing endogenous hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycans (sGAGs), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), transthyretin, tissue inhibitor of metalloproteinase 1 (TIMP-1), aggrecan, or a combination thereof, in an amount effective to reduce the size of Peyronie's disease plaques. (Section 2) Item 10. The composition of claim 1, wherein the aqueous human umbilical cord filtrate has not been subjected to exogenous enzyme digestion. (Section 3) Item 3. The composition according to item 1 or 2, wherein the composition does not contain particles with a diameter exceeding 100 μm. (Section 4) The aqueous human umbilical cord filtrate was prepared by adding acellular Wharton's jelly to a solution containing 3.0 x 10 6 pg / mL ~ 4.0 × 10 8 pg / mL hyaluronan (HA), with a concentration of 2.0 × 10 3 pg / mL ~ 6.0 × 10 6pg / mL insulin growth factor binding protein-1 (IGFBP-1), with a concentration of 2.0 × 10 7 pg / mL ~ 3.0 × 10 9 pg / mL sulfated glycosaminoglycans (sGAGs), with a concentration of 2.0 × 10 2 pg / mL ~ 4.0 × 10 4 pg / mL interleukin-1 receptor antagonist (IL-1ra), with a concentration of 6.0 × 10 2 pg / mL ~ 6.0 × 10 5 pg / mL transthyretin, concentration 3.0 × 10 3 pg / mL ~ 8.0 × 10 5 pg / mL tissue inhibitor of metalloproteinase 1 (TIMP-1), with concentrations ranging from 0 pg / mL to 8.0 × 10 4 Item 4. The composition according to any one of items 1 to 3, comprising pg / mL aggrecan, or a combination thereof. (Section 5) Item 5. The composition according to any one of Items 1 to 4, wherein the aqueous human umbilical cord filtrate further comprises an isotonic solution. (Section 6) Item 6. The composition according to any one of Items 1 to 5, wherein the isotonic solution is phosphate buffered saline. (Section 7) Item 7. The composition of any one of Items 1 to 6, wherein the aqueous human umbilical cord filtrate comprises particles of human umbilical cord tissue less than 100 μm in diameter therein. (Section 8) Item 8. The composition according to any one of Items 1 to 7, wherein the aqueous human umbilical cord filtrate is non-immunogenic. (Section 9) A kit comprising a vial, ampoule, or pre-filled syringe containing the processed human umbilical cord composition according to any one of items 1 to 8. (Section 10) 10. A method for reducing the size of Peyronie's disease plaques in a human subject in need thereof, comprising: (a) contacting the Peyronie's disease plaques with a predetermined amount of the composition of any one of paragraphs 1 to 9. (Section 11) A method for producing the composition according to any one of items 1 to 9, (a) providing a human umbilical cord; (b) washing the human umbilical cord with an isotonic solution; (c) pulverizing the washed human umbilical cord tissue of step (b) to produce pulverized human umbilical cord tissue; (d) separating the comminuted human umbilical cord tissue of step (c) into a solid residue and an aqueous human umbilical cord supernatant, optionally further processing the solid residue of step (d) into a micronized human umbilical cord composition; (e) filtering the aqueous human umbilical cord supernatant to produce an aqueous human umbilical cord filtrate, the aqueous human umbilical cord filtrate having particles of the comminuted human umbilical cord tissue therein that are less than 100 μm in diameter; Including, The method, wherein none of steps (a)-(e) includes the introduction of an exogenous enzyme that results in exogenous enzymatic degradation / digestion. (Section 12) Item 12. The method according to Item 11, wherein the human umbilical cord is obtained from a subject, and steps (a) to (c) are then carried out within 48 to 72 hours after birth and / or cesarean section. (Section 13) Item 13. The method according to item 11 or 12, further comprising removing thrombus present in the human umbilical cord during steps (a) to (c). (Section 14) Item 14. The method according to any one of items 11 to 13, wherein step (b) is repeated 1 to 5 times by discarding the used isotonic solution, providing new isotonic solution, and washing the human umbilical cord again with the new isotonic solution. (Section 15) 15. The method according to any one of items 11 to 14, wherein in step (a), 15 to 80 grams of human umbilical cord is prepared, and then steps (b) to (f) are carried out. (Section 16) Item 16. The method according to any one of Items 11 to 15, wherein the isotonic solution is phosphate buffered saline supplied in an amount of 300 mL to 1000 mL per washing step (b). (Section 17) 17. The method of any one of paragraphs 11 to 16, wherein step (c) uses a grinding tool configured to grind and / or pulverize the washed human umbilical cord at a speed of 40 to 200 revolutions per minute (RPM) until the cord is completely ground. (Section 18) 18. The method of any one of paragraphs 11 to 17, further comprising, prior to step (d), contacting the comminuted umbilical cord tissue produced in step (c) with a filter and then filtering the comminuted umbilical cord tissue to produce the solid residue remaining on the filter and the aqueous human umbilical cord supernatant of step (d) that has passed through the filter. (Section 19) Item 19. The method according to Item 18, wherein the filter has pores of 100 μm to 200 μm. (Section 20) 20. The method of claim 19, further processing the solid residue, wherein the processing is a crushing process, a freeze-drying process, or a dehydration process that produces the micronized human umbilical cord composition having particles greater than 1 μm and less than 300 μm in size, wherein the particles are polydisperse. (Section 21) 21. The method of claim 20, further processing the solid residue, wherein the processing step is a freeze-grinding process in which the solid residue of step (d) is placed in a liquid nitrogen cooled freeze-grinding chamber and ground therein to produce the micronized human umbilical cord composition having particles of a size greater than 1 μm to 50 μm, wherein the particles are polydisperse. (Section 22) (i) filtering the aqueous human umbilical cord supernatant through a first filter having pores of 30 μm to 40 μm to produce a second human umbilical cord supernatant; (ii) filtering the second human umbilical cord supernatant through a second filter having pores of 12.5 μm to 25 μm to produce a third human umbilical cord supernatant; (iii) filtering the third human umbilical cord supernatant through a third filter having pores of 4 μm to 10 μm to produce the aqueous human umbilical cord filtrate. (Section 23) Item 23. The method of any one of Items 11 to 22, wherein the micronized human umbilical cord composition comprises collagen, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycoaminoglycan (sGAG), exosomes, or any combination thereof. (Section 24) The micronized human umbilical cord composition has a particle size of more than 1 μm to less than 300 μm, and contains collagen, fibronectin, IGFBP-1 at a concentration of 1500 pg / mL to 9000 pg / mL, and 0.1×10 6 pg / ml ~ 3.0 × 10 7 pg / mL sGAG, concentration 1.5 × 10 9 Particles / mL~4.0×10 9 Item 24. The method according to any one of Items 11 to 23, wherein the dried micronized human umbilical cord tissue comprises exosomes having a particle size of 50 nm to 200 nm, or any combination thereof. (Section 25) The aqueous human umbilical cord filtrate was prepared using Wharton's jelly, a solution of 3.0 x 10 6 pg / mL ~ 4.0 × 10 8 pg / mL hyaluronan (HA), with a concentration of 2.0 × 10 3 pg / mL ~ 6.0 × 10 6 pg / mL insulin growth factor binding protein-1 (IGFBP-1), with a concentration of 2.0 × 10 7 pg / mL ~ 3.0 × 10 9 pg / mL sulfated glycosaminoglycans (sGAGs), with a concentration of 2.0 × 10 2 pg / mL ~ 4.0 × 10 4 pg / mL interleukin-1 receptor antagonist (IL-1ra), with a concentration of 6.0 × 10 2 pg / mL ~ 6.0 × 10 5 pg / mL transthyretin, concentration 3.0 × 10 3 pg / mL ~ 8.0 × 10 5 pg / mL tissue inhibitor of metalloproteinase 1 (TIMP-1), with concentrations ranging from 0 pg / mL to 8.0 × 10 4Item 25. The method according to any one of items 11 to 24, comprising: pg / mL aggrecan; or a combination thereof. (Section 26) Item 26. The method of any one of Items 11 to 25, wherein the micronized human umbilical cord composition and the aqueous human umbilical cord filtrate are both sterile. (Section 27) Item 27. The method of any one of Items 11 to 26, wherein the micronized human umbilical cord composition and the aqueous human umbilical cord filtrate are both non-immunogenic. (Section 28) 1. A method for reducing the size of Peyronie's disease plaques in a human subject in need of treatment, comprising: A method comprising: (a) contacting a Peyronie's disease plaque with a predetermined amount of a composition, the composition comprising aqueous human umbilical cord filtrate containing endogenous hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycans (sGAGs), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), transthyretin, tissue inhibitor of metalloproteinase 1 (TIMP-1), aggrecan, or a combination thereof, in an amount effective to reduce the size of the Peyronie's disease plaque. (Section 29) 29. The composition of claim 28, wherein the aqueous human umbilical cord filtrate has not been subjected to exogenous enzyme digestion. (Section 30) Item 30. The composition according to items 28 to 29, wherein the composition does not contain particles with a diameter exceeding 100 μm. (Section 31) The aqueous human umbilical cord filtrate was prepared by adding acellular Wharton's jelly to a solution containing 3.0 x 10 6 pg / mL ~ 4.0 × 10 8 pg / mL hyaluronan (HA), with a concentration of 2.0 × 10 3 pg / mL ~ 6.0 × 10 6 pg / mL insulin growth factor binding protein-1 (IGFBP-1), with a concentration of 2.0 × 10 7 pg / mL ~ 3.0 × 10 9pg / mL sulfated glycosaminoglycans (sGAGs), with a concentration of 2.0 × 10 2 pg / mL ~ 4.0 × 10 4 pg / mL interleukin-1 receptor antagonist (IL-1ra), with a concentration of 6.0 × 10 2 pg / mL ~ 6.0 × 10 5 pg / mL transthyretin, 3.0 × 10 3 pg / mL ~ 8.0 × 10 5 pg / mL tissue inhibitor of metalloproteinase 1 (TIMP-1), with concentrations ranging from 0 pg / mL to 8.0 × 10 4 Item 31. The composition according to any one of items 28 to 30, comprising pg / mL aggrecan, or a combination thereof. (Section 32) Item 32. The composition according to any one of Items 28 to 31, wherein the aqueous human umbilical cord filtrate further comprises an isotonic solution. (Section 33) Item 33. The composition according to any one of Items 28 to 32, wherein the isotonic solution is phosphate buffered saline. (Section 34) Item 34. The composition of any one of Items 28 to 33, wherein the aqueous human umbilical cord filtrate comprises particles of human umbilical cord tissue therein that are less than 100 μm in diameter. (Section 35) Item 35. The composition of any one of Items 28 to 34, wherein the aqueous human umbilical cord filtrate is non-immunogenic.

Claims

1. 1. A processed human umbilical cord composition for use in treating Peyronie's disease by internal injection of an effective amount into a subject in need of treatment, comprising: (a) providing an aqueous human umbilical cord filtrate containing endogenous hyaluronic acid (HA) and / or hyaluronan, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycosaminoglycans (sGAGs), exosomes, interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), transthyretin, tissue inhibitor of metalloproteinase 1 (TIMP-1), aggrecan, or a combination thereof, in an amount effective to reduce the size of Peyronie's disease plaques; composition.

2. 2. The composition of claim 1, wherein the aqueous human umbilical cord filtrate has not been subjected to exogenous enzyme digestion.

3. 3. The composition of claim 1, wherein the composition does not contain particles with a diameter exceeding 100 μm.

4. The aqueous human umbilical cord filtrate was prepared by adding acellular Wharton's jelly to a solution containing 3.0 x 10 ６ pg / mL ~ 4.0×10 ８ pg / mL hyaluronan (HA), with a concentration of 2.0 × 10 ３ pg / mL ~ 6.0×10 ６ pg / mL insulin growth factor binding protein-1 (IGFBP-1), with a concentration of 2.0 × 10 ７ pg / mL ~ 3.0×10 ９ pg / mL sulfated glycosaminoglycan (sGAG), at a concentration of 2.0 × 10 ２ pg / mL ~ 4.0×10 ４ pg / mL interleukin-1 receptor antagonist (IL-1ra), with a concentration of 6.0 × 10 ２ pg / mL ~ 6.0×10 ５ pg / mL transthyretin, with a concentration of 3.0 × 10 ３ pg / mL ~ 8.0×10 ５ pg / mL tissue inhibitor of metalloproteinase 1 (TIMP-1), concentrations ranging from 0 pg / mL to 8.0 × 10 ４ The composition for use according to any one of claims 1 to 3, comprising pg / mL aggrecan, pg / mL aggrecan, or a combination thereof.

5. The composition for use according to any one of claims 1 to 4, wherein the aqueous human umbilical cord filtrate further comprises an isotonic solution.

6. The composition used according to any one of claims 1 to 5, wherein the isotonic solution is phosphate buffered saline.

7. The composition for use according to any one of claims 1 to 6, wherein the aqueous human umbilical cord filtrate comprises particles of human umbilical cord tissue therein that are less than 100 μm in diameter.

8. The composition for use according to any one of claims 1 to 7, wherein the aqueous human umbilical cord filtrate is non-immunogenic.

9. 1. A kit for use in treating Peyronie's disease, comprising: a vial, ampoule, or pre-filled syringe containing the processed human umbilical cord composition of any one of claims 1 to 8; kit.

10. 1. A method of reducing the size of Peyronie's disease plaques in a human subject in need thereof, comprising: (a) contacting Peyronie's disease plaque with a predetermined amount of the composition of any one of claims 1 to 9, method.

11. A method for producing the composition of claims 1 to 9, comprising: (a) providing a human umbilical cord; (b) flushing the human umbilical cord with an isotonic solution; (c) pulverizing the washed human umbilical cord tissue of step (b) to produce pulverized human umbilical cord tissue; (d) separating the comminuted human umbilical cord tissue of step (c) into a solid residue and an aqueous human umbilical cord supernatant, optionally further processing the solid residue of step (d) into a micronized human umbilical cord composition; (e) filtering the aqueous human umbilical cord supernatant to produce an aqueous human umbilical cord filtrate, the aqueous human umbilical cord filtrate having particles of the comminuted human umbilical cord tissue therein that are less than 100 μm in diameter; Including, None of steps (a) to (e) includes the introduction of exogenous enzymes that result in exogenous enzymatic degradation / digestion; method.

12. 12. The method of claim 11, wherein the human umbilical cord is obtained from a subject, and steps (a)-(c) are then performed within 48-72 hours after birth and / or cesarean section.

13. 13. The method of claim 11 or 12, further comprising removing thrombus present in the human umbilical cord during steps (a) to (c).

14. 14. The method of any one of claims 11 to 13, wherein step (b) is repeated 1 to 5 times by discarding the used isotonic solution, providing new isotonic solution, and re-washing the human umbilical cord with the new isotonic solution.

15. 15. The method of any one of claims 11 to 14, wherein in step (a) 15 to 80 grams of human umbilical cord is provided, and thereafter steps (b) to (f) are performed.

16. 16. The method of any one of claims 11 to 15, wherein the isotonic solution is phosphate buffered saline provided in an amount of 300 mL to 1000 mL per wash step (b).

17. 17. The method of any one of claims 11-16, wherein step (c) uses a comminution tool configured to crush and / or pulverize the washed human umbilical cord at a speed of 40 to 200 revolutions per minute (RPM) until the cord is completely crushed.

18. 18. The method of any one of claims 11-17, further comprising, prior to step (d), contacting the comminuted umbilical cord tissue produced in step (c) with a filter and then filtering the comminuted umbilical cord tissue to produce the solid residue retained on the filter and the aqueous human umbilical cord supernatant of step (d) that has passed through the filter.

19. 19. The method of claim 18, wherein the filter has pores of 100 μm to 200 μm.

20. further processing the solid residue; the processing step is a crushing process, a freeze-drying process, or a dehydration process that produces the micronized human umbilical cord composition having particles greater than 1 μm and less than 300 μm in size; 20. The method of claim 19, wherein the particles are polydisperse.

21. further processing the solid residue; the processing step is a freeze-grinding process in which the solid residue of step (d) is placed in a liquid nitrogen cooled freeze-grinding chamber and ground therein to produce the micronized human umbilical cord composition having particles of a size greater than 1 μm to 50 μm; 21. The method of claim 20, wherein the particles are polydisperse.

22. (i) filtering the aqueous human umbilical cord supernatant through a first filter having pores of 30 μm to 40 μm to produce a second human umbilical cord supernatant; (ii) filtering the second human umbilical cord supernatant through a second filter having pores between 12.5 μm and 25 μm to produce a third human umbilical cord supernatant; 22. The method of claims 20 and 21, further comprising multiple filtration steps comprising: (iii) filtering the third human umbilical cord supernatant through a third filter having pores of 4 μm to 10 μm to produce the aqueous human umbilical cord filtrate.

23. 23. The method of any one of claims 11-22, wherein the micronized human umbilical cord composition comprises collagen, fibronectin, insulin growth factor binding protein-1 (IGFBP-1), sulfated glycoaminoglycans (sGAGs), exosomes, or any combination thereof.

24. The micronized human umbilical cord composition has a particle size of more than 1 μm to less than 300 μm, and contains collagen, fibronectin, IGFBP-1 at a concentration of 1500 pg / mL to 9000 pg / mL, and 0.1×10 ６ pg / ml~3.0×10 ７ pg / mL sGAG, concentration 1.5 x 10 ９ Particles / mL ~ 4.0×10 ９ 24. The method of claim 11, wherein the exosomes are dried micronized human umbilical cord tissue, the exosomes having a particle size of 50 nm to 200 nm, or any combination thereof.

25. The aqueous human umbilical cord filtrate was prepared using Wharton's jelly, with a concentration of 3.0 x 10 ６ pg / mL ~ 4.0×10 ８ pg / mL hyaluronan (HA), with a concentration of 2.0 × 10 ３ pg / mL ~ 6.0×10 ６ pg / mL insulin growth factor binding protein-1 (IGFBP-1), with a concentration of 2.0 × 10 ７ pg / mL ~ 3.0×10 ９ pg / mL sulfated glycosaminoglycan (sGAG), at a concentration of 2.0 × 10 ２ pg / mL ~ 4.0×10 ４ pg / mL interleukin-1 receptor antagonist (IL-1ra), with a concentration of 6.0 × 10 ２ pg / mL ~ 6.0×10 ５ pg / mL transthyretin, with a concentration of 3.0 × 10 ３ pg / mL ~ 8.0×10 ５ pg / mL tissue inhibitor of metalloproteinase 1 (TIMP-1), concentrations ranging from 0 pg / mL to 8.0 × 10 ４ 25. The method of any one of claims 11 to 24, comprising: pg / mL aggrecan; pg / mL aggrecan; or a combination thereof.

26. 26. The method of any one of claims 11 to 25, wherein the micronized human umbilical cord composition and the aqueous human umbilical cord filtrate are both sterile.

27. 27. The method of any one of claims 11-26, wherein the micronized human umbilical cord composition and the aqueous human umbilical cord filtrate are both non-immunogenic.

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