Mesodermal compositions and methods of use thereof

Abstract

The present disclosure provides biomaterials and methods for preventing and minimizing the progression of cartilage and / or connective tissue damage. Also provided herein are biomaterials and methods for mitigating and / or reducing the risk of developing arthritis (e.g., osteoarthritis) associated with joint injury and / or joint surgery.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority from U.S. Provisional Application No. 63 / 413,399, filed October 5, 2022. The disclosure of the prior application is considered part of (and is incorporated by reference into) the disclosure of this application.

[0002] Statement Regarding Federally Sponsored Research This invention was made with government support under award W81XWH-17-2-0016 from the U.S. Department of the Army. The government has certain rights in this invention.

[0003] This document relates to biomaterials and methods for maintaining cartilage health, preventing or reducing the progression of cartilage damage, and reducing and / or decreasing the risk of developing arthritis, including early to mid-stage osteoarthritis. [Background technology]

[0004] Articular cartilage within human joints often undergoes a steady process of deterioration, leading to osteoarthritis in the later decades of life. Minor injuries typically lead to further damage. Injuries to other tissues within the joint (e.g., ligaments) can accelerate the deterioration of articular cartilage, sometimes leading to post-traumatic arthritis within 15 years of the initial injury. Extra-articular tissues can heal by forming fibrin clots, which connect the ends of ruptured tissue and then remodel to form a healing scar. However, synovial fluid within the joint naturally prevents fibrin clot formation. Therefore, in synovial joints such as the knee, fibrin clots do not form or are dissolved quickly after injury. This fibrinolytic process inevitably progresses, even with minor injuries, as the fibrin clot scaffold is prematurely degraded, inhibiting the healing process within the joint and intra-articular tissues.

[0005] Osteoarthritis (OA) is a condition characterized by the gradual breakdown of collagen within articular cartilage, transforming the normally smooth surface into one with multiple fissures, cracks, and defects, resulting in stiff, painful joints. Osteoarthritis is highly prevalent, with an estimated 13% of men and 19% of women over the age of 60 having symptomatic osteoarthritis (Cieza et al., The Lancet, 396(10267):2006-2017, 2020). Posttraumatic osteoarthritis (PTOA) is defined as the development of osteoarthritis in an injured joint. PTOA can develop soon after injury or remain asymptomatic for 10 to 20 years after injury. Joint injury significantly increases the risk of OA, and this risk increases with age at the time of injury and the time since injury. For example, anterior cruciate ligament (ACL) injury, even if the initial injury does not damage the cartilage itself, increases the risk of developing early OA by fivefold (Ajuied et al., Am J Sports Med, 42(9):2242-2252, 2014) and accelerates symptomatic OA by 15 years (Roos et al., Osteoarthritis and Cartilage, 3(4):261-267, 1995). Because many patients who injure their joints are high school or college athletes, those who develop PTOA are, on average, much younger than those without the injury, leading to a "young patient, old knee." This "old knee" can have serious consequences later in life, including a 50% increase in myocardial infarction (Meehan et al., Am J Cardiol, 122(11):1879-1884, 2018), likely due to the relative inactivity caused by the painful joint.

[0006] Early-to-mid-stage osteoarthritis causes pain and disability, yet no disease-modifying treatments are available. Symptomatic treatments, such as oral anti-inflammatory drugs, intra-articular corticosteroid injections, and viscosupplementation, have significant drawbacks. For example, oral anti-inflammatory drugs carry the risk of gastrointestinal and systemic toxicity, especially with long-term use. Although intra-articular corticosteroid injections provide pain relief, they have been shown to accelerate OA progression (McAlindon and LaValley, JAMA 318(12):1185-1186 2017; and Zeng et al., Osteoarthritis and Cartilage, 27(6):855-862, 2019). Joint supplementation with hyaluronic acid (a protein found in normal joints) has been much debated due to a lack of proven efficacy in patients. End-stage osteoarthritis, where the cartilage has completely worn away, can be treated with total joint replacement, in which the ends of the bone are removed and replaced with metal and plastic. However, because artificial joints wear down over time, joint replacement is not recommended for early- to mid-stage osteoarthritis ("Surgical Management of Osteoarthritis of the Knee: Appropriate Use Criteria," 2016; available online at aaos.org / smoakauc). Furthermore, many patients experience pain and disability from the early and mid-stages of osteoarthritis, often for decades before progressing to the end-stage stage. Therefore, there is a significant need for treatments that can relieve pain and / or slow or reverse disease progression in patients with early- to mid-stage osteoarthritis. Summary of the Invention

[0007] This document is based, at least in part, on the development of compositions comprising biomaterials (e.g., mesodermal extracellular matrix (ECM) proteins and other components) and methods of using the compositions to treat arthritis and / or reduce the risk of developing arthritis (e.g., PTOA). Accordingly, provided herein are methods and materials that can be used to treat, reduce the likelihood of, and / or inhibit the development of arthritis (e.g., OA) following trauma or injury to a joint (e.g., ACL tear, meniscus injury, minor cartilage damage, or joint surgery). The methods and materials described herein offer several advantages over current standard treatments, including limiting impact activity and subsequent treatment of established cartilage lesions, preserving the complex morphology and architecture of hyaline cartilage by reducing or preventing the progression of damage to injured tissue, and restoring the biochemical makeup of cartilage at the joint site.

[0008] Thus, this document provides methods and materials that can be used to treat, prevent, or reduce the likelihood of developing OA, which may include administering the materials during routine medical examinations. Mixing a powder containing mesodermal ECM proteins and other ingredients with blood and injecting the mixture into joints affected by or at risk for OA can also reduce or reverse the effects of OA. Note that this document sometimes refers to the composition / blood mixture as a "two-part mixture" or simply as a "mixture." Injecting the mixture provided herein into an injured joint can prevent or delay the onset of OA that would otherwise occur naturally after injury.

[0009] In a first aspect, this document features a composition including a powdered extracellular matrix (ECM) component and a fluid, where the ECM component includes mesodermal proteins including collagen, and the concentration of the powdered ECM component in the fluid is about 50 mg / mL to about 200 mg / mL. The concentration of the powdered ECM component in the fluid can be about 67 mg / mL. The concentration of the powdered ECM component in the fluid can be about 100 to about 150 mg / mL (e.g., about 133 mg / mL). The fluid can be blood. The fluid can be saline. The powdered ECM component can have an average particle size of less than about 0.3 mm. The powdered ECM component can have an average particle size of about 0.1 mm to about 1 mm (e.g., about 0.3 mm to about 0.6 mm). The composition can further include growth factors, platelets, leukocytes, stem cells, a crosslinking agent, a neutralizing agent, or any combination thereof. The composition can further include calcium. The composition can be substantially free of one or more of nucleic acids, glycosaminoglycans (GAGs), phospholipids, active pepsin, and active viruses.

[0010] In another aspect, this document features a method for making a composition including a fluid and powdered ECM components, including mesodermal proteins, including collagen. The method can include, or consist essentially of, providing a syringe containing the powdered ECM components and injecting (optionally under vacuum) an aliquot of the fluid into the syringe such that the concentration of the powdered ECM components in the fluid is about 50 mg / mL to about 200 mg / mL. The method can include injecting an aliquot of the fluid into the syringe such that the concentration of the powdered ECM components in the fluid is about 67 mg / mL. The method can include injecting an aliquot of the fluid into the syringe such that the concentration of the powdered ECM components in the fluid is about 100 to about 150 mg / mL (e.g., about 133 mg / mL). The fluid can be blood. The fluid can be saline. The powdered ECM components can have an average particle size of less than about 0.3 mm. The powdered ECM components may have an average particle size of about 0.1 mm to about 1 mm (e.g., about 0.3 mm to about 0.6 mm). The ECM composition may further include growth factors, platelets, leukocytes, stem cells, a crosslinking agent, a neutralizing agent, or any combination thereof. The composition may further include calcium. The composition may be substantially free of one or more of nucleic acids, GAGs, phospholipids, active pepsin, and active viruses.

[0011] In another aspect, this document features a method of treating a mammal. The method can include, or consist essentially of, administering to a joint of a mammal having or at risk of developing arthritis an effective amount of a composition including powdered ECM components and a fluid, wherein the powdered ECM components include mesodermal proteins including collagen, and the powdered ECM components have a concentration of about 50 mg / mL to about 200 mg / mL in the fluid. The powdered ECM components can have a concentration of about 67 mg / mL. The powdered ECM components can have a concentration of about 100 to about 150 mg / mL (e.g., about 133 mg / mL). The fluid can be blood. The fluid can be saline. The powdered ECM components can have an average particle size of less than about 0.3 mm. The powdered ECM components can have an average particle size of about 0.1 mm to about 1 mm (e.g., about 0.3 mm to about 0.6 mm). The ECM composition can further include growth factors, platelets, leukocytes, stem cells, a crosslinking agent, a neutralizing agent, or any combination thereof. The composition may further comprise calcium. The composition may be substantially free of one or more of nucleic acids, GAGs, phospholipids, active pepsin, and active viruses. The mammal may have acute joint injury. The arthritis may be osteoarthritis. The arthritis may be post-traumatic arthritis (e.g., post-traumatic arthritis associated with intra-articular injury or arthroscopic surgery). The intra-articular injury may be selected from the group consisting of anterior cruciate ligament tear, anterior cruciate ligament rupture, meniscus injury, and cartilage injury. The mammal may have undergone surgical treatment for torn, fractured, strained, bruised, or ruptured intra-articular tissue in the joint at least one day prior to administration of the composition. The joint may be a joint of the hand, elbow, wrist, hip, knee, foot, shoulder, ankle, temporomandibular joint, or spine. The mammal may have injury associated with the development of arthritis. Administration may include direct injection into a joint. The mammal may be a human.

[0012] In yet another aspect, this document features a method for treating a mammal having an intra-articular tissue defect. The method can include, or consist essentially of, administering to the defect after visualizing the defect with an arthroscope, an effective amount of a composition including powdered ECM components and a fluid, wherein the ECM components include mesodermal proteins including collagen, and the concentration of the powdered ECM components in the fluid is about 50 mg / mL to about 200 mg / mL relative to the defect. The concentration of the powdered ECM components in the fluid can be about 67 mg / mL. The concentration of the powdered ECM components in the fluid can be about 100 to about 150 mg / mL (e.g., about 133 mg / mL). The fluid can be blood. The fluid can be saline. The powdered ECM components can have an average particle size of less than about 0.3 mm. The powdered ECM components can have an average particle size of about 0.1 mm to about 1 mm (e.g., about 0.3 mm to about 0.6 mm). The ECM composition may further comprise growth factors, platelets, leukocytes, stem cells, crosslinking agents, neutralizing agents, or any combination thereof. The composition may further comprise calcium. The composition may be substantially free of one or more of nucleic acids, GAGs, phospholipids, active pepsin, and active viruses. The defect may be an acute injury in a joint. The defect may be selected from the group consisting of anterior cruciate ligament rupture, anterior cruciate ligament rupture, meniscus injury, and cartilage injury. The defect may have an injury associated with the development of arthritis. The administration may include direct injection into the joint. The mammal may be a human.

[0013] In another aspect, this document features a method for producing a powder composition containing mesodermal extracellular matrix (ECM) proteins. The method can include, or consist essentially of, decellularizing a tissue sample containing tissue originating from mammalian mesoderm; treating the tissue sample with a composition containing peracetic acid before or after decellularization; lyophilizing the decellularized tissue sample; and grinding the lyophilized tissue into a powder. The composition containing peracetic acid can include about 0.1% peracetic acid. The method can include treating the tissue sample, before or after decellularization, with a composition containing peracetic acid for about 5 to about 30 minutes. The composition containing peracetic acid can further include hydrogen peroxide (e.g., about 1% hydrogen peroxide). The method can further include treating the decellularized tissue sample with an enzyme prior to lyophilization, thereby removing species-specific ends of collagen molecules. The method can further include treating the powder with supercritical carbon dioxide (scCO2). The powder may have an average particle size of about 0.1 mm to about 1 mm (e.g., about 0.3 mm to about 0.6 mm). The composition may further include growth factors, platelets, white blood cells, stem cells, a crosslinking agent, a neutralizing agent, or any combination thereof. The composition may further include calcium. The composition may be substantially free of one or more of nucleic acids, GAGs, phospholipids, active pepsin, and active viruses.

[0014] In another aspect, this document features a method for producing a powder composition containing mesodermal ECM proteins. The method can include, or consist essentially of, decellularizing a tissue sample containing tissue from mammalian mesoderm; lyophilizing the decellularized tissue; grinding the lyophilized tissue slurry to a powder; and treating the powder with scCO2. The method can further include treating the decellularized tissue sample with an enzyme prior to lyophilization, thereby removing species-specific ends of collagen molecules. The method can further include treating the tissue sample with a composition containing peracetic acid before or after decellularization. The peracetic acid-containing composition can include about 0.1% peracetic acid. The method can include treating the tissue sample before or after decellularization with the peracetic acid-containing composition for about 5 to 30 minutes. The peracetic acid-containing composition can further include hydrogen peroxide (e.g., about 1% hydrogen peroxide). The powder can have an average particle size of about 0.1 mm to about 1 mm (e.g., about 0.3 mm to about 0.6 mm). The composition may further comprise growth factors, platelets, white blood cells, stem cells, cross-linking agents, neutralizing agents, or any combination thereof. The composition may further comprise calcium. The composition may be substantially free of one or more of nucleic acids, GAGs, phospholipids, active pepsin, and active viruses.

[0015] In another aspect, this document features a method for making a composition including blood and powdered mesodermal ECM components, including collagen. The method can include, or consist essentially of, providing a syringe containing the powdered ECM components; contacting the blood sample with an anticoagulant; injecting a volume of blood into the syringe containing the powdered ECM components so that the concentration of the powdered ECM components in the blood is about 50 mg / mL to about 200 mg / mL; and adding a calcium chloride solution to the syringe, thereby inactivating the anticoagulant. The method can also include injecting a volume of blood into the syringe so that the concentration of the powdered ECM components in the blood is about 100 to about 150 mg / mL (e.g., about 133 mg / mL). The powdered ECM components can have an average particle size of about 0.1 mm to about 1 mm (e.g., about 0.3 mm to about 0.6 mm). The composition can further include growth factors, platelets, leukocytes, stem cells, a crosslinking agent, a neutralizing agent, or any combination thereof. The powdered mesodermal ECM component can be substantially free of one or more of nucleic acids, GAGs, phospholipids, active pepsin, and active viruses. The calcium chloride solution can have a concentration of about 35 mM to about 45 mM. The method can include adding the calcium chloride solution to a syringe to obtain a 1:9 calcium chloride solution to blood mixture.

[0016] Unless otherwise defined, all technical and scientific terms used in this document have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although the present invention can be practiced using methods and materials similar or equivalent to those provided in this document, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned in this document are incorporated by reference in their entirety. In case of conflict, the present document, including definitions, will control. Additionally, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0017] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. [Brief explanation of the drawings]

[0018] [Figure 1] Representative images are included showing X-ray images of guinea pig knee joints 6 weeks after anterior cruciate ligament (ACL) transection, including a control knee (no surgery; left panel), a knee injected with PBS after ACL transection (placebo; center panel), and a knee injected with mesodermal protein composition and blood after ACL transection (treatment group; right panel). [Figure 2] Representative images from histological studies of the medial tibial plateau of guinea pigs 6 weeks after anterior cruciate ligament transection are included, including control knees (no surgery; left panel), knees injected with PBS after ACL transection (placebo; center panel), and knees injected with mesodermal protein composition and blood after ACL transection (treatment group; right panel). [Figure 3] Representative images are included from a histological study of the medial tibial plateau of guinea pigs treated with a mixture of mesodermal protein composition and blood 1 week (left panel), 2 weeks (middle panel), and 4 weeks (right panel) after treatment, as assessed by toluidine blue staining. [Figure 4] Representative images from histological studies of the medial tibial plateau of treated guinea pigs 1 week (left panel), 2 weeks (middle panel), and 4 weeks (right panel) after injection of a mixture of mesodermal proteins and blood were assessed by Masson's trichrome staining. [Figure 5] 1 is a graph plotting the change in basal surface of support (BOS) between animals that did not receive an injection (control group) and animals that were injected with a mixture of mesodermal protein composition and blood (treatment group). [Figure 6] This set of images includes hydrogels containing ECM proteins, including mesodermal proteins, including collagen, produced with powder containing "fine" particles less than 0.3 mm in diameter (left panel) or "coarse" particles greater than 0.3 mm in diameter (right panel). The use of fine particles resulted in a more uniform dispersion of the particles in the diluent. [Figure 7]1 is a graph plotting the % weight remaining after collagenase treatment for hydrogel plugs containing the mesodermal protein powder provided herein at the indicated amounts and particle sizes. *p<0.01. [Figure 8] 1 is a graph plotting collagenase testing results of mesodermal protein gels, showing displacement over time. Samples containing a high concentration of mesodermal protein powder (400 mg / 3 mL) were more resistant to degradation than samples containing a lower concentration of powder (200 mg / 3 mL). [Figure 9] Figure 1 is a graph plotting collagenase testing results on mesodermal protein gels, showing normalized % weight retention after collagenase treatment at 32°C for 2 hours. The 400 mg sample retained more weight than the 200 mg sample. **p<0.01, ***p<0.001. [Figure 10] Images show powder containing ECM proteins, including mesodermal proteins including collagen, mixed with whole blood without vacuum assistance (left panel) and PRP mixed with the powder with vacuum assistance (right panel). Vacuum-assisted mixing resulted in more uniform dispersion of the powder in the diluent. Top panel: 40x magnification; bottom panel: 400x magnification. [Figure 11A] 1 is an image showing a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) assay of aseptically produced mesoderm protein powder before sterilization. [Figure 11B] 1 is an image showing an SDS-PAGE assay of e-beam sterilized powder. [Figure 12] 1 is a graph plotting the dry mass fraction of collagen in consolidated BEAR samples and consolidated powder samples as indicated. [Figure 13A] Graphs plotting shape scores (FIG. 13A) and cleavage scores (FIG. 13B) for the 200 mg / 3 mL samples and shape scores (FIG. 13C) and cleavage scores (FIG. 13D) for the 400 mg / 3 mL samples shown. [Figure 13B]Graphs plotting shape scores (FIG. 13A) and cleavage scores (FIG. 13B) for the 200 mg / 3 mL samples and shape scores (FIG. 13C) and cleavage scores (FIG. 13D) for the 400 mg / 3 mL samples shown. [Figure 13C] Graphs plotting shape scores (FIG. 13A) and cleavage scores (FIG. 13B) for the 200 mg / 3 mL samples and shape scores (FIG. 13C) and cleavage scores (FIG. 13D) for the 400 mg / 3 mL samples shown. [Figure 13D] Graphs plotting shape scores (FIG. 13A) and cleavage scores (FIG. 13B) for the 200 mg / 3 mL samples and shape scores (FIG. 13C) and cleavage scores (FIG. 13D) for the 400 mg / 3 mL samples shown. [Figure 14A] 14A is a graph plotting the diameter of mesodermal protein hydrogel plugs after compression testing for the following powders produced at the indicated pH conditions: 200 mg / 3 mL fine particles (FIG. 14A). [Figure 14B] 14B is a graph plotting the diameter of mesodermal protein hydrogel plugs after compression testing for the following powders produced at the indicated pH conditions: 200 mg / 3 mL coarse particle (FIG. 14B); [Figure 14C] 14C is a graph plotting the diameter of mesodermal protein hydrogel plugs after compression testing for the following powders produced at the indicated pH conditions: 400 mg / 3 mL fine particles (FIG. 14C); [Figure 14D] 14D is a graph plotting the diameter of mesodermal protein hydrogel plugs after compression testing for the following powders produced at the indicated pH conditions: 400 mg / 3 mL coarse particle (FIG. 14D); [Figure 15A] 15A and 15B are graphs plotting the results of enzyme degradation tests as a percentage of the initial mass of powders produced under the indicated pH conditions for 200 mg / 3 mL and 400 mg / 3 mL fine and coarse particle mesoderm protein powder samples. Results are shown for collagenase (FIG. 15A), after collagenase degradation (FIG. 15B), and control (FIG. 15C), normalized to the control. [Figure 15B] 15A and 15B are graphs plotting the results of enzyme degradation tests as a percentage of the initial mass of powders produced under the indicated pH conditions for 200 mg / 3 mL and 400 mg / 3 mL fine and coarse particle mesoderm protein powder samples. Results are shown for collagenase (FIG. 15A), after collagenase degradation (FIG. 15B), and control (FIG. 15C), normalized to the control. [Figure 15C] 15A and 15B are graphs plotting the results of enzyme degradation tests as a percentage of the initial mass of powders produced under the indicated pH conditions for 200 mg / 3 mL and 400 mg / 3 mL fine and coarse particle mesoderm protein powder samples. Results are shown for collagenase (FIG. 15A), after collagenase degradation (FIG. 15B), and control (FIG. 15C), normalized to the control. [Figure 16] 1 is a graph plotting the elastic modulus of mesodermal protein hydrogels containing 200 mg or 400 mg of mesodermal protein powder in 3 mL of PBS. [Figure 17A] Figure 17A shows the dynamic compression test results (Figure 17B) and normalized dynamic compression test results (Figure 17B) for mesoderm protein hydrogels prepared at concentrations of 200 mg / 3 mL PBS or 400 mg / 3 mL PBS and subjected to 45 compression cycles at a frequency of 1 Hz and strains ranging from 8% to 12%. For each sample, the average of the maximum (MAX) and minimum (MIN) stress response readings for the first five load cycles was used as the "start" maximum and minimum stress response, and the average of the maximum (MAX) and minimum (MIN) stress response readings for the last five load cycles (cycles 41-45) was used to represent the "end" stress response range. [Figure 17B]Figure 17A shows the dynamic compression test results (Figure 17B) and normalized dynamic compression test results (Figure 17B) for mesoderm protein hydrogels prepared at concentrations of 200 mg / 3 mL PBS or 400 mg / 3 mL PBS and subjected to 45 compression cycles at a frequency of 1 Hz and strains ranging from 8% to 12%. For each sample, the average of the maximum (MAX) and minimum (MIN) stress response readings for the first five load cycles was used as the "start" maximum and minimum stress response, and the average of the maximum (MAX) and minimum (MIN) stress response readings for the last five load cycles (cycles 41-45) was used to represent the "end" stress response range. [Figure 18A] 18A and 18B are graphs plotting stress relaxation over 10 minutes (FIG. 18A) and extrapolated to 100 minutes (FIG. 18B) for hydrogels containing 200 mg or 400 mg of mesodermal protein powder in 3 mL PBS. [Figure 18B] 18A and 18B are graphs plotting stress relaxation over 10 minutes (FIG. 18A) and extrapolated to 100 minutes (FIG. 18B) for hydrogels containing 200 mg or 400 mg of mesodermal protein powder in 3 mL PBS. [Figure 19] 1 is a graph plotting the Poisson's ratio of hydrogels containing 200 mg or 400 mg of mesodermal protein powder in 3 mL PBS. [Figure 20] Figure 1 is a graph plotting total soluble collagen content compared to untreated controls. Mesodermal tissue was treated for 1, 5, and 10 minutes (double the treatment time for hydrogen peroxide), as indicated by the three bars from left to right. Groups with significantly lower collagen content than controls are annotated based on p-values: *p<0.05, **p<0.001, ++p<0.0001. [Figure 21] 1 is a graph plotting total GAG content compared to untreated controls. Mesodermal tissue was treated for 1, 5, and 10 minutes (double the treatment time for hydrogen peroxide), as indicated by the three bars from left to right. Groups with significantly lower GAGs than controls are marked with a p-value of ++p<0.0001. [Figure 22] This graph plots gelation scores compared to untreated controls. Mesodermal tissue was digested with pepsin to form a slurry and then incubated in a 37°C incubator for 30 seconds. Groups with significantly lower gelation scores than the control were marked with a p-value of ++p<0.0001. The gelation scoring criteria were: 0, no mold shape maintained; 1, gel maintained less than half of its original center height when cast onto a Petri dish; 2, gel maintained more than half of its original height when cast onto a Petri dish; 3, mold shape with rounded corners maintained but not stable after cutting; 4, mold shape with rounded corners maintained and stable after cutting; and 5, mold shape with sharp corners maintained when cut. [Figure 23] This image shows an SDS-PAGE analysis to compare proteolysis by chemical pretreatment. Differences in band patterns were minimal, and no observable differences in proteolysis were observed. The major bands represent (a) type I collagen α polypeptide, (b) α polypeptide dimer, and (c) α polypeptide trimer (collagen triple molecule). [Figure 24] 1 is a graph plotting collagen concentration after treatment with various PAA protocols. [Figure 25] 1 is a graph plotting GAG content and GAG / collagen ratio after treatment with various PAA protocols. [Figure 26] 12 shows representative Western blot images of ECM-derived powder samples treated with the conditions shown in Table 12. M, standard protein ladder; lane 1, 0.1% PAA 1 hour; lane 2, 0.2% PAA 1 hour; lane 3, 0.1% PAA 18 hours; lane 4, 0.2% PAA 18 hours; lane C, control; lane 5, 7.5% hydrogen peroxide 10 minutes; lane 6, 1% CIP-100 10 minutes; lane 7, SPOR-KLENZ® 10 minutes; lane 8, 0.2% PAA 10 minutes. [Figure 27]1 is a graph plotting gelation data for ECM-derived powder samples treated with the indicated PAA protocols. [Figure 28] 1 is an image of SDS-PAGE using an ECM-derived powder protein composition. [Figure 29] 1 is a graph plotting collagen content in samples treated with the indicated sterilization methods, with data representing the mean ± standard deviation. [Figure 30] 1 is a graph plotting GAG content in samples treated with the indicated sterilization methods. Data represent the mean ± standard deviation. [Figure 31] 1 is a graph plotting DNA content in samples treated with the indicated sterilization methods. [Figure 32] 1 is a graph plotting phospholipid content in samples treated with the indicated sterilization methods. Data represent the mean ± standard deviation. [Figure 33] 1 is a graph plotting residual pepsin activity in samples treated with the indicated sterilization methods. Data represent the mean ± standard deviation. [Figure 34-1] 1 is an image of an SDS-PAGE gel for samples treated with the indicated sterilization methods, with a standard ladder provided in the left lane and a collagen reference in the far right lane. [Figure 34-2] This is a continuation of Figure 34. [Figure 35] A set of graphs is included plotting the % weight remaining in the control and treatment groups after enzymatic degradation (left) and the normalized % treated / control weight remaining for the electron beam and supercritical carbon dioxide (scCO2) treated powder-derived gels (right). [Figure 36] 1 is a graph plotting the elastic modulus of samples containing the indicated amounts of mesodermal ECM-derived powder and sterilized in the indicated manner, mean ± standard deviation. [Figure 37] From left to right, frontal and lateral views are shown after opening the porcine knee joint and removing the synovium and meniscus, along with images showing a central histological section of the ACL at low and high magnification. [Figure 38] 1 is a graph plotting ACL volume in pigs treated with BEAR® scaffold or mesodermal protein powder after anterior cruciate ligament transection. [Figure 39] 1 is a graph plotting ACL histology scores for pigs treated with BEAR® scaffold or mesodermal protein powder after anterior cruciate ligament transection. [Figure 40] From left to right, dorsal and lateral views of the sheep shoulder joint after opening are shown, along with images showing a central tissue section of the RCT at low and high magnification. [Figure 41] 1 is a graph plotting RCT volume in sheep treated with sutures only, mesodermal protein powder, mesodermal protein sheet, or BEAR® scaffold after RCT amputation. [Figure 42] 1 is a graph plotting RCT histology scores in sheep treated with sutures only, mesodermal protein powder, mesodermal protein sheet, or BEAR® scaffold after RCT amputation. DETAILED DESCRIPTION OF THE INVENTION

[0019] Detailed Description This document provides materials, such as methods and compositions, that can be used to treat arthritis and / or reduce the risk of developing it. Generally, the compositions provided herein can include proteins and other components derived from mesodermal extracellular matrix (ECM) (e.g., proteins such as collagen and fibrillin). The compositions can also include components such as laminin, salts, and / or calcium. The methods provided herein can be used, for example, to fill cartilage defects, treat OA (e.g., reversing osteoarthritic gait changes in subjects with early to mid-stage osteoarthritic disease), and prevent the progression of post-traumatic osteoarthritis following joint injury, even when the compositions provided herein are administered a significant amount of time after the initial injury.

[0020] Mesodermal ECM compositions (e.g., proteins and other components) can be prepared as powders. Briefly, the powders can be prepared by (1) decellularizing tissue derived from mammalian mesoderm (hereinafter "tissue"), (2) enzymatically digesting the decellularized tissue, (3) lyophilizing the decellularized tissue, or (4) grinding the lyophilized tissue into a powder.

[0021] The composition may be administered via direct injection from a syringe in some cases. Generally, the procedure for administering the compositions provided herein may include: (1) adding the powder composition to a syringe or providing a syringe containing the powder composition; (2) injecting blood into the syringe containing the composition; (3) mixing the blood with the composition; and (4) injecting the blood / composition mixture into the joint. In some cases, the powder composition may be hydrated with a saline solution or water before injecting the blood into the syringe.

[0022] This document also provides an article of manufacture having a compartment containing the powder composition, optionally a compartment containing an aqueous solution ("hydration solution"), optionally a device for mixing the composition and optional hydration solution with blood (e.g., autologous blood from a patient), which mixing device can be controlled from outside the container, and optionally an internal mixing chamber large enough to accommodate the powder, optional hydration solution, and blood.

[0023] In a first aspect, this document provides a composition comprising a powder. The powder is optionally combined with a fluid (e.g., blood or other fluid containing blood cells). Thus, in some cases, the compositions provided herein can be a two-part mixture, with the first part comprising powdered ECM components (e.g., proteins) found in mesodermal tissue. The mesoderm is the mammalian germ layer from which limbs develop and gives rise to multiple tissues. Proteins typically found in the mesoderm include collagen, elastin, fibrillin, and other glycoproteins. The powder included in the compositions provided herein (also referred to as "powder compositions") can be produced by processing tissue derived from the mesoderm to isolate these proteins. The second part can comprise, for example, autologous blood from the patient to be treated.

[0024] The first portion of the composition provided herein may be derived from tissues arising from mammalian mesoderm, and may contain, for example, collagen and / or fibrillin. Examples of tissues arising from mammalian mesoderm include muscle (e.g., skeletal muscle and smooth muscle), connective tissue (e.g., skin, bone, ligament, tendon, bursa, synovium, loose connective tissue, fascia, isolated fascia, and capillary / subcutaneous fascia), blood vessels (e.g., aorta, vena cava, artery, vein, and capillary), bone, cartilage (e.g., articular cartilage, fibrocartilage, hyaline cartilage, and elastic cartilage), kidney, adipose tissue, and urogenital organs. In some cases, the powder composition may be derived from elastic tissue (e.g., ligamentum nuchale, artery, dermis of skin, loose connective tissue, adipose tissue, lung, etc.). These tissues may be useful as sources of powder compositions because they have high concentrations of collagen and, optionally, fibrillin.

[0025] The powder portion of the compositions provided herein can be produced by decellularizing tissue (e.g., elastic tissue) and breaking down the tissue's original structure into a fluid form. As described above, the tissue can be derived from any of a variety of sources, including blood vessels, ligamentum nuchale, fascia, bursae, synovial sheaths, skeletal muscle, and / or smooth muscle. Furthermore, the tissue can be derived from humans or non-human animal sources, including bovine, porcine, caprine, or other mammalian species. The tissue can be from skeletally mature animals or animals still growing. For example, in some cases, the tissue can be from animals aged 1 week to 1 year, 3 months to 6 months, or less than 6 months. In some cases, the tissue (e.g., elastic tissue) can be derived from recombinant technology or other manufacturing methods for protein production.

[0026] In some cases, tissues may be treated with compounds designed to remove bacterial, fungal, and / or viral contamination prior to further processing. Such treatments may include the use of chemicals such as sodium hypochlorite, peracetic acid, hydrogen peroxide, antibiotics, and / or acetic acid. In some cases, treatments may include physical cleaning of the tissue, exposure to high or low pH, ultraviolet light, heat, steam, gamma or electron beam irradiation, or treatment with gases (e.g., ethylene oxide or supercritical CO2) or induced free oxygen radicals to remove or inactivate infectious compounds that may be introduced into the tissue when it is harvested. For example, tissues derived from mammalian mesoderm can be sterilized using supercritical CO for an appropriate time (e.g., about 2 to about 16 hours, about 3 to about 14 hours, about 4 to about 12 hours, about 6 to about 10 hours, about 2 to about 4 hours, about 4 to about 6 hours, about 6 to about 8 hours, about 8 to about 10 hours, about 10 to about 12 hours, about 12 to about 14 hours, about 14 to about 16 hours, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 hours). Such tissue pretreatments to remove or reduce bioburden can be used alone or in various combinations. In some cases, hydrogen peroxide, peracetic acid (PAA), or a combination thereof can be used to treat the tissue prior to processing into a powder composition.

[0027] When treating a tissue sample with peracetic acid and / or hydrogen peroxide, the treatment can be carried out for any suitable time, and the peracetic acid and / or hydrogen peroxide can be used at any suitable concentration. In some cases, for example, the tissue sample can be treated with peracetic acid, hydrogen peroxide, or a combination thereof for 5 to 30 minutes (e.g., 5 to 10 minutes, 10 to 15 minutes, 15 to 20 minutes, 20 to 25 minutes, 25 to 30 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, or about 30 minutes). In some cases, the tissue sample may be treated with a composition comprising about 0.01% to about 1% peracetic acid (e.g., about 0.01% to about 0.05%, about 0.05 to about 0.1%, about 0.1 to about 0.15%, about 0.15 to about 0.25%, about 0.25 to about 0.5%, about 0.5 to about 1%, about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.5%, or about 1% peracetic acid). In some cases, tissue samples may be treated with a composition comprising about 0.1 to about 10% hydrogen peroxide (e.g., about 0.1 to about 0.2%, about 0.2 to about 0.5%, about 0.5 to about 1%, about 1 to about 2%, about 2 to about 5%, about 5 to about 10%, about 0.1%, about 0.5%, about 1%, about 2%, about 5%, or about 10% hydrogen peroxide). In some cases, tissue samples may be treated with a composition comprising about 0.01% to about 1% (e.g., about 0.1%) peracetic acid and about 0.01 to about 10% (e.g., about 1%) hydrogen peroxide.

[0028] Generally, the powder compositions provided herein may contain proteins expressed during early mesodermal development. These proteins may signal stem cells in the blood and in the tissue containing the defect to assemble at the defect site and generate new tissue. The strategy of recruiting native cells from surrounding tissues and assembling them into a provided protein scaffold to fill the tissue defect is significantly different from transplanting tissue-specific mature cells within a mature matrix into the defect site.

[0029] Collagen is the major structural protein of the extracellular matrix (ECM) of connective tissues in the body. It is the most abundant protein in mammals and is found in developing, healing, and mature tissues, as well as in the mesoderm of mammalian embryos. The six most common types of collagen are types I, II, III, IV, V, and VI. Type I collagen is found in mature and healing skin, tendons, blood vessels, organs, intervertebral discs, and bone. Type I collagen is composed of two collagen α-1(I) chains and one collagen α-2(I) chain; therefore, proteomic analyses such as mass spectrometry reveal a 1:1 ratio of type I collagen molecules to collagen α-2(I) chains. Type II collagen is the major form of collagen found in articular cartilage. Type III collagen is most commonly found in healing fibrous tissues, including healing ligaments, tendons, and skin, where it is found together with type I collagen. Type IV collagen is found in the basement membranes of tissues, while type V collagen is found on cell surfaces and in hair and the placenta. Type VI collagen is found in the extracellular matrix of skeletal muscle. Mass spectrometry can detect proteins by determining spectral counts using peptides unique to the protein. In some cases, the compositions provided herein can contain 10% to 90% (e.g., 10% to 50%, 10% to 40%, or 10% to 20%) type I collagen, as quantified by mass spectrometry using normalized spectral counts of collagen α-2(I) chain. The remaining proteins can, in some cases, include fibrillin and / or other types of collagen.

[0030] The powder compositions provided herein may optionally contain fibrillin. Fibrillin is an important glycoprotein in the mesoderm and is essential for the formation of elastic fibers in connective tissues. Because elastic fibers are important components of tissues such as articular cartilage, skin, blood vessels, ligaments, tendons, bone, and intervertebral discs, the presence of fibrillin in the compositions provided herein may aid in the repair of these tissues. Three forms of fibrillin are known in mammals: fibrillin-1, fibrillin-2, and fibrillin-3. While both fibrillin-1 and fibrillin-2 are thought to play important roles in the development of elastic tissues, fibrillin-3 is primarily found in the brain. While fibrillin-1 is thought to provide force-bearing structural support in tissues, fibrillin-2 is thought to induce elastic fiber formation.

[0031] In some cases, the powder compositions provided herein may contain fibrillin-1 and fibrillin-2. Proteins in compositions containing multiple proteins may also be determined using mass spectrometry. In some cases, the spectral count of fibrillin-1 in a composition may be 1% to 99% (e.g., 10% to 90%, 10% to 50%, or 10% to 25%) of the total spectral count of the powder composition. The remaining proteins may include, for example, collagen family members or other fibrillins. In some cases, the powder composition may contain similar spectral counts of fibrillin-1 and type I collagen as measured by spectral counts of collagen α-2(I) chain. In some cases, the powder composition may contain 2 to 20 times more collagen than fibrillin, or 2 to 4 times more type I collagen than fibrillin protein, as determined by spectral counts. In some cases, the spectral count of fibrillin-1 in the powder composition may be 15-25% of the total spectral count, and the spectral count of collagen α-2(I) chain may be 10-20% of the total spectral count of the powder composition.

[0032] In some cases, the powder composition may comprise primarily collagen and fibrillin, with the collagen and fibrillin comprising 10% to 100% of the composition. In some cases, the collagen and fibrillin may comprise 20% to 90% of the composition (e.g., 30% to 80% of the composition), or may comprise at least 50% of the composition. In some cases, the collagen may comprise at least 40% of the composition, and the fibrillin may comprise at least 20% of the composition.

[0033] In addition to proteins, the powder compositions provided herein may contain components such as, but not limited to, one or more of laminin, salts, growth factors, crosslinkers, neutralizing agents, or any combination thereof. For example, laminin is a major component of basement membranes and is an important part of the protein network infrastructure of many organs. Laminin can affect cell differentiation, migration, and adhesion. In some cases, the spectral count of laminin (determined by mass spectrometry) may be 0.1-2% of the total spectral count of the powder compositions provided herein.

[0034] Salts, including sodium chloride, can be useful in the composition for hydrating powders because they can create a powder slurry with an osmotic pressure similar to that of blood, ensuring that blood cells maintain the same size when added to a hydrated powder. Adding blood cells to a solution with low osmotic pressure can cause them to swell to the point of bursting, potentially hindering their ability to function in physiological pathways and promote tissue healing. In some cases, dry salts can be added to powder compositions to ensure that the osmotic pressure of the hydrated powder is similar to that of blood, ranging from about 200 to about 350 mOsm (e.g., about 280 to about 320 mOsm). In some cases, salts can be added to powder compositions in liquid form, such as phosphate-buffered saline, unbuffered saline, or saline solutions with non-normal concentrations (e.g., semi-normal saline, or salt solutions containing other concentrations of salt or other solutes). In some cases, the osmotic pressure of powders hydrated with physiologically buffered saline can be 280 to 320 mOsm.

[0035] In some cases, the compositions provided herein may contain calcium or a calcium salt (e.g., calcium chloride). The calcium chloride may be in solid or liquid form. For example, the salt may be added in solid form to a powder composition before mixing with another liquid (e.g., saline, water, or blood), or the salt may be added in liquid form to a powder composition or a hydrated powder composition. In some cases, the composition may contain calcium at a concentration sufficient to reverse the effects of anticoagulants that act by sequestering calcium (e.g., sodium citrate or citrate dextrose). The calcium may be included in a solution containing salt. When a solution containing salt and calcium is added to a powder composition, the osmolality of the resulting mixture may be close to the physiological osmolality of blood, to the extent that mixing does not adversely affect blood cells, and the calcium concentration may be sufficient to reverse the effects of calcium-sequestering anticoagulants. In some cases, the combination of powder, calcium, salt, and water, prepared immediately before adding a patient's blood, may have an osmolality in the range of about 250 to about 350 mOsm. In some cases, a calcium solution can be mixed with a salt solution before being added to the powder composition, and the resulting combination can have (1) a calcium concentration sufficient to reverse the effects of a calcium-sequestering anticoagulant when added to autologous blood and (2) an osmolality of about 250 to about 350 mOsm (e.g., about 280 to about 320 mOsm). In some cases, a solution containing calcium chloride at a concentration of about 30 mM to about 50 mM (e.g., about 35 mM to about 45 mM, about 38 mM to about 43 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, or about 45 mM) can be used. The calcium chloride solution can be mixed with anticoagulated blood in any suitable ratio. For example, a solution containing about 35 mM to about 45 mM calcium chloride may be combined with blood in a ratio of calcium chloride solution to blood of about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, or about 1:12.

[0036] In some cases, the compositions provided herein may be substantially free of one or more of nucleic acids, glycosaminoglycans (GAGs), phospholipids, active pepsin, and active viruses. A composition that is "substantially free" of a particular component is one that contains less than about 1% by weight (e.g., less than 0.8%, less than 0.5%, or less than 0.1%) of that component.

[0037] This document also provides methods for making the compositions disclosed herein, including powder compositions and two-part compositions comprising a powder and a fluid (e.g., blood). Generally, the method for making the powder includes (1) decellularizing tissue derived from mammalian mesoderm (hereinafter "tissue"), (2) treating the tissue with an enzyme to remove species-specific ends of collagen molecules to obtain a slurry, (3) freeze-drying the tissue slurry, and (4) grinding the freeze-dried tissue slurry to form a powder.

[0038] Decellularization of tissue can be achieved using any suitable agent(s). For example, tissue can be decellularized using one or more detergents, enzymes, salts, or other suitable physical or chemical methods to generate ECM. In some cases, the decellularization method can reduce the DNA content of the tissue, resulting in a DNA content of less than about 20,000 ng / g powder in the final powder composition. The decellularization method can also reduce the phospholipid content of the tissue, resulting in a phospholipid concentration of less than about 3000 μM / g powder in the final powder composition. In some cases, the DNA content of the powder can be less than about 50,000 ng / g powder. In some cases, the phospholipid content of the powder can be less than about 300 μM / g. After the decellularization procedure, the tissue can be washed (e.g., with water or an aqueous solution such as saline) to remove residual chemicals, enzymes, or excess salts.

[0039] In some cases, decellularized tissue (ECM) can be treated with pepsin in an acidic solution (typically pH less than 5) to create a fluid protein slurry. Pepsin or another enzyme can remove telopeptides at the ends of collagen molecules to produce atelocollagen. Atelocollagen is a less immunogenic collagen derivative obtained by removing N- and C-terminal telopeptide components that may induce antigenicity in humans. Therefore, in some cases, pepsin is used to cleave collagen present in the tissue and convert it to atelocollagen. However, it should be noted that other enzymes capable of digesting proteinaceous tissues (such as, but not limited to, collagenase, trypsin, and elastase) can also be used to create the protein slurry. After digestion with pepsin or other suitable enzymes, the resulting protein slurry can be neutralized to a pH greater than 8.5 to inactivate the pepsin or other enzyme(s). Neutralization can be achieved by adding a base (e.g., NaOH) or a buffer (e.g., phosphate buffer). In some cases, a basic solution can be added to the protein slurry to bring the pH of the solution above 7.5, followed by the addition of an acid or buffer to bring the pH of the solution back to 7.0-7.4. This final pH of the slurry can be particularly useful because, when the slurry is freeze-dried and milled into a powder, the powder can be easily mixed with blood without changing the pH of the blood. Furthermore, if the powder is hydrated before mixing with blood, the pH of the hydrated powder solution will be closer to the physiological pH of the blood to which the hydrated powder is added. In some cases, after treatment with pepsin at an acidic pH, the tissue slurry can be neutralized to a pH of 7.5-8.5, followed by the addition of an acid to bring the pH back to 6.5-8.5. In some cases, the tissue slurry can be kept at a temperature below 4°C during this process. Additionally, in some cases, the decellularized tissue may be enzymatically treated with pepsin at a pH below 4.0, the resulting slurry may be adjusted to a pH above 8.5 using a base (e.g., NaOH), and the slurry may then be neutralized to a pH of 7.0-7.4 before lyophilization.

[0040] The step of lyophilizing a mesodermal ECM composition in slurry form typically involves cooling the slurry to a temperature at which the water in the slurry freezes, then applying a vacuum to the frozen slurry to sublimate the water from the composition, resulting in a dry porous sheet. The dried sheet can be ground into a powder by milling, grinding, blending, or other suitable methods. For example, a lyophilized ECM sheet can be ground into a powder by maintaining the temperature of the composition below about 4°C and grinding the composition while it cools. The particles in the resulting powder can have any suitable size. For example, the particles may be about 0.1 mm to about 1 mm (e.g., about 0.1 mm, about 0.2 mm, about 0.3 mm, about 0.4 mm, about 0.5 mm, about 0.6 mm, about 0.7 mm, about 0.8 mm, about 0.9 mm, about 1 mm, less than about 0.3 mm, less than about 0.4 mm, less than about 0.2 mm, about 0.1 to about 0.2 mm, about 0.2 to about 0.3 mm, about 0.3 to about The powder composition may have an average diameter of about 0.4 mm, about 0.4 to about 0.5 mm, about 0.5 to about 0.6 mm, about 0.6 to about 0.7 mm, about 0.7 to about 0.8 mm, about 0.8 to about 0.9 mm, about 0.9 to about 1 mm, about 0.3 to about 0.5 mm, about 0.4 to about 0.6 mm, about 0.3 to about 0.6 mm, about 0.4 to about 0.7 mm, or about 0.5 to about 0.8 mm. In some cases, the powder composition may be filled into a syringe, which may be packaged and / or sterilized prior to use as needed. Alternatively, the powder composition may be placed in some other type of suitable container for storage or use.

[0041] In some cases, the compositions provided herein can be prepared as a slurry suspension, liquid, or semi-liquid by combining mesodermal ECM powder with a cell-free fluid. Such a "hydration composition" can be prepared by mixing a certain amount of the powder composition with a certain amount of a hydration solution. The hydration solution can be, for example, water or a solution containing a salt such as sodium chloride (e.g., saline, phosphate-buffered saline, or a mixture of water and saline). In some cases, the hydration solution can contain calcium, glucose, phosphate, other salts, and / or an anesthetic. For example, the hydration solution can contain calcium at a concentration sufficient to reverse the effects of calcium-reversible anticoagulants such as sodium citrate or citrate dextrose. In some cases, the hydration solution can be a solution containing blood cells or proteins, such as plasma (e.g., autologous or non-autologous plasma).

[0042] In some cases, the proteins in the mesodermal ECM powder compositions provided herein can self-assemble to form a gel within a certain period of time (e.g., about 20 minutes) after mixing with a hydration solution or blood, provided the mixture is at an appropriate temperature (e.g., about 30°C to about 39°C). As used herein, the term "self-assembly" refers to the ability of a composition to change from a viscous liquid to a solid material that can be removed from a container or cut with a knife and does not lose its shape. In some cases, the proteins in a hydrated mesodermal ECM powder composition can self-assemble to form a gel within about 5 minutes after mixing with a hydration solution or blood cells (e.g., red blood cells, white blood cells, platelets, or platelet-rich plasma). In some cases, the compositions provided herein can self-assemble to form a gel within about 10 minutes after mixing with water or blood cells, provided the mixture is at about 32°C. In some cases, when the composition is hydrated so that the osmolality of the resulting solution is between about 270 and 330 mOsm and the pH is between 6.8 and 7.4, the composition can self-assemble at temperatures approximating the temperature inside a human knee.

[0043] This document also provides an article of manufacture comprising the powder composition described herein in any suitable container. In some cases, the powder composition can be in a container having a single chamber containing the composition. Alternatively, the powder composition can be stored in a first chamber of a container that also has a second chamber for containing a hydration solution (e.g., water, saline, calcium solution, or other substance used to hydrate the composition before adding blood components). Alternatively, within a kit, the powder composition can be in a first container and the hydration solution can be in a second container. The containers or chambers can be connected within the kit or can be separate but connectable (e.g., at the time of administration of the composition). In some cases, the powder composition can be in a first syringe, and the hydration solution, if present, can be in a second syringe or other container. In some cases, the kit can include a first syringe containing the composition, a second syringe containing water, and a connector. Prior to administration, the two syringes can be removed from the kit and connected via the connector. In some cases, the hydration solution may be moved into the syringe containing the powder by operating a syringe plunger from outside the chamber containing the powder and solution, and the resulting suspension may be shuttled back and forth between the two syringes to promote uniform mixing. It should be noted that while syringes are particularly useful, any suitable container, connector, and mixing mechanism may be used.

[0044] Additionally, the container can be configured to aid in mixing of the blood with the powder or hydrating composition. The container can be larger than the volume of the composition and any hydrating material, and can accommodate up to 20 cc of autologous blood. In some cases, the container can include a mechanism for mixing the composition (powder and hydrating solution or powder only) with the blood at the point of care. Suitable mechanisms include, but are not limited to, a syringe connector such as a Luer lock, a rupturable membrane between chambers to allow mixing by externally shaking or shaking the container, and an internally collapsible auger controlled by an external plunger. In some cases, the autologous blood can be injected into a sterile syringe, as described herein, and the syringe containing the blood can then be connected to the syringe containing the powder or hydrating composition. The syringe plunger can be used to mix the resulting composition before administering the mixture to the patient.

[0045] The composition, whether hydrated or powdered, can be sterilized before administration to a patient. If the hydration fluid is mixed with the powder composition before the addition of blood, the combined powder and hydration fluid can be sterilized before administration to a patient. This can be accomplished by sterilizing each component separately before placing them in a sterile container, by sterilizing each component in separate containers and mixing them using aseptic techniques, or by sterilizing both components in a single sterile container, with or without a separating partition. Suitable sterilization methods that can be used to reduce the bioburden of the composition include, but are not limited to, radiation (e.g., gamma irradiation or electron beam irradiation), sterilization using free oxygen radicals, gas sterilization (e.g., with ethylene oxide or supercritical CO2), and ultraviolet irradiation. When radiation is used, a dose of 15 to 25 kGy (e.g., 17.5 to 22.5 kGy) can be used. The radiation dose is typically sufficient to reduce both bacterial and viral loads in tissues by 10% during the manufacturing process. -6 In some cases, the compositions provided herein can be sterilized in their final packaging using supercritical CO or electron beam irradiation at a dose of 20 kGy.

[0046] Prior to administration to a patient, the powdered mesodermal ECM compositions provided herein (also referred to as "powder" or "powder composition") are combined with blood or other fluids (e.g., processed blood samples) containing blood cells (e.g., red blood cells). In some cases, the blood is autologous to the patient being treated. However, in some cases, the blood can be from a third-party donor, including other human or animal donors. When blood from other donors is used, it can be processed to reduce the antigenicity of the blood to the recipient prior to administration.

[0047] Blood may be collected immediately (e.g., in a laboratory) or collected on-site during a consultation, processed if desired, and then brought to the administering clinician for mixing / injection. In some cases, whole blood may be used. However, in other cases, a fluid containing blood cells (e.g., red blood cells) (e.g., a processed blood sample) may be used, and the fluid is not whole blood. The fluid may contain plasma proteins, platelets, and / or white blood cells in combination with red blood cells. In some cases, the fluid may contain progenitor cells or stem cells, particularly those normally found in circulating blood. Red blood cells may be present in the fluid at a concentration similar to that normally found in mammalian (e.g., human) blood, or the blood may be processed to result in a red blood cell concentration higher or lower than that in normal mammalian blood. In some cases, the red blood cell concentration may be within about 10% of the concentration in the circulating blood of the patient to whom the composition is administered.

[0048] In some cases, a blood sample drawn from a patient may be processed before mixing with the powder or hydrated powder composition. For example, the blood sample may be processed by centrifugation, filtration, and / or passage through a cell separation column to separate specific cell types in the blood. In some cases, platelets may be separated using continuous centrifugation and then resuspended in plasma at a higher concentration than in unprocessed blood (thereby producing platelet-rich plasma "PRP"). Enrichment of other types of cells, such as white blood cells or specific subpopulations of white blood cells, such as stem cells, may also be performed to enhance the effectiveness of the final composition at the tissue defect being treated. In some cases, blood containing physiological levels of blood cells and plasma proteins (whole blood) may be used.

[0049] Any suitable phlebotomy method may be used to obtain a blood sample from a mammal (e.g., a human patient). Typically, blood may be obtained from the mammal using a needle. The needle may have any suitable size, typically 14 gauge to 22 gauge (e.g., 16 gauge to 20 gauge). In some cases, the needle may be an 18 gauge needle, which is typically the smallest size that does not significantly damage cells in the blood. The skin of the mammal from which blood is to be drawn may be cleansed in preparation to remove bacteria from the skin. A tourniquet may be used proximal to the blood collection site to increase the size and visibility of the vessel from which the blood sample is to be taken. A needle (e.g., 18 gauge or larger) may be placed through the skin into the vessel, and blood may be expelled through the needle into a syringe or tube. In some cases, blood may be first pumped into a tube that is under vacuum to draw the required amount of blood into the tube.

[0050] In some cases, blood from a mammal (e.g., a human patient) can be injected into a syringe or tube containing an anticoagulant (e.g., a liquid or solid anticoagulant). The anticoagulant can be an anticoagulant that can be reversed by the addition of calcium, such that if the powder composition contains calcium, adding blood to the powder composition reverses the anticoagulant effect. Examples of this type of anticoagulant include, but are not limited to, sodium citrate and acid citrate dextrose. In some cases, blood can be injected into a tube containing liquid acid citrate dextrose, with the volume of blood being 10 times the volume of the acid citrate dextrose. In some cases, blood can be injected into a syringe, tube, or other container containing a calcium-chelating anticoagulant (e.g., acid citrate dextrose) in an amount sufficient to prevent clotting of the blood. In some cases, when a calcium-chelating anticoagulant is used, a calcium solution can be added to the powdered ECM composition or the blood immediately before mixing the blood with the ECM composition to reverse the anticoagulant effect and allow the blood to clot. If no anticoagulant is used, the blood may be mixed with the powder composition (or hydrated powder composition) within 5 minutes of venipuncture. The blood may be stored at room temperature until use.

[0051] The blood and powdered mesodermal ECM composition may be mixed in any suitable amount. In some cases, the compositions provided herein may be hydrogels containing mesodermal ECM powder and blood, with the powder present at a concentration of about 50 mg / mL to about 200 mg / mL (e.g., about 50 to about 100 mg / mL, about 100 to about 150 mg / mL, or about 150 to about 200 mg / mL). In some cases, for example, 200 mg of the powder composition may be mixed with 3 mL of blood, with the powder concentration in the mixture being about 67 mg / mL. In some cases, 400 mg of the powder composition may be mixed with 3 mL of blood, with the powder concentration in the mixture being about 133 mg / mL.

[0052] Furthermore, the blood and powdered mesodermal ECM composition can be mixed in any suitable manner. For example, in some cases, blood can be poured into a container containing the powdered mesodermal ECM composition and mixed by stirring. In some cases, blood can be injected into a syringe containing the powdered mesodermal ECM composition by actuating the syringe plunger. In some cases, a vacuum can be generated in a first syringe containing the powdered mesodermal ECM composition, and a second syringe containing blood can be connected to the first syringe (e.g., via a connector with a luer lock or valve). When the valve is opened so that the chambers of the first and second syringes are in fluid communication with each other, the vacuum can draw blood into the first syringe. Furthermore, the vacuum can facilitate mixing of the powder and blood.

[0053] In some cases, the powdered mesodermal ECM composition may be hydrated with an acellular fluid (a "hydration solution") before mixing with blood (or other fluid containing blood cells). The hydration fluid may include, for example, water and one or more of sodium, chloride, calcium, phosphate, glucose, and / or other molecules typically found in injectable saline or phosphate-buffered saline. The ratio of composition to fluid used to hydrate the composition may be such that when the hydrated powder is mixed with blood, the resulting mixture forms a gel-like matrix.

[0054] In some cases, the powder composition may be mixed with a hydrating fluid to disperse the powder particles before mixing with blood. The powder-to-water ratio may range from about 100 mg powder to 0.1 mL water to about 100 mg powder to 5 mL water. For example, 100 mg powder to 0.5 mL water or saline may be used. In some cases, the fluid may contain a level of calcium that may be sufficient to reverse the effects of calcium-binding anticoagulants, such as sodium citrate or acid citrate dextrose. In some cases, 600 mg of the powder composition may be mixed with 3 mL of water, and the hydrated composition may then be mixed with 3 mL of autologous blood infused without an anticoagulant (resulting in a composition with a powder concentration of 100 mg / mL). In other cases, 600 mg of the powder composition may be combined with 3 mL of water containing calcium, and the hydrated composition may then be mixed with 3 mL of blood anticoagulated with acid citrate dextrose (again resulting in a composition with a powder concentration of 100 mg / mL).

[0055] This document also provides methods for treating, preventing, or reducing the likelihood of onset or progression of arthritis (e.g., OA). In some cases, the methods provided herein can be performed during a routine office visit, in a room specially designed for such procedures (including injections), or in an operating room. The methods can be performed after administering topical, local, oral, regional, general, or painkiller anesthesia to the patient, or without anesthesia. The methods can include delivering a composition provided herein without visualization of the defect to be treated, or visualization with the direct naked eye, or with imaging techniques such as ultrasound, MRI, X-ray, arthroscopy, or computed tomography (CT) scan. In some cases, for example, the methods provided herein can be performed using local anesthesia in a clinic setting.

[0056] Injecting a composition containing ECM proteins derived from tissues of mesodermal origin and blood into a joint may reduce or reverse the effects of OA. Combining the powder composition provided herein with a patient's blood may promote the formation of copolymers between proteins from the powder (e.g., collagen and fibrillin) and proteins in the patient's blood (e.g., fibronectin). These copolymers may bind to exposed collagen in cartilage damaged by early OA, providing a provisional scaffold for cartilage healing. The solidified gel-like material may serve as an effective scaffold for cells from surrounding joint tissue and cartilage to populate and remodel into functional cartilage. Without being bound by theory, proteins found in the developing mammalian mesoderm (e.g., collagen and fibrillin) may enable stem cells from surrounding tissues to heal the defect. Preclinical animal studies have shown that this technique repairs the joint surface and enables more normal walking in animals with arthritis within weeks of injection (see the Examples section of this document). Furthermore, treatment using the methods provided herein has been shown to be capable of filling cartilage defects, reversing osteoarthritis-related gait changes, and slowing (and in some cases halting) the progression of PTOA as seen on radiographs, even when treatment is performed a significant amount of time after injury.

[0057] The methods provided herein do not require surgery and can be used to treat mammals (e.g., humans) with various types of arthritis. For example, mammals (e.g., humans) with idiopathic arthritis, inflammatory arthritis, rheumatoid arthritis, PTOA, or other subtypes of arthritis, or cartilage damage can be treated using the compositions and methods provided herein.

[0058] In some cases, the methods and compositions provided herein can be used to fill tissue defects. The tissue defect can be in any tissue. For example, the tissue defect can be in musculoskeletal connective tissue, including, but not limited to, articular cartilage, meniscus, bone, ligament, tendon, skin, and discs (e.g., spinal discs and temporomandibular joint discs). The defect can be a full-thickness defect or a partial-thickness defect, and can be a visible defect or a microscopic defect such as that seen in tendinopathy. For intervertebral discs, the defect can involve the annulus fibrosis. The defect can be a tissue defect in a joint (intra-articular) or outside of a joint. In some cases, the methods provided herein can be used to fill a tissue defect that causes pain or injury to the mammal being treated.

[0059] In some cases, the methods and compositions provided herein can be used to fill articular cartilage defects. The articular cartilage defect can extend to the subchondral bone, extend to a cartilage tidemark, or be on the surface of a tidemark. The defect can be a partial-thickness defect or a full-thickness defect, can include a fissure and / or a shoulder or non-shoulder lesion, and can range in size from about 0.1 mm to the entire articular surface (e.g., about 0.1 mm to about 0.3 mm, about 0.3 to about 0.5 mm, about 0.5 mm to about 1 mm, about 1 mm to about 2 mm, about 2 mm to about 4 mm, about 3 mm to about 5 mm, or greater than 5 mm). The joint being treated can have one articular cartilage defect or more than one articular cartilage defect (e.g., two, three, four, five, or more than five defects).

[0060] The joints treated may be in the upper or lower limbs (also known as appendicular joints), the spine, or elsewhere in the body. Examples of appendicular joints include, but are not limited to, the knee, tibiotalar joint, subtalar joint, joints of the midfoot, metatarsophalangeal joint, metacarpal joint, metacarpal-phalangeal joint, other joints of the hand, wrist joint, elbow joint, and shoulder joint. Examples of spinal joints include facet joints. In some cases, the methods provided herein may be used to treat multiple partial-thickness defects in articular cartilage of the knee joint.

[0061] Thus, after mixing the powder composition (either hydrated or non-hydrated) with blood (or a fluid containing blood cells), the resulting mixture can be injected into the joint, tissue defect, or injury to be treated (e.g., an injury to a ligament, tendon, bone, or cartilage, such as a meniscus, labrum, or disc). In some cases, joint degeneration can be treated without damaging the cartilage, ligament, tendon, meniscus, labrum, disc, or bone. Tissue defects that can be treated according to the methods provided herein can be intra-articular or extra-articular. In some cases, the tissue defect to be treated may be one that does not heal as quickly as the patient or clinician would like, including, for example, cartilage damage or degeneration, fractures requiring internal fixation, open fractures, intra-articular fractures, rotator cuff tendon injury, meniscus tears, labral tears, intervertebral disc herniations, degenerate temporomandibular disc injuries, and injuries to the triangular fibrocartilage construct of the wrist.

[0062] In some cases, hydrogels generated from powdered mesodermal ECM and blood may be delivered via injection rather than incision. Such delivery may be advantageous in particular clinical situations, including, but not limited to, the following: treatment of delayed union of fractures (additional incisions may further compromise the local blood supply necessary to achieve healing), treatment of partial-thickness rotator cuff tears (where the morbidity of deep muscle approaches may outweigh the potential benefits of repair), treatment of Achilles tendon rupture (where additional incisions may further compromise the local blood supply and increase wound healing problems), and treatment of tendinopathy without tissue tear. In some cases, the hydrogel compositions provided herein may be injected into the knee joint to treat partial-thickness cartilage defects.

[0063] In some cases, the hydrogel generated from powdered mesodermal extracellular matrix (ECM) and blood can be administered by direct injection from a syringe or other suitable method. For example, the hydrogel can be administered through an arthroscopic cannula or portal, or through an incision. In some cases, the hydrogel can be administered using imaging techniques such as ultrasound, magnetic resonance imaging, computed tomography, x-ray, fluoroscopy, or needle arthroscope.

[0064] In some cases, tissue defects other than cartilage defects may also be treated using the compositions and methods provided herein. For example, partial or complete tears of tendons or ligaments, meniscus tears, and labrum tears may be treated by injection of the hydrogel compositions provided herein, as may microscopic tissue injuries (e.g., tendinosis, tendinopathy, sprains, and ligament and tendon strains).

[0065] The methods provided herein may be used to treat those at risk for OA, including, in some cases, patients with previous injury or surgery. The methods provided herein may be used to treat mammals (e.g., humans) with any type of cartilage injury or arthritis. For example, the methods provided herein may be used to treat single cartilage defects, multiple cartilage defects, and cartilage damage caused by rheumatoid arthritis, gout, inflammatory arthritis, psoriatic arthritis, or infection. These methods may also be used to prevent or reduce the risk of future arthritis development. For example, the methods provided herein may be used to prevent or reduce the likelihood of post-traumatic arthritis, in which certain joint injuries can lead to cartilage damage and arthritis years later. This may occur, for example, when a patient tears the anterior cruciate ligament in their knee and, despite having the ligament surgically repaired, develops early arthritis at a much younger age than uninjured patients. These methods may also be applied to patients with other knee injuries, such as meniscus injuries, bone injuries, or cartilage damage. Furthermore, these methods may be used in joints other than the injured knee. In some cases, the powder compositions provided herein may be mixed with blood and administered by injection to a mammal (e.g., a human patient) with mild cartilage damage to reduce the likelihood that the damage will progress to OA.

[0066] During surgical repair of a joint, accidental damage to the cartilage within the joint can occur, potentially leading to early arthritis. In some cases, the methods provided herein can include administering a composition comprising mesodermal ECM powder and blood via injection through an arthroscopic portal, through closed skin, or through an open incision at the completion of a surgical procedure on a joint to reduce the likelihood that cartilage damage occurring during surgery will progress to arthritis. In some cases, the methods provided herein can include administering the mesodermal ECM powder / blood composition during a surgical procedure performed to treat other tissues (e.g., to support cartilage after ACL or meniscus surgery). The surgical procedure can be, for example, a partial meniscectomy, meniscus repair, ACL reconstruction, ACL repair, labral resection or repair of the hip or shoulder joint, treatment of osteochondritis dissecans in any joint, or other surgical procedure performed on a joint.

[0067] In some cases, the methods provided herein can be used to complement surgical repair of cartilage or other tissues. For example, if a microfracture procedure is performed as the primary treatment for an articular cartilage defect, the powdered mesodermal ECM and blood composition provided herein can be administered to the site after the microfracture procedure to aid healing. As another example, after mosaicplasty using a bone and cartilage plug, the composition provided herein can be administered to aid healing of the plug and fill the gap between the surgically implanted plug and the surrounding cartilage. In some cases, if a patch is placed to cover a cartilage defect, the composition provided herein can be injected under, over, or near the patch to help promote healing. In some cases, when cells are injected into a cartilage defect, the cells can first be mixed with the composition provided herein, and then the mixture can be injected into the defect. In such cases, the powdered mesodermal ECM / blood composition can aid in localizing the cells delivered to the injured tissue site. As another example, when a rotator cuff tendon is repaired with sutures and / or bone fixation, a composition provided herein can be administered to the repaired tissue defect to improve healing. Administration can occur before placing sutures, or after placing sutures but before tightening the sutures to reapproximate the tendon ends or tendon to the bone, or after placing and ligating the sutures. In some cases, when a meniscus is repaired with sutures, a composition provided herein can be administered to the repaired tissue defect to improve healing. Administration can occur before placing sutures, or after placing sutures but before tightening the sutures to close the wound gap, or after placing and ligating the sutures.Similarly, the compositions provided herein can be used to augment the repair of the shoulder labrum, hip labrum, ligaments (including, but not limited to, the anterior cruciate ligament, posterior cruciate ligament, medial collateral ligament, lateral collateral ligament, talofibular ligament, and ulnar collateral ligament), and tendons (including, but not limited to, the Achilles tendon, flexor tendons of the hand, tendons attaching to the lateral epicondyle of the distal humerus, quadriceps tendon or patellar tendon, and biceps tendon). For mechanically stable defects, including partial tears of ligaments or tendons, the methods provided herein can be used to treat these defects without the use of sutures.

[0068] Illustrative Embodiments Embodiment 1 is a composition comprising a powdered extracellular matrix (ECM) component and a fluid, wherein the ECM component comprises collagen, and the concentration of the powdered ECM component in the fluid is about 50 mg / mL to about 200 mg / mL.

[0069] Embodiment 2 is the composition of embodiment 1, wherein the powdered ECM component has a concentration in the fluid of about 100 to about 150 mg / mL.

[0070] Embodiment 3 is the composition of embodiment 1 or embodiment 2, wherein the fluid is blood.

[0071] Embodiment 4 is the composition of any one of embodiments 1-3, wherein the powdered ECM component has an average particle size of about 0.1 mm to about 1 mm.

[0072] Embodiment 5 is the composition of any one of embodiments 1-4, further comprising a growth factor, platelets, white blood cells, stem cells, a crosslinking agent, a neutralizing agent, or any combination thereof.

[0073] Embodiment 6 is the composition of any one of embodiments 1-5, further comprising calcium.

[0074] Embodiment 7 is the composition of any one of embodiments 1-6, wherein the composition is substantially free of one or more of nucleic acids, glycosaminoglycans (GAGs), phospholipids, active pepsin, and active viruses.

[0075] Embodiment 8 is a method of making a composition comprising a fluid and a powdered ECM component comprising collagen, comprising the steps of: providing a syringe containing a powdered ECM component; Inject a certain amount of fluid into the syringe so that the concentration of powdered ECM components in the fluid is approximately 50 mg / mL to approximately 200 mg / mL. The method includes:

[0076] Example 9 is the method of Example 8, comprising injecting a volume of fluid into the syringe such that the concentration of the powdered ECM component in the fluid is about 100 to about 150 mg / mL.

[0077] Embodiment 10 is the method of embodiment 8 or embodiment 9, wherein the fluid is blood.

[0078] Embodiment 11 is the method of any one of embodiments 8-10, wherein the powdered ECM component has an average particle size of about 0.1 mm to about 1 mm.

[0079] Embodiment 12 is the method of any one of embodiments 8 to 11, wherein the ECM composition further comprises growth factors, platelets, leukocytes, stem cells, a crosslinking agent, a neutralizing agent, or any combination thereof.

[0080] Embodiment 13 is the method of any one of embodiments 8 to 12, wherein the composition further comprises calcium.

[0081] Embodiment 14 is the method of any one of embodiments 8 to 13, wherein the composition is substantially free of one or more of nucleic acids, GAGs, phospholipids, active pepsin, and active viruses.

[0082] Embodiment 15 is a method of treating a mammal, the method comprising administering to a joint of the mammal suffering from or at risk of developing arthritis an effective amount of a composition comprising powdered ECM components and a fluid, wherein the ECM components comprise collagen, and the powdered ECM components have a concentration in the fluid of about 50 mg / mL to about 200 mg / mL.

[0083] Embodiment 16 is the method of embodiment 15, wherein the concentration of the powdered ECM components in the fluid is about 100 to about 150 mg / mL.

[0084] Embodiment 17 is the method of embodiment 15 or embodiment 16, wherein the fluid is blood.

[0085] Embodiment 18 is the method of any one of embodiments 15-17, wherein the powdered ECM component has an average particle size of about 0.1 mm to about 1 mm.

[0086] Embodiment 19 is the method of any one of embodiments 15-18, wherein the ECM composition further comprises growth factors, platelets, leukocytes, stem cells, a crosslinking agent, a neutralizing agent, or any combination thereof.

[0087] Embodiment 20 is the method of any one of embodiments 15-19, wherein the composition further comprises calcium.

[0088] Embodiment 21 is the method of any one of embodiments 15-20, wherein the composition is substantially free of one or more of nucleic acids, GAGs, phospholipids, active pepsin, and active viruses.

[0089] Embodiment 22 is the method of any one of embodiments 15 to 21, wherein the mammal has an acute injury to a joint.

[0090] Embodiment 23 is the method of any one of embodiments 15 to 21, wherein the arthritis is osteoarthritis.

[0091] Embodiment 24 is the method of any one of embodiments 15 to 21, wherein the arthritis is post-traumatic arthritis.

[0092] Embodiment 25 is the method of embodiment 24, wherein the post-traumatic arthritis is associated with an intra-articular injury or arthroscopic surgery.

[0093] Embodiment 26 is the method of embodiment 25, wherein the intra-articular injury is selected from the group consisting of anterior cruciate ligament rupture, anterior cruciate ligament rupture, meniscus injury, and cartilage injury.

[0094] Embodiment 27 is the method of any one of embodiments 15 to 26, wherein the mammal has undergone a surgical procedure for torn, fractured, strained, bruised, or ruptured intra-articular tissue in a joint at least one day prior to administering the composition.

[0095] Embodiment 28 is the method of any one of embodiments 15 to 26, wherein the joint is a joint of the hand, elbow, wrist, hip, knee, foot, shoulder, ankle, temporomandibular joint, or spine.

[0096] Embodiment 29 is the method of any one of embodiments 15 to 28, wherein the mammal has an injury associated with the development of arthritis.

[0097] Embodiment 30 is the method of any one of embodiments 15 to 29, wherein administering comprises direct injection into the joint.

[0098] Embodiment 31 is the method of any one of embodiments 15 to 30, wherein the mammal is a human.

[0099] Embodiment 32 is a method of treating a mammal having an intra-articular tissue defect, comprising visualizing the defect with an arthroscope and then administering to the defect an effective amount of a composition comprising powdered ECM components and a fluid, wherein the ECM components comprise collagen, and the concentration of the powdered ECM components in the fluid is from about 50 mg / mL to about 200 mg / mL relative to the defect.

[0100] Embodiment 33 is the method of embodiment 32, wherein the concentration of the powdered ECM components in the fluid is about 100 to about 150 mg / mL.

[0101] Embodiment 34 is the method of embodiment 32 or embodiment 33, wherein the fluid is blood.

[0102] Embodiment 35 is the method of any one of embodiments 32-34, wherein the powdered ECM component has an average particle size of about 0.1 mm to about 1 mm.

[0103] Embodiment 36 is the method of any one of embodiments 32-35, wherein the ECM composition further comprises growth factors, platelets, leukocytes, stem cells, a crosslinking agent, a neutralizing agent, or any combination thereof.

[0104] Embodiment 37 is the method of any one of embodiments 32-36, wherein the composition further comprises calcium.

[0105] Embodiment 38 is the method of any one of embodiments 32-37, wherein the composition is substantially free of one or more of nucleic acids, GAGs, phospholipids, active pepsin, and active viruses.

[0106] Embodiment 39 is the method of any one of embodiments 32 to 38, wherein the defect is an acute injury in a joint.

[0107] Embodiment 40 is the method of embodiment 39, wherein the defect is selected from the group consisting of anterior cruciate ligament rupture, anterior cruciate ligament rupture, meniscus injury, and cartilage injury.

[0108] Embodiment 41 is the method of any one of embodiments 32 to 40, wherein the defect is an injury associated with the development of arthritis.

[0109] Embodiment 42 is the method of any one of embodiments 32 to 41, wherein the administering comprises direct injection into the defect.

[0110] Embodiment 43 is the method of any one of embodiments 32 to 42, wherein the mammal is a human.

[0111] Embodiment 44 is a method for producing a powder composition comprising mesodermal extracellular matrix (ECM) proteins, comprising the following steps: decellularizing a tissue sample comprising tissue from mammalian mesoderm; treating the tissue sample with a composition comprising peracetic acid before or after decellularization; freeze-drying the decellularized tissue sample; and grinding the freeze-dried tissue into a powder.

[0112] Embodiment 45 is the method of embodiment 44, wherein the composition comprising peracetic acid comprises about 0.1% peracetic acid.

[0113] Embodiment 46 is the method of embodiment 44 or embodiment 45, comprising treating the tissue sample with a composition comprising peracetic acid for about 5 to 30 minutes before or after decellularization.

[0114] Embodiment 47 is the method of any one of embodiments 44-46, wherein the composition comprising peracetic acid further comprises hydrogen peroxide.

[0115] Embodiment 48 is the method of embodiment 47, wherein the composition comprises about 1% hydrogen peroxide.

[0116] Embodiment 49 is the method of any one of embodiments 44 to 48, further comprising treating the decellularized tissue sample with an enzyme prior to freeze-drying, thereby removing species-specific ends of the collagen molecules.

[0117] Embodiment 50 is the method of any one of embodiments 44 to 49, further comprising treating the powder with supercritical carbon dioxide (scCO2).

[0118] Embodiment 51 is the method of any one of embodiments 44 to 50, wherein the powder has an average particle size of about 0.1 mm to about 1 mm.

[0119] Embodiment 52 is the method of any one of embodiments 44-51, wherein the composition further comprises a growth factor, platelets, white blood cells, stem cells, a crosslinking agent, a neutralizing agent, or any combination thereof.

[0120] Embodiment 53 is the method of any one of embodiments 44-52, wherein the composition further comprises calcium.

[0121] Embodiment 54 is the method of any one of embodiments 44 to 52, wherein the composition is substantially free of one or more of nucleic acids, GAGs, phospholipids, active pepsin, and active viruses.

[0122] Embodiment 55 is a method of producing a powder composition comprising mesodermal ECM proteins, comprising the steps of decellularizing a tissue sample comprising tissue from mammalian mesoderm; freeze-drying the decellularized tissue sample; grinding the freeze-dried tissue slurry into a powder; and treating the powder with scCO2.

[0123] Embodiment 56 is the method of embodiment 55, further comprising treating the decellularized tissue sample with an enzyme prior to lyophilization, thereby removing species-specific ends of collagen molecules.

[0124] Embodiment 57 is the method of embodiment 55 or embodiment 56, further comprising treating the tissue sample with a composition comprising peracetic acid before or after decellularization.

[0125] Embodiment 58 is the method of embodiment 57, wherein the composition comprising peracetic acid comprises about 0.1% peracetic acid.

[0126] Embodiment 59 is the method of embodiment 57 or embodiment 58, comprising treating the tissue sample with a composition comprising peracetic acid for about 5 to 30 minutes before or after decellularization.

[0127] Embodiment 60 is the method of any one of embodiments 57-59, wherein the composition comprising peracetic acid further comprises hydrogen peroxide.

[0128] Embodiment 61 is the method of embodiment 60, wherein the composition comprises about 1% hydrogen peroxide.

[0129] Embodiment 62 is the method of any one of embodiments 55 to 61, wherein the powder has an average particle size of about 0.1 mm to about 1 mm.

[0130] Embodiment 63 is the method of any one of embodiments 55 to 62, wherein the composition further comprises a growth factor, platelets, white blood cells, stem cells, a crosslinking agent, a neutralizing agent, or any combination thereof.

[0131] Embodiment 64 is the method of any one of embodiments 55 to 63, wherein the composition further comprises calcium.

[0132] Embodiment 65 is the method of any one of embodiments 55-64, wherein the composition is substantially free of one or more of nucleic acids, GAGs, phospholipids, active pepsin, and active viruses.

[0133] Embodiment 66 is a method for producing a composition comprising blood and powdered mesodermal ECM components including collagen, the method comprising the following steps: providing a syringe containing the powdered ECM components; contacting the blood sample with an anticoagulant; injecting a certain amount of blood into the syringe containing the powdered ECM components so that the concentration of the powdered ECM components in the blood is about 50 mg / mL to about 200 mg / mL; and adding a calcium chloride solution to the syringe, thereby inactivating the anticoagulant.

[0134] Embodiment 67 is the method of embodiment 66, comprising injecting a volume of blood into the syringe such that the concentration of the powdered ECM components in the blood is about 100 to about 150 mg / mL.

[0135] Embodiment 68 is the method of embodiment 66 or embodiment 67, wherein the powdered ECM component has an average particle size of about 0.1 mm to about 1 mm.

[0136] Embodiment 69 is the method of any one of embodiments 66 to 68, wherein the ECM composition further comprises growth factors, platelets, leukocytes, stem cells, a crosslinking agent, a neutralizing agent, or any combination thereof.

[0137] Embodiment 70 is the method of any one of embodiments 66-69, wherein the composition is substantially free of one or more of nucleic acids, GAGs, phospholipids, active pepsin, and active viruses.

[0138] Embodiment 71 is the method of any one of embodiments 66 to 70, wherein the calcium chloride solution has a concentration of about 35 mM to about 45 mM.

[0139] Embodiment 72 is the method of any one of embodiments 66-71, comprising adding calcium chloride solution to the syringe to obtain a 1:9 calcium chloride solution to blood mixture.

[0140] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims. [Example]

[0141] Example 1 - Delaying post-traumatic osteoarthritis in vivo Sixty Lewis rats were randomly assigned to one of three groups (20 rats per group), and each group underwent unilateral surgery as follows: (1) a capsular resection but no ACL injury / manipulation (sham group); (2) the ACL was resected and injected with PBS (placebo group); or (3) the ACL was resected and injected with a composition containing multiple mesodermal proteins, including collagen and fibrillin (treatment group).

[0142] Six weeks after surgery, knees were evaluated for the presence or absence of PTOA evident by radiographic (radiographic) and microscopic evaluation (histology) changes in the cartilage.

[0143] The mesodermal protein composition was aseptically produced from decellularized bovine elastic tissue. Bovine elastic tissue (Maverick BioSciences, New Zealand) was incubated in an antibiotic solution to inactivate infectious particles introduced during collection, and the tissue was decellularized using Triton X-102. The tissue was washed and treated with pepsin digestion and hydrochloric acid solubilization. The resulting mesodermal protein slurry was lyophilized and subsequently rehydrated to a concentration of at least 45 mg / g collagen. The concentrated slurry was neutralized with NaOH and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, and the osmolality of the slurry was adjusted to 295 mOsm with aqueous CaCl2. The slurry was lyophilized and milled into a powder. A single dose of 20 mg of the powder composition was filled into each 1 mL syringe. The filled syringes were stored at room temperature and protected from light.

[0144] During animal surgery, the ACL was exposed via an anteromedial approach, sharply transected under direct vision, and then the capsule was closed. In the sham surgery group, only the ACL was exposed. In the placebo group, the ACL was transected and the capsule was closed, followed by an ultrasound-guided saline injection. In the treatment group, 20 mg of a protein powder composition containing collagen and fibrillin was first rehydrated with 400 μL of water and mixed between two syringes to create a hydrated composition. Next, 200 μL of the hydrated composition was mixed with 200 μL of autologous blood to form a homogeneous mixture, and 100 μL of the two-part mixture was injected into the synovial fluid of the knee using a 25-gauge 5 / 8-inch needle. During injection, the cartilage was not visualized, and the injection was injected into the intraarticular fluid rather than directly into the cartilage.

[0145] Mass spectrometry analysis confirmed the presence of multiple mesodermal proteins in the mesodermal protein powder composition used in the treatment groups, including type I collagen, type III collagen, fibrillin-1, type VI collagen, type II collagen, type V collagen, type VI collagen, fibrillin-2 precursor, and laminin. The collagen concentration in the composition was approximately 500,000 μg / g powder, the DNA content was approximately 40,000 ng / g powder, and the residual pepsin content was less than 122 mg / g powder (the lower limit of measurable value of the assay). The sterility of the mesodermal protein powder composition was confirmed using bioburden testing. Gelation of the aseptically processed mesodermal protein composition occurred at room temperature and 37°C, and there was no blurring of the cut surface when the sample was removed from the mold and cut with a straight blade.

[0146] Six weeks after surgery and injection, radiographs of the knees were taken. Representative images are shown in Figure 1. In the sham-operated group (left panel), where no ACL transection was performed, there was no loss of joint space, the subchondral bone appeared normal, and no osteophytes were observed. In the placebo group (middle panel), knees that received PBS injections after ACL transection showed significant narrowing of the joint space, accompanied by sclerosis of the subchondral bone and osteophyte formation, by six weeks after surgery. In contrast, in knees that received an injection of a mixture of sterilely processed mesodermal protein composition and autologous blood after ACL transection, the knee joint space was maintained (black arrows) at six weeks after surgery, and there was no subchondral bone thickening or osteophyte formation. These results demonstrate that ACL transection treated with placebo injections in a rat model leads to radiographic findings of PTOA six weeks after surgery. Injection of a two-part mixture of mesodermal protein composition and blood at the time of trauma (ACL rupture) prevented the development of post-traumatic osteoarthritis.

[0147] Figure 2 contains images showing the histology of the medial tibial plateau of a representative knee that did not undergo surgery (left panel), a representative knee that underwent ACL transection 6 weeks prior and received a saline placebo injection (center panel), and a knee that underwent ACL transection and received a two-part mixture injection 6 weeks prior to the ACL transection (right panel). The knee that did not undergo surgery had normal-appearing cartilage, while the knee that underwent ACL transection and placebo injection had an irregular cartilage surface and matrix loss (center panel). In contrast, the knee that underwent ACL transection and was treated with the mesodermal protein and blood mixture had normal-appearing cartilage. Thus, injection of the mesodermal protein composition after ACL injury prevented the development of PTOA.

[0148] Quantitative histological examination using the Mankin structural score revealed severe arthritic changes (defined as a score of 7 or 8) in 11% of knees without ACL transection (sham-operated group), 21% of knees treated with PBS, and 5% of knees injected with a mesodermal protein composition mixed with blood after ACL transection.

[0149] Example 2 - Treatment of Osteoarthritis (OA) without Prior Injury The Dunkin-Hartley guinea pig spontaneously develops knee OA between 6 and 9 months of age and is a widely recognized and utilized model of spontaneous OA. While spontaneous OA typically develops over decades in humans, it develops within a few months in Dunkin-Hartley animals.

[0150] In these experiments, 20 6-month-old Dunkin'-Hartley guinea pigs were divided into two experimental groups. One group (n = 5) received no injections (control group). The remaining 15 animals were treated with a single injection of mesodermal protein composition and blood into both knees at week 0 (treatment group). Five animals were euthanized at weeks 1, 2, and 4 after injection, and their knees were evaluated histologically. Mesodermal protein powder was produced by decontaminating bovine elastic tissue with a solution containing hydrogen peroxide, decellularizing the tissue, washing the tissue, and freeze-drying it. After freeze-drying, the tissue was digested with pepsin in an acid solution. The resulting slurry was neutralized, freeze-dried, and ground into a powder, which was then filled into 10 mL syringes at 125 mg doses. The syringes were sealed and packaged, and the powder was terminally sterilized using electron beam irradiation at a dose of at least 20 kGy.

[0151] Mass spectrometry analysis confirmed the presence of multiple mesodermal proteins in the mesodermal protein powder used in the treatment groups, including type I collagen, type III collagen, fibrillin-1, type VI collagen, type II collagen, type V collagen, type VI collagen, fibrillin-2 precursor, and laminin, as well as other extracellular matrix proteins. The collagen concentration in the composition was approximately 500,000 μg / g powder, the DNA content was approximately 40,000 ng / g powder, and the residual pepsin content was below 122 mg / g powder (the lower limit of measurable value of the assay). The sterility of the mesodermal protein powder after sterilization was confirmed. The self-assembly ability of the composition was assessed by mixing a 125 mg powder sample with 0.5 mL of water. The ability of the contained proteins to self-assemble and form a gel was confirmed by placing the mixture sample in a cylindrical tube at 32°C (knee joint temperature). The resulting mixture transformed from a liquid composition to a solid gel within 60 minutes, indicating successful self-assembly. The gel was easily removed from the tube and maintained its cylindrical shape after being placed on a flat surface. Gel stability was tested by cutting a cylinder of gel with a straight blade and assessing its ability to retain its shape. Cutting the test sample with a straight blade did not result in blurring of the cut surface or loss of column integrity.

[0152] The two-part mixture of mesodermal protein composition and blood was injected by adding 0.5 mL of water to a prepared syringe containing 125 mg of lyophilized extracellular matrix protein to form a hydrated composition. 100 μL of the hydrated composition was placed in a 1 mL syringe. Autologous blood was collected from each animal into a syringe containing anticoagulant. Calcium chloride was added to the blood immediately before mixing with the hydrated composition to inactivate the anticoagulant. 200 μL of autologous blood was mixed with 100 μL of the hydrated composition to form a homogenous mixture. After mixing with the autologous blood, the hydrated composition was injected into the synovial fluid of the knee without visualizing the cartilage. Histological examination of the medial tibial plateau was performed on knees 1, 2, and 4 weeks after injection. Gait assessment (base of support) was performed on the control and treatment groups at baseline (time 0) and 1, 2, and 4 weeks after injection.

[0153] Histological analysis of articular cartilage revealed that one week after injection, the area of ​​the medial tibial plateau where superficial cartilage had been lost due to spontaneous arthritis had been filled with mesodermal protein composition / blood mixture material. Toluidine blue stains glycosaminoglycans, and as shown in Figure 3, areas of mesodermal protein composition / blood mixture (which had significantly less glycosaminoglycan than the native cartilage) appeared as light blue areas on the surface of the injured cartilage. At one week, no cells were observed in the mesodermal protein composition / blood mixture (left panel). By two weeks after injection, some of the light blue mesodermal protein composition / blood mixture had been replaced by articular cartilage (which had a high glycosaminoglycan content and contained chondrocytes), but areas of acellular mesodermal protein composition / blood mixture with less glycosaminoglycan remained (middle panel, lighter areas). By four weeks after injection of the mesodermal protein composition / blood mixture, the entire scaffold was engrafted with chondrocytes, and the superficial cartilage was repaired with a glycosaminoglycan-rich material consistent with articular cartilage and containing chondrocytes (right panel). The lamina splendens also appeared intact.

[0154] Figure 4 shows histology of the medial tibial plateau of guinea pigs 1 week (left panel), 2 weeks (middle panel), and 4 weeks (right panel) after injection of the mesodermal protein composition / blood mixture. Masson's trichrome staining was used, showing collagen and bone as blue, cytoplasm as light red / pink, and nuclei as black. In animals after 1 week, the superficial layer of lost cartilage had been filled with acellular mesodermal protein composition / blood mixture (lighter material and black arrows, left panel). By 2 weeks, surrounding chondrocytes began to reconstitute the superficial layer of cartilage where the mesodermal protein composition / blood mixture had previously been located (black arrow, middle panel). By 4 weeks after injection, the mesodermal protein composition / blood mixture had been completely replaced by chondrocytes and a GAG-rich matrix, and the superficial layer of cartilage (including the lamina splendens) had been reconstituted by native chondrocytes (right panel).

[0155] Gait analysis was performed to assess changes in base of support (BOS) in untreated animals and animals treated with the mesodermal protein composition / blood mixture. Treated animals had a higher mean BOS than control animals at 2 and 4 weeks post-injection (Figure 5), indicating long-term improvement in osteoarthritis after injection.

[0156] Taken together, these studies demonstrate that in a spontaneous arthritis model, injection of a mesodermal protein composition / blood mixture containing fibrillin and collagen can fill articular cartilage defects, providing a protected space for surrounding native cells to produce new articular cartilage, effectively resurfacing the joint and treating arthritis. This cartilage repair resulted in improved gait in animals, similar to that described elsewhere with anti-inflammatory drug treatment in the same model (Santangelo et al., Arthritis, 2014(2):503519, doi:10.1155 / 2014 / 503519). However, the effect of the mesodermal protein composition / blood mixture lasted for 4 weeks, while the anti-inflammatory drug treatment lasted for less than 24 hours. Since guinea pigs have a lifespan of approximately 4-8 years, one guinea pig year corresponds to approximately 10 human years, and four weeks of improvement in the guinea pig model could equate to more than six months of relief in a human patient. These studies demonstrated that mesodermal protein compositions containing collagen and fibrillin can be used to fill osteoarthritic defects and allow cartilage healing, thereby reversing the cartilage and gait changes seen in osteoarthritis and effectively treating spontaneous osteoarthritis.

[0157] Example 3 - Powder vs. large single scaffolds A study was conducted to evaluate the absorbed fluid retention rate of the powder formulation provided herein compared with a porous bridge-enhanced anterior cruciate ligament repair (BEAR) scaffold after centrifugation. Mesodermal protein powder was produced by decontaminating bovine elastic tissue with a solution containing hydrogen peroxide, decellularizing the tissue, washing the tissue, and freeze-drying it. After freeze-drying, the tissue was digested with pepsin in an acid solution, and the resulting slurry was neutralized, freeze-dried, and ground into a powder with a particle size of 0.1–0.6 mm. In this study, 250 mg of powder was mixed with 3 mL of phosphate-buffered saline and centrifuged. The porous scaffold was soaked in phosphate-buffered saline before centrifugation. These studies demonstrated that hydrogels produced from the powder formulation retained more than 99% of their absorbed fluid, even after high-speed centrifugation. This was not observed with the porous scaffold, which lost more than 20% of its fluid during centrifugation. The results are shown in Tables 1A and 1B.

[0158] [Table 1A]

[0159] In Table 1A, the mean value (99.9%) represents the weight retained after centrifugation of the gelled composite compared to the weight of the initial gelled composite ("plug"). These data indicate that the centrifugal force generated by sustained centrifugation (20,000 rcf for 10 minutes) does not cause the liquid to separate from the powder. In contrast, as shown in Table 1B, when pieces of scaffold (BEAR) were immersed in fluid until saturated and then centrifuged, the liquid separated after being subjected to centrifugal force (20,000 rcf for 10 minutes), leaving less than 80% of the wet weight.

[0160] [Table 1B]

[0161] Example 4 - Effect of particle size (unsterilized powder) A series of studies was conducted to determine whether finer particle size altered the gelation or mechanical properties of hydrogels made using sterile (non-electron beam sterilized) powders. Fine particles (<0.3 mm in diameter) were compared with coarse particles (0.3 to 0.6 mm in diameter). 400 mg of each powder was mixed with 3 mL of PRP. Both fine and coarse particles demonstrated excellent gelation with a score of 6 / 6 and possessed similar mechanical properties. Gelation was evaluated based on a combined score of shape (score 0 = no shape retention to score 3 = sharp edges of shape) and cutting surface (score 0 = gel too soft to cut to score 3 = sharp edges of cutting). The differences between particles were more evident in histological evaluation (Figure 6), where fine particles (left panel) were more uniformly dispersed in the diluent than coarse particles (right panel). Furthermore, the use of finer particle sizes resulted in more rapid dissolution by collagenase. Table 2 contains data from a study in which 9 mm plugs created by adding 200 mg or 400 mg of coarse or fine powder to PRP were digested with 200 u / mL collagenase at 32° C. for 2 hours.

[0162] [Table 2]

[0163] When normalized using the percent weight retention of the control sample, the 200 mg fine powder sample retained 86% of its weight, the 200 mg coarse powder sample retained 89% of its weight, the 400 mg fine powder sample retained 84% of its weight, and the 400 mg coarse powder sample retained 90% of its weight. The PRP sample retained the least weight at 83%. Comparison of individual groups using one-way analysis of variance (ANOVA) revealed no statistically significant differences. However, when the fine and coarse powder groups were combined, the mean percentage weight loss was greater for the fine powder group than for the coarse powder group, a statistically significant difference (p<0.01). The values ​​are plotted in Figure 7.

[0164] The above experiments suggest that particle sizes less than 0.3 mm in diameter are likely to be more useful in applications where faster absorption and a more uniform product are desired, while particle sizes between 0.3 mm and 0.6 mm are likely to be more useful in applications where collagenase resistance is desired. Furthermore, it is noted that finer powders have a smaller volume when filling syringes, increasing the upper limit of the powder / liquid ratio.

[0165] Example 5 - Effect of powder concentration (unsterilized powder) Gel shape test Gel shape tests were performed to evaluate the effect of powder concentration on shape retention. Gels were prepared in 5 mL syringes using 200 mg or 400 mg of powder in 3 mL of PRP, with PRP serving as a control. 9 mm plugs from each sample were incubated in a 32°C water bath for 2 hours and then evaluated for shape retention and cutting ability. PRP samples did not maintain their original cylindrical shape during incubation; rather, they shrunk to cylinders approximately 3 mm in diameter. These samples were included in the collagenase resistance test but excluded from subsequent gel shape and compression analyses.

[0166] Gel forms of 200 mg and 400 mg of powder per 3 mL of PRP sample were tested on the first 9 mm plugs from four syringes for each powder amount and scored according to the categories shown in Table 3. The test results are shown in Table 4.

[0167] The 400 mg sample remained cylindrical and maintained its shape perfectly after cutting, resulting in a significantly stronger gel with a clearly defined cut surface. The 200 mg sample was noticeably softer and showed a slight sagging of the rounded cylindrical shape. After cutting, the cut surface was less defined than that of the 400 mg sample.

[0168] [Table 3]

[0169] [Table 4]

[0170] Gel compression test A study was conducted to determine the effect of powder concentration on the resistance of the gel to compression. Gel compression tests using 200 mg and 400 mg of powder in 3 mL of PRP were performed using the second 9 mm plug from each of four syringes and scored according to the criteria shown in Table 5. The measured diameter and compression scores are shown in Table 6.

[0171] [Table 5]

[0172] Compression tests revealed that the 400 mg sample exhibited significantly greater resistance to deformation than the 200 mg sample. The diameter of the 200 mg sample increased from 11.5 mm to approximately 15 mm within 1 minute under a load of 0.05 N, increased to approximately 17 mm at 0.15 N, and plateaued after 2 to 5 minutes. This deformation was plastic. The diameter of the 400 mg sample increased to approximately 13.5 mm under a load of 0.05 N and stabilized at 15 mm under a load of 0.15 N. This deformation was also plastic. The sample displacement curves are shown in Figure 8.

[0173] [Table 6]

[0174] Gel collagenase test To assess the effect of concentration on resistance to collagenase digestion, two quarters of the first 9 mm disks from each syringe were digested in 1 mL of 200 μg / mL collagenase solution at 32°C for 2 hours, then centrifuged at 20,000 × g for 10 minutes and the supernatant was decanted. The weights of the quarter fragments were recorded before and after digestion and compared to controls incubated in buffer without collagenase. The percent weight remaining for the control samples was averaged, and the percent weight remaining for the collagenase-treated samples was normalized by the mean control percentage. Table 7 shows the statistical findings for each group.

[0175] [Table 7]

[0176] When normalized by the percent weight retention of the control sample, the 200 mg sample retained 66% of its weight, and the 400 mg sample retained 79%. The PRP sample retained the most weight, at 83%. Statistically significant differences were observed between the 200 mg and 400 mg groups (p<0.001) and between the 200 mg and PRP groups (p<0.01). Values ​​are plotted in Figure 9.

[0177] Example 6 - Effect of Vacuum Mixing (Non-Sterile Powder) Studies were conducted to evaluate the effect of vacuum-assisted mixing on various hydrogel properties. These studies showed, for example, that vacuum-assisted mixing improves hydration uniformity. Coarse powder was mixed with blood with and without vacuum assistance. Images of the resulting mixture are shown in Figure 10. The large, smooth, bright areas in the left panel are unmixed particles, and the dark, granular areas are red blood cells. In the right panel, vacuum mixing was used to hydrate the powder with PRP that had been stripped of red blood cells. Fewer areas of pure blood were observed, instead revealing a more uniform mixing pattern.

[0178] Example 7 - Sterile Powder Material Data composition Terminal sterilization processes are generally harsh and may alter the protein structure of the underlying ECM-derived material, resulting in changes in composition and mechanical properties. For aseptically manufactured materials with bioburden levels below 45 colony-forming units (CFU), electron beam sterilization in the range of 20-25 kGy is recommended to achieve a Sterility Assurance Level (SAL) of 10. -6 The irradiation (electron beam)-induced changes in protein structure can be visualized by SDS-PAGE. For example, Figure 11A shows an image of an SDS-PAGE evaluation of aseptically manufactured powder before sterilization. Figure 11B shows an SDS-PAGE of electron beam sterilized powder at the same sample dilution used in Figure 11A. The rightmost lane in Figure 11B ("COLL STD") contained a collagen standard dilution from the Sircol collagen assay to represent collagen α-monomer, β-dimer, and γ-trimer as a reference. In Figure 11B, the bands appear smeared due to chain scission of collagen molecules, which is a result of the high-energy irradiation of the electron beam treatment.

[0179] Despite the altered collagen profile indicated by SDS-PAGE testing, the quantitative Sircol soluble collagen assay, which uses a dye that binds to the helical portion of soluble collagen, was able to detect and measure collagen levels exceeding 500 mg / g (>50% dry weight), indicating that collagen molecules were not appreciably degraded. Unlike BEAR scaffolds, in which the slurry is made isotonic with calcium chloride and buffered with HEPES before being freeze-dried into scaffolds, the powder provided here was simply adjusted to a neutral pH of 7–7.4 using dilute sodium hydroxide and hydrochloric acid (referred to here as "pH 8.5→7"). Therefore, the salt content of the powder was much lower than that of BEAR scaffolds, and the protein and collagen mass fractions (dry wt / wt) were higher. This is the primary reason for the higher collagen content of the powder provided herein (above 500 mg / g, compared to average levels below 500 mg / g for BEAR scaffolds). The lot-average values ​​for collagen dry weight mass fraction for BEAR scaffolds and the powder provided herein were pooled and statistically analyzed using an unpaired parametric t-test with Welch's correction. On average, the collagen content of the powder was 73 mg / g higher than that of BEAR scaffolds, and this difference was statistically significant (Figure 12).

[0180] Mechanical properties A study of the mechanical properties of electron beam sterilized powders showed that particle size and pH adjustment during tissue digestion and final freeze-drying did not significantly affect the mechanical integrity of the reconstituted gel (powder mixed with buffered saline and incubated at knee joint temperature), but the powder-to-fluid ratio was found to affect mechanical integrity.

[0181] Variables / Groups 1. Neutralization method a. pH 8.5 → 7 b. pH 6, freeze-dried on the same day c. pH 6, left to stand for 3 days before lyophilization 2. Powder size a. Fine = passes through 50 mesh size (<0.3 mm) b. Coarse = passes through a 30 mesh but not a 50 mesh (0.3 mm > powder > 0.6 mm) 3. Powder / Phosphate Buffered Saline (PBS) Ratio a. Powder 200mg / PBS 3mL b. Powder 400mg / PBS 3mL

[0182] Test Method - Gelation 1. Shape / Cutting Score (Table 8) 2. Compression test under load for 15 minutes 3. Resistance to enzymatic degradation

[0183] [Table 8]

[0184] Semiquantitative shape and cut scores showed results similar to those of the non-sterile powders in the study described in Example 5. All 400 mg / 3 mL groups achieved the highest shape scores (3 / 3) (Figure 13A) and cut scores (Figure 13B), higher than the corresponding samples prepared at 200 mg / 3 mL, regardless of neutralization method or particle size. For the 200 mg / 3 mL samples, all groups achieved shape scores (Figure 13C) and cut scores (Figure 13D) of approximately 2. Therefore, samples with higher powder concentrations demonstrated improved shape retention.

[0185] Compression Test The higher-concentration powder sample (400 mg / 3 mL; Figures 14C and 14D) showed a significant delay in compression during the first 2 minutes of loading, and all groups resisted compression better than the lower-concentration sample (200 mg / 3 mL; Figures 14A and 14B). The maximum displacement for all groups was greater than 200%, compared to 150% for the weakest non-sterile group (200 mg / 3 mL). Particle size and pH adjustment method did not significantly affect the gel's ability to resist compression.

[0186] Resistance to enzymatic degradation Sterilized samples containing different powder concentrations were evaluated using the collagenase assay described above. The 400 mg / 3 mL samples were more resistant to enzymatic degradation than the 200 mg / 3 mL samples, regardless of neutralization method and particle size (Figures 15A-15C). No consistent trend was observed between particle size and resistance to enzymatic degradation. However, in the pH 8.5→7 group, coarser powders were more resistant to degradation, as observed with the unsterilized powders.

[0187] Example 8 - Mechanical testing using an Instron Additional studies were conducted to more accurately characterize the mechanical integrity of the gels formed after hydration and homogenization in an isotonic PBS solution using a vacuum mixing method. Specifically, the mechanical integrity of the powders was tested by examining three key variables: (1) powder particle size, (2) pH adjustment / neutralization method after pepsin digestion, and (3) the powder-to-PBS ratio during gel preparation (200 mg powder:3 mL PBS vs. 400 mg powder:3 mL PBS). As described below, these studies indicated that the powder:PBS ratio was the primary variable affecting the mechanical integrity of the gelled composites. However, the mechanical testing results in these studies were crude, relying mostly on semiquantitative shape / cutting scores based on qualitative observations and compression tests, which relied on the diameter increase of gel plugs of equal dimensions when loaded with equal weights. More robust and accurate characterization can be achieved by utilizing an INSTRON® ELECTROPULS™ E1000 (Instron; Norwood, MA) to measure the mechanical properties of the gels.

[0188] In those studies, the neutralization method and powder size were kept constant (initial slurry pH 2 was increased to pH 6.5 by adding sodium hydroxide solution, and powder size was maintained between 0.3 mm and 0.6 mm using sieves), and the powder to PBS ratio was 200 mg:3 mL or 400 mg:3 mL. In those studies, PBS was used instead of whole blood as an isotonic surrogate to isolate the mechanical contribution of gelation without adding the contribution of blood clotting. Targeted results included elastic modulus, stress relaxation curve, dynamic compression curve, and Poisson's ratio (Table 9).

[0189] [Table 9]

[0190] The powder was manufactured according to PS-002 to PS-008 Revision B. Briefly, mesodermal protein powder was produced by decontaminating bovine elastic tissue with a solution containing hydrogen peroxide, decellularizing the tissue, washing, and freeze-drying it. After freeze-drying, the tissue was digested with pepsin in an acidic solution, and the resulting slurry was neutralized. However, instead of adjusting the pH to 8.5 and then back to pH 7, we neutralized the tissue from pH 2 to pH 6.5 to inactivate pepsin while avoiding premature gelation due to pH. The powder was milled and sieved to produce coarse particles with a controlled diameter of 0.3 mm to 0.6 mm. Prefilled syringes were sterilized with electron beam at 20–25 kGy.

[0191] Samples for each group were prepared by transferring powder from a terminally sterilized 500 mg syringe into a specimen container to obtain enough powder to fill four 5 mL syringes per group (two for the 200 mg / 3 mL group and four for the 400 mg / 3 mL group). A 5 mL Luer-Lock syringe was filled with 200 mg or 400 mg of powder, and the plunger was pulled to the 5 mL position, ensuring that no particles were trapped between the plunger head and the syringe wall (as this could interfere with the vacuum during the mixing procedure). The plunger was secured in place with a plastic cutout. A 60 mL syringe was connected to the powder-containing syringe via a two-way valve. With the valve in the open position (parallel to the syringe), 60 mL of air was drawn into the 60 mL syringe, and the valve was then twisted to the closed position (perpendicular to the syringe). This process was repeated twice to create a vacuum within the 5 mL syringe. The 60 mL syringe was removed and the valve was left in the closed position.

[0192] To mix the PBS and powder, a blunt 16-gauge needle was placed on the tip of an empty 5 mL syringe, and 3.3 mL of PBS was drawn into the syringe (the extra 0.3 mL was lost through the two-way valve). The syringe was held upright, and any air bubbles trapped in the plunger were removed by gently tapping the side of the syringe. All trapped air bubbles were expelled through the needle. To remove air bubbles, several drops of PBS were expelled into the open end of the two-way valve, after which the syringe containing the powder was connected to the valve. The valve was opened, allowing the PBS to be absorbed into the powder. The PBS syringe and the two-way valve were then removed, and the syringe connector was connected to the syringe containing the powder and PBS, and the empty PBS syringe was connected to the connector. The contents were mixed by pumping back and forth 10 times, and finally, all of the contents were forced into the syringe that originally contained the powder.

[0193] For the gelation test, the syringes were placed in a 32°C water bath for 2 hours. After 2 hours of incubation, the syringes were removed one by one from the water bath. Using an industrial razor blade, the tip was cut off at the 0 mL mark, simultaneously ensuring a flat end for the slurry plug. 1 mL of gel was gently dispensed onto a clear Petri dish. A razor blade was moistened with PBS and used to cut the plugs. This process was repeated to create three cylindrical plugs from each syringe, which were positioned so that the circular surface of the plug was parallel to the surface of the Petri dish. Each plug was approximately 9 mm long and 12 mm in diameter.

[0194] The plug to be mechanically tested was gently transferred to a small glass slide with tweezers and positioned so that the circular face was parallel to the surface of the slide. The initial height and diameter of the plug were measured and recorded. The glass slide was placed on the stand inside the Instron® instrument, and the machine door was secured. The load cell was balanced with the uncompressed plug resting on the Instron® stand. The crosshead lock was securely fastened, and the strain gauge platform was at its lowest position. The actuator was lowered at full power until it lightly touched the plug. The strain gauge platform was raised and lightly clamped onto the strain gauge until the channel read approximately 0.1 mm. The strain gauge was then balanced. The "Start" button on the Instron® instrument was pressed, initiating a program that compressed the plug at a rate of 0.05 mm / s until a strain of 10% was reached. The maximum diameter of the compressed plug was measured and recorded when the test was paused. The machine door was then re-locked, and the program continued to perform the dynamic compression test (8-12% strain, 45 cycles at 1 Hz) and stress relaxation test (10% strain held for 10 minutes). Data files were saved to an encrypted storage device for analysis.

[0195] The elastic modulus is a measure of stiffness and was used to compare the resistance of gelled plugs to axial compression in the linear (elastic) region. The gel plugs were compressed at a uniform rate (0.05 mm / s) to a strain of 10% (approximately 0.8 mm). The elastic modulus represents the ratio of stress (applied force / cross-sectional area) divided by strain (% change in length compared to the initial length).

[0196] The mean modulus of the 400 mg / 3 mL gel was more than three times that of the 200 mg / 3 mL gel (8.11 ± 0.98 kPa vs. 2.57 ± 0.27 kPa, mean ± SD; Table 10 and Figure 16). The modulus of the 400 mg / 3 mL gel was more variable than that of the 200 mg / 3 mL gel, as indicated by the larger range and standard deviation around the mean (range: 2.54 vs. 0.68, standard deviation: 0.98 vs. 0.27). An unpaired t-test with Welch's correction for unequal variances showed a two-sided p-value of less than 0.001, indicating that the difference in means was statistically significant. The distribution of the sample data was assumed to be normal.

[0197] Dynamic compression testing provided insight into the susceptibility of hydrogels to fatigue. Under cyclic / oscillatory loading, the internal structure of the hydrogel gradually changed due to the formation and propagation of cracks in the polymer network and the loss and incomplete reformation of supporting noncovalent intermolecular bonds within the polymer / water network during each loading cycle. To monitor changes in the response to compression within hydrogels prepared at concentrations of 200 mg / 3 mL PBS or 400 mg / 3 mL PBS, each gel sample was subjected to 45 compression cycles at 8–12% strain, spaced at 1 Hz (one load cycle per second). For each sample, the average of the maximum and minimum stress response readings from the first five load cycles was used to represent the "start" maximum and minimum stress response, and the average of the maximum and minimum stress readings from the last five load cycles (cycles 41–45) was used to represent the "end" stress response range. The difference between the start and end stress readings was calculated for each sample and used to represent key parameters of fatigue.

[0198] [Table 10]

[0199] The stress response (resistance to compression) was higher in all 400 mg groups compared with the corresponding 200 mg groups (Figure 17A). The maximum initial and maximum final stress responses (stress response at 12% strain) were 3.4- and 3.5-fold greater in the 400 mg group than in the 200 mg group, respectively. Meanwhile, the minimum initial and minimum final stress responses (strain at 8% strain) were 4.2- and 5.6-fold greater in the 400 mg group than in the 200 mg group, respectively.

[0200] To compare the differences in initial and final stress responses (fatigue) between the corresponding 200 mg and 400 mg groups, the final stress responses were normalized by their respective initial stress responses (e.g., the mean final and maximum stress responses of the 200 mg groups were normalized by the mean initial maximum stress response of the 200 mg group). Normalized values ​​were then analyzed between the 200 mg and 400 mg hydrogel groups (Figure 17B). The decrease in the normalized maximum stress response (stress response at 12% strain) was greater for the 200 mg powder / 3 mL PBS hydrogel sample than for the 400 mg powder / 3 mL PBS sample, with retention of the initial stress of 90.5% and 93.6%, respectively. Therefore, mechanical fatigue was greater for the 200 mg preparation. This difference was not statistically significant by one-way ANOVA with Tukey's correction for multiple comparisons (adjusted p = 0.896). Similar results were observed for fatigue at the lowest stress response (8% strain), with the 400 mg group retaining an average of 88.0% of their starting stress response, while the 200 mg group retained only 67.6% of their starting stress response. This difference was statistically significant (adjusted p = 0.002).

[0201] Stress relaxation is an important indicator of mechanical properties in viscoelastic materials such as hydrogels, where the elastic behavior of the polymer is complemented by viscous flow within the hydrated network. When a constant external load is applied, a time-dependent decrease in the stress response is observed due to viscous flow within the gel. To monitor stress relaxation in gels prepared with 200 mg or 400 mg of powder, a 10% strain was applied and maintained, and the stress response was recorded over a 10-minute period. The stress relaxation curves exhibited R values ​​ranging from 0.976 to 0.989. 2 value, modeled by a negative exponential function: stress = a*e -bt , where a is the initial stress at t = 0 s and t is the time in seconds.

[0202] There were observable differences in the stress-relaxation curves between hydrogels prepared with 200 mg and 400 mg powder. Figure 18A shows the modeled stress response over 10 minutes (total time measured), and Figure 18B shows the extrapolation of the predicted model over 100 minutes. The initial stress response was higher for the 400 mg hydrogel (0.19 ± 0.03 kPa) than for the 200 mg hydrogel (0.05 ± 0.01 kPa). After 10 minutes, the stress response of the 400 mg hydrogel remained higher than that of the 200 mg hydrogel (0.13 ± 0.02 kPa vs. 0.02 ± 0.01 kPa, respectively).

[0203] Poisson's ratio provides information about the deformation of a material under tensile or compressive load. When a hydrogel plug is compressed axially, the plug expands in the transverse plane perpendicular to the loading direction. Therefore, Poisson's ratio is used as the ratio of transverse strain to longitudinal strain; the longitudinal strain in these studies was 10%. The deformation in the 200 mg hydrogel was significantly greater than that in the 400 mg hydrogel, with a Poisson's ratio that was more than twice as large on average (0.99 ± 0.13 vs. 0.40 ± 0.15, respectively; Figure 19). This difference was statistically significant (p < 0.001, unpaired t-test with Welch's correction for heteroscedasticity).

[0204] Taken together, the comprehensive mechanical testing demonstrated that hydrogel preparations containing 400 mg of powder mixed with 3 mL of PBS had significantly higher mechanical integrity than hydrogels prepared with 200 mg of powder per 3 mL of PBS. The 400 mg powder group was stiffer, with a modulus more than three times that of the 200 mg group. The 400 mg powder group also exhibited greater resistance to fatigue over 45 loading cycles, exhibiting more than twice the stress response of the 200 mg group during the final loading cycle. The stress relaxation curves were well modeled with a negative exponential function. While both groups exhibited viscous properties, the 400 mg group maintained higher stress readings throughout the entire 10-minute measurement period and predicted higher stress readings when the function was extrapolated to 100 minutes. The 400 mg group was also more resistant to deformation under compressive load, with a Poisson's ratio less than half that of the 200 mg group and significantly less transverse strain when subjected to a longitudinal strain of 10%.

[0205] Example 9 – Effect of tissue disinfectants on the properties of mesodermal ECM-derived powders The effects of three different disinfectants (CIP-100, SPOR-KLENZ®, and hydrogen peroxide) on mesodermal tissue-derived ECM powder were evaluated. SPOR-KLENZ® is a combination of 1% hydrogen peroxide, 0.08% peracetic acid, and less than 10% acetic acid. Collagen content was unaffected by any of the disinfection treatments (Figure 20), but each agent significantly reduced GAGs in the product (Figure 21). This loss of GAGs did not appear to affect the gelling properties (Figure 22) or protein content (Figure 23) of the SPOR-KLENZ® or hydrogen peroxide agents.

[0206] Example 10 – Effect of tissue pretreatment protocol on viral reduction efficacy For medical devices, 10 viruses that may be present during the manufacturing process are counted for four virus classes (enveloped DNA viruses, enveloped RNA viruses, non-enveloped DNA viruses, and non-enveloped RNA viruses). 6A reduction of 6 logs is a characteristic required by the FDA before product approval. This has been successfully achieved for enveloped viruses (DNA and RNA) by a combination of surfactants and irradiation (see Table 11). For non-enveloped viruses such as REO3 (an RNA non-enveloped virus) and PPV (a DNA non-enveloped virus), the use of a surfactant step in manufacturing was ineffective. Furthermore, irradiation at 15 kGy reduced REO3 by approximately 10 5 Although the REO3 was reduced to 0.05% by 0.1% and the acid-pepsin step reduced REO3 by an additional 3 logs (i.e., a total reduction of 6 logs for the combined manufacturing process), pepsin / acid reduction of PPV viral load was not effective. Therefore, further studies were conducted to identify processing steps effective in reducing PPV. These studies showed that the use of hydrogen peroxide and PAA was effective in reducing PPV by approximately 4 logs, and that combining hydrogen peroxide and PAA with terminal electron beam sterilization (reducing PPV by 2 logs at 15 kGy) could reduce PPV by 6 logs. Other processes were less effective. Therefore, these studies demonstrated that tissue pretreatment with a combination of hydrogen peroxide and PAA is effective in a parvovirus model without adversely affecting the material.

[0207] [Table 11]

[0208] Example 11 – Effect of PAA concentration on ECM-derived powder properties Because PAA was the only treatment with the greatest effectiveness against PPV, reducing PPV by 6 logs during manufacturing, studies were conducted to determine whether varying the PAA concentration or exposure time would result in changes in the physical properties of the ECM-derived powder, including collagen concentration, GAG content, and gelation. As shown in Table 12, the following treatments were performed: 0.1% PAA for 1 hour, 0.2% PAA for 1 hour, 0.1% PAA for 18 hours, 0.2% PAA for 18 hours, 0.2% PAA for 10 minutes, 7.5% hydrogen peroxide for 10 minutes, 1% CIP-100 for 10 minutes, and SPOR-KLENZ® (RTU) for 10 minutes.

[0209] [Table 12]

[0210] Collagen Concentration: The mean collagen concentration and standard deviation for each treatment are shown in Table 12 and Figure 24. Chemical pretreatment with 0.2% PAA, SPOR-KLENZ®, or CIP-100 for 10 minutes resulted in significantly lower collagen concentrations compared to the control group (adjusted p-value = 0.0314).

[0211] GAG content: Mean GAG concentrations and standard deviations, as well as the percentage of GAG content per collagen content, are reported in Table 14 and Figure 25. All chemically pretreated groups showed significant decreases in absolute GAG ​​content and GAG / collagen percentage compared to the control group (adjusted p-values ​​<0.0001 for all comparisons).

[0212] Protein composition: Visual comparison of protein band positions suggested no significant changes in the molecular weight of individual proteins (Figure 26). However, the PAA chemical treatment group could show strong staining at the top of the gel, which may be consistent with insufficient digestion of the source tissue by pepsin.

[0213] [Table 13]

[0214] [Table 14]

[0215] Gelation ability: The width / height ratio of gelation, cut resistance after 10 minutes, and shape stability after 5 minutes of cutting were significantly improved for the chemically pretreated samples compared to the control group (Table 15 and Figure 27; adjusted p-values ​​<0.0001 for all comparisons).

[0216] [Table 15]

[0217] These studies showed that in all PAA protocols, chemical pretreatment of the source tissues resulted in a significant decrease in GAG content compared to control samples, which did not differ between groups, suggesting that GAG loss likely occurred within 10 minutes of exposure.

[0218] The loss of GAGs did not affect the gelling ability, a key function of ECM-derived powders. In fact, PAA-treated samples maintained their gel shape better over time. This unexpected result was evident in the reduced height loss of the gel cylinder over 10 minutes, the increased resistance to gel cutting, and the gel's shape stability after cutting. Furthermore, this result was consistent across exposures to PAA ranging from 10 minutes at low concentrations (0.08% PAA with 1% hydrogen peroxide and less than 10% acetic acid: SPOR-KLENZ® group) to 18 hours at 0.2% PAA.

[0219] However, prolonged exposure of tissue to PAA and the use of higher concentrations of PAA altered the ability of pepsin to solubilize collagen, as evidenced by stronger staining at the top of the gel bands where higher molecular weight proteins are located in the PAA-only group during gel electrophoresis. Although the protein banding pattern appeared unchanged overall, the higher staining intensity of high molecular weight proteins and the lower staining intensity of low molecular weight proteins may indicate less complete degradation of collagen proteins during pepsin digestion, which is consistent with the finding that PAA-treated tissues took longer to digest than control samples.

[0220] Taken together, these studies showed that the use of 0.1% PAA with 1% hydrogen peroxide for 10 minutes (as seen in the SPOR-KLENZ® group) improved gelation, did not interfere with pepsin solubilization, and resulted in an approximately 4 log reduction in parvovirus.

[0221] Example 12 - Effect of anticoagulant use in vitro In vitro studies of the reactivation of anticoagulated blood showed that adding calcium to anticoagulated blood restored the blood's clotting ability. However, blood anticoagulated with sodium citrate (NaC) clots more rapidly than blood anticoagulated with acid citrate dextrose (ACD). Furthermore, lower concentrations of calcium chloride appeared to restore clotting more quickly in anticoagulated blood. All tested CaCl2 amounts (8 mg, 10 mg, and 12 mg, equivalent to 1 mL of 72 mM, 90 mM, and 108 mM CaCl2 solutions) were able to rescue clotting in 4 mL of anticoagulated blood in all groups (Table 16). In Group 1, mesodermal protein powder was directly rehydrated with CaCl2 solutions of various concentrations before mixing with the anticoagulated blood. In Group 2, an equivalent dry weight of CaCl2 was mixed with the mesodermal protein powder before mixing the powder with the blood. In Group 3, various CaCl2 solutions were dried in the syringe before adding mesodermal protein powder. In all three groups, clotting began earlier in NaC-anticoagulated blood than in ACD-anticoagulated blood. The clotting time of NaC-anticoagulated blood was faster in Group 1 when rehydrated with low and medium concentrations of CaCl2, suggesting the existence of an optimal concentration, whereas no difference in clotting time was observed in Groups 2 and 3 for NaC-anticoagulated blood. The highest CaCl2 concentration resulted in a shorter clotting time for ACD-anticoagulated blood, consistent with the fact that the theoretical mass of CaCl2 required to saturate free citrate was closest to the highest concentration tested (12 mg). It should be noted that the ACD- and NaC-anticoagulated blood used in these studies was obtained from different donors, so natural variations in individual clotting kinetics are likely to affect the results.

[0222] [Table 16A]

[0223] Further studies were conducted to determine the optimal concentration of calcium chloride for use as a rehydration solution for ACD-anticoagulated blood to restore the blood's clotting ability prior to injection. The ratio of rehydration solution to blood was set to mimic the ratio used to prepare the final ECM-derived powder composite (1 part rehydrated powder to 2 parts blood by volume). The solution was prepared as shown in Table 16B.

[0224] Human whole blood was collected and mixed with 10% or 15% ACD. 2 mL of each anticoagulated blood sample was dispensed into two serum collection tubes coated with a clot activator. 1 mL of diluted calcium chloride solution (either 41 mM or 59 mM) was added to each ACD-blood group to create the matrix shown in Table 16C. The time from mixing to clotting was observed and recorded in Table 16C.

[0225] The clotting time for blood not anticoagulated with ACD was 6 minutes. As shown in Table 16C, for both ACD preparations, lower CaCl concentrations surprisingly resulted in faster clotting, with the fastest time to clotting observed for 10% ACD mixed with 41 mM CaCl. Thus, a 41 mM CaCl solution successfully restored clotting ability when mixed with anticoagulated blood at a 1:9 ratio. This calcium chloride solution and mixing ratio was also used in a guinea pig study (Example 13) using anticoagulated blood for rehydration and intra-articular injection of ECM-derived powder.

[0226] [Table 16B]

[0227] [Table 16C]

[0228] Example 13 – Effects of anticoagulant use in vivo A live animal in vivo study of this product, compatible with the use of anticoagulated blood, was conducted in a guinea pig model. Gait analysis and micro-CT analysis of subchondral bone were evaluated, and no adverse effects associated with the use of anticoagulated blood as an additive to the ECM-derived powder were observed. Briefly, no statistically significant differences were observed in hindlimb weight bearing between the groups of animals injected with mesodermal protein powder mixed with anticoagulated blood or fresh blood. Similarly, subchondral bone mineral density, an indicator of osteoarthritic changes, showed no significant differences between groups.

[0229] Example 14 - Effect of moisture-proof packaging A foil / film construction was determined to be a suitable moisture-proof packaging option. Burst and bubble tests, as well as peel tests of the seals, were performed according to industry standards. Peel force results showed that the seal was consistent over three consecutive sealings (Table 17), indicating acceptable operational and performance qualities.

[0230] [Table 17]

[0231] Example 15 - Product characterization (quantitative MS / ELISA / SDS-PAGE) To further characterize the protein composition of the ECM-derived powder, SDS-PAGE analysis was performed (Figure 28). Intense bands were distinguishable at the expected positions of collagen type 1 dimer (270 kDa) and collagen type 1 monomer (130 kDa), highlighting that collagen was the major protein in the powder. A slight smear was observed around and below the bands, indicating protein degradation due to electron beam irradiation.

[0232] A more detailed protein analysis of the powder was performed using mass spectrometry. A total of 1,072 peptides were identified. 032201 sterile powder was used as a control (because it is derived from the historically used tissue), and the output list was sorted from highest to lowest relative peak intensity within this group. The top 20 peptides (mean, SD) are shown in Table 18. This analysis considered individual peptides of fibrous proteins and their higher-order quaternary structure subunits separately. The most abundant peptides identified within each group were type I collagen, followed by type III collagen, and minor amounts of type II collagen, fibrillin I and 2, and keratin.

[0233] [Table 18]

[0234] Example 16 - Sterilization Method (Supercritical CO 2 ) Identification and verification In single- and multiple-injection studies using a guinea pig model of spontaneous osteoarthritis, electron beam-sterilized ECM-derived powder did not result in improved histological cartilage destruction or gait. Therefore, supercritical CO2 was evaluated as an alternative sterilization method. E-beam-sterilized ECM-derived powder was compared with scCO2-sterilized ECM-derived powder, and the collagen content, GAG content, DNA content, phospholipid content, pepsin activity, protein content, resistance to enzymatic degradation, and physical properties were evaluated.

[0235] Collagen Content: To compare the dry mass fraction of collagen in powders produced from the same lot after each type of terminal sterilization, the Sircol collagen assay was performed on two samples from six e-beam-sterilized syringes (n = 12 total, 20–25 kGy) and 12 samples of powder sterilized using scCO2. Each sample was prepared by dissolving 30 ± 3 mg of powder in 50 mL of dilute hydrochloric acid (HCl) at pH 2 (n = 12 per group). The mean mass fraction of collagen in the dried ECM-derived powders sterilized with either e-beam or scCO2 was 578 mg / g and 668 mg / g, respectively, corresponding to dry weight (w / w) percentages of 58% and 67% (Figure 29). The 9% difference in total dry weight percentage was statistically significant, with a p-value of < 0.001 by an unpaired, two-tailed t-test with Welch's correction. The standard deviations for the electron beam and scCO2 groups were 41 mg / g and 39 mg / g, respectively, and the variability within each group was comparable.

[0236] GAG content: To compare the dry mass fraction of glycosaminoglycans in powders produced from the same lot after each type of terminal sterilization, the Blyscan GAG assay was performed on two samples from six e-beam-sterilized syringes (total n = 12, 20–25 kGy) and 12 samples of powder sterilized using scCO2. Each sample was prepared by dissolving 30 ± 3 mg of powder in 1 mL of papain buffer (n = 12 per group). The mean mass fraction of GAGs in the e-beam-sterilized and scCO2-sterilized dried ECM-derived powders was 2.1 mg / g and 2.0 mg / g, respectively, corresponding to dry weight (w / w) percentages of 2.1% and 2.0% (Figure 30). A 0.1% difference in total dry weight percentage was not statistically significant, with a p-value of 0.323 by an unpaired, two-tailed t-test with Welch's correction. The standard deviations for the e-beam and scCO2 groups were 0.18 mg / g and 0.10 mg / g, respectively, with greater variability in the e-beam group.

[0237] DNA content: To compare the dry mass fraction of DNA in powders produced from the same lot after each type of terminal sterilization, we performed the Picogreen dsDNA assay on two samples from six e-beam-sterilized syringes (n = 12 total, 20–25 kGy) and 12 samples of powder sterilized using scCO2. Each sample was prepared by dissolving 30 ± 3 mg of powder in 1 mL of papain buffer (n = 12 per group). DNA content was determined as an indicator of effective decellularization from the extracellular matrix (ECM) and subsequent removal of residual cellular material, with an acceptable limit of 50,000 ng / g. The average mass fraction of DNA in powders derived from e-beam- or scCO2-sterilized dried ECM was 11,406 ng / g and 11,275 ng / g, corresponding to dry weight (w / w) percentages of 0.00114% and 0.00113%, respectively (Figure 31). The difference in percentage total dry weight was not statistically significant with a p-value of 0.700 by unpaired two-tailed t-test with Welch's correction. The standard deviations for the e-beam and scCO2 groups were 896 and 746 ng / g, respectively, with similar variability within each group but slightly greater in the e-beam group.

[0238] Phospholipid content: To compare the dry molar fraction of phospholipids in powders produced from the same lot after each type of terminal sterilization, the ENZYCHRO™ Phospholipid Assay was performed on two samples from six e-beam-sterilized syringes (n = 12 total, 20-25 kGy) and 12 samples of powder sterilized using scCO2. Each sample was prepared by dissolving 10 ± 1 mg of powder in 1 mL of Tris-buffered Triton-X 102 solution (n = 12 per group). Phospholipid content is a secondary indicator of effective decellularization and subsequent removal of residual cellular material from the extracellular matrix. The mean molar fractions of phospholipids in the e-beam-sterilized and scCO2-sterilized dry ECM-derived powders were 745 mmol / g and 588 mmol / g, respectively (Figure 32). The 157 mmol / g difference in molar fraction was statistically significant with a p-value of < 0.001 by an unpaired, two-tailed t-test with Welch's correction. The standard deviations for the e-beam and scCO2 groups were 101 mmol / g and 66 mmol / g, respectively, with slightly greater variability in the e-beam group.

[0239] Pepsin activity: To compare the residual pepsin activity in powders manufactured from the same lot after each type of terminal sterilization, two samples from six e-beam-sterilized syringes (n = 12 total, 20–25 kGy) and 12 samples of powder sterilized using scCO2 were used. Each sample was prepared by dissolving 20 ± 2 mg of powder in 1 mL of dilute pH 2 hydrochloric acid (n = 12 per group). The mean residual pepsin activity in dried ECM-derived powders sterilized with e-beam or scCO2 was 160 u / g and 167 u / g, respectively (Figure 33). The difference in molar fraction of 7 u / g was not statistically significant, with a p-value of 0.250 by unpaired, two-tailed t-test with Welch's correction. The standard deviations for the e-beam and scCO2 groups were 18 and 11 u / g, respectively, with slightly greater variability in the e-beam group.

[0240] SDS-PAGE: To compare protein banding patterns in powders from the same lot after terminal sterilization of each format, two samples from six e-beam-sterilized syringes (n = 12 total, 20-25 kGy) and 12 samples of powder sterilized using scCO2 were used. Each sample was prepared by dissolving 30 ± 3 mg of powder in 50 mL of dilute hydrochloric acid (HCl) at pH 2 (n = 12 per group). A 3-8% Tris-acetate gel was used, filling each well to two-thirds capacity. A reference of 250 μg / mL type I collagen from bovine skin (the standard used in the Sircol collagen assay) was prepared and run in the far right lane of the gel to indicate the location of collagen α-monomer, β-dimer, and γ-trimer in each lane (Figure 34). The gel of the e-beam-treated sample (Figure 34, upper panel) showed clear banding at the α1 and α2 monomer positions, faint bands at the α1 + α1 and α1 + α2 dimer positions, and almost no banding at the trimer position. A broad smear was observed across each lane, indicating extensive nonspecific cleavage of the proteins in the powder. In the scCO2-treated powder sample (Figure 34, lower panel), all major bands, including type I collagen monomer, dimer, and trimer, were present and distinct. There was no smear, and three other bands were observed at higher molecular weights. These high-molecular-weight bands were also observed in the collagen reference lane.

[0241] Resistance to enzymatic degradation: Three gel disc samples (diameter = 12 mm, height = 9 mm) were prepared using both e-beam sterilized and scCO2 sterilized powders at a powder concentration of 400 mg / 3 mL PBS. The discs were cut into quarters to create a total of 12 samples. Six quarter-sections from each group served as control samples and were incubated in enzyme-free buffer (PBS containing calcium and magnesium), while six served as treatment samples and were incubated in collagenase type I (200 activity units / mL) for 2 hours at 32°C on a rocker table set at 100 rpm. Gels were weighed before and after incubation, and the percent weight remaining was used as the primary indicator for group comparison. The mean (±SD) weight retention of control samples of powders treated with e-beam or scCO2 sterilization was 120±2% and 111±5%, respectively (Figure 35, left panel), suggesting that one-quarter of the gel in both groups absorbed liquid from its surroundings throughout the 2-hour incubation in enzyme-free buffer. The mean weight retention of e-beam control samples was 9% greater than that of the scCO2 group, and this difference was significant (p = 0.004) using an unpaired, two-tailed t-test with Welch's correction. The mean (±SD) weight retention of treated samples of powders treated with e-beam or scCO2 sterilization was 46±3% and 82±4%, respectively. This difference was also statistically significant (p < 0.001). The normalized weight retention (% e-beam treated / control e-beam) of e-beam gel samples was approximately two-fold lower than that of the scCO2 samples, at 38% and 74%, respectively (Figure 35, right panel).

[0242] Mechanical Testing: A series of uniaxial compression tests were performed to characterize the mechanical properties of ECM-derived powder / PBS hydrogels. To isolate the mechanical contribution of gelation without adding the contribution of coagulation, PBS was used as an isotonic replacement for whole blood in this study. Six samples were tested for each preparation method (200 mg E-beam, 200 mg scCO2, 400 mg E-beam, or 400 mg scCO2 powder vacuum-mixed with 3 mL of PBS and incubated at 32°C for at least 1 hour) using powder from a single ECM-derived powder lot and sieved to a particle size of 0.3–0.6 mm.

[0243] Elastic modulus: Elastic modulus is a measure of stiffness that can be used to compare the resistance of gelled plugs to axial compression in the linear (elastic) region. Gel plugs were compressed at a uniform rate (0.05 mm / s) to 10% strain (approximately 0.8 mm). Elastic modulus represents the ratio of stress (applied force / cross-sectional area) divided by strain (percent change in length relative to the initial length). The mean (±SD) elastic moduli of gels prepared at a powder concentration of 200 mg / 3 mL by e-beam sterilization or scCO2 sterilization were 2.5 ± 0.8 kPa and 6.9 ± 1.5 kPa, respectively (Figure 36). This 4.4 kPa difference was statistically significant, with a p-value of < 0.001 by an unpaired, two-tailed t-test with Welch's correction. The mean (±SD) moduli of gels prepared using either e-beam sterilization or scCO2 at a powder concentration of 400 mg / 3 mL were 9.9 ± 5.6 kPa and 16.0 ± 4.2 kPa, respectively. This difference of 6.1 kPa was not statistically significant, with a p-value of 0.059, according to an unpaired, two-tailed t-test with Welch's correction.

[0244] Briefly, the first part of these studies focused on the biochemical composition of ECM-derived powders sterilized by 20–25 kGy e-beam irradiation or supercritical carbon dioxide with an additive (NOVAKIL™, NovaSterilis, Inc., Lansing, NY). Although the GAG ​​content of each group was very similar, the measurable collagen content of the scCO2-treated powder was significantly higher than that of the e-beam-treated powder. The phospholipid content of the scCO2-treated powder was significantly lower than that of the e-beam-treated powder, but the DNA content of each group was comparable, suggesting no differences related to the degradation of potentially antigenic cellular material. The second part of this study focused on the physical properties of the e-beam- or scCO2-treated powders when hydrogels were prepared at two powder concentrations: 200 mg and 400 mg in 3 mL of PBS. Differences in mechanical integrity, as measured by stiffness (elastic modulus), fatigue resistance (dynamic compression), viscosity (stress relaxation), and deformation (Poisson's ratio), as well as resistance to enzymatic degradation, were observed. For example, gels derived from scCO2-sterilized powders were significantly more resistant to enzymatic degradation than gels derived from e-beam-sterilized powders, retaining nearly twice the initial weight when compared to controls incubated in buffer alone. The scCO2 gels were also significantly stiffer, with mechanical properties less susceptible to internal viscous flow at both concentrations, exhibiting moduli 1.5 times higher than e-beam gels and lower stress relaxation rates over periods longer than 10 min. Dynamic compression tests revealed that the scCO2 gels were more susceptible to fatigue; however, the stress response to achieve the same strain remained higher for the scCO2 gels at all time points at both concentrations. Strain-dependent Poisson's ratios indicated that compressed gels derived from powders processed by either sterilization method responded to similar strains in the cross section. It was clear that the overall mechanical integrity of gels derived from scCO2-sterilized powders was higher than that of gels derived from e-beam-sterilized powders.As suggested by biochemical composition studies, proteins, especially type I collagen, were more highly preserved, which may have allowed the ECM powder to better maintain its inherent ability to undergo fibrillogenesis in an isosmotic environment and maintain a more organized and mechanically resilient structure through a higher degree of intermolecular interactions.

[0245] Example 17 – ACL Repair Study A study was conducted to compare the healing of torn ACLs using the established BEAR® scaffold (Miach Orthopedics, Westborough, MA) sterilized by electron beam irradiation with the mesodermal protein powder described in this paper. The mesodermal protein powder was terminally sterilized using scCO2 at NovaSterilis (Lansing, NY). Prior to injection, 600 mg of ECM-derived powder was placed in a syringe and mixed with 6 mL of fresh autologous blood from the pig being operated on. To simulate ACL injury, eight (n = 8) adolescent (3-month-old) male and female Yorkshire pigs underwent unilateral ACL resection. Knee stability was restored with a suture stent, and sutures were secured to the tibial ACL stump to approximate the femoral stump. ACL repair was assisted in half of the animals (n = 4) by the blood-soaked BEAR® scaffold or the mesodermal protein powder mixed with blood (n = 4). Groups were randomly assigned to contain equal numbers of animals of the same sex and operated knees on the same side. Animals were euthanized 6 weeks after the initial ACL repair procedure, and the macroscopic and histological appearance of the repaired ACL was evaluated. Statistical comparisons between the two study groups were performed using Student's t-tests when the assumptions of normality of data distribution and homogeneity of variance were met. A p-value of 0.05 was considered statistically significant.

[0246] Six weeks after the initial ACL repair, successful reattachment of the femoral and tibial ACL stumps was observed in all specimens (Figure 37). No obvious differences in morphology or size were observed between ACLs repaired with the aid of either mesodermal protein powder or BEAR® scaffold. Macroscopic measurements of ACL volume showed no statistically significant differences between ACLs repaired with the aid of either mesodermal protein powder or BEAR® scaffold (Figure 38), and histological analysis of the repaired ACLs showed no significant differences between ACLs repaired with the aid of either mesodermal protein powder or BEAR® scaffold (Figure 39). Thus, in a large animal model of ACL transection, the use of either mesodermal protein powder or BEAR® scaffold to aid in the healing of ACL injuries resulted in comparable scar tissue formation at both the macroscopic and microscopic levels between repaired ACL stumps after 6 weeks.

[0247] Example 18 – ACL Repair Study A study was conducted to compare the healing of ruptured rotator cuff tendons (RCTs) using suture repair alone, mesodermal protein powder, mesodermal protein sheets, or established BEAR® sheets. Twelve adolescent (3-month-old) male / female sheep underwent unilateral RCT amputations. Equal numbers (n = 3) of sheep underwent suture repair alone, or treatment with mesodermal protein powder, mesodermal protein sheets, or established BEAR® sheets as described herein. Mesodermal protein powder was terminally sterilized using scCO2 at NovaSterilis (Lansing, NY). Prior to injection, 600 mg of mesodermal protein powder was placed in a syringe and mixed with 6 mL of fresh autologous blood from the sheep undergoing surgery. Groups were randomly assigned to include equal numbers of animals of the same sex and operated knees on the same side. Animals were euthanized 8 weeks after the initial RCT repair procedure, and shoulder stability and range of motion were assessed, as well as the macroscopic and histological appearance of the repaired RCT. Statistical comparisons between the two study groups were performed using one-way ANOVA, provided the assumptions of normality of data distribution and homogeneity of variance were met. A p value of 0.05 was considered statistically significant.

[0248] Eight weeks after initial RCT repair, sufficient reconnection between the proximal and distal RCT stumps was observed in all specimens (Figure 40). Macroscopic measurements of RCT thickness revealed no statistically significant differences between RCTs repaired with suture repair alone, mesodermal protein powder, mesodermal protein sheets, or the established BEAR® sheet (Figure 41). Histological analysis of the repaired RCTs also revealed no significant differences between RCTs repaired with suture repair alone, mesodermal protein powder, mesodermal protein sheets, or the established BEAR® sheet (Figure 42). The results of this pilot study demonstrate that using suture repair alone, mesodermal protein powder, mesodermal protein sheets, or the established BEAR® sheet to aid in the healing of RCT injuries in a large animal model of RCT amputation results in similar scar tissue formation between the repaired RCT stumps after 8 weeks, both at the macroscopic and microscopic levels.

[0249] Other embodiments While the present invention has been described in conjunction with its detailed description, it is understood that the foregoing description is intended to be illustrative, and not limiting, of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A composition comprising a powdered extracellular matrix (ECM) component and a fluid, wherein the ECM component comprises mesodermal proteins including collagen, and the powdered ECM component has a concentration in the fluid of about 50 mg / mL to about 200 mg / mL.

2. 10. The composition of claim 1, wherein the powdered ECM component has a concentration in the fluid of about 100 to about 150 mg / mL.

3. The composition of claim 1 , wherein the fluid is blood.

4. 10. The composition of claim 1, wherein the powdered ECM component has an average particle size of about 0.1 mm to about 1 mm.

5. 10. The composition of claim 1, further comprising a growth factor, a platelet, a white blood cell, a stem cell, a crosslinking agent, a neutralizing agent, or any combination thereof.

6. The composition of claim 1 further comprising calcium.

7. 10. The composition of claim 1, wherein the composition is substantially free of one or more of nucleic acids, glycosaminoglycans (GAGs), phospholipids, active pepsin, and active viruses.

8. 1. A method for producing a composition comprising a fluid and a powdered ECM component comprising mesodermal proteins including collagen, the method comprising the steps of: providing a syringe containing a powdered ECM component; Injecting a volume of fluid into the syringe so that the concentration of the powdered ECM component in the fluid is between about 50 mg / mL and about 200 mg / mL. A method comprising:

9. 9. The method of claim 8, comprising injecting a volume of fluid into the syringe such that the concentration of the powdered ECM component in the fluid is about 100 to about 150 mg / mL.

10. The method of claim 8 , wherein the fluid is blood.

11. 9. The method of claim 8, wherein the powdered ECM component has an average particle size of about 0.1 mm to about 1 mm.

12. 9. The method of claim 8, wherein the ECM composition further comprises growth factors, platelets, leukocytes, stem cells, a crosslinking agent, a neutralizing agent, or any combination thereof.

13. The method of claim 8 , wherein the composition further comprises calcium.

14. 9. The method of claim 8, wherein the composition is substantially free of one or more of nucleic acids, GAGs, phospholipids, active pepsin, and active viruses.

15. 1. A method of treating a mammal, comprising administering to a joint of the mammal having or at risk of developing arthritis an effective amount of a composition comprising powdered ECM components and a fluid, wherein the ECM components comprise mesodermal proteins including collagen, and the powdered ECM components have a concentration in the fluid of about 50 mg / mL to about 200 mg / mL.

16. 16. The method of claim 15, wherein the powdered ECM component is present in a fluid at a concentration of about 100 to about 150 mg / mL.

17. 16. The method of claim 15, wherein the fluid is blood.

18. 16. The method of claim 15, wherein the powdered ECM component has an average particle size of about 0.1 mm to about 1 mm.

19. 16. The method of claim 15, wherein the ECM composition further comprises growth factors, platelets, leukocytes, stem cells, a crosslinking agent, a neutralizing agent, or any combination thereof.

20. 16. The method of claim 15, wherein the composition further comprises calcium.

21. 16. The method of claim 15, wherein the composition is substantially free of one or more of nucleic acids, GAGs, phospholipids, active pepsin, and active viruses.

22. 16. The method of claim 15, wherein the mammal has an acute injury to a joint.

23. 16. The method of claim 15, wherein the arthritis is osteoarthritis.

24. 16. The method of claim 15, wherein the arthritis is post-traumatic arthritis.

25. 25. The method of claim 24, wherein the post-traumatic arthritis is associated with an intra-articular injury or arthroscopic surgery.

26. 26. The method of claim 25, wherein the intra-articular injury is selected from the group consisting of anterior cruciate ligament rupture, anterior cruciate ligament rupture, meniscus injury, and cartilage injury.

27. 16. The method of claim 15, wherein the mammal has undergone a surgical procedure for torn, fractured, strained, bruised, or ruptured intra-articular tissue in a joint at least one day prior to administration of the composition.

28. 16. The method of claim 15, wherein the joint is a joint of the hand, elbow, wrist, hip, knee, foot, shoulder, ankle, temporomandibular joint, or spine.

29. 16. The method of claim 15, wherein the mammal has a lesion associated with the development of arthritis.

30. 16. The method of claim 15, wherein the administration comprises direct injection into the joint.

31. 16. The method of claim 15, wherein the mammal is a human.

32. 1. A method for treating a mammal having an intra-articular tissue defect, comprising: visualizing the defect with an arthroscope; and administering to the defect an effective amount of a composition comprising powdered ECM components and a fluid, wherein the ECM components comprise mesodermal proteins including collagen, and the concentration of the powdered ECM components in the fluid is from about 50 mg / mL to about 200 mg / mL relative to the defect.

33. 33. The method of claim 32, wherein the powdered ECM component is present in the fluid at a concentration of about 100 to about 150 mg / mL.

34. 33. The method of claim 32, wherein the fluid is blood.

35. 33. The method of claim 32, wherein the powdered ECM component has an average particle size of about 0.1 mm to about 1 mm.

36. 33. The method of claim 32, wherein the ECM composition further comprises growth factors, platelets, leukocytes, stem cells, a crosslinking agent, a neutralizing agent, or any combination thereof.

37. 33. The method of claim 32, wherein the composition further comprises calcium.

38. 33. The method of claim 32, wherein the composition is substantially free of one or more of nucleic acids, GAGs, phospholipids, active pepsin, and active viruses.

39. 33. The method of claim 32, wherein the defect is an acute injury in a joint.

40. 40. The method of claim 39, wherein the defect is selected from the group consisting of anterior cruciate ligament rupture, anterior cruciate ligament rupture, meniscus injury, and cartilage injury.

41. 33. The method of claim 32, wherein the defect is damage associated with the development of arthritis.

42. 33. The method of claim 32, wherein administering comprises direct injection into the defect.

43. 33. The method of claim 32, wherein the mammal is a human.

44. 1. A method for producing a powder composition comprising mesodermal extracellular matrix (ECM) proteins, the method comprising the steps of: decellularizing a tissue sample comprising tissue arising from mammalian mesoderm; treating the tissue sample with a composition comprising peracetic acid before or after decellularization; Lyophilizing the decellularized tissue sample; and Grinding freeze-dried tissue into a powder A method comprising:

45. 45. The method of claim 44, wherein the composition comprising peracetic acid comprises about 0.1% peracetic acid.

46. 45. The method of claim 44, comprising treating the tissue sample with a composition comprising peracetic acid for about 5 to 30 minutes before or after decellularization.

47. 45. The method of claim 44, wherein the composition comprising peracetic acid further comprises hydrogen peroxide.

48. 48. The method of claim 47, wherein the composition comprises about 1% hydrogen peroxide.

49. 45. The method of claim 44, further comprising treating the decellularized tissue sample with an enzyme prior to freeze-drying, thereby removing species-specific ends of collagen molecules.

50. 45. The method of claim 44, further comprising treating the powder with supercritical carbon dioxide (scCO2).

51. 45. The method of claim 44, wherein the powder has an average particle size of about 0.1 mm to about 1 mm.

52. 45. The method of claim 44, wherein the composition further comprises a growth factor, a platelet, a white blood cell, a stem cell, a crosslinking agent, a neutralizing agent, or any combination thereof.

53. 45. The method of claim 44, wherein the composition further comprises calcium.

54. 45. The method of claim 44, wherein the composition is substantially free of one or more of nucleic acids, GAGs, phospholipids, active pepsin, and active viruses.

55. 1. A method for producing a powder composition comprising mesodermal ECM proteins, comprising the steps of: decellularizing a tissue sample comprising tissue from mammalian mesodermal tissue; freeze-drying the decellularized tissue sample; grinding the freeze-dried tissue slurry into a powder; and Treating the powder with scCO2 A method comprising:

56. 56. The method of claim 55, further comprising treating the decellularized tissue sample with an enzyme prior to freeze-drying, thereby removing species-specific ends of collagen molecules.

57. 56. The method of claim 55, further comprising treating the tissue sample with a composition comprising peracetic acid before or after decellularization.

58. 58. The method of claim 57, wherein the composition comprising peracetic acid comprises about 0.1% peracetic acid.

59. 58. The method of claim 57, comprising treating the tissue sample with a composition comprising peracetic acid for about 5 to 30 minutes before or after decellularization.

60. 58. The method of claim 57, wherein the composition comprising peracetic acid further comprises hydrogen peroxide.

61. 61. The method of claim 60, wherein the composition comprises about 1% hydrogen peroxide.

62. 56. The method of claim 55, wherein the powder has an average particle size of about 0.1 mm to about 1 mm.

63. 56. The method of claim 55, wherein the composition further comprises a growth factor, a platelet, a white blood cell, a stem cell, a crosslinking agent, a neutralizing agent, or any combination thereof.

64. 56. The method of claim 55, wherein the composition further comprises calcium.

65. 56. The method of claim 55, wherein the composition is substantially free of one or more of nucleic acids, GAGs, phospholipids, active pepsin, and active viruses.

66. 1. A method for producing a composition comprising blood and a powdered mesodermal ECM component including collagen, the method comprising the steps of: providing a syringe containing a powdered ECM component; contacting the blood sample with an anticoagulant; injecting a volume of blood into the syringe containing the powdered ECM component such that the concentration of the powdered ECM component in the blood is between about 50 mg / mL and about 200 mg / mL; and Adding calcium chloride solution to the syringe, thereby inactivating the anticoagulant A method comprising:

67. 67. The method of claim 66, comprising injecting a volume of blood into the syringe such that the concentration of the powdered ECM components in the blood is about 100 to about 150 mg / mL.

68. 67. The method of claim 66, wherein the powdered ECM composition has an average particle size of about 0.1 mm to about 1 mm.

69. 67. The method of claim 66, wherein the ECM composition further comprises growth factors, platelets, white blood cells, stem cells, a crosslinking agent, a neutralizing agent, or any combination thereof.

70. 67. The method of claim 66, wherein the composition is substantially free of one or more of nucleic acids, GAGs, phospholipids, active pepsin, and active viruses.

71. 67. The method of claim 66, wherein the calcium chloride solution has a concentration of about 35 mM to about 45 mM.

72. 67. The method of claim 66, comprising adding calcium chloride solution to a syringe to obtain a 1:9 ratio mixture of calcium chloride solution to blood.

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