Porcine scaffolds and methods of preparation

Abstract

To provide an improved porcine scaffold comprising decellularized porcine placental extracellular matrix. Further provided are methods of preparing the improved decellularized porcine placental extracellular matrix as well as methods of treatment.SOLUTION: Provided are porcine scaffolds comprising a decellularized porcine placental extracellular matrix, wherein the porcine scaffold comprises at least about 0.5% w / w hyaluronic acid based on the total weight of the porcine scaffold, and methods of preparing the porcine scaffolds.SELECTED DRAWING: Figure 9

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application Publication No. 62 / 979,731, filed February 21, 2020. [Background technology]

[0002] The extracellular matrix (ECM) is a secreted molecule that creates a microenvironment for cells and provides shape and strength to tissues (Brew, Dinakarpandian, & Nagase, 2000; Young, Holle, & Spatz, 2016). Biological scaffolds created from ECM are becoming increasingly popular for the treatment of various medical conditions (Hussey, Dziki, & Badylak, 2018). Commercially available scaffolds have been created from a wide range of sources; multiple species and tissue types have been successfully engineered into biological scaffolds, including porcine small intestinal submucosa, bovine pericardium, porcine urinary bladder, and human dermis (Agmon & Christman, 2016). In most commercially available ECMs, the structural component is primarily collagen, with the majority containing type 1 collagen (Badylak, Freytes, & Gilbert, 2009). Additional fibrin collagen species, types III, V, and XI, as well as nonfibrin collagen forms, types IV and VIII, exist depending on the source material and tissue type (Theocharis, Skandalis, Gialeli, & Karamanos, 2016). In addition to collagen content, ECMs can contain several adhesion molecules, such as elastin, fibronectin, and laminin (Badylak et al., 2009), which give each tissue type a unique ECM related to tissue function. The third major structural component of ECM (Badylak et al., 2009) is proteoglycan (PG), a basic protein bound to one or more glycosaminoglycans (GAGs). Some common extracellular proteoglycans are aggrecan, versican, and decorin, but like other structural molecules, proteoglycan content varies based on source material and tissue type ( Theocharis et al., 2016 ).

[0003] When creating biological scaffolds to repair damaged tissue, many factors influence the effectiveness of the final scaffold; starting material, tissue type, quality of the tissue source, tissue location, donor age, recovery time, decellularization, manufacturing process, and sterilization. The placental membrane, with its crucial role in defense and nutrient supply during fetal development, offers many unique properties that make it an ideal source tissue for biological scaffolds (Shaifur Ra, Islam, Asaduzzama, & Shahedur R, 2015). For this reason, human placental membranes (e.g., amniotic and / or chorionic tissue) have been used for various types of reconstructive surgery since the early 1900s. The membranes serve as substrates and are more commonly referred to as biological dressings or wound covers. Typically, human placental membranes are collected after cesarean section and minimally processed to ensure that the manufacturing process does not alter the membrane's original, relevant characteristics relevant to its utility for reconstruction, repair, or replacement.

[0004] The primary role of the placental membrane is to provide a physiological barrier to prevent desiccation (Mamede et al., 2012) and an immunological barrier for the fetus. Placental membrane tissue exhibits antimicrobial properties due to the presence of beta-3 defensins, which act to prevent microbial colonization of epithelial surfaces (Chopra & Thomas, 2013; Niknejad et al., 2008). The anti-inflammatory properties of placental membranes are based on the presence of interleukin-4 (IL-4), interleukin-10 (IL-10), TIMP-1, TIMP-2, and TIMP-4 (Hortensius & Harley, 2016; Mamede et al., 2012), which may also be beneficial for tissue healing. Placental membranes have shown clinical evidence of epithelialization (Dua, Gomes, King, & Maharajan, 2004 ; Subrahmanyam, 1995 ; Ward & Bennett, 1984 ) and the potential for scar-free healing (Leavitt et al., 2016 ).

[0005] The quality of source material for biological scaffolds can be challenging because all naturally occurring materials have some inherent variability (Cardinal, 2015). This challenge is particularly prevalent with human source material. Variation in gene expression between individuals is well documented (Genomes Project et al., 2010; International HapMap et al., 2010), and genetic variation has been demonstrated in individual tissues (O'Huallachain, Karczewski, Weissman, Urban, & Snyder, 2012). In addition to genetic variability, tissues can also be affected by numerous environmental and behavioral risk factors. For example, harvested human amniotic tissue can be affected by the maternal lifestyle (Day et al., 2015). Increased expression of cytochrome P450 enzymes, a family of enzymes responsible for metabolizing toxic compounds, is a marker of oxidative stress in human tissues (Strolin-Benedetti, Brogin, Bani, Oesch, & Hengstler, 1999). Human placental tissue has been shown to have increased cytochrome P450 levels in smokers (Huuskonen et al., 2016), drug users (Paakki et al., 2000), mothers with a BMI > 30 (DuBois et al., 2012), diabetic patients (McRobie, Glover, & Tracy, 1998), and alcohol abusers (Collier, Tingle, Paxton, Mitchell, & Keelan, 2002). Delivery and gestational age have been shown to alter the expression of cytochrome P450 ( Collier et al., 2002 ) and growth factors ( Lopez-Valladares et al., 2010 ) in human placental tissue. Despite the wide variability associated with human placenta products, growing awareness of the healing properties associated with such products has increased demand over the past decade, thus driving increased sales. However, there remains a need in the art for wound healing treatments with high clinical efficacy that overcome the inconsistencies of commercially available human-sourced products. [Prior art documents] [Patent documents]

[0006] [Non-Patent Document 1] Placenta,2019,Vol.81,p.32-41 Summary of the Invention

[0007] The present disclosure is generally directed to porcine scaffolds and processes for making such porcine scaffolds. The porcine scaffolds disclosed herein exhibit various regenerative properties. The porcine scaffolds provided herein can be cut to various sizes that may be preferred for specific applications. The porcine scaffolds may also be applied to other areas of the body that have persistent damage but have not undergone surgical procedures. A porcine scaffold is provided. According to one embodiment, the porcine scaffold comprises decellularized porcine placental extracellular matrix comprising at least about 0.5% w / w hyaluronic acid based on the total weight of the porcine scaffold. According to one embodiment, the placental extracellular matrix is ​​substantially devoid of intact cells. According to one embodiment, the placental extracellular matrix comprises up to about 1200 ng of dsDNA per mg of porcine scaffold. According to one embodiment, the porcine scaffold is formulated as a membrane-based construct. According to one embodiment, the placental extracellular matrix comprises at least one placental membrane, at least one amniotic membrane, at least one chorion, or a combination thereof. According to one embodiment, the porcine scaffold comprises at least about 1.0% w / w hyaluronic acid based on the total weight of the porcine scaffold. According to one embodiment, the porcine scaffold comprises at least about 1.5% w / w hyaluronic acid based on the total weight of the porcine scaffold. According to one embodiment, the porcine scaffold comprises at least about 2.0% w / w hyaluronic acid based on the total weight of the porcine scaffold. According to one embodiment, the porcine scaffold comprises at least about 2.5% w / w hyaluronic acid based on the total weight of the porcine scaffold. According to one embodiment, the porcine scaffold retains at least about 50% w / w of any hyaluronic acid present in a native porcine placental membrane. According to one embodiment, the porcine scaffold retains at least about 45% of any sulfated glycosaminoglycans present in a native porcine placental membrane. According to one embodiment, the porcine scaffold retains at least about 66% of any elastin present in a native porcine placental membrane. According to one embodiment, the porcine scaffold comprises up to about 1650 ng of fibronectin per gram of porcine scaffold.

[0008] According to one aspect, a porcine scaffold is provided that includes decellularized porcine placental extracellular matrix that has been treated with a surfactant and an alkaline solution. According to one aspect, a porcine scaffold is provided that comprises a decellularized porcine placental extracellular matrix comprising up to about 85% w / w total collagen based on the total mass of the porcine scaffold. According to one aspect, a porcine scaffold is provided that comprises decellularized porcine placental extracellular matrix comprising up to about 10% w / w elastin based on the total mass of the porcine scaffold. According to one aspect, a porcine scaffold is provided that comprises decellularized porcine placental extracellular matrix comprising up to about 3% w / w hyaluronic acid based on the total mass of the porcine scaffold. According to one aspect, a porcine scaffold is provided that comprises a decellularized porcine placental extracellular matrix comprising up to about 2% w / w sulfated glycosaminoglycans based on the total mass of the porcine scaffold. According to one aspect, a porcine scaffold is provided that comprises a decellularized porcine placental extracellular matrix comprising up to about 99% w / w total collagen, elastin, hyaluronic acid, and sulfated glycosaminoglycans, based on the total mass of the porcine scaffold.

[0009] According to one aspect, a porcine scaffold is provided that comprises a decellularized porcine placental extracellular matrix comprising up to about 1650 ng of fibronectin per gram of porcine scaffold. According to one aspect, a porcine scaffold is provided that includes a surface defining one or more fenestrations. Also provided is a wound dressing comprising the porcine scaffold provided herein. According to one embodiment, the wound dressing can include a surface defining one or more fenestrations. According to one aspect, a method of preparing a porcine scaffold is provided, comprising processing a porcine placental membrane to form the porcine scaffold. According to one embodiment, the method provided herein results in a porcine scaffold that retains at least about 50% w / w of all hyaluronic acid present in a native porcine placental membrane. According to one embodiment, the method provided herein results in a porcine scaffold that retains at least about 45% w / w of all sulfated glycosaminoglycans present in a native porcine placental membrane. According to one embodiment, the method provided herein results in a porcine scaffold that retains at least about 66% w / w of all elastin present in a native porcine placental membrane. According to one embodiment, the method provided herein results in a porcine scaffold that retains at least about 50% of sulfated and non-sulfated glycosaminoglycans present in a native porcine placental membrane.

[0010] According to one embodiment, the step of decellularizing the porcine placental extracellular matrix comprises treating the porcine placental membrane with a detergent solution, wherein the detergent solution comprises at least one protease enzyme. According to one embodiment, the detergent solution further comprises at least one anionic detergent. According to one embodiment, the step of decellularizing the porcine placental extracellular matrix comprises treating the porcine placental membrane with a virus inactivation solution, wherein the virus inactivation solution comprises at least one alkaline solution. According to one embodiment, the alkaline solution comprises sodium hydroxide in an amount of about 1 mL to about 50 mL of about 0.1 M to about 3.0 M sodium hydroxide per gram of porcine placental membrane. According to one aspect, a method for treating a defect is provided. The method for treating a defect includes administering a porcine scaffold provided herein to the defect. The defect may be located on or in a mammal in need of treatment. According to one embodiment, the defect is selected from partial-thickness wounds, full-thickness wounds, pressure ulcers, venous ulcers, diabetic ulcers, chronic duct ulcers, tunneling or digging wounds, surgical wounds, wound dehiscence, abrasions, lacerations, second-degree burns, skin lacerations, and draining wounds. According to one embodiment, the defect is a wound or ulcer. According to another aspect, there is provided a method of treating a wound comprising providing a powder-based construct as provided herein and applying the powder-based construct to or around the wound. According to one embodiment, the wound is an ulcer, an abrasion, or a burn. [Brief explanation of the drawings]

[0011] [Figure 1] Figure 1 shows postoperative wound dehiscence in a 63-year-old male patient. [Figure 2] FIG. 2 shows the postoperative surgical wound of FIG. 1 after two weeks of standard of care treatment. [Figure 3] FIG. 3 shows the post-operative wound of FIG. 1 after five consecutive weeks of treatment with one embodiment of a porcine scaffold. [Figure 4] FIG. 4 shows the post-operative wound of FIG. 1 after five consecutive weeks of treatment with one embodiment of a porcine scaffold. [Figure 5] FIG. 5 shows the post-operative wound of FIG. 1 after five consecutive weeks of treatment with one embodiment of a porcine scaffold. [Figure 6] FIG. 6 shows the post-operative wound of FIG. 1 after five consecutive weeks of treatment with one embodiment of a porcine scaffold. [Figure 7] FIG. 7 shows the post-operative wound of FIG. 1 after five consecutive weeks of treatment with one embodiment of a porcine scaffold. [Figure 8] FIG. 8 shows the post-operative wound of FIG. 1 after the fifth application of the porcine scaffold. [Figure 9] FIG. 9 shows the post-operative wound of FIG. 8 one month after the fifth application of the porcine scaffold. [Figure 10] Figure 10 shows postoperative wound dehiscence in a 77-year-old female patient. [Figure 11] FIG. 11 shows the post-operative wound of FIG. 10 after 4 weeks of standard of care treatment. [Figure 12] FIG. 12 shows the post-operative wound of FIG. 10 after one week of treatment with one embodiment of a porcine scaffold. [Figure 13] FIG. 13 shows the post-operative wound of FIG. 10 after six weeks of treatment with one embodiment of a porcine scaffold. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present disclosure will now be described more fully hereinafter with reference to exemplary embodiments thereof. These exemplary embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art. Indeed, this disclosure may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. As used herein and in the appended claims, the words "optional" or "optionally" mean that the event or circumstance described thereafter may or may not occur. As used herein, the term "birth tissue" includes, but is not limited to, elements of mammalian birth tissue, such as, for example, placental membranes (amnion and chorion), Wharton's gel, umbilical cord, umbilical artery, umbilical vein, and amniotic fluid.

[0013] As used herein, the term "commercialized ECM product" refers to a commercialized extracellular matrix product composed of porcine small intestinal submucosa source material. As used herein, the terms "fenestrated" and "fenestrated" may be used interchangeably and refer to a placental membrane-based construct that has been further modified to include at least one or more pre-prepared through-holes (fenestration) in the construct. Such holes allow exudate to pass through the construct. Furthermore, the number and size of the holes are predetermined to ensure that the fenestration is appropriately spaced to allow sufficient opportunity for exudate generated by the wound to pass through the construct, while also maintaining sufficient construct surface area to effectively treat the wound. As used herein, the term "placental membrane" refers to the complete, intact placental membrane, including the amniotic and chorionic layers, obtained from a mammal, eg, a pig or a human. As used herein, the term "membrane" refers to at least one placental membrane, at least one amniotic membrane, at least one chorionic membrane, or any combination thereof. The membranes referred to herein may be obtained from a mammal, such as, for example, a pig or a human.

[0014] As used herein, the terms "pig" and "porcine" may be used interchangeably. As used herein, the terms "porcine scaffold" and "powder-based construct" are constructs that are applied onto or around an injured area of ​​the mammalian body. As used herein, the terms "defect" and "wound" may be used interchangeably and refer to an area in need of treatment, such as an injured area of ​​a mammalian body. As used herein, the term "decellularization" refers to a process in which all or substantially all of the intact cells and cell nuclei are removed from the placental membrane, leaving behind the placental extracellular matrix derived from the original placental membrane. As used herein, the term "placental extracellular matrix" refers to decellularized placental membrane from which all or substantially all intact cells and cell nuclei have been removed, leaving behind a three-dimensional meshwork of extracellular macromolecules, such as collagen, elastin, glycosaminoglycans, laminin, and fibronectin, which provides both structure and biochemical support when used in a patient's body.

[0015] As used herein, the term "scaffold" refers to a decellularized extracellular matrix structure that allows a patient's cells to infiltrate to aid in the healing cascade and regeneration of damaged tissue. The scaffolds provided herein include extracellular matrix derived from birth tissue, such as porcine placental membrane or other mammalian placental membrane. As used herein, the term "native" refers to the state of a tissue after it has been procured from a mammal but before it has been subjected to the preparation steps provided herein. The present disclosure provides extracellular matrix scaffolds prepared from mammalian birth tissue. The present disclosure particularly provides porcine scaffolds prepared from porcine birth tissue. However, the methods and uses provided herein may be applied to birth tissue sourced from any mammal that forms a scaffold suitable for regenerative purposes. Suitable mammals include, but are not limited to, humans, cows, horses, goats, or sheep. Pig placenta provides a unique source material for extracellular matrix scaffolds while overcoming the inherent challenges associated with the human placenta. Pig placenta lacks the social factors (e.g., obesity, tobacco, alcohol, and drug consumption) that profoundly impact the availability and quality of human-source tissues. In contrast, Purposebred sows have a fully structured lifestyle in which the sow's age, diet, exercise regimen / activity level, and health are fully controlled by the breeder, thereby reducing the variability of the porcine placenta starting material. For example, unlike the widely adopted age standard for human placental membranes, which encompasses all women of childbearing age regardless of age, sows are reared from 1 year to approximately 6 years of age, which corresponds to a human age range of 16 to 35 years. Sows give birth to piglets after a gestation period of approximately 114 days. The combination of a low birth age and gestational age reduces the likelihood of stress markers (e.g., cytochrome P450 enzymes) being present in porcine tissues.

[0016] The porcine scaffolds and methods provided herein attempt to address unmet needs in the current human placental membrane market by providing an alternative source material that overcomes the inherent challenges associated with the human placenta, as specifically mentioned herein. Aside from the fact that porcine-sourced material is free of the same age, health, and lifestyle issues that can affect human products, current porcine scaffolds allow host cells to infiltrate the scaffold and the affected area (e.g., defect), deposit collagen, and easily and rapidly reconstruct the defect. The porcine scaffolds provided herein can aid in the healing cascade or healing process of a mammalian defect, such as a wound or ulcer. The porcine scaffold can be completely resorbed by the mammalian body during the healing process. A method is provided for aseptically processing a placental membrane to prepare a porcine scaffold. According to one embodiment, a porcine placental extracellular matrix placenta is prepared from a placental membrane that remains intact in that it retains the amniotic and chorionic layers and any intermediate layers. According to one embodiment, the placental membrane is processed in a manner such that all natural layers are retained except for Wharton's gelatinous substance.

[0017] The porcine scaffold provided herein may be formulated as a membrane-based construct. According to one embodiment, the porcine scaffold comprises one or more layers of porcine placental membrane, including an intact placental membrane or one or more layers of isolated amniotic or chorionic membrane. According to one embodiment, the porcine placental membrane comprises the dehydrated, decellularized porcine placental extracellular matrix provided herein. According to another embodiment, the porcine scaffold provided herein may also be formulated as a powder, gel, liquid, or spray. According to one embodiment, when formulated as a membrane-based scaffold, the placental membrane selected to be processed to form the scaffold may be treated to provide for delivery of various antibodies, anti-inflammatory agents, growth factors, and / or other specialized proteins or small molecules. Additionally, the resulting membrane-based scaffold may be combined with or covered with a base layer (sterile gauze, sterile polymeric substance, or other tissue or biomaterial) to increase the strength of the porcine scaffold for suturing or to extend the longevity of the implant.

[0018] The scaffolds described herein may be made by processing mammalian birth tissue according to any or all of the steps provided herein as applied to birth tissue. According to certain embodiments, the porcine scaffolds described herein may be made by processing porcine birth tissue according to the steps provided herein. According to one aspect, a porcine scaffold is provided. According to one embodiment, the porcine scaffold comprises a decellularized porcine placental extracellular matrix including one or more of collagen I, collagen III, collagen IV, elastin, laminin, fibronectin, hyaluronic acid, and sulfated glycosaminoglycans. According to one embodiment, each of one or more of collagen I, collagen III, collagen IV, elastin, laminin, fibronectin, hyaluronic acid, and sulfated glycosaminoglycans is present in an amount different from that of a native porcine placental membrane that has not been processed according to one or more of the processing steps provided herein.

[0019] According to one embodiment, the porcine scaffold provided herein comprises a decellularized porcine placenta extracellular matrix comprising at least about 0.5% w / w hyaluronic acid based on the total mass of the porcine scaffold. According to one embodiment, the porcine scaffold comprises at least about 1.0% w / w hyaluronic acid based on the total mass of the porcine scaffold. According to one embodiment, the porcine scaffold comprises at least about 1.5% w / w hyaluronic acid based on the total mass of the porcine scaffold. According to one embodiment, the porcine scaffold comprises at least about 2.0% w / w hyaluronic acid based on the total mass of the porcine scaffold. According to one embodiment, the porcine scaffold comprises a decellularized porcine placenta extracellular matrix comprising at least about 2.5% w / w hyaluronic acid based on the total mass of the porcine scaffold. According to one embodiment, the porcine scaffold retains at least about 50% w / w of any hyaluronic acid present in native porcine membrane. According to one embodiment, the placental extracellular matrix forming the porcine scaffold is substantially devoid of intact cells. According to one embodiment, the placental extracellular matrix comprises up to about 1200 ng of dsDNA per mg of porcine scaffold.

[0020] According to one embodiment, the porcine scaffold is formulated as a membrane-based construct or scaffold. According to one embodiment, the placental extracellular matrix comprises at least one dehydrated placental membrane, at least one dehydrated amniotic membrane, at least one dehydrated chorionic membrane, or a combination thereof. According to one embodiment, the placental extracellular matrix is ​​chemically dehydrated. According to one embodiment, the porcine scaffold retains at least about 45% of all sulfated glycosaminoglycans present in native porcine placental membrane. According to one embodiment, the porcine scaffold retains at least about 66% of all elastin present in native porcine placental membrane. According to one embodiment, the porcine scaffold comprises up to about 1650 ng of fibronectin per gram of porcine scaffold.

[0021] According to one embodiment, a porcine scaffold is provided, comprising decellularized porcine placental extracellular matrix treated with a surfactant and an alkaline solution. According to one embodiment, a porcine scaffold is provided, comprising decellularized porcine placental extracellular matrix comprising up to about 85% w / w total collagen based on the total mass of the porcine scaffold. According to one embodiment, a porcine scaffold is provided, comprising decellularized porcine placental extracellular matrix comprising up to about 10% w / w elastin based on the total mass of the porcine scaffold. According to one embodiment, a porcine scaffold is provided, comprising decellularized porcine placental extracellular matrix comprising up to about 3% w / w hyaluronic acid based on the total mass of the porcine scaffold. According to one embodiment, a porcine scaffold is provided, comprising decellularized porcine placental extracellular matrix comprising up to about 2% w / w sulfated glycosaminoglycans based on the total mass of the porcine scaffold. According to one embodiment, there is provided a porcine scaffold comprising decellularized porcine placental extracellular matrix comprising up to about 99% w / w total collagen, elastin, hyaluronic acid, and sulfated glycosaminoglycans based on the total mass of the porcine scaffold. According to one embodiment, there is provided a porcine scaffold comprising decellularized porcine placental extracellular matrix comprising up to about 1650 ng fibronectin per gram of porcine scaffold.

[0022] According to one embodiment, a porcine scaffold is provided that includes a surface defining one or more fenestrations. A wound dressing is also provided. The wound dressing comprises a porcine scaffold provided herein. According to one embodiment, the wound dressing can include a surface defining one or more fenestrations. According to one embodiment, the wound dressing can be combined with or covered by a base layer or non-adherent secondary dressing (sterile gauze, sterile polymeric substance, or other tissue or biomaterial) to increase the strength of the porcine scaffold for suturing or to extend the life of the graft.

[0023] A method of preparing a porcine scaffold. According to one embodiment, the method includes processing a porcine placental membrane to form a porcine scaffold, wherein the porcine placental scaffold retains at least about 50% w / w of any hyaluronic acid present in native porcine placental membrane. According to one embodiment, the method includes processing a porcine placental membrane to form a porcine scaffold, wherein the porcine scaffold retains at least about 45% w / w of any sulfated glycosaminoglycans present in native porcine placental membrane. According to one embodiment, the method includes processing a porcine placental membrane to form a porcine scaffold, wherein the porcine placental scaffold retains at least about 66% w / w of any elastin present in native porcine placental membrane. According to one embodiment, the method includes processing a porcine placental membrane to form a porcine scaffold, wherein the porcine placental scaffold retains at least about 40% w / w of any sulfated glycosaminoglycans present in native porcine placental membrane. According to one embodiment, processing a porcine placental membrane to form a porcine scaffold includes treating the porcine placental membrane with a detergent solution, wherein the detergent solution comprises at least one protease enzyme. According to one embodiment, the detergent solution further comprises at least one anionic detergent. According to one embodiment, processing a porcine placental membrane to form a porcine scaffold includes treating the porcine placental membrane with a virus inactivation solution, wherein the virus inactivation solution comprises at least one alkaline solution. According to one embodiment, the alkaline solution comprises sodium hydroxide in an amount of about 1 mL to about 50 mL of about 0.1 M to about 3.0 M sodium hydroxide per gram of porcine placental membrane. According to one embodiment, the porcine scaffold retains at least about 50% of the sulfated and non-sulfated glycosaminoglycans present in native porcine placental membrane.

[0024] According to one embodiment, a method of preparing a pig scaffold is provided. The method includes collecting birth tissues, including umbilical cord, placental membranes (amnion and chorion), and amniotic fluid, from a sow. According to one embodiment, the method includes collecting birth tissues, including umbilical cord, placental membranes (amnion and chorion), and amniotic fluid, from a sow. According to one embodiment, the sow has not been genetically modified to silence or reduce expression of a functional alpha-1,3 galactosyltransferase gene. According to one embodiment, the placental membrane includes an attached umbilical cord. Potential birth tissue donors are screened and tested to eliminate any donors that may present a health risk. According to one embodiment, the birth tissue is collected from a full-term birth of one or more offspring, such as infants or piglets. According to one embodiment, the method may include rinsing the birth tissues, including the umbilical cord and placental membranes (amnion and chorion), by methods known to those skilled in the art. According to one embodiment, the method further comprises placing the birth tissue, including the umbilical cord and placental membranes (amnion and chorion), in a transport container. According to one embodiment, the method further comprises placing the birth tissue, including the umbilical cord and placental membranes (amnion and chorion), in a transport container containing a transport solution.

[0025] According to one embodiment, the method optionally includes freezing the umbilical cord and placental membranes by methods known to those skilled in the art. According to one embodiment, the umbilical cord and placental membranes may remain frozen until further processing is required. According to one embodiment, the method further includes removing the frozen, bagged umbilical cord and placental membranes from the freezer and thawing them in a refrigerator. According to one embodiment, the method further includes thawing the umbilical cord and placental membranes at ambient temperature. According to one embodiment, the method optionally includes placing any retained and frozen amniotic fluid in a container. According to one embodiment, the method includes rinsing the umbilical cord and placental membranes with water. According to one embodiment, the method includes draining the umbilical cord and placental membranes. According to one embodiment, the method includes separating the placental membranes from the umbilical cord.

[0026] According to one embodiment, the method includes dividing the placental membrane into fragments. According to one embodiment, the fragments are cut using a rotary cutter or other suitable cutter. When formulated as a membrane-based construct, the birth tissue can be cut into various sizes, thicknesses, and shapes. The placental membrane fragments are preferably of a size and shape sufficient to be applied onto or around a wound on or in the body of a mammalian patient. The thickness of the placental membrane can vary depending on the application, the type of membrane, and the number of membrane layers. According to one embodiment, the method includes removing Wharton's gel and excess fluid from the placental membrane to produce a clean placental membrane. According to one embodiment, the method includes treating the placental membrane with a bioburden-reducing solution. According to a preferred embodiment, the bioburden-reducing solution is sodium chloride. According to one embodiment, the method includes adding about 1 mL to about 100 mL of 0.1 M to about 0.5 M sodium chloride solution per gram of placental membrane. According to one embodiment, the method includes soaking the placental membrane in the sodium chloride solution for about 15 minutes to about 8 hours. According to one embodiment, the method includes agitating the placental membrane in the sodium chloride solution at about 20 RPM to about 100 RPM for about 15 minutes to about 8 hours.

[0027] According to one embodiment, the method includes decanting the sodium chloride. According to one embodiment, the method includes rinsing the placental membrane with water. According to one embodiment, the placental membrane is rinsed once with water. According to one embodiment, the rinsing step is performed multiple times with water. According to one embodiment, the placental membrane is rinsed about two to about five times with water. According to one embodiment, the method includes placing the placental membrane in about 1 mL to about 100 mL of a surfactant solution. According to one embodiment, the surfactant is present at a concentration of about 0.1% to about 10% w / v. According to one embodiment, the surfactant solution includes at least one ionic surfactant. According to certain embodiments, the surfactant solution includes at least one anionic surfactant. According to certain embodiments, the surfactant solution includes at least one anionic surfactant and at least one protease enzyme. According to one embodiment, the surfactant solution contains phosphate, and the phosphorus content is about 7.5%. According to one embodiment, the surfactant solution includes phosphate, carbonate, sodium linear alkylaryl sulfonate, and one protease enzyme. According to one embodiment, the method includes soaking the placental membrane in the surfactant solution for about 15 minutes to about 8 hours. According to one embodiment, the method includes agitating the placental membrane in the surfactant solution for about 15 minutes to about 8 hours at about 20 RPM to about 100 RPM.

[0028] According to one embodiment, the method includes decanting the surfactant solution. According to one embodiment, the method includes rinsing the placental membrane with water. According to one embodiment, the placental membrane is rinsed with water once. According to one embodiment, the placental membrane is rinsed with water multiple times. According to one embodiment, the placental membrane is rinsed with water about two to about five times. According to one embodiment, the method includes treating the placental membrane with a virus inactivation solution, such as, for example, sodium hydroxide, hydrogen peroxide, ethanol, or supercritical carbon dioxide. In a preferred embodiment, the virus inactivation solution is sodium hydroxide. According to one embodiment, the method includes adding or introducing about 1 mL to about 50 mL of about 0.1 M to about 3.0 M sodium hydroxide per gram of placental membrane. According to one embodiment, the method includes soaking the placental membrane in sodium hydroxide for about 1 minute to about 120 minutes. According to one embodiment, the method includes shaking the placental membrane in sodium hydroxide for about 1 minute to about 120 minutes at about 20 RPM to about 100 RPM. The sodium hydroxide may then be decanted. According to one embodiment, the steps of adding sodium hydroxide, shaking, and decanting may be repeated as many times as necessary to inactivate any viruses present in the placental membrane and produce a placental membrane that is substantially virus-free. According to one embodiment, the steps of adding sodium hydroxide, shaking, and decanting may be repeated once. According to one embodiment, the steps of adding sodium hydroxide, shaking, and decanting may be repeated up to five times. According to a preferred embodiment, the method includes adding or introducing about 5 mL to about 15 mL of 0.25 M sodium hydroxide per gram of placental membrane. According to a preferred embodiment, the method includes adding or introducing about 10 mL of 0.25 M sodium hydroxide for about 20 minutes, shaking, decanting, and repeating the process once. According to this preferred embodiment, the resulting porcine scaffold is substantially free of viruses, but much of the extracellular matrix composition, including a substantial proportion of glycosaminoglycans, is preserved. According to one embodiment, the method includes rinsing the placental membrane with water.

[0029] According to one embodiment, the method includes adding or introducing about 1 mL to about 50 mL of buffer solution per gram of placental membrane. According to one embodiment, the method includes immersing the placental membrane in the buffer solution. According to one embodiment, the method includes shaking the placental membrane in the buffer solution for about 1 minute to about 120 minutes at about 20 RPM to about 100 RPM. The buffer solution may then be decanted. In a preferred embodiment, the buffer solution is a phosphate buffer solution. According to one embodiment, the method includes measuring the pH of the placental membrane after buffer solution treatment. According to one embodiment, the steps of adding buffer solution, shaking, and decanting may be repeated until the pH of the placental membrane is about 6.8 to about 7.2. According to one embodiment, the method includes rinsing the placental membrane with water. According to one embodiment, the placental membrane is washed once with water. According to one embodiment, the rinsing step is performed multiple times with water. According to one embodiment, the placental membrane is rinsed with water about two to about five times.

[0030] When preparing a membrane-based construct, the placental membrane may be wet or dehydrated. According to one embodiment, the placental membrane may be dehydrated by any method known in the art, including, but not limited to, chemical dehydration (e.g., organic solvents), lyophilization, drying, oven dehydration, and air drying. According to a preferred embodiment, the method includes adding or introducing alcohol to the placental membrane to cover the entire surface of the placental membrane (i.e., submerging the placental membrane). According to one embodiment, the method includes adding or introducing about 1 mL to about 100 mL of alcohol per gram of placental membrane. According to one embodiment, the placental membrane is completely submerged in alcohol for about 10 minutes to about 24 hours. The alcohol may be any alcohol that is safe and suitable for contact with the placental membrane. According to a specific embodiment, the alcohol is ethanol. According to one embodiment, the method includes decanting or emptying the alcohol from the placental membrane.

[0031] According to one embodiment, the method includes spreading the placental membrane on a drying table (e.g., a Delrin drying table). According to one embodiment, the placental membrane may be blotted dry with a microfiber wipe or the like. The placental membrane may be spread in a manner to completely dehydrate the placental membrane while ensuring the absence of wrinkles or air bubbles. When preparing a membrane-based construct, the method includes cutting the placental membrane to a predetermined or desired size. According to one embodiment, the placental membrane is cut to size using a rotary cutter or other suitable instrument. According to one embodiment, cutting is performed using a scalpel blade. According to another embodiment, the method includes forming one or more (e.g., multiple) fenestrations in the placental membrane. The resulting scaffold thus includes a surface defining one or more fenestrations (e.g., through-holes). According to one embodiment, the placental membrane may be fenestrated using a scalpel blade or other instrument, the one or more fenestrations being appropriately spaced to allow sufficient opportunity for exudate generated by the wound to pass through the placental membrane, while also maintaining sufficient placental membrane surface area to effectively treat a defect such as a wound or ulcer.

[0032] The processing methods provided herein result in mammalian scaffolds comprising decellularized placental extracellular matrix, such as decellularized porcine placental extracellular matrix derived from placental membrane. According to one embodiment, the method includes placing the cut scaffolds in one or more packaging materials. According to one embodiment, the method includes terminally sterilizing the packaged scaffold. According to one embodiment, the method of terminal sterilization can be e-beam irradiation, gamma irradiation, peracetic acid treatment, vaporized peracetic acid (VPA) treatment, any combination thereof, or any other terminal sterilization method known in the art. According to another embodiment, the scaffold is formulated as a powder-based construct. When prepared as a powder-based construct, the scaffold may be wet or dehydrated. According to one embodiment, the scaffold may be dehydrated by any method known in the art, including, but not limited to, chemical dehydration (e.g., organic solvents), lyophilization, drying, oven dehydration, and air drying. According to one embodiment, the method includes cutting the scaffold into strips. According to one embodiment, the scaffold strips may then be placed in a grinder and ground into a powder to form the powder-based construct. According to one embodiment, the scaffold may consist of the entire placental membrane, or portions thereof, which may be placed in a grinder and ground into a powder to form the powder-based construct. According to one embodiment, the powder-based construct may then be placed in a suitable container or vial at a desired concentration. According to one embodiment, the method for preparing a powder-based construct includes lyophilizing the ground / ground powder-based construct in a vial to remove residual moisture. The vial containing the powder-based construct is then subjected to terminal sterilization. According to one embodiment, the method of terminal sterilization may be e-beam irradiation, gamma irradiation, peracetic acid treatment, vaporized peracetic acid (VPA) treatment, any combination thereof, or any other terminal sterilization method known in the art.

[0033] A method of treating a defect is also provided. According to one embodiment, the method includes administering a porcine scaffold provided herein. The porcine scaffold is then administered to (e.g., placed on or around) the defect. The defect may be a soft tissue defect, including, for example, a wound such as a burn, a cut, or an abrasion. According to one embodiment, the defect is selected from a partial-thickness wound, a full-thickness wound, a pressure ulcer, a venous ulcer, a diabetic ulcer, a chronic ductal ulcer, a tunneling or digging wound, a surgical wound, a wound dehiscence, an abrasion, a laceration, a second-degree burn, a skin laceration, and a draining wound. The defect may be any ulcer. According to one embodiment, the wound may be a surgical site anywhere on or in a mammalian body. The porcine scaffold may be placed over the surgical site or held in place by the patient's musculature or skin. Sutures or staples may be used to hold the membrane-based porcine scaffold in place. The porcine scaffold may be hydrated at the application site during a procedure. The porcine scaffold may be used as an implant. The porcine scaffold may also be used to cover an implant or other device that may be placed on or within a mammal.

[0034] According to one embodiment, the porcine scaffolds provided herein are useful in conjunction with common surgical procedures to aid in the healing cascade, reduce adhesions, and reduce pain / inflammation. Such common surgical procedures include, but are not limited to, breast reconstruction, hernia repair / abdominal wall reconstruction / fascial reconstruction, and vascular bypass graft sites. According to one embodiment, the porcine scaffolds provided herein are useful as hemostatic or biological adhesives. According to one embodiment, the porcine scaffolds provided herein are useful for treating, reducing, and preventing scar formation. Such scar formation may be the result of trauma or a surgical procedure. A surgical procedure includes any procedure that may result in scarring. According to one embodiment, the porcine scaffolds provided herein are useful in neurosurgery to serve as dura substitutes, nerve conduits, nerve wraps, aiding nerve regeneration or repair, or in conjunction with aneurysm repair. According to one embodiment, the porcine scaffolds provided herein are useful in orthopedic surgery (e.g., sports-related injuries to muscles, ligaments, and tendons; bone-related surgery (e.g., spine), total joint replacement, laminectomy (anti-adhesion barrier), tendon / ligament repair, nerve repair, osteoarthritis, cartilage repair, and bone grafting). According to one embodiment, the porcine scaffolds provided herein are useful in colorectal surgery, such as colonic anastomosis or fistula repair. According to one embodiment, the porcine scaffolds provided herein are useful in cosmetic surgery as dermal fillers or to aid in skin wrinkle reduction, skin resurfacing, skin rejuvenation, and other cosmetic purposes.

[0035] According to one embodiment, the porcine scaffolds provided herein are useful in cardiovascular surgery in conjunction with pericardial patches, heart valve leaflets, or vascular grafts. According to one embodiment, the porcine scaffolds provided herein are useful in pulmonology for lung repair. According to one embodiment, the porcine scaffolds provided herein are useful for the treatment and reduction of existing scars (e.g., scarplasty). In particular, the porcine scaffolds provided herein may be used to improve or reduce the appearance of scars, restore skin function, and correct skin changes (impaired appearance), such as those caused by injury, wound, or previous surgery. According to one embodiment, the porcine scaffolds provided herein can be used as dressings to aid in the healing and prevention of scars, such as those associated with cancer resection (e.g., Mohs surgery).

[0036] According to one embodiment, the porcine scaffolds provided herein are useful for treating defects in the ear, nose, mouth, or throat, such as in treating oral fistulas or septum repair. In some embodiments, the porcine scaffolds provided herein are useful for treating dental defects, such as wrapping dental implants, treating advanced gingival recession defects, soft palate reconstruction, periodontal defects, or in guiding tissue repair. According to one embodiment, the porcine scaffolds provided herein are useful for treating ophthalmic conditions (e.g., ocular surface repair, keratitis, corneal ulcers / occlusions, or pterygium). According to one embodiment, the porcine scaffolds provided herein are useful for treating various gynecological or urological applications, such as in ureter repair, hysterectomy, uterine fibrosis, urinary incontinence, or vaginal prolapse. Although specific embodiments of the present invention have been illustrated and described in detail herein, the present invention is not limited thereto. The above detailed description is provided as an example of the present invention and should not be construed as constituting any limitation of the present invention. Modifications will be obvious to those skilled in the art, and all modifications that do not depart from the spirit of the present invention are intended to be included within the scope of the appended claims.

[0037] biochemical analysis One embodiment of the porcine scaffold provided herein underwent extensive biochemical analysis. The results showed that the porcine scaffold was primarily composed of four major extracellular matrix components: collagen, elastin, hyaluronic acid, and sulfated glycosaminoglycans (sGAGs). Specific amounts are listed in Table I below. [Table 1]

[0038] The ratio of collagen I to collagen III in the porcine scaffolds and the resulting collagen I and III mass contents are provided below in Table II. For this analysis, it was assumed that the 77% total collagen content provided in Table II was primarily comprised of collagen I and III. [Table 2]

[0039] An ELISA test was used to quantify the fibronectin found in the porcine scaffolds (Example 5). The results showed that the porcine scaffolds contained an average amount of 1521.3±112.8 ng of fibronectin per gram of porcine scaffold. Biochemical composition analysis of one embodiment of a porcine scaffold produced by the methods provided herein demonstrated an extracellular matrix with optimal regenerative potential. Those skilled in the art recognize the evolution of regenerative wound care products from products made from purified collagen to those made from more complex extracellular matrix structures with intact biologically important extracellular matrix components (e.g., elastin, fibronectin, glycosaminoglycans, and other extracellular matrix molecules). Purified collagen products, by definition (Cassel & Kanagy, 1949), have limited regenerative potential because an increase in collagen content necessarily results in a decrease in non-collagenous extracellular matrix molecules with regenerative potential. Non-collagenous extracellular matrix molecules such as elastin, fibronectin, hyaluronic acid, sulfated GAGs, and other molecules can act in concert or alone to create a more favorable wound healing environment. Those skilled in the art recognize that biomaterials derived from natural extracellular matrix sources (e.g., tendons, dermis) with very dense collagen are mechanically strong but offer limited regenerative potential, while biomaterials derived from natural extracellular matrix sources with lower collagen density and higher levels of non-collagenous extracellular matrix components (e.g., bladder, pericardium, peritoneum) have more regenerative properties due to their higher levels of non-collagenous extracellular matrix components. To this end, the porcine scaffolds provided herein retain a large amount of non-collagenous extracellular matrix components compared to other commercialized biomaterials derived from natural extracellular matrix sources, which aids in the wound healing capabilities of the porcine scaffolds. For example, the average total collagen (the sum of soluble and insoluble collagen) of the present porcine scaffolds is approximately 77% w / w. The average total collagen of commercialized ECM products is approximately 94% w / w.Although both can be considered collagen biomaterials, as each is primarily composed of collagen, the fact that the porcine scaffold contains less collagen and more complex non-collagenous major extracellular matrix components, including hyaluronic acid, sulfated glycosaminoglycans, elastin, and fibronectin, indicates that it is a more potent regenerative biomaterial compared to commercial ECM products.

[0040] Non-collagenous molecules in porcine scaffolds Glycosaminoglycans (GAGs) are complex carbohydrates that are ubiquitously and abundantly expressed on cell surfaces and in the extracellular matrix. Their structural diversity allows them to interact with a wide range of biomolecules, facilitating numerous functions in the extracellular matrix, including cell proliferation, cell adhesion, growth factor signaling, immune cell function, and regulating tissue mechanical properties, including collagen structure. Five linear, complex, and polydisperse GAGs are produced in mammalian systems. Hyaluronic acid (HA) is a non-sulfated GAG, while sulfated GAGs include chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and keratan sulfate (KS). Sulfated GAGs are attached to protein cores to form proteoglycans (PGs), and non-sulfated GAGs, such as hyaluronic acid, can exist as GAG chains.

[0041] Hyaluronic acid (non-sulfated GAG) Hyaluronic acid is a natural linear carbohydrate composed of repeating disaccharide units of β-1,3-N-acetylglucosamine and β-1,4-glucuronic acid, with a molecular weight of up to 6 MDa (WO 2010 / 003797). Hyaluronic acid is widely distributed throughout connective, epithelial, and neural tissues and is known for its ability to play an important role in promoting wound healing and tissue regeneration (Chen Wound Rep Reg 1999; Litwiniuk Wounds 2016; Kessiena Wound Rep Reg 2014). Hyaluronic acid is also involved in all stages of extracellular tissue repair: inflammation, granulation, re-epithelialization, and wound healing (EP 1948200). Hyaluronic acid is also known as hyaluronan, hyaluronate, or HA. The terms hyaluronic acid and HA may be used interchangeably herein. The glycosaminoglycan (GAG) content of porcine placenta throughout the second half of gestation was found to be approximately 60 μg / mg (0.06 g / g), and at the end of gestation, hyaluronan accounted for approximately 64% of the total GAG content (Steele 1980). Using this data, the mean hyaluronan content of porcine placenta at the end of term was typically about 38.4 μg / mg (0.038 g / g), with the remaining sulfated GAGs accounting for 21.6 μg / mg (0.022 g / g) at the end of term.

[0042] The provided porcine scaffolds have been shown to retain at least about 0.02 g of hyaluronic acid per gram of porcine scaffold (see Example 6). This measurement corresponds to a retention of about 50% of the hyaluronic acid content of the porcine scaffold after processing. Thus, the porcine scaffolds provided herein retain at least about 50% w / w of any hyaluronic acid present in native porcine placental membranes. The retention of hyaluronic acid content in the porcine scaffolds represents a surprising result in light of the preparation methods provided herein. Decellularization of the extracellular matrix can be achieved by various chemical treatments known in the art, some of which are known to remove GAGs; alkali / acid; non-ionic detergents (e.g., Triton X-100); ionic detergents (e.g., sodium dodecyl sulfate (SDS)); and certain enzymes (e.g., nucleases) during the decellularization process (Gilbert Biom 2006). According to the methods disclosed herein, a porcine scaffold may be prepared by a method comprising treating a porcine placental membrane with an ionic detergent. According to the methods disclosed herein, a porcine scaffold may be prepared by a method comprising treating a porcine placental membrane with an alkali / acid (alkali-sodium hydroxide solution) treatment step. Despite these preparation methods disclosed herein, a high content (at least about 50% w / w) of hyaluronic acid is preserved from the raw porcine placental tissue in the resulting porcine scaffold.

[0043] The porcine scaffold provided herein contains a significantly increased level of hyaluronic acid compared to other commercially available extracellular matrix products.For example, the porcine scaffold provided herein contains approximately 10 times more hyaluronic acid per gram (w / w) than other commercially available extracellular matrix products, including, for example, extracellular matrix products sourced from porcine small intestine submucosa (commercialized ECM products) and extracellular matrix products sourced from human birth tissue.Commercialized ECM products contain approximately 0.002g of hyaluronic acid per gram of product (see Example 6); dehydrated human amniotic membrane / chorion allograft also contains approximately 0.002g of hyaluronic acid per gram of product according to manufacturer's published data (Lei Adv Wound Care 2016).

[0044] Sulfated GAGs Sulfated GAG side chains on proteoglycans are crucial macromolecules in all stages of wound healing (Ghatak, S. et al.). During the proliferative phase of wound healing, fibroblasts and mesenchymal cells invade the inflamed wound in response to growth factors, which are necessary to stimulate cell proliferation. Fibroblasts synthesize collagen and proteoglycans, a process that continues for several weeks, resulting in a proportional increase in collagen. During this period, endothelial cells form capillaries, and GAG (hyaluronic acid, chondroitin sulfate (CS), and dermatan sulfate (DS)) levels also change. During the first two weeks, hyaluronic acid is synthesized in large quantities by fibroblasts, followed by an increase in the levels of DS and CS proteoglycans. Gradually, as cell proliferation plateaus, heparan sulfate (HS) proteoglycan levels increase in the wound. Sulfated proteoglycans containing CS and DS support collagen polymerization, and HS proteoglycans on cells can anchor to the surrounding matrix. Proteoglycan degradation by proteases in wounds can release GAG-peptide fragments, which can regulate the wound healing process. For example, CS and DS can regulate growth factor activity and stimulate nitric oxide production, which in turn can regulate angiogenesis, while HS can stimulate the release of IL-1, IL-6, PGE2, and TGF-β, contributing to the regulation of their proangiogenic effects in tissues. Furthermore, sulfated GAGs as part of proteoglycans are thought to play a role in collagen fibril and fiber assembly due to electrostatic interactions between positively charged sites on collagen molecules and negatively charged GAGs (Michelacci, YM).

[0045] The porcine scaffolds provided herein contain or otherwise maintain approximately 0.01 g of sulfated GAGs per gram of porcine scaffold (see Example 7), which corresponds to at least about 45% retention of sulfated GAG content after processing. Thus, the porcine scaffolds retain at least about 45% of any sulfated GAGs present in native porcine placental membranes. Similar to the hyaluronic acid retention outlined above, approximately half (e.g., at least about 50% w / w) of the sulfated GAG content is retained in the final porcine scaffold. Thus, processing porcine placental membranes according to the methods provided herein preserves at least about 50% w / w of the initial content of both sulfated and non-sulfated GAGs from native porcine placental membranes in the final porcine scaffold.

[0046] Elastin Elastin is an extracellular matrix protein that provides elasticity and resilience to many tissue types and plays a crucial role in wound healing, including inducing a range of cellular activities, including cell migration and proliferation, matrix synthesis, and protease production (Almine, Wise, & Weiss, 2012). Given its mechanical and signaling properties, elastin plays a multifunctional role in wound healing (Almine et al., 2013). The porcine scaffolds provided herein contain or otherwise maintain approximately 0.09 g of elastin per gram of porcine scaffold, which corresponds to the retention of at least about 66% of the elastin content after processing (see Example 4). Thus, the porcine scaffolds retain at least about 66% of any elastin present in native porcine placental membranes.

[0047] fibronectin Fibronectin is an adhesive glycoprotein that plays a crucial role in wound healing, particularly in extracellular matrix formation and re-epithelialization (Lenselink, 2013). The porcine scaffolds provided herein contain or otherwise maintain approximately 1520 ng of fibronectin per gram of porcine scaffold (see Example 5). A commercialized ECM product contains approximately 139 ng of fibronectin per gram of commercialized ECM product, which is equivalent to approximately 9% of the average fibronectin concentration found in porcine scaffolds. Thus, the porcine scaffolds have more than 10 times the fibronectin per ng / g (w / w) compared to the commercialized ECM product. The porcine scaffolds provided herein retain a large amount of non-collagenous extracellular matrix components when compared to other commercialized biomaterials derived from natural extracellular matrix sources, which aids in the wound healing capabilities of the porcine scaffolds. The porcine scaffolds provided herein contain higher levels of several non-collagenous extracellular matrix molecules, including hyaluronic acid, elastin, and fibronectin, suggesting a more robust regenerative biomaterial compared to commercialized ECM products.

[0048] Decellularization of porcine scaffolds Adequate decellularization of extracellular matrix is ​​critical to the functionality of the final extracellular matrix product. The goal of decellularization is to remove cells and residual genetic material while preserving extracellular matrix components and retaining the characteristics of the underlying tissue (Gilpin & Yang 2017). Incomplete decellularization has been shown to decrease the ratio of M2-activated macrophages to M1-activated macrophages compared to more complete decellularization methods (Keane et al., 2012). Further research has demonstrated that a higher ratio of M2-activated to M1-activated macrophages promotes greater immunomodulatory, constitutive mechanisms, and remodeling activities (Sicari et al., 2014).

[0049] Those skilled in the art recognize that cells can be removed from tissue using physical, chemical, enzymatic, protease inhibitor, or antibiotic methods. Regardless of the removal method, the efficiency of decellularization is determined. A common method for verifying cell removal is through the use of histological stains. Hematoxylin and eosin (H&E) and DAPI (4',6-diamidino-2-phenylindole) have been successfully used to quantify the remaining cellular and nuclear content, respectively, of processed tissues (Gilbert et al., 2006; Oliveira et al., 2013). In addition to the porcine scaffolds, cell debris testing was performed on raw, washed porcine placenta to characterize the ability of the porcine scaffold manufacturing process to remove cellular material and generate "clean" tissue.

[0050] Hematoxylin and eosin (H&E) staining was used to determine the presence of cells and cell debris. 4',6-diamidino-2-phenylindole (DAPI) staining was also used to assess intact cell nuclei. The postnatal porcine placenta tissue contained abundant intact cells and cell nuclei throughout the tissue. In contrast, the porcine scaffolds contained no visible intact cells or cell nuclei. Therefore, the porcine scaffold fabrication process is an effective decellularization process. Furthermore, the remaining DNA / nucleic acids within the porcine scaffolds were quantified using an IT PicoGreen assay, resulting in a negligible amount of biochemical residue (1115.0±137.0 ng / mg porcine scaffold). Detailed testing methods are discussed in Examples 2 and 3. [Example]

[0051] Example 1 Herovici analysis - semi-quantitative analysis of collagen I to collagen III ratio The objectives of this study were to evaluate the collagen I to collagen III ratio in porcine scaffolds prepared from three pig breeds (Breed 1, Breed 2, and Breed 3); and in commercialized ECM products. The collagen I to collagen III ratio was assessed by semiquantitative analysis of Herovici staining. Testing was performed in accordance with key requirements of Good Laboratory Practice (GLP). method Samples from each specimen were fixed in 10% neutral buffered formalin, embedded in paraffin, and 5 μm sections were cut onto slides using methods known in the art. Four 5 μm sections per specimen were cut for each slide. The slides were stained. Two sets of slides were stained using the Herovici protocol, with each set stained as an independent batch. The pink (collagen I) to blue (collagen III) ratio was quantified for four sections on each slide using a CriNuance FX multispectral imaging system under a Nikon Eclipse E600 microscope with a 20x objective. The results are summarized in Tables III and IV.

[0052] result [Table 3] [Table 4]

[0053] conclusion Herovici staining provided a method for semiquantitative analysis of the collagen I to collagen III ratio in tissue samples (Turner et al., 2013). Herovici quantification of the specimens showed that the collagen I to collagen III ratio was 2.15 ± 0.22 for the porcine scaffold and 2.61 ± 0.49 for the commercialized ECM product. A Kruskal-Wallis one-way analysis of variance (Dunn's method) based on ranks with full pairwise multiple comparisons was performed on the data and showed that there were no statistically significant differences in collagen I to collagen III ratios between the commercialized ECM product and any of the three groups of porcine scaffolds prepared from breed 1, breed 2, and breed 3.

[0054] Example 2 Assessment of cell debris the purpose The purpose of this study was to evaluate the degree of cellular debris present in (a) porcine scaffolds manufactured from placentas derived from one of three pig breeds (Breed 1, Breed 2, and Breed 3) and (b) commercialized ECM products; cellular material present in raw, washed placentas derived from the three breeds (Breed 1, Breed 2, and Breed 3) served as tissue controls. Testing was performed in accordance with key requirements of Good Laboratory Practice (GLP).

[0055] method Samples from each study were fixed in 10% neutral buffered formalin, embedded in paraffin, and 5 μm-thick sections were cut onto slides. Two 5 μm sections per study were cut to fit each slide. One slide from each sample was deparaffinized and stained with hematoxylin and eosin (H&E); one slide from each sample type was deparaffinized and stained with DAPI-Fluoromount-G slide mounting medium (Southern Biotech #010020). DAPI (4',6-diamidino-2-phenylindole) is a blue-fluorescent DNA dye that exhibits approximately 20-fold enhancement of fluorescence upon binding to AT regions of dsDNA. Slides were imaged on a Zeiss Observer Z1 microscope using an Axiocam MRc camera.

[0056] result Specimens were stained with H&E and imaged at 32x magnification to assess the presence of cells and cellular debris. Additionally, specimens were stained with DAPI and imaged at 10x and 32x to assess the presence of intact cell nuclei. Analysis of the H&E-stained slides demonstrated that (i) raw, washed porcine placenta samples from three pig breeds (Breed 1, Breed 2, and Breed 3) contained cellular material appearing as intact cells throughout the tissue, as expected for native placental tissue; (ii) porcine scaffold specimens fabricated from placentas from one of the three pig breeds (Breed 1, Breed 2, and Breed 3) contained cellular material in certain areas but not throughout the tissue; and (iii) commercialized ECM products contained cellular material throughout the tissue. While much of the cellular material observed in the commercialized ECM product appeared to be the expected size and shape for intact cells, the cellular material observed in the porcine scaffold specimens from Breed 1, Breed 2, and Breed 3 appeared to be smaller than intact cells.

[0057] Analysis of DAPI-stained slides showed that (i) raw, washed placenta samples from the three breeds contained intact cell nuclei throughout the tissue, as expected for native placental tissue; (ii) porcine scaffold specimens manufactured from the three breeds (Breed 1, Breed 2, and Breed 3) had no visible intact cell nuclei; and (iii) commercialized ECM products contained intact cell nuclei throughout the tissue. Analysis of H&E and DAPI stained tissues showed that the cellular material present in the porcine scaffold specimens was cellular debris and did not contain intact cells, and that the commercialized ECM product contained intact cells throughout the tissue. Conclusion: Comparing cellular debris that may pose immunogenic or inflammatory risks, the porcine scaffold is considered a safer material than the commercialized ECM product. The porcine scaffold showed no intact cells or cell nuclei, while the commercialized ECM product contained numerous intact cells and cell nuclei throughout its structure.

[0058] Example 3 Nucleic Acid Quantification and Analysis the purpose The objectives of this study were to quantify nucleic acids (by DNA content) in (i) porcine scaffolds manufactured from placentas of one of three different breeds of pigs (Breed 1, Breed 2, and Breed 3) and (ii) commercialized ECM products. Testing was performed in accordance with key requirements of Good Laboratory Practice (GLP). method: DNA was extracted from samples according to methods known in the art. Double-stranded DNA (dsDNA) was quantified using the Quant-IT PicoGreen assay (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. All samples were assayed in triplicate. Additionally, test group samples were run simultaneously to ensure data acceptability due to assay sensitivity. Results are summarized in Table V.

[0059] result A.PicoGreen quantification results [Table 5]

[0060] conclusion Each porcine scaffold from one of three pig breeds (Breed 1, Breed 2, and Breed 3) contains approximately 1000-1200 ng of dsDNA per mg of dry mass. The commercialized ECM product contains over 2300 ng of dsDNA per mg of dry mass. The dsDNA content of each of the three porcine scaffold samples is approximately half the dsDNA content of the commercialized ECM product. These results, combined with those from Example 2 (Evaluation of Cell Debris), indicate that the porcine scaffold manufacturing process is a more effective decellularization process than commercial ECM products. Compared to both raw porcine placenta material and commercial ECM products, the porcine scaffold is both cleaner and safer with respect to residual cellular and nucleic acid material.

[0061] Example 4 Elastin quantification the purpose The objectives of this study were to measure elastin content in (i) porcine scaffolds manufactured from placentas of one of three different breeds of pigs (Breed 1, Breed 2, and Breed 3), (ii) raw, washed placentas from the same three breeds, and (iii) commercialized ECM products. Testing was performed in accordance with key requirements of Good Laboratory Practice (GLP). method Each porcine scaffold test sample was prepared into powder from a pool of porcine scaffolds from six individual sows from the same breed. Each raw, washed placenta test sample was prepared into powder from a pool of placentas from three individual sows from the same breed. Raw placenta material was prepared for testing from frozen placenta material: frozen porcine placentas were thawed, briefly rinsed three times with deionized water, trimmed, lyophilized, and finally powdered. All lyophilized material was powdered using a Wiley Mini Mill through a #40 mesh filter by methods commonly known in the art. One extract was prepared from each pooled sample, and each extract was tested in triplicate. All samples were quantified for elastin using the Fastin Elastin Assay Kit from Biocolor (Biocolor #F2000; batch code #BB087). Elastin was extracted from 6.3 mg of each sample. Sample extraction and quantification methods followed the manufacturer's instructions. Results are summarized in Table VI.

[0062] result [Table 6]

[0063] conclusion Elastin was quantified in (i) porcine scaffolds manufactured from three breeds (breed 1, breed 2, and breed 3); (ii) raw, washed placentas from the same three breeds; and (iii) commercialized ECM products. The porcine scaffolds contained approximately 66-97 μg of elastin per mg of tissue dry mass. Raw, washed placenta contained approximately 118-139 μg of elastin per mg of tissue dry mass. It can be concluded that the porcine scaffolds retain a large proportion of the elastin present in the native tissue. The porcine scaffolds provided herein contain or otherwise maintain approximately 0.09 g of elastin per gram of porcine scaffold, which corresponds to retention of at least about 66% of the elastin content after processing.

[0064] Example 5 Fibronectin quantification the purpose The objective of this study was to quantify the concentration of fibronectin in (i) porcine scaffolds produced from one of three pig breeds (Breed 1, Breed 2, and Breed 3), (ii) raw, washed placentas from the same three pig breeds, and (iii) commercialized ECM products. Testing was performed in accordance with key requirements of Good Laboratory Practice (GLP). method Each porcine scaffold test sample was prepared into powder from a pool of porcine scaffolds from six individual sows from the same breed. Each raw, washed placenta sample was prepared into powder from a pool of placentas from three individual sows from the same breed. Raw placenta material was prepared for testing from frozen placenta: frozen porcine placentas were thawed, briefly rinsed three times with deionized water, trimmed, freeze-dried, and finally powdered. All freeze-dried material was powdered using a Wiley Mini Mill through a #40 mesh filter. One extract was prepared from each pooled sample, and each extract was tested in triplicate.

[0065] Samples were quantified for fibronectin by enzyme-linked immunosorbent assay (ELISA). PBS and urea-heparin extracts were prepared from all samples, and preliminary ELISAs were performed to determine which preparation (PBS extract or urea-heparin extract) was more efficient at quantifying the solubilization of each molecule. Samples were then quantified from the appropriate extract. Fibronectin was quantified from the urea-heparin extract of the samples using LSBio Pig FN1 / Fibronectin ELISA, LSBio #LS-F8531. All quantification assays were performed in triplicate and according to the manufacturer's instructions. The results are summarized in Table VII.

[0066] result Fibronectin (FN) quantification [Table 7]

[0067] conclusion Fibronectin was quantified in (i) porcine scaffolds from three breeds (Breed 1, Breed 2, and Breed 3), (ii) raw, washed placentas from the three breeds; and (iii) commercialized ECM products. Raw, washed porcine placenta contained 32,000–35,500 ng of FN per gram of tissue dry mass. FN was still present in porcine scaffolds prepared from three breeds (Breed 1, Breed 2, and Breed 3), ranging from 1,409–1,634 ng of FN per gram of tissue dry mass. Commercialized ECM products contained 139 ng of FN per gram of tissue dry mass, an average FN concentration of approximately 9% in porcine scaffolds. Thus, porcine scaffolds have more than 10 times the FN of commercialized ECM products.

[0068] Example 6 Hyaluronic acid quantification the purpose: The objectives of this study were to measure hyaluronic acid (HA) content in (i) porcine scaffolds, each manufactured from placentas derived from one of three pig breeds (Breed 1, Breed 2, and Breed 3); (ii) commercialized ECM products; and (iii) porcine urinary bladder extracellular matrix (UECM) as an assay control. Testing was performed in accordance with key requirements of Good Laboratory Practice (GLP). method Each porcine scaffold test sample was prepared into powder from a pool of porcine scaffolds from six individual sows from the same breed. One extract was prepared from each pooled sample, and each extract was tested in triplicate. All samples were quantified for HA using the Purple-Jelley Hyaluronan Assay Kit from Biocolor (Biocolor #H1000; batch code #BB031), prepared according to the manufacturer's instructions. The results are summarized in Table VIII.

[0069] result [Table 8]

[0070] Conclusion: The porcine scaffold, commercial ECM product, and UECM each contained measurable amounts of hyaluronic acid (HA). Three porcine scaffolds, each prepared from a different breed of pig (Breed 1, Breed 2, and Breed 3), contained high levels of HA. The porcine scaffold prepared from Breed 1 contained 17.0 mg of HA per gram of tissue dry mass. The porcine scaffold prepared from Breed 2 contained 22.3 mg of HA per gram of tissue dry mass. The porcine scaffold prepared from Breed 3 contained 11.8 mg of HA per gram of tissue dry mass. These concentrations revealed that HA accounted for 1.7%, 2.2%, and 1.2% of the total dry mass of the porcine scaffolds from Breed 1, Breed 2, and Breed 3, respectively. The commercialized ECM product contained 1.58 mg of HA per gram of tissue dry mass, indicating that HA accounted for 0.16% of the commercialized ECM product dry mass. The UECM (control) contained 0.562 mg of HA per gram of dry mass, indicating that HA accounted for 0.06% of the tissue dry mass. The average amount of HA in the porcine scaffolds was approximately 0.02 g of HA per gram of porcine scaffold, and the average amount of HA in the commercialized ECM products was approximately 0.002 g of HA per gram of commercialized ECM products, indicating that the porcine scaffolds had approximately 10 times more hyaluronic acid than the commercialized ECM products.

[0071] Example 7 Sulfated glycosaminoglycans (GAGs) quantification the purpose The objectives of this study were to measure sulfated glycosaminoglycan (sGAG) content in (i) porcine scaffolds, each manufactured from placentas derived from one of three pig breeds (Breed 1, Breed 2, and Breed 3); (ii) commercialized ECM products; and (iii) porcine urinary bladder extracellular matrix (UECM) as an assay control. Testing was performed in accordance with key requirements of Good Laboratory Practice (GLP). method All samples were digested with 0.1 mg / ml proteinase K at 50 mg / ml in 2 ml. Each porcine scaffold test sample was prepared into powder from a pool of porcine scaffolds from six individual sows from the same breed. One extract was prepared from each pooled sample, and each extract was tested in triplicate. Sulfated glycosaminoglycans were quantified using the Blyscan Sulfated Glycosaminoglycan Assay Kit from Biocolor (Biocolor #B1000; batch code #BB053) according to the manufacturer's instructions. The results are summarized in Table IX.

[0072] result [Table 9]

[0073] conclusion The porcine scaffolds, commercialized ECM products, each prepared from placentas from one of three pig breeds (Breed 1, Breed 2, and Breed 3), and UECM each contained measurable amounts of sulfated glycosaminoglycans (sGAG). The average concentration of sGAG in the porcine scaffolds was 10.12 mg sGAG per gram of dry tissue mass. The concentration of sGAG in the commercialized ECM products was 11.93 mg sGAG per gram of dry tissue mass. The concentration of sGAG in the UECM was 9.94 mg sGAG per gram of dry tissue mass. Thus, the average amount of sGAG in the porcine scaffolds was approximately 0.01 g sGAG per gram of porcine scaffold, and the average amount of sGAG in the commercialized ECM products was similarly 0.01 g sGAG per gram of commercialized ECM product.

[0074] Example 8 Collagen quantification (soluble-insoluble) the purpose The objectives of this study were to measure collagen content in (i) porcine scaffolds, each manufactured from placentas derived from one of three pig breeds (Breed 1, Breed 2, and Breed 3); (ii) commercialized ECM products; and (iii) porcine urinary bladder extracellular matrix (UECM) as an assay control. Testing was performed in accordance with key requirements of Good Laboratory Practice (GLP). method Each porcine scaffold test sample was prepared into powder from a pool of porcine scaffolds from six individual sows from the same breed. Cold acid pepsin-soluble collagen was extracted from each sample with 10 mg / ml sample in 0.1 mg / ml pepsin / 0.5 M acetic acid overnight at 4°C, and each extract was assayed in triplicate. One extract was prepared from each pooled sample, and each extract was assayed in triplicate. From the residue of each extract remaining after cold acid pepsin extraction, the collagen that was not cold acid pepsin-soluble was heat solubilized and then assayed in triplicate. Collagen was quantified using the Sircol dye-binding method with a collagen assay kit from Biocolor (Biocolor #S4000 = CLRS1000 (Sircol soluble assay) and CLRS2000 (Sircol insoluble assay) batch code #BB084). Extraction and quantification methods followed the manufacturer's instructions. Results are summarized in Table X.

[0075] result [Table 10] conclusion In the porcine scaffold test samples, soluble collagen was approximately 1.6%–2.7% of the total tissue dry mass, while insoluble collagen ranged from 70%–85% of the total tissue dry mass. Commercialized ECM products had lower concentrations of soluble collagen and higher concentrations of insoluble collagen compared to the porcine scaffold samples.

[0076] Example 9 Immunolabeling for collagen IV and laminin the purpose The objectives of this study were to (i) evaluate the presence of collagen IV and laminin in porcine scaffolds, each fabricated from placentas derived from one of three pig breeds (Breed 1, Breed 2, and Breed 3); and (ii) evaluate the presence of collagen IV and laminin in raw, washed placentas derived from the three breeds. Testing was performed in accordance with key requirements of Good Laboratory Practice (GLP). method Immunolabeling was used to identify the presence of collagen IV and laminin. Test specimens were hydrated in type 1 water for 1 hour, then fixed in 10% neutral buffered formalin (NBF) for at least 72 hours, embedded in paraffin, sectioned at 5 μm thickness, and mounted on glass slides. The antibodies used for immunolabeling of collagen IV and laminin are summarized in Table XI.

[0077] [Table 11] When each section of the test article was exposed to blocking solution containing the primary antibody, a background control section on the same microscope slide was treated with blocking solution alone ("primary deletion control"). All sections were then exposed to secondary antibodies diluted in blocking solution. Following immunolabeling, slides were imaged on a Zeiss Observer Z1 microscope using an Axiocam MRc camera and a 20x objective.

[0078] conclusion The presence of collagen IV (Col IV) and laminin (Lam) in (i) porcine scaffolds fabricated from placentas from one of three pig breeds (Breed 1, Breed 2, and Breed 3) and (ii) raw, washed placentas from the three breeds was assessed by immunolabeling. The immunolabeling studies evaluated the presence of Col IV and Lam recognized by the primary antibodies used for immunolabeling and did not detect Col IV or Lam that had been altered during processing so that the epitopes recognized by the antibodies were no longer intact. Immunolabeling results revealed that Col IV was present in raw porcine placentas from all three breeds (Breed 1, Breed 2, and Breed 3) and localized to the expected regions, particularly those rich in basement membrane. Furthermore, Col IV was present in porcine scaffolds prepared from Breed 1; Breed 2; and Breed 3. Lam immunolabeling results show that Lam was present in raw porcine placentas from all three breeds (Breed 1, Breed 2, and Breed 3) and localized in the expected regions, i.e., basement membrane and blood vessel-rich areas. Furthermore, Lam was present in porcine scaffolds prepared from Breed 1; Breed 2; and Breed 3.

[0079] Example 10 Treatment of post-surgical incision dehiscence A 63-year-old male patient was found to have a postoperative surgical incision dehiscence approximately 3 weeks after a Syme amputation. Comorbidities included diabetes, coronary artery disease, and stage 3 kidney disease. The patient was found to have a postoperative surgical wound dehiscence measuring 4 cm in length and 1.5 cm in height (see Figure 1). Initial treatment of the wound dehiscence included standard care procedures, including sharp debridement with perimeter cleansing and Betadine wet-dry dressing changes. The wound did not resolve after 2 weeks of continuous treatment. Following two weeks of standard care treatment, the wound underwent sharp debridement and cleaning, leaving a 4 cm long, 1 cm high wound (see Figure 2). The wound was covered with a porcine scaffold prepared according to the methods provided herein. A non-adhesive secondary dressing and gauze were placed over the porcine scaffold. The patient underwent four consecutive weeks of treatment, each of which included wound debridement, application of the porcine scaffold, and placement of a non-adhesive secondary dressing and gauze (see Figures 3-7). The wound showed progress in healing during the five weeks of porcine scaffold application. One week following the fifth application of the porcine scaffold, the wound exhibited re-epithelialization and was considered healed (see Figure 8). At the four-week follow-up appointment, the wound remained closed and healing (see Figure 9).

[0080] Example 11 Treatment of post-surgical incision dehiscence A 77-year-old female patient was found to have a postoperative surgical incision dehiscence approximately two weeks after tarsal resection. Comorbidities included diabetes, diabetic neuropathy, and Charcot joints. The patient was found to have a postoperative surgical wound dehiscence measuring 1 cm in length and 0.2 cm in height (see Figure 10). Initial treatment for the wound dehiscence included standard care treatment, including sharp debridement with antiseptic and circumferential cleansing. The wound did not resolve after four weeks of continuous treatment. Following four weeks of standard care treatment, the wound underwent sharp debridement and irrigation, leaving a 1 cm in length and 0.2 cm in height wound (see Figure 11). The wound was covered with a porcine scaffold prepared according to the methods provided herein. A non-adhesive secondary dressing and gauze were then applied over the porcine scaffold. The patient was required to wear a walking shoe and bear weight. At the 1-week follow-up appointment, the wound had closed (see Figure 12) and the patient had transitioned to a diabetic shoe with a custom orthotic. At the 6-week follow-up appointment, the wound remained closed and healing (see Figure 13).

Claims

1. A method for producing a porcine extracellular matrix scaffold, below: (a) A step of treating the porcine placental membrane with an aqueous sodium chloride solution to reduce bioburden; (b) After step (a), the porcine placental membrane is treated with a surfactant solution containing at least one protease enzyme and at least one anionic surfactant; (c) After step (b), the porcine placental membrane is treated with a virus inactivation solution containing sodium hydroxide; (d) After step (c), the step of contacting the porcine placental membrane with a phosphate buffer solution until the pH of the porcine placental membrane becomes 6.8 to 7.2; and (e) After step (d), dehydrate the porcine placental membrane to obtain a porcine extracellular matrix scaffold. Includes, Here, steps (a) through (d) are carried out continuously without intermediate drying.

2. The method according to claim 1, wherein the sodium chloride solution has a concentration of 0.1 M to 5 M.

3. The method according to claim 1 or 2, wherein the sodium chloride solution is used in an amount of 1 mL to 100 mL per gram of porcine placental membrane.

4. The method according to any one of claims 1 to 3, wherein step (a) is carried out for 15 minutes to 8 hours.

5. The method according to any one of claims 1 to 4, wherein the surfactant solution has a surfactant concentration of 0.1 to 10% (w / v).

6. The method according to any one of claims 1 to 5, wherein the surfactant solution comprises a phosphate, a carbonate, and a sodium linear alkylaryl sulfonate.

7. The method according to any one of claims 1 to 6, wherein the sodium hydroxide has a concentration of 0.1 M to 3.0 M and is used in an amount of 1 mL to 50 mL per gram of porcine placental membrane.

8. The method according to any one of claims 1 to 7, wherein the sodium hydroxide solution contains 0.25 M sodium hydroxide and is used in an amount of 5 mL to 15 mL per gram of the porcine placental membrane.

9. The method according to claim 8, wherein step (c) is performed twice.

10. The method according to any one of claims 1 to 9, wherein step (d) comprises repeating contact with the phosphate buffer solution until the pH becomes 6.8 to 7.

2.

11. The method according to any one of claims 1 to 10, wherein step (e) includes chemical dehydration using ethanol.

12. The method according to claim 11, wherein the pig placental membrane is immersed in ethanol for 10 minutes to 24 hours.

13. The method according to any one of claims 1 to 12, further comprising the step of packaging and final sterilizing the porcine extracellular matrix scaffold by e-beam irradiation, gamma-ray irradiation, peracetic acid treatment, vaporized peracetic acid treatment, or any combination thereof.

14. The method according to any one of claims 1 to 13, further comprising the step of pulverizing the dehydrated porcine placental membrane to form a powder-based extracellular matrix scaffold.

15. The method according to claim 14, further comprising the steps of filling a container with the powder-based extracellular matrix scaffold and freeze-drying the powder-based extracellular matrix scaffold in the container.

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