Modified porcine scaffolds and methods of preparation

Abstract

A porcine scaffold is provided that includes a decellularized porcine placental extracellular matrix that has been processed to include one or more cytokines in amounts that differ from those of native porcine placental membranes.Methods of treating wounds are also provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application Publication No. 62 / 704,724, filed May 26, 2020, the entire contents of which are incorporated herein. [Background technology]

[0002] The extracellular matrix (ECM) is a set of secreted molecules that create the microenvironment for cells and provide structure and strength to tissues (Brew, Dinakarpandian, & Nagase, 2000; Young, Holle, & Spatz, 2016). Biological scaffolds made from ECM are becoming increasingly popular for the treatment of various medical conditions (Hussey, Dziki, & Badylak, 2018). Commercially available scaffolds have been produced 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 types, 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 provide each tissue type with a unique ECM that is relevant to tissue function. The third major structural component of ECM (Badylak et al., 2009) is proteoglycan (PG), a basic protein linked 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] Functional components of the ECM are a range of cytokines that contribute to its function through autocrine, juxtacrine, and paracrine signaling (Wallner, 1998). Functional cytokines can be divided into categories based on their action: growth factors, interleukins, and tissue inhibitors of metalloproteinases (TIMPs) (Engin, 2017; Hussey, 2018; Theocharis, 2016; Kim, 2011). Growth factors are signaling proteins that direct cellular activity and are present throughout the body, contributing to the proper function of tissues. Common growth factors include vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), and transforming growth factor β (TGFβ) (Kim, 2011; Taipale, 1997). Interleukins are a group of proteins primarily responsible for intercellular communication in the immune system and are important for regulating both immune and inflammatory responses. More than 50 interleukins have been identified (Brocker, 2010). TIMPs help regulate tissue function by inhibiting the activity of matrix metalloproteinases, a group of enzymes capable of degrading various matrix proteins (Brew, 2000). All of these functional molecules work synergistically to maintain normal and repair activity in tissues (Schultz, 2011).

[0004] Studies have investigated the role of multiple molecules in the wound healing process. Broughton and Li provided an extensive overview of the involvement of growth factors and ECM proteins and macromolecules in the wound healing process (Broughton, 2006; Li, 2007). A review dedicated to growth factors involved in wound healing was published by Dinh et al. (2015), summarizing studies demonstrating that TGFβ, PDGF, basic fibroblast growth factor (bFGF), and epidermal growth factor all play important roles in wound healing. The multiple roles of VEGF as a cell mitogen, chemotactic agent, and vascular permeability inducer were examined in a study by Bao et al. (Bao, 2009). Studies have also examined the important role that collagen plays in wound repair (Brett, 2008; Madden, 1971). Hyaluronic acid has been shown to play a crucial role in wound healing during inflammation and matrix synthesis (Aya, 2014; Chen, 1999). Both fibronectin (Grinnell, 1981; Sethi, 2002) and laminin (Malinda, 2008; Ishihara, 2018) have been shown to bind crucial growth factors and enhance wound healing and cellular activity.

[0005] When creating biological scaffolds to repair damaged tissue, many factors affect 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 manufacturing processes do not alter the membrane's original, relevant characteristics relevant to its utility for reconstruction, repair, or replacement.

[0006] 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 ).

[0007] 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. Summary of the Invention

[0008] According to one aspect, a porcine scaffold is provided, comprising decellularized porcine placental extracellular matrix to form the porcine scaffold. The porcine scaffold comprises at least one cytokine present in an amount different from that of a native porcine placental membrane. According to one embodiment, the at least one cytokine is decorin present in an amount of at least about 70 pg / mg. According to one embodiment, the at least one cytokine is MIF present in an amount less than about 50 pg / mg. According to one embodiment, the at least one cytokine is PDGF-BB present in an amount of at least about 75 pg / mg. According to one embodiment, the at least one cytokine is TIMP-2 present in an amount less than about 175 pg / mg. According to one embodiment, the at least one cytokine is VEGF present in an amount of at least about 3 pg / mg. According to one embodiment, the at least one cytokine is PIGF-2 present in an amount less than about 30 pg / mg. According to one embodiment, the at least one cytokine is TGF-β1 in an amount of at least about 82 pg / mg. According to one embodiment, the at least one cytokine is IGF-2 in an amount of at least about 7 pg / mg. According to one embodiment, the dehydrated porcine placental membranes are treated with a bioburden reduction step, a detergent rinse step, and a viral inactivation step.

[0009] According to one aspect, a porcine scaffold is provided, comprising decellularized porcine placental extracellular matrix to form the porcine scaffold. The porcine scaffold comprises at least one cytokine for increasing angiogenesis at a wound site. The porcine scaffold comprises at least one cytokine present in an amount different from that of a native porcine placental membrane. According to one embodiment, the at least one cytokine is decorin present in an amount of at least about 70 pg / mg. According to one embodiment, the at least one cytokine is VEGF present in an amount of at least about 3 pg / mg. According to one embodiment, the at least one cytokine is PIGF-2 in an amount less than about 30 pg / mg. According to one aspect, a porcine scaffold is provided, comprising decellularized porcine placental extracellular matrix to form the porcine scaffold. The porcine scaffold comprises at least one cytokine for controlling cell growth and division. The porcine scaffold comprises at least one cytokine present in an amount different from that of a native porcine placental membrane. According to one embodiment, the at least one cytokine is PDGF-BB present in an amount of at least about 75 pg / mg. According to one embodiment, the at least one cytokine is TGF-β1 in an amount of at least about 82 pg / mg. According to one embodiment, the at least one cytokine is IGF-2 in an amount of at least about 7 pg / mg.

[0010] According to one aspect, a porcine scaffold is provided, comprising decellularized porcine placental extracellular matrix to form the porcine scaffold. The porcine scaffold comprises at least one cytokine that inhibits matrix metalloproteinase activity. The porcine scaffold comprises at least one cytokine present in an amount different from that of a native porcine placental membrane. According to one embodiment, the cytokine is TIMP-2 in an amount less than about 175 pg / mg. According to one aspect, a porcine scaffold is provided, comprising decellularized porcine placental extracellular matrix to form the porcine scaffold. The porcine scaffold comprises at least one cytokine for inflammatory modulation. The porcine scaffold comprises at least one cytokine present in an amount different from that of a native porcine placental membrane. According to one embodiment, the cytokine is MIF present in an amount less than about 50 pg / mg. According to one aspect, a porcine scaffold is provided, comprising decellularized porcine placental extracellular matrix for forming the porcine scaffold. The porcine scaffold comprises at least one cytokine for stimulating collagen production. The porcine scaffold comprises at least one cytokine present in an amount different from that of a native porcine placental membrane. According to one embodiment, the cytokine is PIGF-2 present in an amount less than about 30 pg / mg.

[0011]

[0003] A method for treating a defect is provided. The method includes administering a porcine scaffold provided herein to the defect. The defect may be a partial thickness wound, a full thickness wound, a pressure ulcer, a venous ulcer, a diabetic ulcer, a chronic ductal ulcer, a tunneling or excavation wound, a surgical wound, a wound dehiscence, an abrasion, a laceration, a second-degree burn, a skin laceration, and a draining wound, or any combination thereof.

[0009] A wound dressing is provided. The wound dressing includes a porcine scaffold provided herein. According to one embodiment, the porcine scaffold includes a surface defining one or more fenestrations. 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, e.g., 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. 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.

[0014] 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. 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.

[0015] As used herein, the term "native" refers to the state of the tissue after it has been procured from a mammal, such as a pig, 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 standards for human placental membranes, which encompass 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. Methods are provided for aseptically processing placental membranes to prepare porcine scaffolds. According to one embodiment, porcine placental extracellular matrix is ​​prepared from placental membranes that remain intact in that the placental membrane 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 native layers are retained except for Wharton's gelatinous substance.

[0017] The porcine scaffolds provided herein may be formulated as membrane-based constructs. According to one embodiment, the porcine scaffolds comprise 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 a dehydrated, decellularized porcine placental extracellular matrix provided herein. According to another embodiment, the porcine scaffolds 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. The scaffolds described herein may be made by processing mammalian birth tissue according to any or all of the steps provided herein that apply 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.

[0018] A porcine scaffold is provided. According to one embodiment, the porcine scaffold comprises a decellularized porcine placental extracellular matrix comprising 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. According to one embodiment, the porcine scaffold comprises four major extracellular matrix components: collagen, elastin, hyaluronic acid, and sulfated glycosaminoglycans (sGAGs). According to one embodiment, the porcine scaffold comprises at least one cytokine in an amount different from that found in unmodified, raw, or native porcine birth tissue, such as Eotaxin-1, EPO, FGF-21, Galectin-9, IFNb, IGF-2, IL-21, IL-28B, PIGF-2, SCF, ANG-1, IL-17F, MIF, OPG, PDGF-BB, RANTES, TGFa, TIMP-1, TIMP-2, VEGF, Decorin, GASP-1, IGFBP-5, IL-15, IL-22, Insulin, IP-10, MCP-1, NCAM-1, TWEAK. R, CCL3L1, IFNa, IL-1a, IL-1ra, IL-13, IL-17a, IL-18, MIG, MIP-1b, PECAM-1, IL-1b, IL-4, IL-6, IL-8, IL-10, IL-12p40p70, GM-CSF, IFNg, TGF-β1, and TNFa.

[0019] A porcine scaffold is provided that includes a decellularized porcine placental extracellular matrix to form a porcine scaffold, the porcine scaffold including at least one cytokine present in an amount different from that of a native porcine placental membrane. According to one embodiment, the at least one cytokine is decorin present in an amount of at least about 70 pg / mg. According to one embodiment, the at least one cytokine is decorin present in an amount of at least about 75 pg / mg. According to one embodiment, the at least one cytokine is decorin present in an amount of at least about 80 pg / mg. According to one embodiment, the at least one cytokine is decorin present in an amount of at least about 85 pg / mg. According to one embodiment, the at least one cytokine is decorin present in an amount of at least about 90 pg / mg. According to one embodiment, the at least one cytokine is decorin present in an amount of at least about 95 pg / mg. According to one embodiment, the at least one cytokine is decorin present in an amount of at least about 100 pg / mg.

[0020] According to one embodiment, the at least one cytokine is MIF present in an amount of less than about 50 pg / mg. According to one embodiment, the at least one cytokine is MIF present in an amount of less than about 40 pg / mg. According to one embodiment, the at least one cytokine is MIF present in an amount of less than about 30 pg / mg. According to one embodiment, the at least one cytokine is MIF present in an amount of less than about 20 pg / mg. According to one embodiment, the at least one cytokine is MIF present in an amount of less than about 10 pg / mg. According to one embodiment, the at least one cytokine is MIF present in an amount of less than about 5 pg / mg. According to one embodiment, the at least one cytokine is PDGF-BB present in an amount of at least about 75 pg / mg. According to one embodiment, the at least one cytokine is PDGF-BB present in an amount of at least about 80 pg / mg. According to one embodiment, the at least one cytokine is PDGF-BB present in an amount of at least about 85 pg / mg. According to one embodiment, the at least one cytokine is PDGF-BB present in an amount of at least about 90 pg / mg. According to one embodiment, the at least one cytokine is PDGF-BB present in an amount of at least about 95 pg / mg.

[0021] According to one embodiment, the at least one cytokine is TIMP-2 present in an amount less than about 175 pg / mg. According to one embodiment, the at least one cytokine is TIMP-2 present in an amount less than about 150 pg / mg. According to one embodiment, the at least one cytokine is TIMP-2 present in an amount less than about 125 pg / mg. According to one embodiment, the at least one cytokine is TIMP-2 present in an amount less than about 100 pg / mg. According to one embodiment, the at least one cytokine is TIMP-2 present in an amount less than about 75 pg / mg. According to one embodiment, the at least one cytokine is TIMP-2 present in an amount less than about 50 pg / mg. According to one embodiment, the at least one cytokine is TIMP-2 present in an amount less than about 25 pg / mg. According to one embodiment, the at least one cytokine is TIMP-2 present in an amount less than about 12 pg / mg. According to one embodiment, the at least one cytokine is TIMP-2 present in an amount less than about 6 pg / mg. According to one embodiment, the at least one cytokine is TIMP-2 present in an amount less than about 3 pg / mg.

[0022] According to one embodiment, the at least one cytokine is VEGF present in an amount of at least about 3 pg / mg. According to one embodiment, the at least one cytokine is VEGF present in an amount of at least about 10 pg / mg. According to one embodiment, the at least one cytokine is VEGF present in an amount of at least about 20 pg / mg. According to one embodiment, the at least one cytokine is VEGF present in an amount of at least about 40 pg / mg. According to one embodiment, the at least one cytokine is VEGF present in an amount of at least about 80 pg / mg. According to one embodiment, the at least one cytokine is VEGF present in an amount of at least about 150 pg / mg. According to one embodiment, the at least one cytokine is PIGF-2 present in an amount less than about 30 pg / mg. According to one embodiment, the at least one cytokine is PIGF-2 present in an amount less than about 25 pg / mg. According to one embodiment, the at least one cytokine is PIGF-2 present in an amount less than about 20 pg / mg. According to one embodiment, the at least one cytokine is PIGF-2 present in an amount less than about 15 pg / mg. According to one embodiment, the at least one cytokine is PIGF-2 present in an amount less than about 7 pg / mg.

[0023] According to one embodiment, the at least one cytokine is TGF-β1 in an amount of at least about 82 pg / mg. According to one embodiment, the at least one cytokine is TGF-β1 in an amount of at least about 100 pg / mg. According to one embodiment, the at least one cytokine is TGF-β1 in an amount of at least about 200 pg / mg. According to one embodiment, the at least one cytokine is TGF-β1 in an amount of at least about 300 pg / mg. According to one embodiment, the at least one cytokine is TGF-β1 in an amount of at least about 400 pg / mg. According to one embodiment, the at least one cytokine is TGF-β1 in an amount of at least about 500 pg / mg. According to one embodiment, the at least one cytokine is TGF-β1 in an amount of at least about 600 pg / mg. According to one embodiment, the at least one cytokine is TGF-β1 in an amount of at least about 700 pg / mg. According to one embodiment, the at least one cytokine is IGF-2 in an amount of at least about 7 pg / mg. According to one embodiment, the at least one cytokine is IGF-2 in an amount of at least about 10 pg / mg. According to one embodiment, the at least one cytokine is IGF-2 in an amount of at least about 100 pg / mg. According to one embodiment, the at least one cytokine is IGF-2 in an amount of at least about 200 pg / mg. According to one embodiment, the at least one cytokine is IGF-2 in an amount of at least about 300 pg / mg. According to one embodiment, the at least one cytokine is IGF-2 in an amount of at least about 400 pg / mg.

[0024] According to one embodiment, dehydrated porcine placental membranes are treated with a bioburden reduction step, a detergent rinse step, and a viral inactivation step. A porcine scaffold is provided that includes decellularized porcine placental extracellular matrix to form the porcine scaffold. The porcine scaffold includes at least one cytokine for increasing angiogenesis at a wound site. The porcine scaffold includes at least one cytokine present in an amount different from that of a native porcine placental membrane. According to one embodiment, the at least one cytokine is decorin present in an amount of at least about 70 pg / mg, 75 pg / mg, 80 pg / mg, 85 pg / mg, 90 pg / mg, 95 pg / mg, or 100 pg / mg. According to one embodiment, the at least one cytokine is VEGF present in an amount of at least about 3 pg / mg, 10 pg / mg, 20 pg / mg, 40 pg / mg, 80 pg / mg, or 150 pg / mg. According to one embodiment, the at least one cytokine is PIGF-2 present in an amount less than about 30 pg / mg, 25 pg / mg, 20 pg / mg, 15 pg / mg, or 7 pg / mg, According to one embodiment, the at least one cytokine is a combination of one or more of decorin, VEGF, and PIGF-2 in the aforementioned amounts.

[0025] A porcine scaffold is provided that includes decellularized porcine placental extracellular matrix to form the porcine scaffold. The porcine scaffold includes at least one cytokine for controlling cell growth and division. The porcine scaffold includes at least one cytokine present in an amount different from that of a native porcine placental membrane. According to one embodiment, the at least one cytokine is PDGF-BB present in an amount of at least about 75 pg / mg, 80 pg / mg, 85 pg / mg, 90 pg / mg, or 95 pg / mg. According to one embodiment, the at least one cytokine is TGF-β1 in an amount of at least about 82 pg / mg, 100 pg / mg, 200 pg / mg, 300 pg / mg, 400 pg / mg, 500 pg / mg, 600 pg / mg, or 700 pg / mg. According to one embodiment, the at least one cytokine is IGF-2 in an amount of at least about 7 pg / mg, 10 pg / mg, 100 pg / mg, 200 pg / mg, 300 pg / mg, or 400 pg / mg. According to one embodiment, the at least one cytokine is a combination of one or more of PDGF-BB, TGF-β1, or IGF-2 in the aforementioned amounts.

[0026] A porcine scaffold is provided that includes decellularized porcine placental extracellular matrix to form the porcine scaffold. The porcine scaffold includes at least one cytokine that regulates the activity of matrix metalloproteinases. The porcine scaffold includes at least one cytokine present in an amount different from that of a native porcine placental membrane. According to one embodiment, the cytokine is TIMP-2, present in an amount of less than about 175 pg / mg, 100 pg / mg, 50 pg / mg, 25 pg / mg, 12 pg / mg, 6 pg / mg, or 3 pg / mg. A porcine scaffold is provided that includes decellularized porcine placental extracellular matrix to form the porcine scaffold. The porcine scaffold includes at least one cytokine for regulating inflammation. The porcine scaffold includes at least one cytokine present in an amount different from that of a native porcine placental membrane. According to one embodiment, the cytokine is MIF present in an amount less than about 50 pg / mg, 40 pg / mg, 30 pg / mg, 20 pg / mg, 10 pg / mg, or 5 pg / mg.

[0027] A porcine scaffold is provided that includes decellularized porcine placental extracellular matrix to form the porcine scaffold. The porcine scaffold includes at least one cytokine for stimulating collagen production. The porcine scaffold includes at least one cytokine present in an amount different from that of a native porcine placental membrane. According to one embodiment, the cytokine is PIGF-2 present in an amount less than about 30 pg / mg, 25 pg / mg, 20 pg / mg, 15 pg / mg, or 7 pg / mg. Any of the porcine scaffolds provided herein may include a surface defining one or more fenestrations.

[0010] 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 implant.

[0028] A method for preparing a porcine scaffold is provided. According to one embodiment, processing a porcine placental membrane to form a porcine scaffold includes treating the porcine placental membrane with a surfactant solution, the surfactant solution including at least one protease enzyme. According to one embodiment, the surfactant solution further includes at least one anionic surfactant. 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, the virus inactivation solution including at least one alkaline solution. According to one embodiment, the alkaline solution includes 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, a method of preparing a porcine 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.

[0029] According to one embodiment, the method optionally includes freezing the umbilical cord and placental membranes by methods known to those of skill 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. 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 may be cut into various sizes, thicknesses, and shapes. The placental membrane fragments are preferably of a size and shape sufficient to be applied on or around a wound on or in the body of a mammalian patient. The thickness of the placental membrane may vary depending on the application, the type of membrane, and the number of membrane layers.

[0030] 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. 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.

[0031] 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. 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.

[0032] 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.

[0033] 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.

[0034] 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 particular embodiment, the alcohol is ethanol. According to one embodiment, the method includes decanting or emptying the alcohol from the placental membrane. 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 that no wrinkles or air bubbles are present.

[0035] 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. 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 into 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.

[0036] 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 comminuted to 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 comminuted to 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 the powder-based construct includes one or more steps of lyophilizing the comminuted / comminuted powder-based construct in a vial to remove residual moisture. The vials containing the powder-based constructs are then terminally sterilized. 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.

[0037]

[0003] Methods of treating defects are 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 partial-thickness wounds, full-thickness wounds, pressure ulcers, venous ulcers, diabetic ulcers, chronic ductal ulcers, tunneling or excavation wounds, surgical wounds, wound dehiscences, abrasions, lacerations, second-degree burns, skin lacerations, and draining wounds. 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.

[0038] 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.

[0039] 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).

[0040] 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 in 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 ureteral 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. [Example]

[0041] Example 1 Porcine cytokine detection and quantification Native porcine placental membrane versus porcine scaffold Cytokine quantification was performed on: (1) porcine scaffolds prepared from porcine placental membranes isolated from three sows from each of three breeds; and (2) native porcine placental membranes isolated from three different sows from the same three different breeds. The porcine scaffold samples were prepared according to the methods provided herein. Porcine samples were prepared for quantification by creating a lysate for each individual sample. For each sample, the tissue was placed in an Eppendorf tube with 6 ml of lysis buffer containing protease inhibitors (RayBiotech, Norcross, Georgia, USA) and incubated overnight at 4°C. The tissue was homogenized for 2 minutes at 4°C. The Eppendorf tube was then centrifuged at 10,400 rpm for 20 minutes at 4°C. The resulting supernatant was transferred to a microfuge tube. The supernatant was subjected to protein quantification using a BCA Protein Quantification Kit (RayBiotech, Norcross, Georgia, USA) to assess the total protein content in the supernatant solution. Samples were diluted to standard concentrations before use for cytokine quantification.

[0042] Cytokine quantification was performed using Quantibody® arrays from RayBiotech, Norcross, Georgia, USA. Quantibody® arrays were specifically used to detect standard cytokines believed to be present in the porcine placental membranes used to generate porcine scaffolds when processed according to the methods provided herein. The minimum level of detection (referred to as "LOD") and maximum limit of detection (referred to as "MAX") for each cytokine tested on the Quantibody® array are provided in Table 1.

[0043] [Table 1-1] [Table 1-2]

[0044] The Quantibody® Array multiplex sandwich ELISA-based quantitative array platform was able to simultaneously determine the concentrations of multiple cytokines typically present in porcine tissue. The Quantibody® Array utilized a pair of cytokine-specific antibodies for detection. The capture antibody was first bound to a glass surface. After incubation with the sample porcine tissue, the target cytokine was trapped on the solid surface. A second biotin-labeled detection antibody capable of recognizing a different epitope of the target cytokine was then added. A laser scanner was then used to visualize the cytokine-antibody-biotin complex through the addition of a dye equivalent to Cy3 conjugated to streptavidin.

[0045] Quantification results for each cytokine detected in the porcine scaffolds are provided in Tables 2 and 3 for each of the nine sows (Sow A to Sow I). [Table 2-1] [Table 2-2]

[0046] [Table 3-1] [Table 3-2]

[0047] Quantification results for each cytokine detected in native porcine placental membranes are provided in Tables 4 and 5 for each of the nine sows (Sow J to Sow R). [Table 4-1] [Table 4-2]

[0048] [Table 5-1] [Table 5-2]

[0049] Because (i) each individual sample had a unique amount of total protein extracted during processing; and (ii) the resulting protein solution was diluted before placement on the Quantibody® array, to compare quantification results for porcine scaffolds to native porcine placental membranes, pg / ml concentrations were converted to pg / mg mass-to-mass concentrations. Using the known mass of starting tissue for each sow, protein extract concentration, and volume of dilution, the exact pg / mg concentration of each cytokine per starting mass of tissue for each sample was calculated. The results of these calculations are provided in Tables 6-9.

[0050] [Table 6-1] [Table 6-2]

[0051] [Table 7-1] [Table 7-2]

[0052] [Table 8-1] [Table 8-2]

[0053] [Table 9-1] [Table 9-2]

[0054] Using mass-to-mass values, cytokine concentrations in the porcine scaffolds were statistically compared to native porcine placental membranes. Six cytokines detectable in all 18 samples had the majority of samples in the most reliable range of the Quantibody® array, demonstrating statistical significance (p<0.05) (see Table 10). [Table 10]

[0055] The process used to create porcine scaffolds from native porcine placental membranes significantly increases the concentrations of decorin, platelet-derived growth factor-BB (PDGF-BB), and vascular endothelial growth factor (VEGF). Conversely, the process used to create porcine constructs from native porcine placental membranes significantly decreases the concentrations of macrophage migration inhibitory factor (MIF), tissue inhibitor of metalloproteinase 2 (TIMP-2), and placental growth factor 2 (PIGF-2). All six of these factors are known to affect skin wound healing. Decorin, a proteoglycan, binds to type 1 collagen and plays a role in extracellular matrix assembly. Studies examining the role of decorin in dermal wound healing have shown increased angiogenesis and vascular connective tissue ingrowth in implanted sponges (Jarvelainen 2006). Increasing the concentration of decorin in porcine scaffolds provides a stimulatory effect on wound healing after placement by increasing angiogenesis at the wound site.

[0056] PDGF-BB is a growth factor known to regulate cell growth and division. Recombinant protein has been successfully used to treat chronic wounds (Wieman 1998). Studies examining cytokine activity and concentrations in non-healing and healing chronic leg ulcers have found that higher concentrations of PDGF were found in healing wounds compared to non-healing wounds (Trengrove, 2001). Studies have shown that PDGF affects platelets, keratinocytes, macrophages, endothelial cells, and fibroblasts (Barrientos 2008), but specifically influences matrix formation and remodeling (Heldin 1999, Lederle 2006, Uutela 2004). Increasing the concentration of PDGF-BB in porcine scaffolds may affect multiple cell types that play a role in the healing response. VEGF is a growth factor known to stimulate new blood vessel formation. Studies examining cytokine activity and concentrations in non-healing and healing chronic leg ulcers found that higher concentrations of VEGF were found in healing wounds compared to non-healing wounds (Trengrove, 2001). Studies have shown that VEGF affects platelets, neutrophils, macrophages, endothelial cells, and fibroblasts (Barrientos 2008), but specifically influences angiogenesis (Johnson 2014, Jazwa 2006, Thomas 1996) and granulation tissue formation (Yerba 1996, Nissen 1998). Increasing the concentration of VEGF in porcine scaffolds may affect multiple cell types in the early regenerative response.

[0057] MIF is an inflammatory cytokine and regulator of innate immunity that plays a complex role in skin wound healing. Studies have shown that MIF can induce both regenerative and inflammatory responses (Gilliver 2010). Studies have demonstrated that MIF plays an important role in the early inflammatory response, but high levels in the later stages of wound healing can inhibit the process (Ashcroft 2003, Shimizu 2005). Reducing the concentration of MIF in porcine scaffolds may have a positive effect on regulating inflammation during wound healing. TIMP-2 is an inhibitor of matrix metalloproteinase (MMP) activity and plays an important role in extracellular matrix homeostasis by preventing MMPs from degrading ECM proteins. TIMP-2, unlike other TIMPs, has been shown to inhibit endothelial cell proliferation (Murphy 1993). Reducing the concentration of TIMP-2 in porcine scaffolds may allow the inhibitor to act on MMPs without negative effects on endothelial cell proliferation. PIGF-2 is a growth factor that is a member of the VEGF subfamily and plays an important role in angiogenesis and vasculogenesis, especially during embryogenesis. Studies have shown that PIGF is produced by keratinocytes during wound healing (Failla 2000) and stimulates collagen production by fibroblasts (Arief 2000). Reducing the concentration of PIGF-2 in porcine scaffolds, but not completely eliminating it, can stimulate collagen production during wound healing.

[0058] It is also noted that the process used to create the porcine scaffold statistically significantly increases the concentrations of transforming growth factor-β1 (TGF-β1) and insulin-like growth factor-2 (IGF-2), such that they are detectable in the porcine scaffold assay but below the level of detection or highest confidence range in the native porcine placental membrane assay. These two growth factors are known to affect skin wound healing. A comparison of the two cytokines is provided in Table 11. [Table 11]

[0059] The TGF-β superfamily of growth factors, including TGF-β1, is known to regulate multiple cellular functions, including cell growth, proliferation, and differentiation. Studies have examined the effects of TGF-β1 on various aspects of wound healing and demonstrated that increased in vitro proliferation of fibroblasts occurred with growth factor application (Rolfe 2007). A second study highlighted the role of TGF-β in stimulating the formation of granulation tissue (Singer 1999). Studies have shown that TGF-β affects platelets, keratinocytes, macrophages, lymphocytes, and fibroblasts (Barrientos 2008), but specifically influences extracellular matrix formation and remodeling (Tsunawaki 1988, Riedel 2007). Increasing the concentration of TGF-β1 in porcine scaffolds could affect multiple cell types in the healing response.

[0060] IGF-2 is a growth factor known to regulate cell growth, particularly during fetal development. Studies have demonstrated that IGF-2 expression increases from the initial injury until 10–15 days after injury in a rat model of wound healing (Gartner 1992). A second study confirmed that IGF-2 expression increases from the initial injury and is expressed at higher levels in diabetic animals. Studies have also localized IGF-2 to the epithelium of healing wounds (Brown 1997). Increasing the concentration of IGF-2 in porcine scaffolds could affect epithelial healing. Another aspect of the present invention may be as follows. [1] Decellularized porcine placental extracellular matrix A porcine scaffold comprising: A porcine scaffold comprising at least one cytokine present in an amount different from that of a native porcine placental membrane. [2] The porcine scaffold according to [1], wherein at least one cytokine is decorin present in an amount of at least about 70 pg / mg. [3] The porcine scaffold according to [1], wherein at least one cytokine is MIF present in an amount of less than about 50 pg / mg. [4] The porcine scaffold according to [1], wherein at least one cytokine is PDGF-BB present in an amount of at least about 75 pg / mg. [5] The pig scaffold according to [1], wherein at least one cytokine is TIMP-2 present in an amount of less than about 175 pg / mg. [6] The porcine scaffold according to [1], wherein at least one cytokine is VEGF present in an amount of at least about 3 pg / mg. [7] The porcine scaffold according to [1], wherein at least one cytokine is PIGF-2 present in an amount of less than about 30 pg / mg. [8] The pig scaffold according to [1], wherein at least one cytokine is TGF-β1 in an amount of at least about 82 pg / mg. [9] The pig scaffold according to [1], wherein at least one cytokine is IGF-2 in an amount of at least about 7 pg / mg.

[10] The porcine scaffold described in [1], wherein the dehydrated porcine placental membrane is treated with a bioburden reduction step, a surfactant rinsing step, and a viral inactivation step.

[11] Decellularized porcine placental extracellular matrix A porcine scaffold comprising: A porcine scaffold comprising at least one cytokine for increasing angiogenesis at a wound site.

[12] The pig scaffold according to

[11] , wherein at least one cytokine is decorin present in an amount of at least about 70 pg / mg.

[13] The pig scaffold according to

[11] , wherein at least one cytokine is VEGF present in an amount of at least about 3 pg / mg.

[14] The porcine scaffold according to

[11] , wherein at least one cytokine is PIGF-2 present in an amount of less than about 30 pg / mg.

[15] Decellularized porcine placental extracellular matrix A porcine scaffold comprising: A porcine scaffold comprising at least one cytokine for controlling cell growth and division.

[16] The porcine scaffold according to

[15] , wherein at least one cytokine is PDGF-BB present in an amount of at least about 75 pg / mg.

[17] The pig scaffold according to

[15] , wherein at least one cytokine is TGF-β1 in an amount of at least about 82 pg / mg.

[18] The pig scaffold according to

[15] , wherein at least one cytokine is IGF-2 in an amount of at least about 7 pg / mg.

[19] Decellularized porcine placental extracellular matrix A porcine scaffold comprising: A porcine scaffold comprising at least one cytokine that inhibits matrix metalloproteinase activity.

[20] The pig scaffold according to

[19] , wherein the cytokine is TIMP-2 present in an amount of less than about 175 pg / mg.

[21] Decellularized porcine placental extracellular matrix A porcine scaffold comprising: A porcine scaffold comprising at least one cytokine for regulating inflammation.

[22] The pig scaffold according to

[21] , wherein the cytokine is MIF present in an amount of less than about 50 pg / mg.

[23] Decellularized porcine placental extracellular matrix A porcine scaffold comprising: A porcine scaffold comprising at least one cytokine for stimulating collagen production.

[24] The pig scaffold according to

[23] , wherein the cytokine is PIGF-2 present in an amount of less than about 30 pg / mg.

[25] A method for treating a defect, comprising: A method comprising a step of administering the porcine scaffold provided in any one of [1] to

[24] to a defect.

[26] The method according to

[25] above, wherein the defect is selected from partial thickness wounds, full thickness wounds, pressure ulcers, venous ulcers, diabetic ulcers, chronic duct ulcers, tunneling or perforating wounds, surgical wounds, wound dehiscence, abrasions, lacerations, second-degree burns, skin lacerations, and draining wounds.

[27] A wound dressing comprising the porcine scaffold according to any one of [1] to

[24] above.

[28] The wound dressing described in

[27] , wherein the porcine scaffold comprises a surface defining one or more fenestrations.

Claims

1. 1. A porcine scaffold comprising decellularized porcine placental extracellular matrix, A porcine scaffold comprising at least one cytokine present in an amount different from that of a native porcine placental membrane.

2. 2. The porcine scaffold of claim 1, wherein the at least one cytokine is decorin present in an amount of at least 70 pg / mg.

3. 2. The porcine scaffold of claim 1, wherein the at least one cytokine is MIF present in an amount of less than 50 pg / mg.

4. 2. The porcine scaffold of claim 1, wherein the at least one cytokine is PDGF-BB present in an amount of at least 75 pg / mg.

5. 2. The porcine scaffold of claim 1, wherein the at least one cytokine is TIMP-2 present in an amount less than 175 pg / mg.

6. 2. The porcine scaffold of claim 1, wherein the at least one cytokine is VEGF present in an amount of at least 3 pg / mg.

7. 2. The porcine scaffold of claim 1, wherein the at least one cytokine is PIGF-2 present in an amount less than 30 pg / mg.

8. 2. The porcine scaffold of claim 1, wherein the at least one cytokine is TGF-β1 in an amount of at least 82 pg / mg.

9. 2. The porcine scaffold of claim 1, wherein the at least one cytokine is IGF-2 in an amount of at least 7 pg / mg.

10. 1. A method for producing a porcine scaffold obtained from a porcine placental membrane, comprising: Dehydrating the porcine placental membrane; and a bioburden reduction step of treating the dehydrated porcine placental membrane with a bioburden reduction solution, a surfactant rinsing step of treating the dehydrated porcine placental membrane with a surfactant solution, and a virus inactivation step of treating the dehydrated porcine placental membrane with a virus inactivation solution; The porcine scaffold comprises a decellularized porcine placental extracellular matrix and comprises at least one cytokine that is present in an amount different from that of a native porcine placental membrane. Method for manufacturing porcine scaffolds.

11. Decellularized porcine placental extracellular matrix A porcine scaffold comprising: A porcine scaffold comprising at least one cytokine for increasing angiogenesis at the wound site.

12. 12. The porcine scaffold of claim 11, wherein the at least one cytokine is decorin present in an amount of at least 70 pg / mg.

13. 12. The porcine scaffold of claim 11, wherein the at least one cytokine is VEGF present in an amount of at least 3 pg / mg.

14. 12. The porcine scaffold of claim 11, wherein the at least one cytokine is PIGF-2 present in an amount less than 30 pg / mg.

15. Decellularized porcine placental extracellular matrix A porcine scaffold comprising: A porcine scaffold comprising at least one cytokine for controlling cell growth and division.

16. 16. The porcine scaffold of claim 15, wherein the at least one cytokine is PDGF-BB present in an amount of at least 75 pg / mg.

17. 16. The porcine scaffold of claim 15, wherein the at least one cytokine is TGF-β1 in an amount of at least 82 pg / mg.

18. 16. The porcine scaffold of claim 15, wherein the at least one cytokine is IGF-2 in an amount of at least 7 pg / mg.

19. Decellularized porcine placental extracellular matrix A porcine scaffold comprising: A porcine scaffold comprising at least one cytokine that inhibits matrix metalloproteinase activity.

20. 20. The porcine scaffold of claim 19, wherein the cytokine is TIMP-2 present in an amount of less than 175 pg / mg.

21. Decellularized porcine placental extracellular matrix A porcine scaffold comprising: A porcine scaffold comprising at least one cytokine for regulating inflammation.

22. 22. The porcine scaffold of claim 21, wherein the cytokine is MIF present in an amount of less than 50 pg / mg.

23. Decellularized porcine placental extracellular matrix A porcine scaffold comprising: A porcine scaffold comprising at least one cytokine for stimulating collagen production.

24. 24. The porcine scaffold of claim 23, wherein the cytokine is PIGF-2 present in an amount less than 30 pg / mg.

25. 1. A porcine scaffold for use in treating a defect, comprising: the porcine scaffold comprises decellularized porcine placental extracellular matrix, the porcine scaffold comprises at least one cytokine that is present in an amount different from that of a native porcine placental membrane, and 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 perforating wound, a surgical wound, a wound dehiscence, an abrasion, a laceration, a second degree burn, a skin laceration, and a draining wound. Pig scaffold.

26. A wound dressing comprising the porcine scaffold according to any one of claims 1 to 9 and 11 to 25.

27. 27. The wound dressing of claim 26, wherein the porcine scaffold comprises a surface defining one or more fenestrations.

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