A sterilized human placental allograft having a plurality of slits, openings, and / or windows formed thereon

Abstract

A method of preparing a sterilized human placental allograft, comprising: providing human placental tissue from a donor within 72 hours but at least 24 hours after childbirth; removing any visible blood, blood clots, and / or blood components from the human placental tissue without scraping or scrubbing the human placental tissue to preserve the structural integrity of the human placental tissue; washing the human placental tissue in an isotonic solution while maintaining the structural integrity of the human placental tissue; dehydrating the human placental tissue to form a dehydrated human placental tissue; sizing the dehydrated human placental tissue into dehydrated human placental tissue portions having a predetermined size; and sterilizing the dehydrated human placental tissue portions of step (e) to form the sterilized human placental allograft. Also disclosed is a sterilized human placental allograft produced by the method.

Description

Technical Field

[0001] The present invention generally relates to the field of human placental tissue, and more particularly to a growth factor-rich sterilized human placental allograft and a method of making such allografts having various dental, cosmetic, and wound healing uses. In certain embodiments, the sterilized human placental allograft has slits, openings, and / or windows formed thereon, and the slits, openings, and / or windows are configured to allow wound exudate from a wound to pass through the sterilized human placental allograft away from the wound, facilitating wound drainage, wound healing, or a combination thereof.

Background Art

[0002] The use of the human placenta for medical purposes dates back to 1593 when Li Shizhen wrote about the use of "zi he chi" (human placenta) in the Compendium of Materia Medica. However, the advantages of placental membranes for skin grafting were first formally documented in the medical literature in 1910.

[0003] The use of placental membranes for clinical purposes has continued to the present day. To date, placental membrane processing has mainly focused on the preservation of cells within the tissue or the removal of all non-solid components for the purpose of delivering stem cells and / or providing a substrate for regenerative growth. These placental membrane processing techniques often result in the loss of endogenous growth factors found in human placental tissue and / or result in an endogenous degradation process that further results in the loss of endogenous growth factors found in human placental tissue, thereby resulting in a reduction in the effectiveness of the final use (e.g., reduced wound healing) for any allografts obtained from these processes.

Summary of the Invention

[0004] The object of this invention is to provide a safe, biocompatible, and growth factor-rich sterilized human placental allograft. The methods disclosed herein achieve these biocompatible and growth factor-rich sterilized human placental allografts while maintaining a growth factor profile that mimics the human placenta in vivo. These methods purify and prepare human placental tissue in a gentle and effective manner while minimizing the risk of including biological tissues and / or growth factors for subsequent implantation / transplantation of the allograft. In particular, the disclosed methods use cold physiological buffers, gentle handling, dehydration at ambient or physiological temperature, and mild sterilization conditions for the human placental allografts disclosed herein, various mild purification techniques, preservation techniques, and mild sterilization techniques, to obtain sterilized human placental allografts that maintain a growth factor profile that mimics the human placenta in vivo. These sterilized human placental allografts have a variety of different clinical and cosmetic purposes and can achieve improved wound healing, for example, due to their growth factor profile compared to conventional human placental allografts. Further, the allografts disclosed herein meet or exceed all requirements set by the US Food and Drug Administration (FDA) and the American Association of Tissue Banks (AATB).

[0005] In certain embodiments, disclosed herein is a method for preparing a sterilized human placental allograft, the method comprising: (a) providing human placental tissue from a donor within 24 to 72 hours after delivery; (b) removing any visible blood, blood clots, and / or blood components from the human placental tissue without scraping or scrubbing the human placental tissue to preserve the structural integrity of the human placental tissue; (c) washing the human placental tissue in an isotonic solution while maintaining the structural integrity of the human placental tissue; (d) dehydrating the human placental tissue to form a dehydrated human placental tissue; (e) sizing the dehydrated human placental tissue into dehydrated human placental tissue portions having a predetermined size; and (f) sterilizing the dehydrated human placental tissue portions of step (e) to form the sterilized human placental allograft.

[0006] In certain embodiments, either before or during step (a), the human placental tissue may be disinfected with at least one of a bactericidal composition, a mycobactericidal composition, a fungicidal composition, a virucidal composition, or any combination thereof. In certain embodiments, the at least one of a bactericidal composition, a mycobactericidal composition, a fungicidal composition, a virucidal composition, or any combination thereof comprises an alcohol solution, more preferably isopropyl alcohol at a concentration of 70% or more and 100% or less. In a preferred embodiment, the alcohol concentration ranges from 70% to 75%, and in a most preferred embodiment, the alcohol is 70% isopropyl alcohol. Isopropyl alcohol is preferred over other commercially available laboratory grade and / or pharmaceutical grade alcohols such as ethanol because isopropyl alcohol advantageously disinfects and purifies the placental tissue without damaging the human placental tissue (e.g., initiating excessive dehydration, apoptosis processes, and / or necrosis processes). In certain embodiments, at least one of a bactericidal composition, a mycobactericidal composition, a fungicidal composition, a virucidal composition, or any combination thereof in a predetermined volume may be used, and the predetermined volume ranges from 250 mL to 1000 mL, more preferably from 400 mL to 600 mL.

[0007] In certain embodiments, step (b) comprises manual hematopoietic reduction by manually removing any visible blood, blood clots, blood components, and / or other debris from the human placental tissue.

[0008] In certain embodiments, the isotonic solution comprises at least one of 1× phosphate buffered saline, isotonic saline solution, lactated Ringer's, Plasma-Lyte® (at pH 7.4, 5.26 g / L NaCl, 0.37 g / L KCl, 0.30 g / L magnesium chloride hexahydrate, 3.68 g / L sodium acetate trihydrate, 5.02 g / L sodium gluconate), Normosol® (at pH 7.4, 5.26 g / L NaCl, 0.37 g / L KCl, 0.30 g / L magnesium chloride, 2.22 g / L sodium acetate anhydrous, 5.02 g / L sodium gluconate), or any combination thereof.

[0009] In certain embodiments, the method further comprises repeating step (c) a predetermined number of times.

[0010] In certain embodiments, each washing step (c) is carried out at a temperature in the range of 4°C to 15°C for a time period of 5 minutes to 15 minutes.

[0011] In certain embodiments, the dehydrating step (d) is carried out at a temperature in the range of 20°C to 40°C for a time period of 60 minutes to 4.5 hours, thereby resulting in dehydrated (or dried) human placental tissue having a residual water content of about 10% to 15%. In certain preferred embodiments, the dehydrating step (d) is carried out at a temperature in the range of 30°C to 40°C for a time period of 60 minutes to 4.5 hours, thereby resulting in dehydrated (or dried) human placental tissue having a residual water content of about 10% to 15%.

[0012] In certain embodiments, for example, during step (e), the dehydrated human placental tissue is sized to a predetermined size within the range of 1 cm × 1 cm to 8 cm × 8 cm for human placental tissue portions having a square shape and a rectangular shape. In a further embodiment, the human placental tissue portions having a square shape and a rectangular shape may include 2 cm × 2 cm, 2 cm × 3 cm, 2 cm × 4 cm, 4 cm × 4 cm, 4 cm × 6 cm, 4 cm × 8 cm, 6 cm × 6 cm, 8 cm × 8 cm, where any endpoint within any of these ranges can serve as an endpoint for any additional range that falls within it. In certain embodiments, the predetermined size may include human placental tissue portions having a round shape and / or a circular shape with a diameter in the range of 0.5 mm to 50 mm, where any endpoint within any of these ranges can serve as an endpoint for any additional range that falls within it. In a further embodiment, these sizes may include human placental tissue portions having a round shape and / or a circular shape with a diameter of 2 mm, 4 mm, 6 mm, 8 mm, 10 mm, 12 mm, 14 mm, 16 mm, 20 mm, 30 mm, 40 mm, or 50 mm.

[0013] In certain embodiments, the placental tissue portion has a predetermined shape and is configured for dental use, cosmetic use, and / or wound healing use.

[0014] In certain embodiments, step (f) of sterilizing includes sterilizing by electron beam irradiation.

[0015] In certain embodiments, step (f) involves subjecting the dehydrated human placental tissue portion to electron beam irradiation to a sterility assurance level (SAL) of 10 -6 to form the sterilized human placental allograft.

[0016] In certain embodiments, the human placental tissue includes intact placental tissue. In this embodiment, the dehydrated human placental tissue of step (d) and the portion of the dehydrated human placental tissue having a predetermined size of step (e) are intact in cross-section, and in this cross-section, the human amniotic layer and the human chorionic layer have an intact human intermediate sponge layer positioned between the human amniotic layer and the human chorionic layer to connect the human amniotic layer to the human chorionic layer. In this embodiment, the sterilized human placental allograft of step (f) is intact in cross-section, and in this cross-section, the human amniotic layer and the human chorionic layer have an intact human intermediate sponge layer positioned between the human amniotic layer and the human chorionic layer to connect the human amniotic layer to the human chorionic layer.

[0017] In certain embodiments, for example, the sterilized human placental allograft produced by the above method is configured for dental, cosmetic, and / or wound healing applications.

[0018] Also, in certain embodiments, prior to step (f) (preferably after step (d) and after step (e), or after step (d) and before step (e)), slits, openings, and / or windows are formed in the human placental allograft (and through it) and / or within the dehydrated human placental portion, such that the sterilized human placental allograft (of step (f)) includes a plurality of slits, openings, or windows (such as flaps) formed in the upper surface of the allograft and corresponding (such as aligned and / or axially aligned) slits, openings, or windows (flaps) formed in the lower surface of the allograft, such that the slits, openings, or windows (such as flaps) formed in the upper surface of the allograft are in fluid communication with the corresponding slits, openings, or windows (such as flaps) formed in the lower surface of the allograft, such that during use the allograft allows wound exudate to flow from the wound through the allograft from the lower surface of the allograft in a direction away from the wound to the upper surface of the allograft, thereby facilitating wound drainage, wound healing, or a combination thereof. In certain embodiments, the sterilized human placental allograft comprises a plurality of windows formed in the sterilized human placental allograft, the plurality of windows being configured such that wound exudate flows from the wound through the sterilized human placental allograft in a direction from the lower surface of the sterilized human placental allograft to the upper surface of the sterilized human placental allograft, thereby facilitating wound drainage, wound healing, or a combination thereof. In this embodiment, each window is a flap configured to be open when wound exudate and / or other body fluids from the wound are flowing through the sterilized human placental allograft and closed when wound exudate and / or other body fluids are not flowing through the sterilized human placental allograft. The flap moves in a direction away from the lower and upper surfaces of the sterilized human placental allograft when in use and in an open configuration.In certain embodiments, the window has a predetermined shape, which includes a "V" shape, a "U" shape, a half "U" shape, a half square shape, a half rectangular shape, or combinations thereof.

[0019] Also disclosed herein are sterilized human placental allografts. In certain embodiments, these sterilized human placental allografts are produced by the methods disclosed herein. The sterilized human placental allografts disclosed herein are preferably non-immunogenic - there is little or no resulting immune response after implantation / transplantation into a human recipient.

[0020] In certain embodiments, the sterilized human placental allograft comprises the following: (a) interleukin-1 receptor antagonist (IL-1ra) in the range of 200 pg / cm 2 or more and 7800 pg / cm 2 or less; (b) hepatocyte growth factor (HGF) in the range of 500 pg / cm 2 or more and 8500 pg / cm 2 or less; (c) vascular endothelial growth factor receptor 1 (VEGFR1) in the range of 800 pg / cm 2 or more and 2750 pg / cm 2 or less; (d) hyaluronic acid (HA) in the range of 5.0×10 6 pg / cm 2 or more and 1.5×10 8 pg / cm 2 or less; and (e) 150 pg / cm 2 or more and 400 pg / cm 2A platelet-derived growth factor B subunit homodimer (PDGF-BB) within the following range, and at least two of (f) glycosaminoglycan (GAG), (g) collagen, and (h) exosome, wherein any endpoint within any of the above ranges can serve as an endpoint for any additional range falling within it. In this aspect, the sterilized human placental allograft is intact in cross-section, and in the cross-section, the human amniotic layer and the human chorionic layer have an intact human intermediate sponge layer positioned between the human amniotic layer and the human chorionic layer to connect the human amniotic layer to the human chorionic layer. In certain embodiments, the human placental allograft has the following: (a) interleukin-1 receptor antagonist (IL-1ra) in the range of 200 pg / cm 2 or more and 7800 pg / cm 2 or less, (b) hepatocyte growth factor (HGF) in the range of 500 pg / cm 2 or more and 8500 pg / cm 2 or less, (c) vascular endothelial growth factor receptor 1 (VEGFR1) in the range of 800 pg / cm 2 or more and 2750 pg / cm 2 or less, (d) hyaluronic acid (HA) in the range of 5.0×10 6 pg / cm 2 or more and 1.5×10 8 pg / cm 2 or less, (e) platelet-derived growth factor B subunit homodimer (PDGF-BB) in the range of 150 pg / cm 2 or more and 400 pg / cm 2 or less, and may include at least three, at least four, at least five, at least six, at least seven, or at least eight of (f) glycosaminoglycan (GAG), (g) collagen, and (h) exosome, wherein any endpoint within any of the above ranges can serve as an endpoint for any additional range falling within it. In this aspect, the sterilized human placental allograft is intact in cross-section, and in the cross-section, the human amniotic layer and the human chorionic layer have an intact human intermediate sponge layer positioned between the human amniotic layer and the human chorionic layer to connect the human amniotic layer to the human chorionic layer.

[0021] In certain embodiments, the sterilized human placental allograft is configured for dental use, cosmetic use, and / or wound healing use.

[0022] In certain embodiments, the sterilized human placental allograft has a predetermined size of 1 cm × 1 cm or more and 8 cm × 8 cm or less for square and rectangular sterilized human placental allografts. In a further embodiment, the square and rectangular sterilized human placental allografts may include 2 cm × 2 cm, 2 cm × 3 cm, 2 cm × 4 cm, 4 cm × 4 cm, 4 cm × 6 cm, 4 cm × 8 cm, 6 cm × 6 cm, 8 cm × 8 cm, where any endpoint within these ranges can serve as an endpoint for any additional range that falls within them. In certain embodiments, the predetermined size may include round and / or circular sterilized human placental allografts with a diameter in the range of 0.5 mm or more and 50 mm or less, where any endpoint within these ranges can serve as an endpoint for any additional range that falls within them. In a further embodiment, the size may include round and / or circular sterilized human placental tissue portions with a diameter of 2 mm, 4 mm, 6 mm, 8 mm, 10 mm, 12 mm, 14 mm, 16 mm, 20 mm, 30 mm, 40 mm, or 50 mm.

[0023] In certain embodiments, the sterilized human placental allograft has a circular shape, a square shape, a rectangular shape, an oval shape, a triangular shape, or any combination thereof.

[0024] In certain embodiments, the upper surface and / or the lower surface of the sterilized human placental allograft is planar.

[0025] In certain embodiments, the upper surface and the lower surface of the sterilized human placental allograft are planar.

[0026] In certain embodiments, the sterilized human placental allograft includes a plurality of slits, openings, or windows (e.g., flaps) formed on the upper surface of the allograft, and further includes corresponding slits, openings, or windows (flaps) formed on the lower surface of the allograft, such that the slits, openings, or windows (e.g., flaps) formed on the upper surface of the allograft are in fluid communication with the corresponding slits, openings, or windows (e.g., flaps) formed on the lower surface of the allograft, thereby enabling the allograft to allow wound exudate to flow away from the wound and pass through the allograft in a direction from the lower surface of the allograft to the upper surface of the allograft during use, thereby facilitating wound drainage, wound healing, or a combination thereof. In certain embodiments, the sterilized human placental allograft includes a plurality of windows formed in the sterilized human placental allograft, and the plurality of windows are configured to allow wound exudate to flow away from the wound and pass through the sterilized human placental allograft in a direction from the lower surface of the sterilized human placental allograft to the upper surface of the sterilized human placental allograft, thereby facilitating wound drainage, wound healing, or a combination thereof. In this embodiment, each window is a flap configured to be open when wound exudate and / or other body fluids are flowing through the sterilized human placental allograft from the wound and to be closed when wound exudate and / or other body fluids are not flowing through the sterilized human placental allograft. The flap moves in a direction away from the lower surface and the side surface of the sterilized human placental allograft when open during use and while exudate is flowing through the allograft. In certain embodiments, the window has a predetermined shape, and the shape includes a "V" shape, a "U" shape, a semi-"U" shape, a semi-square shape, a semi-rectangular shape, or a combination thereof.

[0027] In certain embodiments, the sterilized human placental allograft consists essentially of human amnion, and in a further embodiment, the sterilized human placental allograft consists of the human amnion. In each of these embodiments, the sterilized human placental allograft (consisting essentially of and / or consisting of amnion) has the following: (a) interleukin-1 receptor antagonist (IL-1ra) in the range of 150 pg / cm 2 or more and 1800 pg / cm 2 or less; (b) hepatocyte growth factor (HGF) in the range of 75 pg / cm 2 or more and 400 pg / cm 2 or less; (c) vascular endothelial growth factor receptor 1 (VEGFR1) in the range of 50 pg / cm 2 or more and 250 pg / cm 2 or less; (d) hyaluronic acid (HA) in the range of 1.1×10 6 pg / cm 2 or more and 2.0×10 7 pg / cm 2 or less; (e) 200 pg / cm 2 or more and 500 pg / cm 2A platelet-derived growth factor B subunit homodimer (PDGF-BB) within the following range, and at least two of (f) glycosaminoglycan (GAG), (g) collagen, and (h) exosome, wherein any endpoint within any of the above ranges can serve as an endpoint for any additional range that falls within it. In this aspect, the sterilized human placental allograft is configured for dental use, cosmetic use, and / or wound healing use. In this aspect, the sterilized human placental allograft has a predetermined size in the range of 1 cm × 1 cm or more and 8 cm × 8 cm or less for rectangular-shaped and / or square-shaped human placental allografts, where any endpoint within any of these ranges can serve as an endpoint for any additional range that falls within it, and has a predetermined size in the range of 2 mm in diameter or more and 50 mm in diameter or less for circular and / or round sterilized human placental allografts, where any endpoint within any of these ranges can serve as an endpoint for any additional range that falls within it. In this aspect, the sterilized human placental allograft has a circular shape, square shape, rectangular shape, oval shape, triangular shape, or any combination thereof. In this aspect, the upper surface and / or the lower surface of the sterilized human placental allograft is planar. In this aspect, the upper surface and the lower surface of the sterilized human placental allograft are planar.

[0028] Also disclosed herein is a kit comprising the sterilized human placental allograft packaged in a sterilized container. In this aspect, the sterilized human placental allograft is produced by the method disclosed herein.

[0029] Embodiments of this invention can include one or more or any combination of the above features and configurations.

[0030] Additional features, aspects, and advantages of the present invention will be set forth in the detailed description which follows, and in part will be obvious to those skilled in the art from that description, or can be learned by practice of the invention as described herein. It is to be understood that both the foregoing general description and the following detailed description are exemplary and intended to provide an overview or framework for understanding the nature and character of the invention as claimed. The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification.

Brief Description of the Drawings

[0031] These and other features, aspects, and advantages of the present invention will be better understood when the following detailed description of the invention is read with reference to the accompanying drawings.

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DETAILED DESCRIPTION OF THE INVENTION

[0048] Next, the present invention will be described in more detail hereinafter with reference to the accompanying drawings showing exemplary embodiments of the invention. However, the invention may be embodied in many different forms and should not be construed as limited to the representative embodiments set forth herein. The exemplary embodiments are provided so that this disclosure will be thorough and complete and will fully convey the scope of the invention to enable those skilled in the art to make, use, and practice the invention.

[0049] It should be noted that as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0050] Concentrations, amounts, and other numerical data may be expressed or presented in a range format in this specification. It should be understood that such a range format is merely used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly stated as the limits of the range, but also all of the individual numerical values or sub-ranges encompassed within that range, as if each numerical value and sub-range were explicitly stated. As an example, a numerical range of "about 1 to 5" should be interpreted to include not only the explicitly stated values of about 1 to about 5, but also the individual values and sub-ranges within the indicated range. Thus, included within this numerical range are the individual values such as 2, 3, and 4, and sub-ranges such as not only 1, 2, 3, 4, and 5 individually, but also 1 to 3, 2 to 4, and 3 to 5, etc. The same principle applies to ranges that specify only a single numerical value as the minimum or maximum value. Further, such an interpretation should apply regardless of the breadth of the range or the nature of the trait being described.

[0051] "Standardization," as referred to herein, enables the accurate quantification of growth factors and components within the disclosed compositions for subsequent use and treatment in the subject in need thereof (e.g., treatment for wound healing). The Applicant has calculated the range per centimeter of the factor to be delivered as that which is delivered to the patient (the subject in need). This means that the amounts of IL-1ra, HGF, VEGFR1, HA, etc. delivered to the patient / subject in need are measured as the amount eluted from 1 cm of the sterilized human placental allograft. The quantification process used for the standardization of the disclosed sterilized human placental allograft is used to accurately reflect the relationship between the human placental allograft and the growth factors and other chemical components therein. The calculation of the standardization is derived from the following formula. 2 The amount eluted from 1 cm of the sterilized human placental allograft is measured as the amount delivered to the patient / subject in need. The quantification process used for the standardization of the disclosed sterilized human placental allograft is used to accurately reflect the relationship between the human placental allograft and the growth factors and other chemical components therein. The calculation of the standardization is derived from the following formula. 2 The quantification process used for the standardization of the disclosed sterilized human placental allograft is used to accurately reflect the relationship between the human placental allograft and the growth factors and other chemical components therein. The standardization calculation is derived from the following formula. Factor analyzed (pg / ml) · ml of buffer / cm of sample 2 = pg of factor per 1 cm 2 of the factor per centimeter This is accomplished by taking a sample having a known cm 2 (a biopsy punch is preferred). The sample / punch is placed in buffer at 37° C. for 48 to 72 hours. The contents are collected and analyzed for the desired components. Multiply these results by the volume of buffer collected. Divide this number by the cm 2 of the sample / punch. This can be utilized for any volume of eluate used and any sample size. The tests presented in this invention utilized a 10 mm biopsy punch (78.54 cm 2 ) placed in buffer (1×PBS) at 37° C. for 72 hours, mimicking the application of a sterile human placental allograft within an internal or external wound space. The eluate was collected and analyzed, mimicking what elutes from a sterile human placental allograft into a wound. This results in a controlled and quantifiable amount of tested sterile human placental allografts, and a controlled, quantifiable amount of growth factors and stromal components.

[0052] The terms "human placental tissue" and "placental tissue" are used interchangeably herein to refer to human placental tissue. Unless otherwise expressly stated herein, "human placental tissue" and "placental tissue" refer to either an intact human placenta (having a human umbilical cord, human amnion, human chorion, and an intact human intermediate sponge layer positioned between the human amnion layer and the human chorion layer and connecting the human amnion layer and the human chorion layer), or human placental tissue that is intact in cross-section (i.e., comprising only human amnion, human chorion, and an intact human intermediate sponge layer), wherein in the cross-section, the human amnion layer and the human chorion layer have an intact human intermediate sponge layer positioned between the human amnion layer and the human chorion layer and connecting the human amnion layer to the human chorion layer. In certain expressly stated examples herein, "human placental tissue" and / or placental tissue consists essentially of or consists only of human amnion separated from all other placental tissues. Similarly, unless otherwise expressly stated, a "sterile human placental allograft" is an allograft having human placental tissue that is intact in cross-section (i.e., comprising only human amnion, human chorion, and an intact human intermediate sponge layer), wherein in the cross-section, the human amnion layer and the human chorion layer have an intact human intermediate sponge layer positioned between the human amnion layer and the human chorion layer and connecting the human amnion layer to the human chorion layer. In the expressly stated examples herein, a "sterile human placental allograft" refers to an allograft that consists essentially of or consists only of human amnion separated from all other placental tissues.

[0053] Disclosed herein are a safe, biocompatible, and growth factor-rich sterile human placental allograft and a method for preparing / producing the same. The allografts and methods disclosed herein achieve these biocompatible and growth factor-rich sterile human placental allografts while mimicking the human placenta in vivo and maintaining an improved growth factor profile compared to conventional allografts, thereby achieving better therapeutic outcomes for dental applications and wound healing. (Preparation of Sterilized Human Placenta Allografts)

[0054] As suggested above, it is an object to prepare sterilized human placenta allografts that maintain a growth factor profile mimicking the human placenta in vivo. The methods disclosed herein utilize mild conditions to ensure that various endogenous growth factors in human placenta tissue, including but not limited to IL-1ra, HGF, VEGFR1, HA, PDGF-BB, GAG, and collagen, are preserved (and / or their loss is minimized), ultimately resulting in the sterilized human placenta allografts disclosed herein. As generally shown in FIG. 1, the disclosed methods purify and prepare human placenta tissue in a gentle and effective manner while minimizing the risk of involvement in the destruction of biological tissue for subsequent implantation / transplantation of the allograft. In particular, and as further disclosed in detail below, the disclosed methods use various mild purification techniques, preservation techniques, and mild sterilization techniques that use cold physiological buffer, gentle handling, dehydration at ambient or physiological temperature, and the mild sterilization conditions for the human placenta allografts disclosed herein to obtain sterilized human placenta allografts that maintain a growth factor profile mimicking the human placenta in vivo. These sterilized human placenta allografts have various different clinical and cosmetic purposes and can achieve, for example, improved wound healing compared to conventional human placenta allografts. Additionally, the allografts disclosed herein meet or exceed all requirements set by the U.S. Food and Drug Administration (FDA) and the American Association of Tissue Banks (AATB).

[0055] In view of FIG. 1, and prior to subjecting human placental tissue to step (a) of FIG. 1, all human placental tissue undergoes donor prescreening and screening processes. Donor prescreening includes screening the donor's medical and social history, interviewing the donor, and physically examining the donor to exclude high-risk donors from the potential donor pool. During or after the prescreening process, cultures of the donor's tissue and donor blood specimens are collected for comprehensive serological testing, which is subsequently performed in a laboratory registered with the FDA and certified by CLIA to further determine the viability of human donor placental tissue for the methods disclosed herein. Serological testing includes screening the donor and / or human placental tissue for the following infectious diseases. · Hepatitis: · Hepatitis B surface antigen · Hepatitis B core antibody · Antibody to hepatitis C virus · Hepatitis B nucleic acid test · Hepatitis C nucleic acid test · Human immunodeficiency virus (HIV): · Antibody to HIV-1 · Antibody to HIV-2 · HIV nucleic acid test · Syphilis - rapid plasma reagin · Leukemia / lymphoma · Antibody to human T-lymphotropic virus type 1 · Antibody to human T-lymphotropic virus type 2 · West Nile virus In addition to the above serological tests, and prior to subjecting human placental tissue to step (a) of FIG. 1, bioburden samples are collected to assess the number of viable microorganisms on or in the human placental tissue. In addition, environmental monitoring samples are collected extensively to evaluate surface and air quality. The above collections ensure that subsequent human placental tissue and processing are subject to consistent microbiological control.

[0056] Figure 1 schematically depicts a method for preparing a sterilized human placental allograft. This method involves (a) providing human placental tissue from a donor within 24 hours or more and within 72 hours after childbirth, and optionally (a i ) separating the desired components from the placenta (amnion, chorion, amnion / chorion together, umbilical cord, etc.), (b) removing any visible blood, blood clots, blood components, and / or debris from the human placental tissue without scraping or scrubbing the human placental tissue to preserve the structural integrity of the human placental tissue, (c) washing the human placental tissue in an isotonic solution while maintaining the structural integrity of the human placental tissue, (d) dehydrating the human placental tissue to thereby form the dehydrated human placental tissue, (e) sizing the dehydrated human placental tissue into dehydrated human placental tissue portions having a predetermined size, and (f) sterilizing the dehydrated human placental tissue portion of step (e) to thereby form the sterilized human placental allograft.

[0057] After obtaining human placental tissue from a donor, during step (a), the human placental tissue is disinfected. In particular, the human placental tissue is placed in a bactericidal, mycobactericidal, fungicidal, and / or virucidal composition. In particular, the human placental tissue is placed in an alcohol solution of 250 mL or more and 1000 mL (preferably 500 mL) or less to wash and remove any exogenous non-placental components and / or any other potential contaminants. The alcohol solution is preferably isopropyl alcohol at a concentration of 70% or more and 100% or less. In a preferred embodiment, the alcohol concentration ranges from 70% to 75%. In the most preferred embodiment, the alcohol is 70% isopropyl alcohol. Isopropyl alcohol is preferred over other commercially available laboratory grade and / or pharmaceutical grade alcohols such as ethanol because, advantageously, isopropyl alcohol can disinfect and purify the placental tissue without damaging the human placental tissue (e.g., initiating excessive dehydration, apoptosis processes, and / or necrosis processes) over a period of 60 seconds or more and 120 seconds (preferably 90 seconds) or less at 4°C or more and 20°C or less, thereby reducing and / or eliminating the presence of microorganisms, fungi, and viruses (preferably, any pathogenic microorganisms, fungi, and / or viruses if present) on and / or within the human placental tissue. Step (a) uses bactericidal, mycobactericidal, fungicidal, and virucidal disinfection with minimal damage, in which the placental tissue is placed in an effective and minimally damaging disinfectant for the required time to gently reduce and / or eliminate any external contamination, including but not limited to mycobacteria, fungi, herpes, HIV, influenza, hepatitis B and C, Ebola, and many viruses including SARS.

[0058] After step (a), the disinfected human placental tissue from step (a) undergoes step (b), where in step (b), the disinfected human placental tissue is visually inspected to remove any visible blood, blood clots, blood components, and / or unwanted debris without scraping or scrubbing the human placental tissue, the purpose being to preserve not only the endogenous growth factors within the human placental tissue but also the structural integrity of the human placental tissue. In certain preferred embodiments, step (b) of FIG. 1 is a manual hematopoietic reduction step that uses forceps and / or tweezers, soft wet gauze, and a scalpel to remove any visible blood, blood clots, blood components, and / or unwanted debris. The hematopoietic components are removed by hand without any excessive scraping or scrubbing and / or without performing any excessive scraping or scrubbing, to preserve the integrity of the membrane of the disinfected human placental tissue and to further preserve the various endogenous growth factors therein (e.g., IL-1ra, HGF, VEGFR1, HA, PDGF-BB, GAG, and collagen). As should be recognized, the presence of blood, blood clots, blood components, and / or any other unwanted debris induces an immunogenic response. Thus, the removal of blood, blood clots, blood components, and / or any other unwanted debris is essential to reduce the immunogenicity of the sterilized human allograft obtained as a final result and / or, if present, the probability of an immunogenic response when implanted / transplanted into the human subject in need.

[0059] After step (b), the human placental tissue is subjected to at least one purification / washing step as depicted in step (c) of FIG. 1. During step (c), the human placental tissue is placed in an isotonic solution (and / or isosmotic solution) to wash and remove any exogenous non-human placental components and / or any other potential contaminants. In certain embodiments, the isotonic solution comprises at least one of 1× phosphate buffered saline, isotonic saline solution, lactated Ringer's, Plasma-Lyte® (at pH 7.4, 5.26 g / L NaCl, 0.37 g / L KCl, 0.30 g / L magnesium chloride hexahydrate, 3.68 g / L sodium acetate trihydrate, 5.02 g / L sodium gluconate), Normosol® (at pH 7.4, 5.26 g / L NaCl, 0.37 g / L KCl, 0.30 g / L magnesium chloride, 2.22 g / L sodium acetate anhydrous, 5.02 g / L sodium gluconate), or any combination thereof. The washing step of step (c) may occur once and / or each washing step occurs once, twice, three times, or four times in an isotonic solution at a temperature in the range of 4° C. to 20° C. in a predetermined volume (e.g., 250 mL or more and 1000 mL or less, preferably 500 mL) for 5 minutes or more and 15 minutes or less. Step (c) preferably occurs at low temperature using a pH-adjusted isotonic solution, which more advantageously reduces and / or eliminates the degradation of the human placental tissue and / or endogenous growth factors (e.g., IL-1ra, HGF, VEGFR1, HA, PDGF-BB, GAG, and collagen) during step (c). Although the above-described isotonic solutions may be used, it should be further recognized that strong chemical substances / solutions such as strong organic acids or strong inorganic acids, strong bases, and / or strong peroxides (e.g., NaOH, carboxylic acids, HCl, H2O2) are not used during step (c) or at any time during the method depicted in FIG. 1.In particular, the use of strong organic acids, strong inorganic acids, strong bases, or strong peroxides can result in the degradation of endogenous growth factors and, in a harmful manner, the degradation of human placental tissue, and for these reasons should be avoided.

[0060] After step (c) and as further shown in Figure 1, the human placental tissue is subjected to a dehydration / drying step (d). In particular, the human placental tissue is gently laid flat in a biological safety cabinet having a circulation fan at ambient temperature for 2 to 12 hours to dehydrate / dry the human placental tissue, or placed in a dehydrator for 0.5 to 6 hours, more preferably 1 to 4 hours (at a temperature in the range of 30°C to 40°C) to obtain dehydrated human placental tissue. The slow dehydration / drying of the human placental tissue advantageously avoids the damage often observed from freezing (e.g., freeze-drying) and / or drying at high temperatures (e.g., accelerated drying) of the human placental tissue, while preserving the tissue structure, integrity, and endogenous growth factors therein. The resulting dehydrated human placental tissue has a maximum water content in the range of 10 wt% to 15 wt%, which reduces the likelihood of any endogenous degradation process that depends on the presence of water, thereby further minimizing the degradation of growth factors during the method of Figure 1.

[0061] After step (d), the dehydrated human placental tissue is sized during step (e) into a dehydrated human placental tissue portion having a predetermined size and a predetermined shape. When sizing the dehydrated human placental tissue into the dehydrated human placental tissue portion, a die having a predetermined size and / or dimensions and / or shape may be used to precisely size the dehydrated human placental tissue into the dehydrated human placental tissue portion to the desired shape, thereby ensuring the optimal tissue size, shape, and quality for each of the sterilized human placental allografts obtained as a final result produced by the methods disclosed herein. In certain embodiments, the dehydrated human placental tissue portion is sized into a circular shape, a square shape, a rectangular shape, a rhomboid shape, an oval shape, a triangular shape, or any combination thereof. Each of these shapes may have various different sizes and / or dimensions for various different end uses and / or applications. In certain embodiments, the predetermined size of the dehydrated placental tissue portion ranges from 1 cm × 1 cm to 8 cm × 8 cm for a rectangular-shaped and / or square-shaped dehydrated placental tissue portion, where any endpoint within these ranges can serve as an endpoint for any additional range that falls within them. In certain embodiments, the predetermined size of the dehydrated placental tissue portion ranges from 8 mm in diameter to 50 mm in diameter for a circular-shaped dehydrated placental tissue portion, where any endpoint within these ranges can serve as an endpoint for any additional range that falls within them. In certain embodiments, the dehydrated human placental tissue portion may further include an upper surface of the plane or a lower surface of the plane, or the human placental tissue portion includes both an upper surface of the plane and a lower surface of the plane.

[0062] After step (e), the dehydrated human placental tissue portion of step (e) (the sized portion having the desired predetermined shape and size) is subsequently sterilized during step (f), thereby forming a sterilized human placental allograft. Also, and as shown in FIGS. 1 and 12A - 14D, in certain embodiments, prior to step (f) (i.e., after step (d) and after step (e), or after step (d) and before step (e)), slits, openings, and / or windows are formed on (and through) the human placental allograft and / or within the dehydrated human placental portion, such that the sterilized human placental allograft (resulting after step (f)) includes a plurality of slits, openings, or windows (e.g., flaps) formed on the upper surface of the allograft, and corresponding (e.g., aligned and / or axially aligned) slits, openings, or windows (flaps) are formed on the lower surface of the allograft, such that the slits, openings, or windows (e.g., flaps) formed on the upper surface of the allograft are in fluid communication with the corresponding slits, openings, or windows (e.g., flaps) formed on the lower surface of the allograft, thereby enabling the allograft, in use, to allow wound exudate to flow from the wound through the allograft in a direction from the lower surface of the allograft to the upper surface of the allograft, thereby facilitating wound drainage, wound healing, or a combination thereof.In view of the above, FIG. 12A depicts a top view of a sterilized human placental allograft disclosed herein, in an unexpanded configuration, having a plurality of slits (110 or 210) formed thereon. FIG. 12B depicts a top view of the human placental allograft (120 or 220) of FIG. 12A, in an expanded configuration, wherein the openings (110 or 210 formed from the slits upon expansion of the allograft) are arranged in a predetermined pattern (e.g., a grid pattern, and / or a grid pattern having openings in the shape of elongated rectangles or semi-elongated rectangles), and the openings are configured such that wound exudate flows from the wound and passes through the allograft from the lower surface of the allograft to the upper surface of the allograft. FIG. 13 depicts a top view of a sterilized human placental allograft (100 or 200) having a plurality of openings (130 or 230) formed therein, such that the lower and upper surfaces of the allograft are in fluid communication with each other, and the openings are configured such that wound exudate flows from the wound and passes through the allograft from the lower surface of the allograft to the upper surface of the allograft. In certain embodiments, the slits and / or openings may be formed on the allograft using conventional devices such as dies or cutting devices.

[0063] In certain embodiments, as shown in FIGS. 14A-14D, 16A, and 16B, the human sterilized placental allograft (100 or 200) includes a plurality of windows (140 or 240, 150 or 250, 160 or 260) formed in the sterilized human placental allograft, the plurality of windows being configured such that wound exudate flows from the wound, through the sterilized human placental allograft in a direction from the lower surface of the sterilized human placental allograft to the upper surface of the sterilized human placental allograft (100 or 200), facilitating wound drainage, wound healing, or a combination thereof. In this embodiment, as shown in FIGS. 14C and 14D, each window is a flap configured to be open when wound exudate and / or other body fluids are flowing through the sterilized human placental allograft and to be closed when wound exudate and / or other body fluids are not flowing through the sterilized human placental allograft. Each flap is spaced sufficiently apart from one another to allow wound exudate to flow therethrough without obstructing the opening and without closing any adjacent flap.

[0064] Figures 14A - 14C depict an allograft with a window formed thereon in a closed and / or closed configuration. Figure 14D is a schematic depiction of a cross - sectional and / or side view of the allograft (100 or 200) when in use and positioned over a wound (500), showing that as wound exudate and / or other body fluids originate from the wound (500) and flow away from the wound (500) and the lower surface (104 or 204) and upper surface (102 or 202) of the sterilized human placental allograft, the open flap (160 or 260) is moved in direction (D1), thereby facilitating wound drainage, wound healing, or a combination thereof. In certain embodiments, and as further shown in Figures 14A - 14D, the window has a predefined shape, which includes "V" shapes (140 or 240), "U" shapes (150 or 250), semi - "U" shapes (160 or 260), semi - square shapes, semi - rectangular shapes, or combinations thereof. Also, Figure 16A is a photograph of a sterilized human placental allograft with multiple windows formed thereon, and Figure 16B is another photograph of a sterilized human placental allograft with multiple windows formed thereon. The windows (e.g., flaps) discussed above are formed by planar pressing or roll pressing of the allograft (preferably prior to sterilization, e.g., electron beam sterilization) on a die (600) or cutting instrument having a predefined shape formed thereon, such as shown in Figure 15, to cut and form the windows within the allograft, and the windows have a predefined shape (e.g., notches in a "V", "U", or semi - "U" shape).

[0065] In certain embodiments, step (f) of sterilizing comprises subjecting the dehydrated human placental tissue portion to electron beam irradiation to a sterility assurance level (SAL) of 10 -6 as calculated by the dose distribution from an electron beam manufacturer, thereby forming a sterilized human placental allograft, wherein the SAL expressed as 10 -N is the expected probability of remaining bioburden after processing (e.g., the expected probability of any remaining microorganisms after sterilization is 10 -6The following. Before sterilization, the dehydrated human placental tissue portion of step (e) (the sized portion having the desired predetermined shape and size) may first be placed within either a primary packaging and / or a secondary packaging and then subjected to low-dose electron beam irradiation to produce a sterilized human placental allograft, which is in a ready-to-use situation for final use (after removal from the primary or secondary packaging). Alternatively, the dehydrated human placental tissue portion of step (e) (the sized portion having the desired predetermined shape and size) may first be subjected to low-dose electron beam irradiation to produce a sterilized human placental allograft, and then subsequently placed within a sterile packaging while maintaining the sterility of the sterilized human placental allograft for its subsequent final use.

[0066] Low-dose electron beam irradiation is particularly preferred over other sterilization methods because these other methods require direct access to the human placental tissue and leave residual contaminants (e.g., ethylene oxide) on the resulting allograft, which can result in an immunogenic response upon use of the resulting allograft or require exposure of the tissue to higher doses of irradiation (e.g., gamma irradiation) over an extended period, which can unduly result in a decrease in the structural integrity of the placental tissue and degradation of endogenous growth factors.

[0067] As further shown in FIGS. 10A and 11A, the sterilized human placental allografts produced by the above method have a predetermined shape and size. As suggested above, the sterilized human placental allografts can be of circular shape, square shape, rectangular shape, oblong shape, oval shape, triangular shape, or any combination thereof. Each of these shapes can have various different sizes and / or dimensions for various different end uses and / or applications. In certain embodiments, the predetermined size of the sterilized human placental allograft ranges from 1 cm×1 cm to 8 cm×8 cm for rectangular-shaped and / or square-shaped sterilized human placental allografts, where any endpoint within these ranges can serve as an endpoint for any additional range that falls within it, and ranges from 2 mm in diameter to 50 mm in diameter for circular and / or round sterilized human placental allografts, where any endpoint within these ranges can serve as an endpoint for any additional range that falls within it. In certain embodiments, the sterilized human placental allograft can further include an upper surface of the plane or a lower surface of the plane, or the sterilized human placental allograft includes both an upper surface of the plane and a lower surface of the plane. Also, as shown in FIGS. 10A and 11A, the sterilized human placental allograft can have a thickness in the range of 0.01 mm to 0.2 mm and can further have a translucent appearance.

[0068] Upon terminating step (f), all sterilized human placental allografts are subjected to quality control and lot control. All records from the human placental tissue processing from the above method are finally reviewed by the medical director and the on-site quality leader to ensure a thorough quality assurance review and compliance of the resulting sterilized human allografts using applicable government regulations, compliance, and procedures. (Sterilized human placental allograft)

[0069] The sterilized human placental allografts disclosed herein can be produced by the methods disclosed herein. The allografts disclosed herein are safe, biocompatible, and growth factor-rich sterilized human placental allografts that have a much greater amount of growth factors than conventional allografts in the art, and the sterilized human placental allografts mimic the growth factor profile of the human placenta better in vivo than other conventional allografts, thereby achieving better therapeutic outcomes for dental applications and wound healing, and / or, depending on the application, better cosmetic / aesthetic outcomes. The sterilized human placental allografts disclosed herein are preferably non-immunogenic - there is little or no resulting immune response after implantation / transplantation into a human recipient and / or after application to a wound on the body and / or body surface of a subject in need thereof.

[0070] The sterilized human placental allograft disclosed in this specification is particularly rich in interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), vascular endothelial growth factor receptor 1 (VEGFR1), hyaluronic acid (HA), platelet-derived growth factor B subunit homodimer (PDGF-BB), glycosaminoglycan (GAG), and collagen, as compared with conventional allografts. Further, FIG. 6 schematically depicts that various human endogenous pathways activated by vascular endothelial growth factor receptor 1 (VEGFR1), interleukin-1 receptor antagonist (IL-1ra), hepatocyte growth factor (HGF), hyaluronic acid (HA), glycosaminoglycan (GAG), and platelet-derived growth factor B subunit homodimer (PDGF-BB) are associated with in vivo cell remodeling, wound healing, and other human endogenous pathways. In particular, IL-1ra non-productively binds to the IL-1 (inflammatory cytokine) receptor and prevents signal transduction by IL-1 (inflammatory molecule). This results in the modulation of various IL-1-related immune responses and inflammatory reactions. HGF is a factor for cell growth, motility, and morphogenesis secreted by mesenchymal cells. HGF has been shown to play an important role in embryonic organogenesis, specifically, myogenesis, adult organ regeneration, and wound healing. Importantly, an increase in HGF expression has been associated with the enhanced and scarless wound healing ability of fibroblasts isolated from oral mucosal tissue. VEGFR1 acts as a cell surface receptor for VEGF-A, VEGF-B, and PGF for the development of the embryonic vascular system, regulation of angiogenesis, cell survival, and cell migration. VEGFR1 can promote endothelial cell proliferation, survival, and angiogenesis in adulthood. The function of VEGFR1 to promote cell proliferation seems to be cell type-specific. For example, VEGFR1 promotes PGF-mediated proliferation of endothelial cells but not normal fibroblast proliferation (in vitro). PDGF-BB is a potent promoter of cell proliferation and plays a crucial role in angiogenesis (vascular formation), including the growth of blood vessels from existing vascular tissue.PDGF-BB promotes the proliferation and directional migration of mesenchymal cells, significantly enhances the influx of inflammatory cells, accelerates the formation of extracellular matrix and collagen, and thus shortens the total wound healing period. Glycosaminoglycan (GAG) exists in all mammalian tissues, where it interacts with other ECM components to organize and form a structural scaffold suitable for remodeling. GAG also modulates cell growth and proliferation, cell adhesion, anticoagulation, and wound repair. Hyaluronic acid (HA) is a well-documented major component of the extracellular matrix. HA is a non-sulfated glycosaminoglycan that provides a backbone for sulfated glycosaminoglycans, in particular. HA also binds integrins to result in a stabilized ECM and absorbs water, another major component of the ECM. High-molecular-weight HA results in unique biophysical properties such as high viscoelasticity and high colloidal osmotic pressure. Additionally, HA leads to the stabilization of the extracellular matrix, water retention, and regulation of protein distribution. Collagen is the most abundant protein in the body and is a major component of the extracellular matrix. During wound healing, collagen attracts fibroblasts and promotes not only the binding but also the deposition of new collagen and inactivates excessive matrix metalloproteinases (degradation). Exosomes are vesicles in the range of 30 nm or more and 200 nm or less that enable cell-to-cell communication without direct cell-to-cell contact. Exosomes are released via the cell membrane [Keller 2006, Zomer 2010] (placental cells in this case), taken up by tissues, and release signals from the placental tissue in those tissues. As a result, signals for development and regeneration are delivered to the cells of the damaged tissue. Exosomes are rich in small RNA species including miRNA and have been demonstrated to be functional within the recipient cells [Montecalvo 2008]. Exosomes are gaining momentum as a safe and effective therapy for skin wounds / injuries [Subhan 2021].

[0071] In certain embodiments, as further shown in FIGS. 3A-3F, FIGS. 5A and 5B, and FIG. 10A(100), the sterilized human placental allograft is intact in cross-section, and in the cross-section, the human amnion layer and the human chorionic layer have an intact human intermediate sponge layer positioned between the human amnion layer and the human chorionic layer and connecting the human amnion layer to the human chorionic layer. These allografts have the following: (a) interleukin-1 receptor antagonist (IL-1ra) in the range of 200 pg / cm 2 or more and 7800 pg / cm 2 or less, more preferably 1000 pg / cm 2 or more and 6000 pg / cm 2 or less; (b) hepatocyte growth factor (HGF) in the range of 500 pg / cm 2 or more and 8500 pg / cm 2 or less, more preferably 1000 pg / cm 2 or more and 7500 pg / cm 2 or less; (c) vascular endothelial growth factor receptor 1 (VEGFR1) in the range of 1000 pg / cm 2 or more and 2750 pg / cm 2 or less, more preferably 900 pg / cm 2 or more and 2500 pg / cm 2 or less; (d) hyaluronic acid (HA) in the range of 5.0×10 6 pg / cm 2 or more and 1.5×10 8 pg / cm 2 or less, more preferably 1.5×10 7 pg / cm 2 or more and 1.0×10 8 pg / cm 2 or less; and (e) in the range of 150 pg / cm 2 or more and 00 pg / cm 2 or less, more preferably 200 pg / cm 2 or more and 375 pg / cm 2At least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight of platelet-derived growth factor B subunit homodimer (PDGF-BB), (f) glycosaminoglycan (GAG), (g) collagen, and (h) exosome, within the following ranges, where any endpoint within any of the above-mentioned ranges can serve as an endpoint for any additional range that falls within it. The concentrations mentioned above were obtained / calculated by the "standardization" calculations and methods disclosed herein. It should further be recognized that these allografts are configured for specific dental, wound healing, and cosmetic purposes, and are preferably implantable / transplantable into the subject in need or applicable to shallow wounds (e.g., diabetic ulcers). In this regard, these sterilized human placental allografts have a predetermined shape and size, and in that shape and size, the predetermined shape can include, but is not limited to, circular shape, square shape, rectangular shape, rhomboid shape, oval shape, triangular shape, or any combination thereof. Also, the predetermined size of the sterilized human placental allografts includes dimensions in the range of 1 cm × 1 cm or more and 8 cm × 8 cm or less, where any endpoint within any of these ranges can serve as an endpoint for any additional range that falls within it, and / or includes a diameter in the range of 8 mm diameter or more and 50 mm diameter or less, where any endpoint within any of these ranges can serve as an endpoint for any additional range that falls within it. These sterilized human placental allografts can further include the upper surface or the lower surface of a plane, depending on their specific use, or these sterilized human placental allografts can include both the upper surface and the lower surface of a plane. The sterilized human placental allografts can have a thickness in the range of 0.08 mm or more and 0.2 mm or less, depending on their specific use, and can further have a translucent appearance, for example, as shown in FIG. 10A.

[0072] In certain embodiments, as further shown in FIGS. 2A - 2F, FIGS. 4A and 4B, and FIG. 11A(200), the sterilized human placental allograft consists essentially of human amnion and / or, in a further embodiment, the sterilized human placental allograft consists of human amnion. In each of these embodiments, the sterilized human placental allograft (consisting essentially of amnion and / or consisting of amnion) has the following: (a) interleukin - 1 receptor antagonist (IL - 1ra) in the range of 150 pg / cm 2 or more and 1800 pg / cm 2 or less, more preferably in the range of 200 pg / cm 2 or more and 1500 pg / cm 2 or less; (b) hepatocyte growth factor (HGF) in the range of 75 pg / cm 2 or more and 400 pg / cm 2 or less, more preferably in the range of 150 pg / cm 2 or more and 350 pg / cm 2 or less; (c) vascular endothelial growth factor receptor 1 (VEGFR1) in the range of 50 pg / cm 2 or more and 250 pg / cm 2 or less, more preferably in the range of 75 pg / cm 2 or more and 200 pg / cm 2 or less; (d) hyaluronic acid (HA) in the range of 1.1×10 6 pg / cm 2 or more and 2.0×10 7 pg / cm 2 or less, more preferably in the range of 1.5×10 6 pg / cm 2 or more and 1.8×10 7 pg / cm 2 or less; and (e) in the range of 200 pg / cm 2 or more and 500 pg / cm 2 or less, more preferably in the range of 250 pg / cm 2 or more and 450 pg / cm 2At least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight of platelet-derived growth factor B subunit homodimer (PDGF-BB), (f) glycosaminoglycan (GAG), (g) collagen, and (h) exosome, within the following ranges, where any endpoints within any of the above ranges can serve as endpoints for any additional ranges that fall within them. The concentrations mentioned above were obtained by the "standardization" calculations and methods disclosed herein. It should further be recognized that these allografts are configured for specific dental, wound healing, and cosmetic purposes, and preferably are implantable / transplantable into the subject in need or can be applied to shallow wounds (e.g., diabetic ulcers). In this aspect, these sterilized human placental allografts have a predetermined shape and size, and in that shape and size, the predetermined shape can include, but is not limited to, a circular shape, a square shape, a rectangular shape, a rhomboid shape, an oval shape, a triangular shape, or any combination thereof. Also, the predetermined size of the sterilized human placental allografts includes dimensions in the range of 1 cm × 1 cm or more and 8 cm × 8 cm or less, where any endpoints within any of these ranges can serve as endpoints for any additional ranges that fall within them, and / or includes a diameter in the range of 8 mm in diameter or more and 50 mm in diameter or less, where any endpoints within any of these ranges can serve as endpoints for any additional ranges that fall within them. These sterilized human placental allografts can further include an upper surface of a plane or a lower surface of a plane, depending on their specific use, or these sterilized human placental allografts can include both an upper surface of a plane and a lower surface of a plane. The sterilized human placental allografts can have a thickness in the range of 0.01 mm or more and 0.05 mm or less, depending on their specific use, and can further have a translucent appearance, for example, as shown in FIG. 11A.

[0073] Also, as shown in FIGS. 1 and 12A - 14D, in certain embodiments, the allografts disclosed herein may include slits, openings, and / or windows formed on (and through) a human placental allograft, such that a sterilized human placental allograft includes a plurality of slits, openings, or windows (e.g., flaps) formed on the upper surface of the allograft, and corresponding (e.g., aligned and / or axially aligned) slits, openings, or windows (flaps) are formed on the lower surface of the allograft, such that the slits, openings, or windows (e.g., flaps) formed on the upper surface of the allograft are in fluid communication with the corresponding slits, openings, or windows (e.g., flaps) formed on the lower surface of the allograft, such that in use, the allograft allows wound exudate to flow from the wound through the allograft in a direction from the lower surface of the allograft to the upper surface of the allograft, thereby facilitating wound drainage, wound healing, or a combination thereof. In view of the above, FIG. 12A depicts a top view of a sterilized human placental allograft disclosed herein with a plurality of slits (110 or 210) formed thereon in a non - expanded configuration, and FIG. 12B depicts a top view of the human placental allograft (120 or 220) of FIG. 12A in an expanded configuration, wherein the openings (110 or 210 formed from the slits upon expansion of the allograft) are arranged in a predetermined pattern (e.g., a grid pattern, and / or a grid pattern having openings in the shape of elongated rhombuses or half - elongated rhombuses), and the openings are configured such that wound exudate flows from the wound through the allograft in a direction from the lower surface of the allograft to the upper surface of the allograft.Figure 13 depicts a sterilized human placental allograft (100 or 200) having a plurality of openings (130 or 230) formed therein such that the lower and upper surfaces of the allograft are in fluid communication with each other and such that these openings are configured for wound exudate to flow from the wound, through the allograft in a direction from the lower surface of the allograft to the upper surface of the allograft. In certain embodiments, the slits and / or openings may be formed on the allograft using conventional devices such as dies or cutting apparatuses.

[0074] In certain embodiments, as shown in FIGS. 14A - 14D, 16A, and 16B, the human sterilized placental allograft (100 or 200) includes a plurality of windows (140 or 240, 150 or 250, 160 or 260) formed in the sterilized human placental allograft, and the plurality of windows are configured such that wound exudate flows from the wound, through the sterilized human placental allograft in a direction from the lower surface to the upper surface of the sterilized human placental allograft, facilitating wound drainage, wound healing, or a combination thereof. In this embodiment, as shown in FIGS. 14C and 14D, each window is a flap configured to be open when wound exudate and / or other body fluids are flowing through the sterilized human placental allograft and closed when wound exudate and / or other body fluids are not flowing through the sterilized human placental allograft. Each flap is spaced sufficiently apart from one another to allow wound exudate to flow therethrough without obstructing the openings and without closing any adjacent flaps. For example, the windows (e.g., flaps) are spaced approximately 14 mm from the center of one window (flap) to the center of another window (flap). Depending on the overall size of the allograft window (flap), the spacing may vary. For example, a 5 cm x 10 cm allograft may have 28 notches, where each window (flap) is spaced approximately 14 mm (the spacing as described immediately above) from another window (flap), and each window (flap) is approximately 7.5 mm wide. Advantageously, the ability of the windows (flaps) to allow fluid in the wound environment to exit the space bears the risk of this loss of surface area and disruption of the matrix. The windows (flaps) address these issues because the allograft does not lose integrity and / or the matrix does not become disrupted, which allows fluid to exit the wound without loss of surface area. Additionally, an allograft having windows (flaps) as disclosed herein advantageously retains the matrix orientation and organization, thus promoting ECM deposition and stabilization while providing factors that play a decisive role in ECM composition and wound healing.

[0075] Figures 14A - 14C depict an allograft with a window formed thereon that is closed and / or in a closed configuration. Figure 14D is a schematic depiction of a cross - sectional and / or side view of an allograft (100 or 200), and in use, the open flap (160 or 260) is moved in direction (D 1 ) by wound exudate and / or other body fluids arising from the wound flowing away from the wound (500) and from the lower (104 or 204) and upper (102 or 202) surfaces of the sterilized human placental allograft, thereby facilitating wound drainage, wound healing, or a combination thereof. In certain embodiments, and as further shown in Figures 14A - 14D, the window has a predefined shape, which includes "V" shapes (140 or 240), "U" shapes (150 or 250), semi - "U" shapes (160 or 260), or combinations thereof. Also, Figure 16A is a photograph of a sterilized human placental allograft with a plurality of windows formed thereon, and Figure 16B is another photograph of a sterilized human placental allograft with a plurality of windows formed thereon. The windows (e.g., flaps) discussed above are formed by a planar or roll press of the allograft on a die (600) having a predefined shape formed thereon to cut and form the window in the allograft (preferably before sterilization (e.g., electron beam sterilization)), and the window has a predefined shape (e.g., a notch in the shape of a V - shape, U - shape, or semi - U - shape). (Kit and method of use)

[0076] Also disclosed herein is a kit comprising a sterilized human placental allograft as disclosed herein, packaged within a sterilized container. Upon opening the sterilized container, the sterilized human placental allograft packaged therein is in a ready-to-use situation for the desired final use. The sterilized human allograft is configured for use in orthodontic, wound healing, and / or cosmetic applications, and is preferably implantable / transplantable into a subject in need thereof or applicable to superficial wounds (e.g., diabetic ulcers). As suggested above, the sterilized human placental allograft disclosed herein is preferably non-immunogenic - resulting in little or no immune response following implantation / transplantation into a human recipient and / or following topical application to a human recipient.

[0077] In certain embodiments, further disclosed is a method of treating a wound in a human subject in need thereof, the method comprising contacting the wound with a sterilized human placental allograft for a predetermined period of time to facilitate wound healing in the human subject in need thereof. The predetermined period of time includes from 3 days to 30 days for implant uses and from 1 day to 5 days for topical uses. In certain embodiments, the sterilized human placental allograft is implanted into the human subject in need thereof, where the wound is an internal wound and / or a dental wound in the subject in need thereof, thereby facilitating wound healing by elution / diffusion of growth factors from the sterilized human placental allograft into the wound. Further, the sterilized human placental allograft remains in the subject in need thereof and acts as a scaffold for cell and tissue remodeling after elution / diffusion of growth factors from the sterilized human placental allograft into the wound, and the allograft is eventually degraded and absorbed in the subject in need thereof. In certain other embodiments, the sterilized human placental allograft is topically applied to the wound of the human subject in need thereof. The topical application of the sterilized human placental allograft can be used to treat, for example, diabetic ulcers (such as diabetic foot ulcers) and / or other defined or undefined topical wounds (extending to the epidermis, dermis, and / or subcutaneous tissue) in the human subject in need thereof. The sterilized human placental allograft is topically placed on the wound of the subject in need thereof for a predetermined period of time, thereby facilitating wound healing by elution / diffusion of growth factors from the sterilized human placental allograft onto and into the wound.

[0078] In one embodiment, the method further comprises disinfecting the human placental tissue with at least one of a bactericidal composition, a mycobactericidal composition, a fungicidal composition, a virucidal composition, or any combination thereof, either before or during step (a), wherein the at least one of a bactericidal composition, a mycobactericidal composition, a fungicidal composition, a virucidal composition, or any combination thereof comprises isopropyl alcohol at a concentration of 70% or more and 100% or less, and / or each washing step (c) is carried out at a temperature in the range of 4°C or more and 15°C or less for a time period of 5 minutes or more and 15 minutes or less, and / or the dehydration step (d) is carried out at a temperature in the range of 20°C to 40°C for a time period of 60 minutes or more and 4.5 hours or less, and / or the sterilization step (f) comprises sterilization by electron beam irradiation.

[0079] In one aspect, the present invention relates to a method for preparing a sterilized human placental allograft, the method comprising (a) providing human placental tissue from a donor within 24 hours or more and 72 hours or less after childbirth, disinfecting the human placental tissue with at least one of a bactericidal composition, a mycobactericidal composition, a fungicidal composition, a virucidal composition, or any combination thereof, either before or during step (a), wherein the at least one of a bactericidal composition, a mycobactericidal composition, a fungicidal composition, a virucidal composition, or any combination thereof comprises isopropyl alcohol at a concentration of 70% or more and 100% or less, (b) removing any visible blood, blood clots, and / or blood components from the human placental tissue without scraping or scrubbing the human placental tissue to preserve the structural integrity of the human placental tissue; (c) washing the human placental tissue in an isotonic solution at a temperature in the range of 4°C or more and 15°C or less for a time period of 5 minutes or more and 15 minutes or less while maintaining the structural integrity of the human placental tissue; (d) At a temperature in the range of 20°C or higher and 40°C or lower, dehydrate the human placental tissue over a time period of 60 minutes or longer and 4.5 hours or shorter, thereby forming a dehydrated human placental tissue. (e) Resize the dehydrated human placental tissue into a dehydrated human placental tissue portion having a predetermined size. (f) Sterilize the dehydrated human placental tissue portion of step (e) by electron beam irradiation, thereby forming the sterilized human placental allograft. comprises.

[0080] In one aspect, the present invention relates to a sterilized human placental allograft for use in treating wounds (preferably according to the present invention).

[0081] In one aspect, the present invention relates to a sterilized human placental allograft for use in treating a wound of a human subject in need thereof (preferably according to the present invention), the treatment comprising contacting the wound with the aforementioned sterilized human placental allograft over a predetermined time period to facilitate wound healing of the human subject in need thereof.

[0082] A sterilized human placental allograft for use in the application of implanting into a subject in need thereof. A sterilized human placental allograft for use in the case where the wound is an internal wound and / or a dental wound in a subject in need thereof. A sterilized human placental allograft for use in the application of locally applying to a wound of a human subject in need thereof. A sterilized human placental allograft for use in the case where the wound is a diabetic ulcer. A sterilized human placental allograft for use in the case where the wound is a diabetic foot ulcer.

Examples

[0083] The following examples are submitted to provide those skilled in the art with a complete disclosure and description of how the compounds, compositions, and methods described and claimed herein are made and evaluated, are purely intended to be exemplary, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some errors and deviations should be accounted for. Unless otherwise indicated, parts are by weight, temperature is in °C, or ambient temperature if not listed, and pressure is at or near atmospheric pressure. There are numerous variations and combinations of conditions, such as component concentrations, temperatures, pressures, and other reaction ranges and conditions that can be used to optimize the purity and yield of the products obtained from the described processes. Only reasonable and routine experimentation will be required to optimize such process conditions.

[0084] Figures 8A - 11B provide graphs and photographs of exemplary sterilized human allografts (Examples 1 and 2) prepared by the methods / processes disclosed herein, further compared with various commercially available sterilized human allografts (Comparative Example 1, Comparative Example 2, and Comparative Example 3). These graphs and photographs further demonstrate not only dramatically different growth factor profiles but also visual differences among the multiple exemplary sterilized human allografts disclosed herein when compared with various commercially available sterilized human allografts.

[0085] Figures 8A, 8B, and 8C are graphs depicting the PDGF-BB concentration, HGF concentration, and HA concentration within a commercially available sterilized human placental allograft product having dehydrated amnion and dehydrated chorion (i.e., Example 1 (a sterilized human placental allograft disclosed herein that is intact in cross-section, wherein in the cross-section, the human amnion layer and the human chorion layer are positioned between the human amnion layer and the human chorion layer and connect the human amnion layer to the human chorion layer, and having an intact human intermediate sponge layer), compared to Comparative Example 1 ("Comp Ex.1") and Comparative Example 2 ("Comp.Ex.2")). Figure 10A is a photograph of the sterilized human placental allograft of Figure 8A (i.e., Example 1), Figure 10B is a photograph of the commercially available product of Figure 8B (i.e., Comp Ex.1), and Figure 10C is a photograph of the commercially available product of Figure 8C (i.e., Comp Ex.2).

[0086] It should be noted that Comparative Example 1 is a commercially available sterilized human placental allograft composition (NuShield® by Organogenesis https: / / organogenesis.com / surgical-sports-medicine / nushield / (accessed August 1, 2022)) prepared by the Tutoplast® process (https: / / www.rtix.com / en_us / operational-excellence / patient-safety / tissue-biologics / tutoplast-tissue-sterilization-process (accessed August 1, 2022)). The Tutoplast® process uses a number of intensive washing processes to remove all lipids, bacteria, and soluble proteins, and final sterilization is performed by gamma irradiation, resulting in a final product that is a structural allograft with depleted various growth factors.

[0087] It should be noted that Comparative Example 2 is a commercially available sterile human placenta allograft composition (EpiFix® by Mimedx, https: / / www.mimedx.com / products / epifix / (accessed August 1, 2022)) that was prepared by the Purion® process (https: / / www.mimedx.com / purion-process / (accessed August 1, 2022) and further reported by Koob et al. 2013, 2014), which separates and purifies the human amniotic and chorionic membrane layers, and then reattaches (stacks) the layers prior to dehydration.

[0088] As shown in Figures 8A, 8B, and 8C, the sterile human placenta allograft of Example 1 had higher PDGF-BB, HGF, and HA (pg / cm) than either Comparative Example 1 or Comparative Example 2. 2 The graphs provided in Figures 8A, 8B, and 8C show that the 1 cm 2 Average weight of amnion / chorion per cubic metre (7.72 mg / cm 2 ) from mg to cm 2 As further shown in Figures 8A, 8B, and 8C, Example 1 contained, on average, 320.31% more PDGF-BB than Comparative Examples 1 and 2, 1142.41% more HGF than Comparative Examples 1 and 2, and 1389.23% more HA than Comparative Example 1. These results clearly indicate that various endogenous growth factors are retained and preserved in the sterile human placenta allograft of Example 1 when compared to similar products on the market. Figures 10A-10C further support these results, in which Figure 10A (100) is translucent but more opaque than Comparative Examples 1 and 2 (Figures 10B and 10C, respectively), further indicating that various endogenous growth factors are retained and preserved in the sterile human placenta allograft of Example 1 when compared to similar products on the market.

[0089] FIG. 9 is a graph depicting the IL-1ra concentration in Example 2 (a sterilized human placental allograft disclosed herein that consists essentially of human amnion) compared to a commercial product containing only dehydrated amnion (i.e., Comparative Example 3 (“Comp Ex. 3”)). In particular, Comparative Example 3 is AmnioFix® amnion by Mimedx (https: / / www.petepetit.com / pete-petit-professional-blog / 2015 / mimedx-products-amniofix.html (accessed August 1, 2022)), and the amnion is prepared by the Purion® process (https: / / www.mimedx.com / purion-process / (accessed August 1, 2022), and further reported by Koob et al. 2013, 2014).

[0090] As shown in FIG. 9, the sterilized human placental allograft of Example 1 contains, on average, 220.88% more IL-1ra than Comparative Example 3, which indicates that various endogenous growth factors are retained and preserved in the human sterilized placental allograft of Example 2 (i.e., the sterilized human placental allograft disclosed herein prepared by the method / process disclosed herein and consisting essentially of human amnion) when compared to a similar commercial product of Comparative Example 3. FIGS. 11A and 11B further support these results, where FIG. 11A (200) is opaque, even though translucent, compared to the highly processed translucent visual appearance of Comparative Example 3 (FIG. 11B), which further indicates that various endogenous growth factors are retained and preserved in the human sterilized placental allograft of Example 2 when compared to a similar commercial product.

[0091] The foregoing description provides only embodiments of this invention by way of example. It is contemplated that other embodiments may perform similar functions and / or achieve similar results. All such equivalent embodiments and examples are within the scope of this invention and are intended to be covered by the appended claims.

Claims

Claim 1 A method for preparing a sterilized human placental allograft, comprising: (a) providing human placental tissue from a donor within 24 hours or less and within 72 hours after childbirth; (b) removing from said human placental tissue any visible blood, blood clots, and / or blood components without scraping or scrubbing said human placental tissue to preserve the structural integrity of said human placental tissue; (c) washing said human placental tissue in an isotonic solution while maintaining the structural integrity of said human placental tissue; (d) dehydrating said human placental tissue to thereby form a dehydrated human placental tissue; (e) sizing said dehydrated human placental tissue into dehydrated human placental tissue portions having a predetermined size; (f) sterilizing said dehydrated human placental tissue portion of step (e) to thereby form said sterilized human placental allograft. A method comprising the above steps. Claim 2 The method according to claim 1, further comprising disinfecting said human placental tissue with at least one of a bactericidal composition, a mycobactericidal composition, a fungicidal composition, a virucidal composition, or any combination thereof, either before or during step (a). Claim 3 The method according to claim 1 or 2, wherein step (b) comprises manual hematopoietic system reduction by manually removing any visible blood, blood clots, and / or blood components from said human placental tissue. Claim 4 The method according to any one of claims 1 to 3, wherein said isotonic solution comprises at least one of 1× phosphate buffered saline, isotonic saline solution, lactated Ringer's solution, Plasma-Lyte® (at pH 7.4, 5.26 g / L NaCl, 0.37 g / L KCl, 0.30 g / L magnesium chloride hexahydrate, 3.68 g / L sodium acetate trihydrate, 5.02 g / L sodium gluconate), Normosol® (at pH 7.4, 5.26 g / L NaCl, 0.37 g / L KCl, 0.30 g / L magnesium chloride, 2.22 g / L sodium acetate anhydride, 5.02 g / L sodium gluconate), or any combination thereof. Claim 5 The method according to any one of claims 1 to 4, further comprising repeating step (c) a predetermined number of times. Claim 6 The method according to any one of claims 1 to 5, wherein each washing step (c) is carried out at a temperature in the range of 4°C or more and 20°C or less for a time period of 5 minutes to 15 minutes.

7. The method according to any one of claims 1 to 6, wherein the dehydrating step (d) is carried out at a temperature in the range of 20°C or more and 40°C or less for a time period of 60 minutes or more and 4.5 hours or less.

8. The method according to any one of claims 1 to 7, wherein the predetermined size of the placental tissue portion is 1 cm × 1 cm or more and 8 cm × 8 cm or less for a placental tissue portion having a rectangular shape and / or a square shape, and 8 mm or more and 50 mm or less in diameter for a placental tissue portion having a circular shape.

9. The method according to any one of claims 1 to 8, wherein the placental tissue portion has a predetermined shape.

10. The method according to any one of claims 1 to 9, wherein the sterilizing step (f) includes sterilization by electron beam irradiation.

11. Step (f) is to perform electron beam irradiation on the dehydrated human placental tissue portion to a sterility assurance level (SAL) of 10 as calculated by the dose distribution to form the sterilized human placental allograft. The method according to any one of claims 1 to 10 includes this step. －６ The method according to any one of claims 1 to 10, comprising: performing electron beam irradiation on the dehydrated human placental tissue portion to a sterility assurance level (SAL) of 10 as calculated by the dose distribution to form the sterilized human placental allograft.

12. The method according to any one of claims 1 to 11, wherein the human placental tissue includes intact placental tissue.

13. The dehydrated human placental tissue in step (d) and the dehydrated human placental tissue portion having a predetermined size in step (e) are intact in cross-section, and in the cross-section, the human amniotic layer and the human chorionic layer are positioned between the human amniotic layer and the human chorionic layer to connect the human amniotic layer to the human chorionic layer, and have an intact human intermediate sponge layer. The method according to any one of claims 1 to 12.

14. The sterilized human placental allograft in step (f) is intact in cross-section, and in the cross-section, the human amniotic layer and the human chorionic layer are positioned between the human amniotic layer and the human chorionic layer to connect the human amniotic layer to the human chorionic layer, and have an intact human intermediate sponge layer. The method according to any one of claims 1 to 13.

15. The method according to any one of claims 1 to 14, wherein the sterilized human placental allograft is configured for dental use, cosmetic use, and / or wound healing use.

16. The sterilized human placental allograft includes a plurality of slits, openings, or windows formed on the upper surface of the sterilized human placental allograft, and corresponding slits, openings, or windows formed on the lower surface of the sterilized human placental allograft, and the plurality of slits, openings, or windows, and the corresponding slits, openings, or windows are configured such that wound exudate flows from the wound and passes through the sterilized human placental allograft in a direction from the lower surface of the sterilized human placental allograft to the upper surface of the allograft, facilitating wound drainage, wound healing, or a combination thereof. The method according to any one of claims 1 to 15.

17. The plurality of slits, openings, or windows formed on the upper surface of the sterilized human placental allograft are axially aligned with the corresponding slits, openings, or windows formed on the lower surface of the sterilized human placental allograft. The method according to claim 16.

18. The human sterilized placental allograft includes a plurality of windows formed in the sterilized human placental allograft, and the plurality of windows are configured such that wound exudate flows from the wound and passes through the sterilized human placental allograft in a direction from the lower surface of the sterilized human placental allograft to the upper surface of the sterilized human placental allograft, facilitating wound drainage, wound healing, or a combination thereof. The method according to claim 16.

19. Each window is a flap configured to be open when wound exudate and / or other body fluids are flowing through the sterilized human placental allograft and to be closed when wound exudate and / or other body fluids are not flowing through the sterilized human placental allograft. The method according to claim 18.

20. The flap moves in a direction away from the lower surface and the upper surface of the sterilized human placental allograft when open. The method according to claim 18 or 19.

21. The window has a predetermined shape. The method according to any one of claims 18 to 20.

22. The predetermined shape is a "V" shape, a "U" shape, a semi-"U" shape, or a combination thereof. The method according to claim 22.

23. The method further includes disinfecting the human placental tissue with at least one of a bactericidal composition, a mycobactericidal composition, a fungicidal composition, a virucidal composition, or any combination thereof, either before or during step (a), and the at least one of a bactericidal composition, a mycobactericidal composition, a fungicidal composition, a virucidal composition, or any combination thereof contains isopropyl alcohol at a concentration of 70% or more and 100% or less. Each washing step (c) is carried out at a temperature in the range of 4°C or more and 15°C or less for a time period of 5 minutes or more and 15 minutes or less. The dehydration step (d) is carried out at a temperature in the range of 20°C or more and 40°C or less for a time period of 60 minutes or more and 4.5 hours or less. The sterilization step (f) includes sterilization by electron beam irradiation. The method according to any one of claims 1 to 22.

24. A sterilized human placental allograft produced by the method according to any one of claims 1 to 23.

25. The sterilized human placental allograft has the following: (a) 200 pg / cm ２ above 7800 pg / cm ２ an interleukin-1 receptor antagonist (IL-1ra) in the following range, and (b) 500 pg / cm ２ 8500 pg / cm or more ２ Hepatocyte growth factor (HGF) within the following range, and (c) 800 pg / cm ２ above 2750 pg / cm ２ vascular endothelial growth factor receptor 1 (VEGFR1) within the following range, and (d) 5.0 × 10 ６ pg / cm ２ or more 1.5 × 10 ８ pg / cm ２ hyaluronic acid (HA) within the following range, and (e) 150 pg / cm ２ 400 pg / cm or less above ２ Platelet-derived growth factor B subunit homodimer (PDGF-BB) within the following range, and (f) glycosaminoglycan (GAG); (g) collagen; (h) exosome; The sterilized human placental allograft according to claim 24, which contains at least two of them.

26. The sterilized human placental allograft is intact in cross-section, and in the cross-section, the human amniotic layer and the human chorionic layer are positioned between the human amniotic layer and the human chorionic layer to connect the human amniotic layer to the human chorionic layer, and has an intact human intermediate sponge layer. The sterilized human placental allograft according to claim 24 or 25.

27. The sterilized human placental allograft is configured for dental use, cosmetic use, and / or wound healing use. The sterilized human placental allograft according to any one of claims 24 to 26.

28. The sterilized human placental allograft has a predetermined size of 1 cm × 1 cm or more and 8 cm × 8 cm or less for a rectangular and / or square-shaped sterilized human placental allograft, and 8 mm or more and 50 mm or less in diameter for a circular-shaped sterilized human placental allograft. The sterilized human placental allograft according to any one of claims 24 to 27.

29. The sterilized human placental allograft according to any one of claims 24 to 28, having a circular shape, a square shape, a rectangular shape, an oval shape, a triangular shape, or any combination thereof.

30. The sterilized human placental allograft according to any one of claims 24 to 29, wherein the upper surface and / or the lower surface of the sterilized human placental allograft is a flat surface.

31. The sterilized human placental allograft according to any one of claims 24 to 30, wherein the upper surface and the lower surface of the sterilized human placental allograft are flat surfaces.

32. The sterilized human placental allograft according to any one of claims 24 to 31, comprising a plurality of slits, openings, or windows formed on the upper surface of the sterilized human placental allograft and corresponding slits, openings, or windows formed on the lower surface of the sterilized human placental allograft, wherein the plurality of slits, openings, or windows and the corresponding slits, openings, or windows are configured such that wound exudate flows from the wound through the sterilized human placental allograft in a direction from the lower surface of the sterilized human placental allograft to the upper surface of the allograft, facilitating wound drainage, wound healing, or a combination thereof.

33. The sterilized human placental allograft according to any one of claims 24 to 32, wherein the plurality of slits, openings, or windows formed on the upper surface of the sterilized human placental allograft are axially aligned with the corresponding slits, openings, or windows formed on the lower surface of the sterilized human placental allograft.

34. The sterilized human placental allograft according to any one of claims 24 to 33, comprising a plurality of windows formed in the sterilized human placental allograft, wherein the plurality of windows are configured such that wound exudate flows from the wound through the sterilized human placental allograft in a direction from the lower surface of the sterilized human placental allograft to the upper surface of the sterilized human placental allograft, facilitating wound drainage, wound healing, or a combination thereof.

35. Each window is configured to be open when wound exudate and / or other body fluids are flowing through the sterilized human placental allograft and to be closed when wound exudate and / or other body fluids are not flowing through the sterilized human placental allograft, the sterilized human placental allograft according to any one of claims 24 to 34.

36. The flap moves in a direction away from the lower surface and the upper surface of the sterilized human placental allograft when open, the sterilized human placental allograft according to any one of claims 24 to 35.

37. The window has a predetermined shape, the sterilized human placental allograft according to any one of claims 24 to 36.

38. The predetermined shape is a "V" shape, a "U" shape, a semi-"U" shape, or a combination thereof, the sterilized human placental allograft according to any one of claims 24 to 37.

39. The sterilized human placental allograft consists essentially of the amnion, the sterilized human placental allograft according to any one of claims 24 to 38.

40. The sterilized human placental allograft is as follows, (a) 150 pg / cm ２ 1800 pg / cm or more ２ an interleukin-1 receptor antagonist (IL-1ra) within the following range, and (b) 75 pg / cm ２ 400 pg / cm or less ２ Hepatocyte growth factor (HGF) within the following range, and (c) 50 pg / cm ２ 250 pg / cm or less above ２ vascular endothelial growth factor receptor 1 (VEGFR1) within the following range, and (d) 1.1 × 10 ６ pg / cm ２ above 2.0 × 10 ７ pg / cm ２ hyaluronic acid (HA) in the following range, and (e) 200 pg / cm ２ 500 pg / cm or less above ２ A platelet-derived growth factor B subunit homodimer (PDGF-BB) within the following range, and (f) glycosaminoglycan (GAG), (g) collagen, (h) exosome, and includes at least two of them, the sterilized human placental allograft according to any one of claims 24 to 39.

41. The sterilized human placental allograft is configured for dental use, cosmetic use, and / or wound healing use, the sterilized human placental allograft according to any one of claims 24 to 40.

42. The sterilized human placental allograft has a predetermined size of 1 cm × 1 cm or more and 8 cm × 8 cm or less for a rectangular and / or square sterilized human placental allograft and 8 mm diameter or more and 50 mm diameter or less for a circular sterilized human placental allograft, the sterilized human placental allograft according to any one of claims 24 to 41.

43. The sterilized human placental allograft has a circular shape, a square shape, a rectangular shape, an oval shape, a triangular shape, or any combination thereof, the sterilized human placental allograft according to any one of claims 24 to 42.

44. The upper surface and / or the lower surface of the sterilized human placental allograft is planar, the sterilized human placental allograft according to any one of claims 24 to 43.

45. The sterilized human placenta allograft according to any one of claims 24 to 44, wherein the upper surface and the lower surface of the sterilized human placenta allograft are flat.

46. A kit comprising the sterilized human placenta allograft produced by the method of claim 1 and packaged in a sterilized container, and / or a kit comprising the sterilized human placenta allograft according to any one of claims 24 to 45.

47. A method for treating a wound of a human subject in need thereof, the method comprising contacting the wound with the sterilized human placenta allograft according to any one of claims 24 to 45 for a predetermined period of time to facilitate wound healing of the human subject in need thereof.

48. The method according to claim 47, wherein the sterilized human placenta allograft is implanted into the subject in need thereof.

49. The method according to claim 48, wherein the wound is an internal wound and / or a dental wound within the subject in need thereof.

50. The method according to claim 47, wherein the sterilized human placenta allograft is applied locally to the wound in the human subject in need thereof.

51. The method according to claim 47, wherein the wound is a diabetic ulcer.

52. The following, (a) 200 pg / cm ２ 7800 pg / cm or less ２ an interleukin-1 receptor antagonist (IL-1ra) within the following range, and (b) 500 pg / cm ２ 8500 pg / cm or more ２ hepatocyte growth factor (HGF) within the following range, and (c) 800 pg / cm ２ 2750 pg / cm or more ２ Vascular endothelial growth factor receptor 1 (VEGFR1) within the following range, and (d) 5.0 × 10 ６ pg / cm ２ above 1.5 × 10 ８ pg / cm ２ and hyaluronic acid (HA) in the following range (e) 150 pg / cm ２ 400 pg / cm or less above ２ A platelet-derived growth factor B subunit homodimer (PDGF-BB) within the following range, and (f) glycosaminoglycan (GAG), (g) collagen, (h) exosome, A sterilized human placenta allograft comprising at least two of them.

53. The sterilized human placenta allograft according to claim 52, wherein the sterilized human placenta allograft is intact in cross-section, and in the cross-section, a human amniotic layer and a human chorionic layer are positioned between the human amniotic layer and the human chorionic layer to connect the human amniotic layer to the human chorionic layer, and has an intact human intermediate sponge layer.

54. The sterilized human placenta allograft according to claim 52 or 53, wherein the sterilized human placenta allograft is configured for dental use, cosmetic use, and / or wound healing use.

55. The sterilized human placenta allograft according to any one of claims 52 to 54, wherein the sterilized human placenta allograft having a rectangular shape and / or a square shape has a predetermined size of 1 cm × 1 cm or more and 8 cm × 8 cm or less, and the sterilized human placenta allograft having a circular shape has a diameter of 8 mm or more and 50 mm or less.

56. The sterilized human placental allograft according to any one of claims 52 to 55, having a circular shape, a square shape, a rectangular shape, an oval shape, a triangular shape, or any combination thereof.

57. The sterilized human placental allograft according to any one of claims 52 to 56, wherein the upper surface and / or the lower surface of the sterilized human placental allograft is a flat surface.

58. The sterilized human placental allograft according to any one of claims 52 to 57, wherein the upper surface and the lower surface of the sterilized human placental allograft are flat surfaces.

59. The sterilized human placental allograft according to any one of claims 52 to 58, comprising a plurality of slits, openings, or windows formed on the upper surface of the sterilized human placental allograft and corresponding slits, openings, or windows formed on the lower surface of the sterilized human placental allograft, wherein the plurality of slits, openings, or windows and the corresponding slits, openings, or windows are configured such that wound exudate flows from the wound and passes through the sterilized human placental allograft in a direction from the lower surface of the sterilized human placental allograft to the upper surface of the allograft, facilitating wound drainage, wound healing, or a combination thereof.

60. The sterilized human placental allograft according to any one of claims 52 to 59, wherein the plurality of slits, openings, or windows formed on the upper surface of the sterilized human placental allograft are axially aligned with the corresponding slits, openings, or windows formed on the lower surface of the sterilized human placental allograft.

61. The sterilized human placental allograft according to any one of claims 52 to 60, comprising a plurality of windows formed in the sterilized human placental allograft, wherein the plurality of windows are configured such that wound exudate flows from the wound and passes through the sterilized human placental allograft in a direction from the lower surface of the sterilized human placental allograft to the upper surface of the sterilized human placental allograft, facilitating wound drainage, wound healing, or a combination thereof.

62. Each window is configured to be open when wound exudate and / or other body fluids are flowing through the sterilized human placental allograft, and to be closed when wound exudate and / or other body fluids are not flowing through the sterilized human placental allograft, the sterilized human placental allograft according to any one of claims 52 to 61.

63. The flap moves in a direction away from the lower surface and the upper surface of the sterilized human placental allograft when open, the sterilized human placental allograft according to any one of claims 52 to 62.

64. The window has a predetermined shape, the sterilized human placental allograft according to any one of claims 52 to 63.

65. The predetermined shape is a "V" shape, a "U" shape, a semi-"U" shape, or a combination thereof, the sterilized human placental allograft according to any one of claims 52 to 64.

66. The sterilized human placental allograft consists essentially of the amnion, the sterilized human placental allograft according to any one of claims 52 to 65.

67. The sterilized human placental allograft is as follows, (a) 150 pg / cm ２ above 1800 pg / cm ２ an interleukin-1 receptor antagonist (IL-1ra) in the following range, and (b) 75 pg / cm ２ 400 pg / cm or less above ２ hepatocyte growth factor (HGF) within the following range, and (c) 50 pg / cm ２ 250 pg / cm or less above ２ vascular endothelial growth factor receptor 1 (VEGFR1) within the following range, and (d) 1.1 × 10 ６ pg / cm ２ or more 2.0 × 10 ７ pg / cm ２ and hyaluronic acid (HA) in the following range, (e) 200 pg / cm ２ 500 pg / cm or less above ２ Platelet-derived growth factor B subunit homodimer (PDGF-BB) within the following range, and (f) glycosaminoglycan (GAG), (g) collagen, (h) exosome, and contains at least two of them, the sterilized human placental allograft according to claim 66.

68. The sterilized human placental allograft is configured for dental use, cosmetic use, and / or wound healing use, the sterilized human placental allograft according to any one of claims 52 to 67.

69. The sterilized human placental allograft has a predetermined size of 1 cm × 1 cm or more and 8 cm × 8 cm or less for rectangular-shaped and / or square-shaped sterilized human placental allografts, and 8 mm in diameter or more and 50 mm in diameter or less for circular-shaped sterilized human placental allografts, the sterilized human placental allograft according to any one of claims 52 to 68.

70. The sterilized human placental allograft has a circular shape, a square shape, a rectangular shape, an oval shape, a triangular shape, or any combination thereof, the sterilized human placental allograft according to any one of claims 52 to 69.

71. The upper surface and / or the lower surface of the sterilized human placental allograft is flat, the sterilized human placental allograft according to any one of claims 52 to 70.

72. The sterilized human placental allograft according to any one of claims 52 to 71, wherein the upper surface and the lower surface of the sterilized human placental allograft are flat.

73. The following, (a) 200 pg / cm ２ 7800 pg / cm or less ２ an interleukin-1 receptor antagonist (IL-1ra) within the following range, and (b) 500 pg / cm ２ 8500 pg / cm or more ２ hepatocyte growth factor (HGF) within the following range, and (c) 800 pg / cm ２ 2750 pg / cm or more ２ Vascular endothelial growth factor receptor 1 (VEGFR1) within the following range, and (d) 5.0 × 10 ６ pg / cm ２ or more and 1.5 × 10 ８ pg / cm ２ or less of hyaluronic acid (HA), and (e) 150 pg / cm ２ 400 pg / cm or less above ２ A platelet-derived growth factor B subunit homodimer (PDGF-BB) within the following range, and (f)Glycosaminoglycan (GAG), (g)Collagen, (h)Exosomes, The sterilized human placental allograft according to any one of claims 52 to 72, comprising at least three of them.

74. The following, (a) 200 pg / cm ２ up to 7800 pg / cm ２ an interleukin-1 receptor antagonist (IL-1ra) in the following range, and (b) 500 pg / cm ２ 8500 pg / cm or more ２ Hepatocyte growth factor (HGF) within the following range, and (c) 800 pg / cm ２ 2750 pg / cm or more ２ Vascular endothelial growth factor receptor 1 (VEGFR1) within the following range, and (d) 5.0 × 10 ６ pg / cm ２ or more and 1.5 × 10 ８ pg / cm ２ or less of hyaluronic acid (HA), and (e) 150 pg / cm ２ above 400 pg / cm ２ Platelet-derived growth factor B subunit homodimer (PDGF-BB) within the following range, and (f)Glycosaminoglycan (GAG) (g)Collagen, (h)Exosomes, The sterilized human placental allograft according to any one of claims 52 to 73, comprising at least four of them.

75. The following, (a) 200 pg / cm ２ 7800 pg / cm or more ２ an interleukin-1 receptor antagonist (IL-1ra) within the following range, and (b) 500 pg / cm ２ 8500 pg / cm or more ２ Hepatocyte growth factor (HGF) within the following range, and (c) 800 pg / cm ２ 2750 pg / cm or more ２ vascular endothelial growth factor receptor 1 (VEGFR1) within the following range, and (d) 5.0 × 10 ６ pg / cm ２ above 1.5 × 10 ８ pg / cm ２ hyaluronic acid (HA) in the following range, and (e) 150 pg / cm ２ above 400 pg / cm ２ a platelet-derived growth factor B subunit homodimer (PDGF-BB) within the following range, and (f)Glycosaminoglycan (GAG), (g)Collagen, (h)Exosomes, The sterilized human placental allograft according to any one of claims 52 to 74, comprising at least five of them.

76. The following, (a) 200 pg / cm ２ 7800 pg / cm or more ２ an interleukin-1 receptor antagonist (IL-1ra) in the following range, and (b) 500 pg / cm ２ 8500 pg / cm or more ２ hepatocyte growth factor (HGF) within the following range, and (c) 800 pg / cm ２ 2750 pg / cm or less above ２ Vascular endothelial growth factor receptor 1 (VEGFR1) within the following range, and (d) 5.0 × 10 ６ pg / cm ２ or more and 1.5 × 10 ８ pg / cm ２ or less of hyaluronic acid (HA), and (e) 150 pg / cm ２ 400 pg / cm or less ２ platelet-derived growth factor B subunit homodimer (PDGF-BB) within the following range, and (f)Glycosaminoglycan (GAG), (g)Collagen, (h)Exosomes, The sterilized human placental allograft according to any one of claims 52 to 75, comprising at least six of them.

77. The following, (a) 200 pg / cm ２ 7800 pg / cm or more ２ an interleukin-1 receptor antagonist (IL-1ra) within the following range, and (b) 500 pg / cm ２ 8500 pg / cm or more ２ Hepatocyte growth factor (HGF) within the following range, and (c) 800 pg / cm ２ 2750 pg / cm or more ２ Vascular endothelial growth factor receptor 1 (VEGFR1) within the following range, and (d) 5.0 × 10 ６ pg / cm ２ above 1.5 × 10 ８ pg / cm ２ hyaluronic acid (HA) within the following range, and (e) 150 pg / cm ２ 400 pg / cm or less above ２ Platelet-derived growth factor B subunit homodimer (PDGF-BB) within the following range, and (f)Glycosaminoglycan (GAG), (g)Collagen, (h)Exosomes, The sterilized human placental allograft according to any one of claims 52 to 76, comprising at least seven of them.

78. The following, (a) 200 pg / cm ２ 7800 pg / cm or more ２ an interleukin-1 receptor antagonist (IL-1ra) in the following range, and (b) 500 pg / cm ２ 8500 pg / cm or more ２ Hepatocyte growth factor (HGF) within the following range, and (c) 800 pg / cm ２ 2750 pg / cm or more ２ Vascular endothelial growth factor receptor 1 (VEGFR1) within the following range, and (d) 5.0 × 10 ６ pg / cm ２ or more 1.5 × 10 ８ pg / cm ２ hyaluronic acid (HA) within the following range, and (e) 150 pg / cm ２ 400 pg / cm or less above ２ Platelet-derived growth factor B subunit homodimer (PDGF-BB) within the following range, and (f)Glycosaminoglycan (GAG), (g)Collagen, (h)Exosomes, The sterilized human placental allograft according to any one of claims 52 to 77, comprising at least eight of them.

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