Sterilized human placental allograft and method for producing the same

Abstract

A method for preparing a sterilized human placental allograft, comprising providing human placental tissue from a donor within 72 hours and more than 24 hours after childbirth; removing, without scraping or scrubbing the human placental tissue, any visible blood, blood clots, and / or blood components from the human placental tissue while preserving 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 thereby 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 thereby 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 the allograft, wherein the allograft has various dental, cosmetic, and wound healing applications.

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 within human placental tissue and / or result in an endogenous degradation process that further results in the loss of endogenous growth factors found within human placental tissue, thereby resulting in a reduction in the effectiveness of the final use (e.g., reduced wound healing) for any allograft 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 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 placental allografts disclosed herein 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 various 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. Furthermore, 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 hours or more and 72 hours or less after delivery; (b) removing from the human placental tissue any visible blood, blood clots, and / or blood components 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 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 thereby form the sterilized human placental allograft.

[0006] In certain embodiments, 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, either before or during step (a). 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 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 isopropyl alcohol advantageously disinfects and purifies the placental tissue without damaging the human placental tissue (e.g., causing excessive dehydration, initiating apoptosis and / or necrosis processes). In certain embodiments, the at least one of a bactericidal composition, a mycobactericidal composition, a fungicidal composition, a virucidal composition, or any combination thereof, may be used in a predetermined volume, wherein 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, 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 anhydrous sodium acetate, 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 placenta 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 placenta tissue having a residual water content of about 10% to 15%.

[0012] In certain embodiments, the predetermined size of the human placental tissue portion is in the range of 1 cm × 1 cm or more and 8 cm × 8 cm or less for square-shaped and rectangular-shaped human placental tissue portions. In a further embodiment, the square-shaped and rectangular-shaped human placental tissue portions can 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 ranges that fall within it. In certain embodiments, the predetermined size can include round-shaped and / or circular-shaped human placental tissue portions with a diameter in the range of 0.5 mm or more and 50 mm or less, where any endpoint within any of these ranges can serve as an endpoint for any additional ranges that fall within it, and in a further embodiment, these sizes can include round-shaped and / or circular-shaped 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.

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

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

[0015] In certain embodiments, step (f) 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 by the electron beam manufacturer 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 allografts produced by the above method are configured for dental, cosmetic, and / or wound healing applications.

[0018] 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.

[0019] In certain embodiments, the sterilized 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) 5.0×10 6 pg / cm 2 or more and 1.5×108 pg / cm 2 hyaluronic acid (HA) in the following range, and (e) 150 pg / cm 2 or more and 400 pg / cm or less 2 platelet-derived growth factor B subunit homodimer (PDGF-BB) in the following range, and (f) glycosaminoglycan (GAG), (g) collagen, (h) exosome, and includes at least two of them, where 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 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.

[0020] In a particular aspect, the sterilized human placental allograft is configured for dental use, cosmetic use, and / or wound healing use.

[0021] 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.

[0022] In certain embodiments, the sterilized human placental allograft has a circular, square, rectangular, oval, triangular, or any combination thereof.

[0023] In certain embodiments, the upper surface and / or lower surface of the sterilized human placental allograft is planar.

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

[0025] In certain embodiments, the sterilized human placental allograft consists essentially of human amnion. 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 amnion and / or consisting of amnion) has the following: (a) 150 pg / cm 2 or more and 1800 pg / cm2 Interleukin-1 receptor antagonist (IL-1ra) in the following range, and (b) 75 pg / cm 2 400 pg / cm or more 2 Hepatocyte growth factor (HGF) in the following range, and (c) 50 pg / cm 2 250 pg / cm or more 2 Vascular endothelial growth factor receptor 1 (VEGFR1) in the following range, and (d) 1.1×10 6 pg / cm 2 2.0×10 or more 7 pg / cm 2 Hyaluronic acid (HA) in the following range, and (e) 200 pg / cm 2 500 pg / cm or more 2 Platelet-derived growth factor B subunit homodimer (PDGF-BB) in the following range, and (f) glycosaminoglycan (GAG), (g) collagen, and (h) exosome, containing at least two of them, where 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 lower surface of the sterilized human placental allograft is flat. In this aspect, the upper surface and the lower surface of the sterilized human placental allograft are flat.

[0026] Also disclosed herein is a kit comprising the sterilized human placental allograft, packaged within a sterilized container. In this embodiment, the sterilized human placental allograft is produced by the method disclosed herein.

[0027] Embodiments of this invention can include one or more or any combination of the above features and configurations.

[0028] Additional features, aspects, and advantages of this invention will be set forth in the detailed description that follows, and in part will be readily apparent to those skilled in the art from that description, or can be learned by practicing the invention as described herein. It is to be understood that both the foregoing general description and the following detailed description are presented 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

[0029] 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 in conjunction with the accompanying drawings.

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Best Mode for Carrying Out the Invention

[0041] Next, with reference to the accompanying drawings showing exemplary embodiments of the present invention, the present invention will be described more fully hereinafter. However, the present invention may be embodied in many different forms and should not be construed as limited to the exemplary embodiments set forth herein. The exemplary embodiments are provided so that this disclosure will be both 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.

[0042] 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.

[0043] Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. It should be understood that such range formats are used merely for convenience and brevity and are thus to be interpreted in a flexible manner to include not only the explicitly recited values as the limits of the range but also all the individual values or sub-ranges subsumed within that range as if each were explicitly recited. By way of example, the numerical range "about 1 to 5" should be interpreted to include not only the explicitly recited values of about 1 to about 5 but also the individual values and sub-ranges within the indicated range. Thus, included in this numerical range are the individual values such as 2, 3, and 4, and sub-ranges such as 1 to 3, 2 to 4, and 3 to 5, etc. The same principle applies to ranges where only a single numerical value is recited as the minimum or maximum. Further, such interpretation should apply regardless of the breadth of the range or the nature of the recited feature.

[0044] "Standardization," as referred to in this specification, 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 calculates the range per 1 cm 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 amounts eluted from 1 cm of a sterilized human placental allograft. The quantification process used for the standardization of the disclosed sterilized human placental allografts 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 Applicant calculates the range per 1 cm 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 amounts eluted from 1 cm of a sterilized human placental allograft. 2 The quantification process used for the standardization of the disclosed sterilized human placental allografts 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. Analyzed factor (pg / ml) · ml of buffer / cm of sample 2 = pg of factor per 1 cm 2 This is accomplished by taking a sample (a biopsy punch is preferred) with a known cm. The said 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 amount of buffer collected. Divide this number by the cm of the sample / punch. This can be utilized for any volume of eluent used and any sample size. The tests presented in this invention utilized biopsy punches of 10 mm (78.54 cm This is accomplished by taking a sample (a biopsy punch is preferred) with a known cm. 2 The quantification process used for the standardization of the disclosed sterilized human placental allografts 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 This is accomplished by taking a sample (a biopsy punch is preferred) with a known cm. The said 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 amount of buffer collected. Divide this number by the cm of the sample / punch. This can be utilized for any volume of eluent used and any sample size. 2 ) placed in buffer (1×PBS) at 37°C for 72 hours, mimicking the application of a sterilized human placental allograft within an internal or external wound space. The eluate was collected and analyzed, mimicking what elutes from the sterilized human placental allograft into the wound. This consequently results in a controlled and quantifiable amount of the tested sterilized human placental allograft and a controlled, quantifiable, and associable amount of growth factors and stromal components.

[0045] The terms "human placental tissue" and "placental tissue" are used interchangeably herein to refer to human placental tissue. Unless otherwise explicitly stated herein, "human placental tissue" and "placental tissue" refer to either a completely 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., including only human amnion, human chorion, and the 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 specific explicitly 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 explicitly stated, "sterile human placental allograft" refers to an allograft having human placental tissue that is intact in cross-section (i.e., including only human amnion, human chorion, and the 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 explicitly stated examples herein, "sterile human placental allograft" refers to an allograft that consists essentially of or consists only of human amnion separated from all other placental tissues.

[0046] Disclosed herein are safe, biocompatible, and growth factor-rich sterile human placental allografts and methods 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)

[0047] As suggested above, it is an object to prepare sterilized human placenta allografts that maintain a growth factor profile that mimics the human placenta in vivo. The methods disclosed herein utilize mild conditions to ensure that various endogenous growth factors in human placental 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 placental 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 disclosed in further detail below, the disclosed methods use various mild purification techniques, preservation techniques, and mild sterilization techniques that employ cold physiological buffers, 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 that mimics 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. 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).

[0048] In view of FIG. 1, and prior to subjecting human placental tissue to step (a) of FIG. 1, all human placental tissue undergoes a donor pre-screening and screening process. A brief description of FIG. 1 is as follows. Step (a) of FIG. 1: Disinfection with minimal damage imparted. Immerse the membrane in an effective bactericidal, anti-tuberculosis, anti-fungal, and anti-viral solution to eliminate pathogens. Step (ai) of FIG. 1: Manual membrane separation. Separate the membrane into any desired component. Step (b) of FIG. 1: Manual hematopoietic system reduction. Manually remove blood and hematopoietic components without scraping or scrubbing to preserve the membrane structure. Step (c) of FIG. 1: Cold isotonic purification. Remove the remaining debris while minimizing the destruction of tissues and growth factors by a series of washings in a clean, low-temperature, pH-adjusted solution with gentle stirring. Step (d) of FIG. 1: Gentle gradual dehydration. Preserve the tissue structure and natural growth factors and prevent damage from freezing or high temperatures by slow drying of the membrane using physiological temperature. Step (e) of FIG. 1: Precise placement of cutting dies. Manually and precisely placing the cutting dies helps ensure optimal tissue quality for each individual allograft. Step (f) of FIG. 1: Final sterilization by low-dose electron beam. Individually package the grafts and perform final sterilization via an electron beam to avoid harmful by-products or excessive irradiation associated with other methods. 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 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 against hepatitis C virus · Hepatitis B nucleic acid test · Hepatitis C nucleic acid test · Human immunodeficiency virus (HIV): · Antibodies against HIV-1 · Antibodies against HIV-2 · HIV nucleic acid test · Syphilis - Rapid Plasma Reagin · Leukemia / Lymphoma · Antibodies against Human T-lymphotropic virus type 1 · Antibodies against Human T-lymphotropic virus type 2 · West Nile virus In addition to the above serological tests, and before providing human placental tissue in step (a) of FIG. 1, bio-burden samples are collected to evaluate the number of viable microorganisms on or within the human placental tissue. Additionally, environmental monitoring samples are collected extensively to evaluate the surface and air quality. By the above collection, it is ensured that subsequent human placental tissue and processing comply with consistent microbiological control.

[0049] FIG. 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 to 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 form a dehydrated human placental tissue, (e) sizing the dehydrated human placental tissue into dehydrated human placental tissue parts having a predetermined size, and (f) sterilizing the dehydrated human placental tissue part in step (e) to form the sterilized human placental allograft.

[0050] 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, tuberculocidal, 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 with a concentration of 70% or more and 100% or less. In a preferred embodiment, the alcohol concentration ranges from 70% to 75%, and 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 process, and / or necrosis process) 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, all pathogenic microorganisms, fungi, and / or viruses if present) on and / or within the human placental tissue. Step (a) uses bactericidal, tuberculocidal, 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.

[0051] 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 hematopoiesis reduction step that uses forceps and / or tweezers, soft moist 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 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 resulting sterilized human allograft when implanted / transplanted into the human subject in need and / or, if present, the probability of an immunogenic response.

[0052] 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 with an isotonic solution at a temperature in the range of 4° C. to 20° C. of 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, and may be repeated 1, 2, 3, or 4 times. Step (c) preferably occurs at low temperature using a pH-adjusted isotonic solution, which more advantageously reduces and / or eliminates the degradation of 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 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 strongly stimulating organic acids, strongly stimulating inorganic acids, strongly stimulating bases, or strongly stimulating peroxides can result in the decomposition of endogenous growth factors as well as the decomposition of human placental tissue in a harmful manner and should be avoided for these reasons.

[0053] After step (c) and as further shown in FIG. 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 1 / 2 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 FIG. 1.

[0054] 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 a 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 a dehydrated human placental tissue portion and into a desired shape, thereby ensuring the optimal tissue size, shape, and quality for each of the resulting sterile human placental allografts 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 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 a plane or a lower surface of a plane, or the human placental tissue portion includes both an upper surface of a plane and a lower surface of a plane.

[0055] 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 sterile human placental allograft. In certain embodiments, the sterilization step (f) subjects the dehydrated human placental tissue portion to 10 as calculated by the dose distribution by an electron beam maker. -6sterilizing by electron beam irradiation to an aseptic assurance level (SAL) of, thereby forming a sterilized human placental allograft, in which the SAL expressed as 10 -N The SAL expressed as is the expected probability of remaining organisms after treatment (for example, the expected probability of any remaining microorganisms after sterilization is 10 -6 or less). 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 into either a primary package and / or a secondary package, 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 end use (after removal from the primary or secondary package). 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 into a sterile package while maintaining the sterility of the sterilized human placental allograft for subsequent end use.

[0056] 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 (such as ethylene oxide) on the resulting allograft, which can result in an immunogenic response upon use of the resulting allograft, or may require exposure of the tissue to higher doses of irradiation (such as gamma irradiation) over an extended period, which causes an undue reduction in the structural integrity of the placental tissue and degradation of endogenous growth factors.

[0057] 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 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 them, 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 them. 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.

[0058] Upon completion of 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 thorough quality assurance review and compliance of the resulting sterilized human allografts using applicable government regulations, compliance, and procedures. (Sterilized human placental allograft)

[0059] 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 significantly 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 surface of a subject in need thereof.

[0060] 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. Also, Figure 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 consequently 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 embryonic vascular system development, 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 fibroblasts (in vitro). PDGF-BB is a potent promoter of cell proliferation and plays a crucial role in angiogenesis (blood vessel 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 structurally suitable scaffold 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, maintenance of moisture, and regulation of protein distribution. Collagen is the most abundant protein in the body and 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 placental tissues in those tissues. As a result, signals for development and regeneration are delivered to the cells of damaged tissues. Exosomes are rich in small RNA species including miRNA and have been demonstrated to be functional within recipient cells [Montecalvo 2008]. Exosomes are gaining momentum as a safe and effective therapy for skin wounds / injuries [Subhan 2021].

[0061] In certain embodiments, as further shown in FIGS. 3A-3F, FIGS. 5A and 5B, and FIG. 10A, the sterilized human placental allograft is intact in cross-section, and in that 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. 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 in the range of 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 in the range of 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 in the range of 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 in the range of 1.5×10 7 pg / cm 2 or more and 1.0×10 8 pg / cm 2 or less; and (e) hyaluronic acid (HA) in the range of 150 pg / cm 2 or more and 400 pg / cm 2 or less, more preferably in the range of 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 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. Further to be recognized, these allografts are configured for specific dental, wound healing, and cosmetic purposes, and preferably are implantable / transplantable into a subject in need or can be applied to shallow wounds (e.g., diabetic ulcers). In this regard, these sterilized human placental allografts have a predetermined shape and size, and in said shape and said 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 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 in diameter or more and 50 mm in 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 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 0.08 mm or more and 0.2 mm or less, depending further on their specific use, and can further have a translucent appearance, for example, as shown in FIG. 10A.

[0062] In certain embodiments, as further shown in FIGS. 2A-2F, FIGS. 4A and 4B, and FIG. 11A, 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 1.1×106 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 endpoint within any of the above ranges can serve as an endpoint for any additional range that falls within it. The concentrations mentioned above were obtained by the "standardization" calculations and methods disclosed herein. Further to be recognized, 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, 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 an upper surface of a plane or a lower surface of a plane depending on their specific use, or these sterilized human placental allografts 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. (Kit and method of use)

[0063] Also disclosed herein is a kit comprising a sterilized human placental allograft packaged within a sterile container. Upon opening the sterile container, the sterilized human placental allograft packaged therein is in a ready-to-use situation for the desired end use. The sterilized human allograft is configured for use for specific, dental, wound healing, and / or cosmetic purposes, and preferably is implantable / transplantable into a subject in need thereof, or can be applied to a shallow wound (e.g., diabetic ulcer). As suggested above, the sterilized human placental allograft disclosed herein is preferably non-immunogenic - there is little or no resulting immune response after implantation / transplantation into a human recipient and / or after topical application to a human recipient.

[0064] 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 over a predetermined time period to facilitate wound healing in the human subject in need thereof. The predetermined time period includes from 3 days to 30 days for implant applications and from 1 day to 5 days for topical applications. 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. 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 (e.g., 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 over the wound of the subject in need thereof over a predetermined time period, thereby facilitating wound healing by elution / diffusion of growth factors from the sterilized human placental allograft onto and into the wound.

[0065] 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 contains 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 or more and 40°C or less 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.

[0066] 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, wherein the human placental tissue is disinfected 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; (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. It includes.

[0067] In one aspect, the present invention relates to a sterilized human placental allograft for use in treating wounds (preferably according to the present invention).

[0068] In one aspect, the present invention relates to a sterilized human placental allograft for use in treating wounds 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.

[0069] A sterilized human placental allograft for use in implanting in a subject in need thereof. A sterilized human placental allograft for use in cases 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 cases where the sterilized human placental allograft is locally applied to the wound of a human subject in need thereof. A sterilized human placental allograft for use in cases where the wound is a diabetic ulcer. A sterilized human placental allograft for use in cases where the wound is a diabetic foot ulcer.

Example

[0070] 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 parts by weight, temperatures are in °C, or are ambient temperature if not listed, and pressures are at or near atmospheric pressure. Numerous variations and combinations of conditions exist, 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.

[0071] 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 to 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 when compared to various commercially available sterilized human allografts, but also visual differences among the multiple exemplary sterilized human allografts disclosed herein.

[0072] Figures 8A, 8B, and 8C are graphs depicting the PDGF-BB concentration, HGF concentration, and HA concentration in 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 with 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).

[0073] 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 on 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 on 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.

[0074] 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.

[0075] 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 commercially available products. Figures 10A-10C further support these results, in which Figure 10A, although translucent, is 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 commercially available products.

[0076] Figure 9 is a graph depicting the IL-1ra concentration within 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 Aug. 1, 2022)), and the amnion is prepared by the Purion® process (https: / / www.mimedx.com / purion-process / (accessed Aug. 1, 2022), and further reported by Koob et al 2013, 2014).

[0077] As shown in Figure 9, the sterilized human placental allograft of Example 2 contains on average 220.88% more IL-1ra than Comparative Example 3, which indicates that various endogenous growth factors are retained and preserved within the human sterilized placental allograft of Example 2 (i.e., the sterilized human placental allograft disclosed herein that is prepared by the methods / processes disclosed herein and consists essentially of human amnion) when compared to a similar commercial product of Comparative Example 3. Figures 11A and 11B further support these results, where Figure 11A is opaque even though translucent, as compared to the highly processed translucent visual appearance of Comparative Example 3 (Figure 11B), which further indicates that various endogenous growth factors are retained and preserved within the human sterilized placental allograft of Example 2 when compared to a similar commercial product.

[0078] The foregoing description provides only examples of embodiments of this invention. It is contemplated that other embodiments may perform similar functions and / or achieve similar results. All such equivalent embodiments and examples are intended to be within the scope of this invention and are intended to be covered by the appended claims.

Claims

1. 1. A method for preparing a sterile human placental allograft, comprising: (a) providing human placental tissue from a donor not more than 24 hours but not more than 72 hours postpartum, and further comprising, either before or during step (a), disinfecting the human placental tissue with at least one of a bactericidal composition, a tuberculocidal composition, a fungicidal composition, a virucidal composition, or any combination thereof, wherein at least one of the bactericidal composition, the tuberculocidal composition, the fungicidal composition, the virucidal composition, or any combination thereof comprises isopropyl alcohol at a concentration of not less than 70% and not more than 100%; (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 a predetermined number of times while maintaining the structural integrity of the human placental tissue, each washing step (c) being at a temperature in the range of not less than 4°C and not more than 15°C for a time period of not less than 5 minutes and not more than 15 minutes; (d) dehydrating the human placental tissue, thereby forming dehydrated human placental tissue, said dehydrating step (d) comprising dehydrating the human placental tissue at a temperature ranging from 20° C. to 40° C. for a period of 60 minutes to 4.5 hours; (e) sizing the dehydrated human placental tissue into dehydrated human placental tissue portions having a predetermined size; (f) sterilizing the dehydrated human placental tissue portion of step (e), thereby forming the sterile human placental allograft, wherein said sterilizing step (f) comprises sterilizing by electron beam irradiation; A method comprising:

2. 10. The method of claim 1, wherein step (b) comprises manual hematopoietic reduction by manually removing any visible blood, blood clots, and / or blood components from the human placental tissue.

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

4. the predetermined size of the dehydrated human placental tissue portion is between 1 cm x 1 cm and 8 cm x 8 cm for rectangular and / or square shaped dehydrated human placental tissue portions, and between 8 mm and 50 mm in diameter for circular shaped dehydrated human placental tissue portions; and / or the dehydrated human placental tissue portion has a predetermined shape; The method of claim 1.

5. The sterilizing step (f) is performed at a dose distribution of 10 to 150 μg / cm² for the dehydrated human placental tissue portion. －６ 2. The method of claim 1, comprising: subjecting the sterile human placenta allograft to electron beam irradiation to a sterility assurance level (SAL) of 0.1% to form the sterile human placenta allograft.

6. The method of claim 1 , wherein the human placental tissue comprises intact human placental tissue.

7. the dehydrated human placental tissue of step (d) and the dehydrated human placental tissue portion having a predetermined size of step (e) are intact in cross-section, and in the cross-section, a human amnion layer and a human chorion layer have an intact human intermediate sponge layer positioned between the human amnion layer and the human chorion layer, connecting the human amnion layer to the human chorion layer; and / or the sterile human placental allograft of step (f) is intact in cross-section, and in said cross-section, the human amnion layer and the human chorion layer have an intact human intermediate sponge layer positioned between and connecting the human amnion layer to the human chorion layer; and / or 10. The method of claim 1, wherein the sterile human placental allograft is configured for dental, cosmetic, and / or wound healing applications.

8. A sterile human placenta allograft produced by the method of any one of claims 1 to 7.

9. 9. The sterile human placental allograft of claim 8, The sterile human placental allograft comprises: (a) 200 pg / cm ２ More than 7800 pg / cm ２ an interleukin-1 receptor antagonist (IL-1ra) in the range: (b) 500 pg / cm ２ More than 8500 pg / cm ２ Hepatocyte growth factor (HGF) in the following ranges: (c) 800 pg / cm ２ More than 2750 pg / cm ２ Vascular endothelial growth factor receptor 1 (VEGFR1) in the range: (d) 5.0 x 10 ６ 1.5x10 or more ８ pg / cm ２ Hyaluronic acid (HA) in the following ranges: (e) 150 pg / cm ２ More than 400 pg / cm ２ Platelet-derived growth factor B subunit homodimer (PDGF-BB) in the following ranges: (f) glycosaminoglycans (GAGs); (g) collagen, and (h) exosomes; A sterile human placenta allograft comprising at least two of the following:

10. the sterile human placental allograft is intact in cross-section, wherein in the cross-section, a human amnion layer and a human chorion layer have an intact human intermediate sponge layer positioned between and connecting the human amnion layer to the human chorion layer; and / or the sterile human placenta allograft is configured for dental, cosmetic, and / or wound healing applications; and / or the sterile human placental allograft has a predetermined size of at least 1 cm x 1 cm and at most 8 cm x 8 cm for rectangular and / or square shaped sterile human placental allografts, and at least 8 mm diameter and at most 50 mm diameter for circular shaped sterile human placental allografts; and / or the sterile human placental allograft has a circular shape, a square shape, a rectangular shape, an oval shape, a triangular shape, or any combination thereof; 9. The sterile human placental allograft of claim 8.

11. the upper and / or lower surfaces of the sterile human placental allograft are flat; and / or the sterile human placental allograft consists essentially of amniotic membrane; 9. The sterile human placental allograft of claim 8.

12. The sterile human placental allograft comprises: (a) 150 pg / cm ２ More than 1800 pg / cm ２ an interleukin-1 receptor antagonist (IL-1ra) in the range: (b) 75 pg / cm ２ More than 400 pg / cm ２ Hepatocyte growth factor (HGF) in the following ranges: (d) 1.1 x 10 ６ Above 2.0 x 10 ７ pg / cm ２ Hyaluronic acid (HA) in the following ranges: (e) 200 pg / cm ２ More than 500 pg / cm ２ Platelet-derived growth factor subunit B homodimer (PDGF-BB) in the range: (f) glycosaminoglycans (GAGs); and (g) collagen; (h) exosomes; 11. The sterile human placental allograft of claim 10, comprising at least two of:

13. 10. A kit comprising the sterile human placental allograft of claim 8 packaged in a sterile container.

14. 1. A sterile human placenta allograft for use in a method of treating a wound in a human subject, comprising: contacting the wound with the sterile human placental allograft for a predetermined period of time, or implanting the sterile human placental allograft into the human subject in need to facilitate wound healing in the human subject in need; and / or the wound is an internal wound and / or a dental wound in the human subject; and / or 10. The sterile human placental allograft of claim 8, wherein the sterile human placental allograft is applied topically to the wound in the human subject in need thereof.

15. 15. A sterile human placenta allograft for use in the method of claim 14, wherein the wound is a diabetic ulcer or a diabetic foot ulcer.

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