Lyophilized perforated placental membrane tissue grafts

Abstract

A dehydrated, partially perforated placental tissue allograft that can be used to treat wounds. Specifically, the dehydrated, partially perforated placental tissue allograft has the property of being able to be washed during processing without destroying the structural integrity of the graft or reducing the concentration of growth factors found within the completed graft. The dehydrated, partially perforated placental tissue allograft is reconstituted prior to its application to a subject and is substantially free of undesirable maternal blood contaminants.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 362,540, filed April 6, 2022, all teachings of which are incorporated by reference herein in their entirety.

[0002] FIELD OF THEINVENTION The present invention relates to freeze-dried perforated placental membrane tissue grafts for use in the treatment of a variety of chronic wounds. [Background technology]

[0003] Human placental membranes (e.g., amniotic membrane or tissue) have been used in various types of reconstructive surgery since the early 1900s. The membranes function as a matrix material, more commonly referred to as biological dressings or patch grafts. Such membranes have also been used extensively in ophthalmic procedures in the United States and Southern Hemisphere countries. Typically, such membranes are frozen or dried for storage and preservation until needed for surgery.

[0004] Such placental tissue is typically harvested after elective Caesarean section surgery or natural birth. The placenta has three major tissue layers, including the amnion, the intermediate layer (or sponge layer), and the chorion. The amnion is a non-vascular tissue that is the innermost layer of the placenta and consists of a single layer attached to a basement membrane. Histological evaluation shows that the membranous layer of the amnion consists of epithelial cells, a thin reticular fiber (basement membrane), a thick stratum densa, and a fibroblast layer. The fibrous layer of the amnion (i.e., basement membrane) contains cell-anchored collagen types IV, V, and VII. The chorion is also considered part of the fetal membrane. However, the amnion and chorion layers are separate separable entities. The chorion includes a reticular layer, a basement membrane, and a trophoblast layer. The intermediate layer is found between the amnion and chorion, adjacent to the fibroblast layer of the amnion and the reticular layer of the chorion, loosely connecting the two.

[0005] Amniotic membrane offers unique graft properties when used in surgical procedures including providing a matrix for cell migration / growth, providing a natural biological barrier, being non-immunogenic, promoting increased self-healing, being easy to fix in place using different techniques including fibrin glue or suturing, and such grafts, when properly prepared, can be stored at room temperature for extended periods of time without the need for refrigeration or freezing until needed for a surgical procedure.

[0006] Known clinical treatments or uses for such placental membrane grafts include Schneiderian membrane repair (i.e., sinus lift), guided tissue regeneration (GTR), general wound care, and primary closure membranes. Known clinical treatments or uses for such placental membrane grafts include biological wound dressings.

[0007] A detailed review of the history and procedures for harvesting "living" amniotic tissue for use in surgical procedures, as well as methods for harvesting and freezing amniotic tissue grafts for ophthalmic procedures, is provided in U.S. Pat. No. 6,152,142 issued to Tseng, which is incorporated herein by reference in its entirety.

[0008] However, placental membranes may present challenges during processing, such as removing residual maternal blood from the middle layer of the placental membrane, which may result in immune reactions in patients. In addition, the presence of blood spots and blood clots may result in a final product that is not aesthetically pleasing to end users, such as patients or medical professionals. Therefore, manufacturers have developed various techniques to cleanse the tissue to reduce the chance of immune reactions and create more aesthetically pleasing products that appeal to patients who receive placental tissue grafts as well as medical professionals who apply them in the field. Summary of the Invention

[0009] An embodiment of the present invention is a placental tissue graft comprising an amniotic membrane layer, an intermediate membrane layer, and a chorionic membrane layer, the intermediate layer is fully or partially perforated; The amniotic membrane is a non-perforated placental tissue graft.

[0010] An embodiment of the present invention is a placental tissue graft comprising an amniotic membrane layer, an intermediate membrane layer, and a chorionic membrane layer, the intermediate layer includes a first region and a second region, the first region adjacent to the amnion and the second region adjacent to the chorion; The second area is completely perforated, The first region is non-perforated, the placental tissue graft.

[0011] An embodiment of the invention is a method of preparing a placental tissue graft comprising an amniotic membrane, an intermediate membrane, and a chorion, the method comprising: a. separating the amniotic layer from the chorionic layer to obtain an amniotic layer with some intermediate layer attached and a chorionic layer with some intermediate layer attached; b. perforating the intermediate layer attached to the chorion layer; c. washing the amniotic and chorionic membrane layers; d. contacting the intermediate layer attached to the amniotic membrane layer with the intermediate layer attached to the chorionic membrane layer, This method results in the production of a placental tissue graft comprising the amniotic membrane, the intermediate membrane, and the chorion.

[0012] In certain embodiments, the subject matter described herein is directed to dehydrated, selectively perforated placental membrane tissue grafts, particularly human allografts, that are treated to preserve the spongy interlayer while also removing undesirable blood residues and blood clots. In addition, the perforated placental membrane tissue graft is dehydrated to increase its shelf life and durability.

[0013] After harvesting, the placental membrane tissue is processed in several steps to obtain the products described herein. For example, the amniotic and chorionic layers are separated from other placental tissues, such as umbilical cord and discoid placental tissue (other tissues may be retained for other purposes). All components are sourced from a single donor. Once the amniotic and chorionic membranes are separated from other tissue components, the amniotic layer is physically separated from the chorionic layer to obtain isolated amniotic and chorionic layers. The separated layers are then washed with sterile water.

[0014] After rinsing the amniotic and chorionic layers with sterile water, they are placed on a flat surface. The chorionic layer is placed with the spongy middle layer facing up. Blood, clots and other debris should be removed from the amniotic and chorionic layers by using gauze, forceps and / or gloved hands. Once the tissue layers are properly positioned, the middle layer attached to the chorionic layer is perforated, full or partial thickness, using a specialized rotating pinwheel tool.

[0015] The amniotic tissue is gently cleaned and minimally manipulated to preserve the inherent growth factors and proteins within the tissue. Notable growth factors in amniotic tissue include transforming growth factor beta (TGF-β), basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF AA and BB), and vascular endothelial growth factor (VEGF)14,15, which are known to regulate wound healing.

[0016] Once the cleaning and perforation steps are complete, the amniotic and chorionic layers, with the intermediate layer still attached, must be further rinsed in sterile water. After the sterile water rinse is complete, the amniotic and chorionic layers are subjected to a decontamination step in which they are rinsed in a solution containing one or more broad-spectrum antibiotics, such as streptomycin sulfate, gentamicin sulfate, polymyxin B sulfate, and / or bacitracin.

[0017] After the rinsing and decontamination steps have been performed, the selected layers of placental tissue are reassembled and then subjected to a drying process, which may involve any type of commercially acceptable process known in the art, including, but not limited to, air drying, chemical drying, or freeze drying of the amniotic and chorionic layers.

[0018] In one embodiment, the selected tissue layer is dried on a fixture, a surface of the drying fixture having a plurality of grooves defining an outer contour of each of the plurality of placental membrane tissue grafts, and the cutting step includes cutting the selected layer along the grooves.

[0019] The finished product is packaged in a sterile container and reconstituted with an excipient acceptable to the end user before the implant is applied to the subject's wound site. Alternatively, the implant may be applied directly to the wound site and reconstituted with a combination of excipients and the patient's own bodily fluids that may be present at the wound site. [Brief description of the drawings]

[0020] Further features and advantages of the present invention will become apparent from the detailed description of the preferred embodiments thereof, taken in conjunction with the following drawings, in which like elements are referred to with like reference numerals and in which: [Figure 1] 1 is a photograph of an exemplary specialized rotating pinwheel micro-needling tool. [Diagram 2] FIG. 1 is a schematic diagram of an implant according to the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] One challenge encountered in the field is that placental tissue grafts can retain blood, blood clots, and other undesirable contaminants that can adversely affect the appearance of the finished product and may also trigger an undesirable immune response in the patient to whom the graft is applied. Manufacturers have developed several methods to help remove these blood contaminants during processing.

[0022] Therefore, there is a need in the market for an efficient method of removing as much blood, blood clots, and other undesirable contaminants as possible, especially from the intermediate layer, without compromising the structural integrity of the tissue itself or the concentration of growth factors remaining in the tissue graft after the tissue has been subjected to the manufacturing process.

[0023] It is to be understood that the invention is not limited to the particular embodiments described, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the invention will be limited only by the appended claims.

[0024] The detailed description of the present invention is divided into various sections for the convenience of the reader only, and the disclosures found in any section may be combined with those of another section. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the manufacture, practice, or testing of the present invention, the preferred methods and materials are described herein. All patents and publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials related to the cited publications.

[0025] Each embodiment disclosed herein is contemplated to be applicable to each of the other disclosed embodiments. All combinations and subcombinations of the various elements described herein are within the scope of the embodiments.

[0026] When a parameter range is provided, it is understood that all integers and ranges within that range, as well as tenths and hundredths thereof, are also provided by the embodiment. For example, "5-10%" includes 5%, 6%, 7%, 8%, 9%, and 10%, 5.0%, 5.1%, 5.2%...9.8%, 9.9%, and 10.0%, and 5.00%, 5.01%, 5.02%...9.98%, 9.99%, and 10.00%, as well as 6-9%, 5.1%-9.9%, and 5.01%-9.99%. This is also true for ratios. For example, a recited ratio range of "1:100 to 200:1" includes ratios such as 1:50, 1:1, and 100:1, as well as ranges such as 1:100 to 1:1, 1:50 to 50:1, and 1:1 to 200:1.

[0027] As used herein, "about" in the context of a numerical value or range means within ±1%, ±5%, or ±10% of the recited or claimed numerical value or range.

[0028] The invention illustratively disclosed herein may suitably be practiced in the absence of any element not specifically disclosed herein.

[0029] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0030] definition As used herein, the following terms have the following meanings.

[0031] "Placental tissue" or "placenta" refers to the placental amnion, the placental intercalary membrane, and the placental chorion.

[0032] "Comprising" or "comprises" is intended to mean that the compositions (e.g., media) and methods include the recited elements, but do not exclude other elements. "Consisting essentially of," when used to define a method, is intended to mean excluding other elements that have any essential significance to the combination for the purpose described. "Consisting of" is intended to mean excluding additional substantial method steps. Embodiments defined by each of these transition terms are within the scope of the invention.

[0033] By "dehydrated" it is meant that tissue has had substantially all of its water removed (i.e., greater than 85%, greater than 90%, greater than 95%, greater than 99% or 100% of its water removed).

[0034] "Substantially uniform" with respect to the thickness of the intermediate layer means that the thickness is ±20%, ±15%, ±10%, ±5%, or ±1% throughout the implant.

[0035] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes cases where the event or circumstance occurs and cases where it does not occur.

[0036] As used herein, the term "subject" is any vertebrate organism, including, but not limited to, mammalian subjects such as humans, livestock, pets, etc. The term "patient" may be used interchangeably with "subject."

[0037] The term "treating" in reference to a wound means reducing the time it would take for a wound to heal in the absence of medical intervention of any kind.

[0038] A "fully perforated" tissue layer has at least one microneedle penetrating the entire thickness of the layer. A "partially perforated" tissue layer has at least one microneedle penetrating a portion of the total thickness of the layer. A fully or partially perforated tissue layer may or may not retain the perforations after further processing of the tissue layer.

[0039] As used herein, the term "microneedle" refers to any device that can fully or partially penetrate tissue without removing any surface area of ​​the tissue. Non-limiting examples of microneedles include needles and pins.

[0040] An embodiment of the present invention is a placental tissue graft comprising an amniotic membrane layer, an intermediate membrane layer, and a chorionic membrane layer, the intermediate layer is fully or partially perforated; The amniotic membrane is a non-perforated placental tissue graft.

[0041] In an embodiment, the intermediate layer is completely perforated. In an embodiment, the intermediate layer is partially perforated.

[0042] An embodiment of the present invention is a placental tissue graft comprising an amniotic membrane layer, an intermediate membrane layer, and a chorionic membrane layer, the intermediate layer includes a first region and a second region, the first region adjacent to the amnion and the second region adjacent to the chorion; The second area is completely perforated, The first region is non-perforated, the placental tissue graft.

[0043] In embodiments, the chorion is partially perforated. In embodiments, the chorion is partially non-perforated. In embodiments, the chorion is non-perforated.

[0044] In an embodiment, the intermediate layer is substantially uniform throughout the implant.

[0045] In embodiments, the placental tissue allograft is dehydrated. In embodiments, the placental tissue allograft is freeze-dried.

[0046] In embodiments, the placental tissue allograft is contained in a sealed pouch. In embodiments, the sealed pouch is deoxygenated.

[0047] An embodiment of the present invention is a method of preparing a placental tissue graft comprising an amniotic membrane, an intermediate membrane, and a chorion, the method comprising: a. separating the amniotic layer from the chorionic layer to obtain an amniotic layer with some intermediate layer attached and a chorionic layer with some intermediate layer attached; b. perforating the intermediate layer attached to the chorion layer; c. washing the amniotic and chorionic membrane layers; d. contacting the intermediate layer attached to the amniotic membrane layer with the intermediate layer attached to the chorionic membrane layer; This method results in the production of a placental tissue graft comprising the amniotic membrane, the intermediate membrane, and the chorion.

[0048] In embodiments, the method further comprises dehydrating the placental tissue graft after step d. In embodiments, the dehydration comprises freeze-drying.

[0049] In embodiments, the amniotic membrane is not perforated during the method.

[0050] In embodiments, the method further comprises manipulating the intermediate layer attached to the amniotic membrane and / or chorion to cause the intermediate layer to be more evenly distributed, in embodiments, causing the intermediate layer to be more evenly distributed resulting in a substantially evenly distributed intermediate layer throughout the graft.

[0051] In an embodiment, steps b and c are each performed two or more times.

[0052] In an embodiment, the method further comprises disrupting the epithelial layer of the amniotic membrane.In an embodiment, the method further comprises substantially removing the epithelial layer of the amniotic membrane.

[0053] An embodiment is an implant prepared by any of these methods.

[0054] Also an embodiment of the invention is a method of treating or covering a wound comprising contacting the wound with an implant as described herein.

[0055] Manufacturing method Initial tissue collection Collection of placental tissue begins in the hospital and is taken during a Caesarean section or natural birth. A donor refers to a mother about to give birth, who voluntarily undergoes a comprehensive screening process designed to provide the safest possible tissue for transplantation. This screening process preferably uses conventional serological tests to test for antibodies to human immunodeficiency virus types 1 and 2 (anti-HIV-1 and anti-HIV-2), hepatitis B surface antigen (HBsAg), hepatitis C virus (anti-HCV), human T-lymphotropic virus types I and II (anti-HTLV-I and anti-HTLV-II), CMV, and syphilis. The above list of tests is merely exemplary, and as will be appreciated by those skilled in the art, more, less, or different tests may be desired or required over time or based on the intended use of the graft.

[0056] Based on a review of the donor's information and screening test results, the donor is either deemed acceptable or not. Additionally, at the time of delivery, cultures are taken to determine the presence or absence of, for example, Clostridium or Streptococcus. If the donor's information, screening test, and delivery cultures are all negative (i.e., indicating no risk or an acceptable level of risk), the donor is approved and the tissue specimen is initially designated as eligible for further processing and evaluation.

[0057] Human placentas that meet the above selection criteria are preferably individually bagged in saline in sterile shipping bags and stored in a container of ice water for transport to a processing site or laboratory for further processing.

[0058] Material Check-in and Evaluation Upon arrival at the processing center or laboratory, the shipment is opened and verified to ensure the sterile shipping bag / container remains sealed and intact, that ice or other refrigerant is present, that the contents are chilled, that appropriate donor paperwork is present and that the donor number on that paperwork matches the number on the sterile shipping bag containing the tissue. The sterile shipping bag containing the tissue is then stored in a refrigerator until ready for further processing. All appropriate forms are completed, as are chain of custody and processing logs.

[0059] Whole tissue processing When the tissue is ready for further processing, the sterile supplies needed for further processing of the placental tissue are assembled in a staging area within the controlled environment and prepared for introduction to the critical environment. If the critical environment is a production hood, the sterile supplies are opened and placed in the hood using conventional sterilization techniques. If the critical environment is a clean room, the sterile supplies are opened and placed on a cart that is covered with a sterile drape. All work surfaces are draped with a piece of sterile drape using conventional sterilization techniques, and the sterile supplies and processing equipment are placed on this sterile drape, again using conventional sterilization techniques.

[0060] If placental tissue is collected prior to the completion or receipt of the results of screening tests and delivery cultures, such tissue will be labeled and stored in quarantine. The tissue will be approved for further processing only after the necessary screening evaluations and delivery cultures have been satisfied to declare the tissue safe for handling and use.

[0061] The processing equipment is decontaminated according to conventional industry accepted decontamination procedures and then introduced into the critical environment, where it is strategically located to minimize proximity to and the possibility of inadvertent contamination by tissue specimens.

[0062] The placenta is then removed from the sterile shipping bag and aseptically transferred to a sterile processing bath in a critical environment. The bath contains sterile water, preferably at or near room temperature. The placenta is gently massaged to aid in the separation of blood clots and to allow the temperature of the placental tissue to reach room temperature, which facilitates the separation of the amniotic and chorionic layers from each other, as discussed below. After warming to ambient temperature (approximately 10-30 minutes), the placenta is removed from the sterile processing bath and laid flat, with the amniotic layer facing down, on a processing tray for examination.

[0063] The placental tissue is inspected and the results of the inspection recorded on the "Raw Tissue Evaluation Form." The placental tissue is inspected for discoloration, debris or other contamination, odor, and signs of damage. The size of the tissue is also noted. At this point a determination is made as to whether the tissue is acceptable for further processing.

[0064] Next, if the placental tissue is deemed acceptable for further processing, the amniotic and chorionic layers of the placental tissue are carefully separated. Materials and equipment used in this procedure include a processing tray, sterile water, a sterile 4×4 inch sponge, and two sterile Nalgene jars. The placental tissue is then closely examined to locate an area (usually a corner) where the amniotic layer can be separated from the chorionic layer. The amniotic membrane appears as a thin, opaque layer on the chorion.

[0065] With the placenta tissue in the processing tray with the amniotic layer facing down, the chorion layer is gently lifted from the amniotic layer in a slow, continuous motion, taking care to avoid tearing the amniotic membrane. If tearing begins, it is recommended to restart the separation process from a different location to minimize tearing of either layer of tissue. The separation process continues manually without the use of a sponge, taking care not to tear either the amniotic or chorion layers. After separation, the majority of the middle layer is on the chorion, and the remaining part of the middle layer is on the amniotic membrane.

[0066] Care is then taken to remove blood clots and other extraneous tissue from each layer of tissue until the amniotic tissue and chorion are clean and ready for further processing. More specifically, the amniotic and chorionic tissues are placed on a processing tray and blood clots are carefully removed by gently scraping the blood using a blunt instrument, finger, or sterile non-particulate gauze until the blood clots are freed from the amniotic stromal tissue and chorionic trophoblastic tissue. The amniotic stromal layer is the side of the amniotic membrane that faces the mother. In contrast, the basement membrane layer is the side of the amniotic membrane that faces the fetus.

[0067] Any remaining debris or contaminants are also removed using a blunt instrument such as forceps or sterile gauze. Again, this step must be performed with great care to avoid tearing the amniotic or chorionic tissue and to avoid destroying or removing the middle layer from the amniotic or chorionic membrane. The amniotic membrane is cleaned when the amniotic tissue appears smooth and opaque white and the middle layer is clean. Over-cleaning of the chorionic membrane may remove the opaque layer. If an area of ​​chorionic membrane is cleaned too vigorously and appears clear, it is unacceptable and ultimately discarded.

[0068] Microneedling of the chorionic layer Once the amniotic and chorionic layers have been separated and washed, a microneedling tool is used to pierce the intermediate layer that is attached to the chorionic layer. In embodiments, the microneedling tool is applied at a 45 degree angle from the surface of the chorion about 10-15 times, about 2 cm apart. In embodiments, the chorionic layer is then rotated 90 degrees from its original orientation, and the tool is again applied about 2 cm apart, about 10-15 times. It is important that the microneedling tool is applied with enough pressure to penetrate the intermediate layer, but should not completely penetrate the entire chorionic layer. In some embodiments, the microneedling tool does not penetrate the basement membrane of the chorion.

[0069] Optional Epithelial Cell Removal Step In some embodiments, the epithelial layer present on the amniotic membrane is substantially removed to expose the basement membrane of the amniotic membrane. The term "substantially removed" with respect to the amount of epithelium removed is defined herein as removing more than 90%, more than 95%, or more than 99% of the epithelial cells from the amniotic membrane. In some embodiments, the majority (more than 50%) of the epithelial cells are removed. In some embodiments, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, or more than 85% of the epithelial cells are removed. The presence or absence of epithelial cells remaining on the amniotic membrane layer can be evaluated using techniques known in the art. For example, after removal of the epithelial cell layer, a representative tissue sample from the processing lot is placed on a standard microscopy slide. The tissue sample is then stained with Eosin Y stain and evaluated as described below. The sample is then covered and left to stand. After sufficient time has passed to stain, visual observation is performed under magnification.

[0070] The epithelial layer can be removed by techniques known in the art in some embodiments. For example, the epithelial layer can be scraped off the amniotic membrane using a cell scraper. Other techniques include, but are not limited to, freezing the membrane, physical removal using a cell scraper, or exposing the epithelial cells to non-ionic detergents, anionic detergents, and nucleases. The de-epithelialized tissue is then evaluated to determine that the basement membrane remains intact and intact. This step is performed after the completion of the processing steps and before the tissue is dehydrated. For example, a representative sample graft is removed for microscopic analysis. The tissue sample is placed on a standard slide, stained with Eosin Y, and observed under a microscope. If epithelium is present, it will appear as a cobblestone of cells. In embodiments in which the epithelial layer is removed, it is understood that the epithelial layer is removed in a manner that does not result in the removal of the middle layer.

[0071] Chemical Decontamination Process Each membrane is then placed into a sterile Nalgene bottle for the next step of chemical decontamination.

[0072] Each Nalgene bottle is then aseptically filled with sterile water and sealed (or capped), and the bottles are then placed on a rocker platform and agitated for 30-90 minutes to further cleanse the tissue of any contaminants.

[0073] If the rocker platform is not in a critical environment (e.g., a production hood), the Nalgene bottle is returned to the critical / sterile environment and unsealed. Using sterile forceps, the tissue is gently removed from the Nalgene bottle containing the sterile water and placed into an empty Nalgene bottle. This empty Nalgene bottle containing the tissue is then aseptically filled with a premixed antibiotic solution. Preferably, the premixed antibiotic solution is comprised of a cocktail of antibiotics such as streptomycin sulfate and gentamicin sulfate. Other antibiotics such as polymyxin B sulfate and bacitracin or similar antibiotics currently available or available in the future are also suitable. Additionally, the antibiotic solution is preferably at room temperature when added so as not to change the temperature of the tissue or damage it. This bottle or container containing the tissue and antibiotic is then sealed or closed, placed on a rocker platform, and agitated, preferably for 60-90 minutes. Such rocking or agitation of the tissue in the antibiotic solution further washes contaminants and bacteria from the tissue.

[0074] Again, if the rocker platform is not within the critical environment (e.g., production hood), the jar or container with the tissue and antibiotic is returned to the critical / sterile environment and unsealed. Using sterile forceps, gently remove the tissue from the jar or container and place into a sterile bowl containing sterile water or normal saline (0.9% saline). Allow the tissue to soak in the sterile water / normal saline for at least 10-15 minutes. The tissue may be slightly agitated to facilitate removal of the antibiotic solution and any other contaminants from the tissue. After at least 10-15 minutes, the tissue is dehydrated and ready to be further processed. Alternatively, the microneedling and / or chemical decontamination steps may be repeated at this point to remove further contaminants.

[0075] Optional destruction of the epithelial layer In embodiments where an epithelial layer is present, the epithelial layer of the amniotic membrane may be disrupted, in embodiments this involves gently scraping the epithelial layer with a sterile device such as a ruler.

[0076] Recombination process In embodiments, the amniotic membrane and chorion are reattached prior to dehydration. The stromal side of the amniotic membrane (with some intermediate layer attached) is placed against the plexiform side of the chorion (with some intermediate layer attached). In embodiments, the intermediate layer on one or both of the amniotic membrane and chorion is manipulated to make it more uniform and / or to move the intermediate layer to portions of the graft that have little or no intermediate layer. This manipulation may be performed with any suitable sterile tool, including but not limited to a ruler. In embodiments, this manipulation is performed prior to reattachment of the amniotic membrane and chorion. In embodiments, this manipulation is performed after reattachment of the amniotic membrane and chorion.

[0077] The reattached graft is shown in schematic form (not to scale) in FIG. 2. The middle layer is found between the amnion and the chorion. There are two regions in the middle layer, a first region adjacent to the amnion (the "amniotic region") and a second region adjacent to the chorion (the "chorionic region"). The first region is not perforated during processing and formation of the graft. The second region is completely perforated during processing and formation of the graft.

[0078] Dehydration process Preferably, the placental tissue is placed into individual sealed Tyvek pouches (or other commercially available pouches) and placed into a commercially available freeze-drying chamber. Any freeze-drying process known to those of skill in the art can be used, so long as the placental tissue is substantially dehydrated upon completion of the freeze-drying process.

[0079] Other methods may be used to sufficiently dehydrate the placental tissue. Such techniques may include, but are not limited to, chemical dehydration, or placing the placental tissue in a low humidity / high temperature environment for a sufficient period of time until optimal dehydration of the placental tissue is achieved. Such dehydration techniques are generally well known to those of skill in the art.

[0080] sterile The resulting inner and outer pouches along with the dehydrated placental tissue graft are subjected to a terminal sterilization step, which is accomplished by exposing the dehydrated placental tissue graft to high-energy penetrating ionizing radiation, such as electron beam or gamma irradiation, while the product is in its final packaging unit.

[0081] 3. Reconstitution of Dehydrated, Perforated Placental Tissue Grafts To administer the placental tissue graft to a subject, the end user can first reconstitute the graft by rehydrating the graft. Optimally, the rehydration agent is 0.9% saline, although any suitable excipient may be used.

[0082] Administration of perforated placental tissue grafts Once the placental tissue graft has been reconstituted with the desired rehydration agent, it is then applied to the wound site. The graft may also be hydrated at the wound site using rehydration agent or blood present from the wound bed preparation. The reconstituted placental tissue graft may also have a pattern applied to it that indicates whether the amniotic or chorionic layer is facing the wound site.

[0083] Efforts have been made to ensure accuracy with respect to numbers used (eg amounts, temperature, etc.) but some experimental errors and deviations should be accounted for.

[0084] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this subject matter belongs and are consistent with Singleton et al. (1994) Dictionary of Microbiology and Molecular Biology, 2nd Ed., J. Wiley & Sons, New York, NY, and Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immunobiology, 5th Ed., Garland Publishing, New York.

[0085] Many variations and other embodiments described herein will come to mind to one skilled in the art to which this subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. It is to be understood, therefore, that the subject matter is not to be limited to the specific embodiments disclosed, and that variations and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein that could be used in implementing the subject matter described herein. The present disclosure is in no way limited to only the methods and materials described.

Claims

1. A placental tissue graft comprising the amnional layer, the mesolayer, and the chorionic layer, The aforementioned intermediate layer is completely or partially perforated. The amniotic membrane is not perforated, and the placental tissue graft is intact.

2. The placental tissue graft according to claim 1, wherein the intermediate layer is completely perforated.

3. The placental tissue graft according to claim 1, wherein the intermediate layer is partially perforated.

4. A placental tissue graft comprising the amnional layer, the mesolayer, and the chorionic layer, The intermediate layer comprises a first region and a second region, the first region being adjacent to the amnion and the second region being adjacent to the chorionic membrane. The aforementioned second region is completely perforated, The first region is not perforated. The amniotic membrane is not perforated, and the placental tissue graft is intact.

5. The placental tissue graft according to any one of claims 1 to 4, wherein the chorionic membrane is partially perforated.

6. The placental tissue graft according to any one of claims 1 to 4, wherein the chorionic membrane is not partially perforated.

7. The placental tissue graft according to claim 1, wherein the intermediate layer is substantially uniform throughout the entire graft.

8. The placental tissue graft according to claim 1 or 4, wherein the placental tissue graft is dehydrated.

9. The placental tissue graft according to claim 1 or 4, wherein the placental tissue graft is rehydrated.

10. The placental tissue graft according to claim 1 or 4, wherein the graft is contained within a sealed pouch.

11. The placental tissue graft according to claim 10, wherein the sealed pouch is deoxygenated.

12. A method for preparing a placental tissue graft comprising the amnion, mesolayer, and chorion, wherein the method is: a. A step of separating the amnional layer from the chorionic layer to obtain an amnional layer with some intermediate layers attached and a chorionic layer with some intermediate layers attached, b. A step of perforating the intermediate layer attached to the chorionic membrane layer, c. A step of washing the amniotic layer and the chorionic layer, d. The step of bringing the intermediate layer attached to the amniotic layer into contact with the intermediate layer attached to the chorionic layer, A method for producing a placental tissue graft comprising the amnion, mesolayer, and chorion.

13. The method according to claim 12, further comprising dehydrating the placental tissue graft after step d.

14. The method according to claim 13, wherein the dehydration includes freeze-drying.

15. The method according to any one of claims 12 to 14, wherein the amniotic membrane is not perforated during the method.

16. The method according to claim 12, further comprising the step of manipulating the intermediate layer attached to the amnion and / or chorion to distribute the intermediate layer more uniformly.

17. The method according to claim 12, wherein steps b and c are each performed two or more times.

18. The method according to claim 12, further comprising destroying the epithelial layer of the amnion.

19. The method according to claim 12, further comprising substantially removing the epithelial layer of the amnion.

20. A graft prepared by the method described in claim 12.

21. A method for treating a wound, the method comprising bringing the wound into contact with a graft according to any one of claims 1, 4, and 20.

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