Multilayer amniotic membrane products, related devices, and related methods

Abstract

A multi-layer amniotic membrane product may include a plurality of layers oriented over one another, where the plurality of layers includes at least one chorion layer and at least two amniotic membrane layers. The plurality of layers may include at least four and less than ten layers. At least two of the at least two amniotic layers may form an outermost layer of the plurality of layers. Each amnion layer may include an epithelial layer, a basement membrane, and a fibroblast layer, and each chorion layer may include a mesh layer and a basement membrane.

Description

Cross-references to related literature

[0001] This patent application claims the benefit of priority to U.S. non-provisional patent application No. 18 / 347,279, filed on July 5, 2023, which application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 367,940, filed on July 8, 2022, under 35 U.S.C. §120, the entire contents of which are incorporated herein by reference. Technical Field

[0002] Various embodiments of the present disclosure generally relate to tissue sheets, coverings, wraps, connectors, grafts or protectors (collectively referred to as "tissue products"), and more specifically relate to multi-layer tissue products, such as amniotic tissue products formed from amniotic tissue or amniotic tissue and chorionic tissue, and related manufacturing methods and use methods. Background Art

[0003] After one or more tissue injuries, tissue repair and / or tissue reconstruction, protecting the damaged tissue area can promote the healing process. For example, in the case of nerve tissue, it is impossible to cover and / or isolate the nerve repair or nerve injury site, which can cause unwanted axon growth to the surrounding area, which may cause the attachment and scar of soft tissue. By protecting the nerve repair or injury site (for example, by covering and isolating), it is possible to suppress (for example, reduce or eliminate) unwanted axon growth, and in some cases, axon growth can be guided to a better nerve regeneration site rather than a non-target area, which can reduce the healing time. In addition, this technology can also help to provide reinforcement to the nerve repair or injury site, and help to suppress the separation of the nerves joined (coapted). In order to provide protection and coverage at the nerve repair or injury site, a tube, a duct, a sheet (i.e., a coating) for coating or other forms of membrane tissue grafts can be used to support and strengthen the microsurgical repair of the damaged nerve. As described below, multilayer amniotic membrane products can be used to help repair tissue to assist in repairing nerves. Summary of the invention

[0004] The present invention is directed to multilayer tissue products and methods of making such products. The multilayer tissue product can be any type of tissue, such as amniotic membrane or amniotic membrane and chorionic tissue. According to various aspects of the present disclosure, multilayer amniotic membrane products can be formed and / or used to help repair tissue (e.g., nerves).

[0005] In one aspect, the dehydrated multi-layer amniotic membrane product may include a plurality of layers oriented on top of each other, wherein the plurality of layers is at least four layers and less than ten layers, consisting of at least one chorionic layer and at least two amniotic layers, and wherein at least two of the at least two amniotic layers form the outermost layers of the plurality of layers. Each of the amniotic layers includes an epithelial layer, a basement membrane, and a fibroblastic layer, and each of the chorionic layers includes a reticular layer and a basement membrane.

[0006] In other aspects, the multiple layers may include five, six or seven layers; the multi-layer amniotic membrane product may include at least three amniotic membrane layers, wherein at least two of the at least three amniotic membrane layers are adjacent to each other and the adjacent amniotic membrane layers are oriented such that the epithelial layer of the first adjacent amniotic membrane layer is oriented facing the fibroblast layer of the second adjacent amniotic membrane layer; the multi-layer amniotic membrane product may include at least three amniotic membrane layers, wherein at least two of the at least three amniotic membrane layers are adjacent to each other and the adjacent amniotic membrane layers are oriented such that the fibroblast layer of the first adjacent amniotic membrane layer is oriented facing the fibroblast layer of the second adjacent amniotic membrane layer; each of the outermost amniotic membrane layers of the multiple layers is oriented such that the epithelial layer faces outward; and the at least two amniotic membrane layers and at least one chorionic layer may be obtained from a human source or a non-human source.

[0007] In certain aspects, a method of making a multi-layer amniotic membrane product may include: washing amniotic and chorionic layers, wherein the amniotic and chorionic layers are obtained from an amniotic sac and a placenta; cutting the amniotic and chorionic layers; and overlaying the washed and cut amniotic and chorionic layers on each other to form a multi-layer sheet having at least four layers and less than ten layers, wherein the washed and cut amniotic and chorionic layers are oriented such that the outermost layer of the multi-layer sheet is the amniotic layer. The method may also include drying the multi-layer sheet to form a dehydrated sheet; and sterilizing the multi-layer sheet to form a multi-layer amniotic membrane product.

[0008] Different aspects of the method may include: wherein cleaning the amnion and chorion layers includes using two cleaning solutions; the first cleaning solution of the two cleaning solutions may include about 0.5% Tween 20 (v / v), about 0.05% polyhexamethylene biguanide (w / v) or alternatively about 0.5% (w / v) lohexidine; about 0.9% NaCl (w / v), about 10 mM 1,3-bis (tris (hydroxymethyl) methylamino) propane (Bis-Tris propane), and a solution of about pH 6.7 + / - 0.1; the first cleaning solution of the two cleaning solutions may include about 0.5% Tween 20 (v / v) and about 10.0% NaCl (w / v), and the pH value is about 4.5 to 7.0; the second cleaning solution of the two cleaning solutions may include about 0.144 g / L KH 2 PO 4, about 9 g / L NaCl and about 0.795 g / L Na 2 HPO 4 The second cleaning solution of the two cleaning solutions comprises about 0.9% NaCl (w / v) and has a pH of about 4.5 to 7.0; at least two adjacent amniotic membrane layers can be oriented adjacent to each other so that the fibroblast layer of the first adjacent amniotic membrane layer is oriented to face the fibroblast layer of the second adjacent amniotic membrane layer, or the fibroblast layer of the first adjacent amniotic membrane layer is oriented to face the epithelial layer of the second adjacent amniotic membrane layer; the multilayer sheet comprises five, six or seven layers; the outermost amniotic membrane layer can be oriented so that the epithelial layer of each of the outermost amniotic membrane layers facing outward; collecting the amnion layer and the chorion layer from the amniotic sac and the placenta may include separating the amnion layer in the spongy layer, separating the chorion layer from the decidua of the amniotic sac in the trophoblast layer, and substantially removing the trophoblast cells remaining on the chorion layer; cutting the amnion and the chorion layer may occur before washing the amnion and the chorion layer; the multi-layer sheet may include five layers, oriented as AACCA, AACCA, AACAA, AAACA, or ACCCA, wherein each A represents an amnion layer (amnion layer layer) and each C represents a chorion layer; the multi-layer sheet may include six layers oriented as AACCAA, AAACCA, AAAACA, AACAAA, ACCCAA, or ACCCCA, wherein each A represents an amniotic layer and each C represents a chorion layer; the multi-layer sheet may include seven layers oriented as AAACAAA, AAAACAA, AAAAACA, AACCAAA, AACCCAA, AAACCCA, AACCCCA, or ACCCCCA, wherein each A represents an amniotic layer and each C represents a chorion layer; sterilizing the multi-layer sheet includes using electron beam radiation to achieve about 10 -6 Sterility is ensured; drying the multilayer sheet to form a dehydrated sheet includes using a vacuum pressure of about 50 mbar to about 350 mbar at a temperature of about 22 degrees Celsius to about 35 degrees Celsius; the method may further include winding the multilayer sheet to form a tube.

[0009] Various aspects of the present disclosure may relate to a method of manufacturing a multi-layer amniotic membrane product, comprising: washing a multi-layer amniotic membrane layer, wherein the amniotic membrane layer is obtained from an amniotic sac; cutting the amniotic membrane layer; and overlaying the washed and cut amniotic membrane layers on each other to form a multi-layer sheet having at least four layers and less than ten layers, wherein the washed and cut amniotic membrane layers are oriented such that the outermost layer of the multi-layer sheet is the amniotic membrane layer, and the outermost amniotic membrane layer is oriented such that the epithelial layer of each of the outermost amniotic membrane layers faces outward. The method may further include drying the multi-layer sheet to form a dehydrated sheet and sterilizing the multi-layer sheet to form a multi-layer amniotic membrane product.

[0010] In different aspects, the multilayer sheet may include five, six or seven layers; adjacent amniotic layers may be oriented so that the epithelial layer of a first adjacent amniotic layer is oriented facing the fibroblast layer of a second adjacent amniotic layer; or adjacent amniotic layers may be oriented so that the fibroblast layer of a first adjacent amniotic layer is oriented facing the fibroblast layer of a second adjacent amniotic layer.

[0011] In certain aspects, the present disclosure may relate to a dehydrated multi-layer amniotic membrane product comprising multiple layers oriented above each other, wherein the multiple layers include at least four amniotic membrane layers, wherein each of the amniotic membrane layers includes an epithelial layer, a basement membrane, and a fibroblast layer, and wherein the outermost layer of the multiple layers is an amniotic membrane layer, and wherein the outermost amniotic membrane layer is oriented such that the epithelial layer faces outward.

[0012] In different aspects, the plurality of layers may consist of five amniotic layers; the plurality of layers may include four, five, six or seven layers; the adjacent amniotic layers are oriented such that the epithelial layer of a first adjacent amniotic layer is oriented facing the fibroblast layer of a second adjacent amniotic layer; the adjacent amniotic layers are oriented such that the fibroblast layer of a first adjacent amniotic layer is oriented facing the fibroblast layer of a second adjacent amniotic layer; or the amniotic layers may be obtained from a human or non-human source.

[0013] Other objects and advantages of the disclosed embodiments will be partially listed in the following description and may be obvious from the description or may be learned by practicing the disclosed embodiments. The objects and advantages of the disclosed embodiments will be realized and achieved by the elements and combinations particularly pointed out in the attached claims.

[0014] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not restrictive of the disclosed embodiments, as claimed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate various exemplary embodiments and, together with the description, serve to explain the principles of the disclosed embodiments.The drawings contained herein may not be drawn to scale.

[0016] Figure 1 A cross-sectional view of a human amniotic sac.

[0017] Figure 2 is a perspective view of a multi-layer amniotic membrane product according to one or more embodiments.

[0018] Figure 3A The human placenta and amniotic sac are shown, and Figures 3B to 3E Histological images showing hematoxylin and eosin staining of various parts or layers of the harvested amniotic sac.

[0019] Figure 4A graph showing various properties of different multi-layer amniotic membrane products having different numbers of layers.

[0020] Figure 5 A flow chart of an exemplary method for forming a multilayer amniotic membrane product.

[0021] Figure 6 A flow chart of another exemplary method of forming a multilayer amniotic membrane product. DETAILED DESCRIPTION

[0022] The present invention provides multilayer tissue products and methods for making such products. The multilayer tissue product can be any type of tissue, such as a membrane tissue graft for tissue repair, reconstruction or protection. Various embodiments of the present disclosure are generally related to multilayer amniotic membrane products that can be used to help repair tissue to assist in repairing nerves.

[0023] In the present disclosure, the singular forms "a", "an" and "the" include plural referents unless the context indicates otherwise. The term "exemplary" means "example" rather than "ideal". The terms "include", "contain", "comprises" or other variations thereof are intended to cover non-exclusive inclusion, so that a composition, method or process containing a list of components or steps does not necessarily include only those components or steps, but may include other components or steps not explicitly listed or inherent to such composition, method or process. Unless otherwise stated in the specification, relative terms such as "about" and "approximately" are generally used to indicate a possible variation of ±10% of the stated or understood value. In addition, the term "between" used to describe a range of values ​​is intended to include the minimum and maximum values ​​described herein. The use of the term "or" in the claims and the specification is used to mean "and / or", unless it is explicitly stated that only alternatives are referred to or the alternatives are mutually exclusive, although the present disclosure supports definitions that only refer to alternatives and "and / or". "Another" as used herein may mean at least a second or more.

[0024] Embodiments of the present disclosure may relate to dehydrated multi-layered placental amnion or amnion / chorion products, such as sheets, which can be configured to be placed around damaged tissue, such as damaged nerves, including damaged peripheral nerves. Multi-layered amnion or amnion / chorion products, such as sheets, can be used as tissue coverings to act as anatomical barriers, helping to provide protection from surrounding environmental influences.

[0025] Examples of tissues of the multilayer amniotic membrane or amniotic / chorionic membrane products described herein that can be used include neural tissues, such as peripheral neural tissues or central nervous system tissues. Other types of tissues suitable for use in the present disclosure include, but are not limited to, epithelial tissues, connective tissues, muscle tissues, tendon tissues, ligamentous tissues, vascular tissues, intestinal tissues (including but not limited to forestomach), dermal tissues, and cardiac tissues.

[0026] It is noteworthy that, although most of the present disclosure discusses multilayer products formed by amnion or amnion and chorion, the present disclosure is not limited thereto. For example, the multilayer product may be formed by one or more different types of tissues, such as layers including but not limited to the various types of tissues mentioned in the preceding paragraphs above. In addition, the tissue may include mammalian tissue, including human tissue and tissues of other primates, rodent tissue, horse tissue, canine tissue, rabbit tissue, porcine tissue, sheep or other ruminant tissues. In addition, the tissue may be a non-mammalian tissue selected from fish, amphibian or insect tissue. For the recipient of the implant, the tissue may be allogeneic or xenogeneic. The tissue may be a synthetic tissue, such as but not limited to laboratory grown or 3D printed tissue. These tissue types can be used to replace the amnion or amnion and chorion tissue layers, or in addition to the amnion or amnion and chorion tissue layers, these tissue types are used in addition, such as those described herein. Products made of the alternative tissue types discussed in this paragraph and / or the preceding paragraphs may include a number of layers similar to the number of layers described herein for the multilayer amnion product.

[0027] Multilayer amnion (MLA) product can be a dehydrated sheet composed of at least four layers, such as four to ten layers, four to seven layers, at least four layers and less than ten layers of placental sac amnion or amnion and chorion, as will be further discussed below. The term multilayer amnion or MLA refers to a tissue product composed of all amnion layers or a tissue product composed of amnion layers and chorion layers. In these two product types, the outermost layer (e.g., top layer and bottom layer) is an amnion layer. In some aspects, the multilayer amnion product can be a dehydrated sheet composed of at least five layers, such as five to ten layers, five to seven layers, or five to six layers of placental sac amnion or amnion and chorion. Placental sac amnion and / or chorion can be obtained from human sources, or alternatively, non-human sources, or a mixture of human and non-human sources. In some embodiments, these layers can be alternating human placental sac amnion and chorion.

[0028] The amniotic membrane layer and the chorionic membrane layer of the present invention are Figure 1 The constituent layers of a cross-section of a human amniotic placental sac 100 are shown. As used herein, the amniotic membrane layer 102 refers to Figure 1The portion shown includes an epithelial layer 104, a basement membrane 106, and a fibroblast layer 108. The chorionic layer 110 used herein includes a reticular layer 112 and optionally a basement membrane 114, but ideally omits most (if not all) cells of the trophoblast layer 116. For example, during harvesting, the chorionic layer 110 can be separated from the cells of the trophoblast layer 116. As shown, the dense layer 120 can separate the basement membrane 106 of the amniotic layer 102 from the fibroblast layer 108. In addition, the spongy layer 118 can be located between the amniotic layer 102 and the chorionic layer 110 (e.g., between the fibroblast layer 108 and the reticular layer 112). The fibroblast layer 108, the spongy layer 118, and the reticular layer 112 are interstitial cells 122. The first side of the placental sac 100, for example Figure 1 The top side of the placental sac 100 (e.g., the outer portion of the epithelial cells in the epithelial layer 104 of the amniotic layer 102) can be adjacent to the amniotic cavity 140. The second side of the placental sac 100, e.g. Figure 1 The bottom side (eg, the outer portion of the decidua layer 130 ) may be adjacent to the uterus 150 .

[0029] For example, the amniotic placental sac 100 ( Figure 3A ) The amniotic layer 102 is harvested. The chorionic layer 110 can be harvested from the decidual layer 130 of the amniotic sac 116 by physical separation at the trophoblast layer 116. The trophoblast cells remaining on the harvested chorionic layer 110 can be substantially removed. To harvest the layers, the placenta can be oriented so that the amniotic layer faces outward. Manual debridement can then be performed to separate the amniotic membrane. If the chorionic layers are also to be harvested, they can also be manually separated from the placental tissue. A cell scraper or other suitable tool can also be used during the debridement.

[0030] The removed amniotic layer 102 and chorionic layer 110 can be cleaned after being separated from the placental sac 100. In some embodiments, the amniotic layer 102 or the chorionic layer 110 can be cut before cleaning. For example, one or more templates can be used to identify portions of the layer that can be cut into appropriate sizes for use, and the tissue layer can be cut according to the template. The amniotic membrane or the amniotic membrane and chorionic layer can be cut using a scalpel blade, a die cutter, a pneumatic press, or any other suitable instrument. In some embodiments, the amniotic layer 102 or the chorionic layer 110 can be cut after cleaning. In other embodiments, the amniotic layer 102 or the chorionic layer 110 can be cut before and after cleaning. In some embodiments, the amniotic layer 102 or the chorionic layer 110 can be cleaned before and after cutting.

[0031] After harvesting, the amniotic layer 102 and the chorionic layer 110 may be cleaned using one or more solutions to clean the amniotic layer 102 and the chorionic layer 110. In one embodiment, two solutions may be used to clean one or more of the amniotic layer 102 or the chorionic layer 110. The first cleaning solution may contain about 0.5% polyoxyethylenesorbitol ester such as Tween 20 (v / v), about 0.05% polyhexamethylenebiguanide (PHMB) (w / v) or alternatively about 0.5% lohexidine (w / v), about 0.9% NaCl (w / v), about 10 mM 1,3-bis(tris(hydroxymethyl)methylamino)propane, and a solution of about pH 6.7+ / -0.1. In another example, the first solution may contain about 0.5% polyoxyethylene sorbitol ester such as Tween 20 (v / v) and about 10.0% NaCl (w / v), and the pH value is about 4.5 to 7.0 (e.g., pH 5.6). The second cleaning solution may include about 0.144 g / L KH2PO4, about 9 g / L NaCl, and about 0.795 g / L Na2HPO4. In another example, the second solution may contain about 0.9% NaCl (w / v) and the pH value is about 4.5 to 7.0 (e.g., pH 5.6).

[0032] For example, if the amnion or the amnion and chorion layers are not cut before cleaning, the amnion or the amnion and chorion layers can be cut after cleaning and superimposed on each other to produce a multi-layer amnion product 200, for example Figure 2 In some aspects, the multi-layer amniotic membrane product may include a minimum of four total layers of amnion or amnion and chorion. In some embodiments, the amnion or amnion and chorion layers may be superimposed on each other to produce four to ten layers, or four to seven layers, such as four layers, five layers, six layers, or seven layers. The number of layers of the multi-layer amniotic membrane product may affect the handling characteristics of the multi-layer amniotic membrane product, as described below with reference to Figure 4 One or more support structures (eg, a layering tool) can be used to help cover and / or position the amnion or amnion and chorion layers.

[0033] The multilayer amniotic membrane product may contain an amniotic membrane layer 102 on one exposed side, which is oriented so that the epithelial layer 104 faces outward. The opposite exposed side of the multilayer amniotic membrane product may contain another amniotic membrane layer 102, which may also be oriented so that the epithelial layer 104 faces outward. Therefore, the orientation of the outermost layer of the MLA product may make the amniotic membrane layer 102 the outermost layer on both sides of the multilayer amniotic membrane product. The outermost amniotic membrane layer 102 on both sides of the multilayer amniotic membrane product, such as a sheet, may be oriented so that the epithelial layer 104 faces outward. Therefore, for a sheet having a top surface and a bottom surface, the top surface may include epithelial cells, and the bottom surface may include epithelial cells. In these aspects, the epithelial layer 104 may be smoother and / or more slippery than the layers facing inward of the amniotic membrane layer 102 and the chorionic layer 110. Having a top surface and a bottom surface that include epithelial cells from the epithelial layer 104 allows the multilayer amniotic membrane product to not have "sidedness". For example, multiple layers of amniotic membrane can be applied with the top or bottom surface facing toward or away from the tissue to which it is applied during use.

[0034] The amniotic membrane layer has two sides. One side contains epithelial cells and the other side contains a fibroblast layer. When the amniotic membrane layers are overlapped to form a multi-layer amniotic membrane product, the adjacent amniotic membrane layers can be oriented so that the epithelial portion of the first amniotic membrane layer is adjacent to the fibroblast portion of the second amniotic membrane layer, or so that the fibroblast portion of the first amniotic membrane layer is adjacent to the fibroblast portion of the second amniotic membrane layer. The adjacent first and second amniotic membrane layers may not be layered in an epithelial-epithelial direction to avoid delamination.

[0035] In some embodiments, the organization of the layers within a multi-layer amniotic membrane product can be such that the amniotic and chorionic layers are repeated, i.e., similar layers are organized adjacent to each other. For example, the combination of layers of a five-layer, six-layer, or seven-layer MLA product can be organized as follows:

[0036] Fifth level: AACCA, AACAA, AAACA, ACACA, AAAAA or ACCCA.

[0037] Sixth level: AACCAA, AAACCA, AAAACA, AACAAA, ACCCCAA, ACCCCA or AAAAAA.

[0038] Seven levels: AAACAAA, AAAACAA, AAAACA, AACCAAA, AACCCAA, AAACCCA, AACCCCA, ACCCCA or AAAAAAA.

[0039] In the above examples, each A represents an amniotic layer (e.g., amniotic layer 102) and each C represents a chorionic layer (e.g., chorionic layer) 110. Each amniotic layer may include an epithelial layer (e.g., epithelial layer 104), a basement membrane (e.g., basement membrane 106), and a fibroblast layer (e.g., fibroblast layer 108), as described above. Each chorionic layer may include a reticular layer (e.g., reticular layer 112). In some embodiments, each chorionic layer may include a basal layer in addition to the reticular layer. In some embodiments, one or more chorionic layers, but not all chorionic layers, include a basal layer (e.g., basement membrane 114) in addition to the reticular layer. In any of the above embodiments, one or more chorionic layers may be substantially free of trophoblast cells (e.g., trophoblast 116).

[0040] In some embodiments, the covering layer of tissue, including but not limited to the covering layer of amnion or the covering layer of amnion and chorion, can then be dried to form a dehydrated sheet. For example, although any suitable drying method can be used, the covering layer can be dried using a vacuum at a temperature of about 18 degrees Celsius to about 35 degrees Celsius, such as about 22 degrees Celsius to about 35 degrees Celsius (e.g., at a pressure less than atmospheric pressure) to dry the multi-layered tissue or, for example, multi-layered amniotic membrane product. Exemplary vacuum pressures for drying can be, for example, about 50 millibars to about 350 millibars.

[0041] The subsequent dehydrated tissue sheet consisting of the amnion or amnion and chorion layers may then be sterilized using a suitable method. In one embodiment, electron beam radiation may be used to achieve a sterility assurance of about 10-6.

[0042] The method of forming a multi-layer amniotic membrane product may include separating the amniotic membrane and the chorionic membrane layer from the placenta and the amniotic sac, washing the amniotic membrane and the chorionic membrane layer, stacking them on each other in an alternating or repeated order, and dehydrating the amniotic membrane and the chorionic membrane layer after stacking them on each other. In an embodiment where only an amniotic membrane layer is used, the method of forming a multi-layer amniotic membrane product may include separating the amniotic membrane layer from the placenta and the amniotic sac, washing the amniotic membrane layer, stacking them on each other, and dehydrating the amniotic membrane layer after stacking them on each other. For example, the method of forming a multi-layer amniotic membrane product may include collecting the amniotic sac and the placenta, and separating the chorionic membrane layer from the decidua at the trophoblast. The amniotic membrane layer may include all of the spongy layer, at least some of the spongy layer, or no spongy layer. If a chorionic membrane layer is used, the trophoblast cells on the chorionic membrane layer may be substantially removed. For example, the chorionic membrane layer used for the multi-layer amniotic membrane product may not include trophoblast cells. In other aspects, the chorionic membrane layer used for the multi-layer amniotic membrane product may include some trophoblast cells. Furthermore, in various aspects, the chorion layer can include all of the sponge layer, at least some of the sponge layer, or no sponge layer.

[0043] The method may also include using one or more cleaning solutions, such as two cleaning solutions, examples of which are as described above, to clean the amnion or the amnion and chorion layers. The cut and cleaned layers may be overlaid on each other to form a multilayer amnion product such as a sheet. The multilayer amnion product may include at least four or at least five layers, such as four to ten layers, or four to seven layers, five to ten layers, five to seven layers, or five to six layers, for example, four layers, five layers, six layers or seven layers. These layers may be oriented so that the amnion layer is the outermost layer on both sides of the multilayer sheet. The outermost amnion layers on both sides (e.g., the top layer and the bottom layer) may be oriented so that the epithelial layer faces outward. Insofar as there are adjacent amnion layers in the multilayer amnion product, the adjacent amnion layers may be oriented so that the epithelial layer of the first adjacent amnion layer faces the fibroblast layer of the second adjacent amnion layer, or so that the fibroblast layer of the first adjacent amnion layer faces the fibroblast layer of the second adjacent amnion layer. The multilayer amnion product may then be dried to form a dehydrated sheet. In some embodiments, drying the multilayer amniotic membrane product may include vacuum drying at a vacuum pressure of about 50 mbar to about 350 mbar and a temperature of about 22 degrees C to about 35 degrees C. The multilayer amniotic membrane product may be sterilized, for example, using electron beam radiation or other suitable sterilization methods.

[0044] Figure 2 A sample seven-layer multi-layer amniotic membrane prototype 200 is shown. Figure 3A A human placental amniotic sac, such as human amniotic placental sac 100 , is shown. Figure 3B A hematoxylin and eosin-stained histological image of a harvested amniotic placental sac 100 is shown, which has all layers, for example, including at least an amniotic layer 102 , a chorionic layer 110 , and a decidua layer 130 . Figure 3C A histological image of the harvested, separated amniotic membrane layer 102 stained with hematoxylin and eosin is shown. Figure 3D A histological image of the separated reticular layer 112 of the harvested chorionic layer 110 is shown by hematoxylin and eosin staining. Figure 3E Histological image showing hematoxylin and eosin staining of a five-layer multi-layer amniotic membrane product 300. As described above, a multi-layer amniotic membrane product may include multiple amniotic membrane layers 102 alone or in combination with one or more chorionic layers 110 (eg, reticular layers 112 of chorionic layers 110).

[0045] Figure 4A chart comparing various properties of multilayer amniotic membrane products with different numbers of layers. As shown in the figure, the multilayer amniotic membrane products tested include four-layer MLA products, five-layer MLA products and six-layer MLA products. Four-layer MLA products, five-layer MLA products and six-layer MLA products can be formed through the steps and procedures discussed in this article. The four-layer MLA product is formed by the amniotic membrane layer and the chorionic membrane layer oriented by AACA, wherein A represents the amniotic membrane, and C represents the chorionic membrane. The five-layer MLA product is formed by the amniotic membrane layer and the chorionic membrane layer oriented by AACAA and AACCA, wherein A represents the amniotic membrane, and C represents the chorionic membrane. The six-layer MLA product is formed by the amniotic membrane layer and the chorionic membrane layer oriented by AAACCA and ACACAA, wherein A represents the amniotic membrane, and C represents the chorionic membrane. Dehydrated umbilical cord membrane products were also tested for comparison. The dehydrated umbilical cord membrane product is approximately 3cm x 6cm, while the four-layer, five-layer and six-layer MLA products are approximately 4cmx 8cm.

[0046] As shown in the figure, the visual appearance, layering, transparency, conformability, repositionability, durability, sutureability and overall impression of each multi-layer amniotic membrane product were evaluated or assessed. Each multi-layer amniotic membrane product was rated according to, for example, very good (e.g., 4), acceptable (e.g., 3), slightly unacceptable (e.g., 2) and unacceptable (e.g., 1, not shown). Figure 4 ) levels are evaluated.

[0047] The evaluation of visual appearance is based on the user's visual inspection of the tissue layers of the multi-layer amniotic membrane product. For example, the tissue layer should be light yellow or white, and judge whether there is any discoloration. Visually inspect whether there are red blood streaks, whether the tissue color is consistent, whether the tissue has holes, tears, etc.

[0048] Delamination is evaluated based on whether the tissue layers of the multi-layer amniotic membrane product are delaminated (e.g., separated from adjacent tissue layers) when handled. In these aspects, less delamination can help ensure that the multi-layer amniotic membrane product maintains its shape and inter-layer connections when handled. Delamination may negatively impact the user's ability to handle and / or apply the multi-layer amniotic membrane product. A score of 4 corresponds to no delamination; a score of 3 corresponds to some visible delamination, but the tissue is generally unchanged (e.g., the corners or edges of the tissue product separate, but the tissue product is still usable overall); a score of 2 corresponds to significant delamination; and a score of 1 corresponds to complete delamination (the tissue product is unusable and the layers are completely separated).

[0049] The transparency assessment is based on the transparency of tissue (e.g., tissue forming a multi-layer amniotic membrane product) after, for example, hydration. Transparency can help a user visualize and / or locate tissue (e.g., nerve tissue) beneath and / or around the multi-layer amniotic membrane product. Thus, transparency can make the multi-layer amniotic membrane product easier to use. A score of 4 corresponds to transparent tissue; a score of 3 corresponds to slightly transparent tissue; a score of 2 corresponds to slightly opaque tissue; and a score of 1 corresponds to opaque tissue. During use, transparency can allow a user to see that the MLA product is applied or wrapped around the surrounding underlying structures. This can make the MLA product easier to use in situations such as nerve attachment because the user is able to visualize the location of the nerve endings within the MLA product and relative to each other.

[0050] The conformability rating is based on the satisfaction with the conformability or drapability of the tissue (e.g., drapability across the midline). A greater degree of conformability or drapability can help the multilayer amniotic membrane product wrap around or conform to the shape or size of tissue (e.g., neural tissue). For example, a score of 4 corresponds to satisfactory, complete drapability; a score of 3 corresponds to somewhat satisfactory drapability; a score of 2 corresponds to somewhat unsatisfactory drapability; and a score of 1 corresponds to unsatisfactory drapability, such as a stiff sheet.

[0051] The evaluation of repositionability is based on the ability of tissue (e.g., tissue forming a multi-layer amniotic membrane product) to be straightened and / or repositioned after being folded. For example, a score of 4 corresponds to no folding or clumping; a score of 3 corresponds to some folding or clumping, but the folding or clumping is relatively easy to handle; a score of 2 corresponds to partial folding or clumping of the tissue; and a score of 1 corresponds to the tissue being completely folded on itself. In these aspects, a greater degree of repositionability can help the multi-layer amniotic membrane product be straightened and / or repositioned, such as being more appropriately positioned on the tissue.

[0052] The evaluation of durability is based on the assessed durability of tissue (e.g., tissue forming a multi-layer amniotic membrane product). For example, the evaluation of the durability of the tissue includes pulling on the tissue (e.g., pulling on a suture that has been passed through the tissue) and evaluating the ability of the tissue to withstand tearing of the suture passing through the tissue. For example, a score of 4 corresponds to a very durable tissue; a score of 3 corresponds to a durable tissue; a score of 2 corresponds to a slightly durable tissue (e.g., some tearing is seen); and a score of 1 corresponds to a non-durable tissue (e.g., the tissue falls apart). In these aspects, a greater degree of durability can help the multi-layer amniotic membrane product be positioned, sutured, pulled, and / or otherwise manipulated multiple times and / or in multiple directions before, during, or after implantation in a patient.

[0053] The evaluation of sutureability is based on the assessed sutureability of tissue (e.g., tissue forming a multi-layer amniotic membrane product). For example, the assessed sutureability of the tissue includes whether the suture can pass through the tissue and / or whether the tissue is torn during suture. A high score corresponds to the ability to push the suture through the tissue product and the ability to resist tearing or unraveling once the suture passes through. For example, 4 points correspond to satisfactory sutures (e.g., sutures easily pass through and then remain in place and do not tear the tissue product); 3 points correspond to slightly satisfactory sutures (sutures are slightly more difficult to pass through and / or slightly torn); 2 points correspond to slightly unsatisfactory sutures (sutures are more difficult to pass through and / or more torn); 1 point corresponds to unsatisfactory sutures (e.g., sutures do not pass through and / or pass directly through and tear). In these aspects, a greater degree of sutureability can help suture the multi-layer amniotic membrane product to tissue (e.g., neural tissue) and / or itself, for example, to surround the tissue.

[0054] The evaluation of the overall impression is based on the assessed overall treatment of the multilayer amniotic membrane product. For example, the assessed overall impression includes an evaluation of the usability of the multilayer amniotic membrane product. For example, a score of 4 corresponds to a very usable tissue product; a score of 3 corresponds to a usable tissue; a score of 2 corresponds to a slightly usable tissue; and a score of 1 corresponds to a non-usable tissue. In these aspects, a greater degree of usability can help the multilayer amniotic membrane product be used to treat tissue (e.g., neural tissue).

[0055] like Figure 4 As shown, the five-layer MLA product showed consistent high scores in each category, for example, the scores of each category were at least higher than 3.5. These scores are based on a large number of evaluations of various types of multilayer amniotic membrane products by users (e.g., surgeons). In addition, users said that four-layer, five-layer and six-layer MLA products feel thinner and / or easier to operate than dehydrated umbilical cord membrane products. The thickness of an exemplary MLA product may be about 30 microns to about 120 microns, for example, about 90 microns to about 100 microns, or about 60 microns to about 80 microns. In addition, users said that due to the thickness and other aspects being inconsistent with dehydrated umbilical cord membrane products, dehydrated umbilical cord membrane products do not use sutures, but use microclips, fibrin glue or by wrapping themselves to fix the dehydrated umbilical cord membrane products. In comparison, four-layer, five-layer and six-layer MLA products show very good or very satisfactory suture scores.

[0056] In other aspects, the four-layer, five-layer, and six-layer MLA products were more consistent in appearance and / or transparency than the dehydrated umbilical cord lining products. In addition, the four-layer, five-layer, and six-layer MLA products had limited or no delamination issues. In these aspects, delamination may result in separation of adjacent layers in the MLA product, which may negatively impact the operability, durability, and / or one or more other properties of the MLA product. The four-layer MLA product was rated slightly unacceptable in terms of repositionability and overall impression. The five-layer and six-layer MLA products received highly acceptable ratings in all categories or attributes. Based on this evaluation, the five-layer MLA product received the best overall feedback, for example, balancing the trade-off between processing quality and the amount of implanted / resorbed tissue (i.e., number of layers).

[0057] In general, it was found that if too many layers are used in a multi-layer amniotic membrane product, the resulting product will not drape or conform properly. Alternatively, if too few layers are used, workability may be reduced because the resulting product may tear or rip and may lack durability, repositionability, and / or sutureability. These properties may not be as important for external wound care products, but embodiments of the present disclosure may be applicable to internal tissues such as nerves. Therefore, forming a product with a greater number of layers makes it easier for surgeons to handle, position, and / or secure the multi-layer amniotic membrane product of the present disclosure relative to delicate tissue structures such as nerves, while still maintaining sufficient drape to, for example, wrap around a nerve.

[0058] In these aspects, the four-layer, five-layer, and six-layer MLA products have the trophoblast 116 of the chorionic layer 110 substantially removed. Thus, the four-layer, five-layer, and six-layer MLA products are composed of birth tissue membranes and minimal unorganized tissue. Most other publicly available amniotic membrane products are not composed of birth tissue membranes with little or no unorganized tissue. The MLA products of the present disclosure may be stronger, have a more consistent thickness, and / or may be thicker than commercially available products, and may retain their shape better than commercially available products that may shrink and have low or no durability.

[0059] Lamination tools can be used to help support and / or maintain various layers of tissue, such as to form the MLA products discussed herein. For example, four or more layers of amnion and / or chorion can be positioned sequentially one above the other.

[0060] Figure 5Flowchart of an exemplary method 500 for forming or manufacturing a multilayer amniotic membrane product. Although not shown, method 500 may include one or more initial and / or optional steps. For example, method 500 may include an initial step of collecting placental tissue and clearing it. The initial step may include collecting amnion or amnion and chorion from the placental membrane. The initial step may include clearing chorionic tissue (if included) to retain the reticular layer and basement membrane while also substantially removing the trophoblast. The initial step may include determining the size of the amnion and chorion from the placental membrane. For example, an amnion layer or an amnion layer and a chorion layer may be positioned above a lamination tool.

[0061] Method 500 includes step 502, which includes washing or otherwise performing a solution treatment procedure on the amniotic layer or the amniotic membrane and chorionic tissue. As described above, washing may include using one or more washing solutions. For example, the first washing solution may contain about 0.5% polyoxyethylene sorbitol esters such as Tween 20 (v / v), about 0.05% polyhexamethylene biguanide (PHMB) (w / v) or alternatively about 0.5% lohexidine (w / v), about 0.9% NaCl (w / v), about 10mM 1,3-bis (tris (hydroxymethyl) methylamino) propane and a solution of about pH 6.7 + / - 0.1. In another example, the first solution may contain about 0.5% polyoxyethylene sorbitol esters such as Tween 20 (v / v) and about 10.0% NaCl (w / v), and the pH value is about 4.5 to 7.0 (e.g., pH 5.6). The second cleaning solution may contain about 0.144 g / L of KH 2 PO 4 , about 9 g / L NaCl and about 0.795 g / L Na 2 HPO 4 In another example, the second solution may contain about 0.9% NaCl (w / v) and have a pH value of about 4.5 to 7.0 (eg, pH value of 5.6).

[0062] Step 502 may include, for example, thawing the frozen amnion or amnion and chorion fragments in a solution. Thawing may be performed at ambient temperature (e.g., 15° C. to 30° C.) for at least four hours. For example, thawing may be performed for about 4 hours to about 18 hours, such as about 4 hours to about 12 hours, about 14 hours to about 18 hours, or about 6 hours to about 8 hours. In some aspects, the frozen amnion or amnion and chorion fragments may be thawed at a refrigerated temperature (e.g., 2° C. to 10° C.) for about 48 hours to about 72 hours.

[0063] In addition, step 502 may include, for example, washing the amnion or the amnion and the chorion layer in a saline solution one or more times. Step 502 may include culturing and / or stirring the amnion or the amnion and the chorion layer in one or more solutions. For example, in some aspects, step 502 may include culturing the amnion or the amnion and the chorion layer in the first solution at ambient temperature (e.g., between about 15 degrees Celsius and about 30 degrees Celsius) for a period of 15 minutes to about 2 hours, for example, 20 minutes to about 2 hours, 30 minutes to about 1.5 hours, about 1 hour to about 1.5 hours. Cultivation may also include, for example, stirring at about 80rpm to about 100rpm, for example, about 90rpm to about 95rpm. Step 502 may also include an optional holding step in an incubator, for example, for about 30 minutes to about 60 minutes, for example, about 40 minutes.

[0064] In addition, in some aspects, step 502 may include one or more additional culture procedures. For example, step 502 may also include culturing the amnion or the amnion and the chorionic layer in the second solution for a period of about 5 minutes to about 1 hour, for example, about 15 minutes to about 40 minutes, at ambient temperature (e.g., between about 15 degrees Celsius and about 30 degrees Celsius). Cultivation in the second solution may also include, for example, stirring at about 80rpm to about 100rpm, for example, about 90rpm to about 95rpm. Step 502 may also include an optional holding step of, for example, about 30 minutes to about 60 minutes, for example, about 40 minutes, using the second solution in an incubator. One or more of these culture procedures may be repeated, for example, the second culture procedure may be repeated, for example, repeated three times or more additional times.

[0065] Method 500 includes step 504, which includes cutting the amnion or the amnion and the chorion layer. The amnion or the amnion and the chorion layer can be cut into a desired size and / or shape. In some aspects, one or more templates can be arranged on the tissue, and the tissue can then be cut according to the template arrangement. Example template dimensions can be, for example, about 8×8 cm, about 10×10 cm, or about 12×12 cm. The amnion or the amnion and the chorion layer can be cut using a scalpel blade, a die cutter, a pneumatic press, or any other suitable instrument. In some aspects, the cutting described herein can be performed before the cleaning step 502. Alternatively, a multilayer amnion product can be formed (e.g., via the various steps of the method discussed herein), and the multilayer amnion product can then be cut into a desired size and / or shape.

[0066] Method 500 also includes step 506, which includes covering the cleaned and cut amnion or amnion and chorion layers to form a product having at least four layers or at least five layers, such as four to ten layers, four to seven layers, or five to seven layers. A stacking tool can be used to help support the amnion or amnion and chorion layers to form a multi-layer amnion product. In some aspects, a layer of biocompatible non-placental material can be positioned between the bottom plate of the stacking tool and the first amnion layer. The first amnion layer can be oriented so that the epithelial layer faces downward, opposite to the position where the layer of more tissue will be stacked.

[0067] If two amniotic membrane layers are stacked on top of each other, the adjacent amniotic membrane layers can be oriented so that the epithelial portion of one adjacent amniotic membrane layer faces the fibroblastic portion of another adjacent amniotic membrane layer, or so that the fibroblastic portion of one adjacent amniotic membrane layer faces the fibroblastic portion of another adjacent amniotic membrane layer. Adjacent amniotic membrane layers cannot be stacked so that the epithelial portions face each other to avoid delamination. It is worth noting that the middle layer of the chorion does not need to be oriented in a specific direction or arrangement. The last amniotic membrane layer can then be positioned on top of the other layers of the amnion or amnion and chorion. The last amniotic membrane layer can be oriented so that the epithelial layer faces upward, away from the preceding tissue layers.

[0068] In some aspects, the top plate of the stacking tool can then be positioned on the top of the amniotic layer or the amniotic and chorionic layers, for example to help maintain and / or compress the stacked layers. In some aspects, a layer of non-placental material of biocompatibility can be positioned between the top plate and the multilayer amniotic product. In some aspects, at least four or at least five layers of amniotic or amniotic and chorionic can be covered, and the epithelial cells of the outermost amniotic layer are facing outward on both sides of the covered layer. In some embodiments, each layer can be positioned on a biocompatible material sheet, and the layer and the biocompatible material sheet can be positioned on the stacking tool. In some aspects, after each layer is positioned on the stacking tool, the biocompatible material sheet can be removed.

[0069] Method 500 includes step 508, which includes drying the amnion or the stacked layers of amnion and chorion to form a dehydrated sheet of a multilayer amnion product. Drying can be performed via vacuum drying. In these aspects, the amnion layer or the amnion layer and the chorion layer can be positioned in a drying or vacuum bag. As an example, suitable drying or vacuum bags and drying methods are described in U.S. Provisional Application No. 63 / 477,566, filed on December 29, 2022, the entire contents of which are incorporated herein by reference.

[0070] In one or more aspects, drying can be performed for about 16 hours to about 24 hours (e.g., one day and / or overnight). Drying can be performed at a temperature between about 15 degrees Celsius and about 35 degrees Celsius. Drying can be performed at an exhaust flow rate between about 30 SLPH and about 340 SLPH, for example, about 50 SLPH. Drying can be performed at a vacuum pressure between about 10 inHg and about 28 inHg. In some aspects, drying can be performed at a vacuum pressure greater than 25 inHg. It is worth noting that the above-mentioned drying steps and procedures are merely exemplary, and one or more different drying steps or procedures can be performed to dry the multilayer amniotic membrane product. In addition, although step 504 is shown as before steps 506 and 508, it should be noted that step 504 can optionally be performed after step 506 or after step 508. In some aspects, cutting step 504 can be performed again after step 508 or after step 510, as described below. For example, a first step 504 may be performed to cut the amnion and / or chorion layers to overlap relative to each other, and a second step 504 may be performed after the drying step 508 or after step 510 to cut the MLA product into a specific product shape and / or size.

[0071] In addition, method 500 includes step 510, which includes sterilizing the multilayer amniotic membrane product, for example after drying the multilayer amniotic membrane product. For example, after sufficient drying, the multilayer amniotic membrane product can be sterilized, for example, using electron beam radiation. Step 510 can also include placing (e.g., sealing) the multilayer amniotic membrane product in, for example, at least one bag. For example, the multilayer amniotic membrane product can be placed in one or more foil bags. In some aspects, the multilayer amniotic membrane product can be placed in an inner foil bag, which can then be placed in an outer foil bag.

[0072] In some aspects, method 500 may include a second cutting step 504 after step 508 or 510. The dried and / or sterilized multilayer amniotic membrane product may be cut into, for example, one or more pieces. For example, the multilayer amniotic membrane product may be cut into an inner portion and a sacrificial portion. For example, the multilayer amniotic membrane product may be cut using a scalpel blade, a cutting die, a pneumatic die, or other suitable cutting device. In addition, the multilayer amniotic membrane product may be cut into different sizes or shapes, for example, using one or more cutting dies. The size or shape may correspond to the appropriate size or shape of the final tissue product. The cutting die may have different sizes and / or shapes, such as approximately 4 cm×approximately 6 cm, approximately 3 cm×approximately 6 cm, approximately 3 cm×approximately 4 cm, approximately 2 cm×approximately 4 cm, approximately 2 cm×approximately 2 cm, etc. In some aspects, various cutting procedures may be performed on the cutting surface.

[0073] like Figure 6As shown, method 600 includes the various steps of method 500 that can be performed using only amniotic membrane layers, for example, to form or manufacture a multilayer amniotic membrane product without any chorionic membrane layer. In these aspects, method 600 includes step 602, which includes cleaning the amniotic membrane tissue layer. Method 600 also includes step 604, which includes cutting the amniotic membrane layer. Method 600 includes step 606, which includes overlapping the cleaned and cut amniotic membrane layers to form a multilayer amniotic membrane product having at least four layers or at least five layers. In addition, method 600 includes step 608, which includes drying the multilayer sheet to form a dehydrated sheet. In addition, method 600 includes step 610, which includes sterilizing the resulting multilayer amniotic membrane product. One or more steps of method 600 can be performed in a different order and / or can be repeated (e.g., step 604), as described above with respect to method 500. Method 600 can be performed in a similar manner to method 500, except that method 600 can be applied to amniotic membrane-only multi-layer products.

[0074] The multilayer amniotic membrane products (e.g., multilayer amniotic membrane products dehydrated) disclosed herein can be used as tissue coverings placed around damaged tissues, such as damaged nerves, such as peripheral nerves. The multilayer amniotic membrane products can be composed of, for example, four to ten, four to seven, five to ten, or five to seven layers of separated amniotic membrane layers, or amniotic membrane and chorionic membrane layers that have been collected from, for example, human amniotic sacs. As discussed above, the multilayer amniotic membrane product can be composed of five layers of separated human amniotic membranes, or amniotic membrane and chorionic membrane layers collected from human amniotic sacs or alternatively from non-human sources. The amniotic membrane layers may include an epithelial layer, a basement membrane, and a fibroblast layer. The chorionic membrane layer (if included) may include a reticular layer and a basement membrane. In some aspects, these membranes can be stacked into a multilayer graft in various arrangements as described above.

[0075] The multi-layer amniotic membrane product can be configured to be placed around damaged peripheral nerves as a tissue covering to act as an anatomical barrier to help provide protection from the surrounding environment. The multi-layer amniotic membrane product can be formed into a sheet and then rolled into a tube, cap, or other structure before placement around tissue, or can be wrapped around tissue during placement. The multi-layer amniotic membrane product can have any suitable size or shape, which can depend at least in part on the intended use, such as the size or type of tissue with which the product is intended to be used.

[0076] A method for repairing tissue using a multilayer amniotic membrane product according to any embodiment disclosed herein may include the step of orienting the MLA product at the junction or injury site of two adjacent severed tissue ends or adjacent damaged tissue. If the MLA product has not yet been formed into a tube, the method may also include wrapping the MLA product around the damaged tissue into a tubular shape. Once positioned around the damaged tissue, the MLA product can be fixed to the damaged tissue. In some aspects, the damaged or impaired tissue may be neural tissue, such as peripheral neural tissue.

[0077] Compared to other membranes based on dehydrated amniotic sacs, multilayer amniotic membrane products may have superior handling characteristics around tissues such as peripheral nerves. Exemplary superior handling characteristics may include one or more of the following: conformability around tissues such as nerves, ability to hold sutures, ability to move freely as a sheet in a dehydrated or rehydrated state, ability to be repositioned after placement on one or more tissues, etc. Such handling characteristics may not be as important when used for external purposes such as for external wounds, but may be particularly important when wrapping around or integrating with internal tissues such as nerves. For example, similar to Figure 4 The properties tested in the present invention may allow surgeons to position, cover, reposition, fix, visualize, etc. the multilayer amniotic membrane product when positioning the multilayer amniotic membrane product relative to tissue in the body. The embodiments of the present disclosure may be applicable to in vivo tissue rather than just external wound care. For these reasons, existing products may not be suitable for delicate internal tissues, such as nerves.

[0078] In addition, the multilayer amniotic membrane products of the present disclosure may have resorption kinetics suitable for use with internal tissues in the body, such as with nerves. Embodiments of the present disclosure may be resorbed after about four weeks, after about five weeks, after about six weeks, or after about seven weeks, such as about four to eight weeks, or about six to eight weeks. While products for application to wounds can be reapplied at regular time periods, products applied internally, such as to nerves, cannot be reapplied without surgical intervention. Therefore, embodiments of the present disclosure may have resorption times that allow them to remain in place long enough for tissue healing.

[0079] The method and multi-layer amniotic membrane product can also achieve one or more of the following: (1) clearing and separating placental membrane layers without damaging the placental membrane layers; (2) stacking the amnion or the amnion and chorion layers together in an effective manner; (3) determining the direction of the amnion layers (epithelial side or sponge side); (4) maximizing the yield of overlapping tissue layers (for example, amnion layers or amnion layers and chorion layers having four or more overlapping layers).

[0080] Other embodiments of the present disclosure will be apparent to those skilled in the art by considering the specification and practice of the invention disclosed herein. The specification and embodiments are intended to be considered as exemplary only, with the actual scope and spirit of the present invention being indicated by the appended claims.

Claims

1. A dehydrated multi-layer amniotic membrane product, characterized in that: include: a plurality of layers oriented on top of each other, wherein the plurality of layers is at least four layers and less than ten layers, consisting of at least one chorionic layer and at least two amniotic layers, and wherein the at least two amniotic layers form the outermost layers of the plurality of layers; Each of the amniotic membrane layers comprises an epithelial layer, a basement membrane and a fibroblast layer, and Each of the chorionic membrane layers includes a reticular layer and a basement membrane.

2. The multilayer amniotic membrane product of claim 1, wherein the plurality of layers comprises five, six or seven layers.

3. A multi-layer amniotic membrane product as described in claim 1 or 2, comprising at least three amniotic membrane layers, wherein at least two of the at least three amniotic membrane layers are adjacent to each other, and the adjacent amniotic membrane layers are oriented so that the epithelial layer of the first adjacent amniotic membrane layer is oriented to face the fibroblast layer of the second adjacent amniotic membrane layer.

4. A multi-layer amniotic membrane product as described in any of the preceding claims, comprising at least three amniotic membrane layers, wherein at least two of the at least three amniotic membrane layers are adjacent to each other, and the adjacent amniotic membrane layers are oriented so that the fibroblast layer of the first adjacent amniotic membrane layer is oriented to face the fibroblast layer of the second adjacent amniotic membrane layer.

5. The multilayer amniotic membrane product of any of the preceding claims, wherein each of the outermost amniotic membrane layers of the plurality of layers is oriented such that the epithelial layer faces outward.

6. The multilayer amniotic membrane product of any of the preceding claims, wherein the at least two amniotic membrane layers and the at least one chorionic membrane layer are obtained from human sources.

7. The multilayer amniotic membrane product of any of the preceding claims, wherein at least two of the amniotic membrane layers and at least one of the chorionic membrane layers are obtained from non-human sources.

8. A method for manufacturing a multilayer amniotic membrane product, characterized in that: include: Cleaning the amniotic membrane and the chorionic layer, wherein the amniotic membrane and the chorionic layer are obtained from the amniotic sac and the placenta; cutting the amnion and chorion layers; overlaying the cleaned and cut amnion and chorion layers onto each other to form a multilayer sheet having at least four layers and less than ten layers, wherein the cleaned and cut amnion and chorion layers are oriented such that the outermost layer of the multilayer sheet is the amnion layer; drying the multilayer sheet to form a dehydrated sheet; and The multi-layer sheet is sterilized to form the multi-layer amniotic membrane product.

9. The method of claim 8, wherein cleaning the amniotic and chorionic layers comprises using two cleaning solutions.

10. The method of claim 9, wherein a first cleaning solution of the two cleaning solutions comprises: About 0.5% Tween 20 (v / v); about 0.05% polyhexamethylene biguanide (w / v) or alternatively about 0.5% (w / v) lohexidine; About 0.9% NaCl (w / v); About 10 mM 1,3-bis(tris(hydroxymethyl)methylamino)propane; and A solution of approximately pH 6.7 + / - 0.

1.

11. The method of claim 9, wherein a first cleaning solution of the two cleaning solutions comprises: About 0.5% Tween 20 (v / v); and About 10.0% NaCl (w / v), The pH value of the first cleaning solution is about 4.5 to 7.

0.

12. The method according to any one of claims 9 to 11, wherein the second cleaning solution of the two cleaning solutions comprises: About 0.144 g / L KH2PO4; About 9 g / L of NaCl; and About 0.795 g / L of Na2HPO4.

13. The method of any one of claims 9-11, wherein a second cleaning solution of the two cleaning solutions comprises about 0.9% NaCl (w / v) and has a pH of about 4.5 to 7.

0.

14. A method as described in any one of claims 8-13, wherein the at least two adjacent amniotic layers are oriented to be adjacent to each other, so that the fibroblast layer of the first adjacent amniotic layer is oriented to face the fibroblast layer of the second adjacent amniotic layer, or the fibroblast layer of the first adjacent amniotic layer is oriented to face the epithelial layer of the second adjacent amniotic layer.

15. The method of any one of claims 8 to 14, wherein the multilayer sheet comprises five, six or seven layers.

16. The method of any one of claims 8-15, wherein the outermost amniotic layers are oriented such that the epithelial layer of each of the outermost amniotic layers faces outward.

17. The method according to any one of claims 8 to 16, further comprising The amniotic layer and the chorionic layer are collected from the amniotic sac and the placenta, wherein the collecting comprises: Separating the amniotic membrane layer from the sponge layer; separating the chorionic layer from the decidua of the amniotic sac at the trophoblast layer; and Trophoblastic cells remaining on the chorionic layer are substantially removed.

18. The method of any one of claims 8-17, wherein cutting the amniotic and chorionic layers occurs before washing the amniotic and chorionic layers.

19. The method of any one of claims 8-18, wherein the multilayer sheet comprises five layers, and wherein overlaying the cleaned and cut amnion and chorion layers upon each other comprises orienting the amnion and chorion layers in one of the following arrangements: AACCA; ACACA; AACAA; AAACA; or ACCCA, Therein, each A represents the amniotic layer and each C represents the chorionic layer.

20. The method of any one of claims 8-18, wherein the multilayer sheet comprises six layers, and wherein overlaying the cleaned and cut amnion and chorion layers upon each other comprises orienting the amnion and chorion layers in one of the following arrangements: AACCAA; AAACCA; AAAACA; AACAAA; ACCCAA; or ACCCCA, Therein, each A represents the amniotic layer and each C represents the chorionic layer.

21. The method of any one of claims 8-18, wherein the multilayer sheet comprises seven layers, and wherein overlaying the cleaned and cut amnion and chorion layers upon each other comprises orienting the amnion and chorion layers in one of the following arrangements: AAAAAA; AAAACAA; AAAAACA; AACCAAA; AACCCAA; AAACCCA; AACCCCA; or ACCCCCA, Therein, each A represents the amniotic layer and each C represents the chorionic layer.

22. The method of claim 8, wherein sterilizing the multilayer sheet comprises using electron beam radiation to achieve about 10 -6 Sterility guaranteed.

23. The method of any one of claims 8-22, wherein drying the multilayer sheet to form a dewatered sheet comprises using a vacuum pressure of about 50 mbar to about 350 mbar at a temperature of about 22 degrees Celsius to about 35 degrees Celsius.

24. The method of any one of claims 8-23, further comprising winding the multilayer sheet to form a tube.

25. A method for manufacturing a multi-layer amniotic membrane product, characterized in that: include: washing multiple layers of amniotic membrane, wherein the layers of amniotic membrane are obtained from an amniotic sac; cutting the amniotic membrane layer; overlaying the washed and cut amniotic membrane layers onto each other to form a multilayer sheet having at least four layers and less than ten layers, wherein the washed and cut amniotic membrane layers are oriented such that the outermost layers of the multilayer sheet are amniotic membrane layers, and the outermost amniotic membrane layers are oriented such that the epithelial layer of each of the outermost amniotic membrane layers faces outward; drying the multilayer sheet to form a dehydrated sheet; and The multi-layer sheet is sterilized to form the multi-layer amniotic membrane product.

26. The method of claim 25, wherein the multilayer sheet comprises five, six or seven layers.

27. The method of claim 25 or 26, wherein adjacent amniotic membrane layers are oriented such that the epithelial layer of a first adjacent amniotic membrane layer is oriented facing the fibroblastic layer of a second adjacent amniotic membrane layer.

28. The method of any one of claims 25-27, wherein adjacent amniotic membrane layers are oriented such that the fibroblast layer of a first adjacent amniotic membrane layer is oriented facing the fibroblast layer of a second adjacent amniotic membrane layer.

29. A dehydrated multi-layer amniotic membrane product, characterized in that: include: a plurality of layers oriented upon one another, wherein the plurality of layers comprises at least four amniotic layers, Each of the amniotic membrane layers comprises an epithelial layer, a basement membrane and a fibroblast layer, and wherein an outermost layer of the plurality of layers is an amniotic layer, and wherein the outermost amniotic layer is oriented such that the epithelial layer faces outward.

30. The multi-layer amniotic membrane product of claim 29, wherein the plurality of layers consists of five amniotic membrane layers.

31. The multilayer amniotic membrane product of claim 29, wherein the plurality of layers comprises four, five, six, or seven layers.

32. The multilayer amniotic membrane product of any one of claims 29-31, wherein adjacent amniotic membrane layers are oriented such that the epithelial layer of a first adjacent amniotic membrane layer is oriented facing the fibroblastic layer of a second adjacent amniotic membrane layer.

33. The multilayer amniotic membrane product of any one of claims 29-32, wherein adjacent amniotic membrane layers are oriented such that the fibroblast layer of a first adjacent amniotic membrane layer is oriented facing the fibroblast layer of a second adjacent amniotic membrane layer.

34. The multilayer amniotic membrane product of any one of claims 29-33, wherein the amniotic membrane layers are obtained from a human source.

35. The multilayer amniotic membrane product of any one of claims 29-33, wherein the amniotic membrane layers are obtained from a non-human source.

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