This patent application claims priority from U.S. non-provisional patent application Ser. No. 18/347,279, filed on 7.5, 2023, which is hereby incorporated by reference in its entirety, in accordance with 35U.S. C. ≡120 claiming priority from U.S. provisional patent application Ser. No. 63/367,940, filed on 7.8, 2022.
Detailed Description
The present invention provides a multi-layered tissue product and a method of making such a product. The multi-layered tissue product may be any type of tissue, such as a membranous tissue graft for tissue repair, reconstruction, or protection. Various embodiments of the present disclosure are generally directed to multi-layered amniotic membrane products that may be used to aid in repairing tissue to assist in repairing nerves.
In the present disclosure, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. The term "exemplary" refers to "exemplar" rather than "ideal". The terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a composition, method, or process that comprises a list of elements or steps does not include only those elements or steps, but may include other elements or steps not expressly listed or inherent to such composition, method, or process. Unless otherwise indicated in the specification, relative terms such as "about" and "about" are generally used to indicate possible variations of ±10% of the stated or understood value. In addition, the term "between" as used to describe a range of values is intended to encompass the minimum and maximum values described herein. The use of the term "or" in the claims and specification is used to mean "and/or" unless explicitly indicated to refer to only the alternative or to the alternative being mutually exclusive, although the disclosure supports definitions of only the alternative and/or. As used herein, "another" may mean at least a second or more.
Embodiments of the present disclosure may relate to dehydrated multilayer placental amniotic membrane or amniotic/chorionic products, such as sheets, which may be configured to be placed around damaged tissue, such as damaged nerves, including damaged peripheral nerves. A multi-layered amniotic membrane or amniotic/chorionic product, such as a sheet, may be used as a tissue covering to serve as an anatomical barrier to help provide protection from environmental effects.
Examples of tissues of the multi-layered amniotic membrane or amniotic membrane/chorionic product described herein that may be used include neural tissues, such as peripheral neural tissues or central nervous system tissues. Other types of tissue suitable for use in the present disclosure include, but are not limited to, epithelial tissue, connective tissue, muscle tissue, tendon tissue, ligament tissue, vascular tissue, intestinal tissue (including but not limited to the anterior stomach), dermal tissue, and cardiac tissue.
Notably, although much of the disclosure discusses a multi-layer product formed from amniotic membrane or amniotic membrane and chorion, the disclosure is not so limited. For example, the multi-layer product may be formed from one or more different types of tissue, such as layers including, but not limited to, the various types of tissue mentioned in the preceding paragraphs. Furthermore, the tissue may include mammalian tissue, including human tissue and other primate tissue, rodent tissue, horse tissue, dog tissue, rabbit tissue, pig tissue, sheep or other ruminant tissue. In addition, the tissue may be a non-mammalian tissue selected from fish, amphibian or insect tissue. For the subject into which the graft is implanted, 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 may be used in place of or in addition to the amniotic or amniotic and chorionic tissue layers, such as those described herein. The products made from the alternative tissue types discussed in this and/or the preceding paragraphs may include layers similar to those described herein with respect to the multi-layer amniotic product.
The multi-layer amnion (MLA) product may be a dehydrated sheet composed of at least four layers, e.g., four to ten layers, four to seven layers, at least four and less than ten layers of placenta-sac amniotic membrane or amniotic membrane and chorion, as will be discussed further below. The term multi-layered amniotic membrane or MLA refers to a tissue product consisting of all amniotic layers or a tissue product consisting of amniotic layers and chorionic layers. In both product types, the outermost layers (e.g., top and bottom layers) are amniotic layers. In some aspects, the multi-layered amniotic membrane product may 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 placenta-sac amniotic membrane or amniotic membrane and chorion. The placenta-derived amniotic membrane and/or chorion may be obtained from human sources, or alternatively, from non-human sources, or from a mixture of human and non-human sources. In some embodiments, the layers may be alternating human placenta-derived amniotic membrane and chorion.
The amniotic membrane layer of the present disclosure is a constituent layer of the cross section of the human amniotic placenta sac 100 shown in fig. 1. The amniotic membrane layer 102, as used herein, refers to the portion shown in fig. 1, and includes an epithelial layer 104, a basement membrane 106, and a fibroblast layer 108. Chorion layer 110, as used herein, includes reticulum layer 112 and optional basement membrane 114, but desirably omits most, if not all, of the cells of trophoblast 116. For example, during harvesting, chorion layer 110 may separate from cells of trophoblast 116. As shown, the dense layer 120 may separate the basement membrane 106 of the amniotic membrane layer 102 from the fibroblast layer 108. Additionally, the sponge layer 118 may be positioned between the amniotic membrane layer 102 and the chorion layer 110 (e.g., between the fibroblast layer 108 and the mesh layer 112). The fibroblast layer 108, the sponge layer 118 and the reticulum layer 112 are interstitial cells 122. A first side of the placenta bladder 100, such as the topside in fig. 1 (e.g., the outer portion of epithelial cells in the epithelial layer 104 of the amniotic layer 102) may be adjacent to the amniotic cavity 140. A second side of the placenta capsule 100, such as the bottom side in fig. 1 (e.g., the outer portion of the decidua layer 130), may be adjacent to the uterus 150.
For example, the amniotic layer 102 may be harvested from the amniotic placenta sac 100 (fig. 3A) by physical separation at the sponge layer 118. Chorion layer 110 may be harvested from decidua layer 130 of amniotic sac 116 by physical separation at trophoblast 116. Trophoblast cells remaining on the harvested villus layer 110 may be substantially removed. To harvest the layers, the placenta may be oriented such that the amniotic membrane layer faces outward. Manual removal manual debridement may then be performed to isolate the amniotic membrane. If chorion layers are also harvested, they may also be manually separated from the placental tissue. A cell scraper or other suitable tool may also be used during the removal.
The removed amniotic membrane layer 102 and chorion layer 110 may be washed after separation from the placenta capsule 100. In some embodiments, the amniotic membrane layer 102 or chorion layer 110 may be cut prior to washing. For example, one or more templates can be used to identify portions of a layer that can be cut to a suitable size for use, and a layer of tissue can be cut according to the templates. The amniotic membrane or amniotic membrane and chorion layers may be cut using a scalpel blade, a die cutter (die cutter), a pneumatic press, or any other suitable instrument. In some embodiments, the amniotic membrane layer 102 or chorion layer 110 may be cut after washing. In other embodiments, the amniotic membrane layer 102 or chorion layer 110 may be cut before and after washing. In some embodiments, the amniotic membrane layer 102 or chorion layer 110 may be washed before and after cutting.
After harvesting, the amniotic membrane layer 102 and chorion layer 110 may be cleaned using one or more solutions to clean the amniotic membrane layer 102 and chorion layer 110. In one embodiment, two solutions may be used to clean one or more of the amniotic membrane layer 102 or the chorion layer 110. The first wash solution may contain about 0.5% polyoxyethylene sorbitol ester (polyoxyethylene sorbitol ester) such as Tween 20 (v/v), about 0.05% polyhexamethylene biguanide (polyhexamethylene biguanide, PHMB) (w/v), or alternatively a solution of about 0.5% lohuxetine (w/v), about 0.9% NaCl (w/v), about 10mM 1, 3-bis (tris (hydroxymethyl) methylamino) propane, and 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 a pH of about 4.5 to 7.0 (e.g., pH 5.6). The second wash solution may include about 0.144g/L KH2PO4, about 9g/L NaCl, and about 0.795g/L Na2HPO4. In another example, the second solution may contain about 0.9% NaCl (w/v) and have a pH of about 4.5 to 7.0 (e.g., pH 5.6).
For example, if the amniotic membrane or amniotic membrane and chorion layers are not cut prior to washing, the amniotic membrane or amniotic membrane and chorion layers may be cut after washing and superimposed on each other to produce a multi-layered amniotic membrane product 200, as shown in fig. 2. In some aspects, the multi-layered amniotic membrane product may include a minimum of four total layers of amniotic membrane or amniotic membrane and chorion. In some embodiments, the amniotic membrane or amniotic membrane and chorion layers may be superimposed on each other to produce four to ten layers, or four to seven layers, for example four, five, six, or seven layers. The number of layers of the multi-layer amniotic product may affect the handling characteristics of the multi-layer amniotic product, as further described below with reference to fig. 4. One or more support structures (e.g., lamination tools (layering tool)) may be used to help cover and/or position the amniotic membrane or amniotic membrane and chorion layers.
The multi-layer amniotic membrane product may contain an amniotic membrane layer 102 on one exposed side, which is oriented such that the epithelial layer 104 faces outwardly. The opposite exposed side of the multi-layer amniotic membrane product may contain another amniotic membrane layer 102, which may also be oriented with the epithelial layer 104 facing outward. Thus, the orientation of the outermost layers of the MLA product may be such that the amniotic membrane layer 102 is the outermost layer on both sides of the multi-layer amniotic membrane product. The outermost amniotic layers 102 on both sides of a multi-layer amniotic product, e.g., sheet, may be oriented such that the epithelial layers 104 face outward. Thus, for a sheet having a top surface and a bottom surface, the top surface may comprise epithelial cells and the bottom surface may comprise epithelial cells. In these aspects, the epithelial layer 104 may be smoother and/or more slippery than the inward facing layers of the amniotic layer 102 and the chorion layer 110. Having a top surface and a bottom surface that contain epithelial cells from the epithelial layer 104 may allow the multi-layer amniotic membrane product to have no "sidedness". For example, the multi-layer amniotic membrane can be applied with the top or bottom surface facing toward or away from the tissue to be applied during use.
The amniotic membrane layer has two sides. One side contains epithelial cells and the other side contains a fibroblast layer. When the amniotic layers are overlapped to form a multi-layer amniotic product, the adjacent amniotic layers may be oriented such that the epithelial portion of the first amniotic layer is adjacent to the fibroblast portion of the second amniotic layer, or such that the fibroblast portion of the first amniotic layer is adjacent to the fibroblast portion of the second amniotic layer. The adjacent first and second amniotic membrane layers may not be layered in the epithelial-epithelial direction to avoid delamination (delamination).
In some embodiments, the organization of layers within the multi-layer amniotic membrane product may be such that the amniotic and chorionic layers are repeated, i.e., similar layers are organized adjacent. For example, a combination of layers of a five-, six-, or seven-layer MLA product may be organized as follows:
five layers AACCA, AACAA, AAACA, ACACA, AAAAA or ACCCA.
Six layers AACCAA, AAACCA, AAAACA, AACAAA, ACCCCAA, ACCCCA or AAAAAA.
Seven layers AAACAAA, AAAACAA, AAAACA, AACCAAA, AACCCAA, AAACCCA, AACCCCA, ACCCCCA or AAAAAAA.
In the above examples, each a represents an amniotic membrane layer (e.g., amniotic membrane layer 102) and each C represents a chorion layer (e.g., chorion layer) 110. Each amniotic membrane 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 chorion layer may include a mesh layer (e.g., mesh layer 112). In some embodiments, each chorion layer may include a substrate layer in addition to the mesh layer. In some embodiments, one or more, but not all, of the chorion layers include a substrate layer (e.g., the basement membrane 114) in addition to the mesh layer. In any of the above embodiments, one or more chorion layers may be substantially depleted of trophoblast cells (e.g., trophoblast 116).
In some embodiments, the cover of tissue including, but not limited to, the cover of amniotic membrane or the cover of amniotic membrane and chorion may then be dried to form a dehydrated sheet. For example, while any suitable drying method may be used, the overlay layer may be dried (e.g., at a pressure less than atmospheric pressure) 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, to dry the multi-layer tissue or, for example, the multi-layer amniotic membrane product. Exemplary vacuum pressures for drying may be, for example, about 50 millibars to about 350 millibars.
Subsequent dehydrated tissue sheets composed of amniotic membrane or amniotic membrane and chorion layers may then be sterilized using appropriate methods. In one embodiment, electron beam irradiation may be used to achieve sterility assurance of about 10-6.
Methods of forming a multi-layered amniotic product may include separating amniotic membrane and chorion layers from placenta and amniotic sac, washing the amniotic membrane and chorion layers, stacking them on top of each other in an alternating or repeating order, and dehydrating the amniotic membrane and chorion layers after stacking on top of each other. In embodiments where only the amniotic layer is used, the method of forming the multi-layered amniotic product may include separating the amniotic layer from the placenta and the amniotic sac, washing the amniotic layer, stacking them on top of each other, and dehydrating the amniotic layer after stacking. For example, a method of forming a multi-layered amniotic product may include one or more of harvesting the amniotic sac and placenta, and separating the chorion layer from the decidua at the trophoblast. The amniotic membrane layer may include all, at least some, or none of the layers. If a chorion layer is used, trophoblast cells on the chorion layer can be substantially removed. For example, chorion layers for multi-layer amniotic products may not include trophoblast cells. In other aspects, the chorion layer for the multi-layer amniotic membrane product may include some trophoblast cells. Further, in various aspects, the chorion layer may include all, at least some, or none of the sponge layer.
The method may further comprise washing the amniotic membrane or amniotic membrane and chorion layers with one or more cleaning solutions, for example two cleaning solutions, examples of which are described above. The cut and cleaned layers may be overlaid on top of each other to form a multi-layer amniotic membrane product such as a sheet. The multi-layer amniotic membrane product may comprise at least four or at least five layers, for example, 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. The layers may be oriented such that the amniotic membrane layer is the outermost layer on both sides of the multiwall sheet. The outermost amniotic layers (e.g., top and bottom layers) on both sides may be oriented with the epithelial layers facing outward. To the extent that adjacent amniotic layers are present within the multi-layered amniotic product, the adjacent amniotic layers may be oriented such that the epithelial layer of a first adjacent amniotic layer faces the fibroblast layer of a second adjacent amniotic layer, or such that the fibroblast layer of a first adjacent amniotic layer faces the fibroblast layer of a second adjacent amniotic layer. The multi-layer amniotic membrane product may then be dried to form a dehydrated sheet. In some embodiments, drying the multi-layer 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 celsius to about 35 degrees celsius. The multi-layer amniotic membrane product may be sterilized, for example, using electron beam radiation or other suitable sterilization methods.
Fig. 2 shows a seven-layer multi-layer amniotic membrane prototype 200 of the sample. Fig. 3A shows a human placental amniotic sac, for example, human amniotic placental sac 100. Fig. 3B shows a histological image of hematoxylin and eosin staining of harvested amniotic placenta sac 100 with all layers, e.g., including at least amniotic membrane layer 102, chorion layer 110 and decidua layer 130. Fig. 3C shows histological images of hematoxylin and eosin staining of the harvested isolated amniotic membrane layer 102. Fig. 3D shows a histological image of hematoxylin and eosin staining of the separated reticular layer 112 of the harvested chorionic layer 110. Fig. 3E shows a histological image of hematoxylin and eosin staining of a five-layer amniotic membrane product 300. As described above, the multi-layer amniotic membrane product may include only multiple amniotic membrane layers 102 or be combined with one or more chorion layers 110 (e.g., mesh layer 112 of chorion layers 110).
Fig. 4 is a graph comparing various properties of multi-layer amniotic membrane products having different layers. As shown, the multi-layer amniotic membrane product tested included a four-layer MLA product, a five-layer MLA product, and a six-layer MLA product. Four-layer MLA products, five-layer MLA products, and six-layer MLA products may be formed via the steps and procedures discussed herein. The four-layer MLA product is formed from an AACA-oriented amniotic membrane layer and a chorion layer, where A represents the amniotic membrane and C represents the chorion. Five-layer MLA products were formed from AACAA and AACCA oriented amniotic and chorionic layers, where a represents amniotic membrane and C represents chorionic membrane. The six-layer MLA product is formed from AAACCA and ACACAA oriented amniotic and chorionic layers, where a represents amniotic membrane and C represents chorionic membrane. The dehydrated umbilical cord membrane products were also tested for comparison. The dehydrated umbilical cord membrane product was about 3cm x 6cm, while the four, five, six layer MLA product was about 4cm x 8cm.
As shown, the visual appearance, layering, clarity, integrity, repositionability, durability, seamability, and overall impression of each multi-layered amniotic membrane product were evaluated or assessed. Each multi-layered amniotic membrane product was rated on a scale between, for example, very good (e.g., 4), acceptable (e.g., 3), slightly unacceptable (e.g., 2), and unacceptable (e.g., 1, not shown in fig. 4).
The visual appearance is assessed based on visual inspection of the tissue layers of the multi-layered amniotic membrane product by the user. For example, the tissue layer should be pale yellow or white and determine if there is any discoloration. Visual inspection of whether there are red blood filaments, whether the tissue color is consistent, whether there are holes, tears, etc. in the tissue.
The layering is evaluated based on whether the tissue layers of the multi-layered amniotic product are layered (e.g., separated from adjacent tissue layers) during processing. In these aspects, fewer layers may help ensure that the multi-layer amniotic membrane product retains its shape and interlayer connection during processing. Delamination may negatively impact the ability of the user to handle and/or apply the multi-layered amniotic membrane product. Score 4 corresponds to no layering, score 3 corresponds to some visible layering, but the organization is substantially unchanged (e.g., the corners or edges of the organization product separate, but the organization product remains available as a whole), score 2 corresponds to significant layering, and score 1 corresponds to complete layering (organization product cannot be used and layers are completely separated).
The evaluation of clarity is based on the clarity of the tissue (e.g., the tissue forming the multi-layered amniotic membrane product) after, for example, hydration. The transparency can help a user visualize and/or locate tissue (e.g., neural tissue) under and/or around the multi-layered amniotic membrane product. Thus, clarity may make the multi-layer amniotic membrane product easier to use. A score of 4 corresponds to tissue being transparent, a score of 3 corresponds to tissue being slightly transparent, a score of 2 corresponds to tissue being slightly opaque, and a score of 1 corresponds to tissue being opaque. In use, the transparency may allow a user to see the underlying structure to which the MLA product is applied or wrapped around. This may make the MLA product easier to use in situations such as nerve engagement, as the user is able to visualize the location of nerve endings within the MLA product as well as relative to each other.
The assessment of integration is based on satisfaction of tissue integration or sagging (drapability) (e.g., sagging across the midline). The greater degree of integration or drape may help the multi-layered amniotic membrane product to surround or integrate the shape or size of tissue (e.g., neural tissue). For example, a score of 4 corresponds to satisfactory, fully drawdown, a score of 3 corresponds to slightly satisfactory drawdown, a score of 2 corresponds to slightly unsatisfactory drawdown, and a score of 1 corresponds to unsatisfactory drawdown, such as stiff sheet.
The assessment of repositionability is based on the ability of tissue (e.g., tissue forming a multi-layered amniotic membrane product) to be straightened and/or repositioned after folding. For example, a score of 4 corresponds to no folding or clumping occurring, a score of 3 corresponds to some folding or clumping occurring, but folding or clumping is relatively easy to handle, a score of 2 corresponds to tissue portions folding or clumping together, and a score of 1 corresponds to tissue fully folding upon itself. In these aspects, a greater degree of repositionability may help the multi-layer amniotic membrane product to be straightened and/or repositioned, e.g., more properly positioned on the tissue.
The evaluation of durability is based on the assessed durability of the tissue (e.g., the tissue forming the multi-layered amniotic membrane product). For example, the assessment of the durability of tissue includes pulling the tissue (e.g., pulling a suture that has passed through the tissue) and assessing the ability of the tissue to withstand tearing of the suture through the tissue. For example, a score of 4 corresponds to the tissue being very durable, a score of 3 corresponds to the tissue being durable, a score of 2 corresponds to the tissue being somewhat durable (e.g., some tearing is seen), and a score of 1 corresponds to the tissue being not durable (e.g., tissue spreading). In these aspects, the greater degree of durability can facilitate the multi-layered amniotic membrane product being positioned, sutured, pulled, and/or otherwise manipulated multiple times before, during, or after implantation in a patient and/or in multiple directions.
The evaluation of the stitchability is based on the assessed stitchability of the tissue (e.g., the tissue forming the multi-layered amniotic membrane product). For example, assessed suturability of tissue includes whether a suture can pass through the tissue, and/or whether the tissue tears during suturing. A high score corresponds to the ability to push the suture through the tissue product and to resist tearing or unraveling once the suture is passed. For example, score 4 corresponds to a satisfactory suture (e.g., suture is easy to pass and then hold in place and does not tear tissue product), score 3 corresponds to a slightly satisfactory suture (suture is slightly more difficult to pass and/or slightly tear), score 2 corresponds to a slightly unsatisfactory suture (suture is more difficult to pass and/or more tear), and score 1 corresponds to an unsatisfactory suture (e.g., suture does not pass and/or passes directly and tears). In these aspects, a greater degree of stitchability may facilitate suturing the multi-layered amniotic membrane product to tissue (e.g., nerve tissue) and/or itself, e.g., to surround tissue.
The evaluation of the overall impression is based on the assessed overall treatment of the multi-layered amniotic membrane product. For example, the assessed overall impression includes an assessment of the usability of the multi-layered amniotic membrane product. For example, a score of 4 corresponds to a tissue product having a very good availability, a score of 3 corresponds to a tissue having a good availability, a score of 2 corresponds to a tissue having a slight availability, and a score of 1 corresponds to the tissue not having a good availability. In these aspects, a greater degree of availability may aid in the use of the multi-layered amniotic membrane product for treating tissue (e.g., neural tissue).
As shown in fig. 4, five-layer MLA products exhibit consistent high scores in each category, e.g., a score of at least 3.5 for each category. These scores are based on a user (e.g., surgeon) extensive assessment of each type of multi-layered amniotic membrane product. Furthermore, the user means that four, five and six layer MLA products feel thinner and/or easier to handle than dehydrated umbilical cord membrane products. Exemplary MLA products may have a thickness of about 30 microns to about 120 microns, such as about 90 microns to about 100 microns, or about 60 microns to about 80 microns. Furthermore, the user indicates that the dehydrated umbilical cord membrane product does not use sutures, but rather uses mini-clips (microclip), fibrin glue, or secures the dehydrated umbilical cord membrane product by coating itself, due to less consistency in thickness and other aspects with the dehydrated umbilical cord membrane product. In contrast, four, five and six layer MLA products exhibited very good or very satisfactory stitch scores.
In other aspects, four, five and six layer MLA products are more consistent in appearance and/or clarity than dehydrated umbilical film products. In addition, the delamination problem is limited or absent for four, five and six layer MLA products. 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 characteristics of the MLA product. Four-layer MLA products were rated as slightly unacceptable in terms of repositionability and overall impression. Five-layer and six-layer MLA products achieved a highly acceptable assessment of all categories or attributes. Based on this evaluation, a five-layer MLA product achieves optimal overall feedback, e.g., balancing the trade-off between treatment quality and the number of implanted/resorbed tissues (i.e., layers).
Overall, it was found that if too many layers were used in a multi-layer amniotic membrane product, the resulting product would not properly hang or fit. Or if too few layers are used, the handleability may be reduced because the resulting product may tear or tear and may lack durability, repositionability, and/or seamability. These characteristics may be less important for external wound care products, but embodiments of the present disclosure may be applicable to internal tissues such as nerves. Thus, forming a product with a greater number of layers makes it easier for a surgeon to treat, position, and/or fix the multi-layered amniotic membrane product of the present disclosure with respect to delicate tissue structures such as nerves, while still maintaining sufficient drape to, for example, wrap around the nerves.
In these aspects, four, five and six layer MLA products have the trophoblast 116 of the chorion layer 110 substantially removed. Thus, four, five and six layer MLA products consist of birth tissue (birth tissue) membranes and minimal unstructured tissue. Other commercially available amniotic membrane products do not consist for the most part of a childbirth tissue membrane with little or no unstructured 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 be better able to retain their shape than commercially available products that may shrink and have low or no durability.
The layering tool may be used to help support and/or hold various layers of tissue, for example, to form the MLA products discussed herein. For example, four or more layers of amniotic membrane and/or chorion may be sequentially positioned over one another.
Fig. 5 is a flow chart of an exemplary method 500 of forming or manufacturing a multi-layer amniotic membrane product. Although not shown, the 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 removing it. The initial step may comprise collecting the amniotic membrane or amniotic membrane and chorion from the placental membrane. The initial step may include removing chorionic tissue, if included, to retain the reticular and basal membranes while also substantially removing the trophoblast. The initial step may comprise determining the dimensions of the amniotic membrane and chorion from the placental membrane. For example, the amniotic or chorion layer may be positioned over the layering tool.
The method 500 includes a step 502 that includes washing or otherwise performing a solution treatment procedure on the amniotic membrane layer or amniotic membrane and chorion tissue. As described above, cleaning may include the use of one or more cleaning solutions. For example, the first wash solution may contain about 0.5% polyoxyethylene sorbitol esters such as Tween20 (v/v), about 0.05% polyhexamethylene biguanide (PHMB) (w/v), or alternatively about 0.5% lohucidine (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 ester such as Tween20 (v/v) and about 10.0% NaCl (w/v), and a pH of about 4.5 to 7.0 (e.g., pH 5.6). The second cleaning solution may contain about 0.144g/L KH2PO4, about 9g/L NaCl, and about 0.795g/L Na2HPO4. In another example, the second solution may contain about 0.9% NaCl (w/v) and have a pH of about 4.5 to 7.0 (e.g., pH 5.6).
Step 502 may comprise thawing frozen amniotic membrane or chorionic membrane fragments, for example, in solution. Thawing may be performed at ambient temperature (e.g., 15 ℃ to 30 ℃) 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 amniotic membrane or chorion fragment may be thawed at a refrigeration temperature (e.g., 2 ℃ to 10 ℃) for about 48 hours to about 72 hours.
Additionally, step 502 may include, for example, washing the amniotic membrane or amniotic membrane and chorion layers one or more times in an aqueous saline solution. Step 502 may comprise culturing and/or agitating the amniotic membrane or amniotic membrane chorion layers in one or more solutions. For example, in some aspects, step 502 may comprise incubating the amniotic membrane or amniotic membrane and chorion layers in a first solution at ambient temperature (e.g., between about 15 degrees celsius and about 30 degrees celsius) for a period of time of 15 minutes to about 2 hours, e.g., 20 minutes to about 2 hours, 30 minutes to about 1.5 hours, about 1 hour to about 1.5 hours. Culturing may also include, for example, stirring at about 80rpm to about 100rpm, such as about 90rpm to about 95 rpm. Step 502 may also comprise an optional holding step in the incubator, for example, for about 30 minutes to about 60 minutes, for example about 40 minutes.
Further, in some aspects, step 502 may include one or more additional incubation procedures. For example, step 502 may further comprise incubating the amniotic membrane or amniotic membrane and chorion layer in the second solution at ambient temperature (e.g., between about 15 degrees celsius and about 30 degrees celsius) for a period of time of about 5 minutes to about 1 hour, such as about 15 minutes to about 40 minutes. Culturing in the second solution may also include stirring, for example, at about 80rpm to about 100rpm, for example, about 90rpm to about 95 rpm. Step 502 may also include an optional holding step in the incubator with the second solution, for example, for about 30 minutes to about 60 minutes, for example, about 40 minutes. One or more of these culturing procedures may be repeated, for example, a second culturing procedure may be repeated, for example, three or more additional times.
The method 500 includes step 504, which includes cutting the amniotic membrane or amniotic membrane and chorion layers. The amniotic membrane or amniotic membrane and chorion layers may be cut to a desired size and/or shape. In some aspects, one or more templates may be arranged on the tissue, and the tissue may then be cut according to the template arrangement. Example template dimensions may be, for example, about 8 x 8cm, about 10 x 10cm, or about 12 x 12cm. The amniotic membrane or amniotic membrane and chorion layers may be cut using a scalpel blade, a die cutter, a pneumatic press, or any other suitable instrument. In some aspects, the cutting described herein may be performed prior to the cleaning step 502. Alternatively, a multi-layer amniotic membrane product may be formed (e.g., via the various steps of the methods discussed herein), and the multi-layer amniotic membrane product may then be cut to a desired size and/or shape.
The method 500 also includes a step 506 that includes covering the cleaned and cut amniotic membrane or amniotic membrane and chorion layers to form a membrane having at least four or at least five layers, such as four to ten layers, four to seven layers, or five to seven layers. Lamination tools can be used to help support the amniotic membrane or amniotic membrane layers to form a multi-layered amniotic membrane product. In some aspects, a layer of biocompatible non-placental material can be positioned between the base plate of the lamination tool and the first amniotic membrane layer. The first amniotic membrane layer may be oriented with the epithelial layer facing downward, opposite the location where the layers of more tissue are to be stacked.
If two layers of amniotic membrane are stacked on top of each other, adjacent layers of amniotic membrane may be oriented such that the epithelial portion of one adjacent layer of amniotic membrane faces the fibroblast portion of another adjacent layer of amniotic membrane, or oriented such that the fibroblast portion of one adjacent layer of amniotic membrane faces the fibroblast portion of another adjacent layer of amniotic membrane. Adjacent amniotic layers cannot be layered so that the epithelial portions face each other to avoid delamination. Notably, the intermediate layers of the chorion need not be oriented in a particular direction or arrangement. The final amniotic membrane layer may then be positioned over the amniotic membrane or other layers of the amniotic membrane and chorion. The last amniotic membrane layer may be oriented with the epithelial layer facing upward, away from the anterior tissue layer.
In some aspects, the top plate of the lamination tool may then be positioned on top of the amniotic membrane layer or amniotic and chorionic layers, for example, to help hold and/or compress the stacked layers. In some aspects, a layer of biocompatible non-placental material can be positioned between the top sheet and the multi-layer amniotic membrane product. In some aspects, at least four or at least five layers of amniotic membrane or amniotic membrane and chorion may be covered, with the epithelial cells of the outermost amniotic layer facing outward on both sides of the covered layer. In some embodiments, the layers may be positioned on a biocompatible sheet of material, and the layers and the biocompatible sheet of material may be positioned on a lamination tool. In some aspects, the sheet of biocompatible material may be removed after positioning the layers on the lamination tool.
The method 500 includes step 508, which includes drying the amniotic membrane or stacked layers of the amniotic membrane and chorion to form a dehydrated sheet of a multi-layered amniotic membrane product. Drying may be performed via vacuum drying. In these aspects, the amniotic membrane layer or layers, or chorion layers, may be positioned within a dry or vacuum bag. Suitable drying or vacuum bags and drying methods are described, by way of example, in U.S. provisional application No.63/477566 filed on month 29 of 2022, the entire contents of which are incorporated herein by reference.
In one or more aspects, drying may be performed for about 16 hours to about 24 hours (e.g., one day and/or overnight). Drying may be performed at a temperature between about 15 degrees celsius and about 35 degrees celsius. Drying can be performed at an exhaust flow rate of between about 30SLPH and about 340SLPH, such as about 50 SLPH. The drying may be performed at a vacuum pressure of between about 10inHg and about 28 inHg. In some aspects, drying may be performed at a vacuum pressure of greater than 25 inHg. It is noted that the above drying steps and procedures are merely exemplary, and that one or more different drying steps or procedures may be performed to dry the multi-layered amniotic membrane product. Further, although step 504 is shown before steps 506 and 508, it should be noted that step 504 can alternatively be performed after step 506 or after step 508. In some aspects, the cutting step 504 may be performed again after step 508 or after step 510, as described below. For example, the first step 504 may be performed to cut the amniotic membrane and/or chorion layers to stack with respect to each other, and the second step 504 may be performed after the drying step 508 or after the step 510 to cut the MLA product into a specific product shape and/or size.
Furthermore, the method 500 comprises a step 510, the step 510 comprising sterilizing the multi-layered amniotic membrane product, for example, after drying the multi-layered amniotic membrane product. For example, after sufficient drying, the multi-layer amniotic membrane product may be sterilized, for example, using electron beam radiation. Step 510 may also include placing (e.g., sealing) the multi-layer amniotic membrane product in, for example, at least one pouch. For example, the multi-layer amniotic membrane product may be placed in one or more foil bags. In some aspects, the multi-layer amniotic membrane product may be placed in an inner foil pouch, which may then be placed in an outer foil pouch.
In some aspects, the method 500 may include a second cutting step 504 after step 508 or 510. The dried and/or sterilized multi-layered amniotic membrane product may be cut into, for example, one or more pieces. For example, the multi-layer amniotic membrane product may be cut into an inner portion and a sacrificial portion (SACRIFICIAL PORTION). For example, the multi-layer amniotic membrane product may be cut using a scalpel blade, a cutting die, a pneumatic die, or other suitable cutting device. In addition, the multi-layer 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 about 4cm by about 6cm, about 3cm by about 4cm, about 2cm by about 2cm, etc. In some aspects, various cutting procedures may be performed on the cutting surface.
As shown in fig. 6, method 600 includes the various steps of method 500 that may be performed using only the amniotic membrane layer, for example, to form or manufacture a multi-layer amniotic membrane product without any chorion layer. In these aspects, the method 600 comprises a step 602, the step 602 comprising washing the amniotic membrane tissue layer. The method 600 further comprises step 604, step 604 comprising cutting the amniotic membrane layer. The method 600 includes step 606, step 606 comprising overlapping the washed and cut amniotic membrane layers to form a multi-layer amniotic membrane product having at least four or at least five layers. Additionally, the method 600 includes a step 608, the step 608 including drying the multilayer sheet to form a dehydrated sheet. In addition, the method 600 comprises step 610, step 610 comprising sterilizing the resulting multi-layered 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 previously described with respect to method 500. Method 600 can be performed in a similar manner as method 500, except that method 600 can be applied to an amniotic membrane-only multi-layer product.
The multi-layer amniotic membrane products of the present disclosure (e.g., multi-layer amniotic membrane dehydrated products) may be used as a tissue covering for placement around damaged tissue, e.g., damaged nerves, such as peripheral nerves. The multi-layered amniotic membrane product may consist of, for example, four to ten, four to seven, five to ten, or five to seven separate amniotic membrane layers or amniotic and chorionic membrane layers which have been harvested from, for example, human amniotic sacs. As discussed above, the multi-layered amniotic membrane product may consist of five layers of isolated human amniotic membrane, or amniotic membrane and chorion layers harvested from human amniotic sacs or alternatively from a non-human source. The amniotic membrane layer may include an epithelial layer, a basement membrane, and a fibroblast layer. The chorion layer (if included) may include a mesh layer and a basement membrane. In some aspects, these films can be laminated into a multilayer graft in various arrangements as described above.
The multi-layered amniotic membrane product may be configured to be placed around the peripheral nerve of the lesion as a tissue covering to serve as an anatomical barrier to help provide protection from the surrounding environment. The multi-layer amniotic membrane product may be formed as a sheet and then rolled into a tube, cap or other structure prior to placement around tissue, or may be wrapped around tissue during placement. The multi-layer amniotic membrane product may have any suitable size or shape, which may depend at least in part on the intended use, such as the size or type of tissue intended for use with the product.
Methods of repairing tissue using a multi-layer amniotic membrane product according to any of the embodiments 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 injured tissue. If the MLA product has not been formed into a tube, the method may further comprise cladding the MLA product into a tube around the damaged tissue. Once positioned around the damaged tissue, the MLA product may be secured to the damaged tissue. In some aspects, the damaged or injured tissue may be neural tissue, such as peripheral neural tissue.
The multi-layered amniotic membrane product may have superior handling characteristics around tissues such as peripheral nerves compared to other membranes based on dehydrated amniotic sacs. Exemplary superior handling characteristics may include one or more of, for example, integration around tissue of a nerve, the ability to hold a suture, the ability to move freely as a sheet in a dehydrated or rehydrated state, the ability to be repositioned after placement on one or more tissues, and the like. Such handling characteristics may be less important when used for external purposes, such as for external wounds, but may be particularly important when surrounding or integrated into internal tissues such as nerves. For example, such properties similar to those tested in fig. 4 may allow a surgeon to locate, coat, reposition, fix, visualize, etc. the multi-layer amniotic product when locating the multi-layer amniotic product with respect to tissue within the body. 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 fragile internal tissues, such as nerves.
In addition, the multi-layered amniotic membrane products of the present disclosure may have resorption kinetics (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, for example, about four to eight weeks, or about six to eight weeks. Although products for application to wounds can be reapplied at regular time periods, products applied internally, for example for nerves, cannot be reapplied without surgical intervention. Thus, embodiments of the present disclosure may have a resorption time that allows them to remain in place long enough for the tissue to heal.
The methods and multi-layered amniotic products may also achieve one or more of (1) removing and separating placental membrane layers without damaging the placental membrane layers, (2) layering the amniotic membrane or amniotic membrane and chorion together in an efficient manner, (3) determining the orientation (epithelial side or spongy side) of the amniotic membrane layers, (4) maximizing the yield of overlapping tissue layers (e.g., amniotic membrane layers or amniotic membrane layers and chorion layers having four or more layers stacked).
Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.