MANUFACTURE

FIELD OF THE INVENTION

[0001] The disclosure relates generally to the field of acellular regenerative products, methods of their manufacture, and use in treatment.

BACKGROUND

[0002] Human placental membrane, such as amniotic membrane or tissue, has been used for various types of reconstructive surgical procedures since the early 1900s. The membrane can serve as a substrate material, more commonly referred to as a biological dressing or graft patch. Typically, such membrane is either frozen or dried for preservation and storage until needed for surgery, and is in the form of a sheet of material, which may have been processed through thinning or chemical treatments, including adding growth promoters or other biological agents. Several drawbacks of using large intact membranes are non-optimal wound coverage, adhesion, and release of factors into relevant tissues, as well as reduced production efficiency and storage stability.

[0003] Due to the drawbacks of intact membranes, cell-based treatments have also been used. These treatments take placental membranes and extract intact cells, stabilizing them for future applications, e.g. wound treatment. However, conventional cell treatments, such as recovered stem cells and placental cells, have low effectiveness because it is difficult to extract, maintain, and deliver viable whole cells in a medical context. In many cases, the cells form a barrier between key therapeutic cellular agents and a wound site.

SUMMARY

[004] An acellular product may be derived from human placenta and may be used in various scenarios for wound healing. Because the product may be acellular, the product may be processed for storage and transportation with minimal degradation. The product may include various scaffolding such as biomaterials or human tissue, and the scaffolding may be infused with various plasmas and agents. The cell-free treatment may maintain the biological activity of many therapeutic agents found within cells and may possess multiple structural components to support cellular attachment. The structural components or scaffolds may function as a reservoir of highly diffusible chemotactic and cellular-programming factors that may be useful to treat injury and disease. In many cases, fibrinogen may be absent, which may reduce scarring.

[005] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

[006] FIG. 1 depicts the effect of removal of water content from platelet poor plasma (PPP) to 10% of the original volume (PPPc), with increased pg/mL of growth factors TFG-Bl and VEGF. [007] FIG. 2 depicts the effect of activation of whole blood or platelet rich plasma with thrombin followed by the removal of water content from platelet poor plasma to 10% of the original volume (A-W.B.c), with further increased pg/mL of growth factors TGF-B1 and VEGF.

[008] FIG. 3A depicts a histological analysis of a porcine study treating porcine dermal incision injuries with the product having 0.5mL amniotic fluid per square centimeter amniotic membrane. Full thickness dermal regeneration and reductions in scar tissue and fibrosis were observed, with significant difference from sutured, non-product controls at both 2 and 10 days post operation. FIG. 3B shows histological analysis of the same study, but for the rear incisions made during the study. FIG. 3C shows the combined results of the study.

[009] FIG. 4A depicts a histological analysis of a porcine study using two embodiments.

LotlA is 0.5mL amniotic fluid per square centimeter amniotic membrane. LotlB is 2.0mL amniotic fluid per square centimeter amniotic membrane. The figure shows improved scar tissue across experimental samples and particularly at the 0.5mL/cm ² concentration. FIG. 4B shows improved scar tissue and fibrosis across experimental samples, and particularly at the 0.5mL/cm ² concentration. FIG. 4C shows reduced neovascularization across experimental samples at 31 days, and particularly at the 0.5mL/cm ² concentration.

DETAILED DESCRIPTION

Overview

[0010] An acellular product may be derived from human placenta and may be used in various scenarios for wound healing. Because the product may be acellular, the product may be processed for storage and transportation with minimal degradation. The product may include various scaffolding such as biomaterials or human tissue, and the scaffolding may be infused with various plasmas and agents. The cell-free treatment may maintain the biological activity of many therapeutic agents found within cells. The product may possess multiple structural components to support cellular attachment, and these structural components or scaffolds may function as well as a reservoir of highly diffusible chemotactic and cellular-programming factors that may be useful to treat injury and disease. In many cases, fibrinogen may be absent, which may reduce scarring.

[0011] Scaffolds may be selected for a number of properties, such as biocompatibility, biodegradability, mechanical properties, and architectural properties such as degree of porosity. Exemplary scaffolds include human extracellular matrix derived from amniotic cells, which may contain a favorable substrate for cell migration and subsequent wound repair. One example of scaffolds may comprise the intermediate layer of an amniotic membrane. Other exemplary scaffolds are natural polymer scaffolds, synthetic scaffolds such as those made from synthetic polymers, and ceramic scaffolds.

[0012] The scaffold may be biocompatible with human cells, and may allow cells to adhere, function normally, and migrate through the surface and into the structure of the scaffold.

Scaffolds may also be biodegradable within the body, which may allow the body's cells to eventually replace the foreign scaffold with extracellular components appropriate to the specific local cell types. Various scaffolds may provide several functions, including maintaining growth factors in a stable conformation, supplying a platform for progenitor cell interaction, maintaining hydration of a wound-bed, mitigating fibrotic cell-type interaction, aiding in tissue formation and contractile collagen formation, as well as other functions.

[0013] Scaffolds may be infused with plasmas that may have super-physiological levels of natively interacting growth factors. Plasmas may be selected for a number of properties, such as including developmental components and factors. Plasmas may be human, animal, or of other composition. Plasmas may be derived from developmental tissues such as amniotic tissues, or may be derived from other tissues, such as blood. Plasmas may contain non-scaffold

components. These components may include a pool of free-state factors that may diffuse into the wound bed. These factors may include growth factors or other factors that may promote or may accelerate wound healing.

[0014] The process of generating the product may maintain many of the proteins, peptides, saccharides and anabolic free-state lipids in non-cross-linked, non-denatured states while mitigating the toxicity of unwanted solutes or salts. The product may or may not contain additives such as dimethyl sulfoxide (DMSO) or various detergents. Many processing sequences may not include a freezing step and may not be lyophilized.

[0015] Methods of manufacturing an acellular product may include processing an amniotic serum separately from processing an amniotic membrane, and combining the processed products. Methods may include removing residual cell content from a membrane, adding an amniotic fluid to create a suspension, and morselizing the membrane. In some embodiments, water content may be removed be removed through centrifugation or other process.

[0016] The product's combination of factors may trigger local progenitor cells of injured tissue to respond to injury as a "developmental-void", and the product may reduce inflammatory responses, fibrosis and scar tissue while accelerating wound closure with native soft tissue. This may give an advantage not only in dermal tissue repair but also in the repair of other soft tissues, including cornea, muscle, tendon and ligaments. In some embodiments, this may promote skin healing, and may be useful in cosmetic surgery, reconstructive surgery, treatment of burn wounds or other uses. In other embodiments, the product may be used to treat joint or bone ailments, and may be useful in treatment of arthritic conditions, fractures, or other ailments. The product may provide advantages in ease of use and application to wound-beds, simple storage requirements, and versatility during delivery, which may include solid, gel or liquid forms.

[0017] The plasma portion and particulate biological membrane portion may be derived from human tissue. When derived from human tissue, the plasma portion may be human plasma, and may comprise extracellular matrix or an amniotic plasma. The particulate biological membrane may comprise human developmental tissue, and may be a human amniotic membrane.

[0018] The product may comprise one or more adjuvant factors. These factors may be, for example, soluble or insoluble compounds found in the plasma, such as growth factors.

[0019] In some embodiments, the product may contain substantially no fibrinogen or fibrin.

[0020] The scaffold may comprise material from an amniotic membrane intermediate layer. This material may be amnion, chorion, or a mixture thereof. This particulate human amniotic membrane may be impregnated with human plasma-derived proteins.

[0021] The products can be in various forms, and in some embodiments are dry-solids. In some embodiments, the dry-solid is capable of gelling upon contact with water.

[0022] A method of manufacturing an acellular product may include processing an amniotic serum, processing an amniotic membrane, and combining the processed products. In some embodiments, processing an amniotic serum may comprise obtaining an amniotic serum, mechanically separating the serum into fractions, and isolating the serum fraction from the cellular fraction.

[0023] In other embodiments, processing the amniotic membrane may comprise obtaining an amniotic membrane, determining that the chorion may be completely or substantially removed, optionally removing any additional chorion, removing residual cell content, adding an amniotic fluid to create a suspension, and morselizing the membrane.

[0024] The process may additionally comprise adding material from an amniotic membrane intermediate layer. In other embodiments, the intermediate layer alone may be used as the amniotic membrane portion in the composition. In yet other embodiments, the supernatant suspension of the combined product may be removed, and any remaining water content may be removed through centrifugation or other process.

[0025] Methods of treating disease and injury may use the product to promote skin healing, and is useful in cosmetic surgery, reconstructive surgery, the treatment of burn wounds, or other dermal uses. The product may be used to treat joint or bone ailments, and may be useful in arthritic conditions, fractures, or other orthopedic uses. In some embodiments, a gelling form factor allows improved treatment of deep and narrow wounds.

[0026] The following definitions are used within this specification and claims:

[0027] "Placental tissue" means a tissue derived from a placenta, whether in whole or in part. Placental tissue may include, for example, chorion, amnion, a chorion and amniotic membrane, such as an amniochorion, Wharton's jelly, umbilical cord, placental cotyledons or combinations thereof. The placental tissue may also be dissected, digested, or otherwise treated to remove portions, membranes, or structures.

[0028] "Placental cells" refer to any cells that may be obtained from a placenta, and may include, for example, mesenchymal stem cells, endometrial stromal cells, placenta-derived mesenchymal progenitor cells, placental mesenchymal stem cells, fibroblasts, epithelial cells, macrophages, and the like. [0029] "Growth factor" means any factor or factors contributing to cellular or bodily growth, repair, or maintenance, and includes, without limitation, angiogenic factors, chemokines, cytokines, growth hormones, growth signals, protease, protease inhibitor, or matrix components. Exemplary growth factors include matrix metalloproteinases, tissue inhibitors of

metalloproteinases, thrombospondins, transforming growth factors, fibroblast growth factors, platelet-derived growth factors, human growth factors, vascular endothelial growth factors, fibronectin, interleukins and interleukin receptors, angiogenins/angiopoietins, and insulin-like growth factors and insulin-like growth factor-binding proteins. Additional classes of proteins may also be considered growth factors, and are included herein without limitation.

[0030] "Acellular" means materials and mixtures with significantly reduced intact cell content. For example, acellular as applicable to plasma may indicate low or no cellular content as compared to commonly available isolated red blood cell plasma. Acellular products may be generated by any means known in the art, such as one or more of mechanical or chemical lysis followed by a process such as centrifugation and supernatant removal. Acelluar products may include some intact cells or remnants of cells, however, the effective agents within the product may be predominantly acellular components.

[0031] "Particulate" means any matter in particulate form or subjected to a process for generating said matter. As specifically applied to biological membranes and other components, particulate refers to material that may have been significantly altered in structure through mechanical processes such as cutting, fracturing, or perforation, chemical processes, or other processing, resulting in material comprising an increased number of smaller fragments.

[0032] "Biological membrane" and "membrane" means any enclosing or separating membrane that may act as a selectively permeable barrier within an organism, and may also be referred to as a biomembrane. In some examples, the biological membrane may be derived from human reproductive tissue, such as a placenta. Such tissue may be of any suitable origin, e.g. human or porcine, without limitation. In some embodiments, the membrane may be a developmental membrane, and may be a placental membrane.

[0033] "Plasma" means a liquid component of cells as known in the art, for example amniotic cells or blood cells, and may comprise the extracellular matrix of these cells. In some embodiments, the plasma may be derived from blood cells, and may be a human or other plasma. In other embodiments, the plasma may be derived from amniotic cells, and may be a human or other plasma, and such embodiments may use the term "amniotic serum". Typical plasma content may include various proteins and other components, for example serum albumins, globulins, fibrinogen, glucose, clotting factors, hormones, electrolytes, and carbon dioxide. Plasma may be generated by any method known in the art, and in typical embodiments may be a human blood plasma. Amniotic plasma may contain particularly useful components in some acellular products.

[0034] "Scaffold" means any structure that may be capable of acting as a porous structural component for use in a biomedical product, and may be of biological or non-biological origin. Scaffolds may be selected for a number of properties, such as biocompatibility, biodegradability, mechanical properties, and architectural properties such as degree of porosity. The scaffold may be biocompatible with human cells, and may allow cells to adhere, function normally, and migrate through the surface and into the structure of the scaffold. Scaffolds may also be biodegradable within the body, which may allow the body's cells to eventually replace the foreign scaffold with extracellular components appropriate to the specific local cell types.

Exemplary scaffolds provided herein include human extracellular matrix derived from amniotic cells, which may contain a favorable substrate for cell migration and subsequent wound repair. One example of scaffolds may comprise the intermediate layer of an amniotic membrane. Other exemplary scaffolds are natural polymer scaffolds, synthetic scaffolds such as those made from synthetic polymers, and ceramic scaffolds.

Extracellular-Matrix Directed Treatments

[0035] Extracellular matrix may be a network of highly organized connective proteins, and is often referred to as a scaffold. The extracellular matrix provides tissues and organs with their native mechanical characteristics, such as elasticity and density. Biologically, the extracellular matrix may be the structure that interact with the resident cells of a tissue, and may provide the signal that a cell is in the proper location for its purpose. Extracellular matrix may also bind growth factors that may be produced by the resident cells. The growth factors may have many effects, and may provide a cue that allows resident cell maintenance, function, and growth. Under normal conditions, the extracellular matrix may be a highly organized structure that may bind resident cells, but may also allow the resident cells to migrate to some extent.

[0036] When an extracellular matrix is damaged, non-native factors may be introduced to the environment via blood perfusion, and specific cell types may be activated to respond to the damage. The introduction of non-native factors and cells may reduce the ability of the area to return to a pre-injury state. In many cases, scar tissue may form.

[0037] In a normal wound healing process, homeostasis may be followed by inflammation, fibroblast-based proliferation, maturation into scar tissue, and finally slow remodeling of the scar tissue. Homeostasis may be achieved when active bleeding stops. Inflammation may be caused by the migration of white blood cells in to the site to clear any pathogens, such as bacteria. Proliferation may happen when fibroblasts migrate to the site and divide and spread out across the wound and are stimulated by the fibrin matrix and may produce rigid collagens.

[0038] Maturation may then happen, with fibroblasts pulling on collagen fibers to contract and close the wound, causing de-vascularization and active remodeling of the collagen network, forming scar tissue. On a more detailed level, the damage response may involve the creation of greater amounts of fibrin, which may form a much more tightly-woven extracellular matrix. Non-native cells may also be introduced into the matrix. The tighter extracellular matrix may be small enough to bind platelets and prevent red blood cell loss, stopping bleeding, but may signal scar formation.

[0039] Scar tissue may have a number of undesirable properties, mostly attributed with its inability to function as an original local cell, and its varying structural properties. When scarring is sufficiently debilitating towards normal function, a wound may have to be additionally surgically altered to restore function, resulting in additional treatment risk and expense.

[0040] Extracellular matrix-directed treatments may attempt to prevent or mitigate the cascade that causes scar tissue formation. In some cases, such treatments may tend to accelerate conversion of scar tissue into functional tissue.

[0041] A scaffold-directed wound repair may use human developmental tissue to facilitate a developmental response by, in some cases, impregnating tissues with human developmental proteins to attract and activate regenerative cells. Such a treatment may saturate natural interaction sites on the tissue with plasma factors to beyond natural physiological levels (super saturated), which may accelerate healing response. In some embodiments, the inclusion of additional diffusible factors such as growth factors, cytokines, chemokines, and other molecule classes, may strongly influence the overall course of wound healing. Further, in some embodiments, the absence of fibrin and fibrinogen in the final product may reduce inflammation and scarring, thereby inhibiting the damage response that may lead to scar tissue formation.

[0042] The coupling of scaffold and plasma sources may cause a native interaction that may have tremendous implications for wound-healing. Saturating the growth factor binding sites within the scaffold with the free substrates of the plasma may have multiple additive effects. The result of using the techniques described herein may minimize post-trauma complication such as infection, an enhanced rate of recovery, and the redevelopment of functional tissue with significantly reduced amounts of scar tissue.

Acellular product and treatment

[0043] The acellular product comprises a plasma portion and a biological membrane portion. In many cases, the biological membrane portion may be particulated and impregnated with the plasma portion. In some embodiments, the biological membrane may be derived from developmental tissue, and in some embodiments it may be derived from human tissue.

[0044] The general properties and benefits of using human amniotic membranes in treatment include improved wound stabilization, which is believed to be observed because of the non- specificity of the cells and components found in the amniotic membranes. This may mitigate undesirable immune responses that may slow down healing and may contribute to scar tissue.

[0045] The particulate amniotic membrane may be delivered as a liquid, gel, or powder. These forms offer the medical professional several ways of improving wound penetration and coverage. The particulated membrane products can be relatively stable and easy to transport and use.

Additionally, particulating the amniotic membrane may not significantly damage structural components, leaving the components as still easily recruited for use in wound healing. [0046] In some embodiments, the plasma portion may be derived from human tissue, and in typical embodiments the plasma may be human or other animal's blood or amniotic plasma. The concentration of proteins and other factors in plasma may provide a natural blueprint for specific physiological responses.

[0047] Previous generation products may suffer from their inclusion of factors such as fibrinogen, which may promote scar tissue formation, reducing or eliminating the effectiveness of treatments incorporating a plasma portion. In some embodiments, the products may be substantially free of fibrin and fibrinogen.

[0048] Figure 1 shows the effect of water removal from plasma, illustrating some of the advantages with respect to increased growth factor concentration. Specifically, water content is inversely related to growth factor levels (pg/mL). Plasma products shown range in processing level from platelet poor plasma (PPP) to platelet rich plasma (A-PRP), platelet lysate (PL), and platelet poor plasma that has water levels reduced to 10% of the original content (PPPc). The method used to remove water content in these samples was lyophilization. The effect of water removal from plasma to 10% of the original content increases the concentration of TGF-Bl and VEGF approximately 9-10 fold. This also decreases the volume of plasma compositions, making them more useful for treatments.

[0049] Lyophilization or freeze-drying is used in regenerative medicine techniques to reduce the water content of plasma samples, for example in the activated platelet (A-PRP) and platelet lysate (PL) samples. However, activated platelets and platelet lysates are primary contributors to the inefficiency of previous products and methods, contributing to scarring and poor wound remodeling. [0050] Figure 2 depicts the thrombin activation of platelet poor plasma reduced in water content by lyophilization to 10% of the original volume (A-W.B.c). The large bar indicates high levels of thrombin. While lyophilization techniques further concentrate growth factors, thrombin takes a primary role in the undesirable clotting and immune responses seen when plasma is administered. Products herein may achieve high concentrations of growth factors while avoiding inclusion of platelets and platelet lysates that may contribute to thrombin activation.

[0051] Centrifugation may remove fibrin, fibrinogen, and other cellular content from the product. Centrifugation may concentrate desirable growth factors while removing

fibrin/fibrinogen and any cellular content that might be present in a plasma sample. Thus, centrifugation processing of the product may remove the pro-inflammatory or clotting factors and may deliver a high concentration of growth factors.

[0052] The combination of the morselized membrane and plasma portions may have synergistic effects that aid in treatment. Specifically, impregnating the scaffold (membrane) sites with factors found in plasma may provide several benefits. One such benefit may be that the product may have a two-stage diffusion. Upon delivery to a treatment site, such as a wound, free factors may be immediately released into the site, while others remain in the scaffold. Initial factor delivery may aid in immediate wound treatment by preventing clinically-negative immune responses and promoting cellular recruitment and healing. Slower factor delivery from the scaffold may continue to support cellular recruitment and healing, and can extend the time over which such factors are available at the wound site. Finally, factors remaining in the scaffold fragments may be later utilized by migrating cells, contributing to further wound healing and remodeling. In some embodiments, different factors may be provided at each release stage. [0053] In some embodiments, the product may have the ratio of 0.01-3.0 mL amniotic serum per square centimeter of amniotic membrane. The surface area of the amniotic membrane may be measured prior to particulation or other processing. This ratio may saturate or impregnate the native binding sites on the scaffold to their complementary growth factors, as well as may provide residual factors in an unbound, free state which will be subject to diffusion at the time of application.

[0054] In a typical embodiment, the processed amniotic serum may be added to the micronized amniotic membrane at a ratio of 0.01-0.1 mL amniotic serum per square centimeter of amniotic membrane. In a specific embodiment, the processed amniotic serum may be added to the micronized amniotic membrane at approximately 0.5 mL amniotic serum per square centimeter of amniotic membrane.

[0055] This ratio may contribute to the improved effectiveness of the product by both ensuring saturation of the membrane in later steps and providing a different set of factors that

synergistically aid in the desirable cellular processes.

[0056] Figures 3A, 3B, and 3C depict a histological analysis of a porcine study treating porcine dermal incision injuries with the product having, 0.5mL amniotic fluid per square centimeter amniotic membrane. Full thickness dermal regeneration and reductions in scar tissue and fibrosis were observed, with significant difference from sutured, non-product controls at both 2 and 10 days post operation. The experimental sites had significantly reduced fibrosis and scarring across all samples as compared to controls, illustrating the effectiveness of treatment with the product.

[0057] Figure 4A depicts a histological analysis of the porcine study using two embodiments. LotlA is 0.5mL amniotic fluid per square centimeter amniotic membrane. LotlB is 2.0mL amniotic fluid per square centimeter amniotic membrane. Figure 4A shows improved scar tissue across experimental samples and particularly at the 0.5mL/cm ² concentration. The control sample had histological scoring averaging 3 out of 5, or poor, with significant scarring found in the wound. The 0.5mL concentration experimental sample showed very strong results, with scarring averaging 1 out of 5, or very good, with little scarring found in the wound. The 2.0mL concentration sample was less effective than the 0.5mL concentration sample, but still reduced average scarring to 2.5 out of 5.

[0058] Figure 4B shows improved scar tissue and fibrosis across experimental samples. The control sample had histological scoring averaging 3 out of 5, or intermediate scarring and fibrosis. 0.5mL/cm ² concentration (Lotl A) showed very strong results, with scarring and fibrosis averaging 1 out of 5, or very good, with little scarring or fibrosis found in the wound. The 2.0mL concentration sample was less effective than the 0.5mL concentration sample, but still reduced average scarring and fibrosis to 2.0 out of 5.

[0059] Figure 4C shows reduced neovascularization across porcine experimental samples at 31 days. As compared to the control having scoring of nearly 5 out of 5, or extremely poor, the 0.5mL/cm ² concentration (LotlA) had greatly improved average scoring of 2.5 out of 5. The 2.0mL /cm ² concentratoin (LotlB) also had improved average scoring of less than 4 out of 5.

[0060] Figures 4A, 4B, 4C illustrate that certain increased concentrations of amniotic serum may offer improved wound healing scoring and reduced scar tissue, fibrosis, and neovascularization compared to control samples. Further, optimization of this effect may be linked to idealized ratios of amniotic serum to amniotic membrane.

[0061] In some embodiments, an amniotic intermediate layer may also be incorporated into the product or may serve as the exclusive or primary scaffold. The amniotic intermediate layer may bind a different array of factors as compared to the amniotic membrane, and may allow additional saturation with growth factors from amniotic plasma or other sources. Further, the amniotic intermediate layer may be contain proteoglycans and hyaluronic acid, which may be capable of holding water within the wound bed and simultaneously acting as a point-of- interaction/docking for stem cells via CD44. Such an embodiment may provide polymers which may be catabolized to provide resources that may be used to deposit new skin. The intermediate layer can be separated using a scalpel and added at any stage of processing.

[0062] In some embodiments, the product may comprise additional treatments or be delivered with additional treatments. For example, in some embodiments medicaments are incorporated into the product.

Processes for generating an acellular product

[0063] Manufacturing an acellular product may include processing an amniotic serum, processing an amniotic membrane, and combining those products.

[0064] Amniotic serum may be obtained in any number of forms, and may require different levels of processing. Typically, fresh placental tissues may be obtained from a suitable provider, and typically may be stored for less than 12 hours before being packaged on dry ice packs and shipped for processing, with the time period from recovery to reception by the manufacturer occurring generally less than 24 hours. Typically, tissues may be tested for infectious agents or quality before use. Tissues may be processed immediately, or may be maintained for up to 24 hours at 4°C.

[0065] Processing the amniotic serum to remove fibrin and red blood cells may improve the effectiveness of the final product, and may be done by any known technique, such as by filtering or mass-based techniques. In some embodiments, amniotic serum may be centrifuged to remove fibrin and red blood cells. Centrifugation may be done, for example, for approximately 10 minutes at around 200 times gravity, or at any speed or for any length of time that allows fibrin clots and red blood cells to migrate to the bottom of the centrifugation column. In some embodiments, inspection of the centrifugation column may indicate a need for further centrifugation to obtain a clear interface between coagulated mass, red blood cells, and plasma. Failure of a sample to form a red blood cell and serum interface may disqualify the sample from further processing.

[0066] The plasma portion may be isolated from processed amniotic serum, as outlined above, or may be obtained by any other method. In some embodiments, the plasma portion may be obtained from the centrifugation column by simple pipetting, which may be done without drawing red blood cells and fibrin into the sample.

[0067] The centrifuged serum supernantant or alternatively, whole amniotic fluid component may be placed in dialysis tubing, closed and immersed in deionized distilled water. Such a step may lyse any smaller "formed bodies" such as. WBCs, platelets, red blood cells, and may enable the rapid diffusion of ions/salts from the mixture.

[0068] Alternatively, the serum or total amniotic fluid can be placed in dialysis tubing and submerged in a saline solution considered to be hypertonic (> 5%) for any period of time. Such a method may rapidly and efficiently remove a substantial amount of the water content. The serum may become hypertonic and may destroy formed bodies in this fashion, and may serve as a step to prepare the soluble proteins for collection in future steps. The increased salt content of the solution (with respect to water content) may decrease the solubility of proteins, which might be otherwise soluble in water and may otherwise be more difficult to collect. Therefore, this step may reduce the processing time and gravitational force required during future ultracentrifugation steps.

[0069] Processing the amniotic membrane may be performed to obtain a decellularized material. In some embodiments, the amniotic membrane may be obtained as an intact membrane, and in other embodiments may be pre-processed into smaller pieces. In a typical embodiment, sheets of amniotic membrane may be removed intact from a saline bath and then processed. In some embodiments, the total surface area of the amniotic membrane and intermediate layers may be measured. In additional embodiments, the chorion layer may be removed prior to processing, which may be performed by shaving or some other technique.

[0070] Note that in specific embodiments, an intermediate layer may be used in place of an intact amniotic membrane. In these embodiments, the intermediate layer may be used in the same manner as the membrane.

[0071] The amniotic membrane may be typically further decellularized, which may be performed by any method, and in typical embodiments is washed with ethanol to remove residual cell content. The membrane may then optionally be cut or dissected.

[0072] In typical embodiments, the amniotic fluid may be added to the membrane in a suitable container, and the membrane may be morselized in the amniotic fluid suspension using any suitable means, such as a needle or punch, though any method of morselization is contemplated.

[0073] In some embodiments, the processed amniotic serum may be added to the micronized amniotic membrane at a ratio of 0.01-3.0 mL amniotic serum per square centimeter of amniotic membrane. In a typical embodiment, the processed amniotic serum may be added to the micronized amniotic membrane at a ratio of 0.01-0.1 mL amniotic serum per square centimeter of amniotic membrane. In a specific embodiment, the processed amniotic serum may be added to the micronized amniotic membrane at approximately 0.5 mL amniotic serum per square centimeter of amniotic membrane.

[0074] A mechanical method of particulating portions of the amniotic membrane may offer improvements over a freezing and fracturing methods or chemical methods. A chemical method may involve subjecting the proteins of the amniotic membrane to fewer denaturing forces.

Typically, membrane particles ranging from approximately 10-400 microns in size are produced, however in some embodiments membrane particles may be produced in a variety of larger and smaller sizes.

[0075] An additional benefit of the morselization step is that the membrane may now be able to interact with a larger number of amniotic fluid factors via the several different layers within the now exposed membrane. The moselization may improve saturation with amniotic fluid and its various factors, which may improve the ability of the final product to deliver factors and aid in treatment.

[0076] In some embodiments, additional material may be added from an amniotic intermediate layer. The amniotic intermediate layer may contain additional growth factors and other compounds that further promote cellular migration, differentiation, growth, and maturation. The intermediate layer may be separated from an amniotic sample by shaving or removal with a scalpel. The intermediate layer may then be added to the morselized membrane particulate and amniotic serum mixture. Note that in some embodiments, the intermediate layer may be used as the membrane portion itself.

[0077] After mixing the intermediate layer into the morselized membrane particulate (as applicable) and amniotic serum mixture, the products may be centrifuged. In some

embodiments, centrifugation may be high-speed and low temperature, for example at 1-8 hours at approximately 50,000-100,000,000 times gravity at 4°C. The centrifugation step may pelletize proteins and may separate them from the water and salt content. Further, scaffolds such as membrane and/or intermediate layer may be forced to interact with diffuse proteins in suspension and will allow impregnation of the scaffolds with these factors. After a protein pellet forms at the base of the tube, the supernatant of salt and water may be removed, by pipetting or other mechanism. This step may remove greater than 95% of the water and salt content.

[0078] In another embodiment, the serum may be centrifuged at 50,000 - 1,000,0000 times gravity for 2 hours or more without a scaffold component. The insoluble proteins and cellular debris may be removed from the soluble fraction. The supernatant can then be combined with the micronized amnion, and/or intermediate layer for impregnation with an 8 hour

ultracentrifugation cycle.

[0079] In yet another embodiment, the amniotic serum may have the saline content removed via dialysis for a period of 2 hours in a low molecular weight dialysis tubing submerged in deionized-distilled water. This process may also ablate viable cell populations. The serum may then be centrifuged to remove the cellular debris and insoluble factors. The supernatant which may be collect may be recombined with the scaffold without cellular components or salts.

[0080] The product may then be prepared to various specifications, and in typical embodiments may be dried through centrifugal evaporation. Such as step may remove remaining water content without the freezing and freeze-drying, thereby preserving protein and scaffold viability. The product may then be terminally sterilized using an irradiation method, peracetic acid method, or other method.

[0081] In another embodiment, a short method of processing may involve using high-speed centrifugation and/or ultracentrifugation to remove a bulk of the water content in a fraction of the time it requires to perform the initial centrifugal evaporation step. This method may have the benefit of saturating and impregnating the interaction sites of the scaffold with the appropriate constituents.

[0082] In the alternative processing embodiment, a human serum source devoid of fibrinogen may be filtered to remove contaminating cell populations and/or filtered to remove bacterial cells. The serum solution may then be mixed with a biological or synthetic scaffold of a particular size. In some embodiments, the scaffold may comprise a biological scaffold micronized to a range of 50-500um. In some embodiments, a ratio of amniotic fluid (mL) to amniotic membrane (cm ² ) may be approximately 0.5 mL/cm ² In other embodiments, the ratio may be approximately 0.01-3.0 mL/cm ² . In yet other embodiments, the ratio may be approximately 0.01-0.1 mL/cm ² .

[0083] In some embodiments, the total solution created may be added with or without establishing a gradient for separation of proteins from lipids. The total solution may then be centrifuged under refrigerated conditions, for example for at least 8 hours at a gravity of at least 50,000 times gravity. The resulting centrifugation pellet may be the desirable scaffold/serum protein product, and additionally includes exosome and microvesicles. The interface formed at the top most portion of the centrifugation column may be the lipid layer, which may be collected by any method, such as pipetting, and the remaining solution may be removed from the centrifuge column and discarded. This step may remove the bulk aqueous content from the desirable protein and scaffold materials.

[0084] In such embodiments, the protein pellet embedded into the scaffold by centrifugation may then be collected. In some embodiments, the pellet may be placed into a suitable container for storage and therapeutic use. In other embodiments, residual water may be removed from the pellet through any known method, including but not limited to gentle lyophilization. Because the majority of the water content may have already been removed by centrifugation, the denaturation of desirable proteins and cost of lyophilization may be significantly reduced. Alternatively or subsequently, the pellet may be further centrifuged to remove residual water content and packaged and sterilized for use.

[0085] In yet another embodiment, passive impregnation of scaffold and serum may be achieved by mixing the scaffold and processed serum and removing water content by any means. In some embodiments, water content may be removed by nitrogen convection or cryogenic methods. In other embodiments, water content may be removed by centrifugal evaporation.

[0086] A final product may be a crystalline solid capable of phase-change with the addition of water. Such a product may have high treatment potential compared to the use of intact tissues.

[0087] When dehydrated, the product may be highly shelf stable at room temperature, and may be expected to last 5 years without significant degradation. This is another benefit of the product, as many previous generation products require complex storage and preparation.

[0088] In some embodiments, additional treatments or medicaments may be incorporated into one or more of the scaffold, serum, or mixture. For example, additional chemical or medical factors may be added to the scaffold, serum, or mixture prior to processing. In other

embodiments, the additional treatments or medicaments are provided after processing.

Methods of treatment

[0089] The products may be useful to treat a number of injuries and diseases. In some embodiments, an acellular product may be used as a medicament in the treatment of dermal wounds. In some embodiments, the dermal wound may be an incision wound, such as a surgical site wound.

[0090] The product may be applied in a number of forms. One version may be as powder or gel, which may be reconstituted from a powder by a practitioner. Typically, application of the product occurs prior to wound closure, however in some embodiments the product may be applied after wound closure. In some embodiments, the product may be applied only once to the wound site, while in other embodiments is the product may be applied more than one time over a certain time period. If applied over a time period, the product may be applied, for example, hourly, two or more times a day, daily, weekly, bi-weekly, or monthly.

[0091] In some embodiments, the product may be used to treat a human subject. In other embodiments, the product may be used to treat an animal, such as a domesticated pet, work, or food animal.

[0092] In some embodiments, the product may be mixed with or otherwise delivered or used with other compounds or treatments.

[0093] In dermal treatment embodiments, treatment with the product may improve one or more disease or injury states. Exemplary metrics include wound healing time, prognosis, outcome, cost of treatment, or evaluation of markers such as scar tissue evaluation and quality of tissue healing.

EXAMPLES

[0094] The following examples are provided to illustrate aspects of the invention, and in no instance should be considered to limit the scope of the claims.

Example 1 : Obtaining placental tissue and quality assurance [0095] Placental tissue may be obtained by any known method. Fresh placental tissue specimens may be obtained from providers. Ideally, specimens are to be stored for less than 12 hours before being packaged on dry ice packs and shipped for processing, with the time period from recovery to reception by the manufacturer occurring in no more than 24 hours. In all cases, tissues are to be handled in accordance with the validated protocols of the processing facility, and are to be tested for infectious agents prior to use. The integrity of the biological containment vessels and contents are verified prior to use, and tissues are processed immediately or maintained for up to 24 hours at 4°C to ensure safety and effectiveness.

Example 2: Processing and decellularization of amniotic serum

[0096] Note that all of the processing use sterile/aseptic techniques and containment to ensure safety and effectiveness.

[0097] During shipping, the amniotic fluid may typically be placed in dialysis tubing and submerged in a stabilization media of glucose, saline, ethanol, sucrose, water or a combination thereof in the range of 0.5% - 10% of any component. The media may remove water content, increase solubility of factors in the serum, or challenge the integrity of formed bodies (cells). Alternatively or additionally, low-temperature (less than about 4°C) dialysis in ethanol may be used. Dialysis techniques may also adjust the isoelectric constant and increase protein collection efficiency if desired.

[0098] Amniotic serum is aliquoted into 50mL centrifuge tubes, avoiding any coagulated fibrin. The samples may be loaded into a centrifuge according to standard laboratory procedure, and ran for at least 10 minutes at approximately 200xg. [0099] The tubes may be carefully removed and placed in a tube rack so as to not disrupt the red blood cell and serum interface. The quality of the interface may be inspected, and verified to be distinct with no intermediate space. If the interface is not distinct, the sample may be centrifuged at approximately 1500RPM for an additional 3 minutes. Further failure to form a red blood cell and serum interface requires considering the disqualification of the sample.

[00100] Tubes and tube rack may be sprayed with 70%+ isopropanol alcohol diluted in distilled water and transferred into the prepped/cleaned work space. The entire plasma portion from each tube may be isolated using a serological pipette (5 mL or 10 mL capacity), taking caution to not draw any red blood cells into the pipette. The remaining non-plasma portion contains the remaining cellular content and fibrin.

Example 3 : Processing of amniotic membrane

[00101] Containers may be prepped according to standard safety protocols (70% isopropanol) and transferred to work space, then the membranes may be removed from the saline bath in which they typically arrive.

[00102] The total surface area of the amniotic membrane and intermediate layers may be measured and inspected to ensure that the chorion is removed. Gently wash each layer with 70% ethanol to remove residual red blood cells and decellularize the membranes.

[0103] Cut the membrane into quarters and transfer each quarter of the membrane into a 50 mL conical tube.

[0104] Add previously obtained amniotic fluid up to 40 rriLs. In two examples generated herein, amniotic fluid was added at 0.5mL per square centimeter of membrane and 2.0mL per square centimeter of membrane. [0105] Morselize the membrane in the amniotic fluid suspension using an OmniTip for 2 minutes. This allows the production of particulate/particulated amniotic membrane without freezing it. The membrane also interacts with a larger number of amniotic fluid factors via the several different layers within the now exposed membrane.

[0106] Membrane particles ranging from approximately 10-400 microns in size are produced. Example 4: Processing of fluid/scaffold particulate mixture

[0107] The morselized membrane particulate generated in Example 3 may be transferred to total amniotic serum at a ratio of 0.01-O. lmL amniotic serum per square centimeter of micronized amniotic membrane as determined in Example 3.

[0108] The intermediate layer may be added into the mixture in the same sized aliquot as the amniotic membrane particulate. Aliquot the mixture into 50mL conical tubes and centrifuge for approximately 8 hours at 80,000-100,000 times gravity at 4°C. Ensure that a protein pellet has formed at the base of the tube, and if so pipette the remaining supernatant solution out.

[0109] Aliquot the remaining wet pellet into individual serum vials (typically ranging from 0.5- l.Og size) and cover the vials with a tyvex barrier before subjecting to centrifugal evaporation for 3-6 hours.

Example 5: Alternative processing method

[0110] Amniotic serum is aliquoted into 50mL centrifuge tubes, avoiding any coagulated fibrin. Filter to remove any contaminating cell populations and optionally to remove bacterial cells. The samples are then mixed with a scaffold such as particulated amniotic membrane or intermediate membrane without particulated amniotic membrane. This may be centrifuged under refrigerated conditions for >8 hours at 50,000 times gravity.

[0111] Verify that the topmost (lipid) interface is formed. Remove the topmost layer via pipetting. Decant or pipette the remaining solution from the centrifuge tube and discard, removing the bulk aqueous content.

[0112] Collect the remaining material and evaluate aqueous content. Either carry out centrifugal evaporation as above or directly store as above.

Example 6: Terminal sterilization, storage, and distribution

[0113] Remove the vials generated as in examples 3 or 4 and terminally sterilize using irradiation or paracetic acid.

[0114] Discard waste products in appropriate biohazard containers, and ensure lot number and manufacture dates are documented. The product may be stored at ambient temperature for up to 5 years.

Example 7: Treatment of dermal incision wounds using the product

[0115] Three full dermal surgical incisions 5cm in length were made on each of the shoulder and rear areas of anesthetized porcine subjects using a surgical blade and full dermal thickness biopsies were introduced. Incisions were examined for consistency, ensuring that the incision penetrated the full dermal thickness. One site on each front and rear area was labeled a control site, and the other two on each front and rear area were immediately treated with concentrations of the acellular product wound covering. [0116] The wound sites were observed and recorded at 2 days post operation and 10 days post operation. At 10 days post operation, the porcine subjects were culled, and the wound areas were subjected to histological analysis using hematoxylin and eosin staining. As shown in FIG. 3 A, 3B, and 3C, the experimental sites had significantly reduced fibrosis and scarring across all samples as compared to controls.

[0117] The acellular product-treated shoulder incisions had markedly reduced scar tissue and fibrosis, with an overall score of around 1, while the control incision had a score of around 3. The acellular product -treated rear incisions also had an overall score of below 1, while the control incision had a score of over 2. With respect to scar tissue alone, experimental sites had overall scar-tissue scores of 0.5 and 1.0, while the control site had a scar-tissue score of 3.5. Additionally, reduced neovascularization was noted for experimental sites.

[0118] This experiment illustrates the accelerated and improved healing of dermal wounds treated with a single application the products disclosed herein.

[0119] The preceding description is presented for purposes of illustration and description, and does not limit the scope of the invention to the disclosures, examples, and embodiments provided therein. On the contrary, a number of modifications and variations are possible based on the above teachings, and alternative embodiments are included to the full scope allowable by the prior art.