Compositions Comprising Collagen and Micronized Placental Tissue and Methods of Preparation and Use Thereof

Abstract

To provide compositions useful in applications such as wound healing, repairing damaged tendons, cosmetic applications, and covering biocompatible materials and / or devices.SOLUTION: Provided is a composition comprising water, soluble collagen, and micronized placental tissue components such that the composition is an integrated admixture having apparent homogeneity.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to compositions comprising water, collagen, and micronized placental tissue components, and methods for preparing the same. In certain embodiments, the compositions of the present invention are useful in applications such as wound healing, repair of injured tendons, cosmetic applications, and coatings for biocompatible materials and / or devices. [Background technology]

[0002] Human placental membranes (e.g., amniotic membranes) have been used for various reconstructive surgeries since the early 1900s. These membranes function as substrates and are more commonly referred to as biological dressings or tissue grafts. Such membranes have also been used in ophthalmic procedures and more recently in dental regeneration procedures and ulcer treatment, where they are used as an adhesion barrier. Typically, these membranes are frozen or dried for preservation and storage.

[0003] Placental tissue is typically retrieved after an elective cesarean section. The placenta consists of the amniotic sac, which may be intended to include the umbilical cord and amniotic sac. The amniotic sac contains amniotic fluid, providing a protective environment for the fetus.

[0004] The amniotic sac, commonly referred to as the amniotic membrane, has two major tissue layers: the amnion and the chorion, separated by a thin connective layer, sometimes called the middle layer. The amnion is the innermost layer of the amniotic sac and is in direct contact with the amniotic fluid. Histological evaluation has shown that the membranous layer of the amnion consists of a single layer of epithelial cells, a thin reticular fiber (basement membrane), a thick stratum densa, and a fibroblast layer. The fibrous layer of the amniotic membrane (i.e., basement membrane) contains types IV, V, and VII collagens and cell adhesion bioactive factors such as fibronectin and laminin.

[0005] Placental tissues, such as the amnion membrane, offer unique properties when used for surgical and wound healing procedures, such as providing a substrate for cell migration / growth, providing a natural biological barrier, being non-immunogenic, and containing numerous bioactive molecules. Placental tissues can be used as membranes to support tissue regeneration and improve healing outcomes in numerous applications. The amnion membrane has the ability to self-adhere or can alternatively be secured in place using different techniques, such as fibrin glue or sutures.

[0006] Placental tissue grafts are used in a variety of medical applications, including wound care and cosmetics. However, these tissue grafts lack flexibility and / or cohesion, especially in environments where shear forces are common. Therefore, there is a need to provide placental tissue compositions with improved flexibility and / or cohesion.

[0007] Micronized placental tissue components have previously been disclosed and used as aqueous suspensions for injection into injured body components such as tendons, ligaments, etc. However, despite the beneficial properties they impart in healing such wounds, it would be desirable to provide a composition that would retain its position when injected into the body. Summary of the Invention

[0008] The present invention is based, at least in part, on the discovery that aqueous suspensions of micronized placental tissue components and collagen provide solid masses with unexpected properties, including improved flexibility and / or cohesion. It was further surprising that such properties can be improved upon application of suitable shear and force, such as upon ejection from a syringe. It was also unexpected that by varying the ratio of the major components (water, collagen, and micronized placental tissue), a desired level of consistency and cohesion of the composition can be targeted. The resulting solid mass compositions are particularly suitable for wound healing, tendon repair, cosmetic applications, and coating biocompatible materials and / or devices.

[0009] Without being bound by theory, it is believed that the combination of aqueous collagen and micronized placental tissue provides a more effective composition for tissue regeneration and / or stem cell recruitment than either component alone. For example, collagen provides scaffolding and / or bulking to the area where the composition is applied, while micronized placental tissue provides biological activity (growth factors, stem cell recruitment, etc.). Furthermore, the compositions described herein are particularly suitable for cosmetic applications.

[0010] In one aspect of the present invention, a composition is provided that comprises water, collagen, and micronized placental tissue components, such that the composition is an integrated mixture having an apparent homogeneity. In another aspect, a composition is provided that comprises collagen and micronized placental tissue components, such that the composition is an apparent homogeneous, yet cohesive and pliable mass.

[0011] In one aspect of the invention, a method is provided for converting an aqueous / gelatinous collagen mass into a cohesive and flexible composition, the method comprising incorporating sufficient micronized placental tissue into an aqueous collagen material and subjecting the composition to sufficient force and shear to convert the composition into a cohesive and flexible mass.

[0012] The compositions of the present invention can be used to aid in wound healing, such as injured tendons. The compositions of the present invention can also be used in cosmetic applications or to coat biocompatible materials and / or devices.

[0013] In one embodiment, the micronized placental tissue component comprises micronized amniotic membrane, micronized chorion, micronized Wharton's jelly, or any combination thereof. In one embodiment, the micronized component has a particle size of less than 400 μm.

[0014] In one embodiment, the micronized placental tissue component is obtained by micronizing a tissue graft comprising an amniotic layer and a chorionic layer, wherein the chorionic layer is layered directly on the amniotic layer, and wherein the amniotic layer further has an exposed basement membrane and an intact fibroblast component. In another embodiment, the micronized placental tissue comprises an amniotic layer and a chorionic layer, wherein the chorionic layer is layered directly on the amniotic layer, and wherein the amniotic layer further has an intact epithelial cell layer and an intact fibroblast component.

[0015] In one embodiment, the collagen is human collagen, for example, human placental collagen. In one embodiment, the collagen is an aqueous collagen material comprising about 0.1% to about 2% collagen.

[0016] In one embodiment, the weight ratio of micronized placental tissue to collagen is about 3:1 to about 100:1. In another embodiment, the weight ratio of micronized placental tissue components to collagen is about 100:1 to about 300:1. In a preferred embodiment, the weight ratio of micronized placental tissue to collagen is about 50:1.

[0017] In one embodiment, the composition is cohesive and pliable under physiological conditions, eg, at a temperature range of about 15°C to about 45°C.

[0018] In another embodiment, the composition is obtained by releasing a precursor composition comprising aqueous / gelatinous collagen and micronized placental tissue components, hi yet other embodiments, the precursor composition comprising an aqueous / gelatinous mass is in the form of a gel, putty, or paste.

[0019] The advantages of the present invention will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the aspects described hereinafter. The advantages described below will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive.

[0020] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects described below. [Brief explanation of the drawings]

[0021] [Figure 1] 1 is a schematic flow chart of a process for preparing a cohesive and flexible hydrated collagen composition described herein. [Figure 2] 2A and 2B show the aqueous collagen composition (FIG. 2A) and the composition of collagen and micronized placental tissue (FIG. 2B) as they are expelled from a 27-gauge needle. [Figure 3] 3A and 3B show the lack of tensile strength of a hydrated collagen composition (FIG. 3A) compared to a composition comprising collagen and micronized placental tissue (FIG. 3B). [Figure 4] Figures 1A-1C show sequential addition of micronized placental tissue components to aqueous collagen materials. In Panel A, 32.3 mg of micronized placental tissue components were mixed with 1 mL of 0.469% (4.69 mg) neutralized collagen. In Panel B, 33.1 mg of micronized placental tissue components were mixed with the composition in Panel A. In Panel C, 27.7 mg of micronized placental tissue components were mixed with the composition in Panel B. In Panel D, 33.9 mg of micronized placental tissue components were mixed with the composition in Panel C. In Panel E, 33.2 mg of micronized placental tissue components were mixed with the composition in Panel D. In Panel F, 37.5 mg of micronized placental tissue components were mixed with the composition in Panel E. In Panel G, 36.3 mg of micronized placental tissue components were mixed with the composition in Panel F. The composition of Panel G contains a total of 234 mg of micronized placental tissue components and 4.69 mg of collagen in 1 mL of aqueous solution. [Figure 5] Panel G in Figure 4 illustrates the process of applying sufficient force and shear to the composition by passing it through a 27 gauge needle to convert it into a coherent and flexible mass. DETAILED DESCRIPTION OF THE INVENTION

[0022] Before the present invention is disclosed and described, it is to be understood that the embodiments described below are not limited to specific compositions, synthetic methods, or uses, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting of the invention.

[0023] In this specification and in the claims that follow, you will refer to a number of terms that shall have the following meanings.

[0024] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a cross-linking agent" includes mixtures of two or more such agents, and the like.

[0025] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes cases where the event or circumstance occurs and cases where it does not occur. For example, the phrase "optional washing step" means that a washing step may or may not be performed.

[0026] As used herein, the term "subject" refers to any vertebrate organism, including mammals, e.g., domestic animals and primates, such as humans. In a preferred embodiment, the subject is a human.

[0027] As used herein, the term "about" indicates that a value may vary by ±5%, such as ±5%, ±2% or ±1%.

[0028] The terms "coherent," "coherency," and "cohesion" are used herein to refer to a composition that retains its homogenous mass after formation at a temperature of at least 0°C to about 40°C. That is, once formed, the mass does not separate into individual components or break down into smaller particles when maintained in vitro at a temperature of at least 0°C to about 40°C. In other words, the composition has sufficient coherence such that it does not break, tear, disintegrate, or fragment before or during introduction into or onto a subject's body. One of skill in the art can determine the appropriate coherence based on the purpose of introduction, the amount of placental tissue composition used, the mode of administration / introduction, and the specific body part for administration.

[0029] The compositions are cohesive upon administration, but naturally endogenous enzymes such as collagenases and proteases break down these compositions. During breakdown, growth factors and other biological factors are released from the micronized placental tissue over time, thereby providing a sustained release of such factors at the site of administration / introduction.

[0030] The terms "flexible" and "flexibility" are used interchangeably herein to refer to a material's ability to change shape when a force is applied. A material that is more flexible than another is capable of greater shape change. The degree of flexibility of the present compositions varies depending on the application, the amount of placental tissue component used, etc. For applications requiring less flexibility, the flexibility is reduced by decreasing the amount of placental tissue component (e.g., a composition comprising a micronized placental tissue component:collagen weight ratio of at least 100:1). For compositions requiring less flexibility, the micronized placental tissue component:collagen weight ratio is increased to about 300:1. One of skill in the art will be able to determine the optimal flexibility of the present compositions for a particular application. The flexibility of the present collagen and micronized placental tissue compositions can be altered by varying the ratio of collagen to micronized placental tissue.

[0031] For applications requiring greater flexibility, flexibility is increased by decreasing the amount of micronized placental tissue component (e.g., a cohesive composition comprising a micronized placental tissue component:collagen weight ratio of less than 100:1). The greater the amount of micronized placental tissue, the less flexible the composition.

[0032] Compositions having a weight ratio of micronized placental tissue component to collagen of about 1:1 to less than about 100:1 maintain a gel-like composition, wherein the gel-like composition has an apparent homogeneity of the micronized placental tissue component throughout the mass. As will be apparent, each embodiment has uses suited to specific circumstances. For example, a gelatinous mass having an apparent homogeneity can be easily injected into joints, a cohesive yet flexible mass can be used for cosmetic purposes such as wrinkle reduction, and a cohesive but less flexible mass can be used as a wound care dressing for burns.

[0033] As used herein, the term "apparent homogeneity" refers to a composition of collagen and micronized placental tissue components in which there is no substantial distinction between the collagen and placental tissue components and in which the placental tissue components are uniformly dispersed throughout. For example, there may be no substantial distinction between the collagen and placental tissue components when visualized without magnification. In other compositions, there may be no substantial distinction between the collagen and placental tissue components when visualized with magnification. The apparent homogeneity of the collagen and micronized placental tissue components may be induced by force and / or shear applied to the composition. For example, force and / or shear may be applied by pressure in a mold or press, ejection from a syringe, or other methods common in the art. The apparent homogeneity of the collagen and micronized placental tissue components may be achieved by mixing the composition until there is no substantial distinction between the collagen and placental tissue components and the placental tissue components are uniformly dispersed throughout.

[0034] As used herein, the term "amniotic membrane" includes an isolated egg membrane from which intermediate tissue layers have been substantially removed. The amniotic membrane is preferably obtained from a placenta. In some embodiments, the amniotic membrane is obtained from an umbilical cord.

[0035] The term "placental tissue" refers to any and all known components of the placenta, including, but not limited to, the amniotic membrane, the chorion, and, if the umbilical cord is considered part of the placenta, Wharton's jelly (but not the entire placenta). In a preferred embodiment, the placental tissue does not include any of the umbilical cord components (e.g., Wharton's jelly, umbilical vein, umbilical artery, and surrounding membranes). In preferred embodiments, the placental tissue is typically modified by removing the intermediate layer between the amniotic membrane and the chorion, such that the resulting composition does not include the intermediate layer.

[0036] As can be seen from the above, processing placental tissue requires steps to prevent cross-contamination from one processed placental tissue to the next. Such steps require the assembly of sterile supplies and equipment at a staging area in a controlled, sterile environment to prepare the placental tissue for introduction into the sterile, controlled environment described herein. If the controlled environment is a production hood, the sterile supplies are unpacked and placed in the hood using conventional aseptic technique. If the controlled environment is a clean room or similar sterile room, the sterile supplies are unpacked and placed on a cart covered with a sterile drape. Using conventional aseptic technique, all work surfaces are covered with a single sterile drape, and the sterile supplies and processing equipment are placed on the sterile drape, again using conventional aseptic technique. Also, prior to use of processing equipment for subsequent non-autologous placental tissue (i.e., placental tissue from a different donor), any processing equipment utilized with the initial placental tissue must be decontaminated according to conventional, industry-accepted decontamination procedures, where the equipment must be removed, decontaminated, and then reintroduced into the controlled environment.

[0037] The term "placental tissue graft" refers to any combination of placental tissue, including either the amniotic or chorionic membrane layer and, optionally, additional layers that may or may not be derived from the placenta. Preferably, the placental tissue graft does not include an intermediate layer. A single layer of amniotic membrane or chorionic membrane can be used, although multiple layers of amniotic membrane and / or chorionic membrane in the tissue graft are typically dehydrated and preferably laminated together. The amniotic membrane can optionally be partially or completely decellularized, e.g., by removing substantially all of the epithelial and / or fibroblast layers. Examples of placental tissue grafts suitable for the present invention include, by way of example only, those described in U.S. Pat. Nos. 8,323,701 and 8,372,437 and U.S. Patent Application Publication Nos. 2014 / 0052247, 2014 / 0067058, 2014 / 0205646, and 2013 / 0202676, the disclosures of each of which are incorporated herein by reference in their entirety.

[0038] The term "tensile strength" as used herein refers to the resistance of a composition to longitudinal stress as measured by the minimum amount of longitudinal stress required to break the material. Tensile strength can be measured by any method known in the art. For example, Young's modulus, also known as the tensile modulus of elasticity, is a measure of the stiffness of a material. It is defined as the ratio of uniaxial stress to uniaxial strain in the stress range where Hooke's law is applicable. Tensile strength may be measured using a uniaxial electromechanical material testing system, such as an Instron Model 5565 or an Instron Model 1122.

[0039] As used herein, the term "non-autologous tissue" refers to placental tissue from a different placental donor.

[0040] Titles or subtitles may be used herein for the convenience of the reader, but they do not affect the scope of the invention. Additionally, some terms used herein are more specifically defined below.

[0041] Collagen and micronized placental tissue compositions and methods for preparing same - Patent Application 20070122997 Described herein is a cohesive and flexible composition consisting of hydrated collagen and micronized placental tissue components. In one embodiment, the composition comprises water, collagen, and micronized placental tissue components. For example, preferred compositions can be prepared by the following route: For example, such compositions are prepared from micronized placental tissue components as described in PCT Application WO 2012112410 and U.S. Patent Application Publication Nos. 2013 / 0344162 and 2014 / 0050788. The entire disclosures of these applications are expressly incorporated herein by reference.

[0042] Of course, the term "micronized" is intended to include placental tissue particles of micron and submicron size.

[0043] Certain embodiments of the process for collecting, processing, and preparing placental material for subsequent use as a collagen and micronized placental tissue composition are outlined below and illustrated in FIG. 1. Following is a more detailed description and discussion of each individual step. First, placental tissue is collected from a consenting patient following an elective cesarean section (step 110). The material is preserved using conventional tissue preservation methods and transported to a suitable processing location or facility for reception and evaluation (step 120). Macroscopic processing, handling, and separation of the amniotic membrane and chorion then occur (step 130). The acceptable tissue is then decontaminated (step 140) and dehydrated (step 145). Following decontamination and dehydration, the placental tissue components (e.g., amniotic membrane, Wharton's jelly, and / or chorion, either individually or as grafts) are then micronized (step 150). Following micronization, the micronized placental tissue components are incorporated into aqueous collagen (step 160). Each step is described in detail below.

[0044] Initial tissue collection (step 110) The components used to produce the micronized placental tissue are derived from the placenta. The placenta source may vary. In one embodiment, the placenta is derived from a mammal, such as a human. Other animals, including but not limited to, cows and pigs, can be used herein. In the case of humans, the placenta is collected in a hospital, where it is collected during a Caesarean section delivery. The donor (referring to the mother about to give birth) voluntarily undergoes a comprehensive screening process designed to obtain the safest tissue available for transplantation. The screening process preferably uses conventional serology tests to test for antibodies against human immunodeficiency virus types 1 and 2 (anti-HIV-1 and anti-HIV-2), hepatitis B virus (anti-HBV), hepatitis B surface antigen (HBsAg), hepatitis C virus (anti-HCV), human T-lymphotropic virus types 1 and 2 (anti-HTLV-I, anti-HTLV-II), CMV, and syphilis, with nucleic acid testing for human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV). As will be appreciated by those skilled in the art, the above list of tests is exemplary only, as more, fewer, or different tests may be desired or required over time or based on the intended use of the placental component.

[0045] A decision is made as to whether the donor is acceptable based on a review of the donor information and the results of the screening test. Additionally, cultures are taken at the time of delivery to determine the presence of bacteria, such as Clostridium or Streptococcus. If the donor information, screening test, and delivery culture are all satisfactory (i.e., they indicate no risk or an acceptable level of risk), the donor is approved by the medical director, and the tissue specimen is initially designated eligible for further processing and evaluation.

[0046] Human placentas that meet the above selection criteria are placed in a sterile shipping bag, preferably containing saline, and stored in a container containing wet ice for shipment to a processing site or laboratory for further processing.

[0047] If placental tissue is collected prior to obtaining screening test results and delivery cultures, such tissue will be labeled and stored in isolation. Such placentas will be approved for further processing only after the necessary screening evaluations and delivery cultures, which indicates that the tissue is safe and satisfactory for handling and use, and that final approval has been obtained from the Medical Director.

[0048] Material Reception and Evaluation (Step 120) Upon arrival at the processing center or laboratory, the shipment is opened and verified to ensure the sterile shipping bag / container is still sealed and in refrigerant, that the appropriate donor documentation is present, and that the donor number on the documentation matches the number on the sterile shipping bag containing the tissue. The sterile shipping bag containing the tissue is then stored in a refrigerator until ready for further processing.

[0049] Gross Tissue Processing (Step 130) Once the tissue is prepared for further processing, the sterile supplies needed to process the placenta tissue are assembled at a staging area in a controlled (i.e., aseptic) environment and prepared for introduction into the controlled environment. In one embodiment, the placenta is processed at room temperature. If the controlled environment is a production hood, the sterile supplies are unpacked and placed in the hood using conventional aseptic technique. If the controlled environment is a clean room, the sterile supplies are unpacked and placed on a cart covered with a sterile drape. Using conventional aseptic technique, all work surfaces are covered with a single sterile drape, and the sterile supplies and processing equipment are placed on the sterile drape, again using conventional aseptic technique.

[0050] Processing equipment is decontaminated according to conventional, industry-accepted decontamination procedures and then placed in a controlled environment. This equipment is strategically placed in the controlled environment to minimize the chance of the equipment being in close proximity to or inadvertently contaminated by tissue specimens.

[0051] The placenta is then removed from the sterile shipping bag and aseptically transferred to a sterile processing basin in a controlled environment. The sterile basin contains hypertonic saline (e.g., 18% NaCl) at or near room temperature. The placenta is gently massaged to aid in the separation of blood clots and allow the placental tissue to reach room temperature, thereby facilitating the separation of placental components (e.g., amniotic membrane and chorion) from each other. After warming to ambient temperature (e.g., approximately 10–30 minutes), the placenta is then removed from the sterile processing basin and placed flat on a processing tray with the amniotic membrane facing down for examination.

[0052] The placenta is examined for discoloration, debris or other contamination, odor, and signs of damage. The size of the tissue is also noted. At this point, a decision is made as to whether the tissue is acceptable for further processing.

[0053] The amniotic membrane and chorion are then carefully separated. In one embodiment, the materials and equipment used in this procedure include a processing tray, 18% saline, 4x4 sterile sponges, and two sterile Nalgene wide-mouth bottles. The placental tissue is then examined closely to find an area (typically a corner) where the amniotic membrane can be separated from the chorion. The amniotic membrane appears as a thin, opaque layer on the chorion.

[0054] A piece of sterile gauze or cotton swab is used to gently touch both sides of the amniotic membrane to identify the fibroblast layer. The fibroblast layer will adhere to the test material. The amniotic membrane is placed in a processing tray with the basement membrane layer facing down. Any remaining blood is also removed using a blunt instrument, cell scraper, or sterile gauze. This step must again be performed with appropriate care to avoid tearing the amniotic membrane. The amniotic membrane is cleaned when it appears smooth and opaque white.

[0055] In certain embodiments, the intermediate tissue layer, also known as the spongy layer, is substantially removed from the amniotic membrane to expose the fibroblast layer. The term "substantially remove," with respect to the amount of intermediate tissue layer removed, is defined herein as removing more than 90%, more than 95%, or more than 99% of the intermediate tissue layer from the amniotic membrane. This can be accomplished by peeling the intermediate tissue layer from the amniotic membrane. Alternatively, the intermediate tissue layer can be removed from the amniotic membrane by wiping it with gauze or other suitable wipes. The resulting amniotic membrane can then be decontaminated using the methods described below. While not wishing to be bound by theory, removal of the intermediate layer can accelerate the drying of the tissue graft, especially when multiple amniotic membranes are used to generate the graft.

[0056] The methods described herein allow for the retention or removal of substantially all or a portion of the cellular components of the amniotic membrane layer. Removal of cellular components is a technique referred to in the art as "decellularization." Decellularization generally involves the physical and / or chemical removal of all cells present in the amniotic membrane, including epithelial cells and fibroblasts. In certain embodiments, the amniotic membrane is completely decellularized (e.g., removal of epithelial cells and fibroblasts). In other embodiments, only the epithelial layer or a portion thereof is removed. In still other embodiments, only the fibroblast layer is removed. Optionally, all cellular components are present.

[0057] In certain embodiments, a portion of the epithelial layer present on the amniotic membrane or substantially all of the epithelial layer is removed to expose a portion or a majority of the basal layer of the amniotic membrane. The term "substantially removed," with respect to the amount of epithelium removed, is defined herein as removing more than 90%, more than 95%, or more than 99% of the epithelial cells from the amniotic membrane. The presence or absence of remaining epithelial cells in the amniotic membrane layer can be assessed using techniques known in the art. For example, after removal of the epithelial cell layer, a representative tissue sample from a processing lot is placed on a standard microscope slide. The tissue sample is then stained using Eosin Y staining and evaluated as described below. The sample is then covered and left to stand. After an appropriate length of time to allow for staining, visual observation is performed under magnification.

[0058] In one embodiment, the epithelial layer can be removed by techniques known in the art. For example, the epithelial layer can be scraped off the amniotic membrane using a cell scraper. Other techniques include, but are not limited to, freezing the membrane, physical removal using a cell scraper, or exposing the epithelial cells to non-ionic detergents, anionic detergents, and nucleases. The amount of epithelial layer removed can range from 0% (i.e., the epithelial layer is retained) to removal of substantially all of the epithelial layer. Intermediate amounts of epithelial layer removal can be achieved by preparing rows in which the epithelial layer has been removed mixed with rows in which the epithelial layer has been retained. Alternatively, a portion of the epithelial layer can be removed while retaining the remainder.

[0059] The epithelium-depleted tissue is then evaluated to determine whether the basement membrane remains intact. This step is performed after the processing steps are completed but before the tissue is dehydrated, as described in the next section. For example, a representative sample explant is removed for microscopic analysis. The tissue sample is placed on a standard slide, stained with eosin Y, and viewed under a microscope. If epithelium is present, it will appear as cobblestone cells.

[0060] Chemical Decontamination (Step 140) The isolated placental tissue components can be chemically decontaminated using the techniques described below. In one embodiment, the amniotic membrane and / or chorion are decontaminated at room temperature. In one embodiment, the amniotic membrane produced in step 130 can be placed in a sterile Nalgene bottle for further processing. In one embodiment, the amniotic membrane can be washed using the following procedure: A Nalgene bottle is aseptically filled with 18% hypertonic saline and sealed (or alternatively sealed with a cap). The bottle is then placed on a shaking table and agitated for 30-90 minutes, which further removes contaminants from the amniotic membrane. If the shaking table is not located in a critical environment (e.g., a manufacturing hood), the Nalgene bottle is returned to a controlled / sterile environment before being opened. The amniotic membrane is gently removed from the Nalgene bottle containing 18% hypertonic saline using sterile forceps or by aseptically decanting the contents and placed into the empty Nalgene bottle. The empty Nalgene wide-mouth bottle containing the amniotic membrane is then aseptically filled with a premixed antibiotic solution. In one embodiment, the premixed antibiotic solution consists of a mixture of antibiotics, such as streptomycin sulfate and gentamicin sulfate. Other antibiotics, such as polymyxin B sulfate and bacitracin, or similar antibiotics currently or in the future, are also suitable. Furthermore, it is preferable that the antibiotic solution be at room temperature during addition to avoid changing the temperature of the amniotic membrane, which could otherwise damage it. The wide-mouth bottle or container containing the amniotic membrane and antibiotics is then sealed or closed and placed on a shaking table, preferably for 60-90 minutes, and agitated. Such agitation or agitation of the amniotic membrane in the antibiotic solution further removes contaminants and bacteria from the tissue. Optionally, the amniotic membrane can be washed with a detergent. In one embodiment, the amniotic membrane can be washed with a 0.1-10%, 0.1-5%, 0.1-1%, or 0.5% Triton-X wash solution.

[0061] If a shaking table is not located within the critical environment (e.g., a production hood), the wide-mouth bottle or container containing the amniotic membrane and antibiotics is returned to the critical sterile environment before being opened. Using sterile forceps, gently remove the amniotic membrane from the wide-mouth bottle or container and place it in a sterile basin containing sterile water or normal saline (0.9% saline). Leave the amniotic membrane submerged in the sterile water / normal saline for at least 10-15 minutes. The amniotic membrane may be slightly agitated to facilitate removal of the antibiotic solution and any other contaminants from the tissue. After at least 10-15 minutes, the amniotic membrane is ready for dehydration and further processing.

[0062] For chorionic membranes, the following exemplary procedure can be used: After separating the chorion from the amniotic membrane and removing the blood clot from the fibrous layer, the chorion is rinsed with 18% saline for 15 to 60 minutes. During the first rinse cycle, the 18% saline solution is heated in a sterile container using a laboratory heating plate so that the solution temperature is approximately 48°C. The solution is decanted, and the chorionic tissue is placed in a sterile container, and the decanted saline is poured into the container. The container is sealed and placed on a shaking table and agitated for 15 to 60 minutes. After the 1-hour agitation bath, the chorionic tissue is removed and placed in a second heated agitation bath for an additional 15 to 60 minutes of rinsing cycle. Optionally, the chorionic tissue can be washed with a detergent (e.g., Triton-X washing solution) as described above for decontaminating the amniotic membrane. The container is sealed and agitated for 15 to 120 minutes without heating. The chorionic tissue is then washed in deionized water (4 x 250 mL deionized water), using vigorous agitation after each rinse. The tissue is removed and placed in a container containing 1 x PBS w / EDTA solution. The container is sealed and agitated for 1 hour at controlled temperature for 8 hours. The chorionic tissue is removed and rinsed with sterile water. A visual inspection is performed to remove any remaining discolored fibrous blood material from the chorionic tissue. The chorionic tissue should have a creamy white appearance without any visible brownish discoloration.

[0063] Dehydration(145) In one embodiment, the amniotic membrane, chorion, Wharton's jelly, or any combination thereof can be processed into a tissue graft (i.e., a laminate) that is then micronized. In another embodiment, individual amniotic membrane, chorion, or Wharton's jelly layers can be independently dehydrated and then micronized, either individually or as a mixture of components. In one embodiment, the tissue (i.e., individual membrane or graft) is dehydrated by chemical dehydration and then freeze-dried. In one embodiment, the chemical dehydration step is carried out by contacting the amniotic membrane, chorion, and / or Wharton's jelly with a polar organic solvent for a time and in an amount sufficient to substantially (i.e., greater than 90%, 95%, or greater than 99%) or completely remove any residual water present in the tissue (i.e., dehydrate the tissue). The solvent can be protic or aprotic. Examples of polar organic solvents useful herein include, but are not limited to, alcohols, ketones, ethers, aldehydes, or any combination thereof. Specific, non-limiting examples include DMSO, acetone, tetrahydrofuran, ethanol, 2-propanol, or any combination thereof. In one embodiment, the placental tissue is contacted with the polar organic solvent at room temperature. No further steps are required, and the tissue can be directly lyophilized as described below.

[0064] After chemical dehydration, the tissue is freeze-dried to remove any remaining water and polar organic solvents. In one embodiment, the amniotic membrane, chorion, and / or Wharton's jelly can be placed on a suitable drying fixture before freeze-drying. For example, one or more strips of amniotic membrane can be placed on a suitable drying fixture. The chorion is then placed on top of the amniotic membrane. In this embodiment, an amniotic / chorionic tissue graft is produced. Alternatively, a strip of amniotic membrane can be placed on a first drying fixture, and a strip of chorion can be placed on a second drying fixture. The drying fixture is preferably large enough to accommodate the fully flattened placental tissue. In one embodiment, the drying fixture is made of Teflon® or Delrin (a trade name for an acetal resin engineering plastic invented and sold by DuPont and also commercially available from Werner Machine, Marietta, Georgia). Any other suitable heat-resistant and cut-resistant material that can be formed into an appropriate shape to accommodate wet tissue can also be used for the drying fixture.

[0065] Once the tissue is placed on the drying fixture, the drying fixture is placed into a freeze-dryer. Using a freeze-dryer to dehydrate the tissue allows for more efficient and thorough dehydration compared to other techniques, such as thermal dehydration. It is generally desirable to avoid ice crystal formation in placental tissue, as this can damage the extracellular matrix within the tissue. Chemically dehydrating the placental tissue prior to freeze-drying can circumvent this problem.

[0066] In another embodiment, heat is applied to the tissue during the dehydration process. In one embodiment, the amniotic membrane, chorion, and / or Wharton's jelly are placed on a suitable desiccant (either as individual strips or in the laminate described above), and the desiccant is placed in a sterile Tyvex (or similar breathable, heat-resistant, sealable material) dehydration bag and sealed. The breathable dehydration bag prevents the tissue from drying out too quickly. When multiple desiccant devices are processed simultaneously, each device is placed in its own Tyvex bag or in a suitable mounting frame designed to hold multiple desiccant frames above it, and the entire frame is then placed in a single, larger, sterile Tyvex dehydration bag and sealed.

[0067] The Tyvex dehydration bag containing one or more drying fixtures is then placed in a non-vacuum desiccator or incubator that has been preheated to approximately 35-50°C. The Tyvex bag is left in the desiccator for 30-120 minutes. In one embodiment, the heating step can be carried out for 45 minutes at a temperature of approximately 45°C to dry the tissue sufficiently but without over-drying or burning. The specific temperature and time for any particular desiccator will need to be calibrated and adjusted based on other factors such as altitude, size of the desiccator, accuracy of the desiccator temperature, material used for the desiccators, number of desiccators to be dried simultaneously, and whether single or multiple frames of desiccators are dried simultaneously.

[0068] Preparation of Micronized Composition (Step 150) Once the amniotic membrane, chorion, and / or Wharton's jelly layer are dehydrated, either individually or in the form of a tissue graft, the dehydrated tissue is micronized. The micronized compositions can be produced using equipment known in the art. For example, a Retsch Oscillating Mill MM400 can be used to produce the micronized compositions described herein. The particle size of the materials in the micronized compositions can also vary depending on the intended use of the micronized composition. In one embodiment, the micronized composition has particles less than 500 μm, less than 400 μm, less than 300 μm, or between 25 μm and 300 μm, 25 μm and 200 μm, or 25 μm or 150 μm. In certain embodiments, particles with larger diameters (e.g., between 150 μm and 350 μm) are desirable. Those skilled in the art will appreciate that the particle sizes and size ranges of the materials in the micronized compositions of the present invention are average.

[0069] In one embodiment, micronization is achieved by mechanical grinding or crushing. In another embodiment, micronization is achieved by cryogenic grinding. In this embodiment, the grinding bottle containing the tissue is constantly cooled with liquid nitrogen from an integrated cooling system before and during the grinding process. This makes the sample brittle and preserves volatile components. Furthermore, denaturation of proteins in the amniotic membrane, Wharton's jelly, and / or chorion is minimized or prevented. In one embodiment, a Retsch CryoMill can be used in this embodiment.

[0070] The choice of placental tissue component used to prepare the compositions described herein may vary depending on the end use of the composition. For example, individual components of placental tissue, i.e., amniotic membrane, chorion, intermediate tissue layer, Wharton's jelly, or any combination thereof, can be mixed together and then micronized. In another embodiment, one or more tissue grafts consisting of one or more placental tissues, amniotic membrane, chorion, Wharton's jelly layer, or any combination thereof (i.e., laminates) can be micronized. In a further embodiment, one or more tissue grafts consisting of one or more amniotic membranes, chorion, Wharton's jelly layer, or any combination thereof can be mixed with the individual components amniotic membrane, chorion, Wharton's jelly layer, or any combination thereof, and then micronized.

[0071] The amounts of different components used to prepare the micronized compositions described herein may vary depending on the intended use of the micronized composition. In one embodiment, when the micronized composition comprises amniotic membrane (with or without an intermediate tissue layer) and Wharton's jelly, the weight ratio of amniotic membrane to Wharton's jelly is 10:1 to 1:10, 9:1 to 1:1, 8:1 to 1:1, 7:1 to 1:1, 6:1 to 1:1, 5:1 to 1:1, 4:1 to 1:1, 3:1 to 1:1, 2:1 to 1:1, or about 1:1. In another embodiment, when the micronized composition comprises amniotic membrane (with or without an intermediate tissue layer) and chorion, the weight ratio of chorion:amniotic membrane is 10:1 to 1:10, 9:1 to 1:1, 8:1 to 1:1, 7:1 to 1:1, 6:1 to 1:1, 5:1 to 1:1, 4:1 to 1:1, 3:1 to 1:1, 2:1 to 1:1, or about 1:1.

[0072] For example, particle size separation can be achieved by fractionation of micronized material in sterile water by forming a suspension of particles. The top of the suspension contains primarily the smallest particles, and the bottom of the suspension contains primarily the heaviest particles. Fractionation results in particle size separation, and repeated fractionation results in separation of the micronized particles into various particle sizes.

[0073] The separated particles can be recombined in a desired particle size ratio that is most appropriate for the preparation and desired use of the collagen and micronized placental tissue composition. As will be appreciated by those skilled in the art, particles of different sizes will result in different flexibility and cohesiveness of the compositions described herein.

[0074] In further embodiments, the placental tissue can be crosslinked. For example, a crosslinking agent can be added to the composition (e.g., amniotic membrane, chorion, Wharton's jelly, or any combination thereof as individual components and / or tissue grafts) before and / or after micronization. Generally, crosslinking agents are non-toxic and non-immunogenic. When the amniotic membrane, Wharton's jelly, and chorion (or tissue grafts thereof) are treated with a crosslinking agent, the crosslinking agents can be the same or different. In one embodiment, the amniotic membrane, Wharton's jelly, and chorion can be treated separately with a crosslinking agent, or in another method, the amniotic membrane, Wharton's jelly, and chorion can be treated together with the same crosslinking agent. In certain embodiments, the amniotic membrane, Wharton's jelly, and chorion can be treated with two or more different crosslinking agents. The conditions for treating the amniotic membrane, Wharton's jelly, and chorion can be different. In other embodiments, the amniotic membrane, Wharton's jelly, and / or chorion can be micronized, and the micronized composition can then be treated with a crosslinking agent. In one embodiment, the concentration of the cross-linking agent is 0.1M to 5M, 0.1M to 4M, 0.1M to 3M, 0.1M to 2M, or 0.1M to 1M.

[0075] Crosslinking agents generally have two or more functional groups that can react with proteins to form covalent bonds. In one embodiment, crosslinking agents have a group that can react with the amino group present in proteins. Examples of such functional groups include, but are not limited to, hydroxyl groups, substituted or unsubstituted amino groups, carboxyl groups, and aldehyde groups. In one embodiment, the crosslinking agent can be a dialdehyde, such as glutaraldehyde. In another embodiment, the cross-linking agent may be a carbodiimide such as, for example, N-(3-dimethylaminopropyl)-N'-ethyl-carbodiimide (EDC). In other embodiments, the cross-linking agent may be oxidized dextran, p-azidobenzoylhydrazide, N-[α-maleimidoacetoxy]succinimide ester, p-azidophenylglyoxal monohydrate, bis-[β-(4-azidosalicylamido)ethyl]disulfide, bis-[sulfosuccinimidyl]suberate, dithiobis[succinimidyl]propionate, disuccinimidyl suberate, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride, a bifunctional oxirane (OXR), or ethylene glycol diglycidyl ether (EGDE).

[0076] In one embodiment, a sugar is a cross-linking agent, wherein the sugar can react with proteins present in the amniotic membrane, Wharton's jelly, and / or chorion to form a covalent bond. For example, the sugar can react with proteins via the Maillard reaction, which begins with the non-enzymatic glycosylation of an amino group on the protein with a reducing sugar, followed by the formation of a covalent bond. Examples of sugars useful as cross-linking agents include, but are not limited to, D-ribose, glycerol, altrose, talose, erythrose, glucose, lyxose, mannose, xylose, gulose, arabinose, idose, allose, galactose, maltose, lactose, sucrose, cellobiose, gentiobiose, melibiose, turanose, trehalose, isomaltose, or any combination thereof.

[0077] In certain embodiments, the micronized compositions described herein can be formulated with any excipient that can be tolerated by a biological system or entity to produce a pharmaceutical composition. Examples of such excipients include, but are not limited to, water, aqueous hyaluronic acid solution, physiological saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions. Non-aqueous vehicles such as fixed oils, vegetable oils (e.g., olive oil and sesame oil), triglycerides, propylene glycol, polyethylene glycol, and injectable organic esters (e.g., ethyl oleate) can also be used. Other useful formulations include suspensions containing viscosity-enhancing agents such as sodium carboxymethylcellulose, sorbitol, or dextran. The excipient may also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer, and Tris buffer, and examples of preservatives include thimerosal, cresol, formalin, and benzyl alcohol. In certain embodiments, the pH can be varied depending on the mode of administration. Furthermore, the pharmaceutical compositions may include carriers, thickeners, diluents, preservatives, surfactants, and the like in addition to the compounds described herein.

[0078] Preparation of a Composition of Collagen and Micronized Placental Tissue (Step 160) Once the placental tissue is micronized, the micronized placental tissue components are ready for incorporation into aqueous / gelatinous collagen. In one aspect, the collagen is human collagen, and in a preferred embodiment, the collagen is human placental collagen.

[0079] Preferably, the aqueous / gelatinous collagen is soluble (solubilized) collagen. Soluble collagen is significantly different from naturally occurring collagen, which exists in highly ordered fibrils. Collagen molecules aggregate to form collagen fibrils and fibrils, which then undergo a natural cross-linking process. These are insoluble crystalline structures. In contrast, collagen in aqueous solution exists as individual collagen molecules or can aggregate to form collagen fibrils but is not cross-linked, and can be resolubilized unless cross-linked (i.e., solubility is reversible until the collagen is cross-linked).

[0080] The composition can be prepared using techniques known in the art. As used herein, the term "incorporate" or "incorporated" means "take in," "contain," "absorb," or "mix," etc. For example, in one embodiment, the composition is prepared by mixing a micronized placental tissue component described herein with collagen. The term "mixing" is defined as mixing two or more components (e.g., collagen and a micronized placental tissue component) together such that there is no chemical reaction or physical interaction. The term "mixing" also includes a chemical reaction or physical interaction between two or more compounds (e.g., between collagen and a micronized placental tissue component).

[0081] Of course, the actual preferred amount of micronized placental tissue component in the specified examples will vary depending on the specific utility of the composition, the particular composition formulated, the mode of application, and the particular location and subject being treated. The dosage for a given subject can be determined, for example, by appropriate conventional pharmaceutical procedures, using conventional studies, such as routine comparisons of the activity difference between the subject compound and known drugs. Physicians and formulators skilled in the art of determining the dosage of pharmaceutical compounds will have no difficulty determining the dosage in accordance with standard recommendations (Physician's Desk Reference, Barnhart Publishing (1999)). Generally, the amount of micronized placental tissue component used is sufficient to convert the gelatinous aqueous collagen into a flexible and cohesive composition. Without being bound by theory, it is believed that the micronized placental tissue component absorbs the maximum amount of water contained in the hydrated collagen, thereby causing the gelatinous composition to be converted into a flexible and cohesive composition.

[0082] The amount of micronized placental tissue component and collagen used to prepare the compositions described herein can vary depending on the application. In one embodiment, the weight ratio of micronized placental tissue component to collagen is about 300:1 to about 1:1. In one embodiment, the weight ratio of micronized placental tissue component to collagen is about 100:1 to about 300:1. Such ratios result in a cohesive mass. Furthermore, the flexibility of the mass is inversely proportional to the amount of micronized placental tissue component used. The greater the amount of micronized placental tissue component, the less flexible the mass. This allows one of skill in the art to select the degree of flexibility appropriate for the intended use.

[0083] In some embodiments, the weight ratio of micronized placental tissue component to collagen is about 300:1 to about 50:1, about 300:1 to about 75:1, about 300:1 to about 100:1, about 300:1 to about 150:1, about 300:1 to about 200:1, or about 300:1 to about 250:1. In some embodiments, the weight ratio of micronized placental tissue component to collagen is about 100:1 to about 300:1, about 100:1 to about 250:1, about 100:1 to about 200:1, or about 100:1 to about 150:1. The weight ratio may be any value or subrange included in any of the recited ranges, including the endpoints.

[0084] In another aspect, the weight ratio of micronized placental tissue component to collagen is from 1:1 to less than 100:1. At these ratios, the gelatinous nature of the composition is maintained, with the understanding that the composition will be enhanced by the growth factors and cytokines then contained in the micronized placental tissue component. In some embodiments, the weight ratio of micronized placental tissue component to collagen is from about 1:1 to about 100:1, from about 1:1 to about 90:1, from about 1:1 to about 80:1, from about 1:1 to about 70:1, from about 1:1 to about 60:1, from about 1:1 to about 50:1, from about 1:1 to about 40:1, from about 1:1 to about 30:1, from about 1:1 to about 20:1, or from about 1:1 to about 10:1. In some embodiments, the weight ratio of micronized placental tissue component to collagen is about 10:1 to about 100:1, about 20:1 to about 100:1, about 30:1 to about 100:1, about 40:1 to about 100:1, about 50:1 to about 100:1, about 60:1 to about 100:1, about 70:1 to about 100:1, about 80:1 to about 100:1, or about 90:1 to about 100:1. The weight ratio may be any value or subrange included in any of the recited ranges, including the endpoints.

[0085] Those skilled in the art will appreciate that the amount of collagen in the present compositions ranges from about 0.1% to about 2% collagen. In preferred embodiments, the aqueous collagen contains about 0.5% collagen. In some embodiments, the aqueous collagen contains about 0.1% to about 1.8% collagen, about 0.1% to about 1.6% collagen, about 0.1% to about 1.5% collagen, about 0.1% to about 1.4% collagen, about 0.1% to about 1.2% collagen, about 0.1% to about 1.0% collagen, about 0.1% to about 0.8% collagen, about 0.1% to about 0.6% collagen, about 0.1% to about 0.5% collagen, about 0.1% to about 0.4% collagen, or about 0.1% to about 0.2% collagen. In some embodiments, the aqueous collagen comprises about 0.5% to about 2% collagen, about 0.8% to about 2%, about 1% to about 2%, about 1.2% to about 2%, about 1.5% to about 2%, or about 1.8% to about 2% collagen. The amount of collagen may be any value or subrange included within any of the recited ranges, including the endpoints.

[0086] In certain embodiments, compositions comprising collagen and micronized placental tissue components exhibited increased flexibility and cohesiveness upon application of sufficient force and shear (e.g., via a syringe) when compared to an aqueous / gelatinous solution of collagen in the absence of the micronized placental tissue components. Of course, any type of force and / or shear that causes a composition to change from gelatinous to a cohesive and flexible composition is encompassed by the present invention. In a preferred embodiment, the force and shear is achieved by ejection from the tip of a needle. Alternatively, a press or mold can be used as well. It was surprising and unexpected that the addition of micronized placental tissue components to collagen resulted in a composition that ejected from the tip of a 27-gauge needle that was cohesive and capable of sticking to the needle when lifted after injection (see Figure 3B). That is, the modified placental tissue alters the ejected mass to form a more flexible and / or more cohesive structure.

[0087] In certain embodiments, the composition is cohesive and flexible under physiological conditions, e.g., at a temperature ranging from about 15°C to about 45°C. In certain embodiments, the composition may be a gel at about 37°C (e.g., upon injection into a subject). Without being bound by theory, it is believed that the collagen and micronized placental tissue component composition remains at the injection site for a longer period of time compared to injectable compositions of micronized placental tissue component that do not contain collagen (e.g., AmnioFix® or EpiFix®). Furthermore, without being bound by theory, it is believed that the collagen and micronized placental tissue component composition breaks down more slowly compared to collagen-only compositions. The collagen and micronized placental tissue component composition is also expected to have more growth factors and other factors that aid in healing.

[0088] In certain embodiments, additional ingredients may be added to the collagen and micronized placental tissue component composition. The additional ingredients may be fillers, bioactive agents, adhesives, stabilizers, buffers, pharmaceutical ingredients, colorants, disintegrants, etc. The additional ingredients may be added to the collagen and / or micronized placental tissue component before or after mixing the collagen and micronized placental tissue component.

[0089] In one embodiment, a bulking agent can be added. Examples of bulking agents include, but are not limited to, allograft pericardium, allograft acellular dermis, Wharton's jelly isolated from vasculature (i.e., umbilical veins and arteries) and surrounding membranes, purified xenograft type I collagen, biocellulose polymer or copolymer, biocompatible synthetic polymer or copolymer film, purified small intestinal submucosa, acellular matrix of the urinary bladder, cadaveric fascia, or any combination thereof.

[0090] In another embodiment, bioactive agents can be added to the composition before and / or after micronization. Examples of bioactive agents include, but are not limited to, naturally occurring growth factors derived from platelet concentrates obtained using either autologous blood collection and separation products or from expired stored blood, stem cells derived from bone marrow aspirate, enriched human placental umbilical cord blood stem cells, enriched amniotic fluid stem cells, or stem cells expanded in a bioreactor, or antibiotics. When the composition containing the bioactive agent is applied to the area of ​​interest, the bioactive agent is delivered to the area over time. Thus, the micronized particles described herein are useful as delivery devices for bioactive agents and other pharmaceuticals when administered to a subject. Among other things, the release profile can be tailored based on the selection of ingredients used to prepare the micronized composition and the particle size of the particles.

[0091] In other embodiments, one or more adhesives can be mixed with the collagen and micronized placenta composition. Examples of such adhesives include, but are not limited to, fibrin sealants, cyanoacrylates, gelatin and thrombin products, polyethylene glycol polymers, albumin and glutaraldehyde products. Without wishing to be bound by theory, a composition consisting of smaller micronized particles produces a denser composition that can withstand mechanical loads. Alternatively, larger micronized particles produce a less dense and compressible construct. This feature can be useful in cavity filling under no load, especially when it is desirable to have a composition that can conform to irregular shapes. The three-dimensional construct can include one or more bioactive agents as described herein.

[0092] In one embodiment, a coloring agent is added to facilitate locating and properly placing the collagen and micronized placental tissue component composition at the intended treatment site, hi another embodiment, a disintegrant alters the rate at which the collagen and micronized placental tissue component composition breaks down or disintegrates in vivo after introduction into a subject.

[0093] In yet another embodiment, the collagen and micronized placental tissue component composition is mixed with at least one plasticizer. One skilled in the art would select a suitable plasticizer based on its biocompatibility, its effect on the rate of degradation or destruction of the placental tissue graft in vivo, and its effect on the properties of the mixture that promote flexibility and cohesion of the collagen and micronized placental tissue component composition. Exemplary plasticizers include, but are not limited to, polyethylene glycol, glucose monoesters, and partial fatty acid esters.

[0094] The collagen and micronized placental tissue component compositions described herein can be administered in a number of ways depending on the area to be treated, whether local or systemic treatment is desired. In one embodiment, administration can be by injection. In another embodiment, the composition can be formulated to be administered internally to a subject. In another embodiment, the composition can be administered locally (e.g., intraocularly, intravaginally, intrarectally, intranasally, orally, or directly to the skin).

[0095] Optional Preparation of Topical Compositions In one embodiment, the composition can be formulated as a topical composition that can be applied directly to the skin.The formulation for topical administration can include emulsion, cream, aqueous solution, oil, ointment, putty, paste, gel, lotion, emulsion and suspension.In one embodiment, the topical composition can include one or more surfactants and / or emulsifiers.

[0096] Surfactants (or surface-active substances) that may be present are anionic, nonionic, cationic and / or amphoteric surfactants. Typical examples of anionic surfactants include, but are not limited to, soaps, alkyl benzene sulfonates, alkane sulfonates, olefin sulfonates, alkyl ether sulfonates, glycerin ether sulfonates, α-methyl ester sulfonates, sulfofatty acids, alkyl sulfates, fatty alcohol ether sulfates, glycerin ether sulfates, fatty acid ether sulfates, hydroxy mixed ether sulfates, monoglyceride (ether) sulfates, fatty acid amide (ether) sulfates, mono- and dialkyl sulfosuccinates, mono- and dialkyl sulfosuccinamates, sulfotriglycerides, amide soaps, ether carboxylic acids and their salts, fatty acid isethionates, fatty acid sarcosinates, fatty acid taurides. Examples of nonionic surfactants include fatty alcohol polyglycol ethers, alkylphenol polyglycol ethers, fatty acid polyglycol esters, fatty acid amide polyglycol ethers, fatty amine polyglycol ethers, alkoxylated triglycerides, mixed ethers or mixed formals, optionally partially oxidized alkyl(enyl) oligoglycosides or glucuronic acid derivatives, fatty acid N-alkylglucamides, protein hydrolysates (especially wheat-based plant products), polyol fatty acid esters, sugar esters, sorbitan esters, polysorbates, and amine oxides. Examples of amphoteric or zwitterionic surfactants include, but are not limited to, alkylbetaines, alkylamidobetaines, aminopropionates, aminoglycinates, imidazolinium betaines, and sulfobetaines.

[0097] In one aspect, the surfactant may be a fatty alcohol polyglycol ether sulfate, monoglyceride sulfate, mono- and / or di-alkyl sulfosuccinate, fatty acid isethionate, fatty acid sarcosinate, fatty acid tauride, fatty acid glutamate, α-olefin sulfonate, ether carboxylic acid, alkyl oligoglucoside, fatty acid glucamide, alkylamidobetaine, amphoacetal, and / or protein fatty acid concentrate.

[0098] Examples of zwitterionic surfactants include N-alkyl-N,N-dimethylammonium glycinates, such as cocoalkyldimethylammonium glycinate, N-acylaminopropyl-N,N-dimethylammonium glycinates, for example cocoacylaminopropyldimethylammonium glycinate and 2-alkyl-3-carboxymethyl-3-hydroxyethylimidazolines, in each case having 8 to 18 carbon atoms in the alkyl or acyl group, and betaines such as cocoacylaminoethylhydroxyethyl-carboxymethylglycinate.

[0099] In one embodiment, the emulsifier is selected from the group consisting of straight chain fatty alcohols having 8 to 22 carbon atoms, fatty acids having 12 to 22 carbon atoms, adducts of 2 to 30 moles of ethylene oxide and / or 0 to 5 moles of propylene oxide to alkylphenols having 8 to 15 carbon atoms in the alkyl group and alkylamines having 8 to 22 carbon atoms in the alkyl radical, alkyl and / or alkenyl oligoglycosides having 8 to 22 carbon atoms in the alkyl(enyl) radical and their ethoxylated analogs, castor oil, and / or hydrogenated castor oil with 1 to 15 moles of ethylene oxide adduct, castor oil and / or hydrogenated castor oil with 15 to 60 moles of ethylene oxide adduct, partial esters of glycerin and / or sorbitan with unsaturated linear or saturated branched fatty acids having 12 to 22 carbon atoms and / or hydroxycarboxylic acids having 3 to 18 carbon atoms and their 1 to 30 moles of ethylene oxide adduct, polyglycerin (average self-condensation degree: 2 to 8), trimethylolpropane, pentaerythritol, sugar alcohol The surfactant may be a nonionic surfactant selected from the group consisting of ethanol (e.g., sorbitol), alkylglucosides (e.g., methyl glucoside, butyl glucoside, lauryl glucoside) and polyglucosides (e.g., cellulose) with saturated and / or unsaturated linear or branched fatty acids having 12 to 22 carbon atoms and / or hydroxycarboxylic acids having 3 to 18 carbon atoms and their adducts with 1 to 30 moles of ethylene oxide, pentaerythritol, mixed esters of fatty acids, citric acid and fatty alcohols and / or mixed esters of fatty acids having 6 to 22 carbon atoms, methyl glucose and polyols, preferably glycerin or polyglycerin, mono-, di- and / or tri-alkyl phosphates and mono-, di- and / or tri-PEG alkyl phosphates and their salts, wool wax alcohol, polysiloxane-polyalkyl-polyether copolymers and corresponding derivatives, and block copolymers, for example, polyethylene glycol-30 dipolyhydroxystearate.In one embodiment, the emulsifier is a polyalkylene glycol, such as polyethylene glycol or polypropylene glycol, hi another embodiment, the emulsifier is a polyethylene glycol having a molecular weight of 100 Da to 5,000 Da, 200 Da to 2,500 Da, 300 Da to 1,000 Da, 400 Da to 750 Da, 550 Da to 650 Da, or about 600 Da.

[0100] In another embodiment, the emulsifier is a poloxamer. In one embodiment, a poloxamer is a nonionic triblock copolymer consisting of a central hydrophobic chain of polyoxypropylene (e.g., poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (e.g., poly(ethylene oxide)). In one embodiment, a poloxamer has the formula: HO(CHO) b (C3H6O) a (C2H4O) b It has OH.

[0101] wherein a is 10-100, 20-80, 25-70, 25-70, or 50-70, and b is 5-250, 10-225, 20-200, 50-200, 100-200, or 150-200. In another embodiment, the poloxamer has a molecular weight of 2,000-15,000, 3,000-14,000, or 4,000-12,000. Poloxamers useful herein are sold under the trade name Pluronic®, manufactured by BASF. Non-limiting examples of poloxamers useful herein include, but are not limited to, Pluronic® F68, P103, P105, P123, F127, and L121.

[0102] In another embodiment, the emulsifier comprises one or more fatty alcohols. In one embodiment, the fatty alcohol is a linear or branched C6-C 35Examples of fatty alcohols include, but are not limited to, caprylic alcohol (1-octanol), 2-ethylhexanol, pelargonic alcohol (1-nonanol), capric alcohol (1-decanol, decyl alcohol), undecyl alcohol (1-undecanol, undecanol, hendecanol), lauryl alcohol (dodecanol, 1-dodecanol), tridecyl alcohol (1-tridecanol, tridecanol, isotridecanol), myristyl alcohol (1-tetradecanol), pentadecyl alcohol (1-pentadecanol, pentadecanol), cetyl alcohol (1-hexadecanol), palmitoleyl alcohol (palmitoleyl alcohol), and the like. alcohol) (cis-9-hexadecen-1-ol), heptadecyl alcohol (1-n-heptadecanol, heptadecanol), stearyl alcohol (1-octadecanol), isostearyl alcohol (16-methylheptadecan-1-ol), elaidyl alcohol (9E-octadecen-1-ol), oleyl alcohol (cis-9-octadecen-1-ol), linoleyl alcohol (9Z,12Z-octadecadien-1-ol), elaidolinoleyl alcohol (9E,12E-octadecadien-1-ol), linolenyl alcohol (9Z,12Z,15Z-octadecatrien-1-ol), elaidolinolenyl alcohol (9E,12E,15-E-octadecatrien-1-ol), ricinoleyl alcohol (12-hydroxy-9-octadecen-1-ol), nonadecyl alcohol (1-nonadecanol), arachidyl alcohol (1-eicosanol), heneicosyl alcohol (1-heneicosanol), behenyl alcohol (1-docosanol), erucyl alcohol (cis-13-docosen-1-ol), lignoceryl alcohol (1-tetracosanol), seryl alcohol (1-hexacosanol), montanyl alcohol, cluytyl alcohol (1-octacosanol), myricyl alcohol, melissyl alcohol (1-triacontanol), geddyl alcohol (1-tetratriacontanol), or cetearyl alcohol.

[0103] In one embodiment, the carrier used to form the topical composition is a mixture of polyethylene and one or more fatty alcohols. For example, the carrier is comprised of 50% to 99%, 75% to 99%, 90% to 99%, or about 95% by weight of polyethylene glycol and 1% to 50%, 1% to 25%, 1% to 10%, or about 5% by weight of a fatty alcohol. In a further embodiment, the carrier is a mixture of polyethylene glycol and cetyl alcohol.

[0104] The topical composition can also include additional ingredients typically found in such compositions. In one embodiment, the topical composition can include one or more of the following ingredients: fats, waxes, pearlescent waxes, bodying agents, thickeners, superfatting agents, stabilizers, polymers, silicone compounds, lecithin, phospholipids, bioactive ingredients, deodorants, antibacterial agents, antiperspirants, swelling agents, insect repellents, hydrotropes, solubilizers, preservatives, fragrance oils, and dyes. Examples of each of these ingredients are disclosed in U.S. Patent No. 8,067,044, the disclosure of which is incorporated by reference.

[0105] Topical compositions comprising the collagen and micronized placental tissue component compositions described herein can be prepared by mixing the composition with a carrier. In cases where the carrier comprises two or more components, the components can be mixed together before adding the collagen and micronized placental tissue component composition. The amount of collagen and micronized placental tissue component composition present in the topical composition can vary depending on the application. In one embodiment, the collagen and micronized placental tissue component composition is 0.5% to 20%, 1% to 10%, 2% to 5%, or about 3% by weight of the topical composition.

[0106] Once prepared, the collagen and micronized placental tissue component composition can be used directly or can be appropriately packaged for storage and later use.

[0107] Optional preparation of 3D constructs In one embodiment, after the collagen and micronized placental tissue component composition is prepared, a shaped three-dimensional construct may be generated by filling the collagen and micronized placental tissue component composition into a mold having specific dimensions.

[0108] In one embodiment, the collagen and micronized placental tissue component are treated with the cross-linking agent and then placed into a mold having specific dimensions. Alternatively, the collagen and micronized placental tissue component composition is placed into a mold and then treated with the cross-linking agent. In one embodiment, the collagen and micronized placental tissue component composition is molded without cross-linking. In one embodiment, the collagen and micronized placental tissue component composition is formed by hand into any desired shape.

[0109] Optional preparation of tissue allografts In one embodiment, after the collagen and micronized placental tissue component composition is prepared, the collagen and micronized placental tissue component composition may be applied to one or more sides of a biocompatible material and / or device to create a coated device.

[0110] In one embodiment, the biocompatible material is a biocompatible mesh that can be made from a non-absorbable material such as, but not limited to, a biocompatible metal (e.g., titanium alloy, stainless steel, cobalt-chromium alloy, and nickel-titanium alloy). In another embodiment, the layer of the biocompatible mesh can be made from a non-absorbable polymeric material such as, but not limited to, a thermoplastic, polyethylene, ultra-high molecular weight polyethylene, high molecular weight polyolefin, uncoated monofilament polypropylene, polyetheretherketone, polyethylene terephthalate, polytetrafluoroethylene, expanded polytetrafluoroethylene, nylon, any polymer or aliphatic hydrocarbon with one or more double bonds, any other suitable porous material, or any other suitable porous material that can be bent or otherwise shaped.

[0111] In another embodiment, the biocompatible mesh may be composed of synthetic or bioresorbable polymeric materials such as, but not limited to, polyglycolic acid, poly-L-lactic acid (PLLA), poly-D,L-lactic acid (PDLA), trimethylene carbonate (TMC), poly-ε-caprolactone, poly-P-dioxanone, copolymers of lactide and glycolide (PLGA), poly(3-hydroxybutyric acid), collagen, hyaluronic acid, silk, biocellulose, other protein-based polymers, polysaccharides, poly(DTE carbonate), polyarylate, blends of PLLA, PLDA, or PLGA with TMC, and other combinations of these polymers.

[0112] In one embodiment, a biocompatible mesh is coated on one or both sides with a placental tissue graft and / or the present collagen and micronized placental tissue component composition to form a reinforced tissue graft. Examples of reinforced placental tissue allografts and methods of making are described in U.S. Patent Application Publication No. 2014 / 0067058, the entire disclosure of which is incorporated herein by reference.

[0113] In some aspects, the present disclosure relates to a tissue graft comprising one or more layers of placental tissue combined with one or more layers of the present collagen and micronized placental tissue composition. In one embodiment, the placental tissue graft comprising the present collagen and micronized placental tissue composition is a placental tissue graft comprising one or more layers of amniotic membrane, one or more layers of chorion, or one or more layers of amniotic membrane and chorion. Examples of tissue grafts that can be modified to include the collagen and micronized placental tissue composition described herein are described in U.S. Patent Nos. 8,623,421, 8,709,494, 8,357,403, 8,409,626, and 9,186,382, and U.S. Patent Application Publication Nos. 2014 / 0205646, 2014 / 0343688, and 2013 / 020676, the entire disclosures of each of which are incorporated herein by reference.

[0114] Uses of Collagen and Micronized Placental Tissue Compositions The collagen and micronized placental tissue component compositions described herein can be used in a number of medical applications in a subject, including wound healing, repair of damaged tendons, and cosmetic applications. The collagen and micronized placental tissue component compositions described herein can also be used to coat biocompatible materials and / or devices.

[0115] In one embodiment, the collagen and micronized placental tissue component composition is used in various medical applications without first subjecting the composition to sufficient force and shear, while in other embodiments, the composition is subjected to sufficient force and shear to convert the composition into a coherent and pliable mass prior to use in various medical applications.

[0116] In one aspect, the collagen and micronized placental tissue component compositions described herein are useful in enhancing or improving wound healing. There are many different types of wounds seen by physicians every day. Acute wounds are caused by surgical interventions, trauma, and burns. Chronic wounds are wounds that are slow to close compared to healing in otherwise healthy individuals. Examples of types of chronic wounds that plague patients include diabetic foot ulcers, venous leg ulcers, pressure ulcers, arterial ulcers, and infected surgical wounds.

[0117] The physician's goal when treating traumatic wounds is to heal the wound while minimizing scar formation and infection, allowing the patient to maintain natural function in the wound area. If the wound becomes infected, it can lead to loss of limb or life. Large wounds, such as burns, are particularly difficult to treat because they expose a large surface area to the environment (and therefore to infection risk), making them prone to potentially painful and debilitating scars. Physicians treating chronic wounds are primarily concerned with closing the wound as quickly as possible to minimize the risk of infection, which can lead to loss of limb or life. Chronic wounds are wounds in patients with comorbidities that complicate or delay the healing cascade. In one aspect, the compositions described herein can function as a tissue regeneration template that delivers essential wound healing factors, extracellular matrix proteins, and inflammatory mediators to help reduce inflammation, enhance healing, and reduce scar tissue formation.

[0118] The collagen and micronized placental tissue component compositions described herein also function as wound dressings. The compositions may be placed over a skin wound (which would otherwise be exposed to the environment) to act as a barrier. The compositions may be in substantial direct contact with the wound to cover most of the wound, maintaining sufficient surface area. Thus, the compositions may provide bioactive factors, scaffolding, and the like, as needed. "Substantially direct contact" refers to contact with the wound, but may also be placed over exudate, drugs, biocompatible mesh, or any other article or agent deemed by a clinician to be beneficial for wound healing, by way of non-limiting example.

[0119] In one embodiment, the collagen and micronized placental tissue component composition can be used to repair injured tendons. The composition can be placed over the injured tendon to prevent scar formation on the tendon during healing. The composition can also provide a protective, sealing environment to allow for successful repair.

[0120] In other embodiments, the collagen and micronized placental tissue component compositions described herein can be used in cosmetic applications, such as cosmetic surgery. Scar revision surgery is surgery to improve or reduce the appearance of scars. It also restores function and corrects skin changes (disfigurement) caused by trauma, wounds, or previous surgery. Scar tissue forms as the skin heals after trauma or surgery. The amount of scarring can be determined by the size, depth, and location of the wound, the person's age, genetics, and skin characteristics such as skin color (pigmentation). Surgery involves excision of the scar and careful closure of the defect. In one embodiment, the collagen and micronized placental tissue component compositions described herein can be used as patches to aid in scar healing and prevention, as well as revision / removal of keloids or carcinomas where careful approximation of soft tissue edges is not achievable and scar tissue may result. Additionally, the anti-inflammatory properties of the micronized placental tissue component can enhance healing.

[0121] In another embodiment, the collagen and micronized placental tissue component compositions described herein can help prevent or reduce the formation of wrinkles in a subject. In yet another embodiment, the compositions can promote re-epithelialization of the superficial layer of skin after laser resurfacing. The compositions described herein can be used alone or in combination with other skin treatments, such as moisturizers, vitamin A creams, vitamin E, recombinant hyaluronic acid, or human and / or animal and / or natural oils (e.g., tea tree oil). Compositions containing micronized placental tissue component microparticles having particle sizes of about 20 μm to about 100 μm or about 25 μm to about 75 μm can be effective in cosmetic applications, such as preventing or reducing the formation of wrinkles in a subject.

[0122] In one embodiment, the composition is injected or applied at or near the site of a cosmetic defect, such as, for example, a wrinkle, fine line, abrasion, scar, etc. Additional cosmetic defects that can be treated using the compositions described herein are described, for example, in U.S. Patent Application Publication No. 2015 / 0086634, the entire disclosure of which is incorporated herein by reference.

[0123] Without being bound by theory, it is believed that the compositions of the present invention remain at the injection or application site longer and degrade more slowly than compositions of either collagen or micronized placental tissue component alone.

[0124] In another embodiment, the collagen and micronized placental tissue component compositions described herein can be applied to biocompatible materials and / or devices, such as, for example, implantable medical devices. Implantable medical devices can be coated with one or more of the collagen and micronized placental tissue component compositions described herein to provide the device with beneficial properties when used in biological tissue (e.g., improved wound healing and prevention of scar formation).

[0125] Examples of suitable implantable devices that can be coated with the collagen and micronized placental tissue component compositions described herein include, but are not limited to, coronary stents, peripheral stents, implants (e.g., dental, orthopedic, spinal), catheters, arteriovenous grafts, bypass grafts, pacemaker and defibrillator leads, vascular anastomosis clips, arterial closure devices, patent orifice ovale closure devices, and drug delivery balloons.The implantable devices can be made of any suitable biocompatible material, such as biostable and bioabsorbable materials.Suitable biocompatible metallic materials include, but are not limited to, stainless steel, tantalum, titanium alloys (including nitinol), and cobalt alloys (including cobalt-chromium-nickel and cobalt-chromium-tungsten alloys). Suitable biocompatible non-metallic materials include, but are not limited to, polyamides, fluoropolymers, polyolefins (i.e., polypropylene, polyethylene, etc.), non-absorbable polyesters (i.e., polyethylene terephthalate), and bioabsorbable aliphatic polyesters (i.e., homopolymers and copolymers of lactic acid, glycolic acid, lactide, glycolide, para-dioxanone, trimethylene carbonate, ε-caprolactone, etc., and combinations thereof).

[0126] In other embodiments, the collagen and micronized placental tissue component compositions described herein can be applied to a wound healing device. For example, the wound healing device can be a bandage, wrap, gauze, suture, or any other device used to treat wounds. In these embodiments, the collagen and micronized placental tissue component compositions can be coated on and / or impregnated into the device. In other embodiments, the wound healing device can be a membrane or implant used in wound healing applications, where the micronized composition is coated on one or more sides of the membrane or implant. For example, any of the tissue implants described herein that are precursors to the micronized composition can be coated with the composition. [Example]

[0127] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to prepare and evaluate the compositions and methods described and claimed herein, and are merely illustrative and not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some errors and deviations should be accounted for. Unless otherwise specified, parts are parts by weight, temperature is in degrees Celsius (°C) or is ambient, and pressure is at or near atmospheric. Numerous variations and combinations of reaction conditions, such as component concentrations, desired solvents, solvent mixtures, temperatures, pressures, and other reaction ranges and product purity and yields obtained in the processes described herein, exist. Only reasonable and routine experimentation will be required to optimize such process conditions.

[0128] Example 1 The micronized placental tissue component used herein was produced by the process described in U.S. Patent Application Publication No. 2013 / 0344162, the disclosure of which is incorporated by reference in its entirety.

[0129] A composition of approximately 5 mg of aqueous / gelatinous placental collagen alone retained the same aqueous / gelatinous properties when released through a 27-gauge needle (Figure 2A). Such a composition also had low tensile strength, such that the composition broke off from the tip of the needle when the needle was lifted at a 90° angle (Figure 3A). In contrast, when micronized placental tissue was added to 1 mL of approximately 0.5% human placental collagen (approximately 5 mg of collagen) in successive additions (up to a total of approximately 230 mg of micronized placental tissue; see Figure 4) and released through a 27-gauge needle, a compositional change occurred, whereby the gelatinous material transformed into a flexible and cohesive composition (Figure 2B). Surprisingly, this flexible and cohesive composition also had a greater tensile strength than the aqueous / gelatinous placental collagen composition, such that the composition remained attached when the needle was lifted at a 90° angle (Figures 3B and 5).

[0130] The compounds, compositions, and methods described herein can be modified and varied in many ways. Other aspects of the compounds, compositions, and methods described herein will be apparent from consideration of the specification and practice of the compounds, compositions, and methods disclosed herein. The specification and examples are intended to be illustrative.

Claims

1. 1. A composition comprising an aqueous collagen material containing 0.1% to 2% soluble collagen and a micronized placental tissue component, such that the aqueous collagen material is an integrated mixture having apparent homogeneity, the composition is an aqueous or gelatinous composition that forms a cohesive and flexible mass upon application of force and / or shear; the weight ratio of the micronized placental tissue component to the collagen is 100:1 to 300:1; the micronized placental tissue component comprises micronized amniotic membrane, the micronized amniotic membrane comprising epithelial cells and / or fibroblasts; composition.

2. 10. The composition of claim 1, wherein the micronized placental tissue component further comprises micronized chorion, micronized Wharton's jelly, or any combination thereof.

3. 3. The composition of claim 2, wherein said micronized placental tissue component has a particle size of less than 400 μm.

4. 4. The composition of any one of claims 1-3, wherein the micronized placental tissue component comprises a micronized tissue explant comprising an amniotic membrane layer and a chorionic membrane layer, the chorionic membrane layer being layered directly on the amniotic membrane layer, and the amniotic membrane layer further having an exposed basement membrane and an intact fibroblast component.

5. 4. The composition of any one of claims 1-3, wherein the micronized placental tissue component comprises a micronized tissue explant comprising an amniotic membrane layer and a chorionic membrane layer, the chorionic membrane layer being layered directly on the amniotic membrane layer, and the amniotic membrane layer further having an intact epithelial cell layer and an intact fibroblast cell component.

6. The composition according to any one of claims 1 to 3, wherein the collagen is human collagen.

7. The composition according to any one of claims 1 to 3, wherein the collagen is human placental collagen.

8. 1. A method for converting an aqueous / gelatinous collagen mass into a cohesive and flexible composition, comprising: a. providing an aqueous or gelatinous collagen mass comprising an aqueous collagen material comprising 0.1% to 2% soluble collagen and a micronized placental tissue component, such that the composition is an integrated mixture having an apparent homogeneity, wherein the weight ratio of the micronized placental tissue component to the collagen is 100:1 to 300:1, and the micronized placental tissue component comprises micronized amniotic membrane, which comprises epithelial cells and / or fibroblasts; b. subjecting said composition to sufficient force and shear to convert said composition into a coherent and flexible mass; A method comprising:

9. 9. The method of claim 8, wherein the micronized placental tissue component further comprises micronized chorion, micronized Wharton's jelly, or any combination thereof.

10. 10. The method of claim 9, wherein the placental tissue component has a particle size of less than 400 μm.

11. 11. The method of any one of claims 8-10, wherein the micronized placental tissue component comprises a micronized tissue explant comprising an amniotic membrane layer and a chorionic membrane layer, the chorionic membrane layer being layered directly on the amniotic membrane layer, and the amniotic membrane layer further having an exposed basement membrane and an intact fibroblast component.

12. 11. The method of any one of claims 8-10, wherein the micronized placental tissue component comprises a micronized tissue explant comprising an amniotic membrane layer and a chorionic membrane layer, the chorionic membrane layer being layered directly on the amniotic membrane layer, and the amniotic membrane layer further comprising an intact epithelial cell layer and an intact fibroblast cell component.

13. The method according to any one of claims 8 to 10, wherein the collagen is human collagen.

14. The method according to any one of claims 8 to 10, wherein the collagen is human placental collagen.

15. 10. The method of claim 8, wherein the composition is obtained by expelling from a syringe a precursor composition comprising aqueous / gelatinous collagen and micronized placental tissue components.

16. 10. The composition of claim 1 for use in wound healing.

17. 10. The composition of claim 1 for use in repairing injured tendons.

18. 10. The composition of claim 1 for use in cosmetic applications.

19. 10. Use of the composition of claim 1 for coating a biocompatible material or device.

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