Placental tissue component compositions for the treatment of skin defects and methods of using same

Abstract

To provide a method that treats wrinkles, scarring and other deformities via subdermal injection, and an injectable composition.SOLUTION: The present disclosure provides methods of treating defects such as wrinkles or scars via a subdermal injection of placental tissue components. It also provides placental tissue compositions maintained within the barrel of a syringe. The placental tissue compositions may be for use in treating such defects.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 673,528, filed May 18, 2018, which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates generally to methods of treating age-related skin conditions, such as wrinkles, by injecting placental tissue, for example, a combination of amniotic membrane, chorion, umbilical cord, and / or placental disc. [Background technology]

[0003] Wrinkles and other unsightly skin conditions are often the result of dermatological breakdown, such as the loss of collagen and other connective tissue. While such breakdown may be commonly referred to as wrinkles or scars (e.g., acne scars), their development is indicative of an underlying condition. The resulting wrinkles and / or scars are often accompanied by emotional and / or psychological concerns by patients.

[0004] Forbes estimated that the appearance industry generated $445 billion in sales last year. This volume of sales is strong evidence of the high demand for products that can help improve patients' appearance, especially in the facial area.

[0005] One particular area where appearance-related products are particularly popular is in wrinkle and scar therapy. Products in this area are generally aimed at reducing the natural effects of dermatological breakdown. On the face, dermatological breakdown typically causes loss of muscle tone and thinning of the skin, which can give the face a loose or saggy appearance. Skin also begins to dry out and lose fat with age, resulting in a loss of plumpness and smoothness. Prolonged exposure to environmental factors, such as smoking and sunlight, tends to increase and intensify the appearance of wrinkles. Wrinkles tend to be most noticeable around the corners of the eyes and nasolabial folds. Age spots and dark spots may become more noticeable as well.

[0006] Several different products have been developed to combat the appearance of wrinkles and scars. Some of these products can be applied directly to the skin, while others are injected directly into the wrinkle or scar area. Botox, a bacterial toxin, can be used to temporarily paralyze certain facial muscles so they don't pull on the skin and increase the appearance of wrinkles. Other injectable products are administered via subcutaneous injection into the area of ​​wrinkles or scars, acting as a filler to fill in the subcutaneous voids and reduce the appearance of wrinkles or scars. The North American and European dermal filler market is expected to reach $2.8 billion in sales by 2026. Some common wrinkle filler products include hyaluronic acid, collagen, silicone, and autologous fat. While these injectable fillers are effective to varying degrees, they can cause patients to experience side effects such as allergic reactions, swelling, bruising, disfigurement, nerve palsies, and skin discoloration. Furthermore, many of these injectable fillers are eventually absorbed by the body at different rates, requiring multiple follow-up injections to maintain patients' original results. Furthermore, the use of injectable compositions is problematic as most patients suffer from inflammation, swelling, redness at the injection site, and discomfort that can last for up to several days.

[0007] Therefore, there is a need for a product that reduces wrinkles and scarring and reduces inflammation at the injection site. Summary of the Invention

[0008] The present invention relates to injectable compositions that treat wrinkles, scars and other deformities while also providing anti-inflammatory properties to address the inflammation, swelling and redness associated with the injection(s).

[0009] In one embodiment, the present invention provides a rehydration composition comprising at least one placental tissue component. In one embodiment, the composition comprises a placental and / or umbilical cord component selected from the group consisting of placental disc, amniotic tissue, chorionic tissue, and umbilical cord tissue. In a further embodiment, the placental and / or umbilical cord component has a size of 150 microns or less. In one embodiment, the composition, when reconstituted with an aqueous solution, provides a paste having pseudothixotropic properties such that the composition has a flow rate of about 0.05 mL / sec to 0.75 mL / sec when passed through a 27-gauge needle at room temperature.

[0010] Human placental membranes (e.g., amniotic membranes or tissue) have been used in various types of reconstructive surgery since the early 1900s. These membranes function as substrate materials, more commonly referred to as biological bandages or patch grafts. Such membranes are also widely used in ophthalmic surgery. Typically, these membranes are frozen or dried for preservation and storage until needed for surgery.

[0011] Such placental tissue is typically harvested after an elective cesarean section. The placenta consists of the umbilical cord and amniotic sac. The amniotic sac, commonly referred to as the amnion, has two major tissue layers: the amnion and the chorion. The amniotic tissue is the innermost layer of the amniotic sac and is in direct contact with the amniotic fluid. The amniotic sac contains amniotic fluid and protects the fetus's environment. Histological evaluation has shown that the membranous layer of the amniotic membrane 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., the basement membrane) contains types IV, V, and VII collagen, as well as cell adhesion bioactive factors such as fibronectin and laminin.

[0012] After collection, the placenta tissue is separated into three distinct components: the umbilical cord, the amnion / chorion, and the placental disc. All components are derived from a single donor. Each of these components undergoes a specialized process in which each component is rinsed with an antibiotic solution and then rinsed again to remove any residual antibiotics.

[0013] Preferably, the placental tissue component is washed with a hypertonic solution, the hypertonic solution comprising an NaCl concentration in the range of about 30% to about 10%.

[0014] In some embodiments, the method further comprises physically washing the selected layer after separating the chorionic tissue layer from the amniotic membrane layer to remove blood clots and other contaminants. Specifically, the spongy intermediate layer found between the amniotic and chorionic membrane layers in naturally occurring placental tissue is removed from these layers during processing and, in some embodiments, discarded.

[0015] The umbilical cord is then treated by removing the veins and arteries, gently cleaning the remaining cord tissue, and minimally manipulating it to preserve the tissue's inherent growth factors and proteins. Notable growth factors in the umbilical cord include transforming growth factor beta (TGF-β), basic fibroblast growth factor (bFGF), platelet-derived growth factors (PDGF AA and BB), and vascular endothelial growth factor (VEGF). 14, 15 These factors are known to regulate wound healing.

[0016] The amniotic membrane and chorion are gently washed and minimally manipulated to preserve the inherent growth factors (over 200) and proteins within the tissue. Notable growth factors in amniotic membrane and chorion include epidermal growth factor (EGF), transforming growth factor alpha and beta (TGF-α and β), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF AA and BB), and vascular endothelial growth factor (VEGF).

[0017] The placental disc tissue is subjected to the same cleaning process as the membrane and umbilical cord tissue described above, as well as an additional decellularization step. The decellularization process aims to remove antigenic materials from the placental tissue by significantly reducing the presence of maternal DNA. Decellularization also reduces the presence of growth factors and other soluble or intracellular proteins while retaining essential extracellular matrix components.

[0018] Once each component is decontaminated and rinsed (or, in the case of placental disc tissue, decellularized), the components are recombined and dehydrated by freeze-drying. After the tissue is dehydrated, it is ground to an appropriate size, allowing the processed tissue to be injected into the wrinkle site.

[0019] The finished product is packaged in a sterile container and reconstituted with an acceptable excipient by the end user.

[0020] In some embodiments, particle size is altered to affect the viscosity and stability of the resulting injectable product.

[0021] In some embodiments, the relative proportions of placental disc tissue, amniotic membrane / chorion, and umbilical cord tissue are adjusted to affect the viscosity and stability of the resulting injectable product.

[0022] In some aspects, the present disclosure provides therapeutic methods for administering placental tissue-based injectables to reduce the appearance of wrinkles in a patient.

[0023] In some aspects, the present disclosure provides an apparatus for administering a placental tissue-based injectable to reduce the appearance of wrinkles in a patient. [Brief explanation of the drawings]

[0024] [Figure 1A-1B]

[0013] Figure 1A shows one embodiment of a reconstitution device that can be used to mix acceptable excipients with dried placental tissue-based injectables prior to injection. Figure 1A shows two syringes, a Luer connector, and a microvial. Figure 1B shows the assembled device.

[0025] [Figure 2A-2B] Photographs of a pig carcass showing the wrinkled appearance before (Figure 2A) and after (Figure 2B) administration of an injectable agent based on placental tissue. DETAILED DESCRIPTION OF THE INVENTION

[0026] It is to be understood that the invention is not limited to the particular embodiments described, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0027] The detailed description of the present invention is divided into various sections for the convenience of the reader only, and disclosures found in any section may be combined with those of another section. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the manufacture, practice, or testing of the present invention, the preferred methods and materials are described herein. All patents and publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials for which the publications are cited.

[0028] Each embodiment disclosed herein is contemplated as being applicable to each other disclosed embodiment. All combinations and subcombinations of the various elements described herein are within the scope of the embodiments.

[0029] Where a parameter range is provided, it is understood that all integers and ranges within that range, and tenths and hundredths thereof, are also provided by embodiments. For example, "5-10%" includes 5%, 6%, 7%, 8%, 9%, and 10%; 5.0%, 5.1%, 5.2%, 9.8%, 9.9%, and 10.0%; and 5.00%, 5.01%, 5.02%, 9.98%, 9.99%, and 10.00%, as well as, for example, 6-9%, 5.1%-9.9%, and 5.01%-9.99%.

[0030] As used herein, "about" in the context of a numerical value or range means within ±1%, ±5%, or ±10% of the recited or claimed numerical value or range.

[0031] The inventions illustratively disclosed herein may suitably be practiced in the absence of any element not specifically disclosed herein.

[0032] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pluripotent stem cell" includes a plurality of pluripotent stem cells.

[0033] definition As used herein, the following terms have the following meanings:

[0034] "Comprising" or "comprise" is intended to mean that compositions (e.g., media) and methods include the recited elements, but do not exclude other elements. "Consisting essentially of," when used to define a method, means excluding other elements of any essential significance to the combination for the purpose described. "Consisting of" is intended to mean excluding substantial method steps. Embodiments defined by each of these transition terms are within the scope of the present invention.

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

[0036] "Rehydration" refers to a composition, particle, or other material that was previously dehydrated but is now not dehydrated. Rehydration can be achieved by placing the dehydrated material in an aqueous solution (such as 0.9% saline) or by other means known in the art.

[0037] As used herein, the term "subject" is any vertebrate organism, including, but not limited to, mammalian subjects such as humans, livestock, pets, etc. The term "patient" may be used interchangeably with "subject."

[0038] The term "placental tissue" refers to any and all of the well-known components of the placenta, including, but not limited to, the amniotic membrane, chorion, stratum intermedia, Wharton's jelly, placental disc, umbilical cord, and the like.

[0039] The term "treatment" in relation to a defect means reducing the severity of the defect or eliminating the defect entirely.

[0040] The term "defect" refers to an undesirable aesthetic feature, including but not limited to, a wrinkle, a crease, a crease, a line, or a scar, which may be present in a patient, particularly in the patient's facial area. Details of such defects, types of wrinkles, and wrinkle severity can be found in "A Classification of Facial Wrinkles," Gottried et al.; Cosmetic; 2001, the contents of which are incorporated herein by reference.

[0041] Manufacturing method Initial tissue collection Placental tissue collection begins in the hospital, with collection at the time of birth via cesarean section. The donor, the expectant mother, voluntarily undergoes a comprehensive screening process designed to provide the safest tissue possible for transplantation. This screening process preferably uses conventional serological tests to test for antibodies to human immunodeficiency virus types 1 and 2 (anti-HIV-1 and anti-HIV-2), hepatitis B surface antigen (HBsAg), hepatitis C virus (anti-HCV), human T-lymphotropic virus types I and H (anti-HTLV-I and anti-HTLV-II), CMV, and syphilis. The above list of tests is merely exemplary; as will be understood by those skilled in the art, more, fewer, or different tests may be desired or required over time or based on the intended use of the graft.

[0042] Based on a review of the donor's information and screening tests, the donor is either deemed acceptable or not. Additionally, at the time of delivery, cultures are performed to determine the presence of, for example, Clostridium or Streptococcus. If the donor's information, screening tests, and delivery cultures are all negative (i.e., indicating no risk or an acceptable level of risk), the donor is approved and the tissue specimen is designated as initially eligible for further processing and evaluation.

[0043] Human placentas that meet the above selection criteria are preferably individually bagged in saline in sterile shipping bags and stored in a container of ice water for transport to a processing site or laboratory for further processing.

[0044] Material check-in and evaluation Upon arrival at the processing center or laboratory, the shipment is opened and verified to ensure the sterile shipping bag / container remains sealed and intact, that ice or other refrigerant is present, that the contents are chilled, that appropriate donor documentation is present, and that the donor number on that 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. All appropriate forms are completed, as are chain of custody and processing logs.

[0045] Whole tissue processing When the tissue is ready for further processing, the sterile supplies needed for further processing of the placental tissue are assembled in a staging area within the controlled environment and prepared for introduction into the critical environment. If the critical environment is a production hood, the sterile supplies are opened and placed within the hood using conventional sterilization techniques. If the critical environment is a clean room, the sterile supplies are opened and placed on a cart covered with a sterile drape. All work surfaces are draped with a single sterile drape using conventional sterilization techniques, and the sterile supplies and processing equipment are placed onto this drape, again using conventional sterilization techniques.

[0046] If placental tissue is collected prior to the completion or availability of screening tests and delivery cultures, such tissue will be labeled and stored in quarantine. The tissue will only be approved for further processing if it meets the screening assessments and delivery cultures necessary to declare it safe for handling and use.

[0047] The processing equipment is decontaminated according to conventional industry-accepted decontamination procedures and then introduced into the critical environment, where it is strategically located to minimize proximity to and the possibility of inadvertent contamination by tissue specimens.

[0048] The placenta is then removed from the sterile shipping bag and aseptically transferred to a sterile processing bath in a critical environment. The bath preferably contains an 18% NaCl (hypertonic saline) solution at or near room temperature. The placenta is gently massaged to separate blood clots and allow the placental tissue to reach room temperature, which facilitates separation of the amniotic and chorionic layers from each other, as described below. After warming to ambient temperature (approximately 10-30 minutes), the placenta is removed from the sterile processing bath and placed flat, with the amniotic layer facing down, on a processing tray for examination.

[0049] Next, if the placental tissue is deemed acceptable for further processing, the amniotic and chorionic layers of the placental tissue are carefully separated. Materials and equipment used in this procedure include a processing tray, 18% saline, sterile 4x4 sponges, and two sterile Nalgene jars. Next, the placental tissue is closely examined to find an area (usually a corner) where the amniotic layer can be separated from the chorionic layer. The amniotic membrane appears as a thin, opaque layer on the chorion.

[0050] With the placenta tissue in the processing tray, with the amniotic layer facing down, the chorionic layer is slowly lifted from the amniotic layer in a slow, continuous motion, taking care to prevent tearing of the amniotic membrane. If tearing begins, it is recommended to restart the separation process from a different location to minimize tearing of either layer of tissue. The separation process continues manually without the use of a sponge, taking care not to tear either the amniotic or chorionic layers.

[0051] Care is then taken to remove blood clots and other extraneous tissue from each layer of tissue until the amniotic tissue and chorion are clean and ready for further processing. More specifically, the amniotic and chorionic tissues are placed on a processing tray, and the clot is carefully removed by gently scraping the clot with a blunt instrument, fingers, or sterile, non-particulate gauze until it is freed from the amniotic stromal tissue and chorionic trophoblastic tissue. The amniotic stromal layer is located on the maternal-facing side of the amnion. In contrast, the basement membrane layer is located on the fetal-facing side of the amnion.

[0052] Any remaining debris or contaminants are removed using a blunt instrument, cell scraper, or sterile gauze. This step must also be performed with great care to avoid tearing the amniotic or chorionic tissue. The amniotic membrane is cleaned when it is smooth and opaque white. Over-cleaning the amniotic membrane may remove the opaque layer. If an area of ​​the amniotic membrane is cleaned too vigorously and appears transparent, it is unacceptable and should ultimately be discarded.

[0053] After the amnion and chorion are separated from the placenta, the remaining components of the placenta are also processed. Specifically, the umbilical cord is removed from the placenta. The cord is incised and the umbilical vein and artery (containing maternal DNA and antigens) are removed. The remaining tissue is retained and consists of the umbilical cord and Wharton's jelly. After the umbilical cord and amnion / chorion are removed from the placenta, the remaining placental disc tissue is also retained and set aside for further processing. All retained tissue components undergo a chemical decontamination process.

[0054] Chemical decontamination process The retained placental tissue components (amniotic membrane / chorion, placental disc, and umbilical cord) are then placed in a sterile Nalgene bottle for the next step of chemical decontamination. Undesirable placental tissue components are discarded in an appropriate biohazard container.

[0055] Each Nalgene bottle is then aseptically filled with 18% saline and sealed (or closed at the top). The bottles are then placed on a rocker platform and agitated for 30-90 minutes to further remove tissue contaminants.

[0056] If the rocker platform is not located within a critical environment (e.g., a manufacturing hood), the Nalgene bottle is returned to the critical / sterile environment and unsealed. Using sterile forceps, the placental tissue components are gently removed from the Nalgene bottle containing the 18% hypertonic saline solution and placed into an empty Nalgene bottle. The empty Nalgene bottle containing the tissue is then aseptically filled with a premixed antibiotic solution. Preferably, the premixed antibiotic solution consists of a cocktail of antibiotics, such as streptomycin sulfate and gentamicin sulfate. Other antibiotics, such as polymyxin B sulfate and bacitracin, or similar antibiotics currently or to become available in the future, are also suitable. Furthermore, the antibiotic solution is preferably at room temperature when added to avoid altering the tissue's temperature or damaging it. The bottle or container containing the tissue and antibiotics is then sealed or closed, placed on a rocker platform, and agitated, preferably for 60-90 minutes. This rocking or agitation of the tissue in the antibiotic solution further cleanses the placental tissue components of contaminants and bacteria.

[0057] Again, if the rocker platform is not in a critical environment (e.g., a production hood), the jar or container containing the tissue or antibiotic is returned to the critical / sterile environment and opened. Using sterile forceps, the placental tissue components are gently removed from the jar or container and placed in a sterile bath containing sterile water or normal saline (0.9% saline solution). The placental tissue components are left in place in the sterile water / normal saline solution for at least 10-15 minutes. The placental tissue components may be slightly agitated to facilitate removal of the antibiotic solution and any other contaminants from the placental tissue components. After at least 10-15 minutes, the tissue is dehydrated and ready for further processing.

[0058] Decellularization of placental disc components After the chemical decontamination process, the placental disc portion of the placental tissue component should preferably be decellularized due to the presence of maternal antigens in this tissue, which may cause an undesirable immune response from the end-user patient. Any decellularization process known to those skilled in the art may be used, such as the decellularization method disclosed in U.S. Patent Application Publication No. 2002 / 0160510 or U.S. Patent No. 8,071,135. Decellularization can also be performed by methods such as temperature methods, electrical breakdown, chemical decellularization, or enzymatic decellularization. Preferably, the process used to decellularize the placental disc tissue does not destroy the natural composition of the proteins that make up the placental disc tissue.

[0059] Dehydration / lyophilization process After all components have been properly decontaminated and decellularized (as appropriate for the placental disc component), the components are recombined and a dehydration step is performed.

[0060] Preferably, the separate placental components are placed in individually sealed Tyvek pouches (or other commercially available pouches) and placed in a commercially available freeze-drying chamber. Any freeze-drying process known to those skilled in the art may be used, so long as the placental components are substantially dehydrated at the end of the freeze-drying process.

[0061] Other methods may be used to sufficiently dehydrate the ingredients. Such techniques may include, but are not limited to, chemical dehydration or placing the ingredients in a low humidity / high temperature environment for an appropriate period of time until optimal dehydration of the ingredients is achieved. Such dehydration techniques are generally well known to those skilled in the art.

[0062] Recombination and crushing process Once the placental components are completely dehydrated, they are recombined and then ground into smaller particles. In one embodiment, the ratio of the recombined placental components is selected to provide a paste with the aforementioned flow rate. It is understood that the specific ratio of these components is less important than the resulting flow rate, so that the composition is not only easily injectable but also remains stationary upon injection. The recombined placental components are fed into a commercially available grinding device. The placental components are then ground at a speed of approximately 8,000 rpm through a sieve size ranging from 50 to 150 μm. It is understood that the term "tissue" is the same as the term "component" as used herein.

[0063] This grinding process optimally results in a particle size of the placental component in the range of 70 to 150 μm.

[0064] In preferred embodiments, the recombined placental tissue component should be comprised of about 50-98% by weight placental disc tissue, about 1-30% by weight umbilical cord tissue, if present, and about 1-20% by weight amniotic / chorionic tissue, if present. In one embodiment, the recombined placental tissue component should be comprised of about 60-95%, about 65-90%, about 70-85%, or about 75-80% by weight placental disc tissue. In one embodiment, the recombined placental tissue component should be comprised of about 1-25%, about 5-20%, or about 10-15% by weight umbilical cord tissue. In one embodiment, the recombined placental tissue component should be comprised of about 1-15%, about 4-12%, or about 8-10% by weight amniotic / chorionic tissue. In one embodiment, the recombined placental tissue component should be comprised of about 1-15%, about 4-12%, or about 8-10% amniotic tissue by weight. In one embodiment, the recombined placental tissue component should be comprised of about 1-15%, about 4-12%, or about 8-10% chorionic tissue by weight.

[0065] When the placental disc, amnion, chorion, and umbilical cord are all obtained from a single donor, the recombined, dehydrated, and resulting recombined placental tissue component contains approximately 11-30% by weight of non-placental disc components (i.e., amnion, chorion, and / or umbilical cord).

[0066] In one embodiment, the recombined placental tissue component comprises about 11-30% by weight of non-placental disc components, hi one embodiment, the recombined placental tissue component comprises about 15-25% by weight or about 18-22% by weight of non-placental disc components.

[0067] In one embodiment, the weight percent is determined before dehydration. In another embodiment, the weight percent is determined after dehydration.

[0068] In one embodiment, the recombined placental tissue component comprises a placental disc and amniotic tissue. In one embodiment, the recombined placental tissue component comprises a placental disc and chorionic tissue. In one embodiment, the recombined placental tissue component comprises a placental disc, amniotic tissue, and chorionic tissue.

[0069] In one embodiment, the recombined placental tissue component comprises a placental disc and umbilical cord tissue. Sieving process

[0070] The milled particles then undergo a sieving process. The milled particles are placed on a commercially available sifter and placed on a sieve. The sieve size ranges from 50 to 150 μm. The sifter is turned on and the particles are sieved for a period of 5 to 20 minutes. This sieving process results in a powder of particles with a uniform particle size of 50 to 150 μm. Once the desired particle size is achieved, the powder may be aseptically transferred into vials, holding doses of 75 to 600 mg, and sealed.

[0071] In alternative embodiments, the particles may be 50-500 μm, 100-400 μm, 150-300 μm, or 200-250 μm. In alternative embodiments, the particles may be ≦50 μm, ≦100 μm, ≦150 μm, ≦200 μm, ≦250 μm, ≦300 μm, ≦350 μm, ≦400 μm, ≦450 μm, and ≦500 μm. In alternative embodiments, the particles may be ≧50 μm, ≧100 μm, ≧150 μm, ≧200 μm, ≧250 μm, ≧300 μm, ≧350 μm, ≧400 μm, or ≧450 μm. In one embodiment, the particles may have a range of particle sizes.

[0072] Reconstruction To administer the particulate placental component to a subject, the end user must first reconstitute the powder by rehydrating it. Optimally, the rehydrating agent is 0.9% saline, although any suitable excipient may be used.

[0073] The powder is combined with 0.9% saline solution at a ratio of 100-150 mg of powder per cc of 0.9% saline solution. In one embodiment, the desired volume of 0.9% saline solution is first transferred into a syringe with an 18-gauge needle. Once the 0.9% saline solution has been transferred, the needle tip is removed. The desired amount of dehydrated powder is then transferred from the vial into a second syringe. The two syringes are connected together by a female-female Luer connector.

[0074] Once the syringes are connected, the saline solution and dehydrated powder are mixed by alternately pressing the plungers of each syringe. Complete reconstitution should require 10 mixing strokes (a stroke is defined as one full compression of the saline plunger followed by one full compression of the powder plunger). The individual components of this reconstitution device embodiment are shown in Figure 1A, and an assembled reconstitution device embodiment is shown in Figure 1B.

[0075] Once the powder is sufficiently rehydrated, the connector between the two syringes is removed and the empty syringe is discarded. A 27-gauge needle is then attached to the syringe containing the reconstituted powder.

[0076] In one embodiment, the composition further comprises hyaluronic acid.

[0077] Administration of placental components to a subject Once the powder is rehydrated and a 27-gauge needle is attached, the placental component can be administered to the subject. Depending on the size and severity of the wrinkled area, a dose of 75 to 600 mg can be administered. After this dose is administered, the subject's wrinkles should have a filled-in appearance. Figures 2A and 2B show the appearance of wrinkles on a pig carcass before and after administration of a dose consisting of 100 mg of powder in 1 mL of 0.9% saline solution, respectively. Depending on the results, the patient should return 6 to 9 months after administration for a follow-up injection.

[0078] In one embodiment, the placental component is administered in combination with a conventional product for treating defects, such as hyaluronic acid, collagen, silicone, and autologous fat. The placental component may be administered simultaneously with the conventional product (i.e., in a single composition) or contemporaneously (i.e., separately but at the same time, or sufficiently close together to produce an additive or synergistic effect). In one embodiment, the combined administration of the injectable composition of the present invention and the conventional product produces a synergistic effect.

Claims

1. 1. A method of preparing an injectable placental tissue composition, comprising: a) decellularizing the placental disc; b) dehydrating the placental disc; c) dehydrating one or more of the amniotic membrane, chorion, and umbilical cord; d) combining the dehydrated placental disc with one or more of the dehydrated amniotic membrane, chorion, and umbilical cord; and e) milling the composition formed in step d) to form dehydrated particles. A method comprising:

2. The method of claim 1, further comprising sieving the dehydrated particles using a sieve to obtain dehydrated particles having a particle size of 50 to 500 μm.

3. 3. The method of claim 2, further comprising rehydrating the sieved dehydrated particles to form a rehydrated composition.

4. The method of claim 3, further comprising placing the rehydrated composition in a syringe.

5. 10. The method of claim 1, wherein the dehydrated particles, if present, are about 1-20% by weight dehydrated amniotic membrane and / or chorion particles, if present, about 1-30% by weight dehydrated umbilical cord particles, and about 50-98% by weight dehydrated decellularized placental disc particles.

6. The method of claim 1 , wherein the particles comprise a placental disc and an amniotic membrane.

7. The method of claim 1 , wherein the particles comprise placental discs and chorionic membranes.

8. The method of claim 1 , wherein the particles comprise placental disc, amniotic membrane, and chorion.

9. The method of claim 1 , wherein the particles comprise placental discs and umbilical cords.

10. The method of claim 1 , wherein the particles comprise a placental disc, an amniotic membrane, a chorion, and an umbilical cord.

11. The method of claim 1, wherein the particles have a size of about 50 to 150 μm.

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