TECHNICAL FIELD

[0001] The present invention relates generally to the field of compositions prepared from placental membranes, and more specifically to a method of standardizing a dehydrated isolated human placental membrane allograft dosage by quantifying endogenous components therein.

BACKGROUND

[0002] The medical benefits of placental membranes have become increasingly apparent over the past several years. A variety of membrane products and combinations have been developed for applications ranging from cosmetic to invasive surgery. Some are ideal for protection from external insults (e.g. contamination) while others provide an extensive array of growth factors.

[0003] The placenta is the organ that develops in the uterus shortly after implantation of a fertilized egg, attaching the egg to the wall of the uterus. The placenta is composed of parenchyma, chorion, amnion, and umbilical cord; the latter of which connects to the fetus. During fetal development, the amnion and chorion come together to form the chorio-amniotic membrane that encases the amniotic fluid and the fetus. The most common components of the placenta under exploration for regenerative use are the amnion, chorion, amniotic fluid, and the umbilical cord.

[0004] The fetal membrane (amniotic sac) full thickness measures approximately 0.25 mm at term. This includes the layers of the innermost amnion and layers of the outer chorion. The amnion is between 0.02 and 0.05 mm thick. It is avascular with no nerve or lymph. Hence, it receives nutrients through diffusion. The amnion is in direct contact with the amniotic fluid. Amnion has three layers: the epithelial layer, the thick basement membrane, and the avascular mesenchymal tissue. These contain type I and type III- VII collagen and high concentrations of proteoglycans and glycoproteins along with fibronectin and laminin. There are 2 main cell types in amnion: amniotic epithelial cells (AECs) and amniotic mononuclear mesenchymal cells. The functions of amniotic membrane include physical protection of the fetus, protection from bacterial infection, regulation of pH, and secreting growth factors and other molecules. These serve antimicrobial and anti-inflammatory functions.

[0005] In 1910, John Davis was the first to report the use of amnion membrane (AM) as a surgical material in skin transplantation. He showed that in skin grafting, amniotic membrane resulted in better outcomes than xenograft or cadaveric dressing. Since then, human amniotic membrane has been used widely in regenerative medicine due to continued discovery of its favorable biological and mechanical properties. Numerous applications benefit from these properties. For example, amnion membrane for management and treatment of skin burns is well documented and has been utilized in over 200 clinical trials. Similarly, amniotic membrane has shown advantageous outcomes when used for chronic wounds, urinary system issues, dental and oral treatments, ophthalmic indications and orthopedic treatments. Many other applications for amniotic membrane have been studied. Of particular interest is its documented antimicrobial activity.

[0006] The chorion is the outer layer in contact with the mother’s cells. It forms tail-like structures called the chorionic villi which provide blood and nutrients from the mother to the baby. The amnion and chorion are separated by a jellylike matrix (chorionic fluid) until the two membranes ‘‘fuse” at 11-13 weeks post-conception. The chorion is 3-4 times thicker than the amnion and has three layers: the intermediate/reticular layer (contacts the amnion), the basement membrane, and the trophoblast layer. The reticular layer contains type I, type III, type IV, type V and type VI collagen. The basement membrane contains type IV collagen along with fibronectin and laminin. Chorion by itself is not commonly used in medicine for regenerative applications. But the combination of amnion and chorion (AC) has become very' popular. When amnion and chorion are used together, the chorion, which is thicker, is reported to be responsible for 75% of the growth factors present. Various preparations focus on exploiting the available growth factors and adding strength to AC. These include lamination, cryopreservation, dehydration and freeze drying. Each processing and preservation method results in the opti mization of different properties of the membrane. Some focus on retaining cellular viability' while others focus on providing an optima] substrate for protection and endogenous cell adhesion. Still others prefer to provide natural growth factors.

[0007] Like AM, AC in various forms have been used for numerous applications. The primary application to date is for wound healing. For example, in 2015 Zelen et al reported that use of laminated dehydrated human AC resulted in better and faster healing in wound care compared to other products. Other reports include AC in dental and oral treatments, urogenital system issues and orthopedic treatments. As seen with AM, there is documented antimicrobial activity from AC.

[0008] Although there is a vast array of information collected on amnion and amnion chorion membranes, most reports are collected from fresh or cryopreserved tissues. Unprocessed tissue may be more advantageous to utilize, but unprocessed tissue cannot be used for clinical application due to transmittable disease risk and storage time before end use. Hence, not currently FDA cleared for use. Cryopreserved tissue is stored in and absorbs cryopreservant, usually DMSO, which is accepted, but not desirable, for human applications. The few studies conducted on dehydrated or freeze-dried membranes utilize rehydrated, macerated, enzymatically’ digested, and/or centrifuged extracts. In the course of treatment with membrane product, the tissue is intact, usually sterile, and laid on a wound or treatment area. The factors present which are available to the treatment area from the applied product need to be elucidated and presented, [0009] Characterization of AM and AC in its native state is reported. But this information does not properly describe the membranes as delivered products for medical use. As mentioned previously, the processing method has a substantial effect on the properties of final product membranes. F urthermore, the methods of characteri zati on may not be appropriate for the indicated end use. For example, using the digested, pre-processed membrane to release all proteins gives us a clear look at the potential of the membrane, but does not allow us to determine the factors available to a patient. In addition, grinding, digesting or homogenizing a membrane that is ready for use, to extract all factors present for analysis does not clearly indicate what would be released when the membrane is placed intact on a wound. There exists a need for determining a standardized dosage of the delivered products in a given area of end use placental membrane tissue,

SUMMARY

[0010] To address the above shortcomings, disclosed herein are methods for quantifying and characterizing endogenous components (e.g,, growth factors and other endogenous compounds) from human placental allografts, dehydrated human placental membrane allografts, and/or dehydrated isolated human placental membrane allografts that are eluted from a specific area (per square centimeter) of the allograft(s) as would be eluted within patient tissue thereby advantageously achieving more consistent and predictable therapeutic outcomes.. This information can be advantageously applied to allografts having different sizes and construction to to provide consistent dosing. Further disclosed herein are methods of collecting human placental membranes subsequently preparing dehydrated isolated human placental membrane allografts and/or dehydrated human placental membrane allografts therefrom in which each have a standardized dosage of endogenous components therein

[001 1] In certain aspects, disclosed herein is a method of standardizing placenta] membrane allograft dosage (e.g., dehydrated isolated human placental membrane allografts and dosage thereof) including (a) preparing a dehydrated human placental membrane allograft (e.g., a dehydrated isolated human placental membrane allograft), and (b) determining a standardized dosage per square centimeter (cm ² ) of one or more endogenous components in the allograft for subsequent treatment in a human subject. Step (b) includes: (i) placing a predetermined size of the dehydrated human placental membrane allograft (e.g., dehydrated isolated human placental membrane allografts) into a predetermined volume of isotonic elution solution for a predetermined time period forming an eluate comprising at least one endogenous component eluted from the dehydrated human placental membrane allograft (e.g., dehydrated isolated human placental membrane allografts), (ii) determ ining a concentration of the at least one endogenous component in the eluate, and (iii) calculating the standardized dosage of the at least one endogenous component by multiplying the concentration of the at least one endogenous component in the eluate by the predetermined volume of isotonic elution solution and dividing by the predetermined size of the dehydrated placental membrane allograft (e.g., dehydrated isolated human placental membrane allografts).

[0012] In certain aspects, calculating the standardized dosage of the at least one endogenous component includes: [0013] In certain aspects, the dehydrated human placental allograft (e.g., dehydrated isolated human placental membrane allografts) includes an isolated human amnion layer (e.g,, an isolated dehydrated human amnion layer). Additionally, or alternatively, the dehydrated human placental allograft comprises an isolated human chorion layer (e.g., an isolated dehydrated human chorion layer). Additionally, or alternatively, the dehydrated human placental allograft (e.g., dehydrated isolated human placental membrane allografts) includes an amnion layer and a chorion layer having an intact intermediate spongy layer, when viewed in cross-section, which is positioned there between directly connecting the amnion layer to the chorion layer.

[0014] In certain aspects, the isotonic elution solution includes at least one of 1 X phosphate buffered saline, isotonic saline, lactated ringers, Plasma-Lyte® (NaCl 5.26 g/L, KC1 0.37 g/L, Magnesium Chloride hexahydrate 0.30 g/L, Sodium Acetate trihydrate 3.68 g/L, Sodium Gluconate 5.02 g/L at pH 7.4), Normosol® (NaCl 5.26 g/L, KC1 0.37 g/L, Magnesium Chloride 0.30 g/L, Sodium Acetate anhydrous 2.22 g/L, Sodium Gluconate 5.02 g/L at pH 7.4), or any combination thereof.

[0015] In certain aspects, the at least one endogenous component in the dehydrated placental membrane allograft (e.g., dehydrated isolated human placental membrane allografts) includes at least one of: collagen, fibronectin, glycosaminoglycans (GAGs), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), hyaluronic acid (HA), hepatocyte growth factor (HGF), Interleukin- 1 receptor antagonist (IL-IRa), vascular endothelial growth factor receptor 1 (VEGFR1), macrophage inflammatory protein- l a (MIP-la/CCL3), regulated on activation, normal T cell expressed and secreted (RANTES/CCL5), platelet derived growth factor subunit B homodimer (PDGF-BB), extracellular vesicles, or any combination thereof. [0016] In certain aspects, the at least one endogenous component in the dehydrated placental membrane allograft (e.g., dehydrated isolated human placental membrane allografts) includes at least two of: collagen, fibronectin, glycosaminoglycans (GAGs), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), hyaluronic acid (HA), hepatocyte growth factor (HGF), Interleukin- 1 receptor antagonist (IL-IRa), vascular endothelial growth factor receptor I (VEGFR.1), macrophage inflammatory protein- la (MIP-la/CCL3), regulated on activation, norma] T cell expressed and secreted (RANTES/CCL5), platelet derived growth factor subunit B homodimer (PDGF-BB), extracellular vesicles, or any combination thereof.

[0017] In certain aspects, the at least one endogenous component in the dehydrated placenta] membrane allograft (e.g., dehydrated isolated human placental membrane allografts) includes at least three of: collagen, fibronectin, glycosaminoglycans (GAGs), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), hyaluronic acid (HA), hepatocyte growth factor (HGF), Interleukin- 1 receptor antagonist (IL-I Ra), vascular endothelial growth factor receptor 1 (VEGFR1), macrophage inflammatory protein- la (MIP-la. CCL3), regulated on activation, norma] T cell expressed and secreted (RANTES/CCL5), platelet derived growth factor subunit B homodimer (PDGF-BB), extracellular vesicles, or any combination thereof.

[0018] In certain aspects, the at least one endogenous component in the dehydrated placenta] membrane allograft (e.g., dehydrated isolated human placental membrane allografts) includes at least four of: collagen, fibronectin, glycosaminoglycans (GAGs), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), hyaluronic acid (HA), hepatocyte growth factor (HGF), Interleukin- 1 receptor antagonist (IL-IRa), vascular endothelial growth factor receptor 1 (VEGFR1), macrophage inflammatory protein-la (MIP-lcvCCL3), regulated on activation, normal T cell expressed and secreted (R.ANTES/CCL5), platelet derived growth factor subunit B homodimer (PDGF-BB), extracellular vesicles, or any combination thereof.

[0019] In certain aspects, the at least one endogenous component in the dehydrated placental membrane allograft (e.g., dehydrated isolated human placental membrane allografts) includes at least five of; collagen, fibronectin, glycosaminoglycans (GAGs), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), hyaluronic acid (HA), hepatocyte growth factor (HGF), Interleukin- 1 receptor antagonist (IL-l Ra), vascular endothelial growth factor receptor 1 (VEGFR1), macrophage inflammatory protein- la (MIP-la/CCL3), regulated on activation, normal T cell expressed and secreted (RANTES/CCL5), platelet derived growth factor subunit B homodimer (PDGF-BB), extracellular vesicles, or any combination thereof.

[0020] In certain aspects, the at least one endogenous component in the dehydrated placental membrane allograft (e.g., dehydrated isolated human placental membrane allografts) includes at least six of: collagen, fibronectin, glycosaminoglycans (GAGs), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), hyaluronic acid (HA), hepatocyte growth factor (HGF), Interleukin- 1 receptor antagonist (IL-l Ra), vascular endothelial growth factor receptor 1 (VEGFR1), macrophage inflammatory protein- la (MIP-la/CCL3), regulated on activation, normal T cell expressed and secreted (RANTES/CCL5), platelet derived growth factor subunit B homodimer (PDGF-BB), extracellular vesicles, or any combination thereof.

[0021] In certain aspects, the at least one endogenous component in the dehydrated placental membrane allograft (e.g., dehydrated isolated human placental membrane allografts) includes at least seven of: collagen, fibronectin, glycosaminoglycans (GAGs), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), hyaluronic acid (HA), hepatocyte growth factor (HGF), Interleukin- 1 receptor antagonist (IL-lRa), vascular endothelial grorvth factor receptor 1 (VEGFR1), macrophage inflammatory protein- l a (MIP- l a CCL3 ), regulated on activation, normal T cell expressed and secreted (RANTES/CCL5), platelet derived growth factor subunit B homodimer (PDGF-BB), extracellular vesicles, or any combination thereof.

[0022] In certain aspects, the standardized dosage of the at least one endogenous component per square cent imeter of the dehydrated isolated human placental membrane allograft (e.g., dehydrated isolated human placental membrane allograft) includes approximately 15 pg/cm ² to about 200 pg/cm ² basic fibroblast growth factor (bFGF), approximately 3.0 pg/cm ² to 15 pg/cm ² epidermal growth factor (EGF), approximately 1.3 x 10 ¹ ' pg/cm- to about 1.5 x 10 ^s pg/cm- hyaluronic acid (HA), approximately 1 10 pg/cm ² to about 8500 pg/cm ² hepatocyte growth factor (HGF), approximately 200 pg/cm ² to about 7800 pg/cm ² Interleukin- 1 receptor antagonist (IL- IRa), approximately 60 pg/cm ² to about 2750 pg/cm ² vascular endothelial growth factor receptor I (VEGFR 1 ), approximately 10 pg/cm ² to 40 pg/cm ² macrophage inflammatory protein- l a (MIP- l a/CCL3), approximately 5 pg/cm ² to 95 pg/cm ² regulated on activation, norma! T cell expressed and secreted (RANTES/CCL5), approximately 150 pg/cm ² to about 400 pg'cm ² platelet derived growth factor subunit B homodimer (PDGF-BB), from approximately 3.0 x 10 ⁵ to about 2.5 x 10 ^s particles/cm ² extracellular vesicles having a particle diameter ranging from 50 to 200 nm, or any combination thereof.

[0023] In certain aspects, the standardized dosage of the at least one endogenous component per square centimeter of the dehydrated isolated human placental membrane allograft (e.g., dehydrated isolated human placental membrane allograft) includes at least two of: approximately 15 pg/cm ² to about 200 pg/cm ² basic fibroblast growth factor (bFGF), approximately 3.0 pg/cm ² to 15 pg/cm ² epidermal growth factor (EGF), approximately 1.3 x 10 ⁶ pg/cm ² to about 1.5 x 10 ^s pg/cm ² hyaluronic acid (HA), approximately 1 10 pg/cm ² to about 8500 pg/cm ² hepatocyte growth factor (HGF), approximately 200 pg/cm ² to about 7800 pg/cm ³ Interleukin-1 receptor antagonist (IL-IRa), approximately 60 pg/cm ² to about 2750 pg/cm ² vascular endothelial growth factor receptor 1 ( VEGFR1), approximately 10 pg/cnr to 40 pg/cnr macrophage inflammatory protein- 1 a (MIP-la/CCL3), approximately 5 pg/cm ² to 95 pg/cm ² regulated on activation, normal T cell expressed and secreted (RANTES/CCL5), approximately 150 pg'cm ² to about 400 pg/cnr platelet derived growth factor subunit B homodimer (PDGF-BB), from approximately 3.0 x 10 ⁵ to about 2.5 x 10 ^s particles/cm ² extracellular vesicles having a particle diameter ranging from 50 to 200 nm, or any combination thereof.

[0024] In certain aspects, the standardized dosage of the at least one endogenous component per square centimeter of the dehydrated isolated human placental membrane allograft (e.g., dehydrated isolated human placental membrane allograft) includes at least three of: approximately 15 pg/cm ² to about 200 pg/cm ² basic fibroblast growth factor (bFGF), approximately 3.0 pg/cm ² to 15 pg/cm ² epidermal growth factor (EGF), approximately 1 .3 x K) ⁶ pg/cm ³ to about 1.5 x 10 ^s pg/cm ² hyaluronic acid (HA), approximately 110 pg/cm ² to about 8500 pg/cm ² hepatocyte growth factor (HGF), approximately 200 pg/cm ² to about 7800 pg/cm ² Interleukin- 1 receptor antagonist (IL-lRa), approximately 60 pg/cm ² to about 2750 pg/cm ² vascular endothelial growth factor receptor 1 (VEGFR1), approximately 10 pg/cm ² to 40 pg/cm ² macrophage inflammatory protein- la (MIP-la/CCL3), approximately 5 pg/cm ² to 95 pg/cm ² regulated on activation, normal T cell expressed and secreted (RANTES/CCL5), approximately 150 pg/cm ² to about 400 pg/cm ² platelet derived growth factor subunit B homodimer (PDGF-BB), from approximately 3.0 x 10 ⁵ to about 2.5 x 10 ^s particles/cm ² extracellular vesicles having a particle diameter ranging from 50 to 200 nm, or any combination thereof. [0025] In certain aspects, the standardized dosage of the at least one endogenous component per square centimeter of the dehydrated isolated human placental membrane allograft (e.g., dehydrated isolated human placental membrane allograft) includes at least four of: approximately 15 pg/cm ² to about 200 pg/cm ² basic fibroblast growth factor (bFGF), approximately 3.0 pg/cm ² to 15 pg/cm ² epidermal growth factor (EGF), approximately 1.3 x I0 ⁶ pg/cm ² to about 1 .5 x 10 ⁸ pg/cm ² hyaluronic acid (HA), approximately 110 pg/cm ² to about 8500 pg/cm ² hepatocyte growth factor (HGF), approximately 200 pg/cm ² to about 7800 pg/cm ² Interleukin-1 receptor antagonist (IL-IRa), approximately 60 pg/cm ² to about 2750 pg/cm ² vascular endothelial growth factor receptor 1 (VEGFR1), approximately 10 pg/cm ² to 40 pg/cm ² macrophage inflammatory protein- la (MIP-la/CCL3), approximately 5 pg/cm ² to 95 pg/cm ² regulated on activation, normal T cell expressed and secreted (RANTES/CCL5), approximately 150 pg/cm ² to about 400 pg/cm ² platelet derived growth factor subunit B homodimer (PDGF-BB), from approximately 3.0 x 10 ^J to about 2.5 x 10 ^s particles/cnr extracellular vesicles having a particle diameter ranging from 50 to 200 nm, or any combination thereof.

[0026] In certain aspects, the standardized dosage of the at least one endogenous component per square centimeter of the dehydrated isolated human placental membrane allograft (e.g., dehydrated isolated human placental membrane allograft) includes at least five of: approximately 15 pg/cnr to about 200 pg/cm ² basic fibroblast growth factor (bFGF), approximately 3.0 pg/cm ² to 15 pg/cm ² epidermal growth factor (EGF), approximately 1.3 x IO ⁶ pg/cm ² to about 1.5 x 10 ^s pg/cm ² hyaluronic acid (HA), approximately 1 10 pg/cm ² to about 8500 pg/cm ² hepatocyte growth factor (HGF), approximately 200 pg/cm ² to about 7800 pg/cm ² Interleukin- 1 receptor antagonist (IL-IRa), approximately 60 pg/cm ² to about 2750 pg/cm ² vascular endothelial growth factor receptor I (VEGFR1), approximately 10 pg/cm ² to 40 pg/cm ² macrophage inflammatoiy protein- la (MIP-la/CCL3), approximately 5 pg/cm ² to 95 pg/cm ² regulated on activation, normal T cell expressed and secreted (RANEES C( '1.5), approximately 150 pg cm' to about 400 pg/cm ² platelet derived growth factor subunit B homodimer (PDGF-BB), from approximately 3.0 x 10 ⁵ to about 2.5 x 10 ^s particles/cm ² extracellular vesicles having a particle diameter ranging from 50 to 200 nm, or any combination thereof.

[0027] In certain aspects, the standardized dosage of the at least one endogenous component per square centimeter of the dehydrated isolated human placental membrane allograft (e.g., dehydrated isolated human placental membrane allograft) includes at least six of: approximately 25 pg/cm ² to about 100 pg/cm ² basic fibroblast growth factor (bFGF), approximately 5.0 x 10 ⁶ pg/cm ² to about 1.5 x 10 ⁸ pg/cm ² hyaluronic acid (HA), approximately 500 pg/cm ² to about 8500 pg/cm ² hepatocyte growth factor (HGF), approximately 200 pg/cm ² to about 7800 pg/cm ² Interleukin- 1 receptor antagonist (IL- IRa), approximately 800 pg/cm ² to about 2750 pg/cm ² vascular endothelial growth factor receptor 1 ( VEGFR1 ), approximately 150 pg/cm ² to about 400 pg/cm ² platelet derived growth factor subunit B homodimer (PDGF-BB), from approximately 3.0 x 10 ^s to about 2.5 x 10 ^s particles/cm ² extracellular vesicles having a particle diameter ranging from 50 to 200 nm, or any combination thereof.

[0028] In certain aspects, the standardized dosage of the at least one endogenous component per square centimeter of the dehydrated isolated human placental membrane allograft (e.g., dehydrated isolated human placental membrane allograft) includes at least seven of: approximately 25 pg/cm ² to about 100 pg/cm ² basic fibroblast growth factor (bFGF), approximately 5.0 x 10 ⁶ pg/cm ² to about 1.5 x 10 ^s pg/cm ² hyaluronic acid (HA), approximately 500 pg/cm ² to about 8500 pg/cm ² hepatocyte growth factor (HGF), approximately 200 pg/cm ² to about 7800 pg/cm ² Interleukin- 1 receptor antagonist (IL- IRa), approximately 800 pg cm to about 2750 pg/cm ² vascular endothelial growth factor receptor 1 (VEGFRl), approximately 150 pg/cm ² to about 400 pg/cm ² platelet derived growth factor subunit B homodimer (PDGF-BB), from approximately 3.0 x 10’ to about 2.5 x 10 ⁸ particles/cm ² extracellular vesicles having a particle diameter ranging from 50 to 200 nm, or any combination thereof.

[0029] In certain aspects, step (a) comprises: step (i) providing a human placental tissue from a donor within 24 to 72 hours post-childbirth, and step (ii) isolating a desired human placental membrane from the human placental tissue thereby forming the isolated human placental membrane.

[0030] In certain aspects, step (a) further comprises: step (iii) removing immunogenic components from the isolated human placental membrane (e.g., removing visible blood, blood clots, and/or blood components from the human placental tissue without scraping or scrubbing the human placental tissue to preserve structural integrity of the human placental tissue).

[0031] In certain aspects, step (a) further comprises: step (iv) dehydrating the isolated human placental tissue thereby forming the dehydrated isolated human placental tissue (dehydrating the isolated human placental membrane thereby forming a dehydrated human placental membrane allograft), and step (v) resizing the dehydrated isolated human placental tissue into dehydrated human placental tissue portions having a predetermined size (resizing the dehydrated isolated human placenta] membrane allograft into a predetermined size and/or a predetermined shape for subsequent use in or on a subject in need thereof).

[0032] In certain aspects, the predetermined size of the dehydrated placental membrane allograft (e.g., dehydrated isolated human placental membrane allografts) ranges from 0.7 mm ² to

64.0 cm ² . In preferred aspects, the predetermined size of the dehydrated placental membrane allograft (e.g., dehydrated isolated human placental membrane allografts) ranges from 0.5 cm ² to 1.13 cm ² .

[0033] In certain aspects, the predetermined volume of isotonic elution solution ranges from 50 p L to 50 mL. In preferred aspects, the predetermined volume of isotonic elution solution ranges from 1 mL to 5 mL.

[0034] Also disclosed herein is a dehydrated human placental membrane allograft having a standardized dosage (of endogenous components) prepared by the steps of: (a) providing a human placental tissue from a donor, (b) isolating a desired placental membrane from the human placental tissue, (c) dehydrating the desired placental membrane thereby forming the dehydrated placental membrane allograft, and (d) determining a standardized dosage per square centimeter of the dehydrated human placental membrane allograft for subsequent treatment in a human subject in need thereof. Step (d), includes: (i) placing the dehydrated human placental membrane allograft in a predetermined volume of isotonic elution solution for a predetermined period of time to form an eluate comprising at least one endogenous component eluted from the dehydrated placental membrane allograft, (ii) determining a concentration of the at least one endogenous component in the eluate, and (iii) calculating the standardized dosage of the at least one endogenous component per square centimeter in the dehydrated human placental membrane allograft by multiplying the concentration of the at least one endogenous component in the eluate by the predetermined volume of isotonic elution solution, and dividing by the predetermined size per square centimeter of the dehydrated placental membrane allograft.

[0035] In certain aspects, step (iii) calculating the standardized dosage of the dehydrated human placental membrane allograft includes:

100361 In certain aspects, the dehydrated human placental allograft includes an amnion layer (i.e., an isolated amnion layer). Additionally, or alternatively, the dehydrated human placental allograft includes a chorion layer (i.e., an isolated chorion layer). Additionally, or alternatively, the dehydrated human placental allograft includes an amnion layer and a chorion layer having an intact intermediate spongy layer, for example when viewed in cross-section, that is positioned there between connecting (e.g., directly connecting) the amnion layer to the chorion layer.

[0037] In certain aspects, dehydrated human placental membrane allograft (e.g., dehydrated isolated human placental membrane allografts) comprises at least one of: collagen, fibronectin, glycosaminoglycans (GAGs), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), hyaluronic acid (HA), hepatocyte growth factor (I IGF). Interleukin- 1 receptor antagonist (IL-lRa), vascular endothelial growth factor receptor 1 (VEGFR1), macrophage inflammatory protein-la (MIP-la/CCL3), regulated on activation, normal T cell expressed and secreted (R.ANTES/CCL5), platelet derived growth factor subunit B homodimer (PDGF-BB), extracellular vesicles, or any combination thereof.

[0038] In certain aspects, the dehydrated human placental membrane allograft (e.g., dehydrated isolated human placental membrane allografts) comprises at least two of: collagen, fibronectin, glycosaminoglycans (GAGs), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), hyaluronic acid (HA), hepatocyte growth factor (HGF), Interleukin- 1 receptor antagonist ( ll. -I Ra), vascular endothelial growth factor receptor 1 (VEGFR 1), macrophage inflammatory protein- l a (MIP-la/CCL3), regulated on activation, normal T cell expressed and secreted (RANTES/CCL5), platelet derived growth factor subunit B homodimer (PDGF-BB), extracellular vesicles, or any combination thereof.

[0039] In certain, aspects, the dehydrated human placental membrane allograft (e.g., dehydrated isolated human placental membrane allografts) comprises at least three of: collagen, fibronectin, glycosaminoglycans (GAGs), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), hyaluronic acid (HA), hepatocyte growth factor (HGF), Interleukin- 1 receptor antagonist (IL-IRa), vascular endothelial growth factor receptor 1 (VEGFR1), macrophage inflammatory protein- 1 a (MIP-la/CCL3), regulated on activation, normal T cell expressed and secreted (RANTES/CCL5), platelet derived growth factor subunit B homodimer (PDGF-BB), extracellular vesicles, or any combination thereof.

[0040] In certain aspects, the dehydrated human placental membrane allograft (e.g., dehydrated isolated human placental membrane allografts) comprises at least four of: collagen, fibronectin, glycosaminoglycans (GAGs), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), hyaluronic acid (HA), hepatocyte growth factor (HGF), Interleukin- 1 receptor antagonist (IL-IRa), vascular endothelial growth factor receptor I (VEGFR1), macrophage inflammatory protein-la (MIP-la CCL3), regulated on activation, normal T cell expressed and secreted (RANTES/CCL5), platelet derived growth factor subunit B homodimer (PDGF-BB), extracellular vesicles, or any combination thereof.

[0041] In certain aspects, the dehydrated human placental membrane allograft (e.g., dehydrated isolated human placental membrane allografts) comprises at least five of: collagen, fibronectin, glycosaminoglycans (GAGs), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), hyaluronic acid (HA), hepatocyte growth factor (HGF), Interleukin- 1 receptor antagonist (IL-IRa), vascular endothelial growth factor receptor 1 (VEGFR1), macrophage inflammatory protein- l a (MlP-1 a/CCL3), regulated on activation, normal T cell expressed and secreted (RANTES/CCL5), platelet derived growth factor subunit B homodimer (PDGF-BB), extracellular vesicles, or any combination thereof,

[0042] In certain aspects, the dehydrated human placental membrane allograft ha(e.g., dehydrated isolated human placental membrane allografts) comprises at least six of; collagen, fibronectin, glycosaminoglycans (GAGs), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), hyaluronic acid (HA), hepatocyte growth factor (HGF), Interleukin- 1 receptor antagonist (IL-IRa), vascular endothelial growth factor receptor I (VEGFR1), macrophage inflammatory protein-la (MIP-1 a/CCL3), regulated on activation, normal T cell expressed and secreted (RANTES/CCL5), platelet derived growth factor subunit B homodimer (PDGF-BB), extracellular vesicles, or any combination thereof.

[0043] In certain aspects, the dehydrated human placental membrane allograft (e.g., dehydrated isolated human placental membrane allografts) comprises at least seven of: collagen, fibronectin, glycosaminoglycans (GAGs), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), hyaluronic acid (HA), hepatocyte growth factor (HGF), Interleukin- 1 receptor antagonist (IL-IRa), vascular endothelial growth factor receptor 1 (VEGFR1), macrophage inflammatory' protein- la (MIP-1 a/CCL3), regulated on activation, normal T cell expressed and secreted (RANTES/CCL5), platelet derived growth factor subunit B homodimer (PDGF-BB), extracellular vesicles, or any combination thereof.

[0044] In certain aspects, the dehydrated human placental membrane allograft (e.g., dehydrated isolated human placental membrane allografts) comprises at least one of: approximately 25 pg/cm ² to about 100 pg/cm ² basic fibroblast growth factor (bFGF), approximately 5.0 x 10 ⁶ pg/cm ² to about 1.5 x 10 ⁸ pg/cm ² hyaluronic acid (HA), approximately' 500 pg/cm ² to about 8500 pg/cm ² hepatocyte growth factor (HGF), approximately 200 pg/cm ² to about 7800 pg/cm ² Interleukin- 1 receptor antagonist (TL- IRa), approximately 800 pg/cm ² to about 2750 pg/cm ² vascular endothelial growth factor receptor 1 (VEGFRl ), approximately 150 pg/cm ² to about 400 pg/cm ² platelet derived growth factor subunit B homodimer (PDGF-BB), from approximately 3.0 x 10 ⁵ to about 2.5 x 10 ^s particles/cm ² extracellular vesicles having a particle diameter ranging from 50 to 200 mn, or any combination thereof.

[0045] In certain aspects, the dehydrated human placental membrane allograft (e.g., dehydrated isolated human placental membrane allografts) comprises at least two of: approximately 25 pg/cm ² to about 100 pg/cm ² basic fibroblast growth factor (bFGF), approximately 5.0 x 10 ⁶ pg/cm ² to about 1.5 x 10 ⁸ pg/cm ² hyaluronic acid (HA), approximately 500 pg/cm ² to about 8500 pg/cm ² hepatocyte growth factor (HGF), approximately 200 pg/cm ² to about 7800 pg/cm ² lnterleukin-1 receptor antagonist (IL- IRa), approximately 800 pg/cm ² to about 2750 pg/cm ² vascular endothelial growth factor receptor 1 ( VEGFRl ), approximately 150 pg/cm ² to about 400 pg/cm ² platelet derived growth factor subunit B homodimer (PDGF-BB), from approximately 3.0 x 10 ^s to about 2.5 x 10 ^s particles/cm ² extracellular vesicles having a particle diameter ranging from 50 to 200 nm, or any combination thereof.

[0046] In certain aspects, the dehydrated human placental membrane allograft (e.g., dehydrated isolated human placental membrane allografts) comprises at least three of: approximately 25 pg/cm ² to about 100 pg/cm ² basic fibroblast growth factor (bFGF), approximately 5.0 x 10° pg/cm ² to about 1.5 x 10 ⁸ pg/cm ² hyaluronic acid (HA), approximately 500 pg/cm ² to about 8500 pg/cm ² hepatocyte growth factor (HGF), approximately 200 pg/cm ² to about 7800 pg/cm ² Interleukin- 1 receptor antagonist (IL- 1 Ra), approximately 800 pg/cm ² to about 2750 pg/cm ² vascular endothelial growth factor receptor 1 (VEGFRl ), approximately 150 pg/cm ² to about 400 pg/cm ² platelet derived growth factor subunit B homodimer (PDGF-BB), from approximately 3,0 x 10 ⁵ to about 2.5 x 10 ^s particles/cm ² extracellular vesicles having a particle diameter ranging from 50 to 200 nm, or any combination thereof.

[0047] In certain aspects, the dehydrated human placental membrane allograft (e.g., dehydrated isolated human placental membrane allografts) comprises at least four of; approximately 25 pg/cm ² to about 100 pg cnr basic fibroblast growth factor (bFGF), approximately 5.0 x 10 ⁶ pg/cm ² to about 1.5 x 10 ⁸ pg/cm ² hyaluronic acid (HA), approximately 500 pg/cm- to about 8500 pg/cm ² hepatocyte growth factor (HGF), approximately 200 pg/cm ² to about 7800 pg/cm ² Interleukin- 1 receptor antagonist (IL- 1 Ra), approximately 800 pg/cm ² to about 2750 pg/cm ² vascular endothelial growth factor receptor 1 (VEGFR1), approximately 150 pg/cm ² to about 400 pg/cm ^z platelet derived growth factor subunit B homodimer (PDGF-BB), from approximately 3.0 x 10" to about 2.5 x 10 ^s particles/cm ² extracellular vesicles having a particle diameter ranging from 50 to 200 nm, or any combination thereof.

[0048] In certain aspects, the dehydrated human placental membrane allograft (e.g., dehydrated isolated human placental membrane allografts) comprises at least five of: approximately 25 pg/cm ² to about 100 pg/cm ² basic fibroblast growth factor (bFGF), approximately 5.0 x 10 ⁶ pg/cm ² to about 1.5 x 10 ⁸ pg/cm ² hyaluronic acid (HA), approximately 500 pg/cm ² to about 8500 pg/cm ² hepatocyte growth factor (HGF), approximately 200 pg/cm ² to about 7800 pg/cm ² Interleukin- 1 receptor antagonist (IL- 1 Ra), approximately 800 pg/cm ² to about 2750 pg/cm ² vascular endothelial growth factor receptor 1 (VEGFR1 ), approximately 150 pg/cm ² to about 400 pg/cm ² platelet derived growth factor subunit B homodimer (PDGF-BB), from approximately 3.0 x 10 ^s to about 2.5 x 10 ⁸ particles/cm ² extracellular vesicles having a particle diameter ranging from 50 to 200 nm, or any combination thereof. [0049] In certain aspects, the dehydrated human placental membrane allograft ha(e.g., dehydrated isolated human placental membrane allografts) comprises at least six of; approximately

25 pg/cm ² to about 100 pg/cm ² basic fibroblast growth, factor (bFGF), approximately 5.0 x IO ⁶ pg/cm ² to about 1.5 x 10 ^s pg/cm ² hyaluronic acid (HA), approximately 500 pg/cm ² to about 8500 pg/cm ² hepatocyte growth factor (HGF), approximately 200 pg/cm ² to about 7800 pg/cm ² Interleukin- 1 receptor antagonist (IL-IRa), approximately 800 pg/cm ² to about 2750 pg/cm ² vascular endothelial growth factor receptor 1 (VEGFR1), approximately 150 pg/cm ² to about 400 pg/cm- platelet derived growth factor subunit B homodimer (PDGF-BB), from approximately 3.0 x 10° to about 2.5 x 10 ⁸ particles/cm ² extracellular vesicles having a particle diameter ranging from 50 to 200 nm, or any combination thereof.

[0050] Tn certain aspects, the dehydrated human placental membrane allograft (e.g., dehydrated isolated human placental membrane allografts) comprises at least seven of: approximately 25 pg/cm ² to about 100 pg/cm ² basic fibroblast growth factor (bFGF), approximately 5.0 x 10 ⁶ pg/cm ² to about 1.5 x 10 ^s pg/cm ² hyaluronic acid (HA), approximately 500 pg/cm ² to about 8500 pg/cm ² hepatocyte growth factor (HGF), approximately 200 pg/cm ² to about 7800 pg/cm ² Interleukin- 1 receptor antagonist (IL-1 Ra), approximately 800 pg/cm ² to about 2750 pg cm vascular endothelial growth factor receptor 1 (VEGFR1 ), approximately 150 pg/cm ² to about 400 pg/cm ² platelet derived growth factor subunit B homodimer (PDGF-BB), from approximately 3.0 x 10 to about 2.5 x 10 ⁸ particles/cm ² extracellular vesicles having a particle diameter ranging from 50 to 200 nm, or any combination thereof.

[0051] Also disclosed herein is an in vitro method of quantifying an administered dosage of at least one endogenous component available from a human placental membrane allograft to a subject in need thereof in vivo during a predetermined time period. The method includes: (i) placing a predetermined size of the human placental membrane allograft in a predetermined volume of an isotonic elution solution for a predetermined period of time thereby forming an eluate comprising at least one endogenous component eluted from the human placental membrane allograft; (ii) determining a concentration of the at least one endogenous component in the eluate; (iii) calculating a standardized dosage of the at least one endogenous component per square centimeter in the human placental membrane allograft by multiplying the concentration of the at least one endogenous component in the eluate by the predetermined volume of isotonic elution solution, and dividing by the predetermined size of the human placental membrane allograft; and (iv) correlating the calculated standardized dosage in step (iii) to the estimated administered dosage of the at least one endogenous component in vivo in a subject in need thereof during the predetermined time period,

[0052] In certain aspects, step (iii) calculating the standardized dosage of the human placental membrane allograft includes:

[0053] In certain aspects, the human placental allograft includes an amnion layer (i.e., an isolated amnion layer). Additionally, or alternatively, the human placental allograft includes a chorion layer (i.e., an isolated chorion layer). Additionally, or alternatively, the human placental allograft includes an amnion layer and a chorion layer having an intact intermediate spongy layer, for example when viewed in cross-section, that is positioned there between connecting (e.g., directly connecting) the amnion layer to the chorion layer.

[0054] In certain aspects, the isotonic elution solution includes at least one of IX phosphate buffered saline, isotonic saline, lactated ringers, Plasma-Lyte® (NaCl 5.26 g/L, KC1 0.37 g/L, Magnesium Chloride hexahydrate 0.30 g/L, Sodium Acetate trihydrate 3.68 g/L, Sodium Gluconate 5.02 g/L at pH 7.4), Nomiosol® (Naf'l 5.26 g/L, KC1 0.37 g/L, Magnesium Chloride 0.30 g/L, Sodium Acetate anhydrous 2.22 g/L, Sodium Gluconate 5.02 g/L at pH 7.4), or any combination thereof.

[0055] In certain aspects, human placental membrane allograft having a standardized dosage comprises at least one of: collagen, fibronectin, glycosaminoglycans (GAGs), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), hyaluronic acid (HA), hepatocyte growth factor (HGF), Interleukin- 1 receptor antagonist (IL-IRa), vascular endothelial growth factor receptor 1 (VEGFR1), macrophage inflammatory protein- la (MIP-la/CCL3), regulated on activation, normal T cell expressed and secreted (RANTES/CCL5), platelet derived growth factor subunit B homodimer (PDGF-BB), extracellular vesicles, or any combination thereof.

[0056] In certain aspects, the human placental membrane allograft having a standardized dosage comprises at least two of: collagen, fibronectin, glycosaminoglycans (GAGs), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), hyaluronic acid (HA), hepatocyte growth factor (HGF), Interleukin- 1 receptor antagonist (IL-I Ra), vascular endothelial growth factor receptor 1 (VEGFR1 ), macrophage inflammatory protein-la (MIP-l a/CCL3), regulated on activation, normal T cell expressed and secreted (RANTES/CCL5), platelet derived growth factor subunit B homodimer (PDGF-BB), extracellular vesicles, or any combination thereof.

[0057] In certain aspects, the human placental membrane allograft having a standardized dosage comprises at least three of: collagen, fibronectin, glycosaminoglycans (GAGs), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), hyaluronic acid (HA), hepatocyte growth factor (HGF), Interleukin- 1 receptor antagonist (IL-IRa), vascular endothelial growth factor receptor 1 (VEGFRI), macrophage inflammatory protein- l a (MIP-la/CCL3), regulated on activation, normal T cell expressed and secreted (RANTES/CCL5), platelet derived growth factor subunit B homodimer (PDGF-BB), extracellular vesicles, or any combination thereof.

[0058] In certain aspects, the human placental membrane allograft having a standardized dosage comprises at least four of: collagen, fibronectin, glycosaminoglycans (GAGs), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), hyaluronic acid (HA), hepatocyte growth factor (HGF), Interleukin- 1 receptor antagonist (IL-IRa), vascular endothelial growth factor receptor 1 (VEGFR1), macrophage inflammatory protein- 1 a (MIP-la/CCL3), regulated on activation, normal T cell expressed and secreted (RANTES/CCL5), platelet derived growth factor subunit B homodimer (PDGF-BB), extracellular vesicles, or any combination thereof.

[0059] In certain aspects, the human placental membrane allograft having a standardized dosage comprises at least five of: collagen, fibronectin, glycosaminoglycans (GAGs), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), hyaluronic acid (HA), hepatocyte growth factor (HGF), Interleukin- 1 receptor antagonist (IL-IRa), vascular endothelial growth factor receptor 1 (VEGFR1), macrophage inflammatory ⁷ protein- la (MIP- 1 cx/CCL3), regulated on activation, normal T cell expressed and secreted (RANTES/CCL5), platelet derived growth factor subunit B homodimer (PDGF-BB), extracellular vesicles, or any combination thereof.

[0060]

[0061] In certain aspects, the standardized dosage comprises at least six of: collagen, fibronectin, glycosaminoglycans (GAGs), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), hyaluronic acid (FI A), hepatocyte growth factor (HGF), Interleukin- 1 receptor antagonist (IL-IRa), vascular endothelial growth factor receptor 1 (VEGFRl), macrophage inflammatory protein- la (MIP-1 a/CCL3), regulated on activation, normal T cell expressed and secreted (RANTES/CCL5), platelet derived growth factor subunit B homodimer (PDGF-BB), extracellular vesicles, or any combination thereof.

[0062] In certain aspects, the standardized dosage comprises at least seven of: collagen, fibronectin, glycosaminoglycans (GAGs), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), hyaluronic acid (HA), hepatocyte growth factor (HGF), Interleukin- 1 receptor antagonist (IL-lRa), vascular endothelial growth factor receptor 1 (VEGFR1), macrophage inflammatory protein- 1 a (MIP-l a/CCL3), regulated on activation, normal T cell expressed and secreted (RANTES/CCL5), platelet derived growth factor subunit B homodimer (PDGF-BB), extracellular vesicles, or any combination thereof.

[0063] In certain aspects, the standardized dosage comprises at least one of: approximately 25 pg/cm ² to about 100 pg/cm ² basic fibroblast growth factor (bFGF), approximately 5.0 x 10 ⁶ pg/cm ² to about 1.5 x 10 ^s pg/cm ² hyaluronic acid (HA), approximately 500 pg/cm ² to about 8500 pg/cm ² hepatocyte growth factor (HGF), approximately 200 pg/cm ² to about 7800 pg/cm ² Interleukin- 1 receptor antagonist (IL-lRa), approximately 800 pg/cm ² to about 2750 pg/cm ² vascular endothelial growth factor receptor 1 (VEGFR 1), approximately 150 pg/cm ² to about 400 pg/cm ² platelet derived growth factor subunit B homodimer (PDGF-BB), from approximately 3.0 x 10’ to about 2.5 x 10 ⁸ particles/cm ² extracellular vesicles having a particle diameter ranging from 50 to 200 nm, or any combination thereof.

[0064] In certain aspects, the standardized dosage comprises at least two of: approximately 25 pg/cm ² to about 100 pg/cm ² basic fibroblast growth factor (bFGF), approximately 5.0 x 10 ⁶ pg/cm ² to about 1 .5 x 10 ⁸ pg/cm ² hyaluronic acid (HA), approximately 500 pg/cm ² to about 8500 pg/cm ² hepatocyte growth factor (HGF), approximately 200 pg/cm ² to about 7800 pg/cm ² Interleukin-1 receptor antagonist (IL-lRa), approximately 800 pg/cm ² to about 2750 pg/cm ² vascular endothelial growth factor receptor 1 (VEGFR1), approximately 150 pg/cm ² to about 400 pg/cm ² platelet derived growth factor subunit B homodimer (PDGF-BB), from approximately 3.0 x l() ⁵ to about 2.5 x 10 ^s particles/cm ² extracellular vesicles having a particle diameter ranging from 50 to 200 nm, or any combination thereof.

[0065] In certain aspects, the standardized dosage comprises at least three of; approximately 25 pg/cm ² to about 100 pg'cni ² basic fibroblast growth factor (bFGF), approximately 5.0 x 10 ⁶ pg/cm ² to about 1.5 x 10 ⁸ pg/cm ² hyaluronic acid (HA), approximately 500 pg/cm- to about 8500 pg/cm ² hepatocyte growth factor (HGF), approximately 200 pg/cm ² to about 7800 pg/cm ² Interleukin- 1 receptor antagonist (IL- 1 Ra), approximately 800 pg/cm ² to about 2750 pg/cm ² vascular endothelial growth factor receptor 1 (VEGFR1), approximately 150 pg/cm ² to about 400 pg/cm ^z platelet derived growth factor subunit B homodimer (PDGF-BB), from approximately 3.0 x 10" to about 2.5 x 10 ^s particles/cm ² extracellular vesicles having a particle diameter ranging from 50 to 200 run, or any combination thereof.

[0066] In certain aspects, the standardized dosage comprises at least four of: approximately 25 pg/cm ² to about 100 pg/cm ² basic fibroblast growth factor (bFGF), approximately 5.0 x 10 ⁶ pg/cm ² to about 1.5 x 10 ⁸ pg/cm ² hyaluronic acid (HA), approximately 500 pg/cm ² to about 8500 pg/cm ² hepatocyte growth factor (HGF), approximately 200 pg/cm ² to about 7800 pg/cm ² Interleukin- 1 receptor antagonist (IL- I Ra), approximately 800 pg/cm ² to about 2750 pg/cm ² vascular endothelial growth factor receptor 1 (VEGFR1), approximately 150 pg/cm ² to about 400 pg/cm ² platelet derived growth factor subunit B homodimer (PDGF-BB), from approximately 3.0 x IO ⁵ to about 2.5 x 10 ⁸ particles/cm ² extracellular vesicles having a particle diameter ranging from 50 to 200 nm, or any combination thereof. [0067] In certain aspects, the standardized dosage comprises at least five of: approximately 25 pg/cm ² to about 100 pg/cm ² basic fibroblast growth factor (bFGF), approximately 5.0 x 10 ⁶ pg/cm ² to about 1.5 x 10* pg/cm ² hyaluronic acid (HA), approximately 500 pg/cm ² to about 8500 pg/cm ² hepatocyte growth factor (HGF), approximately 200 pg/cm ² to about 7800 pg/cm" Interleukin- 1 receptor antagonist (IL-IRa), approximately 800 pg/cm ² to about 2750 pg/cm ² vascular endothelial growth factor receptor 1 (VEGFR1 ), approximately 150 pg/cm ² to about 400 pg/cm ² platelet derived growth factor subunit B homodimer (PDGF-BB), from approximately 3.0 x IO ⁵ to about 2.5 x 10* particles/cm ² extracellular vesicles having a particle diameter ranging from 50 to 200 nm, or any combination thereof.

[0068] In certain aspects, the standardized dosage comprises at least six of: approximately 25 pg/cm ² to about 100 pg/cm ² basic fibroblast growth factor (bFGF), approximately 5.0 x 10 ⁶ pg/cm ² to about 1.5 x 10 ^s pg/cm ² hyaluronic acid (HA), approximately 500 pg/cm ² to about 8500 pg/cm ² hepatocyte growth factor (HGF), approximately 200 pg/cm ² to about 7800 pg/cm ² Interleukin- 1 receptor antagonist (IL-IRa), approximately ⁷ 800 pg/cm ² to about 2750 pg/cm ² vascular endothelial growth factor receptor 1 (VEGFR1), approximately 150 pg/cm ² to about 400 pg/cm ² platelet derived growth factor subunit B homodimer (PDGF-BB), from approximately 3.0 x 10’ to about 2.5 x 10 ⁸ particles/cm ² extracellular vesicles having a particle diameter ranging from 50 to 200 nm, or any combination thereof.

[0069] In certain aspects, the standardized dosage comprises at least seven of: approximately ⁷ 25 pg/cm ² to about 100 pg/cm ² basic fibroblast growth factor (bFGF), approximately 5.0 x 10 ⁶ pg/cm ² to about 1.5 x 10 ^s pg/cm ² hyaluronic acid (HA), approximately 500 pg/cm ² to about 8500 pg/cm ² hepatocyte growth factor (HGF), approximately 200 pg/cm ² to about 7800 pg/cm ² Interleukin- 1 receptor antagonist (IL- IRa), approximately 800 pg cm to about 2750 pg/cm ² vascular endothelial growth factor receptor 1 (VEGFRl), approximately 150 pg/cm ² to about 400 pg/cm ² platelet derived growth factor subunit B homodimer (PDGF-BB), from approximately 3.0 x 10’ to about 2.5 x 10 ⁸ particles/cm ² extracellular vesicles having a particle diameter ranging from 50 to 200 nm, or any combination thereof.

[0070] In certain aspects, the predetermined size of the dehydrated placental membrane allograft (e.g., dehydrated isolated human placental membrane allografts) ranges from 0.7 mm ² to 64.0 cm ² . In preferred aspects, the predetermined size of the dehydrated placental membrane allograft (e.g., dehydrated isolated human placental membrane allografts) ranges from 0.5 cm ² to 1.13 cm ² .

[0071] In certain aspects, the predetermined volume of isotonic elution solution ranges from 50 p L to 50 mL. In preferred aspects, the predetermined volume of isotonic elution solution ranges from 1 mL to 5 mL.

[0072] Additional features, aspects and advantages of the invention will be set forth in the detailed description which follows, and in part will be readily apparent to those skilled in the art from that description or recognized by practicing the invention as described herein. It is to be understood that both the foregoing general description and the following detailed description present various embodiments of the invention, and are intended to provide an overview or framework for understanding the nature and character of the invention as it is claimed. The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] The drawings described herein are for illustrative purposes for selected embodiments and not all possible implementations, and are not intended to li mit the scope of the present disclosure.

[0074] FIG. 1 schematically depicts the steps in the method of collecting and quantifying elements per square centimeter of tissue, as disclosed herein.

[0075] FIGs. 2A. 2B, 2C, 2D, 2E and 2F each show microscopic images depicting the histology’ and presence of collagen, and glycosaminoglycans of the dehydrated placental membrane obtained from the method disclosed herein.

[0076] FIGs. 3A, 3B, 3C, 3D, 3E, 3F, 3G, and 3H each show microscopic images depicting the histology and presence of fibronectin, laminin, collagen I and collagen III of the placental membrane obtained from the method disclosed herein.

[0077] FIG. 4A shows the concentrations of elements per square centimeter of amniotic membrane obtained from the method disclosed herein.

[0078] FIG. 4B shows the concentrations of dements per square centimeter of amnion/chorion obtained from the method disclosed herein.

[0079] FIG. 5A shows the concentrations of growth factors per square centimeter of amniotic membrane obtained from the method disclosed herein.

[0080] FIG. 5B shows the concentrations of growth factors per square centimeter of amnion/chorion obtained from the method disclosed herein.

[0081] FIG. 6 shows the concentrations of vascular endothelial growth factors per square centimeter of amniotic membrane obtained from the method disclosed herein. [0082] FIG. 7 shows the concentrations of vascular endothelial growth factors per square centimeter of amnion’ chorion obtained from the method disclosed herein.

[0083] FIG. 8 shows the concentrations of hyaluronic acid per square centimeter of amniotic membrane obtained from the method disclosed herein.

[0084] FIG. 9 shows the concentrations of hyaluronic acid per square centimeter of amnion/chorion obtained from the method disclosed herein.

DETAILED DESCRIPTION

[0085] The present invention will now be described more fully hereinafter with reference to the accompanying drawings in which exemplary embodiments of the invention are shown. However, the invention may be embodied in many different forms and should not be construed as limited to the representative embodiments set forth herein. The exemplary embodiments are provided so that this disclosure will be both thorough and complete, and will fully convey the scope of the invention and enable one of ordinary skill in the art to make, use and practice the invention.

[0086] It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0087] Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within the ranges as if each numerical value and sub-range is explicitly recited. As an illustration, a numerical range of “about 1 to 5’' should be interpreted to include not only the explicitly recited values of about 1 to about 5, but also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 2, 3, and 4 and sub-ranges such as from 1 -3, from 2-4, and from 3-5, etc. as well as 1, 2, 3, 4, and 5, individually. The same principle applies to ranges reciting only one numerical value as a minimum or a maximum. Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.

[0088] The phrases “human placental tissue” and “placental tissue” are used interchangeably herein to indicate human placental tissue. Unless otherwise explicitly stated herein, “human placental tissue” and “placental tissue” refer to either the completely intact human placenta (having a human umbilical cord, human amnion, human chorion, and intact human intermediate spongy layer positioned between and connecting the human amnion layer and human chorion layer) or intact human placenta tissue (i.e., only including human amnion, human chorion, and intact human intermediate spongy layer) in cross-section in which a human amnion layer and a human chorion layer have an intact human intermediate spongy layer positioned there between connecting the human amnion layer to the human chorion layer. In certain explicitly stated instances herein, “human placental tissue” and/or placental tissue consists essentially of or only consist of the human amnion that is separated from all other placental tissues. Likewise, unless explicitly stated otherwise, “sterile human placental allograft” refers allografts having intact human placenta tissue in cross-section (i.e., only including human amnion, human chorion, and intact human intermediate spongy layer) in which a human amnion layer and a human chorion layer have an intact human intermediate spongy layer positioned there between connecting the human amnion layer to the human chorion layer. In the explicitly stated instances herein, “sterile human placental allograft” refers to an allograft consisting essentially of or only consist of the human amnion that is separated from all other placental tissues.

[0089] Disclosed herein is a standardized placental membrane allograft dosage and a method of standardizing the dosage of the placental membrane allograft. The allografts and methods disclosed herein achieve these biocompatible, growth factor-rich sterile human placental allografts grafts while maintaining a growth factor profile that mimics the human placenta in vivo and that is improved compared to conventional allografts. The allografts and methods for standardizing their dosage provide a more predictable and controlled therapeutic method for a variety of applications including, but not limited to, dental applications and wound healing.

[0090] The placenta] membrane allografts disclosed herein are particularly rich in interleukin- 1 receptor antagonist (IL- Ira), hepatocyte growth factor (HGF), vascular endothelial growth factor receptor I (VEGFR1), hyaluronic acid (HA), platelet derived growth factor subunit B homodimer (PDGF-BB), glycosaminoglycans (GAGs); and collagen when compared with conventional allografts. Moreover, various human endogenous pathways activated by vascular endothelial growth factor receptor 1 (VEGFR 1), interleukin-1 receptor antagonist (IL- Ira), hepatocyte growth factor (HGF), hyaluronic acid (HA), glycosaminoglycans (GAGs) and that ), platelet derived growth factor subunit B homodimer (PDGF-BB) are associated with cellular remodeling, wound healing, and other human endogenous pathways in vivo. In particular, IL-lra binds non -productively to the IL-1 (pro-inflammatory cytokine) receptor, preventing IL-1 (inflammatory molecule) from sending a signal. This results in modulation of a variety of IL-1 related immune and inflammatory responses. HGF is cellular growth, motility and morphogenic factor secreted by mesenchymal cells. It has been shown to have a major role in embryonic organ development, specifically in myogenesis, in adult organ regeneration, and in wound healing. Importantly, increased expression of HGF has been associated with the enhanced and scarless wound healing capabilities of fibroblast cells isolated from the oral mucosa tissue. VEGFR1 acts as a cell-surface receptor for VEGF-A, VEGF-B and PGF for the development of embryonic vasculature, the regulation of angiogenesis, cell survival and cell migration. VEGFR1 can promote endothelial cell proliferation, survival and angiogenesis in adulthood. Its function in promoting cell proliferation seems to be cell-type specific. For example, it promotes PGF-mediated proliferation of endothelial cells, but does not promote proliferation of normal fibroblasts (in vitro). PDGF-BB is a powerful promoter of cell proliferation and plays a significant role in angiogenesis (blood vessel formation), including the growth of blood vessels from already-existing blood vessel tissue. PDGF-BB promotes the proliferation and directed migration of mesenchymal cells and significantly augments the influx of inflammatory cells, accelerating extracellular matrix and collagen formation and thus reducing overall wound healing time. Glycosaminoglycans (GAGs) are present in all mammalian tissues where they interact with other ECM components to organize and form structural scaffolding suitable for remodeling. GAGs also modulate cell growth and proliferation, cell adhesion, anticoagulation, and wound repair. Hyaluronic acid (HA) is a well- documented chief component of the extracellular matrix. HA is a non-sulfated glycosaminoglycan that, among other things, provides a backbone for sulfated glycosaminoglycans. FLA. also binds integrins resulting in stabilized ECM and absorbs water which is another chief component of the ECM. The high molecular mass of HA results in unique biophysical properties, such as, high viscoelasticity and high colloid osmotic pressure. Additionally, HA leads to extracellular matrix stabilization, water maintenance and regulation of protein distribution. Collagen is the most abundant protein in the body and a key component of the extracellular matrix, and during wound healing, collagen attract fibroblasts and encourage deposition of new collagen as well as bind and inactivate excessive matrix metalloproteinase (degradation). Extracellular vesicles and, more specifically, exosomes, are vesicles ranging from 30 to 200 nm that enable intercellular communication without direct cell-cell contact. They are released through the membrane of cells [Keller 2006, Zomer 2010] (in this case, placental cells) and taken up by the tissues where they release signals that came from the placenial tissue. As a result, signals for development and regeneration are delivered to cells of the damaged tissue. Exosomes are enriched in small RNA species including miRNAs, which have been demonstrated to be functional in the recipient cells [Montecalvo 2008]. Exosomes have gained momentum as a safe and effective therapy for skin wounds/ disorders [Subhan 2021],

[0091 ] Method of Standardizing Placental Membrane Allograft Dosage

[0092] It is an object of this disclosure to provide a method of standardizing a placental membrane allograft dosage. As illustrated in FIG. 1, the method includes, at step (a), preparing a dehydrated placental membrane allograft. The method disclosed herein is configured to maintain a growth factor profile that mimics the human placenta in vivo. To that end, the methods disclosed herein utilize mild conditions to ensure that various endogenous growth factors in the human placental tissue including, but not limited to, IL- Ira, HGF, VEGFR 1, HA, PDGF-BB, GAGs, and collagen are preserved (and/or loss thereof is minimized) in the end resulting sterile human placental allografts disclosed herein.

[0093] Prior to subjecting the human placental tissue to step (a) of FIG. 1, all human placental tissue goes through a donor pre-screening and screening process. Donor pre-screening includes screening the donor’s medical and social history, interviewing the donor, and physically examining the donor to eliminate high-risk donors from the potential donor pooh Either during pre-screening process or after concluding the pre-screening process, cultures of the donor’s tissue and donor blood specimens are collected for comprehensive serological testing, which are subsequently performed in FDA registered or CLIA certified laboratories. Serological testing includes screening the donor and/or human placental tissue for the following transmissible diseases: Hepatitis via Hepatitis-B Surface Antigen, Hepatitis-B Core Antibody, Antibodies to the Hepatitis-C Virus, and/or Hepatitis-B Nucleic Acid Test; Human Immunodeficiency Virus (HIV) via Antibodies to HIV-1, Antibodies to HIV-2, and/or H I V Nucleic Acid Test; Syphilis via Rapid Plasma Regain; Leukemia/Lymphoma via antibodies to Human T-Lymphotropic virus 1, and/or Antibodies to Human T-Lyniphotropic Virus-2; and West Nile Virus.

[0094] In addition to the above serological testing and before subjecting the human placental tissue to step (a) of FIG. 1, bioburden samples are collected to evaluate the number of viable microorganisms on or in the human placental tissue. In addition, environmental monitoring samples are collected throughout to evaluate the quality of surfaces and air. The above-mentioned collections ensure that the human placental tissue and processing thereafter meet consistent microbiological control(s).

[0095] Preparing the dehydrated isolated placental membrane allograft may include steps (a)(i)-(a)(v) as depicted schematically in FIG. 1. Preparing the placental membrane allograft may include (a)(i) providing a human placental tissue from a donor within 24 hours to 72 hours postchildbirth; optionally (a)(ii) separating desired components (amnion, chorion, amnion/ch orion together, umbilical cord, etc) from the placenta. The dehydrated placental membrane allograft may be the amnion layer of the placenta. Alternatively, the dehydrated placental membrane allograft maybe the chorion layer of the placenta. Additionally, the dehydrated isolated placental membrane allograft may be the amnion layer and the chorion layer with an intact spongy layer between the amnion and chorion layers when viewed in cross-section. In certain aspects, the desired components may be separated, cleaned, and then reattached to form an isolated placental membrane laminate. The placental membrane laminate may comprise components from one placenta. The placental membrane laminate may be cross-linked via the use of a cross-linking agent. Cross-linking agents may contain sugars and dialdehyde, epoxide, hydrazide or carbodiimide.

[0096] After step (a)(ii), the placental tissue from step (a)(ii) is visually inspected and cleaned by (a)(iii) removing any immunogenic components, such as components containing heme including: visible blood, blood clots, blood components, and/or debris from the human placental membrane without scraping or scrubbing the human placental membrane to preserve structural integrity of the human placental membrane. Tn a preferred aspect, step (a)(iii) includes a manual hematopoietic reduction step in which forceps and/or tweezers, soft wet gauze and scalpels are used to remove visible blood, blood clots, blood components, and/or undesirable debris. Hematopoietic components may also be displaced by hand, without any and/or without excess scraping or scrubbing to preserve the disinfected human placental tissue’s membrane integrity and to further preserve various endogenous growth factors (e.g., IL- Ira, HGF, VEGFR1 , HA, PDGF- BB, GAGs, and collagen) therein. As should be appreciated, the presence of blood, blood clots, blood components, and/or any other undesirable debris induces immunogenic responses. Therefore, removal of blood, blood clots, blood components, and/or any other undesirable debris is imperative to reduce immunogenicity and/or the probability of an immunogenic response, if any, of the end resulting sterilized human allograft when implanted/transplanted into a human subject in need thereof. Optionally, after step (a)(iii), a cleaning/washing step is performed. During the optional cleaning/washing step, the human placental tissue is placed into an isotonic washing solution (and/or an iso-osmolar solution) to rinse away any extraneous non-human placental components and/or any other potential contaminants. In certain aspects, the isotonic washing solution comprises at least one of lx phosphate buffered saline, isotonic saline, lactated ringers, Plasma-Lyte® (NaCl 5.26 g/L, KC1 0,37 g/L, Magnesium Chloride hexahydrate 0.30 g/L, Sodium Acetate trihydrate 3.68 g/L, Sodium Gluconate 5.02 g/L at pH 7.4), Normosol® (NaCl 5.26 g/L, KC1 0.37 g/L, Magnesium Chloride 0.30 g/L, Sodium Acetate anhydrous 2.22 g/L, Sodium Gluconate 5.02 g/L at pH 7.4), or any combination thereof. The washing step may occur once and/or may be repeated one time, two times, three times, or four times with each wash step occurring for 5 to 15 minutes with a predetermined volume (e.g., 300 mL to 1000 niL, preferably 500 mL) of the isotonic washing solution at a temperature ranging from 4 °C to 20 °C. The washing step preferably occurs at 1 ow-temperature with the isotonic washing solutions having balanced pH, which further advantageously reduces and/or eliminates human placental tissue degradation and/or endogenous growth factor degradation (e.g., IL-lra, HGF, VEGFR.1 , HA, PDGF-BB, GAGs, and collagen). Although the above-mentioned isotonic washing solutions may be used, it should be further appreciated that no harsh chemicals/solutions such as harsh organic or harsh inorganic acids, harsh bases, and/or harsh peroxides (e.g., NaOH, carboxylic acids, HC1, H2O2) are used during the washing step. In particular, the use of harsh organic or harsh inorganic acids, harsh bases, or harsh peroxides can detrimentally result in human placental tissue degradation as well as endogenous growth factor degradation and should be avoided for these reasons.

[0097] After step (a)(ii), optional cleaning/washing, and step (a)(iii), step (a)(iv) includes dehydrating the isolated human placental membrane thereby forming the dehydrated isolated human placental membrane. In particular, the isolated human placental tissue is gently laid flat at ambient temperature for 2-12 hours in the biosafety cabinet with a circulating fan to dehydrate/dry the human placental tissue, or placed in a dehydrator for ’// hour to 6 hours, more preferably from I hours to 4 hours (at a temperature ranging 30° C to 40° C) to obtain the dehydrated isolated human placental tissue. Slow dehydrating/drying of the human placental tissue advantageously preserves tissue structure, integrity, and the endogenous growth factors therein while advantageously avoiding damage frequently observed from freezing (e.g. freeze-drying) and/or drying at high temperatures (e.g. accelerated drying) human placental tissues. The end resulting dehydrated human placental tissue has a maximum water content ranging from 5 wt% to 20 wt%, which reduces the likelihood of any endogenous degradative processes reliant on the presence of water thereby further minimizing growth factor degradation during the method of FIG. 1.

[0098] After dehydration, step (a)(v) includes resizing the dehydrated isolated human placenta] membrane into dehydrated isolated human placental membrane portions having predetermined sizes. When resizing the dehydrated human placental tissue into the dehydrated isolated human placental tissue portions, a die having predetermined size(s) and/or dimension(s) and/or shape(s) may be used to precisely resize the dehydrated isolated human placental tissue into dehydrated isolated human placental tissue portions into the desired shape thereby ensuring for optimal tissue size, shape, and quality for each end resulting sterile human placental allografts produced by the methods disclosed herein. Additionally, a cutting apparatus such as a punch, scalpel, or automated cutting machine may be used to precisely resize the dehydrated isolated human placental tissue into the desired shape and size. In certain aspects, the dehydrated isolated human placental tissue portions is resized into a circular shape, a square shape, a rectangular shape, a rhomboid shape, an ovoid shape, a triangular shape, or any combination thereof. Each of these shapes may have various different sizes and/or dimensions for varied end uses and/or applications. In certain aspects, the predetermined sizes of the dehydrated isolated human placental tissue portions range from 1 cm x 1 cm to 8 cm x 8 cm for the rectangular and/or square shaped dehydrated isolated human placental tissue portions in which any endpoints within any of these ranges may serve as endpoints for any additional ranges falling therein. In certain aspects, the predetermined sizes of the dehydrated isolated human placental tissue portions range from, 8 mm diameter to 12 mm diameter for circular shaped dehydrated isolated human placental tissue portions in which any endpoints within any of these ranges may serve as endpoints for any additional ranges falling therein. In certain aspects, the dehydrated isolated human placental tissue portions may further include a planar upper or lower surface, or the dehydrated isolated human placental tissue portion includes both planar upper and lower surfaces. Further depending on the specific use, the sterile human placental allografts may have a thickness ranging from 0,08 mm to 0.2 mm. In certain aspects, the predetermined size range is from about 0.7 mm ² to about 64 cm ² in which any endpoints within any of these ranges may serve as endpoints for any additional ranges falling therein.

[0099] Optionally, before or after step (a)(v), the dehydrated isolated human placenta] membrane may be sterilized. In certain aspects, sterilizing includes sterilizing the dehydrate human placental tissue portions with e-beam irradiation to a sterility assurance level (SAL) of 10‘ ⁶ , as determined by dose mapping by E-beam vendor, thereby forming the sterile dehydrated isolated human placental allograft in which the SAL, expressed as 10 " ^N , is the expected probability of surviving organisms post treatment (e.g., expected probability of any surviving microorganism after sterilization is 10 ^b or less). Before sterilization, the dehydrated isolated human placental tissue portions of step (a)(v) (resized portions having the desired predetermined shape(s) and size(s)) may be first placed into either into primary and/or secondary packaging and then subjected to low-dose E-beam irradiation to produce the sterile human placental allograft, which is ready for end use (after removal from the primary or secondary packaging). Alternatively, the dehydrated isolated human placental tissue portions of step (a)(v) (resized portions having the desired predetermined shape(s) and size(s)) may be first subjected to low-dose E-beam irradiation to produce the sterile human placental allograft and then may be subsequently placed into sterile packaging while maintaining sterility of the sterile dehydrated isolated human placental allograft for subsequent end use. Low-dose E-beani irradiation is particularly preferred to other sterilization methods because these other methods require direct access to the human placental tissue and leave residual contaminants (e.g. ethylene oxide) on the end resulting allograft, which may result in an immunogenic response when the resulting allograft is used, or require exposure of the tissue to extended periods of irradiation (e.g. gamma irradiation), which unnecessarily degrades structural integrity and endogenous growth factors of the placental tissue.

[0100] After preparation of the dehydrated isolated human placental membrane allograft at step (a), the standardized dosage per square centimeter (cm ² ) of dehydrated isolated human placental membrane allograft is determined at step (b). Determining the standardized dosage includes steps (b)(i)-(b)(iii). Step (b)(i) includes placing a predetermined size of dehydrated isolated human placental membrane allograft into a predetermined volume of isotonic elution solution to form an eluate. The isotonic elution solution may be IX phosphate buffered saline, isotonic saline, lactated ringers, Plasma-Lyte® (NaCl 5.26 g/L, KC1 0.37 g/L, Magnesium Chloride hexahydrate 0.30 g/L, Sodium Acetate trihydrate 3.68 g/L, Sodium Gluconate 5.02 g/L at pH 7.4), Normosol® (NaCl 5.26 g/L, KC1 0.37 g/L, Magnesium Chloride 0.30 g/L, Sodium Acetate anhydrous 2.22 g/L, Sodium Gluconate 5.02 g/L at pH 7.4), or any combination thereof. The predetermined volume of isotonic elution solution may be determined based on the predetermined size of the dehydrated isolated human placental membrane. In certain aspects, the volume of the isotonic elution solution ranges from about 50 pL to about 50 mL, in which any endpoints within any of these ranges may serve as endpoints for any additional ranges falling therein.

[0101] The eluate wi ll contain various endogenous components eluted from the dehydrated isolated human placental membrane allograft. The endogenous components may include one or more of: collagen, fibronectin, glycosaminoglycans (GAGs), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), hyaluronic acid (HA), hepatocyte growth factor (HGF), Interleukin- 1 receptor antagonist (IL-I Ra), vascular endothelial growth factor receptor 1 (VEGFR1), macrophage inflammatory’ protein- la (MIP-l a/CCL3), regulated on activation, normal T cell expressed and secreted (RANTES/CCL5), platelet derived growth factor subunit B homodimer (PDGF-BB), extracellular vesicles, or any combination thereof.

[0102] Step (b)(ii) includes determining a concentration of an endogenous component in the eluate. Determining concentrations of the endogenous component, or components may include the use of an assay kit, for example the Bio-Plex Pro Human Cytokine, Chemokine, and Growth Factor Assays marketed and sold by Bio-Rad; ELISA Kits, for example VEGFR and VEGFR1 ELISA kits manufactured and sold by Invitrogen; and/or Immunoassays, for example the Hyaluronan immunoassay manufactured and sold by R&D Systems.

[0103] Step (b)( iii) includes calculating the standardized dosage of the endogenous component per cm ² of the dehydrated isolated human placental membrane allograft. This calculation includes multiplying the concentration of the endogenous component in the eluate by the predetermined volume and dividing by the predetermined size of the dehydrated placental membrane allograft. This may include the use of equation 1 , below.

[ ^L 0104] ^J [0105] The standardized dosage of the endogenous components per cm ² of the dehydrated isolated human placental membrane allograft may include; approximately 25 pg/cm ² to about 100 pg/cm ² basic fibroblast growth factor (bFGF), approximately 5.0 x I0 ⁶ pg/cm ² to about 1.5 x 10 ⁸ pg/cm ² hyaluronic acid (HA), approximately 500 pg/cm ² to about 8500 pg/cm ² hepatocyte growth factor (HGF), approximately 200 pg'cm ² to about 7800 pg/cm ² Interleukin- 1 receptor antagonist (IL- I fta), approximately 800 pg/cm ² to about 2750 pg/cm ² vascular endothelial growth factor receptor 1 (VEGFR1), approximately 150 pg/cm" to about 400 pg/cm ² platelet derived growth factor subunit B homodimer (PDGF-BB), approximately 3.0 x 10 ⁵ 50-200 nm particles/cm ² to about 2.5 x 10 ^s 50-200 nm particles/cm ² extracellular vesicles, or any combination thereof.

[0106] Dehydrated Human Placental Membrane Allograft with Standardized Dosage

[0107] Disclosed herein are dehydrated human placental membrane allograft having a standardized dosage. The dehydrated human placental membrane allograft having a standardized dosage are prepared from fresh human placental tissue, harvested and processed within 48 to 72 hours of extraction from the human subject. The placental membrane allografts described herein are not subjected to biochemical and/or enzymatic digestion, resulting in a placental membrane allograft that retains a si gnificant portion of the extracellular profile compared to the endogenous profile of human placental tissue in vivo. The dehydrated human placental membrane allograft having a standardized dosage described herein may be used for a variety of medical purposes and medical procedures, which include, but are not limited to dental, wound healing, and cosmetic purposes. The dehydrated human placental membrane allograft having a standardized dosage are preferably implantable/transplantable in a subject in need thereof or may be applied to superficial wounds (e.g., diabetic ulcers). [0108] The dehydrated human placental membrane allograft having a standardized dosage may be prepared as described above. The dehydrated human placental membrane allograft having a standardized dosage is prepared by providing a human placental tissue from a donor, isolating a desired placental membrane from the human placental tissue, dehydrating the desired placental membrane, and determining a standardized dosage per squ are centimeter of the dehydrated human placental membrane allograft.

[0109] As described above, prior to providing the human placental tissue from the donor, the donor may be pre-screened for Hepatitis, HIV, Leukemia/Lymphoma, and/or West Nile Vims. Isolating a desired placental membrane from the human placental tissue, as described above, may include separating amnion, chorion, amnion., chorion together (with an intact spongy layer there between), umbilical cord etc. Additionally, this step may include the preparation of placental membrane laminates, as described above. Prior to dehydrating the placental membrane, the placental tissue may be visually inspected and cleaned to remove blood, blood clots, blood components, and/or debris as described above. Additionally, the tissue may washed with an isotonic washing solution to remove any other potential contaminants, as described above. Dehydrating the human placental membrane may include the use of a biosafety cabinet and/or dehydrator as described above. After dehydrating, the dehydrated placental membrane may be resized into portions having a predetermined size, as described above.

[01 10] Determining the standardized dosage per square centimeter includes the steps of: placing the dehydrated placental membrane allograft into a predetermined volume of isotonic elution solution for a predetermined time period to form an eluate comprising one or more endogenous components from the dehydrated placental membrane allograft, determining the concentration of the endogenous component(s) in the eluate, and calculating a standardized dosage of the endogenous component per square centimeter of the dehydrated placental membrane allograft.

[01 1 1] The isotonic elution solution may be at least one of IX phosphate buffered saline, isotonic saline, lactated ringers, Plasma- Lyte® (NaCl 5.26 g/L, KC1 0.37 g/L, Magnesium Chloride hexahydrate 0.30 g/L, Sodium Acetate trihydrate 3.68 g/L, Sodium Gluconate 5.02 g/L at pH 7.4), Normosol® (NaCl 5.26 g/L, KC1 0.37 g/L, Magnesium Chloride 0.30 g/L, Sodium Acetate anhydrous 2.22 g'L, Sodium Gluconate 5.02 g/L at pH 7.4), or any combination thereof. The predetermined volume of isotonic elution solution may be determined based on the size of the dehydrated placental membrane allograft, as described above.

[01 12] After determining the concentration of one or more endogenous components in the eluate as described above, the standardized dosage is calculated. This calculation includes multiplying the concentration of the endogenous component in the eluate by the predetermined volume and dividing by the predetermined size of the dehydrated placental membrane allograft. This may include the use of equation 1 (above).

[0113] The dehydrated human placental membrane allograft having a standardized dosage described herein may include endogenous components such as: collagen, fibronectin, glycosaminoglycans (GAGs), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), hyaluronic acid (HA), hepatocyte growth factor (HGF), Interleukin- 1 receptor antagonist (IL-1 Ra), vascular endothelial growth factor receptor I (VEGFR1 ), macrophage inflammatory protein- la (MIP-1 a/CCL3), regulated on activation, normal T cell expressed and secreted (RANTES/CCL5), platelet derived growth factor subunit B homodimer (PDGF-BB), extracellular vesicles, or any combination thereof. [0114] The dehydrated human placental membrane allograft having a standardized dosage comprising an amnion layer, a chorion layer, or an amnion layer and a chorion layer with an intact spongy layer there between may have: approximately 25 pg/cm ² to about 100 pg/cm ² basic fibroblast growth factor fbFGF), approximately 5,0 x 10 ⁶ pg/cm ² to about 1.5 x 10 ⁸ pg/cm ² hyaluronic acid (HA), approximately 500 pg/cm ² to about 8500 pg/cm ² hepatocyte growth factor { H( iF). approximately 200 pg/cm ² to about 7800 pg/cm ² Interleukin-1 receptor antagonist (IL- IRa), approximately 800 pg/cm ² to about 2750 pg/cm ² vascular endothelial growth factor receptor I (VEGFR1), approximately 150 pg/cm ² to about 400 pg/cm ² platelet derived growth factor subunit B homodimer (PDGF-BB), approximately 3.0 x 10’ 50-200 nm particles/cm ² to about 2.5 x 10 ⁸ 50-200 nm particles/cm ² extracellular vesicles, or any combination thereof.

[0115] In Vitro Method of Quantifying Estimated Administered Dosage Eluted In Vivo From a Human Placental Membrane Allograft

[01 16] Disclosed herein is an in vitro method of quantifying an estimated administered dosage of one or more endogenous components available from a human placental membrane allograft to a subject in need thereof in vivo during a predetermined time period. The in vitro method includes step (b)(i) placing a predetermined size of human placental membrane allograft into a predetermined volume of isotonic elution solution to form an eluate. The isotonic elution solution may be IX phosphate buffered saline, isotonic saline, lactated ringers. Plasma- Lyte® (NaCI 5,26 g/L, KC1 0.37 g/L, Magnesium Chloride hexahydrate 0.30 g/L, Sodium Acetate trihydrate 3.68 g/L, Sodium Gluconate 5.02 g/L at pH 7.4), Normosol® (NaCI 5.26 g/L, KC1 0.37 g/L, Magnesium Chloride 0.30 g/L, Sodium Acetate anhydrous 2.22 g/L, Sodium Gluconate 5.02 g/L at pH 7.4), or any combination thereof. The predetermined volume of isotonic elution solution may be determined based on the predetermined size of the dehydrated isolated human placental membrane. In certain aspects, the volume of the isotonic elution solution ranges from about 50 pL to about 50 mL, in which any endpoints within any of these ranges may serve as endpoints for any additional ranges falling therein.

[0117] Prior to step (b)(i ), the human placental membrane allograft may be prepared in the same manner as described above. For example, human placental tissue may be provided from a donor within 24 to 72 hours post-childbirth. A desired human placental membrane from the human placenta] tissue may be isolated forming an isolated human placental membrane. Immunogenic components may be removed from the human placental membrane as described previously. The human placental membrane allograft may be dehydrated. The human placental membrane allograft may also be resized into a predetermined size and/or a predetermined shape.

[01 18] The eluate will contain various endogenous components eluted from the human placental membrane allograft. The endogenous components may include one or more of: collagen, fibronectin, glycosaminoglycans (GAGs). epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), hyaluronic acid (HA), hepatocyte growth factor (HGF), Interleukin- 1 receptor antagonist (IL-IRa), vascular endothelial growth factor receptor 1 (VEGFR1), macrophage inflammatory protein-la (MIP-la/CCL3), regulated on activation, normal T cell expressed and secreted (RANTES/CCL5), platelet derived growth factor subunit B homodimer (PDGF-BB), extracellular vesicles, or any combination thereof.

[01 19] Step (b)(ii) includes determining a concentration of an endogenous component in the eluate. Determining concentrations of the endogenous component, or components may include the use of an assay kit, for example the Bio-Plex Pro Human Cytokine, Chemokine, and Growth Factor Assays marketed and sold by Bio-Rad; ELISA Kits, for example VEGFR and VEGFR1 ELISA kits manufactured and sold by Invitrogen; and/or Immunoassays, for example the Hyaluronan immunoassay manufactured and sold by R&D Systems.

[0120] Step (b)(iii) includes calculating a standardized dosage of the endogenous component per cm ² of the human placental membrane allograft. This calculation includes multiplying the concentration of the endogenous component in the eluate by the predetermined eluate volume and dividing by the predetermined size of the dehydrated placental membrane allograft used for the elution. This may include the use of equation 1 , below.

[0121]

[0122] After calculating the standardized dosage of the endogenous component per cm ² , step (b)(iv) includes correlating the standardized dosage per cm ² to an estimated administered dosage of the endogenous component eluted in vivo from the dehydrated placental membrane allograft to a subject in need thereof during the predetermined time period.

[0123] The standardized dosage of the endogenous components per cm ^z of the human placental membrane allograft may include; approximately 25 pg/ cm ² to about 100 pg/cm ² basic fibroblast growth factor (bFGF), approximately 5.0 x IO ⁶ pg/cm ² to about 1.5 x 10 ^M pg/cm ² hyaluronic acid (HA), approximately 500 pg/cm ² to about 8500 pg/cm ² hepatocyte growth factor (FIGF), approximately 200 pg/cm ² to about 7800 pg/cm ² Interleukin-1 receptor antagonist (IL- IRa), approximately 800 pg/cm ² to about 2750 pg/cm ² vascular endothelial growth factor receptor 1 (VEGFR1), approximately 150 pg/cm ² to about 400 pg/cm ² platelet derived growth factor subunit B homodimer (PDGF-BB), approximately 3.0 x IO’ 50-200 nm particles 'cm ² to about 2.5 x 10 ⁸ 50-200 nm particles/cm ² extracellular vesicles, or any combination thereof.

[0124] The foregoing description provides embodiments of the invention by way of example only. It is envisioned that other embodiments may perform similar functions and/or achieve similar results. Any and all such equivalent embodiments and examples are within the scope of the present invention and are intended to be covered by the appended claims.

[0125] Working Examples

[0126] Collection of Placental Membrane(s)

[0127] Donated human placentas were acquired from accredited Gift of Life tissue recovery agencies (e.g. Telegen) after planned cesarean sections with informed consent. All donations and processing were completed in accordance with FDA Good Tissue Practices (GTP) and American Association of Tissue Banks (AATB) standards. Donors were screened for medical issues, social issues, and communicable diseases, as well as infectious diseases, including human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV), hepatitis B and C, syphilis, and cytomegalovirus (CMV). Additionally, grafts are terminally sterilized by electron beam sterilization (Steri-tek, Fremont, CA).

[0128] Amnion or amnion with chorion were isolated from the placenta and processed with a proprietary procedure as disclosed herein according to FIG. 1 step (a). The resulting dehydrated amniotic membrane or dehydrated amnion/chorion was cut to preferred sizes to obtain dehydrated human placental membrane allografts, packaged, and sent out for E-beam sterilization. The final sterilized products were used for all tests. Dehydrated human placental membrane allografts prepared from amnion/chorion are processed intact without separation of layers.

[0129] Histology

[0130] Histology was performed by HistoWiz Inc. (histowiz.com) using a Standard Operating Procedure and fully automated workflow'. Samples were processed, embedded in paraffin, and sectioned at 4pm. Immunohistochemistry was performed on a Bond Rx autostainer (Leica Biosystems) with enzyme treatment (1 :1000) using standard protocols. Antibodies used were rat monoclonal F4/80 primary antibody (eBioscience, 14-4801, 1 :200) and rabbit anti-rat secondary (Vector, 1:100). Bond Polymer Refine Detection (Leica Biosystems) was used according to the manufacturer's protocol. After staining, sections were dehydrated and film coverslipped using a TissueTek-Prisma and Coverslipper (Sakura). Whole slide scanning (40x) was performed on an Aperio AT2 (Leica Biosystems). Four separate lots of dehydrated human placental membrane allografts prepared from dehydrated amniotic membrane and four separate lots of dehydrated human placental membrane allografts prepared from dehydrated amnion/chorion were sent in biopsy cages with 50% paraformaldehyde/PBS. Stains include H&E for structure and nuclei, Van Gieson for elastin and collagen, Alcian blue for polysaccharides, and immunohistochemistry for Collagen I, Collagen HI, fibronectin and laminin.

[0131] Since one of the main benefits of using placental tissues for healing and regeneration is the existence of numerous structural components that promote endogenous cell migration and proliferation, total glycosaminoglycans were visualized with Alcian blue, elastin and total collagens with Van Gieson, and nuclei against extracellular matrix (ECM) by Hematoxylin and eosin (H&E) staining. H&E staining clearly shows the retention of structure for dehydrated human placental membrane allografts prepared from dehydrated amniotic membrane as shown in FIG. 2A and dehydrated human placental membrane allografts prepared from dehydrated amnion/chorion , in FIG. 2B after dehydration and sterilization. Alcian Blue staining shows glycosaminoglycans distributed evenly below the basement membrane in dehydrated human placental membrane allografts prepared from dehydrated amniotic membrane (FIG. 2C) and strongly between the two basement membranes in dehydrated human placental membrane allografts prepared from dehydrated amnion/chorion , as shown in FIG. 2D, also strongly evident in the lower portion of the chorion. Van Geison indicates collagen distribution throughout the dehydrated human placental membrane allografts prepared from dehydrated amniotic membrane, in FIG. 2E, with elastic fibers distributed sparsely. In the dehydrated human placental membrane allografts prepared from dehydrated amnion/chorion, as seen in FIG. 2F, collagen is also seen throughout and elastin more strongly dispersed below the chorionic basement membrane.

[0132] The localization of fibronectin, collagen I, collagen III and laminin was evaluated. There was a robust overall distribution of fibronectin in dehydrated human placental membrane allografts prepared from dehydrated amniotic membrane , shown in FIG. 3A, and dehydrated human placental membrane allografts prepared from dehydrated amnion/chorion, in FIG. 3B, and its presumed binding to other extracellular matrix proteins (e.g. collagen). Although expressed throughout, Collagen I was strongly expressed in the dehydrated human placental membrane allografts prepared from dehydrated amniotic membrane, in FIG. 3C below' the basement membrane, and in intermediate layer of the dehydrated human placental membrane allografts prepared from dehydrated amnion 'chorion, in FIG. 3D. Less intense expression was also seen throughout the chorion below the trophoblast layer. Collagen III was lightly expressed in the dehydrated human placental membrane allografts prepared from dehydrated amniotic membrane with the strongest expression in the intermediate layer of the dehydrated human placenta] membrane allografts prepared from dehydrated amniotic membrane , in FIG. 3E and dehydrated human placental membrane allografts prepared from dehydrated amnion'ch orion, in FIG. 3F, along the connective tissue tendrils. There was minimal expression of laminin in the dehydrated human placental membrane allografts prepared from dehydrated amniotic membrane, shown in FIG. 3G, which maybe an effect of the processing. In contrast, laminin was expressed throughout the dehydrated human placental membrane allografts prepared from dehydrated amnion/chorion, as seen in FIG. 3 FL with even distribution in the compact layer of the amnion. It is conceivable that keeping the layers together during processing protects the laminin within the tissue.

[0133] Quantification of growth factors

[0134] To determine the elution of growth factors from the membranes, 8 mm biopsy punches from 5 separate lots of dehydrated human placental membrane allografts prepared from dehydrated amniotic membrane and dehydrated human placental membrane allografts prepared from dehydrated amnion/chorion were placed in 500 pl DPBS at 37° C for 24 hours. For the hyaluronic acid (HA) assay, dehydrated human placental membrane allografts prepared from dehydrated amniotic membrane and dehydrated human placental membrane allografts prepared from dehydrated amnion/chorion were placed in 1 ml DPBS at 37 °C for 3 days. The supernatant was collected and stored at -80 °C until use. A 30-plex cytokine assay (Bio-Plex Pro, Bio-Rad, Hercules, California) was performed on three separate lots of dehydrated human placental membrane allografts prepared from dehydrated amniotic membrane and dehydrated human placental membrane allografts prepared from dehydrated amnion/chorion. A custom 5-plex panel (Bio-Plex Pro, Bio-Rad, Hercules, California) was designed to determine the concentration of hepatocyte growth factor (HGF), IL-1 receptor antagonist (IL- I ra), basic fibroblast growth factor (bFGF), interleukin 10 (IL- 10) and platelet derived growth factor B homodimer (PDGF-BB). Assays were performed with a wash station and analyzed on a Bio-Plex 200 multiplex system (Bio-Rad, Hercules, California) with high-throughput fluids. All samples were run in duplicate. Results were obtained using 5-parameter logistic standard curves and a plate blank. Vascular endothelial growth factor receptor 1 (VEGFR1 ) was assessed with the Human VEGFR1 ELISA Kit (Invitrogen #BMS268-3). Hyaluronic acid (HA) was assessed with the Hyaluronan Immunoassay (R&D Systems #DHYALO) and vascular endothelial growth factor (VEGF) was assessed with the Human VEGF ELISA Kit (Invitrogen #KHG0111). All ELISAs were calculated from a 4-parameter logistic standard curve.

[0135] The 8mm punches have an area of 0.503 cm ² . The average pg'ml of growth factor was multiplied by the milliliters of eluate. This total GF content in the eluate was divided by 0.503 to reveal the pg of growth factor in 1 cm ² of tissue. Equation 1 can be utilized for any volume of eluate used and any sample punch size.

[0136]

[0137] Of the 30 cytokines tested, the most highly expressed factors in dehydrated human placental membrane allografts prepared from dehydrated amniotic membrane, as shown in FIG. 4A, were HGF and IL- Ira (200.74 pg/cm ² and 56.17 pg/cm ² , respectively). Of the 30 cytokines, HGF, VEGF, IL-7, IL-2R, TNFa, INFg, GM-CSF, MIP-lb, IL-17a, IL- 12, IFNa, IL-5 and IL-6 were all out of range. The dehydrated human placental membrane allografts prepared from dehydrated amnion/chorion (FIG. 4B) were found to express IL- Ira and IL-8 (680.48 pg/cm ² and 206.04 pg'cm ² , respectively). Of the 30 cytokines, HGF, VEGF, IL-7, IL-2R, TNFa, IP-10, INFg, GM-CSF, G-CSF, IL- 17a, IL- 12, 1FN a and IL-5 were all out of range. Results were calculated and reported by BioPlex Manager software as average pg/ml of 3 lots with standard deviation. Data was converted to cm ² according to the present invention.

[0138] Based on these results, 5 growth factors (HGF, IL-lra, bFGF, IL-10 and PDGF- BB) were tested with a more specific 5-plex. bFGF and IL- 10 were also tested, even though they were not significantly expressed, because they have immunoregulatory and anti-inflammatory properties which could provide a substantial effect at lo w concentration on regeneration and wound healing. The sample size was increased to 5 lots for increased accuracy and calculation. This provided a sample size appropriate for additional error calculation. Raw results were collected by BioPlex Manager software and the standard deviation was divided by the square root of the sample size for standard error of the mean. Data was converted to cm ² according to the present invention. The 5-plex confirmed strong elution of IL- Ira and HGF from dehydrated human placenta] membrane allografts prepared from dehydrated amniotic membrane , in FIG. 5 A, and dehydrated human placental membrane allografts prepared from dehydrated amnion chorion, in FIG. 5B, with absence of IL-10. In addition, due to the increased specificity and reduced cross-reactivity of using only 5 antibodies, consistent results were obtained that shed light on the variability between the 5 donors. PGDF-BB and FGF-2 were also expressed in dehydrated human placental membrane allografts prepared from dehydrated amniotic membrane and dehydrated human placental membrane allografts prepared from dehydrated amnion/chorion, as see in FIGS. 5A and 5B. To expand on the available factors eluted from these membranes, the eluate was tested for VEGF, VEGFR1, and HA separately from 5 donors by ELISA. VEGF was detected in dehydrated human placental membrane allografts prepared from dehydrated amniotic membrane, in FIG. 6, but not in dehydrated human placental membrane allografts prepared from dehydrated amnion/chorion, shown in FIG. 7. VEGFR1 was eluted from dehydrated human placental membrane allografts prepared from dehydrated amniotic membrane, as seen in FIG. 6 and, to a much greater extent, from dehydrated human placental membrane allografts prepared from dehydrated amnion chori on, in FIG. 7. Tn addition, HA was highly expressed by both dehydrated human placental membrane allografts prepared from dehydrated amniotic membrane, as shown in FIG. 8, and dehydrated human placental membrane allografts prepared from dehydrated amnion/chorion, in FIG. 9.