WO2014126931A1

WO2014126931A1 - Stable platelet- rich-plasma compositions and methods of use

Info

Publication number: WO2014126931A1
Application number: PCT/US2014/015844
Authority: WO
Inventors: Steven VICTOR
Original assignee: Victor Steven
Priority date: 2013-02-15
Filing date: 2014-02-11
Publication date: 2014-08-21

Abstract

Stable compositions comprising platelet-rich-plasma (PRP) for therapeutic and cosmetic uses which are stable days or months.

Description

STABLE PLATELET-RICH-PLASMA COMPOSITIONS AND METHODS OF USE

FIELD OF THE INVENTION

[0001] The present invention relates to compositions comprising platelet-rich-plasma (PRP) which are stable for days or months and therapeutic and cosmetic uses thereof.

BACKGROUND OF THE INVENTION

[0002] Platelets are cytoplasmic portions of marrow megakaryocytes and lack a nucleus.

Platelets have an expected lifetime of about five to nine days. Platelets are involved in the hemostatic process and release several initiators of the coagulation cascade. Platelets also release cytokines, stored in alpha granules, involved with initiating wound healing. In response to platelet to platelet aggregation or contact with connective tissue, the cell membrane of the platelet is "activated" to secrete the contents of the alpha granules. This release of agents is referred to as the "releasate" (e.g., the internal contents of the platelet, including cytokines.) The alpha granules release cytokines via active secretion through the platelet cell membrane as histones and carbohydrate side chains are added to the protein backbone to form the complete cytokine. Platelet disruption or fragmentation does not result in release of the complete cytokine. Ruggeri & Mendolichio (2007) Circ. Res. 100: 1673-1685.

[0003] A large number of cytokines are released by activated platelets including but not limited to platelet derived growth factor (PDGF), transforming growth factor-beta (TGF-β), platelet- derived angiogenesis factor (PDAF) and platelet derived endothelial cell growth factor (PD- ECGF) and insulin-like growth factor (IGF). These cytokines serve different functions in the healing process, including helping to stimulate cell division at an injury site. They also work as powerful chemotactic factors for mesenchymal cells, monocytes, and fibroblasts. These growth factors function to assist in healing by stimulating stem cells to regenerate new tissue and by promoting vascularization. D. Podd, Platelet-Rich Plasma Therapy: Origins and Applications Investigated, 25 J. Am. Acad. Physician Assts. 44 (2012).

[0004] The platelet-rich plasma (PRP) is blood product with a concentrated number of platelets in a small volume of plasma. PRP may contain about 2,500,000 platelets^L compared to a baseline concentration of platelets in whole blood of about 328,00 platelets^L. The PRP mimics the last step of the coagulation cascade, leading to the formation of a fibrin clot, which consolidates and adheres to the application site in a short period of time. [0005] Platelet-rich plasma (PRP) is obtained from whole blood or plasma by concentrating the platelets from the blood. Whole blood may contain about 95% red blood cells, about 5% platelets and less than 1 % white blood cells. PRP, by contrast, may contain about 95% platelets with about 4% red blood cells, and less than about 1% white blood cells. PRP also contains fibrin, fibronectin, and vitronectin. Nagata, et al. (2010) European Journal of Dentistry 4: 395-402.

[0006] The isolation of platelet-rich-plasma is known in the art. For example, PRP may be prepared by the discontinuous cell separation method from a blood volume of approximately 450 mL. Whitman, et al. (1997) J Oral Maxillofac Surg 55: 1294-1299 and Marx, et al. (1998) Oral Surg Oral Med Pathol Oral Radiol 85: 638-646. Several simplified protocols for the preparation of PRP are also known. See, also, Landesberg, et al. (2000) J Oral Maxillofac Surg 58: 297; Efeoglu, et al. (2004) J Oral Maxillofac Surg 61(11): 1403-7; and Nagata, et al. (2010)

European Journal of Dentistry 4: 395-402. Further, neutrophil-depleted platelet rich plasma may be obtained using the methods described in U.S. Patent No. 8,142,993.

[0007] PRP has hemostatic and healing properties including being able to hold tissues or materials in a required configuration for healing and repair. PRP's biocompatible and biodegradable properties prevent the PRP from inducing foreign body reactions, tissue necrosis, or extensive fibrosis. Absorption of the fibrin clot is achieved during wound healing within weeks following application. Gimeno, et al. (2006) Thrombosis Journal 4: 18 and Moodell-May, et al. (2005) Journal of Carniofacial Surgery 16: 749. Activation of PRP results in release of the various cytokines and also creates a clotting reaction within various constituents of the plasma fraction. The clotting reaction rapidly forms a platelet gel (PG) which can be applied to various wound surfaces for purposes of hemostasis, sealing, and adhesion. PRP has been used to form a fibrin tissue adhesive through activation of the PRP using thrombin and calcium. See U.S. Patent Nos. 5,165,938 and 5,599,558.

[0008] PRP has been used in humans in different kinds of transplant procedures such as dentistry, orthopedics, maxillofacial surgery, plastic surgery and ophthalmology. In addition, PRP may be used as a carrier for biological active agents and a healing substance causing less post-surgical pain. Gimeno, et al. (2006) Thrombosis Journal 4: 18 and Moodell-May, et al. (2005) Journal of Carniofacial Surgery 16: 749. In addition to use in the treatment of chronic skin and soft tissue ulcerations, publications regarding the use of PRP include periodontal and oral surgery, maxillofacial surgery, orthopedic and trauma surgery, cosmetic and plastic surgery, spinal surgery, heart bypass surgery, and burns. Lacci, et al. (2010) Yale Journal of Biology and Medicine 83: 1-9.

[0009] PRP has also been used in bone grafting and dental implant applications and as part of a composition as a surgical adhesive. For example, U.S. Patent No. 6,322,785 discloses an autologous platelet gel that includes PRP for bone grafts and dental implants. The PRP may be activated by collagen and applied topically to promote wound healing. U.S. Patent No.

5,585,007 discloses a preparation of PRP and its use as a tissue sealant. U.S. Patent No.

5,614,214 discloses a biopolymer that optionally includes PRP and its use to temporarily block arteries and veins. U.S. Patent No. 5,599,558 discloses a platelet releasate product, which includes platelets buffered to approximately pH 6.5, for use in a topical application to wounds. See also U.S. Patent Application Publication 2003/0152639.

[0010] Although platelet-rich plasma is useful in medicine, it must be maintained at room temperature and used within 8-12 hours of preparation due to platelet instability, which limits its use. Thus, there exists a need in the art for stable compositions of platelet-rich-plasma (PRP) for uses in cosmetics and medicine.

SUMMARY OF THE INVENTION

[0011] A stable composition comprising platelet-rich-plasma (PRP). In one embodiment, the composition may be stable for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. In one embodiment, the composition may be stable for at least about 1, 2, 3, 4, 5, or 6 months. In one embodiment, the composition may be stable for at least about 3 months.

[0012] In one embodiment, the composition may further comprise hematopoietic elements.

[0013] In one embodiment, the composition may comprise at least about 250,000 to about 7,000,000 platelets per microliter. In another embodiment, the composition may have a platelet count of at least about 250,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000;

900,000; 1,000,000; 1,100,000; 1,250,000; 1,500,000; 1,750,000; 2,000,000; 2,500,000;

2,600,000; 3,000,000; 3,500,000; 4,000,000; 5,000,000; 5,200,000; 6,000,000; or 7,000,000 platelets per microliter. In another embodiment, the composition may have a platelet count of at least about 500,000-7,000,000 platelets per microliter. In another embodiment, the composition may have a platelet count of at least about 500,000-700,000; 700,000-900,000; 900,000- 1,000,000; 1 ,000,000-1,250,000; 1,250,000-1,500,000; 1,500,000-2,500,000; 2,500,000- 5,000,000; or 5,000,000-7,000,000 platelets, optionally about 2,600,000 or 5,200,000 platelets.

[0014] In one embodiment, the platelet-rich-plasma may be activated, optionally by calcium chloride or thrombin.

[0015] In one embodiment, the composition may be in a form selected from the group consisting of a balm, solution, suspension, emulsion, ointment, foam, paste, gel, cream, lotion, powder, salve, soap, surfactant-containing cleansing, oil, serum, drops, liposomes, nanoparticles, nanoboots, and spray. In another embodiment, the composition may be a cream, lotion, or solution. In another embodiment, the composition may be impregnated or made part of a bandage. In another embodiment, the bandage may be a surgical dressing, a plaster bandage, an adhesive bandage, or a gauze.

[0016] In one embodiment, the composition may further comprise a component selected from the group consisting of monocytes, stem cells, gene therapy products, vitamins, palmitate retinol, tocoferil acetate, sodium ascorbil phosphate, D-panthenol, peptides, recombinant growth factors, micronized human-identical hormones, aminoacids, phyto-extracts, antioxidants, lipoic acid, DMAE, collagen, GAG, hyaluronic acid, proteoglycans, adenine, guanine, cytosine, thymine, trace elements, minerals, proteases, ceramides, polisaccarides, algae, and marine extracts.

[0017] In one embodiment, the composition may further comprise white blood cells (WBCs) at a higher concentration than white blood cells in whole blood. In another embodiment, the composition may comprise about 8,000-10,000; 10,000-15,000; 15,000-20,000; 20,000-30,000; 30,000-50,000; 50,000-75,000; or 75,000-100,000 white blood cells per microliter.

[0018] In one embodiment, the composition may further comprise lymphocytes at a higher concentration than lymphocytes in whole blood. In another embodiment, the composition may comprise about 5,000-20,000 lymphocytes per microliter.

[0019] In one embodiment, the composition may further comprise monocytes at a higher concentration than monocytes in whole blood. In another embodiment, the composition may comprise about 1,000-5,000 per monocytes microliter.

[0020] In one embodiment, the composition may further comprise eosinophils at a higher concentration than eosinophil in whole blood. In another embodiment, the composition comprises about 200-1,000 eosinophils per microliter. [0021] In one embodiment, the composition may further comprise neutrophils at a lower concentration than neutrophils in whole blood. In another embodiment, the composition comprises about 200-1,000 neutrophils per microliter. In another embodiment, the neutrophil content of the platelet-rich plasma has been reduced by 50-99% as compared to whole blood, optionally reduced by 50%, 60%, 70%, 80%, 90%, 95%, or 99% as compared to whole blood.

[0022] In one embodiment, the composition may further comprise red blood cells at a lower concentration than red blood cells in whole blood. In another embodiment, the composition comprises about 4,600-5,200 red blood cells per microliter.

[0023] In one embodiment, the composition may further comprise hemoglobin at a lower concentration than hemoglobin in whole blood. In another embodiment, the composition may comprise a hemoglobin concentration at about 1 g/dL or less, between about 1-5 g dL, about 5-10 g/dL, about 10-15 g/dL, or about 15-20 g/dL.

[0024] In one embodiment, the composition may further comprise fibrin, fibronectin, or vitronectin.

[0025] In one embodiment, the platelet-rich-plasma may be autologous.

[0026] In one embodiment, the composition may be a pharmaceutical composition further comprising an pharmaceutically acceptable excipient. In another embodiment, the excipient may be isotonic sodium chloride solution, physiological saline, phosphate buffered saline, potassium buffered saline, normal saline, dextrose 5% in water, dextrose 10% in water, Ringer solution, lactated Ringer solution, Ringer lactate, or Ringer lactate solution.

[0027] In one embodiment, the composition may have a pH of about 6.0-8.0, optionally a pH of about 7.4.

[0028] A cosmetic cream comprising the PRP compositions described herein.

[0029] A cosmetic lotion comprising the PRP compositions described herein

[0030] A cosmetic solution comprising the PRP compositions described herein.

[0031] A pharmaceutical composition comprising the PRP compositions described herein and an pharmaceutically acceptable excipient.

[0032] A medical device comprising the PRP compositions described herein.

[0033] An implant comprising the PRP compositions described herein.

[0034] A biocompatible polymer comprising the PRP compositions described herein. [0035] In one embodiment, the PRP compositions described herein may be used in the manufacture of cosmetic surgery products, optionally comprising dermal fillers.

[0036] In one embodiment, a method for cosmetic surgery may comprise administering the the PRP compositions described herein.

[0037] In one embodiment, a composition for cosmetic surgery may comprise an effective amount of the PRP compositions described herein.

[0038] In one embodiment, the PRP compositions described herein may be used in the manufacture of cosmetic surgery products, optionally comprising dermal fillers, for the reduction of a skin defect.

[0039] In one embodiment, a method for reducing a skin defect may comprise administering the PRP compositions described herein.

[0040] In one embodiment, a composition for reduction of a skin defect may comprise an effective amount of the PRP compositions described herein.

[0041] In one embodiment, the PRP compositions described herein may be used in the manufacture of cosmetic surgery products, optionally comprising dermal fillers, for reducing wrinkles, improving rosacea, increase skin thickness, increase skin tome, improve skin texture, and tighten skin.

[0042] In one embodiment, a method for reducing wrinkles, improving rosacea, increase skin thickness, increase skin tome, improve skin texture, and tighten skin may comprise administering the PRP compositions described herein.

[0043] In one embodiment, a composition for reducing wrinkles, improving rosacea, increase skin thickness, increase skin tome, improve skin texture, and tighten skin may comprise an effective amount of the PRP compositions described herein. In another embodiment, the use, method, or composition may be for the reduction of a skin defect, the skin defect may be a dynamic wrinkle, a fine wrinkles or a static wrinkle. In another embodiment, the dynamic wrinkle may be a forehead crease, a brow burrow or an eye line (crow's feet). In another embodiment, the static wrinkle may be a skin fold wrinkle resulting from sagging skin. In another embodiment, the skin defect may be a medical condition selected from the group consisting of an acne scar, optionally a "rolling" scar, a "boxcar" scar or an "ice pick" scar, a surgical scar, trauma scar, a large pore and a soft tissue contour defect. In another embodiment, the wrinkle or scar may be the result of loss of collagen and hyaluronic acid in the skin during the aging process. In another embodiment, the wrinkle or scar may be the result of premature aging, optionally premature aging caused by overexposure to sunlight, overexposure to environmental pollutants, smoking tobacco products, exposure to cigarette smoke, poor nutrition, and skin disorders.

[0044] In one embodiment, the PRP compositions described herein may be used in the manufacture of a medicament for the treatment of a disease.

[0045] In one embodiment, a method of treating a disease comprising administering the PRP compositions described herein.

[0046] In one embodiment, a composition for treating a disease may comprise an effective amount of the PRP compositions described herein.

[0047] In one embodiment, a method for treating a disease may comprise administering the PRP compositions described herein.

[0048] In one embodiment, a method for treating a disease may comprise administering the PRP compositions described herein.

[0049] In one embodiment, a pharmaceutical composition for the treatment of a disease may comprise the PRP compositions described herein. In another embodiment, a pharmaceutical composition for the treatment of a disease may comprise the PRP compositions described herein. In another embodiment, the PRP compositions described herein may be used in the manufacture of a medicament for treating an injury.

[0050] In one embodiment, a method for treating an injury may comprise administering the PRP compositions described herein.

[0051] In one embodiment, a composition for treating an injury may comprise an effective amount of the PRP compositions described herein. In another embodiment, the injury may be a shoulder injury, optionally Rotator Cuff Tendinitis or Tear, Rotator Cuff Impingement Syndrome or Bursitis, Bicipital Tendinitis, labrum tears, arthritis, and instability. In another embodiment, the injury may be a wrist hand injury optionally DeQuervaine's Tenosynovitis, arthritis, other wrist or finger tendinitis, ligament tears, or dysfunction of the fingers. In another embodiment, the injury may be an elbow injury, optionally medial and lateral epicondylitis (tennis & golfers elbow). In another embodiment, the injury may be hip injury, optionally Illiotibial Band Tendinitis (ΓΓΒ Syndrome), Psoas Tendinitis and bursitis, Greater Trochanteric Bursitis, Hip labrum tears, Piriformis Syndrome, Sacroiliac Joint Dysfunction, and arthritis. In another embodiment, the injury may be a knee injury, optionally Patellar Tendinitis, Patellar Femoral Syndrome, chondromalacia patella, partially torn or strained major ligaments of knee

(ACL/LCL/MCL), meniscus tears, arthritis, and patellar instability. In another embodiment, the injury may be an ankle/foot injury, optionally Achilles Tendinitis, Peroneal Tendinitis, arthritis, recurrent ankle sprains, other foot or ankle tendinitis. In another embodiment, the injury may be a neck injury optionally Whiplash injuries, headaches related to the neck, and arthritis. In another embodiment, the injury may be a back injury, optionally facet joint arthritis, rib problems, or pain associated with scoliosis. In another embodiment, the disease or injury may be gum recession, loss of bone, including the jaw, bone fractures, dermal treatment for burns and non-healing wounds, post-laser treatment burns, enterocutaneous fistula (HULPUTC), gingival gum regeneration, hair loss (in both men and women), gum recession, orthopedic problems, osteoarthritis, plantar fascitis, recto-vaginal fistula, rotator cuff injuries, sports medicine injuries, optionally tears and sprains of the ligaments and tendons, tennis elbow, ulcers, and non-healing wounds.

[0052] In one embodiment, the PRP compositions described herein may be used in the manufacture of an medicament for skin repair, skin regeneration, skin protection, preventing hair loss, for promoting the physiological growth of hair, reducing photoaging, reducing skin oxidative stress, regenerating the hair bulb cells and scalp.

[0053] In one embodiment, a method for skin repair, skin regeneration, skin protection, preventing hair loss, for promoting the physiological growth of hair, reducing photoaging, reducing skin oxidative stress, regenerating the hair bulb cells and scalp may comprise administering the PRP compositions described herein.

[0054] In one embodiment, a composition for skin repair, skin regeneration, skin protection, preventing hair loss, for promoting the physiological growth of hair, reducing photoaging, reducing skin oxidative stress, regenerating the hair bulb cells and scalp may comprise an effective amount of the PRP compositions described herein.

[0055] In one embodiment, the PRP compositions described herein may be used in the manufacture of a medicament for the treatment of a Escherichia coli, Staphylococcus aureus, optionally methicillin-resistant Staphylococcus aureus, Candida albicans, or Cryptococcus neoformans infection. [0056] In one embodiment, a method for the treatment of a Escherichia coli, Staphylococcus aureus, optionally methicillin-resistant Staphylococcus aureus, Candida albicans, or

Cryptococcus neoformans infection may comprise administering an effective amount of the PRP compositions described herein.

[0057] In one embodiment, a composition the treatment of a Escherichia coli, Staphylococcus aureus, optionally methicillin-resistant Staphylococcus aureus, Candida albicans, or

Cryptococcus neoformans infection may comprise an effective amount of the PRP compositions described herein.

[0058] In one embodiment, a method for formulating a topical skin cream comprising isolating platelet-rich-plasma from a patient and compounding to form a topical skin cream may comprise autologous platelet-rich plasma. In another embodiment, the PRP composition may be administered topically. In another embodiment, the PRP composition may be administered via microneedles. In another embodiment, the platelet-rich-plasma may be used in a cosmetic surgery application, to promote wound healing, are used in a tissue filler, tissue engineering, or burn treatment. In another embodiment, the use, method, or composition may comprise an anesthetic, optionally anbesol, benzocaine, lidocaine, procaine, or bupivicaine. In another embodiment, the use, method, or composition may result in the release of cytokines by PRP. In another embodiment, the cytokines may be selected from the group consisting of platelet-derived growth factor, transforming growth factor beta, fibroblast growth factor, insulin-like growth factor 1 (IGF1), insulin-like growth factor 2 (IGF2), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), interleukin 8 (IL-8), keratinocyte growth factor (KGF), and connective tissue growth factor. In another embodiment, the platelet-rich-plasma may be autologous. In another embodiment, the use, method, or composition may further comprise administering a platelet activator selected from the group consisting of thrombin, calcium, collagen, epinephrine, adenosine diphosphate, and mixtures thereof. In another embodiment, the thrombin may be autologous to the subject. In another embodiment, the administering a platelet activator may further comprise administering fibrinogen. In another embodiment, the use, method, or composition may further comprise or comprise administering an isolated angiogenic factor selected from the group consisting of angiogenin, angiopoietin-1, del-1 protein, acidic fibroblast growth factor (aFGF or FGF-1), basic fibroblast growth factor (bFGF or FGF- 2), follistatin, granulocyte colony-stimulating factor (G-CSF), hepatocyte growth factor (HGF), interleukin-8 (IL-8), leptin, midkine, placental growth factor, platelet-derived endothelial growth factor (PD-ECGF), platelet-derived growth factor (PDGF), pleiotrophin (PTN), progranulin, proliferin, transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-β), tumor necrosis factor alpha (TNF-a), vascular endothelial growth factor (VEGF), vascular permeability factor (VPF), and combinations thereof. In another embodiment, the use, method, or composition may comprise treatment alone or in combination with tissue fillers. In another embodiment, the PRP may comprise activated platelets that release cytokines, optionally IL-IB, IL-6, TNF-A, chemokines, optionally ENA-78 (CXCL5), IL-8 (CXCL8), MCP-3 (CCL7), MflP- 1A (CCL3), NAP-2 (CXCL7), PF4 (CXCL4), RANTES (CCL5), inflammatory mediators, optionally PGE2, and growth factors, optionally Angiopoitin-1, bFGF, EGF, FGF, HGF, IGF-I, IGF-II, PDAF, PDEGF, PF-4, PDGF AA and BB, TGF-beta 1, 2, and 3, and VEGF.

[0059] In one embodiment, an anti-aging serum, rosacea serum, treatment mask, post-laser treatment mask, and cleansing solution for skin brushes may comprise the PRP compositions described herein. In another embodiment, the PRP may be autologous.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0060] In order that the invention herein described may be fully understood, the following detailed description is set forth.

Definitions

[0061] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this invention belongs.

[0062] As used in the description herein and throughout the claims that follow, the meaning of "a," "an," and "the" includes plural reference unless the context clearly dictates otherwise.

[0063] "About," will be understood by persons of ordinary skill in the art and will vary to some extent based on the context in which it is used.

[0064] "Allogeneic," as used herein, refers broadly to any material derived from a different mammal of the same species from genetically different individuals.

[0065] "Allograft," as used herein, refers broadly to a tissue graft from a donor genetically unrelated to the recipient. [0066] "Allotransplantation," as used herein, refers broadly to the transplantation of cells, tissues, or organs, to a recipient from a (genetically non-identical) donor from the same species. Allotransplants may be referred to an allograft, allogeneic transplant, or homograft in the art.

[0067] "Applicator," as used herein, refers broadly to any device including, but not limited to, a hypodermic syringe, a pipette, for administering the compounds and compositions of the invention.

[0068] "Autograft," as used herein, refers broadly to a tissue transplanted from one site to another on the same patient (e.g., removal of blood, preparation of PRP, and administration to another site on the same patient).

[0069] "Autologous," as used herein, refers broadly to any material derived from the same individual to which it is later to be re-introduced.

[0070] "Baseline concentration," as used herein, refers broadly to the concentration of the specified cell type found in the patient's blood which would be the same as the concentration of that cell type found in a blood sample from that patient without manipulation of the sample by laboratory techniques such as cell sorting, centrifugation or filtration. Where blood samples are obtained from more than one source, baseline concentration means the concentration found in the mixed blood sample from which the PRP is derived without manipulation of the mixed sample by laboratory techniques such as cell sorting, centrifugation or filtration.

[0071] "Central nervous system," as used herein, refers broadly to include brain and/or the spinal cord of a mammal. The term may also include the eye and optic nerve in some instances.

[0072] "Cosmetically or aesthetically effective amount," as used herein, refers broadly to the amount of PRP sufficient to provide a cosmetically or aesthetically beneficial effect to the subject to which the PRP is administered such as skin rejuvenation, enhancement in plumpness or volume or appearance of treated tissue. Also, as used herein, a "cosmetically effective amount" is the amount of PRP which is sufficient to provide a beneficial effect to the subject to which the PRP is administered.

[0073] "Effective amount," as used herein, refers broadly to the amount of PRP sufficient to provide a beneficial effect to the subject to which the PRP is administered.

[0074] "Endogenous," as used herein, refers broadly to any material from or produced inside an organism, cell or system. [0075] "Exogenous," as used herein, refers broadly to any material introduced from or produced outside an organism, cell, or system. In particular exogenous may refer to a material that is not present in the treated adipose tissue.

[0076] "Injury," as used herein, refers broadly to any tissue damage including a wound, trauma, lesion, or any tissue degeneration.

[0077] "Isolated," as used herein, refers broadly to material removed from its original environment in which it naturally occurs, and thus is altered by the hand of man from its natural environment. Isolated material may be, for example, exogenous nucleic acid included in a vector system, exogenous nucleic acid contained within a host cell, or any material which has been removed from its original environment and thus altered by the hand of man (e.g. , "isolated cell"). For example, "isolated" or "purified," as used herein, refers broadly to a protein, cell, DNA, antibody, RNA, or biologically active portion thereof, that is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the biological substance is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. For example, "isolated cell" refers broadly to a cell which has been separated from other components and/or cells which naturally accompany the isolated cell in a tissue or mammal.

[0078] "Isograft," as used herein, refers broadly to a transplanted organ or tissue from a genetically identical donor (i.e. , identical twin).

[0079] "Graft," as used herein, refers broadly to a cell, tissue, or organ that is implanted into an individual, typically to replace, correct or otherwise overcome a defect. A graft may further comprise a scaffold. The tissue or organ may consist of cells that originate from the same individual; this graft is referred to herein by the following interchangeable terms: "autograft", "autologous transplant", "autologous implant" and "autologous graft". A graft comprising cells from a genetically different individual of the same species is referred to herein by the following interchangeable terms: "allograft", "allogeneic transplant", "allogeneic implant" and "allogeneic graft". A graft from an individual to his identical twin is referred to herein as an "isograft", a "syngeneic transplant", a "syngeneic implant" or a "syngeneic graft".

[0080] "Immunophenotype," as used herein, refers broadly to cell is used herein to refer to the phenotype of a cell in terms of the surface protein profile of a cell. [0081] "Mammal," as used herein, refers broadly to any and all warm-blooded vertebrate animals of the class Mammalia, including humans, characterized by a covering of hair on the skin and, in the female, milk-producing mammary glands for nourishing the young. Examples of mammals include but are not limited to alpacas, armadillos, capybaras, cats, camels,

chimpanzees, chinchillas, cattle, dogs, elephants, goats, gorillas, hamsters, horses, humans, lemurs, llamas, mice, non-human primates, pigs, rats, sheep, shrews, squirrels, tapirs, and voles. Mammals include but are not limited to bovine, canine, equine, feline, murine, ovine, porcine, primate, and rodent species. Mammal also includes any and all those listed on the Mammal Species of the World maintained by the National Museum of Natural History, Smithsonian Institution in Washington DC.

[0082] "Platelet-rich plasma (PRP)," as used herein, refers broadly to a concentration of platelets greater than the peripheral blood concentration suspended in a solution of plasma, or other excipient suitable for administration to a human or non-human animal including, but not limited to isotonic sodium chloride solution, physiological saline, normal saline, dextrose 5% in water, dextrose 10% in water, Ringer solution, lactated Ringer solution, Ringer lactate, or Ringer lactate solution. Platelet counts may range from 500,000 to 7,000,000 per milliliter. PRP is formed from the concentration of platelets from whole blood, and may be obtained using autologous, allogenic, or pooled sources of platelets and/or plasma. PRP may be formed from a variety of animal sources, including human sources. In preferred embodiments, PRP according to the invention is buffered to physiological pH (e.g., pH 7.4).

[0083] "Treat," as used herein, refers broadly to reduce the frequency of the disease or disorder, or reducing the frequency with which a symptom of the one or more symptoms disease or disorder is experienced by an animal.

Platelet-Rich-Plasma (PRP)

[0084] Wound healing is a complex and growing science. Many cell types, growth factors and other proteins interact with one another to bring about timely and efficient repair of wounds. Researchers continue to study various growth factors to determine the actual role and mechanism of each growth factor in healing. On vessel injury and exposure of subendothelial tissue to blood (either due toaccident or surgical manipulation),platelets begin to stick to exposed collagen proteins. Once platelets stick to collagen, they release granules containing adenosine

diphosphate, serotonin and thromboxane, all of which contribute to the hemostatic mechanism and the clotting cascade. Additional platelets are drawn to the area and contribute to the formation of a platelet plug. The resultant plug is strengthened by an insoluble protein fiber meshwork known as fibrin that is formed as a result of the clotting cascade.

[0085] This platelet plug and the initiation of the clotting cascade once were thought to be the extent of a platelet' s role in wound healing. Platelets actively extrude several growth factors involved in initiating and sustaining wound repair. The two most important of these growth factors are platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β). PDGF is chemotactic for polymorphonucleocytes, macrophages, fibroblasts and smooth muscle cells. PDGF also stimulates cell replication of important stem cells for fibroblasts and endothelial cells (increasing budding of new capillaries), stimulates production of fibronectin— a cell adhesion molecule used in cellular proliferation and migration during healing, including osteoconduction— and hyaluronic acid and helps bring about wound contraction and

remodeling. TGF-β stimulates fibroblast chemotaxis and the production of collagen and fibronectin by cells, while inhibiting collagen degradation by decreasing proteases and increasing protease inhibitors, all of which favor fibrogenesis.

[0086] One of the causes of ageing skin is inflammation and the PRP via immodulation helps reverse and halt this inflammation and helps make the skin younger and stay younger longer. Also there is decreased vascularity in aged skin and the growth factor in PRP via VGEF help to increase the vascularity of the skin and return the glow to the skin. The tissue growth factors will also stimulate new collagen and elastin growth via the growth factors TGF-β in the PRP and help tighten the skin.

[0087] Each growth factor has the capability to induce a unique response in the enhancement of healing. It has been shown that the topical application of these growth factors to healing sites can accelerate repair and wound maturation. One such study by Pierce and colleagues examined the composition, quantity and rate of extracellular matrix deposition within growth factor-treated rabbit ear excisional wounds. Full-thickness, excisional punch biopsies were performed on a rabbit ear to bare cartilage. Individual growth factors were applied to wounds a single time, were followed and then were compared with control wounds that had no growth factor added to them. Researchers found that PDGF accelerated wound closure primarily through augmenting connective-tissue matrix deposition at the leading edge of new granulation tissue. New collagen accumulation did not occur until later in the healing process. Pierce, et al. (1992) American Journal of Pathology 140(6): 1375-1388. Thus, this study indicated that PDGF accelerates early wound closure primarily via enhanced glycosaminoglycan, hyaluronic acid and fibronectin deposition. TGF-β, on the other hand, stimulated new collagen deposition and maturation into large bundles at the leading edge of the wound, creating a mature fibroblastic wound directly and likely bypassing some of the acute inflammatory phase of wound repair.

[0088] It also has been demonstrated that PDGF and TGF-β specifically stimulated significant new granulation tissue in vivo, thus suggesting their importance in the healing of full-thickness dermal wounds. Each growth factor has the capability to induce a unique response in the enhancement of healing, especially having inductive effects on cells entering the wound.

[0089] Platelet-rich plasma (PRP) is the portion of the plasma fraction of whole blood having a platelet concentration above baseline. PRP also has been referred to as platelet-enriched plasma, platelet-rich concentrate, autologous platelet gel, and platelet releasate. PRP serves as a growth factor agonist and has both mitogenic and chemotactic properties. PRP contains a high level of platelets and a full complement of clotting and growth factors.

[0090] PRP functions as a tissue sealant and drug delivery system, with the platelets initiating wound repair by releasing locally acting growth factors via a-granules degranulation. The secretory proteins contained in the a-granules of platelets include platelet-derived growth factor (PDGF-AA, BB, and AB isomers), transforming growth factor-β (TGF-β), platelet factor 4 (PF4), interleukin-1 (IL-1), platelet-derived angiogenesis factor (PDAF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), platelet-derived endothelial growth factor (PDEGF), epithelial cell growth factor (ECGF), insulin-like growth factor (IGF), osteocalcin (Oc), osteonectin (On), fibrinogen (Ff), vitronectin (Vn), fibronectin (Fn), and thrombospondin-1 (TSP-1). These growth factors aid healing by attracting un-differentiated cells in the newly formed matrix and triggering cell division. PRP may suppress cytokine release and limit inflammation, interacting with macrophages to improve tissue healing and regeneration, promote new capillary growth, and accelerate epithelialization in chronic wounds. See

Wrobleewski, et al. (2010) Operative Techniques in Orthopaedics 20(2): 98-105.

[0091] Platelets in PRP also play a role in host defense mechanism at the wound site by producing signaling proteins that attract macrophages. PRP also may contain a small number of leukocytes that synthesize interleukins as part of a non-specific immune response. PRP has antimicrobial activity against Escherichia coli, Staphylococcus aureus, including methicillin- resistant Staphylococcus aureus, Candida albicans, and Cryptococcus neoformans. Lacci, et al. (2010) Yale Journal of Biology and Medicine 83: 1-9.

[0092] The inventor surprisingly discovered that platelet-rich-plasma (PRP) may be isolated and admixed to form a stable composition that may be stable for at least 30 days and used in cosmetic and therapeutic methods. This was unexpected because platelet-rich-plasma (PRP) has only been isolated and maintained for a brief period of time {e.g., hours) before it must be used. PRP Preparations

[0093] A therapeutic concentration of PRP may be five times the platelet count in normal blood. The platelet-rich-plasma composition may be prepared using any conventional method of isolating platelets from whole blood or platelet-containing blood fractions. These include centrifugal methods, filtration, and affinity columns. Conventional methods of obtaining PRP known in the art may be utilized. See U.S. Patent Nos. 5,585,007 and 5,788,662.

[0094] A wide range of commercially available systems that produce various concentrations of platelets are known in the art. These systems involve centrifugation of whole blood to separate red blood cells from the leukocytes and platelets. The commonly used techniques for PRP preparation include gravitational platelet sequestration, standard cell separating, and autologous plateletpheresis.

[0095] During the centrifugation process, variations in force and duration may produce different levels of platelet concentration. After centrifugation, the red blood cells have been separated from the leukocytes and platelets, the plasma is able to be classified as platelet-poor and platelet- rich portions (Hall, et al. (2009) Journal of the American Academy of Orthopaedic Surgeons 17(10): 602-608). According to Lopez- Vidriero, et al. (2010) Arthroscopy: The Journal of Arthroscopic and Related Surgery 26(2): 269-278, the end product is visible as three layers: red layer (bottom layer that contains erythrocytes), white layer (middle layer that contains leukocytes and inflammatory cytokines), and yellow layer (top layer that contains plasma, platelets, and growth factors). The yellow or top layer is the platelet-rich-plasma (PRP).

[0096] Platelet-rich plasma (PRP) is a concentration of platelets greater than the peripheral blood concentration suspended in a solution of plasma, or other excipient suitable for administration to a human or non-human animal including, but not limited to isotonic sodium chloride solution, physiological saline, phosphate buffered saline, potassium buffered saline, normal saline, dextrose 5% in water, dextrose 10% in water, Ringer solution, lactated Ringer solution, Ringer lactate, or Ringer lactate solution. PRP compositions may be an autologous preparation from whole blood taken from the subject to be treated. Also, the PRP compositions may be prepared from a whole blood sample taken from a single donor source or from whole blood samples taken from multiple donor sources. In general, PRP compositions comprise platelets at a platelet concentration that is higher than the baseline concentration of the platelets in whole blood.

[0097] The pH of the PRP composition may be adjusted using a variety of pH adjusting agents, including physiologically tolerated buffers, but may also include other agents that modify pH including agents that modify lactic acid production by stored platelets. The pH adjustment agent may comprise sodium bicarbonate. Physiological pH ranges from about 7.35-7.45. pH adjusting agents useful in the practice of this invention include but are not limited to bicarbonate buffers (e.g. , sodium bicarbonate), calcium gluconate, choline chloride, dextrose (d-glucose), ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid (HEPES), maleic acid, 4-morpholinepropanesulfonic acid

(MOPS), l,4-piperazinebis(ethanesulfonic acid) (PIPES), sucrose, N-tris(hydroxymethyl)methyl- 2-aminoethanesulfonic acid (TES), tris(hydroxymethyl)aminomethane (TRIS BASE), tris(hydroxymethyl)aminomethane hydrochloride (TRIS-HCl), and urea. The pH adjusting agent may be a bicarbonate buffer, optionally sodium bicarbonate.

[0098] Whole blood can be fractionated into platelet rich plasma (PRP). PRP compositions may generally comprise platelet rich plasma that includes a specific concentration of platelets, red blood cells, and white blood cells. The PRP compositions may be characterized relative to a baseline concentration of the platelets, red blood cells, and/or white blood cells of the whole blood from which the compositions are directly or indirectly derived. The PRP compositions may comprise concentrated platelets and white blood cells which are higher than the baseline levels of the whole blood from which the PRP composition was derived. Formulations of PRP that may comprise an increased concentration of platelets and monocytes and or lymphocytes in comparison to neutrophils (e.g., granulocyte or neutrophil-depleted platelet rich plasma.) The neutrophil-depleted PRP composition may be depleted in neutrophils at a level of 0 to 0.999 of the concentration of whole blood or be completely eliminated of the neutrophils from the composition.

[0099] Further, white blood cells have activity that prevents infiltration by bacteria or harmful microorganisms, have immunological functions or bactericidal functions, and promote tissue repair by protecting damaged tissue. It is known that among white blood cells, monocytes, macrophages also play an important role in tissue repair. Thus, the PRP composition may comprise white blood cells, monocytes, macrophages.

[0100] The PRP may comprise a small volume, optionally 1-3 mL. The PRP may comprise a small volume of the compositions, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mL. The PRP may comprise a small volume of the compositions, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nL.

[0101] The PRP compositions described herein may be a serum, lotion, or cream.

[0102] The PRP compositions described herein is more stable than prior-art PRP preparations. For example, the PRP compositions described herein may be stable for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. The PRP compositions described herein may be stable for at least about 1 , 2, 3, 4, 5, or 6 months.

[0103] The PRP compositions may comprise platelet cells at a concentration higher than the concentration of platelets in whole blood. The concentration of platelets in human whole blood is about 328,000 platelets per microliter. The platelet concentration in the PRP compositions may be between about 500,000-7,500,000 platelets per microliter.

[0104] The PRP compositions may comprise about 500,000-7,000,000 platelets per microliter. The PRP compositions may comprise about 500,000-700,000; 700,000-900,000; 900,000- 1,000,000; 1,000,000-1,250,000; 1,250,000-1,500,000; 1,500,000-2,500,000; 2,500,000- 5,000,000; or 5,000,000-7,000,000 platelets per microliter.

[0105] The platelet-rich-plasma (PRP) compositions described herein may comprise about 2,600,000 platelets per mL. About 1-2 mL of a platelet composition comprising about

2,600,000 platelets per mL may be used to manufacture a composition. A platelet-rich-plasma composition may comprise about 2,600,000-5,200,000 platelets.

[0106] The platelet-rich-plasma (PRP) compositions described herein may have a platelet concentration of about 200-500% greater than the same volume of whole blood, optionally 300- 400% greater than the same volume of whole blood.

% platelet increase over whole blood= platelet count of PRP - platelet count of whole blood/platelet count of whole blood X 100.

Platelet concentration = platelet count of PRP/platelet count of whole blood X 100. [0107] The platelet-rich-plasma (PRP) compositions described herein may have a platelet count of at least about 500,000-7,000,000 platelets per microliter. The platelet-rich-plasma (PRP) compositions described herein may have a platelet count of at least about 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,100,000; 1,250,000; 1,500,000; 1,750,000; 2,000,000; 3,000,000; 4,000,000; 5,000,000; 6,000,000; or 7,000,000 platelets per microliter.

[0108] The PRP compositions may further comprise white blood cells (WBCs) at a higher concentration than white blood cells in whole blood. The concentration of white blood cells in human whole blood is about 4,000-11,000 white blood cells per microliter. The WBC count in a microliter of the PRP composition may be between about 8,000-10,000; about 10,000-15,000; about 15,000-20,000; about 15,000-50,000; about 20,000-30,000; about 30,000-50,000; about 50,000-75,000; or 75,000-100,000 white blood cells per microliter.

[0109] The PRP compositions may further comprise lymphocytes at a higher concentration than lymphocytes in whole blood. The concentration of lymphocytes in human whole blood is about 500-4,800 lymphocytes per microliter. The lymphocyte concentration may be between about 5,000-20,000 lymphocytes per microliter.

[0110] The PRP compositions may further comprise monocytes at a higher concentration than monocytes in whole blood. The concentration of monocytes in human whole blood is about 200- 800 monocytes per microliter. The monocyte concentration is between about 1,000-5,000 per monocytes microliter.

[0111] The PRP compositions may further comprise eosinophils at a higher concentration than eosinophils in whole blood. The concentration of eosinophils in human whole blood is about 100-500 monocytes per microliter. The eosinophil concentration may be between about 200- 1,000 eosinophils per microliter.

[0112] The PRP compositions may further comprise neutrophils at a lower concentration than neutrophils in whole blood. The concentration of neutrophils in human whole blood is about 3,000-12,000 neutrophils per microliter. The neutrophil concentration may be between about 200-1,000 neutrophils per microliter. The PRP composition may include neutrophils at a concentration of 50-70%, 30-50%, 10-30%, 5-10%, 1-5%, 0.5-1%, or 0.1-0.5% of levels of neutrophils found in whole blood. The neutrophils may have been depleted by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. The neutrophils in the platelet rich plasma may be substantially removed. The neutrophils may have been eliminated or non-detectable in the PRP composition. Methods for depleting PRP of neutrophils are known in the art. U.S. Patent No. 7,462,268.

[0113] The PRP compositions may further comprise red blood cells at a lower concentration than red blood cells in whole blood. The concentration of red blood cells in human whole blood is about 4,600,000-5,200,000 red blood cells per microliter. Whole blood drawn from a male patient may have an RBC count of at least 4,300,000 to 4,500,000 and up to 5,900,000 to 6,200,000 per microliter. Whole blood from a female patient may have an RBC count of at least 3,500,000 to 3,800,000 and up to 5,500,000 to 5,800,000 per microliter. The red blood cell concentration of the PRP composition may be between about 4,600-5,200 red blood cells per microliter. The red blood cells may have been eliminated or non-detectable in the PRP composition.

[0114] The PRP compositions may further comprise hemoglobin at a lower concentration than hemoglobin in whole blood. The concentration of hemoglobin in human whole blood is ranges from about 11.9-17.7 g/dL. The hemoglobin of the PRP composition may be between about 5- 10 g/dL or 5 g/dL. The hemoglobin concentration may be about 1 g/dL or less, between about 1- 5 g/dL, about 5-10 g/dL, about 10-15 g/dL, or about 15-20 g/dL.

Activation of Platelets

[0115] Platelet-rich plasma is activated by endogenous or exogenous pathways (i.e., calcium chloride or thrombin). The platelets collected in PRP may be activated by thrombin and calcium chloride to induce the release of these growth factors from alpha granules. PRP can be activated exogenously by thrombin, calcium chloride, or mechanical trauma. Once PRP is activated, a fibrin network begins to form, solidifying the plasma and creating a fibrin clot or membrane. If PRP is activated too strongly, the fibrin network will be a bivalent, unstable network. If it is activated in a more physiologic manner, a tetramolecular stable network forms that enhances enmeshment of cells and growth factors.

[0116] However, exogenous or extra activators need not be administered to a patient. Collagen, a major component of connective tissues, is a strong activator of platelets. Thus, when the platelet- rich-plasma composition is introduced into and/or around connective tissue, platelets in the PRP composition may bind to the collagen and be activated. This reduces or eliminates the need for administering an exogenous activator (e.g., thrombin.) Other strong activators (e.g., calcium ions) can cause severe pain, unintentional clotting, and other undesirable side effects. Thus, no or substantially no exogenous activator may be present or added as part of the platelet-rich plasma composition, or is used in the preparation of the platelet-rich plasma composition. Exogenous activators may still be employed if a physician determines that they are medically necessary or desirable.

[0117] A PRP composition may be activated exogenously with thrombin and/or calcium to produce a gel that can be applied to an area to be treated. The process of exogenous activation, however, results in immediate release of growth factors. The in vivo activation of PRP results in slower growth factor release at the desired site.

[0118] The PRP compositions described herein comprises activated platelets which may release active agents including, but are not limited to, cytokines (e.g., IL-IB, IL-6, TNF-A), chemokines (e.g., ENA-78 (CXCL5), IL-8 (CXCL8), MCP-3 (CCL7), MIP-1A (CCL3), NAP-2 (CXCL7), PF4 (CXCL4), RANTES (CCL5)), inflammatory mediators (e.g., PGE2), and growth factors (e.g., Angiopoitin-1, bFGF, EGF, FGF, HGF, IGF-I, IGF-II, PDAF, PDEGF, PDGF AA and BB, TGF-beta 1 , 2, and 3, and VEGF).

[0119] Compositions may be stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high PRP concentration. The carrier can be a solvent or dispersion medium containing, for example, water, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0120] Other compounds which can be included by admixture are, for example, medically inert ingredients (e.g., solid and liquid diluent), such as lactose, dextrosesaccharose, cellulose, starch, calcium phosphate, olive oil, ethyl oleate, water, or vegetable oil; lubricating agents such as silica, talc, stearic acid, magnesium or calcium stearate and/or polyethylene glycols; gelling agents such as colloidal clays; thickening agents such as gum tragacanth or sodium alginate; and other therapeutically acceptable accessory ingredients, such as humectants, preservatives, buffers and antioxidants, which are known additives for such formulations.

[0121] Similarly, compositions for liquid preparations include solutions, emulsions, dispersions, suspensions, syrups, and elixirs, with suitable carriers and additives including but not limited to water, oils, glycols, and suspending agents. Typical preparations for parenteral administration comprise the active ingredient with a carrier such as sterile water or parenterally acceptable oil including but not limited to polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil, with other additives for aiding solubility or preservation may also be included. For dispersions and suspensions, appropriate carriers and additives include aqueous gums, celluloses, silicates, or oils.

[0122] The platelet-rich plasma composition may be obtained from the patient and thus, may be autologous.

[0123] When the composition is for cosmetic use: the platelet-rich plasma concentration is from 0.1 to 5.0% (w/w) of the total weight of the composition. When the composition is for pharmaceutical use: the platelet-rich plasma concentration is from5.1 to 50.0% (w/w) of the total weight of the composition. The emulsion structure was studied and optimized to obtain a functional cream, which is not too oily and permits the maximum vitality to the platelets. It has characteristics of perfect skin compatibility and skin similarity with structures available in the skin tissue, as well as in the body. This cream also moisturizes, replenishes and stabilizes the lipid content. This was achieved by adding natural emolients, emulsifiers, surfactants and humectant sorbitol.

[0124] The platelet-rich-plasma compositions described herein may be used to compound a topical skin cream comprising a patient's own growth factors derived from platelet rich plasma from the same patient (autologous). The platelet-rich-plasma compositions described herein, optionally a topical skin cream, may be patient specific and compounded by the physician at the time of the patient visit.

[0125] The platelet-rich-plasma compositions described herein may be used to formulate an antiaging serum, rosacea serum, treatment mask, post-laser treatment mask, and cleansing solution for skin brushes. These compositions may be blended by doctor and custom for the patient {e.g., comprising autologous PRP). The base creams, lotions and serum may be loaded into a syringe and then attached to the syringe with the PRP with a plastic transfer device (»>) and the PRP and base cream, lotion, or serum may be mixed by moving the contents of the two syringes back and forth. The final product may be delivered to the patient in the syringe or the final product can be placed into any type of container and then dispensed to the patient.

Cytokines (Growth Factors)

[0126] The PRP compositions described herein may comprise increased concentrations of growth factors and other cytokines. The PRP compositions may include increased concentrations of at least one of platelet-derived growth factor (PDFG), transforming growth factor beta (TGF- β), fibroblast growth factor (FGF), insulin-like growth factor (IGF-1), insulin-like growth factor 2 (IGF-2), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), interleukin-8 (IL-8), keratinocyte growth factor (KGF), and connective tissue growth factor. Secondary Agents

[0127] The methods disclosed herein may further comprise mixing into the platelet-rich plasma composition at least one of the ingredients selected from thrombin, epinephrine, collagen, calcium salts, and pH adjusting agents. Also useful are materials to promote degranulation or preserve platelets, additional growth factors or growth factor inhibitors, small molecule pharmaceuticals {e.g., NSAIDS), steroids, and anti-infective agents. The platelet-rich plasma composition may be substantially free from exogenous activators.

[0128] The platelet-rich plasma composition may comprise fibrinogen and other blood components, e.g., white blood cells. Platelets fulfill an important role when adhering to damaged subendothelial tissue and coagulating to form thrombi and arrest hemorrhage, or when storing and releasing substances that induce migration and differentiation of other cells. Fibrin is generated when thrombin acts on fibrinogen in blood plasma, and is therefore a substance that is involved in the final stage of blood coagulation. It is also important as a scaffold for infiltration and cytodifferentiation by a cell to conduct tissue repair.

[0129] The PRP compositions described herein may be applied alone or in combination with other cells, tissue, tissue fragments, demineralized bone, growth factors (e.g., insulin or drugs, e.g., members of the thiaglitazone family), biologically active compounds, biologically inert compounds, resorbable plastic scaffolds, or other additive intended to enhance the delivery, efficacy, tolerability, or function of the population.

[0130] Immunosuppressive agents may be administered to the patient receiving the PRP composition to reduce, and preferably prevent, rejection of the transplant. The

immunosuppressive agents may be administered before, simultaneously, or after the

administration of the PRP composition. Examples of immunosuppressive agents suitable with the methods disclosed herein include agents that inhibit T-cell/B-cell costimulation pathways, such as agents that interfere with the coupling of T-cells and B-cells via the CTLA4 and B7 pathways. U.S. Patent Application Publication No. 2002/0182211. Other examples include but are not limited to cyclosporin, myophenylate mofetil, rapamicin, and anti-thymocyte globulin. [0131] Methods of administering the composition including platelet-rich plasma can further comprise administering thrombin, fibrinogen, and calcium to the subject. The fibrinogen or calcium may be combined with the thrombin or these components may be applied separately. In some cases, applying the tissue sealant comprises co-administering a first solution comprising fibrinogen and the platelet-rich plasma composition, and a second solution comprising thrombin and calcium. The first solution and second solution are kept separate until administered so that the thrombin does not activate the fibrinogen to form a fibrin matrix until after the solutions are mixed and applied at the treatment site. The solutions may be mixed just before application to the treatment site or may be mixed at the treatment site. For example, a dual barreled syringe may be used to simultaneously administer the two solutions. The thrombin, fibrinogen, and calcium can also be combined together to initiate the clotting cascade at a time prior to application to the site on the subject. As the clotting cascade progresses, a gel-like material starts to form, which can then be applied to the site for use as a tissue sealant with the platelet-rich plasma composition. Where the blood component includes a chelating agent, such as citrate, the composition comprising platelet-rich plasma can include excess calcium to overwhelm the chelating agent and provide free calcium ions for the blood-clotting cascade.

Cryopreserved Preparations of platelet-rich-plasma (PRP) compositions

[0132] The platelet-rich-plasma (PRP) compositions may be frozen for storage. The platelet- rich-plasma (PRP) compositions may be stored by any appropriate method known in the art (e.g., cryogenically frozen) and may be frozen at any temperature appropriate for storage of the cells. For example, the cells may be frozen at about -20°C, -80°C, -120°C, -130°C, -135°C, -140°C, -150°C, -160°C, -170°C, -180°C, -190°C, -196°C, at any other temperature appropriate for storage of cells. Cryogenically frozen platelet-rich-plasma (PRP) compositions may be stored in appropriate containers and prepared for storage to reduce risk of cell damage and maximize the likelihood that the cells will survive thawing. The platelet-rich-plasma (PRP) compositions may also be maintained at room temperature, or refrigerated at, for example, about 4°C.

[0133] Cryopreserved PRP preparations described herein may comprise at least about 50,000- 100,000 platelets per microliter. The cryopreserved platelet-rich-plasma (PRP) composition may also comprise at least about 500,000-700,000; 700,000-900,000; 900,000-1,000,000;

1,000,000-1,250,000; 1 ,250,000-1 ,500,000; 1,500,000-2,500,000; 2,500,000-5,000,000; or 5,000,000-7,000,000 platelets per microliter. [0134] The cryopreserved PRP preparations described herein may comprise at least about 500,000-1,000,000 platelets per microliter. The cryopreserved PRP preparations may also comprise at least about 200,000-500,000 platelets per microliter. Also, the cryopreserved PRP preparations may comprise at least about 500,000, 600,000, 750,000, 800,000, or 1,000,000 platelets per microliter. The cryopreserved PRP preparations may comprise at least about lxlO⁶, 2xl0⁶, 3xl0⁶, 4xl0⁶, 5xl0⁶, 6xl0⁶, 7xl0⁶, 8xl0⁶, 9xl0⁶, lxlO⁷, 2xl0⁷, 3xl0⁷, 4xl0⁷, 5xl0⁷, 6xl0⁷, 7xl0⁷, 8xl0⁷, 9xl0⁷, lxlO⁸, 2xl0⁸, 3xl0⁸, 4xl0⁸, 5xl0⁸, 6xl0⁸, 7xl0⁸, 8xl0⁸, 9xl0⁸, lxlO⁹, 2xl0⁹, 3xl0⁹, 4xl0⁹, 5xl0⁹, 6xl0⁹, 7xl0⁹, 8xl0⁹, 9xl0⁹, lxlO¹⁰, 2xl0¹⁰, 3xl0¹⁰, 4xl0¹⁰, 5xl0¹⁰, 6xl0¹⁰, 7xl0¹⁰, 8xl0¹⁰, or 9xl0¹⁰ platelets per microliter. The platelets of the

cryopreserved PRP preparations may be mammalian platelets, including human platelets.

[0135] The platelets of the invention may be recovered from storage following cryopreservation. The platelets recovered from cryopreservation also maintain their viability. For example, at least about 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the platelets may retain viability following cryopreservation. Further, the platelets of the invention may be cryopreserved and maintain their viability after being stored for at least about 1, 2, 3, 4, 5, 6, or 7 days. The platelets of the invention may also be cryopreserved and maintain their viability after being stored for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. The platelets of the invention may be cryopreserved and maintain their viability after being stored for at least about 1, 2, 3, 4, 5, 6, or 7 years. The cryopreservation preparation comprising platelets may be substantially free of DMSO.

[0136] The invention also provides a method of cryopreserving PRP comprising (a) isolating PRP from whole blood and (b) resuspending said PRP in 10% DMSO/90% FBS solution, optionally at least about 10⁶ platelets per μL.

Methods of Making PRP

[0137] The PRP composition may comprise a PRP derived from a human or animal source of whole blood. The PRP may be prepared in methods known in the art. See, e.g., European Patent Application EP1547606; U.S. Patent Application Publication No. 2005/0170327; U.S. Patent No. 8,105,495. The PRP may be prepared from an autologous source, an allogeneic source, a single source, or a pooled source of platelets and/or plasma. To derive the PRP, whole blood may be collected, for example, using a blood collection syringe. The amount of blood collected may depend on a number of factors, including, for example, the amount of PRP desired, the health of the patient, the severity or location of the tissue damage, the availability of prepared PRP, or any suitable combination of factors. Any suitable amount of blood may be collected and processed to isolate PRP.

[0138] The PRP may be prepared from whole blood using a centrifuge. The whole blood may or may not be cooled after being collected. Isolation of platelets from whole blood depends upon the density difference between platelets and red blood cells. The platelets and white blood cells are concentrated in the layer (i.e., the "buffy coat") between the platelet depleted plasma (top layer) and red blood cells (bottom layer). For example, a bottom buoy and a top buoy may be used to trap the platelet-rich layer between the upper and lower phase.

[0139] In one method of isolating PRP, Benton Dickerson PST plasma separation tubes may be used, the patient's blood is drawn into the PST vacutainer tubes and then centrifuged in swing out rotor centrifuge at about 1100-1300 rpm for about 6-10 minutes and then draw out PRP with needle (16 g) into 10 cc syringe. In another method, blood may be drawn from a patient using PST vacutainer. The PST vacutainer tubes are inverted 8-10 times and then centrifuge in horizontal swing arm centrifuge for 6-12 minutes and then the PRP is withdrawn using a 16g needle.

[0140] In a two-step process, whole blood from the patient is first centrifuged to separate the plasma from packed red blood cells and then further centrifuged to separate PRP from platelet-poor plasma.

[0141] For example, about 1-150 cc (mL) of blood may be drawn (e.g., about 27-1 10 cc or about 27-55 cc of blood may be drawn). The blood is collected from a patient. PRP made from a patient's own blood may significantly reduce the risk of adverse reactions or infection (e.g., autologous PRP). Autologous Platelet-Rich Plasma (PRP) may be produced from blood, by removing red blood cells through apheresis based centrifugation, yielding a high concentration of platelets, immune cells, and endogenous growth factors. Thus, the PRP composition may be autologous.

[0142] About 55 cc of blood may be withdrawn into a 60 cc syringe (or another suitable syringe) that contains about 5 cc of an anticoagulant, such as a citrate dextrose solution. The syringe may be attached to an apheresis needle, and primed with the anticoagulant. Blood (about 27-55 cc) may be drawn from the patient using standard aseptic practice. A local anesthetic including but not limited to anbesol, benzocaine, lidocaine, procaine, bupivicaine, or any appropriate anesthetic known in the art may be used to anesthetize the insertion area.

[0143] The blood may then be centrifuged using a gravitational platelet system, such as the Cell Factor Technologies GPS System® centrifuge. The blood- filled syringe containing between about 20-150 mL of blood (e.g., about 55 mL of blood) about 5 mL citrate dextrose may be slowly transferred to a disposable separation tube which may be loaded into a port on the GPS centrifuge. The sample may be capped and placed into the centrifuge. The centrifuge may be counterbalanced with about 60 mL sterile saline, placed into the opposite side of the centrifuge. Alternatively, if two samples are prepared, two GPS disposable tubes may be filled with equal amounts of blood and citrate dextrose. The samples may then be spun to separate platelets from blood and plasma. The samples may be spun at about 2000-5000 rpm for about 5-30 minutes. For example, centrifugation may be performed at 3200 rpm for extraction from a side of the separation tube and then isolated platelets may be suspended in about 3-5 mL of plasma by agitation. The PRP may then be extracted from a side port using, for example, a 10 mL syringe. From about 55 mL of blood, about 5 mL of PRP may be obtained.

[0144] This platelet-rich layer may then be withdrawn using a syringe or pipette. Generally, at least 60% or at least 80%) of the available platelets within the blood sample can be captured. These platelets may be resuspended in a volume that may be about 3-20% or about 5-10% of the sample volume.

[0145] This concentrate is then activated with the addition of thrombin or calcium, resulting in a gelatinous platelet gel. An effective amount of PRP in a composition may comprise at least one million platelets per microliter. Lesser concentrations cannot be relied on to enhance wound healing, and greater concentrations have not been shown to increase wound healing. Using known methods, the PRP must be isolated from the whole blood within 6 hours of drawing the blood and PRP must be used within 8-12 hours of isolation. Lacci, et al. (2010) Yale Journal of Biology and Medicine 82: 1-9. In contrast, the PRP compositions described herein have a longer shelf-life of at least about 1-30 days, or up to 3 months.

[0146] The blood material used to prepare platelet-rich plasma can be mixed with an

anticoagulant prior to preparation of the platelet-rich plasma or at one or more points during preparation. Suitable anticoagulants include those known in the art, such as heparin, citrate phosphate dextrose (CPD), ethylenediaminetetraacetic acid (EDTA), acid citrate dextrose solution (ACD), and mixtures thereof. The anticoagulant may include a chelating agent (e.g., citrate, EDTA) to complex free calcium ions. For example, the anticoagulant may be placed in a syringe used for drawing blood from the subject, or may be mixed with the blood after it is drawn.

[0147] Platelet-rich plasma may also be isolated using any of a variety of devices described in the art, including those in U.S. Patent Nos. 6,398,972; 6,649,072; 6,790,371 ; 7,011 ,852;

7,179,391 ; 7,374,678; 7,223,346; and 7,708,152.

Methods of Making PRP Compositions

[0148] The PRP may be admixed with a solution, buffer, diluent, solvent, or stablizier to form a composition. Examples of the additional agents include, but are not limited to, thrombin, epinephrine, collagen, calcium salts, pH adjusting agents, materials to promote degranulation or preserve platelets, additional growth factors or growth factor inhibitors, NSAIDS, steroids, anti- infective agents, and mixtures and combinations of the foregoing.

[0149] The PRP may be autologous, i.e. , derived from the same donor. The donor may be the same patient who is to be treated with the PRP composition (e.g., autograft). The harvested cells can be stored in cryostorage and be used for the patient at a later date. For example, the PRP may be stored and be readily available to treat patients for a multitude of injuries (e.g., orthopedic, post myocardiacal infarction). The PRP may be stored at +4°C, -20°C, or -70°C. The platelet-rich-plasma (PRP) compositions described herein may be used in autograft methods. For example, a patient may have whole blood removed, the PRP may be isolated from the blood, using methods known in the art, admixed to form a PRP composition as described herein and then administered to the same patient.

[0150] The PRP may be buffered using an alkaline buffering agent to a physiological pH. The buffering agent may be a biocompatible buffer such as HEPES, TRIS, monobasic phosphate, monobasic bicarbonate, or any suitable combination thereof that may be capable of adjusting the PRP to physiological pH between about 6.5-8.0. The physiological pH is from about 7.3-7.5 (e.g., about 7.4.) For example, the buffering agent may be an 8.4% sodium bicarbonate solution. For each mL of PRP isolated from whole blood, 0.05 mL of 8.4% sodium bicarbonate may be added. The syringe may be gently shaken to mix the PRP and bicarbonate.

[0151] The PRP composition may be delivered as a liquid, a solid, a semi-solid (e.g., a gel), or some combination thereof. When the PRP is delivered as a liquid, it may comprise a solution, an emulsion, or a suspension. A PRP semi-solid or gel may be prepared by adding a clotting agent (e.g., thrombin, epinephrine, calcium salts) to the PRP. The gel may be more viscous than a solution and therefore may better preserve its position once it is delivered to target tissue. The PRP composition is delivered without a clotting agent.

[0152] It may be desirable to deliver the PRP composition as a liquid and have it gel or harden in situ. For example, the PRP compositions may include, for example, collagen, cyanoacrylate, adhesives that cure upon injection into tissue, liquids that solidify or gel after injection into tissue, suture material, agar, gelatin, light-activated dental composite, other dental composites, silk-elastin polymers, Matrigel® gelatinous protein mixture (BD Biosciences), hydrogels and/or other suitable biopolymers. Alternatively, the above mentioned agents need not form part of the PRP composition. For example, the above mentioned agents may be delivered to the target tissue before or after the PRP has been delivered to the target tissue to cause the PRP to gel. The PRP composition may harden or gel in response to one or more environmental or chemical factors such as temperature, H, and proteins.

[0153] The compositions comprising platelet-rich-plasma (PRP) may be used in the manufacture of creams, lotions, and solutions.

[0154] The platelet-rich-plasma (PRP) compositions described herein may comprise about 2,600,000 platelets per mL. About 1-2 mL of a platelet composition comprising about

2,600,000 platelets per mL may be used to manufacture a composition. A platelet-rich-plasma composition may comprise about 2,600,000-5,200,000 platelets. Provided herein are exemplary compositions that may further comprise about 2,600,000-5,200,000 platelets. These

compositions are stable for at least about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, optionally up to about 3 months.

Composition #1: "ANTI-AGING CLEANSER"

INGREDIENT CORRECT INCI INCI DESIGNATION / MISSING NAMES IN % BY

(TRADE NAME) NAME RED WEIGHT

WATER WATER (AQUA) 52.64

TETRASODIUM EDTA TETRASODIUM EDTA 0.10

CARBOWAX PEG 0.15

C ARBOW AX PEG 300 PEG-6 0.15

STAND APOL ES 1 SODIUM LAURETH SULFATE 4.75

_{ΛΠπ νλΙ} . , PEG 150 DECYL ALCOHOL/SMDI

ACUL™ 44 COPOLYMER ⁱ

TAURANOL WS SODIUM METHYL COCOYL TAURATE 2.00 CONC.

GLYCERIN 96% GLYCERIN 1.00 VELVETEX BK 35 COCAMIDOPROPYL BETAINE 5.00 STEARIC ACID XXX STEARIC ACID 7.00 MYRISTIC ACID MYRISTIC ACID 9.00 COCONUT FATTY

COCONUT ACID 4.00 ACID

TOCOPHERYL ACETATE CAPRYLIC/CAPRIC TRIGLYCERIDES

MACADAMIA TERNIFOLIA SEED OIL RETINYL PALMITATE ZEA MAYS (CORN)

OIL UBIQUINONE PALMITIC ACID

SCAVENOL J BETA CAROTENE 0.15 SHEA BUTTER BUTYROSPERMUM PARKII (SHEA BUTTER) 1.50 CERASYNT Q GLYCERYL STEARATE SE 0.75 POTASSIUM

POTASSIUM HYDROXIDE 6.36 HYDROXIDE

GLUCOSAMINE HCI ALGAE EXTRACT

UGL COMPLEX YEAST EXTRACT UREA 3.00

METHYLCHLOROISOTHIAZOLINONE

KATYHON CG METHYLISOTHIAZOLINONE 0.05

FUCOGEL 1000 BP C BIOSACCHARIDE GUM- 1 1.00 BOUQUET # 29840 FRAGRANCE (PARFUM) 0.20

MANNITOL CELLULOSE HYDROXYPROPYL METHYLCELLULOSE CHROMIUM HYDROXIDE GREEN (CI 77289)

TOCOPHERYL ACETATE ASCORBYL

UNISPHERES NT 2304 PALMITATE 0.20 TOTAL 100.00

Composition #2 "EYE REPAIR LOTION"

INGREDIENT

(TRADE NAME) INCI DESIGNATION

WATER WATER (AQUA)

DISODIUM EDTA DISODIUM EDTA GLYCERIN 96% GLYCERIN

MP DIOL METHYLPROPANEDIOL ARLATONE 2121 SORB IT AN STEARATE SUCROSE COCOATE

PHENOXYETHANOL METHYLPARABEN

ETHYLPARABEN BUTYLPARABERN

PHENONIP PROPYLPARABEN ISOBUTYLPARABEN CRODACOL CS50 CETEARYL ALCOHOL

LANETTE 22 BEHENYL ALCOHOL

JEECHEM OP ETHYLHEXYL PALMITATE CERAPHYL 424 MYRISTYL MYRISTATE MYRISTYL LAURATE

C12-15 ALKYL BENZOATE TRD3EHENIN

CERAMIDE 2 PEG- 10 RAPESEED STEROL

DERMAXYL PALMITOYL OLIGOPEPTIDE JEESILC IDD ISODODECANE DIMETHICONE CROSSPOLYMER-3

POLYGLYCERYL 3 METHYLGLUCOSE

TEGOCARE 450

DISTEARATE

PURESYN 2 HYDROGENATED POLYDECENE

DC 200 100 CS DIMETHICONE SANTALUM ALBUM (SANDALWOOD) EXTRACT

PHELLODENDRON AMURENSE BARK EXTRACT

BOIS II HORDEUM DISTICHON (BARLEY) EXTRACT 1.000 LECINOL S10 HYDROGENATED LECITHIN 0.500

HYDROXYETHYL ACRYLATE/SODIUM

ACRYLOYLDIMETHYL TAURATE COPOLYMER

SIMULGEL NS SQUALANE POLYSORBATE 60 2.500 WATER WATER (AQUA) 24.000

HYDROLYZED HIBISCUS ESCULENTUS EXTRACT

MYOXINOL LS 9736 3.000

DEXTRIN DERMOCHLORELLA D WATER CHLORELLA VULGARIS EXTRACT 3.000

WATER PHOSPHOLIPIDS TOCOPHERYL ACETATE

BROOKOSOME ACE RETINYL PALMITATE ASCORBYL PALMITATE 0.500

WATER GLYCERIN HESPERIDIN METHYL CHALCONE STERETH-20 DIPEPTIDE-2

EYELISS PALMITYOL TETRAPEPTIDE-3 1.500

BOUQUET #29840B FRAGRANCE (PARFUM) 0.150

TOTAL 100.000

Composition #3 "Growth Stimulating Factor"

INGREDIENT % BY (TRADE NAME) FUNCTION INCI DESIGNATION WEIGHT

WATER WATER (AQUA) 68.972

DISODIUM EDTA DISODIUM EDTA 0.100 KELTROL T XANTHAN GUM 0.400 GIVOBIO GPNa SODIUM GLYCEROPHOSPHATE 0.200 HEXYLENE

HEXYLENE GLYCOL 1.000 GLYCOL MP DIOL METHYLPROPANEDIOL 1.000

PHENOXYETHANOL

CHLORPHENESIN

METHYLPARABEN

GERMAZIDE PMP PROPYLPARABEN 1.000 IMMUCELL GLYCOPROTEINS 3.000

GLYCERYL

POLYMETHACRYLATE PROPYLENE GLYCOL

BIOPEPTIDE CL PALMITOYL OLIGOPEPTIDE 2.000

GLUCOSAMINE HCI ALGAE EXTRACT YEAST EXTRACT

UGL COMPLEX UREA 3.000 SD ALCOHOL 40 2

SD ALCOHOL 40-2

190 PROOF 12.500 MENTHOL MENTHOL 0.200 ZENICONE DMC 1 PEG-8 DIMETHICONE 2.000

PEG-40 HYDROGENATED

CREMOPHOR RH40 2.500

CASTOR OIL

BOUQUET # 29840B FRAGRANCE 0.400

TOCOPHERYL ACETATE CAPRYLIC/CAPRIC

TRIGLYCERIDES MACADAMIA TERNIFOLIA

SCAVENOL J SEED OIL RETINYL 0.500 PALMITATE ZEA MAYS

(CORN) OIL UBIQUINONE

PALMITIC ACID BETA

CAROTENE

NIACINAMIDE NIACINAMIDE 0.100 CITRIC ACID (20% SOL'N) CITRIC ACID 0.000 NAOH 20% SOLN SODIUM HYDROXIDE 0.000

D&C GREEN #5 0.1 % SOLN GREEN 5 CI 61570 0.510 FD&C YELLOW #5 1.0% SOLN YELLOW 5 CI 19140 0.274 FD&C BLUE #1 0.1% SOLN BLUE 1 CI 42090 0.344

TOTAL 100.000

Composition # 4 "INTENSIVE WRINKLE REPAIR LOTION"

INGREDIENT % BY (TRADE NAME) INCI NAME INCI DESIGNATION WEIGHT

WATER WATER (AQUA) 54.75

DISODIUM EDTA DISODIUM EDTA 0.10 GLYCERIN 96% GLYCERIN 2.00 KELTROL T XANTHAN GUM 0.20

PHENOXYETHANOL

METHYLPARABEN

ETHYLPARABEN

BUTYLPARABERN

PROPYLPARABEN

PHENONIP ISOBUTYLPARABEN 1.00

BEHENYL ALCOHOL GLYCERYL BEHENYL ALCOHOL STEARATE GLYCERYL GLYCERYL STEARATE CITRATE STEARATE CITRATE SODIUM SODIUM DICOCOYLETHYLENEDIAMINE DICOCOYLETHYLENEDIAMINE

CERALUTION H PEG- 15 SLFATE PEG- 15 SULFATE 5.00 DC 200 200 CS DIMETHICONE 0.50 PURESYN 2 HYDROGENATED POLYDECENE 2.00

ISOHEXADECANE DIMETHICONE CROSSPOLYMER

JEESILC IHD 3 2.50 CRODACOL C70 CETYL ALCOHOL 1.00 CERASYNT SD GLYCERYL STEARATE 1.00 LIPOWAX D CETEARYL ALCOHOL

4.00 CETEARETH-20

LIPONATE GC CAPRYLIC/CAPRIC

1.50 TRIGLYCERIDE

ZENIGLOSS CASTOR ISOSTEARATE

1.00 SUCCINATE

CERAPHYL 424 MYRISTYL MYRISTATE

0.50 MYRISTYL LAURATE

SYMDIOL 68 1,2-HEXANDIOL CAPRYLYL

0.50 GLYCOL

JEECHEM OP ETHYLHEXYL PALMITATE 3.00 WATER WATER (AQUA) 12.00

HYDROLYZED HIBISCUS

MYOXINOL LS ESCULENTUS EXTRACT

9736 DEXTRIN 3.00

SEPIGEL 305 POLYACRYLAMIDE CI 3- 14 2.00 INGREDIENT % BY (TRADE NAME) INCI NAME INCI DESIGNATION WEIGHT

ISOPARAFFIN LAURETH-7

WATER PHOSPHOLIPIDS TOCOPHERYL ACETATE

BROOKOSOME RETINYL PALMITATE ACE ASCORBYL PALMITATE 0.50

EDELWEISS GC LEONTOPODIUM ALPINUM

0.25 EXTRACT

HYDROLYZED RICE BRAN PROTEIN SOY PEPTIDES

REGU AGE OXIDO REDUCTASES 1.25 ACTIPHYTE JAP. PROPYLENE GLYCOL WATER

GREEN TEA ACT. CAMELLIA OLEIFERA LEAF

ORG #300 230-11 EXTRACT 0.20 BOUQUET #

FRAGRANCE (PARFUM) 0.25 29840B

TOTAL 100.00

Composition #5 "BIO-NUTRITIVE LUXURY CREAM"

INGREDIENT % BY (TRADE NAME) INCI DESIGNATION INCI NAME WEIGHT

WATER WATER (AQUA) 55.58

DISODIUM EDTA DISODIUM EDTA 0.10 GLYCERIN 96% GLYCERIN 2.00 JEECHEM BUGL BUTYLENE GLYCOL 2.00 LIPONIC EG 1 GLYCERETH-26 1.50

PHENOXYETHANOL

METHYLPARABEN

ETHYLPARABEN

BUTYLPARABEN

PROPYLPARABEN

PHENONIP ISOBUTYLPARABEN 1.00 CARBOPOL ULTREZ ACRYLATES/C 10-30 ALKYL

21 ACRYLATE CROSSPOLYMER 0.30

KELTROL T XANTHAN GUM 0.20

BEHENYL ALCOHOL

GLYCERYL STEARATE

CITRATE SODIUM

DICOCOYLETHYLENEDIAMINE

CERALUTION H PEG- 15 SULFATE 4.00 BRIJ 721 STEARETH-21 0.80 BRIJ 72 STEARETH-2 0.25 JEECHEM DO DECYL OLEATE 3.00 JEECHEM OP ETHYLHEXYL PALMITATE 2.00

CYCLOPENTASILO XANE

POLYSILICONE-1 1

CYCLOPENTASILOXANE CYCLOMETHICON

GRANSIL GCM 5 POLYSILICONE-11 E 1.50 CERAPHYL 424 MYRISTYL MYRISTATE 0.50 INGREDIENT % BY (TRADE NAME) INCI DESIGNATION INCI NAME WEIGHT

MYRISTYL LAURATE CAPRYLIC/CAPRIC

LIPONATE GC TRIGLYCERIDE 3.00 DC 200 100 CS DIMETHICONE 2.00

HYDROGENATED PURESYN 2

POLYDECENE 2.00 BHT BHT 0.05

C12-15 ALKYL BENZOATE

TRIBEHENIN CERAMIDE 2

PEG- 10 RAPESEED STEROL

DERMAXYL PALMITOYL OLIGOPEPTIDE 3.00

PPG-3 BENZYL ETHER

CRODAMOL STS

MYRISTATE 3.00

CASTOR ISOSTEARATE

ZENIGLOSS SUCCINATE 0.25

BUTYROSPERMUM PARKII

SHEA BUTTER

(SHEA BUTTER) 0.50

FLAMENCO SUPERPEARL 120C MICA TITANIUM DIOXIDE 1.00 WATER WATER (AQUA) 1.50

POLYMETHYL

GANZPEARL GM0600

METHACRYLATE 0.50

NaOH

SODIUM HYDROXIDE 0.64 GLYCERIN WATER SODIUM POLYACRYLATE PVM/MA

ZILGEL OIL COPOLYMER 0.50

BUTYLENE GLYCOL WATER ETHOXYDIGLYCOL MUSA SAPIENTUM (BANANA)

FLOWER EXTRACT CENTELLA

MATURINE ASIATICA EXTRACT 5.00

WATER ZEA MAYS (CORN)

PHYTOVITYL C KERNEL EXTRACT 1.00

BUTYLENE GLYCOL

ACTIPHYTE OF CIMICIFUGA RACEMOSA ROOT BLACK COHOSH BG EXTRACT 0.10

BUTYLENE GLYCOL

ACTIPHYTE OF DIOSCOREA VILLOSA (WILD

WILD YAMS YAM) ROOT EXTRACT 0.10

ACTIPHYTE JAP. PROPYLENE GLYCOL WATER

GREEN TEA ACT. CAMELLIA OLEIFERA LEAF

ORG #300 230-11 EXTRACT 0.10

BUTYLENE GLYCOL WATER CHAMOMILLA RECUTITA

ACTIPHYTE OF (MATRICARIA) FLOWER

CHAMOMILE BG50P EXTRACT 0.10 GLUADIN SOY HYDROLYZED SOY PROTEIN 0.10 WATER FRESH 428-

0.15 039 FRAGRANCE

COSMEDIA SP SODIUM POLYACRYLATE 0.25 INGREDIENT % BY (TRADE NAME) INCI DESIGNATION INCI NAME WEIGHT

0.1%

D&C RED #33 SOL 0.20

N RED 33 RED 33 (CI 17200)

0.1 %

FD&C YELLOW #5 SOL YELLOW 5 (CI 0.23

N YELLOW 5 19140)

100.00

TOTAL

Composition #6 "MIRACLE SERUM"

TRADE NAME INCI NAME % BY

WEIGHT

Dow Corning 1401 CYCLOMETHICONE DIMETHICONOL 75.00

Dow Corning 345 CYCLOMETHICONE 21.30

CYCLOMETHICONE DIMETHICONOL DIMETHICONE OCTYL COCOATE PHENYLTRIMETHICONE LECITHIN GLYCOLIPIDS PHENOXYETHANOL METHYLPARABEN ETHYLPARABEN PROPYLPARABEN BUTYLPARABEN

Hydrophobic Sphingolipid Complex ISOBUTYLPARABEN 2.00

Vitamin E Acetate TOCOPHERYL ACETATE 0.50

Fitoderm SQUALANCE 0.20

Borage Oil BORAGO OFFICINALIS SEED OIL 1.00

100.0

TOTAL

[0155] The exemplary compositions #1-6 may further comprise about 2,600,000-5,200,000 platelets, optionally in about 1-2 mL volume. These compositions are stable for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, optionally up to about 3 months. The PRP may be autologous. The compositions #1-6 may be admixed using autologous PRP specific for the patient.

Cosmetic & Therapeutic Methods of Use

[0156] The platelet-rich-plasma (PRP) compositions described herein may be used in numerous applications including use in reconstructive and aesthetic plastic surgery, and therapies. The PRP compositions described herein may be used in therapeutic and cosmetic uses. The inventor surprisingly found that the platelet-rich-plasma (PRP) compositions described herein are stable for longer than other PRP preparations as compared to prior art methods. This was unexpected because known methods of producing PRP resulted in PRP that had to be used within about 8-12 hours of isolation. Further, the composition comprising platelet- rich-plasma (PRP) described herein unexpectedly maintain their effectiveness in therapeutic uses, methods, and compositions for an extended period of time (e.g., longer than 30 days). The PRP compositions may be stable for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, or 70. The PRP compositions may be stable for at least 1, 2, 3, 4, 5, or 6 months. The PRP compositions may be stable for at least 30 days or 3 months.

[0157] The PRP composition may be delivered at any suitable dose. The dose may be between in a volume of about 1-3 cc (mL), between about 3-5 cc, between about 5-10 cc, between about 10-20 cc. The dose may be delivered according to a medical procedure (e.g., at specific points in a procedure) and/or according to a schedule. For example, prior to an elective surgical procedure, the PRP composition may be delivered about 24 hours, about 12 hours, about 6 hours, about 2 hours, and/ or about 1 hour before the procedure begins. The PRP may be admixed with the appropriate base (e.g., cream, lotion, serum, or powder) to form an autologous PRP composition and then administered to the patient.

[0158] The composition comprising platelet-rich-plasma (PRP) described herein may be used in methods of treating wrist/hand injuries including but not limited to DeQuervaine's tenosynovitis, arthritis, other wrist or finger tendinitis, ligament tears or dysfunction of the fingers, optionally the PRP composition may be applied topically.

[0159] The compositions comprising platelet-rich-plasma (PRP) described herein may be used in methods of treating elbow injuries including but not limited to medial and lateral epicondylitis (tennis & golfers elbow), optionally the PRP composition may be applied topically.

[0160] The compositions comprising platelet-rich-plasma (PRP) described herein may be used in methods of treating hip injuries including but not limited to illiotibial band tendinitis (ΓΓΒ syndrome), psoas tendinitis and bursitis, greater trochanteric bursitis, hip labrum tears, piriformis syndrome, sacroiliac joint dysfunction, and arthritis, optionally the PRP composition may be applied topically.

[0161] The compositions comprising platelet-rich-plasma (PRP) described herein may be used in methods of treating knee injuries including but not limited to patellar tendinitis, patellar femoral syndrome, chondromalacia patella, partially torn or strained major ligaments of knee

(ACL/LCL/MCL), meniscus tears, arthritis, and patellar instability, optionally the PRP composition may be applied topically. [0162] The compositions comprising platelet-rich-plasma (PRP) described herein may be used in methods of treating ankle/foot injuries including but not limited to Achilles tendinitis, peroneal tendinitis, arthritis, recurrent ankle sprains, other foot or ankle tendinitis, optionally the PRP composition may be applied topically.

[0163] The compositions comprising platelet-rich-plasma (PRP) described herein may be used in methods of treating neck injuries including but not limited to whiplash injuries, headaches related to the neck, and arthritis, optionally the PRP composition may be applied topically.

[0164] The compositions comprising platelet-rich-plasma (PRP) described herein may be used in methods of treating back injuries including but not limited to facet joint arthritis, rib problems, pain associated with scoliosis, optionally the PRP composition may be applied topically.

[0165] The platelet-rich-plasma (PRP) compositions described herein may be used for reducing wrinkles, improving rosacea, increase skin thickness, increase skin tome, improve skin texture, and tighten skin, optionally the PRP composition may be applied topically.

[0166] The platelet-rich-plasma (PRP) compositions described herein may be used in patients such as for tissue reconstruction, tissue regeneration, or wound healing. In particular, the platelet-rich-plasma (PRP) compositions described herein may be used with or in lieu of tissue fillers, e.g., for treating gum recession, loss of bone, including e.g., the jaw. Further, the platelet- rich-plasma (PRP) compositions described herein may be used in methods of treating orthopedic problems, nerve injury, tendinitis, osteoarthritis, cardiac muscle injury, bone repair and regeneration, arthritis, sports injuries, multiple sclerosis, wounds, ulcers, ischemic heart failure, rheumatoid arthritis, post-infarct remodeling, plantar fascitis, rotator cuff injuries, and tennis elbow, optionally the PRP composition may be applied topically.

[0167] The compositions comprising platelet-rich-plasma (PRP) may be used in plastic surgery or oral surgery.

[0168] The platelet-rich-plasma (PRP) compositions described herein may be used to promote wound healing or tissue engineering.

[0169] The platelet-rich-plasma (PRP) compositions described herein may be used in methods of treating burns, optionally post-laser treatment burns comprising topically administering an effective amount of a PRP composition described herein.

[0170] The platelet-rich-plasma (PRP) compositions described herein may be used in methods of treating rosacea, psoriasis, acne, eczema, and atopic dermatitis, optionally the PRP composition may be applied topically. Further, the platelet-rich-plasma (PRP) compositions described herein may be used in cosmetic methods treat wrinkles, tone, text, large pores, dullness, or loose skin, optionally the PRP composition may be applied topically.

[0171] The compositions comprising platelet-rich-plasma (PRP) may be used in a method of augmenting soft tissue to provide of a skin defect. The compositions comprising platelet-rich- plasma (PRP) may be used in a method of augmenting soft tissue to provide reduction of a skin defect comprising topically applying to the skin defect a composition comprising platelet-rich- plasma (PRP).

[0172] The compositions comprising platelet-rich-plasma (PRP) may be used in may be applied topically to the skin defect in a method of augmenting soft tissue to provide reduction of a skin defect. The skin defect may be a result of loss of collagen and hyaluronic acid in the skin during the aging process. The skin defect may be a result of premature aging, said premature aging caused by overexposure to sunlight, overexposure to environmental pollutants, smoking tobacco products, exposure to cigarette smoke, poor nutrition, and skin disorders.

[0173] The compositions comprising platelet-rich-plasma (PRP) may be used in a method of augmenting soft tissue to provide reduction of a skin defect, the skin defect may be a dynamic wrinkle, a fine wrinkles or a static wrinkle. The dynamic wrinkle may be a forehead crease, a brow burrow or an eye line (crow's feet). The static wrinkle may be a skin fold wrinkle resulting from sagging skin. The skin defect is a medical condition selected from the group consisting of an acne scar, for example, a "rolling" scar, a "boxcar" scar or an "ice pick" scar, a surgical scar, trauma scar, a large pore and a soft tissue contour defect. The medical condition may be a deformity that requires re-contouring, such as a small tissue defect (e.g., after animal bite(s)) or a deformity related to trauma where the deformity is cosmetically unappealing. The augmentation may be after plastic surgery to achieve symmetry or a desired result.

[0174] The method of augmenting soft tissue to provide long-term reduction of a skin defect, a "long-term" reduction of a skin defect is of a duration of at least one year. A long-term reduction of a skin defect is of a duration of from at least one year to about five years. A long- term reduction of a skin defect is of a duration from about five years to about ten years. A long- term reduction of a skin defect is of a duration from about ten years or longer. [0175] The compositions comprising platelet-rich-plasma (PRP) may be used in a method of reducing wrinkles and scars comprising administering a composition comprising platelet-rich- plasma (PRP) described herein beneath the skin defect.

[0176] The PRP compositions described herein may be used to treat numerous diseases, including, and not limited to, osteoarthritis, sports medicine injuries, including but not limited to tears and sprains of the ligaments and tendons, gingival gum regeneration, dermal treatment of burns and non-healing wounds, rheumatoid arthritis, multiple sclerosis, ALS disease, and bone fractures.

[0177] Additionally, the PRP compositions described herein may be used to formulate compositions for the treatment of loss of bone, optionally the jaw, amyotrophic lateral sclerosis (ALS), arthritis, optionally dermal treatment for burns and non-healing wounds, enterocutaneous fistula (HULPUTC), gingival gum regeneration, gum recession, plantar fascitis, recto-vaginal fistula, rheumatoid arthritis, or wounds.

[0178] Also, the PRP compositions described herein may be used in methods of treatment of the loss of bone, optionally the jaw, amyotrophic lateral sclerosis (ALS), arthritis, optionally dermal treatment for burns and non-healing wounds, enterocutaneous fistula (HULPUTC), gingival gum regeneration, gum recession, plantar fascitis, recto-vaginal fistula, rheumatoid arthritis, or wounds.

[0179] The PRP compositions described herein may be used therapeutic treatment of a number of human conditions and diseases including repair of cartilage in vivo. The PRP compositions described herein may be used in methods of treatment of use with or in lieu of tissue fillers, as a gum recession, loss of bone, including the jaw, treatment of orthopedic problems, treatment of arthritis, treatment of multiple sclerosis, treatment of wounds treatment of plantar fascitis, treatment of rotator cuff, and treatment of tennis elbow.

Devices, Implants, and Biocompatible Polymer Compositions

[0180] The PRP compositions described herein may be used for treatment of a disease, including transplantation, in combination with a device, implant, or biocompatible polymer. The compositions comprising platelet-rich-plasma (PRP) may be used in use of any of the compositions described herein with other suitable compositions, including as mixtures or as separately administered substances, as would be recognized by persons skilled in the art based upon the guidance provided herein. The present invention contemplates the use of the compositions as medical devices or in conjunction with suitable medical devices, including implantable and non-implantable medical devices.

[0181] Biocompatible polymers, for example, include but are not limited to, homopolymers, copolymers, block polymers, cross-linkable or crosslinked polymers, photoinitiated polymers, chemically initiated polymers, biodegradable polymers, and nonbiodegradable polymers. In some embodiments, the PRP compositions described herein may comprise a polymer matrix that is nonpolymerized, to allow it to be combined with a tissue, organ, or engineered tissue in a liquid or semi-liquid state, for example, by injection. In other embodiments, the PRP

compositions described herein may comprise a liquid matrix that may polymerize or

substantially polymerize in situ. In still other embodiments, the PRP compositions described herein may be admixed with the polymer, polymerized or substantially polymerized prior to injection. Such injectable compositions are prepared using conventional materials and methods know in the art. See, e.g. , Knapp, et al. (1977) Plastic and Reconstr. Surg. 60:389-405; Fagien (2000) Plastic and Reconstr. Surg. 105: 362-73 and 2526- 28; Klein, et al. (1984) J. Dermatol. Surg. Oncol. 10: 519-22; Klein (1983) J. Amer. Acad. Dermatol. 9: 224-28; Watson, et al. (1983) Cutis 31 : 543^16; Klein (2001) Dermatol. Clin. 19: 491-508; Klein (1999) Pedriat. Dent. 21 : 449-50; Skorman (1987) J. Foot Surg. 26: 51 15; Burgess (1992) Facial Plast. Surg. 8: 176- 82; Laude, et al. (2000) J. Biomech. Eng. 122: 231-35; Frey, et al. (1995) J. Urol. 154: 812-15; Rosenblatt, et al. (1994) Biomaterials 15: 985-95; Griffey, et al. (2001) J. Biomed. Mater. Res. 58: 10-15; Stenburg, et al. (1999) Scfand. J. Urol. Nephroi. 33: 355-61 ; Sclafani, et al. (2000) Facial Plast. Surg. 16: 29-34; Spira, et al. (1993) Clin. Plast. Surg. 20: 18188; Ellis, et al. (2001) Facila Plast. Surg. Clin. North Amer. 9: 405-11 ; Alster, et al. (2000) Plastic Reconstr. Surg. 105: 2515-28; and U.S. Patent Nos. 3,949,073 and 5,709,854.

[0182] The polymerized or nonpolymerized matrix may comprise collagen, including but not limited to contracted and non-contracted collagen gels, hydrogels comprising, for example, but not limited to, fibrin, alginate, agarose, gelatin, hyaluronate, polyethylene glycol (PEG), dextrans, including dextrans that are suitable for chemical crosslinking, photocrosslinking, or both, albumin, polyacrylamide, polyglycolyic acid, polyvinyl chloride, polyvinyl alcohol, poly(n-vinyl-2-pyrollidone), poly(2-hydroxy ethyl methacrylate), hydrophilic polyurethanes, acrylic derivatives, or pluronics (e.g., polypropylene oxide and polyethylene oxide copolymer.) The fibrin or collagen is autologous or allogeneic with respect to the intended recipient. The skilled artisan will appreciate that the matrix may comprise non-degradable materials, for example, but not limited to, expanded polytetrafluoroethylene (ePTFE), polytetrafluoroethylene (PTFE), polyethyleneterephthalate (PET), polyurethane, polyethylene, polycabonate, polystyrene, silicone, or selectively degradable materials (e.g., poly (lactic-co-glycolic acid; PLGA), PLA, or PGA). See also, Middleton, et al. (2000) Biomaterials 21 : 2335-2346;

Middleton, et al. Medical Plastics and Biomaterials (March/ April 1998) pages 30-37; Handbook of Biodegradable Polymers, Domb, Kost, and Domb, Eds., 1997, Harwood Academic Publishers, Australia; Rogalla (1997) Minim. Invasive Surg. Nurs. 1 1: 67-69; Klein (2001) Facial Plast. Surg. Clin. North Amer. 9: 205-18; Klein, et al. (1985) J. Dermatol. Surg. Oncol. 1 1 : 337-39; Frey, et al. (1995) J. Urol. 154: 812-15; Peters, et al. (1998) J. Biomed. Mater. Res. 43: 422-27; and Kuijpers, et al. (2000) J. Biomed. Mater. Res. 51: 136-45.

[0183] The PRP compositions described herein may be used in methods of making

bioremodelable graft prostheses prepared from cleaned tissue material derived from animal sources. The bioengineered graft prostheses of the invention are prepared using methods that preserve cell compatibility, strength, and bioremodelability of the processed tissue matrix. The bioengineered graft prostheses are used for implantation, repair, or use in a mammalian host. U.S. Patent 6,986,735.

[0184] The PRP compositions described herein may be used in an implantable biodegradable device containing a fibrous matrix, the fibrous matrix being constructed from fibers A and fibers B, wherein fibers A biodegrade faster than fibers B, fibers A and fibers B are present in relative amounts and are organized such that the fibrous matrix is provided with properties useful in repair and/or regeneration of mammalian tissue. U.S. Patent No 7,192,604.

[0185] The platelet-rich-plasma (PRP) compositions described herein may be placed into the recipient and surrounded by a resorbable plastic sheath such as that manufactured by MacroPore Biosurgery, Inc. U.S. Patent Nos. 6,269,716 and 5,919,234. In this setting the sheath would prevent prolapse of muscle and other soft tissue into the area of a bone fracture thereby allowing the emplaced PRP composition to promote repair of the fracture. The beneficial effect may be enhanced by supplementation with additional components such as pro-osteogenic protein growth factors, biological scaffolds, or artificial scaffolds.

[0186] The PRP composition may be delivered to an individual in need thereof by injection using a syringe or catheter. The PRP composition may also be delivered via delivery device such as a dermal patch, a spray device, sutures, stents, screws, plates, or some other implantable medical device such as bioresorbable tissue patch. The PRP composition may be used as a coating or incorporated into the delivery device. The PRP delivery device may be incubated with PRP prior to use. Incubation times may be from a few seconds up to any convenient time such as a few seconds to hours before use, such as less than 1 minute, 5-10 minutes, 10-60 minutes, 1-3 hours, 4-12 hours, 13-24 hours, 1-3 days, or 3-31 days. PRP compositions may be used in conjunction with an ointment, bone graft, or drug.

[0187] The PRP compositions described herein may be used as a tissue repair implant comprising: a tissue carrier matrix comprising a plurality of biocompatible, bioresorbable granules and an effective amount of a PRP composition in association with the tissue carrier matrix, wherein the tissue carrier matrix is in the form of an injectable suspension, and wherein an average maximum outer diameter of the granules is in a range of about 150 to about 600 μηι. U.S. Patent No. 7,316,822 and 7,875,296.

[0188] The PRP compositions described herein may be administered alone or in combination with tissue fillers {e.g., Juvederm) or scaffolds or matrices used to promote tissue regeneration or reconstruction. Injectable dermal fillers provide a noninvasive option for providing a scaffold for PRP compositions in therapeutic methods, uses, and compositions. The PRP compositions may be used in cosmetic compositions used for topical application to the skin to effect rejuvenation and promote radiance, and reduce wrinkling, optionally in combination with a dermal filler. For example, collagen, Autologen® (autologous collagen dispersion), Isolagen® (autologous fibroblast composition), Dermalogen® (injectable human dermal implant material), hyaluronic acid, calcium hydroxyapatite, and/or synthetic poly-lactic acid may be used as a scaffold for PRP compositions in therapeutic methods, uses, and compositions. Additionally, a dermal filler composition comprising an acrylate/methacrylate copolymer may be used as scaffold for PRP compositions in therapeutic methods, uses, and compositions. See U.S. Patent No. 7,910,134. Another permanent microsphere-based injectable dermal filler contains larger non-resorbable microspheres made of polymethyl methacrylate (PMMA), each having a diameter of between 30 and 42 μηι and a smooth surface, and a highly purified bovine collagen gel in a ratio of 20% PMMA to 80% bovine collagen. Routes of Administration

[0189] The PRP compositions described herein may be administered in any of the following routes: epicutaneous, infusion, intraarterial, intracardial, intradermal, intramuscular,

intraperitoneal, intrathecal, intravenous, oral, parenteral, pulmonary, rectally via an enema or suppository, subcutaneous, subdermal, sublingual, topically, via microneedles, and transdermal. The administration can be local, where the composition is administered directly, close to, in the locality, near, at, about, or in the vicinity of, the site(s) of disease or injury. Local administration can be administration to the cell, tissue, organ, and/or organ system, which encompasses and/or is affected by the disease or injury, and/or where the disease signs and/or symptoms are active or are likely to occur (e.g. , topically). Administration can be topical with a local effect, composition is applied directly where its action is desired.

[0190] The PRP composition may be delivered to an individual in need thereof by convention means which include injection using a syringe or catheter. The PRP composition may also be delivered via a dermal patch, a spray device or in combination with an ointment, bone graft or drug. It may further be used as a coating on suture, stents, screws, plates or some other implantable medical device. Finally, it may be used in conjunction with a bioresorbable drug or device.

[0191] The PRP compositions described herein may be administered directly into the patient. In other words, the PRP may be administered to the patient without being removed from the system or exposed to the external environment of the system before being administered to the patient. Providing a closed system reduces the possibility of contamination of the material being administered to the patient. Thus, processing the tissue in a closed system provides advantages because the PRP is more likely to be sterile. The only time the PRP is exposed to the external environment, or removed from the system, is when the PRP are being withdrawn into an application device and admixed to form a PRP composition before being administered to the patient. The application device can also be part of the closed system. For example, the blood may be drawn, centrifuged, and admixed to form a PRP composition and then administered to the patient. Thus, the PRP used need not be stored, transported, or cryopreserved.

[0192] The site of delivery of the platelet-rich plasma composition is at or near the site of tissue injury and/or damage. The site of tissue injury or damage is determined by well-established methods including imaging studies and patient feedback or a combination thereof. The preferred imaging study used is determined by the tissue type. Commonly used imaging methods include, but are not limited to MRI, X-ray, CT scan, Positron Emission tomography (PET), Single Photon Emission Computed Tomography (SPECT), Electrical Impedance Tomography (ΕΓΓ), Electrical Source Imaging (ESI), Magnetic Source Imaging (MSI), laser optical imaging and ultrasound techniques. The patient may also assist in locating the site of tissue injury or damage by pointing out areas of particular pain and/or discomfort.

[0193] The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

EXAMPLES

Example 1

Preparation of PRP

[0194] 2-20 cc (mL) of blood may be drawn from a patient into PST plasma separation blood tubes. The blood may be centrifuged at appropriate centrifuge speed and time (5-20 min at 1100-1300 rpm). The PRP may be withdrawn using a 3 cc syringe. This method may be used to produce PRP to be admixed to form a PRP composition described herein.

Example 2

Preparation of PRP

[0195] PRP may be prepared using a centrifuge unit made by Harvest (Plymouth, Mass.).

(Similar units are available as The Biomet GPS system, the Depuy Symphony machine and the Medtronic Magellan machine.) Approximately 55 cc of blood may be drawn from a patient using a standard sterile syringe, combined with 5 cc of a citrate dextrose solution for anticoagulation, and then centrifuged to isolate the platelets. These platelets may then resuspended in

approximately 3 cc of plasma. The resulting platelet rich plasma solution (PRP) may be acidic and may be neutralized with using approximately 0.05 cc of an 8.4% sodium bicarbonate buffer per cc of PRP under sterile conditions to approximately physiologic pH of 7.4. This method may be used to produce PRP to be admixed to form a PRP composition described herein.

Example 3

Preparation of PRP [0196] A 5 mL syringe containing 0.35 mL of 10% sodium citrate may be used to collect 3.15 mL of blood. The blood may be centrifuged at 160 G for 20 minutes at 22°C. After

centrifugation, a red lower fraction (red cell component) and an upper yellow turbid fraction (serum component) may be observed. A point may be marked below the line dividing the two fractions and all of the content about this point may be removed and transferred to another 5 mL tube. The sample may then be centrifuged at 400 G for 15 minutes resulting in two components: a top platelet-poor plasma (PPP) and below a platelet-rich plasma (PRP). The PRP may be removed and activated using 0.05 mL of 10% calcium chloride solution for each 1 mL of PRP. See Messora, et al. (2011) RSBO 8(3): 299-304. This method may be used to produce PRP to be admixed to form a PRP composition described herein.

Example 4

Preparation of Neutrophil-depleted PRP Releasate

[0197] Fifty cc of whole blood may be drawn from a patient, and prepared according to the method of described in U.S. Patent No. 5,165,938. The PRP may be activated using recombinant human thrombin. The degranulated platelets may be spun down and the releasate containing supernatant is recovered. Neutrophils may be removed from the PRP composition by methods known in the art. The neutrophil-depleted releasate may be optionally pH adjusted to a pH of 7.4 using sodium bicarbonate buffer. U.S. Patent 8,142,993. This method may be used to produce neutrophil-depleted PRP to be admixed to form a neutrophil-depleted PRP composition described herein.

Example 5

Preparation of Neutrophil-depleted PRP

[0198] Thirty mL of whole blood may be drawn from a patient. A platelet composition may be obtained using methods known in the art except that no alginate is added to the platelet composition. Neutrophils may be removed from the PRP composition by methods known in the art obtain neutrophil-depleted PRP. U.S. Patent 8,142,993. This method may be used to produce neutrophil-depleted PRP to be admixed to form a neutrophil-depleted PRP composition described herein.

Example 6

Preparation of PRP Skin Care Serum Formulas [0199] About 2-20 cc of blood may be drawn from a patient into PST plasma separation blood tubes. The tube may be spun at an appropriate centrifuge speed and time (e.g., 5-20 min at 1100-1300 rpm). The PRP may be collected using a 3 cc syringe. A predetermined amount of PRP may be used to compound skin care serum formulas. The skin care serum formula may be autologous.

Example 7

Treatment of lateral epicondylitis (tennis elbow)

[0200] A PRP composition may be topically administered to patients presenting with lateral epicondylitis (tennis elbow). The patients may be evaluated to obtain a visual pain score, a Mayo Elbow Score, and grip strength. For the visual analog pain score, zero equals "no pain" and 100 equals "the worst pain possible." The Mayo elbow score is an overall function score with a higher score indicating better overall function. The values of the two scores are statistically evaluated using a paired sample T test with significance set at p<0.05.

[0201] Approximately 3-5 mL of the PRP composition described herein may be applied topically to the patient's elbow. After the administration of the PRP composition, the patient's arm may be immobilized at about 90% of flexion without elevation of the arm or hand. The patient asked not to move their arm for 30 minutes. Each patients' neurovascular, pain and function status may evaluated shortly after the injection, and at 30 minutes following the end of the procedure. Each patient may be given oral narcotic pain medication as needed for the first 24-48 hours after the procedure. A formal postoperative stretching and strengthening program may be initiated at 2-3 days after the procedure. The visual pain score, Mayo Elbow Score, and grip strength may be monitored post-procedure. Thus, the PRP compositions described herein may be used to treat lateral epicondylitis (tennis elbow).

[0202] All publications (e.g., Non-Patent Literature), patents, patent application publications, and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All such publications (e.g. , Non-Patent Literature), patents, patent application publications, and patent applications are herein incorporated by reference to the same extent as if each individual publication, patent, patent application publication, or patent application was specifically and individually indicated to be incorporated by reference. [0203] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

We claim:

1. A stable composition comprising platelet-rich-plasma (PRP).

2. The composition of claim 1, wherein said composition is stable for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days.

3. The composition of claim 1, wherein said composition is stable for at least about 1, 2, 3, 4, 5, or 6 months.

4. The composition of claim 1, wherein said composition is stable for at least about 3 months.

5. The composition of claim 1, wherein said composition further comprises hematopoietic elements.

6. The composition of claim 1 , wherein said composition has a platelet count of at least about 250,000 to about 7,000,000 platelets per microliter.

7. The composition of claim 1, wherein said composition has a platelet count of at least about 250,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,100,000; 1 ,250,000; 1,500,000; 1,750,000; 2,000,000; 2,500,000; 2,600,000; 3,000,000; 3,500,000; 4,000,000; 5,000,000; 5,200,000; 6,000,000; or 7,000,000 platelets per microliter.

8. The composition of claim 1, wherein said composition has a platelet count of at least about 500,000-7,000,000 platelets per microliter.

9. The composition of claim 1, wherein said composition has a platelet count of at least about 500,000-700,000; 700,000-900,000; 900,000-1,000,000; 1,000,000-1 ,250,000;

1,250,000-1,500,000; 1,500,000-2,500,000; 2,500,000-5,000,000; or 5,000,000-7,000,000 platelets, optionally about 2,600,000 or 5,200,000 plateletsOO.

10. The composition of claim 1, wherein said platelet-rich-plasma is activated, optionally by calcium chloride or thrombin.

11. The composition of claim 1, wherein said composition is in a form selected from the group consisting of a balm, solution, suspension, emulsion, ointment, foam, paste, gel, cream, lotion, powder, salve, soap, surfactant-containing cleansing, oil, serum, drops, liposomes, nanoparticles, nanoboots, and spray.

12. The composition of claim 11, wherein said composition is a cream, lotion, or solution.

13. The composition of claim 1, wherein said composition is impregnated or made part of a bandage.

14. The composition of claim 13, wherein the bandage is a surgical dressing, a plaster bandage, an adhesive bandage, or a gauze.

15. The composition of claim 1, wherein said composition further comprises a component selected from the group consisting of monocytes, stem cells, gene therapy products, vitamins, palmitate retinol, tocoferil acetate, sodium ascorbil phosphate, D-panthenol, peptides, recombinant growth factors, micronized human-identical hormones, aminoacids, phyto-extracts, antioxidants, lipoic acid, DMAE, collagen, GAG, hyaluronic acid, proteoglycans, adenine, guanine, cytosine, thymine, trace elements, minerals, proteases, ceramides, polisaccarides, algae, and marine extracts.

16. The composition of any one of claims 1-15, wherein said composition further comprises white blood cells (WBCs) at a higher concentration than white blood cells in whole blood.

17. The composition of claim 16, wherein said composition comprises about 8,000-10,000;

10,000-15,000; 15,000-20,000; 20,000-30,000; 30,000-50,000; 50,000-75,000; or 75,000-100,000 white blood cells per microliter.

18. The composition of any one of claims 1-15, wherein said composition further comprises lymphocytes at a higher concentration than lymphocytes in whole blood.

19. The composition of claim 18, wherein said composition comprises about 5,000-20,000 lymphocytes per microliter.

20. The composition of any one of claims 1-15, wherein said composition further comprises monocytes at a higher concentration than monocytes in whole blood.

21. The composition of claim 20, wherein said composition comprises about 1,000-5,000 per monocytes microliter.

22. The composition of any one of claims 1-15, wherein said composition further comprises eosinophils at a higher concentration than eosinophil in whole blood.

23. The composition of claim 22, wherein said composition comprises about 200-1,000

eosinophils per microliter.

24. The composition of any one of claims 1-15, wherein said composition further comprises neutrophils at a lower concentration than neutrophils in whole blood.

25. The composition of claim 24, wherein said composition comprises about 200-1,000 neutrophils per microliter.

26. The composition of any one of claims 1-15, wherein the neutrophil content of the platelet- rich plasma has been reduced by 50-99% as compared to whole blood, optionally reduced by 50%, 60%, 70%, 80%, 90%, 95%, or 99% as compared to whole blood.

27. The composition of any one of claims 1-15, wherein said composition further comprises red blood cells at a lower concentration than red blood cells in whole blood.

28. The composition of claim 27, wherein said composition comprises about 4,600-5,200 red blood cells per microliter.

29. The composition of any one of claims 1-15, wherein said composition further comprises hemoglobin at a lower concentration than hemoglobin in whole blood.

30. The composition of claim 29, wherein said composition comprises a hemoglobin

concentration at about 1 g/dL or less, between about 1-5 g/dL, about 5-10 g/dL, about 10- 15 g/dL, or about 15-20 g/dL.

31. The composition of any one of claims 1-30, wherein said composition further comprises fibrin, fibronectin, or vitronectin.

32. The composition of any one of claims 1-31, wherein said platelet-rich-plasma is

autologous.

33. The composition of any one of claims 1-31, wherein said composition is a pharmaceutical composition further comprising an pharmaceutically acceptable excipient.

34. The composition of claim 33, wherein said excipient is isotonic sodium chloride solution, physiological saline, phosphate buffered saline, potassium buffered saline, normal saline, dextrose 5% in water, dextrose 10% in water, Ringer solution, lactated Ringer solution, Ringer lactate, or Ringer lactate solution.

35. The composition of any one of claims 1-34, wherein said composition has a pH of about 6.0-8.0, optionally a pH of about 7.4.

36. A cosmetic cream comprising the composition of any one of claims 1-36.

37. A cosmetic lotion comprising the composition of any one of claims 1-36.

38. A cosmetic solution comprising the composition of any one of claims 1-36.

39. A pharmaceutical composition comprising the composition of any one of claims 1-36 and an pharmaceutically acceptable excipient.

40. A medical device comprising the composition of any one of claims 1-36.

41. An implant comprising the composition of any one of claims 1-36.

42. A biocompatible polymer comprising the composition of any one of claims 1-36.

43. Use of the composition of any one of claims 1-39 in the manufacture of cosmetic surgery products, optionally comprising dermal fillers.

44. A method for cosmetic surgery comprising administering the composition of any one of claims 1-39.

45. A composition for cosmetic surgery comprising an effective amount of the composition of any one of claims 1-39.

46. Use of the composition of any one of claims 1-39 in the manufacture of cosmetic surgery products, optionally comprising dermal fillers, for the reduction of a skin defect.

47. A method for reducing a skin defect comprising administering the composition of any one of claims 1-39.

48. A composition for reduction of a skin defect comprising an effective amount of the

composition of any one of claims 1-39.

49. Use of the composition of any one of claims 1-39 in the manufacture of cosmetic surgery products, optionally comprising dermal fillers, for reducing wrinkles, improving rosacea, increase skin thickness, increase skin tome, improve skin texture, and tighten skin.

50. A method for reducing wrinkles, improving rosacea, increase skin thickness, increase skin tome, improve skin texture, and tighten skin comprising administering the composition of any one of claims 1-39.

51. A composition for reducing wrinkles, improving rosacea, increase skin thickness, increase skin tome, improve skin texture, and tighten skin comprising an effective amount of the composition of any one of claims 1-39.

52. The use, method, or composition of any one of claims 43-51, wherein said use, method, or composition is for the reduction of a skin defect, the skin defect may be a dynamic wrinkle, a fine wrinkles or a static wrinkle.

53. The use, method, or composition of any one of claims 43-51 , wherein the dynamic wrinkle is a forehead crease, a brow burrow or an eye line (crow's feet).

54. The use, method, or composition of any one of claims 43-51, wherein the static wrinkle is a skin fold wrinkle resulting from sagging skin.

55. The use, method, or composition of any one of claims 43-51, wherein the skin defect is a medical condition selected from the group consisting of an acne scar, optionally a "rolling" scar, a "boxcar" scar or an "ice pick" scar, a surgical scar, trauma scar, a large pore and a soft tissue contour defect.

56. The use, method, or composition of any one of claims 43-51, wherein the wrinkle or scar is the result of loss of collagen and hyaluronic acid in the skin during the aging process.

57. The use, method, or composition of any one of claims 43-51, wherein the wrinkle or scar is the result of premature aging, optionally premature aging caused by overexposure to sunlight, overexposure to environmental pollutants, smoking tobacco products, exposure to cigarette smoke, poor nutrition, and skin disorders.

58. Use of the composition of any one of claims 1-39 in the manufacture of a medicament for the treatment of a disease.

59. A method of treating a disease comprising administering the composition of any one of claims 1-39.

60. A composition for treating a disease comprising an effective amount of the composition of any one of claims 1-39.

61. A method for treating a disease comprising administering the composition of any one of claims 1-39.

62. A method for treating a disease comprising administering the composition of any one of claims 1-39.

63. A pharmaceutical composition for the treatment of a disease comprising the composition of any one of claims 1-39.

64. A pharmaceutical composition for the treatment of a disease the composition of any one of claims 1-39.

65. Use of the composition of any one of claims 1-39 in the manufacture of a medicament for treating an injury.

66. A method for treating an injury comprising administering the composition of any one of claims 1-39.

67. A composition for treating an injury comprising an effective amount of the composition of any one of claims 1-39.

68. The use, method, or composition of any one of claims 65-67, wherein the injury is a shoulder injury, optionally Rotator Cuff Tendinitis or Tear, Rotator Cuff Impingement Syndrome or Bursitis, Bicipital Tendinitis, labrum tears, arthritis, and instability.

69. The use, method, or composition of any one of claims 65-67, wherein the injury is a

wrist/hand injury optionally DeQuervaine's Tenosynovitis, arthritis, other wrist or finger tendinitis, ligament tears, or dysfunction of the fingers.

70. The use, method, or composition of any one of claims 65-67, wherein the injury is an

elbow injury, optionally medial and lateral epicondylitis (tennis & golfers elbow).

71. The use, method, or composition of any one of claims 65-67, wherein the injury is hip

injury, optionally Illiotibial Band Tendinitis (ΓΤΒ Syndrome), Psoas Tendinitis and bursitis, Greater Trochanteric Bursitis, Hip labrum tears, Piriformis Syndrome, Sacroiliac Joint Dysfunction, and arthritis.

72. The use, method, or composition of any one of claims 65-67, wherein the injury is a knee injury, optionally Patellar Tendinitis, Patellar Femoral Syndrome, chondromalacia patella, partially torn or strained major ligaments of knee (ACL LCL MCL), meniscus tears, arthritis, and patellar instability.

73. The use, method, or composition of any one of claims 65-67, wherein the injury is an

ankle/foot injury, optionally Achilles Tendinitis, Peroneal Tendinitis, arthritis, recurrent ankle sprains, other foot or ankle tendinitis.

74. The use, method, or composition of any one of claims 65-67, wherein the injury is a neck injury optionally Whiplash injuries, headaches related to the neck, and arthritis.

75. The use, method, or composition of any one of claims 65-67, wherein said injury is a back injury, optionally facet joint arthritis, rib problems, or pain associated with scoliosis.

76. The use, method, or composition of any one of claims 65-67, wherein said disease or injury is gum recession, loss of bone, including the jaw, bone fractures, dermal treatment for burns and non-healing wounds, post-laser treatment burns, enterocutaneous fistula

(HULPUTC), gingival gum regeneration, hair loss, optionally in men and/or women, gum recession, orthopedic problems, osteoarthritis, plantar fascitis, recto-vaginal fistula, rotator cuff injuries, sports medicine injuries, optionally tears and sprains of the ligaments and tendons, tennis elbow, ulcers, and non-healing wounds.

77. Use of the composition of any one of claims 1-39 in the manufacture of an medicament for skin repair, skin regeneration, skin protection, preventing hair loss, for promoting the physiological growth of hair, reducing photoaging, reducing skin oxidative stress, regenerating the hair bulb cells and scalp.

78. A method for skin repair, skin regeneration, skin protection, preventing hair loss, for

promoting the physiological growth of hair, reducing photoaging, reducing skin oxidative stress, regenerating the hair bulb cells and scalpcomprising administering the composition of any one of claims 1-39.

79. A composition for skin repair, skin regeneration, skin protection, preventing hair loss, for promoting the physiological growth of hair, reducing photoaging, reducing skin oxidative stress, regenerating the hair bulb cells and scalp comprising an effective amount of the composition of any one of claims 1-39.

80. Use of the composition of any one of claims 1-39 in the manufacture of a medicament for the treatment of a Escherichia coli, Staphylococcus aureus, optionally methicillin-resistant Staphylococcus aureus, Candida albicans, or Cryptococcus neoformans infection.

82. A method for the treatment of a Escherichia coli, Staphylococcus aureus, optionally

methicillin-resistant Staphylococcus aureus, Candida albicans, or Cryptococcus neoformans infection comprising administering an effective amount of the composition of any one of claims 1-39.

84. A composition the treatment of a Escherichia coli, Staphylococcus aureus, optionally methicillin-resistant Staphylococcus aureus, Candida albicans, or Cryptococcus neoformans infection comprising an effective amount of the composition of any one of claims 1-39.

85. A method for formulating a topical skin cream comprising isolating platelet-rich-plasma from a patient and compounding to form a topical skin cream comprising autologous platelet-rich plasma.

86. The use, method, or composition of any one of claims 43-85, wherein said PRP

composition is administered topically.

87. The use, method, or composition of any one of claims 43-85, wherein said PRP

composition is administered via microneedles.

88. The use, method, or composition of any one of claims 43-85, wherein the platelet-rich- plasma are used in a cosmetic surgery application, to promote wound healing, are used in a tissue filler, tissue engineering, or burn treatment.

89. The use, method, or composition of any one of claims 43-85, wherein said use, method, or composition comprises an anesthetic, optionally anbesol, benzocaine, lidocaine, procaine, or bupivicaine.

90. The use, method, or composition of any one of claims 43-85, wherein said use, method, or composition results in the release of cytokines by PRP.

91. The use, method, or composition of claim 86, wherein said cytokines are selected from the group consisting of platelet-derived growth factor, transforming growth factor beta, fibroblast growth factor, insulin-like growth factor 1 (IGF1), insulin-like growth factor 2 (IGF2), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), interleukin 8 (IL-8), keratinocyte growth factor (KGF), and connective tissue growth factor.

92. The use, method, or composition of any one of claims 43-85, wherein said platelet-rich- plasma is autologous.

93. The use, method, or composition of any one of claims 43-85, further comprising

administering a platelet activator selected from the group consisting of thrombin, calcium, collagen, epinephrine, adenosine diphosphate, and mixtures thereof.

94. The use, method, or composition of claim 89, wherein the thrombin is autologous to the subject.

95. The use, method, or composition of claim 89, wherein administering a platelet activator further comprises administering fibrinogen.

96. The use, method, or composition of any one of claims 43-85, further comprises or

comprises administering an isolated angiogenic factor selected from the group consisting of angiogenin, angiopoietin-l, del-1 protein, acidic fibroblast growth factor (aFGF or FGF-1), basic fibroblast growth factor (bFGF or FGF-2), follistatin, granulocyte colony-stimulating factor (G-CSF), hepatocyte growth factor (HGF), interleukin-8 (IL-8), leptin, midkine, placental growth factor, platelet-derived endothelial growth factor (PD-ECGF), platelet- derived growth factor (PDGF), pleiotrophin (PTN), progranulin, proliferin, transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-β), tumor necrosis factor alpha (TNF-a), vascular endothelial growth factor (VEGF), vascular permeability factor (VPF), and combinations thereof.

97. The use, method, or composition of any one of claims 43-85, wherein said use, method, or composition comprises treatment alone or in combination with tissue fillers.

98. The use, composition, or method of any one of claims 43-85, wherein the PRP comprises activated platelets that release cytokines, optionally IL-1B, IL-6, TNF-A, chemokines, optionally ENA-78 (CXCL5), IL-8 (CXCL8), MCP-3 (CCL7), ΜΓΡ-1Α (CCL3), NAP-2 (CXCL7), PF4 (CXCL4), RANTES (CCL5), inflammatory mediators, optionally PGE2, and growth factors, optionally Angiopoitin-1, bFGF, EGF, FGF, HGF, IGF-I, IGF-II, PDAF, PDEGF, PF-4, PDGF AA and BB, TGF-beta 1 , 2, and 3, and VEGF.

99. An anti-aging serum, rosacea serum, treatment mask, post-laser treatment mask,

and cleansing solution for skin brushes comprising the composition of any one of claims 1- 39.

100. The anti-aging serum, rosacea serum, treatment mask, post-laser treatment mask,

and cleansing solution for skin brushes of claim 99, wherein said PRP is autologous.